Nadine Wiper-Bergeron

Stimulation of preadipocyte differentiation by steroid through targeting of an HDAC1 complex

Glucocorticoids potentiate the early steps of preadipocyte differentiation and promote obesity in Cushings syndrome and during prolonged steroid therapy. We show that glucocorticoids stimulate 3T3 L1 preadipocyte differentiation through a non‐transcriptional mechanism mediated through the ligand‐binding domain of the glucocorticoid receptor. This enhanced the onset of CCAAT/enhancer binding protein (C/EBPα) expression by potentiating its initial transcriptional activation by C/EBPβ. In the absence of steroid, C/EBPβ associated with a transcriptional corepressor complex containing mSin3A and histone deacetylase 1 (HDAC1), but lacking HDAC2 and RbAp46/48. HDAC1/mSin3A were recruited to the C/EBPα promoter with C/EBPβ and promoted the deacetylation of histone H4. Steroid induced the specific depletion of this corepressor by targeting the HDAC1 within the complex for degradation through the 26S proteasome. Treatment with histone deacetylase inhibitors replaced the effects of steroid treatment on preadipocyte differentiation and C/EBPα expression, while overexpression of HDAC1 abrogated the stimulatory effects of steroid. Recapitulation of the glucocorticoid effect by progestin treatment in the presence of the progesterone receptor ligand‐binding domain suggests a conserved mechanism relevant to many aspects of steroid‐mediated differentiation.

Glucocorticoid-stimulated preadipocyte differentiation is mediated through acetylation of C/EBPβ by GCN5

Preadipocyte differentiation in culture is driven by an insulin and cAMP dependant transcriptional cascade which induces the bzip transcription factors C/EBPβ and C/EBPδ. We have previously shown that glucocorticoid treatment, which strongly potentiates this differentiation pathway, stimulates the titration of the corepressor histone deacetylase 1 (HDAC1) from C/EBPβ. This results in a dramatic enhancement of C/EBPβ-dependent transcription from the C/EBPα promoter, concomitant with potentiation of preadipocyte differentiation. Here, we show that C/EBPβ is acetylated by GCN5 and PCAF within a cluster of lysine residues between amino acids 98–102 and that this acetylation is strongly induced by glucocorticoid treatment. Arginine substitution of the lysine residues within the acetylation motif of C/EBPβ prevented acetylation and blocked the ability of glucocorticoids to enhance C/EBPβ-directed transcription and to potentiate C/EBPβ-dependent preadipocyte differentiation. Moreover, acetylation of C/EBPβ appeared to directly interfere with the interaction of HDAC1 with C/EBPβ, suggesting that PCAF/GCN5-dependent acetylation of C/EBPβ serves as an important molecular switch in determining the transcriptional regulatory potential of this transcription factor.

Transcription Factor Smad3 Is Required for the Inhibition of Adipogenesis by Retinoic Acid

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 β (C/EBPβ) expression. C/EBPβ then acts to up-regulate the expression of adipogenic factors such as C/EBPα, which control the late stage of adipogenesis. Retinoic acid (RA) is a potent inhibitor of adipogenesis, and its action appears to block C/EBPβ transcriptional potential early during differentiation. Using preadipocytes and mesenchymal stem cell models, we show that RA specifically blocks the occupancy of C/EBPβ of the Cebpa promoter, thereby abrogating the differentiation process. RA does not act directly on C/EBPβ but rather stimulates the expression of the transforming growth factor β-effector protein Smad3, which can interact with C/EBPβ via its Mad homology 1 domain and can interfere with C/EBPβ 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/EBPβ 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/EBPβ DNA occupancy suggesting that Smad3 is necessary to convey the inhibitory effects of retinoic acid during adipogenesis.

Retinoic acid-induced Smad3 expression is required for the induction of osteoblastogenesis of mesenchymal stem cells

Mesenchymal stem cells are pluripotent precursor cells that can differentiate into osteoblasts, adipocytes, chondrocytes and myocytes. Despite their important therapeutic potential little is known about the transcriptional cascades that govern lineage decisions in these cells. Treatment of C3H10T1/2 mouse mesenchymal stem cells with retinoic acid (RA) inhibits adipogenesis and enhances osteoblastogenesis. In particular, RA treatment stimulates the expression of the osteoblast master regulator, runt-related transcription factor 2 (Runx2), whose expression is necessary for the formation of bone. We have shown previously in mesenchymal stem cells that RA acts to stimulate osteoblastogenesis by interfering with the actions of the bzip transcription factor CCAAT/Enhancer Binding Protein beta (C/EBPβ), where it binds to a negative regulatory element within the Runx2 promoter and inhibits its expression. Herein we show that Smad3, whose expression is stimulated by RA, relays the effects of RA on differentiation by initiating the displacement of C/EBPβ from the Runx2 promoter. In addition to stimulating Smad3 expression, RA also stimulated the nuclear localization of this factor, such that in the absence of RA, ectopic Smad3 was unable to drive osteoblastogenesis. While not sufficient to promote osteoblastogenesis, knockdown of Smad3 using a specific shRNA prevented the RA-mediated stimulation of differentiation and displacement of C/EBPβ from the Runx2 P1 promoter. Taken together, these data indicate that Smad3 is an important mediator of RA activity during mesenchymal stem cell differentiation and is necessary for the stimulation of osteoblastogenesis.

Hedgehog Signaling Regulates MyoD Expression and Activity

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. The inhibition of MyoD expression is important for obtaining muscle progenitors that can replenish the satellite cell niche during muscle repair. Progenitors could be derived from either embryonic stem cells or satellite cells. Hedgehog (Hh) signaling is important for MyoD expression during embryogenesis and adult muscle regeneration. To date, the mechanistic understanding of MyoD regulation by Hh signaling is unclear. Here, we demonstrate that the Hh effector, Gli2, regulates MyoD expression and associates with MyoD gene elements. Gain- and loss-of-function experiments in pluripotent P19 cells show that Gli2 activity is sufficient and required for efficient MyoD expression during skeletal myogenesis. Inhibition of Hh signaling reduces MyoD expression during satellite cell activation in vitro. In addition to regulating MyoD expression, Hh signaling regulates MyoD transcriptional activity, and MyoD activates Hh signaling in myogenic conversion assays. Finally, Gli2, MyoD, and MEF2C form a protein complex, which enhances MyoD activity on skeletal muscle-related promoters. We therefore link Hh signaling to the function and expression of MyoD protein during myogenesis in stem cells.

Skeletal myosin light chain kinase regulates skeletal myogenesis by phosphorylation of MEF2C

The MEF2 factors regulate transcription during cardiac and skeletal myogenesis. MEF2 factors establish skeletal muscle commitment by amplifying and synergizing with MyoD. While phosphorylation is known to regulate MEF2 function, lineage‐specific regulation is unknown. Here, we show that phosphorylation of MEF2C on T80 by skeletal myosin light chain kinase (skMLCK) enhances skeletal and not cardiac myogenesis. A phosphorylation‐deficient MEF2C mutant (MEFT80A) enhanced cardiac, but not skeletal myogenesis in P19 stem cells. Further, MEFT80A was deficient in recruitment of p300 to skeletal but not cardiac muscle promoters. In gain‐of‐function studies, skMLCK upregulated myogenic regulatory factor (MRF) expression, leading to enhanced skeletal myogenesis in P19 cells and more efficient myogenic conversion. In loss‐of‐function studies, MLCK was essential for efficient MRF expression and subsequent myogenesis in embryonic stem (ES) and P19 cells as well as for proper activation of quiescent satellite cells. Thus, skMLCK regulates MRF expression by controlling the MEF2C‐dependent recruitment of histone acetyltransferases to skeletal muscle promoters. This work identifies the first kinase that regulates MyoD and Myf5 expression in ES or satellite cells.

CCAAT/enhancer binding protein beta is expressed in satellite cells and controls myogenesis

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/EBPβ) is expressed in the satellite cells of healthy muscle. C/EBPβ expression is regulated during myogenesis such that C/EBPβ is rapidly and massively downregulated upon induction to differentiate. Furthermore, persistent expression of C/EBPβ 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/EBPβ‐overexpressing 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/EBPβ inhibits myogenic differentiation and that its levels must be reduced to allow for activation of MyoD target genes and the progression of differentiation. STEM CELLS 2012;30:2619–2630

Mdm2 Promotes Myogenesis through the Ubiquitination and Degradation of CCAAT/Enhancer Binding Protein β

Background: CCAAT/Enhancer-binding Protein β (C/EBPβ) inhibits differentiation of muscle satellite cells and is rapidly down-regulated in early myogenesis. Results: The E3 ubiquitin ligase Mouse double minute 2 homolog (Mdm2) targets C/EBPβ for degradation thereby promoting entry into myogenesis. Conclusion: Mdm2 expression is necessary for entry into myogenesis. Significance: Establishes a new role for Mdm2 in cellular differentiation. Myogenesis is a tightly regulated differentiation process during which precursor cells express in a coordinated fashion the myogenic regulatory factors, while down-regulating the satellite cell marker Pax7. CCAAT/Enhancer-binding protein β (C/EBPβ) is also expressed in satellite cells and acts to maintain the undifferentiated state by stimulating Pax7 expression and by triggering a decrease in MyoD protein expression. Herein, we show that C/EBPβ protein is rapidly down-regulated upon induction of myogenesis and this is not due to changes in Cebpb mRNA expression. Rather, loss of C/EBPβ protein is accompanied by an increase in Mdm2 expression, an E3 ubiquitin ligase. We demonstrate that Mdm2 interacts with, ubiquitinates and targets C/EBPβ for degradation by the 26 S proteasome, leading to increased MyoD expression. Knockdown of Mdm2 expression in myoblasts using a shRNA resulted in high C/EBPβ levels and a blockade of myogenesis, indicating that Mdm2 is necessary for myogenic differentiation. Primary myoblasts expressing the shMdm2 construct were unable to contribute to muscle regeneration when grafted into cardiotoxin-injured muscle. The differentiation defect imposed by loss of Mdm2 could be partially rescued by loss of C/EBPβ, suggesting that the regulation of C/EBPβ turnover is a major role for Mdm2 in myoblasts. Taken together, we provide evidence that Mdm2 regulates entry into myogenesis by targeting C/EBPβ for degradation by the 26 S proteasome.

CCAAT/enhancer binding protein beta protects muscle satellite cells from apoptosis after injury and in cancer cachexia

CCAAT/enhancer binding protein beta (C/EBPβ), a transcription factor expressed in muscle satellite cells (SCs), inhibits the myogenic program and is downregulated early in differentiation. In a conditional null model in which C/EBPβ expression is knocked down in paired box protein 7+ (Pax7+) SCs, cardiotoxin (CTX) injury is poorly repaired, although muscle regeneration is efficient in control littermates. While myoblasts lacking C/EBPβ can differentiate efficiently in culture, after CTX injury poor regeneration was attributed to a smaller than normal Pax7+ population, which was not due to a failure of SCs to proliferate. Rather, the percentage of apoptotic SCs was increased in muscle lacking C/EBPβ. Given that an injury induced by BaCl2 is repaired with greater efficiency than controls in the absence of C/EBPβ, we investigated the inflammatory response following BaCl2 and CTX injury and found that the levels of interleukin-1β (IL-1β), a proinflammatory cytokine, were robustly elevated following CTX injury and could induce C/EBPβ expression in myoblasts. High levels of C/EBPβ expression in myoblasts correlated with resistance to apoptotic stimuli, while its loss increased sensitivity to thapsigargin-induced cell death. Using cancer cachexia as a model for chronic inflammation, we found that C/EBPβ expression was increased in SCs and myoblasts of tumor-bearing cachectic animals. Further, in cachectic conditional knockout animals lacking C/EBPβ in Pax7+ cells, the SC compartment was reduced because of increased apoptosis, and regeneration was impaired. Our findings indicate that the stimulation of C/EBPβ expression by IL-1β following muscle injury and in cancer cachexia acts to promote SC survival, and is therefore a protective mechanism for SCs and myoblasts in the face of inflammation.

Expression of CCAAT/Enhancer Binding Protein Beta in Muscle Satellite Cells Inhibits Myogenesis in Cancer Cachexia

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.