Structural Requirement of hA5G18 Peptide (DDFVFYVGGYPS) from Laminin α5 Chain for Amyloid-like Fibril Formation and Cell Adhesion.

The hA5G18 peptide (DDFVFYVGGYPS) identified from the human laminin α5 chain G domain promotes cell attachment and spreading when directly coated on a plastic plate, but does not show activity when it is conjugated on a chitosan matrix. Here, we focused on the structural requirement of hA5G18 for activity. hA5G18 was stained with Congo red and formed amyloid-like fibrils. A deletion analysis of hA5G18 revealed that FVFYV was a minimum active sequence for the formation of amyloid-like fibrils, but FVFYV did not promote cell attachment. Next, we designed functional fibrils using FVFYV as a template for amyloid-like fibrils. When we conjugated an integrin binding sequence Arg-Gly-Asp (RGD) to the FVFYV peptide with Gly-Gly (GG) as a spacer, FVFYVGGRGD promoted cell attachment in a plate coat assay, but a negative control sequence RGE conjugated peptide, FVFYVGGRGE, also showed activity. However, when the peptides were conjugated to Sepharose beads, the FVFYVGGRGD beads showed cell attachment activity, but the FVFYVGGRGE beads did not. These results suggest that RGD and RGE similarly contribute to cell attachment activity in amyloid-like fibrils, but only RGD contributes the activity on the Sepharose beads. Further, we conjugated a basic amino acid (Arg, Lys, and His) to the FVFYV peptide. Arg or Lys-conjugated FVFYV peptides, FVFYVGGR and FVFYVGGK, showed cell attachment activity when they were coated on a plate, but a His-conjugated FVFYV peptide FVFYVGGH did not show activity. None of the basic amino acid-conjugated peptides showed cell attachment in a Sepharose bead assay. The cell attachment and spreading on FVFYVGGR and FVFYVGGK were inhibited by an anti-integrin ß1 antibody. These results suggest that the Arg and Lys residues play critical roles in the interaction with integrins in amyloid-like fibrils. FVFYV is useful to use as a template for amyloid-like fibrils and to develop multi-functional biomaterials.

Lung fibrosis is a novel therapeutic target to suppress lung metastasis of osteosarcoma.

The prognosis of patients with metastatic and recurrent osteosarcoma has not improved over the last 30 years because no effective treatment strategy has been established for lung metastases. Although molecular-targeted drugs that modify the extracellular environment, such as antifibrotic agents, have been developed for cancer treatment, the suppressive effects of antifibrotic agents on osteosarcoma lung metastasis are unclear. Osteosarcomas need to adapt to considerable changes with respect to the stiffness of the environment and fibrosis during lung metastasis and may thus be vulnerable to fibrotic suppression as they originate at the site of a stiff bone with considerable fibrosis. In our study, we investigated whether fibrosis was a therapeutic target for suppressing osteosarcoma metastasis. Lung tissue samples from patients and a mouse model (LM8-Dunn model) showed that lung metastatic colonization of osteosarcoma cells proceeded with massive lung fibrosis. Metastatic osteosarcoma LM8 cells proliferated in a scaffold-dependent manner; the proliferation was less dependent on YAP-mediated mechanotransduction on soft polyacrylamide gels. The antifibrotic agents pirfenidone and nintedanib suppressed lung metastasis in the LM8-Dunn model. The osteosarcoma cells did not show increased proliferation, as reported in breast cancer, after continuous culture in a soft environment. We speculated that the antifibrotic agents were effective because the osteosarcoma cells remained scaffold-dependent in the soft tissue environment. Thus, antifibrotic strategies may be useful in suppressing lung metastasis of bone and soft tissue tumors with stiff primary sites such as those in osteosarcoma.

Conformational dependence of integrin-binding peptides derived from homologous loop regions in the laminin α chains.

Laminin α chains (α1-α5 chains) are expressed in a tissue- and developmental stage-specific manner and have diverse chain-specific biological functions. Especially, laminin globular (LG) modules (LG1-LG5) located at the C-terminus of the α chains play a critical role in the biological activities of laminins. Each LG module is composed of a 14-stranded ß-sheet (A-N) sandwich structure. We previously screened cell attachment activity of the loop regions between the E and F strands in the LG modules using 17 homologous peptides (EF peptides) and found that four active EF peptides bind to integrin α2ß1. One of the four peptides, G4EF1 demonstrated improved cell attachment activity when cyclized. Here, we focused on the remaining three integrin α2ß1-binding EF peptides (G5EF1, G3EF3, and G5EF5) and analyzed the relationship between their peptide conformation and cell attachment activity. First, we determined their active core sequences and found that G5EF1z (IGLEIVDGKVLFHVNN), G3EF3z (LLVTLEDGHIALST), and G5EF5z (KVLTEQVL) are the core sequences. Cyclic peptides of the core sequences (cycloG5EF1z, cycloG3EF3z, and cycloG5EF5z) enhanced integrin-mediated cell adhesion activity compared with their linear peptides. The results indicated that cell adhesion activity of the integrin α2ß1-binding EF peptides is conformation dependent and that the loop structure is critical for their activity. This suggests that conformation of the loop regions plays an important role for the activities of the LG modules.

Molecular evidence of IGFBP-3 dependent and independent VD3 action and its nonlinear response on IGFBP-3 induction in prostate cancer cells.

BACKGROUND: Clinical trials have been conducted to clarify the beneficial effects of VD3 (1α,25-dihydroxy vitamin D3, also known as calcitriol) treatment in prostate cancer. However, the molecular mechanisms underlying these effects are not fully understood. Recent studies on IGFBP-3 have indicated its intracellular functions in cell growth and apoptosis. The aim of this study was to confirm the benefits of low-dose VD3 treatment and clarify the molecular mechanisms underlying these beneficial effects in prostate cancer cells. METHODS: The molecular effects of simultaneous treatment of LNCaP cells and their genetically modified cell lines with low concentration of docetaxel and VD3 were biologically and biochemically analyzed. To further determine the effects of VD3 treatment on IGFBP-3 induction system, cells were temporarily treated with VD3 in combination with a transcriptional inhibitor or protein synthesis inhibitor. Bcl-2 protein and its mRNA behavior were also observed in Igfbp-3 expression-modified LNCaP cells to determine the involvement of IGFBP-3 in the suppression of Bcl-2 by VD3 treatment. RESULTS: Changes in IGFBP-3 expression levels in LNCaP cells indicated that it mediated the inhibition of cell growth induced by VD3 treatment. IGFBP-3 was also found to be a mediator of the enhanced cytotoxicity of prostate cancer cells to VD3 in combination with the anti-cancer drug. We further identified the distinct property of the IGFBP-3 induction system, wherein temporal VD3 stimulation-induced prolonged IGFBP-3 expression and VD3 treatment-induced increase in IGFBP-3 expression were optimized based on the protein concentration rather than the mRNA concentration. Meanwhile, Bcl-2 expression was down-regulated by VD3 treatment in an IGFBP-3-independent manner. CONCLUSION: These findings indicate the molecular mechanisms of IGFBP-3 induction stimulated by VD3 and IGFBP-3 independent Bcl-2 suppression by VD3 treatment in prostate cancer cells. The results could prompt a re-evaluation of VD3 usage in therapy for patients with prostate cancer.

Identification of active sequences in human laminin α5 G domain.

Human laminin-511 (α5ß1γ1) and its truncated protein, laminin-511 E8 fragment, bind to integrin α6ß1 and have been widely used for embryonic stem cell and induced pluripotent stem cell culture under feeder-free conditions. In this study, we focused on human laminin α5 chain G domain, which is thought to be critical for the biological functions of laminin-511, and screened its biologically active sequences using a synthetic peptide library. We synthesized 115 peptides (hA5G1-hA5G115) covering the entire laminin α5 chain G domain and evaluated cell attachment activity using both the peptide-coated plate and peptide-chitosan matrix (peptide-ChtM) assays. Seventeen peptides demonstrated cell attachment activity in the assays. Both hA5G18 and hA5G26-coated plates and hA5G74-ChtMs promoted integrin ß1-mediated cell attachment. These findings are useful for the study of molecular mechanisms of laminin-511, and the active peptides have a potential for use as a molecular probe for cell adhesion receptors.

Characterization of dystroglycan binding in adhesion of human induced pluripotent stem cells to laminin-511 E8 fragment.

Human induced pluripotent stem cells (hiPSCs) grow indefinitely in culture and have the potential to regenerate various tissues. In the development of cell culture systems, a fragment of laminin-511 (LM511-E8) was found to improve the proliferation of stem cells. The adhesion of undifferentiated cells to LM511-E8 is mainly mediated through integrin α6ß1. However, the involvement of non-integrin receptors remains unknown in stem cell culture using LM511-E8. Here, we show that dystroglycan (DG) is strongly expressed in hiPSCs. The fully glycosylated DG is functionally active for laminin binding, and although it has been suggested that LM511-E8 lacks DG binding sites, the fragment does weakly bind to DG. We further identified the DG binding sequence in LM511-E8, using synthetic peptides, of which, hE8A5-20 (human laminin α5 2688-2699: KTLPQLLAKLSI) derived from the laminin coiled-coil domain, exhibited DG binding affinity and cell adhesion activity. Deletion and mutation studies show that LLAKLSI is the active core sequence of hE8A5-20, and that, K2696 is a critical amino acid for DG binding. We further demonstrated that hiPSCs adhere to hE8A5-20-conjugated chitosan matrices. The amino acid sequence of DG binding peptides would be useful to design substrata for culture system of undifferentiated and differentiated stem cells.

Biological activities of laminin-111-derived peptide-chitosan matrices in a primary culture of rat cortical neurons.

Cell adhesive biomaterials have been used for various cells in culture, especially for primary cultures of neurons. Here we examined laminin-111 and its active peptides conjugated to chitosan matrices (ChtMs) for primary culture of rat cortical neurons. Laminin-111 on poly-d-lysine substrate promoted neuronal cell attachment and differentiation. The biological activity of six active laminin-111-derived peptides was examined using a peptide-ChtM construct. When the syndecan-binding peptides, AG73 (RKRLQVQLSIRT, mouse laminin α1 chain 2719-2730) and C16 (KAFDITYVRLKF, laminin γ1 chain 139-150), were conjugated to chitosan, AG73-ChtM and C16-ChtM showed potent neuronal cell attachment activity and promoted axon extension by primary cultured rat cortical neurons. However, the remaining peptides, including integrin-binding peptides, did not show activity when conjugated to ChtM. AG73-ChtM and C16-ChtM also supported neuron survival for at least 4 weeks in serum-free medium without a glia feeder layer. These data suggest that AG73-ChtM and C16-ChtM are useful for primary cultures of central nervous system neurons and have a potential for use as functional biomaterials for tissue engineering in the central nervous system.

Identification of laminin α5 short arm peptides active for endothelial cell attachment and tube formation.

Laminin-511, a major component of endothelial basement membrane, consists of α5, ß1, and γ1 chains. The short arm region of the α5 chain is a structural feature of endothelial laminins. In this study, we identified active sequences for human umbilical vein endothelial cells (HUVECs) using recombinant proteins and synthetic peptides. The short arm of the α5 chain contains three globular domains [laminin N-terminal globular domain, laminin 4 domain a, and laminin 4 domain b (LN, L4a, and L4b)] and three rod-like elements [laminin epidermal growth factor-like domain a, b, and c (LEa, LEb, and LEc)]. The cell attachment assay using recombinant proteins showed that RGD-independent cell attachment sites were localized in the α5LN-LEa domain. Further, we synthesized 70 peptides covering the amino acid sequences of the α5LN-LEa domain. Of the 70 peptides, A5-16 (mouse laminin α5 230-243: LENGEIVVSLVNGR) potently exhibited endothelial cell attachment activity. An active sequence analysis using N-terminally and C-terminally truncated A5-16 peptides showed that the nine-amino acid sequence IVVSLVNGR was critical for the endothelial cell attachment activity. Cell adhesion to the peptides was dependent on both cations and heparan sulfate. Further, the A5-16 peptide inhibited the capillary-like tube formation of HUVECs with the cells forming small clumps with short tubes. The eight-amino acid sequence EIVVSLVN in the A5-16 peptide was critical to inhibit HUVEC tube formation. This amino acid sequence could be useful for grafts and thus modulate endothelial cell behavior for vascular surgery. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Biological activity of peptide-conjugated polyion complex matrices consisting of alginate and chitosan.

Peptide-conjugated polysaccharide matrices using bioactive laminin-derived peptides are useful biomaterials for tissue and cell engineering. Here, we demonstrate an easy handling preparation method for peptide-polysaccharide matrices using polyion complex with both alginate and chitosan. First, aldehyde-alginate was synthesized by oxidization of alginate using NaIO4 , and then, reacted with Cys-peptides. Next, the peptide-alginate solution was added to a chitosan-coated plate, and the peptide-polyion complex matrices (peptide-PCMs) were prepared. The peptide-PCMs using an integrin αvß3-binding peptide (A99a: ALRGDN, mouse laminin α1 chain 1145-1150) and an integrin α2ß1-binding peptide (EF1XmR: RLQLQEGRLHFXFD, X = Nle, mouse laminin α1 chain 2751-2763) showed strong cell attachment activity in a dose-dependent manner. When we examined the effect of various spacers on the biological activity of A99a-PCM, hydrophobic and long spacers enhanced the cell attachment activity. Further, the A99a-PCM with the spacers strongly promoted neurite outgrowth. The polyion complex method is an easy way to obtain insolubilized matrix and is widely applicable for various polysaccharides. The peptide-PCM is useful as a biomaterial for cell and tissue engineering.

Effect of spacer length and type on the biological activity of peptide-polysaccharide matrices.

Peptide-polysaccharide matrices can mimic extracellular matrix structure and function and are useful for tissue and cell engineering. The spacer between the peptide and the polysaccharide is important for both peptide conformation and the interaction between the peptide and receptors. Here, the effect of a spacer on the biological activity of peptide-polysaccharide matrices using various lengths of spacers consisting of glycine, ß-alanine, and ε-aminocaproic acid has been examined. Active laminin-derived peptides, including a syndecan-binding peptide (AG73: RKRLQVQLSIRT), an integrin αvß3-binding peptide (A99a: ALRGDN), and an integrin α6ß1-binding peptide (A2G10: SYWYRIEASRTG), were used as the peptide ligands and chitosan was used as a polysaccharide matrix. The spacers did not influence the biological activity of the AG73-chitosan matrix. In contrast, the integrin-binding peptide-chitosan matrices showed spacer-dependent activity. Hydrophobic spacers enhanced the cell attachment activity of the A99a-chitosan matrix. A four-glycine spacer showed the strongest effect for the biological activity of the A2G10-chitosan matrix. These results suggested that spacer-optimization for each peptide is important for designing effective peptide-polysaccharide matrices. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 512-520, 2016.

Identification of fibronectin binding sites in dermatopontin and their biological function.

BACKGROUND: Dermatopontin (DP) is a 22kDa acidic extracellular matrix (ECM) protein that plays a critical role in both ECM structure and wound healing. Previously, we demonstrated that DP interacts with fibronectin (Fn) and promotes formation of insoluble Fn fibrils that are called activated Fn (Kato et al., J Biol Chem 2011;286:14861-9). OBJECTIVE: Details of the interaction between DP and Fn are investigated to further examine the biological functions of DP. METHODS: Interactive sites between Fn and DP were examined by a solid-phase assay using Fn, DP, and their respective recombinant proteins and synthetic peptides. The effect of the DP peptides on insoluble Fn fibril formation was examined by both electrophoresis and electron microscopy. RESULTS: A binding site in DP for Fn was identified as the DP-4 (PHGQVVVAVRS) peptide, and the major binding site in Fn for DP was the 14th type III repeat (III14) domain. Further, the major DP binding site in the III14 domain was located around the B- and C-strands and their connecting loop region. A synthetic cyclic peptide mimicking the Fn loop structure enhanced DP binding activity. The DP-4 peptide induced Fn polymerization but the morphology was different from that of Fn fibrils formed by full length DP. The Fn fibrils with DP-4 enhanced integrin α5ß1-mediated cell adhesion and spreading. CONCLUSION: Interactive sites between Fn and DP were identified. The DP-4 peptide activated Fn and enhanced cell adhesion activity. DP-4 has the potential to be used for therapeutic applications, such as a wound treatment.

Functional peptide of dermatopontin produces fibrinogen fibrils and modifies its biological activity.

BACKGROUND: Dermatopontin (DP), a small extracellular matrix protein, interacts with both fibrinogen and fibrin. DP accelerates fibrin fibril formation and enhances cell adhesion to fibrin fibrils but DP does not influence fibrinogen fibril formation. We have previously demonstrated that DP-4 (PHGQVVVAVRS) is a functional dermatopontin peptide (Wu et al., 2014). OBJECTIVE: Identification of biological functions of DP-4. METHODS: Protein-protein interactions were examined by solid-phase assay. The kinetics of fibrinogen/fibrin polymer formation was monitored by turbidity change, SDS-PAGE, and electron microscopy. A cell adhesion assay was performed using human umbilical vein endothelial cells. RESULTS: Although DP promoted fibrin formation, the DP-4 peptide promoted fibrinogen polymerization but did not apparently affect fibrin formation. The polymerized fibrinogen formed straight solid fibrils comparable to the normally formed fibrin fibrils. A minimum functional sequence of the DP-4 peptide was determined to be VVVAVRS. An αC domain in fibrinogen was involved in the fibril formation. Fibrinogen fibrils made by DP-4 enhanced endothelial cell adhesion and spreading in a dose-dependent manner. This cell adhesion was inhibited by heparin and by anti-αvß3 and ß1 integrin antibodies. CONCLUSION: DP-4 did not reproduce the full functional biological activities of DP with fibrin but DP-4 did promote fibrinogen fibril formation. The fibrinogen fibrils produced by DP-4 are useful as a novel synthetic biomaterial for therapeutic applications.