Connective tissue growth factor mediates transforming growth factor beta-induced collagen synthesis: down-regulation by cAMP.
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.
Duncan
M R
MR
Department of Cell Biology and Anatomy, University of Miami School of Medicine, Miami, Florida 33136, USA.
Frazier
K S
KS
Abramson
S
S
Williams
S
S
Klapper
H
H
Huang
X
X
Grotendorst
G R
GR
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D002772:Cholera Toxin / Q000494:pharmacology; D003094:Collagen / Q000096:biosynthesis*; D055513:Connective Tissue Growth Factor; D000242:Cyclic AMP / Q000494:pharmacology*; D016373:DNA, Antisense; D015536:Down-Regulation; D004347:Drug Interactions; D005347:Fibroblasts; D005355:Fibrosis / Q000209:etiology; D015870:Gene Expression / Q000187:drug effects; D017930:Genes, Reporter; D006097:Granulation Tissue / Q000187:drug effects; D006133:Growth Substances / Q000235:genetics / Q000276:immunology / Q000494:pharmacology*; D006801:Humans; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D051379:Mice; D008822:Mice, Transgenic; D051381:Rats; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
C526317:CCN2 protein, human; C526318:CCN2 protein, mouse; C526316:CCN2 protein, rat; D016373:DNA, Antisense; D006133:Growth Substances; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D016212:Transforming Growth Factor beta; D055513:Connective Tissue Growth Factor; D015124:8-Bromo Cyclic Adenosine Monophosphate; D003094:Collagen; D002772:Cholera Toxin; D000242:Cyclic AMP