Glucocorticoids potentiate the early steps of preadipocyte differentiation and promote obesity in Cushing's 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/EBPa) expression by potentiating its initial transcriptional activation by C/EBPb. In the absence of steroid, C/EBPb 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/EBPa promoter with C/EBPb and promoted the deacetylation of histone H4. Steroid induced the speci®c 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/EBPa 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. Keywords: CAAT enhancer binding protein b function/ histone deacetylase 1/initiation of preadipocyte differentiation/26S proteasome/steroid hormone action IntroductionThe glucocorticoid receptor (GR) is a ligand-activated nuclear hormone receptor that regulates gene expression primarily through direct interaction with DNA response elements (Mangelsdorf et al., 1995). Glucocorticoids provide an adipogenic stimulus that is most obvious in the truncal obesity of patients with Cushing's syndrome (Peeke and Chrousos, 1995). Weight gain is also a sideeffect of immunosuppressive glucocorticoid therapies (Pijl and Meinders, 1996). In rodents, the weight loss that follows adrenalectomy is prevented by glucocorticoid replacement (Freedman et al., 1986;Sainsbury et al., 2001). However, gene-targeted mice in which GR is compromised for DNA binding are of normal weight and do not display any overt signs of alterations in adipogenesis, suggesting that the effects of glucocorticoids on adipogenesis may be mediated through a non-genomic mechanism (Reichardt et al., 2000a).The adipocytes that constitute white fat originate from committed precursor cells, which differentiate in response to a series of cues including insulin and inducers of cAMP. In primary preadipocytes and most cell culture models, glucocorticoids strongly potentiate differentiation (Gregoire et al., 1998). The early responses to insulin and cAMP include the transient induction of CCAAT/enhancer binding protein (C/EBP) b and C/EBPd, and overexpression of C/EBPb is suf®cient to force preadipocyte differentiation in culture (Yeh et al., 1995). In vivo, the stimulatory effect of C/EBPb activity is complemented by the action of C/...

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.

Preadipocyte differentiation in culture is driven by an insulin and cAMP dependant transcriptional cascade which induces the bzip transcription factors C/EBPbeta and C/EBPdelta. 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/EBPbeta. This results in a dramatic enhancement of C/EBPbeta-dependent transcription from the C/EBPalpha promoter, concomitant with potentiation of preadipocyte differentiation. Here, we show that C/EBPbeta 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/EBPbeta prevented acetylation and blocked the ability of glucocorticoids to enhance C/EBPbeta-directed transcription and to potentiate C/EBPbeta-dependent preadipocyte differentiation. Moreover, acetylation of C/EBPbeta appeared to directly interfere with the interaction of HDAC1 with C/EBPbeta, suggesting that PCAF/GCN5-dependent acetylation of C/EBPbeta serves as an important molecular switch in determining the transcriptional regulatory potential of this transcription factor.

Background: CCAAT/Enhancer-binding Protein ␤ (C/EBP␤) 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/EBP␤ 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.

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.

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 T 80 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 lossof-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.

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.