[Show abstract][Hide abstract] ABSTRACT: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a powerful platform for uncovering disease mechanisms and assessing drugs for efficacy/toxicity. However, the accuracy with which hiPSC-CMs recapitulate the contractile and remodeling signaling of adult cardiomyocytes is not fully known. We used β-adrenergic receptor (β-AR) signaling as a prototype to determine the evolution of signaling component expression and function during hiPSC-CM maturation. In "early" hiPSC-CMs (less than or equal to d 30), β2-ARs are a primary source of cAMP/PKA signaling. With longer culture, β1-AR signaling increases: from 0% of cAMP generation at d 30 to 56.8 ± 6.6% by d 60. PKA signaling shows a similar increase: 15.7 ± 5.2% (d 30), 49.8 ± 0.5% (d 60), and 71.0 ± 6.1% (d 90). cAMP generation increases 9-fold from d 30 to 60, with enhanced coupling to remodeling pathways (e.g., Akt and Ca(2+)/calmodulin-dependent protein kinase type II) and development of caveolin-mediated signaling compartmentalization. By contrast, cardiotoxicity induced by chronic β-AR stimulation, a major component of heart failure, develops much later: 5% cell death at d 30 vs. 55% at d 90. Moreover, β-AR maturation can be accelerated by biomechanical stimulation. The differential maturation of β-AR functional vs. remodeling signaling in hiPSC-CMs has important implications for their use in disease modeling and drug testing. We propose that assessment of signaling be added to the indices of phenotypic maturation of hiPSC-CMs.-Jung, G., Fajardo, G., Ribeiro, A. J. S., Kooiker, K. B., Coronado, M., Zhao, M., Hu, D.-Q., Reddy, S., Kodo, K., Sriram, K., Insel, P. A., Wu, J. C., Pruitt, B. L., Bernstein, D. Time-dependent evolution of functional vs. remodeling signaling in induced pluripotent stem cell-derived cardiomyocytes and induced maturation with biomechanical stimulation.
Full-text · Article · Dec 2015 · The FASEB Journal
[Show abstract][Hide abstract] ABSTRACT: In this volume, the Annual Review of Pharmacology and Toxicology (ARPT) continues a feature that began with the prior volume: a series of review articles organized around a Theme. The Editors and Editorial Committee members of ARPT seek to identify a Theme for each volume. Our goal is to assemble for readers a bundled group of reviews that emphasize current and emerging aspects in pharmacology and toxicology and that provide complementary insights regarding the topic of the Theme. "Cancer Pharmacology," the Theme in this volume, includes articles that emphasize fundamental aspects of pharmacology and toxicology and others that address translational and clinical features of cancer therapeutics. We believe that these articles capture for readers the vitality and excitement of research in cancer biology and, especially, in the treatment of cancer. Of note, some of the articles are an extension of last year's Theme, "Precision Medicine and Prediction in Pharmacology." Indeed, cancer therapeutics is the area of clinical medicine that is moving the fastest toward personalized treatment (based, in particular, on genetic features of tumors) in the United States (in part, via the Precision Medicine Initiative introduced in 2015 by President Obama) and abroad. The Editors have cast a wide net in choosing 12 articles in this volume that fit into the "Cancer Pharmacology" Theme, but readers may identify others that are relevant to this area. We hope that the reviews we have identified will be of interest not only to readers who work in cancer pharmacology but also to those less familiar with evolving discoveries related to this Theme. Expected final online publication date for the Annual Review of Pharmacology and Toxicology Volume 56 is January 06, 2016. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
Full-text · Article · Nov 2015 · Annual Review of Pharmacology
[Show abstract][Hide abstract] ABSTRACT: Cyclic AMP/protein kinase A (cAMP/PKA) and glucocorticoids promote the death of many cell types, including cells of hematopoietic origin. In wild-type (WT) S49 T-lymphoma cells, signaling by cAMP and glucocorticoids converges on the induction of the proapoptotic B-cell lymphoma-family protein Bim to produce mitochondria-dependent apoptosis. Kin(-), a clonal variant of WT S49 cells, lacks PKA catalytic (PKA-Cα) activity and is resistant to cAMP-mediated apoptosis. Using sorbitol density gradient fractionation, we show here that in kin(-) S49 cells PKA-Cα is not only depleted but the residual PKA-Cα mislocalizes to heavier cell fractions and is not phosphorylated at two conserved residues (Ser(338) or Thr(197)). In WT S49 cells, PKA-regulatory subunit I (RI) and Bim coimmunoprecipitate upon treatment with cAMP analogs and forskolin (which increases endogenous cAMP concentrations). By contrast, in kin(-) cells, expression of PKA-RIα and Bim is prominently decreased, and increases in cAMP do not increase Bim expression. Even so, kin(-) cells undergo apoptosis in response to treatment with the glucocorticoid dexamethasone (Dex). In WT cells, glucorticoid-mediated apoptosis involves an increase in Bim, but in kin(-) cells, Dex-promoted cell death appears to occur by a caspase 3-independent apoptosis-inducing factor pathway. Thus, although cAMP/PKA-Cα and PKA-R1α/Bim mediate apoptotic cell death in WT S49 cells, kin(-) cells resist this response because of lower levels of PKA-Cα and PKA-RIα subunits as well as Bim. The findings for Dex-promoted apoptosis imply that these lymphoma cells have adapted to selective pressure that promotes cell death by altering canonical signaling pathways.
No preview · Article · Sep 2015 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: In this Perspectives, former and current editors of Molecular Pharmacology together with the guest editors for this 50 year Anniversary Issue provide a historical overview of the Journal since its founding in 1965. The substantial impact the journal has had on the field of pharmacology as well as on biomedical science is discussed, as is the broad scope of the journal. The authors conclude that, true to the original goals for the Journal, Molecular Pharmacology today remains an ideal venue for work that provides a mechanistic understanding of drugs, molecular probes, and their biological targets.
The American Society for Pharmacology and Experimental Therapeutics.
Full-text · Article · May 2015 · Molecular pharmacology
[Show abstract][Hide abstract] ABSTRACT: G protein-coupled receptors (GPCRs), the largest family of signaling receptors in the human genome, are also the largest class of targets of approved drugs. Are the optimal GPCRs (in terms of efficacy and safety) currently targeted therapeutically? Especially given the large number (~120) of orphan GPCRs (which lack known physiologic agonists), it is likely that previously unrecognized, especially orphan, GPCRs regulate cell function and can be therapeutic targets. Knowledge is limited regarding the GPCRs expressed by native cells that are activated by endogenous ligands (endoGPCRs). Here, we review approaches to define their expression in tissues and cells and results from studies using these approaches. We identify problems with the available data and suggest future ways to identify and validate the physiologic and therapeutic roles of previously unrecognized GPCRs. We propose that a particularly useful approach to identify functionally important GPCRs with therapeutic potential will be to focus on receptors that show selective increases in expression in diseased cells from patients and experimental animals.
The American Society for Pharmacology and Experimental Therapeutics.
Full-text · Article · Mar 2015 · Molecular pharmacology
[Show abstract][Hide abstract] ABSTRACT: Objective
The trigeminovascular system plays a central role in migraine, a condition in need of new treatments. The neuropeptide, calcitonin gene-related peptide (CGRP), is proposed as causative in migraine and is the subject of intensive drug discovery efforts. This study explores the expression and functionality of two CGRP receptor candidates in the sensory trigeminal system.Methods
Receptor expression was determined using Taqman G protein-coupled receptor arrays and immunohistochemistry in trigeminal ganglia (TG) and the spinal trigeminal complex of the brainstem in rat and human. Receptor pharmacology was quantified using sensitive signaling assays in primary rat TG neurons.ResultsmRNA and histological expression analysis in rat and human samples revealed the presence of two CGRP-responsive receptors (AMY1: calcitonin receptor/receptor activity-modifying protein 1 [RAMP1]) and the CGRP receptor (calcitonin receptor-like receptor/RAMP1). In support of this finding, quantification of agonist and antagonist potencies revealed a dual population of functional CGRP-responsive receptors in primary rat TG neurons.InterpretationThe unexpected presence of a functional non-canonical CGRP receptor (AMY1) at neural sites important for craniofacial pain has important implications for targeting the CGRP axis in migraine.
[Show abstract][Hide abstract] ABSTRACT: The inductive role of dendritic cells (DC) in Th2 differentiation has not been fully defined. We addressed this gap in knowledge by focusing on signaling events mediated by the heterotrimeric GTP binding proteins Gαs, and Gαi, which respectively stimulate and inhibit the activation of adenylyl cyclases and the synthesis of cAMP. We show here that deletion of Gnas, the gene that encodes Gαs in mouse CD11c(+) cells (Gnas(ΔCD11c) mice), and the accompanying decrease in cAMP provoke Th2 polarization and yields a prominent allergic phenotype, whereas increases in cAMP inhibit these responses. The effects of cAMP on DC can be demonstrated in vitro and in vivo and are mediated via PKA. Certain gene products made by Gnas(ΔCD11c) DC affect the Th2 bias. These findings imply that G protein-coupled receptors, the physiological regulators of Gαs and Gαi activation and cAMP formation, act via PKA to regulate Th bias in DC and in turn, Th2-mediated immunopathologies.
Preview · Article · Jan 2015 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Epac, a guanine nucleotide exchange factor for the low molecular weight G protein Rap, is an effector of cAMP signaling and
has been implicated to have roles in numerous diseases, including diabetes mellitus, heart failure, and cancer. We used a
computational molecular modeling approach to predict potential binding sites for allosteric modulators of Epac and to identify
molecules that might bind to these regions. This approach revealed that the conserved hinge region of the cyclic nucleotide-binding
domain of Epac1 is a potentially druggable region of the protein. Using a bioluminescence resonance energy transfer-based
assay (CAMYEL, cAMP sensor using YFP-Epac-Rluc), we assessed the predicted compounds for their ability to bind Epac and modulate
its activity. We identified a thiobarbituric acid derivative, 5376753, that allosterically inhibits Epac activity and used
Swiss 3T3 and HEK293 cells to test the ability of this compound to modulate the activity of Epac and PKA, as determined by
Rap1 activity and vasodilator-stimulated phosphoprotein phosphorylation, respectively. Compound 5376753 selectively inhibited
Epac in biochemical and cell migration studies. These results document the utility of a computational approach to identify
a domain for allosteric regulation of Epac and a novel compound that prevents the activation of Epac1 by cAMP.
[Show abstract][Hide abstract] ABSTRACT: Increases in cyclic AMP (cAMP) are pro-apoptotic in numerous cell types, but the mechanisms of cAMP-promoted apoptosis are poorly defined. We have used murine S49 T-lymphoma cells as a model to provide insight into these mechanisms. Increases in cAMP in wild-type (WT) S49 cells were first noted to kill these cells in the 1970 s, but only in recent years, it was shown that this occurs by the intrinsic (mitochondria-dependent) apoptotic pathway. The apoptotic response does not occur in protein kinase A-null (kin-) clonal variants of WT S49 cells and thus is mediated by protein kinase A (PKA). A second S49 clonal variant, cAMP-Deathless (D-), has PKA activity but lacks cAMP-promoted apoptosis. Apoptosis in WT S49 cells occurs many hours after cAMP/PKA-promoted G1 cell cycle arrest and involves increased expression of Bim, a pro-apoptotic member of the Bcl-2 (B-cell lymphoma-2) family. This increase in Bim expression does not occur in kin- or D- S49 cells and knockdown of Bim blunts cAMP-mediated apoptosis in WT cells. Cytotoxic T lymphocyte antigen-2 also appears to contribute to cAMP/PKA-promoted apoptosis of S49 cells. Based on time-dependent differences in gene expression between WT, D- and kin- S49 cells following incubation with 8-(4-chlorophenylthio)-cAMP, additional genes and proteins are likely involved in this apoptosis. Studies with S49 cells should reveal further insight regarding the mechanisms of cAMP/PKA-promoted cell death, including the identification of proteins that are targets to enhance (e. g., in cancer) or inhibit (e. g., cardiac failure) apoptosis in response to hormones, neurotransmitters, and drugs.
No preview · Article · Jul 2014 · Hormone and Metabolic Research
[Show abstract][Hide abstract] ABSTRACT: Caveolae, flask-like invaginations of the plasma membrane, were discovered nearly 60 years ago. Originally regarded as fixation artifacts of electron microscopy, the functional role for these structures has taken decades to unravel. The discovery of the caveolin protein in 1992 (by the late Richard G.W. Anderson) accelerated progress in defining the contribution of caveolae to cellular physiology and pathophysiology. The three isoforms of caveolin (caveolin-1, -2, and -3) are caveolae-resident structural and scaffolding proteins that are critical for the formation of caveolae and their localization of signaling entities. A PubMed search for "caveolae" reveals ∼280 publications from their discovery in the 1950s to the early 1990s, whereas a search for "caveolae or caveolin" after 1990, identifies ∼7000 entries. Most work on the regulation of biological responses by caveolae and caveolin since 1990 has focused on caveolae as plasma membrane microdomains and the function of caveolin proteins at the plasma membrane. By contrast, our recent work and that of others has explored the localization of caveolins in multiple cellular membrane compartments and in the regulation of intracellular signaling. Cellular organelles that contain caveolin include mitochondria, nuclei and the endoplasmic reticulum. Such intracellular localization allows for a complexity of responses to extracellular stimuli by caveolin and the possibility of novel organelle-targeted therapeutics. This review focuses on the impact of intracellular localization of caveolin on signal transduction and cell regulation.-Fridolfsson, H. N., Roth, D. M., Insel, P. A., Patel, H. P. Regulation of intracellular signaling and function by caveolin.
[Show abstract][Hide abstract] ABSTRACT: The signaling molecule cAMP primarily mediates its effects by activating PKA and/or Epac. Epac has been implicated in many responses in cells but its precise roles have been difficult to define in the absence of Epac inhibitors. Epac, a guanine nucleotide exchange factor for the low molecular weight G protein Rap, is directly activated by cAMP. Using a BRET-based assay (CAMYEL) to examine modulators of Epac activity, we took advantage of its intramolecular movement that occurs upon cAMP binding to assess Epac activation. We found that the use of CAMYEL can detect the binding of cAMP analogs to Epac and their modulation of its activity and can distinguish between agonists (cAMP), partial agonists (8-CPT-cAMP), and super-agonists (8-CPT-2'-O-Me-cAMP). The CAMYEL assay can also identify competitive and uncompetitive Epac inhibitors, e.g., Rp-cAMPS and CE3F4, respectively. To confirm the results with the CAMYEL assay, we used Swiss 3T3 cells and assessed the ability of cyclic nucleotide analogs to modulate the activity of Epac or PKA, determined by Rap1 activity or VASP phosphorylation, respectively. We used computational molecular modeling to analyze the interaction of analogs with Epac1. The results reveal a rapid means to identify modulators (potentially including allosteric inhibitors) of Epac activity that also provides insight into the mechanisms of Epac activation and inhibition.
Preview · Article · Feb 2014 · Journal of Biological Chemistry