Eur. J. Biochem. 268, 3797±3806 (2001) q FEBS 2001 Regulation of laminin b2 chain gene expression in human cancer cell lines Marian E. Durkin 1, *, Finn C. Nielsen 2 , Frosty Loechel 1 , Reidar Albrechtsen 1 and Ulla M. Wewer 1 1 Institute of Molecular Pathology, University of Copenhagen, Denmark; 2 Department of Clinical Biochemistry, Rigshospitalet, Copenhagen, Denmark The laminin b2 chain is a basement membrane component expressed in a tissue- and developmental stage-specific manner. In this report we have examined the transcriptional and post-transcriptional regulation of the human laminin b2 chain in human tumor cell lines. Both the A204 rhabdo- myosarcoma and clone A colon carcinoma cells express the laminin b2 chain mRNA, but only the A204 cells secrete laminin heterotrimers containing the b2 chain. Segments of the b2 chain gene promoter region were cloned into luciferase reporter vectors, and their ability to stimulate transcription was tested by transient transfection. Sequences downstream of the transcription start site between nucleotides 191 and 1120 were found to be essential for luciferase activity in the two cell lines. Additional positive regulatory regions were present further upstream, between nucleotides 2164 to 2667 and between nucleotides 2667 to 21724. Genomic DNA at the 3 0 end of the gene also appeared to have enhancer activity, as a 1.1-kb fragment located downstream of the last exon stimulated the luciferase activity of the nucleotides 2667/1297 promoter segment < threefold. Alternative splicing of the first intron of the human laminin b2 chain gene generates two isoforms of the 5 0 untranslated region of the b2 chain mRNA. The trans- lational efficiencies of the two laminin b2 chain leaders did not differ significantly, when assayed by polysome profile analysis of endogenous clone A cell b2 chain mRNA, transient transfection of chimeric b2 chain leader/luciferase expression plasmids in clone A cells, and translation of in vitro synthesized RNAs in rabbit reticulocyte lysates. Keywords: laminin; gene expression; promoter; trans- lational control. Laminin is one of the principal structural components of basement membranes and a major cell-binding protein [1]. The laminin molecule initially extracted from rodent tumor extracellular matrices was a heterotrimer composed of three homologous but nonidentical subunits, now known as the a1, b1 and g1 chains [2]. Subsequent studies revealed that rodent tumor laminin is only one member of a growing family of structurally and functionally diverse laminin isoforms. At the present time five a, three b and three g chains have been identified, which could potentially assemble into at least 30 different laminin heterotrimers [3,4]. The 190-kDa laminin b2 chain (s-laminin) is highly homologous to the laminin b1 chain at both the amino-acid sequence and gene structure levels [5±8], and it can substitute for the b1 chain in laminin heterotrimers containing the five known a chains, the g1, and g3 chains [9±13]. Targeted inactivation of the gene in mice demon- strated that the laminin b2 chain is necessary for postnatal survival [14]. The defects in the neuromuscular junction, kidney glomerulus, and retina of mice lacking the laminin b2 chain indicate that in these sites the b2 chain performs essential functions that cannot be compensated for by the other b chains [14±16]. The b1 and b2 chain-containing laminin isoforms differ in their ability to support the adhesion of some cell types [17], and changes in laminin b2 chain levels may thus alter basement membrane±cell interactions. The expression of the laminin b2 chain gene displays a complex developmental pattern. During the maturation of the kidney glomerulus, arterial smooth muscle, and peri- pheral nerve perineurium basement membranes, the b2 chain appears at a later stage than the b1 chain and even- tually replaces it [18±21]. In extrasynaptic skeletal muscle and visceral smooth muscle basement membranes, the b2 chain is coexpressed with the b1 chain in adults but is absent during development [20,22]. In contrast, the b2 chain is present in the developing epidermis and liver, but is downregulated after birth [23,24]. Changes in the levels of the laminin b2 chain have been found in pathological conditions such as cancer [6,25], diabetes [26], and neuro- muscular disorders [27,28]. While one study found that TGF-b up-regulated laminin b2 mRNA levels in rat alveolar cells [29], the factors controlling laminin b2 chain gene expression remain largely unknown. We have previously determined the exon±intron organ- ization of the 12-kb human laminin b2 chain gene and characterized the surrounding genomic DNA on chromo- some 3p21.3 [8,30]. In this report, we have studied the control of laminin b2 chain gene expression at both the transcriptional and post-transcriptional level in human cancer cell lines. Transient transfection assays were used to analyze the promoter activity of segments of the 5 0 Correspondence to U. M. Wewer, Institute of Molecular Pathology, University of Copenhagen, Frederik V's vej 11, DK-2100 Copenhagen, Denmark. Fax: 1 45 35326081, Tel.: 1 45 35326056, E-mail: ullaw@pai.ku.dk Abbreviations: DMEM, Dulbecco's modified Eagle's medium; RNP, ribonucleoprotein particle; RT, reverse transcription. *Present address: National Cancer Institute, NIH, Bethesda, MD, 20892, USA (Received 3 April 2001, accepted 11 May 2001)