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Biomedical subjects

Kenneth M Yamada

Publications and source records attributed to Kenneth M Yamada.

46 records · Page 3Linked to original sources
PubMed2002

A scaffold protein in the c-Jun N-terminal kinase signaling pathway is associated with focal adhesion kinase and tyrosine-phosphorylated.

Focal adhesion kinase (FAK) becomes activated and tyrosine-phosphorylated in response to cell adhesion to extracellular matrix proteins in a variety of cell types, and associates with a number of signaling molecules, structural proteins, and beta integrin cytoplasmic domains. Here we demonstrated that c-Jun N-terminal kinase (JNK)/stress activated protein kinase-associated protein 1 (JSAP1), a scaffold factor in the mitogen-activated protein kinase (MAPK) cascades, forms a complex with the N-terminus of FAK. The complex formation was further stimulated by c-Src, in which JSAP1 was tyrosine-phosphorylated and other FAK/Src signaling molecules were recruited. Fibronectin (FN) stimulation of cells expressing JSAP1 induced its tyrosine phosphorylation concomitant with association with FAK. Expression of JSAP1 in Hela cells facilitated formation of well-organized focal contacts and actin stress fibers, and promoted cell spreading onto FN. Taken together, these results suggest that JSAP1 is involved an integrin-mediated signaling pathway through FAK/Src by recruiting other signaling molecules, resulting in promotion of cell spreading onto FN.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Synergistic activity of fibronectin and fibroblast growth factor receptors on neuronal adhesion and neurite extension through extracellular signal-regulated kinase pathway.

Fibroblast growth factor (FGF) is an important modulator of cell growth and differentiation of various cells including neuron. Cells need to adhere specifically to cellular and extracellular components of their environment to carry out diverse physiological functions. Here, we examined whether fibronectin (FN) and FGF can cooperate for neuronal adhesion and neurite outgrowth. Using recombinant FN peptide (FNIII9-10), we found that FNIII9-10-mediated adhesion promotes the effect of FGF1 on neurite outgrowth of PC12 cells, while FGF1 enhances the FNIII9-10-mediated neuronal adhesion of PC12 cells. This collaboration of FNIII9-10 and FGF1 was the result of the sustained activation of extracellular signal-regulated kinase (ERK)-type MAP kinase. Finally, the synergistic activity of FGF1 and FN was inhibited by PD98059, an MEK inhibitor. Taken together, these findings indicate that FN-mediated signaling can collaborate with FGFRs signaling for neurite outgrowth through selective activation of ERK-type MAP kinase in PC12 cells, and suggest that FN and FGF act in concert to regulate cell differentiation in the nervous system.

Animals↗
PubMed2002

Involvement of integrin alphavbeta3 in the pathogenesis of human immunodeficiency virus type 1 infection in monocytes.

Attachment of HIV to macrophages is a critical early event in the establishment of infection. In the present study, we demonstrate the involvement of integrin alphavbeta3 (vitronectin receptor) in HIV infection of peripheral blood monocyte-derived macrophages. Culturing monocytes in the presence of M-CSF for 3 days upregulated expression of the alphav-containing integrins, alphavbeta3 and alphavbeta5. The increase in alphavbeta3 expression was accompanied by increased HIV-1 replication by monocytes. Immunoblot analysis showed that purified HIV-gp120 protein interacted with CD4 and alphavbeta3 in immunoprecipitation experiments. Neutralizing antibodies against the alphavbeta3 integrin interfered with the coprecipitation of alphavbeta3 with an anti-gp120 antibody and substantially inhibited HIV infection of monocytes. Neutralizing antibodies against alphavbeta5 or beta1 integrins did not significantly affect HIV infection. These results indicate that HIV infection of primary monocytes requires differentiation of these cells and may involve alphavbeta3 interaction with the HIV-1 envelope protein gp120 for productive infection.

Antibodies↗
PubMed2002

Integrin clustering induces kinectin accumulation.

Integrin receptors mediate the formation of adhesion complexes and play important roles in signal transduction from the extracellular matrix. Integrin-based adhesion complexes (IAC) contain proteins that link integrins to the cytoskeleton and recruit signaling molecules, including vinculin, paxillin, focal adhesion kinase, talin and alpha-actinin. In this study, we describe a approximately 160 kDa protein that is markedly enriched at IAC induced by clustering integrins with fibronectin-coated beads. Protein sequence analysis reveals that this approximately 160 kDa protein is kinectin. Kinectin is an integral membrane protein found in endoplasmic reticulum, and it serves as a receptor for the motor protein kinesin. Fibronectin-induced IAC sequestered over half of the total cellular content of kinectin within 20 minutes. In addition, two other ER-resident proteins, RAP [low-density lipoprotein receptor-related protein (LRP) receptor-associated protein] and calreticulin, were found to be clustered at IAC, whereas kinesin was not. Our results identify a novel class of constituents of IAC.

Amino Acid Sequence↗
PubMed2002

Laminin-10/11 and fibronectin differentially prevent apoptosis induced by serum removal via phosphatidylinositol 3-kinase/Akt- and MEK1/ERK-dependent pathways.

Cell adhesion to the extracellular matrix inhibits apoptosis, but the molecular mechanisms underlying the signals transduced by different matrix components are not well understood. Here, we examined integrin-mediated antiapoptotic signals from laminin-10/11 in comparison with those from fibronectin, the best characterized extracellular adhesive ligand. We found that the activation of protein kinase B/Akt in cells adhering to laminin-10/11 can rescue cell apoptosis induced by serum removal. Consistent with this, wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase, or ectopic expression of a dominant-negative mutant of Akt selectively accelerated cell death upon serum removal. In contrast to laminin-10/11, fibronectin rescued cells from serum depletion-induced apoptosis mainly through the extracellular signal-regulated kinase pathway. Cell survival on fibronectin but not laminin was significantly reduced by treatment with PD98059, a specific inhibitor of mitogen- or extracellular signal-regulated kinase kinase-1 (MEK1) and by expression of a dominant-negative mutant of MEK1. Laminin-10/11 was more potent than fibronectin in preventing apoptosis induced by serum depletion. These results, taken together, demonstrate laminin-10/11 potency as a survival factor and demonstrate that different extracellular matrix components can transduce distinct survival signals through preferential activation of subsets of multiple integrin-mediated signaling pathways.

Apoptosis↗
PubMed2002

Cell interactions with three-dimensional matrices.

Signaling and other cellular functions differ in three-dimensional compared with two-dimensional systems. Cell adhesion structures can evolve in vitro towards in-vivo-like adhesions with distinct biological activities. In this review, we examine recent advances in studies of interactions of fibroblasts with collagen gels and fibronectin-containing matrices that mimic in vivo three-dimensional microenvironments. These three-dimensional systems are illuminating mechanisms of cell-matrix interactions in living organisms.

Animals↗
PubMed2002

Cell-cell adhesion and RhoA-mediated actin polymerization are independent phenomena in microtubule disrupted keratinocytes.

E-cadherin-mediated adherens junction formation and maintenance are thought to involve actin filament rearrangements through the action of small GTPases. Recently, we demonstrated that microtubule disruption in normal human epidermal keratinocytes grown in low calcium media conditions induces cell-cell adhesion by redistribution of endogenous E-cadherin, and it promotes stress fiber formation. This actin rearrangement was apparently mediated by RhoA activation. This model system therefore provides a tool with which to dissect relationships between cell-cell adhesion and Rho-mediated stress fiber formation. In this study, we have demonstrated in normal human epidermal keratinocytes that disruption of actin structures including stress fibers does not interfere with E-cadherin redistribution during microtubule-induced cell-cell adhesion. Moreover, this cell-cell adhesion could not be blocked by RhoA inactivation at the level for inhibition of stress fiber formation. Additionally, in the immortalized HaCaT keratinocyte cell line, which does not undergo cell-cell adhesion after microtubule disruption in low calcium conditions, expression of dominant-active RhoA could induce stress fiber formation without inducing adhesion. On the other hand, a variant of the HaCaT cell line, HC-R1, showed microtubule-disruption-induced cell-cell adhesion without stress fiber formation. Together, our results suggest that, in keratinocytes, the process of cell adhesion can occur independently of RhoA-mediated stress fiber formation.

Actins↗
PubMed2000

Activation of DNA synthesis and AP-1 by profilin, an actin-binding protein, via binding to a cell surface receptor in cultured rat mesangial cells.

Profilin is known to bind to actin monomers (to regulate actin polymerization) and to phosphatidylinositol-4,5-bisphosphate (to inhibit hydrolysis by unphosphorylated phospholipase C-gammal). It was recently reported that profilin is overexpressed in glomerular mesangial cells (MC) of rats with anti-Thy-1.1-induced glomerulonephritis and is accumulated in the extracellular space around MC. In this study, the biologic activities of extracellular profilin were examined. Scatchard analysis indicated the existence of a single class of cell surface binding sites, with similar equilibrium dissociation constants for purified splenic profilin and recombinant profilin, in cultured rat MC. Profilin increased [(3)H]thymidine incorporation in a dose-dependent manner and produced additive effects on platelet-derived growth factor-induced [(3)H]thymidine incorporation. Profilin increased AP-1 DNA-binding activity in a concentration-dependent (ED(50) = 30 nM) and time-dependent manner after transient c-jun gene expression, as measured using gel-shift assays and competitive reverse transcription-PCR. Pretreatment of profilin with an anti-profilin inhibitory antibody suppressed profilin-induced AP-1 activation and [(3)H]thymidine incorporation. Furthermore, profilin induced rapid transient activation of protein kinase C, and staurosporine and H-7 reduced the profilin-induced activation of AP-1, suggesting protein kinase C-dependent activation of AP-1. These findings indicate that profilin in the extracellular space can bind to cell surface receptors of MC and act as an inducer of signal transduction. These results suggest that extracellular profilin may be involved in the progression of glomerular diseases, by affecting cell growth.

Animals↗
PubMedDate not supplied

High-throughput investigation of osteoblast response to polymer crystallinity: influence of nanometer-scale roughness on proliferation.

A high-throughput method for analyzing cellular response to crystallinity in a polymer material is presented. Variations in crystallinity lead to changes in surface roughness on nanometer length scales, and it is shown that cells are exquisitely sensitive to these changes. Gradients of polymer crystallinity were fabricated on films of poly(L-lactic acid) using a gradient in annealing temperature. The resultant morphologies were characterized using an atomic force microscope. Root-mean-square (rms) roughness values ranging from 0.5 to 13 nm were created on a single sample. MC3T3-E1 osteoblastic cells were cultured for 1, 3 and 5 d, and the number of cells was measured using automated fluorescence microscopy. It is shown that the rate of proliferation on the smooth regions of the films is much greater than that on the rough regions, and a monotonic variation in rate is observed as a function of roughness. The critical rms roughness, above which a statistically significant reduction in rate of proliferation occurs, was approximately 1.1 nm. Fluorescence microscopy measurements on immunostained cells indicate there is no significant change in cell area, the number or type of adhesions formed, or the degree of actin polymerization. Results from enzyme-linked immunofluorescence assays indicated that there was no detectable change in adhesion protein accessibility, suggesting the cells directly respond to substrate topography. The use of the gradient library approach yielded the functional dependence of cell proliferation on nanometer-scale roughness and gave a sensitive estimate of the critical roughness for which a decrease in proliferation is observed.

3T3 Cells↗