Loss of Myeloid Cell-Specific ß2-Adrenergic Receptor Expression Ameliorates Cardiac Function and Remodeling after Acute Ischemia.

Myeloid cells, including neutrophils, monocytes and macrophages, accumulate quickly after ischemic injury in the heart where they play integral roles in the regulation of inflammation and repair. We previously reported that deletion of ß2-adrenergic receptor (ß2AR) in all cells of hematopoietic origin resulted in generalized disruption of immune cell responsiveness to injury, but with unknown impact on myeloid cells specifically. To investigate this, we crossed floxed ß2AR (F/F) mice with myeloid cell-expressing Cre (LysM-Cre) mice to generate myeloid cell-specific ß2AR knockout mice (LB2) and subjected them to myocardial infarction (MI). Via echocardiography and immunohistochemical analyses, LB2 mice displayed better cardiac function and less fibrotic remodeling after MI than the control lines. Despite similar accumulation of myeloid cell subsets in the heart at 1-day post-MI, LB2 mice displayed reduced numbers of Nu by 4 days post-MI, suggesting LB2 hearts have enhanced capacity for Nu efferocytosis. Indeed, bone marrow-derived macrophage (BMDM)-mediated efferocytosis of Nu was enhanced in LB2-versus F/F-derived cells in vitro. Mechanistically, several pro-efferocytosis-related genes were increased in LB2 myeloid cells, with annexin A1 ( Anxa1 ) in particular elevated in several myeloid cell types following MI. Accordingly, shRNA-mediated knockdown of Anxa1 in LB2 bone marrow prior to transplantation into irradiated LB2 mice reduced Mac-induced Nu efferocytosis in vitro and prevented the ameliorative effects of myeloid cell-specific ß2AR deletion on cardiac function and fibrosis following MI in vivo. Altogether, our data reveal a previously unrecognized role for ß2AR in the regulation of myeloid cell-dependent efferocytosis in the heart following injury.

Myeloid cell-specific deletion of epidermal growth factor receptor aggravates acute cardiac injury.

Myeloid cells, including macrophages, play important roles as first responders to cardiac injury and stress. Epidermal growth factor receptor (EGFR) has been identified as a mediator of macrophage responsiveness to select diseases, though its impact on cardiac function or remodeling following acute ischemic injury is unknown. We aimed to define the role of myeloid cell-specific EGFR in the regulation of cardiac function and remodeling following acute myocardial infarction (MI)-induced injury. Floxed EGFR mice were bred with homozygous LysM-Cre (LMC) transgenic mice to yield myeloid-specific EGFR knockout (mKO) mice. Via echocardiography, immunohistochemistry, RNA sequencing and flow cytometry, the impact of myeloid cell-specific EGFR deletion on cardiac structure and function was assessed at baseline and following injury. Compared with LMC controls, myeloid cell-specific EGFR deletion led to an increase in cardiomyocyte hypertrophy at baseline. Bulk RNASeq analysis of isolated cardiac Cd11b+ myeloid cells revealed substantial changes in mKO cell transcripts at baseline, particularly in relation to predicted decreases in neovascularization. In response to myocardial infarction, mKO mice experienced a hastened decline in cardiac function with isolated cardiac Cd11b+ myeloid cells expressing decreased levels of the pro-reparative mediators Vegfa and Il10, which coincided with enhanced cardiac hypertrophy and decreased capillary density. Overall, loss of EGFR qualitatively alters cardiac resident macrophages that promotes a low level of basal stress and a more rapid decrease in cardiac function along with worsened repair following acute ischemic injury.

Epidermal growth factor receptor-dependent maintenance of cardiac contractility.

AIMS: Epidermal growth factor receptor (EGFR) is essential to the development of multiple tissues and organs and is a target of cancer therapeutics. Due to the embryonic lethality of global EGFR deletion and conflicting reports of cardiac-overexpressed EGFR mutants, its specific impact on the adult heart, normally or in response to chronic stress, has not been established. Using complimentary genetic strategies to modulate cardiomyocyte-specific EGFR expression, we aim to define its role in the regulation of cardiac function and remodelling. METHODS AND RESULTS: A floxed EGFR mouse model with α-myosin heavy chain-Cre-mediated cardiomyocyte-specific EGFR downregulation (CM-EGFR-KD mice) developed contractile dysfunction by 9 weeks of age, marked by impaired diastolic relaxation, as monitored via echocardiographic, haemodynamic, and isolated cardiomyocyte contractility analyses. This contractile defect was maintained over time without overt cardiac remodelling until 10 months of age, after which the mice ultimately developed severe heart failure and reduced lifespan. Acute downregulation of EGFR in adult floxed EGFR mice with adeno-associated virus 9 (AAV9)-encoded Cre with a cardiac troponin T promoter (AAV9-cTnT-Cre) recapitulated the CM-EGFR-KD phenotype, while AAV9-cTnT-EGFR treatment of adult CM-EGFR-KD mice rescued the phenotype. Notably, chronic administration of the ß-adrenergic receptor agonist isoproterenol effectively and reversibly compensated for the contractile dysfunction in the absence of cardiomyocyte hypertrophy in CM-EGFR-KD mice. Mechanistically, EGFR downregulation reduced the expression of protein phosphatase 2A regulatory subunit Ppp2r3a/PR72, which was associated with decreased phosphorylation of phospholamban and Ca2+ clearance, and whose re-expression via AAV9-cTnT-PR72 rescued the CM-EGFR-KD phenotype. CONCLUSIONS: Altogether, our study highlights a previously unrecognized role for EGFR in maintaining contractile homeostasis under physiologic conditions in the adult heart via regulation of PR72 expression.

Epidermal growth factor receptor association with ß1-adrenergic receptor is mediated via its juxtamembrane domain.

ß1-adrenergic receptor (ß1AR)-mediated transactivation of epidermal growth factor receptor (EGFR) engages downstream signaling events that impact numerous cellular processes including growth and survival. While association of these receptors has been shown to occur basally and be important for relaying transactivation-specific intracellular events, the mechanism by which they do so is unclear and elucidation of which would aid in understanding the consequence of disrupting their interaction. Using fluorescence resonance energy transfer (FRET) and immunoprecipitation (IP) analyses, we evaluated the impact of C-terminal truncations of EGFR on its ability to associate with ß1AR. While loss of the last 230 amino acid C-terminal phosphotyrosine-rich domain did not disrupt the ability of EGFR to associate with ß1AR, truncation of the entire intracellular domain of EGFR resulted in almost complete loss of its interaction with ß1AR, suggesting that either the kinase domain or juxtamembrane domain (JMD) may be required for this association. Treatment with the EGFR antagonist gefitinib did not prevent ß1AR-EGFR association, however, treatment with a palmitoylated peptide encoding the first 20 amino acids of the JMD domain (JMD-A) disrupted ß1AR-EGFR association over time and prevented ß1AR-mediated ERK1/2 phosphorylation, both in general and specifically in association with EGFR. Conversely, neither a mutated JMD-A peptide nor a palmitoylated peptide fragment consisting of the subsequent 18 amino acids of the JMD domain (JMD-B) were capable of doing so. Altogether, the proximal region of the JMD of EGFR is responsible for its association with ß1AR, and its disruption prevents ß1AR-mediated transactivation, thus providing a new tool to study the functional consequences of disrupting ß1AR-EGFR downstream signaling.

G protein-coupled receptor kinase 2 contributes to impaired fatty acid metabolism in the failing heart.

Increased G protein-coupled receptor kinase (GRK)2 is central to heart failure (HF) pathogenesis, via desensitization of ß-adrenergic receptors and loss of contractile reserve. Since GRK2 has been shown to compromise fatty acid (FA) oxidation, this kinase may link metabolic and contractile defects in HF. The aim of this study was to investigate the mechanistic role of GRK2 in FA metabolism and bioenergetics in the heart. For that purpose, we measured FA uptake and cluster of differentiation (CD)36 expression, phosphorylation, and ubiquitination in mice with cardiac-specific overexpression of GRK2 (TgGRK2) or expression of its c-terminus (GRK2 inhibitor- TgßARKct) or in global heterozygous GRK2 knockout (GRK2+/-) mice. Cellular bioenergetics were also measured in isolated cardiomyocytes following adenoviral delivery of exogenous GRK2, ßARKct, or short hairpin GRK2 (shGRK2). Additionally, CD36 expression and phosphorylation were evaluated following transverse aortic constriction (TAC) in wild type (WT) and GRK2+/- mice. Our results show a 33% ±â€¯0.81 reduction in FA uptake rate, accompanied by 51% ±â€¯0.17 lower CD36 protein, and 70% ±â€¯0.23 and 69% ±â€¯0.18 increases in CD36 phosphorylation and ubiquitination, respectively, in the TgGRK2 mice. Moreover, an in vitro kinase assay suggests that GRK2 directly phosphorylates CD36. In isolated cardiomyocytes, GRK2 overexpression induced a 26% ±â€¯2.21 decrease in maximal respiration, which was enhanced (20% ±â€¯4.02-5.14) with inhibition of the kinase. Importantly, in hearts with systolic dysfunction, notable reductions in CD36 mRNA and protein, as well as a significant increase in CD36 phosphorylation were normalized in the GRK2+/- mice post-TAC. Thus, we propose that GRK2 up-regulation in HF is, at least partly, responsible for reduced FA uptake and oxidation and may be a nodal link between metabolic and contractile defects.

Leukocyte-Expressed ß2-Adrenergic Receptors Are Essential for Survival After Acute Myocardial Injury.

BACKGROUND: Immune cell-mediated inflammation is an essential process for mounting a repair response after myocardial infarction (MI). The sympathetic nervous system is known to regulate immune system function through ß-adrenergic receptors (ßARs); however, their role in regulating immune cell responses to acute cardiac injury is unknown. METHODS: Wild-type (WT) mice were irradiated followed by isoform-specific ßAR knockout (ßARKO) or WT bone-marrow transplantation (BMT) and after full reconstitution underwent MI surgery. Survival was monitored over time, and alterations in immune cell infiltration after MI were examined through immunohistochemistry. Alterations in splenic function were identified through the investigation of altered adhesion receptor expression. RESULTS: ß2ARKO BMT mice displayed 100% mortality resulting from cardiac rupture within 12 days after MI compared with ≈20% mortality in WT BMT mice. ß2ARKO BMT mice displayed severely reduced post-MI cardiac infiltration of leukocytes with reciprocally enhanced splenic retention of the same immune cell populations. Splenic retention of the leukocytes was associated with an increase in vascular cell adhesion molecule-1 expression, which itself was regulated via ß-arrestin-dependent ß2AR signaling. Furthermore, vascular cell adhesion molecule-1 expression in both mouse and human macrophages was sensitive to ß2AR activity, and spleens from human tissue donors treated with ß-blocker showed enhanced vascular cell adhesion molecule-1 expression. The impairments in splenic retention and cardiac infiltration of leukocytes after MI were restored to WT levels via lentiviral-mediated re-expression of ß2AR in ß2ARKO bone marrow before transplantation, which also resulted in post-MI survival rates comparable to those in WT BMT mice. CONCLUSIONS: Immune cell-expressed ß2AR plays an essential role in regulating the early inflammatory repair response to acute myocardial injury by facilitating cardiac leukocyte infiltration.

ß-arrestin-biased signaling through the ß2-adrenergic receptor promotes cardiomyocyte contraction.

ß-adrenergic receptors (ßARs) are critical regulators of acute cardiovascular physiology. In response to elevated catecholamine stimulation during development of congestive heart failure (CHF), chronic activation of Gs-dependent ß1AR and Gi-dependent ß2AR pathways leads to enhanced cardiomyocyte death, reduced ß1AR expression, and decreased inotropic reserve. ß-blockers act to block excessive catecholamine stimulation of ßARs to decrease cellular apoptotic signaling and normalize ß1AR expression and inotropy. Whereas these actions reduce cardiac remodeling and mortality outcomes, the effects are not sustained. Converse to G-protein-dependent signaling, ß-arrestin-dependent signaling promotes cardiomyocyte survival. Given that ß2AR expression is unaltered in CHF, a ß-arrestin-biased agonist that operates through the ß2AR represents a potentially useful therapeutic approach. Carvedilol, a currently prescribed nonselective ß-blocker, has been classified as a ß-arrestin-biased agonist that can inhibit basal signaling from ßARs and also stimulate cell survival signaling pathways. To understand the relative contribution of ß-arrestin bias to the efficacy of select ß-blockers, a specific ß-arrestin-biased pepducin for the ß2AR, intracellular loop (ICL)1-9, was used to decouple ß-arrestin-biased signaling from occupation of the orthosteric ligand-binding pocket. With similar efficacy to carvedilol, ICL1-9 was able to promote ß2AR phosphorylation, ß-arrestin recruitment, ß2AR internalization, and ß-arrestin-biased signaling. Interestingly, ICL1-9 was also able to induce ß2AR- and ß-arrestin-dependent and Ca(2+)-independent contractility in primary adult murine cardiomyocytes, whereas carvedilol had no efficacy. Thus, ICL1-9 is an effective tool to access a pharmacological profile stimulating cardioprotective signaling and inotropic effects through the ß2AR and serves as a model for the next generation of cardiovascular drug development.

Temporal and gefitinib-sensitive regulation of cardiac cytokine expression via chronic ß-adrenergic receptor stimulation.

Chronic stimulation of ß-adrenergic receptors (ßAR) can promote survival signaling via transactivation of epidermal growth factor receptor (EGFR) but ultimately alters cardiac structure and contractility over time, in part via enhanced cytokine signaling. We hypothesized that chronic catecholamine signaling will have a temporal impact on cardiac transcript expression in vivo, in particular cytokines, and that EGFR transactivation plays a role in this process. C57BL/6 mice underwent infusion with vehicle or isoproterenol (Iso)±gefitinib (Gef) for 1 or 2 wk. Cardiac contractility decreased following 2 wk of Iso treatment, while cardiac hypertrophy, fibrosis, and apoptosis were enhanced at both timepoints. Inclusion of Gef preserved contractility, blocked Iso-induced apoptosis, and prevented hypertrophy at the 2-wk timepoint, but caused fibrosis on its own. RNAseq analysis revealed hundreds of cardiac transcripts altered by Iso at each timepoint with subsequent RT-quantitative PCR validation confirming distinct temporal patterns of transcript regulation, including those involved in cardiac remodeling and survival signaling, as well as numerous cytokines. Although Gef infusion alone did not significantly alter cytokine expression, it abrogated the Iso-mediated changes in a majority of the ßAR-sensitive cytokines, including CCL2 and TNF-α. Additionally, the impact of ßAR-dependent EGFR transactivation on the acute regulation of cytokine transcript expression was assessed in isolated cardiomyocytes and in cardiac fibroblasts, where the majority of Iso-dependent, and EGFR-sensitive, changes in cytokines occurred. Overall, coincident with changes in cardiac structure and contractility, ßAR stimulation dynamically alters cardiac transcript expression over time, including numerous cytokines that are regulated via EGFR-dependent signaling.

ß-adrenergic receptor-mediated cardiac contractility is inhibited via vasopressin type 1A-receptor-dependent signaling.

BACKGROUND: Enhanced arginine vasopressin levels are associated with increased mortality during end-stage human heart failure, and cardiac arginine vasopressin type 1A receptor (V1AR) expression becomes increased. Additionally, mice with cardiac-restricted V1AR overexpression develop cardiomyopathy and decreased ß-adrenergic receptor (ßAR) responsiveness. This led us to hypothesize that V1AR signaling regulates ßAR responsiveness and in doing so contributes to development of heart failure. METHODS AND RESULTS: Transaortic constriction resulted in decreased cardiac function and ßAR density and increased cardiac V1AR expression, effects reversed by a V1AR-selective antagonist. Molecularly, V1AR stimulation led to decreased ßAR ligand affinity, as well as ßAR-induced Ca(2+) mobilization and cAMP generation in isolated adult cardiomyocytes, effects recapitulated via ex vivo Langendorff analysis. V1AR-mediated regulation of ßAR responsiveness was demonstrated to occur in a previously unrecognized Gq protein-independent/G protein receptor kinase-dependent manner. CONCLUSIONS: This newly discovered relationship between cardiac V1AR and ßAR may be informative for the treatment of patients with acute decompensated heart failure and elevated arginine vasopressin.

ß-adrenergic receptor-dependent alterations in murine cardiac transcript expression are differentially regulated by gefitinib in vivo.

ß-adrenergic receptor (ßAR)-mediated transactivation of epidermal growth factor receptor (EGFR) has been shown to promote cardioprotection in a mouse model of heart failure and we recently showed that this mechanism leads to enhanced cell survival in part via regulation of apoptotic transcript expression in isolated primary rat neonatal cardiomyocytes. Thus, we hypothesized that this process could regulate cardiac transcript expression in vivo. To comprehensively assess cardiac transcript alterations in response to acute ßAR-dependent EGFR transactivation, we performed whole transcriptome analysis of hearts from C57BL/6 mice given i.p. injections of the ßAR agonist isoproterenol in the presence or absence of the EGFR antagonist gefitinib for 1 hour. Total cardiac RNA from each treatment group underwent transcriptome analysis, revealing a substantial number of transcripts regulated by each treatment. Gefitinib alone significantly altered the expression of 405 transcripts, while isoproterenol either alone or in conjunction with gefitinib significantly altered 493 and 698 distinct transcripts, respectively. Further statistical analysis was performed, confirming 473 transcripts whose regulation by isoproterenol were significantly altered by gefitinib (isoproterenol-induced up/downregulation antagonized/promoted by gefinitib), including several known to be involved in the regulation of numerous processes including cell death and survival. Thus, ßAR-dependent regulation of cardiac transcript expression in vivo can be modulated by the EGFR antagonist gefitinib.

ß-Adrenergic receptor-mediated transactivation of epidermal growth factor receptor decreases cardiomyocyte apoptosis through differential subcellular activation of ERK1/2 and Akt.

ß-Adrenergic receptor (ßAR)-mediated transactivation of epidermal growth factor receptor (EGFR) has been shown to relay pro-survival effects via unknown mechanisms. We hypothesized that acute ßAR-mediated EGFR transactivation in the heart promotes differential subcellular activation of ERK1/2 and Akt, promoting cell survival through modulation of apoptosis. C57BL/6 mice underwent acute i.p. injection with isoproterenol (ISO)±AG 1478 (EGFR antagonist) to assess the impact of ßAR-mediated EGFR transactivation on the phosphorylation of ERK1/2 (P-ERK1/2) and Akt (P-Akt) in distinct cardiac subcellular fractions. Increased P-ERK1/2 and P-Akt were observed in cytosolic, plasma membrane and nuclear fractions following ISO stimulation. Whereas the P-ERK1/2 response was EGFR-sensitive in all fractions, the P-Akt response was EGFR-sensitive only in the plasma membrane and nucleus, results confirmed in primary rat neonatal cardiomyocytes (RNCM). ßAR-mediated EGFR-transactivation also decreased apoptosis in serum-depleted RNCM, as measured via TUNEL as well as caspase 3 activity/cleavage, which were sensitive to the inhibition of either ERK1/2 (PD184352) or Akt (LY-294002) signaling. Caspase 3 activity/cleavage was also sensitive to the inhibition of transcription, which, with an increase in nuclear P-ERK1/2 and P-Akt in response to ISO, suggested that ßAR-mediated EGFR transactivation may regulate apoptotic gene transcription. An Apoptosis PCR Array identified tnfsf10 (TRAIL) to be altered by ISO in an EGFR-sensitive manner, results confirmed via RT-PCR and ELISA measurement of both membrane-bound and soluble cardiomyocyte TRAIL levels. ßAR-mediated EGFR transactivation induces differential subcellular activation of ERK1/2 and Akt leading to increased cell survival through the modulation of caspase 3 activity and apoptotic gene expression in cardiomyocytes.

Quantification of Gi-mediated inhibition of adenylyl cyclase activity reveals that UDP is a potent agonist of the human P2Y14 receptor.

The P2Y14 receptor was initially identified as a G protein-coupled receptor activated by UDP-glucose and other nucleotide sugars. We have developed several cell lines that stably express the human P2Y14 receptor, allowing facile examination of its coupling to native Gi family G proteins and their associated downstream signaling pathways (J Pharmacol Exp Ther 330:162-168, 2009). In the current study, we examined P2Y14 receptor-dependent inhibition of cyclic AMP accumulation in human embryonic kidney (HEK) 293, C6 glioma, and Chinese hamster ovary (CHO) cells stably expressing this receptor. Not only was the human P2Y14 receptor activated by UDP-glucose, but it also was activated by UDP. The apparent efficacies of UDP and UDP-glucose were similar, and the EC50 values (74, 33, and 29 nM) for UDP-dependent activation of the P2Y14 receptor in HEK293, CHO, and C6 glioma cells, respectively, were similar to the EC50 values (323, 132, and 72 nM) observed for UDP-glucose. UDP and UDP-glucose also stimulated extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in P2Y14 receptor-expressing HEK293 cells but not in wild-type HEK293 cells. A series of analogs of UDP were potent P2Y14 receptor agonists, but the naturally occurring nucleoside diphosphates, CDP, GDP, and ADP exhibited agonist potencies over 100-fold less than that observed with UDP. Two UDP analogs were identified that selectively activate the P2Y14 receptor over the UDP-activated P2Y6 receptor, and these molecules stimulated phosphorylation of ERK1/2 in differentiated human HL-60 promyeloleukemia cells, which natively express the P2Y14 receptor but had no effect in wild-type HL-60 cells, which do not express the receptor. We conclude that UDP is an important cognate agonist of the human P2Y14 receptor.

Synthesis and P2Y receptor activity of nucleoside 5'-phosphonate derivatives.

Ribose-based nucleoside 5'-diphosphates and triphosphates and related nucleotides were compared in their potency at the P2Y receptors with the corresponding nucleoside 5'-phosphonate derivatives. Phosphonate derivatives of UTP and ATP activated the P2Y(2) receptor but were inactive or weakly active at P2Y(4) receptor. Uridine 5'-(diphospho)phosphonate was approximately as potent at the P2Y(2) receptor as at the UDP-activated P2Y(6) receptor. These results suggest that removal of the 5'-oxygen atom from nucleotide agonist derivatives reduces but does not prevent interaction with the P2Y(2) receptor. Uridine 5'-(phospho)phosphonate as well as the 5'-methylenephosphonate equivalent of UMP were inactive at the P2Y(4) receptor and exhibited maximal effects at the P2Y(2) receptor that were 50% of that of UTP suggesting novel action of these analogues.

Gi-dependent cell signaling responses of the human P2Y14 receptor in model cell systems.

Eight G protein-coupled receptors comprise the P2Y receptor family of cell signaling proteins. The goal of the current study was to define native cell signaling pathways regulated by the uridine nucleotide sugar-activated P2Y(14) receptor (P2Y(14)-R). The P2Y(14)-R was stably expressed in human embryonic kidney (HEK) 293 and C6 rat glioma cells by retroviral infection. Nucleotide sugar-dependent P2Y(14)-R activation was examined by measuring inhibition of forskolin-stimulated cAMP accumulation. The effect of P2Y(14)-R activation on mitogen-activated protein kinase signaling also was studied in P2Y(14)-HEK293 cells and in differentiated HL-60 human myeloid leukemia cells. UDP-Glc, UDP-galactose, UDP-glucuronic acid, and UDP-N-acetylglucosamine promoted inhibition of forskolin-stimulated cAMP accumulation in P2Y(14)-HEK293 and P2Y(14)-C6 cells, and this signaling effect was abolished by pretreatment of cells with pertussis toxin. Inhibition of cAMP formation by nucleotide sugars also was observed in direct assays of adenylyl cyclase activity in membranes prepared from P2Y(14)-C6 cells. UDP-Glc promoted concentration-dependent and pertussis toxin-sensitive extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in P2Y(14)-HEK293 cells. P2Y(14)-R mRNA was not observed in wild-type HL-60 cells but was readily detected in dimethyl sulfoxide-differentiated cells. Consistent with this observation, no effect of UDP-Glc was observed in wild-type HL-60 cells, but UDP-Glc-promoted pertussis toxin-sensitive activation of ERK1/2 occurred after differentiation. These results illustrate that the human P2Y(14)-R signals through G(i) to inhibit adenylyl cyclase, and P2Y(14)-R activation also leads to ERK1/2 activation. This work also identifies two stable P2Y(14)-R-expressing cell lines and differentiated HL-60 cells as model systems for the study of P2Y(14)-R-dependent signal transduction.

Distinct phosphodiesterase-4D variants integrate into protein kinase A-based signaling complexes in cardiac and vascular myocytes.

Numerous cAMP-elevating agents regulate events required for efficient migration of arterial vascular smooth muscle cells (VSMCs). Interestingly, when the impact of cAMP-elevating agents on individual migration-related events is studied, these agents have been shown to have distinct, and sometimes unexpected, effects. For example, although cAMP-elevating agents inhibit overall migration, they promote VSMC adhesion to extracellular matrix proteins and the formation of membrane extensions, which are both events that are essential for and promote migration. Herein, we extend previous observations that identified phosphodiesterase-4D3 (PDE4D3) as an integral component of a PKA/A kinase-anchoring protein (AKAP) complex in cultured/hypertrophied rat cardiac myocytes to the case for nonhypertrophied cardiac myocytes. Moreover, we show that while rat aortic VSMCs also express PDE4D3, this protein is not detected in PKA/AKAP complexes isolated from these cells. In contrast, we show that another PDE4D splice variant expressed in arterial vascular myocytes, namely, PDE4D8, integrates into PKA/AKAP-based signaling complexes in VSMCs. Consistent with the idea that a PDE4D8/PKA/AKAP complex regulates specific VSMC functions, PKA and PDE4D8 were each recruited to leading-edge structures in migrating VSMCs, and inhibition of PDE4D8 recruitment to pseudopodia of migrating cells caused localized changes in actin dynamics. Our data are presented in the context that cardiac myocytes and arterial VSMCs may use distinct PDE4D variants to regulate selected pools of targeted PKA activity and that disruption of this complex may allow selective regulation of cAMP-dependent events between these two cardiovascular cell types.

Numerous distinct PKA-, or EPAC-based, signalling complexes allow selective phosphodiesterase 3 and phosphodiesterase 4 coordination of cell adhesion.

By activating two distinct classes of effector enzymes, namely Protein Kinases A [PKA] or Exchange Proteins Activated by cAMP [EPAC], the ubiquitous second messenger cAMP selectively coordinates numerous events simultaneously in virtually all cells. Studies focused on dissecting the manner by which cAMP simultaneously regulates multiple cellular events have shown that cAMP activates its effectors non-uniformly in cells and that this localized cAMP-mediated signalling is made possible, at least in part, by anchoring of cAMP effectors to selected subcellular structures. In the work described here, we report that HEK293T cells ["293T"] contain several PKA- and EPAC1-based signalling complexes. Interestingly, our data do not identify signalling complexes in which both PKA and EPAC are each present but rather are consistent with the idea that these two effectors operate in distinct complexes in these cells. Similarly, we report that while individual PKA- or EPAC-containing complexes can contain either phosphodiesterase 3B, [PDE3B] or phosphodiesterase 4D [PDE4D], they do not contain both these phosphodiesterases. Indeed, although PDE4D enzymes were identified in both PKA- and EPAC-based complexes, PDE3B was largely identified in EPAC-based complexes. Using a combination of approaches, we identified that integration of PDE3B into EPAC-based complexes occurred through its amino terminal fragment [PDE3B(AT)]. Consistent with the idea that integration of PDE3B within EPAC-based complexes was dynamic and regulated PDE3 inhibitor-mediated effects on cellular functions, expression of PDE3B(AT) competed with endogenous PDE3B for integration into EPAC-based complexes and antagonized PDE3 inhibitor-based cell adhesion. Our data support the concept that cells can contain several non-overlapping PKA- and EPAC-based signalling complexes and that these complexes may also represent sites within cells were the effects of family-selective PDE inhibitors could be integrated to affect cell functions, including adhesion.

Both protein kinase A and exchange protein activated by cAMP coordinate adhesion of human vascular endothelial cells.

cAMP regulates integrin-dependent adhesions of vascular endothelial cells (VECs) to extracellular matrix proteins, their vascular endothelial cadherin-dependent intercellular adhesions, and their proliferation and migration in response to growth and chemotactic factors. Previously, we reported that cAMP-elevating agents differentially inhibited migration of human VECs isolated from large vascular structures (macro-VECs, human aortic endothelial cells [HAECs]) or small vascular structures (micro-VECs, human microvascular endothelial cells [HMVECs]) and that cAMP hydrolysis by phosphodiesterase (PDE)3 and PDE4 enzymes was important in coordinating this difference. Here we report that 2 cAMP-effector enzymes, namely protein kinase (PK)A and exchange protein activated by cAMP (EPAC), each regulate extracellular matrix protein-based adhesions of both macro- and micro-VECs. Of interest and potential therapeutic importance, we report that although specific pharmacological activation of EPAC markedly stimulated adhesion of micro-VECs to extracellular matrix proteins when PKA was inhibited, this treatment only modestly promoted adhesion of macro-VECs. Consistent with an important role for cAMP PDEs in this difference, PDE3 or PDE4 inhibitors promoted EPAC-dependent adhesions in micro-VECs when PKA was inhibited but not in macro-VECs. At a molecular level, we identify multiple, nonoverlapping, PKA- or EPAC-based signaling protein complexes in both macro- and micro-VECs and demonstrate that each of these complexes contains either PDE3B or PDE4D but not both of these PDEs. Taken together, our data support the concept that adhesion of macro- and micro-VECs is differentially regulated by cAMP and that these differences are coordinated through selective actions of cAMP at multiple nonoverlapping signaling complexes that contain PKA or EPAC and distinct PDE variants.

Formation of extracellular matrix-digesting invadopodia by primary aortic smooth muscle cells.

Invasion of the subendothelial space by vascular smooth muscle cells (VSMCs) contributes to the development and progression of diverse cardiovascular diseases. In this report we show that the expression of activated versions of Src, Cdc42 and Rac1, or a kinase-dead but open form of the p21-activated kinase (PAK1), induces primary rat aorta VSMCs to form extracellular matrix-degrading actin-rich protrusions that are morphologically similar to the invadopodia formed by highly invasive tumor cells. The matrix-degrading structures are enriched in known markers for invadopodia, including cortactin and tyrosine-phosphorylated cortactin and contain the matrix metalloproteinases MMP-9 and MT1-MMP and the urokinase plasminogen activator receptor (uPAR). In contrast to other cell types, invadopodia formation in VSMCs is only weakly supported by the phorbol ester PBDu. Invadopodia formation by Src was dependent on Cdc42, Rac, and ERK, but not on p38 MAPK. Invadopodia formation induced by kinase-dead PAK1 required Src and ERK activity and a direct interaction with the exchange factor PIX. VSMCs embedded in a three-dimensional collagen matrix formed actin- and cortactin-rich extensions that penetrated through holes in the matrix, suggesting that invadopodia-like structures are formed in a three-dimensional environment.

Protein kinase A phosphorylation of human phosphodiesterase 3B promotes 14-3-3 protein binding and inhibits phosphatase-catalyzed inactivation.

Recent studies confirm that intracellular cAMP concentrations are nonuniform and that localized subcellular cAMP hydrolysis by cyclic nucleotide phosphodiesterases (PDEs) is important in maintaining these cAMP compartments. Human phosphodiesterase 3B (HSPDE3B), a member of the PDE3 family of PDEs, represents the dominant particulate cAMP-PDE activity in many cell types, including adipocytes and cells of hematopoietic lineage. Although several previous reports have shown that phosphorylation of HSPDE3B by either protein kinase A (PKA) or protein kinase B (PKB) activates this enzyme, the mechanisms that allow cells to distinguish these two activated forms of HSPDE3B are unknown. Here we report that PKA phosphorylates HSPDE3B at several distinct sites (Ser-73, Ser-296, and Ser-318), and we show that phosphorylation of HSPDE3B at Ser-318 activates this PDE and stimulates its interaction with 14-3-3 proteins. In contrast, although PKB-catalyzed phosphorylation of HSPDE3B activates this enzyme, it does not promote 14-3-3 protein binding. Interestingly, we report that the PKA-phosphorylated, 14-3-3 protein-bound, form of HSPDE3B is protected from phosphatase-dependent dephosphorylation and inactivation. In contrast, PKA-phosphorylated HSPDE3B that is not bound to 14-3-3 proteins is readily dephosphorylated and inactivated. Our data are presented in the context that a selective interaction between PKA-activated HSPDE3B and 14-3-3 proteins represents a mechanism by which cells can protect this enzyme from deactivation. Moreover, we propose that this mechanism may allow cells to distinguish between PKA- and PKB-activated HSPDE3B.