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A more recent version of this article appeared on November 9, 2001
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M108130200v1
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Papers In Press, published online ahead of print September 10, 2001
J. Biol. Chem, 10.1074/jbc.M108130200
Submitted on August 23, 2001
Revised on September 5, 2001
Accepted on September 6, 2001

Kruppel-like factor 4 regulates laminin alpha 3A expression in mammary epithelial cells

Kristi A. Miller, Elizabeth A. Eklund, Marie L. Peddinghaus, Zhengjin Cao, Nisha Fernandes, Patrick W. Turk, Bayar Thimmapaya, and Sigmund A. Weitzman

Department of Medicine, Northwestern University Medical School, Chicago, IL 60611

Corresponding Author: s-weitzman{at}northwestern.edu

Laminin-5, the major extracellular matrix protein produced by mammary epithelial cells, is composed of three chains (designated alpha 3A, beta 3 and gamma 2), each encoded by a separate gene. Laminin-5 is markedly downregulated in breast cancer cells. Little is known about the regulation of laminin gene transcription in normal breast cells, nor about the mechanism underlying the downregulation seen in cancer. In the present study, we cloned the promoter of the gene for the human laminin alpha 3A 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 (EMSA) 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 alpha 3A in breast cancer cells appears, at least in part, attributable to the lack of KLF4 activity.


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