ATP1A1 Mutant in Aldosterone-Producing Adenoma Leads to Cell Proliferation.

The molecular mechanisms by which ATP1A1 mutation-mediated cell proliferation or tumorigenesis in aldosterone-producing adenomas (APAs) have not been elucidated. First, we investigated whether the APA-associated ATP1A1 L104R mutation stimulated cell proliferation. Second, we aimed to clarify the molecular mechanisms by which the ATP1A1 mutation-mediated cell proliferated. We performed transcriptome analysis in APAs with ATP1A1 mutation. ATP1A1 L104R mutation were modulated in human adrenocortical carcinoma (HAC15) cells (ATP1A1-mutant cells), and we evaluated cell proliferation and molecular signaling events. Transcriptome and immunohistochemical analysis showed that Na/K-ATPase (NKA) expressions in ATP1A1 mutated APA were more abundant than those in non-functioning adrenocortical adenoma or KCNJ5 mutated APAs. The significant increase of number of cells, amount of DNA and S-phase population were shown in ATP1A1-mutant cells. Fluo-4 in ATP1A1-mutant cells were significantly increased. Low concentration of ouabain stimulated cell proliferation in ATP1A1-mutant cells. ATP1A1-mutant cells induced Src phosphorylation, and low concentration of ouabain supplementation showed further Src phosphorylation. We demonstrated that NKAs were highly expressed in ATP1A1 mutant APA, and the mutant stimulated cell proliferation and Src phosphorylation in ATP1A1-mutant cells. NKA stimulations would be a risk factor for the progression and development to an ATP1A1 mutant APA.

Development of a panel of monoclonal antibodies against the mineralocorticoid receptor.

Mineralocorticoid receptors (MR) bind both mineralocorticoids and glucocorticoids. They are expressed in multiple tissues and mediate diverse functions. Less is known about MR regulation and function compared with other major steroid receptors, although its importance has become increasingly apparent. A significant obstacle to such studies has been the dearth of specific high-affinity MR antibodies. We have produced monoclonal antibodies against 10 different peptide conjugates, six from the N terminus (A/B domain) and four from the C terminus (steroid binding domain), with the anticipation that their individual affinities for the MR would differ depending upon its conformation, which in turn, is dependent upon the location of the receptor within the cell and the proteins associated with it. Hybridoma clones with high titers to the cognate peptide ELISA were analyzed by Western blots using protein from Chinese hamster ovary cells transfected with enhanced green fluorescent protein-rat MR cDNA and from hippocampal cytosol from adrenalectomized rats. Immunohistochemistry was done on kidney, heart, colon, and brain. Antibodies that proved to be most useful for Western blot analysis and immunohistochemistry include those raised against peptides comprising amino acids 1-18, 64-82, 79-97, and 365-381. The intensity of immunoreactivity in the cytosol compared with nucleus in the same cells differed between antibodies, suggesting that certain receptor epitopes were more or less exposed depending on the location of the receptor within the cell. In summary, several antibodies are described that recognize different parts of the MR that should facilitate the study of this important mediator of two classes of steroid hormone action.

The myosin binding protein is a novel mineralocorticoid receptor binding partner.

The mineralocorticoid receptor (MR) plays a role in congestive heart failure; however, the molecular mechanism(s) remains undefined. We hypothesized that interaction of the MR with a cardiac protein modulates the transcriptional activation function of the MR within the heart. We used the yeast two-hybrid technique to screen a human heart library and found an aldosterone-dependent interaction between the hMR and the cardiac myosin binding protein (cMBP-c). The EC(50) of the hMR-MBP-c interaction was approximately 80nM, and the cMBP-c did not interact with the glucocorticoid receptor (GR). The GST pull-down technique was used to confirm an interaction between the MR and the cMBP-c as well as the lack of interaction with the GR. Spironolactone partially blocked this interaction, further suggesting MR specificity. We also determined the cMBP-c binding site lies within the C-terminus of the MR. We propose that interaction of the MR with cMBP-c may play a role in cardiac remodeling.

Glycyrrhizic acid suppresses type 2 11 beta-hydroxysteroid dehydrogenase expression in vivo.

Licorice-derivatives such as glycyrrhizic acid (GA) competitively inhibit 11 beta-hydroxysteroid dehydrogenase(11 beta-HSD) type 2 (11-HSD2) enzymatic activity, and chronic clinical use often results in pseudoaldosteronism. Since the effect of GA on 11-HSD2 expression remains unknown, we undertook in vivo and in vitro studies. Male Wistar rats were given 30, 60 or 120 mg/kg of GA twice a day for 2 weeks. Plasma corticosterone was decreased in those given the 120 mg dose, while urinary corticosterone excretion was increased in those given the 30 and 60 mg doses but decreased in those given 120 mg GA. NAD(+)-dependent dehydrogenase activity in kidney microsomal fraction was decreased in animals receiving doses of 60 and 120 mg GA. The 11-HSD2 protein and mRNA levels were decreased in those given 120 mg GA. In contrast, in vitro studies using mouse kidney M1 cells revealed that 24h treatment with glycyrrhetinic acid did not affect the 11-HSD2 mRNA expression levels. Thus, in addition to its role as a competitive inhibitor of 11-HSD2, the chronic high dose of GA suppresses mRNA and protein expression of 11-HSD2 possibly via indirect mechanisms. These effects may explain the prolonged symptoms after cessation of GA administration in some pseudoaldosteronism patients.