CD40 stimulation induces Pax5/BSAP and EBF activation through a APE/Ref-1-dependent redox mechanism.
CD40 is a member of the growing tumor necrosis factor receptor family that has been shown to play important roles in T cell-mediated B lymphocyte activation. Ligation of B cell CD40 by CD154, mainly expressed on activated T cells, stimulates B cell proliferation, differentiation, isotype switching, up-regulation of surface molecules contributing to antigen presentation, development of the germinal center, and the humoral memory response. In this study we demonstrate that the redox factor APE/Ref-1 acts as a key signaling intermediate in response to CD40-mediated B cell activation. The transcription factors Pax5a or BSAP (B cell lineage-specific activator protein) and EBF (early B cell factor) are constitutively expressed in spleen B cells and CD40 cross-linking induces increases in Pax5a and EBF binding activity compared with nonstimulated B cells. We show that upon CD40 antibody-mediated cross-linking, APE/Ref-1 translocates from the cytoplasm to the nucleus of activated B cells, where it modulates the DNA binding activity of both Pax5a and EBF. Moreover, we show that the repression of APE/Ref-1 protein production is able to block CD40-mediated Pax5a activation. We also provide evidence that APE/Ref-1 can modulate the cooperative activation of the blk promoter operated by Pax5a and EBF and that APE/Ref-1 might directly regulate EBF functional activity. Finally, we show that the interaction between Pax5a and EBF enhances EBF binding activity to its consensus sequence, suggesting that Pax5a can physically interact with EBF and modulate its DNA binding activity.
Dipartimento di Scienze e Tecnologie Biomediche, M.A.T.I. Center of Excellence, Piazzale Kolbe 4, Università degli Studi di Udine, 33100 Udine, Italy.
Sonia
S
Merluzzi
Massimo
M
Moretti
Sandro
S
Altamura
Patty
P
Zwollo
Mikael
M
Sigvardsson
Gaetano
G
Vitale
Carlo
C
Pucillo
D021581:Active Transport, Cell Nucleus; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D051017:Apoptosis Regulatory Proteins; D019013:CD40 Antigens / Q000502:physiology*; D002352:Carrier Proteins / Q000235:genetics; D004247:DNA / Q000378:metabolism; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C; D024101:Mitochondrial Proteins; D010084:Oxidation-Reduction; D051757:PAX5 Transcription Factor; D011401:Promoter Regions, Genetic; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; C111741:Bik protein, mouse; D019013:CD40 Antigens; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C489921:Ebf1 protein, mouse; D024101:Mitochondrial Proteins; D051757:PAX5 Transcription Factor; C497282:Pax5 protein, mouse; D015534:Trans-Activators; D014157:Transcription Factors; D004247:DNA; C486216:Apex1 protein, mouse; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase