Estrogen-induced Tgfbr1 and Bmpr1a Expression Repressed via Estrogen Receptor Beta in MC3T3-E1 Cells

Abstract

Background:Estrogen, as an important hormone in human physiological process, is closely related to bone metabolism. The aim of this study was to investigate the mechanism of estrogen on osteoblasts metabolism in MC3T3-E1 cells.Methods:We treated the MC3T3-E1 cells with different concentrations of β-estradiol (0.01, 0.1, 1, and 10 nmol/L), observed the morphological changes of the cells, and detected the cell's proliferation and apoptosis of MC3T3-E1 cells. Two transcriptome libraries were constructed and sequenced. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to confirm the differentially expressed genes (DEGs), and then treated the MC3T3-E1 cells with estrogen receptor (ER) inhibitors α and β, respectively, and then examined the expression of Tgfbr1 and Bmpr1a genes. The promoter of Tgfbr1 and Bmpr1a gene was analyzed, and the ER response elements were identified. Finally, ChIP was used to verify the binding of ER to Tgfbr1 and Bmpr1a promoter.Results:In the high-concentration β-estradiol treatment group (1 nmol/L and 10 nmol/L), there was no significant difference in the morphology of the cells under the microscope, 1 nmol/L and 10 nmol/L treated group appeared statistically significant difference in cell apoptosis and proliferation (P < 0.05 and P < 0.01, respectively). We found 460 DEGs compared with the control group. Among the DEGs, there were 66 upregulated genes and 394 downregulated genes. Gene ontology classification and Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that many bone metabolism-related biological processes and cell signaling pathways were disordered. The qRT-PCR verification showed that the expressions of Tgfbr1- and Bmpr1a-related genes in bone metabolism pathway in the 10 nmol/L treatment group were significantly decreased (P < 0.05). ER β was involved in the inhibitory effect of Tgfbr1 and Bmpr1a genes. The bioinformatics of the promoter found that there were three ER response elements in the promoter of Tgfbr1, and there were two ER response elements in Bmpr1a promoter regions. ChIP experiments showed that estrogen could enhance the binding of ERs to Tgfbr1 and Bmpr1a genes.Conclusions:Estrogen can promote the apoptosis and proliferation of osteoblasts simultaneously, and the mechanism may be the joint action of transforming growth factor-beta, Wnt, mitogen-activated protein kinase, and nuclear factor-kappaB bone metabolism-related signaling pathway. Estrogen inhibits the expression of Tgfbr1 and Bmpr1a genes through ER β and affects the metabolism of MC3T3-E1 osteoblasts.

Effect of estradiol on high glucose‑induced osteoblast injury

“…Different concentrations of E2 were added to mouse osteoblasts, and the results revealed that 0.1 µM E2 significantly increased MC3T3-E1 cell viability. This result was consistent with the study of He et al (31) that high concentration of E2 can improve the cell viability of MC3T3-E1.…”

“…Gopalakrishnan et al revealed that insulin and E2 increased the number of mineralized nodules, the area stained for collagen and mineralization, as well as the proliferation of rat bone marrow stromal cells under HG (30). Estrogen-induced transforming growth factor β receptor 1 and bone morphogenetic protein receptor type 1A expression levels were suppressed by estrogen receptor β activation in MC3T3-E1 cells (31). The present study demonstrated that E2 may serve an important protective role in HG-induced osteogenic injury.…”

Estradiol Ameliorates Acute Kidney Ischemia-Reperfusion Injury by Inhibiting the TGF-βRI-SMAD Pathway

“…Estrogen inhibits inflammation through its receptors ERα, ERβ, and GPER ( 52 ), and a clinical study comparing the expression of ERα in the renal tissues of patients with immunoglobulin A nephropathy (IgAN) suggested that the expression of ERα in the glomeruli of IgAN renal tissue decreased gradually as the severity of renal dysfunction increased, as indicated by the estimated glomerular filtration rate (eGFR), Scr clearance rate, and pathological grade ( 53 ). More importantly, several ER response elements are reported to exist in the promoter region of the TGF-βRI gene, and E 2 treatment reduces the mRNA expression of TGF-βRI in MC3T3−E1 cells ( 29 ). As male and OVX rats suffered more damage after IRI than normal female rats, E 2 is hypothesized to exert a protective effect during IRI.…”

“…TGF-βRI-mRNA-expressing cells were also shown to exhibit ERα immunoreactivity in the anteroventral periventricular nucleus, medial preoptic nucleus, and arcuate nucleus ( 28 ). In MC3T3-E1 cells, a clonal preosteoblastic cell line, ERβ is involved in the estrogen-mediated repression of TGF-βRI by binding to EREs located in the TGF-βRI promoter region ( 29 ), suggesting a close relationship between ERs and the TGF-βRI/SMAD pathway. As mentioned previously, E 2 plays a dominant role in AKI, and the mechanisms by which ERs influence the TGF-βRI/SMAD pathway are therefore worthy of further exploration.…”