Thyroid Hormone Receptor Isoform-specific Modification by Small Ubiquitin-like Modifier (SUMO) Modulates Thyroid Hormone-dependent Gene Regulation

Thyroid hormone receptor (TR) α and β mediate thyroid hormone action at target tissues. TR isoforms have specific roles in development and in adult tissues. The mechanisms underlying TR isoform-specific action, however, are not well understood. We demonstrate that posttranslational modification of TR by conjugation of small SUMO to TRα and TRβ plays an important role in triiodothyronine (T3) action and TR isoform specificity. TRα was sumoylated at lysines 283 and 389, and TRβ at lysines 50, 146, and 443. Sumoylation of TRβ was ligand-dependent, and sumoylation of TRα was ligand-independent. TRα-SUMO conjugation utilized the E3 ligase PIASxβ and TRβ-SUMO conjugation utilized predominantly PIAS1. SUMO1 and SUMO3 conjugation to TR was important for T3-dependent gene regulation, as demonstrated in transient transfection assay and studies of endogenous gene regulation. The functional role of SUMO1 and SUMO3 in T3 induction in transient expression assays was closely matched to the pattern of TR and cofactor recruitment to thyroid hormone response elements (TREs) as determined by ChIP assays. SUMO1 was required for the T3-induced recruitment of the co-activator CREB-binding protein (CBP) and release of nuclear receptor co-repressor (NCoR) on a TRE but had no significant effect on TR DNA binding. SUMO1 was required for T3-mediated recruitment of NCoR and release of CBP from the TSHβ-negative TRE. SUMO3 was required for T3-stimulated TR binding to the TSHβ-negative TRE and recruitment of NCoR. These findings demonstrate that conjugation of SUMO to TR has a TR-isoform preference and is important for T3-dependent gene induction and repression.     

Group X Secretory Phospholipase A2 Regulates the Expression of Steroidogenic Acute Regulatory Protein (StAR) in Mouse Adrenal Glands

We developed C57BL/6 mice with targeted deletion of group X secretory phospholipase A₂ (GX KO). These mice have ∼80% higher plasma corticosterone concentrations compared with wild-type (WT) mice under both basal and adrenocorticotropic hormone (ACTH)-induced stress conditions. This increased corticosterone level was not associated with increased circulating ACTH or a defect in the hypothalamic-pituitary axis as evidenced by a normal response to dexamethasone challenge. Primary cultures of adrenal cells from GX KO mice exhibited significantly increased corticosteroid secretion compared with WT cells. Conversely, overexpression of GX secretory phospholipase A₂ (sPLA₂), but not a catalytically inactive mutant form of GX sPLA₂, significantly reduced steroid production 30–40% in Y1 mouse adrenal cell line. This effect was reversed by the sPLA₂ inhibitor, indoxam. Silencing of endogenous M-type receptor expression did not restore steroid production in GX sPLA₂-overexpressing Y1 cells, ruling out a role for this sPLA₂ receptor in this regulatory process. Expression of steroidogenic acute regulatory protein (StAR), the rate-limiting protein in corticosteroid production, was ∼2-fold higher in adrenal glands of GX KO mice compared with WT mice, whereas StAR expression was suppressed in Y1 cells overexpressing GX sPLA₂. Results from StAR-promoter luciferase reporter gene assays indicated that GX sPLA₂ antagonizes StAR promoter activity and liver X receptor-mediated StAR promoter activation. In summary, GX sPLA₂ is expressed in mouse adrenal glands and functions to negatively regulate corticosteroid synthesis, most likely by negatively regulating StAR expression.     

Nuclear receptor interactions in methotrexate induction of human dehydroepiandrosterone sulfotransferase (hSULT2A1)

Cytosolic sulfotransferases (SULTs) are a major family of phase II drug-metabolizing enzymes. SULT-catalyzed sulfonation regulates hormone activities metabolizes drugs detoxifies xenobiotic toxicants bioactivates carcinogens. Human dehydroepiandrosterone sulfotransferase (hSULT2A1 DHEA-ST) plays a very important role in sulfating endogenous hydroxysteroids and exogenousxenobiotics. Our recent studies have shown that methotrexate can induce hSULT2A1 expression. To investigate the molecular mechanism involved in hSULT2A1 induction we generated the promoter sequence of hSULT2A1 by PCR and constructed a reporter gene vector. Both reporter gene assay and endogenous induction results suggested that human constitutive active receptor (hCAR) mediates the methotrexate induction of hSULT2A1 in both Caco-2 and Hep G2 cells. Human vitamin D receptor (hVDR) also upregulated hSULT2A1 gene expression while human pregnane X receptor (hPXR) downregulated it. Human pregnane X receptor suppressed hCAR-mediated methotrexate induction of hSULT2A1 in both Caco-2 and Hep G2 cells. hVDR competed with hCAR for the hSULT2A1 promoter in Caco-2 cells. hCAR inhibited hVDR-mediated vitamin D3 induction of hSULT2A1 but not methotrexate induction of hSULT2A1. These results strongly support the hypothesis that cross-talk occurs among nuclear receptors in the signal transduction pathway of hSULT2A1 and that interactions among nuclear receptors also depend on ligands (inducers) in the system.     

Discovery of the Selective Androgen Receptor Modulator MK-0773 Using a Rational Development Strategy Based on Differential Transcriptional Requirements for Androgenic Anabolism Versus Reproductive Physiology

Selective androgen receptor modulators (SARMs) are androgen receptor (AR) ligands that induce anabolism while having reduced effects in reproductive tissues. In various experimental contexts SARMs fully activate, partially activate, or even antagonize the AR, but how these complex activities translate into tissue selectivity is not known. Here, we probed receptor function using >1000 synthetic AR ligands. These compounds produced a spectrum of activities in each assay ranging from 0 to 100% of maximal response. By testing different classes of compounds in ovariectomized rats, we established that ligands that transactivated a model promoter 40–80% of an agonist, recruited the coactivator GRIP-1 <15%, and stabilized the N-/C-terminal interdomain interaction <7% induced bone formation with reduced effects in the uterus and in sebaceous glands. Using these criteria, multiple SARMs were synthesized including MK-0773, a 4-aza-steroid that exhibited tissue selectivity in humans. Thus, AR activated to moderate levels due to reduced cofactor recruitment, and N-/C-terminal interactions produce a fully anabolic response, whereas more complete receptor activation is required for reproductive effects. This bimodal activation provides a molecular basis for the development of SARMs.     

GPRC6A mediates the non-genomic effects of steroids

The identity of the putative G-protein coupled receptor (GPCR) that mediates the non-genomic effects of androgens is unknown. We present in vitro and in vivo evidence that the orphan GPRC6A receptor, a widely expressed calcium and amino acid sensing GPCR, transduces the non-genomic effects of testosterone and other steroids. Overexpression of GPRC6A imparts the ability of extracellular testosterone to illicit a rapid, non-genomic signaling response in HEK-293 cells lacking the androgen receptor. Conversely, testosterone-stimulated rapid signaling and phosphorylation of ERK is attenuated in bone marrow stromal cells derived from GPRC6A(-/-) mice and in 22Rv1 prostate cancer cells after siRNA-mediated knockdown of GPRC6A. Compared with wild-type controls, GPRC6A(-/-) null mice exhibit significantly less ERK activation and Egr-1 expression in both bone marrow and testis in response to pharmacological doses of testosterone in vivo. In addition, testosterone administration results in suppression of luteinizing hormone in wild-type male mice, but paradoxically stimulates serum luteinizing hormone levels in GPRC6A(-/-) null mice. These results suggest that GPRC6A is functionally important in regulating non-genomic effects of androgens in multiple tissues.