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.
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.
Bianco
Caterina
C
Tumor Growth Factor Section, Basic Research Laboratory, National Cancer Institute/NIH,, Bldg 10, Rm 5B39, 9000 Rockville Pike, Bethesda, MD 20892, USA.
Normanno
Nicola
N
De Luca
Antonella
A
Maiello
Monica Rosaria
MR
Wechselberger
Christian
C
Sun
Youping
Y
Khan
Nadia
N
Adkins
Heather
H
Sanicola
Michele
M
Vonderhaar
Barbara
B
Cohen
Bruce
B
Seno
Masaharu
M
Salomon
David
D
D000818:Animals; D019556:COS Cells; D002364:Caseins / Q000037:antagonists & inhibitors; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000187:drug effects; D004815:Epidermal Growth Factor; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005260:Female; D058851:GPI-Linked Proteins; D006728:Hormones / Q000494:pharmacology; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics; D007150:Immunohistochemistry; D036341:Intercellular Signaling Peptides and Proteins; D007774:Lactation / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology; D008321:Mammary Glands, Animal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008562:Membrane Glycoproteins; D051379:Mice; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D011247:Pregnancy; D011485:Protein Binding / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D014162:Transfection
D002364:Caseins; D058851:GPI-Linked Proteins; D006728:Hormones; D007141:Immunoglobulin Fc Fragments; D036341:Intercellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins; D011993:Recombinant Fusion Proteins; C070636:TDGF1 protein, human; C489450:Tdgf1 protein, mouse; D004815:Epidermal Growth Factor