Nucleic Acids Research, 2000, Vol. 28, No. 5 1099-1105
© 2000 Oxford University Press
An ‘environment to nucleus’ signaling system operates in B lymphocytes: redox status modulates BSAP/Pax-5 activation through Ref-1 nuclear translocation
1Dipartimento di Scienze e Tecnologie Biomediche, Università di Udine, P. le Kolbe 4, I-33100 Udine, Italy, 2Dipartimento di Biochimica, Biofisica e Chimica delle Macromolecole, Università di Trieste, Via Giorgieri 1, I-34127 Trieste, Italy, 3Divisione di Oncologia Sperimentale 2, Centro di Riferimento Oncologico di Aviano, I-33081 Aviano, Italy and 4Department of Pediatrics and Biochemistry and Molecular Biology, Herman B. Wells Center for Pediatric Research at Indiana University School of Medicine, Indianapolis, IN, USA
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 H2O2 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.
* To whom correspondence should be addressed. Tel: +39 0432 494340; Fax: +39 0432 494301; Email: cpucillo@makek.dstb.uniud.it
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