Cross-regulation between Egr-1 and APE/Ref-1 during early response to oxidative stress in the human osteoblastic HOBIT cell line: evidence for an autoregulatory loop.
The Early Growth Response protein (Egr-1) is a C(2)H(2)-zinc finger-containing transcriptional regulator involved in the control of cell proliferation and apoptosis. Its DNA-binding activity is redox regulated in vitro through the oxidation-reduction of Cys residues within its DNA-binding domain. APE/Ref-1 is a DNA-repair enzyme with redox modulating activities on several transcription factors. In this study, by evaluating the effects of different stimuli, we found a similar timing of activation being suggestive for a common and co-linear regulation for the two proteins. Indeed, we show that APE/Ref-1 increases the Egr-1 DNA-binding activity in unstimulated osteoblastic HOBIT cells. H(2)O(2) stimulation induces a strong interaction between Egr-1 and APE/Ref-1 at early times upon activation, as assayed by immunoprecipitation experiments. By using a cell transfection approach, we demonstrated the functional role of this interaction showing that two specific Egr-1 target genes, the PTEN phosphatase and the thymidine kinase (TK) genes promoters, are activated by contransfection of APE/Ref-1. Interestingly, by using a cell transfection approach and Chromatin immunoprecipitation assays, we were able to demonstrate that Egr-1 stimulates the transcriptional activity of APE/Ref-1 gene promoter by a direct interaction with specific DNA-binding site on its promoter. Taken together, our data delineate a new molecular mechanism of Egr-1 activation occurring soon after H(2)O(2) stimulation in osteoblastic cells and suggest a model for a positive loop between APE/Ref-1 and Egr-1 that could explain the early transcriptional activation of APE/Ref-1 gene expression.
Pines
Alex
A
Dipartimento di Biochimica, Biofisica e Chimica delle Macromolecole, University of Trieste, via Giorgieri 1, 34127 Trieste, Italy.
Bivi
Nicoletta
N
Romanello
Milena
M
Damante
Giuseppe
G
Kelley
Mark R
MR
Adamson
Eileen D
ED
D'Andrea
Paola
P
Quadrifoglio
Franco
F
Moro
Luigi
L
Tell
Gianluca
G
D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D049109:Cell Proliferation; D047369:Chromatin Immunoprecipitation; D004247:DNA / Q000378:metabolism; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D051766:Early Growth Response Protein 1; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D051059:PTEN Phosphohydrolase; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D013937:Thymidine Kinase / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; D017874:Immediate-Early Proteins; D016877:Oxidants; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D004247:DNA; D006861:Hydrogen Peroxide; D013937:Thymidine Kinase; D010744:Phosphoric Monoester Hydrolases; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase