Abstract
Transforming growth factor beta (TGF-beta) regulates many biological processes, and aberrant TGF-beta signaling is implicated in tumor development. Smad3 is a central component of the TGF-beta signaling pathway, and once activated, Smad3 forms complexes with Smad4 or other receptor-regulated Smads, which accumulate in the nucleus to transcriptionally regulate TGF-beta target genes. Because Smad3 plays a significant role in mediating the activities of TGF-beta, we examined its regulation during tumor development using a well characterized tumor model. We demonstrate that Smad3 levels are dramatically reduced in the tumorigenic cell line transformed with activated H-Ras compared with the normal parental epithelial cells. Interestingly, we also observe a cell cycle-dependent regulation of Smad3 in both cell types, with high Smad3 levels in quiescent cells and a significant drop in Smad3 protein levels in proliferating cells. Smad3 is regulated at the mRNA level and at the level of protein stability. In addition, functional analysis indicates that down-regulation of Smad3 levels is required for the tumor cells to proliferate in the presence of TGF-beta, because ectopic expression of Smad3 in the tumorigenic cell line restores the growth inhibitory response to TGF-beta. In contrast, expression of high levels of Smad3 did not interfere with the ability of these cells to undergo epithelial to mesenchymal transition upon TGF-beta stimulation. Altogether, our results suggest that the level of Smad3 protein is an important determinant of the progression of tumorigenesis. High levels of Smad3 are required for the tumor suppressor activities of TGF-beta, whereas lower levels are sufficient for the tumor promoting functions.
Highlights
Signals predominantly via a receptor complex comprising the type I receptor ALK5 and the type II receptor TRII, both of which are serine/threonine kinases [1]
At early stages of tumorigenesis, TGF- signaling is thought to act as a tumor suppressor, as a result of its ability to arrest the growth of epithelial cells, and in some cases induce apoptosis [5, 6]
We demonstrate that ectopic expression of Smad3 in EpRas cells restores the growth inhibitory response to TGF- but has no effect on the ability of these cells to undergo epithelial to mesenchymal transition (EMT)
Summary
Cell Culture, Treatments, Transfections, and Generation of Cell Lines—All cell lines were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, and in the case of EpRas cells, 500 g/ml G418. Antigen detection was performed using anti-E-cadherin (BD Biosciences), anti-ZO1 (Zymed Laboratories Inc.) or Vimentin (clone LN-6; Sigma) followed by Alexa Fluor 488conjugated goat anti-mouse IgG antibody or Texas Red conjugated goat anti-rabbit IgG antibody as appropriate. Extraction of RNA, probe preparation, and RNase protection assays were performed as described [26, 27]. Cell Cycle Analysis and EMT Studies—For EpH4 and EpRas cell G1/S progression studies, cells were seeded at high density, arrested in G0/G1 by confluency for 2 days, released by trypsinization, and replated at low density for up to 24 h before harvesting for FACS analysis and/or Western blotting. The growth inhibitory assays in EpH4, EpRas cells, and EpRas cell clones were performed essentially as described previously [29]. Cells were fixed in 70% ethanol at 4 °C, treated with hydrochloric acid, incubated with an anti-BrdUrd antibody (Becton Dickinson) and secondary fluorescein isothiocyanateconjugated antibody (DAKO) before FACS analysis
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
