Issue published November 15, 2001

C ardiac hypertrophy is a major cause of morbidity and mortality worldwide. The hypertrophic process is mediated, in part, by small G proteins of the Rho family. We hypothesized that statins, inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase, inhibit cardiac hypertrophy by blocking Rho isoprenylation. We treated neonatal rat cardiac myocytes with angiotensin II (AngII) with and without simvastatin (Sim) and found that Sim decreased AngII-induced protein content, [³H] leucine uptake, and atrial natriuretic factor (ANF) promoter activity. These effects were associated with decreases in cell size, membrane Rho activity, superoxide anion (O₂·̄) production, and intracellular oxidation, and were reversed with L-mevalonate or geranylgeranylpyrophosphate, but not with farnesylpyrophosphate or cholesterol. Treatments with the Rho inhibitor C3 exotoxin and with cell-permeable superoxide dismutase also decreased AngII-induced O₂·̄ production and myocyte hypertrophy. Overexpression of the dominant-negative Rho mutant N17Rac1 completely inhibited AngII-induced intracellular oxidation and ANF promoter activity, while N19RhoA partially inhibited it, and N17Cdc42 had no effect. Indeed, Sim inhibited cardiac hypertrophy and decreased myocardial Rac1 activity and O₂·̄ production in rats treated with AngII infusion or subjected to transaortic constriction. These findings suggest that statins prevent the development of cardiac hypertrophy through an antioxidant mechanism involving inhibition of Rac1.

A series of events initiated by glutamate-receptor interaction perturbs cellular homeostasis resulting in elevation of intracellular free calcium and cell death. Cells subject to such environmental change express stress proteins, which contribute importantly to maintenance of metabolic homeostasis and viability. We show that an inducible chaperone present in endoplasmic reticulum (ER), the 150-kDa oxygen-regulated protein (ORP150), is expressed both in the human brain after seizure attack and in mouse hippocampus after kainate administration. Using mice heterozygous for ORP150 deficiency, exposure to excitatory stimuli caused hippocampal neurons to display exaggerated elevation of cytosolic calcium accompanied by activation of μ-calpain and cathepsin B, as well as increased vulnerability to glutamate-induced cell death in vitro and decreased survival to kainate in vivo. In contrast, targeted neuronal overexpression of ORP150 suppressed each of these events and enhanced neuronal and animal survival in parallel with diminished seizure intensity. Studies using cultured hippocampal neurons showed that ORP150 regulates cytosolic free calcium and activation of proteolytic pathways causing cell death in neurons subject to excitatory stress. Our data underscore a possible role for ER stress in glutamate toxicity and pinpoint a key ER chaperone, ORP150, which contributes to the stress response critical for neuronal survival.

A therosclerosis is an inflammatory disease of large arteries that is initiated through the activation of endothelium by proinflammatory mediators. CD40 receptor stimulation has been implicated in the pathogenesis of atherosclerosis. One of the most important atheroprotective stimuli is the viscous drag (shear stress) generated by the streaming blood acting on the endothelial monolayer. Here, we demonstrate that shear stress prevents CD40 ligand–induced endothelial cell activation, and we identify upregulation of TNF receptor–associated factor-3 (TRAF-3) as a potent CD40-inhibitory mechanism. Shear stress specifically upregulates TRAF-3 in cultured endothelial cells. Moreover, in the endothelial cells overlying human atherosclerotic plaques, TRAF-3 expression is upregulated in areas with high shear stress. Overexpression of TRAF-3 inhibits endothelial expression of proinflammatory cytokines and tissue factor and blocks DNA-binding activity of the transcription factor AP-1; it thereby prevents CD40-induced endothelial activation. Thus, upregulation of TRAF-3 represents a novel mechanism for preserving the functional integrity of the endothelial monolayer.

T he gp130 cytokine receptor activates a cardiomyocyte survival pathway during the transition to heart failure following the biomechanical stress of pressure overload. Although gp130 activation is observed transiently during transverse aortic constriction (TAC), its mechanism of inactivation is largely unknown in cardiomyocytes. We show here that suppressor of cytokine signaling 3 (SOCS3), an intrinsic inhibitor of JAK, shows biphasic induction in response to TAC. The induction of SOCS3 was closely correlated with STAT3 phosphorylation, as well as the activation of an embryonic gene program, suggesting that cardiac gp130-JAK signaling is precisely controlled by this endogenous suppressor. In addition to its cytoprotective action, gp130-dependent signaling induces cardiomyocyte hypertrophy. Adenovirus-mediated gene transfer of SOCS3 to ventricular cardiomyocytes completely suppressed both hypertrophy and antiapoptotic phenotypes induced by leukemia inhibitory factor (LIF). To our knowledge, this is the first clear evidence that these two separate cardiomyocyte phenotypes induced by gp130 activation lie downstream of JAK. Three independent signaling pathways, STAT3, MEK1-ERK1/2, and AKT activation, that are coinduced by LIF stimulation were completely suppressed by SOCS3 overexpression. We conclude that SOCS3 is a mechanical stress–inducible gene in cardiac muscle cells and that it directly modulates stress-induced gp130 cytokine receptor signaling as the key molecular switch for a negative feedback circuit for both myocyte hypertrophy and survival.

H ost defenses are profoundly compromised in HIV-infected hosts due to progressive depletion of CD4⁺ T lymphocytes. Moreover, deficient CD4⁺ T lymphocytes impair vaccination approaches to prevent opportunistic infection. Therefore, we investigated a CD4⁺ T cell–independent vaccine approach to a prototypic AIDS-defining infection, Pneumocystis carinii (PC) pneumonia. Here, we demonstrate that bone marrow–derived dendritic cells (DCs) expressing the murine CD40 ligand, when pulsed ex vivo by PC antigen, elicited significant titers of anti-PC IgG in CD4-deficient mice. Vaccinated animals demonstrated significant protection from PC infection, and this protection was the result of an effective humoral response, since adoptive transfer of CD4-depleted splenocytes or serum conferred this protection to CD4-deficient mice. Western blot analysis of PC antigen revealed that DC-vaccinated, CD4-deficient mice predominantly reacted to a 55-kDa PC antigen. These studies show promise for advances in CD4-independent vaccination against HIV-related pathogens.

M embers of the signal transducer and activator of transcription (STAT) family are transcription factors that mediate many of the effects of pro- and anti-inflammatory cytokines. The progressive systemic inflammatory response induced by endotoxin is mediated by overzealous cytokine production. Here we identify STAT4 and STAT6 as critical regulators of the systemic inflammatory response to endotoxin. Mice deficient for STAT4 or STAT6 were highly susceptible to lethal endotoxemia. In STAT4^–/– mice, antibody blockade of IL-12 prevented mortality, suggesting that STAT4 confers protection, while another signaling pathway mediates the detrimental effects of IL-12. In STAT6^–/– mice we observed dysregulated activation of the transcription factor NF-κB, resulting in augmented production of proinflammatory cytokines and chemokines. Furthermore, STAT6^–/– mice displayed increased organ accumulation of leukocytes and significant hepatocellular injury. These findings demonstrate that STAT4 and STAT6 confer protection against endotoxin-induced death and that for STAT6 these protective effects occur through the regulation of NF-κB activation and subsequent production of proinflammatory cytokines and chemokines.

D ietary proteins are mostly absorbed as di- and tripeptides by the intestinal proton-dependent transporter PepT1. We have examined the effects of leptin on PepT1 function in rat jejunum and in monolayers of the human enterocyte-like 2 cell Caco-2. Leptin is produced by the stomach and secreted in the gut lumen. We show here that PepT1 and leptin receptors are expressed in Caco-2 and rat intestinal mucosal cells. Apical (but not basolateral) leptin increased Caco-2 cell transport of cephalexin (CFX) and glycylsarcosine (Gly-Sar), an effect that was associated with increased Gly-Sar uptake, increased membrane PepT1 protein, decreased intracellular PepT1 content, and no change in PepT1 mRNA levels. The maximal velocity (V_max) for Gly-Sar transport was significantly increased by leptin, whereas the apparent Michaelis-Menten constant (K_m) did not change. Furthermore, leptin-stimulated Gly-Sar transport was completely suppressed by colchicine, which disrupts cellular translocation of proteins to plasma membranes. Intrajejunal leptin also induced a rapid twofold increase in plasma CFX after jejunal perfusion with CFX in the rat, indicating enhanced intestinal absorption of CFX. These data revealed an unexpected action of gastric leptin in controlling ingestion of dietary proteins.

I n hepatocytes, Na⁺ influx through nonselective cation (NSC) channels represents a key point for regulation of cell volume. Under basal conditions, channels are closed, but both physiologic and pathologic stimuli lead to a large increase in Na⁺ and water influx. Since osmotic stimuli also activate mitogen-activated protein (MAP) kinase pathways, we have examined regulation of Na⁺ permeability and cell volume by MAP kinases in an HTC liver cell model. Under isotonic conditions, there was constitutive activity of p38 MAP kinase that was selectively inhibited by SB203580. Decreases in cell volume caused by hypertonic exposure had no effect on p38, but increases in cell volume caused by hypotonic exposure increased p38 activity tenfold. Na⁺ currents were small when cells were in isotonic media but could be increased by inhibiting constitutive p38 MAP kinase, thereby increasing cell volume. To evaluate the potential inhibitory role of p38 more directly, cells were dialyzed with recombinant p38α and its upstream activator, MEK-6, which substantially inhibited volume-sensitive currents. These findings indicate that constitutive p38 activity contributes to the low Na⁺ permeability necessary for maintenance of cell volume, and that recombinant p38 negatively modulates the set point for volume-sensitive channel opening. Thus, functional interactions between p38 MAP kinase and ion channels may represent an important target for modifying volume-sensitive liver functions.

S mooth muscle cell (SMC) accumulation is a key event in the development of atherosclerosis, including vein bypass graft arteriosclerosis. Because members of the protein kinase C (PKC) family signal cells to undergo proliferation, differentiation, or apoptosis, we generated PKCδ knockout mice and performed vein bypass grafts on these animals. PKCδ^–/– mice developed normally and were fertile. Vein segments from PKCδ^–/– mice isografted to carotid arteries of recipient mice of either genotype led to a more severe arteriosclerosis than was seen with PKCδ^+/+ vein grafts. Arteriosclerotic lesions in PKCδ^–/– mice showed a significantly higher number of SMCs than were found in wild-type animals; this was correlated with decreased SMC death in lesions of PKCδ^–/– mice. SMCs derived from PKCδ^–/– aortae were resistant to cell death induced by any of several stimuli, but they were similar to wild-type SMCs with respect to mitogen-stimulated cell proliferation in vitro. Furthermore, pro-apoptotic treatments led to diminished caspase-3 activation, poly(ADP-ribose) polymerase cleavage, and cytochrome c release in PKCδ^–/– relative to wild-type SMCs, suggesting that their apoptotic resistance involves the loss of free radical generation and mitochondrial dysfunction in response to stress stimuli. Our data indicate that PKCδ maintains SMC homeostasis and that its function in the vessel wall per se is crucial in the development of vein graft arteriosclerosis.

N ADPH oxidase is upregulated in smooth muscle cells (SMCs) in response to growth factor stimulation, concomitant with increased reactive oxygen species (ROS) production. We investigated the role of ROS production by NADPH oxidase in SMC responses to growth factors and in atherosclerotic lesion formation in ApoE^–/– mice. SMCs from wild-type, p47phox^–/–, and gp91phox^–/– mice differed markedly with respect to growth factor responsiveness and ROS generation. p47phox^–/– SMCs had diminished superoxide production and a decreased proliferative response to growth factors compared with wild-type cells, whereas the response of gp91phox^–/– SMCs was indistinguishable from that of wild-type SMCs. The relevance of these in vitro observations was tested by measuring atherosclerotic lesion formation in genetically modified (wild-type, p47phox^–/–, ApoE^–/–, and ApoE^–/–/p47phox^–/–) mice. ApoE^–/–/p47phox^–/– mice had less total lesion area than ApoE^–/– mice, regardless of whether mice were fed standard chow or a high-fat diet. Together, these studies provide convincing support for the hypothesis that superoxide generation in general, and NADPH oxidase in particular, have a requisite role in atherosclerotic lesion formation, and they provide a rationale for further studies to dissect the contributions of ROS to vascular lesion formation.

T he X protein (HBX) of the hepatitis B virus (HBV) has been shown to be important for the establishment of HBV infection in vivo. Our previous studies suggested that interaction of HBX with the proteasome complex may underlie the pleiotropic functions of HBX. In this study, we generated a series of woodchuck hepatitis virus (WHV) X mutants, including mutants of the domain interacting with the proteasome, and studied their infectivity in woodchucks. Many of the mutants were defective in transactivation but none of them were completely replication defective in vitro. In vivo, all the wild-type and some X mutant–transfected animals demonstrated evidence of infection with anti-WHc and/or anti-WHs seroconversion. Most of the wild-type– and X mutant–transfected animals had transient viremia. Some animals were later challenged with infectious WHV. Animals inoculated with X mutants, including those with no serologic evidence of infection, were protected from the challenge, suggesting previous infection with resulting protective immunity. Our study demonstrates that the previously described functional domains of HBX are biologically important and the X-defective mutants, possibly as attenuated viruses, are not completely replication defective in vivo.

O besity is associated with increased cardiovascular morbidity and mortality and with elevated circulating levels of the satiety factor leptin. This study provides evidence for a direct link between leptin and the risk for thrombotic complications in obese individuals. For example, although arterial injury provokes thrombosis in both lean and obese (ob/ob) mice, the time to complete thrombotic occlusion is significantly delayed in the ob/ob mice, and the thrombi formed are unstable and frequently embolize. The ob/ob mice lack leptin, and intraperitoneal administration of leptin to these mice before injury restores the phenotype of lean mice by shortening the time to occlusion, stabilizing the thrombi, and decreasing the patency rate. The thrombi that form when leptin receptor-deficient obese (db/db) mice are injured also are unstable. However, in this instance, leptin has no effect. Platelets express the leptin receptor, and leptin potentiates the aggregation of platelets from ob/ob but not db/db mice in response to known agonists. These results reveal a novel receptor-dependent effect of leptin on platelet function and hemostasis and provide new insights into the molecular basis of cardiovascular complications in obese individuals. The results suggest that these prothrombotic properties should be considered when developing therapeutic strategies based on leptin.

A rsenic is effective in the treatment of acute promyelocytic leukemia. Paradoxically, it is also carcinogenic. In the process of elucidating a mechanism of arsenic resistance in a leukemia cell line, NB4, we discovered that arsenic exposure causes chromosomal abnormalities, with a preponderance of end-to-end fusions. These chromosomal end fusions suggested that telomerase activity may be inhibited by arsenic. We found that arsenic inhibits transcription of the hTERT gene, which encodes the reverse transcriptase subunit of human telomerase. This effect may in part be explained by decreased c-Myc and Sp1 transcription factor activities. Decreased telomerase activity leads to chromosomal end lesions, which promote either genomic instability and carcinogenesis or cancer cell death. These phenomena may explain the seemingly paradoxical carcinogenic and antitumor effects of arsenic.