TGF-beta 1-induced PAI-1 expression is E box/USF-dependent and requires EGFR signaling.
Transforming growth factor-beta1 (TGF-beta1) transcriptionally regulates the expression of genes that encode specific proteins (e.g., plasminogen activator inhibitor-1; PAI-1) important in stromal remodeling and cellular invasion. Definition of molecular events underlying TGF-beta1-initiated PAI-1 transcription, therefore, may lead to the identification of new therapeutic targets for diseases associated with elevated PAI-1 synthesis (e.g., tissue fibrosis, vascular disorders, tumor progression). An intact upstream stimulatory factor (USF)-binding E box motif (5'-(-165)CACGTG(-160)-3') at the HRE-2 site in the rat PAI-1 gene was required for PAI-1 transcription in TGF-beta1-treated cells. Mutation of the CA dinucleotide to TC at position -165/-164 in a reporter construct driven by 764 bp of PAI-1 promoter sequence decreased TGF-beta1-dependent CAT activity by >80% indicating the necessity for a consensus hexanucleotide E box motif in induced expression. The same CA --> TC substitution eliminated USF binding to an 18-bp HRE-2 DNA target highlighting the importance of site occupancy to transcriptional activation. Transfection of a dominant-negative USF construct, moreover, completely inhibited formation of USF/HRE-2 probe complexes, attenuated PAI-1 promoter-driven luciferase activity and reduced the response of the endogenous PAI-1 gene to TGF-beta1 (to that approximating quiescent controls). Maximal immediate-early PAI-1 induction upon exposure to TGF-beta1 required EGFR, p21ras, MEK and pp60(c-src) signaling as pharmacologic or dominant-negative inhibition of any of the four intermediates (EGFR, p21ras, MEK, pp60(c-src)) virtually eliminated TGF-beta1-augmented PAI-1 levels. U0126 titering experiments, furthermore, revealed that the same MEK inhibitor concentration that blocked the TGF-beta1 increase in ERK1/2 phosphorylation (20 microM) also effectively attenuated the PAI-1 inductive response suggesting a requirement for stimulated ERK signaling in TGF-beta1-mediated PAI-1 expression. These data suggest a model whereby TGF-beta1 activates a complex signaling cascade to affect PAI-1 gene control and involves USF occupancy of a critical E box motif at the HRE-2 site in the PAI-1 gene.
Kutz
Stacie M
SM
Center for Cell Biology and Cancer Research, Albany Medical College, MC-165, 47 New Scotland Avenue, Albany, NY 12208, USA.
Higgins
Craig E
CE
Samarakoon
Rohan
R
Higgins
Stephen P
SP
Allen
Rosalie R
RR
Qi
Li
L
Higgins
Paul J
PJ
D000818:Animals; D002460:Cell Line; D024721:E-Box Elements / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000502:physiology*; D006801:Humans; D020930:MAP Kinase Kinase Kinases / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D017395:Plasminogen Activator Inhibitor 1 / Q000096:biosynthesis* / Q000235:genetics; D011401:Promoter Regions, Genetic; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000502:physiology; D011799:Quinazolines; D051381:Rats; D015398:Signal Transduction / Q000502:physiology*; D016212:Transforming Growth Factor beta / Q000502:physiology*; D053773:Transforming Growth Factor beta1; D020032:Tyrphostins / Q000494:pharmacology
D004791:Enzyme Inhibitors; D017395:Plasminogen Activator Inhibitor 1; D011799:Quinazolines; C508960:TGFB1 protein, human; C508962:Tgfb1 protein, rat; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D020032:Tyrphostins; C101044:RTKI cpd; D066246:ErbB Receptors; D016392:Proto-Oncogene Proteins pp60(c-src); D020930:MAP Kinase Kinase Kinases