Blockade of Smad4 in transformed keratinocytes containing a Ras oncogene leads to hyperactivation of the Ras-dependent Erk signalling pathway associated with progression to undifferentiated carcinomas.
Smad4 functions as a transcription factor in TGF-beta signalling. We have investigated the role of Smad4 in the TGF-beta1 cell responses of transformed PDV keratinocytes, which contain a Ras oncogene, and of non-tumorigenic MCA3D keratinocytes, by transfecting both cell lines with a dominant-negative Smad4 construct. Smad4 mediates TGF-beta1-induced up-regulation of p21Cip1 and growth arrest in MCA3D cells. However, in PDV keratinocytes, Smad4 is only partially involved in TGF-beta1-induced growth inhibition, and does not mediate enhancement of p21Cip1 levels by the growth factor. TGF-beta1 activates Ras/Erk signalling activity in both cell lines. PD098059, a specific inhibitor of MEK, disminishes TGF-beta1-induced p21Cip1 levels in PDV but not in MCA3D cells, suggesting an involvement of Erk in up-regulation of p21Cip1 by TGF-beta1 in PDV cells. PDV dominant-negative Smad4 cell transfectants, but not MCA3D transfectants, showed constitutive hyperactivation of the Ras/Erk signalling pathway, increased secretion of urokinase, higher motility properties, and a change to a fibroblastoid cell morphology associated in vivo with the transition from a well differentiated to a poorly differentiated tumour phenotype. Infection of MCA3D control and dominant negative Smad4 cell transfectants with retroviruses carrying a Ras oncogene led to enhanced p21Cip1 and urokinase secreted levels, independently of TGF-beta1 stimulation, that were reduced by PD098059. These results suggest that Smad4 acts inhibiting Ras-dependent Erk signalling activity in Ras-transformed keratinocytes. Loss of Smad4 function in these cells results in hyperactivation of Erk signalling and progression to undifferentiated carcinomas. Oncogene (2000) 19, 4134 - 4145
Iglesias
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Instituto de Investigaciones Biomédicas Alberto Sols CSIC-UAM, Arturo Duperier 4, 28029-Madrid, Spain.
Frontelo
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Gamallo
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Quintanilla
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D000818:Animals; D015153:Blotting, Western; D002277:Carcinoma / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D002461:Cell Line, Transformed; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D005455:Fluorescent Antibody Technique; D015603:Keratinocytes / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D008819:Mice, Nude; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D015398:Signal Transduction; D012878:Skin Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D051901:Smad4 Protein; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D015854:Up-Regulation; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
C493957:Cdkn1a protein, mouse; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004268:DNA-Binding Proteins; D051901:Smad4 Protein; C490810:Smad4 protein, mouse; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D020928:Mitogen-Activated Protein Kinases; D014568:Urokinase-Type Plasminogen Activator; D018631:ras Proteins