| Glucocorticoids (GCs) regulate a variety of biological processes, including cell growth, differentiation and apoptosis. GCs have well-documented effects on bone metabolism. Physiological concentrations of glucocorticoids promote the development or maturation of osteoblast cells, and continued exposure of the skeletal tissue to pharmacological dose of glucocorticods can cause osteoporosis. GC-induced osteoporosis is characterized histologically by a decreased bone formation rate, decreased trabecular wall thickness, and depleted osteoblast numbers, all indicators of a deficient osteoblast population. It has been known that GCs exert antiproliferative effect in most osteoblast cell contexts including G-292, osteoblast-like cancer cells through activating the glucocorticoid receptor (GR), which is a ligand-dependent transcriptional regulator that transduces the hormonal signal into the nucleus to alter the expression of target genes. But the down-stream effector proteins of GR mediated the antiproliferative action of these compounds on osteoblast cells, however, is not fully understood.Small GTPases of the Rho subfamily have been implicated in many physiological and pathological cellular processes, including cell adhesion, motility, proliferation, survival and inflammation. The Rho subfamily mainly includes RhoA, RhoB and RhoC proteins. RhoB is quite different from RhoA and RhoC in many aspects although it shares-90% homology to RhoA and RhoC. For example, RhoB has a tumor-suppressive role, including inhibiting cell proliferation and inducing apoptosis in several human cancer cells, and inhibiting tumor growth in a nude mouse xenograft model, while RhoA activation promotes cell malignant transformation, cell proliferation, invasion and metastasis. Furthermore, RhoB, unlike RhoA which is constitutively expressed, has been shown to be induced by genotoxic stress, such as UV, chemotherapeutic drugs (e.g. cisplatin and 5-FU), and some growth factors such as EGF, PDGF.We have demonstrated previously that RhoB is also upregulated by Dexmethasone (a synthesis glucocorticoid, Dex), and RhoB signaling is involved in Dex-induced proliferation inhibition of human ovarian cancer HO-8910 cells. However, RhoB is not induced by Dex in human fibrosarcoma cell HT-AR1, indicating that the effect of glucocorticoid on RhoB expression is cell specific. So, we want to know whether RhoB is regulated by Dex in human osteosarcoma cells and plays a role in Dex-induced cell growth inhibition and differentiation.We demonstrated that Dex could induce both mRNA transcription and protein expression of RhoB in osteosarcoma MG-63 cell line. The up-regulation of RhoB mRNA by Dex may mainly due to Dex's effect on the stabilization of RhoB mRNA and RhoB protein instead of enhancement of transcripts because RhoB promoter (-1765 to+111) contains no functional GRE. Induction of RhoB expression by Dex depend on new protein synthesis, both PI-3K/Akt and p38 MAPK signaling are involved in the RhoB expression by Dex, but JNK and ERK signaling are not. Once again we confirmed the inhibition effect of Dex to MG-63 cells time and dose dependently. Overexpression of RhoB repressed the growth of osteosarcoma cell line MG-63 and enhanced Dex-induced cell growth inhibition, but has no effect on cell differentiation. While interfering of RhoB expression facilitated cell growth and reversed partially Dex-induced proliferation inhibition. Furthermore, we reported that Dex can enhance the adhesive activity of MG-63 cells to fibronection and RhoB signaling is involved in the adhesion enhancement of MG-63 cells by Dex.Transforming growth factorβ1 (TGF-β1) is one of the most highly expressed cytokines in osteoblast cells. Like Dex, TGFβalso have multiply biology effects in cell growth, migration, differentiation and apoptosis. It has been reported that TGF-β1 can inhibit the growth of osteoblast cells including MG-63, G-292, and osteoblast-like cancer cells. On the other hand, articles declared that TGF-β1 induce the expression of RhoB in many cell types. We wonder whether TGF-β1 can induce the expression of RhoB in MG-63 cells and whether RhoB signaling are involved in the grow inhibition and adhesion of MG-63 cells by TGF-β1.We found that TGF-β1 treatment could also increases the expression of RhoB in MG-63 cells like Dex. However, TGF-β1 can enhance the transcriptional activity of the human RhoB promoter (-1765/+111) in MG-63 cells. We also demonstrate that PI-3K/Akt but not p38 MAPK signaling is involved in the RhoB expression by TGF-β1. Moreover, we confirmed the inhibition effect of TGF-β1 to MG-63 cells and demonstrated the enhancement of adhesive ability of MG-63 cells to TGF-β1.The relationship between Dex and TGF-β1 is complex. Our previous study showed that the co-treatment of Dex with TGF-β1 could significantly enhance the adhesion of HO-8910 cells to ECM and increased the synthesis of extracellular matrix (ECM). Given that both Dex and TGF-β1 can induce the expression of RhoB, and both Dex and TGF-β1 can inhibit the grow and enhance the adhesion of MG-63 cells through the involvement of RhoB, whether co-treatment of Dex with TGF-β1 has synergetic effect on the expression of RhoB, adhesion and proliferation of MG-63 cells, and whether RhoB is involved in these processes are ready to be elucidated.We found the synergetic effect of Dex and TGF-β1 to upregulate the expression of RhoB in MG-63 cells, and the synergetic effect of Dex and TGF-β1 to the adhesive ability of MG-63 cells on fibronectin. However, no synergetic effect of Dex and TGF-β1 was found to the growth inhibition effect of MG-63 cells. Furthermore, we found the secretion of TGF-β1 was not facilitated by Dex on MG-63 cells with ELISA method. Maybe different singnaling pathways are involved in the adhesion process by Dex and TGF-β1 and same singnaling pathway in the growth inhibition effect. |
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