Up-Regulation of 14-3-3and#963; in Hacat upon Co-Cultivation with HEPM

Background: To generate an in vitro system of the dermal epithelial-mesenchymal interaction we co？￠？€？‘cultured HaCaT (keratinocytes) and HEPM (embryonic mesenchyme). This model allowed a local disjunction with preserved cell-cell communication. Methods: For the analysis of different protein expression patterns between co- and pure-cultured HaCaTs we performed 2D-electrophoresis and mass spectrometry. Afterwards the mass spectromertically identified protein 14-3- 3σ was silenced by siRNA in HaCaT cells. Results: We analyzed 28 spots and found 17 different expressed mainly metabolic and cytoskeletal proteins. Interestingly, stratifin (14-3-3σ), maspin and Profilin-1 (PFN1) were up-regulated, whereas Peroxiredoxin-5 (PRDX5), PDZ-and-LIM-domain-protein-1 (CLIM1) and Annexin A1 (ANXA1) were decreased in co-cultured HaCaTs. The specific knock-down of 14-3-3σ resulted in a down-regulation of RhoA, Rac1/2/3, LIMK1 and phosphorylated cofilin, which are involved in cytoskeleton dynamics. Furthermore, reduction of 14-3-3σ coincided with significantly decreased proliferation rates and levels of Ki67. Conclusions: We concluded that the interaction with mesenchymal cells may initiate the alternation of epidermal precursor cells to differentiated keratinocytes, what was confirmed by the up-regulation of different keratins. Furthermore, 14-3-3σ may influence the proliferation-rate of keratinocytes via Rho GTPases