Distinct Roles of the Adaptor Protein Shc and Focal Adhesion Kinase in Integrin Signaling to ERK

It has been proposed that integrins activate ERK through the adaptor protein Shc independently of focal adhesion kinase (FAK) or through FAK acting on multiple target effectors, including Shc. We show that disruption of the actin cytoskeleton by cytochalasin D causes a complete inhibition of FAK but does not inhibit Shc signaling and activation of ERK. We have then generated primary fibroblasts carrying a targeted deletion of the segment of beta(1) subunit cytoplasmic domain required for activation of FAK. Analysis of these cells indicates that FAK is not necessary for efficient tyrosine phosphorylation of Shc, association of Shc with Grb2, and activation of ERK in response to matrix adhesion. In addition, integrin-mediated activation of FAK does not appear to be required for signaling to ERK following growth factor stimulation. To examine if FAK could contribute to the activation of ERK in a cell type-specific manner through the Rap1/B-Raf pathway, we have used Swiss-3T3 cells, which in contrast to primary fibroblasts express B-Raf. Dominant negative studies indicate that Shc mediates the early phase and peak, whereas FAK, p130(CAS), Crk, and Rap1 contribute to the late phase of integrin-dependent activation of ERK in these cells. In addition, introduction of B-Raf enhances and sustains integrin-mediated activation of ERK in wild-type primary fibroblasts but not in those carrying the targeted deletion of the beta(1) cytoplasmic domain. Thus, the Shc and FAK pathways are activated independently and function in a parallel fashion. Although not necessary for signaling to ERK in primary fibroblasts, FAK may enhance and prolong integrin-mediated activation of ERK through p130(CAS), Crk, and Rap1 in cells expressing B-Raf

The coupling of α 6 β 4 integrin to Ras-MAP kinase pathways mediated by Shc controls keratinocyte proliferation

The signaling pathways linking integrins to nuclear events are incompletely understood. We have examined intracellular signaling by the alpha6beta4 integrin, a laminin receptor expressed in basal keratinocytes and other cells. Ligation of alpha6beta4 in primary human keratinocytes caused tyrosine phosphorylation of Shc, recruitment of Grb2, activation of Ras and stimulation of the MAP kinases Erk and Jnk. In contrast, ligation of the laminin- and collagen-binding integrins alpha3beta1 and alpha2beta1 did not cause these events. While the stimulation of Erk by alpha6beta4 was suppressed by dominant-negative Shc, Ras and RhoA, the activation of Jnk was inhibited by dominant-negative Ras and Rac1 and by the phosphoinositide 3-kinase inhibitor Wortmannin. Adhesion mediated by alpha6beta4 induced transcription from the Fos serum response element and promoted cell cycle progression in response to mitogens. In contrast, alpha3beta1- and alpha2beta1-dependent adhesion did not induce these events. These findings suggest that the coupling of alpha6beta4 integrin to the control of cell cycle progression mediated by Shc regulates the proliferation of basal keratinocytes and possibly other cells which are in contact with the basement membrane in vivo

Downregulation of urokinase plasminogen activator receptor expression inhibits Erk signalling with concomitant suppression of invasiveness due to loss of uPAR–β1 integrin complex in colon cancer cells

Cancer invasion is regulated by cell surface proteinases and adhesion molecules. Interaction between specific cell surface molecules such as urokinase plasminogen activator receptor (uPAR) and integrins is crucial for tumour invasion and metastasis. In this study, we examined whether uPAR and beta1 integrin form a functional complex to mediate signalling required for tumour invasion. We assessed the expression of uPAR/beta1 integrin complex, Erk signalling pathway, adhesion, uPA and matrix metalloproteinase (MMP) expression, migration/invasion and matrix degradation in a colon cancer cell line in which uPAR expression was modified. Antisense inhibition of the cell surface expression of uPAR by 50% in human colon carcinoma HCT116 cells (A/S) suppressed Erk-MAP kinase activity by two-fold. Urokinase plasminogen activator receptor antisense treatment of HCT116 cells was associated with a 1.3-fold inhibition of adhesion, approximately four-fold suppression of HMW-uPA secretion and inhibition of pro-MMP-9 secretion. At a functional level, uPAR antisense resulted in a four-fold decline in migration/invasion and abatement of plasmin-mediated matrix degradation. In empty vector-transfected cells (mock), uPA strongly elevated basal Erk activation. In contrast, in A/S cells, uPA induction of Erk activation was not observed. Urokinase plasminogen activator receptor associated with beta1 integrin in mock-transfected cells. Disruption of uPAR-beta1 integrin complex in mock-transfected cells with a specific peptide (P25) inhibited uPA-mediated Erk-MAP kinase pathway and inhibited migration/invasion and plasmin-dependent matrix degradation through suppression of pro-MMP-9/MMP-2 expression. This novel paradigm of uPAR-integrin signalling may afford opportunities for alternative therapeutic strategies for the treatment of cancer

Restoration of caveolin-1 expression suppresses growth and metastasis of head and neck squamous cell carcinoma

Caveolin-1 (Cav-1) plays an important role in modulating cellular signalling, but its role in metastasis is not well defined. A significant reduction in Cav-1 levels was detected in lymph node metastases as compared with primary tumour of head and neck squamous cell carcinoma (HNSCC) specimens (P<0.0001), confirming the downregulation of Cav-1 observed in a highly metastatic M4 cell lines derived from our orthotopic xenograft model. To investigate the function of Cav-1 in metastasis of HNSCC, we compared stable clones of M4 cells carrying human cav-1 cDNA (CavS) with cells expressing an empty vector (EV) in vitro and in the orthotopic xenograft model. Overexpression of Cav-1 suppressed growth of the CavS tumours compared with the EV tumours. The incidence of lung metastases was significantly lower in animals carrying CavS tumours than those with EV tumours (P=0.03). In vitro, CavS cells displayed reduced cell growth, invasion, and increased anoikis compared with EV cells. In CavS cells, Cav-1 formed complex with integrin β1 and Src. Further application of integrin β1 neutralising antibody or Src inhibitor PP2 to EV cells illustrated similar phenotypes as CavS cells, suggesting that Cav-1 may play an inhibitory role in tumorigenesis and lung metastasis through regulating integrin β1- and Src-mediated cell–cell and cell–matrix interactions

Stanniocalcin-1 Regulates Re-Epithelialization in Human Keratinocytes

Stanniocalcin-1 (STC1), a glycoprotein hormone, is believed to be involved in various biological processes such as inflammation, oxidative responses and cell migration. Riding on these emerging evidences, we hypothesized that STC1 may participate in the re-epithelialization during wound healing. Re-epithelialization is a critical step that involves keratinocyte lamellipodia (e-lam) formation, followed by cell migration. In this study, staurosporine (STS) treatment induced human keratinocyte (HaCaT) e-lam formation on fibronectin matrix and migration via the activation of focal adhesion kinase (FAK), the surge of intracellular calcium level [Ca2+]i and the inactivation of Akt. In accompanied with these migratory features, a time- and dose-dependent increase in STC1 expression was detected. STC1 gene expression was found not the downstream target of FAK-signaling as illustrated by FAK inhibition using PF573228. The reduction of [Ca2+]i by BAPTA/AM blocked the STS-mediated keratinocyte migration and STC1 gene expression. Alternatively the increase of [Ca2+]i by ionomycin exerted promotional effect on STS-induced STC1 gene expression. The inhibition of Akt by SH6 and GSK3β by lithium chloride (LiCl) could respectively induce and inhibit the STS-mediated e-lam formation, cell migration and STC1 gene expression. The STS-mediated e-lam formation and cell migration were notably hindered or induced respectively by STC1 knockdown or overexpression. This notion was further supported by the scratched wound assay. Collectively the findings provide the first evidence that STC1 promotes re-epithelialization in wound healing

Detection of delirium by nurses among long-term care residents with dementia

<p>Abstract</p> <p>Background</p> <p>Delirium is a prevalent problem in long-term care (LTC) facilities where advanced age and cognitive impairment represent two important risk factors for this condition. Delirium is associated with numerous negative outcomes including increased morbidity and mortality. Despite its clinical importance, delirium often goes unrecognized by nurses. Although rates of nurse-detected delirium have been studied among hospitalized older patients, this issue has been largely neglected among demented older residents in LTC settings. The goals of this study were to determine detection rates of delirium and delirium symptoms by nurses among elderly residents with dementia and to identify factors associated with undetected cases of delirium.</p> <p>Methods</p> <p>In this prospective study (N = 156), nurse ratings of delirium were compared to researcher ratings of delirium. This procedure was repeated for 6 delirium symptoms. Sensitivity, specificity, positive and negative predictive values were computed. Logistic regressions were conducted to identify factors associated with delirium that is undetected by nurses.</p> <p>Results</p> <p>Despite a high prevalence of delirium in this cohort (71.5%), nurses were able to detect the delirium in only a minority of cases (13%). Of the 134 residents not identified by nurses as having delirium, only 29.9% of them were correctly classified. Detection rates for the 6 delirium symptoms varied between 39.1% and 58.1%, indicating an overall under-recognition of symptoms of delirium. Only the age of the residents (≥ 85 yrs) was associated with undetected delirium (OR: 4.1; 90% CI: [1.5–11.0]).</p> <p>Conclusion</p> <p>Detection of delirium is a major issue for nurses that clearly needs to be addressed. Strategies to improve recognition of delirium could result in a reduction of adverse outcomes for this very vulnerable population.</p