Cloning and characterization of human oncostatin M promoter
Y Ma1, R J Streiff, J Liu
1Department of Veterans Affairs Medical Center, Boise, ID 83702, USA.
Abstract:
Oncostatin M (OSM), an IL-6 subfamily cytokine, inhibits proliferation and causes morphological changes in many tumor cell lines. GM-CSF, phorbol-12-myristate-13-acetate (PMA), and lipopolysaccharide (LPS) induce OSM expression. To investigate the mechanisms governing OSM promoter activity, we have cloned and partially sequenced an 8.5 kb fragment of human genomic DNA immediately 5' of the OSM coding region and mapped the transcription start site. Transient transfection assays with a series of 5' deletion plasmids demonstrated maximal reporter activity in U937 cells with a minimum 304 bp construct. The 5'-proximal region of the human OSM gene contains a C/EBP consensus element around -45 bp and several GC-rich regions around -60, each of which is responsible for basal promoter activity. Electrophoretic mobility shift assay coupled with supershift analysis confirmed the presence of a cis -acting binding site for activated STAT5 complexes following GM-CSF treatment. Furthermore, transient transfection studies demonstrated a loss of GM-CSF responsiveness in reporter constructs containing mutations within this STAT element. Our results establish that C/EBP and an as yet unidentified GC-rich binding transcription factor are responsible for basal OSM promoter activity, while GM-CSF-stimulated OSM expression is driven by activated STAT5 complexes binding to a cis -acting STAT element on the OSM promoter.
Insights
Researchers identified key factors controlling Oncostatin M (OSM) gene activity. C/EBP and a GC-rich factor drive basal activity, while STAT5 activation by GM-CSF stimulates OSM expression in tumor cells.
Area of Science:
- Molecular Biology
- Immunology
- Cancer Research
Background:
- Oncostatin M (OSM), an IL-6 subfamily cytokine, impacts tumor cell proliferation and morphology.
- OSM expression is induced by various stimuli including GM-CSF, PMA, and LPS.
Purpose of the Study:
- To elucidate the regulatory mechanisms of the human OSM gene promoter.
- To identify transcription factors and DNA elements involved in basal and stimulated OSM expression.
Main Methods:
- Cloning and sequencing of the 5' flanking region of the human OSM gene.
- Transient transfection assays using deletion constructs and reporter genes.
- Electrophoretic mobility shift assays (EMSA) and supershift analysis.
Main Results:
- Maximal reporter activity was observed with a 304 bp construct, indicating a minimal promoter region.
- C/EBP and GC-rich elements are crucial for basal OSM promoter activity.
- GM-CSF stimulation leads to STAT5 binding to a specific cis-acting element, driving OSM expression.
Conclusions:
- Basal OSM promoter activity is mediated by C/EBP and an unidentified GC-rich binding factor.
- GM-CSF-induced OSM expression is dependent on STAT5 activation and binding to the OSM promoter.
- These findings provide insights into the transcriptional regulation of Oncostatin M in cancer contexts.
