The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Hynda K Kleinman - One of the best experts on this subject based on the ideXlab platform.
-
identification of a potent peptide antagonist to an active Laminin 1 sequence that blocks angiogenesis and tumor growth
Cancer Research, 2003Co-Authors: Lourdes M Ponce, Mayumi Mochizuki, Motoyoshi Nomizu, Suguru Hibino, Agata M Lebioda, Hynda K KleinmanAbstract:The extracellular matrix plays an important role in many physiological processes. We have identified >20 angiogenic sites in the extracellular matrix protein Laminin-1. The most potent sites are A13 (RQVFQVAYIIIKA) and C16 (KAFDITYVRLKF), which are present in homologous NH(2)-terminal domains of the alpha 1 and gamma 1 chains, respectively. We reported recently that a scrambled C16 sequence, C16S (DFKLFAVTIKYR), acts as an antagonist to both peptides. Here, we have identified a stronger antiangiogenic peptide, C16Y (C16S with a T to Y substitution), with potent activity in several biological assays including tumor growth. C16Y is more potent in promoting endothelial cell attachment and inhibiting attachment to Laminin-1 than either C16 or C16Y. Disruption of tube formation by C16Y is also observed at concentrations at least five times lower than C16S. The minimal active sequence was found to be DFKLFAVY. C16Y is more potent in blocking C16-induced chick chorioallantoic membrane angiogenesis than C16S. Tumor growth studies on the chick chorioallantoic membrane showed that C16Y reduces breast cancer cell growth without affecting cell proliferation. This result suggests that angiogenesis is being inhibited by the peptide. In vivo animal studies demonstrated that C16Y treatment significantly reduced tumor growth and decreased tumor vessel number, as compared with controls, additionally suggesting that angiogenesis was affected. These results indicate that we have identified a more potent antiangiogenesis inhibitor peptide that may be used as a therapeutic to treat cancer.
-
the b16f10 cell receptor for a metastasis promoting site on Laminin 1 is a heparan sulfate chondroitin sulfate containing proteoglycan
Cancer Research, 2002Co-Authors: Jean A Engbring, Matthew P Hoffman, Arezo J Karmand, Hynda K KleinmanAbstract:Exposure to AG73, a synthetic peptide (LQVQLSIR) from the COOH-terminal region of the Laminin α1 chain, induces a malignant phenotype in B16F10 melanoma cells. Coinjection of this peptide with the cells results in an increase of lung tumors and also the formation of liver tumors in ∼50% of the mice (W. H. Kim et al. , Int. J. Cancer, 77: 632–639, 1998). Here we have characterized the cell surface receptor and its functional groups on B16F10 cells. Peptide affinity chromatography identified a cell surface protein eluting with 1 m NaCl, which ran in SDS gels as a broad band of M r ∼150,000–200,000. Digestion with heparitinase and chondroitinase produced a core protein of lower molecular weight ( M r ∼90,000). Involvement of the glycosaminoglycan (GAG) side chains was demonstrated by inhibition of cell binding to the peptide by heparin, heparan sulfate, and chondroitin sulfate B, but not by chondroitin sulfates A or C, or hyaluronic acid. The IC50 for heparin was the lowest, followed by heparan sulfate, then chondroitin sulfate B, suggesting that the overall sulfation of the GAG side chain is critical. This was confirmed by inhibition of attachment with chemically modified heparin and heparan sulfate, which also showed that N or O linkages were not important for function. Using sized heparin fragments to inhibit cell binding to the peptide demonstrated that 16-mer is the minimum length required. B16F10 cells form a network when grown on Matrigel, and this is prevented by addition of the AG73 peptide. The GAGs alone did not affect network formation, but heparin, heparan sulfate, and chondroitin sulfate B reversed the inhibitory effect of the peptide, whereas other GAGs were inactive. Furthermore, removal of cell surface GAGs inhibited cell attachment to the peptide. Cells treated with glycosidases and coinjected with the peptide formed liver tumors equal to the control group receiving no peptide, suggesting that the GAGs play an early role in peptide-mediated tumor metastasis. These data indicate that the B16F10 cell receptor for a Laminin metastasis-promoting sequence is a heparan sulfate/chondroitin sulfate-containing proteoglycan, and these GAG side chains are functionally important in the cell-peptide interaction.
-
Laminin 1 and α6β1 integrin regulate acinar morphogenesis of normal and malignant human prostate epithelial cells
The Prostate, 2001Co-Authors: Diana Bellodeocampo, Hynda K Kleinman, Nestor D Deocampo, Mukta M WebberAbstract:Background Cell–matrix interactions via integrin receptors are critical for acinar morphogenesis. The non-tumorigenic, human prostate epithelial cell line RWPE-1 was used in a three-dimensional (3D) cell culture model to identify the matrix protein and its integrin receptor required for acinar morphogenesis. Methods 3D cultures, immunostaining, confocal microscopy, and Western blot analysis were used to examine acinar formation on matrix proteins and to determine integrin receptor expression. Results RWPE-1 cells differentiate into acini of polarized cells with a distinct lumen in 3D Matrigel culture. In contrast, the malignant WPE1-NB26 prostate epithelial cells form solid cell masses. In 3D gels of Laminin-1, type IV collagen, or fibronectin, RWPE-1 cells form acini only in Laminin-1. Anti-Laminin-1 antibody reduces acinar formation in a dose-dependent manner. Polarized RWPE-1 cells showed basal expression of α6 and β1 integrin subunits. Blocking antibodies to α6 or β1 reduced acinar formation to 9 and 6? of control, respectively. The β1 integrin colocalized with focal adhesion kinase (FAK). Inhibition of extracellular signal-regulated kinase kinase activity significantly reduced acinar formation to 38? of control, suggesting that β1 integrin-mediated signal transduction may be regulated through a FAK pathway. Conclusions While basal expression of α6β1 integrin in RWPE-1 cells correlates with their ability to polarize and form acini, a decrease or loss of α6, and diffused β1 expression in WPE1-NB26 cells correlates with loss of acinar-forming ability. Results show that Laminin-1 and a functional α6β1 integrin receptor are required for acinar morphogenesis. This novel 3D cell culture model is useful for elucidating regulation of acinar morphogenesis and its loss during prostate carcinogenesis. Prostate 46:142–153, 2001. © 2001 Wiley-Liss, Inc.
-
neural cell response to multiple novel sites on Laminin 1
Journal of Neuroscience Research, 2000Co-Authors: Sharon K Powell, Yoshihiko Yamada, Motoyoshi Nomizu, Jayashree Rao, Eva Roque, Yuichiro Kuratomi, Hynda K KleinmanAbstract:The basement membrane protein Laminin-1 is a potent stimulator of neurite outgrowth for a variety of neuronal cell types. Previous studies have identified neurite outgrowth activity in several distinct regions of the Laminin-1 molecule. In this study, 545 overlapping 12- to 14-mer synthetic peptides, corresponding to most of the amino acid sequence of the alpha1, beta1, and gamma1 chains of Laminin-1, were screened for cell attachment and neurite outgrowth activity using primary cultures of mouse cerebellar granule neurons and two neuronal cell lines. We identified 48 peptides derived from novel regions of the Laminin-1 molecule that were positive for neural cell adhesion activity. Only the cerebellar cells were found to have true neurite outgrowth activity with certain of the peptides, whereas some peptides induced short spike-like process with the cell lines. Although 23 of these peptides were active on all 3 cell types screened, 25 others showed cell-type specificity in their activity. These studies show that (1) there are multiple and distinct sites on Laminin-1 for cell adhesion and neurite-like outgrowth and (2) that there are neural cell-type-specific active domains. The multiple active sites found explains, in part, the potent activity of Laminin-1 on neurite outgrowth.
-
identification of Laminin α1 and β1 chain peptides active for endothelial cell adhesion tube formation and aortic sprouting
The FASEB Journal, 1999Co-Authors: Katherine M Malinda, Yoshihiko Yamada, Motoyoshi Nomizu, Hynda K Kleinman, Melissa Chung, Mucio Delgado, Yuchiro Kuratomi, Lourdes M PonceAbstract:Laminin-1 is a basement membrane glycoprotein that promotes several biological activities including cell attachment, tumor metastasis, and angiogenesis. Angiogenesis plays an important role in tissue formation, reproduction, wound healing, and several pathological conditions. In this study, we screened 405 synthetic peptides from the α1 and β1 chains to identify potential sites on Laminin-1 active with endothelial cells. Peptides were initially screened by testing both endothelial cell adhesion to peptide-coated wells and tube formation on Matrigel in the presence of soluble peptide. Twenty active peptides were identified in these screens. A secondary screen using the rat aortic ring sprouting assay identified 13 of the 20 peptides that stimulated endothelial sprouting. Several of these active peptides were also found to stimulate human umbilical vein endothelial cell migration in Boyden chamber assays. Differences in the amount of peptide needed for the response and in the resultant morphologies/response...
Motoyoshi Nomizu - One of the best experts on this subject based on the ideXlab platform.
-
identification of multiple amyloidogenic sequences in Laminin 1
Biochemistry, 2007Co-Authors: Shingo Kasai, Kentaro Hozumi, Naoki Ichikawa, Yoshihiko Yamada, Yuichi Kadoya, Norio Nishi, Shunsuke Urushibata, Fumiharu Yokoyama, Nobuhisa Watanabe, Motoyoshi NomizuAbstract: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.
-
identification of a potent peptide antagonist to an active Laminin 1 sequence that blocks angiogenesis and tumor growth
Cancer Research, 2003Co-Authors: Lourdes M Ponce, Mayumi Mochizuki, Motoyoshi Nomizu, Suguru Hibino, Agata M Lebioda, Hynda K KleinmanAbstract:The extracellular matrix plays an important role in many physiological processes. We have identified >20 angiogenic sites in the extracellular matrix protein Laminin-1. The most potent sites are A13 (RQVFQVAYIIIKA) and C16 (KAFDITYVRLKF), which are present in homologous NH(2)-terminal domains of the alpha 1 and gamma 1 chains, respectively. We reported recently that a scrambled C16 sequence, C16S (DFKLFAVTIKYR), acts as an antagonist to both peptides. Here, we have identified a stronger antiangiogenic peptide, C16Y (C16S with a T to Y substitution), with potent activity in several biological assays including tumor growth. C16Y is more potent in promoting endothelial cell attachment and inhibiting attachment to Laminin-1 than either C16 or C16Y. Disruption of tube formation by C16Y is also observed at concentrations at least five times lower than C16S. The minimal active sequence was found to be DFKLFAVY. C16Y is more potent in blocking C16-induced chick chorioallantoic membrane angiogenesis than C16S. Tumor growth studies on the chick chorioallantoic membrane showed that C16Y reduces breast cancer cell growth without affecting cell proliferation. This result suggests that angiogenesis is being inhibited by the peptide. In vivo animal studies demonstrated that C16Y treatment significantly reduced tumor growth and decreased tumor vessel number, as compared with controls, additionally suggesting that angiogenesis was affected. These results indicate that we have identified a more potent antiangiogenesis inhibitor peptide that may be used as a therapeutic to treat cancer.
-
Laminin-1 peptide-conjugated chitosan membranes as a novel approach for cell engineering.
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 2003Co-Authors: Mayumi Mochizuki, Masanori Yamada, Yuichi Kadoya, Yoko Wakabayashi, Kozue Kato, Ikuko Okazaki, Taku Sato, Nobuo Sakairi, Norio Nishi, Motoyoshi NomizuAbstract: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.
-
an association of igg anti Laminin 1 autoantibodies with endometriosis in infertile patients
Human Reproduction, 2003Co-Authors: Junko Inagaki, Motoyoshi Nomizu, Mayumi Sugiuraogasawara, Mikiya Nakatsuka, Katsuo Ikuta, Nobuharu Suzuki, Keiko Kaihara, Kazuko Kobayashi, Tatsuji Yasuda, Yehuda ShoenfeldAbstract:Background Laminin-1, a multifunctional glycoprotein of the basement membrane, is thought to be important in embryogenesis, embryonic implantation, and placentation. We recently showed that serum IgG anti-Laminin-1 autoantibodies (auto-Abs) are associated with recurrent first-trimester miscarriages. The present study assessed the clinical significance of anti-Laminin-1 Abs with infertility, accompanied with or without endometriosis. Methods Sixty-eight infertile patients who underwent laparoscopy or laparotomy and 39 healthy non-pregnant women were tested for IgG anti-Laminin-1 Abs. The association between the Abs and endometriosis was analysed. The presence of Laminin-1 mRNA was detected in endometriotic lesions. Results Twenty infertile patients were positive for anti-Laminin-1 Abs. The Ab levels in those patients were significantly higher than those in healthy non-pregnant women (P = 0.0005). The presence of the Abs was significantly associated with endometriosis in those patients (P = 0.0096). The Abs recognized a particular domain, i.e., the Laminin-alpha1 chain G domain. mRNA encoding Laminin-alpha1, -beta1, and -gamma1 chains was expressed in 90% of endometriotic lesions. Conclusions IgG anti-Laminin-1 Abs were significantly associated with endometriosis in infertile patients. The Abs might be clinically important in the development of autoimmune-mediated reproductive failures and the assessment of the Abs may provide a novel non-invasive diagnosis of endometriosis.
-
neural cell response to multiple novel sites on Laminin 1
Journal of Neuroscience Research, 2000Co-Authors: Sharon K Powell, Yoshihiko Yamada, Motoyoshi Nomizu, Jayashree Rao, Eva Roque, Yuichiro Kuratomi, Hynda K KleinmanAbstract:The basement membrane protein Laminin-1 is a potent stimulator of neurite outgrowth for a variety of neuronal cell types. Previous studies have identified neurite outgrowth activity in several distinct regions of the Laminin-1 molecule. In this study, 545 overlapping 12- to 14-mer synthetic peptides, corresponding to most of the amino acid sequence of the alpha1, beta1, and gamma1 chains of Laminin-1, were screened for cell attachment and neurite outgrowth activity using primary cultures of mouse cerebellar granule neurons and two neuronal cell lines. We identified 48 peptides derived from novel regions of the Laminin-1 molecule that were positive for neural cell adhesion activity. Only the cerebellar cells were found to have true neurite outgrowth activity with certain of the peptides, whereas some peptides induced short spike-like process with the cell lines. Although 23 of these peptides were active on all 3 cell types screened, 25 others showed cell-type specificity in their activity. These studies show that (1) there are multiple and distinct sites on Laminin-1 for cell adhesion and neurite-like outgrowth and (2) that there are neural cell-type-specific active domains. The multiple active sites found explains, in part, the potent activity of Laminin-1 on neurite outgrowth.
Matthew P Hoffman - One of the best experts on this subject based on the ideXlab platform.
-
the b16f10 cell receptor for a metastasis promoting site on Laminin 1 is a heparan sulfate chondroitin sulfate containing proteoglycan
Cancer Research, 2002Co-Authors: Jean A Engbring, Matthew P Hoffman, Arezo J Karmand, Hynda K KleinmanAbstract:Exposure to AG73, a synthetic peptide (LQVQLSIR) from the COOH-terminal region of the Laminin α1 chain, induces a malignant phenotype in B16F10 melanoma cells. Coinjection of this peptide with the cells results in an increase of lung tumors and also the formation of liver tumors in ∼50% of the mice (W. H. Kim et al. , Int. J. Cancer, 77: 632–639, 1998). Here we have characterized the cell surface receptor and its functional groups on B16F10 cells. Peptide affinity chromatography identified a cell surface protein eluting with 1 m NaCl, which ran in SDS gels as a broad band of M r ∼150,000–200,000. Digestion with heparitinase and chondroitinase produced a core protein of lower molecular weight ( M r ∼90,000). Involvement of the glycosaminoglycan (GAG) side chains was demonstrated by inhibition of cell binding to the peptide by heparin, heparan sulfate, and chondroitin sulfate B, but not by chondroitin sulfates A or C, or hyaluronic acid. The IC50 for heparin was the lowest, followed by heparan sulfate, then chondroitin sulfate B, suggesting that the overall sulfation of the GAG side chain is critical. This was confirmed by inhibition of attachment with chemically modified heparin and heparan sulfate, which also showed that N or O linkages were not important for function. Using sized heparin fragments to inhibit cell binding to the peptide demonstrated that 16-mer is the minimum length required. B16F10 cells form a network when grown on Matrigel, and this is prevented by addition of the AG73 peptide. The GAGs alone did not affect network formation, but heparin, heparan sulfate, and chondroitin sulfate B reversed the inhibitory effect of the peptide, whereas other GAGs were inactive. Furthermore, removal of cell surface GAGs inhibited cell attachment to the peptide. Cells treated with glycosidases and coinjected with the peptide formed liver tumors equal to the control group receiving no peptide, suggesting that the GAGs play an early role in peptide-mediated tumor metastasis. These data indicate that the B16F10 cell receptor for a Laminin metastasis-promoting sequence is a heparan sulfate/chondroitin sulfate-containing proteoglycan, and these GAG side chains are functionally important in the cell-peptide interaction.
-
Laminin 1 and Laminin 2 g domain synthetic peptides bind syndecan 1 and are involved in acinar formation of a human submandibular gland cell line
Journal of Biological Chemistry, 1998Co-Authors: Matthew P Hoffman, Motoyoshi Nomizu, Eva Roque, Dale W Jung, Y Yamada, Hynda K KleinmanAbstract:Abstract The culture of human submandibular gland (HSG) cells on Laminin-1 induces acinar differentiation. We identified a site on Laminin involved in acinar differentiation using synthetic peptides derived from the C-terminal G-domain of the Laminin α1 and α2 chains. The α1 chain peptide AG73 (RKRLQVQLSIRT) decreases the size of acini formed on Laminin-1. Cells cultured with either AG73 or the homologous α2 chain peptide MG73 (KNRLTIELEVRT) form structures that appear acinar-like, but the cell nuclei are not polarized to the basal surface and no lumen formation occurs, indicating that additional sites on Laminin are required for complete differentiation. The G-domain of Laminin-1 contains both integrin and heparin binding sites, and anti-β1-integrin antibodies disrupt acinar formation. Cell adhesion to the peptides and to E3, an elastase digest fragment of Laminin-1 containing AG73, is specific, since other Laminin peptides or EDTA do not compete the binding. Heparin and heparan sulfate decrease cell adhesion to AG73 and MG73 but anti-β1-integrin antibodies have no effect. Treating the cell surface with heparitinase inhibits adhesion to both AG73 and MG73. We isolated cell surface ligands using both peptide affinity chromatography and Laminin-1 affinity chromatography. Treating the material bound to the affinity columns with heparitinase and chondroitinase enriches for a core protein identified as syndecan-1 by Western blot analysis, thus identifying a syndecan-1 binding site in the globular domain of Laminin-1 and Laminin-2. In summary, multiple interactions between Laminin and HSG cells contribute to acinar differentiation, involving both β1-integrins and syndecan-1.
-
Laminin 1 and Laminin 2 g domain synthetic peptides bind syndecan 1 and are involved in acinar formation of a human submandibular gland cell line
Journal of Biological Chemistry, 1998Co-Authors: Matthew P Hoffman, Motoyoshi Nomizu, Eva Roque, Dale W Jung, Y Yamada, Sharon Lee, Hynda K KleinmanAbstract:The culture of human submandibular gland (HSG) cells on Laminin-1 induces acinar differentiation. We identified a site on Laminin involved in acinar differentiation using synthetic peptides derived from the C-terminal G-domain of the Laminin alpha1 and alpha2 chains. The alpha1 chain peptide AG73 (RKRLQVQLSIRT) decreases the size of acini formed on Laminin-1. Cells cultured with either AG73 or the homologous alpha2 chain peptide MG73 (KNRLTIELEVRT) form structures that appear acinar-like, but the cell nuclei are not polarized to the basal surface and no lumen formation occurs, indicating that additional sites on Laminin are required for complete differentiation. The G-domain of Laminin-1 contains both integrin and heparin binding sites, and anti-beta1-integrin antibodies disrupt acinar formation. Cell adhesion to the peptides and to E3, an elastase digest fragment of Laminin-1 containing AG73, is specific, since other Laminin peptides or EDTA do not compete the binding. Heparin and heparan sulfate decrease cell adhesion to AG73 and MG73 but anti-beta1-integrin antibodies have no effect. Treating the cell surface with heparitinase inhibits adhesion to both AG73 and MG73. We isolated cell surface ligands using both peptide affinity chromatography and Laminin-1 affinity chromatography. Treating the material bound to the affinity columns with heparitinase and chondroitinase enriches for a core protein identified as syndecan-1 by Western blot analysis, thus identifying a syndecan-1 binding site in the globular domain of Laminin-1 and Laminin-2. In summary, multiple interactions between Laminin and HSG cells contribute to acinar differentiation, involving both beta1-integrins and syndecan-1.
-
role of Laminin 1 and tgf beta 3 in acinar differentiation of a human submandibular gland cell line hsg
Journal of Cell Science, 1996Co-Authors: Matthew P Hoffman, Maura C Kibbey, John J Letterio, Hynda K KleinmanAbstract:Previous studies show that culturing an immortalized human submandibular gland cell line (HSG) on Matrigel, a basement membrane extract, induces cytodifferentiation. We have further defined this model system and identified factors involved in HSG cell acinar development and cyto-differentiation. Acinar development is marked by cell migration into multi-cellular spherical structures, cell proliferation and apoptosis of the centrally localized cells. In addition, functional differentiation was determined by indirect immunofluorescence and immunoblot analysis for cystatin, a salivary gland acinar cell-specific protein found to be produced by differentiated HSG cells. Matrigel contains multiple extracellular matrix proteins, however, Laminin-1 was identified as the major matrix component that induced HSG cell acinar development and cytodifferentiation. Antibodies against specific components of Matrigel and against cell surface adhesion molecules were added to cells in culture to identify components important for HSG cell acinar differentiation. Immunostaining of HSG cell acini identified TGF-beta 2 and beta 3 as the predominant isoforms within the cells. Neutralizing antibodies directed against TGF-beta 3 significantly decreased (P
-
role of Laminin 1 and tgf beta 3 in acinar differentiation of a human submandibular gland cell line hsg
Journal of Cell Science, 1996Co-Authors: Matthew P Hoffman, Maura C Kibbey, John J Letterio, Hynda K KleinmanAbstract:Previous studies show that culturing an immortalized human submandibular gland cell line (HSG) on Matrigel, a basement membrane extract, induces cytodifferentiation. We have further defined this model system and identified factors involved in HSG cell acinar development and cyto-differentiation. Acinar development is marked by cell migration into multi-cellular spherical structures, cell proliferation and apoptosis of the centrally localized cells. In addition, functional differentiation was determined by indirect immunofluorescence and immunoblot analysis for cystatin, a salivary gland acinar cell-specific protein found to be produced by differentiated HSG cells. Matrigel contains multiple extracellular matrix proteins, however, Laminin-1 was identified as the major matrix component that induced HSG cell acinar development and cytodifferentiation. Antibodies against specific components of Matrigel and against cell surface adhesion molecules were added to cells in culture to identify components important for HSG cell acinar differentiation. Immunostaining of HSG cell acini identified TGF-beta 2 and beta 3 as the predominant isoforms within the cells. Neutralizing antibodies directed against TGF-beta 3 significantly decreased (P < or = 0.0002) the size of acini formed. These results indicate that multiple components, including Laminin-1 and TGF-beta 3, contribute to HSG cell acinar development. This model system will be useful to study acinar differentiation and salivary gland-specific protein expression in vitro.
Yoshihiko Yamada - One of the best experts on this subject based on the ideXlab platform.
-
binding of Laminin 1 to monosialoganglioside gm1 in lipid rafts is crucial for neurite outgrowth
Journal of Cell Science, 2009Co-Authors: Naoki Ichikawa, Kumiko Ishii, Kentaro Hozumi, Toshihide Kobayashi, Kazuhisa Iwabuchi, Nobutaka Hattori, Hidetake Kurihara, Takako Sasaki, Yoshikuni Mizuno, Yoshihiko YamadaAbstract: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.
-
identification of multiple amyloidogenic sequences in Laminin 1
Biochemistry, 2007Co-Authors: Shingo Kasai, Kentaro Hozumi, Naoki Ichikawa, Yoshihiko Yamada, Yuichi Kadoya, Norio Nishi, Shunsuke Urushibata, Fumiharu Yokoyama, Nobuhisa Watanabe, Motoyoshi NomizuAbstract: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.
-
neural cell response to multiple novel sites on Laminin 1
Journal of Neuroscience Research, 2000Co-Authors: Sharon K Powell, Yoshihiko Yamada, Motoyoshi Nomizu, Jayashree Rao, Eva Roque, Yuichiro Kuratomi, Hynda K KleinmanAbstract:The basement membrane protein Laminin-1 is a potent stimulator of neurite outgrowth for a variety of neuronal cell types. Previous studies have identified neurite outgrowth activity in several distinct regions of the Laminin-1 molecule. In this study, 545 overlapping 12- to 14-mer synthetic peptides, corresponding to most of the amino acid sequence of the alpha1, beta1, and gamma1 chains of Laminin-1, were screened for cell attachment and neurite outgrowth activity using primary cultures of mouse cerebellar granule neurons and two neuronal cell lines. We identified 48 peptides derived from novel regions of the Laminin-1 molecule that were positive for neural cell adhesion activity. Only the cerebellar cells were found to have true neurite outgrowth activity with certain of the peptides, whereas some peptides induced short spike-like process with the cell lines. Although 23 of these peptides were active on all 3 cell types screened, 25 others showed cell-type specificity in their activity. These studies show that (1) there are multiple and distinct sites on Laminin-1 for cell adhesion and neurite-like outgrowth and (2) that there are neural cell-type-specific active domains. The multiple active sites found explains, in part, the potent activity of Laminin-1 on neurite outgrowth.
-
identification of Laminin α1 and β1 chain peptides active for endothelial cell adhesion tube formation and aortic sprouting
The FASEB Journal, 1999Co-Authors: Katherine M Malinda, Yoshihiko Yamada, Motoyoshi Nomizu, Hynda K Kleinman, Melissa Chung, Mucio Delgado, Yuchiro Kuratomi, Lourdes M PonceAbstract:Laminin-1 is a basement membrane glycoprotein that promotes several biological activities including cell attachment, tumor metastasis, and angiogenesis. Angiogenesis plays an important role in tissue formation, reproduction, wound healing, and several pathological conditions. In this study, we screened 405 synthetic peptides from the α1 and β1 chains to identify potential sites on Laminin-1 active with endothelial cells. Peptides were initially screened by testing both endothelial cell adhesion to peptide-coated wells and tube formation on Matrigel in the presence of soluble peptide. Twenty active peptides were identified in these screens. A secondary screen using the rat aortic ring sprouting assay identified 13 of the 20 peptides that stimulated endothelial sprouting. Several of these active peptides were also found to stimulate human umbilical vein endothelial cell migration in Boyden chamber assays. Differences in the amount of peptide needed for the response and in the resultant morphologies/response...
-
the α chain of Laminin 1 is independently secreted and drives secretion of its β and γ chain partners
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Peter D Yurchenco, Yoshihiko Yamada, Yong Quan, Holly Colognato, Todd Mathus, David G Harrison, Julian J OrearAbstract:A mammalian recombinant strategy was established to dissect rules of basement membrane Laminin assembly and secretion. The α-, β-, and γ-chain subunits of Laminin-1 were expressed in all combinations, transiently and/or stably, in a near-null background. In the absence of its normal partners, the α chain was secreted as intact protein and protein that had been cleaved in the coiled-coil domain. In contrast, the β and γ chains, expressed separately or together, remained intracellular with formation of ββ or βγ, but not γγ, disulfide-linked dimers. Secretion of the β and γ chains required simultaneous expression of all three chains and their assembly into αβγ heterotrimers. Epitope-tagged recombinant α subunit and recombinant Laminin were affinity-purified from the conditioned medium of αγ and αβγ clones. Rotary-shadow electron microscopy revealed that the free α subunit is a linear structure containing N-terminal and included globules with a foreshortened long arm, while the trimeric species has the typical four-arm morphology of native Laminin. We conclude that the α chain can be delivered to the extracellular environment as a single subunit, whereas the β and γ chains cannot, and that the α chain drives the secretion of the trimeric molecule. Such an α-chain-dependent mechanism could allow for the regulation of Laminin export into a nascent basement membrane, and might serve an important role in controlling basement membrane formation.
Rupert Timpl - One of the best experts on this subject based on the ideXlab platform.
-
tropoelastin binding to fibulins nidogen 2 and other extracellular matrix proteins
FEBS Letters, 1999Co-Authors: Takako Sasaki, Joel Rosenbloom, William R Abrams, Walter Gohring, Nicolai Miosge, Rupert TimplAbstract:Elastic fibers in vessel walls and other tissues consist of cross-linked tropoelastin in association with several microfibrillar proteins. In order to understand the molecular basis of these structures, we examined the binding of recombinant human tropoelastin to other extracellular matrix ligands in solid phase binding and surface plasmon resonance assays. These studies demonstrated a particularly high affinity (Kd about 1 nM) of tropoelastin for microfibrillar fibulin-2 and the recently described nidogen-2 isoform. More moderate affinities were observed for fibulin-1, Laminin-1 and perlecan, while several other ligands such as collagens, nidogen-1, fibronectin and BM-40 showed little or no binding. In immunogold staining of mouse aortic media, elastic fibers were heavily decorated with tropoelastin, fibulin-2 and nidogen-2, while the reaction with fibulin-1 was lower. The colocalization of these proteins emphasizes the potential for in vivo interactions.
-
deficiency of beta 1 integrins in teratoma interferes with basement membrane assembly and Laminin 1 expression
Experimental Cell Research, 1998Co-Authors: Takako Sasaki, Erik Forsberg, Wilhelm Bloch, Klaus Addicks, Reinhard Fassler, Rupert TimplAbstract:Subcutaneous injection of beta 1 integrin-deficient embryonic stem cells in mice causes the formation of teratomas although they occur with a lower frequency and are smaller than wild-type cells. Immunofluorescence analysis of these deficient tumors indicates a disorganized deposition of several basement membrane proteins. This was confirmed by electron microscopy which demonstrated frequent gaps in cell-associated basement membranes or loss of close contacts to the cells. Further aberrant features were multilaminar structures and amorphous deposits, indicating a strong impairment of correct basement membrane assembly. Quantitative radioimmunoassays were used to determine the levels of specific proteins in successive tissue extracts with neutral buffer in the absence and presence of EDTA and with 6 M guanidine. This demonstrated a more than 90% decrease in the content of Laminin-1 (alpha 1 beta 1 gamma 1) and a 70% decrease in nidogen in the beta 1 integrin-deficient teratomas. No significant changes were detected for other matrix proteins (perlecan, fibronectin, fibulins). This selective change impaired the formation of Laminin-nidogen complex and enhanced nidogen degradation. Northern blots also demonstrated a distinctly reduced expression of Laminin alpha 1, beta 1, and gamma 1 chains. Similar reductions were also observed in cultured embryonic stem cells prior to any differentiation. No or only smaller changes were observed for Laminin alpha 2 and beta 2 chain, nidogen, and perlecan mRNA. These data emphasize a distinct role of beta 1 integrins in the correct assembly of basement membranes which may occur through direct ligand binding and/or regulatory events at the transcriptional level.
-
hnk 1 carbohydrate mediated cell adhesion to Laminin 1 is different from heparin mediated and sulfatide mediated cell adhesion
FEBS Journal, 1997Co-Authors: Heike Hall, Rupert Timpl, Rainer Deutzmann, Lloyd Vaughan, Brigitte Schmitz, Melitta SchachnerAbstract:The sulfated HNK- 1 carbohydrate present on glycolipids and on several neural recognition molecules has been shown to mediate the adhesion of murine small cerebellar neurons and astrocytes to the extracellular matrix molecule Laminin-1. In this study, we characterized the binding of the HNK-1 carbohydrate to Laminin-1 extracted from the Engelbreth-Holm-Swarm (EHS) sarcoma and distinguished it unequivocally from binding sites for other sulfated carbohydrates. Electron microscopic analysis of rotary shadowed complexes of Laminin-1 and a HNK-1 neoglycoprotein revealed a major binding site on the G domain that comprises the C-terminal globule of the Laminin a1 chain. The HNK-1 carbohydrate also interacted with placental Laminin isoforms containing an a chain variant. It bound to the proteolytic Laminin-1 fragment E8 comprising the domains GI-G3, but not to fragment E3 that carries the major heparin-binding site on domains G4-G5. No binding was observed to the short arm containing fragments ElXNd or P1. Binding studies with native or denatured Laminin E8 fragments and proteolytic or recombinant fragments of the G domain localized the HNK-1 carbohydrate binding site to domain G2. The binding could be clearly distinguished from binding sites for other sulfated carbohydrates such as heparin and sulfatides. Further, the binding could not be abolished by reduction and alkylation or by urea treatment of Laminin-1 and was independent of the native conformation of Laminin-I and of Ca”. The G2 domain is also involved in the adhesion of HNK-1 carbohydrate expressing early postnatal cerebellar neurons and is different from heparin- and sulfatide-mediated cell adhesion to Laminin-I. HNK-1 carbohydrate-mediated cell adhesion appears, however, to be dependent on the native conformation of Laminin-I indicating a more complex cellular recognition process.
-
binding of purified collagen receptors alpha 1 beta 1 alpha 2 beta 1 and rgd dependent integrins to Laminins and Laminin fragments
FEBS Journal, 1994Co-Authors: Martin Pfaff, Walter Gohring, Judith C Brown, Rupert TimplAbstract:Integrins alpha 1 beta 1 and alpha 2 beta 1 when purified by collagen affinity chromatography, showed distinct binding to mouse tumor Laminin-1, which has the chain composition alpha 1 beta 1 gamma 1. The binding was, however, about 10-fold lower than to collagen IV. Only little (alpha 1 beta 1) or no binding (alpha 2 beta 1) was observed to two different Laminin isoforms (alpha 2 beta 1 gamma 1, alpha 2 beta 2 gamma 1) from human placenta. Binding to Laminin-1 was abolished by EDTA and could be specifically inhibited by antibodies to the respective integrin alpha subunit. These antibodies also inhibited cell adhesion to collagens. The binding of soluble integrins was weaker than that of immobilized integrins but could be enhanced by an activating anti(beta 1 integrin). No enhancement was observed for immobilized integrins. Studies with Laminin-1 fragments demonstrated lack of binding to the major cell-adhesive fragment E8 from the long arm, fragments E3 and E4, involved in heparin-binding and self-assembly, respectively, and fragment P1, corresponding to the inner segments of the short arms. A larger short-arm fragment (E1XNd), which lacks the N-terminal beta 1 chain domains V and VI, was as active as Laminin. Together, these results, suggested the localization of the binding sites for alpha 1 beta 1 and alpha 2 beta 1 to the N-terminal region of the Laminin alpha 1 chain. Fragment P1 but not intact Laminin-1 bound to alpha V beta 3 integrin in an EDTA-sensitive and RGD-sensitive manner, underscoring previous data on the cryptic nature of the RGD site in Laminin-1. Further analyses by surface plasmon resonance assays demonstrated a KD = 50 nM for alpha 2 beta 1/Laminin-1 binding and a KD = 450 nM for alpha V beta 3/fragment P1 binding and confirmed the anti-beta 1-mediated increase in affinity for alpha 2 beta 1.
