Abstract. Branching epithelial morphogenesis requiresinteractions between the surrounding mesenchyme and the epithelium, as well as interactions between basement membrane components and the epithelium. Embryonic submandibular gland was used to study the roles of two mesenchymal proteins, epimorphin and tenascin-C, as well as the epithelial protein laminin-1 and one of its integrin receptors on branching morphogenesis. Laminin-1 is a heterotrimer composed of an al chain and two smaller chains (/31 and ,y1). Immunofluorescence revealed a transient expression of laminin al chain in the epithelial basement membrane during early stages of branching morphogenesis. Other laminin-1 chains and a6, /~1, and/34 integrin subunits seemed to be expressed constitutively. Expression of epimorphin, but not tenascin-C, was seen in the mesenchyme during early developmental stages, but a mAb against epimorphin did not perturb branching morphogenesis of this early epithelium. In contrast, inhibition of branching morphogenesis was seen with a mAb against the carboxy terminus of laminin cd chain, the E3 domain. An inhibition of branching was also seen with a mAb against the integrin oL6 subunit. The antibodies against laminin cd chain and integrin a6 subunit perturbed development in distinct fashions. Whereas treatment with the anti-E3 resulted in discontinuities of the basement membrane at the tips of the branching epithelium, treatment with the mAb against a6 integrin subunit seemed to leave the basement membrane intact. We suggest that the laminin E3 domain is involved in basement membrane formation, whereas ot6fll integrin binding to laminin-1 may elicit differentiation signals to the epithelial cells.

Laminin ␣ chains (␣ 1 -␣ 5 chains) have diverse chainspecific biological functions. The LG4 modules of laminin ␣ chains consist of a 14-stranded ␤-sheet (A-N) sandwich structure. Several biologically active sequences have been identified in the connecting loop regions. Here, we evaluated the biological activities of the loop regions of the E and F strands in the LG4 modules using five homologous peptides from each of the mouse ␣ chains (EF-1: DYATLQLQEGRLHFMFDLG, ␣ 1 chain 2747-2765; EF-2: DFGTVQLRNGFPFFSYDLG, ␣ 2 chain 2808 -2826; EF-3: RDSFVALYLSEGHVIFALG, ␣ 3 chain 2266 -2284; EF-4: DFMTLFLAHGRLVFMFNVG, ␣ 4 chain 1511-1529; EF-5: SPSLVLFLNHGHFVAQTEGP, ␣ 5 chain 3304 -3323). These homologous peptides showed chainspecific cell attachment and neurite outgrowth activities. Well organized actin stress fibers and focal contacts with vinculin accumulation were observed in fibroblasts attached on EF-1, whereas fibroblasts on EF-2 and EF-4 showed filopodia with ruffling. Fibroblast attachment to EF-2 and EF-4 was mediated by syndecan-2. In contrast, EF-1 promoted ␣ 2 ␤ 1 integrin-mediated fibroblast attachment and inhibited fibroblast attachment to a recombinant laminin ␣ 1 chain LG4-5. The receptors for EF-3 and EF-5 are unknown. Further, when the active core sequence of EF-1 was cyclized, utilizing two additional cysteine residues at both the N and C termini through a disulfide bridge, the cyclic peptide significantly enhanced integrin-mediated cell attachment. These results indicate that integrin-mediated cell attachment to the EF-1 sequence is conformation-dependent and that the loop structure is important for the activity. The homologous peptides, which promote either integrin-or syndecan-mediated cell attachment, may be useful for understanding the cell type-and chain-specific biological activities of the laminins.

Laminin, a major component of the basement membrane, has diverse biological activities. Recently, we identified various biologically active sequences on laminin-1 by using a large set of synthetic peptides. Chitosan, a polysaccharide, is biodegradable and has been used as a biomaterial. Here, we conjugated several biologically active laminin peptides onto chitosan membranes and measured the cell attachment activity of peptide-conjugated chitosan membranes with various cell types. The active laminin peptide-conjugated chitosan membranes promoted cell attachment with cell type specificity. A99 (AGTFALRGDNPQG)-chitosan membrane promoted cell attachment with well-organized actin stress fibers. This adhesion was inhibited by EDTA but not by heparin. AG73 (RKRLQVQLSIRT)-chitosan membrane promoted cell attachment with filopodia formation, and this adhesion was inhibited by heparin but not by EDTA. These data suggest that the A99-chitosan membrane interacted with an integrin cellular receptor and that the AG73-chitosan membrane promoted proteoglycan-mediated cell attachment, as previously reported. Furthermore, both AG73-chitosan and A99-chitosan membranes effectively promoted neurite outgrowth with PC12 rat pheochromocytoma cells. We conclude that conjugation on a chitosan membrane is applicable for testing quantitatively the biological activity of synthetic peptides and that these constructs have a potential ability to serve as bioadhesive materials for tissue regeneration and engineering.

The Ile-Lys-Val-Ala-Val (IKVAV) containing peptide, A208 (AASIKVAVSADR, mouse laminin alpha1 chain 2097-2108), was recently found to form amyloid-like fibrils. Fibril formation is critical for its biological activities, including promotion of cell adhesion and neurite outgrowth. In the present study, we designed multifunctional peptide fibrils using the A208 peptide and an Arg-Gly-Asp (RGD)-containing fibronectin active sequence for biomedical applications. The fibronectin active sequence GRGDS (FN) or a scrambled sequence RSGGD (SC) were conjugated to either A208 or to A208S (AASVVIAKSADR), a scrambled peptide of A208, with a glycine as a spacer. The FN-A208 and SC-A208 peptides formed a gel and were stained with Congo red similar to that of A208, but FN-A208S and SC-A208S did not form a gel. These results indicate that FN-A208 and SC-A208 form amyloid-like fibrils similar to A208. A208 and SC-A208 promoted cell attachment with filopodia formation, and this adhesion was inhibited by the IKVAV-containing peptide, but not by EDTA or a GRGDS peptide. FN-A208 promoted cell attachment with well-organized actin stress fibers, and this adhesion was partially inhibited by either EDTA, GRGDS, or IKVAV. These data suggest that A208 binds to only IKVAV receptor(s) while the FN-A208 interacts with both integrins and the IKVAV receptor(s). We conclude that multifunctional peptide fibrils can be designed by conjugation of active peptides on A208 and that this construct has potential to serve as a bioadhesive for tissue regeneration and engineering.

The Ile-Lys-Val-Ala-Val (IKVAV) sequence derived from laminin-1 promotes cell adhesion, neurite outgrowth, and tumor growth and metastasis. Here, we examined amyloid formation of an IKVAV-containing peptide (LAM-L: AASIK-VAVSADR, mouse laminin K K1 chain 2097^2108). The LAM-L peptide was stained with Congo red and exhibited ¢brils in electron microscopy with a characteristic cross-L L X-ray di¡rac-tion pattern. Further, infrared spectra of LAM-L suggested a L L-sheet structure. These results indicate that LAM-L forms amyloid-like ¢brils. We also examined amyloid-like ¢bril formation of LAM-L analogs. The neurite outgrowth activity of the LAM-L analogs was closely related to their amyloid-like ¢bril formation.