Linc-RAM is required for FGF2 function in regulating myogenic cell differentiation

Abstract

Myogenic differentiation of skeletal muscle stem cells, also known satellite cells, is tightly orchestrated by extrinsic and intrinsic regulators. Basic fibroblast growth factor (FGF2) is well documented to be implicated in satellite cell self-renewal and differentiation by repressing MyoD. We recently identified a MyoD-regulated and skeletal muscle-specifically expressed long non-coding RNA Linc-RAM which enhances myogenic differentiation by facilitating MyoD/Baf60c/Brg1 complex assembly. Herein, we investigated the transcriptional regulation and intracellular signaling pathway in mediating Linc-RAM gene expression during muscle cell differentiation. Firstly, we demonstrate Linc-RAM is negatively regulated by FGF2 via Ras/Raf/Mek/Erk signaling pathway in muscle cells. Overexpression of MyoD significantly attenuates repression of Linc-RAM promoter activities in C2C12 cells treated with FGF2. Knockout of MyoD abolishes FGF2-mediated repression of Linc-RAM gene transcription in satellite cells sorted from skeletal muscle of MyoD^-/-;Pax7-nGFP mice, suggesting inhibition of MyoD is required for FGF2-mediated expression of Linc-RAM. For the functional significance, we show that overexpression of Linc-RAM rescues FGF2-induced inhibition of C2C12 cell differentiation, indicating inhibition of Linc-RAM is required for FGF2-mediated suppression of myogenic differentiation. Consistently, we are able to further corroborate the requirement of Linc-RAM inhibition for FGF2-modulated repression of myogenic differentiation by using an ex vivo cultured single fiber system and satellite cells sorted from Linc-RAM^-/-;Pax7-nGFP knockout mice. Collectively, the present study not only reveals the intracellular signaling in FGF2-mediated Linc-RAM gene expression but also demonstrate the functional significance of Linc-RAM in FGF2-mediated muscle cell differentiation.

Keywords: FGF2; Linc-RAM; Long non-coding RNA; MyoD; muscle cell differentiation.

Figure 1.

Linc-RAM is negatively regulated by FGF2 in muscle cells. A. C2C12 cells were treated with FGF2 in differentiation medium (DM) for the indicated times. The expressions of MyoD, MyoG, and Linc-RAM were examined by real-time quantitative RT-PCR (RT-qPCR). B. Satellite cells sorted from Pax7-nGFP mice were treated with FGF2 in growth medium (GM) for 24hr, and the expressions of Pax7, MyoD, MyoG, and Linc-RAM were examined by RT-qPCR. C. Satellite cells sorted from Pax7-nGFP mice were treated with FGF2 in GM for 48hr, and the expressions of Pax7, MyoD, MyoG, and Linc-RAM were examined by RT-qPCR. D. The responsiveness of the Linc-RAM gene promoter to FGF2 was examined in C2C12 cells using a luciferase reporter gene assay. C2C12 cells were treated with FGF2 and then transfected with promoter-reporter plasmids for Linc-RAM (RAM). The MyoD and MyoG gene promoters served as positive controls. Promoter activity is expressed as luciferase activity relative to that in the vehicle control group (defined as 1). Values are means ± s.e.m. of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. All P values were generated using the two-tailed Student's t-test.

Figure 2.

FGF2 regulates Linc-RAM gene expression via the Ras/Raf/Mek/Erk signaling pathway. A. C2C12 cells in DM were treated with FGF2, PD98059 (PD, a Mek inhibitor), or PD plus (30 min later) FGF2. After 24 hr, the phosphorylations of Erk and Mek were detected by Western blotting. Total Erk and Mek were examined as controls. The blots shown are representative of the results obtained from three independent experiments. B. The expressions of Linc-RAM and MyoD, MyoG in cells treated as described in a were examined by RT-qPCR. C. The FGF2 responsiveness of the Linc-RAM gene promoter was examined in C2C12 cells by luciferase reporter gene assay. C2C12 cells were treated with FGF2, PD, or PD (for 1 h) plus FGF2, and then transfected with promoter reporter plasmids for Linc-RAM. The MyoD gene promoter served as positive controls. Promoter activity is expressed as luciferase activity relative to that in the vehicle control group (defined as 1). D. C2C12 cells in DM were treated with FGF2, FTA (a Ras inhibitor), or FTA (for 30 min) plus FGF2. After 24 hr, the expressions of Linc-RAM and MyoD, MyoG were examined by RT-qPCR. E. C2C12 cells in DM were treated with FGF2, GW (a Raf inhibitor), or GW (for 30 min) plus FGF2. After 24 hr, the expressions of Linc-RAM and MyoD, MyoG were examined by RT-qPCR. Values are means ± s.e.m. of three independent experiments. *P< 0.05, **P< 0.01, ***P < 0.001, as assessed by the two-tailed Student's t-test.

Figure 3.

MyoD is required for FGF2-mediated Linc-RAM gene expression. A. Expression of Linc-RAM in satellite cells isolated from Pax7-nGFP;MyoD^−/−(KO) and Pax7-nGFP (WT) mice. Cells were cultured in GM for 48 hr in the presence and absence of FGF2. The expression of MyoG served as positive controls. B. The responsiveness of the Linc-RAM gene promoter (luciferase activity) was examined in C2C12 cells co-transfected with a MyoD-encoding plasmid and treated with or without FGF2. Promoter activity is expressed as luciferase activity relative to that in the control group (defined as 1). C. The relative enrichment of MyoD in the Linc-RAM gene promoter region was assessed by chromatin immunoprecipitation (ChIP) with a MyoD antibody. C2C12 cells cultured in DM for 24 hr in the presence or absence of FGF2 were subjected to ChIP experiments. The miR-206 and MyoG promoters served as positive controls. Values are means ± s.e.m. of three independent experiments. *P< 0.05, **P< 0.01, ***P < 0.001, as assessed by the two-tailed Student's t-test.

Figure 4.

Overexpression of Linc-RAM rescues the FGF2-induced inhibition of muscle cell differentiation. A. C2C12 cells stably overexpressing Linc-RAM (Linc-OE) and control C2C12 cells were treated with FGF2 in DM. After 24 and 48 hr, differentiation was examined by immunostaining for MyoG and MHC, respectively. Scale bar, 100 μm. The images are representative of the results obtained from three independent experiments. B. The numbers of MyoG⁺ cells per microscopic view in A were counted. C. The numbers of MHC⁺ cells per microscopic view in A were counted. Values are means ± s.e.m. of three independent experiments. ***P < 0.001, as obtained using the two-tailed Student's t-test.

Figure 5.

Deletion of Linc-RAM attenuates satellite cell differentiation and primes for self-renewal. A. Representative images of satellite cells from ex vivo-cultured single fibers immunostained for Pax7 (red) and MyoD (green). The single fibers were isolated from extensor digitorum longus (EDL) muscles and ex vivo cultured for 72 hr. DAPI was used to visualize nuclei (blue). Scale bar, 50μm.The lower panel shows magnified views of the upper panels. B. Percentages of Pax7⁺/MyoD⁺, Pax7^–/MyoD⁺ and Pax7⁺/MyoD^– cells immunostained as described in A Ex vivo-cultured single fibers were isolated from Linc-RAM^−/− and WT mice. The data are from three mice of each genotype, and more than 150 myofibers were analyzed per mice. C. The relative expressions of Linc-RAM in FACS-sorted satellite cells cultured in the presence or absence of FGF2 were determined by RT-qPCR. The satellite cells were sorted from Linc-RAM^−/−;Pax7-nGFP(KO) and Pax7-nGFP(WT) control mice (n = 3 animals for each genotype). D. Representative images of MyoG immunostaining (red) for FACS-resolved satellite cells cultured in GM for 48 hr in the presence or absence of FGF2. DAPI was used to visualize nuclei (blue). Scale bar, 100 μm. E. The percentages of MyoG-positive cells in d were calculated from three independent experiments. F. The relative expressions of MyoG in the cells described in D were determined by RT-qPCR. Values are means ± s.e.m. *P < 0.05, as assessed by the two-tailed Student's t-test.