Endothelin-1 stimulates heat shock protein 27 induction in osteoblasts: involvement of p38 MAP kinase

Abstract

We previously reported that endothelin-1 (ET-1) activates p42/p44 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells and consequently induces synthesis of interleukin-6. In the present study, we investigated the effect of ET-1 on the induction of heat shock protein 27 (HSP 27) in MC3T3-E1 cells. ET-1 time and dose dependently stimulated HSP 27 accumulation. ET-1 induced an increase in the levels of mRNA for HSP 27. Both staurosporine and calphostin C, inhibitors of protein kinase C (PKC), suppressed the ET-1-induced HSP 27 accumulation. 12-O-tetradecanoylphorbol 13-acetate (TPA), a PKC activator, induced the HSP 27 accumulation and the expression of mRNA for HSP 27. The ET-1-stimulated HSP 27 accumulation was reduced in PKC-downregulated MC3T3-E1 cells. The HSP 27 accumulation by ET-1 was not suppressed by PD-98059, an inhibitor of the upstream kinase that activates p42/p44 MAP kinase. ET-1 or TPA induced the phosphorylation of p38 MAP kinase. SB-203580, an inhibitor of p38 MAP kinase, reduced the ET-1-stimulated HSP 27 accumulation. Calphostin C and U-73122, a phospholipase C inhibitor, suppressed the ET-1-induced phosphorylation of p38 MAP kinase. U-73122 and propranolol, a phosphatidic acid phosphohydrolase inhibitor, reduced the ET-1-stimulated HSP 27 accumulation. SB-203580 suppressed the ET-1-stimulated increase in the mRNA levels for HSP 27. These results strongly suggest that ET-1 stimulates HSP 27 induction in osteoblasts and that p38 MAP kinase activation is involved in the HSP 27 induction.

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

“…The maximum effect of SP600125 on HSP27 accumulation was observed at 10 mmol/l, which caused about an 80% reduction in the ET-1 effect. We previously reported that ET-1 increased the levels of HSP27 with a Western blot analysis using the antibodies for HSP27 utilized in the immunoassay (14). We confirmed that the ET-1-increased HSP27 levels were similarly detectable using the commercially obtained antibodies for HSP27 (Fig.…”

“…It is recognized that HSP27 phosphorylation is catalyzed by the MAP kinases such as p38 MAP kinase (1). We previously reported that the phosphorylation of p38 MAP kinase induced by ET-1 occurs within 10 min and returns to the basal level until 60 min after the ET-1 stimulation, suggesting that the ET-1-activated p38 MAP kinase affects their substrates within 1 h (14). On the other hand, the levels of HSP27 in unstimulated MC3T3-E1 cells were quite low (, 1 ng/mg.protein) (14).…”

“…Three major MAP kinases, p44/p42 MAP kinase, p38 MAP kinase, and SAPK/JNK, are recognized to transduce signals in mammalian cells (27). We earlier reported that ET-1 activates p44/p42 MAP kinase and p38 MAP kinase in osteoblast-like MC3T3-E1 cells and that p38 MAP kinase, but not p44/p42 MAP kinase, plays a role in ET-1-stimulated HSP27 induction (13,14). We have recently demonstrated that ET-1 activates SAPK/JNK in addition to p44/p42 MAP kinase and p38 MAP kinase in MC3T3-E1 cells (15).…”

“…We have previously shown that SB203580, a specific inhibitor of p38 MAP kinase (25), significantly suppressed ET-1-induced accumulation of HSP27 and the mRNA levels for HSP27 (14). We therefore examined the combination effect of SP600125 and SB203580 on ET-1-stimulated HSP27 accumulation.…”

“…We have shown that ET-1 stimulates phosphatidylcholine hydrolysis by PLD in osteoblast-like MC3T3-E1 cells (12). In addition, we have reported that ET-1 activates both p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase in these cells, and that p38 MAP kinase, but not p44/p42 MAP kinase, plays a role in ET-1-stimulated HSP27 induction (13,14). Recently, we demonstrated that ET-1 activates stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), another member of the MAP kinase family (15).…”