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. 2011 Apr 22;286(16):13805-14.
doi: 10.1074/jbc.M110.192625. Epub 2011 Feb 15.

TGF-beta regulates miR-206 and miR-29 to control myogenic differentiation through regulation of HDAC4

Catherine E Winbanks  1 , Bo Wang, Claudia Beyer, Phillip Koh, Lloyd White, Phillip Kantharidis, Paul Gregorevic
Affiliations

TGF-beta regulates miR-206 and miR-29 to control myogenic differentiation through regulation of HDAC4

Catherine E Winbanks et al. J Biol Chem. .

Abstract

MicroRNAs (miRs) are emerging as prominent players in the regulation of many biological processes, including myogenic commitment and skeletal muscle formation. Members of the TGF-β family can influence the proliferation and myogenic differentiation of cells, although it is presently not clear what role miRNAs play in the TGF-β-mediated control of myogenic differentiation. Here, we demonstrate in the myogenic C2C12 cell line, and in primary muscle cells, that miR-206 and miR-29-two miRs that act on transcriptional events implicated in muscle differentiation are down-regulated by TGF-β. We further demonstrate that TGF-β treatment of myogenic cells is associated with increased expression of histone deacetylase 4 (HDAC4), a key inhibitor of muscle differentiation that has been identified as a target for regulation by miR-206 and miR-29. We confirmed that increased expression of miR-206 and miR-29 resulted in the translational repression of HDAC4 in the presence or absence of TGF-β via interaction with the HDAC4 3'-untranslated region. Importantly, we found that miR-206 and miR-29 can attenuate the inhibitory actions of TGF-β on myogenic differentiation. Furthermore, we present evidence that the mechanism by which miR-206 and miR-29 can inhibit the TGF-β-mediated up-regulation of HDAC4 is via the inhibition of Smad3 expression, a transducer of TGF-β signaling. These findings identify a novel mechanism of interaction between TGF-β and miR-206 and -29 in the regulation of myogenic differentiation through HDAC4.

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Figures

FIGURE 1.
FIGURE 1.
Up-regulation of miRNAs during C2C12 differentiation is altered by TGF-β exposure. a, C2C12 cells were differentiated in medium supplemented with 2% horse serum (differentiation medium, DM) or left in medium containing 10% FBS (growth medium, GM) for 48 h. b, treatment of C2C12 cells in DM with 5 ng/ml TGF-β for 48 h resulted in the suppression of all members of the miR-29 family (*, p < 0.05), miR-206 (*, p < 0.001), miR-133a (*, p < 0.001), and miR-133b (*, p < 0.005). TGF-β did not significantly affect members of the miR-200 family. c, RNA obtained from C2C12 cells treated with TGF-β in c was analyzed for the expression of pri-miR-206 and pri-miR-29c. TGF-β similarly suppressed levels of pri-miR-206 (*, p < 0.001) and pri-miR-29 (*, p < 0.001). d, the effect of TGF-β was tested on primary cells, which displayed increased proliferation with treatment (panel 2 versus panel 1). Similarly to its effect on C2C12 cells, TGF-β suppressed levels of miR-29a (*, p = 0.01) and miR-29c (*, p < 0.05) and the primary form of miR-29a (*, p < 0.05). Furthermore, the mature (*, p < 0.001) and primary forms of miR-206 (*, p < 0.001) were also suppressed by TGF-β in murine primary muscle cells. Con, control.
FIGURE 2.
FIGURE 2.
TGF-β exposure mediates HDAC4 expression via miR action upon the HDAC4 3′-UTR. a, because miR-206 and miR-29 target sites feature within the HDAC4 3′-UTR, we cloned the first 500 bp (containing the miR-29 target) and also the full-length 3′-UTR into a luciferase reporter construct. b, TGF-β treatment at 5 ng/ml for 48 h induces luciferase activity in cells transfected with either the short or full-length HDAC4 3′-UTR luciferase constructs, compared with transfected cells receiving only vehicle (*, p < 0.005). c, treatment with increasing doses of TGF-β for 48 h induces HDAC4 protein levels (detected by Western blot) in a dose-dependent manner up to doses of 1.25 ng/ml TGF-β, after which HDAC4 protein induction was maximal (Fig. 2c; *, p < 0.05 versus control). We also tested the ability of TGF-β to regulate HDAC4 protein levels in primary muscle cells and found that TGF-β at doses of 0.5 ng/ml could maximally induce HDAC4 protein levels (Fig. 2c; *, p < 0.05 versus control). d, in TGF-β-treated C2C12 cells exhibiting increased HDAC expression, we observed no change in HDAC4 gene expression, although other known TGF-β responsive genes, such as plasminogen activator inhibitor (30, 31), were strongly induced (*, p < 0.001). RLU, relative light units.
FIGURE 3.
FIGURE 3.
TGF-β regulates HDAC4 through canonical TGF-β signaling. a, although TGF-β1 increases activity of a luciferase construct featuring a HDAC4 3′-UTR, TGF-β2 also has a similar effect (*, p < 0.005 versus NC; #, p < 0.05 versus NC). BMP7 and activin-A, however, had no significant effect on the expression of luciferase. b, the increased expression of Luc-HDAC4 3′-UTR in cells treated with TGF-β (*, p < 0.001) was markedly inhibited when cells were co-transfected with a Smad7 expression cassette (compared with a nonfunctional cassette). c, furthermore, although Luc-HDAC4 3′-UTR activity was induced in response to TGF-β (*, p < 0.005 versus control), the response was inhibited by treatment with the type I receptor inhibitor SB431542 at 1 μm (SB; #, p < 0.01 versus TGF-β) or 10 μm (+, p = 0.001 versus TGF-β), where induction was prevented. RLU, relative light units.
FIGURE 4.
FIGURE 4.
The effect of TGF-β on HDAC4 activity is attenuated in the presence of miR-206 and miR-29. a and b, cells were transfected with miR-29 or miR-206 mimics for 48 h in the presence or absence of 5 ng/ml TGF-β. Compared with nontreated NC, TGF-β induced HDAC4 luciferase activity (a, *, p = 0.01 versus NC; b, *, p < 0.01 versus NC), whereas overexpression of either miR-29 (#, p = 0.05 versus NC) or miR-206 (#, p < 0.001 versus NC) reduced HDAC4 3′-UTR luciferase activity. Both miR-29abc and miR-206 also prevented induction of luciferase activity by TGF-β. c and d, transfection of cells with either miR-29 inhibitors or miR-206 inhibitors increases Luc-HDAC4 3′-UTR activity (c, #, p = 0.05 versus NC; *, p < 0.05 versus NC; and d, *, p < 0.001 versus NC; #, p < 0.001 versus NC). e, in C2C12 cells transfected with either miR-206 or miR-29 for 48 h, miR-29 inhibits basal Smad3 expression, whereas miR-206 does not (*, p < 0.01 versus control). f, C2C12 cells transfected with either miR-206 or miR-29 and treated with TGF-β for 48 h exhibit markedly reduced ability of TGF-β to induce Smad3 (*, p < 0.05 versus NC; +, p < 0.005 versus NC). RLU, relative light units.
FIGURE 5.
FIGURE 5.
The regulation of HDAC4 by TGF-β and miRs -206 and -29 is through post-transcriptional mechanisms. Cells treated with TGF-β alone or in combination with either miR-206 (a) or miR-29 (b) do not exhibit significant changes in HDAC4 transcription (HDAC4 mRNA levels are expressed relative to the negative control), even though the cells treated with TGF-β alone exhibit increased quantities of HDAC4 protein. Overexpression of either miR-206 (c) or miR-29 (d) degrades protein levels of HDAC4 and suppresses the induction of HDAC4 in the presence of TGF-β (*, p < 0.01 versus NC; +, p < 0.05 versus NC).
FIGURE 6.
FIGURE 6.
The ability of TGF-β to inhibit differentiation is impaired in the presence of miR-29 and miR-206. a and b, C2C12 cells were differentiated for 72 h in the presence or absence of TGF-β with or without miR-29/miR-206. Although TGF-β inhibits myogenic differentiation (panel 1 versus panel 2) and miR-29 and miR-206 both promote this process (panel 1 versus panel 3), the ability of TGF-β to inhibit differentiation in the presence of miR-29 or miR-206 is reduced (panel 2 versus panel 4). c and d, the mRNA levels of MyoD were increased in cells transfected with miR-29 (*, p = 0.005 versus NC; #, p < 0.005 versus NC; +, p < 0.05 versus NC) or miR-206 (*, p < 0.01 versus NC; #, p < 0.001 versus NC; +, p = NS versus NC). e and f, furthermore, the inhibitory effects of TGF-β on protein levels of differentiation markers, myosin heavy chain (MyHC) (*, p < 0.01 versus control; #, p < 0.01 versus control; +, p < 0.01 versus TGF-β), and Mef-2 (*, p < 0.05 versus control; #, p < 0.05 versus control; +, p < 0.05 versus TGF-β) were attenuated in the presence of these miRNAs.
FIGURE 7.
FIGURE 7.
A proposed model of the mechanisms by which TGF-β inhibits myogenic commitment. The actions of TGF-β suppress levels of miR-206 and miR-29, which relieves translational suppression of HDAC4. Increased HDAC4 protein levels contribute to the inhibition of MRFs, thereby attenuating transcriptional activity that otherwise facilitates commitment to myogenic differentiation. The stimulatory effect of TGF-β upon HDAC4 expression can be prevented by inhibiting receptor dependent signaling such as Smad7 or by expressing pathway-specific ubiquitin ligases. Increased concentrations of miR-206 and miR-29 can reduce expression of Smad3, thereby regulating the potential of TGF-β to exert effects via this signaling protein. In addition to TGF-β, myostatin and IGF signaling can also act to control muscle cell differentiation through substrates including Smad3 and Akt/mTOR (47), the latter of which can regulate expression of miR-1 (48) and both of which can exert effects on HDAC4 and the myogenic transcriptional elements Mef-2 and MyoD (9, 23, 24, 49).
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