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W W Chin

Publications and source records attributed to W W Chin.

At least 55 records · Page 3Linked to original sources
PubMed1998

An inhibitory region of the DNA-binding domain of thyroid hormone receptor blocks hormone-dependent transactivation.

We have employed a chimeric receptor system in which we cotransfected yeast GAL4 DNA-binding domain/retinoid X receptor beta ligand-binding domain chimeric receptor (GAL4RXR), thyroid hormone receptor-beta (TRbeta), and upstream activating sequence-reporter plasmids into CV-1 cells to study repression, derepression, and transcriptional activation. In the absence of T3, unliganded TR repressed transcription to 20% of basal level, and in the presence of T3, liganded TRbeta derepressed transcription to basal level. Using this system and a battery of TRbeta mutants, we found that TRbeta/RXR heterodimer formation is necessary and sufficient for basal repression and derepression in this system. Additionally, an AF-2 domain mutant (E457A) mediated basal repression but not derepression, suggesting that interaction with a putative coactivator at this site may be critical for derepression. Interestingly, a mutant containing only the TRbeta ligand binding domain (LBD) not only mediated derepression, but also stimulated transcriptional activation 10-fold higher than basal level. Studies using deletion and domain swap mutants localized an inhibitory region to the TRbeta DNA-binding domain. Titration studies further suggested that allosteric changes promoting interaction with coactivators may account for enhanced transcriptional activity by LBD. In summary, our findings suggest that TR heterodimer formation with RXR is important for repression and derepression, and coactivator interaction with the AF-2 domain may be needed for derepression in this chimeric system. Additionally, there may be an inhibitory region in the DNA-binding domain, which reduces TR interaction with coactivators, and prevents full-length wild-type TRbeta from achieving transcriptional activation above basal level in this chimeric receptor system.

Allosteric Regulation↗
PubMed1997

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.

Adipocytes↗
PubMed1997

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, 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.

Amino Acid Sequence↗
PubMed1997

Specific activation of retinoic acid receptors (RARs) and retinoid X receptors reveals a unique role for RARgamma in induction of differentiation and apoptosis of S91 melanoma cells.

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.

Apoptosis↗
PubMed1997

Immunohistochemical expression of retinoid X receptor isoforms in human pituitaries and pituitary adenomas.

Retinoid X receptors (RXRs) are transcriptional factors that belong to the steroid/thyroid hormone receptor superfamily. There are 3 RXR isoforms-alpha, beta, gamma-known to bind 9-cis-retinoic acid as their ligand. The expression of RXRs in human pituitary glands and pituitary adenomas has not been extensively investigated. To determine whether specific RXR isoforms may play roles in the state of differentiation of pituitary adenomas, we have investigated the immunohistochemical expression of RXR alpha and RXR gamma in 6 nontumorous pituitaries and in 60 different pituitary adenomas using isoform-specific antibodies. In the nontumorous pituitaries. RXR alpha was expressed in the nuclei of almost all cells, while RXR gamma was only expressed in thyrotropin (TSH) cells and in some cells positive for growth hormone (GH) and glycoprotein alpha-subunit (alpha SU) but not in luteinizing hormone (LH) beta-subunit, follicle-stimulating hormone (FSH) beta-subunit, prolactin (PRL) or adrenocorticotropin (ACTH) cells by double immunostaining. All 60 adenomas were RXR alpha positive, and 39 of 60 adenomas (65%) were positive for RXR gamma. The incidence of RXR gamma immunoreactivity in the different adenoma types was: 13 of 16 GH-producing adenomas (81.3%), 9 of 14 PRL-secreting adenomas (64.3%), 6 of 6 TSH-secreting adenomas (100%), 2 of 5 ACTH-secreting adenomas (40%) and 9 of 19 nonfunctioning adenomas (47.4%) including immunohistochemically gonadotropin-subunit-positive adenomas. The colocalization of RXR gamma with the TSH beta subunit, GH and alpha SU in the same adenoma cells was frequently observed, and sometimes RXR gamma was colocalized with PRL, ACTH, FSH beta or LH beta as shown by double immunostaining. We conclude that RXR alpha is expressed in both human pituitaries and pituitary adenomas. In contrast, RXR gamma is expressed more broadly in pituitary adenomas than in normal pituitaries and thus may play a role in the differentiation-specific cell types in the human pituitary both under physiological and pathological conditions.

Adenoma↗
PubMed1997

Differential effects of gonadotropin-releasing hormone (GnRH) pulse frequency on gonadotropin subunit and GnRH receptor messenger ribonucleic acid levels in vitro.

The hypothalamic hormone, GnRH, is released and transported to the anterior pituitary in a pulsatile manner, where it binds to specific high-affinity receptors and regulates gonadotropin biosynthesis and secretion. The frequency of GnRH pulses changes under various physiological conditions, and varying GnRH pulse frequencies have been shown to regulate differentially the secretion of LH and FSH and the expression of the gonadotropin alpha, LH beta, and FSH beta subunit genes in vivo. We demonstrate differential effects of varying GnRH pulse frequency in vitro in superfused primary monolayer cultures of rat pituitary cells. Cells were treated with 10 nM GnRH pulses for 24 h at a frequency of every 0.5, 1, 2, or 4 h. alpha, LH beta, and FSH beta messenger RNA (mRNA) levels were increased by GnRH at all pulse frequencies alpha and LH beta mRNA levels and LH secretion were stimulated to the greatest extent at a GnRH pulse frequency of every 30 min, whereas FSH beta mRNA levels and FSH secretion were stimulated maximally at a lower GnRH pulse frequency, every 2 h. GnRH receptor (GnRHR) mRNA levels also were increased by GnRH at all pulse frequencies and were stimulated maximally at a GnRH pulse frequency of every 30 min. Similar results were obtained when the dose of each pulse of GnRH was adjusted to maintain a constant total cumulative dose of GnRH over 24 h. These data show that gonadotropin subunit gene expression is regulated differentially by varying GnRH pulse frequencies in vitro, suggesting that the differential effects of varying GnRH pulse frequencies on gonadotropin subunit gene expression occur directly at the level of the pituitary. The pattern of regulation of GnRHR mRNA levels correlated with that of alpha and LH beta but was different from that of FSH beta. This suggests that alpha and LH beta mRNA levels are maximally stimulated when GnRHR levels are relatively high, whereas FSH beta mRNA levels are maximally stimulated at lower levels of GnRHR expression, and that the mechanism for differential regulation of the gonadotropins by varying pulse frequencies of GnRH may involve levels of GnRHR. Furthermore, these data suggest that the mechanisms whereby varying GnRH pulse frequencies stimulate alpha LH beta, and GnRHR gene expression are similar, whereas the stimulation of FSH beta mRNA levels may be different.

Animals↗
PubMed1997

Deletion of the thyroid hormone beta1 receptor increases basal and triiodothyronine-induced growth hormone messenger ribonucleic acid in GH3 cells.

The conserved diversity, restricted distribution, and differential regulation of the thyroid hormone receptor (TR) isoforms raise the possibility of isoform-specific functions. We have addressed the roles of individual TRs in GH gene expression in GH3 cells by using an isoform-specific antisense RNA to delete TRbeta1. An antisense RNA vector, directed against the isoform-specific coding sequence of the parent TRbeta1 complementary DNA, was constructed. Stable transfected GH3-derived cell lines expressing this construct were established. Appropriate control cell lines were established in parallel. Depletion of TRbeta1 in cells expressing the antisense construct was confirmed at both the messenger RNA and protein levels. Total TR expression was maintained in these cells by a reciprocal increase in TRbeta2 levels. This perturbation of the TR population was associated with a 10.5-fold increase in basal and a 5.0-fold increase in T3-stimulated GH gene expression, but no increase in total T3 binding of nuclear extracts. In transient cotransfection experiments, there were no differences between control cells and those expressing the antisense construct in either basal or T3-stimulated expression of reporters containing a variety of thyroid hormone response elements. Depletion of TRbeta1 in GH3 cells results in a reciprocal increase in TRbeta2. These changes are associated with increased basal and T3-stimulated GH gene expression, which are not due to a nonspecific enhancement of basal or hormone-stimulated transcription. We demonstrate that TRbeta1 is not required for T3 induction of the GH gene in GH3 cells and that TRbeta1 and TRbeta2 are not equivalent in their effects on basal repression of the GH promoter. The data illustrate the potential for isoform- and promoter-specific dissociation of the repression and activation properties of the TRs.

Animals↗
PubMed1997

Interactions of estrogen- and thyroid hormone receptors on a progesterone receptor estrogen response element (ERE) sequence: a comparison with the vitellogenin A2 consensus ERE.

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.

Animals↗
PubMed1997

Mutant and wild-type androgen receptors exhibit cross-talk on androgen-, glucocorticoid-, and progesterone-mediated transcription.

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.

Androgens↗
PubMed1997

3,5-Diiodo-L-thyronine (T2) has selective thyromimetic effects in vivo and in vitro.

Recent data have suggested that the iodothyronine, 3,5-diiodo-l-thyronine (T2), has selective thyromimetic activity. In vivo, T2 has been shown to suppress TSH levels at doses that do not produce significant peripheral manifestations of thyroid hormone activity. Furthermore, T2 has been shown to produce smaller increments in peripheral indices of thyroid status than does T3, when doses resulting in equivalent suppression of circulating TSH are compared. We have assessed the selective thyromimetic activity of T2 in vivo and in vitro, and performed in vitro studies to assess the potential molecular basis for these selective properties. T2 was 100-fold less potent than T3 in stimulating GH mRNA levels in GH3 cells. In contrast, the iodothyronines were almost equivalent in their ability to downregulate TRbeta2 mRNA levels in this cell line. Both 3,3'-diiodo-L-thyronine and thyronine exhibited no significant thyromimetic effects on either process. In vivo, doses of T2 and T3 that were equivalent in their induction of hepatic malic enzyme (ME) mRNA did not produce equivalent suppression of circulating TSH, with T2 being only 27% as effective as T3. T2 was up to 500-fold less potent than T3 in displacing [125I]-T3 from in vitro translated specific nuclear receptors (TRs) and GH3 cell nuclear extracts. Electrophoretic mobility shift assays, assessing the ability of T2 to produce dissociation of TRbeta1 homodimers from inverted palindrome T3 response elements, indicated that T2 was also 1000-fold less potent than T3 in this respect. These data confirm that T2 has significant thyromimetic activity, and that this activity is selective both in vivo and in vitro. However, there are no data to support a selective central effect, T2 being relatively more potent in stimulating hepatic ME mRNA than in suppression of TSH in vivo. The basis for this differential thyromimetic activity is not selective affinity of the different TR isoforms for T2, or divergent properties of T2 in competitive binding and functional assays in vitro.

Animals↗
PubMed1996

Estrogen and thyroid hormone interaction on regulation of gene expression.

Estrogen receptor (ER) and thyroid hormone receptors (TRs) are ligand-dependent nuclear transcription factors that can bind to an identical half-site, AGGTCA, of their cognate hormone response elements. By in vitro transfection analysis in CV-1 cells, we show that estrogen induction of chloramphenicol acetyltransferase (CAT) activity in a construct containing a CAT reporter gene under the control of a minimal thymidine kinase (tk) promoter and a copy of the consensus ER response element was attenuated by cotransfection of TR alpha 1 plus triiodothyronine treatment. This inhibitory effect of TR was ligand-dependent and isoform-specific. Neither TR beta 1 nor TR beta 2 cotransfection inhibited estrogen-induced CAT activity, although both TR alpha and TR beta can bind to a consensus ER response element. Furthermore, cotransfection of a mutated TR alpha 1 that lacks binding to the AGGTCA sequence also inhibited the estrogen effect. Thus, the repression of estrogen action by liganded TR alpha 1 may involve protein-protein interactions although competition of ER and TR at the DNA level cannot be excluded. A similar inhibitory effect of liganded TR alpha 1 on estrogen induction of CAT activity was observed in a construct containing the preproenkephalin (PPE) promoter. A study in hypophysectomized female rats demonstrated that the estrogen-induced increase in PPE mRNA levels in the ventromedial hypothalamus was diminished by coadministration of triiodothyronine. These results suggest that ER and TR may interact to modulate estrogen-sensitive gene expression, such as for PPE, in the hypothalamus.

Animals↗
PubMed1996

Different DNA elements can modulate the conformation of thyroid hormone receptor heterodimer and its transcriptional activity.

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 TRalpha 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.

Binding Sites↗
PubMed1996

Molecular identification of a major retinoic-acid-synthesizing enzyme, a retinaldehyde-specific dehydrogenase.

Retinoic acid, a developmental signal implicated in the formation of the neural axis, is present at high levels in the early embryonic trunk region, where it is synthesized by a novel dehydrogenase. Here we show that the same enzyme is inducible by retinoic acid in P19 teratocarcinoma cells, and we report the cloning from P19 cells of a cDNA encoding a novel dehydrogenase, named retinaldehyde dehydrogenase-2 (RALDH-2). Expression in COS cells shows RALDH-2 to be highly effective in oxidation of retinaldehyde, with no detectable activity on any other aldehyde tested. In situ hybridization histochemistry on the embryonic trunk reveals RALDH-2 mRNA both in mesoderm and neuroectoderm, with highest neuroectodermal expression in the ventral horn of the spinal cord at two restricted locations along the anteroposterior axis, presumably the subpopulation of motoneurons that innervate the limbs.

Aging↗
PubMed1996

Urea inducibility of egr-1 in murine inner medullary collecting duct cells is mediated by the serum response element and adjacent Ets motifs.

The renal medullary solute urea increases transcription and protein expression of the zinc finger-containing transcription factor Egr-1 in a renal epithelial cell-specific fashion. Transient transfection of mIMCD3 cells with a luciferase reporter gene driven by 1.2 kilobases of the murine egr-1 5'-flanking sequence showed 4-fold increase in reporter gene activity with 200 mM urea treatment. The effect of impermeant solutes such as NaCl was much less pronounced, whereas the permeant solute glycerol had no effect. In addition, this same sequence, minus the egr-1 minimal promoter, conferred urea responsiveness to a heterologous (thymidine kinase) promoter. Whereas deletion of two putative AP-1 sites from the sequence had no effect upon urea inducibility, elimination of the five putative serum response elements (SREs) abolished the urea effect. Progressive deletion of the SREs caused a corresponding diminution in urea effect. Two key tandem SREs (SRE-3 and SRE-4), in conjunction with their two adjacent clusters of Ets motifs, were sufficient to confer urea responsiveness to a reporter gene. This response was markedly attenuated in the absence of either cluster of Ets motifs and was abolished if both clusters were deleted. By electrophoretic mobility shift assay, formation of the ternary complex was constitutive and was demonstrable in vitro despite the presence of 200 mosm urea or NaCl. Therefore urea-inducible egr-1 transcription in renal medullary cells is mediated through the SRE and adjacent Ets motifs; ternary complex formation is not inhibited even in the presence of physiological hyperosmolality.

Animals↗
PubMed1996

Thyroid hormone modulates the interaction between iron regulatory proteins and the ferritin mRNA iron-responsive element.

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.

Animals↗
PubMed1996

Vitamin D receptors repress basal transcription and exert dominant negative activity on triiodothyronine-mediated transcriptional activity.

We have examined vitamin D receptor (VDR), thyroid hormone receptor (TR), and retinoid X receptor beta (RXR beta) binding to vitamin D response elements (VDREs), two thyroid hormone response elements (TREs) (DR4 and F2), and a retinoic acid response element (DR5). VDR/RXR bound well to the VDREs and to DR4 and DR5 using the electrophoretic mobility shift assay. Surprisingly, VDR/RXR also bound well to F2, which contains half-sites arranged as an inverted palindrome. In co-transfection experiments using CV-1 cells, we observed that VDR repressed basal transcription in the absence of ligand on DR3 and osteopontin VDREs and F2, but had no effect on DR4 or DR5. VDR selectively mediated ligand-dependent transcription on only VDREs. VDR also exhibited dominant negative activity as it blocked triiodothyronine (T3)-mediated transcriptional activity on DR4 and F2. These results demonstrate that VDR/RXR heterodimers can bind promiscuously to a wide range of hormone response elements, including inverted palindromes. Moreover, they show that unliganded VDRs, similar to TRs and retinoic acid receptors, can repress basal transcription. Last, they also suggest a novel repressor function of VDR on T3-mediated transcription which may be significant in tissues where VDR and TR are co-expressed.

Animals↗
PubMed1996

Urea signaling in cultured murine inner medullary collecting duct (mIMCD3) cells involves protein kinase C, inositol 1,4,5-trisphosphate (IP3), and a putative receptor tyrosine kinase.

Urea, in concentrations unique to the renal medulla, increases transcription and protein expression of several immediate-early genes (IEGs) including the zinc finger-containing transcription factor, Egr-1. In the present study, the proximal 1.2 kb of the murine Egr-1 5' -flanking sequence conferred urea-responsiveness to a heterologous luciferase reporter gene when transiently transfected into renal medullary mIMCD3 cells,and this effect was comparable with that of the extremely potent immediate-early gene inducer, O-tetradecanoylphorbol 13-acetate (TPA). Urea inducibility of Egr-1 expression was protein kinase C (PKC)-dependent because staurosporine and calphostin C abrogated the urea effect, and down-regulation of PHC through chronic TPa treatment inhibited both urea-inducible Egr-1 protein expression and gene transcription. In addition, hyperosmotic urea increased inositol 1,4,5-trisphosphate (IP3) release from mIMCD3 cells and induced tyrosine phosphorylation of the receptor tyrosine kinase-specific phospholipase C (PLC) isoform, PLC-gamma. Importantly, urea-inducible Egr-1 expression was strongly genistein-sensitive, to a much greater extent than the comparable TPA-inducible Egr-1 expression. These data suggest that urea-inducible Egr-1 expression is a consequence of sequential PLC-gamma activation, IP3 release, and PKC activation. Urea-inducible PLC-gamma activation, in conjunction with the genistein-sensitivity of urea-inducible Egr-1 expression suggest the possibility of a cell surface or cytoplasmic urea-sensing receptor tyrosine kinase.

Alkaloids↗