Lipid homeostasis is controlled by the peroxisome proliferator-activated receptors (PPARalpha, -beta/delta, and -gamma) that function as fatty acid-dependent DNA-binding proteins that regulate lipid metabolism. In vitro and in vivo genetic and pharmacological studies have demonstrated PPARalpha regulates lipid catabolism. In contrast, PPARgamma regulates the conflicting process of lipid storage. However, relatively little is known about PPARbeta/delta in the context of target tissues, target genes, lipid homeostasis, and functional overlap with PPARalpha and -gamma. PPARbeta/delta, a very low-density lipoprotein sensor, is abundantly expressed in skeletal muscle, a major mass peripheral tissue that accounts for approximately 40% of total body weight. Skeletal muscle is a metabolically active tissue, and a primary site of glucose metabolism, fatty acid oxidation, and cholesterol efflux. Consequently, it has a significant role in insulin sensitivity, the blood-lipid profile, and lipid homeostasis. Surprisingly, the role of PPARbeta/delta in skeletal muscle has not been investigated. We utilize selective PPARalpha, -beta/delta, -gamma, and liver X receptor agonists in skeletal muscle cells to understand the functional role of PPARbeta/delta, and the complementary and/or contrasting roles of PPARs in this major mass peripheral tissue. Activation of PPARbeta/delta by GW501516 in skeletal muscle cells induces the expression of genes involved in preferential lipid utilization, beta-oxidation, cholesterol efflux, and energy uncoupling. Furthermore, we show that treatment of muscle cells with GW501516 increases apolipoprotein-A1 specific efflux of intracellular cholesterol, thus identifying this tissue as an important target of PPARbeta/delta agonists. Interestingly, fenofibrate induces genes involved in fructose uptake, and glycogen formation. In contrast, rosiglitazone-mediated activation of PPARgamma induces gene expression associated with glucose uptake, fatty acid synthesis, and lipid storage. Furthermore, we show that the PPAR-dependent reporter in the muscle carnitine palmitoyl-transferase-1 promoter is directly regulated by PPARbeta/delta, and not PPARalpha in skeletal muscle cells in a PPARgamma coactivator-1-dependent manner. This study demonstrates that PPARs have distinct roles in skeletal muscle cells with respect to the regulation of lipid, carbohydrate, and energy homeostasis. Moreover, we surmise that PPARbeta/delta agonists would increase fatty acid catabolism, cholesterol efflux, and energy expenditure in muscle, and speculate selective activators of PPARbeta/delta may have therapeutic utility in the treatment of hyperlipidemia, atherosclerosis, and obesity.

The overlapping expression profile of MEF2 and the class-II histone deacetylase, HDAC7, led us to investigate the functional interaction and relationship between these regulatory proteins. HDAC7 expression inhibits the activity of MEF2 (-A, -C, and -D), and in contrast MyoD and Myogenin activities are not affected. Glutathione S-transferase pulldown and immunoprecipitation demonstrate that the repression mechanism involves direct interactions between MEF2 proteins and HDAC7 and is associated with the ability of MEF2 to interact with the N-terminal 121 amino acids of HDAC7 that encode repression domain 1. The MADS domain of MEF2 mediates the direct interaction of MEF2 with HDAC7. MEF2 inhibition by HDAC7 is dependent on the N-terminal repression domain and surprisingly does not involve the C-terminal deacetylase domain. HDAC7 interacts with CtBP and other class-I and -II HDACs suggesting that silencing of MEF2 activity involves corepressor recruitment. Furthermore, we show that induction of muscle differentiation by serum withdrawal leads to the translocation of HDAC7 from the nucleus into the cytoplasm. This work demonstrates that HDAC7 regulates the function of MEF2 proteins and suggests that this class-II HDAC regulates this important transcriptional (and pathophysiological) target in heart and muscle tissue. The nucleocytoplasmic trafficking of HDAC7 and other class-II HDACs during myogenesis provides an ideal mechanism for the regulation of HDAC targets during mammalian development and differentiation.Skeletal muscle has become a model for understanding many fundamental principles of development. Differentiation of precursor cells into skeletal muscle cells involves two events, determination into myoblasts and the formation of postmitotic, multinucleated myotubes with contractile phenotype. These processes are under control of members of the MyoD family of basic-helix-loop-helix (bHLH) 1 transcription factors (MyoD, Myf5, Myogenin, and MRF4). These proteins can inhibit cell proliferation, regulate a cascade of muscle-specific gene expression, auto-and cross-regulate their own and each other's expression, and induce muscle differentiation in nonmuscle cells (1-3). Myogenic bHLH proteins activate transcription of muscle-specific genes by forming heterodimers with other, ubiquitously expressed bHLH proteins known as E2A proteins (alternatively spliced products of the E2A gene) (4 -7). These heterodimers bind to the E box motif (CANNTG), which functions as the cognate binding site in the regulatory regions of most muscle genes (1,3,8). MyoD and Myf5 are required for determination of precursor cells into myoblasts (9), whereas Myogenin is specifically required for differentiation (10, 11). Therefore myoD and myf5 are expressed in proliferating myoblasts and are markers for commitment, whereas the expression of myogenin is a marker of terminal-differentiation.Even though members of the MyoD family are the key regulators of muscle differentiation, the activation of muscle-specific genes is dependent on the associat...

Corepressors are involved in gene silencing by various transcriptional repressor proteins such as MAD/MAX and MxiI (2), YY1 (74), KRAB domain proteins (27), NGF1-A, KROX 20 (59, 63) and some members of the nuclear hormone receptor (NHR) superfamily, such as thyroid hormone receptor (TR) and retinoic acid receptor (RAR) (8,19,38,50). Both TR and RAR repress gene activity in the absence of hormone in vivo (3, 4, 21, 73) and in vitro (28, 67, 68). This repression is mediated by a silencing domain in the carboxy terminus, encompassing about 250 amino acids (aa) (3,33,46,58). In addition to the silencing function, TR and RAR harbor several other functions C-terminal to their DNA binding domain (DBD) including dimerization, hormone binding and hormone-dependent transactivation. These activities can be transferred to heterologous proteins and therefore represent functional domains (for reviews, see references 6, 50, and 65).Gene silencing by NHRs is relieved by addition of the cognate ligand, which induces a conformational change and transforms the receptor into a transcriptional activator. In this way, both hormone binding and the small conserved receptor activation domain, AF2/AF2-AD/4/c (8, 11, 12, 22, 49, 50), representing helix 12 (14, 38, 55, 57, 71), are required to dissociate corepressors from the receptors (8,9,19,38). It is yet unknown why so many different coactivators are involved in transcriptional activation by NHRs. Gene silencing by TR, RAR, Rev-erbA␣, and COUP-TF is mediated, at least in part, by corepressors in vivo (8, 10, 19, 25, 38, 61) and in vitro (68), which bind to the unliganded (apo) receptors. Only one class of nuclear receptor corepressors has been identified, which exhibit hormone-sensitive interaction. This class contains two related members, 38). These corepressors were isolated by the yeast twohybrid system and bind to the silencing domains of TR and RAR only in the absence of ligand. Hormone binding by the receptor leads to dissociation of these corepressors. Furthermore, SMRT and N-CoR are localized in the cell nucleus and harbor an autonomous silencing function when bound to DNA (19,38). The mechanism of repression by the SMRT/N-CoR class involves interaction with SIN3 and a histone deacetylase function (1,35,52).Here, we describe a novel corepressor, Alien, which is unrelated to SMRT and N-CoR and is highly conserved from humans to Drosophila. Conserved sequences are even found in Ricinus communis and Caenorhabditis elegans. Alien interacts with TR only in the absence of hormone and does not interact with RAR, retinoid X receptor (RXR), or glucocorticoid

Vitamin D acts through the immature osteoblast to stimulate osteoclastogenesis. Transgenic elevation of VDR in mature osteoblasts was found to inhibit osteoclastogenesis associated with an altered OPG response. This inhibition was confined to cancellous bone. This study indicates that vitamin D-mediated osteoclastogenesis is regulated locally by OPG production in the mature osteoblast.Introduction: Vitamin D stimulates osteoclastogenesis acting through its nuclear receptor (VDR) in immature osteoblast/stromal cells. This mobilization of calcium stores does not occur in a random manner, with bone preferentially removed from cancellous bone. The process whereby the systemic, humoral regulator is targeted to a particular region of the skeleton is unclear. Materials and Methods: Bone resorption was assessed in mice with vitamin D receptor transgenically elevated in mature osteoblasts (OSVDR). Vitamin D-mediated osteoclastogenesis was examined in vitro using OSVDR osteoblasts and osteoblastic RANKL: osteoprotegerin (OPG) examined in vivo and in vitro after vitamin D treatment. Results: Vitamin D-mediated osteoclastogenesis was reduced in OSVDR mice on chow and calciumrestricted diets, with effects confined to cancellous bone. OSVDR osteoblasts had a reduced capacity to support osteoclastogenesis in culture. The vitamin D-mediated reduction in OPG expression was reduced in OSVDR osteoblasts in vivo and in vitro, resulting in a reduced RANKL/OPG ratio in OSVDR compared with wildtype, after exposure to vitamin D. Conclusions: Mature osteoblasts play an inhibitory role in bone resorption, with active vitamin D metabolites acting through the VDR to increase OPG. This inhibition is less active in cancellous bone, effectively targeting this region for resorption after the systemic release of activated vitamin D metabolites.

Posttranscriptional gene silencing (PTGS) of transgenes involves abundant 21-nucleotide small interfering RNAs (siRNAs) and lowabundance 22-nucleotide siRNAs produced from double-stranded RNA (dsRNA) by DCL4 and DCL2, respectively. However, DCL2 facilitates the recruitment of RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) to ARGONAUTE 1-derived cleavage products, resulting in more efficient amplification of secondary and transitive dsRNA and siRNAs. Here, we describe a reporter system where RDR6-dependent PTGS is initiated by restricted expression of an inverted-repeat dsRNA specifically in the Arabidopsis (Arabidopsis thaliana) root tip, allowing a genetic screen to identify mutants impaired in RDR6-dependent systemic PTGS. Our screen identified dcl2 but not dcl4 mutants. Moreover, grafting experiments showed that DCL2, but not DCL4, is required in both the source rootstock and the recipient shoot tissue for efficient RDR6-dependent systemic PTGS. Furthermore, dcl4 rootstocks produced more DCL2-dependent 22-nucleotide siRNAs than the wild type and showed enhanced systemic movement of PTGS to grafted shoots. Thus, along with its role in recruiting RDR6 for further amplification of PTGS, DCL2 is crucial for RDR6-dependent systemic PTGS.