Increased Β1 Integrin Expression Research Articles

TIMP1 protects against blood-brain barrier disruption after subarachnoid haemorrhage by inhibiting ubiquitination of astrocytic β1-integrin

BackgroundAstrocytes regulate blood-brain barrier (BBB) integrity, whereas subarachnoid haemorrhage (SAH) results in astrocyte dysregulation and BBB disruption. Here, we explored the involvement of tissue inhibitor of matrix metalloprotease-1 (TIMP1) in...

Inhibition of PKC/MEK pathway suppresses β1-integrin and mitigates breast cancer cells proliferation.

Prostaglandin E2 (PGE2) and β1-integrin have been correlated with breast cancer, where both could enhance progression and metastasis. Protein kinase C (PKC) and MEK have played a vital role in breast cancer development. Our study was conducted to elucidate the effect of inhibition of E-prostanoid receptor 1 (EP1)/ PKC/ MEK/ β1-integrin pathway in mitigating breast cancer progression and to evaluate the role of the intermediate signals FOXC2, E2F1, NF-ҡB and survivin. MCF7 cells were treated with 17 -PT-PGE2, an EP1 agonist, for 24 h, and β1-integrin was measured. To MCF7 cells treated with 17-PT-PGE2, inhibitors of either EP1, MEK, PKC or NF-ҡB were added followed by measurement of β1-integrin gene expression and cell proliferation in each case. Addition of 17- PT-PGE2 to MCF7 cells showed enhancement of both cell proliferation, and cell cycle transition from G1 to S phase. In addition, activation of EP1 receptor increased β1-integrin expression. On the contrary, inhibition of EP1 receptor showed a decrease in the cell proliferation, β1-integrin expression and cells transition to S phase, but increased cell count in apoptotic phase. Selective inhibition of each of MEK, PKC, and NF-ҡB suppressed 17 -PT-PGE2-mediated β1-integrin expression as well as cell proliferation. Furthermore, FOXC2, phosphorylated NF-ҡB, E2F1, and survivin levels were upregulated with 17- PT-PGE2 and suppressed by MEK, PKC and NF-ҡB inhibitors. Targeting the biochemical mediators of PKC/MEK pathway may be of value in developing new chemical entities for cancer treatment.

Reveal the Neutrophil: Elucidating the Role of a Neutrophil-Specific JAK2-V617F Mutation

Introduction: Myeloproliferative neoplasms (MPN) are characterized by monoclonal proliferation of myeloid cells. The clinical presentation of classic BCR-ABL negative MPN includes blood count changes, splenomegaly, chronic inflammation and, prognostically relevant, thrombosis. Within polycythemia vera, essential thrombocythemia and primary myelofibrosis, the most frequent genetic alteration is the point mutation V617F (VF) in the Jak2 gene. Due to an activation of leucocyte β1 and β2 integrins, in particular on neutrophils, JAK2-VF is driving thrombus formation (Edelmann et al., JCI, 2018). Thus, understanding the role of neutrophils within JAK2-VF-positive MPN is of special interest. We developed a murine JAK2-VF Catchup model of MPN that is characterized by the neutrophil-specific expression of JAK2-VF in combination with the fluorescent protein tdTomato allowing in-vivo two-photon microscopy. Neutrophil-specificity was achieved through a knock-in allele of Cre recombinase and tdTomato replacing exon 1 of Ly6G (Hasenberg et al., Nat Methods, 2015). Methods: The murine JAK2-VF Catchup model was developed by crossing JAK2+/loxP-VF-loxP mice and Ly6G+/Cre-tdTomato CatchupIVM-red mice. Experiments were performed in 12 weeks old JAK2+/loxP-VF-loxP Ly6G+/Cre-tdTomato CatchupIVM-red (JAK2+/VF Catchup) mice (n=5) and JAK2+/+ Ly6G+/Cre-tdTomato CatchupIVM-red (JAK2+/+ Catchup) mice (n=5) as wildtype (WT) controls. Whole blood cell counts and spleen weight were determined. Bone marrow (BM) cells were harvested from hind limbs followed by isolation of granulocytes using negative selection. In order to determine hematopoietic stem and progenitor cells (HSPCs), BM and spleen cell suspensions were analyzed by flow cytometry (FC). Using ICAM-1, VCAM-1, E-selectin and P-selectin, each with and without Fc-tag, adhesion properties of the isolated granulocytes were examined by either FC or microplate reader analysis. The expression of β1 integrin was further analyzed by FC. Results and Discussion: JAK2+/VF Catchup mice showed a slight but significant increase in platelet counts [1544±58.02 Gpt/l; WT: 1190±90.50 Gpt/l; p=0.011], with the mean value still remaining within the normal range. Hematocrit, red blood cell, white blood cell and neutrophil count as well as spleen weight were also within the normal range showing no differences from their WT controls. Interestingly, neutrophil and platelet counts of JAK2+/VF Catchup mice significantly correlated with one another [p=0.0251; R2=0.8529]. MPN-typical changes in number and composition of c-Kit+ HSPCs in either BM or spleen could not be found in JAK2+/VF Catchup mice. Directly compared to reliably MPN-developing Vav-1-Cre JAK2+/VF mice, JAK2+/VF Catchup mice showed significantly less megakaryocyte-erythroid progenitor (MEP), Lin- Sca1+ c-Kit+ (LSK) and multipotent progenitor cells (MPP) in the BM. In spleen of JAK2+/VF Catchup mice, the fractions of Lin- c-Kit+ (LK) and granulocyte-macrophage progenitor cells (GMP) were significantly lower compared to Vav-1-Cre JAK2+/VF mice. Thus, the neutrophil-specific JAK2-VF mutation does not lead to apparent numerical or compositional changes of HSPCs. These results show that a neutrophil-specific JAK2-VF mutation is not sufficient to induce a characteristic phenotype of JAK2-VF-positive MPN. The expression of β1 integrin on granulocytes was significantly increased in JAK2+/VF Catchup mice [fold change vs. control; 1.126±0.041; WT: 1.0±0.011; p=0.0194]. Unexpectedly, despite increased β1 integrin expression, the binding of granulocytes from JAK2+/VF Catchup mice to either ICAM-1, VCAM-1, E-selectin or P-selectin corresponded to that of their WT controls. Static adhesion and soluble ligand binding assays showed similar results. Since adhesion of granulocytes appeared unchanged in-vitro, we hypothesize that expression of JAK2-VF in neutrophils is not sufficient to overactivate integrins but secondary extrinsic signals (e.g. cytokines) may play a dominant role. Further experimentation is under way to elucidate this molecular mechanism. Conclusions: In order to dissect the properties of JAK2-VF-positive neutrophils, the JAK2-VF Catchup model represents a promising murine model of MPN. Herewith, the initiation of thrombosis and the development of other granulocyte-driven disease manifestations of JAK2-VF induced MPN can be thoroughly examined. Disclosures No relevant conflicts of interest to declare.

Cyclooxygenase-2 induced β1-integrin expression in NSCLC and promoted cell invasion via the EP1/MAPK/E2F-1/FoxC2 signal pathway.

Cyclooxygenase-2 (COX-2) has been implicated in cell invasion in non-small-cell lung cancer (NSCLC). However, the mechanism is unclear. The present study investigated the effect of COX-2 on β1-integrin expression and cell invasion in NSCLC. COX-2 and β1-integrin were co-expressed in NSCLC tissues. COX-2 overexpression or Prostaglandin E2 (PGE2) treatment increased β1-integrin expression in NSCLC cell lines. β1-integrin silencing suppressed COX-2-mediated tumour growth and cancer cell invasion in vivo and in vitro. Prostaglandin E Receptor EP1 transfection or treatment with EP1 agonist mimicked the effect of PGE2 treatment. EP1 siRNA blocked PGE2-mediated β1-integrin expression. EP1 agonist treatment promoted Erk1/2, p38 phosphorylation and E2F-1 expression. MEK1/2 and p38 inhibitors suppressed EP1-mediated β1-integrin expression. E2F-1 silencing suppressed EP1-mediated FoxC2 and β1-integrin upregulation. ChIP and Luciferase Reporter assays identified that EP1 agonist treatment induced E2F-1 binding to FoxC2 promotor directly and improved FoxC2 transcription. FoxC2 siRNA suppressed β1-integrin expression and EP1-mediated cell invasion. Immunohistochemistry showed E2F-1, FoxC2, and EP1R were all highly expressed in the NSCLC cases. This study suggested that COX-2 upregulates β1-integrin expression and cell invasion in NSCLC by activating the MAPK/E2F-1 signalling pathway. Targeting the COX-2/EP1/PKC/MAPK/E2F-1/FoxC2/β1-integrin pathway might represent a new therapeutic strategy for the prevention and treatment of this cancer.

Acquisition of epithelial-mesenchymal transition phenotype in the tamoxifen-resistant breast cancer cell: a new role for G protein-coupled estrogen receptor in mediating tamoxifen resistance through cancer-associated fibroblast-derived fibronectin and β1-integrin signaling pathway in tumor cells

IntroductionAcquired tamoxifen resistance remains the major obstacle to breast cancer endocrine therapy. β1-integrin was identified as one of the target genes of G protein-coupled estrogen receptor (GPER), a novel estrogen receptor recognized as an initiator of tamoxifen resistance. Here, we investigated the role of β1-integrin in GPER-mediated tamoxifen resistance in breast cancer.MethodsThe expression of β1-integrin and biomarkers of epithelial-mesenchymal transition were evaluated immunohistochemically in 53 specimens of metastases and paired primary tumors. The function of β1-integrin was investigated in tamoxifen-resistant (MCF-7R) subclones, derived from parental MCF-7 cells, and MCF-7R β1-integrin-silenced subclones in MTT and Transwell assays. Involved signaling pathways were identified using specific inhibitors and Western blotting analysis.ResultsGPER, β1-integrin and mesenchymal biomarkers (vimentin and fibronectin) expression in metastases increased compared to the corresponding primary tumors; a close expression pattern of β1-integrin and GPER were in metastases. Increased β1-integrin expression was also confirmed in MCF-7R cells compared with MCF-7 cells. This upregulation of β1-integrin was induced by agonists of GPER and blocked by both antagonist and knockdown of it in MCF-7R cells. Moreover, the epidermal growth factor receptor/extracellular regulated protein kinase (EGFR/ERK) signaling pathway was involved in this transcriptional regulation since specific inhibitors of these kinases also reduced the GPER-induced upregulation of β1-integrin. Interestingly, silencing of β1-integrin partially rescued the sensitivity of MCF-7R cells to tamoxifen and the α5β1-integrin subunit is probably responsible for this phenomenon. Importantly, the cell migration and epithelial-mesenchymal transition induced by cancer-associated fibroblasts, or the product of cancer-associated fibroblasts, fibronectin, were reduced by knockdown of β1-integrin in MCF-7R cells. In addition, the downstream kinases of β1-integrin including focal adhesion kinase, Src and AKT were activated in MCF-7R cells and may be involved in the interaction between cancer cells and cancer-associated fibroblasts.ConclusionsGPER/EGFR/ERK signaling upregulates β1-integrin expression and activates downstream kinases, which contributes to cancer-associated fibroblast-induced cell migration and epithelial-mesenchymal transition, in MCF-7R cells. GPER probably contributes to tamoxifen resistance via interaction with the tumor microenvironment in a β1-integrin-dependent pattern. Thus, β1-integrin may be a potential target to improve anti-hormone therapy responses in breast cancer patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0579-y) contains supplementary material, which is available to authorized users.

CXCL12/SDF‐1 Stimulates Megakaryocyte β‐1,4‐Galactosyltransferase 1 Activity to Enhance Platelet Production in Vivo

Platelet recovery following bone marrow (BM) transplant and chemotherapy is crucial to prevent bleeding complications. The chemokine SDF‐1 promotes megakaryocyte (MK) migration towards BM sinusoids increasing platelet production. Here we show that SDF‐1 upregulates expression of the glycan structure Type‐2‐Lactosaminoglycan (LacNAc), synthesized by β4Galactosyltransferase1 (β4galt1). We hypothesized that SDF‐1 increases LacNAc synthesis promoting platelet production. Supporting this hypothesis, β4galt1–/– mice had severe macrothrombocytopenia (80% reduction) with increased BMMK numbers (35%), which localized poorly with BM sinusoids (50%), compared to WT MKs (72%). β4galt1–/– hematopoietic stem cells (HSCs) transplanted into lethally irradiated WT mice restored BMMKs, which failed to migrate to sinusoids and produce circulating platelets. We next treated WT and β4galt1–/– mice with the myeloablative chemotherapeutic agent 5‐Fluorouracil (5‐FU), which decreased platelet counts by 50% in both phenotypes. SDF‐1 injections increased platelet counts by 70% in 5‐FU treated WT mice but not in β4galt1–/– mice. Importantly, WT but not β4galt1–/– platelets had increased LacNAc expression after SDF‐1 injections.β4galt1–/– BMMKs had increased β1 integrin expression. Expression of laminin, β1 integrin ligand, was upregulated in β4galt1–/– MKs and sinusoids. Our results suggest that SDF‐1 upregulates β4GalT1‐dependentLacNAc expression promoting MK interaction with BM sinusoids, likely regulating β1 integrin expression and interaction with components of the extracellular matrix, specifically laminin.

Prostaglandin E2 stimulates β1-integrin expression in hepatocellular carcinoma through the EP1 receptor/PKC/NF-κB pathway.

Prostaglandin E2 (PGE2) has been implicated in cell invasion in hepatocellular carcinoma (HCC), via increased β1-integrin expression and cell migration; however, the mechanism remains unclear. PGE2 exerts its effects via four subtypes of the E prostanoid receptor (EP receptor 1–4). The present study investigated the effect of EP1 receptor activation on β1-integrin expression and cell migration in HCC. Cell migration increased by 60% in cells treated with 17-PT-PGE2 (EP1 agonist), which was suppressed by pretreatment with a β1-integrin polyclonal antibody. PGE2 increased β1-integrin expression by approximately 2-fold. EP1 receptor transfection or treatment with 17-PT-PGE2 mimicked the effect of PGE2 treatment. EP1 siRNA blocked PGE2-mediated β1-integrin expression. 17-PT-PGE2 treatment induced PKC and NF-κB activation; PKC and NF-κB inhibitors suppressed 17-PT-PGE2-mediated β1-integrin expression. FoxC2, a β1-integrin transcription factor, was also upregulated by 17-PT-PGE2. NF-κB inhibitor suppressed 17-PT-PGE2-mediated FoxC2 upregulation. Immunohistochemistry showed p65, FoxC2, EP1 receptor and β1-integrin were all highly expressed in the HCC cases. This study suggested that PGE2 upregulates β1-integrin expression and cell migration in HCC cells by activating the PKC/NF-κB signaling pathway. Targeting PGE2/EP1/PKC/NF-κB/FoxC2/β1-integrin pathway may represent a new therapeutic strategy for the prevention and treatment of this cancer.

Prostaglandin E2 upregulates β1 integrin expression via the E prostanoid 1 receptor/nuclear factor κ-light-chain-enhancer of activated B cells pathway in non-small-cell lung cancer cells

The prostaglandin E2 (PGE2) E prostanoid (EP)1 receptor shown to be associated with lung cancer cell invasion. However, the mechanism of EP1 receptor-mediated cell migration remains to be elucidated. β1 integrin is an essential regulator of the tumorigenic properties of non-small-cell lung carcinoma (NSCLC) cells. To date, little is known regarding the association between the EP1 receptor and β1 integrin expression. The present study investigated the effect of EP1 receptor activation on β1 integrin expression and cell migration in NSCLC cells. A total of 34 patients with clinical diagnosis of NSCLC and 10 patients with benign disease were recruited for the present study. The expression levels of the EP1 receptor and β1 integrin expression were studied in resected lung tissue using immunohistochemistry. A statistical analysis was performed using Stata se12.0 software. The effects of PGE2, EP1 agonist 17-phenyl trinor-PGE2 (17-PT-PGE2) and the nuclear factor κ-B (NF-κB) inhibitor on β1 integrin expression were investigated on A549 cells. The expression of β1 integrin and the phosphorylation of NF-κB‑p65 Ser536 was investigated by western blot analysis. Cell migration was assessed by a transwell assay. The results demonstrated that β1 integrin and EP1 receptor expression exhibited a positive correlation of evident significance in the 44 samples. The in vitro migration assay revealed that cell migration was increased by 30% when the cells were treated with 5 µM 17-PT-PGE2 and that the pre-treatment of β1 integrin monoclonal antibody inhibited 17-PT-PGE2‑mediated cell migration completely. PGE2 and 17-PT-PGE2 treatment increased β1 integrin expression. RNA interference against the EP1 receptor blocked the PGE2-mediated β1 integrin expression in A549 cells. Treatment with 17-PT-PGE2 induced NF-κB activation, and the selective NF-κB inhibitor pyrrolidinedithiocarbamate inhibited 17-PT-PGE2-mediated β1 integrin expression. In conclusion, the present study indicated that the PGE2 EP1 receptor regulates β1 integrin expression and cell migration in NSCLC cells by activating the NF-κB signaling pathway. Targeting the PGE2/EP1/β1 integrin signaling pathway may aid in the development of new therapeutic strategies for the prevention and treatment of this type of cancer.

β1 Integrin Targeting Potentiates Antiangiogenic Therapy and Inhibits the Growth of Bevacizumab-Resistant Glioblastoma

Antiangiogenic therapies like bevacizumab offer promise for cancer treatment, but acquired resistance, which often includes an aggressive mesenchymal phenotype, can limit the use of these agents. Upregulation of β1 integrin (ITGB1) occurs in some bevacizumab-resistant glioblastomas (BRG) whereby, mediating tumor-microenvironment interactions, we hypothesized that it may mediate a mesenchymal-type resistance to antiangiogenic therapy. Immunostaining analyses of β1 integrin and its downstream effector kinase FAK revealed upregulation in 75% and 86% of BRGs, respectively, compared with pretreatment paired specimens. Furthermore, flow cytometry revealed eight-fold more β1 integrin in primary BRG cells compared with cells from bevacizumab-naïve glioblastomas (BNG). Fluorescence recovery after photobleaching of cells engineered to express a β1-GFP fusion protein indicated that the mobile β1 integrin fraction was doubled, and half-life of β1 integrin turnover in focal adhesions was reduced markedly in BRG cells compared with bevacizumab-responsive glioblastoma multiforme cells. Hypoxia, which was increased with acquisition of bevacizumab resistance, was associated with increased β1 integrin expression in cultured BNG cells. BRGs displayed an aggressive mesenchymal-like phenotype in vitro. We found that growth of BRG xenograft tumors was attenuated by the β1 antibody, OS2966, allowing a 20-fold dose reduction of bevacizumab per cycle in this model. Intracranial delivery of OS2966 through osmotic pumps over 28 days increased tumor cell apoptosis, decreased tumor cell invasiveness, and blunted the mesenchymal morphology of tumor cells. We concluded that β1 integrin upregulation in BRGs likely reflects an onset of hypoxia caused by antiangiogenic therapy, and that β1 inhibition is well tolerated in vivo as a tractable strategy to disrupt resistance to this therapy.

Continuous resistance training alters exercise‐induced MMPs activation and expression of ECM components

Recent studies indicate that matrix metalloproteinases (MMPs) and skeletal muscle extracellular matrix (ECM), especially basal lamina, components play a role in muscle mass regulation, although the effects of resistance training (RT) on those proteins are unclear. Thus, the purpose of the study was to investigate the effects of RT on activities and protein expression of MMPs and ECM. Ten male SD rats were divided into 1‐exercise bout (1B) or 18‐exercise bouts (18B) group. This study was conducted in conformance with the FASEB Statement of Principles for the use of animals in research and education. The right gastrocnemius muscle was isometrically trained via percutaneous electrical stimulation (maximum contraction for 5 sec × 5, 5 sets) every other day, whereas the left leg served as a control. MMP‐2 and MMP‐9 activities (via gelatin zymography) were increased 10 min after acute exercise with no change in expression of tissue inhibitors of metalloproteinases in 1B group. However, repeated bouts attenuated exercise‐induced MMPs activation in 18B group. MMPs expressions were significantly higher in trained muscles. RT increased β1‐integrin expression but not the other ECM proteins. Our results suggest that resistance exercise activates MMPs during initial phase of RT but this response is attenuated with continuation of RT.This study was supported by grant from Meiji Yasuda Life Foundation of Health and Welfare.

Fibrillar Type I Collagen Matrices Enhance Metastasis/Invasion of Ovarian Epithelial Cancer Via β1 Integrin and PTEN Signals

This study investigated the involvement of fibrillar collagen in remodeling extracellular matrices (ECM) and its significant impact on the metastasis/invasion of epithelial ovarian cancer cells via β1 integrin/phosphatase and tensin homolog (PTEN) signaling. Normal ovarian surface epithelium tissues (n = 13), ovarian cancer tissues (n = 28), ovarian cancer cell lines, and a 3-dimensional model of fibrillar type I collagen that mimicked pathological ECM in vivo were used in the study. We explored the specific mechanisms behind ECM remodeling and the cellular signals that affected the invasion of ovarian cancer cells. The data showed that increased β1 integrin expression in ovarian cancer cells led to enhance migration/invasion of ovarian cancer cells via regulation of PTEN/protein kinase B (Akt) signal in response to fibrillar type I collagen matrices. Low PTEN activity corresponded to the following: (1) increased PTEN degradation and (2) phosphorylation of PTEN. Decreased protein phosphatase 2A activity was detected in ovarian cancer. Protein phosphatase 2A might play a role in enhancing the progression of ovarian cancer through regulating PTEN/Akt signal. These findings indicate that fibrillar type I collagen, by modulating integrin-PTEN/PI3K/Akt signaling pathway in remodeling ECM, is very important in affecting the invasion of aggressive ovarian cancer cells. Moreover, these data provide direct evidence for pathological ECM remodeling and cell signaling networks involved in the invasion of ovarian cancer cells.

Involvement of β1‐integrin via PIP complex and FAK/paxillin in dexamethasone‐induced human mesenchymal stem cells migration

Although glucocorticoids strongly affect numerous biological processes including cell growth, development, and homeostasis, their effects on migration of human mesenchymal stem cells (hMSCs) are unclear. Therefore, we investigated the role of dexamethasone (DEX) and its related signaling pathways on migration of hMSCs. We found that DEX, at 10(-8) to 10(-6) M, significantly increased migration after a 24 h incubation, and DEX (10(-6) M) increased migration at >12 h. Moreover, DEX (10(-6) M) increased the level of glucocorticoid receptor (GR)-α mRNA and protein expression, but not GR-β mRNA. The increases in DEX-induced migration were inhibited by the GR antagonist mifepristone (10(-7) M). In addition, DEX increased integrin-linked kinase (ILK) and α-parvin expression but did not change PINCH-1/2 expression in lysate. DEX also increased formations of complex with ILK and α-parvin, and ILK and PINCH-1/2 as shown by immunoprecipitation, which were all inhibited by mifepristone. DEX-induced migration was blocked by ILK and α-parvin small interfering(si)RNAs. In addition, DEX increased focal adhesion kinase (FAK) and paxillin expression, which were attenuated by ILK and α-parvin siRNAs. DEX-induced cell migration was inhibited by FAK/paxillin siRNAs. DEX also increased β1-integrin expression, which was blocked by FAK/paxillin siRNAs. In addition, DEX-induced cell migration was inhibited by β1-integrin siRNA. Downregulation of ILK, α-parvin, FAK/paxillin and β1-integrin expression by siRNAs decreased DEX-induced filamentous(F)-actin organization and migration of hMSCs. In conclusion, DEX partially stimulates hMSC migration by the expression of β1-integrin through formation of a PINCH-1/2/ILK/α-parvin complex (PIP complex), and FAK and paxillin expression.

Topical HDL administration reduces vein graft atherosclerosis in apo E deficient mice

ObjectiveUse of autologous vein grafts for surgical revascularisation is limited by vein graft failure. Topical high-density lipoprotein (HDL) administration on the adventitial side of vein grafts was evaluated as a new therapeutic modality to improve vein graft patency and function. MethodsCaval veins of C57BL/6 apo E−/− mice were grafted to the right carotid arteries of recipient 3 month-old C57BL/6 TIE2-LacZ/apo E−/− mice. HDL (200μg/ml; 50μl) in 20% pluronic F-127 gel was applied on the adventitial side of vein grafts. ResultsTopical HDL application reduced intimal area by 55% (p<0.001) at day 28 compared to control mice. Blood flow quantified by micro magnetic resonance imaging at day 28 was 2.8-fold (p<0.0001) higher in grafts of topical HDL treated mice than in control mice. Topical HDL potently reduced intimal inflammation and resulted in enhanced endothelial regeneration as evidenced by a 1.9-fold (p<0.05) increase in the number of CD31 positive endothelial cells. HDL potently enhanced migration and adhesion of endothelial colony-forming cells (ECFCs) in vitro, and these effects were dependent on signaling via scavenger receptor-BI, extracellular signal-regulated kinases, and NO, and on increased β1 integrin expression. Correspondingly, the number of CD31 β-galactosidase double positive cells, reflecting incorporated circulating progenitor cells, was 3.9-fold (p<0.01) higher in grafts of HDL treated mice than in control grafts. ConclusionsTopical HDL administration on the adventitial side of vein grafts attenuates vein graft atherosclerosis via increased incorporation of circulating progenitor cells in the endothelium, enhanced endothelial regeneration, and reduced intimal inflammation.

Abstract SY35-03: eIF4E in breast cancer is a master regulator of tumor cell invasion through increased translation of beta-1 integrin mRNA and TGF-beta activation

Abstract eIF4E is frequently overexpressed in tumors of the breast, colon, prostate, and lung, and expression correlates with disease progression, increased tissue invasion and metastasis. It has been suggested that overexpression of eIF4E contributes to malignancy by selectively increasing translation of mRNAs containing significant secondary structure in the their 5' noncoding regions. A limited group of mRNAs encode key proteins involved in cellular growth, survival, angiogenesis and malignancy and contain long GC-rich highly structured 5' noncoding regions. The effect of increased levels of eIF4E in tumor cells, however, is still not well understood, nor is the role of its mTOR regulated repressor, 4E-BP1. Here we demonstrate the surprising finding that overexpression of eIF4E acts primarily to increase breast cancer cell invasion by significantly increasing translation of a single mRNA: β1 integrin. eIF4E overexpression at levels found in breast cancers (3-4 fold) results in a 10-fold increased level of tumor cell invasion. Analysis of polysomal mRNA found increased translation of β1 integrin mRNA with physiological levels of eIF4E overexpression observed in breast cancers. We found that increased β1 integrin expression was due to increased read-through of the complex GC-rich 5' noncoding region of β1 integrin mRNA following eIF4E overexpression, the first time this has been described physiologically, and that this effect is blocked by co-overexpression of 4E-BP1. The modest increase in β1 integrin protein levels is amplified by a disproportionately large increase in the formation of the cell surface heterodimer α3β1, an integrin frequently implicated in breast tumor invasion. These and other results can account for the large increase in invasive ability in eIF4E overexpressing breast cancer cells, suggesting that β1 integrin levels may be limiting in the cell. In addition, we found that increased expression of eIF4E results in increased transcription and consequent protein levels of many canonical TGFβ-responsive genes, including β1 integrin. TGFβ is partially responsible for the increased invasion seen with eIF4E overexpression, although cells are still 4.5 fold more invasive even in the absence of the cytokine, consistent with the TGFβ-independent increase in β1 integrin levels observed with eIF4E overexpresison. The TGFβ transcriptional effect is likely due to an increased rate of activation of latent TGFβ (typically present in serum or the stroma), mediated by the eIF4E-driven increased levels of β1 integrin and its subsequent heterodimerization with α integrins capable of activating TGFβ, including αvβ1. TGFβ transcriptional effects are due to TGFβ ligation with its receptor, and not eIF4E-mediated increased translation of a downstream TGFβ effector, as they are blocked with the use of a TGFβ receptor I kinase inhibitor. Additionally, eIF4E overexpression does not facilitate the translation of TGFβ mRNA, as protein levels of TGFβ are not increased with elevated levels of eIF4E. Co-overexpression of 4E-BP1 blocks all TGFβ effects. Our results suggest a new but prevalent model whereby eIF4E level is a master regulator that determines the direction of TGFβ activity. Increased expression of eIF4E at typical human breast cancer levels mediates increased tumor cell invasion through the selective increased translation of β1 integrin mRNA. Higher levels of β1 integrin promotes increased heterodimerization with α-integrins which activate latent TGFβ present in the breast stroma, thereby directing TGFβ to pro-invasive function. Citation Format: Celine Mestel, Mary-Helen Barcelos-Hoff, Jiri Zavadil, Silvia C. Formenti, Robert J. Schneider. eIF4E in breast cancer is a master regulator of tumor cell invasion through increased translation of beta-1 integrin mRNA and TGF-beta activation [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr SY35-03

Human leiomyoma cell proliferation and extracellular matrix expression is inhibited by integrin signaling

OBJECTIVE: The integrin family of extracellular matrix (ECM) receptors mediates cell-matrix interactions, differentiation and survival. Increased β1 integrin expression has been demonstrated in leiomyomas. Integrins can mediate their activity through RhoA activation. The objective of this study was to determine if functional inhibition of integrin signaling might affect leiomyoma cell proliferation and ECM production.DESIGN: Experimental interventionMATERIALS AND METHODS: Immortalized uterine leiomyoma and myometrial cell lines were incubated with function inhibiting antibodies to α2, α3, α6 and β1 integrins to determine the effect on proliferation. Rho activity was measured using the RhoA G-LISA kit. Proteins were visualized by immunofluorescence. ECM gene expression was evaluated by qRTPCR.RESULTS: After 72hrs of incubation and at 1μg/ml β1 integrin antibody, we observed a 45% growth inhibition of leiomyoma cells compared to 25% inhibition of myometrial cells. Functional inhibition of the α-integrins did not significantly effect proliferation of either cell lines. Leiomyoma cells also showed decreased surface attachment. Higher levels of Rho activity and FAK expression were measured in leiomyoma cells. Following β1 antibody treatment, we observed inhibition of Rho activity (1.5 fold) and serum induced F-actin. Leiomyoma cells expressed a 3-fold higher expression of fibronectin gene compared to myometrial cells. At lower concentration of β1 antibody, leiomyoma cells showed a 3.16+0.32 fold (p<0.05) decreased fibronectin expression. Myometrial cells demonstrated an upregulation of fibronectin gene at all concentrations. Similar expression patterns were observed with collagen 1A1 and versican genes.CONCLUSIONS: Inhibition of β1 integrin signaling resulted in a reduction in leiomyoma cell growth and Rho activity. Furthermore, increased levels of collagen 1A1, versican and fibronectin were dependent upon β1 integrin signaling in leiomyoma cells, suggesting the possibility of a novel pathway to regulate fibroid growth. OBJECTIVE: The integrin family of extracellular matrix (ECM) receptors mediates cell-matrix interactions, differentiation and survival. Increased β1 integrin expression has been demonstrated in leiomyomas. Integrins can mediate their activity through RhoA activation. The objective of this study was to determine if functional inhibition of integrin signaling might affect leiomyoma cell proliferation and ECM production. DESIGN: Experimental intervention MATERIALS AND METHODS: Immortalized uterine leiomyoma and myometrial cell lines were incubated with function inhibiting antibodies to α2, α3, α6 and β1 integrins to determine the effect on proliferation. Rho activity was measured using the RhoA G-LISA kit. Proteins were visualized by immunofluorescence. ECM gene expression was evaluated by qRTPCR. RESULTS: After 72hrs of incubation and at 1μg/ml β1 integrin antibody, we observed a 45% growth inhibition of leiomyoma cells compared to 25% inhibition of myometrial cells. Functional inhibition of the α-integrins did not significantly effect proliferation of either cell lines. Leiomyoma cells also showed decreased surface attachment. Higher levels of Rho activity and FAK expression were measured in leiomyoma cells. Following β1 antibody treatment, we observed inhibition of Rho activity (1.5 fold) and serum induced F-actin. Leiomyoma cells expressed a 3-fold higher expression of fibronectin gene compared to myometrial cells. At lower concentration of β1 antibody, leiomyoma cells showed a 3.16+0.32 fold (p<0.05) decreased fibronectin expression. Myometrial cells demonstrated an upregulation of fibronectin gene at all concentrations. Similar expression patterns were observed with collagen 1A1 and versican genes. CONCLUSIONS: Inhibition of β1 integrin signaling resulted in a reduction in leiomyoma cell growth and Rho activity. Furthermore, increased levels of collagen 1A1, versican and fibronectin were dependent upon β1 integrin signaling in leiomyoma cells, suggesting the possibility of a novel pathway to regulate fibroid growth.

BCR/ABL Expression of Myeloid Progenitors Increases β1-Integrin Mediated Adhesion to Stromal Cells

The expression of the fusion protein BCR/ABL is a hallmark of chronic myeloid leukemia. BCR/ABL is a constitutively active tyrosine kinase influencing cell proliferation, apoptosis, and differentiation. To what extent and by which mechanism BCR/ABL affects the adhesion of leukemic cells to bone marrow stromal cells (BMSC) is controversial. To characterize adhesion of BCR/ABL-transformed 32D cells (32D-BCR/ABL) to the BMSC line M2-10B4, we used washing assays and single-cell force spectroscopy (SCFS). Compared to control 32D cells (32D-V), 32D-BCR/ABL developed threefold higher adhesion forces. This enhanced cell adhesion could be reduced to control levels after specifically inhibiting the activity of the tyrosine kinase BCR/ABL using imatinib mesylate (IM). SCFS showed that the adhesion forces of 32D-BCR/ABL were strongest to fibronectin and collagen type I, suggesting that β1-integrin has a major role in mediating the adhesion of leukemic cells to BMSC. Indeed, the β1-integrin blocking antibody Ha2/5 abrogated the attachment of 32D-V and 32D-BCR/ABL cells to BMSC. Although 32D-BCR/ABL cells show significantly increased β1-integrin expression, no significant difference of β1-integrin mRNA levels could be detected, indicating a post-transcriptional regulation of β1-comprising integrin heterodimers by BCR/ABL. The data presented here argue that the interaction of β1-integrin and extracellular matrix components is functionally important in leukemic cells expressing high-levels of BCR/ABL, and could provide a rationale for the development of optimized targeted therapies.

Expression and function of the chemokine receptor CCR7 in thyroid carcinomas

The chemokine receptor CCR7 plays a critical role in lymphocyte and dendritic cell trafficking into and within lymph nodes, the preferential metastatic site for papillary (PTC) and medullary (MTC) thyroid carcinomas. In order to determine a possible role for CCR7 in mediating the metastatic behaviour of thyroid carcinomas, we analysed its expression in normal and tumoral thyroid tissues of different histotypes and studied the in vitro effects of its activation by the CCR7 ligand, CCL21. Using real-time quantitative-PCR, we observed that CCR7 expression was higher in PTCs and MTCs than in follicular and poorly differentiated thyroid carcinomas. CCR7 expression was ninefold higher in classic compared with follicular variants of PTCs, and its expression in MTCs was significantly correlated with lymph node metastases. Immunohistochemical staining for CCR7 showed protein expression in neoplastic thyroid cells, with higher intensity in PTCs, MTCs and their lymph node metastases (LNMs). We further showed that CCL21 stimulation of a CCR7-expressing thyroid tumour cell line (TPC-1) promotes cell proliferation and migration, and the chemotactic effect of CCL21 in these cells involves actin polymerization, increased beta1-integrin expression and increased matrix metalloproteinase secretion. Taken together, our results demonstrate that CCR7 activation on thyroid carcinoma cells by CCL21 - a chemokine abundantly expressed in lymph nodes - favours tissue invasion and cell proliferation, and therefore may promote thyroid carcinoma growth and LNM.

Extracellular matrix regulates glomerular epithelial cell survival and proliferation.

Glomerular epithelial cell (GEC) injury and apoptosis may contribute to sclerosis in glomerulonephritis. The present study addresses signals that regulate survival of GEC in culture and in the acute puromycin aminonucleoside nephrosis (PAN) model of GEC injury in vivo. Compared with GEC on plastic substratum, adhesion to collagen increased activation of focal adhesion kinase (FAK), c-Src, and ERK and facilitated survival (prevented apoptosis). GEC on plastic exhibited increased caspase-8 and -9 activities, increased expression of the proapoptotic protein, Bax, and decreased the antiapoptotic protein, Bcl-XL, compared with collagen. Stable expression of constitutively active mutants of FAK (CD2-FAK) or MEK (R4F-MEK) activated the ERK pathway and supplanted the requirement of collagen for survival. In contrast, expression of a Ras mutant that activates phosphatidylinositol 3-kinase but blocks ERK activation or pharmacological inhibition of the ERK pathway decreased survival on collagen. Glomeruli isolated from rats with PAN revealed increased beta1-integrin expression, along with increased activation of FAK, c-Src, and ERK, compared with controls. EGF receptor activation was undetectable in PAN. Therefore, adhesion to collagen, resulting in activation of FAK and the Ras-ERK pathway, supports GEC survival. Analogous signals for GEC survival are activated in PAN.

Expression of β1 Integrins by Cultured Articular Chondrocytes and in Osteoarthritic Cartilage

Expression of β1 integrins was studied in vitro as articular chondrocytes reestablished a matrix in culture and in situ in a nonhuman primate model of osteoarthritis in order to investigate a potential role for integrins in mediating cell-extracellular matrix interactions in cartilage. Chondrocytes were found to express α1β1, α3β1, and α5β1 integrins both in vitro and in situ. Cell surface expression of β1 integrins increased as chondrocytes were maintained in culture from 3 to 7 days. Increased β1 integrin expression was also observed in osteoarthritic cartilage compared with normal cartilage. The greatest relative increase in both systems was noted for the α1β1 integrin. The increase in chondrocyte β1 integrin expression in vitro was noted in both monolayer and alginate cultures and occurred prior to detectable changes in the differentiated phenotype of the chondrocyte. Disruption of the cytoskeleton with the drug dihydrocytochalasin B inhibited the cell culture induced increase in integrin expression, while treatment of cultured cells with TGF-β resulted in increased expression of the α5β1 integrin. The modulation of β1 integrin expression noted in vitro and in situ indicates that chondrocytes are capable of regulated expression of β1 integrins and suggests that β1 integrins may play an important role in mediating chondrocyte-extracellular matrix interactions in cartilage.