LNK (SH2B3) is a key regulator of integrin signaling in endothelial cells and targets α-parvin to control cell adhesion and migration.

Focal adhesion (FA) formation and disassembly play an essential role in adherence and migration of endothelial cells. These processes are highly regulated and involve various signaling molecules that are not yet completely identified. Lnk [Src homology 2-B3 (SH2B3)] belongs to a family of SH2-containing proteins with important adaptor functions. In this study, we showed that Lnk distribution follows that of vinculin, localizing Lnk in FAs. Inhibition of Lnk by RNA interference resulted in decreased spreading, whereas sustained expression dramatically increases the number of focal and cell-matrix adhesions. We demonstrated that Lnk expression impairs FA turnover and cell migration and regulates ß1-integrin-mediated signaling via Akt and GSK3ß phosphorylation. Moreover, the α-parvin protein was identified as one of the molecular targets of Lnk responsible for impaired FA dynamics and cell migration. Finally, we established the ILK protein as a new molecular partner for Lnk and proposed a model in which Lnk regulates α-parvin expression through its interaction with ILK. Collectively, our results underline the adaptor Lnk as a novel and effective key regulator of integrin-mediated signaling controlling endothelial cell adhesion and migration.

Goblet cells are derived from a FOXJ1-expressing progenitor in a human airway epithelium.

The overproduction of mucus is a key pathology associated with respiratory diseases, such as asthma and chronic obstructive pulmonary disease. These conditions are characterized by an increase in the number of mucus-producing goblet cells in the airways. We have studied the cellular origins of goblet cells using primary human bronchial epithelial cells (HBECs), which can be differentiated to form a stratified epithelium containing ciliated, basal and goblet cells. Treatment of differentiated HBEC cultures with the cytokine IL-13, an important mediator in asthma, increased the numbers of goblet cells and decreased the numbers of ciliated cells. To determine whether ciliated cells act as goblet cell progenitors, ciliated cells in HBEC cultures were hereditably labeled with enhanced green fluorescent protein (EGFP) using two lentiviral vectors, one which contained Cre recombinase under the control of a FOXJ1 promoter and a second Cytomegalovirus (CMV)-floxed-EGFP construct. The fate of the EGFP-labeled ciliated cells was tracked in HBEC cultures. Treatment with IL-13 reduced the numbers of EGFP-labeled ciliated cells compared with untreated cultures. In contrast, IL-13 treatment significantly increased the numbers of EGFP-labeled goblet cells. This study demonstrates that goblet cells formed in response to IL-13 treatment are in part or wholly derived from progenitors that express the ciliated cell marker, FOXJ1.

[The adaptor protein Lnk modulates endothelial cell activation]. / La protéine adaptatrice Lnk module l'activation des cellules endothéliales.

Lnk is an adaptator protein involved in B lymphocytes and platelet differentiation and in T lymphocyte activation. We previously reported on Lnk expression and regulation in endothelial cells (ECs) upon activation. In the present study, the involvement of Lnk in the tumor necrosis factor alpha (TNFalpha) pathway was investigated in vitro through Lnk overexpression in primary cultures of human endothelial cells. Using a recombinant adenovirus encoding human Lnk, we first demonstrated that Lnk overexpression does not induce vascular cell adhesion molecule-1 (VCAM-1) suggesting that Lnk does not promote ECs activation. However, Lnk overexpression significantly reduced TNFalpha-mediated expression of VCAM-1 (at mRNA and protein levels) in activated EC as compared with controls. Western blot analysis showed that Lnk overexpression in HUVEC was associated with phosphorylation of Akt kinase (at Ser 473) with no effect on IkappaBalpha, the specific inhibitor of NFkappaB, indicating that Lnk promotes activation of the phosphatidylinositol 3-kinase (PI3-kinase) pathway in ECs. Altogether, these results suggest that, in ECs, Lnk may participate to a regulatory pathway involving the PI3-kinase and modulating the inflammatory response.

Exogenous tissue inhibitor of metalloproteinase-1 promotes endothelial cell survival through activation of the phosphatidylinositol 3-kinase/Akt pathway.

Control of molecular targets and signaling pathways which improve endothelial cell survival may be an attractive concept for interfering with dysregulated vascular injury and remodeling, a key mechanism for transplant arteriosclerosis and chronic allograft rejection. In addition to inhibiting matrix metalloproteinase activity, it has been suggested by recent studies that the tissue inhibitor of metalloproteinase (TIMP)-1 may inhibit apoptosis in various cell types. The present work examines the possibility that TIMP-1 belongs to a protective pathway via antiapoptotic properties and investigates the signaling pathway mediated by TIMP-1 in human ECs. We demonstrate that exogenous, recombinant, TIMP-1 efficiently prevents apoptosis induced by TNFalpha in cycloheximide-sensitized ECs. The antiapoptotic effect of TIMP-1 was dose-dependent and a maximal effect of TIMP-1 (30% protection) was reached using 250 ng/mL of recombinant TIMP-1. We present evidence that TIMP-1 induces activation of PI3-kinase but not NFkappaB pathway in ECs. Our findings further indicate that TIMP-1-induced EC survival is mediated through activation of PI3-kinase pathway and the downstream phosphorylation of Akt kinase. Blocking the PI3-kinase pathway with wortmannin or LY294002 restores TNFalpha-mediated EC death. In conclusion, our findings suggest that TIMP-1, generated upon inflammation, acts as an antiapoptotic molecule that can prevent EC apoptosis through activation of the PI3-kinase and phosphorylation of the Akt kinase.

The adaptor molecule Lnk negatively regulates tumor necrosis factor-alpha-dependent VCAM-1 expression in endothelial cells through inhibition of the ERK1 and -2 pathways.

Lnk, with APS and SH2-B (Src homology 2-B), belongs to a family of SH2-containing proteins with potential adaptor functions. Lnk regulates growth factor and cytokine receptor-mediated pathways implicated in lymphoid, myeloid, and platelet homeostasis. We have previously shown that Lnk is expressed and up-regulated in vascular endothelial cells (ECs) in response to tumor necrosis factor-alpha (TNFalpha). In this study, we have shown that, in ECs, Lnk down-regulates the expression, at both mRNA and protein levels, of the proinflammatory molecules VCAM-1 and E-selectin induced by TNFalpha. Mechanistically, our data indicated that, in response to TNFalpha, NFkappaB/p65 phosphorylation and translocation as well as IkappaBalpha phosphorylation and degradation were unchanged, suggesting that Lnk does not modulate NFkappaB activity. However, Lnk activates phosphatidylinositol 3-kinase (PI3K) as reflected by Akt phosphorylation. Our results identify endothelial nitric-oxide synthase as a downstream target of Lnk-mediated activation of the PI3K/Akt pathway and HO-1 as a new substrate of Akt. We found that sustained Lnk-mediated activation of PI3K in TNFalpha-activated ECs correlated with the inhibition of ERK1/2 phosphorylation, whereas phosphorylation of p38 and c-Jun NH(2)-terminal kinase (JNK) mitogen-activated protein kinases (MAPKs) was unchanged. ERK1/2 inhibition decreases VCAM-1 expression in TNFalpha-treated ECs. Collectively, our results identify the adaptor Lnk as a negative regulator in the TNFalpha-signaling pathway mediating ERK inhibition and suggest a role for Lnk in the interplay between PI3K and ERK triggered by TNFalpha in ECs.