Immunosuppression With FTY720 Reverses Cardiac Dysfunction in Hypomorphic ApoE Mice Deficient in SR-BI Expression That Survive Myocardial Infarction Caused by Coronary Atherosclerosis.

AIMS: We recently reported that immunosuppression with FTY720 improves cardiac function and extends longevity in Hypomorphic ApoE mice deficient in scavenger receptor Type-BI expression, also known as the HypoE/SR-BI(­/­) mouse model of diet-induced coronary atherosclerosis and myocardial infarction (MI). In this study, we tested the impact of FTY720 on cardiac dysfunction in HypoE/SR-BI(­/­) mice that survive MI and subsequently develop chronic heart failure. METHODS/RESULTS: HypoE/SR-BI(­/­) mice were bred to Mx1-Cre transgenic mice, and offspring were fed a high-fat diet (HFD) for 3.5 weeks to provoke hyperlipidemia, coronary atherosclerosis, and recurrent MIs. In contrast to our previous study, hyperlipidemia was rapidly reversed by inducible Cre-mediated gene repair of the HypoE allele and switching mice to a normal chow diet. Mice that survived the period of HFD were subsequently given oral FTY720 in drinking water or not, and left ventricular (LV) function was monitored using serial echocardiography for up to 15 weeks. In untreated mice, LV performance progressively deteriorated. Although FTY720 treatment did not initially prevent a decline of heart function among mice 6 weeks after Cre-mediated gene repair, it almost completely restored normal LV function in these mice by 15 weeks. Reversal of heart failure did not result from reduced atherosclerosis as the burden of aortic and coronary atherosclerosis actually increased to similar levels in both groups of mice. Rather, FTY720 caused systemic immunosuppression as assessed by reduced numbers of circulating T and B lymphocytes. In contrast, FTY720 did not enhance the loss of T cells or macrophages that accumulated in the heart during the HFD feeding period, but it did enhance the loss of B cells soon after plasma lipid lowering. Moreover, FTY720 potently reduced the expression of matrix metalloproteinase-2 and genes involved in innate immunity-associated inflammation in the heart. CONCLUSIONS: Our data demonstrate that immunosuppression with FTY720 prevents postinfarction myocardial remodeling and chronic heart failure.

High-density lipoprotein determines adult mouse cardiomyocyte fate after hypoxia-reoxygenation through lipoprotein-associated sphingosine 1-phosphate.

The lipid mediator sphingosine 1-phosphate (S1P) confers survival benefits in cardiomyocytes and isolated hearts subjected to oxidative stress. High-density lipoprotein (HDL) is a major carrier of S1P in the serum, but whether HDL-associated S1P directly mediates survival in a preparation composed exclusively of cardiomyocytes has not been demonstrated. Accordingly, we tested the hypothesis that signal activation and survival during simulated ischemia-reperfusion injury in response to HDL require lipoprotein-associated S1P. As a model, we used adult mouse cardiomyocytes subjected to hypoxia-reoxygenation. Cells were treated or not with autologous mouse HDL, which significantly increased myocyte viability as measured by trypan blue exclusion. This survival effect was abrogated by the S1P(1) and SIP(3) receptor antagonist VPC 23019. The selective S1P(3) antagonist CAY10444, the G(i) antagonist pertussis toxin, the MEK (MAPK/ERK) kinase inhibitor PD-98059, and the phosphoinositide-3 kinase inhibitor wortmannin also inhibited the prosurvival effect of HDL. We observed that HDL activated both Akt (protein kinase B) and the MEK1/2-ERK1/2 pathway and also stimulated phosphorylation of glycogen synthase kinase-3beta. ERK1/2 activation was through an S1P(1) subtype receptor-G(i) protein-dependent pathway, whereas the activation of Akt was inhibited by CAY10444, indicating mediation by S1P(3) subtype receptors. We conclude that HDL, via its cargo of S1P, can directly protect cardiomyocytes against simulated oxidative injury in the absence of vascular effects and that prosurvival signal activation is dependent on both S1P(1) and S1P(3) subtype receptors.

Cardiomyocyte S1P1 receptor-mediated extracellular signal-related kinase signaling and desensitization.

We examined the ability of sphingosine-1-phosphate (S1P) to desensitize extracellular signal-related kinase (ERK), a mitogen-activated protein kinase linked to antiapoptotic responses in the heart. In isolated adult mouse cardiomyocytes, S1P (10 nM-5 microM) induced ERK phosphorylation in a time- and dose-dependent manner. S1P stimulation of ERK was completely inhibited by an S1P1/3 subtype receptor antagonist (VPC23019), by a Gi protein inhibitor (pertussis toxin) and by a mitogen-activated protein kinase/ERK kinase inhibitor (PD98059). A selective S1P3 receptor antagonist (CAY10444) had no effect on S1P-induced ERK activation. The selective S1P1 agonist SEW2871 also induced ERK phosphorylation. Activation of ERK by restimulation with 100 nM S1P was suppressed after 1 hour of preincubation with 100 nM S1P but recovered fully the next day, suggesting receptor recycling. Similar results were obtained in protein kinase C epsilon-null cardiomyocytes. Treatment with the nonselective S1P receptor agonist FTY720 for 1 hour also reduced phospho-ERK expression in response to subsequent S1P stimulation. In contrast to S1P, some desensitization to FTY720 persisted after overnight exposure. Cell death induced by hypoxia/reoxygenation was reduced by pretreatment with exogenous S1P. This enhanced survival was abrogated by pretreatment with PD98059, VPC23019, or pertussis toxin. Thus, exogenous S1P induces rapid and reversible S1P1-mediated ERK phosphorylation. S1P-induced adult mouse cardiomyocyte survival requires ERK activation mediated via an S1P1-Gi pathway.

ADP-ribosylation factor 6 regulates tumorigenic and invasive properties in vivo.

This study shows that the small GTP-binding protein ADP-ribosylation factor 6 (ARF6) is an important regulator of tumor growth and metastasis. Using spontaneous melanoma tumor growth assays and experimental metastasis assays in nude mice, we show that sustained activation of ARF6 reduces tumor mass growth but significantly enhances the invasive capacity of tumor cells. In contrast, mice injected with tumor cells expressing a dominantly inhibitory ARF6 mutant exhibited a lower incidence and degree of invasion and lung metastasis compared with control animals. Effects on tumor growth correlate with reduced cell proliferation capacity and are linked at least in part to alterations in mitotic progression induced by defective ARF6 cycling. Furthermore, phospho-ERK levels in subcultured cells from ARF6(GTP) and ARF6(GDP) tumor explants correlate with invasive capacity. ARF6-induced extracellular signal-regulated kinase (ERK) signaling leads to Rac1 activation to promote invadopodia formation and cell invasion. These findings document an intricate role for ARF6 and the regulation of ERK activation in orchestrating mechanisms underlying melanoma growth, invasion, and metastases.

A sphingosine kinase 1 mutation sensitizes the myocardium to ischemia/reperfusion injury.

OBJECTIVE: Sphingosine kinase (SphK) is a key enzyme in the synthesis of sphingosine 1-phosphate (S1P), a bioactive sphingolipid. SphK is involved in ischemic preconditioning (IPC). To date no studies in genetically altered animals have examined the role of SphK1 in myocardial ischemia/reperfusion (IR) injury and IPC. METHODS AND RESULTS: Wild-type and SphK1 null mouse hearts were subjected to IR (50 min global ischemia and 40 min reperfusion) in a Langendorff apparatus. IPC consisted of 2 min of global ischemia and 2 min of reperfusion for two cycles. At baseline, there were no differences in left ventricular developed pressure (LVDP), +/-dP/dtmax, and LV end-diastolic pressure (EDP) between SphK1 mutant and wild-type (WT) mouse hearts. In the mutants, total SphK enzyme activity was reduced by 44% and S1P levels were decreased by 41%. SphK1 null hearts subjected to IR exhibited more cardiac damage compared with WT: LVDP and +/-dP/dtmax decreased, LVEDP increased, and infarct size increased (n=6, P<0.05). Apoptosis was markedly enhanced in SphK1 mutant IR mouse hearts. IPC was cardioprotective in WT hearts, but this protection appeared to be ineffective in SphK1 null hearts. There was no change in infarct size in the IPC+IR group compared to the IR group in the null hearts (50.1+/-5.0% vs 45.0+/-3.8%, n=6, P=NS). IPC remained ineffective in the null hearts even when the index ischemia time was shortened by 10 min. CONCLUSIONS: Deletion of the SphK1 gene sensitizes the myocardium to IR injury and appears to impair the protective effect of IPC. These data provide the first genetic evidence that the SphK1-S1P pathway is a critical mediator of IPC and cell survival.

Investigating the role of ADP-ribosylation factor 6 in tumor cell invasion and extracellular signal-regulated kinase activation.

Tumor cell invasion is a coordinated process involving the formation of invadopodia and the localized degradation of the extracellular matrix (ECM). The process of cell invasion is regulated by cell-signaling proteins such as Ras-related GTPases and members of the mitogen-activated protein kinase (MAPK) family. Our studies have focused on the role of the ADP-ribosylation factor 6 (ARF6) GTPase in the process of tumor cell invasion. Using activated and dominant negative mutants of ARF6 in a tumor cell culture model, our laboratory has demonstrated that the GTPase cycle of ARF6 regulates invadopodia formation and matrix degradation. Furthermore, ARF6-mediated cell invasion was found to be dependent on the activation of the extracellular signal-regulated kinase (ERK). These findings demonstrate a critical role for ARF6 in ERK activation and tumor cell invasion. To investigate the role of ARF6 in tumor cell invasion and ERK activation, a number of methods were employed. These procedures include transfection of LOX cells, in vitro matrix-degradation assays, immunofluorescence microscopy, and biochemical assays. These approaches can be applied effectively to measure the degree of invasiveness fostered by ARF6 and/or other GTPases and to examine the subcellular distribution of the molecular players that are trafficked or recruited to sites of cell invasion.

Mimicking phosphorylation of alphaB-crystallin on serine-59 is necessary and sufficient to provide maximal protection of cardiac myocytes from apoptosis.

AlphaB-crystallin (alphaBC), a small heat shock protein expressed in high levels in the heart, is phosphorylated on Ser-19, 45, and 59 after stress. However, it is not known whether alphaBC phosphorylation directly affects cell survival. In the present study, constructs were prepared that encode forms of alphaBC harboring Ser to Ala (blocks phosphorylation) or Ser to Glu (mimics phosphorylation) mutations at positions 19, 45, and 59. The effects of each form on apoptosis of cultured cardiac myocytes after hyperosmotic or hypoxic stress were assessed. Compared with controls, cells that expressed alphaBC with Ser to Ala substitutions at all three positions, alphaBC(AAA), exhibited more stress-induced apoptosis. Cells expressing either alphaBC(AAE) or (EEE) exhibited 3-fold less apoptosis than cells expressing alphaBC(AAA), indicating that phosphorylation of Ser-59 confers protection. alphaBC is known to bind to procaspase-3 and to decrease caspase-3 activation. Compared with cells expressing alphaBC(AAA), the activation of caspase-3 was decreased by 3-fold in cells expressing alphaBC(AAE). These results demonstrate that mimicking the phosphorylation of alphaBC on Ser-59 is necessary and sufficient to confer caspase-3 inhibition and protection of cardiac myocytes against hyperosmotic or hypoxic stress. These findings provide direct evidence that alphaBC(S59P) contributes to the cardioprotection observed after physiologically relevant stresses, such as transient hypoxia. Identifying the targets of alphaBC(S59P) will reveal important details about the mechanism underlying the cytoprotective effects of this small heat shock protein.

Coordination of ATF6-mediated transcription and ATF6 degradation by a domain that is shared with the viral transcription factor, VP16.

ATF6 is a 670-amino acid endoplasmic reticulum (ER) transmembrane protein that is cleaved in response to ER stress. The resulting N-terminal fragment of approximately 400 amino acids translocates to the nucleus and activates selected ER stress-inducible genes, such as GRP-78 and sarco/endoplasmic reticulum ATPase, which are required for cell survival. In studying the mechanism of ATF6-activated transcription, we found that when HeLa cells were transfected with a plasmid encoding ATF6-(1-373), ER stress-inducible reporter gene activation was high, but ATF6-(1-373) expression was low, unless a proteasome inhibitor was added. In contrast, transfection with a plasmid encoding ATF6-(94-373) resulted in low reporter activation and high expression of ATF6-(94-373), which was independent of the proteasome inhibitor. Thus, the information responsible for transcriptional activation and proteasomal degradation must lie within the N-terminal 93 amino acids of ATF6. This portion of ATF6 was found to be homologous to the herpes simplex viral protein, VP16. One 8-amino acid domain of particular interest in this region of ATF6 is 75% identical to the VN8 region in VP16. VN8 is required for VP16-mediated transcription, as well as rapid degradation of VP16 by proteasomes. Point mutations in the VN8-like region of ATF6 caused a loss of transcription, increased expression levels, and an increase in half-life. Thus, the potent transcriptional activities and rapid degradation of ATF6 and VP16 require the VN8 domains in each protein. Homology searches indicate that ATF6 is the only eukaryotic protein known that possesses an active VN8 domain, raising questions about how this domain evolved and the functional importance underlying its appearance in only these two transcription factors.