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Human protection policies require favorable risk–benefit judgments prior to launch of clinical trials. For phase I and II trials, evidence for such judgment often stems from preclinical efficacy studies (PCESs). We undertook a systematic investigation of application materials (investigator brochures [IBs]) presented for ethics review for phase I and II trials to assess the content and properties of PCESs contained in them. Using a sample of 109 IBs most recently approved at 3 institutional review boards based at German Medical Faculties between the years 2010–2016, we identified 708 unique PCESs. We then rated all identified PCESs for their reporting on study elements that help to address validity threats, whether they referenced published reports, and the direction of their results. Altogether, the 109 IBs reported on 708 PCESs. Less than 5% of all PCESs described elements essential for reducing validity threats such as randomization, sample size calculation, and blinded outcome assessment. For most PCESs (89%), no reference to a published report was provided. Only 6% of all PCESs reported an outcome demonstrating no effect. For the majority of IBs (82%), all PCESs were described as reporting positive findings. Our results show that most IBs for phase I/II studies did not allow evaluators to systematically appraise the strength of the supporting preclinical findings. The very rare reporting of PCESs that demonstrated no effect raises concerns about potential design or reporting biases. Poor PCES design and reporting thwart risk–benefit evaluation during ethical review of phase I/II studies., Author summary To make a clinical trial ethical, regulatory agencies and institutional review boards have to judge whether the trial-related benefits (the knowledge gain) outweigh the trial-inherent risks. For early-phase human research, these risk–benefit assessments are often based on evidence from preclinical animal studies reported in so-called “investigator brochures.” However, our analysis shows that the vast majority of such investigator brochures lack sufficient information to systematically appraise the strength of the supporting preclinical findings. Furthermore, the very rare reporting of preclinical efficacy studies that demonstrated no effect raises concerns about potential design and/or reporting biases. The poor preclinical study design and reporting thwarts risk–benefit evaluation during ethical review of early human research. Regulators should develop standards for the design and reporting of preclinical efficacy studies in order to support the conduct of ethical clinical trials.

To study the role of the thyroid hormone (TH) in cerebellar development, we generated transgenic mice expressing a dominant-negative TH receptor (TR) in cerebellar Purkinje cells. A mutant human TRβ1 (G345R), which binds to the TH-response element but cannot bind to T3, was subcloned into exon 4 of the full-length L7/Pcp-2 gene, which is specifically expressed in Purkinje and retinal rod bipolar cells. The transgene was specifically expressed in Purkinje cells in the postnatal cerebellum. Purkinje cell dendrite arborization was significantly delayed in the transgenic mice. Surprisingly, granule cell migration was also significantly delayed. In the primary cerebellar culture, TH-induced Purkinje cell dendrite arborization was also suppressed. In quantitative real-time RT-PCR analysis, the expression levels of several TH-responsive genes were altered. The expression levels of inositol trisphosphate receptor type 1 and retinoic acid receptor-related orphan receptorα mRNAs, which are mainly expressed in Purkinje cells, and brain-derived neurotrophic factor mRNA, which is expressed in both Purkinje and granule cells, were significantly decreased. The expression levels of neurotrophin-3 and hairless mRNAs, which are mainly expressed in granule cells, and myelin basic protein mRNA, which is mainly expressed in oligodendrocytes, were also decreased. The motor coordination of transgenic mice was significantly disrupted. These results indicate that TH action through its binding to TR in Purkinje cells is required for the normal cerebellar development. TH action through TR in Purkinje cells is also important for the development of other subsets of cerebellar cells such as granule cells and oligodendrocytes.

Modern approaches for research with human biospecimens employ a variety of substantially different types of ethics approval and informed consent. In most cases, standard ethics reporting such as “consent and approval was obtained” is no longer meaningful. A structured analysis of 120 biospecimen studies recently published in top journals revealed that more than 85% reported on consent and approval, but in more than 90% of cases, this reporting was insufficient and thus potentially misleading. Editorial policies, reporting guidelines, and material transfer agreements should include recommendations for meaningful ethics reporting in biospecimen research. Meaningful ethics reporting is possible without higher word counts and could support public trust as well as networked research., This Perspective shows that while ethics reporting on consent and approval for biospecimen research is widespread, for the majority of cases it is not meaningful; the authors make pragmatic suggestions of substantial changes needed to remedy this.

Vitamin D receptor (VDR) ligands are therapeutic agents for the treatment of psoriasis, osteoporosis, and secondary hyperparathyroidism. VDR ligands also show immense potential as therapeutic agents for autoimmune diseases and cancers of skin, prostate, colon, and breast as well as leukemia. However, the major side effect of VDR ligands that limits their expanded use and clinical development is hypercalcemia that develops as a result of the action of these compounds mainly on intestine. In order to discover VDR ligands with less hypercalcemia liability, we sought to identify tissue-selective VDR modulators (VDRMs) that act as agonists in some cell types and lack activity in others. Here, we describe LY2108491 and LY2109866 as nonsecosteroidal VDRMs that function as potent agonists in keratinocytes, osteoblasts, and peripheral blood mononuclear cells but show poor activity in intestinal cells. Finally, these nonsecosteroidal VDRMs were less calcemic in vivo, and LY2108491 exhibited more than 270-fold improved therapeutic index over the naturally occurring VDR ligand 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in an in vivo preclinical surrogate model of psoriasis.

Steroid receptor coactivator-1 (SRC-1) plays a crucial role in nuclear receptor-mediated transcription including thyroid hormone receptor (TR)-dependent gene expression. Interaction of the TR-ligand binding domain and SRC-1 through LXXLL motifs is required for this action. However, potential interactions between the TRbeta1-N terminus (N) and SRC-1 have not been explored and thus are examined in this manuscript. Far-Western studies showed that protein construct containing TRbeta1-N + DNA binding domain (DBD) bound to nuclear receptor binding domain (NBD)-1 (amino acid residue, aa 595-780) of SRC-1 without ligand. Mammalian two-hybrid studies showed that NBD-1, as well as SRC-1 (aa 595-1440), bound to TRbeta1-N+DBD in the absence of ligand in CV-1 cells. However, NBD-2 (aa 1237-1440) did not bind to this protein. Glutathione-S-transferase pull-down studies showed that TRbeta1-N (aa 1-105) bound to the broad region of SRC-1-C terminus. Expression vectors encoding a series of truncations and/or point mutations of TRbeta1 were used in transient transfection-based reporter assays in CV-1 cells. N-terminal truncated TRbeta1 (DeltaN-TRbeta1) showed lower activity than that of wild-type in both artificial F2-thyroid hormone response element and native malic enzyme response element. These results suggest that there is the interaction between N terminus of TRbeta1 and SRC-1, which may serve a full activation of SRC-1, together with activation function-2 on TRbeta1-mediated transcription.

To effectively direct targeted repression, the class I histone deacetylases (HDACs) associate with many important regulatory proteins. In this paper we describe the molecular characterization of a member of the Jumonji domain 2 (JMJD2) family of proteins, and demonstrate its binding to both class I HDACs and the retinoblastoma protein (pRb). JMJD2 proteins are characterized by the presence of two leukemia-associated protein/plant homeodomain (LAP/PHD) zinc fingers, one JmjN, one JmjC (containing an internal retinoblastoma-binding protein 2 (RBBP2)-like sequence), and two Tudor domains. The first member of this group, JMJD2A, is widely expressed in human tissues and cell lines, and high endogenous expression of JMJD2A mRNA was found in several cell types, including human T-cell lymphotropic virus 1 (HTLV-1)-infected cell lines. JMJD2A and JMJD2B exhibit cell type-specific responses to the HDAC inhibitor trichostatin A. We show that the JMJD2A protein associates in vivo with pRb and class I HDACs, and mediates repression of E2F-regulated promoters. In HTLV-1 virus-infected cells, we find that JMJD2A binds to the viral Tax protein. Antibodies to JMJD2A recognize the native protein but also a half-sized protein fragment, the latter up-regulated in THP-1 cells during the G(2)/M phase of the cell cycle. The ability of JMJD2A to associate with pRb and HDACs and potentiate pRb-mediated repression of E2F-regulated promoters implies an important role for this protein in cell proliferation and oncogenesis.

The gonadotropin-releasing hormone receptor (GnRHR) is expressed primarily in the gonadotropes of the anterior pituitary. Pituitary homeobox 1 (Pitx-1) has been shown to activate pituitary-specific gene expression by direct DNA binding and/or protein-protein interaction with other transcription factors. We hypothesized that Pitx-1 might also dictate tissue-specific expression of the mouse GnRHR (mGnRHR) gene in a similar manner. Pitx-1 activated the mGnRHR gene promoter, and transactivation was localized to sequences between −308 and −264. Pitx-1 bound to this region only with low affinity. This region includes an activating protein 1 (AP-1) site, which was previously shown to be important for mGnRHR gene expression. Further characterization indicated that an intact AP-1 site was required for full Pitx-1 responsiveness. Furthermore, Pitx-1 and AP-1 were synergistic in the activation of the mGnRHR gene promoter. A Pitx-1 homeodomain (HD) point mutation, which eliminated DNA binding ability, caused only a partial reduction of transactivation, whereas deletion of the HD completely prevented transactivation. Pitx-1 interacted directly with c-Jun, and the HD was sufficient for this interaction. While the point mutation in the Pitx-1 HD did not affect interaction with c-Jun, deletion of the HD eliminated the interaction. Taken together, our studies indicate that Pitx-1 can direct transactivation of the mGnRHR gene, in part by DNA binding and in part by an action of Pitx-1 as a cofactor for AP-1, augmenting AP-1 activity through a novel protein-protein interaction between c-Jun and the HD of Pitx-1.

We have recently shown that in colon cancer cells, Vitamin D receptor (VDR) interacts with the catalytic subunit of Ser/Thr protein phosphatases, PP1c and PP2Ac, and induces their enzymatic activity in a ligand-dependent manner. The VDR-PP1c and VDR-PP2Ac interactions were ligand independent in vivo, and 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3))-mediated increase in VDR-associated phosphatase activity resulted in dephosphorylation and inactivation of p70S6 kinase in colon cancer cells. Here, we demonstrate that in myeloid leukemia cells, 1,25(OH)(2)D(3) treatment increased the Thr389 phosphorylation of p70S6 kinase. Accordingly, 1,25(OH)(2)D(3) decreased VDR-associated Ser/Thr protein phosphatase activity by dissociating VDR-PP1c and VDR-PP2Ac interactions. Further, 1,25(OH)(2)D(3) increased the association between VDR and Thr389 phosphorylated p70S6 kinase. Finally, by using non-secosteroidal VDR ligands, we demonstrate a separation between transactivation and p70S6 kinase phosphorylation activities of VDR and show pharmacologically that p70S6 kinase phosphorylation correlates with HL-60 cell differentiation.

The vitamin D receptor (VDR) belongs to the thyroid hormone/retinoid receptor subfamily of nuclear receptors and functions as a heterodimer with retinoid X receptor (RXR). The RXR-VDR heterodimer, in contrast to other members of the class II nuclear receptor subfamily, is nonpermissive where RXR does not bind its cognate ligand, and therefore its role in VDR-mediated transactivation by liganded RXR-VDR has not been fully characterized. Here, we show a unique facet of the intermolecular RXR-VDR interaction, in which RXR actively participates in vitamin D3-dependent gene transcription. Using helix 3 and helix 12 mutants of VDR and RXR, we provide functional evidence that liganded VDR allosterically modifies RXR from an apo (unliganded)- to a holo (liganded)-receptor conformation, in the absence of RXR ligand. As a result of the proposed allosteric modification of RXR by liganded VDR, the heterodimerized RXR shows the "phantom ligand effect" and thus acquires the capability to recruit coactivators steroid receptor coactivator 1, transcriptional intermediary factor 2, and amplified in breast cancer-1. Finally, using a biochemical approach with purified proteins, we show that RXR augments the 1,25-dihydroxyvitamin D3-dependent recruitment of transcriptional intermediary factor 2 in the context of RXR-VDR heterodimer. These results confirm and extend the previous observations suggesting that RXR is a significant contributor to VDR-mediated gene expression and provide a mechanism by which RXR acts as a major contributor to vitamin D3-dependent transcription.

Nuclear receptors mediate gene activation through ligand-dependent interaction with coactivators. We previously cloned and characterized thyroid hormone receptor-binding protein, TRBP (NcoA6: AIB3/ASC-2/RAP250/PRIP/TRBP/NRC), as an LXXLL-containing coactivator that associates with coactivator complexes through its C terminus. To search for protein factors involved in TRBP action, we identified a distinct set of proteins from HeLa nuclear extract that interacts with the C terminus of TRBP. Analysis by mass spectrometric protein sequencing revealed a DNA-dependent protein kinase (DNA-PK) complex including its catalytic subunit and regulatory subunits, Ku70 and Ku86. DNA-PK is a heterotrimeric nuclear phosphatidylinositol 3-kinase that functions in DNA repair, recombination, and transcriptional regulation. DNA-PK phosphorylates TRBP at its C-terminal region, which directly interacts with Ku70 but not Ku86 in vitro. In addition, in the absence of DNA, TRBP itself activates DNA-PK, and the TRBP-stimulated DNA-PK activity has an altered phosphorylation pattern from DNA-stimulated activity. An anti-TRBP antibody inhibits TRBP-induced kinase activity, suggesting that protein content of TRBP is responsible for the stimulation of DNA-independent kinase activity. Furthermore, in DNA-PK-deficient scid cells, TRBP-mediated transactivation is significantly impaired, and nuclear localization of TRBP is altered. The activation of DNA-PK in the absence of DNA ends by the coactivator TRBP suggests a novel mechanism of coactivator-stimulated DNA-PK phosphorylation in transcriptional regulation.

Fenofibrate is clinically successful in treating hypertriglyceridemia and mixed hyperlipidemia presumably through peroxisome proliferator-activated receptor alpha (PPARalpha)-dependent induction of genes that control fatty acid beta-oxidation. Lipid homeostasis and cholesterol metabolism also are regulated by the nuclear oxysterol receptors, liver X receptors alpha and beta (LXRalpha and LXRbeta). Here we show that fenofibrate ester, but not fenofibric acid, functions as an LXR antagonist by directly binding to LXRs. Likewise, ester forms, but not carboxylic acid forms, of other members of the fibrate class of molecules antagonize the LXRs. The fibrate esters display greater affinity for LXRs than the corresponding fibric acids have for PPARalpha. Thus, these two nuclear receptors display a degree of conservation in their recognition of ligands; yet, the acid/ester moiety acts as a chemical switch that determines PPARalpha versus LXR specificity. Consistent with its LXR antagonistic activity, fenofibrate potently represses LXR agonist-induced transcription of hepatic lipogenic genes. Surprisingly, fenofibrate does not repress LXR-induced transcription of various ATP-binding cassette transporters either in liver or in macrophages, suggesting that fenofibrate manifests variable biocharacter in the context of differing gene promoters. These findings provide not only an unexpected mechanism by which fenofibrate inhibits lipogenesis but also the basis for examination of the pharmacology of an LXR ligand in humans.

The nuclear receptor superfamily comprises a large number of ligand-activated transcription factors that are involved in numerous biological processes such as cell proliferation, differentiation, and homeostasis. ROR(alpha) (NR1F1) and Reverb(alpha) (NR1D1) are two members of this family whose biological functions are largely unknown. In addition, no ligand has been yet identified for these two receptors; therefore, they are referred as orphan receptors. Here, we show that ROR(alpha) and Reverb(alpha) are expressed with a similar tissue distribution and are both induced during the differentiation of rat L6 myoblastic cells. Ectopic expression of ROR(alpha)1 in L6 cells significantly induces Reverb(alpha) expression as demonstrated by Northern blot analysis. Using reverse transcription-PCR to analyze Reverb(alpha) gene expression from staggerer mice, we found that there was a significant reduction of Reverb(alpha) mRNA in the skeletal muscle comparing it with the wild-type mice, which suggests that ROR(alpha) is involved in the regulation of Reverb(alpha) gene expression. Transient transfection assays using the Reverb(alpha) promoter demonstrate that ROR(alpha) regulates the Reverb(alpha) gene at the transcriptional level. Furthermore, mutagenesis experiments indicate that ROR(alpha) regulates Reverb(alpha) transcription via a monomeric ROR response element located in the Reverb(alpha) gene promoter. Electrophoretic mobility shift assays show that ROR(alpha) binds strongly to this site in a specific-manner. Finally, overexpression of GRIP-1/TIF-2, but not SRC-1, potentiates ROR(alpha)-stimulated Reverb(alpha) promoter activity in transient transfection experiments. Together, our results identify Reverb(alpha) as a novel target gene for ROR(alpha).

We provide evidence of a cross-talk between nuclear receptor and Ser/Thr protein phosphatases and show that vitamin D receptor (VDR) interacts with the catalytic subunit of protein phosphatases, PP1c and PP2Ac, and induces their enzymatic activity in a ligand-dependent manner. PP1c specifically interacts with VDR but not retinoic acid receptor alpha and retinoid X receptor alpha in yeast. Although VDR-PP1c and VDR-PP2Ac interaction is ligand-independent in vivo, 1alpha,25-dihydroxy-vitamin D(3) induces VDR-associated phosphatase activity. Further, VDR modulation of PP1c/PP2Ac activity results in a rapid and specific dephosphorylation and inactivation of their substrate, p70 S6 kinase (p70(S6k)). Finally, we demonstrate that the endogenous VDR, PP1c or PP2Ac, and p70(S6k) are present in a ternary complex in vivo, and the interaction of p70(S6k) with the VDR-PP complex is modulated by the phosphorylation state of the kinase. Since p70(S6k) is essential for G(1)-S transition, our results provide a molecular basis of 1alpha,25-dihydroxyvitamin D(3)-induced G(1) block in colon cancer cells.

Ligand-dependent gene transcription mediated by the nuclear receptors involves the recruitment of transcriptional coactivators to the ligand-binding domain (LBD), which leads to interaction with the basal transcription machinery, and ultimately with RNA polymerase II. Although most of these coactivators are ubiquitously expressed, a tissue-selective coactivator, PGC-1, has recently been characterized. Because PGC-1 and the retinoid X receptors (RXRs) possess an overlapping tissue distribution, we investigated whether PGC-1 is a coactivator for the retinoid X receptors. In a transient transfection assay, PGC-1 augments ligand-stimulated RXR transcription. Furthermore, PGC-1 efficiently enhances the RXR element-driven reporter gene transcription by all three RXR isoforms. An immunoprecipitation assay reveals that PGC-1 and RXRalpha interact in vivo. In addition, a glutathione S-transferase pull-down assay showed that this interaction requires the presence of the LXXLL motif of PGC-1. We demonstrate further, in a mammalian two-hybrid assay, that this physical interaction also requires the presence of the AF-2 region of RXR to interact with the LXXLL motif of PGC-1, which is consistent with our protein-protein interaction results. A time-resolved fluorescence assay shows that a peptide within the NR box of PGC-1 is efficiently recruited by a ligand-bound RXRalpha in vitro. Finally, PGC-1 and TIF2 synergistically enhance ligand-activated RXRalpha transcriptional activity. Taken together, these results indicate that PGC-1 is a bona fide coactivator for RXRalpha.

Nuclear receptors are ligand-activated transcription factors, which have the potential to integrate internal metabolic events in an organism, with consequences for control of behaviour. Previous studies from this laboratory have shown that thyroid hormone receptor (TR) isoforms can inhibit oestrogen receptor (ER)alpha-mediated induction of preproenkephalin (PPE) gene expression in the hypothalamus. Also, thyroid hormone administration inhibits lordosis, a behaviour facilitated by PPE expression. We have examined the effect of multiple ligand-binding TR isoforms on the ER-mediated induction of the PPE gene in transient transfection assays in CV-1 cells. On a natural PPE gene promoter fragment containing two putative oestrogen response elements (EREs), both ER alpha and beta isoforms mediate a four to five-fold induction by oestrogen. Cotransfection of TR alpha 1 along with ER alpha inhibited the ER alpha transactivation of PPE by approximately 50%. However, cotransfection with either TR beta 1 or TR beta 2 expression plasmids produced no effect on the ER alpha or ER beta mediated induction of PPE. Therefore, under these experimental conditions, interactions with a single ER isoform are specific to an individual TR isoform. Transfection with a TR alpha 1 DNA-binding mutant could also inhibit ER alpha transactivation, suggesting that competition for binding on the ERE may not be the exclusive mechanism for inhibition. Data with the coactivator, SRC-1, suggested that coactivator squelching may participate in the inhibition. In dramatic contrast, when ER beta is cotransfected, TR alpha 1 stimulated ER beta-mediated transactivation of PPE by approximately eight-fold over control levels. This is the first study revealing specific interactions among nuclear receptor isoforms on a neuroendocrine promoter. These data also suggest that the combinatorics of ER and TR isoforms allow multiple forms of flexible gene regulations in the service of neuroendocrine integration.

Thyroid hormone (TH) plays an important role in brain development. The rodent cerebellum has been an excellent model for the study of the molecular mechanisms of TH action in brain. However, most studies have utilized the rat rather than the mouse. Considering the usefulness of mice with regard to diverse genetic models, the study of TH effect on mouse cerebellar development is needed. Thus, we examined the effect of perinatal hypothyroidism on the expression of neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) genes, which play critical roles in cerebellar development. Newborn mice were rendered hypothyroid by administering methimazole and perchlorate in drinking water to their mothers. The growth of hypothyroid mice was retarded, which was reversed by daily thyroxine administration. NT-3 and BDNF gene expression was depressed in the perinatal hypothyroid cerebellum. Furthermore, the expression of retinoid-receptor-related orphan nuclear hormone receptor-alpha (RORalpha), an orphan nuclear receptor that plays critical roles in Purkinje cell development, was also decreased. Morphologically, disappearance of the external granule cell layer was retarded and arborization of Purkinje cell dendrite was decreased, events that were also observed in hypothyroid rats. These results indicate that the mouse cerebellum is comparable to the rat cerebellum as a model for the examination of the molecular mechanisms of TH action in brain development.

Estrogen receptors (ER) and thyroid hormone receptors (TR) are members of the nuclear receptor family of transcription factors that induce or repress the expression of target genes. Previous behavioral studies in female rodents have demonstrated that thyroid hormones can antagonize the effects of estrogen in the central nervous system (CNS), particularly by attenuating estrogen's ability to facilitate reproductive behaviors. Additional molecular studies have suggested a mechanism for this antagonism by showing that ligand-activated ER alpha and TRs have the potential to interact in their transcriptional controls. Although the expression patterns of ER alpha and TRs in the rodent brain appear to overlap in behaviorally relevant areas, it remained to be determined whether these two classes of proteins coexist in vivo at the level of single neurons. To address this possibility, we employed a highly sensitive double-label in situ hybridization technique using digoxigenin and (35)S-labeled cRNA probes to analyze, in detail, the expression of ER alpha mRNA with TR alpha 1 and TR alpha 2 mRNAs in the same neurons of the ovariectomized (OVX) adult mouse brain. Our results demonstrate that a large majority of the ER alpha-positive neurons also expresses TR alpha 1 and TR alpha 2 mRNAs. Quantitative examination of the cellular expression in the ventromedial and arcuate nuclei of the hypothalamus (VMH and Arc) showed that 81.5% and 80.5% of the neurons endowed with ER alpha mRNA also contain TR alpha 1 and TR alpha 2 mRNAs, respectively. In the amygdala, more than 60.5% and 67% of ER alpha-positive cells also contain TR alpha 1 and TR alpha 2 mRNAs, respectively. These findings provide the first anatomical evidence that ER and TR can be found in the same neurons, including hypothalamic neurons. This coexpression of ER alpha and TR provides the cellular basis for a new level of neuronal integration in a brain region where estrogens control female reproductive behaviors.

Thyroid hormone receptor (TR), a ligand-mediated transcription factor, binds to a DNA sequence known as a thyroid-hormone response element (TRE) to activate or repress transcription of target genes. Recently, studies have shown that Ca2+/calmodulin-dependent protein kinases (CaMKs) may be involved in regulating gene transcription via phosphorylation of specific transcription factors, including RORalpha, a retinoic acid-related orphan nuclear hormone receptor. In this light, we examined the effect of CaMK type IV (CaMKIV) and RORalpha, which also shown to influence thyroid hormone action, on TR-mediated transcription using a transient transfection assay. Expression vectors containing TR, vitamin D receptor (VDR), and estrogen receptor (ER) were cotransfected in CV-1 cells with RORalpha and/or constitutively active CaMKIV and thymidine kinase promotor-luciferase reporter vector containing their cognate response elements. When CaMKIV or RORalpha was co-transfected with TR, the T3-induced transcription was significantly augmented compared to that induced by TR alone. When both were co-transfected with TR, T3-induced transcription was augmented additively. In contrast, the augmentation by CaMKIV or ROR on ligand-induced transcription was not detected with VDR and ER. Hence, these results indicate that the augmentation mediated by CaMKIV and RORalpha is specific for TR-mediated transcription on TRE. Our results suggest that CaMKIV, as well as RORalpha, play important roles in TR-mediated transcription on TREs.

Nuclear hormone receptors activate gene transcription through ligand-dependent association with coactivators. Specific LXXLL sequence motifs present in these cofactors are sufficient to mediate these ligand-induced interactions. A thyroid hormone receptor (TR)-binding protein (TRBP) was cloned by a Sos-Ras yeast two-hybrid system using TRβ1-ligand binding domain as bait. TRBP contains 2063 amino acid residues, associates with TR through a LXXLL motif, and is ubiquitously expressed in a variety of tissues and cells. TRBP strongly transactivates through TRβ1 and estrogen receptor in a dose-related and ligand-dependent manner, and also exhibits coactivation through AP-1, CRE, and NFκB-response elements, similar to the general coactivator CBP/p300. The C terminus of TRBP binds to CBP/p300 and DRIP130, a component of the DRIP/TRAP/ARC complex, which suggests that TRBP may activate transcription by means of such interactions. Further, the association of TRBP with the DNA-dependent protein kinase (DNA-PK) complex and DNA-independent phosphorylation of TRBP C terminus by DNA-PK point to a potential connection between transcriptional control and chromatin architecture regulation.

Thyroid hormone (TH) plays an important role in the postnatal development of the rodent cerebellum, particularly within the first 2 weeks of postnatal life. This action is exerted through the regulation of specific genes during development and is mediated by coactivator and corepressor proteins that determine transcriptional repression or activation, respectively. Thus, we hypothesized that the effect of TH on rodent cerebellar development could be influenced by the relative amounts of coactivator and corepressor proteins in vivo. These ratios might be modulated in an age-specific manner and/or by hormones to generate the "critical period" of TH action. To examine this hypothesis, we cloned rat complementary DNA fragments corresponding to coactivators (SRC1, TIF2 and TRAM1) and corepressors (N-CoR and SMRT), and studied the ontogenic changes in their corresponding messenger RNAs in rat cerebellum of normal and hypothyroid rats during postnatal development, using a RNase protection assay. We found an increased expression of SRC1 and TIF2, as well as of N-CoR, during rat cerebellar development but no change in the expression of SMRT and TRAM1 genes. However, thyroid hormone status did not affect the expression of coactivator and corepressor genes in the cerebellum. These results indicate that coactivator and corepressor messenger RNAs exhibit differential expression through cerebellar development but are not regulated by TH during this period.

Thyroid hormones influence both bone formation and bone resorption. Clinical data and animal studies provide evidence of skeletal site heterogeneity (hip vs. spine) of bone responses to thyroid hormones. In vitro studies also demonstrate direct effects of thyroid hormones on cells of the osteoblast lineage. Transcriptional regulation by thyroid hormone is mediated by ligand-dependent transcription factors called thyroid hormone receptors (TRs). Two genes, c-ErbAalpha and c-ErbAbeta, generate at least four TR isoforms in the rat: TRalpha(1), c-erbAalpha(2), TRbeta(1), and TRbeta(2). Although functional TRs have been identified in cells of the osteoblast lineage, it is still not known if TR isoform expression in bone differs depending upon which skeletal site is examined. We have used ribonuclease protection assay and Northern blot analysis to simultaneously examine the expression of TR isoform mRNAs in adult rat femoral and vertebral bone. TRalpha(1), c-erbAalpha(2), and TRbeta(1) are expressed in both femur and vertebra whole bone. Bone marrow cells from both skeletal sites were also cultured under conditions whereby the osteoprogenitors differentiated into osteoblasts and formed a mineralized extracellular matrix. TRalpha(1), c-erbAalpha(2), and TRbeta(1) mRNAs are each expressed in both femoral and vertebral osteoblast cultures. The presence of TRalpha(1), c-erbAalpha(2), and beta(1) proteins was confirmed by Western analysis of nuclear protein extracts from femoral and vertebral cell cultures. These results indicate that the three predominant TR isoforms are highly expressed in bone and osteoblasts from femurs and vertebrae. Whether there are distinct mechanisms of thyroid hormone action mediated by TRalpha(1), c-erbAalpha(2), and TRbeta(1) at these separate skeletal sites remain to be shown.

Thyroid hormone (TH) plays a critical role in normal cerebellar development. However, the molecular mechanisms of TH action in the developing cerebellum are not fully understood. This action could be exerted in part through brain-derived neurotropic factor (BDNF), as cerebellar BDNF messenger RNA (mRNA) expression is lower, and replacement of BDNF partially reverses the abnormal neurogenesis in the hypothyroid rat. The rat BDNF gene consists of four noncoding exons (exons I–IV), each of which is linked to a different promoter, and a protein-coding exon (exon V). To study promoter-specific regulation of the BDNF gene by TH, ribonuclease protection assay of each exon mRNA was performed using total developing rat cerebellar RNA. During cerebellar development, all exon mRNAs were detected, but with different expression patterns; among noncoding exon mRNAs, exon II mRNA was the most abundant. Daily TH replacement induced a 3-fold increase in exon II mRNA on postnatal day (P) 15. On P30, exon II mRNA was still much greater in the TH-replaced animal. Exon I mRNA was detected on P2 and P7. However, in contrast to exon II mRNA, TH treatment suppressed the expression of exon I mRNA on P2. Exon III and IV mRNAs were not detected on P2 and P7, but small amounts were observed starting on P15 in TH-replaced animals. They were not detected by P30 in hypothyroid animals. In contrast, in the cerebral cortex, although all exons are differentially regulated during development, the expression of each mRNA was not significantly altered by TH. These results indicate that TH regulates BDNF gene expression in a promoter-, developmental stage-, and brain region-specific manner, which may play an important role in region- and stage-specific regulation of brain development by TH.

GnRH plays a critical role in regulating mammalian reproductive development and function. At the level of the anterior pituitary, GnRH binds to the GnRH receptor (GnRHR) on the cell surface of pituitary gonadotropes. Here, it activates intracellular signal transduction pathways to effect both the synthesis and intermittent release of the gonadotropins LH and FSH. These hormones then enter the systemic circulation to regulate gonadal function, including steroid hormone synthesis and gametogenesis. The response of pituitary gonadotropes to GnRH correlates directly with the concentration of GnRHR on the cell surface, which is mediated, at least in part, at the level of gene expression. A number of endocrine, paracrine, and autocrine factors are known to regulate GnRHR gene expression. This article reviews in detail the role of the GnRHR in the hypothalamic-pituitary-gonadal axis and the factors mediating expression of this gene. A better understanding of the molecular mechanisms that regulate transcription of the GnRHR gene will further our knowledge about the role of this receptor in mammalian reproductive physiology in health and disease.

Ligand-dependent nuclear hormone receptors (NRs), such as retinoic acid and thyroid hormone receptors, play critical roles in diverse aspects of development. They enhance or repress transcription by recruiting an array of coactivator and corepressor proteins, which function as signaling intermediates between the NRs and the basal transcriptional machinery. To study the possible involvement of these cofactors on tissue-specific regulation of gene expression by NRs, we examined the expression of the coactivator SRC-1 mRNA during mouse embryogenesis by in situ hybridization (ISH). 35S-labeled riboprobe specific for SRC-1 mRNA was used for analysis. The distribution of this transcript was studied from 8.5 to 18.5 embryonic days (E8.5-E18.5) and in postnatal day 15 (P15). The SRC-1 transcript was largely ubiquitously expressed, even on E8.5. At E14.5 and E18.5, highest levels of SRC-1 transcript was found in the olfactory epithelium. Significant SRC-1 hybridization signal was also detected in the neocortex, anterior pituitary and heart. We conclude that (1) SRC-1 mRNA is widely expressed in the developing embryo, and (2) SRC-1 mRNA is expressed at the highest level in the olfactory epithelium, suggesting that this coactivator may be involved in the development and/or function of the olfactory system.

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 PPARalpha, while inhibiting PPARgamma under certain conditions. However, it was hitherto unclear whether the stimulatory effect of insulin on PPARalpha was direct and by which mechanism it occurs. We now demonstrate that amino acids 1-92 of hPPARalpha 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 PPARalpha 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 PPARalpha, 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 PPARgamma2, which was previously shown to be phosphorylated at a single site in a motif that is not homologous to the sites now described in PPARalpha. Although the molecular details involved in the phosphorylation-dependent enhancement of the transcriptional activity of PPARalpha remain to be elucidated, we demonstrate that the effect of insulin on the AF-1 region of PPARalpha can be mimicked by the addition of triiodothyronine receptor beta1, a strong binder of corepressor proteins. In addition, a triiodothyronine receptor beta1 mutant deficient in interacting with corepressors is unable to activate PPARalpha. These observations suggest that the AF-1 region of PPARalpha is partially silenced by corepressor proteins, which might interact in a phosphorylation-dependent manner.

Inactivation of the melanocortin-4 receptor (MC4-R) by gene-targeting results in mice that develop maturity-onset obesity, hyperinsulinemia, and hyperglycemia. These phenotypes resemble common forms of human obesity, which are late-onset and frequently accompanied by NIDDM. It is not clear whether sequence variation of the MC4-R gene contributes to obesity in humans. Therefore, we examined the human MC4-R gene polymorphism in 190 individuals ascertained on obesity status. Three allelic variants were identified, including two novel ones, Thr112Met and Ile137Thr. To analyze possible functional alterations, the variants were cloned and expressed in vitro and compared with the wild-type receptor. One of the novel variants, Ile137Thr, identified in an extremely obese proband (BMI 57), was found to be severely impaired in ligand binding and signaling, raising the possibility that it may contribute to development of obesity. Furthermore, our results also suggest that sequence polymorphism in the MC4-R coding region is unlikely to be a common cause of obesity in the population studied, given the low frequency of functionally significant mutations.

This study is designed to clarify the role of an orphan nuclear hormone receptor, ROR alpha, on thyroid hormone (TH) receptor (TR)-mediated transcription on a TH-response element (TRE). A transient transfection study using various TREs [i.e., F2 (chick lysozyme TRE), DR4 (direct repeat), and palindrome TRE] and TR and ROR alpha1 was performed. When ROR alpha1 and TR were cotransfected into CV1 cells, ROR alpha1 enhanced the transactivation by liganded-TR on all TREs tested without an effect on basal repression by unliganded TR. By electrophoretic mobility shift assay, on the other hand, although ROR alpha bound to all TREs tested as a monomer, no (or weak) TR and ROR alpha1 heterodimer formation was observed on various TREs except when a putative ROR-response element was present. The transactivation by ROR alpha1 on a ROR-response element, which does not contain a TRE, was not enhanced by TR. The effect of ROR alpha1 on the TREs is unique, because, whereas other nuclear hormone receptors (such as vitamin D receptor) may competitively bind to TRE to exert dominant negative function, ROR alpha1 augmented TR action. These results indicate that ROR alpha1 may modify the effect of liganded TR on TH-responsive genes. Because TR and ROR alpha are coexpressed in cerebellar Purkinje cells, and perinatal hypothyroid animals and ROR alpha-disrupted animals show similar abnormalities of this cell type, cross-talk between these two receptors may play a critical role in Purkinje cell differentiation.

Recent in vivo and in vitro studies have implicated the orphan nuclear receptor, steroidogenic factor-1 (SF-1), and the early growth response protein 1 (Egr-1) in the transcriptional regulation of the luteinizing hormone beta-subunit (LHbeta) gene. We have previously demonstrated the ability of SF-1 to bind to and transactivate the rat LHbeta gene promoter acting at a consensus gonadotrope-specific element (GSE) located at position -127. We have now identified a second functional GSE site at position -59. In addition, based on electrophoretic mobility shift assay, in vitro translated Egr-1 is shown to bind to two putative Egr-1 binding sites (positions -112 and -50), which appear to be paired with the identified GSE sites. By transient transfection assay in pituitary-derived GH3 cells, it was seen that Egr-1 increases promoter activity of region -207/+5 of the rat LHbeta gene promoter through action at both Egr-1 sites. Furthermore, LHbeta gene promoter activity is markedly augmented in the presence of both factors together relative to activity in the presence of SF-1 or Egr-1 alone (150-fold versus 14-fold and 12-fold, respectively). These data define two composite SF-1-Egr-1 response-elements in the proximal LHbeta gene promoter and suggest that SF-1 and Egr-1 act synergistically to increase expression of the LHbeta gene in the gonadotrope.

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 patterns of these genes. We show that T3 and E2 regulate the expression of steroid receptor coactivator-1 messenger RNA in the anterior pituitary in addition to a gender-related difference. These tissue variations may have physiological implications for heterogeneity of hormone responses that are observed in normal and malignant tissues.

Deficiency of thyroid hormone (TH) during the perinatal period results in severe neurological abnormalities in rodent cerebellar development. However, the molecular mechanisms of TH action in the developing cerebellum are not fully understood. Of note, a mutant mouse, staggerer, in which the orphan nuclear hormone receptor ROR alpha gene is disrupted, exhibits cerebellar abnormalities similar to those seen in the hypothyroid animals, despite normal thyroid function. We, therefore, speculated that TH (tetraiodo-L-thyronine; T4) may regulate ROR alpha gene expression, which then may regulate genes essential for normal brain development. To test this hypothesis, we studied the changes in ROR alpha gene expression in perinatal hypothyroid rat cerebellum and the effect of TH replacement using Northern blot analysis, ribonuclease protection assay and in situ hybridization histochemistry. During cerebellar development, an approximately 3-fold increase in the cerebellar content of ROR alpha messenger RNA (mRNA) was seen in both propylthiouracil-treated, and propylthiouracil-treated and T4-replaced animals. However, the increase was accelerated when T4 was injected, although the ROR alpha mRNA content was identical, with or without T4, by 30 days after birth (P30). In contrast, T4 treatment suppressed the TH receptor alpha1 and c-erbA alpha2 mRNA content by P30; retinoic acid X receptor-beta mRNA content was not influenced by thyroid status. A significant hybridization signal for ROR alpha mRNA was seen only over Purkinje cells in the cerebellar cortex by in situ hybridization histochemistry. These results indicate that TH alters the timing of expression of the ROR alpha gene in the Purkinje cells of the cerebellar cortex, which may, in turn, influence Purkinje cell differentiation.

The regulation of LH and FSH subunit gene expression is under the control of GnRH. Physiological changes in the frequency of pulsatile GnRH release from the hypothalamus result in differential stimulation of α-, LHβ-, and FSHβ-gene expression. Previous studies indicate that the GnRH receptor couples to G proteins of the Gq/11 family, with phosphoinositide turnover and its resultant increase in intracellular calcium concentration and protein kinase C (PKC) activation, to stimulate secretion of LH and FSH. However, the molecular mechanisms by which GnRH mediates its transcriptional effects remain largely unknown. We used GH3 cells, constitutively expressing the rat GnRH receptor (GGH3-1′ cells) and transiently transfected with a luciferase reporter gene controlled by either the α, LHβ, or FSHβ gene regulatory region (αLUC, LHβLUC, and FSHβLUC, respectively), to examine the roles of several signal transduction pathways in the GnRH-mediated stimulation of gonadotropin subunit gene expression. Activation of PK...

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.

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, a thyroid hormone receptor activator molecule, a approximately 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 TRbeta1 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.

The identification of hormone response elements in the promoter regions of hormonally regulated genes has revealed a striking similarity between the half-site of the estrogen-response element (ERE) and a consensus sequence constituting the thyroid hormone-response element. Because of the potential for thyroid hormone (T3) to affect estrogen (E)- and progesterone-dependent female reproductive behavior via EREs, we have begun to investigate the activity of an ERE identified in the progesterone receptor (PR) proximal promoter and its interactions with the estrogen receptor (ER) and thyroid hormone receptors (TR). In addition, we have compared ER and TR interactions on the PR ERE construct with that of the vitellogenin A2 (vit A2) consensus ERE. Electrophoretic mobility shift assays demonstrated that TR binds to the PR ERE as well as to the consensus ERE sequence in vitro. Further, these two EREs were differentially regulated by T3 in the presence of TR. T3 in the presence of TR alpha increased transcription from a PR ERE construct approximately 5-fold and had no inhibitory effect on E induction. Similarly, T3 also activated a beta-galactosidase reporter construct containing PR promoter sequences spanning -1400 to +700. In addition, the TR isoforms beta1 and beta2 also stimulated transcription from the PR ERE construct by 5- to 6-fold. A TR alpha mutant lacking the ability to bind AGGTCA sequences in vitro failed to activate transcription from the PR ERE construct, demonstrating dependence on DNA binding. In contrast to its actions on the PR ERE construct, TR alpha did not activate transcription from the vit A2 consensus ERE but rather attenuated E-mediated transcriptional activation. Attenuation from the vit A2 consensus ERE is not necessarily dependent on DNA binding as the TR alpha DNA binding mutant was still able to inhibit E-dependent transactivation. In contrast to TR alpha, the isoforms TRbeta1 and TRbeta2 failed to inhibit E-induced activation from the vit A2 consensus ERE. These results demonstrate that the PR ERE construct differs from the vit A2 consensus ERE in its ability to respond to TRs and that divergent pathways exist for activation and inhibition by TR. Since ERs, PRs, and TRs are all present in hypothalamic neurons, these findings may be significant for endocrine integration, which is important for reproductive behavior.

Retinoic acid (RA) and 9-cis-RA induce growth arrest and differentiation of S91 melanoma cells. RA activates retinoic acid receptors (RARs), whereas 9-cis-RA activates both RARs and retinoid X receptors (RXRs). Both classes of receptors function as ligand-dependent transcription factors. S91 melanoma cells contain mRNA for RXRalpha, RXRbeta, RARalpha, RARgamma, and RARbeta in low levels. Among these, only RARbeta gene transcription is induced by retinoids. However, at present the individual role(s) for each RXR and RAR isoform in these processes is unclear. We assessed the function of all isoforms in the S91 melanoma model by using RXR and RAR isoform-specific retinoids to study their effects on cell growth, RARbeta expression, and differentiation. Activation of each of the endogenous RXR or RAR isoforms induces RARbeta gene expression, and blocks cellular proliferation. However, only the RARgamma-ligands cause additional differentiation toward a melanocytic phenotype, which coincides with substantial apoptosis well before morphological changes are apparent. Apoptosis is completely dependent on de novo protein synthesis but cannot be induced by changes in activities of AP-1, protein kinase C, and protein kinase A, nor can it be blocked by the presence of the antioxidant glutathione. These results argue against a specific role for RARbeta, but suggest that RARgamma has a critical role in a genetic switch between melanocytes and melanoma, and induction of ligand-dependent apoptosis.

Androgen, glucocorticoid, and progesterone receptors (ARs, GRs, and PRs) often can regulate transcription via composite hormone response elements in target genes. We have used artificial and natural mutant ARs from patients with androgen resistance to study their effects on dominant negative activity on wild type AR, GR, and PR function on mouse mammary tumor virus (MMTV) and tyrosine aminotransferase (TAT) promoters. Artificial ARs that contained internal deletions within the amino-terminal region had minimal transcriptional activity but blocked ligand-mediated transcription by wild type AR. Mutants containing deletions of the DNA-binding and ligand-binding domains had minimal or weak dominant negative activity. We then tested the ability of wild type and mutant ARs to modulate GR- and PR-mediated transcriptional activity. The amino-terminal deletion mutants exerted dominant negative effects on GR- and PR-mediated activity, both in the absence and presence of testosterone. Surprisingly, wild type AR, which had approximately 20% of the maximal transcriptional activity of GR on the MMTV promoter, also had dominant negative activity on dexamethasone-regulated transcription mediated by GR. This dominant negative activity likely involves DNA binding because a point mutation in the DNA-binding domain abrogated such activity of an amino-terminal deletion mutant. Additionally, natural human AR mutants from patients with androgen resistance, which do not bind either DNA or ligand, did not block dexamethasone-mediated transcription. In summary, these studies suggest that mutant and wild type ARs can display dominant negative activity on other steroid hormone receptors that bind to a composite hormone response element This cross-regulation may be important in regulating maximal transcriptional activity in tissues where these receptors are coexpressed and may contribute to the phenotype of patients with steroid hormone resistance.

Autosomal dominant inheritance is exhibited by about 10% of cases of amyotrophic lateral sclerosis (ALS), a paralytic disorder characterized by the death of motor neurons in the brain and spinal cord. A subgroup of these familial cases are linked to mutations in the gene which codes for Cu/Zn superoxide dismutase (SOD1). We report three additional mutations occurring in the SOD1 gene in ALS patients and two single base pair variant changes. The single base pair change in an ALS family causes a glycine 93 to valine substitution, which is the fifth distinct amino acid change reported for the glycine 93 residue. One missense mutation in exon 5 would substitute neutral valine for the negatively-charged aspartate 124 (aspartate 124 to valine). An individual with an apparently sporadic case of ALS carries a three base pair deletion in exon 5 of the SOD1 gene. These three mutations bring to 38 the total number of distinct SOD1 mutations associated with familial ALS.

Thyroid-hormone receptors (TRs) form heterodimers with retinoid-X receptors (RXRs) on thyroid-hormone-response elements (TREs). However, it is not known whether the formation of liganded TR/RXR heterodimer on a TRE alone is sufficient to dictate transcriptional activity. We designed several mutated DR4s (half-sites arranged as direct repeats with a nucleotide gap of 4) that bound TR/RXR heterodimers preferentially, and employed them to characterize functional and biochemical properties of the heterodimers on DNA. Although TR/RXR heterodimer binding was similar on some of the mutated DR4s, transient transfection assays showed that TRα failed to support triiodothyronine (T3)-stimulated transcription on “inactive” DR4s but mediated basal repression on both “active” and inactive mutated DR4. T3 binding assays showed that the mutated DR4s did not affect T3 binding to the heterodimer. Finally, partial proteolysis studies revealed that binding of active DR4 elements and T3 to the heterodimer synergistically enhanced heterodimerization-induced protease resistance of TR, but not RXR, in the heterodimer. These results suggest that: 1) liganded TR/RXR heterodimer binding to a DR4 is not sufficient for transcriptional activation of the target gene, and 2) DNA sequences in specific TREs may modify T3-mediated transcription by affecting the conformation of the liganded heterodimer.

The cytoplasmic iron regulatory protein (IRP) modulates iron homeostasis by binding to iron-responsive elements (IREs) in the transferrin receptor and ferritin mRNAs to coordinately regulate transferrin receptor mRNA stability and ferritin mRNA translational efficiency, respectively. These studies demonstrate that thyroid hormone (T3) can modulate the binding activity of the IRP to an IRE in vitro and in vivo. T3 augmented an iron-induced reduction in IRP binding activity to a ferritin IRE in RNA electrophoretic mobility shift assays using cytoplasmic extracts from human liver hepatoma (HepG2) cells. Hepatic IRP binding to the ferritin IRE also diminished after in vivo administration of T3 with iron to rats. In transient transfection studies using HepG2 cells and a human ferritin IRE-chloramphenicol acetyltransferase (H-IRE-CAT) construct, T3 augmented an iron-induced increase in CAT activity by approximately 45%. RNase protection analysis showed that this increase in CAT activity was not due to a change in the steady state level of CAT mRNA. Nuclear T3-receptors may be necessary for this T3-induced response, because the effect could not be reproduced by the addition of T3 directly to cytoplasmic extracts and was absent in CV-1 cells which lack T3-receptors. We conclude that T3 can functionally regulate the IRE binding activity of the IRP. These observations provide evidence of a novel mechanism for T3 to up-regulate hepatic ferritin expression, which may in part contribute to the elevated serum ferritin levels seen in hyperthyroidism.

It has been reported that activin A stimulates the synthesis of the GnRH receptors (GnRHR) in rat pituitary cultures. However, the role of activin A in the regulation of the GnRHR gene at the molecular level is not known. In the present work, we have studied the regulation of the GnRHR gene by activin A in the gonadotrope cell line, alpha T3-1, where the GnRHR gene is highly expressed. First, we demonstrate that these cells express the mRNAs of three types of activin receptors: I, II, and IIB. Activin A increases GnRHR mRNA levels in a dose-and time-dependent manner, with maximal stimulation (2.5 +/- 0.5-fold) occurring with a dose of 20 ng/ml after 36 h of incubation. To ascertain whether this effect occurs at the transcriptional level, we performed nuclear run-off experiments in alpha T3-1 cells, which demonstrate a 1.6-fold increase in the levels of newly synthesized GnRHR mRNA in response to activin A. To investigate further the effect of activin A on the transcription of the GnRHR gene, alpha T3-1 cells were transiently transfected with a mouse GnRHR promoter/luciferase reporter gene (GnRHR-Luc) and challenged with activin A. Luciferase activity increases in response to activin A to the same extent (2.4 +/- 0.4-fold) and with similar dose-response and time-course profiles as the mRNA levels. Follistatin (100 ng/ml), a well known activin antagonist, completely abolishes the activin A effect on both mRNA levels and GnRHR-Luc activity. Follistatin also decreases the basal expression of the GnRHR gene by 33% as determined by GnRHR-Luc activity. This, together with our demonstration of the presence of the inhibin beta B-subunit mRNA in alpha T3-1 cells, suggests a potential paracrine/autocrine role of endogenous activin B on the regulation of the GnRHR gene in these cells. To provide evidence for biological significance of activin A stimulation of GnRHR gene expression, the response of a human gonadotropin alpha-subunit promoter/luciferase reporter gene (alpha Gon-Luc) to GnRH was assessed in alpha T3-1 cells pretreated with activin A. Activin enhances the stimulation of alpha Gon-Luc activity by GnRH by 1.6 +/- 0.4-fold. These data demonstrate that activin A can stimulate the expression of the GnRHR gene at the transcriptional level. Furthermore, transfection studies localize the activin responsive element to 1.2 kb of the 5'-flanking region of the GnRHR gene. Transcriptional activation of the GnRHR gene by activin A may serve as a mechanism for the modulation of gonadotrope responsiveness to GnRH.

The orphan nuclear receptor, steroidogenic factor-1 (SF-1), is expressed in the pituitary and in the gonadotrope precursor cell line, alphaT3-1, where it is believed to enhance expression of the common gonadotropin alpha-subunit gene through transactivation of the gonadotrope-specific element (GSE). Sequence analysis of the rat luteinizing hormone beta-subunit (LH beta) gene promoter revealed the presence of a consensus GSE at -127 to -119 (TGACCTTGT). We have demonstrated the ability of SF-1 to bind specifically to this putative GSE sequence by electrophoretic mobility shift assay, utilizing both alphaT3-1 nuclear extracts and in vitro translated SF-1. In addition, mutation of the putative LHbeta-GSE (TGAAATTGT) eliminated specific DNA binding. To examine the ability of SF-1 to enhance LHbeta promoter activity, CV-1 cells, which lack endogenous SF-1, were cotransfected with an SF-1-containing expression vector and an LHbeta-luciferase reporter construct. When cotransfected with -209/+5 of the LHbeta promoter, SF-1 increased luciferase activity by 56-fold. SF-1 responsiveness was markedly diminished with loss of the putative GSE region in deletion constructs and in the presence of a two base pair mutation, analogous to the mutation which eliminated DNA binding. Finally, the LHbeta-GSE was able to confer SF-1 responsiveness on a heterologous minimal growth hormone promoter, GH50 (57-fold). We conclude that SF-1 both binds to and transactivates the rat LHbeta promoter. These data suggest that SF-1 may participate in the expression of the LHbeta gene by the gonadotrope.

Two genes, c-ErbA alpha and c-ErbA beta, generate at least three functional T3 receptor (TR) isoforms in the rat: TR alpha-1, TR beta-1, and TR beta-2. The latter is an N-terminal splice variant of TR beta-1 whose expression is high in the pituitary gland, whereas the other isoforms are more widely expressed. It is believed that TR beta-2 might play an important role in the pituitary, but no specific biological activities have been defined. Using in vitro translated receptors, we were unable to detect striking isoform-specific differences in T3 binding, DNA binding, homodimerization and heterodimerization activities with retinoid X receptor in the electrophoretic mobility assay, or T3-dependent repression and activation of basal transcription in transfection assays. The N-terminus of TR beta is completely dispensible for these activities. However, we found that unliganded TR alpha-1 and TR beta-1, but not TR beta-2, can repress glucocorticoid receptor-mediated transcriptional activation if the reporter gene promoter contains binding sites for both receptor species. TR beta-2 also synergizes most efficiently with the glucocorticoid receptor in the presence for the of T3. The TR beta-2-specific N-terminus is required for these effects. This result indicates that the relative abundance of specific TR isoforms may determine the with the glucocorticoid receptor in the presence of T3. The TR beta-2 specific N-terminus is required for these effects. This result indicates that the relative abundance of specific TR isoforms may determine the quantitative response of a gene that is regulated by T3 and glucocorticoids. In particular, pituitary TR beta-2 may be an important mediator of GH gene expression, which is regulated by both of these hormones.

Thyroid hormone receptors (TRs) recently have been produced in E. coli by several laboratories. We produced E. coli-expressed human TR beta using the histidine/fusion protein system. Surprisingly, we observed that reticulocyte lysate, nonspecific proteins, and 1% Triton X dramatically increased both the T3- and DNA-binding activities of human TR beta. These studies demonstrate that there are a number of factors that will enhance ligand and DNA binding of E. coli-expressed TR beta. Addition of these factors to reaction samples containing E. coli-expressed TRs will help to optimize measurement conditions. These findings also suggest that experiments in which cellular proteins are added to highly purified TR preparations may require controls to eliminate contributions by nonspecific proteins.

Thyroid hormone receptors (TRs) are ligand-dependent transcription factors that regulate target gene transcription. Interestingly, in the absence of ligand, TRs also can repress basal transcription of positively regulated target genes, suggesting that unliganded TR may have a distinct role in gene regulation. In this paper, DNA binding, truncation, and natural human TR beta mutants were used in cotransfection and electrophoretic mobility shift assays to study various aspects of TR-mediated basal repression. Presently, little is known about the role(s) of natural human TR beta mutants on basal repression. These results show that: 1) TR binding to DNA likely is required for basal repression; 2) the amino-terminal region of TR is not required for basal repression; 3) TR homodimer binding is not absolutely required for basal repression, as TR mutants that selectively form TR-retinoid X receptor heterodimers can mediate basal repression; and 4) TR mutants with poor T3-binding affinity likely have constitutive basal repression, even in the presence of ligand. These findings provide new insight on the mechanism of basal repression by unliganded TRs.

Rat GH (rGH) gene expression is increased by both thyroid hormone (T3) and all-trans retinoic acid (RA) via a composite hormone response element (HRE) containing three putative half-sites (rGH-HRE). However, it is not known whether 9-cis RA (9cRA) also can regulate rGH gene expression. In this study, we performed a Northern blot analysis that demonstrated that 9cRA, as well as T3 and RA, increased rGH messenger RNA expression in rat pituitary GH3 cells. Transient transfection studies in GH3 cells, using reporter plasmids containing the rGH-HRE and mutated half-sites, revealed that 9cRA-stimulation of rGH transcription was mediated by the rGH-HRE and that all three half-sites are necessary. Additionally, we performed cotransfection studies to elucidate the particular receptor complexes involved in 9cRA regulation of rGH gene expression using CV-1 cells, which contain little or no endogenous RA receptors, and thyroid hormone receptors. Interestingly, in the presence of either retinoid X receptor alone, RA receptors alone, or both receptors, 9cRA caused similar induction of transcriptional activity. However, cotransfection of thyroid hormone receptors with these receptors repressed basal and blocked 9cRA-induced transcriptional activity in the absence of T3. Our data suggest that 9cRA-stimulation of rGH transcription is likely mediated by 9cRA-bound retinoid X receptor- and/or RA receptor-containing complexes but not by thyroid hormone receptor-containing complexes. Our studies provide evidence that several different members of the nuclear hormone receptor family can interact on this composite DNA element, with transcription stimulated or blocked depending on the presence or absence of cognate ligands.

Four cell lines, stably transfected with rat GnRH receptor complementary DNA, have been prepared from the lactotropic GH3 cell line. All four lines (as well as the parent line and a line transfected with the vector DNA) show extensive rosettes of circular polyribosomes, characteristic of high protein synthetic activity, although secretory granules are virtually absent; the rough endoplasmic reticulum (rER) cisternae were short and straight. Instances were observed in which the ER reaches to the plasma membrane, suggesting a possible nongranular secretory route. All four lines (but not the parent or a control transfected line) expressed GnRH receptors that were down-regulated (1-5 h, depending on the cell line) after exposure to 10 nM GnRH; receptors then recovered (2-7 h). This pattern is reminiscent of the GnRH receptor in the primary gonadotrope cell cultures. All cell lines released PRL (4-96 h) in response to a GnRH agonist (D-tBuSer6-des-Gly10-Pro9-ethylamide-GnRH), an event that was inhibited by all three major classes of Ca+2 ion channel antagonists (methoxyverapamil, 1,4-dihydropyridines, and diltiazem); in contrast, GnRH-stimulated LH release from pituitary-derived primary cultures is only inhibited by methoxyverapamil. One line became refractory to GnRH analog stimulation after 24 h, although the other three released PRL vigorously up to the longest time point examined (96 h). All four lines responded substantially more robustly to 1 microgram/ml Buserelin than to 1 microgram/ml TRH. All four lines produced inositol phosphate metabolites and released immunoassayable cAMP (24 h) in response to treatment with Buserelin. These cell lines are good models for understanding the mechanisms by which the GnRH receptor is coupled to second messenger systems and for comparing these mechanisms with TRH-receptor coupling in the same cell.

It was previously shown that when cells of renal epithelial origin are exposed to hyperosmotic urea in concentrations unique to the renal medulla, abundance of mRNA encoding the immediate-early gene product Egr-1 is up-regulated. This phenomenon appears restricted to cells of renal epithelial origin. In the present study, the newly isolated murine renal inner medullary cell line mIMCD3 was used to determine whether the urea-induced increase in Egr-1 mRNA abundance is associated with increased expression of functional protein product, and whether this increase is transcriptionally mediated. In Western analysis, urea (200 mM) increased Egr-1 immunoreactivity 3-fold relative to sham-treated cells. [35S]Methionine pulse-labeling followed by immunoprecipitation confirmed that this increased immunoreactivity was associated with increased de novo Egr-1 protein synthesis. Electrophoretic mobility shift assay demonstrated that urea treatment induced a commensurate increase in specific DNA binding activity for the Egr-1 consensus sequence. In addition, the increase in Egr-1 mRNA expression accompanying urea treatment was a consequence of enhanced transcription, as determined by nuclear run-off assay. Taken together, these data indicate that hyperosmotic urea increases both Egr-1 transcription and new protein synthesis in renal epithelial cells in culture, and that this newly synthesized Egr-1 is a functional DNA-binding protein. To our knowledge, this is the first example of urea-inducible gene transcription. In addition, Egr-1 represents the first eukaryotic transcription factor transcriptionally activated by a hyperosmotic stressor.

TRH and GnRH receptors are each coupled to G proteins of the Gq/11 family. Activation of each of these receptors by their respective ligands results in the stimulation of phospholipase C activity, leading to calcium mobilization and protein kinase C activation. Thus, the effects of TRH and GnRH may be mediated through the same intracellular signal transduction pathway. To compare responses to TRH and GnRH directly within one cell type, we have stably transfected the rat pituitary GH3 lactotrope cell line, which expresses the endogenous TRH receptor, with an expression vector containing rat GnRH receptor cDNA. Transfected cells specifically bound GnRH with high affinity and responded to GnRH stimulation with an increase in PRL mRNA levels, analogous to their response to TRH stimulation. Stably transfected GH3 cells, which were then transiently transfected with luciferase reporter constructs containing either the PRL or the glycoprotein hormone alpha-subunit promoter, responded to either GnRH or TRH stimulation with an increase in luciferase activity in a time- and dose-dependent fashion. The stimulatory effects of maximally effective concentrations of TRH and GnRH were additive on PRL, but not alpha-subunit, gene expression. These data, coupled with evidence of cross-desensitization of alpha-subunit, but not PRL, promoter activity stimulation by TRH and GnRH, suggest that there may be differences in the signal transduction pathways activated by TRH and GnRH receptors in the regulation of PRL and alpha-subunit gene expression.