Extracellular Role Research Articles

Role of HMGB1 in TLR3-mediated inflammation (180.10)

Abstract Nuclear high mobility group box 1 (HMGB1), a DNA binding protein, belongs to the superfamily of HMG proteins and mediates structural and functional changes to chromatin. HMGB1 can be released either passively by necrotic cells or actively by stimulated cells. The extracellular role of HMGB1 as a cytokine has been intensively studied and shown to be important as a danger-associated molecular pattern protein. HMGB1 co-operates with other pro-inflammatory molecules to induce inflammation. HMGB1 forms complexes with various Toll-like receptor (TLR) ligands of LPS and CpG, enhancing their cytokine-inducing function. In this study, we demonstrated that HMGB1 binds to TLR3 both directly and indirectly in cooperation with poly(I:C), using immunoprecipitation and surface plasmon resonance assays. Exogenous HMGB1 treatment to HEK293/TLR3 cells was co-localized in intracytoplasmic endosomes containing TLR3 followed by NF-κB activation. In conclusion, HMGB1 is a novel ligand of TLR3 and is involved in TLR3-mediated inflammation.

Granzyme B Cleaves Decorin, Biglycan and Soluble Betaglycan, Releasing Active Transforming Growth Factor-β1

ObjectiveGranzyme B (GrB) is a pro-apoptotic serine protease that contributes to immune-mediated target cell apoptosis. However, during inflammation, GrB accumulates in the extracellular space, retains its activity, and is capable of cleaving extracellular matrix (ECM) proteins. Recent studies have implicated a pathogenic extracellular role for GrB in cardiovascular disease, yet the pathophysiological consequences of extracellular GrB activity remain largely unknown. The objective of this study was to identify proteoglycan (PG) substrates of GrB and examine the ability of GrB to release PG-sequestered TGF-β1 into the extracellular milieu.Methods/ResultsThree extracellular GrB PG substrates were identified; decorin, biglycan and betaglycan. As all of these PGs sequester active TGF-β1, cytokine release assays were conducted to establish if GrB-mediated PG cleavage induced TGF-β1 release. Our data confirmed that GrB liberated TGF-β1 from all three substrates as well as from endogenous ECM and this process was inhibited by the GrB inhibitor 3,4-dichloroisocoumarin. The released TGF-β1 retained its activity as indicated by the induction of SMAD-3 phosphorylation in human coronary artery smooth muscle cells.ConclusionIn addition to contributing to ECM degradation and the loss of tissue structural integrity in vivo, increased extracellular GrB activity is also capable of inducing the release of active TGF-β1 from PGs.

Matrix architecture defines the preferential localization and migration of T cells into the stroma of human lung tumors

Appropriate localization and migration of T cells is a prerequisite for antitumor immune surveillance. Studies using fixed tumor samples from human patients have shown that T cells accumulate more efficiently in the stroma than in tumor islets, but the mechanisms by which this occurs are unknown. By combining immunostaining and real-time imaging in viable slices of human lung tumors, we revealed that the density and the orientation of the stromal extracellular matrix likely play key roles in controlling the migration of T cells. Active T cell motility, dependent on chemokines but not on β1 or β2 integrins, was observed in loose fibronectin and collagen regions, whereas T cells migrated poorly in dense matrix areas. Aligned fibers in perivascular regions and around tumor epithelial cell regions dictated the migratory trajectory of T cells and restricted them from entering tumor islets. Consistently, matrix reduction with collagenase increased the ability of T cells to contact cancer cells. Thus, the stromal extracellular matrix influences antitumor immunity by controlling the positioning and migration of T cells. Understanding the mechanisms by which this collagen network is generated has the potential to aid in the development of new therapeutics.

15-Lipoxygenase eicosanoids are the putative ligands for vanilloid receptors and peroxisome proliferator-activated receptors (PPARs)

We read the article by Zhao et al. (1), which showed that epithelial 15-lipoxygenase 1 (15LO1) and its product 15-hydroxyeicosatetraenoic acid phosphatidylethanolamine interact with phosphatidylethanolamine-binding protein (PEBP1) to enhance MAPK signaling (ERK activation) in human airway epithelia. This can then be seen as an internal mechanism in airway epithelium for adjusting the gain settings of critical inflammatory pathways integral to asthma pathogenesis. However, this model rests critically on three assumptions: (i) Epithelial 15LO1 is the major form of 15LO1 and the source of 15S-hydroxyeicosatetraenoic acid (15S-HETE), (ii) metabolites or eicosanoids of 15LO1 such as 15S-HETE are not released extracellularly, and (iii) these eicosanoids do not influence MAPK signaling through any other receptor. The authors explicitly stated that “15HETE is not released extracellularly” and that “unlike other eicosanoids, 15LO1 does not appear to function through receptor-mediated mechanisms, and, indeed, no receptor has been identified.” In this model, all these signaling events (15LO1–15-hydroxyeicosatetraenoic acid-phosphatidylethanolamine-ERK) are direct and exclusively occur inside the airway epithelia. This is potentially misleading. First, a minor discrepancy in the data, whereby 15LO1 was nearly undetectable in fresh bronchial epithelia isolated from normal and air–liquid interface (ALI)-cultured human bronchial epithelial cells without IL-13 induction [figures 1a and 2b (1)] but strongly expressed even without IL-13 stimulation in other supposed ALI epithelial cultures [figure 6a (1)], could be secondary to possible contamination of ALI cultures with macrophages. It has been reported that macrophages are also important sources of 15LO1 and 15(S)-HETE in the airway, with uptake and incorporation of 15(S)-HETE by epithelial cells (2). As their Materials and Methods (1) did not report the use of any technique to confirm the purity of isolated airway epithelia or cultured epithelia and to exclude macrophage contamination, we wonder if this possibility has been excluded. Second, eicosanoids of 15LO1, including 15S-HETE, have been found in biological fluids such as BAL fluid supernatants and sputum supernatants (2), indicating they also could be secretory with an extracellular role. Third, eicosanoids of 15LO directly bind with vanilloid receptors and PPARs (3, 4), indicating that 15S-HETE also could lead to receptor-mediated signaling for MAPK activation. TRPV1, one of the vanilloid receptors that may be important in refractory asthma, was found to be expressed in bronchial epithelia rather than macrophages, which indicates a possible crosstalk between macrophage and epithelium through secreted 15S-HETE from macrophages. Indeed, 15S-HETE has been considered as a 15LO2 metabolite rather than a 15LO1 metabolite (13S-HODE) (4), which is contradictory to the title of the article by Zhao et al. (1). Exogenous induction with metabolites of 15LO1 and 15LO2 differentially regulate MAPK signaling involving PPAR (4).

DJ-1 promotes angiogenesis and osteogenesis by activating FGF receptor-1 signaling

Communication between osteoblasts and endothelial cells is essential for bone fracture repair, but the molecular identities of such communicating factors are not well defined. Here we identify DJ-1 as a novel mediator of the cross-talk between osteoblasts and endothelial cells through an unbiased screening of molecules secreted from human mesenchymal stem cells during osteogenesis. We show that DJ-1 stimulates the differentiation of human mesenchymal stem cells to osteoblasts and that DJ-1 induces angiogenesis in endothelial cells through activation of fibroblast growth factor receptor-1 signalling. In a rodent model of bone fracture repair, extracellular application of DJ-1 enhances bone regeneration in vivo by stimulating the formation of blood vessels and new bones. Both these effects are blocked by antagonizing fibroblast growth factor receptor-1 signalling. These findings uncover previously undefined extracellular roles of DJ-1 to promote angiogenesis and osteogenesis, suggesting DJ-1 may have therapeutic potential to stimulate bone regeneration.

The impact of extracellular syntaxin4 on HaCaT keratinocyte behavior

Syntaxin4 belongs to t-SNARE protein family and functions as a vesicular fusion mediator in the plasma membrane in a wide variety of cell types. This protein resembles another family member, epimorphin, a subpopulation of which has been shown to be secreted extracellularly in order to exert signaling functions. Here, we demonstrate the secretion of syntaxin4 via a non-classical pathway and its extracellular functions by using the functionally normal keratinocyte HaCaT. Extracellularly presented syntaxin4 appeared to elicit many cell responses similar to epimorphin with an important exception: it clearly facilitated keratinocyte cornification. The circularized peptide ST4n1 was synthesized from the putative functional core of syntaxin4 (a.a. 103–108), which is equivalent to the previously generated antagonist of epimorphin, and neutralized this contradictory effect. Intriguingly, an epimorphin mutant (EP4M) in which the functional core was replaced by that of syntaxin4 behaved like epimorphin, which was again antagonized by ST4n1. Electrophoresis-based analyses demonstrated the distinct structure of syntaxin4 compared to epimorphin or EP4M. These results revealed, for the first time, the extracellular role of syntaxin4 and shed light on the division of the extracellular effects exerted by epimorphin and syntaxin4 on keratinocyte cornification.

Role of CD1A and HSP60 in the antitumoral response of oesophageal cancer

Oesophageal cancer (OC) is one of the most common and severe forms of tumor. A wider knowledge of molecular mechanisms which lead to a normal epithelium becoming a neoplasm may reveal new strategies to improve treatment and outcome of this disease. In this review, we report recent findings concerning molecular events which take place during carcinogenesis of the oesophagus. In particular, we focus on the role of two molecules, CD1a and Hsp60, which are overexpressed in oesophageal and many other types of tumor. Both molecules may present tumor antigens and promote in situ the stimulation of an antitumoral immune activity. We suggest there is a synergistic action between these molecules. Further knowledge about their intracellular pathways and extracellular roles may help develop new antitumoral tools for OC.

Heparan Sulfate Is Essential for High Mobility Group Protein 1 (HMGB1) Signaling by the Receptor for Advanced Glycation End Products (RAGE)

In a proteomic search for heparan sulfate-binding proteins on monocytes, we identified HMGB1 (high mobility group protein B1). The extracellular role of HMGB1 as a cytokine has been studied intensively and shown to be important as a danger-associated molecular pattern protein. Here, we report that the activity of HMGB1 depends on heparan sulfate. Binding and competition studies demonstrate that HMGB1 interacts with CHO and endothelial cell heparan sulfate. By site-directed mutagenesis, we identified a loop region that connects the A-box and B-box domains of HMGB1 as responsible for heparan sulfate binding. HMGB1-induced Erk1/2 and p38 phosphorylation is abolished when endothelial heparan sulfate is removed or blocked pharmacologically, resulting in decreased HMGB1-induced endothelial sprouting. However, mutated HMGB1 that lacks the heparan sulfate-binding site retained its signaling activity. We show the major receptor for HMGB1, receptor for advanced glycation end products (RAGE), also binds to heparan sulfate and that RAGE and heparan sulfate forms a complex. Our data establishes that the functional receptor for HMGB1 consists of a complex of RAGE and cell surface heparan sulfate.

Specific Protein Patterns Characterize Metastatic Potential of Advanced Bladder Cancer

The prognosis in patients with metastasized bladder cancer is still poor. Clinical and histopathological parameters have limited ability to predict the risk of tumor progression. Thus, we identified specific protein patterns associated with tumor progression to differentiate specimens with and without metastasis. We analyzed 46 metastasized and 42 nonmetastasized muscle invasive bladder cancers by ProteinChip® technology surface enhanced laser desorption/ionization time of flight mass spectrometry. Cell lysis was done after laser capture microdissection from cryostat sections to achieve high tumor cell purity. Surface enhanced laser desorption/ionization time of flight mass spectrometry was completed with 2 matrices (Q10 and CM10). Bioinformatic analysis was performed by XLMiner® clustering using the Fuzzy c-means method. Differentially expressed proteins were identified and verified by 2-dimensional gel electrophoresis, tryptic in gel digest, peptide mapping, immunodepletion assay and Western blot analysis. By combining data on 2 chip surfaces (Q10 and CM10) results showed 86% sensitivity and 89% specificity in the training set, and 63% sensitivity and 88% specificity in the validation set. The relevant protein peaks 10.83, 14.68, 16.15 and 27.85 Da were identified as S100A8, MAP-1LC3, MUC-1S1 and GST-M1, respectively. We defined specific protein patterns with ProteinChip technology using bioinformatic evaluation software, which allowed differentiation between nonmetastasized and metastasized bladder tumor samples with high sensitivity and specificity. We identified 4 differentially expressed proteins. Thus, it seems possible to identify patients at high metastasized risk even at a clinically localized stage, leading to individual therapy decisions.

S100A14 Stimulates Cell Proliferation and Induces Cell Apoptosis at Different Concentrations via Receptor for Advanced Glycation End Products (RAGE)

S100A14 is an EF-hand containing calcium-binding protein of the S100 protein family that exerts its biological effects on different types of cells. However, exact extracellular roles of S100A14 have not been clarified yet. Here we investigated the effects of S100A14 on esophageal squamous cell carcinoma (ESCC) cell lines. Results demonstrated that low doses of extracellular S100A14 stimulate cell proliferation and promote survival in KYSE180 cells through activating ERK1/2 MAPK and NF-κB signaling pathways. Immunoprecipitation assay showed that S100A14 binds to receptor for advanced glycation end products (RAGE) in KYSE180 cells. Inhibition of RAGE signaling by different approaches including siRNA for RAGE, overexpression of a dominant-negative RAGE construct or a RAGE antagonist peptide (AmphP) significantly blocked S100A14-induced effects, suggesting that S100A14 acts via RAGE ligation. Furthermore, mutation of the N-EF hand of S100A14 (E39A, E45A) virtually reduced 10 µg/ml S100A14-induced cell proliferation and ERK1/2 activation. However, high dose (80 µg/ml) of S100A14 causes apoptosis via the mitochondrial pathway with activation of caspase-3, caspase-9, and poly(ADP-ribose) polymerase. High dose S100A14 induces cell apoptosis is partially in a RAGE-dependent manner. This is the first study to demonstrate that S100A14 binds to RAGE and stimulates RAGE-dependent signaling cascades, promoting cell proliferation or triggering cell apoptosis at different doses.

Guanosine is neuroprotective against oxygen/glucose deprivation in hippocampal slices via large conductance Ca2+-activated K+ channels, phosphatidilinositol-3 kinase/protein kinase B pathway activation and glutamate uptake

Guanine derivatives (GD) have been implicated in many relevant brain extracellular roles, such as modulation of glutamate transmission and neuronal protection against excitotoxic damage. GD are spontaneously released to the extracellular space from cultured astrocytes and during oxygen/glucose deprivation (OGD). The aim of this study has been to evaluate the potassium channels and phosphatidilinositol-3 kinase (PI3K) pathway involvement in the mechanisms related to the neuroprotective role of guanosine in rat hippocampal slices subjected to OGD. The addition of guanosine (100 μM) to hippocampal slices subjected to 15 min of OGD and followed by 2 h of re-oxygenation is neuroprotective. The presence of K+ channel blockers, glibenclamide (20 μM) or apamin (300 nM), revealed that neuroprotective effect of guanosine was not dependent on ATP-sensitive K+ channels or small conductance Ca2+-activated K+ channels. The presence of charybdotoxin (100 nM), a large conductance Ca2+-activated K+ channel (BK) blocker, inhibited the neuroprotective effect of guanosine. Hippocampal slices subjected to OGD and re-oxygenation showed a significant reduction of glutamate uptake. Addition of guanosine in the re-oxygenation period has blocked the reduction of glutamate uptake. This guanosine effect was inhibited when hippocampal slices were pre-incubated with charybdotoxin or wortmanin (a PI3K inhibitor, 1 μM) in the re-oxygenation period. Guanosine promoted an increase in Akt protein phosphorylation. However, the presence of charybdotoxin blocked such effect. In conclusion, the neuroprotective effect of guanosine involves augmentation of glutamate uptake, which is modulated by BK channels and the activation of PI3K pathway. Moreover, neuroprotection caused by guanosine depends on the increased expression of phospho-Akt protein.

Extracellular Trypanosoma cruzi calreticulin in the host–parasite interplay

Calreticulin (CRT) from vertebrates is a calcium-binding protein present mainly in the endoplasmic reticulum (ER). There, it directs the conformation of proteins and controls calcium levels. This review will focus on several extracellular roles of Trypanosoma cruzi CRT (TcCRT) in relation to its capacity to inhibit the complement system, mediate parasite infectivity, interfere with angiogenesis and, as a possible consequence, with tumor growth. The TcCRT antiangiogenic effect parallels with the capacity of T. cruzi infection to inhibit tumor development in vivo. Thus, the TcCRT, complement, and endothelial cell interactions seem to be an evolutionary adaptation to promote prolonged parasite-host relationships.

HMGB1 promotes lymphangiogenesis of human lymphatic endothelial cells in vitro

High-mobility group box 1 (HMGB1), a proinflammatory cytokine, plays an important role in inflammatory diseases, including severe sepsis and arthritis. This recent discovery of the extracellular role of HMGB1 as a multifunctional cytokine involved in tumorigenesis, tumor angiogenesis as well as metastasis has opened up a new field of research to study the role of HMGB1 in tumors. However, its molecular mechanism in lymphangiogenesis remains poorly understood. In this study, human lymphatic endothelial cells (LECs) were treated with human recombinant HMGB1 (rHMGB1) and recombinant VEGF-C (rVEGF-C). Changes in cell proliferation, migration, and the capillary-like tube formation were assessed by MTT assay, transwell chamber assay, and a Matrigel model, respectively. Human rHMGB1 induced LEC proliferation, migration, and tube formation in a dose- and time-dependent manner with the maximal effect at a concentration of 2 μg/ml. As a specific lymphangiogenes factor, the role of rVEGF-C in promoting lymphangiogenesis was significant. These data indicate, for the first time, that HMGB1 might contribute to tumor lymphangiogenesis and lymphatic metastasis, and it might ultimately represent a therapeutic target in tumor patients.

Temporal and Spatial Characterization of Hyaluronic Acid and Associated Transcriptional Activity During Early Porcine Embryo Development.

Hyaluronic acid (HA), a member of the glycoaminoglycan family, has been shown to have positive effects on in vitro embryo development in mice, cows, and pigs. Furthermore, its addition to culture media has been shown to increase embryo development following transfer in mice and improve pregnancy rates resulting from embryo transfer in human populations with multiple previous failures. However, the mechanisms by which HA affects early embryo development is yet unknown, a situation further complicated by changing effects in response to both the concentration and molecular weight of the HA used. The objective of this experiment was to determine the normal status of HA within the porcine reproductive tract and resolve any temporal and spatial variations within the system which may affect embryo viability up to the blastocyst stage. Primiparous sows (n=21), on their second estrus following weaning, were randomly designated for sample collection at seven periods corresponding to the germinal vesicle (GV), metaphase II (MII), 2cell, 4cell, early morula, morula, and blastocyst stages of embryonic development. Timing of collection for each the post-ovulatory stages was based on detection of ovulation by real time transcutaneous ovarian ultrasound at 4 hour intervals; while the staging of the GV group was based on detection of standing estrus. A terminal hysterectomy was performed, and flushing of the oviduct and lower, middle, and upper thirds of the uterine horns relative to cervix carried out. Tissue samples from each of the uterine sections along with the ampula, isthmus and ampulary-ismic junction were collected and flash frozen in liquid nitrogen, while embryos were isolated from the flushings, imaged and frozen in pools of five embryos at like stages. HA content in each segment was determined through quantitative ELISA and concentrations adjusted to account for flush volume. Total RNA was extracted from tissue samples and embryos, DNase treated, and analyzed using real time PCR for determination of relative gene expression of HAS1, 2 and 3 and CD44 using 18S as an endogenous control. Expression was analysed using the ΔCT method for endometrial samples, and the residuals of 18S as a covariate for embryos. ELISA data was log transformed for normality and all data was analysed for temporal and spatial variation using the general linear model of SAS. Minimal spatial variation was found in the post-ovulatory stages, however temporal differences in HA concentration and gene expression were identified. ELISA results show elevated concentrations of HA at the GV stage that decrease rapidly at the 2cell stage and then increase toward the blastocyst stage. HAS1 and HAS3 were identified as the predominantly transcribed synthase forms in both the reproductive tract and embryos, while low level expression of HAS2 was detected in the reproductive tract irrespective of embryonic stage. Synthase gene expression appears to be reciprocal between the uterus and embryos, with highest expression peaking at the GV and 4 cell stages respectively. These results suggest HA does not play a significant role in the development of spatial asynchrony in the reproductive tract, but may play a significant role in temporal asynchrony. Furthermore, the complex interplay between embryonic and uterine expression profiles suggests an extracellular signaling role for HA during early development. (poster)

Matrix Metalloproteinase-9 Is Upregulated in Nucleophosmin-Anaplastic Lymphoma Kinase–Positive Anaplastic Lymphomas and Activated at the Cell Surface by the Chaperone Heat Shock Protein 90 to Promote Cell Invasion

Many anaplastic large cell lymphomas (ALCL) express the chimeric oncogene NPM-ALK, which drives malignant transformation and invasion. In this study, we show that NPM-ALK expression increases matrix metalloproteinase-9 (MMP-9) expression. Accordingly, we found that 100% of a large panel of ALK(+) ALCL biopsies examined were also MMP-9(+), in contrast to only 36.3% of ALK(-) tumors. Mechanistic studies revealed that Rac1 drove MMP-9 secretion. The MMP inhibitor GM6001 and MMP-9 blocking antibodies abolished the invasiveness of NPM-ALK(+) cells. Interestingly, the hyaluronan receptor CD44 acted as a docking surface for MMP-9 and the chaperone heat shock protein 90 on the cell surface, where MMP-9 was cleaved and activated. Membrane-associated MMP-9 was localized to invadopodia, which display a strong gelatinase activity. Taken together, our observations strengthen the concept that chaperones have a major extracellular role in the regulation of protein activation status, and reveal new factors that are crucial for spreading and invasion of ALK(+) ALCL. They also point out new factors crucial for ALK(+) ALCL.

The HMGB protein gene family in zebrafish: Evolution and embryonic expression patterns

The High-Mobility Group Box (HMGB) proteins are highly abundant proteins with both nuclear and extracellular roles in key biological processes. In mammals, three family members are present: HMGB1, HMGB2 and HMGB3. We characterized the HMGB family in zebrafish and report a detailed phylogenetic analysis of HMGB proteins. The B1, B2, and B3 subfamilies are present in cartilaginous fish, bony fish, and tetrapods, while jawless fish sequences emerge as basal to the gene family expansion. Two co-orthologs of each mammalian HMGB gene are present in zebrafish. All six zebrafish hmgb genes are maternally expressed, but huge differences in expression levels exist during embryonic development. The hmgb2a/hmgb2b genes are the most highly expressed, while hmgb3b is expressed at the lowest level. Remarkably, hmgb3 genes are not present in fugu, medaka, Tetraodon and stickleback. Our analysis highlights substantial overlaps, but also subtle differences and specificities in the expression patterns of the zebrafish hmgb genes.

The C-terminal acidic tail is responsible for the inhibitory effects of HMGB1 on efferocytosis

HMGB1 was described originally as a nuclear protein involved in DNA binding and transcriptional regulation. However, HMGB1 also has an extracellular role as a potent mediator of inflammation and can diminish the uptake of apoptotic cells by phagocytes, a process called efferocytosis. To explore the mechanism responsible for the ability of HMGB1 to inhibit efferocytosis, we examined the role of the C-terminal acidic tail, a region of HMGB1 that has been shown to participate in specific intramolecular interactions. Deletion of the C-terminal tail abrogated the ability of HMGB1 to decrease murine macrophage ingestion of apoptotic neutrophils and to diminish phagocytosis-induced activation of Erk and Rac-1 in macrophages. We found that RAGE plays a major role in efferocytosis, and deletion of the C-terminal tail of HMGB1 prevented binding of HMGB1 to RAGE but not to other macrophage receptors involved in efferocytosis, such as the α(V)β(3) integrin. Whereas HMGB1 decreased ingestion of apoptotic neutrophils significantly by alveolar macrophages under in vivo conditions in the lungs of mice, this effect was lost when the C-terminal acidic tail was absent from HMGB1. These results demonstrate that the HMGB1 C-terminal tail is responsible for the inhibitory effects of HMGB1 on phagocytosis of apoptotic neutrophils under in vitro and in vivo conditions.

Intracellular and Extracellular Effects of S100B in the Cardiovascular Response to Disease

S100B, a calcium-binding protein of the EF-hand type, exerts both intracellular and extracellular functions. S100B is induced in the myocardium of human subjects and an experimental rat model following myocardial infarction. Forced expression of S100B in neonatal rat myocyte cultures and high level expression of S100B in transgenic mice hearts inhibit cardiac hypertrophy and the associated phenotype but augments myocyte apoptosis following myocardial infarction. By contrast, knocking out S100B, augments hypertrophy, decreases apoptosis and preserves cardiac function following myocardial infarction. Expression of S100B in aortic smooth muscle cells inhibits cell proliferation and the vascular response to adrenergic stimulation. S100B induces apoptosis by an extracellular mechanism via interaction with the receptor for advanced glycation end products and activating ERK1/2 and p53 signaling. The intracellular and extracellular roles of S100B are attractive therapeutic targets for the treatment of both cardiac and vascular diseases.

Crystal Structure of the 2-Oxoglutarate- and Fe(II)-Dependent Lysyl Hydroxylase JMJD6

Lysyl and prolyl hydroxylations are well-known post-translational modifications to animal and plant proteins with extracellular roles. More recent work has indicated that the hydroxylation of intracellular animal proteins may be common. JMJD6 catalyses the iron- and 2-oxoglutarate-dependent hydroxylation of lysyl residues in arginine–serine-rich domains of RNA-splicing-related proteins. We report crystallographic studies on the catalytic domain of JMJD6 in complex with Ni(II) substituting for Fe(II). Together with mutational studies, the structural data suggest how JMJD6 binds its lysyl residues such that it can catalyse C-5 hydroxylation rather than Nɛ-demethylation, as for analogous enzymes.

Distribution of NTPDase5 and NTPDase6 and the regulation of P2Y receptor signalling in the rat cochlea

Membrane-bound ectonucleoside triphosphate diphosphohydrolases (E-NTPDases) in the inner ear regulate complex extracellular purinergic type-2 (P2) receptor signalling pathways through hydrolysis of extracellular nucleoside 5'-triphosphates and diphosphates. This study investigated the distribution of NTPDase5 and NTPDase6, two intracellular members of the E-NTPDase family, and linked this to regulation of P2 receptor signalling in the adult rat cochlea. These extracellular ectonucleotidases preferentially hydrolyse nucleoside 5'-diphosphates such as UDP and GDP. Expression of both enzymes at mRNA and protein level was detected in cochlear tissues and there was in vivo release of soluble NTPDase5 and 6 into cochlear fluids. Strong NTPDase5 immunostaining was found in the spiral ganglion neurones and supporting Deiters' cells of the organ of Corti, while NTPDase6 was confined to the inner hair cells. Upregulation of NTPDase5 after exposure to loud sound indicates a dynamic role for NTPDase5 in cochlear response to stress, whereas NTPDase6 may have more limited extracellular roles. Noise-induced upregulation of co-localised UDP-preferring P2Y(6) receptors in the spiral ganglion neurons further supports the involvement of NTPDase5 in regulation of P2Y receptor signalling. Noise stress also induced P2Y(14) (UDP- and UDP-glucose preferring) receptor expression in the root processes of the outer sulcus cells, but this was not associated with localization of the E-NTPDases.