• Angiotensin II Regulation of Aldosterone Synthase

      Nogueira, Edson da F.; Department of Physiology (2009-07)
      Angiotensin II (Ang II) is the major physiological regulator of aldosterone production acting acutely to stimulate aldosterone biosynthesis and chronically to increase the capacity of the adrenals to produce aldosterone. Aldosterone is principally synthesized in the zona glomerulosa of the adrenal by a series of enzymatic reactions leading to the conversion of cholesterol to aldosterone. The major goal of our study was to define the Ang II-induced mechanisms regulating the expression of aldosterone synthase (CYP11B2) in adrenocortical cells. We approached the analysis of the protein synthesis-dependent regulation of this enzyme by defining, through microarray and real time PCR analysis, the transcription factors that are rapidly induced by Ang II incubation of adrenocortical cell models from three species (human, bovine, and rat). The gene list generated by this comparison included: ATF3, BTG2, NR4A1, NR4A2, NR4A3, EGR1, FOS, FOSB, and JUNB. Importantly, pretreatment of H295R cells with cycloheximide had no effect on Ang II induction of these genes, suggesting that they are direct targets of Ang II signaling. Co-transfection studies, used to investigate the role of these transcription factors in the regulation of CYP11B2, determined that out of the nine transcription factors listed above, only the NGFI-B family members (NGFI-B, NURR1, and NOR1) increased expression of CYP11B2. The importance of NGFI-B in the regulation of CYP11B2 was confirmed by the decrease in CYP11B2 expression in the presence of a dominant-negative (DN)- NGFI-B. A pharmacological approach used to characterize the Ang II pathways regulating transcription of NGFI-B family genes suggested that Ang II binding to the AT1R increases activity of protein kinase C (PKC), Ca -dependent calmodulin kinases (CaMK), and SRC kinase (SRC), which act to regulate the expression of the family of NGFI-B genes as well as CYP11B2. In the current study we also analyzed protein synthesis-independent mechanisms regulating CYP11B2 expression. We studied the role of the ATF/CREB family of transcription factors (ATF1, ATF2, CREB, and CREM), which may bind the cAMP response element (CRE) in the promoter region of the CYP11B2 gene. Importantly, analysis of these transcription factors in the human H295R adrenocortical cell line revealed very low expression of CREB in comparison to the other CRE-binding proteins herein studied. We investigated Ang II-induced phosphorylation of these transcription factors, their binding to the promoter region of CYP11B2, and their effect on CYP11B2 expression. Ang II time-dependently induced phosphorylation of ATF1, ATF2, and CREM in H295R cells. The association of these transcription factors with the CYP11B2 promoter region was induced by Ang II and K+. Transfection of siRNA for ATF1, ATF2, and CREM significantly reduced CYP11B2 expression in Ang II-stimulated conditions. Expression of NURR-1 alone or with constitutively active ATF1, ATF2, CREB, and CREM increased the promoter activity of CYP11B2 in H295R cells. In summary, Ang II rapidly induces expression of newly synthesized transcription factors as well as the phosphorylation of transcription factors already present in the adrenocortical cell. These events are followed by increased CYP11B2 expression and, therefore, represent important mechanisms to increase the adrenal capacity to produce aldosterone.
    • Genetic mutations cause primary aldosteronism

      Hattangady, Namita G; Department of Physiology (2014-10)
      The human adrenal glands are complex endocrine organs that are physiologically located above the kidney. The cortex of the adrenal gland may be considered as a combination of three different steroidogenic tissue-types which form concentric zones within each adrenal. The three cortical zones include zona glomerulosa (ZG), zona fasciculata (ZF) and zona reticularis (ZR). Each zone, under independent regulation, produces unique steroid(s) which exhibit specific functions. The outermost ZG layer secretes the steroid, aldosterone due to ZG specific expression of aldosterone synthase (CYP11B2). Aldosterone regulates sodium reabsorption, and therefore, blood pressure. Aldosterone production is tightly regulated by the renin-angiotensin-aldosterone system. Thus, aldosterone levels are in direct proportion with renin levels. Other known physiological regulators of aldosterone production include serum K+ and adrenocorticotrophic hormone. A type of endocrine hypertension termed ‘Primary Aldosteronism’ (PA), is characterized by aldosterone secretion under suppressed renin levels. PA accounts for almost 10 % of hypertension. More recently, genetic mutations in an inward rectifying K+ channel (KCNJ5) that occur as both, somatic and germline cases, have been implicated in the pathology of PA. The goal of this dissertation is to define the role of KCNJ5 mutations in PA. In this dissertation, I will summarize my studies that describe the acute and chronic events involved in mutated KCNJ5 mediated aldosterone excess. In addition, I will define a novel mutation in KCNJ5 of germline nature identified at Georgia Regents University. Finally, I will also describe some interesting lessons we learnt from the expression of mutated KCNJ5 in primary cultures of human adrenals. The prevalence of a hereditary form of PA termed as Familial Hyperaldosteronism type III (FH III) is very rare. Thus far, only a few mutations in the KCNJ5 gene, including T158A, G151R, G151E and I157S, are confirmed as causing FH III, following Mendelian genetics. Perhaps the most interesting feature of this disease is the varied phenotype between the different mutations. T158A-affected patients present with massive hyperplasia and require bilateral adrenalectomy. In contrast, patients affected by the G151E mutation have more severe hypertension, although their adrenals are near normal in appearance. In this study we identify a new germline mutation (Y152C). The index case was a 61 year old woman who underwent unilateral adrenalectomy. The patient with the Y152C mutation exhibited a milder hypertension phenotype (like the G151E-affected patient) with extensive hyperplasia (as seen in the T158A-affected patient). In vitro analyses of the Y152C mutation indicated a pathology similar to other known mutations in KCNJ5, including change in conductance to Na+ ions and elevated calcium levels, and increase in CYP11B2 mRNA and aldosterone production. The inherent challenge presented by current studies utilizing constitutive expression of KCNJ5 mutations is the limitation in studying acute temporal events such as post translational modifications of steroidogenic enzymes and transcription factors. To address this issue, we generated a doxycycline inducible cell model system for the T158A harboring KCNJ5 transgene. Herein, we demonstrate a useful system that was amenable to the study of acute and chronic events involved in mutant-KCNJ5 mediated aldosterone excess. Our findings suggest that mutant KNCJ5 increases CYP11B2 expression through the activation of transcriptional activators of CYP11B2. Additionally, this is the first study to demonstrate that mutant KCNJ5 also activates steroidogenic acute regulatory protein (StAR) at the levels of translation and post translational phosphorylation. We also demonstrate calcium channel blocker, verapamil as an efficient blocker of mKCNJ5 mediated aldosterone production. Finally, one of the sharp advantages of our study was the use of primary cultures of human adrenal cells to confirm the effects of mutated KCNJ5. Interestingly, transduction of cells with constitutive viruses for mutant KCNJ5, confirmed an increase in KCNJ5 mRNA, although no change in CYP11B2 expression levels was observed. Pilot data including treatment of primary cells with calcium ionophores indicated that ZF/ZR cells may have a phenotype that is ‘muted’ for calcium mediated pathways. We could also speculate that this may disprove some current hypotheses that APA harboring KCNJ5 mutations may originate from the ZF. Overall, this study has improved our knowledge regarding the pathogenesis of PA caused by KCNJ5 mutations and has identified verapamil as a potentially effective therapeutic strategy in the inhibition of aldosterone excess in this type of PA.
    • The Mechanisms Underlying VLDL-Induced Aldosterone Production

      Tsai, Ying-Ying; Department of Physiology (2014-03)
      Aldosterone is responsible for sodium retention, thus increased blood volume and pressure. Excessive production of aldosterone results in high blood pressure, as well as renal disease, stroke, and visual loss via its effects on blood pressure. Although weight gain is associated with increased blood pressure, it remains unclear how excess fat deposits increase blood pressure. Indeed, overweight and obesity issues are correlated with serious health risks. In addition to hypertension, obese patients typically have high lipoprotein levels; moreover, some studies have suggested that aldosterone levels are also elevated and represent a link between obesity and hypertension. Very low density lipoprotein (VLDL) functions to transport triglycerides from the liver to peripheral tissues. Previous studies have demonstrated that VLDL can stimulate aldosterone production. By analogy with the signaling pathways activated by Angll, including the finding that VLDL increases cytosolic calcium levels, here we show that both phospholipase C (PLC) and phospholipase D (PLD) are involved in VLDL-induced aldosterone production. The effects of VLDL on steroidogeneses are mediated via an ability of these signaling pathways to result in the induction of steroidogenic acute regulatory (StAR) protein and aldosterone synthase (CYP11B2) expression, the early and late limiting steps in aldosterone biosynthesis, presumably byincreasing the phosphorylation (activation) of their regulatory transcription factors, such as the cAMP response element binding (CREB) protein family of transcription factors.
    • Protein Kinase D Restrains Angiotensin II-Induced Aldosterone Secretion in Primary Adrenal Glomerulosa Cells

      Shapiro, Brian A.; Department of Physiology (2007-07)
      Misregulation of the renin-angiotensin II (Angll)-aldosterone (Aldo) system is a key feature of cardiovascular disease. A focus of study in this system is the Angll-elicited secretion of Aldo from the adrenocortical zona glomerulosa. An excellent model in which to study this phenomenon is primary cultures of bovine adrenal glomerulosa (AG) cells. These cells secrete detectable quantities of Aldo in response to secretagogues, such as Angll, elevated potassium (K+), adrenocorticotrophic hormone (ACTH) and phorbol 12-myristate 13-acetate (PMA), within 30 minutes. The serine (Ser)/threonine kinase protein kinase D (PKD) is reported to be activated by Angll in several systems, including the adrenocortical carcinoma cell line NCI H295R, and is thought to have a positive role in chronic (24 hours) Angll-evoked Aldo secretion. Because the role of PKD in acute Angll-elicited Aldo secretion has never been examined in a primary culture system, we undertook to study the role of PKD in acute (minutes to one hour) Aldo secretion. Thus, Angll (10 nM) and PMA (100 nM), but not elevated K+ (15 mM) and ACTH (10 nM), induced phosphorylation of PKD on Ser910, a marker of PKD activation, in primary bovine AG cells. This finding was confirmed by an in vitro kinase activity assay. Angll and PMA were also able to induce PKD activation in H295R cells. Furthermore, this activation was concentration dependent, and was rapidly induced (by 5 min). PKD activation was dependent on Angll type 1 (AT-1), but not AT-2 receptor, signaling, and was independent of tyrosine kinase signaling. Finally, we introduced, via adenovirus transduction, wild-type PKDwt and dominant negative PKDS738/742A constructs into primary AG cells and monitored Angll-evoked Aldo secretion. PKDwt -transduced AG cells exhibited decreased Angll-stimulated Aldo secretion, while in the PKDS738A742A - infected AG cells Angll-stimulated Aldo was enhanced. Thus, we hypothesize that PKD has an anti-secretory role in Angll-induced acute Aldo secretion.