This application claims the benefit of U.S. Provisional Patent Application Ser. No. 60/625,881, filed Nov. 8, 2004, which is hereby incorporated by reference in its entirety.
The subject matter of this application was made with support from the United States Government under National Institutes of Health Grant No. RO1 HL 44721, HL-66919, and GM-071485-01A1. The U.S. Government may have certain rights.
The present invention relates generally to transgenic non-human animal models of ischemic reperfusion damage and the use thereof to identify potential therapeutics for inhibiting reperfusion damage following an ischemic event.
The sodium/hydrogen exchanger (NHE) family regulates intracellular pH (pHi). Among the plasma membrane isoforms only NHE1 is expressed at significant levels in the heart. Numerous experimental studies show that NHE1 activity plays a critical role in acute cardiac ischemia and reperfusion (I/R) injury. Pharmacological strategies that inhibit NHE1 activity dramatically reduce infarct size and improve cardiac function (Karmazyn, M., “Amiloride Enhances Postischemic Ventricular Recovery: Possible Role of Na + -H + Exchange,” Am J Physiol 255:H608-615 (1988)). Several compounds, including amiloride, eniporide (EMD-85131), and cariporide (HOE642), are well known as specific NHE1 inhibitors. Using cariporide in an experimental I/R model, infarct size was reduced and cardiac cell death was improved (Miura et al., “Infarct Size Limitation by a New Na + -H + Exchange Inhibitor, Hoe 642: Difference From Preconditioning in the Role of Protein Kinase C.,” J Am Coll Cardiol 29:693-701 (1997); Chakrabarti et al., “A Rapid Ischemia-Induced Apoptosis in Isolated Rat Hearts and Its Attenuation by the Sodium-Hydrogen Exchange Inhibitor HOE 642 (Cariporide),” J Mol Cell Cardiol 29:3169-3174 (1997)). This evidence led to the clinical testing of highly selective pharmacological inhibitors of NHE1 as potential therapeutic agents for cardioprotection in acute coronary syndromes and after myocardial infarction. Unfortunately, no clinical benefit was observed in two large clinical trials (Klatte et al., “Increased Mortality After Coronary Artery Bypass Graft Surgery is Associated with Increased Levels of Postoperative Creatine Kinase-Myocardial Band Isoenzyme Release: Results From the GUARDIAN Trial,” J Am Coll Cardiol 38:1070-1077 (2001); Zeymer et al., “The Na + /H + Exchange Inhibitor Eniporide as an Adjunct to Early Reperfusion Therapy for Acute Myocardial Infarction. Results of the Evaluation of the Safety and Cardioprotective Effects of Eniporide in Acute Myocardial Infarction (ESCAMI) Trial,” J Am Coll Cardiol 38:1644-1650 (2001)). One reason may be that the basal, acid stimulated homeostatic function of NHE1 is impaired by cariporide and zoniporide, and this function is likely important for cell survival.
It was previously reported that transfection of HEK293 cells with wild-type RSK enhanced NHE phosphorylation and activity, while RSK reduced NHE1 (Takahashi et al., “p90RSK is a Serum-Stimulated NHE Kinase: Regulatory Phosphorylation of Serine 703 of Na + /H + Exchanger Isoform-1,” J Biol Chem 274:20206-20214 (1999)). Furthermore, it was found that RSK phosphorylated S703 on the C-terminus of NHE1 and the adapter protein 14-3-3 bound to phospho-S703, which increased NHE1 activity (Lehoux et al., “14-3-3 Binding to Na + /H + Exchanger Isoform-1 is Associated With Serum-Dependent Activation of Na + /H + Exchange,” J Biol Chem 276:15794-15800 (2001); Cavet et al., “14-3-3beta is a p90 Ribosomal S6 Kinase (RSK) Isoform 1-Binding Protein That Negatively Regulates RSK Kinase Activity,” J Biol Chem 278:18376-18383 (2003)).
Takeishi et al. reported that RSK and ERK1/2 were activated in patients with late phase dilated cardiomyopathy (Takeishi et al., “Activation of Mitogen-Activated Protein Kinases and p90 Ribosomal S6 Kinase in Failing Human Hearts with Dilated Cardiomyopathy,” Cardiovasc Res 53:131-137 (2002)). Also, Seko et al. reported that RSK and ERK1/2 were activated by Raf-1-MAPK cascade in neonatal rat cardiomyocytes stimulated by VEGF (Seko et al., “Vascular Endothelial Growth Factor (VEGF) Activates Raf-1, Mitogen-Activated Protein (MAP) Kinases, and S6 Kinase (p90rsk) in Cultured Rat Cardiac Myocytes,” J Cell Physiol 175:239-246 (1998)). Additionally, RSK and ERK1/2 were activated by Raf-1 stimulation following hypoxia oxygenation in neonatal rat cardiomyocytes (Seko et al., “Hypoxia and Hypoxia/Reoxygenation Activate Raf-1, Mitogen-Activated Protein Kinase, Mitogen-Activated Protein Kinases, and S6 Kinase in Cultured Rat Cardiac Myocytes,” Circ Res 78:82-90 (1996)). Based on these reports, it is proposed herein that NHE1 is activated in the myocardium after I/R by a cascade including ERK1/2, RSK, and NHE1.
The renin-angiotensin and kallikrein-kinin systems are important regulators of blood pressure and atherosclerosis. Renin is an enzyme that converts the circulating substrate angiotensinogen, abundant in many tissues and the circulating blood, into the decapeptide angiotensin I (ang I) in plasma and tissue. Angiotensin-converting enzyme (ACE), present in vascular endothelium, particularly in the lungs, mediates the generation of an octapeptide, angiotensin II (ang II), from angiotensin I. Ang II causes increases in systemic vascular resistance and arterial pressure, which can lead to vasoconstriction, and possibly hypertension. Other cellular reactions mediate by ang II include production of endothelin and superoxide, retention of sodium and water, and cellular proliferation. ACE and ang II inhibitors are well-known post myocardial infarction (MI) therapeutics.
Diabetes is an independent risk factor for both mortality and morbidity after myocardial infarction (Grundy et al., “Diabetes and Cardiovascular Disease: a Statement for Healthcare Professionals From the American Heart Association,” Circulation 100(10):1134-1146 (1999)). A nrber of clinical studies show that post-MI left ventricular function is significantly worse in diabetic patients compared with non-diabetic patients (Zuanetti et al., “Effect of the ACE Inhibitor Lisinopril On Mortality in Diabetic Patients With Acute Myocardial Infarction: Data From the GISSI-3 Study,” Circulation 96(12):4239-4245 (1997); Gustafsson et al., “Effect of the Angiotensin-Converting Enzyme Inhibitor Trandolapril On Mortality and Morbidity in Diabetic Patients With Left Ventricular Dysfunction After Acute Myocardial Infarction,” Trace Study Group J Am Coll Cardiol 34(1):83-89 (1999)). In addition, several clinical studies strongly indicate that activation of the renin-angiotensin system (RAS) in diabetic patients is a critical factor to developing heart failure after MI (Zuanetti et al., “Effect of the ACE Inhibitor Lisinopril On Mortality in Diabetic Patients With Acute Myocardial Infarction: Data From the GISSI-3 Study,” Circulation 96(12):4239-4245 (1997); Gustafsson et al., “Effect of the Angiotensin-Converting Enzyme Inhibitor Trandolapril On Mortality and Morbidity in Diabetic Patients With Left Ventricular Dysfunction After Acute Myocardial Infarction Trace Study Group,” J Am Coll Cardiol 34(1):83-89 (1999)). Although these clinical studies indicated that there is greater benefit for ACE inhibitor treatment post-MI in diabetic patients than nondiabetic patients, the molecular basis for this difference is unclear. Over the past several decades, a number of laboratories have examined the levels and activity of elements of the renin-angiotensin system (RAS) in plasma and in various tissues during diabetes. The measurements of angiotensin (Ang) II and its upstream components of the RAS have been complicated by the rapid degradation of these peptides (Al-Merani et al., “The Half-Lives of Angiotensin II, Angiotensin II-Amide, Angiotensin III, Sar1-Ala8-Angiotensin II and Renin in the Circulatory System of the Rat,” J Physiol 278:471-490 (1978); Chapman et al., “Half-Life of Angiotensin II in the Conscious and Barbiturate-Anaesthetized Rat,” Br J Anaesth 52(4):389-393 (1980)), and the local regulation of this production within specific vascular tissue and lesions (Takai et al., “Induction of Chymase That Forms Angiotensin II in the Monkey Atherosclerotic Aorta,” FEBS Lett 412(1):86-90 (1997)). Therefore, reports on the effects of diabetes on plasma and tissue RAS including ang II levels are controversial (Nakayama et al., “Adrenal Renin-Angiotensin-Aldosterone System in Streptozotocin-Diabetic Rats,” Horm Metab Res 30(1):12-15 (1998); Cronin et al., “Reduced Plasma Aldosterone Concentrations in Randomly Selected Patients With Insulin-Dependent Diabetes Mellitus,” Diabet Med 12(9):809-815 (1995); Price et al., “The Paradox of the Low-Renin State in Diabetic Nephropathy,” J Am Soc Nephrol 10(11):2382-2391 (1999)), and interpretation of these changes is limited by the potential downstream modulation of RAS production and stability.
The importance of PKCP activation during diabetes has been demonstrated by studies reporting that the specific PKCP inhibitor, LY333531, inhibited many abnormalities such as renal mesangial expansion, cardiomyopathy, and monocyte activation in diabetic rats (King et al., “Biochemical and Molecular Mechanisms in the Development of Diabetic Vascular Complications,” Diabetes 3:S105-108 (1996); Tuttle et al., “A Novel Potential Therapy for Diabetic Nephropathy and Vascular Complications: Protein Kinase C beta Inhibition,” Am J Kidney Dis 42(3):456-465 (2003)). It has also been reported that cardiac-specific overexpression of PKCβII, but not PKCε, in transgenic mice decreased cardiac function (Takeishi et al., “Transgenic Overexpression of Constitutively Active Protein Kinase C Epsilon Causes Concentric Cardiac Hypertrophy,” Circ Res 86(12):1218-1223 (2000)). Previously it was shown that H 2 O 2 -mediated p90RSK activation is partially dependent on PKC activation in Jurkat cells (Abe et al., “Reactive Oxygen Species Activate p90 Ribosomal S6 Kinase Via fyn and ras,” J Biol Chem 275(3):1739-1748 (2000)). Interestingly, p90RSK activation is specifically up-regulated in overexpression of PKCβII transgenic mice, which is thought to be a diabetic cardiomyopathy model (Itoh et al., “Role of p90 Ribosomal S6 Kinase (p90RSK) in Reactive Oxygen Species and Protein Kinase C β (PKC β)-mediated Cardiac Troponin I Phosphorylation,” J Biol Chem 280(25):24135-24142 (2005)).
p90RSK is a serine/threonine kinase, and is involved in activation of nuclear factor-κB by phosphorylation of IK-B (Ghoda et al., “The 90-kDa Ribosomal S6 Kinase (pp90rsk) Phosphorylates the N-terminal Regulatory Domain of IkappaBalpha and Stimulates Its Degradation In Vitro,” J Biol Chem 272(34):21281-21288 (1997)), or phosphorylation of transcription factors, including c-Fos (Chen et al., “Regulation of pp 90rsk Phosphorylation and S6 Phosphotransferase Activity in Swiss 3T3 Cells by Growth Factor-, Phorbol Ester-, and Cyclic AMP-mediated Signal Transduction,” Mol Cell Biol 11(4):1861-1867 (1991)), Nur77 (Fisher et al., “Evidence for Two Catalytically Active Kinase Domains in pp90rsk,” Mol Cell Biol 16(3):1212-1219 (1996)), and CREB (Xing et al., “Coupling of the RAS-MAPK Pathway to Gene Activation by RSK2, a Growth Factor-regulated CREB Kinase,” Science 273(5277):959-963 (1996)). However, the role of p90RSK and its relation with RAS in diabetic hearts remains largely unknown.
What is needed now is a method to treat I/R injury that involves specifically targeting inhibition of RSK and reduction of NHE1 activity in response to agonists such as H 2 O 2 and/or other reactive oxygen species, while preserving basal Na + /H + exchange function. Such a method would provide a tremendous benefit for prevention of and recovery from myocardial infarction, stroke, and other debilitating and potentially fatal I/R injury-related conditions for which no such treatment currently exists. Also needed is a model for the study of diabetic cardiomyopathy, and a greater understanding of the functional role(s) of p90RSK and PRECE induction in ischemic and diabetic myocardium, which may provide an alternative therapeutic approach to treat diabetic cardiomyopathy.
The present invention is directed to overcoming these and other deficiencies in the art.
A first aspect of the present invention relates to a transgenic non-human animal having a transgene encoding a mutant p90 ribosomal S6 kinase (RSK) that is rendered kinase inactive for phosphorylation of NHE1, particularly though not exclusively, phosphorylation at S703. A method of generating the transgenic animal is also disclosed.
A second aspect of the present invention relates to an isolated, recombinant cell comprising a transgene encoding a mutant p90 ribosomal S6 kinase (RSK) that is rendered kinase inactive for phosphorylation of NHE1, particularly though not exclusively, phosphorylation at S703. A method of generating the transgenic animal is also disclosed.
A third aspect of the present invention relates to a method of treating an individual to inhibit reperfusion damage following an ischemic event. This method involves administering to an individual an agent that inhibits p90RSK-induced activation of NHE1, thereby inhibiting activated NHE1-induced reperfusion damage associated with the ischemic event.
A fourth aspect of the present invention relates to a method of identifying an agent capable of inhibiting p90RSK-induced activation of NHE1. This method involves providing a cell culture having cells that express p90RSK and NHE1, treating the cells with a drug to be tested, exposing the cells to an agonist that normally causes RSK-induced activation of NHE1, and determining the level of p90RSK-induced activation of NHE1 in the treated cells A reduction in the level of p90RSK-induced activation of NHE1 occurring in the treated cells, as compared to the untreated cells, indicates the efficacy of the agent.
A fifth aspect of the present invention relates to a method of identifying an agent that modulates ischemic reperfusion (I/R) injury resulting from an ischemic event. This method involves providing a transgenic non-human animal whose genome comprises a transgene encoding a mutant p90 ribosomal S6 kinase (p90RSK) that is rendered kinase inactive for phosphorylation, preferably S703 phosphorylation, of NHEL; exposing the transgenic non-human animal to conditions effective to produce an ischemic event in the transgenic non-human animal; administering to the transgenic non-human animal an agent to be tested; and determining whether the agent modulates the ischemic reperfusion injury resulting from the ischemic event in the transgenic non-human animal (i.e., as compared to a non-human animal lacking the transgene).
A sixth aspect of the present invention relates to an isolated nucleic acid molecule encoding a mutant p90 ribosomal S6 kinase (p90RSIC), where the mutant p90RSK is a K94A/K447A mutant of a wild type p90RSK amino acid sequence. Also provided in the present invention are expression vectors and hosts including a K94A/K447A p90RSK mutant.
A seventh aspect of the present invention relates to a second transgenic non-human animal. This transgenic non-human animal includes a transgene that encodes for cardiac-specific overexpression of wild type p90RSK compared to a non-transgenic animal.
An eighth aspect of the present invention relates to an isolated, recombinant cell comprising a transgene that encodes for cardiac-specific overexpression of wildtype p90RSK.
A ninth aspect of the present invention relates to a method of treating an individual to inhibit ischemia reperfusion injury associated with an ischemic event. This method involves administering to an individual an effective amount of an agent that inhibits p90 ribosomal S6 kinase (p90RSK)-induced activation of pro-renin converting enzyme (PRECE), thereby inhibiting ischemia reperfusion injury associated with an ischemic event.
A tenth aspect of the present invention relates to a method of identifying an agent that modulates ischemic reperfusion injury resulting from an ischemic event. This method involves providing a transgenic non-human animal whose genome comprises a transgene encoding for cardiac-specific overexpression of wild type p90 ribosomal S6 kinase (p90RSK); exposing the transgenic non-human animal to conditions effective to produce an ischemic event in the transgenic non-human animal; administering to the transgenic non-human animal an agent to be tested; and determining whether the agent modulates the ischemic reperfusion (I/R) injury resulting from the ischemic event in the transgenic non-human animal.
The present invention provides two transgenic non-human animals useful for the study of I/R injury and the development of therapeutics and methods of treatment for I/R injury that are directed to new pathological mediators of I/R injury in the heart. Also provided is an improved and much needed method of preventing functional derangement and cell death in cells that have been, or may be, subjected to I/R injury.
FIG. 1 is a western blot showing wild type (WT-RSK) and double negative mutant p90 ribosomal S6 kinase (DN-RSK) expression in neonatal rat cardiomyocytes. An adenoviral expression vector containing the DN-RSK gene (Ad.DN-RSK) was transduced into neonatal rat cardiomyocytes. Transduction was for 3 hrs incubated without serum, and cells were harvested after 48 hrs. Cell lysates were prepared and western blot performed with an antibody to RSK that detects both endogenous RSK isoforms (RSK 1 and RSK2) and the transduced DN-RSK.
FIGS. 2A-D are graphs showing that H 2 O 2 -stimulated intracellular pH (pHi) recovery is inhibited by Ad.DN-RSK. Neonatal rat cardiac myocytes transduced with adenovirus were acid-loaded by NH 4 Cl prepulse, plus H 2 O 2 treatment for 10 min. Results are average of >10 individual cell recordings. The rate of pHi recovery was measured with BCECF-AM (2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein , acetoxymethyl ester). FIG. 2A shows results in Ad.LacZ-transduced cells. FIG. 2B shows the results with Ad.DN-RSK-transduced cells. FIG. 2C shows recovery rate, calculated from the first 60 sec of each recovery curve (n=5). FIG. 2D shows the rate of H + efflux (J H ) during pHi recovery, calculated in H 2 O 2 stimulated cells. Results are mean ±S.E., n=5,*p<0.05 vs. vehicle-control, †p<0.05 vs. H 2 O 2 -lacZ.
FIGS. 3A-E show analysis of cardiac RSK expression, endogenous cardiomyocyte RSK phosphorylation and the effect of Ad.DN-RSK on apoptosis (cell death). Endogenous cardiomyocyte RSK phosphorylation was analyzed by western blot analysis using an antibody specific for phospho-RSK (p-RSK). Isolated cardiomyocytes were subjected to A/R (12 hr/10 min). Cell lysates were prepared and subjected to SDS-PAGE (20 μg total protein) followed by western blotting for p-RSK (n=3, *p<0.05). Western blot results are shown in FIG. 3A. FIG. 3B is graph showing increase of p-RSK expression in A/R cells vs. control cells. FIGS. 3C-D are graphs showing effects of Ad.DN-RSK on cell death. Cells were transduced with Ad.LacZ or Ad.DN-RSK for two hr and cultured one day after changing the medium. Apoptosis was induced by 12 hrs anoxia/24 hrs reoxygenation (A/R). FIG. 3C shows quantitation of cardiomyocytes apoptosis performed with a TUNEL (Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling) assay. FIG. 3D shows cells death quantitated by anti-DNA fragmentation ELISA. Data are mean ±S.E. (n=5 for each group from 3 independent experiments; *p<0.05). FIG. 3E shows WT-RSK enhanced A/R induced apoptosis in H9c2 cells via NHE1 activity. H9c2 rat embryonic cardiac myoblasts were transduced with cDNAs expressing EGFP alone, WT-RSK, NHE1-WT or NHE1-S703A. The latter three were co-transfected with EGFP to identify transfected cells. Cells were exposed to experimental conditions 48 hrs after transfection. Conditions included EIPA alone (5 μM), A/R (12 hr/24 hr) or both EIPA and A/R. Transfected cells only were counted for analysis and were identified by expression of EGFP. To analyze apoptosis, 100 TUNEL positive cells were measured for each condition. Data are mean ±S.E (n=5 for each group from 3 independent experiments). *p<0.05 vs. Control (no A/R), **p<0.05 vs. A/R, †p<0.05 vs. A/R WT-NHE ††p<0.05 vs. A/R and A/R+RSK.
FIGS. 4A-C show results of treatment consisting of 45 min ischemia/24 hrs reperfusion in non-transgenic littermate controls (NLC) and DN-RSK TG mice. FIG. 4A shows RSK expression detected by western blotting (top panel) and PCR (bottom panel) performed as described in the Examples. FIG. 4B are representative photographs of midventricular myocardium, showing infarct size, from transgenic (TG) DN-RSK mouse and NLC. FIG. 4C is a graph showing quantitation of infarct size (1S) in area at risk (AAR) ratio in NLC (n=11) and DN-RSK TG (n=11, *p<0.05) following treatment as described.
FIGS. 5A-B show a time course of endogenous RSK activation by I/R. Hearts made ischemic by coronary ligation for 45 min followed by the indicated reperfusion times (0, 20, 120, 360 min). After reperfusion, hearts were saline perfused, stained with Evans blue, sectioned, and the ischemic area harvested for western blotting. The phospho-specific p90RSK antibody was used to recognize activated RSK by virtue of binding to phospho-Thr359/Ser363.
FIG. 5A shows the peak of endogenous RSK phosphorylation at 20 min reperfusion. FIG. 5B shows quantitation by densitometry. Results were normalized by arbitrarily setting the baseline value (I/R=0/0) to 1.0 (n=4).
FIGS. 6A-C show results of NHE1 binding to 14-3-3 β in I/R heart tissue. FIG. 6A shows samples from sham and I/R hearts lysed and immunoprecipitated with 14-3-3 β antibody and immunoblotted for NHE1 (upper panel) and 14-3-3 P (middle panel). Total cell lysate was immunoblotted with NHE1 antibody (lower panel). FIG. 6B shows densitometric analysis of NHE1 binding to 14-3-3 after normalizing NLC to 1.0 (n=4), p=0.01). FIG. 6C shows in vitro RSK kinase activity of samples from FIG. 6A.
FIGS. 7A-C are comparisons of DN-RSK-Tg (TG) and control (NLC) hearts after I/R (I=45 min, R=2 wks). FIG. 7A shows H&E (hematoxylin and eosin) and Masson trichrome staining section of mid-ventricular myocardium from TG and NLC mice. FIG. 7B is a fibrosis area measurement from the Masson trichrome staining of FIG. 7A. Values are group means ±S.E, n=11*P<0.05. FIG. 7C shows representative M-mode echocardiographic images of intact beating hearts after reperfusion for 2 weeks, NLC (upper panel) TG (lower panel).
FIGS. 8A-D are western blots of ERK1/2 and PKCα/βII activity in STZ-mediated hyperglycemic mice. FIG. 8A shows result with a PKCα/βII antibody. FIG. 8B shows results with a PKCβII antibody. FIG. 8C shows results with phosphor-specific ERK1/2 antibody. FIG. 8D shows results with anti-ERK1/2. These results demonstrate that PKCα/βII and p90RSK activation, but not ERK1/2, were increased in STZ-mediated hyperglycemic mice.
FIG. 9 is a graph showing p90RSK activation in STZ-mediated hyperglycemic mice. p90RSK activity was detected by in vitro kinase assay using S6 kinase substrate peptide as described below. Data (n=3) were expressed as mean ±S.D. **p<0.01
FIGS. 10A-B are immunoblots of lysates prepared from 10-week-old NLC and WT-p90RSK-Tg mice hearts showing the cardiac selective expression of WT-p90RSK. FIG. 10A shows results using a p90RSK antibody.
FIG. 10B shows actin control on same lysates.
FIGS. 11A-D show effects of ischemia on cardiac function and enzyme production. FIG. 11A are measurements of left ventricular developed pressure before, during, and after global (no-flow) ischemia followed by reperfusion. FIG. 11B are measurements of left ventricular dP/dtmax before, during, and after global (no-flow) ischemia followed by reperfusion. All experimental values calculated for NLC (n=5) and WT-p90RSK-Tg hearts (n=5) are represented as mean ±S.D. FIGS. 11C-D shows creative kinase (CK) and lactate dehydrogenase (LDH) cardiac enzymes, respectively, measured in the superfusate from the heart after ischemia (n=4) and reported as mean units/L±S.D.
FIGS. 12A-B are protein expression profiles of NLC and WT-p90RSK-Tg mice hearts. FIG. 12A upper and lower panels, are 2-D gels of NLC (upper) and WT-p90RSK-Tg (lower) cardiac proteins, stained with silver staining; IPG NL 4-7; 10% SDS-PAGE. After staining with silver staining, gel images were compared. Spots were selected that were significantly increased in WT-p90RSK-Tg samples, and digested with trypsin, then analyzed with MALDI-TOF mass spectrometry. Analysis of MALDI-TOF mass spectrometry demonstrates the 40% matching with PRECE-2 (mKLK26) amino acid sequence (SEQ ID NO: 12), shown in FIG. 12B. Bold characters in mouse PRECE-2 amino acid sequence indicate matched amino acids.
FIGS. 13A-B show PRECE expression in INT-p90RSH-Tg vs. NLC mice.
FIG. 13A shows results of relative quantitative RT-PCR analysis, showing PRECE mRNA expression increased in WT-p90RSK-Tg mice hearts. 18S rRNA was used as internal control. FIG. 13B is densitometric analysis of PRECE mRNA expression in NLC and WT-p90RSK-Tg mouse hearts. Results were normalized for all experiments by arbitrary setting the mean densitometry of NLC heart samples to 1.0 (shown in mean ±S.D., n=3, **p<0.01).
FIGS. 14A-B are analysis of angiotensinogen level in NLC and WT-p90RSK-Tg mice after perfusion. FIG. 14A shows immunoblot of lysates prepared from 10-week-old NLC and WT-p90RSK-Tg mice hearts and contacted with angiotensinogen (upper panel) and tubulin (bottom panel) antibodies. FIG. 14B shows densitometric analysis of serial angiotensinogen protein level in NLC and WT-p90RSK-Tg mouse hearts after perfusion. Results were normalized for all experiments by arbitrary setting the mean densitometry of NLC heart samples to 1.0 at 3 min after KH buffer perfusion (shown in mean ±S.D., n=4, *p<0.01).
FIGS. 15A-B show diabetes-mediated PRECE mRNA expression inhibited in DN-p90RSK-Tg mouse hearts. FIG. 15A shows STZ injection-mediated diabetes increased PRECE mRNA expression after 2 weeks of STZ injection, which was inhibited in DN-p90RSK-Tg mouse hearts. 18S rRNA was used as internal control. FIG. 15B is densitometric analysis of PRECE mRNA expression in STZ-injected diabetic NLC and DN-p90RSK-Tg mice. Results were normalized for all experiments by arbitrary setting the densitometry of control heart samples to 1.0 (shown in mean ±S.D., n=4, *p<0.05).
FIGS. 16A-H demonstrate ACE inhibitor (captopril 50 μM) protected WT-p90RSK-Tg hearts but not NLC hearts from I/R-induced contractile dysfunction. FIGS. 16A-D show measurements of left ventricular developed pressure and dP/dtmax before, during, and after global (no-flow) ischemia followed by reperfusion with vehicle or captopril (50 μM) pretreatment in NLC hearts. Short 20 min (FIG. 16A-B) or prolonged 40 min (FIG. 16C-D) ischemia was performed. FIGS. 16E-F shows measurements of left ventricular developed pressure and dP/dtmax, respectively, before, during, and after global (no-flow) ischemia followed by reperfusion with vehicle or captopril (50 μM) pretreatment in WT-p90RSK-Tg mouse hearts after 20 min ischemia. FIGS. 16G-H show measurements of left ventricular developed pressure and dP/dtmax, respectively, after prolonged 40 min (FIG. 16G) ischemia in NLC hearts and short 20 min (FIG. 16H) ischemia in WT-p90RSK-Tg hearts followed by 25 min reperfusion with vehicle or captopril (50 μM) pretreatment (shown in mean ±S.D., n=5, **p<0.01).
FIGS. 17A-B demonstrate ACE inhibitor (captopril 50 μM) protected WT-p90RSK-Tg hearts but not NLC hearts from I/R-induced cardiac injury. Cardiac enzymes were measured in the superfusate from the NLC hearts after prolonged 40 min ischemia (n=4) and p90RSK-Tg mouse hearts after short 20 min ischemia (n=4). FIG. 17A shows results of creatine kinase (CK) release.
FIG. 17B shows results of lactate dehydrogenase (LDH) release values reported as mean units/L ±S.D. (*p<0.05, **p<0.01).
FIGS. 18A-C are hemodynamic measurements in NLC (n=6) and WT-p90RSK-Tg (n=6) mice at age of 10 months old. All data are expressed as mean ±S.D. (**p<0.01, *p<0.05).
FIG. 19 are representative M-mode echocardiographic images of contracting hearts in 10 months old NLC and WT-p90RSK-Tg mice, showing cardiac dysfunction in WT-p90RSK-Tg mice.
FIGS. 20A-B shows percent fractional shorting (% FS) and velocity of circumferential fiber shortening (Vcfs), respectively, in 3 and 10 months old NLC (n=6), and WT-p90RSK-Tg (n=5) mice. Values (mean ±SEM) were determined by echocardiography. **p<0.01 between groups.
FIGS. 21A-C show detection of apoptosis by TUNEL assay. FIG. 21A shows results with NLC mice. FIG. 21B shows results with WT-p90RSK-Tg mice. Green fluorescence shows apoptotic cardiomyocytes stained with TUNEL, nuclei were counterstained with Hoechst33342 staining (blue), and cardiomyocytes were stained with anti-α-actin (sarcomeric) (clone EA-53, red). Overlay images were shown. FIG. 21C is quantitative analysis of apoptotic cells. The vertical axis indicates the % ratio of TUNEL-positive cell number relative to that of Hoechst33342-positive nuclei, which were clearly overlaid with EA-53 staining (indicated by arrows). Cells which did not counter stained clearly with EA-53 staining (indicated by asterisk) were not counted. More than 1000 cells were screened per section.
FIG. 22 shows Bcl-2 expression in NLC and WT-p90RSK-Tg mice. Lysates were prepared from 10-months-old NLC and WT-p90RSK-Tg mice hearts and immunoblot with a Bcl-2 (upper panel) and actin (lower panel) antibodies.
FIG. 23 shows ratios of heart weight to body weight (HW/BW) in 3 and 10 months old NLC and WT-p90RSK-Tg mice. Results demonstrate increase in cardiac hypertrophy over time.
FIG. 24A-B are blots showing atrial natriuretic factor (ANF) and brain natriuretic protein respectively (BNP). The upper panels in FIGS. 24A-B show mRNA expression in 10 months old NLC and WT-p90RSK-Tg mice. ANF and BNP mRNA levels were determined by relative quantitative RT-PCR. 18S rRNA was used as internal control. FIG. 24A-B, bottom panel, shows densitometric analysis of ANF and BNP mRNA expression, as marked. Results were normalized for all experiments by arbitrary setting the densitometry of NLC 10 months old heart samples to 1.0 (shown in mean ±S.D., n=4, **p<0.01).
FIG. 25 is representative image of NLC and WT-p90RSK-Tg hearts at 10 months of age.
FIGS. 26A-B are histological images (at 200×, Masson's trichrome) of hearts from a NLC and WT-p90RSK-Tg, respectively, at 10 months old, indicating interstitial fibrosis with apoptosis in WT-p90RSH-Tg mice.
FIGS. 27A-E demonstrate AT1 receptor blocker (olmesartan 10 μM) protected WT-p90RSK-Tg but not NCL hearts from I/R-induced contractile dysfunction. FIGS. 27A-B are graphs of measurements of left ventricular developed pressure and dP/dtmax, respectively, before, during, and after global (no-flow) ischemia followed by reperfusion with vehicle or olmesartan (an AT 1 receptor blocker) (10 μM) pretreatment in NLC hearts. Prolonged 40 min ischemia was performed. FIGS. 27C-D are graphs of measurements of left ventricular developed pressure and dP/dtmax, respectively, before, during, and after global (no-flow) ischemia followed by reperfusion with vehicle or olmesartan (10 μM) pretreatment in WT-p90RSK-Tg mouse hearts after 20 min ischemia. FIG. 27E is a graph of the measurement of left ventricular developed pressure after prolonged 40 min ischemia in NLC hearts and short 20 min ischemia in WT-p90RSK-Tg hearts followed by 25 min reperfusion with vehicle or olmesartan pretreatment (shown in mean ±S.D., n=5, **p<0.01).
FIG. 28 is a VISTA plot of the mouse KLK26 (PRECE-2) region (chromosome7; 38,077,009-38,091,292) on human genome (chromosome19: 56,049,788-56,073,634) detailing conserved regions between human and mouse. Peaks represent conserved regions, peak width represents the size of the conserved region, and peak height represents the percentage identity between human and mouse sequences. The positions of the exons are indicated by the blue boxes above the upper axis. The shaded regions indicate the conserved regions with the identity above 75%.
Applicants have identified the role that p90 ribosomal S6 Kinase (RSK or p90RSK, which are used interchangeably herein) plays in the activation of NHE1. In particular, applicants have demonstrated that inhibiting RSK activation of NHE1 can minimize ischemic-reperfusion injury while not otherwise modifying basal NHE1 exchange activity.
One aspect of the present invention relates to a method of (i.e., an assay for) identifying an agent (e.g., a drug) capable of inhibiting p90RSK-induced activation of NHE1. This method involves providing a cell culture having cells that express RSK and NHE1, treating the cells with an agent to be tested, exposing the cells to an agonist that normally causes RSK-induced activation of NHE1, and determining the level of RSK-induced activation of NHE1 in the treated cells. A reduction in the level of RSK-induced activation of NHE1 occurring in the treated cells, as compared to untreated cells exposed to the same agonist, indicates efficacy of the agent.
In one embodiment of this assay, exposure to the agonist precedes treatment of the cells in culture with the agent to be tested.
In another embodiment, the assay involves exposing the cells in culture to an agonist after treating the cells with the drug to be tested.
In yet another embodiment, the assay can be carried out with exposure to the agonist and treatment of the cells with the agent being performed concurrently.
In all aspects of this assay, the cells may be exposed to an agonist. This can be carried out by directly or indirectly by adding a reactive oxygen species to the cell culture. Suitable reactive oxygen species include, without limitation, H 2 O 2 , a molecule that generates H 2 O 2 , or any other reactive oxygen species.
Determining the level of p90RSK-induced activation of NHE1 in the treated cells may be carried out by any suitable method known in the art, including, without limitation, measuring H + efflux from the cells, measuring the binding of 14-3-3 proteins to NHE1 in the cells, measuring the S703 phosphorylation or dephosphorylation of NHE1 in the cells (e.g., using an antibody specific to phosphorylated or dephosphorylated NHE1 S703), measuring the changes in intracellular pH in the cells, measuring the changes in sodium fluxes in the cells, as well as any combination thereof.
Cells suitable for use in the cell culture of this aspect of the present invention are any cells that undergo functional derangement and cell death in response to ischemia/reperfusion, reactive oxygen species or oxidative stress, including, without limitation, cardiac muscle cells, smooth muscle cells, skeletal muscle cells, neuronal cells, and other cell types where reactive oxygen species, ischemia/reperfusion, and oxidative stress contribute to tissue dysfunction, cell impairment, and cell death. Preferably such cells are mammalians cells, including, without limitation, rodent and human.
The present invention also relates to a method of treating an individual to inhibit reperfusion damage following an ischemic event. This method involves administering to an individual an agent that inhibits p90RSK-induced activation of NHE1, thereby inhibiting activated NHE1-induced reperfusion damage associated with the ischemic event. In this aspect of the present invention, the agent that is administered preferably inhibits RSK-induced activation of NHE1 selectively, without altering basal Na + /H + exchange activity in the subject.
As described in greater detail herein below, pharmacological and genetic studies indicate that the Na + /H + exchanger isoform 1 (NHE1) plays a critical role in myocardial ischemia and reperfusion (I/R) injury. p90RSK phosphorylates the serine at position 703 of NHE1, stimulating the binding of NHE1 to the 14-3-3 protein, which, in turn, activates NHE1, leading to functional degradation and ultimately to apoptosis (cell death) of the NHE1-activated cells. Because the I/R injury results from a series of steps, I/R-mediated injury, i.e., reperfusion damage following an ischemic event, can be prevented or ameliorated by inhibiting the ability of RSK to phosphorylate NHE1, by decreasing the level of phosphorylation that NHE1 undergoes, or by interfering with the binding of the 14-3-3 protein with NHE1. As used herein “inhibition of RSK-induced activation of NHE1” is intended to mean the inhibition of the step of activating NHE1 as well as interfering with maintenance or function of the activated NHE1. Therefore, in one embodiment, the method of treating an individual to inhibit reperfusion damage following an ischemic event involves administering an agent that inhibits RSK phosphorylation of NHE1 S703. In another embodiment, this method involves administering an agent that accelerates the dephosphorylation of NHE1 S703. In yet another embodiment, this method involves administering an agent that accelerates the dissociation of a 14-3-3 protein from phosphorylated NHE1 S703.
Ischemic events suitable for treatment according to the present invention include, without limitation, heart attack (myocardial infarction), acute coronary syndrome, coronary artery bypass surgery, stroke, gastrointestinal ischemia, peripheral vascular disease, and surgical procedures associated with tissue ischemia.
All mammals are suitable individuals for treatment using this method of the present invention. Exemplary mammals include humans, non-human primates, rodents such as mice, rats, and guinea pigs, dogs, cats, etc.
In all aspects of this method of the present invention, suitable methods of “administering” the agent include, without limitation, oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, or intranasal. Preferred routes of administration deliver the active agent (e.g. drug) directly to the site of the ischemic event, thereby regulating the activation of NHE1 within the affected tissues. The agents may be administered alone or with suitable pharmaceutical carriers, and can be in solid or liquid form such as, tablets, capsules, powders, solutions, suspensions, or emulsions.
The active compounds of the present invention may be orally administered, for example, with an inert diluent, or with an assimilable edible carrier, or they may be enclosed in hard or soft shell capsules, compressed into tablets, or incorporated directly with the food of the diet. For oral therapeutic administration, the agents of the present invention may be incorporated with excipients and used in the form of tablets, capsules, elixirs, suspensions, syrups, and the like. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained.
The tablets, capsules, and the like may also contain a binder such as gum tragacanth, acacia, corn starch, or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose, or saccharin. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a fatty oil.
Various other materials may be present as coatings or to modify the physical form of the dosage unit. For instance, tablets may be coated with shellac, sugar, or both. A syrup may contain, in addition to active ingredient, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye, and flavoring such as cherry or orange flavor.
These active compounds may also be administered parenterally. Solutions or suspensions of these active compounds can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil. In general, water, saline, aqueous dextrose and related sugar solution, and glycols such as, propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.
In all aspects of this method, administration of the agent of the present invention may occur at the time of presentation of the ischemic event (i.e., soon after its occurrence), prior to presentation of the ischemic event, or concurrently with the ischemic event. In addition, such administration can be carried out in combination with other known therapeutic agents or hereafter developed therapeutic agents for the treatment of the ischemic event.
The present invention also relates to a transgenic non-human animal having a transgene encoding a mutant p90RSK that is rendered kinase inactive for cellular substrates including, without limitation, serine 703 (S703) phosphorylation of NHE1. According to one embodiment, the transgenic non-human animal is bred to contain both somatic and germ cells that harbor the RSK mutant transgene. In another embodiment, the transgenic non-human animal of the present invention is a somatic mosaic (i.e, harbors the RSK mutation in a subpopulation of somatic cells that have been transformed so as to express the transgene).
As used herein, kinase inactive forms of p90RSK are those that exhibit less than 25% activity (as compared to the rat p90RSK of SEQ ID NO:1) preferably less than 10% activity, more preferably less than 5% activity (including complete absence of activity).
Regardless of the embodiment, the transgenic non-human animal of the present invention is prepared so as to express the mutant p90RSK protein in one or more of cardiac muscle cells, smooth muscle cells, skeletal muscle cells, neuronal cells, and other cell types where reactive oxygen species, ischemia/reperfusion, and oxidative stress contribute to tissue dysfunction, cell impairment, and cell death.
In one aspect of the present invention the transgene is inserted into a suitable vector under the control of a tissue-specific nucleic acid promoter. An exemplary promoter is the α-myosin heavy chain promoter region (α-MHC), which allows expression preferentially in myosin-containing tissues, e.g., in the heart.
The term transgenic animal refers to an animal in which there has been a deliberate modification of the genome, i.e, the material responsible for inheritance. Foreign DNA is introduced into the animal, using recombinant DNA technology, and then must be transmitted through the germ line so that every cell, including germ cells, of the animal contains the same modified genetic material. The application of targeted gene modification and production of transgenic animals is a powerful tool for studying gene function in the context of a whole animal. Transgenic animals can be created by several methods that include either microinjection or viral infection of embryos, or through the manipulation in culture of embryonic stem cells that are subsequently incorporated back into the embryo for insertion into the germ line. Any of these techniques is useful for altering the expression of endogenous proteins by transfer of recombinant genes into cells in culture and into live animals to produce transgenic animals harboring the desired gene (Evans, M. J., “Potential for Genetic Manipulation of Mammals,” Mol Biol Med 6:557-565 (1989); Mansour, S. L., “Gene Targeting in Murine Embryonic Stem Cells: Introduction of Specific Alterations into the Mammalian Genome,” Genet Anal Tech Appl 7:219-227 (1990), which are hereby incorporated by reference in their entirety).
The transgenic non-human animal of the present invention may be made, for example, by DNA microinjection (Gordon et al., “Integration and Stable Germ Line Transformation of Genes injected into Mouse Pronuclei,” Science 214:1244-1246 (1981), which is hereby incorporated by reference in its entirety), a method used initially for mice, but has since been applied to many animal species. Briefly, this method involves the direct microinjection of a chosen gene construct (a single gene or a combination of genes) from another member of the same species or from a different species, into the pronucleus of a fertilized ovum. Microinjection of nucleic acid molecules into fertilized eggs (pronuclear stage) can be carried using an inverted microscope, micromanipulation equipment, and injection/holding devices. The pronuclear microinjection method of producing a transgenic animal results in the introduction of DNA sequences into the chromosomes of the fertilized eggs. The animal arising from the injected egg will carry the new gene and subsequently transmit this gene and its effect to offspring. If this transferred genetic material is integrated into one of the embryonic chromosomes, the animal will be born with a copy of this new information in every cell. The modified nucleic acid molecule must be integrated into the genome prior to the doubling of the genetic material that precedes the first cleavage. If this does not occur, only a few cells will integrate the gene. Because the germline of mammals is well protected against the incorporation of foreign genetic material, early embryonic stages (i.e., before the cells differentiate into the precursors of body and germ cells) are best suited for genetic manipulation. For this reason, the desired nucleic acid molecule is introduced into the fertilized egg at the earliest stage, which is the pronuclear period immediately following fertilization. The microinjected eggs are placed into a foster recipient and a normal pregnancy ensues.
Some of the resulting offspring animals in the litter will be somatic mosaics, in that a fraction of their somatic (body) cells will be hemizygous (have only one copy of the desired modified/mutated gene). These animals are identified, for example, by using polymerase chain reaction (PCR) for detection of the transgene. A fraction of the animals in this group will also be mosaic in their germ lines, which is determined by testing for progeny that are purely hemizygous. Chimeric offspring purely hemizygous for the desired trait are then mated to obtain homozygous individuals, and colonies characterized by the presence of the desired mutant protein are established.
In accordance with the invention, a nucleic acid molecule encoding a mutant RSK protein of the present invention is introduced in vivo using microinjection techniques, as describe above, and in Example 1, below, to produce a transgenic DN-RSK mutant non-human animal.
In one embodiment of the present invention, the transgenic non-human animal of the present invention is a somatic mosaic (i.e, harbors the RSK transgene of choice in a subpopulation of somatic cells only). In this aspect, the transgenic animal is prepared using standard DNA transformation techniques to incorporate the RSK mutant or wild type nucleic acid molecule into the sornatic cells of the animal. This involves, briefly, adding the desired nucleic acid molecule to cells other than egg or sperm cells. This can be carried out by preparing the desired RSK mutation nucleic acid molecule, combining it with suitable regulatory nucleic acid molecules, and inserting it into a host animal using any number of suitable methods. Recombinant molecules can be introduced into cells, without limitation, via direct injection of “naked” DNA into the animal using, e.g., electroporation or by gene gun; or incorporation into the host animal using viral vectors (transduction) or liposomal vectors containing the desired RSK mutant nucleic acid molecule, or using any other methods known in the art (e.g., as described by Sambrook et al., Molecular Cloning: A Laboratory Manual , Second Edition, Cold Springs Laboratory, Cold Springs Harbor, N.Y. (1989), which is hereby incorporated by reference in its entirety).
Suitable hosts are all non-human mammals, including, without limitation, rodents, such as mice or rats, as well as those identified above.
In one aspect of the present invention the transgenic non-human animal contains a nucleic acid molecule encoding a p90RSK mutant protein. A “p90RSK mutant” as used herein means a protein or polypeptide wherein specific amino acid substitutions to the mature wild-type RSK protein have been made that render the protein substantially inactive (preferably fully inactive) for kinase activity toward the ribosomal protein S6 peptide. “Wild-type RSK,” as used herein means a RSK protein or variant thereof, including but not limited to, that of rat, mouse, or human (e.g., SEQ. ID. No. 3; GenBank Accession No. M99169; Swiss-Pro Accession No. P18653; GenBank Accession No AF090421) that retains at least 75%, preferably 85-115%, more preferably 95-100% of normal activity. In a preferred embodiment of the present invention, the RSK mutant contains two separate amino acid substitutions, namely, a lysine to alanine substitution at peptide 94 (K94A) and a lysine to alanine substitution at peptide 447 (K447A) of the native RSK polypeptide, making the preferred K94A/K447A RSK mutant of the present invention, which is inactive for cellular substrates including serine 703. In one aspect of the present invention, the mutant p90RSK protein is a rat protein, made by selected amino acid substitutions made to the wild type rat p90RSK-1 ( R. norvegicus , sp:Q63531—K6A1_RAT Ribosomal protein S6 kinase alpha 1), SEQ ID NO: 1, as follows:
| Met Pro Leu Ala Gln Leu Lys Glu Pro Trp Pro Leu Met Glu Leu Val | |
| 1 5 10 15 | |
| Pro Leu Asp Pro Glu Asn Gly Gln Ala Ser Gly Glu Glu Ala Gly Leu | |
| 20 25 30 | |
| Gln Pro Ser Lys Asp Glu Gly Ile Leu Lys Glu Ile Ser Ile Thr His | |
| 35 40 45 | |
| His Val Lys Ala Gly Ser Glu Lys Ala Asp Pro Ser His Phe Glu Leu | |
| 50 55 60 | |
| Leu Lys Val Leu Gly Gln Gly Ser Phe Gly Lys Val Phe Leu Val Arg | |
| 65 70 75 80 | |
| Lys Val Thr Arg Pro Asp Asn Gly His Leu Tyr Ala Met Lys Val Leu | |
| 85 90 95 | |
| Lys Lys Ala Thr Leu Lys Val Arg Asp Arg Val Arg Thr Lys Met Glu | |
| 100 105 110 | |
| Arg Asp Ile Leu Ala Asp Val Asn His Pro Phe Val Val Lys Leu His | |
| 115 120 125 | |
| Tyr Ala Phe Gln Thr Glu Gly Lys Leu Tyr Leu Ile Leu Asp Phe Leu | |
| 130 135 140 | |
| Arg Gly Gly Asp Leu Phe Thr Arg Leu Ser Lys Glu Val Met Phe Thr | |
| 145 150 155 160 | |
| Glu Glu Asp Val Lys Phe Tyr Leu Ala Glu Leu Ala Leu Gly Leu Asp | |
| 165 170 175 | |
| His Leu His Ser Leu Gly Ile Ile Tyr Arg Asp Leu Lys Pro Glu Asn | |
| 180 185 190 | |
| Ile Leu Leu Asp Glu Glu Gly His Ile Lys Leu Thr Asp Phe Gly Leu | |
| 195 200 205 | |
| Ser Lys Glu Ala Ile Asp His Glu Lys Lys Ala Tyr Ser Phe Cys Gly | |
| 210 215 220 | |
| Thr Val Glu Tyr Met Ala Pro Glu Val Val Asn Arg Gln Gly His Thr | |
| 225 230 235 240 | |
| His Ser Ala Asp Trp Trp Ser Tyr Gly Val Leu Met Phe Glu Met Leu | |
| 245 250 255 | |
| Thr Gly Ser Leu Pro Phe Gln Gly Lys Asp Arg Lys Glu Thr Met Thr | |
| 260 265 270 | |
| Leu Ile Leu Lys Ala Lys Leu Gly Met Pro Gln Phe Leu Ser Thr Glu | |
| 275 280 285 | |
| Ala Gln Ser Leu Leu Arg Ala Leu Phe Lys Arg Asn Pro Ala Asn Arg | |
| 290 295 300 | |
| Leu Gly Ser Gly Pro Asp Gly Ala Glu Glu Ile Lys Arg His Ile Phe | |
| 305 310 315 320 | |
| Tyr Ser Thr Ile Asp Trp Asn Lys Leu Tyr Arg Arg Glu Ile Lys Pro | |
| 325 330 335 | |
| Pro Phe Lys Pro Ala Val Ala Gln Pro Asp Asp Thr Phe Tyr Phe Asp | |
| 340 345 350 | |
| Thr Glu Phe Thr Ser Arg Thr Pro Arg Asp Ser Pro Gly Ile Pro Pro | |
| 355 360 365 | |
| Ser Ala Gly Ala His Gln Leu Phe Arg Gly Phe Ser Phe Val Ala Thr | |
| 370 375 380 | |
| Gly Leu Met Glu Asp Asp Ser Lys Pro Arg Ala Thr Gln Ala Pro Leu | |
| 385 390 395 400 | |
| His Ser Val Val Gln Gln Leu His Gly Lys Asn Leu Val Phe Ser Asp | |
| 405 410 415 | |
| Gly Tyr Ile Val Lys Glu Thr Ile Gly Val Gly Ser Tyr Ser Val Cys | |
| 420 425 430 | |
| Lys Arg Cys Val His Lys Ala Thr Asn Met Glu Tyr Ala Val Lys Val | |
| 435 440 445 | |
| Ile Asp Lys Ser Lys Arg Asp Pro Ser Glu Glu Ile Glu Ile Leu Leu | |
| 450 455 460 | |
| Arg Tyr Gly Gln His Pro Asn Ile Ile Thr Leu Lys Asp Val Tyr Asp | |
| 465 470 475 480 | |
| Asp Ser Lys His Val Tyr Leu Val Thr Glu Leu Met Arg Gly Gly Glu | |
| 485 490 495 | |
| Leu Leu Asp Lys Ile Leu Arg Gln Lys Phe Phe Ser Glu Arg Glu Ala | |
| 500 505 510 | |
| Ser Phe Val Leu Tyr Thr Ile Ser Lys Thr Val Glu Tyr Leu His Ser | |
| 515 520 525 | |
| Gln Gly Val Val His Arg Asp Leu Lys Pro Ser Asn Ile Leu Tyr Val | |
| 530 535 540 | |
| Asp Glu Ser Gly Asn Pro Glu Cys Leu Arg Ile Cys Asp Phe Gly Phe | |
| 545 550 555 560 | |
| Ala Lys Gln Leu Arg Ala Glu Asn Gly Leu Leu Met Thr Pro Cys Tyr | |
| 565 570 575 | |
| Thr Ala Asn Phe Val Ala Pro Glu Val Leu Lys Arg Gln Gly Tyr Asp | |
| 580 585 590 | |
| Glu Gly Cys Asp Ile Trp Ser Leu Gly Val Leu Leu Tyr Thr Met Leu | |
| 595 600 605 | |
| Ala Gly Tyr Thr Pro Phe Ala Asn Gly Pro Ser Asp Thr Pro Glu Glu | |
| 610 615 620 | |
| Ile Leu Thr Arg Ile Ser Ser Gly Lys Phe Thr Leu Ser Gly Gly Asn | |
| 625 630 635 640 | |
| Trp Asn Thr Val Ser Glu Thr Ala Lys Asp Leu Val Ser Lys Met Leu | |
| 645 650 655 | |
| His Val Asp Pro His Gln Arg Leu Thr Ala Lys Gln Val Leu Gln His | |
| 660 665 670 | |
| Pro Trp Ile Thr Gln Lys Asp Lys Leu Pro Gln Ser Gln Leu Ser His | |
| 675 680 685 | |
| Gln Asp Leu Gln Leu Val Lys Gly Gly Met Ala Ala Thr Tyr Ser Ala | |
| 690 695 700 | |
| Leu Ser Ser Ser Lys Pro Thr Pro Gln Leu Lys Pro Ile Glu Ser Ser | |
| 705 710 715 720 | |
| Ile Leu Ala Gln Arg Arg Val Arg Lys Leu Pro Ser Thr Thr Leu | |
| 725 730 735 |
An exemplary mutant RSK of the present invention is the K94A/K447A RSK mutant, having an amino acid sequence of SEQ ID NO: 2 as follows:
| Met Pro Leu Ala Gln Leu Lys Glu Pro Trp Pro Leu Met Glu Leu Val | |
| 5 10 15 15 | |
| Pro Leu Asp Pro Glu Asn Gly Gln Ala Ser Gly Glu Glu Ala Gly Leu | |
| 20 25 30 | |
| Gln Pro Ser Lys Asp Glu Gly Ile Leu Lys Glu Ile Ser Ile Thr His | |
| 35 40 45 | |
| His Val Lys Ala Gly Ser Glu Lys Ala Asp Pro Ser His Phe Glu Leu | |
| 50 55 60 | |
| Leu Lys Val Leu Gly Gln Gly Ser Phe Gly Lys Val Phe Leu Val Arg | |
| 65 70 75 80 | |
| Lys Val Thr Arg Pro Asp Asn Gly His Leu Tyr Ala Met Ala Val Leu | |
| 85 90 95 | |
| Lys Lys Ala Thr Leu Lys Val Arg Asp Arg Val Arg Thr Lys Met Glu | |
| 100 105 110 | |
| Arg Asp Ile Leu Ala Asp Val Asn His Pro Phe Val Val Lys Leu His | |
| 115 120 125 | |
| Tyr Ala Phe Gln Thr Glu Gly Lys Leu Tyr Leu Ile Leu Asp Phe Leu | |
| 130 135 140 | |
| Arg Gly Gly Asp Leu Phe Thr Arg Leu Ser Lys Glu Val Met Phe Thr | |
| 145 150 155 160 | |
| Glu Glu Asp Val Lys Phe Tyr Leu Ala Glu Leu Ala Leu Gly Leu Asp | |
| 165 170 175 | |
| His Leu His Ser Leu Gly Ile Ile Tyr Arg Asp Leu Lys Pro Glu Asn | |
| 180 185 190 | |
| Ile Leu Leu Asp Glu Glu Gly His Ile Lys Leu Thr Asp Phe Gly Leu | |
| 195 200 205 | |
| Ser Lys Glu Ala Ile Asp His Glu Lys Lys Ala Tyr Ser Phe Cys Gly | |
| 210 215 220 | |
| Thr Val Glu Tyr Met Ala Pro Glu Val Val Asn Arg Gln Gly His Thr | |
| 225 230 235 240 | |
| His Ser Ala Asp Trp Trp Ser Tyr Gly Val Leu Met Phe Glu Met Leu | |
| 245 250 255 | |
| Thr Gly Ser Leu Pro Phe Gln Gly Lys Asp Arg Lys Glu Thr Met Thr | |
| 260 265 270 | |
| Leu Ile Leu Lys Ala Lys Leu Gly Met Pro Gln Phe Leu Ser Thr Glu | |
| 275 280 285 | |
| Ala Gln Ser Leu Leu Arg Ala Leu Phe Lys Arg Asn Pro Ala Asn Arg | |
| 290 295 300 | |
| Leu Gly Ser Gly Pro Asp Gly Ala Glu Glu Ile Lys Arg His Ile Phe | |
| 305 310 315 320 | |
| Tyr Ser Thr Ile Asp Trp Asn Lys Leu Tyr Arg Arg Glu Ile Lys Pro | |
| 325 330 335 | |
| Pro Phe Lys Pro Ala Val Ala Gln Pro Asp Asp Thr Phe Tyr Phe Asp | |
| 340 345 350 | |
| Thr Glu Phe Thr Ser Arg Thr Pro Arg Asp Ser Pro Gly Ile Pro Pro | |
| 355 360 365 | |
| Ser Ala Gly Ala His Gln Leu Phe Arg Gly Phe Ser Phe Val Ala Thr | |
| 370 375 380 | |
| Gly Leu Met Glu Asp Asp Ser Lys Pro Arg Ala Thr Gln Ala Pro Leu | |
| 385 390 395 400 | |
| His Ser Val Val Gln Gln Leu His Gly Lys Asn Leu Val Phe Ser Asp | |
| 405 410 415 | |
| Gly Tyr Ile Val Lys Glu Thr Ile Gly Val Gly Ser Tyr Ser Val Cys | |
| 420 425 430 | |
| Lys Arg Cys Val His Lys Ala Thr Asn Met Glu Tyr Ala Val Ala Val | |
| 435 440 445 | |
| Ile Asp Lys Ser Lys Arg Asp Pro Ser Glu Glu Ile Glu Ile Leu Leu | |
| 450 455 460 | |
| Arg Tyr Gly Gln His Pro Asn Ile Ile Thr Leu Lys Asp Val Tyr Asp | |
| 465 470 475 480 | |
| Asp Ser Lys His Val Tyr Leu Val Thr Glu Leu Met Arg Gly Gly Glu | |
| 485 490 495 | |
| Leu Leu Asp Lys Ile Leu Arg Gln Lys Phe Phe Ser Glu Arg Glu Ala | |
| 500 505 510 | |
| Ser Phe Val Leu Tyr Thr Ile Ser Lys Thr Val Glu Tyr Leu His Ser | |
| 515 520 525 | |
| Gln Gly Val Val His Arg Asp Leu Lys Pro Ser Asn Ile Leu Tyr Val | |
| 530 535 540 | |
| Asp Glu Ser Gly Asn Pro Glu Cys Leu Arg Ile Cys Asp Phe Gly Phe | |
| 545 550 555 560 | |
| Ala Lys Gln Leu Arg Ala Glu Asn Gly Leu Leu Met Thr Pro Cys Tyr | |
| 565 570 575 | |
| Thr Ala Asn Phe Val Ala Pro Glu Val Leu Lys Arg Gln Gly Tyr Asp | |
| 580 585 590 | |
| Glu Gly Cys Asp Ile Trp Ser Leu Gly Val Leu Leu Tyr Thr Met Leu | |
| 595 600 605 | |
| Ala Gly Tyr Thr Pro Phe Ala Asn Gly Pro Ser Asp Thr Pro Glu Glu | |
| 610 615 620 | |
| Ile Leu Thr Arg Ile Ser Ser Gly Lys Phe Thr Leu Ser Gly Gly Asn | |
| 625 630 635 640 | |
| Trp Asn Thr Val Ser Glu Thr Ala Lys Asp Leu Val Ser Lys Met Leu | |
| 645 650 655 | |
| His Val Asp Pro His Gln Arg Leu Thr Ala Lys Gln Val Leu Gln His | |
| 660 665 670 | |
| Pro Trp Ile Thr Gln Lys Asp Lys Leu Pro Gln Ser Gln Leu Ser His | |
| 675 680 685 | |
| Gln Asp Leu Gln Leu Val Lys Gly Gly Met Ala Ala Thr Tyr Ser Ala | |
| 690 695 700 | |
| Leu Ser Ser Ser Lys Pro Thr Pro Gln Leu Lys Pro Ile Glu Ser Ser | |
| 705 710 715 720 | |
| Ile Leu Ala Gln Arg Arg Val Arg Lys Leu Pro Ser Thr Thr Leu | |
| 725 730 735 |
The K94A/K447A RSK mutation makes the RSK protein a “dominant negative” RSK mutant (DN-RSK). A dominant negative mutation creates a gene product (protein or polypeptide) that adversely affects the normal, wild-type gene product within the same cell, usually by dimerizing with the wild-type protein or polypeptide. The mutant p90RSK of the present invention may be made from any mammal including, but not limited to, rat, mouse, and human (including but not limited to Genbank Accession Nos. M99169, Swiss-Pro P16853, and Genbank Accession No.AF09042, which are hereby incorporated by reference in their entirety.)
Additional RSK mutants of the present invention include those known in the art or which may be characterized by amino acid insertions, deletions, substitutions, and modifications at one or more sites in or at the other residues of the native RSK polypeptide chain. (Spring et al., “Deletion of 11 Amino Acids in p90(rsk-mo-1) Abolishes Kinase Activity,” Mol Cell Biol 19(1):317-20 (1999); Roux et al., “Phosphorylation of p90 Ribosomal S6 Kinase (RSK) Regulated Extracellular Signal-Regulated Kinase Docking and RSK Activity,” Mol Cell Biol 23(14):4796-804 (2003); which are hereby incorporated by reference in their entirety). In accordance with this invention any such insertions, deletions, substitutions, and modifications should result in an RSK mutant that is rendered kinase inactive for cellular substrates including serine 703 (S703) phosphorylation of NHE1. Preferably, additional RSK mutants made according to the present invention would also be dominant negative mutants of RSK or would mimic the functional effects of an RSK mutant with regard to activation of p90RSK.
The RSK mutants of the present invention can be produced by any suitable method known in the art. Such methods include constructing a DNA sequence encoding the RSK mutants of the present invention and expressing those sequences in a suitably transformed host. This method will produce recombinant mutants of this invention. This technique is well known (Mourez et al., “Mapping Dominant-Negative Mutations of Anthrax Protective Antigen by Scanning Mutagenesis,” Proc. Natl. Acad. Sci. USA 100(24):13803-13808 (2003); Mark et al., “Site-specific Mutagenesis of The Human Fibroblast Interferon Gene,” Proc. Natl. Acad. Sci. USA 81:5662-66 (1984); U.S. Pat. No. 4,588,585, which are hereby incorporated by reference in their entirety).
Chemical synthesis can also be used to construct a DNA sequence encoding the RSK mutants of the present invention. For example, a nucleic acid molecule which encodes the desired RSK mutant may be synthesized by chemical means using an oligonucleotide synthesizer. Such oligonucleotides are designed based on the amino acid sequence of the desired RSK mutant, and preferably selecting those codons that are favored in the host cell in which the recombinant mutant will be produced. In this regard, it is well recognized that the genetic code is degenerate, i.e., that an amino acid may be coded for by more than one codon. Accordingly, it will be appreciated by one skilled in the art that for a given DNA sequence encoding a particular RSK mutant, there will be many degenerate DNA sequences that will code for that mutant. These degenerate DNA sequences are considered within the scope of this invention. Therefore, the present invention also encompasses suitable RSK mutants and degenerate variants thereof, which, in the context of this invention means all DNA sequences that code for a particular mutant.
Additional standard methods may be applied to synthesize a nucleic acid molecule encoding an RSK mutant of the present invention. For example, the complete amino acid sequence may be used to construct a back-translated gene. A DNA oligomer containing a nucleotide sequence coding for RSK mutant may be synthesized. For example, several small oligonucleotides coding for portions of the desired polypeptide may be synthesized and then ligated. The individual oligonucleotides typically contain 5′ or 3′ overhangs for complementary assembly.
The mutants of this invention may also be produced by a combination of chemical synthesis and recombinant DNA technology.
As used herein, comparison of the mutant p90RSK proteins can be made to wild type proteins. The wild type proteins can be naturally occurring variants of p90RSK as well as modified p90RSK proteins or polypeptides that possess substantially the same activity as the human or rat p90RSK of GenBank Accession Nos. AF090421 and M99169; which are hereby incorporated by reference in its entirety. By substantially the same, it is intended that the modified protein have at least 75%, preferably 85-115%, more preferably 95-100% of normal activity. The nucleic acid sequence encoding a RSK mutant of the present invention, whether prepared by site-directed mutagenesis, chemical synthesis, or other methods, may or may not also include DNA sequences that encode a signal sequence. Such signal sequence, if present, should be one recognized by the cell chosen for expression of the RSK mutant. It may be prokaryotic, eukaryotic or a combination of the two. It may also be the signal sequence of native RSK. The inclusion of a signal sequence depends on whether it is desired to secrete the RSK mutant from the recombinant cells in which it is made. If the chosen cells are prokaryotic, it generally is preferred that the DNA sequence not encode a signal sequence but include an N-terminal methionine to direct expression. If the chosen cells are eukaryotic, it generally is preferred that a signal sequence be encoded and most preferably that the wild-type RSK mutant signal sequence be used.
Once assembled (by synthesis, site-directed mutagenesis or another method), the nucleic acid sequences encoding an RSK mutant of this invention will be inserted into an expression vector and operatively linked to an expression control sequence appropriate for expression of the RSK mutant in the desired transformed host. Proper assembly may be confirmed by nucleotide sequencing, restriction mapping, and expression of a biologically active polypeptide in a suitable host. As is well known in the art, to obtain high expression levels of a transfected gene in a host, the gene must be operatively linked to transcriptional and translational expression control sequences that are functional in the chosen expression host.
The preparation of the nucleic acid constructs of the present invention including a nucleic acid molecule encoding a mutant RSK protein is carried out using methods well known in the art. U.S. Pat. No. 4,237,224 to Cohen and Boyer, which is hereby incorporated by reference in its entirety, describes the production of expression systems in the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase. These recombinant plasmids are then introduced by means of transformation and replicated in unicellular cultures including prokaryotic organisms and eukaryotic cells grown in tissue culture. Other vectors are also suitable.
Suitable vectors include, but are not limited to, vectors such as lambda vector system gt11, gt WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKC101, SV 40, pBluescript II SK +/−or KS +/−(see “Stratagene Cloning Systems” Catalog (1993) from Stratagene, La Jolla, Calif., which is hereby incorporated by reference in its entirety), pQE, pIH821, pGEX, pET series (see F. W. Studier et. al., “Use of T7 RNA Polymerase to Direct Expression of Cloned Genes,” Gene Expression Technology Vol. 185 (1990), which is hereby incorporated by reference in its entirety), and any derivatives thereof. Several viral systems including murine retrovirus, adenovirus, parvovirus (adeno-associated virus), vaccinia virus, and herpes virus, such as herpes simplex virus and Epstein-Barr virus, and retroviruses, such as MoMLV have been developed as therapeutic gene transfer vectors (Nienhuis et al., Hematology , Vol. 16 : Viruses and Bone Marrow , N. S. Young (ed.), pp. 353-414 (1993), which is hereby incorporated by reference in its entirety). Viral vectors provide a more efficient means of transferring genes into cells as compared to other techniques such as calcium phosphate or DEAE-dextran-mediated transfection, electroporation, or microinjection. It is believed that the efficiency of viral transfer is due to the fact that the transfer of DNA is a receptor-mediated process (i.e., the virus binds to a specific receptor protein on the surface of the cell to be infected.) Among the viral vectors that have been cited frequently for use in preparing transgenic mammal cells are adenoviruses (U.S. Pat. No. 6,203,975 to Wilson). In one embodiment of the present invention, the nucleic acid encoding the desired mutant RSK protein of the present invention is incorporated into an adenovirus expression vector.
Once a suitable expression vector is selected, the desired nucleic acid sequence(s) cloned into the vector using standard cloning procedures in the art, as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory , Cold Springs Harbor, N.Y. (1989), or U.S. Pat. No. 4,237,224 to Cohen and Boyer, which are hereby incorporated by reference in their entirety. The vector is then introduced to a suitable host. Thus, another aspect of the present invention is a p90RSK mutant nucleic acid molecule incorporated into an expression vector and a host. In a preferred embodiment this mutant is the K94A/K447A mutant nucleic acid molecule described herein above.
A variety of host-vector systems may be utilized to express the recombinant protein or polypeptide inserted into a vector as described above. Primarily, the vector system must be compatible with the host used. Host-vector systems include, without limitation, the following: bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containing yeast vectors; mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); and plant cells infected by bacteria. The expression elements of these vectors vary in their strength and specificities. Depending upon the host-vector system utilized, any one of a number of suitable transcription and translation elements can be used to carry out this and other aspects of the present invention.
Different genetic signals and processing events control many levels of gene expression (e.g., DNA transcription and messenger RNA (“mRNA”) translation). Transcription of DNA is dependent upon the presence of a promoter, which is a DNA sequence that directs the binding of RNA polymerase, and thereby promotes mRNA synthesis. The DNA sequences of eukaryotic promoters differ from those of prokaryotic promoters. Furthermore, eukaryotic promoters and accompanying genetic signals may not be recognized in, or may not function in, a prokaryotic system, and, further, prokaryotic promoters are not recognized and do not function in eukaryotic cells.
Similarly, translation of mRNA in prokaryotes depends upon the presence of the proper prokaryotic signals which differ from those of eukaryotes. Efficient translation of mRNA in prokaryotes requires a ribosome binding site called the Shine-Dalgarno (“SD”) sequence on the mRNA. This sequence is a short nucleotide sequence of mRNA that is located before the start codon, usually AUG, which encodes the amino-terminal methionine of the protein. The SD sequences are complementary to the 3′-end of the 16S rRNA (ribosomal RNA) and probably promote binding of mRNA to ribosomes by duplexing with the rRNA to allow correct positioning of the ribosome. For a review on maximizing gene expression see Roberts and Lauer, Methods in Enzymology, 68:473 (1979), which is hereby incorporated by reference in its entirety.
Promoters vary in their “strength” (i.e., their ability to promote transcription). For the purposes of expressing a cloned gene, it is desirable to use strong promoters in order to obtain a high level of transcription and, hence, expression of the gene. Depending upon the host system utilized, any one of a number of suitable promoters may be used. For instance, when cloning in E. coli , its bacteriophages, or plasmids, promoters such as the T7 phage promoter, lac promoter, trp promoter, recA promoter, ribosomal RNA promoter, the PR and PL promoters of coliphage lambda and others, including but not limited, to lacUV5, ompF, bla, lpp, and the like, may be used to direct high levels of transcription of adjacent DNA segments. Additionally, a hybrid trp-lacUV5 (tac) promoter or other E. coli promoters produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene.
Bacterial host strains and expression vectors may be chosen which inhibit the action of the promoter unless specifically induced. In certain operons, the addition of specific inducers is necessary for efficient transcription of the inserted DNA. For example, the lac operon is induced by the addition of lactose or IPTG (isopropylthio-beta-D-galactoside). A variety of other operons, such as trp, pro, etc., are under different controls.
Common promoters suitable for directing expression in mammalian cells include, without limitation, SV40, MMTV, metallothionein-1, adenovirus E1a, CMV, immediate early, immunoglobulin heavy chain promoter and enhancer, and RSV-LTR. Preferred promoters are cardiac-specific promoters. Exemplary cardiac-specific promoters include, without limitation, the α-myosin heavy chain promoter.
When multiple nucleic acid molecules are inserted, the multiple nucleic acid molecules may all be placed under a single 5′ regulatory region and a single 3′ regulatory region, where the regulatory regions are of sufficient strength to transcribe and/or express the nucleic acid molecules as desired.
Specific initiation signals are also required for efficient gene transcription and translation in prokaryotic cells. These transcription and translation initiation signals may vary in “strength” as measured by the quantity of gene specific messenger RNA and protein synthesized, respectively. The nucleic acid expression vector, which contains a promoter, may also contain any combination of various “strong” transcription and/or translation initiation signals. For instance, efficient translation in E. coli requires a Shine-Dalgamo (“SD”) sequence about 7-9 bases 5′ to the initiation codon (ATG) to provide a ribosome binding site. Thus, any SD-ATG combination that can be utilized by host ribosomes may be employed. Such combinations include but are not limited to the SD-ATG combination from the cro gene or the N gene of coliphage lambda, or from the E. coli tryptophan E, D, C, B or A genes. Additionally, any SD-ATG combination produced by recombinant DNA or other techniques involving incorporation of synthetic nucleotides may be used. Depending on the vector system and host utilized, any number of suitable transcription and/or translation elements, including constitutive, inducible, and repressible promoters, as well as minimal 5′ promoter elements, enhancers or leader sequences may be used.
Typically, when a recombinant host is produced, an antibiotic or other compound useful for selective growth of the transgenic cells only is added as a supplement to the media. The compound to be used will be dictated by the selectable marker element present in the plasmid with which the host was transformed. Suitable genes are those which confer resistance to gentamycin, G418, hygromycin, streptomycin, spectinomycin, tetracycline, chloramphenicol, and the like. Similarly, “reporter genes,” which encode enzymes providing for production of an identifiable compound identifiable, or other markers which indicate relevant information regarding the outcome of gene delivery, are suitable. For example, various luminescent or phosphorescent reporter genes are also appropriate, such that the presence of the heterologous gene may be ascertained visually.
An example of a marker suitable for the present invention is the green fluorescent protein (GFP) gene. The isolated nucleic acid molecule encoding a green fluorescent protein can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA, including messenger RNA or mRNA), genomic or recombinant, biologically isolated or synthetic. The DNA molecule can be a cDNA molecule, which is a DNA copy of a messenger RNA (mRNA) encoding the GFP. In one embodiment, the GFP can be from Aequorea victoria (Prasher et al., “Primary Structure of the Aequorea Victoria Green-Fluorescent Protein,” Gene 111(2):229-233 (1992); U.S. Pat. No. 5,491,084 to Chalfie et al., which are hereby incorporated by reference in their entirety). A plasmid encoding the GFP of Aequorea victoria is available from the ATCC as Accession No. 75547. Mutated forms of GFP that emit more strongly than the native protein, as well as forms of GFP amenable to stable translation in higher vertebrates, are commercially available from Clontech Laboratories, Inc. (Palo Alto, Calif.) and can be used for the same purpose. The plasmid designated pTα1-GFPh (ATCC Accession No. 98299, which is hereby incorporated by reference in its entirety) includes a humanized form of GFP. Indeed, any nucleic acid molecule encoding a fluorescent form of GFP can be used in accordance with the subject invention. Standard techniques are then used to place the nucleic acid molecule encoding GFP under the control of the chosen cell specific promoter.
The selection marker employed will depend on the target species and/or host or packaging cell lines compatible with a chosen vector.
A nucleic acid molecule encoding the desired RSK-encoding nucleic acid molecule (wild type or mutant) of the present invention, a promoter molecule of choice, including, without limitation, enhancers, and leader sequences; a suitable 3′ regulatory region to allow transcription in the host, and any additional desired components, such as reporter or marker genes, are cloned into the vector of choice using standard cloning procedures in the art, such as described in Sambrook et al., Molecular Cloning: A Laboratory Manual , Cold Spring Laboratory, Cold Spring Harbor, N.Y. (1989); Ausubel et al., “ Short Protocols in Molecular Biology ,” New York:Wiley (1999), and U.S. Pat. No. 4,237,224 to Cohen and Boyer, which are hereby incorporated by reference in their entirety.
Once the isolated nucleic acid molecule encoding a suitable nucleic acid molecule has been cloned into an expression vector, it is ready to be incorporated into a host. Recombinant molecules can be introduced into cells, without limitation, via transformation (if the host is a prokaryote), transfection (if the host is a eukaryote), transduction (if the host is a virus), conjugation, mobilization, or electroporation, lipofection, protoplast fusion, mobilization, particle bombardment, or electroporation, using standard cloning procedures known in the art, as described by Sambrook et al., Molecular Cloning: A Laboratory Manual , Second Edition, Cold Springs Laboratory, Cold Springs Harbor, N.Y. (1989), which is hereby incorporated by reference in its entirety. Suitable hosts include, but are not limited to, bacteria, virus, yeast, and mammalian cells, including, without limitation, mouse, and used to prepare the transgenic non-human animal of the present invention.
Alternatively, the RSK mutant-encoding nucleic acid molecule of the present invention may be inserted into a host cell and used as for studying RSK phosphorylation/NHE1 activation in vitro. Accordingly, another aspect of the present invention relates to a method of making a recombinant cell. Basically, this method is carried out by transforming a host with a nucleic acid construct of the present invention under conditions effective to yield transcription of the nucleic acid molecule in the host. Preferably, a nucleic acid construct containing a suitable nucleic acid molecule of the present invention is stably inserted into the genome of the recombinant host as a result of the transformation. Suitable host cells for the for the RSK mutant of the present invention includes, without limitation, cardiac muscle cells, smooth muscle cells, skeletal muscle cells, neuronal cells, and other cell types where reactive oxygen species, ischemia/reperfusion, and oxidative stress contribute to tissue dysfunction, cell impairment, and cell death. The cells may be from any mammalian species, including human. Suitable hosts for expression or other uses are bacterial or yeast cells, and viruses, as described herein above.
Transient expression allows quantitative studies of gene expression since the population of cells is very high (on the order of 106). To deliver DNA inside mammalian cells, several methodologies have been proposed, among them electroporation (Neumann et al., “Gene Transfer into Mouse Lyoma Cells by Electroporation in High Electric Fields,” EMBO J. 1:841-45 (1982); Wong et al., “Electric Field Mediated Gene Transfer,” Biochem Biophys Res Commun 30:107(2):584-7 (1982); Potter et al., “Enhancer-Dependent Expression of Human Kappa Immunoglobulin Genes Introduced into Mouse pre-B Lymphocytes by Electroporation,” Proc. Natl. Acad. Sci. USA 81:7161-65 (1984), which are hereby incorporated by reference in their entirety) and polyethylene glycol (PEG) mediated DNA uptake, Sambrook et al., Molecular Cloning: A Laboratory Manual , Chap. 16, Second Edition, Cold Springs Laboratory, Cold Springs Harbor, N.Y. (1989), which is hereby incorporated by reference in its entirety). During electroporation, the DNA is introduced into the cell by means of a reversible change in the permeability of the cell membrane due to exposure to an electric field. PEG transformation introduces the DNA by changing the elasticity of the membranes. Unlike electroporation, PEG transformation does not require any special equipment and transformation efficiencies can be equally high. Another appropriate method of introducing the nucleic acid construct of the present invention into a host is fusion of nucleic acid-containing vectors with other entities, either minicells, cells, lysosomes, or other fusible lipid-surfaced bodies that contain the chimeric gene (Fraley, et al., Proc Natl Acad Sci USA 79:1859-63 (1982), which is hereby incorporated by reference in its entirety).
Stable transformants are preferable for the methods of the present invention, which can be achieved by using variations of the methods above as describe in Sambrook et al., Molecular Cloning: A Laboratory Manual , Chap. 16, Second Edition, Cold Springs Laboratory, Cold Springs Harbor, N.Y. (1989), Ausubel et al., “ Short Protocols in Molecular Biology ,” New York:Wiley (1999), and U.S. Pat. No. 4,237,224 to Cohen and Boyer, which are hereby incorporated by reference in their entirety, and other methods known to those in the art.
The present invention provides a second transgenic non-human animal for the investigation of I/R injury and therapeutics for the prevention and treatment of I/R injury. This second transgenic non-human animal includes a transgene that encodes for cardiac-specific overexpression of wild type p90RSK compared to a non-transgenic animal.
The WT-p90RSK transgenic animal (WT-p90RSK-Tg) of the present invention overexpresses a wild-type RSK protein as a result of the introduction of a wild-type RSK-encoding nucleic acid molecule operably linked to an α-MHC promoter region for cardiac