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

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.

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