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E.G. Tall, S.K. Masur; Extracellular Matrix Influences CTGF Secretion in Response to TGF–Beta . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5038.
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© ARVO (1962-2015); The Authors (2016-present)
After corneal wounding, there are dramatic changes in the extracellular matrix (ECM) and in growth factor expression by corneal fibroblasts. One of the growth factors, Transforming Growth Factor–beta (TGF–ß), induces the secretion of Connective Tissue Growth Factor (CTGF). CTGF functions both as a growth factor and as a matricellular protein. Because of its ability to regulate proliferation, migration, and differentiation, CTGF has been implicated in fibrosis. We investigated whether TGF–ß’s regulation of CTGF secretion is influenced by the changing composition of ECM after wounding.
Human corneal fibroblasts were plated on either fibronectin (FN), vitronectin (VN), or collagen (CL) and grown in supplemented serum–free media with either TGF–ß (0.5 ng/mL) or Fibroblast Growth Factor 2 (FGF, 20 ng/mL) and heparin (5 µg/mL). After 4–72 hrs, cells were lysed in 1% Triton X–100 lysis buffer. After SDS–PAGE, Western blot analysis using antibody to CTGF was performed on the Triton–soluble and Triton–insoluble fractions, which represent newly synthesized CTGF (in the secretory pathway) and ECM–associated CTGF, respectively.
We found that the amount and pattern of CTGF synthesis induced by TGF–ß is matrix dependent. On FN, cells synthesized and secreted CTGF with an early peak response in 8–12 hours that diminished by 24 hrs. Cells on CL showed a more gradual rise in CTGF levels that reached a similar peak by 72 hrs. Continuously increasing amounts of alpha–smooth muscle–actin (α–SMA) were detected in TGF–ß–treated cells on FN and CL, indicating myofibroblast differentiation. Cells plated on VN synthesized CTGF over the narrowest time range (peaking sharply at 8 hours), and did not synthesize significant α–SMA before 72 hours. Treatment with FGF/heparin on all matrices inhibited new CTGF synthesis and myofibroblast differentiation.
The matrix effect on TGF–ß–induced CTGF secretion suggests cross–talk between growth factor and integrin signaling pathways. Identifying this cross–talk is important for understanding the context–dependent signaling that occurs after wounding. Such understanding could be used to promote healing and limit fibrosis.
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