The five known members of the sphingosine-1-phosphate (S1P) receptor family exhibit diverse tissue expression profiles and couple to distinct G-protein-mediated signalling pathways. S1P1, S1P2, and ...S1P3 receptors are all present in the heart, but the ratio of these subtypes differs for various cardiac cells. The goal of this review is to summarize data concerning which S1P receptor subtypes regulate cardiac physiology and pathophysiology, which G-proteins and signalling pathways they couple to, and in which cell types they are expressed. The available information is based on studies using a lamentably limited set of pharmacological agonists/antagonists, but is complemented by work with S1P receptor subtype-specific knockout mice and sphingosine kinase knockout mice. In cardiac myocytes, the S1P1 receptor subtype is the predominant subtype expressed, and the activation of this receptor inhibits cAMP formation and antagonizes adrenergic receptor-mediated contractility. The S1P3 receptor, while expressed at lower levels, mediates the bradycardic effect of S1P agonists. Studies using knockout mice indicate that S1P2 and S1P3 receptors play a major role in mediating cardioprotection from ischaemia/reperfusion injury in vivo. S1P receptors are also involved in remodelling, proliferation, and differentiation of cardiac fibroblasts, a cell type in which the S1P3 receptor predominates. Receptors for S1P are also present in endothelial and smooth muscle cells where they mediate peripheral vascular tone and endothelial responses, but the role of this regulatory system in the cardiac vasculature is unknown. Further understanding of the contributions of each cell and receptor subtype to cardiac function and pathophysiology should expedite consideration of the endogenous S1P signalling pathway as a therapeutic target for cardiovascular disease.
A-kinase anchoring proteins (AKAPs) tether the cAMP-dependent protein kinase (PKA) to intracellular sites where they preferentially phosphorylate target substrates. Most AKAPs exhibit nanomolar ...affinity for the regulatory (RII) subunit of the type II PKA holoenzyme, whereas dual-specificity anchoring proteins also bind the type I (RI) regulatory subunit of PKA with 10–100-fold lower affinity. A range of cellular, biochemical, biophysical, and genetic approaches comprehensively establish that sphingosine kinase interacting protein (SKIP) is a truly type I-specific AKAP. Mapping studies located anchoring sites between residues 925–949 and 1,140–1,175 of SKIP that bind RI with dissociation constants of 73 and 774 nM, respectively. Molecular modeling and site-directed mutagenesis approaches identify Phe 929 and Tyr 1,151 as RI-selective binding determinants in each anchoring site. SKIP complexes exist in different states of RI-occupancy as single-molecule pull-down photobleaching experiments show that 41 ± 10% of SKIP sequesters two YFP-RI dimers, whereas 59 ± 10% of the anchoring protein binds a single YFP-RI dimer. Imaging, proteomic analysis, and subcellular fractionation experiments reveal that SKIP is enriched at the inner mitochondrial membrane where it associates with a prominent PKA substrate, the coiled-coil helix protein ChChd3.
1 Biomedical Sciences Graduate Program and Departments of 2 Pharmacology and 3 Medicine, University of California, San Diego, La Jolla, California; and 4 Department of Molecular Biology, The Scripps ...Research Institute, La Jolla, California
Submitted 5 December 2006
; accepted in final form 6 February 2007
Sphingosine 1-phosphate (S1P) is released at sites of tissue injury and effects cellular responses through activation of G protein-coupled receptors. The role of S1P in regulating cardiomyocyte survival following in vivo myocardial ischemia-reperfusion (I/R) injury was examined by using mice in which specific S1P receptor subtypes were deleted. Mice lacking either S1P 2 or S1P 3 receptors and subjected to 1-h coronary occlusion followed by 2 h of reperfusion developed infarcts equivalent to those of wild-type (WT) mice. However, in S1P 2,3 receptor double-knockout mice, infarct size following I/R was increased by >50%. I/R leads to activation of ERK, JNK, and p38 MAP kinases; however, these responses were not diminished in S1P 2,3 receptor knockout compared with WT mice. In contrast, activation of Akt in response to I/R was markedly attenuated in S1P 2,3 receptor knockout mouse hearts. Neither S1P 2 nor S1P 3 receptor deletion alone impaired I/R-induced Akt activation, which suggests redundant signaling through these receptors and is consistent with the finding that deletion of either receptor alone did not increase I/R injury. The involvement of cardiomyocytes in S1P 2 and S1P 3 receptor mediated activation of Akt was tested by using cells from WT and S1P receptor knockout hearts. Akt was activated by S1P, and this was modestly diminished in cardiomyocytes from S1P 2 or S1P 3 receptor knockout mice and completely abolished in the S1P 2,3 receptor double-knockout myocytes. Our data demonstrate that activation of S1P 2 and S1P 3 receptors plays a significant role in protecting cardiomyocytes from I/R damage in vivo and implicate the release of S1P and receptor-mediated Akt activation in this process.
cardioprotection; mitogen-activated kinase; G protein-coupled receptors; infarct
Address for reprint requests and other correspondence: J. H. Brown, Dept. of Pharmacology, Univ. of California, San Diego, 9500 Gilman Dr., La Jolla, CA, 92093-0636 (e-mail: jhbrown{at}ucsd.edu )
Abstract Sphingosine-1-phosphate (S1P), a bioactive lysophospholipid, is generated and released at sites of tissue injury in the heart and can act on S1P1 , S1P2 , and S1P3 receptor subtypes to ...affect cardiovascular responses. We established that S1P causes little phosphoinositide hydrolysis and does not induce hypertrophy indicating that it does not cause receptor coupling to Gq . We previously demonstrated that S1P confers cardioprotection against ischemia/reperfusion by activating RhoA and its downstream effector PKD. The S1P receptor subtypes and G proteins that regulate RhoA activation and downstream responses in the heart have not been determined. Using siRNA or pertussis toxin to inhibit different G proteins in NRVMs we established that S1P regulates RhoA activation through Gα13 but not Gα12 , Gαq , or Gαi . Knockdown of the three major S1P receptors using siRNA demonstrated a requirement for S1P3 in RhoA activation and subsequent phosphorylation of PKD, and this was confirmed in studies using isolated hearts from S1P3 knockout (KO) mice. S1P treatment reduced infarct size induced by ischemia/reperfusion in Langendorff perfused wild-type (WT) hearts and this protection was abolished in the S1P3 KO mouse heart. CYM-51736, an S1P3 -specific agonist, also decreased infarct size after ischemia/reperfusion to a degree similar to that achieved by S1P. The finding that S1P3 receptor- and Gα13 -mediated RhoA activation is responsible for protection against ischemia/reperfusion suggests that selective targeting of S1P3 receptors could provide therapeutic benefits in ischemic heart disease.
PKA is retained within distinct subcellular environments by the association of its regulatory type II (RII) subunits with A-kinase anchoring proteins (AKAPs). Conventional reagents that universally ...disrupt PKA anchoring are patterned after a conserved AKAP motif. We introduce a phage selection procedure that exploits high-resolution structural information to engineer RII mutants that are selective for a particular AKAP. Selective RII (RSelect) sequences were obtained for eight AKAPs following competitive selection screening. Biochemical and cell-based experiments validated the efficacy of RSelect proteins for AKAP2 and AKAP18. These engineered proteins represent a new class of reagents that can be used to dissect the contributions of different AKAP-targeted pools of PKA. Molecular modeling and high-throughput sequencing analyses revealed the molecular basis of AKAP-selective interactions and shed new light on native RII-AKAP interactions. We propose that this structure-directed evolution strategy might be generally applicable for the investigation of other protein interaction surfaces.
Background: PKA is confined to subcellular compartments by A-kinase anchoring proteins (AKAPs).
Results: A structure-based phage-directed evolution strategy has yielded modified PKA regulatory type II subunits with AKAP-selective binding properties.
Conclusion: Engineered RSelect proteins preferentially target particular AKAP-PKA interfaces
Significance: RSelect subunits are tools to distinguish and manipulate subpopulations of anchored PKA.
Sphingosine-1-phosphate (S1P), a bioactive lysophospholipid, is generated and released at sites of tissue injury in the heart and can act on S1P
, S1P
, and S1P
receptor subtypes to affect ...cardiovascular responses. We established that S1P causes little phosphoinositide hydrolysis and does not induce hypertrophy indicating that it does not cause receptor coupling to G
. We previously demonstrated that S1P confers cardioprotection against ischemia/reperfusion by activating RhoA and its downstream effector PKD. The S1P receptor subtypes and G proteins that regulate RhoA activation and downstream responses in the heart have not been determined. Using siRNA or pertussis toxin to inhibit different G proteins in NRVMs we established that S1P regulates RhoA activation through Gα
but not Gα
, Gα
, or Gα
. Knockdown of the three major S1P receptors using siRNA demonstrated a requirement for S1P
in RhoA activation and subsequent phosphorylation of PKD, and this was confirmed in studies using isolated hearts from S1P
knockout (KO) mice. S1P treatment reduced infarct size induced by ischemia/reperfusion in Langendorff perfused wild-type (WT) hearts and this protection was abolished in the S1P
KO mouse heart. CYM-51736, an S1P
-specific agonist, also decreased infarct size after ischemia/reperfusion to a degree similar to that achieved by S1P. The finding that S1P
receptor- and Gα
-mediated RhoA activation is responsible for protection against ischemia/reperfusion suggests that selective targeting of S1P
receptors could provide therapeutic benefits in ischemic heart disease.
Although poor nutritional status and weight loss in cancer patients is known to affect outcomes, little is known about malnutrition differences based on geographic location. We investigated ...nutritional and inflammatory status of 220 newly diagnosed adults with solid tumors at the University of Kentucky's Markey Cancer Center during December 2008 through October 2011. Chi-square tests were used to determine any associations between suboptimal nutritional levels and rural–urban areas of residence. Out of the 13 lab values collected, the only significant difference between rural and urban participants was found for vitamin D resulting in more rural subjects (67.4%) having a suboptimal vitamin D status as compared to those residing in urban areas (53.3%, P = 0.04). Controlling for baseline demographics including age, race, sex, body mass index, nutritional status, and type of cancer, logistic regression analyses concluded those in rural areas had nearly a twofold increase in the odds of having a suboptimal vitamin D level compared to those in urban areas (odd's ratio = 1.97; 95% confidence interval = 1.04, 3.74). Further investigation into the rural–urban differences in vitamin D needs to be investigated in order to improve outcomes during cancer treatment.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, OILJ, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK, VSZLJ
The effect of the lysophospholipid, lysophosphatidic acid (LPA), on signaling and hypertrophy of neonatal rat ventricular cardiomyocytes was examined. Myocytes express mRNA for all three ...G-protein-coupled LPA receptor subtypes (LPA(1)/Edg-2, LPA(2)/Edg-4, and LPA(3)/Edg-7) as indicated by RT-PCR analysis. LPA inhibits isoproterenol-stimulated cyclic AMP accumulation with an IC(50) approximately 40 nM and promotes phosphorylation of ERK-1/2. LPA also elicits a small, slow onset, and activation of phosphoinositide hydrolysis with EC(50) approximately 400 nM, and stimulates a marked increase in the extent of Rho activation. Longer-term treatment with LPA induces a hypertrophic response in myocytes as indicated by increases in cell size, actin organization, ANF staining of the perinuclear region and activation of ANF promoter-luciferase gene expression. Pretreatment of myocytes with pertussis toxin (PTX) not only blocks the capacity of LPA to inhibit cyclic AMP formation and stimulate ERK phosphorylation, but also inhibits hypertrophic changes in cell morphology and ANF-luciferase gene expression. Neither phospholipase C nor Rho activation is PTX sensitive. The hypertrophic effects of LPA on myocytes are also inhibited by treatment with C3 exoenzyme or by transfection of plasmids expressing either C3 exoenzyme or dominant-negative Rho to block Rho function. Inhibition of ERK activation with PD98059 blocks LPA-induced hypertrophy while inhibitors of phospholipase C (U73122), PKC (GF109203X), or p38MAPK (SB203580) do not. These data suggest that LPA induces cardiomyocyte hypertrophy via a pathway different from the conventional G(q) pathway utilized by phenylephrine, endothelin, and PGF2 alpha and involving activation of a PTX-sensitive G(i)/ERK pathway in conjunction with activation of Rho-mediated signals.
ABSTRACT—Expression of the wild-type α subunit of Gq stimulates phospholipase C and induces hypertrophy in cardiomyocytes. Addition of Gq-coupled receptor agonists additionally activates ...phospholipase C, as does expression of a constitutively active mutant form of Gαq. Under these conditions, hypertrophy is rapidly succeeded by apoptotic cellular and molecular changes, including myofilament disorganization, loss of mitochondrial membrane potential, alterations in Bcl-2 family protein levels, DNA fragmentation, increased caspase activity (≈4-fold), cytochrome c redistribution, and nuclear chromatin condensation in ≈12% of the cells. We used various interventions to define the molecular relationships between these events and identify potential sites at which these features of apoptosis could be rescued. Treatment with caspase inhibitors prevented DNA fragmentation and promoted myocyte survival; however, cytochrome c release and loss of mitochondrial membrane potential still occurred. In contrast, treatment with bongkrekic acid, an inhibitor of the mitochondrial permeability transition pore, not only prevented DNA fragmentation and reduced nuclear chromatin condensation but also preserved mitochondrial membrane potential and limited cytochrome c redistribution to only ≈2% of cells. These data demonstrate the central role of mitochondrial membrane potential in initiation of caspase activation and downstream apoptotic events and suggest that preservation of mitochondrial integrity is crucial for prolonging the life and function of cardiomyocytes exposed to pathological levels of stress.
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