Keratinocytes secrete many mitogenic or melanogenic molecules for melanocytes. Among several keratinocyte-derived cytokines, SCF and ET-1 are regarded as main mediators involved in skin pigmentation. Protease-activated receptor (PAR)-2 is a member of G-protein coupled seven transmembrane receptor family. PAR-2 is known to be involved in the production of inflammatory mediators as well as melanosomes transfer. In this study, we would like to investigate whether direct activation of PAR-2 by PAR-2 agonist can induce the production of SCF and ET-1 in keratinocytes.
The expression of SCF and ET-1 in HaCaT cells was examined after PAR-2 activation with PAR-2 activating peptide (PAR-2 AP, SLIGKV-NH2) and kallikrein 5 in western blotting and real time RT-PCR. After treatment of PAR-2 AP and kallikrein 5, SCF and ET-1 in HaCaT cells were increased in the levels of protein and mRNA. To confirm if the increase of SCF and ET-1 by PAR-2 AP and kallikrein 5 was directly associated with PAR-2 activation, incubation of PAR-2 AP and kallikrein 5 after pretreatment of PAR-2 antagonist and knockdown of PAR-2 expression by PAR-2 siRNA technique were performed. After treatment with PAR-2 antagonist, increased SCF and ET-1 after kallikrein 5 was not observed. In addition, treatment with PAR-2 AP did not increase SCF and ET-1 in PAR-2 siRNA-treated HaCaT cells contrary to scrambled siRNA-treated HaCaT cells.
In conclusion, our study suggested that PAR-2, which is known to act as a gate of melanin transfer might directly mediate skin pigmentation via production of keratinocyte-derived mediators such as SCF and ET-1.