TGF-β-stimulated CTGF production enhanced by collagen and associated with biogenesis of a novel 31-kDa CTGF form in human corneal fibroblasts.
OBJECTIVE
Connective tissue growth factor (CTGF) is induced by transforming growth factor-beta (TGF-β) after corneal wounding. This study addressed the role of the extracellular matrix in the induction of CTGF by TGF-β.


METHODS
Human corneal fibroblasts (HCFs) were grown on fibronectin (FN), vitronectin (VN), or collagen (CL) in supplemented serum-free media alone or with TGF-β1 or fibroblast growth factor plus heparin. CTGF mRNA was analyzed by qPCR and protein expression by Western blot analysis of Triton X-100 (TX-100)-soluble and TX-100-insoluble cell lysates using antibodies to N-terminal, mid, and C-terminal CTGF regions. Immunocytochemistry was performed on nonconfluent or scrape-wounded confluent HCFs.


RESULTS
TGF-β-treated HCFs grown on CL produced five times more 38-kDa CTGF than untreated controls (72 hours). TGF-β-treated HCFs on CL secreted twofold more CTGF than those on FN or VN. Furthermore, a 31-kDa CTGF form, lacking the N-terminal domain, was detected in Triton X-100 insoluble fractions in Western blot analysis. Immunodetectable extracellular CTGF formed linear arrays parallel to, but not colocalized with, CL or FN. It also did not colocalize with FAK, vinculin, or integrins α(v)β(3) and α(5)β(1). Intracellular CTGF was detected in the Golgi apparatus and vesicles, including endosomes.


CONCLUSIONS
Enhanced CTGF secretion induced by TGF-β in CL-grown cells may contribute to positive feedback in which CL is overexpressed in CTGF-induced fibrosis. N-terminal CTGF fragments in the plasma of patients with severe fibrotic disease may be a product of CTGF proteolysis that also produces the newly identified 31-kDa CTGF that remains cell associated and may have its impact by non-integrin signaling pathways.
Tall
Edward G
EG
Department of Ophthalmology, Mount Sinai School of Medicine, New York, New York 10029-6574, USA.
Bernstein
Audrey M
AM
Oliver
Noelynn
N
Gray
Julia L
JL
Masur
Sandra K
SK
D000328:Adult; D000368:Aged; D015153:Blotting, Western; D002478:Cells, Cultured; D003094:Collagen / Q000494:pharmacology*; D055513:Connective Tissue Growth Factor / Q000096:biosynthesis* / Q000235:genetics; D003315:Cornea / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D004357:Drug Synergism; D005346:Fibroblast Growth Factors / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism; D005353:Fibronectins / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D008875:Middle Aged; D008970:Molecular Weight; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014019:Tissue Donors; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*; D019096:Vitronectin / Q000494:pharmacology; D055815:Young Adult
C526317:CCN2 protein, human; D005353:Fibronectins; D012333:RNA, Messenger; D053773:Transforming Growth Factor beta1; D019096:Vitronectin; D055513:Connective Tissue Growth Factor; D005346:Fibroblast Growth Factors; D003094:Collagen
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