Connective tissue growth factor (CTGF, CCN2) contributes to fibrotic responses in diabetic retinopathy, both before clinical manifestations occur in the pre-clinical stage of diabetic retinopathy (PCDR) and in proliferative diabetic retinopathy (PDR), the late clinical stage of the disease. CTGF is a secreted protein that modulates the actions of many growth factors and extracellular matrix (ECM) proteins, leading to tissue reorganization, such as ECM formation and remodeling, basal lamina (BL) thickening, pericyte apoptosis, angiogenesis, wound healing and fibrosis. In PCDR, CTGF contributes to thickening of the retinal capillary BL and is involved in loss of pericytes. In this stage, CTGF expression is induced by advanced glycation end products, and by growth factors such as vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-β. In PDR, the switch from neovascularization to a fibrotic phase - the angio-fibrotic switch - in PDR is driven by CTGF, in a critical balance with vascular endothelial growth factor (VEGF). We discuss here the roles of CTGF in the pathogenesis of DR in relation to ECM remodeling and wound healing mechanisms, and explore whether CTGF may be a potential novel therapeutic target in the clinical management of early as well as late stages of DR.

In scleroderma (systemic sclerosis, SSc), persistent activation of myofibroblast leads to severe skin and organ fibrosis resistant to therapy. Increased mechanical stiffness in the involved fibrotic tissues is a hallmark clinical feature and a cause of disabling symptoms. Myocardin Related Transcription Factor-A (MRTF-A) is a transcriptional co-activator that is sequestered in the cytoplasm and translocates to the nucleus under mechanical stress or growth factor stimulation. Our objective was to determine if MRTF-A is activated in the disease microenvironment to produce more extracellular matrix in progressive SSc. Immunohistochemistry studies demonstrate that nuclear translocation of MRTF-A in scleroderma tissues occurs in keratinocytes, endothelial cells, infiltrating inflammatory cells, and dermal fibroblasts, consistent with enhanced signaling in multiple cell lineages exposed to the stiff extracellular matrix. Inhibition of MRTF-A nuclear translocation or knockdown of MRTF-A synthesis abolishes the SSc myofibroblast enhanced basal contractility and synthesis of type I collagen and inhibits the matricellular profibrotic protein, connective tissue growth factor (CCN2/CTGF). In MRTF-A null mice, basal skin and lung stiffness was abnormally reduced and associated with altered fibrillar collagen. MRTF-A has a role in SSc fibrosis acting as a central regulator linking mechanical cues to adverse remodeling of the extracellular matrix.

Skin hair follicles (HF) contain bulge stem cells (SC) that regenerate HFs during hair cycles, and repair skin epithelia following injury. As natural aging is associated with decreased skin repair capacity in humans, we have investigated the impact of age on human scalp HF bulge cell number and function. Here, we isolated human bulge cells, characterized as CD200+/KRT15+/KRT19+ cells of the HF, by dissection-combined CD200 selection in young and aged human skin. Targeted transcriptional profiling indicates that KRT15, KRT19, Dkk3, Dkk4, Tcf3, S100A4, Gas1, EGFR and CTGF/CCN2 are also preferentially expressed by human bulge cells, compared to differentiated HF keratinocytes (KC). Our results demonstrate that aging does not alter expression or localization of these HF SC markers. In addition, we could not detect significant differences in HF density or bulge cell number between young and aged human scalp skin. Interestingly, hedgehog (Hh) signaling is activated in human bulge cells in vivo, and down-regulated in differentiated HF KCs, both in young and aged skin. In addition, activation of Hh signaling by lentivirus-mediated overexpression of transcription factor Gli1 induces transcription of HF SC markers KRT15, KRT19, and Gas1, in cultured KCs. Together with previously reported knock-out mouse results, these data suggest a role for Hh signaling in maintaining bulge cell phenotype in young and aged human skin.

Connective tissue growth factor (CTGF or CCN2), which belongs to the CCN family, is a secreted protein. It has been implicated in various biological processes, such as cell proliferation, migration, angiogenesis, and tumorigenesis. In this study, we found that CTGF expression level was elevated in primary papillary thyroid carcinoma (PTC) samples and correlated with clinical features, such as metastasis, tumor size, and clinical stage. Overexpression of CTGF in PTC cells accelerated their growth in liquid culture and soft agar as well as protecting PTC cells from apoptosis induced by IFN-gamma treatment. Downregulation of CTGF in PTC cells inhibits cell growth in liquid culture and soft agar and induces the activation of caspase pathway and sensitized PTC cells to apoptosis. Our data suggest that CTGF plays an important role in PTC progression by supporting tumor cell survival and drug resistance, and CTGF may be used as a potential tumor marker for PTC diagnosis.

Connective tissue growth factor (CTGF, CCN2) is overexpressed in pancreatic cancer. We mapped the minimal CCN2 promoter active in PANC-1 cells, a human pancreatic cancer cell line. Within this region, Sp1, BCE-1 and Ets elements were important for the activity of the CCN2 promoter. Constitutive hyperactivated ras is a hallmark of cancers, including that of the pancreas. Treatment of PANC-1 cells with the MEK inhibitor U0126 or the Sp1 inhibitor mithramycin reduced CCN2 mRNA and promoter activity. Mutation of the BCE-1, but not Sp1 or Ets, site abolished the responsiveness of the CCN2 promoter to U0126. Overexpressing constitutively active MEK1 or ras activated CCN2 promoter activity. Thus CCN2 is likely to act downstream of ras in PANC-1 cells. CCN2 is overexpressed in cancer cells. Activated ras/MEK/ERK is a hallmark of cancer, and we have shown that the elevated CCN2 expression in pancreatic cancer cells is dependent on this pathway.

The origin of fibrotic cells within connective tissue is unclear. For example, the extent to which microvascular pericytes contribute to the number of myofibroblasts present in dermal fibrosis in uncertain. Connective tissue growth factor (CTGF/CCN2) is a marker and mediator of fibrosis. In this report, we use an antibody recognizing CCN2 to assess the cell types in mouse dermis which express CCN2 in the bleomycin model of skin scleroderma. Control (PBS injected) and fibrotic (bleomycin-injected) dermis was examined for CCN2, alpha-smooth muscle actin (alpha-SMA) (to detect myofibroblasts), and NG2 (to detect pericytes) expression. Consistent with previously published data, CCN2 expression was largely absent in the dermis of control mice. However, upon exposure to bleomycin, CCN2 was observed in the dermis. Cells that expressed CCN2 were alpha-SMA-expressing myofibroblasts. Approximately 85% of myofibroblasts were NG2-positive, CCN2-expressing pericytes, indicating that pericytes significantly contributed to the presence of myofibroblasts in sclerotic dermis. Thus CCN2 is induced in fibrotic skin, correlating with the induction of myofibroblast induction. Moreover, CCN2-expressing pericytes significantly contribute to the appearance of myofibroblasts in bleomycin-induced skin scleroderma.

Connective tissue growth factor (CTGF/CCN2) is involved in extracellular matrix production, tumor cell proliferation, adhesion, migration, and metastasis. Recent studies have shown that CTGF expression is elevated in precursor B-acute lymphoblastic leukemia (ALL) and that increased expression of CTGF is associated with inferior outcome in B-ALL. In this study, we characterized the functional role and downstream signaling pathways of CTGF in ALL cells. First, we utilized lentiviral shRNA to knockdown CTGF in RS4;11 and REH ALL cells expressing high levels of CTGF mRNA. Silencing of CTGF resulted in significant suppression of leukemia cell growth compared to control vector, which was associated with AKT/mTOR inactivation and increased levels of cyclin-dependent kinase inhibitor p27. CTGF knockdown sensitized ALL cells to vincristine and methotrexate. Treatment with an anti-CTGF monoclonal antibody, FG-3019, significantly prolonged survival of mice injected with primary xenograft B-ALL cells when co-treated with conventional chemotherapy (vincristine, L-asparaginase and dexamethasone). Data suggest that CTGF represents a targetable molecular aberration in B-ALL, and blocking CTGF signaling in conjunction with administration of chemotherapy may represent a novel therapeutic approach for ALL patients.

CCN2/connective tissue growth factor (CTGF) can be induced by hypoxia and promotes tumor angiogenesis. Our previous studies revealed that hypoxia-induced gene expression of human ccn2 mRNA is regulated post-transcriptionally in human chondrosarcoma-derived cell line, HCS-2/8, in which a minimal cis-element, entitled CAESAR, in the 3'-untranslated region (UTR) of ccn2 mRNA and a 35-kDa protein counterpart play an important role by determining the stability of ccn2 mRNA. In the present study, we identified this corresponding protein as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by utilizing RNA affinity chromatography combined with mass spectrometry. The results of an RNA binding assay revealed the specific binding of GAPDH to this cis-element. To further characterize the interaction between GAPDH and ccn2 mRNA, we examined the roles of redox conditions and glycolytic coenzyme in the binding of GAPDH to the ccn2 mRNA. An oxidizing agent, diamide, abolished the GAPDH-RNA interaction in a concentration-dependent manner; whereas this effect could be reversed by subsequent treatment with 2-mercaptoethanol (2-ME). In addition, nicotinamide-adenine dinucleotide (NAD), a coenzyme of GAPDH, inhibited the GAPDH-RNA binding. Taken together, these findings suggest that the glycolytic enzyme GAPDH regulates the gene expression of ccn2 mRNA in trans by acting as a sensor of oxidative stress and redox signals, leading to CCN2 overexpression under the condition of hypoxia and promotion of angiogenesis.

OBJECTIVE

The investigation of the expression and localization of connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24/CCN family member 2 (CTGF/Hcs24/CCN2) in normal and osteoarthritic (OA) cartilage, and quantification of CTGF/Hcs24-positive cells.

METHODS

Cartilage samples of patients (n=20) with late stage OA were obtained at total joint replacement surgery. Morphologically normal cartilage was harvested for comparison purposes from the femoral heads of 6 other patients with femoral neck fracture. Paraffin-embedded sections were stained by Safranin O. The severity of the OA lesions was divided into four stages (normal, early, moderate, and severe). The localization of protein and mRNA for CTGF/Hcs24 was investigated by immunohistochemistry and in situ hybridization, respectively. The population of CTGF/Hcs24-positive chondrocytes in OA cartilage and chondro-osteophyte was quantified by counting the number of the cells under light microscopy.

RESULTS

Signals for CTGF/Hcs24 were detected in a small percentage of chondrocytes throughout the layers of normal cartilage. In early stage OA cartilage, the CTGF/Hcs24-positive chondrocytes were localized mainly in the superficial layer. In moderate to severe OA cartilage, intense staining for CTGF/Hcs24 was observed in proliferating chondrocytes forming cell clusters next to the cartilage surface. In chondro-osteophyte, strong signals were found in the chondrocytes of the proliferative and hypertrophic zones.

CONCLUSIONS

CTGF/Hcs24 expression was detected in both normal and OA chondrocytes of human samples. The results of the current study suggested that expression of CTGF/Hcs24 was concomitant with development of OA lesions and chondrocyte differentiation in chondro-osteophyte.

OBJECTIVE

Myocardial CCN2/CTGF is induced in heart failure of various etiologies. However, its role in the pathophysiology of left ventricular (LV) remodeling after myocardial infarction (MI) remains unresolved. The current study explores the role of CTGF in infarct healing and LV remodeling in an animal model and in patients admitted for acute ST-elevation MI.

RESULTS

Transgenic mice with cardiac-restricted overexpression of CTGF (Tg-CTGF) and non-transgenic littermate controls (NLC) were subjected to permanent ligation of the left anterior descending coronary artery. Despite similar infarct size (area of infarction relative to area at risk) 24 hours after ligation of the coronary artery in Tg-CTGF and NLC mice, Tg-CTGF mice disclosed smaller area of scar tissue, smaller increase of cardiac hypertrophy, and less LV dilatation and deterioration of LV function 4 weeks after MI. Tg-CTGF mice also revealed substantially reduced mortality after MI. Remote/peri-infarct tissue of Tg-CTGF mice contained reduced numbers of leucocytes, macrophages, and cells undergoing apoptosis as compared with NLC mice. In a cohort of patients with acute ST-elevation MI (n = 42) admitted to hospital for percutaneous coronary intervention (PCI) serum-CTGF levels (s-CTGF) were monitored and related to infarct size and LV function assessed by cardiac MRI. Increase in s-CTGF levels after MI was associated with reduced infarct size and improved LV ejection fraction one year after MI, as well as attenuated levels of CRP and GDF-15.

CONCLUSIONS

Increased myocardial CTGF activities after MI are associated with attenuation of LV remodeling and improved LV function mediated by attenuation of inflammatory responses and inhibition of apoptosis.

Low-density lipoprotein receptor-related protein 1 (LRP1) is known to be a receptor for signal transmission and endocytosis. We have previously reported that LRP1 regulates WNT-β-catenin and protein kinase C signaling in chondrocytes, represses the hypertrophy of chondrocytes during endochondral ossification and that LRP1 is colocalized with a ligand, CCN family member 2 (CCN2; also known as connective tissue growth factor, CTGF), which conducts endochondral ossification, in chondrocytes. However, the role of LRP1 in the endocytic transport of CCN2 in chondrocytes is not yet understood. In the present study, we investigated the interaction between LRP1 and CCN2 during endocytic trafficking. Small interfering RNA (siRNA)-mediated knockdown of LRP1 in chondrocytic HCS-2/8 cells showed that the amount of exogenous CCN2 binding and/or incorporation was decreased in the LRP1 downregulated cells. Importantly, we observed that CCN2 internalization in chondrocytes was dependent on clathrin, and internalizated CCN2 was colocalized with an early or recycling endosome marker. Transcytosis of CCN2 through HCS-2/8 cells was confirmed by performing experiments with a trans-well apparatus, and the amount of transcytosed CCN2 was decreased by an LRP1 antagonist. These findings rule out possible leakage and confirm the crucial involvement of LRP1 during experimental transcytosis. Moreover, under hypoxic conditions that mimic the cartilaginous microenvironment, the level of LRP1 and the amount of transcytosed CCN2 increased, and these increases were neutralized by treatment with the LRP1 antagonist. The distribution of LRP1 and its antagonist in the growth plate in vivo was consistent with that of CCN2 in this tissue, which is produced by and transported by LRP1 from the chondrocytes in the prehypertrophic layer. These findings suggest that LRP1 mediates the transcytosis of CCN2, which might be a crucial event that determines the distribution of CCN2 in cartilage.

Diabetic cardiomyopathy is characterized by interstitial fibrosis and cardiomyocyte hypertrophy and apoptosis. Also known as CCN2, connective tissue growth factor (CTGF) is implicated in the fibrosis; however, whether it contributes to cardiomyocytes changes and adverse effects of high glucose and lipids on these cells remains unknown. Hearts from streptozotocin-induced diabetic rats had elevated CTGF and changes of pathological myocardial hypertrophy, fibrosis, and cardiomyocyte apoptosis. Rat H9c2 cardiomyocytes were then treated with recombinant human (rh)CTGF, high glucose, or the saturated free fatty acid palmitate. Each reagent induced cell hypertrophy, as indicated by the ratio of total protein to cell number, cell size, and gene expression of cardiac hypertrophy marker genes atrial natriuretic peptide (ANP), and alpha-skeletal actin. Each treatment also caused apoptosis measured by increased caspase3/7 activity, apoptotic cells by transferase-mediated dUTP nick end labeling (TUNEL) assay, and lower viable cell number. Further studies showed CTGF mRNA was rapidly induced by high glucose and palmitate in H9c2 cells and in mouse neonatal cardiomyocyte primary cultures. small interfering RNA against CTGF blocked the high glucose and palmitate induction of hypertrophy and apoptosis. In addition, these CTGF effects were through the tyrosine kinase A (TrkA) receptor with tyrosine kinase activity, which has previously been implicated in CTGF signaling: TrkA was phosphorylated by CTGF, and a specific TrkA blocker abrogated CTGF-induced effects on hypertrophy and apoptosis. For the first time in any system, fatty acid is newly identified as a regulator of CTGF, and this work implicates autocrine CTGF as a mediator of adverse effects of high glucose and fatty acids in cardiomyocytes.

Connective tissue growth factor (CTGF)/CCN family member 2 (CCN2) is a CCN family member of matricellular signaling modulators. It has been shown that CCN2/CTGF mediates cell adhesion, aggregation and migration in a large variety of cell types, including vascular endothelial cells, fibroblasts, epithelial cells, aortic smooth muscle and also pluripotent stem cells. Others matricellular proteins are capable of interacting with CCN2/CTGF to mediate its function. Cell migration is a key feature for tumor cell invasion and metastasis. CCN2/CTGF seems to be a prognostic marker for cancer. In addition, here we intend to discuss recent discoveries and a new strategy to develop therapies against CCN2/CTGF, in order to treat cancer metastasis.

Hippo pathway controls the organ size by modulating cell proliferation and apoptosis. However, the upstream regulation of hippo signaling by actin cytoskeleton is not clear. To elucidate the role of actin as an upstream regulator of Hippo signaling, the levels of F (filamentous)-actin in cells were elevated using jasplakinolide, an actin-stabilizing drug. Induction of F-actin formation in HeLa cells resulted in decreased phosphorylation of YAP, a key effector molecule for Hippo signaling. The activated YAP is localized to the cell nucleus and YAP increase was associated with increased expression of downstream CCN growth factors CCN1/CYR61 and CCN2/CTGF. The effect of the actin-stabilizing drug was blocked when YAP levels were suppressed in YAP "knock-down" cells. In summary, using an actin-stabilizing drug we show that actin cytoskeleton is one of the upstream regulators of Hippo signaling capable of activating YAP and increasing its downstream CCN growth factors.

The von Hippel-Lindau (VHL) protein serves as a negative regulator of hypoxia-inducible factor (HIF)-alpha subunits. Since HIF regulates critical angiogenic factors such as vascular endothelial growth factor (VEGF) and lesions in VHL gene are present in a majority of the highly vascularized renal cell carcinoma (RCC), it is believed that deregulation of the VHL-HIF pathway is crucial for the proangiogenic activity of RCC. Although VEGF has been confirmed as a critical angiogenic factor upregulated in VHL-mutant cells, the efficacy of antiangiogenic therapy specifically targeting VEGF signaling remains modest. In this study, we developed a three-dimensional in vitro assay to evaluate the ability of RCC cells to promote cord formation by the primary human dermal microvascular endothelial cells (HDMECs). Compared with VHL wild-type cells, VHL-mutant RCC cells demonstrated a significantly increased proangiogenic activity, which correlated with increased secretion of cysteine-rich 61 (Cyr61)/cysteine-rich 61-connective tissue growth factor-nephroblastoma overexpressed (CCN) 1, connective tissue growth factor (CTGF)/CCN2 and VEGF in conditioned culture medium. Both CCN proteins are required for HDMEC cord formation as shown by RNA interference knockdown experiments. Importantly, the proangiogenic activities conferred by the CCN proteins and VEGF are additive, suggesting non-overlapping functions. Expression of the CCN proteins is at least partly dependent on the HIF-2alpha function, the dominant HIF-alpha isoform expressed in RCC. Finally, immunohistochemical staining of Cyr61/CCN1 and CTGF/CCN2 in RCC tissue samples showed that increased expression of these proteins correlates with the loss of VHL protein expression. These findings strengthened the notion that the hypervascularized phenotype of RCC is afforded by multiple proangiogenic factors that function in parallel pathways.

Recent studies have revealed an unexpected synergism between two seemingly unrelated protein families: CCN matricellular proteins and the tumor necrosis factor (TNF) family of cytokines. CCN proteins are dynamically expressed at sites of injury repair and inflammation, where TNF cytokines are also expressed. Although TNFalpha is an apoptotic inducer in some cancer cells, it activates NFkappaB to promote survival and proliferation in normal cells, and its cytotoxicity requires inhibition of de novo protein synthesis or NFkappaB signaling. The presence of CCN1, CCN2, or CCN3 overrides this requirement and unmasks the apoptotic potential of TNFalpha, thus converting TNFalpha from a proliferation-promoting protein into an apoptotic inducer. These CCN proteins also enhance the cytotoxicity of other TNF cytokines, including LTalpha, FasL, and TRAIL. Mechanistically, CCNs function through integrin alpha(6)beta(1) and the heparan sulfate proteoglycan (HSPG) syndecan-4 to induce reactive oxygen species (ROS) accumulation, which is essential for apoptotic synergism. Mutant CCN1 proteins defective for binding alpha(6)beta(1)-HSPGs are unable to induce ROS or apoptotic synergism with TNF cytokines. Further, knockin mice that express an alpha(6)beta(1)-HSPG-binding defective CCN1 are blunted in TNFalpha- and Fas-mediated apoptosis, indicating that CCN1 is a physiologic regulator of these processes. These findings implicate CCN proteins as contextual regulators of the inflammatory response by dictating or enhancing the cytotoxicity of TNFalpha and related cytokines.

Activation of fibroblasts and their differentiation into myofibroblasts, excessive collagen production and fibrosis occurs in a number of bladder diseases. Similarly, conversion of epithelial cells into mesenchymal cells (EMT) has been shown to increase fibroblasts like cells. TGF-β1 can induce the EMT and the role of TGF-β1-induced EMT during bladder injury leading to fibrosis and possible organ failure is gaining increasing interest. Here we show that EMT and fibrosis in porcine bladder urothelial (UC) cells are Smad dependent. Fresh normal porcine bladder urothelial cells were grown in culture with or without TGF-β1 and EMT markers were assessed. TGF-β1 treatment induced changes in cellular morphology as depicted by a significant decrease in the expression of E-cadherin and corresponding increase in N-cadherin and α-SMA. We knocked down Smad2 and Smad3 by Smad specific siRNA. Downregulation of E-cadherin expression by TGF-β1 was Smad3-dependent, whereas N-cadherin and α-SMA were dependent on both Smad2 and Smad3. Connective tissue growth factor (CTGF/CCN2), matrix metalloproteinase-2 and -9 (MMP-2, MMP-9) has been shown to play important roles in the pathogenesis of fibrosis. Induction of these genes by TGF-β1 was found to be time dependent. Upregulation of CTGF/CCN2 by TGF-β1 was Smad3 dependent; whereas MMP-2 was Smad2 dependent. Smad2 and Smad3 both participated in MMP-9 expression. TGF-β1 reprogrammed mesenchymal fibroblast like cells robustly expressed collagen I and III and these was inhibited by SB-431542, a TGF-β receptor inhibitor. Our results indicate that EMT of porcine bladder UC cells is TGF-β1 dependent and is mediated through Smad2 and Smad3. TGF-β1 may be an important factor in the development of bladder fibrosis via an EMT mechanism. This identifies a potential amenable therapeutic target.

Gradual occlusion of coronary arteries may result in reversible loss of cardiomyocyte function (hibernating myocardium), which is amenable to therapeutic neovascularization. The role of myocardin-related transcription factors (MRTFs) co-activating serum response factor (SRF) in this process is largely unknown. Here we show that forced MRTF-A expression induces CCN1 and CCN2 to promote capillary proliferation and pericyte recruitment, respectively. We demonstrate that, upon G-actin binding, thymosin ß4 (Tß4), induces MRTF translocation to the nucleus, SRF-activation and CCN1/2 transcription. In a murine ischaemic hindlimb model, MRTF-A or Tß4 promotes neovascularization, whereas loss of MRTF-A/B or CCN1-function abrogates the Tß4 effect. We further show that, in ischaemic rabbit hindlimbs, MRTF-A as well as Tß4 induce functional neovascularization, and that this process is inhibited by angiopoietin-2, which antagonizes pericyte recruitment. Moreover, MRTF-A improves contractile function of chronic hibernating myocardium of pigs to a level comparable to that of transgenic pigs overexpressing Tß4 (Tß4tg). We conclude that MRTF-A promotes microvessel growth (via CCN1) and maturation (via CCN2), thereby enabling functional improvement of ischaemic muscle tissue.

OBJECTIVE

Connective tissue growth factor (CTGF) has been implicated in regulation of chondrocyte differentiation at multiple steps and has been implicated in the progression of diseases such as scleroderma and osteoarthritis. However, the pathways mediating the expression of CTGF/CCN2 and related factors in cartilage are not fully understood. We have previously shown that the Rho family of proteins and the actin cytoskeleton regulate both early and late chondrocyte differentiation.

RESULTS

Here we demonstrate that several CTGF/Cyr61/Nov (CCN) family members are differentially affected by either inhibition of actin polymerization (cytochalasin D treatment), promotion of actin polymerization (jasplakinolide treatment), inhibition of RhoA/rho kinase (ROCK) signaling (Y27632 treatment) and Rac1 signaling. We also show that the Smad site in the CTGF/CCN2 promoter is responsive to both Rac1 inhibition and cytochalasin D treatment, suggesting a role of TGFbeta/Smad signaling in mediating the effects of actin dynamics and Rac1.

CONCLUSIONS

Collectively, these data show that Rac1 and actin pathways control CTGF/CCN2 expression in chondrocytes which might be relevant to both skeletal development and associated diseases such as osteoarthritis.

Cardiomyopathy caused by lamin A/C gene mutations (LMNA cardiomyopathy) is characterized by increased myocardial fibrosis, which impairs left ventricular relaxation and predisposes to heart failure, and cardiac conduction abnormalities. While we previously discovered abnormally elevated extracellular signal-regulated kinase 1/2 (ERK1/2) activities in heart in LMNA cardiomyopathy, its role on the development of myocardial fibrosis remains unclear. We now showed that transforming growth factor (TGF)-β/Smad signaling participates in the activation of ERK1/2 signaling in LMNA cardiomyopathy. ERK1/2 acts on connective tissue growth factor (CTGF/CCN2) expression to mediate the myocardial fibrosis and left ventricular dysfunction. Studies in vivo demonstrate that inhibiting CTGF/CCN2 using a specific antibody decreases myocardial fibrosis and improves the left ventricular dysfunction. Together, these findings show that cardiac ERK1/2 activity is modulated in part by TGF-β/Smad signaling, leading to altered activation of CTGF/CCN2 to mediate fibrosis and alter cardiac function. This identifies a novel mechanism in the development of LMNA cardiomyopathy.

Hippo signaling pathway consists of conserved serine/threonine kinases to maintain optimal organ sizes. Studies have demonstrated that fragmentation of murine ovaries increases actin polymerization and disrupts Hippo signaling, leading to nuclear translocation of Hippo signaling effector Yes-associated protein (YAP) in ovarian follicles and follicle growth. For patients with polycystic ovarian syndrome showing follicle arrest, ovarian wedge resection and laser drilling promote follicle growth. Because these damaging procedures likely involve actin polymerization, we tested whether actin polymerization-promoting drugs could promote YAP translocation and stimulate follicle growth. Treatment of murine ovaries with μM Jasplakinolide (JASP), an actin polymerization-promoting cyclic peptide, or sphingosine-1-phosphate (S1P), a follicular fluid constituent known to promote actin polymerization, increased the conversion of globular actin to the filamentous form, followed by increased nuclear YAP and expression of downstream connective tissue growth factor (CCN2). After short-term treatments with JASP or S1P, in vitro cultured and in vivo grafted ovaries showed follicle growth. Furthermore, induction of constitutively active YAP in ovarian grafts of transgenic mice enhanced follicle development, whereas treatment of human ovarian cortices with JASP or S1P increased CCN2 expression. Thus, JASP and S1P stimulate follicle growth and are potential therapeutic agents for treating polycystic ovarian syndrome and other ovarian disorders.

The structural integrity of human skin is largely dependent on the quality of the dermal extracellular matrix (ECM), which is produced, organized, and maintained by dermal fibroblasts. Normally, fibroblasts attach to the ECM and thereby achieve stretched, elongated morphology. A prominent characteristic of dermal fibroblasts in aged skin is reduced size, with decreased elongation and a more rounded, collapsed morphology. Here, we show that reduced size of fibroblasts in mechanically unrestrained three-dimensional collagen lattices coincides with reduced mechanical force, measured by atomic force microscopy. Reduced size/mechanical force specifically down-regulates TGF-β type II receptor (TβRII) and thus impairs TGF-β/Smad signaling pathway. Both TβRII mRNA and protein were decreased, resulting in 90% loss of TGF-β binding to fibroblasts. Down-regulation of TβRII was associated with significantly decreased phosphorylation, DNA-binding, and transcriptional activity of its key downstream effector Smad3 and reduced expression of Smad3-regulated essential ECM components type I collagen, fibronectin, and connective tissue growth factor (CTGF/CCN2). Restoration of TβRII significantly increased TGF-β induction of Smad3 phosphorylation and stimulated expression of ECM components. Reduced expression of TβRII and ECM components in response to reduced fibroblast size/mechanical force was fully reversed by restoring size/mechanical force. Reduced fibroblast size was associated with reduced expression of TβRII and diminished ECM production, in aged human skin. Taken together, these data reveal a novel mechanism that provides a molecular basis for loss of dermal ECM, with concomitant increased fragility, which is a prominent feature of human skin aging.

CCN2 plays a critical role in the development of mesenchymal tissues such as cartilage and bone, and the binding of CCN2 to various cytokines and receptors regulates their signaling.By screening a protein array, we found that CCN2 could bind to fibroblast growth factor receptors (FGFRs) 2 and 3, with a higher affinity toward FGFR2.We ascertained that FGFR2 bound to CCN2 and that the binding of FGFR2 to FGF2 and FGF4 was enhanced by CCN2.CCN2 and FGF2 had a collaborative effect on the phosphorylation of ERK and the differentiation of osteoblastic cells.The present results indicate the biological significance of the binding of CCN2 to FGFR2 in bone metabolism.

Connective tissue growth factor (CTGF/CCN2) has long been associated with human cancers. The role it plays in these neoplasms is diverse and tumour specific. Recurring patterns in clinical outcome, histological desmoplasia and mechanisms of action have been found. When CTGF is overexpressed compared to low-expressing normal tissue or is underexpressed compared to high-expressing normal tissue, the functional outcome favours tumour survival and disease progression. CTGF acts by altering proliferation, drug resistance, angiogenesis, adhesion and migration contributing to metastasis. The pattern of CTGF expression and tumour response helps to clarify the role of this matricellular protein across a multitude of human cancers.