Study Of Proliferation, Differentiation And RSK2 Expression Mediated By FGFR1 In Osteoblasts Exposed To Estrogen

Backgrounds:Osteoprosis is one of the common skeletal degenerative diseases, which happened because of the dynamic balance of bone reconstruction was broken. Estrogen can affect osteoblasts function by complex molecular mechanism and promote bone reconstruction. FGF/FGFR signal is an essential regulatory factor in the process of osteogenesis which has important physiological significance for bone development, formation and repair process. Fibroblast growth factor receptor 1 (FGFR1) is the main FGFRs expressed in osteoblasts after birth. P90 ribosomal S6 kinase 2 (p90RSK2) is an important effector molecule lying in downstream of the Raf-MEK-ERK cascade signaling pathways, and it can phosphorylate kinds of intracellular protein to regulate cell division, differentiation and survival, etc. In the previous experiment of our project,17-β estradiol (10-8 mol/L) was applied on MC3T3-E1 cells and cultured the cells for 5 days, the results of microarray showed that the expression of FGFR1 and RSK2 was elevated significantly, demonstrating that FGFR1 and RSK2 may play important roles in the response of osteoblasts to 17-β estradiol. Therefore, to investigate the relationship and communication of FGFR1 and RSK2 further in osteoblasts exposed to estrogen can reveal the mechanism of the bone formation and reconstruction, which provide important targets for alveolar bone reconstruction, osteoporosis and associated bone disease.Objective:This study is to explore the role and mechanism of FGFR1 signal adopting specific FGFR1 inhibitors PD166866 in the estradiol-induced biological response in MC3T3-E1 cells, and to make clear whether the estradiol affect ostoblasts proliferation and differation through RSK2 mediated by FGFR1 using MTT、ALP、 Westernblot �'� RT-PCR. Thereby, effective strategies can be made for this process and mechanism and new ideas can be provide for the treatment of bone disease concerned.Methods:1. MC3T3-E1 cells were cultured in vitro.2. Cells were divided into four groups randomly, which were control group, FGFR1 inhibitor (5 × 10-8mol/L, PD166866) group, estrogen (10-8mol/L,17-β estradiol) group, and estrogen (10-8mol/L,17-β estradiol group) combined with FGFRl inhibitor (5×10-8mol/, PD166866) group.3. MTT assay and ALP were adopted to test the proliferation and differentiation of the four groups of osteoblasts.4. Western blot was applied to detect the protein expression level of RSK2, p-RSK2 and OCN after corresponding treatment.5. RT-PCR was applied to detect the mRNA expression level of Runx2.6. Statistical analyses were conducted by SPSS 17.0 software.Results:1. MC3T3-E1 cells grew well.2. Cells proliferation:Compared to control group,17-β estradiol increased the MTT activity (P<0.05); while the MTT activity decreased when applied with FGFR1 inhibitor PD166866 both in 17-β estradiol and control groups (P<0.05).3. Cells differention:Compared to control group,17-β estradiol increased the ALP activity (P<0.05); and the ALP activity also increased when applied with FGFR1 inhibitor PD166866 both in 17-β estradiol and control groups (P<0.05).4. Westernblot was applied to detect the expression of RSK2、p-RSK2 and OCN: Results showed that there was no significant difference of the expression of RSK2(P>0.05) in the four groups; 17-β estradiol increased the protein expression level of p-RSK2 and OCN compared to the control group (P<0.05); treatment of FGFR1 inhibitor leaded to an increase of p-RSK2 and OCN as well; the expression level of the two proteins increased slightly in the 17-β estradiol+FGFR1 inhibitor groups compared to FGFR1 inhibitor group or 17-0 estradiol group, which is statistically significant (P<0.05).5. The osteogenetic differentiation related genes Runx2 mRNA expression: Results showed that the expression level of Runx2 mRNA increased respectively in 17-β estradiol group and FGFR1 inhibitor group compared to control groups (P<0.05); the expression level of Runx2mRNA increased in the 17-β estradiol+FGFR1 inhibitor groups compared to FGFR1 inhibitor group or 17-β estradiol group (P<0.05).Conclusions:1. FGFR1 participated in the regulation of osteoblasts activity under the treatment of estrogen; FGFR1 promoted proliferation and inhibited differentiation of osteoblasts.2. when FGFR1 was inhibited, the expression of p-RSK2 increased,the mechanism of estrogen regulating bone forming through FGFR1 and RSK2 needs further study.