Surface Expression Of Beta1 Integrins Research Articles

Extracellular matrix-specific Caveolin-1 phosphorylation on tyrosine 14 is linked to augmented melanoma metastasis but not tumorigenesis.

Caveolin-1 (CAV1) is a scaffolding protein that plays a dual role in cancer. In advanced stages of this disease, CAV1 expression in tumor cells is associated with enhanced metastatic potential, while, at earlier stages, CAV1 functions as a tumor suppressor. We recently implicated CAV1 phosphorylation on tyrosine 14 (Y14) in CAV1-enhanced cell migration. However, the contribution of this modification to the dual role of CAV1 in cancer remained unexplored. Here, we used in vitro [2D and transendothelial cell migration (TEM), invasion] and in vivo (metastasis) assays, as well as genetic and biochemical approaches to address this question in B16F10 murine melanoma cells. CAV1 promoted directional migration on fibronectin or laminin, two abundant lung extracellular matrix (ECM) components, which correlated with enhanced Y14 phosphorylation during spreading. Moreover, CAV1-driven migration, invasion, TEM and metastasis were ablated by expression of the phosphorylation null CAV1(Y14F), but not the phosphorylation mimicking CAV1(Y14E) mutation. Finally, CAV1-enhanced focal adhesion dynamics and surface expression of beta1 integrin were required for CAV1-driven TEM. Importantly, CAV1 function as a tumor suppressor in tumor formation assays was not altered by the Y14F mutation. In conclusion, our results provide critical insight to the mechanisms of CAV1 action during cancer development. Specific ECM-integrin interactions and Y14 phosphorylation are required for CAV1-enhanced melanoma cell migration, invasion and metastasis to the lung. Because Y14F mutation diminishes metastasis without inhibiting the tumor suppressor function of CAV1, Y14 phosphorylation emerges as an attractive therapeutic target to prevent metastasis without altering beneficial traits of CAV1.

Involvement of integrin up‐regulation in RANKL/RANK pathway of chondrosarcomas migration

Invasion of tumor cells is the primary cause of therapeutic failure in malignant chondrosarcomas treatment. Receptor activator of nuclear factor-kappaB ligand (RANKL) and its receptor, RANK, play a key roles in osteoclastogenesis and tumor metastasis. We found that the RANKL and RANK expression in human chondrosarcoma tissues was higher than that in normal cartilage. We also found that RANKL directed the migration and increased cell surface expression of beta1 integrin in human chondrosarcoma cells (JJ012 cells). Pretreatment of JJ012 cells with MAPK kinase (MEK) inhibitors, PD98059 or U0126, inhibited the RANKL-induced migration and integrin expression. Stimulation of cells with RANKL increased the phosphorylation of MEK and extracellular signal-regulating kinase (ERK). In addition, NF-kappaB inhibitor (PDTC) or IkappaB protease inhibitor (TPCK) also inhibited RANKL-induced cells migration and integrin up-regulation. Taken together, these results suggest that the RANKL acts through MEK/ERK, which in turn activates IKKalpha/beta and NF-kappaB, resulting in the activation of beta1 integrin and contributing to the migration of human chondrosarcoma cells.

Nm23-H1 suppresses hepatocarcinoma cell adhesion and migration on fibronectin by modulating glycosylation of integrin beta1

BackgroundNm23 gene was isolated as a metastatic suppressor gene. The antimetastatic effect of Nm23 has been an enigma for more than 10 years. Little is known about its molecular mechanisms. In this study we overexpressed Nm23-H1 in H7721 cells and observed reduction of cell adhesion, migration and extension of actin stress fibers in cells stimulated by fibronectin (Fn).MethodspcDNA3/Nm23-H1 was introduced into H7721 cells, and expression of Nm23-H1 was monitored by RT-PCR and western blot. Cell adhesion, actin extension and wound-induced migration assays were done on dishes coated with fibronectin. Phosphorylation of focal adhesion kinase (FAK) and total amount of integrin alpha5 and beta1 in Nm23-H1 transfected cells and control cells were measured by western blot. Flow cytometry was used to detect expression of surface alpha5 and beta1 integrin. N-glycosylation inhibitor tunicamycin was used to deglycosylate the integrin beta1 subunit.ResultsOverexpression of nm23-H1 in H7721 cells reduced cell adhesion, migration and extension of actin stress fibers on dishes coated with Fn. Phosphorylation of FAK in Nm23-H1 transfected cells was also attenuated. Integrin alpha5 and beta1 gene messages were unaltered in nm23-H1 overexpressed cells as detected by RT-PCR. However, while cell surface integrin alpha5 was unchanged, surface expression of beta1 integrin was downregulated. Western blot also showed that the total amounts of integrin alpha5 and beta1 were unaltered, but the level of mature integrin beta1 isoform was decreased significantly. Furthermore, partially glycosylated precursor beta1 was increased, which indicated that the impaired glycosylation of integrin beta1 precursor might contribute to the loss of cell surface integrin beta1 in nm23-H1 overexpressed cells.ConclusionThese results suggest that by modulating glycosylation of integrin beta1, nm23-H1 down-regulates integrin beta1 subunit on cell surface and mediates intracellular signaling and subsequent suppression of the invasive process, including cell adhesion and migration.

CD98 activation increases surface expression and clustering of β1 integrins in MCF-7 cells through FAK/Src- and cytoskeleton-independent mechanisms

CD98, a disulfide-linked 125-kDa heterodimeric type II transmembrane glycoprotein, regulates beta1 integrin- mediated cell adhesion. However, the molecular mechanisms underlying CD98-mediated activation of beta1 integrin are presently unclear. In this study, the effects of CD98 signaling on the expression and clustering of beta1 integrin were investigated. Activation of CD98 augmented surface expression of beta1 integrin on MCF-7 cells. Cross-linking CD98 induced clustering of beta1 integrins. Inhibition of phosphorylation of focal adhesion kimase (FAK) by PP2, an inhibitor of Src family kinase, reduced cell-extracellular matrix adhesion, but not surface expression and clustering of beta1 integrin on MCF-7 cells. This result was confirmed by over-expression of dominant negative forms of FAK. In addition, phalloidin or cytochalasin D inhibited CD98-mediated induction of cell-ECM adhesion, but not surface expression and clustering of beta1 integrins. The inhibitory effects of PP2, cytochalasin D or phalloidin on CD98-stimulated cell adhesion were diminished by pretreatment of cells with Mn2+, which is shown to induce conformational change of integrins. These results provide the first evidence that CD98 activation increases not only beta1 integrin affinity but also its surface expression and clustering and the latter is independent of FAK/Src and cytoskeleton.

Differential Regulation of Soluble Guanylyl Cyclase Expression and Signaling by Collagens

Glomerular diseases are characterized by an abnormal synthesis of extracellular matrix proteins, such as collagen type I. Evidence that growth on collagen type I downregulates soluble guanylyl cyclase (sGC) expression and the responsiveness of human mesangial cells to nitric oxide (NO) by activating specific integrin signals involving integrin-linked kinase (ILK) is presented. Human mesangial cells were grown on collagen type I or IV for 24 to 72 h. Compared with collagen IV, growth on collagen I reduced the protein expression and NO-stimulated enzyme activity of sGC. This downregulation was effected at the level of transcription, because steady-state sGC mRNA expression was reduced on collagen I, but inhibition of transcription with Actinomycin D revealed no differences in transcript stability between the two culture conditions. Collagen I also reduced the capacity of cells to relax in response to NO after H2O2 challenge and inhibited NO-induced phosphorylation of vasodilator-activated phosphoprotein, a target of cyclic guanine monophosphate-dependent protein kinase. Examination of the surface expression of integrins, the receptors for extracellular matrix components, revealed that alpha1 and alpha2 integrin subunits were more abundant on cells that were grown on collagen IV and that surface expression of beta1 integrin did not vary with collagen type. However, growth on collagen I induced beta1 integrin to adopt its active conformation, and this activation of beta1 integrin was accompanied by increased activity of its downstream effector ILK. Dominant-negative suppression of ILK signaling relieved the suppression of sGC expression and NO-induced vasodilator-activated phosphoprotein phosphorylation induced by collagen I.

Activated Notch4 inhibits angiogenesis: role of beta 1-integrin activation.

Notch4 is a member of the Notch family of transmembrane receptors that is expressed primarily on endothelial cells. Activation of Notch in various cell systems has been shown to regulate cell fate decisions. The sprouting of endothelial cells from microvessels, or angiogenesis, involves the modulation of the endothelial cell phenotype. Based on the function of other Notch family members and the expression pattern of Notch4, we postulated that Notch4 activation would modulate angiogenesis. Using an in vitro endothelial-sprouting assay, we show that expression of constitutively active Notch4 in human dermal microvascular endothelial cells (HMEC-1) inhibits endothelial sprouting. We also show that activated Notch4 inhibits vascular endothelial growth factor (VEGF)-induced angiogenesis in the chick chorioallantoic membrane in vivo. Activated Notch4 does not inhibit HMEC-1 proliferation or migration through fibrinogen. However, migration through collagen is inhibited. Our data show that Notch4 cells exhibit increased beta1-integrin-mediated adhesion to collagen. HMEC-1 expressing activated Notch4 do not have increased surface expression of beta 1-integrins. Rather, we demonstrate that Notch4-expressing cells display beta1-integrin in an active, high-affinity conformation. Furthermore, using function-activating beta 1-integrin antibodies, we demonstrate that activation of beta1-integrins is sufficient to inhibit VEGF-induced endothelial sprouting in vitro and angiogenesis in vivo. Our findings suggest that constitutive Notch4 activation in endothelial cells inhibits angiogenesis in part by promoting beta 1-integrin-mediated adhesion to the underlying matrix.

Alteration of cell adhesion and cell cycle properties of ES cells by an inducible dominant interfering Myb mutant.

The Myb transcription factors, c-Myb, A-Myb, and B-Myb, regulate cell differentiation and/or proliferation. To investigate the role of B-Myb in embryogenesis, we introduced an inducible dominant interfering Myb protein (MERT) into embryonic stem (ES) cells, which express B-Myb as an exclusive member of Myb family. Disruption of normal B-Myb function by the conditional activation of MERT caused a drastic morphological alteration of ES cells and G(1)-S cell cycle arrest. The inhibition of B-Myb function by MERT dissociated tightly packed ES cell colonies into dispersed single cells that subsequently detached from the culture dish. Cell adhesion analyses revealed that suppression of B-Myb function reduced the adhesion with extracellular matrix proteins, such as laminin, collagen, and fibronectin. This reduction was presumably due to decreased cell surface expression of beta1 integrin. Embryoid body formation was also severely retarded by the activation of MERT. This impairment was attributed to reduced expression of E-cadherin, which functions as a homophilic intercellular adhesion molecule. Simultaneously, blocking B-Myb function did not alter the expression of differentiation markers. Our data indicate that B-Myb plays important roles in regulating cell adhesion and cell cycle progression. These results are well consistent with the recent report on the phenotype of B-Myb null mice and show that the regulation of cell adhesion is an important B-Myb function that has not yet been assumed.

Involvement of the adapter protein CRKL in integrin-mediated adhesion.

CRKL, an SH2-SH3-SH3 adapter protein, is one of the major tyrosine phosphoproteins detected in primary leukemic neutrophils from patients with CML. CRKL binds directly to BCR/ABL through its N-terminal SH3 domain, suggesting it may be involved in BCR/ABL signal transduction. However, the biological function of CRKL in either normal or leukemic cells is still largely unknown. In this study, we have examined the effects of overexpressing full length or deletion mutants of CRKL in hematopoietic cell lines. Full length, SH2- and SH3(N)-domain deletion mutants of CRKL were transfected into an interleukin-3-dependent hematopoietic cell line, Ba/F3, and 3-5 individual sublines which stably overexpressed each transgene were obtained [Ba/F-CRKL, Ba/F-CRKL deltaSH2, and Ba/F-CRKL deltaSH3(N)]. The growth properties of these transfected cells in the presence or absence of IL-3 were not different from mock transfected or untransfected Ba/F3 cells. However, Ba/F3 cells overexpressing full length CRKL, but not deletion mutants of CRKL, were found to have an increase in their ability to bind to fibronectin-coated surfaces. Further, expression of full length, but not deltaSH2- or deltaSH3-CRKL deletion mutants, was found to alter cell morphology on fibronectin-coated plates, an effect which was further enhanced by certain kinds of stress stimuli, such as ionizing radiation. Similar results were obtained when CRKL was transiently overexpressed in Ba/F3 cells, and were also obtained in a second IL-3 dependent hematopoietic cell line, 32Dcl3. Adhesion to fibronectin was blocked by anti-beta1 integrin monoclonal antibody, but overexpression of CRKL did not affect surface expression of beta1 integrins, nor did it spontaneously induce expression of the beta1 integrin 'activation' epitope recognized by the 9EG7 monoclonal antibody. These data suggest a role for CRKL in signaling pathways which regulate adhesion to fibronectin.

Beta1 integrins are critically involved in neutrophil locomotion in extravascular tissue In vivo.

Recruitment of leukocytes from blood to tissue in inflammation requires the function of specific cell surface adhesion molecules. The objective of this study was to identify adhesion molecules that are involved in polymorphonuclear leukocyte (PMN) locomotion in extravascular tissue in vivo. Extravasation and interstitial tissue migration of PMNs was induced in the rat mesentery by chemotactic stimulation with platelet-activating factor (PAF; 10(-7) M). Intravital time-lapse videomicroscopy was used to analyze migration velocity of the activated PMNs, and the modulatory influence on locomotion of locally administered antibodies or peptides recognizing various integrin molecules was examined. Immunofluorescence flow cytometry revealed increased expression of alpha4, beta1, and beta2 integrins on extravasated PMNs compared with blood PMNs. Median migration velocity in response to PAF stimulation was 15.5 +/- 4.5 micron/min (mean +/- SD). Marked reduction (67 +/- 7%) in motility was observed after treatment with mAb blocking beta1 integrin function (VLA integrins), whereas there was little, although significant, reduction (22 +/- 13%) with beta2 integrin mAb. Antibodies or integrin-binding peptides recognizing alpha4beta1, alpha5beta1, or alphavbeta3 were ineffective in modulating migration velocity. Our data demonstrate that cell surface expression of beta1 integrins, although limited on blood PMNs, is induced in extravasated PMNs, and that members of the beta1 integrin family other than alpha4beta1 and alpha5beta1 are critically involved in the chemokinetic movement of PMNs in rat extravascular tissue in vivo.