CCN2) expression in nucleus pulposus cells

Accepted Manuscript Osmolarity and calcium regulate connective tissue growth factor (CTGF/CCN2) expression in nucleus pulposus cells

Wenbo Lin, Changgui Shi, Weiheng Wang, Huiqiao Wu, Chen Yang, An Wang, Xiaolong Shen, Ye Tian, Peng Cao, Wen Yuan PII: DOI: Reference:

S0378-1119(19)30366-X https://doi.org/10.1016/j.gene.2019.04.020 GENE 43779

To appear in:

Gene

Received date: Revised date: Accepted date:

21 February 2019 26 March 2019 5 April 2019

Please cite this article as: W. Lin, C. Shi, W. Wang, et al., Osmolarity and calcium regulate connective tissue growth factor (CTGF/CCN2) expression in nucleus pulposus cells, Gene, https://doi.org/10.1016/j.gene.2019.04.020

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ACCEPTED MANUSCRIPT Osmolarity and Calcium Regulate Connective Tissue Growth Factor （CTGF/CCN2）Expression in Nucleus Pulposus Cells Running title：The orchestra of CCN2 expression in NP cells Wenbo Lin a,1, Changgui Shi a,1, Weiheng Wang a, 1, Huiqiao Wu a,, Chen Yang a, An Wang b, Xiaolong Shen a, Ye Tian a, Peng Cao a, *, Wen Yuan a, *

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Department of Orthopaedics, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China b

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These authors contributed equally to this work.

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Department of Orthopaedics, Shanghai Armed Police Force Hospital, Shanghai 201103, China,

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Wenbo Lin, [email protected];

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*Correspondence should be addressed to Peng Cao and Wen Yuan, Department of Orthopaedics, Changzheng Hospital, Second Military Medical University, 415 Fengyang Road, Huangpu, Shanghai 200003, P. R. China. Tel/fax: +86-021-81886806

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Changgui Shi, [email protected]; Weiheng Wang, [email protected]; Huiqiao Wu, [email protected];

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Chen Yang, [email protected]; An Wang, [email protected];

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Xiaolong Shen, [email protected]; Ye Tian, [email protected]; Peng Cao, [email protected]; Wen Yuan, [email protected]

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Abstract Objective The objective of our study was to verify the hypothesis that the expression of connective tissue growth factor (CTGF/CCN2), a key molecule essential for the maintenance of nucleus pulposus (NP) matrix homeostasis, is regulated by osmolarity and intracellular calcium in NP cells. Methods Gene and protein expression levels of CCN2 were assessed using quantitative real-time PCR and western blot. Transfections and dual luciferase assays were performed to measure the effect of hyperosmolarity, tonicity enhancer binding protein (TonEBP) and Ca2+-calcineurin (Cn)-NFAT signaling on CCN2 promoter activity. Results Cultured in hyperosmotic media, there was a significant decrease in the levels of CCN2 promoter activity, gene and protein expression in NP cells. The JASPAR database was used to analyze the construction of human CCN2 promoter, we found conserved TonE and NFAT binding sites. We then investigated whether TonEBP controlled CCN2 expression. Forced expression of TonEBP in NP cells showed that TonEBP negatively regulated CCN2 promoter activity, while suppression of TonEBP induced CCN2 promoter activity and expression. We then examined if Ca2+-Cn-NFAT signaling participated in the regulation of CCN2 expression. Co-expression of CCN2 reporter with individual NFAT1–4 expression plasmids and/or calcineurin A/B constructs suggested this signaling pathway played a role in the regulation of CCN2expression in NP cells. Conclusions Results of these studies illustrated that the expression of CCN2 in NP cells was regulated by the NFAT family through a signaling pathway network involving both activator (Ca2+-CnNFAT signaling) and suppressor (Hyperosmolarity-TonEBP) molecules.

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Keywords: CCN2, Osmolarity, Nucleus Pulposus Cells, TonEBP, NFAT, Calcineurin

ACCEPTED MANUSCRIPT Introduction

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Low back pain leads to personal disability and reduced productivity, which is causing a serious socio-economic burden both in developed and developing countries(Luo et al., 2004). While numerous factors and potential pain generators are reported to be correlated, intervertebral disc degeneration (IDD) is the most documented cause (Luoma et al., 2000; de Schepper et al., 2010; Livshits et al., 2011). Although many research groups and programs have spared no efforts in the research of IDD in these decades, the accurate etiology and pathogenesis of IDD remains unclear(Kepler et al., 2013; Vergroesen et al., 2015). Located between the two adjoining vertebral bones, the intervertebral disc functions as a robust hydrodynamic system and maintains the normal spine function(Wang et al., 2007). It consists of outer annulus fibrosus (AF), gel-like nucleus pulposus (NP) tissue and cartilaginous endplates. Surrounded by the outer AF and end plates, the healthy NP is characterized with avascularity, low cellularity, low oxygen tension, and hyperosmolarity(Bartels et al., 1998; Kepler et al., 2013). Raised by the interaction between cations and charged sulfated glycosaminoglycan sidechains on the aggrecan molecule, the osmolarity of the extracellular environment of the nucleus pulposus cells ranges from 450 to 550 mosm/kg(Urban, 2002), fluctuating with diurnal cycle(Matsumura et al., 2009). This unusually high extracellular osmotic milieu in the disc plays a critical role in both cell function and matrix composition(Haschtmann et al., 2006; Johnson et al., 2014). Numerous studies indicated that NP cells adapt to survive in this hyperosmotic microenvironment through osmoadaptive response of the transcription factor tonicity-responsive enhancer binding protein (TonEBP), also known as nuclear factor of activated T-cells 5 (NFAT5)(Johnson et al., 2014). TonEBP maintains the balance of intracellular and extracellular osmotic pressure by controlling the plasma membrane transportation of organic nonionic osmolites, such as taurine, aldose reductase, sodium myoinositol, and betaine(Tsai et al., 2006). Besides, TonEBP induces transcription of matrix homeostasis related genes, including aggrecan, β1,3-glucoronosyltransferase 1 (GlcAT-I), and aquaporin 2 to autoregulate and adapt to the extracellular hyperosmotic environment(Gajghate et al., 2009; Hiyama et al., 2009).

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Connective tissue growth factor (CTGF/CCN2), a most investigated member of the CCN matricellular proteins family, plays important roles in embryonic development, inflammation, wound healing, and injury repair(Jun and Lau, 2011). Over the past decade, extensive studies have revealed that CCN2 responds to numerous stimulating factors, including growth factors, oxygen tension and mechanical impacts, demonstrating highly diverse and context-dependent function in various microenvironments through signaling crosstalk with multiple matrix biomolecules(Abreu et al., 2002; Ivkovic et al., 2003; Higgins et al., 2004; Hoshijima et al., 2006; Nishida et al., 2007; Maeda et al., 2009; Nishida et al., 2009; Maeda-Uematsu et al., 2014; Kubota and Takigawa, 2015). In skeletal system, CCN2 acts as a hub by manipulating extracellular signaling molecules to promote cellular proliferation, adhesion, migration, and extracellular matrix production, conducting the harmonized development of cartilage and bone(Ivkovic et al., 2003; Hoshijima et al., 2006; Nishida et al., 2007; Maeda et al., 2009; Maeda-Uematsu et al., 2014). In the developing and mature NP, CCN2 is expressed in the AF and enriched in the NP. It is proved to be an anabolic factor and serves to be crucial in the

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regulation of cell activity, extracellular matrix composition and maintaining tissue homeostasis(Erwin et al., 2006; Bedore et al., 2013; Bedore et al., 2014). Several in vitro observations verified that CCN2 interacts with environmental stimuli and growth factors, including hypoxia, hypoxia-inducible factor (HIF), transforming growth factor β (TGF-β) and interleukin1β (IL-1β)(Tran et al., 2010; Tran et al., 2013; Tran et al., 2014). However, whether the CCN2 expression is regulated by hyperosmolarity in the NP cells remains to be determined. Given the specific hyperosmolarity in the disc microenvironment and the significant role of CCN2 in disc cell homeostasis, it is critical to explore the regulation mechanism of CCN2 level in local hyperosmotic milieu.

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The objective of this study was to explore the assumption that the expression of CCN2, a key molecule crucial for the maintenance of matrix homeostasis, is regulated by osmotic stress and calcium signaling through transcription factors of the NFAT family in NP cells. Results of our study clearly demonstrate that expression of CCN2 is down-regulated through TonEBP under hyperosmotic conditions. Meanwhile, we also find that Ca2+-Cn-NFAT signaling serves as a positive regulator in the regulation of CCN2 expression.

Materials and Methods

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2.1. Isolation, culture and treatments of NP cells

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Male Sprague Dawley rats (180-200 g) were euthanized with CO2. NP cells were isolated from lumbar discs of rats using the previously published protocols reported by Risbud et al(Risbud et al., 2006). The isolated cells were then maintained in medium containing low glucose (1 g/L) Dulbecco’s modified Eagle’s medium (DMEM), 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and 100 U/mL penicillin (Gibco), 100 μg/mL streptomycin (Gibco). Cells were seeded in 10-cm dishes and the second passage were used for all the further experiments. When reached confluence in the plates, cells were washed and incubated in a range of osmolarity conditions (330,450,500 mosmol/kg)(Tsai et al., 2007) for a period of 8 to 24 h. In subsequent experiments, cells were treated with 1 μM calcium ionophore ionomycin (Sigma，St. Louis, MO, USA) and 100 ng/ml phorbol 12‐ myristate 13‐ acetate (PMA) with or without 10 μM BAPTA-AM or 10 ng/ml Cn inhibitors FK506 and 1 μg/ml cyclosporine A (CsA). All the cells were cultured at 37 °C in 5% CO2 and 21% O2 humidified incubator. The research proposal was approved by the Animal Ethics Committee of the Second Military Medical University (permit number 20166000108), Shanghai, China. 2.2. RNA Extraction and quantitative RT-PCR analysis Following treatments, total RNA in NP cells (5×105 cells/plate) was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the standard instructions. NanoDrop (NanoDrop, Wilmington, DE, USA) was used to measure the RNA concentrations. Complementary DNA was subsequently synthesized using a High-Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA). Real-time RT-PCR was performed using

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SYBR® Premix Ex Taq™ II (TaKaRa, Otsu, Japan) with the ABI 7500 Real-Time PCR System (Applied Biosystems). The relative quantification of mRNA in the control and treatment groups were calculated using the comparative Ct (2−ΔΔCt) method. GAPDH was used as internal control to normalize gene expression. All Primers of the target genes were designed and synthesized by Sangon (Sangon Biotech, Shanghai, China). The sequences of primers used to assay rat CCN2 were as follows: forward 5′-ATCCAATCGAGACCCTGGTG-3′ and reverse 5′-ATCTCTCCTATGTGCTGGCC-3′. The primers for GAPDH were as follows: forward 5′-TTC TCT TGT GAC AAA GTG GACAT-3′ and reverse 5′-GAA GGG GCG GAG ATG ATGAC-3′.

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2.3. Total Cell Protein Extraction and Western Blot

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Following treatments, NP Cells were placed on ice immediately and washed with 4° precooled phosphate-buffered saline (PBS) three times. Then cells were harvested at 4 °C in RIPA lysis buffer (Beyotime, Shanghai, China) containing protease inhibitor cocktail (Pierce Biotechnology, IL, USA). All of the wash buffers and final cell resuspension buffer contained 1×protease inhibitor mixture (Pierce, IL, USA), 5 mM NaF, and 200 μm Na3VO4. BCA protein assay (P0009, Beyotime, Shanghai, China) was utilized to quantify the protein concentrations. Total protein was resolved on 5–10% sodium dodecyl sulfate-polyacrylamide gels (SDSPAGE) and subsequently transferred by electroblotting to PVDF membrane (Bio-Rad, CA, USA). The membranes were blocked with 5% bovine serum albumin in TBST (50 mM Tris, pH 7.6, 150 mM NaCl, 0.1% Tween 20) and then incubated overnight at 4 °C in TBST with anti-CCN2(1:500, sc-34772, Santa Cruz Biotechnology, CA, USA)，anti-TonEBP (1:3000, ab3446, Abcam, MA, USA) and anti-β-tubulin(1:2000, number2146, Cell Signaling, Danvers, MA, USA) antibodies. After being washed three times with TBST, the membrane was incubated for 2h with horseradish peroxidase -conjugated secondary antibody (1:2000, Abcam) at 37°C. Pierce ECL Western Blotting Substrate (Pierce Biotechnology, Rockford, IL, USA) was used to detect immunolabeling. Image J software (National Institutes of Health, Bethesda, MD, USA) was used to measure the relative expression levels by quantitative densitometric analysis and normalized to the expression of β-tubulin. Data were from three independent experiments. 2.4. Immunofluorescence Microscopy Cells were cultured in 24-well plates (2×104/well) and incubated in a range of osmolarity conditions for 24 hours. After treatments, cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 in PBS for 10 min. Next, the cells were blocked with PBS containing 5% BSA and incubated with primary antibody against CCN2(1:1000, sc34772, Santa Cruz Biotechnology, CA, USA) at 4 °C overnight. Cells were incubated with isotype IgG as a negative control. After washing with PBS three times, the cells were then reacted with Alexa Fluor-488-conjugated anti-goat secondary antibody (Invitrogen) at a dilution of 1:5000 and 0.1 μg/ml DAPI (4′-6-diamid-ino-2-phenylindole) for DNA

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Reporter and expression plasmids were kindly provided by Dr. George Yang from Stanford University (Human CTGF reporter) and Dr. Ben C. Ko of University of Hong Kong (pTonEBP and dominant-negative TonEBP/DN-TonEBP). Plasmids for NFAT1 (number 11100) and NFAT2 (number 11102), NFAT4 (number 11790) developed by Dr. Anjana Rao, NFAT3 (number 10961), developed by Dr. Toren Finkel, and 3×NFAT-Luc (number 17870), catalytic subunit (CnA, number 17871) and regulatory subunit (CnB, number 17872) were purchased from Addgene (Cambridge, USA). We used the pRL-TK plasmid (Promega, Madison, WI, USA) as transfection control. NP Cells at the second passage were transferred to 24-well plates at a density of 5 × 104 cells/well 1 day before transfection. Plasmids were premixed with the transfection reagent, Lipofectamine 2000 (Invitrogen) for each transfection. For determining the effect of hyperosmolarity on CCN2 promoter activity, NP cells co-transfected with 500 ng of CCN2 reporter plasmid and 500 ng of pRL-TK plasmid were treated with media of different osmolarity. To measure the effect of TonEBP on CCN2, cells were co-transfected with 100 to 300 ng of pTonEBP or DN-TonEBP or with appropriate amount of backbone vector pcDNA3.1 with 250 ng of pRL-TK plasmid and 250 ng of CCN2 reporter. To investigate the effect of ionomycin on CCN2 reporter activity, cells were treated 24 h after transfection with ionomycin (1 μM) and PMA (100 ng) with or without BAPTA-AM (10 μM) or FK506 (10 ng/ml) and CsA (1 μg/ml) in isotonic media. Following treatments, cells were harvested and a Dual-Luciferase™ reporter assay (Promega) was used for measurements of firefly and renilla luciferase activities. Independent transfections were performed at least three times, and all analyses were performed in triplicate.

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All experiments were replicated independently at least three times. Data are presented as mean ± standard deviation. GraphPad Prism 5.0 software (GraphPad Software, CA, USA) were utilized for statistical analyses. Data were analyzed using the Student's t-test and Oneway ANOVA, P-values < 0.05 were considered statistically significant.

ACCEPTED MANUSCRIPT Results

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Effect of hyperosmolarity on CCN2 expression in NP cells. To explore the assumption that hyperosmolarity regulates CCN2 expression, NP cells were treated with isotonic (330 mosmol/kg) and hypertonic conditions (450 and 500 mosmol/kg) for 8-24h, and CCN2 mRNA expression was analyzed using real-time RT-PCR. As is shown in Fig.1A, the NP cells presented a significant osmolarity-dependent decrease in CCN2 mRNA expression. When cells were incubated in hypertonic medium (500 mosmol/kg), there was a time-correlated decrease in CCN2 mRNA transcription (Fig. 1B). Compared with the isotonic group, the transcriptional level of CCN2 decreased significantly after treatment in hyperosmotic medium for 8 h and was maintained at 24 h. In addition, western blot analyses demonstrated a concomitant CCN2 protein expression decrease in hypertonic medium (Fig. 1C), densitometric analysis showed that the suppression of CCN2 in cell lysates was pronounced in the 500 mosmol/kg group (Fig. 1D). Additionally, immunofluorescence microscopy was used for further evaluation, the results confirmed a weaker staining of CCN2 when compared with untreated control, indicating decreased CCN2 protein expression in response to hyperosmotic treatment (Fig. 1E)

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Fig. 1 Hyperosmotic medium decreases CCN2 expression in NP cells. A-B Real-time RT-PCR results of CCN2 mRNA expression in NP cells indicate an osmolaritydependent (330, 450, 500 mosmol/kg) and time-dependent (8h and 24h) decrease in mRNA expression under different conditions. C-D Western blots and densitometric analysis of NP cells cultured in hyperosmolarity for 24 h reveal a decreased expression of CCN2 protein. E Immunofluorescent staining was further used to detect CCN2 protein expression of NP cells cultured 24 h in different osmotic media, CCN2 staining is decreased after treatment. Magnification×40. Scale bar = 20 μm. Results in A, B, and D are presented as mean ± S.D of three independent replicated experiments, *p< 0.05; ns, nonsignificant.

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Osmolarity regulates CCN2 promoter activity through TonEBP. Because TonEBP is an important and only transcription factor of osmolarity signaling, we investigated whether hyperosmolarity-reduced CCN2 expression in NP cells involved TonEBP pathway. We used a 2.0-kb human CCN2 luciferase reporter containing TonEBP and NFAT binding sites. The human CCN2 promoter structure and binding sites of possible transcription factor was presented in Fig.2A. Analysis of the JASPAR database revealed that the CCN2 promoter contains a conserved TonE (TTTCCA) motif from -1892 to -1896 bp, and 3 NFAT (GGAAA or TTTCC) motif in the human CCN2 promoter, from -1194 to -1198 bp, from -1059 to -1063 bp, and from -126 to -131 bp. To study the influence of osmolarity on promoter activity of CCN2 in NP cells, cells were transfected with CCN2 reporter and then cultured in isotonic and hyperosmotic media. CCN2 reporter activities were significantly decreased after cultured in hypertonic media for 24 h (Fig.2B). We then examined the changes of TonEBP expression levels in NP cells under hyperosmolarity. Western blot results confirmed a robust induction of TonEBP protein (Fig.2C-D). Further studies were performed to verify whether the hyperosmotic reduction of CCN2 promoter activity was mediated through TonEBP. We transfected the second passage NP cells with full-length TonEBP and DN-TonEBP expression plasmids, and cultured in different osmotic conditions. Under isotonic conditions, forced expression of pTonEBP resulted in a dose-dependent decrease in CCN2 promoter activities, a significant inhibitory effect on CCN2 promoter activity was observed when the cells were transfected with 100 ng pTonEBP plasmids, which was further reinforced when the dose of transfected plasmid was increased to 300 ng (Fig.2E). On the contrary, when the medium was hypertonic, transfection of cells with DN-TonEBP expression plasmids resulted in a rescue of the compromised CCN2 promoter activity caused by hypertonicity (Fig.2F).

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Fig.2 Osmolarity regulates CCN2 promoter activity through TonEBP. A Schematic diagram of proximal promoter structure of the human CCN2 gene, displaying the binding sites of several major transcription factor. The transcription start location is marked as +1. The TonE binding motif is shown with a black flattened circle, and the NFAT binding sites are shown with black rectangles. B NP cells were transfected with CCN2 reporter plasmids along with pRL-TK backbone vector. Cells were incubated in different osmotic medium (450–500mosmol/kg) for 24 h, and luciferase assay system was utilized to detect reporter activity. Treatment resulted in reduction in CCN2 reporter activity. C-D Western blot and densitometric analysis of the TonEBP protein expression level in NP cells treated with different osmotic medium. TonEBP protein was up-regulated when medium osmolarity rise from 330 to 500 mosmol/kg. E When pTonEBP was co-transfected with CCN2 reporter under isotonic conditions, luciferase analysis demonstrated a dose-correlated decrease in CCN2 reporter activity under isotonic conditions. F CCN2 reporter plasmids with different dose of DN-TonEBP or appropriate amount of backbone vector were transfected into NP cells. Luciferase activity was evaluated and showed that DN-TonEBP overexpression leaded to a complete rescue of CCN2 promoter activity suppressed by hypertonicity. Results shown are mean ± SD of three independent replicated experiments, *p< 0.05; ns, nonsignificant.

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Ca2+- calcineurin signaling increased the expression of CCN2 in NP cells. To investigate whether Ca2+- calcineurin signaling plays a part in the regulation of CCN2 expression, we treated NP cells with calcineurin-mediated signaling activator, calcium ionophore, which is a chemical cocktail containing ionomycin and PMA. Treatment with ionomycin resulted in increased CCN2 protein expression (Fig.3A-B). However, addition of the BAPTA-AM (calcium chelator) diminished ionomycin-mediated induction in CCN2 reporter activity. This phenomenon indicated that the induction of CCN2 was mediated by increasing intracellular Ca2+ concentration. While the calcineurin signaling inhibitors, FK506 and CsA, completely blocked ionomycin-mediated induction of CCN2, this result implied the significant role of calcineurin in the regulation of CCN2. Luciferase analyses confirmed the role of Ca2+- calcineurin signaling on the CCN2 promoter activity (Fig.3C). To delineate the role of calcineurin signaling in CCN2 expression in the NP cells, we co-transfected NP cells with catalytic (CnA) and regulatory (CnB) subunits along with CCN2 reporter plasmid and determined CCN2 reporter activity. Fig.3D shows that calcineurin overexpression had a significant dose-dependent inductive effect on CCN2 promoter activity in transfected cells. Meanwhile, the effect of calcineurin on CCN2 protein level in NP cells was further investigated by western blot and densitometric analysis. As expected, transfected cells exhibit an increase in CCN2 protein expression level (Fig.3E-F). The densitometric analysis confirmed a significant increase in CCN2 levels after the treatment.

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Fig.3 Involvement of calcium and calcineurin signaling in the regulation of CCN2 expression in NP cells. A-B Western blot and densitometric analysis of different group of NP cells treated with ionomycin and PMA(I+P) with or without BAPTA-AM (B) or FK506 and CsA (F+C). The expression of CCN2 was induced by ionomycin and PMA, note, BAPTA-AM inhibit ionomycin-mediated CCN2 induction. While FK506 and CsA resulted in a thorough block of the CCN2 induction caused by ionomycin. C Effect of calcium ions on CCN2 promoter activity. D The CCN2 reporter was co-transfected with CnA/CnB expression plasmids or empty vector into NP cells to investigate the role of calcineurin in the regulation of CCN2. The co-expression of calcineurin subunits increased CCN2 reporter activity in transfected cells. E-F Western blot and densitometric analysis of CCN2 protein expression in NP cells after transfected with CnA/CnB. Results shown are mean ± SD of three independent replicated experiments, *p< 0.05; ns, nonsignificant.

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NFAT signaling participates in the regulation of CCN2 promoter activity in NP cells. To explore the role of NFAT signaling in the regulation of CCN2 expression in NP cells, we transfected cells with NFAT1–4 plasmids with or without Cn (CnA and CnB), and measured with luciferase assay system to determine the activity of the CCN2 reporter. NFAT1 (Fig.4A), NFAT2 (Fig.4B), NFAT3 (Fig.4C) alone or with Cn significantly increased the CCN2 promoter activity. However, when 100 ng NFAT4 (Fig.4D) was overexpressed in NP cells, CCN2 reporter activity was significantly decreased. Transfection of CnA/CnB alone induced CCN2 reporter activity rapidly. In contrast, co-transfection of CnA/CnB and NFAT4 expression plasmids diminished the induced CCN2 activity by CnA/CnB. Therefore, NFATs is a positive regulator of CCN2 expression with or without CnA/CnB, except for NFAT4. To confirm the functionality of all the transfected NFAT plasmids in NP cells, NFAT1, NFAT2, NFAT3 and NFAT4 were separately co-transfected with a NFAT responsive reporter. Figure 4E showed significant induction of 3×NFAT reporter activity with each of the expression plasmids, indicating functionality of expressed proteins.

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Fig.4 Effect of NFATs and/or CnA/CnB on CCN2 promoter activity of NP cells. A-C individual NFAT expression plasmid or with CnA/CnB resulted in significantly increase of the CCN2 reporter activity in NP cells. D NFAT4 significantly decreased CCN2 reporter activity. E NP cells were co-transfected with 3×NFAT reporter and individual NFAT, reporter activity was measured. Co-expression of all the four individual NFATs resulted in a significant induction in the 3×NFAT reporter activity. Results shown are mean ± SD of three independent replicated experiments, *p< 0.05; ns, nonsignificant.

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Fig.5 A proposed model of CCN2 regulation by osmolarity and Ca2+-Cn-NFAT signaling in NP cells. Based on these experiments, we proposed a working model of CCN2 regulation by hyperosmolarity and Ca2+-Cn-NFAT signaling in NP cells (Fig.5). Under condition of hyperosmolarity, the CCN2 transcriptional level was reduced by TonEBP (NFAT5). In contrast, Ca2+-Cn-NFAT signaling functioned as a positive regulator of CCN2 expression.

Discussion

We demonstrated for the first time that hyperosmolarity decreased CCN2 promoter activity and expression through TonEBP (NFAT5), an intensively investigated osmotic related transcription factor that was proved to be essential for the NP cells to survive in the hyperosmotic microenvironment. Another important observation was that, aside from osmolarity, Ca2+-Cn-NFAT signaling functioned as a positive regulator of CCN2 expression in NP. From the above, this study illustrates that within the hydrodynamically stressed and hyperosmotic intervertebral disc niche, expression of CCN2 in NP cells is tightly regulated through a precise regulation network comprising both activator (Ca2+-Cn-NFAT signaling)

ACCEPTED MANUSCRIPT and suppressor (Hyperosmolarity-TonEBP) molecules. Whereas CCN2 is continuously expressed in the developing, mature and degenerated disc, and serves as a vital role in the maintenance of matrix homeostasis in this hyperosmotic niche, this kind of regulatory mechanism would contribute to achieving optimal control of CCN2 expression to maintain its normal biological function and to prevent excessive accumulation.

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Considerable research efforts and studies have shown that the NP cells reside in a hyperosmotic environment, we firstly illuminated the influence of different osmolarity on the CCN2 expression in NP cells. It was well documented that hyperosmolarity is a specific biological nature that maintains normal function of spine and regulates cell survival and homeostasis related genes in this unique tissue(Hiyama et al., 2009; Johnson et al., 2014; Li et al., 2016; Walter et al., 2016; Choi et al., 2018). Our findings suggest that CCN2 transcription and protein expression in NP cells were also regulated by the hyperosmolarity conditions. Tran (Tran et al., 2010)and Peng et al (Peng et al., 2009) demonstrated increasing CTGF expression in the degenerated discs compared with tissues in the normal control discs. One possible explanation for this phenomenon is that the decreasing osmotic pressure during the degenerative process, thereby losing its regulatory inhibition on the CCN2 expression. Further research in CCN2 function is needed to shed more lights on its role in the biology and pathology of disc tissue.

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The mechanism of CCN2 regulation by osmolarity in NP cells has been investigated. TonEBP is the only known transcription factor regulated by tonicity in mammals(Burg et al., 1997; Tsai et al., 2006; Lee et al., 2011; Cheung and Ko, 2013). With respect to the activation of TonEBP, as expected, we confirmed that hyperosmotic treatments resulted in a significantly increase in TonEBP protein. This observation was in line with the results of previous studies (Tsai et al., 2006; Tsai et al., 2007; Gajghate et al., 2009). Further evidence for the importance of TonEBP in regulating CCN2 promoter activity was derived from gain and loss of function experiments performed using pTonEBP and DN-TonEBP. Under isotonic conditions, pTonEBP decreased CCN2 reporter expression; suppression of TonEBP activity rescued repression of CCN2 in hypertonic media. Therefore, regulation of CCN2 in the hyperosmotic microenvironment is mediated via TonEBP. In recent studies, TonEBP was proved to be critical in the activation of TNF-α-induced inflammatory gene expression and increased the transient receptor potential vallinoid-4 signaling, enhancing the production of proinflammatory cytokines during disc degeneration(Walter et al., 2016; Johnson et al., 2017). Taken together, these functional experiments demonstrated that, in addition to the regulation of targets that allow for survival under hypertonic stress, TonEBP serves to be a center for information exchange within and outside the NP cells under multiple stimuli and plays an essential role in the maintenance of cell function and process of disc degeneration. Intracellular calcium is a highly versatile intracellular signal that regulates many important processes and functions, including cell differentiation, gene expression, and tissue synthesis(Berridge et al., 2003). Pritchard et al.(Pritchard et al., 2002) reported that the shifts in Ca2+ and calcium transients exposed to the fluctuation of osmotic stress served as key

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transducers of applied biomechanical loads in the disc. Changes in Ca2+ ion concentration also mediated the regulation of tissue synthesis by dynamic loading in chondrocyte cells(Lv et al., 2017). Additionally, calcium flux regulated the expression of GlcAT-I and enhanced the glycosaminoglycans biosynthesis in NP cells (Hiyama et al., 2009). Future research is warranted to examine if Ca2+ signaling contributes to the regulation of CCN2 expression in a calcineurin-NFAT fashion in addition to osmolarity in NP cells. Noteworthily, treatment of NP cells with calcium ionophore resulted in a small but significant induction in CCN2 mRNA and protein expression. The addition of BAPTA-AM significantly moderated the upward trend, suggesting that the increase of CCN2 promoter activity was induced by the increased concentration of intracellular Ca2+. Thus, intracellular Ca2+ may serve as a positive regulator for CTGF expression in NP cells. The mechanism of Ca2+ induced CCN2 expression was not immediately obvious. It was well established that Ca2+ penetrates the cell membrane upon receptors induction and activates calcineurin by binding to calmodulin, which is the regulatory subunit of calcineurin (Stemmer and Klee, 1994). Calcineurin removes several phosphate residues from the N-terminus of the NFAT protein, exposing the nuclear localization sequence in the NFAT protein, resulting in its rapid entry into the nucleus and DNA binding(Beals et al., 1997). In cardiac myofibroblasts, calcineurin cooperated with the protein kinase C epsilon (PKCε) to suppress the expression of fibrosis markers including CCN2, fibronectin, and collagens(Mesquita et al., 2014). On the other hand, Angiotensin II increased the expression of CCN2 and induced fibrosis in kidney and heart tissue via blood pressure and calcineurindependent pathways(Finckenberg et al., 2003). To further elucidate whether calcineurin signaling is involved in the regulation of CCN2 expression, we treated NP cells with ionomycin and PMA with CsA and FK506(Lopez-Rodriguez et al., 1999), induction of CCN2 was completely blocked. We consider the calcineurin signaling may be needed for the transcription of CCN2 promoter. More supporting evidence for the role of calcineurin in regulating CCN2 expression in NP cells was forthcoming from overexpression of catalytic (CnA) and regulatory (CnB) subunits, which clearly showed a significant stimulation in the CCN2 reporter activity. Accordingly, we advance the notion that calcineurin signaling serves as a positive regulator of CCN2 and stimulation of CCN2 expression is caused by the change of intracellular concentration of Ca2+ through calcineurin signaling.

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TonEBP is identical to NFAT5, a transcription factor of the NFAT family. The classification of this family was based on the similar structure and function of DNA-binding domains(Hogan et al., 2003). Whereas we have shown that TonEBP profoundly influenced CCN2 expression, the following question and possibility remains to be identified. What are the roles of other NFAT proteins in the regulation of CCN2 promoter activity? Related to the mechanism of activation and function of NFAT family, Rao et al. proved that TonEBP(NFAT5) is a transcription factor essential for cellular responses to hypertonic stress(Lopez-Rodriguez et al., 2001), while the remaining four NFAT proteins (NFAT1–NFAT4) are commonly regulated by Ca2+-calcineurin signaling(Hogan et al., 2003; Crabtree and Schreiber, 2009). It was well documented that the NFAT transcription factor family acts as a central regulator of chondrocyte physiology and pathology in cartilage biology (Sitara and Aliprantis, 2010). Besides, the presence of three putative NFAT motifs in the CCN2 promoter indicated the

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possible functional involvement of these factors in controlling transcription. The expression of each NFAT in NP cells has been reported previously(Hiyama et al., 2009). To elucidate the role of NFATs in the regulation of CCN2, we transfected the NP cells with individual NFAT plasmid and determined the promoter activity of CCN2. NFAT1, -2, and -3 increased CCN2 promoter activity, while NFAT 4 showed the opposite function. We then confirmed the functional expression of NFATs in NP cells with co-transfection of each NFATs with the 3× NFAT luciferase reporter. Based on the positive effect of Ca2+-calcineurin signaling on the regulation of CCN2 and the pivotal role of calcineurin for NFATs proteins entry to the nucleus(Crabtree and Schreiber, 2009), it is concluded that the CCN2 promoter activity is positively regulated by the Ca2+-Cn-NFAT signaling. This observation further supported the results reported above and illustrated that intracellular calcium signaling participate in the regulation of tissue synthesis and microenvironment homeostasis in local osmotic microenvironment in the disc. Given the critical functional importance of CCN2 in NP cells, it is not unreasonable to conclude that, concurrently with TonEBP, there exist some induction factors that positively regulate the CCN2 expression, thereby counterbalancing the TonEBPmediated repression. This regulation mode is similar to the one that has been reported in the regulation of AQP2 and GlcAT-I by calcium and osmolarity in NP cells(Gajghate et al., 2009; Hiyama et al., 2009). Whether this mode is specified for the intervertebral disc awaits further study.

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This study has several limitations. NP cell phenotypes and the regulation of CCN2 expression by osmolarity in passage 2 NP cells might be different from that of degenerative IVD. We would try to harvest NP cells from developed degenerative rat models for future research. In addition, all the cell experiments were performed under normoxia condition, which may exert pressure on the cells. However, the primary aim of this study was to examine the osmolarity and intracellular calcium regulation of CCN2 in NP cells. Therefore, we did not account for the effect of normaxia, which warrants future research. In this study, we used in vitro cells and set up the groups mimicking the osmotic pressures of severe degenerative, mild degenerative, and healthy IVD. However, this method cannot reproduce the exact complex environment in vivo IVD. Future in vivo experiments are needed to verify our findings.

Results of our study and findings illustrate that within the hyperosmotic microenvironment of the intervertebral disc, expression of CCN2 in NP cells is precisely regulated by the NFAT family through a signaling pathway network involving both activator (Ca2+-Cn-NFAT signaling) and suppressor (Hyperosmolarity-TonEBP) molecules. Our study provides a new vision of the regulatory mechanism of a key matricellular protein necessary for NP cell survival and function.

Data Availability The supporting data are available from the first author or corresponding author upon request.

Competing Interests

ACCEPTED MANUSCRIPT The authors declare no competing financial interests.

Acknowledgements This study was funded by the Natural Science Foundation of China (No. 81501918 and 81601928) and Shanghai Committee of Science and Technology (201440513). The authors would like to thank Xiaoxing Xie and Zhihao Hu of Changzheng Hospital for helping on experimental technique.

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Author Contributions

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W.L., C.S. and W.W. carried out the cell and molecular studies, and drafted manuscript, H.W, X.S. and Y.T. participated in isolation of NP cells, luciferase assay and revision of the manuscript. C.Y., A.W. carried out the immunofluorescence staining. P.C. and W.Y. conceived of the study, and participated in the design of the study. All authors read and approved the final manuscript.

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Abreu, J.G., Ketpura, N.I., Reversade, B. and De Robertis, E.M., 2002. Connective-tissue growth factor (CTGF) modulates cell signalling by BMP and TGF-beta. Nat Cell Biol 4, 599-604. Bartels, E.M., Fairbank, J.C., Winlove, C.P. and Urban, J.P., 1998. Oxygen and lactate concentrations measured in vivo in the intervertebral discs of patients with scoliosis and back pain. Spine (Phila Pa 1976) 23, 1-7; discussion 8. Beals, C.R., Clipstone, N.A., Ho, S.N. and Crabtree, G.R., 1997. Nuclear localization of NF-ATc by a calcineurin-dependent, cyclosporinsensitive intramolecular interaction. Genes Dev 11, 824-34. Bedore, J., Leask, A. and Seguin, C.A., 2014. Targeting the extracellular matrix: matricellular proteins regulate cell-extracellular matrix communication within distinct niches of the intervertebral disc. Matrix Biol 37, 124-30. Bedore, J., Sha, W., McCann, M.R., Liu, S., Leask, A. and Seguin, C.A., 2013. Impaired intervertebral disc development and premature disc degeneration in mice with notochord-specific deletion of CCN2. Arthritis Rheum 65, 2634-44. Berridge, M.J., Bootman, M.D. and Roderick, H.L., 2003. Calcium signalling: dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol 4, 517-29. Burg, M.B., Kwon, E.D. and Kultz, D., 1997. Regulation of gene expression by hypertonicity. Annu Rev Physiol 59, 437-55. Cheung, C.Y. and Ko, B.C., 2013. NFAT5 in cellular adaptation to hypertonic stress - regulations and functional significance. J Mol Signal 8, 5. Choi, H., Chaiyamongkol, W., Doolittle, A.C., Johnson, Z.I., Gogate, S.S., Schoepflin, Z.R., Shapiro, I.M. and Risbud, M.V., 2018. COX-2

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

SC

RI

PT

expression mediated by calcium-TonEBP signaling axis under hyperosmotic conditions serves osmoprotective function in nucleus pulposus cells. J Biol Chem 293, 8969-8981. Crabtree, G.R. and Schreiber, S.L., 2009. SnapShot: Ca2+-calcineurin-NFAT signaling. Cell 138, 210, 210 e1. de Schepper, E.I., Damen, J., van Meurs, J.B., Ginai, A.Z., Popham, M., Hofman, A., Koes, B.W. and Bierma-Zeinstra, S.M., 2010. The association between lumbar disc degeneration and low back pain: the influence of age, gender, and individual radiographic features. Spine (Phila Pa 1976) 35, 531-6. Erwin, W.M., Ashman, K., O'Donnel, P. and Inman, R.D., 2006. Nucleus pulposus notochord cells secrete connective tissue growth factor and up-regulate proteoglycan expression by intervertebral disc chondrocytes. Arthritis Rheum 54, 3859-67. Finckenberg, P., Inkinen, K., Ahonen, J., Merasto, S., Louhelainen, M., Vapaatalo, H., Muller, D., Ganten, D., Luft, F. and Mervaala, E., 2003. Angiotensin II induces connective tissue growth factor gene expression via calcineurin-dependent pathways. Am J Pathol 163, 35566. Gajghate, S., Hiyama, A., Shah, M., Sakai, D., Anderson, D.G., Shapiro, I.M. and Risbud, M.V., 2009. Osmolarity and intracellular calcium regulate aquaporin2 expression through TonEBP in nucleus pulposus cells of the intervertebral disc. J Bone Miner Res 24, 992-1001. Haschtmann, D., Stoyanov, J.V. and Ferguson, S.J., 2006. Influence of diurnal hyperosmotic loading on the metabolism and matrix gene expression of a whole-organ intervertebral disc model. J Orthop Res 24, 1957-66. Higgins, D.F., Biju, M.P., Akai, Y., Wutz, A., Johnson, R.S. and Haase, V.H., 2004. Hypoxic induction of Ctgf is directly mediated by Hif-1. Am J Physiol Renal Physiol 287, F1223-32. Hiyama, A., Gajghate, S., Sakai, D., Mochida, J., Shapiro, I.M. and Risbud, M.V., 2009. Activation of TonEBP by calcium controls {beta}1,3-glucuronosyltransferase-I expression, a key regulator of glycosaminoglycan synthesis in cells of the intervertebral disc. J Biol Chem 284, 9824-34. Hogan, P.G., Chen, L., Nardone, J. and Rao, A., 2003. Transcriptional regulation by calcium, calcineurin, and NFAT. Genes Dev 17, 2205-32. Hoshijima, M., Hattori, T., Inoue, M., Araki, D., Hanagata, H., Miyauchi, A. and Takigawa, M., 2006. CT domain of CCN2/CTGF directly interacts with fibronectin and enhances cell adhesion of chondrocytes through integrin alpha5beta1. FEBS Lett 580, 1376-82. Ivkovic, S., Yoon, B.S., Popoff, S.N., Safadi, F.F., Libuda, D.E., Stephenson, R.C., Daluiski, A. and Lyons, K.M., 2003. Connective tissue growth factor coordinates chondrogenesis and angiogenesis

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

SC

RI

PT

during skeletal development. Development 130, 2779-91. Johnson, Z.I., Doolittle, A.C., Snuggs, J.W., Shapiro, I.M., Le Maitre, C.L. and Risbud, M.V., 2017. TNF-alpha promotes nuclear enrichment of the transcription factor TonEBP/NFAT5 to selectively control inflammatory but not osmoregulatory responses in nucleus pulposus cells. J Biol Chem 292, 17561-17575. Johnson, Z.I., Shapiro, I.M. and Risbud, M.V., 2014. Extracellular osmolarity regulates matrix homeostasis in the intervertebral disc and articular cartilage: evolving role of TonEBP. Matrix Biol 40, 10-6. Jun, J.I. and Lau, L.F., 2011. Taking aim at the extracellular matrix: CCN proteins as emerging therapeutic targets. Nat Rev Drug Discov 10, 945-63. Kepler, C.K., Ponnappan, R.K., Tannoury, C.A., Risbud, M.V. and Anderson, D.G., 2013. The molecular basis of intervertebral disc degeneration. Spine J 13, 318-30. Kubota, S. and Takigawa, M., 2015. Cellular and molecular actions of CCN2/CTGF and its role under physiological and pathological conditions. Clin Sci (Lond)5.629 128, 181-96. Lee, S.D., Choi, S.Y., Lim, S.W., Lamitina, S.T., Ho, S.N., Go, W.Y. and Kwon, H.M., 2011. TonEBP stimulates multiple cellular pathways for adaptation to hypertonic stress: organic osmolyte-dependent and independent pathways. Am J Physiol Renal Physiol 300, F707-15. Li, P., Gan, Y., Xu, Y., Li, S., Song, L., Li, S., Li, H. and Zhou, Q., 2016. Osmolarity affects matrix synthesis in the nucleus pulposus associated with the involvement of MAPK pathways: A study of ex vivo disc organ culture system. J Orthop Res 34, 1092-100. Livshits, G., Popham, M., Malkin, I., Sambrook, P.N., Macgregor, A.J., Spector, T. and Williams, F.M., 2011. Lumbar disc degeneration and genetic factors are the main risk factors for low back pain in women: the UK Twin Spine Study. Ann Rheum Dis 70, 1740-5. Lopez-Rodriguez, C., Aramburu, J., Jin, L., Rakeman, A.S., Michino, M. and Rao, A., 2001. Bridging the NFAT and NF-kappaB families: NFAT5 dimerization regulates cytokine gene transcription in response to osmotic stress. Immunity 15, 47-58. Lopez-Rodriguez, C., Aramburu, J., Rakeman, A.S. and Rao, A., 1999. NFAT5, a constitutively nuclear NFAT protein that does not cooperate with Fos and Jun. Proc Natl Acad Sci U S A 96, 7214-9. Luo, X., Pietrobon, R., Sun, S.X., Liu, G.G. and Hey, L., 2004. Estimates and patterns of direct health care expenditures among individuals with back pain in the United States. Spine (Phila Pa 1976) 29, 7986. Luoma, K., Riihimaki, H., Luukkonen, R., Raininko, R., Viikari-Juntura, E. and Lamminen, A., 2000. Low back pain in relation to lumbar disc

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

SC

RI

PT

degeneration. Spine (Phila Pa 1976) 25, 487-92. Lv, M., Zhou, Y., Chen, X., Han, L., Wang, L. and Lu, X.L., 2017. Calcium signaling of in situ chondrocytes in articular cartilage under compressive loading: Roles of calcium sources and cell membrane ion channels. J Orthop Res. Maeda-Uematsu, A., Kubota, S., Kawaki, H., Kawata, K., Miyake, Y., Hattori, T., Nishida, T., Moritani, N., Lyons, K.M., Iida, S. and Takigawa, M., 2014. CCN2 as a novel molecule supporting energy metabolism of chondrocytes. J Cell Biochem 115, 854-65. Maeda, A., Nishida, T., Aoyama, E., Kubota, S., Lyons, K.M., Kuboki, T. and Takigawa, M., 2009. CCN family 2/connective tissue growth factor modulates BMP signalling as a signal conductor, which action regulates the proliferation and differentiation of chondrocytes. J Biochem 145, 207-16. Matsumura, Y., Kasai, Y., Obata, H., Matsushima, S., Inaba, T. and Uchida, A., 2009. Changes in water content of intervertebral discs and paravertebral muscles before and after bed rest. J Orthop Sci 14, 45-50. Mesquita, R.F., Paul, M.A., Valmaseda, A., Francois, A., Jabr, R., Anjum, S., Marber, M.S., Budhram-Mahadeo, V. and Heads, R.J., 2014. Protein kinase Cepsilon-calcineurin cosignaling downstream of toll-like receptor 4 downregulates fibrosis and induces wound healing gene expression in cardiac myofibroblasts. Mol Cell Biol 34, 574-94. Nishida, T., Kawaki, H., Baxter, R.M., Deyoung, R.A., Takigawa, M. and Lyons, K.M., 2007. CCN2 (Connective Tissue Growth Factor) is essential for extracellular matrix production and integrin signaling in chondrocytes. J Cell Commun Signal 1, 45-58. Nishida, T., Kondo, S., Maeda, A., Kubota, S., Lyons, K.M. and Takigawa, M., 2009. CCN family 2/connective tissue growth factor (CCN2/CTGF) regulates the expression of Vegf through Hif-1alpha expression in a chondrocytic cell line, HCS-2/8, under hypoxic condition. Bone 44, 24-31. Peng, B., Chen, J., Kuang, Z., Li, D., Pang, X. and Zhang, X., 2009. Expression and role of connective tissue growth factor in painful disc fibrosis and degeneration. Spine (Phila Pa 1976) 34, E178-82. Pritchard, S., Erickson, G.R. and Guilak, F., 2002. Hyperosmotically induced volume change and calcium signaling in intervertebral disk cells: the role of the actin cytoskeleton. Biophys J 83, 2502-10. Risbud, M.V., Guttapalli, A., Stokes, D.G., Hawkins, D., Danielson, K.G., Schaer, T.P., Albert, T.J. and Shapiro, I.M., 2006. Nucleus pulposus cells express HIF-1 alpha under normoxic culture conditions: a metabolic adaptation to the intervertebral disc microenvironment. J Cell Biochem 98, 152-9. Sitara, D. and Aliprantis, A.O., 2010. Transcriptional regulation of bone

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

SC

RI

PT

and joint remodeling by NFAT. Immunol Rev 233, 286-300. Stemmer, P.M. and Klee, C.B., 1994. Dual calcium ion regulation of calcineurin by calmodulin and calcineurin B. Biochemistry 33, 685966. Tran, C.M., Fujita, N., Huang, B.L., Ong, J.R., Lyons, K.M., Shapiro, I.M. and Risbud, M.V., 2013. Hypoxia-inducible factor (HIF)-1alpha and CCN2 form a regulatory circuit in hypoxic nucleus pulposus cells: CCN2 suppresses HIF-1alpha level and transcriptional activity. J Biol Chem 288, 12654-66. Tran, C.M., Markova, D., Smith, H.E., Susarla, B., Ponnappan, R.K., Anderson, D.G., Symes, A., Shapiro, I.M. and Risbud, M.V., 2010. Regulation of CCN2/connective tissue growth factor expression in the nucleus pulposus of the intervertebral disc: role of Smad and activator protein 1 signaling. Arthritis Rheum 62, 1983-92. Tran, C.M., Schoepflin, Z.R., Markova, D.Z., Kepler, C.K., Anderson, D.G., Shapiro, I.M. and Risbud, M.V., 2014. CCN2 suppresses catabolic effects of interleukin-1beta through alpha5beta1 and alphaVbeta3 integrins in nucleus pulposus cells: implications in intervertebral disc degeneration. J Biol Chem 289, 7374-87. Tsai, T.T., Danielson, K.G., Guttapalli, A., Oguz, E., Albert, T.J., Shapiro, I.M. and Risbud, M.V., 2006. TonEBP/OREBP is a regulator of nucleus pulposus cell function and survival in the intervertebral disc. J Biol Chem 281, 25416-24. Tsai, T.T., Guttapalli, A., Agrawal, A., Albert, T.J., Shapiro, I.M. and Risbud, M.V., 2007. MEK/ERK signaling controls osmoregulation of nucleus pulposus cells of the intervertebral disc by transactivation of TonEBP/OREBP. J Bone Miner Res 22, 965-74. Urban, J.P., 2002. The role of the physicochemical environment in determining disc cell behaviour. Biochem Soc Trans 30, 858-64. Vergroesen, P.P., Kingma, I., Emanuel, K.S., Hoogendoorn, R.J., Welting, T.J., van Royen, B.J., van Dieen, J.H. and Smit, T.H., 2015. Mechanics and biology in intervertebral disc degeneration: a vicious circle. Osteoarthritis Cartilage. Walter, B.A., Purmessur, D., Moon, A., Occhiogrosso, J., Laudier, D.M., Hecht, A.C. and Iatridis, J.C., 2016. Reduced tissue osmolarity increases TRPV4 expression and pro-inflammatory cytokines in intervertebral disc cells. Eur Cell Mater 32, 123-36. Wang, D.L., Jiang, S.D. and Dai, L.Y., 2007. Biologic response of the intervertebral disc to static and dynamic compression in vitro. Spine (Phila Pa 1976) 32, 2521-8.

ACCEPTED MANUSCRIPT List of abbreviations

nucleus pulposus

CTGF/CCN2

connective tissue growth factor

TonEBP/NFAT5

tonicity enhancer binding protein/ nuclear factor of activated T-cells 5

Cn

calcineurin

IDD

intervertebral disc degeneration

AF

annulus fibrosus

GlcAT-I

β1,3-glucoronosyltransferaseⅠ

HIF

hypoxia-inducible factor

TGF-β

transforming growth factor β

IL-1β

interleukin1β

DMEM

dulbecco’s modified eagle’s medium

I

ionomycin

P/PMA

phorbol 12‐ myristate 13‐ acetate

C/CsA

cyclosporine A

B

BAPTA-AM

PBS

phosphate-buffered saline

CnA

catalytic subunit

CnB

regulatory subunit

PKCε

protein kinase C epsilon

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Highlights 1. Hyperosmolarity decreases CCN2 expression in nucleus pulposus cells via TonEBP. 2. Ca2+-calcineurin signaling increased CCN2 expression in nucleus pulposus cells. 3. NFATs regulate CCN2 promoter activity in nucleus pulposus cells. 4. Expression of CCN2 is tightly regulated in nucleus pulposus cells.