Integrin signalling in directed cell migration

Migrating cells tend to continue moving in the same direction, a property called persistence. During migration, cells, by definition, form new adhesions at their front and break old adhesions at the rear. We hypothesize that the distinction between new adhesions at the front and older adhesions at the rear plays a major role in directional persistence. We propose specific mechanisms of persistence on the basis of known properties of integrin signals, in hope of stimulating investigation of these ideas.     

Integrin beta1 over-expression associates with resistance to tyrosine kinase inhibitor gefitinib in non-small cell lung cancer

The epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) such as gefitinib and erlotinib have been widely used in treating patients with advanced non-small cell lung cancer (NSCLC). However, acquired resistance to EGFR TKI almost occurs in every patient eventually. To identify its potential mechanism, we established a human NSCLC cell line PC9/AB2 which was 576-fold decrease in gefitinib sensitivity compared with its parental PC9 cell lines. No EGFR-T790M mutation or abnormal expression of c-Met protein was found in PC9/AB2 cells. Over-expression of integrin β1 was found, accompanied with increase of the cells' adhesion and migration. To further confirm the role of integrin β1 in gefitinib acquired resistance, we transferred its siRNA-expressing plasmid and its whole cDNA expressing plasmid into PC9/AB2 and into PC9 cells, respectively. The sensitivity of NSCLC cells to gefitinib was negatively correlated with integrin β1 expression levels. All these data suggest that up-regulation of integrin β1 might be an important factor for gefitinib resistance in NSCLC cell line PC9/AB2.     

The integrin alpha9beta1 mediates adhesion to activated endothelial cells and transendothelial neutrophil migration through interaction with vascular cell adhesion molecule-1

The integrin alpha9beta1 has been shown to be widely expressed on smooth muscle and epithelial cells, and to mediate adhesion to the extracellular matrix proteins osteopontin and tenascin-C. We have found that the peptide sequence this integrin recognizes in tenascin-C is highly homologous to the sequence recognized by the closely related integrin alpha4beta1, in the inducible endothelial ligand, vascular cell adhesion mole-cule-1 (VCAM-1). We therefore sought to determine whether alpha9beta1 also recognizes VCAM-1, and whether any such interaction would be biologically significant. In this report, we demonstrate that alpha9beta1 mediates stable cell adhesion to recombinant VCAM-1 and to VCAM-1 induced on human umbilical vein endothelial cells by tumor necrosis factor-alpha. Furthermore, we show that alpha9beta1 is highly and selectively expressed on neutrophils and is critical for neutrophil migration on VCAM-1 and tenascin-C. Finally, alpha9beta1 and alpha4 integrins contribute to neutrophil chemotaxis across activated endothelial monolayers. These observations suggest a possible role for alpha9beta1/VCAM-1 interactions in extravasation of neutrophils at sites of acute inflammation.     

Intermediate-affinity LFA-1 binds alpha-actinin-1 to control migration at the leading edge of the T cell

T lymphocytes use LFA-1 to migrate into lymph nodes and inflammatory sites. To investigate the mechanisms regulating this migration, we utilize mAbs selective for conformational epitopes as probes for active LFA-1. Expression of the KIM127 epitope, but not the 24 epitope, defines the extended conformation of LFA-1, which has intermediate affinity for ligand ICAM-1. A key finding is that KIM127-positive LFA-1 forms new adhesions at the T lymphocyte leading edge. This LFA-1 links to the cytoskeleton through alpha-actinin-1 and disruption at the level of integrin or actin results in loss of cell spreading and migratory speed due to a failure of attachment at the leading edge. The KIM127 pattern contrasts with high-affinity LFA-1 that expresses both 24 and KIM127 epitopes, is restricted to the mid-cell focal zone and controls ICAM-1 attachment. Identification of distinctive roles for intermediate- and high-affinity LFA-1 in T lymphocyte migration provides a biological function for two active conformations of this integrin for the first time.     

TGF-beta control of cell proliferation

This article focuses on recent findings that the type V TGF-beta receptor (TbetaR-V), which co-expresses with other TGF-beta receptors (TbetaR-I, TbetaR-II, and TbetaR-III) in all normal cell types studied, is involved in growth inhibition by IGFBP-3 and TGF-beta and that TGF-beta activity is regulated by two distinct endocytic pathways (clathrin- and caveolar/lipid-raft-mediated). TGF-beta is a potent growth inhibitor for most cell types, including epithelial and endothelial cells. The signaling by which TGF-beta controls cell proliferation is not well understood. Many lines of evidence indicate that other signaling pathways, in addition to the prominent TbetaR-I/TbetaR-II/Smad2/3/4 signaling cascade, are required for mediating TGF-beta-induced growth inhibition. Recent studies revealed that TbetaR-V, which is identical to LRP-1, mediates IGF-independent growth inhibition by IGFBP-3 and mediates TGF-beta-induced growth inhibition in concert with TbetaR-I and TbetaR-II. In addition, IRS proteins and a Ser/Thr-specific protein phosphatase(s) are involved in the TbetaR-V-mediated growth inhibitory signaling cascade. The TbetaR-V signaling cascade appears to cross-talk with the TbetaR-I/TbetaR-II, insulin receptor (IR), IGF-I receptor (IGF-IR), integrin and c-Met signaling cascades. Attenuation or loss of the TbetaR-V signaling cascade may enable carcinoma cells to escape from TGF-beta growth control and may contribute to the aggressiveness and invasiveness of these cells via promoting epithelial-to-mesenchymal transdifferentiation (EMT). Finally, the ratio of TGF-beta binding to TbetaR-II and TbetaR-I is a signal controlling TGF-beta partitioning between two distinct endocytosis pathways and resultant TGF-beta responsiveness. These recent studies have provided new insights into the molecular mechanisms underlying TGF-beta-induced cellular growth inhibition, cross-talk between the TbetaR-V and other signaling cascades, the signal that controls TGF-beta responsiveness and the role of TbetaR-V in tumorigenesis.     

Regulation of the Type II Hemidesmosomal Plaque Assembly in Intestinal Epithelial Cells

Hemidesmosomes (HDs) are cellular junctions that anchor epithelial cells to the extracellular matrix (ECM) and are associated morphologically with the cytoskeleton. Hemidesmosomal molecular components include two proteins involved in linking intermediate filaments, HD1/plectin and BP230, and two transmembrane proteins, BP180 and the alpha6beta4 integrin, a laminin receptor. In cells lacking BP230 and BP180, HD1/plectin still associates with alpha6beta4 integrin, forming HD-like structures, called type II HDs. In the present study, we used an intestinal epithelial cell line that expresses HD1/plectin and the alpha6beta4 integrin to investigate the regulation of assembly of these proteins in type II HDs. These compounds were found to be clustered at sites of cell-ECM contact and their polarized localization was influenced by either cell confluency or extracellular matrix deposition. Conventional and immunoelectron microscopy showed that HD1/plectin and the beta4 integrin subunit are colocalized in an adhesion structure. Using cytoskeleton-disrupting drugs and confocal microscopy, we demonstrated that type II HDs are made up of numerous individual plaques whose assembly into a cluster requires actin filaments, but not microtubules.     

Integrin alphaVbeta6-mediated activation of latent TGF-beta requires the latent TGF-beta binding protein-1

Transforming growth factor-betas (TGF-beta) are secreted as inactive complexes containing the TGF-beta, the TGF-beta propeptide, also called the latency-associated protein (LAP), and the latent TGF-beta binding protein (LTBP). Extracellular activation of this complex is a critical but incompletely understood step in TGF-beta regulation. We have investigated the role of LTBP in modulating TGF-beta generation by the integrin alphaVbeta6. We show that even though alphavbeta6 recognizes an RGD on LAP, LTBP-1 is required for alphaVbeta6-mediated latent TGF-beta activation. The domains of LTBP-1 necessary for activation include the TGF-beta propeptide-binding domain and a basic amino acid sequence (hinge domain) with ECM targeting properties. Our results demonstrate an LTBP-1 isoform-specific function in alphaVbeta6-mediated latent TGF-beta activation; LTBP-3 is unable to substitute for LTBP-1 in this assay. The results reveal a functional role for LTBP-1 in latent TGF-beta activation and suggest that activation of specific latent complexes is regulated by distinct mechanisms that may be determined by the LTBP isoform and its potential interaction with the matrix.     

Stimulation-dependent recycling of integrin beta1 regulated by ARF6 and Rab11

In comparison to the internalization pathways of endocytosis, the recycling pathways are less understood. Even less defined is the process of regulated recycling, as few examples exist and their underlying mechanisms remain to be clarified. In this study, we examine the endocytic recycling of integrin β1, a process that has been suggested to play an important role during cell motility by mediating the redistribution of integrins to the migrating front. External stimulation regulates the endocytic itinerary of β1, mainly at an internal compartment that is likely to be a subset of the recycling endosomes. This stimulation-dependent recycling is regulated by ARF6 and Rab11, and also requires the actin cytoskeleton in an ARF6-dependent manner. Consistent with these observations being relevant for cell motility, mutant forms of ARF6 that affect either actin rearrangement or recycling inhibit the motility of a breast cancer cell line.     

Integrin signaling and cell spreading mediated by phorbol 12-myristate 13-acetate treatment

Spreading of SNU16mAd gastric carcinoma cells was previously shown to be regulated via a signaling network from transforming growth factor beta1 (TGFbeta1) to integrins signaling, through a mediation of protein kinase C delta (PKCdelta). However, in the previous study, the roles of PKCdelta appeared complicated. In this study to clarify the roles of PKCdelta in the spreading of the gastric carcinoma cells, we questioned if PKC activation via phorbol 12-myristate 13-acetate (PMA) treatment could mimic the TGFbeta1 effects. An acute PMA treatment increased phosphorylations of focal adhesion (FA) kinase, paxillin, c-Src, and cofilin, just as TGFbeta1 did. Furthermore, cell spreading mediated by TGFbeta1- or acute PMA treatment correlated with activation of RhoA, which regulates actin reorganization and FA formation. However, stress fiber formation was prominent in TGFbeta1-treated cells, compared to cortical actin organization in PMA-treated cells. Altogether, these observations indicate that acute PMA treatment could mimic the TGFbeta1 mechanisms for cell spreading through subtly different effects on actin reorganization.     

Effect of cyclophosphamide and 61.22 GHz millimeter waves on T-cell, B-cell, and macrophage functions

The present study was undertaken to investigate whether millimeter waves (MMWs) at 61.22 GHz can modulate the effect of cyclophosphamide (CPA), an anti-cancer drug, on the immune functions of mice. During the exposure each mouse's nose was placed in front of the center of the antenna aperture (1.5 x 1.5 cm) of MMW generator. The device produced 61.22 +/- 0.2 GHz wave radiation. Spatial peak Specific Absorption Rate (SAR) at the skin surface and spatial peak incident power density were measured as 885 +/- 100 W/kg and 31 +/- 5 mW/cm(2), respectively. Duration of the exposure was 30 min each day for 3 consecutive days. The maximum temperature elevation at the tip of the nose, measured at the end of 30 min, was 1 degrees C. CPA injection (100 mg/kg) was given intraperitoneally on the second day of exposure to MMWs. The animals were sacrificed 2, 5, and 7 days after CPA administration. MMW exposure caused upregulation in tumor necrosis factor-alpha (TNF-alpha) production in peritoneal macrophages suppressed by CPA administration. MMWs also caused a significant increase in interferon-gamma (IFN-gamma) production by splenocytes and enhanced proliferative activity of T-cells. Conversely, no changes were observed in interleukin-10 (IL-10) level and B-cell proliferation. These results suggest that MMWs accelerate the recovery process selectively through a T-cell-mediated immune response.     

Phloroglucinol derivative MCPP induces cell apoptosis in human colon cancer

This study is the first to investigate the anticancer effects of the new phloroglucinol derivative (3,6-bis(3-chlorophenylacetyl)phloroglucinol; MCPP) in human colon cancer cells. MCPP induced cell death and antiproliferation in three human colon cancer, HCT-116, SW480, and Caco-2 cells, but not in primary human dermal fibroblast cells. MCPP-induced concentration-dependent apoptotic cell death in colon cancer cells was measured by fluorescence-activated cell sorter (FACS) analysis. Treatment of HCT-116 human colon cancer cells with MCPP was found to induce a number of signature endoplasmic reticulum (ER) stress markers; and up-regulation of CCAAT/enhancer-binding protein homologous protein (CHOP) and glucose-regulated protein (GRP)-78, phosphorylation of eukaryotic initiation factor-2α (eIF-2α), suggesting the induction of ER stress. MCPP also increased GSK3α/β(Tyr270/216) phosphorylation and reduced GSK3α/β(Ser21/9) phosphorylation time-dependently. Transfection of cells with GRP78 or CHOP siRNA, or treatment of GSK3 inhibitor SB216163 reduced MCPP-mediated cell apoptosis. Treatment of MCPP also increased caspase-7, caspase-9, and caspase-3 activity. The inhibition of caspase activity by z-DEVE-FMK or z-VAD-FMK significantly reduced MCPP-induced apoptosis. Furthermore, treatment of GSK3 inhibitor SB216763 also dramatically reversed MCPP-induced GRP and CHOP up-regulation, and pro-caspase-3 and pro-caspase-9 degradation. Taken together, the present study provides evidences to support that GRP78 and CHOP expression, and GSK3α/β activation in mediating the MCPP-induced human colon cancer cell apoptosis.     

Glycogen synthase kinase-3beta regulates DeltaNp63 gene transcription through the beta-catenin signaling pathway

Overexpression of DeltaNp63 has been observed in a number of human cancers, suggesting a role for DeltaNp63 in carcinogenesis. In the present study, we show that inhibition of glycogen synthase kinase-3beta (GSK-3beta) by lithium chloride (LiCl) elicited a stimulatory effect on DeltaNp63 promoter activity in HEK 293T cells. Exposure to LiCl induced DeltaNp63 promoter activation as well as DeltaNp63 protein expression in the cells. The effect of GSK-3beta on DeltaNp63 expression was further confirmed by the use of two highly specific GSK-3beta inhibitors, SB216763 and SB415286. Further study showed the presence of a putative beta-catenin responsive element (beta-catenin-RE) in the DeltaNp63 promoter region, and the stimulation of DeltaNp63 promoter activity by GSK-3beta inhibitor is markedly abolished by mutation or deletion of the putative beta-catenin-RE. Data are also presented to show that beta-catenin acts together with Lef-1 to influence DeltaNp63 promoter activity and protein expression.     

RIFLE: a novel ring zinc finger-leucine-rich repeat containing protein, regulates select cell adhesion molecules in PC12 cells

Cell adhesion molecules play a critical role in cell contacts, whether cell-cell or cell-matrix, and are regulated by multiple signaling pathways. In this report, we identify a novel ring zinc finger-leucine-rich repeat containing protein (RIFLE) and show that RIFLE, expressed in PC12 cells, enhances the Serine (Ser)21/9 phosphorylation of glycogen synthase kinase-3alpha/beta (GSK-3alpha/beta) resulting in the inhibition of GSK-3 kinase activity and increase of beta-catenin levels. RIFLE expression also is associated with elevated E-cadherin protein levels but not N-cadherin. The regulation of these cell adhesion-associated molecules by RIFLE is accompanied by a significant increase in cell-cell and cell-matrix adhesion. Moreover, increase in cell-cell adhesion but not cell-matrix adhesion by RIFLE can be mimicked by selective inhibition of GSK-3. Our results suggest that RIFLE represents a novel signaling protein that mediates components of the Wnt/wingless signaling pathway and cell adhesion in PC12 cells.     

Ultrastructural localization of integrin subunits beta4 and alpha3 within the migrating epithelial tongue of in vivo human wounds

Subsequent to wounding, keratinocytes must quickly restore barrier function. In vitro wound models have served to elucidate mechanisms of epithelial closure and key roles for integrins alpha6beta4 and alpha3beta1. To extrapolate in vitro data to in vivo human tissues, we used ultrathin cryomicrotomy to simultaneously observe tissue ultrastructure and immunogold localization in unwounded skin and acute human cutaneous wounds. Localization of the beta4 integrin subunit in unwounded skin shows dominant hemidesmosomal association and minor basal keratinocyte lateral filopodic cell-cell expression. After wounding, beta4 dominantly localized to cytokeratin-rich regions (trailing edge hemidesmosomes) and minor association with lamellipodia (leading edge). beta4 colocalizes with alpha3 within filopodia juxtaposed to wound matrix, and increased concentrations of beta4 were found in cytoplasmic vesicles within basal keratinocytes of the migrating tongue. alpha3 integrin subunit dominantly localized to filopodia within basal keratinocyte lateral cell-cell interfaces in unwounded skin and both cell-cell and cell-matrix filopodic interactions in wounded skin. This study indicates that beta4 interacts with the extracellular environment through both stable and transient interactions and may be managed through a different endosomal trafficking pathway than alpha3. alpha3 integrin, despite its ability to respond to alternate ligands after wounding, does so through a single structure, the filopodia.     

Palladin regulates cell and extracellular matrix interaction through maintaining normal actin cytoskeleton architecture and stabilizing beta1-integrin

Cell and extracellular matrix (ECM) interaction plays an important role in development and normal cellular function. Cell adhesion and cell spreading on ECM are two basic cellular behaviors related to cell-ECM interaction. Here we show that palladin, a novel actin cytoskeleton-associated protein, is actively involved in the regulation of cell-ECM interaction. It was found that palladin-deficient mouse embryonic fibroblasts (MEFs) display decreased cell adhesion and compromised cell spreading on various ECMs. Disorganized actin cytoskeleton architecture characterized by faint stress fibers, less lamellipodia and focal adhesions can account for the weakened cell-ECM interaction in palladin(-/-) MEFs. Furthermore, decreased polymerized filament actin and increased globular actin can be observed in palladin(-/-) MEFs, strongly suggesting that palladin is essential for the formation or stabilization of polymerized filament actin. Elevated phospho-cofilin level and proper responses in cofilin phosphorylation to either Rho signal agonist or antagonist in palladin(-/-) MEFs indicate that disrupted stress fibers in palladin(-/-) MEFs is not associated with cofilin phosphorylation. More interestingly, the protein level of ECM receptor beta1-integrin is dramatically decreased in MEFs lacking palladin. Down-regulation of beta1-integrin protein can be restored by proteasome inhibitor MG-132 treatment. All these data implicate that palladin is essential for cell-ECM interaction through maintaining normal actin cytoskeleton architecture and stabilizing beta1-integrin protein.     

Endothelial cell and macrophage regulation of vascular smooth muscle cell calcification modulated by cholestane-3beta, 5alpha, 6beta-triol

The cellular and molecular mechanisms that mediate vascular calcification remain poorly understood. In our previous study, oxysterol cholestane-3beta, 5alpha, 6beta-triol (Triol) was shown to promote vascular smooth muscle cells (VSMCs) calcification. In this study, by using direct coculture, non-contact transwell coculture, and culture with conditioned media, we investigated the roles of endothelial cells (ECs) and macrophages in the regulation of VSMCs calcification in the absence or presence of Triol. In vitro calcification was induced by incubation of VSMCs with beta-glycerophosphate. The results showed that ECs inhibited VSMCs calcification, as manifested by the reduction of calcium deposition in extracellular matrix. This effect of ECs on calcification was via the secreted soluble factors. Furthermore, the stimulation of ECs by Triol had no influence on ECs inhibition of calcification. On the other hand, macrophages promoted VSMCs calcification via the secreted soluble factors such as reactive oxygen species, which was further enhanced by Triol. Our results supported the roles for ECs and macrophages in vascular calcification, modulated by oxysterols in atherosclerotic plaque.