The nuclear hormone receptor coactivator SRC-1 is a specific target of p300.

p300 and its family member, CREB-binding protein (CBP), function as key transcriptional coactivators by virtue of their interaction with the activated forms of certain transcription factors. In a search for additional cellular targets of p300/CBP, a protein-protein cloning strategy, surprisingly identified SRC-1, a coactivator involved in nuclear hormone receptor transcriptional activity, as a p300/CBP interactive protein. p300 and SRC-1 interact, specifically, in vitro and they also form complexes in vivo. Moreover, we show that SRC-1 encodes a new member of the basic helix-loop-helix-PAS domain family and that it physically interacts with the retinoic acid receptor in response to hormone binding. Together, these results implicate p300 as a component of the retinoic acid signaling pathway, operating, in part, through specific interaction with a nuclear hormone receptor coactivator, SRC-1.

Functional analysis of retinoid Z receptor beta, a brain-specific nuclear orphan receptor.

The retinoid Z receptor beta (RZR beta), an orphan receptor, is a member of the retinoic acid receptor (RAR)/thyroid hormone receptor (TR) subfamily of nuclear receptors. RZR beta exhibits a highly restricted brain-specific expression pattern. So far, no natural RZR beta target gene has been identified and the physiological role of the receptor in transcriptional regulation remains to be elucidated. Electrophoretic mobility shift assays reveal binding of RZR beta to monomeric response elements containing the sequence AnnTAGGTCA, but RZR beta-mediated transactivation of reporter genes is only achieved with two property spaced binding sites. We present evidence that RZR beta can function as a cell-type-specific transactivator. In neuronal cells, GaI-RZR beta fusion proteins function as potent transcriptional activators, whereas no transactivation can be observed in nonneuronal cells. Mutational analyses demonstrate that the activation domain (AF-2) of RZR beta and RAR alpha are functionally interchangeable. However, in contrast to RAR and TR, the RZR beta AF-2 cannot function autonomously as a transactivation domain. Furthermore, our data define a novel repressor function for the C-terminal part of the putative ligand binding domain. We propose that the transcriptional activity of RZR beta is regulated by an interplay of different receptor domains with coactivators and corepressors.

A protein that interacts with members of the nuclear hormone receptor family: identification and cDNA cloning.

In search of proteins which interact with activated steroid hormone receptors, we screened a human liver lambda gt11 expression library with the glucocorticoid receptor. We identified and cloned a cDNA sequence of 1322 bp that encodes a protein of 274 aa. This protein consists predominantly of hydrophilic amino acids and contains a putative bipartite nuclear localization signal. The in vitro translated receptor-associating protein runs in SDS/polyacrylamide gels with an apparent molecular mass of 46 kDa. By use of the bacterially expressed fusion protein with glutathione S-transferase we have found that interaction is not limited to the glucocorticoid receptor but included other nuclear receptors--most notably, the estrogen and thyroid receptors. Binding also occurs with the glucocorticoid receptor complexed with the antiglucocorticoid RU 38486, with the estrogen receptor complexed with the antiestrogen 4-hydroxytamoxifen or ICI 164,384, and even with receptors not complexed with ligand. Association with steroid hormone receptors depends on prior receptor activation--i.e., release from heat shock proteins. The sequence identified here appears to be a general partner protein for nuclear hormone receptors, with the gene being expressed in a variety of mammalian tissues.

Isolation, regulation, and DNA-binding properties of three Drosophila nuclear hormone receptor superfamily members.

We have designed a rapid cloning and screening strategy to identify new members of the nuclear hormone receptor superfamily that are expressed during the onset of Drosophila metamorphosis. Using this approach, we isolated three Drosophila genes, designated DHR38, DHR78, and DHR96. All three genes are expressed throughout third-instar larval and prepupal development. DHR38 is the Drosophila homolog of NGFI-B and binds specifically to an NGFI-B response element. DHR78 and DHR96 are orphan receptor genes. DHR78 is induced by 20-hydroxyecdysone (20E) in cultured larval organs, and its encoded protein binds to two AGGTCA half-sites arranged as either direct or palindromic repeats. DHR96 is also 20E-inducible, and its encoded protein binds selectively to the hsp27 20E response element. The 20E receptor can bind to each of the sequences recognized by DHR78 and DHR96, indicating that these proteins may compete with the receptor for binding to a common set of target sequences.

A nuclear hormone receptor-associated protein that inhibits transactivation by the thyroid hormone and retinoic acid receptors.

Nuclear hormone receptors are transcription factors that require multiple protein-protein interactions to regulate the expression of their target genes. Using the yeast two-hybrid system, we identified a protein, thyroid hormone receptor uncoupling protein (TRUP), that specifically interacts with a region of the human thyroid hormone receptor (TR) consisting of the hinge region and the N-terminal portion of the ligand binding domain in a hormone-independent manner. Interestingly, TRUP inhibits transactivation by TR and the retinoic acid receptor but has no effect on the estrogen receptor or the retinoid X receptor in mammalian cells. We also demonstrate that TRUP exerts its action on TR and retinoic acid receptor by interfering with their abilities to interact with their DNA. TRUP represents a type of regulatory protein that modulates the transcriptional activity of a subclass of the nuclear hormone receptor superfamily by preventing interaction with their genomic response elements.

The yeast nuclear import receptor is required for mitosis.

The nuclear import system is highly conserved among eukaryotes. Here we report the effects of a conditional mutation in SRP1, which encodes a Saccharomyces cerevisiae homolog of the vertebrate nuclear import receptor importin. Importin was isolated as a factor required for the initial targeting step of a nuclear import substrate to the nuclear envelope in a mammalian in vitro assay. We show that yeast Srp1 is similarly required for protein import. In addition, Srp1 is also required for the execution of mitosis: we demonstrate that cells containing a conditional mutation of SRP1 arrest with a G2/M phenotype in a manner analogous to classic cdc mutants. This defect may be due to the failure of the mutant to degrade the mitotic cyclin Clb2 and other proteins required for mitosis. The requirement of a nuclear import receptor for cell cycle-regulated proteolysis implies that import of cell cycle regulators into the nucleus is critical for cell cycle progression.

Nur77 regulates lipolysis in skeletal muscle cells - Evidence for cross-talk between the beta-adrenergic and an orphan nuclear hormone receptor pathway

Skeletal muscle is a major mass peripheral tissue that accounts for similar to 40% of total body weight and 50% of energy expenditure and is a primary site of glucose disposal and fatty acid oxidation. Consequently, muscle has a significant role in insulin sensitivity, obesity, and the blood-lipid profile. Excessive caloric intake is sensed by the brain and induces beta-adrenergic receptor (beta-AR)- mediated adaptive thermogenesis. beta-AR null mice develop severe obesity on a high fat diet. However, the target gene(s), target tissues(s), and molecular mechanism involved remain obscure. We observed that 30 - 60 min of beta-AR agonist ( isoprenaline) treatment of C2C12 skeletal muscle cells strikingly activated (> 100-fold) the expression of the mRNA encoding the nuclear hormone receptor, Nur77. In contrast, the expression of other nuclear receptors that regulate lipid and carbohydrate metabolism was not induced. Stable transfection of Nur77-specific small interfering RNAs (siNur77) into skeletal muscle cells repressed endogenous Nur77 mRNA expression. Moreover, we observed attenuation of gene and protein expression associated with the regulation of energy expenditure and lipid homeostasis, for example AMP-activated protein kinase gamma 3, UCP3, CD36,adiponectin receptor 2, GLUT4, and caveolin-3. Attenuation of Nur77 expression resulted in decreased lipolysis. Finally, in concordance with the cell culture model, injection and electrotransfer of siNur77 into mouse tibialis cranialis muscle resulted in the repression of UCP3 mRNA expression. This study demonstrates regulatory cross-talk between the nuclear hormone receptor and beta-AR signaling pathways. Moreover, it suggests Nur77 modulates the expression of genes that are key regulators of skeletal muscle lipid and energy homeostasis. In conclusion, we speculate that Nur77 agonists would stimulate lipolysis and increase energy expenditure in skeletal muscle and suggest selective activators of Nur77 may have therapeutic utility in the treatment of obesity.

Regulation of endocytosis, nuclear translocation, and signaling of fibroblast growth factor receptor 1 by E-cadherin

Fibroblast growth factor (FGF) receptors (FGFRs) signal to modulate diverse cellular functions, including epithelial cell morphogenesis. In epithelial cells, E-cadherin plays a key role in cell-cell adhesion, and its function can be regulated through endocytic trafficking. In this study, we investigated the location, trafficking, and function of FGFR1 and E-cadherin and report a novel mechanism, based on endocytic trafficking, for the coregulation of E-cadherin and signaling from FGFR1. FGF induces the internalization of surface FGFR1 and surface E-cadherin, followed by nuclear translocation of FGFR1. The internalization of both proteins is regulated by common endocytic machinery, resulting in cointernalization of FGFR1 and E-cadherin into early endosomes. By blocking endocytosis, we show that this is a requisite, initial step for the nuclear translocation of FGFR1. Overexpression of E-cadherin blocks both the coendocytosis of E-cadherin and FGFR1, the nuclear translocation of FGFR1 and FGF-induced signaling to the mitogen-activated protein kinase pathway. Furthermore, stabilization of surface adhesive E-cadherin, by overexpressing p120(ctn), also blocks internalization and nuclear translocation of FGFR1. These data reveal that conjoint endocytosis and trafficking is a novel mechanism for the coregulation of E-cadherin and FGFR1 during cell signaling and morphogenesis.

FRAP analysis of nucleocytoplasmic dynamics of the vitamin D receptor splice variant VDRB1: preferential targeting to nuclear speckles

Although the key components of the cellular nuclear transport machinery have largely been characterized through extensive efforts in recent years, in vivo measurements of the kinetics of nuclear protein import/export are patently few. The present study applies the approach of FRAP (fluorescence recovery after photobleaching) to examine the nucleocytoplasmic flux of a novel human VDRB1 (vitamin D receptor B I) isoform in living cells. Through an N-terminal extension containing a consensus nuclear targeting sequence, VDRB1 is capable of localizing in nuclear speckles adjacent to SC-35 (35 kDa splicing component)containing speckles as well as in the nucleoplasm, dependent on ligand. Investigation of VDRB1 nucleocytoplasmic transport using FRAP indicates for the first time that the VDRB1 has a serum-modulated, active nuclear-import mechanism. There is no evidence of an efficient, active export mechanism for VDRB1, probably as a result of nuclear retention. VDRB1 nuclear import in the absence of serum occurred more rapidly and to a greater extent to nuclear speckles compared with import to other nuclear sites. This preferential transport from the cytoplasm to and accumulation within nuclear speckles is consistent with the idea that the latter represent dynamic centres of VDRB1 interaction with other nuclear proteins. The results are consistent with the existence of specialized pathways to target proteins to nuclear subdomains.

Autoantibody from women with preeclampsia induces soluble Fms-like tyrosine kinase-1 production via angiotensin type 1 receptor and calcineurin/nuclear factor of activated T-cells signaling

Preeclampsia is a pregnancy-specific hypertensive syndrome that causes substantial maternal and fetal morbidity and mortality. Recent evidence indicates that maternal endothelial dysfunction in preeclampsia results from increased soluble Fms-like tyrosine kinase-1 (sFlt-1), a circulating antiangiogenic protein. Factors responsible for excessive production of sFlt-1 in preeclampsia have not been identified. We tested the hypothesis that angiotensin II type 1 (AT1) receptor activating autoantibodies, which occur in women with preeclampsia, contribute to increased production of sFlt-1. IgG from women with preeclampsia stimulates the synthesis and secretion of sFlt-1 via AT1 receptor activation in pregnant mice, human placental villous explants, and human trophoblast cells. Using FK506 or short-interfering RNA targeted to the calcineurin catalytic subunit mRNA, we determined that calcineurin/nuclear factor of activated T-cells signaling functions downstream of the AT1 receptor to induce sFlt-1 synthesis and secretion by AT1-receptor activating autoantibodies. AT1-receptor activating autoantibody–induced sFlt-1 secretion resulted in inhibition of endothelial cell migration and capillary tube formation in vitro. Overall, our studies demonstrate that an autoantibody from women with preeclampsia induces sFlt-1 production via angiotensin receptor activation and downstream calcineurin/nuclear factor of activated T-cells signaling. These autoantibodies represent potentially important targets for diagnosis and therapeutic intervention.

CTCF modulates Estrogen Receptor function through specific chromatin and nuclear matrix interactions

Enhancer regions and transcription start sites of estrogen-target regulated genes are connected by means of Estrogen Receptor long-range chromatin interactions. Yet, the complete molecular mechanisms controlling the transcriptional output of engaged enhancers and subsequent activation of coding genes remain elusive. Here, we report that CTCF binding to enhancer RNAs is enriched when breast cancer cells are stimulated with estrogen. CTCF binding to enhancer regions results in modulation of estrogen-induced gene transcription by preventing Estrogen Receptor chromatin binding and by hindering the formation of additional enhancer-promoter ER looping. Furthermore, the depletion of CTCF facilitates the expression of target genes associated with cell division and increases the rate of breast cancer cell proliferation. We have also uncovered a genomic network connecting loci enriched in cell cycle regulator genes to nuclear lamina that mediates the CTCF function. The nuclear lamina and chromatin interactions are regulated by estrogen-ER. We have observed that the chromatin loops formed when cells are treated with estrogen establish contacts with the nuclear lamina. Once there, the portion of CTCF associated with the nuclear lamina interacts with enhancer regions, limiting the formation of ER loops and the induction of genes present in the loop. Collectively, our results reveal an important, unanticipated interplay between CTCF and nuclear lamina to control the transcription of ER target genes, which has great implications in the rate of growth of breast cancer cells.

Nuclear localization of NPR1 is required for activation of PR gene expression

Systemic acquired resistance (SAR) is a broad-spectrum resistance in plants that involves the upregulation of a battery of pathogenesis-related (PR) genes. NPR1 is a key regulator in the signal transduction pathway that leads to SAR. Mutations in NPR1 result in a failure to induce PR genes in systemic tissues and a heightened susceptibility to pathogen infection, whereas overexpression of the NPR1 protein leads to increased induction of the PR genes and enhanced disease resistance. We analyzed the subcellular localization of NPR1 to gain insight into the mechanism by which this protein regulates SAR. An NPR1–green fluorescent protein fusion protein, which functions the same as the endogenous NPR1 protein, was shown to accumulate in the nucleus in response to activators of SAR. To control the nuclear transport of NPR1, we made a fusion of NPR1 with the glucocorticoid receptor hormone binding domain. Using this steroid-inducible system, we clearly demonstrate that nuclear localization of NPR1 is essential for its activity in inducing PR genes.

Signal transduction mechanisms underlying growth hormone receptor action

Our understanding of the mechanisms of action of GH and its receptor, the GHR, has advanced significantly in the last decade and has provided some important surprises. It is now clear that the GH-GHR axis activates a number of inter-related signalling pathways, not all of which are dependent on the intracellular tyrosine kinase, JAK2 as originally postulated. JAK2-independent pathways, mediated via the Src family kinases, together with a number of negative regulators of GH signalling and emerging cross-talk mechanisms with other growth factor receptors, provide a complex array of mechanisms that are capable of fine-tuning responses to GH in a cell context dependent manner. Additionally, it is also now clear that GH and the GHR can translocate to the nucleus of target cells and initiate, as yet not well defined, nuclear responses. Continued emphasis on elucidation of these complex mechanisms is critical to provide further insights into the diverse physiological and pathophysiological effects of GH.