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TGF-beta1 modulates focal adhesion kinase expression in rat intestinal epithelial IEC-6 cells via stimulatory and

Mary F Walsh1, Dinakar R Ampasala, Arun K Rishi

  • 1Department of Surgery, Wayne State University, Detroit 48201-1932, USA.

Biochimica Et Biophysica Acta
|December 9, 2008
PubMed

Insights

Transforming growth factor-beta (TGF-beta) regulates Focal Adhesion Kinase (FAK) gene transcription in intestinal epithelial cells through complex Smad and non-Smad signaling pathways. Key regulatory elements and signaling molecules were identified, revealing intricate control mechanisms.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Transforming growth factor-beta (TGF-beta) and Focal Adhesion Kinase (FAK) are critical regulators of intestinal epithelial cell functions, including migration, differentiation, proliferation, and apoptosis.
  • TGF-beta is known to promote FAK transcription in these cells via both Smad-dependent and independent pathways.

Purpose of the Study:

  • To characterize the basal and TGF-beta-mediated FAK gene transcription in IEC-6 cells using a FAK promoter-luciferase construct.
  • To elucidate the roles of Smad and non-Smad signaling pathways, including JNK, Akt, ERK, p53, and AML1a, in regulating FAK transcription.

Main Methods:

  • Utilized a 1320 bp FAK promoter-luciferase construct in IEC-6 cells.
  • Employed inhibitor studies targeting JNK, Akt, and ERK signaling pathways.
  • Performed co-transfection with Smad4 and Smad7.
  • Conducted serial promoter deletions and mutational analyses of Smad binding elements (SBE) and p53 sites.
  • Applied Chromatin Immunoprecipitation (ChIP) assays to assess DNA binding of key proteins.

Main Results:

  • Inhibition of JNK or Akt abolished TGF-beta-stimulated FAK promoter activity, while ERK inhibition increased basal activity.
  • Co-transfection with Co-Smad4 enhanced the TGF-beta response, and Smad7 abolished it.
  • The two most distal SBEs were identified as positive regulators, and SBE3 as a negative regulator of FAK transcription.
  • Mutational deletion of p53 sites enhanced basal activity but did not affect TGF-beta-stimulated increases.
  • ChIP assays confirmed TGF-beta-induced DNA binding of Smad4, phospho-Smad2/3, and Runx1/AML1a to the promoter region.
  • Point mutation of SBE1, but not AML1a sites, ablated the TGF-beta-mediated increase in promoter activity.

Conclusions:

  • TGF-beta regulation of FAK transcription in intestinal epithelial cells is a complex process involving intricate interactions between Smad and non-Smad signaling pathways.
  • Specific Smad binding elements (SBEs) and non-Smad factors like AML1a play crucial roles, with SBE1 being essential for the TGF-beta response.
  • The regulatory mechanism is independent of p53 and AML1a binding sites for the TGF-beta-induced transcriptional increase.

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