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Platelet-derived growth factor B-chain gene expression in mesangial cells: effect of phorbol ester on gene
B Bhandari1, K Woodruff, H E Abboud
1Department of Medicine, University of Texas Health Science Center at San Antonio.
Abstract:
We have investigated the effect of phorbol 12-myristate 13-acetate (PMA) on platelet-derived growth factor (PDGF) B-chain gene transcription as well as on mRNA stability in cultured human mesangial cells. Addition of actinomycin to cells stimulated with PMA decreases steady state levels of PDGF-B chain mRNA analysed by solution hybridization assay. PDGF-B chain gene transcription was also assayed directly by measuring elongation of transcripts in isolated nuclei followed by hybridization of labeled RNA transcripts to a cDNA encoding for PDGF-B chain. Our data show that PMA induces PDGF-B chain gene transcription by approximately 2-fold. alpha-Amanitin, an RNA polymerase II inhibitor, blocked transcription by more than 70%. In addition, we determined the effect of PMA on the halflife of PDGF-B chain mRNA directly by pulse chase method. In human mesangial cells, the PDGF-B chain mRNA exhibited halflife of approximately 105 min. In the presence of PMA, the halflife of PDGF-B chain mRNA was reduced to approximately 72 min. These studies indicate that regulation of PDGF-B chain gene by PMA in human mesangial cells involves a coordinate effort at the level of transcription and mRNA stability.
Insights
Phorbol 12-myristate 13-acetate (PMA) influences platelet-derived growth factor (PDGF) B-chain gene expression in human mesangial cells. PMA increases PDGF-B gene transcription and reduces mRNA stability, impacting gene regulation.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- Platelet-derived growth factor (PDGF) plays a crucial role in cellular processes.
- Understanding the regulation of PDGF-B chain gene expression is vital in human mesangial cells.
Purpose of the Study:
- To investigate the impact of phorbol 12-myristate 13-acetate (PMA) on PDGF-B chain gene transcription.
- To determine the effect of PMA on PDGF-B chain mRNA stability in human mesangial cells.
Main Methods:
- Solution hybridization assay to analyze mRNA levels.
- Isolated nuclei assay to measure gene transcription.
- Pulse-chase method to determine mRNA half-life.
- Inhibition studies using actinomycin and alpha-amanitin.
Main Results:
- PMA treatment increased PDGF-B chain gene transcription by approximately 2-fold.
- Alpha-amanitin significantly inhibited PMA-induced transcription, indicating RNA polymerase II involvement.
- PMA reduced the half-life of PDGF-B chain mRNA from approximately 105 minutes to 72 minutes.
Conclusions:
- PMA coordinately regulates PDGF-B chain gene expression at both transcriptional and post-transcriptional (mRNA stability) levels.
- These findings elucidate a novel regulatory mechanism of PDGF-B chain in human mesangial cells.