The Salmonella invasin SipB induces macrophage apoptosis by binding to caspase-1

Recently, Salmonella spp. were shown to induce apoptosis in infected macrophages. The mechanism responsible for this process is unknown. In this report, we establish that the Inv-Spa type III secretion apparatus target invasin SipB is necessary and sufficient for the induction of apoptosis. Purified SipB microinjected into macrophages led to cell death. Binding studies show that SipB associates with the proapoptotic protease caspase-1. This interaction results in the activation of caspase-1, as seen in its proteolytic maturation and the processing of its substrate interleukin-1β. Caspase-1 activity is essential for the cytotoxicity. Functional inhibition of caspase-1 activity by acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone blocks macrophage cytotoxicity, and macrophages lacking caspase-1 are not susceptible to Salmonella-induced apoptosis. Taken together, the data demonstrate that SipB functions as an analog of the Shigella invasin IpaB.

Metalloprotease-mediated ligand release regulates autocrine signaling through the epidermal growth factor receptor

Ligands that activate the epidermal growth factor receptor (EGFR) are synthesized as membrane-anchored precursors that appear to be proteolytically released by members of the ADAM family of metalloproteases. Because membrane-anchored EGFR ligands are thought to be biologically active, the role of ligand release in the regulation of EGFR signaling is unclear. To investigate this question, we used metalloprotease inhibitors to block EGFR ligand release from human mammary epithelial cells. These cells express both transforming growth factor α and amphiregulin and require autocrine signaling through the EGFR for proliferation and migration. We found that metalloprotease inhibitors reduced cell proliferation in direct proportion to their effect on transforming growth factor α release. Metalloprotease inhibitors also reduced growth of EGF-responsive tumorigenic cell lines and were synergistic with the inhibitory effects of antagonistic EGFR antibodies. Blocking release of EGFR ligands also strongly inhibited autocrine activation of the EGFR and reduced both the rate and persistence of cell migration. The effects of metalloprotease inhibitors could be reversed by either adding exogenous EGF or by expressing an artificial gene for EGF that lacked a membrane-anchoring domain. Our results indicate that soluble rather than membrane-anchored forms of the ligands mediate most of the biological effects of EGFR ligands. Metalloprotease inhibitors have shown promise in preventing spread of metastatic disease. Many of their antimetastatic effects could be the result of their ability to inhibit autocrine signaling through the EGFR.

Calcineurin and vacuolar-type H+-ATPase modulate macrophage effector functions

While effector molecules produced by activated macrophages (including nitric oxide, tumor necrosis factor α, interleukin 1, etc.) help to eliminate pathogens, high levels of these molecules can be deleterious to the host itself. Despite their importance, the mechanisms modulating macrophage effector functions are poorly understood. This work introduces two key negative regulators that control the levels and duration of macrophage cytokine production. Vacuolar-type H+-ATPase (V-ATPase) and calcineurin (Cn) constitutively act in normal macrophages to suppress expression of inflammatory cytokines in the absence of specific activation and to inhibit macrophage cytokine responses induced by bacterial lipopolysaccharide (V-ATPase), interferon γ (V-ATPase and Cn), and calcium (Ca2+) flux (Cn). Cn and V-ATPase modulate effector gene expression at the mRNA level by inhibiting transcription factor NF-κB. This negative regulation by Cn is opposite to its crucial positive role in T cells, where it activates NFAT transcription factor(s) leading to expression of interleukin 2, tumor necrosis factor α, and other cytokine genes. The negative effects of V-ATPase and Cn on NF-κB-dependent gene expression are not limited to the macrophage lineage, as similar effects have been seen with a murine fibroblast cell line and with primary astrocytes.

Salicylates and sulfasalazine, but not glucocorticoids, inhibit leukocyte accumulation by an adenosine-dependent mechanism that is independent of inhibition of prostaglandin synthesis and p105 of NFκB

The antiinflammatory action of aspirin generally has been attributed to direct inhibition of cyclooxygenases (COX-1 and COX-2), but additional mechanisms are likely at work. These include aspirin’s inhibition of NFκB translocation to the nucleus as well as the capacity of salicylates to uncouple oxidative phosphorylation (i.e., deplete ATP). At clinically relevant doses, salicylates cause cells to release micromolar concentrations of adenosine, which serves as an endogenous ligand for at least four different types of well-characterized receptors. Previously, we have shown that adenosine mediates the antiinflammatory effects of other potent and widely used antiinflammatory agents, methotrexate and sulfasalazine, both in vitro and in vivo. To determine in vivo whether clinically relevant levels of salicylate act via adenosine, via NFκB, or via the “inflammatory” cyclooxygenase COX-2, we studied acute inflammation in the generic murine air-pouch model by using wild-type mice and mice rendered deficient in either COX-2 or p105, the precursor of p50, one of the components of the multimeric transcription factor NFκB. Here, we show that the antiinflammatory effects of aspirin and sodium salicylate, but not glucocorticoids, are largely mediated by the antiinflammatory autacoid adenosine independently of inhibition of prostaglandin synthesis by COX-1 or COX-2 or of the presence of p105. Indeed, both inflammation and the antiinflammatory effects of aspirin and sodium salicylate were independent of the levels of prostaglandins at the inflammatory site. These experiments also provide in vivo confirmation that the antiinflammatory effects of glucocorticoids depend, in part, on the p105 component of NFκB.

A mutant allele of essential, general translation initiation factor DED1 selectively inhibits translation of a viral mRNA

Positive-strand RNA virus genomes are substrates for translation, RNA replication, and encapsidation. To identify host factors involved in these functions, we used the ability of brome mosaic virus (BMV) RNA to replicate in yeast. We report herein identification of a mutation in the essential yeast gene DED1 that inhibited BMV RNA replication but not yeast growth. DED1 encodes a DEAD (Asp-Glu-Ala-Asp)-box RNA helicase required for translation initiation of all yeast mRNAs. Inhibition of BMV RNA replication by the mutant DED1 allele (ded1–18) resulted from inhibited expression of viral polymerase-like protein 2a, encoded by BMV RNA2. Inhibition of RNA2 translation was selective, with no effect on general cellular translation or translation of BMV RNA1-encoded replication factor 1a, and was independent of p20, a cellular antagonist of DED1 function in translation. Inhibition of RNA2 translation in ded1–18 yeast required the RNA2 5′ noncoding region (NCR), which also conferred a ded1–18-specific reduction in expression on a reporter gene mRNA. Comparison of the similar RNA1 and RNA2 5′ NCRs identified a 31-nucleotide RNA2-specific region that was required for the ded1–18-specific RNA2 translation block and attenuated RNA2 translation in wild-type yeast. Further comparisons and RNA structure predictions suggest a modular arrangement of replication and translation signals in RNA1 and RNA2 5′ NCRs that appears conserved among bromoviruses. The 5′ attenuator and DED1 dependence of RNA2 suggest that, despite its divided genome, BMV regulates polymerase translation relative to other replication factors, just as many single-component RNA viruses use translational read-through and frameshift mechanisms to down-regulate polymerase. The results show that a DEAD-box helicase can selectively activate translation of a specific mRNA and may provide a paradigm for translational regulation by other members of the ubiquitous DEAD-box RNA helicase family.

Calcium-triggered acrosomal exocytosis in human spermatozoa requires the coordinated activation of Rab3A and N-ethylmaleimide-sensitive factor

The acrosome reaction of spermatozoa is a complex, calcium-dependent, regulated exocytosis. Fusion at multiple sites between the outer acrosomal membrane and the cell membrane causes the release of the acrosomal contents and the loss of the membranes surrounding the acrosome. However, very little is known about the molecules that mediate and regulate this unique fusion process. Here, we show that N-ethylmaleimide-sensitive factor (NSF), a protein essential for most fusion events, is present in the acrosome of several mammalian spermatozoa. Moreover, we demonstrate that calcium-dependent exocytosis of permeabilized sperm requires active NSF. Previously, we have shown that the addition of the active (GTP-bound) form of the small GTPase Rab3A triggers exocytosis in permeabilized spermatozoa. In the present report we show that Rab3A is necessary for calcium-dependent exocytosis. The activation of Rab3A protects NSF from N-ethylmaleimide inhibition and precludes the exchange of the endogenous protein with recombinant dominant negative mutants of NSF. Furthermore, Rab3A activation of acrosomal exocytosis requires active NSF. Our results suggest that, upon calcium stimulation, Rab3A switches to its active GTP-bound form, triggering the formation of a protein complex in which NSF is protected. This process is suggested to be an essential part of the molecular mechanism of membrane fusion leading to the release of the acrosomal contents.

CYR61, a product of a growth factor-inducible immediate early gene, promotes angiogenesis and tumor growth

CYR61 is a secreted, cysteine-rich, heparin-binding protein encoded by a growth factor-inducible immediate–early gene. Acting as an extracellular, matrix-associated signaling molecule, CYR61 promotes the adhesion of endothelial cells through interaction with the integrin αVβ3 and augments growth factor-induced DNA synthesis in the same cell type. In this study, we show that purified CYR61 stimulates directed migration of human microvascular endothelial cells in culture through an αVβ3-dependent pathway and induces neovascularization in rat corneas. Both the chemotactic and angiogenic activities of CYR61 can be blocked by specific anti-CYR61 antibodies. Whereas most human tumor-derived cell lines tested express CYR61, the gastric adenocarcinoma cell line RF-1 does not. Expression of the CYR61 cDNA under the regulation of a constitutive promoter in RF-1 cells significantly enhances the tumorigenicity of these cells as measured by growth in immunodeficient mice, resulting in tumors that are larger and more vascularized than those produced by control RF-1 cells. Taken together, these results identify CYR61 as an angiogenic inducer that can promote tumor growth and vascularization; the results also suggest potential roles for CYR61 in physiologic and pathologic neovascularization.

Inhibition of β-catenin-mediated transactivation by cadherin derivatives

We studied the effect of N-cadherin, and its free or membrane-anchored cytoplasmic domain, on the level and localization of β-catenin and on its ability to induce lymphocyte enhancer-binding factor 1 (LEF-1)-responsive transactivation. These cadherin derivatives formed complexes with β-catenin and protected it from degradation. N-cadherin directed β-catenin into adherens junctions, and the chimeric protein induced diffuse distribution of β-catenin along the membrane whereas the cytoplasmic domain of N-cadherin colocalized with β-catenin in the nucleus. Cotransfection of β-catenin and LEF-1 into Chinese hamster ovary cells induced transactivation of a LEF-1 reporter, which was blocked by the N-cadherin-derived molecules. Expression of N-cadherin and an interleukin 2 receptor/cadherin chimera in SW480 cells relocated β-catenin from the nucleus to the plasma membrane and reduced transactivation. The cytoplasmic tails of N- or E-cadherin colocalized with β-catenin in the nucleus, and suppressed the constitutive LEF-1-mediated transactivation, by blocking β-catenin–LEF-1 interaction. Moreover, the 72 C-terminal amino acids of N-cadherin stabilized β-catenin and reduced its transactivation potential. These results indicate that β-catenin binding to the cadherin cytoplasmic tail either in the membrane, or in the nucleus, can inhibit β-catenin degradation and efficiently block its transactivation capacity.

Glucocorticoid-mediated repression of nuclear factor-κBdependent transcription involves direct interference with transactivation

Glucocorticoids exert multiple anti-inflammatory activities, one of which is the inhibition of transcription dependent on the nuclear factor (NF)-κB. It has been suggested that the effect of dexamethasone (DEX), a glucocorticoid analog, is attributed to an increased production of the inhibitory IκB molecule, which in turn would bind and remove activated, DNA-bound NF-κB complexes in the cell nucleus. Upon investigating DEX-mediated repression of interleukin-6 expression induced by tumor necrosis factor, DEX treatment was found to act directly on NF-κB-dependent transcription, without changing the expression level of IκB. Neither the mRNA of IκB nor the protein was significantly elevated by a combined treatment with tumor necrosis factor and DEX of murine endothelial or fibroblast cells. The DNA-binding activity of induced NF-κB also remained unchanged after stimulation of cells with DEX. Evidence for a direct nuclear mechanism of action was obtained by analysis of cell lines stably expressing a fusion protein between the DNA-binding domain of the yeast Gal4 protein and the transactivating p65 subunit of NF-κB. Expression of a Gal4-dependent luciferase reporter gene activated by this nuclear fusion protein was also strongly repressed after addition of DEX. Because the DNA-binding activity of the Gal4 fusion protein was not affected by DEX, it can be concluded that the reduction of gene activation was caused by interference of the activated glucocorticoid receptor with the transactivation potential of the NF-κB p65 subunit.

Angiogenesis promoted by vascular endothelial growth factor: Regulation through α1β1 and α2β1 integrins

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is a cytokine of central importance for the angiogenesis associated with cancers and other pathologies. Because angiogenesis often involves endothelial cell (EC) migration and proliferation within a collagen-rich extracellular matrix, we investigated the possibility that VEGF promotes neovascularization through regulation of collagen receptor expression. VEGF induced a 5- to 7-fold increase in dermal microvascular EC surface protein expression of two collagen receptors—the α1β1 and α2β1 integrins—through induction of mRNAs encoding the α1 and α2 subunits. In contrast, VEGF did not induce increased expression of the α3β1 integrin, which also has been implicated in collagen binding. Integrin α1-blocking and α2-blocking antibodies (Ab) each partially inhibited attachment of microvascular EC to collagen I, and α1-blocking Ab also inhibited attachment to collagen IV and laminin-1. Induction of α1β1 and α2β1 expression by VEGF promoted cell spreading on collagen I gels which was abolished by a combination of α1-blocking and α2-blocking Abs. In vivo, a combination of α1-blocking and α2-blocking Abs markedly inhibited VEGF-driven angiogenesis; average cross-sectional area of individual new blood vessels was reduced 90% and average total new vascular area was reduced 82% without detectable effects on the pre-existing vasculature. These data indicate that induction of α1β1 and α2β1 expression by EC is an important mechanism by which VEGF promotes angiogenesis and that α1β1 and α2β1 antagonists may prove effective in inhibiting VEGF-driven angiogenesis in cancers and other important pathologies.

ATM-dependent expression of the insulin-like growth factor-I receptor in a pathway regulating radiation response

The ATM gene is mutated in the syndrome of ataxia telangiectasia (AT), associated with neurologic dysfunction, growth abnormalities, and extreme radiosensitivity. Insulin-like growth factor-I receptor (IGF-IR) is a cell surface receptor with tyrosine kinase activity that can mediate mitogenesis, cell transformation, and inhibition of apoptosis. We report here that AT cells express low levels of IGF-IR and show decreased IGF-IR promoter activity compared with wild-type cells. Complementation of AT cells with the ATM cDNA results in increased IGF-IR promoter activity and elevated IGF-IR levels, whereas expression in wild-type cells of a dominant negative fragment of ATM specifically reduces IGF-IR expression, results consistent with a role for ATM in regulating IGF-IR expression at the level of transcription. When expression of IGF-IR cDNA is forced in AT cells via a heterologous viral promoter, near normal radioresistance is conferred on the cells. Conversely, in ATM cells complemented with the ATM cDNA, specific inhibition of the IGF-IR pathway prevents correction of the radiosensitivity. Taken together, these results establish a fundamental link between ATM function and IGF-IR expression and suggest that reduced expression of IGF-IR contributes to the radiosensitivity of AT cells. In addition, because IGF-I plays a major role in human growth and metabolism and serves as a survival and differentiation factor for developing neuronal tissue, these results may provide a basis for understanding other aspects of the AT syndrome, including the growth abnormalities, insulin resistance, and neurodegeneration.

pRB induces Sp1 activity by relieving inhibition mediated by MDM2

pRB activates transcription by a poorly understood mechanism that involves relieving negative regulation of the promoter specificity factor Sp1. We show here that MDM2 inhibits Sp1-mediated transcription, that MDM2 binds directly to Sp1 in vitro as well as in vivo, and that MDM2 inhibits the DNA-binding activity of Sp1. Forced expression of pRB relieves MDM2-mediated repression, and interaction of pRB with the MDM2-Sp1 complex releases Sp1 and restores DNA binding. These results suggest a model in which the opposing activities of MDM2 and pRB regulate Sp1 DNA-binding and transcriptional activity.

Prevention of ischemia-induced retinopathy by the natural ocular antiangiogenic agent pigment epithelium-derived factor

Aberrant blood vessel growth in the retina that underlies the pathology of proliferative diabetic retinopathy and retinopathy of prematurity is the result of the ischemia-driven disruption of the normally antiangiogenic environment of the retina. In this study, we show that a potent inhibitor of angiogenesis found naturally in the normal eye, pigment epithelium-derived growth factor (PEDF), inhibits such aberrant blood vessel growth in a murine model of ischemia-induced retinopathy. Inhibition was proportional to dose and systemic delivery of recombinant protein at daily doses as low as 2.2 mg/kg could prevent aberrant endothelial cells from crossing the inner limiting membrane. PEDF appeared to inhibit angiogenesis by causing apoptosis of activated endothelial cells, because it induced apoptosis in cultured endothelial cells and an 8-fold increase in apoptotic endothelial cells could be detected in situ when the ischemic retinas of PEDF-treated animals were compared with vehicle-treated controls. The ability of low doses of PEDF to curtail aberrant growth of ocular endothelial cells without overt harm to retinal morphology suggests that this natural protein may be beneficial in the treatment of a variety of retinal vasculopathies.

Transforming Growth Factor-β1 Mediates Epithelial to Mesenchymal Transdifferentiation through a RhoA-dependent Mechanism

Transforming growth factor-β1 (TGF-β) can be tumor suppressive, but it can also enhance tumor progression by stimulating the complex process of epithelial-to-mesenchymal transdifferentiaion (EMT). The signaling pathway(s) that regulate EMT in response to TGF-β are not well understood. We demonstrate the acquisition of a fibroblastoid morphology, increased N-cadherin expression, loss of junctional E-cadherin localization, and increased cellular motility as markers for TGF-β–induced EMT. The expression of a dominant-negative Smad3 or the expression of Smad7 to levels that block growth inhibition and transcriptional responses to TGF-β do not inhibit mesenchymal differentiation of mammary epithelial cells. In contrast, we show that TGF-β rapidly activates RhoA in epithelial cells, and that blocking RhoA or its downstream target p160ROCK, by the expression of dominant-negative mutants, inhibited TGF-β–mediated EMT. The data suggest that TGF-β rapidly activates RhoA-dependent signaling pathways to induce stress fiber formation and mesenchymal characteristics.

Targeted inhibition of calcineurin attenuates cardiac hypertrophy in vivo

The Ca2+-calmodulin-activated Ser/Thr protein phosphatase calcineurin and the downstream transcriptional effectors of calcineurin, nuclear factor of activated T cells, have been implicated in the hypertrophic response of the myocardium. Recently, the calcineurin inhibitory agents cyclosporine A and FK506 have been extensively used to evaluate the importance of this signaling pathway in rodent models of cardiac hypertrophy. However, pharmacologic approaches have rendered equivocal results necessitating more specific or genetic-based inhibitory strategies. In this regard, we have generated Tg mice expressing the calcineurin inhibitory domains of Cain/Cabin-1 and A-kinase anchoring protein 79 specifically in the heart. ΔCain and ΔA-kinase-anchoring protein Tg mice demonstrated reduced cardiac calcineurin activity and reduced hypertrophy in response to catecholamine infusion or pressure overload. In a second approach, adenoviral-mediated gene transfer of ΔCain was performed in the adult rat myocardium to evaluate the effectiveness of an acute intervention and any potential species dependency. ΔCain adenoviral gene transfer inhibited cardiac calcineurin activity and reduced hypertrophy in response to pressure overload without reducing aortic pressure. These results provide genetic evidence implicating calcineurin as an important mediator of the cardiac hypertrophic response in vivo.