Bitopic Sphingosine 1-Phosphate Receptor 3 (S1P3) Antagonist Rescue from Complete Heart Block: Pharmacological and Genetic Evidence for Direct S1P3 Regulation of Mouse Cardiac Conduction.

The molecular pharmacology of the G protein-coupled receptors for sphingosine 1-phosphate (S1P) provides important insight into established and new therapeutic targets. A new, potent bitopic S1P3 antagonist, SPM-354, with in vivo activity, has been used, together with S1P3-knockin and S1P3-knockout mice to define the spatial and functional properties of S1P3 in regulating cardiac conduction. We show that S1P3 is a key direct regulator of cardiac rhythm both in vivo and in isolated perfused hearts. 2-Amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol in vivo and S1P in isolated hearts induced a spectrum of cardiac effects, ranging from sinus bradycardia to complete heart block, as measured by a surface electrocardiogram in anesthetized mice and in volume-conducted Langendorff preparations. The agonist effects on complete heart block are absent in S1P3-knockout mice and are reversed in wild-type mice with SPM-354, as characterized and described here. Homologous knockin of S1P3-mCherry is fully functional pharmacologically and is strongly expressed by immunohistochemistry confocal microscopy in Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 4 (HCN4)-positive atrioventricular node and His-Purkinje fibers, with relative less expression in the HCN4-positive sinoatrial node. In Langendorff studies, at constant pressure, SPM-354 restored sinus rhythm in S1P-induced complete heart block and fully reversed S1P-mediated bradycardia. S1P3 distribution and function in the mouse ventricular cardiac conduction system suggest a direct mechanism for heart block risk that should be further studied in humans. A richer understanding of receptor and ligand usage in the pacemaker cells of the cardiac system is likely to be useful in understanding ventricular conduction in health, disease, and pharmacology.

The organization of the sphingosine 1-phosphate signaling system.

The understanding of the role of the sphingosine 1-phosphate signaling system in immunology and host defense has deepened exponentially over the past 12 years since the discovery that lymphocyte egress was reversibly modulated by sphingosine 1-phosphate receptors, and with the development of fingolimod, a prodrug of a nonselective S1P receptor agonist, for therapeutic use in the treatment of relapsing, remitting multiple sclerosis. Innovative genetic and chemical approaches, together with structural biology, now provide a more detailed molecular understanding of a regulated lysophospholipid ligand with a variety of autocrine, paracrine, and systemic effects in physiology and pathology, based upon selective interactions with a high affinity and selective evolutionary cluster of G-protein-coupled receptors.

S1P signaling: new therapies and opportunities.

Development of sphingosine-1-phosphate receptor 1 (S1P1) modulators to dampen inflammation and its sequelae is becoming increasingly promising for treating medical conditions characterized by significant immunopathology. As shown by the non-selective S1P receptor modulator FTY720 (fingolimod [Gilenya(®)]) in the treatment of relapsing-remitting multiple sclerosis (MS), the ability to use S1P1 modulation to precisely block immune cell traffic-immunomodulation-while maintaining immunosurveillance, has opened therapeutic opportunities in various other immune-derived chronic pathologies, including inflammatory bowel disease (IBD), lupus, psoriasis, as well as, potentially, in early acute viral respiratory infection. Proof-of-concept studies across validated animal models with S1P receptor modulators highly selective for S1P1, such as BAF-312 (Siponimod), KRP-203, ONO-4641 (Ceralifimod), ponesimod and RPC-1063, and emerging clinical trials for safety and efficacy in humans, particularly in MS, ulcerative colitis (UC) and psoriasis, have set the stage for us to consider additional testing in various other autoimmune diseases.

Sphingosine 1-phosphate receptor 1 (S1P(1)) upregulation and amelioration of experimental autoimmune encephalomyelitis by an S1P(1) antagonist.

Sphingosine 1-phosphate receptor 1 (S1P(1)) is a G protein-coupled receptor that is critical for proper lymphocyte development and recirculation. Agonists to S1P(1) are currently in use clinically for the treatment of multiple sclerosis, and these drugs may act on both S1P(1) expressed on lymphocytes and S1P(1) expressed within the central nervous system. Agonists to S1P(1) and deficiency in S1P(1) both cause lymphocyte sequestration in the lymph nodes. In the present study, we show that S1P(1) antagonism induces lymphocyte sequestration in the lymph nodes similar to that observed with S1P(1) agonists while upregulating S1P(1) on lymphocytes and endothelial cells. Additionally, we show that S1P(1) antagonism reverses experimental autoimmune encephalomyelitis in mice without acting on S1P(1) expressed within the central nervous system, demonstrating that lymphocyte sequestration via S1P(1) antagonism is sufficient to alleviate autoimmune pathology.

Real-time differential labeling of blood, interstitium, and lymphatic and single-field analysis of vasculature dynamics in vivo.

Lymph nodes are highly organized structures specialized for efficient regulation of adaptive immunity. The blood and lymphatic systems within a lymph node play essential roles by providing functionally distinct environments for lymphocyte entry and egress, respectively. Direct imaging and measurement of vascular microenvironments by intravital multiphoton microscopy provide anatomical and mechanistic insights into the essential events of lymphocyte trafficking. Lymphocytes, blood endothelial cells, and lymphatic endothelial cells express sphingosine 1-phosphate receptor 1, a key G protein-coupled receptor regulating cellular egress and a modulator of endothelial permeability. Here we report the development of a differential vascular labeling (DVL) technique in which a single intravenous injection of a fluorescent dextran, in combination with fluorescent semiconductor quantum dot particles, differentially labels multiple blood and lymphatic compartments in a manner dependent on the size of the fluorescent particle used. Thus DVL allows measurement of endothelial integrity in multiple vascular compartments and the affects or pharmacological manipulation in vascular integrity. In addition, this technique allows for real-time observation of lymphocyte trafficking across physiological barriers differentiated by DVL. Last, single-field fluid movement dynamics can be derived, allowing for the simultaneous determination of fluid flow rates in diverse blood and lymphatic compartments.

S1P(1) receptor modulation with cyclical recovery from lymphopenia ameliorates mouse model of multiple sclerosis.

Multiple sclerosis (MS) therapies modulate T-cell autoimmunity in the central nervous system (CNS) but may exacerbate latent infections. Fingolimod, a nonselective sphingosine-1-phosphate (S1P) receptor agonist that induces sustained lymphopenia and accumulates in the CNS, represents a new treatment modality for MS. We hypothesized that sustained lymphopenia would not be required for efficacy and that a selective, CNS-penetrant, peripherally short-acting, S1P(1) agonist would show full efficacy in a mouse MS model. Using daily treatment with 10 mg/kg 2-(4-(5-(3,4-diethoxyphenyl)-1,2,4-oxadiazol-3-yl)-2,3-dihydro-1H-inden-1-yl amino)ethanol (CYM-5442) at the onset of clinical signs in myelin oligodendrocyte glycoprotein MOG(35-55)- induced experimental allergic encephalomyelitis (EAE), we assessed clinical scores, CNS cellular infiltration, demyelination, and gliosis for 12 days with CYM-5442, vehicle, or fingolimod. CYM-5442 levels in CNS and plasma were determined at experiment termination, and blood lymphopenia was measured 3 and 24 h after the last injection. Plasma levels of cytokines were assayed at the end of the protocol. Changes in S1P(1)-enhanced green fluorescent protein expression on neurons and astrocytes during active EAE and upon CYM-5442 treatment were quantified with flow cytometry and Western blotting by using native-locus enhanced green fluorescent protein-tagged S1P(1) mice. S1P(1) agonism alone reduced pathological features as did fingolimod (maximally lymphopenic throughout), despite full reversal of lymphopenia within each dosing interval. CYM-5442 levels in CNS but not in plasma were sustained. Neuronal and astrocytic S1P(1) expression in EAE was suppressed by CYM-5442 treatment, relative to vehicle, and levels of key cytokines, such as interleukin 17A, were also significantly reduced in drug-treated mice. S1P(1)-selective agonists that induce reversible lymphopenia while persisting in the CNS may be effective MS treatments.

Actions of a picomolar short-acting S1P1 agonist in S1P1-eGFP knock-in mice.

Sphingosine 1-phosphate receptor 1 (S1P(1)) is critical for lymphocyte recirculation and is a clinical target for treatment of multiple sclerosis. By generating a short-duration S1P(1) agonist and mice in which fluorescently tagged S1P(1) replaces wild-type receptor, we elucidate physiological and agonist-perturbed changes in expression of S1P(1) at a subcellular level in vivo. We demonstrate differential downregulation of S1P(1) on lymphocytes and endothelia after agonist treatment.

Sphingosine 1-phosphate receptor signaling.

The sphingosine 1-phosphate (S1P) receptor signaling system is a productive model system. A hydrophobic zwitterionic lysophospholipid ligand with difficult physical properties interacts with five high-affinity G protein-coupled receptors to generate multiple downstream signals. These signals modulate homeostasis and pathology on a steep agonist concentration-response curve. Ligand presence is essential for vascular development and endothelial integrity, while acute increases in ligand concentrations result in cardiac death. Understanding this integrated biochemical system has exemplified the impact of both genetics and chemistry. Developing specific tools with defined biochemical properties for the reversible modulation of signals in real time has been essential to complement insights gained from genetic approaches that may be irreversible and compensated. Despite its knife-edge between life and death, this system, based in part on receptor subtype-selectivity and in part on differential attenuation of deleterious signals, now appears to be on the cusp of meaningful therapy for multiple sclerosis.

Full pharmacological efficacy of a novel S1P1 agonist that does not require S1P-like headgroup interactions.

Strong evidence exists for interactions of zwitterionic phosphate and amine groups in sphingosine-1 phosphate (S1P) to conserved Arg and Glu residues present at the extracellular face of the third transmembrane domain of S1P receptors. The contribution of Arg(120) and Glu(121) for high-affinity ligand-receptor interactions is essential, because single-point R(120)A or E(121)A S1P(1) mutants neither bind S1P nor transduce S1P function. Because S1P receptors are therapeutically interesting, identifying potent selective agonists with different binding modes and in vivo efficacy is of pharmacological importance. Here we describe a modestly water-soluble highly selective S1P(1) agonist [2-(4-(5-(3,4-diethoxyphenyl)-1,2,4-oxadiazol-3-yl)-2,3-dihydro-1H-inden-1-yl amino) ethanol (CYM-5442)] that does not require Arg(120) or Glu(121) residues for activating S1P(1)-dependent p42/p44 mitogen-activated protein kinase phosphorylation, which defines a new hydrophobic pocket in S1P(1). CYM-5442 is a full agonist in vitro for S1P(1) internalization, phosphorylation, and ubiquitination. It is noteworthy that CYM-5442 was a full agonist for induction and maintenance of S1P(1)-dependent blood lymphopenia, decreasing B lymphocytes by 65% and T lymphocytes by 85% of vehicle. Induction of CYM-5442 lymphopenia was dose- and time-dependent, requiring serum concentrations in the 50 nM range. In vitro measures of S1P(1) activation by CYM-5442 were noncompetitively inhibited by a specific S1P(1) antagonist [(R)-3-amino-(3-hexylphenylamino)-4-oxobutylphosphonic acid (W146)], competitive for S1P, 2-amino-2-(4-octylphenethyl)propane-1,3-diol (FTY720-P), and 5-[4-phenyl-5-(trifluoromethyl)-2-thienyl]-3-[3-(trifluoromethyl)phenyl]-1,2, 4-oxadiazole (SEW2871). In addition, lymphopenia induced by CYM-5442 was reversed by W146 administration or upon pharmacokinetic agonist clearance. Pharmacokinetics in mice also indicated that CYM-5442 partitions significantly in central nervous tissue. These data show that CYM-5442 activates S1P(1)-dependent pathways in vitro and to levels of full efficacy in vivo through a hydrophobic pocket separate from the orthosteric site of S1P binding that is headgroup-dependent.

Ligand-binding pocket shape differences between sphingosine 1-phosphate (S1P) receptors S1P1 and S1P3 determine efficiency of chemical probe identification by ultrahigh-throughput screening.

We have studied the sphingosine 1-phosphate (S1P) receptor system to better understand why certain molecular targets within a closely related family are much more tractable when identifying compelling chemical leads. Five medically important G-protein-coupled receptors for S1P regulate heart rate, coronary artery caliber, endothelial barrier integrity, and lymphocyte trafficking. Selective S1P receptor agonist probes would be of great utility to study receptor subtype-specific function. Through systematic screening of the same libraries, we identified novel selective agonist chemotypes for each of the S1P1 and S1P3 receptors. Ultrahigh-throughput screening (uHTS) for S1P1 was more effective than that for S1P3, with many selective, low nanomolar hits of proven mechanism emerging. Receptor structure modeling and ligand docking reveal differences between the receptor binding pockets, which are the basis for subtype selectivity. Novel selective agonists interact primarily in the hydrophobic pocket of the receptor in the absence of headgroup interactions. Chemistry-space and shape-based analysis of the screening libraries in combination with the binding models explain the observed differential hit rates and enhanced efficiency for lead discovery for S1P1 versus S1P3 in this closely related receptor family.

Modulating tone: the overture of S1P receptor immunotherapeutics.

SUMMARY: Modulation of complex functions within the immune system has proven to be surprisingly sensitive to alterations in the lysophospholipid sphingosine 1-phosphate (S1P) receptor-ligand rheostat. This has become increasingly evident from both chemical and genetic manipulation of the S1P system, with pharmacological effects upon lymphoid cells, dendritic cell function, as well as vascular interfaces. The integrated immune system, perhaps as a result of its relatively recent evolutionary ontogeny, has selected for a number of critical control points regulated by five distinct high affinity G-protein-coupled receptor subtypes with a shared ligand, with receptors distributed on lymphocytes, dendritic cells, and endothelium. All of these cellular components of the axis are capable of modulating immune responses in vivo, with the impact on the immune response being very different from classical immunosuppressants, by virtue of selective spatial and temporal sparing of humoral and myeloid elements of host defense. Pharmacological subversion of the S1P rheostat is proving to be clinically efficacious in multiple sclerosis, and both the scope and limitations of therapeutic modulation of the S1P axis in immunotherapy are becoming clearer as understanding of the integrated chemical physiology of the S1P system emerges.

Tipping the gatekeeper: S1P regulation of endothelial barrier function.

The lysophospholipid sphingosine 1-phosphate (S1P) is a pleiotropic signaling lipid present constitutively in plasma, and secreted locally at elevated concentrations at sites of inflammation. S1P maintains essential variable homeostatic functions in addition to inducing pathophysiology through the activation of five specific high-affinity G-protein-coupled receptors. Therefore, S1P can function as an extracellular rheostat regulating tonic and acutely evoked functions. Although S1P receptors can regulate lymphoid development and lymphocyte trafficking, and different opinions exist on the roles of receptor agonism and functional antagonism in regulating lymphocyte recirculation, this personal perspective highlights the pivotal control points regulated by constitutive and induced S1P receptor tone at vascular endothelial and lymphatic endothelial barriers, through which S1P agonism impacts on both innate and adaptive immunity. We also emphasize how specific, proof-of-concept chemical tools complement genetic approaches by enabling reversible perturbation of the S1P-S1P(1) receptor axis and, thus, clarifying in vivo mechanisms in the absence of developmental compensations.

Mapping pathways downstream of sphingosine 1-phosphate subtype 1 by differential chemical perturbation and proteomics.

Sphingosine 1-phosphate subtype 1 (S1P(1)) receptor agonists alter lymphocyte trafficking and endothelial barrier integrity in vivo. Among these is the potent, non-selective agonist, FTY720-P, whose mechanism of action has been suggested to correlate with S1P(1) down-regulation. Discovery of the in vivo active S1P(1)-selective agonist, SEW2871, has broadened our understanding of minimal requirements for S1P(1) function while highlighting differences regarding agonist effect on S1P(1) fate, because SEW2871 does not degrade S1P(1). To further understand the mechanism of agonist-induced S1P(1) down-regulation, we compared signaling and fate of human S1P(1)-green fluorescent protein (GFP) in stable 293 cells, using AFD-R, a chiral analog of FTY720-P, SEW2871, and S1P. Although all agonists acutely internalized S1P(1) to late endosomal vesicles and activated GTPgammaS(35) binding and pERK to similar maxima, only AFD-R led to significant S1P(1) down-regulation, as shown by GFP immunoprecipitation studies. Down-regulation was time- and concentration-dependent, was partially blocked by proteasomal inhibition and reversed by chloroquine and an antagonist to S1P(1). All agonists induced a receptor-associated increase in ubiquitination, with AFD-R inducing 3-fold more accumulation than S1P and being 3-4 logs more potent than SEW2871. The formation of AFD-R-receptor ubiquitin complex was inhibited by antagonist and chloroquine and was enhanced by proteasomal inhibition. Identification of proteins by PAGE liquid chromatography-tandem mass spectrometry in cells treated with AFD-R confirmed the co-migration of ubiquitin peptides with those of S1P(1) and GFP, relative to vehicle alone. These data suggest that the hierarchy of ubiquitin recruitment to S1P(1) (AFD-R > S1P > SEW2871) correlates with the efficiency of lysosomal receptor degradation and reflects intrinsic differences between agonists.

Enhancement of capillary leakage and restoration of lymphocyte egress by a chiral S1P1 antagonist in vivo.

Sphingosine 1-phosphate (S1P, 1) regulates vascular barrier and lymphoid development, as well as lymphocyte egress from lymphoid organs, by activating high-affinity S1P1 receptors. We used reversible chemical probes (i) to gain mechanistic insights into S1P systems organization not accessible through genetic manipulations and (ii) to investigate their potential for therapeutic modulation. Vascular (but not airway) administration of the preferred R enantiomer of an in vivo-active chiral S1P1 receptor antagonist induced loss of capillary integrity in mouse skin and lung. In contrast, the antagonist did not affect the number of constitutive blood lymphocytes. Instead, alteration of lymphocyte trafficking and phenotype required supraphysiological elevation of S1P1 tone and was reversed by the antagonist. In vivo two-photon imaging of lymph nodes confirmed requirements for obligate agonism, and the data were consistent with the presence of a stromal barrier mechanism for gating lymphocyte egress. Thus, chemical modulation reveals differences in S1P-S1P1 'set points' among tissues and highlights both mechanistic advantages (lymphocyte sequestration) and risks (pulmonary edema) of therapeutic intervention.

S1P1-selective in vivo-active agonists from high-throughput screening: off-the-shelf chemical probes of receptor interactions, signaling, and fate.

The essential role of the sphingosine 1-phosphate (S1P) receptor S1P(1) in regulating lymphocyte trafficking was demonstrated with the S1P(1)-selective nanomolar agonist, SEW2871. Despite its lack of charged headgroup, the tetraaromatic compound SEW2871 binds and activates S1P(1) through a combination of hydrophobic and ion-dipole interactions. Both S1P and SEW2871 activated ERK, Akt, and Rac signaling pathways and induced S1P(1) internalization and recycling, unlike FTY720-phosphate, which induces receptor degradation. Agonism with receptor recycling is sufficient for alteration of lymphocyte trafficking by S1P and SEW2871. S1P(1) modeling and mutagenesis studies revealed that residues binding the S1P headgroup are required for kinase activation by both S1P and SEW2871. Therefore, SEW2871 recapitulates the action of S1P in all the signaling pathways examined and overlaps in interactions with key headgroup binding receptor residues, presumably replacing salt-bridge interactions with ion-dipole interactions.

Differential regulation of the cell cycle by alpha1-adrenergic receptor subtypes.

Alpha(1)-Adrenergic receptors have been implicated in growth-promoting pathways. A microarray study of individual alpha(1)-adrenergic receptor subtypes (alpha(1A), alpha(1B), and alpha(1D)) expressed in Rat-1 fibroblasts revealed that epinephrine altered the transcription of several cell cycle regulatory genes in a direction consistent with the alpha(1A)- and alpha(1D)-adrenergic receptors mediating G(1)-S cell cycle arrest and the alpha(1B-)mediating cell-cycle progression. A time course indicated that in alpha(1A) cells, epinephrine stimulated a G(1)-S arrest, which began after 8 h of stimulation and maximized at 16 h, at which point was completely blocked with cycloheximide. The alpha(1B)-adrenergic receptor profile also showed unchecked cell cycle progression, even under low serum conditions and induced foci formation. The G(1)-S arrest induced by alpha(1A)- and alpha(1D)-adrenergic receptors was associated with decreased cyclin-dependent kinase-6 and cyclin E-associated kinase activities and increased expression of the cyclin-dependent kinase inhibitor p27(Kip1), all of which were blocked by prazosin. There were no differences in kinase activities and/or expression of p27(Kip1) in epinephrine alpha(1B)-AR fibroblasts, although the microarray did indicate differences in p27(Kip1) RNA levels. Cell counts proved the antimitotic effect of epinephrine in alpha(1A) and alpha(1D) cells and indicated that alpha(1B)-adrenergic receptor subtype expression was sufficient to cause proliferation of Rat-1 fibroblasts independent of agonist stimulation. Analysis in transfected PC12 cells also confirmed the alpha(1A)- and alpha(1B)-adrenergic receptor effect. The alpha(1B)-subtype native to DDT1-MF2 cells, a smooth muscle cell line, caused progression of the cell cycle. These results indicate that the alpha(1A)- and alpha(1D)-adrenergic receptors mediate G(1)-S cell-cycle arrest, whereas alpha(1B)-adrenergic receptor expression causes a cell cycle progression and may induce transformation in sensitive cell lines.

The alpha(1B)-adrenergic receptor decreases the inotropic response in the mouse Langendorff heart model.

OBJECTIVE: alpha(1)-Adrenergic receptors (ARs) are known mediators of a positive inotropy in the heart, which may play even more important roles in heart disease. Due to a lack of sufficiently selective ligands, the contribution of each of the three alpha(1)-AR subtypes (alpha(1A), alpha(1B) and alpha(1D)) to cardiac function is not clearly defined. In this study, we used a systemically expressing mouse model that overexpresses the alpha(1B)-AR to define the role of this subtype in cardiac function. METHODS: We used the mouse Langendorff heart model to assess changes in contractility under basal and phenylephrine-induced conditions. RESULTS: We find that a 50% increase of the alpha(1B)-AR in the heart does not change basal cardiac parameters compared to age-matched normals (heart rate, +/-dP/dT and coronary flow). However, the inotropic response to phenylephrine is blunted. The same results were obtained in isolated adult myocytes. The difference in inotropy could be blocked by the selective alpha(1A)-AR antagonist, 5-methylurapidil, which correlated with decreases in alpha(1A)-AR density, suggesting that the alpha(1B)-AR had caused a compensatory downregulation of the alpha(1A)-AR. CONCLUSIONS: These results suggest that the alpha(1B)-AR does not have a major role in the positive inotropic response in the mouse myocardium but may negatively modulate the response of the alpha(1A)-AR.

Gene expression profile of neurodegeneration induced by alpha1B-adrenergic receptor overactivity: NMDA/GABAA dysregulation and apoptosis.

The alpha1-adrenergic receptors (alpha1ARs) play an important role in mediating sympathetic neurotransmission in peripheral organ systems; however, central alpha1ARs are not well characterized. Additionally, due to the lack of sufficiently subtype-selective drugs or high avidity antibodies, the contribution of each alpha1AR subtype to various central functions is currently unclear. Transcription regulation through alpha1AR subtypes in the CNS is also unknown. Of interest, transgenic mice that systemically overexpress the alpha1BAR show central symptoms that include age-progressive impaired mobility, neurodegeneration and susceptibility to epileptic seizure. To investigate the molecular basis of this phenotype, oligonucleotide microarray studies of whole brains of various ages were carried out to compare gene expression profiles between transgenic and normal brains. The results indicated changes in expression of apoptotic, calcium regulatory, neurodegenerative and genes involved in neurotransmission. Defects in regulation of intracellular calcium are known to play a role in cell death; thus, these genes may provide clues as to the molecular basis of alpha1BAR-induced neurodegeneration. Epilepsy is a disorder that can be caused by an imbalance between excitatory (e.g. glutamate) and inhibitory (e.g. GABA) signals. Microarray analysis of transgenic brains showed increased N-methyl-d-aspartate (NMDA) receptors and decreased GABAA, which were confirmed with immunohistochemistry, western blot and radioligand binding studies. The alpha1BAR also co-localized with the glutamatergic distribution, suggesting a glutamate imbalance as a molecular rationale for the epileptic seizures.

Genetic profiling of alpha 1-adrenergic receptor subtypes by oligonucleotide microarrays: coupling to interleukin-6 secretion but differences in STAT3 phosphorylation and gp-130.

Alpha(1)-adrenoceptor subtypes (alpha(1A)-, alpha(1B)-, alpha(1D)-) are known to couple to similar signaling pathways, although differences among the subtypes do exist. As a more sensitive assay, we used oligonucleotide microarrays to identify gene expression changes in Rat-1 fibroblasts stably expressing each individual subtype. We report the gene expressions that change by at least a factor of 2 or more. Gene expression profiles significantly changed equally among all three subtypes, despite the unequal efficacy of the inositol phosphate response. Gene expressions were clustered into cytokines/growth factors, transcription factors, enzymes, and extracellular matrix proteins. There were also a number of individual subtype-specific changes in gene expression, suggesting a link to independent pathways. In addition, all three alpha(1)-AR subtypes robustly stimulated the transcription of the prohypertrophic cytokine interleukin (IL)-6, but differentially altered members of the IL-6 signaling pathway (gp-130 and STAT3). This was confirmed by measurement of secreted IL-6, activated STAT3, and gp-130 levels. Activation of STAT3 Tyr705 phosphorylation by the alpha(1)-ARs was not through IL-6 activation but was synergistic with IL-6, suggesting direct effects. Interestingly, alpha(1B)-AR stimulation caused the dimerization-dependent phosphorylation of Tyr705 on STAT3 but did not activate the transcriptional-dependent phosphorylation of Ser727. The alpha(1B)-AR also constitutively down-regulated the protein levels of gp-130. These results suggest that the alpha(1B)-AR has differential effects on the phosphorylation status of the STAT3 pathway and may not be as prohypertrophic as the other two subtypes.

Gene expression profiling of alpha(1b)-adrenergic receptor-induced cardiac hypertrophy by oligonucleotide arrays.

OBJECTIVE: Cardiac hypertrophy is closely associated with the development of cardiomyopathies that lead to heart failure. The alpha(1B) adrenergic receptor (alpha(1)-AR) is an important regulator of the hypertrophic process. Cardiac hypertrophy induced by systemic overexpression of the alpha(1b)-AR in a mouse model does not progress to heart failure. We wanted to explore potential gene expression differences that characterize this type of hypertrophy that may identify genes that prevent progression to heart failure. METHODS: Transgenic and normal mice (B6CBA) representing two time points were compared; one at 2-3 months of age before disease manifests and the other at 12 months when the hypertrophy is significant. Age-matched hearts were removed, cRNA prepared and biotinylated. Aliquots of the cRNA was subjected to hybridization with Affymetrix chips representing 12,656 murine genes. Gene expression profiles were compared with normal age-matched controls as the baseline and confirmed by Northern and Western analysis. RESULTS: The non-EST genes could be grouped into five functional classifications: embryonic, proliferative, inflammatory, cardiac-related, and apoptotic. Growth response genes involved primarily Src-related receptors and signaling pathways. Transgenic hearts also had a 60% higher Src protein content. There was an inflammatory response that was verified by an increase in IgG and kappa-chained immunoglobulins by western analysis. Apoptosis may be regulated by cell cycle arrest through a p53-dependent mechanism. Cardiac gene expression was decreased for common hypertrophy-inducing proteins such as actin, collagen and GP130 pathways. CONCLUSIONS: Our results suggest a profile of gene expression in a case of atypical cardiac hypertrophy that does not progress to heart failure. Since many of these altered gene expressions have not been linked to heart failure models, our findings may provide a novel insight into the particular role that the alpha(1B)AR plays in its overall progression or regression.