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

Laminin-5, the major extracellular matrix protein produced by mammary epith elial cells, is composed of three chains (designated alpha 3A, beta3, and g amma2), each encoded by a separate gene. Laminin-5 is markedly down-regulat ed in breast cancer cells. Little is known about the regulation of laminin gene transcription in normal breast cells, nor about the mechanism underlyi ng the down-regulation seen in cancer. In the present study, we cloned the promoter of the gene for the human laminin alpha 3A chain (LAMA3A) and inve stigated its regulation in functionally normal MCF10A breast epithelial cel ls and several breast cancer cell lines. Using site-directed mutagenesis of promoter-reporter constructs in transient transfection assays in MCF10A ce lls, we find that two binding sites for Kruppel-like factor 4 (KLF4/GKLF/EZ F) are required for expression driven by the LAMA3A promoter. Electrophoret ic mobility shift assays reveal absence of KLF4 binding activity in extract s 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 contain ing duplicate KLF4-binding sites in its promoter is expressed at high level s in MCF10A cells but at negligible levels in breast cancer cells. Thus, KL F4 is required for LAMA3A expression and absence of laminin alpha 3A in bre ast cancer cells appears, at least in part, attributable to the lack of KLF 4 activity.