Paul A. Insel - US grants

The principal goal of this project is to examine how catecholamine-responsive cells regulate their number of alpha- and beta-adrenergic receptors. Recent work in my laboratory using radioligand binding techniques and functional assays has shown that two cell types, BC3H-1 muscle cells and MDCK renal tubular epithelial cells, co-express alphal-and beta2-adrenergic receptors. These two cell types thus offer a unique opportunity to test directly whether alpha- and beta-adrenergic receptors present in the same cell are separate molecular species; to resolve this issue we propose experiments of solubilized receptors analyzed by density gradient centrifugation, gel filtration chromatography, and SDS polyacrylamide gel electrophoresis. Moreover, we will compare the "life cycle" of these two types of receptors in the same cell by using BC3H-1 and MDCK cells to assess biosynthesis and degradation of alphal- and beta2-adrenergic receptors. We will establish two separate methods--one involving "heavy amino acids" and the other receptor-specific, irreversible antagonists--in order to define the kinetics and mechanisms of receptor formation and turnover. In additional studies of mechanisms regulating receptor metabolism, we will use inhibitors of processes implicated in such regulation and will monitor the fate of receptors labeled with irreversible antagonists. In further experiments we will relate the appearance of surface receptors to the acquisition of functional activity and will define the contribution of changes in receptor synthesis and degradation to agonist-mediated "down regulation." The experiments in this proposal have been designed to assess alpha- and beta-adrenergic receptor metabolism in target cells using methods that only minimally perturb cellular function. Accordingly, the results are likely to yield new and potentially important information regarding cellular events that receptors on catecholamine-responsive cells. Because of the wide range of tissues capable of responding to catecholamines and the importance of adrenergic drugs in therapy of many clinical disorders, the results are likely to prove applicable not only to the two principal cell types that we will study--muscle and kidney epitheliuim--but also to a large number of other cells that possess functional adrenergic receptors. Information gained in these studies should have particular relevance to cardiovascular and pulmonary diseases.

Considerable data have documented the key role the kidney plays in the pathophysiology of hypertension and of the likelihood that alterations in adrenergic response occur in experimental and clinical hypertension. We propose to test the hypothesis that changes in renal adrenergic receptors contribute to pathophysiologic manifestations of hypertension. In our recent work we have demonstrated that we can use radioligand binding techniques to identify and study regulation of the 4 principal subtypes of adrenergic receptors (Alpha1, Alpha2, Beta1, Beta2) in rat renal cortical membranes. In addition, we have developed in vitro autoradiography methods to study adrenergic receptors in kidney slices. Our aims in this proposal are to examine changes in receptor expression in vivo by using radioligand binding, in vitro autoradiography, kinetic modelling of binding data, and assessment of biochemical and functional responses. Other studies will examine agonist-mediated uncoupling of receptors and covalent modification in receptors by photoaffinity labelling. We will then use those techniques to assess changes in renal adrenergic receptors in 3 rat models of hypertension: SHR, DOCA-saline, and salt-sensitive Dahl hypertension, each of which may provide unique insights into the role of renal adrenergic receptors in this disease. Our long term goal is to define mechanisms whereby target cells regulate and are regulated by adrenergic receptors. The proposed studies whould provide new insights into mechanisms that operate in vivo to regulate adrenergic receptors and in particular to mechanisms regulating renal receptors in normal and hypertensive animals.

This project is based on the premise that studies of hormone and drug receptors in intact cells can provides physiologically relevant information regarding regulation of receptor expression and function. The principal focus of the proposal is on beta- adrenergic receptors of wild-type S49 lymphoma cells and S49 variants having lesions in the pathway of beta-adrenergic receptors/Gs/adenylate cyclase/cAMP dependent protein kinase. I propose to use several different approaches to define various features of the beta-adrenergic receptor "life cycle in the S49 system. These approaches includes use of a new irreversible probe, BIM, to block beta-receptors selectively and then to measure recovery of receptor binding and function (cAMP generation) to control levels. Kinetic analysis of recovery data will allow us to estimate rates of receptor appearance and disappearance. Studies with S49 variants will determine the role of distal components in the beta-receptor pathway in regulation of receptor appearance and disappearance and experiments with various inhibitors will help dissect out the contribution of particular processes to calculated rates of receptor appearance and his disappearance. Further studies will involve preparation of antireceptor antibodies using synthetic peptides and chimeric proteins derived from transient expression of restriction fragments of beta2-receptor cDNA in E. Coli. Antibodies will be used to define additional aspects of beta-receptor synthesis and turnover. Methods will also be developed to quanititate beta2 receptor mRNA in S49 cells. In additional studies we will use antipeptide antibodies that we have generated to the Gs protein to examine formation and turnover of this protein in wild-type and variant S49 cells. Other experiments will be directed at defining the basis for the depletion of beta-adrenergic receptors in two S49 variants, beta p and beta d, which lack -50% and -85% of cellular beta- receptors, respectively. Taken together these studies should provide new insights into the mechanisms by which cells regulate their expression of beta-adrenergic receptors. Because of the wide distribution of those receptors, the results should be of relevance to beta-receptors in several organ systems, perhaps most importantly in the cardiovascular and pulmonary systems, and should provide new insights into the molecular pharmacology of beta- adrenergic drug responses.

This project continues my laboratory's studies of cellular actions of alpha1-adrenergic receptors using a clonal isolate of Madin Darby kidney (MDCK) cells. In recent studies our laboratory has shown that in these cells alpha1-adrenergic receptors may be of a unique subtype that links to a guanine nucleotide binding (G) protein and in turn to multiple phospholipases, including one or more phospholipases A2, C and D. Regulation of phospholipase A2 by alpha1-adrenergic receptors occurs in part via protein kinase C, in particular, by the alpha isoform of this enzyme. the current studies are designed to test hypotheses related to alpha-adrenergic receptor structure, phospholipase regulation, and protein kinase C isoforms in MDCK-D1 cells. Pharmacological methods will be used to define alpha1-receptor subtypes and subsequent molecular biological methods will be used to characterize the subtype(s) present in MDCK D1 cells. Studies of phospholipases will involve biochemical characterization of phospholipase D and phospholipase A2. The third aim is to assess the role of isoforms of protein kinase C in cell regulation, especially in regulation of phospholipase A2. The methods to be used in these latter studies include treatment of cells with antisense oligonucleotides and transient and stable transfection with antisense cDNA's to generate cells null with respect to individual C-kinase isoforms. Other approaches will involve microscopy with immunological probes for particular isoforms. Taken together, the studies proposed should provide new information regarding alpha1-adrenergic receptor action and the role of protein kinase C isoforms in regulation of cellular function. Given the importance of alpha1-adrenergic receptors and protein kinase C for a wide variety of cellular functions, the results may have importance to disease settings such as cancer and several types of cardiovascular disorders.

Increased adrenergic activation provides an important means of increasing cardiac function in the normal heart. However when ventricular function has substantially deteriorated, the effects of sustained adrenergic drive may no longer be desirable. Thus, adrenergic activation serves as an important compensatory mechanism for decreased cardiac function, but ultimately this mechanism exacerbates the problem and may contribute to morbidity and mortality. Our purpose will be to explore the molecular pathogenesis of abnormal adrenergic responsiveness in the hart and in cardiac myocytes during the development of congestive heart failure (CHF). We will use several complementary approaches - assessment of signal transduction mechanisms in an animal model of dilated cardiomyopathy and in isolated myocytes, as well as generation of transgenic mice. We will focus on betaAR signalling and on the molecular mechanism for alterations in signal transduction. We will evaluate the physiological impact of these changes in conscious animals by sequential hemodynamic and ventricular functional assessments. A strength of our laboratories is that we are able to wed physiological and molecular studies of adrenergic function in the heart. Substantial work has been performed regarding changes in beta-adrenergic receptor-mediated signalling in the failing heart, and thus we will focus upon three key areas that have received little attention but that are likely to provide important new mechanistic information; 1) precise assessment of alterations in the quantify and function of the catalytic subunit of adenylyl cyclase (C), and molecular mechanisms for changes in this protein associated with CHF; 2) the molecular pathogenesis and physiological impact of altered levels of beta-adrenergic receptor kinase (betaARK) on adrenergic signalling in the failing heart; and 3) the pathogenesis of decreased cardiac Gsalpha, the alpha subunit of the stimulatory guanine nucleotide regulatory protein, and the role of the intracellular redistribution of cardiac Gsalpha in CHF. We hypothesize that exposure of cardiac myocytes to increased levels of catecholamines in CHF will lead to specific alteration in C, betaARK and Gsalpha. Specific hypotheses regarding each of these are; 1. In CHF the amount and function of one or more isoforms of C, most likely types V and VI, will be decreased. 2. In CHF the amount and function of beta- adrenergic receptor kinase (betaARK) will be increased, thereby promoting the uncoupling of receptors from Gsalpha. 3. In CHF the distribution of Gsalpha will be altered so that less is found in the sarcolemma and a greater portion is found in intracellular compartments, thereby decreasing the likelihood that remaining cell surface receptors activate adenylyl cyclase. Complementary studies on human tissue from failing and control hearts will examine content of the catalytic subunit of adenylyl cyclase, quantity betaARK levels and cellular Gs content and its compartmentation. These investigation should provide new information regarding the molecular pathogenesis for altered myocardial adrenergic signalling in heart failure. Data from these studies, we hope, will provide fundamental information leading to new therapeutic strategies for patients with congestive heart failure.

The physiological catecholamines, epinephrine and norepinephrine, and many clinical useful pharmacological agonists and antagonists act on beta-adrenergic receptors. Although much new knowledge has recently been provided regarding beta-adrenergic receptor structure, much remains unknown regarding regulation of receptor expression and function in target cells. In this project we propose to continue studies that emphasize cellular and molecular mechanisms that regulate signal transduction by beta-adrenergic receptors in intact cells. My colleagues and I will continue to study wild-type S49 lymphoma cells and S49 variants in the pathway for beta receptor signalling via the guanine nucleotide regulatory protein, G(s) and in turn, adenylyl cyclase and cAMP-dependent protein kinase. We propose three specific aims designed to characterize the life cycles of beta-adrenergic receptors, Galpha(s), and the catalyst (C) of adenylyl cyclase. Antibodies to these components will be used to characterize biosynthesis and turnover and to assess the role of phosphorylation of beta-receptors and C. Other studies are designed to use molecular biological techniques (such as antisense technology and transfection with cDNAs) to help define the role of the beta-adrenergic receptor kinase (beta-ARK) in S49 cells and to evaluate stoichiometry between alpha(s) and C. Additional experiments will use biochemical and micrologic techniques to assess the subcellular localization of Galpha(s). Taken together, experimental approaches should provide new information regarding the key components involved in beta-adrenergic receptor signal transduction. As such, the results should be relevant to a variety of clinical settings, in particular cardiovascular disorders, in which betaadrenergic receptor function may be altered. In addition, these studies should help advance understanding of the molecular basis of pharmacological action at beta-adrenergic receptors and at other receptors that activate G(s) and adenylyl cyclase.

adrenergic receptor; biomedical facility; biological signal transduction; receptor binding; nucleic acid sequence; tissue /cell culture; human tissue;

DESCRIPTION (the applicant's description verbatim): This proposal is designed to test hypotheses related to gene transfer of adenylyl cyclases to modulate agonist-specific cyclic AMP generation of cardiovascular cells. Adenylyl cyclase (AC) expression appears to be the limiting component for maximal generation of cyclic AMP by agonists, such as beta-adrenergic agonists, that promote such generation. Our preliminary data in cardiac myocytes demonstrate that increased expression of AC-6 (one of the ten isoforms of this enzyme) dramatically enhances beta-adrenergic response without enhancing response to several other stimulatory agonists but while retaining and perhaps enhancing ability of the muscarinic cholinergic agonist carbachol and endothelin to inhibit cAMP generation. We propose to test the impact of increasing expression of AC-6 (using an adenoviral construct) on cyclic AMP formation in key cardiovascular cells, endothelial cells, and vascular smooth muscle cells, in addition to cardiac myocytes, and in particular, to characterize the patterns of stimulation and inhibition of AC-6 in such cells. In other Aims we will test hypotheses developed to explain the selective enhancement of beta-adrenergic response by overexpressed AC-6: 1) preferential coupling of beta-adrenergic receptors to AC-6 compared to certain other AC isoforms (by testing impact of adenoviral transfer of AC-4 and AC-8, each with its own unique pattern of regulation, to cardiac cells); 2) inhibitory regulation of AC-6 that may contribute to patterns of receptor stimulation of cAMP synthesis; and 3) targeting of AC isoforms to membrane domains enriched in certain receptors and other components that regulate cyclic AMP formation and action. Results of these studies should yield new information regarding the role of AC expression as a limiting component in cAMP formation, the impact of gene transfer of AC isoforms to several key cardiovascular cells, and provide evidence that this approach may provide a novel means to alter signaling in such cells.

The overriding goal of this Unit is to provide a rationale and an initial test of what we believe may be a new and potentially useful approach for the treatment of heart failure. In the past 5 years we have shown that expression of the catalyst of adenylyl cyclase (AC) is the key limiting component in the generation of cyclic AMP by beta-adrenergic receptor (betaAR) activation. Thus, modulating expression of Ac is a rational strategy for regulating cardiac responsiveness. We have tested the utility of over-expression of AC/VI, a predominant AC isoform in mammalian cardiac myocytes, as a means to increase cardiac responsiveness to catecholamine stimulation. We have demonstrated that this strategy is a highly effective means to increase intracellular cAMP and cardiac function in response to catecholamine stimulation. Over-expressing AC does not alter transmembrane signaling except when receptors are activated, in distinction with receptor/G protein over-expression, which yield continuous activation and the detrimental consequences. Preliminary experiments indicate that expressing AC in the background of cardiomyopathy improves cardiac function and adrenergic responsiveness, These data indicate that cardiac over-expression of AC may be a safe effective means to treat heart failure. We propose four Specific Aims, each of a hypothesis-testing nature, as follows: 1) To test the hypothesis that increased AN/VI expression provides relative betaAR -selective amplification of transmembrane signaling; 2) To test the hypothesis that compartmentation of AC/VI provides betaAR-selective amplification of transmembrane signaling; 3) To test the hypothesis that cardiac over-expression of AC/VI improves cardiac function and responsiveness in murine dilated cardiomyopathy; 4) To test the hypothesis that cardiac over-expression of AC/VI improves cardiac function of betaAR responsiveness in a large animal model of heart failure.

neurotransmitter receptor; genetic polymorphism; biomedical facility; tissue /cell culture; pharmacogenetics; transfection; cooperative study; chromaffin cells;

The overriding of this hypothesis is that gene transfer of isoforms of adenylyl cyclase (CA) can be used to increase cyclic AMP (cAMP) synthesis and action in pulmonary artery smooth muscle cells (PASMC). PASMC, whose function is altered in a variety of important clinical conditions associated with pulmonary arterial hypertension, will be used with adenoviral or other viral vectors to assess gene transfer of AC type 6 and 8, two AC isoforms with unique patterns of regulation. We will assess impact o increased AC expression on cAMP generation and on "downstream" responses including activation of cAMP-dependent protein kinase, metabolic, contractile and growth responses, and on ion channel activity and expression. Other studies will assess compartmentation of AC expression and function of PASMC cells derived from patients with primary pulmonary hypertension. Taken together, the proposed studies should provide new information regarding gene transfer of AC to PASMC and impact of increased AC expression of PASMC function. Successful completion of these studies would provide an experimental basis to initiate clinical studies in patients with abnormal PASMC function.

DESCRIPTION (provided by applicant): We propose to continue our long-standing studies in the B-adrenergic receptor (BetaAR) signalling pathway of S49 lymphoma cells. These cells provide a unique model system in which to characterize the role of particular components in this pathway in which BAR increase cellular levels of cyclic AMP (cAMP), activate protein kinase A (PKA), and trigger events that ultimately lead to growth arrest and death of the cells. Feedback regulation of signalling occurs via a G protein receptor kinase (GRK), as well as PKA. Our preliminary studies indicate that cAMP-mediated cell death of S49 cells occurs via an apoptotic mechanism that requires PKA. Moreover, other recent findings implicate a key role of PKA desensitization of the BAR pathway. We propose to utilize wild-type S49 cells and known variants of S49 cells with lesions in the BAR response pathway (including an S49 cell line which does not undergo apoptosis in spite of apparently normal "upstream" signalling components) to define the role of cAMP generation/PKA activation and mechanisms involved in apoptosis and in addition the contribution of G protein receptor kinase and PKA to ongoing feedback regulation of events involved in cAMP formation and action. Based on other preliminary results, we will also conduct studies of GRK and PKA using Chinese hamster ovary (CHO) cells, both wild-type and PKA-absent CHO cells. Data in the CHO system regarding receptor desensitization should complement studies in the S49 cell system. By using a combined approach that involves techniques of cell and molecular biology, biochemistry, and pharmacology, we should obtain new information regarding BAR signalling. Since BAR play an important physiologic role in many organ systems, in particular the cardiovascular, renal, and pulmonary systems, and BAR are targets of drug therapy in many clinically important disorders (e.g., hypertension, asthma, myocardial ischemia, chronic obstructive pulmonary disease, congestive heart failure), our results may provide useful new insights regarding both physiological regulation as well as drug therapy with agents that stimulate or block BetaAR signalling. Moreover, the results should prove applicable to the many other hormone and neurotransmitter receptors that link to G proteins.

DESCRIPTION (provided by applicant): This application is a revised proposal to assess aspects of adrenergic receptor and P2Y purinergic receptor signalling. Our recent, preliminary data for this application include the identification and cloning of five different P2Y receptors in MDCK-D1 cells and discovery of a key role of ATP release as an autocrine/paracrine mechanism for establishing basal levels of signal transduction in these cells. The proposed studies will focus on studies in MDCK-D1 cells, designed to define information regarding nucleotide release and autocrine/paracrine signalling by P2Y-receptors, stoichiometry and compartmentation of G protein-coupled receptors/G protein and effector molecules, agonist-promoted regulation of adrenergic and P2[unreadable]-receptors, and in parallel studies using murine knockouts, the role of P2Y receptors and adrenergic receptors in regulation of renal function. The studies will test several hypotheses, including the possibilities that: 1) nucleotide release is a critical factor for the set point of multiple signal transduction pathways; 2) P2Y receptors, A1-AR and (B2-AR differentially target in polarized MDCK cells, and create compartmentalized signalling microdomains; and 3) adrenergic and P2Y receptors will show evidence of"cross regulation." The latter may contribute to modulation of response via sympathetic postganglionic neurons in the kidney and other cells. In additional experiments, we will attempt to define the functional activity of adrenergic and P2[unreadable] receptors in vivo by the studies of mice with knockouts of certain receptors. Overall, the data should provide new insights into regulation of epithelial cells, in particular renal epithelial cells, by adrenergic and P2Y receptors and should help define the cell and renal physiological role of those receptors. In addition, the findings may have pathophysiologic and therapeutic implications for diseases such as cardiovascular, renal, and other disorders that involve the sympathetic nervous system or in which cell injury leads to release of nucleotides.

Abstract not provided

Cardiac fibrosis, a pathological consequence of cardiac injury, can be manifest either as localized scar or[unreadable] more diffuse interstitial fibrosis. The cells responsible for cardiac fibrosis are cardiac fibroblasts, which[unreadable] numerically are the most prevalent cells in the heart. Cardiac fibroblasts are regulated by hormones, many of[unreadable] which act via plasma membrane receptors, including ones that are linked to heterotrimeric G proteins and G-protein-[unreadable] regulated effectors. One such effector, adenylyl cyclase, catalyzes the formation of cyclic AMP from[unreadable] ATP and is the focus of this proposal. Cyclic AMP has anti-fibrotic actions that include inhibition of the[unreadable] transformation of "resting" cardiac fibroblasts to pro-fibrogenic myofibroblasts. This proposal will test several[unreadable] hypotheses related to the ability of increased cyclic AMP formation, produced by enhanced expression of[unreadable] adenylyl cyclase-6 (AC-6), to alter biochemical and functional activities of cardiac fibroblasts. Studies will be[unreadable] conducted using primary cultures of cardiac fibroblasts and, in addition, will involve the use of an animal[unreadable] model (angiotensin infusion in rats and mice) that produces cardiac fibrosis. The experiments focus on AC-6[unreadable] with an emphasis on its compartmentation with proximal and distal signaling components as well as impact[unreadable] of increased AC-6 expression on the fibrotic response, as assessed by several phenotypic characteristics.[unreadable] The results should provide proof-of-principle data regarding the potential for therapeutic targeting of cardiac[unreadable] fibroblasts by gene transfer with AC-6 and potentially other AC isoforms. It is likely that targeting of AC-6 to[unreadable] cardiac myocytes via intracoronary delivery of recombinant viral vectors (Project 1) will increase AC-6[unreadable] expression in fibroblasts. The general hypothesis of Project 2 is that increased expression of AC-6 in cardiac[unreadable] fibroblasts will alter the fibrotic response and favorably modify cardiac remodeling to improve cardiac[unreadable] function in the failing heart.

DESCRIPTION (provided by applicant): This application requests funding for the 2009 Gordon Research Conference on Molecular Pharmacology to be held at Il Ciocco Conference Center in Barga (Pisa), Italy, May 31st-June 5th 2009. The Molecular Pharmacology Gordon Research Conference has a long history of attracting the best scientists in the field. It is a unique forum for the gathering of scientists from academia, government, and industry to discuss recent, cutting-edge advances in signal transduction research, receptors and other drug targets. The broad goal of the Conference has been, and continues to be, to advance progress by providing an integrated approach among important "growth areas" in Molecular Pharmacology. The 2009 Conference seeks to integrate two main aspects of research in molecular pharmacology, i.e. basic and translational, by highlighting recent advances in molecular mechanisms and pathophysiological implications of drug receptors and drug action through the use of experimental and clinically relevant models. Although speakers and session chairs will provide an introduction suited for the less-acquainted attendee, presentations will be geared to late-breaking, novel and unpublished findings. The conference is expected to attract leading investigators from the field and expected to have ~120 participants of whom ~85% will be academic researchers. Approximately 25% are expected to be graduate students and postdoctoral researchers. About 25% of the participants will speak;the majority of the remainder will present posters. Among the specific aims of the 2009 Conference is to encourage the participation of students and post-docs and to pay particular attention to the inclusion of women and members of underrepresented minorities. The 2009 program will cover three main aspects: i) structural features of receptors and monitoring of receptor function at single molecule level, ii) signaling and regulatory networks in cancer, viral infections, endocrine and cardiovascular disorders, and iii) murine models that explore behavior, drugs of addiction and regulation of metabolism. PUBLIC HEALTH RELEVANCE: The Conference proposed in this application addresses basic research questions that can have direct clinical relevance and applications. A bidirectional transfer of information exists between molecular pharmacology and clinical medicine: molecular pharmacology elucidates fundamental aspects of drug action and provides new pharmacological tools useful in patient care while in parallel, pathophysiological and therapeutic problems in clinical medicine raise new questions and trigger new hypotheses for testing in basic research studies. Topics at the 2009 Conference are relevant to a number of areas including cancer, viral infections, cardiovascular and endocrine disorders as well as behavior and regulation of metabolism.

DESCRIPTION (provided by applicant): Molecular Pharmacology focuses on the mechanisms of drug action at a molecular level. The use of drugs as clinical diagnostic and therapeutic agents and in studying mechanisms in physiology and disease makes molecular pharmacology a very dynamic discipline in experimental biology and medicine. Molecular pharmacology is an interface discipline that integrates approaches from fields that include molecular, cellular and structural biology, genomics, chemistry and systems biology. Research in molecular pharmacology seeks to elucidate fundamental aspects of drug action and provide new pharmacological tools and therapies. In addition, progress in clinical medicine has raised new questions regarding the molecular basis of drug action. The placement of molecular pharmacology at the interface of experimental biology and translational research makes it an exciting area of discovery that seeks to provide answers to questions of relevance to clinical medicine. These ideas underlie this application for NIH support for the 2011 Molecular Pharmacology Gordon Research Conference (GRC), which seeks to integrate basic and translational research and to highlight recent advances regarding molecular mechanisms and pathophysiological aspects of drug receptors in cell systems and animal models. Topics at this GRC will be in areas that include signaling and drug action in cancer, infection, cardiovascular and endocrine disorders as well as evolving information regarding critical molecular aspects that contribute to behavior, drugs of addiction and the regulation of multiple organ systems. PUBLIC HEALTH RELEVANCE: Beyond its role in updating information within the field, the Molecular Pharmacology GRC contributes to the training and creative development of the next generation of scientists who will drive the field forward. The intimate, informal atmosphere of the conference gives trainees (e.g., graduate students and post-doctoral fellows) an opportunity to experience the excitement of cutting-edge research and to interact with senior scientists.

DESCRIPTION (provided by applicant): Pancreatic cancer, in particular pancreatic ductal adenocarcinoma (PDAC), is the fourth leading cause of cancer death in the U.S. and has a 5-year survival of ~ 5%. A key contributor to this poor survival is the extensive desmoplasia (abundant fibrotic stroma) of PDAC, which can make it difficult to surgically remove the tumor and can limit access of therapeutic drugs to the tumor cells. Cancer-associated fibroblasts (CAFs), myofibroblast-like cells that produce extracellular matrix proteins, are responsible for the desmoplasia in PDAC. Pancreatic stellate cells (PSCs) and fibroblasts (PFs) are key progenitors of CAFs. Blocking the expression and activity of CAFs may be a means to improve the therapy and prognosis of PDAC. This proposal posits that G-protein-coupled receptors (GPCRs) are an important, understudied aspect of pancreatic CAFs and may be novel targets for the treatment of pancreatic cancer. GPCRs are the largest family of cell surface signaling receptors (3% of the human genome) and regulate many physiological processes. As plasma membrane proteins, GPCRs are accessible from the extracellular space; GPCRs are selectively expressed on cell types and tissues and show specificity in ligand interaction-factors that help explain why GPCRs are the targets for ~30% of current therapeutic agents albeit not in oncology. This R21 seeks to be transformative by identifying, quantifying and validating the expression of GPCRs by human pancreatic CAFs, and by assessing the therapeutic potential of GPCRs selectively expressed by CAFs (compared to PSCs and PFs). The project uses patient-derived pancreatic CAFs as a model and will use several genomic strategies: a GPCRomic approach to quantify gene expression of all non-chemosensory GPCRs, use of RNA interference knockdown GPCRs expressed by CAFs and identify the GPCRs that mediate key functional responses and gain-of-function studies by using normal pancreatic fibroblasts or pancreatic stellate cells and seeking to recapitulate the fibrotic phenotype of CAFs by enhancing expression of GPCRs in PSCs and PFs. The goal is to use genomic approaches to identify GPCRs as potential targets for the treatment of pancreatic cancer through their ability to block expression and pro-fibrotic activity of CAFs. The strategy that we will employ- GPCRomics of members of the GPCR gene family and gain of function and loss of function studies with members of this family in a human disease (in this case, in pancreatic cancer-associated fibroblasts)-is a novel one that should be readily applicable to CAFs in other cancers and to other disease settings. In addition, a further novel and clinically important aspect of this proposl is its initiation of efforts to test the idea that GPCRs on CAFs (in particular pancreatic CAFs) may be druggable targets for a disease that is in desperate need of new therapies.

Project Summary/Abstract This application seeks to assess a new mechanism for aging-related alterations in cell and tissue function. The proposed studies will determine if our recent discovery and preliminary data regarding a previously unrecognized mechanism for signal transduction---direct, membrane-delimited activation of matrix metalloproteinase 14 (MMP14, also known as MMT1-MMP [membrane type-1 matrix metalloproteinase] by GTP and GTP?S acting via heterotrimeric (???) GTP binding (G) proteins?contribute to such alterations, in particular in cardiac cells and the heart. We find that GTP-promoted activation of MMP14 releases (?sheds?) proteins from the membrane, including heparin binding epidermal growth factor (HB-EGF) and leads to activation of EGF receptors, thus identifying a new mechanism by which G-protein coupled receptor (GPCR)/heterotrimeric G-proteins transactivate receptor tyrosine kinases. Our preliminary data indicate that MMP14 activity and its activation by GTP?S is increased in cardiac cell membranes from aged mice. We propose that this increase in MMP14 activity is a consequence of age-related alterations in the plasma membrane, in particular a loss in caveolin proteins, which scaffold signaling molecules. The Aims of the studies proposed in this R21 application are to: 1) Define the heterotrimeric G-protein subunit(s) that activate MMP14, the mechanism of its activation by the G-proteins, and of the aging-related increase in MMP14 activity through co-localization with caveolins and loss of caveolin- mediated inhibition of MMP14 and 2) Determine the ability of MMP14 activation by G-proteins to release membrane proteins and activate extracellular proteins, (e.g., soluble MMPs) and to assess the role of MMP14 in aging-related loss in cardiac cell function, including in isolated hearts. Our working hypothesis, which the proposed studies will test, is that MMP14 is part of a GPCR/G-protein/MMP14/MMP14 targets signaling module that is altered in aging as a consequence of aging-related changes in membrane structure. Our preliminary data provide evidence in support of this idea. We believe that those data and our past efforts related to G-protein- mediated signaling and combined efforts of those involved in this project will facilitate success in the proposed studies. In addition, we believe that the results will provide novel information of relevance to biochemical and physiological changes that occur in aging as well as results of interest to multiple disciplines (biochemistry, cell biology, physiology/pathophysiology and pharmacology). Moreover, the findings may also be relevant to other tissues and cell types besides the cardiovascular cells that we will study in the proposed experiments and may identify new targets for the prevention and/or treatment of aging-related disorders.

Project Summary/Abstract Cardiovascular disease is the major cause of death in Americans over the age of 65. Mechanisms of cardiovascular aging are not well defined nor are effective treatments available. This project proposes a novel hypothesis: age-related loss in the expression by cardiac myocytes of caveolae and the protein caveolin-3 with the resultant stimulation of pro-fibrotic activity of cardiac fibroblasts, thereby enhancing production of extracellular matrix, cardiac fibrosis, diastolic dysfunction, and heart failure, especially heart failure with preserved ejection fraction (HFpEF). HFpEF is a clinical problem for which no effective therapies exist. This hypothesis derives from our preliminary data which show that cardiac myocytes of aged mice have a loss in expression of caveolae and their key resident protein, caveolin-3 and that cardiac fibroblasts isolated from aged animals have increased pro- fibrotic activity, which we detect in fibroblasts cultured ex vivo. Our proposed studies will test if caveolin-3 in cardiac myocytes has cardioprotective properties in aging by reducing the profibrotic state of cardiac fibroblasts, thus blunting the development and progression of aging-related cardiac fibrosis. In addition, we will determine if restoration of the loss in caveolin-3 with advanced age can reduce age-related cardiac fibrosis. Our two specific aims will ask: 1) Does caveolin-3 expression in cardiac myocytes regulate the pro-fibrotic state of cardiac fibroblasts and cardiac fibrosis in aging? and 2) Does restoration of caveolin-3 in cardiac myocytes reduce age- related cardiac fibrosis? In Aim 1, we will assess cardiac function and the fibrotic activity of cardiac fibroblasts isolated from young (2-3 month) and aged (18-22 month) wild-type, caveolin-3- knockout, and cardiac myocyte- targeted caveolin-3 over-expressing mice. We will also conduct RNA-seq studies of cardiac myocytes and cardiac fibroblasts and will use conditioned media from cardiac myocytes to assess exosomes and soluble proteins released into the media by young and old hearts. In addition to studies of physiological aging, we will subject mice to transaortic constriction, which induces cardiac fibrosis, so as to increase the dynamic range of fibrotic changes. In Aim 2, we will test the impact of AAV constructs engineered to increase caveolin-3 expression in cardiac myocytes on cardiac function, cardiac myocytes and cardiac fibroblasts using the approaches from Aim 1. Based on our preliminary data and past efforts, the proposed studies should be highly feasible even though they are of the high risk/high reward type that is sought in R21 applications. We believe that the results will advance understanding of aging-related cardiac fibrosis and dysfunction and may identify a therapeutic approach?increasing caveolin-3 expression in the heart?for a major contributor to morbidity and mortality in the aged population.