Smad3 has a critical role in TGF-beta-mediated growth inhibition and apoptosis in colonic epithelial cells

J Surg Res. 2004 Apr;117(2):296-305. doi: 10.1016/S0022-4804(03)00335-4.

Abstract

Background: Smad proteins play a key role in TGF-beta signaling that regulates cell proliferation, differentiation, and apoptosis. Mice deficient in Smad3 develop colonic adenocarcinoma.

Materials and methods: We developed a Smad3-deficient colonocyte cell line that was used to study TGF-beta-mediated growth inhibition and induction of apoptosis was compared to young adult mouse colonocyte (YAMC) control cells. Growth inhibition was assessed by cell count and ((3)H)-thymidine incorporation assay. Transcriptional response to TGF-beta was measured by transfecting the reporters p3TP-Lux and p(CAGA)(9)-MLP-luc. TGF-beta-induced apoptosis was assessed using ELISA and Hoechst staining. Mediators of cell-cycle arrest and apoptosis were assayed by Western blot.

Results: Smad3-/- cells were resistant to TGF-beta-mediated growth inhibition compared to control cells. Ninety-eight percent of cell count growth inhibition observed in YAMC cells, while 34% inhibition was observed in Smad3-/- cells after TGF-beta treatment. ((3)H)-thymidine incorporation was inhibited by 61% in YAMC cells, while Smad3-/- cells showed 25% inhibition after TGF-beta treatment. Smad3-/- cells were deficient in luciferase reporter induction by TGF-beta. TGF-beta induced apoptosis 8-fold in YAMC cells, but had no effect on apoptosis in Smad3-/- cells. p21(Cip11) and PAI-1 are induced in YAMC cells by TGF-beta, but unchanged in Smad3-/- cells. TGF-beta decreases cyclin D1 levels in YAMC cells but does not affect levels in Smad3-/- cells.

Conclusions: Our findings suggest that the loss of Smad3 contributes to resistance of TGF-beta growth inhibition and apoptosis in colonic epithelium. This may represent a mechanism by which cells are able to escape antiproliferative controls and embark on a pathway toward neoplasia.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Apoptosis / physiology*
  • Cell Division / physiology
  • Cell Line
  • Colon / physiology*
  • DNA-Binding Proteins / deficiency
  • DNA-Binding Proteins / physiology*
  • Growth Inhibitors / physiology*
  • Intestinal Mucosa / physiology*
  • Mice
  • Mice, Transgenic
  • Mutation
  • Promoter Regions, Genetic / physiology
  • Smad3 Protein
  • Trans-Activators / deficiency
  • Trans-Activators / physiology*
  • Transforming Growth Factor beta / physiology*

Substances

  • DNA-Binding Proteins
  • Growth Inhibitors
  • Smad3 Protein
  • Smad3 protein, mouse
  • Trans-Activators
  • Transforming Growth Factor beta