Effect of Connective Tissue Growth Factor on Protein Kinase Expression and Activity in Human Corneal Fibroblasts

Abstract

Abstract: : Purpose: To evaluate expression patterns of protein kinases and protein kinase activities in human corneal fibroblasts treated with connective tissue growth factor (CTGF). Methods: Human corneal fibroblast cultures were grown to confluence and treated with CTGF for 0, 5, and 15 minutes. Cytoplasmic protein extracts were obtained; protein kinase expression and activity arrays were performed using Kinetworks^TM analysis (screening for expression of 75 different protein kinases and 31 different phosphoproteins). Further studies using extended time courses of CTGF exposure in corneal fibroblasts (0, 1, 2, 3, 4, 5, 10, 15, 30, and 60 minutes) were performed using immunoblot analysis to detect expression of protein kinase A catalytic subunit (PKA–cat), and focal adhesion kinase (FAK). All western analysis results were normalized by comparison to beta–actin. Results: After 5 minutes of exposure to CTGF, levels of active proteins increased for 21 of the 75 kinases analyzed. Some notable protein kinases that were induced include: death–associated kinase 1, focal adhesion kinase (FAK), G–protein coupled receptor kinase 2, protein kinase A catalytic subunit (PKA–cat), protein kinase B alpha, and protein kinase C (ε, µ, and ζ, subunits). Extended time course analysis of PKA–cat and FAK showed statistically significant increases in expression following CTGF stimulation within 15 minutes. In addition, several second messengers were phosphorylated after 5 minutes of exposure to CTGF such as extracellular regulated kinases 1,2 (ERK1&2), MAP kinase kinase 1,2 (MEK1/2), signal transducer and activator of transcription 3 (STAT3), and stress–activated protein kinase (JNK). Conclusions: CTGF increased the levels of active protein kinases in human corneal fibroblast cultures, including PKA–cat and FAK after 5 minutes of exposure. CTGF also increases phosphorylation of second messengers including some members of the ERK/MEK pathway. These results further our understanding of the signal transduction mechanism activated by CTGF in corneal fibroblasts and suggest that CTGF mediates processes such as cell proliferation and collagen synthesis by altering levels of protein kinases and phosphorylation of second messengers.

Keywords: growth factors/growth factor receptors • signal transduction • cornea: stroma and keratocytes