P. aeruginosa augments irradiation injury via 15-lipoxygenase-catalyzed generation of 15-HpETE-PE and induction of theft-ferroptosis.

Total body irradiation (TBI) targets sensitive bone marrow hematopoietic cells and gut epithelial cells, causing their death and inducing a state of immunodeficiency combined with intestinal dysbiosis and nonproductive immune responses. We found enhanced Pseudomonas aeruginosa (PAO1) colonization of the gut leading to host cell death and strikingly decreased survival of irradiated mice. The PAO1-driven pathogenic mechanism includes theft-ferroptosis realized via (a) curbing of the host antiferroptotic system, GSH/GPx4, and (b) employing bacterial 15-lipoxygenase to generate proferroptotic signal - 15-hydroperoxy-arachidonoyl-PE (15-HpETE-PE) - in the intestines of irradiated and PAO1-infected mice. Global redox phospholipidomics of the ileum revealed that lysophospholipids and oxidized phospholipids, particularly oxidized phosphatidylethanolamine (PEox), represented the major factors that contributed to the pathogenic changes induced by total body irradiation and infection by PAO1. A lipoxygenase inhibitor, baicalein, significantly attenuated animal lethality, PAO1 colonization, intestinal epithelial cell death, and generation of ferroptotic PEox signals. Opportunistic PAO1 mechanisms included stimulation of the antiinflammatory lipoxin A4, production and suppression of the proinflammatory hepoxilin A3, and leukotriene B4. Unearthing complex PAO1 pathogenic/virulence mechanisms, including effects on the host anti/proinflammatory responses, lipid metabolism, and ferroptotic cell death, points toward potentially new therapeutic and radiomitigative targets.

Methylated HNRNPK acts on RPS19 to regulate ALOX15 synthesis in erythropoiesis.

Post-transcriptional control is essential to safeguard structural and metabolic changes in enucleated reticulocytes during their terminal maturation to functional erythrocytes. The timely synthesis of arachidonate 15-lipoxygenase (ALOX15), which initiates mitochondria degradation at the final stage of reticulocyte maturation is regulated by the multifunctional protein HNRNPK. It constitutes a silencing complex at the ALOX15 mRNA 3' untranslated region that inhibits translation initiation at the AUG by impeding the joining of ribosomal 60S subunits to 40S subunits. To elucidate how HNRNPK interferes with 80S ribosome assembly, three independent screens were applied. They consistently demonstrated a differential interaction of HNRNPK with RPS19, which is localized at the head of the 40S subunit and extends into its functional center. During induced erythroid maturation of K562 cells, decreasing arginine dimethylation of HNRNPK is linked to a reduced interaction with RPS19 in vitro and in vivo. Dimethylation of residues R256, R258 and R268 in HNRNPK affects its interaction with RPS19. In noninduced K562 cells, RPS19 depletion results in the induction of ALOX15 synthesis and mitochondria degradation. Interestingly, residue W52 in RPS19, which is frequently mutated in Diamond-Blackfan Anemia (DBA), participates in specific HNRNPK binding and is an integral part of a putative aromatic cage.

Lipoxygenase Inhibition by Plant Extracts.

Lipoxygenases are widespread enzymes that catalyze oxidation of polyunsaturated fatty acids (linoleic, linolenic, and arachidonic acid) to produce hydroperoxides. Lipoxygenase reactions can be desirable, but also lipoxygenases can react in undesirable ways. Most of the products of lipoxygenase reactions are aromatic compounds that can affect food properties, especially during long-term storage. Lipoxygenase action on unsaturated fatty acids could result in off-flavor/off-odor development, causing food spoilage. In addition, lipoxygenases are present in the human body and play an important role in stimulation of inflammatory reactions. Inflammation is linked to many diseases, such as cancer, stroke, and cardiovascular and neurodegenerative diseases. This review summarized recent research on plant families and species that can inhibit lipoxygenase activity.

Enhanced 15-Lipoxygenase 1 Production is Related to Periostin Expression and Eosinophil Recruitment in Eosinophilic Chronic Rhinosinusitis.

BACKGROUND: The pathological features of chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP) tissues include an eosinophilic infiltration pattern (eosinophilic CRS (ECRS)) or a less eosinophilic pattern (non-ECRS). Recently, it has been suggested that 15-lipoxygenase 1 (15-LOX-1) may have significant roles in allergic disease; however, the significance of 15-LOX-1 in CRS is not well understood. The objective of this study was to demonstrate the expression of 15-LOX-1 in CRS. METHODS: The mRNA expression levels of 15-LOX-1 and periostin in nasal tissues were measured by quantitative real-time polymerase chain reaction. We also performed an immunofluorescence study of nasal tissues. Cells of the Eol-1 eosinophilic leukemic cell line were stimulated with interleukin-33 to test the induction of 15-LOX-1. RESULTS: The expression level of 15-LOX-1 mRNA in nasal polyps (NPs) was significantly higher in ECRS patients than in non-ECRS patients. The immunofluorescence study revealed that both airway epithelial cells and eosinophils in NPs expressed 15-LOX-1. A significant correlation was seen between the number of eosinophils and the mRNA expression levels of 15-LOX-1 and periostin in nasal polyps. Moreover, interleukin-33 enhanced 15-LOX-1 expression in Eol-1 cells. CONCLUSIONS: 15-LOX-1 was shown to be a significant molecule that facilitates eosinophilic inflammation in ECRS.

Effects of peripheral administration of GH and IGF-I on gene expression in the hippocampus of hypophysectomised rats.

OBJECTIVE: Growth hormone (GH) increases insulin-like growth factor I (IGF-I) production and both hormones affect hippocampal plasticity. We have previously shown that Hbb and Alas2 in the rat hippocampus were robustly regulated by GH-infusions for six days, whereas other transcripts were weakly affected. Here, we explored the effects of prolonged GH administration on transcripts linked to neuroprotection and investigated whether serum IGF-I administration may exert similar effects. DESIGN: Hypophysectomised female rats were infused with GH or IGF-I for 19 days. Hbb, Alas2 and seven additional GH- and IGF-I-related transcripts were quantified by Q-RT-PCR in rat hippocampus. RESULTS: Three transcripts, Hbb, Alas2, and Alox15 were increased by both GH and IGF-I administration. The other transcripts were marginally affected. CONCLUSION: The 19-day GH-infusion induced similar effects as those reported after 6-day GH treatment, with the addition of the regulation of transcript Alox15. IGF-I induced altered gene expression in relation to its effect on weight gain. This study underlines that there is an entity of transcripts involved in neuroprotection and vascular tone that is regulated by both systemic GH and IGF-I. For other transcripts, the longer duration of this study did not significantly enhance the marginal effects of GH administration seen previously.

Platelet-type 12-lipoxygenase deletion provokes a compensatory 12/15-lipoxygenase increase that exacerbates oxidative stress in mouse islet ß cells.

In type 1 diabetes, an autoimmune event increases oxidative stress in islet ß cells, giving rise to cellular dysfunction and apoptosis. Lipoxygenases are enzymes that catalyze the oxygenation of polyunsaturated fatty acids that can form lipid metabolites involved in several biological functions, including oxidative stress. 12-Lipoxygenase and 12/15-lipoxygenase are related but distinct enzymes that are expressed in pancreatic islets, but their relative contributions to oxidative stress in these regions are still being elucidated. In this study, we used mice with global genetic deletion of the genes encoding 12-lipoxygenase (arachidonate 12-lipoxygenase, 12S type [Alox12]) or 12/15-lipoxygenase (Alox15) to compare the influence of each gene deletion on ß cell function and survival in response to the ß cell toxin streptozotocin. Alox12-/- mice exhibited greater impairment in glucose tolerance following streptozotocin exposure than WT mice, whereas Alox15-/- mice were protected against dysglycemia. These changes were accompanied by evidence of islet oxidative stress in Alox12-/- mice and reduced oxidative stress in Alox15-/- mice, consistent with alterations in the expression of the antioxidant response enzymes in islets from these mice. Additionally, islets from Alox12-/- mice displayed a compensatory increase in Alox15 gene expression, and treatment of these mice with the 12/15-lipoxygenase inhibitor ML-351 rescued the dysglycemic phenotype. Collectively, these results indicate that Alox12 loss activates a compensatory increase in Alox15 that sensitizes mouse ß cells to oxidative stress.

The role of expression and activity of 15-Lipoxygenase isoforms and related cytokines in patients with Multiple Sclerosis and healthy controls.

BACKGROUND: Multiple Sclerosis (MS) as an inflammatory multifactorial auto immune nervous system disease imposes devastating burden of morbidity worldwide. Among environmental and genetic factors, the relevance of inflammatory mediators in MS pathogenesis is well documented. 15-Lipoxygense enzyme and its derived products have received attention as possible mediators of inflammatory responses. The involvement of 15-Lipoxygense pathway in the pathogenesis of inflammatory diseases such as MS has yet to be illustrated which is perused in the current study. METHODS: The expression level of 15-Lipoxygense isoforms was assessed via Real-Time PCR in the peripheral blood mononuclear cells separated from patients with MS and healthy subjects. The level of 15-Lipoxygense products (15(S) HETE, 13(S) HODE) and related cytokines (IL4 and IL13) were evaluated using enzyme immunoassay kits in serum samples. RESULTS: Our results demonstrated that 15-Lipoxygense-1 and 15-Lipoxygense-2 expression levels were increased in patients suffering from MS comparing to healthy subjects which were more obvious in Relapsing-Remitting MS. The elevated levels of 15-Lipoxygense isoforms were accompanied with 15(S) HETE and 13(S) HODE enhancement in serum of patients and the IL 13 elevation but not IL4 was consistent with higher expression of 15-Lipoxygense. The diagnostic value of 15-Lipoxygense isoforms and products were considerable between patients and healthy groups. CONCLUSION: The possible effect of 15-Lipoxygense pathway in the regulation of inflammatory events may light up new therapeutic possibilities regarding MS pathogenesis.

Antineoplastic effects of 15(S)-hydroxyeicosatetraenoic acid and 13-S-hydroxyoctadecadienoic acid in non-small cell lung cancer.

BACKGROUND: Previous studies have shown that the levels of 15-lipoxygenase 1 (15-LOX-1) and 15-LOX-2 as well as their metabolites 13-S-hydroxyoctadecadienoic acid (13(S)-HODE) and 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) are significantly reduced in smokers with non-small cell lung carcinoma (NSCLC). Furthermore, animal model experiments have indicated that the reduction of these molecules occurs before the establishment of cigarette smoking carcinogen-induced lung tumors, and this suggests roles in lung tumorigenesis. However, the functions of these molecules remain unknown in NSCLC. METHODS: NSCLC cells were treated with exogenous 13(S)-HODE and 15(S)-HETE, and then the ways in which they affected cell function were examined. 15-LOX-1 and 15-LOX-2 were also overexpressed in tumor cells to restore these 2 enzymes to generate endogenous 13(S)-HODE and 15(S)-HETE before cell function was assessed. RESULTS: The application of exogenous 13(S)-HODE and 15(S)-HETE significantly enhanced the activity of peroxisome proliferator-activated receptor γ (PPARγ), inhibited cell proliferation, induced apoptosis, and activated caspases 9 and 3. The overexpression of 15-LOX-1 and 15-LOX-2 obviously promoted the endogenous levels of 13(S)-HODE and 15(S)-HETE, which were demonstrated to be more effective in the inhibition of NSCLC. CONCLUSIONS: This study has demonstrated that exogenous or endogenous 13(S)-HODE and 15(S)-HETE can functionally inhibit NSCLC, likely by activating PPARγ. The restoration of 15-LOX activity to increase the production of endogenous 15(S)-HETE and 13(S)-HODE may offer a novel research direction for molecular targeting treatment of smoking-related NSCLC. This strategy can potentially avoid side effects associated with the application of synthetic PPARγ ligands.

STAT-dependent upregulation of 12/15-lipoxygenase contributes to neuronal injury after stroke.

Oxidative stress is a major brain injury mechanism after ischemic stroke. 12/15-lipoxygenase (12/15-LOX) is a key mediator of oxidative stress, contributing to neuronal cell death and vascular leakage. Nonetheless, the mechanism leading to its upregulation is currently unknown. We show here that Signal Transducers and Activators of Transcription (STATs), specifically STAT6 and possibly STAT1, increase transcription of 12/15-LOX in neuronal cells. Both p-STAT6 and -1 bound to specific STAT binding sites in the mouse 12/15-LOX promoter. Small interfering RNA (siRNA) knockdown showed STAT6 to be the dominant regulator, reducing 12/15-LOX promoter activation and cell death in oxidatively stressed HT22 cells. STAT6 siRNA efficiently prevented the increase of 12/15-LOX in murine primary neurons, both after induction of oxidative stress and after oxygen-glucose deprivation. Early activation of STAT6 and STAT1 in mice was consistent with a role in regulating 12/15-LOX in focal ischemia. Brains of human stroke patients showed increased p-STAT6 and p-STAT1 in the peri-infarct region, along with 12/15-LOX and markers of apoptosis. These results link STAT6 and STAT1 to the 12/15-LOX damage pathway and suggest disregulation of STAT-dependent transcription as injury mechanism in stroke. Selectively targeting STATs may thus be a novel therapeutic approach to reducing brain injury after a stroke.

Correlation of ALOX15 expression with eosinophilic or reflux esophagitis in a cohort of pediatric patients with esophageal eosinophilia.

The differential diagnosis between eosinophilic esophagitis (EoE) and gastroesophageal reflux disease (GERD) is often challenging. We recently showed that the ALOX15 protein is expressed in 95% of esophageal biopsies from patients with a definitive diagnosis of EoE. Here we correlated ALOX15 expression with the clinical classification of EoE or GERD in a cohort of consecutive pediatric patients (n = 62) with at least 1 esophageal biopsy containing at least 15 eosinophils per high-power field (eos/HPF). The patients were categorized into the following groups: (1) at least 15 eos/HPF in the distal esophagus only (n = 24), (2) at least 15 eos/HPF in the proximal esophagus only (n = 5), and (3) at least 15 eos/HPF in the distal and proximal biopsies (n = 33). Control groups included patients with GERD with biopsies containing 6 to 15 eos/HPF (n = 9), patients with GERD with 5 eos/HPF or less (n = 15), patients with candida esophagitis (n = 15), and patients with normal biopsies (n = 15). ALOX15 was positive in 90.5% of patients with EoE (13/16 in group 1, 4/4 in group 2, 31/33 in group 3) versus 44% of patients with GERD (4/8 in group 1, 0/1 in group 2, and 0/0 in group 3), 2 of 9 (22%) of patients with 6 to 15 eos/HPF, and was negative in all patients with GERD with biopsies containing 5 eos/HPF or less, all patients with candida esophagitis, and all normal controls. In conclusion, ALOX15 is a sensitive marker of EoE; however, subpopulations of patients with GERD with >5 eos/HPF also express ALOX15. Positive ALOX15 expression is more prevalent in EoE than in GERD and may prove to be a useful diagnostic marker in patients with discrepant biopsy findings between the proximal and distal esophagus.

15-LOX-1 suppression of hypoxia-induced metastatic phenotype and HIF-1α expression in human colon cancer cells.

The expression of 15-lipoxygenase-1 (15-LOX-1) is downregulated in colon cancer and other major cancers, and 15-LOX-1 reexpression in cancer cells suppresses colonic tumorigenesis. Various lines of evidence indicate that 15-LOX-1 expression suppresses premetastatic stages of colonic tumorigenesis; nevertheless, the role of 15-LOX-1 loss of expression in cancer epithelial cells in metastases continues to be debated. Hypoxia, a common feature of the cancer microenvironment, promotes prometastatic mechanisms such as the upregulation of hypoxia-inducible factor (HIF)-1α, a transcriptional master regulator that enhances cancer cell metastatic potential, angiogenesis, and tumor cell invasion and migration. We have, therefore, tested whether restoring 15-LOX-1 in colon cancer cells affects cancer cells' hypoxia response that promotes metastasis. We found that 15-LOX-1 reexpression in HCT116, HT29LMM, and LoVo colon cancer cells inhibited survival, vascular endothelial growth factor (VEGF) expression, angiogenesis, cancer cell migration and invasion, and HIF-1α protein expression and stability under hypoxia. These findings demonstrate that 15-LOX-1 expression loss in cancer cells promotes metastasis and that therapeutically targeting ubiquitous 15-LOX-1 loss in cancer cells has the potential to suppress metastasis.

12/15-lipoxygenase expression is increased in oligodendrocytes and microglia of periventricular leukomalacia.

Oxidative stress involving premyelinating oligodendrocytes (OLs) is a major factor in the pathogenesis of preterm white matter injury. In animal and cell culture studies, activation of the lipid-oxidizing enzyme 12/15-lipoxygenase (12/15-LOX) plays a central role as an inflammatory mediator in the pathology of oxidative stress and OL cell death, as well as ischemia and neuronal death. The role of 12/15-LOX, however, is unclear in the developing human brain. The mechanism of 12/15-LOX involves the production of reactive oxygen species through the metabolism of arachidonic acid, as well as direct detrimental effects on organelle membranes. Here we tested the hypothesis that the density of 12/15-LOX-expressing cells is increased in periventricular leukomalacia (PVL). Using immunocytochemistry (ICC) in human paraffin-embedded tissue, 12/15-LOX expression was seen in macrophages of the focally necrotic lesions in the periventricular white matter, as well as in glial cells throughout the surrounding white matter with reactive gliosis. Interestingly, no significant 12/15-LOX expression was detected in neurons in the cerebral cortex overlying the damaged white matter. Using a scoring system from 0 to 3, we assessed the density of 12/15-LOX-expressing cells in diffusely gliotic white matter from 20 to 43 postconceptional (PC) weeks in 19 PVL cases (median = 36 PC weeks) and 10 control (non-PVL) cases (median = 34 PC weeks). The density of 12/15-LOX-positive cells was significantly increased in the diffuse component of PVL (score = 1.17 ± 0.15) compared to controls (score = 0.48 ± 0.21; p = 0.014). Using double-label ICC, 12/15-LOX was observed in PVL in OLs of the O4 and O1 premyelinating stages, as well as in mature OLs as determined with the mature OL marker adenomatous polyposis coli (APC). In addition, 12/15-LOX expression was present in a population of CD68-positive activated microglia. There was no 12/15-LOX expression in reactive astrocytes. Finally we observed terminal deoxynucleotide transferase dUTP nick end-labeling-positive cells within the white matter of PVL that expressed 12/15-LOX and/or within close proximity of 12/15-LOX-positive cells. Our data support a role for 12/15-LOX activation as an inflammatory mediator of injury in PVL, with a contribution of 12/15-LOX to PVL-induced damage to or cell death of OLs, including those at the O1 and O4 stages.

Heme oxygenase-1 induces 15-lipoxygenase expression during hypoxia-induced pulmonary hypertension.

We previously reported that 15-lipoxygenase (15-LO) induced by hypoxia catalyzed the conversion of arachidonic acid (AA) into 15-hydroxyeicosatetraenoic acid (15-HETE), which plays an essential role in the development of hypoxic pulmonary arterial hypertension (HPH). However, the mechanisms by which hypoxia up-regulated 15-LO are still unclear. Heme oxygenase-1 (HO-1), an oxygen-dependent enzyme regulating vascular tone and cell proliferation, was implicated in HPH and was promoted by hypoxia. Therefore, the present study was carried out to determine whether hypoxia induced the expression of 15-LO via the HO-1 pathway. To test this hypothesis, we studied the role of HO-1 in HPH and 15-LO/15-HETE expression We found increased right ventricular systolic pressure and pulmonary arteries (PAs) reactivity to vasoconstrictors as well as intima-to-media ratio of PAs in HO-1 overexpressing transgenic mice. Moreover, HO-1 up-regulated 15-LO transcription and translation as well as 15-HETE in both transgenic mice and cultured pulmonary arterial smooth muscle cells (PASMCs). Results from immunoprecipitation and immunocytochemistry showed the interaction and colocalization of HO-1 and 15-LO. Together, these data suggest that HO-1 is an important upstream mediator in the hypoxia-induced 15-LO up-regulation during HPH. Unveiling the relevance of HO-1 signaling in PHP provides attractive treatment targets for HPH.

Distinct responses of lung and liver macrophages to acute endotoxemia: role of toll-like receptor 4.

Exposure to excessive quantities of bacterial-derived lipopolysaccharide (LPS) is associated with injury to the lung and the liver. Macrophages are thought to play a key role in the pathogenic response to LPS by releasing proinflammatory/cytotoxic mediators. Macrophage responses to LPS are mediated in large part by toll-like receptor 4 (TLR4). In the present studies we used C3H/HeJ mice, which possess a mutated nonfunctional TLR4, to examine its role in lung and liver macrophage responses to acute endotoxemia induced by LPS administration. Treatment of control C3H/HeOuJ mice with LPS (3 mg/ml, i.p.) was associated with a significant increase in the number of macrophages in both the lung and the liver. This was most prominent after 48 h, and was preceded by expression of proliferating cell nuclear antigen (PCNA), suggesting that macrophage proliferation contributes to the response. In liver, but not lung macrophages, LPS administration resulted in a rapid (within 3 h) increase in mRNA expression of Mn superoxide dismutase (SOD) and heme oxygenase-1 (HO-1), key enzymes in antioxidant defense. In contrast, HO-1 protein expression decreased 3 h after LPS administration in liver macrophages, while in lung macrophages it increased. mRNA expression of enzymes mediating the biosynthesis of eicosanoids, including cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase-1 (mPGES-1), but not 12/15-lipoxygenase (LOX), was upregulated in liver macrophages 3-24 h after LPS, with no effect on lung macrophages. However, COX-2 protein expression increased in both cell types. Loss of functional TLR4 significantly blunted the effects of LPS. Thus, no major changes were observed after LPS administration in the number of lung and liver macrophages recovered from TLR4 mutant mice, or on expression of PCNA. Increases in HO-1, MnSOD, COX-2 and PGES-1 mRNA expression in liver macrophages were also reduced in these mice. Conversely, in lung macrophages, loss of functional TLR4 resulted in increased expression of COX-2 protein and 12/15-LOX mRNA. These results demonstrate distinct lung and liver macrophage responses to acute endotoxemia are mediated, in part, by functional TLR4.

Inducible endothelium-derived hyperpolarizing factor: role of the 15-lipoxygenase-EDHF pathway.

Endothelium-derived hyperpolarizing factors (EDHFs) regulate vascular tone by contributing to the vasorelaxations to shear stress and endothelial agonists such as bradykinin and acetylcholine. 15(S)-Hydroxy-11,12-epoxyeicosatrienoic acid (15-H-11,12-EETA) and 11(R),12(S),15(S)-trihydroxyeicosatrienoic acid (11,12,15-THETA) are endothelial metabolites of the 15-lipoxygenase (15-LO) pathway of arachidonic acid metabolism and are EDHFs. 11,12,15-THETA activates small conductance, calcium-activated potassium channels on smooth muscle cells causing membrane hyperpolarization, and relaxation. Expression levels of 15-LO in the endothelium regulate the activity of the 15-LO/15-H-11,12-EETA/11,12,15-THETA pathway and its contribution to vascular tone. Regulation of its expression is by transcriptional, translational, and epigenetic mechanisms. Hypoxia, hypercholesterolemia, atherosclerosis, anemia, estrogen, interleukins, and possibly other hormones increase 15-LO expression. An increase in 15-LO results in increased synthesis of 15-H-11,12-EETA and 11,12,15-THETA, increased membrane hyperpolarization, and enhanced contribution to relaxation by endothelial agonists. Thus, the 15-LO pathway represents the first example of an inducible EDHF. In addition to 15-LO metabolites, a number of chemicals have been identified as EDHFs and their contributions to vascular tone vary with species and vascular bed. The reason for multiple EDHFs has evaded explanation. However, EDHF functioning as constitutive EDHFs or inducible EDHFs may explain the need for chemically and biochemically distinct pathways for EDHF activity and the variation in EDHFs between species and vascular beds. This new EDHF classification provides a framework for understanding EDHF activity in physiological and pathological conditions.

Increased invasion of malignant gliomas after 15-LO-1 and HSV-tk/ganciclovir combination gene therapy.

Recent clinical trials for malignant glioma have drawn attention to the potential therapeutic efficacy of herpes simplex virus-thymidine kinase (HSV-tk) suicide gene therapy. Nevertheless, because of the nature of these tumors, it is believed that no single treatment alone is able to combat this fatal disease. Combination therapies may provide a solution to further improve therapies against malignant gliomas. We have recently demonstrated that 15-lipoxygenase-1 (15-LO-1) is able to inhibit tumor angiogenesis as well as enhance apoptosis in tumors. As a result, we studied the potential additive/synergistic effects of 15-LO-1 gene therapy when combined with HSV-tk gene therapy for the treatment of malignant gliomas. For that, BT4C malignant glioma cells were implanted into BDIX male rats. Fourteen days after tumor cell implantation, animals were transduced using adenoviral vectors either with HSV-tk alone or in combination with 15-LO-1. The results show that the combination gene therapy neither improved inhibition of tumor growth nor did it show any benefit on survival. Instead, a profound effect on the migratory properties of the tumor cells was found, resulting in decreased survival. Similar to conventional therapies, the combination of two therapeutic genes may result in unexpected side effects, not seen when given alone.

High expression of arachidonate 15-lipoxygenase and proinflammatory markers in human ischemic heart tissue.

A common feature of the ischemic heart and atherosclerotic plaques is the presence of hypoxia (insufficient levels of oxygen in the tissue). Hypoxia has pronounced effects on almost every aspect of cell physiology, and the nuclear transcription factor hypoxia inducible factor-1α (HIF-1α) regulates adaptive responses to low concentrations of oxygen in mammalian cells. In our recent work, we observed that hypoxia increases the proinflammatory enzyme arachidonate 15-lipoxygenase (ALOX15B) in human carotid plaques. ALOX15 has recently been shown to be present in the human myocardium, but the effect of ischemia on its expression has not been investigated. Here we test the hypothesis that ischemia of the heart leads to increased expression of ALOX15, and found an almost 2-fold increase in HIF-1α mRNA expression and a 17-fold upregulation of ALOX15 mRNA expression in the ischemic heart biopsies from patients undergoing coronary bypass surgery compared with non ischemic heart tissue. To investigate the effect of low oxygen concentration on ALOX15 we incubated human vascular muscle cells in hypoxia and showed that expression of ALOX15 increased 22-fold compared with cells incubated in normoxic conditions. We also observed increased mRNA levels of proinflammatory markers in ischemic heart tissue compared with non-ischemic controls. In summary, we demonstrate increased ALOX15 in human ischemic heart biopsies. Furthermore we demonstrate that hypoxia increases ALOX15 in human muscle cells. Our results yield important insights into the underlying association between hypoxia and inflammation in the human ischemic heart disease.

Hck is a key regulator of gene expression in alternatively activated human monocytes.

IL-13 is a Th2 cytokine that promotes alternative activation (M2 polarization) in primary human monocytes. Our studies have characterized the functional IL-13 receptor complex and the downstream signaling events in response to IL-13 stimulation in alternatively activated monocytes/macrophages. In this report, we present evidence that IL-13 induces the activation of a Src family tyrosine kinase, which is required for IL-13 induction of M2 gene expression, including 15-lipoxygenase (15-LO). Our data show that Src kinase activity regulates IL-13-induced p38 MAPK tyrosine phosphorylation via the upstream kinases MKK3 or MKK6. Our findings also reveal that the IL-13 receptor-associated tyrosine kinase Jak2 is required for the activation of both Src kinase as well as p38 MAPK. Further, we found that Src tyrosine kinase-mediated activation of p38 MAPK is required for Stat1 and Stat3 serine 727 phosphorylation in alternatively activated monocytes/macrophages. Additional studies identify Hck as the specific Src family member, stimulated by IL-13 and involved in regulating both p38 MAPK activation and p38 MAPK-mediated 15-LO expression. Finally we show that the Hck regulates the expression of other alternative state (M2)-specific genes (Mannose receptor, MAO-A, and CD36) and therefore conclude that Hck acts as a key regulator controlling gene expression in alternatively activated monocytes/macrophages.

Monocyte 15-lipoxygenase gene expression requires ERK1/2 MAPK activity.

IL-13 induces profound expression of 15-lipoxygenase (15-LO) in primary human monocytes. Our studies have defined the functional IL-13R complex, association of Jaks with the receptor components, and the tyrosine phosphorylation of several Stat molecules in response to IL-13. Furthermore, we identified both p38MAPK and protein kinase Cδ as critical regulators of 15-LO expression. In this study, we report an ERK1/2-dependent signaling cascade that regulates IL-13-mediated 15-LO gene expression. We show the rapid phosphorylation/activation of ERK1/2 upon IL-13 exposure. Our results indicate that Tyk2 kinase is required for the activation of ERK1/2, which is independent of the Jak2, p38MAPK, and protein kinase Cδ pathways, suggesting bifurcating parallel regulatory pathways downstream of the receptor. To investigate the signaling mechanisms associated with the ERK1/2-dependent expression of 15-LO, we explored the involvement of transcription factors, with predicted binding sites in the 15-LO promoter, in this process including Elk1, early growth response-1 (Egr-1), and CREB. Our findings indicate that IL-13 induces Egr-1 nuclear accumulation and CREB serine phosphorylation and that both are markedly attenuated by inhibition of ERK1/2 activity. We further show that ERK1/2 activity is required for both Egr-1 and CREB DNA binding to their cognate sequences identified within the 15-LO promoter. Furthermore, by transfecting monocytes with the decoy oligodeoxyribonucleotides specific for Egr-1 and CREB, we discovered that Egr-1 and CREB are directly involved in regulating 15-LO gene expression. These studies characterize an important regulatory role for ERK1/2 in mediating IL-13-induced monocyte 15-LO expression via the transcription factors Egr-1 and CREB.

Decreased oxidative stress and greater bone anabolism in the aged, when compared to the young, murine skeleton with parathyroid hormone administration.

Because of recent insights into the pathogenesis of age-related bone loss, we investigated whether intermittent parathyroid hormone (PTH) administration antagonizes the molecular mechanisms of the adverse effects of aging on bone. Parathyroid hormone produced a greater increase in vertebral trabecular bone mineral density and bone volume as well as a greater expansion of the endocortical bone surface in the femur of 26- when compared to 6 -month-old female C57BL/6 mice. Moreover, PTH increased trabecular connectivity in vertebrae, and the toughness of both vertebrae and femora in old, but not young, mice. Parathyroid hormone also increased the rate of bone formation and reduced osteoblast apoptosis to a greater extent in the old mice. Most strikingly, PTH reduced reactive oxygen species, p66(Shc) phosphorylation, and expression of the lipoxygenase Alox15, and it increased glutathione and stimulated Wnt signaling in bone of old mice. Parathyroid hormone also antagonized the effects of oxidative stress on p66(Shc) phosphorylation, Forkhead Box O transcriptional activity, osteoblast apoptosis, and Wnt signaling in vitro. In contrast, administration of the antioxidants N-acetyl cysteine or pegylated catalase reduced osteoblast progenitors and attenuated proliferation and Wnt signaling. These results suggest that PTH has a greater bone anabolic efficacy in old age because in addition to its other positive actions on bone formation, it antagonizes the age-associated increase in oxidative stress and its adverse effects on the birth and survival of osteoblasts. On the other hand, ordinary antioxidants cannot restore bone mass in old age because they slow remodeling and attenuate osteoblastogenesis by interfering with Wnt signaling.