Cripto-1 (CR-1), an epidermal growth factor-CFC (EGF-CFC) family member, has a demonstrated role in embryogenesis and mammary gland development and is overexpressed in several human tumors. Recently, EGF-CFC proteins were implicated as essential signaling cofactors for Nodal, a transforming growth factor β family member whose expression has previously been defined as embryo specific. To identify a receptor for CR-1, a human brain cDNA phage display library was screened using CR-1 protein as bait. Phage inserts with identity to ALK4, a type I serine/threonine kinase receptor for Activin, were identified. CR-1 binds to cell surface ALK4 expressed on mammalian epithelial cells in fluorescence-activated cell sorter analysis, as well as by coimmunoprecipitation. Nodal is coexpressed with mouse Cr-1 in the mammary gland, and CR-1 can phosphorylate the transcription factor Smad-2 in EpH-4 mammary epithelial cells only in the presence of Nodal and ALK4. In contrast, CR-1 stimulation of mitogen-activated protein kinase and AKT in these cells is independent of Nodal and ALK4, suggesting that CR-1 may modulate different signaling pathways to mediate its different functional roles.

The EGF-CFC gene family encodes a group of structurally related proteins that serve as important competence factors during early embryogenesis in Xenopus, zebrafish, mice and humans. This multigene family consists of Xenopus FRL-1, zebrafish one-eyed-pinhead (oep), mouse cripto (Cr-1) and cryptic, and human cripto (CR-1) and criptin. FRL-1, oep and mouse cripto are essential for the formation of mesoderm and endoderm and for correct establishment of the anterior/posterior axis. In addition, oep and cryptic are important for the establishment of left-right (L/R) asymmetry. In zebrafish, there is strong genetic evidence that oep functions as an obligatory co-factor for the correct signaling of a transforming growth factor-beta (TGFbeta)-related gene, nodal, during gastrulation and during L/R asymmetry development. Expression of Cr-1 and cryptic is extinguished in the embryo after day 8 of gestation except for the developing heart where Cr-1 expression is necessary for myocardial development. In the mouse, cryptic is not expressed in adult tissues whereas Cr-1 is expressed at a low level in several different tissues including the mammary gland. In the mammary gland, expression of Cr-1 in the ductal epithelial cells increases during pregnancy and lactation and immunoreactive and biologically active Cr-1 protein can be detected in human milk. Overexpression of Cr-1 in mouse mammary epithelial cells can facilitate their in vitro transformation and in vivo these Cr-1-transduced cells produce ductal hyperplasias in the mammary gland. Recombinant mouse or human cripto can enhance cell motility and branching morphogenesis in mammary epithelial cells and in some human tumor cells. These effects are accompanied by an epithelial-mesenchymal transition which is associated with a decrease in beta-catenin function and an increase in vimentin expression. Expression of cripto is increased several-fold in human colon, gastric, pancreatic and lung carcinomas and in a variety of different types of mouse and human breast carcinomas. More importantly, this increase can first be detected in premalignant lesions in some of these tissues. Although a specific receptor for the EGF-CFC proteins has not yet been identified, oep depends upon an activin-type RIIB and RIB receptor system that functions through Smad-2. Mouse and human cripto have been shown to activate a ras/raf/MAP kinase signaling pathway in mammary epithelial cells. Activation of phosphatidylinositol 3-kinase and Akt are also important for the ability of CR-1 to stimulate cell migration and to block lactogenic hormone-induced expression of beta-casein and whey acidic protein. In mammary epithelial cells, part of these responses may depend on the ability of CR-1 to transactivate erb B-4 and/or fibroblast growth factor receptor 1 through an src-like tyrosine kinase.