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K M Yamada

Publications and source records attributed to K M Yamada.

At least 73 records · Page 4Linked to original sources
PubMed1997

Evidence that beta1 integrins in keratinocyte cell-cell junctions are not in the ligand-occupied conformation.

Integrins are a family of heterodimeric cell surface molecules that function as adhesion receptors in cell-cell and cell-extracellular matrix contact. Integrins of the beta1 family are found on keratinocytes clustered at sites of cell-cell junctions both in culture and in normal skin. The possibility that these integrins function in cell-cell adhesion has been both supported and refuted in recent conflicting publications. Rather than testing further for the presence or absence of an interaction, we present evidence to show that beta1 integrins in keratinocyte cell-cell junctions are in the non-ligand-occupied conformation. We transfected keratinocytes with a construct that expresses a chimeric cell surface molecule containing the integrin beta1 cytoplasmic tail. This chimera is thought to mimic the ligand-occupied receptor and has previously been shown to be actively localized to focal adhesions in fibroblasts. We find that keratinocytes are also able to localize this chimera in focal adhesions but do not localize it to areas of cell-cell junctions. A monoclonal anti-beta1 antibody that has been previously shown to preferentially recognize ligand-occupied beta1 receptors was used to stain keratinocytes. This antibody showed staining of focal adhesions, with little or no staining of cell-cell junctions. In contrast, four other anti-beta1 antibodies showed strong, preferential staining at cell-celljunctions. Double staining confirmed that both the conformation-specific monoclonal antibody and a pan-beta1 antibody were capable of recognizing the same focal adhesions. Taken together, these data indicate that integrins in cell- cell junctions of keratinocytes are in the non-ligand-occupied conformation. Although we do not directly prove the absence of an integrin-integrin interaction at this site, we show that any such interaction does not induce the ligand-occupied conformation and, therefore, is less likely to play a major role in cytoskeletal re-organization or signal transduction.

Antibodies, Monoclonal↗
PubMed1997

The regulation of expression of integrin receptors.

The integrins are a family of cell surface receptors that mediate cell-extracellular matrix and cell-cell interactions. The quantities and activities of these receptors are modulated during a wide variety of biological processes. A variety of agents have been found to affect expression of integrins and their function. These include cytokines, hormones, and pharmacologic agents. Mechanisms regulating integrin expression and function include regulation of protein levels by transcriptional or posttranscriptional mechanisms, alteration of protein structure by alternative splicing of mRNA, mobilization to the cell surface of preexisting intracellular stores of integrins, and modulation of receptor activity (inside-out signaling). We review studies that assess the effects of external agents on integrin levels using the cytokine TGFbeta as an example. We also review studies that analyze integrin regulation with an emphasis on the control of integrin gene transcription. This review shows that the strategies for integrin modulation are quite complex. This regulatory sophistication is likely necessary, given the critical role that integrins play in the myriad social interactions of cells.

Animals↗
PubMed1996

HIV-1-Tat protein promotes chemotaxis and invasive behavior by monocytes.

Monocytes are susceptible to HIV infection and to activation by a regulatory gene product of the HIV genome, HIV-Tat. Recently, we have demonstrated that treatment with HIV-Tat up-regulates monocyte adhesion to the endothelium and increases metalloproteinase production. in the present study, we have examined the ability of the HIV-Tat protein to alter the migratory and invasive behavior of monocytes. Monocytes pretreated for 24 h with 10 ng/ml HIV-Tat exhibited enhanced migratory behavior compared with untreated monocytes in chemotaxis assays, both in the absence of a chemoattractant as well as in response to FMLP. in addition, HIV-Tat itself induced the migration of both untreated and HIV-Tat pretreated monocytes. Checkerboard analysis showed that monocytes migrated in response to an HIV-Tat concentration gradient, thus confirming the chemotactic characteristics of the HIV-Tat protein. Pretreatment of monocytes with 10 ng/ml HIV-Tat for 24 h also increased their ability to invade reconstituted extracellular membrane (Matrigel)-coated filters by 5-fold in the absence of chemoattractant. The presence of FMLP or HIV-Tat further enhanced invasion by both untreated and HIV-Tat-pretreated monocytes by more than 10-fold. Monocyte invasion was partially inhibited by the inclusion of anti-beta integrin Ab or tissue inhibitor of metalloproteinase (TIMP). Thus, for the first time, we present evidence that HIV-Tat can enhance the chemotactic and invasive behaviors of monocytes and propose an active role for HIV-Tat in the recruitment of monocytes into extravascular tissues, a process which may contribute to the destruction of tissues and cellular architecture often seen in patients with acquired immunodeficiency syndrome.

Chemotaxis, Leukocyte↗
PubMed1996

Integrin-dependent signal transduction.

Integrins are receptor molecules for extracellular matrix molecules (e.g., the beta(1) family), serum components (alpha(v) family) and immunoglobulin family adhesion molecules (beta(2) family). Integrin-dependent adhesion has also been shown to have metabolic consequences. Adhesion to a variety of extracellular matrix proteins, such as fibronectin, collagen, and laminin, is a potent regulator of cell growth, differentiation, and gene expression. Ligand binding or aggregation of integrin receptors initiates a number of metabolic changes including activation of serine/threonine and tyrosine kinases, increased Ca2+ influx, increased cytoplasmic alkalinization, and altered inositol lipid metabolism. In some instances activation of transcription factors and induction of gene expression have also been demonstrated. Components of key signaling pathways involving integrins are beginning to be identified. Some studies have shown that integrins form multi-component complexes with signal transduction molecules. Elucidating the interactions of the signal transduction molecules with each other and with the integrin cytoplasmic domains will be key to understanding the initial events of signal transduction through the integrins.

Animals↗
PubMed1996

Integrin alpha v beta 5-dependent serine phosphorylation of paxillin in cultured human macrophages adherent to vitronectin.

The macrophage colony-stimulating factor (M-CSF) is able to induce the expression of the alpha v beta 5 integrin receptor on the surface of cultured human macrophages (De Nichilo, M. O., and Burns, G. F. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 2517-2521). In the present study, we establish that the adhesion of M-CSF-treated macrophages to vitronectin is mediated by the integrin alpha v beta 5, and show by indirect immunofluorescence analysis that alpha v beta 5 and the cytoskeletal protein paxillin localize to focal contacts upon adhesion to vitronectin. Immunoprecipitation and Western blot analysis revealed that M-CSF-treated macrophages do not express focal adhesion kinase (FAK), thereby providing direct evidence for integrin-dependent localization of paxillin to focal contacts in the absence of FAK expression. Investigation of paxillin phosphorylation by two-dimensional phosphoamino acid analysis indicates that paxillin is 99% phosphorylated on serine residue(s) in response to vitronectin adhesion, and only 1% phosphorylated on tyrosine. Stimulation of protein kinase C (PKC) activity with the phorbol ester phorbol 12-myristate 13-acetate enhances paxillin phosphorylation, while two selective inhibitors of PKC, GF109203X and chelerythrine chloride, effectively block the phosphorylation of paxillin induced in response to vitronectin adhesion. Taken together, these data demonstrate that in M-CSF-treated macrophages adherent to vitronectin, paxillin localizes to focal contacts in the absence of FAK expression and is predominantly phosphorylated on serine residue(s) in a PKC-dependent manner.

Cell Adhesion↗
PubMed1996

A two-domain mechanism for group A streptococcal adherence through protein F to the extracellular matrix.

Streptococcus pyogenes binds to the extracellular matrix (ECM) and a variety of host cells and tissues, causing diverse human diseases. Protein F, a S.pyogenes adhesin that binds fibronectin (Fn), contains two binding domains. A repeated domain (RD2) and an additional domain (UR), located immediately N-terminal to RD2. Both domains are required for maximal Fn binding. In this study, we characterize RD2 and UR precisely and compare their functions and binding sites in Fn. The minimal functional unit of RD2 is of 44 amino acids, with contributions from two adjacent RD2 repeats flanked by a novel 'MGGQSES' motif. RD2 binds to the N-terminal fibrin binding domain of Fn. UR contains 49 amino acids, of which six are from the first repeat of RD2. It binds to Fn with higher affinity than RD2, and recognizes a larger fragment that contains fibrin and collagen binding domains. Expression of UR and RD2 independently on the surface-exposed region of unrelated streptococcal protein demonstrates that both mediate adherence of the bacteria to the ECM. We describe here a mechanism of adherence of a pathogen that involves two pairs of sites located on a single adhesin molecule and directed at the same host receptor.

Adhesins, Bacterial↗
PubMed1996

Full-length sequence, localization, and chromosomal mapping of ameloblastin. A novel tooth-specific gene.

We report the full-length sequencing, cell type-specific expression, and immunolocalization of a novel gene expressed in rat incisors, which we have designated ameloblastin. Northern blot analysis of RNA from multiple rat and mouse tissues demonstrated high levels of expression of two distinct transcripts of approximately 2.0 and 1.6 kilobase pairs that were expressed only in teeth. In situ hybridization using a digoxigenin-labeled RNA probe showed that the tissue distribution of ameloblastin was limited to the ameloblast in rat incisors. Immunohistochemical staining of rat incisors using a polyclonal antibody raised against a fusion protein revealed a unique localization pattern. Ameloblastin was found to be expressed during the differentiation of inner enamel epithelium into ameloblasts, with intense localization in the Tomes' processes of secretory ameloblasts. In contrast to amelogenin, only modest amounts of ameloblastin were detected in enamel matrix. The ameloblastin gene encodes an open reading frame of 422 amino acids corresponding to a putative protein of 45 kDa. The predicted protein is acidic (pI = 5.54) and the most abundant amino acids are Pro (15.2%), Gly (9.9%), and Leu (9.9%). We have also mapped the ameloblastin gene, Ambn, to a locus on mouse chromosome 5 near other genes associated with mineralized tissues. Thus, ameloblastin represents a unique ameloblast-specific gene product that may be important in enamel matrix formation and mineralization.

Amelogenin↗
PubMed1996

HIV-1-Tat modulates the function of monocytes and alters their interactions with microvessel endothelial cells. A mechanism of HIV pathogenesis.

Monocytes are major targets of HIV infection in patients with AIDS. In vitro infection of monocytes with HIV is associated with increased expression of beta 2 integrins, which increases both monocyte aggregation and monocyte/endothelial adhesion as well as monocyte metalloproteinase (MMP-9) expression. Treatment of primary monocytes with soluble HIV-Tat protein mimicked many of the properties of HIV infection of monocytes. Tat treatment up-regulated the expression of the beta 2 integrins, which was associated with the formation of large aggregates of monocytes and increased adhesion to endothelial monolayers. Treatment of monocytes with Tat increased their adhesion to both untreated and TNF-alpha-treated endothelial monolayers, and adhesion was inhibited by inclusion of anti-beta 2 and anti-ICAM-1 Abs. The increased adhesion of activated monocytes was accompanied by substantial disruption of the endothelial monolayers, with retraction or detachment of individual endothelial cells. Tat treatment of monocytes up-regulated the synthesis and release of the protease MMP-9, providing a potential mechanism to explain endothelial cell/basement membrane detachment. Thus, extracellular Tat is capable of activating monocytes even in the absence of HIV infection. Our studies demonstrate that many of the effects of HIV infection on monocyte homotypic and heterotypic adhesion, protease secretion, and disruption of the endothelium can be mimicked by treatment with HIV-Tat protein alone. These results suggest a mechanism where monocytes could be inappropriately activated by HIV-Tat, secreted by HIV-infected cells, causing them to extravasate into underlying tissues and ultimately contribute to tissue damage as seen during the progression of AIDS.

Cell Adhesion↗
PubMed1996

Integrins can collaborate with growth factors for phosphorylation of receptor tyrosine kinases and MAP kinase activation: roles of integrin aggregation and occupancy of receptors.

Integrins mediate cell adhesion, migration, and a variety of signal transduction events. These integrin actions can overlap or even synergize with those of growth factors. We examined for mechanisms of collaboration or synergy between integrins and growth factors involving MAP kinases, which regulate many cellular functions. In cooperation with integrins, the growth factors EGF, PDGF-BB, and basic FGF each produced a marked, transient activation of the ERK (extracellular signal-regulated kinase) class of MAP kinase, but only if the integrins were both aggregated and occupied by ligand. Transmembrane accumulation of total tyrosine-phosphorylated proteins, as well as nonsynergistic MAP kinase activation, could be induced by simple integrin aggregation, whereas enhanced transient accumulation of the EGF-receptor substrate eps8 required integrin aggregation and occupancy, as well as EGF treatment. Each type of growth factor receptor was itself induced to aggregate transiently by integrin ligand-coated beads in a process requiring both aggregation and occupancy of integrin receptors, but not the presence of growth factor ligand. Synergism was also observed between integrins and growth factors for triggering tyrosine phosphorylation of EGF, PDGF, and FGF receptors. This collaborative response also required both integrin aggregation and occupancy. These studies identify mechanisms in the signal transduction response to integrins and growth factors that require various combinations of integrin aggregation and ligands for integrin or growth factor receptors, providing opportunities for collaboration between these major regulatory systems.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1996

Ameloblastin expression in rat incisors and human tooth germs.

We recently identified ameloblastin as an ameloblast-specific gene product from a rat incisor cDNA library (Krebsbach et al., J. Biol. Chem. 271: 4431-4435, 1996). Here we report the developmental pattern of expression of ameloblastin in rat incisors and human tooth germs as visualized by in situ hybridization and immunochemistry. Compared to the expression of amelogenin, the major ameloblast product, ameloblastin mRNA was more widely expressed in ameloblasts from the presecretory to the late maturation stage of development. Ameloblastin mRNA was first observed in the juxtanuclear cytoplasm or presecretory stage ameloblasts, gradually increased in the distal cytoplasm of secretory stage ameloblasts and was found throughout the cytoplasm of early to late maturation stage ameloblasts. The immunostaining of ameloblastin, using a monospecific antibody raised against a recombinant protein, showed intense reactivity in Tomes' processes of secretory stage ameloblasts and surrounding enamel. The immunoreaction was concentrated in the juxtanuclear cytoplasm of late maturation stage ameloblasts. High-resolution colloidal gold immunocytochemistry established the presence of ameloblastin antigenicity in the Golgi apparatus, secretory granules in Tomes' process and enamel. Human tooth germs in early to late bell stage also expressed ameloblastin mRNA and ameloblastin antigenicity in the ameloblasts. Western blot analysis of protein extracts from rat incisor tissues indicated that ameloblastin can be found in the enamel epithelial tissue and in mineralized enamel, as well as in the EDTA decalcification solution. These data indicate that ameloblastin is an ameloblast secretory product which is sequentially expressed from the presecretory to the late maturation stage in rat and human teeth. This unique developmental pattern suggests that ameloblastin may have a broader role in amelogenesis than amelogenin and tuftelin.

Ameloblasts↗
PubMed1995

p190-B, a new member of the Rho GAP family, and Rho are induced to cluster after integrin cross-linking.

p120GAP forms distinct complexes with two phosphoproteins, p62 and p190. Here we have cloned a cDNA encoding a protein with 51% amino acid identity to p190 (hereafter designated p190-A) and have designated it p190-B. The N-terminal portion of p190-B contained several motifs characteristic of a GTPase domain, while its C terminus contained a Rho GAP domain. A recombinant Rho GAP domain polypeptide showed GAP activity for RhoA, Rac1, and G25K/CDC42Hs. Immunoprecipitation and immunofluorescence studies demonstrated that p190-B protein was expressed in a variety of cells and was localized diffusely in the cytoplasm and in fibrillar patterns that co-localized with the alpha 5 beta 1 integrin receptor for fibronectin. Adhesion of fibronectin-coated latex beads to cells resulted in recruitment of significant amounts of p190-B and Rho to the plasma membrane beneath the site of bead binding. In contrast, beads coated with polylysine or concanavalin A were unable to recruit p190-B or Rho. Additionally, anti-beta 1 or anti-alpha 5 integrin antibody-coated beads were also able to recruit large amounts of p190-B and Rho. These results identify a novel second member of the p190 family and establish the existence of a novel transmembrane link between integrins and a new protein p190-B and Rho.

Amino Acid Sequence↗
PubMed1995

Requirement for the synergy site for cell adhesion to fibronectin depends on the activation state of integrin alpha 5 beta 1.

We investigated the influence of the activation state of integrin alpha 5 beta 1 on its dependence on the PHSRN synergy site for binding to RGD in fibronectin. K562 and MV3 cells lacked alpha v beta 3 expression and adhered to fibronectin through alpha 5 beta 1. Mel57 cells adhered through alpha v beta 3 and alpha 5 beta 1. A recombinant fibronectin polypeptide, containing five type III repeats from the central cell binding domain 3Fn6-10, and a mutated polypeptide lacking the synergy site were equally effective in promoting Mel57 adhesion. For K562 and MV3, the mutated polypeptide was not or poorly active compared to the control polypeptide. Expression of alpha v beta 3 in MV3 induced strong adhesion to the mutated polypeptide. TS2/16 stimulatory beta 1-integrin antibodies or Mn2+ induced alpha 5 beta 1-mediated adhesion of K562 and MV3 to GRGDSP. In the presence of TS2/16 or Mn2+, alpha 5 beta 1-mediated MV3 adhesion to the mutated polypeptide was equally strong as adhesion to the control polypeptide. Mn2+ or TS2/16 induced weak K562 binding to the mutated polypeptide, and in the presence of a combination of phorbol 12-myristate 13-acetate, Mn2+, and TS2/16, alpha 5 beta 1-mediated K562 adhesion to the mutated and control polypeptide was equally strong. Our findings demonstrate that requirement for the PHSRN synergy site for alpha 5 beta 1-mediated adhesion to RGD in fibronectin depends on the activation state of the integrin.

Amino Acid Sequence↗
PubMed1995

Synergistic roles for receptor occupancy and aggregation in integrin transmembrane function.

Integrin receptors mediate cell adhesion, signal transduction, and cytoskeletal organization. How a single transmembrane receptor can fulfill multiple functions was clarified by comparing roles of receptor occupancy and aggregation. Integrin occupancy by monovalent ligand induced receptor redistribution, but minimal tyrosine phosphorylation signaling or cytoskeletal protein redistribution. Aggregation of integrins by noninhibitory monoclonal antibodies on beads induced intracellular accumulations of pp125FAK and tensin, as well as phosphorylation, but no accumulation of other cytoskeletal proteins such as talin. Combining antibody-mediated clustering with monovalent ligand occupancy induced accumulation of seven cytoskeletal proteins, including alpha-actinin, talin, and F-actin, thereby mimicking multivalent interactions with fibronectin or polyvalent peptides. Integrins therefore mediate a complex repertoire of functions through the distinct effects of receptor aggregation, receptor occupancy, or both together.

Amino Acid Sequence↗
PubMed1995

Alternatively spliced juxtamembrane domain of a tyrosine kinase receptor is a multifunctional regulatory site. Deletion alters cellular tyrosine phosphorylation pattern and facilitates binding of phosphatidylinositol-3-OH kinase to the hepatocyte growth factor receptor.

The hepatocyte growth factor (HGF) receptor is a tyrosine kinase receptor that mediates signal transduction upon ligand stimulation. This receptor is present in mouse tissues as two major isoforms differing by a 47-amino acid segment in the juxtamembrane domain, an alternatively spliced cytoplasmic region adjacent to the transmembrane domain of the receptor. We report here that the juxtamembrane domain of the receptor is involved in the regulation of downstream signal transduction. The two receptor isoforms were transiently expressed in COS-7 cells. Both exogenous receptors underwent autophosphorylation and subsequently stimulated a set of protein tyrosine phosphorylations that were not present in control cells. Comparisons of phosphotyrosine profiles of transfected cell lysates induced by receptor isoforms demonstrated that at least three phosphorylated proteins of approximately 62, approximately 35, and approximately 30 kDa were differentially induced by the receptor isoforms, suggesting that the juxtamembrane domain of a kinase receptor can play a role in selective signal transduction. Furthermore, the p85 subunit of phosphatidylinositol-3-OH kinase (PI3 kinase) co-precipitated with the small isoform of the HGF receptor, and this association was dramatically inhibited by treatment with 12-O-tetradecanoylphorbol-13-acetate. Since removal of the juxtamembrane domain facilitates the binding of p85 to the receptor, it is likely that the juxtamembrane region plays a role in negative regulation of the binding of PI3 kinase to the HGF receptor. Our study establishes novel molecular sequelae of alternative splicing of an intracellular domain of the HGF receptor.

Alternative Splicing↗
PubMed1995

HIV-1 infection alters monocyte interactions with human microvascular endothelial cells.

HIV infection of monocytes resulted in twofold elevation of adhesion molecule LFA-1 (both alpha L/CD11a and beta 2/CD18 subunits) and LFA-3 (CD58), with no apparent increase in LFA-2 (CD2) or various beta 1-integrins. Homotypic aggregation of monocytes was evident 2 h after exposure to virus and was inhibited by mAbs to both the alpha L- and beta 2-subunits of LFA-1. HIV-infected monocytes also showed a marked increase in adherence to human capillary endothelial cell monolayers derived from brain, lung, and skin. This adherence was inhibited by mAb to either LFA-1 subunit and by mAb to the counter-receptor intercellular adhesion molecule-1. Cocultivation of HIV-infected monocytes with endothelial cells increased permeability of endothelial cell monolayers to 125I albumin in transwell assay systems. The increased endothelial permeability induced by HIV-infected monocytes was associated with a substantial disruption of the endothelial cell monolayer. Morphologic disruption was not a direct toxic effect on endothelial cells, but appeared to be secondary to changes in endothelial cell-cell or cell-matrix interactions. Northern blot analysis showed increased expression of gelatinase B (92-kDa gelatinase), tissue inhibitor of metalloproteinase TIMP-1, and TIMP-2 in the HIV-infected monocytes. Consistent with these Northern analyses, secretion of gelatinase activity in culture fluids of HIV-infected monocytes was also increased and was dependent on the stage of virus replication. Incubation of HIV-infected monocytes with the proteinase inhibitors TIMP-1 and TIMP-2 inhibited the increased permeability of endothelial cell monolayers to 125I albumin. These results suggest possible mechanisms for extravasation of HIV-infected monocytes through vascular endothelium into tissue in early stages of HIV disease.

Antigens, CD↗
PubMed1995

Posterior extension of the chick nephric (Wolffian) duct: the role of fibronectin and NCAM polysialic acid.

The nephric duct of the chick embryo starts to form at about stage 10 of Hamburger and Hamilton ([1951] J. Morphol. 88:49-92) and extends posteriorly, fusing with the cloaca at about the end of the third day of incubation (HH stage 17). Evidence from the literature suggests that the extension involves active migration of the posterior tip. This investigation concerned some molecules that might control this migration: fibronectin, vitronectin, the beta 1 integrin receptor, and NCAM polysialic acid. The concentration of fibronectin in the extracellular matrix was found by immunocytochemistry to be negligible at the posterior end of the duct; treatment of the living embryo with GRGDS failed to halt further extension of the duct; SEM examination of embryos treated with the synthetic peptides of fibronectin GRGDS, GRDGS, SDGR, and GRGES, or with vitronectin, revealed negligible morphological effects on the duct. It is concluded that there is yet no evidence that fibronectin is an important factor in duct migration. NCAM polysialic acid had a similar distribution to fibronectin, but treatment of the living embryo with Endo-N caused cessation of extension of the duct. Endo-N is an enzyme that specifically degrades PSA without affecting the NCAM polypeptide itself. It is suggested therefore that PSA may play an important role in duct extension. The synthetic peptides of fibronectin each produced distinctive patterns of blebbing on the surfaces of cells in trunk mesoderm, but the duct cells were unaffected. GRGES and SDGR caused blebbing on cells in the somites and the anterior segmental plate, though not on cells in the posterior segmental plate. This suggests that integrin receptors change in the anterior segmental plate as the mesoderm forms somites from somitomeres.

Amino Acid Sequence↗