Effect of tacrolimus on the expression and function of protease-activated receptor 2 in human keratinocytes
- VernacularTitle: 他克莫司对人角质形成细胞蛋白酶活化受体2表达及功能的影响
- Author:
Shangshang WANG
1
;
Chunya NI
;
Ying ZOU
;
Wei LI
;
Jinhua XU
Author Information
- Publication Type:Journal Article
- Keywords: Tacrolimus; Keratinocytes; Calcium signaling; Receptors, thrombin; Receptor, PAR-2
- From: Chinese Journal of Dermatology 2019;52(10):747-752
- CountryChina
- Language:Chinese
-
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 (allP < 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 (bothP < 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 the PAR-2 protein expression was observed between the 10-8- or 10-9-mol/L tacrolimus group and the control group (bothP > 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: 1 463 ± 283, 1 455 ± 270, 1 423 ± 291 respectively) than in the control group (1 602 ± 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 (1 649 ± 379, 1 633 ± 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.
