Dataset Information

Retinoic acid promotes myogenesis in myoblasts by antagonizing transforming growth factor-beta signaling via C/EBPβ.

ABSTRACT: BACKGROUND: The effects of transforming growth factor-beta (TGFβ) are mediated by the transcription factors Smad2 and Smad3. During adult skeletal myogenesis, TGFβ signaling inhibits the differentiation of myoblasts, and this can be reversed by treatment with retinoic acid (RA). In mesenchymal stem cells and preadipocytes, RA treatment can function in a non-classical manner by stimulating the expression of Smad3. Smad3 can bind to and prevent the bzip transcription factor CCAAT/enhancer-binding protein beta (C/EBPβ) from binding DNA response elements in target promoters, thereby affecting cell differentiation. In skeletal muscle, C/EBPβ is highly expressed in satellite cells and myoblasts and is downregulated during differentiation. Persistent expression of C/EBPβ in myoblasts inhibits their differentiation. METHODS: Using both C2C12 myoblasts and primary myoblasts, we examined the regulation of C/EBPβ expression and activity following treatment with TGFβ and RA. RESULTS: We demonstrate that treatment with RA upregulates Smad3, but not Smad2 expression in myoblasts, and can partially rescue the block of differentiation induced by TGFβ. RA treatment reduces C/EBPβ occupancy of the Pax7 and Smad2 promoters and decreased their expression. RA also inhibits the TGFβ-mediated phosphorylation of Smad2, which may also contribute to its pro-myogenic activities. TGFβ treatment of C2C12 myoblasts stimulates C/EBPβ expression, which in turn can stimulate Pax7 and Smad2 expression, and inhibits myogenesis. Loss of C/EBPβ expression in myoblasts partially restores differentiation in the presence of TGFβ. CONCLUSIONS: TGFβ acts, at least in part, to inhibit myogenesis by upregulating the expression of C/EBPβ, as treatment with RA or loss of C/EBPβ can partially rescue differentiation in TGFβ-treated cells. This work identifies a pro-myogenic role for Smad3, through the inhibition of C/EBPβ's actions in myoblasts, and reveals mechanisms of crosstalk between RA and TGFβ signaling pathways.

SUBMITTER: Lamarche E

PROVIDER: S-EPMC4397812 | biostudies-other | 2015

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:Transforming growth factor beta (TGF?) plays a major role in the regulation of tumor initiation, progression, and metastasis. It is depended on the type II TGF? receptor (T?RII) for signaling. Previously, we have shown that deletion of T?RII in mammary epithelial of MMTV-PyMT mice results in shortened tumor latency and increased lung metastases. However, active TGF? signaling increased the number of circulating tumor cells and metastases in MMTV-Neu mice. In the current study, we describe a newly discovered connection between attenuated TGF? signaling and human epidermal growth factor receptor 2 (HER2) signaling in mammary tumor progression.All studies were performed on MMTV-Neu mice with and without dominant-negative T?RII (DNIIR) in mammary epithelium. Mammary tumors were analyzed by flow cytometry, immunohistochemistry, and immunofluorescence staining. The levels of secreted proteins were measured by enzyme-linked immunosorbent assay. Whole-lung mount staining was used to quantitate lung metastasis. The Cancer Genome Atlas (TCGA) datasets were used to determine the relevance of our findings to human breast cancer.Attenuated TGF? signaling led to a delay tumor onset, but increased the number of metastases in MMTVNeu/DNIIR mice. The DNIIR tumors were characterized by increased vasculogenesis, vessel leakage, and increased expression of vascular endothelial growth factor (VEGF). During DNIIR tumor progression, both the levels of CXCL1/5 and the number of CD11b+Gr1+ cells and T cells decreased. Analysis of TCGA datasets demonstrated a significant negative correlation between TGFBR2 and VEGF genes expression. Higher VEGFA expression correlated with shorter distant metastasis-free survival only in HER2+ patients with no differences in HER2-, estrogen receptor +/- or progesterone receptor +/- breast cancer patients.Our studies provide insights into a novel mechanism by which epithelial TGF? signaling modulates the tumor microenvironment, and by which it is involved in lung metastasis in HER2+ breast cancer patients. The effects of pharmacological targeting of the TGF? pathway in vivo during tumor progression remain controversial. The targeting of TGF? signaling should be a viable option, but because VEGF has a protumorigenic effect on HER2+ tumors, the targeting of this protein could be considered when it is associated with attenuated TGF? signaling.

Project description:IntroductionTransforming growth factor beta (TGF-β) has a dual role during tumor progression, initially as a suppressor and then as a promoter. Epithelial TGF-β signaling regulates fibroblast recruitment and activation. Concurrently, TGF-β signaling in stromal fibroblasts suppresses tumorigenesis in adjacent epithelia, while its ablation potentiates tumor formation. Much is known about the contribution of TGF-β signaling to tumorigenesis, yet the role of TGF-β in epithelial-stromal migration during tumor progression is poorly understood. We hypothesize that TGF-β is a critical regulator of tumor-stromal interactions that promote mammary tumor cell migration and invasion.MethodsFluorescently labeled murine mammary carcinoma cells, isolated from either MMTV-PyVmT transforming growth factor-beta receptor II knockout (TβRII KO) or TβRIIfl/fl control mice, were combined with mammary fibroblasts and xenografted onto the chicken embryo chorioallantoic membrane. These combinatorial xenografts were used as a model to study epithelial-stromal crosstalk. Intravital imaging of migration was monitored ex ovo, and metastasis was investigated in ovo. Epithelial RNA from in ovo tumors was isolated by laser capture microdissection and analyzed to identify gene expression changes in response to TGF-β signaling loss.ResultsIntravital microscopy of xenografts revealed that mammary fibroblasts promoted two migratory phenotypes dependent on epithelial TGF-β signaling: single cell/strand migration or collective migration. At epithelial-stromal boundaries, single cell/strand migration of TβRIIfl/fl carcinoma cells was characterized by expression of α-smooth muscle actin and vimentin, while collective migration of TβRII KO carcinoma cells was identified by E-cadherin+/p120+/β-catenin+ clusters. TβRII KO tumors also exhibited a twofold greater metastasis than TβRIIfl/fl tumors, attributed to enhanced extravasation ability. In TβRII KO tumor epithelium compared with TβRIIfl/fl epithelium, Igfbp4 and Tspan13 expression was upregulated while Col1α2, Bmp7, Gng11, Vcan, Tmeff1, and Dsc2 expression was downregulated. Immunoblotting and quantitative PCR analyses on cultured cells validated these targets and correlated Tmeff1 expression with disease progression of TGF-β-insensitive mammary cancer.ConclusionFibroblast-stimulated carcinoma cells utilize TGF-β signaling to drive single cell/strand migration but migrate collectively in the absence of TGF-β signaling. These migration patterns involve the signaling regulation of several epithelial-to-mesenchymal transition pathways. Our findings concerning TGF-β signaling in epithelial-stromal interactions are important in identifying migratory mechanisms that can be targeted as recourse for breast cancer treatment.

Dataset Information

Retinoic acid promotes myogenesis in myoblasts by antagonizing transforming growth factor-beta signaling via C/EBPβ.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets