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PNAS 104 (1): 145-150

Copyright © 2007 by the National Academy of Sciences.

Bcl10 plays a critical role in NF-{kappa}B activation induced by G protein-coupled receptors

Donghai Wang*,{dagger}, Yun You*, Pei-Chun Lin*, Liquan Xue{ddagger}, Stephan W. Morris{ddagger}, Hu Zeng§, Renren Wen§, and Xin Lin*,

*Department of Molecular and Cellular Oncology, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030; {ddagger}Departments of Pathology and Hematology–Oncology, St. Jude Children's Research Hospital, Memphis, TN 38105; and §Blood Research Institute, Blood Center of Wisconsin, Milwaukee, WI 53201


Figure 1
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  		Fig. 1. Effect of Bcl10 siRNA on G{alpha}-induced NF-{kappa}B activation. Human embryo kidney 293T cells were treated with transfection reagent alone or siRNA against Bcl10 (50 nM) for 48 h, then cells were transfected with NF-{kappa}B-dependent luciferase reporter together with G{alpha}q(Q209L), G{alpha}12(Q229), or pCMV5 vector DNA. At 6 h after transfection, the cells were starved for {approx}16–18 h. Whole-cell lysates were prepared from these cells. (A) Luciferase activities were determined by dual-luciferase reporter assay. Data are the mean of triplicates from a representative experiment. (B) Cell lysates were subjected to 10% acrylamide SDS/PAGE followed by Western blot using the indicated antibodies.

 

Figure 2
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  		Fig. 2. Bcl10 is required for LPA-induced NF-{kappa}B activation. (A) Bcl10+/– or Bcl10–/– MEF cells were stimulated with LPA or TNF-{alpha} for 30 or 60 min. Nuclear extracts were prepared from these cells and used for EMSA with 32P-labeled NF-{kappa}B or AP-1 probes. Med, Medium. (B) Bcl10+/– or Bcl10–/– MEF cells were pretreated with cycloheximide for 30 min and then stimulated with LPA or TNF-{alpha} for the indicated time points. Cell lysates were prepared from these cells and subjected to SDS/PAGE and Western blot analysis with the antibodies as indicated.

 

Figure 3
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  		Fig. 3. Retroviral-transduced Bcl10 rescues the defect in Bcl10–/– cells. (A) Schematic presentation of retroviral vectors encoding GFP or Bcl10–IRES–GFP. (B) Bcl10–/– MEF cells were infected with retrovirus encoding GFP (Mock) or Bcl10–IRES–GFP (Bcl10). Cell lysates were prepared and subjected to immunoblot with anti-Bcl10 antibodies. (C) Nuclear extracts were prepared from the retroviral-infected Bcl10–/–MEFs or Bcl10+/– MEF cells after LPA stimulation. NF-{kappa}B and Oct-1 activities were examined by EMSA with 32P-labeled NF-{kappa}B and AP-1 probes. N/A, noninfected.

 

Figure 4
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  		Fig. 4. LPA-induced NF-{kappa}B activation depends on Bcl10, but not PI3K activation. (A) Bcl10+/– or Bcl10–/– MEF cells were stimulated with LPA and TNF-{alpha} in the absence or presence of Wortmannin (WM). Nuclear extracts and cell lysates were prepared for EMSA with 32P-labeled NF-{kappa}B, AP-1, or Oct-1 probes. Med, medium. (B) Lysates of these cells were subjected to Western blot analysis with the antibodies indicated. (C) Bcl10+/– or Bcl10–/– MEF cells were stimulated with LPA or PMA plus ionomycin (P/I) for various time points. Cell lysates were prepared and subjected to Western blot analysis with the antibodies indicated.

 

Figure 5
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  		Fig. 5. Bcl10 is required for NF-{kappa}B activation induced by ET-1, LPA, and PMA/ionomycin, but not by LPS, TNF-{alpha}, and integrins. (A) Bcl10+/– or Bcl10–/– MEF cells were stimulated with or without ET-1, LPA, PMA (20 ng/ml) plus ionomycin (PMA+Iono; 200 ng/ml), or TNF-{alpha} (10 ng/ml). Nuclear extracts were prepared from these cells and subjected to EMSA with 32P-labeled NF-{kappa}B or Oct-1 probes. Med, medium. (B) Bcl10+/– or Bcl10–/– MEFs were plated onto dishes precoated with poly-L-Lys (PLL), osteopontin (OPN), or fibronectin (FBN) for 2 h. For TNF-{alpha} and LPS stimulation, cells were plated onto PLL-coated dishes for 1.5 h and then stimulated with TNF-{alpha} (10 ng/ml) or LPS (10 ng/ml) for 30 min. Nuclear extracts were prepared from these cells and subjected to EMSA with 32P-labeled NF-{kappa}B or Oct-1 probes.

 

Figure 6
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  		Fig. 6. LPA-induced MIP-2 production depends on Bcl10. (A) Bcl10+/– or Bcl10–/– MEF cells were subcultured into 10-cm dishes in DMEM supplemented with 10% FCS and antibiotics for 48 h. The cell cultures were stimulated with or without LPA for another 24 h. Cultured media were collected and cleared by centrifugation for ELISA analysis. MIP-2 level was determined by using the MIP-2 Quantikine kit from R&D Systems. Med, medium. (B) A schematic model of GPCR-induced and T cell receptor (TCR)-induced signaling cascades. In the GPCR pathway, proximal signaling events lead to activation of PKC, which, in turn, may regulate CARMA3 and Bcl10. The activated CARMA3 and Bcl10 may directly or indirectly regulate the IKK complex. In contrast, in the TCR pathway, PKC phosphorylates CARMA1, which induces the formation of the CARMA1–Bcl10–MALT1 complex, leading to activation of the IKK complex.

 

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