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Laminin α5 is an extracellular matrix protein containing multiple domains implicated in various biological processes, such as embryogenesis and renal function. In this study, we used recombinant proteins and synthetic peptides to identify amino acid residues within the short arm region of α5 that were critical for neurite outgrowth activity. The short arm of α5 contains three globular domains (LN, L4a, and L4b) and three rodlike elements (LEa, LEb, and LEc). Recombinant proteins comprised of the α5 short arm fused with a Fc tag produced in 293 cells were assayed for PC12 (pheochromocytoma) cell adhesion and neurite outgrowth activities. Although it did not have cell attachment activity, neurite outgrowth was promoted by the recombinant protein. To narrow the region involved in neurite outgrowth activity, two truncated recombinant proteins were produced in 293 cells. A recombinant protein lacking L4a and LEb lost activity. Furthermore, we synthesized 78 partially overlapping peptides representing most of the amino acid sequences of L4a and LEb. Of the peptides, A5-76 [mouse laminin α5 928-939 (TSPDLFRLVFRY) in L4a] exhibited neurite outgrowth activity. Mutagenesis studies showed that Phe(933) and Arg(934) were involved in neurite outgrowth activity. Moreover, inhibition assays using anti-integrin monoclonal antibodies showed that neurite outgrowth on the α5 short arm was partially mediated by integrin α1β1. However, the antibodies to integrin α1 and β1 did not inhibit neurite elongation on the A5-76 peptide. These results suggest that in addition to cellular interactions with the active site in the L4a domain, the binding of integrin α1β1 seems to modulate neurite elongation on the short arm of α5.

Laminin-111, a multifunctional matrix protein, has diverse biological functions. Previously, we have identified various biologically active sequences in laminin-111 by a systematic peptide screening. We also demonstrated that peptide-conjugated chitosan matrices enhance the biological functions of the active sequences and are useful as a scaffold. Here, we conjugated sixty biologically active laminin-111 peptides onto chitosan matrices. The twenty-nine peptide-chitosan matrices promoted various biological activities, including cell attachment, spreading, and neurite outgrowth. The biological activities of peptide-chitosan matrices depend on the peptide. These peptide-chitosan matrices are categorized into six groups depending on their biological activities. Next, we conjugated five active peptides, which showed strong cell attachment activity in the each group, onto a single chitosan matrix to mimic the multiple activities of laminin-111. The mixed peptides-chitosan matrix significantly promoted cell attachment and cell spreading over that observed with the individual peptides. We also demonstrated that a mixed peptides-chitosan matrix, using four neurite outgrowth-promoting peptides each from a different group, enhanced the activity. These data suggest that the mixed peptides synergistically induce laminin-like biological activities on a chitosan matrix. The active peptides-chitosan matrices described here have potential for use as biomaterial for tissue engineering and regeneration.

Cell adhesive peptides derived from extracellular matrix components are potential candidates to afford bio-adhesiveness to cell culture scaffolds for tissue engineering. Previously, we covalently conjugated bioactive laminin peptides to polysaccharides, such as chitosan and alginate, and demonstrated their advantages as biomaterials. Here, we prepared functional polysaccharide matrices by mixing laminin active peptides and agarose gel. Several laminin peptide/agarose matrices showed cell attachment activity. In particular, peptide AG73 (RKRLQVQLSIRT)/agarose matrices promoted strong cell attachment and the cell behavior depended on the stiffness of agarose matrices. Fibroblasts formed spheroid structures on the soft AG73/agarose matrices while the cells formed a monolayer with elongated morphologies on the stiff matrices. On the stiff AG73/agarose matrices, neuronal cells extended neuritic processes and endothelial cells formed capillary-like networks. In addition, salivary gland cells formed acini-like structures on the soft matrices. These results suggest that the peptide/agarose matrices are useful for both two- and three-dimensional cell culture systems as a multifunctional biomaterial for tissue engineering.

Cell-adhesive peptides derived from extracellular matrix (ECM) proteins are potential candidates for incorporating cell-binding activities into materials for tissue engineering. We have identified a number of cell adhesive peptides from laminins, which are major components of basement membrane ECM. Our goal is the development of synthetic basement membranes using the peptides on scaffolds. We review peptide–polysaccharide complexes, which were prepared by conjugation of the peptides to chitosan and alginate, and the biological activities of the resulting matrices. The peptide–polysaccharide matrices can also be used as a biomaterial for cell transplantation. These studies suggest that the peptide–polysaccharide complexes have the potential to mimic the multifunctional basement membrane and may be useful for tissue engineering.

Perlecan (Hspg2) is a heparan sulfate proteoglycan expressed in basement membranes and cartilage. Perlecan deficiency (Hspg2(-/-)) in mice and humans causes lethal chondrodysplasia, which indicates that perlecan is essential for cartilage development. However, the function of perlecan in endochondral ossification is not clear. Here, we report the critical role of perlecan in VEGF signaling and angiogenesis in growth plate formation. The Hspg2(-/-) growth plate was significantly wider but shorter due to severely impaired endochondral bone formation. Hypertrophic chondrocytes were differentiated in Hspg2(-/-) growth plates; however, removal of the hypertrophic matrix and calcified cartilage was inhibited. Although the expression of MMP-13, CTGF, and VEGFA was significantly upregulated in Hspg2(-/-) growth plates, vascular invasion into the hypertrophic zone was impaired, which resulted in an almost complete lack of bone marrow and trabecular bone. We demonstrated that cartilage perlecan promoted activation of VEGF/VEGFR by binding to the VEGFR of endothelial cells. Expression of the perlecan transgene specific to the cartilage of Hspg2(-/-) mice rescued their perinatal lethality and growth plate abnormalities, and vascularization into the growth plate was restored, indicating that perlecan in the growth plate, not in endothelial cells, is critical in this process. These results suggest that perlecan in cartilage is required for activating VEGFR signaling of endothelial cells for vascular invasion and for osteoblast migration into the growth plate. Thus, perlecan in cartilage plays a critical role in endochondral bone formation by promoting angiogenesis essential for cartilage matrix remodeling and subsequent endochondral bone formation.

Cell-adhesive peptides derived from extracellular matrix (ECM) proteins are potential candidates for incorporating cell-binding activities into materials for tissue engineering. We have identified a number of cell adhesive peptides from laminins, which are major components of basement membrane ECM. Our goal is the development of synthetic basement membranes using the peptides on scaffolds. We review peptide–polysaccharide complexes, which were prepared by conjugation of the peptides to chitosan and alginate, and the biological activities of the resulting matrices. The peptide–polysaccharide matrices can also be used as a biomaterial for cell transplantation. These studies suggest that the peptide–polysaccharide complexes have the potential to mimic the multifunctional basement membrane and may be useful for tissue engineering.

Collagen has been widely used for tissue engineering. Here, we applied bioactive laminin-derived peptides as an additive for collagen, laminin-111, and fibronectin matrices resulting in peptide/collagen, peptide/laminin-111, and peptide/fibronectin matrices. Several syndecan-binding peptides, including AG73 (RKRLQVQLSIRT), enhanced the cell attachment activity of collagen matrices. AG73 synergistically enhanced not only cell attachment but also cell spreading on collagen matrices. AG73 also enhanced integrin-binding to the collagen matrices, including organization of actin stress fibers and promotion of Tyr397-focal adhesion kinase (FAK) phosphorylation. Additionally, AG73 enhanced neurite outgrowth on collagen matrices. These results suggest that the integrin-mediated biological activity of collagen matrices is synergistically enhanced by the syndecan-mediated cellular function of AG73. Further, cell attachment and spreading activity of laminin-111 and fibronectin matrices was also synergistically enhanced by AG73. The syndecan-binding peptides are useful to enhance the integrin-mediated biological activities of extracellular matrix (ECM) proteins, such as collagen, laminin-111, and fibronectin. The peptide/matrix mixed method is a new concept for biomaterial fabrication and has the potential for wide use in cell and tissue engineering.

The B133 peptide (DSITKYFQMSLE, mouse laminin beta1 chain 1319-1330) promotes cell attachment, and forms amyloid-like fibrils. Here, we evaluated the active core sequences using B133 deletion peptides. B133a, lacking the N-terminal Asp residue, promoted cell spreading via integrin alpha2beta1, whereas B133g, lacking the C-terminal Glu residue, lost the activity. Congo red analysis using the truncated peptides determined that B133g forms amyloid-like fibrils but B133a did not. These results suggest that the N- and C-terminal amino acids contribute to integrin alpha2beta1 binding and to fibril formation, respectively. Further analyses using the truncated peptides showed that the C-terminal eight residues (B133d: KYFQMSLE) are a minimum active sequence for integrin alpha2beta1-mediated cell attachment and the N-terminal nine residues (B133i: DSITKYFQM) are critical for amyloid-like fibril formation. These results suggest that peptide B133 is multifunctional with two different active core sequences: integrin alpha2beta1-mediated cell attachment and amyloid-like fibril formation. Moreover, alanine substitution analysis of B133a indicated that six amino acids, Ile, Thr, Tyr, Phe, Met, and Glu, are important for cell attachment activity. When the Ser residue at the 9th position of B133a was replaced with Ala, the cell attachment activity was enhanced. Further mutation analysis at the 9th position of B133a using various amino acids suggests that hydrophobic amino acids are effective for the integrin alpha2beta1-mediated cell attachment activity. These findings define multifunctional and overlapping sites on the B133 peptide and are useful for designing multifunctional synthetic molecules.

Tribenoside has been used clinically for hemorrhoidal disease associated with coagulation, inflammation, and wounds. However, the pharmacological mechanism of tribenoside activity has never been clear. In this study we examined whether tribenoside affected expression and deposition of laminins that are required for reconstruction of basement membranes (BMs) during wound healing in hemorrhoidal disease. HaCaT cells, which are derived from human epidermis, were treated in growth media supplemented with tribenoside. Reverse transcriptase-polymerase chain reaction (RT-PCR) using primers specific for laminin chains showed that HaCaT cells constitutively expressed laminin alpha3, alpha5, beta1, beta3, gamma1, and gamma2 chains. Tribenoside treatment of HaCaT cells did not induce expression of other laminin chains. We also quantified the expression of laminin chains in tribenoside-treated cells using real-time PCR. The expression level of laminin alpha3, beta1, beta3, gamma1, and gamma2 chains was not affected. In contrast, the expression of laminin alpha5 in the tribenoside-treated cells was four times higher than that of control cells. Immunocytochemistry also showed that tribenoside accelerated the focal deposition of laminin-332 (alpha3, beta3, gamma2). These results suggest that tribenoside interacts with epidermal cells and regulates the expression and localization of laminins to help reconstruct BMs in wound healing of hemorrhoids.

Laminin alpha2 chain is specifically expressed in the basement membrane surrounding muscle and nerve. We screened biologically active sequences in the mouse laminin alpha2 chain G domain using 110 soluble peptides by the peptide-coated plate and the peptide-conjugated Sepharose bead assays. Fourteen peptides showed cell attachment activity in either or both assays. Cell attachment to A2G94 (YFDGTGFAKAVG) was inhibited by anti-integrin beta1 antibody, suggesting that the peptide promotes an integrin beta1-mediated cell attachment. Five peptides promoted PC12 cell neurite outgrowth. Since A2G10 (SYWYRIEASRTG) promoted strong cell attachment in the bead assay but showed slight activity in the plate assay, we conjugated A2G10 to chitosan membranes which increase cell attachment activity of the peptides via conformational stability. A2G10-chitosan membrane promoted an integrin alpha6beta1-mediated cell attachment and spreading with well-organized actin stress fibers and neurite outgrowth. These active peptides are useful for evaluating the molecular mechanisms of laminin-receptor interactions.

Scaffolds are used for bioengineering to regulate cellular functions. Previously, we developed laminin-derived peptide–conjugated chitosan membranes for cell engineering. Here, we determined whether changes in the chitosan scaffold altered the cellular response. When an αvβ3 integrin-binding peptide A99a (ALRGDN) was conjugated on chitosan membranes of varying density (1.5–1500 ng/mm 2 ), cell adhesion was altered depending on the amount of chitosan. 3 or 30 ng/mm 2 of the A99a-chitosan membrane effectively promoted cell attachment, cell spreading with well-organized actin stress fibers, phosphorylation of FAK Tyr397, and neurite outgrowth. In contrast, syndecan-binding peptide AG73 (RKRLQVQLSIRT) conjugated chitosan membranes density (1.5–1500 ng/mm 2 ) promoted similar biological activities at all of the concentrations tested. These results suggest that integrin-mediated cell adhesion is sensitive to the scaffold condition. To improve the function of integrin-mediated biological activities on a large amount of scaffold, we designed an A99a/AG73 mixed peptide–chitosan membrane. The mixed peptide–chitosan membrane promoted the strongest biological activities at 150–1500 ng/mm 2 of chitosan membrane. We conclude that the A99a/AG73 mixed peptide–chitosan membrane effectively interacts with both integrins and syndecans and is a useful multi-functional biomaterial.

Collagen is an abundantly distributed extracellular matrix protein in mammalian bodies that maintains structural integrity of the organs and tissues. Besides its function as a structural protein, collagen has various biological functions which regulate cell adhesion, migration and differentiation. In order to develop totally synthetic collagen-surrogates, we recently reported a basic concept for preparing collagen-like triple helical supramolecules based on the self-assembly of staggered trimeric peptides with self-complementary shapes. In this paper, we add one of the specific cellular functions of the native collagen to the collagen-mimetic supramolecule. We synthesized a self-assembling peptide unit containing the integrin-binding sequence Gly-Phe-Hyp-Gly-Glu-Arg. The supramolecule carrying the sequence exhibited significant binding activity to human dermal fibroblasts. The supramolecular structure was found to be essential for function in in vitro cell culture. Cell adhesion was shown to be comparable to that of native collagen, and was further demonstrated to be mediated solely by integrin alpha 2 beta 1. Well-grown focal contacts and stress fibers were observed in cells spread on the supramolecular collagen-mimetic. The results demonstrate the potential of peptide-based artificial collagen as a biomaterial for regulating specific cellular function and fate.

We have designed multifunctional peptide fibrils using bioactive laminin-derived peptides and evaluated their potential as a biomedical material for tissue engineering. The Leu-Arg-Gly-Asp-Asn (LRGDN) peptide derived from laminin-111, which contains an RGD sequence bound to integrin alphavbeta3, was added to the N-terminus of the four amyloidogenic cell-adhesive laminin-derived peptides (A119: LSNIDYILIKAS, AG97: SAKVDAIGLEIV, B133: DISTKYFQMSLE, and B160: VILQQSAADIAR). The RGD-conjugated peptides were stained with Congo red and exhibited amyloid-like fibril formation in the electron microscopic. The RGD-conjugated peptides promoted human dermal fibroblasts spreading with well-organized actin stress fibers and focal contacts. Human dermal fibroblast attachment to the RGD-conjugated peptides was inhibited by anti-alphav integrin antibody. Further, cell attachment to B133 was inhibited by anti-alpha2 and anti-beta1 integrin antibodies, whereas attachment to RGD-B133 was inhibited by anti-alphav and anti-beta1 integrin antibodies. These results suggest that the RGD-conjugated peptides interact with integrin alphavbeta3 and that RGD-B133 interacts with both integrin alphavbeta3 and integrin beta1. The RGD-conjugated peptide fibrils promoted neurite outgrowth in a peptide-dependent manner. These results support that biologically active sequence-conjugated peptide fibrils interact in a receptor-specific manner with cells and promote multifunctional activities. These fibrils may have use as biological supports for cell-specific tissue engineering.

Syndecans function as receptors for extracellular matrix (ECM) with integrins in cell spreading. However, the molecular mechanism of their specific involvement in cell migration or in wound healing has not been elucidated yet. Here, we report that a synthetic peptide, PEP75, which contains the syndecan-binding sequence of the laminin alpha 3LG4 module, induces keratinocyte migration in in vitro and in vivo. Soluble PEP75 induced the clustering of syndecan-4 and conformation-modified integrin beta1 colocalized with syndecan-4 in soluble PEP75-induced clusters. Treatment of cells in solution with PEP75 resulted in the exposure of the P4G11 antibody epitope of integrin beta1 in immunostaining as well as in flow cytometry and augmented integrin beta1-dependent cell adhesion to ECM. Pulldown assays demonstrated that PEP75 bound to syndecan-4, but not to integrin beta1. A siRNA study revealed a role for syndecan-4 in PEP75-induced up-regulation of P4G11 antibody binding and migration of HaCaT cells. We conclude that binding of soluble PEP75 to syndecan-4 induces the coupling of integrin beta1, which is associated with integrin beta1-conformational changes and activation, and leads to keratinocyte migration. To activate integrin function through syndecans could be a novel therapeutic approach for chronic wound.

Purpose To investigate the effect of a peptide (PHSRN) corresponding to the second cell-binding site of fibronectin on the expression of ZO-1 in cultured human corneal epithelial (HCE) cells. Methods The effects of the PHSRN peptide on the expression of ZO-1, -2, and -3; claudin; and occludin were determined by reverse transcription-polymerase chain reaction (RT-PCR), immunoblot, and immunofluorescence analyses. Phosphorylation of mitogen-activated protein kinases (MAPKs) and the transcription factor c-Jun was assessed with a multiplex analysis system and immunoblot analysis. The barrier function of cultured HCE cells was evaluated by measurement of transepithelial electrical resistance. Results RT-PCR and immunoblot analyses revealed the PHSRN peptide increased the amounts of ZO-1 mRNA and protein in HCE cells in a concentration- and time-dependent manner. The PHSRN peptide had no effect on the expression of ZO-2, ZO-3, claudin, or occludin. Immunofluorescence microscopy showed that the PHSRN peptide did not affect the localization of ZO-1 at the interfaces of neighboring cells. The PHSRN peptide induced the phosphorylation of the MAPKs ERK, p38, and JNK as well as that of c-Jun. The upregulation of ZO-1 expression by the PHSRN peptide was blocked by inhibitors of signaling by ERK (PD098059), p38 (SB203580), or JNK (JNK inhibitor II). The PHSRN peptide had no effect on the transepithelial electrical resistance of cultured HCE cells. Conclusions The PHSRN peptide upregulated the expression of ZO-1 through activation of MAPK signaling pathways in HCE cells. This effect of the PHSRN sequence of fibronectin may contribute to the formation of tight junctions and play an important role in the differentiation of corneal epithelial cells.

Laminin-1, an extracellular matrix molecule, promotes neurite outgrowth through the interaction of integrin and actin. Monosialoganglioside GM1 in the lipid rafts associates with and activates the NGF receptor TrkA, and enhances neurite outgrowth. However, the role of GM1 in laminin-1-induced neurite outgrowth was still unclear. Here, we describe that laminin-1 binds to GM1 through a carbohydrate moiety and a specific conformation of GM1, induces focal formation of large clusters of GM1, and enhances the relocation of TrkA in the membrane of dorsal root ganglion (DRG) and PC12 cells. We found that laminin-1-mediated clustering of GM1 causes the translocation and enrichment of β1 integrin in lipid rafts – where TrkA colocalizes with β1 integrin – and the activation of Lyn, Akt and MAPK to promote the outgrowth of neurites. Our results suggest that the binding of laminin-1 to GM1 facilitates the formation of a focal microdomain in the membrane, and enhances signal transduction that promotes neurite outgrowth by linking NGF-TrkA signaling with the laminin-integrin signaling pathways.

Laminin alpha1 chain LG4 module is multifunctional and interacts with syndecans and integrin alpha2beta1 via AG73 (RKRLQVQLSIRT) and EF-I (DYATLQLQEGRLHFMFDLG) sites, respectively. Here, we conjugated the AG73 and EF1zz (ATLQLQEGRLHFXFDLGKGR, X: Nle) peptides on a chitosan membrane in various ratios to develop an LG4 mimic biomaterial. The AG73-chitosan membrane promoted strong cell attachment with membrane ruffling and the EF1zz-chitosan membrane promoted integrin-mediated cell adhesion with well-organized actin stress fibers. When AG73 and EF1zz were conjugated on a chitosan membrane with 1:9 molar ratio, the mixed peptide-chitosan membrane promoted the strong cell attachment and neurite outgrowth similar to that on the recombinant LG4 protein. Well-organized actin stress fibers and vinculin accumulated focal contacts were observed in the cells attached on the AG73:EF1zz (molar ratio=1:9)-chitosan membrane. These results suggest that the mixed peptide-chitosan membrane interacts with both syndecans and integrin alpha2beta1 and mimics the cell adhesion of a multifunctional LG4 protein. The mixed peptide-chitosan approach has potential as a multifunctional biomaterial for cell and tissue engineering.

Amyloid fibril formation is associated with several pathologies, including Alzheimer's disease, Parkinson's disease, type II diabetes, and prion diseases. Recently, a relationship between basement membrane components and amyloid deposits has been reported. The basement membrane protein, laminin, may be involved in amyloid-related diseases, since laminin is present in amyloid plaques in Alzheimer's disease and binds to amyloid precursor protein. Recently, we showed that peptide A208 (AASIKVAVSADR), the IKVAV-containing peptide, formed amyloid-like fibrils. We previously identified 60 cell adhesive sequences in laminin-1 using a total of 673 12-mer synthetic peptides. Here, we screened for additional amyloidogenic sequences among 60 cell adhesive peptides derived from laminin-1. We first examined amyloid-like fibril formation by the 60 active peptides with Congo red, a histological dye binding to many amyloid-like proteins. Thirteen peptides were stained with Congo red. Four of the 13 peptides promoted cell attachment and neurite outgrowth like the IKVAV-containing peptide. The four peptides also showed amyloid-like fibril formation in both X-ray diffraction and electron microscopic analyses. The amyloidogenic peptides contain consensus amino acid components, including both basic and acidic amino acids and Ser and Ile residues. These results indicate that at least five laminin-derived peptides can form amyloid-like fibrils. We conclude that the laminin-derived amyloidogenic peptides have the potential to form amyloid-like fibrils in vivo, possibly when laminin-1 is degraded.

Extracellular matrix (ECM) plays an important role in tissue regeneration by promoting cell adhesion, migration, proliferation, and differentiation. ECM mimetics are of importance for tissue engineering because of their functions as scaffolds for cells. Previously, we developed bioactive laminin-derived peptide-conjugated chitosan membranes and demonstrated their cell- and peptide-type specific functions. Here, we conjugated twelve integrin-binding peptides derived from ECM proteins onto chitosan membranes and examined biological activity. Seven peptide-chitosan membranes promoted human foreskin fibroblast attachment. Additionally, FIB1 (YAVTGRGDSPAS; from fibronectin), A99 (AGTFALRGDNPQG; from laminin alpha1 chain), EF1zz (ATLQLQEGRLHFXFDLGKGR, X = Nle; from laminin alpha1 chain), and 531 (GEFYFDLRLKGDKY; from collagen alpha1 (IV) chain) conjugated chitosan membranes promoted integrin-dependent cell adhesion. Various integrins, including alphav, beta1, and beta3, were involved in the cell adhesion to the peptide-chitosan membranes. Further, only the FIB1- and A99-chitosan membranes promoted neurite outgrowth with PC12 rat pheochromocytoma cells. These data demonstrate that peptide-chitosan membranes can regulate specific integrin-mediated cell responses and are useful constructs as ECM mimetics.

Nucleoplasmin is the most abundant protein in the Xenopus laevis oocyte nucleus. It is able to assemble nucleosomes by binding histones and transferring them to DNA in vitro, and the first protein named as molecular chaperone [1]. It is an acidic, thermostable protein and forms a stable pentamer. Recently, it was reported that nucleoplasmin removed sperm nuclear binding proteins, protamines, from the sperm nuclei of Xenopus laevis and converted into nucelosome in vitro. Thus nucleoplasmin plays a leading role in the whole fertilization process and may act as both of an assembly and a disassembly factors for remodeling of sperm chromatin in vivo [2, 3]. We have studied the interaction of nucleoplasmin with DNA-salmine complex as a simple model of salmon sperm nuclei in a molecular level, and reported that a poly glutamic acid tract in the carboxyl terminus side of nucleoplasmin is mainly related to the removal of protamine from sperm nuclei [4]. In the present study, the interaction of nucleoplasmin with histone octamer, fractionated histones and also with DNA were investigated by fluorescence to obtain the further information on the mechanism of the conversion into nucleosome by nucleoplasmin.

Amidine-type peptide bond isosteres were designed based on the substitution of the peptide bond carbonyl (C=O) group with an imino (C=NH) group. The positively-charged property of the isosteric part resembles a reduced amide-type peptidomimetic. The peptidyl amidine units were synthesized by the reduction of a key amidoxime (N-hydroxyamidine) precursor, which was prepared from nitrile oxide components as an aminoacyl or peptidyl equivalent. This nitrile oxide-mediated C-N bond formation was also used for peptide macrocyclization, in which the amidoxime group was converted to peptide bonds under mild acidic conditions. Syntheses of the cyclic RGD peptide and a peptidomimetic using both approaches, and their inhibitory activity against integrin-mediated cell attachment, are presented.

Integrins and syndecans mediate cell adhesion to extracellular matrix and their synergistic cooperation is implicated in cell adhesion processes. We previously identified two active peptides, AG73 and EF1, from the laminin alpha1 chain LG4 module, that promote cell attachment through syndecan- and alpha2beta1 integrin-binding, respectively. Here, we examined time-dependent cell attachment on the mixed peptides AG73/EF1. The AG73/EF1 promoted stronger and more rapid cell attachment, spreading, FAK phosphorylation that reached a maximum at 20 min than that on AG73 (40 min) or EF1 (90 min) supplied singly. Thus, the syndecan- and alpha2beta1 integrin-binding peptides synergistically affect cells and accelerate cell adhesion.