Integrin Fibronectin Receptor Research Articles

ITGB1-Expressing Effector Memory CD4 + T-Cell Response Associates with Clinical Recovery in Childhood Immune Thrombocytopenia

Background: The autoimmune bleeding disorder childhood immune thrombocytopenia (ITP) features antibody and T-cell immune responses against platelet self-antigens. This involves activation of B-cells, CD4 + T-cell help, and CD8 + T-cells. Novel prognostic biomarkers are needed to predict recovery and treatment responses. Compared to healthy children, changes in total lymphocytes, CD4 +, regulatory CD4 +, CD8 +T-cells, CD19 + B-cells, and NK cells were described in childhood ITP. Children with transient ITP were also found to have a fewer CD25 hi CD4 + T-cells than those who develop chronic ITP. In contrast, the platelet-stimulated peripheral blood mononuclear cells (PBMC) of children with chronic ITP produced more IL-2 than those of transient ITP. At present, none of these biomarkers have been validated for ITP prognosis, and consequently they are not in clinical use. This emphasizes the need for clinical-grade biomarkers to predict clinical responses in childhood ITP. Aims: To predict spontaneous recovery and IVIg response in newly diagnosed ITP by (1) validating previously suggested changes in immune cell frequencies, and (2) identifying novel immune cell subsets as predictors. Methods: Children with newly diagnosed ITP were randomized 1:1 to observation or IVIg treatment (TIKI trial). Recovery was determined by platelet counts according to IWG criteria 1, 4, 13, 26 and 52 weeks after the diagnosis. For validation, the CD4 +, regulatory CD4 + (CD25 +/CD127 lo), CD8 +, CD19 +, and NK cells were quantified at the time of diagnosis in a centralized laboratory by flow cytometry. For identification of novel predictors, CD4 + central (CD45RO + CD27 +) and effector memory (CD45RO + CD27 -) frequencies were determined. The primary clinical outcome was longitudinal recovery, either spontaneous or after IVIg (adjusted Cox-proportional hazard model). Secondary outcomes included the mean age-adjusted difference between transient/persistent and chronic ITP by multivariate regression, and comparison to age-appropriate healthy control data. Additionally, PBMC (N=6) were analyzed at diagnosis by single-cell RNA sequencing (scRNA-seq) combined with T- and B- cell receptor sequencing (scTCR-seq and scBCR-seq). Results: For validation of previously suggested predictors, the absolute CD3 +, CD4 +, CD8 +, CD19 +, and NK cell counts of newly diagnosed ITP patients were within the age-appropriate healthy reference range (N=158); they were not associated with recovery, and there were no age-adjusted differences between transient/persistent (N=139) and chronic ITP (N=19). The regulatory CD4 + T cell frequency was not reduced compared to age-appropriate reference data; not associated with recovery, and not different between transient/persistent and chronic ITP patients. For identification of novel predictors, we found that high CD4 +effector memory cells numbers were associated with a reduced recovery rate, with an adjusted hazard ratio for complete recovery over a one-year follow-up to be 0.55 (95% CI, 0.35 - 0.85; ≥ median; adjusted for age and treatment; N=150). Chronic ITP patients displayed a mean age-adjusted increase in effector memory CD4 +cells of 1.4 % (95% CI, 0.4 - 2.4; P= 0.005). The association with recovery was independent of a preceding infection, total leukocyte and lymphocyte counts, and the presence of anti-platelet IgG or IgM autoantibodies. ScRNA-seq analysis of 7965 PBMC also showed an effector memory CD4 + T-cell cluster that was expanded in chronic ITP, present in at a frequency of 8.1 ± 1.4 % (mean ± sem) in transient vs 14.8 ± 1.2 % in chronic ITP. This cluster also expressed high levels of fibronectin receptor integrin b1 ( ITGB1) interleukin 32 (IL32) interleukin 7 receptor (IL7R) andlymphotoxin beta (LTB). The effector memory phenotype of CD4 + ITGB1 + T-cells was confirmed by flow cytometry analysis in healthy individuals and ITP patients. Conclusions: Previously suggested changes in T-, B-, or NK cell frequencies could not be validated as predictors of spontaneous recovery or IVIg response. However, we identified the frequency of ITGB1-expressing effector memory CD4 + T cells as an independent predictor of spontaneous recovery and IVIg response. Thus, T cell phenotyping at the time of diagnosis may be suitable to determine prognosis and personalize treatment decisions in newly diagnosed childhood ITP.

Targeting integrin α5β1 in urological tumors: opportunities and challenges.

Urological tumors, such as prostate cancer, renal cell carcinoma, and bladder cancer, have shown a significant rise in prevalence in recent years and account for a significant proportion of malignant tumors. It has been established that metastasis to distant organs caused by urological tumors is the main cause of death, although the mechanisms underlying metastasis have not been fully elucidated. The fibronectin receptor integrin α5β1 reportedly plays an important role in distant metastasis and is closely related to tumor development. It is widely thought to be an important cancer mediator by interacting with different ligands, mediating tumor adhesion, invasion, and migration, and leading to immune escape. In this paper, we expound on the relationship and regulatory mechanisms of integrin α5β1 in these three cancers. In addition, the clinical applications of integrin α5β1 in these cancers, especially against treatment resistance, are discussed. Last but not least, the possibility of integrin α5β1 as a potential target for treatment is examined, with new ideas for future research being proposed.

IDENTIFICATION AND FUNCTIONAL CHARACTERIZATION IMBALANCED OSTEOARTHRITIS ASSOCIATED FIBRONECTIN SPLICE VARIANTS

Purpose: Transcriptome-wide analyses demonstrated FN1 is the highest expressed gene in articular cartilage and is significantly upregulated with ongoing OA pathophysiology. FN1 encodes fibronectin, a dimeric glycoprotein, particularly localized in the pericellular matrix directly surrounding the chondrocytes. Fibronectin mediates a wide variety of cellular interactions by binding to extra cellular matrix proteins and to chondrocytes via integrins. The main integrin-binding domain of fibronectin is the RGD motif, which binds multiple integrin heterodimers, including the classic fibronectin receptor integrin α5β1.

Co-localization of Fibronectin Receptors LILRB4/gp49B and Integrin on Dendritic Cell Surface.

A myeloid immune checkpoint, leukocyte immunoglobulin-like receptor (LILR) B4 (B4, also known as ILT3/CD85k in humans and gp49B in mice) is expressed on dendritic cells (DCs). However, a mode of regulation of DCs by B4/gp49B is not identified yet in relation to the ligand(s) as well as to the counteracting, activation-type receptor. Our recent identification of the physiological/pathological ligand for B4/gp49B as the fibronectin (FN) N-terminal 30-kDa domain poses the question of the relationship between B4/gp49B and a classical FN receptor/cellular activator, integrin, on DCs. Here we showed that FN is not constitutively tethered on the surface of bone marrow-derived cultured DCs (BMDCs) or splenic DCs, even though the FN receptor integrin and gp49B are co-expressed on these cells. Confocal laser scanning microscopic analysis, however, revealed weak correlation of fluorescent signals between gp49B and integrin β1, suggesting their partial co-localization on the BMDC surface even in the absence of FN. We found that the plating of BMDCs onto immobilized FN induced tyrosine phosphorylation of focal adhesion kinase (FAK) and spleen tyrosine kinase (Syk). In the absence of gp49B, while the FAK phosphorylation level was virtually unchanged, that of phosphorylation of Syk was markedly augmented. These results suggested that the immobilized FN induced a crosstalk between gp49B and integrin in terms of the intracellular signaling of BMDCs, in which gp49B suppressed the integrin-mediated pro-inflammatory cascade. Our observations may provide a clue for elucidating the mechanism of the therapeutic efficacy of B4/gp49B blocking in autoimmune disease and cancer.

Integrin α5β1 nano-presentation regulates collective keratinocyte migration independent of substrate rigidity.

Nanometer-scale properties of the extracellular matrix influence many biological processes, including cell motility. While much information is available for single-cell migration, to date, no knowledge exists on how the nanoscale presentation of extracellular matrix receptors influences collective cell migration. In wound healing, basal keratinocytes collectively migrate on a fibronectin-rich provisional basement membrane to re-epithelialize the injured skin. Among other receptors, the fibronectin receptor integrin α5β1 plays a pivotal role in this process. Using a highly specific integrin α5β1 peptidomimetic combined with nanopatterned hydrogels, we show that keratinocyte sheets regulate their migration ability at an optimal integrin α5β1 nanospacing. This efficiency relies on the effective propagation of stresses within the cell monolayer independent of substrate stiffness. For the first time, this work highlights the importance of extracellular matrix receptor nanoscale organization required for efficient tissue regeneration.

Membranous Expression of Integrin α5β1 is Elevated in Endothelial Cells of Pulmonary Arterial Hypertension

Pulmonary Arterial Hypertension (PAH) is an incurable pathology characterized by remodeling of small pulmonary arteries leading to increased pulmonary vascular resistance and right ventricular heart failure. The endothelial dysfunction and endothelial-to-mesenchymal transition (EndoMT) observed in PAH contribute to the formation of plexiform lesions. Fibronectin is a commonly used molecular marker of EndoMT and is abundantly present in the plexiform lesions observed in human PAH. We have previously observed that pulmonary artery endothelial cells (PAECs) from the lesion forming Sugen/hypoxia rat model of PAH demonstrate increased proliferation and angiogenesis compared to PAECs from normoxic control animals. As the fibronectin receptor integrin α5β1 promotes endothelial proliferation and angiogenesis, we aimed to determine whether the expression of integrin α5β1 was elevated in PAH PAECs. Sugen/hypoxia rat PAECs (PAH PAECs) or normoxic control cells were received from the University of South Alabama. Initially, we observed an increase in mRNA expression of integrins α5, αV and β1 in PAH PAECs compared to control cells using a PCR screening array for molecular markers of epithelial-to-mesenchymal transition. Increased protein expression of the integrin α5, αV and β1 monomers were also observed in membrane fractions of PAH PAECs via western blot, compared to the cell membranes of control PAECs following a cell fractionation assay. To confirm the presence of the integrin α5β1 heterodimer at the membranes of PAH PAECs, co-immunoprecipitation of integrins α5 and β1 was performed from the membrane fractions of PAH and control PAECs. Following immunoprecipitation, western blot analysis demonstrated a strong coprecipitation of integrin α5 and β1 in membrane fractions of PAH PAECs, compared to weak coprecipitation in control PAECs. To confirm the increased membranous expression of integrin α5β1 in human tissue, human pulmonary endothelial cells from patients with idiopathic PAH (IPAH), familial PAH (FPAH) and control cells from failed donors (FD) were analyzed with an integrin-mediated cell adhesion array for cell surface expression of alpha and beta integrins. Human cells were provided by PHBI under the Pulmonary Hypertension Breakthrough Initiative (PHBI)*. Monomeric α5- and β1-mediated adhesion and heterodimeric α5β1-mediated adhesion were increased in IPAH and FPAH cells compared to FD control pulmonary endothelial cells. Collectively, these data strongly indicate that protein levels of the integrin α5β1 fibronectin receptor are increased at the cell membranes of pulmonary endothelial cells in PAH.

Cancer-associated fibroblast migration in non-small cell lung cancers is modulated by increased integrin α11 expression.

Cancer‐associated fibroblasts (CAFs) regulate cancer progression through the modulation of extracellular matrix (ECM) and cancer cell adhesion. While undergoing a series of phenotypic changes, CAFs control cancer–stroma interactions through integrin receptor signaling. Here, we isolated CAFs from patients with non‐small‐cell lung cancer (NSCLC) and examined their gene expression profiles. We identified collagen type XI α1 (COL11A1), integrin α11 (ITGA11), and the ITGA11 major ligand collagen type I α1 (COL1A1) among the 390 genes that were significantly enriched in NSCLC‐associated CAFs. Increased ITGA11 expression in cancer stroma was correlated with a poor clinical outcome in patients with NSCLC. Increased expression of fibronectin and collagen type I induced ITGA11 expression in CAFs. The cellular migration of CAFs toward collagen type I and fibronectin was promoted via ERK1/2 signaling, independently of the fibronectin receptor integrin α5β1. Additionally, ERK1/2 signaling induced ITGA11 and COL11A1 expression in cancer stroma. We, therefore, propose that targeting ITGA11 and COL11A1 expressing CAFs to block cancer–stroma interactions may serve as a novel, promising anti‐tumor strategy.

Abstract 13606: Cardiac Fibroblast Derived Matrix-educated Macrophages Express VEGF and IL-6, and Recruit Mesenchymal Stromal Cells

Introduction: Polarizing monocytes into macrophages that possess anti-inflammatory and angiogenic properties could be cardio-reparative following myocardial infarction. Co-culturing cardiac fibroblasts (CF) with monocytes is observed to lead to alternatively activated macrophages. We previously showed that CF produce a unique cardiac matrix (CX) that is fibronectin enriched. Whether decellularized CX can drive differentiation of monocytes to macrophages with angiogenic and anti-inflammatory properties is unknown. Hypotheses: (i) CX educated macrophages (CXMq) that secrete VEGF and IL-6, which have known angiogenic and anti-inflammatory properties respectively, (ii) CXMq can recruit mesenchymal stromal cells (MSC), which have known anti-inflammatory properties and (iii) fibronectin is responsible for this effect. Methods: Human monocytes (CD14 + ) were cultured on CX, plastic and gelatin. After 3 days, flow expression of the macrophage markers CD14, CD16, CD163, CD206, PDL1, CD86, and HLA-DR was measured. VEGF and IL-6 levels were measured from the spent media. A transwell migration assay was performed by placing monocytes on CX in the lower chamber and MSC in the upper chamber. CX was enzymatically dispersed and flow cytometry was performed for CD14 (macrophage marker) and CD73 (MSC marker). Migration was defined as the % of MSCs reaching the lower chamber (see Figure). Controls included monocytes plated on plastic, and monocyte/CX combination cultured with and without fibronectin integrin receptor inhibitor. Results: CXMq secreted high levels of VEGF and IL-6 and also had low expression of inflammatory markers CD16, CD86, HLADR. Further, CXMq recruited MSC more robustly than monocytes and CX alone (see Figure). Selective fibronectin integrin receptor blockade prevented MSC recruitment (*p<0.05). Conclusions: CXMq has anti-inflammatory and pro-angiogenic properties that is potentially mediated by fibronectin.

Fibronectin Promotes Cell Growth and Migration in Human Renal Cell Carcinoma Cells

The prognostic and therapeutic values of fibronectin have been reported in patients with renal cell carcinoma (RCC). However, the underlying mechanisms of malignancy in RCC are not completely understood. We found that silencing of fibronectin expression attenuated human RCC 786-O and Caki-1 cell growth and migration. Silencing of potential fibronectin receptor integrin α5 and integrin β1 decreased 786-O cell ability in movement and chemotactic migration. Biochemical examination revealed a reduction of cyclin D1 and vimentin expression, transforming growth factor-β1 (TGF-β1) production, as well as Src and Smad phosphorylation in fibronectin-silenced 786-O and Caki-1 cells. Pharmacological inhibition of Src decreased 786-O cell growth and migration accompanied by a reduction of cyclin D1, fibronectin, vimentin, and TGF-β1 expression, as well as Src and Smad phosphorylation. In 786-O cells, higher activities in cell growth and migration than in Caki-1 cells were noted, along with elevated fibronectin and TGF-β1 expression. The additions of exogenous fibronectin and TGF-β1 promoted Caki-1 cell growth and migration, and increased cyclin D1, fibronectin, vimentin, and TGF-β1 expression, as well as Src and Smad phosphorylation. These findings highlight the role of fibronectin in RCC cell growth and migration involving Src and TGF-β1 signaling.

The immunohistochemical localization of the fibronectin receptor (Integrin β1) and laminin in the bitch uterus during the estrous cycle

In mammals, the purpose of the estrous cycle is the successful progression of implantation and pregnancy. The endometrium undergoes several changes during the estrous cycle. The extracellular matrix proteins have a likely role in the maintenance of a healthy endometrium. In this study, we investigated the localization of the fibronectin receptor (Integrin β 1 ), a member of the integrin family, and laminin, an extracellular matrix protein, in the bitch uterus throughout the estrous cycle. In the bitch uterus, immunoreactions of similar intensity were observed for the fibronectin receptor (Integrin β 1 ) in the myometrium, perimetrium, peritoneal mesothelial cells and uterine vasculature, and for laminin in the peritoneal mesothelial cells, throughout the estrous cycle. Laminin and the fibronectin receptor (Integrin β 1 ) were found to be present at high intensity in the endometrium of the bitch uterus at the luteal stage.

Integrin Targeting and Toxicological Assessment of Peptide‐Conjugated Liposome Delivery Systems to Activated Endothelial Cells

Utilization of functionalized liposomes as the means of targeted delivery of therapeutics may enhance specific transport of biologically active drugs to target tissues, while avoiding or reducing undesired side effects. In the present investigation, peptide-conjugated cationic liposomes were constructed with the aim of targeting integrins (i.e. vitronectin and/or fibronectin receptors) on activated endothelial cells. The peptide-conjugated liposomes induced only cytotoxicity at the highest concentration in non-activated or activated endothelial cells, as well as in co-culture of endothelial cells and macrophages. There was unaltered secretion of cytokines after exposure of peptide-conjugated liposomes to endothelial cells, indicating that the materials were not inflammogenic. Liposomes with a peptide targeting the fibronectin receptor (integrin α5β1) were more effective in targeting of activated endothelial cells, as compared to a liposome with a peptide that targeted both the fibronectin and vitronectin receptors, as well as liposomes with a control peptide. The liposome targeted to the fibronectin receptor also displayed uptake in endothelial cells in co-culture with activated macrophages. Therefore, this study demonstrates the feasibility of constructing a peptide-conjugated cationic liposome, which displays targeting to activated endothelial cells at concentrations that are not cytotoxic or inflammogenic to the cells.

The metastasis suppressor CD82/KAI1 inhibits fibronectin adhesion-induced epithelial-to-mesenchymal transition in prostate cancer cells by repressing the associated integrin signaling.

The transmembrane protein CD82/KAI1 suppresses the metastatic potential of various cancer cell types. Moreover, decrease or loss of CD82 expression is closely associated with malignancy and poor prognosis in many human cancers including prostate cancer. Despite intense scrutiny, the mechanisms underlying the metastasis-suppressing role of CD82 are still not fully understood. Here, we found that a fibronectin matrix induced mesenchymal phenotypes in human prostate cancer cells with no or low CD82 expression levels. However, high CD82 expression rendered prostate cancer cells to have intensified epithelial characteristics upon fibronectin engagement, along with decreased cell motility and invasiveness. The CD82 function of inhibiting fibronectin-induced epithelial-to-mesenchymal transition (EMT) was dependent not only on CD82 interactions with fibronectin-binding α3β1/α5β1 integrins but also on the integrin-mediated intracellular signaling events. Notably, CD82 attenuated the FAK-Src and ILK pathways downstream of the fibronectin-receptor integrins. Immunofluorescence staining of human prostate cancer tissue specimens illustrated a negative association of CD82 with EMT-related gene expression as well as prostate malignancy. Altogether, these results suggest that CD82 suppresses EMT in prostate cancer cells adhered to the fibronectin matrix by repressing adhesion signaling through lateral interactions with the associated α3β1 and α5β1 integrins, leading to reduced cell migration and invasive capacities.

Interaction between integrin α5 and PDE4D regulates endothelial inflammatory signalling.

Atherosclerosis is primarily a disease of lipid metabolism and inflammation; however, it is also closely associated with endothelial extracellular matrix (ECM) remodelling, with fibronectin accumulating in the laminin-collagen basement membrane. To investigate how fibronectin modulates inflammation in arteries, we replaced the cytoplasmic tail of the fibronectin receptor integrin α5 with that of the collagen/laminin receptor integrin α2. This chimaera suppressed inflammatory signalling in endothelial cells on fibronectin and in knock-in mice. Fibronectin promoted inflammation by suppressing anti-inflammatory cAMP. cAMP was activated through endothelial prostacyclin secretion; however, this was ECM-independent. Instead, cells on fibronectin suppressed cAMP via enhanced phosphodiesterase (PDE) activity, through direct binding of integrin α5 to phosphodiesterase-4D5 (PDE4D5), which induced PP2A-dependent dephosphorylation of PDE4D5 on the inhibitory site Ser651. In vivo knockdown of PDE4D5 inhibited inflammation at athero-prone sites. These data elucidate a molecular mechanism linking ECM remodelling and inflammation, thereby identifying a new class of therapeutic targets.

Therapeutic inhibition of breast cancer bone metastasis progression and lung colonization: breaking the vicious cycle by targeting α5β1 integrin.

At diagnosis, 10% of breast cancer patients already have locally advanced or metastatic disease; moreover, metastasis eventually develops in at least 40% of early breast cancer patients. Osteolytic bone colonization occurs in 80-85% of metastatic breast cancer patients and is thought to be an early step in metastatic progression. Thus, breast cancer displays a strong preference for metastasis to bone, and most metastatic breast cancer patients will experience its complications. Our prior research has shown that the α5β1 integrin fibronectin receptor mediates both metastatic and angiogenic invasion. We invented a targeted peptide inhibitor of activated α5β1, Ac-PHSCN-NH2 (PHSCN), as a validated lead compound to impede both metastatic invasion and neovascularization. Systemic PHSCN monotherapy prevented disease progression for up to 14months in Phase I clinical trial. Here, we report that the next-generation construct, Ac-PhScN-NH2 (PhScN), which contains D-isomers of histidine (h) and cysteine (c), is greater than 100,000-fold more potent than PHSCN at blocking basement membrane invasion. Moreover, PhScN is also up to 10,000-fold more potent than PHSCN at inhibiting lung extravasation and colonization in athymic mice for both MDA-MB-231 metastatic and SUM149PT inflammatory breast cancer cells. Furthermore, we show that systemic treatment with 50mg/kg PhScN monotherapy reduces established intratibial MDA-MB-231 bone colony progression by 80%. Thus, PhScN is a highly potent, well-tolerated inhibitor of both lung colonization and bone colony progression.

A Sawtooth Pattern of Cadherin 2 Stability Mechanically Regulates Somite Morphogenesis

Differential cadherin (Cdh) expression is a classical mechanism for in vitro cell sorting. Studies have explored the roles of differential Cdh levels in cell aggregates and during vertebrate gastrulation, but the role of differential Cdh activity in forming in vivo tissue boundaries and boundary extracellular matrix (ECM) is unclear. Here, we examine the interactions between cell-cell and cell-ECM adhesion during somitogenesis, the formation of the segmented embryonic precursors of the vertebral column and musculature. We identify a sawtooth pattern of stable Cdh2 adhesions in which there is a posterior-to-anterior gradient of stable Cdh2 within each somite, while there is a step-like drop in stable Cdh2 along the somite boundary. Moreover, we find that the posterior somite boundary cells with high levels of stable Cdh2 have the most columnar morphology. Cdh2 is required for maximal cell aspect ratio and thus full epithelialization of the posterior somite. Loss-of-function analysis also indicates that Cdh2 acts with the fibronectin (FN) receptor integrin α5 (Itgα5) to promote somite boundary formation. Using genetic mosaics, we demonstrate that differential Cdh2 levels are sufficient to induce boundary formation, Itgα5 activation, and FN matrix assembly in the paraxial mesoderm. Elevated cytoskeletal contractility is sufficient to replace differential Cdh2 levels in genetic mosaics, suggesting that Cdh2 promotes ECM assembly by increasing cytoskeletal and tissue stiffness along the posterior somite boundary. Throughout somitogenesis, Cdh2 promotes ECM assembly along tissue boundaries and inhibits ECM assembly in the tissue mesenchyme.

Metastasis suppressor NME1 regulates melanoma cell morphology, self-adhesion and motility via induction of fibronectin expression.

Expression of the metastasis suppressor NME1 in melanoma is associated with reduced cellular motility and invasion in vitro and metastasis in vivo, but the underlying molecular mechanisms are not completely understood. Herein, we report a novel mechanism through which NME1 controls melanoma cell morphology via upregulation of the extracellular matrix (ECM) protein fibronectin. Expression of NME1 strongly suppressed cell motility in melanoma cell lines 1205LU and M14. The resulting sedentary phenotype was associated with a more flattened appearance and marked increases in actin stress fibre and focal adhesion formation. NME1‐induced focal adhesions were colocalized with dense deposits of fibronectin, which were absent or minimal in the corresponding NME1‐deficient parental lines. NME1 was a strong inducer of fibronectin mRNA and protein expression, shown with reciprocal approaches of forced NME1 expression and shRNA‐mediated knock‐down. Increased synthesis and ECM deposition of fibronectin was necessary for NME1‐induced cell spreading, as knock‐down of fibronectin opposed the effects of NME1 on cell morphology. Fibronectin knock‐down also reversed the ability of NME1 to promote aggregation when cells were plated on a non‐adherent substratum. Similarly, inhibiting activation of the fibronectin receptor integrin α4β1 with an anti‐α4 antibody reversed the motility‐suppressing effect of NME1. A positive correlation was observed between NME1 and fibronectin mRNA in clinical biopsies of normal skin, benign nevi and primary melanomas, but not in metastatic forms, suggesting the NME1/fibronectin axis represents a barrier to melanoma progression. In summary, these findings indicate fibronectin is an important effector of the motility‐suppressing function of NME1 in melanoma cells.

Integrin-based meningioma cell migration is promoted by photon but not by carbon-ion irradiation.

Sublethal doses of photon irradiation (IR) are suspected to increase tumor cell migration and support locoregional recurrence of disease, which has already been shown in other cell lines. This manuscript describes the effect of photon and carbon-ion IR on WHO class I meningioma cell migration and provides an approach to the underlying cellular mechanisms. Meningioma cells were gained operatively at the university hospital in Homburg/Saar, Germany. For migration, membranes (8-µm pore sizes) were coated with collagen I, with collagen IV, and with fibronectin. Cells were analyzed in migration experiments with or without serum stimulation, with or without photon and carbon IR 24h prior to experiments, and with or without integrin antibodies. Fluorescence-activated cell sorting (FACS) analyses of the integrins ανβ1, ανβ3, and ανβ5 were performed without IR and 6, 12 and 24h after IR. Enzyme-linked immunosorbent assay (ELISA) analyses of matrix metalloproteinases (MMP)-2 and MMP-9 were realized with and without IR after cells were cultured on collagen I, collagen IV, or fibronectin for 24h. Cells and supernatants for FACS and ELISA were stored at - 18 °C. The significance level was set at 5 % using both Student's t test and two-way ANOVA. Migration of meningioma cells was serum-inducible (p < 0.001). It could be increased by photon IR (p < 0.02). The integrins ανβ1 and ανβ5 showed a 21 and 11 % higher expression after serum stimulation (not significant), respectively, and ανβ1 expression was raised by 14 % (p = 0.0057) after photon IR. Antibody blockage of the integrins ανβ1 and ανβ5 inhibited serum- and photon-induced migration. Expression of MMP-2 and MMP-9 remained unchanged after both IR and fetal bovine serum (FBS). Carbon-ion IR left both integrin expression and meningioma cell migration unaffected. Photon but not carbon-ion IR promotes serum-based meningioma cell migration. Fibronectin receptor integrin ανβ1 signaling can be identified as an important mechanism for serum- and photon-induced migration of WHO class I meningioma cells.

Combinatorial fibronectin and laminin signaling promote highly efficient cardiac differentiation of human embryonic stem cells.

Cardiomyocytes (CMs) differentiated from human embryonic stem cells (hESCs) are a promising and potentially unlimited cell source for myocardial repair and regeneration. Recently, multiple methodologies—primarily based on the optimization of growth factors—have been described for efficient cardiac differentiation of hESCs. However, the role of extracellular matrix (ECM) signaling in CM differentiation has not yet been explored fully. This study examined the role of ECM signaling in the efficient generation of CMs from both H7 and H9 ESCs. The hESCs were differentiated on ECM substrates composed of a range of fibronectin (FN) and laminin (LN) ratios and gelatin and evaluated by the fluorescence activated cell scanning (FACS) analysis on day 14. Of the ECM substrates examined, the 70:30 FN:LN reproducibly generated the greatest numbers of CMs from both hESC lines. Moreover, the LN receptor integrin β4 (ITGB4) and FN receptor integrin β5 (ITGB5) genes, jointly with increased phosphorylated focal adhension kinase and phosphorylated extracellular signal-regulated kinases (p-ERKs), were up-regulated over 13-fold in H7 and H9 cultured on 70:30 FN:LN compared with gelatin. Blocking studies confirmed the role of all these molecules in CM specification, suggesting that the 70:30 FN:LN ECM promotes highly efficient differentiation of CMs through the integrin-mediated MEK/ERK signaling pathway. Lastly, the data suggest that FN:LN-induced signaling utilizes direct cell-to-cell signaling from distinct ITGB4+ and ITGB5+ cells.

Die genomische Integration von HPV16-DNS begünstigt eine photonenstimulierte Integrinexpression und Zellmigration

Persistent human papilloma virus 16 (HPV16) infections are a major cause of cervical cancer. The integration of the viral DNA into the host genome causes E2 gene disruption which prevents apoptosis and increases host cell motility. In cervical cancer patients, survival is limited by local infiltration and systemic dissemination. Surgical control rates are poor in cases of parametrial infiltration. In these patients, radiotherapy (RT) is administered to enhance local control. However, photon irradiation itself has been reported to increase cell motility. In cases of E2-disrupted cervical cancers, this phenomon would impose an additional risk of enhanced tumor cell motility. Here, we analyze mechanisms underlying photon-increased migration in keratinocytes with differential E2 gene status. Isogenic W12 (intact E2 gene status) and S12 (disrupted E2 gene status) keratinocytes were analyzed in fibronectin-based and serum-stimulated migration experiments following single photon doses of 0, 2, and 10 Gy. Quantitative FACS analyses of integrin expression were performed. Migration and adhesion are increased in E2 gene-disrupted keratinocytes. E2 gene disruption promotes attractability by serum components, therefore, effectuating the risk of local infiltration and systemic dissemination. In S12 cells, migration is further increased by photon RT which leads to enhanced expression of fibronectin receptor integrins. HPV16-associated E2 gene disruption is a main predictor of treatment-refractory cancer virulence. E2 gene disruption promotes cell motility. Following photon RT, E2-disrupted tumors bear the risk of integrin-related infiltration and dissemination.

Glycosylation of the laminin receptor (α3β1) regulates its association with tetraspanin CD151: Impact on cell spreading, motility, degradation and invasion of basement membrane by tumor cells

Invasion is the key requirement for cancer metastasis. Expression of β1,6 branched N-oligosaccharides associated with invasiveness, has been shown to promote adhesion to most Extra Cellular Matrix (ECM) and basement membrane (BM) components and haptotactic motility on ECM (fibronectin) but attenuate it on BM (laminin/matrigel) components. To explore the mechanism and to evaluate the significance of these observations in terms of invasion, highly invasive B16BL6 cells were compared with the parent (B16F10) cells or B16BL6 cells in which glycosylation was inhibited. We demonstrate that increased adhesion to matrix components induced secretion of MMP-9, important for invasion. Further, both the subunits of integrin receptors for fibronectin (α5β1) and laminin (α3β1) on B16BL6 cells were shown to carry these oligosaccharides. Although, glycosylation of receptors had no effect on their surface expression, it had same differential effect on cell spreading as haptotactic motility. Absence of correlation between invasiveness and expression of most tetraspanins (major regulators of integrin function) hints at an alternate mechanism. Here we show that glycosylation on α3β1 impedes its association with CD151 and modulates spreading and motility of cells apparently to reach an optimum required for invasion of BM. These studies demonstrate the complex mechanisms used by cancer cells to be invasive.