Granulocyte-macrophage colony-stimulating factor expression is regulated at transcriptional and posttranscriptional levels in a murine bone marrow stromal cell line.
We have reported modulation, by cytokines interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) and by hormonal cyclic-adenosine-monophosphate (cAMP) agonists, of hematopoietic growth factor production in the murine marrow adherent cell line +/+(-)1.LDA11. Previously, we reported that increased intracellular cAMP levels inhibited bioactive granulocyte-macrophage colony-stimulatory factor (GM-CSF) production stimulated by IL-1 or by the synergistic stimulus of IL-1 plus TNF-alpha. On the other hand, increased intracellular cAMP stimulated IL-6 synthesis in +/+(-)1.LDA11 cells. In addition, cAMP was additive with either IL-1 or IL-1 plus TNF-alpha in inducing production of soluble IL-6. In the present study, these observations were pursued mechanistically at the level of messenger RNA (mRNA) production. Northern blot analysis of steady-state mRNA for GM-CSF revealed induction by treatment of +/+(-)1.LDA11 cells with IL-1 or with TNF-alpha. The combined stimulation by IL-1 plus TNF-alpha resulted in supra-additive increases in GM-CSF expression by +/+(-)1.LDA11. Addition to stromal cells of the soluble cAMP agonist 8-bromo-cAMP (8BrcAMP) at 0.5 to 1 mM stimulated IL-6 mRNA expression acting alone, and it was additive with IL-1 or IL-1 plus TNF-alpha in stimulating IL-6 expression. On the other hand, 8BrcAMP inhibited GM-CSF mRNA expression stimulated by IL-1 or IL-1 plus TNF-alpha. Inhibition of GM-CSF mRNA by 8BrcAMP was time-dependent, starting 120 to 180 minutes posttreatment. In addition, inhibition of GM-CSF transcript expression in +/+(-)1.LDA11 by 8BrcAMP required the expression of a labile protein. Nuclear run-on assays revealed that GM-CSF and IL-6 genes were transcriptionally induced in +/+(-)1.LDA11 by incubation with IL-1 plus TNF-alpha. IL-6 transcription was further enhanced by 8BrcAMP co-incubation. More sensitive experiments using a luciferase reporter vector containing the GM-CSF promoter region were necessary to convincingly establish the role of TNF-alpha and 8BrcAMP on transcriptional induction of the GM-CSF gene in +/+(-)1.LDA11 stromal cells. Considering these results and an effect of 8BrcAMP on decreasing GM-CSF transcript stability in actinomycin-D (act-D) decay experiments, we conclude that the inhibitory effect of 8BrcAMP on GM-CSF expression is exerted at the posttranscriptional level. These data demonstrate that the intracellular level of cAMP has an important discriminatory role on expression of the cytokines GM-CSF and IL-6 in a model stromal cell line.
Derigs
H G
HG
Division of Hematology/Oncology, Indiana University School of Medicine, Indianapolis.
Reifel-Miller
A
A
Kaushansky
K
K
Hromas
R A
RA
Boswell
H S
HS
D000818:Animals; D015152:Blotting, Northern; D001853:Bone Marrow / Q000378:metabolism / Q000502:physiology*; D001854:Bone Marrow Cells; D002460:Cell Line; D000242:Cyclic AMP / Q000032:analysis / Q000378:metabolism / Q000502:physiology; D015536:Down-Regulation / Q000187:drug effects; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D007375:Interleukin-1 / Q000494:pharmacology; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D051379:Mice; D011401:Promoter Regions, Genetic / Q000235:genetics; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D013997:Time Factors; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D007375:Interleukin-1; D015850:Interleukin-6; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D000242:Cyclic AMP