Regulation Of Glucocorticoid Signaling Pathway During Osteoblast Differentiation

Background:Osteoporosis is the most common metabolic disease characterized by loss of the normal density of bone, resulting in fragile bone and a higher risk of fractures in response to relatively minor injuries. Glucocorticoids (GCs) are naturally-produced steroid hormones, or synthetic compounds, that inhibit the process of inflammation or autoimmune disorders. Clinically, both of the natural or synthetic glucocorticoids can lead to the side effects of bone loss or osteoporosis while they are administrated especially of over dose and in long term. Patients under glucocorticoids treatment are one of the main groups of persons who suffer from osteoporosis. Glucocorticoids-induced Osteoporosis (GCOP) is the most common cause of adult osteoporosis in population aged from 20 to 45 and iatrogenic osteoporosis. Though the synthetic glucocorticoids such as prednisone and dexamethasone (DEX) have severe GCOP complication, they are still widely used in treatment of allergy, rheumatoid arthritis, and allograft rejection.Although bone resorption has been suggested to cause GIO, recent studies have revealed the main mechanism underlying long-term GCs-induced bone loss is the impairment of osteoblast function and bone formation. Glucocorticoids may inhibit osteoblasts through multiple mechanisms with the results of significant reduction in bone formation. Bone morphogenetic protein 2 (BMP-2) is proven to inducing bone formation and currently clinically used in orthopedic surgery. Glucocorticoid is revealed to inhibit osteoblast cell differentiation through the repression of bone morphogenetic protein 2, which leads to a decrease in bone formation. Wnt signaling pathway, a complex network of proteins most well known for their roles in embryogenesis and cancer, is also another key pathway for promoting osteoblastogenesis. In studies, glucocorticoids inhibit osteoblast cell differentiation by opposing Wnt-b-catenin signaling.Moreover, glucocorticoids are able to redirect differentiation of bone marrow stromal cells from the osteoblastic lineage to the adipocyte lineage. The underlying mechanisms involve the regulation of nuclear factors of the CAAT enhancer-binding protein family, which might also indirectly reduce osteoblast proliferation and decrease IGF-I transcription, and the induction of peroxisome proliferator-activated receptor y2 (PPARy2). However, the glucocorticoid signaling pathway during osteoblast differentiation remains unclear.Transcriptional coactivator with PDZ-binding motif (TAZ), aβ-catenin-like molecule, drives mesenchymal stem cells (MSCs) to differentiate into osteoblast lineage through co-activation of Runx2-dependent gene transcription and repression of peroxisome proliferator-activated receptor y (PPARy)-dependent gene transcription. Dexamethasone (DEX), a synthetic and widely used glucocorticoid, affects osteogenesis. However, the signaling pathway by which DEX affects osteoblastic differentiation remains obscure.Stable-isotope labeling by amino acids in cell culture (SILAC) enables metabolic incorporation of isotope mass tags into proteins [1]. The cells representing two biological conditions are grown in normal medium (light medium) and isotope amino acid labeled medium (heavy medium), respectively. After grown for six cell divisions in labeled medium to reach 100% of the incorporation of the isotope amino acid, the cells are processed for further treatment, followed by liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) analysis. Therefore the relative intensity ratio of the two pairs of "light" and "heavy "peaks in the peptide mass spectrum can reflect the protein quantitative expression levels under different conditions [1]. Materials and methods:The bone marrow cells from sixteen four-week-old virgin female Sprague-Dawley rats were harvested. The primary-MSCs were isolated and identified through detection of surface antigens and multi-differentiation ability. The passage 1 MSCs were reseeded in the density of 5x105/cm2 in six-well-plates. To induce osteoblast differentiation, the MSCs were cultured in different differentiation mediums. The calcium deposition ability (AR-S staining) and ALP activity were detected to evaluate the osteoblast differentiation ability of the MSCs. In addition, we carried out RT-PCR, Real-time PCR, Western blot, and immunochemistry to measure the mRNA and protein levels of TAZ, ALP, BMP-2, and Runx-2 during the osteoblast differentiation.Results:In this study, we found that isolated adhere cells in our study were CD54 positive and CD90 positive, but CD14 negative and CD34 negative. In addition, the MSCs possess the abilities of differentiating into osteoblast, adipocyte, and chondrocyte. Osteoprogenitor MC3T3-E1 cells were treated with or without 10-6 M DEX for 7 days and the differentiation ability, proliferation, the cells were measured. The protein level changes were analyzed using SILAC and liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS). Therefore, the surface antigen identification and mutilineage differentiation ability supported that these isolated cells were MSCs. Present study showed that 10-8 M DEX increased calcium formation in MSCs at day 14 and the effect was synergized by AA andβ-GP. Real-time PCR and immunobloting analysis showed that TAZ mRNA level and protein expression were enhanced in MSCs treated with 10-8 M DEX at day 7. In addition, the number of TAZ-positive MSCs after 10-8 M DEX treatment significantly increased compared to control using immunochemistry assay. The stimulative effect of 10-8 M DEX on TAZ was blocked by 10-5M RU486, an antagonist of glucocorticoid receptor and MSCs were treated with AA orβ-GP did not show increased TAZ level. In the present study,DEX at 10-8 M and 10-9 M significantly elevated ALP activity (P< 0.01) whereas 10-7 M DEX (P<0.05) and 10-6M DEX (P< 0.01) inhibited ALP activity in rat MSCs. We also demonstrated that higher concentration of DEX (10-7M) decreased TAZ mRNA level and ALP activity from day 3 to day 7, which indicates that TAZ level is closely related with osteoblast differentiation ability. In this study,10-6 M DEX inhibited both osteoblast differentiation and proliferation but induced apoptosis in osteoprogenitor MC3T3-E1 cells on day 7. We found that 10-6 M DEX increased the levels of tubulins (TUBA1A, TUBB2B, and TUBB5), IQGAP1, and S100 proteins (S100A11, S100A6, S100A4, and S100A10), while inhibited the protein levels of G3BP-1 and Ras related proteins (Rab-1A, Rab-2A and Rab-7) in MC3T3-E1 cells.Conclusions:These findings suggests that TAZ is not only involved in the signal pathway of BMP-2 induced osteoblastic differentiation, but also involved in the signaling pathway of DEX-induced osteoblastic differentiation, supporting the notion that TAZ is a convergence point of two signaling pathways, BMP-2 signaling pathway and Wnt-β-catenin signaling pathway.10-6 M DEX inhibited the osteoblast differentiation and proliferation in MC3T3-E1 cells on day 7. The upregulated levels of tubulin, IQGAP1, and S100 proteins and the downregulated protein levels of G3BP-1 and Ras related proteins (Rab-1A, Rab-2A and Rab-7) may be critical in the mechanism of the DEX-induced results. Such protein expression changes may be of pathological significance in coping with GCOP.