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Mechanism of c-fos induction by active oxygen
P A Amstad1, G Krupitza, P A Cerutti
1Department of Carcinogenesis, Swiss Institute for Experimental Cancer Research, Lausanne.
Cancer Research
|July 15, 1992
Summary
Active oxygen, serum, and phorbol ester all induce c-fos transcription via shared regulatory elements. However, active oxygen uniquely requires poly-ADP-ribosylation for c-fos induction, potentially linked to DNA repair.
Area of Science:
- Molecular Biology
- Cell Signaling
- Gene Regulation
Background:
- The c-fos gene is a key immediate-early gene involved in cellular responses to various stimuli.
- Understanding the distinct signaling pathways that converge on c-fos regulation is crucial for deciphering cellular responses to inflammation and other stresses.
Purpose of the Study:
- To compare the transcriptional induction mechanisms of the c-fos gene by active oxygen, serum, and phorbol ester in mouse epidermal cells.
- To investigate the role of poly-ADP-ribosylation in c-fos gene induction by these different stimuli.
Main Methods:
- Utilized mouse epidermal JB6 cells (clone 30) stimulated with active oxygen, serum, or phorbol ester.
- Employed protein kinase and synthesis inhibitors (H7, cycloheximide) to probe signaling pathways.
- Constructed stable transfectants with fos 5' upstream regulatory sequences linked to a reporter gene (HSV-tk-chloramphenicol-acetyl-transferase).
- Assessed poly-ADP-ribosylation using specific inhibitors (benzamide, 3-amino-benzamide) and analyzed protein-DNA interactions via electrophoretic mobility shift assays.
Main Results:
- All three inducers (active oxygen, serum, phorbol ester) triggered c-fos induction, inhibited by H7 and enhanced by cycloheximide.
- The joint dyad symmetry element-AP-1 motifs in the fos 5' upstream region were critical for induction by active oxygen and serum.
- Only active oxygen induction of c-fos necessitated poly-ADP-ribosylation, suppressing c-fos mRNA elongation and the synthesis of transcription factors like c-Fos and c-Jun.
- Active oxygen, unlike serum or phorbol ester, induced DNA breakage, suggesting a role for poly-ADP-ribosylation in DNA repair during transcription.
Conclusions:
- Signal transduction pathways for active oxygen, serum, and phorbol ester converge on common 5' regulatory sequences of the c-fos gene.
- Poly-ADP-ribosylation is a unique requirement for active oxygen-mediated c-fos induction, likely involved in repairing DNA breaks that impede transcription.
- While Fos protein modification by poly-ADP-ribosylation was observed, its functional significance remains to be elucidated.