The fact that was determined in RT-PCR analyses (Fig. 9, C and D). Only the

The fact that was determined in RT-PCR analyses (Fig. 9, C and D). Only the the RT-PCR analyses on the HaCaT cells utilised resulted in a silencing of TRPC6 diminished the 7696-12-0 Autophagy hyperforin-induced cal- broad profile of expressed TRPC channels, we lastly decided to cium influx (Fig. 9A), delivering additional proof for the central knock down all TRPC channels in parallel experimental set ups. function of TRPC6 channels in keratinocyte physiology. Further- This broad method clearly showed that the hyperforin-medimore, we tested the effect of TRPC knockdown on higher ated effect in HaCaT cells were mediated by TRPC6. As well as the acute effects on intracellular ion concen[Ca2 ]o-induced calcium influx. In contrast to the clear outcome on the hyperforin-induced calcium entry, the part of TRPC tration, hyperforin is also inducing differentiation in HaCaT channels in higher [Ca2 ]o-induced calcium influx is considerably extra and hPK cells by means of TRPC6 channels. Disturbed keratinocyte difDECEMBER 5, 2008 VOLUME 283 Quantity 49 JOURNAL OF BIOLOGICAL CHEMISTRYTRPC6 Channel Function in Human Keratinocytesdifferentiation. Furthermore, the TRPC6 expression pattern is linked towards the differentiation state influenced by hyperforin or Ca2 . In addition, we proved the ex vivo relevance of TRPC6 channels in human skin explants. Ca2 and hyperforin induced to a similar extent the expression of TRPC6 in hPKs and in quick term cultured human skin explants. Inside the skin explants, TRPC6 was primarily expressed by 58652-20-3 site stratum spinosum and stratum granulosum keratinocytes and not in basal keratinocytes, supporting our findings that keratinocyte epidermal differentiation will depend on TRPC6 expression. Part of TRPC Channels in Keratinocyte Differentiation–Because their expression levels alter inside a differentiation-dependent manner, functional properties of TRPC channels in keratinocytes have already been suggested to become involved in differentiation, that is regulated by Ca2 FIGURE 9. Role of other TRPC channels in hyperforin- and higher calcium-induced effects in keratinocytes. influx (12, 14, 15). Lately, TRPC1 HaCaT keratinocytes were transfected with handle siRNA and the respective siRNA for every single TRPC channel. has been implicated inside the Ca2 -inAfter an incubation period of 48 h, HaCaT cells were loaded with fura-2 and have been stimulated with hyperforin (A) or Ca2 2 mM (B) (n six; , p 0.1; , p 0.01, unpaired t test; ns, nonsignificant). C, the effectiveness from the duced terminal differentiation of respective RNAi transfection was analyzed in RT-PCR experiments. D, histogram showing the relative expres- human keratinocytes in vitro (14, sion on the TRPC channels, compared with their normalized expression levels in untransfected, untreated 24). However, silencing TRPC1 did HaCaT cells (n three). not totally block keratinocyte differentiation, suggesting that ferentiation and proliferation have been detected in many skin other TRPC channels may perhaps also be involved, particularly illnesses like AD and psoriasis (5). Many TRPC channels simply because they are identified to type multimers in vivo. TRPC4 and such as TRPC6 are discussed as playing a major function for the TRPV6 have also been reported to take element in keratinocyte Ca2 -mediated regulation of keratinocyte differentiation (12). differentiation (15, 25). Our final results regarding the involvement of However, investigating their individual function was hampered by TRPC1, TRPC3, TRPC4, and TRPC6 in the high [Ca2 ]o-inthe lack of precise stimulation or inhibitors. For the reason that we have.