Opposing effects of protein kinase Calpha and protein kinase Cepsilon on collagen expression by human lung fibroblasts are mediated via MEK/ERK and caveolin-1 signaling

The roles of MEK, ERK, the epsilon and alpha isoforms of protein kinase C (PKC), and caveolin-1 in regulating collagen expression were studied in normal lung fibroblasts. Knocking down caveolin-1 gave particularly striking results. A 70% decrease caused a 5-fold increase in MEK/ERK activation and collagen expression. The combined data reveal a branched signaling pathway. In its central portion MEK activates ERK, leading to increased collagen expression. Two branches converge on MEK/ERK. In one, increased PKCepsilon leads to MEK/ERK activation. In another, increased PKCalpha induces caveolin-1 expression, which in turn inhibits MEK/ERK activation and collagen expression. Lung fibroblasts from scleroderma patients with pulmonary fibrosis showed altered signaling. Consistent with their overexpression of collagen, scleroderma lung fibroblasts contain more activated MEK/ERK and less caveolin-1 than normal lung fibroblasts. Because cutaneous fibrosis is the hallmark of scleroderma, we also studied dermal fibroblasts. As in lung, there was more activated MEK/ERK in cells from scleroderma patients than in control cells, and MEK inhibition decreased collagen expression. However, the distinctive levels of PKCepsilon, PKCalpha, and caveolin-1 in lung and dermal fibroblasts from scleroderma patients and control subjects indicate that the links between these signaling proteins and MEK/ERK must function differently in the four cell types. Finally, we confirmed the relevance of these signaling cascades in vivo. The combined results demonstrate that a branched signaling pathway involving MEK, ERK, PKCepsilon, PKCalpha, and caveolin-1 regulates collagen expression in normal lung tissue and is perturbed during fibrosis.     

Constitutive ALK5-independent c-Jun N-terminal kinase activation contributes to endothelin-1 overexpression in pulmonary fibrosis: evidence of an autocrine endothelin loop operating through the endothelin A and B receptors

The signal transduction mechanisms generating pathological fibrosis are almost wholly unknown. Endothelin-1 (ET-1), which is up-regulated during tissue repair and fibrosis, induces lung fibroblasts to produce and contract extracellular matrix. Lung fibroblasts isolated from scleroderma patients with chronic pulmonary fibrosis produce elevated levels of ET-1, which contribute to the persistent fibrotic phenotype of these cells. Transforming growth factor beta (TGF-beta) induces fibroblasts to produce and contract matrix. In this report, we show that TGF-beta induces ET-1 in normal and fibrotic lung fibroblasts in a Smad-independent ALK5/c-Jun N-terminal kinase (JNK)/Ap-1-dependent fashion. ET-1 induces JNK through TAK1. Fibrotic lung fibroblasts display constitutive JNK activation, which was reduced by the dual ETA/ETB receptor inhibitor, bosentan, providing evidence of an autocrine endothelin loop. Thus, ET-1 and TGF-beta are likely to cooperate in the pathogenesis of pulmonary fibrosis. As elevated JNK activation in fibrotic lung fibroblasts contributes to the persistence of the myofibroblast phenotype in pulmonary fibrosis by promoting an autocrine ET-1 loop, targeting the ETA and ETB receptors or constitutive JNK activation by fibrotic lung fibroblasts is likely to be of benefit in combating chronic pulmonary fibrosis.     

Endothelin-1 promotes myofibroblast induction through the ETA receptor via a rac/phosphoinositide 3-kinase/Akt-dependent pathway and is essential for the enhanced contractile phenotype of fibrotic fibroblasts

The endothelins are a family of endothelium-derived peptides that possess a variety of functions, including vasoconstriction. Endothelin-1 (ET-1) is up-regulated during tissue repair and promotes myofibroblast contraction and migration, hence contributing to matrix remodeling during tissue repair. Here, we show that addition of ET-1 to normal lung fibroblasts induces expression of proteins that contribute to a contractile phenotype, including alpha-smooth muscle actin (alpha-SMA), ezrin, moesin, and paxillin. We confirm that ET-1 enhances the ability of lung fibroblasts to contract extracellular matrix, a function essential for tissue repair, through induction of de novo protein synthesis. Blockade of the Akt/phosphoinositide 3-kinase (PI3-kinase) pathway with LY294002 and wortmannin prevents the ability of ET-1 to induce alpha-SMA, ezrin, paxillin, and moesin and to promote matrix contraction. Dominant negative rac and Akt blocked the ability of ET-1 to promote formation of alpha-SMA stress fibers. Using specific ET-1 receptor inhibitors, we show that ET-1 induces collagen matrix contraction through the ETA, but not the ETB, receptor. Relative to normal pulmonary fibroblasts, fibroblasts cultured from scars of patients with the fibrotic disease systemic sclerosis (scleroderma) show enhanced ET-1 expression and binding. Systemic sclerosis lung fibroblasts show increased ability to contract a collagen matrix and elevated expression of the procontractile proteins alpha-SMA, ezrin, paxillin, and moesin, which are greatly reduced by antagonizing endogenous ET-1 signaling. Thus, blocking ET-1 or the PI3-kinase/Akt cascades might be beneficial in reducing scar formation in pulmonary fibrosis.     

CCN2 (connective tissue growth factor) promotes fibroblast adhesion to fibronectin

In vivo, CCN2 (connective tissue growth factor) promotes angiogenesis, osteogenesis, tissue repair, and fibrosis, through largely unknown mechanisms. In vitro, CCN2 promotes cell adhesion in a variety of systems via integrins and heparin sulfate proteoglycans (HSPGs). However, the physiological relevance of CCN2-mediated cell adhesion is unknown. Here, we find that HSPGs and the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase cascade are required for adult human dermal fibroblasts to adhere to CCN2. Endogenous CCN2 directly binds fibronectin and the fibronectin receptors integrins alpha4 beta1 and alpha5 and syndecan 4. Using Ccn2-/- mouse embryonic fibroblasts, we show that loss of endogenous CCN2 results in impaired spreading on fibronectin, delayed alpha-smooth muscle actin stress fiber formation, and reduced ERK and focal adhesion kinase phosphorylation. These results suggest that a physiological role of CCN2 is to potentiate the ability of fibroblasts to spread on fibronectin, which may be important in modulating fibroblast adhesion to the provisional matrix during tissue development and wound healing. These results are consistent with the notion that a principal function of CCN2 is to modulate receptor/ligand interactions in vivo.     

CXCL12 Induces Connective Tissue Growth Factor Expression in Human Lung Fibroblasts through the Rac1/ERK,JNK, and AP-1 Pathways

CXCL12 (stromal cell-derived factor-1, SDF-1) is a potent chemokine for homing of CXCR4⁺ fibrocytes to injury sites of lung tissue, which contributes to pulmonary fibrosis. Overexpression of connective tissue growth factor (CTGF) plays a critical role in pulmonary fibrosis. In this study, we investigated the roles of Rac1, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and activator protein-1 (AP-1) in CXCL12-induced CTGF expression in human lung fibroblasts. CXCL12 caused concentration- and time-dependent increases in CTGF expression and CTGF-luciferase activity. CXCL12-induced CTGF expression was inhibited by a CXCR4 antagonist (AMD3100), small interfering RNA of CXCR4 (CXCR4 siRNA), a dominant negative mutant of Rac1 (RacN17), a mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor (PD98059), a JNK inhibitor (SP600125), a p21-activated kinase inhibitor (PAK18), c-Jun siRNA, and an AP-1 inhibitor (curcumin). Treatment of cells with CXCL12 caused activations of Rac1, Rho, ERK, and c-Jun. The CXCL12-induced increase in ERK phosphorylation was inhibited by RacN17. Treatment of cells with PD98059 and SP600125 both inhibited CXCL12-induced c-Jun phosphorylation. CXCL12 caused the recruitment of c-Jun and c-Fos binding to the CTGF promoter. Furthermore, CXCL12 induced an increase in α-smooth muscle actin (α-SMA) expression, a myofibroblastic phenotype, and actin stress fiber formation. CXCL12-induced actin stress fiber formation and α-SMA expression were respectively inhibited by AMD3100 and CTGF siRNA. Taken together, our results suggest that CXCL12, acting through CXCR4, activates the Rac/ERK and JNK signaling pathways, which in turn initiates c-Jun phosphorylation, and recruits c-Jun and c-Fos to the CTGF promoter and ultimately induces CTGF expression in human lung fibroblasts. Moreover, overexpression of CTGF mediates CXCL12-induced α-SMA expression.     

Proteomic analysis of CTGF-activated lung fibroblasts: identification of IQGAP1 as a key player in lung fibroblast migration

Connective tissue growth factor (CTGF, CCN2) is overexpressed in lung fibroblasts isolated from patients with interstitial lung disease (ILD) and systemic sclerosis (SSc, scleroderma) and is considered to be a molecular marker of fibrosis. To understand the significance of elevated CTGF, we investigated the changes in lung fibroblast proteome in response to CTGF overexpression. Using 2-dimensional gel electrophoresis followed by in-gel proteolytic digestion and mass spectrometric analysis, we identified 13 proteins affected by CTGF. Several of the CTGF-induced proteins, such as pro-alpha (I) collagen and cytoskeletal proteins vinculin, moesin, and ezrin, are known to be elevated in pulmonary fibrosis, whereas 9 of 13 proteins have not been studied in pulmonary fibrosis and are, therefore, novel CTGF-responsive molecules that may have important roles in ILD. Our study demonstrates that 1 of the novel CTGF-induced proteins, IQ motif containing GTPase activating protein (IQGAP) 1, is elevated in lung fibroblasts isolated from scleroderma patients with ILD. IQGAP1 is a scaffold protein that plays a pivotal role in regulating migration of endothelial and epithelial cells. Scleroderma lung fibroblasts and normal lung fibroblasts treated with CTGF demonstrated increased rate of migration in a wound healing assay. Depletion of IQGAP1 expression by small interfering RNA inhibited CTGF-induced migration and MAPK ERK1/2 phosphorylation in lung fibroblasts. MAPK inhibitor U0126 decreased CTGF-induced cell migration and did not interfere with CTGF-induced IQGAP1 expression, suggesting that MAPK pathway is downstream of IQGAP1. These findings further implicate the importance of CTGF in lung tissue repair and fibrosis and propose that CTGF-induced migration of lung fibroblasts to the damaged tissue is mediated via IQGAP1 and MAPK signaling pathways.     

Loss of PTEN expression by mouse fibroblasts results in lung fibrosis through a CCN2-dependent mechanism

Elevated adhesive signaling promotes fibrosis. Protein phosphatase and tensin homologue (PTEN) dephosphorylates focal adhesion kinase and suppresses the activation of Akt and hence suppresses adhesive signaling. Loss of PTEN expression is associated with lung fibrosis, but whether PTEN expression by type I collagen-expressing cells controls lung fibrosis is unclear. Here, we use mice expressing tamoxifen-dependent cre recombinase expressed under the control of a COL1A2 promoter/enhancer and mice harboring floxed-PTEN and/or floxed-CCN2 alleles to assess whether loss of PTEN expression by type I collagen producing cells results in lung fibrosis in a CCN2-dependent fashion. In vivo, loss of PTEN expression resulted in the overexpression of both collagen type I and the pro-adhesive matricellular protein connective tissue growth factor (CTGF/CCN2). However, α-smooth muscle actin expression was unaffected. Loss of CCN2 expression by lung fibroblasts rescues this phenotype; i.e.., mice deficient in both PTEN and CCN2 in collagen type I-expressing cells do not develop significant collagen deposition in the lung. PTEN expression by collagen type I-expressing cells controls collagen deposition; therapeutic strategies blocking CCN2 may be of benefit in blocking excessive collagen deposition in fibrosis.     

The gene expression profile induced by Wnt 3a in NIH 3T3 fibroblasts

Wnt proteins play important roles in regulating cell differentiation, proliferation and polarity. Wnts have been proposed to play roles in tissue repair and fibrosis, yet the gene expression profile of fibroblasts exposed to Wnts has not been examined. We use Affymetrix genome-wide expression profiling to show that a 6-h treatment of fibroblasts of Wnt3a results in the induction of mRNAs encoding known Wnt targets such as the fibrogenic pro-adhesive molecule connective tissue growth factor (CTGF, CCN2). Wnt3a also induces mRNAs encoding potent pro-fibrotic proteins such as TGFβ and endothelin-1 (ET-1). Moreover, Wnt3a promotes genes associated with cell adhesion and migration, vasculature development, cell proliferation and Wnt signaling. Conversely, Wnt3a suppresses gene associated with skeletal development, matrix degradation and cell death. Results were confirmed using real-time polymerase chain reaction of cells exposed to Wnt3a and Wnt10b. These results suggest that Wnts induce genes promoting fibroblast differentiation towards angiogenesis and matrix remodeling, at the expense of skeletal development.     

The gene expression profile induced by Wnt 3a in NIH 3T3 fibroblasts

Wnt proteins play important roles in regulating cell differentiation, proliferation and polarity. Wnts have been proposed to play roles in tissue repair and fibrosis, yet the gene expression profile of fibroblasts exposed to Wnts has not been examined. We use Affymetrix genome-wide expression profiling to show that a 6-h treatment of fibroblasts of Wnt3a results in the induction of mRNAs encoding known Wnt targets such as the fibrogenic pro-adhesive molecule connective tissue growth factor (CTGF, CCN2). Wnt3a also induces mRNAs encoding potent pro-fibrotic proteins such as TGFbeta and endothelin-1 (ET-1). Moreover, Wnt3a promotes genes associated with cell adhesion and migration, vasculature development, cell proliferation and Wnt signaling. Conversely, Wnt3a suppresses gene associated with skeletal development, matrix degradation and cell death. Results were confirmed using real-time polymerase chain reaction of cells exposed to Wnt3a and Wnt10b. These results suggest that Wnts induce genes promoting fibroblast differentiation towards angiogenesis and matrix remodeling, at the expense of skeletal development.     

CCN2 is necessary for the function of mouse embryonic fibroblasts

CCN2 is expressed by mesenchymal cells undergoing active tissue remodeling, and is characteristically overexpressed in connective tissue pathologies such as fibrosis and cancer. However, the physiological roles and mechanism of action of CCN2 are largely unknown. Here, we probe the contribution of CCN2 to the biology of mouse embryonic fibroblasts (MEFs) using genome-wide mRNA expression profiling, proteomic and functional bioassay analyses. We show that ccn2-/- mouse embryonic fibroblasts (MEFs) have significantly reduced the expression of pro-adhesive, pro-inflammatory and pro-angiogenic genes such as interleukin-6 (IL-6), ceruloplasmin, thrombospondin-1, lipocalin-2 and syndecan 4. Anti-syndecan 4 antibody reduced ERK phosphorylation in ccn2+/+ MEFs. In ccn2+/+ MEFs, the MEK inhibitor U0126 and dominant negative ras reduced expression of IL-6 and lipocalin-2. Overexpressing syndecan 4 in ccn2-/- MEFs restored IL-6 and lipocalin-2 mRNA expression. Syndecan 4 has been shown to mediate cell migration. We found that ccn2+/+ MEFs migrated significantly faster than ccn2-/- MEFs; anti-syndecan 4 antibody and U0126 reduced the migration of ccn2+/+ MEFs to that of ccn2-/- MEFs. These results collectively support the notion that syndecan 4 acts downstream of CCN2 in MEFs, and that reduced syndecan 4 expression contributes to at least part of the ccn2-/- phenotype. Further, these results suggest that CCN2 is required for MEFs to contribute to aspects of tissue remodeling. Consistent with this notion, whereas ccn2+/+ MEFs displayed actin stress fibers and focal adhesions at the cell periphery consistent with a migratory phenotype, ccn2-/- MEFs displayed reduced focal adhesions and actin stress fibers, and a reduced ability to transduce forces across a collagen gel matrix. Collectively, these results suggest that CCN2 supplies essential, non-redundant functions required for fibroblasts to properly participate in features of embryogenesis, and further suggest that CCN2 may play essential roles in adult wound healing, tissue repair and fibrogenesis.     

Fibroblast adhesion results in the induction of a matrix remodeling gene expression program

Fibrosis is believed to occur through the failure to terminate the normal tissue remodeling program. Tissue repair intimately involves the ability of fibroblasts to attach to extracellular matrix (ECM), resulting in cell migration and ECM contraction. Elevated, activated adhesive signaling is a key phenotypic hallmark of fibrotic cells. The precise contribution of adhesion to tissue remodeling and repair and fibrotic responses in fibroblasts is unclear, but involves focal adhesion kinase (FAK). FAK signals downstream of integrin-mediates attachment of fibroblasts to extracellular matrix. In this report, we show that FAK is required for the expression of a cohort of mRNAs encoding ECM and matrix remodeling genes including CCN2, alpha-smooth muscle actin (SMA) and type I collagen. Adhesion of fibroblasts to fibronectin, a component of the provisional matrix deposited in the initial phases of tissue repair, also resulted in the induction of CCN2, alpha-SMA and type I collagen mRNAs. Endothelin-1 (ET-1), a key inducer of pro-fibrotic gene expression, was also induced upon fibroblast attachment to ECM, and antagonism of the ET-1 receptors significantly reduced the ability of adhesion to induce expression of CCN2, alpha-SMA and type I collagen mRNAs. These results suggest that adhesion of fibroblasts to matrix during the initial phases of tissue remodeling and repair may actively contribute to the tissue repair program through the induction of pro-fibrotic gene expression.     

Induction by IL-1 beta of tissue inhibitor of metalloproteinase-1 in human orbital fibroblasts: modulation of gene promoter activity by IL-4 and IFN-gamma

Thyroid-associated ophthalmopathy (TAO), an autoimmune component of Graves' disease, is associated with profound connective tissue remodeling and fibrosis that appear to involve the selective activation of orbital fibroblasts. Accumulation of extracellular matrix molecules is a hallmark of this process. Here we report that orbital fibroblasts treated with IL-1beta express high levels of tissue inhibitor of metalloproteinase-1 (TIMP-1), an important modulator of matrix metalloproteinase activity. These high levels are associated with increased TIMP-1 activity. The induction is mediated at the pretranslational level and involves activating the TIMP-1 gene promoter. IL-1beta activates the ERK 1/2 pathway in these fibroblasts and interrupting this signaling either with PD98059, a chemical inhibitor of MEK, or by transfecting cells with a dominant negative ERK 1 plasmid results in the attenuation of TIMP-1 induction. Surprisingly, treatment with IL-4 or IFN-gamma could also block the IL-1beta induction by attenuating TIMP-1 gene promoter activity. These findings suggest that TIMP-1 expression in orbital fibroblasts following activation with IL-1beta could represent an important therapeutic target for modifying the proteolytic environment. This might alter the natural course of tissue remodeling in TAO.     

Cyr61 increases matrix metalloproteinase-3 expression and cell motility in human oral squamous cell carcinoma cells

Oral squamous cell carcinoma (OSCC) has a striking tendency to migrate and metastasize. Cysteine-rich 61 (Cyr61), from the CCN gene family, is a secreted and matrix-associated protein, which is involved in many cellular activities such as growth and differentiation. However, the effects of Cyr61 on human OSCC cells are largely unknown. In this study, we found that Cyr61 increased the migration and the expression of matrix metalloproteinases-3 (MMP)-3 in human OSCC cells. αvβ5 or α6β1 monoclonal antibody (mAb), focal adhesion kinase (FAK) inhibitor, and mitogen-activated protein kinase (MEK) inhibitors (PD98059 and U0126) inhibited the Cyr61-induced increase of the migration and MMP-3 up-regulation of OSCC cells. Cyr61 stimulation increased the phosphorylation of FAK, MEK, and extracellular signal-regulated kinase (ERK). In addition, NF-κB inhibitors suppressed the cell migration and MMP-3 expression enhanced by Cyr61. Moreover, Cyr61 increased NF-κB luciferase activity and binding of p65 to the NF-κB element on the MMP-3 promoter. Taken together, our results indicate that Cyr61 enhances the migration of OSCC cells by increasing MMP-3 expression through the αvβ3 or α6β1 integrin receptor, FAK, MEK, ERK, and NF-κB signal transduction pathway.     

Wnt 10b activates the CCN2 promoter in NIH 3T3 fibroblasts through the Smad response element

Wnt proteins elevate expression of the CCN family. For example, Wnt10b induces the fibrogenic pro-adhesive molecule connective tissue growth factor (CTGF, CCN2) in NIH 3T3 fibroblasts. Wnt10b activates the CCN2 minimal promoter. In this report, we map the Wnt10b response element in the CCN2 minimal promoter to the previously identified Smad response element. These results suggest that Wnts may cross-talk with the Smad signaling pathway to induce fibrotic responses in fibroblasts.     

Activation of PERK in ET-1- and thrombin-induced pulmonary fibroblast differentiation: Inhibitory effects of curcumin

In the present study, we investigated the role of PKR-like endoplasmic reticular kinase (PERK), an endoplasmic reticulum (ER) stress kinase, in endothelin 1 (ET-1)- and thrombin-induced pulmonary fibrosis (PF), and the preventive effects of curcumin (CUR). Using the human embryonic WI-38 lung fibroblast cell line, ET-1 and thrombin induced the expression of ER stress-related proteins (CCAAT-enhancer-binding protein homologous protein, PERK, and binding immunoglobulin protein), a profibrogenic factor (cellular communication network factor 2 [CCN2]), and differentiation markers including α-smooth muscle actin (α-SMA), collagen I (Col I), and Col IV. Knockdown of PERK expression via small interfering RNA (siRNA) significantly reduced the increases in CCN2, α-SMA, Col I, and Col IV proteins in WI-38 cells according to western blot analysis and immunohistochemistry (IHC). Activation of c-Jun N-terminal kinase (JNK) was observed in ET-1- and thrombin-treated WI-38 cells, and the addition of a JNK inhibitor (SP) suppressed the induction of the indicated proteins by ET-1 and thrombin. Thapsigargin (TG), an ER stress inducer, elevated expressions of PERK and ER stress-related proteins with increased differentiation of WI-38 cells. Knockdown of PERK by siRNA or the PERK inhibitor glycogen synthesis kinase reduced expressions of the differentiation markers, α-SMA and Col IV, in WI-38 cells. CUR concentration-dependently inhibited ET-1- or thrombin-induced CCN2, α-SMA, and vimentin proteins with decreased levels of phosphorylated mitogen-activated protein kinase and PERK in WI-38 cells. An in vivo bleomycin-induced PF study showed that an intraperitoneal injection of CUR (30 mg/kg) reduced expressions of α-SMA, CCN2, Col IV, and vimentin in lung tissues via IHC staining using specific antibodies. This study is the first to demonstrate that PERK activation contributes to pulmonary fibroblast differentiation elicited by ET-1 or thrombin, and the inhibitory activity of CUR against PF is demonstrated herein.     

CCN2 expression and localization in melanoma cells

The matricellular protein connective tissue growth factor (CTGF, CCN2) is overexpressed in several forms of cancer and may represent a novel target in anti-cancer therapy. However, whether CCN2 is expressed in melanoma cells is unknown. The highly metastatic murine melanoma cell line B16(F10) was used for our studies. Real time polymerase chain reaction analysis was used to detect mRNA expression of CCN1, CCN2, CCN3 and CCN4 in Western blot and immunofluorescence analyses were used to detect CCN2 protein. Inhibitors of signal transduction cascades were used to probe the mechanism underlying CCN2 expression in B16(F10) cells. CCN2 was expressed in B16(F10) cells, and was reduced by the FAK/src inhibitor PP2 and the MEK/ERK inhibitor U0126 indicating that CCN2 acts downstream of these pathways in B16(F10) murine melanoma cells. Expression of CCN1, CCN3 and CCN4 was not reduced by PP2 or U0126; in fact, expression of CCN4 mRNA was elevated by PP2 or U0126 treatment. To our surprise, CCN2 protein was detected in the nuclei of B16(F10) cells, and was undetectable in the cytoplasm. CCN2 was expressed in B16(F10) melanoma cells, adding to the list of cancer cells in which CCN2 is expressed. Of the CCN family members tested, only CCN2 is downstream of the highly oncogenic MEK/ERK pathway. CCN2 should be further evaluated for a possible role in melanoma growth and progression.     

ALK5 inhibition blocks TGFβ-induced CCN1 expression in human foreskin fibroblasts

The potent profibrotic cytokine TGFβ induces connective tissue growth factor (CCN2/CTGF) is induced in fibroblasts in a fashion sensitive to SB-431542, a specific pharmacological inhibitor of TGFβ type I receptor (ALK5). In several cell types, TGFβ induces CCN1 but suppresses CCN3, which opposes CCN1/CCN2 activities. However, whether SB-431542 alters TGFβ-induced CCN1 or CCN3 in human foreskin fibroblasts in unclear. Here we show that TGFβ induces CCN1 but suppresses CCN3 expression in human foreskin fibroblasts in a SB-431542-sensitive fashion. These results emphasize that CCN1/CCN2 and CCN3 are reciprocally regulated and support the notion that blocking ALK5 or addition of CCN3 may be useful anti-fibrotic approaches.     

Regulatory mechanisms of TGF‐β1‐induced fibrogenesis of human alveolar epithelial cells

Pulmonary fibrosis is characterized by an extensive activation of fibrogenic cells and deposition of extracellular matrix (ECM). Transforming growth factor (TGF)‐β1 plays a pivotal role in the pathogenesis of pulmonary fibrosis, probably through the epithelial‐ to‐mesenchymal transition (EMT) and ECM production. The present study investigates potential mechanism by which TGF‐β1 induces EMT and ECM production in the fibrogenesis of human lung epithelial cells during pulmonary fibrosis. The expression of EMT phenotype and other proteins relevant to fibrogenesis were measured and the cell bio‐behaviours were assessed using Cell‐IQ Alive Image Monitoring System. We found that TGF‐β1‐induced EMT was accompanied with increased collagen I deposition, which may be involved in the regulation of connective tissue growth factor (CTGF) and phosphoinositide 3‐kinase (PI3K) signalling pathway. Treatment with PI3K inhibitors significantly attenuated the TGF‐β1‐ induced EMT, CTGF expression and collagen I synthesis in lung epithelial cells. The interference of CTGF expression impaired the basal and TGF‐β1‐stimulated collagen I deposition, but did not affect the process of EMT. Our data indicate that the signal pathway of TGF‐β1/PI3K/CTGF plays an important role in the fibrogenesis of human lung epithelial cells, which may be a novel therapeutic approach to prevent and treat pulmonary fibrosis.     

CCN6 enhances ICAM-1 expression and cell motility in human chondrosarcoma cells

Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. CCN6 is a cysteine-rich protein that belongs to the CCN (Cyr61, CTGF, and Nov) family of matricellular proteins. However, the effects of CCN6 on human chondrosarcoma cells are largely unknown. In this study, we found that CCN6 increased the migration and the expression of intercellular adhesion molecule-1 (ICAM-1) in human chondrosarcoma cells. αvβ3 and αvβ5 integrin monoclonal antibody and mitogen-activated protein kinase (MEK) inhibitors (PD98059 and U0126) inhibited the CCN6-induced increase of the migration and ICAM-1 up-regulation of chondrosarcoma cells. CCN6 stimulation increased the phosphorylation of focal adhesion kinase (FAK), MEK, and extracellular signal-regulated kinase (ERK). In addition, activator protein-1 (AP-1) inhibitors suppressed the cell migration and ICAM-1 expression enhanced by CCN6. Moreover, CCN6 increased AP-1 luciferase activity and binding of c-Jun to the AP-1 element on the ICAM-1 promoter. Taken together, our results indicate that CCN6 enhances the migration of chondrosarcoma cells by increasing ICAM-1 expression through the αvβ3 and αvβ5 integrin receptor, FAK, MEK, ERK, c-Jun, and AP-1 signal transduction pathway.