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Variable osteogenic performance of MC3T3-E1 subclones impacts their utility as models of osteoblast biology.

ABSTRACT: The spontaneously immortalized murine calvarial cell line MC3T3-E1 and its derivative subclones are widely used models of osteoblast biology. Many investigators have reported conflicting data under seemingly similar experimental conditions, though the specific subclone studied is often not specified. The purpose of this study was to directly compare the commercially available MC3T3-E1 subclones 4, 14, and 24 in terms of responsiveness to osteogenic induction media and/or stimulation with rhPTH[1-34]. We assayed osteogenic gene expression, capacity to deposit and mineralize a collagenous matrix, and the expression and signaling function of PTH1R. Our data demonstrate that each subclone bears little functional resemblance to the others, or to primary calvarial osteoblasts. Specifically, whereas subclone 4 is responsive to PTH stimulation and capable of matrix mineralization, subclones 14 and 24 do not faithfully replicate these key aspects of osteoblast biology. Furthermore, little overlap was observed between the gene expression profile of subclone 4 and primary calvarial osteoblasts. Our experience working with these cell lines demonstrates that the MC3T3-E1 derived cell lines are imperfect models of osteoblast biology, and reinforce the importance of clearly articulating selection and reporting of research materials.

SUBMITTER: Hwang PW

PROVIDER: S-EPMC6549152 | biostudies-other | 2019 Jun

REPOSITORIES: biostudies-other

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Variable osteogenic performance of MC3T3-E1 subclones impacts their utility as models of osteoblast biology.

Hwang Phillip W PW Horton Jason A JA

Scientific reports 20190605 1

The spontaneously immortalized murine calvarial cell line MC3T3-E1 and its derivative subclones are widely used models of osteoblast biology. Many investigators have reported conflicting data under seemingly similar experimental conditions, though the specific subclone studied is often not specified. The purpose of this study was to directly compare the commercially available MC3T3-E1 subclones 4, 14, and 24 in terms of responsiveness to osteogenic induction media and/or stimulation with rhPTH[1 ...[more]

PMID: 31165768

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Project description:BackgroundIn mouse, it was discovered that resveratrol (Res) enhanced osteoporosis (OP) by boosting osteogenesis. Besides, Res can also have an impact on MC3T3-E1 cells, which are crucial for the control of osteogenesis and thus increase osteogenesis. Although some articles have discovered that Res enhanced autophagy to promote the value-added differentiation of MC3T3, it is unclear exactly how this affects the process of osteogenesis in mouse. Therefore, we will show that Res encourages MC3T3-E1 proliferation and differentiation in mouse pre-osteoblasts and further investigate the autophagy-related mechanism for this impact.Methods(1) MC3T3-E1 cells were separated into blank control group and various concentrations (0.01, 0.1, 1, 10, 100µmol/L) of group in order to determine the ideal Res concentration. In the Res group, Cell Counting Kit-8 (CCK-8) was used to measure the proliferation activity of pre-osteoblasts in mice in each group after resveratrol intervention. Alkaline Phosphatase (ALP) and alizarin red staining were used to gauge the degree of osteogenic differentiation, and RT-qPCR was used to measure the expression levels of Runx2 and OCN in the osteogenic differentiation ability of the cells. (2) In the experiment, four groups were set up: the control group, 3MA group, Res group, and Res + 3MA group. To examine cell mineralization, ALP and alizarin red staining were utilized. RT-qPCR and Western blot detection of cell autophagy activity levels and osteogenic differentiation capacity in each group following intervention.Results(1) Resveratrol might increase the number of mice pre-osteoblast, with the impact being most pronounced at 10µmol/L (P < 0.05). The nodules developed substantially more often than in the blank control group, and Runx2 and OCN expressions significantly increased (P < 0.05). (2) In contrast to the Res group, after 3MA purine blocked autophagy, the Res + 3MA group's alkaline phosphatase staining and the development of mineralized nodules were reduced. Runx2, OCN, LC3II / LC3I expression decreased, p62 expression increased (P < 0.05).ConclusionThe present study partially or indirectly demonstrated that Res may, through increased autophagy, induce osteogenic differentiation of MC3T3-E1 cells.

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.

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.

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Variable osteogenic performance of MC3T3-E1 subclones impacts their utility as models of osteoblast biology.

Publications

Variable osteogenic performance of MC3T3-E1 subclones impacts their utility as models of osteoblast biology.

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