CCN2 Production Research Articles

Effect of Angiotensin II on Chondrocyte Degeneration and Protection via Differential Usage of Angiotensin II Receptors.

The renin–angiotensin system (RAS) controls not only systemic functions, such as blood pressure, but also local tissue-specific events. Previous studies have shown that angiotensin II receptor type 1 (AT1R) and type 2 (AT2R), two RAS components, are expressed in chondrocytes. However, the angiotensin II (ANG II) effects exerted through these receptors on chondrocyte metabolism are not fully understood. In this study, we investigated the effects of ANG II and AT1R blockade on chondrocyte proliferation and differentiation. Firstly, we observed that ANG II significantly suppressed cell proliferation and glycosaminoglycan content in rat chondrocytic RCS cells. Additionally, ANG II decreased CCN2, which is an anabolic factor for chondrocytes, via increased MMP9. In Agtr1a-deficient RCS cells generated by the CRISPR-Cas9 system, Ccn2 and Aggrecan (Acan) expression increased. Losartan, an AT1R antagonist, blocked the ANG II-induced decrease in CCN2 production and Acan expression in RCS cells. These findings suggest that AT1R blockade reduces ANG II-induced chondrocyte degeneration. Interestingly, AT1R-positive cells, which were localized on the surface of the articular cartilage of 7-month-old mice expanded throughout the articular cartilage with aging. These findings suggest that ANG II regulates age-related cartilage degeneration through the ANG II–AT1R axis.

Possible reparative effect of low-intensity pulsed ultrasound (LIPUS) on injured meniscus.

Menisci are a pair of crescent-shaped fibrocartilages, particularly of which their inner region of meniscus is an avascular tissue. It has characteristics similar to those of articular cartilage, and hence is inferior in healing. We previously reported that low-intensity pulsed ultrasound (LIPUS) treatment stimulates the production of CCN2/CTGF, a protein involved in repairing articular cartilage, and the gene expression of major cartilage matrices such as type II collagen and aggrecan in cultured chondrocytes. Therefore, in this present study, we investigated whether LIPUS has also favorable effect on meniscus cells and tissues. LIPUS applied with a 60mW/cm2 intensity for 20min stimulated the gene expression and protein production of CCN2 via ERK and p38 signaling pathways, as well as gene expression of SOX9, aggrecan, and collagen type II in human inner meniscus cells in culture, and slightly stimulated the gene expression of CCN2 and promoted the migration in human outer meniscus cells in culture. LIPUS also induced the expression of Ccn2, Sox9, Col2a1, and Vegf in rat intact meniscus. Furthermore, histological evaluations showed that LIPUS treatment for 1 to 4weeks promoted healing of rat injured lateral meniscus, as evidenced by better and earlier angiogenesis and extracellular matrix synthesis. The data presented indicate that LIPUS treatment might prevent meniscus from degenerative change and exert a reparative effect on injured meniscus via up-regulation of repairing factors such as CCN2 and that it might thus be useful for treatment of an injured meniscus as a non-invasive therapy.

Activation of cancer-associated fibroblasts is required for tumor neovascularization in a murine model of melanoma

Metastatic melanoma is highly fatal. Within the tumor microenvironment, the role of cancer-associated fibroblasts (CAFs) in melanoma metastasis and progression is relatively understudied. The matricellular protein CCN2 (formerly termed connective tissue growth factor, CTGF) is overexpressed, in a fashion independent of BRAF mutational status, by CAFs in melanoma. Herein, we find, in human melanoma patients, that CCN2 expression negatively correlates with survival and positively correlates with expression of neovascularization markers. To assess the role of CAFs in melanoma progression, we used C57BL/6 mice expressing a tamoxifen-dependent cre recombinase expressed under the control of a fibroblast-specific promoter/enhancer (COL1A2) to delete CCN2 postnatally in fibroblasts. Mice deleted or not for CCN2 in fibroblasts were injected subcutaneously with B16-F10 melanoma cells. Loss of CCN2 in CAFs resulted in reduced CAF activation, as detected by staining with anti-α-smooth muscle actin antibodies, and reduced tumor-induced neovascularization, as detected by micro-computed tomography (micro-CT) and staining with anti-CD31 antibodies. CCN2-deficient B16(F10) cells were defective in a tubule formation/vasculogenic mimicry assay in vitro. Mice deleted for CCN2 in CAFs also showed impaired vasculogenic mimicry of subcutaneously-injected B16-F10 cells in vivo. Our results provide new insights into the cross-talk among different cell types in the tumor microenvironment and suggest CAFs play a heretofore unappreciated role by being essential for tumor neovascularization via the production of CCN2. Our data are consistent with the hypothesis that activated CAFs are essential for melanoma metastasis and that, due to its role in this process, CCN2 is a therapeutic target for melanoma.

Regulatory mechanism of CCN2 production by serotonin (5-HT) via 5-HT2A and 5-HT2B receptors in chondrocytes.

Serotonin (5-hydroxytryptamine: 5-HT) is recognized as a neurotransmitter in the central nerve system and as a regulator of systemic blood pressure in the peripheral tissues. Recently, it was reported that 5-HT2 receptors (5-HT2Rs) were expressed in cartilage tissues lacking both vessels and neurons, suggesting possible novel functions of 5-HT during cartilage development and regeneration. Our previous data indicated that CCN family protein 2/connective tissue growth factor (CCN2/CTGF) plays a central role in cartilage development and regeneration. Therefore, the aim of this study was to investigate the effect of 5-HT on the production of CCN2 in chondrocytes. Firstly, we showed that the mRNAs of 5-HT2R subtypes 5-HT2AR and 5-HT2BR, were expressed in a human chondrocytic cell line, HCS-2/8; however, 5-HT2CR mRNA was not detected. In addition, exogenously added 5-HT did not affect the 5-HT2AR and 5-HT2BR expressions. Next, we demonstrated that CCN2 production was increased by treatment with a 5-HT2AR agonist and the combination of 5-HT and 5-HT2BR antagonist. In contrast, treatment with a 5-HT2BR agonist and the combination of 5-HT and 5-HT2AR antagonist decreased CCN2 production. Furthermore, we showed that phosphorylation of Akt and p38 MAPK were increased by treatment with 5-HT2AR agonist, and that phosphorylation of PKCε, PKCζ, ERK1/2 and JNK were increased by treatment with 5-HT2BR agonist. Finally, we found that 5-HT2AR was localized in the growth plate, whereas 5-HT2BR was localized in the articular cartilage. These findings suggest that 5-HT promotes CCN2 production through the 5-HT2AR in growth plates, and that it represses CCN2 production through the 5-HT2BR in articular cartilage for harmonized development of long bones.

Substance P increases CCN2 dependent on TGF-beta yet Collagen Type I via TGF-beta1 dependent and independent pathways in tenocytes

ABSTRACTTransforming growth factor beta 1 (TGFbeta-1) and connective tissue growth factor (CCN2) are important mediators of tissue repair and fibrosis, with CCN2 functioning as a downstream mediator of TGFβ-1. Substance P (SP) is also linked to collagen production in tenocytes. A link between SP, TGFbeta-1 and CCN2 has yet to be established in tenocytes or fibrogenic processes. We sought to determine whether SP induces tenocyte proliferation, CCN2, or collagen production via TGFbeta-1 signaling or independently in rat primary tenocytes. Tenocytes were isolated from rat tendons, cultured and stimulated by SP and/or TGFbeta-1. Cultured cells expressed proteins characteristic of tenocytes (vimentin and tenomodulin) and underwent increased proliferation dose dependently after SP and TGFbeta-1 treatments, alone or combined (more than SP alone when combined). SP induced TGFbeta-1 expression in tenocytes in both dose- and time-dependent manners. SP and TGFbeta-1, alone or combined, stimulated CCN2 expression in tenocytes and their supernatants after both 24 and 48 h of stimulation; a response blocked with addition of a TGFbeta-1 receptor inhibitor. In contrast, SP potentiated collagen type I secretion by tenocytes, a response abrogated by the TGFbeta-1 receptor inhibitor after 48 h of stimulation, but not after the shorter 24 h of stimulation. Our findings suggest that both SP and TGFbeta-1 can stimulate tenocyte fibrogenic processes, albeit differently. TGFbeta-1 pathway signaling was involved in CCN2 production at all time points examined, while SP induced collagen type I production independently prior to the onset of signaling through the TGFbeta-1 pathway.

Compressive force-produced CCN2 induces osteocyte apoptosis through ERK1/2 pathway

Osteocytes produce various factors that mediate the onset of bone formation and resorption and play roles in maintaining bone homeostasis and remodeling in response to mechanical stimuli. One such factor, CCN2, is thought to play a significant role in osteocyte responses to mechanical stimuli, but its function in osteocytes is not well understood. Here, we showed that CCN2 induces apoptosis in osteocytes under compressive force loading. Compressive force increased CCN2 gene expression and production, and induced apoptosis in osteocytes. Application of exogenous CCN2 protein induced apoptosis, and a neutralizing CCN2 antibody blocked loading-induced apoptosis. We further examined how CCN2 induces loaded osteocyte apoptosis. In loaded osteocytes, extracellular signal-regulated kinase 1/2 (ERK1/2) was activated, and an ERK1/2 inhibitor blocked loading-induced apoptosis. Furthermore, application of exogenous CCN2 protein caused ERK1/2 activation, and the neutralizing CCN2 antibody inhibited loading-induced ERK1/2 activation. Therefore, this study demonstrated for the first time to our knowledge that enhanced production of CCN2 in osteocytes under compressive force loading induces apoptosis through activation of ERK1/2 pathway. © 2014 American Society for Bone and Mineral Research.

Fibrogenic Signaling Is Suppressed in Hepatic Stellate Cells through Targeting of Connective Tissue Growth Factor (CCN2) by Cellular or Exosomal MicroRNA-199a-5p

Pathways of liver fibrosis are controlled by connective tissue growth factor (CCN2). In this study, CCN2 was identified as a target of miR-199a-5p, which was principally expressed in quiescent mouse hepatic stellate cells (HSCs) and directly suppressed production of CCN2. Up-regulated CCN2 expression in fibrotic mouse livers or in activated primary mouse HSCs was associated with miR-199a-5p down-regulation. MiR-199a-5p in quiescent mouse HSCs inhibited the activity of a wild-type CCN2 3' untranslated region (3'-UTR) but not of a mutant CCN2 3'-UTR lacking the miR-199a-5p-binding site. In activated mouse HSCs, CCN2, α-smooth muscle actin, and collagen 1(α1) were suppressed by a miR-199a-5p mimic, whereas in quiescent mouse HSCs, the inhibited CCN2 3'-UTR activity was blocked by a miR-199a-5p antagomir. CCN2 3'-UTR activity in human HSCs was reduced by a miR-199a-5p mimic. MiR-199a-5p was present at higher levels in exosomes from quiescent versus activated HSCs. MiR-199a-5p-containing exosomes were shuttled from quiescent mouse HSCs to activated mouse HSCs in which CCN2 3'-UTR activity was then suppressed. Exosomes from quiescent HSCs caused miR-199a-5p-dependent inhibition of CCN2, α-smooth muscle actin, or collagen 1(α1) in activated HSCs invitro andbound to activated HSCs invivo. Thus, CCN2 suppression by miR-199a-5p accounts, in part, for low-level fibrogenic gene expression in quiescent HSCs and causes dampened gene expression in activated HSCs after horizontal transfer of miR-199a-5p in exosomes from quiescent HSCs.

Prevention of Phenytoin-Induced Gingival Overgrowth by Lovastatin in Mice

Drug-induced gingival overgrowth is caused by the antiseizure medication phenytoin, calcium channel blockers, and ciclosporin. Characteristics of these drug-induced gingival overgrowth lesions differ. We evaluate the ability of a mouse model to mimic human phenytoin-induced gingival overgrowth and assess the ability of a drug to prevent its development. Lovastatin was chosen based on previous analyses of tissue-specific regulation of CCN2 production in human gingival fibroblasts and the known roles of CCN2 in promoting fibrosis and epithelial to mesenchymal transition. Data indicate that anterior gingival tissue overgrowth occurred in phenytoin-treated mice based on gross tissue observations and histomorphometry of tissue sections. Molecular markers of epithelial plasticity and fibrosis were regulated by phenytoin in gingival epithelial tissues and in connective tissues similar to that seen in humans. Lovastatin attenuated epithelial gingival tissue growth in phenytoin-treated mice and altered the expressions of markers for epithelial to mesenchymal transition. Data indicate that phenytoin-induced gingival overgrowth in mice mimics molecular aspects of human gingival overgrowth and that lovastatin normalizes the tissue morphology and the expression of the molecular markers studied. Data are consistent with characterization of phenytoin-induced human gingival overgrowth in vivo and in vitro characteristics of cultured human gingival epithelial and connective tissue cells. Findings suggest that statins may serve to prevent or attenuate phenytoin-induced human gingival overgrowth, although specific human studies are required.

Regulation of CCN2 and CCN3 in bone marrow through myloid zinc finger‐1 and its medical implication in hematopoiesis (1005.4)

CCN2, also known as CTGF, the connective tissue growth factor, has recently been found to be in high abundance in platelets and released upon activation, an effect inhibited by aspirin, suggesting additional role in blood clotting and/or wound healing. It has been demonstrated that another member of the CCN family, the Nephroblastoma overexpressed gene (NOV, CCN3) functions in hematopoietic potential, blood cell formation, and helps to maintain a competent immune system. Taken together, these two CCN family members represent interesting new areas of research in blood cell biology, with dramatic clinical potential.Recent research in our lab discovered that the transcription factor MZF‐1 regulates both the CCN2 and CCN3 genes. Specifically, we found that MZF‐1 binds to the promoter of both CTGF (CCN2) and NOV (CCN3). It is known that both vitamin D (calcitriol) and vitamin A (all‐trans retinoic acid) stimulate transcription of the MZF1 gene. We have thus discovered a potential new pathway in which Vitamins A and D induce MZF‐1 to upregulate CCN3 and CCN2 resulting in increased hematopoietic potential and increased differentiation of HSCs and their progeny. It is known that the presence of CTGF in platelets is the result of endocytosis from extracellular environment. However, we have confirmed that treatments with either vitamin A or D activate the MZF‐1 pathway and results in increased production of CCN2 in stromal fibroblasts, which in turn results in enhanced loading of CCN2 into developing platelets in vitro.Furthermore, the regulation of CCN3 by MZF‐1 represents a testable new clinical treatment. We have observed that both vitamin A and vitamin D induce CCN3 expression, through MZF‐1, in multiple blood cell types. We are currently testing if this will lead to enhanced hematopoietic potential. This work could impact the efficacy of blood donation and transfusion, bone marrow transplants, and the treatment of bleeding and clotting diseases as well as lymphomas and leukemias.

Elevated YAP and Its Downstream Targets CCN1 and CCN2 in Basal Cell Carcinoma: Impact on Keratinocyte Proliferation and Stromal Cell Activation

Elevated YAP and Its Downstream Targets CCN1 and CCN2 in Basal Cell Carcinoma: Impact on Keratinocyte Proliferation and Stromal Cell Activation

Connective tissue growth factor (CCN2) and microRNA-21 are components of a positive feedback loop in pancreatic stellate cells (PSC) during chronic pancreatitis and are exported in PSC-derived exosomes

Pancreatitis is an inflammatory condition of the pancreas which, in its chronic form, involves tissue destruction, exocrine and endocrine insufficiency, increased risk of pancreatic cancer, and an extensive fibrotic pathology which is due to unrelenting collagen deposition by pancreatic stellate cells (PSC). In response to noxious agents such as alcohol-excessive consumption of which is a major cause of pancreatitis in the West-normally quiescent PSC undergo a phenotypic and functional transition to activated myofibroblasts which produce and deposit collagen at high levels. This process is regulated by connective tissue growth factor (CCN2), expression of which is highly up-regulated in activated PSC. We show that CCN2 production by activated PSC is associated with enhanced expression of microRNA-21 (miR-21) which was detected at high levels in activated PSC in a murine model of alcoholic chronic pancreatitis. A positive feedback loop between CCN2 and miR-21 was identified that resulted in enhancement of their respective expression as well as that of collagen α1(I). Both miR-21 and CCN2 mRNA were present in PSC-derived exosomes, which were characterized as 50-150nm CD9-positive nano-vesicles. Exosomes from CCN2-GFP- or miR-21-GFP-transfected PSC were taken up by other PSC cultures, as shown by direct fluorescence or qRT-PCR for GFP. Collectively these studies establish miR-21 and CCN2 as participants in a positive feedback loop during PSC activation and as components of the molecular payload in PSC-derived exosomes that can be delivered to other PSC. Thus interactions between cellular or exosomal miR-21 and CCN2 represent novel aspects of fibrogenic regulation in PSC. Summary Chronic injury in the pancreas is associated with fibrotic pathology which is driven in large part by CCN2-dependent collagen production in pancreatic stellate cells. This study shows that CCN2 up-regulation in PSC is associated with increased expression of miR-21 which, in turn, is able to stimulate CCN2 expression further via a positive feedback loop. Additionally miR-21 and CCN2 were identified in PSC-derived exosomes which effected their delivery to other PSC. The cellular and exosomal miR-21-CCN2 axis is a novel component in PSC fibrogenic signaling.

Compressive Force-Produced CCN2 Induces Osteocyte Apoptosis Through ERK1/2 Pathway

Osteocytes produce various factors that mediate the onset of bone formation and resorption and play roles in maintaining bone homeostasis and remodeling in response to mechanical stimuli. One such factor, CCN2, is thought to play a significant role in osteocyte responses to mechanical stimuli, but its function in osteocytes is not well understood. Here, we showed that CCN2 induces apoptosis in osteocytes under compressive force loading. Compressive force increased CCN2 gene expression and production, and induced apoptosis in osteocytes. Application of exogenous CCN2 protein induced apoptosis, and a neutralizing CCN2 antibody blocked loading-induced apoptosis. We further examined how CCN2 induces loaded osteocyte apoptosis. In loaded osteocytes, extracellular signal-regulated kinase 1/2 (ERK1/2) was activated, and an ERK1/2 inhibitor blocked loading-induced apoptosis. Furthermore, application of exogenous CCN2 protein caused ERK1/2 activation, and the neutralizing CCN2 antibody inhibited loading-induced ERK1/2 activation. Therefore, this study demonstrated for the first time to our knowledge that enhanced production of CCN2 in osteocytes under compressive force loading induces apoptosis through activation of ERK1/2 pathway.

EGCG blocks TGFβ1-induced CCN2 by suppressing JNK and p38 in buccal fibroblasts

Transforming growth factor β (TGFβ) has been suggested as the main trigger for the increased collagen production and decreased matrix degradation pathways in oral submucous fibrosis (OSF). Connective tissue growth factor (CTGF/CCN2) and cyclooxygenase-2 (COX-2) were found to overexpress in OSF. The aim of this study was to investigate the molecular mechanism underlying the TGFβ-induced CCN2 expressions in human buccal mucosal fibroblasts (BMFs) to identify the potential targets for drug intervention or chemoprevention of OSF. TGFβ-induced CCN2 expression and its signaling pathways were assessed by Western blot analyses in BMFs. TGFβ1 stimulated CCN2 synthesis in BMFs. Pretreatment with c-Jun NH(2)-terminal kinase (JNK) inhibitor SP600125, p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580, and activin receptor-like kinase 5 (ALK5) inhibitor SB431542 significantly reduced TGFβ1-induced CCN2 synthesis. Epigallocatechin-3-gallate (EGCG) completely blocked TGFβ1-induced CCN2 synthesis by inhibiting the phosphorylation of JNK and p38 MAPK. Prostaglandin E(2) (PGE(2)) inhibited the TGFβ1-induced CCN2 synthesis in human fetal lung fibroblasts IMR90 but not in BMFs. The TGFβ1-induced CCN2 synthesis in BMFs could be mediated by the ALK5, JNK, and p38 MAPK pathways. EGCG blocks TGFβ1-induced CCN2 by suppressing JNK and p38 in BMFs. The exceptional signal transduction pathways of TGFβ1-induced CCN2 production in BMFs contribute to the resistance of PGE(2) downregulation of CCN2 expression; therefore, the CTGF/CCN2 levels are maintained in the OSF tissues in the presence of COX-2. EGCG may serve as a useful agent in controlling OSF.

Tissue factor–thrombin signaling enhances the fibrotic activity of myofibroblasts in systemic sclerosis through up‐regulation of endothelin receptor A

The extrinsic coagulation cascade is involved in the fibrotic process, via thrombin-dependent induction of CCN2 (connective tissue growth factor) expression. Given the previously reported activation of the coagulation system in systemic sclerosis (SSc), we undertook the present study to investigate the involvement of cross-talk between the tissue factor (TF)-thrombin axis and endothelin 1 (ET-1) signaling in the fibrotic activity of SSc. Human colonic myofibroblasts (HCMFs) from 6 patients with SSc and gastrointestinal symptoms and from 6 control subjects were isolated and cultured under various conditions. Messenger RNA and protein levels of TF, CCN2, and endothelin receptor A (ET(A) ) were investigated. Collagen production and migratory activity of HCMFs were further assessed. HCMFs from SSc patients demonstrated increased basal CCN2 production, collagen deposition, and migration rate, in a thrombin-dependent manner. Increased TF expression was also observed in SSc HCMFs. Subsequent activation of the extrinsic coagulation system resulted in thrombin-dependent enhancement of ET(A) expression. ET(A) overexpression led to further increases in both TF expression and fibrotic activity in HCMFs. Moreover, inhibition of ET-1 signaling by bosentan abolished the TF-mediated fibrotic capacity of HCMFs. Tissue factor-thrombin signaling is involved in the increased fibrotic activity of HCMFs from patients with SSc. Moreover, the up-regulation of ET(A) expression by thrombin and the effect of ET-1 in the induction of TF expression indicate an amplification loop for enhanced collagen deposition. Therapeutic interventions targeting the extrinsic coagulation system or ET-1 signaling may provide clinical benefit by breaking this vicious circle.

Thrombopoietic‐mesenchymal interaction that may facilitate both endochondral ossification and platelet maturation via CCN2

CCN2 plays a central role in the development and growth of mesenchymal tissue and promotes the regeneration of bone and cartilage in vivo. Of note, abundant CCN2 is contained in platelets, which is thought to play an important role in the tissue regeneration process. In this study, we initially pursued the possible origin of the CCN2 in platelets. First, we examined if the CCN2 in platelets was produced by megakaryocyte progenitors during differentiation. Unexpectedly, neither megakaryocytic CMK cells nor megakaryocytes that had differentiated from human haemopoietic stem cells in culture showed any detectable CCN2 gene expression or protein production. Together with the fact that no appreciable CCN2 was detected in megakaryocytes in vivo, these results suggest that megakaryocytes themselves do not produce CCN2. Next, we suspected that mesenchymal cells situated around megakaryocytes in the bone marrow were stimulated by the latter to produce CCN2, which was then taken up by platelets. To evaluate this hypothesis, we cultured human chondrocytic HCS-2/8 cells with medium conditioned by differentiating megakaryocyte cultures, and then monitored the production of CCN2 by the cells. As suspected, CCN2 production by HCS-2/8 was significantly enhanced by the conditioned medium. We further confirmed that human platelets were able to absorb/uptake exogenous CCN2 in vitro. These findings indicate that megakaryocytes secrete some unknown soluble factor(s) during differentiation, which factor stimulates the mesenchymal cells to produce CCN2 for uptake by the platelets. We also consider that, during bone growth, such thrombopoietic-mesenchymal interaction may contribute to the hypertrophic chondrocyte-specific accumulation of CCN2 that conducts endochondral ossification.

Susceptibility to liver fibrosis in mice expressing a connective tissue growth factor transgene in hepatocytes # †

Connective tissue growth factor (CCN2) is a matricellular protein that is up-regulated in many fibrotic disorders and coexpressed with transforming growth factor beta. CCN2 promotes fibrogenesis and survival in activated hepatic stellate cells, and injured or fibrotic liver contains up-regulated levels of CCN2 that are produced by a variety of different cell types, including hepatocytes. To investigate CCN2 action in vivo, transgenic FVB mice were created in which the human CCN2 gene was placed under the control of the albumin enhancer promoter to elevate hepatocyte CCN2 levels. Production of human CCN2 (hCCN2) messenger RNA and elevated CCN2 protein levels was demonstrated in transgenic livers, whereas levels of endogenous mouse CCN2 were comparable between transgenic and wild-type mice. Liver histology and liver function tests were unaffected in transgenic animals. However, after chronic administration of CCl(4), alpha-smooth muscle actin (alpha-SMA)-expressing cells and collagen deposition were increased as a function of the dosage of the hCCN2 transgene (hccn2(+/+) > hccn2(+/-) > hccn2(-/-)). Moreover, CCl(4)-induced serum hyaluronic acid, hepatic tissue levels of alpha-SMA or acid-soluble collagen, and messenger RNA expression of alpha-SMA, collagen alpha1 (I), matrix metalloprotease-2, or tissue inhibitor of metalloprotease-1 were greater in transgenic mice than in wild-type mice. Transgenic mice also exhibited enhanced hepatic deposition of collagen 2 weeks after bile duct ligation. Production of elevated CCN2 levels in hepatocytes of transgenic mice in vivo does not cause hepatic injury or fibrosis per se but renders the livers more susceptible to the injurious actions of other fibrotic stimuli. These studies support a central role of CCN2 in hepatic fibrosis and demonstrate a role of the microenvironment in regulating the profibrotic action of CCN2.

CCN3 (NOV) Is a Negative Regulator of CCN2 (CTGF) and a Novel Endogenous Inhibitor of the Fibrotic Pathway in an in Vitro Model of Renal Disease

Fibrosis is a major cause of end-stage renal disease, and although initiation factors have been elucidated, uncertainty concerning the downstream pathways has hampered the development of anti-fibrotic therapies. CCN2 (CTGF) functions downstream of transforming growth factor (TGF)-beta, driving increased extracellular matrix (ECM) accumulation and fibrosis. We examined the possibility that CCN3 (NOV), another CCN family member with reported biological activities that differ from CCN2, might act as an endogenous negative regulator of ECM and fibrosis. We show that cultured rat mesangial cells express CCN3 mRNA and protein, and that TGF-beta treatment reduced CCN3 expression levels while increasing CCN2 and collagen type I activities. Conversely, either the addition of CCN3 or CCN3 overexpression produced a marked down-regulation of CCN2 followed by virtual blockade of both collagen type I transcription and its accumulation. This finding occurred in both growth-arrested and CCN3-transfected cells under normal growth conditions after TGF-beta treatment. These effects were not attributable to altered cellular proliferation as determined by cell cycle analysis, nor were they attributable to interference of Smad signaling as shown by analysis of phosphorylated Smad3 levels. In conclusion, both CCN2 and CCN3 appear to act in a yin/yang manner to regulate ECM metabolism. CCN3, acting downstream of TGF-beta to block CCN2 and the up-regulation of ECM, may therefore serve to naturally limit fibrosis in vivo and provide opportunities for novel, endogenous-based therapeutic treatments.

Strategies for blocking the fibrogenic actions of connective tissue growth factor (CCN2): From pharmacological inhibition in vitro to targeted siRNA therapy in vivo.

Connective tissue growth factor (CCN2) is a major pro-fibrotic factor that frequently acts downstream of transforming growth factor beta (TGF-β)-mediated fibrogenic pathways. Much of our knowledge of CCN2 in fibrosis has come from studies in which its production or activity have been experimentally attenuated. These studies, performed both in vitro and in animal models, have demonstrated the utility of pharmacological inhibitors (e.g. tumor necrosis factor alpha (TNF-α), prostaglandins, peroxisome proliferator-activated receptor-gamma (PPAR-γ) agonists, statins, kinase inhibitors), neutralizing antibodies, antisense oligonucleotides, or small interfering RNA (siRNA) to probe the role of CCN2 in fibrogenic pathways. These investigations have allowed the mechanisms regulating CCN2 production to be more clearly defined, have shown that CCN2 is a rational anti-fibrotic target, and have established a framework for developing effective modalities of therapeutic intervention in vivo.

Regulation of chondrocytic phenotype by micro RNA 18a: Involvement of Ccn2/Ctgf as a major target gene

We searched for miRNAs that were down-regulated in chondrocytic cells and predicted to target CCN2/connective tissue growth factor (CCN2/CTGF) that promotes endochondral ossification. Among them, expression of miR-18a was most strongly repressed in chondrocytic cells. Reporter gene analysis confirmed the functionality of an miR-18a target in the 3′-untranslated region of Ccn2 mRNA, which was predicted in silico. Indeed, introduction of miR-18a efficiently repressed the CCN2 production from chondrocytic cells. Finally, transfected miR-18a significantly repressed the mature chondrocytic phenotype. Our present study revealed a regulatory role for miR-18a in chondrocytic differentiation through CCN2.

Ethanol-mediated expression of connective tissue growth factor (CCN2) in mouse pancreatic stellate cells

Activated pancreatic stellate cells (PSC) play a central role in the pathogenesis of pancreatic fibrosis, a common feature of chronic pancreatitis which is often caused by excessive alcohol consumption. In view of the central role of connective tissue growth factor (CCN2) in fibrosis, we investigated the mechanisms by which CCN2 is regulated in PSC following their exposure to ethanol or acetaldehyde. Primary cultures of PSC from Balb/c mice were treated with 0–50 mM ethanol or 0–200 μM acetaldehyde in the presence or absence of 4-methylpyrazole (4MP; an inhibitor of alcohol dehydrogenase), diallyl sulfide (DAS; an inhibitor of cytochrome P4502E1) or anti-oxidant catalase or vitamin D. CCN2 production, assessed by reverse-transcriptase polymerase chain reaction to measure CCN2 mRNA levels or by fluorescence activated cell sorting to assess CCN2 protein, was enhanced in a dose-dependent manner by ethanol or acetaldehyde. In the presence of 4MP, DAS, or the anti-oxidants vitamin D or catalase, there was a substantial decrease in the ability of ethanol to stimulate CCN2 mRNA expression and a concomitant decrease in CCN2-positive PSC. Accumulation of reactive oxygen species in PSC after exposure to ethanol was verified by loading the cells with dichlorofluorescin diacetate and showing that there was a stimulation of its oxidized fluorescent product, the latter of which was diminished in the presence of catalase or vitamin D. These results show the production of acetaldehyde and oxidant stress in mouse PSC are the cause of increased CCN2 mRNA and protein production after exposure of the cells to ethanol. The potential therapeutic effects of inhibitors of ethanol metabolism or anti-oxidants in alcoholic pancreatitis may arise in part through their ability to attenuate CCN2 production by PSC.