EVIDENCE FOR THE PRESENCE OF A PROTEASE-ACTIVATED RECEPTOR DISTINCT FROM THE THROMBIN RECEPTOR IN HUMAN KERATINOCYTES

Thrombin receptor activation was explored in human epidermal keratinoc ytes and human dermal fibroblasts, cells that are actively involved in skin tissue repair, The effects of thrombin, trypsin, and the recepto r agonist peptides SFLLRN and TFRIFD were assessed in inositolphosphol ipid hydrolysis and calcium mobilization studies, Thrombin and SFLLRN stimulated fibroblasts in both assays to a similar extent, whereas TFR IFD was less potent, Trypsin demonstrated weak efficacy in these assay s in comparison with thrombin. Results in fibroblasts were consistent with human platelet thrombin receptor activation, Keratinocytes, howev er, exhibited a distinct profile, with trypsin being a far better acti vator of inositolphospholipid hydrolysis and calcium mobilization than thrombin, Furthermore, SFLLRN was more efficacious than thrombin, whe reas no response was observed with TFRIFD. Since our data indicated th at keratinocytes possess a trypsin-sensitive receptor, we addressed th e possibility that these cells express the human homologue of the newl y described murine protease-activated receptor, PAR-2 [Nystedt, S., Em ilsson, K,, Wahlestedt, C, & Sundelin, J, (1994) Proc, Natl. Acad, Sci . USA 91, 9208-9212], PAR-2 is activated by nanomolar concentrations o f trypsin and possesses the tethered ligand sequence SLIGRL, SLIGRL wa s found to be equipotent with SFLLRN in activating keratinocyte inosit olphospholipid hydrolysis and calcium mobilization, Desensitization st udies indicated that SFLLRN, SLIGRL, and trypsin activate a common rec eptor, PAR-2, Northern blot analyses detected a transcript of PAR-2 in total RNA from keratinocytes but not fibroblasts, Levels of thrombin receptor message were equivalent in the two cell types, Our results in dicate that human keratinocytes possess PAR-2, suggesting a potential role for this receptor in tissue repair and/or skin-related disorders.