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PubMed2000

CD44 and beta1 integrins mediate ovarian carcinoma cell migration toward extracellular matrix proteins.

Epithelial cancer of the ovary spreads by implantation of tumor cells onto the mesothelial cells that line the peritoneal cavity. The aim of this study was to identify the cell-matrix interactions that mediate ovarian carcinoma cell migration toward components of the mesothelial cell-associated extracellular matrix. The human ovarian carcinoma cell lines NIH:OVCAR5 and SKOV3 were analyzed by flow cytometry for the expression of cell surface receptors. The ability of those receptors to mediate ovarian carcinoma cell migration toward fibronectin, type IV collagen, and laminin was determined. A monoclonal antibody against the beta1 integrin subunit abrogated the migration of both cell lines toward the extracellular matrix proteins. Blocking antibodies against alpha integrin subunits suggest that ovarian carcinoma cell migration toward fibronectin is primarily mediated by the alpha5beta1 integrin, type IV collagen by the alpha2beta1 integrin, and laminin by the alpha6beta1 integrin. These results suggest that ovarian carcinoma cell migration is regulated by multiple beta1 integrin-matrix interactions. Significant reduction of cell migration was observed with a monoclonal antibody against CD44 that blocks the hyaluronan-binding site of CD44, but not with an antibody that binds at an alternate site on CD44. Intact hyaluronan and/or hyaluronan oligomers also inhibited cell migration, suggesting that the CD44-hyaluronan interaction provides an integrin-independent mechanism of control for ovarian carcinoma cell migration. These results suggest that ovarian carcinoma cell migration is regulated by both integrin-dependent mechanisms, involving the interaction of beta1 integrins with extracellular matrix proteins, and an integrin-independent mechanism that involves the interaction of CD44 and hyaluronan.

Antibodies, Monoclonal↗

PubMed1997

Calreticulin is essential for integrin-mediated calcium signalling and cell adhesion.

Integrins are important mediators of cell adhesion to extracellular ligands and can transduce biochemical signals both into and out of cells. The cytoplasmic domains of integrins interact with several structural and signalling proteins and consequently participate in the regulation of cell shape, motility, growth and differentiation. It has been shown that calreticulin associates with the cytoplasmic domains of integrin alpha-subunits and that this interaction can influence integrin-mediated cell adhesion to extracellular matrix. We have now developed calreticulin-deficient embryonic stem (ES) cells and isolated embryonic fibroblasts from calreticulin mutant mice. We find that in both cell types integrin-mediated adhesion is severely impaired, although integrin expression is unaltered. Expression of recombinant calreticulin in double knockout ES cells by complementary DNA transfection rescued integrin-mediated adhesion. In wild-type cells, engagement of surface integrins induced a transient elevation in cytosolic calcium concentration owing to influx of extracellular calcium. This calcium transient was absent in calreticulin-deficient cells. In contrast, the amount of calcium in endomembrane stores, which is sensitive to both inositol 1,4,5-trisphosphate and thapsigargin, was indistinguishable in the two cell types. Our results indicate that calreticulin is an essential modulator both of integrin adhesive functions and integrin-initiated signalling, but that it may not play a significant role in the storage of luminal calcium.

Animals↗

PubMed1999

AlphaVbeta5 integrin: a co-receptor for adeno-associated virus type 2 infection.

Understanding the primary steps of viral entry can have important implications for strategies to prevent infection of known viral pathogens as well as determining parameters for efficient gene delivery using viral vectors. Recently, a two-step process for viral infection involving attachment of virus to a primary receptor (coxsackievirus adenovirus receptor and heparan sulfate proteoglycan) and subsequent mediation of virus entry by a co-receptor (alphaV integrins and HVEM) has been determined for both adenovirus and HSV, respectively. Heparan sulfate proteoglycan serves as a primary attachment receptor for adeno-associated virus type 2 (AAV-2)(ref. 5). Here we determined that alphaVbeta5 integrin plays a part in efficient AAV infection. Experiments using the chelating agent EDTA to disrupt integrin function resulted in a corresponding decrease in AAV infection, consistent with the possibility that integrin mediates infection. Viral overlay experiments on purified plasma membrane proteins as well as immunoprecipitated integrin beta5 subunit demonstrated that AAV directly associates with the beta5 subunit of alphaVbeta5 integrin. Genetically defined cells expressing alphaVbeta5 integrin showed increased susceptibility to AAV infection, demonstrating a biological role of this integrin in AAV infection. Finally, viral binding and internalization studies indicate that alphaVbeta5 integrin is not a primary attachment receptor for AAV-2, but is instead involved in facilitating virus internalization. This study supports the idea that alphaVbeta5 integrin serves as a co-receptor for AAV-2 virions, and should have a substantial effect on the use of AAV vectors in human gene therapy.

Cell Fractionation↗

PubMed1997

Effects of integrins on proliferation and apoptosis of renal epithelial cells after acute injury.

We sought to determine the importance of integrins for recovery after acute tubular injury and to investigate the signal transduction pathways for integrin effects on cell cycle regulation involving proliferation and apoptosis. Primary cultures of rat renal proximal tubule epithelial cells were exposed to a superoxide-generating system to induce injury in the absence of overt necrosis. Integrin function was antagonized by the integrin recognition sequence tetrapeptide Gly-Arg-Gly-Asp (GRGD) or monoclonal antibody to beta 1-integrin. Injured cells had reduced thymidine uptake compared with normal cells. The presence of GRGD during recovery from injury caused a further 44% reduction in DNA synthesis but did not affect DNA synthesis in normal cells. Injured cells had an increased proportion of apoptosis that was further accentuated by exposed to GRGD during recovery. Integrin antagonism also stimulated apoptosis in uninjured cells. To investigate signal transduction mechanisms for this effect of integrins, inhibitors and activators of protein tyrosine kinase (PTK) and protein kinase C (PKC) were evaluated. Activation of PKC stimulated cellular proliferation, whereas inhibitors of PKC and PTK had no significant effect. Genistein, a PTK inhibitor, induced apoptosis in normal cells, mimicking the effect of integrin inhibition. On the other hand, PMA, an activator of PKC, prevented cells from becoming apoptotic when exposed to injury plus GRGD. The phosphorylation status of intracellular proteins was evaluated by immunoblotting with antiphosphotyrosine antibody. A similar pattern of decreased phosphorylation was observed after either integrin inhibition, injury, both, or PTK inhibition. These findings suggest that kinase cascades are involved in the effects of integrins on renal epithelial cell proliferation and apoptosis. After injury, an interaction between cells and the extracellular matrix is required for cells to proliferate and contribute to repair rather than to enter an apoptotic pathway.

Animals↗

PubMed2001

alpha(4) integrin-dependent eosinophil recruitment in allergic but not non-allergic inflammation.

1. Although anti-alpha(4) integrin mAbs reduce eosinophil accumulation in several models of allergic inflammation, it is not clear whether this occurs via a direct action to block eosinophil alpha(4) integrins or indirectly on another cell type. The role of alpha(4) integrins on the accumulation of (111)In-labelled eosinophils in allergic and non-allergic inflammation in guinea-pig skin was therefore investigated. 2. Intradermal injection of antigen in sensitized skin sites induced accumulation of (111)In-eosinophils that was reduced up to 70% by two anti-alpha(4) integrin mAbs. In contrast, accumulation of (111)In-eosinophils to intradermal chemoattractants was unaffected by the same mAbs. 3. Accumulation of (111)In-eosinophils in allergic and non-allergic conditions was partly inhibited by a low dose of an anti-beta(2) integrin mAb. In combination with anti-alpha(4) integrin mAb, responses were not further reduced suggesting that these adhesion pathways are not additive or synergic. 4. Pretreating skin sites with antiserum or contaminating LPS did not reveal an alpha(4) integrin dependent pathway for chemoattractant-induced (111)In-eosinophil accumulation. These data suggest that alpha(4) integrins are involved in the response to antigen in sensitized skin sites. 5. Pretreating (111)In-eosinophil with alpha(4) integrin mAb blocked their adhesion to fibronectin in vitro but did not inhibit their accumulation in allergic inflammation suggesting that the blocking effect in vivo was eosinophil independent. 6. These data support the concept that targeting alpha(4) integrins on cells other than eosinophils could control eosinophil accumulation and have therapeutic potential in allergic diseases such as asthma and atopic dermatitis.

Animals↗

PubMed1992

Integrins as a primary signal transduction molecule regulating monocyte immediate-early gene induction.

Integrins are cell surface receptors found on monocytes that facilitate adhesion to both cellular and extracellular substrates. These integrins are thought to be involved in the selective gene induction observed after monocyte adhesion to various extracellular matrices. To investigate this hypothesis, we stimulated monocytes with monoclonal antibodies to different integrin receptors to specifically mimic the integrin receptor-ligand interactions. Engagement of the common beta chain of the beta 1 subfamily of integrins resulted in expression of the inflammatory mediator genes, interleukin 1 beta, interleukin 1 receptor antagonist, and monocyte adherence-derived inflammatory gene 6 (MAD-6), whereas engagement of the common beta chain of the beta 2 family did not. Furthermore, to characterize integrin-mediated gene induction, we examined the ability of antibodies to the alpha chain of integrin receptors to regulate gene expression. Engagement of the very late antigen 4 (VLA-4) receptor resulted in induction of all the mediator genes. Receptor crosslinking was required because individual Fab fragments were unable to stimulate gene induction whereas the divalent F(ab')2 fragment and the whole IgG molecule could. Interleukin 1 beta secretion was dependent on the anti-integrin antibody used. Some antibodies required a second signal and, for others, direct engagement was sufficient for protein production. In conclusion, engagement of integrin receptors regulated the production of both inflammatory mediator mRNA and protein. These results suggest that integrin-dependent recognition and adherence may provide the key signals for initiation of the inflammatory response during monocyte diapedesis.

Antibodies, Monoclonal↗

PubMed1997

A genetic analysis of integrin function: Glanzmann thrombasthenia in vitro.

Glanzmann thrombasthenia, an inherited bleeding disorder, can be caused by a defect or deficiency in platelet integrin alphaIIb beta3 (GPIIb-IIIa). Studies of thrombasthenia variants have facilitated identification of sites involved in the functions of alphaIIb beta3 and other integrins. Such sites include those that bind ligand and those that participate in the "activation" of alphaIIb beta3 required for high affinity binding of ligands such as fibrinogen or PAC1, a monoclonal antibody. Here we describe the isolation of such variants, created in vitro with Chinese hamster ovary cells that express an activated form of alphaIIb beta3. These cells were exposed to a mutagen, ethyl methane sulfonate, and variants that lost the capacity to bind PAC1 were isolated by fluorescence-activated cell sorting. These variants were grouped into three phenotypic classes. One comprised integrin mutations that disrupt ligand binding function; a second comprised mutations that interfere with the capacity of cells to activate the integrin. Most of these activation-defective mutations were in the integrin cytoplasmic domain, but surprisingly, several were caused by mutations affecting three closely spaced residues in the beta3 extracellular domain. A third class of mutants exhibited a defect in integrin activation not ascribable to changes in the integrin sequence. Thus, these may represent mutated signaling molecules required for integrin activation. This unbiased genetic approach provides new insights into the structural basis of integrin function and may assist in identifying the cellular events that regulate integrin function.

Amino Acid Sequence↗

PubMed1997

Oncogenic Ki-ras but not oncogenic Ha-ras blocks integrin beta1-chain maturation in colon epithelial cells.

Human colorectal tumors commonly contain mutations in Ki-ras but rarely, if ever, in Ha-ras. The selectivity for Ki-ras mutations in this tumor was explored using the HD6-4 colon epithelial cell line which contains no ras mutations. After adhesion to an extracellular matrix, HD6-4 cells polarize into columnar goblet cells with distinct apical and basal regions. Stable HD6-4 transfectants were made with mini-gene constructs of the oncogenic cellular Ki-ras4BG12V gene, the oncogenic Ha-rasG12V gene, or mini-gene constructs of wild-type Ki-ras4B as a control. Ki-ras mutations, but not Ha-ras mutations, disrupted colon epithelial cell apicobasal polarity and adhesion to collagen I and laminin. Three Ha-ras transfectants and three Ki-ras transfectants exhibited Ras proteins expressing the Val-12 mutation by Western blotting with pan-rasG12V antibody. Only wild-type Ki-ras transfectant cells and oncogenic Ha-ras transfectant cells synthesized the mature, fully glycosylated forms of beta1 integrin. Instead of the mature integrin beta1-chain, a faster migrating beta1-chain intermediate was detected on the cell surface and in the cytoplasm of the oncogenic Ki-ras transfectants. Expression of the oncogenic Ki-ras gene caused the altered beta1 integrin maturation because phosphorothiolated antisense oligonucleotides to Ki-ras reduced expression of both the mutant Ki-Ras protein and the aberrant integrin beta1-chain and increased expression of the mature integrin beta1-chain. Altered glycosylation generated the new beta1 integrin form since integrin core beta1-chain proteins of the same molecular weight were yielded in Ki-ras, Ha-ras, and control transfectants after removal of sugar residues with endoglycosidase F or following tunicamycin treatment to inhibit glycosylation. The selective effect of oncogenic Ki-ras on beta1 integrin glycosylation was not due to selective activation of mitogen-activated protein kinases because both mutated Ki- and Ha-ras genes activated this pathway and increased cell proliferation. Since blocking the glycosylation of integrin beta1-chain inhibited the adherence, polarization, and subsequent differentiation of colon epithelial cells, the selective effects of the oncogenic cellular Ki-ras gene on integrin beta1-chain glycosylation may account, at least in part, for the selection of Ki-ras mutations in human colon tumors.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed2002

Loss of p53 compensates for alpha v-integrin function in retinal neovascularization.

alpha(v)-Integrin antagonists block neovascularization in various species, whereas 20% of alpha(v)-integrin null mice are born with many normal looking blood vessels. Given that blockade of alpha(v)-integrins during angiogenesis induces p53 activity, we utilized p53 null mice to elucidate whether loss of p53 can compensate for alpha(v)-integrin function in neovascularization of the retina. Murine retinal vascularization was inhibited by systemic administration of an alpha(v)-integrin antagonist. In contrast, mice lacking p53 were refractory to this treatment, indicating that neovascularization in normal mice depends on alpha(v)-integrin-mediated suppression of p53. Blockade of alpha(v)-integrins during neovascularization resulted in an induction of p21(CIP1) in wild type and, surprisingly, in p53 null retinas, indicating that alpha(v)-integrin ligation regulates p21(CIP1) levels in a p53-independent manner. In conclusion, we demonstrate for the first time an in vivo intracellular mechanism for compensation of integrin function and that p53 and alpha(v)-integrins act in concert during retinal neovascularization.

Animals↗

PubMed2002

The tetraspan protein epithelial membrane protein-2 interacts with beta1 integrins and regulates adhesion.

The growth arrest-specific-3 (GAS3)/PMP22 proteins are members of the four-transmembrane (tetraspan) superfamily. Although the function of these proteins is poorly understood, GAS3/PMP22 proteins have been implicated in the control of growth and progression of certain cancers. Epithelial membrane protein-2 (EMP2), a GAS3/PMP22 family member, was recently identified as a putative tumor suppressor gene. Here, we addressed the normal function of EMP2 by testing the prediction that it influences integrin-related cell functions. We observed that EMP2 associates with the beta(1) integrin subunit. Co-immunoprecipitation and immunodepletion experiments indicated that approximately 60% of beta(1) integrins and EMP2 can be isolated in common protein complexes. Whereas this association between EMP2 and beta(1) integrin may be direct or indirect, it has features of integrin heterodimer selectivity. Thus, by laser confocal microscopy, EMP2 colocalized with alpha(6)beta(1) but not alpha(5)beta(1) integrin. Increased expression of EMP2 also influenced the integrin heterodimer repertoire present on the plasma membrane. EMP2 specifically increased the surface expression of the alpha(6)beta(1) integrin while decreasing that of the alpha(5)beta(1) protein. Reciprocally, reduction in EMP2 expression using a specific ribozyme decreased surface expression of alpha(6)beta(1) integrin. Accordingly, these EMP2-mediated changes resulted in a dramatic alteration in cellular adhesion to extracellular matrix proteins. This study demonstrates for the first time the interaction of a GAS3/PMP22 family member with an integrin protein and suggests that such interactions and their functional consequences are a physiologic role of GAS3/PMP22 proteins.

3T3 Cells↗

PubMed2003

Low density lipoprotein receptor-related protein-1 promotes beta1 integrin maturation and transport to the cell surface.

Low density lipoprotein receptor-related protein-1 (LRP-1) mediates the endocytosis of multiple plasma membrane proteins and thereby models the composition of the cell surface. LRP-1 also functions as a catabolic receptor for fibronectin, limiting fibronectin accumulation in association with cells. The goal of the present study was to determine whether LRP-1 regulates cell surface levels of the beta(1) integrin subunit. We hypothesized that LRP-1 may down-regulate cell surface beta(1) by promoting its internalization; however, unexpectedly, LRP-1 expression was associated with a substantial increase in cell surface beta(1) integrin in two separate cell lines, murine embryonic fibroblasts (MEFs) and CHO cells. The total amount of beta(1) integrin was unchanged because LRP-1-deficient cells retained increased amounts of beta(1) in the endoplasmic reticulum (ER). Expression of human LRP-1 in LRP-1-deficient MEFs reversed the shift in subcellular beta(1) integrin distribution. Metabolic labeling experiments demonstrated that the precursor form of newly synthesized beta(1) integrin (p105) is converted into mature beta(1) (p125) more slowly in LRP-1-deficient cells. Although low levels of cell surface beta(1) integrin, in LRP-1-deficient MEFs, were associated with decreased adhesion to fibronectin, the subcellular distribution of beta(1) integrin was most profoundly dependent on LRP-1 only after the cell cultures became confluent. A mutagen-treated CHO cell line, in which LRP-1 is expressed but retained in the secretory pathway, also demonstrated nearly complete ER retention of beta(1) integrin. These studies support a model in which LRP-1 either directly or indirectly promotes maturation of beta(1) integrin precursor and thereby increases the level of beta(1) integrin at the cell surface.

Animals↗

PubMed2005

Integrin alpha5/beta1 expression mediates HER-2 down-regulation in colon cancer cells.

HER-2 is constitutively activated and overexpressed in many cancers, and its inhibition in colon cancer cells diminishes tumorigenicity and induces apoptosis. Little is known about the regulation of HER-2 signaling in colon cancer cells. Integrin alpha5/beta1 expression is frequently lost in colorectal cancer cells compared with normal intestinal epithelium, and colon cancer cells lacking integrin alpha5/beta1 expression utilize HER-2 signaling for proliferation and tumorigenicity. Re-expression of integrin alpha5/beta1 in colon cancer cells abrogated their tumorigenicity, but how this occurs is not well known. Stable expression of integrin alpha5/beta1 in colon cancer cells with little or no detectable integrin alpha5/beta1 protein expression resulted in the post-transcriptional down-regulation of HER-2 protein. Integrin alpha5/beta1 was found to interact with HER-2, and the cytoplasmic domain of integrin alpha5/beta1 was sufficient to mediate HER-2 down-regulation. Integrin alpha5/beta1-mediated down-regulation of HER-2 was the result of increased lysosomal targeting. The inhibition of HER-2 signaling represents a potential mechanism by which integrin alpha5/beta1 exerts its tumor suppressor-like activity in colon cancer cells. These results also suggest that a novel function for integrin alpha5/beta1 is the control of HER-2 expression.

Agar↗

PubMed2005

A streptococcal collagen-like protein interacts with the alpha2beta1 integrin and induces intracellular signaling.

The streptococcal collagen-like proteins Scl1 and Scl2 are prokaryotic members of a large protein family with domains containing the repeating amino acid sequence (Gly-Xaa-Yaa)(n) that form a collagen-like triple-helical structure. Here, we test the hypothesis that Scl variant might interact with mammalian collagen-binding integrins. We show that the recombinant Scl protein p176 promotes adhesion and spreading of human lung fibroblast cells through an alpha2beta1 integrin-mediated interaction as shown in cell adhesion inhibition assays using anti-alpha2beta1 and anti-beta1 integrins monoclonal antibodies. Accordingly, C2C12 cells stably expressing alpha2beta1 integrin as the only collagen-binding integrin show productive cell adhesion activities on p176 that can be blocked by an anti-alpha2beta1 integrin antibody. In addition, p176 promotes tyrosine phosphorylation of p125(FAK) of C2C12 cells expressing alpha2beta1 integrin, whereas parental cells do not. Furthermore, C2C12 adhesion of human lung fibroblast cells to p176 induces phosphorylation of p125FAK, p130CAS, and p68Paxillin proteins. In a domain swapping experiment, we show that integrin binds to the collagenous domain of the Scl protein. Moreover, the recombinant inserted domain of the alpha2 integrin interacts with p176 with a relatively high affinity (K(D) = 17 nm). Attempts to identify the integrin sites in p176 suggest that more than one site may be involved. These studies, for the first time, suggest that the collagen-like proteins of prokaryotes retained not only structural but also functional characteristics of their eukaryotic counterparts.

Animals↗

PubMed2004

AlphaPIX associates with calpain 4, the small subunit of calpain, and has a dual role in integrin-mediated cell spreading.

Binding of integrins to the extracellular matrix results in actin cytoskeletal rearrangements, e.g. during cell spreading, by regulating the activity of Rho GTP-ases. We have shown previously that alphaPIX (Cool-2 or ARHGEF6), a Rac1/Cdc42-specific guanine nucleotide exchange factor (GEF), binds to beta-parvin/affixin and colocalizes with integrin-linked kinase in actively spreading cells, suggesting that alphaPIX is involved in integrin-induced signaling leading to activation of Rac1/Cdc42. Here we report calpain 4, the small subunit of the proteases mu-calpain and m-calpain, as a novel binding partner of alphaPIX. This association was identified by the CytoTrap system and confirmed by coimmunoprecipitation and glutathione S-transferase pull-down assays. The alphaPIX triple domain SH3-DH-PH was found to be required for calpain 4 binding. During integrin-dependent spreading of CHO-K1 cells, alphaPIX colocalized with mu- and m-calpain, integrin-linked kinase, and beta1 integrin in early integrin-containing clusters. Overexpression of alphaPIX wild type but not the GEF-deficient mutant (L386R/L387S) resulted in enhanced formation of characteristic cellular protrusions during cell spreading, suggesting that alphaPIX GEF activity is necessary for this specific actin cytoskeletal reorganization. The calpain inhibitors calpeptin and calpain inhibitor IV significantly inhibited integrin-dependent cell spreading. However, concomitant overexpression of alphaPIX wild type or the L386R/L387S mutant restored cell spreading. Together, these data suggest that alphaPIX is a component of early integrin clusters and plays a dual role in integrin-dependent cell spreading. Whereas alphaPIX GEF activity contributes to enhanced formation of cellular protrusions, the GEF-independent association with calpain 4 leads to induction of a yet unknown signaling cascade resulting in cell spreading.

Amino Acid Substitution↗

PubMed2006

Activated epidermal growth factor receptor induces integrin alpha2 internalization via caveolae/raft-dependent endocytic pathway.

Elevated expression or activity of the epidermal growth factor (EGF) receptor is common in ovarian cancer and is associated with poor patient prognosis. Our previous studies demonstrated that expression of the constitutively active mutant form of the EGF receptor (EGFRvIII) in ovarian cancer cells led to reduction in integrin alpha2 surface expression, defects in cell spreading, and disruption of focal adhesions. Inhibition of EGFRvIII catalytic activity reversed the response, suggesting that EGF receptor activation regulates integrin alpha2. In this study we found that EGF treatment resulted in a transient loss of integrin alpha2 from the cell surface. Before EGF stimulation, integrin alpha2 and EGF receptors were associated based on biochemical and immuno-colocalization approaches. After EGF treatment, EGF receptor and integrin alpha2 were internalized and segregated into different compartments. Integrin alpha2, but not EGF receptor, was associated with caveolin-1 and GM1 (Gal_1,3GalNAc_1,4(Neu5Ac-_ 2,3)Gal_1,4Glc_1,1-ceramide) gangliosides, suggesting caveolae-mediated endocytosis. Moreover, integrin alpha2 was subsequently targeted to the Golgi apparatus and the endoplasmic reticulum. Together, these findings demonstrate that activated EGF receptor transiently modulates integrin alpha2 cell surface expression and stimulates integrin alpha2 trafficking via caveolae/raft-mediated endocytosis, representing a novel mechanism by which the EGF receptor may regulate integrin-mediated cell behavior.

Caveolae↗

PubMed1999

Intermediate filament, laminin and integrin expression in lacrimal gland acinar cells: comparison of an immortalized cell line to primary cells, and their response to retinoic acid.

PURPOSE: The goal of this study was to characterize intermediate filament, integrin and laminin expression by rabbit lacrimal gland acinar cells in culture, to determine whether retinoic acid (RA) alters expression of these proteins and to compare primary cells to an immortalized rabbit lacrimal gland acinar cell line using flow cytometric analysis. METHODS: Primary cells, maintained in serum free medium, were exposed to 10(-6) M retinoic acid for 24 hours. Immortalized cells were grown in defined medium with Nu-Serum and exposed to retinoic acid. Cells were labeled with monoclonal antibodies to cytokeratins (AE1, AE2, AE3, AE5, CK10/13, CK18), integrins (alpha(3), alpha(6), alpha(V), beta(1), beta(2), beta(3) and beta(4)), laminin, or vimentin and with FITC-conjugated secondary antibodies. Cells were analyzed for antigen expression by flow cytometry and immunocytochemistry. RESULTS: Primary and immortalized cells expressed type I and type II epithelial cytokeratins (AE1 and AE3), cytokeratin 18, and cytokeratin 3 (AE5) Both cell types were negative to AE2 and CK10/13. Primary and immortalized cells expressed vimentin in culture, with immortalized cells expressing this protein at higher levels. Lacrimal acinar cells appear to synthesize laminin which was detected intracellularly in both cells types. Integrins alpha(6) (CD49f) and alpha(V) (CD51) were expressed by primary and immortalized cells. Expression of integrin alpha(6) was 10-fold higher in immortalized cells compared to primary cells. Retinoic acid increased integrin alpha( V) expression by primary and immortalized cells 1.3-fold and 3-fold, respectively, and caused a slight increase in integrin alpha(6) expression by primary cells. Both cell types also expressed integrins beta( 1), beta(2) and beta(3), but beta(4) was detected only in immortalized cells. Lacrimal acinar cells do not express integrin alpha(3). CONCLUSIONS: Expression of cytokeratins, laminin and integrins by primary and immortalized cells was similar, suggesting that the immortalized cell line is a good model for the study of lacrimal structure and function. Since retinoic acid up-regulated only integrin alpha(V), but not cytokeratins, these cells appear to be highly differentiated. Flow cytometry is a useful method for analysis of protein expression by lacrimal acinar cells.

Animals↗

PubMedDate not supplied

Angiotensin II stimulates endothelial integrin beta3 expression via nuclear factor-kappaB activation.

The present study examined the effect and part mechanism of angiotensin II-stimulated integrin beta3 gene expression in human umbilical vein endothelial cells. Protein level and mRNA level of integrin beta3 expression were determined using enzyme-linked immunoadsorbent assay and reverse transcription-polymerase chain reaction. Four plasmids of 5'-different deletion of integrin beta3 gene promoter were constructed to transiently transfected into cells to uncover the region in response to angiotensin II. Blockade of nuclear factor-kappaB (NF-kappaB) signaling pathway effect on integrin beta3 expression was analyzed by cotransfection with mutant plasmids for NF-kappaB-inducing kinase, inhibitory proteins alpha and beta of NF-kappaB kinase, respectively, together with the integrin beta3 plasmid including the sequence -1486 approximately - 900. The study found that 10(-8) mol/L,10(-7) mol/L, 10(-6) mol/L, and 10(-5) mol/L angiotensin II increased integrin beta(3) protein level by 45%, 52%, 62%, and 73% respectively. Angiotensin II at 10(-6) mol/L increased integrin beta3 mRNA level by 67%. The luciferase activity of the integrin beta3 plasmid PGL3 - 1486 approximately - 900 increased by 84.72% in response to angiotensin II. N-acetylcysteine blocked angiotensin II-induced NF-kappaB activity and integrin beta3 expression. Blockade of NF-kappaB signaling pathway abolished the stimulation of angiotensin II. These results suggest that angiotensin II stimulates integrin beta3 expression partly by NF-kappaB activation.

Acetylcysteine↗

PubMed1989

Dynamic cytoskeleton-integrin associations induced by cell binding to immobilized fibronectin.

We have examined the early events of cellular attachment and spreading (10-30 min) by allowing chick embryonic fibroblasts transformed by Rous sarcoma virus to interact with fibronectin immobilized on matrix beads. The binding activity of cells to fibronectin beads was sensitive to both the mAb JG22E and the GRGDS peptide, which inhibit the interaction between integrin and fibronectin. The precise distribution of cytoskeleton components and integrin was determined by immunocytochemistry of frozen thin sections. In suspended cells, the distribution of talin was diffuse in the cytoplasm and integrin was localized at the cell surface. Within 10 min after binding of cells and fibronectin beads at 22 degrees C or 37 degrees C, integrin and talin aggregated at the membrane adjacent to the site of bead attachment. In addition, an internal pool of integrin-positive vesicles accumulated. The mAb ES238 directed against the extracellular domain of the avian beta 1 integrin subunit, when coupled to beads, also induced the aggregation of talin at the membrane, whereas ES186 directed against the intracellular domain of the beta 1 integrin subunit did not. Cells attached and spread on Con A beads, but neither integrin nor talin aggregated at the membrane. After 30 min, when many of the cells were at a more advanced stage of spreading around beads or phagocytosing beads, alpha-actinin and actin, but not vinculin, form distinctive aggregates at sites along membranes associated with either fibronectin or Con A beads. Normal cells also rapidly formed aggregates of integrin and talin after binding to immobilized fibronectin in a manner that was similar to the transformed cells, suggesting that the aggregation process is not dependent upon activity of the pp60v-src tyrosine kinase. Thus, the binding of cells to immobilized fibronectin caused integrin-talin coaggregation at the sites of membrane-ECM contact, which can initiate the cytoskeletal events necessary for cell adhesion and spreading.

Animals↗