Cellular communication network factor 2 (CCN2) is a cysteine-rich secreted matricellular protein that regulates various cellular functions including cell differentiation. CCN2 is highly expressed under several types of mechanical stress, such as stretch, compression, and shear stress, in mesenchymal cells including chondrocytes, osteoblasts, and fibroblasts. In particular, CCN2 not only promotes cell proliferation and differentiation of various cells but also regulates the stability of mRNA of TRPV4, a mechanosensitive ion channel in chondrocytes. Of note, CCN2 behaves like a biomarker to sense suitable mechanical stress, because CCN2 expression is down-regulated when chondrocytes are subjected to excessive mechanical stress. These findings suggest that CCN2 is a mechano-sensing regulator. CCN2 expression is regulated by the activation of various mechano-sensing signaling pathways, e.g., mechanosensitive ion channels, integrin-focal adhesion-actin dynamics, Rho GTPase family members, Hippo-YAP signaling, and G protein-coupled receptors. This review summarizes the characterization of mechanoreceptors involved in CCN2 gene regulation and discusses the role of CCN2 as a mechano-sensing regulator of mesenchymal cell differentiation, with particular focus on chondrocytes.

Keywords: Cellular communication network factor 2 (CCN2); Chondrocytes; Mechanical stress; Mechanoreceptors.

CCN2, formerly known as connective tissue growth factor, was discovered in 1991. Soon after its discovery, CCN2 was shown to be upregulated in response to the profibrotic cytokine transforming growth factor (TGF) β and to be constitutively overexpressed in fibrotic conditions. These early observations led to the hypothesis that CCN2 was a key regulator of fibrosis. However, only recently have data been generated that directly demonstrate this hypothesis. This review summarizes these observations and suggests a mechanism of action for CCN2.

Fibroblast growth factor 1 (FGF-1) is a classical member of the FGF family and is produced by chondrocytes cultured from osteoarthritic patients. Also, this growth factor was shown to bind to CCN family protein 2 (CCN2), which regenerates damaged articular cartilage and counteracts osteoarthritis (OA) in an animal model. However, the pathophysiological role of FGF-1 in cartilage has not been well investigated. In this study, we evaluated the effects of FGF-1 in vitro and its production in vivo by use of an OA model. Treatment of human chondrocytic cells with FGF-1 resulted in marked repression of genes for cartilaginous extracellular matrix components, whereas it strongly induced matrix metalloproteinase 13 (MMP-13), representing its catabolic effects on cartilage. Interestingly, expression of the CCN2 gene was dramatically repressed by FGF-1, which repression eventually caused the reduced production of CCN2 protein from the chondrocytic cells. The results of a reporter gene assay revealed that this repression could be ascribed, at least in part, to transcriptional regulation. In contrast, the gene expression of FGF-1 was enhanced by exogenous FGF-1, indicating a positive feedback system in these cells. Of note, induction of FGF-1 was observed in the articular cartilage of a rat OA model. These results collectively indicate a pathological role of FGF-1 in OA development, which includes an insufficient cartilage regeneration response caused by CCN2 down regulation.

The CCN family of matricellular proteins (CYR61/CCN1, CTGF/CCN2, NOV/CCN3 and WISP1-2-3/CCN4-5-6) are essential players in the key pathophysiological processes of angiogenesis, wound healing and inflammation. These proteins are well recognized for their important roles in many cellular processes, including cell proliferation, adhesion, migration and differentiation, as well as the regulation of extracellular matrix differentiation. Substantial evidence implicates four of the proteins (CCN1, CCN2, CCN3 and CCN4) in the inflammatory pathologies of rheumatoid arthritis (RA) and osteoarthritis (OA). A smaller evidence base supports the involvement of CCN5 and CCN6 in the development of these diseases. This review focuses on evidence providing insights into the involvement of the CCN family in RA and OA, as well as the potential of the CCN proteins as therapeutic targets in these diseases.

Keywords: CCN family; CCN proteins; juvenile idiopathic arthritis; osteoarthritis; rheumatoid arthritis.

Background: Fibrosis is a major cause of loss of renal function in autosomal dominant polycystic kidney disease (ADPKD). In this study, we examined whether vasopressin type-2 receptor (V2R) activity in cystic epithelial cells can stimulate interstitial myofibroblasts and fibrosis in ADPKD kidneys.

Methods: We treated Pkd1 gene knockout (Pkd1KO) mice with dDAVP, a V2R agonist, for 3 days and evaluated the effect on myofibroblast deposition of extracellular matrix (ECM). We also analyzed the effects of conditioned media from primary cultures of human ADPKD cystic epithelial cells on myofibroblast activation. Because secretion of the profibrotic connective tissue growth factor (CCN2) increased significantly in dDAVP-treated Pkd1KO mouse kidneys, we examined its role in V2R-dependent fibrosis in ADPKD as well as that of yes-associated protein (YAP).

Results: V2R stimulation using dDAVP increased the renal interstitial myofibroblast population and ECM deposition. Similarly, conditioned media from human ADPKD cystic epithelial cells increased myofibroblast activation in vitro, suggesting a paracrine mechanism. Renal collecting duct-specific gene deletion of CCN2 significantly reduced cyst growth and myofibroblasts in Pkd1KO mouse kidneys. We found that YAP regulates CCN2, and YAP inhibition or gene deletion reduces renal fibrosis in Pkd1KO mouse kidneys. Importantly, YAP inactivation blocks the dDAVP-induced increase in myofibroblasts in Pkd1KO kidneys. Further in vitro studies showed that V2R regulates YAP by an ERK1/2-dependent mechanism in human ADPKD cystic epithelial cells.

Conclusions: Our results demonstrate a novel mechanism by which cystic epithelial cells stimulate myofibroblasts in the pericystic microenvironment, leading to fibrosis in ADPKD. The V2R-YAP-CCN2 cell signaling pathway may present a potential therapeutic target for fibrosis in ADPKD.

Keywords: polycystic kidney disease; renal fibrosis; vasopressin receptor.

Gingival connective tissue often has a composition resembling that of scar surrounding dental implant abutments. Increased cell adhesion, α-smooth muscle actin (α-SMA) expression and increased extracellular matrix deposition are a hallmark of fibrotic cells, but how topographic features influence gingival fibroblast adhesion and adoption of the α-SMA positive myofibroblast phenotype associated with scarring is unknown. The purpose of the present study was to demonstrate whether implant topographies that limit adhesion formation would reduce myofibroblast differentiation and extracellular matrix deposition. Human gingival fibroblasts were cultured on PT (smooth) and SLA (roughened) titanium discs for varying time-points. At 1 and 2 weeks after seeding, incorporation of α-SMA into stress-fibre bundles and fibronectin deposition was significantly higher on PT than SLA surfaces indicating differentiation of the cells towards a myofibroblast phenotype. Analysis of adhesion formation demonstrated that cells formed larger adhesions and more stable adhesions on PT, with more nascent adhesions observed on SLA. Gene expression analysis identified up-regulation of 15 genes at 24 hrs on SLA versus PT associated with matrix remodelling. Pharmacological inhibition of Src/FAK signalling in gingival fibroblasts on PT reduced fibronectin deposition and CCN2 expression. We conclude that topographical features that reduce focal adhesion stability could be applied to inhibit myofibroblast differentiation in gingival fibroblasts.

Lymphatic absorption in the peritoneal cavity may contribute to ultrafiltration failure in peritoneal dialysis (PD). Lymphatic vessels develop during PD-related peritoneal fibrosis. Connective tissue growth factor (CTGF, also called CCN2) is an important determinant of fibrotic tissue remodeling, but little is known about its possible involvement in lymphangiogenesis. In this study, we investigated the relationship between CTGF and peritoneal lymphangiogenesis. A positive correlation was observed between vascular endothelial growth factor-C (VEGF-C), a major lymphangiogenic growth factor, and the CTGF concentration in human PD effluents. CTGF expression was positively correlated with expression of lymphatic markers and VEGF-C in human peritoneal biopsies. We found a positive correlation between the increase in CTGF and the increase in VEGF-C in cultured human peritoneal mesothelial cells (HPMCs) treated with transforming growth factor-β1 (TGF-β1). The diaphragm is a central player in peritoneal lymphatic absorption. CTGF expression was also correlated with expression of VEGF-C and lymphatics in a rat diaphragmatic fibrosis model induced by chlorhexidine gluconate (CG). Furthermore, CTGF gene deletion reduced VEGF-C expression and peritoneal lymphangiogenesis in the mouse CG model. Inhibition of CTGF also reduced VEGF-C upregulation in HPMCs treated with TGF-β1. Our results suggest a close relationship between CTGF and PD-associated lymphangiogenesis.

Pulmonary fibrosis is a severely disabling disease often leading to death. CCN2 (Cellular Communication Network factor 2, also known as CTGF) is a known mediator of fibrosis and clinical trials studying anti-CCN2 efficacy in pulmonary fibrosis are currently underway. Fork head box D1 (FoxD1) transcription factor is transiently expressed in several mesenchymal cell types, including those of fetal lungs. Differentiation of FoxD1-progenitor derived pericytes into myofibroblasts involves CCN2 expression and contributes importantly to maladaptive tissue remodeling in for example kidney and lung fibrosis models. To generate a model for studying the contribution of CCN2 expression in FoxD1-progenitor derived cells to development of fibrotic tissue remodeling, we set out to establish a FoxD1Cre - CCN2^flox/flox mouse colony. However, all double-transgenic mice died soon after birth due to asphyxia. Histopathological examination revealed a reduction in alveolar space and lung weight, and subtle axial (thoracic and cervical) skeletal deformities. Together with the previously reported association of a FoxD1 containing locus with human adolescent idiopathic scoliosis, our data suggest that the fatal pulmonary hypoplasia resulting from selective deletion of CCN2 from FoxD1-progenitor derived mesenchymal cells developed secondary to impaired breathing movements due to aberrant axial skeletogenesis.

Bronchopulmonary dysplasia (BPD) is a chronic lung disease that often occurs in preterm infants. However, there is still no effective treatment for BPD. Recent studies demonstrated that connective tissue growth factor (CTGF) is involved in the development of BPD in experimental models. CTGF, also known as CCN2, is the second member of the CCN family and is necessary for normal lung development. The expression of CTGF is increased in lung tissues in infants with BPD. Hyperoxia, inflammation and mechanic ventilation increase CTGF expression which may promote fibroblast proliferation, matrix production and vascular remodeling. Conditional overexpression of CTGF in alveolar epithelial type II cells disrupts alveolarization and vascular development, induces vascular remodeling, and results in pulmonary hypertension, the pathological hallmarks of severe BPD. Further studies have shown that inhibition of CTGF by a CTGF monoclonal antibody improved alveolarization and vascular development, and decreased pulmonary vascular remodeling and pulmonary hypertension in a rodent model of BPD induced by hyperoxia. CTGF may be a novel target for BPD therapy in preterm infants.

OBJECTIVE

Persistent transforming growth factor beta (TGF-beta) signaling is the major factor contributing to scleroderma (SSc) fibrosis. This review will summarize recent progress on the noncanonical TGF-beta signaling pathways and their role in SSc fibrosis.

RESULTS

Canonical TGF-beta signaling involves activation of the TGF-beta receptors and downstream signal transducers Smad2/3. The term noncanonical TGF-beta signaling includes a variety of intracellular signaling pathways activated by TGF-beta independently of Smad2/3 activation. There is evidence that these pathways play important role in SSc fibrosis. In a subset of SSc fibroblasts, a multiligand receptor complex consisting of TGF-beta and CCN2 receptors drives constitutive activation of the Smad1 pathway. CCN2 is also a primary effector of this pathway, thus establishing an autocrine loop that amplifies TGF-beta signaling. SSc fibroblasts also demonstrate reduced expression of endogenous antagonists of TGF-beta signaling including transcriptional repressors, Friend leukemia integration-1 and perixosome proliferator-activated receptor-gamma, as well as inhibitor of Smad3 phosphorylation, PTEN. PTEN is a key mediator of the cross-talk between the sphingosine kinase and the TGF-beta pathways.

CONCLUSIONS

Discovery of the role of noncanonical TGF-beta signaling in fibrosis offers new molecular targets for the antifibrotic therapies. Due to the heterogeneous nature of SSc, knowledge of these pathways could help to tailor the therapy to the individual patient depending on the activation status of a specific profibrotic pathway.

In this Editorial, I would like to provide our readers with a brief mid-year update about our activities and efforts to bring together researchers working on intercellular signaling proteins at international meetings. The roots emerged about 20 years ago in the discovery of three genes originally designated cyr61, ctgf, and nov. The proteins encoded by these genes were first proposed to constitute a family of proteins (CCN) which now comprises 6 members (CCN1, CCN2, CCN3, CCN4-6) including the wisp proteins. These proteins were recognized to share a striking structural organization and a high degree of identity although they exhibited quite distinct biological properties. After historical considerations regarding the reasons for using the CCN acronym, and how the ICCNS publishing landscape that drove the ICCNS from Cell Communication and Signaling to the Journal of Cell Communication and Signaling, this short update will focus on the 7th edition of the International Workshop on the CCN family of genes to be held in Nice, Oct 16-19, 2013.

Connective tissue growth factor (CTGF, also named CCN2) plays an important role in the development of tubulointerstitial fibrosis, which most critically determines the progression to end-stage renal failure in autosomal-dominant polycystic kidney disease (ADPKD), the most common genetically caused renal disease. We determined CTGF expression in a well-characterized animal model of human ADPKD, the PKD/Mhm (cy/+) rat. Kidneys of 12 weeks old (cy/+) as well as (+/+) non-affected rats were analyzed for CTGF RNA and protein expression by RT-PCR, Northern and Western blot analyses, in situ hybridization, and IHC. Besides the established expression of CTGF in glomerular cells in kidneys of wild-type (+/+) animals, in (cy/+) rats, CTGF mRNA and protein were robustly expressed in interstitial, stellate-shaped cells, located in a scattered pattern underlying the cystic epithelium and in focal areas of advanced tubulointerstitial remodeling. Renal CTGF mRNA and protein expression levels were significantly higher in (cy/+) rats compared with their (+/+) littermates. Detection of CTGF expression in cells adjacent to cystic epithelium and in areas of marked fibrosis suggests a role in the local response to cyst development and indicates that CTGF may be a relevant factor contributing to tubulointerstitial fibrosis in polycystic kidney disease.

A recent paper in this journal, presented a novel method by topical application of growth factors in stimulating diabetic cutaneous wound healing that caught our attention. We believe that the experimental method in the article is efficient and creative, but it also has some controversies and shortcomings to be discussed. We noted that the authors used "Tegaderm" as a semiocclusive dressing film and stated that it exerted a "splinting effect" on the wound margins and controlled contraction. Indeed, the "Tegaderm" itself can serve as a dressing film to isolate the wound bed with outside environments while the "splinting effect" is mainly achieved by adding silicone splints around the wound. Considering the unique properties of silicone splints and "Tegaderm," our experimental group propose an alternative method named "combined-suturing" technique that is not only suturing the silicone splints but also securing the "Tegaderm" around the wound. The specific reasons and operative procedures are explained in detail in this letter.

CCN2 (connective tissue growth factor [CTGF]/hypertrophic chondrocyte-specific gene product 24 [Hcs24]) is regulated at the transcriptional and posttranscriptional level. For example, an element in the its 3' untranslated region (3'-UTR) of the CCN2 mRNA controls message stability in chondrocytes. In a recent study, Mukudai et al. (Mol Cell Biol 28:6134-6147, 2008) purified and identified a trans-factor protein binding to the minimal repressive cis element in the 3'-UTR of ccn2 mRNA and identify this protein as the multifunctional nucleolar phosphoprotein nucleophosmin (NPM) This commentary summarizes these observations.

BACKGROUND

Joint haemorrhage is the principal clinical manifestation of haemophilia frequently leading to advanced arthropathy and arthrofibrosis, resulting in severe disability. The degree and prevalence of arthrofibrosis in hemophilic arthropathy is more severe than in other forms of arthropathy. Expression of connective tissue growth factor (CTGF) has been linked to many fibrotic diseases, but has not been studied in the context of haemophilic arthropathy.

OBJECTIVE

We aim to compare synovial tissues histologically from haemophilia and osteoarthritis patients with advanced arthropathy in order to compare expression of proteins that are possibly aetiologic in the development of arthrofibrosis.

METHODS

Human synovial tissues were obtained from 10 haemophilia and 10 osteoarthritis patients undergoing joint surgery and processed for histology and immunohistochemistry.

RESULTS

All samples from haemophilia patients had synovitis with hypertrophy and hyperplasia of synovial villi. Histologically, synovial tissues contained hyperplastic villi with increased cellularity and abundant haemosiderin- and ferritin-pigmented macrophage-like cells (HMCs), with a perivascular localization in the sub-surface layer. CTGF staining was observed in the surface layer and sub-surface layer in all haemophilia patients, exclusively co-localizing with HMCs. Quantification showed that the extent of CTGF-positive areas was correlated with the degree of detection of HMCs. CTGF was not observed in any of the samples from osteoarthritis patients.

CONCLUSIONS

Using histological analysis, we showed that CTGF expression is elevated in haemophilia patients with arthrofibrosis and absent in patients with osteoarthritis. Additionally, we found that CTGF is always associated with haemosiderin-pigmented macrophage-like cells, which suggests that CTGF is produced by synovial A cells following the uptake of blood breakdown products.

Promoter analysis is the most basics in the analysis of gene regulation. Luciferase gene is the most commonly used reporter gene in promoter analysis. Luciferase is an enzyme that is used when firefly and Renilla reniformis (sea pansy) emit light. The first experimental step in this reporter gene assay is to connect a particular DNA segment to a luciferase gene. The second step is to transfect the reporter construct into the cells. Thereafter, stable luciferase will be produced with the help of transcriptional machinery, mRNA transporters, and translational machinery in the cells. Luciferase assay measures the quantity of light that is emitted by luciferin-luciferase reaction. Consistent with the fact that CCN2 expression has been shown to be altered by a variety of stimuli, the CCN2 promoter region also haa been shown to be bound and regulated by multiple transcription factors such as Smad, MMP3, NF-κB, AP1, TCF/LEF, and Sox9.

To investigate mechanical-dependent bone remodeling, we had previously applied various types of mechanical loading onto the teeth of rats and mice. In vitro cultured bone cells were then used to elucidate the mechanisms underlying the specific phenomenon revealed by in vivo experiments. This review describes the techniques used to upregulate CCN2 expression in bone cells produced by different types of mechanical stress, such as fluid shear stress and substrate strain in vitro, and compression or tension force in vivo.

OBJECTIVE

To investigate the relationship between connective tissue growth factor (CCN5) and hepatic stellate cell (HSC) activation as well as the mechanism of action.

METHODS

As the research object, LX-2 cells were stimulated with transforming growth factor-beta1 ( TGF-(beta1), and the protein expression levels of CCN5 and CCN2 were determined by Western blot; Hepatocyte high expression system of CCN5 was constructed and transfected hepatic stellate cells (HSC) to make CCN5 overexpression; The expression levels of alpha-smooth muscle actin (alpha-SMA) and collagen I were determined by RT-PCR and Western blot. To further study its mechanism of action, Smad2 and phosphorylation level of Smad2 were determined by RT-PCR and Western blot.

RESULTS

Under normal circumstances, CCN2 expression levels were much higher than CCN5 in LX-2 cells, while CCN2 expression was significantly higher than CCN5 if LX-2 cells were stimulated by TGF-beta1. However, there was no change for CCN5. Compared with the control group and the vector group, CCN5 was successfully overexpressed in the transfection group, and mRNA and protein levels of alpha-SMA and collagen I were significantly decreased (P < 0.01). Meanwhile, phosphorylation level of Smad2 was also significantly decreased (P < 0.01).

CONCLUSIONS

CCN5, which has the function that inhibits HSC activation, has the opposite role compared with CCN2, therefore, a new idea was proposed for the prevention and treatment of liver fibrosis.

Connective tissue growth factor (CCN2) regulates diverse cellular functions, including tooth development. In order to delineate the precise role of CCN2 in the epithelium during odontogenesis, we investigated how it is expressed and what roles it may have in primary cultures of epithelial cells derived from developing tooth germ of the bovine fetus. Ccn2 mRNA and protein were strongly expressed in the inner dental epithelium, which is consistent with the expression of transforming growth factor-β2 mRNA and proliferating cell nuclear antigen. Bone morphogenetic protein 4 (BMP4) and fibroblast growth factor 2 (FGF2) were also expressed in the inner dental epithelium, indicating that CCN2 functionally interacts with these factors in the epithelium. The stimulatory effects of FGF2 on cell proliferation and BMP4 on cell differentiation were additively up-regulated by CCN2 in a newly-established dental epithelium cell culture. Taken together, our data provide clear evidence that CCN2 is synthesized by inner dental epithelial cells, and appears to act as an autocrine factor, which regulates dental epithelial cell proliferation and differentiation in concert with growth factors.

OBJECTIVE

Transforming growth factor-β (TGF-β) plays an important role in the pathogenesis of cyclosporine A (CsA)-induced gingival overgrowth (GO). Connective tissue growth factor (CTGF/CCN2) acts as a cofactor with TGF-β to induce the maximal profibrotic effects of TGF-β. We investigated the effects of CsA on CCN2 expression in human gingival fibroblasts (HGFs) and the potential chemopreventive agent for CsA-induced GO.

METHODS

Western blot analyses were used to examine the signaling pathways of CsA-induced CCN2 expression in HGFs and whether epigallocatechin-3-gallate (EGCG), curcumin, or lovastatin can inhibit CsA-induced CCN2 expression.

RESULTS

CsA significantly stimulated CCN2 synthesis in HGFs. This effect can be inhibited by c-Jun NH(2)-terminal kinase (JNK) and Smad3 inhibitors but not by TGF-β neutralizing antibody and TGF-β type I receptor inhibitor. Furthermore, EGCG completely blocked CsA-induced CCN2 expression.

CONCLUSIONS

CsA-induced CCN2 protein expression is mediated through JNK and Smad signaling. CsA may contribute to the pathogenesis of GO through upregulation of CCN2 expression in HGFs. EGCG could be an adjuvant for the prevention of CsA-induced GO.

The development of obesity and its metabolic complications is associated with dysregulation of various intrinsic mechanisms, which control basic metabolic processes via changes in the expression of numerous regulatory genes. We studied the expression of the subset of genes, which responsible for control of cell growth and glucose metabolism, in blood cells of obese boys with normal and impaired insulin sensitivity as well as in normal (control) individuals. It was shown that obesity with normal insulin sensitivity enhances the expression of IRS1, RIPK2, IL13RA2, RSPO1, IQSEC, and CCN2 genes but decreases the expression level IRS2 and DNAJC15 genes in the blood cells as compared to control group. Insulin resistance in obese boys leads to up-regulation of IRS2, RSPO1, and DNAJC15 gene expressions as wells to down-regulation of IRS1 and RIPK2 genes in the blood cells versus obese patients with normal insulin sensitivity. Results of this study provide evidence that obesity affects the expression of the subset of genes related to cell growth and glucose metabolism in blood cells and that insulin resistance in obesity is associated with changes in the expression level of IRS1, IRS2, RIPK2, RSPO1, and DNA JC15 genes, which contribute to the development of insulin resistance and glucose intolerance and possibly reflect some changes in fat tissue.

CCN2 (connective tissue growth factor, CTGF), a member of the CCN family is overexpressed in fibrotic disease and is essential for the development of experimental fibrosis. Drugs targeting CCN2 action may therefore prove to be useful anti-fibrotic approaches. CCN2 acts via integrins and heparan sulfate-containing proteoglycans (HSPGs). In a recent study, Vial and colleagues (2011) show that decorin can bind CCN2. A peptide corresponding to the leucine rich repeats peptide 12 region of decorin can neutralize CCN2-mediated activity on C2C12 cells in vitro. Thus it is conceivable that this peptide could be used in the future as a novel antifibrotic approach.

To identify proteins that cooperate with cellular communication network factor 2 (CCN2), we carried out GAL4-based yeast two-hybrid screening using a cDNA library derived from the chondrocytic cell line HCS-2/8. Rab14 GTPase (Rab14) polypeptide was selected as a CCN2-interactive protein. The interaction between CCN2 and Rab14 in HCS-2/8 cells was confirmed using the in situ proximity ligation assay. We also found that CCN2 interacted with Rab14 through its IGFBP-like domain among the four domains in CCN2 protein. To detect the colocalization between CCN2 and Rab14 in the cells in detail, CCN2, wild-type Rab14 (Rab14WT), a constitutive active form (Rab14CA), and a dominant negative form (Rab14DN) of Rab14 were overexpressed in monkey kidney-tissue derived COS7 cells. Ectopically overexpressed Rab14 showed a diffuse cytosolic distribution in COS7 cells; however, when Rab14WT was overexpressed with CCN2, the Rab14WT distribution changed to dots that were evenly distributed within the cytosol, and both Rab14 and CCN2 showed clear colocalization. When Rab14CA was overexpressed with CCN2, Rab14CA and CCN2 also showed good localization as dots, but their distribution was more widespread within cytosol. The coexpression of Rab14DN and CCN2 also showed a dotted codistribution but was more concentrated in the perinuclear area. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis revealed that the reduction in RAB14 or CCN2 mRNA by their respective siRNA significantly enhanced the expression of ER stress markers, BIP and CHOP mRNA in HCS-2/8 chondrocytic cells, suggesting that ER and Golgi stress were induced by the inhibition of membrane vesicle transfer via the suppression of CCN2 or Rab14. Moreover, to study the effect of the interaction between CCN2 and its interactive protein Rab14 on proteoglycan synthesis, we overexpressed Rab14WT or Rab14CA or Rab14DN in HCS-2/8 cells and found that the overexpression of Rab14DN decreased the extracellular proteoglycan accumulation more than the overexpression of Rab14WT/CA did in the chondrocytic cells. These results suggest that intracellular CCN2 is associated with Rab14 on proteoglycan-containing vesicles during their transport from the Golgi apparatus to endosomes in chondrocytes and that this association may play a role in proteoglycan secretion by chondrocytes.