Involvement of stress-activated protein kinase/c-Jun N-terminal kinase in endothelin-1-induced heat shock protein 27 in osteoblasts

Objective: We have reported that endothelin-1 (ET-1) activates p38 mitogen-activated protein (MAP) kinase through protein kinase C in osteoblast-like MC3T3-E1 cells, and that p38 MAP kinase plays a role in the ET-1-induced heat shock protein 27 (HSP27). Recently, we found that stress-activated protein kinase/c-lun N-terminal kinase (SAPK/JNK) is activated by ET-I in these cells. In the present study, we have investigated the involvement of SAPK/JNK in ET-1-induced HSP27 in MC3T3-E1 cells. Methods: The concentration of HSP27 in soluble extracts of the cells, the expression of mRNA for HSP2 7, and the phosphorylation of SAPK/JNK were determined by an enzyme immunoassay, Northern blot analysis, and Western blot analysis respectively. Results: SP600125, a specific inhibitor of SAPK/JNK, markedly reduced ET-1-stimulated HSP27 accumulation. The inhibitory effect of SP600125 was dose dependent in the range between I and 50 muM. SP60012 5 reduced the ET-1-increased level of HSP2 7 mRNA. Calphostin C and Go 69 76, inhibitors of protein kinase C, reduced the ET-1-induced phosphorylation of SAPK/JNK. 12-O-Tetra-decanoylphorbol-13-acetate, a direct activator of protein kinase C, induced SAPK/JNK phosphorylation, which was suppressed by SP600125. A combination of SP600125 and p38 MAP kinase inhibitor such as SB203580 and PD169316 additively reduced the ET-1-stimulated accumulation of HSP27. Conclusions: These results strongly suggest that JNK plays a part in ET-1-induced HSP2 7 in addition to p38 MAP kinase in osteoblasts.