Upon injury, muscle satellite cells become activated and produce skeletal muscle precursors that engage in myogenesis. We demonstrate that the transcription factor CCAAT/ enhancer binding protein beta (C/EBPb) is expressed in the satellite cells of healthy muscle. C/EBPb expression is regulated during myogenesis such that C/EBPb is rapidly and massively downregulated upon induction to differentiate. Furthermore, persistent expression of C/EBPb in myoblasts potently inhibits differentiation at least in part through the inhibition of MyoD protein function and stability. As a consequence, myogenic factor expression, myosin heavy chain expression, and fusogenic activity were reduced in C/EBPboverexpressing cells. Using knockout models, we demonstrate that loss of Cebpb expression in satellite cells results in precocious differentiation of myoblasts in growth conditions and greater cell fusion upon differentiation. In vivo, loss of Cebpb expression in satellite cells resulted in larger muscle fiber cross-sectional area and improved repair after muscle injury. Our results support the notion that C/EBPb inhibits myogenic differentiation and that its levels must be reduced to allow for activation of MyoD target genes and the progression of differentiation.
The process of adipocyte differentiation is driven by a highly coordinated cascade of transcriptional events that results in the development of the mature adipocyte and in lipid accumulation. One of the early events of differentiation is the up-regulation of CCAAT/enhancer-binding protein  (C/EBP) expression. C/EBP then acts to up-regulate the expression of adipogenic factors such as C/EBP␣, which control the late stage of adipogenesis. Retinoic acid (RA) is a potent inhibitor of adipogenesis, and its action appears to block C/EBP transcriptional potential early during differentiation. Using preadipocytes and mesenchymal stem cell models, we show that RA specifically blocks the occupancy of C/EBP of the Cebpa promoter, thereby abrogating the differentiation process. RA does not act directly on C/EBP but rather stimulates the expression of the transforming growth factor -effector protein Smad3, which can interact with C/EBP via its Mad homology 1 domain and can interfere with C/EBP DNA binding. The RA-induced increase in Smad3 expression results in increased cytoplasmic and nuclear Smad3, an important event as ectopic expression of Smad3 in preadipocytes in the absence of RA treatment only modestly inhibits adipogenesis and C/EBP DNA binding, suggesting that Smad3 alone is not sufficient to completely recapitulate the effects of retinoic acid treatment during differentiation. However, in the absence of Smad3, RA is not able to inhibit adipocyte differentiation or to elicit a decrease in C/EBP DNA occupancy suggesting that Smad3 is necessary to convey the inhibitory effects of retinoic acid during adipogenesis.
Runx2/CBFA1/AML3 is a master regulator of the osteoblast lineage and has been shown to directly control the transcription of numerous osteoblast-specific genes including alkaline phosphatase, osteopontin, and type I collagen. In its absence, ossification does not occur during development resulting in animals with cartilaginous skeletons and no osteoblasts. In humans, loss of one copy of Runx2 causes cleidocranial dysplasia characterized by malformations of the facial and cranial bones and the clavicle. Despite its important role in osteoblast biology, relatively little is known about the transcriptional regulation of the Runx2 gene. In the present study, we show that CCAAT/enhancer binding protein beta (C/EBPbeta) is a negative regulator of Runx2 expression and acts by directly binding a C/EBP element located at -591/-576 within the osteoblast-specific Runx2 P1 promoter. Ectopic expression of C/EBPbeta in C3H10T1/2 cells causes a reduction in Runx2 expression concomitant with a decrease in osteogenic potential during all-trans retinoic acid (ATRA)-induced differentiation. In nondifferentiating cells, C/EBPbeta can be found occupying the C/EBP negative response element within the Runx2 P1 promoter. ATRA, the effects of which are mediated by retinoic acid receptor alpha and gamma in C3H10T1/2 cells, stimulates the dissociation of C/EBPbeta from this element and promotes Runx2 expression. Thus, ATRA initiates osteoblastic differentiation of C3H10T1/2 cells, at least in part, by triggering the dissociation of C/EBPbeta from the Runx2 promoter.
Cancer cachexia is a paraneoplastic syndrome that causes profound weight loss and muscle mass atrophy and is estimated to be the cause of up to 30% of cancer deaths. Though the exact cause is unknown, patients with cancer cachexia have increased muscle protein catabolism. In healthy muscle, injury activates skeletal muscle stem cells, called satellite cells, to differentiate and promote regeneration. Here, we provide evidence that this mechanism is inhibited in cancer cachexia due to persistent expression of CCAAT/Enhancer Binding Protein beta (C/EBPβ) in muscle myoblasts. C/EBPβ is a bzip transcription factor that is expressed in muscle satellite cells and is normally downregulated upon differentiation. However, in myoblasts exposed to a cachectic milieu, C/EBPβ expression remains elevated, despite activation to differentiate, resulting in the inhibition of myogenin expression and myogenesis. In vivo, cancer cachexia results in increased number of Pax7+ cells that also express C/EBPβ and the inhibition of normal repair mechanisms. Loss of C/EBPβ expression in primary myoblasts rescues differentiation under cachectic conditions without restoring myotube size, indicating that C/EBPβ is an important inhibitor of myogenesis in cancer cachexia.
Background: Hedgehog (Hh) signaling regulates skeletal myogenesis; however, the molecular mechanisms involved are not fully understood.Results: Gli2, a transactivator of Hh signaling, associates with MyoD gene elements, regulating MyoD expression, and binds to MyoD protein, regulating its ability to induce myogenesis.Conclusion: Hh signaling is linked to MyoD gene expression and MyoD protein function.Significance: Novel mechanistic insight is gained into the Hh-regulated myogenesis.
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