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Biomedical subjects

D Sheppard

Publications and source records attributed to D Sheppard.

At least 19 recordsLinked to original sources
PubMed2001

Expression of the alphavbeta6 integrin promotes migration and invasion in squamous carcinoma cells.

The integrin alphavbeta6 is a fibronectin receptor whose expression is not detectable on normal oral epithelium but is increased significantly in healing and in oral epithelial dysplasia and oral squamous cell carcinoma, suggesting it may promote changes associated with tumor development. To study whether alphavbeta6 may drive invasive behavior we have used transfection and retroviral infection to create a panel of epithelial cell lines expressing various levels of alphavbeta6. We report that increased expression of alphavbeta6 in malignant keratinocytes promotes invasion and leads to an increased capacity for migration towards fibronectin. alphavbeta6 expression may have a significant role in contributing to the malignant behavior of epithelial cells.

Antigens, Neoplasm↗
PubMed2001

The cytoplasmic domain of the integrin alpha9 subunit requires the adaptor protein paxillin to inhibit cell spreading but promotes cell migration in a paxillin-independent manner.

The integrin alpha9 subunit forms a single heterodimer, alpha9beta1. The alpha9 subunit is most closely related to the alpha4 subunit, and like alpha4 integrins, alpha9beta1 plays an important role in leukocyte migration. The alpha4 cytoplasmic domain preferentially enhances cell migration and inhibits cell spreading, effects that depend on interaction with the adaptor protein, paxillin. To determine whether the alpha9 cytoplasmic domain has similar effects, a series of chimeric and deleted alpha9 constructs were expressed in Chinese hamster ovary cells and tested for their effects on migration and spreading on an alpha9beta1-specific ligand. Like alpha4, the alpha9 cytoplasmic domain enhanced cell migration and inhibited cell spreading. Paxillin also specifically bound the alpha9 cytoplasmic domain and to a similar level as alpha4. In paxillin(-/-) cells, alpha9 failed to inhibit cell spreading as expected but surprisingly still enhanced cell migration. Further, mutations that abolished the alpha9-paxillin interaction prevented alpha9 from inhibiting cell spreading but had no effect on alpha9-dependent cell migration. These findings suggest that the mechanisms by which the cytoplasmic domains of integrin alpha subunits enhance migration and inhibit cell spreading are distinct and that the alpha9 and alpha4 cytoplasmic domains, despite sequence and functional similarities, enhance cell migration by different intracellular signaling pathways.

Amino Acid Motifs↗
PubMed2001

Role of the cytoplasmic domain of the beta-subunit of integrin alpha(v)beta6 in infection by foot-and-mouth disease virus.

Field isolates of foot-and-mouth disease virus (FMDV) are believed to use RGD-dependent integrins as cellular receptors in vivo. Using SW480 cell transfectants, we have recently established that one such integrin, alpha(v)beta6, functions as a receptor for FMDV. This integrin was shown to function as a receptor for virus attachment. However, it was not known if the alpha(v)beta6 receptor itself participated in the events that follow virus binding to the host cell. In the present study, we investigated the effects of various deletion mutations in the beta6 cytoplasmic domain on infection. Our results show that although loss of the beta6 cytoplasmic domain has little effect on virus binding, this domain is essential for infection, indicating a critical role in postattachment events. The importance of endosomal acidification in alpha(v)beta6-mediated infection was confirmed by experiments showing that infection could be blocked by concanamycin A, a specific inhibitor of the vacuolar ATPase.

Acids↗
PubMed2001

Interleukin-13 induces dramatically different transcriptional programs in three human airway cell types.

Interleukin (IL)-13, a cytokine released by T lymphocytes during immediate hypersensitivity responses, is a central mediator of asthma. Because IL-13 induces phenotypic features of asthma in mice deficient in T and B lymphocytes, it is likely that this cytokine contributes to the development of asthma by acting directly on resident airway cells. To analyze the global effects of IL-13 on gene expression in airway cells that could contribute to the phenotypic features of asthma, we used Genechip HuGene FL arrays (Affymetrix, Santa Clara, CA) that contain probes for approximately 6,500 human genes. Despite activating a common signaling pathway, IL-13 induced dramatically different patterns of gene expression in primary cultures of airway epithelial cells, airway smooth muscle cells, and lung fibroblasts, with little overlap among cell types. The most prominent effects of IL-13 were on airway smooth muscle, but several genes induced in airway epithelial cells and fibroblasts are also candidates that may contribute to phenotypic features of asthma. These results suggest that the in vivo response to IL-13 in the airways likely results from a combination of distinct effects on each of several resident airway cell types.

Cells, Cultured↗
PubMed2001

TGF-beta is a critical mediator of acute lung injury.

We have shown that the integrin alphavbeta6 activates latent TGF-beta in the lungs and skin. We show here that mice lacking this integrin are completely protected from pulmonary edema in a model of bleomycin-induced acute lung injury (ALI). Pharmacologic inhibition of TGF-beta also protected wild-type mice from pulmonary edema induced by bleomycin or Escherichia coli endotoxin. TGF-beta directly increased alveolar epithelial permeability in vitro by a mechanism that involved depletion of intracellular glutathione. These data suggest that integrin-mediated local activation of TGF-beta is critical to the development of pulmonary edema in ALI and that blocking TGF-beta or its activation could be effective treatments for this currently untreatable disorder.

Animals↗
PubMed2001

Expression of the beta6 integrin subunit is associated with sites of neutrophil influx in lung epithelium.

Inhalation of ozone by Rhesus monkeys results in epithelial injury and granulocyte influx in both conducting airways and respiratory bronchioles. We have reported that ozone-induced neutrophil recruitment and subsequent epithelial repair can be inhibited in vivo with a CD18 antibody. The antibody-mediated effect is abrogated by local instillation of C5a (a CD18-independent neutrophil chemoattractant), thereby demonstrating a role for neutrophils in lung epithelial repair processes. As an extension of this study, we examined the effect of ozone and neutrophil influx on epithelial expression of the beta6 integrin, an adhesion molecule associated with proliferation and repair. Expression of beta6 integrin was determined by immunohistochemistry for ozone-exposed monkeys treated with either control immunoglobulins or a CD18 antibody. The tracheal epithelium of ozone-exposed monkeys treated with control immunglobulins expressed the beta6 integrin. In contrast, the tracheal epithelium of ozone-exposed monkeys treated with CD18 antibody exhibited very low to undetectable expression of beta6 integrin. In association with C5a instillation and neutrophil influx, beta6 integrin was also observed in respiratory bronchiolar epithelium from both control and ozone-exposed animals. These findings cumulatively suggest that lung epithelial cell expression of beta6 integrin is associated with sites of neutrophil recruitment.

Administration, Inhalation↗
PubMed2001

Increased expression of tenascin-C-binding epithelial integrins in human bullous keratopathy corneas.

We previously found an abnormal deposition of an extracellular matrix glycoprotein, tenascin-C (TN-C), in human corneas with pseudophakic/aphakic bullous keratopathy (PBK/ABK). In this work, we studied cellular TN-C receptors in normal and PBK/ABK corneas. Cryostat sections of normal and PBK/ABK corneas were stained by immuno-fluorescence for TN-C receptors: alpha2, alpha8, alpha9, alphaVbeta3, beta1, and beta6 integrins, and annexin II. Beta6 integrin mRNA levels were assessed by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) using beta2-microglobulin gene to normalize the samples. In PBK/ABK compared to normal corneas, relatively minor changes were observed for alpha2 and beta1 integrins, and for annexin II. Alpha8, alpha9, and beta6 subunits of TN-C receptors, alpha8beta1 alpha9beta1, and alphaVbeta6, respectively, were absent from normal central corneas but were found in the central epithelium of PBK/ABK corneas. Beta6 integrin showed the most significant accumulation. It correlated best with the expression of TN-C rather than with the expression of other alphaVbeta6 ligands, fibronectin, and vitronectin. RT-PCR analysis also showed elevated levels of beta6 mRNA in PBK/ABK compared to normal corneas. Therefore, accumulation of TN-C in PBK/ABK corneas was accompanied by an increased expression of its three binding integrins, especially alphaVbeta6 in the corneal epithelium. The interaction of tenascin-C with these integrins may contribute to the fibrotic process that occurs in PBK/ABK corneas.

Corneal Diseases↗
PubMed2001

Integrin-mediated activation of transforming growth factor-beta(1) in pulmonary fibrosis.

The integrin alphavbeta6 is restricted to epithelial cells and is dramatically induced in response to injury and inflammation. Mice expressing a null mutation of this integrin develop exaggerated inflammation of the lungs and skin, but are dramatically protected from bleomycin-induced pulmonary fibrosis. This phenotype led to the identification of a unique role for this integrin in binding to and activating latent extracellular complexes of the anti-inflammatory, profibrotic cytokine, transforming growth factor-beta(1). This integrin-mediated activation is tightly spatially restricted and appears to require direct presentation of the activated cytokine to receptors on adjacent cells. The process also requires distinct regions of the beta6-subunit cytoplasmic domain and an intact actin cytoskeleton, suggesting the existence of additional cellular mechanisms to regulate this process. If this mechanism is found to be as important in humans as it is in mice, the integrin and as yet to be identified pathways for cellular regulation of this process could be exciting new targets for intervention in fibrotic diseases of the lung and other epithelial organs.

Animals↗
PubMed2000

RGD-independent binding of integrin alpha9beta1 to the ADAM-12 and -15 disintegrin domains mediates cell-cell interaction.

ADAMs (a disintegrin and metalloproteases) mediate several important processes (e.g. tumor necrosis factor-alpha release, fertilization, and myoblast fusion). The ADAM disintegrin domains generally lack RGD motifs, and their receptors are virtually unknown. Here we show that integrin alpha(9)beta(1) specifically interacts with the recombinant ADAMs-12 and -15 disintegrin domains in an RGD-independent manner. We also show that interaction between ADAM-12 or -15 and alpha(9)beta(1) supports cell-cell interaction. Interestingly, the cation requirement and integrin activation status required for alpha(9)beta(1)/ADAM-mediated cell adhesion and cell-cell interaction is similar to those required for known integrin-extracellular matrix interaction. These results are quite different from recent reports that ADAM-2/alpha(6)beta(1) interaction during sperm/egg fusion requires an integrin activation status distinct from that for extracellular matrix interaction. These results suggest that alpha(9)beta(1) may be a major receptor for ADAMs that lack RGD motifs, and that, considering a wide distribution of ADAMs and alpha(9)beta(1), this interaction may be of potential biological and pathological significance.

ADAM Proteins↗
PubMed2000

Inhibitory effects of MLDG-containing heterodimeric disintegrins reveal distinct structural requirements for interaction of the integrin alpha 9beta 1 with VCAM-1, tenascin-C, and osteopontin.

The integrin alpha9beta1 is expressed on epithelial cells, smooth muscle cells, skeletal muscle, and neutrophils and recognizes at least three distinct ligands: vascular cell adhesion molecule 1 (VCAM-1), tenascin-C, and osteopontin. The alpha9 subunit is structurally similar to the integrin alpha4 subunit, and alpha9beta1 and alpha4beta1 both recognize VCAM-1 as a ligand. We therefore examined whether the disintegrin EC3, which we have recently shown specifically inhibits the binding of alpha4 integrins to ligands, would also be a functional inhibitor of alpha9beta1. EC3 and a novel heterodimeric disintegrin that we identified, EC6, both were potent inhibitors of alpha9beta1-mediated adhesion to VCAM-1 and of neutrophil migration across tumor necrosis factor-activated endothelial cells. A peptide containing a novel MLDG motif shared by both of these disintegrins also inhibited alpha9beta1- and alpha4beta1-mediated adhesion to VCAM-1. Surprisingly though, concentrations of EC3 that completely inhibited adhesion of alpha9-transfected cells to VCAM-1 had little or no effect on adhesion to either of the other alpha9beta1 ligands, osteopontin and tenascin-C. Furthermore, peptides AEIDGIEL and SVVYGLR, which we have previously shown inhibit binding of alpha9beta1-expressing cells to tenascin-C and osteopontin, respectively, had no effect on adhesion to VCAM-1. These data suggest that there are structurally distinct requirements for interactions of the alpha9beta1 integrin with VCAM-1 and the extracellular matrix ligands osteopontin and tenascin-C.

Amino Acid Motifs↗
PubMed2000

Tissue transglutaminase, coagulation factor XIII, and the pro-polypeptide of von Willebrand factor are all ligands for the integrins alpha 9beta 1 and alpha 4beta 1.

We previously reported that MOLT-3 human lymphocyte-like leukemia cells adhere to tissue-type transglutaminase (tTG) through the integrin alpha(4)beta(1). We now report that G-361 human melanoma cells also adhere to tTG, although they do not express alpha(4)beta(1). G-361 cells utilize two additional integrins, alpha(9)beta(1) and alpha(5)beta(1) to adhere to tTG. Furthermore, blood coagulation factor XIII (FXIII), another member of the transglutaminase family that is highly homologous to tTG, and propolypeptide of von Willebrand factor (pp-vWF) also promoted cell adhesion through alpha(9)beta(1) or alpha(4)beta(1) in G-361 or MOLT-3 cells, respectively. In the case of pp-vWF, alpha(9)beta(1) and alpha(4)beta(1) both bind to the same site, comprised of 15 amino acid residues and designated T2-15. Moreover, SW480 human colon cancer cells stably transfected to express alpha(9)beta(1), but not mock transfectants, adhered to tTG, FXIII, pp-vWF, and T2-15/bovine serum albumin conjugate. These data identify tTG, FXIII, and pp-vWF as shared ligands for the integrins alpha(9)beta(1) and alpha(4)beta(1). This report is the first to unambiguously show that these two integrins share the same cell adhesion site within one protein and provides strong support for classifying alpha(9)beta(1-) and alpha(4)-integrins as functionally related members of an integrin subfamily.

Amino Acid Sequence↗
PubMed2000

Plasmin-sensitive dibasic sequences in the third fibronectin-like domain of L1-cell adhesion molecule (CAM) facilitate homomultimerization and concomitant integrin recruitment.

L1 is a multidomain transmembrane neural recognition molecule essential for neurohistogenesis. While moieties in the immunoglobulin-like domains of L1 have been implicated in both heterophilic and homophilic binding, the function of the fibronectin (FN)-like repeats remains largely unresolved. Here, we demonstrate that the third FN-like repeat of L1 (FN3) spontaneously homomultimerizes to form trimeric and higher order complexes. Remarkably, these complexes support direct RGD-independent interactions with several integrins, including alpha(v)beta(3) and alpha(5)beta(1). A pep- tide derived from the putative C-C' loop of FN3 (GSQRKHSKRHIHKDHV(852)) also forms trimeric complexes and supports alpha(v)beta(3) and alpha(5)beta(1) binding. Substitution of the dibasic RK(841) and KR(845) sequences within this peptide or the FN3 domain limited multimerization and abrogated integrin binding. Evidence is presented that the multimerization of, and integrin binding to, the FN3 domain is regulated both by conformational constraints imposed by other domains and by plasmin- mediated cleavage within the sequence RK( downward arrow)HSK( downward arrow)RH(846). The integrin alpha(9)beta(1), which also recognizes the FN3 domain, colocalizes with L1 in a manner restricted to sites of cell-cell contact. We propose that distal receptor ligation events at the cell-cell interface may induce a conformational change within the L1 ectodomain that culminates in receptor multimerization and integrin recruitment via interaction with the FN3 domain.

Amino Acid Sequence↗
PubMed2000

Global analysis of gene expression in pulmonary fibrosis reveals distinct programs regulating lung inflammation and fibrosis.

The molecular mechanisms of pulmonary fibrosis are poorly understood. We have used oligonucleotide arrays to analyze the gene expression programs that underlie pulmonary fibrosis in response to bleomycin, a drug that causes lung inflammation and fibrosis, in two strains of susceptible mice (129 and C57BL/6). We then compared the gene expression patterns in these mice with 129 mice carrying a null mutation in the epithelial-restricted integrin beta6 subunit (beta6(-/-)), which develop inflammation but are protected from pulmonary fibrosis. Cluster analysis identified two distinct groups of genes involved in the inflammatory and fibrotic responses. Analysis of gene expression at multiple time points after bleomycin administration revealed sequential induction of subsets of genes that characterize each response. The availability of this comprehensive data set should accelerate the development of more effective strategies for intervention at the various stages in the development of fibrotic diseases of the lungs and other organs.

Animals↗
PubMed2000

In vivo functions of integrins: lessons from null mutations in mice.

The integrin family (Hynes, R.O., 1992. Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69, 11-25) is composed of at least 24 heterodimers formed from eight beta subunits and 18 alpha subunits. Thus far, mice expressing null mutations of seven of the eight beta subunits and 13 of the 18 known alpha subunits have been generated, With only a few exceptions, the phenotypes of each of the knockout lines are quite distinct. Studies utilizing integrin knockout mice and cells derived from these mice have provided considerable and sometimes surprising insights into unique functions of individual members of this family.

Animals↗
PubMed2000

Mapping of the cryptic integrin-binding site in osteopontin suggests a new mechanism by which thrombin can regulate inflammation and tissue repair.

The integrin alpha9beta1 mediates neutrophil migration across several ligands that are enriched at sites of inflammation. In one of these ligands, the acidic phosphoprotein osteopontin, the alpha9beta1 binding site is cryptic, but is revealed after thrombin cleavage. We have recently mapped the alpha9beta1 binding site in osteopontin to the linear peptide sequence, SVVYGLR, immediately adjacent to the thrombin cleavage site. Interestingly, this site is also adjacent to a sequence (RGD) through which five other integrins bind to osteopontin. These findings suggest a novel mechanism by which thrombin can modulate integrin signaling at sites of tissue injury.

Binding Sites↗
PubMed2000

The epithelial integrin alphavbeta6 is a receptor for foot-and-mouth disease virus.

Field isolates of foot-and-mouth disease virus (FMDV) have been shown to use the RGD-dependent integrin alphavbeta3 as a cellular receptor on cultured cells. However, several other RGD-dependent integrins may have the potential to act as receptors for FMDV in vivo. Of these, alphavbeta6 is a likely candidate for use as a receptor by FMDV as it is expressed on epithelial cells, which correlates with the tissue tropism of the virus. In this report, we show that human colon carcinoma cells (SW480) that are normally nonpermissive for FMDV become susceptible to infection as a result of transfection with the integrin beta6 subunit and expression of alphavbeta6 at the cell surface. Integrin alphavbeta6 is the major site for virus attachment on the beta6-transfected cells, and binding to alphavbeta6 serves to increase the rate of virus entry into these cells. In addition, we show that virus binding and infection of the beta6-transfected cells is mediated through an RGD-dependent interaction that is specifically inhibited by a monoclonal antibody (10D5) that recognizes alphavbeta6. These studies establish a role for alphavbeta6 as a cellular receptor for FMDV.

Animals↗
PubMed2000

Fatal bilateral chylothorax in mice lacking the integrin alpha9beta1.

Members of the integrin family of adhesion receptors mediate both cell-cell and cell-matrix interactions and have been shown to play vital roles in embryonic development, wound healing, metastasis, and other biological processes. The integrin alpha9beta1 is a receptor for the extracellular matrix proteins osteopontin and tenacsin C and the cell surface immunoglobulin vascular cell adhesion molecule-1. This receptor is widely expressed in smooth muscle, hepatocytes, and some epithelia. To examine the in vivo function of alpha9beta1, we have generated mice lacking expression of the alpha9 subunit. Mice homozygous for a null mutation in the alpha9 subunit gene appear normal at birth but develop respiratory failure and die between 6 and 12 days of age. The respiratory failure is caused by an accumulation of large volumes of pleural fluid which is rich in triglyceride, cholesterol, and lymphocytes. alpha9(-/-) mice also develop edema and lymphocytic infiltration in the chest wall that appears to originate around lymphatics. alpha9 protein is transiently expressed in the developing thoracic duct at embryonic day 14, but expression is rapidly lost during later stages of development. Our results suggest that the alpha9 integrin is required for the normal development of the lymphatic system, including the thoracic duct, and that alpha9 deficiency could be one cause of congenital chylothorax.

Animals↗