Characterization of TGF-? signaling in a human organotypic skin model reveals that loss of TGF-?RII induces invasive tissue

Transforming growth factor-beta (TGF-beta) signaling regulates various aspects of cell growth and differentiation and is often dysregulated in human cancers. We combined genetic engineering of a human organotypic three-di-mensional (3D) skin model with global quantitative proteomics and phosphoproteomics to dissect the impor-tance of essential components of the TGF-beta signaling pathway, including the ligands TGF-beta 1, TGF-beta 2, and TGF-beta 3, the receptor TGF-beta RII, and the intracellular effector SMAD4. Consistent with the antiproliferative effects of TGF-beta signaling, the loss of TGF-beta 1 or SMAD4 promoted cell cycling and delayed epidermal differentiation. The loss of TGF-beta RII, which abrogates both SMAD4-dependent and SMAD4-independent downstream signaling, more strongly affected cell proliferation and differentiation than did loss of SMAD4, and it induced invasive growth. TGF-beta RII knockout reduced cell-matrix interactions, and the production of matrix proteins increased the production of cancer-associated cell-cell adhesion proteins and proinflammatory mediators and increased mitogen-activated protein kinase (MAPK) signaling. Inhibiting the activation of the ERK and p38 MAPK pathways blocked the development of the invasive phenotype upon the loss of TGF-beta RII. This study provides a framework for exploring TGF-beta signaling pathways in human epithelial tissue homeostasis and transformation using genetic engineering, 3D tissue models, and high-throughput quantitative proteomics and phosphoproteomics.