An Keratinocytesnormalization clearly show that incubation inside the presence of higher [Ca2 ]o also as hyperforin enhanced the transcription of early and late keratinocyte differentiation markers. Hyperforin Inhibits Proliferation in HaCaT Keratinocytes– Along with differentiation, proliferation of keratinocytes is also controlled by intracellular free Ca2 concentration. Hence, we performed proliferation measurements with the bromodeoxyuridine immunoassay kit (Chemicon). Synchronized HaCaT keratinocytes incubated with high [Ca2 ]o for three days showed significantly decreased proliferation (Fig. 2A). Notably, hyperforin (1 M) also inhibited the proliferation of keratinocytes, as shown in Fig. 2A. To confirm these findings, we analyzed the expression of the nuclear proliferation marker protein Ki-67 by Western blotting. Ki-67 is expressed in cells undergoing the S/G2/M transition and serves as a well established marker to ascertain proliferating cells (21). As shown in Fig. 2B, protein expression of Ki-67 is similarly decreased in HaCaT cells treated Sorbinil Biological Activity either with hyperforin or high [Ca2 ]o. To exclude toxic effects induced by FIGURE 3. Hyperforin induces nonselective cation influx in HaCaT keratinocytes. A, representative time hyperforin, we performed MTT two traces show hyperforin-induced alterations in [Ca ]i in fura-2-loaded HaCaT and hPK cells. Hyperforin (Hyp, ten assay (Fig. 2C). The test showed M) was added 50 s just after the start out with the experiment. B, HaCaT cells and hPKs were stimulated with various concentration of hyperforin (n six). clearly that hyperforin had no influ-FIGURE four. Carbachol-, 1-oleoyl-2-acetyl-sn-glycerol-, and hyperforin-induced current in HaCaT keratinocytes. Whole cell recording of unselective cation currents in HaCaT cells had been obtained in response to 1-oleoyl-2-acetyl-sn-glycerol (OAG, A), carbachol (CCh, B), and hyperforin (hyp, C). The data are gathered from voltage ramp from 100 to one hundred mV. Left panels, currents measured at one hundred and one hundred mV are plotted with time. The presence of the drugs is shown by horizontal bars. Middle panels, shown would be the corresponding I relationships on the cells inside the left panels measured ahead of and through maximal agonist response. Proper panels, the imply current amplitudes are presented as bars (n 8 for one hundred M 1-oleoyl-2-acetyl-sn-glycerol, n six for 100 M carbachol, n 13 for 20 M hyperforin). Ctr, control.DECEMBER 5, 2008 VOLUME 283 NUMBERJOURNAL OF BIOLOGICAL CHEMISTRYTRPC6 Channel Function in Human Keratinocytescurrents in keratinocytes (Fig. 4). As shown in Fig. 3A, hyperforin (10 M) reproducibly induced fast and transiently elevations of calcium-dependent fluorescence in fura-2-loaded HaCaT keratinocytes and in hPKs. The response was L-Glucose Cancer suppressed in the presence of Ca2 -free measuring buffer (supplemental Fig. S1), indicating that the hyperforin-induced impact is mainly mediated by an influx across the plasma membrane. The hyperforin-mediated adjustments in fluorescence have been concentration-dependent, and in some cases at low concentrations (1 M) significant elevations were reproducibly detectable (Fig. 3B). For further characterization, we substituted calcium in the buffer by barium or strontium ions, resulting in enhanced fluorescence upon the application of hyperforin (supplemental Fig. S1). Moreover, the hyperforin-mediated alterations in fluorescence have been suppressed within the presence of several compounds (gadolinum chloride, lanthanum chloride, SK F 96365, 2-aminophenoxyborate, and N-(p-amylcinnamoyl).
