Scientific Report 2003-2004 - Cleveland Clinic Lerner Research ...

The Department of Cell BiologyTGF-β Signaling PathwaysTHE HOWELABORATORYThe central focus of the work in mylaboratory is to determine the molecularsignaling pathway used by transforminggrowth factor β (TGFβ). We have recentlyidentified an adaptor molecule termed disabled-2(Dab2) as a mediator of TGFβ signaling. Dab2serves as a link between activated cell surfaceTGFβ receptors and their downstream signalingmediators, the Smad proteins. Currently, twospecific aspects of Dab2 are under investigation.We have recently determined that Dab2 binds toseveral key regulatory molecules, Dvl-3 and axin,that function in Wnt-mediated signalingtransduction. We have shown that Dab2 servesas a negative regulator of the Wnt signalingpathway and results in modulation of nuclear β-catenin levels. We are currently investigating, at amolecular level, the mechanism through whichthe binding of Dab2 to Dvl-3 and axin ultimatelyregulates β-catenin levels. In a related project, weare examining the role of Dab2 in mediatingTGFβ-induced epithelial to mesenchymal transdifferentiation(EMT) in mouse mammaryepithelial cells. We have observed that duringTGFβ-induced EMT in these cells, there is aconcomitant upregulation of the p96 form ofDab2 and a downregulation in the p67 form ofDab2. This effect of TGFβ on the respectiveDab2 proteins is due to its effects on alternativepre-mRNA splicing. We are therefore examiningwhether Dab2 alternative splicing regulates thetransdifferentiation of epithelium into mesenchyme.Our second major area of interest focuseson determining the molecular mechanisms bywhich TGFβ aids in maintenance of selftolerance through its induction of apoptosis in Blymphocytes. We have obtained preliminaryresults demonstrating that TGFβ-inducedapoptosis in B lymphocytes is mediated throughthe induction of the pro-apoptotic Bcl-2 familymember Bim. It appears that TGFβ-mediatedinduction of Bim protein is regulated at both thetranscriptional and post-transcriptional levels. Weare investigating the signal transduction pathwaysthat mediate post-transcriptional effects on Bimprotein and the 5′ promoter region of Bim forTGFβ-regulated transactivation.INVESTIGATORSBarbara A. Hocevar, Ph.D.Gary L. Wildey, Ph.D.POSTDOCTORAL FELLOWSCeline Prunier, Ph.D.Xiaojun Qi, Ph.D.TECHNICAL ASSISTANTJessica RennoldsCOLLABORATORSJonathan Cooper, Ph.D. 1Edward B. Leof, Ph.D. 2Xiangxi (Mike) Xu, Ph.D. 31Fred Hutchinson Cancer Fndn.,Seattle, WA2Dept of Biochemistry, MayoClinic, Rochester, MN3Dept. of Biochemistry, EmoryUniv., Atlanta, GAPhilip H. Howe, Ph.D.Hocevar, B.A., Brown, T.L., and P.H. Howe (1999) TGF-β- induces fibronectin synthesis through a c-Jun N-terminal kinasedependent,Smad4-independent pathway. EMBO J. 18:1345-1356.Brown, T.L., Patil, S., Cianci, C.D., Morrow, J.S., and P.H. Howe (1999) Transforming growth factor β induces caspase 3-independent cleavage of α II-spectrin (α-fodrin) coincident with apoptosis. J. Biol. Chem. 274:23256-23262.Patil, S., Wildey, G.M., Brown, T.L., Choy, L., Derynck, .R, and P.H. Howe (2000) Smad7 is induced by CD40 and protectsWEHI 231 B-lymphocytes from transforming growth factor-β-induced growth inhibition and apoptosis. J. Biol. Chem.275:38363-38370.Hocevar, B.A., Smine, A., Xu, X.X., and P.H. Howe (2001) The adaptor molecule Disabled-2 links the transforming growthfactor β receptors to the Smad pathway. EMBO J. 20:2789-2801.Wildey, G.M., Patil, S., and P.H. Howe (2003) Smad3 potentiates transforming growth factor β (TGFβ)-induced apoptosisand expression of the BH3-only protein Bim in WEHI 231 B lymphocytes. J. Biol. Chem. 278:18069-18077.Hocevar, B.A., Mou, F., Rennolds, J.L., Morris, S.M., Cooper, J.A., and P.H. Howe (2003) Regulation of the Wnt signalingpathway by disabled-2 (Dab2). EMBO J. 22:3084-3094.Prunier, C., Pessah, M., Ferrand, N., Howe, P.H., and A. Atfi (2003) The oncoprotein Ski acts as an antagonist of TGFβsignaling by suppressing Smad2 phosphorylation. J. Biol. Chem. (in press).75