Cell Adhesion Response Research Articles

Hsa-miR-183-5p Modulates Cell Adhesion by Repression of ITGB1 Expression in Prostate Cancer.

Prostate cancer is a major health problem worldwide. MiR-183 is an oncomiR and a candidate biomarker in prostate cancer, affecting various pathways responsible for disease initiation and progression. We sought to discover the most relevant processes controlled by miR-183 through an unbiased transcriptomic approach using prostate cell lines and patient tissues to identify miR-183 responsive genes and pathways. Gain of function experiments, reporter gene assays, and transcript and protein measurements were conducted to validate predicted functional effects and protein mediators. A total of 135 candidate miR-183 target genes overrepresenting cell adhesion terms were inferred from the integrated transcriptomic analysis. Cell attachment, spreading assays and focal adhesion quantification of miR-183-overexpressing cells confirmed the predicted reduction in cell adhesion. ITGB1 was validated as a major target of repression by miR-183 as well as a mediator of cell adhesion in response to miR-183. The reporter gene assay and PAR-CLIP read mapping suggest that ITGB1 may be a direct target of miR-183. The negative correlation between miR-183 and ITGB1 expression in prostate cancer cohorts supports their interaction in the clinical set. Overall, cell adhesion was uncovered as a major pathway controlled by miR-183 in prostate cancer, and ITGB1 was identified as a relevant mediator of this effect.

EMT Transcription Factors Are Involved in the Altered Cell Adhesion under Simulated Microgravity Effect or Overloading by Regulation of E-cadherin.

In order to study the effect of stress changes on cell adhesion, HUVEC, and MCF-7 cells were treated with simulated microgravity effect (SMG) and overloading (OL). Methods: Rotating Wall Vessel (2D-RWVS) bioreactor was used to create different culture conditions. In addition, the alteration of cell adhesion states, adhesion proteins, and relating factors of adhesion molecules under these two conditions were detected using cell adhesion assay, immunofluorescence, western blot, and qRT-PCR technology. Results: The results showed that the adhesion of cells decreased under SMG, while increased under OL. The expressions of integrin β1, paxillin, and E-cadherin under SMG condition were down-regulated as compared to that of the control group showing a time-dependent pattern of the decreasing. However, under OL condition, the expressions of adhesion proteins were up-regulated as compared to that of the control group, with a time-dependent pattern of increasing. EMT transcription factors Snail, twist, and ZEB1 were up-regulated under SMG while down-regulated under OL. Conclusion: Collectively our results indicated that cells could respond to stress changes to regulate the expressions of adhesion proteins and adapt their adhesion state to the altered mechanical environment. The altered cell adhesion in response to the mechanical stress may involve the changed expression of EMT-inducing factors, Snail, Twist, and ZEB1under the SMG/OL conditions.

Deciphering the transcription factor-microRNA-target gene regulatory network associated with graphene oxide cytotoxicity

Graphene oxide (GO) has recently emanated as a promising material in cancer treatment. To unveil the underlying mechanisms of microRNAs (miRNAs) and potential target genes involved in GO cytotoxicity, we firstly compiled GO-related miRNAs and genes in human cancer cell lines treated with GO from public databases and published works. Besides miRNAs as post-transcriptional regulators of gene expression, transcription factors (TFs) are also the main regulators at the transcriptional level. In the following, we explored the regulatory relationships between miRNAs, target genes, and TFs. Thereafter, a gene regulatory network consisting of GO-responsive miRNAs, GO-responsive genes, and known human TFs was constructed. Then, 3-node regulatory motif types were detected in the resulting network. Among them, miRNA-FFL (feed-forward loop) was identified as a significant motif type. A total of 184 miRNA-FFLs were found and merged to generate a regulatory sub-network. Pathway analysis of the resulting sub-network highlighted adherens junction, focal adhesion, and TGFβ signaling pathways as the major pathways that previous studies demonstrate them to be the affected pathways in GO-treated cells. Functional investigations displayed that miRNAs might be involved in the control of apoptosis through disruption of cell adhesion in response to cytotoxicity. Moreover, GO-cell interactions can lead to miRNA targeting of genes (i.e. Rac1 and RhoA) involved in the cytoskeleton assembly process. These specific toxic properties support biomedical applications of GO, especially for cancer therapy.

SRF'ing and SAP'ing - the role of MRTF proteins in cell migration.

Actin-based cell migration is a fundamental cellular activity that plays a crucial role in a wide range of physiological and pathological processes. An essential feature of the remodeling of actin cytoskeleton during cell motility is the de novo synthesis of factors involved in the regulation of the actin cytoskeleton and cell adhesion in response to growth-factor signaling, and this aspect of cell migration is critically regulated by serum-response factor (SRF)-mediated gene transcription. Myocardin-related transcription factors (MRTFs) are key coactivators of SRF that link actin dynamics to SRF-mediated gene transcription. In this Review, we provide a comprehensive overview of the role of MRTF in both normal and cancer cell migration by discussing its canonical SRF-dependent as well as its recently emerged SRF-independent functions, exerted through its SAP domain, in the context of cell migration. We conclude by highlighting outstanding questions for future research in this field.

The force-sensitive protein Ajuba regulates cell adhesion during epithelial morphogenesis.

The reorganization of cells in response to mechanical forces converts simple epithelial sheets into complex tissues of various shapes and dimensions. Epithelial integrity is maintained throughout tissue remodeling, but the mechanisms that regulate dynamic changes in cell adhesion under tension are not well understood. In Drosophila melanogaster, planar polarized actomyosin forces direct spatially organized cell rearrangements that elongate the body axis. We show that the LIM-domain protein Ajuba is recruited to adherens junctions in a tension-dependent fashion during axis elongation. Ajuba localizes to sites of myosin accumulation at adherens junctions within seconds, and the force-sensitive localization of Ajuba requires its N-terminal domain and two of its three LIM domains. We demonstrate that Ajuba stabilizes adherens junctions in regions of high tension during axis elongation, and that Ajuba activity is required to maintain cell adhesion during cell rearrangement and epithelial closure. These results demonstrate that Ajuba plays an essential role in regulating cell adhesion in response to mechanical forces generated by epithelial morphogenesis.

YAP and MRTF-A, transcriptional co-activators of RhoA-mediated gene expression, are critical for glioblastoma tumorigenicity.

The role of YAP (Yes associated protein 1 gene) and MRTF-A (myocardin-related transcription factor A), two transcriptional co-activators regulated downstream of GPCRs (G-protein coupled receptors) and RhoA, in growth of glioblastoma cells and in vivo glioblastoma multiforme (GBM) tumor development was explored using human glioblastoma cell lines and tumor initiating cells derived from patient derived xenografts (PDX). Knockdown of these co-activators in GSC-23 PDX cells using shRNA significantly attenuated in vitro self-renewal capability assessed by limiting dilution, oncogene expression and neurosphere formation. Orthotopic xenografts of the MRTF-A and YAP knockdown PDX cells formed significantly smaller tumors and were of lower morbidity than wild-type cells. In vitro studies used PDX and 1321N1 glioblastoma cells to examine functional responses to sphingosine 1-phosphate (S1P), a GPCR agonist that activates RhoA signaling, demonstrated that YAP signaling was required for cell migration and invasion whereas MRTF-A was required for cell adhesion; both YAP and MRTF-A were required for proliferation. Gene expression analysis by RNA-sequencing of S1P-treated MRTF-A or YAP knockout cells identified 44 genes that were induced through RhoA and highly dependent on YAP, MRTF-A, or both. Knockdown of F3 (tissue factor gene; TF), a target gene regulated selectively through YAP, blocked cell invasion and migration, whereas knockdown of HBEGF (Heparin binding EGF-like growth factor), a gene selectively induced through MRTF-A, prevented cell adhesion in response to S1P. Proliferation was sensitive to knockdown of target genes regulated through either or both YAP and MRTF-A. Expression of TF and HBEGF was also selectively decreased in tumors from PDX cells lacking YAP or MRTF-A, indicating that these transcriptional pathways are regulated in preclinical GBM models and suggesting that their activation through GPCRs and RhoA contributes to growth and maintenance of human GBM.

Label-free cell-substrate adhesion imaging on plasmonic nanocup arrays.

Cell adhesion is a crucial biological and biomedical parameter defining cell differentiation, cell migration, cell survival, and state of disease. Because of its importance in cellular function, several tools have been developed in order to monitor cell adhesion in response to various biochemical and mechanical cues. However, there remains a need to monitor cell adhesion and cell-substrate separation with a method that allows real-time measurements on accessible equipment. In this article, we present a method to monitor cell-substrate separation at the single cell level using a plasmonic extraordinary optical transmission substrate, which has a high sensitivity to refractive index changes at the metal-dielectric interface. We show how refractive index changes can be detected using intensity peaks in color channel histograms from RGB images taken of the device surface with a brightfield microscope. This allows mapping of the nonuniform refractive index pattern of a single cell cultured on the plasmonic substrate and therefore high-throughput detection of cell-substrate adhesion with observations in real time.

Super-complexes of adhesion GPCRs and neural guidance receptors.

Latrophilin adhesion-GPCRs (Lphn1–3 or ADGRL1–3) and Unc5 cell guidance receptors (Unc5A–D) interact with FLRT proteins (FLRT1–3), thereby promoting cell adhesion and repulsion, respectively. How the three proteins interact and function simultaneously is poorly understood. We show that Unc5D interacts with FLRT2 in cis, controlling cell adhesion in response to externally presented Lphn3. The ectodomains of the three proteins bind cooperatively. Crystal structures of the ternary complex formed by the extracellular domains reveal that Lphn3 dimerizes when bound to FLRT2:Unc5, resulting in a stoichiometry of 1:1:2 (FLRT2:Unc5D:Lphn3). This 1:1:2 complex further dimerizes to form a larger ‘super-complex' (2:2:4), using a previously undescribed binding motif in the Unc5D TSP1 domain. Molecular dynamics simulations, point-directed mutagenesis and mass spectrometry demonstrate the stability and molecular properties of these complexes. Our data exemplify how receptors increase their functional repertoire by forming different context-dependent higher-order complexes.

Non-human Primate and Rat Cardiac Fibroblasts Show Similar Extracellular Matrix-related and Cellular Adhesion Gene Responses to Substance P

The sensory nerve neuropeptide substance P (SP) regulates cardiac fibrosis in rodents under pressure overload conditions. Interestingly, SP induces transient increased expression of specific genes in isolated rat cardiac fibroblasts, without resultant changes in cell function. This suggests that SP 'primes' fibroblasts, but does not directly activate them. We investigated whether these unusual findings are specific to rodent fibroblasts or are translatable to a larger animal model more closely related to humans. We compared the effects of SP on genes associated with extracellular matrix (ECM) regulation, cell-cell adhesion, cell-matrix adhesion and ECM in cardiac fibroblasts isolated from a non-human primate and Sprague-Dawley rats. We found that rodent and non-human primate cardiac fibroblasts showed similar responses in genes that relate to ECM regulation and cell adhesion in response to SP. There were large discrepancies in ECM component genes, however, this did not result in collagen or laminin synthesis in rat or non-human primate fibroblasts in response to SP. This study further supports the notion that SP serves as a 'primer' for fibroblasts rather than initiating direct effects and suggests that rodent fibroblasts are a suitable model for studying gene and functional responses to SP in the absence of human or non-human primate fibroblasts.

P38MAPK is involved in loss of keratinocyte cohesion induced by plakoglobin but not desmoplakin depletion

Desmosomes are patch‐like cell contact structures that are essential for the integrity of the epidermis by providing adhesive strength between keratinocytes. The core is organized of transmembrane proteins of the desmosomal cadherin family, which bind to their counterparts on the adjacent cell via their extracellular domain and are linked intracellularly to the keratin filament network by adapter proteins such as plakoglobin (Pg) and desmoplakin (Dsp). Since in the severe skin blistering disease pemphigus autoantibodies bind to desmosomal cadherins and induce loss of keratinocyte cohesion at least in part via p38MAPK activation and cause depletion of desmosomal components, we evaluated the roles of Pg and Dsp in p38MAPK‐dependent loss of cell adhesion. siRNA‐mediated silencing of either Pg or Dsp protein expression reduced cohesion of cultured human keratinocytes in dissociation assays. However, Pg but not Dsp silencing induced activation of p38MAPK. Accordingly, cell dissociation was blunted by p38MAPK inhibition under conditions of reduced Pg expression only. Interestingly, keratin filament retraction, a hallmark of pemphigus, was prominent under diminished Pg levels, an effect prevented by p38MAPK inhibition. Our data indicate that p38MAPK‐dependent keratin filament collapse is involved in loss of cell adhesion in response to Pg but not Dsp depletion.

Roles of SLP-76, ADAP and Pyk2 on chemokine-stimulated T cell adhesion mediated by the integrin alpha4-beta1

Event Abstract Back to Event Roles of SLP-76, ADAP and Pyk2 on chemokine-stimulated T cell adhesion mediated by the integrin alpha4-beta1 Soledad Isern De Val1, Ana Dios1 and Joaquín Teixidó1* 1 Centro de Investigaciones Biológicas, Spain Lymphocyte extravasation at sites of tissue injury or infection depends on chemokine-activated adhesion mediated by the alpha4-beta1 and alphaL-beta2 integrins. This activation must be rapid to deliver tight cell attachment to resist the blood shear stress. The chemokine-dependent integrin activation step in lymphocytes requires an inside-out signalling that impinges on the integrin beta subunit cytoplasmic domains, a key event for transmitting conformational changes to the extracellular regions that leads to increase in integrin affinity. Talin is a recipient of this inside-out signalling that promote their binding to the beta cytoplasmic domains, finally stimulating the transition to high-affinity alpha4-beta1 and alphaL-beta2 conformations. A key inside-out molecule that regulates the activation of alpha4-beta1 is the guanine-nucleotide exchange factor Vav1, which associates with talin forming a key complex that controls chemokine-dependent, alpha4-beta1-mediated T cell adhesion. SLP-76 and Pyk2 are Vav1-binding proteins with the potential of influencing alpha4-beta1-mediated T cell adhesion in response to chemokine stimulation. We are investigating the role of SLP-76, its binding partner ADAP, and Pyk2, as possible regulators of this adhesion by inside-out mechanisms arising from chemokine stimulation. Keywords: T cells, Chemokines, leukocyte trafficking, Integrins, chemokine signaling Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Immune receptors and signaling Citation: Isern De Val S, Dios A and Teixidó J (2013). Roles of SLP-76, ADAP and Pyk2 on chemokine-stimulated T cell adhesion mediated by the integrin alpha4-beta1. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00944 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Joaquín Teixidó, Centro de Investigaciones Biológicas, Madrid, Spain, joaquint@cib.csic.es Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Soledad Isern De Val Ana Dios Joaquín Teixidó Google Soledad Isern De Val Ana Dios Joaquín Teixidó Google Scholar Soledad Isern De Val Ana Dios Joaquín Teixidó PubMed Soledad Isern De Val Ana Dios Joaquín Teixidó Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Stroma regulates increased epithelial lateral cell adhesion in 3D culture: a role for actin/cadherin dynamics.

BackgroundCell shape and tissue architecture are controlled by changes to junctional proteins and the cytoskeleton. How tissues control the dynamics of adhesion and cytoskeletal tension is unclear. We have studied epithelial tissue architecture using 3D culture models and found that adult primary prostate epithelial cells grow into hollow acinus-like spheroids. Importantly, when co-cultured with stroma the epithelia show increased lateral cell adhesions. To investigate this mechanism further we aimed to: identify a cell line model to allow repeatable and robust experiments; determine whether or not epithelial adhesion molecules were affected by stromal culture; and determine which stromal signalling molecules may influence cell adhesion in 3D epithelial cell cultures.Methodology/Principal FindingsThe prostate cell line, BPH-1, showed increased lateral cell adhesion in response to stroma, when grown as 3D spheroids. Electron microscopy showed that 9.4% of lateral membranes were within 20 nm of each other and that this increased to 54% in the presence of stroma, after 7 days in culture. Stromal signalling did not influence E-cadherin or desmosome RNA or protein expression, but increased E-cadherin/actin co-localisation on the basolateral membranes, and decreased paracellular permeability. Microarray analysis identified several growth factors and pathways that were differentially expressed in stroma in response to 3D epithelial culture. The upregulated growth factors TGFβ2, CXCL12 and FGF10 were selected for further analysis because of previous associations with morphology. Small molecule inhibition of TGFβ2 signalling but not of CXCL12 and FGF10 signalling led to a decrease in actin and E-cadherin co-localisation and increased paracellular permeability.Conclusions/SignificanceIn 3D culture models, paracrine stromal signals increase epithelial cell adhesion via adhesion/cytoskeleton interactions and TGFβ2-dependent mechanisms may play a key role. These findings indicate a role for stroma in maintaining adult epithelial tissue morphology and integrity.

The Structural and Dynamic Response of MAGI-1 PDZ1 with Noncanonical Domain Boundaries to the Binding of Human Papillomavirus E6

PDZ domains are protein interaction domains that are found in cytoplasmic proteins involved in signaling pathways and subcellular transport. Their roles in the control of cell growth, cell polarity, and cell adhesion in response to cell contact render this family of proteins targets during the development of cancer. Targeting of these network hubs by the oncoprotein E6 of “high-risk” human papillomaviruses (HPVs) serves to effect the efficient disruption of cellular processes. Using NMR, we have solved the three-dimensional solution structure of an extended construct of the second PDZ domain of MAGI-1 (MAGI-1 PDZ1) alone and bound to a peptide derived from the C-terminus of HPV16 E6, and we have characterized the changes in backbone dynamics and hydrogen bonding that occur upon binding. The binding event induces quenching of high-frequency motions in the C-terminal tail of the PDZ domain, which contacts the peptide upstream of the canonical X-[T/S]-X-[L/V] binding motif. Mutations designed in the C-terminal flanking region of the PDZ domain resulted in a significant decrease in binding affinity for E6 peptides. This detailed analysis supports the notion of a global response of the PDZ domain to the binding event, with effects propagated to distal sites, and reveals unexpected roles for the sequences flanking the canonical PDZ domain boundaries.

An Intracellular Signal Pathway That Regulates Cancer Cell Adhesion in Response to Extracellular Forces

Increasing evidence suggests that tumor cells can regulate their own adhesion via intracellular signals that modulate integrin binding affinity. Although the full pathway has not yet been elucidated, the effects of pressure seem likely to require cytoskeletal mechanosensing, Src, phosphatidylinositol 3-kinase, focal adhesion kinase, and Akt-1 activation. Ultimately, activated focal adhesion kinase accumulates at the membrane in association with beta(1)-integrin heterodimers and may modulate integrin binding affinity. This pathway may be a promising target for manipulation to inhibit metastatic cancer cell adhesion.

Id-1 promotes TGF-β1-induced cell motility through HSP27 activation and disassembly of adherens junction in prostate epithelial cells

Id-1 (inhibitor of differentiation or DNA binding-1) has been positively associated with cell proliferation, cell cycle progression, and invasiveness during tumorigenesis. In addition, Id-1 has been shown to modulate cellular sensitivity to TGF-β1 (transforming growth factor β1). Here we demonstrate a novel role of Id-1 in promoting TGF-β1-induced cell motility in a non-malignant prostate epithelial cell line, NPTX. We found that Id-1 promoted F-actin stress fiber formation in response to TGF-β1, which was associated with increased cell–substrate adhesion and cell migration in NPTX cells. In addition, this positive effect of Id-1 on TGF-β1-induced cell motility was mediated through activation of MEK–ERK signaling pathway and subsequent phosphorylation of HSP27 (heat shock protein 27). Furthermore, Id-1 disrupted the adherens junction complex in TGF-β1-treated cells through down-regulation of E-cadherin, redistribution of β-catenin, along with up-regulation of N-cadherin. These lines of evidence reveal a novel tumorigenic role of Id-1 through reorganization of actin cytoskeleton and disassembly of cell–cell adhesion in response to TGF-β1 in human prostate epithelial cells, and suggest that intracellular Id-1 levels might be a determining factor for switching TGF-β1 from a growth inhibitor to a tumor promoter during prostate carcinogenesis.

A Structural Hypothesis for the Transition between Bent and Extended Conformations of the Leukocyte β2 Integrins

Integrins mediate cell adhesion in response to activation signals that trigger conformational changes within their ectodomain. It is thought that a compact bent conformation of the molecule represents its physiological low affinity state and extended conformations its active state. We have determined the structure of two integrin fragments of the beta2 subunit. The first structure, consisting of the plexin-semaphorin-integrin domain, hybrid, integrin-epidermal growth factor 1 (I-EGF1), and I-EGF2 domains (PHE2), showed an L-shaped conformation with the bend located between the I-EGF1 and I-EGF2 domains. The second structure, which includes, in addition, the I-EGF3 domain, showed an extended conformation. The major reorientation of I-EGF2 with respect to the other domains in the two structures is accompanied by a change of torsion angle of the disulfide bond between Cys(461)-Cys(492) by 180 degrees and the conversion of a short alpha-helix (residues Ser(468)-Cys(475)) into a flexible coil. Based on the PHE2 structure, we introduced a disulfide bond between the plexin-semaphorin-integrin domain and I-EGF2 domains in the beta2 subunit. The resultant alphaLbeta2 integrin (leukocyte function-associated antigen-1) variant was locked in a bent state and could not be detected with the monoclonal antibody KIM127 in Mg(2+)/EGTA. However, it retained the binding activity to ICAM-1. These results provide a structural hypothesis for our understanding of the transition between the resting and active states of leukocyte function-associated antigen-1.

Ligation of the beta4 integrin triggers adhesion behavior of human keratinocytes by an "inside-out" mechanism.

Carcinogenesis is considered as a multistep process involving functional changes in the hemidesmosomal organization. In normal skin keratinocytes, expression of the alpha(6)beta(4) integrin is restricted to the proliferative basal layer and mediates stable adhesion to the underlying basement membrane. Observations in carcinoma cells show a functional and spatial dissociation of the alpha(6)beta(4) integrin from the hemidesmosomal complex, which stimulates cell migration and, therefore, may contribute to carcinoma invasion. We now have evaluated the adhesion behavior of epithelial cells at different stages of transformation in response to activation of the beta(4) integrin. It is demonstrated that ligation of the beta(4) integrin augmented adhesion of carcinoma and pre-carcinoma cells to non-modified plastic. In contrast, adhesion behavior of normal human keratinocytes was not influenced by ligation of the beta(4) integrin. In order to explain the mechanism of beta(4)-mediated adhesion, the hypothesis of an "inside-out" activation of integrins was tested. Evidence is given that for cells expressing the alpha(6)beta(4) integrin, ligation of the beta(4) integrin increased beta(1) integrin-mediated adhesion. Furthermore, ligation of the beta(4) integrin led to phosphorylation of PKB/Akt at both phosphorylation sites. Functional blocking of PKB/Akt by dominant-negative overexpression decreased cell adhesion in response to beta(4) integrin ligation. Taken together, the present data establish a link between the ligation of the beta(4) integrin and beta(1) integrin-mediated cell adhesion in carcinoma and pre-carcinoma cells. Hence, these findings provide further insight into the conversion processes during carcinogenesis and show the beta(4) integrin to be a key regulator of cellular adhesion.

Colon cancer cell adhesion in response to Src kinase activation and actin-cytoskeleton by non-laminar shear stress.

Malignant cells shed from tumors during surgical resection or spontaneous metastasis experience physical forces such as shear stress and turbulence within the peritoneal cavity during irrigation, laparoscopic air insufflation, or surgical manipulation, and within the venous or lymphatic system. Since physical forces can activate intracellular signals that modulate the biology of various cell types in vitro, we hypothesized that shear stress and turbulence might increase colon cancer cell adhesion to extracellular matrix, potentiating metastatic implantation. Primary human malignant colon cancer cells isolated from resected tumors and SW620 were subjected to shear stress and turbulence by stirring cells in suspension at 600 rpm for 10 min. Shear stress for 10 min increased subsequent SW620 colon cancer cell adhesion by 40.0 +/- 3.0% (n = 3; P < 0.001) and primary cancer cells by 41.0 +/- 3.0% to collagen I when compared to control cells. In vitro kinase assay (1.5 +/- 0.13 fold) and Western analysis (1.34 +/- 0.04 fold) demonstrated a significant increase in Src kinase activity in cells exposed shear stress. Src kinase inhibitors PP1 (0.1 microM), PP2 (20 microM), and actin-cytoskeleton stabilizer phalloidin (10 microM) prevented the shear stress stimulated cell adhesion to collagen I. Furthermore, PP2 inhibited basal (50.0 +/- 2.8%) and prevented shear stress induced src activation but phalloidin pretreatment did not. These results raise the possibility that shear stress and turbulence may stimulate the adhesion of malignant cells shed from colon cancers by a mechanism that requires both actin-cytoskeletal reorganization an independent physical force activation of Src kinase. Blocking this pathway might reduce tumor metastasis during surgical resection.

Controlling Distribution by Cleavage

Arachidonic acid (AA) stimulates the adhesion of cells, such as the metastatic breast carcinoma cell line MDA-MB-435, to extracellular matrix proteins, such as collagen. Kennett et al. show that exposure of cells to AA stimulates activation of protein kinase Cμ (PKCμ) and the p38 mitogen-activated protein kinase and promotes adhesion of MDA-MB-435 to collagen. Not only was PKCμ activated by phosphorylation in response to AA, it was translocated to the membrane and cleaved by calpain, which resulted in the accumulation of a truncated, activated cytosolic form of PKCμ. Pharmacological inhibition of PKCμ or calpain decreased cell adhesion in response to AA. Stimulation of phosphorylation of PKCμ or p38 was independent of each other, on the basis of pharmacological inhibition experiments. However, inhibition of p38 activity did decrease the calpain-mediated proteolysis of PKCμ. Thus, p38 may provide feedback on the PKCμ arm of the pathway not at the level of activation, but at the level of proteolysis by calpain and subsequent subcellular redistribution of the truncated active form of PKCμ. S. B. Kennett, J. D. Roberts, K. Olden, Requirement of protein kinase Cμ activation and calpain-mediated proteolysis for arachidonic acid-stimulated adhesion of MDA-MB-435 human mammary carcinoma cells to collagen type IV. J. Biol. Chem. 279 , 3300-3307 (2004). [Abstract] [Full Text]

Integrin α4β1 involvement in stromal cell-derived factor-1α-promoted myeloma cell transendothelial migration and adhesion: role of cAMP and the actin cytoskeleton in adhesion

The chemokine stromal cell-derived factor-1alpha (SDF-1α) is expressed by bone arrow (BM) stromal cells and plays key roles in cell homing to and retention into the bone marrow. In multiple myeloma, blood-borne malignant plasma cells home to the BM and accumulate in contact with stromal cells, implicating myeloma cell migration across endothelium. Myeloma cells express the SDF-1α receptor CXCR4, as well as the integrin α4ß1, which mediates their attachment to BM stroma. We show here that SDF-1α promotes transendothelial migration of purified BM myeloma cells and myeloma-derived NCI-H929 cells, involving a transient upregulation of α4ß1-dependent cell adhesion to the endothelium. Characterization of intracellular signaling pathways involved in the modulation by SDF-1α of α4ß1-mediated myeloma cell adhesion revealed that intracellular cAMP amounts associated with the activation of protein kinase A play key roles in this modulation. Furthermore, a functional link between cAMP actions on the dynamics of actin cytoskeleton, RhoA activation, and α4ß1-dependent cell adhesion in response to SDF-1α has been found. The regulation of α4ß1-mediated myeloma cell adhesion by SDF-1α could play key roles during myeloma cell homing into and trafficking inside the BM, and characterization of the molecular events involved in SDF-1α-activated modulation of this adhesion will contribute to a better understanding of mechanisms participating in cell migration.