Bhavani S. Sahu

Coordinated transcriptional regulation of Hspa1a gene by multiple transcription factors: crucial roles for HSF-1, NF-Y, NF-κB, and CREB.

Although the transcript level of inducible heat shock protein 70.3 (Hsp70.3, also known as Hspa1a) is altered in various disease states, its transcriptional regulation remains incompletely understood. Here, we systematically analyzed the Hspa1a promoter to identify major cis elements and transcription factors that may govern the constitutive/inducible gene expression. Computational analyses coupled with extensive in vitro (promoter-reporter activity and electrophoretic mobility shift assays) and in vivo (chromatin immunoprecipitation assays) revealed interaction of several transcription factors with Hspa1a promoter motifs: HSF-1 (heat shock factor 1) at -114/-97 bp and -788/-777bp, NF-Y (nuclear transcription factor Y) at -73/-58 bp, NF-κB (nuclear factor kappa B) at -133/-124 bp, and CREB (cAMP response element binding protein) at -483/-476 bp. Consistently, siRNA (small interfering RNA)-mediated down-regulation of each of these transcription factors caused substantial reduction of endogenous Hspa1a expression. Heat-shock-induced activation of Hspa1a was coordinately regulated by HSF-1 and NF-Y/NF-κB. The Hspa1a expression was augmented by TNF-α (tumor necrosis factor-alpha) and forskolin in NF-κB and CREB-dependent manners, respectively. NF-κB and CREB also activated Hspa1a transcription in cardiac myoblasts upon exposure to ischemia-like conditions. Taken together, this study discovered previously unknown roles for NF-κB and CREB to regulate Hspa1a expression and a coordinated action by several transcription factors for Hspa1a transactivation under heat-shock/ischemia-like conditions and thereby provided new insights into the mechanism of Hspa1a regulation.

Chromogranin A: a novel susceptibility gene for essential hypertension

Chromogranin A (CHGA) is ubiquitously expressed in secretory cells of the endocrine, neuroendocrine, and neuronal tissues. Although this protein has long been known as a marker for neuroendocrine tumors, its role in cardiovascular disease states including essential hypertension (EH) has only recently been recognized. It acts as a prohormone giving rise to bioactive peptides such as vasostatin-I (human CHGA1–76) and catestatin (human CHGA352–372) that exhibit several cardiovascular regulatory functions. CHGA is over-expressed but catestatin is diminished in EH. Moreover, genetic variants in the promoter, catestatin, and 3′-untranslated regions of the human CHGA gene alter autonomic activity and blood pressure. Consistent with these findings, targeted ablation of this gene causes severe arterial hypertension and ventricular hypertrophy in mice. Transgenic expression of the human CHGA gene or exogenous administration of catestatin restores blood pressure in these mice. Thus, the accumulated evidence establishes CHGA as a novel susceptibility gene for EH.

Molecular interactions of the physiological anti-hypertensive peptide catestatin with the neuronal nicotinic acetylcholine receptor

Catestatin (CST), a chromogranin-A-derived peptide, is a potent endogenous inhibitor of the neuronal nicotinic acetylcholine receptor (nAChR). It exerts an anti-hypertensive effect by acting as a ‘physiological brake’ on transmitter release into the circulation. However, the mechanism of interaction of CST with nAChR is only partially understood. To unravel molecular interactions of the wild-type human CST (CST-WT) as well as its naturally occurring variants (CST-364S and CST-370L, which have Gly→Ser and Pro→Leu substitutions, respectively) with the human α3β4 nAChR, we generated a homology-modeled human α3β4 nAChR structure and solution structures of CST peptides. Docking and molecular dynamics simulations showed that ~90% of interacting residues were within 15 N-terminal residues of CST peptides. The rank order of binding affinity of these peptides with nAChR was: CST-370L>CST-WT>CST-364S; the extent of occlusion of the receptor pore by these peptides was also in the same order. In corroboration with computational predictions, circular dichroism analysis revealed significant differences in global structures of CST peptides (e.g. the order of α-helical content was: CST-370L>CST-WT>CST-364S). Consistently, CST peptides blocked various stages of nAChR signal transduction, such as nicotine- or acetylcholine-evoked inward current, rise in intracellular Ca2+ and catecholamine secretion in or from neuron-differentiated PC12 cells, in the same rank order. Taken together, this study shows molecular interactions between human CST peptides and human α3β4 nAChR, and demonstrates that alterations in the CST secondary structure lead to the gain of potency for CST-370L and loss of potency for CST-364S. These findings have implications for understanding the nicotinic cholinergic signaling in humans.

Functional Genetic Variants of the Catecholamine-Release-Inhibitory Peptide Catestatin in an Indian Population: Allele-Specific Effects on Metabolic Traits

Background: Catestatin is emerging as a novel regulator of cardiovascular/metabolic functions. Results: We discovered a common amino acid substitution variant of catestatin that caused profound changes in plasma catecholamines, glucose, and lipid levels. Conclusion: Naturally occurring variants of catestatin peptide seem to alter the risk for metabolic syndrome. Significance: These findings provide new insights into the mechanism of metabolic diseases in humans. Catestatin (CST), a chromogranin A (CHGA)-derived peptide, is a potent inhibitor of catecholamine release from adrenal chromaffin cells and postganglionic sympathetic axons. We re-sequenced the CST region of CHGA in an Indian population (n = 1010) and detected two amino acid substitution variants: G364S and G367V. Synthesized CST variant peptides (viz. CST-Ser-364 and CST-Val-367) were significantly less potent than the wild type peptide (CST-WT) to inhibit nicotine-stimulated catecholamine secretion from PC12 cells. Consistently, the rank-order of blockade of nicotinic acetylcholine receptor (nAChR)-stimulated inward current and intracellular Ca2+ rise by these peptides in PC12 cells was: CST-WT > CST-Ser-364 > CST-Val-367. Structural analysis by CD spectroscopy coupled with molecular dynamics simulations revealed the following order of α-helical content: CST-WT > CST-Ser-364 > CST-Val-367; docking of CST peptides onto a major human nAChR subtype and molecular dynamics simulations also predicted the above rank order for their binding affinity with nAChR and the extent of occlusion of the receptor pore, providing a mechanistic basis for differential potencies. The G364S polymorphism was in strong linkage disequilibrium with several common CHGA genetic variations. Interestingly, the Ser-364 allele (detected in ∼15% subjects) was strongly associated with profound reduction (up to ∼2.1-fold) in plasma norepinephrine/epinephrine levels consistent with the diminished nAChR desensitization-blocking effect of CST-Ser-364 as compared with CST-WT. Additionally, the Ser-364 allele showed strong associations with elevated levels of plasma triglyceride and glucose levels. In conclusion, a common CHGA variant in an Indian population influences several biochemical parameters relevant to cardiovascular/metabolic disorders.

Functional Promoter Polymorphisms Govern Differential Expression of HMG-CoA Reductase Gene in Mouse Models of Essential Hypertension

3-Hydroxy-3-methylglutaryl-coenzyme A [HMG-CoA] reductase gene (Hmgcr) is a susceptibility gene for essential hypertension. Sequencing of the Hmgcr locus in genetically hypertensive BPH (blood pressure high), genetically hypotensive BPL (blood pressure low) and genetically normotensive BPN (blood pressure normal) mice yielded a number of single nucleotide polymorphisms (SNPs). BPH/BPL/BPN Hmgcr promoter-luciferase reporter constructs were generated and transfected into liver HepG2, ovarian CHO, kidney HEK-293 and neuronal N2A cells for functional characterization of the promoter SNPs. The BPH-Hmgcr promoter showed significantly less activity than the BPL-Hmgcr promoter under basal as well as nicotine/cholesterol-treated conditions. This finding was consistent with lower endogenous Hmgcr expression in liver and lower plasma cholesterol in BPH mice. Transfection experiments using 5′-promoter deletion constructs (strategically made to assess the functional significance of each promoter SNP) and computational analysis predicted lower binding affinities of transcription factors c-Fos, n-Myc and Max with the BPH-promoter as compared to the BPL-promoter. Corroboratively, the BPH promoter-luciferase reporter construct co-transfected with expression plasmids of these transcription factors displayed less pronounced augmentation of luciferase activity than the BPL construct, particularly at lower amounts of transcription factor plasmids. Electrophoretic mobility shift assays also showed diminished interactions of the BPH promoter with HepG2 nuclear proteins. Taken together, this study provides mechanistic basis for the differential Hmgcr expression in these mouse models of human essential hypertension and have implications for better understanding the role of this gene in regulation of blood pressure.

The neuropeptide TLQP-21 opposes obesity via C3aR1-mediated enhancement of adrenergic-induced lipolysis

Objectives Obesity is characterized by excessive fat mass and is associated with serious diseases such as type 2 diabetes. Targeting excess fat mass by sustained lipolysis has been a major challenge for anti-obesity therapies due to unwanted side effects. TLQP-21, a neuropeptide encoded by the pro-peptide VGF (non-acronymic), that binds the complement 3a receptor 1 (C3aR1) on the adipocyte membrane, is emerging as a novel modulator of adipocyte functions and a potential target for obesity-associated diseases. The molecular mechanism is still largely uncharacterized. Methods We used a combination of pharmacological and genetic gain and loss of function approaches. 3T3-L1 and mature murine adipocytes were used for in vitro experiments. Chronic in vivo experiments were conducted on diet-induced obese wild type, β1, β2, β3-adrenergic receptor (AR) deficient and C3aR1 knockout mice. Acute in vivo lipolysis experiments were conducted on Sprague Dawley rats. Results We demonstrated that TLQP-21 does not possess lipolytic properties per se. Rather, it enhances β-AR activation-induced lipolysis by a mechanism requiring Ca2+ mobilization and ERK activation of Hormone Sensitive Lipase (HSL). TLQP-21 acutely potentiated isoproterenol-induced lipolysis in vivo. Finally, chronic peripheral TLQP-21 treatment decreases body weight and fat mass in diet induced obese mice by a mechanism involving β-adrenergic and C3a receptor activation without associated adverse metabolic effects. Conclusions In conclusion, our data identify an alternative pathway modulating lipolysis that could be targeted to diminish fat mass in obesity without the side effects typically observed when using potent pro-lipolytic molecules.

Catestatin Gly364Ser Variant Alters Systemic Blood Pressure and the Risk for Hypertension in Human Populations via Endothelial Nitric Oxide Pathway

Catestatin (CST), an endogenous antihypertensive/antiadrenergic peptide, is a novel regulator of cardiovascular physiology. Here, we report case–control studies in 2 geographically/ethnically distinct Indian populations (n≈4000) that showed association of the naturally-occurring human CST-Gly364Ser variant with increased risk for hypertension (age-adjusted odds ratios: 1.483; P=0.009 and 2.951; P=0.005). Consistently, 364Ser allele carriers displayed elevated systolic (up to ≈8 mm Hg; P=0.004) and diastolic (up to ≈6 mm Hg; P=0.001) blood pressure. The variant allele was also found to be in linkage disequilibrium with other functional single-nucleotide polymorphisms in the CHGA promoter and nearby coding region. Functional characterization of the Gly364Ser variant was performed using cellular/molecular biological experiments (viz peptide–receptor binding assays, nitric oxide [NO], phosphorylated extracellular regulated kinase, and phosphorylated endothelial NO synthase estimations) and computational approaches (molecular dynamics simulations for structural analysis of wild-type [CST-WT] and variant [CST-364Ser] peptides and docking of peptide/ligand with &bgr;-adrenergic receptors [ADRB1/2]). CST-WT and CST-364Ser peptides differed profoundly in their secondary structures and showed differential interactions with ADRB2; although CST-WT displaced the ligand bound to ADRB2, CST-364Ser failed to do the same. Furthermore, CST-WT significantly inhibited ADRB2-stimulated extracellular regulated kinase activation, suggesting an antagonistic role towards ADRB2 unlike CST-364Ser. Consequently, CST-WT was more potent in NO production in human umbilical vein endothelial cells as compared with CST-364Ser. This NO-producing ability of CST-WT was abrogated by ADRB2 antagonist ICI 118551. In conclusion, CST-364Ser allele enhanced the risk for hypertension in human populations, possibly via diminished endothelial NO production because of altered interactions of CST-364Ser peptide with ADRB2 as compared with CST-WT.

Role of Clathrin in Dense Core Vesicle Biogenesis

Knocking down clathrin in PC12 cells not only affects the maturation of dense core vesicles, it also renders them essentially incapable of secretagogue-induced exocytosis.

A haplotype variant of the human chromogranin A gene (CHGA) promoter increases CHGA expression and the risk for cardiometabolic disorders

The acidic glycoprotein chromogranin A (CHGA) is co-stored/co-secreted with catecholamines and crucial for secretory vesicle biogenesis in neuronal/neuroendocrine cells. CHGA is dysregulated in several cardiovascular diseases, but the underlying mechanisms are not well established. Here, we sought to identify common polymorphisms in the CHGA promoter and to explore the mechanistic basis of their plausible contribution to regulating CHGA protein levels in circulation. Resequencing of the CHGA promoter in an Indian population (n = 769) yielded nine single-nucleotide polymorphisms (SNPs): G−1106A, A−1018T, T−1014C, T−988G, G−513A, G−462A, T−415C, C−89A, and C−57T. Linkage disequilibrium (LD) analysis indicated strong LD among SNPs at the −1014, −988, −462, and −89 bp positions and between the −1018 and −57 bp positions. Haplotype analysis predicted five major promoter haplotypes that displayed differential promoter activities in neuronal cells; specifically, haplotype 2 (containing variant T alleles at −1018 and −57 bp) exhibited the highest promoter activity. Systematic computational and experimental analyses revealed that transcription factor c-Rel has a role in activating the CHGA promoter haplotype 2 under basal and pathophysiological conditions (viz. inflammation and hypoxia). Consistent with the higher in vitro CHGA promoter activity of haplotype 2, individuals carrying this haplotype had higher plasma CHGA levels, plasma glucose levels, diastolic blood pressure, and body mass index. In conclusion, these results suggest a functional role of the CHGA promoter haplotype 2 (occurring in a large proportion of the world population) in enhancing CHGA expression in haplotype 2 carriers who may be at higher risk for cardiovascular/metabolic disorders.

Clearance kinetics of the VGF-derived neuropeptide TLQP-21

TLQP-21 is a multifunctional neuropeptide and a promising new medicinal target for cardiometabolic and neurological diseases. However, to date its clearance kinetics and plasma stability have not been studied. The presence of four arginine residues led us to hypothesize that its half-life is relatively short. Conversely, its biological activities led us to hypothesize that the peptide is still taken up by adipose tissues effectively. [125I]TLQP-21 was i.v. administered in rats followed by chasing the plasma radioactivity and assessing tissue uptake. Plasma stability was measured using LC-MS. In vivo lipolysis was assessed by the palmitate rate of appearance. RESULTS A small single i.v. dose of [125I]TLQP-21 had a terminal half-life of 110 min with a terminal clearance rate constant, kt, of 0.0063/min, and an initial half-life of 0.97 min with an initial clearance rate constant, ki, of 0.71/min. The total net uptake by adipose tissue accounts for 4.4% of the entire dose equivalent while the liver, pancreas and adrenal gland showed higher uptake. Uptake by the brain was negligible, suggesting that i.v.-injected peptide does not cross the blood-brain-barrier. TLQP-21 sustained isoproterenol-stimulated lipolysis in vivo. Finally, TLQP-21 was rapidly degraded producing several N-terminal and central sequence fragments after 10 and 60 min in plasma in vitro. This study investigated the clearance and stability of TLQP-21 peptide for the first time. While its pro-lipolytic effect supports and extends previous findings, its short half-life and sequential cleavage in the plasma suggest strategies for chemical modifications in order to enhance its stability and therapeutic efficacy.