Study Of The Mechanism Of FGFR1-ERK Pathway In The Promotion Effects Of Estrogen And Fluid Shear Stress On The Proliferation And Differatiation Of MC3T3-E1 Cells

Backgrounds:Estrogen and fluid shear stress play important role in regulating bone metabolism, estrogen deficiency or reduced movement can lead to osteoporosis. As one of growing factors in the surface of MC3T3-E1 osteoblastic cells, FGFR1 can receive extracellular stimulation and activate intracellular signaling pathways to regulate the proliferation and differation of osteoblasts. In our previous experiments, after exposing MC3T3-E1 cells to estrogen, fluid shear stress or both of the two factors, the expression level of FGFR1 changes significantly, which indicated that FGFR1 plays important roles in the response of MC3T3-E1 cells to estrogen, fluid shear stress or both of the two factors. The in-depth study of FGFR1 finds that FGFR1 as an upstream factor of MAPK signal pathway can regulate MAPK signaling cascades. Therefore, the further study of regulatory mechanism of FGFR1 on osteoblasts will provide theoretical basis for the prevention and treatment of osteoporosis and the alveolar bone remoding.Objective:On the basis of the previous studies, under the effects of estrogen, fluid shear stress or both of the two factor, the FGFR1 inhibitor PD166866 in this experiment was used to surpress the activity of FGFR1 in the surface of MC3T3-E1 cells, combining MTT assay, ALP assay and the expression level measurement of Runx2 and OCN to make clear the regulatory founction of FGFR1. Besides, Weastern blot was used to masure the expression level and phosphorylation of the key genes in the MAPK signaling pathway to illuminate the FGFR1 regulatory mechanism on MC3T3-E1 cells.Methods:1. MC3T3-E1 cells were cultured in vitro2. Constructing parallel plate flow chamber system and dividing the cells into eight groups randomly:①blank control group;②inhibitor group (5×10-8mol/l);③estrogen group (10-8mol/l);④estrogen(10-8mol/l)+inhibitor (5×10-8mol/l) group;⑤fluid shear stress (12 dyne/cm2)group;⑥fluid shear stress(12 dyne/cm2)+inhibitor (5×10-8mol/l) group;⑦estrogen (10"8mol/l)+fluid shear stress (12 dyne/cm2) group;⑧estrogen (10-8mol/l)+fluid shear stress (12 dyne/cm2)+inhibitor (5×10-8mol/l) group.3. MTT and ALP assays were used to measure the prolifferention and differatiation activity.4. Western blot was used to compare the expression level of Runx2 and OCN.5. Weastern blot was used to masure the expression level and phosphorylation level of the key genes in the MAPK signaling pathway.6. Imagej and SPSS22.0 softwares were used to analysis the results.Results:1. MC3T3-E1 cells, cultured in vitro, adapt to the fluid shear stress generated by parallel plate flow chamber system very well.2. Under the effects of estrogen, fluid shear stress or both of the two factors, the value of MTT and ALP is higher than the blank control group (P<0.05), and the joint effects of the two factors is the highest (P<0.05); after the FGFR1 inhibitor PD166866 was added, the MTT in groups with inhibitor is lower and ALP is higher than the corresponding non-inhibitor groups(P<0.05). The results of Western blot showed that the expression levels of Runx2 and OCN is higher than the corresponding non-inhibitor groups(P<0.05), which indicate that FGFR1 inhibitors can significantly suppress proliferation and increase differentiation activity of MC3T3-E1 cells responding to estrogen, fluid shear stress and both of the two factors.3. Western blot results show that under the effects of estrogen, fluid shear stress or both of the factors, the expression level of ERK, JNK and P38 has no obvious changes compared with the blank control group, while the phosphorylation levels of ERK, JNK and P38 is higher(P<0.05) and the two factors group is the highest(P<0.05). After the FGFR1 inhibitor PD166866 was added, the phosphorylation levels of ERK is higher than the corresponding non-inhibitor groups(P<0.05), while the phosphorylation levels of JNK and P38 has no obvious changes.Conclusions:1. Parallel plate flow chamber system works stablely and meets the requirements of study of cell mechanics, which provides basis for further studying the mechanism of MC3T3-E1 cells responding to fluid shear stress.2. Estrogen, fluid shear stress, the two factors working together can significantly improve the osteoblast proliferation and differentiation activity of MC3T3-E1 cells, and estrogen and fluid shear stress have synergistic effect.3. FGFR1 has promotive effect on osteoblast proliferation and inhibitory effects on osteoblast differentiation and is explored in the response of MC3T3-E1 cells to the effects of estrogen, fluid shear stress and the double factors.4. ERK, JNK and P38 all participate in the response of MC3T3-E1 cells to the effects of estrogen, fluid shear stress and the double factors, however, among three main MAPK cascades, only ERK signaling pathway plays an important role in the regulatory function of FGFR1 to the proliferation and differentiation activity of osteoblasts under the effects of estrogen, fluid shear stress or both of the two factors.