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


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.
Mithani
Suhail K
SK
Department of Surgery, Vanderbilt University Medical Center, Nashville, TN 37232-2730, USA.
Balch
Glen C
GC
Shiou
Sheng-Ru
SR
Whitehead
Robert H
RH
Datta
Pran K
PK
Beauchamp
R Daniel
RD
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D002455:Cell Division / Q000502:physiology; D002460:Cell Line; D003106:Colon / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000172:deficiency / Q000502:physiology*; D006131:Growth Inhibitors / Q000502:physiology*; D007413:Intestinal Mucosa / Q000502:physiology*; D051379:Mice; D008822:Mice, Transgenic; D009154:Mutation; D011401:Promoter Regions, Genetic / Q000502:physiology; D051900:Smad3 Protein; D015534:Trans-Activators / Q000172:deficiency / Q000502:physiology*; D016212:Transforming Growth Factor beta / Q000502:physiology*
D004268:DNA-Binding Proteins; D006131:Growth Inhibitors; D051900:Smad3 Protein; C497791:Smad3 protein, mouse; D015534:Trans-Activators; D016212:Transforming Growth Factor beta