Modulation Of Integrin Activity Research Articles

MiRNA-200c-3p targets talin-1 to regulate integrin-mediated cell adhesion

The ability of integrins on the cell surface to mediate cell adhesion to the extracellular matrix ligands is regulated by intracellular signaling cascades. During this signaling process, the talin (TLN) recruited to integrin cytoplasmic tails plays the critical role of the major adaptor protein to trigger integrin activation. Thus, intracellular levels of TLN are thought to determine integrin-mediated cellular functions. However, the epigenetic regulation of TLN expression and consequent modulation of integrin activation remain to be elucidated. Bioinformatics analysis led us to consider miR-200c-3p as a TLN1-targeting miRNA. To test this, we have generated miR-200c-3p-overexpressing and miR-200c-3p-underexpressing cell lines, including HEK293T, HCT116, and LNCaP cells. Overexpression of miR-200c-3p resulted in a remarkable decrease in the expression of TLN1, which was associated with the suppression of integrin-mediated cell adhesion to fibronectin. In contrast, the reduction in endogenous miR-200c-3p levels led to increased expression of TLN1 and enhanced cell adhesion to fibronectin and focal adhesion plaques formation. Moreover, miR-200c-3p was found to target TLN1 by binding to its 3′-untranslated region (UTR). Taken together, our data indicate that miR-200c-3p contributes to the regulation of integrin activation and cell adhesion via the targeting of TLN1.

Proliferative vitreoretinopathy prophylaxis : Mission (im)possible

Proliferative vitreoretinopathy (PVR) is one of the most important complications following vitreoretinal surgery. So far, surgical strategies have been the gold standard in treatment. Pharmacological approaches for prevention and treatment of PVR are under clinical investigation and intervene in different phases of the PVR cascade. The relevant literature as well as own data and experience with PVR are discussed in this review article. The most important aspects of pharmacological approaches for PVR prophylaxis and treatment are explained. Aprophylactic use of systemic prednisone administration as an anti-inflammatory substance showed contradictory results, while there was no additional benefit for intravitreal triamcinolone. Orally administered isotretinoin also seems to be able to minimize the formation of PVR after retinal reattachment surgery, whereas there was no improvement in the success rate in established PVR. Cell proliferation inhibitors have already been extensively studied. The combined intravitreal prophylactic approach of 5‑fluorouracil and low molecular weight heparin was recently further investigated in amulticenter, placebo-controlled study and showed apositive effect in some studies. New preclinical and experimental approaches include the inhibition of growth factors, modulation of integrin activity and the induction of apoptosis. Most clinical studies dealt with an anti-inflammatory or antiproliferative approach. So far, no pharmacological substance has been established for the treatment of PVR but there are promising approaches for prophylaxis.

In Vivo Blockade of Beta-1 and Beta-2 Integrin Activity Inhibits Splenomegaly in JAK2-V617F Positive Myeloproliferative Disease

Introduction:Recently, we showed that in chronic myeloproliferative neoplasia (CMN), β1 and β2 integrins are overactivated on leukocytes in patients and in a JAK2-V617F knock-in mouse model (Gupta et al., Leukemia, 2017; Edelmann-Stephan et al., JCI, In-Press Preview) resulting in increased adhesion of leukocytes to vascular cell adhesion molecule 1 (VCAM-1) and to intercellular cell adhesion molecule 1 (ICAM-1). VCAM-1 and ICAM-1 are abundantly expressed on endothelial cells and in spleen tissue. In JAK2-V617F induced CMN, current therapeutic strategies for symptomatic splenomegaly as hydroxyurea, JAK inhibitors or allogeneic stem cell transplantation (SCT) are effective, however, often limited in duration of efficacy or in eligibility for SCT. Therefore, there is a need to deeper understand the pathophysiology of splenomegaly in CMN in order to develop novel therapeutic approaches. We sought to investigate the effects of in vivo blockade of β1 and β2 integrin overactivation on spleen size, cellular composition of spleen and peripheral blood counts.Material and Methods:11 weeks old Vav1-Cre x JAK2+/V617F mice were treated with a combination of 200µg neutralizing anti-α4β1-integrin (VLA4) antibody (BioXCell, USA) plus 200µg neutralizing anti-β2 integrin antibody (BD Pharmingen, USA) (N=6) and with isotype controls (each 200µg) (N=3), respectively. Animals were injected i.p. on day 1 and day 8. Small animal MRI was used to serially assess the development of spleen volume on days 1, 8, and 15. Abdominal MRI-images employing a slice thickness of 1-2 mm were obtained. The volume of spleens were computed according to radiology standards. Peripheral blood was collected via retro-orbital bleeding and blood counts were measured on days 1, 8, and 15. Thereafter, mice were sacrificed by cervical dislocation and spleens were removed for further analysis by FACS.Results and Discussion:The applied antibody injections were well tolerated and all animals survived the planned observation period. At the start of the experiments (day 1), splenomegaly was confirmed by MRI. As expected, the spleen volumes were similar in the cohorts of isotype and β1/β2 integrin antibody treated JAK2+/V617F mice: 550±76 mm3 (mean±SD) and 518±96 mm3, respectively (Fig. 1). At day 8, upon a single injection of β1/β2 integrin antibodies, a significant decrease in mean spleen volume from baseline 518±96 mm3 to 414±32 mm3 (p=0.0302) was observed (Fig. 1). The correspondent mean±SD for the isotype antibody treated animals was 594±63 mm3. Thus, on day 8, a 30.3% reduction in spleen volume was demonstrated upon β1/β2 integrin antibody treatment in comparison to isotype treatment (p=0.0006). On day 15, the mean spleen volumes of isotype-treated and β1/β2 integrin antibody-treated mice were 665±187 mm3 and 460±69 mm3 (p=0.0410). These results demonstrate that in JAK2-V617F induced disease, blocking of overactivated β1/β2 integrins reduces splenomegaly within a short period of time (8 days). Longer application schedules may build up even stronger and stable decrease in spleen size.In addition to MRI imaging, we also analyzed the composition of peripheral blood cells. At the start of the experiments on day 1, blood parameters showed the typical abnormalities of myeloproliferative disease in both cohorts. Mean counts in isotype treated animals were: WBC = 14,300/µL, hematocrit = 80.3 %, PLT = 1283/nL. Mean counts in β1/β2 integrin antibody treated animals were: WBC = 13,850/µL, hematocrit = 76.0 %, PLT = 1348/nL. This confirms that the JAK2-V617F mouse model used indeed resembles polycythemia vera. In general, the changes observed upon β1/β2 integrin antibody treatment in blood were subtle: the WBC slightly decreased from 13,850/µL to 12,520/µL on day 15, whereas RBC, HCT and PLT remained nearly unchanged. This indicates tolerability of β1/β2 integrin antibody treatment at the level of the hematopoietic system. In isotype treated mice, peripheral blood counts also stayed stable. Data obtained by FACS analysis of peripheral blood and of spleen tissue (composition in granulocytes, B-cells and T-cells) will be presented.Overall, our results indicate a novel role of JAK2-V617F induced overactivation of β1/β2 integrins in development of splenomegaly. Thus, modulation of integrin activity may have therapeutic potential to reduce the spleen volume in CMN patients insufficiently responding to standard treatment. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Modulation of integrin activation and signaling by α1/α1′-helix unbending at the junction

How conformational signals initiated from one end of the integrin are transmitted to the other end remains elusive. At the ligand-binding βI domain, the α1/α1'-helix changes from a bent to a straightened α-helical conformation upon integrin headpiece opening. We demonstrated that a conserved glycine at the α1/α1' junction is crucial for maintaining the bent conformation of the α1/α1'-helix in the resting state. Mutations that facilitate α1/α1'-helix unbending rendered integrin constitutively active; however, mutations that block the α1/α1'-helix unbending abolished soluble ligand binding upon either outside or inside stimuli. Such mutations also blocked ligand-induced integrin extension from outside the cell, but had no effect on talin-induced integrin extension from inside the cell. In addition, integrin-mediated cell spreading, F-actin stress fiber and focal adhesion formation, and focal adhesion kinase activation were also defective in these mutant integrins, although the cells still adhered to immobilized ligands at a reduced level. Our data establish the structural role of the α1/α1' junction that allows relaxation of the α1/α1'-helix in the resting state and transmission of bidirectional conformational signals by helix unbending upon integrin activation.

RSK2 Protein Suppresses Integrin Activation and Fibronectin Matrix Assembly and Promotes Cell Migration

Modulation of integrin activation is important in many cellular functions including adhesion, migration, and assembly of the extracellular matrix. RSK2 functions downstream of Ras/Raf and promotes tumor cell motility and metastasis. We therefore investigated whether RSK2 affects integrin function. We report that RSK2 mediates Ras/Raf inactivation of integrins. As a result, we find that RSK2 impairs cell adhesion and integrin-mediated matrix assembly and promotes cell motility. Active RSK2 appears to affect integrins by reducing actin stress fibers and disrupting focal adhesions. Moreover, RSK2 co-localizes with the integrin activator talin and is present at integrin cytoplasmic tails. It is thereby in a position to modulate integrin activation and integrin-mediated migration. Activation of RSK2 promotes filamin phosphorylation and binding to integrins. We also find that RSK2 is activated in response to integrin ligation to fibronectin. Thus, RSK2 could participate in a feedback loop controlling integrin function. These results reveal RSK2 as a key regulator of integrin activity and provide a novel mechanism by which it may promote cell migration and cancer metastasis.

Neutrophil infiltration during inflammation is regulated by PILRα via modulation of integrin activation

Acute inflammatory responses are important in host defense, whereas dysregulated inflammation results in life-threatening complications. Here we found that paired immunoglobulin-like type 2 receptor alpha (PILRα), an inhibitory receptor containing immunoreceptor tyrosine-based inhibitory motifs (ITIMs), negatively regulated neutrophil infiltration during inflammation. Pilra(-/-) mice had increased neutrophil recruitment to inflammatory sites and were highly susceptible to endotoxin shock. Pilra(-/-) neutrophils showed enhanced transmigration ability and increased adhesion to the β(2) integrin ligand ICAM-1. PILRα expressed on neutrophils constitutively associated in cis with its ligands, resulting in clustering of PILRα during stimulation with a chemoattractant. Clustering of PILRα enhanced ITIM-mediated signaling, thus modulating β(2) integrin inside-out activation. These data demonstrate that neutrophil recruitment in inflammatory responses is regulated by PILRα via modulation of integrin activation.

A functional genetic screen reveals new regulators of β1-integrin activity

β1 integrins constitute a large group of widely distributed adhesion receptors, which regulate the ability of cells to interact with their surroundings. This regulation of the expression and activity of integrins is crucial for tissue homeostasis and development and contributes to inflammation and cancer. We report an RNA interference screen to uncover genes involved in the regulation of β1-integrin activity using cell spot microarray technology in cancer cell lines. Altogether, ten cancer and two normal cell lines were used to identify regulators of β1 integrin activity. Cell biological analysis of the identified β1-integrin regulatory genes revealed that modulation of integrin activity can influence cell invasion in a three-dimensional matrix. We demonstrate with loss-of-function and rescue experiments that CD9 activates and MMP8 inactivates β1 integrins and that both proteins associate with β1 integrins in cells. Furthermore, CD9 and MMP8 regulate cancer cell extravasation in vivo. Our discovery of new regulators of β1-integrin activity highlight the complexity of integrin activity regulation and provide a set of new genes involved in regulation of integrin function.

Modulation of Integrin Activation by an Entropic Spring in the β-Knee

We show that the length of a loop in the β-knee, between the first and second cysteines (C1-C2) in integrin EGF-like (I-EGF) domain 2, modulates integrin activation. Three independent sets of mutants, including swaps among different integrin β-subunits, show that C1-C2 loop lengths of 12 and longer favor the low affinity state and masking of ligand-induced binding site (LIBS) epitopes. Shortening length from 12 to 4 residues progressively increases ligand binding and LIBS epitope exposure. Compared with length, the loop sequence had a smaller effect, which was ascribable to stabilizing loop conformation, and not interactions with the α-subunit. The data together with structural calculations support the concept that the C1-C2 loop is an entropic spring and an emerging theme that disordered regions can regulate allostery. Diversity in the length of this loop may have evolved among integrin β-subunits to adjust the equilibrium between the bent and extended conformations at different set points.

JAM-C Induces Endothelial Cell Permeability Through Its Association and Regulation of β 3 Integrins

The molecular mechanisms regulating vascular permeability are only now being elucidated. The junctional adhesion molecule (JAM) JAM-C has been linked to the induction of vascular permeability. We sought to understand the mechanism whereby JAM-C may disrupt junctional integrity in endothelial cells (ECs). We show here that JAM-C alters permeability through modulation of integrin activity. JAM-C overexpression results in an increase in JAM-C at junctions and an increase in permeability. Conversely, knockdown of JAM-C by siRNA results in a reduction in permeability. JAM-C associates with alphavbeta3 integrin and regulates its localization and activity. JAM-C also inhibits the activation state of the beta(1) integrin although it does not associate with this integrin. These changes induced on the integrins are mediated through regulation of the small GTPase, Rap1b but not Rap1a. Thrombin, a powerful inductor of vascular leak, causes localization of JAM-C into the junctions, whereas angiopoietin-1, an inhibitor of permeability, prevents JAM-C translocation. The regulation of EC junctional integrity involves the coordinated and dynamic modification of localization and activity of junctional stabilizers such as the integrin beta(3) and the destabilizer, JAM-C.

Recent advances in vascular biology: Selected highlights from IVBM 2008

Thromb Haemost 2009; 101: 997–998 The contributions encapsulated in this themed issue of Thrombosis and Haemostasis derive from invited lectures at the 15th International Vascular Biology Meeting (IVBM) held in June, 2008 in Sydney, Australia. IVBMs, which are held every two years, have become the most prestigious international biomedical conference in blood vessel biology and disease. The scientific programme featured 130 talks couched in seven plenary sessions (14 keynote speakers), 17 workshops (2–3 invited speakers and 2–3 speakers selected from abstracts), two sponsored symposia, and almost 400 posters. The programme also featured three memorial orations honoring true giants in vascular biology that had passed away since the last IVBM in Amsterdam in 2006, namely Judah Folkman, Tucker Collins and Rudi Busse. The following provides a brief synopsis of six areas covered at IVBM 2008. Angiogenesis underpins normal physiological processes such as reproduction and wound healing, as well as a wide range of vascular disorders such as atherogenesis, and invasive tumour growth and metastasis. Angiogenesis involves the suppression of adhesion receptors, such as ICAM-1, VCAM-1, E-selectin and CD34 in tumour endothelium (1). Angiogenesis inhibitors are able to normalise endothelial adhesion molecule expression in tumour blood vessels, restoring leukocyte vessel wall interactions and enhancing inflammatory infiltration in the tumours, thus making tumours more vulnerable to attack by the immune system. Arjan Griffioen (VU University Medical Center, Amsterdam, The Netherlands) and Florry Vyth-Dreese (Netherlands Cancer Institute, Amsterdam, The Netherlands) have reviewed (2) the potential of angiostasis therapy to stimulate immune-mediated tumour inhibitory activity and speculate on new therapeutic approaches involving angiostasis in immunotherapy. The various mechanisms with which angiogenesis inhibitors might improve immuno-targeted approaches include tumour cell lysis stimulating tumour antigen presentation, reduced inhibitory molecule load that improves immune reactivity, limited release of immunosuppressive cytokines into the tumour microenvironment, and the creation of a pro-inflammatory microenvironment. Blood vessels adjust their calibre size in order to adapt to the demands of tissues or organs for nutrients and oxygen. The mechanisms involved in calibre size determination in blood vessels are not well understood. Nobuyuki Takakura and Hiroyasu Kidoya (Osaka University, Osaka, Japan) recently discovered that apelin, a factor secreted from endothelial cells following activation of Tie2 receptor tyrosine kinase, regulates the calibre size of blood vessels through its cognate endothelial G proteincoupled receptor APJ (3). Takakura and Kidoya have reviewed (4) the cooperative effect of apelin and VEGF on endothelial cell proliferation and capillary tube formation, and apelin’s regulation of cell-to-cell aggregation and cord hollowing. Integrins are heterodimeric cell-surface molecules that mediate molecular interactions between cells and their extracellular matrix (ECM) environment (5). These signalling receptors transmit information in both “inside-out” and “outside-in” directions across the cell membrane. A necessary and sufficient final step in integrin activation is the binding of the cytoskeletal protein talin to the integrin β-subunit. Brian Petrich (University of California, San Diego, USA) has reviewed (6) our current understanding of the mechanisms regulating the interaction of talin and that of numerous other proteins (such as filamin and kindlin) with the β-integrin subunit and modulation of integrin activation. Our growing appreciation of the functional significance of integrin signalling in haemostasis and thrombosis has been facilitated by mutational analysis in recombinant cell lines and very recently by the use of genetically modified mice. These and other advances focusing mainly on integrin αIIbβ3 (GPIIb-IIIa) are reviewed. Antibodies represent the fastest growing class of human therapeutics and the second largest class of drugs after vaccines, and these molecules are widely used as therapeutic agents in a diverse range of clinical settings, including cancer, inflammatory disease and cardiovascular disease. Single-chain antibodies (7) are one of the smallest possible formats for a recombinant antibody. Karlheinz Peter and colleagues (Baker IDI Heart and Diabetes Institute, Melbourne, Australia) have described (8) the © 2009 Schattauer GmbH, Stuttgart

Interactions of TANGO and leukocyte integrin CD11c/CD18 regulate the migration of human monocytes

The TANGO gene was originally identified as a new member of the MIA gene family. It codes for a protein of yet unknown function. TANGO revealed a very broad expression pattern in contrast to the highly restricted expression pattern determined for the other family members. The only cells lacking TANGO expression are cells of the hematopoietic system. One of the major differences between mature hematopoietic cells and other tissue cells is the lack of adhesion until these cells leave the bloodstream. In this study, we observed that TANGO expression was induced after adhesion of human monocytic cells to substrate. To understand the mechanism of TANGO function during monocyte adhesion we isolated interacting proteins and found an interaction between TANGO and the leukocyte-specific integrin CD11c. In functional assays, we observed reduced attachment of human monocytic cells to fibrinogen, ICAM-1 and to human microvascular endothelial cells (HMECs) after stimulation with recombinant TANGO protein. Additionally, the migrating capacity of premonocytic cells through fibrinogen or HMECs was increased after stimulation of these cells with recombinant TANGO. Therefore, we suggest that TANGO reduced the attachment to fibrinogen or other cell adhesion molecules. As TANGO does not compete for CD11c ligand binding directly, we hypothesize TANGO function by modulation of integrin activity. Taken together, the results from this study present TANGO as a novel ligand for CD11c, regulating migratory processes of hematopoietic cells.

PECAM-1 isoform-specific regulation of kidney endothelial cell migration and capillary morphogenesis

Platelet endothelial cell adhesion molecule-1 (PECAM-1) has been implicated in angiogenesis through its involvement in endothelial cell-cell and cell-matrix interactions and signal transduction. Recent studies indicate that the cytoplasmic domain of PECAM-1 plays an important role in its cell adhesive and signaling properties. However, the role PECAM-1 isoforms play during angiogenic events such as cell adhesion and migration requires further delineation. To gain insight into the role PECAM-1 plays during vascular development and angiogenesis, we examined the expression pattern of PECAM-1 isoforms during kidney vascularization. We show that multiple isoforms of PECAM-1 are expressed during renal vascular development with different frequencies. The PECAM-1 that lacks exons 14 and 15 (Delta14&15) was the predominant isoform detected in the renal vasculature. To further study PECAM-1 isoform-specific functions we isolated kidney endothelial cells (EC) from wild-type and PECAM-1-deficient (PECAM-1-/-) mice with B(4)-lectin-coated magnetic beads. PECAM-1-/- kidney EC showed reduced migration, inability to undergo capillary morphogenesis in Matrigel, dense peripheral focal adhesions, and peripheral cortical actin distribution compared with wild-type cells. PECAM-1-/- kidney EC secreted increased amounts of fibronectin and decreased amounts of tenascin-C and thrombospondin-1. Reexpression of Delta14&15, but not full-length, PECAM-1 in PECAM-1-/- kidney EC restored cell migration and capillary morphogenesis defects. Thus PECAM-1 may regulate the adhesive and migratory properties of kidney EC in an isoform-specific fashion through modulation of integrin activity and extracellular matrix protein expression. Our results indicate that regulated expression of specific PECAM-1 isoforms may enable EC to accommodate the different stages of angiogenesis.

Identification of a Putative Tumor Suppressor Gene Rap1GAP in Pancreatic Cancer

Human chromosome 1p35-p36 has long been suspected to harbor a tumor suppressor gene in pancreatic cancer and other tumors. We found that expression of rap1GAP, a gene located in this chromosomal region, is significantly down-regulated in pancreatic cancer. Only a small percentage of preneoplastic pancreatic intraductal neoplasia lesions lost rap1GAP expression, whereas loss of rap1GAP expression occurred in 60% of invasive pancreatic cancers, suggesting that rap1GAP contributes to pancreatic cancer progression. In vitro and in vivo studies showed that loss of rap1GAP promotes pancreatic cancer growth, survival, and invasion, and may function through modulation of integrin activity. Furthermore, we showed a high frequency of loss of heterozygosity of rap1GAP in pancreatic cancer. Collectively, our data identify rap1GAP as a putative tumor suppressor gene in pancreatic cancer.

PEA-15 Binding to ERK1/2 MAPKs Is Required for Its Modulation of Integrin Activation

Activation of Raf-1 suppresses integrin activation, potentially through the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). However, bulk ERK1/2 activation does not correlate with suppression. PEA-15 reverses suppression of integrin activation and binds ERK1/2. Here we report that PEA-15 reversal of integrin suppression depends on its capacity to bind ERK1/2, indicating that ERK1/2 function is indeed required for suppression. Mutations in either the death effector domain or C-terminal tail of PEA-15 that block ERK1/2 binding abrogated the reversal of integrin suppression. Furthermore, we used ERK/p38 chimeras and site-directed mutagenesis to identify ERK1/2 residues required for binding PEA-15. Mutations of residues that precede the alphaG helix and within the mitogen-activated protein kinase insert blocked ERK2 binding to PEA-15, but not activation of ERK2. These ERK2 mutants blocked the ability of PEA-15 to reverse suppression of integrin activation. Thus, PEA-15 regulation of integrin activation depends on its binding to ERK1/2. To directly test the role of ERK1/2 localization in suppression, we enforced membrane association of ERK1 and 2 by joining a membrane-targeting CAAX box sequence to them. Both ERK1-CAAX and ERK2-CAAX were membrane-localized and suppressed integrin activation. In contrast to suppression by membrane-targeted Raf-CAAX, suppression by ERK1/2-CAAX was not reversed by PEA-15. Thus, ERK1/2 are the Raf effectors for suppression of integrin activation, and PEA-15 reverses suppression by binding ERK1/2.

RhoA-dependent switch between alpha2beta1 and alpha3beta1 integrins is induced by laminin-5 during early stage of HT-29 cell differentiation.

Integrin-mediated interactions between the basement membrane and epithelial cells control the differentiation of epithelia. We characterized the modulation of adhesive behaviors to basement membrane proteins and of integrin function in the human colon adenocarcinoma HT-29 cell line, which differentiates into enterocytes after the substitution of galactose for glucose in the medium. We demonstrate an increased capability of these cells to adhere to collagen type IV during the early stage of differentiation. This effect occurs without any changes in integrin cell surface expression but rather results from an alpha2beta1/alpha3beta1 integrin switch, alpha3beta1 integrin becoming the major collagen receptor. The increase in laminin-5 secretion and deposit on the matrix is a key factor in the mechanism regulating cell adhesion, because it is responsible for the activation of alpha3beta1 integrin. Furthermore, down-regulation of RhoA GTPase activity occurs during HT-29 cell differentiation and correlates with the activation of the integrin alpha3beta1. Indeed, C3 transferase, a RhoA GTPase inhibitor, induces a similar alpha2beta1/alpha3beta1 switch in undifferentiated HT-29 cells. These results indicate that the decrease in RhoA activation is the biochemical mechanism underlying this integrin switch observed during cell differentiation. The physiological relevance of such modulation of integrin activity in the functioning of the crypt-villus axis is discussed.

Modulation of integrin activity is vital for morphogenesis.

Cells can vary their adhesive properties by modulating the affinity of integrin receptors. The activation and inactivation of integrins by inside-out mechanisms acting on the cytoplasmic domains of the integrin subunits has been demonstrated in platelets, lymphocytes, and keratinocytes. We show that in the embryo, normal morphogenesis requires the alpha subunit cytoplasmic domain to control integrin adhesion at the right times and places. PS2 integrin (alphaPS2betaPS) adhesion is normally restricted to the muscle termini, where it is required for attaching the muscles to the ends of other muscles and to specialized epidermal cells. Replacing the wild-type alphaPS2 with mutant forms containing cytoplasmic domain deletions results in the rescue of the majority of defects associated with the absence of the alphaPS2 subunit, however, the mutant PS2 integrins are excessively active. Muscles containing these mutant integrins make extra muscle attachments at aberrant positions on the muscle surface, disrupting the muscle pattern and causing embryonic lethality. A gain- of-function phenotype is not observed in the visceral mesoderm, showing that regulation of integrin activity is tissue-specific. These results suggest that the alphaPS2 subunit cytoplasmic domain is required for inside-out regulation of integrin affinity, as has been seen with the integrin alphaIIbbeta3.

Modulatory effects of interferon-γ on the fibronectin receptor function of squamous cell carcinoma cells in vitro

We previously showed that the in vivo invasion of a squamous cell carcinoma induced by the intradermal injection of tumor cells was significantly delayed after the IFN-γ-producing gene transfer to tumor cells. With respect to the mechanism of the delayed invasion, it was suggested that the IFN-γ might inhibit the adhesion of the cells to extracellular matrices (ECM) and the subsequent locomotion. Thus, we examined the effect of IFN-γ on the adhesion of Pam-T cells to ECM. The attachment of Pam-T cells to fibronectin (FN) was significantly higher than that to laminin (LN), collagen type I (COL I) or collagen type IV (COL IV) substrata. The attachment of FN was significantly enhanced specifically by the IFN-γ-treatment of the cells, although the attachment to LN, COL I or COL IV was not altered by IFN-γ. Neither IFN-α nor IFN-β had any effect on the attachment of Pam-T cells to FN. When Pam-T cells were treated with IFN-γ together with a neutralizable anti-IFN-γ antibody, this enhancement was completely abolished. Moreover, the attachment of IFN-γ-treated Pam-T cells as well as non-treated cells to FN was blocked by the synthetic peptide Arg-Gly-Asp-Ser (RGDS), but not by the control peptide Arg-Gly-Glu-Ser. Based on these results, we conclude that IFN-γ specifically enhances the adhesiveness of Pam-T cells to FN substrata by the modulation of integrin activity.