Daniel T. O'Connor

Secretion of Chromogranin A by Peptide-Producing Endocrine Neoplasms

Chromogranin A, the protein that is co-stored and co-released with catecholamines from the adrenal medulla, has recently been identified in a variety of human endocrine tissues, both normal and neoplastic. We investigated the secretion of chromogranin A by peptide hormone-producing human tumors in studies of patients with the following neoplastic disorders: pheochromocytoma, parathyroid adenoma, primary parathyroid hyperplasia, medullary thyroid carcinoma, thyroidal C-cell hyperplasia, carcinoid tumor, oat-cell lung carcinoma, pancreatic islet-cell tumor, and aortic-body tumor. All these patient groups had elevated concentrations of plasma chromogranin A. We distinguished different forms of immunoreactive plasma chromogranin A by size with the use of gel filtration. Plasma chromogranin A levels were not elevated in patients with diverse control conditions--both benign and malignant and both endocrine and nonendocrine--in which peptide hormones are not produced. The sensitivity and specificity of plasma chromogranin A elevations in the diagnosis of peptide-producing endocrine neoplasms were 81 and 100 percent, respectively. The elevation of plasma chromogranin A in our subjects suggests that their neoplasms co-release chromogranin A along with the usual resident hormone of the tumor, that these neoplasms could be characterized as chromograninomas, and that measurement of plasma chromogranin A may be a useful diagnostic procedure in subjects with endocrine tumors, especially multiple endocrine neoplasia.

Radioimmunoassay of Chromogranin a in Plasma as a Measure of Exocytotic Sympathoadrenal Activity in Normal Subjects and Patients with Pheochromocytoma

Chromogranin A is the major soluble protein stored and secreted by exocytosis, along with catecholamines, from vesicles in the adrenal medulla and sympathetic nerves. We investigated the possible use of chromogranin A as a probe of exocytotic sympathoadrenal activity, by developing a radioimmunoassay for chromogranin A purified from pheochromocytoma vesicles. In 18 normal recumbent men chromogranin A was present in plasma at a concentration of 129 +/- 12 ng per milliliter. The concentration varied with physiologic, pharmacologic, and pathologic changes in sympathoadrenal activity. It rose with standing and fell with recumbency, though it was not perturbed by brief dynamic exercise. It rose during brief vasodilation and fell during stimulation of nonexocytotic catecholamine release by tyramine and during ganglionic blockade. The plasma concentration of chromogranin A was elevated in 11 patients with pheochromocytoma (1614 +/- 408 ng per milliliter). The mean plasma half-life was 18.4 minutes. We conclude that both resting and activated sympathoadrenal tone in normal persons, as well as catecholamine secretion by pheochromocytoma, are at least in part exocytotic in mechanism and that chromogranin A may be a useful probe of exocytotic sympathoadrenal activity.

Human Tyrosine Hydroxylase Natural Genetic Variation Delineation of Functional Transcriptional Control Motifs Disrupted in the Proximal Promoter

Background— Tyrosine hydroxylase ( TH ) is the rate-limiting enzyme in catecholamine biosynthesis. Common genetic variation at the human TH promoter predicts alterations in autonomic activity and blood pressure, but how such variation influences human traits and, specifically, whether such variation affects transcription are not yet known.nnMethods and Results— Pairwise linkage disequilibrium across the TH locus indicated that common promoter variants (C-824T, G-801C, A-581G, and G-494A) were located in a single 5′ linkage disequilibrium block in white, black, Hispanic, and Asian populations. Polymorphisms C-824T and A-581G were located in highly conserved regions and were predicted to disrupt known transcriptional control motifs myocyte enhancer factor-2 (MEF2), sex-determining region Y (SRY), and forkhead box D1 (FOXD1) at C-824T and G/C-rich binding factors specificity protein 1 (SP1), activating enhancer-binding protein 2 (AP2)], early growth response protein 1 (EGR1) at A-581G. At C-824T and A-581G, promoter and luciferase reporter plasmids indicated differential allele strength (T>C at C-824T; G>A at A-581G) under both basal circumstances and secretory stimulation. C-824T and A-581G displayed the most pronounced effects on both transcription in cella and catecholamine secretion in vivo. We further probed the functional significance of C-824T and A-581G by cotransfection of trans -activating factors in cella ; MEF2, SRY, and FOXD1 differentially activated C-824T, whereas the G/C-rich binding factors SP1, AP2, and EGR1 differentially activated A-581G. At C-824T, factor MEF2 acted in a directionally coordinate fashion (at T>C) to explain the in vivo trait associations, whereas at A-581G, factors SP1, AP2, and EGR1 displayed similar differential actions (at G>A). Finally, chromatin immunoprecipitation demonstrated that the endogenous factors bound to the motifs in cella .nnConclusion— We conclude that common genetic variants in the proximal TH promoter, especially at C-824T and A-581G, are functional in cella and alter transcription so as to explain promoter marker-on-trait associations in vivo. MEF2, FOXD1, and SRY contribute to functional differences in C-824T expression, whereas SP1, AP2, and EGR1 mediate those of A-581G. The SRY effect on TH transcription suggests a mechanism whereby male and female sex may differ in sympathetic activity and hence blood pressure. These results point to new strategies for diagnostic and therapeutic intervention into disorders of human autonomic function and their cardiovascular consequences.nnReceived August 25, 2009; accepted January 21, 2010. nn# CLINICAL PERSPECTIVE {#article-title-2}Background—Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis. Common genetic variation at the human TH promoter predicts alterations in autonomic activity and blood pressure, but how such variation influences human traits and, specifically, whether such variation affects transcription are not yet known. Methods and Results—Pairwise linkage disequilibrium across the TH locus indicated that common promoter variants (C-824T, G-801C, A-581G, and G-494A) were located in a single 5′ linkage disequilibrium block in white, black, Hispanic, and Asian populations. Polymorphisms C-824T and A-581G were located in highly conserved regions and were predicted to disrupt known transcriptional control motifs myocyte enhancer factor-2 (MEF2), sex-determining region Y (SRY), and forkhead box D1 (FOXD1) at C-824T and G/C-rich binding factors specificity protein 1 (SP1), activating enhancer-binding protein 2 (AP2)], early growth response protein 1 (EGR1) at A-581G. At C-824T and A-581G, promoter and luciferase reporter plasmids indicated differential allele strength (T>C at C-824T; G>A at A-581G) under both basal circumstances and secretory stimulation. C-824T and A-581G displayed the most pronounced effects on both transcription in cella and catecholamine secretion in vivo. We further probed the functional significance of C-824T and A-581G by cotransfection of trans-activating factors in cella; MEF2, SRY, and FOXD1 differentially activated C-824T, whereas the G/C-rich binding factors SP1, AP2, and EGR1 differentially activated A-581G. At C-824T, factor MEF2 acted in a directionally coordinate fashion (at T>C) to explain the in vivo trait associations, whereas at A-581G, factors SP1, AP2, and EGR1 displayed similar differential actions (at G>A). Finally, chromatin immunoprecipitation demonstrated that the endogenous factors bound to the motifs in cella. Conclusion—We conclude that common genetic variants in the proximal TH promoter, especially at C-824T and A-581G, are functional in cella and alter transcription so as to explain promoter marker-on-trait associations in vivo. MEF2, FOXD1, and SRY contribute to functional differences in C-824T expression, whereas SP1, AP2, and EGR1 mediate those of A-581G. The SRY effect on TH transcription suggests a mechanism whereby male and female sex may differ in sympathetic activity and hence blood pressure. These results point to new strategies for diagnostic and therapeutic intervention into disorders of human autonomic function and their cardiovascular consequences.

Common Functional Genetic Variants in Catecholamine Storage Vesicle Protein Promoter Motifs Interact to Trigger Systemic Hypertension

OBJECTIVESnThe purpose of this study is to understand whether naturally occurring genetic variation in the promoter of chromogranin B (CHGB), a major constituent of catecholamine storage vesicles, is functional and confers risk for cardiovascular disease.nnnBACKGROUNDnCHGB plays a necessary (catalytic) role in catecholamine storage vesicle biogenesis. Previously, we found that genetic variation at CHGB influenced autonomic function, with association maximal toward the 5 region.nnnMETHODSnHere we explored transcriptional mechanisms of such effects, characterizing 2 common variants in the proximal promoter, A-296C and A-261T, using transfection/cotransfection, electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation (ChIP). We then tested the effects of promoter variation on cardiovascular traits.nnnRESULTSnThe A-296C disrupted a c-FOS motif, exhibiting differential mobility shifting to chromaffin cell nuclear proteins during EMSA, binding of endogenous c-FOS on ChIP, and differential response to exogenous c-FOS. The A-261T disrupted motifs for SRY and YY1, with similar consequences for EMSA, endogenous factor binding, and responses to exogenous factors. The 2-SNP CHGB promoter haplotypes had a profound (p=3.16E-20) effect on blood pressure (BP) in the European ancestry population, with a rank order of CT<AA<<CA<AT on both systolic blood pressure (SBP) and diastolic blood pressure (DBP), accounting for approximately 2.3% to approximately 3.4% of SBP/DBP variance; the haplotype effects on BP in vivo paralleled those on promoter activity in cella. Site-by-site interactions at A-296C and A-261T yielded highly nonadditive effects on SBP/DBP. The CHGB haplotype effects on BP were also noted in an independent (African ancestry) sample. In normotensive twins, parallel effects were noted for a pre-hypertensive phenotype, BP response to environmental stress.nnnCONCLUSIONSnThe common CHGB promoter variants A-296C and A-261T, and their consequent haplotypes, alter binding of specific transcription factors to influence gene expression in cella as well as BP in vivo. Such variation contributes substantially to risk for human hypertension. Involvement of the sex-specific factor SRY suggests a novel mechanism for development of sexual dimorphism in BP.

Genetic regulation of catecholamine synthesis, storage and secretion in the spontaneously hypertensive rat

Understanding catecholamine metabolism is crucial for elucidating the pathogenesis of hereditary hypertension. Here we integrated transcriptional and biochemical profiling with physiologic quantitative trait locus (eQTL and pQTL) mapping in adrenal glands of the HXB/BXH recombinant inbred (RI) strains, derived from the spontaneously hypertensive rat (SHR) and normotensive Brown Norway (BN.Lx). We found simultaneous down-regulation of five heritable transcripts in the catecholaminergic pathway in young (6 weeks) SHRs. We identified cis-acting eQTLs for Dbh, Pnmt (catecholamine biosynthesis) and Vamp1 (catecholamine secretion); enzymatic activities of Dbh and Pnmt paralleled transcripts, with pQTLs for activities mirroring eQTLs. We also detected trans-regulated expression of Vmat1 and Chga (both involved in catecholamine storage), with co-localization of these trans-eQTLs to the Pnmt locus. Pnmt re-sequencing revealed promoter polymorphisms that result in decreased response of the transfected SHR promoter to glucocorticoid, compared with BN.Lx. Of physiological pertinence, Dbh activity negatively correlated with systolic blood pressure in RI strains, whereas Pnmt activity was negatively correlated with heart rate. The finding of such cis- and trans-QTLs at an age before the onset of frank hypertension suggests that these heritable changes in biosynthetic enzyme expression represent primary genetic mechanisms for regulation of catecholamine action and blood pressure control in this widely studied model of hypertension.

Early Inflammatory and Metabolic Changes in Association With AGTR1 Polymorphisms in Prehypertensive Subjects

BACKGROUNDnThe Seventh Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure in 2003 created a prehypertension category for persons with blood pressures ranging from systolic blood pressure (SBP) of 120-139 mm Hg or diastolic blood pressure (DBP) from 80 to 89 mm Hg, due to increased risk of cardiovascular disease.nnnMETHODSnOur study utilized the University of California-San Diego (UCSD) Twin Hypertension Cohort. We measured comprehensive plasma cholesterol levels and metabolic (glucose, insulin, leptin) and inflammatory markers (interleukin-6 (IL-6), C-reactive protein (CRP), free fatty acids) to determine the differences between normotensive and prehypertensive subjects. Additionally, we determined whether angiotensin II receptor type-1 (AGTR1) polymorphisms, previously associated with hypertension, could predict prehypertension.nnnRESULTSnA total of 455 white subjects were included in the study (mean age 37.1 years). Prehypertensive subjects were older with greater body mass index (BMI) than the normotensives, and after adjusting for sex and age, had greater plasma glucose, insulin, and IL-6. The common AGTR1 A1166C (rs5186) polymorphism in the 3-UTR region, particularly the presence of the 1166C allele, which fails to downregulate gene expression, predicted greater likelihood of being in the prehypertension group and higher SBP. A lesser-studied polymorphism in intron-2 of AGTR1 (A/G; rs2276736) was associated with plasma high-density lipoprotein (HDL) and apolipoprotein A-1. In a subgroup analysis of nonobese subjects (N = 405), similar associations were noted.nnnCONCLUSIONnPrehypertensive subjects already exhibit early pathophysiologic changes putting them at risk of future cardiovascular disease, and AGTR1 may also contribute to this increased risk. Further investigation is needed to confirm these findings and the precise molecular mechanisms of action.

Renal kallikrein excretion and epigenetics in human acute kidney injury: Expression, mechanisms and consequences

BackgroundRenal kallikrein (KLK1) synthesis and urinary excretion are reportedly diminished during AKI (acute kidney injury) in animal models, and provision of kallikrein abrogates renal injury in this setting, but data in human AKI is limited. Therefore we first examined KLK1 renal excretion in human AKI, and then probed potential endocrine and epigenetic mechanisms for its alterations.MethodsKLK1 enzymatic activity excretion was evaluated in urine from patients with established or incipient AKI, versus healthy/non-hospital as well as ICU controls. Endocrine control of KLK1 excretion was then probed by catecholamine and aldosterone measurements in established AKI versus healthy controls. To examine epigenetic control of KLK1 synthesis, we tested blood and urine DNA for changes in promoter CpG methylation of the KLK1 gene, as well as LINE-1 elements, by bisulfite sequencing.ResultsPatients with early/incipient AKI displayed a modest reduction of KLK1 excretion, but unexpectedly, established AKI displayed substantially elevated urine KLK1 excretion, ~11-fold higher than healthy controls, and ~3-fold greater than ICU controls. We then probed potential mechanisms of the change. Established AKI patients had lower SBP, higher heart rate, and higher epinephrine excretion than healthy controls, though aldosterone excretion was not different. Promoter KLK1 CpG methylation was higher in blood than urine DNA, while KLK1 methylation in blood DNA was significantly higher in established AKI than healthy controls, though KLK1 methylation in urine tended to be higher in AKI, directionally consistent with earlier/incipient but not later/established changes in KLK1 excretion in AKI. On multivariate ANOVA, AKI displayed coordinate changes in KLK1 excretion and promoter methylation, though directionally opposite to expectation. Control (LINE-1 repetitive element) methylation in blood and urine DNA was similar between AKI and controls.ConclusionsUnexpectedly, increased KLK1 excretion in AKI patients was found; this increase is likely to be due in part to increments in adrenergic tone during BP depression. Epigenetic changes at KLK1 may also play a role in early changes of KLK1 expression and thus AKI susceptibility or recovery.

Endogenous epinephrine protects against obesity induced insulin resistance

Epinephrine (E) is a hormone released from the adrenal medulla in response to low blood sugar and other stresses. E and related β2-adrenergic agonists are used to treat asthma, but a side effect is high blood sugar. C57BL/6 mice prone to overfeeding induced type II diabetes had the PNMT gene knocked out to prevent E synthesis. These E deficient mice were very similar to control animals on a 14% fat diet. On a 40.6% fat diet they gained 20 to 33% more weight than control animals and increased their blood glucose response to a glucose tolerance test because they became resistant to insulin. Although the short term effect of β2-agonists such as E is to raise blood glucose, some long acting β2-agonists improve muscle glucose uptake. Endogenous E protects against overfeeding induced diabetes. Since adrenal E release can be impaired with aging and diabetes, endogenous E may help prevent adult onset diabetes.

Proteomic Analysis Yields an Unexpected Trans-Acting Point in Control of the Human Sympathochromaffin Phenotype

Background— The secretory protein chromogranin A (CHGA) plays a necessary role in formation of catecholamine storage vesicles and gives rise to a catecholamine release–inhibitory fragment. Because genetic variation in the proximal human CHGA promoter predicts autonomic function and blood pressure, we explored how a common genetic variant alters transcription of the gene. Methods and Results— Bioinformatic analysis suggested that the common G−462A promoter variant (rs9658634) may disrupt as many as 3 transcriptional control motifs: LEF1, COUP-TF, and PPAR&ggr;-RXR&agr;. During electrophoretic mobility shifts, chromaffin cell nuclear proteins bound specifically to the A (though not G) allele of CHGA promoter G−462A. On oligonucleotide affinity chromatography followed by electrospray ionization followed by 2-dimensional (tandem) mass spectrometry analysis of A allele eluates, the transcription factor LEF1 (lymphoid enhancer-binding factor-1) was identified. Interaction of LEF1 with the A allele at G−462A was confirmed by supershift. On cotransfection, LEF1 discriminated between the allelic variants, especially in chromaffin cells. Allele specificity of trans-activation by LEF1 was transferable to an isolated G−462A element fused to a heterologous (SV40) promoter. Because &bgr;-catenin (CTNNB1) can heterodimerize with LEF1, we tested the effect of cotransfection of this factor and again found A allele–specific perturbation of CHGA transcription. Conclusions— Common genetic variation within the human CHGA promoter alters the interaction of specific factors in trans with the promoter, with LEF1 identified by proteomic analysis and confirmed by supershift. Coexpression experiments show functional effects of LEF1 and CTNNB1 on CHGA promoter. The findings document a novel role for components of the immune and WNT pathways in control of human sympathochromaffin phenotypes.