TGF-Beta Increases Proteinase-Activated Receptor 2 (PAR2) Expression and Upregulates PAR2 Activation-Induced Secretion of IL-6 and IL-8 in Endometriotic Stromal Cells.

Proteinase-activated receptor 2 (PAR2) is a G protein-coupled receptor that is activated by serine proteases such as trypsin, tryptase, and coagulation factors VIIa and Xa. A large body of evidence has shown that PAR2 plays an important role in various inflammatory events including endometriosis. We previously reported that PAR2 activation by a specific PAR2 agonist peptide (PAR2AP) in cultured endometriotic stromal cells (ESC) increased secretion of interleukin (IL)-6 and IL-8, inflammatory mediators involved in the development of endometriosis. PAR2 activation also stimulates proliferation of ESC, indicating an important role of PAR2 in the progression of the disease. However, the regulatory mechanism of PAR2 expression in ESC is unknown. Recent studies have shown that transforming growth factor beta (TGF β) increases PAR2 expression in osteoarthritis chondrocyte and fibroblast. Interestingly, TGFβ is expressed in the tissues of ovarian endometrial cyst and its concentration is high in the cyst fluid. In view of these findings, we are prompted to study whether TGFβ has any implication in the development of endometriosis via regulation of PAR2. ESC were isolated from cyst walls of ovarian endometrial cysts and cultured for the following experiments. First, ESC was stimulated with TGFβ (10ng/ml) for various times (3, 6, 12, 24, 48H). PAR2 mRNA levels were examined by real-time reverse transcription-polymerase chain reaction analysis using RNA extracted from the ESC. Secondly, ESC was stimulated with TGFβ for various concentrations (1, 5, 10ng/ml) for 24H, and then incubated with PAR2AP (30 μM) for the following 24H. After the incubation, the concentrations of IL-6 and IL-8 of the supernatants were measured using specific enzyme-linked immunosorbent assay kits. Third, ESC was cultured with SB431542, an inhibitor of TGFβ type I receptor, (10μM) for 24H, and then stimulated with PAR2AP (30μM) for the following 24H. The concentrations of IL-6 and IL-8 of the supernatants were measured. TGFβ increased PAR2 mRNA expression with the maximum level of 3.2 fold of the basal level at 6H. TGFβ also increased PAR2 mRNA expression in a dose-dependent manner, the maximum level obtained at 10ng/ml. PAR2AP alone increased IL-6 secretion to 2.8 fold of the basal level, whereas TGFβ pretreatment dose-dependently upregulated PAR2AP-induced IL6 secretion to 9.8 fold at 10ng/ml. Likewise, PAR2AP alone increased IL-8 secretion to 1.2 fold of the basal level, whereas TGFβ pretreatment dose-dependently upregulated PAR2AP-induced IL8 secretion to 2.3 fold at 10ng/ml. SB431542 decreased PAR2 mRNA level to 13% of the basal level, suggesting the involvement of autocrine/ paracrine regulation of PAR2 expression by endogenous TGFβ form ESC. SB431542 pretreatment suppressed PAR2AP-induced IL-6 secretion to 65% of the control that is without SB431542 and PAR2AP. Similarly, SB431542 pretreatment significantly suppressed PAR2AP-induced IL-8 secretion to 63% of the control, which was substantially the same level without PAR2AP but with SB431542 pretreatment. The findings of the present study indicate that TGFβ stimulates PAR2 expression in ESC and upregulates PAR2 activation-induced IL-6 and IL-8 secretion in ESC. TGFβ may be involved in the development of endometriosis by regulating the expression of PAR2.