Platelet-derived growth factor stimulates Src-dependent mRNA stabilization of specific early genes in fibroblasts.
The Src family of protein-tyrosine kinases (SFKs) participates in a variety of signal transduction pathways, including promotion of cell growth, prevention of apoptosis, and regulation of cell interactions and motility. In particular, SFKs are required for the mitogenic response to platelet-derived growth factor (PDGF). However, it is not clear whether there is a discrete SFK-specific pathway leading to enhanced gene expression or whether SFKs act to generally enhance PDGF-stimulated gene expression. To examine this, we treated quiescent NIH3T3 cells with PDGF in the presence or absence of small molecule inhibitors of SFKs, phosphatidylinositol 3-kinase (PI3K), and MEK1/2. Global patterns of gene expression were analyzed by using Affymetrix Gene-Chip arrays, and data were validated by using reverse transcription-PCR and ribonuclease protection assay. We identified a discrete set of immediate early genes induced by PDGF and inhibited in the presence of the SFK-selective inhibitor SU6656. A subset of these SFK-dependent genes was induced by PDGF even in the presence of the MEK1/2 inhibitor U0126 or the PI3K inhibitor LY294002. By using ribonuclease protection assays and nuclear run-off assays, we further determined that PDGF did not stimulate the rate of transcription of these SFK-dependent immediate early genes but rather promoted mRNA stabilization. Our data suggest that PDGF regulates gene expression through an SFK-specific pathway that is distinct from the Ras-MAPK and PI3K pathways, and that SFKs signal gene expression by enhancing mRNA stability.
Bromann
Paul A
PA
Laboratory of Signal Regulation and Cancer, Van Andel Research Institute, 333 Bostwick NE, Grand Rapids, Michigan 49503, USA.
Korkaya
Hasan
H
Webb
Craig P
CP
Miller
Jeremy
J
Calvin
Tammy L
TL
Courtneidge
Sara A
SA
D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D002070:Butadienes / Q000494:pharmacology; D002465:Cell Movement; D002467:Cell Nucleus / Q000378:metabolism; D049109:Cell Proliferation; D002867:Chromones / Q000494:pharmacology; D016000:Cluster Analysis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism*; D007211:Indoles / Q000494:pharmacology; D048369:MAP Kinase Kinase 1 / Q000378:metabolism; D048370:MAP Kinase Kinase 2 / Q000378:metabolism; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism; D051379:Mice; D009025:Morpholines / Q000494:pharmacology; D041681:NIH 3T3 Cells; D009570:Nitriles / Q000494:pharmacology; D020411:Oligonucleotide Array Sequence Analysis; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010982:Platelet-Derived Growth Factor / Q000378:metabolism*; D011485:Protein Binding; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012260:Ribonucleases / Q000378:metabolism; D015398:Signal Transduction; D013449:Sulfonamides / Q000494:pharmacology; D013997:Time Factors; D014158:Transcription, Genetic; D019061:src-Family Kinases / Q000378:metabolism*
D002070:Butadienes; D002867:Chromones; D004791:Enzyme Inhibitors; D007211:Indoles; D009025:Morpholines; D009570:Nitriles; D010982:Platelet-Derived Growth Factor; D012333:RNA, Messenger; C416927:SU 6656; D013449:Sulfonamides; C113580:U 0126; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D012313:RNA; D019869:Phosphatidylinositol 3-Kinases; D019061:src-Family Kinases; D020930:MAP Kinase Kinase Kinases; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; C482129:Map2k2 protein, mouse; D012260:Ribonucleases