084 Intracellular signalling of phytoestrogens in relation to VEGF receptors expression by hair follicle cells.

S. Lachgar, M. Charveron, M.E. Balana, Y. Gall, Pierre Fabre research Institute, Faculté de Médecine Rangueil, Toulouse, France

Many studies indicate the impact of phytoestrogens (PHO) in normal physiology of humans and animals. They have demonstrated to possess numerous biological properties such as inhibition of tyrosine kinase activity. The mechanisms by which these PHO exert their effect are multifactorial and tissue-specific. The impact of PHO on hair growth was less extensively investigated. Published reports indicate that treatment of mouse keratinocytes with selective PKC inhibitors stimulates their proliferation and in vivo stimulates murine hair growth (Yokoo et al,1999). These data led us to study firstly the effect of PHO on PKC and then after to test the hypothesis that the vascular responses of hair follicle cells in vitro are linked to the effects of PHO on PKC activity and expression. We evaluated by Western- blot analysis the effect of two total plant extracts: soya and pueraria in comparison with two free forms (genistein, daidzein) and two conjugated forms (daidzin,puerarin) on the expression of PKC alpha and delta isoforms. We also evaluated their effect on PKC activity in cultured human hair follicle dermal papilla cells (DPC). In parallel, we evaluated by Western-blot and RT-PCR the effect of PHO on VEGF receptors (flt-1 and Flk-1) expression on DPC. The direct effect of PHO on hair growth was assessed on cultured human isolated hair follicles. Our results showed a dose-dependent inhibition of PKC alpha and delta expression in the presence of PHO and specific inhibitors of PKC isoforms. PKC activity was also seen to be markedly decreased in the presence of genistein, daidzein and soya extracts (42% inhibition for 1 µM genistein and 44% for soya extract at 1µg/ml). The PKC activity was less reduced in the presence of pueraria extract at 1µg/ml (10% inhibition). The inhibition induced by genistein is similar to that of a PKC inhibitor : Calphostin C (45% inhibition). However, we observed an increase of VEGF receptors gene and protein expression in the presence of PHO. Specific inhibitors of PKC isoforms also promoted Flt-1 expression by DPC. An improvement of hair follicle growth is observed in the presence of PHO. Our results showed that Flt-1 and KDR expression within hair DPC is mainly mediated through the PKC pathway. This signalization is modified in the presence of PHO and chemical protein kinase inhibitors : an overexpression of VEGF receptors is observed in DPC. It will be of interest to examine the functional impact and significance of protein kinase C modulation on the established vasculature of the hair follicle.