Mediator Of Migration Research Articles

Interleukin-23 Facilitates Thyroid Cancer Cell Migration and Invasion by Inhibiting SOCS4 Expression via MicroRNA-25.

Interleukin–23 (IL–23) is a conventional proinflammatory cytokine that plays a role in tumor progression by inducing inflammation in the tumor microenvironment. However, the role of IL–23 in thyroid cancer migration and invasion remains unclear. In the present study, we observed that the treatment with IL–23, induced migration and invasion in human thyroid cancer cells. Additional data demonstrate that SOCS4 negatively regulates IL-23-mediated migration and invasion. On investigating the mechanisms involved in IL–23 mediated migration and invasion, we observed that miR–25 promotes the migration and invasion of thyroid cancer cells by directly binding to the 3′-UTR of SOCS4 that leads to the inhibition of SOCS4. In addition, we also demonstrated that IL–23 increases miR–25 expression levels, and overexpressed miR–25 is involved in IL-23-associated SOCS4 inhibition and cell migration and invasion. Together, our data suggest that IL–23 induces migration and invasion in thyroid cancer cells by mediating the miR–25/SOCS4 signaling pathway.

YangZheng XiaoJi exerts anti-tumour growth effects by antagonising the effects of HGF and its receptor, cMET, in human lung cancer cells.

BackgroundHepatocyte growth factor (HGF) is a cytokine that has a profound effect on cancer cells by stimulating migration and invasion and acting as an angiogenic factor. In lung cancer, the factor also plays a pivotal role and is linked to a poor outcome in patients. In particular, HGF is known to work in combination with EGF on lung cancer cells. In the present study, we investigated the effect of a traditional Chinese medicine reported in cancer therapies, namely YangZheng XiaoJi (YZXJ) on lung cancer and on HGF mediated migration and invasion of lung cancer cells.MethodsHuman lung cancer cells, SKMES1 and A549 were used in the study. An extract from the medicine was used. Cell migration was investigated using the EVOS and by ECIS. Cell–matrix adhesion and in vitro invasion were assessed. In vivo growth of lung cancer was tested using an in vivo xenograft tumour model and activation of the HGF receptor in lung tumours by an immunofluorescence method.ResultsBoth lung cancer cells increased their migration in response to HGF and responded to YZXJ by reducing their speed of migration. YZXJ markedly reduced the migration and in vitro invasiveness induced by HGF. It worked synergistically with PHA665752 and SU11274, HGF receptor inhibitors on the lung cancer cells both on HGF receptor activation and on cell functions. A combination of HGF and EGF resulted in a greater increase in cell migration, which was similarly inhibited by YZXJ, and in combination with the HGF receptor and EGF receptor inhibitors. In vivo, YZXJ reduced the rate of tumour growth and potentiated the effects of PHA665752 on tumour growth. It was further revealed that YZXJ significantly reduced the degree of phosphorylation of the HGF receptor in lung tumours.ConclusionYZXJ has a significant role in reducing the migration, invasion and in vivo tumour growth of lung cancer and acts to inhibit the migratory and invasive effects induced by HGF and indeed by HGF/EGF. This effect is likely attributed to the inhibition of the HGF receptor activation. These results indicate that YZXJ has a therapeutic role in lung cancer and that combined strategy with methods to block HGF and EGF should be considered.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0639-1) contains supplementary material, which is available to authorized users.

Abstract 2072: Ormeloxifene attenuates wnt/β-catenin signaling in colon cancer cells by modulation of PKD1 and glycolytic pathways

Abstract Background: Colorectal cancer is the third most commonly diagnosed cancer and third leading cause of cancer-related deaths. Aberrant Wnt/β-catenin signaling is implicated in development and progression of colon cancers. Therefore, modulation of this master regulatory signaling is essential for the prevention and treatment of colon cancer. Protein Kinase D1 (PKD1) which is downregulated in colon cancer, regulates β-catenin transcriptional activity by its phosphorylation. Therefore, drug mediated upregulation of PKD1 may have high clinical significance for the prevention/treatment of Wnt/β-catenin signaling induced cancers. We have identified, ormeloxifene, a synthetic non-steroidal selective estrogen receptor modulator (SERM), as a novel PKD1 modulator. Herein, we have investigated the effect of ormeloxifene on tumorigenic phenotypes of colon cancer cells and its effect on PKD1 and glycolytic pathways mediated oncogenic β-catenin signaling. Materials and Methods: We have used SW480-PKD1-GFP and SW480-GFP colon cancer cells (SW480 has a mutated APC gene leading to stabilization of β-catenin due to lack of degradation) to investigate the effect of PKD1 on β-catenin activity and xenograft mouse model. SW480, SW48, SW620 and Lovo cell lines have been used to observe the effect of ormeloxifene (5-20 μM) on various cellular characteristics, including cell proliferation, clonogenic potential, motility and aggregation. β-catenin/T-cell factor 4 (TCF4) transcription activity was measured by Dual-Glo luciferase reporter assays. The expression and subcellular localization of proteins was determined by immunoblotting and confocal microscopy. Chemokine (C-C) motif ligand 2 (CCL2) and glucose assay were performed using ELISA technique. Results: Ormeloxifene treatment restores PKD1 expression, thereby, attenuates β-catenin transcriptional activity. We have also observed that ormeloxifene treatment effectively inhibits CCL2 expression, glucose uptake, glucose metabolism and Glut1 receptor expression in cancer cells. Ormeloxifene (5-20μM) inhibits cell proliferation and motility of colon cancer cells. Additionally, ormeloxifene effectively inhibited CCL2 mediated cellular migration of colon cancer cells. Furthermore, Western blot results depict that PKD1 inhibits TCF4 activity in colon cancer cells. SW480 PKD1 overexpressing cells clearly delayed tumor formation in nude mice, compared to control cells. In addition, SW480-PKD1-GFP cells showed less hypoxia (Glut1) and increased Vasculature (CD31) in tumors. Conclusion: Our findings demonstrate that ormeloxifene treatment effectively attenuates β-catenin mediated oncogenic signaling and tumorigenic phenotypes of colon cancer cells via upregulation of PKD1 and suppression of glycolytic pathways. Thus it can be used as a potential new preventive/treatment modality for colon cancer. Citation Format: Aditya Ganju, Rishi Gara, Sonam Kumari, Man Mohan Singh, Subhash Chauhan, Meena Jaggi. Ormeloxifene attenuates wnt/β-catenin signaling in colon cancer cells by modulation of PKD1 and glycolytic pathways. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2072. doi:10.1158/1538-7445.AM2015-2072

MicroRNA-141 inhibits migration of gastric cancer by targeting zinc finger E-box-binding homeobox 2.

Human microRNA (miR)-141 is a member of the miR-200 family, which has been reported to be downregulated in gastric cancer, and involved in the proliferation of gastric cancer cells. However, little is currently known regarding its role in the migration of gastric cancer. The present study investigated the function of miR-141 in gastric cancer cell migration, and evaluated the contribution of zinc finger E-box-binding homeobox 1 and 2 (ZEB1/2) in miR-141 mediated migration of gastric cancer cells. The expression levels of miR-141 and its potential ZEB1/2 targets were examined by quantitative polymerase chain reaction (qPCR) and western blotting, respectively. The migration of SGC-7901 and HGC-27 gastric cancer cells, which had been transfected with an miRNA precursor, was examined by cell migration and wound healing assays. A luciferase activity assay was used to validate whether ZEB1/2 was a direct target of miR-141. The results demonstrated that overexpression of miR-141 markedly inhibited the migration of gastric cancer cells in vitro. Forced overexpression of miR-141 significantly reduced the luciferase activity of the 3′-untranslated region of ZEB2 in gastric cancer cells. Furthermore, the mRNA and protein expression levels of ZEB2 were reduced in cells overexpressing miR-141, whereas the protein expression levels of E-cadherin were increased. In gastric tumor samples the expression levels of ZEB2 were inversely correlated with the expression of miR-141. These results suggest that miR-141 may be involved in the inhibition of gastric cancer cell migration, and that ZEB2 is a target gene of miR-141.

ChemInform Abstract: Lab on Upconversion Nanoparticles: Optical Properties and Applications Engineering via Designed Nanostructure

AbstractReview: 268 refs.

Abstract 656: Lipid Phosphate Phosphatase-3 Mediated Inflammation and Angiogenesis

A recent meta-analysis of genome-wide association studies (GWAS) of coronary artery disease (CAD) involving over 86,000 individuals identified PPAP2B (gene that encodes lipid phosphate phosphatase-3; LPP3) as 1 of 13 new loci that associate with CAD. The PPAP2B loci independently predicted CAD (P = 3.81 X 10-19) and lacked association with traditional risk factors. The lipid phosphate phosphatase 3 is a cell surface integral membrane protein that regulates the bio availability of major bioactive lysophospholipids, like lysophosphatidic acid (LPA). Our group also made the surprising discovery that tissue-specific deficiency of LPP3 exaggerates vascular inflammation and promotes the development of neointima after vascular injury. Here we present evidence that enothelial cell LPP3 serves as an intrinsic negative regulator of vascular inflammation, promotes endothelial barrier function. Our results suggest that loss of LPP3 by siRNA mediated knockdown enhanced proliferation with elevated pErk signaling whereas migration was inhibited in human microvascular endothelial cells (HMEC). The endothelial cells that were silenced for LPP3 were further subjected LPA receptor knockdown and we found that although LPA-induced Erk phosphorylation was similar between LPA 1 and LPA 4 silencing, LPA 4 silencing resulted in enhancement of Akt phosphorylation at Thr-308 in response to LPA. Dual inhibition of LPA 1 and LPA 4 abolished LPA mediated migration in LPP3 silenced HMEC suggesting that the signaling response is mediated through the LPA 1 receptor. Rac activation in response to LPA was barely detectable in LPP3 silenced HMEC, which correlated with its weak migratory response. In contrast, a prominent increase in Rac-GTP levels was observed upon LPA 4 silencing under these conditions. Activation of HMEC with LPA increased phosphorylation of MSK downstream of p38, and CREB phosphorylation was inhibited upon LPA 4 receptor blockade in LPP3 silenced endothelial cells.

HIV Tat 101-mediated loss of pericytes at the blood-brain barrier involves PDGF-BB

HIV-1-associated neurocognitive disorders (HAND) affect almost 30-50% of infected individuals, even in the presence of successful control of virus replication by combined antiretroviral therapy (cART).HIV Tat protein, a nuclear trans-activator of viral gene transcription, that is secreted by infected cells and can be taken up by the neighboring cells, is present in various tissues despite the presence of cART, and has been shown to break down the integrity of the blood-brain barrier (BBB). This, in turn, leads to disruption of the neovascular unit, affecting functioning of the brain microvascular endothelial cells as well as astrocytes. Pericytes, yet another important constituent of the BBB, play a critical role in the maintenance of the integrity of the BBB. Loss of pericytes resulting in disruption of BBB has been observed in several pathologies including HAND. Furthermore, while PDGF-BB is essential for pericyte generation, paradoxically, high concentrations of PDGF-BB lead to loss of pericytes in tumor vessels. In this research highlight, we provide a brief review of our recently published finding, which have demonstrated a novel role of PDGF-BB in HIV-Tat mediated migration of pericytes, leading ultimately to loss of pericyte coverage from the endothelium, with a subsequent breach of the BBB. These findings underpin yet another mechanism by which BBB integrity is disrupted in HAND.

The BTK Inhibitor Ibrutinib Blocks SDF1/CXCR4 Mediated Migration of Acute Myeloid Leukemia Cells

IntroductionDespite recent significant progress in the understanding of the biology of acute myeloid leukemia (AML) the clinical outcomes for the majority of patients diagnosed with AML presently remain poor. Consequently, there is an urgent need to identify pharmacological strategies in AML, which are not only effective but can be tolerated by the older, less well patient. Recently our group and others have shown that there is high Bruton’s Tyrosine Kinase (BTK) phosphorylation and RNA expression in AML. Moreover, our recent study described for the first time that ibrutinib and BTK-targeted RNA interference reduced factor-induced proliferation of both AML cell lines and primary AML blasts, as well as reducing AML blast adhesion to bone marrow stromal cells. Inhibition of BTK has been shown to regulate chronic lymphocytic leukemia, mantle cell lymphoma and multiple myeloma cell migration by inhibiting SDF1 (stromal derived factor 1) induced CXCR4 regulated cell trafficking. Here we report that in human AML ibrutinib in addition functions in a similar way to inhibit SDF1/CXCR4-mediated AML migration at concentrations achievable in vivo.MethodsTo investigate the role of BTK in regulating AML migration we used both pharmacological inhibitor ibrutinib and genetic knockdown using a lentivirus mediated BTK targeted miRNA in primary AML blasts and AML cell lines. We examined migration of AML blasts and AML cells to SDF-1 using Transwell permeable plates with 8.0µM pores. Western blotting was used to examine the role of SDF-1 in regulating BTK, AKT and MAPK activation in primary AML blasts.ResultsWe initially examined the expression of CXCR4 in human AML cell lines and found that 4/4 cell lines were positive for CXCR4 expression. Next we examined the effects of ibrutinib on the migration of the AML cell lines U937, MV4-11, HL60 and THP-1 in response to SDF1. We found that ibrutinib can inhibit the migration of all AML cell lines tested. We tested the in-vitro activity of ibrutinib on SDF-1 induced migration in a spectrum of primary AML blasts from a wide age spectrum of adult patients and across a range of WHO AML subclasses and found that ibrutinib significantly inhibits primary AML blast migration (n=12). Next we found that ibrutinib can inhibit SDF-1 induced BTK phosphorylation and downstream MAPK and AKT signalling in primary AML blast. Finally to eliminate the problems associated with off target ibrutinib activity we evaluated migration of AML cells lines using genetic inhibition of BTK. The introduction of BTK-specific miRNA dramatically inhibited the expression of BTK in THP-1 and HL60 and reduced SDF1 mediated migration confirming that BTK is involved in regulating AML migration in response to SDF1.ConclusionsThese results reported here provide a molecular mechanistic rationale for clinically evaluating BTK inhibition in AML patients and suggests that in some AML patients the blasts count may initially rise in response to ibrutinib therapy, analgous to similar clinical observations in CLL. DisclosuresNo relevant conflicts of interest to declare.

Anti-tumor effect of a novel PI3-kinase inhibitor, SF1126, in (12) V-Ha-Ras transgenic mouse glioma model.

BackgroundGrowth factor mediated activation of RAS-MAP-kinase and PI3-kinase-AKT pathways are critical for the pathogenesis of glioblastoma. The attenuation of PI3-kinase/AKT signaling will be effective in regulating the tumorigenic phenotypes of the glioma cells.MethodsGlioma cells derived from the brain of the 12 V-Ha-Ras transgenic mice were used to study the effect of PI-3 kinase inhibitor SF1126 on activation of AKT and ERK signaling, proliferation, vitronectin mediated migration and changes in the distribution of cortical actin on vitronectin in the glioma cells in vitro. The anti-tumor effects of SF1126 were also tested in vivo using pre-established tumors (subcutaneous injection of the glioma cells from 12 V-Ha-Ras transgenic mice) in a mouse xenograft model.ResultsOur results demonstrate that treatment of LacZ+, GFAP + and PCNA + 12 V-Ras Tg transformed astrocytes with SF1126 and LY294002 blocked the activation of AKT as well as EGF-induced phospho-ERK. Most notably, treatment of SF1126 blocked integrin-dependent migration in transwell and scratch assays and caused a significant change in the organization and distribution of cortical actin on vitronectin in the glioma cells. Moreover, SF1126 treatment inhibited in vitro proliferation of these cells and in vivo growth of pre-established subcutaneous tumors in a xenograft model.ConclusionThe present study validate the potent anti-proliferative and anti-migratory activity of SF1126, in a V12 Ras oncogene driven glioma model and suggest that this effect is mediated potentially through a combined attenuation of PI3-kinase and MAP-kinase signaling pathways.

Abstract 2075: Resistin promotes MMP-2 production and migration in human chondrosarcoma cells through AMPK/p38/mir 519d pathway

Abstract Chondrosarcoma is a primary malignant bone cancer, with a potent capacity to invade locally and cause distant metastasis; it has a poor prognosis and shows a predilection for metastasis to the lungs. Resistin the most potent pro-inflammatory and plays a crucial role in migration and metastasis of human cancer cells. However, the effects of resistin on human chondrosarcoma cells are largely unknown. In this study, we found that treatment of human chondrosarcoma cells (JJ012) with resistin increased migration and expression of matrix metalloproteinase (MMP)-2. Resistin-mediated cell migration and MMP-2 expression were reduced by pretreatment with inhibitors of AMP-activated protein kinase (AMPK), and p38 mitogen-activated protein kinases (p38). Resistin treatment induced phosphorylation of AMPK and p38. In addition, resistin reduced miRNA 519d expression. Transfect ion of 519d mimic also reduced resistin mediated migration and MMP2 expression. Furthermore, we analyzed samples from chondrosarcoma patients by immunohistochemical staining. The expression of resistin and MMP2 in chondrosarcoma patients was significantly higher than in normal cartilage. Taken together, these results suggest that resistin increased MMP-2 expression and migration in human chondrosarcoma cells. Citation Format: Ho-Ning Huang, Chih-Hish Tang, Yuan-Li Huang. Resistin promotes MMP-2 production and migration in human chondrosarcoma cells through AMPK/p38/mir 519d pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2075. doi:10.1158/1538-7445.AM2014-2075

Abstract 2478: Glycosylation of tid1 regulates galectin-7 ubiquitination and localization to suppress head and neck cancer metastasis

Abstract Background: Tid1, a DnaJ cochaperone protein mainly located in mitochondria, functions as a tumor suppressor in different types of solid tumors. Recently, we first demonstrate that Tid1 is negatively associated with cell migration, tumor status, and survival prognosis in head and neck squamous cell carcinoma (HNSCC) (J Pathol 2009; 219:347-55). As a cochaperone protein like Tid1, it should be instrumental to find the Tid1 client/interacting proteins, consequently, to understand the physiological function of those Tid1 client/interacting proteins as therapeutic targets for future HNSCC treatment. Methods: First, we applied affinity chromatography and systemic proteomics analysis to identify galectin-7 as one of client/interacting proteins of Tid1. Subsequently, using the metastatic and/or non-metastatic cells determined tumorigenic properties of HNSCC with overexpression and/or knockdown of galectin-7 and/or Tid1. The nuclear and cytosolic subfractions were isolated to determine the role of Tid1 of galectin-7 nuclearization. We also attempted to correlate the expression of Tid1 and galectin-7 with tumor stage of HNSCC patient tissues by immunohistochemical assay. Finally, site-directed mutagenesis was applied to identify the novel N-link glycosylated sites of Tid1, by which to regulate the interaction and stability of galectin-7. Results: We found metastatic cells expressed lower level of Tid1 and higher level of galectin-7 than non-metastatic cells. For in vitro tumorigenic analyses, we observed that overexpression of galectin-7 resulted in elevated migration, invasion and soft agar colony formation abilities in HNSCC. Inversely, co-overexpression of Tid1 reduced the expression of galectin-7, impaired the galectin-7 mediated migration, invasion and soft agar colony formation ability of HNSCC, and retarded nuclearization of galectin-7. Moreover, knockdown of galectin-7 reduced migration, invasion and soft agar colony formation ability of HNSCC metastatic cells. Further, we found that galectin-7 ubiquitination as revealed by antiubiquitin immunoblot analysis in immunoprecipitates. Finally, the negative relationship was between Tid1 and Galectin-7 expression in HNSCC clinical specimen. We found out that Tid1 interacts with galectin-7 through N-linked glycosylation. Tid1 mutants of N-link glycosylated sites restored the level and the tumorigenic function of galectin-7. Conclusion: Our studies first pointed out that galectin-7 plays pivotal roles in HNSCC tumorigenesis. Notably, we identified galectin-7, which is the client/interacting protein of Tid1. We hypothesized that Tid1/ Hsp70 may regulate galectin-7 stability by ubiquitinylation. The molecular mechanism by which Tid1 negatively modulates the galectin-7 warrants further investigation. Citation Format: Yu-Syuan Chen. Glycosylation of tid1 regulates galectin-7 ubiquitination and localization to suppress head and neck cancer metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2478. doi:10.1158/1538-7445.AM2014-2478

Ibrutinib inhibits SDF1/CXCR4 mediated migration in AML.

Pharmacological targeting of BTK using ibrutinib has recently shown encouraging clinical activity in a range of lymphoid malignancies. Recently we reported that ibrutinib inhibits human acute myeloid leukemia (AML) blast proliferation and leukemic cell adhesion to the surrounding bone marrow stroma cells. Here we report that in human AML ibrutinib, in addition, functions to inhibit SDF1/CXCR4-mediated AML migration at concentrations achievable in vivo. It has previously been shown that SDF1/CXCR4-induced migration is dependent on activation of downstream BTK in chronic lymphocytic leukaemia (CLL) and multiple myeloma. Here we show that SDF-1 induces BTK phosphorylation and downstream MAPK signalling in primary AML blast. Furthermore, we show that ibrutinib can inhibit SDF1-induced AKT and MAPK activation. These results reported here provide a molecular mechanistic rationale for clinically evaluating BTK inhibition in AML patients and suggests that in some AML patients the blasts count may initially rise in response to ibrutinib therapy, analgous to similar clinical observations in CLL.

Digitally Enhanced? Mediated Migration and ‘Fourth Wave’ Chileans in Australia

This paper examines the contemporary experience of migration for Chileans arriving in Australia in the last two decades. It specifically explores the increasing role of Information Communication Technologies (ICTs) and its mediating effects on experiences of departure, arrival, ‘being’ and ‘belonging’. In doing so, the paper considers some of the tensions between ‘hyper-digital’ transnationalism and dominant discourses of nationalism. The findings indicate diversity of experience, but highlight that these recent Chilean migrants utilise ICT to suspend and manage physical relocation and to engage both on and offline with wide and shallow networks of Chileans and non-Chileans in local and transnational spaces. At the same time, they reflect a more cultural, issues-based orientation towards the structural and discursive dimensions of migration in both ‘home’ and ‘host’ countries suggesting that ICT is imbricated in wider processes of distancing the ‘migrant’ from the national core. This highlights some of the possibilities and limitations of ‘hyper-digital’ transnationalism.

Low intensity pulsed ultrasound enhanced mesenchymal stem cell recruitment through stromal derived factor-1 signaling in fracture healing.

Low intensity pulsed ultrasound (LIPUS) has been proven effective in promoting fracture healing but the underlying mechanisms are not fully depicted. We examined the effect of LIPUS on the recruitment of mesenchymal stem cells (MSCs) and the pivotal role of stromal cell-derived factor-1/C-X-C chemokine receptor type 4 (SDF-1/CXCR4) pathway in response to LIPUS stimulation, which are essential factors in bone fracture healing. For in vitro study, isolated rat MSCs were divided into control or LIPUS group. LIPUS treatment was given 20 minutes/day at 37°C for 3 days. Control group received sham LIPUS treatment. After treatment, intracellular CXCR4 mRNA, SDF-1 mRNA and secreted SDF-1 protein levels were quantified, and MSCs migration was evaluated with or without blocking SDF-1/CXCR4 pathway by AMD3100. For in vivo study, fractured 8-week-old young rats received intracardiac administration of MSCs were assigned to LIPUS treatment, LIPUS+AMD3100 treatment or vehicle control group. The migration of transplanted MSC to the fracture site was investigated by ex vivo fluorescent imaging. SDF-1 protein levels at fracture site and in serum were examined. Fracture healing parameters, including callus morphology, micro-architecture of the callus and biomechanical properties of the healing bone were investigated. The in vitro results showed that LIPUS upregulated SDF-1 and CXCR4 expressions in MSCs, and elevated SDF-1 protein level in the conditioned medium. MSCs migration was promoted by LIPUS and partially inhibited by AMD3100. In vivo study demonstrated that LIPUS promoted MSCs migration to the fracture site, which was associated with an increase of local and serum SDF-1 level, the changes in callus formation, and the improvement of callus microarchitecture and mechanical properties; whereas the blockade of SDF-1/CXCR4 signaling attenuated the LIPUS effects on the fractured bones. These results suggested SDF-1 mediated MSCs migration might be one of the crucial mechanisms through which LIPUS exerted influence on fracture healing.

Mesenchymal stem cells from osteoporotic patients reveal reduced migration and invasion upon stimulation with BMP-2 or BMP-7

Fractures to the osteoporotic bone feature a delay in callus formation and reduced enchondral ossification. Human mesenchymal stem cells (hMSC), the cellular source of fracture healing, are recruited to the fracture site by cytokines, such as BMP-2 and BMP-7. Aim of the study was to scrutinize hMSC for osteoporosis associated alterations in BMP mediated migration and invasion as well as in extracellular matrix (ECM) binding integrin expression.HMSC were isolated from 18 healthy or osteoporotic donors. Migration was assessed using a collagen IV coated micro-slide linear gradient chamber and time-lapse microscopy. Invasion was analyzed utilizing an ECM coated transmembrane invasion assay. Quantitative real-time RT PCR was performed for the ECM binding integrins α1, α2, α3, α4, α5, α11, αv and β1.HMSC from osteoporotic patients showed a significant increase of migration upon BMP-2 or FCS stimulation, as well as a significant increase of invasion upon BMP-2, BMP-7 or FCS stimulation. Nevertheless, the migration and invasion capacity was significantly decreased compared to healthy controls. Out of all integrins analyzed, collagen binding integrin α2 was significantly downregulated in hMSC from osteoporotic patients.In conclusion, we here demonstrate for the first time osteoporosis associated alterations in BMP mediated hMSC recruitment. These findings may underlie the reduced healing of osteoporotic fractures. Nevertheless, the maintained migration and invasion response upon BMP stimulation illustrates the therapeutic potential of these clinically approved substances in the treatment of osteoporotic fractures. Another therapeutic target may be the downregulation of the collagen binding integrin α2 in hMSC from osteoporotic patients.

77 Toll-Like Receptor (TLR) 2 Agonists Reduced NSAID-Induced Small Intestinal Damages Possibly Through P-Selectin Mediated Leukocytes Migration in the Inflamed Mucosa Induced by Activated Macrophages

77 Toll-Like Receptor (TLR) 2 Agonists Reduced NSAID-Induced Small Intestinal Damages Possibly Through P-Selectin Mediated Leukocytes Migration in the Inflamed Mucosa Induced by Activated Macrophages

TGF-β1–ROS–ATM–CREB signaling axis in macrophage mediated migration of human breast cancer MCF7 cells

Macrophages in the tumor microenvironment play an important role in tumor cell survival. They influence the tumor cell to proliferate, invade into surrounding normal tissues and metastasize to local and distant sites. In this study, we evaluated the effect of conditioned medium from monocytes and macrophages on growth and migration of breast cancer cells. Macrophage conditioned medium (MϕCM) containing elevated levels of cytokines TNF-α, IL-1β and IL-6 had a differential effect on non-invasive (MCF7) and highly invasive (MDA-MB-231) breast cancer cell lines. MϕCM induced the secretion of TGF-β1 in MCF7 cells. This was associated with apoptosis in a fraction of cells and generation of reactive oxygen and nitrogen species (ROS and RNS) and DNA damage in the remaining cells. This, in turn, increased expression of cAMP response element binding protein (CREB) and vimentin resulting in migration of cells. These effects were inhibited by neutralization of TNF-α, IL-1β and IL-6, inhibition of ROS and RNS, DNA damage and siRNA mediated knockdown of ATM. In contrast, MDA-MB-231 cells which had higher basal levels of pCREB were not affected by MϕCM. In summary, we have found that pro-inflammatory cytokines secreted by macrophages induce TGF-β1 in tumor cells, which activate pCREB signaling, epithelial–mesenchymal-transition (EMT) responses and enhanced migration.

Dictyostelium cells migrate similarly on surfaces of varying chemical composition.

During cell migration, cell-substrate binding is required for pseudopod anchoring to move the cell forward, yet the interactions with the substrate must be sufficiently weak to allow parts of the cell to de-adhere in a controlled manner during typical protrusion/retraction cycles. Mammalian cells actively control cell-substrate binding and respond to extracellular conditions with localized integrin-containing focal adhesions mediating mechanotransduction. We asked whether mechanotransduction also occurs during non-integrin mediated migration by examining the motion of the social amoeba Dictyostelium discoideum, which is thought to bind non-specifically to surfaces. We discovered that Dictyostelium cells are able to regulate forces generated by the actomyosin cortex to maintain optimal cell-surface contact area and adhesion on surfaces of various chemical composition and that individual cells migrate with similar speed and contact area on the different surfaces. In contrast, during collective migration, as observed in wound healing and metastasis, the balance between surface forces and protrusive forces is altered. We found that Dictyostelium collective migration dynamics are strongly affected when cells are plated on different surfaces. These results suggest that the presence of cell-cell contacts, which appear as Dictyostelium cells enter development, alter the mechanism cells use to migrate on surfaces of varying composition.

Role of class IA PI 3-kinase isoforms in neointima formation following balloon angioplasty: only p110alpha is important

Growth factor induced proliferation and migration of vascular smooth muscle cells (SMCs) are essential for neointima formation following balloon angioplasty. Growth factors such as platelet-derived growth factor (PDGF) activate receptor tyrosine kinases (RTKs). RTK-induced responses are largely mediated by activation of class IA phosphatidylinositol 3'-kinase (PI3K) isoforms. Class IA PI3Ks comprise one of three different catalytic isoforms (p110alpha, p110beta, p110delta). Here, we sought to clarify, which p110 subunits contribute to neointima formation following balloon injury. Western blot analysis of lysates from cultured human, mouse and rat SMCs revealed that p110alpha, p110beta, and p110delta are expressed in these cells. However, co-immunoprecipitation experiments using an antibody against the PDGF beta receptor indicate that only p110alpha and p110delta bind to activated RTKs. p110beta signals downstream of G-protein coupled receptors (GPCR) as p110beta inhibition by TGX-221 (1μM) completely abrogated LPA (GPCR agonist) induced AKT activation downstream of PI3K. TGX-221 had no influence on RTK induced AKT activation. Inhibition (PIK75, 500 nM) or siRNA mediated knockdown of p110alpha completely abrogated PDGF mediated proliferation (BrdU incorporation assay) and migration (modified Boyden-chamber) of SMCs. p110beta (TGX-221, 3μM) and p110delta (IC87114, 3μM) inhibition had no influence. However, knockdown of p110delta using a specific siRNA significantly reduced PDGF induced SMC proliferation without effecting SMC migration. As expected, siRNA against p110beta was noneffective. By means of SMC-specific p110alpha deficient mice and p110delta knockout mice, we analysed in vivo the relevance of p110alpha and p110delta for restenosis formation following balloon angioplasty. The extent of neointima formation was quantified 4 weeks following balloon angioplasty of carotid arteries from wild-type (WT) and p110alpha ko as well as from WT and p110delta ko mice (n=7, respectively). Whereas p110alpha deficiency largely suppressed neointima formation (p<0.05), p110delta deficiency was noneffective. In conclusion, our results indicate that the p110alpha subunit of PI3K, is crucial for growth factor-mediated proliferation and migration of SMCs in restonosis following balloon angioplasty. Therefore, p110alpha represents a promising therapeutic target.

C-C motif chemokine receptor 9 positive macrophages activate hepatic stellate cells and promote liver fibrosis in mice

Chemokine receptors mediate migration of immune cells into the liver, thereby promoting liver inflammation. C-C motif chemokine receptor (CCR) 9(+) macrophages are crucial in the pathogenesis of acute liver inflammation, but the role and underlying mechanisms of this macrophage subset in chronic liver injury and subsequent liver fibrosis are not fully understood. We confirmed that tumor necrosis factor alpha (TNF-α)-producing CCR9(+) macrophages accumulated during the initiation of carbon tetrachloride (CCl4 )-induced liver injury, and CCR9 deficiency attenuated the degree of liver damage. Accumulation of CCR9(+) macrophages persisted prominently during the process of liver fibrosis induced by repetitive CCl4 or thioacetamide (TAA)/leptin administration. Increased CCR9 expression was also found on activated hepatic stellate cells (HSCs). Importantly, experimental liver fibrosis was significantly ameliorated in CCR9(-/-) mice compared with wild-type (WT) mice, assessed by α-smooth muscle actin (α-SMA) immunostain, Sirius red staining, and messenger RNA (mRNA) expression levels of α-SMA, collagen 1α1, transforming growth factor (TGF)-β1, and tissue inhibitor of metalloproteinase (TIMP)-1. Accumulated CD11b(+) macrophages in CCl4 -treated WT mice showed marked increases in TNF, NO synthase-2, and TGF-β1 mRNA expression compared with CCR9(-/-) mice, implying proinflammatory and profibrogenic properties. Hepatic CD11b(+) macrophages from CCl4 -treated WT mice (i.e., CCR9(+) macrophages), but not CD8(+) T lymphocytes or non-CD11b(+) cells, significantly activated HSCs in vitro compared with those from CCR9(-/-) mice. TNF-α or TGF-β1 antagonism attenuated CCR9(+) macrophage-induced HSC activation. Furthermore, C-C motif chemokine ligand (CCL) 25 mediated migration and, to a lesser extent, activation of HSCs in vitro. Accumulated CD11b(+) macrophages are critical for activating HSCs through the CCR9/CCL25 axis and therefore promote liver fibrosis. CCR9 antagonism might be a novel therapeutic target for liver fibrosis.