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© 2020 by the authors., Membrane-bound proteases play a key role in biology by degrading matrix proteins or shedding adhesion receptors. MT1-MMP metalloproteinase is critical during cancer invasion, angiogenesis, and development. MT1-MMP activity is strictly regulated by internalization, recycling, autoprocessing but also through its incorporation into tetraspanin-enriched microdomains (TEMs), into invadopodia, or by its secretion on extracellular vesicles (EVs). We identified a juxtamembrane positively charged cluster responsible for the interaction of MT1-MMP with ERM (ezrin/radixin/moesin) cytoskeletal connectors in breast carcinoma cells. Linkage to ERMs regulates MT1-MMP subcellular distribution and internalization, but not its incorporation into extracellular vesicles. MT1-MMP association to ERMs and insertion into TEMs are independent phenomena, so that mutation of the ERM-binding motif in the cytoplasmic region of MT1-MMP does not preclude its association with the tetraspanin CD151, but impairs the accumulation and coalescence of CD151/MT1-MMP complexes at actin-rich structures. Conversely, gene deletion of CD151 does not impact on MT1-MMP colocalization with ERM molecules. At the plasma membrane MT1-MMP autoprocessing is severely dependent on ERM association and seems to be the dominant regulator of the enzyme collagenolytic activity. This newly characterized MT1-MMP/ERM association can thus be of relevance for tumor cell invasion., This work has been supported by grants BFU2014-55478-R, REDIEX. SAF2015-71231-REDT and BIO2017-86500-R from Ministerio Español de Economía y Competitividad (MINECO) and by a grant from Fundación Ramón Areces “Ayudas a la Investigación en Ciencias de la Vida y de la Materia, 2014” to M.Y.-M. H.S. was supported by a FPI-UAM fellowship. The CNIC is supported by the Ministry of Ciencia, Innovacion y Universidades and the Pro CNIC Foundation, is a Severo Ochoa Center of Excellence (SEV-2015-0505), also supported by European Regional Development Fund (FEDER) “Una manera de hacer Europa”.

Previous findings indicate that reducing brain insulin-like growth factor I receptor (IGF-IR) activity promotes ample neuroprotection. We now examined a possible action of IGF-IR on brain glucose transport to explain its wide protective activity, as energy availability is crucial for healthy tissue function. Using (18) FGlucose PET we found that shRNA interference of IGF-IR in mouse somatosensory cortex significantly increased glucose uptake upon sensory stimulation. In vivo microscopy using astrocyte specific staining showed that after IGF-IR shRNA injection in somatosensory cortex, astrocytes displayed greater increases in glucose uptake as compared to astrocytes in the scramble-injected side. Further, mice with the IGF-IR knock down in astrocytes showed increased glucose uptake in somatosensory cortex upon sensory stimulation. Analysis of underlying mechanisms indicated that IGF-IR interacts with glucose transporter 1 (GLUT1), the main facilitative glucose transporter in astrocytes, through a mechanism involving interactions with the scaffolding protein GIPC and the multicargo transporter LRP1 to retain GLUT1 inside the cell. These findings identify IGF-IR as a key modulator of brain glucose metabolism through its inhibitory action on astrocytic GLUT1 activity. GLIA 2016;64:1962-1971.

In response to injury, the brain produces different neuroprotective molecules, such as insulin-like growth factor I (IGF-I). However, IGF-I is also taken up by the brain from the circulation in response to physiological stimuli. Herein, we analyzed in mice the relative contribution of circulating and locally produced IGF-I to increased brain IGF-I levels after insult. Traumatic brain injury (TBI) induced by a controlled impact resulted in increased IGF-I levels in the vicinity of the lesion, but mice with low serum IGF-I showed significantly lower increases. Indeed, in normal mice, peripheral IGF-I accumulated at the lesion site after injury, and at the same time serum IGF-I levels decreased. Collectively, these data suggest that serum IGF-I enter into the brain after TBI and contributes to increased brain IGF-I levels at the injury site. This connection between central and circulating IGF-I provides an amenable route for treatment, as subcutaneous administration of IGF-I to TBI mice led to functional recovery. These latter results add further support to the use of systemic IGF-I or its mimetics for treatment of brain injuries.

Inherited neurodegenerative diseases such as Friedreich's ataxia (FRDA), produced by deficiency of the mitochondrial chaperone frataxin (Fxn), shows specific neurological deficits involving different subset of neurons even though deficiency of Fxn is ubiquitous. Because astrocytes are involved in neurodegeneration, we analyzed whether they are also affected by frataxin deficiency and contribute to the disease. We also tested whether insulin-like growth factor I (IGF-I), that has proven effective in increasing frataxin levels both in neurons and in astrocytes, also exerts in vivo protective actions. Using the GFAP promoter expressed by multipotential stem cells during development and mostly by astrocytes in the adult, we ablated Fxn in a time-dependent manner in mice (FGKO mice) and found severe ataxia and early death when Fxn was eliminated during development, but not when deleted in the adult. Analysis of underlying mechanisms revealed that Fxn deficiency elicited growth and survival impairments in developing cerebellar astrocytes, whereas forebrain astrocytes grew normally. A similar time-dependent effect of frataxin deficiency in astrocytes was observed in a fly model. In addition, treatment of FGKO mice with IGF-I improved their motor performance, reduced cerebellar atrophy, and increased survival. These observations indicate that a greater vulnerability of developing cerebellar astrocytes to Fxn deficiency may contribute to cerebellar deficits in this inherited disease. Our data also confirm a therapeutic benefit of IGF-I in early FRDA deficiency., This work was supported by Spanish Ministry of Science (SAF2010–60051/SAF2013–40710-R), by CIBERNED, and by the Elitenetzwerk Bayern.

Reducing insulin-like growth factor I receptor (IGF-IR) levels or administration of IGF-I show beneficial effects in the brain. We now provide evidence to help resolve this paradox. The unliganded IGF-IR inhibits glucose uptake by astrocytes while its stimulation with IGF-I, in concert with insulin activation of the insulin receptor, produces the opposite effect. In vivo imaging showed that shRNA interference of brain IGF-IR increased glucose uptake by astrocytes while pharmacological blockade of IGF-IR reduced it. Brain 18FGlucose-PET of IGF-IR shRNA injected mice confirmed an inhibitory role of unliganded IGF-IR on glucose uptake, whereas glucose-dependent recovery of neuronal activity in brain slices was blunted by pharmacological blockade of IGF-IR. Mechanistically, we found that the unliganded IGF-IR retains glucose transporter 1 (GLUT1), the main glucose transporter in astrocytes, inside the cell while IGF-I, in cooperation with insulin, synergistically stimulates MAPK/PKD to promote association of IGF-IR with GLUT 1 via Rac1/GIPC1 and increases GLUT1 availability at the cell membrane. These findings identify IGF-I and its receptor as antagonistic modulators of brain glucose uptake.

Tumorigenesis involves not only tumor cells that become transformed but also the peritumoral stroma which reacts inducing inflammatory and angiogenic responses. Angiogenesis, the formation of new capillaries from preexisting vessels, is an absolute requirement for tumor growth and metastasis, and it can be induced and modulated by a wide variety of soluble factors. During angiogenesis, quiescent endothelial cells are activated and they initiate migration by degrading the basement membranes through the action of specific proteases, in particular of matrix metalloproteinases (MMPs). Among these, the membrane type 1-matrix metalloproteinase (MT1-MMP) has been identified as a key player during the angiogenic response. In this review, we will summarize the role of MT1-MMP in angiogenesis and the regulatory mechanisms of this protease in endothelial cells. Since our recent findings have suggested that MT1-MMP is not universally required for angiogenesis, we hypothesize that the regulation and participation of MT1-MMP in angiogenesis may depend on the nature of the angiogenic stimulus. Experiments aimed at testing this hypothesis have shown that similarly to the chemokine stromal cell-derived factor-1 (SDF-1)/CXCL12, lipopolysaccharide (LPS) seems to induce the formation of capillary tubes by human or mouse endothelial cells (ECs) in an MT1-MMP-independent manner. The implications of these findings in the potential use of MT1-MMP inhibitors in cancer therapy are discussed.

We have investigated the putative role and regulation of membrane type 1-matrix metalloproteinase (MT1-MMP) in angiogenesis induced by inflammatory factors of the chemokine family. The absence of MT1-MMP from null mice or derived mouse lung endothelial cells or the blockade of its activity with inhibitory antibodies resulted in the specific decrease of in vivo and in vitro angiogenesis induced by CCL2 but not CXCL12. Similarly, CCL2- and CXCL8-induced tube formation by human endothelial cells (ECs) was highly dependent on MT1-MMP activity. CCL2 and CXCL8 significantly increased MT1-MMP surface expression, clustering, activity, and function in human ECs. Investigation of the signaling pathways involved in chemokine-induced MT1-MMP activity in ECs revealed that CCL2 and CXCL8 induced cortical actin polymerization and sustained activation of phosphatidylinositol 3-kinase (PI3K) and the small GTPase Rac. Inhibition of PI3K or actin polymerization impaired CCL2-induced MT1-MMP activity. Finally, dimerization of MT1-MMP was found to be enhanced by CCL2 in ECs in a PI3K- and actin polymerization-dependent manner. In summary, we identify MT1-MMP as a molecular target preferentially involved in angiogenesis mediated by CCL2 and CXCL8, but not CXCL12, and suggest that MT1-MMP dimerization might be an important mechanism of its regulation during angiogenesis.

Loss-of-function mutations in the phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome10) contribute to aberrant cell growth in part through upregulation of the mitogenic IGF-1/PI3 K/Akt pathway. In turn, this pathway exerts a homeostatic feedback over PTEN. Using mutagenesis analysis to explore a possible impact of this mutual control on astrocyte growth, we found that truncation of the C-terminal region of PTEN (Δ51) associates with a marked increase in NFκB activity, a transcription factor overactivated in astrocyte tumors. Whereas mutations of PTEN are considered to lead to a loss-of-function, PTENΔ51, a truncation that comprises a region frequently mutated in human gliomas, displayed a neomorphic (gain-of-function) activity that was independent of its phosphatase activity. This gain-of-function of PTENΔ51 includes stimulation of IGF-1 synthesis through protein kinase A activation of the IGF-1 promoter. Increased IGF-1 originates an autocrine loop that activates Akt and NFκB. Constitutive activation of NFκB in PTENΔ51-expressing astrocytes leads to aberrant cell growth; astrocytes expressing this mutant PTEN generate colonies in vitro and tumors in vivo. Mutations converting a tumor suppressor such as PTEN into a tumor promoter through a gain-of-function involving IGF-1 production may further our understanding of the role played by this growth factor in glioma growth and help us define druggable targets for personalized therapy. © 2014 Macmillan Publishers Limited.

The development of a new vascular network is essential for the onset and progression of many pathophysiologic processes. Cyclooxygenase-2 displays a proangiogenic activity in in vitro and in vivo models, mediated principally through its metabolite prostaglandin E2 (PGE2). Here, we provide evidence for a novel signaling route through which PGE2 activates the Alk5-Smad3 pathway in endothelial cells. PGE2 induces Alk5-dependent Smad3 nuclear translocation and DNA binding, and the activation of this pathway involves the release of active TGFβ from its latent form through a process mediated by the metalloproteinase MT1-MMP, whose membrane clustering is promoted by PGE2. MT1-MMP–dependent transforming growth factor β (TGFβ) signaling through Alk5 is also required for PGE2-induced endothelial cord formation in vitro, and Alk5 kinase activity is required for PGE2-induced neovascularization in vivo. These findings identify a novel signaling pathway linking PGE2 and TGFβ, 2 effectors involved in tumor growth and angiogenesis, and reveal potential targets for the treatment of angiogenesis-related disorders.

Angiogenesis, the formation of new vessels from pre-existing capillaries, is a fundamental physiological process which is also critical for the development of several pathological conditions; thus a diminished angiogenic response is related to ischemic disorders, whereas increased angiogenesis is associated with tumorigenesis and chronic inflammatory diseases. New ways of modulating angiogenesis therefore have potential in the treatment of these diseases. During angiogenesis, normally quiescent endothelial cells (ECs) become migratory and invade the surrounding tissue. To do this, they require a specific enzyme machinery to degrade the tissue barriers presented by the basement membranes and the interstitial matrix. This function is supplied by matrix metalloproteinase (MMP) proteins, a large family of enzymes responsible for degrading a variety of extracellular matrix (ECM) components and for modulating the bioactivity of transmembrane receptors and soluble factors. In this review we examine the participation of MMPs - in particular membrane type 1-matrix metalloproteinase (MT1-MMP) - in the different steps of angiogenesis, and discuss the mechanisms of regulation of MT1-MMP in ECs. Finally, we explore the potential use of MMP inhibitors (MMPI) in the treatment of angiogenesis-related disease, with especial emphasis on novel approaches to the inhibition of MT1-MMP activity in ECs.

4 Figures. The online version of this article contains a data supplement. Nitric oxide (NO) is essential for vascular homeostasis and is also a critical modulator of angiogenesis; however, the molecular mechanisms of NO action during angiogenesis remain elusive. We have investigated the potential relationship between NO and membrane type 1–matrix metalloproteinase (MT1-MMP) during endothelial migration and capillary tube formation. Endothelial NO synthase (eNOS) colocalizes with MT1-MMP at motility-associated structures in migratory human endothelial cells (ECs); moreover, NO is produced at these structures and is released into the medium during EC migration. We have therefore addressed 2 questions: (1) the putative regulation of MT1-MMP by NO in migratory ECs; and (2) the requirement for MT1-MMP in NO-induced EC migration and tube formation. NO upregulates MT1-MMP membrane clustering on migratory human ECs, and this is accompanied by increased degradation of type I collagen substrate. MT1-MMP membrane expression and localization are impaired in lung ECs from eNOS-deficient mice, and these cells also show impaired migration and tube formation in vitro. Inhibition of MT1-MMP with a neutralizing antibody impairs NOinduced tube formation by human ECs, and NO-induced endothelial migration and tube formation are impaired in lung ECs from mice deficient in MT1-MMP. MT1-MMP thus appears to be a key molecular effector of NO during the EC migration and angiogenic processes, and is a potential therapeutic target for NO-associated vascular disorders. National Institutes of Health grant AR47074 (S.A.), Comunidad Autónoma de Madrid grants CAM 08.4/0023/2003 (C.Z.) and GR/SAL/0309/2004 (A.G.A.), Spanish Ministerio de Sanidad y Consumo grant CP03/00 025 (A.M.R.), and Spanish Ministerio de Educación y Ciencia grants SAF2005- 06025 (C.Z.), SAF2006-02410 (S.L.), and SAF2005-02228(A.G.A.). P.G. is a postdoctoral researcher from the “Juan de la Cierva” program (MCyT) and C.Z. is a research investigator from the “Ramón y Cajal” program (MCyT).

MT1-matrix metalloproteinase (MT1-MMP) is one of the most critical factors in the invasion machinery of tumor cells. Subcellular localization to invasive structures is key for MT1-MMP proinvasive activity. However, the mechanism driving this polarized distribution remains obscure. We now report that polarized exocytosis of MT1-MMP occurs during MDA-MB-231 adenocarcinoma cell migration into collagen type I three-dimensional matrices. Polarized trafficking of MT1-MMP is triggered by beta1 integrin-mediated adhesion to collagen, and is required for protease localization at invasive structures. Localization of MT1-MMP within VSV-G/Rab8-positive vesicles, but not in Rab11/Tf/TfRc-positive compartment in invasive cells, suggests the involvement of the exocytic traffic pathway. Furthermore, constitutively active Rab8 mutants induce MT1-MMP exocytic traffic, collagen degradation and invasion, whereas Rab8- but not Rab11-knockdown inhibited these processes. Altogether, these data reveal a novel pathway of MT1-MMP redistribution to invasive structures, exocytic vesicle trafficking, which is crucial for its role in tumor cell invasiveness. Mechanistically, MT1-MMP delivery to invasive structures, and therefore its proinvasive activity, is regulated by Rab8 GTPase.

7 Figures Membrane type 1–matrix metalloproteinase (MT1-MMP) is involved in endothelial and tumor-cell migration, but its putative role in leukocyte migration has not been characterized yet. Here, we demonstrate that anti–MT1-MMP monoclonal antibody (mAb) impaired monocyte chemotactic protein-1 (MCP-1)–stimulated monocyte migration on fibronectin (FN), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1). In addition, monocyte transmigration through tumor necrosis factor-α (TNF-α)–activated endothelium is also inhibited by anti–MT1-MMP mAb. Therefore, regulation of MT1-MMP in human peripheral blood monocytes was investigated. First, MT1-MMP clustering was observed at motility-associated membrane protrusions of MCP-1–stimulated monocytes migrating on FN, VCAM-1, or ICAM-1 and at the leading edge, together with profilin, of monocytes transmigrating through activated endothelial cells. In addition, up-regulation of MT1-MMP expression was induced in human monocytes upon attachment to FN in a manner dependent on α41 and α51 integrins. Binding of monocytes to TNF-α–activated human endothelial cells as well as to VCAM-1 or ICAM-1 also resulted in an increase of MT1-MMP expression. These findings correlated with an enhancement of MT1-MMP fibrinolytic activity in monocytes bound to FN, VCAM-1, or ICAM-1. Our data show that MT1-MMP is required during human monocyte migration and endothelial transmigration and that MT1-MMP localization, expression, and activity are regulated in monocytes upon contact with FN or endothelial ligands, pointing to a key role of MT1-MMP in monocyte recruitment during inflammation. Centro Nacional de Investigaciones Cardiovasculares, Madrid,Spain; Departamento de Inmunología, Hospital Universitario de la Princesa,Madrid, Spain; and the Departamento de Inmunología, Hospital Universitario Gregorio Maranón, Madrid, Spain.

Oxidative stress is a proposed mechanism in brain aging, making the study of its regulatory processes an important aspect of current neurobiological research. In this regard, the role of the aging regulator insulin-like growth factor I (IGF-I) in brain responses to oxidative stress remains elusive as both beneficial and detrimental actions have been ascribed to this growth factor. Because astrocytes protect neurons against oxidative injury, we explored whether IGF-I participates in astrocyte neuroprotection and found that blockade of the IGF-I receptor in astrocytes abrogated their rescuing effect on neurons. The protection mediated by IGF-I against oxidative stress (H 2O 2) in astrocytes is probably needed for these cells to provide adequate neuroprotection. Indeed, in astrocytes but not in neurons, IGF-I helps decrease the pro-oxidant protein thioredoxin-interacting protein 1 and normalizes the levels of reactive oxygen species. Furthermore, IGF-I cooperates with trophic signals produced by astrocytes in response to H 2O 2 such as stem cell factor (SCF) to protect neurons against oxidative insult. After stroke, a condition associated with brain aging where oxidative injury affects peri-infarcted regions, a simultaneous increase in SCF and IGF-I expression was found in the cortex, suggesting that a similar cooperative response takes place in vivo. Cell-specific modulation by IGF-I of brain responses to oxidative stress may contribute in clarifying the role of IGF-I in brain aging. © 2014 Genis L et al., This work was funded by grants of the Spanish Ministry of Science (SAF2010-17036) and Centro Investigacion Biomedica en red Enfermedades Neurodegenerativas (CIBERNED) to IT-A.