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At least 973 records · Page 54Linked to original sources
PubMed2003

Amlodipine modulates THP-1 cell adhesion to vascular endothelium via inhibition of protein kinase C signal transduction.

Inflammatory responses play an important role in atherosclerosis. To critically assess the effect of dihydropyridines in inflammatory reactions, we conducted a monocyte-endothelial adhesion assay with monocytic THP-1 cells treated with amlodipine under flow conditions in vitro. THP-1 cells were incubated in the presence of amlodipine (10 micromol/L) for 48 hours and then perfused over activated (interleukin-1beta, 10 U/mL, 4 hours) human umbilical vein endothelial cells. The adhesion of THP-1 cells was significantly reduced after amlodipine treatment (P<0.001); however, flow cytometric analysis reveled that the expression levels of integrins in THP-1 cells were not significantly altered. Furthermore, Western blotting analysis of THP-1 cell lysates revealed that translocation of RhoA from the cytosol to the membrane was significantly diminished after amlodipine treatment. In addition, activation of protein kinase C-alpha and -beta, as well as intracellular calcium influx, induced by phorbol 12-myristate 13-acetate, was diminished after amlodipine treatment. Pretreatment of THP-1 cells with calphostin C, a potent inhibitor of protein kinase C, significantly reduced THP-1 adhesion to vascular endothelium, whereas activation of beta1-integrin was reduced after amlodipine treatment in THP-1 cells, based on the immunoreactivity of an activation-specific antibody for beta1-integrin. Similar inhibitory effects were observed when we used freshly isolated peripheral blood mononuclear cells. These findings suggest a potential role for amlodipine in monocyte-endothelial interactions by modulation of protein kinase C- and RhoA-dependent mechanisms, which might account for its vascular protective effects.

Amlodipine↗
PubMed2001

Adhesion of epithelial cells to fibronectin or collagen I induces alterations in gene expression via a protein kinase C-dependent mechanism.

Adhesion of human salivary gland (HSG) epithelial cells to fibronectin- or collagen I gel-coated substrates, mediated by beta1 integrins, has been shown to upregulate the expression of more than 30 genes within 3-6 h. Adhesion of HSG cells to fibronectin or collagen I for 6 h also enhanced total protein kinase C (PKC) activity by 1.8-2.3-fold. HSG cells expressed PKC-alpha, gamma, delta, epsilon, mu, and zeta. Adhesion of HSG cells to fibronectin or collagen I specifically activated PKC-gamma and PKC-delta. Cytoplasmic PKC-gamma and PKC-delta became membrane-associated, and immunoprecipitated PKC-gamma and PKC-delta kinase activities were enhanced 2.5-4.0-fold in HSG cells adherent to fibronectin or collagen I. In addition, adhesion of fibronectin-coated beads to HSG monolayers co-aggregated beta1 integrin and PKC-gamma and PKC-delta but not other PKC isoforms. Thus, integrin-dependent adhesion of HSG cells to fibronectin or collagen I activated PKC-gamma and PKC-delta. The role of this PKC upregulation on adhesion-responsive gene expression was then tested. HSG cells were treated with the specific PKC inhibitor bisindolylmaleimide I, cultured on non-precoated, fibronectin- or collagen I-coated substrates, and analyzed for changes in adhesion-responsive gene expression. Bisindolylmaleimide I strongly inhibited the expression of seven adhesion-responsive genes including calnexin, decorin, S-adenosylmethionine decarboxylase, steroid sulfatase, and 3 mitochondrial genes. However, the expression of two adhesion-responsive genes was not affected by bisindolylmaleimide I. Treatment with bisindolylmaleimide I did not affect cell spreading and did not significantly affect the actin cytoskeleton. These data suggest that adhesion of HSG cells to fibronectin or collagen I induces PKC activity and that this induction contributes to the upregulation of a variety of adhesion-responsive genes.

Cell Adhesion↗
PubMed1998

Adhesion to fibronectin or collagen I gel induces rapid, extensive, biosynthetic alterations in epithelial cells.

Extracellular matrix influences many cellular events. In this study, we demonstrate that adhesion of human salivary gland (HSG) epithelial cells to fibronectin- or collagen I gel-coated substrates, mediated by beta1 integrins, results in substantial alterations in protein and RNA expression profiles. The large numbers of changes in expression suggest that simply changing the adhesive substrate has basic effects on the regulation of cellular biosynthesis. Two-dimensional electrophoresis of [35S]methionine-labeled HSG cell proteins identified significant differences in the patterns of protein expression by cells cultured on nonprecoated substrates, collagen I gels or fibronectin. Thirty-two differentially expressed cDNA clones, which included both novel and previously sequenced genes, were up-regulated within 6 hr by culturing HSG cells on fibronectin or collagen I gels. Therefore, adhesion to collagen I or fibronectin resulted in rapid, widespread changes in cellular biosynthetic control. Expression of some genes was induced by ligation of beta1 integrins with antifunctional antibodies, whereas the expression of other genes was not induced. Most of the differentially expressed genes were up-regulated by adhesion to both fibronectin- and collagen I gel-coated substrates, but a few genes were selectively up-regulated on only one substrate. Furthermore, the up-regulated expression of some genes was detected within 3 hr, whereas changes in others required 6 hr. Discrete adhesive substrates and integrin molecules differentially affected the expression of a significant number of genes, suggesting that the cellular responses to adhesion were triggered through several signaling pathways.

Antibodies↗
PubMed2004

Developmental expression of OAP-1/Tspan-3, a member of the tetraspanin superfamily.

OSP/claudin-11-associated protein (OAP-1/Tspan-3), originally isolated by yeast two-hybrid screening using OSP/claudin-11 (oligodendrocyte-specific protein) as bait, is a member of the tetraspanin superfamily and OAP-1/Tspan-3, OSP/claudin-11, and beta1 integrin form a protein complex that seems to be involved in oligodendrocyte proliferation and migration. This study investigated the temporal and regional expression, glycosylation status, and tissue distribution of OAP-1/Tspan-3. OAP-1/Tspan-3 mRNA was expressed as a single transcript throughout brain development, with high levels of expression in the germinal zones. OAP-1/Tspan-3 protein contains N-terminal glycosylation sites in extracellular loop 2 and deglycosylation studies indicated a decrease in apparent molecular weight of OAP-1/Tspan-3, consistent with removal of N-glycans. Similar to OSP/claudin-11, OAP-1/Tspan-3 is expressed in all stages of oligodendrocyte development and in the myelin sheath. Unlike OSP/claudin-11, however, it is expressed in all cell types tested in the central nervous system (CNS), including neurons and astrocytes. The association of OAP-1/Tspan-3 with OSP/claudin-11 and beta1 integrin, its subcellular distribution as a cell surface, membrane-spanning glycoprotein, and its widespread distribution supports its potential role in cell migration, proliferation, and interactions between cells and extracellular matrix.

Animals↗
PubMed2000

Increased density of interstitial mast cells in amyloid A renal amyloidosis.

Renal interstitial fibrosis is the final common pathway leading to end-stage renal disease in various nephropathies including renal amyloidosis. However, the role of mast cells (MCs) in the fibrotic process of renal amyloidosis is not fully understood. We compared the distribution of MCs in renal biopsies from 30 patients with AA type renal amyloidosis and 20 control cases. Immunoreactivity of renal MCs to anti-tryptase and anti-chymase was studied. Interstitial myofibroblasts were stained with anti-alpha-smooth muscle actin (alpha-SMA) antibody, and inflammatory cells were identified by anti-CD45, -CD20, and -CD68 mAbs. Positively stained cells were counted, and the relative interstitial and fractional areas of anti-alpha-SMA stained cells were measured. Anti-CD29 mAb was used to detect beta1 integrin and anti-basic fibroblast growth factor (bFGF) mAb for the growth factor on MCs. MCs were rarely found in control samples. In contrast, samples showing amyloid deposition contained numerous tryptase-positive (MCT) (940.17 +/- 5.4 versus 6.74 +/- 1.1/mm2) but fewer chymase-positive (MCTC) cells (20.7 +/- 2.86 versus 1.7 +/- 0.76/mm2) in the renal interstitium. There was a significant relationship between interstitial MCT and creatinine clearance (r = -0.72), and between interstitial MCT and glomerular amyloid-index (GAI) (r = 0.723) and interstitial amyloid area (r = 0.824). Accumulation of MCs correlated significantly with the number of T lymphocytes (MCT: r = 0.694). There was also a significant relationship between mast cell (MC) number and the fractional area of alpha-SMA positive interstitium (r = 0.733) and interstitial fibrotic area (r = 0.6). Double immunostaining demonstrated intracytoplasmic presence of beta1 integrin on 87% of MCT and correlated significantly with the interstitial amyloid area (r = 0.818, P = .001) and T-cell number (r = 0.639, P = .002). bFGF was also detected on 85.5% of MCTC correlating well with the interstitial alpha-SMA-area (r = 0.789). Our results indicate that MCs constitute an integral part of the overall inflammatory process and play a crucial role in interstitial fibrosis in renal amyloidosis.

Actins↗
PubMed2000

Signaling of mechanical stretch in human keratinocytes via MAP kinases.

Cells within human skin are permanently exposed to mechanical stretching. Here we present evidence that alterations in cell shape trigger biochemical signaling via MAP kinases in human keratinocytes. In an in vitro attempt we demonstrate a fast but transient activation of extracellular signal-regulated kinases 1/2 in response to cell stretch. This activation is reversed by preincubation with functional blocking antibodies directed towards beta1-integrins. As a second member of MAP kinases, stress-activated protein kinase/c-JUN NH2-terminal kinase was activated in a slower fashion, peaking at 1 h after the initial stimulus. The delay in signal transmission suggests that extracellular signal-regulated kinases 1/2 and stress-activated protein kinase/c-JUN NH2-terminal kinase do not share the same signaling pathway. p38 was not activated by cell stretching. The contribution of cytoskeletal elements in signal perception and transduction was evaluated by selective disruption of either actin filaments, microtubules, or keratin filaments but showed no clear effect on stretch-induced activation of extracellular signal-regulated kinases 1/2 and stress-activated protein kinase/c-JUN NH2-terminal kinase. In conclusion we found evidence of a cell-shape-dependent activation of MAP kinases in human keratinocytes disclosing beta1-integrins as putative mechano-transducers. It is likely that alterations of skin mechanics in vivo underlying pathogenic processes like wound formation and healing trigger physiologic responses via the MAP kinase pathway.

Cell Line↗
PubMed1998

The involvement of the fibronectin type II-like modules of human gelatinase A in cell surface localization and activation.

Recombinant collagen-binding domain (rCBD) comprising the three fibronectin type II-like modules of human gelatinase A was found to compete the zymogen form of this matrix metalloproteinase from the cell surface of normal human fibroblasts in culture. Upon concanavalin A treatment of cells, the induced cellular activation of gelatinase A was markedly elevated in the presence of the rCBD. Therefore, the mechanistic aspects of gelatinase A binding to cells by this domain were further studied using cell attachment assays. Fibroblasts attached to rCBD-coated microplate wells in a manner that was inhibited by soluble rCBD, blocking antibodies to the beta1-integrin subunit but not the alpha2-integrin subunit, and bacterial collagenase treatment. Addition of soluble collagen rescued the attachment of collagenase-treated cells to the rCBD. As a probe on ligand blots of octyl-beta-D-thioglucopyranoside-solubilized cell membrane extracts, the rCBD bound 140- and 160-kDa protein bands. Their identities were likely procollagen chains being both bacterial collagenase-sensitive and also converted upon pepsin digestion to 112- and 126-kDa bands that co-migrated with collagen alpha1(I) and alpha2(I) chains. A rCBD mutant protein (Lys263 --> Ala) with reduced collagen affinity showed less cell attachment, whereas a heparin-binding deficient mutant (Lys357 --> Ala), heparinase treatment, or heparin addition did not alter attachment. Thus, a cell-binding mechanism for gelatinase A is revealed that does not involve the hemopexin COOH domain. Instead, an attachment complex comprising gelatinase A-native type I collagen-beta1-integrin forms as a result of interactions involving the collagen-binding domain of the enzyme. Moreover, this distinct pool of cell collagen-bound proenzyme appears recalcitrant to cellular activation.

Antibodies↗
PubMed2002

Stage-specific expression of genes associated with rat spermatogenesis: characterization by laser-capture microdissection and real-time polymerase chain reaction.

Spermatogenesis in the rat consists of 14 unique morphologic cellular associations between Sertoli cells and developing germ cells within the seminiferous epithelium. The complexity of the cellular associations leads to difficulty in the isolation of individual cells at a defined stage of development for the study of their unique patterns of gene or protein expression. Thus, laser-capture microdissection is an ideal technique to permit such analysis. This study used laser-capture microdissection and real-time reverse transcription-polymerase chain reaction (RT-PCR) to quantitate the stage-specific expression of a series of genes of functional significance in hormonal regulation and cell-cell interactions in spermatogenesis, including cathepsin-L, CREM-tau, transition protein-1, androgen receptor, beta1-integrin, N-cadherin, and hypoxanthine phosphoribosyltransferase (HPRT). Frozen sections (10 micro m) were obtained from normal adult rat testes. Laser-capture microdissection (LCM) was used to capture all cells in cross-sections of seminiferous tubules that were grouped into stages I-V, VII-VIII, and IX-XIII. Transition protein-1 expression was lowest during stages I-V and increased 5.9-fold during stages VII-VIII and IX-XIII (P < 0.01). Cathepsin-L expression was highest during stages I-V and VII-VIII, falling 4.9-fold during stages IX-XIII (P < 0.05). Similarly, CREM-tau expression was highest during stages I-V and VII-VIII, falling 1.6-fold during stages IX-XIII (P < 0.05). A novel CREM-tau isoform lacking the phosphorylation domain was also characterized but was not stage-specific. beta1-Integrin, N-cadherin, and androgen receptor expression did not change between the spermatogenic stages examined. HPRT housekeeper expression was lowest during stages I-V but increased 1.5-fold during stages VII-VIII and IX-XIII (P < 0.05). This study is the first to apply LCM and real-time RT-PCR analysis to quantitate stage-specific changes in the expression of multiple genes in the seminiferous epithelium.

Animals↗
PubMed2004

Oxidative stress induces IGF-I receptor signaling disturbances in cultured human dermal fibroblasts. A possible mechanism for collagen biosynthesis inhibition.

The effects of oxidative stress on collagen and DNA biosynthesis, beta-galactosidase and prolidase activities, and the expression of prolidase, beta1-integrin receptor, FAK, IGF-IR and MAP-kinases (ERK1, ERK2) were evaluated in human dermal fibroblasts. Subconfluent cells were subjected to repetitive stresses with 30 microM t-BHP for 1 hour per day over the course of 5 days. It was found that oxidative stress induced the inhibition of collagen biosynthesis in these cells in a time-dependent manner. Exposure of the cells to 5 stresses contributed to a decrease in collagen and DNA biosynthesis to about 30% and 50% of the control values, respectively. Prolidase activity and expression were only suppressed in fibroblasts subjected to 1 and 3 stresses. In these cells prolidase activity was decreased by about 20%. As a result of 5 stresses, no further inhibition of prolidase activity occurred; however, expression of the enzyme was slightly increased, as demonstrated by Western blot analysis. It was found that these phenomena were neither related to the expression of beta1-integrin receptor nor to that of FAK. However, the exposure of the cells to 3 and 5 stresses contributed to a distinct decrease in IGF-IR and MAP-kinases (ERK1, ERK2) expression, which is probably responsible for the collagen biosynthesis inhibition.

Cells, Cultured↗
PubMed2005

Adherens junctions of the human detrusor.

OBJECTIVE: To immunohistochemically identify the protein composition of adherens junctions, which couple smooth muscle cells mechanically, and to confirm their decrease in different bladder dysfunctions, as studies in geriatric bladder dysfunction show fewer such junctions in patients with detrusor overactivity (DO) and bladder outlet obstruction (BOO). MATERIAL AND METHODS: Detrusor biopsies were obtained from video-urodynamically evaluated patients with neurogenic DO (NDO, 31 patients), BOO (six patients) and from six patients with stress urinary incontinence (SUI) with stable, unobstructed detrusors (serving as controls). Specimens were fixed, paraffin-embedded, sectioned, stained with a polyclonal pan-cadherin antibody, monoclonal alpha-, beta- and gamma-catenin antibodies, and a monoclonal integrin-beta1 antibody. All antibodies were known to react with proteins of adherens junctions. Two examiners unaware of sample origin evaluated the sections qualitatively and using a semiquantitative scale. The results were correlated with the patient groups. RESULTS: Specific immunohistochemical staining of pan-cadherin, alpha-, beta- and gamma-catenin could not be detected in any detrusor smooth muscle compartment, but was present in the urothelium. There was integrin-beta1 reactivity in the basement membranes of bladder smooth muscle cells in 38 of 43 detrusor biopsies. There were no differences among the three groups. CONCLUSION: The known proteins of cell-cell adherens junctions are not part of the cell-cell junctions of detrusor smooth muscle. The specific staining of integrin-beta1 indicates either the presence of cell-matrix junctions or of cell-cell junctions within the human detrusor. Further studies are needed to identify the complete protein composition of adherens junctions within smooth muscle cells.

Adherens Junctions↗
PubMed2005

Regulation of glioma cell migration by serine-phosphorylated P311.

P311, an 8-kDa polypeptide, was previously shown to be highly expressed in invasive glioma cells. Here, we report the functional characteristics of P311 with regard to influencing glioma cell migration. P311 is constitutively serine-phosphorylated; decreased phosphorylation is observed in migration-activated glioma cells. The primary amino acid sequence of P311 indicates a putative serine phosphorylation site (S59) near the PEST domain. Site-directed mutagenesis of S59A retarded P311 degradation and induced glioma cell motility. In contrast, S59D mutation resulted in the rapid degradation of P311 and reduced glioma cell migration. Coimmunoprecipitation coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis identified Filamin A as a binding partner of P311, and immunofluorescence studies showed that both proteins colocalized at the cell periphery. Moreover, P311-induced cell migration was abrogated by inhibition of beta1 integrin function using TACbeta1A, a dominant-negative inhibitor of beta1 integrin signaling, suggesting that P311 acts downstream of beta1 signaling. Finally, overexpression of P311 or P311 S59A mutant protein activates Rac1 GTPase; small interfering RNA-mediated depletion of Rac1 suppresses P311-induced motility. Collectively, these results suggest a role for levels of P311 in regulating glioma motility and invasion through the reorganization of actin cytoskeleton at the cell periphery.

Amino Acid Sequence↗
PubMed1997

Adhesion and migration are differentially regulated in hematopoietic progenitor cells by cytokines and extracellular matrix.

The conditions that control the migratory status of hematopoietic progenitor cells on extracellular matrix (ECM) and that decide whether a cell migrates or adheres are incompletely understood. We analyzed the migratory behavior of murine hematopoietic progenitor cells factor-dependent-cell-paterson (FDCP)-mix and purified lin-Sca1+ bone marrow cells on ECM. We found that migration on fibronectin (Fn) or laminin (Lam) becomes dependent on beta1-integrins if a surface restraint force is introduced by tilting the ECM-coated culture vessels. Under these conditions, migration specifically occured on Fn and Lam, and was not detected on collagen IV-, hyaluronate-, or bovine serum albumin- coated surfaces. Migration depended on the continuous presence of hematopoietic cytokines interleukin-3 (IL-3), granulocyte colony-stimulating factor (G-CSF), macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), or stem cell factor (SCF), whereas other cytokines, such as IL-8, macrophage inflammatory protein-1alpha, macrophage-chemotactic and activating factor, and erythropoietin resulted in very little or no migratory response. IL-3 induced migration was synergistically enhanced by other CSFs, but was completely inhibited by addition of transforming growth factor-beta1. In contrast to firm local adhesion of previously cytokine depleted progenitors that was rapidly inducible within 1 hour after exposure to cytokines, preincubation on Fn matrix for 4 to 6 hours was required before cytokines could induce migration. A sudden increase of cytokine concentration reversibly inhibited migration and induced a fully adhesive state; this effect could be prolonged by consecutive stimulation with heterologous cytokines. Whereas cytokines activated resting progenitor cells to migrate on ECM, cell migration speed was regulated by Fn concentration. These results indicate that beta1-integrin-mediated progenitor cell adhesion and migration are differentially regulated by external stimuli and suggest that this regulation corresponds to different activation states of beta1-integrins in hematopoietic progenitor cells.

Animals↗
PubMed2000

Opposing effects of engagement of integrins and stimulation of cytokine receptors on cell cycle progression of normal human hematopoietic progenitors.

We evaluated the effect of beta1-integrin receptor engagement on the expression and activity of cell cycle regulatory proteins in CD34(+) cells under conditions that mimic the steady-state marrow microenvironment and in the presence of supraphysiological concentrations of interleukin-3 (IL3) and stem cell factor (SCF). Adhesion of CD34(+) progenitors to fibronectin (FN) was similar whether IL3 or SCF was present or absent. Engagement of beta1-integrins blocked S-phase entry of CD34(+) cells in the absence of IL3 or SCF, whereas addition of 10 ng/mL IL3 or SCF prevented such a block in S-phase entry. In the absence of IL3 or SCF, cyclin-E levels were significantly lower and p27(KIP1) levels significantly higher in FN-adherent than in FN-nonadherent cells, or than in poly-L-lysine (PLL)-adherent or (PLL)-nonadherent cells. Cyclin-dependent-kinase (cdk)-2 activity was decreased and levels of cyclin-E-cdk2 complexes were lower in FN-adherent than in PLL-adherent cells. In contrast, cyclin-E and p27(KIP1) protein levels and cdk2 activity in cells adherent to FN in the presence of IL3 or SCF were similar to those in PLL-adherent and FN-nonadherent or PLL-nonadherent cells. In conclusion, under physiological cytokine conditions, integrin engagement prevents S-phase entrance of CD34(+) cells, which is associated with elevated levels of the contact-dependent cyclin kinase inhibitor p27(KIP1). Supraphysiological concentrations of IL3 or SCF prevent p27(KIP1) elevation and override the integrin-mediated inhibition of entry into S phase.

CDC2-CDC28 Kinases↗
PubMed2003

The influence of the extracellular matrix on the morphology and intracellular pH of cultured astrocytes exposed to media lacking bicarbonate.

In previous work we showed that the polygonal shape of hippocampal astrocytes cultured on poly-L-lysine changes to a stellate morphology with loss of actinomyosin stress fibers on exchanging the culture medium for saline buffered with HEPES [Brain Res 946 (2002)12]. By contrast, in bicarbonate-buffered saline containing Ca(2+) astrocytes remained polygonal and continued to express stress fibers. Evidence suggests that stellation induced by saline buffered with HEPES is related to intracellular acidification due to the absence of bicarbonate. Here we studied the influence of the matrix used in preparing astrocyte cultures. Stellation in HEPES-saline occurred on a matrix of fibronectin, but not on matrices of collagen I or IV provided Ca(2+) was present. Laminin partially prevented stellation in HEPES-saline. Further, the intracellular acidification induced by HEPES-saline observed in astrocytes cultured on polylysine was abolished in cells cultured on collagens and was attentuated on a matrix of laminin. Two observations suggested the involvement of integrins and focal adhesions. (1) Treatment of cultures on collagens with a blocking antibody to the beta1 integrin subunit abolished protection against HEPES-induced stellation. (2) Compared with polylysine, astrocytes cultured on collagens expressed increased contents of phosphotyrosine proteins, focal adhesion proteins vinculin and paxillin, the beta1 integrin subunit and increased numbers of focal adhesions labelled with anti-vinculin. The observation that astrocytes cultured on collagen I or IV, in contrast to polylysine, express stress fibers and a constant intracellular pH in the absence of buffering by bicarbonate may be related to the fact that in the intact brain astrocytic processes (or end-feet) encounter and bind to collagen IV and laminin in the basement membrane of the endothelial cells which surround the cerebral capillaries. It is also possible that astrocytes retain this capacity from early development when fibrous matrix proteins are present.

Actins↗
PubMed2003

Close homolog of L1 is an enhancer of integrin-mediated cell migration.

Close homolog of L1 (CHL1) is a member of the L1 family of cell adhesion molecules expressed by subpopulations of neurons and glia in the central and peripheral nervous system. It promotes neurite outgrowth and neuronal survival in vitro. This study describes a novel function for CHL1 in potentiating integrin-dependent cell migration toward extracellular matrix proteins. Expression of CHL1 in HEK293 cells stimulated their haptotactic migration toward collagen I, fibronectin, laminin, and vitronectin substrates in Transwell assays. CHL1-potentiated cell migration to collagen I was dependent on alpha1beta1 and alpha2beta1 integrins, as shown with function blocking antibodies. Potentiated migration relied on the early integrin signaling intermediates c-Src, phosphatidylinositol 3-kinase, and mitogen-activated protein kinase. Enhancement of migration was disrupted by mutation of a potential integrin interaction motif Asp-Gly-Glu-Ala (DGEA) in the sixth immunoglobulin domain of CHL1, suggesting that CHL1 functionally interacts with beta1 integrins through this domain. CHL1 was shown to associate with beta1 integrins on the cell surface by antibody-induced co-capping. Through a cytoplasmic domain sequence containing a conserved tyrosine residue (Phe-Ile-Gly-Ala-Tyr), CHL1 recruited the actin cytoskeletal adapter protein ankyrin to the plasma membrane, and this sequence was necessary for promoting integrin-dependent migration to extracellular matrix proteins. These results support a role for CHL1 in integrin-dependent cell migration that may be physiologically important in regulating cell migration in nerve regeneration and cortical development.

Cell Adhesion Molecules↗
PubMed2000

Defects in adhesion and migration, but not in proliferation and differentiation, of embryonic stem cells upon replacement of integrin subunit beta1A by beta1D.

Beta1D is a skeletal muscle-specific splice variant of the beta1 integrin subunit, while beta1A integrin subunit has a wide tissue distribution. We have previously shown that replacement of beta1A by beta1D by homologous recombination (knockin) in all mouse tissues was embryonic lethal. Through two successive rounds of homologous recombination, we have now produced embryonic stem (ES) cells expressing beta1D instead of beta1A, and analyzed the ability of beta1D to support ES cell differentiation in vitro and in teratomas in vivo. Beta1D knockin (KI) ES cells grew at a similar rate but as more compact colonies than the beta1A-expressing cells. Increased cell cohesiveness, however, did not appear to involve changes in cadherin activity. Although in both beta1A and beta1D-KI ES cells only one beta1 allele is active; the expression of beta1 integrins in the beta1D-KI ES cells was reduced by 50%, compared with that in the beta1A-expressing cells; this correlated with impaired adhesive and migratory capacities. It appeared that during in vitro cardiac differentiation, in spite of a slight delay in the induction of two cardiac-specific transcripts, the alpha- and beta-myosin heavy chains, contracting cardiomyocytes were detected in similar numbers and at the same time in embryoid bodies (EB) derived from beta1D-KI and from beta1A cells. Furthermore, replacement of beta1A by beta1D in ES cells did not affect neurite differentiation in embryoid bodies in the presence of retinoic acid suggesting that beta1D supports neurogenesis. However, the impaired migration of other cells from the EB, including endodermal cells, prevented the normal outgrowth of neurites in beta1D-KI EB. Finally, injection of beta1D-KI ES cells in the flank of syngeneic mice gave rise to fully developed teratomas containing simple and pluristratified epithelia, muscle, cartilage, blood vessels, and tissues from the neural lineage. These results show that the muscle-specific splice variant beta1D, in spite of its specific cytoplasmic domain, supports the differentiation of many cell types. This further suggests that the embryonic lethality in the beta1D-KI embryos was mainly due to the different ability of beta1 A and beta1D to mediate cell adhesion and migration.

Alternative Splicing↗
PubMed2006

Matrix metalloproteinase-2 (MMP-2) regulates astrocyte motility in connection with the actin cytoskeleton and integrins.

Matrix Metalloproteinases (MMPs) play a role in migration of many cell types outside the central nervous system (CNS). Among neural cells, astrocytes are one of the main sources of MMPs in physiological and postlesional conditions. However, no data are available on the possible role of MMPs in astrocyte motility. Using an in vitro model of 2D migration and broad spectrum and selective MMP inhibitors, the authors demonstrated that MMP-2, but not MMP-9, is a key enzyme for astrocyte migration. In support of these data, the authors found constitutive expression of MMP-2 in astrocytes, while MMP-9 was nearly undetectable by gel zymography and immunocytochemical methods. The inhibition of migration by MMP inhibitors correlated with changes in cell morphology and in the organization of the actin cytoskeleton. In parallel, the characteristic focalized distribution of MMP-2 at the migration front observed in control cells became more diffuse and internalized by treatments that inhibited migration. The disruption of actin by cytochalasin D caused the partial recruitment of MMP-2 and gelatinolytic activity into actin aggregates, indicating a connection between the proteinase and the actin cytoskeleton. Finally, the authors found a co-localization of beta1-integrin with MMP-2 at the leading edge of migrating astrocytes. Altogether, these data provide the first evidence for the implication of MMP-2 in astrocyte motility, probably through the interaction of the proteinase with beta1-integrin that could act as a linker between pericellular proteolysis and the actin cytoskeleton.

Actins↗
PubMed1999

Beta3-integrin-mediated focal adhesion complex formation: adult cardiocytes embedded in three-dimensional polymer matrices.

In vivo studies show that beta3-integrin-mediated focal adhesion formation (FAF) causes recruitment of nonreceptor tyrosine kinases to the cytoskeleton in pressure-overloaded myocardium. To define the mechanism of beta3-integrin-mediated signaling, we developed a cell culture model (adult feline cardiocytes embedded in a 3-dimensional matrix of native type 1 collagen, fibronectin, and vitronectin) wherein beta3-integrin-mediated focal adhesion kinase occurs. Focal adhesion kinase was analyzed immunocytochemically using confocal microscopy. Initial studies suggested that cardiocytes cultured in a 3-dimensional matrix formed focal adhesions consisting of both beta3-integrin and the muscle-specific isoform, beta1-integrin (beta1D). The focal adhesions were associated with focal adhesion kinase on both costameres and intercalated disks. To determine the cause of beta1D-integrin-mediated focal adhesion kinase in this model, time course studies were done. Beta3-integrin-mediated focal adhesion kinase occurred within 30 minutes after embedding cardiocytes and persisted for >24 hours, whereas beta1D-integrin-mediated focal adhesion kinase was present from the outset. Because confocal microscopy showed that laminin was present on the surface of freshly isolated cardiocytes, we hypothesized that this was causative of beta1D-integrin-mediated focal adhesion kinase. Freshly isolated cardiocytes washed with acidic medium (2 minutes, pH 3.0) to remove laminin and then embedded in a 3-dimensional matrix showed complete absence of beta1D-integrin-mediated focal adhesion kinase, but beta3-integrin-mediated focal adhesion kinase occurred with a time course similar to that seen in cultured, unwashed cardiocytes. Acid washing did not alter the binding ability of beta1D-integrin, because acid-washed cardiocytes in the presence of laminin showed beta1D-integrin-mediated focal adhesion kinase. Thus, cardiocytes embedded in a 3-dimensional matrix show beta3-integrin-mediated focal adhesion kinase and provide an in vitro model to study beta3-integrin-mediated signaling in response to hemodynamic cardiac loading.

Animals↗