Denise J. Spring

p90(RSK) is a serum-stimulated Na+/H+ exchanger isoform-1 kinase. Regulatory phosphorylation of serine 703 of Na+/H+ exchanger isoform-1

The Na+/H+exchanger isoform-1 (NHE-1) is the key member of a family of exchangers that regulates intracellular pH and cell volume. Activation of NHE-1 by growth factors is rapid, correlates with increased NHE-1 phosphorylation and cell alkalinization, and plays a role in cell cycle progression. By two-dimensional tryptic peptide mapping of immunoprecipitated NHE-1, we identify serine 703 as the major serum-stimulated amino acid. Mutation of serine 703 to alanine had no effect on acid-stimulated Na+/H+ exchange but completely prevented the growth factor-mediated increase in NHE-1 affinity for H+. In addition, we show that p90 ribosomal S6 kinase (p90RSK) is a key NHE-1 kinase since p90RSK phosphorylates NHE-1 serine 703 stoichiometricallyin vitro, and transfection with kinase-inactive p90RSK inhibits serum-induced phosphorylation of NHE-1 serine 703 in transfected 293 cells. These findings establish p90RSK as a serum-stimulated NHE-1 kinase and a mediator of increased Na+/H+ exchange in vivo.

Inhibition of Toll-like Receptor 4 With Eritoran Attenuates Myocardial Ischemia-Reperfusion Injury

Background— We previously reported that the functional mutation of Toll-like receptor 4 (TLR4) in C3H/HeJ mice subjected to myocardial ischemia-reperfusion (MI/R) injury resulted in an attenuation of myocardial infarction size. To investigate the ligand-activating TLR4 during MI/R injury, we evaluated the effect of eritoran, a specific TLR4 antagonist, on MI/R injury, with the goal of defining better therapeutic options for MI/R injury. Methods and Results— C57BL/6 mice received eritoran (5 mg/kg) intravenously 10 minutes before 30 minutes of in situ of transient occlusion of the left anterior descending artery, followed by 120 minutes of reperfusion. Infarct size was measured using triphenyltetrazoliumchloride staining. A c-Jun NH2-terminal kinase (JNK) activation was determined by Western blotting, nuclear factor (NF)-&kgr;B activity was detected by gel-shift assay, and cytokine expression was measured by ribonuclease protection assay. Mice treated with eritoran developed significantly smaller infarcts when compared with mice treated with vehicle alone (21.0±6.4% versus 30.9±13.9%; P=0.041). Eritoran pretreatment resulted in a reduction in JNK phosphorylation (eritoran versus vehicle: 3.98±0.81 versus 7.01±2.21-fold increase; P=0.020), less nuclear NF-&kgr;B translocation (2.70±0.35 versus 7.75±0.60-fold increase; P=0.00007), and a decrease in cytokine expression (P<0.05). Conclusions— We conclude that inhibition of TLR4 with eritoran in an in situ murine model significantly reduces MI/R injury and markers of an inflammatory response.

Protease-Activated Receptor-1 Contributes to Cardiac Remodeling and Hypertrophy

Background— Protease-activated receptor-1 (PAR-1) is the high-affinity receptor for the coagulation protease thrombin. It is expressed by a variety of cell types in the heart, including cardiomyocytes and cardiac fibroblasts. We have shown that tissue factor (TF) and thrombin contribute to infarct size after cardiac ischemia-reperfusion (I/R) injury. Moreover, in vitro studies have shown that PAR-1 signaling induces hypertrophy of cardiomyocytes and proliferation of cardiac fibroblasts. The purpose of the present study was to investigate the role of PAR-1 in infarction, cardiac remodeling, and hypertrophy after I/R injury. In addition, we analyzed the effect of overexpression of PAR-1 on cardiomyocytes. Methods and Results— We found that PAR-1 deficiency reduced dilation of the left ventricle and reduced impairment of left ventricular function 2 weeks after I/R injury. Activation of ERK1/2 was increased in injured PAR-1−/− mice compared with wild-type mice; however, PAR-1 deficiency did not affect infarct size. Cardiomyocyte-specific overexpression of PAR-1 in mice induced eccentric hypertrophy (increased left ventricular dimension and normal left ventricular wall thickness) and dilated cardiomyopathy. Deletion of the TF gene in cardiomyocytes reduced the eccentric hypertrophy in mice overexpressing PAR-1. Conclusions— Our results demonstrate that PAR-1 contributes to cardiac remodeling and hypertrophy. Moreover, overexpression of PAR-1 on cardiomyocytes induced eccentric hypertrophy. Inhibition of PAR-1 after myocardial infarction may represent a novel therapy to reduce hypertrophy and heart failure in humans.

Protease-Activated Receptor 2 Deficiency Reduces Cardiac Ischemia/Reperfusion Injury

Objective—To investigate the effect of protease-activated receptor (PAR) 2 deficiency on ischemia/reperfusion (I/R) injury–induced infarct size, inflammation, heart remodeling, and cardiac function. Methods and Results—PAR-2 signaling enhances inflammation in different diseases. The effect of PAR-2 deficiency in cardiac I/R injury is unknown. PAR-2−/− mice and wild-type littermates were subjected to 30 minutes of ischemia and up to 4 weeks of reperfusion. Infarct size, oxidative/nitrative stress, phosphorylation of mitogen-activated protein kinases, and inflammatory gene expression were assessed 2 hours after reperfusion. Changes in heart size and function were measured by echocardiography up to 4 weeks after reperfusion. Infarct size was significantly reduced in hearts of PAR-2−/− mice compared with wild-type littermates. In addition, oxidative/nitrative stress, phosphorylation of mitogen-activated protein kinase, and expression of proinflammatory genes were significantly attenuated in injured hearts of PAR-2−/− mice. Finally, PAR-2−/− mice were protected from postinfarction remodeling and showed less impairment in heart function compared with wild-type littermates up to 4 weeks after I/R injury. Conclusion—PAR-2 deficiency reduces myocardial infarction and heart remodeling after I/R injury.

Regulation and actions of activin A and follistatin in myocardial ischaemia–reperfusion injury

Activin A, a member of the transforming growth factor-β superfamily, is stimulated early in inflammation via the Toll-like receptor (TLR) 4 signalling pathway, which is also activated in myocardial ischaemia-reperfusion. Neutralising activin A by treatment with the activin-binding protein, follistatin, reduces inflammation and mortality in several disease models. This study assesses the regulation of activin A and follistatin in a murine myocardial ischaemia-reperfusion model and determines whether exogenous follistatin treatment is protective against injury. Myocardial activin A and follistatin protein levels were elevated following 30 min of ischaemia and 2h of reperfusion in wild-type mice. Activin A, but not follistatin, gene expression was also up-regulated. Serum activin A did not change significantly, but serum follistatin decreased. These responses to ischaemia-reperfusion were absent in TLR4(-/-) mice. Pre-treatment with follistatin significantly reduced ischaemia-reperfusion induced myocardial infarction. In mouse neonatal cardiomyocyte cultures, activin A exacerbated, while follistatin reduced, cellular injury after 3h of hypoxia and 2h of re-oxygenation. Neither activin A nor follistatin affected hypoxia-reoxygenation induced reactive oxygen species production by these cells. However, activin A reduced cardiomyocyte mitochondrial membrane potential, and follistatin treatment ameliorated the effect of hypoxia-reoxygenation on cardiomyocyte mitochondrial membrane potential. Taken together, these data indicate that myocardial ischaemia-reperfusion, through activation of TLR4 signalling, stimulates local production of activin A, which damages cardiomyocytes independently of increased reactive oxygen species. Blocking activin action by exogenous follistatin reduces this damage.

Deletion of 11 Amino Acids in p90rsk-mo-1 Abolishes Kinase Activity

ABSTRACT p90 rsk is a distal member of the mitogen-activated protein kinase signaling pathway. It has been cloned from a variety of species including Xenopus laevis, mouse, chicken, rat, and human. The clone p90 rsk-mo-1, isolated by others from a mouse library, contains a unique 33-nucleotide deletion not found in the p90 rsk clones from any other species that have been examined. When p90 rsk-mo-1 was expressed in Cos-7 cells that were subsequently stimulated with epidermal growth factor, the immunoprecipitated p90 rsk-mo-1 protein showed no measurable kinase activity toward the ribosomal protein S6 peptide. By comparison, expression of rat p90 rsk-1 resulted in significant kinase activity. Deletion of the 33-nucleotide region missing in the p90 rsk-mo-1clone from the p90 rsk-rat-1 cDNA abolished kinase activity in the resulting protein. When these 33 nucleotides were introduced into the p90 rsk-mo-1 cDNA, the expressed protein showed significant kinase activity. Reverse transcription-PCR and direct sequencing of mRNA isolated from several mouse tissues indicated the presence of the full-length form of p90 rsk-1 in the mouse and showed no conclusive evidence for a deletion-containing form. This study indicates the presence of a full-length p90 rsk-1 mRNA in mouse tissues that is homologous to that identified in other species and suggests that the deletion in p90 rsk-mo-1may be a cloning artifact. The findings provide additional support for the conclusion that the first catalytic domain of p90 rsk is responsible for its enzymatic activity toward ribosomal protein S6.