Bone metabolism is finely orchestrated by

Bone metabolism is finely orchestrated by two types of antagonistic functional cells, bone-forming osteoblasts and bone-resorbing osteoclasts [6]. Bone tissue is continuously regenerated through a process “bone remodeling”, resulting in the maintenance of proper bone mass and quality [7]. The imbalance of bone formation and resorption causes metabolic bone diseases including osteoporosis. It is firmly established that osteoblasts play a crucial roles to regulate not only bone formation but also bone resorption through the axis of receptor-activator of nuclear factor-kB (RANK)-RANK-ligand (RANKL)-osteoprotegerin [8]. With regard to HSP27 in osteoblasts, it has been shown that the expression of HSP27 mRNA is transiently upregulated, accompanying with the down-regulation of proliferation [9]. Additionally, estrogen reportedly facilitates the heat-induced HSP27 expression [10]. On the other hand, prostaglandins (PGs) are well known to act as autocrine/paracrine modulators in bone metabolism [11]. As for prostaglandin D2 (PGD2), PGD2 reportedly induces collagen synthesis of osteoblasts during the process of calcification [12]. We have previously shown that PGD2 elicits the induction of HSP27 in osteoblast-like MC3T3-E1 TASIN-1 and that stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and p38 mitogen-activated protein (MAP) kinase positively regulate the HSP27 induction [13], [14]. In addition, we also demonstrated that the calcification of osteoblast-like MC3T3-E1 cells is modulated by HSP27 protein levels and its phosphorylation state [15]. Regarding HSP90 in osteoblasts, bisphosphonate which is one of the most useful medicine for osteoporosis treatment, reportedly induces the expression of HSP90 [16]. It has recently been shown that the levels of HSP90 protein are upregulated by a low-intensity pulsed ultrasound stimulation, related to mineralized nodule formation [17]. In our recent study [18], we have demonstrated that HSP90 inhibitors augment endothelin-1-induced HSP27 through SAPK/JNK but not p38 MAP kinase in osteoblast-like MC3T3-E1 cells. However, the exact roles both of HSP27 and HSP90 in osteoblasts remain unclear. In the present study, we investigated whether HSP90 plays a role in the PGD2-stimulated HSP27 induction in osteoblast-like MC3T3-E1 cells. Our findings strongly show that HSP90 negatively regulates the HSP27 induction stimulated by PGD2 in these cells, and that the suppressive effect of HSP90 is due to the inhibition of p38 MAP kinase and SAPK/JNK.
Materials and methods
Results
Discussion HSP90 is ubiquitously expressed in a variety type of cells including osteoblasts [2], [16]. In our previous study [31], we have shown that HSP90 exists at quite high levels in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether HSP90 plays a role on the induction of HSP27 elicited by PGD2 in these cells. We demonstrated that onalespib, an HSP90 inhibitor [23], significantly enhanced the PGD2-stimulated HSP27 induction in MC3T3-E1 cells. Additionally, we showed that geldanamycin, another type HSP90 inhibitor [24], as well as onalespib augmented the HSP27 induction. It seems likely that onalespib and geldanamycin could release the negative regulation by HSP90 of HSP27 induction in PGD2-stimulated MC3T3-E1 cells. Based on our findings, it is probable that HSP90 plays a role as a suppressor in PGD2-stimulated HSP27 induction in osteoblast-like MC3T3-E1 cells. As for the intracellular signaling mechanism of PGD2 in osteoblasts, we have previously shown that PGD2 elicits HSP27 induction through the activation of SAPK/JNK and p38 MAP kinase in osteoblast-like MC3T3-E1 cells [13]. In order to investigate how HSP90 affects the PGD2-elicited induction of HSP27, we next examined the effects of HSP90 inhibitors on the PGD2-stimulated phosphorylation of SAPK/JNK or p38 MAP kinase in MC3T3-E1 cells. We demonstrated that onalespib and geldanamycin significantly amplified the PGD2-induced levels of phosphorylated SAPK/JNK and phosphorylated p38 MAP kinase. In addition, onalespib hardly affected the levels of HSP27. Based on these findings, it seems likely that the withdrawal of HSP90 action causes the potentiation of PGD2-stimulated activation of SAPK/JNK and p38 MAP kinase without affecting the basal levels of HSP27. Therefore, it is probable that the enhancement by HSP90 inhibitors of PGD2-elicited HSP27 induction is mediated through the upregulation both of SAPK/JNK and p38 MAP kinase in osteoblast-like MC3T3-E1 cells. Furthermore, we clearly showed that both SP600125, an inhibitor of SAPK/JNK [26], and SB203580, an inhibitor of p38 MAP kinase [27], truly reduced the amplification by onalespib of the PGD2-elicited HSP27 induction. These results strongly suggest that the amplifying effects of HSP90 inhibitors on the HSP27 induction by PGD2 are dependent at least in part on the activities both of SAPK/JNK and p38 MAP kinase. Taking our present findings into account as a whole, it is most likely that HSP90 limits the PGD2-elicited HSP27 induction in osteoblast-like MC3T3-E1 cells, and the suppressive effect of HSP90 is exerted through reducing the activities of p38 MAP kinase and SAPK/JNK. Indeed, the reduction of direct target of either p38 MAP kinase such as phospho-ATF2 [32] or SAPK/JNK such as phospho-c-JUN or SMAD4 [33] remains to be elucidated. Thus, it is possible that the increase in phosphorylation of either p38 MAP kinase or SAPK/JNK in the presence of onalespib is due to the release of repression of the upstream activating kinases MKK3/6 (for p38 MAP kinase) or MKK4/7 (for SAPK/JNK). We have recently reported that HSP90 inhibitors augment endothelin-1-induced HSP27 through SAPK/JNK but not p38 MAP kinase in MC3T3-E1 cells [18]. It therefore seems likely that the differences of p38 MAP kinase involvement or not in HSP90-effect related to the HSP27 induction in osteoblasts are due to each stimulator. The potential mechanism underlying the regulation by HSP90 of PGD2-elicited HSP27 induction in osteoblasts is summarized as Fig. 7.