The Study Of Wnt/β-catenin Signaling Pathway In Proliferation Of Osteoblast Cells Stimulated By Estradiol

Background Adolescent idiopathic scoliosis (AIS) is defined as structural deformities of scoliosis during a puberty spurt. It is the most common form of idiopathic scoliosis and generally is regarded as a multi-factorial disorder. From the literature, it was well recognized that AIS primarily affects girls and that the girls with AIS have a growth pattern different from normal controls. Recently, Inoue et al and Wu J reported that estrogen receptor gene polymorphism was associated with AIS, and deoxyribonucleic acid analysis might predict curve progression. Moreover, the estrogen receptor is an important mediator of the hormonal response in estrogen-sensitive tissues, such as the endometrium, breast, and bone. It is the major hormonal regulator of bone metabolism in women and men. Therefore, there is considerable interest in unraveling the pathways by which estrogen exerts its protective effects on bone. While the major consequence of the loss of estrogen is an increase in bone resorption, estrogen deficiency is associated with a gap between bone resorption and formation, indicating that estrogen is also important for maintaining bone formation at the cellular level. In our precious study, we found E2can increase more proliferation of Mc3T3-E1cells with ERβ RNAi than the control group. Therefore, in order to investigate the relationships among estradiol, estrogen receptors and cell cycles, we explained them by three steps.Chapter One Estradiol treatment increases viability andproliferation of mouse osteblastic Mc3T3-E1cellsObject To determine viability and proliferation of the Mc3T3-E1cells treated with different increasing concentrations and time of estradiol.Method Mc3T3-E1cells were treated with different increasing concentrations (DMSO,10-9,10-8,10-7,10-6M) and time (1,3,5,7,9days) of estradiol. Then we examined the proliferation of these cells assessed by MTT. Similarly, we used RT-PCR to estimate levels of ER and Cyclin D1mRNA. Then we examined the proliferation of these cells assessed by MTT. Similarly, we used RT-PCR to estimate levels of ER and Cyclin D1mRNA.Result Subsequently, we found the group that received5days of β-estradiol (10-7M) treatment exhibited the most significant enhancement in cell viability compared with the control group, which was significantly different compared with the other time points and concentrations. The results of RT-PCR demonstrated that the levels of ERs and Cyclin D1mRNA were increased with estradiol treatment. Conclusion Estradiol treatment increases viability and proliferation of mouse osteblastic Mc3T3-E1cells and enhance ERs and Cyclin D1mRNA expression.Chapter Two Estradiol treatment increases viability and proliferation of Mc3T3-E1cells through activation of canonical Wnt signaling pathwayObject To observe the expression of key proteins in canonical Wnt signaling pathway with estradiol treatment.Method Mc3T3-E1cell had been treated with various concentrations of estradiol for120min. Then western blot examined the expression of key proteins (β-catenin, Gsk3β, p-Gsk3β/Ser9and Cyclin D1). Immunofluorescence confocal microscopy tested β-catenin nuclear localization in Mc3T3-El cells. Moreover, the osteoblastic cells were treated with different drug treatments (Blank, E2, Wnt3a, E2+xav939). Then we repeated the previous two experiments to test the expression of the key proteins.Result The western blot results indicated the increased expression levels of β-catenin, p-Gsk3β and Cyclin D1in Mc3T3-E1cells along with increasing concentration of estradiol treatment. β-catenin immunostaining was predominantly localized in the nucleus after treatments with E2. However, the expression levels of P-catenin, p-Gsk3β and Cyclin D1in Mc3T3-E1cells with E2treatment was higher than the blank group, and lower than the Wnt3a treatment group. The expression levels of these key protein was lowest in E2+xav939treatment group.Conclusion E2could activate the canonical Wnt signaling pathway in Mc3T3-E1cells. Chapter Three E2mediated ERa induces preosteoblastic cell proliferation through activation of canonical Wnt signaling pathway Object Detect the relationship between the estrogen receptors and the canonical Wnt singaling pathway in Mc3T3-E1cells with estradiol treatment.Method Constructed plasmid vector (plentilox3.7) of RNA interference specific for estrogen receptors (ER α and ERβ) and packaged recombinant lentivirus. The Mc3T3-E1cells had been transfected the modified virous vectors for24h and then been treated with estradiol for120min. Then western blot examined the expression levels of ER α, ER β, β-catenin, p-Gsk3β and Cyclin D1in Mc3T3-E1cells. Cell viability with different interventions (ER α RNAi, ER β RNAi, blank and ER α+β RNAi) was assessed by MTT assay.Result Enzyme digestion analysis and DNA sequencing showed that the ERs targeted RNA interference recombinant plasmid were constructed successfully. Western blot results indicated that the recombined ERs-RNAi plasmid exerted good silencing effect on ER α and ER β, respectively. The expression of β-catenin, p-Gsk3β and Cyclin D1proteins by Western blot, using ERα and ERβ siRNA to interfere the ERs mRNA expression. The results of Western blot indicated the expression of β-catenin, p-Gsk3β and Cyclin D1were lower in ERα siRNA treatment group, while it was higher in ERβ siRNA group. The percentage of viable cells was determined by an MTT cell proliferation assay. The Mc3T3-E1cells proliferation rate in E2treatment group was higher than that in ERα RNA-treatment group, while it was lower than that in the ER β RNAi-treatment group.conculison E2mediated ERα induces preosteoblastic cell proliferation through activation of Wnt/β-catenin signaling.