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PNAS 104 (1): 139-144

Copyright © 2007 by the National Academy of Sciences.

CARMA3/Bcl10/MALT1-dependent NF-{kappa}B activation mediates angiotensin II-responsive inflammatory signaling in nonimmune cells

Linda M. McAllister-Lucas*, Jürgen Ruland{dagger}, Katy Siu{ddagger}, Xiaohong Jin{ddagger}, Shufang Gu*, David S. L. Kim{ddagger}, Peter Kuffa*, Dawn Kohrt*, Tak W. Mak§, Gabriel Nuñez{ddagger}, and Peter C. Lucas{ddagger},

Departments of *Pediatrics and Communicable Diseases and {ddagger}Pathology, University of Michigan Medical School, Ann Arbor, MI 48109; {dagger}Third Medical Department, Technical University of Munich, Klinikum rechts der Isar, Ismaninger Strasse 22, 81675 Munich, Germany; and §Campbell Family Institute for Breast Cancer Research, Toronto, ON, Canada M5G 2C1


Figure 1
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  		Fig. 1. CARMA3, Bcl10, and MALT1 are all expressed in Ang II-sensitive tissues. (A–E) Total RNA was extracted from mouse tissues and used for real-time, quantitative RT-PCR. mRNA levels for the AT1R, CARMA3, Bcl10, MALT1, and CARMA1 were determined and then normalized against beta-actin mRNA. For each gene, an mRNA expression value was arbitrarily set at 100 for the tissue with the greatest normalized mRNA level. Relative expression then was calculated for the remaining tissues. Results represent the mean ± SEM of three determinations. (F) Total protein was extracted from mouse spleen, thymus, and liver and subjected to Western blotting by using antisera to CARMA3, Bcl10, MALT1, or GAPDH. Results are representative of two independent experiments.

 

Figure 2
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  		Fig. 2. CARMA3 is required for Ang II-dependent NF-{kappa}B activation. (A Upper) A schematic depicting the domain structure of WT CARMA3 and the dominant-negative mutant of CARMA3 (CARMA3{Delta}CARD) is shown. (A Lower) HepG2 cells were transfected with an NF-{kappa}B-responsive luciferase reporter, a constitutively active Renilla reporter, and an AT1AR expression vector, either in the presence or absence of CARMA3{Delta}CARD. Twenty-four hours after transfection, cells were stimulated overnight with 500 nM Sar1-Ang II or 10 ng/ml TNF{alpha}, and NF-{kappa}B activation was determined by measuring the luciferase/Renilla ratio. Results represent the mean ± SEM for at least three experiments. (B) Hairpin sequence for the human-specific CARMA3 shRNA. (C) The CARMA3 shRNA vector was transfected into HepG2 cells in a dose-dependent fashion along with expression vectors for Flag-tagged CARMA3 and IKK{gamma}. Forty-eight hours after transfection, cells were harvested and assayed for CARMA3 and IKK{gamma} proteins by Western blot. In parallel experiments, cells were transfected with a vector containing an unrelated, control shRNA sequence. (D) HepG2 cells were transfected with either the CARMA3 or control shRNA vectors. Forty-eight hours later, cells were treated for an additional 12 h with either 500 nM Sar1-Ang II or 10 ng/ml TNF{alpha}, and NF-{kappa}B activation was assessed as described previously. Results represent the mean ± SEM for at least three experiments. (E) HepG2 cells were similarly transfected with vector encoding CARMA3 shRNA, with or without an expression vector encoding the WT mouse CARMA3 protein. Forty-eight hours later, cells were treated with 500 nM Sar1-Ang II for an additional 12 h. NF-{kappa}B activation was measured as above. Results represent the mean ± SEM of three to four determinations.

 

Figure 3
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  		Fig. 3. In hepatocytes, Bcl10 is essential for Ang II-dependent NF-{kappa}B activation and cytokine induction. (A Upper) A schematic depicting the domain structure of WT Bcl10 and the dominant-negative mutant of Bcl10 (Bcl10{Delta}107–119). (A Lower) HepG2 cells were transfected as described above with control vector or with expression vector encoding Bcl10{Delta}107–119. Forty-eight hours later, cells were stimulated with either 500 nM Sar1-Ang II or 10 ng/ml TNF{alpha}, and NF-{kappa}B activation was assessed. Results are expressed as the absolute luciferase/Renilla ratio and reflect the mean ± SEM of at least three determinations. (B) Primary hepatocytes were isolated from livers of WT and Bcl10–/– mice and cultured overnight in serum-free medium. Cells then were transfected with an NF-{kappa}B-responsive luciferase reporter, a constitutively active Renilla reporter, and an AT1AR expression vector. Cells were treated with either 500 nM Ang II or 10 ng/ml TNF{alpha} for an additional 16 h before NF-{kappa}B activation was measured. Results represent the mean ± SEM for 17–26 determinations; **, significance level of P < 0.001 (as analyzed by one-tailed Student's t test). (C) WT and Bcl10–/– mice received an i.p. injection of PBS or Ang II (6.4 µg/g of body weight). One hour after injection, mice were killed, and a sample of liver was obtained from each. Total RNA was isolated, and IL-6 mRNA levels were determined by real-time quantitative RT-PCR, normalizing for beta-actin. Results are expressed as fold induction of IL-6 for mice receiving Ang II as compared with PBS and reflect the mean ± SEM of seven to nine mice. ***, significance level of P < 0.03 (as analyzed by one-tailed Student's t test).

 

Figure 4
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  		Fig. 4. Ang II treatment induces IKK{gamma} ubiquitination. (A) HepG2-AR cells were transfected with expression vectors encoding myc-tagged IKK{gamma} and HA-tagged ubiquitin. Cells were treated overnight with 500 nM Ang II or 10 ng/ml TNF{alpha} before harvesting. IKK{gamma} then was immunoprecipitated and subjected to Western blotting as indicated. (B) HepG2-AR cells were transfected and treated as described above but in the presence or absence of coexpressed CARMA3{Delta}CARD. IKK{gamma} ubiquitination was assayed as above. (C) HepG2-AR cells were transfected with HA-ubiquitin only. After treatment with Ang II, endogenous IKK{gamma} was immunoprecipitated and subjected to Western blotting as indicated. Results from all ubiquitination assays shown in A–C are representative of at least three separate experiments. (D) HepG2-AR cells were transfected transiently with or without 100 nM RNAi duplexes specifically targeting the human MALT1 RNA sequence. Forty-eight hours after transfection, cells were transfected again with an NF-{kappa}B-responsive luciferase reporter and a constitutively active Renilla reporter. Cells then were treated with either 500 nM Ang II or 10 ng/ml TNF{alpha} for 16 h, such that cell lysates could be prepared and assayed for NF-{kappa}B activation 96 h after the initial transfection with RNAi. Extracts used to measure NF-{kappa}B activation also were subjected to Western blotting as indicated, to assess effective knockdown of MALT1 protein (–, no RNAi; C, scrambled control RNAi; M, MALT1 RNAi). Results represent the mean ± SEM of five separate experiments.

 

Figure 5
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  		Fig. 5. Model: A similar CARMA-Bcl10-MALT1 signaling pathway mediates NF-{kappa}B activation in response to antigen receptor stimulation in lymphocytes and AT1R stimulation in liver cells. (Left) In lymphocytes, antigen-dependent NF-{kappa}B activation leads to cytokine production and cellular proliferation. (Right) In liver, Ang II-dependent NF-{kappa}B activation leads to a variety of proinflammatory effects that are implicated in the development of hepatic fibrosis/cirrhosis.

 

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