Ayano Murai-Takeda

Coactivation of the N-terminal Transactivation of Mineralocorticoid Receptor by Ubc9

Molecular mechanisms underlying mineralocorticoid receptor (MR)-mediated gene expression are not fully understood. Various transcription factors are post-translationally modified by small ubiquitin-related modifier-1 (SUMO-1). We investigated the role of the SUMO-1-conjugating enzyme Ubc9 in MR transactivation. Yeast two-hybrid, GST-pulldown, and coimmunoprecipitation assays showed that Ubc9 interacted with N-terminal MR-(1-670). Endogenous Ubc9 is associated with stably expressing MR in 293-MR cells. Transient transfection assays in COS-1 cells showed that Ubc9 increased MR transactivation of reporter constructs containing MRE, ENaC, or MMTV promoter in a hormone-sensitive manner. Moreover, reduction of Ubc9 protein levels by small interfering RNA attenuated hormonal activation of a reporter construct as well as an endogenous target gene by MR. A sumoylation-inactive mutant Ubc9(C93S) similarly interacted with MR and potentiated aldosterone-dependent MR transactivation. An MR mutant in which four lysine residues within sumoylation motifs were mutated into arginine (K89R/K399R/K494R/K953R) failed to be sumoylated, but Ubc9 similarly enhanced transactivation by the mutant MR, indicating that sumoylation activity is dispensable for coactivation capacity of Ubc9. Coexpression of Ubc9 and steroid receptor coactivator-1 (SRC-1) synergistically enhanced MR-mediated transactivation in transient transfection assays. Indeed, chromatin immunoprecipitation assays demonstrated that endogenous MR, Ubc9, and SRC-1 were recruited to an endogenous ENaC gene promoter in a largely aldosterone-dependent manner. Coimmunoprecipitation assays showed a complex of MR, Ubc9, and SRC-1 in mammalian cells, and the endogenous proteins were colocalized in the nuclei of the mouse collecting duct cells. These findings support a physiological role of Ubc9 as a transcriptional MR coactivator, beyond the known SUMO E2-conjugating enzyme.

NF-YC Functions as a Corepressor of Agonist-bound Mineralocorticoid Receptor

The role of aldosterone has been implicated in the metabolic syndrome and cardiovascular diseases. The biological actions of aldosterone are mediated through mineralocorticoid receptor (MR). Nuclear receptor-mediated gene expression is regulated by dynamic and coordinated recruitment of coactivators and corepressors. To identify new coregulators of the MR, full-length MR was used as bait in yeast two-hybrid screening. We isolated NF-YC, one of the subunits of heterotrimeric transcription factor NF-Y. Specific interaction between MR and NF-YC was confirmed by yeast two-hybrid, mammalian two-hybrid, coimmunoprecipitation assays, and fluorescence subcellular imaging. Transient transfection experiments in COS-7 cells demonstrated that NF-YC repressed MR transactivation in a hormone-sensitive manner. Moreover, reduction of NF-YC protein levels by small interfering RNA potentiated hormonal activation of endogenous target genes in stably MR-expressing cells, indicating that NF-YC functions as an agonist-dependent MR corepressor. The corepressor function of NF-YC is selective for MR, because overexpression of NF-YC did not affect transcriptional activity mediated by androgen, progesterone, or glucocorticoid receptors. Chromatin immunoprecipitation experiments showed that endogenous MR and steroid receptor coactivator-1 were recruited to an endogenous ENaC gene promoter in a largely aldosterone-dependent manner, and endogenous NF-YC was sequentially recruited to the same element. Immunohistochemistry showed that endogenous MR and NF-YC were colocalized within the mouse kidney. Although aldosterone induces interaction of the N and C termini of MR, NF-YC inhibited the N/C interaction. These findings indicate that NF-YC functions as a new corepressor of agonist-bound MR via alteration of aldosterone-induced MR conformation.

Coactivation of SF-1-Mediated Transcription of Steroidogenic Enzymes by Ubc9 and PIAS1

Steroidogenic factor-1 (SF-1) is a nuclear orphan receptor, which is essential for adrenal development and regulation of steroidogenic enzyme expression. SF-1 is posttranslationally modified by small ubiquitin-related modifier-1 (SUMO-1), thus mostly resulting in attenuation of transcription. We investigated the role of sumoylation enzymes, Ubc9 and protein inhibitors of activated STAT1 (PIAS1), in SF-1-mediated transcription of steroidogenic enzyme genes in the adrenal cortex. Coimmunoprecipitation assays showed that both Ubc9 and PIAS1 interacted with SF-1. Transient transfection assays in adrenocortical H295R cells showed Ubc9 and PIAS1 potentiated SF-1-mediated transactivation of reporter constructs containing human CYP17, CYP11A1, and CYP11B1 but not CYP11B2 promoters. Reduction of endogenous Ubc9 and PIAS1 by introducing corresponding small interfering RNA significantly reduced endogenous CYP17, CYP11A1, and CYP11B1 mRNA levels, indicating that they normally function as coactivators of SF-1. Wild type and sumoylation-inactive mutants of Ubc9 and PIAS1 can similarly enhance the SF-1-mediated transactivation of the CYP17 gene, indicating that the coactivation potency of Ubc9 and PIAS1 is independent of sumoylation activity. Chromatin immunoprecipitation assays demonstrated that SF-1, Ubc9, and PIAS1 were recruited to an endogenous CYP17 gene promoter in the context of chromatin in vivo. Immunohistochemistry and Western blotting showed that SF-1, Ubc9, and PIAS1 were expressed in the nuclei of the human adrenal cortex. In cortisol-producing adenomas, the expression pattern of SF-1 and Ubc9 were markedly increased, whereas that of PIAS1 was decreased compared with adjacent normal adrenals. These results showed the physiological roles of Ubc9 and PIAS1 as SF-1 coactivators beyond sumoylation enzymes in adrenocortical steroidogenesis and suggested their possible pathophysiological roles in human cortisol-producing adenomas.

Gastrointestinal pseudo-obstruction after debulking surgery of malignant pheochromocytoma, improved by intravenous administration of α-adrenergic receptor blocker, phentolamine

Gastrointestinal pseudo-obstruction after debulking surgery of malignant pheochromocytoma, improved by intravenous administration of α-adrenergic receptor blocker, phentolamine

Intestinal Mineralocorticoid Receptor Contributes to Epithelial Sodium Channel–Mediated Intestinal Sodium Absorption and Blood Pressure Regulation

Background Mineralocorticoid receptor (MR) has pathological roles in various cell types, including renal tubule cells, myocytes, and smooth muscle cells; however, the role of MR in intestinal epithelial cells (IECs) has not been sufficiently evaluated. The intestine is the sensing organ of ingested sodium; accordingly, intestinal MR is expected to have essential roles in blood pressure (BP) regulation. Methods and Results We generated IEC‐specific MR knockout (IEC‐MR‐KO) mice. With a standard diet, fecal sodium excretion was 1.5‐fold higher in IEC‐MR‐KO mice, with markedly decreased colonic expression of β‐ and γ‐epithelial sodium channel, than in control mice. Urinary sodium excretion in IEC‐MR‐KO mice decreased by 30%, maintaining sodium balance; however, a low‐salt diet caused significant reductions in body weight and BP in IEC‐MR‐KO mice, and plasma aldosterone exhibited a compensatory increase. With a high‐salt diet, intestinal sodium absorption markedly increased to similar levels in both genotypes, without an elevation in BP. Deoxycorticosterone/salt treatment elevated BP and increased intestinal sodium absorption in both genotypes. Notably, the increase in BP was significantly smaller in IEC‐MR‐KO mice than in control mice. The addition of the MR antagonist spironolactone to deoxycorticosterone/salt treatment eliminated the differences in BP and intestinal sodium absorption between genotypes. Conclusions Intestinal MR regulates intestinal sodium absorption in the colon and contributes to BP regulation. These regulatory effects are associated with variation in epithelial sodium channel expression. These findings suggest that intestinal MR is a new target for studying the molecular mechanism of hypertension and cardiovascular diseases.

High Glucose Stimulates Mineralocorticoid Receptor Transcriptional Activity Through the Protein Kinase C β Signaling

Activation of mineralocorticoid receptor (MR) is shown in resistant hypertension including diabetes mellitus. Although protein kinase C (PKC) signaling is involved in the pathogenesis of diabetic complications, an association between PKC and MR is not known. Activation of PKCα and PKCβ by TPA (12-O-Tetradecanoylphorbol 13-acetate) increased MR proteins and its transcriptional activities in HEK293-MR cells. In contrast, a high glucose condition resulted in PKCβ but not PKCα activation, which is associated with elevation of MR protein levels and MR transcriptional activities. Reduction of endogenous PKCβ by siRNA decreased those levels. Interestingly, high glucose did not affect MR mRNA levels, but rather decreased ubiquitination of MR proteins. In db/db mice kidneys, levels of phosphorylated PKCβ2, MR and Sgk-1 proteins were elevated, and the administration of PKC inhibitor reversed these changes compared to db/+ mice. These data suggest that high glucose stimulates PKCβ signaling, which leads to MR stabilization and its transcriptional activities.

OS 25-01 MR SENSITIVITY IS ENHANCED IN HIGH FAT DIET MICE UNDER EQUALLY-ADJUSTED SALT INTAKE.

Objective: It has been reported that obesity-related high blood pressure was partly associated with increased aldosterone secretion and/or activity, but detailed mechanisms are not known. Our previous study suggested that different salt contents in each diet would affect the aldosterone levels. In this study, we investigated the change of aldosterone secretion and MR sensitivity of obesity mice by high fat diet under equally-adjusted salt intake. Design and Method: We prepared normal fat diet (NFD, 10% lard) and high fat diet (HFD, 60% lard) with equal amount of salt based on the amount of food intake of C57BL/6J mice. The 4-week-old male C57BL/6J mice were fed with NFD or HFD until sacrificed for analysis. Animals were monitored with various physiological parameters and measured plasma renin and aldosterone, urinary albumin, electrolytes, and aldosterone. Messenger RNA levels of steroidogenic enzyme genes in adrenal glands and MR target genes in kidneys were quantitated by real-time RT-PCR assay. Results: We confirmed that salt intake was equally-adjusted in two groups by measuring amount of food intake and urine sodium excretion. There was no significant difference in plasma aldosterone concentrations between HFD mice and NFD mice at any time point. Cyp11b2 expression was increased in 8-week HFD mice, however, not changed in 16-week and older HFD mice. Body weight and systolic BP were expectedly higher in HFD mice than in NFD mice. Sgk1 expression and urinary albumin were increased in 28-week and 42-week HFD mice compared with NFD mice. Conclusions: Sgk1 expression was increased in HFD mice though there was no difference in aldosterone secretion between NFD mice and HFD mice. Our salt-adjusted models in this study revealed that MR sensitivity is enhanced in HFD mice. Therefore they can be good models to study detailed mechanisms of enhanced MR sensitivity in obesity-related hypertension, which is a clinically observed hypertension well-controlled by MR antagonists.OBJECTIVE It has been reported that obesity-related high blood pressure was partly associated with increased aldosterone secretion and/or activity, but detailed mechanisms are not known. Our previous study suggested that different salt contents in each diet would affect the aldosterone levels. In this study, we investigated the change of aldosterone secretion and MR sensitivity of obesity mice by high fat diet under equally-adjusted salt intake. DESIGN AND METHOD We prepared normal fat diet (NFD, 10% lard) and high fat diet (HFD, 60% lard) with equal amount of salt based on the amount of food intake of C57BL/6J mice. The 4-week-old male C57BL/6J mice were fed with NFD or HFD until sacrificed for analysis. Animals were monitored with various physiological parameters and measured plasma renin and aldosterone, urinary albumin, electrolytes, and aldosterone. Messenger RNA levels of steroidogenic enzyme genes in adrenal glands and MR target genes in kidneys were quantitated by real-time RT-PCR assay. RESULTS We confirmed that salt intake was equally-adjusted in two groups by measuring amount of food intake and urine sodium excretion. There was no significant difference in plasma aldosterone concentrations between HFD mice and NFD mice at any time point. Cyp11b2 expression was increased in 8-week HFD mice, however, not changed in 16-week and older HFD mice. Body weight and systolic BP were expectedly higher in HFD mice than in NFD mice. Sgk1 expression and urinary albumin were increased in 28-week and 42-week HFD mice compared with NFD mice. CONCLUSIONS Sgk1 expression was increased in HFD mice though there was no difference in aldosterone secretion between NFD mice and HFD mice. Our salt-adjusted models in this study revealed that MR sensitivity is enhanced in HFD mice. Therefore they can be good models to study detailed mechanisms of enhanced MR sensitivity in obesity-related hypertension, which is a clinically observed hypertension well-controlled by MR antagonists.