Regulation of human Cripto-1 gene expression by TGF-beta1 and BMP-4 in embryonal and colon cancer cells.
Human Cripto-1 (CR-1) is a cell membrane protein that is overexpressed in several different types of human carcinomas. In the present study we investigated the mechanisms that regulate the expression of CR-1 gene in cancer cells. We cloned a 2,481 bp 5'-flanking region of the human CR-1 gene into a luciferase reporter vector and transfected NTERA-2 human embryonal carcinoma cells and LS174-T colon cancer cells to test for promoter activity. Activity of CR-1 promoter in both cell lines was modulated by two TGF-beta family members, TGF-beta1 and BMP-4. In particular, TGF-beta1 significantly up-regulated CR-1 promoter activity, whereas a dramatic reduction in CR-1 promoter activity was observed with BMP-4 in NTERA-2 and LS174-T cells. Changes in the CR-1 promoter activity following TGF-beta1 and BMP-4 treatments correlated with changes in CR-1 mRNA and protein expression in NTERA-2 and LS174-T cells. We also identified three Smad binding elements (SBEs) within the CR-1 promoter and point mutation of SBE1 (-2,197/-2,189) significantly reduced response of the CR-1 promoter to both TGF-beta1 and BMP-4 in NTERA-2 and LS174-T cells. Chromatin immunoprecipitation assay also demonstrated binding of Smad-4 to a CR-1 promoter DNA sequence containing SBE1 in LS174-T cells. Finally, BMP-4 inhibited migration of LS174-T cells and F9 mouse embryonal carcinoma cells by downregulation of CR-1 protein. In conclusion, these results suggest a differential modulation of CR-1 gene expression in embryonal and colon cancer cells by two different members of the TGF-beta family.
Mancino
Mario
M
Tumor Growth Factor Section, Mammary Biology and Tumorigenesis Laboratory, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20832, USA.
Strizzi
Luigi
L
Wechselberger
Christian
C
Watanabe
Kazuhide
K
Gonzales
Monica
M
Hamada
Shin
S
Normanno
Nicola
N
Salomon
David S
DS
Bianco
Caterina
C
D024506:5' Flanking Region; D000818:Animals; D001483:Base Sequence; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology*; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D003110:Colonic Neoplasms / Q000235:genetics* / Q000473:pathology; D054278:Embryonal Carcinoma Stem Cells / Q000378:metabolism* / Q000473:pathology; D004815:Epidermal Growth Factor / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D058851:GPI-Linked Proteins; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009363:Neoplasm Proteins / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051785:Smad Proteins / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*
C525809:BMP4 protein, human; C525810:Bmp4 protein, mouse; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins; D058851:GPI-Linked Proteins; D036341:Intercellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D051785:Smad Proteins; C070636:TDGF1 protein, human; C489450:Tdgf1 protein, mouse; D053773:Transforming Growth Factor beta1; D004815:Epidermal Growth Factor