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Sample records for a2b1 integrin transgenic

  1. β1-integrins signaling and mammary tumor progression in transgenic mouse models: implications for human breast cancer

    PubMed Central

    2011-01-01

    Consistent with their essential role in cell adhesion to the extracellular matrix, integrins and their associated signaling pathways have been shown to be involved in cell proliferation, migration, invasion and survival, processes required in both tumorigenesis and metastasis. β1-integrins represent the predominantly expressed integrins in mammary epithelial cells and have been proven crucial for mammary gland development and differentiation. Here we provide an overview of the studies that have used transgenic mouse models of mammary tumorigenesis to establish β1-integrin as a critical mediator of breast cancer progression and thereby as a potential therapeutic target for the development of new anticancer strategies. PMID:22264244

  2. Skeletal Phenotype of Transgenic Mice Expressing the Beta1 Integrin Cytoplasmic Tail In Osteoblasts

    NASA Technical Reports Server (NTRS)

    Globus, R. K.; vanderMeulen, M. C. H.; Damsky, D.; Kim, J.-B.; Amblard, D.; Amblard, D.; Nishimura, Y.; Almeida, E.; Iwaniec, U. T.; Wronski, T. J.; hide

    2002-01-01

    To define the physiologic role of beta1 integrin in bone formation and mechanical loading, transgenic mice were generated by expressing the cytoplasmic tall and transmembrane domain of Beta1 integrin under the control of the osteocalcin promoter. In cultured cells, this truncated fragment of Beta1 can act as a dominant negative. Previously, the matrix of calvariae was shown to be abnormal in transgenic (TG) compared to wildtype (WT) mice. In this study, we analyzed appendicular bone in TG and WT, male and female mice at 14, 35, 63, 90 and 365 days old (n=8-12/gp). To assess beta1 integrin function in mechanical loading, a pilot study using hindlimb unloading by tail suspension was performed. 35d old TG and WT females were hindlimb unloaded for 4 wks (n=3-5). Body mass, bone mineral content, histomorphometric (distal femur) and biomechanical parameters were analyzed. Statistical significance (P less than.05) was defined by ANOVA using the Tukey-Kramer post-hoc test. We confirmed transgene expression by immunoprecipitating then immunoblotting bone lysates using an antibody against the beta1 tail. Body masses of TG mice at 63, 90 and 365d old were greater (16-25%) than WT. Some TG female mice at 365d appeared obese; mean abdominal fat mass was 415% greater in TG than WT mice. Tibiae were longer (5-7%) in TG than WT mice at 63 and 90d. Tibial mineral mass of 35d males was 7% lower in TG than WT mice, but at 63d was 21% higher. The % osteoblast surface in 35d TG mice was 20% higher than WT, and at 63d was 17% lower, while % osteoclast surface did not differ. In 365d mice, cancellous bone volume (125%) and endocortical mineral apposition rate (40%) were greater in TG than WT males but not females. In WT mice, hindlimb unloading caused a reduction in mineral mass of tibiae (-20%) and lumbar vertebrae (-22%) relative to normally loaded controls. Surprisingly, hindlimb unloading also caused a relative reduction (-13%) in humerus mass. The effects of hindlimb unloading on

  3. Psoriasiform skin disease in transgenic pigs with high-copy ectopic expression of human integrins α2 and β1

    PubMed Central

    Staunstrup, Nicklas Heine; Stenderup, Karin; Mortensen, Sidsel; Primo, Maria Nascimento; Steiniche, Torben; Liu, Ying; Li, Rong; Schmidt, Mette; Purup, Stig; Dagnæs-Hansen, Frederik; Schrøder, Lisbeth Dahl; Svensson, Lars; Petersen, Thomas Kongstad; Callesen, Henrik; Bolund, Lars

    2017-01-01

    ABSTRACT Psoriasis is a complex human-specific disease characterized by perturbed keratinocyte proliferation and a pro-inflammatory environment in the skin. Porcine skin architecture and immunity are very similar to that in humans, rendering the pig a suitable animal model for studying the biology and treatment of psoriasis. Expression of integrins, which is normally confined to the basal layer of the epidermis, is maintained in suprabasal keratinocytes in psoriatic skin, modulating proliferation and differentiation as well as leukocyte infiltration. Here, we generated minipigs co-expressing integrins α2 and β1 in suprabasal epidermal layers. Integrin-transgenic minipigs born into the project displayed skin phenotypes that correlated with the number of inserted transgenes. Molecular analyses were in good concordance with histological observations of psoriatic hallmarks, including hypogranulosis and T-lymphocyte infiltration. These findings mark the first creation of minipigs with a psoriasiform phenotype resembling human psoriasis and demonstrate that integrin signaling plays a key role in psoriasis pathology. PMID:28679670

  4. Skeletal phenotype of growing transgenic mice that express a function-perturbing form of beta1 integrin in osteoblasts

    NASA Technical Reports Server (NTRS)

    Globus, R. K.; Amblard, D.; Nishimura, Y.; Iwaniec, U. T.; Kim, J-B; Almeida, E. A. C.; Damsky, C. D.; Wronski, T. J.; van der Meulen, M. C. H.

    2005-01-01

    Skeletal modeling entails the deposition of large amounts of extracellular matrix (ECM) to form structures tailored to withstand increasing mechanical loads during rapid growth. Specific ECM molecules bind to integrin receptors on the cell surface, thereby triggering a cascade of signaling events that affect critical cell functions. To evaluate the role of integrins during skeletal growth, transgenic mice were engineered to express a function-perturbing fragment of beta1 integrin consisting of the transmembrane domain and cytoplasmic tail under the control of the osteocalcin promoter (TG mice). Thus, transgene expression was targeted to mature cells of the osteoblast lineage, and herein we show that cultured cells resembling osteocytes from 90-day-old TG mice display impaired adhesion to collagen I, a ligand for beta1 integrin. To determine the influence of beta1 integrin on bones that are responsible for providing structural support during periods of rapid growth, we examined the phenotype of the appendicular skeleton in TG mice compared to wild type (WT) mice. According to radiographs, bones from mice of both genotypes between 14 and 90 days of age appeared similar in gross structure and density, although proximal tibiae from 35-90 days old TG mice were less curved than those of WT mice (72-92% TG/WT). Although there were only mild and transient differences in absolute bone mass and strength, once normalized to body mass, the tibial dry mass (79.1% TG/WT females), ash mass (78.5% TG/WT females), and femoral strength in torsion (71.6% TG/WT females) were reduced in TG mice compared to WT mice at 90 days of age. Similar effects of genotype on bone mass and curvature were observed in 1-year-old retired breeders, indicating that these phenotypic differences between TG and WT mice were stable well into adulthood. Effects of genotype on histomorphometric indices of cancellous bone turnover were minimal and evident only transiently during growth, but when present they

  5. Development of Transgenic Cloned Pig Models of Skin Inflammation by DNA Transposon-Directed Ectopic Expression of Human β1 and α2 Integrin

    PubMed Central

    Staunstrup, Nicklas Heine; Madsen, Johannes; Primo, Maria Nascimento; Li, Juan; Liu, Ying; Kragh, Peter M.; Li, Rong; Schmidt, Mette; Purup, Stig; Dagnæs-Hansen, Frederik; Svensson, Lars; Petersen, Thomas K.; Callesen, Henrik; Bolund, Lars; Mikkelsen, Jacob Giehm

    2012-01-01

    Integrins constitute a superfamily of transmembrane signaling receptors that play pivotal roles in cutaneous homeostasis by modulating cell growth and differentiation as well as inflammatory responses in the skin. Subrabasal expression of integrins α2 and/or β1 entails hyperproliferation and aberrant differentiation of keratinocytes and leads to dermal and epidermal influx of activated T-cells. The anatomical and physiological similarities between porcine and human skin make the pig a suitable model for human skin diseases. In efforts to generate a porcine model of cutaneous inflammation, we employed the Sleeping Beauty DNA transposon system for production of transgenic cloned Göttingen minipigs expressing human β1 or α2 integrin under the control of a promoter specific for subrabasal keratinocytes. Using pools of transgenic donor fibroblasts, cloning by somatic cell nuclear transfer was utilized to produce reconstructed embryos that were subsequently transferred to surrogate sows. The resulting pigs were all transgenic and harbored from one to six transgene integrants. Molecular analyses on skin biopsies and cultured keratinocytes showed ectopic expression of the human integrins and localization within the keratinocyte plasma membrane. Markers of perturbed skin homeostasis, including activation of the MAPK pathway, increased expression of the pro-inflammatory cytokine IL-1α, and enhanced expression of the transcription factor c-Fos, were identified in keratinocytes from β1 and α2 integrin-transgenic minipigs, suggesting the induction of a chronic inflammatory phenotype in the skin. Notably, cellular dysregulation obtained by overexpression of either β1 or α2 integrin occurred through different cellular signaling pathways. Our findings mark the creation of the first cloned pig models with molecular markers of skin inflammation. Despite the absence of an overt psoriatic phenotype, these animals may possess increased susceptibility to severe skin damage

  6. Induction of protection against foot-and-mouth disease virus in cell culture and transgenic suckling mice by miRNA targeting integrin αv receptor.

    PubMed

    Du, Junzheng; Guo, Xinbing; Gao, Shandian; Luo, Jihuai; Gong, Xiuli; Hao, Chunxia; Yang, Bo; Lin, Tong; Shao, Junjun; Cong, Guozheng; Chang, Huiyun

    2014-10-10

    Foot-and-mouth disease virus (FMDV) is an RNA virus that causes a highly contagious disease in domestic and wild cloven-hoofed animals. Although vaccination has been used to protect animals against FMDV, there are shortcomings in the efficacy of the available vaccines. RNA interference (RNAi) is triggered by small RNA molecules, including short interfering RNAs and microRNAs (miRNAs), and the use of RNAi-based methods have demonstrated promise as an alternative method of controlling the transmission of FMDV. However, the method of delivery, short duration of siRNA and miRNA in vivo, and the genetic variability of FMDV confound the use of RNAi-based strategies for FMDV control. FMDV has been shown to exploit host-cell integrins as cell-surface receptors to initiate infection. We selected the gene for the integrin αv subunit as an RNAi target, and constructed three αv-specific miRNA expression plasmids. The effects of these miRNAs on FMDV infection were examined in PK-15 cells and transgenic suckling mice. In PK-15 cells, the expression of the αv-specific miRNAs significantly inhibited the expression of integrin αv receptor and decreased FMDV infection. The transgenic mice were generated by integrating the αv-specific miRNA expression cassette using pronuclear microinjection. When challenged with a dose of FMDV ten times greater than the LD50, the survival rate of transgenic suckling mice was approximately six-fold higher than that of their non-transgenic littermates, indicating that the interference of the miRNAs significantly reduced FMDV infection in the transgenic mice. This is the first report of limiting FMDV attachment to cellular receptors using miRNA-mediated gene knock down of cell-surface receptors to significantly reduce FMDV infection in cell culture and transgenic suckling mice. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Inhibition of Inflammation and Bone Erosion by RNA Interference-Mediated Silencing of Heterogeneous Nuclear RNP A2/B1 in Two Experimental Models of Rheumatoid Arthritis.

    PubMed

    Herman, Sonja; Fischer, Anita; Presumey, Jessy; Hoffmann, Markus; Koenders, Marije I; Escriou, Virginie; Apparailly, Florence; Steiner, Guenter

    2015-09-01

    The nuclear protein heterogeneous nuclear RNP A2/B1 (hnRNP A2/B1) is involved in posttranscriptional regulation of gene expression. It is constitutively expressed in lymphoid organs and highly up-regulated in the synovial tissue of patients with rheumatoid arthritis (RA), who may also generate autoantibodies to this protein. This study was undertaken to investigate the potential involvement of hnRNP A2/B1 in the pathogenesis of autoimmune arthritis, by silencing hnRNP A2/B1 expression in 2 animal models of RA. Collagen-induced arthritis (CIA) and the K/BxN serum-transfer model were used as animal models of RA. Efficient silencing of hnRNP A2/B1 was achieved using a liposome-based carrier system for delivery of small interfering RNAs. Expression of hnRNP A2/B1 was analyzed by flow cytometry, reverse transcription-quantitative polymerase chain reaction, Western blotting, and immunohistochemistry. The number of osteoclasts was determined by tartrate-resistant acid phosphatase staining. Cytokine levels and anticollagen antibody levels were measured by enzyme-linked immunosorbent assay. Efficient silencing of hnRNP A2/B1 was achieved in all lymphoid organs. In both experimental models, the incidence and severity of arthritis were largely reduced and bone erosion was not detectable as compared to the control groups. Down-modulation of hnRNP A2/B1 significantly interfered with the production of proinflammatory cytokines from monocyte/macrophages, but not from T cells. Consistent with these findings, production of T cell cytokines was not impaired when cells were restimulated in vitro with type II collagen. Furthermore, levels of anticollagen antibodies were not affected by hnRNP A2/B1 silencing. Our findings suggest that hnRNP A2/B1 has an important role in regulation of the innate immune system, especially at the level of monocyte/macrophage activation. Therefore, down-modulation of hnRNP A2/B1 seems to affect primarily the effector phase of autoimmune arthritis. © 2015

  8. β-Asarone Inhibits Invasion and EMT in Human Glioma U251 Cells by Suppressing Splicing Factor HnRNP A2/B1.

    PubMed

    Li, Li; Wu, Mingxia; Wang, Chengqiang; Yu, Zanyang; Wang, Hongmei; Qi, Hongyi; Xu, Xiaoyu

    2018-03-16

    β-asarone, the main component in the volatile oil of Acori tatarinowii Rhizoma, has been found to possess antitumor activity. However, its effect and mechanisms against tumor invasion and epithelial-mesenchymal transition (EMT) are still unclear. In this study, no or less cytotoxicity was caused by β-asarone within 0-120 μM in human glioma U251 cells for 48 h. β-asarone (30 and 60 μM) inhibited the migration of U251 cells in the wound healing assay, suppressed the invasion of U251 cells in the Boyden chamber invasion assay, and inhibited the adhesion of U251 cells onto the Matrigel. Moreover, β-asarone suppressed EMT with the up-regulation of E-cadherin and the down-regulation of vimentin. HnRNP A2/B1, a well-characterized oncogenic protein, was shown at a high basal level in U251 cells and β-asarone reduced hnRNP A2/B1 expression in a concentration and time-dependent way. Importantly, hnRNP A2/B1 overexpression significantly counteracted the inhibition of β-asarone on the migration, invasion, and adhesion of U251 cells and reversed the modulation of EMT markers by β-asarone. Additionally, β-asarone decreased the MMP-9 and p-STAT3 in U251 cells, which was also reversed by hnRNP A2/B1 overexpression. Together, our results suggest that hnRNP A2/B1 may be a potential molecular target underlying the inhibitory effect of β-asarone on invasion and EMT in glioma cells.

  9. VHL genetic alteration in CCRCC does not determine de-regulation of HIF, CAIX, hnRNP A2/B1 and osteopontin.

    PubMed

    Nyhan, Michelle J; El Mashad, Shereen M; O'Donovan, Tracey R; Ahmad, Sarfraz; Collins, Chris; Sweeney, Paul; Rogers, Eamonn; O'Sullivan, Gerald C; McKenna, Sharon L

    2011-06-01

    Von Hippel-Lindau (VHL) tumour suppressor gene inactivation is associated with clear cell renal cell carcinoma (CCRCC) development. The VHL protein (pVHL) has been proposed to regulate the expression of several proteins including Hypoxia Inducible Factor-α (HIF-α), carbonic anhydrase (CA)IX, heterogeneous nuclear ribonucleoprotein (hnRNP)A2/B1 and osteopontin. pVHL has been characterized in vitro, however, clinical studies are limited. We evaluated the impact of VHL genetic alterations on the expression of several pVHL protein targets in paired normal and tumor tissue. The VHL gene was sequenced in 23 CCRCC patients and VHL transcript levels were evaluated by Real-Time RT-PCR. Expression of pVHL's protein targets were determined by Western blotting in 17 paired patient samples. VHL genetic alterations were identified in 43.5% (10/23) of CCRCCs. HIF-1α, HIF-2α and CAIX were up-regulated in 88.2% (15/17), 100% (17/17) and 88.2% (15/17) of tumors respectively and their expression is independent of VHL status. hnRNP A2/B1 and osteopontin expression was variable in CCRCCs and had no association with VHL genetic status. As expression of these proposed pVHL targets can be achieved independently of VHL mutation (and possibly by hypoxia alone), this data suggests that other pVHL targets may be more crucial in renal carcinogenesis.

  10. hnRNP A2/B1 interacts with influenza A viral protein NS1 and inhibits virus replication potentially through suppressing NS1 RNA/protein levels and NS1 mRNA nuclear export

    SciTech Connect

    Wang, Yimeng; Zhou, Jianhong; Du, Yuchun, E-mail: ydu@uark.edu

    2014-01-20

    The NS1 protein of influenza viruses is a major virulence factor and exerts its function through interacting with viral/cellular RNAs and proteins. In this study, we identified heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) as an interacting partner of NS1 proteins by a proteomic method. Knockdown of hnRNP A2/B1 by small interfering RNA (siRNA) resulted in higher levels of NS vRNA, NS1 mRNA, and NS1 protein in the virus-infected cells. In addition, we demonstrated that hnRNP A2/B1 proteins are associated with NS1 and NS2 mRNAs and that knockdown of hnRNP A2/B1 promotes transport of NS1 mRNA from the nucleus to themore » cytoplasm in the infected cells. Lastly, we showed that knockdown of hnRNP A2/B1 leads to enhanced virus replication. Our results suggest that hnRNP A2/B1 plays an inhibitory role in the replication of influenza A virus in host cells potentially through suppressing NS1 RNA/protein levels and NS1 mRNA nucleocytoplasmic translocation. - Highlights: • Cellular protein hnRNP A2/B1 interacts with influenza viral protein NS1. • hnRNP A2/B1 suppresses the levels of NS1 protein, vRNA and mRNA in infected cells. • hnRNP A2/B1 protein is associated with NS1 and NS2 mRNAs. • hnRNP A2/B1 inhibits the nuclear export of NS1 mRNAs. • hnRNP A2/B1 inhibits influenza virus replication.« less

  11. Integrin-mediated short-term memory in Drosophila.

    PubMed

    Grotewiel, M S; Beck, C D; Wu, K H; Zhu, X R; Davis, R L

    1998-01-29

    Volado is a new memory mutant of Drosophila. The locus encodes two isoforms of a new alpha-integrin, a molecule that dynamically mediates cell adhesion and signal transduction. The Volado gene is expressed preferentially in mushroom body cells, which are neurons known to mediate olfactory learning in insects. Volado proteins are concentrated in the mushroom body neuropil, brain areas that contain mushroom body processes in synaptic contact with other neurons. Volado mutants display impaired olfactory memories within 3 min of training, indicating that the integrin is required for short-term memory processes. Conditional expression of a Volado transgene during adulthood rescues the memory impairment. This rescue of memory is reversible, fading over time along with expression of the transgene. Thus the Volado integrin is essential for the physiological processes underlying memory. We propose a model in which integrins act as dynamic regulators of synapse structure or the signalling events underlying short-term memory formation.

  12. Vavilosides A1/A2-B1/B2, new furostane glycosides from the bulbs of Allium vavilovii with cytotoxic activity.

    PubMed

    Zolfaghari, Behzad; Sadeghi, Masoud; Troiano, Raffaele; Lanzotti, Virginia

    2013-04-01

    A phytochemical analysis of the bulbs of Allium vavilovii M. Pop. & Vved. was attained for the first time extensively, affording to the isolation of four new furostanol saponins, named vavilosides A1/A2-B1/B2 (1a/b-2a/2b), as two couple of isomers in equilibrium, together with ascalonicoside A1/A2 (3a/3b) and 22-O-methyl ascalonicoside A1/A2 (4a/4b), previously isolated from shallot, Allium ascalonicum. High concentrations of kaempferol, kaempferide, and kaempferol 4(I)-glucoside were also isolated. The chemical structures of the new compounds, established through a combination of extensive nuclear magnetic resonance, mass spectrometry and chemical analyses, were identified as (25R)-furost-5(6)-en-1β,3β,22α,26-tetraol 1-O-α-L-rhamnopyranosyl-(1→2)-O-β-D-galactopyranosyl 26-O-α-L-rhamnopyranoside (vaviloside A1), (25R)-furost-5(6)-en-1β,3β,22β,26-tetraol 1-O-α-L-rhamnopyranosyl-(1→2)-O-β-D-galactopyranosyl 26-O-α-L-rhamnopyranoside (vaviloside A2), (25R)-furost-5(6)-en-1β,3β,22α,26-tetraol 1-O-α-L-rhamnopyranosyl-(1→2)-O-β-D-xylopyranosyl 26-O-α-L-rhamnopyranoside (vaviloside B1), (25R)-furost-5(6)-en-1β,3β,22β,26-tetraol 1-O-α-L-rhamnopyranosyl-(1→2)-O-β-d-xylopyranosyl 26-O-α-L-rhamnopyranoside (vaviloside B2). The isolated saponins showed cytotoxic activity on J-774, murine monocyte/macrophage, and WEHI-164, murine fibrosarcoma, cell lines with the following rank: vaviloside B1/B2>ascalonicoside A1/A2>vaviloside A1/A2. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Mechanotransduction through Integrins

    NASA Technical Reports Server (NTRS)

    Ingber, Donald

    2004-01-01

    The goal of this project was to characterize the molecular mechanism by which cells recognize and respond to physical forces in their local environment. The project was based on the working hypothesis that cells sense mechanical stresses through cell surface integrin receptors and through their interconnections with the underlying cytoskeleton. Work completed and published in past funding period had provided direct support for this hypothesis. In particular, we demonstrated that application of mechanical stresses to activated integrin receptors (but not inactive integrins or other control transmembrane receptors) resulted in stress-dependent activation of the CAMP signaling pathway leading to gene transcription. We also showed that this form of mechanotransduction requires activation of heterotrimeric G proteins. In this grant, our specific aims included: 1) to characterize the signal processing capabilities of different integrins and other cell surface receptors, 2) to identify heterotrimeric G proteins that mediate CAMP signaling by stresses applied to integrins, 3) to identify molecules that mediate transmembrane mechanochemical coupling between integrins and G proteins, and 4) to use genome-wide gene expression profiling techniques to identify other genes and signaling pathways that are activated by mechanical forces transmitted over specific cell surface receptors. Elucidation of the mechanism by which cells sense mechanical stresses through integrins and translate them into a biochemical response should help us to understand the molecular basis of the cellular response to gravity as well as many other forms of mechanosensation and tissue regulation.

  14. Suppression of HPV-16 late L1 5′-splice site SD3632 by binding of hnRNP D proteins and hnRNP A2/B1 to upstream AUAGUA RNA motifs

    PubMed Central

    Li, Xiaoze; Johansson, Cecilia; Glahder, Jacob; Mossberg, Ann-Kristin; Schwartz, Stefan

    2013-01-01

    Human papillomavirus type 16 (HPV-16) 5′-splice site SD3632 is used exclusively to produce late L1 mRNAs. We identified a 34-nt splicing inhibitory element located immediately upstream of HPV-16 late 5′-splice site SD3632. Two AUAGUA motifs located in these 34 nt inhibited SD3632. Two nucleotide substitutions in each of the HPV-16 specific AUAGUA motifs alleviated splicing inhibition and induced late L1 mRNA production from episomal forms of the HPV-16 genome in primary human keratinocytes. The AUAGUA motifs bind specifically not only to the heterogeneous nuclear RNP (hnRNP) D family of RNA-binding proteins including hnRNP D/AUF, hnRNP DL and hnRNP AB but also to hnRNP A2/B1. Knock-down of these proteins induced HPV-16 late L1 mRNA expression, and overexpression of hnRNP A2/B1, hnRNP AB, hnRNP DL and the two hnRNP D isoforms hnRNP D37 and hnRNP D40 further suppressed L1 mRNA expression. This inhibition may allow HPV-16 to hide from the immune system and establish long-term persistent infections with enhanced risk at progressing to cancer. There is an inverse correlation between expression of hnRNP D proteins and hnRNP A2/B1 and HPV-16 L1 production in the cervical epithelium, as well as in cervical cancer, supporting the conclusion that hnRNP D proteins and A2/B1 inhibit HPV-16 L1 mRNA production. PMID:24013563

  15. Do Integrins Mediate the Skeletal Response to Altered Loading?

    NASA Technical Reports Server (NTRS)

    vanderMeulen, Marjolein C. H.

    2004-01-01

    In vivo experiments were performed to examine the role of B1 integrin in skeletal adaptation to reduced and increased loading. Transgenic mice were generated with a dominant negative form of the B1 integrin cytoplasmic domain with expression driven by the osteocalcin promoter (pOCb1DN). This fragment consists of the transmembrane and intracellular domains and interferes with endogenous integrin signalling in vitro. This promoter targets expression of the transgene to mature bone cells. Expression of the transgene was confirmed by immunoprecipitation and western blotting. Reduced loading was generated by hindlimb suspension and increased loading the resumption of normal loading following hindlimb suspension. Two groups of female 35-day old mice were examined: poCb1DN transgenic mice (TG) and wild-type littermate controls (WT). Animals were hindlimb suspended for 1 week (HU, n = l0/gp) or 4 weeks (HU, n = 4 - 7/gp) or suspended for 4 weeks followed by reloading by normal ambulation for 4 weeks (RL, n = l0/gp). Age-matched controls (CT) were pairfed based on the HU food intake. The protocols were approved by the NASA Ames Research Center IACUC. Upon completion of the experimental protocol, body mass was recorded and tissues of interest removed and analyzed following standard procedures. Femoral whole bone structural behavior was measured in torsion to failure to obtain whole bone strength (failure torque) and torsional rigidity. Ash content (ash) and fraction (% ash) were determined for the tibia. Total ash is indicative of bone size whereas %ash is a material property. Tibial curvature was measured from microradiographs. For each experiment, the effects of genotype (TG, WT) and treatment (CT, HU/RL) were assessed by two-factor ANOVA followed by the Tukey-Kramer posthoc to identify significant differences at an alpha level of 0.05. Our goal was to understand differences resulting from altered integrin function in the adaptation to altered loading.

  16. The tail of integrin activation

    PubMed Central

    Anthis, Nicholas J; Campbell, Iain D

    2010-01-01

    Integrins are essential adhesion receptors found on the surfaces of all metazoan cells. As regulators of cell migration and extracellular matrix assembly, these membrane-spanning heterodimers are critical for embryonic development, tissue repair and immune responses. Signals transmitted by integrins from outside to inside the cell promote cell survival and proliferation, but integrin affinity for extracellular ligands can also be controlled by intracellular cues. This bidirectional signaling is mediated by the short cytoplasmic tails of the two integrin subunits. Recent structural and functional studies of various integrin fragments and complexes between the cytoplasmic tails and intracellular proteins, such as talin, have provided new insight into the signaling processes centered around the tails, particularly inside-out integrin activation. PMID:21216149

  17. The Use of PET Imaging for Prognostic Integrin α2β1 Phenotyping to Detect Non-Small Cell Lung Cancer and Monitor Drug Resistance Responses

    PubMed Central

    Huang, Chiun-Wei; Hsieh, Wen-Chuan; Hsu, Shih-Ting; Lin, Yi-Wen; Chung, Yi-Hsiu; Chang, Wen-Chi; Chiu, Han; Lin, Yun Han; Wu, Chung-Pu; Yen, Tzu-Chen; Huang, Feng-Ting

    2017-01-01

    PURPOSE: Growing evidence has demonstrated that aberrant expression of integrin α2β1 might contribute to the invasion, metastasis and drug resistance of non-small cell lung cancer (NSCLC). Thus, the integrin α2β1 targeting 68Ga-DOTA-A2B1 tracer was validated in NSCLC in contrast to accumulation of the clinically used 18F-FDG PET tracer to see if 68Ga-DOTA-A2B1-PET imaging can offer a valuable and critical diagnostic imaging criterion for the identification of phenotypes of aggressive lung cancer. METHODS: To verify the prognostic value of integrin α2β1, several quantitative and functional in vitro assays were validated in different NSCLC cell lines (CL1-0, CL1-5, A549 and selected A549++ cells). Positron emission tomography (PET) imaging studies using both standard 18F-FDG and a newly developed 68Ga-labeled integrin α2β1 (68Ga-DOTA-A2B1) tracer were sequentially performed on mice with lung tumor xenografts in different anatomic locations (subcutaneous, orthotopic and osseous) to validate the targeting capability of the 68Ga-DOTA-A2B1 tracers. Treatment responses were monitored by injecting animals with metastatic bone tumors with 5 mg/kg doxorubicin. All in vivo treatment responses in each treatment subgroup were monitored with a PET imaging system to evaluate the up-regulation of integrin expression at the earliest stage of treatment (6 h). RESULTS: The PET and computed tomography (CT) images from NSCLC xenograft animals unambiguously demonstrated accumulation of the integrin tracer 68Ga-DOTA-A2B1 in the tumor lesions at all locations. The average tumor uptake and tumor-to-normal (T/N) ratio were 2.51 ± 0.56 %ID/g and T/N = 2.82, 3.40 ± 0.42 %ID/g and T/N = 1.52, and 1.58 ± 0.108 %ID/g and T/N = 2.31 in subcutaneous, orthotopic and osseous tumors, respectively (n = 5; p < 0.05). The xenograft tumors were all clearly visible. In contrast, the accumulation of 18F-FDG reached 3.6 ± 0.76 %ID/g, 1.39 ± 0.075 %ID/g and 3.78 ± 0.73 %ID/g in subcutaneous

  18. Integrin affinity modulation in angiogenesis

    PubMed Central

    Mahabeleshwar, Ganapati H.; Chen, Juhua; Feng, Weiyi; Somanath, Payaningal R.; Razorenova, Olga V.; Byzova, Tatiana V.

    2008-01-01

    Integrins, transmembrane glycoprotein receptors, play vital roles in pathological angiogenesis, but their precise regulatory functions are not completely understood and remain controversial. This study aims to assess the regulatory functions of individual beta subunits of endothelial integrins in angiogenic responses induced by vascular endothelial growth factor (VEGF). Inhibition of expression of β1, β3 or β5 integrins in endothelial cells resulted in down regulation of EC adhesion and migration on the primary ligand for the corresponding integrin receptor, while no effects on the recognition of other ligands were detected. Although inhibition of expression of each subunit substantially affected capillary growth stimulated by VEGF, the loss of β3 integrin was the most inhibitory. EC stimulation by VEGF induced formation of the high affinity (activated) state of αvβ3 in a monolayer and activated αvβ3 was co-localized with VEGF receptor-2 (VEGFR-2). Inhibition of expression of β1, β3 or β5 did not affect expression levels of VEGFR-2 in EC. However, inhibition of β3, but not β1 or β5, resulted in substantial inhibition of VEGFR-2 phosphorylation stimulated by VEGF. Exogenous stimulation of αvβ3 integrin with activating antibodies augmented VEGF-dependent phosphorylation of VEGFR-2, whereas integrin blockade suppressed this response. Most importantly, activated αvβ3 was detected on endothelial cells of tumor vasculature. Activation of αvβ3 was substantially increased in highly-vascularized tumors as compared to normal tissues. Moreover, activated αvβ3 was co-localized with VEGFR-2 on endothelial cells of proliferating blood vessels. Together, these results show the unique role of αvβ3 integrin in cross-talk with VEGFR-2 in the context of pathological angiogenesis. PMID:18287811

  19. Mass spectroscopy identifies the splicing-associated proteins, PSF, hnRNP H3, hnRNP A2/B1, and TLS/FUS as interacting partners of the ZNF198 protein associated with rearrangement in myeloproliferative disease

    SciTech Connect

    Kasyapa, Chitta S.; Kunapuli, Padmaja; Cowell, John K.

    2005-09-10

    ZNF198 is fused with FGFR1 in an atypical myeloproliferative disease that results in constitutive activation of the kinase domain and mislocalization to the cytoplasm. We have used immunoprecipitation of a GFP-tagged ZNF198 combined with MALDI-TOF mass spectroscopy to identify interacting proteins. P splicing factor (PSF) was identified as one of the proteins and this interaction was confirmed by Western blotting. Other proteins identified were the spliceosomal components hnRNP A2/B1, hnRNP H3, and TLS/FUS. PSF is also known to interact with PTB, another member of the hnRNP family of proteins, and we further demonstrated that PTB interacts with ZNF198. The interactionmore » between TLS/FUS and ZNF198 was confirmed using Western blot analysis. In 293 cells expressing the ZNF198/FGFR1 fusion protein, neither PSF nor PTB binds to the fusion protein, possibly because of their differential localization in the cell.« less

  20. Increased expression of the coxsackie and adenovirus receptor downregulates αvβ3 and αvβ5 integrin expression and reduces cell adhesion and migration.

    PubMed

    Majhen, Dragomira; Stojanović, Nikolina; Špeljko, Tea; Brozovic, Anamaria; De Zan, Tihana; Osmak, Maja; Ambriović-Ristov, Andreja

    2011-08-15

    Coxsackie and adenovirus receptor (CAR) is a tumor suppressor and a primary receptor for adenovirus type 5 (Ad5). Our study aims to examine the influence of forced expression of CAR in rhabdomyosarcoma cells (RD) on expression levels of integrins implicated in Ad5 entry, and the effect of CAR on cell-extracellular matrix adhesion and migration. CAR expressing clones were established from RD cells by stable transfection. Flow cytometry was used to evaluate the expression of CAR and integrins. Adhesion was measured in plates previously coated with vitronectin or fibronectin. Boyden chambers were used to investigate migration. Transfection of cells with siRNA was used to achieve integrin silencing. Ad5-mediated transgene expression was measured by β-gal staining. Increased expression of CAR in RD cells reduces the expression of αvβ3 and αvβ5 integrins. Cells overexpressing CAR exhibit significantly reduced adhesion to vitronectin and fibronectin, and reduced cell migration. Specifically silencing αvβ3 integrin in RD cells reduced cell migration indicating that reduced migration could be the consequence of αvβ3 integrin downregulation. This study also demonstrates the negative effect of reduced levels of αvβ3 and αvβ5 integrins on Ad5-mediated transgene expression with Ad5 retargeted to αv integrins. The pharmacological upregulation of CAR aimed to increase Ad5-mediated transgene expression may actually downregulate αvβ3 and αvβ5 integrins and thus alter Ad5-mediated gene transfer. The mechanism of decreased cell migration, a prerequisite for metastasis and invasion, due to increased CAR expression may be explained by reduced αvβ3 integrin expression. Copyright © 2011 Elsevier Inc. All rights reserved.

  1. Leukocyte integrins and their ligand interactions

    PubMed Central

    Hyun, Young-Min; Lefort, Craig T.; Kim, Minsoo

    2010-01-01

    Although critical for cell adhesion and migration during normal immune-mediated reactions, leukocyte integrins are also involved in the pathogenesis of diverse clinical conditions including autoimmune diseases and chronic inflammation. Leukocyte integrins therefore have been targets for anti-adhesive therapies to treat the inflammatory disorders. Recently, the therapeutic potential of integrin antagonists has been demonstrated in psoriasis and multiple sclerosis. However, current therapeutics broadly affect integrin functions and, thus, yield unfavorable side effects. This review discusses the major leukocyte integrins and the anti-adhesion strategies for treating immune diseases. PMID:19184539

  2. THE TMEFF2 TUMOR SUPPRESSOR MODULATES INTEGRIN EXPRESSION, RHOA ACTIVATION AND MIGRATION OF PROSTATE CANCER CELLS

    PubMed Central

    Chen, Xiaofei; Corbin, Joshua M.; Tipton, Greg J.; Yang, Li V.; Asch, Adam S.; Ruiz-Echevarría, Maria J.

    2014-01-01

    Cell adhesion and migration play important roles in physiological and pathological states, including embryonic development and cancer invasion and metastasis. The type I transmembrane protein with epidermal growth factor and two follistatin motifs 2 (TMEFF2) is expressed mainly in brain and prostate and its expression is deregulated in prostate cancer. We have previously shown that TMEFF2 can function as a tumor suppressor by inhibiting cell migration and invasion of prostate cells. However, the molecular mechanisms involved in this inhibition are not clear. In this study we demonstrate that TMEFF2 affects cell adhesion and migration of prostate cancer cells and that this effect correlates with changes in integrin expression and RhoA activation. Deletion of a 13 basic-rich amino acid region in the cytoplasmic domain of TMEFF2 prevented these effects. Overexpression of TMEFF2 reduced cell attachment and migration on vitronectin and caused a concomitant decrease in RhoA activation, stress fiber formation and expression of αv, β1 and β3 integrin subunits. Conversely, TMEFF2 interference in 22Rv1 prostate cancer cells resulted in increased integrin expression. Results obtained with a double TRAMP/TMEFF2 transgenic mouse also indicated that TMEFF2 expression reduced integrin expression in the mouse prostate. In summary, the data presented here indicate an important role of TMEFF2 in regulating cell adhesion and migration that involves integrin signaling and is mediated by its cytoplasmic domain. PMID:24632071

  3. Expression of an Activated Integrin Promotes Long-Distance Sensory Axon Regeneration in the Spinal Cord

    PubMed Central

    Cheah, Menghon; Chew, Daniel J.; Moloney, Elizabeth B.; Verhaagen, Joost; Fässler, Reinhard

    2016-01-01

    After CNS injury, axon regeneration is blocked by an inhibitory environment consisting of the highly upregulated tenascin-C and chondroitin sulfate proteoglycans (CSPGs). Tenascin-C promotes growth of axons if they express a tenascin-binding integrin, particularly α9β1. Additionally, integrins can be inactivated by CSPGs, and this inhibition can be overcome by the presence of a β1-binding integrin activator, kindlin-1. We examined the synergistic effect of α9 integrin and kindlin-1 on sensory axon regeneration in adult rat spinal cord after dorsal root crush and adeno-associated virus transgene expression in dorsal root ganglia. After 12 weeks, axons from C6–C7 dorsal root ganglia regenerated through the tenascin-C-rich dorsal root entry zone into the dorsal column up to C1 level and above (>25 mm axon length) through a normal pathway. Animals also showed anatomical and electrophysiological evidence of reconnection to the dorsal horn and behavioral recovery in mechanical pressure, thermal pain, and ladder-walking tasks. Expression of α9 integrin or kindlin-1 alone promoted much less regeneration and recovery. SIGNIFICANCE STATEMENT The study demonstrates that long-distance sensory axon regeneration over a normal pathway and with sensory and sensory–motor recovery can be achieved. This was achieved by expressing an integrin that recognizes tenascin-C, one of the components of glial scar tissue, and an integrin activator. This enabled extensive long-distance (>25 mm) regeneration of both myelinated and unmyelinated sensory axons with topographically correct connections in the spinal cord. The extent of growth and recovery we have seen would probably be clinically significant. Restoration of sensation to hands, perineum, and genitalia would be a significant improvement for a spinal cord-injured patient. PMID:27383601

  4. Integrins and epithelial cell polarity

    PubMed Central

    Lee, Jessica L.; Streuli, Charles H.

    2014-01-01

    ABSTRACT Cell polarity is characterised by differences in structure, composition and function between at least two poles of a cell. In epithelial cells, these spatial differences allow for the formation of defined apical and basal membranes. It has been increasingly recognised that cell–matrix interactions and integrins play an essential role in creating epithelial cell polarity, although key gaps in our knowledge remain. This Commentary will discuss the mounting evidence for the role of integrins in polarising epithelial cells. We build a model in which both inside-out signals to polarise basement membrane assembly at the basal surface, and outside-in signals to control microtubule apical–basal orientation and vesicular trafficking are required for establishing and maintaining the orientation of epithelial cell polarity. Finally, we discuss the relevance of the basal integrin polarity axis to cancer. This article is part of a Minifocus on Establishing polarity. For further reading, please see related articles: ‘ERM proteins at a glance’ by Andrea McClatchey (J. Cell Sci. 127, 3199–3204). ‘Establishment of epithelial polarity – GEF who's minding the GAP?’ by Siu Ngok et al. (J. Cell Sci. 127, 3205–3215). PMID:24994933

  5. Transgenic Animals.

    ERIC Educational Resources Information Center

    Jaenisch, Rudolf

    1988-01-01

    Describes three methods and their advantages and disadvantages for introducing genes into animals. Discusses the predictability and tissue-specificity of the injected genes. Outlines the applications of transgenic technology for studying gene expression, the early stages of mammalian development, mutations, and the molecular nature of chromosomes.…

  6. Integrin targeted delivery of gene therapeutics.

    PubMed

    Juliano, Rudy L; Ming, Xin; Nakagawa, Osamu; Xu, Rongzuo; Yoo, Hoon

    2011-03-02

    Integrins have become key targets for molecular imaging and for selective delivery of anti-cancer agents. Here we review recent work concerning the targeted delivery of antisense and siRNA oligonucleotides via integrins. A variety of approaches have been used to link oligonucleotides to ligands capable of binding integrins with high specificity and affinity. This includes direct chemical conjugation, incorporating oligonucleotides into lipoplexes, and use of various polymeric nanocarriers including dendrimers. The ligand-oligonucleotide conjugate or complex associates selectively with the integrin, followed by internalization into endosomes and trafficking through subcellular compartments. Escape of antisense or siRNA from the endosome to the cytosol and nucleus may come about through endogenous trafficking mechanisms, or because of membrane disrupting capabilities built into the conjugate or complex. Thus a variety of useful strategies are available for using integrins to enhance the pharmacological efficacy of therapeutic oligonucleotides.

  7. Small molecule antagonists of integrin receptors.

    PubMed

    Perdih, A; Dolenc, M Sollner

    2010-01-01

    The complex and widespread family of integrin receptors is involved in numerous physiological processes, such as tissue remodeling, angiogenesis, development of the immune response and homeostasis. In addition, their key role has been elucidated in important pathological disorders such as cancer, cardiovascular diseases, osteoporosis, autoimmune and inflammatory diseases and in the pathogenesis of infectious diseases, making them highly important targets for modern drug design campaigns. In this review we seek to present a concise overview of the small molecule antagonists of this diverse and highly complex receptor family. Integrin antagonists are classified according to the targeted integrin receptor and are discussed in four sections. First we present the fibrinogen alpha(IIb)beta3 and the vitronectin alpha (V)beta(3) receptor antagonists. The remaining selective integrin antagonists are examined in the third section. The final section is dedicated to molecules with dual or multiple integrin activity. In addition, the use of antibodies and peptidomimetic approaches to modulate the integrin receptors are discussed, as well providing the reader with an overall appreciation of the field.

  8. Tumour exosome integrins determine organotropic metastasis

    PubMed Central

    Hoshino, Ayuko; Costa-Silva, Bruno; Shen, Tang-Long; Rodrigues, Goncalo; Hashimoto, Ayako; Mark, Milica Tesic; Molina, Henrik; Kohsaka, Shinji; Di Giannatale, Angela; Ceder, Sophia; Singh, Swarnima; Williams, Caitlin; Soplop, Nadine; Uryu, Kunihiro; Pharmer, Lindsay; King, Tari; Bojmar, Linda; Davies, Alexander E.; Ararso, Yonathan; Zhang, Tuo; Zhang, Haiying; Hernandez, Jonathan; Weiss, Joshua M.; Dumont-Cole, Vanessa D.; Kramer, Kimberly; Wexler, Leonard H.; Narendran, Aru; Schwartz, Gary K.; Healey, John H.; Sandstrom, Per; Labori, Knut Jørgen; Kure, Elin H.; Grandgenett, Paul M.; Hollingsworth, Michael A.; de Sousa, Maria; Kaur, Sukhwinder; Jain, Maneesh; Mallya, Kavita; Batra, Surinder K.; Jarnagin, William R.; Brady, Mary S.; Fodstad, Oystein; Muller, Volkmar; Pantel, Klaus; Minn, Andy J.; Bissell, Mina J.; Garcia, Benjamin A.; Kang, Yibin; Rajasekhar, Vinagolu K.; Ghajar, Cyrus M.; Matei, Irina; Peinado, Hector; Bromberg, Jacqueline; Lyden, David

    2015-01-01

    Ever since Stephen Paget’s 1889 hypothesis, metastatic organotropism has remained one of cancer’s greatest mysteries. Here we demonstrate that exosomes from mouse and human lung-, liver- and brain-tropic tumour cells fuse preferentially with resident cells at their predicted destination, namely lung fibroblasts and epithelial cells, liver Kupffer cells and brain endothelial cells. We show that tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. Treatment with exosomes from lung-tropic models redirected the metastasis of bone-tropic tumour cells. Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins α6β4 and α6β1 were associated with lung metastasis, while exosomal integrin αvβ5 was linked to liver metastasis. Targeting the integrins α6β4 and αvβ5 decreased exosome uptake, as well as lung and liver metastasis, respectively. We demonstrate that exosome integrin uptake by resident cells activates Src phosphorylation and pro-inflammatory S100 gene expression. Finally, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis. PMID:26524530

  9. Tumour exosome integrins determine organotropic metastasis.

    PubMed

    Hoshino, Ayuko; Costa-Silva, Bruno; Shen, Tang-Long; Rodrigues, Goncalo; Hashimoto, Ayako; Tesic Mark, Milica; Molina, Henrik; Kohsaka, Shinji; Di Giannatale, Angela; Ceder, Sophia; Singh, Swarnima; Williams, Caitlin; Soplop, Nadine; Uryu, Kunihiro; Pharmer, Lindsay; King, Tari; Bojmar, Linda; Davies, Alexander E; Ararso, Yonathan; Zhang, Tuo; Zhang, Haiying; Hernandez, Jonathan; Weiss, Joshua M; Dumont-Cole, Vanessa D; Kramer, Kimberly; Wexler, Leonard H; Narendran, Aru; Schwartz, Gary K; Healey, John H; Sandstrom, Per; Labori, Knut Jørgen; Kure, Elin H; Grandgenett, Paul M; Hollingsworth, Michael A; de Sousa, Maria; Kaur, Sukhwinder; Jain, Maneesh; Mallya, Kavita; Batra, Surinder K; Jarnagin, William R; Brady, Mary S; Fodstad, Oystein; Muller, Volkmar; Pantel, Klaus; Minn, Andy J; Bissell, Mina J; Garcia, Benjamin A; Kang, Yibin; Rajasekhar, Vinagolu K; Ghajar, Cyrus M; Matei, Irina; Peinado, Hector; Bromberg, Jacqueline; Lyden, David

    2015-11-19

    Ever since Stephen Paget's 1889 hypothesis, metastatic organotropism has remained one of cancer's greatest mysteries. Here we demonstrate that exosomes from mouse and human lung-, liver- and brain-tropic tumour cells fuse preferentially with resident cells at their predicted destination, namely lung fibroblasts and epithelial cells, liver Kupffer cells and brain endothelial cells. We show that tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. Treatment with exosomes from lung-tropic models redirected the metastasis of bone-tropic tumour cells. Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins α6β4 and α6β1 were associated with lung metastasis, while exosomal integrin αvβ5 was linked to liver metastasis. Targeting the integrins α6β4 and αvβ5 decreased exosome uptake, as well as lung and liver metastasis, respectively. We demonstrate that exosome integrin uptake by resident cells activates Src phosphorylation and pro-inflammatory S100 gene expression. Finally, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis.

  10. Tumour exosome integrins determine organotropic metastasis

    DOE PAGES

    Hoshino, Ayuko; Costa-Silva, Bruno; Shen, Tang-Long; ...

    2015-10-28

    Ever since Stephen Paget’s 1889 hypothesis, metastatic organotropism has remained one of cancer’s greatest mysteries. In this paper, we demonstrate that exosomes from mouse and human lung-, liver- and brain-tropic tumour cells fuse preferentially with resident cells at their predicted destination, namely lung fibroblasts and epithelial cells, liver Kupffer cells and brain endothelial cells. We show that tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. Treatment with exosomes from lung-tropic models redirected the metastasis of bone-tropic tumour cells. Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins α 6β 4 and α 6β 1 weremore » associated with lung metastasis, while exosomal integrin α vβ 5 was linked to liver metastasis. Targeting the integrins α 6β 4 and α vβ 5 decreased exosome uptake, as well as lung and liver metastasis, respectively. We demonstrate that exosome integrin uptake by resident cells activates Src phosphorylation and pro-inflammatory S100 gene expression. In conclusion, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis.« less

  11. Natalizumab: AN 100226, anti-4alpha integrin monoclonal antibody.

    PubMed

    2004-01-01

    Natalizumab [AN 100226, anti-alpha4 integrin monoclonal antibody, Antegren] is a humanised monoclonal antibody that blocks alpha4beta1 integrin-mediated leukocyte migration. Natalizumab is in phase III trials for the treatment of multiple sclerosis in North America and the UK, and for the treatment of Crohn's disease also in the UK. It may have potential in the treatment of other immune-related inflammatory disease. Elan Corporation intends to examine the potential of natalizumab in rheumatoid arthritis and ulcerative colitis. 4beta1 integrin on circulating leukocytes binds to vascular cell adhesion molecule-1, which is expressed at high levels in the blood vessels in the CNS during exacerbations of multiple sclerosis. This allows leukocytes expressing alpha4beta1 integrin (very late antigen-4) to move from the peripheral blood into the CNS. Inflammatory proteins and other factors released from lymphocytes in the brain lead to the progression of symptoms. A limitation of natalizumab is that it must be injected and cannot be administered orally. Scientists have transformed the large anti-alpha4 monoclonal antibody into much smaller, drug-like molecules suitable for oral administration. Protein Design Labs has granted a worldwide nonexclusive licence under its antibody humanisation patents to Elan Pharmaceuticals for natalizumab. Biogen Inc. has entered into an agreement with Elan for a worldwide exclusive collaboration to develop, manufacture and commercialise natalizumab for multiple sclerosis and Crohn's disease and rheumatoid arthritis. Development of natalizumab is also being funded, in part, by Axogen (acquired by Elan in 1999). In November 2003, Biogen and IDEC Pharmaceuticals merged to form Biogen Idec. Elan repurchased royalty rights on a package of products, including natalizumab, from Autoimmune Disease Research Company. Elan and Genzyme Transgenics Corporation signed an agreement to produce natalizumab in GTC's genetically engineered goats, which will

  12. A color-coded imaging model of the interaction of αv integrin-GFP expressed in osteosarcoma cells and RFP expressing blood vessels in Gelfoam® vascularized in vivo.

    PubMed

    Uehara, Fuminari; Tome, Yasunori; Yano, Shuya; Miwa, Shinji; Mii, Sumiyuki; Hiroshima, Yukihiko; Bouvet, Michael; Maehara, Hiroki; Kanaya, Fuminori; Hoffman, Robert M

    2013-04-01

    The integrin family of proteins has been shown to be involved in the malignant behavior of cells. We report here development of a color-coded imaging model that can visualize the interaction between αv integrin linked to green fluorescent protein (GFP) in osteosarcoma cells and blood vessels in Gelfoam® vascularized after implantation in red fluorescent protein (RFP) transgenic nude mice. Human 143B osteosarcoma cells expressing αv integrin-GFP were generated by transfection with an αv integrin-GFP vector. Gelfoam® (5×5 mm) was transplanted subcutaneously in transgenic RFP nude mice. The implanted Gelfoam® became highly vascularized with RFP vessels within 14 days. Skin flaps were made at days 7, 14, 21, 28 after transplantation of Gelfoam® for observing vascularization of the Gelfoam® using fluorescence imaging. Gelfoam® is a useful tool to observe angiogenesis in vivo. 143B cells (5 × 10(5)) expressing αv integrin-GFP were injected into the Gelfoam® seven days after transplantation of Gelfoam®. Seven days after cancer-cell injection, cancer cells and blood vessels were observed in the Gelfoam® by color-coded confocal microscopy via the skin flap. The 143B cells expressing αv integrin-GFP proliferated into the Gelfoam®, which contained RFP-expressing blood vessels. Strong expression of αv integrin-GFP in 143B cells was observed near RFP vessels in the Gelfoam®. The observation of the behavior of αv integrin-GFP and blood vessels will allow further understanding of the role of αv integrin in cancer cells.

  13. Sarcospan integration into laminin-binding adhesion complexes that ameliorate muscular dystrophy requires utrophin and α7 integrin

    PubMed Central

    Marshall, Jamie L.; Oh, Jennifer; Chou, Eric; Lee, Joy A.; Holmberg, Johan; Burkin, Dean J.; Crosbie-Watson, Rachelle H.

    2015-01-01

    Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene that result in loss of the dystrophin–glycoprotein complex, a laminin receptor that connects the myofiber to its surrounding extracellular matrix. Utrophin, a dystrophin ortholog that is normally localized to the neuromuscular junction, is naturally upregulated in DMD muscle, which partially compensates for the loss of dystrophin. Transgenic overexpression of utrophin causes broad sarcolemma localization of utrophin, restoration of laminin binding and amelioration of disease in the mdx mouse model of DMD. We previously demonstrated that overexpression of sarcospan, a dystrophin- and utrophin-binding protein, ameliorates mdx muscular dystrophy. Sarcospan boosts levels of utrophin to therapeutic levels at the sarcolemma, where attachment to laminin is restored. However, understanding the compensatory mechanism is complicated by concomitant upregulation of α7β1 integrin, which also binds laminin. Similar to the effects of utrophin, transgenic overexpression of α7 integrin prevents DMD disease in mice and is accompanied by increased abundance of utrophin around the extra-synaptic sarcolemma. In order to investigate the mechanisms underlying sarcospan ‘rescue’ of muscular dystrophy, we created double-knockout mice to test the contributions of utrophin or α7 integrin. We show that sarcospan-mediated amelioration of muscular dystrophy in DMD mice is dependent on the presence of both utrophin and α7β1 integrin, even when they are individually expressed at therapeutic levels. Furthermore, we found that association of sarcospan into laminin-binding complexes is dependent on utrophin and α7β1 integrin. PMID:25504048

  14. Integrin signaling is critical for pathological angiogenesis

    PubMed Central

    Mahabeleshwar, Ganapati H.; Feng, Weiyi; Phillips, David R.; Byzova, Tatiana V.

    2006-01-01

    The process of postnatal angiogenesis plays a crucial role in pathogenesis of numerous diseases, including but not limited to tumor growth/metastasis, diabetic retinopathy, and in tissue remodeling upon injury. However, the molecular events underlying this complex process are not well understood and numerous issues remain controversial, including the regulatory function of integrin receptors. To analyze the role of integrin phosphorylation and signaling in angiogenesis, we generated knock-in mice that express a mutant β3 integrin unable to undergo tyrosine phosphorylation. Two distinct models of pathological angiogenesis revealed that neovascularization is impaired in mutant β3 knock-in mice. In an ex vivo angiogenesis assay, mutant β3 knock-in endothelial cells did not form complete capillaries in response to vascular endothelial growth factor (VEGF) stimulation. At the cellular level, defective tyrosine phosphorylation in mutant β3 knock-in cells resulted in impaired adhesion, spreading, and migration of endothelial cells. At the molecular level, VEGF stimulated complex formation between VEGF receptor-2 and β3 integrin in wild-type but not in mutant β3 knock-in endothelial cells. Moreover, phosphorylation of VEGF receptor-2 was significantly reduced in cells expressing mutant β3 compared to wild type, leading to impaired integrin activation in these cells. These findings provide novel mechanistic insights into the role of integrin–VEGF axis in pathological angiogenesis. PMID:17030947

  15. Characterization of Laminin Binding Integrin Internalization in Prostate Cancer Cells†

    PubMed Central

    Das, Lipsa; Anderson, Todd A.; Gard, Jaime M.C.; Sroka, Isis C.; Strautman, Stephanie R.; Nagle, Raymond B.; Morrissey, Colm; Knudsen, Beatrice S.; Cress, Anne E.

    2017-01-01

    Laminin binding integrins α6 (CD49f) and α3 (CD49c) are persistently but differentially expressed in prostate cancer (PCa). Integrin internalization is an important determinant of their cell surface expression and function. Using flow cytometry, and first order kinetic modelling, we quantitated the intrinsic internalization rates of integrin subunits in a single cycle of internalization. In PCa cell line DU145, α6 integrin internalized with a rate constant (kactual) of 3.25min−1, 3-fold faster than α3 integrin (1.0 min−1), 1.5-fold faster than the vitronectin binding αv integrin (CD51) (2.2 min−1), and significantly slower than the unrelated transferrin receptor (CD71) (15 min−1). Silencing of α3 integrin protein expression in DU145, PC3 and PC3B1 cells resulted in up to a 1.71-fold increase in kactual for α6 integrin. The internalized α6 integrin was targeted to early endosomes but not to lamp1 vesicles. Depletion of α3 integrin expression resulted in redistribution of α6β4 integrin to an observed cell-cell staining pattern that is consistent with a suprabasal distribution observed in epidermis and early PIN lesions in PCa. Depletion of α3 integrin increased cell migration by 1.8 fold, which was dependent on α6β1 integrin. Silencing of α6 integrin expression however, had no significant effect on the kactual of α3 integrin or its distribution in early endosomes. These results indicate that α3 and α6 integrins have significantly different internalization kinetics and that coordination exists between them for internalization. This article is protected by copyright. All rights reserved PMID:27509031

  16. Characterization of Laminin Binding Integrin Internalization in Prostate Cancer Cells.

    PubMed

    Das, Lipsa; Anderson, Todd A; Gard, Jaime M C; Sroka, Isis C; Strautman, Stephanie R; Nagle, Raymond B; Morrissey, Colm; Knudsen, Beatrice S; Cress, Anne E

    2017-05-01

    Laminin binding integrins α6 (CD49f) and α3 (CD49c) are persistently but differentially expressed in prostate cancer (PCa). Integrin internalization is an important determinant of their cell surface expression and function. Using flow cytometry, and first order kinetic modeling, we quantitated the intrinsic internalization rates of integrin subunits in a single cycle of internalization. In PCa cell line DU145, α6 integrin internalized with a rate constant (k actual ) of 3.25 min -1 , threefold faster than α3 integrin (1.0 min -1 ), 1.5-fold faster than the vitronectin binding αv integrin (CD51) (2.2 min -1 ), and significantly slower than the unrelated transferrin receptor (CD71) (15 min -1 ). Silencing of α3 integrin protein expression in DU145, PC3, and PC3B1 cells resulted in up to a 1.71-fold increase in k actual for α6 integrin. The internalized α6 integrin was targeted to early endosomes but not to lamp1 vesicles. Depletion of α3 integrin expression resulted in redistribution of α6β4 integrin to an observed cell-cell staining pattern that is consistent with a suprabasal distribution observed in epidermis and early PIN lesions in PCa. Depletion of α3 integrin increased cell migration by 1.8-fold, which was dependent on α6β1 integrin. Silencing of α6 integrin expression however, had no significant effect on the k actual of α3 integrin or its distribution in early endosomes. These results indicate that α3 and α6 integrins have significantly different internalization kinetics and that coordination exists between them for internalization. J. Cell. Biochem. 118: 1038-1049, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  17. Transgenic resistance.

    PubMed

    Cillo, Fabrizio; Palukaitis, Peter

    2014-01-01

    Transgenic resistance to plant viruses is an important technology for control of plant virus infection, which has been demonstrated for many model systems, as well as for the most important plant viruses, in terms of the costs of crop losses to disease, and also for many other plant viruses infecting various fruits and vegetables. Different approaches have been used over the last 28 years to confer resistance, to ascertain whether particular genes or RNAs are more efficient at generating resistance, and to take advantage of advances in the biology of RNA interference to generate more efficient and environmentally safer, novel "resistance genes." The approaches used have been based on expression of various viral proteins (mostly capsid protein but also replicase proteins, movement proteins, and to a much lesser extent, other viral proteins), RNAs [sense RNAs (translatable or not), antisense RNAs, satellite RNAs, defective-interfering RNAs, hairpin RNAs, and artificial microRNAs], nonviral genes (nucleases, antiviral inhibitors, and plantibodies), and host-derived resistance genes (dominant resistance genes and recessive resistance genes), and various factors involved in host defense responses. This review examines the above range of approaches used, the viruses that were tested, and the host species that have been examined for resistance, in many cases describing differences in results that were obtained for various systems developed in the last 20 years. We hope this compilation of experiences will aid those who are seeking to use this technology to provide resistance in yet other crops, where nature has not provided such.

  18. PIP2 and Talin Join Forces to Activate Integrin.

    PubMed

    Orłowski, Adam; Kukkurainen, Sampo; Pöyry, Annika; Rissanen, Sami; Vattulainen, Ilpo; Hytönen, Vesa P; Róg, Tomasz

    2015-09-24

    Integrins are major players in cell adhesion and migration, and malfunctions in controlling their activity are associated with various diseases. Nevertheless, the details of integrin activation are not completely understood, and the role of lipids in the process is largely unknown. Herein, we show using atomistic molecular dynamics simulations that the interplay of phosphatidylinositol 4,5-bisphosphate (PIP2) and talin may directly alter the conformation of integrin αIIbβ3. Our results provide a new perspective on the role of PIP2 in integrin activation and indicate that the charged PIP2 lipid headgroup can perturb a clasp at the cytoplasmic face of the integrin heterodimer.

  19. α5β1-Integrin promotes tension-dependent mammary epithelial cell invasion by engaging the fibronectin synergy site

    PubMed Central

    Miroshnikova, Y. A.; Rozenberg, G. I.; Cassereau, L.; Pickup, M.; Mouw, J. K.; Ou, G.; Templeman, K. L.; Hannachi, E.-I.; Gooch, K. J.; Sarang-Sieminski, A. L.; García, A. J.; Weaver, V. M.

    2017-01-01

    Tumors are fibrotic and characterized by abundant, remodeled, and cross-linked collagen that stiffens the extracellular matrix stroma. The stiffened collagenous stroma fosters malignant transformation of the tissue by increasing tumor cell tension to promote focal adhesion formation and potentiate growth factor receptor signaling through kinase. Importantly, collagen cross-linking requires fibronectin (FN). Fibrotic tumors contain abundant FN, and tumor cells frequently up-regulate the FN receptor α5β1 integrin. Using transgenic and xenograft models and tunable two- and three-dimensional substrates, we show that FN-bound α5β1 integrin promotes tension-dependent malignant transformation through engagement of the synergy site that enhances integrin adhesion force. We determined that ligation of the synergy site of FN permits tumor cells to engage a zyxin-stabilized, vinculin-linked scaffold that facilitates nucleation of phosphatidylinositol (3,4,5)-triphosphate at the plasma membrane to enhance phosphoinositide 3-kinase (PI3K)-dependent tumor cell invasion. The data explain why rigid collagen fibrils potentiate PI3K activation to promote malignancy and offer a perspective regarding the consistent up-regulation of α5β1 integrin and FN in many tumors and their correlation with cancer aggression. PMID:28877984

  20. Distinct Recycling of Active and Inactive β1 Integrins

    PubMed Central

    Arjonen, Antti; Alanko, Jonna; Veltel, Stefan; Ivaska, Johanna

    2012-01-01

    Integrin trafficking plays an important role in cellular motility and cytokinesis. Integrins undergo constant endo/exocytic shuttling to facilitate the dynamic regulation of cell adhesion. Integrin activity toward the components of the extracellular matrix is regulated by the ability of these receptors to switch between active and inactive conformations. Several cellular signalling pathways have been described in the regulation of integrin traffic under different conditions. However, the interrelationship between integrin activity conformations and their endocytic fate have remained incompletely understood. Here, we have investigated the endocytic trafficking of active and inactive β1 integrins in cancer cells. Both conformers are endocytosed in a clathrin- and dynamin-dependent manner. The net endocytosis rate of the active β1 integrins is higher, whereas endocytosis of the inactive β1 integrin is counteracted by rapid recycling back to the plasma membrane via an ARF 6- and early endosome antigen 1-positive compartment in an Rab 4a- and actin-dependent manner. Owing to these distinct trafficking routes, the two receptor pools display divergent subcellular localization. At steady state, the inactive β1 integrin is mainly on the plasma membrane, whereas the active receptor is predominantly intracellular. These data provide new insights into the endocytic traffic of integrins and imply the possibility of a previously unappreciated crosstalk between pathways regulating integrin activity and traffic. PMID:22222055

  1. VEGF-integrin interplay controls tumor growth and vascularization

    NASA Astrophysics Data System (ADS)

    de, Sarmishtha; Razorenova, Olga; McCabe, Noel Patrick; O'Toole, Timothy; Qin, Jun; Byzova, Tatiana V.

    2005-05-01

    Cross-talk between the major angiogenic growth factor, VEGF, and integrin cell adhesion receptors has emerged recently as a critical factor in the regulation of angiogenesis and tumor development. However, the molecular mechanisms and consequences of this intercommunication remain unclear. Here, we define a mechanism whereby integrin v3, through activation, clustering, and signaling by means of p66 Shc (Src homology 2 domain containing), regulates the production of VEGF in tumor cells expressing this integrin. Tumors with "activatable" but not "inactive" 3 integrin secrete high levels of VEGF, which in turn promotes extensive neovascularization and augments tumor growth in vivo. This stimulation of VEGF expression depends upon the ability of v3 integrin to cluster and promote phosphorylation of p66 Shc. These observations identify a link between 3 integrins and VEGF in tumor growth and angiogenesis and, therefore, may influence anti-integrin as well as anti-VEGF therapeutic strategies. activation | angiogenesis | Src homology 2 domain containing

  2. Clustered Integrin Ligands as a Novel Approach for the Targeting of Non-Viral Vectors

    NASA Astrophysics Data System (ADS)

    Ng, Quinn Kwan Tai

    ligand clusters compared to the reacted amounts on the surface of the particle was studied. This provided us the ability to control the size of the clusters formed and the spacing between the integrins for gold nanoparticles of various sizes. We then applied the clustered ligand binding system for targeting of DNA/PEI polyplexes and demonstrated that the use of RGD nanoclusters enhances gene transfer up to 35-fold which was dependent on the density of alphavbeta3 integrins on the cell surface. Cell integrin sensitivity was shown in which cells with higher alpha vbeta3 densities resulting in higher luciferase transgene expression. The targeting of RGD nanoclusters for DNA/PEI polyplexes was further shown in vivo using PET/CT technology which displayed improved targeting towards high level alphavbeta3 integrin expression (U87MG) tumors over medium level alphavbeta 3 integrin expression (HeLa). In addition to studying the clustered integrin binding system, the current non-viral vectors used suffer from stability and toxicity issues in vitro and in vivo. We have applied a new chemistry for synthesizing nanogels utilizing a Traut's reagent initiated Michael addition reaction for modification of diamine containing crosslikers which will allow for the development of stable and cell demanded release of oligonucleotides. We have shown bulk gels made were capable of encapsulating and holding DNA within the gel and were able to synthesize them into nanogels. The combined research shown here using clustered integrin ligands and a new type of nanogel synthesis provides an ideal system for gene delivery in the future.

  3. Neuroanatomy and transgenic technologies

    USDA-ARS?s Scientific Manuscript database

    This is a short review that introduces recent advances of neuroanatomy and transgenic technologies. The anatomical complexity of the nervous system remains a subject of tremendous fascination among neuroscientists. In order to tackle this extraordinary complexity, powerful transgenic technologies a...

  4. AMPK negatively regulates tensin-dependent integrin activity

    PubMed Central

    Lilja, Johanna; Guzmán, Camilo; Yan, Yan; Lerche, Martina; Mäkelä, Tomi P.

    2017-01-01

    Tight regulation of integrin activity is paramount for dynamic cellular functions such as cell matrix adhesion and mechanotransduction. Integrin activation is achieved through intracellular interactions at the integrin cytoplasmic tails and through integrin–ligand binding. In this study, we identify the metabolic sensor AMP-activated protein kinase (AMPK) as a β1-integrin inhibitor in fibroblasts. Loss of AMPK promotes β1-integrin activity, the formation of centrally located active β1-integrin– and tensin-rich mature fibrillar adhesions, and cell spreading. Moreover, in the absence of AMPK, cells generate more mechanical stress and increase fibronectin fibrillogenesis. Mechanistically, we show that AMPK negatively regulates the expression of the integrin-binding proteins tensin1 and tensin3. Transient expression of tensins increases β1-integrin activity, whereas tensin silencing reduces integrin activity in fibroblasts lacking AMPK. Accordingly, tensin silencing in AMPK-depleted fibroblasts impedes enhanced cell spreading, traction stress, and fibronectin fiber formation. Collectively, we show that the loss of AMPK up-regulates tensins, which bind β1-integrins, supporting their activity and promoting fibrillar adhesion formation and integrin-dependent processes. PMID:28289092

  5. αV-class integrins exert dual roles on α5β1 integrins to strengthen adhesion to fibronectin.

    PubMed

    Bharadwaj, Mitasha; Strohmeyer, Nico; Colo, Georgina P; Helenius, Jonne; Beerenwinkel, Niko; Schiller, Herbert B; Fässler, Reinhard; Müller, Daniel J

    2017-01-27

    Upon binding to the extracellular matrix protein, fibronectin, αV-class and α5β1 integrins trigger the recruitment of large protein assemblies and strengthen cell adhesion. Both integrin classes have been functionally specified, however their specific roles in immediate phases of cell attachment remain uncharacterized. Here, we quantify the adhesion of αV-class and/or α5β1 integrins expressing fibroblasts initiating attachment to fibronectin (≤120 s) by single-cell force spectroscopy. Our data reveals that αV-class integrins outcompete α5β1 integrins. Once engaged, αV-class integrins signal to α5β1 integrins to establish additional adhesion sites to fibronectin, away from those formed by αV-class integrins. This crosstalk, which strengthens cell adhesion, induces α5β1 integrin clustering by RhoA/ROCK/myosin-II and Arp2/3-mediated signalling, whereas overall cell adhesion depends on formins. The dual role of both fibronectin-binding integrin classes commencing with an initial competition followed by a cooperative crosstalk appears to be a basic cellular mechanism in assembling focal adhesions to the extracellular matrix.

  6. αV-class integrins exert dual roles on α5β1 integrins to strengthen adhesion to fibronectin

    PubMed Central

    Bharadwaj, Mitasha; Strohmeyer, Nico; Colo, Georgina P.; Helenius, Jonne; Beerenwinkel, Niko; Schiller, Herbert B.; Fässler, Reinhard; Müller, Daniel J.

    2017-01-01

    Upon binding to the extracellular matrix protein, fibronectin, αV-class and α5β1 integrins trigger the recruitment of large protein assemblies and strengthen cell adhesion. Both integrin classes have been functionally specified, however their specific roles in immediate phases of cell attachment remain uncharacterized. Here, we quantify the adhesion of αV-class and/or α5β1 integrins expressing fibroblasts initiating attachment to fibronectin (≤120 s) by single-cell force spectroscopy. Our data reveals that αV-class integrins outcompete α5β1 integrins. Once engaged, αV-class integrins signal to α5β1 integrins to establish additional adhesion sites to fibronectin, away from those formed by αV-class integrins. This crosstalk, which strengthens cell adhesion, induces α5β1 integrin clustering by RhoA/ROCK/myosin-II and Arp2/3-mediated signalling, whereas overall cell adhesion depends on formins. The dual role of both fibronectin-binding integrin classes commencing with an initial competition followed by a cooperative crosstalk appears to be a basic cellular mechanism in assembling focal adhesions to the extracellular matrix. PMID:28128308

  7. Proteomic analysis of integrin adhesion complexes.

    PubMed

    Byron, Adam; Humphries, Jonathan D; Bass, Mark D; Knight, David; Humphries, Martin J

    2011-04-05

    Integrin receptors regulate cell fate by coupling the binding of extracellular adhesion proteins to the assembly of intracellular cytoskeletal and signaling complexes. A detailed, integrative view of adhesion complexes will provide insight into the molecular mechanisms that control cell morphology, survival, movement, and differentiation. To date, membrane receptor-associated signaling complexes have been refractory to proteomic analysis because of their inherent lability and inaccessibility. We developed a methodology to isolate ligand-induced integrin adhesion complexes, and we used this technique to analyze the composition of complexes associated with multiple receptor-ligand pairs and define core and receptor-specific subnetworks. In particular, we identified regulator of chromosome condensation-2 (RCC2) as a component of fibronectin-activated signaling pathways that regulate directional cell movement. The development of this proteomics pipeline provides the means to investigate the molecular composition and function of various adhesion complexes.

  8. Molecular Analyses of Transgenic Plants.

    PubMed

    Trijatmiko, Kurniawan Rudi; Arines, Felichi Mae; Oliva, Norman; Slamet-Loedin, Inez Hortense; Kohli, Ajay

    2016-01-01

    One of the major challenges in plant molecular biology is to generate transgenic plants that express transgenes stably over generations. Here, we describe some routine methods to study transgene locus structure and to analyze transgene expression in plants: Southern hybridization using DIG chemiluminescent technology for characterization of transgenic locus, SYBR Green-based real-time RT-PCR to measure transgene transcript level, and protein immunoblot analysis to evaluate accumulation and stability of transgenic protein product in the target tissue.

  9. Integrins are required for cardioblast polarisation in Drosophila

    PubMed Central

    2012-01-01

    Background The formation of a tubular organ, such as the heart, requires the communication of positional and polarity signals between migratory cells. Key to this process is the establishment of a new luminal domain on the cell surface, generally from the apical domain of a migratory cell. This domain will also acquire basal properties, as it will produce a luminal extracellular matrix. Integrin receptors are the primary means of cell adhesion and adhesion signaling with the extracellular matrix. Here we characterise the requirement of Integrins in a genetic model of vasculogenesis, the formation of the heart in Drosophila. Results As with vertebrates, the Drosophila heart arises from lateral mesoderm that migrates medially to meet their contralateral partners, to then assemble a midline vessel. During migration, Integrins are among the first proteins restricted to the presumptive luminal domain of cardioblasts. Integrins are required for normal levels of leading edge membrane motility. Apical accumulation of Integrins is enhanced by Robo, and reciprocally, apicalisation of luminal factors like Slit and Robo requires Integrin function. Integrins may provide a template for the formation of a lumen by stabilising lumen factors like Robo. Subsequent to migration, Integrin is required for normal cardioblast alignment and lumen formation. This phenotype is most readily modified by other mutations that affect adhesion, such as Talin and extracellular matrix ligands. Conclusion Our findings reveal an instructive role for Integrins in communicating polarising information to cells during migration, and during transition to an epithelial tube structure. PMID:22353787

  10. Integrin-Directed Modulation of Macrophage Responses to Biomaterials

    PubMed Central

    Zaveri, Toral D.; Lewis, Jamal S.; Dolgova, Natalia V.; Clare-Salzler, Michael J.; Keselowsky, Benjamin G.

    2014-01-01

    Macrophages are the primary mediator of chronic inflammatory responses to implanted biomaterials, in cases when the material is either in particulate or bulk form. Chronic inflammation limits the performance and functional life of numerous implanted medical devices, and modulating macrophage interactions with biomaterials to mitigate this response would be beneficial. The integrin family of cell surface receptors mediates cell adhesion through binding to adhesive proteins nonspecifically adsorbed onto biomaterial surfaces. In this work, the roles of integrin Mac-1 (αMβ2) and RGD-binding integrins were investigated using model systems for both particulate and bulk biomaterials. Specifically, the macrophage functions of phagocytosis and inflammatory cytokine secretion in response to a model particulate material, polystyrene microparticles were investigated. Opsonizing proteins modulated microparticle uptake, and integrin Mac-1 and RGD-binding integrins were found to control microparticle uptake in an opsonin-dependent manner. The presence of adsorbed endotoxin did not affect microparticle uptake levels, but was required for the production of inflammatory cytokines in response to microparticles. Furthermore, it was demonstrated that integrin Mac-1 and RGD-binding integrins influence the in vivo foreign body response to a bulk biomaterial, subcutaneously implanted polyethylene terephthalate. A thinner foreign body capsule was formed when integrin Mac-1 was absent (~30% thinner) or when RGD-binding integrins were blocked by controlled release of a blocking peptide (~45% thinner). These findings indicate integrin Mac-1 and RGD-binding integrins are involved and may serve as therapeutic targets to mitigate macrophage inflammatory responses to both particulate and bulk biomaterials. PMID:24462356

  11. Technical Advance: Function and efficacy of an {alpha}4-integrin antagonist using bioluminescence imaging to detect leukocyte trafficking in murine experimental colitis.

    PubMed

    Murphy, Carola T; Moloney, Gerard; Macsharry, John; Haynes, Andrea; Faivre, Emilie; Quinlan, Aoife; McLean, Peter G; Lee, Kevin; O'Mahony, Liam; Shanahan, Fergus; Melgar, Silvia; Nally, Kenneth

    2010-12-01

    Leukocyte trafficking is a therapeutic target in IBD. The integrins α₄β and α₄β₁ regulate leukocyte migration into tissues and lymphoid organs. Current strategies rely on biologics, such as mAb, to inhibit leukocyte recruitment. Here we show the in vivo therapeutic effects of a small molecule α4-integrin antagonist (GSK223618A) in a leukocyte-trafficking model and a murine model of colitis. Leukocytes isolated from MLNs of transgenic β-actin-luc+ mice were injected i.v. into recipients with DSS-induced colitis. Recipient mice were orally gavaged with vehicle or an α₄-integrin antagonist 1 h pre-adoptive transfer, followed by bioluminescence whole body and ex vivo organ imaging 4 h post-transfer. To confirm its therapeutic effect, the α₄-integrin antagonist was given orally twice daily for 6 days to mice with DSS-induced colitis, starting on Day 3. Clinical, macroscopic, and histological signs of inflammation were assessed and gene-expression profiles analyzed. Using bioluminescence imaging, we tracked and quantified leukocyte migration to the inflamed gut and demonstrated its inhibition by a small molecule α₄-integrin antagonist. Additionally, the therapeutic effect of the antagonist was confirmed in DSS-induced colitis in terms of clinical, macroscopic, and histological signs of inflammation. Gene expression analysis suggested enhancement of tissue healing in compound-treated animals. Inhibition of leukocyte trafficking using small molecule integrin antagonists is a promising alternative to large molecule biologics. Furthermore, in vivo bioluminescence imaging is a valuable strategy for preclinical evaluation of potential therapeutics that target leukocyte trafficking in inflammatory diseases.

  12. Generation of transgenic mice.

    PubMed

    Cho, Andrew; Haruyama, Naoto; Kulkarni, Ashok B

    2009-03-01

    This unit describes detailed step-by-step protocols, reagents, and equipment required for successful generation of transgenic mice using pronuclear injection. The experimental methods and practical tips given here will help guide beginners in understanding what is required and what to avoid in these standard protocols for efficiently generating transgenic mice. Copyright 2009 by John Wiley & Sons, Inc.

  13. Generation of Transgenic Mice

    PubMed Central

    Cho, Andrew; Haruyama, Naoto; Kulkarni, Ashok B.

    2009-01-01

    This unit describes detailed step-by-step protocols, reagents, and equipment required for successful generation of transgenic mice using pronuclear injection. The experimental methods and practical tips given here will help guide beginners in understanding what is required and what to avoid in these standard protocols for efficiently generating transgenic mice. PMID:19283729

  14. Integrins protect cardiomyocytes from ischemia/reperfusion injury

    PubMed Central

    Okada, Hideshi; Lai, N. Chin; Kawaraguchi, Yoshitaka; Liao, Peter; Copps, Jeffrey; Sugano, Yasuo; Okada-Maeda, Sunaho; Banerjee, Indroneal; Schilling, Jan M.; Gingras, Alexandre R.; Asfaw, Elizabeth K.; Suarez, Jorge; Kang, Seok-Min; Perkins, Guy A.; Au, Carol G.; Israeli-Rosenberg, Sharon; Manso, Ana Maria; Liu, Zheng; Milner, Derek J.; Kaufman, Stephen J.; Patel, Hemal H.; Roth, David M.; Hammond, H. Kirk; Taylor, Susan S.; Dillmann, Wolfgang H.; Goldhaber, Joshua I.; Ross, Robert S.

    2013-01-01

    Ischemic damage is recognized to cause cardiomyocyte (CM) death and myocardial dysfunction, but the role of cell-matrix interactions and integrins in this process has not been extensively studied. Expression of α7β1D integrin, the dominant integrin in normal adult CMs, increases during ischemia/reperfusion (I/R), while deficiency of β1 integrins increases ischemic damage. We hypothesized that the forced overexpression of integrins on the CM would offer protection from I/R injury. Tg mice with CM-specific overexpression of integrin α7β1D exposed to I/R had a substantial reduction in infarct size compared with that of α5β1D-overexpressing mice and WT littermate controls. Using isolated CMs, we found that α7β1D preserved mitochondrial membrane potential during hypoxia/reoxygenation (H/R) injury via inhibition of mitochondrial Ca2+ overload but did not alter H/R effects on oxidative stress. Therefore, we assessed Ca2+ handling proteins in the CM and found that β1D integrin colocalized with ryanodine receptor 2 (RyR2) in CM T-tubules, complexed with RyR2 in human and rat heart, and specifically bound to RyR2 amino acids 165–175. Integrins stabilized the RyR2 interdomain interaction, and this stabilization required integrin receptor binding to its ECM ligand. These data suggest that α7β1D integrin modifies Ca2+ regulatory pathways and offers a means to protect the myocardium from ischemic injury. PMID:24091324

  15. Visualization of integrin Mac-1 in vivo

    PubMed Central

    Lim, Kihong; Hyun, Young-Min; Lambert-Emo, Kris; Topham, David J; Kim, Minsoo

    2015-01-01

    β2 integrins play critical roles in migration of immune cells and in the interaction with other cells, pathogens, and the extracellular matrix. Among the β2 integrins, Mac-1 (Macrophage antigen-1), composed of CD11b and CD18, is mainly expressed in innate immune cells and plays a major role in cell migration and trafficking. In order to image Mac-1-expressing cells both in live cells and mouse, we generated a knock-in (KI) mouse strain expressing CD11b conjugated with monomeric yellow fluorescent protein (mYFP). Expression of CD11b-mYFP protein was confirmed by Western blot and silver staining of CD11b-immunoprecipitates and total cell lysates from the mouse splenocytes. Mac-1-mediated functions of the KI neutrophils were comparable with those in WT cells. The fluorescence intensity of CD11b-mYFP was sufficient to image CD11b expressing cells in live mice using intravital two-photon microscopy. In vitro, dynamic changes in the intracellular localization of CD11b molecules could be measured by epifluorescent microscopy. Finally, CD11b-expressing immune cells from tissue were easily detected by flow cytometry without anti-CD11b antibody staining. PMID:26342259

  16. Visualization of integrin Mac-1 in vivo.

    PubMed

    Lim, Kihong; Hyun, Young-Min; Lambert-Emo, Kris; Topham, David J; Kim, Minsoo

    2015-11-01

    β2 integrins play critical roles in migration of immune cells and in the interaction with other cells, pathogens, and the extracellular matrix. Among the β2 integrins, Mac-1 (Macrophage antigen-1), composed of CD11b and CD18, is mainly expressed in innate immune cells and plays a major role in cell migration and trafficking. In order to image Mac-1-expressing cells both in live cells and mouse, we generated a knock-in (KI) mouse strain expressing CD11b conjugated with monomeric yellow fluorescent protein (mYFP). Expression of CD11b-mYFP protein was confirmed by Western blot and silver staining of CD11b-immunoprecipitates and total cell lysates from the mouse splenocytes. Mac-1-mediated functions of the KI neutrophils were comparable with those in WT cells. The fluorescence intensity of CD11b-mYFP was sufficient to image CD11b expressing cells in live mice using intravital two-photon microscopy. In vitro, dynamic changes in the intracellular localization of CD11b molecules could be measured by epifluorescent microscopy. Finally, CD11b-expressing immune cells from tissue were easily detected by flow cytometry without anti-CD11b antibody staining. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Human gingival fibroblast integrin subunit expression on titanium implant surfaces.

    PubMed

    Oates, Thomas W; Maller, Steven C; West, Jason; Steffensen, Bjorn

    2005-10-01

    Implant surface characteristics have been shown to modify cell behavior and regulate integrin expression. Integrin expression and resultant integrin-mediated cellular activity are essential components of tissue healing and homeostasis. Although both osseous and soft tissue healing around dental implants are critical to clinical success, there is limited information available on the effect of implant surfaces on integrin expression in soft tissues. Therefore, the aim of this study was to examine integrin expression for gingival fibroblasts on titanium surfaces and the influence of titanium surface roughness on integrin expression and cell morphology. Human gingival fibroblasts were cultured on smooth (polished) and rough (sand-blasted acid-etched) titanium surfaces and a cell culture plastic (control) surface. To analyze integrin expression, total RNA was isolated from experimental and control cells, and levels of integrin subunit mRNA were assessed by reverse transcription-polymerase chain reaction (RT-PCR) using primers specific for the alpha2, alpha4, alpha5, alpha(v), and beta1 integrin subunits and aldolase (internal control). PCR products were analyzed by polyacrylamide gel electrophoresis (PAGE), confirmed via DNA sequencing, and quantified using computer-assisted densitometry. The expression of the integrin subunits was analyzed at the protein level using flow cytometry, as well as fluorescence and confocal laser microscopy. Cell morphology was evaluated using scanning electron microscopy (SEM). Our experiments demonstrated cellular expression of the alpha2, alpha4, alpha5, alpha(v), and beta1 integrin subunits at both mRNA and protein levels on all surfaces. In addition, the alpha4 and beta1 mRNA levels were significantly increased on smooth titanium relative to plastic surfaces (P <.05) with intermediate mRNA levels found on the rough titanium surfaces. The smooth titanium surfaces exhibited a flat monolayer of cells, while rough titanium surfaces showed

  18. Immunolocalization of integrin-like proteins in Arabidopsis and Chara

    NASA Technical Reports Server (NTRS)

    Katembe, W. J.; Swatzell, L. J.; Makaroff, C. A.; Kiss, J. Z.

    1997-01-01

    Integrins are a large family of integral plasma membrane proteins that link the extracellular matrix to the cytoskeleton in animal cells. As a first step in determining if integrin-like proteins are involved in gravitropic signal transduction pathways, we have used a polyclonal antibody against the chicken beta1 integrin subunit in western blot analyses and immunofluorescence microscopy to gain information on the size and location of these proteins in plants. Several different polypeptides are recognized by the anti-integrin antibody in roots and shoots of Arabidopsis and in the internodal cells and rhizoids of Chara. These cross-reactive polypeptides are associated with cellular membranes, a feature which is consistent with the known location of integrins in animal systems. In immunofluorescence studies of Arabidopsis roots, a strong signal was obtained from labeling integrin-like proteins in root cap cells, and there was little or no immunolabel in other regions of the root tip. While the antibody stained throughout Chara rhizoids, the highest density of immunolabel was at the tip. Thus, in both Arabidopsis roots and Chara rhizoids, the sites of gravity perception/transduction appear to be enriched in integrin-like molecules.

  19. Canine malignant melanoma alpha-3 integrin binding peptides

    PubMed Central

    Aina, Olulanu H.; Maeda, Yoshiko; Harrison, Matthew; Zwingenberger, Allison L.; Walker, Naomi J.; Lam, Kit S.; Kent, Michael S.

    2014-01-01

    There is a need to develop novel targeted imaging and therapeutic agents that can aid in early diagnosis, detection of metastasis and treatment of melanoma. Alpha-3 integrin is overexpressed in 82% of metastatic melanomas in humans and may be a potential target for peptide ligands carrying therapeutic agents. Five melanoma cell lines were generated from canine primary oral and metastatic canine tumors, grown in mice, and validated with melanoma markers Melan A, S-100, Micropthalmia transcription factor (MITF), Tyrosinase, and MART-1. The melanoma cell lines were tested for binding affinity to previously published alpha-3 integrin-binding peptides containing the cdGXGXXc motif. Fluorescent conjugates of the alpha-3 integrin binding OA02 peptide were used to quantify receptor affinity in the cell lines, a specimen of canine primary oral melanoma, and melanoma xenografts. Alpha-3 integrin was expressed by all 5 canine melanoma cell lines. Four of the 5 lines as well as the primary canine tumor showed affinity to alpha-3 integrin binding peptides with the cdGXGXXc motif. Optical imaging of canine melanoma xenografts in nude mice indicates rapid, strong uptake of the optical tracer in the tumor with an average persistence of approximately 48 hours. Ex vivo images showed high tumor-to-background ratio, with tumor signals more than twice that of the kidney and other vital organs. We propose that integrin alpha-3 integrin binding ligands could potentially become useful probes for imaging and delivery of cytotoxic agents for the treatment of melanoma. PMID:21722969

  20. Weeding with transgenes.

    PubMed

    Duke, Stephen O

    2003-05-01

    Transgenes promise to reduce insecticide and fungicide use but relatively little has been done to significantly reduce herbicide use through genetic engineering. Recently, three strategies for transgene utilization have been developed that have the potential to change this. These are the improvement of weed-specific biocontrol agents, enhancement of crop competition or allelopathic traits, and production of cover crops that will self-destruct near the time of planting. Failsafe risk mitigation technologies are needed for most of these strategies.

  1. Focal adhesion kinase in integrin-mediated signaling.

    PubMed

    Cary, L A; Guan, J L

    1999-01-15

    Integrins serve as adhesion receptors for extracellular matrix proteins and also transduce biochemical signals into the cell. These signaling events regulate such cellular processes as proliferation, apoptosis, migration and spreading. Focal adhesion kinase (FAK) is an important protein tyrosine kinase which mediates several integrin signaling pathways. Putative mechanisms of integrin-mediated FAK activation and localization to focal adhesions are discussed here. FAK interacts with a number of signaling and cytoskeletal proteins, including Src, phosphatidylinositol 3-kinase, Grb2, p130Cas and paxillin. Both the mechanisms and outcomes of these interactions are also presented. Finally, FAK's roles in the regulation of several integrin-mediated cellular events are discussed, including the promotion of cell migration, proliferation and spreading, and the prevention of cell apoptosis.

  2. Engineered microenvironments for synergistic VEGF - Integrin signalling during vascularization.

    PubMed

    Moulisová, Vladimíra; Gonzalez-García, Cristina; Cantini, Marco; Rodrigo-Navarro, Aleixandre; Weaver, Jessica; Costell, Mercedes; Sabater I Serra, Roser; Dalby, Matthew J; García, Andrés J; Salmerón-Sánchez, Manuel

    2017-05-01

    We have engineered polymer-based microenvironments that promote vasculogenesis both in vitro and in vivo through synergistic integrin-growth factor receptor signalling. Poly(ethyl acrylate) (PEA) triggers spontaneous organization of fibronectin (FN) into nanonetworks which provide availability of critical binding domains. Importantly, the growth factor binding (FNIII 12-14 ) and integrin binding (FNIII 9-10 ) regions are simultaneously available on FN fibrils assembled on PEA. This material platform promotes synergistic integrin/VEGF signalling which is highly effective for vascularization events in vitro with low concentrations of VEGF. VEGF specifically binds to FN fibrils on PEA compared to control polymers (poly(methyl acrylate), PMA) where FN remains in a globular conformation and integrin/GF binding domains are not simultaneously available. The vasculogenic response of human endothelial cells seeded on these synergistic interfaces (VEGF bound to FN assembled on PEA) was significantly improved compared to soluble administration of VEGF at higher doses. Early onset of VEGF signalling (PLCγ1 phosphorylation) and both integrin and VEGF signalling (ERK1/2 phosphorylation) were increased only when VEGF was bound to FN nanonetworks on PEA, while soluble VEGF did not influence early signalling. Experiments with mutant FN molecules with impaired integrin binding site (FN-RGE) confirmed the role of the integrin binding site of FN on the vasculogenic response via combined integrin/VEGF signalling. In vivo experiments using 3D scaffolds coated with FN and VEGF implanted in the murine fat pad demonstrated pro-vascularization signalling by enhanced formation of new tissue inside scaffold pores. PEA-driven organization of FN promotes efficient presentation of VEGF to promote vascularization in regenerative medicine applications. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  3. Function of the alpha 6 Integrins in Breast Carcinoma

    DTIC Science & Technology

    2001-10-01

    motility on laminin-1. Time-lapse was resistant to solubilization with Triton X-100. Cy- videomicroscopy of clone A cells on laminin-1 revealed...represent SEM. The Integrin a6/34 Participates in the Dynamic Formation of Actin-based Motility Structures: Filopodia lapse videomicroscopy in serum-free...threefold greater time-lapse videomicroscopy to understand how the cu6P34 than on an equivalent concentration of collagen type I, integrin contributes to

  4. Neutrophil integrin affinity regulation in adhesion, migration, and bacterial clearance.

    PubMed

    Langereis, Jeroen D

    2013-01-01

    During an infection, neutrophils are the first immune cells to arrive armed to clear the invading pathogen. In order to do so, neutrophils need to transmigrate from the peripheral blood through the endothelial layer toward the site of inflammation. This process is in most cases dependent on integrins, adhesion molecules present on all immune cells. These molecules are functionally regulated by "inside-out" signaling, where stimulus-induced signaling pathways act on the intracellular integrin tail to regulate the activity of the receptor on the outside. Both a change in conformation (affinity) and clustering (avidity/valency) of the receptors occurs and many factors have been linked to regulation of integrins on neutrophils. Control of integrin conformation and clustering is of pivotal importance for proper cell adhesion, migration, and bacterial clearance. Recently, gelsolin was found to be involved in β 1-integrin affinity regulation and cell adhesion. Here, I summarize the role of neutrophil integrin regulation in the essential steps to reach the site of inflammation and clearance of bacterial pathogens.

  5. Signal co-operation between integrins and other receptor systems.

    PubMed

    Streuli, Charles H; Akhtar, Nasreen

    2009-03-15

    The multicellular nature of metazoans means that all cellular processes need to be tuned by adhesive interactions between cells and their local microenvironment. The spatial organization of cells within tissues requires sophisticated networks of extracellular signals to control their survival and proliferation, movements and positioning, and differentiated function. These cellular characteristics are mediated by multiple inputs from adhesion systems in combination with soluble and developmental signals. In the present review we explore how one class of adhesion receptor, the integrins, co-operate with other types of receptor to control diverse aspects of cell fate. In particular we discuss: (i) how beta3 and beta1 integrins work together with growth factors to control angiogenesis; (ii) how alpha6beta4 integrin co-operates with receptor tyrosine kinases in normal epithelial function and cancer; (iii) the interplay between beta1 integrins and EGF (epidermal growth factor) receptor; (iv) signal integration connecting integrins and cytokine receptors for interleukins, prolactin and interferons; and (v) how integrins and syndecans co-operate in cell migration.

  6. Integrin-targeted cancer immunotherapy elicits protective adaptive immune responses

    PubMed Central

    Zhu, Eric F.; Sugito, Harun R.; Ma, Botong; Delaney, Mary K.; Murphy, Patrick A.; Kauke, Monique J.; Angelini, Alessandro; Momin, Noor; Mehta, Naveen K.; Maragh, Alecia M.; Dranoff, Glenn

    2017-01-01

    Certain RGD-binding integrins are required for cell adhesion, migration, and proliferation and are overexpressed in most tumors, making them attractive therapeutic targets. However, multiple integrin antagonist drug candidates have failed to show efficacy in cancer clinical trials. In this work, we instead exploit these integrins as a target for antibody Fc effector functions in the context of cancer immunotherapy. By combining administration of an engineered mouse serum albumin/IL-2 fusion with an Fc fusion to an integrin-binding peptide (2.5F-Fc), significant survival improvements are achieved in three syngeneic mouse tumor models, including complete responses with protective immunity. Functional integrin antagonism does not contribute significantly to efficacy; rather, this therapy recruits both an innate and adaptive immune response, as deficiencies in either arm result in reduced tumor control. Administration of this integrin-targeted immunotherapy together with an anti–PD-1 antibody further improves responses and predominantly results in cures. Overall, this well-tolerated therapy achieves tumor specificity by redirecting inflammation to a functional target fundamental to tumorigenic processes but expressed at significantly lower levels in healthy tissues, and it shows promise for translation. PMID:28473400

  7. The Integrin Receptor in Biologically Relevant Bilayers: Insights from Molecular Dynamics Simulations.

    PubMed

    Kalli, Antreas C; Rog, Tomasz; Vattulainen, Ilpo; Campbell, Iain D; Sansom, Mark S P

    2017-08-01

    Integrins are heterodimeric (αβ) cell surface receptors that are potential therapeutic targets for a number of diseases. Despite the existence of structural data for all parts of integrins, the structure of the complete integrin receptor is still not available. We have used available structural data to construct a model of the complete integrin receptor in complex with talin F2-F3 domain. It has been shown that the interactions of integrins with their lipid environment are crucial for their function but details of the integrin/lipid interactions remain elusive. In this study an integrin/talin complex was inserted in biologically relevant bilayers that resemble the cell plasma membrane containing zwitterionic and charged phospholipids, cholesterol and sphingolipids to study the dynamics of the integrin receptor and its effect on bilayer structure and dynamics. The results of this study demonstrate the dynamic nature of the integrin receptor and suggest that the presence of the integrin receptor alters the lipid organization between the two leaflets of the bilayer. In particular, our results suggest elevated density of cholesterol and of phosphatidylserine lipids around the integrin/talin complex and a slowing down of lipids in an annulus of ~30 Å around the protein due to interactions between the lipids and the integrin/talin F2-F3 complex. This may in part regulate the interactions of integrins with other related proteins or integrin clustering thus facilitating signal transduction across cell membranes.

  8. Epidermal Expression of Intercellular Adhesion Molecule 1 is Not a Primary Inducer of Cutaneous Inflammation in Transgenic Mice

    NASA Astrophysics Data System (ADS)

    Williams, Ifor R.; Kupper, Thomas S.

    1994-10-01

    Keratinocytes at sites of cutaneous inflammation have increased expression of intercellular adhesion molecule 1 (ICAM-1), a cytokine-inducible adhesion molecule which binds the leukocyte integrins LFA-1 and Mac-1. Transgenic mice were prepared in which the expression of mouse ICAM-1 was targeted to basal keratinocytes by using the human K14 keratin promoter. The level of constitutive expression attained in the transgenic mice exceeded the peak level of ICAM-1 expression induced on nontransgenic mouse keratinocytes in vitro by optimal combinations of interferon γ and tumor necrosis factor α or in vivo by proinflammatory stimuli such as phorbol 12-myristate 13-acetate. In vitro adhesion assays demonstrated that cultured transgenic keratinocytes were superior to normal keratinocytes as a substrate for the LFA-1-dependent binding of mouse T cells, confirming that the transgene-encoded ICAM-1 was expressed in a functional form. However, the high level of constitutive ICAM-1 expression achieved on keratinocytes in vivo in these transgenic mice did not result in additional recruitment of CD45^+ leukocytes into transgenic epidermis, nor did it elicit dermal inflammation. Keratinocyte ICAM-1 expression also did not potentiate contact-hypersensitivity reactions to epicutaneous application of haptens. The absence of a spontaneous phenotype in these transgenic mice was not the result of increased levels of soluble ICAM-1, since serum levels of soluble ICAM-1 were equal in transgenic mice and controls. We conclude that elevated ICAM-1 expression on keratinocytes cannot act independently to influence leukocyte trafficking and elicit cutaneous inflammation.

  9. Canine malignant melanoma alpha-3 integrin binding peptides.

    PubMed

    Aina, Olulanu H; Maeda, Yoshiko; Harrison, Matthew; Zwingenberger, Allison L; Walker, Naomi J; Lam, Kit S; Kent, Michael S

    2011-09-15

    There is a need to develop novel targeted imaging and therapeutic agents that can aid in early diagnosis, detection of metastasis and treatment of melanoma. Alpha-3 integrin is overexpressed in 82% of metastatic melanomas in humans and may be a potential target for peptide ligands carrying therapeutic agents. Five melanoma cell lines were generated from canine primary oral and metastatic canine tumors, grown in mice, and validated with melanoma markers Melan A, S-100, Micropthalmia transcription factor (MITF), Tyrosinase, and MART-1. The melanoma cell lines were tested for binding affinity to previously published alpha-3 integrin-binding peptides containing the cdGXGXXc motif. Fluorescent conjugates of the alpha-3 integrin binding OA02 peptide were used to quantify receptor affinity in the cell lines, a specimen of canine primary oral melanoma, and melanoma xenografts. Alpha-3 integrin was expressed by all 5 canine melanoma cell lines. Four of the 5 lines as well as the primary canine tumor showed affinity to alpha-3 integrin binding peptides with the cdGXGXXc motif. Optical imaging of canine melanoma xenografts in nude mice indicates rapid, strong uptake of the optical tracer in the tumor with an average persistence of approximately 48 h. Ex vivo images showed high tumor-to-background ratio, with tumor signals more than twice that of the kidney and other vital organs. We propose that integrin alpha-3 integrin binding ligands could potentially become useful probes for imaging and delivery of cytotoxic agents for the treatment of melanoma. Copyright © 2011 Elsevier B.V. All rights reserved.

  10. Transgenic Crops for Herbicide Resistance

    USDA-ARS?s Scientific Manuscript database

    Since their introduction in 1995, crops made resistant to the broad-spectrum herbicides glyphosate and glufosinate with transgenes are widely available and used in much of the world. As of 2008, over 80% of the transgenic crops grown world-wide have this transgenic trait. This technology has had m...

  11. Transgenic animal technology.

    PubMed

    Wall, R J; Hyman, P; Kerr, D; Pintado, B; Wells, K

    1997-01-01

    There are numerous tools available to modify the genetic makeup of animals. They are being used to good advantage for studying basic biological phenomena. Within the decade, biomedical products derived from transgenic animals will be available, but the use of this technology for enhancing the quality and efficiency of livestock production will await further refinements in the technology.

  12. [Progress on transgenic mosquitoes].

    PubMed

    Yang, Pin

    2011-04-30

    The genetically modified mosquitoes have been developed aiming to control mosquito-borne diseases by either reducing population sizes or replacing existing populations with vectors unable to transmit the disease. introduces some progress on the generation of transgenic mosquitoes and their fitness in wild population. This paper

  13. Apoptosis of adherent cells by recruitment of caspase-8 to unligated integrins

    PubMed Central

    Stupack, Dwayne G.; Puente, Xose S.; Boutsaboualoy, Souphaphone; Storgard, Chris M.; Cheresh, David A.

    2001-01-01

    Integrin-mediated adhesion promotes cell survival in vitro, whereas integrin antagonists induce apoptosis of adherent cells in vivo. Here, we demonstrate that cells adherent within a three-dimensional extracellular matrix undergo apoptosis due to expression of unligated integrins, the β subunit cytoplasmic domain, or its membrane proximal sequence KLLITIHDRKEF. Integrin-mediated death requires initiator, but not stress, caspase activity and is distinct from anoikis, which is caused by the loss of adhesion per se. Surprisingly, unligated integrin or β integrin tails recruit caspase-8 to the membrane, where it becomes activated in a death receptor–independent manner. Integrin ligation disrupts this integrin–caspase containing complex and increases survival, revealing an unexpected role for integrins in the regulation of apoptosis and tissue remodeling. PMID:11684710

  14. Pro32Pro33 Mutations in the Integrin β3 PSI Domain Result in αIIbβ3 Priming and Enhanced Adhesion: Reversal of the Hypercoagulability Phenotype by the Src Inhibitor SKI-606

    PubMed Central

    Oliver, Kendra H.; Jessen, Tammy; Crawford, Emily L.; Chung, Chang Y.; Sutcliffe, James S.

    2014-01-01

    The plasma-membrane integrin αIIbβ3 (CD41/CD61, GPIIbIIIa) is a major functional receptor in platelets during clotting. A common isoform of integrin β3, Leu33Pro is associated with enhanced platelet function and increased risk for coronary thrombosis and stroke, although these findings remain controversial. To better understand the molecular mechanisms by which this sequence variation modifies platelet function, we produced transgenic knockin mice expressing a Pro32Pro33 integrin β3. Consistent with reports utilizing human platelets, we found significantly reduced bleeding and clotting times, as well as increased in vivo thrombosis, in Pro32Pro33 homozygous mice. These alterations paralleled increases in platelet attachment and spreading onto fibrinogen resulting from enhanced integrin αIIbβ3 function. Activation with protease-activated receptor 4– activating peptide, the main thrombin signaling receptor in mice, showed no significant difference in activation of Pro32Pro33 mice as compared with controls, suggesting that inside-out signaling remains intact. However, under unstimulated conditions, the Pro32Pro33 mutation led to elevated Src phosphorylation, facilitated by increased talin interactions with the β3 cytoplasmic domain, indicating that the αIIbβ3 intracellular domains are primed for activation while the ligand-binding domain remains unchanged. Acute dosing of animals with a Src inhibitor was sufficient to rescue the clotting phenotype in knockin mice to wild-type levels. Together, our data establish that the Pro32Pro33 structural alteration modifies the function of integrin αIIbβ3, priming the integrin for outside-in signaling, ultimately leading to hypercoagulability. Furthermore, our data may support a novel approach to antiplatelet therapy by Src inhibition where hemostasis is maintained while reducing risk for cardiovascular disease. PMID:24695082

  15. The therapeutic potential of I-domain integrins.

    PubMed

    Brennan, Marian; Cox, Dermot

    2014-01-01

    Due to their role in processes central to cancer and autoimmune disease I-domain integrins are an attractive drug target. Both antibodies and small molecule antagonists have been discovered and tested in the clinic. Much of the effort has focused on αLβ2 antagonists. Maybe the most successful was the monoclonal antibody efalizumab, which was approved for the treatment of psoriasis but subsequently withdrawn from the market due to the occurrence of a serious adverse effect (progressive multifocal leukoencephalopathy). Other monoclonal antibodies were tested for the treatment of reperfusion injury, post-myocardial infarction, but failed to progress due to lack of efficacy. New potent small molecule inhibitors of αv integrins are promising reagents for treating fibrotic disease. Small molecule inhibitors targeting collagen-binding integrins have been discovered and future work will focus on identifying molecules selectively targeting each of the collagen receptors and identifying appropriate target diseases for future clinical studies.

  16. Role of Integrin in Mechanical Loading of Osteoblasts

    NASA Technical Reports Server (NTRS)

    Globus, Ruth; Demsky, Caroline

    2000-01-01

    Mechanical forces generated by gravity, weightbearing, and muscle contraction play a key role in the genesis and maintenance of skeletal structure. The molecular mechanisms that mediate changes in osteoblast activity in response to altered patterns of skeletal loading are not known, and a better understanding of these processes may be essential for developing effective treatment strategies to prevent disuse osteoporosis. We have elucidated specific integrin/ECM (extracellular matrix) interactions that are required for osteoblast differentiation and survival and have developed a useful loading system to further explore the molecular basis of mechano-sensitivity of osteoblasts. The long term goal of our collaborative research is to understand how the ECM and cell adhesion proteins and integrins interaction to mediate the response of osteoblasts and their progenitors to mechanical loading. We suggest that integrin/ECM interactions are crucial for basic cellular processes, including differentiation and survival, as well as to participate in detecting and mediating cellular responses to mechanical stimuli.

  17. Active and inactive β1 integrins segregate into distinct nanoclusters in focal adhesions.

    PubMed

    Spiess, Matthias; Hernandez-Varas, Pablo; Oddone, Anna; Olofsson, Helene; Blom, Hans; Waithe, Dominic; Lock, John G; Lakadamyali, Melike; Strömblad, Staffan

    2018-04-09

    Integrins are the core constituents of cell-matrix adhesion complexes such as focal adhesions (FAs) and play key roles in physiology and disease. Integrins fluctuate between active and inactive conformations, yet whether the activity state influences the spatial organization of integrins within FAs has remained unclear. In this study, we address this question and also ask whether integrin activity may be regulated either independently for each integrin molecule or through locally coordinated mechanisms. We used two distinct superresolution microscopy techniques, stochastic optical reconstruction microscopy (STORM) and stimulated emission depletion microscopy (STED), to visualize active versus inactive β1 integrins. We first reveal a spatial hierarchy of integrin organization with integrin molecules arranged in nanoclusters, which align to form linear substructures that in turn build FAs. Remarkably, within FAs, active and inactive β1 integrins segregate into distinct nanoclusters, with active integrin nanoclusters being more organized. This unexpected segregation indicates synchronization of integrin activities within nanoclusters, implying the existence of a coordinate mechanism of integrin activity regulation. © 2018 Spiess et al.

  18. The Talin Head Domain Reinforces Integrin-Mediated Adhesion by Promoting Adhesion Complex Stability and Clustering

    PubMed Central

    Ellis, Stephanie J.; Lostchuck, Emily; Goult, Benjamin T.; Bouaouina, Mohamed; Fairchild, Michael J.; López-Ceballos, Pablo; Calderwood, David A.; Tanentzapf, Guy

    2014-01-01

    Talin serves an essential function during integrin-mediated adhesion in linking integrins to actin via the intracellular adhesion complex. In addition, the N-terminal head domain of talin regulates the affinity of integrins for their ECM-ligands, a process known as inside-out activation. We previously showed that in Drosophila, mutating the integrin binding site in the talin head domain resulted in weakened adhesion to the ECM. Intriguingly, subsequent studies showed that canonical inside-out activation of integrin might not take place in flies. Consistent with this, a mutation in talin that specifically blocks its ability to activate mammalian integrins does not significantly impinge on talin function during fly development. Here, we describe results suggesting that the talin head domain reinforces and stabilizes the integrin adhesion complex by promoting integrin clustering distinct from its ability to support inside-out activation. Specifically, we show that an allele of talin containing a mutation that disrupts intramolecular interactions within the talin head attenuates the assembly and reinforcement of the integrin adhesion complex. Importantly, we provide evidence that this mutation blocks integrin clustering in vivo. We propose that the talin head domain is essential for regulating integrin avidity in Drosophila and that this is crucial for integrin-mediated adhesion during animal development. PMID:25393120

  19. Focal adhesion disassembly requires clathrin-dependent endocytosis of integrins

    PubMed Central

    Chao, Wei-Ting; Kunz, Jeannette

    2009-01-01

    Cell migration requires the controlled disassembly of focal adhesions, but the underlying mechanisms remain poorly understood. Here, we show that adhesion turnover is mediated through dynamin- and clathrin-dependent endocytosis of activated β1 integrins. Consistent with this, clathrin and the clathrin adaptors AP-2 and disabled-2 (DAB2) distribute along with dynamin 2 to adhesion sites prior to adhesion disassembly. Moreover, knockdown of either dynamin 2 or both clathrin adaptors blocks β1 integrin internalization, leading to impaired focal adhesion disassembly and cell migration. Together, these results provide important insight into the mechanisms underlying adhesion disassembly and identify novel components of the disassembly pathway. PMID:19306879

  20. An integrin from shrimp Litopenaeus vannamei mediated microbial agglutination and cell proliferation.

    PubMed

    Zhang, Ying; Wang, Leilei; Wang, Lingling; Wu, Ning; Zhou, Zhi; Song, Linsheng

    2012-01-01

    Integrins are a family of adhesion receptors which regulate cell proliferation, differentiation, leukocyte migration, and complement receptor-dependent phagocytosis. In invertebrates, as a cell adhesion receptor, β integrins play an important role for the balanced activation of immune defense responses especially during the encounter of infections. The present study attempts to characterize the immune functions of shrimp integrin (LvIntegrin) to have better understanding on the immune system and its regulation mechanisms in shrimps. A shrimp integrin was identified from the Pacific white shrimp Litopenaeus vannamei (designated as LvIntegrin). Its full-length cDNA was of 2621 bp with an open reading frame (ORF) of 2439 bp encoding a polypeptide of 812 amino acids. The mRNA expression of LvIntegrin was significantly up-regulated at 3, 6 and 12 h after Listonella anguillarum challenge. The cDNA fragment encoding β integrin domains (βA and hybrid domain) of LvIntegrin was recombined and expressed in Escherichia coli BL21(DE3)-pLysS. The recombinant protein (rLvIntegrin) could significantly agglutinate the tested microbe including E. coli JM109, L. anguillarum, Micrococcus luteus and Candida dattiladattila in the presence of divalent cations. Moreover, when NIH3T3 cells were cultured with rLvIntegrin, the proliferation rate increased significantly in a dose-dependent manner. LvIntegrin, a shrimp β integrin was identified from L. vannamei, shared several highly conserved features. LvIntegrin exhibited broad-spectrum agglutination activity towards both bacteria and fungi and could improve the proliferation of NIH3T3 cells, indicating that LvIntegrin is involved in the immune response against microbe challenge and regulation of cell proliferation as a cell adhesion receptor in shrimp.

  1. An Integrin from Shrimp Litopenaeus vannamei Mediated Microbial Agglutination and Cell Proliferation

    PubMed Central

    Zhang, Ying; Wang, Leilei; Wang, Lingling; Wu, Ning; Zhou, Zhi; Song, Linsheng

    2012-01-01

    Background Integrins are a family of adhesion receptors which regulate cell proliferation, differentiation, leukocyte migration, and complement receptor-dependent phagocytosis. In invertebrates, as a cell adhesion receptor, β integrins play an important role for the balanced activation of immune defense responses especially during the encounter of infections. The present study attempts to characterize the immune functions of shrimp integrin (LvIntegrin) to have better understanding on the immune system and its regulation mechanisms in shrimps. Methodology A shrimp integrin was identified from the Pacific white shrimp Litopenaeus vannamei (designated as LvIntegrin). Its full-length cDNA was of 2621 bp with an open reading frame (ORF) of 2439 bp encoding a polypeptide of 812 amino acids. The mRNA expression of LvIntegrin was significantly up-regulated at 3, 6 and 12 h after Listonella anguillarum challenge. The cDNA fragment encoding β integrin domains (βA and hybrid domain) of LvIntegrin was recombined and expressed in Escherichia coli BL21(DE3)-pLysS. The recombinant protein (rLvIntegrin) could significantly agglutinate the tested microbe including E. coli JM109, L. anguillarum, Micrococcus luteus and Candida dattiladattila in the presence of divalent cations. Moreover, when NIH3T3 cells were cultured with rLvIntegrin, the proliferation rate increased significantly in a dose-dependent manner. Conclusions LvIntegrin, a shrimp β integrin was identified from L. vannamei, shared several highly conserved features. LvIntegrin exhibited broad-spectrum agglutination activity towards both bacteria and fungi and could improve the proliferation of NIH3T3 cells, indicating that LvIntegrin is involved in the immune response against microbe challenge and regulation of cell proliferation as a cell adhesion receptor in shrimp. PMID:22792387

  2. α6β4 Integrin Regulates the Collective Migration of Epithelial Cells.

    PubMed

    Colburn, Zachary T; Jones, Jonathan C R

    2017-04-01

    α 6 β 4 integrin is localized in a unique punctate distribution at the cell-substratum interface along the leading front of single, front-rear-polarized A549 cells. These puncta are interspersed between focal adhesions and lack association with the actin cytoskeleton. Knockdown of β 4 integrin in A549 cells inhibits their directed migration, with knockdown cells exhibiting large focal adhesions and reduced actin dynamics. Despite these changes, the speed of knockdown cells is equivalent to control cells. Interestingly, in such cells, α 6 integrin retains its punctate distribution. Moreover, in β 4 integrin knockdown cells, we observe a loss of β 1 integrin from focal adhesions and an enhanced association with α 6 integrin. We confirmed the switch in the β integrin binding partner of α 6 integrin in the knockdown cells by immunoprecipitation. We next investigated the role of β 4 integrin in collective cell migration. Wounded monolayers of β 4 integrin knockdown cells exhibit reduced collective migration compared with controls. When we forced expression of β 4 integrin in the leader cells of wounded monolayers, collective migration was restored. Similarly, forced expression of β 4 integrin in primary rat alveolar epithelial cells also promotes collective cell migration. In addition, we interrogated the pathway by which β 4 integrin regulates A549 cell-directed migration. Constitutively active Ras-related C3 botulinum toxin substrate 1 rescues motility defects resulting from β 4 integrin deficiency. Together, our results support the hypothesis that α 6 β 4 integrin is a positive regulator of collective cell migration of A549 cells through influence on signal pathways in leader cells.

  3. Transgenics in crops

    NASA Technical Reports Server (NTRS)

    Li, Y.; Wu, Y. H.; McAvoy, R.; Duan, H.

    2001-01-01

    With rapid world population growth and declining availability of fresh water and arable land, a new technology is urgently needed to enhance agricultural productivity. Recent discoveries in the field of crop transgenics clearly demonstrate the great potential of this technology for increasing food production and improving food quality while preserving the environment for future generations. In this review, we briefly discuss some of the recent achievements in crop improvement that have been made using gene transfer technology.

  4. Exosomes derived from human macrophages suppress endothelial cell migration by controlling integrin trafficking.

    PubMed

    Lee, Hee Doo; Kim, Yeon Hyang; Kim, Doo-Sik

    2014-04-01

    Integrin trafficking, including internalization, recycling, and lysosomal degradation, is crucial for the regulation of cellular functions. Exosomes, nano-sized extracellular vesicles, are believed to play important roles in intercellular communications. This study demonstrates that exosomes released from human macrophages negatively regulate endothelial cell migration through control of integrin trafficking. Macrophage-derived exosomes promote internalization of integrin β1 in primary HUVECs. The internalized integrin β1 persistently accumulates in the perinuclear region and is not recycled back to the plasma membrane. Experimental results indicate that macrophage-derived exosomes stimulate trafficking of internalized integrin β1 to lysosomal compartments with a corresponding decrease in the integrin destined for recycling endosomes, resulting in proteolytic degradation of the integrin. Moreover, ubiquitination of HUVEC integrin β1 is enhanced by the exosomes, and exosome-mediated integrin degradation is blocked by bafilomycin A, a lysosomal degradation inhibitor. Macrophage-derived exosomes were also shown to effectively suppress collagen-induced activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway and HUVEC migration, which are both dependent on integrin β1. These observations provide new insight into the functional significance of exosomes in the regulation of integrin trafficking. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Integrin-Mediated Cell-Matrix Interaction in Physiological and Pathological Blood Vessel Formation

    PubMed Central

    Niland, Stephan; Eble, Johannes A.

    2012-01-01

    Physiological as well as pathological blood vessel formation are fundamentally dependent on cell-matrix interaction. Integrins, a family of major cell adhesion receptors, play a pivotal role in development, maintenance, and remodeling of the vasculature. Cell migration, invasion, and remodeling of the extracellular matrix (ECM) are integrin-regulated processes, and the expression of certain integrins also correlates with tumor progression. Recent advances in the understanding of how integrins are involved in the regulation of blood vessel formation and remodeling during tumor progression are highlighted. The increasing knowledge of integrin function at the molecular level, together with the growing repertoire of integrin inhibitors which allow their selective pharmacological manipulation, makes integrins suited as potential diagnostic markers and therapeutic targets. PMID:21941547

  6. Functional Analysis of Alpha-6 Integrin Cytoplasinic Domains.

    DTIC Science & Technology

    1997-12-01

    directly with cytoskeletal components, e.g., talin (Horwitz et al., 1986; Knezevic et al., 1996). They may also be involved in regulating signaling (Guan...integrin chain in vitro. Biochem. Biophys. Res. Commun. 217, A66-A1A. Knezevic , I., Leisner, T.M., and Lam, S.C.T. (1996). Direct binding of the platelet

  7. Rab25 regulates integrin expression in polarized colonic epithelial cells

    PubMed Central

    Krishnan, Moorthy; Lapierre, Lynne A.; Knowles, Byron C.; Goldenring, James R.

    2013-01-01

    Rab25 is a tumor suppressor for colon cancer in humans and mice. To identify elements of intestinal polarity regulated by Rab25, we developed Caco2-BBE cell lines stably expressing short hairpin RNA for Rab25 and lines rescuing Rab25 knockdown with reexpression of rabbit Rab25. Rab25 knockdown decreased α2-, α5-, and β1-integrin expression. We observed colocalization and direct association of Rab25 with α5β1-integrins. Rab25 knockdown also up-regulated claudin-1 expression, increased transepithelial resistance, and increased invasive behavior. Rab25-knockdown cells showed disorganized brush border microvilli with decreases in villin expression. All of these changes were reversed by reintroduction of rabbit Rab25. Rab25 knockdown altered the expression of 29 gene transcripts, including the loss of α5-integrin transcripts. Rab25 loss decreased expression of one transcription factor, ETV4, and overexpression of ETV4 in Rab25-knockdown cells reversed losses of α5β1-integrin. The results suggest that Rab25 controls intestinal cell polarity through the regulation of gene expression. PMID:23345591

  8. Syndecan-4 phosphorylation is a control point for integrin recycling.

    PubMed

    Morgan, Mark R; Hamidi, Hellyeh; Bass, Mark D; Warwood, Stacey; Ballestrem, Christoph; Humphries, Martin J

    2013-03-11

    Precise spatiotemporal coordination of integrin adhesion complex dynamics is essential for efficient cell migration. For cells adherent to fibronectin, differential engagement of α5β1 and αVβ3 integrins is used to elicit changes in adhesion complex stability, mechanosensation, matrix assembly, and migration, but the mechanisms responsible for receptor regulation have remained largely obscure. We identify phosphorylation of the membrane-intercalated proteoglycan syndecan-4 as an essential switch controlling integrin recycling. Src phosphorylates syndecan-4 and, by driving syntenin binding, leads to suppression of Arf6 activity and recycling of αVβ3 to the plasma membrane at the expense of α5β1. The resultant elevation in αVβ3 engagement promotes stabilization of focal adhesions. Conversely, abrogation of syndecan-4 phosphorylation drives surface expression of α5β1, destabilizes adhesion complexes, and disrupts cell migration. These data identify the dynamic spatiotemporal regulation of Src-mediated syndecan-4 phosphorylation as an essential switch controlling integrin trafficking and adhesion dynamics to promote efficient cell migration. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. Syndecan-4 Phosphorylation Is a Control Point for Integrin Recycling

    PubMed Central

    Morgan, Mark R.; Hamidi, Hellyeh; Bass, Mark D.; Warwood, Stacey; Ballestrem, Christoph; Humphries, Martin J.

    2013-01-01

    Summary Precise spatiotemporal coordination of integrin adhesion complex dynamics is essential for efficient cell migration. For cells adherent to fibronectin, differential engagement of α5β1 and αVβ3 integrins is used to elicit changes in adhesion complex stability, mechanosensation, matrix assembly, and migration, but the mechanisms responsible for receptor regulation have remained largely obscure. We identify phosphorylation of the membrane-intercalated proteoglycan syndecan-4 as an essential switch controlling integrin recycling. Src phosphorylates syndecan-4 and, by driving syntenin binding, leads to suppression of Arf6 activity and recycling of αVβ3 to the plasma membrane at the expense of α5β1. The resultant elevation in αVβ3 engagement promotes stabilization of focal adhesions. Conversely, abrogation of syndecan-4 phosphorylation drives surface expression of α5β1, destabilizes adhesion complexes, and disrupts cell migration. These data identify the dynamic spatiotemporal regulation of Src-mediated syndecan-4 phosphorylation as an essential switch controlling integrin trafficking and adhesion dynamics to promote efficient cell migration. PMID:23453597

  10. Developmental control of integrin expression regulates Th2 effector homing

    USDA-ARS?s Scientific Manuscript database

    Integrin CD18, a component of the LFA-1 complex that also includes CD11a, is essential for Th2, but not Th1, cell homing, but the explanation for this phenomenon remains obscure. In this study, we investigate the mechanism by which Th2 effector responses require the LFA-1 complex. CD11a-deficient T ...

  11. Dystroglycan receptor is involved in integrin activation in intestinal epithelia

    PubMed Central

    Driss, Adel; Charrier, Laetitia; Yan, Yutao; Nduati, Vivienne; Sitaraman, Shanthi; Merlin, Didier

    2009-01-01

    The dystroglycans (α-DG and β-DG), which play important roles in the formation of basement membranes, have been well studied in skeletal muscle and nerve, but their expression and localization in intestinal epithelial cells has not been previously investigated. Here, we demonstrated that the DG complex, composed of α-DG, β-DG, and utrophin, is specifically expressed in the basolateral membrane of the Caco-2-BBE monolayer. The DG complex coprecipitated with β1-integrin, suggesting a possible interaction among these proteins. In addition, we observed that activation of DG receptors by laminin-1 enhanced the interaction between β1-integrin and laminin-1, whereas activation of DG receptors by laminin-2 reduced the interaction between β1-integrin and laminin-2. Finally, we demonstrated that the intracellular COOH-terminal tail of β-DG and its binding to the DG binding domain of utrophin are crucial for the interactions between laminin-1/-2 and β1-integrin. Collectively, these novel results indicate that dystroglycans play important roles in the regulation of interactions between intestinal epithelial cells and the extracellular matrix. PMID:16357060

  12. Multimodality Imaging of Integrin αvβ3 Expression

    PubMed Central

    Zhang, Yin; Yang, Yunan; Cai, Weibo

    2011-01-01

    Over the last decade, integrin αvβ3 has been studied with every single molecular imaging modality. Since no single modality is perfect and sufficient to obtain all the necessary information for a particular question, combination of certain molecular imaging modalities can offer synergistic advantages over any modality alone. This review will focus on multimodality imaging of integrin αvβ3 expression, where the contrast agent used can be detected by two or more imaging modalities, such as combinations of PET and optical, SPECT and fluorescence, PET and MRI, SPECT and MRI, and lastly, MRI and fluorescence. Most of these agents are based on certain type(s) of nanoparticles. Contrast agents that can be detected by more than two imaging modalities are expected to emerge in the future and a PET/MRI/fluorescence agent will likely find the most future biomedical/clinical applications. Big strides have been made over the last decade for imaging integrin αvβ3 expression and several PET/SPECT probes have been tested in human studies. For dualmodality and multimodality imaging applications, a number of proof-of-principle studies have been reported which opened up many new avenues for future research. The next decade will likely witness further growth and continued prosperity of molecular imaging studies focusing on integrin αvβ3, which can eventually impact patient management. PMID:21547156

  13. Expression and Promoter Analysis of a Highly Restricted Integrin Alpha Gene in Vascular Smooth Muscle

    PubMed Central

    Kitchen, Chad M.; Cowan, Sarah L.; Long, Xiaochun; Miano, Joseph M.

    2012-01-01

    Full genome annotation requires gene expression analysis and elucidation of promoter activity. Here, we analyzed the expression and promoter of a highly restricted integrin gene, Itga8. RNase protection and quantitative RT-PCR showed Itga8 to be expressed most abundantly in vascular smooth muscle cells (SMC). Transcription start site mapping of Itga8 revealed the immediate 5' promoter region to be poorly conserved with orthologous sequences in the human genome. Further comparative sequence analysis showed a number of conserved non-coding sequence modules around the Itga8 gene. The immediate promoter region and an upstream conserved sequence module were each found to contain a CArG box, which is a binding site for serum response factor (SRF). Luciferase reporter assays revealed activity of several Itga8 promoter constructs with no apparent restricted activity to SMC types. Further, neither SRF nor its coactivator, Myocardin (MYOCD), was able to induce several distinct Itga8 promoter constructs. Transgenic mouse studies failed to reveal Itga8 promoter activity indicating distal regulatory elements likely control this gene's in vivo expression profile. Interestingly, although the promoter was unresponsive to SRF/MYOCD, the endogenous Itga8 gene showed increases in expression upon ectopic MYOCD expression even though knockdown of SRF both in vitro and in vivo failed to demonstrate a corresponding change in Itga8. Collectively, these data demonstrate that Itga8 expression is CArGSRF independent, but MYOCD dependent through an as yet unknown sequence module that is distal from the promoter region. PMID:23142384

  14. Transgenic algae engineered for higher performance

    DOEpatents

    Unkefer, Pat J; Anderson, Penelope S; Knight, Thomas J

    2014-10-21

    The present disclosure relates to transgenic algae having increased growth characteristics, and methods of increasing growth characteristics of algae. In particular, the disclosure relates to transgenic algae comprising a glutamine phenylpyruvate transaminase transgene and to transgenic algae comprising a glutamine phenylpyruvate transaminase transgene and a glutamine synthetase.

  15. Evidence That Integrin αIIbβ3-dependent Interaction of Mast Cells with Fibrinogen Exacerbates Chronic Inflammation*

    PubMed Central

    Oki, Toshihiko; Eto, Koji; Izawa, Kumi; Yamanishi, Yoshinori; Inagaki, Naoki; Frampton, Jon; Kitamura, Toshio; Kitaura, Jiro

    2009-01-01

    Integrin αIIbβ3 is expressed in mast cells as well as in megakaryocytes/platelets. A recent study has shown that surface expression levels of integrin αVβ3 are elevated in integrin αIIb-deficient bone marrow-derived mast cells (BMMCs) as compared with wild-type (WT) counterparts, but the underlying mechanism remains obscure. Here we demonstrate by transducing integrin αIIb into integrin αIIb-deficient BMMCs that surface expression levels of integrin αVβ3 are inversely related to those of integrin αIIbβ3. Thus, competitive association of integrin β3 with integrin αIIb or integrin αV determines surface expression levels of integrin αIIbβ3 or αVβ3 in mast cells. We compared WT and integrin αIIb-deficient BMMCs as well as integrin αIIb-deficient BMMCs transduced with integrin αIIb(WT) or non-functional αIIb(D163A) mutant and found that enhancement of proliferation, degranulation, cytokine production, and migration of BMMCs through interaction with fibrinogen (FB) depended on integrin αIIbβ3. In addition, elevated surface expression of integrin αVβ3 failed to compensate for loss of FB-associated functions in integrin αIIb-deficient BMMCs while enhancing adhesion to vitronectin or von Willebrand factor. Importantly, integrin αIIb deficiency strongly suppressed chronic inflammation with the remarkable increase of mast cells induced by continuous intraperitoneal administration of FB, although it did not affect acute allergic responses or mast cell numbers in tissues in steady states. Interestingly, soluble FB promoted cytokine production of BMMCs in response to Staphylococcus aureus with FB-binding capacity, through integrin αIIbβ3-dependent recognition of this pathogen. Collectively, integrin αIIbβ3 in mast cells plays an important part in FB-associated, chronic inflammation and innate immune responses. PMID:19755424

  16. Lymphocyte integrin expression differences between SIRS and sepsis patients.

    PubMed

    Heffernan, D S; Monaghan, S F; Ayala, Alfred

    2017-11-01

    Systemic Inflammatory Response Syndrome (SIRS) and sepsis remain leading causes of death. Despite many similarities, the two entities are very distinct clinically and immunologically. T-Lymphocytes play a key pivotal role in the pathogenesis and ultimately outcome following both SIRS and sepsis. Integrins are essential in the trafficking and migration of lymphocytes. They also serve vital roles in efficient wound healing and clearance of infections. Here, we investigate whether integrin expression, specifically β1 (CD29) and β2 (CD18), are disrupted in SIRS and sepsis, and assess differences in integrin expression between these two critically ill clinical categories. T-Lymphocytes were isolated from whole blood collected from ICU patients exhibiting SIRS or sepsis. Samples were analyzed for CD18 (β2) and CD29 (β1) on CD3 + T cells through flow cytometry. Septic patients were stratified into either exclusively abdominal or non-abdominal sources of sepsis. CD18 was almost ubiquitously expressed on CD3 + T cells irrespective of clinical condition. However, CD29 (β1 integrin) was lowest in SIRS patients (20.4% of CD3 + T cells) when compared with either septic patients (35.5%) or healthy volunteers (54.1%). Furthermore, there was evidence of compartmentalization in septic patients, where abdominal sources had a greater percentage of CD3 + CD29 + T cells (41.7%) when compared with those with non-abdominal sources (29.5%). Distinct differences in T-cell integrin expression exists between patients in SIRS versus sepsis, as well as relative to the source of sepsis. Further work is needed to understand cause and effect relative to the progression from SIRS into sepsis.

  17. Redox regulation of ephrin/integrin cross-talk.

    PubMed

    Buricchi, Francesca; Giannoni, Elisa; Grimaldi, Giovanna; Parri, Matteo; Raugei, Giovanni; Ramponi, Giampietro; Chiarugi, Paola

    2007-01-01

    Interactions linking the Eph receptor tyrosine kinase and ephrin ligands transduce short-range repulsive signals regulating several motile biological processes including axon path-finding, angiogenesis and tumor growth. These ephrin-induced effects are believed to be mediated by alterations in actin dynamics and cytoskeleton reorganization. The members of the small Rho GTPase family elicit various effects on actin structures and are probably involved in Eph receptor-induced actin modulation. In particular, some ephrin ligands lead to a decrease in integrin-mediated cell adhesion and spread. Here we show that the ability of ephrinA1 to inhibit cell adhesion and spreading in prostatic carcinoma cells is strictly dependent on the decrease in the activity of the small GTPase Rac1. Given the recognized role of Rac-driven redox signaling for integrin function, reported to play an essential role in focal adhesion formation and in the overall organization of actin cytoskeleton, we investigated the possible involvement of oxidants in ephrinA1/EphA2 signaling. We now provide evidence that Reactive Oxygen Species are an integration point of the ephrinA1/integrin interplay. We identify redox circuitry in which the ephrinA1-mediated inhibition of Rac1 leads to a negative regulation of integrin redox signaling affecting the activity of the tyrosine phosphatase LMW-PTP. The enzyme in turn actively dephosphorylates its substrate p190RhoGAP, finally leading to RhoA activation. Altogether our data suggest a redox-based Rac-dependent upregulation of Rho activity, concurring with the inhibitory effect elicited by ephrinA1 on integrin-mediated adhesion strength.

  18. Redox Regulation of Ephrin/Integrin Cross-Talk

    PubMed Central

    Buricchi, Francesca; Giannoni, Elisa; Grimaldi, Giovanna; Parri, Matteo; Raugei, Giovanni; Ramponi, Giampietro

    2007-01-01

    Interactions linking the Eph receptor tyrosine kinase and ephrin ligands transduce short-range repulsive signals regulating several motile biological processes including axon path-finding, angiogenesis and tumor growth. These ephrin-induced effects are believed to be mediated by alterations in actin dynamics and cytoskeleton reorganization. The members of the small Rho GTPase family elicit various effects on actin structures and are probably involved in Eph receptor-induced actin modulation. In particular, some ephrin ligands lead to a decrease in integrin-mediated cell adhesion and spread. Here we show that the ability of ephrinA1 to inhibit cell adhesion and spreading in prostatic carcinoma cells is strictly dependent on the decrease in the activity of the small GTPase Rac1. Given the recognized role of Rac-driven redox signaling for integrin function, reported to play an essential role in focal adhesion formation and in the overall organization of actin cytoskeleton, we investigated the possible involvement of oxidants in ephrinA1/EphA2 signaling. We now provide evidence that Reactive Oxygen Species are an integration point of the ephrinA1/integrin interplay. We identify redox circuitry in which the ephrinA1-mediated inhibition of Rac1 leads to a negative regulation of integrin redox signaling affecting the activity of the tyrosine phosphatase LMW-PTP. The enzyme in turn actively dephosphorylates its substrate p190RhoGAP, finally leading to RhoA activation. Altogether our data suggest a redox-based Rac-dependent upregulation of Rho activity, concurring with the inhibitory effect elicited by ephrinA1 on integrin-mediated adhesion strength. PMID:19262085

  19. Plant biotechnology: transgenic crops.

    PubMed

    Shewry, Peter R; Jones, Huw D; Halford, Nigel G

    2008-01-01

    Transgenesis is an important adjunct to classical plant breeding, in that it allows the targeted manipulation of specific characters using genes from a range of sources. The current status of crop transformation is reviewed, including methods of gene transfer, the selection of transformed plants and control of transgene expression. The application of genetic modification technology to specific traits is then discussed, including input traits relating to crop production (herbicide tolerance and resistance to insects, pathogens and abiotic stresses) and output traits relating to the composition and quality of the harvested organs. The latter include improving the nutritional quality for consumers as well as the improvement of functional properties for food processing.

  20. Titin-Based Nanoparticle Tension Sensors Map High-Magnitude Integrin Forces within Focal Adhesions.

    PubMed

    Galior, Kornelia; Liu, Yang; Yehl, Kevin; Vivek, Skanda; Salaita, Khalid

    2016-01-13

    Mechanical forces transmitted through integrin transmembrane receptors play important roles in a variety of cellular processes ranging from cell development to tumorigenesis. Despite the importance of mechanics in integrin function, the magnitude of integrin forces within adhesions remains unclear. Literature suggests a range from 1 to 50 pN, but the upper limit of integrin forces remains unknown. Herein we challenge integrins with the most mechanically stable molecular tension probe, which is comprised of the immunoglobulin 27th (I27) domain of cardiac titin flanked with a fluorophore and gold nanoparticle. Cell experiments show that integrin forces unfold the I27 domain, suggesting that integrin forces exceed ∼30-40 pN. The addition of a disulfide bridge within I27 "clamps" the probe and resists mechanical unfolding. Importantly, incubation with a reducing agent initiates SH exchange, thus unclamping I27 at a rate that is dependent on the applied force. By recording the rate of S-S reduction in clamped I27, we infer that integrins apply 110 ± 9 pN within focal adhesions of rat embryonic fibroblasts. The rates of S-S exchange are heterogeneous and integrin subtype-dependent. Nanoparticle titin tension sensors along with kinetic analysis of unfolding demonstrate that a subset of integrins apply tension many fold greater than previously reported.

  1. Beyond the Matrix: The Many Non-ECM Ligands for Integrins

    PubMed Central

    LaFoya, Bryce; Munroe, Jordan A.; Miyamoto, Alison; Detweiler, Michael A.; Crow, Jacob J.; Gazdik, Tana

    2018-01-01

    The traditional view of integrins portrays these highly conserved cell surface receptors as mediators of cellular attachment to the extracellular matrix (ECM), and to a lesser degree, as coordinators of leukocyte adhesion to the endothelium. These canonical activities are indispensable; however, there is also a wide variety of integrin functions mediated by non-ECM ligands that transcend the traditional roles of integrins. Some of these unorthodox roles involve cell-cell interactions and are engaged to support immune functions such as leukocyte transmigration, recognition of opsonization factors, and stimulation of neutrophil extracellular traps. Other cell-cell interactions mediated by integrins include hematopoietic stem cell and tumor cell homing to target tissues. Integrins also serve as cell-surface receptors for various growth factors, hormones, and small molecules. Interestingly, integrins have also been exploited by a wide variety of organisms including viruses and bacteria to support infectious activities such as cellular adhesion and/or cellular internalization. Additionally, the disruption of integrin function through the use of soluble integrin ligands is a common strategy adopted by several parasites in order to inhibit blood clotting during hematophagy, or by venomous snakes to kill prey. In this review, we strive to go beyond the matrix and summarize non-ECM ligands that interact with integrins in order to highlight these non-traditional functions of integrins. PMID:29393909

  2. LFA-1 and Mac-1 integrins bind to the serine/threonine-rich domain of thrombomodulin

    SciTech Connect

    Kawamoto, Eiji; Emergency and Critical Care Center, Mie University Hospital, 2-174 Edobashi, Tsu 514-8507; Okamoto, Takayuki, E-mail: okamotot@doc.medic.mie-u.ac.jp

    2016-05-13

    LFA-1 (αLβ2) and Mac-1 (αMβ2) integrins regulate leukocyte trafficking in health and disease by binding primarily to IgSF ligand ICAM-1 and ICAM-2 on endothelial cells. Here we have shown that the anti-coagulant molecule thrombomodulin (TM), found on the surface of endothelial cells, functions as a potentially new ligand for leukocyte integrins. We generated a recombinant extracellular domain of human TM and Fc fusion protein (TM-domains 123-Fc), and showed that pheripheral blood mononuclear cells (PBMCs) bind to TM-domains 123-Fc dependent upon integrin activation. We then demonstrated that αL integrin-blocking mAb, αM integrin-blocking mAb, and β2 integrin-blocking mAb inhibited the binding ofmore » PBMCs to TM-domains 123-Fc. Furthermore, we show that the serine/threonine-rich domain (domain 3) of TM is required for the interaction with the LFA-1 (αLβ2) and Mac-1 (αMβ2) integrins to occur on PBMCs. These results demonstrate that the LFA-1 and Mac-1 integrins on leukocytes bind to TM, thereby establishing the molecular and structural basis underlying LFA-1 and Mac-1 integrin interaction with TM on endothelial cells. In fact, integrin-TM interactions might be involved in the dynamic regulation of leukocyte adhesion with endothelial cells. - Highlights: • LFA-1 and Mac-1 integrins bind to the anti-coagulant molecule thrombomodulin. • The serine/threonine-rich domain of thrombomodulin is essential to interact with the LFA-1 and Mac-1 integrins on PBMCs. • Integrin-TM interactions might be involved in the dynamic regulation of leukocyte adhesion with endothelial cells.« less

  3. αv-Integrin isoform expression in primary human tumors and brain metastases.

    PubMed

    Vogetseder, Alexander; Thies, Svenja; Ingold, Barbara; Roth, Patrick; Weller, Michael; Schraml, Peter; Goodman, Simon L; Moch, Holger

    2013-11-15

    To determine whether metastasis to brain is associated with altered expression patterns of integrins, we investigated the expression of αvβ3, αvβ5, αvβ6 and αvβ8 integrins in primary malignancies and metastases to brain of breast, lung and renal carcinomas and in malignant melanoma. Inhibitors of αv integrins are currently in clinical trials for glioblastoma. The role of integrins in the process of brain metastasis from other human tumors is unknown. Immunohistochemistry with novel integrin subtype specific rabbit monoclonal antibodies was performed on tissue microarrays of archival material of surgical biopsies taken from primary tumors and brain metastases. Integrin αvβ3 expression was increased in brain metastases compared to primary tumors of breast adenocarcinoma, non-small cell lung cancer, renal clear cell cancer and malignant cutaneous melanoma (all p < 0.01). Similarly, integrin αvβ8 expression was increased in brain metastases compared to primary tumors of breast cancer (p < 0.0001), lung cancer (p < 0.01) and renal cancer (p < 0.0001), with a similar trend in metastatic melanoma. Integrin αvβ5 was expressed in most primary tumors (98% breast cancer; 67% lung cancer; 90% renal cancer; 89% melanoma) and showed a stronger expression in brain metastases compared to primary tumors from lung cancer and melanoma (p < 0.05). Also integrin αvβ6 expression was increased in brain metastases compared to primary breast cancer (p < 0.001). The stronger αv-integrin expression in brain metastases, especially of αvβ3 and αvβ8 integrins, suggests that certain αv integrin are involved in the process of brain metastasis. αv Integrins may be therapeutic targets for patients with metastatic cancer in brain. Copyright © 2013 UICC.

  4. Integrin α3β1 Binding to Fibronectin Is Dependent on the Ninth Type III Repeat*

    PubMed Central

    Brown, Ashley C.; Dysart, Marilyn M.; Clarke, Kimberly C.; Stabenfeldt, Sarah E.; Barker, Thomas H.

    2015-01-01

    Fibronectin (Fn) is a promiscuous ligand for numerous cell adhesion receptors or integrins. The vast majority of Fn-integrin interactions are mediated through the Fn Arg-Gly-Asp (RGD) motif located within the tenth type III repeat. In the case of integrins αIIbβ3 and α5β1, the integrin binds RGD and the synergy site (PHSRN) located within the adjacent ninth type III repeat. Prior work has shown that these synergy-dependent integrins are exquisitely sensitive to perturbations in the Fn integrin binding domain conformation. Our own prior studies of epithelial cell responses to recombinant fragments of the Fn integrin binding domain led us to hypothesize that integrin α3β1 binding may also be modulated by the synergy site. To explore this hypothesis, we created a variety of recombinant variants of the Fn integrin binding domain: (i) a previously reported (Leu → Pro) stabilizing mutant (FnIII9′10), (ii) an Arg to Ala synergy site mutation (FnIII9R→A10), (iii) a two-Gly (FnIII92G10) insertion, and (iv) a four-Gly (FNIII94G10) insertion in the interdomain linker region and used surface plasmon resonance to determine binding kinetics of integrin α3β1 to the Fn fragments. Integrin α3β1 had the highest affinity for FnIII9′10 and FnIII92G10. Mutation within the synergy site decreased integrin α3β1 binding 17-fold, and the four-Gly insertion decreased binding 39-fold compared with FnIII9′10. Cell attachment studies demonstrate that α3β1-mediated epithelial cell binding is greater on FnIII9′10 compared with the other fragments. These studies suggest that the presence and spacing of the RGD and synergy sites modulate integrin α3β1 binding to Fn. PMID:26318455

  5. Transgenic horticultural crops in Asia

    USDA-ARS?s Scientific Manuscript database

    Modern biotechnology applications, including genetic engineering, are a powerful tool to complement the conventional methods of crop improvement. Asia currently has three countries cultivating biotech/transgenic crops – China, India, and the Philippines, but only China commercially grows a transgen...

  6. Discoidin Domain Receptors Promote α1β1- and α2β1-Integrin Mediated Cell Adhesion to Collagen by Enhancing Integrin Activation

    PubMed Central

    Xu, Huifang; Bihan, Dominique; Chang, Francis; Huang, Paul H.; Farndale, Richard W.; Leitinger, Birgit

    2012-01-01

    The discoidin domain receptors, DDR1 and DDR2, are receptor tyrosine kinases that bind to and are activated by collagens. Similar to collagen-binding β1 integrins, the DDRs bind to specific motifs within the collagen triple helix. However, these two types of collagen receptors recognize distinct collagen sequences. While GVMGFO (O is hydroxyproline) functions as a major DDR binding motif in fibrillar collagens, integrins bind to sequences containing Gxx’GEx”. The DDRs are thought to regulate cell adhesion, but their roles have hitherto only been studied indirectly. In this study we used synthetic triple-helical collagen-derived peptides that incorporate either the DDR-selective GVMGFO motif or integrin-selective motifs, such as GxOGER and GLOGEN, in order to selectively target either type of receptor and resolve their contributions to cell adhesion. Our data using HEK293 cells show that while cell adhesion to collagen I was completely inhibited by anti-integrin blocking antibodies, the DDRs could mediate cell attachment to the GVMGFO motif in an integrin-independent manner. Cell binding to GVMGFO was independent of DDR receptor signalling and occurred with limited cell spreading, indicating that the DDRs do not mediate firm adhesion. However, blocking the interaction of DDR-expressing cells with collagen I via the GVMGFO site diminished cell adhesion, suggesting that the DDRs positively modulate integrin-mediated cell adhesion. Indeed, overexpression of the DDRs or activation of the DDRs by the GVMGFO ligand promoted α1β1 and α2β1 integrin-mediated cell adhesion to medium- and low-affinity integrin ligands without regulating the cell surface expression levels of α1β1 or α2β1. Our data thus demonstrate an adhesion-promoting role of the DDRs, whereby overexpression and/or activation of the DDRs leads to enhanced integrin-mediated cell adhesion as a result of higher integrin activation state. PMID:23284937

  7. Mechanotransduction: all signals point to cytoskeleton, matrix, and integrins

    NASA Technical Reports Server (NTRS)

    Alenghat, Francis J.; Ingber, Donald E.

    2002-01-01

    Mechanical stresses modulate cell function by either activating or tuning signal transduction pathways. Mechanotransduction, the process by which cells convert mechanical stimuli into a chemical response, occurs both in cells specialized for sensing mechanical cues and in parenchymal cells whose primary function is not mechanosensory. However, common among the various responses to mechanical stress is the importance of direct or indirect connections between the internal cytoskeleton, the extracellular matrix (ECM), and traditional signal transducing molecules. In many instances, these elements converge at focal adhesions, sites of structural attachment between the cytoskeleton and ECM that are anchored by cell surface integrin receptors. Alenghat and Ingber discuss the accumulating evidence for the central role of cytoskeleton, ECM, and integrin-anchored focal adhesions in several mechanotransduction pathways.

  8. Cell Adhesion on Amyloid Fibrils Lacking Integrin Recognition Motif*

    PubMed Central

    Jacob, Reeba S.; George, Edna; Singh, Pradeep K.; Salot, Shimul; Anoop, Arunagiri; Jha, Narendra Nath; Sen, Shamik; Maji, Samir K.

    2016-01-01

    Amyloids are highly ordered, cross-β-sheet-rich protein/peptide aggregates associated with both human diseases and native functions. Given the well established ability of amyloids in interacting with cell membranes, we hypothesize that amyloids can serve as universal cell-adhesive substrates. Here, we show that, similar to the extracellular matrix protein collagen, amyloids of various proteins/peptides support attachment and spreading of cells via robust stimulation of integrin expression and formation of integrin-based focal adhesions. Additionally, amyloid fibrils are also capable of immobilizing non-adherent red blood cells through charge-based interactions. Together, our results indicate that both active and passive mechanisms contribute to adhesion on amyloid fibrils. The present data may delineate the functional aspect of cell adhesion on amyloids by various organisms and its involvement in human diseases. Our results also raise the exciting possibility that cell adhesivity might be a generic property of amyloids. PMID:26742841

  9. β1 Integrins as Therapeutic Targets to Disrupt Hallmarks of Cancer

    PubMed Central

    Blandin, Anne-Florence; Renner, Guillaume; Lehmann, Maxime; Lelong-Rebel, Isabelle; Martin, Sophie; Dontenwill, Monique

    2015-01-01

    Integrins belong to a large family of αβ heterodimeric transmembrane proteins first recognized as adhesion molecules that bind to dedicated elements of the extracellular matrix and also to other surrounding cells. As important sensors of the cell microenvironment, they regulate numerous signaling pathways in response to structural variations of the extracellular matrix. Biochemical and biomechanical cues provided by this matrix and transmitted to cells via integrins are critically modified in tumoral settings. Integrins repertoire are subjected to expression level modifications, in tumor cells, and in surrounding cancer-associated cells, implicated in tumor initiation and progression as well. As critical players in numerous cancer hallmarks, defined by Hanahan and Weinberg (2011), integrins represent pertinent therapeutic targets. We will briefly summarize here our current knowledge about integrin implications in those different hallmarks focusing primarily on β1 integrins. PMID:26635609

  10. Laminin121 – Recombinant expression and interactions with integrins

    PubMed Central

    Sasaki, Takako; Takagi, Junichi; Giudici, Camilla; Yamada, Yoshihiko; Arikawa-Hirasawa, Eri; Deutzmann, Rainer; Timpl, Rupert; Sonnenberg, Arnoud; Bächinger, Hans Peter; Tonge, David

    2010-01-01

    Laminin-121, previously referred as to laminin-3, was expressed recombinantly in human embryonic kidney (HEK) 293 cells by triple transfection of full-length cDNAs encoding mouse laminin α1, β2 and γ1 chains. The recombinant laminin-121 was purified using Heparin-Sepharose followed by molecular sieve chromatography and shown to be correctly folded by electron microscopy and circular dichroism (CD). The CD spectra of recombinant laminin-121 were very similar to those of laminin-111 isolated from Engelbreth-Holm Swarm tumor (EHS-laminin) but its Tm value was smaller than EHS-laminin and recombinant lamnin-111 suggesting that the replacement of the β chain reduced the stability of the coiled-coil structure of laminin-121. Its binding to integrins was compared with EHS-laminin, laminin-3A32 purified from murine epidermal cell line and recombinantly expressed laminins-111, -211 and -221. Laminin-121 showed the highest affinity to α6β1 and α7β1 integrins and furthermore, laminin-121 most effectively supported neurite outgrowth. Together, this suggests that the β2 laminins have higher affinity for integrins than the β1 laminins. PMID:20566382

  11. Integrin Alpha-v and HER2 in Breast Cancer Brain Metastasis

    DTIC Science & Technology

    2015-10-01

    chemo-resistant to cisplatin , 5-fluorourical, paclitaxel, and temozolomide than αv-integrin negative cells with same genetic and molecular origin. Pre...treatment of αv-integrin positive MDA-MB231BR cells with intetumumab (10 µg/mL) for 1 hr in vitro enhanced chemo-induced cytotoxicity of cisplatin ...5-fluorourical and paclitaxel but not temozolomide. In addition, cisplatin increased cellular αv-integrin level of MDA-MB231BR cells in dose

  12. Leukocyte integrins: Role in leukocyte recruitment and as therapeutic targets in inflammatory disease

    PubMed Central

    Kourtzelis, Ioannis; Ziogas, Athanassios; Hajishengallis, George; Chavakis, Triantafyllos

    2014-01-01

    Infection or sterile inflammation triggers site-specific attraction of leukocytes. Leukocyte recruitment is a process comprising several steps orchestrated by adhesion molecules, chemokines, cytokines and endogenous regulatory molecules. Distinct adhesive interactions between endothelial cells and leukocytes and signalling mechanisms contribute to the temporal and spatial fine-tuning of the leukocyte adhesion cascade. Central players in the leukocyte adhesion cascade include the leukocyte adhesion receptors of the β2-integrin family, such as the αLβ2 and αMβ2 integrins, or of the β1-integrin family, such as the α4β1- integrin. Given the central involvement of leukocyte recruitment in different inflammatory and autoimmune diseases, the leukocyte adhesion cascade in general, and leukocyte integrins in particular, represent key therapeutic targets. In this context, the present review focuses on the role of leukocyte integrins in the leukocyte adhesion cascade. Experimental evidence that has implicated leukocyte integrins as targets in animal models of inflammatory disorders, such as experimental autoimmune encephalomyelitis, psoriasis, inflammatory bone loss and inflammatory bowel disease as well as preclinical and clinical therapeutic applications of antibodies that target leukocyte integrins in various inflammatory disorders are presented. Finally, we review recent findings on endogenous inhibitors that modify leukocyte integrin function, which could emerge as promising therapeutic targets. PMID:25448040

  13. Combination of integrin siRNA and irradiation for breast cancer therapy

    SciTech Connect

    Cao Qizhen; Cai Weibo; Li Tianfang

    2006-12-22

    Up-regulation of integrin {alpha}{sub v}{beta}{sub 3} has been shown to play a key role in tumor angiogenesis and metastasis. In this study, we evaluated the role of integrin {alpha}{sub v}{beta}{sub 3} in breast cancer cell resistance to ionizing irradiation (IR) and tested the anti-tumor efficacy of combining integrin {alpha}{sub v} siRNA and IR. Colonogenic survival assay, cell proliferation, apoptosis, and cell cycle analysis were carried out to determine the treatment effect of siRNA, IR, or combination of both on MDA-MB-435 cells (integrin {alpha}{sub v}{beta}{sub 3}-positive). Integrin {alpha}{sub v}{beta}{sub 3}-negative MCF-7 cells exert more radiosensitivity than MDA-MB-435 cells. IR up-regulates integrinmore » {alpha}{sub v}{beta}{sub 3} expression in MDA-MB-435 cells and integrin {alpha}{sub v} siRNA can effectively reduce both {alpha}{sub v} and {alpha}{sub v}{beta}{sub 3} integrin expression, leading to increased radiosensitivity. Integrin {alpha}{sub v} siRNA also promotes IR-induced apoptosis and enhances IR-induced G2/M arrest in cell cycle progression. This study, with further optimization, may provide a simple and highly efficient treatment strategy for breast cancer as well as other integrin {alpha}{sub v}{beta}{sub 3}-positive cancer types.« less

  14. Activated β2Integrins Restrict Neutrophil Recruitment during Murine Acute Pseudomonal Pneumonia.

    PubMed

    Wilson, Zachary S; Ahn, Lawrence B; Serratelli, William S; Belley, Matthew D; Lomas-Neira, Joanne; Sen, Mehmet; Lefort, Craig T

    2017-05-01

    Rapid neutrophil recruitment is critical for the efficient clearance of bacterial pathogens from the lungs. Although β 2 integrins and their activation are required for neutrophil recruitment from postcapillary venules of the systemic circulation into inflamed tissues, the involvement of integrins in neutrophil recruitment in response to respiratory infection varies among bacterial pathogens. For stimuli eliciting β 2 integrin-dependent neutrophil influx, including Pseudomonas aeruginosa, it remains unclear whether the activation of β 2 integrins is an essential step in this process. In the current study, we analyze neutrophil trafficking within the lungs of mice infected with Pseudomonas aeruginosa and evaluate the role of β 2 integrin activation through genetic deletion of talin-1 or Kindlin-3 or by pharmacological inhibition of high-affinity β 2 integrins using a small molecule allosteric antagonist. We observe that attenuation of high-affinity β 2 integrins leads to an enhancement of neutrophil emigration into lung interstitium and airspaces. Neutrophil effector functions, including the production of reactive oxygen species and the phagocytosis of bacteria, are only partially dependent on high-affinity β 2 integrins. These results reveal a mechanism by which activated β 2 integrins limit neutrophil entry into the lung tissue and airspaces during acute pseudomonal pneumonia and suggest potential strategies for modulating neutrophil-mediated host defense.

  15. Transmembrane and cytoplasmic domains in integrin activation and protein-protein interactions (review)

    PubMed Central

    WEGENER, KATE L.; CAMPBELL, IAIN D.

    2010-01-01

    Integrins are heterodimeric membrane-spanning adhesion receptors that are essential for a wide range of biological functions. Control of integrin conformational states is required for bidirectional signalling across the membrane. Key components of this control mechanism are the transmembrane and cytoplasmic domains of the α and β subunits. These domains are believed to interact, holding the integrin in the inactive state, while inside-out integrin activation is accompanied by domain separation. Although there are strong indications for domain interactions, the majority of evidence is insufficient to precisely define the interaction interface. The current best model of the complex, derived from computational calculations with experimental restraints, suggests that integrin activation by the cytoplasmic protein talin is accomplished by steric disruption of the α/β interface. Better atomic-level resolution structures of the α/β transmembrane/cytoplasmic domain complex are still required for the resting state integrin to corroborate this. Integrin activation is also controlled by competitive interactions involving the cytoplasmic domains, particularly the β-tails. The concept of the β integrin tail as a focal adhesion interaction ‘hub’ for interactions and regulation is discussed. Current efforts to define the structure and affinity of the various complexes formed by integrin tails, and how these interactions are controlled, e.g. by phosphorylation and localisation, are described. PMID:18654929

  16. Relevance of BAC transgene copy number in mice: transgene copy number variation across multiple transgenic lines and correlations with transgene integrity and expression

    PubMed Central

    Chandler, Kelly J.; Chandler, Ronald L.; Broeckelmann, Eva M.; Hou, Yue; Southard-Smith, E. Michelle; Mortlock, Douglas P.

    2011-01-01

    Bacterial artificial chromosomes (BACs) are excellent tools for manipulating large DNA fragments and, as a result, are increasingly utilized to engineer transgenic mice by pronuclear injection. The demand for BAC transgenic mice underscores the need for careful inspection of BAC integrity and fidelity following transgenesis, which may be crucial for interpreting transgene function. Thus, it is imperative that reliable methods for assessing these parameters are available. However, there are limited data regarding whether BAC transgenes routinely integrate in the mouse genome as intact molecules, how BAC transgenes behave as they are passed through the germline across successive generations, and how variation in BAC transgene copy number relates to transgene expression. To address these questions, we used Taqman real-time PCR to estimate BAC transgene copy number in BAC transgenic embryos and lines. Here we demonstrate the reproducibility of copy number quantification with this method, and describe the variation in copy number across independent transgenic lines. In addition, polymorphic marker analysis suggests the majority of BAC transgenic lines contain intact molecules. Notably, all lines containing multiple BAC copies also contain all BAC-specific markers. Three of 23 founders analyzed contained BAC transgenes integrated into more than one genomic location. Finally, we show increased BAC transgene copy number correlates with increased BAC transgene expression. In sum, our efforts have provided a reliable method for assaying BAC transgene integrity and fidelity, and data that should be useful for researchers using BACs as transgenic vectors. PMID:17882484

  17. Deletion of integrin-linked kinase from skeletal muscles of mice resembles muscular dystrophy due to alpha 7 beta 1-integrin deficiency.

    PubMed

    Gheyara, Ania L; Vallejo-Illarramendi, Ainara; Zang, Keling; Mei, Lin; St-Arnaud, Rene; Dedhar, Shoukat; Reichardt, Louis F

    2007-12-01

    Integrin-linked kinase (Ilk) is a serine/threonine kinase and an adaptor protein that links integrins to the actin cytoskeleton and to a number of signaling pathways involved in integrin action. We hypothesized that Ilk may act as an important effector of integrins in skeletal muscle, where these receptors provide a critical link between the sarcolemma and the extracellular matrix. Using the cre/lox system, we deleted Ilk from skeletal muscles of mice. The resulting mutants developed a progressive muscular dystrophy with multiple degenerating and regenerating muscle fibers, increased central nuclei, and endomysial fibrosis. These defects were widespread but were most severe near myofascial junctions where Ilk mutants showed displacement of focal adhesion-related proteins, including vinculin, paxillin, focal adhesion kinase, dystrophin, and the alpha 7 beta 1D-integrin subunits. Distal ends of mutant muscle fibers appeared irregular, and there was restructuring of the actin cytoskeleton. These findings resemble those seen in humans and mice lacking the alpha 7-integrin subunit and suggest that Ilk may act as a cytoplasmic effector of alpha 7 beta1-integrin in the pathogenesis of these deficiencies.

  18. Transgenic mice in developmental toxicology

    SciTech Connect

    Woychik, R.P.

    1992-01-01

    Advances in molecular biology and embryology are being utilized for the generation of transgenic mice, animals that contain specific additions, deletions, or modifications of genes or sequences in their DNA. Mouse embryonic stem cells and homologous recombination procedures have made it possible to target specific DNA structural alterations to highly localized region in the host chromosomes. The majority of the DNA structural rearrangements in transgenic mice can be passed through the germ line and used to establish new genetic traits in the carrier animals. Since the use of transgenic mice is having such an enormous impact on so many areasmore » of mammalian biological research, including developmental toxicology, the objective of this review is to briefly describe the fundamental methodologies for generating transgenic mice and to describe one particular application that has direct relevance to the field of genetic toxicology.« less

  19. Transgenic mice in developmental toxicology

    SciTech Connect

    Woychik, R.P.

    1992-12-31

    Advances in molecular biology and embryology are being utilized for the generation of transgenic mice, animals that contain specific additions, deletions, or modifications of genes or sequences in their DNA. Mouse embryonic stem cells and homologous recombination procedures have made it possible to target specific DNA structural alterations to highly localized region in the host chromosomes. The majority of the DNA structural rearrangements in transgenic mice can be passed through the germ line and used to establish new genetic traits in the carrier animals. Since the use of transgenic mice is having such an enormous impact on so many areasmore » of mammalian biological research, including developmental toxicology, the objective of this review is to briefly describe the fundamental methodologies for generating transgenic mice and to describe one particular application that has direct relevance to the field of genetic toxicology.« less

  20. Targeting ILK and {beta}4 integrin abrogates the invasive potential of ovarian cancer

    SciTech Connect

    Choi, Yoon Pyo; Kim, Baek Gil; Department of Pathology, Yonsei University College of Medicine, Seoul

    2012-10-26

    Highlights: Black-Right-Pointing-Pointer The potential of targeting ILK and integrins for highly aggressive ovarian cancer. Black-Right-Pointing-Pointer Unanticipated synergistic effect for the combination of ILK/{beta}4 integrin. Black-Right-Pointing-Pointer Combination of ILK/{beta}4 integrin effectively inhibited the PI3K/Akt/Rac1 cascade. Black-Right-Pointing-Pointer Targeting of {beta}4 integrin/ILK had potent inhibitory effects in ovarian cancer. -- Abstract: Integrins and integrin-linked kinase (ILK) are essential to cancerous invasion because they mediate physical interactions with the extracellular matrix, and regulate oncogenic signaling pathways. The purpose of our study is to determine whether deletion of {beta}1 and {beta}4 integrin and ILK, alone or in combination, has antitumoral effects in ovarian cancer. Expressionmore » of {beta}1 and {beta}4 integrin and ILK was analyzed by immunohistochemistry in 196 ovarian cancer tissue samples. We assessed the effects of depleting these molecules with shRNAs in ovarian cancer cells by Western blot, conventional RT-PCR, cell proliferation, migration, invasion, and in vitro Rac1 activity assays, and in vivo xenograft formation assays. Overexpression of {beta}4 integrin and ILK in human ovarian cancer specimens was found to correlate with tumor aggressiveness. Depletion of these targets efficiently suppresses ovarian cancer cell proliferation, migration, and invasion in vitro and xenograft tumor formation in vivo. We also demonstrated that single depletion of ILK or combination depletion of {beta}4 integrin/ILK inhibits phosphorylation of downstream signaling targets, p-Ser 473 Akt and p-Thr202/Tyr204 Erk1/2, and activation of Rac1, as well as reduce expression of MMP-2 and MMP-9 and increase expression of caspase-3 in vitro. In conclusion, targeting {beta}4 integrin combined with ILK can instigate the latent tumorigenic potential and abrogate the invasive potential in ovarian cancer.« less

  1. Integrin expression and glycosylation patterns regulate cell-matrix adhesion and alter with breast cancer progression.

    PubMed

    Singh, Chandrajeet; Shyanti, Ritis K; Singh, Virendra; Kale, Raosaheb K; Mishra, Jai P N; Singh, Rana P

    2018-05-05

    Integrins are the major cell adhesion glycoproteins involved in cell-extracellular matrix (ECM) interaction and metastasis. Further, glycosylation on integrin is necessary for its proper folding and functionality. Herein, differential expression of integrins viz., αvβ3 and αvβ6 was examined in MDA-MB-231, MDA-MB-468 and MCF-10A cells, which signify three different stages of breast cancer development from highly metastatic to non-tumorigenic stage. The expression of αvβ3 and αvβ6 integrins at mRNA and protein levels was observed in all three cell lines and the results displayed a distinct pattern of expression. Highly metastatic cells showed enhanced expression of αvβ3 than moderate metastatic and non-tumorigenic cells. The scenario was reversed in case of αvβ6 integrin, which was strongly expressed in moderate metastatic and non-tumorigenic cells. N-glycosylation of αvβ3 and αvβ6 integrins is required for the attachment of cells to ECM proteins like fibronectin. The cell adhesion properties were found to be different in these cancer cells with respect to the type of integrins expressed. The results testify that αvβ3 integrin in highly metastatic cells, αvβ6 integrin in both moderate metastatic and non-tumorigenic cells play an important role in cell adhesion. The investigation typify that N-glycosylation on integrins is also necessary for cell-ECM interaction. Further, glycosylation inhibition by Swainsonine is found to be more detrimental to invasive property of moderate metastatic cells. Conclusively, types of integrins expressed as well as their N-glycosylation pattern alter during the course of breast cancer progression. Copyright © 2018 Elsevier Inc. All rights reserved.

  2. Transgenic plants for phytoremediation.

    PubMed

    Maestri, Elena; Marmiroli, Nelson

    2011-01-01

    Phytoremediation is a green, sustainable and promising solution to problems of environmental contamination. It entails the use of plants for uptake, sequestration, detoxification or volatilization of inorganic and organic pollutants from soils, water, sediments and possibly air. Phytoremediation was born from the observation that plants possessed physiological properties useful for environmental remediation. This was shortly followed by the application of breeding techniques and artificial selection to genetically improve some of the more promising and interesting species. Now, after nearly 20 years of research, transgenic plants for phytoremediation have been produced, but none have reached commercial existence. Three main approaches have been developed: (1) transformation with genes from other organisms (mammals, bacteria, etc.); (2) transformation with genes from other plant species; and (3) overexpression of genes from the same plant species. Many encouraging results have been reported, even though in some instances results have been contrary to expectations. This review will illustrate the main examples with a critical discussion of what we have learnt from them.

  3. Expression and promoter analysis of a highly restricted integrin alpha gene in vascular smooth muscle.

    PubMed

    Kitchen, Chad M; Cowan, Sarah L; Long, Xiaochun; Miano, Joseph M

    2013-01-15

    Full genome annotation requires gene expression analysis and elucidation of promoter activity. Here, we analyzed the expression and promoter of a highly restricted integrin gene, Itga8. RNase protection and quantitative RT-PCR showed Itga8 to be expressed most abundantly in vascular smooth muscle cells (SMC). Transcription start site mapping of Itga8 revealed the immediate 5' promoter region to be poorly conserved with orthologous sequences in the human genome. Further comparative sequence analysis showed a number of conserved non-coding sequence modules around the Itga8 gene. The immediate promoter region and an upstream conserved sequence module were each found to contain a CArG box, which is a binding site for serum response factor (SRF). Luciferase reporter assays revealed activity of several Itga8 promoter constructs with no apparent restricted activity to SMC types. Further, neither SRF nor its coactivator, Myocardin (MYOCD), was able to induce several distinct Itga8 promoter constructs. Transgenic mouse studies failed to reveal Itga8 promoter activity, indicating distal regulatory elements likely control this gene's in vivo expression profile. Interestingly, although the promoter was unresponsive to SRF/MYOCD, the endogenous Itga8 gene showed increases in expression upon ectopic MYOCD expression even though knockdown of SRF both in vitro and in vivo failed to demonstrate a corresponding change in Itga8. Collectively, these data demonstrate that Itga8 expression is CArG-SRF independent, but MYOCD dependent through an as yet unknown sequence module that is distal from the promoter region. Copyright © 2012 Elsevier B.V. All rights reserved.

  4. α4β7 Integrin is essential for contact hypersensitivity by regulating migration of T cells to skin.

    PubMed

    Ohmatsu, Hanako; Kadono, Takafumi; Sugaya, Makoto; Tomita, Manabu; Kai, Hiromichi; Miyagaki, Tomomitsu; Saeki, Hidehisa; Tamaki, Kunihiko; Steeber, Douglas A; Tedder, Thomas F; Sato, Shinichi

    2010-12-01

    β7 Integrin, a cell adhesion molecule, is present in the form of α4β7 integrin or αEβ7 integrin. α4β7 Integrin is expressed on most leucocytes and is essential for their migration to gut-associated lymphoid tissues by interacting with its primary ligand, mucosal addressin cell adhesion molecule-1, which is preferentially expressed in gut-associated lymphoid tissues. Although the importance of α4β7 integrin in intestinal inflammation has been established, its role in cutaneous inflammation remains to be elucidated. We sought to investigate the role of β7 integrin in cutaneous inflammation. We used a murine contact hypersensitivity model and examined the role of β7 integrin by using β7 integrin-deficient and αE integrin-deficient mice. β7 Integrin-deficient mice, not αE integrin-deficient mice, are defective in contact hypersensitivity responses. β7 Integrin deficiency does not affect irritant contact dermatitis. The distribution, migration, and function of antigen presenting cells from β7 integrin-deficient mice are comparable to those from wild-type mice. Moreover, sensitized β7 integrin-deficient T cells are able to respond to antigen stimuli in vitro and elicit contact hypersensitivity responses when directly injected into the skin. However, they are defective in reaching the skin under inflammatory conditions, resulting in reduced contact hypersensitivity responses when intravenously injected. Furthermore, intraperitoneal injection of anti-α4β7 integrin neutralizing antibody elicit impaired contact hypersensitivity responses. α4β7 Integrin contributes to contact hypersensitivity responses by regulating T-cell migration to inflammatory skin. Copyright © 2010 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

  5. Integrin β3 is required in infection and proliferation of classical swine fever virus.

    PubMed

    Li, Weiwei; Wang, Gang; Liang, Wulong; Kang, Kai; Guo, Kangkang; Zhang, Yanming

    2014-01-01

    Classical Swine Fever (CSF) is a highly infectious fatal pig disease, resulting in huge economic loss to the swine industry. Integrins are membrane-bound signal mediators, expressed on a variety of cell surfaces and are known as receptors or co-receptors for many viruses. However, the role of integrin β3 in CSFV infection is unknown. Here, through quantitive PCR, immunofluorescence (IFC) and immunocytohistochemistry (ICC), we revealed that ST (swine testicles epithelial) cells have a prominent advantage in CSFV proliferation as compared to EC (swine umbilical vein endothelial cell), IEC (swine intestinal epithelial cell) and PK (porcine kidney epithelial) cells. Meanwhile, ST cells had remarkably more integrin β3 expression as compared to EC, IEC and PK cells, which was positively correlated with CSFV infection and proliferation. Integrin β3 was up-regulated post CSFV infection in all the four cell lines, while the CSFV proliferation rate was decreased in integrin β3 function-blocked cells. ShRNA1755 dramatically decreased integrin β3, with a deficiency of 96% at the mRNA level and 80% at the protein level. CSFV proliferation was dramatically reduced in integrin β3 constantly-defected cells (ICDC), with the deficiencies of 92.6%, 99% and 81.7% at 24 h, 48 h and 72 h post CSFV infection, respectively. These results demonstrate that integrin β3 is required in CSFV infection and proliferation, which provide a new insight into the mechanism of CSFV infection.

  6. Integrin β3 prevents apoptosis of HL-1 cardiomyocytes under conditions of oxidative stress.

    PubMed

    Shewchuk, Lee J; Bryan, Sean; Ulanova, Marina; Khaper, Neelam

    2010-03-01

    Integrin receptors are essential in the regulation of vital cardiac functions, and impaired integrin activity has been associated with cardiac remodeling. Oxidative stress is known to be involved in apoptosis and cardiac remodeling and thus may profoundly influence cardiac function via integrin modulation. The aim of this study was to determine the expression pattern and functional role of integrins in HL-1 cardiomyocytes under conditions of oxidative stress. Gene expression was studied by end-point and real-time PCR; surface protein expression was studied by flow cytometry; integrin knockdown was accomplished by siRNA gene silencing; and apoptosis was studied by annexin V staining and active caspase-3/7 using flow cytometry. Among the various subunits under study (alphav, alpha5, alpha6, and beta1, beta3, beta4, and beta5), the expression of beta3 integrin was significantly increased at both the mRNA and protein levels in cardiomyocytes exposed to 100 micromol/L hydrogen peroxide for 3 h. Gene silencing of beta3 integrin by using siRNA resulted in a 2-fold increase in cardiomyocyte apoptosis upon treatment with hydrogen peroxide. This increase in apoptosis, as measured by annexin V staining, correlated with an increase in active caspase-3/7. Integrin beta3 plays a vital role in preventing cardiomyocyte apoptosis under conditions of oxidative stress.

  7. Selectins and chemokines use shared and distinct signals to activate β2 integrins in neutrophils.

    PubMed

    Yago, Tadayuki; Zhang, Nan; Zhao, Liang; Abrams, Charles S; McEver, Rodger P

    2018-04-10

    Rolling neutrophils receive signals while engaging P- and E-selectin and chemokines on inflamed endothelium. Selectin signaling activates β2 integrins to slow rolling velocities. Chemokine signaling activates β2 integrins to cause arrest. Despite extensive study, key aspects of these signaling cascades remain unresolved. Using complementary in vitro and in vivo assays, we found that selectin and chemokine signals in neutrophils triggered Rap1a-dependent and phosphatidylinositol-4-phosphate 5-kinase γ (PIP5Kγ90)-dependent pathways that induce integrin-dependent slow rolling and arrest. Interruption of both pathways, but not either pathway alone, blocked talin-1 recruitment to and activation of integrins. An isoform of PIP5Kγ90 lacking the talin-binding domain (PIP5Kγ87) could not activate integrins. Chemokines, but not selectins, used phosphatidylinositol-4,5-bisphosphate 3-kinase γ (PI3Kγ) in cooperation with Rap1a to mediate integrin-dependent slow rolling (at low chemokine concentrations), as well as arrest (at high chemokine concentrations). High levels of chemokines activated β2 integrins without selectin signals. When chemokines were limiting, they synergized with selectins to activate β2 integrins. © 2018 by The American Society of Hematology.

  8. Integrin-based Therapeutics: Biological Basis, Clinical Use and New Drugs

    PubMed Central

    Ley, Klaus; Rivera-Nieves, Jesus; Sandborn, William J.; Shattil, Sanford

    2016-01-01

    Integrins are activatable adhesion and signaling molecules. Of the 24 known human integrins, three are currently targeted therapeutically by monoclonal antibodies, peptides or small molecules. The platelet αIIbβ3 integrin is targeted by Abciximab, Eptifibatide and Tirofiban, all with indications for preventing thrombotic complications after percutaneous coronary interventions. The lymphocyte α4β1 and α4β7 integrins are targeted by Natalizumab with indications in multiple sclerosis and Crohn’s disease. Although efficacious, use of this antibody is limited by a rare but serious complication, progressive multifocal leukoencephalopathy. Vedolizumab is an antibody to a combinatorial epitope in α4β7 that is approved for use in patients with Crohn’s disease or ulcerative colitis in the United States, Canada and Europe. Progressive multifocal leukoencephalopathy has not been observed in the clinical trials or clinical use of vedolizumab. New antibodies and small molecules targeting β7 integrins (α4β7 and αEβ7) and MAdCAM-1 are in clinical development for treatment of these inflammatory bowel diseases. Overall, integrin-based therapeutics have shown clinically significant benefits in many patients, leading to continued medical interest in the further development of novel integrin inhibitors. Of note, almost all integrin antagonists in use or in late-stage clinical trials target the ligand binding site, or the ligand itself. PMID:26822833

  9. Direct Thy-1/alphaVbeta3 integrin interaction mediates neuron to astrocyte communication.

    PubMed

    Hermosilla, Tamara; Muñoz, Daniel; Herrera-Molina, Rodrigo; Valdivia, Alejandra; Muñoz, Nicolás; Nham, Sang-Uk; Schneider, Pascal; Burridge, Keith; Quest, Andrew F G; Leyton, Lisette

    2008-06-01

    Thy-1 is an abundant neuronal glycoprotein of poorly defined function. We recently provided evidence indicating that Thy-1 clusters a beta3-containing integrin in astrocytes to induce tyrosine phosphorylation, RhoA activation and the formation of focal adhesions and stress fibers. To date, the alpha subunit partner of beta3 integrin in DI TNC1 astrocytes is unknown. Similarly, the ability of neuronal, membrane-bound Thy-1 to trigger astrocyte signaling via integrin engagement remains speculation. Here, evidence that alphav forms an alphavbeta3 heterodimer in DI TNC1 astrocytes was obtained. In neuron-astrocyte association assays, the presence of either anti-alphav or anti-beta3 integrin antibodies reduced cell-cell interaction demonstrating the requirement of both integrin subunits for this association. Moreover, anti-Thy-1 antibodies blocked stimulation of astrocytes by neurons but not the binding of these two cell types. Thus, neuron-astrocyte association involved binding between molecular components in addition to the Thy-1-integrin; however, the signaling events leading to focal adhesion formation in astrocytes depended exclusively on the latter interaction. Additionally, wild-type (RLD) but not mutated (RLE) Thy-1 was shown to directly interact with alphavbeta3 integrin by Surface Plasmon Resonance analysis. This interaction was promoted by divalent cations and was species-independent. Together, these results demonstrate that the alphavbeta3 integrin heterodimer interacts directly with Thy-1 present on neuronal cells to stimulate astrocytes.

  10. Radil controls neutrophil adhesion and motility through β2-integrin activation.

    PubMed

    Liu, Lunhua; Aerbajinai, Wulin; Ahmed, Syed M; Rodgers, Griffin P; Angers, Stephane; Parent, Carole A

    2012-12-01

    Integrin activation is required to facilitate multiple adhesion-dependent functions of neutrophils, such as chemotaxis, which is critical for inflammatory responses to injury and pathogens. However, little is known about the mechanisms that mediate integrin activation in neutrophils. We show that Radil, a novel Rap1 effector, regulates β1- and β2-integrin activation and controls neutrophil chemotaxis. On activation and chemotactic migration of neutrophils, Radil quickly translocates from the cytoplasm to the plasma membrane in a Rap1a-GTP-dependent manner. Cells overexpressing Radil show a substantial increase in cell adhesion, as well as in integrin/focal adhesion kinase (FAK) activation, and exhibit an elongated morphology, with severe tail retraction defects. This phenotype is effectively rescued by treatment with either β2-integrin inhibitory antibodies or FAK inhibitors. Conversely, knockdown of Radil causes severe inhibition of cell adhesion, β2-integrin activation, and chemotaxis. Furthermore, we found that inhibition of Rap activity by RapGAP coexpression inhibits Radil-mediated integrin and FAK activation, decreases cell adhesion, and abrogates the long-tail phenotype of Radil cells. Overall, these studies establish that Radil regulates neutrophil adhesion and motility by linking Rap1 to β2-integrin activation.

  11. Integrin shedding as a mechanism of cellular adaptation during cardiac growth.

    PubMed

    Goldsmith, Edie C; Carver, Wayne; McFadden, Alex; Goldsmith, Jack G; Price, Robert L; Sussman, Mark; Lorell, Beverly H; Cooper, Garth; Borg, Thomas K

    2003-06-01

    Integrin-mediated cell-extracellular matrix (ECM) interactions are essential for multiple cellular processes; however, little is known regarding integrin turnover during these events. Recent studies have demonstrated shedding of cell surface molecules and suggested this as a potential mechanism for integrin turnover. Confocal microscopy of mouse hearts under different physiological conditions demonstrated the presence of beta(1)-integrin-immunoreactive material in the interstitium. Culture media from neonatal rat cardiac myocytes and fibroblasts contained a 55-kDa fragment of beta(1)-integrin. Attachment to ECM components, response to phorbol 12-myristate 13-acetate stimulation, and matrix metalloproteinase inhibition assays demonstrated that fibroblasts responded differently to the fragment compared with myocytes. The beta(1)-integrin fragment stimulated myocyte attachment to collagen and the fragment itself bound a variety of ECM proteins. These studies indicate that as myocytes and fibroblasts change size and shape, cellular contacts with the ECM are altered, resulting in the liberation of a beta(1)-integrin fragment from the cell surface. Integrin shedding may represent a novel mechanism of rapidly modifying cell-ECM contacts during various cellular processes.

  12. Integrin αv in the mechanical response of osteoblast lineage cells

    SciTech Connect

    Kaneko, Keiko; Ito, Masako; Naoe, Yoshinori

    2014-05-02

    Highlights: • Deletion of integrin αv in osteoblast lineage results in an impaired SOST response to loading in vivo. • c-Src–p130Cas–JNK–YAP/TAZ is activated via integrin αv on osteoblasts in response to FSS. • Deletion of integrin αv in osteoblasts results in impaired responses to mechanical stimulation. • Integrin αv is a key component of the mechanosensing machinery in bone. - Abstract: Although osteoblast lineage cells, especially osteocytes, are thought to be a primary mechanosensory cell in bone, the identity of the mechano-receptor and downstream mechano-signaling pathways remain largely unknown. Here we show using osteoblastic cell model of mechanical stimulation withmore » fluid shear stress that in the absence of integrin αv, phosphorylation of the Src substrate p130Cas and JNK was impaired, culminating in an inhibition of nuclear translocation of YAP/TAZ and subsequent transcriptional activation of target genes. Targeted deletion of the integrin αv in osteoblast lineage cells results in an attenuated response to mechanical loading in terms of Sost gene expression, indicative of a role for integrin αv in mechanoreception in vivo. Thus, integrin αv may be integral to a mechanosensing machinery in osteoblastic cells and involved in activation of a Src–JNK–YAP/TAZ pathway in response to mechanical stimulation.« less

  13. Beta-1 Integrin Signaling and Function in MLO-Y4 Osteocyte-Like Cells

    NASA Technical Reports Server (NTRS)

    Searby, N. D.; vanderMeulen, M. C. H.; Dovi, J.; Roden, C.; Banerjee, I.; Kim, J.-B.; Damsky, C. D.; Almeida, E. A. C.; Globus, R. K.

    2004-01-01

    In osteocyte-like cells, disruption of beta-1 integrin signaling by the Beta-1 tail construct: 1) Altered cell morphology; 2) Reduced cell motility; 3) Increased proliferation and final cell density; 4) Reduced cell's ability to maintain shape when subjected to uniaxial strain (1%, 30 min). Thus, beta-1 integrin is important in the response of osteocytic cells to mechanical loading.

  14. A quantitative immunohistochemical study of the expression of integrins by nerves in psoriatic and normal skin.

    PubMed

    Andrew, S M; Edwards, B D; Chalmers, R J; O'Driscoll, J B

    1992-10-01

    Qualitative and quantitative assessment of integrin expression by dermal nerves was made by an avidin-biotin immunoperoxidase method on snap-frozen biopsies from affected psoriatic skin, and skin from normal control subjects with no history of skin disease. Nerves expressed alpha 1, alpha 2, alpha 3, alpha 6, beta 1 and beta 4 integrin subunits, and perineural sheaths in the mid-dermis also expressed these subunits, with the exception of alpha 2. There were more upper dermal nerve segments expressing alpha 1 integrin compared with other integrins both in controls and in psoriatic skin. The greater number of nerves expressing alpha 1 integrin compared with other integrins may be due to anatomical or functional differences between groups of nerves. There were significantly more nerves expressing alpha 1, alpha 2, alpha 3, alpha 6 and beta 4 integrins in psoriatic skin compared with control skin. This generalized increase may indicate a secondary trophic effect on all nerves rather than a specific increase in one type of nerve. However, the expression of alpha 2 integrin may be significant in the pathogenesis of the psoriatic plaque, in that it was barely detectable in the normal site-matched biopsies, but much greater in psoriatic plaques. The study of the expression of adhesion molecules by neurones in psoriasis offers a new avenue for investigation of the role of neuronal hypertrophy in the initiation and maintenance of psoriatic plaques.

  15. Clearance of apoptotic photoreceptors: elimination of apoptotic debris into the subretinal space and macrophage-mediated phagocytosis via phosphatidylserine receptor and integrin alphavbeta3.

    PubMed

    Hisatomi, Toshio; Sakamoto, Taiji; Sonoda, Koh-Hei; Tsutsumi, Chikako; Qiao, Hong; Enaida, Hiroshi; Yamanaka, Ichiro; Kubota, Toshiaki; Ishibashi, Tatsuro; Kura, Shinobu; Susin, Santos A; Kroemer, Guido

    2003-06-01

    The effective phagocytotic clearance of apoptotic debris is fundamental to the maintenance of neural tissues during apoptosis. Retinal photoreceptors undergo apoptosis after retinal detachment. Although their induction phase of apoptosis has been well discussed, their phagocytotic process remains quite unclear. We herein demonstrate that apoptotic photoreceptors are selectively eliminated from their physiological localization, the outer nuclear layer, to the subretinal space, and then phagocytosed by monocyte-derived macrophages. This could be shown by an ultrastructural and immunophenotypic analysis. Moreover, in chimera mice expressing transgenic green fluorescent protein in bone marrow-derived cells, the local infiltration of macrophages could be detected after retinal detachment-induced photoreceptor apoptosis. The local injection of an antibody blocking the phosphatidylserine receptor (PSR) or a peptide (GRGDSP)-blocking integrin alphavbeta3 revealed that phagocytotic clearance involves the PSR as well as integrin alphavbeta3 in vivo. Importantly, the level of blockade obtained with these reagents was different. Although anti-PSR increased the frequency of apoptotic cells that fail to bind to macrophages, GRGDSP prevented the engulfment (but not the recognition) of apoptotic photoreceptor cells by macrophages. To our knowledge, this is the first report describing the mechanisms through which apoptotic photoreceptors are selectively eliminated via a directional process in the subretinal space.

  16. A new in vitro mouse oligodendrocyte precursor cell migration assay reveals a role for integrin-linked kinase in cell motility.

    PubMed

    O'Meara, Ryan W; Cummings, Sarah E; Michalski, John-Paul; Kothary, Rashmi

    2016-02-01

    The decline of remyelination in chronic multiple sclerosis (MS) is in part attributed to inadequate oligodendrocyte precursor cell (OPC) migration, a process governed by the extracellular matrix (ECM). Elucidating the mechanisms underlying OPC migration is therefore an important step towards developing new therapeutic strategies to promote myelin repair. Many seminal OPC culture methods were established using rat-sourced cells, and these often need modification for use with mouse OPCs due to their sensitive nature. It is of interest to develop mouse OPC assays to leverage the abundant transgenic lines. To this end, we developed a new OPC migration method specifically suited for use with mouse-derived cells. To validate its utility, we combined the new OPC migration assay with a conditional knockout approach to investigate the role of integrin-linked kinase (ILK) in OPC migration. ILK is a focal adhesion protein that stabilizes cellular adhesions to the extracellular matrix (ECM) by mediating a linkage between matrix-bound integrin receptors and the cytoskeleton. We identified ILK as a regulator of OPC migration on three permissive substrates. ILK loss produced an early, albeit transient, deficit in OPC migration on laminin matrix, while migration on fibronectin and polylysine was heavily reliant on ILK expression. Inclusively, our work provides a new tool for studying mouse OPC migration and highlights the role of ILK in its regulation on ECM proteins relevant to MS.

  17. Integrin Ligation Results in Nephrin Tyrosine Phosphorylation In Vitro

    PubMed Central

    Verma, Rakesh; Venkatareddy, Madhusudan; Kalinowski, Anne; Patel, Sanjeevkumar R.; Garg, Puneet

    2016-01-01

    Nephrin is expressed at the basolateral aspect of podocytes and is an important signaling protein at the glomerular slit diaphragm. In vitro studies have demonstrated that Nephrin phosphorylation-dependent signaling is able to assemble a protein complex that is able to polymerize actin. However, proximal signaling events that result in nephrin tyrosine phosphorylation are not well understood. Nephrin deletion in mice and human nephrin mutations result in developmental failure of the podocyte intercellular junction resutling in proteinuria. This has been presumed to be due to a failure to respond to an external polarized cue in the absence of nephrin or a failure to transduce an outside-in signal in patients with nephrin mutations. The nephrin extracellular domain binds to itself or neph1 across the foot process intercellular junction. Nephrin is tyrosine phosphorylation-silent in healthy glomeruli when presumably the nephrin extracellular domain is in an engaged state. These observations raise the possibility of an alternate proximal signaling mechanism that might be responsible for nephrin tyrosine phosphorylation. Here we present data showing that integrin engagement at the basal aspect of cultured podocytes results in nephrin tyrosine phosphorylation. This is abrogated by incubating podocytes with an antibody that prevents integrin β1 ligation and activation in response to binding to extracellular matrix. Furthermore, nephrin tyrosine phosphorylation was observed in podocytes expressing a membrane-targeted nephrin construct that lacks the extracellular domain. We propose, integrin-activation based signaling might be responsible for nephrin phosphorylation rather than engagment of the nephrin extracellular domain by a ligand. PMID:26848974

  18. Hypergravity Stimulates Osteoblast Proliferation Via Matrix-Integrin-Signaling Pathways

    NASA Technical Reports Server (NTRS)

    Vercoutere, W.; Parra, M.; Roden, C.; DaCosta, M.; Wing, A.; Damsky, C.; Holton, E.; Searby, N.; Globus, R.; Almeida, E.

    2003-01-01

    Extensive characterizations of the physiologic consequences of microgravity and gravity indicate that lack of weight-bearing may cause tissue atrophy through cellular and subcellular level mechanisms. We hypothesize that gravity is needed for the efficient transduction of cell growth and survival signals from the extra-cellular matrix (ECM) in mechanosensitive tissues. Recent work from our laboratory and from others shows that an increase of gravity increases bone cell growth and survival. We found that 50-g hypergravity stimulation increased osteoblast proliferation for cells grown on Collagen Type I and Fibronectin, but not on Laminin or uncoated plastic. This may be a tissue-specific response, because 50-g hypergravity stimulation caused no increase in proliferation for primary rat fibroblasts. These results combined with RT-PCR for all possible integrins indicate that beta1 integrin subunit may be involved. The osteoblast proliferation response on Collagen Type I was greater at 25-g than at 10-g or 50-g; 24-h duration of hypergravity was necessary to see an increase in proliferation. Survival was enhanced during hypergravity stimulation by the presence of matrix. Flow cytometry analysis indicated that cell cycle may be altered; BrdU incorporation in proliferating cells showed an increase in the number of actively dividing cells from about 60% at 1-g to over 90% at 25-g. To further investigate the molecular components involved, we applied fluorescence labeling of cytoskeletal and signaling molecules to cells after 2 to 30 minutes of hypergravity stimulation. While structural components did not appear to be altered, phosphorylation increased, indicating that signaling pathways may be activated. These data indicate that gravity mechanostimulation of osteoblast proliferation involves specific matrix-integrin signaling pathways which are sensitive to duration and g-level.

  19. Vesicle-associated membrane protein 2 mediates trafficking of {alpha}5{beta}1 integrin to the plasma membrane

    SciTech Connect

    Hasan, Nazarul; Hu, Chuan, E-mail: chuan.hu@louisville.edu

    2010-01-01

    Integrins are major receptors for cell adhesion to the extracellular matrix (ECM). As transmembrane proteins, the levels of integrins at the plasma membrane or the cell surface are ultimately determined by the balance between two vesicle trafficking events: endocytosis of integrins at the plasma membrane and exocytosis of the vesicles that transport integrins. Here, we report that vesicle-associated membrane protein 2 (VAMP2), a SNARE protein that mediates vesicle fusion with the plasma membrane, is involved in the trafficking of {alpha}5{beta}1 integrin. VAMP2 was present on vesicles containing endocytosed {beta}1 integrin. Small interfering RNA (siRNA) silencing of VAMP2 markedly reduced cellmore » surface {alpha}5{beta}1 and inhibited cell adhesion and chemotactic migration to fibronectin, the ECM ligand of {alpha}5{beta}1, without altering cell surface expression of {alpha}2{beta}1 integrin or {alpha}3{beta}1 integrin. By contrast, silencing of VAMP8, another SNARE protein, had no effect on cell surface expression of the integrins or cell adhesion to fibronectin. In addition, VAMP2-mediated trafficking is involved in cell adhesion to collagen but not to laminin. Consistent with disruption of integrin functions in cell proliferation and survival, VAMP2 silencing diminished proliferation and triggered apoptosis. Collectively, these data indicate that VAMP2 mediates the trafficking of {alpha}5{beta}1 integrin to the plasma membrane and VAMP2-dependent integrin trafficking is critical in cell adhesion, migration and survival.« less

  20. Alphavbeta integrins play an essential role in BMP-2 induction of osteoblast differentiation.

    PubMed

    Lai, Chung-Fang; Cheng, Su-Li

    2005-02-01

    Both integrins and BMP-2 exert similar effects on osteoblasts. We examined the relationship between the alphav-containing integrins (alphavbeta) and BMP-2 in osteoblast function. BMP-2 stimulates alphavbeta expression. BMP-2 receptors co-localize/overlap with alphavbeta integrins, and the intact function of alphavbeta is essential in BMP-2 activity. Bone morphogenetic protein (BMP)-2 not only induces osteoblast differentiation and bone matrix mineralization, but also stimulates osteoblast migration on and adhesion to bone matrix proteins. The alphavbeta- and beta1- (alphabeta1) containing integrins mediate osteoblast interaction with many bone matrix proteins and play important roles in osteoblast adhesion, migration, and differentiation. Because alphavbeta integrins and BMP-2 share common effects on osteoblasts, we analyzed their relationship in osteoblast function. The effects of BMP-2 on integrin expression were determined by surface labeling/immunoprecipitation and cell adhesion to matrix proteins. Confocal analysis of the immunostained cells and co-immunoprecipitation of cell extracts were used to study the spatial relationship between integrins and BMP-2 receptors. A function-blocking anti-alphavbeta integrin antibody (L230) was employed to investigate the roles of alphavbeta integrins in BMP-2 function. Human osteoblasts (HOBs) express alphabeta1, alphavbeta3, alphavbeta5, alphavbeta6, and alphavbeta8 integrins at focal adhesion sites. BMP-2 increases the levels of these integrins on osteoblast surface and enhances HOB adhesion to osteopontin and vitronectin. Immunoprecipitation and immunostaining analyses show that BMP-2 receptors co-localize or overlap with alphavbeta and alphabeta1 integrins. Incubation of HOBs with L230 abolishes the antiproliferative effect of BMP-2 and reduces the capacity of BMP-2 to stimulate alkaline phosphatase activity and the expression of osteocalcin, osteopontin, and bone sialoprotein. Furthermore, L230 prevents BMP-2 induction

  1. Guiding plant virus particles to integrin-displaying cells

    NASA Astrophysics Data System (ADS)

    Hovlid, Marisa L.; Steinmetz, Nicole F.; Laufer, Burkhardt; Lau, Jolene L.; Kuzelka, Jane; Wang, Qian; Hyypiä, Timo; Nemerow, Glen R.; Kessler, Horst; Manchester, Marianne; Finn, M. G.

    2012-05-01

    Viral nanoparticles (VNPs) are structurally regular, highly stable, tunable nanomaterials that can be conveniently produced in high yields. Unmodified VNPs from plants and bacteria generally do not show tissue specificity or high selectivity in binding to or entry into mammalian cells. They are, however, malleable by both genetic and chemical means, making them useful scaffolds for the display of large numbers of cell- and tissue-targeting ligands, imaging moieties, and/or therapeutic agents in a well-defined manner. Capitalizing on this attribute, we modified the genetic sequence of the Cowpea mosaic virus (CPMV) coat protein to display an RGD oligopeptide sequence derived from human adenovirus type 2 (HAdV-2). Concurrently, wild-type CPMV was modified via NHS acylation and Cu(i)-catalyzed azide-alkyne cycloaddition (CuAAC) chemistry to attach an integrin-binding cyclic RGD peptide. Both types of particles showed strong and selective affinity for several different cancer cell lines that express RGD-binding integrin receptors.Viral nanoparticles (VNPs) are structurally regular, highly stable, tunable nanomaterials that can be conveniently produced in high yields. Unmodified VNPs from plants and bacteria generally do not show tissue specificity or high selectivity in binding to or entry into mammalian cells. They are, however, malleable by both genetic and chemical means, making them useful scaffolds for the display of large numbers of cell- and tissue-targeting ligands, imaging moieties, and/or therapeutic agents in a well-defined manner. Capitalizing on this attribute, we modified the genetic sequence of the Cowpea mosaic virus (CPMV) coat protein to display an RGD oligopeptide sequence derived from human adenovirus type 2 (HAdV-2). Concurrently, wild-type CPMV was modified via NHS acylation and Cu(i)-catalyzed azide-alkyne cycloaddition (CuAAC) chemistry to attach an integrin-binding cyclic RGD peptide. Both types of particles showed strong and selective affinity

  2. Can transgenic mosquitoes afford the fitness cost?

    PubMed

    Lambrechts, Louis; Koella, Jacob C; Boëte, Christophe

    2008-01-01

    In a recent study, SM1-transgenic Anopheles stephensi, which are resistant partially to Plasmodium berghei, had higher fitness than non-transgenic mosquitoes when they were maintained on Plasmodium-infected blood. This result should be interpreted cautiously with respect to malaria control using transgenic mosquitoes because, despite the evolutionary advantage conferred by the transgene, a concomitant cost prevents it from invading the entire population. Indeed, for the spread of a resistance transgene in a natural situation, the transgene's fitness cost and the efficacy of the gene drive will be more crucial than any evolutionary advantage.

  3. Integrin-mediated cell surface recruitment of autotaxin promotes persistent directional cell migration

    PubMed Central

    Wu, Tao; Kooi, Craig Vander; Shah, Pritom; Charnigo, Richard; Huang, Cai; Smyth, Susan S.; Morris, Andrew J.

    2014-01-01

    Autotaxin (ATX) is a secreted lysophospholipase D (lysoPLD) that binds to integrin adhesion receptors. We dissected the roles of integrin binding and lysoPLD activity in stimulation of human breast cancer and mouse aortic vascular smooth muscle cell migration by ATX. We compared effects of wild-type human ATX, catalytically inactive ATX, an integrin binding-defective ATX variant with wild-type lysoPLD activity, the isolated ATX integrin binding N-terminal domain, and a potent ATX selective lysoPLD inhibitor on cell migration using transwell and single-cell tracking assays. Stimulation of transwell migration was reduced (18 or 27% of control, respectively) but not ablated by inactivation of integrin binding or inhibition of lysoPLD activity. The N-terminal domain increased transwell migration (30% of control). ATX lysoPLD activity and integrin binding were necessary for a 3.8-fold increase in the fraction of migrating breast cancer cell step velocities >0.7 μm/min. ATX increased the persistent directionality of single-cell migration 2-fold. This effect was lysoPLD activity independent and recapitulated by the integrin binding N-terminal domain. Integrin binding enables uptake and intracellular sequestration of ATX, which redistributes to the front of migrating cells. ATX binding to integrins and lysoPLD activity therefore cooperate to promote rapid persistent directional cell migration.—Wu, T., Kooi, C. V., Shah, P., Charnigo, R., Huang, C., Smyth, S. S., Morris, A. J. Integrin-mediated cell surface recruitment of autotaxin promotes persistent directional cell migration. PMID:24277575

  4. Involvement of alpha4 integrins in maintenance of cardiac sympathetic axons.

    PubMed

    Wingerd, Kevin L; Wayne, William C; Jackson, David Y; Clegg, Dennis O

    2005-10-30

    Sympathetic neurons extend and maintain axons that innervate the myocardium, and proper innervation is important for cardiac function. However, the molecular basis for axon outgrowth and maintenance is not well understood. We have shown previously that the integrin alpha4beta1 is expressed on developing axons, and the alpha4 function is important for the development of innervation in vivo [Wingerd, K.L., Goodman, N.L., Tresser, J.W., Smail, M.M., Leu, S.T., Rohan, S.J., Pring, J.L., Jackson, D.Y., and Clegg, D.O., 2002. Alpha 4 integrins and vascular cell adhesion molecule-1 play a role in sympathetic innervation of the heart. J. Neurosci. 22,10772-10780]. Here we examine the function of alpha4beta1 integrins in the maintenance of cardiac sympathetic innervation in vitro and in vivo, and investigate integrin expression and function after myocardial infarction and in hypertensive rats. On substrates of vascular cell adhesion molecule-1 (VCAM-1), alpha4beta1 was required for both initial outgrowth and maintenance of neurites in vitro. On fibronectin substrates, initial outgrowth requires only alpha4 integrins, but maintenance requires both alpha4 integrins and RGD-dependent integrins. In vivo, in adult Long Evans rats, inhibition of alpha4 integrins resulted in decreased maintenance of sympathetic fibers innervating the apex of the heart. However, alpha4 integrins were not detected on most sympathetic axons that sprout after myocardial infarction, and alpha4 function was not required for sprouting. Spontaneously hypertensive rats (SHR) have increased numbers of cardiac sympathetic fibers compared to the parental Wistar strain, but many of these lack alpha4 expression, and alpha4 function is not required for maintenance of these fibers in the heart. These results suggest that developing sympathetic axons and sprouting sympathetic axons use different mechanisms of outgrowth, and that maintenance of cardiac sympathetic innervation involves alpha4 integrins in some rat

  5. Integrin-mediated Cell Attachment Induces a PAK4-dependent Feedback Loop Regulating Cell Adhesion through Modified Integrin αvβ5 Clustering and Turnover

    PubMed Central

    Li, Zhilun; Lock, John G.; Olofsson, Helene; Kowalewski, Jacob M.; Teller, Steffen; Liu, Yajuan

    2010-01-01

    Cell-to-extracellular matrix adhesion is regulated by a multitude of pathways initiated distally to the core cell–matrix adhesion machinery, such as via growth factor signaling. In contrast to these extrinsically sourced pathways, we now identify a regulatory pathway that is intrinsic to the core adhesion machinery, providing an internal regulatory feedback loop to fine tune adhesion levels. This autoinhibitory negative feedback loop is initiated by cell adhesion to vitronectin, leading to PAK4 activation, which in turn limits total cell–vitronectin adhesion strength. Specifically, we show that PAK4 is activated by cell attachment to vitronectin as mediated by PAK4 binding partner integrin αvβ5, and that active PAK4 induces accelerated integrin αvβ5 turnover within adhesion complexes. Accelerated integrin turnover is associated with additional PAK4-mediated effects, including inhibited integrin αvβ5 clustering, reduced integrin to F-actin connectivity and perturbed adhesion complex maturation. These specific outcomes are ultimately associated with reduced cell adhesion strength and increased cell motility. We thus demonstrate a novel mechanism deployed by cells to tune cell adhesion levels through the autoinhibitory regulation of integrin adhesion. PMID:20719960

  6. Integrin Based Isolation Enables Purification of Murine Lineage Committed Cardiomyocytes

    PubMed Central

    Tarnawski, Laura; Xian, Xiaojie; Monnerat, Gustavo; Macaulay, Iain C.; Malan, Daniela; Borgman, Andrew; Wu, Sean M.; Fleischmann, Bernd K.; Jovinge, Stefan

    2015-01-01

    In contrast to mature cardiomyocytes which have limited regenerative capacity, pluripotent stem cells represent a promising source for the generation of new cardiomyocytes. The tendency of pluripotent stem cells to form teratomas and the heterogeneity from various differentiation stages and cardiomyocyte cell sub-types, however, are major obstacles to overcome before this type of therapy could be applied in a clinical setting. Thus, the identification of extracellular markers for specific cardiomyocyte progenitors and mature subpopulations is of particular importance. The delineation of cardiomyocyte surface marker patterns not only serves as a means to derive homogeneous cell populations by FACS, but is also an essential tool to understand cardiac development. By using single-cell expression profiling in early mouse embryonic hearts, we found that a combination of integrin alpha-1, alpha-5, alpha-6 and N-cadherin enables isolation of lineage committed murine cardiomyocytes. Additionally, we were able to separate trabecular cardiomyocytes from solid ventricular myocardium and atrial murine cells. These cells exhibit expected subtype specific phenotype confirmed by electrophysiological analysis. We show that integrin expression can be used for the isolation of living, functional and lineage-specific murine cardiomyocytes. PMID:26323090

  7. Integrin Based Isolation Enables Purification of Murine Lineage Committed Cardiomyocytes.

    PubMed

    Tarnawski, Laura; Xian, Xiaojie; Monnerat, Gustavo; Macaulay, Iain C; Malan, Daniela; Borgman, Andrew; Wu, Sean M; Fleischmann, Bernd K; Jovinge, Stefan

    2015-01-01

    In contrast to mature cardiomyocytes which have limited regenerative capacity, pluripotent stem cells represent a promising source for the generation of new cardiomyocytes. The tendency of pluripotent stem cells to form teratomas and the heterogeneity from various differentiation stages and cardiomyocyte cell sub-types, however, are major obstacles to overcome before this type of therapy could be applied in a clinical setting. Thus, the identification of extracellular markers for specific cardiomyocyte progenitors and mature subpopulations is of particular importance. The delineation of cardiomyocyte surface marker patterns not only serves as a means to derive homogeneous cell populations by FACS, but is also an essential tool to understand cardiac development. By using single-cell expression profiling in early mouse embryonic hearts, we found that a combination of integrin alpha-1, alpha-5, alpha-6 and N-cadherin enables isolation of lineage committed murine cardiomyocytes. Additionally, we were able to separate trabecular cardiomyocytes from solid ventricular myocardium and atrial murine cells. These cells exhibit expected subtype specific phenotype confirmed by electrophysiological analysis. We show that integrin expression can be used for the isolation of living, functional and lineage-specific murine cardiomyocytes.

  8. Redox Regulation of Nonmuscle Myosin Heavy Chain during Integrin Engagement.

    PubMed

    Fiaschi, Tania; Cozzi, Giacomo; Chiarugi, Paola

    2012-01-01

    On the basis of our findings reporting that cell adhesion induces the generation of reactive oxygen species (ROS) after integrin engagement, we were interested in identifying redox-regulated proteins during this process. Mass spectrometry analysis led us to identify nonmuscle myosin heavy chain (nmMHC) as a target of ROS. Our results show that, while nmMHC is reduced in detached/rounded cells, it turns towards an oxidized state in adherent/spread cells due to the integrin-engaged ROS machinery. The functional role of nmMHC redox regulation is suggested by the redox sensitivity of its association with actin, suggesting a role of nmMHC oxidation in cytoskeleton movement. Analysis of muscle MHC (mMHC) redox state during muscle differentiation, a process linked to a great and stable decrease of ROS content, shows that the protein does not undergo a redox control. Hence, we propose that the redox regulation of MHC in nonprofessional muscle cells is mandatory for actin binding during dynamic cytoskeleton rearrangement, but it is dispensable for static and highly organized cytoskeletal contractile architecture in differentiating myotubes.

  9. Integrin activation and focal complex formation in cardiac hypertrophy

    NASA Technical Reports Server (NTRS)

    Laser, M.; Willey, C. D.; Jiang, W.; Cooper, G. 4th; Menick, D. R.; Zile, M. R.; Kuppuswamy, D.

    2000-01-01

    Cardiac hypertrophy is characterized by both remodeling of the extracellular matrix (ECM) and hypertrophic growth of the cardiocytes. Here we show increased expression and cytoskeletal association of the ECM proteins fibronectin and vitronectin in pressure-overloaded feline myocardium. These changes are accompanied by cytoskeletal binding and phosphorylation of focal adhesion kinase (FAK) at Tyr-397 and Tyr-925, c-Src at Tyr-416, recruitment of the adapter proteins p130(Cas), Shc, and Nck, and activation of the extracellular-regulated kinases ERK1/2. A synthetic peptide containing the Arg-Gly-Asp (RGD) motif of fibronectin and vitronectin was used to stimulate adult feline cardiomyocytes cultured on laminin or within a type-I collagen matrix. Whereas cardiocytes under both conditions showed RGD-stimulated ERK1/2 activation, only collagen-embedded cells exhibited cytoskeletal assembly of FAK, c-Src, Nck, and Shc. In RGD-stimulated collagen-embedded cells, FAK was phosphorylated only at Tyr-397 and c-Src association occurred without Tyr-416 phosphorylation and p130(Cas) association. Therefore, c-Src activation is not required for its cytoskeletal binding but may be important for additional phosphorylation of FAK. Overall, our study suggests that multiple signaling pathways originate in pressure-overloaded heart following integrin engagement with ECM proteins, including focal complex formation and ERK1/2 activation, and many of these pathways can be activated in cardiomyocytes via RGD-stimulated integrin activation.

  10. Fructan biosynthesis in transgenic plants.

    PubMed

    Cairns, Andrew J

    2003-01-01

    Data from plants transformed to accumulate fructan are assessed in the context of natural concentrations of reserve carbohydrates and natural fluxes of carbon in primary metabolism: Transgenic fructan accumulation is universally reported as an instantaneous endpoint concentration. In exceptional cases, concentrations of 60-160 mg g(-1) fresh mass were reported and compare favourably with naturally occurring maximal starch and fructan content in leaves and storage organs. Generally, values were less than 20 mg g(-1) for plants transformed with bacterial genes and <9 mg g(-1) for plant-plant transformants. Superficially, the results indicate a marked modification of carbon partitioning. However, transgenic fructan accumulation was generally constitutive and involved accumulation over time-scales of weeks or months. When calculated as a function of accumulation period, fluxes into the transgenic product were low, in the range 0.00002-0.03 nkat g(-1). By comparison with an estimated minimum daily carbohydrate flux in leaves for a natural fructan-accumulating plant in field conditions (37 nkat g(-1)), transgenic fructan accumulation was only 0.00005-0.08% of primary carbohydrate flux and does not indicate radical modification of carbon partitioning, but rather, a quantitatively minor leakage into transgenic fructan. Possible mechanisms for this low fructan accumulation in the transformants are considered and include: (i) rare codon usage in bacterial genes compared with eukaryotes, (ii) low transgene mRNA concentrations caused by low expression and/or high turnover, (iii) resultant low expression of enzyme protein, (iv) resultant low total enzyme activity, (v) inappropriate kinetic properties of the gene products with respect to substrate concentrations in the host, (vi) in situ product hydrolysis, and (vii) levan toxicity. Transformants expressing bacterial fructan synthesis exhibited a number of aberrant phenotypes such as stunting, leaf bleaching, necrosis, reduced

  11. How To Produce and Characterize Transgenic Plants.

    ERIC Educational Resources Information Center

    Savka, Michael A.; Wang, Shu-Yi; Wilson, Mark

    2002-01-01

    Explains the process of establishing transgenic plants which is a very important tool in plant biology and modern agriculture. Produces transgenic plants with the ability to synthesize opines. (Contains 17 references.) (YDS)

  12. High αv Integrin Level of Cancer Cells Is Associated with Development of Brain Metastasis in Athymic Rats.

    PubMed

    Wu, Yingjen Jeffrey; Pagel, Michael A; Muldoon, Leslie L; Fu, Rongwei; Neuwelt, Edward A

    2017-08-01

    Brain metastases commonly occur in patients with malignant skin, lung and breast cancers resulting in high morbidity and poor prognosis. Integrins containing an αv subunit are cell adhesion proteins that contribute to cancer cell migration and cancer progression. We hypothesized that high expression of αv integrin cell adhesion protein promoted metastatic phenotypes in cancer cells. Cancer cells from different origins were used and studied regarding their metastatic ability and intetumumab, anti-αv integrin mAb, sensitivity using in vitro cell migration assay and in vivo brain metastases animal models. The number of brain metastases and the rate of occurrence were positively correlated with cancer cell αv integrin levels. High αv integrin-expressing cancer cells showed significantly faster cell migration rate in vitro than low αv integrin-expressing cells. Intetumumab significantly inhibited cancer cell migration in vitro regardless of αv integrin expression level. Overexpression of αv integrin in cancer cells with low αv integrin level accelerated cell migration in vitro and increased the occurrence of brain metastases in vivo. αv integrin promotes brain metastases in cancer cells and may mediate early steps in the metastatic cascade, such as adhesion to brain vasculature. Targeting αv integrin with intetumumab could provide clinical benefit in treating cancer patients who develop metastases. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  13. A Novel α9 Integrin Ligand, XCL1/Lymphotactin, Is Involved in the Development of Murine Models of Autoimmune Diseases.

    PubMed

    Matsumoto, Naoki; Kon, Shigeyuki; Nakatsuru, Takuya; Miyashita, Tomoe; Inui, Kyosuke; Saitoh, Kodai; Kitai, Yuichi; Muromoto, Ryuta; Kashiwakura, Jun-Ichi; Uede, Toshimitsu; Matsuda, Tadashi

    2017-07-01

    The integrin α9β1 is a key receptor involved in the development of autoimmune diseases. However, the detailed mechanism for the association of α9β1 integrin with its ligands remains unclear. In this study, we introduce XCL1/lymphotactin, a member of the chemokine family, as a novel ligand for α9 integrin. Using α9 integrin-overexpressing NIH3T3 cells and endogenously α9 integrin-expressing human rhabdomyosarcoma cells, the interaction between XCL1 and α9 integrin was confirmed by pull-down assays. XCL1 enhanced α9 integrin-dependent cell migration of these cells, thus acting on α9 integrin as a chemoattractant. We also analyzed the in vivo function of XCL1 in the development of anti-type II collagen Ab-induced inflammatory arthritis (CAIA) in BALB/c mice and experimental autoimmune encephalomyelitis in C57BL/6 mice, because α9 integrin is involved in these autoimmune disease models. In CAIA, recombinant XCL1 aggravated the disease and this exacerbation was inhibited by an anti-α9 integrin Ab. An XCL1-neutralizing Ab produced in this study also ameliorated CAIA. Furthermore, the XCL1-neutralizing Ab abrogated the disease progression in experimental autoimmune encephalomyelitis. Therefore, to our knowledge this study provides the first in vitro and in vivo evidence that the interaction between XCL1 and α9 integrin has an important role for autoimmune diseases. Copyright © 2017 by The American Association of Immunologists, Inc.

  14. The role of Protein Disulfide Isomerase and thiol bonds modifications in activation of integrin subunit alpha11.

    PubMed

    Popielarski, Marcin; Ponamarczuk, Halszka; Stasiak, Marta; Michalec, Lidia; Bednarek, Radoslaw; Studzian, Maciej; Pulaski, Lukasz; Swiatkowska, Maria

    2018-01-08

    Integrins belong to a family of transmembrane receptors that mediate cell migration and adhesion to ECM. Extracellular domains of integrin heterodimers contain cysteine-rich regions, which are potential sites of thiol-disulfide exchanges. Rearrangements of extracellular disulfide bonds regulate activation of integrin receptors by promoting transition from an inactive state into a ligand-binding competent state. Modifications of integrin disulfide bonds dependent on oxidation-reduction can be mediated by Protein Disulfide Isomerse (PDI). This paper provides evidences that binding to integrin ligands initiate changes in free thiol pattern on cell surface and that thiol-disulfide exchange mediated by PDI leads to activation of integrin subunit α11. By employing co-immunoprecipitation and confocal microscopy analysis we showed that α11β1 and PDI create complexes bounded by disulfide bonds. Using surface plasmon resonance we provide biochemical evidence that PDI can interact directly with integrin subunit α11. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Dual role of pericyte α6β1-integrin in tumour blood vessels.

    PubMed

    Reynolds, Louise E; D'Amico, Gabriela; Lechertier, Tanguy; Papachristodoulou, Alexandros; Muñoz-Félix, José M; De Arcangelis, Adèle; Baker, Marianne; Serrels, Bryan; Hodivala-Dilke, Kairbaan M

    2017-05-01

    The α6β1-integrin is a major laminin receptor, and formation of a laminin-rich basement membrane is a key feature in tumour blood vessel stabilisation and pericyte recruitment, processes that are important in the growth and maturation of tumour blood vessels. However, the role of pericyte α6β1-integrin in angiogenesis is largely unknown. We developed mice where the α6-integrin subunit is deleted in pericytes and examined tumour angiogenesis and growth. These mice had: (1) reduced pericyte coverage of tumour blood vessels; (2) reduced tumour blood vessel stability; (3) increased blood vessel diameter; (4) enhanced blood vessel leakiness, and (5) abnormal blood vessel basement membrane architecture. Surprisingly, tumour growth, blood vessel density and metastasis were not altered. Analysis of retinas revealed that deletion of pericyte α6-integrin did not affect physiological angiogenesis. At the molecular level, we provide evidence that pericyte α6-integrin controls PDGFRβ expression and AKT-mTOR signalling. Taken together, we show that pericyte α6β1-integrin regulates tumour blood vessels by both controlling PDGFRβ and basement membrane architecture. These data establish a novel dual role for pericyte α6-integrin as modulating the blood vessel phenotype during pathological angiogenesis. © 2017. Published by The Company of Biologists Ltd.

  16. Applications of snake venom components to modulate integrin activities in cell-matrix interactions

    PubMed Central

    Marcinkiewicz, Cezary

    2013-01-01

    Snake venom proteins are broadly investigated in the different areas of life science. Direct interaction of these compounds with cells may involve a variety of mechanisms that result in diverse cellular responses leading to the activation or blocking of physiological functions of the cell. In this review, the snake venom components interacting with integrins will be characterized in context of their effect on cellular response. Currently, two major families of snake venom proteins are considered as integrin-binding molecules. The most attention has been devoted to the disintegrin family, which binds certain types of integrins through specific motifs recognized as a tri-peptide structurally localized on an integrin-binding loop. Other snake venom integrin-binding proteins belong to the C-type lectin family. Snake venom molecules bind to the cellular integrins resulting in a modulation of cell signaling and in consequence, the regulation of cell proliferation, migration and apoptosis. Therefore, snake venom research on the integrin-binding molecules may have significance in biomedicine and basic cell biology. PMID:23811033

  17. Integrin-Generated Forces Lead to Streptavidin-Biotin Unbinding in Cellular Adhesions

    PubMed Central

    Jurchenko, Carol; Chang, Yuan; Narui, Yoshie; Zhang, Yun; Salaita, Khalid S.

    2014-01-01

    The interplay between chemical and mechanical signals plays an important role in cell biology, and integrin receptors are the primary molecules involved in sensing and transducing external mechanical cues. We used integrin-specific probes in molecular tension fluorescence microscopy to investigate the pN forces exerted by integrin receptors in living cells. The molecular tension fluorescence microscopy probe consisted of a cyclic Arg-Gly-Asp-D-Phe-Lys(Cys) (cRGDfK(C)) peptide tethered to the terminus of a polyethylene glycol polymer that was attached to a surface through streptavidin-biotin linkage. A fluorescence resonance energy transfer mechanism was used to visualize tension-driven extension of the polymer. Surprisingly, we found that integrin receptors dissociate streptavidin-biotin tethered ligands in focal adhesions within 60 min of cell seeding. Although streptavidin-biotin binding affinity is described as the strongest noncovalent bond in nature, and is ∼106 - 108 times larger than that of integrin-RGD affinity, our results suggest that individual integrin-ligand complexes undergo a marked enhancement in stability when the receptor assembles in the cell membrane. Based on the observation of streptavidin-biotin unbinding, we also conclude that the magnitude of integrin-ligand tension in focal adhesions can reach values that are at least 10 fold larger than was previously estimated using traction force microscopy-based methods. PMID:24703305

  18. Dual role of pericyte α6β1-integrin in tumour blood vessels

    PubMed Central

    D'Amico, Gabriela; Lechertier, Tanguy; Papachristodoulou, Alexandros; Muñoz-Félix, José M.; De Arcangelis, Adèle; Baker, Marianne; Serrels, Bryan; Hodivala-Dilke, Kairbaan M.

    2017-01-01

    ABSTRACT The α6β1-integrin is a major laminin receptor, and formation of a laminin-rich basement membrane is a key feature in tumour blood vessel stabilisation and pericyte recruitment, processes that are important in the growth and maturation of tumour blood vessels. However, the role of pericyte α6β1-integrin in angiogenesis is largely unknown. We developed mice where the α6-integrin subunit is deleted in pericytes and examined tumour angiogenesis and growth. These mice had: (1) reduced pericyte coverage of tumour blood vessels; (2) reduced tumour blood vessel stability; (3) increased blood vessel diameter; (4) enhanced blood vessel leakiness, and (5) abnormal blood vessel basement membrane architecture. Surprisingly, tumour growth, blood vessel density and metastasis were not altered. Analysis of retinas revealed that deletion of pericyte α6-integrin did not affect physiological angiogenesis. At the molecular level, we provide evidence that pericyte α6-integrin controls PDGFRβ expression and AKT–mTOR signalling. Taken together, we show that pericyte α6β1-integrin regulates tumour blood vessels by both controlling PDGFRβ and basement membrane architecture. These data establish a novel dual role for pericyte α6-integrin as modulating the blood vessel phenotype during pathological angiogenesis. PMID:28289267

  19. Integrin antagonists as potential therapeutic options for the treatment of Crohn’s disease

    PubMed Central

    McLean, Leon P.; Cross, Raymond K.

    2016-01-01

    Introduction Anti-integrin therapy for the treatment of patients with Crohn’s disease is rapidly evolving. Two agents, natalizumab and vedolizumab, are approved by the United States Food and Drug Administration for the treatment of Crohn’s disease, with vedolizumab the primary anti-integrin used due to a more favorable safety profile. Several other anti-integrins are in various stages of development. Areas Covered This review discusses the current state of anti-integrin therapy as well as suggestions for positioning of these agents in clinical practice. Emerging anti-integrin therapies, their underlying mechanisms of action, and available safety and clinical data are also reviewed. Expert Opinion Anti-integrins are effective for the treatment of Crohn’s disease, even in patients refractory to other therapies. Their use should be considered in patients with Crohn’s disease who do not respond to, develop non-response to, or have contraindications to anti-TNF therapy. Anti-integrin therapies can be offered as a first biologic therapy, in particular for older patients, patients with concurrent multiple sclerosis (natalizumab only), and in patients with contraindications to anti-TNF therapy. In patients with more severe symptoms, providers should consider co-induction with corticosteroids if possible to hasten remission. PMID:26822204

  20. Regulation of Macrophage Foam Cell Formation by αVβ3 Integrin

    PubMed Central

    Antonov, Alexander S.; Kolodgie, Frank D.; Munn, David H.; Gerrity, Ross G.

    2004-01-01

    The accumulation of macrophage foam cells in atherosclerotic lesions is associated with both initiation and progression of this disease. Scavenger receptors CD36 and SRA are the primary receptors responsible for conversion of macrophages into foam cells. Integrin αVβ3 plays a role in the differentiation of several cell types, but its involvement in the transition of macrophages into foam cells and the potential role of this receptor in atherosclerosis have not been examined. Using an in vitro model of single surface receptor activation by binding with an immobilized monoclonal antibody specific to αVβ3 integrin we show that ligation of αVβ3 integrin prevents differentiation of blood monocytes and macrophages into the foam cell phenotype via coordinate down-regulation of CD36 and SRA. This effect of αVβ3 integrin ligation can be reproduced by contact with endothelial cells, whereas the inhibition of αVβ3 receptor ligation restores the uptake of oxidized low-density lipoprotein. Moreover, we found that αVβ3 integrin is readily detected in situ on macrophages in early and advanced atherosclerotic lesions and that in vitro exposure to oxidized low-density lipoprotein up-regulates αVβ3 integrin expression. We hypothesize that αVβ3 integrin regulates macrophage functional maturation into foam cells in a persistent manner, and therefore, by targeting αVβ3 receptor it could potentially be possible to regulate progression of atherosclerosis in humans. PMID:15215180

  1. Galectin-3 alters the lateral mobility and clustering of β1-integrin receptors

    PubMed Central

    Yang, Esther H.; Rode, Julia; Howlader, Md. Amran; Eckermann, Marina; Santos, Jobette T.; Hernandez Armada, Daniel; Zheng, Ruixiang; Zou, Chunxia

    2017-01-01

    Glycoprotein receptors are influenced by myriad intermolecular interactions at the cell surface. Specific glycan structures may interact with endogenous lectins that enforce or disrupt receptor-receptor interactions. Glycoproteins bound by multivalent lectins may form extended oligomers or lattices, altering the lateral mobility of the receptor and influencing its function through endocytosis or changes in activation. In this study, we have examined the interaction of Galectin-3 (Gal-3), a human lectin, with adhesion receptors. We measured the effect of recombinant Gal-3 added exogenously on the lateral mobility of the α5β1 integrin on HeLa cells. Using single-particle tracking (SPT) we detected increased lateral mobility of the integrin in the presence of Gal-3, while its truncated C-terminal domain (Gal-3C) showed only minor reductions in lateral mobility. Treatment of cells with Gal-3 increased β1-integrin mediated migration with no apparent changes in viability. In contrast, Gal-3C decreased both cell migration and viability. Fluorescence microscopy allowed us to confirm that exogenous Gal-3 resulted in reorganization of the integrin into larger clusters. We used a proteomics analysis to confirm that cells expressed endogenous Gal-3, and found that addition of competitive oligosaccharide ligands for the lectin altered the lateral mobility of the integrin. Together, our results are consistent with a Gal-3–integrin lattice model of binding and confirm that the lateral mobility of integrins is natively regulated, in part, by galectins. PMID:29016609

  2. Two Distinct Integrin-Mediated Mechanisms Contribute to Apical Lumen Formation in Epithelial Cells

    PubMed Central

    Myllymäki, Satu Marja; Teräväinen, Terhi Piritta; Manninen, Aki

    2011-01-01

    Background Formation of apical compartments underlies the morphogenesis of most epithelial organs during development. The extracellular matrix (ECM), particularly the basement membrane (BM), plays an important role in orienting the apico-basal polarity and thereby the positioning of apical lumens. Integrins have been recognized as essential mediators of matrix-derived polarity signals. The importance of β1-integrins in epithelial polarization is well established but the significance of the accompanying α-subunits have not been analyzed in detail. Principal Findings Here we demonstrate that two distinct integrin-dependent pathways regulate formation of apical lumens to ensure robust apical membrane biogenesis under different microenvironmental conditions; 1) α2β1- and α6β4-integrins were required to establish a basal cue that depends on Rac1-activity and guides apico-basal cell polarization. 2) α3β1-integrins were implicated in positioning of mitotic spindles in cysts, a process that is essential for Cdc42-driven epithelial hollowing. Significance Identification of the separate processes driven by particular integrin receptors clarifies the functional hierarchies between the different integrins co-expressed in epithelial cells and provides valuable insight into the complexity of cell-ECM interactions thereby guiding future studies addressing the molecular basis of epithelial morphogenesis during development and disease. PMID:21573123

  3. Modeled Microgravity Disrupts Collagen I/Integrin Signaling During Osteoblastic Differentiation of Human Mesenchymal Stem Cells

    NASA Technical Reports Server (NTRS)

    Meyers, Valerie E.; Zayzafoon, Majd; Gonda, Steven R.; Gathings, William E.; McDonald, Jay M.

    2004-01-01

    Spaceflight leads to reduced bone mineral density in weight bearing bones that is primarily attributed to a reduction in bone formation. We have previously demonstrated severely reduced osteoblastogenesis of human mesenchymal stem cells (hMSC) following seven days culture in modeled microgravity. One potential mechanism for reduced osteoblastic differentiation is disruption of type I collagen-integrin interactions and reduced integrin signaling. Integrins are heterodimeric transmembrane receptors that bind extracellular matrix proteins and produce signals essential for proper cellular function, survival, and differentiation. Therefore, we investigated the effects of modeled microgravity on integrin expression and function in hMSC. We demonstrate that seven days of culture in modeled microgravity leads to reduced expression of the extracellular matrix protein, type I collagen (Col I). Conversely, modeled microgravity consistently increases Col I-specific alpha2 and beta1 integrin protein expression. Despite this increase in integrin sub-unit expression, autophosphorylation of adhesion-dependent kinases, focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (PYK2), is significantly reduced. Activation of Akt is unaffected by the reduction in FAK activation. However, reduced downstream signaling via the Ras-MAPK pathway is evidenced by a reduction in Ras and ERK activation. Taken together, our findings indicate that modeled microgravity decreases integrin/MAPK signaling, which likely contributes to the observed reduction in osteoblastogenesis.

  4. Expression Profile of the Integrin Receptor Subunits in the Guinea Pig Sclera

    PubMed Central

    Wang, Kevin K.; Metlapally, Ravikanth; Wildsoet, Christine F.

    2017-01-01

    Purpose The ocular dimensional changes in myopia reflect increased scleral remodeling, and in high myopia, loss of scleral integrity leads to biomechanical weakening and continued scleral creep. As integrins, a type of cell surface receptors, have been linked to scleral remodeling, they represent potential targets for myopia therapies. As a first step, this study aimed to characterize the integrin subunits at the messenger RNA level in the sclera of the guinea pig, a more recently added but increasingly used animal model for myopia research. Methods Primers for α and β integrin subunits were designed using NCBI/UCSC Genome Browser and Primer3 software tools. Total RNA was extracted from normal scleral tissue and isolated cultured scleral fibroblasts, as well as liver and lung, as reference tissues, all from guinea pig. cDNA was produced by reverse transcription, PCR was used to amplify products of predetermined sizes, and products were sequenced using standard methods. Results Guinea pig scleral tissue expressed all known integrin alpha subunits except αD and αE. The latter integrin subunits were also not expressed by cultured guinea pig scleral fibroblasts; however, their expression was confirmed in guinea pig liver. In addition, isolated cultured fibroblasts did not express integrin subunits αL, αM, and αX. This difference between results for cultured cells and intact sclera presumably reflects the presence in the latter of additional cell types. Both guinea pig scleral tissue and isolated scleral fibroblasts expressed all known integrin beta subunits. All results were verified through sequencing. Conclusion The possible contributions of integrins to scleral remodeling make them plausible targets for myopia prevention. Data from this study will help guide future ex vivo and in vitro studies directed at understanding the relationship between scleral integrins and ocular growth regulation in the guinea pig model for myopia. PMID:28094579

  5. A NPxY-independent {beta}5 integrin activation signal regulates phagocytosis of apoptotic cells

    SciTech Connect

    Singh, Sukhwinder; D'mello, Veera; Henegouwen, Paul van Bergen en

    2007-12-21

    Integrin receptors are heterodimeric transmembrane receptors with critical functions in cell adhesion and migration, cell cycle progression, differentiation, apoptosis, and phagocytosis of apoptotic cells. Integrins are activated by intracellular signaling that alter the binding affinity for extracellular ligands, so-called inside to outside signaling. A common element for integrin activation involves binding of the cytoskeletal protein talin, via its FERM domain, to a highly conserved NPxY motif in the {beta} chain cytoplasmic tails, which is involved in long-range conformation changes to the extracellular domain that impinges on ligand affinity. When the human beta-5 ({beta}5) integrin cDNA was expressed in {alpha}v positive,more » {beta}5 and {beta}3 negative hamster CS-1 cells, it promoted NPxY-dependent adhesion to VTN-coated surfaces, phosphorylation of FAK, and concomitantly, {beta}5 integrin-EGFP protein was recruited into talin and paxillin-containing focal adhesions. Expression of a NPxY destabilizing {beta}5 mutant (Y750A) abrogated adhesion and {beta}5-Y750A-EGFP was excluded from focal adhesions at the tips of stress fibers. Surprisingly, expression of {beta}5 Y750A integrin had a potent gain-of-function effect on apoptotic cell phagocytosis, and further, a {beta}5-Y750A-EGFP fusion integrin readily bound MFG-E8-coated 10 {mu}m diameter microspheres developed as apoptotic cell mimetics. The critical sequences in {beta}5 integrin were mapped to a YEMAS motif just proximal to the NPxY motif. Our studies suggest that the phagocytic function of {beta}5 integrin is regulated by an unconventional NPxY-talin-independent activation signal and argue for the existence of molecular switches in the {beta}5 cytoplasmic tail for adhesion and phagocytosis.« less

  6. Exclusion of Integrins from CNS Axons Is Regulated by Arf6 Activation and the AIS

    PubMed Central

    Franssen, Elske H. P.; Zhao, Rong-Rong; Koseki, Hiroaki; Kanamarlapudi, Venkateswarlu; Hoogenraad, Casper C.

    2015-01-01

    Integrins are adhesion and survival molecules involved in axon growth during CNS development, as well as axon regeneration after injury in the peripheral nervous system (PNS). Adult CNS axons do not regenerate after injury, partly due to a low intrinsic growth capacity. We have previously studied the role of integrins in axon growth in PNS axons; in the present study, we investigate whether integrin mechanisms involved in PNS regeneration may be altered or lacking from mature CNS axons by studying maturing CNS neurons in vitro. In rat cortical neurons, we find that integrins are present in axons during initial growth but later become restricted to the somato-dendritic domain. We investigated how this occurs and whether it can be altered to enhance axonal growth potential. We find a developmental change in integrin trafficking; transport becomes predominantly retrograde throughout axons, but not dendrites, as neurons mature. The directionality of transport is controlled through the activation state of ARF6, with developmental upregulation of the ARF6 GEF ARNO enhancing retrograde transport. Lowering ARF6 activity in mature neurons restores anterograde integrin flow, allows transport into axons, and increases axon growth. In addition, we found that the axon initial segment is partly responsible for exclusion of integrins and removal of this structure allows integrins into axons. Changing posttranslational modifications of tubulin with taxol also allows integrins into the proximal axon. The experiments suggest that the developmental loss of regenerative ability in CNS axons is due to exclusion of growth-related molecules due to changes in trafficking. PMID:26019348

  7. Redox-Relevant Aspects of the Extracellular Matrix and Its Cellular Contacts via Integrins

    PubMed Central

    de Rezende, Flávia Figueiredo

    2014-01-01

    Abstract Significance: The extracellular matrix (ECM) fulfills essential functions in multicellular organisms. It provides the mechanical scaffold and environmental cues to cells. Upon cell attachment, the ECM signals into the cells. In this process, reactive oxygen species (ROS) are physiologically used as signalizing molecules. Recent Advances: ECM attachment influences the ROS-production of cells. In turn, ROS affect the production, assembly and turnover of the ECM during wound healing and matrix remodeling. Pathological changes of ROS levels lead to excess ECM production and increased tissue contraction in fibrotic disorders and desmoplastic tumors. Integrins are cell adhesion molecules which mediate cell adhesion and force transmission between cells and the ECM. They have been identified as a target of redox-regulation by ROS. Cysteine-based redox-modifications, together with structural data, highlighted particular regions within integrin heterodimers that may be subject to redox-dependent conformational changes along with an alteration of integrin binding activity. Critical Issues: In a molecular model, a long-range disulfide-bridge within the integrin β-subunit and disulfide bridges within the genu and calf-2 domains of the integrin α-subunit may control the transition between the bent/inactive and upright/active conformation of the integrin ectodomain. These thiol-based intramolecular cross-linkages occur in the stalk domain of both integrin subunits, whereas the ligand-binding integrin headpiece is apparently unaffected by redox-regulation. Future Directions: Redox-regulation of the integrin activation state may explain the effect of ROS in physiological processes. A deeper understanding of the underlying mechanism may open new prospects for the treatment of fibrotic disorders. Antioxid. Redox Signal. 20, 1977–1993. PMID:24040997

  8. Structure of Adenovirus Complexed with Its Internalization Receptor, αvβ5 Integrin

    PubMed Central

    Chiu, Charles Y.; Mathias, Patricia; Nemerow, Glen R.; Stewart, Phoebe L.

    1999-01-01

    The three-dimensional structure of soluble recombinant integrin αvβ5 bound to human adenovirus types 2 and 12 (Ad2 and -12) has been determined at ∼21-Å resolution by cryoelectron microscopy (cryo-EM). The αvβ5 integrin is known to promote Ad cell entry. Cryo-EM has shown that the integrin-binding RGD (Arg-Gly-Asp) protrusion of the Ad2 penton base protein is highly mobile (P. L. Stewart, C. Y. Chiu, S. Huang, T. Muir, Y. Zhao, B. Chait, P. Mathias, and G. R. Nemerow, EMBO J. 16:1189–1198, 1997). Sequence analysis indicated that the Ad12 RGD surface loop is shorter than that of Ad2 and probably less flexible, hence more suitable for structural characterization of the Ad-integrin complex. The cryo-EM structures of the two virus-receptor complexes revealed a ring of integrin density above the penton base of each virus serotype. As expected, the integrin density in the Ad2 complex was diffuse while that in the Ad12 complex was better defined. The integrin consists of two discrete subdomains, a globular domain with an RGD-binding cleft ∼20 Å in diameter and a distal domain with extended, flexible tails. Kinetic analysis of Ad2 interactions with αvβ5 indicated ∼4.2 integrin molecules bound per penton base at close to saturation. These results suggest that the precise spatial arrangement of five RGD protrusions on the penton base promotes integrin clustering and the signaling events required for virus internalization. PMID:10400774

  9. Expression Profile of the Integrin Receptor Subunits in the Guinea Pig Sclera.

    PubMed

    Wang, Kevin K; Metlapally, Ravikanth; Wildsoet, Christine F

    2017-06-01

    The ocular dimensional changes in myopia reflect increased scleral remodeling, and in high myopia, loss of scleral integrity leads to biomechanical weakening and continued scleral creep. As integrins, a type of cell surface receptors, have been linked to scleral remodeling, they represent potential targets for myopia therapies. As a first step, this study aimed to characterize the integrin subunits at the messenger RNA level in the sclera of the guinea pig, a more recently added but increasingly used animal model for myopia research. Primers for α and β integrin subunits were designed using NCBI/UCSC Genome Browser and Primer3 software tools. Total RNA was extracted from normal scleral tissue and isolated cultured scleral fibroblasts, as well as liver and lung, as reference tissues, all from guinea pig. cDNA was produced by reverse transcription, PCR was used to amplify products of predetermined sizes, and products were sequenced using standard methods. Guinea pig scleral tissue expressed all known integrin alpha subunits except αD and αE. The latter integrin subunits were also not expressed by cultured guinea pig scleral fibroblasts; however, their expression was confirmed in guinea pig liver. In addition, isolated cultured fibroblasts did not express integrin subunits αL, αM, and αX. This difference between results for cultured cells and intact sclera presumably reflects the presence in the latter of additional cell types. Both guinea pig scleral tissue and isolated scleral fibroblasts expressed all known integrin beta subunits. All results were verified through sequencing. The possible contributions of integrins to scleral remodeling make them plausible targets for myopia prevention. Data from this study will help guide future ex vivo and in vitro studies directed at understanding the relationship between scleral integrins and ocular growth regulation in the guinea pig model for myopia.

  10. Axonal Localization of Integrins in the CNS Is Neuronal Type and Age Dependent

    PubMed Central

    Soleman, Sara; Mason, Matthew R. J.; Verhaagen, Joost; Bensadoun, Jean-Charles; Aebischer, Patrick

    2016-01-01

    The regenerative ability of CNS axons decreases with age, however, this ability remains largely intact in PNS axons throughout adulthood. These differences are likely to correspond with age-related silencing of proteins necessary for axon growth and elongation. In previous studies, it has been shown that reintroduction of the α9 integrin subunit (tenascin-C receptor, α9) that is downregulated in adult CNS can improve neurite outgrowth and sensory axon regeneration after a dorsal rhizotomy or a dorsal column crush spinal cord lesion. In the current study, we demonstrate that virally expressed integrins (α9, α6, or β1 integrin) in the adult rat sensorimotor cortex and adult red nucleus are excluded from axons following neuronal transduction. Attempts to stimulate transport by inclusion of a cervical spinal injury and thus an upregulation of extracellular matrix molecules at the lesion site, or cotransduction with its binding partner, β1 integrin, did not induce integrin localization within axons. In contrast, virally expressed α9 integrin in developing rat cortex (postnatal day 5 or 10) demonstrated clear localization of integrins in cortical axons revealed by the presence of integrin in the axons of the corpus callosum and internal capsule, as well as in the neuronal cell body. Furthermore, examination of dorsal root ganglia neurons and retinal ganglion cells demonstrated integrin localization both within peripheral nerve as well as dorsal root axons and within optic nerve axons, respectively. Together, our results suggest a differential ability for in vivo axonal transport of transmembrane proteins dependent on neuronal age and subtype. PMID:27570822

  11. Human health and transgenic crops

    USDA-ARS?s Scientific Manuscript database

    Under the joint auspices of the Agrochemical and the Agricultural and Food Chemistry Divisions of the American Chemical Society, we organized a short symposium on “Human Health and Transgenic Crops” at the 244th ACS national meeting, held August 19-23, 2012 in Philadelphia, PA, to examine an array o...

  12. Mechanical control of cyclic AMP signalling and gene transcription through integrins

    NASA Technical Reports Server (NTRS)

    Meyer, C. J.; Alenghat, F. J.; Rim, P.; Fong, J. H.; Fabry, B.; Ingber, D. E.

    2000-01-01

    This study was carried out to discriminate between two alternative hypotheses as to how cells sense mechanical forces and transduce them into changes in gene transcription. Do cells sense mechanical signals through generalized membrane distortion or through specific transmembrane receptors, such as integrins? Here we show that mechanical stresses applied to the cell surface alter the cyclic AMP signalling cascade and downstream gene transcription by modulating local release of signals generated by activated integrin receptors in a G-protein-dependent manner, whereas distortion of integrins in the absence of receptor occupancy has no effect.

  13. Fluorescent angiogenesis models using gelfoam® implanted in transgenic mice expressing fluorescent proteins.

    PubMed

    Hoffman, Robert M

    2014-01-01

    Fidler's group described an in vivo angiogenesis assay utilizing Gelfoam(®) sponges impregnated with agarose and proangiogenic factors. Vessels were detected by staining with fluorescent antibodies against CD31. We showed that Gelfoam(®) implanted in transgenic mice expressing the nestin promoter-driven green fluorescent protein (ND-GFP mice) was rapidly vascularized with ND-GFP-expressing nascent blood vessels. Angiogenesis in the Gelfoam(®) was quantified by measuring the total length of ND-GFP-expressing nascent blood vessels in a skin flap by in vivo fluorescence microscopy imaging. The ND-GFP-expressing nascent blood vessels formed a network on the surface of the basic fibroblast growth factor (bFGF)-treated Gelfoam(®). We then developed a color-coded imaging model that can visualize the interaction between αv integrin linked to green fluorescent protein (GFP) in osteosarcoma cells and blood vessels in Gelfoam(®) vascularized after implantation in red fluorescent protein (RFP) transgenic nude mice. The implanted Gelfoam(®) became highly vascularized with RFP-expressing vessels in 14 days. 143B osteosarcoma cells expressing αv integrin-GFP were injected into the Gelfoam(®) after transplantation of Gelfoam(®). After cancer cell injection, cancer cells interacting with blood vessels were observed in the Gelfoam(®) by color-coded confocal microscopy through the skin flap window. We developed another color-coded Gelfoam(®)-based imaging model that can visualize the anastomosis between blood vessels. RFP-expressing vessels in vascularized Gelfoam(®), previously transplanted into RFP transgenic mice, were re-transplanted into ND-GFP mice. Skin flaps were made and anastomosis between the GFP-expressing nascent blood vessels of ND-GFP transgenic nude mice and RFP blood vessels in the transplanted Gelfoam(®) could be imaged. Our results demonstrate that the Gelfoam(®) in vivo angiogenesis model in combination with fluorescent protein labeling of blood

  14. Targeting Integrin-Dependent Adhesion and Signaling with 3-Arylquinoline and 3-Aryl-2-Quinolone Derivatives: A new Class of Integrin Antagonists

    PubMed Central

    Fiorucci, Sandrine; Lin, Xiaochen; Sadoul, Karin; Fournet, Guy; Bouvard, Daniel; Vinogradova, Olga; Joseph, Benoît; Block, Marc R.

    2015-01-01

    We previously reported the anti-migratory function of 3-aryl-2-quinolone derivatives, chemically close to flavonoids (Joseph et al., 2002). Herein we show that 3-arylquinoline or 3-aryl-2-quinolone derivatives disrupt cell adhesion in a dose dependent and reversible manner yet antagonized by artificial integrin activation such as manganese. Relying on this anti-adhesive activity, a Structure-Activity Relationship (SAR) study was established on 20 different compounds to throw the bases of future optimization strategies. Active drugs efficiently inhibit platelet spreading, aggregation, and clot retraction, processes that rely on αllbβ3 integrin activation and clustering. In vitro these derivatives interfere with β3 cytoplasmic tail interaction with kindlin-2 in pulldown assays albeit little effect was observed with pure proteins suggesting that the drugs may block an alternative integrin activation process that may not be directly related to kindlin recruitment. Ex vivo, these drugs blunt integrin signaling assayed using focal adhesion kinase auto-phosphorylation as a read-out. Hence, 3-arylquinoline and 3-aryl-2-quinolone series are a novel class of integrin activation and signaling antagonists. PMID:26509443

  15. Detection of bidirectional signaling during integrin activation and neutrophil adhesion.

    PubMed

    Altman, Stuart M; Dixit, Neha; Simon, Scott I

    2014-01-01

    Neutrophil arrest and migration on inflamed endothelium is dependent upon a conformational shift in CD11a/CD18 (LFA-1) from a low to high affinity and clustered state which determines the strength and lifetime of bond formation with intracellular adhesion molecule 1 (ICAM-1). Cytoskeletal adaptor proteins kindlin-3 and talin-1 anchor clustered LFA-1 to the cytoskeleton and support the transition from neutrophil rolling to arrest. We employ microfluidic flow channels and total internal reflection fluorescence microscopy to evaluate the spatiotemporal regulation of LFA-1 affinity and bond formation that facilitate the transition from neutrophil rolling to arrest. Methodology is presented to correlate the relationship between integrin conformation, bond formation with ICAM-1, and cytoskeletal engagement and adhesion strengthening necessary to achieve a migratory phenotype.

  16. Increased Adenovirus Type 5 Mediated Transgene Expression Due to RhoB Down-Regulation

    PubMed Central

    Majhen, Dragomira; Stojanović, Nikolina; Vukić, Dunja; Pichon, Chantal; Leduc, Chloé; Osmak, Maja; Ambriović-Ristov, Andreja

    2014-01-01

    Adenovirus type 5 (Ad5) is a non-enveloped DNA virus frequently used as a gene transfer vector. Efficient Ad5 cell entry depends on the availability of its primary receptor, coxsackie and adenovirus receptor, which is responsible for attachment, and integrins, secondary receptors responsible for adenovirus internalization via clathrin-mediated endocytosis. However, efficacious adenovirus-mediated transgene expression also depends on successful trafficking of Ad5 particles to the nucleus of the target cell. It has been shown that changes occurring in tumor cells during development of resistance to anticancer drugs can be beneficial for adenovirus mediated transgene expression. In this study, using an in vitro model consisting of a parental cell line, human laryngeal carcinoma HEp2 cells, and a cisplatin-resistant clone CK2, we investigated the cause of increased Ad5-mediated transgene expression in CK2 as compared to HEp2 cells. We show that the primary cause of increased Ad5-mediated transgene expression in CK2 cells is not modulation of receptors on the cell surface or change in Ad5wt attachment and/or internalization, but is rather the consequence of decreased RhoB expression. We propose that RhoB plays an important role in Ad5 post-internalization events and more particularly in Ad5 intracellular trafficking. To the best of our knowledge, this is the first study showing changed Ad5 trafficking pattern between cells expressing different amount of RhoB, indicating the role of RhoB in Ad5 intracellular trafficking. PMID:24466204

  17. Crim1 regulates integrin signaling in murine lens development

    PubMed Central

    Zhang, Ying; Fan, Jieqing; Ho, Joshua W. K.; Hu, Tommy; Kneeland, Stephen C.; Fan, Xueping; Xi, Qiongchao; Sellarole, Michael A.; de Vries, Wilhelmine N.; Lu, Weining; Lachke, Salil A.; Lang, Richard A.; John, Simon W. M.; Maas, Richard L.

    2016-01-01

    The developing lens is a powerful system for investigating the molecular basis of inductive tissue interactions and for studying cataract, the leading cause of blindness. The formation of tightly controlled cell-cell adhesions and cell-matrix junctions between lens epithelial (LE) cells, between lens fiber (LF) cells, and between these two cell populations enables the vertebrate lens to adopt a highly ordered structure and acquire optical transparency. Adhesion molecules are thought to maintain this ordered structure, but little is known about their identity or interactions. Cysteine-rich motor neuron 1 (Crim1), a type I transmembrane protein, is strongly expressed in the developing lens and its mutation causes ocular disease in both mice and humans. How Crim1 regulates lens morphogenesis is not understood. We identified a novel ENU-induced hypomorphic allele of Crim1, Crim1glcr11, which in the homozygous state causes cataract and microphthalmia. Using this and two other mutant alleles, Crim1null and Crim1cko, we show that the lens defects in Crim1 mouse mutants originate from defective LE cell polarity, proliferation and cell adhesion. Crim1 adhesive function is likely to be required for interactions both between LE cells and between LE and LF cells. We show that Crim1 acts in LE cells, where it colocalizes with and regulates the levels of active β1 integrin and of phosphorylated FAK and ERK. The RGD and transmembrane motifs of Crim1 are required for regulating FAK phosphorylation. These results identify an important function for Crim1 in the regulation of integrin- and FAK-mediated LE cell adhesion during lens development. PMID:26681494

  18. Identification of LRP-1 as an endocytosis and recycling receptor for β1-integrin in thyroid cancer cells

    PubMed Central

    Theret, Louis; Jeanne, Albin; Langlois, Benoit; Hachet, Cathy; David, Marion; Khrestchatisky, Michel; Devy, Jérôme; Hervé, Emonard; Almagro, Sébastien; Dedieu, Stéphane

    2017-01-01

    LRP-1 is a large endocytic receptor mediating the clearance of various molecules from the extracellular matrix. LRP-1 was reported to control focal adhesion turnover to optimize the adhesion-deadhesion balance to support invasion. To better understand how LRP-1 coordinates cell-extracellular matrix interface, we explored its ability to regulate cell surface integrins in thyroid carcinomas. Using an antibody approach, we demonstrated that β1-integrin levels were increased at the plasma membrane under LRP1 silencing or upon RAP treatment, used as LRP-1 antagonist. Our data revealed that LRP-1 binds with both inactive and active β1-integrin conformations and identified the extracellular ligand-binding domains II or IV of LRP-1 as sufficient to bind β1-integrin. Using a recombinant β1-integrin, we demonstrated that LRP-1 acts as a regulator of β1-integrin intracellular traffic. Moreover, RAP or LRP-1 blocking antibodies decreased up to 36% the number of β1-integrin-containing endosomes. LRP-1 blockade did not significantly affect the levels of β1-integrin-containing lysosomes while decreasing localization of β1-integrin within Rab-11 positive vesicles. Overall, we identified an original molecular process in which LRP-1 acts as a main regulator of β1-integrin internalization and recycling in thyroid cancer cells. PMID:29108253

  19. Sialylation of Integrin beta1 is Involved in Radiation-Induced Adhesion and Migration in Human Colon Cancer Cells

    SciTech Connect

    Lee, Minyoung; Lee, Hae-June; Seo, Woo Duck

    2010-04-15

    Purpose: Previously, we reported that radiation-induced ST6 Gal I gene expression was responsible for an increase of integrin beta1 sialylation. In this study, we have further investigated the function of radiation-mediated integrin beta1 sialylation in colon cancer cells. Methods and Materials: We performed Western blotting and lectin affinity assay to analyze the expression and level of sialylated integrin beta1. After exposure to ionizing radiation (IR), adhesion and migration of cells were measured by in vitro adhesion and migration assay. Results: IR increased sialylation of integrin beta1 responsible for its increased protein stability and adhesion and migration of colon cancer cells.more » However, for cells with an N-glycosylation site mutant of integrin beta1 located on the I-like domain (Mu3), these effects were dramatically inhibited. In addition, integrin beta1-mediated radioresistance was not observed in cells containing this mutant. When sialylation of integrin beta1 was targeted with a sulfonamide chalcone compound, inhibition of radiation-induced sialylation of integrin beta1 and inhibition of radiation-induced adhesion and migration occurred. Conclusion: The increase of integrin beta1 sialylation by ST6 Gal I is critically involved in radiation-mediated adhesion and migration of colon cancer cells. From these findings, integrin beta1 sialylation may be a novel target for overcoming radiation-induced survival, especially radiation-induced adhesion and migration.« less

  20. Molecular mechanisms of mechanotransduction in integrin-mediated cell-matrix adhesion

    PubMed Central

    Li, Zhenhai; Lee, Hyunjung; Zhu, Cheng

    2016-01-01

    Cell-matrix adhesion complexes are multi-protein structures linking the extracellular matrix (ECM) to the cytoskeleton. They are essential to both cell motility and function by bidirectionally sensing and transmitting mechanical and biochemical stimulations. Several types of cell-matrix adhesions have been identified and they share many key molecular components, such as integrins and actin-integrin linkers. Mechanochemical coupling between ECM molecules and the actin cytoskeleton has been observed from the single cell to the single molecule level and from immune cells to neuronal cells. However, the mechanisms underlying force regulation of integrin-mediated mechanotransduction still need to be elucidated. In this review article, we focus on integrin-mediated adhesions and discuss force regulation of cell-matrix adhesions and key adaptor molecules, three different force-dependent behaviors, and molecular mechanisms for mechanochemical coupling in force regulation. PMID:27720950

  1. Phospho-Tyrosine(s) vs. Phosphatidylinositol Binding in Shc Mediated Integrin Signaling

    PubMed Central

    Lin, Xiaochen; Vinogradova, Olga

    2015-01-01

    The Shc adaptor protein, particularly its p52 isoform, has been identified as a primary signaling partner for the tyrosine(s)-phosphorylated cytoplasmic tails of activated β3 integrins. Inspired by our recent structure of the Shc PTB domain in complex with a bi-phosphorylated peptide derived from β3 cytoplasmic tail, we have initiated the investigation of Shc interaction with phospholipids of the membrane. We are particularly focused on PtdIns and their effects on Shc mediated integrin signaling in vitro. Here we present thermodynamic profiles and molecular details of the interactions between Shc, integrin, and PtdIns, all of which have been studied by ITC and solution NMR methods. A model of p52 Shc interaction with phosphorylated β3 integrin cytoplasmic tail at the cytosolic face of the plasma membrane is proposed based on these data. PMID:25893141

  2. Phospho-Tyrosine(s) vs. Phosphatidylinositol Binding in Shc Mediated Integrin Signaling.

    PubMed

    Lin, Xiaochen; Vinogradova, Olga

    2015-04-01

    The Shc adaptor protein, particularly its p52 isoform, has been identified as a primary signaling partner for the tyrosine(s)-phosphorylated cytoplasmic tails of activated β 3 integrins. Inspired by our recent structure of the Shc PTB domain in complex with a bi-phosphorylated peptide derived from β 3 cytoplasmic tail, we have initiated the investigation of Shc interaction with phospholipids of the membrane. We are particularly focused on PtdIns and their effects on Shc mediated integrin signaling in vitro . Here we present thermodynamic profiles and molecular details of the interactions between Shc, integrin, and PtdIns, all of which have been studied by ITC and solution NMR methods. A model of p52 Shc interaction with phosphorylated β 3 integrin cytoplasmic tail at the cytosolic face of the plasma membrane is proposed based on these data.

  3. EFFECT OF METHYL MERCURY CHLORIDE EXPOSURE ON PC12 CELL INTEGRIN EXPRESSION AND FUNCTION.

    EPA Science Inventory

    Integrins are heterodimeric transmembrane cell adhesion proteins composed of a and b protein subunits. They are important during brain development in a number of critical functions, including cell migration (Georges-Labouesse, et al., 1998), axonal elongation (Murase and Hayashi...

  4. Fibronectin and integrin alpha 5 play requisite roles in cardiac morphogenesis

    PubMed Central

    Mittal, Ashok; Pulina, Maria; Hou, Shuan-Yu; Astrof, Sophie

    2013-01-01

    Summary Fibronectin and its major receptor, integrin α5β1 are required for embryogenesis. These mutants have similar phenotypes, although, defects in integrin α5-deficient mice are milder. In this paper, we examined heart development in those mutants, in which the heart is formed, and discovered that both fibronectin and integrin α5 were required for cardiac morphogenesis, and in particular, for the formation of the cardiac outflow tract. We found that Isl1+ precursors are specified and migrate into the heart in fibronectin- or integrin α5- mutant embryos, however, the hearts in these mutants are of aberrant shape, and the cardiac outflow tracts are short and malformed. We show that these defects are likely due to the requirement for cell adhesion to fibronectin for proliferation of myocardial progenitors and for Fgf8 signaling in the pharyngeal region. PMID:23791818

  5. Method of increasing radiation sensitivity by inhibition of beta one integrin

    DOEpatents

    Park, Catherine [San Francisco, CA; Bissell, Mina J [Berkeley, CA

    2009-11-17

    A method for increasing or monitoring apoptosis in tumor cells by the co-administration of ionizing radiation and an anti-integrin antibody. Increasing apoptosis reduces tumor growth in vivo and in a cell culture model. The antibody is directed against the beta-1 integrin subunit and is inhibitory of beta-1 integrin signaling. Other molecules having an inhibitory effect on beta-1 integrin, either in signaling or in binding to its cognate extracellular receptors may also be used. The present method is particularly of interest in treatment of tumor cells associated with breast cancer, wherein radiation is currently used alone. The present method further contemplates a monoclonal antibody suitable for human administration that may further comprise a radioisotope attached thereto.

  6. R-Ras regulates beta1-integrin trafficking via effects on membrane ruffling and endocytosis.

    PubMed

    Conklin, Matthew W; Ada-Nguema, Aude; Parsons, Maddy; Riching, Kristin M; Keely, Patricia J

    2010-02-18

    Integrin-mediated cell adhesion and spreading is dramatically enhanced by activation of the small GTPase, R-Ras. Moreover, R-Ras localizes to the leading edge of migrating cells, and regulates membrane protrusion. The exact mechanisms by which R-Ras regulates integrin function are not fully known. Nor is much known about the spatiotemporal relationship between these two molecules, an understanding of which may provide insight into R-Ras regulation of integrins. GFP-R-Ras localized to the plasma membrane, most specifically in membrane ruffles, in Cos-7 cells. GFP-R-Ras was endocytosed from these ruffles, and trafficked via multiple pathways, one of which involved large, acidic vesicles that were positive for Rab11. Cells transfected with a dominant negative form of GFP-R-Ras did not form ruffles, had decreased cell spreading, and contained numerous, non-trafficking small vesicles. Conversely, cells transfected with the constitutively active form of GFP-R-Ras contained a greater number of ruffles and large vesicles compared to wild-type transfected cells. Ruffle formation was inhibited by knock-down of endogenous R-Ras with siRNA, suggesting that activated R-Ras is not just a component of, but also an architect of ruffle formation. Importantly, beta1-integrin co-localized with endogenous R-Ras in ruffles and endocytosed vesicles. Expression of dominant negative R-Ras or knock down of R-Ras by siRNA prevented integrin accumulation into ruffles, impaired endocytosis of beta1-integrin, and decreased beta1-integrin-mediated adhesion. Knock-down of R-Ras also perturbed the dynamics of another membrane-localized protein, GFP-VSVG, suggesting a more global role for R-Ras on membrane dynamics. However, while R-Ras co-internalized with integrins, it did not traffic with VSVG, which instead moved laterally out of ruffles within the plane of the membrane, suggesting multiple levels of regulation of and by R-Ras. Our results suggest that integrin function involves integrin

  7. Reducing the native tropism of adenovirus vectors requires removal of both CAR and integrin interactions.

    PubMed

    Einfeld, D A; Schroeder, R; Roelvink, P W; Lizonova, A; King, C R; Kovesdi, I; Wickham, T J

    2001-12-01

    The development of tissue-selective virus-based vectors requires a better understanding of the role of receptors in gene transfer in vivo, both to rid the vectors of their native tropism and to introduce new specificity. CAR and alphav integrins have been identified as the primary cell surface components that interact with adenovirus type 5 (Ad5)-based vectors during in vitro transduction. We have constructed a set of four vectors, which individually retain the wild-type cell interactions, lack CAR binding, lack alphav integrin binding, or lack both CAR and alphav integrin binding. These vectors have been used to examine the roles of CAR and alphav integrin in determining the tropism of Ad vectors in a mouse model following intrajugular or intramuscular injection. CAR was found to play a significant role in liver transduction. The absence of CAR binding alone, however, had little effect on the low level of expression from Ad in other tissues. Binding of alphav integrins appeared to have more influence than did binding of CAR in promoting the expression in these tissues and was also found to be important in liver transduction by Ad vectors. An effect of the penton base modification was a reduction in the number of vector genomes that could be detected in several tissues. In the liver, where CAR binding is important, combining defects in CAR and alphav integrin binding was essential to effectively reduce the high level of expression from Ad vectors. While there may be differences in Ad vector tropism among species, our results indicate that both CAR and alphav integrins can impact vector distribution in vivo. Disruption of both CAR and alphav integrin interactions may be critical for effectively reducing native tropism and enhancing the efficacy of specific targeting ligands in redirecting Ad vectors to target tissues.

  8. Disruption of integrin-fibronectin complexes by allosteric but not ligand-mimetic inhibitors.

    PubMed

    Mould, A Paul; Craig, Susan E; Byron, Sarah K; Humphries, Martin J; Jowitt, Thomas A

    2014-12-15

    Failure of Arg-Gly-Asp (RGD)-based inhibitors to reverse integrin-ligand binding has been reported, but the prevalence of this phenomenon among integrin heterodimers is currently unknown. In the present study we have investigated the interaction of four different RGD-binding integrins (α5β1, αVβ1, αVβ3 and αVβ6) with fibronectin (FN) using surface plasmon resonance. The ability of inhibitors to reverse ligand binding was assessed by their capacity to increase the dissociation rate of pre-formed integrin-FN complexes. For all four receptors we showed that RGD-based inhibitors (such as cilengitide) were completely unable to increase the dissociation rate. Formation of the non-reversible state occurred very rapidly and did not rely on the time-dependent formation of a high-affinity state of the integrin, or the integrin leg regions. In contrast with RGD-based inhibitors, Ca2+ (but not Mg2+) was able to greatly increase the dissociation rate of integrin-FN complexes, with a half-maximal response at ~0.4 mM Ca2+ for αVβ3-FN. The effect of Ca2+ was overcome by co-addition of Mn2+, but not Mg2+. A stimulatory anti-β1 monoclonal antibody (mAb) abrogated the effect of Ca2+ on α5β1-FN complexes; conversely, a function-blocking mAb mimicked the effect of Ca2+. These results imply that Ca2+ acts allosterically, probably through binding to the adjacent metal-ion-dependent adhesion site (ADMIDAS), and that the α1 helix in the β subunit I domain is the key element affected by allosteric modulators. The data suggest an explanation for the limited clinical efficacy of RGD-based integrin antagonists, and we propose that allosteric antagonists could prove to be of greater therapeutic benefit.

  9. Association between α4 integrin cytoplasmic tail and non-muscle myosin IIA regulates cell migration

    PubMed Central

    Rosado, Leslie A. Rivera; Horn, Troy A.; McGrath, Sara C.; Cotter, Robert J.; Yang, Joy T.

    2011-01-01

    α4β1 integrin regulates cell migration via cytoplasmic interactions. Here, we report an association between the cytoplasmic tail of α4 integrin (α4 tail) and non-muscle myosin IIA (MIIA), demonstrated by co-immunoprecipitation of the MIIA heavy chain (HC) with anti-α4-integrin antibodies and pull-down of MIIA-HC with recombinant α4 tail from cell lysates. The association between the α4 tail and MIIA does not require paxillin binding or phosphorylation at Ser988 in the α4 tail. We found that substituting Glu982 in the α4 tail with alanine (E982A) disrupts the α4–MIIA association without interfering with the paxillin binding or Ser988 phosphorylation. By comparing stably transfected CHO cells, we show that the E982A mutation reduces the ability of α4β1 integrin to mediate cell spreading and to promote front–back polarization. In addition, we show that E982A impairs shear-flow-induced migration of the α4-integrin-expressing CHO cells by reducing their migration speed and directional persistence. The E982A mutation also leads to defects in the organization of MIIA filament bundles. Furthermore, when cells are plated on fibronectin and simulated with shear flow, α4β1 integrin forms filament-like patterns that co-align with MIIA filament bundles. These results provide a new mechanism for linking integrins to the actomyosin cytoskeleton and for regulating cell migration by integrins and non-muscle myosin II. PMID:21224395

  10. Binding of integrin α1 to bone morphogenetic protein receptor IA suggests a novel role of integrin α1β1 in bone morphogenetic protein 2 signalling.

    PubMed

    Zu, Yan; Liang, Xudong; Du, Jing; Zhou, Shuai; Yang, Chun

    2015-11-05

    Here, we observed that integrin α1β1 and bone morphogenetic protein receptor (BMPR) IA formed a complex and co-localised in several cell types. However, the molecular interaction between these two molecules was not studied in detail to date and the role of the interaction in BMPR signalling remains unknown; thus, these were investigated here. In a steered molecular dynamics (SMD) simulation, the observed development of the rupture force related to the displacement between the A-domain of integrin α1 and the extracellular domain of BMPR IA indicated a strong molecular interaction within the integrin-BMPR complex. Analysis of the intermolecular forces revealed that hydrogen bonds, rather than salt bridges, are the major contributors to these intermolecular interactions. By using Enzyme-linked immunosorbent assay (ELISA) and co-immunoprecipitation (co-IP) experiments with site-directed mutants, we found that residues 85-89 in BMPR IA play the most important role for BMPR IA binding to integrin α1β1. These residues are the same as those responsible for bone morphogenetic protein 2 (BMP-2)/BMPR IA binding. In our experiments, we also found that the interference of integrin α1β1 up regulated the level of phosphorylated Smad1, 5, 8, which is the downstream of BMP/BMPR signalling. Therefore, our results suggest that integrin α1β1/BMPR IA may block BMP-2/BMPR IA complex information and interfere with the BMP-2 signalling pathway in cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Human neutrophil integrin alpha9beta1: up-regulation by cell activation and synergy with beta2 integrins during adhesion to endothelium under flow.

    PubMed

    Mambole, Agnès; Bigot, Sylvain; Baruch, Dominique; Lesavre, Philippe; Halbwachs-Mecarelli, Lise

    2010-08-01

    Neutrophil beta1 integrin expression and contribution to cell adhesion were revisited in this study. alpha9beta1 and alpha5beta1 appeared here as the main beta1 integrins expressed on the membrane of resting platelet-depleted neutrophils-alpha6beta1 representing <15% and alpha2beta1 undetectable. Neutrophil activation slightly enhanced alpha5 expression, did not change alpha6, but resulted in a two- to threefold increase of alpha9beta1, which then became the major beta1 integrin of the neutrophil membrane. alpha9beta1 was the only beta1 integrin to be up-regulated after transendothelial migration across TNF-alpha-activated HUVECs. As alpha9beta1 binds VCAM-1, we analyzed its participation to neutrophil adhesion to TNF-alpha-activated endothelial cells. Blocking anti-alpha9 mAb had little effect on neutrophil static adhesion, contrasting with the strong inhibition by anti-beta2 mAb. Under flow conditions, the anti-alpha9 mAb had no effect by itself on neutrophil adhesion to activated HUVECs but enhanced the blocking effect of anti-beta2 antibodies significantly and further enhanced the velocity of beta2-blocked rolling neutrophils. In conclusion, we describe here for the first time a nearly exclusive up-regulation of alpha9beta1 expression among all beta1 integrins during neutrophil activation and transendothelial migration and a possibly important synergy between alpha9beta1 and beta2 integrins in stabilizing neutrophil adhesion to endothelium under flow conditions.

  12. RCP induces Slug expression and cancer cell invasion by stabilizing β1 integrin.

    PubMed

    Hwang, M H; Cho, K H; Jeong, K J; Park, Y-Y; Kim, J M; Yu, S-L; Park, C G; Mills, G B; Lee, H Y

    2017-02-23

    Rab coupling protein (RCP)-induced tumor cell migration has been implicated in tumor pathophysiology and patient outcomes. In the present study, we demonstrate that RCP stabilizes β1 integrin leading to increased β1 integrin levels and activation of a signaling cascade culminating in Slug induction, epithelial-to-mesenchymal transition and increased invasion. Ectopic expression of RCP induced Slug expression. Silencing β1 integrin efficiently inhibited RCP-induced Slug expression and subsequent cancer cell invasion. Conversely, ectopic expression of β1 integrin was sufficient to induce Slug expression. Pharmacological inhibition of integrin linked kinase (ILK), EGFR and NF-κB, as well as transfection of a dominant-negative mutant of Ras (RasN17), significantly inhibited RCP-induced Slug expression and cancer cell invasion. Strikingly, ectopic expression of RCP was sufficient to enhance metastasis of ovarian cancer cells to the lung. Collectively, we demonstrate a mechanism by which RCP promotes cancer cell aggressiveness through sequential β1 integrin stabilization, activation of an ILK/EGFR/Ras/NF-κB signaling cascade and subsequent Slug expression.

  13. The influence of surface integrin binding patterns on specific biomaterial-cell interactions

    NASA Astrophysics Data System (ADS)

    Beranek, Maggi Marie

    As the future of biomaterials progresses toward bioactivity, the biomaterial surface must control non-specific protein adsorption and encourage selective protein and cell adsorption. Integrins alphavbeta3, alpha 1beta1, alpha5beta1 and alpha Mbeta2 are expressed on cells involved in endothelialization, inflammation, and intimal hyperplasia. These cellular events play a vital role in biomaterial biocompatibility, especially in the vascular environment. The overall hypothesis of these studies is that biomaterial surfaces exhibit selective integrin binding, which then specifies differential cell binding. To test this hypothesis, four specific aims were developed. The first aim was designed to determine whether metal and polymeric biomaterials exhibit selective integrin binding. The tested materials included 316L stainless steel, nitinol, gold, Elgiloy RTM, poly(D, L-lactide-co-glycolide), polycarbonate urethane and expanded polytetrafluoroethylene. Discrete integrin binding patterns were detected microscopically using integrin specific fluorescent antibodies. Stainless steel exhibited high level integrin alpha1beta 1 and low level integrin alphaMbeta2 binding pattern. This suggests that this metal surface should selectively encourage endothelial cell to inflammatory cell binding. In contrast, gold bound ten times the amount of integrin alphaMbeta2 compared to integrin alpha1beta1, which should encourage inflammatory cell adhesion. The 65/35 poly(D, L-lactide-co-glycolide) was the only polymeric biomaterial tested that had integrin binding levels comparable to metal biomaterials. Based on these observations, a combinational biomaterial with a surface pattern of 65/35 poly(D, L-lactide-co-glycolide) dots on a 316L stainless steel background was created. A pattern of high level integrin alpha1beta1 binding and low level integrin alpha Mbeta2 binding on this combinational surface indicates that this surface should selectively favor endothelial cell binding. In the second

  14. Cellular localization of integrin isoforms in phenylephrine-induced hypertrophic cardiac myocytes.

    PubMed

    Kim, Dae Joong; Park, Sang Hee; Lim, Chol Seung; Chun, Jang-Soo; Kim, Jin-Kyu; Song, Woo Keun

    2003-03-01

    Cardiac hypertrophy is characterized by remodeling of the extracellular matrix (ECM). Integrins are cell-surface molecules that link the ECM to the cellular cytoskeleton where they play roles as signaling molecules and transducers of mechanical force. To clarify the possible roles of integrins in cardiac myocyte hypertrophy, we investigated the cellular localization and expression of ECM proteins and integrins in both normal cardiac myocytes and phenylephrine-induced hypertrophic myocytes. Addition of phenylephrine (PE) to cultured neonatal cardiac myocytes induced sarcomeric organization, increase in cell size, and synthesis of the hypertrophic marker, atrial natriuretic factor (ANF). In particular, fibronectin and collagen underwent dramatic localization changes during PE-induced cardiac hypertrophy. Significant changes were noted in the cellular localization of the respective collagen and fibronectin receptors, integrin alpha1 and alpha5, from diffuse to a sarcomeric banding pattern. Expression levels of integrins were also increased during hypertrophy. Treatment with okadaic acid (OA), an inhibitor of protein phosphatase 2A (PP2A), resulted in inhibition of hypertrophic response. These results suggest that dephosphorylation of integrin beta1 may be important in the induction of cardiac hypertrophy. Copyright 2002 John Wiley & Sons, Ltd.

  15. Outside-In Signal Transmission by Conformational Changes in Integrin Mac-11

    PubMed Central

    Lefort, Craig T.; Hyun, Young-Min; Schultz, Joanne B.; Law, Foon-Yee; Waugh, Richard E.; Knauf, Philip A.; Kim, Minsoo

    2010-01-01

    Intracellular signals associated with or triggered by integrin ligation can control cell survival, differentiation, proliferation, and migration. Despite accumulating evidence that conformational changes regulate integrin affinity to its ligands, how integrin structure regulates signal transmission from the outside to the inside of the cell remains elusive. Using fluorescence resonance energy transfer, we addressed whether conformational changes in integrin Mac-1 are sufficient to transmit outside-in signals in human neutrophils. Mac-1 conformational activation induced by ligand occupancy or activating Ab binding, but not integrin clustering, triggered similar patterns of intracellular protein tyrosine phosphorylation, including Akt phosphorylation, and inhibited spontaneous neutrophil apoptosis, indicating that global conformational changes are critical for Mac-1-dependent outside-in signal transduction. In neutrophils and myeloid K562 cells, ligand ICAM-1 or activating Ab binding promoted switchblade-like extension of the Mac-1 extracellular domain and separation of the αM and β2 subunit cytoplasmic tails, two structural hallmarks of integrin activation. These data suggest the primacy of global conformational changes in the generation of Mac-1 outside-in signals. PMID:19864611

  16. Hydrogels with precisely controlled integrin activation dictate vascular patterning and permeability

    NASA Astrophysics Data System (ADS)

    Li, Shuoran; Nih, Lina R.; Bachman, Haylee; Fei, Peng; Li, Yilei; Nam, Eunwoo; Dimatteo, Robert; Carmichael, S. Thomas; Barker, Thomas H.; Segura, Tatiana

    2017-09-01

    Integrin binding to bioengineered hydrogel scaffolds is essential for tissue regrowth and regeneration, yet not all integrin binding can lead to tissue repair. Here, we show that through engineering hydrogel materials to promote α3/α5β1 integrin binding, we can promote the formation of a space-filling and mature vasculature compared with hydrogel materials that promote αvβ3 integrin binding. In vitro, α3/α5β1 scaffolds promoted endothelial cells to sprout and branch, forming organized extensive networks that eventually reached and anastomosed with neighbouring branches. In vivo, α3/α5β1 scaffolds delivering vascular endothelial growth factor (VEGF) promoted non-tortuous blood vessel formation and non-leaky blood vessels by 10 days post-stroke. In contrast, materials that promote αvβ3 integrin binding promoted endothelial sprout clumping in vitro and leaky vessels in vivo. This work shows that precisely controlled integrin activation from a biomaterial can be harnessed to direct therapeutic vessel regeneration and reduce VEGF-induced vascular permeability in vivo.

  17. Acidic Extracellular pH Promotes Activation of Integrin αvβ3

    PubMed Central

    Paradise, Ranjani K.; Lauffenburger, Douglas A.; Van Vliet, Krystyn J.

    2011-01-01

    Acidic extracellular pH is characteristic of the cell microenvironment in several important physiological and pathological contexts. Although it is well established that acidic extracellular pH can have profound effects on processes such as cell adhesion and migration, the underlying molecular mechanisms are largely unknown. Integrin receptors physically connect cells to the extracellular matrix, and are thus likely to modulate cell responses to extracellular conditions. Here, we examine the role of acidic extracellular pH in regulating activation of integrin αvβ3. Through computational molecular dynamics simulations, we find that acidic extracellular pH promotes opening of the αvβ3 headpiece, indicating that acidic pH can thereby facilitate integrin activation. This prediction is consistent with our flow cytometry and atomic force microscope-mediated force spectroscopy assays of integrin αvβ3 on live cells, which both demonstrate that acidic pH promotes activation at the intact cell surface. Finally, quantification of cell morphology and migration measurements shows that acidic extracellular pH affects cell behavior in a manner that is consistent with increased integrin activation. Taken together, these computational and experimental results suggest a new and complementary mechanism of integrin activation regulation, with associated implications for cell adhesion and migration in regions of altered pH that are relevant to wound healing and cancer. PMID:21283814

  18. Akt1 Signaling Regulates Integrin Activation, Matrix Recognition, and Fibronectin Assembly*S

    PubMed Central

    Somanath, Payaningal R.; Kandel, Eugene S.; Hay, Nissim; Byzova, Tatiana V.

    2009-01-01

    Akt, a serine-threonine kinase, regulates multiple cellular processes in vascular cells. We have previously documented that Akt activates integrins and Akt1 deficiency results in matrix abnormalities in skin and blood vessels in vivo. Based on these observations, we hypothesized that Akt1 is necessary for integrin activation and matrix assembly by fibroblasts. In this study, using various cell systems, we show that Akt1 is essential for the inside-out activation of integrins in endothelial cells and fibroblasts, which in turn, mediates matrix assembly. Fibronectin is a major extracellular matrix component of the skin and the vascular basement membrane, which possesses binding sites for many integrins and extracellular matrix proteins. Akt1−/− fibroblasts and NIH fibroblasts expressing dominant negative Akt1 (K179M-Akt1) showed impaired fibronectin assembly compared with control fibroblasts. In contrast, expression of constitutively active Akt1 (myrAkt1) resulted in enhanced fibronectin assembly. Although increased fibronectin assembly by myrAkt1-expressing human foreskin fibroblasts was abolished by treatment with anti-integrin β1 blocking antibodies, treatment with β1-stimulating antibodies rescued the impaired fibronectin assembly that was due to lack of Akt activity. Finally, expression of myrAkt1 corrected the phenotype of Akt1−/− fibroblasts thus showing that Akt1 regulates fibronectin assembly through activation of integrin α5β1. PMID:17562714

  19. Integrin binding and mechanical tension induce movement of mRNA and ribosomes to focal adhesions

    NASA Technical Reports Server (NTRS)

    Chicurel, M. E.; Singer, R. H.; Meyer, C. J.; Ingber, D. E.

    1998-01-01

    The extracellular matrix (ECM) activates signalling pathways that control cell behaviour by binding to cell-surface integrin receptors and inducing the formation of focal adhesion complexes (FACs). In addition to clustered integrins, FACs contain proteins that mechanically couple the integrins to the cytoskeleton and to immobilized signal-transducing molecules. Cell adhesion to the ECM also induces a rapid increase in the translation of preexisting messenger RNAs. Gene expression can be controlled locally by targeting mRNAs to specialized cytoskeletal domains. Here we investigate whether cell binding to the ECM promotes formation of a cytoskeletal microcompartment specialized for translational control at the site of integrin binding. High-resolution in situ hybridization revealed that mRNA and ribosomes rapidly and specifically localized to FACs that form when cells bind to ECM-coated microbeads. Relocation of these protein synthesis components to the FAC depended on the ability of integrins to mechanically couple the ECM to the contractile cytoskeleton and on associated tension-moulding of the actin lattice. Our results suggest a new type of gene regulation by integrins and by mechanical stress which may involve translation of mRNAs into proteins near the sites of signal reception.

  20. Transmission of integrin β7 transmembrane domain topology enables gut lymphoid tissue development.

    PubMed

    Sun, Hao; Lagarrigue, Frederic; Gingras, Alexandre R; Fan, Zhichao; Ley, Klaus; Ginsberg, Mark H

    2018-04-02

    Integrin activation regulates adhesion, extracellular matrix assembly, and cell migration, thereby playing an indispensable role in development and in many pathological processes. A proline mutation in the central integrin β3 transmembrane domain (TMD) creates a flexible kink that uncouples the topology of the inner half of the TMD from the outer half. In this study, using leukocyte integrin α4β7, which enables development of gut-associated lymphoid tissue (GALT), we examined the biological effect of such a proline mutation and report that it impairs agonist-induced talin-mediated activation of integrin α4β7, thereby inhibiting rolling lymphocyte arrest, a key step in transmigration. Furthermore, the α4β7(L721P) mutation blocks lymphocyte homing to and development of the GALT. These studies show that impairing the ability of an integrin β TMD to transmit talin-induced TMD topology inhibits agonist-induced physiological integrin activation and biological function in development. © 2018 Sun et al.

  1. Structure of an integrin with an [alpha]I domain, complement receptor type 4

    SciTech Connect

    Xie, Can; Zhu, Jianghai; Chen, Xing

    2010-08-13

    We report the structure of an integrin with an {alpha}I domain, {alpha}{sub X}{beta}{sub 2}, the complement receptor type 4. It was earlier expected that a fixed orientation between the {alpha}I domain and the {beta}-propeller domain in which it is inserted would be required for allosteric signal transmission. However, the {alpha}I domain is highly flexible, enabling two {beta}I domain conformational states to couple to three {alpha}I domain states, and greater accessibility for ligand recognition. Although {alpha}{sub X}{beta}{sub 2} is bent similarly to integrins that lack {alpha}I domains, the terminal domains of the {alpha}- and {beta}-legs, calf-2 and {beta}-tail, are oriented differentlymore » than in {alpha}I-less integrins. Linkers extending to the transmembrane domains are unstructured. Previous mutations in the {beta}2-tail domain support the importance of extension, rather than a deadbolt, in integrin activation. The locations of further activating mutations and antibody epitopes show the critical role of extension, and conversion from the closed to the open headpiece conformation, in integrin activation. Differences among 10 molecules in crystal lattices provide unprecedented information on interdomain flexibility important for modelling integrin extension and activation.« less

  2. Abituzumab Targeting of αV-class Integrins Inhibits Prostate Cancer Progression

    PubMed Central

    Jiang, Yuan; Dai, Jinlu; Yao, Zhi; Shelley, Greg; Keller, Evan T.

    2017-01-01

    Integrins that contain an integrin alpha V subunit contribute to multiple functions that promote cancer progression. The goal of this study was to determine if abituzumab (DI17E6, EMD 525797), a humanized monoclonal antibody (mAb) against integrin alpha V impacts, prostate cancer (PCa) progression. To evaluate this, PCa cells were treated with DI17E6 and its effects on proliferation, apoptosis, cell cycle, adhesion, detachment, migration, invasion and phosphorylation of downstream targets, including FAK, Akt and ERK were determined. DI17E6 promoted detachment and inhibited adhesion of PCa cells to several extracellular matrix (ECM) proteins and cells found in the bone microenvironment, but had no impact on cell viability, cell cycle and caspase 3/7 activity. DI17E6 inhibited migration and invasion of PCa cells. Additionally, DI7E6 decreased phosphorylation of FAK, Akt and ERK. These results indicate that inhibition of integrin alpha V with DI17E6 inhibits several pro-metastatic phenotypes of PCa cells and therefore provide a rationale for further evaluation of DI17E6 for diminishing PCa progression. Implications This work identifies that therapeutic targeting of integrins containing an alpha V integrin unit inhibits cancer progression and thus may be of clinical benefit. PMID:28314844

  3. Carbon Ion Irradiation Inhibits Glioma Cell Migration Through Downregulation of Integrin Expression

    SciTech Connect

    Rieken, Stefan, E-mail: Stefan.Rieken@med.uni-heidelberg.de; Habermehl, Daniel; Wuerth, Lena

    2012-05-01

    Purpose: To investigate the effect of carbon ion irradiation on glioma cell migration. Methods and Materials: U87 and Ln229 glioma cells were irradiated with photons and carbon ions. Migration was analyzed 24 h after irradiation. Fluorescence-activated cell sorting analysis was performed in order to quantify surface expression of integrins. Results: Single photon doses of 2 Gy and 10 Gy enhanced {alpha}{sub {nu}}{beta}{sub 3} and {alpha}{sub {nu}}{beta}{sub 5} integrin expression and caused tumor cell hypermigration on both vitronectin (Vn) and fibronectin (Fn). Compared to integrin expression in unirradiated cells, carbon ion irradiation caused decreased integrin expression and inhibited cell migration onmore » both Vn and Fn. Conclusion: Photon radiotherapy (RT) enhances the risk of tumor cell migration and subsequently promotes locoregional spread via photon induction of integrin expression. In contrast to photon RT, carbon ion RT causes decreased integrin expression and suppresses glioma cell migration on both Vn and Fn, thus promising improved local control.« less

  4. Integrin-like proteins are localized to plasma membrane fractions, not plastids, in Arabidopsis

    NASA Technical Reports Server (NTRS)

    Swatzell, L. J.; Edelmann, R. E.; Makaroff, C. A.; Kiss, J. Z.

    1999-01-01

    Integrins are a large family of integral membrane proteins that function in signal transduction in animal systems. These proteins are conserved in vertebrates, invertebrates, and fungi. Evidence from previous research suggests that integrin-like proteins may be present in plants as well, and that these proteins may function in signal transduction during gravitropism. In past studies, researchers have used monoclonal and polyclonal antibodies to localize beta 1 integrin-like proteins in plants. However, there is a disparity between data collected from these studies, especially since molecular weights obtained from these investigations range from 55-120 kDa for integrin-like proteins. To date, a complete investigation which employs all three basic immunolabeling procedures, immunoblotting, immunofluorescence microscopy, and immunogold labeling, in addition to extensive fractionation and exhaustive controls, has been lacking. In this paper, we demonstrate that use of a polyclonal antibody against the cytoplasmic domain of avian beta 1-integrin can produce potential artifacts in immunolocalization studies. However, these problems can be eliminated through use of starchless mutants or proper specimen preparation prior to electrophoresis. We also show that this antibody, when applied within the described parameters and with careful controls, identifies a large (100 kDa) integrin-like protein that is localized to plasma membrane fractions in Arabidopsis.

  5. Nuclear translocation of integrin cytoplasmic domain-associated protein 1 stimulates cellular proliferation.

    PubMed

    Fournier, Henri-Noël; Dupé-Manet, Sandra; Bouvard, Daniel; Luton, Frédéric; Degani, Simona; Block, Marc R; Retta, Saverio Francesco; Albiges-Rizo, Corinne

    2005-04-01

    Integrin cytoplasmic domain-associated protein 1 (ICAP-1) has been shown to interact specifically with the beta1 integrin cytoplasmic domain and to control cell spreading on fibronectin. Interestingly, ICAP-1 also is observed in the nucleus, by immunocytochemical staining, and after biochemical cell fractionation, suggesting that it has additional roles that have yet to be determined. We show that the nucleocytoplasmic shuttling capability of ICAP-1 is dependent on a functional nuclear localization signal. In addition, overexpression of beta1 integrin strongly reduced this nuclear localization, suggesting that integrin activity could modulate ICAP-1 shuttling by sequestering it in the cytoplasm. Indeed, the nuclear localization of ICAP-1 is dependent on the stage of cell spreading on fibronectin, and we also show that ICAP-1 expression stimulates cellular proliferation in a fibronectin-dependent manner. This function is dependent on its nuclear localization. Moreover, ICAP-1 is able to activate the c-myc promoter in vitro. Together, these results demonstrate that ICAP-1 shuttles between the nucleus and cytoplasm in a beta1 integrin-dependent manner. It could act as a messenger that relays information from sites of integrin-dependent cell adhesion to the nucleus for controlling gene expression and cell proliferation.

  6. Measuring Integrin Conformational Change on the Cell Surface with Super-Resolution Microscopy.

    PubMed

    Moore, Travis I; Aaron, Jesse; Chew, Teng-Leong; Springer, Timothy A

    2018-02-13

    We use super-resolution interferometric photoactivation and localization microscopy (iPALM) and a constrained photoactivatable fluorescent protein integrin fusion to measure the displacement of the head of integrin lymphocyte function-associated 1 (LFA-1) resulting from integrin conformational change on the cell surface. We demonstrate that the distance of the LFA-1 head increases substantially between basal and ligand-engaged conformations, which can only be explained at the molecular level by integrin extension. We further demonstrate that one class of integrin antagonist maintains the bent conformation, while another antagonist class induces extension. Our molecular scale measurements on cell-surface LFA-1 are in excellent agreement with distances derived from crystallographic and electron microscopy structures of bent and extended integrins. Our distance measurements are also in excellent agreement with a previous model of LFA-1 bound to ICAM-1 derived from the orientation of LFA-1 on the cell surface measured using fluorescence polarization microscopy. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

  7. Multiscale simulations suggest a mechanism for integrin inside-out activation.

    PubMed

    Kalli, Antreas C; Campbell, Iain D; Sansom, Mark S P

    2011-07-19

    Integrins are large cell-surface adhesion receptors that can be activated to a high affinity state by the formation of an intracellular complex between the integrin β-subunit tail, the membrane, and talin. The F2 and F3 subdomains of the talin head play a key role in formation of this complex. Here, activation of the integrin αIIb/β3 dimer by the talin head domain was probed using multiscale molecular dynamics simulations. A number of novel insights emerge from these studies, including (i) the importance of the integrin αIIb subunit F992 and F993 residues in stabilizing the "off" state of the αIIb/β3 dimer, (ii) a crucial role for negatively charged groups in the F2-F3/membrane interaction, (iii) binding of the talin F2-F3 domain to negatively charged lipid headgroups in the membrane induces a reorientation of the β transmembrane (TM) domain, (iv) an increase in the tilt angle of the β TM domain relative to the bilayer normal helps to destabilize the α/β TM interaction and promote a scissor-like movement of the integrin TM helices. These results, combined with various published experimental observations, suggest a model for the mechanism of inside-out activation of integrins by talin.

  8. Multiscale simulations suggest a mechanism for integrin inside-out activation

    PubMed Central

    Kalli, Antreas C.; Campbell, Iain D.; Sansom, Mark S. P.

    2011-01-01

    Integrins are large cell-surface adhesion receptors that can be activated to a high affinity state by the formation of an intracellular complex between the integrin β-subunit tail, the membrane, and talin. The F2 and F3 subdomains of the talin head play a key role in formation of this complex. Here, activation of the integrin αIIb/β3 dimer by the talin head domain was probed using multiscale molecular dynamics simulations. A number of novel insights emerge from these studies, including (i) the importance of the integrin αIIb subunit F992 and F993 residues in stabilizing the “off” state of the αIIb/β3 dimer, (ii) a crucial role for negatively charged groups in the F2-F3/membrane interaction, (iii) binding of the talin F2-F3 domain to negatively charged lipid headgroups in the membrane induces a reorientation of the β transmembrane (TM) domain, (iv) an increase in the tilt angle of the β TM domain relative to the bilayer normal helps to destabilize the α/β TM interaction and promote a scissor-like movement of the integrin TM helices. These results, combined with various published experimental observations, suggest a model for the mechanism of inside-out activation of integrins by talin. PMID:21730166

  9. Integrin-extracellular matrix interactions in connective tissue remodeling and osteoblast differentiation

    NASA Technical Reports Server (NTRS)

    Globus, R. K.; Moursi, A.; Zimmerman, D.; Lull, J.; Damsky, C.

    1995-01-01

    The differentiaton of bone cells is a complex multistep process. Bone is somewhat unusual in that it is very actively and continually remodeled in the adult and that maintenance of its mass in the mature organism is exquisitely sensitive to mechanical as well as chemical signals. Bone is also unique because it consists of a very large amount of extracellular matrix (ECM) that is mineralized. The integrin family of ECM receptors has been shown to play an important role in tissue morphogenesis in several systems. Our studies on the regulation of matrix remodeling enzymes by integrins in rabbit synovial fibroblasts show that two b1 integrin fibronectin (FN) receptor complexes (alpha 5 beta 1 and alpha 4 beta 1) cooperate in detecting subtle changes in the composition of the ECM. As a result of signal transduction by these integrins, the levels of mRNA and protein for several members of the metalloproteinase family are regulated in these cells. We have also used antibody and RGD peptide perturbation studies to determine the significance of cell/ECM interactions to normal osteogenesis. We found that interactions between the cell binding domain of FN and integrins are required for both normal morphogenesis and gene expression in cultured osteoblasts that differentiate to form bone-like tissue in culture. These data lead us to propose that beta 1 integrins play an important role in osteoblast differentiation as well as in bone remodeling.

  10. CD177 modulates human neutrophil migration through activation-mediated integrin and chemoreceptor regulation.

    PubMed

    Bai, Ming; Grieshaber-Bouyer, Ricardo; Wang, Junxia; Schmider, Angela B; Wilson, Zachary S; Zeng, Liling; Halyabar, Olha; Godin, Matthew D; Nguyen, Hung N; Levescot, Anaïs; Cunin, Pierre; Lefort, Craig T; Soberman, Roy J; Nigrovic, Peter A

    2017-11-09

    CD177 is a glycosylphosphatidylinositol (GPI)-anchored protein expressed by a variable proportion of human neutrophils that mediates surface expression of the antineutrophil cytoplasmic antibody antigen proteinase 3. CD177 associates with β2 integrins and recognizes platelet endothelial cell adhesion molecule 1 (PECAM-1), suggesting a role in neutrophil migration. However, CD177 pos neutrophils exhibit no clear migratory advantage in vivo, despite interruption of in vitro transendothelial migration by CD177 ligation. We sought to understand this paradox. Using a PECAM-1-independent transwell system, we found that CD177 pos and CD177 neg neutrophils migrated comparably. CD177 ligation selectively impaired migration of CD177 pos neutrophils, an effect mediated through immobilization and cellular spreading on the transwell membrane. Correspondingly, CD177 ligation enhanced its interaction with β2 integrins, as revealed by fluorescence lifetime imaging microscopy, leading to integrin-mediated phosphorylation of Src and extracellular signal-regulated kinase (ERK). CD177-driven cell activation enhanced surface β2 integrin expression and affinity, impaired internalization of integrin attachments, and resulted in ERK-mediated attenuation of chemokine signaling. We conclude that CD177 signals in a β2 integrin-dependent manner to orchestrate a set of activation-mediated mechanisms that impair human neutrophil migration. © 2017 by The American Society of Hematology.

  11. Transgenic Arabidopsis Gene Expression System

    NASA Technical Reports Server (NTRS)

    Ferl, Robert; Paul, Anna-Lisa

    2009-01-01

    The Transgenic Arabidopsis Gene Expression System (TAGES) investigation is one in a pair of investigations that use the Advanced Biological Research System (ABRS) facility. TAGES uses Arabidopsis thaliana, thale cress, with sensor promoter-reporter gene constructs that render the plants as biomonitors (an organism used to determine the quality of the surrounding environment) of their environment using real-time nondestructive Green Fluorescent Protein (GFP) imagery and traditional postflight analyses.

  12. Agribusiness Perspectives on Transgenic Wheat.

    PubMed

    Malcolm, Bill

    2017-01-01

    Declining yields of the major human food crops, looming growth in global population and rise of populism, and ill-founded bans on agricultural and horticultural crops and foodstuffs which are genetically modified have potentially serious implications. It makes the chance less than otherwise would be the case that agribusiness value chains in the future will meet the growing demand around the world for more and different foods from more and wealthier people. In the agribusiness value chain, transgenic wheat, meeting a consumer "trigger need" also must meet the "experience" and "credence," risk-related criteria of well-informed consumers. Public policy that rejects science-based evidence about the reductions in costs of production and price of genetically modified agricultural products and the science about the safety of genetically modified foods, including transgenic wheat, has imposed significant costs on producers and consumers. If the science-based evidence is accepted, transgenic wheat has potential to improve significantly the well-being of grain growers and consumers all over the world.

  13. Transgenic mouse offspring generated by ROSI

    PubMed Central

    MOREIRA, Pedro; PÉREZ-CEREZALES, Serafín; LAGUNA, Ricardo; FERNÁNDEZ-GONZALEZ, Raúl; SANJUANBENITO, Belén Pintado; GUTIÉRREZ-ADÁN, Alfonso

    2015-01-01

    The production of transgenic animals is an important tool for experimental and applied biology. Over the years, many approaches for the production of transgenic animals have been tried, including pronuclear microinjection, sperm-mediated gene transfer, transfection of male germ cells, somatic cell nuclear transfer and the use of lentiviral vectors. In the present study, we developed a new transgene delivery approach, and we report for the first time the production of transgenic animals by co-injection of DNA and round spermatid nuclei into non-fertilized mouse oocytes (ROSI). The transgene used was a construct containing the human CMV immediate early promoter and the enhanced GFP gene. With this procedure, 12% of the live offspring we obtained carried the transgene. This efficiency of transgenic production by ROSI was similar to the efficiency by pronuclear injection or intracytoplasmic injection of male gamete nuclei (ICSI). However, ICSI required fewer embryos to produce the same number of transgenic animals. The expression of Egfp mRNA and fluorescence of EGFP were found in the majority of the organs examined in 4 transgenic lines generated by ROSI. Tissue morphology and transgene expression were not distinguishable between transgenic animals produced by ROSI or pronuclear injection. Furthermore, our results are of particular interest because they indicate that the transgene incorporation mediated by intracytoplasmic injection of male gamete nuclei is not an exclusive property of mature sperm cell nuclei with compact chromatin but it can be accomplished with immature sperm cell nuclei with decondensed chromatin as well. The present study also provides alternative procedures for transgene delivery into embryos or reconstituted oocytes. PMID:26498042

  14. Genetic Variability in Platelet Integrin α2β1 Density: Possible Contributor to Plasmodium vivax–induced Severe Thrombocytopenia

    PubMed Central

    Campos, Fernanda M. F.; Santos, Marina L. S.; Kano, Flora S.; Fontes, Cor J. F.; Lacerda, Marcus V. G.; Brito, Cristiana F. A.; Carvalho, Luzia H.

    2013-01-01

    Understanding the pathogenesis of Plasmodium vivax malaria is challenging. We hypothesized that susceptibility to P. vivax-induced thrombocytopenia could be associated with polymorphisms on relevant platelet membrane integrins: integrin α2 (C807T), and integrin β3 (T1565C). Although β3 polymorphism was not related with P. vivax malaria, α2 807T carriers, which show high levels of integrin α2β1, had a higher probability for severe thrombocytopenia than wild-type carriers. This evidence of the association of integrin polymorphism and P. vivax morbidity was further demonstrated by a moderate but significant correlation between clinical disease and surface levels of the integrin α2β1. PMID:23249684

  15. Cross-talk between integrins {alpha}1{beta}1 and {alpha}2{beta}1 in renal epithelial cells

    SciTech Connect

    Abair, Tristin D.; Department of Cancer Biology, Vanderbilt University, Nashville, TN 37232; Sundaramoorthy, Munirathinam

    2008-11-15

    The collagen-binding integrins {alpha}1{beta}1 and {alpha}2{beta}1 have profoundly different functions, yet they are often co-expressed in epithelial cells. When both integrins are expressed in the same cell, it has been suggested that {alpha}1{beta}1 negatively regulates integrin {alpha}2{beta}1-dependent functions. In this study we utilized murine ureteric bud (UB) epithelial cells, which express no functionally detectable levels of endogenous integrins {alpha}1{beta}1 and {alpha}2{beta}1, to determine the mechanism whereby this regulation occurs. We demonstrate that UB cells expressing integrin {alpha}2{beta}1, but not {alpha}1{beta}1 adhere, migrate and proliferate on collagen I as well as form cellular cords in 3D collagen I gels. Substitution ofmore » the transmembrane domain of the integrin {alpha}2 subunit with that of {alpha}1 results in decreased cell adhesion, migration and cord formation. In contrast, substitution of the integrin {alpha}2 cytoplasmic tail with that of {alpha}1, decreases cell migration and cord formation, but increases proliferation. When integrin {alpha}1 and {alpha}2 subunits are co-expressed in UB cells, the {alpha}1 subunit negatively regulates integrin {alpha}2{beta}1-dependent cord formation, adhesion and migration and this inhibition requires expression of both {alpha}1 and {alpha}2 tails. Thus, we provide evidence that the transmembrane and cytoplasmic domains of the {alpha}2 integrin subunit, as well as the {alpha}1 integrin subunit, regulate integrin {alpha}2{beta}1 cell function.« less

  16. Delta-opioid receptors activate ERK/MAP kinase via integrin-stimulated receptor tyrosine kinases.

    PubMed

    Eisinger, Daniela A; Ammer, Hermann

    2008-12-01

    Integrin-mediated cell adherence to extracellular matrix proteins results in stimulation of ERK1/2 activity, a mechanism involving focal adhesion tyrosine kinases (pp125FAK, Pyk-2) and epidermal growth factor receptors (EGFRs). G protein-coupled receptors (GPCRs) may also mediate ERK1/2 activation in an integrin-dependent manner, the underlying signaling mechanism of which still remains unclear. Here we demonstrate that the delta-opioid receptor (DOR), a typical GPCR, stimulates ERK1/2 activity in HEK293 cells via integrin-mediated transactivation of EGFR function. Inhibition of integrin signaling by RGDT peptides, cytochalasin, and by keeping the cells in suspension culture both blocked [D-Ala(2), D-Leu(5)]enkephalin (DADLE)- and etorphine-stimulated ERK1/2 activity. Integrin-dependent ERK1/2 activation does not involve FAK/Pyk-2, because over-expression of the FAK/Pyk-2 inhibitor SOCS-3 failed to attenuate DOR signaling. Exposure of the cells to the EGFR inhibitors AG1478 and BPIQ-I blocked DOR-mediated ERK1/2 activation. Because RGDT peptides also prevented DOR-mediated EGFR activation, the present findings indicate that in HEK293 cells DOR-stimulated ERK1/2 activity is mediated by integrin-stimulated EGFRs. Further studies with the phospholipase C (PLC) inhibitors U73122 and ET-18-OCH(3) revealed that opioid-stimulated integrin activation is sensitive to PLC. In contrast, integrin-mediated transactivation of EGFR function appears to be dependent on PKC-delta, as indicated by studies with rottlerin and siRNA knock-down. A similar ERK1/2 signaling pathway was observed for NG108-15 cells, a neuronal cell line endogenously expressing the DOR. In these cells, the nerve growth factor TrkA receptor replaces the EGFR in connecting DOR-activated integrins to the Ras/Raf/ERK1/2 pathway. Together, these data describe an alternative ERK1/2 signaling pathway in which the DOR transactivates the growth factor receptor associated mitogen-activated protein kinase cascade in an

  17. Characterization of 14-3-3-ζ Interactions with Integrin Tails

    PubMed Central

    Bonet, Roman; Vakonakis, Ioannis; Campbell, Iain D.

    2013-01-01

    Integrins are a family of heterodimeric (α+β) adhesion receptors that play key roles in many cellular processes. Integrins are unusual in that their functions can be modulated from both outside and inside the cell. Inside-out signaling is mediated by binding adaptor proteins to the flexible cytoplasmic tails of the α- and β-integrin subunits. Talin is one well-known intracellular activator, but various other adaptors bind to integrin tails, including 14-3-3-ζ, a member of the 14-3-3 family of dimeric proteins that have a preference for binding phosphorylated sequence motifs. Phosphorylation of a threonine in the β2 integrin tail has been shown to modulate β2/14-3-3-ζ interactions, and recently, the α4 integrin tail was reported to bind to 14-3-3-ζ and associate with paxillin in a ternary complex that is regulated by serine phosphorylation. Here, we use a range of biophysical techniques to characterize interactions between 14-3-3-ζ and the cytoplasmic tails of α4, β1, β2 and β3 integrins. The X-ray structure of the 14-3-3-ζ/α4 complex indicates a canonical binding mode for the α4 phospho-peptide, but unexpected features are also observed: residues outside the consensus 14-3-3-ζ binding motif are shown to be essential for an efficient interaction; in contrast, a short β2 phospho-peptide is sufficient for high-affinity binding to 14-3-3-ζ. In addition, we report novel 14-3-3-ζ/integrin tail interactions that are independent of phosphorylation. Of the integrin tails studied, the strongest interaction with 14-3-3-ζ is observed for the β1A variant. In summary, new insights about 14-3-3-ζ/integrin tail interactions that have implications for the role of these molecular associations in cells are described. PMID:23763993

  18. Optimization of Biofuel Production From Transgenic Microalgae

    DTIC Science & Technology

    2013-02-27

    AFRL-OSR-VA-TR-2013-0145 OPTIMIZATION OF BIOFUEL PRODUCTION FROM TRANSGENIC MICROALGAE Richard Sayre Donald Danforth...Technical 20080815 to 20120630 OPTIMIZATION OF BIOFUEL PRODUCTION FROM TRANSGENIC MICROALGAE FA9550-08-1-0451 Richard Sayre Donald Danforth Plant...BIOFUEL PRODUCTION FROM TRANSGENIC MICROALGAE Grant/Contract Number: FA9550-08-1-0451 Reporting Period: Final Report Abstract: We have compared the

  19. Promoter Sequences for Defining Transgene Expression

    NASA Astrophysics Data System (ADS)

    Jones, Huw D.; Sparks, Caroline A.

    The design of reverse genetic experiments that utilize transgenic approaches often requires transgenes to be expressed in a predefined pattern and there is limited information regarding the gene expression profile for specific promoters. It is important that expression patterns are predetermined in the specific genotype targeted for transformation because the same promoter-transgene construct can produce different expression patterns in different host species. This chapter compares constitutive, targeted, or inducible promoters that have been characterized in specific cereal species.

  20. Alpha1 and Alpha2 Integrins Mediate Invasive Activity of Mouse Mammary Carcinoma Cells through Regulation of Stromelysin-1 Expression

    SciTech Connect

    Lochter, Andre; Navre, Marc; Werb, Zena

    1998-06-29

    Tumor cell invasion relies on cell migration and extracellular matrix proteolysis. We investigated the contribution of different integrins to the invasive activity of mouse mammary carcinoma cells. Antibodies against integrin subunits {alpha}6 and {beta}1, but not against {alpha}1 and {alpha}2, inhibited cell locomotion on a reconstituted basement membrane in two-dimensional cell migration assays, whereas antibodies against {beta}1, but not against a6 or {alpha}2, interfered with cell adhesion to basement membrane constituents. Blocking antibodies against {alpha}1 integrins impaired only cell adhesion to type IV collagen. Antibodies against {alpha}1, {alpha}2, {alpha}6, and {beta}1, but not {alpha}5, integrin subunits reduced invasion of amore » reconstituted basement membrane. Integrins {alpha}1 and {alpha}2, which contributed only marginally to motility and adhesion, regulated proteinase production. Antibodies against {alpha}1 and {alpha}2, but not {alpha}6 and {beta}1, integrin subunits inhibited both transcription and protein expression of the matrix metalloproteinase stromelysin-1. Inhibition of tumor cell invasion by antibodies against {alpha}1 and {alpha}2 was reversed by addition of recombinant stromelysin-1. In contrast, stromelysin-1 could not rescue invasion inhibited by anti-{alpha}6 antibodies. Our data indicate that {alpha}1 and {alpha}2 integrins confer invasive behavior by regulating stromelysin-1 expression, whereas {alpha}6 integrins regulate cell motility. These results provide new insights into the specific functions of integrins during tumor cell invasion.« less

  1. Macrophage integrins modulate response to ultra-high molecular weight polyethylene particles and direct particle-induced osteolysis.

    PubMed

    Zaveri, Toral D; Dolgova, Natalia V; Lewis, Jamal S; Hamaker, Kiri; Clare-Salzler, Michael J; Keselowsky, Benjamin G

    2017-01-01

    Aseptic loosening due to peri-prosthetic osteolysis is one of the primary causes for failure of artificial joint replacements. Implant-derived wear particles, often ultra-high molecular weight polyethylene (UHMWPE) microparticles, initiate an inflammatory cascade upon phagocytosis by macrophages, which leads to osteoclast recruitment and activation, ultimately resulting in osteolysis. Investigation into integrin receptors, involved in cellular interactions with biomaterial-adsorbed adhesive proteins, is of interest to understand and modulate inflammatory processes. In this work, we investigate the role of macrophage integrins Mac-1 and RGD-binding integrins in response to UHMWPE wear particles. Using integrin knockout mice as well as integrin blocking techniques, reduction in macrophage phagocytosis and inflammatory cytokine secretion is demonstrated when these receptors are either absent or blocked. Along this line, various opsonizing proteins are shown to differentially modulate microparticle uptake and macrophage secretion of inflammatory cytokines. Furthermore, using a calvarial osteolysis model it is demonstrated that both Mac-1 integrin and RGD-binding integrins modulate the particle induced osteolysis response to UHMWPE microparticles, with a 40% decrease in the area of osteolysis by the absence or blocking of these integrins, in vivo. Altogether, these findings indicate Mac-1 and RGD-binding integrins are involved in macrophage-directed inflammatory responses to UHMWPE and may serve as therapeutic targets to mitigate wear particle induced peri-prosthetic osteolysis for improved performance of implanted joints. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Macrophage Integrins Modulate Response to Ultra-High Molecular Weight Polyethylene Particles and Direct Particle-Induced Osteolysis

    PubMed Central

    Zaveri, Toral D.; Dolgova, Natalia V.; Lewis, Jamal S.; Hamaker, Kiri; Clare-Salzler, Michael J.; Keselowsky, Benjamin G.

    2016-01-01

    Aseptic loosening due to peri-prosthetic osteolysis is one of the primary causes for failure of artificial joint replacements. Implant-derived wear particles, often ultra-high molecular weight polyethylene (UHMWPE) microparticles, initiate an inflammatory cascade upon phagocytosis by macrophages, which leads to osteoclast recruitment and activation, ultimately resulting in osteolysis. Investigation into integrin receptors, involved in cellular interactions with biomaterial-adsorbed adhesive proteins, is of interest to understand and modulate inflammatory processes. In this work, we investigate the role of macrophage integrins Mac-1 and RGD-binding integrins in response to UHMWPE wear particles. Using integrin knockout mice as well as integrin blocking techniques, reduction in macrophage phagocytosis and inflammatory cytokine secretion is demonstrated when these receptors are either absent or blocked. Along this line, various opsonizing proteins are shown to differentially modulate microparticle uptake and macrophage secretion of inflammatory cytokines. Furthermore, using a calvarial osteolysis model it is demonstrated that both Mac-1 integrin and RGD-binding integrins modulate the particle induced osteolysis response to UHMWPE microparticles, with a 40% decrease in the area of osteolysis by the absence or blocking of these integrins, in vivo. Altogether, these findings indicate Mac-1 and RGD-binding integrins are involved in macrophage-directed inflammatory responses to UHMWPE and may serve as therapeutic targets to mitigate wear particle induced peri-prosthetic osteolysis for improved performance of implanted joints. PMID:27889664

  3. Proper cerebellar development requires expression of β1-integrin in Bergmann glia, but not in granule neurons.

    PubMed

    Frick, Alexandra; Grammel, Daniel; Schmidt, Felix; Pöschl, Julia; Priller, Markus; Pagella, Pierfrancesco; von Bueren, André O; Peraud, Aurelia; Tonn, Jörg-Christian; Herms, Jochen; Rutkowski, Stefan; Kretzschmar, Hans A; Schüller, Ulrich

    2012-05-01

    β1-class integrins play essential roles both in developmental biology as well as in cancer. Particularly, a Nestin-driven deletion of β1-integrin receptors results in severe abnormalities of brain development including a laminar disorganization of cerebellar granule neurons. However, since Nestin is expressed in all kinds of neural precursors, these data do not allow conclusions to be drawn about the role of β1-integrins in distinct neuronal and glial cell types. By generating conditional knockout mice using granule cell-specific Math1-promoter sequences, we show here that the expression of β1-integrins in granule neurons is dispensable for the development of the cerebellum. Also, deletion of β1-integrin from tumors that arise in a mouse model of granule cell precursor-derived medulloblastoma did not result in a significant survival benefit. Last, expression levels of β1-integrin in human medulloblastoma samples did not predict patient's outcome. However, a β1-integrin knockout using hGFAP-promoter sequences led to cerebellar hypoplasia, inappropriate positioning of Bergmann glia cells in the molecular layer, undirected outgrowth of radial glia fibers, and granule cell ectopia. We therefore conclude that β1-integrin expression in cerebellar granule neurons is not essential during normal development or medulloblastoma formation. In fact, it is the expression of β1-integrin in glia that is crucial for the proper development of the cerebellar cortex. Copyright © 2012 Wiley Periodicals, Inc.

  4. Expression of integrin alphavbeta3 in gliomas correlates with tumor grade and is not restricted to tumor vasculature.

    PubMed

    Schnell, Oliver; Krebs, Bjarne; Wagner, Erika; Romagna, Alexander; Beer, Ambros J; Grau, Stefan J; Thon, Niklas; Goetz, Claudia; Kretzschmar, Hans A; Tonn, Jörg-Christian; Goldbrunner, Roland H

    2008-07-01

    In malignant gliomas, the integrin adhesion receptors seem to play a key role for invasive growth and angiogenesis. However, there is still a controversy about the expression and the distribution of alpha(v)beta(3) integrin caused by malignancy. The aim of our study was to assess the extent and pattern of alpha(v)beta(3) integrin expression within primary glioblastomas (GBMs) compared with low-grade gliomas (LGGs). Tumor samples were immunostained for the detection of alpha(v)beta(3) integrin and quantified by an imaging software. The expression of alpha(v)beta(3) was found to be significantly higher in GBMs than in LGGs, whereby focal strong reactivity was restricted to GBMs only. Subsequent analysis revealed that not only endothelial cells but also, to a large extent, glial tumor cells contribute to the overall amount of alpha(v)beta(3) integrin in the tumors. To further analyze the integrin subunits, Western blots from histologic sections were performed, which demonstrated a significant difference in the expression of the beta(3) integrin subunit between GBMs and LGGs. The presented data lead to new insights in the pattern of alpha(v)beta(3) integrin in gliomas and are of relevance for the inhibition of alpha(v)beta(3) integrin with specific RGD peptides and interfering drugs to reduce angiogenesis and tumor growth.

  5. Integrin α3β1 regulates kidney collecting duct development via TRAF6-dependent K63-linked polyubiquitination of Akt

    PubMed Central

    Yazlovitskaya, Eugenia M.; Tseng, Hui-Yuan; Viquez, Olga; Tu, Tianxiang; Mernaugh, Glenda; McKee, Karen K.; Riggins, Karen; Quaranta, Vito; Pathak, Amrita; Carter, Bruce D.; Yurchenco, Peter; Sonnenberg, Arnoud; Böttcher, Ralph T.; Pozzi, Ambra; Zent, Roy

    2015-01-01

    The collecting system of the kidney develops from the ureteric bud (UB), which undergoes branching morphogenesis, a process regulated by multiple factors, including integrin–extracellular matrix interactions. The laminin (LM)-binding integrin α3β1 is crucial for this developmental program; however, the LM types and LM/integrin α3β1–dependent signaling pathways are poorly defined. We show that α3 chain–containing LMs promote normal UB branching morphogenesis and that LM-332 is a better substrate than LM-511 for stimulating integrin α3β1–dependent collecting duct cell functions. We demonstrate that integrin α3β1–mediated cell adhesion to LM-332 modulates Akt activation in the developing collecting system and that Akt activation is PI3K independent but requires decreased PTEN activity and K63-linked polyubiquitination. We identified the ubiquitin-modifying enzyme TRAF6 as an interactor with the integrin β1 subunit and regulator of integrin α3β1–dependent Akt activation. Finally, we established that the developmental defects of TRAF6- and integrin α3–null mouse kidneys are similar. Thus K63-linked polyubiquitination plays a previously unrecognized role in integrin α3β1–dependent cell signaling required for UB development and may represent a novel mechanism whereby integrins regulate signaling pathways. PMID:25808491

  6. Competitive binding of Rab21 and p120RasGAP to integrins regulates receptor traffic and migration.

    PubMed

    Mai, Anja; Veltel, Stefan; Pellinen, Teijo; Padzik, Artur; Coffey, Eleanor; Marjomäki, Varpu; Ivaska, Johanna

    2011-07-25

    Integrin trafficking from and to the plasma membrane controls many aspects of cell behavior including cell motility, invasion, and cytokinesis. Recruitment of integrin cargo to the endocytic machinery is regulated by the small GTPase Rab21, but the detailed molecular mechanisms underlying integrin cargo recruitment are yet unknown. Here we identify an important role for p120RasGAP (RASA1) in the recycling of endocytosed α/β1-integrin heterodimers to the plasma membrane. Silencing of p120RasGAP attenuated integrin recycling and augmented cell motility. Mechanistically, p120RasGAP interacted with the cytoplasmic domain of integrin α-subunits via its GAP domain and competed with Rab21 for binding to endocytosed integrins. This in turn facilitated exit of the integrin from Rab21- and EEA1-positive endosomes to drive recycling. Our results assign an unexpected role for p120RasGAP in the regulation of integrin traffic in cancer cells and reveal a new concept of competitive binding of Rab GTPases and GAP proteins to receptors as a regulatory mechanism in trafficking.

  7. Usage of integrin and heparan sulfate as receptors for mouse adenovirus type 1.

    PubMed

    Raman, Sharmila; Hsu, Tien-Huei; Ashley, Shanna L; Spindler, Katherine R

    2009-04-01

    Adenovirus fiber knobs are the capsid components that interact with binding receptors on cells, while an Arg-Gly-Asp (RGD) sequence usually found in the penton base protein is important for the interaction of most adenoviruses with integrin entry receptors. Mouse adenovirus type 1 (MAV-1) lacks an RGD sequence in the virion penton base protein. We tested whether an RGD sequence found in the MAV-1 fiber knob plays a role in infection. Treatment of cells with a competitor RGD peptide or a purified recombinant RGD-containing fiber knob prior to infection resulted in reduced virus yields compared to those of controls, indicating the importance of the RGD sequence for infection. An investigation of the role of integrins as possible receptors showed that MAV-1 yields were reduced in the presence of EDTA, an inhibitor of integrin binding, and in the presence of anti-alpha(v) integrin antibody. Moreover, mouse embryo fibroblasts that were genetically deficient in alpha(v) integrin yielded less virus, supporting the hypothesis that alpha(v) integrin is a likely receptor for MAV-1. We also investigated whether glycosaminoglycans play a role in MAV-1 infection. Preincubation of MAV-1 with heparin, a heparan sulfate glycosaminoglycan analog, resulted in a decrease in MAV-1 virus yields. Reduced MAV-1 infectivity was also found with cells that genetically lack heparan sulfate or cells that were treated with heparinase I. Cumulatively, our data demonstrate that the RGD sequence in the MAV-1 fiber knob plays a role in infection by MAV-1, alpha(v) integrin acts as a receptor for the virus, and cell surface heparin sulfate glycosaminoglycans are important in MAV-1 infection.

  8. Constant Applied Force Stimulates Osteoblast Proliferation Via Matrix-Integrin-Signaling Pathways

    NASA Technical Reports Server (NTRS)

    Vercoutere, W.; Parra, M.; Roden, C.; DaCosta, M.; Wing, A.; Damsky, C.; Holton, E.; Searby, N.; Globus, R.; Almeida, E. A. C.

    2003-01-01

    Reduced weight-bearing caused by immobilization, bed-rest or microgravity leads to atrophy in mechanosensitive tissue such as muscle and bone. We hypothesize that bone tissue requires earth s gravity (1-g) for the maintenance of extracellular matrix, integrin, and kinase-mediated cell growth and survival pathways. We investigate the role of matrix-integrin signaling in bone cells using cell culture centrifugation to provide different levels of hypergravity mechanostimulation. The 10-50-g range we use also mimics physiological intermedullary pressure (1.2 - 5 kPa). 24 hours at 50-g increased primary rat osteoblast proliferation on collagen Type I and fibronectin, but not laminin or uncoated plastic. BrdU incorporation in primary osteoblasts over 24 h showed hypergravity increased the number of cells actively synthesizing DNA from about 60% at 1-g to over 90% at 25-g. Primary rat fibroblasts grown at 50-g (24 h) showed no proliferation increase, suggesting this is a tissue-specific phenomenon. These results suggest that the betal and alpha4 integrins may be involved. To further test this, we used osteocytic-like MLO-Y4 cells that showed increased proliferation at 1-g with stable expression of a betal integrin cytoplasmic tail and transmembrane domain construct. At 50-g, MLO-Y4/betal cells showed greater MAPK activation than MLO-Y4 vector controls, suggesting that betal integrin is involved in transducing mitogenic signals in response to hypergravity. Preliminary results also show that interfering with the alpha4 integrin in primary osteoblasts grown on fibronectin blocked the proliferation response. These results indicate that cells from mechanosensitive bone tissue can respond to gravity-generated forces, and this response involves specific matrix and integrin-dependent signaling pathways.

  9. Analysis of integrin expression in U2OS cells cultured on various calcium phosphate ceramic substrates.

    PubMed

    de Ruijter, J E; ter Brugge, P J; Dieudonné, S C; van Vliet, S J; Torensma, R; Jansen, J A

    2001-06-01

    Earlier we observed that calcium phosphate (Ca-P)-coated implant substrates stimulated the differentiation of osteoblast-like cells compared to uncoated substrates. This suggests that this difference in osteogenic induction is due to the chemical composition of the substratum. We hypothesized that Ca-P coatings modulate integrin expression patterns, because those receptors are the sensors of the cell. Therefore, in the present study we quantitatively analyzed integrin expression of osteosarcoma cells and their proliferation behavior on various well-defined Ca-P substrates. For this study we used the osteosarcoma cell line U2OS. Five groups of substrates were used: thermanox (Th), uncoated titanium (Ti), dense sintered hydroxyapatite (HA), and two Ca-P-coated titanium discs (TiHA-O% and TiHA-5%). At day 5, cell numbers were significantly lower (p < 0.05) for both types of Ca-P-coated titanium substrates compared to the other substrates. There were no significant differences between HA and uncoated titanium. From day 5 to 8, accumulated cell number was ranking highest to lowest HA > Th = Ti > TiHA-0% > TiHA-5%. Integrin expression at day 5 and day 8 of incubation was analyzed by flow cytometry for integrin subunits beta 1, alpha 3, alpha 4, alpha 5, alpha 6, and alpha v. Fluorescence-activated cell sorting (FACS) analysis showed that the cells express high levels of beta 1, low levels of alpha 4, alpha 5, and alpha 6, and moderate levels of alpha 3 and alpha v integrin subunits on the various biomaterial substrates. Minor differences in integrin expression between the various substrates were seen. Therefore, the observed differences in proliferation between the coatings may reside in modulating the functional properties of integrins.

  10. Integrin activation by chemokines: relevance to inflammatory adhesion cascade during T cell migration.

    PubMed

    Tanaka, Y

    2000-10-01

    The adhesive function of integrins is regulated through cytoplasmic signaling induced by several stimuli, whose process is designated "inside-out signaling". A large number of leukocytes are rapidly recruited to the sites of inflammation where they form an essential component of the response to infection, injury, autoimmune disorders, allergy, tumor invasion, atherosclerosis and so on. The recruitment of leukocytes into tissue is regulated by a sequence of interactions between the circulating leukocytes and the endothelial cells. Leukocyte integrins play a pivotal role in leukocyte adhesion to endothelial cells. During the process, the activation of integrins by various chemoattractants, especially chemokines, is essential for integrin-mediated adhesion in which a signal transduced to the leukocyte converts the functionally inactive integrin to an active adhesive configuration. We have proposed that H-Ras-sensitive activation of phosphoinositide 3 (PI 3)-kinase and subsequent profilin-mediated actin polymerization, can be involved in chemokine-induced integrin-dependent adhesion of T cells. The present review documents the relevance of cytoplasmic signaling and cytoskeletal assembly to integrin-mediated adhesion induced by chemoattractants including chemokines during inflammatory processes. In contrast, various adhesion molecules are known to transduce extracellular information into cytoplasm, which leads to T cell activation and cytokine production from the cells, designated "outside-in signaling". Such a bi-directional "cross-talking" among adhesion molecules and cytokines is most relevant to inflammatory processes by augmenting immune cell migration from circulation into inflamed tissue such as rheumatoid arthritis, tumor invasion, Behçet's disease and atherosclerosis.

  11. Ozone alters the distribution of beta1 integrins in cultured primate bronchial epithelial cells.

    PubMed

    Jabbour, A J; Altman, L C; Wight, T N; Luchtel, D L

    1998-09-01

    The effects of 0.5 ppm ozone exposure for 6 h on the synthesis and distribution of beta1 integrins were examined in bronchial epithelial cells cultured at an air-cell interface. Ozone exposure damaged cilia and caused significant cell loss. Immunocytochemical localization and quantification of the beta1 subunit in the remaining attached cells using scanning laser cytometry demonstrated time-dependent changes in beta1 distribution in response to ozone. Although no changes were detected immediately after exposure, beta1 immunoreactivity increased 23 +/- 5% and 66 +/- 6% at 6 and 24 h, respectively. The increased immunostaining was localized at the apical surfaces and, to a lesser extent, at cell-cell contacts of cultured cells. Furthermore, integrin redistribution was not due to increased messenger RNA (mRNA) levels and protein synthesis because levels of beta1 mRNA and newly synthesized beta1 protein did not change after ozone exposure. However, immunoprecipitation analysis of beta1 integrins in lysates from equal numbers of cells showed that ozone-exposed cells contained 90 +/- 15% more total beta1 subunit at 24 h after exposure. In addition, our results demonstrated the presence of the alpha5beta1 integrin complex in bronchial epithelial cells and that the detergent-soluble amount of its associated beta1 subunit increased 60 +/- 10% in lysates of ozone-exposed cells. In conclusion, ozone altered cellular distribution of beta1 integrins in the remaining attached cells subsequent to cell injury and loss. The changes in beta1 distribution might be due to increased detergent extractibility of beta1 integrins rather than a real increase in the synthesis of beta1 integrins.

  12. A Built-In Strategy for Containment of Transgenic Plants: Creation of Selectively Terminable Transgenic Rice

    PubMed Central

    Zhao, Te; Cheng, Jiaan; Tu, Juming; Ye, Gongyin; Shen, Zhicheng

    2008-01-01

    Plant transgenic technology has been widely utilized for engineering crops for trait improvements and for production of high value proteins such as pharmaceuticals. However, the unintended spreading of commercial transgenic crops by pollination and seed dispersal is a major concern for environmental and food safety. Simple and reliable containment strategies for transgenes are highly desirable. Here we report a novel method for creating selectively terminable transgenic rice. In this method, the gene(s) of interest is tagged with a RNA interference cassette, which specifically suppresses the expression of the bentazon detoxification enzyme CYP81A6 and thus renders transgenic rice to be sensitive to bentazon, a herbicide used for rice weed control. We generated transgenic rice plants by this method using a new glyphosate resistant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene from Pesudomonas putida as the gene of interest, and demonstrated that these transgenic rice plants were highly sensitive to bentazon but tolerant to glyphosate, which is exactly the opposite of conventional rice. Field trial of these transgenic rice plants further confirmed that they can be selectively killed at 100% by one spray of bentazon at a regular dose used for conventional rice weed control. Furthermore, we found that the terminable transgenic rice created in this study shows no difference in growth, development and yield compared to its non-transgenic control. Therefore, this method of creating transgenic rice constitutes a novel strategy of transgene containment, which appears simple, reliable and inexpensive for implementation. PMID:18350155

  13. Crossroads of integrins and cadherins in epithelia and stroma remodeling

    PubMed Central

    Epifano, Carolina; Perez-Moreno, Mirna

    2012-01-01

    Adhesion events mediated by cadherin and integrin adhesion receptors have fundamental roles in the maintenance of the physiological balance of epithelial tissues, and it is well established that perturbations in their normal functional activity and/or changes in their expression are associated with tumorigenesis. Over the last decades, increasing evidence of a dynamic collaborative interaction between these complexes through their shared interactions with cytoskeletal proteins and common signaling pathways has emerged not only as an important regulator of several aspects of epithelial cell behavior, but also as a coordinated adhesion module that senses and transmits signals from and to the epithelia surrounding microenvironment. The tight regulation of their crosstalk is particularly important during epithelial remodeling events that normally take place during morphogenesis and tissue repair, and when defective it leads to cell transformation and aggravated responses of the tumor microenvironment that contribute to tumorigenesis. In this review we highlight some of the interactions that regulate their crosstalk and how this could be implicated in regulating signals across epithelial tissues to sustain homeostasis. PMID:22568988

  14. Structural basis of substrate discrimination and integrin binding by autotaxin

    SciTech Connect

    Hausmann, Jens; Kamtekar, Satwik; Christodoulou, Evangelos

    2013-09-25

    Autotaxin (ATX, also known as ectonucleotide pyrophosphatase/phosphodiesterase-2, ENPP2) is a secreted lysophospholipase D that generates the lipid mediator lysophosphatidic acid (LPA), a mitogen and chemoattractant for many cell types. ATX-LPA signaling is involved in various pathologies including tumor progression and inflammation. However, the molecular basis of substrate recognition and catalysis by ATX and the mechanism by which it interacts with target cells are unclear. Here, we present the crystal structure of ATX, alone and in complex with a small-molecule inhibitor. We have identified a hydrophobic lipid-binding pocket and mapped key residues for catalysis and selection between nucleotide and phospholipid substrates.more » We have shown that ATX interacts with cell-surface integrins through its N-terminal somatomedin B-like domains, using an atypical mechanism. Our results define determinants of substrate discrimination by the ENPP family, suggest how ATX promotes localized LPA signaling and suggest new approaches for targeting ATX with small-molecule therapeutic agents.« less

  15. Affinity-tuning leukocyte integrin for development of safe therapeutics

    NASA Astrophysics Data System (ADS)

    Park, Spencer

    Much attention has been given to the molecular and cellular pathways linking inflammation with cancer and the local tumor environment to identify new target molecules that could lead to improved diagnosis and treatment. Among the many molecular players involved in the complex response, central to the induction of inflammation is intercellular adhesion molecule (ICAM)-1, which is of particular interest for its highly sensitive and localized expression in response to inflammatory signals. ICAM-1, which has been implicated to play a critical role in tumor progression in various types of cancer, has also been linked to cancer metastases, where ICAM-1 facilitates the spread of metastatic cancer cells to secondary sites. This unique expression profile of ICAM-1 throughout solid tumor microenvironment makes ICAM-1 an intriguing molecular target, which holds great potential as an important diagnostic and therapeutic tool. Herein, we have engineered the ligand binding domain, or the inserted (I) domain of a leukocyte integrin, to exhibit a wide range of monovalent affinities to the natural ligand, ICAM-1. Using the resulting I domain variants, we have created drug and gene delivery nanoparticles, as well as targeted immunotherapeutics that have the ability to bind and migrate to inflammatory sites prevalent in tumors and the associated microenvironment. Through the delivery of diagnostic agents, chemotherapeutics, and immunotherapeutics, the following chapters demonstrate that the affinity enhancements achieved by directed evolution bring the affinity of I domains into the range optimal for numerous applications.

  16. A Novel Mutation in β Integrin Reveals an Integrin-Mediated Interaction between the Extracellular Matrix and cki-1/p27KIP1

    PubMed Central

    Cunningham, Jessica; Cram, Erin J.; Lee, Myeongwoo

    2012-01-01

    The cell-extracellular matrix (ECM) interaction plays an essential role in maintaining tissue shapes and regulates cell behaviors such as cell adhesion, differentiation and proliferation. The mechanism by which the ECM influences the cell cycle in vivo is poorly understood. Here we demonstrate that the β integrin PAT-3 regulates the localization and expression of CKI-1, a C. elegans homologue of the cyclin dependent kinase inhibitor p27KIP1. In nematodes expressing wild type PAT-3, CKI-1::GFP localizes primarily to nucleoli in hypodermal cells, whereas in animals expressing mutant pat-3 with a defective splice junction, CKI-1::GFP appears clumped and disorganized in nucleoplasm. RNAi analysis links cell adhesion genes to the regulation of CKI-1. RNAi of unc-52/perlecan, ina-1/α integrin, pat-4/ILK, and unc-97/PINCH resulted in abnormal CKI-1::GFP localization. Additional RNAi experiments revealed that the SCF E3 ubiquitin-ligase complex genes, skpt-1/SKP2, cul-1/CUL1 and lin-23/F-box, are required for the proper localization and expression of CKI-1, suggesting that integrin signaling and SCF E3 ligase work together to regulate the cellular distribution of CKI-1. These data also suggest that integrin plays a major role in maintaining proper CKI-1/p27KIP1 levels in the cell. Perturbed integrin signaling may lead to the inhibition of SCF ligase activity, mislocalization and elevation of CKI-1/p27KIP1. These results suggest that adhesion signaling is crucial for cell cycle regulation in vivo. PMID:22879977

  17. β4-integrin/PI3K Signaling Promotes Tumor Progression through Galectin-3-N-glycan Complex.

    PubMed

    Kariya, Yukiko; Oyama, Midori; Hashimoto, Yasuhiro; Gu, Jianguo; Kariya, Yoshinobu

    2018-03-16

    Malignant transformation is associated with aberrant N-glycosylation, but the role of protein N-glycosylation in cancer progression remains poorly defined. β4-integrin is a major carrier of N-glycans and is associated with poor prognosis, tumorigenesis, and metastasis. Here, N-glycosylation of β4-integrin contributes to the activation of signaling pathways that promote β4-dependent tumor development and progression. Increased expression of β1,6GlcNAc-branched N-glycans was found to be co-localized with β4-integrin in human cutaneous squamous cell carcinoma tissues, and that the β1,6GlcNAc residue was abundant on β4-integrin in transformed keratinocytes. Interruption of β1,6GlcNAc branching formation on β4-integrin with the introduction of bisecting GlcNAc by N-acetylglucosaminyltransferase III overexpression was correlated with suppression of cancer cell migration and tumorigenesis. N-glycan deletion on β4-integrin impaired β4-dependent cancer cell migration, invasion and growth in vitro, and diminished tumorigenesis and proliferation in vivo. The reduced abilities of β4-integrin were accompanied with decreased phosphoinositol-3 kinase (PI3K)/Akt signals, and were restored by the overexpression of the constitutively active p110 PI3K subunit. Binding of galectin-3 to β4-integrin via β1,6GlcNAc-branched N-glycans promoted β4-integrin-mediated cancer cell adhesion and migration. In contrast, a neutralizing antibody against galectin-3 attenuated β4-integrin N-glycan-mediated PI3K activation and inhibited the ability of β4-integrin to promote cell motility. Furthermore, galectin-3 knockdown by shRNA suppressed β4-integrin N-glycan-mediated tumorigenesis. These findings provide a novel role for N-glycosylation of β4-integrin in tumor development and progression, and the regulatory mechanism for β4-integrin/PI3K signaling via the galectin-3-N-glycan complex. N-glycosylation of β4-integrin plays a functional role in promoting tumor development and

  18. Galectin-3 Induces Clustering of CD147 and Integrin-β1 Transmembrane Glycoprotein Receptors on the RPE Cell Surface

    PubMed Central

    Priglinger, Claudia S.; Szober, Christoph M.; Priglinger, Siegfried G.; Merl, Juliane; Euler, Kerstin N.; Kernt, Marcus; Gondi, Gabor; Behler, Jennifer; Geerlof, Arie; Kampik, Anselm; Ueffing, Marius; Hauck, Stefanie M.

    2013-01-01

    Proliferative vitreoretinopathy (PVR) is a blinding disease frequently occurring after retinal detachment surgery. Adhesion, migration and matrix remodeling of dedifferentiated retinal pigment epithelial (RPE) cells characterize the onset of the disease. Treatment options are still restrained and identification of factors responsible for the abnormal behavior of the RPE cells will facilitate the development of novel therapeutics. Galectin-3, a carbohydrate-binding protein, was previously found to inhibit attachment and spreading of retinal pigment epithelial cells, and thus bares the potential to counteract PVR-associated cellular events. However, the identities of the corresponding cell surface glycoprotein receptor proteins on RPE cells are not known. Here we characterize RPE-specific Gal-3 containing glycoprotein complexes using a proteomic approach. Integrin-β1, integrin-α3 and CD147/EMMPRIN, a transmembrane glycoprotein implicated in regulating matrix metalloproteinase induction, were identified as potential Gal-3 interactors on RPE cell surfaces. In reciprocal immunoprecipitation experiments we confirmed that Gal-3 associated with CD147 and integrin-β1, but not with integrin-α3. Additionally, association of Gal-3 with CD147 and integrin-β1 was observed in co-localization analyses, while integrin-α3 only partially co-localized with Gal-3. Blocking of CD147 and integrin-β1 on RPE cell surfaces inhibited binding of Gal-3, whereas blocking of integrin-α3 failed to do so, suggesting that integrin-α3 is rather an indirect interactor. Importantly, Gal-3 binding promoted pronounced clustering and co-localization of CD147 and integrin-β1, with only partial association of integrin-α3. Finally, we show that RPE derived CD147 and integrin-β1, but not integrin-α3, carry predominantly β-1,6-N-actyl-D-glucosamine-branched glycans, which are high-affinity ligands for Gal-3. We conclude from these data that extracellular Gal-3 triggers clustering of CD147 and

  19. Phosphorylated immunoreceptor tyrosine-based activation motifs and integrin cytoplasmic domains activate spleen tyrosine kinase via distinct mechanisms.

    PubMed

    Antenucci, Lina; Hytönen, Vesa P; Ylänne, Jari

    2018-03-30

    Spleen tyrosine kinase (Syk) is involved in cellular adhesion and also in the activation and development of hematopoietic cells. Syk activation induced by genomic rearrangement has been linked to certain T-cell lymphomas, and Syk inhibitors have been shown to prolong survival of patients with B-cell lineage malignancies. Syk is activated either by its interaction with a double-phosphorylated immunoreceptor tyrosine-based activation motif (pITAM), which induces rearrangements in the Syk structure, or by the phosphorylation of specific tyrosine residues. In addition to its immunoreceptor function, Syk is activated downstream of integrin pathways, and integrins bind to the same region in Syk as does pITAM. However, it is unknown whether integrins and pITAM use the same mechanism to activate Syk. Here, using purified Syk protein and fluorescence-based enzyme assay we investigated whether interaction of the integrin β 3 cytoplasmic domain with the Syk regulatory domain causes changes in Syk activity similar to those induced by pITAM peptides. We observed no direct Syk activation by soluble integrin peptide, and integrin did not compete with pITAM-induced activation even though at high concentrations, the integrin cytoplasmic domain peptide competed with Syk's substrate. However, clustered integrin peptides induced Syk activation, presumably via a transphosphorylation mechanism. Moreover, the clustered integrins also activated a Syk variant in which tyrosines were replaced with phenylalanine (Y348F/Y352F), indicating that clustered integrin-induced Syk activation involved other phosphorylation sites. In conclusion, integrin cytoplasmic domains do not directly induce Syk conformational changes and do not activate Syk via the same mechanism as pITAM. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. αvβ3 integrins negatively regulate cellular forces by phosphorylation of its distal NPXY site.

    PubMed

    Milloud, Rachel; Destaing, Olivier; de Mets, Richard; Bourrin-Reynard, Ingrid; Oddou, Christiane; Delon, Antoine; Wang, Irène; Albigès-Rizo, Corinne; Balland, Martial

    2017-03-01

    Integrins are key receptors that allow cells to sense and respond to their mechanical environment. Although they bind the same ligand, β1 and β3 integrins have distinct and cooperative roles in mechanotransduction. Using traction force microscopy on unconstrained cells, we show that deleting β3 causes traction forces to increase, whereas the deletion of β1 integrin results in a strong decrease of contractile forces. Consistently, loss of β3 integrin also induces an increase in β1 integrin activation. Using a genetic approach, we identified the phosphorylation of the distal NPXY domain as an essential process for β3 integrin to be able to modulate traction forces. Loss of β3 integrins also impacted cell shape and the spatial distribution of traction forces, by causing forces to be generated closer to the cell edge, and the cell shape. Our results emphasize the role of β3 integrin in spatial distribution of cellular forces. We speculate that, by modulating its affinity with kindlin, β3 integrins may be able to locate near the cell edge where it can control β1 integrin activation and clustering. Tensional homeostasis at the single cell level is performed by the ability of β3 adhesions to negatively regulate the activation degree and spatial localization of β1 integrins. By combining genetic approaches and new tools to analyze traction distribution and cell morphology on a population of cells we were able to identify the molecular partners involved in cellular forces regulation. © 2016 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.

  1. Integrin suppresses neurogenesis and regulates brain tissue assembly in planarian regeneration

    PubMed Central

    Bonar, Nicolle A.

    2017-01-01

    Animals capable of adult regeneration require specific signaling to control injury-induced cell proliferation, specification and patterning, but comparatively little is known about how the regeneration blastema assembles differentiating cells into well-structured functional tissues. Using the planarian Schmidtea mediterranea as a model, we identify β1-integrin as a crucial regulator of blastema architecture. β1-integrin(RNAi) animals formed small head blastemas with severe tissue disorganization, including ectopic neural spheroids containing differentiated neurons normally found in distinct organs. By mimicking aspects of normal brain architecture but without normal cell-type regionalization, these spheroids bore a resemblance to mammalian tissue organoids synthesized in vitro. We identified one of four planarian integrin-alpha subunits inhibition of which phenocopied these effects, suggesting that a specific receptor controls brain organization through regeneration. Neoblast stem cells and progenitor cells were mislocalized in β1-integrin(RNAi) animals without significantly altered body-wide patterning. Furthermore, tissue disorganization phenotypes were most pronounced in animals undergoing brain regeneration and not homeostatic maintenance or regeneration-induced remodeling of the brain. These results suggest that integrin signaling ensures proper progenitor recruitment after injury, enabling the generation of large-scale tissue organization within the regeneration blastema. PMID:28126842

  2. Integrin activation dynamics between the RGD-binding site and the headpiece hinge.

    PubMed

    Puklin-Faucher, Eileen; Vogel, Viola

    2009-12-25

    Integrins form mechanical links between the extracellular matrix and the cytoskeleton. Although integrin activation is known to be regulated by an allosteric conformational change, which can be induced from the extracellular or intracellular end of the molecule, little is known regarding the sequence of structural events by which signals propagate between distant sites. Here, we reveal with molecular dynamics simulations of the FnIII(10)-bound alpha(V)beta(3) integrin headpiece how the binding pocket and interdomain betaA/hybrid domain hinge on the distal end of the betaA domain are allosterically linked via a hydrophobic T-junction between the middle of the alpha1 helix and top of the alpha7 helix. The key results of this study are: 1) that this T-junction is induced by ligand binding and hinge opening, and thus displays bidirectionality; 2) that formation of this junction can be accelerated by ligand-mediated force; and 3) how formation of this junction is inhibited by Ca(2+) in place of Mg(2+) at the site adjacent to the metal ion-dependent adhesion site ("ADMIDAS"). Together with recent experimental evidence that integrin complexes can form catch bonds (i.e. become strengthened under force), as well as earlier evidence that Ca(2+) at the ADMIDAS results in lower binding affinity, these simulations provide a common structural model for the dynamic process by which integrins become activated.

  3. Integrin Activation Dynamics between the RGD-binding Site and the Headpiece Hinge*

    PubMed Central

    Puklin-Faucher, Eileen; Vogel, Viola

    2009-01-01

    Integrins form mechanical links between the extracellular matrix and the cytoskeleton. Although integrin activation is known to be regulated by an allosteric conformational change, which can be induced from the extracellular or intracellular end of the molecule, little is known regarding the sequence of structural events by which signals propagate between distant sites. Here, we reveal with molecular dynamics simulations of the FnIII10-bound αVβ3 integrin headpiece how the binding pocket and interdomain βA/hybrid domain hinge on the distal end of the βA domain are allosterically linked via a hydrophobic T-junction between the middle of the α1 helix and top of the α7 helix. The key results of this study are: 1) that this T-junction is induced by ligand binding and hinge opening, and thus displays bidirectionality; 2) that formation of this junction can be accelerated by ligand-mediated force; and 3) how formation of this junction is inhibited by Ca2+ in place of Mg2+ at the site adjacent to the metal ion-dependent adhesion site (“ADMIDAS”). Together with recent experimental evidence that integrin complexes can form catch bonds (i.e. become strengthened under force), as well as earlier evidence that Ca2+ at the ADMIDAS results in lower binding affinity, these simulations provide a common structural model for the dynamic process by which integrins become activated. PMID:19762919

  4. Role of β1 Integrin in Tissue Homing of Neutrophils During Sepsis

    PubMed Central

    Sarangi, Pranita P.; Hyun, Young-Min; Lerman, Yelena V.; Pietropaoli, Anthony P.; Kim, Minsoo

    2012-01-01

    Aberrant activation of neutrophils during sepsis results in the widespread release of pro-inflammatory mediators, leading to multi-organ system failure and death. However, aberrant activation of neutrophils during sepsis results in the widespread release of harmful inflammatory mediators causing host tissue injuries that can lead to multi organ system failure and death. One of the pivotal components of neutrophil migration during inflammation is the expression of surface integrins. In this study, we show that administration of a cyclic analog of RGD peptide (Arg-Gly-Asp) significantly reduced the number of tissue-invading neutrophils and the degree of sepsis-induced lethality in mice as compared to control peptide. Secondly, β1 integrin (CD29) was highly up-regulated on the neutrophils isolated from both septic patients and animals. Finally, conditional genetic ablation of β1 integrin from granulocytes also improved survival and bacterial clearance in septic animals Thus, our results indicate that expression of β1 integrin is important for modulating neutrophil trafficking during sepsis, and that therapeutics designed against β1 integrins may be beneficial. PMID:22683734

  5. Platelet Proteome Analysis Reveals Integrin-dependent Aggregation Defects in Patients with Myelodysplastic Syndromes*

    PubMed Central

    Fröbel, Julia; Cadeddu, Ron-Patrick; Hartwig, Sonja; Bruns, Ingmar; Wilk, Christian M.; Kündgen, Andrea; Fischer, Johannes C.; Schroeder, Thomas; Steidl, Ulrich G.; Germing, Ulrich; Lehr, Stefan; Haas, Rainer; Czibere, Akos

    2013-01-01

    Bleeding complications are a significant clinical problem in patients with myelodysplastic syndromes even at sufficient platelet counts (>50,000/μl). However, the underlying pathology of this hemorrhagic diathesis is still unknown. Here, we analyzed the platelet proteome of patients with myelodysplastic syndromes by quantitative two-dimensional difference gel electrophoresis followed by mass spectrometric protein identification. Proteins identified with lower concentrations, such as Talin-1, Vinculin, Myosin-9, Filmain-A, and Actin play critical roles in integrin αIIbβ3 signaling and thus platelet aggregation. Despite normal agonist receptor expression, calcium flux, and granule release upon activation, the activation capacity of integrin αIIbβ3 was diminished in myelodysplastic syndrome platelets. Förster resonance energy transfer analysis showed a reduced co-localization of Talin-1 to the integrin's β3-subunit, which is required for receptor activation and fibrinogen binding. In addition, platelet spreading on immobilized fibrinogen was incomplete, and platelet aggregation assays confirmed a general defect in integrin-dependent platelet aggregation in patients with myelodysplastic syndromes. Our data provide novel aspects on the molecular pathology of impaired platelet function in myelodysplastic syndromes and suggest a mechanism of defective integrin αIIbβ3 signaling that may contribute to the hemorrhagic diathesis observed in these patients. PMID:23382103

  6. Expression, purification and renaturation of truncated human integrin β1 from inclusion bodies of Escherichia coli.

    PubMed

    Shi, Tonglin; Zhang, Lichao; Li, Zhuoyu; Newton, Ian P; Zhang, Quanbin

    2015-03-01

    Integrins are a family of transmembrane receptors and among their members, integrin β1 is one of the best known. It plays a very important role in cell adhesion/migration and in cancer metastasis. Preparation of integrin β1 has a great potential value especially in studies focused on its function. To this end, recombinant plasmids were constructed containing DNA segments representing 454 amino acids of the N-terminal of integrin β1. The recombinant plasmid was transformed into Escherichiacoli BL21 (DE3) cells and after induction by isopropyl-β-D-thiogalactopyranoside (IPTG), the recombinant protein (molecular weight: 53 kD) was expressed, mainly in the form of inclusion bodies. The inclusion bodies were solubilized by 8M urea solution then purified by nickel affinity chromatography. The recombinant protein was renatured by a stepwise dialysis and finally dissolved in phosphate buffered saline. The final yield was approximately 5.4 mg/L of culture and the purity of the renatured recombinant protein was greater than 98% as assessed by SDS-PAGE. The integrity of the protein was shown by Western blot using monoclonal antibodies against his-tag and integrin β1. Its secondary structure was verified as native by circular dichroism spectra and the bioactivity of the recombinant protein was displayed through the conformation switch under Mn(2+) stimulation. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. Citrullination of collagen II affects integrin-mediated cell adhesion in a receptor-specific manner.

    PubMed

    Sipilä, Kalle; Haag, Sabrina; Denessiouk, Konstantin; Käpylä, Jarmo; Peters, Eric C; Denesyuk, Alexander; Hansen, Uwe; Konttinen, Yrjö; Johnson, Mark S; Holmdahl, Rikard; Heino, Jyrki

    2014-08-01

    Citrullinated collagen II (CII) is a well-known autoantigen in rheumatoid arthritis (RA). However, the direct effects of CII citrullination on cell behavior have not been described. To study whether citrullination of CII could affect cellular functions, we measured the adhesion of 3 different cell types (human Saos2 osteosarcoma cells, human synovial fibroblasts, and rat mesenchymal stem cells) with impedance-based technology. The binding of different collagen receptor integrins to citrullinated collagen was studied by CHO cell lines, each overexpressing 1 of the 4 human collagen receptors on the cell surface, and with solid-phase binding assays, using the recombinant human integrin α1I, α2I, α10I, and α11I domains. Collagen citrullination decreased the adhesion of synovial fibroblasts ∼50% (P<0.05) and mesenchymal stem cells ∼40% (P<0.05) by specifically decreasing the binding of integrins α10β1 and α11β1 to arginine-containing motifs, such as GFOGER. In contrast, citrullination had only a minor effect on the function of α1β1 and α2β1 integrins, which have been reported to play a critical role in regulating leukocyte function. Molecular modeling was used to explain the detected functional differences at the structural level. Given that the integrins regulate cell metabolism, proliferation, and migration, we suggest that collagen citrullination modifies the pathogenesis of RA. Here, CII citrullination was shown to decrease the survival of mesenchymal stem cells. © FASEB.

  8. Dynamin2 controls Rap1 activation and integrin clustering in human T lymphocyte adhesion

    PubMed Central

    Eppler, Felix J.

    2017-01-01

    Leukocyte trafficking is crucial to facilitate efficient immune responses. Here, we report that the large GTPase dynamin2, which is generally considered to have a key role in endocytosis and membrane remodeling, is an essential regulator of integrin-dependent human T lymphocyte adhesion and migration. Chemical inhibition or knockdown of dynamin2 expression significantly reduced integrin-dependent T cell adhesion in vitro. This phenotype was not observed when T cells were treated with various chemical inhibitors which abrogate endocytosis or actin polymerization. We furthermore detected dynamin2 in signaling complexes and propose that it controls T cell adhesion via FAK/Pyk2- and RapGEF1-mediated Rap1 activation. In addition, the dynamin2 inhibitor-induced reduction of lymphocyte adhesion can be rescued by Rap1a overexpression. We demonstrate that the dynamin2 effect on T cell adhesion does not involve integrin affinity regulation but instead relies on its ability to modulate integrin valency. Taken together, we suggest a previously unidentified role of dynamin2 in the regulation of integrin-mediated lymphocyte adhesion via a Rap1 signaling pathway. PMID:28273099

  9. Integrin suppresses neurogenesis and regulates brain tissue assembly in planarian regeneration.

    PubMed

    Bonar, Nicolle A; Petersen, Christian P

    2017-03-01

    Animals capable of adult regeneration require specific signaling to control injury-induced cell proliferation, specification and patterning, but comparatively little is known about how the regeneration blastema assembles differentiating cells into well-structured functional tissues. Using the planarian Schmidtea mediterranea as a model, we identify β1-integrin as a crucial regulator of blastema architecture. β1-integrin(RNAi) animals formed small head blastemas with severe tissue disorganization, including ectopic neural spheroids containing differentiated neurons normally found in distinct organs. By mimicking aspects of normal brain architecture but without normal cell-type regionalization, these spheroids bore a resemblance to mammalian tissue organoids synthesized in vitro We identified one of four planarian integrin-alpha subunits inhibition of which phenocopied these effects, suggesting that a specific receptor controls brain organization through regeneration. Neoblast stem cells and progenitor cells were mislocalized in β1-integrin(RNAi) animals without significantly altered body-wide patterning. Furthermore, tissue disorganization phenotypes were most pronounced in animals undergoing brain regeneration and not homeostatic maintenance or regeneration-induced remodeling of the brain. These results suggest that integrin signaling ensures proper progenitor recruitment after injury, enabling the generation of large-scale tissue organization within the regeneration blastema. © 2017. Published by The Company of Biologists Ltd.

  10. Interface Immobilization Chemistry of cRGD-based Peptides Regulates Integrin Mediated Cell Adhesion

    PubMed Central

    Pallarola, Diego; Bochen, Alexander; Boehm, Heike; Rechenmacher, Florian; Sobahi, Tariq R; Spatz, Joachim P; Kessler, Horst

    2014-01-01

    The interaction of specific surface receptors of the integrin family with different extracellular matrix-based ligands is of utmost importance for the cellular adhesion process. A ligand consists of an integrin-binding group, here cyclic RGDfX, a spacer molecule that lifts the integrin-binding group from the surface and a surface anchoring group. c(-RGDfX-) peptides are bound to gold nanoparticle structured surfaces via polyproline, polyethylene glycol or aminohexanoic acid containing spacers of different lengths. Although keeping the integrin-binding c(-RGDfX-) peptides constant for all compounds, changes of the ligand's spacer chemistry and length reveal significant differences in cell adhesion activation and focal adhesion formation. Polyproline-based peptides demonstrate improved cell adhesion kinetics and focal adhesion formation compared with common aminohexanoic acid or polyethylene glycol spacers. Binding activity can additionally be improved by applying ligands with two head groups, inducing a multimeric effect. This study gives insights into spacer-based differences in integrin-driven cell adhesion processes and remarkably highlights the polyproline-based spacers as suitable ligand-presenting templates for surface functionalization. PMID:25810710

  11. Epithelial cell integrin β1 is required for developmental angiogenesis in the pituitary gland

    PubMed Central

    Scully, Kathleen M.; Skowronska-Krawczyk, Dorota; Krawczyk, Michal; Merkurjev, Daria; Taylor, Havilah; Livolsi, Antonia; Tollkuhn, Jessica; Stan, Radu V.; Rosenfeld, Michael G.

    2016-01-01

    As a key component of the vertebrate neuroendocrine system, the pituitary gland relies on the progressive and coordinated development of distinct hormone-producing cell types and an invading vascular network. The molecular mechanisms that drive formation of the pituitary vasculature, which is necessary for regulated synthesis and secretion of hormones that maintain homeostasis, metabolism, and endocrine function, remain poorly understood. Here, we report that expression of integrin β1 in embryonic pituitary epithelial cells is required for angiogenesis in the developing mouse pituitary gland. Deletion of pituitary epithelial integrin β1 before the onset of angiogenesis resulted in failure of invading endothelial cells to recruit pericytes efficiently, whereas deletion later in embryogenesis led to decreased vascular density and lumen formation. In both cases, lack of epithelial integrin β1 was associated with a complete absence of vasculature in the pituitary gland at birth. Within pituitary epithelial cells, integrin β1 directs a large transcriptional program that includes components of the extracellular matrix and associated signaling factors that are linked to the observed non–cell-autonomous effects on angiogenesis. We conclude that epithelial integrin β1 functions as a critical and canonical regulator of developmental angiogenesis in the pituitary gland, thus providing insight into the long-standing systems biology conundrum of how vascular invasion is coordinated with tissue development. PMID:27810956

  12. Epithelial cell integrin β1 is required for developmental angiogenesis in the pituitary gland.

    PubMed

    Scully, Kathleen M; Skowronska-Krawczyk, Dorota; Krawczyk, Michal; Merkurjev, Daria; Taylor, Havilah; Livolsi, Antonia; Tollkuhn, Jessica; Stan, Radu V; Rosenfeld, Michael G

    2016-11-22

    As a key component of the vertebrate neuroendocrine system, the pituitary gland relies on the progressive and coordinated development of distinct hormone-producing cell types and an invading vascular network. The molecular mechanisms that drive formation of the pituitary vasculature, which is necessary for regulated synthesis and secretion of hormones that maintain homeostasis, metabolism, and endocrine function, remain poorly understood. Here, we report that expression of integrin β1 in embryonic pituitary epithelial cells is required for angiogenesis in the developing mouse pituitary gland. Deletion of pituitary epithelial integrin β1 before the onset of angiogenesis resulted in failure of invading endothelial cells to recruit pericytes efficiently, whereas deletion later in embryogenesis led to decreased vascular density and lumen formation. In both cases, lack of epithelial integrin β1 was associated with a complete absence of vasculature in the pituitary gland at birth. Within pituitary epithelial cells, integrin β1 directs a large transcriptional program that includes components of the extracellular matrix and associated signaling factors that are linked to the observed non-cell-autonomous effects on angiogenesis. We conclude that epithelial integrin β1 functions as a critical and canonical regulator of developmental angiogenesis in the pituitary gland, thus providing insight into the long-standing systems biology conundrum of how vascular invasion is coordinated with tissue development.

  13. controls integrin signaling-dependent cytoskeleton dynamics and organization in megakaryocytes.

    PubMed

    Beauchemin, Hugues; Shooshtarizadeh, Peiman; Vadnais, Charles; Vassen, Lothar; Pastore, Yves D; Möröy, Tarik

    2017-03-01

    Mutations in GFI1B are associated with inherited bleeding disorders called GFI1B -related thrombocytopenias. We show here that mice with a megakaryocyte-specific Gfi1b deletion exhibit a macrothrombocytopenic phenotype along a megakaryocytic dysplasia reminiscent of GFI1B -related thrombocytopenia. GFI1B deficiency increases megakaryocyte proliferation and affects their ploidy, but also abrogates their responsiveness towards integrin signaling and their ability to spread and reorganize their cytoskeleton. Gfi1b -null megakaryocytes are also unable to form proplatelets, a process independent of integrin signaling. GFI1B-deficient megakaryocytes exhibit aberrant expression of several components of both the actin and microtubule cytoskeleton, with a dramatic reduction of α-tubulin. Inhibition of FAK or ROCK, both important for actin cytoskeleton organization and integrin signaling, only partially restored their response to integrin ligands, but the inhibition of PAK, a regulator of the actin cytoskeleton, completely rescued the responsiveness of Gfi1b -null megakaryocytes to ligands, but not their ability to form proplatelets. We conclude that Gfi1b controls major functions of megakaryocytes such as integrin-dependent cytoskeleton organization, spreading and migration through the regulation of PAK activity whereas the proplatelet formation defect in GFI1B-deficient megakaryocytes is due, at least partially, to an insufficient α-tubulin content. Copyright© Ferrata Storti Foundation.

  14. Mesodermal expression of integrin α5β1 regulates neural crest development and cardiovascular morphogenesis

    PubMed Central

    Liang, Dong; Wang, Xia; Mittal, Ashok; Dhiman, Sonam; Hou, Shuan-Yu; Degenhardt, Karl; Astrof, Sophie

    2014-01-01

    Integrin α5-null embryos die in mid-gestation from severe defects in cardiovascular morphogenesis, which stem from defective development of the neural crest, heart and vasculature. To investigate the role of integrin α5β1 in cardiovascular development, we used the Mesp1Cre knock-in strain of mice to ablate integrin α5 in the anterior mesoderm, which gives rise to all of the cardiac and many of the vascular and muscle lineages in the anterior portion of the embryo. Surprisingly, we found that mutant embryos displayed numerous defects related to the abnormal development of the neural crest such as cleft palate, ventricular septal defect, abnormal development of hypoglossal nerves, and defective remodeling of the aortic arch arteries. We found that defects in arch artery remodeling stem from the role of mesodermal integrin α5β1 in neural crest proliferation and differentiation into vascular smooth muscle cells, while proliferation of pharyngeal mesoderm and differentiation of mesodermal derivatives into vascular smooth muscle cells was not defective. Taken together our studies demonstrate a requisite role for mesodermal integrin α5β1 in signaling between the mesoderm and the neural crest, thereby regulating neural crest-dependent morphogenesis of essential embryonic structures. PMID:25242040

  15. β1 Integrin is an Adhesion Protein for Sperm Binding to Eggs

    PubMed Central

    Baessler, Keith A.; Lee, Younjoo; Sampson, Nicole S.

    2009-01-01

    We investigated the role of β1 integrin in mammalian fertilization and the mode of inhibition of fertilinβ-derived polymers. We determined that polymers displaying the Glu-Cys-Asp peptide from the fertilinβ disintegrin domain mediate inhibition of mammalian fertilization through a β1 integrin receptor on the egg surface. Inhibition of fertilization is a consequence of competition with sperm binding to the cell surface, not activation of an egg-signaling pathway. The presence of the β1 integrin on the egg surface increases the rate of sperm attachment, but does not alter the total number of sperm that can attach or fuse to the egg. We conclude that the presence of β1 integrin enhances the initial adhesion of sperm to the egg plasma membrane and that subsequent attachment and fusion are mediated by additional egg and sperm proteins present in the β1 integrin complex. Therefore, the mechanisms by which sperm fertilize wild-type and β1 knockout eggs are different. PMID:19338281

  16. Constitutive integrin activation on tumor cells contributes to progression of leptomeningeal metastases1

    PubMed Central

    Brandsma, Dieta; Ulfman, Laurien; Reijneveld, Jaap C.; Bracke, Madelon; Taphoorn, Martin J.B.; Zwaginga, Jaap Jan; Gebbink, Martijn F.B.; de Boer, Hetty; Koenderman, Leo; Voest, Emile E.

    2006-01-01

    Leptomeningeal metastases are a serious neurological complication in cancer patients and associated with a dismal prognosis. Tumor cells that enter the subarachnoid space adhere to the leptomeninges and form tumor deposits. It is largely unknown which adhesion molecules mediate tumor cell adhesion to leptomeninges. We studied the role of integrin expression and activation in the progression of leptomeningeal metastases. For this study, we used a mouse acute lymphocytic leukemic cell line that was grown in suspension (L1210-S cell line) to develop an adherent L1210 cell line (L1210-A) by selectively culturing the few adherent cells in the cell culture. β1, β2, and β3 integrins were in a constitutively high active state on L1210-A cells and in a low, but inducible, active state on L1210-S cells. Expression levels of these integrins were comparable in the two cell lines. Static adhesion levels of L1210-A cells on a leptomeningeal cell layer were significantly higher than those of L1210-S cells. All mice that were injected intrathecally with L1210-A cells died rapidly of leptomeningeal leukemia. In contrast, 45% long-term survival was seen after intrathecal injection of mice with L1210-S cells. Our data indicate that constitutive integrin activation on leukemic cells promotes progression of leptomeningeal leukemia by increased tumor cell adhesion to the leptomeninges. We argue that an aberrantly regulated inside-out signaling pathway underlies constitutive integrin activation on the adherent leukemic cell population. PMID:16533879

  17. Phosphoinositide Regulation of Integrin Trafficking Required for Muscle Attachment and Maintenance

    PubMed Central

    Ribeiro, Inês; Yuan, Lin; Tanentzapf, Guy; Dowling, James J.; Kiger, Amy

    2011-01-01

    Muscles must maintain cell compartmentalization when remodeled during development and use. How spatially restricted adhesions are regulated with muscle remodeling is largely unexplored. We show that the myotubularin (mtm) phosphoinositide phosphatase is required for integrin-mediated myofiber attachments in Drosophila melanogaster, and that mtm-depleted myofibers exhibit hallmarks of human XLMTM myopathy. Depletion of mtm leads to increased integrin turnover at the sarcolemma and an accumulation of integrin with PI(3)P on endosomal-related membrane inclusions, indicating a role for Mtm phosphatase activity in endocytic trafficking. The depletion of Class II, but not Class III, PI3-kinase rescued mtm-dependent defects, identifying an important pathway that regulates integrin recycling. Importantly, similar integrin localization defects found in human XLMTM myofibers signify conserved MTM1 function in muscle membrane trafficking. Our results indicate that regulation of distinct phosphoinositide pools plays a central role in maintaining cell compartmentalization and attachments during muscle remodeling, and they suggest involvement of Class II PI3-kinase in MTM-related disease. PMID:21347281

  18. Nematode neuropeptides as transgenic nematicides

    PubMed Central

    Patten, Cheryl; Fleming, Colin C.; Maule, Aaron G.

    2017-01-01

    Plant parasitic nematodes (PPNs) seriously threaten global food security. Conventionally an integrated approach to PPN management has relied heavily on carbamate, organophosphate and fumigant nematicides which are now being withdrawn over environmental health and safety concerns. This progressive withdrawal has left a significant shortcoming in our ability to manage these economically important parasites, and highlights the need for novel and robust control methods. Nematodes can assimilate exogenous peptides through retrograde transport along the chemosensory amphid neurons. Peptides can accumulate within cells of the central nerve ring and can elicit physiological effects when released to interact with receptors on adjoining cells. We have profiled bioactive neuropeptides from the neuropeptide-like protein (NLP) family of PPNs as novel nematicides, and have identified numerous discrete NLPs that negatively impact chemosensation, host invasion and stylet thrusting of the root knot nematode Meloidogyne incognita and the potato cyst nematode Globodera pallida. Transgenic secretion of these peptides from the rhizobacterium, Bacillus subtilis, and the terrestrial microalgae Chlamydomonas reinhardtii reduce tomato infection levels by up to 90% when compared with controls. These data pave the way for the exploitation of nematode neuropeptides as a novel class of plant protective nematicide, using novel non-food transgenic delivery systems which could be deployed on farmer-preferred cultivars. PMID:28241060

  19. [Progress in transgenic fish techniques and application].

    PubMed

    Ye, Xing; Tian, Yuan-Yuan; Gao, Feng-Ying

    2011-05-01

    Transgenic technique provides a new way for fish breeding. Stable lines of growth hormone gene transfer carps, salmon and tilapia, as well as fluorescence protein gene transfer zebra fish and white cloud mountain minnow have been produced. The fast growth characteristic of GH gene transgenic fish will be of great importance to promote aquaculture production and economic efficiency. This paper summarized the progress in transgenic fish research and ecological assessments. Microinjection is still the most common used method, but often resulted in multi-site and multi-copies integration. Co-injection of transposon or meganuclease will greatly improve the efficiency of gene transfer and integration. "All fish" gene or "auto gene" should be considered to produce transgenic fish in order to eliminate misgiving on food safety and to benefit expression of the transferred gene. Environmental risk is the biggest obstacle for transgenic fish to be commercially applied. Data indicates that transgenic fish have inferior fitness compared with the traditional domestic fish. However, be-cause of the genotype-by-environment effects, it is difficult to extrapolate simple phenotypes to the complex ecological interactions that occur in nature based on the ecological consequences of the transgenic fish determined in the laboratory. It is critical to establish highly naturalized environments for acquiring reliable data that can be used to evaluate the environ-mental risk. Efficacious physical and biological containment strategies remain to be crucial approaches to ensure the safe application of transgenic fish technology.

  20. [New advances in animal transgenic technology].

    PubMed

    Sun, Zhen-Hong; Miao, Xiang-Yang; Zhu, Rui-Liang

    2010-06-01

    Animal transgenic technology is one of the fastest growing biotechnology in the 21st century. It is used to integrate foreign genes into the animal genome by genetic engineering technology so that foreign genes can be expressed and inherited to the offspring. The transgenic efficiency and precise control of gene expression are the key limiting factors on preparation of transgenic animals. A variety of transgenic techniques are available, each of which has its own advantages and disadvantages and still needs further study because of unresolved technical and safety issues. With the in-depth research, the transgenic technology will have broad application prospects in the fields of exploration of gene function, animal genetic improvement, bioreactor, animal disease models, organ transplantation and so on. This article reviews the recently developed animal gene transfer techniques, including germline stem cell mediated method to improve the efficiency, gene targeting to improve the accuracy, RNA interference (RNAi)-mediated gene silencing technology, and the induced pluripotent stem cells (iPS) transgenic technology. The new transgenic techniques can provide a better platform for the study of trans-genic animals and promote the development of medical sciences, livestock production, and other fields.

  1. Inducible Transgenic Models of BRCA1 Function

    DTIC Science & Technology

    1998-10-01

    Northern hybrization analysis of mammary tissue from a large number of MTA transgenic female mice revealed high levels of expression of rtTA in only 50...undertaken. Northern hybrization analysis of mammary tissue from four MTB transgenic female mice and two FVB wild-type controls, revealed high levels of

  2. Accumulation of nickel in transgenic tobacco

    NASA Astrophysics Data System (ADS)

    Sidik, Nik Marzuki; Othman, Noor Farhan

    2013-11-01

    The accumulation of heavy metal Ni in the roots and leaves of four T1 transgenic lines of tobacco (T(1)20E, T(1)24C, T(1)18B1 and T(1)20B) expressing eiMT1 from E.indica was assessed. The aim of the study was to investigate the level of Ni accumulation in the leaves and roots of each transgenic lines and to evaluate the eligibility of the plants to be classified as a phytoremediation agent. All of the transgenic lines showed different ability in accumulating different metals and has translocation factor (TF) less than 1 (TF<1) at all levels of metal treatment. Among the 4 transgenic lines, transgenic line T(1)24C showed the highest accumulation of Ni (251.9 ± 0.014 mg/kg) and the lowest TF value (TFT(1)24C=0.0875) at 60 ppm Ni.

  3. Transgenes and their contributions to epigenetic research.

    PubMed

    Meyer, Peter

    2013-01-01

    Shortly after gene transfer technologies had been established for different plant species, the first reports emerged about transgenes showing unexpected segregation patterns due to unstable expression. Initially, the erratic expression behavior of transgenes was considered a nuisance that impeded the impact and efficiency of a new technology. With the investigation of transgene silencing effects, however, it soon became clear that transgenes had helped us in a rather unexpected way to identify novel molecular pathways that were highly relevant to plant development and evolution. This article gives an account of a journey that started with the analysis of transgene-related silencing events and that led to the discovery of a new molecular world of small RNAs and epigenetic marks that regulate plant gene expression and adaptation to environmental changes.

  4. Alpha 7 integrin preserves the function of the extensor digitorum longus muscle in dystrophin-null mice

    PubMed Central

    Hakim, Chady H.; Burkin, Dean J.

    2013-01-01

    The dystrophin-associated glycoprotein complex (DGC) and the α7β1-integrin complex are two independent protein complexes that link the extracellular matrix with the cytoskeleton in muscle cells. These associations stabilize the sarcolemma during force transmission. Loss of either one of these complexes leads to muscular dystrophy. Dystrophin is a major component of the DGC. Its absence results in Duchenne muscular dystrophy (DMD). Because α7-integrin overexpression has been shown to ameliorate muscle histopathology in mouse models of DMD, we hypothesize that the α7β1-integrin complex can help preserve muscle function. To test this hypothesis, we evaluated muscle force, elasticity, and the viscous property of the extensor digitorum longus muscle in 19-day-old normal BL6, dystrophin-null mdx4cv, α7-integrin-null, and dystrophin/α7-integrin double knockout mice. While nominal changes were found in single knockout mice, contractility and passive properties were significantly compromised in α7-integrin double knockout mice. Our results suggest that DGC and α7β1-integrin complexes may compensate each other to maintain normal skeletal muscle function. α7β1-Integrin upregulation may hold promise to treat not only histological, but also physiological, defects in DMD. PMID:23990247

  5. Alpha 7 integrin preserves the function of the extensor digitorum longus muscle in dystrophin-null mice.

    PubMed

    Hakim, Chady H; Burkin, Dean J; Duan, Dongsheng

    2013-11-01

    The dystrophin-associated glycoprotein complex (DGC) and the α7β1-integrin complex are two independent protein complexes that link the extracellular matrix with the cytoskeleton in muscle cells. These associations stabilize the sarcolemma during force transmission. Loss of either one of these complexes leads to muscular dystrophy. Dystrophin is a major component of the DGC. Its absence results in Duchenne muscular dystrophy (DMD). Because α7-integrin overexpression has been shown to ameliorate muscle histopathology in mouse models of DMD, we hypothesize that the α7β1-integrin complex can help preserve muscle function. To test this hypothesis, we evaluated muscle force, elasticity, and the viscous property of the extensor digitorum longus muscle in 19-day-old normal BL6, dystrophin-null mdx4cv, α7-integrin-null, and dystrophin/α7-integrin double knockout mice. While nominal changes were found in single knockout mice, contractility and passive properties were significantly compromised in α7-integrin double knockout mice. Our results suggest that DGC and α7β1-integrin complexes may compensate each other to maintain normal skeletal muscle function. α7β1-Integrin upregulation may hold promise to treat not only histological, but also physiological, defects in DMD.

  6. Akt1 mediates prostate cancer cell microinvasion and chemotaxis to metastatic stimuli via integrin β3 affinity modulation

    PubMed Central

    Goc, A; Liu, J; Byzova, T V; Somanath, P R

    2012-01-01

    Background: Activation of Akt and increased expression of integrin β3 are the two most important changes that have been linked to the attainment of metastatic potential by prostate cancer cells. However, a direct link between Akt activity and inside-out activation of integrin β3 in mediating prostate cancer cell metastatic properties is not established. Methods: Using functional and biochemical approaches, we examined the role of Akt1 in the affinity modulation of integrin β3 in prostate cancer cells. Results: Although expression of murine TRAMP and human PC3 cells with constitutively active Akt1 (CA-Akt1) enhanced their affinity for integrin αvβ3 specific ligands and motility on various extracellular matrix proteins, the reverse was observed with the expression of dominant-negative Akt1 (DN-Akt1). Although enhanced motility and transendothelial migration of CA-Akt1-expressing cells were blunted by co-expression with DN-integrin β3 or upon pre-treatment with integrin β3-blocking antibodies (LM 609), impaired motility and transendothelial migration of DN-Akt1-expressing cells were rescued by pre-treatment of prostate cancer cells with integrin β3-activating antibodies, AP7.4. Conclusion: Our data is the first to demonstrate a link between Akt1 activity and affinity modulation of integrin β3 in the regulation of prostate cancer cell motility, transendothelial migration and chemotaxis to metastatic stimuli. PMID:22767145

  7. An EGFR/Src-dependent β4 integrin/FAK complex contributes to malignancy of breast cancer.

    PubMed

    Tai, Yu-Ling; Chu, Pei-Yu; Lai, I-Rue; Wang, Ming-Yang; Tseng, Hui-Yuan; Guan, Jun-Lin; Liou, Jun-Yang; Shen, Tang-Long

    2015-11-09

    β4 integrin and focal adhesion kinase (FAK) are often associated with a poor prognosis in cancer patients, and their signaling events have recently been linked to malignant outcomes. Here, we demonstrate, for the first time, physical and functional interactions between β4 integrin and FAK that influence breast cancer malignancy. An amino-terminal linker within FAK is essential for its binding with the cytodomain of β4 integrin. Moreover, EGFR/Src-signaling triggers the tyrosine phosphorylation of β4 integrin, which, in turn, recruits FAK to β4 integrin and leads to FAK activation and signaling. Upon disruption of the β4 integrin/FAK complex, tumorigenesis and metastasis in triple-negative breast cancer were markedly reduced. Importantly, the concomitant overexpression of β4 integrin and FAK significantly correlates with malignant potential in patients with triple-negative breast cancer. This study describes a pro-metastatic EGFR/Src-dependent β4 integrin/FAK complex that is involved in breast cancer malignancy and is a novel therapeutic target for triple-negative breast cancer.

  8. Protein kinase D isoforms are dispensable for integrin-mediated lymphocyte adhesion and homing to lymphoid tissues

    PubMed Central

    Matthews, Sharon A; San Lek, Hwee; Morrison, Vicky L; Mackenzie, Matthew G; Zarrouk, Marouan; Cantrell, Doreen; Fagerholm, Susanna C

    2012-01-01

    Leukocyte function-associated antigen-1 (LFA-1) and very late antigen-4 (VLA-4) integrins are essential for lymphocyte adhesion, trafficking and effector functions. Protein kinase D (PKD) has previously been implicated in lymphocyte integrin regulation through regulation of Rap1 activity. However, the true role of PKD in integrin regulation in primary lymphocytes has not previously been investigated. The major PKD isoform in lymphocytes is PKD2. Here we employed PKD2-deficient mice, a specific PKD kinase inhibitor, as well as PKD-null DT40 B cells to investigate the role of PKD in integrin regulation in lymphocytes. We report that PKD2-deficient lymphocytes bound normally to integrin ligands in static and shear flow adhesion assays. They also homed normally to lymphoid organs after adoptive transfer into wild-type mice. DT40 B cells devoid of any PKD isoforms and primary lymphocytes pretreated with a specific PKD inhibitor bound normally to integrin ligands, indicating that multiple PKD isoforms do not redundantly regulate lymphocyte integrins. In addition, PKD2-deficient lymphocytes, as well as DT40 cells devoid of any PKD isoforms, could activate Rap1 in response to B-cell receptor ligation or phorbol ester treatment. Together, these results show that the PKD family does not play a critical role in lymphocyte integrin-mediated cell adhesion or lymphocyte trafficking in vivo. PMID:22311617

  9. Constitutive Association of Tie1 and Tie2 with Endothelial Integrins is Functionally Modulated by Angiopoietin-1 and Fibronectin

    PubMed Central

    Dalton, Annamarie C.; Shlamkovitch, Tomer; Papo, Niv; Barton, William A.

    2016-01-01

    Functional cross-talk between Tie2 and Integrin signaling pathways is essential to coordinate endothelial cell adhesion and migration in response to the extracellular matrix, yet the mechanisms behind this phenomenon are unclear. Here, we examine the possibility that receptor cross-talk is driven through uncharacterized Tie-integrin interactions on the endothelial surface. Using a live cell FRET-based proximity assay, we monitor Tie-integrin receptor recognition and demonstrate that both Tie1 and Tie2 readily associate with integrins α5ß1 and αVß3 through their respective ectodomains. Although not required, Tie2-integrin association is significantly enhanced in the presence of the extracellular component and integrin ligand fibronectin. In vitro binding assays with purified components reveal that Tie-integrin recognition is direct, and further demonstrate that the receptor binding domain of the Tie2 ligand Ang-1, but not the receptor binding domain of Ang-2, can independently associate with α5ß1 or αVß3. Finally, we reveal that cooperative Tie/integrin interactions selectively stimulate ERK/MAPK signaling in the presence of both Ang-1 and fibronectin, suggesting a molecular mechanism to sensitize Tie2 to extracellular matrix. We provide a mechanistic model highlighting the role of receptor localization and association in regulating distinct signaling cascades and in turn, the angiogenic switch. PMID:27695111

  10. Interaction of kindlin-3 and β2-integrins differentially regulates neutrophil recruitment and NET release in mice.

    PubMed

    Xu, Zhen; Cai, Jiayi; Gao, Juan; White, Gilbert C; Chen, Fangyuan; Ma, Yan-Qing

    2015-07-16

    Kindlin-3 essentially supports integrin activation in blood cells. Absence of kindlin-3 in humans causes leukocyte adhesion deficiency-III characterized with severe bleeding disorder and recurrent infections. Previously, we generated kindlin-3 knock-in (K3KI) mice carrying an integrin-interaction disrupting mutation in kindlin-3 and verified the functional significance of the binding of kindlin-3 to integrin αIIbβ3 in platelets. Here, using K3KI mice, we functionally evaluate the crosstalk between kindlin-3 and β2-integrins in neutrophils. Although the kindlin-3 mutant in K3KI neutrophils is normally expressed, its binding ability to β2-integrins in neutrophils is disabled. In vitro and in vivo analyses disclose that β2-integrin-mediated K3KI neutrophil adhesion and recruitment are significantly suppressed. Interestingly, the ability of releasing neutrophil extracellular traps (NETs) from K3KI neutrophils is also compromised. Substantially, a peptide derived from the integrin β2 cytoplasmic tail that can inhibit the interaction between kindlin-3 and β2-inegrins significantly jeopardizes NET release without affecting neutrophil adhesion and recruitment under the experimental conditions. These findings suggest that crosstalk between kindlin-3 and β2-integrins in neutrophils is required for supporting both neutrophil recruitment and NET release, but the involved regulatory mechanisms in these two cellular events might be differential, thus providing a novel therapeutic concept to treat innate immune-related diseases. © 2015 by The American Society of Hematology.

  11. Absence of αvβ6 Integrin Is Linked to Initiation and Progression of Periodontal Disease

    PubMed Central

    Ghannad, Farzin; Nica, Daniela; Garcia Fulle, Maria I.; Grenier, Daniel; Putnins, Edward E.; Johnston, Sarah; Eslami, Ameneh; Koivisto, Leeni; Jiang, Guoqiao; McKee, Marc D.; Häkkinen, Lari; Larjava, Hannu

    2008-01-01

    Integrin αvβ6 is generally not expressed in adult epithelia but is induced in wound healing, cancer, and certain fibrotic disorders. Despite this generalized absence, we observed that αvβ6 integrin is constitutively expressed in the healthy junctional epithelium linking the gingiva to tooth enamel. Moreover, expression of αvβ6 integrin was down-regulated in human periodontal disease, a common medical condition causing tooth loss and also contributing to the development of cardiovascular diseases by increasing the total systemic inflammatory burden. Remarkably, integrin β6 knockout mice developed classic signs of spontaneous, chronic periodontal disease with characteristic inflammation, epithelial down-growth, pocket formation, and bone loss around the teeth. Integrin αvβ6 acts as a major activator of transforming growth factor-β1 (TGF-β1), a key anti-inflammatory regulator in the immune system. Co-expression of TGF-β1 and αvβ6 integrin was observed in the healthy junctional epithelium. Moreover, an antibody that blocks αvβ6 integrin-mediated activation of TGF-β1 initiated inflammatory periodontal disease in a rat model of gingival inflammation. Thus, αvβ6 integrin is constitutively expressed in the epithelium sealing the gingiva to the tooth and plays a central role in protection against inflammatory periodontal disease through activation of TGF-β1. PMID:18385522

  12. Differential Influence of Components Resulting from Atmospheric-Pressure Plasma on Integrin Expression of Human HaCaT Keratinocytes

    PubMed Central

    Haertel, Beate; Straßenburg, Susanne; Wende, Kristian; von Woedtke, Thomas

    2013-01-01

    Adequate chronic wound healing is a major problem in medicine. A new solution might be non-thermal atmospheric-pressure plasma effectively inactivating microorganisms and influencing cells in wound healing. Plasma components as, for example, radicals can affect cells differently. HaCaT keratinocytes were treated with Dielectric Barrier Discharge plasma (DBD/air, DBD/argon), ozone or hydrogen peroxide to find the components responsible for changes in integrin expression, intracellular ROS formation or apoptosis induction. Dependent on plasma treatment time reduction of recovered cells was observed with no increase of apoptotic cells, but breakdown of mitochondrial membrane potential. DBD/air plasma increased integrins and intracellular ROS. DBD/argon caused minor changes. About 100 ppm ozone did not influence integrins. Hydrogen peroxide caused similar effects compared to DBD/air plasma. In conclusion, effects depended on working gas and exposure time to plasma. Short treatment cycles did neither change integrins nor induce apoptosis or ROS. Longer treatments changed integrins as important for influencing wound healing. Plasma effects on integrins are rather attributed to induction of other ROS than to generation of ozone. Changes of integrins by plasma may provide new solutions of improving wound healing, however, conditions are needed which allow initiating the relevant influence on integrins without being cytotoxic to cells. PMID:23936843

  13. Integrins are required for tissue organization and restriction of neurogenesis in regenerating planarians.

    PubMed

    Seebeck, Florian; März, Martin; Meyer, Anna-Wiebke; Reuter, Hanna; Vogg, Matthias C; Stehling, Martin; Mildner, Karina; Zeuschner, Dagmar; Rabert, Franziska; Bartscherer, Kerstin

    2017-03-01

    Tissue regeneration depends on proliferative cells and on cues that regulate cell division, differentiation, patterning and the restriction of these processes once regeneration is complete. In planarians, flatworms with high regenerative potential, muscle cells express some of these instructive cues. Here, we show that members of the integrin family of adhesion molecules are required for the integrity of regenerating tissues, including the musculature. Remarkably, in regenerating β1-integrin RNAi planarians, we detected increased numbers of mitotic cells and progenitor cell types, as well as a reduced ability of stem cells and lineage-restricted progenitor cells to accumulate at wound sites. These animals also formed ectopic spheroid structures of neural identity in regenerating heads. Interestingly, those polarized assemblies comprised a variety of neural cells and underwent continuous growth. Our study indicates that integrin-mediated cell adhesion is required for the regenerative formation of organized tissues and for restricting neurogenesis during planarian regeneration. © 2017. Published by The Company of Biologists Ltd.

  14. Integrins are required for tissue organization and restriction of neurogenesis in regenerating planarians

    PubMed Central

    Seebeck, Florian; März, Martin; Meyer, Anna-Wiebke; Reuter, Hanna; Vogg, Matthias C.; Stehling, Martin; Mildner, Karina; Zeuschner, Dagmar; Rabert, Franziska

    2017-01-01

    Tissue regeneration depends on proliferative cells and on cues that regulate cell division, differentiation, patterning and the restriction of these processes once regeneration is complete. In planarians, flatworms with high regenerative potential, muscle cells express some of these instructive cues. Here, we show that members of the integrin family of adhesion molecules are required for the integrity of regenerating tissues, including the musculature. Remarkably, in regenerating β1-integrin RNAi planarians, we detected increased numbers of mitotic cells and progenitor cell types, as well as a reduced ability of stem cells and lineage-restricted progenitor cells to accumulate at wound sites. These animals also formed ectopic spheroid structures of neural identity in regenerating heads. Interestingly, those polarized assemblies comprised a variety of neural cells and underwent continuous growth. Our study indicates that integrin-mediated cell adhesion is required for the regenerative formation of organized tissues and for restricting neurogenesis during planarian regeneration. PMID:28137894

  15. α3β1 integrin promotes radiation-induced migration of meningioma cells.

    PubMed

    Gogineni, Venkateswara Rao; Nalla, Arun Kumar; Gupta, Reshu; Gujrati, Meena; Klopfenstein, Jeffrey D; Mohanam, Sanjeeva; Rao, Jasti S

    2011-06-01

    Cell motility is influenced by the microenvironment, signal transduction and cytoskeleton rearrangement. Cancer cells become resistant to these control mechanisms and gain the ability to move throughout the body and invade healthy tissues, which leads to metastatic disease. Integrins respond to context-dependent cues and promote cell migration and survival in cancer cells. In the present study, we analyzed the role of integrins in radiation-induced migration of meningioma cells. Migration and cell proliferation assays revealed that radiation treatment (7 Gy) significantly increased migration and decreased proliferation in two cell lines, IOMM-Lee and CH-157-MN. α3 and β1 integrins were overexpressed at both the protein and transcript levels after radiation treatment and a function-blocking α3β1 antibody inhibited the radiation-induced migration. Immunofluorescence studies illustrated the localization of α3 integrin and F-actin at the migration front of irradiated cells. Further, an increase in phosphorylation of FAK and ERK was observed, while both FAK phosphorylation inhibitor and FAK shRNA inhibited ERK phosphorylation and downregulated uPA and vinculin. In addition to the co-localization of FAK and ERK at the migration front, these FAK-inhibition results link the downstream effects of ERK to FAK. Correspondingly, U0126 quenched ERK phosphorylation and reduced the expression of molecules involved in migration. Furthermore, brain sections of the animals implanted with tumors followed by radiation treatment showed elevated levels of α3 integrin and active ERK. Taken together, our results show that radiation treatment enhances the migration of meningioma cells with the involvement of α3β1 integrin-mediated signaling via FAK and ERK.

  16. Annexin A6 and Late Endosomal Cholesterol Modulate Integrin Recycling and Cell Migration.

    PubMed

    García-Melero, Ana; Reverter, Meritxell; Hoque, Monira; Meneses-Salas, Elsa; Koese, Meryem; Conway, James R W; Johnsen, Camilla H; Alvarez-Guaita, Anna; Morales-Paytuvi, Frederic; Elmaghrabi, Yasmin A; Pol, Albert; Tebar, Francesc; Murray, Rachael Z; Timpson, Paul; Enrich, Carlos; Grewal, Thomas; Rentero, Carles

    2016-01-15

    Annexins are a family of proteins that bind to phospholipids in a calcium-dependent manner. Earlier studies implicated annexin A6 (AnxA6) to inhibit secretion and participate in the organization of the extracellular matrix. We recently showed that elevated AnxA6 levels significantly reduced secretion of the extracellular matrix protein fibronectin (FN). Because FN is directly linked to the ability of cells to migrate, this prompted us to investigate the role of AnxA6 in cell migration. Up-regulation of AnxA6 in several cell models was associated with reduced cell migration in wound healing, individual cell tracking and three-dimensional migration/invasion assays. The reduced ability of AnxA6-expressing cells to migrate was associated with decreased cell surface expression of αVβ3 and α5β1 integrins, both FN receptors. Mechanistically, we found that elevated AnxA6 levels interfered with syntaxin-6 (Stx6)-dependent recycling of integrins to the cell surface. AnxA6 overexpression caused mislocalization and accumulation of Stx6 and integrins in recycling endosomes, whereas siRNA-mediated AnxA6 knockdown did not modify the trafficking of integrins. Given our recent findings that inhibition of cholesterol export from late endosomes (LEs) inhibits Stx6-dependent integrin recycling and that elevated AnxA6 levels cause LE cholesterol accumulation, we propose that AnxA6 and blockage of LE cholesterol transport are critical for endosomal function required for Stx6-mediated recycling of integrins in cell migration. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Integrin engagement mediates the human polymorphonuclear leukocyte response to a fungal pathogen-associated molecular pattern.

    PubMed

    Lavigne, Liz M; O'Brien, Xian M; Kim, Minsoo; Janowski, Jessie W; Albina, Jorge E; Reichner, Jonathan S

    2007-06-01

    Extravasation of leukocytes from peripheral blood is required for an effective inflammatory response at sites of tissue infection. Integrins help mediate extravasation and navigate the leukocyte to the infectious source. A novel role for integrins in regulating the effector response to a cell wall component of fungal pathogens is the subject of the current study. Although phagocytosis is useful for clearance of unicellular fungi, the immune response against large, noningestible hyphae is not well-understood. Fungal beta-glucan, a pathogen-associated molecular pattern, activates production of superoxide anion in leukocytes without the need for phagocytosis. To model polymorphonuclear leukocyte (PMN) recognition of fungi under conditions in which phagocytosis cannot occur, beta-glucan was covalently immobilized onto tissue culture plastic. Plasma membrane-associated respiratory burst was measured by reduction of ferricytochrome C. Results show that the human PMN oxidative burst response to immobilized beta-glucan is suppressed by addition of beta(1) integrin ligands to the beta-glucan matrix. Suppression was dose dependent and steric hindrance was ruled out. beta(1) integrin ligands did not affect respiratory burst to ingestible beta-glucan-containing particles, phorbol esters or live yeast hyphae. Furthermore, in the absence of matrix, Ab activation of VLA3 or VLA5, but not other beta(1) integrins, also prevented beta-glucan-induced respiratory burst. beta(1)-induced suppression was blocked and burst response restored by treating neutrophils with either the cell-binding fragment of soluble human Fn, cyclic RGD peptide, or Ab specific to VLA3 or VLA5. Together these findings extend the functional role of beta(1) integrins to include modulating PMN respiratory burst to a pathogen-associated molecular pattern.

  18. Remanent cell traction force in renal vascular smooth muscle cells induced by integrin-mediated mechanotransduction

    PubMed Central

    Balasubramanian, Lavanya; Lo, Chun-Min; Sham, James S. K.

    2013-01-01

    It was previously demonstrated in isolated renal vascular smooth muscle cells (VSMCs) that integrin-mediated mechanotransduction triggers intracellular Ca2+ mobilization, which is the hallmark of myogenic response in VSMCs. To test directly whether integrin-mediated mechanotransduction results in the myogenic response-like behavior in renal VSMCs, cell traction force microscopy was used to monitor cell traction force when the cells were pulled with fibronectin-coated or low density lipoprotein (LDL)-coated paramagnetic beads. LDL-coated beads were used as a control for nonintegrin-mediated mechanotransduction. Pulling with LDL-coated beads increased the cell traction force by 61 ± 12% (9 cells), which returned to the prepull level after the pulling process was terminated. Pulling with noncoated beads had a minimal increase in the cell traction force (12 ± 9%, 8 cells). Pulling with fibronectin-coated beads increased the cell traction force by 56 ± 20% (7 cells). However, the cell traction force was still elevated by 23 ± 14% after the pulling process was terminated. This behavior is analogous to the changes of vascular resistance in pressure-induced myogenic response, in which vascular resistance remains elevated after myogenic constriction. Fibronectin is a native ligand for α5β1-integrins in VSMCs. Similar remanent cell traction force was found when cells were pulled with beads coated with β1-integrin antibody (Ha2/5). Activation of β1-integrin with soluble antibody also triggered variations of cell traction force and Ca2+ mobilization, which were abolished by the Src inhibitor. In conclusion, mechanical force transduced by α5β1-integrins triggered a myogenic response-like behavior in isolated renal VSMCs. PMID:23325413

  19. Integrin {alpha}6 cleavage: A novel modification to modulate cell migration

    SciTech Connect

    Pawar, Sangita C.; Demetriou, Manolis C.; Nagle, Raymond B.

    2007-04-01

    Integrins play a major role in cell adhesion and migration. Previous work reported that a cleaved form of integrin {alpha}6 ({alpha}6p) was detected in invasive human prostate cancer tissue, absent in normal prostate tissue and was produced by urokinase-type Plasminogen Activator (uPA) in a plasmin-independent manner. Using site-directed mutagenesis we identified amino acid residues R594 and R595, located in the 'stalk' region of integrin {alpha}6, as essential for cleavage. The cleavage site is located on the extracellular region of the protein between the {beta}-barrel domain and the thigh domain. Prostate cancer cells (PC3N) were stably transfected to overexpress the cleavable,more » wild-type (PC3N-{alpha}6-WT) or the non-cleavable form of integrin {alpha}6 (PC3N-{alpha}6-RR). The number of cells invading laminin 111- and laminin 332-coated filters by PC3N-{alpha}6-WT cells increased by threefold as compared to PC3N-{alpha}6-RR cells. Plasminogen activator inhibitor-1 (PAI-1) reduced the invasion of PC3N-{alpha}6-WT cells by approximately 42% through laminin 332-coated filters and plasmin inhibitor aprotinin had no significant effect. Linear cell migration increased production of integrin {alpha}6p in the PC3N-{alpha}6-WT cells and not in the PC3N-{alpha}6-RR cells and 32% of the PC3N-{alpha}6-WT cells migrated on laminin 111 in the linear migration assay as compared to the 5% PC3N-{alpha}6-RR cells. These data taken together suggest that the uPA-mediated cell surface cleavage of the {alpha}6 integrin extracellular domain is involved in tumor cell invasion and migration on laminin.« less

  20. Cryptic 3' mRNA processing signals hinder the expression of Schistosoma mansoni integrins in yeast.

    PubMed

    Parker-Manuel, Richard P; Grevelding, Christoph G; Gelmedin, Verena

    2015-01-01

    The expression of parasite genes has often proven difficult in heterologous systems such as yeast or E. coli. Most often, promoter choice and codon usage were hypothesised to be the main reason for expression failures. The trematode parasite Schistosoma mansoni has five integrin genes named Smα-Int1-4 and Smβ-Int1, which we aimed to express in the yeast Saccharomyces cerevisiae. This has not been achieved, however, as only Smβ-Int1 integrin could be expressed. When the four α integrins were driven by a stronger promoter, this enabled Smα-Int1 to be expressed as well, but the remaining integrins, Smα-Int2-4, still could not be expressed. Evidence from RT-PCR experiments suggested that this was due to premature transcription termination. Using detailed in silico sequence analyses we identified AT-rich stretches in these integrin genes, which have high similarity to yeast mRNA 3'-end processing signals. We hypothesised that these signals were causing the premature truncation. To test this, we designed an optimised version of Smα-Int3, in which the sequence was modified to replace the yeast 3' processing signals. This strategy allowed us to express Smα-Int3 integrin successfully in S. cerevisiae. These findings show that the misinterpretation of AT-rich sequences by yeast 3'-mRNA processing machinery can cause problems when attempting to express genes containing such sequences in this host. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Cytohesin-1 regulates human blood neutrophil adhesion to endothelial cells through β2 integrin activation.

    PubMed

    El azreq, Mohammed-Amine; Bourgoin, Sylvain G

    2011-07-01

    Cytohesin-1 is a guanine nucleotide exchange factor for ADP ribosylation factor 6 (Arf6) in human blood neutrophils and differentiated PLB-985 neutrophil-like cells. Cytohesin-1 regulates adhesion and the transendothelial migration of monocytes, dendritic cells and T lymphocytes through activation of the β2 integrin LFA-1. In this study we investigated the role of cytohesin-1 in neutrophil and neutrophil-like cell adhesion to HUVECs, immobilized ICAM-1, and the α4β1 and α5β1 integrin extracellular matrix ligand fibronectin. We show that cytohesin-1 knockdown or inhibition with secinH3 inhibits fMLF-mediated cell adhesion to HUVECs and immobilized ICAM-1, whereas cytohesin-1 over-expression has the opposing effect. Binding of PLB-985 cells to HUVECs correlated with expression of the high-affinity β2 integrin epitope recognized by mAb24. Adhesion to HUVECs was inhibited by soluble ICAM-1, anti-ICAM-1, anti-CD11a and anti-CD18, but not anti-CD11b, blocking antibodies. We also demonstrate that cytohesin-1 knockdown promotes fMLF-mediated cell adhesion to fibronectin whereas cytohesin-1 over-expression has the opposing effect. Crosstalk between β1 and β2 integrins also exists since inhibition of β1 integrin functions with blocking antibodies enhanced adhesion of PLB-985 over-expressing cytohesin-1 to ICAM-1. We suggest that cytohesin-1 is a key regulator of neutrophil adhesion to endothelial cells and to components of extracellular matrix, which may influence cell emigration through its dual opposing effect on β2 and β1 integrin activation. Copyright © 2011 Elsevier Ltd. All rights reserved.

  2. Importance of Interaction between Integrin and Actin Cytoskeleton in Suspension Adaptation of CHO cells.

    PubMed

    Walther, Christa G; Whitfield, Robert; James, David C

    2016-04-01

    The biopharmaceutical production process relies upon mammalian cell technology where single cells proliferate in suspension in a chemically defined synthetic environment. This environment lacks exogenous growth factors, usually contributing to proliferation of fibroblastic cell types such as Chinese hamster ovary (CHO) cells. Use of CHO cells for production hence requires a lengthy 'adaptation' process to select clones capable of proliferation as single cells in suspension. The underlying molecular changes permitting proliferation in suspension are not known. Comparison of the non-suspension-adapted clone CHO-AD and a suspension-adapted propriety cell line CHO-SA by flow cytometric analysis revealed a highly variable bi-modal expression pattern for cell-to-cell contact proteins in contrast to the expression pattern seen for integrins. Those have a uni-modal expression on suspension and adherent cells. Integrins showed a conformation distinguished by regularly distributed clusters forming a sphere on the cell membrane of suspension-adapted cells. Actin cytoskeleton analysis revealed reorganisation from the typical fibrillar morphology found in adherent cells to an enforced spherical subcortical actin sheath in suspension cells. The uni-modal expression and specific clustering of integrins could be confirmed for CHO-S, another suspension cell line. Cytochalasin D treatment resulted in breakdown of the actin sheath and the sphere-like integrin conformation demonstrating the link between integrins and actin in suspension-adapted CHO cells. The data demonstrates the importance of signalling changes, leading to an integrin rearrangement on the cell surface, and the necessity of the reinforcement of the actin cytoskeleton for proliferation in suspension conditions.

  3. AlphaVBeta3 Integrin expression within uterine endometrium in unexplained infertility: a prospective cohort study.

    PubMed

    Elnaggar, Ahmed; Farag, Amr H; Gaber, Mohamed E; Hafeez, Mohamed Abdel; Ali, Mohamed S; Atef, Alaa M

    2017-09-26

    Implantation defect is one of these contributing factors for unexplained infertility. In the mid-luteal phase, when implantation is expected to happen, Integrins expression is remarkably increased. So, Integrins could potentially serve as markers for the frame of the window of implantation. αVβ3 integrin could have a role as a potential receptor for embryonic attachment. The aim of the current study is to investigate whether the women with unexplained infertility have a pattern of expression of endometrial αvβ3 integrin that could differ from those who have normal fertility or not. Two groups of women have been included in this study. The first group was the Unexplained Infertility Group. This group included women diagnosed with unexplained primary infertility. The second group was the fertile Group, which included fertile parous women presented to the family planning clinic seeking contraception. 2D transvaginal ultrasound scan (TVS) was performed six days after detecting urinary LH surge. (TVS) was used to measure endometrial thickness, and subendometrial blood flow color Doppler Resistance Index (RI). On the same day of transvaginal ultrasound, endometrial samples were taken using the Endocell® office suction sampler for Immunohistochemistry (IHC) study using monoclonal mouse IgG antibodies to detect endometrial αvβ3 integrin. Thirty-five fertile women with a diagnosis of unexplained infertility were included as a group I [Unexplained infertility Group] along with an equal number of fertile women as group II [Fertile Group]. The group of women with a diagnosis of unexplained infertility had a significantly lower αvβ3 integrin score when compared to the fertile group (median score 0, range:0-2 and median score 1, range: 1-3 and for infertile and fertile groups respectively, P < 0.0001). In addition, the unexplained infertility group had significantly higher subendometrial flow RI and Significantly thinner endometrial thickness. This study showed that

  4. Rapid characterization of transgenic and non-transgenic soybean oils by chemometric methods using NIR spectroscopy

    NASA Astrophysics Data System (ADS)

    Luna, Aderval S.; da Silva, Arnaldo P.; Pinho, Jéssica S. A.; Ferré, Joan; Boqué, Ricard

    Near infrared (NIR) spectroscopy and multivariate classification were applied to discriminate soybean oil samples into non-transgenic and transgenic. Principal Component Analysis (PCA) was applied to extract relevant features from the spectral data and to remove the anomalous samples. The best results were obtained when with Support Vectors Machine-Discriminant Analysis (SVM-DA) and Partial Least Squares-Discriminant Analysis (PLS-DA) after mean centering plus multiplicative scatter correction. For SVM-DA the percentage of successful classification was 100% for the training group and 100% and 90% in validation group for non transgenic and transgenic soybean oil samples respectively. For PLS-DA the percentage of successful classification was 95% and 100% in training group for non transgenic and transgenic soybean oil samples respectively and 100% and 80% in validation group for non transgenic and transgenic respectively. The results demonstrate that NIR spectroscopy can provide a rapid, nondestructive and reliable method to distinguish non-transgenic and transgenic soybean oils.

  5. Distinguishing transgenic from non-transgenic Arabidopsis plants by (1)H NMR-based metabolic fingerprinting.

    PubMed

    Ren, Yanfei; Wang, Tao; Peng, Yufa; Xia, Bin; Qu, Li-Jia

    2009-10-01

    We have recently reported the construction of an nuclear magnetic resonance (NMR)-based metabonomics study platform, Automics. To examine the application of Automics in transgenic plants, we performed metabolic fingerprinting analysis, i.e., (1)H NMR spectroscopy and multivariate analysis, on wild-type and transgenic Arabidopsis. We found that it was possible to distinguish wild-type from four transgenic plants by PLS-DA following application of orthogonal signal correction (OSC). Scores plot following OSC clearly demonstrates significant variation between the transgenic and non-transgenic groups, suggesting that the metabolic changes among wild-type and transgenic lines are possibly associated with transgenic event. We also found that the major contributing metabolites were some specific amino acids (i.e., threonine and alanine), which could correspond to the insertion of the selective marker BAR gene in the transgenic plants. Our data suggests that NMR-based metabonomics is an efficient method to distinguish fingerprinting difference between wild-type and transgenic plants, and can potentially be applied in the bio-safety assessment of transgenic plants.

  6. The P2Y2 nucleotide receptor requires interaction with alpha v integrins to access and activate G12.

    PubMed

    Liao, Zhongji; Seye, Cheikh I; Weisman, Gary A; Erb, Laurie

    2007-05-01

    The P2Y2 nucleotide receptor (P2Y2R) interacts with alpha v integrins to activate G(o) and induce chemotaxis in human 1321N1 astrocytoma cells. In this study, it was determined that the P2Y2R also requires interaction with alpha v integrins to activate G12 and associated signaling pathways that control chemotaxis in 1321N1 cells. Mutation of the Arg-Gly-Asp (RGD) integrin-binding sequence in the first extracellular loop of the human P2Y2R to Arg-Gly-Glu (RGE), which prevents integrin interaction, did not inhibit G(q) or ERK1/2 signaling by the P2Y2R agonist UTP but completely inhibited activation of G12 and G12-mediated events, including Rho activation, cofilin and myosin light chain-2 phosphorylation, stress fiber formation and chemotaxis towards UTP. The involvement of G12 in all these events was verified by using a dominant negative G alpha12 construct. G12 activation by the P2Y2R also was inhibited by anti-alpha v beta5 integrin antibodies and alpha v integrin antisense oligonucleotides, suggesting that alpha v integrin activity and expression are required for the P2Y2R to activate G12. Co-immunoprecipitation experiments confirmed that G alpha12 protein associates with the wild-type P2Y2R and with alpha v integrins but not with the RGE mutant P2Y2R or with alpha3 integrins. Collectively, these results suggest that alpha v integrin complexes provide the P2Y2R with access to G12, thereby allowing activation of this heterotrimeric G protein that controls actin cytoskeletal rearrangements required for chemotaxis.

  7. Generation of transgenic Hydra by embryo microinjection.

    PubMed

    Juliano, Celina E; Lin, Haifan; Steele, Robert E

    2014-09-11

    As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among multicellular animals. Hydra is used as a model for the study of regeneration, pattern formation, and stem cells. However, research efforts have been hampered by lack of a reliable method for gene perturbations to study molecular function. The development of transgenic methods has revitalized the study of Hydra biology(1). Transgenic Hydra allow for the tracking of live cells, sorting to yield pure cell populations for biochemical analysis, manipulation of gene function by knockdown and over-expression, and analysis of promoter function. Plasmid DNA injected into early stage embryos randomly integrates into the genome early in development. This results in hatchlings that express transgenes in patches of tissue in one or more of the three lineages (ectodermal epithelial, endodermal epithelial, or interstitial). The success rate of obtaining a hatchling with transgenic tissue is between 10% and 20%. Asexual propagation of the transgenic hatchling is used to establish a uniformly transgenic line in a particular lineage. Generating transgenic Hydra is surprisingly simple and robust, and here we describe a protocol that can be easily implemented at low cost.

  8. Glyphostate-drift but not herbivory alters the rate of transgene flow from single and stacked trait transgenic canola (Brassica napus L.) to non-transgenic B. napus and B. rapa

    EPA Science Inventory

    While transgenic plants can offer agricultural benefits, the escape of transgenes out of crop fields is a major environmental concern. Escape of transgenic herbicide resistance has occurred between transgenic Brassica napus (canola) and weedy species in numerous locations. In t...

  9. Expression of multiple proteins in transgenic plants

    DOEpatents

    Vierstra, Richard D.; Walker, Joseph M.

    2002-01-01

    A method is disclosed for the production of multiple proteins in transgenic plants. A DNA construct for introduction into plants includes a provision to express a fusion protein of two proteins of interest joined by a linking domain including plant ubiquitin. When the fusion protein is produced in the cells of a transgenic plant transformed with the DNA construction, native enzymes present in plant cells cleave the fusion protein to release both proteins of interest into the cells of the transgenic plant. Since the proteins are produced from the same fusion protein, the initial quantities of the proteins in the cells of the plant are approximately equal.

  10. [Transgenic plants: the future of sustainable agriculture].

    PubMed

    Cordeiro, A R

    2000-01-01

    There has been a daily increase of evidence of the advantages of the transgenic plants which have been licensed so far. Every new product offers greater advantages as regards productivity, reduction of environmental impact, vitamin value and so on. The secret of the transgenic method lies in the fact that it enables strict selection in every phase in the laboratory and during field tests. Though it is still not possible to be risk-free, we can certainly trust transgenics better than plants classified as "organic".

  11. N-Glycosylation of integrin α5 acts as a switch for EGFR-mediated complex formation of integrin α5β1 to α6β4

    PubMed Central

    Hang, Qinglei; Isaji, Tomoya; Hou, Sicong; Zhou, Ying; Fukuda, Tomohiko; Gu, Jianguo

    2016-01-01

    N-Glycosylation of integrin α5β1 is involved in multiple cell behaviors. We previously reported that the N-glycosylations of the calf domain on integrin α5 (S3–5,10–14) are essential for its inhibitory effect on EGFR signaling in regulating cell proliferation. However, the importance of the individual N-glycosylation and the underlying mechanisms of inhibition remain unclear. Here, we characterize the S3–5,10–14 mutants in detail and found that the N-glycosylation of site-11 (Asn712) is key for cell growth. The restoration of site-11, unlike the other individual sites, significantly suppressed cell growth and EGFR signaling in a manner that was similar to that of wild-type (WT). Mechanistically, this N-glycosylation inhibited the response abilities upon EGF stimulation and EGFR dimerization. Interestingly, we found this N-glycosylation controlled the EGFR complex formation with integrin α5β1 or α6β4; i.e., the loss of site-11 switched EGFR-α5β1 to EGFR-α6β4, which is well known to promote cellular signaling for cell growth. Moreover, the site-11 N-glycan exhibited a more branching structure compared with other sites, which may be required for EGFR-α5β1 formation. Taken together, these data clearly demonstrate that the site-11 N-glycosylation on α5 is most important for its inhibitory effect on EGFR signaling, which may provide a novel regulatory mechanism for crosstalks between integrins and EGFR. PMID:27641064

  12. Modulation of integrin antagonist signaling by ligand binding of the heparin-binding domain of vitronectin to the alphaVbeta3 integrin.

    PubMed

    Maile, Laura A; Aday, Ariel W; Busby, Walker H; Sanghani, Ravi; Veluvolu, Umadevi; Clemmons, David R

    2008-10-01

    The interaction between the arginine glycine and aspartic acid motif (RGD) of integrin ligands such as vitronectin and the integrin receptor alphaVbeta3 in mediating cell attachment has been well described. Similarly, the ability of disintegrins, small RGD containing peptides, to inhibit cell attachment and other cellular processes has also been studied extensively. Recently, we characterized a second site of interaction between vitronectin and its integrin partner. We determined that amino acids within the heparin-binding domain of vitronectin bind to a cysteine loop (C-loop) region of beta3 and that this interaction is required for the positive effects of alphaVbeta3 ligand occupancy on IGF-I signaling in smooth muscle cells. In this study we examine the signaling events activated following ligand binding of disintegrins to the alphaVbeta3 and the ability of these signals to be regulated by binding of the heparin-binding domain of vitronectin. We demonstrate that disintegrin ligand binding activates a series of events including the sequential activation of the tyrosine kinases c-Src and Syk. This leads to the activation of calpain and the cleavage of the beta3 cytoplasmic tail. Addition of vitronectin or a peptide homologous to the heparin-binding domain inhibited activation of this pathway. Our results suggest that the signaling events that occur following ligand binding to the alphaVbeta3 integrin reflects a balance between the effects mediated through the RGD binding site interaction and the effects mediated by the heparin binding site interaction and that for intact vitronectin the effect of the heparin-binding domain predominates. (c) 2008 Wiley-Liss, Inc.

  13. A simple and rapid method for determining transgenic cotton plants.

    PubMed

    Zhang, Baohong; Wang, Hongmei; Liu, Fang; Wang, Qinglian

    2013-01-01

    Determining transgenic events is a critical step for obtaining transgenic plants as well as the later stage of application. Traditional methods, such as Northern blotting and qRT-PCR, for determining transgenic events either require radioactively labeled substrates, expensive instruments, or long-time commitments, which result in lab and time-consuming as well as expensive costs. These methods also require destroying the transgenic events. In this chapter, we present a simple and rapid method for determining transgenic cotton plants in both laboratory and field conditions. This method is based on the sensitivity of transgenic and non-transgenic plants to a specific chemical, such as antibiotics or herbicides. This method will facilitate the screening of transgenic events, save time, reduce cost, and speed up the application of transgenic technology on cotton breeding and production. More important, this is a nondestructive bioassay method; the transgenic plants can be transferred into greenhouse or field for the later study after the detection process.

  14. CW-EPR studies revealed different motional properties and oligomeric states of the integrin β1a transmembrane domain in detergent micelles or liposomes.

    PubMed

    Yu, Lu; Wang, Wei; Ling, Shenglong; Liu, Sanling; Xiao, Liang; Xin, Yanlong; Lai, Chaohua; Xiong, Ying; Zhang, Longhua; Tian, Changlin

    2015-01-19

    Integrins are heterodimeric membrane proteins that regulate essential processes: cell migration, cell growth, extracellular matrix assembly and tumor metastasis. Each integrin α or β subunit contains a large extracellular domain, a single transmembrane (TM) domain, and a short cytoplasmic tail. The integrin TM domains are important for heterodimeric association and dissociation during the conversion from inactive to active states. Moreover, integrin clustering occurs by homo-oligomeric interactions between the TM helices. Here, the transmembrane and cytoplasmic (TMC) domains of integrin β1a were overexpressed, and the protein was purified in detergent micelles and/or reconstituted in liposomes. To investigate the TM domain conformational properties of integrin β1a, 26 consecutive single cysteine mutants were generated for site-directed spin labeling and continuous-wave electron paramagnetic resonance (CW-EPR) mobility and accessibility analyses. The mobility analysis identified two integrin β1a-TM regions with different motional properties in micelles and a non-continuous integrin β1a-TM helix with high immobility in liposomes. The accessibility analysis verified the TM range (Val737-Lys752) of the integrin β1a-TMC in micelles. Further mobility and accessibility comparisons of the integrin β1a-TMC domains in micelles or liposomes identified distinctively different oligomeric states of integrin β1a-TM, namely a monomer embedded in detergent micelles and leucine-zipper-like homo-oligomeric clusters in liposomes.

  15. Low-affinity binding in cis to P2Y2R mediates force-dependent integrin activation during hantavirus infection

    PubMed Central

    Bondu, Virginie; Wu, Chenyu; Cao, Wenpeng; Simons, Peter C.; Gillette, Jennifer; Zhu, Jieqing; Erb, Laurie; Zhang, X. Frank; Buranda, Tione

    2017-01-01

    Pathogenic hantaviruses bind to the plexin-semaphorin-integrin (PSI) domain of inactive, β3 integrins. Previous studies have implicated a cognate cis interaction between the bent conformation β5/β3 integrins and an arginine-glycine-aspartic acid (RGD) sequence in the first extracellular loop of P2Y2R. With single-molecule atomic force microscopy, we show a specific interaction between an atomic force microscopy tip decorated with recombinant αIIbβ3 integrins and (RGD)P2Y2R expressed on cell membranes. Mutation of the RGD sequence to RGE in the P2Y2R removes this interaction. Binding of inactivated and fluorescently labeled Sin Nombre virus (SNV) to the integrin PSI domain stimulates higher affinity for (RGD)P2Y2R on cells, as measured by an increase in the unbinding force. In CHO cells, stably expressing αIIbβ3 integrins, virus engagement at the integrin PSI domain, recapitulates physiologic activation of the integrin as indicated by staining with the activation-specific mAB PAC1. The data also show that blocking of the Gα13 protein from binding to the cytoplasmic domain of the β3 integrin prevents outside-in signaling and infection. We propose that the cis interaction with P2Y2R provides allosteric resistance to the membrane-normal motion associated with the switchblade model of integrin activation, where the development of tensile force yields physiological integrin activation. PMID:28835374

  16. Proteomic analysis of known and candidate rice allergens between non-transgenic and transgenic plants.

    PubMed

    Satoh, Rie; Nakamura, Rika; Komatsu, Akira; Oshima, Masahiro; Teshima, Reiko

    2011-04-01

    Salt-soluble proteins extracted from non-transgenic and transgenic rice were evaluated for the presence of known and potential allergens by proteomic techniques. The salt-soluble proteins were extracted, separated by 1D and 2D electrophoresis, and analyzed by Western blotting. 1D immunoblot analysis with patients' sera revealed few qualitative differences between the IgE-binding proteins of the non-transgenic and transgenic rice. 1D immunoblot with antigen-specific-animal sera revealed no qualitative or quantitative differences in two known allergens, RAG2 and glyoxalase I, between non-transgenic and transgenic rice. Multiple spots containing known and novel IgE-binding proteins were detected among the salt-soluble proteins of non-transgenic rice by 2D immunoblotting. Two globulin-like proteins, a 52 kDa protein and a 63 kDa protein, were identified as novel IgE-binding proteins that are candidates for rice allergens. These globulin-like proteins were homologous to Cupin superfamily allergens. Quantitative analysis of 19, 52, and 63 kDa globulins with protein-specific-animal sera showed no significant differences in the expression of these proteins between the transgenic rice and non-transgenic rice. These results indicate that none of the known or novel endogenous IgE-binding proteins detected in this study appear to be altered by genetic modification. Copyright © 2011 Elsevier Inc. All rights reserved.

  17. Transgenic plants with enhanced growth characteristics

    DOEpatents

    Unkefer, Pat J.; Anderson, Penelope S.; Knight, Thomas J.

    2018-01-09

    The invention relates to transgenic plants exhibiting dramatically enhanced growth rates, greater seed and fruit/pod yields, earlier and more productive flowering, more efficient nitrogen utilization, increased tolerance to high salt conditions, and increased biomass yields. In one embodiment, transgenic plants engineered to over-express both glutamine phenylpyruvate transaminase (GPT) and glutamine synthetase (GS) are provided. The GPT+GS double-transgenic plants of the invention consistently exhibit enhanced growth characteristics, with T0 generation lines showing an increase in biomass over wild type counterparts of between 50% and 300%. Generations that result from sexual crosses and/or selfing typically perform even better, with some of the double-transgenic plants achieving an astounding four-fold biomass increase over wild type plants.

  18. Transgenic plants with enhanced growth characteristics

    DOEpatents

    Unkefer, Pat J.; Anderson, Penelope S.; Knight, Thomas J.

    2016-09-06

    The invention relates to transgenic plants exhibiting dramatically enhanced growth rates, greater seed and fruit/pod yields, earlier and more productive flowering, more efficient nitrogen utilization, increased tolerance to high salt conditions, and increased biomass yields. In one embodiment, transgenic plants engineered to over-express both glutamine phenylpyruvate transaminase (GPT) and glutamine synthetase (GS) are provided. The GPT+GS double-transgenic plants of the invention consistently exhibit enhanced growth characteristics, with T0 generation lines showing an increase in biomass over wild type counterparts of between 50% and 300%. Generations that result from sexual crosses and/or selfing typically perform even better, with some of the double-transgenic plants achieving an astounding four-fold biomass increase over wild type plants.

  19. AN APPROACH TO TRANSGENIC CROP MONITORING

    EPA Science Inventory

    Remote sensing by aerial or satellite images may provide a method of identifying transgenic pesticidal crop distribution in the landscape. Genetically engineered crops containing bacterial gene(s) that express an insecticidal protein from Bacillus thuringiensis (Bt) are regulated...

  20. Integrin associated proteins differentially regulate neutrophil polarity and directed migration in 2D and 3D

    PubMed Central

    Yamahashi, Yukie; Cavnar, Peter J.; Hind, Laurel E.; Berthier, Erwin; Bennin, David A.; Beebe, David

    2015-01-01

    Directed neutrophil migration in blood vessels and tissues is critical for proper immune function; however, the mechanisms that regulate three-dimensional neutrophil chemotaxis remain unclear. It has been shown that integrins are dispensable for interstitial three-dimensional (3D) leukocyte migration; however, the role of integrin regulatory proteins during directed neutrophil migration is not known. Using a novel microfluidic gradient generator amenable to 2D and 3D analysis, we found that the integrin regulatory proteins Kindlin-3, RIAM, and talin-1 differentially regulate neutrophil polarization and directed migration to gradients of chemoattractant in 2D versus 3D. Both talin-1-deficient and RIAM-deficient neutrophil-like cells had impaired adhesion, polarization, and migration on 2D surfaces whereas in 3D the cells polarized but had impaired 3D chemotactic velocity. Kindlin-3 deficient cells were able to polarize and migrate on 2D surfaces but had impaired directionality. In a 3D environment, Kindlin-3 deficient cells displayed efficient chemotaxis. These findings demonstrate that the role of integrin regulatory proteins in cell polarity and directed migration can be different in 2D and 3D. PMID:26354879

  1. Ataxin-3 Plays a Role in Mouse Myogenic Differentiation through Regulation of Integrin Subunit Levels

    PubMed Central

    Costa, Maria do Carmo; Bajanca, Fernanda; Rodrigues, Ana-João; Tomé, Ricardo J.; Corthals, Garry; Macedo-Ribeiro, Sandra; Paulson, Henry L.; Logarinho, Elsa; Maciel, Patrícia

    2010-01-01

    Background During myogenesis several transcription factors and regulators of protein synthesis and assembly are rapidly degraded by the ubiquitin-proteasome system (UPS). Given the potential role of the deubiquitinating enzyme (DUB) ataxin-3 in the UPS, and the high expression of the murine ataxin-3 homolog in muscle during embryogenesis, we sought to define its role in muscle differentiation. Methodology/Principal Findings Using immunofluorescence analysis, we found murine ataxin-3 (mATX3) to be highly expressed in the differentiated myotome of E9.5 mouse embryos. C2C12 myoblasts depleted of mATX3 by RNA interference exhibited a round morphology, cell misalignment, and a delay in differentiation following myogenesis induction. Interestingly, these cells showed a down-regulation of α5 and α7 integrin subunit levels both by immunoblotting and immunofluorescence. Mouse ATX3 was found to interact with α5 integrin subunit and to stabilize this protein by repressing its degradation through the UPS. Proteomic analysis of mATX3-depleted C2C12 cells revealed alteration of the levels of several proteins related to integrin signaling. Conclusions Ataxin-3 is important for myogenesis through regulation of integrin subunit levels. PMID:20668528

  2. Interendothelial claudin-5 expression depends on cerebral endothelial cell–matrix adhesion by β1-integrins

    PubMed Central

    Osada, Takashi; Gu, Yu-Huan; Kanazawa, Masato; Tsubota, Yoshiaki; Hawkins, Brian T; Spatz, Maria; Milner, Richard; del Zoppo, Gregory J

    2011-01-01

    The hypothesis tested by these studies states that in addition to interendothelial cell tight junction proteins, matrix adhesion by β1-integrin receptors expressed by endothelial cells have an important role in maintaining the cerebral microvessel permeability barrier. Primary brain endothelial cells from C57 BL/6 mice were incubated with β1-integrin function-blocking antibody (Ha2/5) or isotype control and the impacts on claudin-5 expression and microvessel permeability were quantified. Both flow cytometry and immunofluorescence studies demonstrated that the interendothelial claudin-5 expression by confluent endothelial cells was significantly decreased in a time-dependent manner by Ha2/5 exposure relative to isotype. Furthermore, to assess the barrier properties, transendothelial electrical resistance and permeability measurements of the monolayer, and stereotaxic injection into the striatum of mice were performed. Ha2/5 incubation reduced the resistance of endothelial cell monolayers significantly, and significantly increased permeability to 40 and 150 kDa dextrans. Ha2/5 injection into mouse striatum produced significantly greater IgG extravasation than the isotype or the control injections. This study demonstrates that blockade of β1-integrin function changes interendothelial claudin-5 expression and increases microvessel permeability. Hence, endothelial cell–matrix interactions via β1-integrin directly affect interendothelial cell tight junction claudin-5 expression and brain microvascular permeability. PMID:21772312

  3. An αv-RGD Integrin Inhibitor Toolbox: Drug Discovery Insight, Challenges and Opportunities.

    PubMed

    Hatley, Richard J D; Macdonald, Simon J F; Slack, Robert J; Le, Joelle; Ludbrook, Steven B; Lukey, Pauline T

    2018-03-19

    There is a requirement for efficacious and safe medicines to treat diseases with high unmet need. The resurgence in αv-RGD integrin inhibitor drug discovery is poised to contribute to this requirement. However, drug discovery in the αv integrin space is notoriously difficult due to the receptors being structurally very similar as well as the polar zwitterionic nature of the pharmacophore. This Review aims to guide drug discovery research in this field through an αv inhibitor toolbox, consisting of small molecules and antibodies. Small-molecule αv tool compounds with extended profiles in αvβ1, 3, 5, 6 and 8 cell adhesion assays, with key physicochemical properties, have been collated to assist in the selection of the right tool for the right experiment. This should also facilitate an understanding of partial selectivity profiles of compounds generated in different assays across research institutions. Prospects for further αv integrin research and the critical importance of target validation are discussed, where increased knowledge of the selectivity for individual RGD αv integrins is key. Insights into the design of small-molecule RGD chemotypes for topical or oral administration are provided and clinical findings on advanced molecules are examined. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. The oligodendrocyte precursor mitogen PDGF stimulates proliferation by activation of αvβ3 integrins

    PubMed Central

    Baron, Wia; Shattil, Sanford J.; ffrench-Constant, Charles

    2002-01-01

    Central nervous system development requires precise and localized regulation of neural precursor behaviour. Here we show how the interaction between growth factor and integrin signalling pathways provides a mechanism for such precision in oligodendrocyte progenitor (OP) proliferation. While physiological concentrations of platelet-derived growth factor (PDGF) were not in themselves sufficient to promote OP proliferation, they did so on extracellular matrix (ECM) substrates that bind αvβ3 integrin. Upon PDGF-AA exposure and αvβ3 engagement, a physical co-association between both receptors was demonstrated, confirming the interaction between these signalling pathways. Furthermore, we found that PDGFαR stimulated a protein kinase C-dependent activation of integrin αvβ3, which in turn induced OP proliferation via a phosphatidylinositol 3-kinase-dependent signalling pathway. These studies establish a mechanism by which OP proliferation is dependent on the availability of both an ECM ligand and a mitogenic growth factor. Growth factor- mediated integrin activation is the critical integrative step in proliferation signalling, and ensures that the response of neural precursor cells to long-range cues can be regulated by their cellular neighbours, allowing precise control of cell behaviour during development. PMID:11953315

  5. Localized α4 Integrin Phosphorylation Directs Shear Stress-Induced Endothelial Cell Alignment

    PubMed Central

    Goldfinger, Lawrence E.; Tzima, Eleni; Stockton, Rebecca; Kiosses, William B.; Kinbara, Kayoko; Tkachenko, Eugene; Gutierrez, Edgar; Groisman, Alex; Nguyen, Phu; Chien, Shu; Ginsberg1, Mark H.

    2009-01-01

    Vascular endothelial cells respond to laminar shear stress by aligning in the direction of flow, a process which may contribute to athero-protection. Here we report that localized α4 integrin phosphorylation is a mechanism for establishing the directionality of shear stress-induced alignment in microvascular endothelial cells. Within 5 minutes of exposure to a physiological level of shear stress, endothelial α4 integrins became phosphorylated on Ser988. In wounded monolayers, phosphorylation was enhanced at the downstream edges of cells relative to the source of flow. The shear-induced α4 integrin phosphorylation was blocked by inhibitors of cAMP-dependent protein kinase A (PKA), an enzyme involved in the alignment of endothelial cells under prolonged shear. Moreover, shear-induced localized activation of the small GTPase Rac1, which specifies the directionality of endothelial alignment, was similarly blocked by PKA inhibitors. Furthermore, endothelial cells bearing a non-phosphorylatable α4(S988A) mutation failed to align in response to shear stress, thus establishing α4 as a relevant PKA substrate. We thereby show that shear-induced PKA-dependent α4 integrin phosphorylation at the downstream edge of endothelial cells promotes localized Rac1 activation, which in turn directs cytoskeletal alignment in response to shear stress. PMID:18583710

  6. The fibronectin synergy site re-enforces cell adhesion and mediates a crosstalk between integrin classes

    PubMed Central

    Benito-Jardón, Maria; Klapproth, Sarah; Gimeno-LLuch, Irene; Petzold, Tobias; Bharadwaj, Mitasha; Müller, Daniel J; Zuchtriegel, Gabriele; Reichel, Christoph A; Costell, Mercedes

    2017-01-01

    Fibronectin (FN), a major extracellular matrix component, enables integrin-mediated cell adhesion via binding of α5β1, αIIbβ3 and αv-class integrins to an RGD-motif. An additional linkage for α5 and αIIb is the synergy site located in close proximity to the RGD motif. We report that mice with a dysfunctional FN-synergy motif (Fn1syn/syn) suffer from surprisingly mild platelet adhesion and bleeding defects due to delayed thrombus formation after vessel injury. Additional loss of β3 integrins dramatically aggravates the bleedings and severely compromises smooth muscle cell coverage of the vasculature leading to embryonic lethality. Cell-based studies revealed that the synergy site is dispensable for the initial contact of α5β1 with the RGD, but essential to re-enforce the binding of α5β1/αIIbβ3 to FN. Our findings demonstrate a critical role for the FN synergy site when external forces exceed a certain threshold or when αvβ3 integrin levels decrease below a critical level. DOI: http://dx.doi.org/10.7554/eLife.22264.001 PMID:28092265

  7. The fibronectin synergy site re-enforces cell adhesion and mediates a crosstalk between integrin classes.

    PubMed

    Benito-Jardón, Maria; Klapproth, Sarah; Gimeno-LLuch, Irene; Petzold, Tobias; Bharadwaj, Mitasha; Müller, Daniel J; Zuchtriegel, Gabriele; Reichel, Christoph A; Costell, Mercedes

    2017-01-16

    Fibronectin (FN), a major extracellular matrix component, enables integrin-mediated cell adhesion via binding of α5β1, αIIbβ3 and αv-class integrins to an RGD-motif. An additional linkage for α5 and αIIb is the synergy site located in close proximity to the RGD motif. We report that mice with a dysfunctional FN-synergy motif ( Fn1 syn/syn ) suffer from surprisingly mild platelet adhesion and bleeding defects due to delayed thrombus formation after vessel injury. Additional loss of β3 integrins dramatically aggravates the bleedings and severely compromises smooth muscle cell coverage of the vasculature leading to embryonic lethality. Cell-based studies revealed that the synergy site is dispensable for the initial contact of α5β1 with the RGD, but essential to re-enforce the binding of α5β1/αIIbβ3 to FN. Our findings demonstrate a critical role for the FN synergy site when external forces exceed a certain threshold or when αvβ3 integrin levels decrease below a critical level.

  8. Convergence of eicosanoid and integrin biology: Role of Src in 12-LOX activation.

    PubMed

    Dilly, Ashok-Kumar; Tang, Keqin; Guo, Yande; Joshi, Sangeeta; Ekambaram, Prasanna; Maddipati, Krishna Rao; Cai, Yinlong; Tucker, Stephanie C; Honn, Kenneth V

    2017-02-01

    12-Lipoxygenase (12-LOX) metabolizes arachidonic acid to 12(S)-hydroxyeicosatetraenoic acid, or 12(S)-HETE, a proinflammatory bioactive lipid implicated in tumor angiogenesis, growth, and metastasis. The mechanisms underlying 12-LOX-mediated signaling in cancer progression are still ill-defined. In the present study we demonstrate that 12-LOX phosphorylation and subsequent enzymatic activity occurs after integrin β4 stimulation and Src kinase recruitment to the integrin subunit. Inhibition of Src activity by PP2 or Src dominant-negative mutants reduced 12-LOX tyrosine phosphorylation and 12(S)-HETE production in response to integrin β4 stimulation in A431 cells. The pertinent Src-targeted residues for 12-LOX activity were mapped to Y19 and Y614, where 12-LOX mutants Y19F and Y614F showed 70% less enzymatic activity. Furthermore, we have shown that the 12-LOX activity modulated by these residues impacts migration. To our knowledge, this is the first report that c-Src kinase activity is required for β4-integrin-mediated phosphorylation of 12-LOX. Published by Elsevier Inc.

  9. Neutrophil interactions with keratocytes during corneal epithelial wound healing: a role for CD18 integrins.

    USDA-ARS?s Scientific Manuscript database

    The purpose of this study was to determine the role of keratocytes and leukocyte beta(2) (CD18) integrins in neutrophil (PMN) migration through the corneal stroma after epithelial scrape injury. Using C57BL/6 wild-type and CD18(-/-) mice, corneas were excised at 6 hours (wild-type) or 24 hours (CD18...

  10. INTEGRIN-MEDIATED CELL ATTACHMENT SHOWS TIME-DEPENDENT UPREGULATION OF GAP JUNCTION COMMUNICATION.

    EPA Science Inventory


    Integrin-mediated Cell Attachment Shows Time-Dependent Upregulation of Gap Junction
    Communication

    Rachel Grindstaff and Carl Blackman, National Health & Environmental Effects Research
    Laboratory, Office of Research and Development, US EPA, Research Triang...

  11. Effective treatment of HER2-amplified breast cancer by targeting HER3 and β1 integrin.

    PubMed

    Campbell, Marcia R; Zhang, Hui; Ziaee, Shabnam; Ruiz-Saenz, Ana; Gulizia, Nathaniel; Oeffinger, Julie; Amin, Dhara N; Ahuja, Deepika; Moasser, Mark M; Park, Catherine C

    2016-02-01

    The central role of HER2 as the disease driver and HER3 as its essential partner has made them rational targets for the treatment of HER2-amplifed breast cancers, and there is considerable interest in developing highly effective treatment regimens for this disease that consist of targeted therapies alone. Much of these efforts are focused on dual targeting approaches, particularly dual targeting of the HER2-HER3 tumor driver complex itself, or vertical combinations that target downstream PI3K or Akt in addition to HER2. There is also potential in lateral combinations based on evidence implicating cross-talk with other membrane receptor systems, particularly integrins, and such lateral combinations can potentially involve either HER2 or HER3. We established a preclinical model of targeting HER3 using doxycycline-inducible shRNA and determined the efficacy of a β1 integrin inhibitor in combination with targeting HER3. We report that targeting HER3 and β1 integrin provides a particularly effective combination therapy approach for HER2-amplified cancers, surpassing the combination of HER2 and β1 integrin targeting, and evading some of the safety concerns associated with direct HER2-targeting. This further validates HER3 as a major hub mediating the tumorigenic functions of HER2 and identifies it as a high value target for lateral combination therapy strategies.

  12. Altered expression of epithelial integrins and extracellular matrix receptors in oral erythema multiforme.

    PubMed

    Mirowsk, G W; Lozada-Nur, F; Dekker, N P; MacPhail, L A; Regezi, J A

    1996-10-01

    Inflammation and ulceration at the epithelium-connective tissue interface, a characteristic of erythema multiforme (EM), may be associated with altered molecular attachment of basal keratinocytes. To determine the expression of basal keratinocyte-associated integrins and their basement membrane ligands in oral EM, specimens of clinically and microscopically confirmed EM (n = 12) and mucosal controls (n = 7) were stained immunohistochemically for the integrins alpha 3, beta 6, beta 1, and beta 4 and for extracellular matrix proteins laminin 1, laminin 5, collagen IV, and collagen VII using a standard avidin-biotin-peroxidase technique. In EM, results showed increased staining intensity for all integrins studied in basal and suprabasal keratinocytes. Basement membrane-associated staining of a6 and b4 was intense, but disrupted and fragmented. In EM, integrin staining was most marked at the summit of the connective tissue papillae. Laminin 5 staining was more intense than in controls, was frequently fragmented, and extended into the lamina propria. Laminin 1 staining was discontinuous and was frequently less intense than in controls. Collagen IV staining in EM was interrupted along the basement membrane. Collagen VII staining was fragmented but unchanged in intensity. These alterations in interface adhesion molecules suggest that hemidesmosome-associated molecules are important in the pathogenesis of EM. The staining intensities and patterns of expression of these adhesion molecules suggest that oral EM is initially focused in the connective tissue papillae.

  13. Osteoblast mineralization requires beta1 integrin/ICAP-1-dependent fibronectin deposition.

    PubMed

    Brunner, Molly; Millon-Frémillon, Angélique; Chevalier, Genevieve; Nakchbandi, Inaam A; Mosher, Deane; Block, Marc R; Albigès-Rizo, Corinne; Bouvard, Daniel

    2011-07-25

    The morphogenetic and differentiation events required for bone formation are orchestrated by diffusible and insoluble factors that are localized within the extracellular matrix. In mice, the deletion of ICAP-1, a modulator of β1 integrin activation, leads to severe defects in osteoblast proliferation, differentiation, and mineralization and to a delay in bone formation. Deposition of fibronectin and maturation of fibrillar adhesions, adhesive structures that accompany fibronectin deposition, are impaired upon ICAP-1 loss, as are type I collagen deposition and mineralization. Expression of β1 integrin with a mutated binding site for ICAP-1 recapitulates the ICAP-1-null phenotype. Follow-up experiments demonstrated that ICAP-1 negatively regulates kindlin-2 recruitment onto the β1 integrin cytoplasmic domain, whereas an excess of kindlin-2 binding has a deleterious effect on fibrillar adhesion formation. These results suggest that ICAP-1 works in concert with kindlin-2 to control the dynamics of β1 integrin-containing fibrillar adhesions and, thereby, regulates fibronectin deposition and osteoblast mineralization.

  14. Defining the Role of Integrin Alpha 11 in Wound Healing and Fibrosis

    DTIC Science & Technology

    2009-09-01

    Introduction Scleroderma is characterized by fibrosis, the replacement of healthy tissue with collagenous matrix. The collagen-binding integrins are...Itga11 and determine its role in the etiology of fibrosis and scleroderma . Body The heterozygous (Het) phenotype of one intact Itga11 allele and one

  15. Beta-1 integrin is important for the structural maintenance and homeostasis of differentiating fiber cells

    PubMed Central

    Scheiblin, David A.; Gao, Junyuan; Caplan, Jeffrey L.; Simirskii, Vladimir N.; Czymmek, Kirk J.; Mathias, Richard T.; Duncan, Melinda K.

    2014-01-01

    β1-integrin is a heterodimeric transmembrane protein that has roles in both cell-extracellular matrix and cell-cell interactions. Conditional deletion of β1-integrin from all lens cells during embryonic development results in profound lens defects, however, it is less clear whether this reflects functions in the lens epithelium alone or whether this protein plays a role in lens fibers. Thus, a conditional approach was used to delete β1-integrin solely from the lens fiber cells. This deletion resulted in two distinct phenotypes with some lenses exhibiting cataracts while others were clear, albeit with refractive defects. Analysis of “clear” conditional knockout lenses revealed that they had profound defects in fiber cell morphology associated with the loss of the F-actin network. Physiological measurements found that the lens fiber cells had a two-fold increase in gap junctional coupling, perhaps due to differential localization of connexins 46 and 50, as well as increased water permeability. This would presumably facilitate transport of ions and nutrients through the lens, and may partially explain how lenses with profound structural abnormalities can maintain transparency. In summary, β1-integrin plays a role in maintaining the cellular morphology and homeostasis of the lens fiber cells. PMID:24607497

  16. Integrin-α5 Coordinates Assembly of Posterior Cranial Placodes in Zebrafish and Enhances Fgf-Dependent Regulation of Otic/Epibranchial Cells

    PubMed Central

    Bhat, Neha; Riley, Bruce B.

    2011-01-01

    Vertebrate sensory organs develop in part from cranial placodes, a series of ectodermal thickenings that coalesce from a common domain of preplacodal ectoderm. Mechanisms coordinating morphogenesis and differentiation of discrete placodes are still poorly understood. We have investigated whether placodal assembly in zebrafish requires Integrin- α5 (itga5), an extracellular matrix receptor initially expressed throughout the preplacodal ectoderm. Morpholino knockdown of itga5 had no detectable effect on anterior placodes (pituitary, nasal and lens), but posterior placodes developed abnormally, resulting in disorganization of trigeminal and epibranchial ganglia and reduction of the otic vesicle. Cell motion analysis in GFP-transgenic embryos showed that cell migration in itga5 morphants was highly erratic and unfocused, impairing convergence and blocking successive recruitment of new cells into these placodes. Further studies revealed genetic interactions between itga5 and Fgf signaling. First, itga5 morphants showed changes in gene expression mimicking modest reduction in Fgf signaling. Second, itga5 morphants showed elevated apoptosis in the otic/epibranchial domain, which was rescued by misexpression of Fgf8. Third, knockdown of the Fgf effector erm had no effect by itself but strongly enhanced defects in itga5 morphants. Finally, proper regulation of itga5 requires dlx3b/4b and pax8, which are themselves regulated by Fgf. These findings support a model in which itga5 coordinates cell migration into posterior placodes and augments Fgf signaling required for patterning of these tissues and cell survival in otic/epibranchial placodes. PMID:22164214

  17. Regulation of integrin trafficking, cell adhesion and cell migration by WASH and the Arp2/3 Complex

    PubMed Central

    Duleh, Steve N.; Welch, Matthew D.

    2013-01-01

    WASH is a nucleation-promoting factor for the Arp2/3 complex that is implicated in multiple endocytic trafficking pathways including receptor recycling, cargo degradation, and retromer-mediated receptor retrieval. We sought to examine whether WASH plays an important role in trafficking of specialized cargo molecules such as integrins, for which trafficking is highly regulated during cell migration. We observed that subdomains of early/sorting endosomes associated with dynamic WASH and filamentous actin, and α5-integrins trafficked through this population of endosomes. Depletion of WASH caused accumulation of α5-integrins in intracellular compartments, reduction of α5-integrin localization at focal adhesions, and reduction in focal adhesion number. Transport of α5-integrins from internal endocytic structures to focal adhesions was disrupted upon WASH depletion or Arp2/3 complex inhibition. Furthermore, WASH-depleted cells displayed greatly reduced affinity for specific ECM proteins including fibronectin, and impaired cell spreading ability. Interestingly, the reduced adhesion capacity of WASH-depleted cells resulted in their migrating more rapidly than control cells in wound healing assays. Our results define a requirement for WASH, Arp2/3 complex, and actin in specialized trafficking of integrins. These findings highlight a role for actin dynamics in influencing cell adhesion and migration via endocytic trafficking of integrins, in addition to the well-established role of actin in plasma membrane dynamics and contractility. PMID:23012235

  18. p21-activated Kinase 4 Phosphorylation of Integrin β5 Ser-759 and Ser-762 Regulates Cell Migration*

    PubMed Central

    Li, Zhilun; Zhang, Hongquan; Lundin, Lars; Thullberg, Minna; Liu, Yajuan; Wang, Yunling; Claesson-Welsh, Lena; Strömblad, Staffan

    2010-01-01

    Modulation of integrin αvβ5 regulates vascular permeability, angiogenesis, and tumor dissemination. In addition, we previously found a role for p21-activated kinase 4 (PAK4) in selective regulation of integrin αvβ5-mediated cell motility (Zhang, H., Li, Z., Viklund, E. K., and Strömblad, S. (2002) J. Cell Biol. 158, 1287–1297). This report focuses on the molecular mechanisms of this regulation. We here identified a unique PAK4-binding membrane-proximal integrin β5-SERS-motif involved in controlling cell attachment and migration. We also mapped the integrin β5-binding site within PAK4. We found that PAK4 binding to integrin β5 was not sufficient to promote cell migration, but that PAK4 kinase activity was required for PAK4 promotion of cell motility. Importantly, PAK4 specifically phosphorylated the integrin β5 subunit at Ser-759 and Ser-762 within the β5-SERS-motif. Point mutation of these two serine residues abolished the PAK4-induced cell migration, indicating a functional role for these phosphorylations in migration. Our results may give important leads to the functional regulation of integrin αvβ5, with implications for vascular permeability, angiogenesis, and cancer dissemination. PMID:20507994

  19. p21-activated kinase 4 phosphorylation of integrin beta5 Ser-759 and Ser-762 regulates cell migration.

    PubMed

    Li, Zhilun; Zhang, Hongquan; Lundin, Lars; Thullberg, Minna; Liu, Yajuan; Wang, Yunling; Claesson-Welsh, Lena; Strömblad, Staffan

    2010-07-30

    Modulation of integrin alphavbeta5 regulates vascular permeability, angiogenesis, and tumor dissemination. In addition, we previously found a role for p21-activated kinase 4 (PAK4) in selective regulation of integrin alphavbeta5-mediated cell motility (Zhang, H., Li, Z., Viklund, E. K., and Strömblad, S. (2002) J. Cell Biol. 158, 1287-1297). This report focuses on the molecular mechanisms of this regulation. We here identified a unique PAK4-binding membrane-proximal integrin beta5-SERS-motif involved in controlling cell attachment and migration. We also mapped the integrin beta5-binding site within PAK4. We found that PAK4 binding to integrin beta5 was not sufficient to promote cell migration, but that PAK4 kinase activity was required for PAK4 promotion of cell motility. Importantly, PAK4 specifically phosphorylated the integrin beta5 subunit at Ser-759 and Ser-762 within the beta5-SERS-motif. Point mutation of these two serine residues abolished the PAK4-induced cell migration, indicating a functional role for these phosphorylations in migration. Our results may give important leads to the functional regulation of integrin alphavbeta5, with implications for vascular permeability, angiogenesis, and cancer dissemination.

  20. Adhesive F-actin Waves: A Novel Integrin-Mediated Adhesion Complex Coupled to Ventral Actin Polymerization

    PubMed Central

    Case, Lindsay B.; Waterman, Clare M.

    2011-01-01

    At the leading lamellipodium of migrating cells, protrusion of an Arp2/3-nucleated actin network is coupled to formation of integrin-based adhesions, suggesting that Arp2/3-mediated actin polymerization and integrin-dependent adhesion may be mechanistically linked. Arp2/3 also mediates actin polymerization in structures distinct from the lamellipodium, in “ventral F-actin waves” that propagate as spots and wavefronts along the ventral plasma membrane. Here we show that integrins engage the extracellular matrix downstream of ventral F-actin waves in several mammalian cell lines as well as in primary mouse embryonic fibroblasts. These “adhesive F-actin waves” require a cycle of integrin engagement and disengagement to the extracellular matrix for their formation and propagation, and exhibit morphometry and a hierarchical assembly and disassembly mechanism distinct from other integrin-containing structures. After Arp2/3-mediated actin polymerization, zyxin and VASP are co-recruited to adhesive F-actin waves, followed by paxillin and vinculin, and finally talin and integrin. Adhesive F-actin waves thus represent a previously uncharacterized integrin-based adhesion complex associated with Arp2/3-mediated actin polymerization. PMID:22069459

  1. Increased circulating anti-α6-integrin autoantibodies in psoriasis and psoriatic arthritis but not in rheumatoid arthritis.

    PubMed

    Gál, Brigitta; Dulic, Sonja; Kiss, Mária; Groma, Gergely; Kovács, László; Kemény, Lajos; Bata-Csörgő, Zsuzsanna

    2017-04-01

    In psoriatic skin, laminin integrity is altered, which could lead to insufficient laminin integrin interactions, leaving the α6-integrin exposed and possibly accessible for autoantibody production. Therefore we investigated the presence of anti-α6-integrin autoantibodies in the serum of patients with psoriasis vulgaris (Ps), psoriatic arthritis (PsA) and rheumatoid arthritis (RA) in comparison with healthy donors. The level of circulating anti-α6-integrin antibodies was determined by enzyme-linked immunoassay using α6-integrin fragments. Antibodies against at least one recombinant fragment were found in approximately 30% of Ps and PsA patients. In contrast, in RA patients, the frequency of antibodies was similar to healthy controls. Our study shows the presence of anti-α6-integrin antibodies in Ps and PsA but not in RA, which could indicate ongoing abnormal processes in the skin. Anti-α6-integrin autoantibodies may contribute to the formation of micro-wounds in the skin and to the characteristic wound-healing phenotype in psoriasis. © 2016 Japanese Dermatological Association.

  2. Integrin activation modulates NMDA and AMPA receptor function of CA1 cells in a dose-related fashion in vivo.

    PubMed

    Juhász, Gábor; Vass, Gabriella; Bozsó, Zsolt; Budai, Dénes; Penke, Botond; Szegedi, Viktor

    2008-10-03

    The large family of heterodimeric, transmembrane cell adhesion receptors, integrins mediate numerous functions in the immature and adult CNS. Integrins are described to modulate basic synaptic function and plasticity, and to modulate the activity of the two major excitatory ionotrophic receptor subclass, NMDA and AMPA receptors. We further addressed the role of integrin activation in the normal excitatory synaptic function by utilizing in vivo single-unit recordings combined with microiontophoretic drug application in the CA1 region of the rat. Cells were excited by alternating NMDA and AMPA ejection, while integrin activation was achieved by the ejection of an RGD sequence containing pentapeptide in low and high concentration. Low integrin activation resulted in increased NMDA and decreased AMPA induced firing rate, while high RGD concentration enhanced both types of elicited responses. The control pentapeptide, pentaglycine had no effect on NMDA or AMPA evoked firing rate in either low or high concentration. These results suggest a bidirectional, dose dependent action of integrin activation on basic synaptic transmission, which may underlie the long term synaptic plasticity changes modulated by integrins.

  3. Overexpression of β1 integrin contributes to polarity reversal and a poor prognosis of breast invasive micropapillary carcinoma.

    PubMed

    Liu, Bingbing; Zheng, Xia; Meng, Fanfan; Han, Yunwei; Song, Yawen; Liu, Fangfang; Li, Shuai; Zhang, Lanjing; Gu, Feng; Zhang, Xinmin; Fu, Li

    2018-01-12

    Invasive micropapillary carcinoma (IMPC) of the breast is a highly aggressive breast cancer. Polarity reversal exemplified by cluster growth is hypothesized to contribute to the invasiveness and metastasis of IMPC. In this study, we demonstrate that levels of β1 integrin and Rac1 expression were greater in breast IMPC than in invasive breast carcinoma of no specific type and paraneoplastic benign breast tissue. We show that silencing β1 integrin expression using the β1 integrin inhibitor AIIB2 partially restored polarity in IMPC primary cell clusters and downregulated Rac1. Thus, overexpression of β1 integrin upregulates Rac1. Univariate analysis showed that overexpression of β1 integrin and Rac1 was associated with breast cancer cell polarity reversal, lymph node metastasis, and poor disease-free survival in IMPC patients. Multivariate analysis revealed that polarity reversal was an independent predictor of poor disease-free survival. These findings indicate that overexpression of β1 integrin and the resultant upregulation of Rac1 contribute to polarity reversal and metastasis of breast IMPC, and that β1 integrin and Rac1 could be potential prognostic biomarkers and targets for treatment of breast IMPC.

  4. A ligand-specific blockade of the integrin Mac-1 selectively targets pathologic inflammation while maintaining protective host-defense.

    PubMed

    Wolf, Dennis; Anto-Michel, Nathaly; Blankenbach, Hermann; Wiedemann, Ansgar; Buscher, Konrad; Hohmann, Jan David; Lim, Bock; Bäuml, Marina; Marki, Alex; Mauler, Maximilian; Duerschmied, Daniel; Fan, Zhichao; Winkels, Holger; Sidler, Daniel; Diehl, Philipp; Zajonc, Dirk M; Hilgendorf, Ingo; Stachon, Peter; Marchini, Timoteo; Willecke, Florian; Schell, Maximilian; Sommer, Björn; von Zur Muhlen, Constantin; Reinöhl, Jochen; Gerhardt, Teresa; Plow, Edward F; Yakubenko, Valentin; Libby, Peter; Bode, Christoph; Ley, Klaus; Peter, Karlheinz; Zirlik, Andreas

    2018-02-06

    Integrin-based therapeutics have garnered considerable interest in the medical treatment of inflammation. Integrins mediate the fast recruitment of monocytes and neutrophils to the site of inflammation, but are also required for host defense, limiting their therapeutic use. Here, we report a novel monoclonal antibody, anti-M7, that specifically blocks the interaction of the integrin Mac-1 with its pro-inflammatory ligand CD40L, while not interfering with alternative ligands. Anti-M7 selectively reduces leukocyte recruitment in vitro and in vivo. In contrast, conventional anti-Mac-1 therapy is not specific and blocks a broad repertoire of integrin functionality, inhibits phagocytosis, promotes apoptosis, and fuels a cytokine storm in vivo. Whereas conventional anti-integrin therapy potentiates bacterial sepsis, bacteremia, and mortality, a ligand-specific intervention with anti-M7 is protective. These findings deepen our understanding of ligand-specific integrin functions and open a path for a new field of ligand-targeted anti-integrin therapy to prevent inflammatory conditions.

  5. [Detection of transgenic crop with gene chip].

    PubMed

    Huang, Ying-Chun; Sun, Chun-Yun; Feng, Hong; Hu, Xiao-Dong; Yin, Hai-Bin

    2003-05-01

    Some selected available sequences of reporter genes,resistant genes, promoters and terminators are amplified by PCR for the probes of transgenic crop detection gene chip. These probes are arrayed at definite density and printed on the surface of amino-slides by bioRobot MicroGrid II. Results showed that gene chip worked quickly and correctly, when transgenic rice, pawpaw,maize and soybean were applied.

  6. Convergent signalling in the action of integrins, neuropeptides, growth factors and oncogenes.

    PubMed

    Rozengurt, E

    1995-01-01

    These findings have important implications for signal transduction and cell regulation. Most obviously, they suggest that tyrosine phosphorylation of a novel type of tyrosine kinase p125FAK is a point of convergence in the action of integrins, oncogenic forms of pp60src, mitogenic neuropeptides and growth factors (Fig. 3). One inference is that the signal transduction pathways initiated by these diverse groups of molecules have, at least in part, similar consequences for cellular function. The notion of convergence is reinforced by the striking similarity in the overall pattern of tyrosine phosphorylation produced through these different pathways. It is tempting to speculate that p125FAK, paxillin and p130 are components in a common programme of phosphorylation events stimulated by integrins, mitogenic neuropeptides and growth factors. The localization of p125FAK to focal adhesions is clearly consistent with a role for this protein as a junction point in the transduction of signals that regulate cell substrate adhesion and ultimately cell motility and cell shape, as suggested in Fig. 3. The existence of distinct pathways leading to p125FAK phosphorylation raises the possibility of synergistic interactions between integrins and G protein coupled receptors. In fact, integrin mediated p125FAK tyrosine phosphorylation appears to be mediated by a PKC dependent pathway (Vuori and Ruoslathi, 1993). By contrast, bombesin and LPA induce tyrosine phosphorylation of p125FAK and paxillin through a PKC independent pathway (Sinnett-Smith et al, 1993; Zachary et al, 1993; Seufferlein and Rozengurt, 1994). It is possible that tyrosine phosphorylation of p125FAK by bombesin, LPA and pp60v-src bypasses and perhaps mimics the phosphorylation caused by integrin activation. Further experimental work will be required to elucidate whether integrins and neuropeptides increase the autophosphorylation of Tyr-397 in p125FAK, as has been recently demonstrated in src-transformed cells

  7. Interactions between integrin receptors and fibronectin are required for calvarial osteoblast differentiation in vitro

    NASA Technical Reports Server (NTRS)

    Moursi, A. M.; Globus, R. K.; Damsky, C. H.

    1997-01-01

    We previously showed that anti-fibronectin antibodies or soluble fibronectin fragments containing the central cell-binding domain inhibit formation of mineralized nodules by fetal calvarial osteoblasts in vitro. These findings suggest a critical role for fibronectin in osteoblast differentiation and morphogenesis. In this study we tested the hypothesis that fibronectin's effects on osteogenesis are mediated via direct interactions with integrin receptors for fibronectin on osteoblasts. Immunocytochemical analysis identified the integrin fibronectin receptor alpha5ss1 in fetal rat calvarial tissue and in cultured osteoblasts at all stages of differentiation. Three other integrins, alpha3ss1, alpha8ss1 and alphavss3, which can bind fibronectin, as well as other matrix components, were also identified in tissue and at all stages of cell culture. Immunoprecipitation data showed that alpha5ss1 levels are constant throughout osteoblast differentiation whereas levels of alpha3ss1 and alpha8ss1 decline in mature mineralized cultures. To determine whether integrin fibronectin receptors are required for osteoblast formation of mineralized nodules, we examined the extent of nodule formation in the presence and absence of function-perturbing anti-integrin antibodies. The antibodies were present continuously in cultures beginning at confluence (day 3), and nodule formation was measured at days 10 and 20. An anti-alpha5 integrin subunit antibody reduced nodule formation to less than 5% of control values at both time points. Inhibition of nodule formation was reversible and did not affect cell attachment and viability. Function-perturbing antibodies against alpha3ss1 and alpha8ss1 also reduced nodule formation, to less than 20% of control values. In contrast, function-perturbing antibodies to alphavss3 and alphavss5 did not affect nodule formation, indicating that the inhibitions noted were indeed specific. To determine the effect of antibody treatment on gene expression, steady

  8. The synthesis and biological evaluation of integrin receptor targeting molecules as potential radiopharmaceuticals

    NASA Astrophysics Data System (ADS)

    Pellegrini, Paul

    This thesis reports on the synthesis, characterisation and biological evaluation of a number of metal complexes designed to interact with the alphavbeta3 integrin receptor, an important biological target that is heavily involved in angiogenesis, and thus cancer related processes. Two approaches were used to synthesise the integrin-avid targets. The first was to attach a variety of bifunctional chelators (BFC's) for the incorporation of different metal centres to a known integrin antagonist, L-748,415, developed by Merck. The BFC's used were the hydrazinonicotinamide (HYNIC) and monoamine monoamide dithiol (MAMA) systems for coordination to Tc-99m and rhenium of which was used as a characterization surrogate for the unstable Tc core. The 1,4,7,10-tetraazacyclotridecanetetraacetic acid (TRITA) BFC was attached for the inclusion of copper and lutetium. This 'conjugate' approach was designed to yield information on how the BFC and the linker length would affect the affinity for the integrin receptor. The second approach was an 'integrated' method where the chelation moiety was integral to the biologically relevant part of the molecule, which in the case of the alphavbeta3 integrin receptor, is the arginine-glycine-aspartic acid (RGD) mimicking sequence. Two complexes were created with a modified MAMA derivative placed between a benzimidazole moiety (arginine mimick) and the aspartic acid mimicking terminal carboxylic acid to see how it would affect binding while keeping the molecular weight relatively low. The molecules were tested in vitro against purified human alphavbeta3 integrin receptor protein in a solid phase receptor binding assay to evaluate their inhibition constants against a molecule of known high affinity and selectivity in [I125]L-775,219, the I125 labelled alphavbeta3 integrin antagonist. The radiolabelled analogues were also tested in vivo against the A375 human melanoma cell line transplanted into balb/c nude mice as well as Fischer rats implanted

  9. Absence of Preference for Social Novelty and Increased Grooming in Integrin β3 Knockout Mice: Initial Studies and Future Directions

    PubMed Central

    Carter, Michelle D.; Shah, Charisma R.; Muller, Christopher L.; Crawley, Jacqueline N.; Carneiro, Ana M. D.; Veenstra-VanderWeele, Jeremy

    2010-01-01

    Scientific Abstract Elevated whole blood serotonin (5HT), or hyperserotonemia, is a common biomarker in autism spectrum disorder (ASD). The integrin β3 receptor subunit gene (ITGB3) is a quantitative trait locus for whole blood 5-HT levels. Recent work shows that integrin β3 interacts with the serotonin transporter (SERT) in both platelets and in the midbrain. Furthermore, multiple studies have now reported gene-gene interaction between the integrin β3 and SERT genes in association with ASD. Given the lack of previous data on the impact of integrin β3 on brain or behavioral phenotypes, we sought to compare mice with decreased or absent expression of the integrin β3 receptor subunit (Itgb3 +/- and -/-) with wildtype littermate controls in behavioral tasks relevant to ASD. These mice did not show deficits in activity level in the Open Field or anxiety-like behavior on the Elevated Plus Maze, two potential confounds in the evaluation of mouse social behavior. In the three-chamber social test, mice lacking integrin β3 were shown to have normal sociability but did not show a preference for social novelty. Importantly, the absence of integrin β3 did not impair olfaction or the ability to recall familiar social odors. Additionally, mice lacking integrin β3 showed increased grooming behavior in novel environments. These preliminary studies reveal altered social and repetitive behavior in these mice, which suggests that the integrin β3 subunit may be involved in brain systems relevant to ASD. Further work is needed to fully characterize these behavioral changes and the underlying brain mechanisms. PMID:21254450

  10. Absence of preference for social novelty and increased grooming in integrin β3 knockout mice: initial studies and future directions.

    PubMed

    Carter, Michelle D; Shah, Charisma R; Muller, Christopher L; Crawley, Jacqueline N; Carneiro, Ana M D; Veenstra-VanderWeele, Jeremy

    2011-02-01

    Elevated whole blood serotonin 5-HT, or hyperserotonemia, is a common biomarker in autism spectrum disorder (ASD). The integrin β3 receptor subunit gene (ITGB3) is a quantitative trait locus for whole blood 5-HT levels. Recent work shows that integrin β3 interacts with the serotonin transporter (SERT) in both platelets and in the midbrain. Furthermore, multiple studies have now reported gene-gene interaction between the integrin β3 and SERT genes in association with ASD. Given the lack of previous data on the impact of integrin β3 on brain or behavioral phenotypes, we sought to compare mice with decreased or absent expression of the integrin β3 receptor subunit (Itgb3 +/- and -/-) with wildtype littermate controls in behavioral tasks relevant to ASD. These mice did not show deficits in activity level in the open field or anxiety-like behavior on the elevated plus maze, two potential confounds in the evaluation of mouse social behavior. In the three-chamber social test, mice lacking integrin β3 were shown to have normal sociability but did not show a preference for social novelty. Importantly, the absence of integrin β3 did not impair olfaction or the ability to recall familiar social odors. Additionally, mice lacking integrin β3 showed increased grooming behavior in novel environments. These preliminary studies reveal altered social and repetitive behavior in these mice, which suggests that the integrin β3 subunit may be involved in brain systems relevant to ASD. Further work is needed to fully characterize these behavioral changes and the underlying brain mechanisms. Copyright © 2011, International Society for Autism Research, Wiley Periodicals, Inc.

  11. α6β1 and α7β1 integrins are required in Schwann cells to sort axons.

    PubMed

    Pellegatta, Marta; De Arcangelis, Adèle; D'Urso, Alessandra; Nodari, Alessandro; Zambroni, Desirée; Ghidinelli, Monica; Matafora, Vittoria; Williamson, Courtney; Georges-Labouesse, Elisabeth; Kreidberg, Jordan; Mayer, Ulrike; McKee, Karen K; Yurchenco, Peter D; Quattrini, Angelo; Wrabetz, Lawrence; Feltri, Maria Laura

    2013-11-13

    During development, Schwann cells extend lamellipodia-like processes to segregate large- and small-caliber axons during the process of radial sorting. Radial sorting is a prerequisite for myelination and is arrested in human neuropathies because of laminin deficiency. Experiments in mice using targeted mutagenesis have confirmed that laminins 211, 411, and receptors containing the β1 integrin subunit are required for radial sorting; however, which of the 11 α integrins that can pair with β1 forms the functional receptor is unknown. Here we conditionally deleted all the α subunits that form predominant laminin-binding β1 integrins in Schwann cells and show that only α6β1 and α7β1 integrins are required and that α7β1 compensates for the absence of α6β1 during development. The absence of either α7β1 or α6β1 integrin impairs the ability of Schwann cells to spread and to bind laminin 211 or 411, potentially explaining the failure to extend cytoplasmic processes around axons to sort them. However, double α6/α7 integrin mutants show only a subset of the abnormalities found in mutants lacking all β1 integrins, and a milder phenotype. Double-mutant Schwann cells can properly activate all the major signaling pathways associated with radial sorting and show normal Schwann cell proliferation and survival. Thus, α6β1 and α7β1 are the laminin-binding integrins required for axonal sorting, but other Schwann cell β1 integrins, possibly those that do not bind laminins, may also contribute to radial sorting during peripheral nerve development.

  12. Filamin A Is Required for Optimal T Cell Integrin-Mediated Force Transmission, Flow Adhesion, and T Cell Trafficking.

    PubMed

    Savinko, Terhi; Guenther, Carla; Uotila, Liisa M; Llort Asens, Marc; Yao, Sean; Tojkander, Sari; Fagerholm, Susanna C

    2018-05-01

    T cells traffic from the bloodstream into tissues to perform their functions in the immune system and are therefore subjected to a range of different mechanical forces. Integrins are essential for T cell trafficking into the tissues, as they mediate firm adhesion between the T cell and the endothelium under shear flow conditions. In addition, integrins are important for the formation of the contact between the T cell and the APC required for T cell activation. The actin-binding protein filamin A (FlnA) provides an important link between the integrin and the actin cytoskeleton. FlnA has been reported to function as an integrin inhibitor by competing with talin. However, its role in regulating integrin-dependent immune functions in vivo is currently poorly understood. In this study, we have investigated the role of FlnA in T cells, using T cell-specific FlnA knockout mice. We report that FlnA is required for the formation of strong integrin-ligand bonds under shear flow and for the generation of integrin-mediated T cell traction forces on ligand-coated hydrogels. Consequently, absence of FlnA leads to a reduction in T cell adhesion to integrin ligands under conditions of shear flow, as well as reduced T cell trafficking into lymph nodes and sites of skin inflammation. In addition, FlnA is not needed for T cell activation in vivo, which occurs in shear-free conditions in lymphoid organs. Our results therefore reveal a role of FlnA in integrin force transmission and T cell trafficking in vivo. Copyright © 2018 by The American Association of Immunologists, Inc.

  13. Transgene flow: Facts, speculations and possible countermeasures

    PubMed Central

    Ryffel, Gerhart U

    2014-01-01

    Convincing evidence has accumulated that unintended transgene escape occurs in oilseed rape, maize, cotton and creeping bentgrass. The escaped transgenes are found in variant cultivars, in wild type plants as well as in hybrids of sexually compatible species. The fact that in some cases stacked events are present that have not been planted commercially, implies unintended recombination of transgenic traits. As the consequences of this continuous transgene escape for the ecosystem cannot be reliably predicted, I propose to use more sophisticated approaches of gene technology in future. If possible GM plants should be constructed using either site-directed mutagenesis or cisgenic strategies to avoid the problem of transgene escape. In cases where a transgenic trait is needed, efficient containment should be the standard approach. Various strategies available or in development are discussed. Such a cautious approach in developing novel types of GM crops will enhance the sustainable potential of GM crops and thus increase the public trust in green gene technology. PMID:25523171

  14. Transgenic Expression of Dspp Partially Rescued the Long Bone Defects of Dmp1-null Mice

    PubMed Central

    Jani, Priyam H.; Gibson, Monica P.; Liu, Chao; Zhang, Hua; Wang, Xiaofang; Lu, Yongbo; Qin, Chunlin

    2016-01-01

    Dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP) belong to the Small Integrin-Binding Ligand N-linked Glycoprotein (SIBLING) family. In addition to the features common to all SIBLING members, DMP1 and DSPP share several unique similarities in chemical structure, proteolytic activation and tissue localization. Mutations in, or deletion of DMP1, cause autosomal recessive hypophosphatemic rickets along with dental defects; DSPP mutations or its ablation are associated with dentinogenesis imperfecta. While the roles and functional mechanisms of DMP1 in osteogenesis have been extensively studied, those of DSPP in long bones have been studied only to a limited extent. Previous studies by our group revealed that transgenic expression of Dspp completely rescued the dentin defects of Dmp1-null (Dmp1−/−) mice. In this investigation, we assessed the effects of transgenic Dspp on osteogenesis by analyzing the formation and mineralization of the long bones in Dmp1−/− mice that expresses a transgene encoding full-length DSPP driven by a 3.6-kb rat Col1a1 promoter (referred as “Dmp1−/−;Dspp-Tg mice”). We characterized the long bones of the Dmp1−/−;Dspp-Tg mice at different ages and compared them with those from Dmp1−/− and Dmp1+/− (normal control) mice. Our analyses showed that the long bones of Dmp1−/−;Dspp-Tg mice had a significant increase in cortical bone thickness, bone volume and mineral density along with a remarkable restoration of trabecular thickness compared to those of the Dmp1−/− mice. The long bones of Dmp1−/−;Dspp-Tg mice underwent a dramatic reduction in the amount of osteoid, significant improvement of the collagen fibrillar network, and better organization of the lacunocanalicular system, compared to the Dmp1−/− mice. The elevated levels of biglycan, bone sialoprotein and osteopontin in Dmp1−/− mice were also noticeably corrected by the transgenic expression of Dspp. These findings suggest that

  15. Transgenic expression of Dspp partially rescued the long bone defects of Dmp1-null mice.

    PubMed

    Jani, Priyam H; Gibson, Monica P; Liu, Chao; Zhang, Hua; Wang, Xiaofang; Lu, Yongbo; Qin, Chunlin

    2016-01-01

    Dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP) belong to the Small Integrin-Binding Ligand N-linked Glycoprotein (SIBLING) family. In addition to the features common to all SIBLING members, DMP1 and DSPP share several unique similarities in chemical structure, proteolytic activation and tissue localization. Mutations in, or deletion of DMP1, cause autosomal recessive hypophosphatemic rickets along with dental defects; DSPP mutations or its ablation are associated with dentinogenesis imperfecta. While the roles and functional mechanisms of DMP1 in osteogenesis have been extensively studied, those of DSPP in long bones have been studied only to a limited extent. Previous studies by our group revealed that transgenic expression of Dspp completely rescued the dentin defects of Dmp1-null (Dmp1(-/-)) mice. In this investigation, we assessed the effects of transgenic Dspp on osteogenesis by analyzing the formation and mineralization of the long bones in Dmp1(-/-) mice that expresses a transgene encoding full-length DSPP driven by a 3.6-kb rat Col1a1 promoter (referred as "Dmp1(-/-);Dspp-Tg mice"). We characterized the long bones of the Dmp1(-/-);Dspp-Tg mice at different ages and compared them with those from Dmp1(-/-) and Dmp1(+/-) (normal control) mice. Our analyses showed that the long bones of Dmp1(-/-);Dspp-Tg mice had a significant increase in cortical bone thickness, bone volume and mineral density along with a remarkable restoration of trabecular thickness compared to those of the Dmp1(-/-) mice. The long bones of Dmp1(-/-);Dspp-Tg mice underwent a dramatic reduction in the amount of osteoid, significant improvement of the collagen fibrillar network, and better organization of the lacunocanalicular system, compared to the Dmp1(-/-) mice. The elevated levels of biglycan, bone sialoprotein and osteopontin in Dmp1(-/-) mice were also noticeably corrected by the transgenic expression of Dspp. These findings suggest that DSPP and DMP1 may function

  16. Interaction between Fibronectin and β1 Integrin Is Essential for Tooth Development

    PubMed Central

    Yamada, Aya; Yuasa, Kenji; Yoshizaki, Keigo; Iwamoto, Tsutomu; Saito, Masahiro; Nakamura, Takashi; Fukumoto, Satoshi

    2015-01-01

    The dental epithelium and extracellular matrix interact to ensure that cell growth and differentiation lead to the formation of teeth of appropriate size and quality. To determine the role of fibronectin in differentiation of the dental epithelium and tooth formation, we analyzed its expression in developing incisors. Fibronectin mRNA was expressed during the presecretory stage in developing dental epithelium, decreased in the secretory and early maturation stages, and then reappeared during the late maturation stage. The binding of dental epithelial cells derived from postnatal day-1 molars to a fibronectin-coated dish was inhibited by the RGD but not RAD peptide, and by a β1 integrin-neutralizing antibody, suggesting that fibronectin-β1 integrin interactions contribute to dental epithelial-cell binding. Because fibronectin and β1 integrin are highly expressed in the dental mesenchyme, it is difficult to determine precisely how their interactions influence dental epithelial differentiation in vivo. Therefore, we analyzed β1 integrin conditional knockout mice (Intβ1lox-/lox-/K14-Cre) and found that they exhibited partial enamel hypoplasia, and delayed eruption of molars and differentiation of ameloblasts, but not of odontoblasts. Furthermore, a cyst-like structure was observed during late ameloblast maturation. Dental epithelial cells from knockout mice did not bind to fibronectin, and induction of ameloblastin expression in these cells by neurotrophic factor-4 was inhibited by treatment with RGD peptide or a fibronectin siRNA, suggesting that the epithelial interaction between fibronectin and β1 integrin is important for ameloblast differentiation and enamel formation. PMID:25830530

  17. Alpha-v–containing integrins are host receptors for the Plasmodium falciparum sporozoite surface protein, TRAP

    PubMed Central

    Dundas, Kirsten; Shears, Melanie J.; Sun, Yi; Hopp, Christine S.; Crosnier, Cecile; Metcalf, Tom; Girling, Gareth; Sinnis, Photini; Billker, Oliver; Wright, Gavin J.

    2018-01-01

    Malaria-causing Plasmodium sporozoites are deposited in the dermis by the bite of an infected mosquito and move by gliding motility to the liver where they invade and develop within host hepatocytes. Although extracellular interactions between Plasmodium sporozoite ligands and host receptors provide important guidance cues for productive infection and are good vaccine targets, these interactions remain largely uncharacterized. Thrombospondin-related anonymous protein (TRAP) is a parasite cell surface ligand that is essential for both gliding motility and invasion because it couples the extracellular binding of host receptors to the parasite cytoplasmic actinomyosin motor; however, the molecular nature of the host TRAP receptors is poorly defined. Here, we use a systematic extracellular protein interaction screening approach to identify the integrin αvβ3 as a directly interacting host receptor for Plasmodium falciparum TRAP. Biochemical characterization of the interaction suggests a two-site binding model, requiring contributions from both the von Willebrand factor A domain and the RGD motif of TRAP for integrin binding. We show that TRAP binding to cells is promoted in the presence of integrin-activating proadhesive Mn2+ ions, and that cells genetically targeted so that they lack cell surface expression of the integrin αv-subunit are no longer able to bind TRAP. P. falciparum sporozoites moved with greater speed in the dermis of Itgb3-deficient mice, suggesting that the interaction has a role in sporozoite migration. The identification of the integrin αvβ3 as the host receptor for TRAP provides an important demonstration of a sporozoite surface ligand that directly interacts with host receptors. PMID:29632205

  18. Inhibition of osteoporosis by the αvβ3 integrin antagonist of rhodostomin variants.

    PubMed

    Lin, Tzu-Hung; Yang, Rong-Sen; Tu, Huang-Ju; Liou, Houng-Chi; Lin, Yen-Ming; Chuang, Woie-Jer; Fu, Wen-Mei

    2017-06-05

    Integrins are heterodimeric cell surface receptors that mediate cell-cell and cell-matrix interaction. The vitronectin and osteopontin receptor αvβ3 integrin has increased expression levels and is implicated in the adhesion, activation, and migration of osteoclasts on the bone surface as well as osteoclast polarization. αvβ3 integrin plays an important role in osteoclast differentiation and resorption. In addition, Arg-Gly-Asp (RGD)-containing peptides, small molecular inhibitors, and antibodies to αvβ3 integrin have been shown to inhibit bone resorption in vitro and in vivo. Here we examined the effects of a disintegrin HSA-ARLDDL a genetically modified mutant of rhodostomin conjugated with human serum albumin, which is highly selective of αvβ3, on RANKL-induced osteoclastogenesis and ovariectomy (OVX)-induced osteoporosis. In RANKL-induced osteoclastogenesis, HSA-ARLDDL significantly inhibited osteoclast formation, and IC 50 was at nM range. Post-treatment HSA-ARLDDL also inhibits osteoclast formation. Furthermore, weekly administration of HSA-ARLDDL significantly inhibits the increase in serum bone resorption marker levels and decrease in cancellous bone loss in tibia and femur induced by OVX. On the other hand, HSA-ARLDDL did not affect the differentiation and calcium deposition of osteoblasts. These results indicate that the highly selective and long-acting αvβ3 integrin antagonists could be developed as effective drugs for postmenopausal osteoporosis. Copyright © 2017. Published by Elsevier B.V.

  19. Human integrin α(3)β(1) regulates TLR2 recognition of lipopeptides from endosomal compartments.

    PubMed

    Marre, Meghan L; Petnicki-Ocwieja, Tanja; DeFrancesco, Alicia S; Darcy, Courtney T; Hu, Linden T

    2010-09-22

    Toll-like receptor (TLR)-2/TLR1 heterodimers recognize bacterial lipopeptides and initiate the production of inflammatory mediators. Adaptors and co-receptors that mediate this process, as well as the mechanisms by which these adaptors and co-receptors function, are still being discovered. Using shRNA, blocking antibodies, and fluorescent microscopy, we show that U937 macrophage responses to the TLR2/1 ligand, Pam(3)CSK(4), are dependent upon an integrin, α(3)β(1). The mechanism for integrin α(3)β(1) involvement in TLR2/1 signaling is through its role in endocytosis of lipopeptides. Using inhibitors of endosomal acidification/maturation and physical tethering of the ligand, we show that the endocytosis of Pam(3)CSK(4) is necessary for the complete TLR2/1-mediated pro-inflammatory cytokine response. We also show that TLR2/1 signaling from the endosome results in the induction of different inflammatory mediators than TLR2/1 signaling from the plasma membrane. Here we identify integrin α(3)β(1) as a novel regulator for the recognition of bacterial lipopeptides. We demonstrate that induction of a specific subset of cytokines is dependent upon integrin α(3)β(1)-mediated endocytosis of the ligand. In addition, we address an ongoing controversy regarding endosomal recognition of bacterial lipopeptides by demonstrating that TLR2/1 signals from within endosomal compartments as well as the plasma membrane, and that downstream responses may differ depending upon receptor localization. We propose that the regulation of endosomal TLR2/1 signaling by integrin α(3)β(1) serves as a mechanism for modulating inflammatory responses.

  20. Effects of β4 integrin expression on microRNA patterns in breast cancer

    PubMed Central

    Gerson, Kristin D.; Maddula, V. S. R. Krishna; Seligmann, Bruce E.; Shearstone, Jeffrey R.; Khan, Ashraf; Mercurio, Arthur M.

    2012-01-01

    Summary The integrin α6β4 is defined as an adhesion receptor for laminins. Referred to as ‘β4’, this integrin plays a key role in the progression of various carcinomas through its ability to orchestrate key signal transduction events and promote cell motility. To identify novel downstream effectors of β4 function in breast cancer, microRNAs (miRNAs) were examined because of their extensive links to tumorigenesis and their ability to regulate gene expression globally. Two breast carcinoma cell lines and a collection of invasive breast carcinomas with varying β4 expression were used to assess the effect of this integrin on miRNA expression. A novel miRNA microarray analysis termed quantitative Nuclease Protection Assay (qNPA) revealed that β4 expression can significantly alter miRNA expression and identified two miRNA families, miR-25/32/92abc/363/363-3p/367 and miR-99ab/100, that are consistently downregulated by expression of this integrin. Analysis of published Affymetrix GeneChip data identified 54 common targets of miR-92ab and miR-99ab/100 within the subset of β4-regulated mRNAs, revealing several genes known to be key components of β4-regulated signaling cascades and effectors of cell motility. Gene ontology classification identified an enrichment in genes associated with cell migration within this population. Finally, gene set enrichment analysis of all β4-regulated mRNAs revealed an enrichment in targets belonging to distinct miRNA families, including miR-92ab and others identified by our initial array analyses. The results obtained in this study provide the first example of an integrin globally impacting miRNA expression and provide evidence that select miRNA families collectively target genes important in executing β4-mediated cell motility. PMID:23213459

  1. Intracellular Modifiers of Integrin Alpha 6p Production in Aggressive Prostate and Breast Cancer Cell Lines

    PubMed Central

    Kacsinta, Apollo D.; Rubenstein, Cynthia S.; Sroka, Isis C.; Pawar, Sangita; Gard, Jaime M.; Nagle, Raymond B.; Cress, Anne E.

    2014-01-01

    Cancer metastasis is a multi-step process in which tumor cells gain the ability to invade beyond the primary tumor and colonize distant sites. The mechanisms regulating the metastatic process confer changes to cell adhesion receptors including the integrin family of receptors. Our group previously discovered that the α6 integrin (ITGA6/CD49f) is post translationally modified by urokinase plasminogen activator (uPA) and its receptor, urokinase plasminogen activator receptor (uPAR), to form the variant ITGA6p. This variant of ITGA6 is a cleaved form of the receptor that lacks the ligand-binding domain. Although it is established that the uPA/uPAR axis drives ITGA6 cleavage, the mechanisms regulating cleavage have not been defined. Intracellular integrin dependent “inside-out” signaling is a major regulator of integrin function and the uPA/uPAR axis. We hypothesized that intracellular signaling molecules play a role in formation of ITGA6p to promote cell migration during cancer metastasis. In order to test our hypothesis, DU145 and PC3B1 prostate cancer and MDA-MB-231 breast cancer cell lines were treated with small interfering RNA targeting actin and the intracellular signaling regulators focal adhesion kinase (FAK), integrin linked kinase (ILK), and paxillin. The results demonstrated that inhibition of actin, FAK, and ILK expression resulted in significantly increased uPAR expression and ITGA6p production. Inhibition of actin increased ITGA6p, although inhibition of paxillin did not affect ITGA6p formation. Taken together, these results suggest that FAK and ILK dependent “inside-out” signaling, and actin dynamics regulate extracellular production of ITGA6p and the aggressive phenotype. PMID:25450398

  2. Oncofetal Chondroitin Sulfate Glycosaminoglycans are Key Players in Integrin Signaling and Tumor Cell Motility

    PubMed Central

    Clausen, Thomas Mandel; Pereira, Marina Ayres; Al Nakouzi, Nader; Oo, Htoo Zarni; Agerbæk, Mette Ø; Lee, Sherry; Ørum-Madsen, Maj Sofie; Christensen, Anders Riis; El-Naggar, Amal; Grandgenett, Paul M.; Grem, Jean L.; Hollingsworth, Michael A.; Holst, Peter J.; Theander, Thor; Sorensen, Poul H.; Daugaard, Mads; Salanti, Ali

    2016-01-01

    Many tumors express proteoglycans modified with oncofetal chondroitin sulfate glycosaminoglycan chains (ofCS), which are normally restricted to the placenta. However, the role of ofCS in cancer is largely unknown. The function of ofCS in cancer was analyzed using the recombinant ofCS-binding VAR2CSA protein (rVAR2) derived from the malaria parasite, Plasmodium falciparum. We demonstrate that ofCS plays a key role in tumor cell motility by affecting canonical integrin signaling pathways. Binding of rVAR2 to tumor cells inhibited the interaction of cells with extracellular matrix (ECM) components, which correlated with decreased phosphorylation of Src kinase. Moreover, rVAR2 binding decreased migration, invasion and anchorage-independent growth of tumor cells in vitro. Mass spectrometry of ofCS-modified proteoglycan complexes affinity purified from tumor cell lines on rVAR2 columns, revealed an overrepresentation of proteins involved in cell motility and integrin signaling, such as integrin β1 (ITGB1) and integrin α4 (ITGA4). Saturating concentrations of rVAR2 inhibited downstream integrin signaling, which was mimicked by knockdown of the core CS synthesis enzymes Beta-1,3-Glucuronyltransferase 1 (B3GAT1) and Chondroitin Sulfate N-Acetylgalactosaminyltransferase 1 (CSGALNACT1). The ofCS modification was highly expressed in both human and murine metastatic lesions in situ and pre-incubation or early intravenous treatment of tumor cells with rVAR2 inhibited seeding and spreading of tumor cells in mice. This was associated with a significant increase in survival of the animals. These data functionally link ofCS modifications with cancer cell motility and further highlights ofCS as a novel therapeutic cancer target. Implications The cancer specific expression of oncofetal chondroitin sulfate aids in metastatic phenotypes and is a candidate target for therapy. PMID:27655130

  3. The CD157-integrin partnership controls transendothelial migration and adhesion of human monocytes.

    PubMed

    Lo Buono, Nicola; Parrotta, Rossella; Morone, Simona; Bovino, Paola; Nacci, Giulia; Ortolan, Erika; Horenstein, Alberto L; Inzhutova, Alona; Ferrero, Enza; Funaro, Ada

    2011-05-27

    CD157, a member of the CD38 gene family, is an NAD-metabolizing ectoenzyme and a signaling molecule whose role in polarization, migration, and diapedesis of human granulocytes has been documented; however, the molecular events underpinning this role remain to be elucidated. This study focused on the role exerted by CD157 in monocyte migration across the endothelial lining and adhesion to extracellular matrix proteins. The results demonstrated that anti-CD157 antibodies block monocyte transmigration and adhesion to fibronectin and fibrinogen but that CD157 cross-linking is sufficient to overcome the block, suggesting an active signaling role for the molecule. Consistent with this is the observation that CD157 is prevalently located within the detergent-resistant membrane microdomains to which, upon clustering, it promotes the recruitment of β(1) and β(2) integrin, which, in turn, leads to the formation of a multimolecular complex favoring signal transduction. This functional cross-talk with integrins allows CD157 to act as a receptor despite its intrinsic structural inability to do so on its own. Intracellular signals mediated by CD157 rely on the integrin/Src/FAK (focal adhesion kinase) pathway, resulting in increased activity of the MAPK/ERK1/2 and the PI3K/Akt downstream signaling pathways, which are crucial in the control of monocyte transendothelial migration. Collectively, these findings indicate that CD157 acts as a molecular organizer of signaling-competent membrane microdomains and that it forms part of a larger molecular machine ruled by integrins. The CD157-integrin partnership provides optimal adhesion and transmigration of human monocytes.

  4. Integrin alphavbeta3 mediates K1735 murine melanoma cell motility in vivo and in vitro.

    PubMed

    Li, X; Regezi, J; Ross, F P; Blystone, S; Ilić, D; Leong, S P; Ramos, D M

    2001-07-01

    The integrin alphavbeta3 has been shown to be tightly linked to progression of human melanoma. In this study, using two clones from the K1735 murine melanoma system, we investigated the role of alphavbeta3 in metastasis. The highly metastatic K1735M2 cells express the alphavbeta3 integrin, whereas the poorly metastatic K1735C23 cells do not. When transduced with the beta3 integrin subunit cDNA, the K1735C23 cells produced lung lesions and, in two animals, cardiac metastases, whereas the parental C23 cells did not. By contrast, transduction of the full-length beta3 integrin antisense DNA into the K1735M2 cells suppressed metastatic colonization. To specifically investigate the activation of beta3 integrin-mediated pathways, the beta3-positive and the beta3-negative K1735 cells were plated onto vitronectin, a major matrix molecule of both primary and metastatic melanomas. Tyr397 of FAK was phosphorylated several times higher in beta3-expressing K1735 melanoma cells than in beta3-negative cells. To determine whether phosphorylation of FAK was associated with K1735 melanoma motility, we expressed the FAK-related non-kinase (FRNK) in the highly metastatic K1735M2 cells. Exogenous expression of FRNK suppressed phosphorylation of FAK at Tyr397 and decreased the invasive ability of these cells. In addition, expression of a constitutively active mutant Src in poorly metastatic K1735C23 cells increased invasion in vitro; whereas expression of a kinase-inactive Src mutant suppressed invasion. Our results suggest that signals initiated by alphavbeta3 promote metastasis in K1735 melanoma cells through the phosphorylation of FAK and activation of Src.

  5. β1-Integrin Deletion From the Lens Activates Cellular Stress Responses Leading to Apoptosis and Fibrosis

    PubMed Central

    Wang, Yichen; Terrell, Anne M.; Riggio, Brittany A.; Anand, Deepti; Lachke, Salil A.; Duncan, Melinda K.

    2017-01-01

    Purpose Previous research showed that the absence of β1-integrin from the mouse lens after embryonic day (E) 13.5 (β1MLR10) leads to the perinatal apoptosis of lens epithelial cells (LECs) resulting in severe microphthalmia. This study focuses on elucidating the molecular connections between β1-integrin deletion and this phenotype. Methods RNA sequencing was performed to identify differentially regulated genes (DRGs) in β1MLR10 lenses at E15.5. By using bioinformatics analysis and literature searching, Egr1 (early growth response 1) was selected for further study. The activation status of certain signaling pathways (focal adhesion kinase [FAK]/Erk, TGF-β, and Akt signaling) was studied via Western blot and immunohistochemistry. Mice lacking both β1-integrin and Egr1 genes from the lenses were created (β1MLR10/Egr1−/−) to study their relationship. Results RNA sequencing identified 120 DRGs that include candidates involved in the cellular stress response, fibrosis, and/or apoptosis. Egr1 was investigated in detail, as it mediates cellular stress responses in various cell types, and is recognized as an upstream regulator of numerous other β1MLR10 lens DRGs. In β1MLR10 mice, Egr1 levels are elevated shortly after β1-integrin loss from the lens. Further, pErk1/2 and pAkt are elevated in β1MLR10 LECs, thus providing the potential signaling mechanism that causes Egr1 upregulation in the mutant. Indeed, deletion of Egr1 from β1MLR10 lenses partially rescues the microphthalmia phenotype. Conclusions β1-integrin regulates the appropriate levels of Erk1/2 and Akt phosphorylation in LECs, whereas its deficiency results in the overexpression of Egr1, culminating in reduced cell survival. These findings provide insight into the molecular mechanism underlying the microphthalmia observed in β1MLR10 mice. PMID:28763805

  6. A Primer for Using Transgenic Insecticidal Cotton in Developing Countries

    PubMed Central

    Showalter, Ann M.; Heuberger, Shannon; Tabashnik, Bruce E.; Carrière, Yves

    2009-01-01

    Many developing countries face the decision of whether to approve the testing and commercial use of insecticidal transgenic cotton and the task of developing adequate regulations for its use. In this review, we outline concepts and provide information to assist farmers, regulators and scientists in making decisions concerning this technology. We address seven critical topics: 1) molecular and breeding techniques used for the development of transgenic cotton cultivars, 2) properties of transgenic cotton cultivars and their efficacy against major insect pests, 3) agronomic performance of transgenic cotton in developing countries, 4) factors affecting transgene expression, 5) impact of gene flow between transgenic and non-transgenic cotton, 6) non-target effects of transgenic cotton, and 7) management of pest resistance to transgenic cotton. PMID:19613464

  7. Stability of transgenes in long-term micropropagation of plants of transgenic birch (Betula platyphylla).

    PubMed

    Zeng, Fansuo; Qian, Jingjing; Luo, Wei; Zhan, Yaguang; Xin, Ying; Yang, Chuanping

    2010-01-01

    The stability of integration and expression level of transgenes in long-term micropropagation clones of transgenic birch (Betula platyphylla Suk.) was examined. Multiplexed PCR and reverse primer PCR demonstrated stable integration of transgenes into regenerated plants. Expression levels of the bgt and gus genes among shoot plantlets, subcultured 4, 7, 9 and 15 times, were significantly different. The transcriptional expression level of extraneous genes in regenerated plants decreased with increasing subculture number. Transcriptional gene silencing (TGS) occured in regenerated transgenic lines. The silencing rate of GUS in the 5th subculture plants was 22-65%. TGS in regenerated plants could be reactivated with 5-azacytidine (Azac) at 50-200 microM. GUS and BGT protein expression was reactivated in the micropropagated transgenic birch plants when treated with Azac. A decrease in expression level with increasing number of subcultures is thus associated with DNA methylation.

  8. Down-regulation of integrin alpha(v)beta(3) expression and integrin-mediated signaling in glioma cells by adenovirus-mediated transfer of antisense urokinase-type plasminogen activator receptor (uPAR) and sense p16 genes.

    PubMed

    Adachi, Y; Lakka, S S; Chandrasekar, N; Yanamandra, N; Gondi, C S; Mohanam, S; Dinh, D H; Olivero, W C; Gujrati, M; Tamiya, T; Ohmoto, T; Kouraklis, G; Aggarwal, B; Rao, J S

    2001-12-14

    Interaction between the extracellular matrix and integrin receptors on cell surfaces leads not only to cell adhesion but also to intracellular signaling events that affect cell migration, proliferation, and survival. The vitronectin receptor alpha(v)beta(3) integrin is of key importance in glioma cell biology. The expression of urokinase-type plasminogen activator receptor (uPAR) was recently shown to co-regulate with the expression of alpha(v)beta(3) integrin. Moreover, restoration of the p16 protein in glioma cells inhibits the alpha(v)beta(3) integrin-mediated spreading of those cells on vitronectin. Thus we hypothesized that adenovirus-mediated down-regulation of uPAR and overexpression of p16 might down-regulate the expression of alpha(v)beta(3) integrin and the integrin-mediated signaling in glioma cells, thereby defeating the malignant phenotype. In this study, we used replication-deficient adenovirus vectors that contain either a uPAR antisense expression cassette (Ad-uPAR) or wild-type p16 cDNA (Ad-p16) and a bicistronic adenovirus construct in which both the uPAR antisense and p16 sense expression cassettes (Ad-uPAR/p16) are inserted in the E1-deleted region of the vector. Infecting the malignant glioma cell line SNB19 with Ad-uPAR, Ad-p16, or Ad-uPAR/p16 in the presence of vitronectin resulted in decreased alpha(v)beta(3) integrin expression and integrin-mediated biological effects, including adhesion, migration, proliferation, and survival Our results support the therapeutic potential of simultaneously targeting uPAR and p16 in the treatment of gliomas.

  9. Effects of transgenic rootstocks on growth and development of non-transgenic scion cultivars in apple.

    PubMed

    Smolka, Anders; Li, Xue-Yuan; Heikelt, Catrin; Welander, Margareta; Zhu, Li-Hua

    2010-12-01

    Although cultivation of genetic modified (GM) annual crops has been steadily increasing in the recent 10 years, the commercial cultivation of GM fruit tree is still very limited and reports of field trials on GM fruit trees are rare. This is probably because development and evaluation of GM fruit trees require a long period of time due to long life cycles of trees. In this study, we report results from a field trial on three rolB transgenic dwarfing apple rootstocks of M26 and M9 together with non-transgenic controls grafted with five non-transgenic scion cultivars. We intended to investigate the effects of transgenic rootstock on non-transgenic scion cultivars under natural conditions as well as to evaluate the potential value of using the rolB gene to modify difficult-to-root rootstocks of fruit trees. The results showed that all rolB transgenic rootstocks significantly reduced vegetative growth including tree height regardless of scion cultivar, compared with the non-transgenic rootstocks. Flowering and fruiting were also decreased for cultivars grown on the transgenic rootstocks in most cases, but the fruit quality was not clearly affected by the transgenic rootstocks. Cutting experiment and RT-PCR analysis showed that the rolB gene was stably expressed under field conditions. PCR and RT-PCR analyses displayed that the rolB gene or its mRNA were not detectable in the scion cultivars, indicating no translocation of the transgene or its mRNA from rootstock to scion. Our results suggest that rolB modified rootstocks should be used in combination with vigorous scion cultivars in order to obtain sufficient vegetative growth and good yield. Alternatively, the rolB gene could be used to dwarf vigorous rootstocks of fruit trees or produce bonzai plants as it can significantly reduce the vegetative growth of plants.

  10. Integrin α5 Suppresses the Phosphorylation of Epidermal Growth Factor Receptor and Its Cellular Signaling of Cell Proliferation via N-Glycosylation*

    PubMed Central

    Hang, Qinglei; Isaji, Tomoya; Hou, Sicong; Im, Sanghun; Fukuda, Tomohiko; Gu, Jianguo

    2015-01-01

    Integrin α5β1-mediated cell adhesion regulates a multitude of cellular responses, including cell proliferation, survival, and cross-talk between different cellular signaling pathways. Integrin α5β1 is known to convey permissive signals enabling anchorage-dependent receptor tyrosine kinase signaling. However, the effects of integrin α5β1 on cell proliferation are controversial, and the molecular mechanisms involved in the regulation between integrin α5β1 and receptor tyrosine kinase remain largely unclear. Here we show that integrin α5 functions as a negative regulator of epidermal growth factor receptor (EGFR) signaling through its N-glycosylation. Expression of WT integrin α5 suppresses the EGFR phosphorylation and internalization upon EGF stimulation. However, expression of the N-glycosylation mutant integrin α5, S3–5, which contains fewer N-glycans, reversed the suppression of the EGFR-mediated signaling and cell proliferation. In a mechanistic manner, WT but not S3–5 integrin α5 forms a complex with EGFR and glycolipids in the low density lipid rafts, and the complex formation is disrupted upon EGF stimulation, suggesting that the N-glycosylation of integrin α5 suppresses the EGFR activation through promotion of the integrin α5-glycolipids-EGFR complex formation. Furthermore, consistent restoration of those N-glycans on the Calf-1,2 domain of integrin α5 reinstated the inhibitory effects as well as the complex formation with EGFR. Taken together, these data are the first to demonstrate that EGFR activation can be regulated by the N-glycosylation of integrin α5, which is a novel molecular paradigm for the cross-talk between integrins and growth factor receptors. PMID:26483551

  11. Integrin acts upstream of netrin signaling to regulate formation of the anchor cell's invasive membrane in C. elegans.

    PubMed

    Hagedorn, Elliott J; Yashiro, Hanako; Ziel, Joshua W; Ihara, Shinji; Wang, Zheng; Sherwood, David R

    2009-08-01

    Integrin expression and activity have been strongly correlated with developmental and pathological processes involving cell invasion through basement membranes. The role of integrins in mediating these invasions, however, remains unclear. Utilizing the genetically and visually accessible model of anchor cell (AC) invasion in C. elegans, we have recently shown that netrin signaling orients a specialized invasive cell membrane domain toward the basement membrane. Here, we demonstrate that the integrin heterodimer INA-1/PAT-3 plays a crucial role in AC invasion, in part by targeting the netrin receptor UNC-40 (DCC) to the AC's plasma membrane. Analyses of the invasive membrane components phosphatidylinositol 4,5-bisphosphate, the Rac GTPase MIG-2, and F-actin further indicate that INA-1/PAT-3 plays a broad role in promoting the plasma membrane association of these molecules. Taken together, these studies reveal a role for integrin in regulating the plasma membrane targeting and netrin-dependent orientation of a specialized invasive membrane domain.

  12. Cutting edge: egress of newly generated plasma cells from peripheral lymph nodes depends on beta 2 integrin.

    PubMed

    Pabst, Oliver; Peters, Thorsten; Czeloth, Niklas; Bernhardt, Günter; Scharffetter-Kochanek, Karin; Förster, Reinhold

    2005-06-15

    During humoral immune responses, naive B cells differentiate into Ab-secreting plasma cells within secondary lymphoid organs. Differentiating plasma cells egress from their sites of generation and redistribute to other tissues, predominantly the bone marrow and mucosal tissues. In this study, we demonstrate that within peripheral lymph nodes newly generated plasma cells localize to medullary cords which express the beta(2) integrin ligand ICAM-1. In beta(2) integrin-deficient mice plasma cells accumulate inside the lymph nodes, resulting in severely reduced plasma cell numbers in the bone marrow. Since plasma cells isolated from beta(2) integrin-deficient animals migrate efficiently into the bone marrow when transferred i.v., our findings provide profound evidence that beta(2) integrins are required for the egress of plasma cells from peripheral lymph nodes.

  13. A transmembrane polar interaction is involved in the functional regulation of integrin alpha L beta 2.

    PubMed

    Vararattanavech, Ardcharaporn; Chng, Choon-Peng; Parthasarathy, Krupakar; Tang, Xiao-Yan; Torres, Jaume; Tan, Suet-Mien

    2010-05-14

    Integrins are heterodimeric transmembrane (TM) receptors formed by noncovalent associations of alpha and beta subunits. Each subunit contains a single alpha-helical TM domain. Inside-out activation of an integrin involves the separation of its cytoplasmic tails, leading to disruption of alphabeta TM packing. The leukocyte integrin alpha L beta 2 is required for leukocyte adhesion, migration, proliferation, cytotoxic function, and antigen presentation. In this study, we show by mutagenesis experiments that the packing of alpha L beta 2 TMs is consistent with that of the integrin alpha IIb beta 3 TMs. However, molecular dynamics simulations of alpha L beta 2 TMs in lipids predicted a polar interaction involving the side chains of alpha L Ser1071 and beta2 Thr686 in the outer-membrane association clasp (OMC). This is supported by carbonyl vibrational shifts observed in isotope-labeled alpha L beta 2 TM peptides that were incorporated into lipid bilayers. Molecular dynamics studies simulating the separation of alpha L beta 2 tails showed the presence of polar interaction during the initial perturbation of the inner-membrane association clasp. When the TMs underwent further separation, the polar interaction was disrupted. OMC polar interaction is important in regulating the functions of beta2 integrins because mutations that disrupt the OMC polar interaction generated constitutively activated alpha L beta 2, alpha M beta 2, and alpha X beta 2 in 293T transfectants. We also show that the expression of mutant beta2 Thr686Gly in beta2-deficient T cells rescued cell adhesion to intercellular adhesion molecule 1, but the cells showed overt elongated morphologies in response to chemokine stromal-cell-derived factor 1 alpha treatment as compared to wild-type beta2-expressing cells. These two TM polar residues are totally conserved in other members of the beta2 integrins in humans and across different species. Our results provide an example of the stabilizing effect of polar

  14. Biochemical methods to study the interactions between integrins and ion channels.

    PubMed

    Crociani, Olivia

    2010-01-01

    Protein-protein interactions between integrins and ion channels consist in a complicated bidirectional talk, not yet understood in detail, which triggers a downstream signaling network. Such a coordinated process occurs in discrete, localized microcompartments and involves different membrane and cytoplasmic proteins. Since the early nineties, when the first functional association between integrins and ion channels was characterized, the number of similar examples is constantly increasing. Identifying the components of this pathway has general importance for cell physiology and will eventually lead to fully understand the role of ion channels in the physiological processes typically controlled by integrin receptors, such as cell adhesion, migration and proliferation. Here, we detail the main experimental methods currently available to study these processes and discuss their advantages and disadvantages. Biochemical copurification and genetic interaction studies, as well as high-throughput screening, can be performed to initially identify the interacting proteins. Successively, in vitro binding assays such as pull-down and immunoprecipitation-based techniques allow to verify and better characterize these partnerships, possibly in combination with mass spectrometry methods. When transient interactions are involved, more sophisticated techniques, such as photoaffinity labelingprocedures, are necessary to detect the multiprotein complexes by having them covalently bound together as they interact. To provide even more thorough analyses of the formation, function and composition of protein complexes, other technologies such as confocal microscopy, fluorescence resonance energy transfer microscopy and site directed mutagenesis (possibly in murine models) have to be performed. The progressive accumulation of data defining novel protein-protein interactions has been considerably accelerated by the identification of specific sequence motifs that regulate integrin binding to

  15. Differential integrin expression regulates cell sensing of the matrix nanoscale geometry.

    PubMed

    Di Cio, Stefania; Bøggild, Thea M L; Connelly, John; Sutherland, Duncan S; Gautrot, Julien E

    2017-03-01

    The nanoscale geometry and topography of the extra-cellular matrix (ECM) is an important parameter controlling cell adhesion and phenotype. Similarly, integrin expression and the geometrical maturation of adhesions they regulate have been correlated with important changes in cell spreading and phenotype. However, how integrin expression controls the nanoscale sensing of the ECM geometry is not clearly understood. Here we develop a new nanopatterning technique, electrospun nanofiber lithography (ENL), which allows the production of a quasi-2D fibrous nanopattern with controlled dimensions (250-1000nm) and densities. ENL relies on electrospun fibres to act as a mask for the controlled growth of protein-resistant polymer brushes. SEM, AFM and immunofluorescence imaging were used to characterise the resulting patterns and the adsorption of the extra-cellular matrix protein fibronectin to the patterned fibres. The control of adhesion formation was studied, as well as the remodelling and deposition of novel matrix. Cell spreading was found to be regulated by the size of fibres, similarly to previous observations made on circular nanopatterns. However, cell shape and polarity were more significantly affected. These changes correlated with important cytoskeleton reorganisation, with a gradual decrease in stress fibre formation as the pattern dimensions decrease. Finally, the differential expression of αvβ3 and α5β1 integrins in engineered cell lines was found to be an important mediator of cell sensing of the nanoscale geometry of the ECM. The novel nanofiber patterns developed in this study, via ENL, mimic the geometry and continuity of natural matrices found in the stroma of tissues, whilst preserving a quasi-2D character (to facilitate imaging and for comparison with other 2D systems such as micropatterned monolayers and circular nanopatches generated by colloidal lithography). These results demonstrate that the nanoscale geometry of the ECM plays an important role

  16. Use of protein-engineered fabrics to identify design rules for integrin ligand clustering in biomaterials.

    PubMed

    Benitez, Patrick L; Mascharak, Shamik; Proctor, Amy C; Heilshorn, Sarah C

    2016-01-01

    While ligand clustering is known to enhance integrin activation, this insight has been difficult to apply to the design of implantable biomaterials because the local and global ligand densities that enable clustering-enhanced integrin signaling were unpredictable. Here, two general design principles for biomaterial ligand clustering are elucidated. First, clustering ligands enhances integrin-dependent signals when the global ligand density, i.e., the ligand density across the cellular length scale, is near the ligand's effective dissociation constant (KD,eff). Second, clustering ligands enhances integrin activation when the local ligand density, i.e., the ligand density across the length scale of individual focal adhesions, is less than an overcrowding threshold. To identify these principles, we fabricated a series of elastin-like, electrospun fabrics with independent control over the local (0 to 122 000 ligands μm(-2)) and global (0 to 71 000 ligand μm(-2)) densities of an arginine-glycine-aspartate (RGD) ligand. Antibody blocking studies confirmed that human umbilical vein endothelial cell adhesion to these protein-engineered biomaterials was primarily due to αVβ3 integrin binding. Clustering ligands enhanced cell proliferation, focal adhesion number, and focal adhesion kinase expression near the ligand's KD,eff of 12 000 RGD μm(-2). Near this global ligand density, cells on ligand-clustered fabrics behaved similarly to cells grown on fabrics with significantly larger global ligand densities but without clustering. However, this enhanced ligand-clustering effect was not observed above a threshold cut-off concentration. At a local ligand density of 122 000 RGD μm(-2), cell division, focal adhesion number, and focal adhesion kinase expression were significantly reduced relative to fabrics with identical global ligand density and lesser local ligand densities. Thus, when clustering results in overcrowding of ligands, integrin receptors are no longer

  17. The transgenic animal platform for biopharmaceutical production.

    PubMed

    Bertolini, L R; Meade, H; Lazzarotto, C R; Martins, L T; Tavares, K C; Bertolini, M; Murray, J D

    2016-06-01

    The recombinant production of therapeutic proteins for human diseases is currently the largest source of innovation in the pharmaceutical industry. The market growth has been the driving force on efforts for the development of new therapeutic proteins, in which transgenesis emerges as key component. The use of the transgenic animal platform offers attractive possibilities, residing on the low production costs allied to high productivity and quality of the recombinant proteins. Although many strategies have evolved over the past decades for the generation of transgenic founders, transgenesis in livestock animals generally faces some challenges, mainly due to random transgene integration and control over transgene copy number. But new developments in gene editing with CRISPR/Cas system promises to revolutionize the field for its simplicity and high efficiency. In addition, for the final approval of any given recombinant protein for animal or human use, the production and characterization of bioreactor founders and expression patterns and functionality of the proteins are technical part of the process, which also requires regulatory and administrative decisions, with a large emphasis on biosafety. The approval of two mammary gland-derived recombinant proteins for commercial and clinical use has boosted the interest for more efficient, safer and economic ways to generate transgenic founders to meet the increasing demand for biomedical proteins worldwide.

  18. Growth Factor Transgenes Interactively Regulate Articular Chondrocytes

    PubMed Central

    Shi, Shuiliang; Mercer, Scott; Eckert, George J.; Trippel, Stephen B.

    2014-01-01

    Adult articular chondrocytes lack an effective repair response to correct damage from injury or osteoarthritis. Polypeptide growth factors that stimulate articular chondrocyte proliferation and cartilage matrix synthesis may augment this response. Gene transfer is a promising approach to delivering such factors. No single growth factor gene is likely to optimize these cell functions, but multiple growth factor gene transfer remains unexplored. We tested the hypothesis that multiple growth factor gene transfer selectively modulates articular chondrocyte proliferation and matrix synthesis. We tested the hypothesis by delivering combinations of the transgenes encoding insulin-like growth factor I (IGF-I), fibroblast growth factor-2 (FGF-2), transforming growth factor beta1 (TGF-β1), bone morphogenetic protein-2 (BMP-2) and bone morphogenetic protien-7 (BMP-7) to articular chondrocytes and measured changes in the production of DNA, glycosaminoglycan and collagen. The transgenes differentially regulated all these chondrocyte functions. In concert, the transgenes interacted to generate widely divergent responses from the cells. These interactions ranged from inhibitory to synergistic. The transgene pair encoding IGF-I and FGF-2 maximized cell proliferation. The three-transgene group encoding IGF-I, BMP-2 and BMP-7 maximized matrix production and also optimized the balance between cell proliferation and matrix production. These data demonstrate a potentially tunable approach to articular chondrocyte regulation and suggest that certain growth factor gene combinations have potential value for cell-based articular cartilage repair. PMID:23097312

  19. Growth factor transgenes interactively regulate articular chondrocytes.

    PubMed

    Shi, Shuiliang; Mercer, Scott; Eckert, George J; Trippel, Stephen B

    2013-04-01

    Adult articular chondrocytes lack an effective repair response to correct damage from injury or osteoarthritis. Polypeptide growth factors that stimulate articular chondrocyte proliferation and cartilage matrix synthesis may augment this response. Gene transfer is a promising approach to delivering such factors. Multiple growth factor genes regulate these cell functions, but multiple growth factor gene transfer remains unexplored. We tested the hypothesis that multiple growth factor gene transfer selectively modulates articular chondrocyte proliferation and matrix synthesis. We tested the hypothesis by delivering combinations of the transgenes encoding insulin-like growth factor I (IGF-I), fibroblast growth factor-2 (FGF-2), transforming growth factor beta1 (TGF-β1), bone morphogenetic protein-2 (BMP-2), and bone morphogenetic protien-7 (BMP-7) to articular chondrocytes and measured changes in the production of DNA, glycosaminoglycan, and collagen. The transgenes differentially regulated all these chondrocyte activities. In concert, the transgenes interacted to generate widely divergent responses from the cells. These interactions ranged from inhibitory to synergistic. The transgene pair encoding IGF-I and FGF-2 maximized cell proliferation. The three-transgene group encoding IGF-I, BMP-2, and BMP-7 maximized matrix production and also optimized the balance between cell proliferation and matrix production. These data demonstrate an approach to articular chondrocyte regulation that may be tailored to stimulate specific cell functions, and suggest that certain growth factor gene combinations have potential value for cell-based articular cartilage repair. Copyright © 2012 Wiley Periodicals, Inc.

  20. Targeting the αvβ3 Integrin for Small-Animal PET/CT of Osteolytic Bone Metastases

    PubMed Central

    Wadas, Thaddeus J.; Deng, Hongju; Sprague, Jennifer E.; Zheleznyak, Alexander; Weilbaecher, Katherine N.; Anderson, Carolyn J.

    2010-01-01

    This article describes the evaluation of the radiopharmaceutical 64Cu-CB-TE2A-c(RGDyK) (64Cu-RGD) as an imaging agent for osteolytic bone metastases and their associated inflammation by targeting of the αvβ3 integrin on osteoclasts and the proinflammatory cells involved at the bone metastatic site. Methods The 64Cu-RGD radiotracer was evaluated in the transgenic mouse expressing Tax (Tax+), which spontaneously develops osteolytic tumors throughout the vertebrae and hind limbs, using biodistribution studies and small-animal PET/CT. Histologic analysis was also performed on Tax+ mouse tails, using hematoxylin and eosin and tartrate-resistant acid phosphatase to confirm the presence of osteolytic bone lesions and the presence of osteoclasts, respectively. Additionally, a proof-of-principle study was conducted with a small group of Tax+ animals presenting with osteolytic lesions. These animals were treated with the bisphosphonate zoledronic acid and imaged with 64Cu-RGD to determine whether this radiopharmaceutical was sensitive enough to detect a response to the bisphosphonate therapy. Results Biodistribution studies using 64Cu-RGD demonstrated that Tax+ mice between the ages of 6 and 12 mo had a greater accumulation of activity in their tail vertebrae than did the wild-type (WT) cohort (P = 0.013). Additionally, Tax+ mice between the ages of 6 and 12 mo had significantly more tracer activity associated with their tail vertebrae than did Tax+ mice older than 12 mo (P = 0.003), suggesting that earlier bone metastases cause an increased recruitment of αvβ3-expressing cells. Small-animal PET/CT with 64Cu-RGD was conducted on Tax+ and WT mice. On the basis of standardized uptake value analysis, Tax+ mice had approximately 2-fold more tail-associated activity than did WT animals (P = 0.0157). Additionally, decreases in uptake were observed in the tails of Tax+ mice after treatment with the osteoclast inhibitor zoledronic acid, and histologic analysis of Tax+ mouse

  1. Integrin-mediated transactivation of P2X7R via hemichannel-dependent ATP release stimulates astrocyte migration.

    PubMed

    Alvarez, Alvaro; Lagos-Cabré, Raúl; Kong, Milene; Cárdenas, Areli; Burgos-Bravo, Francesca; Schneider, Pascal; Quest, Andrew F G; Leyton, Lisette

    2016-09-01

    Our previous reports indicate that ligand-induced αVβ3 integrin and Syndecan-4 engagement increases focal adhesion formation and migration of astrocytes. Additionally, ligated integrins trigger ATP release through unknown mechanisms, activating P2X7 receptors (P2X7R), and the uptake of Ca(2+) to promote cell adhesion. However, whether the activation of P2X7R and ATP release are required for astrocyte migration and whether αVβ3 integrin and Syndecan-4 receptors communicate with P2X7R via ATP remains unknown. Here, cells were stimulated with Thy-1, a reported αVβ3 integrin and Syndecan-4 ligand. Results obtained indicate that ATP was released by Thy-1 upon integrin engagement and required the participation of phosphatidylinositol-3-kinase (PI3K), phospholipase-C gamma (PLCγ) and inositol trisphosphate (IP3) receptors (IP3R). IP3R activation leads to increased intracellular Ca(2+), hemichannel (Connexin-43 and Pannexin-1) opening, and ATP release. Moreover, silencing of the P2X7R or addition of hemichannel blockers precluded Thy-1-induced astrocyte migration. Finally, Thy-1 lacking the integrin-binding site did not stimulate ATP release, whereas Thy-1 mutated in the Syndecan-4-binding domain increased ATP release, albeit to a lesser extent and with delayed kinetics compared to wild-type Thy-1. Thus, hemichannels activated downstream of an αVβ3 integrin-PI3K-PLCγ-IP3R pathway are responsible for Thy-1-induced, hemichannel-mediated and Syndecan-4-modulated ATP release that transactivates P2X7Rs to induce Ca(2+) entry. These findings uncover a hitherto unrecognized role for hemichannels in the regulation of astrocyte migration via P2X7R transactivation induced by integrin-mediated ATP release. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Tumor suppressor KAI1 affects integrin {alpha}v{beta}3-mediated ovarian cancer cell adhesion, motility, and proliferation

    SciTech Connect

    Ruseva, Zlatna; Geiger, Pamina Xenia Charlotte; Hutzler, Peter

    2009-06-10

    The tetraspanin KAI1 had been described as a metastasis suppressor in many different cancer types, a function for which associations of KAI1 with adhesion and signaling receptors of the integrin superfamily likely play a role. In ovarian cancer, integrin {alpha}v{beta}3 correlates with tumor progression and its elevation in vitro provoked enhanced cell adhesion accompanied by significant increases in cell motility and proliferation in the presence of its major ligand vitronectin. In the present study, we characterized integrin {alpha}v{beta}3-mediated tumor biological effects as a function of cellular KAI1 restoration and proved for the first time that KAI1, besides its already knownmore » physical crosstalk with {beta}1-integrins, also colocalizes with integrin {alpha}v{beta}3. Functionally, elevated KAI1 levels drastically increased integrin {alpha}v{beta}3/vitronectin-dependent ovarian cancer cell adhesion. Since an intermediate level of cell adhesive strength is required for optimal cell migration, we next studied ovarian cancer cell motility as a function of KAI1 restoration. By time lapse video microscopy, we found impaired integrin {alpha}v{beta}3/vitronectin-mediated cell migration most probably due to strongly enhanced cellular immobilization onto the adhesion-supporting matrix. Moreover, KAI1 reexpression significantly diminished cell proliferation. These data strongly indicate that KAI1 may suppress ovarian cancer progression by inhibiting integrin {alpha}v{beta}3/vitronectin-provoked tumor cell motility and proliferation as important hallmarks of the oncogenic process.« less

  3. Retinoic acid receptor gamma impacts cellular adhesion, Alpha5Beta1 integrin expression and proliferation in K562 cells.

    PubMed

    Kelley, Melissa D; Phomakay, Raynin; Lee, Madison; Niedzwiedz, Victoria; Mayo, Rachel

    2017-01-01

    The interplay between cellular adhesion and proliferation is complex; however, integrins, particularly the α5β1 subset, play a pivotal role in orchestrating critical cellular signals that culminate in cellular adhesion and growth. Retinoids modify the expression of a variety of adhesive/proliferative signaling proteins including α5β1 integrins; however, the role of specific retinoic acid receptors involved in these processes has not been elucidated. In this study, the effect of all-trans-retinoic acid receptor (RAR) agonists on K562 cellular adhesion, proliferation, and α5β1 integrin cell surface expression was investigated. RARγ agonist exposure increased K562 cellular adhesion to RGD containing extracellular matrix proteins fibronectin and FN-120 in a time- and concentration dependent manner, while RARα or RARβ agonist treatment had no effect on cellular adhesion. Due to the novel RARγ- dependent cellular adhesion response exhibited by K562 cells, we examined α5 and β1 integrin subunit expression when K562 cells were exposed to retinoid agonists or vehicle for 24, 48, 72 or 96 hours. Our data demonstrates no differences in K562 cell surface expression of the α5 integrin subunit when cells were exposed to RARα, RARβ, or RARγ agonists for all time points tested. In contrast, RARγ agonist exposure resulted in an increase in cell surface β1 integrin subunit expression within 48 hours that was sustained at 72 and 96 hours. Finally, we demonstrate that while exposure to RARα or RARβ agonists have no effect on K562 cellular proliferation, the RARγ agonist significantly dampens K562 cellular proliferation levels in a time- and concentration- dependent manner. Our study is the first to report that treatment with a RARγ specific agonist augments cellular adhesion to α5β1 integrin substrates, increases cell surface levels of the β1 integrin subunit, and dampens cellular proliferation in a time and concentration dependent manner in a human

  4. Retinoic acid receptor gamma impacts cellular adhesion, Alpha5Beta1 integrin expression and proliferation in K562 cells

    PubMed Central

    Phomakay, Raynin; Lee, Madison; Niedzwiedz, Victoria; Mayo, Rachel

    2017-01-01

    The interplay between cellular adhesion and proliferation is complex; however, integrins, particularly the α5β1 subset, play a pivotal role in orchestrating critical cellular signals that culminate in cellular adhesion and growth. Retinoids modify the expression of a variety of adhesive/proliferative signaling proteins including α5β1 integrins; however, the role of specific retinoic acid receptors involved in these processes has not been elucidated. In this study, the effect of all-trans-retinoic acid receptor (RAR) agonists on K562 cellular adhesion, proliferation, and α5β1 integrin cell surface expression was investigated. RARγ agonist exposure increased K562 cellular adhesion to RGD containing extracellular matrix proteins fibronectin and FN-120 in a time- and concentration dependent manner, while RARα or RARβ agonist treatment had no effect on cellular adhesion. Due to the novel RARγ- dependent cellular adhesion response exhibited by K562 cells, we examined α5 and β1 integrin subunit expression when K562 cells were exposed to retinoid agonists or vehicle for 24, 48, 72 or 96 hours. Our data demonstrates no differences in K562 cell surface expression of the α5 integrin subunit when cells were exposed to RARα, RARβ, or RARγ agonists for all time points tested. In contrast, RARγ agonist exposure resulted in an increase in cell surface β1 integrin subunit expression within 48 hours that was sustained at 72 and 96 hours. Finally, we demonstrate that while exposure to RARα or RARβ agonists have no effect on K562 cellular proliferation, the RARγ agonist significantly dampens K562 cellular proliferation levels in a time- and concentration- dependent manner. Our study is the first to report that treatment with a RARγ specific agonist augments cellular adhesion to α5β1 integrin substrates, increases cell surface levels of the β1 integrin subunit, and dampens cellular proliferation in a time and concentration dependent manner in a human

  5. CD151 promotes α3β1 integrin-dependent organization of carcinoma cell junctions and restrains collective cell invasion.

    PubMed

    Zevian, Shannin C; Johnson, Jessica L; Winterwood, Nicole E; Walters, Katherine S; Herndon, Mary E; Henry, Michael D; Stipp, Christopher S

    2015-01-01

    Integrins function in collective migration both as major receptors for extracellular matrix and by crosstalk to adherens junctions. Despite extensive research, important questions remain about how integrin signaling mechanisms are integrated into collective migration programs. Tetraspanins form cell surface complexes with a subset of integrins and thus are good candidates for regulating the balance of integrin functional inputs into cell-matrix and cell-cell interactions. For example, tetraspanin CD151 directly associates with α3β1 integrin in carcinoma cells and promotes rapid α3β1-dependent single cell motility, but CD151 also promotes organized adherens junctions and restrains collective carcinoma cell migration on 2D substrates. However, the individual roles of CD151s integrin partners in CD151s pro-junction activity in carcinoma cells were not well understood. Here we find that CD151 promotes organized carcinoma cell junctions via α3β1 integrin, by a mechanism that requires the a3b1 ligand, laminin-332. Loss of CD151 promotes collective 3D invasion and growth in vitro and in vivo, and the enhanced invasion of CD151-silenced cells is α3 integrin dependent, suggesting that CD151 can regulate the balance between α3β1s pro-junction and pro-migratory activities in collective invasion. An analysis of human cancer cases revealed that changes in CD151 expression can be linked to either better or worse clinical outcomes depending on context, including potentially divergent roles for CD151 in different subsets of breast cancer cases. Thus, the role of the CD151-α3β1 complex in carcinoma progression is context dependent, and may depend on the mode of tumor cell invasion.

  6. Morphogenetic roles of perlecan in the tooth enamel organ: an analysis of overexpression using transgenic mice.

    PubMed

    Ida-Yonemochi, Hiroko; Satokata, Ichiro; Ohshima, Hayato; Sato, Toshiya; Yokoyama, Minesuke; Yamada, Yoshihiko; Saku, Takashi

    2011-09-01

    Perlecan, a heparan sulfate proteoglycan, is enriched in the intercellular space of the enamel organ. To understand the role of perlecan in tooth morphogenesis, we used a keratin 5 promoter to generate transgenic (Tg) mice that over-express perlecan in epithelial cells, and examined their tooth germs at tissue and cellular levels. Immunohistochemistry showed that perlecan was more strongly expressed in the enamel organ cells of Tg mice than in wild-type mice. Histopathology showed wider intercellular spaces in the stellate reticulum of the Tg molars and loss of cellular polarity in the enamel organ, especially in its cervical region. Hertwig's epithelial root sheath (HERS) cells in Tg mice were irregularly aligned due to excessive deposits of perlecan along the inner, as well as on the outer sides of the HERS. Tg molars had dull-ended crowns and outward-curved tooth roots and their enamel was poorly crystallized, resulting in pronounced attrition of molar cusp areas. In Tg mice, expression of integrin β1 mRNA was remarkably higher at E18, while expression of bFGF, TGF-β1, DSPP and Shh was more elevated at P1. The overexpression of perlecan in the enamel organ resulted in irregular morphology of teeth, suggesting that the expression of perlecan regulates growth factor signaling in a stage-dependent manner during each step of the interaction between ameloblast-lineage cells and mesenchymal cells. Copyright © 2011 International Society of Matrix Biology. All rights reserved.

  7. Synchronized cell attachment triggered by photo-activatable adhesive ligands allows QCM-based detection of early integrin binding.

    PubMed

    Iturri, Jagoba; García-Fernández, Luis; Reuning, Ute; García, Andrés J; del Campo, Aránzazu; Salierno, Marcelo J

    2015-03-31

    The Quartz Crystal Microbalance with dissipation (QCM-D) technique was applied to monitor and quantify integrin-RGD recognition during the early stages of cell adhesion. Using QCM-D crystals modified with a photo-activatable RGD peptide, the time point of presentation of adhesive ligand at the surface of the QCM-D crystal could be accurately controlled. This allowed temporal resolution of early integrin-RGD binding and the subsequent cell spreading process, and their separate detection by QCM-D. The specificity of the integrin-RGD binding event was corroborated by performing the experiments in the presence of soluble cyclicRGD as a competitor, and cytochalasin D as inhibitor of cell spreading. Larger frequency change in the QCM-D signal was observed for cells with larger spread area, and for cells overexpressing integrin αvβ3 upon stable transfection. This strategy enables quantification of integrin activity which, in turn, may allow discrimination among different cell types displaying distinct integrin subtypes and expression levels thereof. On the basis of these findings, we believe the strategy can be extended to other photoactivatable ligands to characterize cell membrane receptors activity, a relevant issue for cancer diagnosis (and prognosis) as other several pathologies.

  8. αvβ3 integrin spatially regulates VASP and RIAM to control adhesion dynamics and migration

    PubMed Central

    Worth, Daniel C.; Hodivala-Dilke, Kairbaan; Robinson, Stephen D.; King, Samantha J.; Morton, Penny E.; Gertler, Frank B.; Humphries, Martin J.

    2010-01-01

    Integrins are fundamental to the control of protrusion and motility in adherent cells. However, the mechanisms by which specific members of this receptor family cooperate in signaling to cytoskeletal and adhesion dynamics are poorly understood. Here, we show that the loss of β3 integrin in fibroblasts results in enhanced focal adhesion turnover and migration speed but impaired directional motility on both 2D and 3D matrices. These motility defects are coupled with an increased rate of actin-based protrusion. Analysis of downstream signaling events reveals that loss of β3 integrin results in a loss of protein kinase A–dependent phosphorylation of the actin regulatory protein vasodilator-stimulated phosphoprotein (VASP). Dephosphorylated VASP in β3-null cells is preferentially associated with Rap1-GTP–interacting adaptor molecule (RIAM) both in vitro and in vivo, which leads to enhanced formation of a VASP–RIAM complex at focal adhesions and subsequent increased binding of talin to β1 integrin. These data demonstrate a novel mechanism by which αvβ3 integrin acts to locally suppress β1 integrin activation and regulate protrusion, adhesion dynamics, and persistent migration. PMID:20404115

  9. A dual role for Integrin α6β4 in modulating hereditary neuropathy with liability to pressure palsies.

    PubMed

    Poitelon, Yannick; Matafora, Vittoria; Silvestri, Nicholas; Zambroni, Desirée; McGarry, Claire; Serghany, Nora; Rush, Thomas; Vizzuso, Domenica; Court, Felipe A; Bachi, Angela; Wrabetz, Lawrence; Feltri, Maria Laura

    2018-05-01

    Peripheral myelin protein 22 (PMP22) is a component of compact myelin in the peripheral nervous system. The amount of PMP22 in myelin is tightly regulated, and PMP22 over or under-expression cause Charcot-Marie-Tooth 1A (CMT1A) and Hereditary Neuropathy with Pressure Palsies (HNPP). Despite the importance of PMP22, its function remains largely unknown. It was reported that PMP22 interacts with the β4 subunit of the laminin receptor α6β4 integrin, suggesting that α6β4 integrin and laminins may contribute to the pathogenesis of CMT1A or HNPP. Here we asked if the lack of α6β4 integrin in Schwann cells influences myelin stability in the HNPP mouse model. Our data indicate that PMP22 and β4 integrin may not interact directly in myelinating Schwann cells, however, ablating β4 integrin delays the formation of tomacula, a characteristic feature of HNPP. In contrast, ablation of integrin β4 worsens nerve conduction velocities and non-compact myelin organization in HNPP animals. This study demonstrates that indirect interactions between an extracellular matrix receptor and a myelin protein influence the stability and function of myelinated fibers. © 2018 International Society for Neurochemistry.

  10. β7-Integrin exacerbates experimental DSS-induced colitis in mice by directing inflammatory monocytes into the colon

    PubMed Central

    Schippers, A; Muschaweck, M; Clahsen, T; Tautorat, S; Grieb, L; Tenbrock, K; Gaßler, N; Wagner, N

    2016-01-01

    Leukocyte recruitment is pivotal for the initiation and perpetuation of inflammatory bowel disease (IBD) and controlled by the specificity and interactions of chemokines and adhesion molecules. Interactions of the adhesion molecules α4β7-integrin and mucosal addressin cell-adhesion molecule-1 (MAdCAM-1) promote the accumulation of pathogenic T-cell populations in the inflamed intestine. We aimed to elucidate the significance of β7-integrin expression on innate immune cells for the pathogenesis of IBD. We demonstrate that β7-integrin deficiency protects recombination-activating gene-2 (RAG-2)-deficient mice from dextran sodium sulfate (DSS)-induced colitis and coincides with decreased numbers of colonic effector monocytes. We also show that β7-integrin is expressed on most CD11b+CD64lowLy6C+ bone marrow progenitors and contributes to colonic recruitment of these proinflammatory monocytes. Importantly, adoptive transfer of CD115+ wild-type (WT) monocytes partially restored the susceptibility of RAG-2/β7-integrin double-deficient mice to DSS-induced colitis, thereby demonstrating the functional importance of β7-integrin-expressing monocytes for the development of DSS colitis. We also reveal that genetic ablation of MAdCAM-1 ameliorates experimental colitis in RAG-2-deficient mice as well. In summary, we demonstrate a previously unknown role of α4β7-integrin–MAdCAM-1 interactions as drivers of colitis by directing inflammatory monocytes into the colon. PMID:26349655

  11. Synchronized cell attachment triggered by photo-activatable adhesive ligands allows QCM-based detection of early integrin binding

    PubMed Central

    Iturri, Jagoba; García-Fernández, Luis; Reuning, Ute; García, Andrés J.; Campo, Aránzazu del; Salierno, Marcelo J.

    2015-01-01

    The Quartz Crystal Microbalance with dissipation (QCM-D) technique was applied to monitor and quantify integrin-RGD recognition during the early stages of cell adhesion. Using QCM-D crystals modified with a photo-activatable RGD peptide, the time point of presentation of adhesive ligand at the surface of the QCM-D crystal could be accurately controlled. This allowed temporal resolution of early integrin-RGD binding and the subsequent cell spreading process, and their separate detection by QCM-D. The specificity of the integrin-RGD binding event was corroborated by performing the experiments in the presence of soluble cyclicRGD as a competitor, and cytochalasin D as inhibitor of cell spreading. Larger frequency change in the QCM-D signal was observed for cells with larger spread area, and for cells overexpressing integrin αvβ3 upon stable transfection. This strategy enables quantification of integrin activity which, in turn, may allow discrimination among different cell types displaying distinct integrin subtypes and expression levels thereof. On the basis of these findings, we believe the strategy can be extended to other photoactivatable ligands to characterize cell membrane receptors activity, a relevant issue for cancer diagnosis (and prognosis) as other several pathologies. PMID:25825012

  12. Integrin beta1-mediated matrix assembly and signaling are critical for the normal development and function of the kidney glomerulus.

    PubMed

    Kanasaki, Keizo; Kanda, Yoshiko; Palmsten, Kristin; Tanjore, Harikrishna; Lee, Soo Bong; Lebleu, Valerie S; Gattone, Vincent H; Kalluri, Raghu

    2008-01-15

    The human kidneys filter 180 l of blood every day via about 2.5 million glomeruli. The three layers of the glomerular filtration apparatus consist of fenestrated endothelium, specialized extracellular matrix known as the glomerular basement membrane (GBM) and the podocyte foot processes with their modified adherens junctions known as the slit diaphragm (SD). In this study we explored the contribution of podocyte beta1 integrin signaling for normal glomerular function. Mice with podocyte specific deletion of integrin beta1 (podocin-Cre beta1-fl/fl mice) are born normal but cannot complete postnatal renal development. They exhibit detectable proteinuria on day 1 and die within a week. The kidneys of podocin-Cre beta1-fl/fl mice exhibit normal glomerular endothelium but show severe GBM defects with multilaminations and splitting including podocyte foot process effacement. The integrin linked kinase (ILK) is a downstream mediator of integrin beta1 activity in epithelial cells. To further explore whether integrin beta1-mediated signaling facilitates proper glomerular filtration, we generated mice deficient of ILK in the podocytes (podocin-Cre ILK-fl/fl mice). These mice develop normally but exhibit postnatal proteinuria at birth and die within 15 weeks of age due to renal failure. Collectively, our studies demonstrate that podocyte beta1 integrin and ILK signaling is critical for postnatal development and function of the glomerular filtration apparatus.

  13. Targeted inhibition of {alpha}v{beta}3 integrin with an RNA aptamer impairs endothelial cell growth and survival

    SciTech Connect

    Mi Jing; Zhang Xiuwu; Giangrande, Paloma H.

    2005-12-16

    {alpha}v{beta}3 integrin is a crucial factor involved in a variety of physiological processes, such as cell growth and migration, tumor invasion and metastasis, angiogenesis, and wound healing. {alpha}v{beta}3 integrin exerts its effect by regulating endothelial cell (EC) migration, proliferation, and survival. Inhibiting the function of {alpha}v{beta}3 integrin, therefore, represents a potential anti-cancer, anti-thrombotic, and anti-inflammatory strategy. In this study, we tested an RNA aptamer, Apt-{alpha}v{beta}3 that binds recombinant {alpha}v{beta}3 integrin, for its ability to bind endogenous {alpha}v{beta}3 integrin on the surface of cells in culture and to subsequently affect cellular response. Our data illustrate that Apt-{alpha}v{beta}3 binds {alpha}v{beta}3 integrin expressedmore » on the surface of live HUVECs. This interaction significantly decreases both basal and PDGF-induced cell proliferation as well as inhibition of cell adhesion. Apt-{alpha}v{beta}3 can also reduce PDGF-stimulated tube formation and increase HUVEC apoptosis through inhibition of FAK phosphorylation pathway. Our results demonstrate that by binding to its target, Apt-{alpha}v{beta}3 can efficiently inhibit human EC proliferation and survival, resulting in reduced angiogenesis. It predicts that Apt-{alpha}v{beta}3 could become useful in both tumor imaging and the treatment of tumor growth, atherosclerosis, thrombosis, and inflammation.« less

  14. Fibronectin-bound α5β1 integrins sense load and signal to reinforce adhesion in less than a second

    NASA Astrophysics Data System (ADS)

    Strohmeyer, Nico; Bharadwaj, Mitasha; Costell, Mercedes; Fässler, Reinhard; Müller, Daniel J.

    2017-12-01

    Integrin-mediated mechanosensing of the extracellular environment allows cells to control adhesion and signalling. Whether cells sense and respond to force immediately upon ligand-binding is unknown. Here, we report that during adhesion initiation, fibroblasts respond to mechanical load by strengthening integrin-mediated adhesion to fibronectin (FN) in a biphasic manner. In the first phase, which depends on talin and kindlin as well as on the actin nucleators Arp2/3 and mDia, FN-engaged α5β1 integrins activate focal adhesion kinase (FAK) and c-Src in less than 0.5 s to steeply strengthen α5β1- and αV-class integrin-mediated adhesion. When the mechanical load exceeds a certain threshold, fibroblasts decrease adhesion and initiate the second phase, which is characterized by less steep adhesion strengthening. This unique, biphasic cellular adhesion response is mediated by α5β1 integrins, which form catch bonds with FN and signal to FN-binding integrins to reinforce cell adhesion much before visible adhesion clusters are formed.

  15. Physical and functional interactions between a glioma cation channel and integrin-β1 require α-actinin

    PubMed Central

    Rooj, Arun K.; Liu, Zhiyong; McNicholas, Carmel M.

    2015-01-01

    Major plasma membrane components of the tumor cell, ion channels, and integrins play crucial roles in metastasis. Glioma cells express an amiloride-sensitive nonselective cation channel composed of acid-sensing ion channel (ASIC)-1 and epithelial Na+ channel (ENaC) α- and γ-subunits. Inhibition of this channel is associated with reduced cell migration and proliferation. Using the ASIC-1 subunit as a reporter for the channel complex, we found a physical and functional interaction between this channel and integrin-β1. Short hairpin RNA knockdown of integrin-β1 attenuated the amiloride-sensitive current, which was due to loss of surface expression of ASIC-1. In contrast, upregulation of membrane expression of integrin-β1 increased the surface expression of ASIC-1. The link between the amiloride-sensitive channel and integrin-β1 was mediated by α-actinin. Downregulation of α-actinin-1 or -4 attenuated the amiloride-sensitive current. Mutation of the putative binding site for α-actinin on the COOH terminus of ASIC-1 reduced the membrane localization of ASIC-1 and also resulted in attenuation of the amiloride-sensitive current. Our data suggest a novel interaction between the amiloride-sensitive glioma cation channel and integrin-β1, mediated by α-actinin. This interaction may form a mechanism by which channel activity can regulate glioma cell proliferation and migration. PMID:26108662

  16. The Effect of Conditional Inactivation of Beta 1 Integrins using Twist 2 Cre, Osterix Cre and Osteocalcin Cre Lines on Skeletal Phenotype

    PubMed Central

    Shekaran, Asha; Shoemaker, James T.; Kavanaugh, Taylor E.; Lin, Angela S.; LaPlaca, Michelle C.; Fan, Yuhong; Guldberg, Robert E.; García, Andrés J.

    2014-01-01

    Skeletal development and growth are complex processes regulated by multiple microenvironmental cues, including integrin-ECM interactions. The β1 sub-family of integrins is the largest integrin sub-family and constitutes the main integrin binding partners of collagen I, the major ECM component of bone. As complete β1 integrin integrin knockout results in embryonic lethality, studies of β1 integrin function in vivo rely on tissue-specific gene deletions. While multiple in vitro studies indicate that β1 integrins are crucial regulators of osteogenesis and mineralization, in vivo osteoblast-specific perturbations of β1 integrins have resulted in mild and sometimes contradictory skeletal phenotypes. To further investigate the role of β1 integrins on skeletal phenotype, we used the Twist2-Cre, Osterix-Cre and Osteocalcin-Cre lines to generate conditional β1 integrin deletions, where cre is expressed primarily in mesenchymal condensation, pre-osteoblast, and mature osteoblast lineage cells respectively within these lines. Mice with Twist2-specific β1 integrin disruption were smaller, had impaired skeletal development, especially in the craniofacial and vertebral tissues at E19.5, and did not survive beyond birth. Osterix-specific β1 integrin deficiency resulted in viable mice which were normal at birth but displayed early defects in calvarial ossification, incisor eruption and growth as well as femoral bone mineral density, structure, and mechanical properties. Although these defects persisted into adulthood, they became milder with age. Finally, a lack of β1 integrins in mature osteoblasts and osteocytes resulted in minor alterations to femur structure but had no effect on mineral density, biomechanics or fracture healing. Taken together, our data indicate that β1 integrin expression in early mesenchymal condensations play an important role in skeletal ossification, while β1 integrin-ECM interactions in pre-osteoblast, odontoblast- and hypertrophic

  17. Advances in transgenic animal models and techniques.

    PubMed

    Ménoret, Séverine; Tesson, Laurent; Remy, Séverine; Usal, Claire; Ouisse, Laure-Hélène; Brusselle, Lucas; Chenouard, Vanessa; Anegon, Ignacio

    2017-10-01

    On May 11th and 12th 2017 was held in Nantes, France, the international meeting "Advances in transgenic animal models and techniques" ( http://www.trm.univ-nantes.fr/ ). This biennial meeting is the fifth one of its kind to be organized by the Transgenic Rats ImmunoPhenomic (TRIP) Nantes facility ( http://www.tgr.nantes.inserm.fr/ ). The meeting was supported by private companies (SONIDEL, Scionics computer innovation, New England Biolabs, MERCK, genOway, Journal Disease Models and Mechanisms) and by public institutions (International Society for Transgenic Technology, University of Nantes, INSERM UMR 1064, SFR François Bonamy, CNRS, Région Pays de la Loire, Biogenouest, TEFOR infrastructure, ITUN, IHU-CESTI and DHU-Oncogeffe and Labex IGO). Around 100 participants, from France but also from different European countries, Japan and USA, attended the meeting.

  18. Transgenic Cdx2 induces endogenous Cdx1 in intestinal metaplasia of Cdx2-transgenic mouse stomach.

    PubMed

    Mutoh, Hiroyuki; Hayakawa, Hiroko; Sakamoto, Hirotsugu; Sashikawa, Miho; Sugano, Kentaro

    2009-10-01

    Cdx1 and Cdx2, which are transcription factors regulating normal intestinal development, have been studied as potential key molecules in the pathogenesis of the precancerous intestinal metaplasia of the human stomach. However, the regulation of Cdx1 expression in the intestinal metaplasia is poorly understood. Cdx2-expressing gastric mucosa of Cdx2-transgenic mouse stomach was replaced by intestinal metaplastic mucosa. The aim of this study was to investigate the following: (a) Cdx1 expression in the intestinal metaplastic mucosa of the Cdx2-transgenic mouse stomach; and (b) the relationship between Cdx1 and Cdx2. A mouse model of intestinal metaplasia, the Cdx2-transgenic mouse, was used to investigate Cdx1 gene expression by RT-PCR. DNA methylation profile analysis was performed by bisulfite sequencing, and the interaction of Cdx2 with the Cdx1 promoter was examined by chromatin immunoprecipitation assay, electrophoretic mobility shift assay, and luciferase reporter assays. Cdx2 mRNA was expressed in the Cdx2-transgenic mouse stomach. However, endogenous Cdx2 mRNA was not expressed in the intestinal metaplasia of the Cdx2-transgenic mouse stomach. On the other hand, endogenous Cdx1 mRNA and protein were expressed in the intestinal metaplasia of the Cdx2-transgenic mouse stomach. The Cdx1 promoter was unmethylated in the intestinal metaplasia of the Cdx2-transgenic mouse stomach. Chromatin immunoprecipitation assay and electrophoretic mobility shift assay showed that Cdx2 was bound to the Cdx1 promoter region in the intestinal metaplasia and the normal intestine. Cdx2 upregulated and siRNA-Cdx2 downregulated the transcriptional activity of the Cdx1 gene in the human gastric carcinoma cell lines AGS, MKN45, and MKN74. In conclusion, transgenic Cdx2 induced endogenous Cdx1 through the binding of Cdx2 to the unmethylated Cdx1 promoter region in the intestinal metaplasia of the Cdx2-transgenic mouse stomach.

  19. Phospho-Caveolin-1 Mediates Integrin-Regulated Membrane Domain Internalisation

    PubMed Central

    del Pozo, Miguel A.; Alderson, Nazilla B.; Grande-García, Araceli; Balasubramanian, Nagaraj; Schwartz, Martin A.; Kiosses, William B.; Anderson, Richard G.W.

    2005-01-01

    Growth of normal cells is anchorage-dependent because signalling through multiple pathways including Erk, PI 3-kinase and Rac requires integrin-mediated cell adhesion 1. Components of these pathways localize to low density, cholesterol-rich domains in the plasma membrane named “lipid rafts” 2,3 or “cholesterol enriched membrane microdomains” (CEMM) 4. We previously reported that integrin-mediated adhesion regulates CEMM trafficking such that cell detachment from the extracellular matrix (ECM) triggers CEMM internalisation and clearance from the plasma membrane 5. We now report that this internalisation is mediated by dynamin-2 and caveolin-1. Internalisation requires phosphorylation of caveolin-1 on tyrosine 14. A shift in localisation of phospho-caveolin-1 from focal adhesions to caveolae induces CEMM internalisation upon cell detachment, which mediates inhibition of Erk, PI 3-kinase and Rac. These data define a novel molecular mechanism for growth and tumour suppression by caveolin-1. PMID:16113676

  20. How Osteoblasts Sense their Environment: Integrin-Extracellular Matrix Interactions and Mechanical Loading of Bone

    NASA Technical Reports Server (NTRS)

    Globus, Ruth K.; Dalton, Bonnie (Technical Monitor)

    2002-01-01

    Osteoblasts are the cells responsible for forming and replacing bone throughout life. We know that mechanical stimulation through weight-bearing at I gravity on Earth is needed to maintain healthy bone, and that osteoblasts play a critical role in that process. Over the last 9 years in my laboratory at NASA ARC, we have studied the regulation of osteoblast function by interactions between the extracellular matrix and die cell. Using a cell culture approach, we defined the repertoire of adhesion receptors, called integrins, which are expressed on the osteoblast surface, as well as specific extracellular matrix proteins, which are needed for cellular differentiation and survival. We are now extending these observations to determine if integrin signaling is involved in the skeletal responses to disuse and recovery from disuse using the rodent model of hindlimb unloading by tail suspension. Together, our cell culture and animal studies are providing new insight into the regulation of osteoblast function in bone.

  1. Tetraspanin CD151 regulates alpha6beta1 integrin adhesion strengthening

    NASA Technical Reports Server (NTRS)

    Lammerding, Jan; Kazarov, Alexander R.; Huang, Hayden; Lee, Richard T.; Hemler, Martin E.

    2003-01-01

    The tetraspanin CD151 molecule associates specifically with laminin-binding integrins, including alpha6beta1. To probe strength of alpha6beta1-dependent adhesion to laminin-1, defined forces (0-1.5 nN) were applied to magnetic laminin-coated microbeads bound to NIH 3T3 cells. For NIH 3T3 cells bearing wild-type CD151, adhesion strengthening was observed, as bead detachment became more difficult over time. In contrast, mutant CD151 (with the C-terminal region replaced) showed impaired adhesion strengthening. Static cell adhesion to laminin-1, and detachment of beads coated with fibronectin or anti-alpha6 antibody were all unaffected by CD151 mutation. Hence, CD151 plays a key role in selectively strengthening alpha6beta1 integrin-mediated adhesion to laminin-1.

  2. Canine Chondrodysplasia Caused by a Truncating Mutation in Collagen-Binding Integrin Alpha Subunit 10

    PubMed Central

    Kyöstilä, Kaisa; Lappalainen, Anu K.; Lohi, Hannes

    2013-01-01

    The skeletal dysplasias are disorders of the bone and cartilage tissues. Similarly to humans, several dog breeds have been reported to suffer from different types of genetic skeletal disorders. We have studied the molecular genetic background of an autosomal recessive chondrodysplasia that affects the Norwegian Elkhound and Karelian Bear Dog breeds. The affected dogs suffer from disproportionate short stature dwarfism of varying severity. Through a genome-wide approach, we mapped the chondrodysplasia locus to a 2-Mb region on canine chromosome 17 in nine affected and nine healthy Elkhounds (praw = 7.42×10−6, pgenome-wide = 0.013). The associated locus contained a promising candidate gene, cartilage specific integrin alpha 10 (ITGA10), and mutation screening of its 30 exons revealed a nonsense mutation in exon 16 (c.2083C>T; p.Arg695*) that segregated fully with the disease in both breeds (p = 2.5×10−23). A 24% mutation carrier frequency was indicated in NEs and an 8% frequency in KBDs. The ITGA10 gene product, integrin receptor α10-subunit combines into a collagen-binding α10β1 integrin receptor, which is expressed in cartilage chondrocytes and mediates chondrocyte-matrix interactions during endochondral ossification. As a consequence of the nonsense mutation, the α10-protein was not detected in the affected cartilage tissue. The canine phenotype highlights the importance of the α10β1 integrin in bone growth, and the large animal model could be utilized to further delineate its specific functions. Finally, this study revealed a candidate gene for human chondrodysplasias and enabled the development of a genetic test for breeding purposes to eradicate the disease from the two dog breeds. PMID:24086591

  3. α5β1-Integrin inhibitor (CLT-28643) effective in rabbit trabeculectomy model.

    PubMed

    Schultheiss, Maximilian; Schnichels, Sven; Konrad, Eva-Maria; Bartz-Schmidt, Karl U; Zahn, Grit; Caldirola, Patrizia; Fsadni, Mario G; Caram-Lelham, Ninus; Spitzer, Martin S

    2017-02-01

    Glaucoma filtration surgery (GFS) fails due to fibrosis. The α5β1-integrin plays a pivotal role in fibrosis, angiogenesis and inflammation. This is the first experiment evaluating the prevention of fibrosis after GFS by a specific small molecule α5β1-integrin inhibitor (CLT-28643). Twenty-four rabbits received trabeculectomy on their right eyes. The rabbits were randomized into three groups of eight eyes each. CLT-28643 was given as a single subconjunctival injection intraoperatively to two of the right eye groups followed by postoperative vehicle eye drops (CLT+ group) or CLT-28643 eye drops 4 times daily (CLT++ group). A third group received mitomycin-C (MMC) intraoperatively (sponge application, 0.04%, 2 min) followed by vehicle eye drops postoperatively. The control-surgery group consisted of 12 left eyes having trabeculectomy with no adjunctive therapy. The remaining 12 left eyes formed the untreated group. Clinical assessment included intraocular pressure (IOP) measurement, slit-lamp examination (including bleb survival and morphology) and bleb photography. The rabbits were killed after four weeks for histology. Both CLT-28643-treated groups showed significantly prolonged bleb survival, and better bleb score compared to the control-surgery group. At end of the study, most functioning blebs were found in the MMC group (MMC group 75%; CLT+ group 12.5%, CLT++ group 25%; CLT+ group 12.5%, control-surgery group 0%). CLT-28643 was non-toxic and well tolerated. This rabbit GFS study indicates that inhibition of α5β1-integrin by the novel α5β1-integrin antagonist CLT-28643 significantly improved the outcome. The effect of a single intro-operative application of CLT-28643 seems to be inferior to 0.04% MMC. © 2016 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

  4. Complementary roles of KCa3.1 channels and β1-integrin during alveolar epithelial repair.

    PubMed

    Girault, Alban; Chebli, Jasmine; Privé, Anik; Trinh, Nguyen Thu Ngan; Maillé, Emilie; Grygorczyk, Ryszard; Brochiero, Emmanuelle

    2015-09-04

    Extensive alveolar epithelial injury and remodelling is a common feature of acute lung injury and acute respiratory distress syndrome (ARDS) and it has been established that epithelial regeneration, and secondary lung oedema resorption, is crucial for ARDS resolution. Much evidence indicates that K(+) channels are regulating epithelial repair processes; however, involvement of the KCa3.1 channels in alveolar repair has never been investigated before. Wound-healing assays demonstrated that the repair rates were increased in primary rat alveolar cell monolayers grown on a fibronectin matrix compared to non-coated supports, whereas an anti-β1-integrin antibody reduced it. KCa3.1 inhibition/silencing impaired the fibronectin-stimulated wound-healing rates, as well as cell migration and proliferation, but had no effect in the absence of coating. We then evaluated a putative relationship between KCa3.1 channel and the migratory machinery protein β1-integrin, which is activated by fibronectin. Co-immunoprecipitation and immunofluorescence experiments indicated a link between the two proteins and revealed their cellular co-distribution. In addition, we demonstrated that KCa3.1 channel and β1-integrin membrane expressions were increased on a fibronectin matrix. We also showed increased intracellular calcium concentrations as well as enhanced expression of TRPC4, a voltage-independent calcium channel belonging to the large TRP channel family, on a fibronectin matrix. Finally, wound-healing assays showed additive effects of KCa3.1 and TRPC4 inhibitors on alveolar epithelial repair. Taken together, our data demonstrate for the first time complementary roles of KCa3.1 and TRPC4 channels with extracellular matrix and β1-integrin in the regulation of alveolar repair processes.

  5. Cell-free ligand-binding assays for integrin LFA-1.

    PubMed

    Yuki, Koichi

    2012-01-01

    Integrin LFA-1 plays an important role in leukocyte trafficking to inflammatory sites as well as the interaction of T cells with antigen-presenting cells. Inhibition of the LFA-1:ICAM-1 interactions is a promising therapeutic approach for alleviating inflammation and autoimmune diseases. This chapter describes cell-free assays to study the LFA-1:ICAM-1 interactions. These assays may be used for screening novel LFA-1 antagonists.

  6. Integrin-type signaling has a distinct influence on NMDA-induced cytoskeletal disassembly.

    PubMed

    Bahr, B A

    2000-03-15

    Adhesion responses triggered by integrin-class matrix receptors have been implicated in the synaptic reorganization events necessary for certain types of neuronal plasticity. Hippocampal slice cultures were used to test whether the related structural transformations elicited by NMDA receptor stimulation are regulated by integrin-type signals. Infusing the slices with NMDA for a short period induced the expected disassembly of the cytoskeletal network, measured with antibodies that selectively recognize spectrin cleavage sites targeted by the protease calpain. Marked levels of the 150-kDa breakdown product (BDP) were produced, whereas concentrations of the parent spectrin were not changed. Interestingly, the calpain cleavage events were attenuated by 60% when integrin-type signaling was disrupted with the antagonist Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP). This effect was RGDS-dependent, was largely evident in synapse-dense dendritic areas, particularly in subfield CA1, and was abolished when the NMDA exposure period was >5 min. These findings suggest that only those cytoskeletal alterations associated with brief synaptic activity are regulated by intact contact zones. AMPA-type glutamate receptors also were tested because, like spectrin, they are targets for calpain. Brief NMDA treatment caused a 15% loss of AMPA receptor GluR1 carboxytermini and this modification was augmented to 32% in the presence of GRGDSP. Thus, although blockage of matrix recognition signals decreased spectrin's susceptibility to disassembly, it increased the susceptibility of AMPA receptors to proteolysis. These data indicate that integrin-type signaling complexes are appropriately positioned to govern cytoskeletal reconfiguration while stabilizing the structural nature of AMPA receptors. Copyright 2000 Wiley-Liss, Inc.

  7. Expression of laminin 5, fibronectin, and epithelium-associated integrins in recurrent aphthous ulcers.

    PubMed

    Richards, D W; MacPhail, L A; Dekker, N; Greenspan, D; Greenspan, J S; Lozada-Nur, F; Regezi, J A

    1996-07-01

    Recurrent aphthous ulceration (RAU) is characterized by an ulcerated lesion that persists longer than traumatic ulcers of similar size. This delayed healing phase of the lesion was investigated for extracellular matrix components and matrix receptors (integrins). The hypothesis tested was that aphthous ulcers may lack key extracellular matrix components, or their receptors, that are necessary for the migration of marginal keratinocytes from the ulcer edge. We immunocytochemically stained biopsy specimens of RAUs and non-involved mucosal specimens from HIV+ and non-infected individuals to investigate the presence and distribution of molecules reported to be associated with reepithelialization of mucosal and cutaneous wounds. Fibronectin, laminin type 5 (kalinin), and integrin subunits beta 1, beta 4, alpha 6, and alpha v were consistently found at the margins of RAU, as they are in traumatic ulcers. The alpha 5 and beta 6 subunits were not always present. We also found alpha v in the intact stratified squamous epithelium adjacent to ulcers. Immunohistochemical stains showed distruption in the deposition of laminin 5 and an apparent lack of fibronectin at the edges of some ulcers. Although these tissue results do not determine which integrin subunits are paired with each other, they do show some alterations in their expression in RAU. Absence of one or more of these molecules at the migrating front may contribute to delayed epithelial regeneration. It is likely that the absence or inappropriate expression of keratinocyte integrins or their extracellular matrix receptors occurs after the causative factors (currently unknown) of the lesion are gone. The reason for the altered expression of these molecules may be related to the secretory products (including lymphokines and proteinases) of the lymphocytic infiltrate.

  8. Lectin cDNA and transgenic plants derived therefrom

    DOEpatents

    Raikhel, Natasha V.

    2000-10-03

    Transgenic plants containing cDNA encoding Gramineae lectin are described. The plants preferably contain cDNA coding for barley lectin and store the lectin in the leaves. The transgenic plants, particularly the leaves exhibit insecticidal and fungicidal properties.

  9. Production of homozygous transgenic rainbow trout with enhanced disease resistance

    USDA-ARS?s Scientific Manuscript database

    Previous studies conducted in our laboratory showed that transgenic medaka expressing cecropin B transgenes exhibited resistant characteristic to fish bacterial pathogens, Pseudomonas fluorescens and Vibrio anguillarum. To confirm whether antimicrobial peptide gene will also exhibit antibacterial an...

  10. An Epithelial Integrin Regulates the Amplitude of Protective Lung Interferon Responses against Multiple Respiratory Pathogens

    PubMed Central

    Van de Velde, Nicholas C.; Karlsson, Erik A.; Neale, Geoff; Vogel, Peter; Sharma, Shalini; Duan, Susu; Surman, Sherri L.; Jones, Bart G.; Johnson, Michael D. L.; Bosio, Catharine; Jolly, Lisa; Jenkins, R. Gisli; Hurwitz, Julia L.; Rosch, Jason W.; Sheppard, Dean; Thomas, Paul G.; Murray, Peter J.; Schultz-Cherry, Stacey

    2016-01-01

    The healthy lung maintains a steady state of immune readiness to rapidly respond to injury from invaders. Integrins are important for setting the parameters of this resting state, particularly the epithelial-restricted αVβ6 integrin, which is upregulated during injury. Once expressed, αVβ6 moderates acute lung injury (ALI) through as yet undefined molecular mechanisms. We show that the upregulation of β6 during influenza infection is involved in disease pathogenesis. β6-deficient mice (β6 KO) have increased survival during influenza infection likely due to the limited viral spread into the alveolar spaces leading to reduced ALI. Although the β6 KO have morphologically normal lungs, they harbor constitutively activated lung CD11b+ alveolar macrophages (AM) and elevated type I IFN signaling activity, which we traced to the loss of β6-activated transforming growth factor-β (TGF-β). Administration of exogenous TGF-β to β6 KO mice leads to reduced numbers of CD11b+ AMs, decreased type I IFN signaling activity and loss of the protective phenotype during influenza infection. Protection extended to other respiratory pathogens such as Sendai virus and bacterial pneumonia. Our studies demonstrate that the loss of one epithelial protein, αVβ6 integrin, can alter the lung microenvironment during both homeostasis and respiratory infection leading to reduced lung injury and improved survival. PMID:27505057

  11. A dual role for the βPS integrin myospheroid in mediating Drosophila embryonic macrophage migration

    PubMed Central

    Comber, Kate; Huelsmann, Sven; Evans, Iwan; Sánchez-Sánchez, Besaid J.; Chalmers, Andrew; Reuter, Rolf; Wood, Will; Martín-Bermudo, Maria D.

    2013-01-01

    Summary Throughout embryonic development, macrophages not only act as the first line of defence against infection but also help to sculpt organs and tissues of the embryo by removing dead cells and secreting extracellular matrix components. Key to their function is the ability of embryonic macrophages to migrate and disperse throughout the embryo. Despite these important developmental functions, little is known about the molecular mechanisms underlying embryonic macrophage migration in vivo. Integrins are key regulators of many of the adult macrophage responses, but their role in embryonic macrophages remains poorly characterized. Here, we have used Drosophila macrophages (haemocytes) as a model system to address the role of integrins during embryonic macrophage dispersal in vivo. We show that the main βPS integrin, myospheroid, affects haemocyte migration in two ways; by shaping the three-dimensional environment in which haemocytes migrate and by regulating the migration of haemocytes themselves. Live imaging revealed a requirement for myospheroid within haemocytes to coordinate the microtubule and actin dynamics, and to enable haemocyte developmental dispersal, contact repulsion and inflammatory migration towards wounds. PMID:23704353

  12. Maintenance of Stem Cell Niche Integrity by a Novel Activator of Integrin Signaling

    PubMed Central

    Lee, Joo Yeun; Chang, Karen T.

    2016-01-01

    Stem cells depend critically on the surrounding microenvironment, or niche, for their maintenance and self-renewal. While much is known about how the niche regulates stem cell self-renewal and differentiation, mechanisms for how the niche is maintained over time are not well understood. At the apical tip of the Drosophila testes, germline stem cells (GSCs) and somatic stem cells share a common niche formed by hub cells. Here we demonstrate that a novel protein named Shriveled (Shv) is necessary for the maintenance of hub/niche integrity. Depletion of Shv protein results in age-dependent deterioration of the hub structure and loss of GSCs, whereas upregulation of Shv preserves the niche during aging. We find Shv is a secreted protein that modulates DE-cadherin levels through extracellular activation of integrin signaling. Our work identifies Shv as a novel activator of integrin signaling and suggests a new integration model in which crosstalk between integrin and DE-cadherin in niche cells promote their own preservation by maintaining the niche architecture. PMID:27191715

  13. Activated integrin VLA-4 localizes to the lamellipodia and mediates T cell migration on VCAM-11

    PubMed Central

    Hyun, Young-Min; Chung, Hung-Li; McGrath, James L.; Waugh, Richard E.; Kim, Minsoo

    2009-01-01

    Lymphocyte migration from blood into lymphoid tissues or to sites of inflammation occurs through interactions between cell surface integrins and their ligands expressed on the vascular endothelium and the extracellular matrix. Very Late Antigen-4 (VLA-4, α4β1) is a key integrin in the effective trafficking of lymphocytes. Although it has been well established that integrins undergo functionally significant conformational changes to mediate cell adhesion, there is no mechanistic information that explains how these are dynamically and spatially regulated during lymphocyte polarization and migration. Using dynamic fluorescence resonance energy transfer (FRET) analysis of a novel VLA-4 FRET sensor under total internal reflection fluorescence (TIRF) microscopy, we show that VLA-4 activation localizes to the lamellipodium in living cells. During T cell migration on VCAM-1, VLA-4 activation concurs with spatial redistribution of chemokine receptor and active Rap1 at the leading edge. Selective inhibition of the activated VLA-4 at leading edge with a small molecule inhibitor is sufficient to block T cell migration. These data suggest that a subpopulation of activated VLA-4 is mainly localized to the leading edge of polarized human T cells, and is critical for T cell migration on VCAM-1. PMID:19542447

  14. Crystal Structure of the Extracellular Segment of Integrin αVβ3

    PubMed Central

    Xiong, Jian-Ping; Stehle, Thilo; Diefenbach, Beate; Zhang, Rongguang; Dunker, Reinhardt; Scott, David L.; Joachimiak, Andrzej; Goodman, Simon L.; Arnaout, M. Amin

    2010-01-01

    Integrins are αβ heterodimeric receptors that mediate divalent cation-dependent cell-cell and cell-matrix adhesion through tightly regulated interactions with ligands. We have solved the crystal structure of the extracellular portion of integrin αVβ3 at 3.1 Å resolution. Its 12 domains assemble into an ovoid “head” and two “tails.” In the crystal, αVβ3 is severely bent at a defined region in its tails, reflecting an unusual flexibility that may be linked to integrin regulation. The main inter-subunit interface lies within the head, between a seven-bladed β-propeller from αV and an A domain from β3, and bears a striking resemblance to the Gα/Gβ interface in G proteins. A metal ion–dependent adhesion site (MIDAS) in the βA domain is positioned to participate in a ligand-binding interface formed of loops from the propeller and βA domains. MIDAS lies adjacent to a calcium-binding site with a potential regulatory function. PMID:11546839

  15. β2 integrin mediates hantavirus-induced release of neutrophil extracellular traps

    PubMed Central

    Raftery, Martin J.; Lalwani, Pritesh; Krautkrӓmer, Ellen; Peters, Thorsten; Scharffetter-Kochanek, Karin; Krüger, Renate; Hofmann, Jörg; Seeger, Karl; Krüger, Detlev H.

    2014-01-01

    Rodent-borne hantaviruses are emerging human pathogens that cause severe human disease. The underlying mechanisms are not well understood, as hantaviruses replicate in endothelial and epithelial cells without causing any cytopathic effect. We demonstrate that hantaviruses strongly stimulated neutrophils to release neutrophil extracellular traps (NETs). Hantavirus infection induced high systemic levels of circulating NETs in patients and this systemic NET overflow was accompanied by production of autoantibodies to nuclear antigens. Analysis of the responsible mechanism using neutrophils from β2 null mice identified β2 integrin receptors as a master switch for NET induction. Further experiments suggested that β2 integrin receptors such as complement receptor 3 (CR3) and 4 (CR4) may act as novel hantavirus entry receptors. Using adenoviruses, we confirmed that viral interaction with β2 integrin induced strong NET formation. Collectively, β2 integrin–mediated systemic NET overflow is a novel viral mechanism of immunopathology that may be responsible for characteristic aspects of hantavirus-associated disease such as kidney and lung damage. PMID:24889201

  16. Drosophila PS2 and PS3 integrins play distinct roles in retinal photoreceptors-glia interactions.

    PubMed

    Tavares, Lígia; Pereira, Emiliana; Correia, Andreia; Santos, Marília A; Amaral, Nuno; Martins, Torcato; Relvas, João B; Pereira, Paulo S

    2015-07-01

    Cellular migration and differentiation are important developmental processes that require dynamic cellular adhesion. Integrins are heterodimeric transmembrane receptors that play key roles in adhesion plasticity. Here, we explore the developing visual system of Drosophila to study the roles of integrin heterodimers in glia development. Our data show that αPS2 is essential for retinal glia migration from the brain into the eye disc and that glial cells have a role in the maintenance of the fenestrated membrane (Laminin-rich ECM layer) in the disc. Interestingly, the absence of glial cells in the eye disc did not affect the targeting of retinal axons to the optic stalk. In contrast, αPS3 is not required for retinal glia migration, but together with Talin, it functions in glial cells to allow photoreceptor axons to target the optic stalk. Thus, we present evidence that αPS2 and αPS3 integrin have different and specific functions in the development of retinal glia. © 2015 Wiley Periodicals, Inc.

  17. Bovine leukocyte adhesion deficiency: in vitro assessment of neutrophil function and leukocyte integrin expression.

    PubMed Central

    Olchowy, T W; Bochsler, P N; Neilsen, N R; Welborn, M G; Slauson, D O

    1994-01-01

    Bovine leukocyte adhesion deficiency (BLAD) was identified in a two-month-old Holstein heifer calf using DNA-polymerase chain reaction analysis of the affected calf and other clinical parameters. Neutrophil integrin expression (CD18, CD11a, CD11c), aggregation, and transendothelial migration were studied in vitro. Neutrophils were isolated from the affected calf and from normal, healthy, age-matched control Holstein calves. Neutrophils isolated from the affected BLAD calf had decreased expression of leukocyte integrins on their cell surface, decreased ability to aggregate in response to chemotactic stimuli, and decreased ability to migrate across bovine endothelial cell monolayers in vitro. Transendothelial migration of neutrophils from normal calves was reduced to levels comparable to the BLAD neutrophils by treatment with an anti-CD18 monoclonal antibody (MAb 60.3). Peripheral-blood lymphocytes from the BLAD calf also expressed negligible levels of leukocyte integrins, similar to their neutrophil counterparts. Our experimental findings in vitro correlate well with the clinical observations of decreased leukocyte trafficking and diminished host defense in leukocyte adhesion-deficient animals. The syndrome of BLAD may be a suitable model for one of the human leukocyte adhesion deficiency disorders. Images Fig. 4. PMID:7911733

  18. Integrin-regulated Secretion of Interleukin 4: A Novel Pathway of Mechanotransduction in Human Articular Chondrocytes

    PubMed Central

    Millward-Sadler, S.J.; Wright, M.O.; Lee, H.-S.; Nishida, K.; Caldwell, H.; Nuki, G.; Salter, D.M.

    1999-01-01

    Chondrocyte function is regulated partly by mechanical stimulation. Optimal mechanical stimulation maintains articular cartilage integrity, whereas abnormal mechanical stimulation results in development and progression of osteoarthritis (OA). The responses of signal transduction pathways in human articular chondrocytes (HAC) to mechanical stimuli remain unclear. Previous work has shown the involvement of integrins and integrin-associated signaling pathways in activation of plasma membrane apamin-sensitive Ca2+-activated K+ channels that results in membrane hyperpolarization of HAC after 0.33 Hz cyclical mechanical stimulation. To further investigate mechanotransduction pathways in HAC and show that the hyperpolarization response to mechanical stimulation is a result of an integrin-dependent release of a transferable secreted factor, we used this response. Neutralizing antibodies to interleukin 4 (IL-4) and IL-4 receptor α inhibit mechanically induced membrane hyperpolarization and anti–IL-4 antibodies neutralize the hyperpolarizing activity of medium from mechanically stimulated cells. Antibodies to interleukin 1β (IL-1β) and cytokine receptors, interleukin 1 receptor type I and the common γ chain/CD132 (γ) have no effect on me- chanically induced membrane hyperpolarization. Chondrocytes from IL-4 knockout mice fail to show a membrane hyperpolarization response to cyclical mechanical stimulation. Mechanically induced release of the chondroprotective cytokine IL-4 from HAC with subsequent autocrine/paracrine activity is likely to be an important regulatory pathway in the maintenance of articular cartilage structure and function. Finally, dysfunction of this pathway may be implicated in OA. PMID:10189377

  19. Adenylate Cyclase Toxin Promotes Internalisation of Integrins and Raft Components and Decreases Macrophage Adhesion Capacity

    PubMed Central

    Gómez-Bilbao, Geraxane; Ostolaza, Helena

    2011-01-01

    Bordetella pertussis, the bacterium that causes whooping cough, secretes an adenylate cyclase toxin (ACT) that must be post-translationally palmitoylated in the bacterium cytosol to be active. The toxin targets phagocytes expressing the CD11b/CD18 integrin receptor. It delivers a catalytic adenylate cyclase domain into the target cell cytosol producing a rapid increase of intracellular cAMP concentration that suppresses bactericidal functions of the phagocyte. ACT also induces calcium fluxes into target cells. Biochemical, biophysical and cell biology approaches have been applied here to show evidence that ACT and integrin molecules, along with other raft components, are rapidly internalized by the macrophages in a toxin-induced calcium rise-dependent process. The toxin-triggered internalisation events occur through two different routes of entry, chlorpromazine-sensitive receptor-mediated endocytosis and clathrin-independent internalisation, maybe acting in parallel. ACT locates into raft-like domains, and is internalised, also in cells devoid of receptor. Altogether our results suggest that adenylate cyclase toxin, and maybe other homologous pathogenic toxins from the RTX (Repeats in Toxin) family to which ACT belongs, may be endowed with an intrinsic capacity to, directly and efficiently, insert into raft-like domains, promoting there its multiple activities. One direct consequence of the integrin removal from the cell surface of the macrophages is the hampering of their adhesion ability, a fundamental property in the immune response of the leukocytes that could be instrumental in the pathogenesis of Bordetella pertussis. PMID:21383852

  20. Adenylate cyclase toxin promotes internalisation of integrins and raft components and decreases macrophage adhesion capacity.

    PubMed

    Martín, César; Uribe, Kepa B; Gómez-Bilbao, Geraxane; Ostolaza, Helena

    2011-02-23

    Bordetella pertussis, the bacterium that causes whooping cough, secretes an adenylate cyclase toxin (ACT) that must be post-translationally palmitoylated in the bacterium cytosol to be active. The toxin targets phagocytes expressing the CD11b/CD18 integrin receptor. It delivers a catalytic adenylate cyclase domain into the target cell cytosol producing a rapid increase of intracellular cAMP concentration that suppresses bactericidal functions of the phagocyte. ACT also induces calcium fluxes into target cells. Biochemical, biophysical and cell biology approaches have been applied here to show evidence that ACT and integrin molecules, along with other raft components, are rapidly internalized by the macrophages in a toxin-induced calcium rise-dependent process. The toxin-triggered internalisation events occur through two different routes of entry, chlorpromazine-sensitive receptor-mediated endocytosis and clathrin-independent internalisation, maybe acting in parallel. ACT locates into raft-like domains, and is internalised, also in cells devoid of receptor. Altogether our results suggest that adenylate cyclase toxin, and maybe other homologous pathogenic toxins from the RTX (Repeats in Toxin) family to which ACT belongs, may be endowed with an intrinsic capacity to, directly and efficiently, insert into raft-like domains, promoting there its multiple activities. One direct consequence of the integrin removal from the cell surface of the macrophages is the hampering of their adhesion ability, a fundamental property in the immune response of the leukocytes that could be instrumental in the pathogenesis of Bordetella pertussis.

  1. Demonstration of mechanical connections between integrins, cytoskeletal filaments, and nucleoplasm that stabilize nuclear structure

    NASA Technical Reports Server (NTRS)

    Maniotis, A. J.; Chen, C. S.; Ingber, D. E.

    1997-01-01

    We report here that living cells and nuclei are hard-wired such that a mechanical tug on cell surface receptors can immediately change the organization of molecular assemblies in the cytoplasm and nucleus. When integrins were pulled by micromanipulating bound microbeads or micropipettes, cytoskeletal filaments reoriented, nuclei distorted, and nucleoli redistributed along the axis of the applied tension field. These effects were specific for integrins, independent of cortical membrane distortion, and were mediated by direct linkages between the cytoskeleton and nucleus. Actin microfilaments mediated force transfer to the nucleus at low strain; however, tearing of the actin gel resulted with greater distortion. In contrast, intermediate filaments effectively mediated force transfer to the nucleus under both conditions. These filament systems also acted as molecular guy wires to mechanically stiffen the nucleus and anchor it in place, whereas microtubules acted to hold open the intermediate filament lattice and to stabilize the nucleus against lateral compression. Molecular connections between integrins, cytoskeletal filaments, and nuclear scaffolds may therefore provide a discrete path for mechanical signal transfer through cells as well as a mechanism for producing integrated changes in cell and nuclear structure in response to changes in extracellular matrix adhesivity or mechanics.

  2. Prevention of experimental autoimmune encephalomyelitis by antibodies against α4βl integrin

    NASA Astrophysics Data System (ADS)

    Yednock, Ted A.; Cannon, Catherine; Fritz, Lawrence C.; Sanchez-Madrid, Francisco; Steinman, Lawrence; Karin, Nathan

    1992-03-01

    EXPERIMENTAL autoimmune encephalomyelitis (EAE) is an inflammatory condition of the central nervous system with similarities to multiple sclerosis1,2. In both diseases, circulating leukocytes penetrate the blood-brain barrier and damage myelin, resulting in impaired nerve conduction and paralysis3-5. We sought to identify the adhesion receptors that mediate the attachment of circulating leukocytes to inflamed brain endothelium in EAE, because this interaction is the first step in leukocyte entry into the central nervous system. Using an in vitro adhesion assay on tissue sections, we found that lymphocytes and monocytes bound selectively to inflamed EAE brain vessels. Binding was inhibited by antibodies against the integrin molecule α4βl, but not by antibodies against numerous other adhesion receptors. When tested in vivo, anti-α4 integrin effectively prevented the accumulation of leukocytes in the central nervous system and the development of EAE. Thus, therapies designed to interfere with α4βl integrin may be useful in treating inflammatory diseases of the central nervous system, such as multiple sclerosis.

  3. Molecular basis of CIB binding to the integrin alpha IIb cytoplasmic domain.

    PubMed

    Barry, William T; Boudignon-Proudhon, Christel; Shock, David D; McFadden, Andrew; Weiss, Jonathan M; Sondek, John; Parise, Leslie V

    2002-08-09

    Integrin adhesion receptors appear to be regulated by molecules that bind to their cytoplasmic domains. We previously identified a 22-kDa, EF-hand-containing protein, CIB, which binds to the alpha(IIb) cytoplasmic tail of the platelet integrin, alpha(IIb)beta(3). Here we describe regions within CIB and alpha(IIb) that interact with one another. CIB binding to alpha(IIb) cytoplasmic tail peptides, as measured by intrinsic tryptophan fluorescence, indicates a CIB-binding site within a hydrophobic, 15-amino acid, membrane-proximal region of alpha(IIb). This region is analogous to the alpha-helical targets of other EF-hand-containing proteins, such as calcineurin B or calmodulin. A homology model of CIB based upon calcineurin B and recoverin indicated a conserved hydrophobic pocket within the C-terminal EF-hand motifs of CIB as a potential integrin-binding site. CIB engineered to contain alanine substitutions in the implicated regions retained wild type secondary structure as determined by circular dichroism, yet failed to bind alpha(IIb) in 11 of 12 cases, whereas CIB mutated within the N terminus retained binding activity. Thus, specific hydrophobic residues in the C terminus of CIB appear necessary for CIB binding to alpha(IIb). The identification of essential interacting regions within alpha(IIb) and CIB provides tools for further probing potential interrelated functions of these proteins.

  4. Multiple roles of integrin-α3 at the neuromuscular junction

    PubMed Central

    Ross, Jacob A.; Webster, Richard G.; Lechertier, Tanguy; Reynolds, Louise E.; Turmaine, Mark; Bencze, Maximilien; Jamshidi, Yalda; Cetin, Hakan; Muntoni, Francesco; Beeson, David; Hodilvala-Dilke, Kairbaan

    2017-01-01

    ABSTRACT The neuromuscular junction (NMJ) is the synapse between motoneurons and skeletal muscle, and is responsible for eliciting muscle contraction. Neurotransmission at synapses depends on the release of synaptic vesicles at sites called active zones (AZs). Various proteins of the extracellular matrix are crucial for NMJ development; however, little is known about the identity and functions of the receptors that mediate their effects. Using genetically modified mice, we find that integrin-α3 (encoded by Itga3), an adhesion receptor at the presynaptic membrane, is involved in the localisation of AZ components and efficient synaptic vesicle release. Integrin-α3 also regulates integrity of the synapse – mutant NMJs present with progressive structural changes and upregulated autophagy, features commonly observed during ageing and in models of neurodegeneration. Unexpectedly, we find instances of nerve terminal detachment from the muscle fibre; to our knowledge, this is the first report of a receptor that is required for the physical anchorage of pre- and postsynaptic elements at the NMJ. These results demonstrate multiple roles of integrin-α3 at the NMJ, and suggest that alterations in its function could underlie defects that occur in neurodegeneration or ageing. PMID:28386022

  5. Identification of Equine Lactadherin-derived Peptides That Inhibit Rotavirus Infection via Integrin Receptor Competition*

    PubMed Central

    Civra, Andrea; Giuffrida, Maria Gabriella; Donalisio, Manuela; Napolitano, Lorenzo; Takada, Yoshikazu; Coulson, Barbara S.; Conti, Amedeo; Lembo, David

    2015-01-01

    Human rotavirus is the leading cause of severe gastroenteritis in infants and children under the age of 5 years in both developed and developing countries. Human lactadherin, a milk fat globule membrane glycoprotein, inhibits human rotavirus infection in vitro, whereas bovine lactadherin is not active. Moreover, it protects breastfed infants against symptomatic rotavirus infections. To explore the potential antiviral activity of lactadherin sourced by equines, we undertook a proteomic analysis of milk fat globule membrane proteins from donkey milk and elucidated its amino acid sequence. Alignment of the human, bovine, and donkey lactadherin sequences revealed the presence of an Asp-Gly-Glu (DGE) α2β1 integrin-binding motif in the N-terminal domain of donkey sequence only. Because integrin α2β1 plays a critical role during early steps of rotavirus host cell adhesion, we tested a minilibrary of donkey lactadherin-derived peptides containing DGE sequence for anti-rotavirus activity. A 20-amino acid peptide containing both DGE and RGD motifs (named pDGE-RGD) showed the greatest activity, and its mechanism of antiviral action was characterized; pDGE-RGD binds to integrin α2β1 by means of the DGE motif and inhibits rotavirus attachment to the cell surface. These findings suggest the potential anti-rotavirus activity of equine lactadherin and support the feasibility of developing an anti-rotavirus peptide that acts by hindering virus-receptor binding. PMID:25814665

  6. Structural specializations of α4β7, an integrin that mediates rolling adhesion

    PubMed Central

    Yu, Yamei; Zhu, Jianghai; Mi, Li-Zhi; Walz, Thomas; Sun, Hao; Chen, JianFeng

    2012-01-01

    The lymphocyte homing receptor integrin α4β7 is unusual for its ability to mediate both rolling and firm adhesion. α4β1 and α4β7 are targeted by therapeutics approved for multiple sclerosis and Crohn’s disease. Here, we show by electron microscopy and crystallography how two therapeutic Fabs, a small molecule (RO0505376), and mucosal adhesion molecule-1 (MAdCAM-1) bind α4β7. A long binding groove at the α4–β7 interface for immunoglobulin superfamily domains differs in shape from integrin pockets that bind Arg-Gly-Asp motifs. RO0505376 mimics an Ile/Leu-Asp motif in α4 ligands, and orients differently from Arg-Gly-Asp mimics. A novel auxiliary residue at the metal ion–dependent adhesion site in α4β7 is essential for binding to MAdCAM-1 in Mg2+ yet swings away when RO0505376 binds. A novel intermediate conformation of the α4β7 headpiece binds MAdCAM-1 and supports rolling adhesion. Lack of induction of the open headpiece conformation by ligand binding enables rolling adhesion to persist until integrin activation is signaled. PMID:22232704

  7. JAK tyrosine kinases promote hierarchical activation of Rho and Rap modules of integrin activation.

    PubMed

    Montresor, Alessio; Bolomini-Vittori, Matteo; Toffali, Lara; Rossi, Barbara; Constantin, Gabriela; Laudanna, Carlo

    2013-12-23

    Lymphocyte recruitment is regulated by signaling modules based on the activity of Rho and Rap small guanosine triphosphatases that control integrin activation by chemokines. We show that Janus kinase (JAK) protein tyrosine kinases control chemokine-induced LFA-1- and VLA-4-mediated adhesion as well as human T lymphocyte homing to secondary lymphoid organs. JAK2 and JAK3 isoforms, but not JAK1, mediate CXCL12-induced LFA-1 triggering to a high affinity state. Signal transduction analysis showed that chemokine-induced activation of the Rho module of LFA-1 affinity triggering is dependent on JAK activity, with VAV1 mediating Rho activation by JAKs in a Gαi-independent manner. Furthermore, activation of Rap1A by chemokines is also dependent on JAK2 and JAK3 activity. Importantly, activation of Rap1A by JAKs is mediated by RhoA and PLD1, thus establishing Rap1A as a downstream effector of the Rho module. Thus, JAK tyrosine kinases control integrin activation and dependent lymphocyte trafficking by bridging chemokine receptors to the concurrent and hierarchical activation of the Rho and Rap modules of integrin activation.

  8. Tetraspanin CD151 Stimulates Adhesion-dependent Activation of Ras, Rac, and Cdc42 by Facilitating Molecular Association between β1 Integrins and Small GTPases*

    PubMed Central

    Hong, In-Kee; Jeoung, Doo-Il; Ha, Kwon-Soo; Kim, Young-Myeong; Lee, Hansoo

    2012-01-01

    Tetraspanin CD151 associates with laminin-binding α3β1/α6β1 integrins in epithelial cells and regulates adhesion-dependent signaling events. We found here that CD151 plays a role in recruiting Ras, Rac1, and Cdc42, but not Rho, to the cell membrane region, leading to the formation of α3β1/α6β1 integrin-CD151-GTPases complexes. Furthermore, cell adhesion to laminin enhanced CD151 association with β1 integrin and, thereby, increased complex formation between the β1 family of integrins and small GTPases, Ras, Rac1, and Cdc42. Adhesion receptor complex-associated small GTPases were activated by CD151-β1 integrin complex-stimulating adhesion events, such as α3β1/α6β1 integrin-activating cell-to-laminin adhesion and homophilic CD151 interaction-generating cell-to-cell adhesion. Additionally, FAK and Src appeared to participate in this adhesion-dependent activation of small GTPases. However, engagement of laminin-binding integrins in CD151-deficient cells or CD151-specific siRNA-transfected cells did not activate these GTPases to the level of cells expressing CD151. Small GTPases activated by engagement of CD151-β1 integrin complexes contributed to CD151-induced cell motility and MMP-9 expression in human melanoma cells. Importantly, among the four tetraspanin proteins that associate with β1 integrin, only CD151 exhibited the ability to facilitate complex formation between the β1 family of integrins and small GTPases and stimulate β1 integrin-dependent activation of small GTPases. These results suggest that CD151 links α3β1/α6β1 integrins to Ras, Rac1, and Cdc42 by promoting the formation of multimolecular complexes in the membrane, which leads to the up-regulation of adhesion-dependent small GTPase activation. PMID:22843693

  9. IDENTIFICATION OF ESCAPED TRANSGENIC CREEPING BENTGRASS IN OREGON

    EPA Science Inventory

    When transgenic plants are cultivated near wild species that are sexually compatible with the crop, gene flow between the crop and wild plants is possible. A resultant concern is that transgene flow and transgene introgression within wild populations could have unintended ecologi...

  10. Maize transgenes containing zein promoters are regulated by opaque2

    USDA-ARS?s Scientific Manuscript database

    Transgenes have great potential in crop improvement, but relatively little is known about the epistatic interaction of transgenes with the native genes in the genome. Understanding these interactions is critical for predicting the response of transgenes to different genetic backgrounds and environm...

  11. B-Raf regulation of integrin α4β1-mediated resistance to shear stress through changes in cell spreading and cytoskeletal association in T cells.

    PubMed

    Brown, Wells S; Khalili, Jahan S; Rodriguez-Cruz, Tania G; Lizee, Greg; McIntyre, Bradley W

    2014-08-15

    The regulation of integrin-mediated adhesion is of vital importance to adaptive and innate immunity. Integrins are versatile proteins and mediate T cell migration and trafficking by binding to extracellular matrix or other cells as well as initiating intracellular signaling cascades promoting survival or activation. The MAPK pathway is known to be downstream from integrins and to regulate survival, differentiation, and motility. However, secondary roles for canonical MAPK pathway members are being discovered. We show that chemical inhibition of RAF by sorafenib or shRNA-mediated knockdown of B-Raf reduces T cell resistance to shear stress to α4β1 integrin ligands vascular cell adhesion molecule 1 (VCAM-1) and fibronectin, whereas inhibition of MEK/ERK by U0126 had no effect. Microscopy showed that RAF inhibition leads to significant inhibition of T cell spreading on VCAM-1. The association of α4β1 integrin with the actin cytoskeleton was shown to be dependent on B-Raf activity or expression, whereas α4β1 integrin affinity for soluble VCAM-1 was not. These effects were shown to be specific for α4β1 integrin and not other integrins, such as α5β1 or LFA-1, or a variety of membrane proteins. We demonstrate a novel role for B-Raf in the selective regulation of α4β1 integrin-mediated adhesion. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Expression of Integrin αvβ3 in Gliomas Correlates with Tumor Grade and Is not Restricted to Tumor Vasculature

    PubMed Central

    Schnell, Oliver; Krebs, Bjarne; Wagner, Erika; Romagna, Alexander; Beer, Ambros J; Grau, Stefan J; Thon, Niklas; Goetz, Claudia; Kretzschmar, Hans A; Tonn, Jörg-Christian; Goldbrunner, Roland H

    2008-01-01

    In malignant gliomas, the integrin adhesion receptors seem to play a key role for invasive growth and angiogenesis. However, there is still a controversy about the expression and the distribution of αvβ3 integrin caused by malignancy. The aim of our study was to assess the extent and pattern of αvβ3 integrin expression within primary glioblastomas (GBMs) compared with low-grade gliomas (LGGs). Tumor samples were immunostained for the detection of αvβ3 integrin and quantified by an imaging software. The expression of αvβ3 was found to be significantly higher in GBMs than in LGGs, whereby focal strong reactivity was restricted to GBMs only. Subsequent analysis revealed that not only endothelial cells but also, to a large extent, glial tumor cells contribute to the overall amount of αvβ3 integrin in the tumors. To further analyze the integrin subunits, Western blots from histologic sections were performed, which demonstrated a significant difference in the expression of the β3 integrin subunit between GBMs and LGGs. The presented data lead to new insights in the pattern of αvβ3 integrin in gliomas and are of relevance for the inhibition of αvβ3 integrin with specific RGD peptides and interfering drugs to reduce angiogenesis and tumor growth. PMID:18394009

  13. β3 integrin promotes chemoresistance to epirubicin in MDA-MB-231 through repression of the pro-apoptotic protein, BAD

    SciTech Connect

    Nair, Madhumathy G.; Desai, Krisha; Prabhu, Jyothi S.

    2016-08-01

    Resistance to anthracycline based chemotherapy is a major limitation in the treatment of breast cancer, particularly of the triple negative sub-type that lacks targeted therapies. Resistance that arises from tumor-stromal interaction facilitated by integrins provides the possibility of targeted disruption. In the present study, we demonstrate that integrin β3 signaling inhibits apoptosis induced by a DNA-damaging chemotherapeutic agent, epirubicin, in MDA-MB-231 breast cancer cells. Drug efflux based mechanisms do not contribute to this effect. We show that integrin β3 employs the PI3K-Akt and the MAPK pathway for enabling cell survival and proliferation. Further, our results indicate that integrin β3 helpsmore » inhibit epirubicin induced cytotoxicity by repression of the pro-apoptotic protein BAD, thus promoting an anti-apoptotic response. Myristoylated RGT peptide and a monoclonal antibody against integrin β3 brought about a reversal of this effect and chemosensitized the cells. These results identify β3 integrin signaling via repression of BAD as an important survival pathway used by breast cancer cells to evade chemotherapy induced stress. - Highlights: • Integrin β3 signaling promotes chemoresistance to epirubicin in breast cancer cells. • Integrin β3 promotes cell survival and proliferation in drug treated cells through the PI3K and MAPK pathways. • Integrin signaling helps evade drug induced cytotoxicity by repression of pro-apoptotic molecule; BAD.« less

  14. The effect of conditional inactivation of beta 1 integrins using twist 2 Cre, Osterix Cre and osteocalcin Cre lines on skeletal phenotype.

    PubMed

    Shekaran, Asha; Shoemaker, James T; Kavanaugh, Taylor E; Lin, Angela S; LaPlaca, Michelle C; Fan, Yuhong; Guldberg, Robert E; García, Andrés J

    2014-11-01

    Skeletal development and growth are complex processes regulated by multiple microenvironmental cues, including integrin-ECM interactions. The β1 sub-family of integrins is the largest integrin sub-family and constitutes the main integrin binding partners of collagen I, the major ECM component of bone. As complete β1 integrin knockout results in embryonic lethality, studies of β1 integrin function in vivo rely on tissue-specific gene deletions. While multiple in vitro studies indicate that β1 integrins are crucial regulators of osteogenesis and mineralization, in vivo osteoblast-specific perturbations of β1 integrins have resulted in mild and sometimes contradictory skeletal phenotypes. To further investigate the role of β1 integrins on skeletal phenotype, we used the Twist2-Cre, Osterix-Cre and osteocalcin-Cre lines to generate conditional β1 integrin deletions, where Cre is expressed primarily in mesenchymal condensation, pre-osteoblast, and mature osteoblast lineage cells respectively within these lines. Mice with Twist2-specific β1 integrin disruption were smaller, had impaired skeletal development, especially in the craniofacial and vertebral tissues at E19.5, and did not survive beyond birth. Osterix-specific β1 integrin deficiency resulted in viable mice which were normal at birth but displayed early defects in calvarial ossification, incisor eruption and growth as well as femoral bone mineral density, structure, and mechanical properties. Although these defects persisted into adulthood, they became milder with age. Finally, a lack of β1 integrins in mature osteoblasts and osteocytes resulted in minor alterations to femur structure but had no effect on mineral density, biomechanics or fracture healing. Taken together, our data indicate that β1 integrin expression in early mesenchymal condensations play an important role in skeletal ossification, while β1 integrin-ECM interactions in pre-osteoblast, odontoblast- and hypertrophic chondryocyte

  15. Antimetastatic Action of Pentoxifylline, a Methyl Xanthine Derivative, Through its Effect on PKC Mediated Integrin Transport in B16F10 Melanoma Cells.

    PubMed

    Ratheesh, Aparna; Jain, Meenakashi; Gude, Rajiv P

    2010-10-01

    Integrins are adhesion molecules known to regulate cellular processes like adhesion, migration and proliferation. At the same time role of integrin in progress of cancer metastasis is well established, increased integrin expression is reported to be linked to high metastasis potential of cells. Pentoxifylline a methyl xanthine derivative is a potent antimetastatic agent. Studies on the mechanism of inhibition of lung homing of B16F10 melanoma cells by PTX shows that it can inhibit cell- Extracellular Matrix adhesion, cell surface integrin expression as well as Protein kinase C activity. Previous study from our laboratory have shown PTX treatment can selectively inhibit the cell surface expression of α5 integrin in B16F10 cells without affecting its total cellular protein levels. Numerous studies have documented that differences in surface expression and distribution of integrins affects metastasis. The purpose of present study is to observe the effect of PTX on cellular distribution/ redistribution of integrins and to study the underlying molecular mechanism of PTX action. Integrin internalization and transport was observed using immunofluorescence confocal microscopy. PKC activity was determined using MBP4-14 as a substrate. Immunoprecipitation and western blotting was used to show association between PKC and α5 integrin, cell adhesion assay was performed using fibronectin/fibrinogen as substrate. Immunofluorescence studies showed that PTX treatment caused a redistribution of α5 integrins from the plasma membrane to a perinuclear compartment where it colocalized with Transferrin receptor and Rab-11 GTPase. Rate of integrin internalization and recycling showed that PTX inhibited the recycling of α5 integrins from perinuclear recycling endosomes. PTX is reported to affect kinases; here we showed that PTX inhibited total PKC activity. Association between α5β1 integrin and PKC is studied using Immunoprecipitation which show that PTX affects α5β1 integrin

  16. Bactrian camel (Camelus bactrianus) integrins alphavbeta3 and alphavbeta6 as FMDV receptors: molecular cloning, sequence analysis and comparison with other species.

    PubMed

    Du, Junzheng; Gao, Shandian; Chang, Huiyun; Cong, Guozheng; Lin, Tong; Shao, Junjun; Liu, Zaixin; Liu, Xiangtao; Cai, Xuepeng

    2009-10-15

    Integrins are heterodimeric adhesion receptors that participate in a variety of cell-cell and cell-extracellular matrix protein interactions. Many integrins recognize RGD sequences displayed on extracellular matrix proteins and the exposed loops of viral capsid proteins. Four members of the alphav integrin family of cellular receptors, alphavbeta3, alphavbeta6, alphavbeta1 and alphavbeta8, have been identified as receptors for foot-and-mouth disease virus (FMDV) in vitro, and integrins are believed to be the receptors used to target epithelial cells in the infected animals. To analyse the roles of the alphav integrins from a susceptible species as viral receptors, we have cloned Bactrian camel alphav, beta3 and beta6 integrin cDNAs and compared them to those of other species. The coding sequences for Bactrian camel integrin alphav, beta3 and beta6 were found to be 3165, 2289 and 2367 nucleotides in length, encoding 1054, 762 and 788 amino acids, respectively. The Bactrian camel alphav, beta3 and beta6 subunits share many structural features with homologues of other species, including the ligand binding domain and cysteine-rich region. Phylogenetic trees and similarity analyses showed the close relationships of integrin genes from Bactrian camels, pigs and cattle, which are each susceptible to FMDV infection, that were distinct from the orders Rodentia, Primates, Perissodactyla, Carnivora, Galliformes and Xenopus. We postulate that host tropism of FMDV may in part be related to the divergence in integrin subunits among different species.

  17. Monitoring transgenic plants using in vivo markers

    SciTech Connect

    Stewart, C.N. Jr.

    1996-06-01

    The gene coding for green fluorecent protein (GFP), isolated and cloned from the jellyfish Aequorea victoria, is an ideal transgene for the monitoring of any plant species. It has the ability to fluoresce without added substrate, enzyme, or cofactor; it does not introduce morphological or sexual aberrations when expressed. 7 refs., 1 fig.

  18. Transgenic plants protected from insect attack

    NASA Astrophysics Data System (ADS)

    Vaeck, Mark; Reynaerts, Arlette; Höfte, Herman; Jansens, Stefan; de Beuckeleer, Marc; Dean, Caroline; Zabeau, Marc; Montagu, Marc Van; Leemans, Jan

    1987-07-01

    The Gram-positive bacterium Bacillus thuringiensis produces proteins which are specifically toxic to a variety of insect species. Modified genes have been derived from bt2, a toxin gene cloned from one Bacillus strain. Transgenic tobacco plants expressing these genes synthesize insecticidal proteins which protect them from feeding damage by larvae of the tobacco hornworm.

  19. Transgenic mouse models and prion strains.

    PubMed

    Telling, Glenn C

    2011-01-01

    Here we review the known strain profiles of various prion diseases of animals and humans, and how transgenic mouse models are being used to elucidate basic molecular mechanisms of prion propagation and strain variation and for assessing the zoonotic potential of various animal prion strains.

  20. Viable transgenic goats derived from skin cells.

    PubMed

    Behboodi, Esmail; Memili, Erdogan; Melican, David T; Destrempes, Margaret M; Overton, Susan A; Williams, Jennifer L; Flanagan, Peter A; Butler, Robin E; Liem, Hetty; Chen, Li How; Meade, Harry M; Gavin, William G; Echelard, Yann

    2004-06-01

    The current study was undertaken to evaluate the possibility of expanding transgenic goat herds by means of somatic cell nuclear transfer (NT) using transgenic goat cells as nucleus donors. Skin cells from adult, transgenic goats were first synchronized at quiescent stage (G0) by serum starvation and then induced to exit G0 and proceed into G1. Oocytes collected from superovulated donors were enucleated, karyoplast-cytoplast couplets were constructed, and then fused and activated simultaneously by a single electrical pulse. Fused couplets were either co-cultured with oviductal cells in TCM-199 medium (in vitro culture) or transferred to intermediate recipient goat oviducts (in vivo culture) until final transfer. The resulting morulae and blastocysts were transferred to the final recipients. Pregnancies were confirmed by ultrasonography 25-30 days after embryo transfer. In vitro cultured NT embryos developed to morulae and blastocyst stages but did not produce any pregnancies while 30% (6/20) of the in vivo derived morulae and blastocysts produced pregnancies. Two of these pregnancies were resorbed early in gestation. Of the four recipients that maintained pregnancies to term, two delivered dead fetuses 2-3 days after their due dates, and two recipients gave birth to healthy kids at term. Fluorescence in situ hybridization (FISH) analysis confirmed that both kids were transgenic and had integration sites consistent with those observed in the adult cell line.

  1. Metal resistance sequences and transgenic plants

    DOEpatents

    Meagher, Richard Brian; Summers, Anne O.; Rugh, Clayton L.

    1999-10-12

    The present invention provides nucleic acid sequences encoding a metal ion resistance protein, which are expressible in plant cells. The metal resistance protein provides for the enzymatic reduction of metal ions including but not limited to divalent Cu, divalent mercury, trivalent gold, divalent cadmium, lead ions and monovalent silver ions. Transgenic plants which express these coding sequences exhibit increased resistance to metal ions in the environment as compared with plants which have not been so genetically modified. Transgenic plants with improved resistance to organometals including alkylmercury compounds, among others, are provided by the further inclusion of plant-expressible organometal lyase coding sequences, as specifically exemplified by the plant-expressible merB coding sequence. Furthermore, these transgenic plants which have been genetically modified to express the metal resistance coding sequences of the present invention can participate in the bioremediation of metal contamination via the enzymatic reduction of metal ions. Transgenic plants resistant to organometals can further mediate remediation of organic metal compounds, for example, alkylmetal compounds including but not limited to methyl mercury, methyl lead compounds, methyl cadmium and methyl arsenic compounds, in the environment by causing the freeing of mercuric or other metal ions and the reduction of the ionic mercury or other metal ions to the less toxic elemental mercury or other metals.

  2. The global importance of transgenic cotton

    USDA-ARS?s Scientific Manuscript database

    The origins of transgenic cotton are reviewed including the original objectives, early efforts to establish the technical capabilities, selection of initial traits for development, market place benefits, and global acceptance of the technology. Further consideration is given to cotton’s place in th...

  3. Transgenic plants with increased calcium stores

    NASA Technical Reports Server (NTRS)

    Wyatt, Sarah (Inventor); Tsou, Pei-Lan (Inventor); Robertson, Dominique (Inventor); Boss, Wendy (Inventor)

    2004-01-01

    The present invention provides transgenic plants over-expressing a transgene encoding a calcium-binding protein or peptide (CaBP). Preferably, the CaBP is a calcium storage protein and over-expression thereof does not have undue adverse effects on calcium homeostasis or biochemical pathways that are regulated by calcium. In preferred embodiments, the CaBP is calreticulin (CRT) or calsequestrin. In more preferred embodiments, the CaBP is the C-domain of CRT, a fragment of the C-domain, or multimers of the foregoing. In other preferred embodiments, the CaBP is localized to the endoplasmic reticulum by operatively associating the transgene encoding the CaBP with an endoplasmic reticulum localization peptide. Alternatively, the CaBP is targeted to any other sub-cellular compartment that permits the calcium to be stored in a form that is biologically available to the plant. Also provided are methods of producing plants with desirable phenotypic traits by transformation of the plant with a transgene encoding a CaBP. Such phenotypic traits include increased calcium storage, enhanced resistance to calcium-limiting conditions, enhanced growth and viability, increased disease and stress resistance, enhanced flower and fruit production, reduced senescence, and a decreased need for fertilizer production. Further provided are plants with enhanced nutritional value as human food or animal feed.

  4. Osteogenic differentiation of mesenchymal stem cells from dental bud: Role of integrins and cadherins.

    PubMed

    Di Benedetto, Adriana; Brunetti, Giacomina; Posa, Francesca; Ballini, Andrea; Grassi, Felice Roberto; Colaianni, Graziana; Colucci, Silvia; Rossi, Enzo; Cavalcanti-Adam, Elisabetta A; Lo Muzio, Lorenzo; Grano, Maria; Mori, Giorgio

    2015-11-01

    Several studies have reported the beneficial effects of mesenchymal stem cells (MSCs) in tissue repair and regeneration. New sources of stem cells in adult organisms are continuously emerging; dental tissues have been identified as a source of postnatal MSCs. Dental bud is the immature precursor of the tooth, is easy to access and we show in this study that it can yield a high number of cells with ≥95% expression of mesenchymal stemness makers and osteogenic capacity. Thus, these cells can be defined as Dental Bud Stem Cells (DBSCs) representing a promising source for bone regeneration of stomatognathic as well as other systems. Cell interactions with the extracellular matrix (ECM) and neighboring cells are critical for tissue morphogenesis and architecture; such interactions are mediated by integrins and cadherins respectively. We characterized DBSCs for the expression of these adhesion receptors and examined their pattern during osteogenic differentiation. Our data indicate that N-cadherin and cadherin-11 were expressed in undifferentiated DBSCs and their expression underwent changes during the osteogenic process (decreasing and increasing respectively), while expression of E-cadherin and P-cadherin was very low in DBSCs and did not change during the differentiation steps. Such expression pattern reflected the mesenchymal origin of DBSCs and confirmed their osteoblast-like features. On the other hand, osteogenic stimulation induced the upregulation of single subunits, αV, β3, α5, and the formation of integrin receptors α5β1 and αVβ3. DBSCs differentiation toward osteoblastic lineage was enhanced when cells were grown on fibronectin (FN), vitronectin (VTN), and osteopontin (OPN), ECM glycoproteins which contain an integrin-binding sequence, the RGD motif. In addition we established that integrin αVβ3 plays a crucial role during the commitment of MSCs to osteoblast lineage, whereas integrin α5β1 seems to be dispensable. These data suggest that

  5. Removal of transgene-expressing cells by a specific immune response induced by sustained transgene expression.

    PubMed

    Yin, Yalei; Takahashi, Yuki; Ebisuura, Norifumi; Nishikawa, Makiya; Takakura, Yoshinobu

    2014-01-01

    Induction of the immune response to transgene products is a serious concern in gene therapy, and is generally known to be influenced by the transgene expression profile, as well as the types of cells that express the transgene. However, the exact nature of the association between the transgene expression profile and immune induction following gene transfer is unclear. In the present study, plasmids, pCpG-fLuc or pCMV-fLuc, used for driving long- or short-term expression of firefly luciferase, respectively, were injected into mice by hydrodynamic injections along with a reporter plasmid expressing Gaussia luciferase (pROSA-gLuc) to evaluate the transgene expression profile in the liver. Single pROSA-gLuc administration resulted in stable gLuc activity in serum for more than 1 year; thus, gLuc activity was used for monitoring immune responses to the liver cells expressing both gLuc and fLuc after co-injection. A significant reduction in gLuc activity was observed 2 weeks after co-injection of pROSA-gLuc with pCpG-fLuc, whereas stable gLuc activity was observed when pROSA-gLuc was co-injected with pCMV-fLuc. A high level of fLuc-specific immunoglobulin G was detectable in pCpG-fLuc-injected mice; furthermore, histological analysis of the liver sections of these mice indicated CD8(+) cell infiltration, implying that the transgene-expressing hepatocytes were removed by the infiltrating cells. Our results demonstrate that sustained transgene expression in hepatocytes triggers antigen-specific immune responses, although short-term expression of the same transgene product elicits little, if any, immune response. Copyright © 2014 John Wiley & Sons, Ltd.

  6. Expression of alpha v beta 5 integrin is necessary for efficient adenovirus-mediated gene transfer in the human airway.

    PubMed Central

    Goldman, M J; Wilson, J M

    1995-01-01

    Recombinant adenoviruses are being evaluated for gene therapy of cystic fibrosis lung disease with the goal of reconstituting the expression of the cystic fibrosis transmembrane conductance regulator in pulmonary epithelia by direct administration of the virus into the airway. The therapeutic potential of recombinant adenoviruses is limited in part by the relative inefficiency by which gene transfer occurs. This study uses a human bronchial xenograft model to study adenovirus infection in the human airway in an attempt to define the molecular events that limit gene transfer. Our studies of the human airway confirm previous observations of cell lines that have indicated a two-step process for adenovirus entry, which begins with the binding of the virus to the cell through the fiber protein and continues with internalization via interactions among cellular integrins and an RGD motif (Arg-Gly-Asp) in the penton base. Furthermore, the level of maturity of the epithelia in xenografts has a major impact on gene transfer. Undifferentiated epithelia express high levels of alpha v beta 5 integrins and are easily infected with recombinant adenoviruses; gene transfer is completely inhibited with excess fiber and partially inhibited with RGD peptide and alpha v beta 5 integrin antibody. Pseudostratified epithelia do not express alpha v beta 5 integrin in differentiated columnar cells and are relatively resistant to adenovirus-mediated gene transfer; what little gene transfer occurs is inhibited by fiber but not by RGD peptide or alpha v beta 5 integrin antibody. These studies suggest that the expression of integrins in human airway epithelia limits the efficiency of gene transfer with recombinant adenoviruses. However, low-level gene transfer can occur in fully mature epithelia through alpha v beta 5 integrin-independent pathways. PMID:7545239

  7. Bone marrow-derived mesenchymal stem cells enhance angiogenesis via their α6β1 integrin receptor

    SciTech Connect

    Carrion, Bita; Kong, Yen P.; Kaigler, Darnell

    2013-11-15

    Bone marrow-derived mesenchymal stem cells (BMSCs) facilitate the angiogenic response of endothelial cells (ECs) within three-dimensional (3D) matrices in vivo and in engineered tissues in vitro in part through paracrine mediators and by acting as stabilizing pericytes. However, the molecular interactions between BMSCs and nascent tubules during the process of angiogenesis are not fully understood. In this study, we have used a tractable 3D co-culture model to explore the functional role of the α6β1 integrin adhesion receptor on BMSCs in sprouting angiogenesis. We report that knockdown of the α6 integrin subunit in BMSCs significantly reduces capillary sprouting, and causes theirmore » failure to associate with the nascent vessels. Furthermore, we demonstrate that the BMSCs with attenuated α6 integrin proliferate at a significantly lower rate relative to either control cells expressing non-targeting shRNA or wild type BMSCs; however, despite adding more cells to compensate for this deficit in proliferation, deficient sprouting persists. Collectively, our findings demonstrate that the α6 integrin subunit in BMSCs is important for their ability to stimulate vessel morphogenesis. This conclusion may have important implications in the optimization of cell-based strategies to promote angiogenesis. Highlights: • BMSCs stimulate angiogenesis, but the mechanisms remain unclear. • We silenced the expression of the α6 integrin subunit in BMSCs. • Silencing this receptor subunit significantly inhibited angiogenic sprouting. • Knocking down α6 integrin affected laminin and αSMA expression. • Silencing α6 integrin expression also reduced BMSC proliferation.« less

  8. Covisualization by computational optical-sectioning microscopy of integrin and associated proteins at the cell membrane of living onion protoplasts

    NASA Technical Reports Server (NTRS)

    Gens, J. S.; Reuzeau, C.; Doolittle, K. W.; McNally, J. G.; Pickard, B. G.; Evans, M. L. (Principal Investigator)

    1996-01-01

    Using higher-resolution wide-field computational optical-sectioning fluorescence microscopy, the distribution of antigens recognized by antibodies against animal beta 1 integrin, fibronectin, and vitronectin has been visualized at the outer surface of enzymatically protoplasted onion epidermis cells and in depectinated cell wall fragments. On the protoplast all three antigens are colocalized in an array of small spots, as seen in raw images, in Gaussian filtered images, and in images restored by two different algorithms. Fibronectin and vitronectin but not beta 1 integrin antigenicities colocalize as puncta in comparably prepared and processed images of the wall fragments. Several control visualizations suggest considerable specifity of antibody recognition. Affinity purification of onion cell extract with the same anti-integrin used for visualization has yielded protein that separates in SDS-PAGE into two bands of about 105-110 and 115-125 kDa. These bands are again recognized by the visualization antibody, which was raised against the extracellular domain of chicken beta 1 integrin, and are also recognized by an antibody against the intracellular domain of chicken beta 1 integrin. Because beta 1 integrin is a key protein in numerous animal adhesion sites, it appears that the punctate distribution of this protein in the cell membranes of onion epidermis represents the adhesion sites long known to occur in cells of this tissue. Because vitronectin and fibronection are matrix proteins that bind to integrin in animals, the punctate occurrence of antigenically similar proteins both in the wall (matrix) and on enzymatically prepared protoplasts reinforces the concept that onion cells have adhesion sites with some similarity to certain kinds of adhesion sites in animals.

  9. ADAM disintegrin-like domain recognition by the lymphocyte integrins α4β1 and α4β7

    PubMed Central

    Bridges, Lance C.; Sheppard, Dean; Bowditch, Ron D.

    2004-01-01

    The ADAM (a disintegrin and metalloprotease) family of proteins possess both proteolytic and adhesive domains. We have established previously that the disintegrin domain of ADAM28, an ADAM expressed by human lymphocytes, is recognized by the integrin α4β1. The present study characterizes the integrin binding properties of the disintegrin-like domains of human ADAM7, ADAM28 and ADAM33 with the integrins α4β1, α4β7 and α9β1. Cell-adhesion assays demonstrated that, similar to ADAM28, the ADAM7 disintegrin domain supported α4β1-dependent Jurkat cell adhesion, whereas the ADAM33 disintegrin domain did not. The lymphocyte integrin α4β7 was also found to recognize both disintegrin domains of ADAM7 and ADAM28, but not of ADAM33. This is the first demonstration that mammalian disintegrins are capable of interacting with α4β7. All three disintegrin domains supported α9β1-dependent cell adhesion. Recognition by both α4β1 and α4β7 of ADAM7 and ADAM28 was activation-dependent, requiring either the presence of Mn2+ or an activating monoclonal antibody for cell attachment. Charge-to-alanine mutagenesis experiments revealed that the same residues within an individual ADAM disintegrin domain function in recognizing multiple integrins. However, the residues within a specific region of each ADAM disintegrin-like domain required for integrin binding were distinct. These results establish that ADAM7 and ADAM28 are recognized by the leucocyte integrins α4β1, α4β7 and α9β1. ADAM33 exclusively supported only α9β1-dependent adhesion. PMID:15504110

  10. Improved production of genetically modified fetuses with homogeneous transgene expression after transgene integration site analysis and recloning in cattle.

    PubMed

    Bressan, Fabiana Fernandes; Dos Santos Miranda, Moyses; Perecin, Felipe; De Bem, Tiago Henrique; Pereira, Flavia Thomaz Verechia; Russo-Carbolante, Elisa Maria; Alves, Daiani; Strauss, Bryan; Bajgelman, Marcio; Krieger, José Eduardo; Binelli, Mario; Meirelles, Flavio Vieira

    2011-02-01

    Animal cloning by nuclear transfer (NT) has made the production of transgenic animals using genetically modified donor cells possible and ensures the presence of the gene construct in the offspring. The identification of transgene insertion sites in donor cells before cloning may avoid the production of animals that carry undesirable characteristics due to positional effects. This article compares blastocyst development and competence to establish pregnancies of bovine cloned embryos reconstructed with lentivirus-mediated transgenic fibroblasts containing either random integration of a transgene (random integration group) or nuclear transfer derived transgenic fibroblasts with known transgene insertion sites submitted to recloning (recloned group). In the random integration group, eGFP-expressing bovine fetal fibroblasts were selected by fluorescence activated cell sorting (FACS) and used as nuclei donor cells for NT. In the recloned group, a fibroblast cell line derived from a transgenic cloned fetus was characterized regarding transgene insertion and submitted to recloning. The recloned group had higher blastocyst production (25.38 vs. 14.42%) and higher percentage of 30-day pregnancies (14.29 vs. 2.56%) when compared to the random integration group. Relative eGFP expression analysis in fibroblasts derived from each cloned embryo revealed more homogeneous expression in the recloned group. In conclusion, the use of cell lines recovered from transgenic fetuses after identification of the transgene integration site allowed for the production of cells and fetuses with stable transgene expression, and recloning may improve transgenic animal yields.

  11. Making BAC transgene constructs with lambda-red recombineering system for transgenic animals or cell lines.

    PubMed

    Holmes, Scott; Lyman, Suzanne; Hsu, Jen-Kang; Cheng, JrGang

    2015-01-01

    The genomic DNA libraries based on Bacteria Artificial Chromosomes (BAC) are the foundation of whole genomic mapping, sequencing, and annotation for many species like mice and humans. With their large insert size, BACs harbor the gene-of-interest and nearby transcriptional regulatory elements necessary to direct the expression of the gene-of-interest in a temporal and cell-type specific manner. When replacing a gene-of-interest with a transgene in vivo, the transgene can be expressed with the same patterns and machinery as that of the endogenous gene. This chapter describes in detail a method of using lambda-red recombineering to make BAC transgene constructs with the integration of a transgene into a designated location within a BAC. As the final BAC construct will be used for transfection in cell lines or making transgenic animals, specific considerations with BAC transgenes such as genotyping, BAC coverage and integrity as well as quality of BAC DNA will be addressed. Not only does this approach provide a practical and effective way to modify large DNA constructs, the same recombineering principles can apply to smaller high copy plasmids as well as to chromosome engineering.

  12. Primary transgenic bovine cells and their rejuvenated cloned equivalents show transgene-specific epigenetic differences.

    PubMed

    Alonso-González, Lucia; Couldrey, Christine; Meinhardt, Marcus W; Cole, Sally A; Wells, David N; Laible, Götz

    2012-01-01

    Cell-mediated transgenesis, based on somatic cell nuclear transfer (SCNT), provides the opportunity to shape the genetic make-up of cattle. Bovine primary fetal fibroblasts, commonly used cells for SCNT, have a limited lifespan, and complex genetic modifications that require sequential transfections can be challenging time and cost-wise. To overcome these limitations, SCNT is frequently used to rejuvenate the cell lines and restore exhausted growth potential. We have designed a construct to be used in a 2-step cassette exchange experiment. Our transgene contains a puromycin resistance marker gene and an enhanced green fluorescence protein (EGFP) expression cassette, both driven by a strong mammalian promoter, and flanked by loxP sites and sequences from the bovine β-casein locus. Several transgenic cell lines were generated by random insertion into primary bovine cell lines. Two of these original cell lines were rederived by SCNT and new primary cells, with the same genetic makeup as the original donors, were established. While the original cell lines were puromycin-resistant and had a characteristic EGFP expression profile, all rejuvenated cell lines were sensitive to puromycin, and displayed varied EGFP expression, indicative of various degrees of silencing. When the methylation states of individual CpG sites within the transgene were analyzed, a striking increase in transgene-specific methylation was observed in all rederived cell lines. The results indicate that original transgenic donor cells and their rejuvenated derivatives may not be equivalent and differ in the functionality of their transgene sequences.

  13. Diversity of arthropod community in transgenic poplar-cotton ecosystems.

    PubMed

    Zhang, D J; Lu, Z Y; Liu, J X; Li, C L; Yang, M S

    2015-12-02

    Poplar-cotton agro-ecosystems are the main agricultural planting modes of plain cotton fields in China. Here, we performed a systematic survey of the diversity and population of arthropod communities in four different combination of poplar-cotton eco-systems, including I) non-transgenic poplar and non-transgenic cotton fields; II) non-transgenic poplar and transgenic cotton fields [Bacillus thuringiensis (Bt) cotton]; III) Bt transgenic poplar (high insect resistant strain Pb29) and non-transgenic cotton; and IV) transgenic poplar and transgenic cotton fields, over a period of 3 years. Based on the statistical methods used to investigate community ecology, the effects of transgenic ecosystems on the whole structure of the arthropod community, on the structure of arthropods in the nutritive layer, and on the similarity of arthropod communities were evaluated. The main results were as follows: the transgenic poplar-cotton ecosystem has a stronger inhibitory effect on insect pests and has no impact on the structure of the arthropod community, and therefore, maintains the diversity of the arthropod community. The character index of the community indicated that the structure of the arthropod community of the transgenic poplar-cotton ecosystem was better than that of the poplar-cotton ecosystem, and that system IV had the best structure. As for the abundance of nutritional classes, the transgenic poplar-cotton ecosystem was also better than that of the non-transgenic poplar-cotton ecosystem. The cluster analysis and similarity of arthropod communities between the four different transgenic poplar-cotton ecosystems illustrated that the structure of the arthropod community excelled in the small sample of the transgenic poplar-cotton ecosystems.

  14. Can Transgenic Maize Affect Soil Microbial Communities?

    PubMed Central

    Mulder, Christian; Wouterse, Marja; Raubuch, Markus; Roelofs, Willem; Rutgers, Michiel

    2006-01-01

    The aim of the experiment was to determine if temporal variations of belowground activity reflect the influence of the Cry1Ab protein from transgenic maize on soil bacteria and, hence, on a regulatory change of the microbial community (ability to metabolize sources belonging to different chemical guilds) and/or a change in numerical abundance of their cells. Litter placement is known for its strong influence on the soil decomposer communities. The effects of the addition of crop residues on respiration and catabolic activities of the bacterial community were examined in microcosm experiments. Four cultivars of Zea mays L. of two different isolines (each one including the conventional crop and its Bacillus thuringiensis cultivar) and one control of bulk soil were included in the experimental design. The growth models suggest a dichotomy between soils amended with either conventional or transgenic maize residues. The Cry1Ab protein appeared to influence the composition of the microbial community. The highly enhanced soil respiration observed during the first 72 h after the addition of Bt-maize residues can be interpreted as being related to the presence of the transgenic crop residues. This result was confirmed by agar plate counting, as the averages of the colony-forming units of soils in conventional treatments were about one-third of those treated with transgenic straw. Furthermore, the addition of Bt-maize appeared to induce increased microbial consumption of carbohydrates in BIOLOG EcoPlates. Three weeks after the addition of maize residues to the soils, no differences between the consumption rate of specific chemical guilds by bacteria in soils amended with transgenic maize and bacteria in soils amended with conventional maize were detectable. Reaped crop residues, comparable to post-harvest maize straw (a common practice in current agriculture), rapidly influence the soil bacterial cells at a functional level. Overall, these data support the existence of short

  15. Sulfur mustard disrupts human α3β1-integrin receptors in concert with α6β4-integrin receptors and collapse of the keratin K5/K14 cytoskeleton

    NASA Astrophysics Data System (ADS)

    Werrlein, Robert J.; Braue, Catherine R.

    2004-06-01

    Sulfur mustard (SM; bis(2-chloroethyl) sulfide) is a chemical warfare agent that produces persistent, incapacitating blisters of the skin. The lesions inducing vesication remain elusive, and there is no completely effective treatment. Using mulitphoton microscopy and immunofluorescent staining, we found that exposing human epidermal keratinocytes (HEK) and intact epidermis to SM (400 μm for 5 min) caused progressive collapse of the keratin (K5/K14) cytoskeleton and depletion of α6β integrins. We now report that SM causes concomitant disruption nad collapse of the basal cell's α3β1-integrin receptors. At 1 h postexposure, images of Alexa488-conjugated HEK/α3β1 integrins showed almost complete withdrawal and disappearance of retraction fibers and a progressive loss of polarized mobility. With stero imaging, in vitro expression of this SM effect was characterized by collapse and abutment of adjacent cell membranes. At 2 h postexposure, there was an average 13% dorso-ventral collapse of HEK membranes that paralleled progressive collapse of the K5/K14 cytoskeleton. α3β1 integrin, like α6β4 integrin, is a regulator of cytoskeletal assembly, a receptor for laminin 5 and a mediator of HEK attachment to the basement membrane. Our images indicate that SM disrupts these receptors. We suggest that the progressive disruption destabilizes and potentiates blistering of the epidermal-dermal junction.

  16. α2 Integrin, extracellular matrix metalloproteinase inducer, and matrix metalloproteinase-3 act sequentially to induce differentiation of mouse embryonic stem cells into odontoblast-like cells

    SciTech Connect

    Ozeki, Nobuaki; Kawai, Rie; Hase, Naoko

    2015-02-01

    We previously reported that interleukin 1β acts via matrix metalloproteinase (MMP)-3 to regulate cell proliferation and suppress apoptosis in α2 integrin-positive odontoblast-like cells differentiated from mouse embryonic stem (ES) cells. Here we characterize the signal cascade underpinning odontoblastic differentiation in mouse ES cells. The expression of α2 integrin, extracellular matrix metalloproteinase inducer (Emmprin), and MMP-3 mRNA and protein were all potently increased during odontoblastic differentiation. Small interfering RNA (siRNA) disruption of the expression of these effectors potently suppressed the expression of the odontoblastic biomarkers dentin sialophosphoprotein, dentin matrix protein-1 and alkaline phosphatase, and blocked odontoblast calcification. Our siRNA, western blotmore » and blocking antibody analyses revealed a unique sequential cascade involving α2 integrin, Emmprin and MMP-3 that drives ES cell differentiation into odontoblasts. This cascade requires the interaction between α2 integrin and Emmprin and is potentiated by exogenous MMP-3. Finally, although odontoblast-like cells potently express α2, α6, αV, β1, and β3, integrins, we confirmed that β1 integrin acts as the trigger for ES cell differentiation, apparently in complex with α2 integrin. These results demonstrate a unique and unanticipated role for an α2 integrin-, Emmprin-, and MMP-3-mediated signaling cascade in driving mouse ES cell differentiation into odontoblast-like cells. - Highlights: • Odontoblast differentiation requires activation of α2 integrin, Emmprin and MMP-3. • α2 integrin, Emmprin and MMP-3 form a sequential signaling cascade. • β1 integrin acts a specific trigger for odontoblast differentiation. • The role of these effectors is highly novel and unanticipated.« less

  17. Rhinocetin, a Venom-derived Integrin-specific Antagonist Inhibits Collagen-induced Platelet and Endothelial Cell Functions*

    PubMed Central

    Vaiyapuri, Sakthivel; Hutchinson, E. Gail; Ali, Marfoua S.; Dannoura, Abeer; Stanley, Ronald G.; Harrison, Robert A.; Bicknell, Andrew B.; Gibbins, Jonathan M.

    2012-01-01

    Snaclecs are small non-enzymatic proteins present in viper venoms reported to modulate hemostasis of victims through effects on platelets, vascular endothelial, and smooth muscle cells. In this study, we have isolated and functionally characterized a snaclec that we named “rhinocetin” from the venom of West African gaboon viper, Bitis gabonica rhinoceros. Rhinocetin was shown to comprise α and β chains with the molecular masses of 13.5 and 13 kDa, respectively. Sequence and immunoblot analysis of rhinocetin confirmed this to be a novel snaclec. Rhinocetin inhibited collagen-stimulated activation of human platelets in a dose-dependent manner but displayed no inhibitory effects on glycoprotein VI (collagen receptor) selective agonist, CRP-XL-, ADP-, or thrombin-induced platelet activation. Rhinocetin antagonized the binding of monoclonal antibodies against the α2 subunit of integrin α2β1 to platelets and coimmunoprecipitation analysis confirmed integrin α2β1 as a target for this venom protein. Rhinocetin inhibited a range of collagen-induced platelet functions such as fibrinogen binding, calcium mobilization, granule secretion, aggregation, and thrombus formation. It also inhibited integrin α2β1-dependent functions of human endothelial cells. Together, our data suggest rhinocetin to be a modulator of integrin α2β1 function and thus may provide valuable insights into the role of this integrin in physiological and pathophysiological scenarios, including hemostasis, thrombosis, and envenomation. PMID:22689571

  18. UNC-97/PINCH is involved in the assembly of integrin cell adhesion complexes in Caenorhabditis elegans body wall muscle.

    PubMed

    Norman, Kenneth R; Cordes, Shaun; Qadota, Hiroshi; Rahmani, Poupak; Moerman, Donald G

    2007-09-01

    UNC-97/PINCH is an evolutionarily conserved protein that contains five LIM domains and is located at cell-extracellular matrix attachment sites known as cell adhesion complexes. To understand the role of UNC-97/PINCH in cell adhesion, we undertook a combined genetic and cell biological approach to identify the steps required to assemble cell adhesion complexes in Caenorhabditis elegans. First, we have generated a complete loss of function mutation in the unc-97 coding region. unc-97 null mutants arrest development during embryogenesis and reveal that the myofilament lattice and its attachment structures, which include PAT-4/ILK (integrin-linked kinase) and integrin fail to assemble into properly organized arrays. Although in the absence of UNC-97/PINCH, PAT-4/ILK and integrin fail to organize normally, they are capable of colocalizing together at the muscle cell membrane. Alternatively, in integrin and pat-4 mutants, UNC-97/PINCH fails to localize to the muscle cell membrane and instead is found diffusely throughout the muscle cell cytoplasm. In agreement with mammalian studies, we show that LIM domain 1 of UNC-97/PINCH is required for its interaction with PAT-4/ILK in yeast two-hybrid assays. Additionally, we find, by LIM domain deletion analysis, that LIM1 is required for the localization of UNC-97/PINCH to cell adhesion complexes. Our results provide evidence that UNC-97/PINCH is required for the development of C. elegans and is required for the formation of integrin based adhesion structures.

  19. High-Throughput Screening based Identification of Small Molecule Antagonists of Integrin CD11b/CD18 Ligand Binding

    PubMed Central

    Faridi, Mohd Hafeez; Maiguel, Dony; Brown, Brock T.; Suyama, Eigo; Barth, Constantinos J.; Hedrick, Michael; Vasile, Stefan; Sergienko, Eduard; Schürer, Stephan; Gupta, Vineet

    2010-01-01

    Binding of leukocyte specific integrin CD11b/CD18 to its physiologic ligands is important for the development of normal immune response in vivo. Integrin CD11b/CD18 is also a key cellular effector of various inflammatory and autoimmune diseases. However, small molecules selectively inhibiting the function of integrin CD11b/CD18 are currently lacking. We used a newly described cell-based high throughput screening assay to identify a number of highly potent antagonists of integrin CD11b/CD18 from chemical libraries containing >100,000 unique compounds. Computational analyses suggest that the identified compounds cluster into several different chemical classes. A number of the newly identified compounds blocked adhesion of wild-type mouse neutrophils to CD11b/CD18 ligand fibrinogen. Mapping the most active compounds against chemical fingerprints of known antagonists of related integrin CD11a/CD18 shows little structural similarity, suggesting that the newly identified compounds are novel and unique. PMID:20188705

  20. Oxidation-induced Structural Changes of Ceruloplasmin Foster NGR Motif Deamidation That Promotes Integrin Binding and Signaling

    PubMed Central

    Barbariga, Marco; Curnis, Flavio; Spitaleri, Andrea; Andolfo, Annapaola; Zucchelli, Chiara; Lazzaro, Massimo; Magnani, Giuseppe; Musco, Giovanna; Corti, Angelo; Alessio, Massimo

    2014-01-01

    Asparagine deamidation occurs spontaneously in proteins during aging; deamidation of Asn-Gly-Arg (NGR) sites can lead to the formation of isoAsp-Gly-Arg (isoDGR), a motif that can recognize the RGD-binding site of integrins. Ceruloplasmin (Cp), a ferroxidase present in the cerebrospinal fluid (CSF), contains two NGR sites in its sequence: one exposed on the protein surface (568NGR) and the other buried in the tertiary structure (962NGR). Considering that Cp can undergo oxidative modifications in the CSF of neurodegenerative diseases, we investigated the effect of oxidation on the deamidation of both NGR motifs and, consequently, on the acquisition of integrin binding properties. We observed that the exposed 568NGR site can deamidate under conditions mimicking accelerated Asn aging. In contrast, the hidden 962NGR site can deamidate exclusively when aging occurs under oxidative conditions, suggesting that oxidation-induced structural changes foster deamidation at this site. NGR deamidation in Cp was associated with gain of integrin-binding function, intracellular signaling, and cell pro-adhesive activity. Finally, Cp aging in the CSF from Alzheimer disease patients, but not in control CSF, causes Cp deamidation with gain of integrin-binding function, suggesting that this transition might also occur in pathological conditions. In conclusion, both Cp NGR sites can deamidate during aging under oxidative conditions, likely as a consequence of oxidative-induced structural changes, thereby promoting a gain of function in integrin binding, signaling, and cell adhesion. PMID:24366863

  1. Identification of the alphavbeta3 integrin-interacting motif of betaig-h3 and its anti-angiogenic effect.

    PubMed

    Nam, Ju-Ock; Kim, Jung-Eun; Jeong, Ha-Won; Lee, Sung-Jin; Lee, Byung-Heon; Choi, Je-Yong; Park, Rang-Woon; Park, Jae Yong; Kim, In-San

    2003-07-11

    betaig-h3 is an extracellular matrix protein that mediates adhesion and migration of several cell types through interaction with integrins. In the present study, we tested whether betaig-h3 mediates endothelial cell adhesion and migration, thereby regulating angiogenesis. In this study, we demonstrate that not only betaig-h3 itself but also all four fas-1 domains of betaig-h3 mediate endothelial cell adhesion and migration through interaction with the alphavbeta3 integrin. We found that the alphavbeta3 integrin-interacting motif of the four fas-1 domains of betaig-h3 is the same YH motif that we reported previously to interact with alphavbeta5 integrin. The YH peptide inhibited endothelial cell adhesion and migration in a dose-dependent manner. We demonstrate that the YH peptide has anti-angiogenic activity in vitro and in vivo using an endothelial cell tube formation assay and a Matrigel plug assay, respectively. Our results reveal that betaig-h3 bears alphavbeta3 integrin-interacting motifs that mediate endothelial cell adhesion and migration and, therefore, may regulate angiogenesis.

  2. Amygdalin blocks the in vitro adhesion and invasion of renal cell carcinoma cells by an integrin-dependent mechanism.

    PubMed

    Juengel, Eva; Afschar, Masud; Makarević, Jasmina; Rutz, Jochen; Tsaur, Igor; Mani, Jens; Nelson, Karen; Haferkamp, Axel; Blaheta, Roman A

    2016-03-01

    Information about the natural compound amygdalin, which is employed as an antitumor agent, is sparse and thus its efficacy remains controversial. In this study, to determine whether amygdalin exerts antitumor effects on renal cell carcinoma (RCC) cells, its impact on RCC metastatic activity was investigated. The RCC cell lines, Caki-1, KTC-26 and A498, were exposed to amygdalin from apricot kernels, and adhesion to human vascular endothelium, immobilized collagen or fibronectin was investigated. The influence of amygdalin on chemotactic and invasive activity was also determined, as was the influence of amygdalin on surface and total cellular α and β integrin expression, which are involved in metastasis. We noted that amygdalin caused significant reductions in chemotactic activity, invasion and adhesion to endothelium, collagen and fibronectin. Using FACScan analysis, we noted that amygdalin also induced reductions, particularly in integrins α5 and α6, in all three cell lines. Functional blocking of α5 resulted in significantly diminished adhesion of KTC-26 and A498 to collagen and also in decreased chemotactic behavior in all three cell lines. Blocking α6 integrin significantly reduced chemotactic activity in all three cell lines. Thus, we suggest that exposing R