Molecular Mechanism for Hypertensive Renal Disease: Differential Regulation of Chromogranin A Expression at 3'-Untranslated Region Polymorphism C+87T by MicroRNA-107.

Chromogranin A (CHGA) is coreleased with catecholamines from secretory vesicles in adrenal medulla and sympathetic axons. Genetic variation in the CHGA 3'-region has been associated with autonomic control of circulation, hypertension, and hypertensive nephropathy, and the CHGA 3'-untranslated region (3'-UTR) variant C+87T (rs7610) displayed peak associations with these traits in humans. Here, we explored the molecular mechanisms underlying these associations. C+87T occurred in a microRNA-107 (miR-107) motif (match: T>C), and CHGA mRNA expression varied inversely with miR-107 abundance. In cells transfected with chimeric luciferase/CHGA 3'-UTR reporters encoding either the T allele or the C allele, changes in miR-107 expression levels had much greater effects on expression of the T allele. Cotransfection experiments with hsa-miR-107 oligonucleotides and eukaryotic CHGA plasmids produced similar results. Notably, an in vitro CHGA transcription/translation experiment revealed that changes in hsa-miR-107 expression altered expression of the T allele variant only. Mice with targeted ablation of Chga exhibited greater eGFR. Using BAC transgenesis, we created a mouse model with a humanized CHGA locus (T/T genotype at C+87T), in which treatment with a hsa-miR-107 inhibitor yielded prolonged falls in SBP/DBP compared with wild-type mice. We conclude that the CHGA 3'-UTR C+87T disrupts an miR-107 motif, with differential effects on CHGA expression, and that a cis:trans (mRNA:miR) interaction regulates the association of CHGA with BP and hypertensive nephropathy. These results indicate new strategies for probing autonomic circulatory control and ultimately, susceptibility to hypertensive renal sequelae.

Inflammatory Cytokines Rewire the Proinsulin Interaction Network in Human Islets.

CONTEXT: Aberrant biosynthesis and secretion of the insulin precursor proinsulin occurs in both type I and type II diabetes. Inflammatory cytokines are implicated in pancreatic islet stress and dysfunction in both forms of diabetes, but the mechanisms remain unclear. OBJECTIVE: We sought to determine the effect of the diabetes-associated cytokines on proinsulin folding, trafficking, secretion, and ß-cell function. METHODS: Human islets were treated with interleukin-1ß and interferon-γ for 48 hours, followed by analysis of interleukin-6, nitrite, proinsulin and insulin release, RNA sequencing, and unbiased profiling of the proinsulin interactome by affinity purification-mass spectrometry. RESULTS: Cytokine treatment induced secretion of interleukin-6, nitrites, and insulin, as well as aberrant release of proinsulin. RNA sequencing showed that cytokines upregulated genes involved in endoplasmic reticulum stress, and, consistent with this, affinity purification-mass spectrometry revealed cytokine induced proinsulin binding to multiple endoplasmic reticulum chaperones and oxidoreductases. Moreover, increased binding to the chaperone immunoglobulin binding protein was required to maintain proper proinsulin folding in the inflammatory environment. Cytokines also regulated novel interactions between proinsulin and type 1 and type 2 diabetes genome-wide association studies candidate proteins not previously known to interact with proinsulin (eg, Ataxin-2). Finally, cytokines induced proinsulin interactions with a cluster of microtubule motor proteins and chemical destabilization of microtubules with Nocodazole exacerbated cytokine induced proinsulin secretion. CONCLUSION: Together, the data shed new light on mechanisms by which diabetes-associated cytokines dysregulate ß-cell function. For the first time, we show that even short-term exposure to an inflammatory environment reshapes proinsulin interactions with critical chaperones and regulators of the secretory pathway.

Analysis of p53 and NF-κB signaling in modulating the cardiomyocyte fate during hypertrophy.

Cardiac hypertrophy leading to eventual heart failure is the most common cause of mortality throughout the world. The triggering mechanisms for cardiac hypertrophy are not clear but both apoptosis and cell proliferation have been reported in sections of failing hearts. In this study, we utilized both angiotensin II (AngII) treatment of cardiomyocytes and aortic ligation in rats (Rattus norvegicus, Wistar strain) for induction of hypertrophy to understand the cellular factors responsible for activation of apoptotic or anti-apoptotic pathway. Hypertrophy markers (ANF, ß-MHC), apoptotic proteins (Bax, Bad, Fas, p53, caspase-3, PARP), and anti-apoptotic or cell proliferation marker proteins (Bcl2, NF-κB, Ki-67) were induced significantly during hypertrophy, both in vitro as well as in vivo. Co-localization of both active caspase-3 and Ki-67 was observed in hypertrophied myocytes. p53 and NF-κBp65 binding to co-activator p300 was also increased in AngII treated myocytes. Inhibition of p53 resulted in downregulation of apoptosis, NF-κB activation, and NF-κB-p300 binding; however, NF-κB inhibition did not inhibit apoptosis or p53-p300 binding. Blocking of either p53 or NF-κB by specific inhibitors resulted in decrease in cell proliferation and hypertrophy markers, suggesting that p53 initially binds to p300 and then this complex recruits NF-κB. Thus, these results indicate the crucial role of p53 in regulating both apoptotic and cell proliferation during hypertrophy.

Chromogranin A pathway: from pathogenic molecule to renal disease.

BACKGROUND: Chromogranin A (CHGA) is an index granin protein critical for biogenesis and exocytotic release of catecholamine storage granules. It is elevated in plasma of patients with sympathetic over-activity and kidney dysfunction. Several CHGA polymorphisms are associated with hypertensive kidney disease. Previously, we unraveled the molecular mechanism by which CHGA expression is regulated in African Americans carrying a genetic variation associated with hypertensive chronic kidney disease (CKD). METHOD: Experimental CKD mouse model were created by 5/6th nephrectomy (Npx) using wild-type and Chga-/- knockout mouse strains to delineate the role of CHGA in CKD. RESULT: Wild-type-Npx mice expressing Chga developed exacerbated azotemia and fibrosis as compared with their knockout-Npx counterparts. Gene expression profiling revealed downregulation of mitochondrial respiratory complexes genes consistent with maladaptive mitochondria in wild-type-Npx mice, contrasted to knockout-Npx. In healthy individuals, an inverse relationship between circulating CHGA levels and glomerular function was observed. In vitro, mesangial cells treated with CHGA-triggered nitric oxide release by a signaling mechanism involving scavenger receptor SR-A. The CHGA-treated and untreated mesangial cells displayed differential expression of cytokine, chemokine, complement, acute phase inflammatory and apoptotic pathway genes. Thus, build-up of plasma CHGA because of kidney injury served as an insult to the mesangial cells resulting in expression of genes promoting inflammation, fibrosis, and progression of CKD. CONCLUSION: These findings improve understanding of the role of elevated CHGA in the progression of CKD and reveal novel pathways that could be exploited for therapeutic strategies in hypertensive kidney disease.

Unbiased Profiling of the Human Proinsulin Biosynthetic Interaction Network Reveals a Role for Peroxiredoxin 4 in Proinsulin Folding.

The ß-cell protein synthetic machinery is dedicated to the production of mature insulin, which requires the proper folding and trafficking of its precursor, proinsulin. The complete network of proteins that mediate proinsulin folding and advancement through the secretory pathway, however, remains poorly defined. Here we used affinity purification and mass spectrometry to identify, for the first time, the proinsulin biosynthetic interaction network in human islets. Stringent analysis established a central node of proinsulin interactions with endoplasmic reticulum (ER) folding factors, including chaperones and oxidoreductases, that is remarkably conserved in both sexes and across three ethnicities. The ER-localized peroxiredoxin PRDX4 was identified as a prominent proinsulin-interacting protein. In ß-cells, gene silencing of PRDX4 rendered proinsulin susceptible to misfolding, particularly in response to oxidative stress, while exogenous PRDX4 improved proinsulin folding. Moreover, proinsulin misfolding induced by oxidative stress or high glucose was accompanied by sulfonylation of PRDX4, a modification known to inactivate peroxiredoxins. Notably, islets from patients with type 2 diabetes (T2D) exhibited significantly higher levels of sulfonylated PRDX4 than islets from healthy individuals. In conclusion, we have generated the first reference map of the human proinsulin interactome to identify critical factors controlling insulin biosynthesis, ß-cell function, and T2D.

Role of Proinsulin Self-Association in Mutant INS Gene-Induced Diabetes of Youth.

Abnormal interactions between misfolded mutant and wild-type (WT) proinsulin (PI) in the endoplasmic reticulum (ER) drive the molecular pathogenesis of mutant INS gene-induced diabetes of youth (MIDY). How these abnormal interactions are initiated remains unknown. Normally, PI-WT dimerizes in the ER. Here, we suggest that the normal PI-PI contact surface, involving the B-chain, contributes to dominant-negative effects of misfolded MIDY mutants. Specifically, we find that PI B-chain tyrosine-16 (Tyr-B16), which is a key residue in normal PI dimerization, helps confer dominant-negative behavior of MIDY mutant PI-C(A7)Y. Substitutions of Tyr-B16 with either Ala, Asp, or Pro in PI-C(A7)Y decrease the abnormal interactions between the MIDY mutant and PI-WT, rescuing PI-WT export, limiting ER stress, and increasing insulin production in ß-cells and human islets. This study reveals the first evidence indicating that noncovalent PI-PI contact initiates dominant-negative behavior of misfolded PI, pointing to a novel therapeutic target to enhance PI-WT export and increase insulin production.

Mice overexpressing chromogranin A display hypergranulogenic adrenal glands with attenuated ATP levels contributing to the hypertensive phenotype.

OBJECTIVE: Elevated circulating chromogranin A (CHGA) is observed in human hypertension. CHGA is critical for granulogenesis and exocytosis of catecholamine stores from secretory large dense core vesicles (LDCV). This study aims to understand the morphological, molecular and phenotypic changes because of excess CHGA and the mechanistic link eventuating in hyper-adrenergic hypertension. METHODS: Blood pressure and heart rate was monitored in mouse models expressing normal and elevated level of CHGA by telemetry. Catecholamine and oxidative stress radicals were measured. Adrenal ultrastructure, LDCV content and mitochondrial abundance were compared and respiration analyzed by Seahorse assay. Effect of CHGA dosage on adrenal ATP content, electron transport chain components and uncoupling protein 2 (UCP-2) were compared in vivo and in vitro. RESULTS: Mice with excess-CHGA displayed hypertensive phenotype, higher heart rate and increased sympathetic tone. They had elevated plasma catecholamine and adrenal ROS levels. Excess-CHGA caused an increase in size and abundance of LDCV and adrenal mitochondria. Nonetheless, they had attenuated levels of ATP. Isolated adrenal mitochondria from mice with elevated CHGA showed higher maximal respiration rates in the presence of protonophore, which uncouples oxidative phosphorylation. Elevated CHGA resulted in overexpression of UCP2 and diminished ATP. In vitro in chromaffin cells overexpressing CHGA, concomitant increase in UCP2 protein and decreased ATP was detected. CONCLUSION: Elevated CHGA expression resulted in underlying bioenergetic dysfunction in ATP production despite higher mitochondrial mass. The outcome was unregulated negative feedback of LDCV exocytosis and secretion, resulting in elevated levels of circulating catecholamine and consequently the hypertensive phenotype.

Analysis and validation of traits associated with a single nucleotide polymorphism Gly364Ser in catestatin using humanized chromogranin A mouse models.

OBJECTIVE: The human prohormone chromogranin A (CHGA), an index member of the granin family is processed to generate catestatin, a peptide that is hypotensive in action and modulates catecholamine release within the sympathoadrenal system. Hypertensive patients with excess sympathetic activity have diminished catestatin. Often the study of physiological consequences of human genetic variation is confounded by elements such as other variations in obligatory linkage disequilibrium with the variant being studied. Also the phenotype of the variant may be influenced by genetic background that varies amongst individuals. This study addresses the effects of a human catestatin polymorphism (rs9658667) using humanized CHGA mouse models. METHODS: We created pertinent humanized mouse models wherein the mouse Chga gene locus was replaced by the human ortholog wild-type and the variant versions. This allowed for probing of the effects of catestatin variation in vivo with controls for other variations and global genetic background. RESULTS: Both the wild-type and variant human catestatin expressing mouse models were normotensive. The variant catestatin mouse model recapitulated physiological influence of the polymorphism on autonomic traits. These mice had diminished catecholamine, attenuated stress response and increased baroreceptor slopes that would suggest reduced risk of developing hypertension. Elevated plasma glucose, a trait observed in humans was not observed in mice expressing the variant catestatin. CONCLUSION: This functional genomics approach of creating humanized mouse models to study rs9658667 polymorphism recapitulated and validated many of the human trait associations. This approach can also be applied in the study of other human gene polymorphisms.

Cardiac electrical activity in a genomically "humanized" chromogranin a monogenic mouse model with hyperadrenergic hypertension.

The prohormone chromogranin A (CHGA) is ubiquitously found in vesicles of adrenal chromaffin cells and adrenergic neurons, and it is processed to the hypotensive hormone peptide catestatin (CST). Both CHGA and CST regulate blood pressure and cardiac function. This study addresses their role in cardiac electrical activity. We have generated two genomically "humanized" transgenic mouse strains (Tg31CHGA+/+; Chga-/- (HumCHGA31) and Tg19CHGA+/+; Chga-/- (HumCHGA19)) with varied CHGA expression and the ability to rescue the Chga-/- phenotype (hypertensive, hyperadrenergic with dilated cardiomyopathy). The normotensive HumCHGA31 mice express CHGA at levels comparable to wild-type. In contrast, the hypertensive HumCHGA19 mice have low levels of CHGA. EKG recordings revealed that the QT interval, R-amplitude, and QRS time-voltage integral are markedly longer in HumCHGA19 compared to wild-type and HumCHGA31 mice. These differences are accompanied by increased heart rate and QT variability, indicating that ventricular assault happens in a status of low levels of circulating CST.