Mechanisms underlying the urotensin II-induced proliferation of and α-smooth muscle actin expression in human dermal fibroblasts

Abstract

Objective To evaluate the effects of urotensin Ⅱ on cell proliferation of and α-smooth muscle actin (α-SMA) expression in normal human dermal fibroblasts (NFs) , and to explore their regulatory mechanisms. Methods NFs were isolated from foreskin tissues and subjected to primary culture in vitro. Reverse transcription PCR and Western blot analysis were performed to measure the mRNA and protein expression of urotensin Ⅱ and its receptor, respectively. Cell counting kit-8 (CCK-8) assay was conducted to estimate the proliferation of NFs, which were treated with urotensin Ⅱ at different concentrations of 0, 10-10, 10-9, 10-8, 10-7 and 10-6 mol/L for 0, 6, 12, 24 and 48 hours separately, and then the optimal concentration and duration of urotensin Ⅱ exposure were selected to be 10-8 mol/L and 24 hours respectively. Some cultured NFs were divided into 5 groups: control group receiving no treatment, UⅡgroup treated with 10-8 mol/L urotensin Ⅱ, UⅡ+ nicardipine group treated with 10-8 mol/L urotensin Ⅱ and the calcium channel blocker nicardipine at the concentration of 10-5 mol/L, UⅡ+ PD98059 group treated with 10-8 mol/L urotensin Ⅱ and the mitogen activated protein kinase (MAPK) inhibitor PD98059 at the concentration of 10-5 mol/L, and UⅡ+ cyclosporine group treated with 10-8 mol/L urotensin Ⅱ and the calcium-dependent protein kinase (CaM PK) inhibitor cyclosporine at the concentration of 10-5 mol/L. After24-hour treatment, CCK-8 assay was conducted to evaluate the proliferation of NFs in the above groups, realtime fluorescence-based quantitative PCR and Western blot analysis were performed to determine the mRNA and protein expression of α-SMA respectively. Results Urotensin Ⅱ receptor was expressed in NFs, but urotensin Ⅱ was not. The proliferative activity of NFs significantly differed among the control group, UⅡ group, UⅡ+ nicardipine group, UⅡ+ PD98059 group and UⅡ+ cyclosporine group (the mean absorbance value at 405 nm: 1.036 ± 0.046, 1.405 ± 0.158, 1.121 ± 0.109, 1.192 ± 0.089 and 1.141 ± 0.056, respectively; F = 9.587, P < 0.01) , and the UⅡ group showed significantly higher proliferative activity of NFs compared with the control group, UⅡ + nicardipine group, UⅡ + PD98059 group and UⅡ + cyclosporine group (q = 8.263, 6.355, 4.774 and 5.912, respectively, all P < 0.05) . There were significant differences in the mRNA and protein expression of α-SMA among the 5 groups (F = 6.351, 7.045, both P < 0.01) , and the mRNA and protein expression of α-SMA was significantly higher in the UⅡ group than in the other 4 groups (all P < 0.05) . Conclusion UrotensinⅡ may induce the proliferation of and α-SMA expression in NFs through calcium channels, MAPK and CaM PK pathways. Key words: Urotensins; Fibroblasts; Cell proliferation; Actins; Cicatrix; Receptor, urotensins