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PubMed1997

Expression of integrin cell adhesion receptors during human airway epithelial repair in vivo.

Airway epithelium is subject to injury during inflammation and exposure to a variety of inhaled and infectious agents. Little is known about the expression of integrins during human airway epithelial regeneration and differentiation after injury. We therefore characterized integrin subunit expression after mechanical injury in an in vivo xenograft model of human bronchial epithelium. On the migrating cells at the edges of surface epithelial wounds, there was increased expression of the alpha v-, beta 5-, beta 6-, and alpha 5-integrin subunits. During the later phase of repair, the increased expression of alpha v-, beta 5-, and beta 6-subunits persisted, but the expression of the beta 8-subunits was restricted to basal cells. In addition, there was a redistribution of the alpha 2- and alpha 6-collagen/laminin-binding integrins to suprabasal epithelial layers. There was no expression of the beta 3- or alpha 4-integrin subunit on reparative epithelium. A similar upregulation of alpha v-, beta 5-, and beta 6-integrin receptor subunits was observed in areas of undifferentiated airway from cystic fibrosis patients. Injured epithelium was found to be significantly more susceptible to gene transfer with a recombinant adenovirus, suggesting that the increased integrin expression has implications for the acquisition of adenovirus infections and for lung-directed gene therapy.

Animals↗

PubMed1997

An Arg-Gly-Asp peptide stimulates constriction in rat afferent arteriole.

The potential role of integrins in the myogenic mechanism was studied in the rat afferent arteriole (AA) by fluorescence immunolocalization and microperfusion of isolated AA. Confocal fluorescence images were acquired from frozen sections of rat kidney after indirect immunostaining for various integrin beta- and alpha-subunits. The beta 1-, beta 3-, alpha 3-, alpha 5-, and alpha V-integrins were found on the plasma membrane in smooth muscle of AA, providing the morphological basis for participation of integrins in mechanotransduction. With 1 mM nitro-L-arginine methyl ester (L-NAME) in the luminal perfusate to inhibit endogenous nitric oxide (NO) production from AA, the hexapeptide GRGDSP (10(-7)-10(-3)M) induced immediate vasoconstriction. The constriction was dose dependent and specific or peptides with arginine-glycine-aspartic acid (RGD) motifs, commonly found on the binding sites of extracellular matrix to integrins. In controls, the hexapeptide GRGESP induced no constriction. GRGDSP, 1 mM, induced a 21.6 +/- 2.6% decrease (P < 0.05, n = 6) in lumen diameter for 30 s and an 18.3 +/- 4.1% increase (P < 0.05, n = 6) in smooth muscle intracellular calcium concentration for 18 s, as measured by the emission ratio of Fluo-3/Fura Red. Binding of exogenous RGD motifs with exposed integrins on AA smooth muscle therefore triggers calcium-dependent vasoconstriction. However, the dose response to RGD was not sensitive to the myogenic tone of the vessel, which suggests that the integrin-mediated vasoconstriction is different from myogenic constriction.

Amino Acid Sequence↗

PubMed2004

Amino acid mutagenesis within ligand-binding loops in alpha v confers loss-of-function or gain-of-function phenotype on integrin alpha v beta 3.

The crystal structure of alpha(v)beta(3) in complex with a cyclic RGD-containing ligand has recently been demonstrated. However, the functional significance of each residue within ligand binding loops has not been fully elucidated. Here, by employing alanine-scanning mutagenesis, we have examined the functional role of ligand contact residues in alpha(v). Tyr178 --> Ala substitution (Tyr178Ala) and Asp218Ala abolished a monovalent ligand, WOW-1 Fab binding as well as soluble fibrinogen binding, which is in perfect agreement with the crystallography. However, Asp150Ala showed no or only a modest inhibition of ligand binding. In contrast, Tyr substitution at Ala215 (Ala215Tyr) increased WOW-1 Fab binding, suggesting that the substitution increased the integrin affinity. The adhesion assay to immobilized fibrinogen showed essentially the same data as obtained using soluble ligands. Our present data indicate that Tyr178 and Asp218, but not Asp150 in alpha(v) is critically involved in ligand-binding and that Ala215 could regulate the affinity of alpha(v)beta(3).

Amino Acid Substitution↗

PubMed1997

Inhibition of protein tyrosine kinases attenuates increases in expression of transforming growth factor-beta isoforms and their receptors following arterial injury.

Transforming growth factor-beta 1 (TGF-beta 1) has been implicated in neointima formation in mechanically injured vessels and in restenosis after angioplasty. To further understand the significance of TGF-beta s in neointima formation, we examined the temporal expression of three TGF-beta isoforms (-beta 1, -beta 2, and -beta 3), their receptors (ALK-2, ALK-5, and T beta RII), and two putative TGF-beta responses (elevations in alpha v and beta 3 integrin mRNAs) in balloon catheter-injured rat carotid arteries and their dependency on tyrosine kinase activity. Using a standardized reverse transcriptase-polymerase chain reaction assay optimized to estimate mRNA levels, we observed distinct patterns of mRNA regulation for TGF-beta 1, -beta 2, and -beta 3 during the 48 hours immediately after injury, which were localized to the vessel's media. TGF-beta 1 mRNA increased 10-fold during this time while TGF-beta 3 mRNA also increased almost 2-fold. There were also increases in mRNAs encoding the TGF-beta type I receptors ALK-5 and ALK-2, as well as the type II receptor (T beta RII). Eight hours after the injury, mRNA levels for ALK-2 and ALK-5 were on average 2-fold higher; mRNA encoding the type II receptor increased approximately 3-fold by 24 hours. There were also associated increases in TGF-beta 1, TGF-beta 3, ALK-5, and T beta RII immunoreactive peptide levels. Peak increases in mRNAs for integrins alpha v and beta 3 averaged approximately 2-fold and 2.5-fold, respectively. Perivascular administration of the tyrosine kinase inhibitor genistein at the time of vessel injury markedly (> 85%) inhibited elevations in mRNAs encoding TGF-beta 1, TGF-beta 3, T beta RII, and the two integrins alpha v and beta 3, while application of its inactive chemically similar homologue daidzein did not prevent the injury-induced elevations in mRNA levels. Since the increases in integrins alpha v and beta 3 mRNA could be theoretically attributed to TGF-beta actions despite being dependent on tyrosine kinase activity, we examined whether the observed elevations in integrins alpha v and beta 3 were due to TGF-beta 1 secretion, using cultured rat carotid artery smooth muscle cells. TGF-beta 1 neutralizing antibodies specifically inhibited elevations in integrins alpha v and beta 3 mRNAs due to platelet-derived growth factor-BB and fibroblast growth factor-2. We conclude that multiple components of the TGF-beta system in vessels are activated following injury and influence expression of integrin receptors important for smooth muscle cell migration. Activation of the TGF-beta system appears to be highly dependent on tyrosine kinases.

Activin Receptors, Type I↗

PubMed2004

Endoproteolytic activation of alpha(v) integrin by proprotein convertase PC5 is required for vascular smooth muscle cell adhesion to vitronectin and integrin-dependent signaling.

BACKGROUND: Integrins play an important role for vascular smooth muscle cell (VSMC) migration during the development of atherosclerosis and restenosis. Integrin alpha(v)-subunit consists of disulphide-bound 125-kDa heavy and 25-kDa light chains, which are generated by endoproteolytic cleavage. This type of activation requires the presence of suitable proprotein convertases (PCs). Based on ex vivo and in vitro data, the PC5 isozyme has been suggested to be the major integrin convertase. We have recently demonstrated that PC5 is upregulated during vascular remodeling in rodents, colocalizing with alpha(v) in VSMCs. The aim of this study was to investigate the activation of alpha(v) by PCs in VSMCs and its consequences for alpha(v)-dependent cell functions. METHODS AND RESULTS: Immunoblotting demonstrated that inhibition of PC activity by the specific pharmacological inhibitor dec-CMK inhibits alpha(v) cleavage in VSMCs. These results were confirmed using PC5-specific antisense oligonucleotides. PC5-antisense oligonucleotides and dec-CMK inhibited VSMC adhesion to the alpha(v)beta3/beta5 ligand vitronectin (both P<0.05). Furthermore, PC5-asODNs inhibited VSMC migration on vitronectin-coated wells (P<0.05). Inhibition of PC activity and consequently alpha(v) cleavage inhibited the adhesion-dependent focal adhesion kinase(Y397)-autophosphorylation and subsequent Akt activation, whereas phosphorylation of extracellular signal-regulated kinase 1/2 was not affected. In human endarterectomy lesions, PC5 colocalized with alpha(v) integrin in VSMCs in the atherosclerotic plaques. CONCLUSIONS: The present study demonstrates that alpha(v) endoproteolytic activation is necessary for integrin-mediated adhesion and migration as well as signaling and requires PC5 in VSMCs. The colocalization of PC5 and alpha(v) in human carotid plaques indicates that PC5 might play a key role for alpha(v) activation in vivo.

Animals↗

PubMed2000

Angiotensin II enhances integrin and alpha-actinin expression in adult rat cardiac fibroblasts.

Angiotensin II (Ang II) plays an important role in cardiac remodeling through stimulation of proliferation and extracellular matrix (ECM) production in cardiac fibroblasts. Integrins are a family of transmembrane receptors that mediate the attachment of cells to ECM. We hypothesized that Ang II regulation of integrins further contributes to its role in cardiac remodeling. We cultured adult rat cardiac fibroblasts with and without Ang II (100 nmol/L) to determine the effects on mRNA and protein levels of integrins, as well as alpha-actinin and other cytoskeletal proteins that link to integrins at the site of focal adhesions. Ang II was also added in the presence of irbesartan (10 micromol/L), a specific Ang II type 1 (AT(1)) receptor antagonist, or PD 123319 (10 micromol/L), a specific Ang II type 2 receptor antagonist. To investigate the function of these integrins, we determined the effects of blocking antibodies on Ang II-induced adhesion to ECM. We also treated spontaneously hypertensive rats (SHR) with an AT(1) receptor blocker, losartan, or with hydralazine to investigate integrin and alpha-actinin expression in treated and untreated SHR. Ang II enhanced alpha(v), beta(1), beta(3), and beta(5) integrins; osteopontin; and alpha-actinin mRNA and protein levels in cardiac fibroblasts. All of these effects were inhibited by irbesartan but not by PD 123319. Pretreatment of cardiac fibroblasts with Ang II enhanced cell attachment to ECM proteins and induced focal adhesion kinase phosphorylation. Blocking antibodies to beta(3) and alpha(v)beta(5) attenuated Ang II-induced adhesion. In SHR, ventricular alpha(v) and beta(5) integrin expression and alpha-actinin were increased compared with those in Wistar-Kyoto rats. Although both losartan and hydralazine lowered mean arterial pressure and decreased peripheral vascular resistance, only losartan attenuated the increased integrin, alpha-actinin, fibronectin laminin, and osteopontin expression and the increased left ventricular mass (as determined with echocardiography). Hydralzine had none of these effects. Although both agents attenuated beta-myosin heavy chain expression, a marker of hypertrophy, losartan had a greater effect. These results suggest that integrins and alpha-actinin are upregulated by Ang II and in left ventricular hypertrophy and that the block of expression of these proteins through inhibition of the AT(1) receptor is associated with attenuation of the hypertrophic response. Ang II induces integrin and alpha-actinin expression in cardiac fibroblasts that is associated with adhesion and left ventricular hypertrophy and blocked through inhibition of the AT(1) receptor.

Actinin↗

PubMed2005

Assessment of endogenous and therapeutic arteriogenesis by contrast ultrasound molecular imaging of integrin expression.

BACKGROUND: We hypothesized that molecular imaging with contrast-enhanced ultrasound (CEU) and microbubbles targeted to endothelial integrins could be used to noninvasively assess early angiogenic responses to ischemia and growth factor therapy. METHODS AND RESULTS: Hindlimb ischemia was produced in 48 rats by ligation of an iliac artery. Half of the animals received intramuscular sustained-release fibroblast growth factor-2 (FGF-2). Immediately after ligation and at subsequent intervals from 4 to 28 days, blood flow and oxygen tension in the proximal adductor muscles were measured by CEU perfusion imaging and phosphor quenching, respectively. Targeted CEU imaging of alpha(v)- and alpha5beta1-integrin expression was performed with microbubbles bearing the disintegrin echistatin. Iliac artery ligation produced a 65% to 70% reduction in blood flow and oxygen tension. In untreated ischemic muscle, muscle flow and oxygen tension partially recovered by days 14 to 28. In these animals, signal from integrin-targeted microbubbles was intense and peaked before flow increase (days 4 to 7). In comparison to untreated animals, FGF-2-treated muscle had a greater rate and extent of blood flow recovery and greater signal intensity from integrin-targeted microbubbles, which peaked before maximal recovery of flow. On immunohistology, arteriolar but not capillary density increased in the ischemic limb after ligation, the rate and degree of which were greater in FGF-2-treated rats. Immunofluorescence demonstrated intense staining for alpha(v) in arterioles, the temporal course of which correlated with targeted imaging. CONCLUSIONS: Targeted CEU can be used to assess endogenous and therapeutic arteriogenesis before recovery of tissue perfusion. These results suggest that molecular imaging of integrin expression may be useful for evaluating proangiogenic therapies.

Animals↗

PubMed2002

Nuclear factor-kappaB activation in alveolar macrophages requires IkappaB kinase-beta, but not nuclear factor-kappaB inducing kinase.

Cytokine mediated activation of alveolar macrophages (AMs) is an important event in the pathogenesis of fibrosing alveolitis (FA). Through membrane-associated antigens, cytokines (e.g., tumor necrosis-factor-alpha and interleukin-1) are believed to activate a common kinase cascade that initiates the cytoplasmic degradation of IkappaB and nuclear translocation of "nuclear factor-kappaB" (NF-kappaB). In the nucleus, NF-kappaB promotes the transcription of genes encoding chemokines and cytokines involved in chronic inflammation. Preventing cytokine-mediated NF-kappaB activation is a potential strategy for attenuating the lung injury that occurs in FA. Previously, we have demonstrated that, unlike AMs from healthy volunteers, AMs from patients with inflammatory lung diseases express the coxsackie/adenovirus receptor and the alphav integrins required for adenovirus (Adv) infection. This property allows Adv-mediated transgene delivery to diseased, but not normal, AMs and analysis of molecular pathways involved in gene transcription. In this study, AMs were infected with Adv constructs expressing a defective beta subunit of IkappaB kinase (AdvIKKbetakd) and a defective NF-kappaB inducing kinase (AdvNIKkd) to investigate the contribution of these molecules to NF-kappaB activation. We observed that IKKbeta, but not NIK, was required for NF-kappaB activation. The results of this study identify IKKbeta, but not NIK, as a potential therapeutic target in diseases that involve NF-kappaB-dependent gene transcription.

Adenoviridae↗

PubMed2005

Role of osteopontin in amplification and perpetuation of rheumatoid synovitis.

Osteopontin (OPN) is an extracellular matrix protein of pleiotropic properties and has been recently recognized as a potential inflammatory cytokine. In this study, we demonstrate, for the first time to our knowledge, that overexpression of OPN in synovial T cells is associated with local inflammatory milieu and that OPN acts as an important mediator in amplification and perpetuation of rheumatoid synovitis. The study revealed that mRNA expression of OPN was highly elevated in CD4(+) synovial T cells derived from patients with RA, which correlated with increased OPN concentrations in synovial fluid (SF). The pattern of OPN overexpression was confined to rheumatoid synovium and correlated with coexpression of selected OPN receptors in synovial T cells, including integrins alphav and beta1 and CD44. RA-derived SF stimulated the expression of OPN in T cells, which was attributable to IL-10 present in SF and abrogated by anti-IL-10 antibody. Among the more than 300 autoimmune and inflammatory response genes examined, OPN selectively induced the expression of proinflammatory cytokines and chemokines known to promote migration and recruitment of inflammatory cells. Furthermore, it was evident that OPN activated transcription factor NF-kappaB in mononuclear cells. The study has important implications for understanding the role of OPN in rheumatoid synovitis and other inflammatory conditions.

Adult↗

PubMed1999

CAR-dependent and CAR-independent pathways of adenovirus vector-mediated gene transfer and expression in human fibroblasts.

Primary fibroblasts are not efficiently transduced by subgroup C adenovirus (Ad) vectors because they express low levels of the high-affinity Coxsackie virus and adenovirus receptor (CAR). In the present study, we have used primary human dermal fibroblasts as a model to explore strategies by which Ad vectors can be designed to enter cells deficient in CAR. Using an Ad vector expressing the human CAR cDNA (AdCAR) at high multiplicity of infection, primary fibroblasts were converted from being CAR deficient to CAR sufficient. Efficiency of subsequent gene transfer by standard Ad5-based vectors and Ad5-based vectors with alterations in penton and fiber was evaluated. Marked enhancement of binding and transgene expression by standard Ad5 vectors was achieved in CAR-sufficient fibroblasts. Expression by AdDeltaRGDbetagal, an Ad5-based vector lacking the arginine-glycine-aspartate (RGD) alphaV integrin recognition site from its penton base, was achieved in CAR-sufficient, but not CAR-deficient, cells. Fiber-altered Ad5-based vectors, including (a) AdF(pK7)betagal (bearing seven lysines on the end of fiber) (b) AdF(RGD)betagal (bearing a high-affinity RGD sequence on the end of fiber), and (c) AdF9sK betagal (bearing a short fiber and Ad9 knob), demonstrated enhanced gene transfer in CAR-deficient fibroblasts, with no further enhancement in CAR-sufficient fibroblasts. Together, these observations demonstrate that CAR deficiency on Ad targets can be circumvented either by supplying CAR or by modifying the Ad fiber to bind to other cell-surface receptors.

Adenoviruses, Human↗

PubMed2004

Alpha v beta 3 integrin expression in human odontoblasts and co-localization with osteoadherin.

Integrins are heterodimeric transmembrane receptors which promote cell adhesion, thus contributing to the maintenance of tissue organization in both normal and pathological conditions. To characterize the way odontoblasts may interact with other cells and the extracellular matrix in human teeth, we studied expression of alpha v beta 3 integrin, a putative receptor for osteoadherin. We showed that alpha v beta 3 integrin expression was restricted to odontoblasts, blood vessels, and small rounded cells in sound and carious pulp. Odontoblast staining intensity increased from the apical to the cusp region. Osteoadherin staining was strong in the whole odontoblast layer (with a slight decrease in the cusp region) and in predentin. Odontoblasts differentiating in vitro were stained with the anti-alpha v beta 3 integrin antibody, first at the level of intercellular contacts, then throughout the cell membrane. These results suggest that the alpha v beta 3 integrin could play a role in interodontoblast adhesion and odontoblast binding to the surrounding predentin/dentin/pulp matrix, possibly through osteoadherin.

Adolescent↗

PubMed2005

The extracellular adherence protein (Eap) of Staphylococcus aureus inhibits wound healing by interfering with host defense and repair mechanisms.

Staphylococcus aureus is a major human pathogen interfering with host-cell functions. Impaired wound healing is often observed in S aureus-infected wounds, yet, the underlying mechanisms are poorly defined. Here, we identify the extracellular adherence protein (Eap) of S aureus to be responsible for impaired wound healing. In a mouse wound-healing model wound closure was inhibited in the presence of wild-type S aureus and this effect was reversible when the wounds were incubated with an isogenic Eap-deficient strain. Isolated Eap also delayed wound closure. In the presence of Eap, recruitment of inflammatory cells to the wound site as well as neovascularization of the wound were prevented. In vitro, Eap significantly reduced intercellular adhesion molecule 1 (ICAM-1)-dependent leukocyte-endothelial interactions and diminished the consequent activation of the proinflammatory transcription factor nuclear factor kappaB (NFkappaB) in leukocytes associated with a decrease in expression of tissue factor. Moreover, Eap blocked alphav-integrin-mediated endothelial-cell migration and capillary tube formation, and neovascularization in matrigels in vivo. Collectively, the potent anti-inflammatory and antiangiogenic properties of Eap provide an underlying mechanism that may explain the impaired wound healing in S aureus-infected wounds. Eap may also serve as a lead compound for new anti-inflammatory and antiangiogenic therapies in several pathologies.

Animals↗

PubMed2005

ADAM-9 (MDC-9/meltrin-gamma), a member of the a disintegrin and metalloproteinase family, regulates myeloma-cell-induced interleukin-6 production in osteoblasts by direct interaction with the alpha(v)beta5 integrin.

ADAM-9, a member of the a disintegrin and metalloproteinase family, contains both metalloproteinase and disintegrin domains. Myeloma cell lines express ADAM-9; however, its function and role in the pathophysiology of multiple myeloma is unknown. The aim of this study was to establish whether primary myeloma cells express ADAM-9, whether ADAM-9 regulates IL-6 production in human osteoblasts (hOBs), whether ADAM-9 interacts with specific integrin heterodimers, and the identity of downstream signaling pathways. Primary myeloma cells demonstrated increased expression of ADAM-9 (P < .01). ADAM-9 promoted a 5-fold increase in IL-6, but not IL-1beta mRNA, and a dose- and time-dependent increase in IL-6 production by hOBs (P < .01). IL-6 induction was inhibited by an antibody to the alpha(v)beta5 integrin (P < .01) but not by antibodies to other integrin heterodimers. ADAM-9 was shown to bind directly to the alpha(v)beta5 integrin on hOBs. Antibodies to ADAM-9 and alpha(v)beta5 integrin inhibited myeloma cell-induced IL-6 production by hOBs (P < .01). Furthermore, inhibitors of p38 MAPK and cPLA2, but not NF-kappaB and JAK2, signaling pathways inhibited ADAM-9-induced IL-6 production by hOBs (P < .01). These data demonstrate that ADAM-9, expressed by myeloma cells, stimulates IL-6 production in hOBs by binding the alpha(v)beta5 integrin. This may have important consequences for the growth and survival of myeloma cells in bone.

ADAM Proteins↗

PubMed2004

CD155/PVR plays a key role in cell motility during tumor cell invasion and migration.

BACKGROUND: Invasion is an important early step of cancer metastasis that is not well understood. Developing therapeutics to limit metastasis requires the identification and validation of candidate proteins necessary for invasion and migration. METHODS: We developed a functional proteomic screen to identify mediators of tumor cell invasion. This screen couples Fluorophore Assisted Light Inactivation (FALI) to a scFv antibody library to systematically inactivate surface proteins expressed by human fibrosarcoma cells followed by a high-throughput assessment of transwell invasion. RESULTS: Using this screen, we have identified CD155 (the poliovirus receptor) as a mediator of tumor cell invasion through its role in migration. Knockdown of CD155 by FALI or by RNAi resulted in a significant decrease in transwell migration of HT1080 fibrosarcoma cells towards a serum chemoattractant. CD155 was found to be highly expressed in multiple cancer cell lines and primary tumors including glioblastoma (GBM). Knockdown of CD155 also decreased migration of U87MG GBM cells. CD155 is recruited to the leading edge of migrating cells where it colocalizes with actin and alphav-integrin, known mediators of motility and adhesion. Knockdown of CD155 also altered cellular morphology, resulting in cells that were larger and more elongated than controls when plated on a Matrigel substrate. CONCLUSION: These results implicate a role for CD155 in mediating tumor cell invasion and migration and suggest that CD155 may contribute to tumorigenesis.

Cell Line, Tumor↗

PubMed2006

MUC1 alters oncogenic events and transcription in human breast cancer cells.

INTRODUCTION: MUC1 is an oncoprotein whose overexpression correlates with aggressiveness of tumors and poor survival of cancer patients. Many of the oncogenic effects of MUC1 are believed to occur through interaction of its cytoplasmic tail with signaling molecules. As expected for a protein with oncogenic functions, MUC1 is linked to regulation of proliferation, apoptosis, invasion, and transcription. METHODS: To clarify the role of MUC1 in cancer, we transfected two breast cancer cell lines (MDA-MB-468 and BT-20) with small interfering (si)RNA directed against MUC1 and analyzed transcriptional responses and oncogenic events (proliferation, apoptosis and invasion). RESULTS: Transcription of several genes was altered after transfection of MUC1 siRNA, including decreased MAP2K1 (MEK1), JUN, PDGFA, CDC25A, VEGF and ITGAV (integrin alphav), and increased TNF, RAF1, and MMP2. Additional changes were seen at the protein level, such as increased expression of c-Myc, heightened phosphorylation of AKT, and decreased activation of MEK1/2 and ERK1/2. These were correlated with cellular events, as MUC1 siRNA in the MDA-MB-468 line decreased proliferation and invasion, and increased stress-induced apoptosis. Intriguingly, BT-20 cells displayed similar levels of apoptosis regardless of siRNA, and actually increased proliferation after MUC1 siRNA. CONCLUSION: These results further the growing knowledge of the role of MUC1 in transcription, and suggest that the regulation of MUC1 in breast cancer may be more complex than previously appreciated. The differences between these two cell lines emphasize the importance of understanding the context of cell-specific signaling events when analyzing the oncogenic functions of MUC1, and caution against generalizing the results of individual cell lines without adequate confirmation in intact biological systems.

Antigens, Neoplasm↗

PubMed2006

Adenoviral-encoded antigens are presented efficiently by a subset of dendritic cells expressing high levels of alpha(v)beta3 integrins.

Dendritic cells (DC) play a central role in antigen presentation and are often targeted by adenoviral (Ad)-based gene therapy. However, DC lack the coxsackie-Ad receptor, and little is known about the process by which they acquire and present Ad-encoded antigens. We examined the expression of alpha(v)beta3 integrins (CD51/CD61) on mouse bone marrow-derived DC (BM-DC) and their susceptibility to transduction by Ad vectors. Less than 10% of BM-DC precursors expressed CD51, but expression increased over time in culture with granulocyte macrophage-colony stimulating factor (GM-CSF)/interleukin (IL)-4. After 7 days, 28 +/- 1.7% of CD11c+ DC expressed high levels of CD51 (CD51(hi)), and the remaining DC expressed low levels of CD51 (CD51(lo)). CD51(hi) CD express higher major histocompatibility complex type 1 (MHC I); however, both of the DC subsets expressed similar levels of MHC II and costimulatory molecules. When exposed to a first-generation Ad vector, transgene expression was restricted to the CD51(hi) DC subset and blocked by soluble peptides expressing an arginine, glycine, aspartic acid (RGD) sequence, confirming the role of integrins in viral entry. Consistent with this, a modified Ad expressing an RGD-binding sequence in its fiber knob (Ad-RGD) transduced the CD51(hi) DC subset with significantly higher efficiency. When BM-DC were transduced with an Ad-expressing ovalbumin (Ad-OVA), the CD51(hi) subset proved superior in activating OT-I (T cell receptor-OVA) T cells. Similar to in vitro effects, systemic administration of GM-CSF/IL-4 increased the expression of CD51 on splenic DC and rendered these cells susceptible to Ad transduction. These results suggest that a limited subset of DC expressing high levels of alpha(v)beta3 integrins is preferentially transduced by Ad vectors and activates CD8+ T cell responses against Ad-encoded antigens.

Adenoviridae↗

PubMed2003

Connection between integrins and cell activation in rat adrenal glomerulosa cells: a role for Arg-Gly-Asp peptide in the activation of the p42/p44(mapk) pathway and intracellular calcium.

Integrins are responsible for adhesion and activation of several intracellular cascades. The present study was aimed at determining whether the interaction between fibronectin and integrins could generate pathways involved in physiological functions of rat adrenal glomerulosa cells. Immunofluorescence studies and adhesion assays showed that fibronectin was the best matrix in promoting the formation of focal adhesion. Binding of glomerulosa cells to fibronectin, but not to collagen I or poly-L-lysine, involved the integrin-binding sequence Arg-Gly-Asp (RGD). Activation of glomerulosa cells with Arg-Gly-Asp-Ser (RGDS) induced an increase in [Ca(2+)](i), whereas fibronectin triggered a release of Ca(2+) from InsP(3)-sensitive Ca(2+) stores. Aldosterone secretion induced by ACTH, angiotensin II, and RGDS and proliferation were improved on fibronectin, compared with poly-L-lysine. The RGDS peptide induced a transient increase in the activity of the p42/p44(mapk), independent of phosphatidylinositol-3 kinase and protein kinase C. Integrins alpha(5) and alpha(V) as well as their fibronectin receptor partners beta(1) and beta(3), were identified. These results suggest that in rat adrenal glomerulosa cells, binding of the alpha(5)beta(1), alpha(v)beta(1), or alpha(v)beta(3) integrins to fibronectin is involved in the generation of two important signaling events, increase in intracellular calcium, and activation of the p42/p44(mapk) cascade, leading to cell proliferation and aldosterone secretion.

Adrenal Cortex↗

PubMed1995

Alpha v and alpha 3 integrin subunits are associated with myofibrils during myofibrillogenesis.

The development of the myofibrillar apparatus in skeletal muscle is a process in which transmembrane linkages with adhesion molecules are implicated. Integrins are one class of transmembrane adhesion receptors which appear to mediate these interactions. Two prominent linkages are at the myotendinous junction (MTJ), which residues at the ends of the cell and connects myofibrils to the tendon, and the costameres, which encircle the girth of the cell and connect the Z-disks to the sarcolemma. In this study we report that the alpha v integrin subunit is a prominent component of the costamere. The alpha v subunit is present initially on developing myotubes in a diffuse staining pattern with some concentration along nascent myofibrils. However, it appears in a striated pattern at the costamere and inconsistently at the M-line following the striation of alpha-actinin and titin but before that of desmin. Its recruitment to preformed striation suggests that it is incorporated into a pre-existing structure. The presence of alpha v in the costamere points to a role in lateral myofibrillar anchorage. In addition, we find that the alpha 3 subunit is transiently associated with myofibrils along portions of their lengths and at their ends during myofibrillogenesis. The alpha 3 subunit staining shows a novel localization and junctional structure. As myofibrils become striated the alpha 3 integrin dissociates from the localized pattern and becomes diffuse. This suggests a possible role in the stabilization of nascent myofibrils prior to striation. Antibody-induced perturbation of adhesion mediated by the integrin beta 1 subunit in developing myotubes inhibits assembly of the sarcomeric architecture.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗