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. 2002 Jul 9;99(14):9386-91.
doi: 10.1073/pnas.142294499. Epub 2002 Jun 27.

Role of NF kappa B activator Act1 in CD40-mediated signaling in epithelial cells

Youcun Qian  1 , Zhendong Zhao, Zhengfan Jiang, Xiaoxia Li
Affiliations

Role of NF kappa B activator Act1 in CD40-mediated signaling in epithelial cells

Youcun Qian et al. Proc Natl Acad Sci U S A. .

Abstract

CD40, a cell surface receptor in the tumor necrosis factor receptor family, first identified and functionally characterized on B lymphocytes, is also expressed on epithelial and other cells and is now thought to play a more general role in immune regulation. Overexpression of the NF kappa B activator 1 (Act1) leads to the activation of both NF kappa B and Jun kinase in epithelial cell lines. Endogenous Act1 is recruited to the CD40 receptor in human intestinal (HT29) and cervical (HeLa) epithelial cells upon stimulation with CD40 ligand, indicating that Act1 is involved in this signaling pathway. Act1 also interacts with tumor necrosis factor receptor-associated factor 3, a component involved in CD40-activated pathway. Furthermore, transfection of Act1 into C33A cervical epithelial cells, which do not express it, renders these cells sensitive to CD40 ligand-induced NF kappa B activation and protects them from CD40 ligand-induced apoptosis. We conclude that Act1 plays an important role in CD40-mediated signaling in epithelial cells.

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Figures

Figure 1
Figure 1
Act1 function in IKK-null cells. pCMV-Act1 (Act1, 100 or 250 ng) or control vector (250 ng) was transiently cotransfected with 5 × IP-10-luc (5 × NFκB sites from the IP10 promoter, 100 ng) into wild-type, IKKα-, and IKKβ-null MEFs. After 48 h, the cells were harvested, followed by a luciferase reporter assay. The data are presented as fold induction of luciferase activity of the cells transfected with Act1 compared with wild-type MEFs transfected with control vector.
Figure 2
Figure 2
Interaction of Act1 with TAB1 and TAK1. (A) TAB1: Extract from 293-TK/Zeo cells with or without expression of flag-Act1 was either untreated (−) or treated (IL-1, 10 ng/ml, 30 min), followed by Western analysis with anti-TAB1. (B) TAK1: Extract from 293-TK/Zeo cells was immunoprecipitated with anti-TAK1 and probed with anti-flag (M2). Preimmune serum was used as a negative control. (C) Effect of DN-TAK1 on Act1-mediated NFκB activation: Increasing amounts of the kinase-dead mutant pCMV-TAK (K66-W) were transiently cotransfected with pCMV-Act1 and E-selectin-Luc into 293-TK/Zeo cells followed by a luciferase reporter assay 72 h later. The data presented are averages and SDs from three independent experiments.
Figure 3
Figure 3
Act1-interacting proteins. Flag-tagged TRAFs 1–6 in the pCMV vector were transfected transiently into 293 cells containing HA-Act1. Extracts of these transfected cells were immunoprecipitated with anti-HA and probed with anti-flag (M2) and anti-HA. Whole-cell extracts were examined directly by Western analysis with the above Abs as well, to show the expression of these signaling components after transfection.
Figure 4
Figure 4
Recruitment of Act1 to CD40 after CD40L stimulation. (A) Overexpression of Act1 and CD40: Flag-tagged CD40 was transfected into 293 cells transfected with HA-tagged Act1. Extracts from the transfected cells, untreated (0) or treated with CD40L (30 min), were immunoprecipitated with anti-HA and probed with anti-flag (M2) and anti-HA. (B) Overexpression of CD40: Extracts from HeLa cells transfected with flag-tagged CD40, untreated (0) or treated with CD40L for different times, were immunoprecipitated with anti-Act1 and probed with anti-flag (M2) and anti-Act1. (C) Western analysis of CD40: Extracts from various epithelial cells were analyzed by the Western procedure with anti-CD40. (D) Endogenous Act1 and CD40: Extracts from HeLa and HT29 cells, untreated (0) or treated with CD40L for different times, were immunoprecipitated with anti-Act1 and probed with anti-CD40 and anti-Act1.
Figure 5
Figure 5
Role of Act1 in CD40-mediated signaling. (A) NFκB gel-shift assay: Extracts of C33A, HT29, A549, and HeLa cells untreated (−) or treated with CD40L (+, 30 min) were used for gel-shift assays. An NFκB-binding site from the IP-10 gene was used as a probe (Materials and Methods). (B) Western analysis: Extracts of various cell lines were used for Western analysis with anti-Act1. (C) NFκB gel shift: Extracts of C33A and C33A cells stably transfected with pCMV-Act1 (C33/Act1) and untreated (−) or treated (+, 30 min) with CD40L were used for gel-shift assay. (D) Western analysis: Extracts of C33A and C33A/Act1 cells were used for Western analysis with anti-Act1. (E) DNA fragmentation: Genomic DNA from C33A cells untreated (−) or stimulated (+) with CD40L for 24 h were analyzed on 2% agarose gel and visualized by ethidium bromide staining. (F) Western analysis: Extracts from various cell lines, untreated (−) or treated (+, 30 min) with CD40L for 24 h, were used for Western analysis with an Ab that recognizes the cleaved poly(ADP-ribose) polymerase (PARP).
Figure 6
Figure 6
TRAF3 inhibits Act1-mediated NFκB activation. pCMV-Act1 (250 ng, A) or pTK-IRAK (250 ng, B) was transiently cotransfected with E-selectin-luc and increasing amounts of pCMV-TRAF3 into 293 cells, followed by a luciferase reporter assay. The fold induction is relative to that in cells transfected with vector DNA.
Figure 7
Figure 7
A model of CD40-mediated signaling. TRAFs 2, 3, 5, and 6 are intermediary CD40-binding proteins. Act1 functions as an adapter, linking TRAF proteins to TAK1/IKK to activate NFκB and to mitogen-activated protein kinase complex to activate JNK. TRAF3 inhibits Act1-dependent CD40-mediated NFκB activation and initiates CD40L-induced apoptosis. Act1-dependent CD40-mediated NFκB activation in turn protects the cells from CD40L-induced apoptosis.
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