Interactions with Asialo-Glycoprotein Receptors and Platelets Are Dispensable for CD8+ T Cell Localization in the Murine Liver.

Liver-resident CD8+ T cells can play critical roles in the control of pathogens, including Plasmodium and hepatitis B virus. Paradoxically, it has also been proposed that the liver may act as the main place for the elimination of CD8+ T cells at the resolution of immune responses. We hypothesized that different adhesion processes may drive residence versus elimination of T cells in the liver. Specifically, we investigated whether the expression of asialo-glycoproteins (ASGPs) drives the localization and elimination of effector CD8+ T cells in the liver, while interactions with platelets facilitate liver residence and protective function. Using murine CD8+ T cells activated in vitro, or in vivo by immunization with Plasmodium berghei sporozoites, we found that, unexpectedly, inhibition of ASGP receptors did not inhibit the accumulation of effector cells in the liver, but instead prevented these cells from accumulating in the spleen. In addition, enforced expression of ASGP on effector CD8+ T cells using St3GalI-deficient cells lead to their loss from the spleen. We also found, using different mouse models of thrombocytopenia, that severe reduction in platelet concentration in circulation did not strongly influence the residence and protective function of CD8+ T cells in the liver. These data suggest that platelets play a marginal role in CD8+ T cell function in the liver. Furthermore, ASGP-expressing effector CD8+ T cells accumulate in the spleen, not the liver, prior to their destruction.

A centenary of service: 100 years of Immunology & Cell Biology.

This year marks the 100th year of the publication of Immunology & Cell Biology since it was first published in March 1924 as the Australian Journal of Experimental Biology and Medical Science. In this Editorial, we recount the journal from its founding, to its focus on immunology, through to the modern era.

Acute T-Cell-Driven Inflammation Requires the Endoglycosidase Heparanase-1 from Multiple Cell Types.

It has been accepted for decades that T lymphocytes and metastasising tumour cells traverse basement membranes (BM) by deploying a battery of degradative enzymes, particularly proteases. However, since many redundant proteases can solubilise BM it has been difficult to prove that proteases aid cell migration, particularly in vivo. Recent studies also suggest that other mechanisms allow BM passage of cells. To resolve this issue we exploited heparanase-1 (HPSE-1), the only endoglycosidase in mammals that digests heparan sulfate (HS), a major constituent of BM. Initially we examined the effect of HPSE-1 deficiency on a well-characterised adoptive transfer model of T-cell-mediated inflammation. We found that total elimination of HPSE-1 from this system resulted in a drastic reduction in tissue injury and loss of target HS. Subsequent studies showed that the source of HPSE-1 in the transferred T cells was predominantly activated CD4+ T cells. Based on bone marrow chimeras, two cellular sources of HPSE-1 were identified in T cell recipients, one being haematopoiesis dependent and the other radiation resistant. Collectively our findings unequivocally demonstrate that an acute T-cell-initiated inflammatory response is HPSE-1 dependent and is reliant on HPSE-1 from at least three different cell types.

Potential contrasting effects of platelets on the migration and invasion of sarcomas versus carcinomas.

The ability of platelets to promote carcinoma and melanoma progression has been thoroughly studied and occurs in numerous ways. In contrast, the effect of platelets on sarcomas, tumors arising from mesenchymal cells, has received very little attention. This study was undertaken to simultaneously compare the effects of platelets on murine and human sarcomas and carcinomas. In contrast to their effect on carcinomas, platelets inhibited the invasion of some murine- and all human sarcomas tested in vitro. Further invasion studies with TGFß treatment only partially recapitulated the results seen with whole platelets. In a spontaneous tumor growth and lung metastasis model, platelets promoted 4T1 mammary carcinoma metastasis but not MCA-1 fibrosarcoma metastasis. Gene expression analysis of the platelet-promoted MDA-MB-231 breast carcinoma, and the platelet-inhibited HT1080 fibrosarcoma cell lines revealed that exposure of MDA-MB-231 to platelets, resulted in upregulation of oncogenes and EMT-associated genes whereas in HT1080 a tumor-suppressor gene was significantly upregulated. Thus, this study has revealed a potential diametrically opposing effect of platelets on mesenchymal and epithelial cancers, a finding that warrants further investigation.

Heparanase: Historical Aspects and Future Perspectives.

Heparanase is an endo-ß-glucuronidase that cleaves at a limited number of internal sites the glycosaminoglycan heparan sulfate (HS). Heparanase enzymatic activity was first reported in 1975 and by 1983 evidence was beginning to emerge that the enzyme was a facilitator of tumor metastasis by cleaving HS chains present in blood vessel basement membranes and, thereby, aiding the passage of tumor cells through blood vessel walls. Due to a range of technical difficulties, it took another 16 years before heparanase was cloned and characterized in 1999 and a further 14 years before the crystal structure of the enzyme was solved. Despite these substantial deficiencies, there was steady progress in our understanding of heparanase long before the enzyme was fully characterized. For example, it was found as early as 1984 that activated T cells upregulate heparanase expression, like metastatic tumor cells, and the enzyme aids the entry of T cells and other leukocytes into inflammatory sites. Furthermore, it was discovered in 1989 that heparanase releases pre-existing growth factors and cytokines associated with HS in the extracellular matrix (ECM), the liberated growth factors/cytokines enhancing angiogenesis and wound healing. There were also the first hints that heparanase may have functions other than enzymatic activity, in 1995 it being reported that under certain conditions the enzyme could act as a cell adhesion molecule. Also, in the same year PI-88 (Muparfostat), the first heparanase inhibitor to reach and successfully complete a Phase III clinical trial was patented.Nevertheless, the cloning of heparanase (also known as heparanase-1) in 1999 gave the field an enormous boost and some surprises. The biggest surprise was that there is only one heparanase encoding gene in the mammalian genome, despite earlier research, based on substrate specificity, suggesting that there are at least three different heparanases. This surprising conclusion has remained unchanged for the last 20 years. It also became evident that heparanase is a family 79 glycoside hydrolase that is initially produced as a pro-enzyme that needs to be processed by proteases to form an enzymatically active heterodimer. A related molecule, heparanase-2, was also discovered that is enzymatically inactive but, remarkably, recently has been shown to inhibit heparanase-1 activity as well as acting as a tumor suppressor that counteracts many of the pro-tumor properties of heparanase-1.The early claim that heparanase plays a key role in tumor metastasis, angiogenesis and inflammation has been confirmed by many studies over the last 20 years. In fact, heparanase expression is enhanced in all major cancer types, namely carcinomas, sarcomas, and hematological malignancies, and correlates with increased metastasis and poor prognosis. Also, there is mounting evidence that heparanase plays a central role in the induction of inflammation-associated cancers. The enzymatic activity of heparanase has also emerged in unexpected situations, such as in the spread of HS-binding viruses and in Type-1 diabetes where the destruction of intracellular HS in pancreatic insulin-producing beta cells precipitates diabetes. But the most extraordinary recent discoveries have been with the realization that heparanase can exert a range of biological activities that are independent of its enzymatic function, most notably activation of several signaling pathways and being a transcription factor that controls methylation of histone tails. Collectively, these data indicate that heparanase is a truly multifunctional protein that has the additional property of cleaving HS chains and releasing from ECM and cell surfaces hundreds of HS-binding proteins with a plethora of functional consequences. Clearly, there are many unique features of this intriguing molecule that still remain to be explored and are highlighted in this Chapter.

Heparanase and Type 1 Diabetes.

Type 1 diabetes (T1D) results from autoimmune destruction of insulin-producing beta cells in pancreatic islets. The degradation of the glycosaminoglycan heparan sulfate (HS) by the endo-ß-D-glycosidase heparanase plays a critical role in multiple stages of the disease process. Heparanase aids (i) migration of inflammatory leukocytes from the vasculature to the islets, (ii) intra-islet invasion by insulitis leukocytes, and (iii) selective destruction of beta cells. These disease stages are marked by the solubilization of HS in the subendothelial basement membrane (BM), HS breakdown in the peri-islet BM, and the degradation of HS inside beta cells, respectively. Significantly, healthy islet beta cells are enriched in highly sulfated HS which is essential for their viability, protection from damage by reactive oxygen species (ROS), beta cell function and differentiation. Consequently, mouse and human beta cells but not glucagon-producing alpha cells (which contain less-sulfated HS) are exquisitely vulnerable to heparanase-mediated damage. In vitro, the death of HS-depleted mouse and human beta cells can be prevented by HS replacement using highly sulfated HS mimetics or analogues. T1D progression in NOD mice and recent-onset T1D in humans correlate with increased expression of heparanase by circulating leukocytes of myeloid origin and heparanase-expressing insulitis leukocytes. Treatment of NOD mice with the heparanase inhibitor and HS replacer, PI-88, significantly reduced T1D incidence by 50%, impaired the development of insulitis and preserved beta cell HS. These outcomes identified heparanase as a novel destructive tool in T1D, distinct from the conventional cytotoxic and apoptosis-inducing mechanisms of autoreactive T cells. In contrast to exogenous catalytically active heparanase, endogenous heparanase may function in HS homeostasis, gene expression and insulin secretion in normal beta cells and immune gene expression in leukocytes. In established diabetes, the interplay between hyperglycemia, local inflammatory cells (e.g. macrophages) and heparanase contributes to secondary micro- and macro-vascular disease. We have identified dual activity heparanase inhibitors/HS replacers as a novel class of therapeutic for preventing T1D progression and potentially for mitigating secondary vascular disease that develops with long-term T1D.

Extracellular histones induce erythrocyte fragility and anemia.

Extracellular histones have been shown to play an important pathogenic role in many diseases, primarily through their cytotoxicity toward nucleated cells and their ability to promote platelet activation with resultant thrombosis and thrombocytopenia. In contrast, little is known about the effect of extracellular histones on erythrocyte function. We demonstrate in this study that histones promote erythrocyte aggregation, sedimentation, and using a novel in vitro shear stress model, we show that histones induce erythrocyte fragility and lysis in a concentration-dependent manner. Furthermore, histones impair erythrocyte deformability based on reduced passage of erythrocytes through an artificial spleen. These in vitro results were mirrored in vivo with the injection of histones inducing anemia within minutes of administration, with a concomitant increase in splenic hemoglobin content. Thrombocytopenia and leukopenia were also observed. These findings suggest that histones binding to erythrocytes may contribute to the elevated erythrocyte sedimentation rates observed in inflammatory conditions. Furthermore, histone-induced increases in red blood cell lysis and splenic clearance may be a significant factor in the unexplained anemias seen in critically ill patients.

Chromatin-associated protein kinase C-θ regulates an inducible gene expression program and microRNAs in human T lymphocytes.

Studies in yeast demonstrate that signaling kinases have a surprisingly active role in the nucleus, where they tether to chromatin and modulate gene expression programs. Despite these seminal studies, the nuclear mechanism of how signaling kinases control transcription of mammalian genes is in its infancy. Here, we provide evidence for a hitherto unknown function of protein kinase C-theta (PKC-θ), which physically associates with the regulatory regions of inducible immune response genes in human T cells. Chromatin-anchored PKC-θ forms an active nuclear complex by interacting with RNA polymerase II, the histone kinase MSK-1, and the adaptor molecule 14-3-3ζ. ChIP-on-chip reveals that PKC-θ binds to promoters and transcribed regions of genes, as well as to microRNA promoters that are crucial for cytokine regulation. Our results provide a molecular explanation for the role of PKC-θ not only in normal T cell function, but also in circumstances of its ectopic expression in cancer.

Phase I trial of Lipovaxin-MM, a novel dendritic cell-targeted liposomal vaccine for malignant melanoma.

INTRODUCTION: In this phase I study using a 3 + 3 dose escalation design, the safety, dose-limiting toxicity (DLT), immunogenicity and efficacy of intravenous Lipovaxin-MM-a multi-component dendritic cell-targeted liposomal vaccine against metastatic melanoma-was investigated. METHODS: Twelve subjects with metastatic cutaneous melanoma were recruited in three cohorts. Patients in Cohort A (n = 3) and Cohort B (n = 3) received three doses of 0.1 and 1 mL of Lipovaxin-MM, respectively, every 4 weeks. Patients in Cohort C (n = 6) received four doses of 3 mL vaccine weekly. Immunologic assessments of peripheral blood were made at regular intervals and included leukocyte subsets, cytokine levels, and Lipovaxin-MM-specific T-cell and antibody reactivities. Tumor responses were assessed by RECIST v1.0 at screening, then 8 weekly in Cohorts A and B and 6 weekly in Cohort C. RESULTS: Of a total of 94 adverse events (AEs) reported in ten subjects, 43 AEs in six subjects were considered to be possibly or probably vaccine-related. Most (95%) vaccine-related AEs were grade 1 or 2, two (5%) grade 3 vaccine-related AEs of anemia and lethargy were recorded, and higher grade AEs and DLTs were not observed. No consistent evidence of vaccine-specific humoral or cellular immune responses was found in post-immunization blood samples. One patient had a partial response, two patients had stable disease, and the remaining patients had progressive disease. CONCLUSIONS: Lipovaxin-MM was well tolerated and without clinically significant toxicity. Immunogenicity of Lipovaxin-MM was not detected. Partial response and stable disease were observed in one and two patients, respectively.

The transcriptional repressor Bcl-6 directs T follicular helper cell lineage commitment.

Follicular helper T (Tfh) cells provide selection signals to germinal center B cells, which is essential for long-lived antibody responses. High CXCR5 and low CCR7 expression facilitates their homing to B cell follicles and distinguishes them from T helper 1 (Th1), Th2, and Th17 cells. Here, we showed that Bcl-6 directs Tfh cell differentiation: Bcl-6-deficient T cells failed to develop into Tfh cells and could not sustain germinal center responses, whereas forced expression of Bcl-6 in CD4(+) T cells promoted expression of the hallmark Tfh cell molecules CXCR5, CXCR4, and PD-1. Bcl-6 bound to the promoters of the Th1 and Th17 cell transcriptional regulators T-bet and RORgammat and repressed IFN-gamma and IL-17 production. Bcl-6 also repressed expression of many microRNAs (miRNAs) predicted to control the Tfh cell signature, including miR-17-92, which repressed CXCR5 expression. Thus, Bcl-6 positively directs Tfh cell differentiation, through combined repression of miRNAs and transcription factors.

The role of heparan sulphate in inflammation.

The polysaccharide heparan sulphate is ubiquitously expressed as a proteoglycan in extracellular matrices and on cell surfaces. Heparan sulphate has marked sequence diversity that allows it to specifically interact with many proteins. This Review focuses on the multiple roles of heparan sulphate in inflammatory responses and, in particular, on its participation in almost every stage of leukocyte transmigration through the blood-vessel wall. Heparan sulphate is involved in the initial adhesion of leukocytes to the inflamed endothelium, the subsequent chemokine-mediated transmigration through the vessel wall and the establishment of both acute and chronic inflammatory reactions.

Synthesis and preliminary evaluation of 5,7-dimethyl-2-aryl-3H-pyrrolizin-3-ones as angiogenesis inhibitors.

Sunitinib (Sutent®) is a receptor tyrosine kinase (RTK) and angiogenesis inhibitor approved for the treatment of renal cell carcinomas, gastrointestinal stromal tumours and pancreatic neuroendocrine tumours. A key structural motif retained throughout medicinal chemistry efforts during sunitinib's development was the indoline-2-one group. In the search for new anti-angiogenic scaffolds, we previously reported that non-indoline-2-one-based derivatives of semaxanib (SU5416, a structurally simpler sunitinib predecessor that underwent Phase III trials) are active as angiogenesis inhibitors, indicating that the group is not essential for activity. This Letter describes the synthesis and structure-activity relationships of another class of non-indoline-2-one angiogenesis inhibitors related to sunitinib/semaxanib; the 5,7-dimethyl-2-aryl-3H-pyrrolizin-3-ones. A focussed library of 19 analogues was prepared using a simple novel process, wherein commercially available substituted arylacetic acids activated with an amide coupling reagent (HBTU) were reacted with the potassium salt of 3,5-dimethyl-1H-pyrrole-2-carbaldehyde in one-pot. Screening of the library using a cell-based endothelial tube formation assay identified 6 compounds with anti-angiogenesis activity. Two of the compounds were advanced to the more physiologically relevant rat aortic ring assay, where they showed similar inhibitory effects to semaxanib at 10µg/mL, confirming that 5,7-dimethyl-2-aryl-3H-pyrrolizin-3-ones represent a new class of angiogenesis inhibitors.

Mice deficient in the putative phospholipid flippase ATP11C exhibit altered erythrocyte shape, anemia, and reduced erythrocyte life span.

Transmembrane lipid transporters are believed to establish and maintain phospholipid asymmetry in biological membranes; however, little is known about the in vivo function of the specific transporters involved. Here, we report that developing erythrocytes from mice lacking the putative phosphatidylserine flippase ATP11C showed a lower rate of PS translocation in vitro compared with erythrocytes from wild-type littermates. Furthermore, the mutant mice had an elevated percentage of phosphatidylserine-exposing mature erythrocytes in the periphery. Although erythrocyte development in ATP11C-deficient mice was normal, the mature erythrocytes had an abnormal shape (stomatocytosis), and the life span of mature erythrocytes was shortened relative to that in control littermates, resulting in anemia in the mutant mice. Thus, our findings uncover an essential role for ATP11C in erythrocyte morphology and survival and provide a new candidate for the rare inherited blood disorder stomatocytosis with uncompensated anemia.

Heme oxygenase-1 deficiency alters erythroblastic island formation, steady-state erythropoiesis and red blood cell lifespan in mice.

Heme oxygenase-1 is critical for iron recycling during red blood cell turnover, whereas its impact on steady-state erythropoiesis and red blood cell lifespan is not known. We show here that in 8- to 14-week old mice, heme oxygenase-1 deficiency adversely affects steady-state erythropoiesis in the bone marrow. This is manifested by a decrease in Ter-119(+)-erythroid cells, abnormal adhesion molecule expression on macrophages and erythroid cells, and a greatly diminished ability to form erythroblastic islands. Compared with wild-type animals, red blood cell size and hemoglobin content are decreased, while the number of circulating red blood cells is increased in heme oxygenase-1 deficient mice, overall leading to microcytic anemia. Heme oxygenase-1 deficiency increases oxidative stress in circulating red blood cells and greatly decreases the frequency of macrophages expressing the phosphatidylserine receptor Tim4 in bone marrow, spleen and liver. Heme oxygenase-1 deficiency increases spleen weight and Ter119(+)-erythroid cells in the spleen, although α4ß1-integrin expression by these cells and splenic macrophages positive for vascular cell adhesion molecule 1 are both decreased. Red blood cell lifespan is prolonged in heme oxygenase-1 deficient mice compared with wild-type mice. Our findings suggest that while macrophages and relevant receptors required for red blood cell formation and removal are substantially depleted in heme oxygenase-1 deficient mice, the extent of anemia in these mice may be ameliorated by the prolonged lifespan of their oxidatively stressed erythrocytes.

Extracellular matrix components in the pathogenesis of type 1 diabetes.

Type 1 diabetes (T1D) results from progressive immune cell-mediated destruction of pancreatic ß cells. As immune cells migrate into the islets, they pass through the extracellular matrix (ECM). This ECM is composed of different macromolecules localized to different compartments within and surrounding islets; however, the involvement of this ECM in the development of human T1D is not well understood. Here, we summarize our recent findings from human and mouse studies illustrating how specific components of the islet ECM that constitute basement membranes and interstitial matrix of the islets, and surprisingly, the intracellular composition of islet ß cells themselves, are significantly altered during the pathogenesis of T1D. Our focus is on the ECM molecules laminins, collagens, heparan sulfate/heparan sulfate proteoglycans, and hyaluronan, as well as on the enzymes that degrade these ECM components. We propose that islet and lymphoid tissue ECM composition and organization are critical to promoting immune cell activation, islet invasion, and destruction of islet ß cells in T1D.

PLASMA DYNAMICS OF NEUTROPHIL EXTRACELLULAR TRAPS AND CELL-FREE DNA IN SEPTIC AND NONSEPTIC VASOPLEGIC SHOCK: A PROSPECTIVE COMPARATIVE OBSERVATIONAL COHORT STUDY.

ABSTRACT: Background: The association between neutrophil extracellular traps (NETs) and the requirement for vasopressor and inotropic support in vasoplegic shock is unclear. This study aimed to investigate the dynamics of plasma levels of NETs and cell-free DNA (cfDNA) up to 48 h after the admission to the intensive care unit (ICU) for management of vasoplegic shock of infectious (SEPSIS) or noninfectious (following cardiac surgery, CARDIAC) origin. Methods: This is a prospective, observational study of NETs and cfDNA plasma levels at 0H (admission) and then at 12H, 24H, and 48H in SEPSIS and CARDIAC patients. The vasopressor inotropic score (VIS), the Sequential Organ Failure Assessment (SOFA) score, and time spent with invasive ventilation, in ICU and in hospital, were recorded. Associations between NETs/cfDNA and VIS and SOFA were analyzed by Spearman's correlation (rho), and between NETs/cfDNA and ventilation/ICU/hospitalization times by generalized linear regression. Results: Both NETs and cfDNA remained elevated over 48 h in SEPSIS (n = 46) and CARDIAC (n = 30) patients, with time-weighted average concentrations greatest in SEPSIS (NETs median difference 0.06 [0.02-0.11], P = 0.005; cfDNA median difference 0.48 [0.20-1.02], P < 0.001). The VIS correlated to NETs (rho = 0.3-0.60 in SEPSIS, P < 0.01, rho = 0.36-0.57 in CARDIAC, P ≤ 0.01) and cfDNA (rho = 0.40-0.56 in SEPSIS, P < 0.01, rho = 0.38-0.47 in CARDIAC, P < 0.05). NETs correlated with SOFA. Neither NETs nor cfDNA were independently associated with ventilator/ICU/hospitalization times. Conclusion: Plasma levels of NETs and cfDNA correlated with the dose of vasopressors and inotropes administered over 48 h in patients with vasoplegic shock from sepsis or following cardiac surgery. NETs levels also correlated with organ dysfunction. These findings suggest that similar mechanisms involving release of NETs are involved in the pathophysiology of vasoplegic shock irrespective of an infectious or noninfectious etiology.

Histidine-rich glycoprotein: the Swiss Army knife of mammalian plasma.

Histidine-rich glycoprotein (HRG), also known as histidine-proline-rich glyco-protein, is an abundant and well-characterized protein of vertebrate plasma. HRG has a multidomain structure that allows the molecule to interact with many ligands, including heparin, phospholipids, plasminogen, fibrinogen, immunoglobulin G, C1q, heme, and Zn²(+). The ability of HRG to interact with various ligands simultaneously has suggested that HRG can function as an adaptor molecule and regulate numerous important biologic processes, such as immune complex/necrotic cell/pathogen clearance, cell adhesion, angiogenesis, coagulation, and fibrinolysis. The present review covers the proposed multifunctional roles of HRG with a focus on recent findings that have led to its emergence as a key regulator of immunity and vascular biology. Also included is a discussion of the striking functional similarities between HRG and other important multifunctional proteins found in plasma, such as C-reactive protein, C1q, ß2 glycoprotein I, and thrombospondin-1.

The antiangiogenic properties of sulfated ß-cyclodextrins in anticancer formulations incorporating 5-fluorouracil.

Sulfated ß-cyclodextrins (S-ß-CDs) are useful excipients for improving the solubility of drugs. One such formulation incorporating 5-fluorouracil (5-FU), termed FD(S), showed improved efficacy over 5-FU alone in orthotopic carcinoma xenograft models. S-ß-CDs have heparin-like anticoagulant properties, which may have contributed toward the improved antitumor effect of FD(S). S-ß-CDs have also been reported to modify a number of processes involved in angiogenesis. Although the anticoagulant nature of S-ß-CDs was established, the antiangiogenic properties of S-ß-CDs within FD(S) were unknown. The effect of S-ß-CD and FD(S) on the proliferation and migration of endothelial cells in live-cell kinetic assays, and the reorganization of human umbilical vein endothelial cells into tubule structures in vitro was assessed. The effects of S-ß-CD on angiogenesis in vitro were validated ex vivo using the rat aorta ring assay and the chick embryo chorioallantoic membrane assay. S-ß-CD does not alter proliferative endothelial cell sensitivity to 5-FU cytotoxicity. S-ß-CD alone and within FD(S) significantly inhibited angiogenesis by impeding endothelial cell migration, resulting in the inhibition of tubule formation and hence new vasculature. In addition to the cytotoxic action of the drug 5-FU, therapeutic inhibition of angiogenesis by S-ß-CDs within FD(S) could potentially limit local invasion and metastases. This has important implications for the exploitation of S-ß-CDs for drug formulation improvements or for drug delivery of anticancer biologics.

The yeast homolog of heme oxygenase-1 affords cellular antioxidant protection via the transcriptional regulation of known antioxidant genes.

Heme oxygenase-1 (HO-1) degrades heme and protects cells from oxidative challenge. This antioxidant activity is thought to result from the HO-1 enzymatic activity, manifested by a decrease in the concentration of the pro-oxidant substrate heme, and an increase in the antioxidant product bilirubin. Using a global transcriptional approach, and yeast as a model, we show that HO-1 affords cellular protection via up-regulation of transcripts encoding enzymes involved in cellular antioxidant defense, rather than via its oxygenase activity. Like mammalian cells, yeast responds to oxidative stress by expressing its HO-1 homolog and, compared with the wild type, heme oxygenase-null mutant cells have increased sensitivity toward oxidants that is rescued by overexpression of human HO-1 or its yeast homolog. Increased oxidant sensitivity of heme oxygenase-null mutant cells is explained by a decrease in the expression of the genes encoding γ-glutamylcysteine synthetase, glutathione peroxidase, catalase, and methionine sulfoxide reductase, because overexpression of any of these genes affords partial, and overexpression of all four genes provides complete, protection to the null mutant. Genes encoding antioxidant enzymes represent only a small portion of the 480 differentially expressed transcripts in heme oxygenase-null mutants. Transcriptional regulation may be explained by the nuclear localization of heme oxygenase observed in oxidant-challenged cells. Our results challenge the notion that HO-1 functions simply as a catabolic and antioxidant enzyme. They indicate much broader functions for HO-1, the unraveling of which may help explain the multiple biological responses reported in animals as a result of altered HO-1 expression.

Computational analyses of the catalytic and heparin-binding sites and their interactions with glycosaminoglycans in glycoside hydrolase family 79 endo-ß-D-glucuronidase (heparanase).

Mammalian heparanase is an endo-ß-glucuronidase associated with cell invasion in cancer metastasis, angiogenesis and inflammation. Heparanase cleaves heparan sulfate proteoglycans in the extracellular matrix and basement membrane, releasing heparin/heparan sulfate oligosaccharides of appreciable size. This in turn causes the release of growth factors, which accelerate tumor growth and metastasis. Heparanase has two glycosaminoglycan-binding domains; however, no three-dimensional structure information is available for human heparanase that can provide insights into how the two domains interact to degrade heparin fragments. We have constructed a new homology model of heparanase that takes into account the most recent structural and bioinformatics data available. Heparin analogs and glycosaminoglycan mimetics were computationally docked into the active site with energetically stable ring conformations and their interaction energies were compared. The resulting docked structures were used to propose a model for substrates and conformer selectivity based on the dimensions of the active site. The docking of substrates and inhibitors indicates the existence of a large binding site extending at least two saccharide units beyond the cleavage site (toward the nonreducing end) and at least three saccharides toward the reducing end (toward heparin-binding site 2). The docking of substrates suggests that heparanase recognizes the N-sulfated and O-sulfated glucosamines at subsite +1 and glucuronic acid at the cleavage site, whereas in the absence of 6-O-sulfation in glucosamine, glucuronic acid is docked at subsite +2. These findings will help us to focus on the rational design of heparanase-inhibiting molecules for anticancer drug development by targeting the two heparin/heparan sulfate recognition domains.