We investigated the effect of prostaglandin E2 (PGE2) on the induction of heat shock protein 27 (HSP27) and HSP70, and the mechanism behind the induction in osteoblast-like MC3T3-E1 cells. PGE2 time-dependently increased the level of HSP27 without affecting the level of HSP70. PGE2 stimulated the accumulation of HSP27 dose-dependently in the range between 10 nM and 10 microM. PGE2 stimulated the increase in the level of the mRNA for HSP27. Staurosporine and calphostin C, inhibitors of protein kinase C (PKC), suppressed the PGE2-induced HSP27 accumulation. The effect of PGE2 on HSP27 accumulation was reduced in the PKC down-regulated cells. BAPTA/AM, a chelator of intracellular Ca2+, or TMB-8, an inhibitor of intracellular Ca2+ mobilization, reduced the accumulation of HSP27 induced by PGE2. Dibutyryl cAMP had little effect on the basal level of HSP27. PGE2 induced the phosphorylation of both p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase. PD98059 and U-0126, inhibitors of the upstream kinase of p44/p42 MAP kinase, reduced the accumulation of HSP27 induced by PGE2. SB203580, a specific inhibitor of p38 MAP kinase, suppressed the HSP27 accumulation induced by PGE2. U-73122, an inhibitor of phospholipase C, and calphostin C reduced the PGE2-induced phosphorylation of both p44/p42 MAP kinase and p38 MAP kinase. These results indicate that PGE2 stimulates the induction of HSP27 through PKC-dependent activations of both p44/p42 MAP kinase and p38 MAP kinase in osteoblasts.
H Tokuda, O Kozawa, M Niwa, H Matsuno, K Kato and T Uematsu
K. Tamura, M. Kobayashi, S. Suzuki, Y. Ishii, S. Koyama, H. Yamada, K. Hashimoto, M. Niwa and F. Shibayama
Monoclonal antibodies (McAb) and polyclonal antibodies (PcAb) against human insulin-like growth factor-I (somatomedin C; hIGF-I) were produced. Using these two antibodies, an enzyme-linked immunosorbent assay (ELISA) system for hIGF-I was established. The ELISA system was able to detect hIGF-I at a range of 1–25 μg/l, compared with the range of 1–50 μg/l detected by radioimmunoassay (RIA). Human IGF-II and human insulin could not be recognized in this system. The plasma concentrations of IGF-I found using the ELISA agreed well with those found using RIA after conventional Sep-Pak C18 cartridge pretreatment. Epitopes of hIGF-I to McAb and PcAb were investigated by enzymatic digestion of hIGF-I followed by comparing the affinity of the antibodies to the peptides obtained proteolytically. The epitope to McAb was found to be a peptide containing Leu10-Val11-Asp12 (epitope 2). Five epitopes to PcAb containing the following key fragments were identified: a conformational structure formed by the disulphide bonds between Cys6 and Cys48, and between Cys47 and Cys52 (epitope 1), Leu10-Val11-Asp12 (epitope 2), Val17-Cys18-Gly19-Asp20 (epitope 3), Arg21-Gly22-Phe23-Tyr24 (epitope 4) and Lys68-Ser69-Ala70 (epitope 5). Of these, the peptide containing epitope 5 showed the highest affinity to PcAb. The results indicated that our ELISA system combined recognition by epitope 2 of McAb and recognition by epitope 5 of PcAb to obtain its good specificity.
Journal of Endocrinology (1990) 125, 327–335