The FoxO3/type 2 deiodinase pathway is required for normal mouse myogenesis and muscle regeneration
Monica Dentice, Alessandro Marsili, Raffaele Ambrosio, Ombretta Guardiola, Annarita Sibilio, Ji-Hye Paik, Gabriella Minchiotti, Ronald A. DePinho, Gianfranco Fenzi, P. Reed Larsen, Domenico Salvatore
Monica Dentice, Alessandro Marsili, Raffaele Ambrosio, Ombretta Guardiola, Annarita Sibilio, Ji-Hye Paik, Gabriella Minchiotti, Ronald A. DePinho, Gianfranco Fenzi, P. Reed Larsen, Domenico Salvatore
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Research Article Muscle biology

The FoxO3/type 2 deiodinase pathway is required for normal mouse myogenesis and muscle regeneration

Abstract

The active thyroid hormone 3,5,3′ triiodothyronine (T3) is a major regulator of skeletal muscle function. The deiodinase family of enzymes controls the tissue-specific activation and inactivation of the prohormone thyroxine (T4). Here we show that type 2 deiodinase (D2) is essential for normal mouse myogenesis and muscle regeneration. Indeed, D2-mediated increases in T3 were essential for the enhanced transcription of myogenic differentiation 1 (MyoD) and for execution of the myogenic program. Conversely, the expression of T3-dependent genes was reduced and after injury regeneration markedly delayed in muscles of mice null for the gene encoding D2 (Dio2), despite normal circulating T3 concentrations. Forkhead box O3 (FoxO3) was identified as a key molecule inducing D2 expression and thereby increasing intracellular T3 production. Accordingly, FoxO3-depleted primary myoblasts also had a differentiation deficit that could be rescued by high levels of T3. In conclusion, the FoxO3/D2 pathway selectively enhances intracellular active thyroid hormone concentrations in muscle, providing a striking example of how a circulating hormone can be tissue-specifically activated to influence development locally.

Authors

Monica Dentice, Alessandro Marsili, Raffaele Ambrosio, Ombretta Guardiola, Annarita Sibilio, Ji-Hye Paik, Gabriella Minchiotti, Ronald A. DePinho, Gianfranco Fenzi, P. Reed Larsen, Domenico Salvatore

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Figure 5

D2 expression in myoblasts is under direct FoxO3 control.

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D2 expression in myoblasts is under direct FoxO3 control. (A) C2C12 or w...
(A) C2C12 or wild-type pp6 cells were transiently transfected with a FoxO3 or empty vector as indicated, and Dio2 mRNA measured 48 hours later by RT-PCR. (B) Left: Nuclear extracts from transfected cells with (+) or without (–) FoxO3 plasmid were analyzed by EMSA as indicated. Right: Schematic representation of mouse Dio2 proximal promoter region. Relevant binding sites and their mutant version are shown schematically. FoxO3 C.A, FoxO3 constitutively active; Comp., oligonucleotides used for competition (S, self oligo; U, unrelated oligo). (C) ChIP analysis of the interaction between FoxO3 and the mouse Dio2 promoter. Chromatin extracted from C2C12 cells was immunoprecipitated using the indicated antibodies. Unrel., unrelated genomic region. (D) Left: Dio2 mRNA in pp6 cells from wild-type and Foxo3+/– mice were cultured in proliferative and differentiating conditions and transfected with empty vector or a FoxO3 dominant-negative (FoxO D.N.) expression plasmid. Right: Dio2 mRNA in TA muscles from wild-type and Foxo3+/– mice. (E) Dio2 mRNA levels were measured by RT-PCR in shCTR or shFoxO3 cells. shFoxO3 cells were transfected with an RNAi-resistant FoxO3 plasmid as indicated. (F) pp6 cells from wild-type and Foxo3+/– mice were cultured in proliferative and differentiating conditions and treated with vehicle or 30 nM T3. MyoD mRNA was measured by RT-PCR. (G) MyoD mRNA levels in pp6 wild-type cells transfected with empty vector or FoxO C.A. or FoxO D.N. vector and treated with vehicle, T3, or rT3 as indicated. (H) Myogenin mRNA levels in pp6 from Dio2–/– mice cultured in proliferative and differentiating conditions and transfected with empty vector, FoxO3 C.A., or MyoD expression vectors. Values are mean ± SEM of at least 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.