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Regulation of c-jun expression during induction of monocytic differentiation by okadaic acid
S Kharbanda1, R Datta, E Rubin
1Laboratory of Clinical Pharmacology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115.
Abstract:
The present work has examined the effects of okadaic acid, an inhibitor of type 1 and 2A protein phosphatases, on the regulation of c-jun expression during monocytic differentiation of U-937 leukemia cells. The results demonstrate that okadaic acid treatment is associated with induction of a differentiated monocyte phenotype characterized by: (a) growth arrest; (b) increases in Mac-1 cell surface antigen expression; (c) down-regulation of c-myc transcripts; and (d) induction of tumor necrosis factor gene expression. This induction of monocytic differentiation was associated with transient increases in c-jun mRNA levels, which were maximal at 6 h. Similar effects were obtained for the c-fos gene. Run-on analysis demonstrated detectable levels of c-jun transcription in U-937 cells and that this rate is increased approximately 40-fold following okadaic acid exposure. c-jun mRNA levels were superinduced in cells treated with both okadaic acid and cycloheximide, whereas inhibition of protein synthesis had little, if any, effect on okadaic acid-induced c-jun transcription. The half-life of c-jun mRNA was similar (45-50 min) in both untreated and okadaic acid-induced cells. In contrast, treatment with both okadaic acid and cycloheximide was associated with stabilization (t 1/2 = 90 min) of c-jun transcripts. Taken together, these findings indicate that the induction of c-jun transcription by okadaic acid is controlled primarily by a transcriptional mechanism. Since previous studies have demonstrated that the c-jun gene is autoinduced by Jun/AP-1, we also studied transcription of c-jun promoter (positions -132/+170)-reporter gene constructs with and without a mutated AP-1 element.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Okadaic acid induces monocytic differentiation in U-937 cells by increasing c-jun transcription, primarily through a transcriptional mechanism, not mRNA stabilization.
Area of Science:
- Cell Biology
- Molecular Biology
- Cancer Research
Background:
- U-937 leukemia cells are a model for studying monocytic differentiation.
- Protein phosphatases play critical roles in cellular signaling pathways.
- c-jun gene expression is regulated during cellular differentiation.
Purpose of the Study:
- To investigate the effects of okadaic acid, a protein phosphatase inhibitor, on c-jun expression during monocytic differentiation.
- To elucidate the regulatory mechanisms controlling c-jun gene induction.
Main Methods:
- Treatment of U-937 cells with okadaic acid.
- Analysis of cell surface antigen expression (Mac-1).
- Quantification of c-jun and c-myc mRNA levels.
- Run-on transcription assays.
- Reporter gene assays to study promoter activity.
Main Results:
- Okadaic acid induced monocytic differentiation markers (growth arrest, Mac-1 expression, TNF gene induction).
- Transient increases in c-jun mRNA levels were observed, peaking at 6 hours.
- Okadaic acid treatment increased c-jun transcription approximately 40-fold.
- c-jun transcription induction was primarily transcriptional, with minimal effect from protein synthesis inhibition.
- c-jun mRNA half-life was not significantly altered by okadaic acid alone but was stabilized by co-treatment with cycloheximide.
Conclusions:
- Okadaic acid effectively induces monocytic differentiation in U-937 cells.
- The induction of c-jun transcription by okadaic acid is predominantly controlled at the transcriptional level.
- Further studies are needed to fully understand the role of AP-1 elements in c-jun autoinduction during this process.