title: Kruppel-like factor 4 regulates laminin alpha 3A expression in mammary epithelial cells.
abstract: Laminin-5, the major extracellular matrix protein produced by mammary epithelial cells, is composed of three chains (designated alpha3A, beta3, and gamma2), each encoded by a separate gene. Laminin-5 is markedly down-regulated in breast cancer cells. Little is known about the regulation of laminin gene transcription in normal breast cells, nor about the mechanism underlying the down-regulation seen in cancer. In the present study, we cloned the promoter of the gene for the human laminin alpha3A chain (LAMA3A) and investigated its regulation in functionally normal MCF10A breast epithelial cells and several breast cancer cell lines. Using site-directed mutagenesis of promoter-reporter constructs in transient transfection assays in MCF10A cells, we find that two binding sites for Kruppel-like factor 4 (KLF4/GKLF/EZF) are required for expression driven by the LAMA3A promoter. Electrophoretic mobility shift assays reveal absence of KLF4 binding activity in extracts from T47D, MDA-MB 231, ZR75-1, MDA-MB 436, and MCF7 breast cancer cells. Transient transfection of a plasmid expressing KLF4 activates transcription from the LAMA3A promoter in breast cancer cells. A reporter vector containing duplicate KLF4-binding sites in its promoter is expressed at high levels in MCF10A cells but at negligible levels in breast cancer cells. Thus, KLF4 is required for LAMA3A expression and absence of laminin alpha3A in breast cancer cells appears, at least in part, attributable to the lack of KLF4 activity.
authors.affiliation: Department of Medicine, Northwestern University Medical School, Chicago, Illinois, USA.
authors.forename: K A
authors.initials: KA
authors.lastname: Miller
authors.forename: E A
authors.initials: EA
authors.lastname: Eklund
authors.forename: M L
authors.initials: ML
authors.lastname: Peddinghaus
authors.forename: Z
authors.initials: Z
authors.lastname: Cao
authors.forename: N
authors.initials: N
authors.lastname: Fernandes
authors.forename: P W
authors.initials: PW
authors.lastname: Turk
authors.forename: B
authors.initials: B
authors.lastname: Thimmapaya
authors.forename: S A
authors.initials: SA
authors.lastname: Weitzman
mesh_terms: D001665:Binding Sites; D015153:Blotting, Western; D001940:Breast / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000378:metabolism* / Q000502:physiology*; D015536:Down-Regulation; D004847:Epithelial Cells / Q000378:metabolism*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D007797:Laminin / Q000096:biosynthesis*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D013997:Time Factors; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism* / Q000502:physiology*; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D004268:DNA-Binding Proteins; C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D007797:Laminin; D018808:Transcription Factor AP-1; D014157:Transcription Factors; C095567:laminin alpha 3