William W. Chin

TRAM-1, A Novel 160-kDa Thyroid Hormone Receptor Activator Molecule, Exhibits Distinct Properties from Steroid Receptor Coactivator-1

Nuclear hormone receptors (NRs) are ligand-dependent transcription factors that regulate target gene transcription. We report the molecular cloning and characterization of a novel human cDNA encoding TRAM-1, athyroid hormone receptor activatormolecule, a ∼160-kDa protein homologous with SRC-1/TIF2, by far-Western-based expression screening. TRAM-1 binds to thyroid hormone receptor (TR) and other NRs in a ligand-dependent manner and enhances ligand-induced transcriptional activity of TR. The AF-2 region in NRs has been thought to play a critical role in mediating ligand-dependent transactivation by the interaction with coactivators. Surprisingly, TRAM-1 retains strong ligand-dependent interaction with an AF-2 mutant of TR (E457A), while SRC-1 fails to interact with this mutant. Furthermore, we identified a critical TRAM-1 binding site in rat TRβ1 outside of AF-2, as TRAM-1 shows weak ligand-dependent interaction with a helix 3 ligand binding domain TR mutant (K288A), compared with SRC-1. These results suggest that TRAM-1 is a coactivator that may exhibit its activity by interacting with subdomains of NRs other than the AF-2 region, in contrast to SRC-1/TIF2.

Direct effects of leptin on brown and white adipose tissue.

Leptin is thought to exert its actions on energy homeostasis through the long form of the leptin receptor (OB-Rb), which is present in the hypothalamus and in certain peripheral organs, including adipose tissue. In this study, we examined whether leptin has direct effects on the function of brown and white adipose tissue (BAT and WAT, respectively) at the metabolic and molecular levels. The chronic peripheral intravenous administration of leptin in vivo for 4 d resulted in a 1.6-fold increase in the in vivo glucose utilization index of BAT, whereas no significant change was found after intracerebroventricular administration compared with pair-fed control rats, compatible with a direct effect of leptin on BAT. The effect of leptin on WAT fat pads from lean Zucker Fa/ fa rats was assessed ex vivo, where a 9- and 16-fold increase in the rate of lipolysis was observed after 2 h of exposure to 0.1 and 10 nM leptin, respectively. In contrast, no increase in lipolysis was observed in the fat pads from obese fa/fa rats, which harbor an inactivating mutation in the OB-Rb. At the level of gene expression, leptin treatment for 24 h increased malic enzyme and lipoprotein lipase RNA 1.8+/-0.17 and 1.9+/-0.14-fold, respectively, while aP2 mRNA levels were unaltered in primary cultures of brown adipocytes from lean Fa/fa rats. Importantly, however, no significant effect of leptin was observed on these genes in brown adipocytes from obese fa/fa animals. The presence of OB-Rb receptors in adipose tissue was substantiated by the detection of its transcripts by RT-PCR, and leptin treatment in vivo and in vitro activated the specific STATs implicated in the signaling pathway of the OB-Rb. Taken together, our data strongly suggest that leptin has direct effects on BAT and WAT, resulting in the activation of the Jak/STAT pathway and the increased expression of certain target genes, which may partially account for the observed increase in glucose utilization and lipolysis in leptin-treated adipose tissue.

Thyroid Hormone Action and Brain Development

Thyroid hormone (TH) plays a crucial role in brain development. Developing rodent cerebellum might be an excellent model for studying the molecular mechanisms of TH action in the brain because perinatal hypothyroidism greatly affects its ontogeny. Although the TH-regulated genes that play crucial roles in cerebellar development have not yet been fully characterized, recent studies have provided novel insights into TH action in brain development.

A novel member of the thyroid/steroid hormone receptor family is encoded by the opposite strand of the rat c-erbA alpha transcriptional unit.

A cDNA encoding a novel member of the thyroid/steroid hormone receptor superfamily, called Rev-ErbA alpha, has been isolated from a rat GH3 cell library. Rev-ErbA alpha is an approximately 56-kilodalton protein most similar in structure to the thyroid hormone receptor (c-erbA) and the retinoic acid receptor, but it does not bind either thyroid hormone or retinoic acid. The mRNA encoding Rev-ErbA alpha is present in many tissues and is particularly abundant in skeletal muscle and brown fat. A genomic DNA fragment containing the entire Rev-ErbA alpha cDNA sequence was isolated and characterized. Remarkably, this DNA fragment also contained a portion of the c-erbA alpha gene. r-erbA alpha-1 and r-erbA alpha-2 are alternative splice products of the c-erbA alpha gene and are members of the receptor superfamily. The genes encoding Rev-ErbA alpha and r-erbA alpha-2 overlap, with their coding strands oriented opposite one another. A 269-base-pair segment of the bidirectionally transcribed region is exonic in both the Rev-ErbA alpha and r-erbA alpha-2 genes, resulting in complementary mRNAs. Thus, through alternative splicing and opposite-strand transcription, a single genomic locus codes for three different members of the thyroid/steroid hormone receptor superfamily. Potential implications of this unusual genomic arrangement are discussed.

Gastrin-Releasing Peptide (Mammalian Bombesin) Gene Expression in Health and Disease

In recent years much attention has been focused on gastrin-releasing peptide (GRP), the mammalian homologue of bombesin, both as a neuroregulatory hormone and as a tissue-specific growth factor in normal and neoplastic tissues. This paper will analyze the distribution and role of GRP in normal mammalian tissues and examine the potential involvement of GRP in diverse pathologic processes.

Differential and tissue-specific regulation of the multiple rat c-erbA messenger RNA species by thyroid hormone.

Thyroid hormone (T3) has been shown to regulate the level of its receptor in a number of tissues and cell lines. Recently, proteins encoded by the protooncogene c-erbA have been identified as T3 receptors. In the rat, four c-erbA gene products have been isolated, three of which, r-erbA alpha-1, r-erbA beta-1, and r-erbA beta-2, encode biologically active T3 receptors; the fourth, r-erbA alpha-2, may play an inhibitory role in T3 action. The present work examines the molecular nature of T3 receptor autoregulation using probes specific for each c-erbA mRNA. Rats were rendered hypothyroid with propylthiouracil and then treated with either saline or T3. Northern blot analyses reveal marked tissue-specific and differential regulation of the multiple c-erbA mRNAs by T3. In the pituitary the levels of r-erbA beta-1 mRNA increase, whereas the levels of the pituitary-specific r-erbA beta-2 mRNA decrease with T3 treatment. In heart, kidney, liver, and brain the levels of r-erbA beta-1 are unaffected by thyroidal status. The levels of both r-erbA alpha mRNAs decrease with T3 treatment in all tissues examined except for the brain, where there is no change. In addition, we find that changes in the mRNAs encoding specific subpopulations of T3 receptors do not always parallel changes in total nuclear T3 binding. Differential regulation of the specific c-erbA mRNA species could have important consequences for T3 action.

Expression and Hormonal Regulation of Coactivator and Corepressor Genes

Steroid/thyroid/retinoid receptors are members of the nuclear receptor superfamily and ligand-inducible transcription factors. These receptors modulate transcription of various cellular genes, either positively or negatively, by interacting with specific hormone-response elements located in the target gene promoters. Recent data show that nuclear receptors enhance or inhibit transcription by recruiting an array of coactivator and corepressor proteins to the transcription complex. We examined and compared the expression of four coactivator (steroid receptor coactivator-1 and E1A-associated 300-kDa protein) and corepressor (SMRT and N-CoR) genes in a number of tissues including several endocrine glands and cell lines. We also addressed whether their messenger RNA levels are hormonally regulated by studying the effects of thyroid hormone (T3) and estrogen (E2) treatment in rat pituitary cells (GH3) in vitro and in anterior pituitary in vivo. Our studies show that there are distinct tissue-specific expression...

Isolation and characterization of cDNAs encoding the rat pituitary gonadotropin-releasing hormone receptor

Rat pituitary cDNAs encoding the full peptide coding sequence of the rat gonadotropin-releasing hormone receptor were isolated and characterized. The deduced amino acid sequence encodes a protein of 327 residues with seven putative transmembrane domains characteristic of the family of G-protein coupled receptors. It is 95% identical at the amino acid level with the mouse gonadotropin-releasing hormone receptor. An mRNA of 4.5 Kb was identified in the rat pituitary, ovary, and testis, and in murine alpha T3 cells. In addition, a larger mRNA species of 5.0-5.5 Kb was present in these rat tissues, and a smaller mRNA species of 1.8 Kb was present in the rat pituitary and ovary, and in alpha T3 cells. The receptor mRNA levels were increased in the female rat pituitary after ovariectomy compared to levels in intact female rats.

Regulation of the Transcriptional Activity of the Peroxisome Proliferator-activated Receptor α by Phosphorylation of a Ligand-independent trans-Activating Domain

The peroxisome proliferator-activated receptors (PPARs) are a subgroup of nuclear receptors activated by fatty acids and eicosanoids. In addition, they are subject to phosphorylation by insulin, resulting in the activation of PPARα, while inhibiting PPARγ under certain conditions. However, it was hitherto unclear whether the stimulatory effect of insulin on PPARα was direct and by which mechanism it occurs. We now demonstrate that amino acids 1–92 of hPPARα contain an activation function (AF)-1-like domain, which is further activated by insulin through a pathway involving the mitogen-activated protein kinases p42 and p44. Further analysis of the amino-terminal region of PPARα revealed that the insulin-induced trans-activation occurs through the phosphorylation of two mitogen-activated protein kinase sites at positions 12 and 21, both of which are conserved across evolution. The characterization of a strong AF-1 region in PPARα, stimulating transcription one-fourth as strongly as the viral protein VP16, is compatible with the marked basal transcriptional activity of this isoform in transfection experiments. However, it is intriguing that the activity of this AF-1 region is modulated by the phosphorylation of two serine residues, both of which must be phosphorylated in order to activate transcription. This is in contrast to PPARγ2, which was previously shown to be phosphorylated at a single site in a motif that is not homologous to the sites now described in PPARα. Although the molecular details involved in the phosphorylation-dependent enhancement of the transcriptional activity of PPARα remain to be elucidated, we demonstrate that the effect of insulin on the AF-1 region of PPARα can be mimicked by the addition of triiodothyronine receptor β1, a strong binder of corepressor proteins. In addition, a triiodothyronine receptor β1 mutant deficient in interacting with corepressors is unable to activate PPARα. These observations suggest that the AF-1 region of PPARα is partially silenced by corepressor proteins, which might interact in a phosphorylation-dependent manner.

New advances in understanding the molecular mechanisms of thyroid hormone action

Thyroid hormone regulation o f gene transcription is a complex process. There are multiple thyroid hormone receptors (TRs) encoded on separate genes that bind to thyroid hormone-response elements (TREs) of target genes containing different orientation and spacing of half-sites. Additionally, there are multiple TR complexes-monomers, homodimers, and heterodimers with other related nuclear proteins-which bind to TREs and may play important roles in gene transcription. Recently, it has been shown that DNA binding of these TR complexes can be differentially regulated by either ligand or TR phosphorylation. Diversity among TR complexes and TREs, as well as mechanisms for regulating TR binding to TREs, may enable sensitive and precise transcriptional control of target genes.