Elizabeth E. Martelli

PCSK6-mediated corin activation is essential for normal blood pressure.

Hypertension is the most common cardiovascular disease, afflicting >30% of adults. The cause of hypertension in most individuals remains unknown, suggesting that additional contributing factors have yet to be discovered. Corin is a serine protease that activates the natriuretic peptides, thereby regulating blood pressure. It is synthesized as a zymogen that is activated by proteolytic cleavage. CORIN variants and mutations impairing corin activation have been identified in people with hypertension and pre-eclampsia. To date, however, the identity of the protease that activates corin remains elusive. Here we show that proprotein convertase subtilisin/kexin-6 (PCSK6, also named PACE4; ref. 10) cleaves and activates corin. In cultured cells, we found that corin activation was inhibited by inhibitors of PCSK family proteases and by small interfering RNAs blocking PCSK6 expression. Conversely, PCSK6 overexpression enhanced corin activation. In addition, purified PCSK6 cleaved wild-type corin but not the R801A variant that lacks the conserved activation site. Pcsk6-knockout mice developed salt-sensitive hypertension, and corin activation and pro-atrial natriuretic peptide processing activity were undetectable in these mice. Moreover, we found that CORIN variants in individuals with hypertension and pre-eclampsia were defective in PCSK6-mediated activation. We also identified a PCSK6 mutation that impaired corin activation activity in a hypertensive patient. Our results indicate that PCSK6 is the long-sought corin activator and is important for sodium homeostasis and normal blood pressure.

Gβγ-Independent Recruitment of G-Protein Coupled Receptor Kinase 2 Drives Tumor Necrosis Factor α–Induced Cardiac β-Adrenergic Receptor Dysfunction

Background— Proinflammatory cytokine tumor necrosis factor-&agr; (TNF&agr;) induces &bgr;-adrenergic receptor (&bgr;AR) desensitization, but mechanisms proximal to the receptor in contributing to cardiac dysfunction are not known. Methods and Results— Two different proinflammatory transgenic mouse models with cardiac overexpression of myotrophin (a prohypertrophic molecule) or TNF&agr; showed that TNF&agr; alone is sufficient to mediate &bgr;AR desensitization as measured by cardiac adenylyl cyclase activity. M-mode echocardiography in these mouse models showed cardiac dysfunction paralleling &bgr;AR desensitization independent of sympathetic overdrive. TNF&agr;-mediated &bgr;AR desensitization that precedes cardiac dysfunction is associated with selective upregulation of G-protein coupled receptor kinase 2 (GRK2) in both mouse models. In vitro studies in &bgr;2AR-overexpressing human embryonic kidney 293 cells showed significant &bgr;AR desensitization, GRK2 upregulation, and recruitment to the &bgr;AR complex following TNF&agr;. Interestingly, inhibition of phosphoinositide 3-kinase abolished GRK2-mediated &bgr;AR phosphorylation and GRK2 recruitment on TNF&agr;. Furthermore, TNF&agr;-mediated &bgr;AR phosphorylation was not blocked with &bgr;AR antagonist propranolol. Additionally, TNF&agr; administration in transgenic mice with cardiac overexpression of G&bgr;&ggr;-sequestering peptide &bgr;ARK-ct could not prevent &bgr;AR desensitization or cardiac dysfunction showing that GRK2 recruitment to the &bgr;AR is G&bgr;&ggr; independent. Small interfering RNA knockdown of GRK2 resulted in the loss of TNF&agr;-mediated &bgr;AR phosphorylation. Consistently, cardiomyocytes from mice with cardiac-specific GRK2 ablation normalized the TNF&agr;-mediated loss in contractility, showing that TNF&agr;-induced &bgr;AR desensitization is GRK2 dependent. Conclusions— TNF&agr;-induced &bgr;AR desensitization is mediated by GRK2 and is independent of G&bgr;&ggr;, uncovering a hitherto unknown cross-talk between TNF&agr; and &bgr;AR function, providing the underpinnings of inflammation-mediated cardiac dysfunction.

Gβγ Independent Recruitment of G-Protein Coupled Receptor Kinase 2 Drives TNFα-Induced Cardiac Beta-Adrenergic Receptor Dysfunction

Background— Proinflammatory cytokine tumor necrosis factor-&agr; (TNF&agr;) induces &bgr;-adrenergic receptor (&bgr;AR) desensitization, but mechanisms proximal to the receptor in contributing to cardiac dysfunction are not known. Methods and Results— Two different proinflammatory transgenic mouse models with cardiac overexpression of myotrophin (a prohypertrophic molecule) or TNF&agr; showed that TNF&agr; alone is sufficient to mediate &bgr;AR desensitization as measured by cardiac adenylyl cyclase activity. M-mode echocardiography in these mouse models showed cardiac dysfunction paralleling &bgr;AR desensitization independent of sympathetic overdrive. TNF&agr;-mediated &bgr;AR desensitization that precedes cardiac dysfunction is associated with selective upregulation of G-protein coupled receptor kinase 2 (GRK2) in both mouse models. In vitro studies in &bgr;2AR-overexpressing human embryonic kidney 293 cells showed significant &bgr;AR desensitization, GRK2 upregulation, and recruitment to the &bgr;AR complex following TNF&agr;. Interestingly, inhibition of phosphoinositide 3-kinase abolished GRK2-mediated &bgr;AR phosphorylation and GRK2 recruitment on TNF&agr;. Furthermore, TNF&agr;-mediated &bgr;AR phosphorylation was not blocked with &bgr;AR antagonist propranolol. Additionally, TNF&agr; administration in transgenic mice with cardiac overexpression of G&bgr;&ggr;-sequestering peptide &bgr;ARK-ct could not prevent &bgr;AR desensitization or cardiac dysfunction showing that GRK2 recruitment to the &bgr;AR is G&bgr;&ggr; independent. Small interfering RNA knockdown of GRK2 resulted in the loss of TNF&agr;-mediated &bgr;AR phosphorylation. Consistently, cardiomyocytes from mice with cardiac-specific GRK2 ablation normalized the TNF&agr;-mediated loss in contractility, showing that TNF&agr;-induced &bgr;AR desensitization is GRK2 dependent. Conclusions— TNF&agr;-induced &bgr;AR desensitization is mediated by GRK2 and is independent of G&bgr;&ggr;, uncovering a hitherto unknown cross-talk between TNF&agr; and &bgr;AR function, providing the underpinnings of inflammation-mediated cardiac dysfunction.

G-Protein Coupled Receptor Resensitization - Appreciating the Balancing Act of Receptor Function

G-protein coupled receptors (GPCRs) are seven transmembrane receptors that are pivotal regulators of cellular responses including vision, cardiac contractility, olfaction, and platelet activation. GPCRs have been a major target for drug discovery due to their role in regulating a broad range of physiological and pathological responses. GPCRs mediate these responses through a cyclical process of receptor activation (initiation of downstream signals), desensitization (inactivation that results in diminution of downstream signals), and resensitization (receptor reactivation for next wave of activation). Although these steps may be of equal importance in regulating receptor function, significant advances have been made in understanding activation and desensitization with limited effort towards resensitization. Inadequate importance has been given to resensitization due to the understanding that resensitization is a homeostasis maintaining process and is not acutely regulated. Evidence indicates that resensitization is a critical step in regulating GPCR function and may contribute towards receptor signaling and cellular responses. In light of these observations, it is imperative to discuss resensitization as a dynamic and mechanistic regulator of GPCR function. In this review we discuss components regulating GPCR function like activation, desensitization, and internalization with special emphasis on resensitization. Although we have used β-adrenergic receptor as a proto-type GPCR to discuss mechanisms regulating receptor function, other GPCRs are also described to put forth a view point on the universality of such mechanisms.

Phosphoinositide 3-kinase γ inhibits cardiac GSK-3 independently of Akt

A kinase promotes cardiac hypertrophy through a kinase-independent mechanism. Making a Bigger Heart Pathological cardiac hypertrophy can be fatal because it can cause congestive heart failure and arrhythmias. Glycogen synthase kinase-3 (GSK-3), which inhibits cardiac hypertrophy, is active when dephosphorylated by the protein phosphatase PP2A, the activity of which is stimulated by methylation mediated by the methyltransferase PPMT-1. Mohan et al. found that mice lacking the γ isoform of phosphoinositide 3-kinase (PI3K) had smaller hearts than wild-type mice and showed decreased phosphorylation of GSK-3. In addition, these mice showed increased activity of PP2A and PPMT-1. Biochemical experiments indicated that PI3Kγ inhibited the interaction between PP2A and PPMT-1. Heart size and phosphorylation of GSK-3 were increased, and the association of PP2A with PPMT-1 was decreased in PI3Kγ knockout mice by expression of a catalytically inactive form of PI3Kγ. Thus, PI3Kγ promotes cardiac hypertrophy by attenuating the PP2A–PPMT-1 interaction and the inactivation of GSK-3 in a kinase-independent manner. Activation of cardiac phosphoinositide 3-kinase α (PI3Kα) by growth factors, such as insulin, or activation of PI3Kγ downstream of heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors stimulates the activity of the kinase Akt, which phosphorylates and inhibits glycogen synthase kinase-3 (GSK-3). We found that PI3Kγ inhibited GSK-3 independently of the insulin-PI3Kα-Akt axis. Although insulin treatment activated Akt in PI3Kγ knockout mice, phosphorylation of GSK-3 was decreased compared to control mice. GSK-3 is activated when dephosphorylated by the protein phosphatase 2A (PP2A), which is activated when methylated by the PP2A methyltransferase PPMT-1. PI3Kγ knockout mice showed increased activity of PPMT-1 and PP2A and enhanced nuclear export of the GSK-3 substrate NFATc3. GSK-3 inhibits cardiac hypertrophy, and the hearts of PI3Kγ knockout mice were smaller compared to those of wild-type mice. Cardiac overexpression of a catalytically inactive PI3Kγ (PI3Kγinact) transgene in PI3Kγ knockout mice reduced the activities of PPMT-1 and PP2A and increased phosphorylation of GSK-3. Furthermore, PI3Kγ knockout mice expressing the PI3Kγinact transgene had larger hearts than wild-type or PI3Kγ knockout mice. Our studies show that a kinase-independent function of PI3Kγ could directly inhibit GSK-3 function by preventing the PP2A–PPMT-1 interaction and that this inhibition of GSK-3 was independent of Akt.

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.

Primary Immunoprevention of Epithelial Ovarian Carcinoma by Vaccination against the Extracellular Domain of Anti-Müllerian Hormone Receptor II

Epithelial ovarian carcinoma (EOC) is the most prevalent form of ovarian cancer in the United States, representing approximately 85% of all cases and causing more deaths than any other gynecologic malignancy. We propose that optimized control of EOC requires the incorporation of a vaccine capable of inducing safe and effective preemptive immunity in cancer-free women. In addition, we hypothesize that ovarian-specific self-proteins that are “retired” from autoimmune-inducing expression levels as ovaries age but are expressed at high levels in emerging EOC may serve as vaccine targets for mediating safe and effective primary immunoprevention. Here, we show that expression of the extracellular domain of anti-Müllerian hormone receptor II (AMHR2-ED) in normal tissues is confined exclusively to the human ovary, drops to nonautoimmune inducing levels in postmenopausal ovaries, and is at high levels in approximately 90% of human EOC. We found that AMHR2-ED vaccination significantly inhibits growth of murine EOC and enhances overall survival without inducing oophoritis in aged female mice. The observed inhibition of EOC growth was mediated substantially by induction of AMHR2-ED–specific IgG antibodies that agonize receptor signaling of a Bax/caspase-3–dependent proapoptotic cascade. Our results indicate that AMHR2-ED vaccination may be particularly useful in providing safe and effective preemptive immunity against EOC in women at high genetic or familial risk who have the greatest need for a preventive vaccine and ultimately in cancer-free postmenopausal women who account for 75% of all EOC cases. Cancer Prev Res; 10(11); 612–24. ©2017 AACR. See related editorial by Shoemaker et al., p. 607