Effect of tacrolimus on the expression and function of protease-activated receptor 2 in human keratinocytes / 中华皮肤科杂志

ABSTRACT

Objective To evaluate the in vitro effect of tacrolimus on the expression and function of protease-activated receptor 2(PAR-2)in cultured human keratinocytes. Methods After 24-hour co-culture of human keratinocytes with 10-9-10-5 mol/L tacrolimus, semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR)was performed to determine the mRNA expression of PAR-2, immunofluorescence(IF)staining and Western blot analysis were performed to determine the protein expression of PAR-2 in the keratinocytes, and the fluorescent calcium probe fluo-4 was used to evaluate the effect of tacrolimus at different concentrations on the intracellular calcium concentration after the activation of PAR-2 in the keratinocytes. The group treated without tacrolimus served as control group. One-way analysis of variance was used to compare the PAR-2 expression and calcium concentration in the human keratinocytes in different groups, and least significant difference(LSD)-t test was carried out for multiple comparisons. Results PAR-2 was expressed on both the membrane and cytoplasm of keratinocytes. After 24-hour co-culture of keratinocytes with 10 - 9- 10 - 5 mol/L tacrolimus, the PAR-2 mRNA expression significantly decreased in these cells compared with the control group(all P < 0.05), and was negatively correlated with the tacrolimus concentration(r=-0.962, P=0.009). IF staining and Western blot analysis showed that the PAR-2 protein expression was significantly lower in the 10-5- and 10-6-mol/L tacrolimus groups than in the control group (both P < 0.05), and decreased to a certain extent in the 10-7-mol/L tacrolimus group(IF staining:P<0.05;Western blot analysis:P>0.05). No significant difference in thePAR-2 protein expression was observed between the 10-8-or 10-9-mol/L tacrolimus group and the control group(both P>0.05). After 24-hour co-culture, the peak concentration of intracellular calcium after PAR-2 activation was significantly lower in the 10-5-, 10-6- and 10-7-mol/L tacrolimus groups (peak absorbance:1463 ± 283, 1455 ± 270, 1423 ± 291 respectively)than in the control group(1602 ± 407;t=2.582, 2.821, 2.923, P=0.032, 0.022, 0.019, respectively), while there was no significant difference between the 10-8-or 10-9-mol/L tacrolimus group(1649 ± 379, 1633 ± 415 respectively)and the control group(t=0.846, 0.462, P=0.422, 0.657, respectively). Conclusion Tacrolimus can inhibit PAR-2 expression and suppress calcium mobilization induced by a PAR-2 agonist in keratinocytes.