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ATP6V1H facilitates osteogenic differentiation in MC3T3-E1 cells via Akt/GSK3? signaling pathway.

ABSTRACT: Type 2 diabetes mellitus (T2DM) accounts for approximately 90% of all diabetic patients, and osteoporosis is one of the complications during T2DM process. ATP6V1H (V-type proton ATPase subunit H) displays crucial roles in inhibiting bone loss, but its role in osteogenic differentiation remains unknown. Therefore in this study, we aimed to explore the biological role of ATP6V1H in osteogenic differentiation. OM (osteogenic medium) and HG (high glucose and free fatty acids) were used to induce the MC3T3-E1 cells into osteogenic differentiation in a T2DM simulating environment. CCK8 assay was used to detect cell viability. Alizarin Red staining was used to detect the influence of ATP6V1H on osteogenic differentiation. ATP6V1H expression increased in OM-MC3T3-E1 cells, while decreased in OM+HG-MC3T3-E1 cells. ATP6V1H promoted osteogenic differentiation of OM+HG-MC3T3-E1 cells. Overexpression of ATP6V1H inhibited Akt/GSK3? signaling pathway, while knockdown of ATP6V1H promoted Akt/GSK3? signaling pathway. ATP6V1H overexpression promoted osteogenic differentiation of OM+HG-MC3T3-E1 cells. The role of ATP6V1H in osteogenic differentiation in a T2DM simulating environment involved in Akt/GSK3? signaling pathway. These data demonstrated that ATP6V1H could serve as a potential target for osteogenic differentiation in a T2DM simulating environment.

SUBMITTER: Jiang F

PROVIDER: S-EPMC6668661 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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ATP6V1H facilitates osteogenic differentiation in MC3T3-E1 cells via Akt/GSK3β signaling pathway.

Jiang Fusong F Shan Haojie H Pan Chenhao C Zhou Zubin Z Cui Keze K Chen Yuanliang Y Zhong Haibo H Lin Zhibin Z Wang Nan N Yan Liang L Yu Xiaowei X

Organogenesis 20190704 2

Type 2 diabetes mellitus (T2DM) accounts for approximately 90% of all diabetic patients, and osteoporosis is one of the complications during T2DM process. ATP6V1H (V-type proton ATPase subunit H) displays crucial roles in inhibiting bone loss, but its role in osteogenic differentiation remains unknown. Therefore in this study, we aimed to explore the biological role of ATP6V1H in osteogenic differentiation. OM (osteogenic medium) and HG (high glucose and free fatty acids) were used to induce the ...[more]

PMID: 31272281

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Project description:ObjectiveMicroRNAs (miRNAs) are well-recognized for their abilities to regulate gene expression post-transcriptionally in plants and animals. Recently, miRNA-messenger RNA (mRNA) regulatory relationships have been confirmed during biological processes, including osteogenic differentiation. This study aimed to find out more candidate miRNA-mRNA pairs involved in the osteogenic differentiation of MC3T3-E1 cells.MethodsAn MC3T3-E1-based microarray dataset (accessioned as GSE46400) downloaded from the Gene Expression Omnibus included MC3T3-E1 cells with or without 14-day osteoblast differentiation osteoblast induction. Multiple miRNA-mRNA prediction databases were searched by differentially expressed genes (DEGs) to obtain pairs of a miRNA-DEG regulatory network. The MC3T3-E1 cells were cultured and incubated in the osteogenic differentiation medium for 14 days. The expressions of candidate miRNAs and mRNAs were determined by real-time quantitative PCR(RT-qPCR) in MC3T3-E1 cells. The miRNA-mRNA interactions were verified by dual-luciferase reporter gene assays and experiments using mimics miRNA or their inhibitors.ResultsWe identified 715 upregulated DEGs and 603 downregulated DEGs between MC3T3-E1 cells with and without osteoblast induction by analyzing the raw data of the GSE46400 dataset. There were 7 overlapped miRNA-mRNA pairs identified during osteogenic differentiation of MC3T3-E1 cells, including mmu-miR-204-5p-Arhgap11a, mmu-miR-211-5p-Arhgap11a, mmu-miR-24-3p-H2afx, mmu-miR-3470b-Chek2, mmu-miR-3470b-Dlgap5, mmu-miR-466b-3p-Chek1, and mmu-miR-466c-3p-Chek1. The Arhgap11a, H2afx, Chek2, Dlgap5, and Chek1 were hub genes downregulated in MC3T3-E1 cells after osteogenic differentiation, verified by RT-qPCR results. The RT-qPCR also determined declined expressions of miR-204-5p and miR-24-3p concomitant with elevated expressions of miR-211-5p, miR-3470b, miR-466b-3p, and miR-466c-3p in the MC3T3-E1 cells, with osteoblast induction compared with undifferentiated MC3T3-E1 cells. Dual-luciferase reporter gene assays demonstrated Arhgap11a as the target of miR-211-5p. MiR-211-5p upregulation by its mimic increased Arhgap11a expression in MC3T3-E1 cells.ConclusionOur study characterizes miR-211-5p targeting Arhgap11a promotes the osteogenic differentiation of MC3T3-E1 cells, which provides novel targets to promote the osteogenesis process during bone repair.

Project description:BackgroundWe have previously reported that repeated treatment of human periodontal ligament cells and murine pre-osteoblast MC3T3-E1 cells with transforming growth factor-beta 1 (TGF-β1) inhibited their osteoblastic differentiation because of decreased insulin-like growth factor-1 (IGF-1) secretion. We also found that IGF-1/PI3K signaling plays an important role in osteoblast differentiation induced by TGF-β1 treatment; however, the downstream signaling controlling this remains unknown. The aim of this current study is to investigate whether Akt activation is required for osteoblast differentiation.Methodology/principal findingsMC3T3-E1 cells were cultured in osteoblast differentiation medium (OBM) with or without 0.1 ng/mL TGF-β1. OBM containing TGF-β1 was changed every 12 h to provide repeated TGF-β1 administration. MC3T3-E1 cells were infected with retroviral vectors expressing constitutively active (CA) or dominant-negative (DN)-Akt. Alkaline phosphatase (ALP) activity and osteoblastic marker mRNA levels were substantially decreased by repeated TGF-β1 treatment compared with a single TGF-β1 treatment. However, expression of CA-Akt restored ALP activity following TGF-β1 treatment. Surprisingly, ALP activity increased following multiple TGF-β1 treatments as the number of administrations of TGF-β1 increased. Activation of Akt significantly enhanced expression of osteocalcin, but TGF-β1 treatment inhibited this. Mineralization of MC3T3-E1 cells was markedly enhanced by CA-Akt expression under all medium conditions. Exogenous IGF-1 restored the down-regulation of osteoblast-related gene expression by repeated TGF-β1 administration. However, in cells expressing DN-Akt, these levels remained inhibited regardless of IGF-1 treatment. These findings indicate that Akt activation is required for the early phase of osteoblast differentiation of MC3T3-E1 cells induced by TGF-β1. However, Akt activation is insufficient to reverse the inhibitory effects of TGF-β1 in the late stages of osteoblast differentiation.ConclusionsTGF-β1 could be an inducer or an inhibitor of osteoblastic differentiation of MC3T3-E1 cells depending on the state of Akt phosphorylation. Our results indicate that Akt is the molecular switch for TGF-β1-induced osteoblastic differentiation of MC3T3-E1 cells.

Dataset Information

ATP6V1H facilitates osteogenic differentiation in MC3T3-E1 cells via Akt/GSK3? signaling pathway.

Publications

ATP6V1H facilitates osteogenic differentiation in MC3T3-E1 cells via Akt/GSK3β signaling pathway.

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