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Picosecond laser excitation of vascular-targeted gold nanoparticles activates multiple mechanobiological pathways in the endothelial cells and induces reversible blood–brain barrier opening.

Glioblastomas are heterogeneous, primary brain tumors hiding several sub-populations. Patient-derived xenografts are considered gold-standards to study glioblastoma invasion. However, they present many disadvantages, including time consumption, complex standardization, high cost. To counteract these issues and rapidly identify the most invasive sub-populations, we developed anin vivomimicry platform named SP2G (SPheroid SPreading on Grids). Live imaging of tumor-derived spheroids spreading on gridded micro patterns mimicking the brain vasculature recapitulated 3D motility features observed in brain or 3D matrices. Using patient-derived samples coupled with a semi-automated macro suite, SP2G easily characterized and sorted differences in cell migration and motility modes. Moreover, SP2G exposed the hidden intra-patient heterogeneity in cell motility that correlated molecularly to specific integrins. Thus, SP2G constitute a versatile and potentially pan-cancer workflow to identify the diverse invasive tumor sub-populations in patient-derived specimens. SP2G includes an integrative tool, available as open-source Fiji macro suite, for therapeutic evaluations at single patient level.TeaserCracking the inter and intra-patient diversity in Glioblastoma migration profiles

The blood–brain barrier (BBB) acquires unique properties for regulation of the neuronal function during development. The genesis of the BBB coupled with angiogenesis is orchestrated by the Wnt/β-catenin signaling pathway. Aside from the importance of Wnt/β-catenin signaling, the molecular mechanisms that regulate these processes are poorly understood. Here, we identify the brain endothelial adhesion G-protein–coupled receptor Gpr126 as a novel target gene of Wnt/β-catenin signaling that is required for postnatal BBB development, and its expression is detrimental for ischemic stroke in adults. We show that Gpr126 expression is high in mouse brain endothelium during BBB formation, but decreases in the adult. Inactivation of Gpr126 in postnatal endothelial cells results in vessel enlargement and impairs acquisition of the BBB characteristics, such as increased neurovascular permeability, and reduced basement membrane protein deposition and pericyte coverage. Mechanistically, Gpr126 is required during developmental angiogenesis to promote endothelial cell migration, acting via an interaction between Lrp1 and α3β1-integrin, which couples vessel morphogenesis to BBB formation. Interestingly, in adult mice with an established BBB, the lack of Gpr126 expression in acute ischemic stroke is protective and coupled with reduced microglia activation, which contributes to an improved neurological outcome. These data identify Gpr126 as a promising therapeutic target to treat ischemic stroke.

Glioblastoma multiforme (GBM) is the most prevalent malignant tumor in the central nervous system. It has diverse phenotypes, including diffuse single-cell infiltration in which the tumor cells co-opt the normal microvasculature, and the neovascularization of an expanding tumor mass. The blood-brain-tumor barrier (BBTB) is a significant obstacle to GBM treatment and restricts entry of most FDA-approved effective oncology drugs. Herein, we report that picosecond laser excitation of vascular-targeted plasmonic gold nanoparticles (AuNPs) can non-invasively and reversibly modulate the BBTB permeability (optoBBTB). OptoBBTB enhances the delivery of paclitaxel (Taxol) in two genetically engineered glioma models (GEMM) that span the spectrum of GBM phenotypes. OptoBBTB followed by Taxol delivery effectively suppresses tumor growth and prolongs the survival time of both GEMM. Moreover, our results raise the possibility that paclitaxel, which is amongst the most widely used oncology drugs because of its proven efficacy but has been abandoned for GBM following its failure to efficacy in early phase clinical trials due to poor blood-brain barrier (BBB) penetration, could now be reconsidered in combination with strategies to increase BBB permeability. In summary, optoBBTB is a novel and effective approach to increase the delivery of therapeutics with limited BBB permeability to treat neoplastic and non-neoplastic brain diseases.

The blood-brain barrier (BBB) maintains an optimal environment for brain homeostasis but excludes most therapeutics from entering the brain. Strategies that can reversibly increase BBB permeability will be essential for treating brain diseases and is the focus of significant preclinical and translational interest. Recently, we reported that picosecond laser excitation of molecular-targeted gold nanoparticles (AuNPs) induced a graded and reversible increase in BBB permeability in vivo (OptoBBB). Here we investigate how to increase the targeting efficiency and how picosecond lase stimulation of AuNP leads to an increase in endothelial paracellular permeability. Our results suggest that targeting brain endothelial glycoproteins leads to >20-fold higher targeting efficiency compared with tight junction targeting. We report that OptoBBB is associated with a transient elevation of Ca2+ that propagates to adjacent endothelial cells after laser excitation and extends the region of BBB opening. The Ca2+ response involves both internal Ca2+ depletion and Ca2+ influx. Furthermore, we demonstrate that the involvement of actin polymerization and Ca2+-dependent phosphorylation of ERK1/2 lead to cytoskeletal activation, increasing paracellular permeability. Taken together, we provide mechanistic insight into how excitation of endothelial targeted AuNPs leads to an increase in BBB permeability. These insights will be critical for guiding the future developments of this technology for brain disease treatment.

The blood-spinal cord barrier (BSCB) tightly regulates molecular transport from the blood to the spinal cord. Herein, we present a novel approach for transient modulation of BSCB permeability and localized delivery of peptides into the spinal cord for behavior modulation with high spatial resolution. This approach utilizes optical stimulation of vasculature-targeted nanoparticles and allows delivery of BSCB-nonpermeable molecules into the spinal cord without significant glial activation or impact on animal locomotor behavior. We demonstrate minimally invasive light delivery into the spinal cord using an optical fiber and BSCB permeability modulation in the lumbar region. Our method of BSCB modulation allows delivery of bombesin, a centrally-acting and itch-inducing peptide, into the spinal cord and induces a rapid and transient increase in itching behaviors in mice. This minimally invasive approach enables behavior modulation without genetic modifications and is promising for delivering a wide range of biologics into the spinal cord for behavior modulation and potentially therapy.Significance StatementSpinal cord diseases and disorders are common and cause significant disability, including chronic pain, paralysis, cognitive impairment, and mortality. The blood-spinal cord barrier is a considerable challenge for delivery by systemic therapeutic administration. We developed an optical approach for effectively and safely delivering molecules to the spinal cord to overcome this barrier. The fiberoptic method is minimally invasive and overcomes challenges that previous technologies face, including the complicated bone structure and standing waves that complicate BSCB opening using ultrasound. Optical stimulation offers unprecedented spatial resolution for the precise delivery in intricate spinal cord structures. Significantly, our approach modulates animal behavior (i.e., itch) without genetic modifications and demonstrates the potential for delivery of biologics such as peptides into the spinal cord.

Endothelial-to-mesenchymal transition (EndMT) is the biological process through which endothelial cells transdifferentiate into mesenchymal cells. During embryo development, EndMT regulates endocardial cushion formation via TGFβ/BMP signaling. In adults, EndMT is mainly activated during pathological conditions. Hence, it is necessary to characterize molecular regulators cooperating with TGFβ signaling in driving EndMT, to identify potential novel therapeutic targets to treat these pathologies. Here, we studied YAP, a transcriptional co-regulator involved in several biological processes, including epithelial-to-mesenchymal transition (EMT). As EndMT is the endothelial-specific form of EMT, and YAP (herein referring to YAP1) and TGFβ signaling cross-talk in other contexts, we hypothesized that YAP contributes to EndMT by modulating TGFβ signaling. We demonstrate that YAP is required to trigger TGFβ-induced EndMT response, specifically contributing to SMAD3-driven EndMT early gene transcription. We provide novel evidence that YAP acts as SMAD3 transcriptional co-factor and prevents GSK3β-mediated SMAD3 phosphorylation, thus protecting SMAD3 from degradation. YAP is therefore emerging as a possible candidate target to inhibit pathological TGFβ-induced EndMT at early stages., Journal of Cell Science, 134 (15), ISSN:1477-9137, ISSN:0021-9533

Precise manipulation of protein activity in living systems has broad applications in biomedical sciences. However, it is challenging to use light to manipulate protein activity in living systems without genetic modification. Here, we report a technique to optically switch off protein activity in living cells with high spatiotemporal resolution, referred to as molecular hyperthermia (MH). MH is based on the nanoscale confined heating of plasmonic gold nanoparticles by short laser pulses to unfold and photo-inactivate targeted proteins of interest. Firstly, we show that protease activated receptor 2 (PAR2), a G-protein coupled receptor and an important pathway that leads to pain sensitization, can be photo-inactivated in situ by MH without compromising cell proliferation. PAR2 activity can be switched-off in laser-targeted cells without affecting surrounding cells. Furthermore, we demonstrate the molecular specificity of MH by inactivating PAR2 while leaving other receptors intact. Secondly, we demonstrate that the photo-inactivation of a tight junction protein in brain endothelial monolayers leads to a reversible blood brain barrier opening in vitro. Lastly, the protein inactivation by MH is below the nanobubble generation threshold and thus is predominantly due to the nanoscale heating. MH is distinct from traditional hyperthermia (that induces global tissue heating) in both its time and length scales: nanoseconds versus seconds, nanometers versus millimeters. Our results demonstrate that MH enables selective and remote manipulation of protein activity and cellular behavior without genetic modification.

Cerebral cavernous malformation (CCM) is a neurovascular familial or sporadic disease that is characterised by capillary-venous cavernomas, and is due to loss-of-function mutations to any one of three CCM genes. Familial CCM follows a two-hit mechanism similar to that of tumour suppressor genes, while in sporadic cavernomas only a small fraction of endothelial cells shows mutated CCM genes. We reported that in mouse models and in human patients, endothelial cells lining the lesions have different features from the surrounding endothelium, as they express mesenchymal/stem-cell markers. Here we show that cavernomas originate from clonal expansion of few Ccm3-null endothelial cells that express mesenchymal/stem-cell markers. These cells then attract surrounding wild-type endothelial cells, inducing them to express mesenchymal/stem-cell markers and to contribute to cavernoma growth. These characteristics of Ccm3-null cells are reminiscent of the tumour-initiating cells that are responsible for tumour growth. Our data support the concept that CCM has benign tumour characteristics., Cerebral cavernous malformation is a vascular disease characterized by capillary-venous cavernomas in the central nervous system. Here the authors show that cavernomas display benign tumor characteristics and originate from the clonal expansion of mutated endothelial progenitors which can attract surrounding wild-type cells, inducing their mesenchymal transition and leading to growth of the cavernoma.

In some organs, such as the brain, endothelial cells form a robust and highly selective blood-to-tissue barrier. However, in other organs, such as the intestine, endothelial cells provide less stringent permeability, to allow rapid exchange of solutes and nutrients where needed. To maintain the structural and functional integrity of the highly dynamic blood–brain and gut–vascular barriers, endothelial cells form highly specialized cell-cell junctions, known as adherens junctions and tight junctions. Claudins are a family of four-membrane-spanning proteins at tight junctions and they have both barrier-forming and pore-forming properties. Tissue-specific expression of claudins has been linked to different diseases that are characterized by barrier impairment. In this review, we summarize the more recent progress in the field of the claudins, with particular attention to their expression and function in the blood–brain barrier and the recently described gut–vascular barrier, under physiological and pathological conditions. Abbreviations: 22q11DS 22q11 deletion syndrome; ACKR1 atypical chemokine receptor 1; AD Alzheimer disease; AQP aquaporin; ATP adenosine triphosphate; Aβ amyloid β; BAC bacterial artificial chromosome; BBB blood-brain barrier; C/EBP-α CCAAT/enhancer-binding protein α; cAMP cyclic adenosine monophosphate (or 3ʹ,5ʹ-cyclic adenosine monophosphate); CD cluster of differentiation; CNS central nervous system; DSRED discosoma red; EAE experimental autoimmune encephalomyelitis; ECV304 immortalized endothelial cell line established from the vein of an apparently normal human umbilical cord; EGFP enhanced green fluorescent protein; ESAM endothelial cell-selective adhesion molecule; GLUT-1 glucose transporter 1; GVB gut-vascular barrier; H2B histone H2B; HAPP human amyloid precursor protein; HEK human embryonic kidney; JACOP junction-associated coiled coil protein; JAM junctional adhesion molecules; LYVE1 lymphatic vessel endothelial hyaluronan receptor 1; MADCAM1 mucosal vascular addressin cell adhesion molecule 1; MAPK mitogen-activated protein kinase; MCAO middle cerebral artery occlusion; MMP metalloprotease; MS multiple sclerosis; MUPP multi-PDZ domain protein; PATJ PALS-1-associated tight junction protein; PDGFR-α platelet-derived growth factor receptor α polypeptide; PDGFR-β platelet-derived growth factor receptor β polypeptide; RHO rho-associated protein kinase; ROCK rho-associated, coiled-coil-containing protein kinase; RT-qPCR real time quantitative polymerase chain reactions; PDGFR-β soluble platelet-derived growth factor receptor, β polypeptide; T24 human urinary bladder carcinoma cells; TG2576 transgenic mice expressing the human amyloid precursor protein; TNF-α tumor necrosis factor α; WTwild-type; ZO zonula occludens.

The blood–brain barrier (BBB) is highly selective and acts as the interface between the central nervous system and circulation. While the BBB is critical for maintaining brain homeostasis, it represents a formidable challenge for drug delivery. Here we synthesized gold nanoparticles (AuNPs) for targeting the tight junction specifically and demonstrated that transcranial picosecond laser stimulation of these AuNPs post intravenous injection increases the BBB permeability. The BBB permeability change can be graded by laser intensity, is entirely reversible, and involves increased paracellular diffusion. BBB modulation does not lead to significant disruption in the spontaneous vasomotion or the structure of the neurovascular unit. This strategy allows the entry of immunoglobulins and viral gene therapy vectors, as well as cargo-laden liposomes. We anticipate this nanotechnology to be useful for tissue regions that are accessible to light or fiberoptic application and to open new avenues for drug screening and therapeutic interventions in the central nervous system.

The blood-brain barrier (BBB) tightly regulates the entry of molecules into the brain by tight junctions that seals the paracellular space and receptor-mediated transcytosis. It remains elusive to selectively modulate these mechanisms and to overcome BBB without significant neurotoxicity. Here we report that light stimulation of tight junction-targeted plasmonic nanoparticles selectively opens up the paracellular route to allow diffusion through the compromised tight junction and into the brain parenchyma. The BBB modulation does not impair vascular dynamics and associated neurovascular coupling, or cause significant neural injury. It further allows antibody and adeno-associated virus delivery into local brain regions. This novel method offers the first evidence of selectively modulating BBB tight junctions and opens new avenues for therapeutic interventions in the central nervous system.One Sentence SummaryGentle stimulation of molecular-targeted nanoparticles selectively opens up the paracellular pathway and allows macromolecules and gene therapy vectors into the brain.

Glioblastoma are heterogeneous tumors composed of highly invasive and highly proliferative clones. Heterogeneity in invasiveness could emerge from discrete biophysical properties linked to specific molecular expression. We identified clones of patient-derived glioma propagating cells that were either highly proliferative or highly invasive and compared their cellular architecture, migratory, and biophysical properties. We discovered that invasiveness was linked to cellular fitness. The most invasive cells were stiffer, developed higher mechanical forces on the substrate, and moved stochastically. The mechano-chemical-induced expression of the formin FMN1 conferred invasive strength that was confirmed in patient samples. Moreover, FMN1 expression was also linked to motility in other cancer and normal cell lines, and its ectopic expression increased fitness parameters. Mechanistically, FMN1 acts from the microtubule lattice and promotes a robust mechanical cohesion, leading to highly invasive motility. Ministry of Health (MOH) this work was supported by: IFOM (starting package); the Mechanobiology Institute of Singapore (grant WBSR714-016-007 -271); the Italian Association for Cancer Research (AIRC) (Investigator Grant [IG] 20716 to N.C.G. and doctoral fellowship 3-year fellowship "MilanoMarathon-oggicorroperAIRC" -Rif. 22461 to M.C.); Marie Sklodowska-Curie actions (H2O20 -MSCA individual fellowship 796547 to A.G.); and the Singapore Ministry of Health's National Medical Research Council under its Translational and Clinical Research (TCR) Flagship Programme-Tier 1(NMRC/TCR/016-NNI/2016) to C.T.

Supplemental Digital Content is available in the text., Rationale: Intercellular tight junctions are crucial for correct regulation of the endothelial barrier. Their composition and integrity are affected in pathological contexts, such as inflammation and tumor growth. JAM-A (junctional adhesion molecule A) is a transmembrane component of tight junctions with a role in maintenance of endothelial barrier function, although how this is accomplished remains elusive. Objective: We aimed to understand the molecular mechanisms through which JAM-A expression regulates tight junction organization to control endothelial permeability, with potential implications under pathological conditions. Methods and Results: Genetic deletion of JAM-A in mice significantly increased vascular permeability. This was associated with significantly decreased expression of claudin-5 in the vasculature of various tissues, including brain and lung. We observed that C/EBP-α (CCAAT/enhancer-binding protein-α) can act as a transcription factor to trigger the expression of claudin-5 downstream of JAM-A, to thus enhance vascular barrier function. Accordingly, gain-of-function for C/EBP-α increased claudin-5 expression and decreased endothelial permeability, as measured by the passage of fluorescein isothiocyanate (FITC)-dextran through endothelial monolayers. Conversely, C/EBP-α loss-of-function showed the opposite effects of decreased claudin-5 levels and increased endothelial permeability. Mechanistically, JAM-A promoted C/EBP-α expression through suppression of β-catenin transcriptional activity, and also through activation of EPAC (exchange protein directly activated by cAMP). C/EBP-α then directly binds the promoter of claudin-5 to thereby promote its transcription. Finally, JAM-A–C/EBP-α–mediated regulation of claudin-5 was lost in blood vessels from tissue biopsies from patients with glioblastoma and ovarian cancer. Conclusions: We describe here a novel role for the transcription factor C/EBP-α that is positively modulated by JAM-A, a component of tight junctions that acts through EPAC to up-regulate the expression of claudin-5, to thus decrease endothelial permeability. Overall, these data unravel a regulatory molecular pathway through which tight junctions limit vascular permeability. This will help in the identification of further therapeutic targets for diseases associated with endothelial barrier dysfunction.

Background The tumour microenvironment is a critical regulator of malignant cancer progression. While endothelial cells have been widely studied in the context of tumour angiogenesis, their role as modulators of cancer cell invasion and migration is poorly understood. Methods We have investigated the influence of endothelial cells on the invasive and migratory behaviour of human cancer cells in vitro. Results Upon exposure to culture supernatants of endothelial cells, distinct cancer cells, such as SK-BR-3 cells, showed significantly increased invasion and cell migration concomitant with changes in cell morphology and gene expression reminiscent of an epithelial-mesenchymal transition (EMT). Interestingly, the pro-migratory effect on SK-BR-3 cells was significantly enhanced by supernatants obtained from subconfluent, proliferative endothelial cells rather than from confluent, quiescent endothelial cells. Systematically comparing the supernatants of subconfluent and confluent endothelial cells by quantitative MS proteomics revealed eight candidate proteins that were secreted at significantly higher levels by confluent endothelial cells representing potential inhibitors of cancer cell migration. Among these proteins, nidogen-1 was exclusively expressed in confluent endothelial cells and was found to be necessary and sufficient for the inhibition of SK-BR-3 cell migration. Indeed, SK-BR-3 cells exposed to nidogen-1-depleted endothelial supernatants showed increased promigratory STAT3 phosphorylation along with increased cell migration. This reflects the situation of enhanced SK-BR-3 migration upon stimulation with conditioned medium from subconfluent endothelial cells with inherent absence of nidogen-1 expression. Conclusion The identification of nidogen-1 as an endothelial-derived inhibitor of migration of distinct cancer cell types reveals a novel mechanism of endothelial control over cancer progression. Electronic supplementary material The online version of this article (10.1186/s12885-019-5521-8) contains supplementary material, which is available to authorized users.

The KDEL receptor (KDELR) is a seven-transmembrane-domain protein involved in retrograde transport of protein chaperones from the Golgi complex to the endoplasmic reticulum. Our recent findings have shown that the Golgi-localised KDELR acts as a functional G-protein-coupled receptor by binding to and activating Gs and Gq. These G proteins induce activation of PKA and Src and regulate retrograde and anterograde Golgi trafficking. Here we used an integrated coimmunoprecipitation and mass spectrometry approach to identify prohibitin-1 (PHB) as a KDELR interactor. PHB is a multifunctional protein that is involved in signal transduction, cell-cycle control, and stabilisation of mitochondrial proteins. We provide evidence that depletion of PHB induces intense membrane-trafficking activity at the ER–Golgi interface, as revealed by formation of GM130-positive Golgi tubules, and recruitment of p115,β-COP, and GBF1 to the Golgi complex. There is also massive recruitment of SEC31 to endoplasmic-reticulum exit sites. Furthermore, absence of PHB decreases the levels of the Golgi-localised KDELR, thus preventing KDELR-dependent activation of Golgi-Src and inhibiting Golgi-to-plasma-membrane transport of VSVG. We propose a model whereby in analogy to previous findings (e.g., the RAS-RAF signalling pathway), PHB can act as a signalling scaffold protein to assist in KDELR-dependent Src activation.

VE-cadherin is a component of endothelial cell-to-cell adherens junctions, and it has a key role in the maintenance of vascular integrity. During embryo development, VE-cadherin is required for the organization of a stable vascular system, and in the adult it controls vascular permeability and inhibits unrestrained vascular growth. The mechanisms of action of VE-cadherin are complex and include reshaping and organization of the endothelial cell cytoskeleton and modulation of gene transcription. Here we review some of the most important pathways through which VE-cadherin modulates vascular homeostasis and discuss the emerging concepts in the overall biological role of this protein.

Membrane trafficking involves large fluxes of cargo and membrane across separate compartments. These fluxes must be regulated by control systems to maintain homoeostasis. While control systems for other key functions such as protein folding or the cell cycle are well known, the mechanisms that control secretory transport are poorly understood. We have previously described a signalling circuit operating at the Golgi complex that regulates intra-Golgi trafficking and is initiated by the KDEL receptor (KDEL-R), a protein previously known to mediate protein recycling from the Golgi to the endoplasmic reticulum (ER). Here, we investigated the KDEL-R signalling mechanism. We show that the KDEL-R is predicted to fold like a G-protein-coupled receptor (GPCR), and that it binds and activates the heterotrimeric signalling G-protein Gαq/11 which, in turn, regulates transport through the Golgi complex. These findings reveal an unexpected GPCR-like mode of action of the KDEL-R and shed light on a core molecular control mechanism of intra-Golgi traffic.

Over the last decade, accumulating data have demonstrated the presence of ‘classical’ signalling molecules on endomembranes, including endosomes and the Golgi complex. It is now clear that through these signalling molecules, endomembranes can serve two functions: one is to elaborate and relay signalling initiated at the plasma membrane, and the other is to initiate new signalling in response to stimuli originating from the endomembranes themselves. Here, we have examined this emerging paradigm, with particular emphasis on a novel Golgi-based signalling cascade. This system senses, and is activated by, endoplasmic reticulum chaperones that arrive at the Golgi complex during constitutive secretion; these bind to the KDEL receptor and activate the Src family kinases. These, in turn, positively regulate the intra-Golgi transport machinery. This system thus coordinates the initiating process, the membrane input, with an increased membrane output. In more general terms, it responds with signals to an endogenous event. In this respect, it is similar to the unfolded protein response, a complex cell reaction to the accumulation of unfolded proteins in the endoplasmic reticulum, that is, to our knowledge the only other examples of inter-organelle signalling initiated within the cell. However, in contrast to the Golgi-based signalling pathway, this unfolded protein signalling is generally activated by pathological conditions. We propose that inter-organelle signalling is a mechanism by which different compartments of eukaryotic cells coordinate their functions.

Mesoangioblasts are vessel-associated progenitor cells that show therapeutic promise for the treatment of muscular dystrophy. Mesoangioblasts have the ability to undergo skeletal muscle differentiation and cross the blood vessel wall regardless of the developmental stage at which they are isolated. Here we show that PW1/Peg3 is expressed at high levels in mesoangioblasts obtained from mouse, dog and human tissues and its level of expression correlates with their myogenic competence. Silencing PW1/Peg3 markedly inhibits myogenic potential of mesoangioblasts in vitro through MyoD degradation. Moreover, lack of PW1/Peg3 abrogates mesoangioblast ability to cross the vessel wall and to engraft into damaged myofibres through the modulation of the junctional adhesion molecule-A. We conclude that PW1/Peg3 function is essential for conferring proper mesoangioblast competence and that the determination of PW1/Peg3 levels in human mesoangioblasts may serve as a biomarker to identify the best donor populations for therapeutic application in muscular dystrophies., Mesoangioblasts are mesodermal stem cells with a therapeutic potential for treatment of muscular dystrophy due to their ability to differentiate into skeletal muscle. This study shows that the PW1/Peg3 protein is crucial for mesoangioblast myogenic and migratory potency and is a therapeutically relevant biomarker.

Protein mono-ADP-ribosylation is a reversible post-translational modification of cellular proteins. This scheme of amino-acid modification is used not only by bacterial toxins to attack host cells, but also by endogenous ADP-ribosyltransferases (ARTs) in mammalian cells. These latter ARTs include members of three different families of proteins: the well characterised arginine-specific ecto-enzymes (ARTCs), two sirtuins, and some members of the poly(ADP-ribose) polymerase (PARP/ARTD) family. In the present study, we demonstrate that human ARTC1 is localised to the endoplasmic reticulum (ER), in contrast to the previously characterised ARTC proteins, which are typical GPI-anchored ecto-enzymes. Moreover, using the “macro domain” cognitive binding module to identify ADP-ribosylated proteins, we show here that the ER luminal chaperone GRP78/BiP (glucose-regulated protein of 78 kDa/immunoglobulin heavy-chain-binding protein) is a cellular target of human ARTC1 and hamster ARTC2. We further developed a procedure to visualise ADP-ribosylated proteins using immunofluorescence. With this approach, in cells overexpressing ARTC1, we detected staining of the ER that co-localises with GRP78/BiP, thus confirming that this modification occurs in living cells. In line with the key role of GRP78/BiP in the ER stress response system, we provide evidence here that ARTC1 is activated during the ER stress response, which results in acute ADP-ribosylation of GRP78/BiP paralleling translational inhibition. Thus, this identification of ARTC1 as a regulator of GRP78/BiP defines a novel, previously unsuspected, player in GRP78-mediated ER stress responses.

Classical signal transduction is initiated at the plasma membrane by extracellular signals and propagates to the cytosolic face of the same membrane. Multiple studies have shown that endomembranes can act as signaling platforms for this plasma-membrane-originated signaling. Recent evidence has indicated that endomembranes can also trigger their own signaling cascades that involve some of the molecular players that are classically engaged in signal transduction at the plasma membrane. Endomembrane-initiated signaling is important for synchronization of the functioning of the secretory pathway and coordination of the activities of the secretory organelles with other cellular machineries. However, these endomembrane-initiated regulatory circuits are only partially understood to date. This novel field is slowed by a lack of specific tools and the objective difficulties in the study of signal transduction of endomembrane-localized receptors, as their accessibility is limited. For example, the ligand-binding site of the KDEL receptor (that transduces endomembrane signaling) is positioned in the lumen of the Golgi complex. Here we report some approaches that are suitable for the study of endomembrane-initiated signaling.

SummaryA fundamental property of cellular processes is to maintain homeostasis despite varying internal and external conditions. Within the membrane transport apparatus, variations in membrane fluxes from the endoplasmic reticulum (ER) to the Golgi complex are balanced by opposite fluxes from the Golgi to the ER to maintain homeostasis between the two organelles. Here we describe a molecular device that balances transport fluxes by integrating transduction cascades with the transport machinery. Specifically, ER-to-Golgi transport activates the KDEL receptor at the Golgi, which triggers a cascade that involves Gs and adenylyl cyclase and phosphodiesterase isoforms and then PKA activation and results in the phosphorylation of transport machinery proteins. This induces retrograde traffic to the ER and balances transport fluxes between the ER and Golgi. Moreover, the KDEL receptor activates CREB1 and other transcription factors that upregulate transport-related genes. Thus, a Golgi-based control system maintains transport homeostasis through both signaling and transcriptional networks.

Classical signal transduction is initiated at the plasma membrane by extracellular signals and propagates to the cytosolic face of the same membrane. Multiple studies have shown that endomembranes can act as signaling platforms for this plasma-membrane-originated signaling. Recent evidence has indicated that endomembranes can also trigger their own signaling cascades that involve some of the molecular players that are classically engaged in signal transduction at the plasma membrane. Endomembrane-initiated signaling is important for synchronization of the functioning of the secretory pathway and coordination of the activities of the secretory organelles with other cellular machineries. However, these endomembrane-initiated regulatory circuits are only partially understood to date. This novel field is slowed by a lack of specific tools and the objective difficulties in the study of signal transduction of endomembrane-localized receptors, as their accessibility is limited. For example, the ligand-binding site of the KDEL receptor (that transduces endomembrane signaling) is positioned in the lumen of the Golgi complex. Here we report some approaches that are suitable for the study of endomembrane-initiated signaling.

AIMS: Establishing biochemical markers of pre-hypertension and early hypertension could help earlier diagnostics and therapeutic intervention. We assess dynamics of junctional adhesion molecule-A (JAM-A) expression in rat models of hypertension and test whether JAM-A expression could be driven by angiotensin (ANG) II and whether JAM-A contributes to the progression of hypertension. We also compare JAM-A expression in normo- and hypertensive humans.METHODS AND RESULTS: In pre-hypertensive and spontaneously hypertensive rats (SHRs), JAM-A protein was overexpressed in the brainstem microvasculature, lung, liver, kidney, spleen, and heart. JAM-A upregulation at early and late stages was even greater in the stroke-prone SHR. However, JAM-A was not upregulated in leucocytes and platelets of SHRs. In Goldblatt 2K-1C hypertensive rats, JAM-A expression was augmented before any increase in blood pressure, and similarly JAM-A upregulation preceded hypertension caused by peripheral and central ANG II infusions. In SHRs, ANG II type 1 (AT(1)) receptor antagonism reduced JAM-A expression, but the vasodilator hydralazine did not. Body-wide downregulation of JAM-A with Vivo-morpholinos in juvenile SHRs delayed the progression of hypertension. In the human saphenous vein, JAM-A mRNA was elevated in hypertensive patients with untreated hypertension compared with normotensive patients but reduced in patients treated with renin-angiotensin system antagonists.CONCLUSION: Body-wide upregulation of JAM-A in genetic and induced models of hypertension in the rat precedes the stable elevation of arterial pressure. JAM-A upregulation may be triggered by AT(1) receptor-mediated signalling. An association of JAM-A with hypertension and sensitivity to blockers of ANG II signalling were also evident in humans. We suggest a prognostic and possibly a pathogenic role of JAM-A in arterial hypertension.

Membrane trafficking involves large fluxes of cargo and membrane across separate compartments. These fluxes must be regulated by control systems to maintain homoeostasis. While control systems for other key functions such as protein folding or the cell cycle are well known, the mechanisms that control secretory transport are poorly understood. We have previously described a signalling circuit operating at the Golgi complex that regulates intra-Golgi trafficking and is initiated by the KDEL receptor (KDEL-R), a protein previously known to mediate protein recycling from the Golgi to the endoplasmic reticulum (ER). Here, we investigated the KDEL-R signalling mechanism. We show that the KDEL-R is predicted to fold like a G-protein-coupled receptor (GPCR), and that it binds and activates the heterotrimeric signalling G-protein Gα(q/11) which, in turn, regulates transport through the Golgi complex. These findings reveal an unexpected GPCR-like mode of action of the KDEL-R and shed light on a core molecular control mechanism of intra-Golgi traffic.

As with other complex cellular functions, intracellular membrane transport involves the coordinated engagement of a series of organelles and machineries; however, the molecular basis of this coordination is unknown. Here we describe a Golgi-based signalling system that is activated by traffic and is involved in monitoring and balancing trafficking rates into and out of the Golgi complex. We provide evidence that the traffic signal is due to protein chaperones that leave the endoplasmic reticulum and reach the Golgi complex where they bind to the KDEL receptor. This initiates a signalling reaction that includes the activation of a Golgi pool of Src kinases and a phosphorylation cascade that in turn activates intra-Golgi trafficking, thereby maintaining the dynamic equilibrium of the Golgi complex. The concepts emerging from this study should help to understand the control circuits that coordinate high-order cellular functions.