RAMPs and CRLR expressions in osteoblastic cells after dexamethasone treatment

Adrenomedullin (ADM) is a potent stimulator of osteoblastic activity and promotes bone growth in vivo. ADM receptors are formed by heterodimerization of the CRLR and a RAMP2 or RAMP3 molecule. Since glucocorticoid responsive elements were recently identified in the human CRLR promoter and that glucocorticoids exert a major action in bones, we investigated the acute effect of dexamethasone (Dex) treatment on ADM receptor components in osteoblastic cell types: the MC3T3-E1 cells and calvaria-derived osteoblastic cells. Changes in expression of CRLR and RAMPs molecules were evaluated at mRNA levels using RT-PCR and at protein levels by Western blot analysis. We found that Dex increased expression of RAMP1 and RAMP2 mRNA in a time-dependent but dose-independent manner, while RAMP3 was unchanged. In contrast, Dex decreased the CRLR mRNA expression and these changes were reflected at protein levels. We suggest that Dex, in osteoblastic cells, altered ADM receptor by inhibition of CRLR expression and consequently could impair the ADM anabolic effect on bone. Our findings could explain in part, the detrimental side effects observed at bone level during glucocorticoid therapy.     

Suppressive effect of dexamethasone on TIMP-1 production involves murine osteoblastic MC3T3-E1 cell apoptosis

High dose glucocorticoid (GC) treatment induces osteoporosis partly via increasing osteoblast apoptosis. However, the mechanism of GC-induced apoptosis has not been fully elucidated. Osteoblast-derived tissue inhibitor of metalloproteinase-1 (TIMP-1) was recently reported to be involved in bone metabolism. Our previous study demonstrated that TIMP-1 suppressed apoptosis of the mouse bone marrow stromal cell line MBA-1 (pre-osteoblast) induced by serum deprivation. Therefore, we tested the effect of the GC dexamethasone (Dex) on TIMP-1 production in murine osteoblastic MC3T3-E1 cells and further determined whether this action is associated with Dex-induced osteoblast apoptosis. Dex decreased TIMP-1 production in MC3T3-E1 cells, and this effect was blocked by the glucocorticoid receptor (GR) antagonists, RU486 and RU40555. Recombinant TIMP-1 protein reduced caspase-3 activation and apoptosis induced by Dex in MC3T3-E1 cells. In addition, the pro-apoptotic effect of the Dex was augmented by suppression of TIMP-1 with siRNA. Furthermore, mutant TIMP-1, which has no inhibitory effects on MMPs, yet protects MC3T3-E1 cells against Dex-induced apoptosis. Our study demonstrates that Dex suppresses TIMP-1 production in osteoblasts through GR, and this effect is associated with its induction of osteoblast apoptosis. The anti-apoptotic action of TIMP-1 is independent of its inhibitory effects on MMPs activities. The decrease in TIMP-1 production caused by Dex may contribute to the mechanisms of Dex-induced bone loss.     

Up-regulation of Axin2 by dexamethasone promotes adipocyte differentiation in ROB-C26 mesenchymal progenitor cells

Dexamethasone (Dex) regulates osteoblastic and adipocytic differentiation in mesenchymal progenitor cells through regulation of Wnt/β-catenin signaling. To elucidate the regulatory mechanisms underlying the effects of Dex, we examine the expression of Axin2, which is an intracellular inhibitor of Wnt/β-catenin signaling, in ROB-C26 clonal mesenchymal progenitor cells (C26). We observed the induction of Axin2 mRNA in C26 cells in response to Dex treatment. Treatment with a glucocorticoid receptor (GR) antagonist, mifepristone, showed that Dex-induced up-regulation of Axin2 is mediated by the GR. In the absence of Dex, gene silencing by using Axin2-targeted short hairpin RNA increased the number of alkaline phosphatase (ALP)-positive and nuclear β-catenin-positive cells and ALP activity. In the presence of Dex, Axin2 knockdown resulted in an increased number of ALP-positive and nuclear β-catenin-positive cells. Furthermore, Axin2 knockdown in Dex-treated cells suppressed adipocyte differentiation (as determined by reduced Oil Red O staining), reduced the number of PPARγ-positive and aP2-positive cells and decreased the mRNA expression of PPARγ2 and aP2. These results suggest that Axin2 plays a key role in adipocyte and osteoblastic differentiation by controlling β-catenin expression.     

Dexamethasone inhibits bone resorption by indirectly inducing apoptosis of the bone-resorbing osteoclasts via the action of osteoblastic cells

Although glucocorticoids (GCs) are physiologically essentialfor bone metabolism, it is generally accepted that high dosesof GCs cause bone loss through a combination of decreased boneformation and increased bone resorption. However, the actionof GCs on mature osteoclasts remains contradictory. In thisstudy, we have examined the effect of GCs on osteoclasticbone-resorbing activity and osteoclast apoptosis, by using twodifferent cell types, rabbit unfractionated bone cells andhighly enriched mature osteoclasts (>95% of purity).Dexamethasone (Dex, 10^-10–10^-7 M) inhibited resorption pit formation on a dentine slice by the unfractionated bone cells in a dose- and time-dependent manner.However, Dex had no effect on the bone-resorbing activity of the isolated mature osteoclasts. When the isolated osteoclastswere co-cultured with rabbit osteoblastic cells, the osteoclastic bone resorption decreased in response to Dex,dependent on the number of osteoblastic cells. Like the effecton the bone resorption, Dex induced osteoclast apoptosis in cultures of the unfractionated bone cells, whereas it did not promote the apoptosis of the isolated osteoclasts. An inhibitorof caspases, Z-Asp-CH2-DCB attenuated both the inhibitory effecton osteoclastic bone resorption and the stimulatory effect onthe osteoclast apoptosis. In addition, the osteoblastic cellswere required for the osteoclast apoptosis induced by Dex. These findings indicate that the main target cells of GCs arenon-osteoclastic cells such as osteoblasts and that GCsindirectly inhibit bone resorption by inducing apoptosis ofthe mature osteoclasts through the action of non-osteoclasticcells. This study expands our knowledge about the multifunctional roles of GCs in bone metabolism.     

Aromatase expression in a human osteoblastic cell line increases in response to prostaglandin E(2) in a dexamethasone-dependent fashion

Recent progress supports the importance of local estrogen secretion in human bone tissue to increase and maintain bone-mineral density. In a previous report, we found that forskolin (FSK) synergistically induces aromatase (CYP19: a rate-limiting enzyme for estrogen synthesis) expression in dexamethasone (Dex) dependent manner in a human osteoblastic cell line, SV-HFO [Watanabe M, Ohno S, Nakajin S. Forskolin and dexamethasone synergistically induce aromatase (CYP19) expression in the human osteoblastic cell line SV-HFO. Eur J Endocrinol 2005;152:619-24]. In this report, we investigated whether prostaglandin (PG) E(2) induces estrogen production, in other words, if PGE(2) exerts the same effect as FSK because PGE(2) is the major prostanoid in the bone and is one of the key molecules in the osteoblast. We found PGE(2) up-regulates aromatase activity synergistically, but this up-regulation depends on Dex. CYP19 gene expression was also increased synergistically by Dex and PGE(2). Promoter I.4 was activated synergistically by PGE(2) and Dex. PGE(2) receptor, EP(1), EP(2) and EP(4) were involved in the up-regulation of aromatase activity in response to PGE(2) in a Dex-dependent manner. The cAMP-PKA pathway and Ca(2+) signaling pathway were involved in the up-regulation of aromatase activity in response to PGE(2). Furthermore, glucocorticoid response element on promoter I.4 sequence was an essential minimum requirement for its activity and synergism of PGE(2) and Dex. These findings are the first report on osteoblastic cell line which uses predominantly promoter I.4 to drive aromatase expression. These findings also suggest that endogenous PGE(2) produced in bone mainly may synergistically support local estrogen production in osteoblastic cells in the presence of glucocorticoid.     

Prolonged culture of HOS 58 human osteosarcoma cells with 1,25-(OH)2-D3, TGF-beta, and dexamethasone reveals physiological regulation of alkaline phosphatase, dissociated osteocalcin gene expression, and protein synthesis and lack of mineralization

Cultured rodent osteoblastic cells reiterate the phenotypic differentiation and maturation of osteoblasts seen in vivo. As previously shown, the human osteosarcoma cell line HOS 58 represents a differentiated stage of osteoblast development. The potential of HOS 58 for still further in vitro differentiation suggests the line can serve as a model of osteoblast maturation. Using this cell line, we have investigated the influence of 1,25-(OH)2-D3 (D3), TGF-beta and Dexamethasone (Dex) on proliferation and on the protein and mRNA levels of alkaline phosphatase (AP), procollagen 1 (Col 1), and osteocalcin (Oc), as well as mineralization during 28 days in culture. AP mRNA and protein were highly expressed throughout the culture period with further increase of protein AP activity at constant gene expression levels. A differentiation inhibiting effect of either TGF-beta or Dex was seen. Col 1 was investigated without the use of ascorbic acid and showed only minor changes during culture time or stimulation. The gene expression for Oc increased continually whereas protein synthesis peaked at confluence and decreased thereafter. TGF-beta and Dex treatments decreased Oc mRNA and protein levels. Stimulation by D3 was maximal at day 7 with a decrease thereafter. HOS 58 cells showed no mineralization capacity when stimulated with different agents, as measured by energy-dispersive X-ray microanalysis. This was not due to absence of Cbfa1 expression. In conclusion, the HOS 58 osteosarcoma cell line represents a differentiated cell line with highly expressed and physiologically regulated AP expression during further differentiation in culture. We observed a dissociation between osteocalcin gene expression and protein secretion which may contribute to the lack of mineralization in this cell line.     

Parathyroid hormone stimulation and PKA signaling of latent transforming growth factor-beta binding protein-1 (LTBP-1) mRNA expression in osteoblastic cells

Parathyroid hormone (PTH) regulates bone remodeling and calcium homeostasis by acting on osteoblasts. Recently, the gene expression profile changes in the rat PTH (1-34, 10(-8)M)-treated rat osteoblastic osteosarcoma cell line, UMR 106-01, using DNA microarray analysis showed that mRNA for LTBP-1, a latent transforming growth factor (TGF-beta)-binding protein is stimulated by PTH. Latent TGF-beta binding proteins (LTBPs) are required for the proper folding and secretion of TGF-beta, thus modifying the activity of TGF-beta, which is a local factor necessary for bone remodeling. We show here by real time RT-PCR that PTH-stimulated LTBP-1 mRNA expression in rat and mouse preosteoblastic cells. PTH also stimulated LTBP-1 mRNA expression in all stages of rat primary osteoblastic cells but extended expression was found in differentiating osteoblasts. PTH also stimulated TGF-beta1 mRNA expression in rat primary osteoblastic cells, indicating a link between systemic and local factors for intracellular signaling in osteoblasts. An additive effect on LTBP-1 mRNA expression was found when UMR 106-01 cells were treated with PTH and TGF-beta1 together. We further examined the signaling pathways responsible for PTH-stimulated LTBP-1 and TGF-beta1 mRNA expression in UMR 106-01 cells. The PTH stimulation of LTBP-1 and TGF-beta1 mRNA expression was dependent on the PKA and the MAPK (MEK and p38 MAPK) pathways, respectively in these cells, suggesting that PTH mediates its effects on osteoblasts by several intracellular signaling pathways. Overall, we demonstrate here that PTH stimulates LTBP-1 mRNA expression in osteoblastic cells and this is PKA-dependent. This event may be important for PTH action via TGF-beta in bone remodeling.     

Secretion of MUC5AC mucin from pancreatic cancer cells in response to forskolin and VIP

Bisphosphonates are potent antiresorptive drugs commonly employed in the treatment of metabolic bone diseases. Despite their frequent use, the mechanisms of bisphosphonates on bone cells have largely remained unclear. Receptor activator of nuclear factor-kappaB ligand (RANKL) is essential for osteoclast formation and activation, whereas osteoprotegerin (OPG) neutralizes RANKL. Various osteotropic drugs have been demonstrated to modulate osteoblastic production of RANKL and OPG. In this study, we assessed the effects of the bisphosphonates pamidronate (PAM) and zoledronic acid (ZOL) on OPG mRNA steady-state levels (by semiquantitative RT-PCR) and protein production (by ELISA) in primary human osteoblasts (hOB). PAM increased OPG mRNA levels and protein secretion by hOB by up to 2- to 3-fold in a dose-dependent fashion with a maximum effect at 10(-6) M (P < 0.001) after 72 h. Similarly, ZOL enhanced OPG gene expression and protein secretion by hOB in a dose-dependent fashion with a maximum effect at 10(-8) M after 72 h, consistent with the higher biological potency of ZOL. Time course experiments indicated a stimulatory effect of PAM and ZOL on osteoblastic OPG protein secretion by 6-fold, respectively (P < 0.001). Pretreatment with PAM and ZOL prevented the inhibitory effects of the glucocorticoid dexamethasone on OPG mRNA and protein production. Analysis of cellular markers of osteoblastic differentiation revealed that PAM and ZOL induced type I collagen secretion and alkaline phosphatase activity by 2- and 4-fold, respectively (P < 0.0001 by ANOVA). In conclusion, our data suggest that bisphosphonates modulate OPG production by normal human osteoblasts, which may contribute to the inhibition of osteoclastic bone resorption. Since, OPG production increases with osteoblastic cell maturation, enhancement of OPG by bisphosphonates could be related to their stimulatory effects on osteoblastic differentiation.     

Dexamethasone Mediated Stabilization of Insulin Receptor mRNA

Abstract

To determine the mechanism of glucocorticoid mediated enhancement of insulin receptor (IR) gene expression, we cotransfected a glucocorticoid receptor expression vector and a plasmid containing a reporter gene driven by an MMTV or IR promoter into COS 7 cells. Dexamethasone (Dex) increased MMTV promoter activity by 100% but had no effect on IR promoter activity. In the glucocorticoid responsive breast cancer cell line, MCF-7, Dex increased IR mRNA by 60%, and increased the IR mRNA half-life from approximately 6hrs to >24hrs. No glucocorticoid responsive element could be located in the insulin receptor 3′ untranslated region. Glucocorticoids stabilize IR mRNA.