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New functions of lysosomes in bone cells
Journal of Oral Biosciences ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Review
New functions of lysosomes in bone cells Takayuki Tsukuba a,n, Eiko Sakai a, Kazuhisa Nishishita a, Tomoko Kadowaki b, Kuniaki Okamoto a a b
Departments of Dental Pharmacology, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8588, Japan Departments of Frontier Life Science, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8588, Japan
art ic l e i nf o
a b s t r a c t
Article history: Received 9 November 2016 Received in revised form 8 December 2016 Accepted 25 December 2016
Background: Lysosomes are intracellular acidic organelles that contain approximately 50 hydrolases and 25 species of integral membrane proteins. Although lysosome-like specific compartments, termed lysosome-related organelles (LROs), are found in osteoclasts, their functions in these cells and lysosomal functions in osteoblasts remain to be elucidated. Highlight: Recently, we found that expression of RAB27A is markedly increased during osteoclastic differentiation. RAB27A deficiency causes multinucleation and giant cell formation, characterized by abnormal transport of cell surface receptors and LROs into osteoclasts. Furthermore, we have shown that transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, regulates osteoblastic differentiation. Overexpression of TFEB in preosteoblastic MC3T3-E1 cells enhances osteoblastic differentiation via decreased expression of activating transcription factor 4 (ATF4) and CCAAT/enhancer-binding protein homologous protein (CHOP). These results indicate that the expression of ATF4 and CHOP is essential for differentiation into osteoblasts. Conclusion: RAB27A participates in bone resorption by LROs in osteoclasts. In addition, lysosomal biogenesis modulated by TFEB is necessary for osteoblastic differentiation. & 2017 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.
Keywords: Lysosomes Osteoclasts Osteoblast RAB27A TFEB
Contents 1. Introduction . . . . . . . . . . . 2. LROs in osteoclasts . . . . . 3. RAB27A and osteoclasts . 4. Lysosomes in osteoblasts. 5. TFEB and osteoblasts . . . . 6. Conclusions . . . . . . . . . . . Conflicts of interest . . . . . . . . . Acknowledgments . . . . . . . . . . References . . . . . . . . . . . . . . . .
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1. Introduction Lysosomes are acidic membrane-bound organelles that contain approximately 50 hydrolases, including proteases, glycosidases,
Abbreviations: (BMM), bone marrow macrophage; (LROs), lysosome-related organelles; (M-CSF), macrophage-colony stimulating factor; (siRNA), small interfering RNA; (V-ATPase), vacuolar-type H þ -ATPase. n Correspondence to: Department of Dental Pharmacology, Graduate School of Biomedical Sciences, Nagasaki University, 1-7-1 Sakamoto, Nagasaki 852-8588, Japan. E-mail address: [email protected] (T. Tsukuba).
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1 2 2 3 3 3 4 4 4
lipases, nucleases, phospholipases, phosphatases, and sulfatases [1–3]. Lysosomes contain approximately 25 species of integral membrane proteins. The most enriched membrane proteins are lysosome-associated membrane protein 1 (LAMP1), LAMP2, lysosome integral membrane protein 1 (LIMP1), LIMP2, and CD63 [3]. Lysosomes continuously digest macromolecules via three major pathways: endocytosis, phagocytosis, and autophagy. Defective lysosomal functions cause neurodegenerative diseases, immunesystem-related diseases, infectious diseases, cancer, cell differentiation, and so-called lysosomal storage diseases [4,5]. Although lysosomes are present ubiquitously in mammalian cells, there are cell type-specific compartments, termed lysosome-related
http://dx.doi.org/10.1016/j.job.2017.01.004 1349-0079/& 2017 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.
Please cite this article as: Tsukuba T, et al. New functions of lysosomes in bone cells. J Oral Biosci (2017), http://dx.doi.org/10.1016/j. job.2017.01.004i
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organelles (LROs), which are roughly similar to lysosomes, but morphologically and functionally different from them [6,7]. Examples of LROs include melanosomes in melanocytes [8], lytic granules in lymphocytes [9,10], and delta granules in platelets [11]. In addition to these examples, osteoclasts contain LROs surrounded by the ruffled-border membrane [12,13]. So far, studies on LRO functions in osteoclasts have been scarce as compared to studies on LRO functions in other cell types. Moreover, the function of lysosomes in osteoblasts remains unclear. In this review, we compare and discuss lysosomal functions in osteoclasts and osteoblasts.
2. LROs in osteoclasts Osteoclasts are the bone-resorbing cells that function using LROs [6,7,14]. Osteoclastic LROs contain certain specific components, the most abundant of which are cathepsin K and tartrateresistant acid phosphatase (TRAP). Cathepsin K was initially identified as a lysosomal cysteine protease that is predominantly expressed in osteoclasts [15]. Because cathepsin K has a powerful collagenolytic activity, it is predominantly involved in the degradation of extracellular-matrix components such as collagens, lamin, tenascin, and osteonectin [16,17]. Cathepsin K-deficient mice show osteopetrosis characterized by reduced osteoclastic bone resorption [18]. Mutations in human cathepsin K cause an autosomal recessive disease termed pycnodysostosis, a specific type of osteopetrosis, characterized by osteosclerosis with increased bone fragility [19,20]. TRAP (also known as ACP5) is a metalloenzyme that is abundant in osteoclasts although this enzyme is also expressed in some organs and tissues such as the spleen, liver, gastrointestinal tract lining, lung, thymus, and skin [21]. TRAP is also useful as a biomarker of bone resorption; its serum levels are measured for the clinical diagnosis of metabolic and bone diseases [22,23]. TRAP-deficient mice show early onset of the osteopetrotic bone phenotype because their osteoclasts are less active in bone degradation [24]. In addition to these specific soluble enzymes, several lysosomal membrane proteins are known to be osteoclast specific. Vacuolartype H þ -ATPases (V-ATPases), which help to maintain acidic pH of lysosomes, are proton pumps composed of 13 different subunits [25]. Among these, the a3 subunit is known to be osteoclast specific. Expression of a3 is approximately 100-fold greater in osteoclasts than in other cell types [26]. Indeed, mice deficient in this subunit have severe osteopetrosis [27]. In humans, mutation of the gene encoding the a3 subunit of V-ATPase results in autosomal recessive osteopetrosis caused by impaired osteoclast-mediated bone resorption [28]. The complex of voltage-gated chloride channel 7 (CLC7) and osteopetrosis-associated transmembrane protein 1 (OSTM1) has been reported as an osteoclast-specific protein complex [29]. CLC7 is a 2Cl /1 H þ ion exchanger, and OSTM1 is a lysosomal protein important for the stability of the CLC7–OSTM1 complex [29]. Any defect in the gene of either CLC7 or OSTM1 causes severe malignant infantile osteopetrosis [30–32]. Thus, it is likely that all mutant animals deficient in osteoclastspecific lysosomal proteins have osteopetrosis. Osteoclastic LROs have unique segregation systems for their proteins. Localization of several cathepsins is indeed distinct in osteoclasts, whereas it is similar among other cell types. Our immunocytochemical studies have suggested that cathepsins B, E, and K are present in the ruffled-border membrane and nearby vacuoles, whereas cathepsin D is localized to vacuoles and lysosomes that are detached from the ruffled-border membrane [33–36]. In agreement with our findings, osteoclasts from mice deficient in GlcNAc-1-phosphotransferase α/β-subunits [these animals have a defect in the mannose 6-phosphate (Man-6-P)-dependent pathway] show
enhanced secretion of cathepsin K and TRAP but normal targeting of cathepsin D [37]. In addition to lysosomal enzymes, lysosomal membrane proteins are also segregated in osteoclasts. For example, V-ATPase is present along the actin ring of osteoclasts, whereas cathepsin K is localized to the center of the actin ring as a punctate structure [38]. These findings suggest that LROs in osteoclasts have specific segregation systems.
3. RAB27A and osteoclasts To identify a gene that regulates membrane trafficking of LROs during osteoclastic differentiation, we have conducted a DNA microarray analysis. During the analysis, we observed a marked increase in the expression of RAB27A during osteoclastic differentiation [39]. RAB27A, a member of the RAB family of small GTPases, is exclusively localized to secretory granules in various secretory cells, including endocrine and exocrine cells and various leukocytes [40]. Mutation of RAB27A in humans causes type 2 Griscelli syndrome, which is characterized by hypopigmentation of the skin and eyes and immunodeficiency [9,41]. Nonetheless, the involvement of RAB27A in LROs of osteoclasts has yet to be elucidated. Therefore, we further analyzed the functions of RAB27A using a small interfering RNA (siRNA)-mediated knockdown in a murine macrophagic RAW-D cell line or bone marrow macrophages (BMMs) derived from ashen mice, which carry a mutation in Rab27a. Osteoclasts derived from either RAB27A knockdown RAW-D cells or ashen mice were found to show multinucleation and giant cell formation as key phenotypes when compared with control cells [39]. The ashen mouse osteoclasts showed abnormal actin ring formation, aberrant subcellular localization of LAMP2 and cathepsin K, and impaired resorption activity [39]. In addition to the lysosomal systems, trafficking of cell surface receptors was abnormal in the ashen mouse osteoclasts. The surface amount of c-fms, a macrophage-colony stimulating factor (M-CSF) receptor, was slightly greater in ashen mouse BMMs than in wild-type BMMs. Additionally, downregulation of receptor activator of nuclear factor κ B (RANK; a RANK ligand [RANKL] receptor) was delayed. In fact, although cellular RANK in wild-type BMMs was gradually degraded, RANK in ashen mouse BMMs was found to be upregulated [39]. As a result, upon stimulation with M-CSF and RANKL, phosphorylation levels of extracellular signal-regulated kinase (Erk), proto-oncogene tyrosineprotein kinase (Src), and p38 were slightly enhanced in ashen mouse BMMs in comparison with wild-type BMMs [39]. Thus, Rab27A deficiency causes abnormal transport of cell surface receptors thereby modulating multinucleation and LROs in osteoclasts. As shown in Fig. 1, RAB27A deficiency in osteoclasts causes abnormal transport of cell surface receptor called RANK, eventually enhancing signaling that drives osteoclastic differentiation. Nevertheless, RAB27A deficiency leads to impaired transport of LROs for bone resorption. Given that the phenotype of RAB27A-deficient osteoclasts is unique, this phenotype is different from that of osteoclasts deficient for other RABs. For example, RAB3 is known to modulate exocytosis in secretory cells. Osteoclasts from mice deficient in Rab3D—the Rab3 isoform predominantly expressed in osteoclasts —show an abnormal ruffled-border membrane [42]. In addition, a knockdown of RAB7—a key regulator of biogenesis of late endosomes and lysosomes—disrupts polarization of ruffled-border formation and decreases bone resorption by osteoclasts [43]. Thus, our results indicate that RAB27A plays a unique role in membrane trafficking of osteoclasts as compared with the roles of other RAB proteins.
Please cite this article as: Tsukuba T, et al. New functions of lysosomes in bone cells. J Oral Biosci (2017), http://dx.doi.org/10.1016/j. job.2017.01.004i
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Normal osteoclasts
3
RAB27A-deficient osteoclasts RANKL
RANKL RANK Enhanced RANKL signaling
RANK
Impaired Lysosome transport Lysosome Enzymes
Fig. 1. A schematic diagram of the role of RAB27A in osteoclasts. RAB27A deficiency causes multinucleation and giant cell formation and impairs bone resorption. As these molecular mechanisms indicate, RAB27A deficiency in osteoclasts causes abnormal transport of cell surface receptors, eventually enhancing signaling that drives osteoclastic differentiation. Nonetheless, RAB27A deficiency impairs the transport of LROs involved in bone resorption.
Normal osteoblasts
TFEB-knockdown osteoblasts
ER Normal proteins
ER Aggregated proteins
Aggregated proteins
Normal proteins
Lysosome ATF4-CHOP signal
ATF4-CHOP signal
degradation
Secretion of bone matrix
Lysosome
Enhanced ER stress
Delay of osteoblast differentiation
Fig. 2. A schematic diagram of the role of TFEB in osteoblasts. A TFEB knockdown impairs differentiation into osteoblasts. As these molecular mechanisms indicate, the TFEB knockdown upregulates ATF4 and CHOP. This upregulation is probably due to accumulation of aggregated proteins in the ER, known as “ER stress.” The enhanced ER stress probably inhibits osteoblastic differentiation.
4. Lysosomes in osteoblasts Osteoblasts are mononuclear cells predominantly involved in bone formation [44]. Osteoblasts derive from mesenchymal stem cells and generate the bone matrix by producing large amounts of type I collagen, which is a major organic component, and small amounts of extracellular bone matrix proteins, including osteocalcin and osteopontin [45]. Because the main function of osteoblasts is protein production, studies focused on the lysosomal function of osteoblasts are rare. Several lines of evidence have indicated that lysosomal functions are notably augmented during differentiation into osteoblasts. Indeed, osteoblast precursor cells stimulated by ascorbic acid undergo enhanced endocytosis and show an elevated degradation ability and an increased total number of lysosomes [46]. Moreover, a study involving proteomic and transcriptomic analyses of primary mouse osteoblasts has shown that the most prominently upregulated cluster of proteins is that of endosomal or lysosomal proteins, which constitute approximately 21% of all the upregulated proteins [47]. It is unclear, however, whether the enhanced lysosomal functions are directly involved in osteoblastic differentiation.
5. TFEB and osteoblasts In a recent study, we have shown that transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, regulates osteoblastic differentiation. TFEB is a member of the MiTF/TFE family of basic helix-loop-helix leucine zipper transcription factors [48,49]. TFEB controls the expression of various lysosome-related proteins, including lysosomal hydrolases, lysosomal membrane
proteins, and autophagy-related genes [48]. During osteoblastic differentiation of mouse preosteoblastic MC3T3-E1 cells, the mRNA and protein levels of TFEB as well as those of endosomal/lysosomal proteins were found to be increased [50]. Moreover, cells depleted of TFEB by small interfering RNA (siRNA) showed impaired osteoblastic differentiation as compared with control cells. In contrast, overexpression of TFEB markedly accelerates differentiation into osteoblasts. A knockdown of TFEB increased the mRNA and protein levels of activating transcription factor 4 (ATF4) and CCAAT/enhancer-binding protein homologous protein (CHOP), both of which are essential for osteoblastogenesis [50]. Conversely, overexpression of TFEB in MC3T3-E1 cells enhanced osteoblastic differentiation via decreased expression of ATF4 and CHOP [50]. Given that the expression of ATF4 and CHOP is necessary for osteoblastic differentiation, it can be inferred that the TFEB-mediated signaling pathway is responsible for the ATF4and CHOP-dependent signaling pathway during osteoblastic differentiation. As shown in Fig. 2, a TFEB knockdown upregulates ATF4 and CHOP. This upregulation is probably due to accumulation of aggregated proteins in the endoplasmic reticulum (ER), known as “ER stress.” The enhanced ER stress probably inhibits differentiation into osteoblasts. Therefore, it is speculated that TFEB is required for attenuation of ER stress during osteoblastogenesis. Thus, it is likely that lysosome biogenesis is crucial for osteoblastic differentiation.
6. Conclusions This review describes lysosomal functions in osteoclasts and osteoblasts. In particular, RAB27A deficiency causes abnormal transport of LROs into osteoclasts. Moreover, RAB27A deficiency
Please cite this article as: Tsukuba T, et al. New functions of lysosomes in bone cells. J Oral Biosci (2017), http://dx.doi.org/10.1016/j. job.2017.01.004i
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can lead to increased surface amounts of c-fms and delayed downregulation of RANK in ashen mouse cells, resulting in enhanced signaling via M-CSF and RANKL. Given that RAB27A is a typical regulator of LROs, our findings indicate that osteoclasts also possess LROs. Furthermore, TFEB is a transcription factor necessary for differentiation into osteoblasts. Identification of TFEB as an osteoblastic-differentiation factor is important for the development of new therapies for bone diseases.
Conflicts of interest The authors declare that they have no potential conflicts of interest.
Acknowledgments This work was supported by JSPS KAKENHI (Grant Numbers 16K15790, 15H05298, 16K11863, 16K11510, 16K11480, and 15K11079) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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Please cite this article as: Tsukuba T, et al. New functions of lysosomes in bone cells. J Oral Biosci (2017), http://dx.doi.org/10.1016/j. job.2017.01.004i
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Review
New functions of lysosomes in bone cells Takayuki Tsukuba a,n, Eiko Sakai a, Kazuhisa Nishishita a, Tomoko Kadowaki b, Kuniaki Okamoto a a b
Departments of Dental Pharmacology, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8588, Japan Departments of Frontier Life Science, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8588, Japan
art ic l e i nf o
a b s t r a c t
Article history: Received 9 November 2016 Received in revised form 8 December 2016 Accepted 25 December 2016
Background: Lysosomes are intracellular acidic organelles that contain approximately 50 hydrolases and 25 species of integral membrane proteins. Although lysosome-like specific compartments, termed lysosome-related organelles (LROs), are found in osteoclasts, their functions in these cells and lysosomal functions in osteoblasts remain to be elucidated. Highlight: Recently, we found that expression of RAB27A is markedly increased during osteoclastic differentiation. RAB27A deficiency causes multinucleation and giant cell formation, characterized by abnormal transport of cell surface receptors and LROs into osteoclasts. Furthermore, we have shown that transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, regulates osteoblastic differentiation. Overexpression of TFEB in preosteoblastic MC3T3-E1 cells enhances osteoblastic differentiation via decreased expression of activating transcription factor 4 (ATF4) and CCAAT/enhancer-binding protein homologous protein (CHOP). These results indicate that the expression of ATF4 and CHOP is essential for differentiation into osteoblasts. Conclusion: RAB27A participates in bone resorption by LROs in osteoclasts. In addition, lysosomal biogenesis modulated by TFEB is necessary for osteoblastic differentiation. & 2017 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.
Keywords: Lysosomes Osteoclasts Osteoblast RAB27A TFEB
Contents 1. Introduction . . . . . . . . . . . 2. LROs in osteoclasts . . . . . 3. RAB27A and osteoclasts . 4. Lysosomes in osteoblasts. 5. TFEB and osteoblasts . . . . 6. Conclusions . . . . . . . . . . . Conflicts of interest . . . . . . . . . Acknowledgments . . . . . . . . . . References . . . . . . . . . . . . . . . .
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1. Introduction Lysosomes are acidic membrane-bound organelles that contain approximately 50 hydrolases, including proteases, glycosidases,
Abbreviations: (BMM), bone marrow macrophage; (LROs), lysosome-related organelles; (M-CSF), macrophage-colony stimulating factor; (siRNA), small interfering RNA; (V-ATPase), vacuolar-type H þ -ATPase. n Correspondence to: Department of Dental Pharmacology, Graduate School of Biomedical Sciences, Nagasaki University, 1-7-1 Sakamoto, Nagasaki 852-8588, Japan. E-mail address: [email protected] (T. Tsukuba).
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1 2 2 3 3 3 4 4 4
lipases, nucleases, phospholipases, phosphatases, and sulfatases [1–3]. Lysosomes contain approximately 25 species of integral membrane proteins. The most enriched membrane proteins are lysosome-associated membrane protein 1 (LAMP1), LAMP2, lysosome integral membrane protein 1 (LIMP1), LIMP2, and CD63 [3]. Lysosomes continuously digest macromolecules via three major pathways: endocytosis, phagocytosis, and autophagy. Defective lysosomal functions cause neurodegenerative diseases, immunesystem-related diseases, infectious diseases, cancer, cell differentiation, and so-called lysosomal storage diseases [4,5]. Although lysosomes are present ubiquitously in mammalian cells, there are cell type-specific compartments, termed lysosome-related
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organelles (LROs), which are roughly similar to lysosomes, but morphologically and functionally different from them [6,7]. Examples of LROs include melanosomes in melanocytes [8], lytic granules in lymphocytes [9,10], and delta granules in platelets [11]. In addition to these examples, osteoclasts contain LROs surrounded by the ruffled-border membrane [12,13]. So far, studies on LRO functions in osteoclasts have been scarce as compared to studies on LRO functions in other cell types. Moreover, the function of lysosomes in osteoblasts remains unclear. In this review, we compare and discuss lysosomal functions in osteoclasts and osteoblasts.
2. LROs in osteoclasts Osteoclasts are the bone-resorbing cells that function using LROs [6,7,14]. Osteoclastic LROs contain certain specific components, the most abundant of which are cathepsin K and tartrateresistant acid phosphatase (TRAP). Cathepsin K was initially identified as a lysosomal cysteine protease that is predominantly expressed in osteoclasts [15]. Because cathepsin K has a powerful collagenolytic activity, it is predominantly involved in the degradation of extracellular-matrix components such as collagens, lamin, tenascin, and osteonectin [16,17]. Cathepsin K-deficient mice show osteopetrosis characterized by reduced osteoclastic bone resorption [18]. Mutations in human cathepsin K cause an autosomal recessive disease termed pycnodysostosis, a specific type of osteopetrosis, characterized by osteosclerosis with increased bone fragility [19,20]. TRAP (also known as ACP5) is a metalloenzyme that is abundant in osteoclasts although this enzyme is also expressed in some organs and tissues such as the spleen, liver, gastrointestinal tract lining, lung, thymus, and skin [21]. TRAP is also useful as a biomarker of bone resorption; its serum levels are measured for the clinical diagnosis of metabolic and bone diseases [22,23]. TRAP-deficient mice show early onset of the osteopetrotic bone phenotype because their osteoclasts are less active in bone degradation [24]. In addition to these specific soluble enzymes, several lysosomal membrane proteins are known to be osteoclast specific. Vacuolartype H þ -ATPases (V-ATPases), which help to maintain acidic pH of lysosomes, are proton pumps composed of 13 different subunits [25]. Among these, the a3 subunit is known to be osteoclast specific. Expression of a3 is approximately 100-fold greater in osteoclasts than in other cell types [26]. Indeed, mice deficient in this subunit have severe osteopetrosis [27]. In humans, mutation of the gene encoding the a3 subunit of V-ATPase results in autosomal recessive osteopetrosis caused by impaired osteoclast-mediated bone resorption [28]. The complex of voltage-gated chloride channel 7 (CLC7) and osteopetrosis-associated transmembrane protein 1 (OSTM1) has been reported as an osteoclast-specific protein complex [29]. CLC7 is a 2Cl /1 H þ ion exchanger, and OSTM1 is a lysosomal protein important for the stability of the CLC7–OSTM1 complex [29]. Any defect in the gene of either CLC7 or OSTM1 causes severe malignant infantile osteopetrosis [30–32]. Thus, it is likely that all mutant animals deficient in osteoclastspecific lysosomal proteins have osteopetrosis. Osteoclastic LROs have unique segregation systems for their proteins. Localization of several cathepsins is indeed distinct in osteoclasts, whereas it is similar among other cell types. Our immunocytochemical studies have suggested that cathepsins B, E, and K are present in the ruffled-border membrane and nearby vacuoles, whereas cathepsin D is localized to vacuoles and lysosomes that are detached from the ruffled-border membrane [33–36]. In agreement with our findings, osteoclasts from mice deficient in GlcNAc-1-phosphotransferase α/β-subunits [these animals have a defect in the mannose 6-phosphate (Man-6-P)-dependent pathway] show
enhanced secretion of cathepsin K and TRAP but normal targeting of cathepsin D [37]. In addition to lysosomal enzymes, lysosomal membrane proteins are also segregated in osteoclasts. For example, V-ATPase is present along the actin ring of osteoclasts, whereas cathepsin K is localized to the center of the actin ring as a punctate structure [38]. These findings suggest that LROs in osteoclasts have specific segregation systems.
3. RAB27A and osteoclasts To identify a gene that regulates membrane trafficking of LROs during osteoclastic differentiation, we have conducted a DNA microarray analysis. During the analysis, we observed a marked increase in the expression of RAB27A during osteoclastic differentiation [39]. RAB27A, a member of the RAB family of small GTPases, is exclusively localized to secretory granules in various secretory cells, including endocrine and exocrine cells and various leukocytes [40]. Mutation of RAB27A in humans causes type 2 Griscelli syndrome, which is characterized by hypopigmentation of the skin and eyes and immunodeficiency [9,41]. Nonetheless, the involvement of RAB27A in LROs of osteoclasts has yet to be elucidated. Therefore, we further analyzed the functions of RAB27A using a small interfering RNA (siRNA)-mediated knockdown in a murine macrophagic RAW-D cell line or bone marrow macrophages (BMMs) derived from ashen mice, which carry a mutation in Rab27a. Osteoclasts derived from either RAB27A knockdown RAW-D cells or ashen mice were found to show multinucleation and giant cell formation as key phenotypes when compared with control cells [39]. The ashen mouse osteoclasts showed abnormal actin ring formation, aberrant subcellular localization of LAMP2 and cathepsin K, and impaired resorption activity [39]. In addition to the lysosomal systems, trafficking of cell surface receptors was abnormal in the ashen mouse osteoclasts. The surface amount of c-fms, a macrophage-colony stimulating factor (M-CSF) receptor, was slightly greater in ashen mouse BMMs than in wild-type BMMs. Additionally, downregulation of receptor activator of nuclear factor κ B (RANK; a RANK ligand [RANKL] receptor) was delayed. In fact, although cellular RANK in wild-type BMMs was gradually degraded, RANK in ashen mouse BMMs was found to be upregulated [39]. As a result, upon stimulation with M-CSF and RANKL, phosphorylation levels of extracellular signal-regulated kinase (Erk), proto-oncogene tyrosineprotein kinase (Src), and p38 were slightly enhanced in ashen mouse BMMs in comparison with wild-type BMMs [39]. Thus, Rab27A deficiency causes abnormal transport of cell surface receptors thereby modulating multinucleation and LROs in osteoclasts. As shown in Fig. 1, RAB27A deficiency in osteoclasts causes abnormal transport of cell surface receptor called RANK, eventually enhancing signaling that drives osteoclastic differentiation. Nevertheless, RAB27A deficiency leads to impaired transport of LROs for bone resorption. Given that the phenotype of RAB27A-deficient osteoclasts is unique, this phenotype is different from that of osteoclasts deficient for other RABs. For example, RAB3 is known to modulate exocytosis in secretory cells. Osteoclasts from mice deficient in Rab3D—the Rab3 isoform predominantly expressed in osteoclasts —show an abnormal ruffled-border membrane [42]. In addition, a knockdown of RAB7—a key regulator of biogenesis of late endosomes and lysosomes—disrupts polarization of ruffled-border formation and decreases bone resorption by osteoclasts [43]. Thus, our results indicate that RAB27A plays a unique role in membrane trafficking of osteoclasts as compared with the roles of other RAB proteins.
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Normal osteoclasts
3
RAB27A-deficient osteoclasts RANKL
RANKL RANK Enhanced RANKL signaling
RANK
Impaired Lysosome transport Lysosome Enzymes
Fig. 1. A schematic diagram of the role of RAB27A in osteoclasts. RAB27A deficiency causes multinucleation and giant cell formation and impairs bone resorption. As these molecular mechanisms indicate, RAB27A deficiency in osteoclasts causes abnormal transport of cell surface receptors, eventually enhancing signaling that drives osteoclastic differentiation. Nonetheless, RAB27A deficiency impairs the transport of LROs involved in bone resorption.
Normal osteoblasts
TFEB-knockdown osteoblasts
ER Normal proteins
ER Aggregated proteins
Aggregated proteins
Normal proteins
Lysosome ATF4-CHOP signal
ATF4-CHOP signal
degradation
Secretion of bone matrix
Lysosome
Enhanced ER stress
Delay of osteoblast differentiation
Fig. 2. A schematic diagram of the role of TFEB in osteoblasts. A TFEB knockdown impairs differentiation into osteoblasts. As these molecular mechanisms indicate, the TFEB knockdown upregulates ATF4 and CHOP. This upregulation is probably due to accumulation of aggregated proteins in the ER, known as “ER stress.” The enhanced ER stress probably inhibits osteoblastic differentiation.
4. Lysosomes in osteoblasts Osteoblasts are mononuclear cells predominantly involved in bone formation [44]. Osteoblasts derive from mesenchymal stem cells and generate the bone matrix by producing large amounts of type I collagen, which is a major organic component, and small amounts of extracellular bone matrix proteins, including osteocalcin and osteopontin [45]. Because the main function of osteoblasts is protein production, studies focused on the lysosomal function of osteoblasts are rare. Several lines of evidence have indicated that lysosomal functions are notably augmented during differentiation into osteoblasts. Indeed, osteoblast precursor cells stimulated by ascorbic acid undergo enhanced endocytosis and show an elevated degradation ability and an increased total number of lysosomes [46]. Moreover, a study involving proteomic and transcriptomic analyses of primary mouse osteoblasts has shown that the most prominently upregulated cluster of proteins is that of endosomal or lysosomal proteins, which constitute approximately 21% of all the upregulated proteins [47]. It is unclear, however, whether the enhanced lysosomal functions are directly involved in osteoblastic differentiation.
5. TFEB and osteoblasts In a recent study, we have shown that transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, regulates osteoblastic differentiation. TFEB is a member of the MiTF/TFE family of basic helix-loop-helix leucine zipper transcription factors [48,49]. TFEB controls the expression of various lysosome-related proteins, including lysosomal hydrolases, lysosomal membrane
proteins, and autophagy-related genes [48]. During osteoblastic differentiation of mouse preosteoblastic MC3T3-E1 cells, the mRNA and protein levels of TFEB as well as those of endosomal/lysosomal proteins were found to be increased [50]. Moreover, cells depleted of TFEB by small interfering RNA (siRNA) showed impaired osteoblastic differentiation as compared with control cells. In contrast, overexpression of TFEB markedly accelerates differentiation into osteoblasts. A knockdown of TFEB increased the mRNA and protein levels of activating transcription factor 4 (ATF4) and CCAAT/enhancer-binding protein homologous protein (CHOP), both of which are essential for osteoblastogenesis [50]. Conversely, overexpression of TFEB in MC3T3-E1 cells enhanced osteoblastic differentiation via decreased expression of ATF4 and CHOP [50]. Given that the expression of ATF4 and CHOP is necessary for osteoblastic differentiation, it can be inferred that the TFEB-mediated signaling pathway is responsible for the ATF4and CHOP-dependent signaling pathway during osteoblastic differentiation. As shown in Fig. 2, a TFEB knockdown upregulates ATF4 and CHOP. This upregulation is probably due to accumulation of aggregated proteins in the endoplasmic reticulum (ER), known as “ER stress.” The enhanced ER stress probably inhibits differentiation into osteoblasts. Therefore, it is speculated that TFEB is required for attenuation of ER stress during osteoblastogenesis. Thus, it is likely that lysosome biogenesis is crucial for osteoblastic differentiation.
6. Conclusions This review describes lysosomal functions in osteoclasts and osteoblasts. In particular, RAB27A deficiency causes abnormal transport of LROs into osteoclasts. Moreover, RAB27A deficiency
Please cite this article as: Tsukuba T, et al. New functions of lysosomes in bone cells. J Oral Biosci (2017), http://dx.doi.org/10.1016/j. job.2017.01.004i
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can lead to increased surface amounts of c-fms and delayed downregulation of RANK in ashen mouse cells, resulting in enhanced signaling via M-CSF and RANKL. Given that RAB27A is a typical regulator of LROs, our findings indicate that osteoclasts also possess LROs. Furthermore, TFEB is a transcription factor necessary for differentiation into osteoblasts. Identification of TFEB as an osteoblastic-differentiation factor is important for the development of new therapies for bone diseases.
Conflicts of interest The authors declare that they have no potential conflicts of interest.
Acknowledgments This work was supported by JSPS KAKENHI (Grant Numbers 16K15790, 15H05298, 16K11863, 16K11510, 16K11480, and 15K11079) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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