Abstract
The alpha 2 beta 1 serves as a collagen receptor or a collagen/laminin receptor, depending upon cell type. Expression of the integrin is regulated during normal cellular differentiation and is altered during carcinogenesis. We have previously demonstrated that increased expression of the alpha 2 beta 1 integrin during megakaryocytic differentiation is a consequence of increased alpha 2 mRNA due to transcriptional activation of the alpha 2 integrin gene and that the decreased expression of the integrin in breast adenocarcinoma is due to decreased steady-state levels of alpha 2 mRNA. We now report the identification and characterization of the 5'-flanking region of the alpha 2 integrin gene. The 5'-untranslated region of the alpha 2 mRNA extends 129 base pairs 5' to the site of translation initiation. The promoter region lacks TATA and CAAT boxes but contains an abbreviated initiator sequence and six Sp1 binding sites. Consensus binding sites for AP-1 and AP-2 complexes, a GATA box, a Pu.1 box, and two palindromic motifs with potential to bind the estrogen receptor are also present. A 961-base pair fragment of the 5'-flanking region directs both cell type- and differentiation-specific expression of a reporter gene in T47-D epithelial cells and in pluripotent hematopoietic K562 cells upon megakaryocytic differentiation.
Highlights
MATERIALS AND METHODSCell Cultures and Dansfection Assays”T47-D and K562 cell lines obtained fromthe ATCC were propagated in RPMI1640 medium either with or without 0.2 unitdml insulin, respectively
From the Departments of $Pathology and Wedicine,Washington University School of Medicine, St
We report theidentification of the 5”flanking region of the a2integrin gene, compareits structure to the othekrnown integrin promoters, and demonstrate thcaet ll type-specific pro
Summary
Cell Cultures and Dansfection Assays”T47-D and K562 cell lines obtained fromthe ATCC were propagated in RPMI1640 medium either with or without 0.2 unitdml insulin, respectively. The K562 cell line was transfected by eledroporation using a BTX. Electro Cell Manipulator600 (BTX Inc., San Diego,CA) (20).Approximately 1.0 x lo7cells were transfected in RPMI medium containin1g00 pg/ml salmon sperm DNA, 30 pg of plasmid DNA, and 3 pg of RSVluciferase’ DNA by electroporation at 275 V and 600 pF. The a2 subunit isexpressed on many epithelialcell types including those of the skin, breast, and colon, as well as on fibroblasts, endothelial cells, megakaryocytes, platelets, and activated T cells. Luciferase activity produced by 10 pl of the cell lysate in 190 pl of assay buffer (10 nm Mg[OAclz, nm Tris-MES,pH 7.8, and 2 nm ATP) was analyzed usinga Monolight 2010 luminometer(AnalyticalLuminescenceLaboratory, San Diego,. This article must be herebymarked “aduertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s)reported in this paper has been submitted to the GenBankTMIEMBLData Bank with accession number(s) L24121
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