Tgfbeta signaling directly induces Arf promoter remodeling by a mechanism involving Smads 2/3 and p38 MAPK.
We have investigated how the Arf gene product, p19(Arf), is activated by Tgfβ during mouse embryo development to better understand how this important tumor suppressor is controlled. Taking advantage of new mouse models, we provide genetic evidence that Arf lies downstream of Tgfβ signaling in cells arising from the Wnt1-expressing neural crest and that the anti-proliferative effects of Tgfβ depend on Arf in vivo. Tgfβ1, -2, and -3 (but not BMP-2, another member of the Tgfβ superfamily) induce p19(Arf) expression in wild type mouse embryo fibroblasts (MEFs), and they enhance Arf promoter activity in Arf(lacZ/lacZ) MEFs. Application of chemical inhibitors of Smad-dependent and -independent pathways show that SB431542, a Tgfβ type I receptor (TβrI) inhibitor, and SB203580, a p38 MAPK inhibitor, impede Tgfβ2 induction of Arf. Genetic studies confirm the findings; transient knockdown of Smad2, Smad3, or p38 MAPK blunt Tgfβ2 effects, as does Cre recombinase treatment of Tgfbr2(fl/fl) MEFs to delete Tgfβ receptor II. Chromatin immunoprecipitation reveals that Tgfβ rapidly induces Smads 2/3 binding and histone H3 acetylation at genomic DNA proximal to Arf exon 1β. This is followed by increased RNA polymerase II binding and progressively increased Arf primary and mature transcripts from 24 through 72 h, indicating that increased transcription contributes to p19(Arf) increase. Last, Arf induction by oncogenic Ras depends on p38 MAPK but is independent of TβrI activation of Smad 2. These findings add to our understanding of how developmental and tumorigenic signals control Arf expression in vivo and in cultured MEFs.
Zheng
Yanbin
Y
Department of Pediatrics, Section of Hematology/Oncology and Stem Cell Transplantation, The University of Chicago, Chicago, Illinois 60637, USA.
Zhao
Yi D
YD
Gibbons
Melissa
M
Abramova
Tatiana
T
Chu
Patricia Y
PY
Ash
John D
JD
Cunningham
John M
JM
Skapek
Stephen X
SX
D000818:Animals; D015153:Blotting, Western; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics* / Q000378:metabolism; D004622:Embryo, Mammalian / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D015870:Gene Expression / Q000235:genetics; D008297:Male; D051379:Mice; D057507:Mice, 129 Strain; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008822:Mice, Transgenic; D011401:Promoter Regions, Genetic / Q000235:genetics*; D034622:RNA Interference; D018125:Receptors, Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D051899:Smad2 Protein / Q000235:genetics / Q000378:metabolism*; D051900:Smad3 Protein / Q000235:genetics / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
C417743:Cdkn2a protein, mouse; D019941:Cyclin-Dependent Kinase Inhibitor p16; D004791:Enzyme Inhibitors; D018125:Receptors, Transforming Growth Factor beta; D051899:Smad2 Protein; C497783:Smad2 protein, mouse; D051900:Smad3 Protein; C497791:Smad3 protein, mouse; D016212:Transforming Growth Factor beta; D048051:p38 Mitogen-Activated Protein Kinases
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