An 'environment to nucleus' signaling system operates in B lymphocytes: redox status modulates BSAP/Pax-5 activation through Ref-1 nuclear translocation.
The Ref-1 (also called APE or HAP1) protein is a bifunctional enzyme impacting on a wide variety of important cellular functions. It acts as a major member of the DNA base excision repair pathway. Moreover, Ref-1 stimulates the DNA-binding activity of several transcription factors (TFs) through the reduction of highly reactive cysteine residues. Therefore, it represents a mechanism that regulates eukaryotic gene expression in a fast way. However, it has been demonstrated that external stimuli directly act on Ref-1 by increasing its expression levels, a time-consuming mechanism representing a paradox in terms of rapidity of TF regulation. In this paper we demonstrate that this is only an apparent paradox. Exposure of B lymphocytes to H(2)O(2)induced a rapid and sustained increase in Ref-1 protein levels in the nucleus as evaluated by both western blot analysis and by pulse-chase experiments. A time course, two color in situ immunocytochemistry indicated that the up-regulation of Ref-1 in the nucleus at <30 min was primarily the consequence of translocation of its cytoplasmic form. This early nuclear accumulation is effective in modulating the DNA-binding activity of the B cell-specific activator protein BSAP/Pax-5. In fact, EMSA experiments demonstrate that a transient interaction with Ref-1 up-regulates the DNA-binding activity of BSAP/Pax-5. Moreover, in a co-transfection experiment, Ref-1 increased the BSAP/Pax-5 activating effect on an oligomerized BSAP/Pax-5 binding site of the CD19 promoter by 5- to 8-fold. Thus, Ref-1 mediates its effect by up-regulating the DNA-binding activity of BSAP/Pax-5, accounting for a new and fast outside/inside pathway of signaling in B cells.
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Dipartimento di Scienze e Tecnologie Biomediche, Università di Udine, P. le Kolbe 4, I-33100 Udine, Italy.
Zecca
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A
Pellizzari
L
L
Spessotto
P
P
Colombatti
A
A
Kelley
M R
MR
Damante
G
G
Pucillo
C
C
D001402:B-Lymphocytes / Q000502:physiology*; D001692:Biological Transport / Q000502:physiology; D019757:Carbon-Oxygen Lyases / Q000502:physiology*; D002460:Cell Line; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D004268:DNA-Binding Proteins / Q000502:physiology*; D006801:Humans; D009687:Nuclear Proteins / Q000502:physiology*; D010084:Oxidation-Reduction; D051757:PAX5 Transcription Factor; D015398:Signal Transduction / Q000502:physiology*; D014157:Transcription Factors / Q000502:physiology
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D051757:PAX5 Transcription Factor; C497281:PAX5 protein, human; D014157:Transcription Factors; D019757:Carbon-Oxygen Lyases; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase