Laura Kennedy

CCN2 Is Necessary for Adhesive Responses to Transforming Growth Factor-β1 in Embryonic Fibroblasts

CCN2 is induced by transforming growth factor-β (TGFβ) in fibroblasts and is overexpressed in connective tissue disease. CCN2 has been proposed to be a downstream mediator of TGFβ action in fibroblasts; however, the role of CCN2 in regulating this process unclear. By using embryonic fibroblasts isolated from ccn2–/–mice, we showed that CCN2 is required for a subset of responses to TGFβ. Affymetrix genome-wide expression profiling revealed that 942 transcripts were induced by TGFβ greater than 2-fold in ccn2+/+ fibroblasts, of which 345 were not induced in ccn2–/–fibroblasts, including pro-adhesive and matrix remodeling genes. Whereas TGFβ properly induced a generic Smad3-responsive promoter in ccn2–/–fibroblasts, TGFβ-induced activation of focal adhesion kinase (FAK) and Akt was reduced in ccn2–/–fibroblasts. Emphasizing the importance of FAK and Akt activation in CCN2-dependent transcriptional responses to TGFβ in fibroblasts, CCN2-dependent transcripts were not induced by TGFβ in fak–/–fibroblasts and were reduced by wortmannin in wild-type fibroblasts. Akt1 overexpression in ccn2–/–fibroblasts rescued the TGFβ-induced transcription of CCN2-dependent mRNA. Finally, induction of TGFβ-induced fibroblast adhesion to fibronectin and type I collagen was significantly diminished in ccn2–/–fibroblasts. Thus in embryonic fibroblasts, CCN2 is a necessary cofactor required for TGFβ to activate the adhesive FAK/Akt/phosphatidylinositol 3-kinase cascade, FAK/Akt-dependent genes, and adhesion to matrix.

Matrix Contraction by Dermal Fibroblasts Requires Transforming Growth Factor-β/Activin-Linked Kinase 5, Heparan Sulfate-Containing Proteoglycans, and MEK/ERK: Insights into Pathological Scarring in Chronic Fibrotic Disease

Scarring is characterized by excessive synthesis and contraction of extracellular matrix. Here, we show that fibroblasts from scarred (lesional) areas of patients with the chronic fibrotic disorder diffuse scleroderma [diffuse systemic sclerosis (dSSc)] show an enhanced ability to adhere to and contract extracellular matrix, relative to fibroblasts from unscarred (nonlesional) areas of dSSc patients and dermal fibroblasts from normal, healthy individuals. The contractile abilities of normal and dSSc dermal fibroblasts were suppressed by blocking heparin sulfate-containing proteoglycan biosynthesis or antagonizing transforming growth factor-beta receptor type I [activin-linked kinase (ALK5)] or ras/mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK). Compared with both normal and nonlesional fibroblasts, lesional dSSc fibroblasts overexpressed the heparin sulfate-containing proteoglycan syndecan 4. We also found that the procontractile signals from transforming growth factor (TGF)-beta were integrated through syndecan 4 and MEK/ERK because the ability of TGFbeta to induce contraction of dermal fibroblasts was prevented by MEK antagonism. TGFbeta could not induce a contractile phenotype or phosphorylate ERK in syndecan 4(-/-) dermal fibroblasts. These results suggest that integrating TGFbeta and ERK signals via syndecan 4 is essential for the contractile ability of dermal fibroblasts. We conclude that antagonizing MEK/ERK, TGFbeta1/ALK5, or syndecan 4 may alleviate scarring in chronic fibrotic disease.

The induction of CCN2 by TGFβ1 involves Ets-1

CCN2 is encoded by an immediate-early gene induced in mesenchymal cells during the formation of blood vessels, bone and connective tissue. It plays key roles in cell adhesion and migration, as well as matrix remodeling. CCN2 is overexpressed in fibrosis, arthritis and cancer; thus, an understanding of how to control CCN2 expression is likely to have importance in developing therapies to combat these pathologies. Previously, we found that the promoter sequence GAGGAATG is important for Ccn2 gene regulation in NIH 3T3 fibroblasts. In this report, we show that this sequence mediates activation of the CCN2 promoter by the ETS family of transcription factors. Endogenous Ets-1 binds this element of the CCN2 promoter, and dominant negative Ets-1 and specific Ets-1 small interfering RNA block induction of CCN2 expression by TGFβ. In the absence of added TGFβ1, Ets-1, but not the related fli-1, synergizes with Smad 3 to activate the CCN2 promoter. Whereas the ability of transfected Ets-1 to activate the CCN2 promoter is dependent on protein kinase C (PKC), Ets-1 in the presence of co-transfected Smad3 does not require PKC, suggesting that the presence of Smad3 bypasses the requirement of Ets-1 for PKC to activate target promoter activity. Our results are consistent with the notion that Smad3 and Ets-1 cooperate in the induction of the CCN2 promoter by TGFβ1. Antagonizing Ets-1 might be of benefit in attenuating CCN2 expression in fibrosis, arthritis and cancer, and may be useful in modulating the outcome of these disorders.

Focal Adhesion Kinase/Src Suppresses Early Chondrogenesis CENTRAL ROLE OF CCN2

Adhesive signaling plays a key role in cellular differentiation, including in chondrogenesis. Herein, we probe the contribution to early chondrogenesis of two key modulators of adhesion, namely focal adhesion kinase (FAK)/Src and CCN2 (connective tissue growth factor, CTGF). We use the micromass model of chondrogenesis to show that FAK/Src signaling, which mediates cell/matrix attachment, suppresses early chondrogenesis, including the induction of Ccn2, Agc, and Sox6. The FAK/Src inhibitor PP2 elevates Ccn2, Agc, and Sox6 expression in wild-type mesenchymal cells in micromass culture, but not in cells lacking CCN2. Our results suggest a reduction in FAK/Src signaling is a critical feature permitting chondrogenic differentiation and that CCN2 operates downstream of this loss to promote chondrogenesis.

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.

Connective Tissue Growth Factor Promoter Activity in Normal and Wounded Skin

In skin, connective tissue growth factor (CTGF/CCN2) is induced during tissue repair. However, what the exact cell types are that express CTGF in normal and wounded skin remain controversial. In this report, we use transgenic knock-in mice in which the Pacific jellyfish Aequorea victoria enhanced green fluorescent protein (E-GFP) gene has been inserted between the endogenous CTGF promoter and gene. Unwounded (day 0) and wounded (days 3 and 7) skin was examined for GFP to detect cells in which the CTGF promoter was active, α-smooth muscle actin (α-SMA) to detect myofibroblasts, and NG2 expression to detect pericytes. In unwounded mice, CTGF expression was absent in epidermis and was present in a few cells in the dermis. Upon wounding, CTGF expression was induced in the dermis. Double immunolabeling revealed that CTGF-expressing cells also expressed α-SMA, indicating the CTGF was expressed in myofibroblasts. A subset (~30%) of myofibroblasts were also NG2 positive, indicating that pericytes significantly contributed to the number of myofibroblasts in the wound. Pericytes also expressed CTGF. Collectively, these results indicate that CTGF expression in skin correlates with myofibroblast induction, and that CTGF-expressing pericytes are significant contributors to myofibroblast activity during cutaneous tissue repair.

Rac1 signaling regulates CTGF/CCN2 gene expression via TGFβ/Smad signaling in chondrocytes

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. CONCLUSION 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.

Role of Rac1 in a bleomycin-induced scleroderma model using fibroblast-specific Rac1-knockout mice

OBJECTIVE Activated adhesive signaling is a hallmark of fibroblasts isolated from the scars of scleroderma (systemic sclerosis [SSc]) lesions. Rac1 plays a key role in adhesive signaling. The aim of the present study was to examine the role of Rac1 in bleomycin-induced scleroderma, using mice with a fibroblast-specific deletion of Rac1. METHODS Cutaneous sclerosis was induced by subcutaneous injection of bleomycin. Control groups were treated with phosphate buffered saline. Mice with a fibroblast-specific deletion of Rac1 and control mice were investigated. Dermal thickness, inflammation, collagen production, and the number of alpha-smooth muscle actin-positive cells were determined. The quantity of the collagen-specific amino acid hydroxyproline was also measured. RESULTS Bleomycin treatment induced marked cutaneous thickening, inflammation, and fibrosis in control mice. Conversely, deletion of Rac1 resulted in resistance to bleomycin-induced fibrosis and inflammation. CONCLUSION Rac1 expression by fibroblasts is required for fibrogenesis. Inhibition of Rac1 may be a viable method to alleviate the development of cutaneous sclerosis.

Thrombospondin 1 is a key mediator of transforming growth factor β-mediated cell contractility in systemic sclerosis via a mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK)-dependent mechanism

BackgroundThe mechanism underlying the ability of fibroblasts to contract a collagen gel matrix is largely unknown. Fibroblasts from scarred (lesional) areas of patients with the fibrotic disease scleroderma show enhanced ability to contract collagen relative to healthy fibroblasts. Thrombospondin 1 (TSP1), an activator of latent transforming growth factor (TGF)β, is overexpressed by scleroderma fibroblasts. In this report we investigate whether activation of latent TGFβ by TSP1 plays a key role in matrix contraction by normal and scleroderma fibroblasts.MethodsWe use the fibroblast populated collagen lattices (FPCL) model of matrix contraction to show that interfering with TSP1/TGFβ binding and knockdown of TSP1 expression suppressed the contractile ability of normal and scleroderma fibroblasts basally and in response to TGFβ. Previously, we have shown that ras/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) mediates matrix contraction basally and in response to TGFβ.ResultsDuring mechanical stimulation in the FPCL system, using a multistation tensioning-culture force monitor (mst-CFM), TSP1 expression and p-ERK activation in fibroblasts are enhanced. Inhibiting TSP1 activity reduced the elevated activation of MEK/ERK and expression of key fibrogenic proteins. TSP1 also blocked platelet-derived growth factor (PDGF)-induced contractile activity and MEK/ERK activation.ConclusionsTSP1 is a key mediator of matrix contraction of normal and systemic sclerosis fibroblasts, via MEK/ERK.

Ceramide inhibits CCN2 expression in fibroblasts

Connective tissue growth factor (CTGF, CCN2) is induced in response to TGFβ in fibroblasts. In this report, we show that C2 ceramide reduced the ability of TGFβ to induce CCN2 protein, mRNA and promoter activity in fibroblasts. C2 ceramide reduced the ability of TGFβ to induce the generic Smad responsive promoter/reporter construct SBE-luciferase. These results suggest that C2 ceramide reduces the action of TGFβ in fibroblasts via Smad antagonism.