The Intracellular Form of Notch Blocks Transforming Growth Factor β-Mediated Growth Arrest in Mv1Lu Epithelial Cells

Notch signaling specifies cell fate decisions in a variety of tissues during development (1, 2). Activation of the pathway in vertebrates begins with the binding of Notch to its ligand, Delta or Jagged, which stimulates TACE and presenilin-mediated proteolysis (27). These cleavage events release the intracellular domain of Notch, NICD, from the plasma membrane and allow its transit into the nucleus where it interacts with the DNA binding protein CSL and activates transcription (20). Mutations in Notch pathway components are associated with developmental disorders that affect diverse tissues, including bone, blood, liver, and vascular tissue (19, 22, 23, 28, 32, 33). Notch has also been implicated in human cancer as well as cancers induced by retroviral insertions in mice (11, 18, 30, 34). In the situations described thus far, tumorigenesis is associated with unregulated expression of a form of Notch functionally equivalent to NICD. However, NICD is not a classic oncogene; the effects of Notch are exquisitely cell type dependent, and the pathways that mediate growth and transformation are not known. Several reports have documented the sensitivity of T cells to Notch-mediated transformation (4, 11, 30, 34), and there is increasing evidence that epithelial tissues can also be transformed by activated Notch (9, 18, 46). Notch has also been proposed to be a critical player in the transformation of human cells, including epithelial cells, by activated Ras (43). Transforming growth factor β (TGF-β) is an antimitogenic cytokine. Signaling begins with the engagement of receptors at the cell surface, followed by the phosphorylation of cytoplasmic Smad proteins (R-Smads) (25). The R-Smads then enter the nucleus where they typically activate transcription as heterodimers with Smad4. Although these heterodimers can bind DNA, transcriptional activation usually requires their association with distinct DNA binding proteins. TGF-β arrests cells in the G1 phase of the cell cycle by inducing the expression of the Cdk inhibitors p15INK4B and p21 (10, 15). However, since c-Myc dominantly represses the p15INK4B promoter (12, 37, 39, 42), induction of p15INK4B requires the coordinate down-regulation of c-Myc transcription by TGF-β. In this case, TGF-β induces the formation of a repressive complex at the c-Myc promoter, consisting of E2F4/E2F5, p107, and Smad3 (8). Elevated or constitutive expression of c-Myc negates the ability of TGF-β to induce p15INK4B and thereby blocks TGF-β's cytostatic effects. Epithelial tissues utilize the TGF-β signaling pathway for homeostasis, and a number of epithelial tumors are resistant to the cytostatic effects of TGF-β (26). Accordingly, in this study we sought to determine whether Notch can influence the response of epithelial cells to TGF-β. We show that the intracellular form of Notch, NICD, renders mink lung epithelial cells resistant to the effects of TGF-β without affecting TGF-β signaling per se. Our data are consistent with a model in which a strong Notch signal deregulates expression of c-Myc and thereby renders epithelial cells resistant to growth-inhibitory signals.