Expression of integrin subunits αv and β3 in acute lung inflammation

Integrin subunits v and 3 form a dimer, v3, which is expressed on normal neutrophils and endothelium. We investigated the expression of integrin subunits v and 3 in acute lung inflammation in Sprague-Dawley rats (n=5 each) following intratracheal challenge with Escherichia coli or Streptococcus pneumoniae, which induce neutrophil recruitment through different mechanisms. Control rats (n=5) were given endotoxin-free saline. Both bacterial challenges induced similar levels of recruitment of neutrophils in lungs. Western blots showed lower expression of integrin subunits v and 3 in lungs challenged with E. coli compared to those given S. pneumoniae. Immunohistochemistry and immunogold electron microscopy localized both integrin subunits in neutrophils and endothelium in the control and treated rat lungs. Quantitative immunohistochemistry showed that E. coli-challenged rat lungs contained a lower percentage of neutrophils expressing integrin subunits v and 3 compared to those challenged with S. pneumoniae (P<0.05). We conclude that E. coli infection decreased the percentage of neutrophils expressing integrin subunits v and 3 compared to S. pneumoniae infection. These data lay the foundation for further characterization of these integrin subunits in neutrophil migration specifically in S. pneumoniae infection that utilizes molecules other than 2 integrins for neutrophil recruitment.     

The effect of mechanical load on integrin subunits α5 and β1 in chondrocytes from mature and immature cartilage explants

Articular cartilage is subjected to cyclic compressive stresses during joint loading. There is increasing experimental evidence that this loading is essential for the chondrocytes to maintain the functionality of the cartilage extracellular matrix (ECM) and that members of the integrin family of transmembrane receptors may play an important role in signal mechanotransduction between the ECM and chondrocytes. Of particular interest are the integrin subunits 5 and 1, which are known to form the receptor for fibronectin, an important ECM protein, and to be involved in mechanotransduction as well as in the regulation of cytokine production. In this study, we measured the amounts of the integrin subunits 5 and 1 in chondrocytes from young (immature) and adult (mature) bovine articular cartilage explants which were subjected to a continuously applied cyclic compressive stress of 1 MPa for 6 and 24 h. The integrin content per chondrocyte was measured immediately after load cessation by flow cytometry following matrix digestion to release the cells. We found that a mechanical stress induced an increase in the number of integrin subunit 5 in immature and mature cartilage but not in the integrin subunit 1 content. The integrin contents were greatest after 6 h of loading and returned to control levels after 24 h of unloading. The results of this study supply further experimental evidence that chondrocytes respond to changes in their mechanical environment and that the integrin 51 may act as a mechanical signal transducer between the chondrocyte and the ECM for the modulation of cellular physiology.This work was supported by NIH grant AR45748 to PAT and the HSS MacArthur Cartilage Fund.     

Localization and synthesis of α-fetoprotein in the chicken

Summary The localization and sites of synthesis of -fetoprotein in chick embryos throughout development have been investigated using the combined techniques of immunofluorescence microscopy and tissue culture in the presence of radiolabelled amino acids, followed by immunoautoradiographic analysis.Alpha-fetoprotein is present in a range of embryonic tissues and especially concentrated in the yolk sac, liver and connective tissue. Analysis of culture fluids revealed that the yolk sac is the major site of -fetoprotein synthesis with smaller, but significant quantities being produced by the liver.These results are discussed in relation to mammalian -fetoprotein, and the merits of the chick embryo for studies on the biological function of AFP are considered.Supported by an award from the Science Research Council, to whom grateful acknowledgement is made     

Localization of αvβ3-like integrin in cultivated larval cells of the mussel Mytilus trossulus during neuronal and muscle differentiation

Using immunofluorescence phenotyping, the expression of αvβ3-like integrin was examined during neuronal and muscle differentiation in cell cultures derived from trochophore larvae of the mussel Mytilus trossulus. We have demonstrated that some mussel cells grown on fibronectin in vitro express the extracellular matrix (ECM) αvβ3 integrin-like receptor. At the same time, the distribution of αvβ3-like integrin is not ubiquitous, i.e. it depends on the cell type and the time of cultivation. Using immunohistochemical staining, we have found that only in some cells this integrin is co-localized with molluscan neuronal markers, neurotransmitters serotonin (5-HT) or Phe-Met-Arg-Phe-NH(2) neuropeptide (FMRFamide), and also with filament actin but not with paramyosin. Although we have previously shown that an integrin-dependent mechanism is involved in cell adhesion and differentiation of muscle cells of Mytilus, in this study, αvβ3-like integrin has not been found to participate in fibronectin adhesion of muscle cells but may be a linking agent between the ECM and the neuron-like cells.     

Variants of the α6 β1 Laminin Receptor in Early Murine Development: Distribution,Molecular Cloning and Chromosomal Localization of the Mouse Integrin α6 Subunit

Laminin (A:B1:B2) is a major component of the first basement membrane to appear in the developing mouse embryo. Its effects on morphogenesis and differentiation are mediated by interaction with cell surface receptors that are members of the integrin family. We have studied the expression of the α₆ subunit of murine α₆β₁ and its ligand, laminin, in preimplantation mouse embryos, embryo outgrowths and in embryonic stem (ES) cells and embryonal carcinoma (EC) cells. The α₆ subunit is present in the oocyte and throughout preimplantation development. Laminin A chain appears later than α₆ and has a more restricted distribution until the late blastocyst stage. α₆β₁ is strongly expressed in ES and EC cells; the levels of mRNA expression are not altered by differentiation. Molecular cloning of cDNA for the murine integrin α₆ subunit from a mammary gland γt11 library showed, as in man, an open reading frame encoding two variants of α₆, α₆ and α_6B. The identity of the α₆ amino acid sequence to that in man and chicken is 93% and 73%, respectively. The gene for murine α₆ was mapped to chromosome 2. While undifferentiated ES and EC cells express only α_6B, α_6A is co-expressed in ES cells after differentiation is induced by retinoic acid. α_6B is also the only variant expressed in blastocyst stage embryos, but when blastocysts have grown out in culture both α_6A and α_6B are expressed reflecting the results in the cell lines. We suggest that the deposition of laminin in the embryo is a receptor-mediated process and that the shift in the expression of the variants, as the inner cell mass forms its first differentiated progeny, reflects a change in functional properties.     

Localization of β1-Adrenergic Receptors in the Cochlea and the Vestibular Labyrinth

Sympathetic activation in a “fight or flight reaction” may put the sensory systems for hearing and balance into a state of heightened alert via β₁-adrenergic receptors (β₁-AR). The aim of the present study was to localize β₁-AR in the gerbil inner ear by confocal immunocytochemistry, to characterize β₁-AR by Western immunoblots, and to identify β₁-AR pharmacologically by measurements of cAMP production. Staining for β₁-AR was found in strial marginal cells, inner and outer hair cells, outer sulcus, and spiral ganglia cells of the cochlea, as well as in dark, transitional and supporting cells of the vestibular labyrinth. Receptors were characterized in microdissected inner ear tissue fractions as 55 kDa non-glycosylated species and as 160 kDa high-mannose-glycosylated complexes. Pharmacological studies using isoproterenol, ICI-118551 and CGP-20712A demonstrated β₁-AR as the predominant adrenergic receptor in stria vascularis and organ of Corti. In conclusion, β₁-AR are present and functional in inner ear epithelial cells that are involved in K⁺ cycling and auditory transduction, as well as in neuronal cells that are involved in auditory transmission.     

Ser756 of β2 integrin controls Rap1 activity during inside-out activation of αMβ2

During αMβ2-mediated phagocytosis, the small GTPase Rap1 activates the β2 integrin by binding to a region between residues 732 and 761. Using COS-7 cells transfected with αMβ2, we show that αMβ2 activation by the phorbol ester PMA involves Ser(756) of β2. This residue is critical for the local positioning of talin and biochemically interacts with Rap1. Using the CaM (calmodulin) antagonist W7, we found Rap1 recruitment and the inside-out activation of αMβ2 to be affected. We also report a role for CaMKII (calcium/CaM-dependent kinase II) in the activation of Rap1 during integrin activation. These results demonstrate a distinct physiological role for Ser(756) of β2 integrin, in conjunction with the actions of talin and Rap1, during αMβ2 activation in macrophages.     

Distribution of the collagen-binding integrin α10β1 during mouse development

We have previously identified and characterised the collagen type II-binding integrin subunit alpha10, which is a member of the beta1 family and is expressed by chondrocytes. In the present study, we examined the expression of the alpha10 integrin in various mouse tissues. Immunohistochemical analysis of alpha10 on cryosections from 3-day-old mice demonstrated that alpha10beta1 was present in the hyaline cartilage of joints, vertebral column, trachea and bronchi. In addition, alpha10 was found in the ossification groove of Ranvier, in the aortic and atrioventricular valves of the heart and in the fibrous tissue lining skeletal muscle and ligaments. Overall, the distribution was distinct from that of the collagen-binding integrins alpha1beta1 and alpha2beta1. We also found that alpha10beta1was the dominating collagen-binding integrin during cartilage development. Expression of alpha10 appeared at embryonic day 11.5 (E11.5) at the same time as chondrogenesis started as judged by collagen type II expression. At E13.5, alpha10 was present throughout the anlage as well as in the perichondrium and in mesenchyme just outside the perichondrium, where it localised with collagen type I. Four weeks after birth, alpha10 was prominent both at the articular surface and in the growth plate. In conclusion, we found that integrin alpha10beta1 was a major collagen-binding integrin during cartilage development and in mature hyaline cartilage. In addition, we found that alpha10beta1 was present in some fibrous tissues.     

Soluble guanylate cyclase isoenzymes: The expression of α1, α2, β1, and β2 subunits in the benign and malignant breast tumors

Soluble guanylate cyclase (sGC) encompasses α and β subunits. This study examined the expression of α1, α2, β1, and β2 subunits in the malignant and benign breast tumors using the Western blot analysis. Both benign and malignant tumors showed a significantly higher expression of the α1 subunit in comparison with normal tissues (p < 0.0001). In contrast, the expression of α2 and β2 sGC were significantly lower in these tumors than normal tissues (p < .0015 and p < .001, p < .007 and p < .0001, respectively). The expression level of α1 sGC was significantly correlated with ER + PR+ (p < .0001). A significant correlation was also detected for sGC-α1 and -α2 expression with c-erbB2-negative status (p < .01). However, the expression level of sGC was not associated with tumor stage, tumor grade, or other clinicopathological features. In conclusion, as the expression of α1 sGC is upregulated and α2 and β2 sGC are downregulated in malignant breast tumors. Variations in the expression of sGC isoenzymes may be suggested as an indicator to confirm the enzyme antitumor activity.     

Expression of tPA, LH receptor and inhibin α, β_A subunits during follicular atresia in rats

By using DNA 3'-end labeling, immunocytochemistry and mRNA in situ hybridization detection techniques, the expression of inhibin subunits and LH receptor in the granulosa cells and tissue-type plasminogen activator (tPA) in the oocytes has been studied in relation to follicular development and atresia. The results demonstrated that: (ⅰ) tPA activity in the oocytes of normal developing follicles is undetectable, and increases significantly in the follicle undergoing atresia; (ⅱ) the production of inhibin subunits in granulosa cells is negatively correlated with the expression of oocyte tPA activity, indicating that they may be an important regulator of oocyte tPA production and follicular development; (ⅲ) in atretic follicles, granulosa cells do not express LH receptor and inhibin subunits. It is therefore suggested that tPA may play a role in oocyte self-destruction and clearance in some of atretic follicles, and inhibin of granulosa-origin might be an inhibitory factor for the translation of tPA in     

Expression of tPA, LH receptor and inhibin α, βA subunits during follicular atresia in rats

By using DNA 3′-end labeling, immunocytochemistry and mRNA insitu hybridization detection techniques, the expression of inhibin subunits and LH receptor in the granulosa cells and tissue-type plasminogen activator (tPA) in the oocytes has been studied in relation to follicular development and atresia. The results demonstrated that: (i) tPA activity in the oocytes of normal developing follicles is undetectable, and increases significantly in the follicle undergoing atresia; (ii) the production of inhibin subunits in granulosa cells is negatively correlated with the expression of oocyte tPA activity, indicating that they may be an important regulator of oocyte tPA production and follicular development; (iii) in atretic follicles, granulosa cells do not express LH receptor and inhibin subunits. It is therefore suggested that tPA may play a role in oocyte self-destruction and clearance in some of atretic follicles, and inhibin of granulosa-origin might be an inhibitory factor for the translation of tPA in the oocyte.     

Study of the residues involved in the binding of β1 to β3 subunits in the sodium channel

The voltage-gated sodium channel (VGSC) is a complex, which is composed of one pore-forming α subunit and at least one β subunit. Up to now, five β subunits are known: β1/β1A, β1B, β2, β3, and β4, encoded by four genes (SCN1B∼SCN4B). It is critical to have a deep understanding of the interaction between β1 and β3 subunits, two subunits which frequently appear in many diseases concurrently. In this study, we had screened out the new template of β1 subunit for homology modelling, which shares higher similarity to β3. Docking studies of the β1 and β3 homology model were conducted, and likely β1 and β3 binding loci were investigated. The results revealed that β1–β3 is more likely to form a di-polymer than β1–β1 based on molecular interaction analysis, including potential energy analysis, Van der Waals (VDW) energy analysis and electrostatic energy analysis, and in addition, consideration of the hydrogen bonds and hydrophobic contacts that are involved. Based on these analyses, the residues His122 and Lys140 of β1 and Glu 66, Asn 131, Asp 118, Glu 120, Glu133, Asn135, Ser 137 of β3 were predicted to play a functional role.     

Specificities of β1 integrin signaling in the control of cell adhesion and adhesive strength

Cells exert actomyosin contractility and cytoskeleton-dependent force in response to matrix stiffness cues. Cells dynamically adapt to force by modifying their behavior and remodeling their microenvironment. This adaptation is favored by integrin activation switch and their ability to modulate their clustering and the assembly of an intracellular hub in response to force. Indeed integrins are mechanoreceptors and mediate mechanotransduction by transferring forces to specific adhesion proteins into focal adhesions which are sensitive to tension and activate intracellular signals. α(5)β(1) integrin is considered of major importance for the formation of an elaborate meshwork of fibronectin fibrils and for the extracellular matrix deposition and remodeling. Here we summarize recent progress in the study of mechanisms regulating the activation cycle of β(1) integrin and the specificity of α(5)β(1) integrin in mechanotransduction.     

Expression of α2, α5 and α6 subunits of integrin in de-differentiated NIH3T3 cells by cell-free extract of embryonic stem cells

Generation of patient specific stem cells is among the ultimate goals in regenerative medicine. Such a cell needs to be functional when it transplants. Interaction between the matrix proteins and integrin adjust many cells' function such as adhesion, migration, cell cycle and self renewal in stem cells. In this study, NIH3T3 cells were dedifferentiated by mouse Embryonic Stem Cell (mESC) extract. The expression of pluripotency markers as well as a2, a5 and a6 integrin subunits were determined. NIH3T3 cells treated with mESC extract showed noticeable changes in cell morphology as early as day 2 post-treatment forming colonies similar to typical mESC morphology by day 8, after three passages. Alkaline phosphatase (ALP) assay and immunocytochemistry staining were performed for the induced reprogrammed cells. The results indicated that these colonies showed the ALP activity and they express Sox2 and Nanog. RT-PCR revealed that the colonies also express Oct3/4. NIH3T3 cells, ESC and reprogrammed cells expressed a2 integrin. a5 integrin expression was greatest in reprogrammed cells followed by the expression of this integrin in NIH3T3 which in turn was more than in ESC. a6A integrin was expressed in NIH3T3 cells while a6B integrin was expressed in ESC and in very low quantity was expressed in reprogrammed cells. These data provide evidence for both the generation of ES like cells from differentiated somatic cells and the expression profile of integrins after de-differentiation by mESC extract.     

The increased expression of integrin α6 (ITGA6) enhances drug resistance in EVI1(high) leukemia

Ecotropic viral integration site-1 (EVI1) is one of the candidate oncogenes for human acute myeloid leukemia (AML) with chromosomal alterations at 3q26. High EVI1 expression (EVI1(high)) is a risk factor for AML with poor outcome. Using DNA microarray analysis, we previously identified that integrin α6 (ITGA6) was upregulated over 10-fold in EVI1(high) leukemia cells. In this study, we determined whether the increased expression of ITGA6 is associated with drug-resistance and increased cell adhesion, resulting in poor prognosis. To this end, we first confirmed the expression pattern of a series of integrin genes using semi-quantitative PCR and fluorescence-activated cell sorter (FACS) analysis and determined the cell adhesion ability in EVI1(high) leukemia cells. We found that the adhesion ability of EVI1(high) leukemia cells to laminin increased with the increased expression of ITGA6 and integrin β4 (ITGB4). The introduction of small-hairpin RNA against EVI1 (shEVI1) into EVI1(high) leukemia cells reduced the cell adhesion ability and downregulated the expression of ITGA6 and ITGB4. In addition, the overexpression of EVI1 in EVI1(low) leukemia cells enhanced their cell adhesion ability and increased the expression of ITGA6 and ITGB4. In a subsequent experiment, the introduction of shRNA against ITGA6 or ITGB4 into EVI1(high) AML cells downregulated their cell adhesion ability; however, the EVI1(high) AML cells transfected with shRNA against ITGA6 could not be maintained in culture. Moreover, treating EVI1(high) leukemia cells with neutralizing antibodies against ITGA6 or ITGB4 resulted in an enhanced responsiveness to anti-cancer drugs and a reduction of their cell adhesion ability. The expression of ITGA6 is significantly elevated in cells from relapsed and EVI1(high) AML cases; therefore, ITGA6 might represent an important therapeutic target for both refractory and EVI1(high) AML.     

Modulation of α2β1 integrin changes during mammary gland development by β-oestradiol

In order to study the role of cell–matrix interactions in mammary gland function, temporal changes in α₂β₁ integrin, the major receptor for collagen and the influence of β-oestradiol on its level and distribution in rat mammary gland at different stages of development were studied. The level of α₂β₁ integrin determined by ELISA, was found to be high during different days of pregnancy, while in the lactating stage, it was significantly reduced. By immunocytochemical analysis, α₂β₁ integrin was found to be localized towards the luminal side of acinar cells, both in the virgin and midpregnant stage, while it was not detected in the lactating stage. The possible role of hormones in modulating the level of integrin was examined in both in vitro and in vivo experiments using β-oestradiol. Supplementing β-oestradiol to isolated mammary epithelial cells from both virgin and lactating glands caused a concentration dependent increase in the incorporation of [³⁵S]methionine into α₂β₁ integrin associated with the cells. Administration of β-oestradiol to virgin and lactating glands caused about 1.4–4-fold increase in the level of α₂ integrin, indicating that upregulation of integrin during pregnancy may be due to oestrogen and as the oestrogen level falls during lactating phase, downregulation of α₂β₁ integrin occurs. Treatment with β-oestradiol also resulted in the appearance of α₂β₁ integrin in the acinar region of the lactating tissue, while in the untreated controls no staining for integrin was seen. These results indicate that oestrogen, apart from directly affecting the cellular activity, can influence mammary tissue function by affecting cell–ECM interactions through the modulation of integrin receptors for matrix proteins.     

Dissociation of the α-subunit Calf-2 domain and the β-subunit I-EGF4 domain in integrin activation and signaling

Integrin conformational changes mediate integrin activation and signaling triggered by intracellular molecules or extracellular ligands. Even though it is known that αβ transmembrane domain separation is required for integrin signaling, it is still not clear how this signal is transmitted from the transmembrane domain through two long extracellular legs to the ligand-binding headpiece. This study addresses whether the separation of the membrane-proximal extracellular αβ legs is critical for integrin activation and outside-in signaling. Using a disulfide bond to restrict dissociation of the α-subunit Calf-2 domain and β-subunit I-EGF4 domain, we were able to abolish integrin inside-out activation and outside-in signaling. In contrast, disrupting the interface by introducing a glycosylation site into either subunit activated integrins for ligand binding through a global conformational change. Our results suggest that the interface of the Calf-2 domain and the I-EGF4 domain is critical for integrin bidirectional signaling.     

Identification of multiple integrin β1 homologs in zebrafish (Danio rerio)

Background  

Integrins comprise a large family of α,β heterodimeric, transmembrane cell adhesion receptors that mediate diverse essential biological functions. Higher vertebrates possess a single β1 gene, and the β1 subunit associates with a large number of α subunits to form the major class of extracellular matrix (ECM) receptors. Despite the fact that the zebrafish (Danio rerio) is a rapidly emerging model organism of choice for developmental biology and for models of human disease, little is currently known about β1 integrin sequences and functions in this organism.