BMP-2 and Sonic Hedgehog Have Contrary Effects on Adipocyte-Like Differentiation of C3H10T1/2 Cells

Zehentner, Barbara K.; Leser, Ulrike; Burtscher, Helmut

doi:10.1089/10445490050021186

Cited by 73 publications

(54 citation statements)

References 22 publications

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“…These cells proliferate as undifferentiated fibroblasts, and, like primary mesenchymal progenitor cells, are capable of differentiating into several cell types including adipocytes (29,30), osteoblasts (30,31), myoblasts (32), and chondrocytes (33,34), depending on the environment in which they are cultured. Here C3H10T1/2 cells were induced to undergo chondrogenesis by plating them in high-density micromass cultures in the presence of BMP-2 (33).…”

Section: Nkx32 and Runx2 Genes Exhibit Reciprocal Expression Patternmentioning

confidence: 99%

Nkx3.2-mediated Repression of Runx2 Promotes Chondrogenic Differentiation

Lengner

Hassan

Serra

et al. 2005

Journal of Biological Chemistry

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Runx2, a transcription factor known to be essential for osteoblast maturation and skeletogenesis, is also expressed in pre-cartilaginous mesenchymal condensations in the developing embryo. It is therefore necessary to understand the control and consequential regulatory activity of the Runx2 gene within the context of chondrogenic differentiation of a mesenchymal progenitor cell. We identify the homeodomain protein Nkx3.2 as a potent sequence-specific repressor of the Runx2 promoter that acts through a regulatory element 0.1 kb upstream from the site of transcriptional initiation. The biological significance of this repression is established by utilizing bone morphogenic protein 2 (BMP-2)-induced chondrogenic differentiation of pluripotent C3H10T1/2 cells as a model for the initial events of mesenchymal chondrogenesis. We demonstrate that induction of the chondrogenic phenotype and endogenous Nkx3.2 expression is accompanied by a repression of Runx2 gene activity. Bypassing Runx2 repression by adenoviral-mediated introduction of Runx2 into C3H10T1/2 cells can prevent the induction of chondrogenesis, but cannot reverse the chondrogenic phenotype once it has been initiated, as evidenced by Sox9 and type II collagen expression and extracellular matrix deposition. Our results demonstrate that Runx2 is a direct transcriptional target of Nkx3.2, and that repression of Runx2 at the onset of chondrogenesis is a prerequisite for the activation of a chondrocyte-specific program of gene expression. We postulate that Runx2 is a critical link in BMP-2-mediated initiation of mesenchymal chondrogenesis that results in activation of Sox9 at least in part through the Nkx3.2-dependent repression of Runx2.During mammalian embryogenesis, the axial skeleton is initially formed as a cartilaginous template prior to conversion into mineralized bone through the coordinated actions of osteoclasts and osteoblasts. The mesenchymal progenitor cells responsible for cartilage formation aggregate around the notochord and are induced to proliferate and subsequently to differentiate in response to the secreted signaling molecules Sonic hedgehog (Shh) 1 and bone morphogenic protein 2 (BMP-2) (1-4). The sequential and cooperative action of these molecules results in the induction of the pro-chondrogenic NKrelated homeodomain protein Nkx3.2 in mesenchymal progenitor cells. Nkx3.2 mediates transcriptional repression of target genes through its interactions with BMP-responsive SMAD proteins and histone deacetylase HDAC1 (5). Activation of Nkx3.2 is required for and closely followed by activation of the master chondrogenic transcriptional regulator Sox9. The activation of Sox9 through Nkx3.2 ultimately leads to activation of chondrocyte phenotypic genes, including type II collagen and aggrecan, and formation of a cartilaginous extracellular matrix (1, 6). The importance of Nkx3.2 in this process is evident from Nkx3.2 null mice, in which mesenchymal progenitor cells of the sclerotome fail to differentiate resulting in malformation or absence of axial s...

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Section: Nkx32 and Runx2 Genes Exhibit Reciprocal Expression Patternmentioning

confidence: 99%

Nkx3.2-mediated Repression of Runx2 Promotes Chondrogenic Differentiation

Lengner

Hassan

Serra

et al. 2005

Journal of Biological Chemistry

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“…However, we observed that the addition of BMP4 suppressed adipocyte differentiation, unlike the results of another study on preadipocytic C3H10T1/2 cell lines. 47 We suggest that this difference may be attributed, in part, to the concentration of BMP4 used here. Nevertheless, in addition to BMP signaling, several factors, such as the Wnt signaling pathway, regulate GATA2 expression.…”

Section: Regulation Of Bm-msc Differentiation By Gata2mentioning

confidence: 85%

“…47 Noggin inhibits BMP signaling and promotes osteogenic differentiation. 43 In addition, Hedgehog and Wnt signaling pathways inhibit adipocyte differentiation but promote osteoblastic differentiation.…”

Section: Discussionmentioning

confidence: 99%

“…43 In addition, Hedgehog and Wnt signaling pathways inhibit adipocyte differentiation but promote osteoblastic differentiation. [44][45][46][47][48] Efforts to identify the regulatory mechanisms that control the differentiation of BM-MSC into adipocytes/osteoblasts may lead to the development of new clinical applications in the fields of regenerative medicine and tissue engineering and enhance our understanding of hematopoiesis, since BM-MSC are the primary sources of the hematopoietic microenvironment. 1 Although GATA2 may play an important role in regulating adipocyte differentiation from a mouse preadipocytic cell line, [16][17][18] its role in regulating human BM-MSC differentiation is unknown.…”

Section: Discussionmentioning

confidence: 99%

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GATA2 regulates differentiation of bone marrow-derived mesenchymal stem cells

et al. 2014

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Methods Generation of bone marrow mesenchymal stem cellsTo generate mouse BM-MSC, bone marrow cells from GATA2 conditional knockout mice were cultured in MesenCult MSC Basal Medium supplemented with 20% MSC stimulatory supplements (Stem Cell Technologies). The BM-MSC were transfected with the retroviruses expressing iCre to delete the DNA binding domain of GATA2 by inducing the Cre-loxP system. 21,22 To generate human BM-MSC, bone marrow mononuclear cells from healthy donors were cultured with Dulbecco's modified Eagle's medium (Life Technologies) supplemented with 20% fetal bovine serum (Life Technologies), 10 ng/mL basic fibroblast growth factor (PeproTech), 10 mM HEPES (Life Technologies), and 100 μg/mL penicillin/streptomycin (Invitrogen). [23][24][25] Established BM-MSC were used until the seventh generation.The study was approved by the ethical committee of Tohoku University Graduate School of Medicine. Clinical samples were collected after obtaining written informed consent. The ethics policies of the Declaration of Helsinki were followed. Characterization of bone marrow mesenchymal stem cellsBM-MSC immunophenotypes were determined using a FACSAria II (BD). To induce differentiation into adipocytes, human Mesenchymal Stem Cell Adipogenic Differentiation Medium (Lonza) was used. After 12-16 days, morphological changes were assessed using an inverted microscope. Typical adipocytes were stained with Oil Red O.2 The area of mature adipocytes was determined using HistoQuest software (Novel Science). Quantitative reverse transcriptase polymerase chain reaction analysis and transcription profilingQuantitative reverse transcriptase polymerase chain reaction analysis (RT-PCR) was performed as previously described.26 Primer sequences are available upon request.For transcription profiling, the Human Genome U133 Plus 2.0 Array was used (Affymetrix). Gene ontology analysis was conducted using the DAVID bioinformatics program (http://david.abcc.ncifcrf.gov/). Short interfering RNA-mediated knockdownAnti-GATA2 and control short interfering RNA (siRNA) 26 were transfected into human BM-MSC with Lipofectamine TM RNAiMAX reagent (Life Technologies). Cells were analyzed 48 h after transfection. Viral vectors and cell transductionRetroviral overexpression of GATA2 was performed using the MSCV retrovirus vector, which co-expresses green fluorescent protein (GFP) by internal ribosome entry sites (IRES), transfecting into Platinum Retroviral Packaging Cell Lines (PLAT-F) 27 with FuGENE HD (Roche). Human BM-MSC were pretreated with Retronectin (TAKARA BIO.), and GFP-positive cells were sorted using FACSAria II (BD Biosciences).Co-culture of CD34-positive-enriched cells with a mesenchymal stem cell feeder layer BM-MSC were transfected with control or GATA2-siRNA. On day 3, control and GATA2 knockdowned BM-MSC, respectively, were replaced with serum-free medium containing CD34-positiveenriched cells (RIKEN). Serum-free medium (StemPro-34 SFM: Life Technologies) contained 100 ng/mL stem cell factor, 100 ng/mL interleukin (...

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“…C3H10T1/2 mouse mesenchymal cells, a pluripotential cell line, are able to differentiate into myoblasts (Liu et al, 2001), adipocytes (Zehentner et al, 2000), osteoblasts/ chondroblast (Ju et al, 2000) upon specific inductions. Differentiation into osteoblasts is stimulated with BMP-2 in the presence of L-ascorbic acid and b-glycerophosphate (Ju et al, 2000).…”

Section: Itm2a Expression In C3h10t1/2mentioning

confidence: 99%

In vitro studies on Itm2a reveal its involvement in early stages of the chondrogenic differentiation pathway

plas

Merregaert

2004

Biology of the Cell

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The Integral membrane protein 2A (Itm2A) is a transmembrane protein belonging to a family composed of at least two other members, Itm2B and Itm2C, all of them having a different expression pattern. The Itm2a gene serves as a marker for early stages in endochondral ossification. In order to understand the role of Itm2A in this process, expression of the gene was investigated in different cell systems. In C3H10T1/2 cells, the gene was upregulated early on when the cells were induced to the chondrogenic lineage but less to the osteogenic lineage. In MCT cells, expression was upregulated at permissive temperatures but not at non-permissive temperatures. When induced with insulin, ATDC5 cells expressed Itm2a in early stages but not at late stages. Furthermore, PTH treatment seems to upregulate Itm2a transcription.In order to understand the role of Itm2a in the chondrogenic differentiation process in more detail, we constitutively overexpressed exogenous Itm2A in mouse ATDC5 cells. Two clones expressing high levels of Itm2a were isolated and characterized. Gene expression analysis of the overexpresser clones demonstrated that expression of collagen type X was delayed. These results demonstrate that overexpression of Itm2a in mouse ATDC5 cells impede the transition to hypertrophic cells. Taken together, our observation supports the involvement of Itm2a in the early stages of chondrogenesis in vitro.

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BMP-2 and Sonic Hedgehog Have Contrary Effects on Adipocyte-Like Differentiation of C3H10T1/2 Cells

Cited by 73 publications

References 22 publications

Nkx3.2-mediated Repression of Runx2 Promotes Chondrogenic Differentiation

Nkx3.2-mediated Repression of Runx2 Promotes Chondrogenic Differentiation

GATA2 regulates differentiation of bone marrow-derived mesenchymal stem cells

In vitro studies on Itm2a reveal its involvement in early stages of the chondrogenic differentiation pathway

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