Biomedical subjects

Siobhan A Corbett

Publications and source records attributed to Siobhan A Corbett.

8 recordsLinked to original sources

PubMed2006

Lipopolysaccharide transiently activates THP-1 cell adhesion.

Lipopolysaccharide stimulation of adherent THP-1 cells induces morphological changes that are associated with the reorganization of the actin cytoskeleton. We hypothesized that LPS would also increase THP-1 cell adhesion and sought to determine the signaling mechanisms regulating this response. We show that LPS significantly increases THP-1 cell attachment after 1 h, supporting the idea that LPS can stimulate integrin function. By 4 h however, the number of adherent cells returned to control levels. Importantly, detached cells were determined to be viable by propidium iodide staining, indicating that the increase in cell adhesion was transient. LPS-induced adhesion to fibrinogen- but not fibronectin-coated wells was also transient, suggesting that adhesion reflected beta2 integrin activation. This idea was supported by the fact that LPS-induced adhesion could be blocked by a function-blocking anti-beta2 integrin antibody. Interestingly, the protein tyrosine phosphatase (PTP) inhibitor, phenylarsine oxide, prevented cell detachment. Taken together, these data suggest that LPS-mediated integrin activation is transient and can be regulated by PTP-mediated signaling events.

Arsenicals↗

PubMed2005

The integrin alpha5beta1 regulates alphavbeta3-mediated extracellular signal-regulated kinase activation.

BACKGROUND: Integrin-mediated cell migration is essential for wound repair. Previous studies have shown that the interaction between integrins and the extracellular matrix (ECM) can initiate intracellular signaling pathways to regulate cell movement. Both the focal adhesion kinase (FAK) and the extracellular signal-regulated kinase/activated mitogen-activated protein kinase (ERK/MAPK) signaling pathways are required for efficient cell migration. Our previous work has shown that co-expression of the integrin alpha5beta1 inhibits alphavbeta3-mediated cell migration. We hypothesized that alpha5beta1 may regulate cell migration by modulating these alphavbeta3-mediated intracellular signaling events. METHODS: CHO B3 (alphavbeta3+) and B3C5 (alphavbeta3+/alpha5beta1+) cells were monitored by flow cytometry to determine integrin expression. Cells were allowed to migrate on fibrinogen (FBG)-coated transwells, with or without PD98059, an inhibitor of the ERK activator, mitogen-activated protein kinase kinase (MEK). Fixation, staining, and cell counting were used to quantify cell migration. Cells adherent to FBG were lysed and analyzed for FAK and ERK/MAPK activation by immunoblotting followed by image analysis densitometry. All experiments were repeated in triplicate. RESULTS: Treatment with PD98059 significantly decreased alphavbeta3-mediated cell migration on FBG (P = 0.0001) to a level comparable to untreated B3C5 cells. Following adhesion to FBG, B3 cells demonstrated a marked increase in ERK/MAPK activation compared to B3C5 cells. However, no significant difference was detected in FAK activation. CONCLUSION: Signaling through the ERK/MAPK pathway is required for efficient alphavbeta3-mediated migration on FBG. Inhibition of alphavbeta3-mediated migration by the integrin alpha5beta1 correlates with altered intensity and duration of ERK/MAPK activation, but not FAK activation, in response to adhesion. This suggests a mechanism for the regulatory effect of alpha5beta1 on alphavbeta3-mediated cell migration.

Animals↗

PubMed2004

Fibronectin matrix assembly regulates alpha5beta1-mediated cell cohesion.

Integrin-extracellular matrix (ECM) interactions in two-dimensional (2D) culture systems are widely studied (Goldstein and DiMilla, 2002. J Biomed. Mater. Res. 59, 665-675; Koo et al., 2002. J. Cell Sci. 115, 1423-1433). Less understood is the role of the ECM in promoting intercellular cohesion in three-dimensional (3D) environments. We have demonstrated that the alpha5beta1-integrin mediates strong intercellular cohesion of 3D cellular aggregates (Robinson et al., 2003. J. Cell Sci. 116, 377-386). To further investigate the mechanism of alpha5beta1-mediated cohesivity, we used a series of chimeric alpha5beta1-integrin-expressing cells cultured as multilayer cellular aggregates. In these cell lines, the alpha5 subunit cytoplasmic domain distal to the GFFKR sequence was truncated, replaced with that of the integrin alpha4, the integrin alpha2, or maintained intact. Using these cells, alpha5beta1-integrin-mediated cell aggregation, compaction and cohesion were determined and correlated with FN matrix assembly. The data presented demonstrate that cells cultured in the absence of external mechanical support can assemble a FN matrix that promotes integrin-mediated aggregate compaction and cohesion. Further, inhibition of FN matrix assembly blocks the intercellular associations required for compaction, resulting in cell dispersal. These results demonstrate that FN matrix assembly contributes significantly to tissue cohesion and represents an alternative mechanism for regulating tissue architecture.

Animals↗

PubMed2004

Simvastatin suppresses LPS-induced Akt phosphorylation in the human monocyte cell line THP-1.

BACKGROUND: Activation of the small GTPase, Rac, requires post-translational modification by isoprenylation. Statins interfere with this process by blocking the synthesis of isoprenoid intermediates. The protein kinase Akt is a multifunctional regulator of cell behavior that has been linked to Rac activation. We have shown that lipopolysaccharide (LPS) stimulation leads to Rac activation in THP-1 cells. Therefore, we hypothesized that LPS stimulation would also activate Akt, a downstream effector of Rac, and that this may be blocked by statin pretreatment. MATERIALS AND METHODS: THP-1 cells were maintained in 1% fetal calf serum with or without 20 microM simvastatin for 24 h, followed by LPS stimulation for increasing time. Cytoskeletal changes were observed using Alexa-Phalloidin. Akt was immunoprecipitated from total cell lysate. Activated Akt was detected by immunoblotting with a phospho-Akt antibody and was quantified by image densitometry. RESULTS: LPS stimulation of THP-1 cells results in membrane ruffling and cell polarization. Furthermore, LPS increased Akt activation in THP-1 cells when compared with the nonstimulated controls. Akt phosphorylation peaked after 15 min of LPS stimulation and was suppressed by pretreatment with simvastatin. CONCLUSIONS: These data demonstrate that LPS stimulation leads to increased Akt phosphorylation, which can be suppressed with simvastatin pretreatment. This suggests one possible mechanism through which simvastatin could modulate LPS-induced signaling events in monocytes to improve the host response to Gram-negative infections.

Actins↗

PubMed2003

De novo expression of the integrin alpha5beta1 regulates alphavbeta3-mediated adhesion and migration on fibrinogen.

Recent evidence demonstrates that interactions between different integrins that are present on the cell surface can strongly influence the adhesive function of individual receptors. In this report, we show that Chinese hamster ovary cells that express the integrin alphavbeta3 in the absence of alpha5beta1 demonstrate increased adhesion and migration on fibrinogen. Furthermore, alphavbeta3-mediated adhesion to fibrinogen is not augmented by the soluble agonist, MnCl2, suggesting that alphavbeta3 exists in a higher affinity state in these cells. De novo expression of wild-type alpha5beta1 negatively regulates alphavbeta3-mediated adhesion and migration. This effect is not seen with expression of a chimeric alpha5beta1 integrin in which the cytoplasmic portion of the alpha5 integrin subunit is replaced by the cytoplasmic portion of the alpha4 integrin. In addition, it does not require ligation of alpha5beta1 by fibronectin. Cells that express a constitutively active beta3 integrin that contains a point mutation in the conserved membrane proximal region of the cytoplasmic tail, D723R, are resistant to the effect of alpha5beta1 expression. These data provide additional evidence of "cross-talk" between the integrins alpha5beta1 and alphavbeta3, and support the idea that alpha5beta1 regulates alphavbeta3-mediated ligand binding. This provides a relevant biological mechanism whereby variations in alpha5beta1 expression in vivo may modulate activation of alphavbeta3 to influence its adhesive function.

Animals↗

PubMed2003

Alpha5beta1 integrin mediates strong tissue cohesion.

Integrins and cadherins are considered to have distinct and opposing functions. Integrins are traditionally cited for their role in cell-substratum interactions, whereas cadherins are thought to mediate strong intercellular cohesion. Together, these adhesion systems play crucial roles in a wide variety of cellular and developmental processes including cell migration, morphology, differentiation and proliferation. In this manuscript we present evidence that integrins possess the ability to mediate strong intercellular cohesion when cells are grown as 3D aggregates. Much of the data elucidating the role of integrins as mediators of cell-extracellular matrix (ECM) interactions have been generated using conventional cell culture techniques in which cells are plated onto ECM-coated 2D surfaces. In vivo, cells are embedded in a 3D meshwork of ECM proteins. We hypothesized that, within this meshwork, integrin-ECM interactions may impart cohesivity to an aggregate of cells by linking adjacent cells together. To test this hypothesis, we transfected Chinese hamster ovary (CHO-B2) cells to express alpha5beta1 integrin and found that these cells formed compact, spherical aggregates. We measured aggregate cohesivity using tissue surface tensiometry, a novel technique that quantifies cell-cell cohesivity of spheroids under physiological conditions. We determined that alpha5beta1 integrin is capable of conferring strong cohesivity (sigma=8.22+/-0.68 dynes/cm) to aggregates of alpha5-integrin-transfected cells. This cohesion was found to be independent of cadherin expression and was significantly greater than the cohesivity conferred onto CHO-B2 cells transfected with N-cadherin (sigma=3.14+/-0.20 dynes/cm, P</=0.0001), a more traditional cell-cell cohesion system. Fibronectin-null CHO cells that express alpha5beta1 integrin but do not secrete endogenous fibronectin do not form aggregates in fibronectin-depleted medium. Addition of increasing amounts of exogenous dimeric fibronectin to these cells resulted in a dose-dependent compaction. However, compaction failed to occur in the presence of fibronectin monomers. These data indicate that fibronectin is required for alpha5beta1-mediated compaction and that the dimeric structure of fibronectin is essential for this process. Additionally, aggregate formation of the alpha5 integrin transfectants was inhibited by an RGD peptide thus confirming alpha5beta1 integrin specificity. Collectively, these data confirm our hypothesis that alpha5beta1 integrin acts through fibronectin to link adjacent cells together, thus promoting strong intercellular cohesion in 3D cellular aggregates.

Animals↗

PubMed2003

Mevastatin suppresses lipopolysaccharide-induced Rac activation in the human monocyte cell line THP-1.

BACKGROUND: Rac is a member of the Rho family of small guanosine triphosphatases that regulate the actin cytoskeleton. Activation of Rac requires lipid modification that can be blocked by statins. Lipopolysaccharide-induced rearrangements in the actin cytoskeleton lead to changes in cell adhesion and motility that play a role in the cellular response to infection. The lipid products of phosphatidylinositol-3 kinase (PI3K) modulate Rac effector pathways and have been linked to the activation of the Rac-guanosine triphosphatase. We hypothesize that lipopolysaccharide stimulation leads to Rac activation and that this may be inhibited by statin pretreatment. Furthermore, signaling downstream of Rac is linked to PI3K; therefore, we hypothesize that a signaling complex between PI3K and Rac may be involved. METHODS: THP-1 cells were maintained in 1% fetal calf serum with or without 20 micromol/L mevastatin for 24 hours, followed by lipopolysaccharide stimulation. Active Rac was precipitated from THP-1 total cell lysate and then detected by immunoblotting. The PI3K-Rac complex was immunoprecipitated from total cell lysate, and the p85 regulatory subunit of PI3K was detected by immunoblotting. RESULTS: Lipopolysaccharide stimulation activated Rac. Rac activation was suppressed by pretreatment with mevastatin. The p85 subunit of PI3K was associated with Rac. CONCLUSIONS: Lipopolysaccharide stimulation leads to Rac activation in THP-1 cells, which may be suppressed with mevastatin pretreatment. There is an association between Rac and PI3K that demonstrates a role for PI3K in the activation of downstream Rac effector pathways.

Cell Line↗

PubMed2002

Human toll-like receptor 4 mutations but not CD14 polymorphisms are associated with an increased risk of gram-negative infections.

Human toll-like receptor 4 (hTLR4) and CD14 are known to be components of the lipopolysaccharide receptor complex. Our study investigated the association between TLR4 mutations (Asp299Gly and Thr399Ile) and CD14 polymorphism(s) with outcome in an intensive care unit (ICU) population at risk for sepsis. By use of a polymerase chain reaction-based restriction fragment-length polymorphism analysis technique, the hTLR4 gene was altered in 14 (18%) of 77 ICU patients (all positive for systemic inflammatory response syndrome) and in 5 (13%) of 39 volunteers. There was a significantly higher incidence of gram-negative infection among patients with the mutations (11 [79%] of 14), compared with that in the wild-type population (11 [17%] of 63; P=.004). No association between CD14 polymorphism(s) and the incidence of infection or outcome was observed. These findings indicate that hTLR4 mutations are associated with an increased incidence of gram-negative infections in critically ill patients in a surgical setting.

Adult↗