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Okadaic acid stimulated TRE binding activity in a papilloma producing mouse keratinocyte cell line involves increased
1Department of Radiation Oncology, The University of Arizona Health Sciences Center, Tucson 85724, USA.
Abstract:
The effects of the non-phorbol ester type tumor promoter okadaic acid, a serine-threonine phosphatase inhibitor, on activator protein 1 (AP-1) DNA binding activity were studied in papilloma producing 308 mouse keratinocytes. Okadaic acid increased AP-1 binding to a consensus TPA responsive element (TRE) within 2 h; maximum stimulation was observed at 6 h followed by a gradual decrease to basal levels within 24 h. Jun B, Jun D and Fos B proteins were identified as the major components of the AP-1 complex binding to the TRE element at 6 h. Inhibition of transcription with actinomycin D and inhibition of protein synthesis with cycloheximide abrogated the okadaic acid effect on AP-1 DNA binding, indicating that transcription and translation are required for okadaic acid increased TRE binding activity. Northern and Western blot analyses revealed a correlation between increased AP-1 binding activity and accumulation of jun B, jun D and fos B mRNAs and proteins. These data suggest increased AP-1 expression as principal mechanism of okadaic acid stimulated AP-1 activation in the mouse keratinocytes studied.
Insights
Okadaic acid, a phosphatase inhibitor, activates activator protein 1 (AP-1) DNA binding in mouse keratinocytes. This activation requires new gene transcription and protein synthesis, primarily involving Jun B, Jun D, and Fos B proteins.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Okadaic acid is a non-phorbol ester tumor promoter.
- It acts as a serine-threonine phosphatase inhibitor.
- Activator protein 1 (AP-1) is a crucial transcription factor involved in cellular processes.
Purpose of the Study:
- To investigate the effects of okadaic acid on AP-1 DNA binding activity.
- To identify the specific components of the AP-1 complex induced by okadaic acid.
- To elucidate the molecular mechanisms underlying okadaic acid-induced AP-1 activation.
Main Methods:
- Treatment of 308 mouse keratinocytes with okadaic acid.
- Electrophoretic mobility shift assays (EMSAs) to measure AP-1 DNA binding.
- Inhibition studies using actinomycin D (transcription inhibitor) and cycloheximide (protein synthesis inhibitor).
- Northern and Western blot analyses to assess mRNA and protein levels of AP-1 components.
Main Results:
- Okadaic acid significantly increased AP-1 DNA binding activity within 2 hours, peaking at 6 hours.
- Jun B, Jun D, and Fos B proteins were identified as key components of the activated AP-1 complex.
- Inhibition of transcription and protein synthesis blocked the okadaic acid-induced increase in AP-1 binding.
- Increased AP-1 binding correlated with elevated levels of jun B, jun D, and fos B mRNAs and proteins.
Conclusions:
- Okadaic acid stimulates AP-1 activation in mouse keratinocytes.
- This activation is dependent on de novo gene transcription and protein synthesis.
- Increased expression of Jun B, Jun D, and Fos B is the principal mechanism for okadaic acid-induced AP-1 activation.