Regulation and function of connective tissue growth factor/CCN2 in tissue repair, scarring and fibrosis

Connective tissue growth factor (CTGF, CCN2) is a secreted protein with major roles in angiogenesis, chondrogenesis, osteogenesis, tissue repair, cancer and fibrosis. It is a member of the CCN family of immediate-early gene products which are characterised by four discrete protein modules in which reside growth factor binding domains, functional motifs for integrin recognition, heparin and proteoglycan binding, and dimerization motifs. A primary function of CTGF is to modulate and coordinate signaling responses involving cell surface proteoglycans, key components of the extracellular matrix, and growth factors. Integration of these molecular cues regulates growth factor and receptor interactions, cell motility and mesenchymal cell activation and differentiation in tissue remodelling. Abnormal amplification of CTGF dependent signals results in a failure to terminate tissue repair, leading pathological scarring in conditions such as fibrosis and cancer.     

Overexpression of hepatocyte growth factor receptor in renal carcinoma cells indirectly stimulates tumor growth in vivo

We examined the role of increased expression of HGFR kinase in in vivo growth of renal carcinoma. Human renal carcinoma cell line, ACHN cells, was transfected with plasmid encoding wild-type HGFR gene to generate cell lines with increased HGFR protein. ACHN cells with elevated HGFR expression, denoted clones 8 and 10, respectively, showed higher basal kinase activities of HGFR and PI3-kinase than those of empty-vector (mock)-transfected cells. Clone 8 and 10 cells grew similar to mock cells in culture. In mice, tumors of these clones grew more rapidly than those of mock cells. Microvessel density of clone 8 or 10 tumors was higher than that of mock tumors. Clone 8 and 10 cells secreted vascular endothelial growth factor-A (VEGF-A) more than mock cells and the secretion was PI3-kinase inhibitor, LY294002-sensitive. Anti-VEGF-A neutralizing antibody significantly inhibited tumor growth of clones 8 and 10 in mice. These results indicate for the first time that overexpression of HGFR tyrosine kinase in renal carcinoma cells participates in rapid tumor growth in vivo.     

Transactivation of epidermal growth factor receptor by insulin-like growth factor 1 requires basal hydrogen peroxide

Insulin-like growth factor (IGF-1) plays an important role in prostate cancer development. Recent studies suggest that IGF-1 has mitogenic action through epidermal growth factor receptor (EGFR). However, the mechanism remains largely unknown. Here, we demonstrated in prostate cancer DU145 cells that IGF-1 induced EGFR transactivation, leading to ERK activation. Matrix metalloproteinase-mediated shedding of heparin-binding EGF is involved in this process. Antioxidants and catalase inhibited IGF-1-stimulated EGFR phosphorylation, indicating that H(2)O(2) is required for EGFR activation. However, exogenous H(2)O(2) did not activate EGFR or IGF-1R in DU145 cells. IGF-1 did not induced production of H(2)O(2) in DU145 cells. Our results suggest that transactivation of EGFR by IGF-1 requires basal intracellular H(2)O(2) in DU145 cells.     

Insights into the structure/function of hepatocyte growth factor/scatter factor from studies with individual domains

Hepatocyte growth factor/scatter factor (HGF/SF), the ligand for the receptor tyrosine kinase encoded by the c-Met proto-oncogene, is a multidomain protein structurally related to the pro-enzyme plasminogen and with major roles in development, tissue regeneration and cancer. We have expressed the N-terminal (N) domain, the four kringle domains (K1 to K4) and the serine proteinase homology domain (SP) of HGF/SF individually in yeast or mammalian cells and studied their ability to: (i) bind the Met receptor as well as heparan sulphate and dermatan sulphate co-receptors, (ii) activate Met in target cells and, (iii) map their binding sites onto the beta-propeller domain of Met. The N, K1 and SP domains bound Met directly with comparable affinities (K(d)=2.4, 3.3 and 1.4 microM). The same domains also bound heparin with decreasing affinities (N>K1>SP) but only the N domain bound dermatan sulphate. Three kringle domains (K1, K2 and K4) displayed agonistic activity on target cells. In contrast, the N and SP domains, although capable of Met binding, displayed no or little activity. Further, cross-linking experiments demonstrated that both the N domain and kringles 1-2 bind the beta-chain moiety (amino acid residues 308-514) of the Met beta-propeller. In summary, the K1, K2 and K4 domains of HGF/SF are sufficient for Met activation, whereas the N and SP domains are not, although the latter domains contribute additional binding sites necessary for receptor activation by full length HGF/SF. The results provide new insights into the structure/function of HGF/SF and a basis for engineering the N and K1 domains as receptor antagonists for cancer therapy.     

Inhibition of hepatocyte growth factor induction in human dermal fibroblasts by interleukin-1 and its prevention by interferon-gamma

Hepatocyte growth factor (HGF) is one of the vital factors for liver regeneration. HGF production is induced by the activation of protein kinase A and protein kinase C-mediated pathways, interleukin (IL)-1, tumor necrosis factor (TNF)-alpha, and epidermal growth factor (EGF) in mesenchymal cells. We here report that IL-1 and TNF-alpha, hitherto regarded as HGF inducers, potently inhibited HGF production stimulated by other HGF inducers. IL-1alpha, IL-1beta, and TNF-alpha alone had minimal stimulating effects on HGF production in human dermal fibroblasts, but they strongly inhibited production of HGF induced by cholera toxin, 8-bromo-cAMP, EGF, and phorbol 12-myristate 13-acetate (PMA). Moreover, although the high level of HGF production in MRC-5 cells was enhanced by PMA and less markedly by IL-1beta, HGF production in MRC-5 cells treated with PMA plus IL-1beta was less than that in the cells treated with PMA alone. In the presence of interferon (IFN)-gamma, however, cholera toxin- and 8-bromo-cAMP-induced HGF production was not inhibited by IL-1beta. Pretreatment of cells with IL-1beta suppressed the phosphorylation of cAMP-responsive element-binding protein induced by cholera toxin but not that induced by 8-bromo-cAMP. Taken together, our results indicate that IL-1 inhibited HGF production stimulated by various inducers, including protein kinase A-activating agents, and that IFN-gamma overcame this inhibition of induction of HGF production.     

Synergistic induction of hepatocyte growth factor in human skin fibroblasts by the inflammatory cytokines interleukin-1 and interferon-gamma

Hepatocyte growth factor (HGF) is one of the vital factors for wound healing. HGF expression markedly increases in wounded skin and is mainly localized in dermal fibroblasts. HGF expression level in human dermal fibroblasts in vitro, however, is low and thus may be stimulated by some factors in the process of wound healing. Candidates of the factors are inflammatory cytokines released by polymorphonuclear and mononuclear cells infiltrating the wounded area, but HGF production in human dermal fibroblasts is only slightly induced by interleukin (IL)-1, tumor necrosis factor (TNF)-alpha or interferon (IFN)-gamma. We here report that a combination of IL-1beta and IFN-gamma or a combination of TNF-alpha and IFN-gamma very markedly induced HGF production. The synergistic effect of the former was more marked than that of the latter. Synergistic effects of IL-1beta and IFN-gamma were observed at more than 10 pg/ml and 10 IU/ml, respectively, and were detectable as early as 12 h after addition. Neither IFN-alpha nor IFN-beta was able to replace IFN-gamma. HGF mRNA expression was also synergistically upregulated by IL-1beta and IFN-gamma. IL-1beta plus IFN-gamma-induced synergistic production of HGF was potently inhibited by treatment of cells with the extracellular signal-regulated kinase (ERK) kinase inhibitor PD98059 and the p38 inhibitor SB203580 but not by the c-Jun N-terminal kinase (JNK) inhibitor SP600125. Taken together, our results indicate that a combination of IL-1beta and IFN-gamma synergistically induced HGF production in human dermal fibroblasts and suggest that activation of ERK and p38 but not of JNK is involved in the synergistic effect.     

Noncompetitive inhibition of hepatocyte growth factor-dependent Met signaling by a phage-derived peptide

Dysregulation of hepatocyte growth factor (HGF)-induced signaling via its receptor tyrosine kinase Met results in tumor progression and metastasis. To initiate signaling, pro-HGF must be proteolytically activated to reveal a secondary Met binding site within the serine protease-like β-chain of HGF. Although HGF/Met is a large complex, we sought to discover relatively small antagonists that might interfere with this critical Met binding region. Pools of disulfide-constrained random peptide libraries displayed on phage were selected for binding to HGF, ultimately resulting in a disulfide-constrained 15-mer peptide (VNWVCFRDVGCDWVL) termed HB10, which bound to the recombinant human HGF β-chain (HGF β) and competitively inhibited binding to Met with an IC₅₀ of 450 nM. In MDA-MB435 cells, HB10 reduced HGF-dependent Met phosphorylation by 70%, and phosphorylation of downstream kinases AKT and ERK1/ERK2 by 74% and 69%, respectively. Addition of HB10 also inhibited HGF-dependent migration of these cells with an IC₅₀ of ∼ 20 μM. The 2D ¹H-NMR structure of HB10 revealed a β-hairpin loop stabilized by the disulfide bond and cross-strand pairing of Trp3 and Trp13. HGF β mutants deficient in Met binding also have reduced HB10 binding, suggesting an overlapping binding site. Notably HB10 did not inhibit full length HGF binding to Met. Thus steric hindrance of the interaction between HGF β domain binding to Met is sufficient for inhibiting full-length HGF-dependent Met signaling and cell migration that is consistent with a noncompetitive inhibitory mechanism of Met signal transduction.     

CT domain of CCN2/CTGF directly interacts with fibronectin and enhances cell adhesion of chondrocytes through integrin alpha5beta1

Searching for CCN family protein 2/connective tissue growth factor (CCN2/CTGF) interactive proteins by yeast-two-hybrid screening, we identified fibronectin 1 gene product as a major binding partner of CCN2/CTGF in the chondrosarcoma-derived chondrocytic cell line HCS-2/8. Only the CT domain of CCN2/CTGF bound directly to fibronectin (FN). CCN2/CTGF and its CT domain enhanced the adhesion of HCS-2/8 cells to FN in a dose-dependent manner. The CCN2/CTGF-enhancing effect on cell adhesion to FN was abolished by a blocking antibody against alpha5beta1 integrin (alpha5beta1), but not by one against anti-alphavbeta3 integrin. These findings suggest for the first time that CCN2/CTGF enhances chondrocyte adhesion to FN through direct interaction of its C-terminal CT domain with FN, and that alpha5beta1 is involved in this adhesion.     

Mechanisms of hepatocyte growth factor-mediated signaling in differentiation of pancreatic ductal epithelial cells into insulin-producing cells

Pancreatic ductal epithelial cells (PDECs) were induced to differentiate into insulin-producing cells by hepatocyte growth factor (HGF) in our previous study, but the mechanism through which this induction occurs is still unknown. HGF is a ligand that activates a tyrosine kinase encoded by the c-Met proto-oncogene. This activation is followed by indirect activation of multiple downstream signal transduction pathways (including MAPKs and the PI3K/AKT signaling pathways) that initiate various biological effects. Therefore, we speculated that the differentiation of PDECs is through either the MAPK signaling pathway or the PI3K/AKT signaling pathway. To test this hypothesis, isolated PDECs from adult rats were stimulated by adding HGF to their medium for 28 days. Then, the expression levels of several protein kinases, including MAPKs (ERK1/2, p38, and JNK) and AKT, were determined by Western blotting to determine if specific protein kinases are activated in these pathways. Subsequently, re-isolated from adult rats and cultured PDECs were pre-treated with specific inhibitors of proteins shown to be activated in these signaling pathways; these cells were then induced to differentiate by the addition of HGF. The expression levels of protein kinases were determined by Western blotting, and the differentiation rate of insulin-positive cells was determined by flow cytometry. The change of PDEC differentiation rates were compared between the groups in which cells with or without inhibitors pretreatment to determine the specific signaling pathway(s) that may be involved in HGF-induced differentiation of PDECs. After isolating PDECs and stimulating them with HGF for 28 days, the expression levels of phosphorylated ERK1/2 as well as total and phosphorylated AKT of cultured cells were significantly increased compared to the normal control group (p < 0.05), suggesting that the signaling pathways involving ERK1/2 and Akt (MEK-ERK and PI3K-AKT) are activated during HGF-induced PDEC differentiation. MEK1/2 or PI3K inhibitors were separately added to the culture medium of PDECs pre-treated with HGF. These results show that compared to the HGF-treated group, the differentiation rate of insulin-positive cells was significantly decreased in the HGF/LY294002 (PI3K inhibitor) group (13.47 ± 1.57% vs. 33.47 ± 1.34%, p < 0.05); however, the differentiation rate of insulin-positive cells was not significantly different in the HGF/PD98059 (MEK1/2 inhibitor) group. These data suggest that HGF induces PDECs to differentiate into insulin-producing cells through the PI3K/AKT signaling pathway.     

Engineering the NK1 fragment of hepatocyte growth factor/scatter factor as a MET receptor antagonist

The growth and motility factor hepatocyte growth factor/scatter factor (HGF/SF) and its receptor MET, the tyrosine kinase encoded by the c-MET proto-oncogene, exert major roles in cancer invasion and metastasis and are key targets for therapy. NK1 is an alternative spliced variant of HGF/SF that consists of the N-terminal (N) and first kringle (K1) domains and has partial agonistic activity. NK1 crystallises as a head-to-tail dimer with an extensive inter-protomeric interface resulting from contacts between the two short interdomain linkers and reciprocal contacts between the N and K1 domains. Here we show that a subset of mutants at the NK1 dimer interface, such as the linker mutants Y124A or N127A or the kringle mutant V140A:I142A, bind the MET receptor with affinities comparable to wild-type NK1 but fail to assemble a dimeric, signalling competent NK1-MET complex. These NK1 variants have no detectable agonistic activity on, behave as bona fide receptor antagonists by blocking cell migration and DNA synthesis in target cells and have strong prospects as therapeutics for human cancer.     

Growth differentiation factor‐15 (GDF‐15) suppresses in vitro angiogenesis through a novel interaction with connective tissue growth factor (CCN2)

Growth differentiation factor‐15 (GDF‐15) and the CCN family member, connective tissue growth factor (CCN2), are associated with cardiac disease, inflammation, and cancer. The precise role and signaling mechanism for these factors in normal and diseased tissues remains elusive. Here we demonstrate an interaction between GDF‐15 and CCN2 using yeast two‐hybrid assays and have mapped the domain of interaction to the von Willebrand factor type C domain of CCN2. Biochemical pull down assays using secreted GDF‐15 and His‐tagged CCN2 produced in PC‐3 prostate cancer cells confirmed a direct interaction between these proteins. To investigate the functional consequences of this interaction, in vitro angiogenesis assays were performed. We demonstrate that GDF‐15 blocks CCN2‐mediated tube formation in human umbilical vein endothelial (HUVEC) cells. To examine the molecular mechanism whereby GDF‐15 inhibits CCN2‐mediated angiogenesis, activation of α_Vβ₃ integrins and focal adhesion kinase (FAK) was examined. CCN2‐mediated FAK activation was inhibited by GDF‐15 and was accompanied by a decrease in α_Vβ₃ integrin clustering in HUVEC cells. These results demonstrate, for the first time, a novel signaling pathway for GDF‐15 through interaction with the matricellular signaling molecule CCN2. Furthermore, antagonism of CCN2 mediated angiogenesis by GDF‐15 may provide insight into the functional role of GDF‐15 in disease states. J. Cell. Biochem. 114: 1424–1433, 2013. © 2012 Wiley Periodicals, Inc.     

Transcriptional regulation of connective tissue growth factor by sphingosine 1-phosphate in rat cultured mesangial cells

Connective tissue growth factor (CTGF) is induced by transforming growth factor-beta (TGF-beta) via Smad activation in mesangial cells. We recently reported that sphingosine 1-phosphate (S1P) induces CTGF expression in rat cultured mesangial cells. However, the mechanism by which S1P induces CTGF expression is unknown. The present study revealed that S1P-induced CTGF expression is mediated via pertussis toxin-insensitive pathways, which are involved in the activation of small GTPases of the Rho family and protein kinase C. We also showed by luciferase reporter assays and chromatin immunoprecipitation that S1P induces CTGF expression via Smad activation as TGF-beta does.     

Matriptase is required for the active form of hepatocyte growth factor induced Met,focal adhesion kinase and protein kinase B activation on neural stem/progenitor cell motility

Hepatocyte growth factor (HGF) is a chemoattractant and inducer for neural stem/progenitor (NS/P) cell migration. Although the type II transmembrane serine protease, matriptase (MTP) is an activator of the latent HGF, MTP is indispensable on NS/P cell motility induced by the active form of HGF. This suggests that MTP's action on NS/P cell motility involves mechanisms other than proteolytic activation of HGF. In the present study, we investigate the role of MTP in HGF-stimulated signaling events. Using specific inhibitors of phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt) or focal adhesion kinase (FAK), we demonstrated that in NS/P cells HGF-activated c-Met induces PI3k-Akt signaling which then leads to FAK activation. This signaling pathway ultimately induces MMP2 expression and NS/P cell motility. Knocking down of MTP in NS/P cells with specific siRNA impaired HGF-stimulation of c-Met, Akt and FAK activation, blocked HGF-induced production of MMP2 and inhibited HGF-stimulated NS/P cell motility. MTP-knockdown NS/P cells cultured in the presence of recombinant protein of MTP protease domain or transfected with the full-length wild-type but not the protease-defected MTP restored HGF-responsive events in NS/P cells. In addition to functioning as HGF activator, our data revealed novel function of MTP on HGF-stimulated c-Met signaling activation.     

Hepsin activates pro-hepatocyte growth factor and is inhibited by hepatocyte growth factor activator inhibitor-1B (HAI-1B) and HAI-2

Hepsin, a type II transmembrane serine protease, is highly upregulated in prostate cancer and promotes tumor progression and metastasis. We generated a soluble form of hepsin comprising the entire extracellular domain to show that it efficiently converts single-chain hepatocyte growth factor (pro-HGF) into biologically active two-chain HGF. Hepsin activity was potently inhibited by soluble forms of the bi-Kunitz domain inhibitors HAI-1B (IC(50) 21.1+/-2.7 nM) and HAI-2 (IC(50) 1.3+/-0.3 nM). Enzymatic assays with HAI-1B Kunitz domain mutants (R260A and K401A) further demonstrated that inhibition was due to Kunitz domain-1. The results suggest a functional link between hepsin and the HGF/Met pathway, which may contribute to tumor progression.     

Tumor necrosis factor is markedly synergistic with interleukin 1 and interferon-γ in stimulating the production of nerve growth factor in fibroblasts

A possible interaction between tumor necrosis factor- (TNF) and other cytokines/growth factors in stimulating the production of nerve growth factor (NGF) in Swiss 3T3 cells was studied. TNF's stimulatory activity on fibroblast NGF production was synergized by interleukin-1 (IL-1), IL-1β and interferon-γ (IFN-γ), but was antagonized by transforming growth factor-β (TGF-β). The most remarkable synergistic effect was observed between TNF and IL-1/β; as little as 0.003 ng/ml of IL-1β markedly enhanced TNF's stimulatory activity on NGF production in the cells. These findings reinforce the idea that TNF, in concert with IL-1/β, plays an essential role in regulating the regeneration of peripheral nerves following injury through an indirect mechanism by which it stimulates NGF production in fibroblasts.     

Gadolinium-induced fibrosis is counter-regulated by CCN3 in human dermal fibroblasts: a model for potential treatment of nephrogenic systemic fibrosis

We recently show that CCN3 is a counter-regulatory molecule for the pro-fibrotic protein CCN2, and a potentially novel fibrosis therapy. The goal of this study was to assess the role of CCN3 in fibroproliferative/fibrotic responses in human dermal fibroblasts exposed to Omniscan, one of the gadolinium-based contrast agents associated with development of nephrogenic systemic fibrosis (NSF) a rare but life-threatening disease thought to be complication of NMR diagnostics in renal impaired patients. Human dermal fibroblasts were exposed to Omniscan; or to platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β) as controls. Proliferation was assessed along with matrix metalloproteinase-1, tissue inhibitor of metalloproteinases-1 and type 1 procollagen in the absence and presence of CCN3. In parallel, CCN3 production was assessed in control and Omniscan-treated cells. The results showed that PDGF stimulated fibroblast proliferation, production of Timp-1 and MMP-1 whereas exogenous CCN3 inhibited, in a dose response manner, cell proliferation (approx. 50 % max.) and production of MMP-1 (approx 35 % max.) but had little effect on TIMP-1. TGF-β stimulated type 1 procollagen production but not proliferation, Timp-1 or MMP-1 compared to non-TGF-ß treated control cells, and CCN3 treatment blocked (approx. 80 % max.) this up-regulation. Interestingly, untreated, control fibroblasts produced high constitutive levels of CCN3 and concentrations of Omniscan that induced fibroproliferative/fibrogenic changes in dermal fibroblasts correspondingly suppressed CCN3 production. The use of PDGF and TGF-β as positive controls, and the study of differential responses, including that to Omniscan itself, provide the first evidence for a role of fibroblast-derived CCN3 as an endogenous regulator of pro-fibrotic changes, elucidating possible mechanism(s). In conclusion, these data support our hypothesis of a role for fibroblast-derived CCN3 as an endogenous regulator of pro-fibrotic changes in these cells, and suggest that CCN3 may be an important regulatory molecule in NSF and a target for treatment in this and other fibrotic diseases.     

IL-4 modulates the histamine content of mast cells in a mast cell/fibroblast co-culture through a Stat6 signaling pathway in fibroblasts

IL-4 plays a crucial role in the pathogenesis of allergic diseases, such as the induction of IgE synthesis and the development of mast cells. To further understand the effect of IL-4 on mast cells in skin, we utilized a mast cell/fibroblast co-culture system as an in vitro model of dermal mast cells. IL-4 induced mast cell growth in the culture with fibroblasts. Immunoblot analysis revealed that IL-4 activated Stat6 in both mast cells and fibroblasts. The over-expression of dominant-negative Stat6 in fibroblasts in the presence of IL-4 decreased the histamine content per mast cell, but not the number of mast cells. In contrast, the over-expression of constitutively-active Stat6 in fibroblasts increased the histamine content per mast cell, indicating that the activation of Stat6 in fibroblasts supports the maturation of mast cells co-cultured with fibroblasts.     

c-myb stimulates cell growth by regulation of insulin-like growth factor (IGF) and IGF-binding protein-3 in K562 leukemia cells

c-myb plays an important role in the regulation of cell growth and differentiation, and is highly expressed in immature hematopoietic cells. The human chronic myelogenous leukemia cell K562, highly expresses IGF-I, IGF-II, IGF-IR, and IGF-induced cellular proliferation is mediated by IGF-IR. To characterize the impact of c-myb on the IGF-IGFBP-3 axis in leukemia cells, we overexpressed c-myb using an adenovirus gene transfer system in K562 cells. The overexpression of c-myb induced cell proliferation, compared to control, and c-myb induced cell growth was inhibited by anti-IGF-IR antibodies. c-myb overexpression resulted in a significant increase in the expression of IGF-I, IGF-II, and IGF-IR, and a decrease in IGFBP-3 expression. By contrast, disruption of c-myb function by DN-myb overexpression resulted in significant reduction of IGF-I, IGF-II, IGF-IR, and elevation of IGFBP-3 expression. In addition, exogenous IGFBP-3 inhibited the proliferation of K562 cells, and c-myb induced cell growth was blocked by IGFBP-3 overexpression in a dose-dependent manner. The growth-promoting effects of c-myb were mediated through two major intracellular signaling pathways, Akt and Erk. Activation of Akt and Erk by c-myb was completely blocked by IGF-IR and IGFBP-3 antibodies. These findings suggest that c-myb stimulates cell growth, in part, by regulating expression of the components of IGF-IGFBP axis in K562 cells. In addition, disruption of c-myb function by DN-myb may provide a useful strategy for treatment of leukemia.