Regulation Of Hormone Receptors Research Articles

Acquisition of luteolytic capacity: changes in prostaglandin F2alpha regulation of steroid hormone receptors and estradiol biosynthesis in pig corpora lutea.

The pig corpora lutea (CL) acquires luteolytic capacity at about Day 13 of the estrous cycle, after which luteolysis occurs in response to prostaglandin F2alpha (PGF2alpha) treatment. We postulated that differences in transcription factors such as the steroid hormone receptors may be responsible for the differences in PGF2alpha-induced gene expression after acquisition of luteolytic capacity. In these studies, we evaluated the effect of PGF2alpha on luteal expression of receptors for progesterone (nuclear and membrane progesterone receptor [PR]), estradiol (ERalpha and ERbeta), glucocorticoid, androgens, and two enzymes in estradiol synthesis (P450-17alpha and aromatase). Two experiments were conducted to examine the early (0.5 h, experiment I) and late (10 h, experiment II) effects of PGF(2alpha) on the expression of these receptors in CL with (Day 17) or without (Day 9) luteolytic capacity. PGF2alpha decreased ERalpha mRNA (35%) and increased ERbeta mRNA (558%) and protein (376%) only in Day 17 CL and not Day 9. The estradiol biosynthetic pathway was upregulated by PGF2alpha in Day 17 CL but not Day 9 CL, with a dramatic increase in aromatase mRNA and luteal estradiol content. Nuclear PR was not affected by PGF2alpha, but was greater (176%) in Day 9 CL, while a putative membrane PR was greater (156%) in Day 17 than Day 9 CL. There were no detectable changes in glucocorticoid or androgen receptor mRNA. Thus, luteolytic capacity is associated with upregulation of estradiol biosynthesis, which in conjunction with increased ERbeta expression and altered PR expression may promote luteolysis in the pig CL.

Role of protein kinase A, phospholipase C and phospholipase D in parathyroid hormone receptor regulation of protein kinase Cα and interleukin-6 in UMR-106 osteoblastic cells

Parathyroid hormone (PTH) stimulates both bone formation and resorption by activating diverse osteoblast signalling pathways. Upstream signalling for PTH stimulation of protein kinase C-α (PKCα) membrane translocation and subsequent expression of the pro-resorptive cytokine interleukin-6 (IL-6) was investigated in UMR-106 osteoblastic cells. PTH 1–34, PTH 3–34, PTHrP and PTH 1–31 stimulated PKCα translocation and IL-6 promoter activity. Pharmacologic intervention at the adenylyl cyclase (AC) pathway (forskolin, IBMX, PKI) failed to alter PTH 1–34- or PTH 3–34-stimulated PKCα translocation. The phosphoinositol–phospholipase C (PI–PLC) antagonist U73122 slightly decreased PTH 1–34-stimulated PKCα translocation; however, the control analogue U73343 acted similarly. Propranolol, an inhibitor of phosphatidic acid (PA) phosphohydrolase, decreased diacylglycerol (DAG) formation and attenuated PTH 1–34- and PTH 3–34-stimulated PKCα translocation and IL-6 promoter activity, suggesting a phospholipase D (PLD)-dependent mechanism. This is the first demonstration that PLD-mediated signalling leads to both PKC-α translocation and IL-6 promoter activation in osteoblastic cells.

Emerging concepts in the regulation of female sexual behavior.

Recent advances in the field of steroid hormone action have significantly advanced our understanding of neuroendocrine regulation of sexual behavior in female rodents. The presumed classical steroid receptor-steroid hormone functional relationship has undergone significant modifications to integrate the wider context of cellular signaling mechanisms initiated by sensory and environmental stimuli and their transcriptional regulation of steroid hormone receptors in reproductive behavior. This effort has greatly been aided by recent studies identifying steroid hormone receptors as transcriptional mediators of a variety of ligands, whose functional flexibility is dependent upon their recruitment of coregulators, and the availability of gene knockout animal models. This review provides a framework for current concepts in the field of steroid hormone action in the context of their regulation of female sexual behavior.

Essential Role of the Unusual DNA-binding Motif of BAG-1 for Inhibition of the Glucocorticoid Receptor

The co-chaperone BAG-1 is involved in the regulation of steroid hormone receptors, including the glucocorticoid receptor (GR). More recently, BAG-1 was found in the nucleus where it decreases GR transactivation. Moreover, nonspecific DNA binding of BAG-1 has been reported. We discovered that of the N-terminal part of BAG-1M, the first 8 amino acids are sufficient for DNA binding, containing a stretch of three lysines and a stretch of three arginines. Changing the spacing between these stretches had no effect on DNA binding. Surprisingly, this small, nonsequence-specific DNA binding domain was nonetheless necessary for the inhibitory function of BAG-1 for GR-dependent transcription, whereas the following serine- and threonine-rich E(2)X(4) repeat domain was not. Mutational analysis of these two domains revealed that only mutants retaining DNA binding capability were able to down-regulate GR-mediated transactivation. Intriguingly, lack of DNA binding could not be functionally rescued by BAG-1M harboring a point mutation abolishing interaction with hsp70. Thus, DNA binding and hsp70 interaction are required in cis. We propose that the nonsequence-specific DNA-binding protein BAG-1 acts at specific chromosomal loci by interacting with other proteins.

An in vitro long-term culture model for normal human mammary gland: expression and regulation of steroid receptors.

Steroids and their nuclear receptors play crucial roles in the development and maintenance of normal functions of the human mammary gland (HMG). They have also been implicated in breast carcinogenesis. However, the study of steroid action in normal HMG has been hampered by experimental difficulties. By using a newly established in vitro long-term culture method, we successfully cultured normal HMG tissue for more than 2 months without detriment to its morphology or steroid receptor expression. Expression of the cellular structural and extracellular matrix proteins was similar to that prior to culture, and HMG tissue retained its properties of steroid receptor expression and regulation. Addition of 17-beta estrogen to mammary tissues markedly increased the expression of progesterone receptor (PR) but only slightly affected that of the estrogen receptor (ER). Medroxyprogesterone acetate down-regulated the expression of PR within 24-48 h and also increased the expression of androgen receptor. When HMG tissue was cultured in medium containing normal or dextran-coated charcoal-stripped fetal calf serum or normal human serum, the expression and regulation of steroid hormone receptors were similar, although different in extent. When serum was omitted, the morphology of HMG was normal after 1 week, but the expression and regulation of ER and PR were altered. Thus, as HMGs retain the capacity to express steroid receptors in culture, this long-term culture system is probably a good model for studying the regulation of the mammary gland by steroids.

A Gα s mutation (D 229S) differentially effects gonadotropin-releasing hormone receptor regulation by RGS10, RGS3 and RGS3T

Regulators of G protein signaling (RGS) act as GTPase-activating proteins for Gα i and for Gα q/11. There is recent evidence for interaction of RGS proteins with Gα s, and substitution of Ser for Asp 229 in RGS proteins enhances interactions with G proteins. Site-directed mutagenesis of Asp 229 was used to assess the effect of this site on the gonadotropin-releasing hormone receptor (GnRHR)-Gα s mediated signaling in the absence or presence of over-expressed RGS3, RGS10 or a truncated form of RGS3 (RGS3T). We observed increased cAMP release with the mutant Gα s(D 229S) compared to wt Gα s when GGH 3 cells (GH 3 cells stably expressing the GnRH receptor) were stimulated with a GnRH agonist. In the presence of RGS3, we did not observe any difference in cAMP release with wt Gα s or with Gα s(D 229S) compared to control values; in the presence of RGS3T there was an increase of cAMP release with wt Gα s compared to the control but there was no difference between the Gα s(D 229S) and the control values. When cells co-expressed wt Gα s and RGS10, there was a significant increase of cAMP release compared with cells co-expressing wt Gα s and Lac Z. Cells co-expressing Gα s(D 229S) and RGS10 showed a significant increase of cAMP release compared to control cells. These results indicate differential regulation of the GnRHR-Gα s mediated signaling by a single mutation in Gα s in the presence of RGS10 and RGS3T, but not with RGS3. This is the first report of an effect of the Gα s(D 229S) mutation on G protein-coupled receptor-mediated activation.

31] Ligand-inducible transgene regulation for gene therapy

A synthetic ligand regulable system for gene transfer and expression has been developed in our laboratory based on mechanistic studies of steriod hormone receptor and transcriptional regulation. This gene switch system possesses most of the important features that are required for application of the system in biological research and clinical gene therapy in the future. As the primary ligand tested in this system, mifepristone can effectively turn on the regulatory circuit at doses much lower than those used in the clinic. By modification of the chimeric regulator and its feedback regulatory mode, this system has been optimized to produce very low basal activity with high inducibility in the presence of mifepristone. Also, improvements in regulator composition have been made to minimize immunogenicity and make the system more amenable to human gene therapy. Moreover, incorporation of this gene switch system into the HC-Ad vector system has further enhanced the efficiency of gene transfer and the long-term inducible expression of transgenes. However, for each application within a different biological system, the gene switch needs to be optimized to achieve appropriate inductions. In particular, the method used to deliver the transgenes and adjustment of ligand dosage are critical for in vivo gene expression.

Regulation of pituitary corticotropin releasing hormone receptors

Corticotropin releasing hormone (CRH) stimulates pituitary ACTH secretion through type-1 CRH (CRH1) receptors. Stimulation of the hypothalamic pituitary adrenal (HPA) axis as well as increased corticotroph responsiveness during stress and adrenalectomy are associated with marked pituitary CRH binding downregulation. The presence of CRH1 receptors in the pituitary are essential to maintain ACTH secretion. Downregulation of CRH binding is associated with normal or elevated levels of CRH1 receptor mRNA and this may contribute to the maintainence of permissive levels of CRH1 receptors in the pituitary. Injection of either CRH or glucocorticoids in rats in vivo induces CRH binding and CRH1 receptor mRNA downregulation, whereas their simultaneous administration causes only transient CRH1 receptor mRNA loss. Vasopressin increases CRH1 receptor mRNA levels. This suggest that interactions between CRH, vasopressin and glucocorticoids accounts for CRH1 receptor mRNA upregulation during stress. The lack of correlation between CRH binding and CRH1 receptor mRNA indicates that the major sites for pituitary CRH1 receptor regulation are at the post-transcriptional level.

Differential expression of thyroid hormone receptor isoforms by thyroid hormone and lithium in rat GH3 and B103 cells

Background: The interaction between lithium, a mood stabilizer, and the thyroid axis has been extensively studied; however, the regulation of thyroid hormone receptors by lithium is yet to be investigated.Methods: To test whether lithium affects thyroid hormones at the receptor level, we examined the effects of lithium in combination with triiodothyronine (T3) on gene expression of thyroid hormone receptor isoforms in GH3 and B103 cells.Results: The pattern of expression as well as the magnitude of regulation of the different thyroid hormone receptor isoforms appeared to be cell line specific. Whereas T3 regulated all four isoforms in GH3 cells at both time points, T3 did not alter thyroid hormone receptor TRα1 and TRα2 mRNA in B103 cells. Addition of lithium to thyroid hormone-deficient GH3 cells decreased TRα1, α2, and β2 expression without affecting TRβ1 expression at 2 but not 5 days. Addition of lithium to T3-treated GH3 cells did not further modulate gene expression of TRα1, α2, β1, or β2 when compared to cells treated with T3 alone. The effects of lithium in B103 cells appeared to be isoform specific as well as time dependent, since TRα1 expression was selectively decreased in B103 cells, when treated with T3 in the presence of lithium.Conclusions: The present study provides direct evidence that T3 and/or lithium regulate TR gene expression in vitro in a both time-dependent and cell line-specific manner.

Immunohistologic Analysis of Estrogen Receptor Expression in Breast Carcinoma Precursor Lesions

: The growth of invasive breast carcinoma is facilitated by abnormal expression of estrogen receptor (ER), however, the status of ER during disease histogenesis is poorly defined. The extent of ER immunostaining (expressed as percentage of positive cells) was semiquantitated in the invasive (ICA) and corresponding in situ (CIS) components of 50 formalin-fixed, paraffin-embedded breast carcinomas and then compared to staining in normal terminal duct lobular units (TDLU) or hyperplastic lesions (if present) in the same tissue sections. Morphologically normal TDLUs from most cases (61%) exhibited ER staining in 11–30% of epithelial cells; only 10% of the cases had TDLUs demonstrating immunoreactivity in more than 50% of cells. In contrast, 80% of the hyperplastic lesions showed ER staining in more than 30% of epithelial cells, with 44% demonstrating immuoreactivity in more than 50% of epithelial cell nuclei (p = 0.01, chi-square test). Although most in situ carcinomas showed even more extensive ER staining than hyperplasias (>70% of cells were positive in 50% of cases), a significant subset (28%) of CIS exhibited staining in less than 10% of cells (p = 0.002, chi-square test). Invasive carcinomas were less often extensively immunoreactive (i.e., >50% of cells positive) than CIS (32% versus 58% for CIS) and there were five cases (10%) in which the CIS component was ER positive but the invasive component was ER negative. Finally, ER-positive carcinomas were significantly more often accompained by extensive CIS and/or proliferative breast disease (p = 0.007 and 0.003, respectively). The divergent levels of ER expression observed between premalignant, preinvasive, and invasive lesions suggests that inappropriate regulation of hormone receptors may be a factor that promotes early growth or progression in breast neoplasia, and ER-positive breast carcinomas are characterized by a different, possibly lengthier, histogenesis than ER-negative malignancies with a greater frequency of background hyperplastic lesions and a larger preinvasive component.

Transduction pathways involved in rapid hormone receptor regulation in the mammary epithelium.

Previous studies have shown that the envelope protein of the mouse mammary tumor virus (MMTV) rapidly upregulates prolactin (PRL) receptors by shifting them from internal pools to the cell surface and downregulates epidermal growth factor (EGF) receptors by inducing their internalization and degradation. This study shows that the effect on PRL receptors is mediated by the nitric oxide (NO)/cGMP pathway, since it can be mimicked by an NO donor or 8-bromo-cGMP and can be blocked by an NO synthase inhibitor. In contrast, the effect on EGF receptors is mediated by tyrosine phosphorylation and phosphatidylinositol 3-kinase (PI3K), since it can be blocked by either a tyrosine kinase inhibitor or by a PI3K inhibitor. Both of these pathways can be activated by a calcium ionophore and inhibited by calcium chelation. Therefore, it appears that the mouse mammary tumor virus envelope protein, like other retroviral envelope proteins, initially elevates cytoplasmic calcium, which can then stimulate both the NO/cGMP and the tyrosine phosphorylation/PI3K pathways, leading to PRL receptor upregulation and EGF receptor downregulation, respectively.

Growth hormone (GH) receptors, binding proteins and IGF-I concentrations in the serum of transgenic mice expressing bovine GH agonist or antagonist.

We have examined the regulation of hepatic growth hormone receptors (GH-R) and serum GH binding proteins (GHBP) in transgenic mice expressing an antagonist of bovine growth hormone (bGH), G119K-bGH, and consequently exhibiting a growth suppressed dwarf phenotype. Specific GHBP could be measured in transgenic dwarf mouse serum only by immunological methods (RIA), because these mice have a very high concentration of mutated bGH in circulation (> 1 microgram/ml) and, therefore, almost all GHBP is bound to G119K-bGH and cannot be quantitated in binding assays. The concentrations of GHBP were 0.6 +/- 0.4 nM and 1.7 +/- 0.4 nM for normal and dwarf mice respectively. The concentrations of free GHBP in normal mice and in transgenic mice expressing wild-type GH can be calculated using chromatographic techniques as the dissociation constant (Kd) and the ratio of bound 125I-GH to free 125I-GH in the serum ([GHBP]free = B/F.Kd). In agreement with the assumption that GHBP reflects GH-R status, liver uptake of injected labeled bGH was greatly reduced in transgenic dwarfs in comparison with normal mice or with transgenic mice expressing wild-type bGH (liver/blood ratio of 0.48 +/- 0.21, 2.7 +/- 0.2, and 1.3 +/- 0.3 respectively) indicating that the high concentration of the mutated bGH (G119K-bGH) prevents labeled bGH uptake, as was expected from the dwarf phenotype. 125I-bGH taken up by the liver of transgenic dwarf mice was found in a smaller molecular species than in normal mice, compatible with the presence of 1:1 [(GH-R):GH] complexes instead of the 2:1 [(GH-R)2:GH] or 2:2 [(GHBP)2:(GH)2] complexes found in normal mice. The concentration of IGF-I, the principal mediator of GH activity, in the G119K-bGH transgenic mice was correlated with the concentration of free GHBP. This allowed us to use free GHBP concentration as a marker of the effects of the active endogenous hormone (mGH) on liver receptors in the presence of different concentrations of the antagonist of GH. The levels of GHBP in serum, as well as the concentration of GH-R in liver microsomes from mice expressing the bGH antagonist, are up-regulated by the high concentration of G119K-bGH (85%), but significantly less so than that which could be expected for the same concentration of native GH (220-275%). This up-regulation suggests that the G119K-bGH antagonist is internalized and induces synthesis of the receptor and of the binding protein.

Regulation of GH-releasing hormone receptors.

Regulation of GH-releasing hormone receptors.

Differential in vivo regulation of steroid hormone receptor activation by Cdc37p.

The CDC37 gene is essential for the activity of p60(v-src) when expressed in yeast cells. Since the activation pathway for p60(v-src) and steroid hormone receptors is similar, the present study analyzed the hormone-dependent transactivation by androgen receptors and glucocorticoid receptors in yeast cells expressing a mutant version of the CDC37 gene. In this mutant, hormone-dependent transactivation by androgen receptors was defective at both permissive and restrictive temperatures, although transactivation by glucocorticoid receptors was mildly defective only at the restrictive temperature. Cdc37p appears to function via the androgen receptor ligand-binding domain, although it does not influence receptor hormone-binding affinity. Models for Cdc37p regulation of steroid hormone receptors are discussed.

Insulin and insulin-like growth factor-I acutely inhibit surface translocation of growth hormone receptors in osteoblasts: a novel mechanism of growth hormone receptor regulation.

We previously have demonstrated that insulin and insulin-like growth factor-I (IGF-I) down-regulate growth hormone (GH) binding in osteoblasts by reducing the number of surface GH receptors (GHRs). The present study was undertaken to investigate the mechanism of GHR down-regulation. Treatment with 5 nM insulin or IGF-I for 18 hr significantly decreased surface GH binding to 26.4 +/- 2.9% and 23.0 +/- 2.7% of control (mean +/- SE; P < 0.05), respectively. No corresponding reductions in the mRNA level and total cellular content of GHR were found, nor was the rate of receptor internalization affected. The effects on GHR translocation were assessed by measuring the reappearance of GH binding of whole cells after trypsinization to remove the surface receptors. GH binding of control cultures significantly increased (P < 0.05) over 2 hr after trypsinization, whereas no recovery of binding activity was detected in insulin and IGF-I-treated cultures, indicating that GHR translocation was impaired. Studies on the time course of GHR down-regulation revealed that surface GH binding was reduced significantly by 3-hr treatment (P </= 0.0005), whereas GHR translocation was completely abolished by 75-90 min with insulin and IGF-I. The inhibition of receptor translocation by insulin, but not IGF-I, was attenuated by wortmannin. In conclusion, insulin and IGF-I down-regulated GH binding in osteoblasts by acutely impairing GHR translocation, with their effects exerted through distinct postreceptor signaling pathways.

Post-secretory processing of peptide signals: a novel mechanism for the regulation of peptide hormone receptors.

Post-secretory processing of peptide signals: a novel mechanism for the regulation of peptide hormone receptors.

Differential regulation of hypothalamic pituitary corticotropin releasing hormone receptors during development of adjuvant-induced arthritis in the rat.

The expression of corticotropin releasing hormone (CRH) in the hypothalamic paraventricular nucleus (PVN) and CRH receptor mRNA in the PVN and anterior pituitary was studied during development of adjuvant-induced arthritis in Piebald-Viral-Glaxo rats, using in situ hybridization techniques. As previously shown with i.p. hypertonic saline injection, basal and immobilization stress-stimulated CRH mRNA levels in the PVN were significantly lower than in controls 14 days after adjuvant injection. However, 7 days after injection, preceding the onset of inflammation, the increase of CRH mRNA following immobilization was significantly higher than in control rats. In contrast to other chronic stress paradigms, inflammation stress failed to induce type-1 CRH receptor (CRH-R1) mRNA in the PVN, either at 7 days, or at 14 days after adjuvant injection, when inflammation is present. The ability of acute immobilization to induce CRH-R1 mRNA in the PVN was not affected 14 days after adjuvant injection but parallel to the CRH peptide mRNA response it was markedly potentiated at 7 days. Pro-opiomelanocorpin (POMC) mRNA levels in the anterior pituitary increased significantly 14 days after adjuvant injection, and they were unaffected by 1 h immobilization. While CRH binding in the pituitary decreased significantly 14 days after adjuvant injection, CRH-R1 mRNA was unchanged. This study shows biphasic hypothalamic responses to acute stress during development of adjuvant-induced arthritis, with enhanced CRH peptide and CRH-R1 mRNAs responses at 7 days, preceding the onset of inflammation, and blunted CRH mRNA responses at 14 days at the height of the inflammatory response. The lack of CRH receptor expression in the PVN in this model of chronic inflammation stress associated to low hypothalamic CRH peptide levels supports the view that positive feedback regulation by CRH is necessary to maintain enhanced CRH expression during chronic stress.

Tissue-specific Stabilization of the Thyroid Hormone β1 Nuclear Receptor by Phosphorylation

The present study evaluated the expression and regulation of endogenous thyroid hormone receptors (TRs) in cultured cells. In COS-1 cells, the endogenous TR, subtype beta1 (TRbeta1), but not subtype beta2 or alpha1, was induced to express by okadaic acid (OA) in a concentration-dependent manner. The induced TRbeta1 had immunoreactivity and partial V8 proteolytic maps similar to those of the transfected and in vitro translated human TRbeta1 (h-TRbeta1). The OA-induced expression of endogenous TRbeta1 was, however, not observed in a variety of other cultured cell lines tested, indicating that the induction was cell type-dependent. TRbeta1 induced by OA was a multisite phosphorylated protein, in which serine and threonine in a ratio of 10:1 were phosphorylated. The induced TRbeta1 was functional as it could mediate the thyroid hormone-dependent transcriptional activity via several thyroid hormone response elements. The induction of endogenous TRbeta1 expression by OA was not accompanied by an increase in mRNA levels but was the result of an increase in the stability of the TRbeta1 protein. This is the first report to indicate that one of the mechanisms by which the TR isoforms are differentially expressed is via the tissue-specific stabilization of the TR isoform proteins. Furthermore, this selective stability of TRbeta1 could be conferred by phosphorylation.

Hormone Receptor Regulation of the Human Immunodeficiency Virus Type 1 and Type 2 Long Terminal Repeats.

Both host cell and viral transcription factors regulate the long terminal repeat (LTR) of human immuno-deficiency virus (HIV) activity and viral replication. Using transient transfection, ligand-activated thyroid hormone and 9-cis-retinoic acid receptors (T(3)R and RXR) were found to stimulate HIV-1 and HIV-2 LTR activities. They also stimulated HIV-1 viral production. Drosophila SL2 cells that lack Sp1 and T(3)R were used to study HIV-1 and HIV-2 LTR activities. Both activities were stimulated by cotransfection of SP1 (120- and 180-fold, respectively); HIV LTR activities were also stimulated approximately 5-fold by ligand-activated T(3)R, approximately 10-fold by ligand-activated RXR and 20- to 30-fold by both receptors and their cognate ligands. T(3)R.RXR heterodimers bound to NF-kappaB and Sp1 response elements in both HIV LTRs having highest affinity for the HIV-1 NF-kappaB region. When U937 monocytic cells were cotransfected with HIV-1 viral DNA and T(3)R, RXR and retinoic acid receptor (RAR) expression plasmids, hormonal treatment increased viral replication up to 5-fold. Hormonal signals thus have the potential to regulate HIV transcription and viral production. Copyright 1996 S. Karger AG, Basel

Regulation and expression of hormone receptors

Regulation and expression of hormone receptors