The impermeability of the adult blood-brain barrier (BBB) to lysosomal enzymes impedes the ability to treat the central nervous system manifestations of lysosomal storage diseases. Here, we found that simultaneous stimulation of the alpha1 and alpha2 adrenoreceptor restores in adult mice the high rate of transport for the lysosomal enzyme P-GUS that is seen in neonates but lost with development. Beta adrenergics, other monoamines, and acetylcholine did not restore this transport. A high dose (500 microg/mouse) of clonidine, a strong alpha2 and weak alpha1 agonist, was able to act as monotherapy in the stimulation of P-GUS transport. Neither use of alpha1 plus alpha2 agonists nor the high dose clonidine disrupted the BBB to albumin. In situ brain perfusion and immunohistochemistry studies indicated that adrengerics act on transporters already at the luminal surface of brain endothelial cells. These results show that adrenergic stimulation, including monotherapy with clonidine, could be key for CNS enzyme replacement therapy.

Malic enzyme 2 (ME2) is a mitochondrial enzyme that catalyzes the conversion of malate to pyruvate and CO2 and uses NAD as a cofactor. Higher expression of this enzyme correlates with the degree of cell de-differentiation. We found that ME2 is expressed in K562 erythroleukemia cells, in which a number of agents have been found to induce differentiation either along the erythroid or the myeloid lineage. We found that knockdown of ME2 led to diminished proliferation of tumor cells and increased apoptosis in vitro. These findings were accompanied by differentiation of K562 cells along the erythroid lineage, as confirmed by staining for glycophorin A and hemoglobin production. ME2 knockdown also totally abolished growth of K562 cells in nude mice. Increased ROS levels, likely reflecting increased mitochondrial production, and a decreased NADPH/NADP+ ratio were noted but use of a free radical scavenger to decrease inhibition of ROS levels did not reverse the differentiation or apoptotic phenotype, suggesting that ROS production is not causally involved in the resultant phenotype. As might be expected, depletion of ME2 induced an increase in the NAD+/NADH ratio and ATP levels fell significantly. Inhibition of the malate-aspartate shuttle was insufficient to induce K562 differentiation. We also examined several intracellular signaling pathways and expression of transcription factors and intermediate filament proteins whose expression is known to be modulated during erythroid differentiation in K562 cells. We found that silencing of ME2 leads to phospho-ERK1/2 inhibition, phospho-AKT activation, increased GATA-1 expression and diminished vimentin expression. Metabolomic analysis, conducted to gain insight into intermediary metabolic pathways that ME2 knockdown might affect, showed that ME2 depletion resulted in high orotate levels, suggesting potential impairment of pyrimidine metabolism. Collectively our data point to ME2 as a potentially novel metabolic target for leukemia therapy.

Cytochrome P450 2W1 (CYP2W1) is expressed predominantly in colorectal and also in hepatic tumors, whereas the levels are insignificant in the corresponding normal human adult tissues. CYP2W1 has been proposed as an attractive target for colorectal cancer (CRC) therapy by exploiting its ability to activate duocarmycin prodrugs to cytotoxic metabolites. However, its endogenous function, regulation and developmental pattern of expression remain unexplored. Here we report the CYP2W1 developmental expression in the murine and human gastrointestinal tissues. The gene expression in the colon and small intestine commence at early stages of embryonic life and is completely silenced shortly after the birth. Immunohistochemical analysis of human fetal colon revealed that CYP2W1 expression is restricted to the crypt cells. The silencing of CYP2W1 after birth correlates with the increased methylation of CpG-rich regions in both murine and human CYP2W1 genes. Analysis of CYP2W1 expression in the colon adenocarcinoma cell line HCC2998 revealed that the gene expression can be induced by e.g. the antitumor agent imatinib, linoleic acid and its derivatives. The imatinib mediated induction of CYP2W1 suggests an adjuvant therapy to treatment with duocarmycins that thus would involve induction of tumor CYP2W1 levels followed by the CYP2W1 activated duocarmycin prodrugs. Taken together these data strongly support further exploration of CYP2W1 as a specific drug target in CRC.

P28GST, a 28Kd glutathione S-transferase enzymatic protein derived from a schistosome helminth prevents experimental colitis when administered subcutaneously in the presence of adjuvant by decreasing pro-inflammatory Th1/Th17 response. Given the antioxidant properties of P28GST, we evaluated its anti-inflammatory potential when administered locally after colitis induction in the absence of adjuvant.

Atherosclerotic cardiovascular diseases are the leading causes of morbidity and mortality worldwide. We have previously shown that arachidonate 15-lipoxygenase B (ALOX15B) is highly expressed in atherosclerotic carotid plaques, and elucidation of mechanisms downstream of activated lipoxygenases may be relevant to our understanding of the genesis of atherosclerotic diseases. We examined 120 carotid plaques from patients with symptomatic carotid artery stenosis and showed that the extent of ALOX15B staining was significantly increased in carotid plaques with thrombosis. Impedance aggregometry analyses showed that the ALOX15B enzyme products 15-HETE and 15-HPETE increased platelet aggregation. By using a calibrated automatic thrombin assay, we showed that the ALOX15B products also increased both peak levels of thrombin and the total endogenous thrombin potential. Moreover, platelet aggregation was increased by addition of cell lysates from ischemic human macrophages, whereas platelet aggregation was reduced after knockdown of ALOX15B in human macrophages. Our data show that ALOX15B expression in human carotid plaques is associated with thrombus formation and that enzyme products of ALOX15B increase platelet aggregation and thrombin generation. We therefore propose that activated ALOX15B in macrophages may play a role in the induction of atherothrombotic events by increasing platelet aggregation and thrombin generation.

A hypometabolic state can be induced in mice by 5'-AMP administration. Previously we proposed that an underlying mechanism for this hypometabolism is linked to reduced erythrocyte oxygen transport function due to 5'-AMP uptake altering the cellular adenylate equilibrium. To test this hypothesis, we generated mice deficient in adenosine monophosphate deaminase 3 (AMPD3), the key catabolic enzyme for 5'-AMP in erythrocytes. Mice deficient in AMPD3 maintained AMPD activities in all tissues except erythrocytes. Developmentally and morphologically, the Ampd3(-/-) mice were indistinguishable from their wild type siblings. The levels of ATP, ADP but not 5'-AMP in erythrocytes of Ampd3(-/-) mice were significantly elevated. Fasting blood glucose levels of the Ampd3(-/-) mice were comparable to wild type siblings. In comparison to wild type mice, the Ampd3(-/-) mice displayed a deeper hypometabolism with a significantly delayed average arousal time in response to 5'-AMP administration. Together, these findings demonstrate a central role of AMPD3 in the regulation of 5'-AMP mediated hypometabolism and further implicate erythrocytes in this behavioral response.

Benfotiamine is a synthetic thiamine analogue that stimulates transketolase, a cellular enzyme essential for glucose metabolism. Currently, benfotiamine is used to treat diabetic neuropathy. We recently reported that oral benfotiamine induced a temporary but remarkable recovery from acute myeloid leukemia in an elderly patient who was ineligible for standard chemotherapy due to dementia and renal failure. In the present study we present evidences that benfotiamine possess antitumor activity against leukemia cells. In a panel of nine myeloid leukemia cell lines benfotiamine impaired the viability of HL-60, NB4, K562 and KG1 cells and also inhibited the growing of primary leukemic blasts. The antitumor activity of benfotiamine is not mediated by apoptosis, necrosis or autophagy, but rather occurs though paraptosis cell death induction. Mechanistic studies revealed that benfotiamine inhibited the activity of constitutively active ERK1/2 and concomitantly increased the phosphorylation of JNK1/2 kinase in leukemic cells. In addition, benfotiamine induced the down regulation of the cell cycle regulator CDK3 which resulted in G1 cell cycle arrest in the sensitive leukemic cells. Moreover, combination index studies showed that benfotiamine enhanced the antiproliferative activities of cytarabine against leukemia cells. These findings suggest that benfotiamine has antitumor therapeutic potential.

In vitro studies of drug toxicity and drug-drug interactions are crucial for drug development efforts. Currently, the utilization of primary human hepatocytes (PHHs) is the de facto standard for this purpose, due to their functional xenobiotic response and drug metabolizing CYP450 enzyme metabolism. However, PHHs are scarce, expensive, require laborious maintenance, and exhibit lot-to-lot heterogeneity. Alternative human in vitro platforms include hepatic cell lines, which are easy to access and maintain, and induced pluripotent stem cell (iPSC) derived hepatocytes. In this study, we provide a direct comparison of drug induced CYP3A4 and PXR expression levels of PHHs, hepatic cell lines Huh7 and HepG2, and iPSC derived hepatocyte like cells. Confluently cultured Huh7s exhibited an improved CYP3A4 expression and were inducible by up to 4.9-fold, and hepatocytes differentiated from human iPSCs displayed a 3.3-fold CYP3A4 induction. In addition, an increase in PXR expression levels was observed in both hepatic cell lines and iPSC derived hepatocytes upon rifampicin treatment, whereas a reproducible increase in PXR expression was not achieved in PHHs. Our results indicate that both hepatoma originated cell lines and iPSCs may provide alternative sources to primary hepatocytes, providing reliable and reproducible results for CYP3A4/PXR metabolism, upon in vitro maturation. This study may serve as a guide for the selection of suitable and feasible in vitro platforms for drug-drug interaction and toxicology studies.

Insulin-degrading enzyme (IDE) is an atypical zinc-metalloendopeptidase that hydrolyzes insulin and other intermediate-sized peptide hormones, many of which are implicated in skin health and wound healing. Pharmacological inhibitors of IDE administered internally have been shown to slow the breakdown of insulin and thereby potentiate insulin action. Given the importance of insulin and other IDE substrates for a variety of dermatological processes, pharmacological inhibitors of IDE suitable for topical applications would be expected to hold significant therapeutic and cosmetic potential. Existing IDE inhibitors, however, are prohibitively expensive, difficult to synthesize and of undetermined toxicity. Here we used phage display to discover novel peptidic inhibitors of IDE, which were subsequently characterized in vitro and in cell culture assays. Among several peptide sequences tested, a cyclic dodecapeptide dubbed P12-3A was found to potently inhibit the degradation of insulin (Ki = 2.5 ± 0.31 μM) and other substrates by IDE, while also being resistant to degradation, stable in biological milieu, and highly selective for IDE. In cell culture, P12-3A was shown to potentiate several insulin-induced processes, including the transcription, translation and secretion of alpha-1 type I collagen in primary murine skin fibroblasts, and the migration of keratinocytes in a scratch wound migration assay. By virtue of its potency, stability, specificity for IDE, low cost of synthesis, and demonstrated ability to potentiate insulin-induced processes involved in wound healing and skin health, P12-3A holds significant therapeutic and cosmetic potential for topical applications.

Prolidase is the only human enzyme responsible for the digestion of iminodipeptides containing proline or hydroxyproline at their C-terminal end, being a key player in extracellular matrix remodeling. Prolidase deficiency (PD) is an intractable loss of function disease, characterized by mutations in the prolidase gene. The exact causes of activity impairment in mutant prolidase are still unknown. We generated three recombinant prolidase forms, hRecProl-231delY, hRecProl-E412K and hRecProl-G448R, reproducing three mutations identified in homozygous PD patients. The enzymes showed very low catalytic efficiency, thermal instability and changes in protein conformation. No variation of Mn(II) cofactor affinity was detected for hRecProl-E412K; a compromised ability to bind the cofactor was found in hRecProl-231delY and Mn(II) was totally absent in hRecProl-G448R. Furthermore, local structure perturbations for all three mutants were predicted by in silico analysis. Our biochemical investigation of the three causative alleles identified in perturbed folding/instability, and in consequent partial prolidase degradation, the main reasons for enzyme inactivity. Based on the above considerations we were able to rescue part of the prolidase activity in patients' fibroblasts through the induction of Heath Shock Proteins expression, hinting at new promising avenues for PD treatment.

Vascular mineralization contributes to the high cardiovascular morbidity and mortality in patients who suffer from chronic kidney disease and in individuals who have undergone solid organ transplantation. The immunosuppressive regimen used to treat these patients appears to have an impact on vascular alterations. The effect of 6-mercaptopurine (6-MP) on vascular calcification has not yet been determined. This study investigates the effect of 6-MP on vascular mineralization by the induction of trans-differentiation of rat vascular smooth muscle cells in vitro. 6-MP not only induces the expression of osteo-chondrocyte-like transcription factors and proteins but also activates alkaline phosphatase enzyme activity and produces calcium deposition in in vitro and ex vivo models. These processes are dependent on 6-MP-induced production of reactive oxygen species, intracellular activation of mitogen-activated kinases and phosphorylation of the transcription factor Cbfa1. Furthermore, the metabolic products of 6-MP, 6-thioguanine nucleotides and 6-methyl-thio-inosine monophosphate have major impacts on cellular calcification. These data provide evidence for a possible harmful effect of the immunosuppressive drug 6-MP in vascular diseases, such as arteriosclerosis.

The Hypoxia-inducible Factor (HIF) family of transcriptional regulators coordinates the expression of dozens of genes in response to oxygen deprivation. Mammalian development occurs in a hypoxic environment and HIF-null mice therefore die in utero due to multiple embryonic and placental defects. Mouse embryonic stem cells do not differentiate into placental cells; therefore, trophoblast stem cells (TSCs) are used to study mouse placental development. Consistent with a requirement for HIF activity during placental development in utero, TSCs derived from HIF-null mice exhibit severe differentiation defects and fail to form trophoblast giant cells (TGCs) in vitro. Interestingly, differentiating TSCs induce HIF activity independent of oxygen tension via unclear mechanisms. Here, we show that altering the extracellular matrix (ECM) composition upon which TSCs are cultured changes their differentiation potential from TGCs to multinucleated syncytiotropholasts (SynTs) and blocks oxygen-independent HIF induction. We further find that modulation of Mitogen Activated Protein Kinase Kinase-1/2 (MAP2K1/2, MEK-1/2) signaling by ECM composition is responsible for this effect. In the absence of ECM-dependent cues, hypoxia-signaling pathways activate this MAPK cascade to drive HIF induction and redirect TSC fate along the TGC lineage. In addition, we show that integrity of the microtubule and actin cytoskeleton is critical for TGC fate determination. HIF-2α ensures TSC cytoskeletal integrity and promotes invasive TGC formation by interacting with c-MYC to induce non-canonical expression of Lim domain kinase 1-an enzyme that regulates microtubule and actin stability, as well as cell invasion. Thus, we find that HIF can integrate positional and metabolic cues from within the TSC niche to regulate placental development by modulating the cellular cytoskeleton via non-canonical gene expression.

This study is to investigate the mechanisms by which macrophage-activating lipopeptide-2 (MALP-2) induces heme oxygenase (HO)-1, a cytoprotective enzyme that catalyzes the degradation of heme, in human monocytes.

We evaluated 7 C. muridarum ORFs for their ability to induce protection against chlamydial infection in a mouse intravaginal infection model. These antigens, although encoded in C. muridarum genome, are transcriptionally regulated by a cryptic plasmid that is known to contribute to C. muridarum pathogenesis. Of the 7 plasmid-regulated ORFs, the chlamydial glycogen phosphorylase or GlgP, when delivered into mice intramuscularly, induced the most pronounced protective immunity against C. muridarum intravaginal infection. The GlgP-immunized mice displayed a significant reduction in vaginal shedding of live organisms on day 14 after infection. The protection correlated well with a robust C. muridarum-specific antibody and a Th1-dominant T cell responses, which significantly reduced the severity but not overall incidence of hydrosalpinx. The GlgP-induced partial protection against upper genital tract pathology suggests that GlgP may be considered a component for a multi-subunit vaccine. These results have demonstrated that intramuscular immunization of mice with purified proteins can be used to identify vaccine antigens for preventing intravaginal infection with C. trachomatis in humans.

Protein ubiquitylation is a dynamic process that affects the function and stability of proteins and controls essential cellular processes ranging from cell proliferation to cell death. This process is regulated through the balanced action of E3 ubiquitin ligases and deubiquitylating enzymes (DUB) which conjugate ubiquitins to, and remove them from target proteins, respectively. Our genetic analysis has revealed that the deubiquitylating enzyme DmUsp5 is required for maintenance of the ubiquitin equilibrium, cell survival and normal development in Drosophila. Loss of the DmUsp5 function leads to late larval lethality accompanied by the induction of apoptosis. Detailed analyses at a cellular level demonstrated that DmUsp5 mutants carry multiple abnormalities, including a drop in the free monoubiquitin level, the excessive accumulation of free polyubiquitins, polyubiquitylated proteins and subunits of the 26S proteasome. A shortage in free ubiquitins results in the induction of a ubiquitin stress response previously described only in the unicellular budding yeast. It is characterized by the induction of the proteasome-associated deubiquitylase DmUsp14 and sensitivity to cycloheximide. Removal of DmUsp5 also activates the pro-apoptotic machinery thereby resulting in widespread apoptosis, indicative of an anti-apoptotic role of DmUsp5. Collectively, the pleiotropic effects of a loss of DmUsp5 function can be explained in terms of the existence of a limited pool of free monoubiquitins which makes the ubiquitin-dependent processes mutually interdependent.

In the search of new strategies to fight against obesity, we targeted a gene pathway involved in energy uptake. We have thus investigated the APOB mRNA editing protein (APOBEC1) gene pathway that is involved in fat absorption in the intestine. The APOB gene encodes two proteins, APOB100 and APOB48, via the editing of a single nucleotide in the APOB mRNA by the APOBEC1 enzyme. The APOB48 protein is mandatory for the synthesis of chylomicrons by intestinal cells to transport dietary lipids and cholesterol. We produced transgenic rabbits expressing permanently and ubiquitously a small hairpin RNA targeting the rabbit APOBEC1 mRNA. These rabbits exhibited a moderately but significantly reduced level of APOBEC1 gene expression in the intestine, a reduced level of editing of the APOB mRNA, a reduced level of synthesis of chylomicrons after a food challenge, a reduced total mass of body lipids and finally presented a sustained lean phenotype without any obvious physiological disorder. Interestingly, no compensatory mechanism opposed to the phenotype. These lean transgenic rabbits were crossed with transgenic rabbits expressing in the intestine the human APOBEC1 gene. Double transgenic animals did not present any lean phenotype, thus proving that the intestinal expression of the human APOBEC1 transgene was able to counterbalance the reduction of the rabbit APOBEC1 gene expression. Thus, a moderate reduction of the APOBEC1 dependent editing induces a lean phenotype at least in the rabbit species. This suggests that the APOBEC1 gene might be a novel target for obesity treatment.

Greater than 75% of all hematologic malignancies derive from germinal center (GC) or post-GC B cells, suggesting that the GC reaction predisposes B cells to tumorigenesis. Because GC B cells acquire expression of the highly mutagenic enzyme activation-induced cytidine deaminase (AID), GC B cells may require additional DNA repair capacity. The goal of this study was to investigate whether normal human B cells acquire enhanced expression of DNA repair factors upon AID induction. We first demonstrated that several DNA mismatch repair, homologous recombination, base excision repair, and ATR signaling genes were overexpressed in GC B cells relative to naïve and memory B cells, reflecting activation of a process we have termed somatic hyperrepair (SHR). Using an in vitro system, we next characterized activation signals required to induce AID expression and SHR. Although AID expression was induced by a variety of polyclonal activators, SHR induction strictly required signals provided by contact with activated CD4+ T cells, and B cells activated in this manner displayed reduced levels of DNA damage-induced apoptosis. We further show the induction of SHR is independent of AID expression, as GC B cells from AID-/-mice retained heightened expression of SHR proteins. In consideration of the critical role that CD4+ T cells play in inducing the SHR process, our data suggest a novel role for CD4+ T cells in the tumor suppression of GC/post-GC B cells.

Cigarette smoking is a major pathogenic factor in lung cancer. Macrophages play an important role in host defense and adaptive immunity. These cells display diverse phenotypes for performing different functions. M2 type macrophages usually exhibit immunosuppressive and tumor-promoting characteristics. Although macrophage polarization toward the M2 phenotype has been observed in the lungs of cigarette smokers, the molecular basis of the process remains unclear. In this study, we evaluated the possible mechanisms for the polarization of mouse macrophages that are induced by cigarette smoking (CS) or cigarette smoke extract (CSE). The results showed that exposure to CSE suppressed the production of reactive oxygen species (ROS) and nitric oxide (NO) and down-regulated the phagocytic ability of Ana-1 cells. The CD163 expressions on the surface of macrophages from different sources were significantly increased in in vivo and in vitro studies. The M1 macrophage cytokines TNF-α, IL-12p40 and enzyme iNOS decreased in the culture supernatant, and their mRNA levels decreased depending on the time and concentration of CSE. In contrast, the M2 phenotype macrophage cytokines IL-10, IL-6, TGF-β1 and TGF-β2 were up-regulated. Moreover, phosphorylation of JAK2 and STAT3 was observed after the Ana-1 cells were treated with CSE. In addition, pretreating the Ana-1 cells with the STAT3 phosphorylation inhibitor WP1066 inhibited the CSE-induced CD163 expression, increased the mRNA level of IL-10 and significantly decreased the mRNA level of IL-12. In conclusion, we demonstrated that the M2 polarization of macrophages induced by CS could be mediated through JAK2/STAT3 pathway activation.

When oxygen becomes limiting, cells reduce mitochondrial respiration and increase ATP production through anaerobic fermentation of glucose. The Hypoxia Inducible Factors (HIFs) play a key role in this metabolic shift by regulating the transcription of key enzymes of glucose metabolism. Here we show that oxygen regulates the expression of the muscle glycogen synthase (GYS1). Hypoxic GYS1 induction requires HIF activity and a Hypoxia Response Element within its promoter. GYS1 gene induction correlated with a significant increase in glycogen synthase activity and glycogen accumulation in cells exposed to hypoxia. Significantly, knockdown of either HIF1alpha or GYS1 attenuated hypoxia-induced glycogen accumulation, while GYS1 overexpression was sufficient to mimic this effect. Altogether, these results indicate that GYS1 regulation by HIF plays a central role in the hypoxic accumulation of glycogen. Importantly, we found that hypoxia also upregulates the expression of UTP:glucose-1-phosphate urydylyltransferase (UGP2) and 1,4-alpha glucan branching enzyme (GBE1), two enzymes involved in the biosynthesis of glycogen. Therefore, hypoxia regulates almost all the enzymes involved in glycogen metabolism in a coordinated fashion, leading to its accumulation. Finally, we demonstrated that abrogation of glycogen synthesis, by knock-down of GYS1 expression, impairs hypoxic preconditioning, suggesting a physiological role for the glycogen accumulated during chronic hypoxia. In summary, our results uncover a novel effect of hypoxia on glucose metabolism, further supporting the central importance of metabolic reprogramming in the cellular adaptation to hypoxia.

Influenza infection stimulates protective host immune responses but paradoxically enhances lung indoleamine 2,3 dioxygenase (IDO) activity, an enzyme that suppresses helper/effector T cells and activates Foxp3-lineage regulatory CD4 T cells (Tregs). Influenza A/PR/8/34 (PR8) infection stimulated rapid elevation of IDO activity in lungs and lung-draining mediastinal lymph nodes (msLNs). Mice lacking intact IDO1 genes (IDO1-KO mice) exhibited significantly lower morbidity after sub-lethal PR8 infection, and genetic or pharmacologic IDO ablation led to much faster recovery after virus clearance. More robust influenza-specific effector CD8 T cell responses manifested in lungs of PR8-infected IDO1-KO mice, though virus clearance rates were unaffected by IDO ablation. Similar outcomes manifested in mice infected with a less virulent influenza A strain (X31). IDO induction in X31-infected lungs was dependent on IFN type II (IFNγ) signaling and was restricted to non-hematopoietic cells, while redundant IFN type 1 or type II signaling induced IDO exclusively in hematopoietic cells from msLNs. Memory T cells generated in X31-primed IDO1-KO mice protected mice from subsequent challenge with lethal doses of PR8 (100×LD50). However recall T cell responses were less robust in lung interstitial tissues, and classic dominance of TCR Vβ8.3 chain usage amongst memory CD8(+) T cells specific for influenza nucleoprotein (NP366) did not manifest in IDO1-KO mice. Thus, influenza induced IDO activity in lungs enhanced morbidity, slowed recovery, restrained effector T cell responses in lungs and shaped memory T cell repertoire generation, but did not attenuate virus clearance during primary influenza A infection.