Rapid expression of ornithine decarboxylase mRNA in a macrophage-like cell line: cAMP repression of the requirement for prior protein synthesis.
Ornithine decarboxylase (ODC) activity was rapidly induced in the RAW264 macrophage-like cell line after treatment with bacterial lipopolysaccharide (LPS). ODC mRNA levels were determined by isolating cellular RNA, followed by Northern blot and dot blot analysis using a 32P-labeled cDNA probe. ODC mRNA levels increased within 1 hour of stimulation of RAW264 cells with 1.0 microgram/ml LPS. Transcription rate analysis on isolated nuclei indicated that an increase in transcription rate contributed to this increase in ODC mRNA. ODC mRNA levels continued to rise for 4 hours, peaking at eight times the basal level. ODC mRNA appeared as a single 2.2-kb band prior to stimulation. After stimulation, the 2.2-kb band intensified, and a second (2.7 kb) band was seen by Northern gel analysis. Similar induction was demonstrated when 12-O-tetradecanoyl-phorbol-13-acetate (TPA) was used as the stimulus. The induction of ODC mRNA by either LPS or TPA was blocked by the addition of cycloheximide (25 micrograms/ml) or anisomycin (0.1 mM) to the cellular incubation mixture. This indicated that protein synthesis was required as a prerequisite to LPS or TPA induction of ODC mRNA. Experiments in which cycloheximide addition was delayed after LPS treatment indicated that some of the required protein synthesis occurred within the first 30 minutes and that complete expression of ODC mRNA was possible if protein synthesis continued for at least 2 hours before cycloheximide was added. Stimulation with 8-bromo-cAMP in addition to LPS has been shown to enhance the induction of ODC over that induced by LPS or TPA alone. It was not possible to block ODC mRNA induction with cycloheximide or anisomycin after treatment with the combined stimulus of LPS and cAMP or TPA and cAMP, indicating that protein synthesis was not required when cAMP was used as a coinducer. Thus, we have shown that in the same cell, ODC mRNA can be induced by two different pathways, one requiring protein synthesis and one not requiring protein synthesis.
Shurtleff
S A
SA
Department of Microbiology and Immunology, SUNY Health Science Center, Syracuse 13210.
McElwain
C M
CM
Taffet
S M
SM
D000841:Anisomycin / Q000494:pharmacology; D002461:Cell Line, Transformed; D000242:Cyclic AMP / Q000494:pharmacology*; D003513:Cycloheximide / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000201:enzymology*; D009693:Nucleic Acid Hybridization; D009955:Ornithine Decarboxylase / Q000096:biosynthesis / Q000235:genetics*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors; D014158:Transcription, Genetic
D008070:Lipopolysaccharides; D012333:RNA, Messenger; D000841:Anisomycin; D003513:Cycloheximide; D000242:Cyclic AMP; D009955:Ornithine Decarboxylase; D013755:Tetradecanoylphorbol Acetate