Detection and localization of Cripto‐1 binding in mouse mammary epithelial cells and in the mouse mammary gland using an immunoglobulin–cripto‐1 fusion protein†

Abstract

Human Cripto-1 (CR-1), a member of the epidermal growth factor-CFC (EGF-CFC) family of peptides, is expressed in the developing mouse mammary gland and can modulate mammary epithelial cell migration, branching morphogenesis and milk protein expression in vitro. In order to screen for a CR-1 receptor and to identify potential CR-1 target tissues, we constructed a fusion protein comprising the EGF-like domain of CR-1 and the Fc domain of a human IgG1. The recombinant CR-1 fusion protein (CR-1-Fc) was biologically active as it was able to activate the ras/raf/mitogen activated protein kinase (MAPK) pathway and to inhibit transcription of the milk protein beta-casein in NMuMG and HC-11 mouse mammary epithelial cells. By using immunocytochemistry and by an in situ enzyme-linked immunosorbent assay (ELISA), CR-1-Fc was found to specifically bind to NMuMG and HC-11 cells. Finally, immunohistochemical analysis using CR-1-Fc showed a specific localization of CR-1 binding to tissue sections from mouse mammary gland. In particular, more than 60% of the epithelial cells were intensely stained with the CR-1-Fc fusion protein in the lactating mouse mammary gland, whereas approximately 25% of the mammary epithelial cells were stained in the gland from pregnant mouse. Since expression of mouse cripto-1 (Cr-1) in the pregnant and lactating mouse mammary gland as well as its presence in milk has been previously demonstrated, these data strongly suggest that an autocrine pathway involving Cr-1 and its putative receptor is operating in the mouse mammary gland during pregnancy and lactation.

Cripto-1: a multifunctional modulator during embryogenesis and oncogenesis

“…The mouse Cr-1 protein can be released by treatment of cells with phosphatidylinositol-specific phospholipase C (PI-PLC) (Minchiotti et al, 2000). Removing the COOH-terminal stretch of residues where GPI linkage occurs generates soluble forms of biologically active mouse and human Cripto-1 (Xu et al, 1999;Minchiotti et al, 2001;Bianco et al, 2002bBianco et al, , 2003Yan et al, 2002). Full activity requires the presence of a peptide containing only an intact EGF domain and CFC domain Yan et al, 2002).…”

“…Interestingly, activation of these signaling pathways can be achieved with a soluble GPI-truncated CR-1 recombinant protein Bianco et al, 2002b). Since soluble CR-1 has been found to be secreted in the conditioned medium of several human carcinoma cell lines, it is possible that a functional soluble isoform of CR-1 is naturally cleaved from the cell membrane Minchiotti et al, 2000;Normanno et al, 2004).…”

Cripto-1 Alters Keratinocyte Differentiation via Blockade of Transforming Growth Factor-β1 Signaling: Role in Skin Carcinogenesis

“…A short COOH terminus contains consensus sequences for glycosylphosphatidylinositol linkage to the cell membrane (3). EGF-CFC proteins have activity both when expressed as soluble proteins and when secreted from the cell surface following enzymatic cleavage of their glycosylphosphatidylinositol anchor (4,5).…”

Antibody blockade of the Cripto CFC domain suppresses tumor cell growth in vivo

“…Human Cripto is the original member of the EGF-CFC family of proteins defined by two conserved adjacent functional motifs: a variant EGF ("EGF-like") domain and a unique cysteine-rich domain, the CFC domain (named for Cripto, FRL-1, and Cryptic). Site-directed mutagenesis experiments demonstrated that Cripto binds to Alk4 through its CFC domain to facilitate signaling through the Smad pathway (12,14). Our group has previously shown that fucosylation of Cripto at a unique glycosylation site within the EGF-like domain is essential for Nodal signaling (15), and mutations in the EGF-like domain have been shown to disrupt Cripto-Nodal interactions (12).…”