Abstract
Purpose: :
Corneal fibroblasts respond to wounding by secreting new extracellular matrix and growth factors, including Connective Tissue Growth Factor (CTGF), a matricellular protein. CTGF is implicated in normal and fibrotic wound healing in various cell types where it may regulate proliferation or apoptosis, migration or differentiation, and collagen synthesis. CTGF is comprised of four modules that can interact with numerous binding partners. For insights into its role in human cornea, we evaluated the regulation of CTGF synthesis, post-translational processing, and subcellular localization in human corneal fibroblasts (HCFs).
Methods: :
HCFs were plated on collagen and grown in supplemented serum-free medium with or without TGF-β1 or FGF-2. RT-PCR was performed on mRNA extracted after 24 hrs of treatment. Protein extracted from HCF cultures and their conditioned media treated with TGF-β for 8-72 hrs was subjected to Western blot analysis using antibodies to N-terminal, central, and C-terminal epitopes of CTGF. Immunocytochemistry using the same CTGF antibodies and laser scanning confocal microscopy was performed on TGF-β-treated, non-confluent HCFs (24-72 hrs), or on confluent HCF monolayers that were scrape-wounded (8 hrs). In addition, we immunodetected the same cells for CTGF and endocytic markers (LRP and EEA1), focal adhesion components (FAK and vinculin), uPAR, integrins αvβ3 and α5β1.
Results: :
TGF-β treatment of HCFs induced a dose-dependent increase in CTGF mRNA, whereas FGF inhibited it. At the protein level, TGF-β treatment resulted in post-translational processing that generated several distinct forms of CTGF, including a novel 31 kDa form lacking the N-terminal epitope and enriched in the cytoskeletal/ECM fraction. Antibodies to the central region and the C-terminus detected CTGF in the Golgi apparatus and adjacent vesicles. N-terminal and C-terminal domain antibodies also detected vesicles in the cell’s periphery. In addition many CTGF-containing vesicles co-localized with endosomal markers, LRP and EEA1. Although CTGF co-localized with α5β1, it did not co-localize with FAK, vinculin, uPAR, or αvβ3.
Conclusions: :
We show that TGF-β and FGF, previously implicated in divergent (opposing) effects on fibroblast differentiation, also have contrasting effects on CTGF mRNA regulation in HCFs. The presence of multiple CTGF forms in cell lysates and in cytoplasmic vesicles, including the newly identified 31 kDa form, suggests that processing might unmask new sites on CTGF that, by interacting with multiple receptors and receptor-mediated pathways, may account for the diverse effects of CTGF.
Keywords: cornea: stroma and keratocytes • growth factors/growth factor receptors • wound healing