Role of CEACAM1 isoforms in an in vivo model of mammary morphogenesis: mutational analysis of the cytoplasmic domain of CEACAM1-4S reveals key residues involved in lumen formation

Abstract

CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1) is a type I transmembrane glycoprotein expressed in epithelial cells with three or four extracellular domains (ECDs) and either long or short cytoplasmic domain isoforms. We have previously shown that the four extracellular domains, short cytoplasmic domain isoform, CEACAM1-4S, plays an essential role in lumen formation in an in vitro model of mammary morphogenesis. In this study, we transfected MCF-7 cells with either the long or short cytoplasmic domain isoforms of CEACAM1, and grew the cells in humanized mammary mouse fat pads in NOD/SCID mice. In this in vivo model, only the long cytoplasmic domain isoform, CEACAM1-4L, formed glands with lumen. On the basis of other studies that revealed phosphorylation of key Thr and Ser residues in the short cytoplasmic domain, we introduced phosphorylation mimic (for example, Thr or Ser to Asp) or null (Thr or Ser to Ala) mutations into the cytoplasmic domain of CEACAM1-4S and tested them in the in vivo model. Mutation of either Thr or Ser to Asp or the double mutant Thr+Ser to Asp, but not the null mutants, induced gland formation with a central lumen-containing apoptotic cells. Moreover, the phosphorylation mimic mutants of CEACAM1-4S induced downregulation of beta1-integrin, overexpression of beta2-integrin, inhibited phosphorylation of focal adhesion kinase (pTyr-397) and resulted in myofibroblast differentiation as characterized by expression of vimentin, alpha-smooth muscle actin and beta2-integrin, as well as the production of abundant extracellular matrix.