Thymic medullar conduits-associated podoplanin promotes natural regulatory T cells.

Podoplanin, a mucin-like plasma membrane protein, is expressed by lymphatic endothelial cells and responsible for separation of blood and lymphatic circulation through activation of platelets. Here we show that podoplanin is also expressed by thymic fibroblastic reticular cells (tFRC), a novel thymic medulla stroma cell type associated with thymic conduits, and involved in development of natural regulatory T cells (nTreg). Young mice deficient in podoplanin lack nTreg owing to retardation of CD4(+)CD25(+) thymocytes in the cortex and missing differentiation of Foxp3(+) thymocytes in the medulla. This might be due to CCL21 that delocalizes upon deletion of the CCL21-binding podoplanin from medullar tFRC to cortex areas. The animals do not remain devoid of nTreg but generate them delayed within the first month resulting in Th2-biased hypergammaglobulinemia but not in the death-causing autoimmune phenotype of Foxp3-deficient Scurfy mice.

The urokinase receptor (CD87) represents a central mediator of growth factor-induced endothelial cell migration.

Angiogenesis, the sprouting of blood vessels form pre-existing vasculature after injury or in neoplastic diseases, is initiated by growth factor-induced endothelial cell migration. Recently, the major angiogenic growth factor VEGF165 has become the target of therapeutic interventions. However, this approach has been clinically proven to be of limited efficacy, which might be due to the fact that tumour angiogenesis is not only induced by VEGF, but also by a variety of other growth factors. Thus, the identification of a common downstream mediator of growth-factor-induced endothelial cell migration is mandatory to effectively interfere with (tumour-) angiogenesis. We found that the urokinase-type plasminogen activator (uPA)-system, which affects proteolytic as well as adhesive capacities, represents an essential regulatory mechanism in growth factor-induced endothelial cell migration and invasion. This mechanism was not limited to VEGF165, but mediated pro-angiogenic endothelial cell behaviour induced by various growth factors. Thus, VEGF165, VEGF-E, FGF-2, EGF as well as HGF induced a PI3k-dependent activation of pro-uPA when bound to uPAR, which led to an increase in cell surface fibrinolytic activity. As a consequence, uPAR became internalised and redistributed via LDLR-proteins. Interference with these events led to a reduced migratory response of endothelial cells towards VEGF in vitro as well as endothelial cell invasion in vivo. These data give first evidence that the uPA-system, which represents the only level-of-evidence-1 cancer biomarker system for prognosis and/or prediction in node negative breast cancer, might directly affect (tumour-) angiogenesis.

The interferon stimulated gene 12 inactivates vasculoprotective functions of NR4A nuclear receptors.

RATIONALE: Innate and adaptive immune responses alter numerous homeostatic processes that are controlled by nuclear hormone receptors. NR4A1 is a nuclear receptor that is induced in vascular pathologies, where it mediates protection. OBJECTIVE: The underlying mechanisms that regulate the activity of NR4A1 during vascular injury are not clear. We therefore searched for modulators of NR4A1 function that are present during vascular inflammation. METHODS AND RESULTS: We report that the protein encoded by interferon stimulated gene 12 (ISG12), is a novel interaction partner of NR4A1 that inhibits the transcriptional activities of NR4A1 by mediating its Crm1-dependent nuclear export. Using 2 models of vascular injury, we show that ISG12-deficient mice are protected from neointima formation. This effect is dependent on the presence of NR4A1, as mice deficient for both ISG12 and NR4A1 exhibit neointima formation similar to wild-type mice. CONCLUSIONS: These findings identify a previously unrecognized feedback loop activated by interferons that inhibits the vasculoprotective functions of NR4A nuclear receptors, providing a potential new therapeutic target for interferon-driven pathologies.

VEGF-induced endothelial cell migration requires urokinase receptor (uPAR)-dependent integrin redistribution.

AIMS: Vascular endothelial growth factor (VEGF)-initiated angiogenesis requires coordinated proteolytic degradation of extracellular matrix provided by the urokinase plasminogen activator/urokinase receptor (uPA/uPAR) system and regulation of cell migration provided by integrin-matrix interaction. In this study, we investigated the mechanisms underlying the uPAR-dependent modulation of VEGF-induced endothelial migration. METHODS AND RESULTS: We used flow cytometry to quantify integrins at the cell surface. Stimulation of human and murine endothelial cells with VEGF resulted in internalization of α5ß1-integrins. Micropatterning and immunocytochemistry revealed co-clustering of uPAR and α5ß1-integrins and retrieval via clathrin-coated vesicles. It was also contingent on receptors of the low-density lipoprotein receptor (LDL-R) family. VEGF-induced integrin redistribution was inhibited by elimination of uPAR from the endothelial cell surface or by inhibitory peptides that block the uPAR-integrin interaction. Under these conditions, the migratory response of endothelial cells upon VEGF stimulation was impaired both in vitro and in vivo. CONCLUSIONS: The observations indicate that uPAR is an essential component of the network through which VEGF controls endothelial cell migration. uPAR is a bottleneck through which the VEGF-induced signal must be funnelled for both focused proteolytic activity at the leading edge and for redistribution of integrins.

Constant darkness induces IL-6-dependent depression-like behavior through the NF-κB signaling pathway.

Substantial experimental evidence indicates a major role for the circadian system in mood disorders. Additionally, proinflammatory cytokines have been proposed to be involved in the pathogenesis of depression. However, the molecular elements determining the functional interplay between these two systems in depression have not been described as yet. Here we investigate whether long-term light deprivation in the constant darkness (DD) paradigm affects depression-like behavior in mice and concomitantly modulates the levels of proinflammatory cytokines. We find that after 4 weeks of DD, mice display depression-like behavior, which is paralleled by reduced hippocampal cell proliferation. This chronobiologically induced depressive state is associated with elevated levels of plasma IL-6 (interleukin-6) and IL-6 and Il1-R1 (interleukin 1 receptor, type I) protein levels in the hippocampus and also alters hippocampal protein levels of the clock genes per2 and npas2. Using pharmacological blockers of the NF-κB pathway, we provide evidence that the effects of DD on depression-like behavior, on hippocampal cell proliferation, on altered expressional levels of brain and plasma IL-6, and on the modulation of clock gene expression are mediated through NF-κB signaling. Moreover, NF-κB activity is enhanced in hippocampal tissue of DD mice. Mice with a deletion of IL-6, one of the target genes of NF-κB, are resistant to DD-induced depression-like behavior, which suggests a pivotal role for this cytokine in the constant darkness mouse model of depression. We here first describe some of the molecular elements bridging chronobiological and inflammatory processes in the constant darkness mouse model of depression.

Signal integration and coincidence detection in the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) cascade: concomitant activation of receptor tyrosine kinases and of LRP-1 leads to sustained ERK phosphorylation via down-regulation of dual specificity phosphatases (DUSP1 and -6).

Diverse stimuli can feed into the MAPK/ERK cascade; this includes receptor tyrosine kinases, G protein-coupled receptors, integrins, and scavenger receptors (LDL receptor-related protein (LRP)). Here, we investigated the consequence of concomitant occupancy of the receptor tyrosine kinases (by EGF, basic FGF, VEGF, etc.) and of LRP family members (by LDL or lactoferrin). The simultaneous stimulation of a receptor tyrosine kinase by its cognate ligand and of LRP-1 (by lactoferrin or LDL) resulted in sustained activation of ERK, which was redirected to the cytoplasm. Accordingly, elevated levels of active cytosolic ERK were translated into accelerated adhesion to vitronectin. The sustained ERK response was seen in several cell types, but it was absent in cells deficient in LRP-1 (but not in cells lacking the LDL receptor). This response was also contingent on the presence of urokinase (uPA) and its receptor (uPAR), because it was absent in uPA(-/-) and uPAR(-/-) fibroblasts. Combined stimulation of the EGF receptor and of LRP-1 delayed nuclear accumulation of phosphorylated ERK. This shift in favor of cytosolic accumulation of phospho-ERK was accounted for by enhanced proteasomal degradation of dual specificity phosphatases DUSP1 and DUSP6, which precluded dephosphorylation of cytosolic ERK. These observations demonstrate that the ERK cascade can act as a coincidence detector to decode the simultaneous engagement of a receptor tyrosine kinase and of LRP-1 and as a signal integrator that encodes this information in a spatially and temporally distinct biological signal. In addition, the findings provide an explanation of why chronic elevation of LRP-1 ligands (e.g. PAI-1) can predispose to cancer.

Density enhanced phosphatase-1 down-regulates urokinase receptor surface expression in confluent endothelial cells.

VEGF(165), the major angiogenic growth factor, is known to activate various steps in proangiogenic endothelial cell behavior, such as endothelial cell migration and invasion, or endothelial cell survival. Thereby, the urokinase-type plasminogen activator (uPA) system has been shown to play an essential role not only by its proteolytic capacities, but also by induction of intracellular signal transduction. Therefore, expression of its cell surface receptor uPAR is thought to be an essential regulatory mechanism in angiogenesis. We found that uPAR expression on the surface of confluent endothelial cells was down-regulated compared with subconfluent proliferating endothelial cells. Regulation of uPAR expression was most probably affected by extracellular signal-regulated kinase 1/2 (ERK1/2) activation, a downstream signaling event of the VEGF/VEGF-receptor system. Consistently, the receptor-like protein tyrosine phosphatase DEP-1 (density enhanced phosphatase-1/CD148), which is abundantly expressed in confluent endothelial cells, inhibited the VEGF-dependent activation of ERK1/2, leading to down-regulation of uPAR expression. Overexpression of active ERK1 rescued the DEP-1 effect on uPAR. That DEP-1 plays a biologic role in angiogenic endothelial cell behavior was demonstrated in endothelial cell migration, proliferation, and capillary-like tube formation assays in vitro.

Soluble M6P/IGF2R released by TACE controls angiogenesis via blocking plasminogen activation.

RATIONALE: The urokinase plasminogen activator (uPA) system is among the most crucial pericellular proteolytic systems associated with the processes of angiogenesis. We previously identified an important regulator of the uPA system in the mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R). OBJECTIVE: Here, we wanted to clarify whether and how did the soluble form of M6P/IGF2R (sM6P/IGF2R) contribute to modulation of the uPA system. METHODS AND RESULTS: By using specific inhibitors and RNA interference, we show that the tumor necrosis factor α convertase (TACE, ADAM-17) mediates the release of the ectodomain of M6P/IGF2R from human endothelial cells. We demonstrate further that sM6P/IGF2R binds plasminogen (Plg) and thereby prevents Plg from binding to the cell surface and uPA, ultimately inhibiting in this manner Plg activation. Furthermore, peptide 18-36 derived from the Plg-binding site of M6P/IGF2R mimics sM6P/IGF2R in the inhibition of Plg activation and blocks cancer cell invasion in vitro, endothelial cell invasion in vivo, and tumor growth in vivo. CONCLUSIONS: The interaction of sM6P/IGF2R with Plg may be an important regulatory mechanism to inhibit migration of cells using the uPA/uPAR system.

Involvement of CK2 in activation of electrophilic genes in endothelial cells by oxidized phospholipids.

Oxidized phospholipids (OxPLs) are increasingly recognized as pleiotropic lipid mediators demonstrating a variety of biological activities. In particular, OxPLs induce electrophilic stress response and stimulate expression of NF-E2-related factor 2 (NRF2)-dependent genes. The mechanisms of NRF2 upregulation in response to OxPLs, however, are incompletely understood. Here we show that upregulation of NRF2 by OxPLs depends on the activity of the CK2 protein kinase. Inactivation of CK2 by chemical inhibitors or gene silencing resulted in diminished accumulation of NRF2 and its target genes, GCLM, HMOX1, and NQO1, downstream in response to OxPLs. Furthermore, inhibition of CK2 suppressed NRF2-dependent induction of ATF4 and its downstream gene VEGF. Thus, inactivation of CK2 in OxPL-treated endothelial cells results in inhibition of the NRF2-ATF4-VEGF axis and is likely to produce antiangiogenic effects. This work characterizes novel cross-talk between CK2 and cellular stress pathways, which may provide additional insights into the mechanisms of beneficial action and side-effects of CK2 inhibitors.

Oxidized phospholipids are more potent antagonists of lipopolysaccharide than inducers of inflammation.

Polyunsaturated fatty acids are precursors of multiple pro- and anti-inflammatory molecules generated by enzymatic stereospecific and positionally specific insertion of oxygen, which is a prerequisite for recognition of these mediators by cellular receptors. However, nonenzymatically oxidized free and esterified polyunsaturated fatty acids also demonstrate activities relevant to inflammation. In particular, phospholipids containing oxidized fatty acid residues (oxidized phospholipids; OxPLs) were shown to induce proinflammatory changes in endothelial cells but paradoxically also to inhibit inflammation induced via TLR4. In this study, we show that half-maximal inhibition of LPS-induced elevation of E-selectin mRNA in endothelial cells developed at concentrations of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) 10-fold lower than those required to induce proinflammatory response. Similar concentration difference was observed for other classes and molecular species of OxPLs. Upon injection into mice, OxPAPC did not elevate plasma levels of IL-6 and keratinocyte chemoattractant but strongly inhibited LPS-induced upregulation of these inflammatory cytokines. Thus, both in vitro and in vivo, anti-LPS effects of OxPLs are observed at lower concentrations than those required for their proinflammatory action. Quantification of the most abundant oxidized phosphatidylcholines by HPLC/tandem mass spectrometry showed that circulating concentrations of total oxidized phosphatidylcholine species are close to the range where they demonstrate anti-LPS activity but significantly lower than that required for induction of inflammation. We hypothesize that low levels of OxPLs in circulation serve mostly anti-LPS function and protect from excessive systemic response to TLR4 ligands, whereas proinflammatory effects of OxPLs are more likely to develop locally at sites of tissue deposition of OxPLs (e.g., in atherosclerotic vessels).

Anti-inflammatory properties of the PI3K pathway are mediated by IL-10/DUSP regulation.

Resolution of inflammation is an important hallmark in the course of infectious diseases. Dysregulated inflammatory responses may have detrimental consequences for the affected organism. Therefore, tight regulation of inflammation is indispensable. Among numerous modulatory signaling pathways, the PI3K/PTEN signaling pathway has been proposed recently to be involved in the regulation of innate immune reactions. Here, we attempted to elucidate molecular mechanisms that contribute to the modulatory properties of the PI3K signaling pathway in inflammation. PTEN-deficient macrophages, which harbor constitutively active PI3Ks, were analyzed in response to gram-negative bacteria and PAMPs such as LPS. PTEN-deficient cells showed reduced inflammatory cytokine production, which was accompanied by reduced MAPK signaling activation in early- as well as late-phase activation. Simultaneously, we found increased levels of the MKP DUSP1, as well as the anti-inflammatory cytokine IL-10. Our data suggest that differential DUSP1 regulation coupled with enhanced IL-10 production contributes to the anti-inflammatory properties of the PI3K pathway.

Indirubin-3'-monoxime blocks vascular smooth muscle cell proliferation by inhibition of signal transducer and activator of transcription 3 signaling and reduces neointima formation in vivo.

OBJECTIVE: Our goal was to examine the influence of indirubin-3'-monoxime (I3MO), a natural product-derived cyclin-dependent kinase inhibitor, on vascular smooth muscle cell (VSMC) proliferation in vitro, experimentally induced neointima formation in vivo, and related cell signaling pathways. METHODS AND RESULTS: I3MO dose-dependently inhibited platelet-derived growth factor (PDGF)-BB-induced VSMC proliferation by arresting cells in the G(0)/G(1) phase of the cell cycle as assessed by 5-bromo-2'-deoxyuridine incorporation and flow cytometry. PDGF-induced activation of the kinases Akt, Erk1/2, and p38(MAPK) was not affected. In contrast, I3MO specifically blocked PDGF-, interferon-γ-, and thrombin-induced phosphorylation of signal transducer and activator of transcription 3 (STAT3). Human endothelial cells (EA.hy926) responded to I3MO with increased endothelial nitric oxide synthase activity as assessed via [(14)C]l-arginine/[(14)C]l-citrulline conversion. The specific STAT3 inhibitor Stattic led to decreased VSMC proliferation, and transient expression of a constitutively active form of STAT3 overcame the I3MO-induced cell cycle arrest in mouse embryonic fibroblasts. In a murine femoral artery cuff model, I3MO prevented neointima formation while reducing STAT3 phosphorylation and the amount of proliferating Ki67-positive cells. CONCLUSIONS: I3MO represses PDGF- and thrombin-induced VSMC proliferation and, in vivo, neointima formation, likely because it specifically blocks STAT3 signaling. This profile and its positive effect on endothelial NO production turns I3MO into a promising lead compound to prevent restenosis.

Analysis of oxidized phospholipids by MALDI mass spectrometry using 6-aza-2-thiothymine together with matrix additives and disposable target surfaces.

6-Aza-2-thiothymine (ATT) is introduced as novel matrix system for the analysis of oxidized phospholipids (OxPLs) by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). A systematic evaluation comparing different established and novel matrix substances, especially 2,4,6-THAP matrix (Stubiger, G.; Belgacem O. Anal. Chem. 2007, 79, 3206-3213) as reference compound for phospholipid analysis, and specific matrix additives was performed. Thereby, ATT turned out to be the reagent of choice for MALDI analysis of major biologically relevant OxPL classes (e.g., OxPC, OxPE, and OxPS) in positive and negative ionization mode. ATT used together with specific chaotropic reagents at low concentration (0.5-2 mM) acting as OxPL ionization enhancers revealed an excellent comatrix system for application with MALDI instrument types employing UV- and Nd:YAG laser systems (337 and 355 nm). Moreover, disposable MALDI targets surfaces with specific physicochemical properties (e.g., metallized glass or polymeric substrates) were revealed as superior over stainless steel in terms of reduced chemical background noise ( approximately 10-fold better S/N ratios), increased mass spectral reproducibility, and enhanced sensitivity (LOD approximately 250-500 fg on target). The combination of these parameters offers a significant advantage for highly sensitive OxPL profiling by MALDI-MS of biological samples (e.g., human plasma) at trace levels.

Myeloid PTEN promotes inflammation but impairs bactericidal activities during murine pneumococcal pneumonia.

Phosphatidylinositol 3-kinase has been described as an essential signaling component involved in the chemotactic cell influx that is required to eliminate pathogens. At the same time, PI3K was reported to modulate the immune response, thus limiting the magnitude of acute inflammation. The precise role of the PI3K pathway and its endogenous antagonist phosphatase and tensin homolog deleted on chromosome 10 (PTEN) during clinically relevant bacterial infections is still poorly understood. Utilizing mice lacking myeloid cell-specific PTEN, we studied the impact of PTEN on the immune response to Streptococcus pneumoniae. Survival analysis disclosed that PTEN-deficient mice displayed less severe signs of disease and prolonged survival. The inflammatory response to S. pneumoniae was greatly reduced in macrophages in vitro and in vivo. Unexpectedly, neutrophil influx to the lungs was significantly impaired in animals lacking myeloid-cell PTEN, whereas the additional observation of improved phagocytosis by alveolar macrophages lacking PTEN ultimately resulted in unaltered lung CFUs following bacterial infection. Together, the absence of myeloid cell-associated PTEN and consecutively enhanced PI3K activity dampened pulmonary inflammation, reduced neutrophil influx, and augmented phagocytic properties of macrophages, which ultimately resulted in decreased tissue injury and improved survival during murine pneumococcal pneumonia.

Oxidized phospholipids regulate expression of ATF4 and VEGF in endothelial cells via NRF2-dependent mechanism: novel point of convergence between electrophilic and unfolded protein stress pathways.

OBJECTIVE: The ATF4 arm of the unfolded protein response is increasingly recognized for its relevance to pathology, and in particular to angiogenic reactions. Oxidized phospholipids (OxPLs), known to accumulate in atherosclerotic vessels, were shown to upregulate vascular endothelial growth factor (VEGF) and induce angiogenesis via an ATF4-dependent mechanism. In this study, we analyzed the mechanism of ATF4 upregulation by OxPLs and more specifically the involvement of NRF2, the major transcriptional mediator of electrophilic stress response. METHODS AND RESULTS: Using reverse transcription/real-time polymerase chain reaction and Western blotting, we found that OxPLs induced upregulation of ATF4 mRNA and protein in several types of endothelial cells and that these effects were suppressed by short interfering RNA (siRNA) against NRF2. Electrophilic (iso)prostaglandins and oxidized low-density lipoprotein, similarly to OxPLs, elevated ATF4 mRNA levels in an NRF2-dependent mode. Chromatin immunoprecipitation revealed OxPL-dependent binding of NRF2 to a putative antioxidant response element site in the ATF4 gene promoter. Knockdown of NRF2 inhibited OxPL-induced elevation of VEGF mRNA and endothelial cell sprout formation. CONCLUSION: Our data characterize NRF2 as a positive regulator of ATF4 and identify a novel cross-talk between electrophilic and unfolded protein responses, which may play a role in stress-induced angiogenesis.

Novel function for blood platelets and podoplanin in developmental separation of blood and lymphatic circulation.

During embryonic development, lymph sacs form from the cardinal vein, and sprout centrifugally to form mature lymphatic networks. Separation of the lymphatic from the blood circulation by a hitherto unknown mechanism is essential for the homeostatic function of the lymphatic system. O-glycans on the lymphatic endothelium have recently been suggested to be required for establishment and maintenance of distinct blood and lymphatic systems, primarily by mediating proper function of podoplanin. Here, we show that this separation process critically involves platelet activation by podoplanin. We found that platelet aggregates build up in wild-type embryos at the separation zone of podoplanin(+) lymph sacs and cardinal veins, but not in podoplanin(-/-) embryos. Thus, podoplanin(-/-) mice develop a "nonseparation" phenotype, characterized by a blood-filled lymphatic network after approximately embryonic day 13.5, which, however, partially resolves in postnatal mice. The same embryonic phenotype is also induced by treatment of pregnant mice with acetyl salicylic acid, podoplanin-blocking antibodies, or by inactivation of the kindlin-3 gene required for platelet aggregation. Therefore, interaction of endothelial podoplanin of the developing lymph sac with circulating platelets from the cardinal vein is critical for separating the lymphatic from the blood vascular system.

Targeting of VEGF-dependent transendothelial migration of cancer cells by bevacizumab.

Cancer progression is often associated with the formation of malignant effusions. Vascular endothelial growth factor (VEGF) is a major regulator of vascular permeability and has been implicated as mediator of tumor progression. We examined the production and secretion of VEGF(165) in various primary cancer cells derived from malignant effusions, and the role of exogenous VEGF(165) as a mediator of effusion formation. VEGF(165) was constantly secreted by all cultured tumor cells in an mTOR-dependent manner, as it was inhibited by the mTOR inhibitor rapamycin. Secreted VEGF(165) showed functional activity by inducing endothelial leakiness and tumor cell-transendothelial migration in vitro, effects which could be reverted by the anti-VEGF antibody bevacizumab. Thus, mTOR inhibitors as well as bevacizumab should be considered as potential agents in cancer patients suffering from malignant effusions.

Crosstalk between the NF-kappaB activating IKK-complex and the CSN signalosome.

A great variety of signalling pathways regulating inflammation, cell development and cell survival require NF-kappaB transcription factors, which are normally inactive due to binding to inhibitors, such as IkappaBalpha. The canonical activation pathway of NF-kappaB is initiated by phosphorylation of the inhibitor by an IkappaB kinase (IKK) complex triggering ubiquitination of IkappaB molecules by SCF-type E3-ligase complexes and rapid degradation by 26S-proteasomes. The ubiquitination machinery is regulated by the COP9 signalosome (CSN). We show that IkappaB kinases interact with the CSN-complex, as well as the SCF-ubiquitination machinery, providing an explanation for the rapid signalling-induced ubiquitination and degradation of IkappaBalpha. Furthermore, we reveal that IKK's phosphorylate not only IkappaBalpha, but also the CSN-subunit Csn5/JAB1 (c-Jun activation domain binding protein-1) and that IKK2 influences ubiquitination of Csn5/JAB1. Our observations imply that the CSN complex acts as an inhibitor of constitutive NF-kappaB activity in non-activated cells. Knock-down of Csn5/JAB1 clearly enhanced basal NF-kappaB activity and improved cell survival under stress. The inhibitory effect of Csn5/JAB1 requires a functional MPN(+) metalloprotease domain, which is responsible for cleaving ubiquitin-like Nedd8-modifications. Upon activation of cells with tumour necrosis factor-alpha, the CSN complex dissociates from IKK's allowing full and rapid activation of the NF-kappaB pathway by the concerted action of interacting protein complexes.

Mannose 6-phosphate/insulin-like growth factor 2 receptor limits cell invasion by controlling alphaVbeta3 integrin expression and proteolytic processing of urokinase-type plasminogen activator receptor.

The multifunctional mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) is considered a tumor suppressor. We report here that RNA interference with M6P/IGF2R expression in urokinase-type plasminogen activator (uPA)/urokinase-type plasminogen activator receptor (uPAR) expressing human cancer and endothelial cells resulted in increased pericellular plasminogen activation, cell adhesion, and higher invasive potential through matrigel. M6P/IGF2R silencing led also to the cell surface accumulation of urokinase and plasminogen and enhanced expression of alphaV integrins. Genetic rescue experiments and inhibitor studies revealed that the enhanced plasminogen activation was due to a direct effect of M6P/IGF2R on uPAR, whereas increased cell adhesion to vitronectin was dependent on alphaV integrin expression and not uPAR. Increased cell invasion of M6P/IGF2R knockdown cells was rescued by cosilencing both uPAR and alphaV integrin. Furthermore, we found that M6P/IGF2R expression accelerates the cleavage of uPAR. M6P/IGF2R silencing resulted in an increased ratio of full-length uPAR to the truncated D2D3 fragment, incapable of binding most uPAR ligands. We conclude that M6P/IGF2R controls cell invasion by regulating alphaV integrin expression and by accelerating uPAR cleavage, leading to the loss of the urokinase/vitronectin/integrin-binding site on uPAR.