Stimulation of p42,44 map kinase phosphorylation by trysin in human cultured prostate srtomat cells A Myatt, DR. Harriss, & SJ Hill. Institute of Cell Signalling, Queen's Medical Centre, Nottingham, UK, NG7 2UH.

Print abstract Search PubMed for: Myatt A Harriss DR Hill SJ

The proteinase activated receptor PAR-2 is strongly expressed in human prostate tissue (Nystedt et al., 1995). Trypsin (a PAR-2 selective serine protease) can activate both p42/44 isoforms of MAP Kinase (Macfarlane et al., 2001). The aim of the present study was to investigate the effect of trypsin on p42/44 MAP Kinase phosphorylation in human cultured prostate stromal cells.

Human prostate stromal cells were isolated from benign prostatic tissue and cultured as previously described (Abdul-Hamid et al., 2001). Cells were grown in six well plates, serum starved for 2 hours and then stimulated with agonists for 3-60 min. Cells were then washed with ice cold PBS and lysis buffer was added to solubilize the cells (20mM Tris/HCl (pH 7.4), 1mM EGTA, 0.5% triton-X100, 1mM NaF, 1mM DTT, 70mM ß-glycerophosphate, protease inhibitor cocktail tablet). The lysate was centrifuged at 15000 rpm for 10min at 4°C. 6x loading buffer (70% 4xTris/HCl/SDS pH 6.8, 30% glycerol, 1%SDS, 0.5M DTT) was added to the supernatant and the resultant sample was then boiled for 5 min. Samples (7µg protein - protein determined by the method of Lowry et al., 1951) were resolved by SDS-PAGE and transferred to nitrocellulose membranes by western blotting. Membranes were probed with a mouse monoclonal antibody (Cell Signalling Technology) for the phosporylated form of p42,44 MAP Kinase (P-MAPK). Membranes were further incubated with a horseradish peroxidase-conjugated goat anti-mouse secondary antibody. Immuno-reactivity was detected with enhanced chemiluminescence. The results were analysed with densitometry and Molecular Analyst software. Statistical significance was determined using the ANOVA test.

Trypsin stimulation for 15 min caused a concentration- dependent increase in P-MAPK over basal levels (p<0.01; pEC₅₀ 8.25±0.29, n=4). This effect was time-dependent and the response to trypsin (30nM) was maximal at 5 min and sustained for 30 min. The PAR-2 peptide SLIGKV (Macfarlane et al., 2001) caused a concentration-dependent increase in P-MAPK (p<0.05; pEC₅₀ 4.74±0.33, n=4) that was maximal at 3 min and sustained for 15 min (100µM SLIGKV). A significant stimulation of MAP Kinase phosphorylation was also observed with the PAR-1 peptide A(p-F)FRCha-HarY-NH₂ (Macfarlane et al., 2001) (p<0.05, pEC₅₀; 5.80±0.75, n=3; maximal at 3 min), but not with the PAR-1 peptide SFLLRNPNDKYEPF (Macfarlane et al., 2001; 3min stimulation with 100µM, n=3). Thrombin, however, did not produce a consistent and significant (by densitometry) increase in P-MAPK (10nM; 15 min stimulation, n=3).

These data show that p42,44 MAP Kinase phosphorylation is significantly stimulated by trypsin and SLIGKV in human prostate stromal cells, but not by thrombin or the PAR-1 peptide SFLLRNPNDKYEPF. These data suggest that the effects of trypsin are mediated via stimulation of PAR-2 receptors in this cell system.

Abdul-Hamid M.A. et al. (2001). Br. J. Pharmacol. 134. 173P.
Lowry O.H. et al. (1951). J. Biol. Chem. 193. 265-275.
Macfarlane S.R. et al. (2001). Pharmacol Rev. 53. 245-282.
Nystedt S. et al. (1995). Eur. J. Biochem. 232. 84-89.