Histopathological diagnosis of primary aldosteronism using CYP11B2 immunohistochemistry.

CONTEXT: Although primary aldosteronism (PA) is the most common cause of endocrine hypertension, histopathological methods to reveal the presence and sites of aldosterone overproduction remain to be established. OBJECTIVE: The objective of the study was to investigate the significance of immunohistochemical staining to detect CYP11B2 and CYP11B1 in adrenal tissue of patients with PA. DESIGN AND PATIENTS: Thirty-two patients with PA who underwent unilateral adrenalectomy were studied. Immunohistochemical staining was performed using anti-CYP11B2 and anti-CYP11B1 antibodies on paraffin-embedded sections. We analyzed the expression of each enzyme semiquantitatively by scoring staining intensity and correlating it with clinical findings. RESULTS: Twenty-two patients showed positive CYP11B2 immunostaining in their tumors (aldosterone producing adenoma, APA). Four patients with CYP11B2-negative unilateral adenomas and 4 patients without tumors on computed tomography showed aldosterone-producing cell clusters (APCCs) with CYP11B2 immunostaining in the zona glomerulosa (multiple APCCs). The remaining 2 patients had unilateral multiple adrenocortical micronodules and diffuse adrenocortical hyperplasia, respectively. In APA, CYP11B2 score adjusted for tumor volume was positively correlated with plasma aldosterone and negatively correlated with serum potassium. The APA group was divided into 3 subgroups based on relative CYP11B2 and CYP11B1 immunostaining levels. The CYP11B2/CYP11B1-equivalent and CYP11B1-dominant APA groups showed significantly higher serum cortisol after 1 mg dexamethasone and larger tumor size than the CYP11B2-dominant APA group. CONCLUSIONS: The present study clearly demonstrates that CYP11B2 immunostaining is a powerful tool for histopathological diagnosis of aldosterone overproduction in PA and for subtype classification of APA, multiple APCCs, unilateral multiple adrenocortical micronodules, and diffuse hyperplasia.

Identification of a novel human thyroid hormone receptor beta isoform as a transcriptional modulator.

Thyroid hormone exerts a pleiotropic effect on development and homeostasis. A novel thyroid hormone receptor beta isoform (hereafter referred to as TR beta 4) was cloned using PCR from a human pituitary cDNA library as a template. Analysis of the PCR products revealed a 137-bp insertion, which contains a stop codon in the middle, between the 5th and 6th exons that encode the ligand-binding domain of TR beta. The corresponding sequence of this insertion exists within the 5th intron of the human TR beta gene and consensus splice sequences were found at the junction sites. RACE analysis revealed that TR beta 4 is a carboxyl-terminal splicing variant of TR beta 1. RT-PCR and Northern blot analyses indicate that TR beta 4 mRNA is expressed in various human tissues, and especially abundant in testis and skeletal muscle. The TR beta 4 protein was unable to bind thyroid hormone (T3) and transient transfection assays demonstrate that TR beta 4 construct does not mediate T3-dependent gene regulation. TR beta 4 weakly but significantly inhibited transcription mediated by functional TR. Thus, this novel isoform may modulate hormone action as an endogenous antagonist in the tissue or cellular context.

The retinoid X receptor binding to the thyroid hormone receptor: relationship with cofactor binding and transcriptional activity.

Transcriptional regulation is mediated by thyroid hormone (tri-iodothyronine, T(3)) receptors (TR), which bind to T(3) response elements as heterodimers with retinoid X receptors (RXR). TR binds to corepressor proteins (CoR) in the absence of T(3), which mediate transcriptional repression and to coactivator proteins (CoA) in the presence of T(3), which mediate transcriptional stimulation, by recruiting additional proteins to the promoter. To determine the relationship between TR functions and cofactor bindings, we selected 13 single-point mutants on the ligand binding domain of TR, of which T(3) bindings were well preserved and created VP16 chimeric receptors. Using mammalian two-hybrid assays, RXR binding in the absence of T(3) was almost abolished for Y406K (helix; H10) and L422R (H11), while it was preserved for most other TR mutants. RXR binding was increased for I280K, V284R (H3), and C309K (H6). Addition of T(3) enhanced RXR binding and T(3) restored the RXR binding to Y406K but not to L422R. CoR binding was reduced for P214R, W219K (H1), R316H (H6), D366R (H9), and M423A (H11) in addition to the mutants of which RXR binding was affected, and CoA binding was impaired for I280K, V284R (H3), C309K (H6), and E457K (H12), indicating that sites for CoR, CoA, and RXR binding partially overlap. CoR binding was well correlated with T(3)-independent transcriptional regulation and CoA binding was well correlated with T(3)-dependent regulation, while RXR binding was not correlated with any of these functions among TR mutants, suggesting that transcriptional regulation by TR is mainly mediated by an exchange of CoRs and CoAs.

A selective peroxisome proliferator-activated receptor-gamma modulator, telmisartan, binds to the receptor in a different fashion from thiazolidinediones.

Angiotensin type 1 receptor blockers are widely used for the treatment of hypertension, and one angiotensin type 1 receptor blocker, telmisartan, specifically activates the peroxisome proliferator-activated receptor (PPAR)-gamma. We studied the impact of PPARgamma mutants on transcriptional control and interaction with cofactors to elucidate differences in the molecular mechanism between telmisartan and other PPARgamma agonists, thiazolidinediones (TZDs). We created several amino acid substitutions in the ligand binding domain of PPARgamma that, based on molecular modeling, may affect the binding of these agents. In transient expression experiments, wild-type PPARgamma-mediated transcription stimulated by telmisartan was more than one third of that stimulated by TZDs. The activation stimulated by TZDs was impaired, whereas activation stimulated by telmisartan was retained, in the H323Y, S342A, and H449A mutants. In the Y473A mutant, the TZD-induced activation was further impaired and lower than that of telmisartan-induced activation. Coexpression of coactivators enhanced the activation by both telmisartan and TZDs, but activation by telmisartan always exceeded that of TZDs in the Y473A mutant. Based on a mammalian two-hybrid assay, the interaction with corepressors was retained in Y473A. Telmisartan and TZDs, but not 9cis retinoic acid, dissociated corepressors from the wild-type PPARgamma. Telmisartan most effectively dissociated corepressors from Y473A. The interaction with coactivators was enhanced by TZD activation of wild-type PPARgamma and both telmisartan and TZD activation of Y473A. Thus, the Y473A mutant is selectively stimulated by telmisartan but not TZDs, suggesting that telmisartan and TZDs have differential effects on the transcriptional control. In conclusion, these PPARgamma mutants could be powerful tools for developing novel therapeutic agents that retain the metabolic efficacy of PPARgamma activation with fewer adverse effects, such as the increase in body weight associated with TZDs.

Molecular mechanism of the inhibitory effect of aldosterone on endothelial NO synthase activity.

Although the proinflammatory and profibrotic actions of aldosterone (Aldo) on the vasculature have been reported, the effects and molecular mechanisms of Aldo on endothelial function are yet to be determined. We investigated how Aldo regulates endothelial NO synthase (eNOS) function in human umbilical vein endothelial cells (HUVECs). HUVECs were incubated for 16 hours with Aldo 10(-7) mol/L. The concentration of reactive oxygen species was estimated by measuring 2',7'-dichlorodihydrofluorescein diacetate chemiluminescence. Signal transduction was estimated by Western immunoblots. Real-time RT-PCR was performed to measure expression of transcripts of endogenous GTP cyclohydrolase-1 and components of reduced nicotinamide-adenine dinucleotide phosphate oxidase. To eliminate the possible effect of the glucocorticoid receptor (GR) and to emphasize the role of mineralocorticoid receptor, we used GR small interfering RNA and knocked down GR expression in several experiments. NO output was estimated by intracellular cGMP concentration. Reactive oxygen species production increased significantly in Aldo-treated HUVECs but was abolished by pretreatment with eplerenone. Transcripts of p47(phox) were increased by Aldo treatment. Vascular endothelial growth factor-induced eNOS Ser 1177 but not Akt Ser 473 phosphorylation levels were reduced significantly by pretreatment with Aldo. Pretreatment with either eplerenone or okadaic acid restored phosphorylation levels of eNOS Ser 1177 in Aldo-treated cells, suggesting that protein phosphatase 2A was upregulated by Aldo via mineralocorticoid receptor. The decrease in NO output caused by Aldo pretreatment was reversed significantly by 5,6,7,8-tetrahydrobiopterin, GTP cyclohydrolase-1 overexpression, or p47(phox) knockdown. These results suggest that Aldo inhibits eNOS function through bimodal mechanisms of 5,6,7,8-tetrahydrobiopterin deficiency and protein phosphatase 2A activation.