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Redox control of chondrocyte differentiation and chondrogenesis
Author’s Accepted Manuscript Redox Control of Chondrocyte Differentiation and Chondrogenesis Yun Bai, Xiaoshan Gong, Ce Dou, Zhen Cao, Shiwu Dong www.elsevier.com
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S0891-5849(18)32272-X https://doi.org/10.1016/j.freeradbiomed.2018.10.443 FRB14007
To appear in: Free Radical Biology and Medicine Received date: 15 April 2018 Revised date: 14 October 2018 Accepted date: 26 October 2018 Cite this article as: Yun Bai, Xiaoshan Gong, Ce Dou, Zhen Cao and Shiwu Dong, Redox Control of Chondrocyte Differentiation and Chondrogenesis, Free Radical Biology and Medicine, https://doi.org/10.1016/j.freeradbiomed.2018.10.443 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Redox Control of Chondrocyte Differentiation and Chondrogenesis
Yun Baia1, Xiaoshan Gonga1, Ce Doua, Zhen Caoa, Shiwu Donga,b*
1
Department of Biomedical Materials Science, School of Biomedical Engineering, Third
Military Medical University, Chongqing 400038, China. 2
State Key Laboratory of Trauma, Burns and Combined Injury, Third Military Medical
University, Chongqing 400038, China.
*
Corresponding author. Shiwu Dong, Department of Biomedical Materials Science,
School of Biomedical Engineering, Third Military Medical University, Gaotanyan Street No.30, Chongqing 400038, China. Tel.: +86 2368771270. [email protected]
Abstract Chondrogenesis involves the recruitment and migration of mesenchymal cells, mesenchymal condensation, and chondrocyte differentiation and hypertrophy. Multiple factors precisely regulate chondrogenesis. Recent studies have demonstrated that the redox status of chondrocytes plays an essential role in the regulation of chondrocyte differentiation and chondrogenesis. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are important factors that change the intracellular redox status. Physiological levels of ROS/RNS act as intracellular signals in chondrocytes, and oxidative stress impairs the metabolism of chondrocytes. Under physiological conditions, the balance between ROS/RNS production and elimination
1
These authors contributed equally to this work.
ensures that redox-sensitive signalling proteins function correctly. The redox homeostasis of chondrocytes ensures that they respond appropriately to endogenous and exogenous stimuli. This review focuses on the redox regulation of key signalling pathways and transcription factors that control chondrogenesis and chondrocyte differentiation. Additionally, the mechanism by which ROS/RNS regulate signalling proteins and transcription factors in chondrocytes is also reviewed.
Keywords:
reactive
oxygen
species
(ROS),
chondrogenesis,
chondrocyte
differentiation
1. Introduction Chondrogenesis is the earliest stage of skeletal development. This stage involves the recruitment and migration of mesenchymal cells, mesenchymal condensation, and chondrocyte differentiation and maturation and results in the formation of cartilage and bone during endochondral bone formation [1, 2]. During endochondral ossification, chondrocyte hypertrophy is critical because cells alter the extracellular matrix and induce vascular invasion [3, 4]. This process includes the differentiation and hypertrophic differentiation of chondrocytes, which are precisely controlled by various growth factors and specific gene transcription factors through temporal and spatial activation or inhibition of cell signalling pathways. Chondrocytes may respond differently to growth factors due to their intracellular redox status. In general, reactive oxygen species (ROS) and reactive nitrogen species (RNS) are the major factors that affect intracellular redox status. Redox homeostasis is essential in maintaining the survival and function of chondrocytes. When the balance between ROS generation and elimination is destroyed, oxidative stress occurs. Oxidative stress has long been considered a significant factor in the induction of chondrocyte senescence and apoptosis in diseases such as osteoarthritis (OA) [5, 6], and the role of ROS/RNS in the pathogenesis of osteoarthritis has become a primary concern of researchers [7, 8]. However, many recent studies have begun to concentrate on the role of physiological levels of ROS in chondrocyte differentiation and metabolism. This review mainly
focuses on the redox regulation of signal transduction pathways and transcription factors that control chondrocyte differentiation.
2. Redox Status in Chondrocytes ROS include radical species such as superoxide (O2•−) and hydroxyl radicals (HO•) and non-radical species such as H2O2. These radicals are different from O2, with one, two, and three extra electrons each. Superoxide anion radicals in chondrocytes are produced by the enzyme complex NADPH oxidase, which adds a single electron to molecular oxygen. It is demonstrated that porcine articular chondrocytes can produce ROS using the NADPH-oxidase-like complex [9, 10]. After superoxide production, superoxide dismutase (SOD) dissipates superoxide quickly, producing H2O2 and O2 [11]. The reaction of divalent iron ions with H2O2 produces HO•, which is called the Fenton reaction [12]. RNS include nitric oxide (NO) and peroxynitrite (ONOO•). Nitric oxide is produced by nitric oxide synthase (NOS) and mediates different physiological processes. To date, three forms of NOS have been characterized: neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS). Chondrocytes express both eNOS and iNOS [13]. The eNOS are constitutively expressed and are stimulated by increased intracellular calcium levels to produce small amounts of NO. In chondrocytes, iNOS is expressed is in response to the presence of inflammatory cytokines [14, 15]. Calcium levels do not regulate the activity of iNOS; therefore, even in the presence of low concentrations of calcium, it may produce a significant amount of NO [16]. Nitric oxide is highly reactive with free radical species such as O2•−. O2•− reacts with NO to produce ONOO•, which is highly oxidizing (Figure 1). Chondrocytes, similar to other mammalian cells, have enzymatic and non-enzymatic antioxidant systems to eliminate ROS/RNS and maintain redox balance [17]. Superoxide dismutase (SOD) is a significant class of enzymatic antioxidants that catalyse the dissipation of O2•− to H2O2 [18]. After H2O2 production, another enzyme responsible for H2O2 removal is catalase, which is located in the peroxisome. Hydrogen peroxide is further converted to H2O and O2 by catalase. Through the coupling reaction of glutathione peroxidase (GPX), which catalyses the
conversion of reduced glutathione (GSH) to oxidized glutathione (GSSG), H2O2 can also be converted to O2. Multiple GPXs exist in different cells. Among them, glutathione peroxidase 1 (GPX1) is the primary scavenger of H2O2 and lipid hydroperoxide. Non-enzymatic antioxidants also play essential roles in maintaining the redox balance of cells. GSH is the most abundant peptide in cells and exhibits many functions to scavenge free radicals. GSH can directly remove HO• and singlet oxygen and help antioxidants regenerate into their active forms, such as vitamins C and E [19]. The thioredoxin system is another crucial thiol antioxidant and is composed of thioredoxin (Trx) and thioredoxin reductase. The thioredoxin system, in cooperation with the GSH system, plays a vital role in reducing oxidized thiol-containing proteins [20]. Oxidative stress occurs when the balance between ROS/RNS production and the antioxidant defence system is destroyed. An increase in ROS production or a decrease in ROS scavenging capacity due to exogenous or endogenous metabolic changes can lead to increased intracellular ROS levels and, thus, oxidative stress. Many studies have shown that increased oxidative stress plays an essential role in pathological conditions including cancer, degenerative motor diseases, and cardiovascular diseases [21]. Under normal circumstances, physiological levels of ROS/RNS can reversibly modify biomacromolecules, which play crucial roles in regulating cell function. However, under oxidative stress, excess ROS/RNS continually attack lipids, proteins, and DNA, resulting in severe and irreversible oxidative damage. In osteoarthritis, the over-produced ROS act as second messengers to promote the expression of MMPs, which lead to cartilage degradation. Furthermore, ROS directly degrade matrix components and induce cell death [8].
3. ROS/RNS Functions in Chondrocyte Differentiation and Chondrogenesis Chondrogenesis is a dynamic and precisely regulated process. After the condensation of mesenchymal progenitor cells, these cells differentiate into chondrocytes and secrete cartilage matrix to form cartilage [22]. Cartilage is replaced by bone tissue and bone marrow to generate mature bone, a process known as endochondral ossification. Chondrogenesis is initiated by the condensation of
mesenchymal stem cells, followed by the expression of chondrocyte-specific genes such as transcription factor Sox9 and genes encoding chondrocyte extracellular matrix proteins (collagen II and aggrecan) [23]. Under the control of several growth factors, chondrocytes eventually exit the cell cycle and begin to differentiate into hypertrophic chondrocytes. Late hypertrophic chondrocytes begin to express many genes associated with cartilage matrix degradation and bone formation, such as matrix metalloproteinase 13 (Mmp13) and vascular endothelial growth factor (VEGF) [24]. This process results in the degradation of the hypertrophic cartilage matrix and its replacement by bone tissue, thereby completing the process of endochondral ossification. Therefore, chondrogenesis includes different stages of chondrocyte differentiation such as mesenchymal progenitor cells condensation, cartilage formation,
chondrocyte
proliferation,
and
hypertrophic
differentiation
of
chondrocytes. Recent studies have revealed that ROS function as critical signalling molecules in the regulation of cell growth and differentiation [25-27] (Figure 2). In endochondral ossification, Nox1 and Nox4 are expressed in different regions of the growth plate. Nox4 expression in chondrocytes was observed from the resting zone to the hypertrophy region, while Nox1 expression was restricted from the proliferative zone to the pre-hypertrophic chondrocyte zone [28]. Nox4 is required early in cartilage differentiation, and increasing the expression of Nox1 and decreasing the expression of Nox4 are characteristics of chondrogenesis. Therefore, in chondrocyte hypertrophy, ROS produced by Nox1 may play a major role compared to Nox4. [28, 29]. Therefore, chondrocytes always produce ROS throughout the endochondral ossification and emit large amounts of reactive oxygen species from the proliferative zone to the pre-hypertrophic zone. Importantly, it has been proved that the ROS that are generated in response to insulin stimulation are required for chondrogenic differentiation. Kim et al. demonstrated that the ROS that are required for early chondrogenic differentiation were mainly derived from Nox2 and Nox4. After the knockdown of Nox2 or Nox4 in ATDC5 cells, a chondrogenic cell line, the ROS production was decreased, and the insulin-induced chondrocyte differentiation was blocked. These results proved that ROS generated by NOXs were required for early
chondrogenic differentiation[29]. Chondrocyte hypertrophy is the critical stage in chondrogenesis and endochondral ossification. During this process, the proliferating chondrocytes leave the cell cycle and enter the pre-hypertrophic differentiation phase. There is evidence that ROS play a significant role in chondrocyte hypertrophy [30]. Chondrocyte hypertrophy was inhibited by administration of the antioxidant NAC in ATDC5 cells and mice, indicating that physiological levels of ROS determine chondrocyte hypertrophy [30]. Additionally, during chondrocyte hypertrophy, the expression of NOX1, which is responsible for ROS production, was increased. These results proved that early chondrocyte differentiation and hypertrophy was stimulated by the expression of ROS-producing genes, such as NOXs. As a result, the intracellular ROS that were generated by NOXs can alter the cellular response to growth factor cytokines by mediating the redox-sensitive signalling pathways and expression of transcription factors during chondrogenesis [31]. Similar to ROS, NO has been proved to participate in endochondral ossification. Teixeira et al. showed that excess NO promotes hypertrophic differentiation and apoptosis of chondrocytes in chickens [32, 33]. Wang et al. suggest that the basal levels of NO that are generated by iNOS are required for chondrogenesis [34]. Recent evidence convincingly argues that these ROS/RNS play essential roles in numerous physiological and pathological processes [35]. The central mechanism of the redox regulation of proteins is oxidative modification [36]. The mechanism of redox regulation of proteins is through various types of oxidative modifications on the redox-sensitive sites of proteins, and the result may be the activation or inactivation of protein functions. ROS/RNS can directly oxidize various redox-sensitive amino acids, with cysteine (Cys) being the main target molecule. Mild oxidative stress can induce cysteine modifications such as reversible [37, 38] disulfide bond formation [39] and S-nitrosylation [40, 41]. These reversible redox post-translational modifications have proved to be essential signals in many cells. In contrast, oxidative stress promotes the modification of sulfenic, sulfinic and sulfonic acids in proteins, which are destructive to protein function [42] (Figure 3). To date, it has been demonstrated that redox signalling can control chondrogenesis by regulating transcription factors and signal transduction pathways. ROS/RNS can
modulate the ability of transcription factors to bind to DNA by modifying the Cys residues contained in the DNA-binding sites of transcription factors [39]. Additionally, oxidative modification plays a reversible regulatory role in the function of many protein tyrosine phosphatases, which can regulate chondrogenesis and chondrocyte functions.
4. Redox Control of Signal Pathways Involved in Chondrocyte Differentiation and Chondrogenesis 4.1 MAPK signalling pathway The MAPK family includes three significant members, ERK, JNK and p38 MAPK. Studies have shown that ERK and p38 MAPK are required for chondrocyte survival and endochondral ossification. The p38 MAPK has been demonstrated to be activated in response to and to mediate the effects of several growth factors that are essential for chondrocyte differentiation and function, including TGF-β and bone morphogenetic proteins (BMP) [43-45]. During chondrogenic differentiation in vitro, the p38 MAPK pathway regulates the expression of collagen II and aggrecan, proteoglycan synthesis and cartilage nodule formation [46]. Moreover, p38 MAP kinase signalling is required for hypertrophic chondrocyte differentiation [47]. Consistent with p38, ERK participates in the terminal differentiation of chondrocytes. In mice with ERK1/2 deleted in hypertrophic chondrocytes, the process of endochondral ossification is impaired [48]. It has been reported that elevated ROS during chondrocyte hypertrophy have been found to activate ERK and p38 MAPK. Furthermore, MEK and p38 inhibitors suppressed the H2O2-induced hypertrophic gene expression, indicating that ROS transduce differentiation signals by activating the ERK and p38 MAPK pathways in chondrocytes [30]. However, the mechanism of how ROS activates ERK1/2 and p38 MAPK during chondrogenesis is unclear. In other cells, S-nitrosylation or glutathionylation of Ras has been known to directly activate the protein and initiate the Ras-Raf-MEK/ERK cascade [49, 50]. The p38 MAPK has been proved to be activated by apoptosis signal-regulated kinase 1 (ASK1) under oxidative stress. When ROS oxidizes two cysteine residues in the redox centre of thioredoxin, ASK1 is activated, causing the formation of intramolecular disulfide bonds between Cys-32 and Cys-35 [51, 52]. Evidence that the ROS/ASK1/p38
MAPK pathway transmits differentiation signals in other cells may suggest a mechanism for ROS activation of p38 MAPK in chondrocytes [53].
4.2 PI3K-Akt signalling pathway Studies have demonstrated that PI3K plays a vital role in chondrogenesis and chondrocyte differentiation [4, 54, 55]. It has been shown that the activation of the PI3K-Akt pathway inhibits the transition in chondrocytes from proliferation to hypertrophic differentiation. On the other hand, the PI3K-Akt signalling pathway promotes the terminal differentiation of chondrocytes [54, 56]. In addition to redox regulation of phosphatases, ROS/RNS can also directly oxidize protein kinases (PI3K and Akt). This oxidative modification results in the downregulation of the PI3K/Akt signal [57]. In chondrocytes, insulin-like growth factor 1 (IGF-1) activates the IGF-1 receptor (IGF-1R), which induces activation of the insulin receptor substrate 1 (IRS-1) through tyrosine phosphorylation and, subsequently, activates the PI3K/Akt pathway, leading to increased aggrecan synthesis and collagen II expression. During early chondrogenesis, suppression of ROS generation by knockdown of Nox2 or Nox4 inhibited insulin-induced Akt phosphorylation [29]. This indicates that the Nox-derived ROS are required for Akt phosphorylation during early chondrogenesis. However, Yin et al. showed that oxidative stress also inhibited IGF-1-induced Akt phosphorylation in osteoarthritis chondrocytes [58]. Taken together, these results demonstrate that redox status in chondrocytes can alter intracellular signal transduction in response to growth factors and cytokines. The PI3K/Akt signalling pathway is sensitive to redox modifications, and the redox regulation of PI3K/Akt is mainly conducted by oxidative modification of Cys-dependent phosphatases (CDPs) and protein kinases. [59, 60]. CDPs that regulate PI3K/Akt signalling include PI3-phosphatase, PTEN, and PTPases. It is known that ROS can oxidize Cys residues in CDPs to cause the loss of phosphatase activity. This modification activates the PI3K/Akt signalling pathway. For example, oxidative inactivation of PTEN by S-nitrosylation of Cys residues is an important mechanism of oxidative stress-induced PI3K/Akt activation [61].
4.3 mTOR signalling pathway The mTOR signal pathway has been shown to be essential for chondrocyte proliferation and endochondral ossification [62-64]. It has been demonstrated that mTORC1 activity declines during chondrocyte differentiation in vitro and in vivo. Activation of the mTOR signalling pathway in chondrocytes promotes growth and proliferation but prevents the terminal differentiation of hypertrophic chondrocytes [65]. It is proved that the inhibition of mTOR is required for chondrocyte hypertrophy. Our previous study showed that inhibition of mTOR promotes hypertrophic differentiation in the pellet culture model [3]. It has been proved that Nox-derived ROS are required for Akt phosphorylation during early chondrogenesis. We hypothesized that ROS-induced Akt phosphorylation activates mTOR during early chondrogenic differentiation. In addition, physiological ROS levels are known to activate the mTOR signalling pathway in most cells [66-68]. The mechanism by which ROS activate mTOR is the Cys oxidation of Ras homologue enriched in brain (Rheb) and regulatory-associated protein of mTOR (Raptor) [66, 69, 70]. It has been found that the ROS regulate the Raptor-mTOR complex to control the ribosomal protein S6 kinase (S6K1), an effector of Raptor-mTOR. The ROS activate the Raptor-mTOR pathway through destabilization of the Raptor-mTOR interaction. In contrast, a reducing reagent inhibits this pathway by stabilization of the Raptor-mTOR interaction [66]. Importantly, further experiments need to be performed to verify the specific mechanism of ROS function on the mTOR signalling pathway during chondrogenesis. Furthermore, numerous studies have demonstrated that oxidative stress activates autophagy by inhibiting the mTOR signalling pathway to protect chondrocytes from apoptosis [71, 72]. However, the specific mechanism of oxidative stress in inhibiting mTOR activity needs to be further studied in chondrocytes. 4.4. cGMP signalling pathway It has been demonstrated that cGMP plays an important role in chondrocyte terminal differentiation[33]. The increase in cGMP concentration may lead to the activation of phosphodiesterases (PDE), cGMP-regulated ion channels (ICH) and cGMP-dependent
kinases (cGK). In particular, cGKII has been shown to play an essential role in endochondral bone formation [73, 74]. Possible downstream pathways include the inhibition of nuclear translocation of the transcription factor Sox9 and the repression of FGF signalling and ERK activation through Raf1 [75]. Teixeira et al. demonstrated that the expression of NOS and the generation of NO enhanced alkaline phosphatase and collagen type X expression by increasing cGMP concentration [33]. Nitric oxide could bind to the haem-containing soluble protein guanylate cyclase, causing increased enzymatic activity and formation of cGMP. Depending on the cell type, the increase in cGMP concentration may lead to activation of phosphodiesterases, cGMP-regulated ion channels and cGMP-dependent kinases (cGKs) [76].
5. Redox Control of Transcription Factors Involved in Chondrocyte Differentiation and Chondrogenesis 5.1. HIF-1α Hypoxia-inducible factor 1 alpha subunit (HIF-1α) is a crucial transcription factor for chondrogenesis and chondrocyte differentiation. Specific deletion of HIF-1 within chondrocytes led to the death of hypoxic chondrocytes and promoted chondrocyteproliferation, resulting in delayed hypertrophy in the growth plate [77]. Cartilage and growth plates are avascular tissues. Research has proved that HIF-1α is required for the survival of chondrocytes in the hypoxic region of the growth plate [78]. HIF-1α plays a non-redundant and critical role in the differentiation of mesenchymal cells into chondrocytes. HIF-1α was found to enhance the BMP2-induced chondrogenic expression of Sox9 and downstream markers and inhibit the osteogenic expression of Runx2 and downstream markers in vitro [79]. Lack of HIF-1α in limb bud mesenchyme caused a remarkable delay in chondrogenesis [80, 81]. The HIF-1α protein is oxygen-sensitive through oxygen-dependent hydroxylation of specific residues within its amino acid sequence. In normoxia, the oxygen-dependent degradation domain (ODDD) of HIF-1α is hydroxylated by HIF prolyl 4-hydroxylases [82]. The E3 ubiquitin ligase Von Hippel-Lindau (VHL) binds to hydroxylated HIF-1α. Then, HIF-1α degrades via the ubiquitylation pathway [83, 84]. Under hypoxic
conditions, HIF-1α is not hydroxylated. HIF-1α can then freely migrate to the nucleus and bind to the β subunit, acting as a transcription factor. ROS/RNS exert different effects on the HIF-1α protein in normoxia and hypoxia. In normoxia, ROS/RNS production can stabilize HIF-1α [85, 86]. Reducing ROS/RNS levels by antioxidants or reducing active oxygen producing enzyme activity can reduce HIF-1α expression [85, 87, 88]. It has been shown that ROS/RNS can stabilize HIF-1α in normoxia through multiple pathways [89]. Accumulation of ROS can lead to the inactivation of PHD, leading to the stabilization of HIF-1α [90]. Additionally, direct oxidative modifications such as S-nitrosylation of HIF or VHL proteins have been shown to lead to stabilization of HIF-1α [91, 92]. Under hypoxic conditions, both ROS and RNS inhibit HIF-1α DNA-binding activity and HIF-1α accumulation. Kazuo Yudoh et al. found that IL-1β and H2O2 could promote the expression of HIF-1α mRNA and protein in human articular chondrocytes in vitro under hypoxia (O2<6%) [93]. Under hypoxic conditions, inhibitors of PI3K and p38 MAPK reduced the protein levels of IL-1β-induced HIF-1α mRNA expression. However, further in-depth and systematic studies are required to determine how ROS/RNS regulate the turnover and transcriptional activity of HIF-1α in chondrogenesis.
5.2 AP-1 The c-fos and c-Jun proteins form the activator protein 1 (AP-1) complex. Some studies have shown that the AP-1 family plays a significant role in the hypertrophic differentiation of chondrocyte [94]. As a transcription factor, AP-1 activates genes required for the differentiation of various chondrocytes. Furthermore, recent studies have shown that AP-1 could directly interact with transcription factors such as Sox9 and Runx2, which are crucial chondrogenic differentiation factors, thereby promoting the marker gene for hypertrophic chondrocyte differentiation [94]. Nitric oxide may suppress the DNA-binding activity of AP-1 through S-glutathionylation. In the presence of the physiological sulfhydryl glutathione, nitric oxide modifies the two cysteine residues contained in the DNA-binding module of c-Jun selectively and distinctly. The formation of a mixed disulfide bond with glutathione inhibited the binding of AP-1 to DNA [95]. Furthermore, the intracellular level of AP-1 can be regulated by redox-mediated
mechanisms at the levels of transcription and protein turnover. For example, in bovine articular chondrocytes, TNF-α and essential fibroblast growth factor (bFGF) were found to induce ROS production through an NADPH oxidase enzyme complex, resulting in the upregulation of c-fos expression [96]. Similarly, ROS can regulate IL-1β induction of c-fos expression in the articular chondrocytes [97].
5.3 NK-κB/p65 Transcriptional targets of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) have been recognized as key developmental signalling mediators that regulate endochondral ossification. The involvement of NF-κB/p65 as an indispensable factor during chondrogenic development has been studied in the context of mature chondrocytes. NF-κB/p65 has been identified as a transcription factor for BMP2 and Sox9 in mature chondrocytes during endochondral ossification [98-100]. Moreover, it has been proved that activation of p65 promotes the early stage of chondrocyte differentiation during endochondral ossification [101]. The function of NF-κB can be activated or inhibited at various levels of the activation pathway through different redox-mediated mechanisms. In the nucleus, direct oxidation of Cys in the DNA-binding domain can hinder NF-κB DNA binding activity. In the cytosol, activation of NF-κB can be regulated by phosphorylation of NF-κB itself or phosphorylation of its inhibitor IκB. Under normal conditions, NF-κB and IκB form complexes that are sequestered in the cytosol. Increased ROS can directly activate IκB kinase (IκK) by redox-modification of IκK or can indirectly activate IκB kinase (IκB) by activating Akt and, subsequently, can phosphorylate and activate IκK [102]. Activated IκK phosphorylates IκB and releases active NF-κB from the complex for translocation to the nucleus [101]. R. MClancy et al. proved that nitric oxide and its derivatives could regulate cytokine-induced NF-κB activation in chondrocytes. Their results demonstrated that nitric oxide sustains NF-κB signalling in chondrocytes, most likely via peroxynitrite. Persistent activation of NF-κB signalling in chondrocytes further promotes and amplifies the transcription of inflammatory cytokines, growth factors and metalloproteinases [103].
Conclusion
Chondrogenesis is a continuous and dynamic process that is regulated by many growth factors. The intracellular redox status affects the response of chondrocytes to extracellular stimulation. Our review shows that the redox status controls chondrocyte differentiation and chondrogenesis as an endogenous regulator of signalling kinases and transcription factors. Although ROS/RNS have been studied extensively in other cells as signalling molecules that affect cellular processes, they have not been sufficiently investigated in chondrocytes. The specific state of redox levels in the articular cartilage and growth plate regions remains unclear. Similarly, the detailed mechanisms of how ROS regulate signalling pathways has not been studied in detail in chondrocytes and growth plates.
Acknowledgments This work was funded by grants from the National Key Technology Research and Development Program of China (2017YFC1103300), the Nature Science Foundation of China (31870962), and Major project of Logistics Research Plan of PLA (AWS17J004).
Competing Interests The authors declare that there is no conflict of interests regarding the publication of this paper.
References [1] T. Saito, A. Fukai, A. Mabuchi, T. Ikeda, F. Yano, S. Ohba, N. Nishida, T. Akune, N. Yoshimura, T. Nakagawa, K. Nakamura, K. Tokunaga, U.I. Chung, H. Kawaguchi, Transcriptional regulation of endochondral ossification by HIF-2alpha during skeletal growth and osteoarthritis development, Nature medicine 16(6) (2010) 678-86. [2] N. Ortega, D.J. Behonick, Z. Werb, Matrix remodeling during endochondral ossification, Trends in cell biology 14(2) (2004) 86-93. [3] Y. Bai, C. Liu, L. Fu, X. Gong, C. Dou, Z. Cao, H. Quan, J. Li, F. Kang, J. Dai, C. Zhao, S. Dong, Mangiferin enhances endochondral ossification-based bone repair in massive bone defect by inducing autophagy through activating AMP-activated protein kinase signaling pathway, FASEB journal : official publication of the Federation of American Societies for Experimental Biology (2018) fj201701411R. [4] Z. Cao, C. Dou, J. Li, X. Tang, J. Xiang, C. Zhao, L. Zhu, Y. Bai, Q. Xiang, S. Dong, Cordycepin inhibits chondrocyte hypertrophy of mesenchymal stem cells through PI3K/Bapx1 and Notch signaling pathway, BMB reports 49(10) (2016) 548-553. [5] D. Choudhary, S. Adhikary, N. Ahmad, P. Kothari, A. Verma, P.K. Trivedi, P.R.
Mishra, R. Trivedi, Prevention of articular cartilage degeneration in a rat model of monosodium iodoacetate induced osteoarthritis by oral treatment with Withaferin A, Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie 99 (2018) 151-161. [6] N.M. Khan, I. Ahmad, M.Y. Ansari, T.M. Haqqi, Wogonin, a natural flavonoid, intercalates with genomic DNA and exhibits protective effects in IL-1beta stimulated osteoarthritis chondrocytes, Chemico-biological interactions 274 (2017) 13-23. [7] N.M. Khan, A. Haseeb, M.Y. Ansari, P. Devarapalli, S. Haynie, T.M. Haqqi, Wogonin, a plant derived small molecule, exerts potent anti-inflammatory and chondroprotective effects through the activation of ROS/ERK/Nrf2 signaling pathways in human Osteoarthritis chondrocytes, Free radical biology & medicine 106 (2017) 288-301. [8] P. Lepetsos, A.G. Papavassiliou, ROS/oxidative stress signaling in osteoarthritis, Biochim Biophys Acta 1862(4) (2016) 576-591. [9] L. Grange, M.V. Nguyen, B. Lardy, M. Derouazi, Y. Campion, C. Trocme, M.H. Paclet, P. Gaudin, F. Morel, NAD(P)H oxidase activity of Nox4 in chondrocytes is both inducible and involved in collagenase expression, Antioxidants & redox signaling 8(9-10) (2006) 1485-96. [10] T.S. Hiran, P.J. Moulton, J.T. Hancock, Detection of superoxide and NADPH oxidase in porcine articular chondrocytes, Free radical biology & medicine 23(5) (1997) 736-43. [11] N. Jallali, H. Ridha, C. Thrasivoulou, P. Butler, T. Cowen, Modulation of intracellular reactive oxygen species level in chondrocytes by IGF-1, FGF, and TGF-beta1, Connective tissue research 48(3) (2007) 149-58. [12] Y. Henrotin, G. Deby-Dupont, C. Deby, P. Franchimont, I. Emerit, Active oxygen species, articular inflammation and cartilage damage, Exs 62 (1992) 308-22. [13] B. Fermor, J.B. Weinberg, D.S. Pisetsky, M.A. Misukonis, A.J. Banes, F. Guilak, The effects of static and intermittent compression on nitric oxide production in articular cartilage explants, J Orthop Res 19(4) (2001) 729-37. [14] J. Stadler, M. Stefanovic-Racic, T.R. Billiar, R.D. Curran, L.A. McIntyre, H.I. Georgescu, R.L. Simmons, C.H. Evans, Articular chondrocytes synthesize nitric oxide in response to cytokines and lipopolysaccharide, J Immunol 147(11) (1991) 3915-20. [15] M.A. Cake, R.A. Read, B. Guillou, P. Ghosh, Modification of articular cartilage and subchondral bone pathology in an ovine meniscectomy model of osteoarthritis by avocado and soya unsaponifiables (ASU), Osteoarthritis and cartilage 8(6) (2000) 404-11. [16] H.J. Cho, Q.W. Xie, J. Calaycay, R.A. Mumford, K.M. Swiderek, T.D. Lee, C. Nathan, Calmodulin is a subunit of nitric oxide synthase from macrophages, J Exp Med 176(2) (1992) 599-604. [17] Y. Henrotin, B. Kurz, Antioxidant to treat osteoarthritis: dream or reality?, Current drug targets 8(2) (2007) 347-57. [18] F.M. Faraci, S.P. Didion, Vascular protection: superoxide dismutase isoforms in the vessel wall, Arteriosclerosis, thrombosis, and vascular biology 24(8) (2004) 1367-73. [19] H. Nakamura, K. Nakamura, J. Yodoi, Redox regulation of cellular activation, Annual review of immunology 15 (1997) 351-69. [20] M.D. Carlo, Jr., R.F. Loeser, Increased oxidative stress with aging reduces chondrocyte survival: correlation with intracellular glutathione levels, Arthritis and
rheumatism 48(12) (2003) 3419-30. [21] M. Valko, D. Leibfritz, J. Moncol, M.T. Cronin, M. Mazur, J. Telser, Free radicals and antioxidants in normal physiological functions and human disease, The international journal of biochemistry & cell biology 39(1) (2007) 44-84. [22] G. Shen, The role of type X collagen in facilitating and regulating endochondral ossification of articular cartilage, Orthodontics & craniofacial research 8(1) (2005) 11-7. [23] M.B. Goldring, Chondrogenesis, chondrocyte differentiation, and articular cartilage metabolism in health and osteoarthritis, Therapeutic advances in musculoskeletal disease 4(4) (2012) 269-85. [24] H.P. Gerber, T.H. Vu, A.M. Ryan, J. Kowalski, Z. Werb, N. Ferrara, VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation, Nature medicine 5(6) (1999) 623-8. [25] L. Zhang, G. Wang, X. Chen, X. Xue, Q. Guo, M. Liu, J. Zhao, Formyl peptide receptors promotes neural differentiation in mouse neural stem cells by ROS generation and regulation of PI3K-AKT signaling, Scientific reports 7(1) (2017) 206. [26] T. Liu, Q. Men, G. Wu, C. Yu, Z. Huang, X. Liu, W. Li, Tetrandrine induces autophagy and differentiation by activating ROS and Notch1 signaling in leukemia cells, Oncotarget 6(10) (2015) 7992-8006. [27] N. Sampson, R. Koziel, C. Zenzmaier, L. Bubendorf, E. Plas, P. Jansen-Durr, P. Berger, ROS signaling by NOX4 drives fibroblast-to-myofibroblast differentiation in the diseased prostatic stroma, Mol Endocrinol 25(3) (2011) 503-15. [28] K. Ambe, H. Watanabe, S. Takahashi, T. Nakagawa, Immunohistochemical localization of Nox1, Nox4 and Mn-SOD in mouse femur during endochondral ossification, Tissue Cell 46(6) (2014) 433-8. [29] K.S. Kim, H.W. Choi, H.E. Yoon, I.Y. Kim, Reactive oxygen species generated by NADPH oxidase 2 and 4 are required for chondrogenic differentiation, J Biol Chem 285(51) (2010) 40294-302. [30] K. Morita, T. Miyamoto, N. Fujita, Y. Kubota, K. Ito, K. Takubo, K. Miyamoto, K. Ninomiya, T. Suzuki, R. Iwasaki, M. Yagi, H. Takaishi, Y. Toyama, T. Suda, Reactive oxygen species induce chondrocyte hypertrophy in endochondral ossification, J Exp Med 204(7) (2007) 1613-23. [31] R.K. Studer, Nitric oxide decreases IGF-1 receptor function in vitro; glutathione depletion enhances this effect in vivo, Osteoarthritis and cartilage 12(11) (2004) 863-9. [32] C.C. Teixeira, K. Mansfield, C. Hertkorn, H. Ischiropoulos, I.M. Shapiro, Phosphate-induced chondrocyte apoptosis is linked to nitric oxide generation, American journal of physiology. Cell physiology 281(3) (2001) C833-9. [33] C.C. Teixeira, H. Ischiropoulos, P.S. Leboy, S.L. Adams, I.M. Shapiro, Nitric oxide-nitric oxide synthase regulates key maturational events during chondrocyte terminal differentiation, Bone 37(1) (2005) 37-45. [34] G. Wang, Q. Yan, A. Woods, L.A. Aubrey, Q. Feng, F. Beier, Inducible nitric oxide synthase-nitric oxide signaling mediates the mitogenic activity of Rac1 during endochondral bone growth, Journal of cell science 124(Pt 20) (2011) 3405-13. [35] M. Schieber, N.S. Chandel, ROS function in redox signaling and oxidative stress, Current biology : CB 24(10) (2014) R453-62. [36] K. England, T.G. Cotter, Direct oxidative modifications of signalling proteins in mammalian cells and their effects on apoptosis, Redox report : communications in free
radical research 10(5) (2005) 237-45. [37] N. Chakouri, C. Reboul, D. Boulghobra, A. Kleindienst, S. Nottin, S. Gayrard, F. Roubille, S. Matecki, A. Lacampagne, O. Cazorla, Stress-induced protein S-glutathionylation and phosphorylation crosstalk in cardiac sarcomeric proteins - Impact on heart function, International journal of cardiology 258 (2018) 207-216. [38] P. Ghezzi, Regulation of protein function by glutathionylation, Free radical research 39(6) (2005) 573-80. [39] C.A. O'Brian, F. Chu, Post-translational disulfide modifications in cell signaling--role of inter-protein, intra-protein, S-glutathionyl, and S-cysteaminyl disulfide modifications in signal transmission, Free radical research 39(5) (2005) 471-80. [40] W.H. Hoefnagels, J.I. Drayer, A.G. Smals, P.W. Kloppenborg, Spironolactone and amiloride in hypertensive patients with and without aldosterone excess, Clinical pharmacology and therapeutics 27(3) (1980) 317-23. [41] E.M. Jeong, C.Z. Jin, J.H. Jang, Z.H. Zhao, C.L. Jin, J.H. Lee, K.B. Lee, S.J. Kim, I.G. Kim, Y.H. Zhang, S-nitrosylation of transglutaminase 2 impairs fatty acid-stimulated contraction in hypertensive cardiomyocytes, Experimental & molecular medicine 50(4) (2018) 9. [42] L.B. Poole, P.A. Karplus, A. Claiborne, Protein sulfenic acids in redox signaling, Annual review of pharmacology and toxicology 44 (2004) 325-47. [43] T. Iwai, J. Murai, H. Yoshikawa, N. Tsumaki, Smad7 Inhibits chondrocyte differentiation at multiple steps during endochondral bone formation and down-regulates p38 MAPK pathways, The Journal of biological chemistry 283(40) (2008) 27154-64. [44] L.A. Stanton, S. Sabari, A.V. Sampaio, T.M. Underhill, F. Beier, p38 MAP kinase signalling is required for hypertrophic chondrocyte differentiation, Biochem J 378(Pt 1) (2004) 53-62. [45] X. Zhen, L. Wei, Q. Wu, Y. Zhang, Q. Chen, Mitogen-activated protein kinase p38 mediates regulation of chondrocyte differentiation by parathyroid hormone, The Journal of biological chemistry 276(7) (2001) 4879-85. [46] C.D. Oh, S.H. Chang, Y.M. Yoon, S.J. Lee, Y.S. Lee, S.S. Kang, J.S. Chun, Opposing role of mitogen-activated protein kinase subtypes, erk-1/2 and p38, in the regulation of chondrogenesis of mesenchymes, The Journal of biological chemistry 275(8) (2000) 5613-9. [47] (!!! INVALID CITATION !!! {Stanton, 2004 #85}). [48] Z. Chen, S.X. Yue, G. Zhou, E.M. Greenfield, S. Murakami, ERK1 and ERK2 regulate chondrocyte terminal differentiation during endochondral bone formation, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 30(5) (2015) 765-74. [49] H.M. Lander, A.J. Milbank, J.M. Tauras, D.P. Hajjar, B.L. Hempstead, G.D. Schwartz, R.T. Kraemer, U.A. Mirza, B.T. Chait, S.C. Burk, L.A. Quilliam, Redox regulation of cell signalling, Nature 381(6581) (1996) 380-1. [50] R.J. Mallis, J.E. Buss, J.A. Thomas, Oxidative modification of H-ras: S-thiolation and S-nitrosylation of reactive cysteines, The Biochemical journal 355(Pt 1) (2001) 145-53. [51] M. Saitoh, H. Nishitoh, M. Fujii, K. Takeda, K. Tobiume, Y. Sawada, M. Kawabata, K. Miyazono, H. Ichijo, Mammalian thioredoxin is a direct inhibitor of apoptosis signal-regulating kinase (ASK) 1, The EMBO journal 17(9) (1998) 2596-606.
[52] G. Fujino, T. Noguchi, A. Matsuzawa, S. Yamauchi, M. Saitoh, K. Takeda, H. Ichijo, Thioredoxin and TRAF family proteins regulate reactive oxygen species-dependent activation of ASK1 through reciprocal modulation of the N-terminal homophilic interaction of ASK1, Molecular and cellular biology 27(23) (2007) 8152-63. [53] A. Sato, M. Okada, K. Shibuya, E. Watanabe, S. Seino, Y. Narita, S. Shibui, T. Kayama, C. Kitanaka, Pivotal role for ROS activation of p38 MAPK in the control of differentiation and tumor-initiating capacity of glioma-initiating cells, Stem cell research 12(1) (2014) 119-31. [54] K. Kita, T. Kimura, N. Nakamura, H. Yoshikawa, T. Nakano, PI3K/Akt signaling as a key regulatory pathway for chondrocyte terminal differentiation, Genes to cells : devoted to molecular & cellular mechanisms 13(8) (2008) 839-50. [55] V. Ulici, K.D. Hoenselaar, J.R. Gillespie, F. Beier, The PI3K pathway regulates endochondral bone growth through control of hypertrophic chondrocyte differentiation, BMC developmental biology 8 (2008) 40. [56] T. Fujita, Y. Azuma, R. Fukuyama, Y. Hattori, C. Yoshida, M. Koida, K. Ogita, T. Komori, Runx2 induces osteoblast and chondrocyte differentiation and enhances their migration by coupling with PI3K-Akt signaling, The Journal of cell biology 166(1) (2004) 85-95. [57] H. Murata, Y. Ihara, H. Nakamura, J. Yodoi, K. Sumikawa, T. Kondo, Glutaredoxin exerts an antiapoptotic effect by regulating the redox state of Akt, The Journal of biological chemistry 278(50) (2003) 50226-33. [58] W. Yin, J.I. Park, R.F. Loeser, Oxidative stress inhibits insulin-like growth factor-I induction of chondrocyte proteoglycan synthesis through differential regulation of phosphatidylinositol 3-Kinase-Akt and MEK-ERK MAPK signaling pathways, The Journal of biological chemistry 284(46) (2009) 31972-81. [59] R. Naughton, C. Quiney, S.D. Turner, T.G. Cotter, Bcr-Abl-mediated redox regulation of the PI3K/AKT pathway, Leukemia 23(8) (2009) 1432-40. [60] K. Niwa, O. Inanami, T. Yamamori, T. Ohta, T. Hamasu, M. Kuwabara, Redox regulation of PI3K/Akt and p53 in bovine aortic endothelial cells exposed to hydrogen peroxide, Antioxidants & redox signaling 5(6) (2003) 713-22. [61] C.X. Yu, S. Li, A.R. Whorton, Redox regulation of PTEN by S-nitrosothiols, Molecular pharmacology 68(3) (2005) 847-54. [62] J. Chen, F. Long, mTORC1 signaling controls mammalian skeletal growth through stimulation of protein synthesis, Development 141(14) (2014) 2848-54. [63] C.P. Sanchez, Y.Z. He, Bone growth during rapamycin therapy in young rats, BMC pediatrics 9 (2009) 3. [64] Y. Guan, X. Yang, W. Yang, C. Charbonneau, Q. Chen, Mechanical activation of mammalian target of rapamycin pathway is required for cartilage development, FASEB journal : official publication of the Federation of American Societies for Experimental Biology 28(10) (2014) 4470-81. [65] B. Yan, Z. Zhang, D. Jin, C. Cai, C. Jia, W. Liu, T. Wang, S. Li, H. Zhang, B. Huang, P. Lai, H. Wang, A. Liu, C. Zeng, D. Cai, Y. Jiang, X. Bai, mTORC1 regulates PTHrP to coordinate chondrocyte growth, proliferation and differentiation, Nature communications 7 (2016) 11151. [66] D.D. Sarbassov, D.M. Sabatini, Redox regulation of the nutrient-sensitive raptor-mTOR pathway and complex, The Journal of biological chemistry 280(47) (2005)
39505-9. [67] O. Zhenyukh, E. Civantos, M. Ruiz-Ortega, M.S. Sanchez, C. Vazquez, C. Peiro, J. Egido, S. Mas, High concentration of branched-chain amino acids promotes oxidative stress, inflammation and migration of human peripheral blood mononuclear cells via mTORC1 activation, Free radical biology & medicine 104 (2017) 165-177. [68] B. Steinhorn, J.L. Sartoretto, A. Sorrentino, N. Romero, H. Kalwa, E.D. Abel, T. Michel, Insulin-dependent metabolic and inotropic responses in the heart are modulated by hydrogen peroxide from NADPH-oxidase isoforms NOX2 and NOX4, Free radical biology & medicine 113 (2017) 16-25. [69] S. Yoshida, S. Hong, T. Suzuki, S. Nada, A.M. Mannan, J. Wang, M. Okada, K.L. Guan, K. Inoki, Redox regulates mammalian target of rapamycin complex 1 (mTORC1) activity by modulating the TSC1/TSC2-Rheb GTPase pathway, The Journal of biological chemistry 286(37) (2011) 32651-60. [70] C.T. Hsieh, J.H. Chuang, W.C. Yang, Y. Yin, Y. Lin, Ceramide inhibits insulin-stimulated Akt phosphorylation through activation of Rheb/mTORC1/S6K signaling in skeletal muscle, Cellular signalling 26(7) (2014) 1400-8. [71] N.M. Khan, M.Y. Ansari, T.M. Haqqi, Sucrose, But Not Glucose, Blocks IL1-beta-Induced Inflammatory Response in Human Chondrocytes by Inducing Autophagy via AKT/mTOR Pathway, Journal of cellular biochemistry 118(3) (2017) 629-639. [72] Y.S. Li, F.J. Zhang, C. Zeng, W. Luo, W.F. Xiao, S.G. Gao, G.H. Lei, Autophagy in osteoarthritis, Joint, bone, spine : revue du rhumatisme 83(2) (2016) 143-8. [73] F. Kugimiya, H. Chikuda, S. Kamekura, T. Ikeda, K. Hoshi, T. Ogasawara, K. Nakamura, U.I. Chung, H. Kawaguchi, Involvement of cyclic guanosine monophosphate-dependent protein kinase II in chondrocyte hypertrophy during endochondral ossification, Modern rheumatology 15(6) (2005) 391-6. [74] T. Miyazawa, Y. Ogawa, H. Chusho, A. Yasoda, N. Tamura, Y. Komatsu, A. Pfeifer, F. Hofmann, K. Nakao, Cyclic GMP-dependent protein kinase II plays a critical role in C-type natriuretic peptide-mediated endochondral ossification, Endocrinology 143(9) (2002) 3604-10. [75] D.M. Ornitz, P.J. Marie, Fibroblast growth factor signaling in skeletal development and disease, Genes & development 29(14) (2015) 1463-86. [76] J.W. Denninger, M.A. Marletta, Guanylate cyclase and the .NO/cGMP signaling pathway, Biochimica et biophysica acta 1411(2-3) (1999) 334-50. [77] E. Schipani, Hypoxia and HIF-1 alpha in chondrogenesis, Seminars in cell & developmental biology 16(4-5) (2005) 539-46. [78] E. Schipani, H.E. Ryan, S. Didrickson, T. Kobayashi, M. Knight, R.S. Johnson, Hypoxia in cartilage: HIF-1alpha is essential for chondrocyte growth arrest and survival, Genes & development 15(21) (2001) 2865-76. [79] N. Zhou, N. Hu, J.Y. Liao, L.B. Lin, C. Zhao, W.K. Si, Z. Yang, S.X. Yi, T.X. Fan, W. Bao, X. Liang, X. Wei, H. Chen, C. Chen, Q. Chen, X. Lin, W. Huang, HIF-1alpha as a Regulator of BMP2-Induced Chondrogenic Differentiation, Osteogenic Differentiation, and Endochondral Ossification in Stem Cells, Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology 36(1) (2015) 44-60. [80] R. Amarilio, S.V. Viukov, A. Sharir, I. Eshkar-Oren, R.S. Johnson, E. Zelzer,
HIF1alpha regulation of Sox9 is necessary to maintain differentiation of hypoxic prechondrogenic cells during early skeletogenesis, Development 134(21) (2007) 3917-28. [81] S. Provot, D. Zinyk, Y. Gunes, R. Kathri, Q. Le, H.M. Kronenberg, R.S. Johnson, M.T. Longaker, A.J. Giaccia, E. Schipani, Hif-1alpha regulates differentiation of limb bud mesenchyme and joint development, The Journal of cell biology 177(3) (2007) 451-64. [82] R.K. Bruick, S.L. McKnight, A conserved family of prolyl-4-hydroxylases that modify HIF, Science 294(5545) (2001) 1337-40. [83] M. Ivan, K. Kondo, H. Yang, W. Kim, J. Valiando, M. Ohh, A. Salic, J.M. Asara, W.S. Lane, W.G. Kaelin, Jr., HIFalpha targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing, Science 292(5516) (2001) 464-8. [84] P. Jaakkola, D.R. Mole, Y.M. Tian, M.I. Wilson, J. Gielbert, S.J. Gaskell, A. von Kriegsheim, H.F. Hebestreit, M. Mukherji, C.J. Schofield, P.H. Maxwell, C.W. Pugh, P.J. Ratcliffe, Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation, Science 292(5516) (2001) 468-72. [85] R.S. BelAiba, T. Djordjevic, S. Bonello, D. Flugel, J. Hess, T. Kietzmann, A. Gorlach, Redox-sensitive regulation of the HIF pathway under non-hypoxic conditions in pulmonary artery smooth muscle cells, Biological chemistry 385(3-4) (2004) 249-57. [86] A. Gorlach, I. Diebold, V.B. Schini-Kerth, U. Berchner-Pfannschmidt, U. Roth, R.P. Brandes, T. Kietzmann, R. Busse, Thrombin activates the hypoxia-inducible factor-1 signaling pathway in vascular smooth muscle cells: Role of the p22(phox)-containing NADPH oxidase, Circulation research 89(1) (2001) 47-54. [87] T. Kietzmann, A. Gorlach, Reactive oxygen species in the control of hypoxia-inducible factor-mediated gene expression, Seminars in cell & developmental biology 16(4-5) (2005) 474-86. [88] V.A. Shatrov, V.V. Sumbayev, J. Zhou, B. Brune, Oxidized low-density lipoprotein (oxLDL) triggers hypoxia-inducible factor-1alpha (HIF-1alpha) accumulation via redox-dependent mechanisms, Blood 101(12) (2003) 4847-9. [89] E.L. Bell, N.S. Chandel, Mitochondrial oxygen sensing: regulation of hypoxia-inducible factor by mitochondrial generated reactive oxygen species, Essays in biochemistry 43 (2007) 17-27. [90] J. Pouyssegur, F. Mechta-Grigoriou, Redox regulation of the hypoxia-inducible factor, Biological chemistry 387(10-11) (2006) 1337-46. [91] F. Li, P. Sonveaux, Z.N. Rabbani, S. Liu, B. Yan, Q. Huang, Z. Vujaskovic, M.W. Dewhirst, C.Y. Li, Regulation of HIF-1alpha stability through S-nitrosylation, Molecular cell 26(1) (2007) 63-74. [92] I. Pinchuk, E. Schnitzer, D. Lichtenberg, Kinetic analysis of copper-induced peroxidation of LDL, Biochimica et biophysica acta 1389(2) (1998) 155-72. [93] K. Yudoh, H. Nakamura, K. Masuko-Hongo, T. Kato, K. Nishioka, Catabolic stress induces expression of hypoxia-inducible factor (HIF)-1 alpha in articular chondrocytes: involvement of HIF-1 alpha in the pathogenesis of osteoarthritis, Arthritis research & therapy 7(4) (2005) R904-14. [94] X. He, S. Ohba, H. Hojo, A.P. McMahon, AP-1 family members act with Sox9 to promote chondrocyte hypertrophy, Development 143(16) (2016) 3012-23. [95] P. Klatt, E.P. Molina, S. Lamas, Nitric oxide inhibits c-Jun DNA binding by specifically targeted S-glutathionylation, The Journal of biological chemistry 274(22) (1999) 15857-64.
[96] Y.Y. Lo, T.F. Cruz, Involvement of reactive oxygen species in cytokine and growth factor induction of c-fos expression in chondrocytes, The Journal of biological chemistry 270(20) (1995) 11727-30. [97] Y.Y. Lo, J.A. Conquer, S. Grinstein, T.F. Cruz, Interleukin-1 beta induction of c-fos and collagenase expression in articular chondrocytes: involvement of reactive oxygen species, Journal of cellular biochemistry 69(1) (1998) 19-29. [98] M. Ushita, T. Saito, T. Ikeda, F. Yano, A. Higashikawa, N. Ogata, U. Chung, K. Nakamura, H. Kawaguchi, Transcriptional induction of SOX9 by NF-kappaB family member RelA in chondrogenic cells, Osteoarthritis and cartilage 17(8) (2009) 1065-75. [99] J.Q. Feng, L. Xing, J.H. Zhang, M. Zhao, D. Horn, J. Chan, B.F. Boyce, S.E. Harris, G.R. Mundy, D. Chen, NF-kappaB specifically activates BMP-2 gene expression in growth plate chondrocytes in vivo and in a chondrocyte cell line in vitro, The Journal of biological chemistry 278(31) (2003) 29130-5. [100] S. Wu, J.K. Flint, G. Rezvani, F. De Luca, Nuclear factor-kappaB p65 facilitates longitudinal bone growth by inducing growth plate chondrocyte proliferation and differentiation and by preventing apoptosis, The Journal of biological chemistry 282(46) (2007) 33698-706. [101] M.M. Caron, P.J. Emans, D.A. Surtel, A. Cremers, J.W. Voncken, T.J. Welting, L.W. van Rhijn, Activation of NF-kappaB/p65 facilitates early chondrogenic differentiation during endochondral ossification, PloS one 7(3) (2012) e33467. [102] D. Trachootham, W. Lu, M.A. Ogasawara, R.D. Nilsa, P. Huang, Redox regulation of cell survival, Antioxidants & redox signaling 10(8) (2008) 1343-74. [103] R.M. Clancy, P.F. Gomez, S.B. Abramson, Nitric oxide sustains nuclear factor kappaB activation in cytokine-stimulated chondrocytes, Osteoarthritis and cartilage 12(7) (2004) 552-8.
Fig 1. Major sites of cellular reactive oxygen species (ROS) generation include the mitochondria and the NADPH oxidase (NOX). Nitric oxide synthases (NOS) are key enzymes for production of NO. Gpx, glutathione peroxidase; HO•, hydroxyl radical; NO, nitric oxide; ONOO•, peroxynitrite; SOD, superoxide dismutase; GSH, reduced glutathione; GSSG, oxidized glutathione; NADPH, reduced nicotinamide adenine dinucleotide phosphate. Fig 2. Schematic representation of the distribution of Nox1 and Nox4 on growth plates and the effect of ROS on chondrogenesis. In growth plate, Nox1 and Nox4 are expressed in different regions. Nox4 expression in chondrocytes was observed from the resting zone to the hypertrophy region, while Nox1 expression was restricted from the proliferating zone to the prehypertrophic zone. The Nox4-derived ROS is required for the early stage of chondrocyte differentiation. Nox1 synthesizes major reactive oxygen species compared to Nox4. Therefore, chondrocytes always produce ROS throughout the endochondral ossification and emit large amounts of reactive oxygen species from the proliferative zone to the prehypertrophic zone. Fig 3. Schematic representation of the redox regulation of proteins. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) can oxidize reactive cysteine of the protein. Mild oxidative stress can induce cysteine modifications such as reversible S-glutathionylation, disulfide bond formation and S-nitrosylation. These reversible redox post-translational modifications act as signalling in chondrocyte. In contrast, oxidative stress promotes the irreversible modification of sulfenic, sulfinic and sulfonic acids are destructive to protein function. Fig 4. Redox regulation of PI3K/AKT and MAPK signalling pathway in chondrogenesis. The growth factors and cytokines activate PI3K/AKT signalling, which modulate NADPH oxidases (NOXs) -derived reactive oxygen species (ROS). The NOXs derived ROS can activate PI3K/Akt and MAPK signalling pathway. The ROS activate mTOR through cysteine oxidation of Rheb and Raptor. Increased ROS can directly activate IκK by redox-modifying IκK, or indirectly activate IκK by activating Akt, and then phosphorylate and activate IκK. Activated IκK phosphorylates IκB and releases active NF-κB from the complex for translocation to the nucleus. TGFβ and BMP activate p38
MAPK signalling through TAK1. The ROS may increases and induces p38 MAPK activation through activating ASK1. In the process of chondrocyte hypertrophy, p38 MAPK regulates MEF2C, HDAC4 and RUNX2 in pre-hypertrophic and hypertrophic chondrocytes.
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Graphical Abstract Highlights
The role of redox regulation in specific signal transduction of chondrocyte differentiation and chondrogenesis have also been reviewed
Function of Nox-derived ROS in growth plate development have also been reviewed
The mechanism by which ROS/RNS regulates signaling proteins and transcription factors in chondrocytes have also been reviewed
PII: DOI: Reference:
S0891-5849(18)32272-X https://doi.org/10.1016/j.freeradbiomed.2018.10.443 FRB14007
To appear in: Free Radical Biology and Medicine Received date: 15 April 2018 Revised date: 14 October 2018 Accepted date: 26 October 2018 Cite this article as: Yun Bai, Xiaoshan Gong, Ce Dou, Zhen Cao and Shiwu Dong, Redox Control of Chondrocyte Differentiation and Chondrogenesis, Free Radical Biology and Medicine, https://doi.org/10.1016/j.freeradbiomed.2018.10.443 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Redox Control of Chondrocyte Differentiation and Chondrogenesis
Yun Baia1, Xiaoshan Gonga1, Ce Doua, Zhen Caoa, Shiwu Donga,b*
1
Department of Biomedical Materials Science, School of Biomedical Engineering, Third
Military Medical University, Chongqing 400038, China. 2
State Key Laboratory of Trauma, Burns and Combined Injury, Third Military Medical
University, Chongqing 400038, China.
*
Corresponding author. Shiwu Dong, Department of Biomedical Materials Science,
School of Biomedical Engineering, Third Military Medical University, Gaotanyan Street No.30, Chongqing 400038, China. Tel.: +86 2368771270. [email protected]
Abstract Chondrogenesis involves the recruitment and migration of mesenchymal cells, mesenchymal condensation, and chondrocyte differentiation and hypertrophy. Multiple factors precisely regulate chondrogenesis. Recent studies have demonstrated that the redox status of chondrocytes plays an essential role in the regulation of chondrocyte differentiation and chondrogenesis. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are important factors that change the intracellular redox status. Physiological levels of ROS/RNS act as intracellular signals in chondrocytes, and oxidative stress impairs the metabolism of chondrocytes. Under physiological conditions, the balance between ROS/RNS production and elimination
1
These authors contributed equally to this work.
ensures that redox-sensitive signalling proteins function correctly. The redox homeostasis of chondrocytes ensures that they respond appropriately to endogenous and exogenous stimuli. This review focuses on the redox regulation of key signalling pathways and transcription factors that control chondrogenesis and chondrocyte differentiation. Additionally, the mechanism by which ROS/RNS regulate signalling proteins and transcription factors in chondrocytes is also reviewed.
Keywords:
reactive
oxygen
species
(ROS),
chondrogenesis,
chondrocyte
differentiation
1. Introduction Chondrogenesis is the earliest stage of skeletal development. This stage involves the recruitment and migration of mesenchymal cells, mesenchymal condensation, and chondrocyte differentiation and maturation and results in the formation of cartilage and bone during endochondral bone formation [1, 2]. During endochondral ossification, chondrocyte hypertrophy is critical because cells alter the extracellular matrix and induce vascular invasion [3, 4]. This process includes the differentiation and hypertrophic differentiation of chondrocytes, which are precisely controlled by various growth factors and specific gene transcription factors through temporal and spatial activation or inhibition of cell signalling pathways. Chondrocytes may respond differently to growth factors due to their intracellular redox status. In general, reactive oxygen species (ROS) and reactive nitrogen species (RNS) are the major factors that affect intracellular redox status. Redox homeostasis is essential in maintaining the survival and function of chondrocytes. When the balance between ROS generation and elimination is destroyed, oxidative stress occurs. Oxidative stress has long been considered a significant factor in the induction of chondrocyte senescence and apoptosis in diseases such as osteoarthritis (OA) [5, 6], and the role of ROS/RNS in the pathogenesis of osteoarthritis has become a primary concern of researchers [7, 8]. However, many recent studies have begun to concentrate on the role of physiological levels of ROS in chondrocyte differentiation and metabolism. This review mainly
focuses on the redox regulation of signal transduction pathways and transcription factors that control chondrocyte differentiation.
2. Redox Status in Chondrocytes ROS include radical species such as superoxide (O2•−) and hydroxyl radicals (HO•) and non-radical species such as H2O2. These radicals are different from O2, with one, two, and three extra electrons each. Superoxide anion radicals in chondrocytes are produced by the enzyme complex NADPH oxidase, which adds a single electron to molecular oxygen. It is demonstrated that porcine articular chondrocytes can produce ROS using the NADPH-oxidase-like complex [9, 10]. After superoxide production, superoxide dismutase (SOD) dissipates superoxide quickly, producing H2O2 and O2 [11]. The reaction of divalent iron ions with H2O2 produces HO•, which is called the Fenton reaction [12]. RNS include nitric oxide (NO) and peroxynitrite (ONOO•). Nitric oxide is produced by nitric oxide synthase (NOS) and mediates different physiological processes. To date, three forms of NOS have been characterized: neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS). Chondrocytes express both eNOS and iNOS [13]. The eNOS are constitutively expressed and are stimulated by increased intracellular calcium levels to produce small amounts of NO. In chondrocytes, iNOS is expressed is in response to the presence of inflammatory cytokines [14, 15]. Calcium levels do not regulate the activity of iNOS; therefore, even in the presence of low concentrations of calcium, it may produce a significant amount of NO [16]. Nitric oxide is highly reactive with free radical species such as O2•−. O2•− reacts with NO to produce ONOO•, which is highly oxidizing (Figure 1). Chondrocytes, similar to other mammalian cells, have enzymatic and non-enzymatic antioxidant systems to eliminate ROS/RNS and maintain redox balance [17]. Superoxide dismutase (SOD) is a significant class of enzymatic antioxidants that catalyse the dissipation of O2•− to H2O2 [18]. After H2O2 production, another enzyme responsible for H2O2 removal is catalase, which is located in the peroxisome. Hydrogen peroxide is further converted to H2O and O2 by catalase. Through the coupling reaction of glutathione peroxidase (GPX), which catalyses the
conversion of reduced glutathione (GSH) to oxidized glutathione (GSSG), H2O2 can also be converted to O2. Multiple GPXs exist in different cells. Among them, glutathione peroxidase 1 (GPX1) is the primary scavenger of H2O2 and lipid hydroperoxide. Non-enzymatic antioxidants also play essential roles in maintaining the redox balance of cells. GSH is the most abundant peptide in cells and exhibits many functions to scavenge free radicals. GSH can directly remove HO• and singlet oxygen and help antioxidants regenerate into their active forms, such as vitamins C and E [19]. The thioredoxin system is another crucial thiol antioxidant and is composed of thioredoxin (Trx) and thioredoxin reductase. The thioredoxin system, in cooperation with the GSH system, plays a vital role in reducing oxidized thiol-containing proteins [20]. Oxidative stress occurs when the balance between ROS/RNS production and the antioxidant defence system is destroyed. An increase in ROS production or a decrease in ROS scavenging capacity due to exogenous or endogenous metabolic changes can lead to increased intracellular ROS levels and, thus, oxidative stress. Many studies have shown that increased oxidative stress plays an essential role in pathological conditions including cancer, degenerative motor diseases, and cardiovascular diseases [21]. Under normal circumstances, physiological levels of ROS/RNS can reversibly modify biomacromolecules, which play crucial roles in regulating cell function. However, under oxidative stress, excess ROS/RNS continually attack lipids, proteins, and DNA, resulting in severe and irreversible oxidative damage. In osteoarthritis, the over-produced ROS act as second messengers to promote the expression of MMPs, which lead to cartilage degradation. Furthermore, ROS directly degrade matrix components and induce cell death [8].
3. ROS/RNS Functions in Chondrocyte Differentiation and Chondrogenesis Chondrogenesis is a dynamic and precisely regulated process. After the condensation of mesenchymal progenitor cells, these cells differentiate into chondrocytes and secrete cartilage matrix to form cartilage [22]. Cartilage is replaced by bone tissue and bone marrow to generate mature bone, a process known as endochondral ossification. Chondrogenesis is initiated by the condensation of
mesenchymal stem cells, followed by the expression of chondrocyte-specific genes such as transcription factor Sox9 and genes encoding chondrocyte extracellular matrix proteins (collagen II and aggrecan) [23]. Under the control of several growth factors, chondrocytes eventually exit the cell cycle and begin to differentiate into hypertrophic chondrocytes. Late hypertrophic chondrocytes begin to express many genes associated with cartilage matrix degradation and bone formation, such as matrix metalloproteinase 13 (Mmp13) and vascular endothelial growth factor (VEGF) [24]. This process results in the degradation of the hypertrophic cartilage matrix and its replacement by bone tissue, thereby completing the process of endochondral ossification. Therefore, chondrogenesis includes different stages of chondrocyte differentiation such as mesenchymal progenitor cells condensation, cartilage formation,
chondrocyte
proliferation,
and
hypertrophic
differentiation
of
chondrocytes. Recent studies have revealed that ROS function as critical signalling molecules in the regulation of cell growth and differentiation [25-27] (Figure 2). In endochondral ossification, Nox1 and Nox4 are expressed in different regions of the growth plate. Nox4 expression in chondrocytes was observed from the resting zone to the hypertrophy region, while Nox1 expression was restricted from the proliferative zone to the pre-hypertrophic chondrocyte zone [28]. Nox4 is required early in cartilage differentiation, and increasing the expression of Nox1 and decreasing the expression of Nox4 are characteristics of chondrogenesis. Therefore, in chondrocyte hypertrophy, ROS produced by Nox1 may play a major role compared to Nox4. [28, 29]. Therefore, chondrocytes always produce ROS throughout the endochondral ossification and emit large amounts of reactive oxygen species from the proliferative zone to the pre-hypertrophic zone. Importantly, it has been proved that the ROS that are generated in response to insulin stimulation are required for chondrogenic differentiation. Kim et al. demonstrated that the ROS that are required for early chondrogenic differentiation were mainly derived from Nox2 and Nox4. After the knockdown of Nox2 or Nox4 in ATDC5 cells, a chondrogenic cell line, the ROS production was decreased, and the insulin-induced chondrocyte differentiation was blocked. These results proved that ROS generated by NOXs were required for early
chondrogenic differentiation[29]. Chondrocyte hypertrophy is the critical stage in chondrogenesis and endochondral ossification. During this process, the proliferating chondrocytes leave the cell cycle and enter the pre-hypertrophic differentiation phase. There is evidence that ROS play a significant role in chondrocyte hypertrophy [30]. Chondrocyte hypertrophy was inhibited by administration of the antioxidant NAC in ATDC5 cells and mice, indicating that physiological levels of ROS determine chondrocyte hypertrophy [30]. Additionally, during chondrocyte hypertrophy, the expression of NOX1, which is responsible for ROS production, was increased. These results proved that early chondrocyte differentiation and hypertrophy was stimulated by the expression of ROS-producing genes, such as NOXs. As a result, the intracellular ROS that were generated by NOXs can alter the cellular response to growth factor cytokines by mediating the redox-sensitive signalling pathways and expression of transcription factors during chondrogenesis [31]. Similar to ROS, NO has been proved to participate in endochondral ossification. Teixeira et al. showed that excess NO promotes hypertrophic differentiation and apoptosis of chondrocytes in chickens [32, 33]. Wang et al. suggest that the basal levels of NO that are generated by iNOS are required for chondrogenesis [34]. Recent evidence convincingly argues that these ROS/RNS play essential roles in numerous physiological and pathological processes [35]. The central mechanism of the redox regulation of proteins is oxidative modification [36]. The mechanism of redox regulation of proteins is through various types of oxidative modifications on the redox-sensitive sites of proteins, and the result may be the activation or inactivation of protein functions. ROS/RNS can directly oxidize various redox-sensitive amino acids, with cysteine (Cys) being the main target molecule. Mild oxidative stress can induce cysteine modifications such as reversible [37, 38] disulfide bond formation [39] and S-nitrosylation [40, 41]. These reversible redox post-translational modifications have proved to be essential signals in many cells. In contrast, oxidative stress promotes the modification of sulfenic, sulfinic and sulfonic acids in proteins, which are destructive to protein function [42] (Figure 3). To date, it has been demonstrated that redox signalling can control chondrogenesis by regulating transcription factors and signal transduction pathways. ROS/RNS can
modulate the ability of transcription factors to bind to DNA by modifying the Cys residues contained in the DNA-binding sites of transcription factors [39]. Additionally, oxidative modification plays a reversible regulatory role in the function of many protein tyrosine phosphatases, which can regulate chondrogenesis and chondrocyte functions.
4. Redox Control of Signal Pathways Involved in Chondrocyte Differentiation and Chondrogenesis 4.1 MAPK signalling pathway The MAPK family includes three significant members, ERK, JNK and p38 MAPK. Studies have shown that ERK and p38 MAPK are required for chondrocyte survival and endochondral ossification. The p38 MAPK has been demonstrated to be activated in response to and to mediate the effects of several growth factors that are essential for chondrocyte differentiation and function, including TGF-β and bone morphogenetic proteins (BMP) [43-45]. During chondrogenic differentiation in vitro, the p38 MAPK pathway regulates the expression of collagen II and aggrecan, proteoglycan synthesis and cartilage nodule formation [46]. Moreover, p38 MAP kinase signalling is required for hypertrophic chondrocyte differentiation [47]. Consistent with p38, ERK participates in the terminal differentiation of chondrocytes. In mice with ERK1/2 deleted in hypertrophic chondrocytes, the process of endochondral ossification is impaired [48]. It has been reported that elevated ROS during chondrocyte hypertrophy have been found to activate ERK and p38 MAPK. Furthermore, MEK and p38 inhibitors suppressed the H2O2-induced hypertrophic gene expression, indicating that ROS transduce differentiation signals by activating the ERK and p38 MAPK pathways in chondrocytes [30]. However, the mechanism of how ROS activates ERK1/2 and p38 MAPK during chondrogenesis is unclear. In other cells, S-nitrosylation or glutathionylation of Ras has been known to directly activate the protein and initiate the Ras-Raf-MEK/ERK cascade [49, 50]. The p38 MAPK has been proved to be activated by apoptosis signal-regulated kinase 1 (ASK1) under oxidative stress. When ROS oxidizes two cysteine residues in the redox centre of thioredoxin, ASK1 is activated, causing the formation of intramolecular disulfide bonds between Cys-32 and Cys-35 [51, 52]. Evidence that the ROS/ASK1/p38
MAPK pathway transmits differentiation signals in other cells may suggest a mechanism for ROS activation of p38 MAPK in chondrocytes [53].
4.2 PI3K-Akt signalling pathway Studies have demonstrated that PI3K plays a vital role in chondrogenesis and chondrocyte differentiation [4, 54, 55]. It has been shown that the activation of the PI3K-Akt pathway inhibits the transition in chondrocytes from proliferation to hypertrophic differentiation. On the other hand, the PI3K-Akt signalling pathway promotes the terminal differentiation of chondrocytes [54, 56]. In addition to redox regulation of phosphatases, ROS/RNS can also directly oxidize protein kinases (PI3K and Akt). This oxidative modification results in the downregulation of the PI3K/Akt signal [57]. In chondrocytes, insulin-like growth factor 1 (IGF-1) activates the IGF-1 receptor (IGF-1R), which induces activation of the insulin receptor substrate 1 (IRS-1) through tyrosine phosphorylation and, subsequently, activates the PI3K/Akt pathway, leading to increased aggrecan synthesis and collagen II expression. During early chondrogenesis, suppression of ROS generation by knockdown of Nox2 or Nox4 inhibited insulin-induced Akt phosphorylation [29]. This indicates that the Nox-derived ROS are required for Akt phosphorylation during early chondrogenesis. However, Yin et al. showed that oxidative stress also inhibited IGF-1-induced Akt phosphorylation in osteoarthritis chondrocytes [58]. Taken together, these results demonstrate that redox status in chondrocytes can alter intracellular signal transduction in response to growth factors and cytokines. The PI3K/Akt signalling pathway is sensitive to redox modifications, and the redox regulation of PI3K/Akt is mainly conducted by oxidative modification of Cys-dependent phosphatases (CDPs) and protein kinases. [59, 60]. CDPs that regulate PI3K/Akt signalling include PI3-phosphatase, PTEN, and PTPases. It is known that ROS can oxidize Cys residues in CDPs to cause the loss of phosphatase activity. This modification activates the PI3K/Akt signalling pathway. For example, oxidative inactivation of PTEN by S-nitrosylation of Cys residues is an important mechanism of oxidative stress-induced PI3K/Akt activation [61].
4.3 mTOR signalling pathway The mTOR signal pathway has been shown to be essential for chondrocyte proliferation and endochondral ossification [62-64]. It has been demonstrated that mTORC1 activity declines during chondrocyte differentiation in vitro and in vivo. Activation of the mTOR signalling pathway in chondrocytes promotes growth and proliferation but prevents the terminal differentiation of hypertrophic chondrocytes [65]. It is proved that the inhibition of mTOR is required for chondrocyte hypertrophy. Our previous study showed that inhibition of mTOR promotes hypertrophic differentiation in the pellet culture model [3]. It has been proved that Nox-derived ROS are required for Akt phosphorylation during early chondrogenesis. We hypothesized that ROS-induced Akt phosphorylation activates mTOR during early chondrogenic differentiation. In addition, physiological ROS levels are known to activate the mTOR signalling pathway in most cells [66-68]. The mechanism by which ROS activate mTOR is the Cys oxidation of Ras homologue enriched in brain (Rheb) and regulatory-associated protein of mTOR (Raptor) [66, 69, 70]. It has been found that the ROS regulate the Raptor-mTOR complex to control the ribosomal protein S6 kinase (S6K1), an effector of Raptor-mTOR. The ROS activate the Raptor-mTOR pathway through destabilization of the Raptor-mTOR interaction. In contrast, a reducing reagent inhibits this pathway by stabilization of the Raptor-mTOR interaction [66]. Importantly, further experiments need to be performed to verify the specific mechanism of ROS function on the mTOR signalling pathway during chondrogenesis. Furthermore, numerous studies have demonstrated that oxidative stress activates autophagy by inhibiting the mTOR signalling pathway to protect chondrocytes from apoptosis [71, 72]. However, the specific mechanism of oxidative stress in inhibiting mTOR activity needs to be further studied in chondrocytes. 4.4. cGMP signalling pathway It has been demonstrated that cGMP plays an important role in chondrocyte terminal differentiation[33]. The increase in cGMP concentration may lead to the activation of phosphodiesterases (PDE), cGMP-regulated ion channels (ICH) and cGMP-dependent
kinases (cGK). In particular, cGKII has been shown to play an essential role in endochondral bone formation [73, 74]. Possible downstream pathways include the inhibition of nuclear translocation of the transcription factor Sox9 and the repression of FGF signalling and ERK activation through Raf1 [75]. Teixeira et al. demonstrated that the expression of NOS and the generation of NO enhanced alkaline phosphatase and collagen type X expression by increasing cGMP concentration [33]. Nitric oxide could bind to the haem-containing soluble protein guanylate cyclase, causing increased enzymatic activity and formation of cGMP. Depending on the cell type, the increase in cGMP concentration may lead to activation of phosphodiesterases, cGMP-regulated ion channels and cGMP-dependent kinases (cGKs) [76].
5. Redox Control of Transcription Factors Involved in Chondrocyte Differentiation and Chondrogenesis 5.1. HIF-1α Hypoxia-inducible factor 1 alpha subunit (HIF-1α) is a crucial transcription factor for chondrogenesis and chondrocyte differentiation. Specific deletion of HIF-1 within chondrocytes led to the death of hypoxic chondrocytes and promoted chondrocyteproliferation, resulting in delayed hypertrophy in the growth plate [77]. Cartilage and growth plates are avascular tissues. Research has proved that HIF-1α is required for the survival of chondrocytes in the hypoxic region of the growth plate [78]. HIF-1α plays a non-redundant and critical role in the differentiation of mesenchymal cells into chondrocytes. HIF-1α was found to enhance the BMP2-induced chondrogenic expression of Sox9 and downstream markers and inhibit the osteogenic expression of Runx2 and downstream markers in vitro [79]. Lack of HIF-1α in limb bud mesenchyme caused a remarkable delay in chondrogenesis [80, 81]. The HIF-1α protein is oxygen-sensitive through oxygen-dependent hydroxylation of specific residues within its amino acid sequence. In normoxia, the oxygen-dependent degradation domain (ODDD) of HIF-1α is hydroxylated by HIF prolyl 4-hydroxylases [82]. The E3 ubiquitin ligase Von Hippel-Lindau (VHL) binds to hydroxylated HIF-1α. Then, HIF-1α degrades via the ubiquitylation pathway [83, 84]. Under hypoxic
conditions, HIF-1α is not hydroxylated. HIF-1α can then freely migrate to the nucleus and bind to the β subunit, acting as a transcription factor. ROS/RNS exert different effects on the HIF-1α protein in normoxia and hypoxia. In normoxia, ROS/RNS production can stabilize HIF-1α [85, 86]. Reducing ROS/RNS levels by antioxidants or reducing active oxygen producing enzyme activity can reduce HIF-1α expression [85, 87, 88]. It has been shown that ROS/RNS can stabilize HIF-1α in normoxia through multiple pathways [89]. Accumulation of ROS can lead to the inactivation of PHD, leading to the stabilization of HIF-1α [90]. Additionally, direct oxidative modifications such as S-nitrosylation of HIF or VHL proteins have been shown to lead to stabilization of HIF-1α [91, 92]. Under hypoxic conditions, both ROS and RNS inhibit HIF-1α DNA-binding activity and HIF-1α accumulation. Kazuo Yudoh et al. found that IL-1β and H2O2 could promote the expression of HIF-1α mRNA and protein in human articular chondrocytes in vitro under hypoxia (O2<6%) [93]. Under hypoxic conditions, inhibitors of PI3K and p38 MAPK reduced the protein levels of IL-1β-induced HIF-1α mRNA expression. However, further in-depth and systematic studies are required to determine how ROS/RNS regulate the turnover and transcriptional activity of HIF-1α in chondrogenesis.
5.2 AP-1 The c-fos and c-Jun proteins form the activator protein 1 (AP-1) complex. Some studies have shown that the AP-1 family plays a significant role in the hypertrophic differentiation of chondrocyte [94]. As a transcription factor, AP-1 activates genes required for the differentiation of various chondrocytes. Furthermore, recent studies have shown that AP-1 could directly interact with transcription factors such as Sox9 and Runx2, which are crucial chondrogenic differentiation factors, thereby promoting the marker gene for hypertrophic chondrocyte differentiation [94]. Nitric oxide may suppress the DNA-binding activity of AP-1 through S-glutathionylation. In the presence of the physiological sulfhydryl glutathione, nitric oxide modifies the two cysteine residues contained in the DNA-binding module of c-Jun selectively and distinctly. The formation of a mixed disulfide bond with glutathione inhibited the binding of AP-1 to DNA [95]. Furthermore, the intracellular level of AP-1 can be regulated by redox-mediated
mechanisms at the levels of transcription and protein turnover. For example, in bovine articular chondrocytes, TNF-α and essential fibroblast growth factor (bFGF) were found to induce ROS production through an NADPH oxidase enzyme complex, resulting in the upregulation of c-fos expression [96]. Similarly, ROS can regulate IL-1β induction of c-fos expression in the articular chondrocytes [97].
5.3 NK-κB/p65 Transcriptional targets of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) have been recognized as key developmental signalling mediators that regulate endochondral ossification. The involvement of NF-κB/p65 as an indispensable factor during chondrogenic development has been studied in the context of mature chondrocytes. NF-κB/p65 has been identified as a transcription factor for BMP2 and Sox9 in mature chondrocytes during endochondral ossification [98-100]. Moreover, it has been proved that activation of p65 promotes the early stage of chondrocyte differentiation during endochondral ossification [101]. The function of NF-κB can be activated or inhibited at various levels of the activation pathway through different redox-mediated mechanisms. In the nucleus, direct oxidation of Cys in the DNA-binding domain can hinder NF-κB DNA binding activity. In the cytosol, activation of NF-κB can be regulated by phosphorylation of NF-κB itself or phosphorylation of its inhibitor IκB. Under normal conditions, NF-κB and IκB form complexes that are sequestered in the cytosol. Increased ROS can directly activate IκB kinase (IκK) by redox-modification of IκK or can indirectly activate IκB kinase (IκB) by activating Akt and, subsequently, can phosphorylate and activate IκK [102]. Activated IκK phosphorylates IκB and releases active NF-κB from the complex for translocation to the nucleus [101]. R. MClancy et al. proved that nitric oxide and its derivatives could regulate cytokine-induced NF-κB activation in chondrocytes. Their results demonstrated that nitric oxide sustains NF-κB signalling in chondrocytes, most likely via peroxynitrite. Persistent activation of NF-κB signalling in chondrocytes further promotes and amplifies the transcription of inflammatory cytokines, growth factors and metalloproteinases [103].
Conclusion
Chondrogenesis is a continuous and dynamic process that is regulated by many growth factors. The intracellular redox status affects the response of chondrocytes to extracellular stimulation. Our review shows that the redox status controls chondrocyte differentiation and chondrogenesis as an endogenous regulator of signalling kinases and transcription factors. Although ROS/RNS have been studied extensively in other cells as signalling molecules that affect cellular processes, they have not been sufficiently investigated in chondrocytes. The specific state of redox levels in the articular cartilage and growth plate regions remains unclear. Similarly, the detailed mechanisms of how ROS regulate signalling pathways has not been studied in detail in chondrocytes and growth plates.
Acknowledgments This work was funded by grants from the National Key Technology Research and Development Program of China (2017YFC1103300), the Nature Science Foundation of China (31870962), and Major project of Logistics Research Plan of PLA (AWS17J004).
Competing Interests The authors declare that there is no conflict of interests regarding the publication of this paper.
References [1] T. Saito, A. Fukai, A. Mabuchi, T. Ikeda, F. Yano, S. Ohba, N. Nishida, T. Akune, N. Yoshimura, T. Nakagawa, K. Nakamura, K. Tokunaga, U.I. Chung, H. Kawaguchi, Transcriptional regulation of endochondral ossification by HIF-2alpha during skeletal growth and osteoarthritis development, Nature medicine 16(6) (2010) 678-86. [2] N. Ortega, D.J. Behonick, Z. Werb, Matrix remodeling during endochondral ossification, Trends in cell biology 14(2) (2004) 86-93. [3] Y. Bai, C. Liu, L. Fu, X. Gong, C. Dou, Z. Cao, H. Quan, J. Li, F. Kang, J. Dai, C. Zhao, S. Dong, Mangiferin enhances endochondral ossification-based bone repair in massive bone defect by inducing autophagy through activating AMP-activated protein kinase signaling pathway, FASEB journal : official publication of the Federation of American Societies for Experimental Biology (2018) fj201701411R. [4] Z. Cao, C. Dou, J. Li, X. Tang, J. Xiang, C. Zhao, L. Zhu, Y. Bai, Q. Xiang, S. Dong, Cordycepin inhibits chondrocyte hypertrophy of mesenchymal stem cells through PI3K/Bapx1 and Notch signaling pathway, BMB reports 49(10) (2016) 548-553. [5] D. Choudhary, S. Adhikary, N. Ahmad, P. Kothari, A. Verma, P.K. Trivedi, P.R.
Mishra, R. Trivedi, Prevention of articular cartilage degeneration in a rat model of monosodium iodoacetate induced osteoarthritis by oral treatment with Withaferin A, Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie 99 (2018) 151-161. [6] N.M. Khan, I. Ahmad, M.Y. Ansari, T.M. Haqqi, Wogonin, a natural flavonoid, intercalates with genomic DNA and exhibits protective effects in IL-1beta stimulated osteoarthritis chondrocytes, Chemico-biological interactions 274 (2017) 13-23. [7] N.M. Khan, A. Haseeb, M.Y. Ansari, P. Devarapalli, S. Haynie, T.M. Haqqi, Wogonin, a plant derived small molecule, exerts potent anti-inflammatory and chondroprotective effects through the activation of ROS/ERK/Nrf2 signaling pathways in human Osteoarthritis chondrocytes, Free radical biology & medicine 106 (2017) 288-301. [8] P. Lepetsos, A.G. Papavassiliou, ROS/oxidative stress signaling in osteoarthritis, Biochim Biophys Acta 1862(4) (2016) 576-591. [9] L. Grange, M.V. Nguyen, B. Lardy, M. Derouazi, Y. Campion, C. Trocme, M.H. Paclet, P. Gaudin, F. Morel, NAD(P)H oxidase activity of Nox4 in chondrocytes is both inducible and involved in collagenase expression, Antioxidants & redox signaling 8(9-10) (2006) 1485-96. [10] T.S. Hiran, P.J. Moulton, J.T. Hancock, Detection of superoxide and NADPH oxidase in porcine articular chondrocytes, Free radical biology & medicine 23(5) (1997) 736-43. [11] N. Jallali, H. Ridha, C. Thrasivoulou, P. Butler, T. Cowen, Modulation of intracellular reactive oxygen species level in chondrocytes by IGF-1, FGF, and TGF-beta1, Connective tissue research 48(3) (2007) 149-58. [12] Y. Henrotin, G. Deby-Dupont, C. Deby, P. Franchimont, I. Emerit, Active oxygen species, articular inflammation and cartilage damage, Exs 62 (1992) 308-22. [13] B. Fermor, J.B. Weinberg, D.S. Pisetsky, M.A. Misukonis, A.J. Banes, F. Guilak, The effects of static and intermittent compression on nitric oxide production in articular cartilage explants, J Orthop Res 19(4) (2001) 729-37. [14] J. Stadler, M. Stefanovic-Racic, T.R. Billiar, R.D. Curran, L.A. McIntyre, H.I. Georgescu, R.L. Simmons, C.H. Evans, Articular chondrocytes synthesize nitric oxide in response to cytokines and lipopolysaccharide, J Immunol 147(11) (1991) 3915-20. [15] M.A. Cake, R.A. Read, B. Guillou, P. Ghosh, Modification of articular cartilage and subchondral bone pathology in an ovine meniscectomy model of osteoarthritis by avocado and soya unsaponifiables (ASU), Osteoarthritis and cartilage 8(6) (2000) 404-11. [16] H.J. Cho, Q.W. Xie, J. Calaycay, R.A. Mumford, K.M. Swiderek, T.D. Lee, C. Nathan, Calmodulin is a subunit of nitric oxide synthase from macrophages, J Exp Med 176(2) (1992) 599-604. [17] Y. Henrotin, B. Kurz, Antioxidant to treat osteoarthritis: dream or reality?, Current drug targets 8(2) (2007) 347-57. [18] F.M. Faraci, S.P. Didion, Vascular protection: superoxide dismutase isoforms in the vessel wall, Arteriosclerosis, thrombosis, and vascular biology 24(8) (2004) 1367-73. [19] H. Nakamura, K. Nakamura, J. Yodoi, Redox regulation of cellular activation, Annual review of immunology 15 (1997) 351-69. [20] M.D. Carlo, Jr., R.F. Loeser, Increased oxidative stress with aging reduces chondrocyte survival: correlation with intracellular glutathione levels, Arthritis and
rheumatism 48(12) (2003) 3419-30. [21] M. Valko, D. Leibfritz, J. Moncol, M.T. Cronin, M. Mazur, J. Telser, Free radicals and antioxidants in normal physiological functions and human disease, The international journal of biochemistry & cell biology 39(1) (2007) 44-84. [22] G. Shen, The role of type X collagen in facilitating and regulating endochondral ossification of articular cartilage, Orthodontics & craniofacial research 8(1) (2005) 11-7. [23] M.B. Goldring, Chondrogenesis, chondrocyte differentiation, and articular cartilage metabolism in health and osteoarthritis, Therapeutic advances in musculoskeletal disease 4(4) (2012) 269-85. [24] H.P. Gerber, T.H. Vu, A.M. Ryan, J. Kowalski, Z. Werb, N. Ferrara, VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation, Nature medicine 5(6) (1999) 623-8. [25] L. Zhang, G. Wang, X. Chen, X. Xue, Q. Guo, M. Liu, J. Zhao, Formyl peptide receptors promotes neural differentiation in mouse neural stem cells by ROS generation and regulation of PI3K-AKT signaling, Scientific reports 7(1) (2017) 206. [26] T. Liu, Q. Men, G. Wu, C. Yu, Z. Huang, X. Liu, W. Li, Tetrandrine induces autophagy and differentiation by activating ROS and Notch1 signaling in leukemia cells, Oncotarget 6(10) (2015) 7992-8006. [27] N. Sampson, R. Koziel, C. Zenzmaier, L. Bubendorf, E. Plas, P. Jansen-Durr, P. Berger, ROS signaling by NOX4 drives fibroblast-to-myofibroblast differentiation in the diseased prostatic stroma, Mol Endocrinol 25(3) (2011) 503-15. [28] K. Ambe, H. Watanabe, S. Takahashi, T. Nakagawa, Immunohistochemical localization of Nox1, Nox4 and Mn-SOD in mouse femur during endochondral ossification, Tissue Cell 46(6) (2014) 433-8. [29] K.S. Kim, H.W. Choi, H.E. Yoon, I.Y. Kim, Reactive oxygen species generated by NADPH oxidase 2 and 4 are required for chondrogenic differentiation, J Biol Chem 285(51) (2010) 40294-302. [30] K. Morita, T. Miyamoto, N. Fujita, Y. Kubota, K. Ito, K. Takubo, K. Miyamoto, K. Ninomiya, T. Suzuki, R. Iwasaki, M. Yagi, H. Takaishi, Y. Toyama, T. Suda, Reactive oxygen species induce chondrocyte hypertrophy in endochondral ossification, J Exp Med 204(7) (2007) 1613-23. [31] R.K. Studer, Nitric oxide decreases IGF-1 receptor function in vitro; glutathione depletion enhances this effect in vivo, Osteoarthritis and cartilage 12(11) (2004) 863-9. [32] C.C. Teixeira, K. Mansfield, C. Hertkorn, H. Ischiropoulos, I.M. Shapiro, Phosphate-induced chondrocyte apoptosis is linked to nitric oxide generation, American journal of physiology. Cell physiology 281(3) (2001) C833-9. [33] C.C. Teixeira, H. Ischiropoulos, P.S. Leboy, S.L. Adams, I.M. Shapiro, Nitric oxide-nitric oxide synthase regulates key maturational events during chondrocyte terminal differentiation, Bone 37(1) (2005) 37-45. [34] G. Wang, Q. Yan, A. Woods, L.A. Aubrey, Q. Feng, F. Beier, Inducible nitric oxide synthase-nitric oxide signaling mediates the mitogenic activity of Rac1 during endochondral bone growth, Journal of cell science 124(Pt 20) (2011) 3405-13. [35] M. Schieber, N.S. Chandel, ROS function in redox signaling and oxidative stress, Current biology : CB 24(10) (2014) R453-62. [36] K. England, T.G. Cotter, Direct oxidative modifications of signalling proteins in mammalian cells and their effects on apoptosis, Redox report : communications in free
radical research 10(5) (2005) 237-45. [37] N. Chakouri, C. Reboul, D. Boulghobra, A. Kleindienst, S. Nottin, S. Gayrard, F. Roubille, S. Matecki, A. Lacampagne, O. Cazorla, Stress-induced protein S-glutathionylation and phosphorylation crosstalk in cardiac sarcomeric proteins - Impact on heart function, International journal of cardiology 258 (2018) 207-216. [38] P. Ghezzi, Regulation of protein function by glutathionylation, Free radical research 39(6) (2005) 573-80. [39] C.A. O'Brian, F. Chu, Post-translational disulfide modifications in cell signaling--role of inter-protein, intra-protein, S-glutathionyl, and S-cysteaminyl disulfide modifications in signal transmission, Free radical research 39(5) (2005) 471-80. [40] W.H. Hoefnagels, J.I. Drayer, A.G. Smals, P.W. Kloppenborg, Spironolactone and amiloride in hypertensive patients with and without aldosterone excess, Clinical pharmacology and therapeutics 27(3) (1980) 317-23. [41] E.M. Jeong, C.Z. Jin, J.H. Jang, Z.H. Zhao, C.L. Jin, J.H. Lee, K.B. Lee, S.J. Kim, I.G. Kim, Y.H. Zhang, S-nitrosylation of transglutaminase 2 impairs fatty acid-stimulated contraction in hypertensive cardiomyocytes, Experimental & molecular medicine 50(4) (2018) 9. [42] L.B. Poole, P.A. Karplus, A. Claiborne, Protein sulfenic acids in redox signaling, Annual review of pharmacology and toxicology 44 (2004) 325-47. [43] T. Iwai, J. Murai, H. Yoshikawa, N. Tsumaki, Smad7 Inhibits chondrocyte differentiation at multiple steps during endochondral bone formation and down-regulates p38 MAPK pathways, The Journal of biological chemistry 283(40) (2008) 27154-64. [44] L.A. Stanton, S. Sabari, A.V. Sampaio, T.M. Underhill, F. Beier, p38 MAP kinase signalling is required for hypertrophic chondrocyte differentiation, Biochem J 378(Pt 1) (2004) 53-62. [45] X. Zhen, L. Wei, Q. Wu, Y. Zhang, Q. Chen, Mitogen-activated protein kinase p38 mediates regulation of chondrocyte differentiation by parathyroid hormone, The Journal of biological chemistry 276(7) (2001) 4879-85. [46] C.D. Oh, S.H. Chang, Y.M. Yoon, S.J. Lee, Y.S. Lee, S.S. Kang, J.S. Chun, Opposing role of mitogen-activated protein kinase subtypes, erk-1/2 and p38, in the regulation of chondrogenesis of mesenchymes, The Journal of biological chemistry 275(8) (2000) 5613-9. [47] (!!! INVALID CITATION !!! {Stanton, 2004 #85}). [48] Z. Chen, S.X. Yue, G. Zhou, E.M. Greenfield, S. Murakami, ERK1 and ERK2 regulate chondrocyte terminal differentiation during endochondral bone formation, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 30(5) (2015) 765-74. [49] H.M. Lander, A.J. Milbank, J.M. Tauras, D.P. Hajjar, B.L. Hempstead, G.D. Schwartz, R.T. Kraemer, U.A. Mirza, B.T. Chait, S.C. Burk, L.A. Quilliam, Redox regulation of cell signalling, Nature 381(6581) (1996) 380-1. [50] R.J. Mallis, J.E. Buss, J.A. Thomas, Oxidative modification of H-ras: S-thiolation and S-nitrosylation of reactive cysteines, The Biochemical journal 355(Pt 1) (2001) 145-53. [51] M. Saitoh, H. Nishitoh, M. Fujii, K. Takeda, K. Tobiume, Y. Sawada, M. Kawabata, K. Miyazono, H. Ichijo, Mammalian thioredoxin is a direct inhibitor of apoptosis signal-regulating kinase (ASK) 1, The EMBO journal 17(9) (1998) 2596-606.
[52] G. Fujino, T. Noguchi, A. Matsuzawa, S. Yamauchi, M. Saitoh, K. Takeda, H. Ichijo, Thioredoxin and TRAF family proteins regulate reactive oxygen species-dependent activation of ASK1 through reciprocal modulation of the N-terminal homophilic interaction of ASK1, Molecular and cellular biology 27(23) (2007) 8152-63. [53] A. Sato, M. Okada, K. Shibuya, E. Watanabe, S. Seino, Y. Narita, S. Shibui, T. Kayama, C. Kitanaka, Pivotal role for ROS activation of p38 MAPK in the control of differentiation and tumor-initiating capacity of glioma-initiating cells, Stem cell research 12(1) (2014) 119-31. [54] K. Kita, T. Kimura, N. Nakamura, H. Yoshikawa, T. Nakano, PI3K/Akt signaling as a key regulatory pathway for chondrocyte terminal differentiation, Genes to cells : devoted to molecular & cellular mechanisms 13(8) (2008) 839-50. [55] V. Ulici, K.D. Hoenselaar, J.R. Gillespie, F. Beier, The PI3K pathway regulates endochondral bone growth through control of hypertrophic chondrocyte differentiation, BMC developmental biology 8 (2008) 40. [56] T. Fujita, Y. Azuma, R. Fukuyama, Y. Hattori, C. Yoshida, M. Koida, K. Ogita, T. Komori, Runx2 induces osteoblast and chondrocyte differentiation and enhances their migration by coupling with PI3K-Akt signaling, The Journal of cell biology 166(1) (2004) 85-95. [57] H. Murata, Y. Ihara, H. Nakamura, J. Yodoi, K. Sumikawa, T. Kondo, Glutaredoxin exerts an antiapoptotic effect by regulating the redox state of Akt, The Journal of biological chemistry 278(50) (2003) 50226-33. [58] W. Yin, J.I. Park, R.F. Loeser, Oxidative stress inhibits insulin-like growth factor-I induction of chondrocyte proteoglycan synthesis through differential regulation of phosphatidylinositol 3-Kinase-Akt and MEK-ERK MAPK signaling pathways, The Journal of biological chemistry 284(46) (2009) 31972-81. [59] R. Naughton, C. Quiney, S.D. Turner, T.G. Cotter, Bcr-Abl-mediated redox regulation of the PI3K/AKT pathway, Leukemia 23(8) (2009) 1432-40. [60] K. Niwa, O. Inanami, T. Yamamori, T. Ohta, T. Hamasu, M. Kuwabara, Redox regulation of PI3K/Akt and p53 in bovine aortic endothelial cells exposed to hydrogen peroxide, Antioxidants & redox signaling 5(6) (2003) 713-22. [61] C.X. Yu, S. Li, A.R. Whorton, Redox regulation of PTEN by S-nitrosothiols, Molecular pharmacology 68(3) (2005) 847-54. [62] J. Chen, F. Long, mTORC1 signaling controls mammalian skeletal growth through stimulation of protein synthesis, Development 141(14) (2014) 2848-54. [63] C.P. Sanchez, Y.Z. He, Bone growth during rapamycin therapy in young rats, BMC pediatrics 9 (2009) 3. [64] Y. Guan, X. Yang, W. Yang, C. Charbonneau, Q. Chen, Mechanical activation of mammalian target of rapamycin pathway is required for cartilage development, FASEB journal : official publication of the Federation of American Societies for Experimental Biology 28(10) (2014) 4470-81. [65] B. Yan, Z. Zhang, D. Jin, C. Cai, C. Jia, W. Liu, T. Wang, S. Li, H. Zhang, B. Huang, P. Lai, H. Wang, A. Liu, C. Zeng, D. Cai, Y. Jiang, X. Bai, mTORC1 regulates PTHrP to coordinate chondrocyte growth, proliferation and differentiation, Nature communications 7 (2016) 11151. [66] D.D. Sarbassov, D.M. Sabatini, Redox regulation of the nutrient-sensitive raptor-mTOR pathway and complex, The Journal of biological chemistry 280(47) (2005)
39505-9. [67] O. Zhenyukh, E. Civantos, M. Ruiz-Ortega, M.S. Sanchez, C. Vazquez, C. Peiro, J. Egido, S. Mas, High concentration of branched-chain amino acids promotes oxidative stress, inflammation and migration of human peripheral blood mononuclear cells via mTORC1 activation, Free radical biology & medicine 104 (2017) 165-177. [68] B. Steinhorn, J.L. Sartoretto, A. Sorrentino, N. Romero, H. Kalwa, E.D. Abel, T. Michel, Insulin-dependent metabolic and inotropic responses in the heart are modulated by hydrogen peroxide from NADPH-oxidase isoforms NOX2 and NOX4, Free radical biology & medicine 113 (2017) 16-25. [69] S. Yoshida, S. Hong, T. Suzuki, S. Nada, A.M. Mannan, J. Wang, M. Okada, K.L. Guan, K. Inoki, Redox regulates mammalian target of rapamycin complex 1 (mTORC1) activity by modulating the TSC1/TSC2-Rheb GTPase pathway, The Journal of biological chemistry 286(37) (2011) 32651-60. [70] C.T. Hsieh, J.H. Chuang, W.C. Yang, Y. Yin, Y. Lin, Ceramide inhibits insulin-stimulated Akt phosphorylation through activation of Rheb/mTORC1/S6K signaling in skeletal muscle, Cellular signalling 26(7) (2014) 1400-8. [71] N.M. Khan, M.Y. Ansari, T.M. Haqqi, Sucrose, But Not Glucose, Blocks IL1-beta-Induced Inflammatory Response in Human Chondrocytes by Inducing Autophagy via AKT/mTOR Pathway, Journal of cellular biochemistry 118(3) (2017) 629-639. [72] Y.S. Li, F.J. Zhang, C. Zeng, W. Luo, W.F. Xiao, S.G. Gao, G.H. Lei, Autophagy in osteoarthritis, Joint, bone, spine : revue du rhumatisme 83(2) (2016) 143-8. [73] F. Kugimiya, H. Chikuda, S. Kamekura, T. Ikeda, K. Hoshi, T. Ogasawara, K. Nakamura, U.I. Chung, H. Kawaguchi, Involvement of cyclic guanosine monophosphate-dependent protein kinase II in chondrocyte hypertrophy during endochondral ossification, Modern rheumatology 15(6) (2005) 391-6. [74] T. Miyazawa, Y. Ogawa, H. Chusho, A. Yasoda, N. Tamura, Y. Komatsu, A. Pfeifer, F. Hofmann, K. Nakao, Cyclic GMP-dependent protein kinase II plays a critical role in C-type natriuretic peptide-mediated endochondral ossification, Endocrinology 143(9) (2002) 3604-10. [75] D.M. Ornitz, P.J. Marie, Fibroblast growth factor signaling in skeletal development and disease, Genes & development 29(14) (2015) 1463-86. [76] J.W. Denninger, M.A. Marletta, Guanylate cyclase and the .NO/cGMP signaling pathway, Biochimica et biophysica acta 1411(2-3) (1999) 334-50. [77] E. Schipani, Hypoxia and HIF-1 alpha in chondrogenesis, Seminars in cell & developmental biology 16(4-5) (2005) 539-46. [78] E. Schipani, H.E. Ryan, S. Didrickson, T. Kobayashi, M. Knight, R.S. Johnson, Hypoxia in cartilage: HIF-1alpha is essential for chondrocyte growth arrest and survival, Genes & development 15(21) (2001) 2865-76. [79] N. Zhou, N. Hu, J.Y. Liao, L.B. Lin, C. Zhao, W.K. Si, Z. Yang, S.X. Yi, T.X. Fan, W. Bao, X. Liang, X. Wei, H. Chen, C. Chen, Q. Chen, X. Lin, W. Huang, HIF-1alpha as a Regulator of BMP2-Induced Chondrogenic Differentiation, Osteogenic Differentiation, and Endochondral Ossification in Stem Cells, Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology 36(1) (2015) 44-60. [80] R. Amarilio, S.V. Viukov, A. Sharir, I. Eshkar-Oren, R.S. Johnson, E. Zelzer,
HIF1alpha regulation of Sox9 is necessary to maintain differentiation of hypoxic prechondrogenic cells during early skeletogenesis, Development 134(21) (2007) 3917-28. [81] S. Provot, D. Zinyk, Y. Gunes, R. Kathri, Q. Le, H.M. Kronenberg, R.S. Johnson, M.T. Longaker, A.J. Giaccia, E. Schipani, Hif-1alpha regulates differentiation of limb bud mesenchyme and joint development, The Journal of cell biology 177(3) (2007) 451-64. [82] R.K. Bruick, S.L. McKnight, A conserved family of prolyl-4-hydroxylases that modify HIF, Science 294(5545) (2001) 1337-40. [83] M. Ivan, K. Kondo, H. Yang, W. Kim, J. Valiando, M. Ohh, A. Salic, J.M. Asara, W.S. Lane, W.G. Kaelin, Jr., HIFalpha targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing, Science 292(5516) (2001) 464-8. [84] P. Jaakkola, D.R. Mole, Y.M. Tian, M.I. Wilson, J. Gielbert, S.J. Gaskell, A. von Kriegsheim, H.F. Hebestreit, M. Mukherji, C.J. Schofield, P.H. Maxwell, C.W. Pugh, P.J. Ratcliffe, Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation, Science 292(5516) (2001) 468-72. [85] R.S. BelAiba, T. Djordjevic, S. Bonello, D. Flugel, J. Hess, T. Kietzmann, A. Gorlach, Redox-sensitive regulation of the HIF pathway under non-hypoxic conditions in pulmonary artery smooth muscle cells, Biological chemistry 385(3-4) (2004) 249-57. [86] A. Gorlach, I. Diebold, V.B. Schini-Kerth, U. Berchner-Pfannschmidt, U. Roth, R.P. Brandes, T. Kietzmann, R. Busse, Thrombin activates the hypoxia-inducible factor-1 signaling pathway in vascular smooth muscle cells: Role of the p22(phox)-containing NADPH oxidase, Circulation research 89(1) (2001) 47-54. [87] T. Kietzmann, A. Gorlach, Reactive oxygen species in the control of hypoxia-inducible factor-mediated gene expression, Seminars in cell & developmental biology 16(4-5) (2005) 474-86. [88] V.A. Shatrov, V.V. Sumbayev, J. Zhou, B. Brune, Oxidized low-density lipoprotein (oxLDL) triggers hypoxia-inducible factor-1alpha (HIF-1alpha) accumulation via redox-dependent mechanisms, Blood 101(12) (2003) 4847-9. [89] E.L. Bell, N.S. Chandel, Mitochondrial oxygen sensing: regulation of hypoxia-inducible factor by mitochondrial generated reactive oxygen species, Essays in biochemistry 43 (2007) 17-27. [90] J. Pouyssegur, F. Mechta-Grigoriou, Redox regulation of the hypoxia-inducible factor, Biological chemistry 387(10-11) (2006) 1337-46. [91] F. Li, P. Sonveaux, Z.N. Rabbani, S. Liu, B. Yan, Q. Huang, Z. Vujaskovic, M.W. Dewhirst, C.Y. Li, Regulation of HIF-1alpha stability through S-nitrosylation, Molecular cell 26(1) (2007) 63-74. [92] I. Pinchuk, E. Schnitzer, D. Lichtenberg, Kinetic analysis of copper-induced peroxidation of LDL, Biochimica et biophysica acta 1389(2) (1998) 155-72. [93] K. Yudoh, H. Nakamura, K. Masuko-Hongo, T. Kato, K. Nishioka, Catabolic stress induces expression of hypoxia-inducible factor (HIF)-1 alpha in articular chondrocytes: involvement of HIF-1 alpha in the pathogenesis of osteoarthritis, Arthritis research & therapy 7(4) (2005) R904-14. [94] X. He, S. Ohba, H. Hojo, A.P. McMahon, AP-1 family members act with Sox9 to promote chondrocyte hypertrophy, Development 143(16) (2016) 3012-23. [95] P. Klatt, E.P. Molina, S. Lamas, Nitric oxide inhibits c-Jun DNA binding by specifically targeted S-glutathionylation, The Journal of biological chemistry 274(22) (1999) 15857-64.
[96] Y.Y. Lo, T.F. Cruz, Involvement of reactive oxygen species in cytokine and growth factor induction of c-fos expression in chondrocytes, The Journal of biological chemistry 270(20) (1995) 11727-30. [97] Y.Y. Lo, J.A. Conquer, S. Grinstein, T.F. Cruz, Interleukin-1 beta induction of c-fos and collagenase expression in articular chondrocytes: involvement of reactive oxygen species, Journal of cellular biochemistry 69(1) (1998) 19-29. [98] M. Ushita, T. Saito, T. Ikeda, F. Yano, A. Higashikawa, N. Ogata, U. Chung, K. Nakamura, H. Kawaguchi, Transcriptional induction of SOX9 by NF-kappaB family member RelA in chondrogenic cells, Osteoarthritis and cartilage 17(8) (2009) 1065-75. [99] J.Q. Feng, L. Xing, J.H. Zhang, M. Zhao, D. Horn, J. Chan, B.F. Boyce, S.E. Harris, G.R. Mundy, D. Chen, NF-kappaB specifically activates BMP-2 gene expression in growth plate chondrocytes in vivo and in a chondrocyte cell line in vitro, The Journal of biological chemistry 278(31) (2003) 29130-5. [100] S. Wu, J.K. Flint, G. Rezvani, F. De Luca, Nuclear factor-kappaB p65 facilitates longitudinal bone growth by inducing growth plate chondrocyte proliferation and differentiation and by preventing apoptosis, The Journal of biological chemistry 282(46) (2007) 33698-706. [101] M.M. Caron, P.J. Emans, D.A. Surtel, A. Cremers, J.W. Voncken, T.J. Welting, L.W. van Rhijn, Activation of NF-kappaB/p65 facilitates early chondrogenic differentiation during endochondral ossification, PloS one 7(3) (2012) e33467. [102] D. Trachootham, W. Lu, M.A. Ogasawara, R.D. Nilsa, P. Huang, Redox regulation of cell survival, Antioxidants & redox signaling 10(8) (2008) 1343-74. [103] R.M. Clancy, P.F. Gomez, S.B. Abramson, Nitric oxide sustains nuclear factor kappaB activation in cytokine-stimulated chondrocytes, Osteoarthritis and cartilage 12(7) (2004) 552-8.
Fig 1. Major sites of cellular reactive oxygen species (ROS) generation include the mitochondria and the NADPH oxidase (NOX). Nitric oxide synthases (NOS) are key enzymes for production of NO. Gpx, glutathione peroxidase; HO•, hydroxyl radical; NO, nitric oxide; ONOO•, peroxynitrite; SOD, superoxide dismutase; GSH, reduced glutathione; GSSG, oxidized glutathione; NADPH, reduced nicotinamide adenine dinucleotide phosphate. Fig 2. Schematic representation of the distribution of Nox1 and Nox4 on growth plates and the effect of ROS on chondrogenesis. In growth plate, Nox1 and Nox4 are expressed in different regions. Nox4 expression in chondrocytes was observed from the resting zone to the hypertrophy region, while Nox1 expression was restricted from the proliferating zone to the prehypertrophic zone. The Nox4-derived ROS is required for the early stage of chondrocyte differentiation. Nox1 synthesizes major reactive oxygen species compared to Nox4. Therefore, chondrocytes always produce ROS throughout the endochondral ossification and emit large amounts of reactive oxygen species from the proliferative zone to the prehypertrophic zone. Fig 3. Schematic representation of the redox regulation of proteins. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) can oxidize reactive cysteine of the protein. Mild oxidative stress can induce cysteine modifications such as reversible S-glutathionylation, disulfide bond formation and S-nitrosylation. These reversible redox post-translational modifications act as signalling in chondrocyte. In contrast, oxidative stress promotes the irreversible modification of sulfenic, sulfinic and sulfonic acids are destructive to protein function. Fig 4. Redox regulation of PI3K/AKT and MAPK signalling pathway in chondrogenesis. The growth factors and cytokines activate PI3K/AKT signalling, which modulate NADPH oxidases (NOXs) -derived reactive oxygen species (ROS). The NOXs derived ROS can activate PI3K/Akt and MAPK signalling pathway. The ROS activate mTOR through cysteine oxidation of Rheb and Raptor. Increased ROS can directly activate IκK by redox-modifying IκK, or indirectly activate IκK by activating Akt, and then phosphorylate and activate IκK. Activated IκK phosphorylates IκB and releases active NF-κB from the complex for translocation to the nucleus. TGFβ and BMP activate p38
MAPK signalling through TAK1. The ROS may increases and induces p38 MAPK activation through activating ASK1. In the process of chondrocyte hypertrophy, p38 MAPK regulates MEF2C, HDAC4 and RUNX2 in pre-hypertrophic and hypertrophic chondrocytes.
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Graphical Abstract Highlights
The role of redox regulation in specific signal transduction of chondrocyte differentiation and chondrogenesis have also been reviewed
Function of Nox-derived ROS in growth plate development have also been reviewed
The mechanism by which ROS/RNS regulates signaling proteins and transcription factors in chondrocytes have also been reviewed