Analysis and quantitation of splicing variants of the TPA-inducible PGHS-1 mRNA in rat tracheal epithelial cells.
Previously we reported that 12-myristate 13-acetate diester (TPA) and dexamethasone regulate the expression of a putative prostaglandin G/H synthase-1 (PGHS-1) gene in a transformed, immortalized rat tracheal epithelial cell line (EGV-6). Here we report the cloning and sequencing of the cDNA for this gene. Two transcripts of similar size but differing in their 5' ends were detected. One transcript contains the complete 5' coding and noncoding regions, while the other form has an apparently intronic sequence in place of these regions. This aberrantly spliced form lacks codons 1-36. Northern analysis and quantitative PCR indicated that more than 90% of the PGHS-1 mRNA in EGV-6 cells has the aberrantly spliced 5' end. TPA treatment increases the expression of only the complete PGHS-1 mRNA, elevating it to 50% of the total PGHS-1 transcript pool. Levels of the aberrant mRNA are not affected by TPA treatment. A comparison among EGV-6 cells, primary rat tracheal epithelial (RTE) cultures, and rat fibroblasts showed that all three cell lines have similar levels of the aberrantly spliced PGHS-1 mRNA. RTE cells and fibroblasts, unlike EGV-6 cells, also contain properly spliced PGHS-1 mRNA, at levels 100-fold higher than the level of aberrant PGHS-1 mRNA. TPA does not regulate either of the PGHS-1 transcripts in RTE or rat fibroblast cells. These results confirm the induction of functional PGHS-1 mRNA by TPA only in EGV-6 cells.
Kitzler
J
J
Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709.
Hill
E
E
Hardman
R
R
Reddy
N
N
Philpot
R
R
Eling
T E
TE
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004847:Epithelial Cells; D005784:Gene Amplification; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D014644:Genetic Variation; D016678:Genome; D008969:Molecular Sequence Data; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics*; D012326:RNA Splicing / Q000235:genetics*; D051381:Rats; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014132:Trachea / Q000166:cytology / Q000378:metabolism*
D011451:Prostaglandin-Endoperoxide Synthases; D013755:Tetradecanoylphorbol Acetate