Background: Laminin-5 (ln-5), a large heterotrimeric glycoprotein consisting of an a3, P33, and y2 chain, is a component of epithelial cell basement membranes that functions as a ligand of the a3,31 and a6f34 integrins to regulate cell adhesion, migration, and morphogenesis. The ln-5 chains show tissue-specific patterns of regulation in tumors derived from different tissues. For example, ln-5 is often up-regulated in gliomas, gastric carcinomas, and squamous carcinomas and down-regulated in prostate and basal cell carcinomas. Ln-5 expression patterns may represent useful tumor markers and help to elucidate the role of ln-5 in tumor progression in different tissue types. Materials and Methods: We have studied In-5 expression patterns in the breast. mRNA levels were examined in tumor and normal breast epithelial cell lines, tissue samples, and immunomagnetically sorted primary cultures using differential display, Northern blotting, and hybridization arrays. Protein levels were examined by immunoprecipitation. Gene integrity was assessed by Southern blotting of representative cell types. Results: Ln-5 a3, ,B3, and y2 mRNA expression was found to be markedly down-regulated in a panel of breast tumor cell lines when compared with normal breast epithelial cells. Ln-5 mRNA was expressed at relatively high levels in MCF-1OA immortal normal breast epithelial cells, long-term cultures of normal breast cells, and sorted primary cultures of normal breast luminal epithelial and myoepithelial cells. Reduced, but detectable, levels of ln-5 tended to be expressed in cell lines derived from early-stage breast tumors, whereas expression was generally not detected in cell lines derived from later-stage tumors. In breast tumor tissue specimens, expression of ln (x3 and j33 mRNAs tended to be reduced relative to levels observed in adjacent nontumor tissue, whereas in y2 levels were elevated in specimens with increased amounts of myoepithelial cells. These ln-5 expression changes could not be attributed to large-scale mutations or gene rearrangements. Ln-5 protein levels were found to reflect mRNA levels in representative cell lines. At senescence, a growth state believed to suppress tumorigenesis, expression of all three In-5 mRNAs was up-regulated. Conclusion: The down-regulation of ln-5 mRNA expression in breast tumors cells provides a new molecular marker and suggests that In-5 functions to control tumor progression in the breast.

For a number of growth factors and cytokines, ligand dimerization is believed to be central to the formation of an active signaling complex. In the case of fibroblast growth factor-2 (FGF2) signaling, heparin/heparan sulfate-like glycosaminoglycans (HLGAGs) are involved through interaction with both FGF2 and its receptors (FGFRs) in assembling a tertiary complex and modulating FGF2 activity. Biochemical data have suggested different modes of HLGAG-induced FGF2 dimerization involving specific protein-protein contacts. In addition, several recent x-ray crystallography studies of FGF⅐FGFR and FGF⅐FGFR⅐HLGAG complexes have revealed other modes of molecular assemblage, with no FGF-FGF contacts. All these different biochemical and structural findings have clarified less and in fact raised more questions as to which mode of FGF2 dimerization, if any, is essential for signaling. In this study, we address the issue of FGF2 dimerization in signaling using a combination of biochemical, biophysical, and site-directed mutagenesis approaches. Our findings presented here provide direct evidence of FGF2 dimerization in mediating FGF2 signaling.Fibroblast growth factors (FGFs) 1 are involved in a wide range of physiological processes, including morphogenesis, as well as disease processes such as tumor angiogenesis (1-3). The FGF family consists of at least 20 members, including the well characterized acidic FGF (FGF1) and basic FGF (FGF2), both of which are potent mitogens of many cell types (4, 5). FGF signaling is mediated primarily through high affinity interactions with cell-surface FGF receptors (FGFRs), transmembrane polypeptides composed of immunoglobulin-like and tyrosine kinase domains (6, 7). FGF binding to different isoforms of FGFR is believed to trigger receptor dimerization, followed by transphosphorylation of specific tyrosine residues (8). In turn, phosphorylated tyrosine residues activate other signaling proteins, leading to cell proliferation, migration, and survival.For proper presentation to FGFR, FGF2 and other members of the FGF family interact with heparin/heparan sulfate-like glycosaminoglycans (HLGAGs). Consisting of a disaccharide repeat of glucosamine and uronic acid, HLGAGs are heterogeneous in length (10 -100 disaccharide units) and chemical composition (including differential sulfation, acetylation, and epimerization of each disaccharide unit) (9 -12). Found in the extracellular matrix and on the cell surface as part of proteoglycans, HLGAGs modulate FGF2 activity by low affinity interactions with specific FGF2-and FGFR-binding sites (13-15), facilitating FGF2 binding to FGFR. HLGAGs promote FGF2-induced activation of FGFR through a number of mechanisms, including regulating the diffusion rate of FGF2 (16, 17) and possibly dictating the specificity of FGF2-FGFR binding through interactions with both .Another hypothesis is that FGF2 binding to HLGAGs induces ligand oligomerization, which in turn induces dimerization and transphosphorylation of FGFR. Biochemical studies have demonstrated that HLGAGs...

Fibroblast growth factor (FGF) signalling is involved in a wide range of important biological activities with differential effects in various cell types. The activity of FGF is modulated by heparin/heparan sulphate-like glycosaminoglycans (HSGAGs), found both in the extracellular matrix and on the cell surface. HSGAGs affect FGF signalling by interacting with both the growth factor and the FGF receptor (FGFR). In this study we sought to investigate whether HSGAGs at the cell surface of bovine aortic endothelial cells (BAEC) and smooth muscle cells (SMC) can differentially modulate FGF signalling in these cell types and modulate their differential response to FGF. We find that SMC and BAEC express the same FGFR isoforms and bind FGF2 with equal affinity at the cell surface, yet FGF has a markedly higher proliferative effect on SMC than on BAEC. Isolated HSGAGs from these two cell types were found to elicit distinct patterns of proliferation in chlorate-treated cells. Furthermore, examination of focal sequences reveals that HSGAGs from SMC, but not those from BAEC, retain the sulphation pattern necessary to induce FGF2 activity. As such, the differences in FGF2-mediated proliferation can be explained by the distinct cell surface HSGAGs of the two cell types. We conclude that the focal sequences of cell surface HSGAGs from SMC and BAEC govern, at least in part, the differential activity of FGF2 on these two cell types.