Angiotensin II (Ang II) is a peptide hormone that, like many cytokines, acts as a proinflammatory agent and growth factor. After injury to the liver, the hormone assists in tissue repair by stimulating hepatocytes and hepatic stellate cells to synthesize extracellular matrix proteins and secrete secondary cytokines and by stimulating myofibroblasts to proliferate. However, under conditions of chronic liver injury, all of these effects conspire to promote pathologic liver fibrosis. Much of this effect of Ang II results from activation of the proinflammatory NF-B transcription factor in response to stimulation of the type 1 Ang II receptor, a G protein-coupled receptor. Here, we characterize a previously undescribed signaling pathway mediating Ang II-dependent activation of NF-B, which is composed of three principal proteins, CARMA3, Bcl10, and MALT1. Blocking the function of any of these proteins, through the use of either dominant-negative mutants, RNAi, or gene targeting, effectively abolishes Ang II-dependent NF-B activation in hepatocytes. In addition, Bcl10 ؊/؊ mice show defective hepatic cytokine production after Ang II treatment. Evidence also is presented that this pathway activates NF-B through ubiquitination of IKK␥, the regulatory subunit of the I B kinase complex. These results elucidate a concrete series of molecular events that link ligand activation of the type 1 Ang II receptor to stimulation of the NF-B transcription factor. These findings also uncover a function of the CARMA, Bcl10, and MALT1 proteins in cells outside the immune system. G protein-coupled receptor ͉ hepatocyte ͉ IkB kinase ͉ inflammation ͉ ubiquitination

Proper regulation of nuclear factor-κB (NF-κB) transcriptional activity is required for normal lymphocyte function, and deregulated NF-κB signaling can facilitate lymphomagenesis. We demonstrate that the API2-MALT1 fusion oncoprotein created by the recurrent t(11;18)(q21;q21) in mucosa-associated lymphoid tissue (MALT) lymphoma induces proteolytic cleavage of NF-κB inducing kinase (NIK) at Arg325. NIK cleavage requires the concerted actions of both fusion partners and generates a C-terminal NIK fragment that retains kinase activity and is resistant to proteasomal degradation. The resulting deregulated NIK activity is associated with constitutive noncanonical NF-κB signaling, enhanced B-cell adhesion, and apoptosis resistance. Our study reveals the gain-of-function proteolytic activity of a fusion oncoprotein and highlights the importance of the noncanonical NF-κB pathway in B-lymphoproliferative disease.

Objective-The direct role of leptin in vascular disease remains controversial. The objective of this study was to examine the effects of leptin treatment on atherosclerosis and thrombosis in atherosclerotic-prone mice. Methods and Results-Sixteen-week-old, male apolipoprotein E-deficient mice were treated with injections of recombinant leptin (125 g per day IP; nϭ10) or vehicle (nϭ10) 4 and platelets. 5 Plasma leptin levels have also been correlated with cardiovascular complications in humans, an effect independent of body mass index and traditional risk factors. 6 -8 Nevertheless, the direct role of leptin in vascular disease remains controversial. Atherosclerotic mouse models with complete deficiency of leptin suggest that the absence of leptin might promote atherosclerosis. 9,10 However, these studies have been confounded by the extreme obesity and dyslipidemia that result from the loss of leptin-mediated central effects. 9,10 As a result, the direct effect of leptin on atherosclerosis has not yet been addressed. Therefore, to examine the direct role of leptin in atherosclerosis, we analyzed the effect of exogenous recombinant murine leptin on atherosclerosis using apolipoprotein E (apoE)-deficient mice.In addition, we tested the effects of chronic leptin therapy on thrombosis in these atherosclerotic-prone mice to determine whether potential metabolic improvements achieved with leptin therapy would outweigh the acute prothrombotic effect of leptin described previously. 11,12 Methods MiceMale apoE-deficient mice were purchased from the Jackson Laboratory (stock No. 002052; Bar Harbor, Maine) and provided Western chow (TD88137; Harlan Teklad) from 14 to 20 weeks of age. All procedures complied with the principles of laboratory and animal care established by the National Society for Medical Research and were approved by the University of Michigan committee on use and care of animals. Leptin TreatmentBeginning at 16 weeks of age, mice received 200 L intraperitoneal injections daily of either 125 g of recombinant murine leptin (R & D Systems) or vehicle control (nϭ10 per group). The dose of leptin chosen was based on a protocol used to achieve weight loss and fertility in leptin-deficient mice. 13 We determined that a 4-week duration of injections at this dose would be adequate to test the hypothesis that leptin promotes atherosclerosis in this particular model of hyperlipidemia. 14 http://atvb.ahajournals.org/ Downloaded from esthesia (100 mg/kg). Mice were perfused with saline and fixed using formalin with a 25-gauge needle inserted into the left ventricle at a rate of 1 mL/min. After formalin fixation, the arterial tree was meticulously dissected from the carcass and placed in 70% ethanol for Ն72 hours. The surface area occupied by atherosclerosis was then quantitated at the thoracic aorta and major branches, including the brachiocephalic, carotid, and subclavian arteries, via Oil Red O staining and quantitative morphometry, as described previously. 15 The lesion area was calculated for the control and treat...

Thrombin is a potent modulator of endothelial function and, through stimulation of NF-B, induces endothelial expression of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). These cell surface adhesion molecules recruit inflammatory cells to the vessel wall and thereby participate in the development of atherosclerosis, which is increasingly recognized as an inflammatory condition. The principal receptor for thrombin on endothelial cells is protease-activated receptor-1 (PAR-1), a member of the G protein-coupled receptor superfamily. Although it is known that PAR-1 signaling to NF-B depends on initial PKC activation, the subsequent steps leading to stimulation of the canonical NF-B machinery have remained unclear. Here, we demonstrate that a complex of proteins containing CARMA3, Bcl10, and MALT1 links PAR-1 activation to stimulation of the IB kinase complex. IB kinase in turn phosphorylates IB, leading to its degradation and the release of active NF-B. Further, we find that although this CARMA3⅐Bcl10⅐MALT1 signalosome shares features with a CARMA1-containing signalosome found in lymphocytes, there are significant differences in how the signalosomes communicate with their cognate receptors. Specifically, whereas the CARMA1-containing lymphocyte complex relies on 3-phosphoinositide-dependent protein kinase 1 for assembly and activation, the CARMA3-containing endothelial signalosome functions completely independent of 3-phosphoinositide-dependent protein kinase 1 and instead relies on ␤-arrestin 2 for assembly. Finally, we show that thrombin-dependent adhesion of monocytes to endothelial cells requires an intact endothelial CARMA3⅐Bcl10⅐MALT1 signalosome, underscoring the importance of the signalosome in mediating one of the most significant pro-atherogenic effects of thrombin.

The CARMA1, Bcl10, and MALT1 proteins together constitute a signaling complex (CBM signalosome) that mediates antigen-dependent activation of NF-B in lymphocytes, thereby representing a cornerstone of the adaptive immune response. Although CARMA1 is restricted to cells of the immune system, the analogous CARMA3 protein has a much wider expression pattern. Emerging evidence suggests that CARMA3 can substitute for CARMA1 in non-immune cells to assemble a CARMA3-Bcl10-MALT1 signalosome and mediate G protein-coupled receptor activation of NF-B. Here we show that one G proteincoupled receptor, the type 1 receptor for angiotensin II, utilizes this mechanism for activation of NF-B in endothelial and vascular smooth muscle cells, thereby inducing pro-inflammatory signals within the vasculature, a key factor in atherogenesis. Further, we demonstrate that Bcl10-deficient mice are protected from developing angiotensin-dependent atherosclerosis and aortic aneurysms. By uncovering a novel vascular role for the CBM signalosome, these findings illustrate that CBM-dependent signaling has functions outside the realm of adaptive immunity and impacts pathobiology more broadly than previously known.

Mucosa-associated lymphoid tissue (MALT) lymphoma is the most common extranodal lymphoid neoplasm. Chromosomal translocation t(11;18)(q21,q21) is found in 30% of gastric MALT lymphomas and is associated with a failure to respond to standard treatment and a tendency to disseminate. This translocation generates a chimeric protein composed of N-terminal sequences of Inhibitor of Apoptosis 2 (API2, also known as BIRC3 and cIAP2) fused to C-terminal sequences of MALT1. API2-MALT1 promotes cell survival and proliferation via activation of nuclear factor-jB (NF-jB). Here, we investigate the mechanism by which the API2 moiety contributes to NF-jB stimulation. We find that the API2 moiety mediates oligomerization of API2-MALT1 as well as interaction with tumor necrosis factor receptor-associated factor 2 (TRAF2). Surprisingly, oligomerization does not occur via homotypic interaction; rather, the API2 moiety of one monomer interacts with the MALT1 moiety of another monomer. Further, the specific region of the API2 moiety responsible for mediating oligomerization is distinct from that mediating TRAF2 binding. Although deletion or mutation of the TRAF2 binding site does not inhibit oligomerization, it does lead to dramatically decreased NF-jB activation. Deletion of both TRAF2 binding and oligomerization regions results in nearcomplete loss of NF-jB activation. Thus, API2 moietymediated heterotypic oligomerization and TRAF2 binding both contribute to maximal API2-MALT1-dependent NF-jB stimulation.

Background-␣-Galactosidase A (Gla) deficiency leads to widespread tissue accumulation of neutral glycosphingolipids and is associated with premature vascular complications such as myocardial infarction and stroke. Glycosphingolipids have been shown to accumulate in human atherosclerotic lesions, although their role in atherogenesis is unclear. Methods and Results-To determine whether Gla affects the progression of atherosclerosis, mice were generated with combined deficiencies of apolipoprotein E and Gla. At 45 weeks of age, Gla-deficient mice had developed more atherosclerosis than mice with normal Gla expression (25.1Ϯ14.0 versus 12.3Ϯ9.3 mm 2 of total lesion area, PϽ0.02). This increase in atherosclerosis was associated with the presence of increased Gb3, enhanced inducible nitric oxide synthase expression, and increased nitrotyrosine staining. Conclusions-These findings suggest that deficiency of Gla leads to increased inducible nitric oxide synthase expression and accelerated atherosclerosis. (Fabry disease) is an X-linked disorder that leads to widespread tissue accumulation of neutral glycosphingolipids with ␣-galactosyl linkages consisting primarily of globotriaosylceramide (Gb3). 1 Clinical manifestations of Fabry disease include renal failure, painful neuropathies, angiokeratoma, myocardial infarction, and stroke, which lead to premature mortality. 1 Although premature vascular complications are more common in subjects with Fabry disease, 2-4 the effect of Gla deficiency on atherogenesis is unknown. Glycosphingolipids have been shown to accumulate in atherosclerotic plaques even in subjects without Fabry disease, which suggests they may play a role in atherogenesis. 5 A mouse model of Gla deficiency has been generated by targeted disruption of the Gla gene. 6 These mice accumulate glycosphingolipids in multiple organs with age, including the large blood vessels 7,8 ; thus, they provide a useful model to study the vascular consequences of Gla deficiency. The present study was designed to test the consequences of Gla deficiency in vascular disease with a mouse model of atherosclerosis. Methods MiceTo test the consequences of Gla deficiency on vascular disease, we examined the development of lipid lesions between apolipoprotein E-deficient (ApoE Ϫ/Ϫ ) littermate mice with genetic variation in Gla. ApoE Ϫ/Ϫ mice on the C57BL6/J background were purchased from Jackson Laboratory (Bar Harbor, Me). Gla-deficient (Gla Ϫ/0 or Gla Ϫ/Ϫ ) mice were bred from mice provided by Drs Ashok Kulkarni and Roscoe Brady (National Institutes of Health, Bethesda, Md). The "0" in Gla Ϫ/0 denotes the absence of the second X chromosome of the male mice in this X-linked disease. These mice were backcrossed at least 6 generations to the C57BL6/J strain before being bred to Analysis of AtherosclerosisAt 45 weeks of age, mice were euthanized via exsanguination while under intraperitoneal pentobarbital anesthesia (100 mg/kg). The mice were perfused with phosphate-buffered saline and fixed with formalin with a 25-gauge needle, insert...

Background— Activated protein C resistance due to factor V Leiden (FVL) is a common genetic risk factor for venous thrombosis in humans. Although the impact of FVL on the development of venous thrombosis is well established, its effect on arterial thrombosis and atherosclerosis is controversial. Methods and Results— To determine the effect of the FVL mutation on arterial thrombosis in the mouse, wild-type ( Fv +/+ ), heterozygous FVL ( Fv Q /+ ), and homozygous FVL ( Fv Q/Q ) mice underwent photochemical carotid arterial injury to induce occlusive thrombosis. Fv Q/Q mice formed occlusive thromboses 27±3 minutes (n=7) after the onset of injury, which was significantly shorter than that observed for Fv +/+ mice (56±7 minutes, n=9, P <0.01), whereas Fv Q /+ mice (41±7 minutes, n=5) were intermediate ( P =0.5, compared with Fv +/+ ). To determine the source of FVL relevant to the enhanced vascular thrombosis, bone marrow transplantation experiments were performed between Fv +/+ and Fv Q/Q mice. Fv Q/Q mice transplanted with Fv +/+ bone marrow formed occlusive thromboses at 35±5 minutes (n=7, P <0.05 compared with Fv +/+ mice), whereas Fv +/+ mice transplanted with Fv Q/Q bone marrow occluded at 59±7 minutes (n=6, P <0.001 compared with Fv Q/Q mice). To assess the effect of the FVL mutation on the development of atherosclerosis, Fv Q/Q mice were crossed with the atherosclerosis-prone apolipoprotein E (ApoE)–deficient strain ( ApoE −/− ) to generate Fv Q/Q ,ApoE −/− mice. By 52 weeks of age, Fv Q/Q ,ApoE −/− mice (n=8) had developed more aortic atherosclerosis (40±6% lesion area) compared with Fv +/+ ,ApoE −/− mice (15±3% lesion area; n=12, P <0.02). Conclusions— In conclusion, homozygosity for the FVL mutation in mice leads to enhanced arterial thrombosis and atherosclerosis. The source of the FVL leading to accelerated thrombosis appears to be circulating, non–platelet-derived plasma FVL.