Matthew R. Duncan

Connective tissue growth factor (CTGF) is a cysteine-rich peptide synthesized and secreted by fibroblastic cells after activation with transforming growth factor beta (TGF-beta) that acts as a downstream mediator of TGF-beta-induced fibroblast proliferation. We performed in vitro and in vivo studies to determine whether CTGF is also essential for TGF-beta-induced fibroblast collagen synthesis. In vitro studies with normal rat kidney (NRK) fibroblasts demonstrated CTGF potently induces collagen synthesis and transfection with an antisense CTGF gene blocked TGF-beta stimulated collagen synthesis. Moreover, TGF-beta-induced collagen synthesis in both NRK and human foreskin fibroblasts was effectively blocked with specific anti-CTGF antibodies and by suppressing TGF-beta-induced CTGF gene expression by elevating intracellular cAMP levels with either membrane-permeable 8-Br-cAMP or an adenylyl cyclase activator, cholera toxin (CTX). cAMP also inhibited collagen synthesis induced by CTGF itself, in contrast to its previously reported lack of effect on CTGF-induced DNA synthesis. In animal assays, CTX injected intradermally in transgenic mice suppressed TGF-beta activation of a human CTGF promoter/lacZ reporter transgene. Both 8-Br-cAMP and CTX blocked TGF-beta-induced collagen deposition in a wound chamber model of fibrosis in rats. CTX also reduced dermal granulation tissue fibroblast population increases induced by TGF-beta in neonatal mice, but not increases induced by CTGF or TGF-beta combined with CTGF. Our data indicate that CTGF mediates TGF-beta-induced fibroblast collagen synthesis and that in vivo blockade of CTGF synthesis or action reduces TGF-beta-induced granulation tissue formation by inhibiting both collagen synthesis and fibroblast accumulation.

Fibroblast proliferation, differentiation into myofibroblasts, and increased collagen synthesis are key events during both normal wound repair and fibrotic lesion formation. Here we report that these biological responses to TGF-beta by fibroblasts are regulated via a CTGF-dependent pathway in concert with either EGF or IGF-2. Our studies indicate these responses to TGF-beta are mutually exclusive, and cells that are proliferating do not express alpha-SMA or elevated levels of collagen synthesis. Cells expressing alpha-SMA do not exhibit DNA synthesis but do coexpress higher levels of types I and III collagen mRNA. Thus, fibroblast proliferation and differentiation are controlled by combinatorial signaling pathways involving not only components of the TGF-beta/CTGF pathway, but also signaling events induced by EGF and IGF-2-activated receptors. Collectively, our studies indicate TGF-beta functions as a classic embryonic inducer, initiating a cascade that is controlled by other factors in the cellular environment. We propose a model for this process with regard to wound repair and fibrotic lesion formation that is likely applicable to other instances of CTGF action during embryogenesis.

Human peripheral blood mononuclear cells activated with concanavalin A (Con A) or lipopolysaccharide (LPS) produce, respectively, lymphokines (LK) of 50,000 Mr or monokines (MK) of 20,000 Mr that inhibit the growth and collagen production of cultured human dermal fibroblasts. Because antigenic typing of the antiviral activity of these LK and MK preparations revealed that LK contained mainly gamma interferon (IFN-gamma), and MK, primarily IFN-beta, we investigated if any of the fibroblast-inhibiting activities could be attributed to human IFN. Unlike LK and MK, which act within 24 h to inhibit the growth of subconfluent foreskin and adult dermal fibroblasts, samples of purified, natural derived IFN-alpha, -beta, and -gamma and recombinant DNA-derived IFN-alpha 2 and -gamma were ineffective inhibitors at 24 h and required 48-72 h to significantly inhibit growth. However, all IFN did mimic LK/MK action in causing concentration-dependent inhibition of collagen production by confluent fibroblast microcultures. Furthermore, the collagen production-inhibiting activity of Con A-induced LK supernatant and its 50,000 Mr fraction was completely suppressed by 10(3) neutralizing U/ml of either polyclonal or monoclonal antibody to IFN-gamma, while polyclonal antibodies to IFN-alpha and -beta had no effect. Similarly, the collagen production-inhibiting activity of LPS-induced MK supernatant and its 20,000 Mr fraction was suppressed by polyclonal anti-IFN-beta but not by anti-IFN-alpha or -gamma. Anti-IFN failed to reverse early-acting LK or MK growth-inhibiting activities. These data suggest collagen production-inhibiting LK and MK are IFN-gamma and IFN-beta, respectively, and that early acting, growth-inhibiting LK and MK are not IFN.

ABSTRACT All members of the Ctgf, Cyr61, and Nov (CCN) family share a high degree of sequence homology and conservation of structural motifs and domains. Here, we present data about a structure function analysis of connective tissue growth factor (CTGF), a prototypic member of the CCN family, which has been shown to be a downstream mediator of transforming growth factor‐β activities on fibroblasts. Our findings demonstrate the two domains of CTGF function to mediate two distinct biological effects. The N‐terminal domain of CTGF mediates myofibroblast differentiation and collagen synthesis. The C‐terminal domain of CTGF mediates fibroblast proliferation. These data provide a molecular basis for the divergence of CTGF actions on connective tissue cell types and suggest a model for functional analysis of all of the CCN family gene products.—Grotendorst, G. R., Duncan, M. R. Individual domains of connective tissue growth factor regulate fibroblast proliferation and myofibroblast differentiation.—Grotendorst, G. R., Duncan, M. R. Individual domains of connective tissue growth factor regulate fibroblast proliferation and myofibroblast differentiation. FASEB J . 19, 729–738 (2005)