Expression of protease-activated receptor-2 in the immortalized human sebocyte cell line SZ95 and its role in sebaceous lipid synthesis and inflammation

Abstract

It has been shown recently that the expression of protease-activated receptor-2 (PAR-2) and protease activity are increased in the follicular keratinocytes in comedones of patients with acne, suggesting the role of PAR-2 in acne. PAR-2 is a sensor for various proteases and mediates numerous physiological responses in the skin. But, little is known about the role of PAR-2 on sebocyte biology. In this study, we examined the presence of functional PAR-2 in the immortalized human sebaceous gland cell line SZ95 by analyzing PAR-2 gene expression, in situ immunodetection, and PAR-2-mediated intracellular Ca2+ ([Ca2+]i) signaling. The effects of PAR-2 activation on sebaceous lipogenesis and the lipogenic signal via PAR-2 were investigated. In addition, the effects of PAR-2 on the gene expression of pro-inflammatory cytokines, antimicrobial peptides, and matrix metalloproteinases in sebocytes were assessed.PAR-2 was identified in SZ95 at mRNA and protein levels and in human sebaceous glands by RT-PCR and immunohistochemical analysis. Real-time PCR studies showed that PAR-2 gene level was higher in differentiated SZ95 cells than in undifferentiated cells. Increased PAR-2 expression was observed in the sebaceous glands of patients with acne vulgaris. Stimulation of PAR-2 by activating peptide (AP) or Propionibacterium acnes (P. acnes) culture supernatant led to increased [Ca2+]i in SZ95 in a dose-dependent manner and this increase was significantly inhibited by repeated treatment of P. acnes supernatant, indicating that PAR-2 on sebocytes was functional. AP treatment increased the intracellular lipid droplets in SZ95 sebocytes. PAR-2 regulated the sebaceous lipogenesis through the induction of sterol response element binding protein (SREBP)-1 via PLC/ IP3 pathway. Stimulation with AP or P. acnes supernatant upregulated mRNA of interleukin -1, -6, -8, tumor necrosis factor-α, human beta defensin-2, LL-37, MMP-1, -3, and -9 in SZ95, which was significantly inhibited by serine protease inhibitor as well as PAR-2 specific antagonist. Taken together, these data indicate that functional PAR-2 is expressed in SZ95 sebocytes and mediates lipogenesis and inflammation in response to various proteases including proteases secreted from P. acnes.