TGF-beta1 modulates focal adhesion kinase expression in rat intestinal epithelial IEC-6 cells via stimulatory and inhibitory Smad binding elements.
TGF-beta and FAK modulate cell migration, differentiation, proliferation and apoptosis, and TGF-beta promotes FAK transcription in intestinal epithelial cells via Smad-dependent and independent pathways. We utilized a 1320 bp FAK promoter-luciferase construct to characterize basal and TGF-beta-mediated FAK gene transcription in IEC-6 cells. Inhibiting JNK or Akt negated TGF-beta-stimulated promoter activity; ERK inhibition did not block the TGF-beta effect but increased basal activity. Co-transfection with Co-Smad4 enhanced the TGF-beta response while the inhibitory Smad7 abolished it. Serial deletions sequentially removing the four Smad binding elements (SBE) in the 5' untranslated region of the promoter revealed that the two most distal SBE's are positive regulators while SBE3 exerts a negative influence. Mutational deletion of two upstream p53 sites enhanced basal but did not affect TGF-beta-stimulated increases in promoter activity. TGF-beta increased DNA binding of Smad4, phospho-Smad2/3 and Runx1/AML1a to the most distal 435 bp containing 3 SBE and 2 AML1a sites by ChIP assay. However, although point mutation of SBE1 ablated the TGF-beta-mediated rise in SV40-promoter activity, mutation of AML1a sites did not. TGF-beta regulation of FAK transcription reflects a complex interplay between positive and negative non-Smad signals and SBE's, the last independent of p53 or AML1a.
Walsh
Mary F
MF
Department of Surgery, Wayne State University, Detroit 48201-1932, USA.
Ampasala
Dinakar R
DR
Rishi
Arun K
AK
Basson
Marc D
MD
D020121:5' Untranslated Regions / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D004847:Epithelial Cells / Q000378:metabolism*; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D007422:Intestines / Q000166:cytology*; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D051381:Rats; D051785:Smad Proteins / Q000235:genetics / Q000378:metabolism*; D051901:Smad4 Protein / Q000235:genetics / Q000378:metabolism; D051906:Smad7 Protein / Q000235:genetics / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*
D020121:5' Untranslated Regions; D051785:Smad Proteins; D051901:Smad4 Protein; D051906:Smad7 Protein; D053773:Transforming Growth Factor beta1; D051416:Focal Adhesion Protein-Tyrosine Kinases