The CARMA3-Bcl10-MALT1 signalosome promotes angiotensin II-dependent vascular inflammation and atherogenesis

Abstract

The CARMA1, Bcl10, and MALT1 proteins together constitute a signaling complex (CBM signalosome) that mediates antigen-dependent activation of NF-kappaB 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-kappaB. Here we show that one G protein-coupled receptor, the type 1 receptor for angiotensin II, utilizes this mechanism for activation of NF-kappaB 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.

FIGURE 1.

Expression of CBM signalosome components in the vasculature. A, levels of mRNA transcript corresponding to the indicated genes were measured by quantitative RT-PCR. For each transcript, a level of 100% was assigned for the tissue with highest expression. Data are expressed as average ± S.E. for at least three determinations. B, extracts were prepared from whole spleen, thymus, and aorta, and immunoblots were performed to compare levels of Bcl10 and MALT1 within the three tissues. Similarly, extracts were prepared from cell lines derived from T lymphocytes (Jurkat), endothelial cells (EA.hy926 or SVEC4–10), and vascular smooth muscle cells (FLTR or PAC1-AR) to compare Bcl10 and MALT1 levels in vascular cells with those seen in lymphocytes. These studies could not be performed for CARMA3 due to the lack of a sensitive and specific antibody. Hu, human; Mu, mouse; Rt, rat.

FIGURE 2.

The CARMA3-Bcl10-MALT1 signalosome mediates Ang II-dependent NF-κB activation in vascular cells. A, PAC1-AR smooth muscle cells were transiently transfected with either control siRNA or siRNA targeting CARMA3. After 48 h, cells were treated with Ang II for varying periods of time (or with TNFα for 5 min) before harvesting and analyzing by Western blot. Knockdown of CARMA3 mRNA was assessed by quantitative RT-PCR (right). Within 15 min, Ang II treatment induces the phosphorylation of IκB, a marker of canonical NF-κB signaling. p-IκBα, phosphorylated IκBα; pERK, phosphorylated ERK. Data are mean ± S.E. B, PAC1-AR cells were transfected with Myc-tagged IKKγ and HA-tagged ubiquitin prior to treatment with either Ang II or TNFα. Cellular extracts were prepared, and IKKγ was immunoprecipitated (IP) with anti-Myc. Precipitates were then analyzed by immunoblotting with anti-HA to reveal evidence of IKKγ polyubiquitination. WB, Western blot. C, PAC1-AR cells were transfected with siRNAs targeting CARMA3, Bcl10, or MALT1 or with a control siRNA. After 48 h, cells were treated with Ang II for 15 min (left) or with IL-1β for 5 min (right) before analyzing by Western blot. Effective knockdown of Bcl10 and MALT1 was monitored by Western blotting, whereas knockdown of CARMA3 was confirmed by quantitative RT-PCR as in panel A (not shown). *, non-specific band. D, the dominant-negative mutant, CARMA3-ΔCARD, was expressed in PAC1-AR cells and the effect on Ang II- or TNFα-dependent NF-κB activation was assessed by NF-κB luciferase reporter assay. Results are expressed as percent of maximal induction (luciferase/control Renilla) achieved in the absence of the CARMA3 dominant-negative mutant. Data (mean ± S.E.) are from at least three separate determinations. E, PAC1-AR cells were transiently transfected with siRNA targeting Bcl10 or control siRNA. After 24 h, cells were transfected a second time with the NF-κB-luciferase and control Renilla plasmids, and the response to Ang II, TNFα, and IL-1β was assessed the next day. Knockdown was confirmed by immunoblot as shown. NF-κB induction was determined as above using the dual luciferase assay system. Data (mean ± S.E.) are from at least three separate determinations. F, primary rat aortic ECs were transiently transfected with Bcl10 siRNA as in previous panels, and Ang II/TNFα-dependent p-IκB generation was assayed as above. G, Ang II-dependent IKKγ polyubiquitination in primary ECs was assayed as described in the legend for panel B.

FIGURE 3.

Bcl10 deficiency protects ApoE^−/− mice from Ang II-dependent atherogenesis. A, aortas from the ApoE^−/− (n = 22) and ApoE^−/−Bcl10^−/− (n = 13) groups following 4 weeks of chronic Ang II infusion (500 ng/kg/min) and Oil Red O staining. The most significantly affected aortic segments from both groups are shown. B, the aortic arch and major arterial branches extending from the arch for a representative mouse in each group are also shown. C, quantification of atherosclerotic lesional area for all mice is shown, presented as the percentage of the surface area of the entire aorta (ascending, arch, and descending to point of the femoral artery bifurcation) stained positively with Oil Red O. The average percentage of area covered by lesion is indicated by an orange bar, and the S.E. is indicated by blue error bars. D, male ApoE^−/− and ApoE^−/−Bcl10^−/− mice (n = 14 and 13, respectively) were infused with 500 ng/kg/min Ang II for 3 days, after which MCP-1 and CXCL1 mRNA levels were quantified from excised aortas. The level of each of these mRNAs in aortas from ApoE^−/− mice, normalized to GAPDH and TATA-binding protein mRNA, was set arbitrarily at 100.