Changes in Bcl2; Bax; JNK, P65 and Smad 7 Genes in Conjunctival Myofibroblasts Exposed to NGF.

Purpose: Nerve Growth Factor (NGF) might be involved in both healing and fibrotic process occurring in conjunctiva as a result of pathological conditions. We previously described the profibrogenic effect of NGF on conjunctival Fibroblasts, partially mediated by the release of Transforming Growth Factor-β1 (TGFβ1). The aim of this study was to quantify and compare caspase, cJun, p65-NFkB and smad7 expression and/or activation in NGF and TGFβ1 exposed myoFBs. Methods: Human conjunctival Fibroblast (Innoprot.com) were expanded and TGFβ1-induced myofibroblast (myoFBs) were developed according to a stabilize setting. Serum-starved myoFBs were exposed to single/repeated increasing doses of NGF (0-200ng/mL) or TGFβ1 (10ng/mL) for 15min and 1, 3, 5, 24, 48 and 72 hrs. MTS and HO342 staining were performed. Pellets were analysed for Bcl2, Bax, smad7 and caspase3 by real-time PCR, FACS and confocal analysis. Both dosing and timing exposure results were statistically compared. Results: NGF effects on myoFBs are strongly dependent on expression ratio of trkANGFR/p75NTR. TGFβ1 by itself does not trigger significant p75NTR expression, retaining a high trkANGFR/p75NTR ratio. Further exposure to NGF shifted trkANGFR/p75NTR ratio in favour to p75NTR expression, with maximum effect at chronic NGF treatment. NGF triggered Bax expression, without affecting significantly Bcl2 expression. Smad7 was up-regulated in those myoFBs treated with NGF (p<.05). By contrary, TGFβ1 inhibited the pro-apoptotic activities and downregulated Bax expression (p<.05). Phosporylation of cJun and p65-NFkB translocation were detected as later as at chronic 100ng/mL NGF exposure. Conclusions: Together these findings suggest that NGF triggers apoptosis selectively in p75NTR-bearing myoFBs, mainly due to a selective p75NTR-dependent p65 translocation and JNK activation. The molecular mechanism behind this effect may also involve a deregulation of TGFβ1 signaling, as NGF exposure resulted also in Smad7 gene expression, a component of the TGFβ1 pathway with inhibitory activities.