Peroxisome proliferator activated receptor-␥ co-activator-1␣ (PGC-1␣) is a transcriptional co-activator that coordinately regulates the expression of distinct sets of metabolism-related genes in different tissues. Here we show that PGC-1␣ expression is reduced in skeletal muscles from mice lacking the sirtuin family deacetylase SIRT1. Conversely, SIRT1 activation or overexpression in differentiated C2C12 myotubes increased PGC-1␣ mRNA expression. The transcription-promoting effects of SIRT1 occurred through stimulation of PGC-1␣ promoter activity and were enhanced by co-transfection of myogenic factors, such as myocyte enhancer factor 2 (MEF2) and, especially, myogenic determining factor (MyoD). SIRT1 bound to the proximal promoter region of the PGC-1␣ gene, an interaction potentiated by MEF2C or MyoD, which also interact with this region. In the presence of MyoD, SIRT1 promoted a positive autoregulatory PGC-1␣ expression loop, such that overexpression of PGC-1␣ increased PGC-1␣ promoter activity in the presence of co-expressed MyoD and SIRT1. Chromatin immunoprecipitation showed that SIRT1 interacts with PGC-1␣ promoter and increases PGC-1␣ recruitment to its own promoter region. Immunoprecipitation assays further showed that SIRT1-PGC-1␣ interactions are enhanced by MyoD. Collectively, these data indicate that SIRT1 controls PGC-1␣ gene expression in skeletal muscle and that MyoD is a key mediator of this action. The involvement of MyoD in SIRT1-dependent PGC-1␣ expression may help to explain the ability of SIRT1 to drive musclespecific gene expression and metabolism. Autoregulatory control of PGC-1␣ gene transcription seems to be a pivotal mechanism for conferring a transcription-activating response to SIRT1 in skeletal muscle.Peroxisome proliferator activated receptor-␥ co-activator-1␣ (PGC-1␣) 2 is a transcriptional co-activator that is recognized as a master controller of the expression of genes involved in metabolic regulation. PGC-1␣ exerts differential effects on metabolism in different tissues. In brown adipose tissue, PGC-1␣ increases the expression of uncoupling protein 1 and genes involved in mitochondrial oxidative pathways. In the liver, PGC-1␣ induces the expression of genes involved in gluconeogenesis and the metabolic response to starvation. In skeletal muscle, PGC-1␣ expression is rapidly induced by exercise in vivo (1), a response considered to be a mechanism for modulating metabolic flux in response to decreased ATP levels (2). Chronic exercise also increases PGC-1␣ expression in association with fiber-type switching toward the more oxidative and high endurance type IIa and type I fibers. The fiber-type switch promoted by PGC-1␣ is characterized by increased mitochondrial density and function, increased oxidative metabolism, increased expression of myofibrillar proteins characteristic of type I and type IIa muscle fibers and a switch in substrate fuel usage (3). Furthermore, greater levels of PGC-1␣ are found in oxidative fibers compared with glycolytic fibers, even in a rested state (3). Mo...

Studies with the myogenic basic helix-loop-helix and MADS box factors suggest that efficient transactivation is dependent on the recruitment of the steroid receptor coactivator (SRC) and the cofactors p300 and p300/CBPassociated factor. SRCs have been demonstrated to recruit CARM1 (coactivator-associated arginine methyltransferase-1), a member of the S-adenosyl-L-methionine-dependent PRMT1-5 (protein-arginine Nmethyltransferase-1-5) family, which catalyzes the methylation of arginine residues. This prompted us to investigate the functional role of CARM1/PRMT4 during skeletal myogenesis. We demonstrate that CARM1 and the SRC cofactor GRIP-1 cooperatively stimulate the activity of myocyte enhancer factor-2C (MEF2C). Moreover, there are direct interactions among MEF2C, GRIP-1, and CARM1. Chromatin immunoprecipitation demonstrated the in vivo recruitment of MEF2 and CARM1 to the endogenous muscle creatine kinase promoter in a differentiation-dependent manner. Furthermore, CARM1 is expressed in somites during embryogenesis and in the nuclei of muscle cells. Treatment of myogenic cells with the methylation inhibitor adenosine dialdehyde or tet-regulated CARM1 "antisense" expression did not affect expression of MyoD. However, inhibition of CARM1 inhibited differentiation and abrogated the expression of the key transcription factors (myogenin and MEF2) that initiate the differentiation cascade. This work clearly demonstrates that the arginine methyltransferase CARM1 potentiates myogenesis and supports the positive role of arginine methylation in mammalian differentiation.Nuclear hormone receptors have served as prototypic models of coactivator recruitment (1). One current hypothesis suggests that the steroid receptor coactivator (SRC) 1 class of cofactors (2, 3) function as primary coactivators by binding directly to ligand-regulated transcription factors. Transcriptional activation also involves the subsequent recruitment of secondary coactivators such as p300, p300/CBP-associated factor, and the protein methyltransferases PRMT1 protein-arginine Nmethyltransferase-1-5 and CARM1/PRMT4 (4 -6). CBP/p300 and PCAF can acetylate histones and others components of the transcription complex, which leads to chromatin remodeling and initiation. PRMT1 and CARM1 are members of the Sadenosyl-L-methionine-dependent PRMT family, each of which catalyzes the methylation of arginine residues on specific proteins. At least five distinct members have been described, including PRMT1, PRMT2, PRMT3, CARM1/PRMT4, and PRMT5/JBP1 (Refs. 7-10 and references therein). A growing number of proteins, including RNA-binding proteins, contain -N G -monomethylarginine and asymmetric -N G ,N G -dimethylarginine residues. Furthermore, the PRMT family interacts with a range of proteins, including heterogeneous nuclear ribonucleoproteins, Np13 mRNA export protein, poly(A)-binding protein, TIS21, Jak2 receptor tyrosine kinase, SRCs, interferon-␣/␤ receptors, interleukin enhancer-binding factor, etc. Hence, the evidence to date implicates PRMT proteins as esse...

To understand the expression and role of thyroid hormone nuclear receptors (TRs) in hepatocarcinogenesis, we characterized the TRs in 16 human hepatocellular carcinoma (HCC) specimens. The full-length cDNAs for the two TR subtypes, alpha1 and beta1, were cloned from several tumors by reverse transcription-polymerase chain reaction. Southern blot analysis indicated that, in addition to the full-length cDNA, truncated TRalpha1 and TRbeta1 cDNAs were present in nine tumors (53%). In addition, point mutations detected by the mismatch RNase cleavage assay in TRalpha1 and TRbeta1 were found in 65% and 76% of the tumors, respectively. The mutations were confirmed by DNA sequencing. Interestingly, most of the TRalpha1 mutations were in amino acid codons 209-228 and 245-256, two hot-spots in HCC patients. However, no hot-spot was detected in TRbeta1. The expression of TRalpha1 and TRbeta1 proteins was determined in the tissue extracts by western blotting. TRbeta1 protein was expressed or elevated in 10 tumors but not in normal livers, whereas the expression of TRalpha1 was variable among tumors. The mutant TR proteins were translated in vitro, and their hormone- and DNA-binding activities were evaluated. Abnormal binding to the thyroid hormone response elements was observed. The proteins' DNA binding activity was either partially impaired or completely lost. The high prevalence of TR mutations found in the tumors of patients with hepatocellular carcinoma suggests that mutant TRs could play an important role in liver carcinogenesis.

The objective of this study was to identify genes regulated by thyroid hormone (T(3)) and associated with tumor invasion. The gene encoding furin, as previously identified by cDNA microarray, is known to be up-regulated by T(3) treatment, and stimulated furin production occurs in thyroidectomized rats after administration of T(3). Presently, by using serial deletion of the promoter and EMSAs, the T(3) response element on the furin promoter was localized to the -6317/-6302 region. T(3)-mediated furin up-regulation was cooperative with TGF-beta because T(3) induction increased after Smad3/4 addition. Furthermore, the invasiveness of HepG2-thyroid hormone receptor (TR) cells was significantly increased by T(3) treatment, perhaps due to furin processing of matrix metalloproteinase-2 and -9. In addition, furin up-regulation either by stable overexpression or T(3) and/or TGF-beta induction was evident in severe-combined immune-deficient mice inoculated with HepG2-TRalpha1 cells. The HepG2-furin mice displayed a higher metastasis index and tumor size than HepG2-neo mice. Notably, the increased liver and lung tumor number or size in the hyperthyroid severe-combined immune-deficient mice as well as TGF-beta mice was attributed specifically to furin overexpression in the HepG2-TRalpha1 cells. Furthermore, this study demonstrated that furin overexpression in some types of hepatocellular carcinomas is TR dependent and might play a crucial role in the development of hepatocellular carcinoma. Thus, T(3) regulates furin gene expression via a novel mechanism or in cooperation with TGF-beta to enhance tumor metastasis in vitro and in vivo.

Recent studies have demonstrated a critical association between disruption of cellular thyroid hormone (TH) signaling and the incidence of hepatocellular carcinoma (HCC), but the underlying mechanisms remain largely elusive. Here, we showed that disruption of TH production results in a marked increase in progression of diethylnitrosamine (DEN)-induced HCC in a murine model, and conversely, TH administration suppresses the carcinogenic process via activation of autophagy. Inhibition of autophagy via treatment with chloroquine (CQ) or knockdown of ATG7 (autophagy-related 7) via adeno-associated virus (AAV) vectors, suppressed the protective effects of TH against DEN-induced hepatic damage and development of HCC. The involvement of autophagy in TH-mediated protection was further supported by data showing transcriptional activation of DAPK2 (death-associated protein kinase 2; a serine/threonine protein kinase), which enhanced the phosphorylation of SQSTM1/p62 (sequestosome 1) to promote selective autophagic clearance of protein aggregates. Ectopic expression of DAPK2 further attenuated DEN-induced hepatoxicity and DNA damage though enhanced autophagy, whereas, knockdown of DAPK2 displayed the opposite effect. The pathological significance of the TH-mediated hepatoprotective effect by DAPK2 was confirmed by the concomitant decrease in the expression of THRs and DAPK2 in matched HCC tumor tissues. Taken together, these findings indicate that TH promotes selective autophagy via induction of DAPK2-SQSTM1 cascade, which in turn protects hepatocytes from DENinduced hepatotoxicity or carcinogenesis.