We have previously reported that various stimuli, including sphingosine 1-phosphate, are able to induce heat shock protein (HSP) 27 in osteoblast-like MC3T3-E1 cells. However, the precise role of HSP27 in bone metabolism has not been satisfactory clarified. In this study, we investigated the effect of HSP27 on osteocalcin synthesis induced by bone morphogenetic protein (BMP)-4 or T₃ in these cells. In MC3T3-E1 cells, pretreatment with sphingosine 1-phosphate, sodium arsenite, or heat stress caused the attenuation of osteocalcin synthesis induced by BMP-4 or T₃ with concurrent HSP27 induction. To further investigate the effect of HSP27, we established stable HSP27-transfected cells. The osteocalcin synthesis was significantly reduced in the stable HSP27-transfected MC3T3-E1 cells and normal human osteoblasts compared with empty-vector transfected cells. On the other hand, anisomycin, a p38 MAPK activator, caused the phosphorylation of HSP27 in both sphingosine 1-phosphate-stimulated untransfected MC3T3-E1 cells and HSP27-transfected MC3T3-E1 cells. An immunofluorescence microscopy study showed that the phosphorylated HSP27 induced by anisomycin concentrated perinuclearly in these cells, in which it colocalized with the endoplasmic reticulum. We also established stable mutant-HSP27-transfected cells. Osteocalcin synthesis induced by either BMP-4 or T₃ was markedly suppressed in the nonphosphorylatable HSP27-overexpressing MC3T3-E1 cells compared with the phosphomimic HSP27-overexpressing cells. In contrast, the matrix mineralization was more obvious in nonphosphorylatable HSP27-overexpressing cells than that in phosphomimic HSP27-overexpressing cells. Taken together, these results strongly suggest that unphosphorylated HSP27 has an inhibitory effect on osteocalcin synthesis, but has a stimulatory effect on mineralization, in osteoblasts.
Heat shock protein 27, one of the low molecular weight stress proteins, is recognized as a molecular chaperone; however, other functions have not yet been well established. Phosphorylated heat shock protein 27 levels inversely correlate with the progression of human hepatocellular carcinoma. This study shows that phosphorylated heat shock protein 27 interferes with cell growth of the hepatocellular carcinoma-derived HuH7 cells in the presence of the proinflammatory cytokine, tumor necrosis factor-␣, via inhibition of the sustained activation of the extracellular signal-regulated kinase signal pathway. The activities of Raf/extracellular signal-regulated kinase and subsequent activator protein-1 transactivation and the induction levels of cyclin D1 were lower in HuH7 cells transfected with phosphorylated heat shock protein 27 than those with unphosphorylated heat shock protein 27. Moreover, phosphorylated heat shock protein 27 up-regulated the levels of p38 mitogen-activated protein kinase and mitogen-activated protein kinase phosphatase-1, an inhibitory protein of extracellular signal-regulated kinase. These results indicate that phosphorylated heat shock protein 27 might suppress the extracellular signal-regulated kinase activity in the hepatocellular carcinoma cells via two separate pathways in an inflammatory state. The extracellular signal-regulated kinase activity is inversely correlated with phosphorylated heat shock protein 27 at serine 15 and also in human hepatocellular carcinoma tissues in vivo. Because the extracellular signal-regulated kinase signal pathway is a major proliferation signal of hepatocellular carcinoma, activator protein-1 activation is an early event in hepatocarcinogenesis. These findings strongly suggest that the control of the phosphorylated heat shock protein 27 levels could be a new therapeutic strategy especially to counter the recurrence of hepatocellular carcinoma.The mammalian small stress protein, heat shock protein (HSP) 2 27, is a widely expressed 27-kDa protein, and it is one of 10 members of the human low molecular weight HSP family. HSPs are classified into high molecular weight HSPs such as HSP70 and HSP90, and low molecular weight HSPs with molecular masses from 10 to 30 kDa based on their apparent molecular sizes. Low molecular weight HSPs have significant similarities in terms of amino acid sequences, known as the ␣-crystallin domain and WDPF motif (1, 2). The high molecular weight HSPs act as molecular chaperones in protein folding, oligomerization, and translocation (1). Although the functions of low molecular weight HSPs are not as well characterized as those of the high molecular weight HSPs, it is recognized that they may have chaperone activities (1). The functions of HSP27 are regulated by post-translational modifications such as phosphorylation (3, 4). Mouse HSP27 is phosphorylated at two sites (Ser-15 and Ser-82), whereas human HSP27 is phosphorylated at three sites (Ser-15, Ser-78, and Ser-82) (3). Ser-78 and Ser-82 of HSP27 are adjacent to the amino-termin...
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