Assessing the double-edged of extracellular signal-regulated kinase/CCAAT-enhancer-binding protein beta signaling pathway in arsenic-induced skin damage and its potential foodborne interventions.

Arsenic exposure is a major environmental public health challenge worldwide. As typical manifestations for arsenic exposure, the pathogenesis of arsenic-induced skin lesions has not been fully elucidated, as well as the lack of effective control measures. In this study, we first determined the short-term and high-dose arsenic exposure can increase the apoptosis rates, while long-term low-dose arsenic exposure decrease the apoptosis rates. Then, the HaCaT cells with knockdown and overexpression of CCAAT-enhancer-binding protein ß (CEBPB) and extracellular signal-regulated kinase (ERK) were constructed. The results demonstrate that knockdown of CEBPB and ERK can reduce NaAsO2 -induced cell apoptosis by inhibiting ERK/CEBPB signaling pathway and vice versa. Further cells were treated with Kaji-Ichigoside F1 (KF1). The results clearly show that KF1 can decrease the arsenic-induced cell apoptosis rates and the expression of ERK/CEBPB signaling pathway-related genes. These results provide evidence that ERK/CEBPB signaling pathway acts as a double-edged sword in arsenic-induced skin damage. Another interesting finding was that KF1 can alleviate arsenic-induced skin cell apoptosis by inhibiting the ERK/CEBPB signaling pathway. This study will contribute to a deeper understanding of the mechanisms of arsenic-induced skin cell apoptosis, and our findings will help to identify a potential food-borne intervention in arsenic detoxification.

Mechanistic Study of Macranthoside B Effects on Apoptotic Cell Death in Human Cervical Adenocarcinoma Cells.

Macranthoside B (MB) is a triterpenoid saponin extracted from Lonicera macranthoides, a traditional Chinese medicine. In the current study, we investigated the anticancer potential of MB in various cancer cells and elucidated its underlying mechanisms. MB exposure inhibited cell proliferation, induced mitochondrial membrane potential (MMP) loss, increased sub-G1 accumulation, and resulted in cleavage of caspase-3 and PARP, which are reflective of apoptosis. In HeLa cells, MB induced down-regulation of SOD2 and GPx1, phosphorylation of Akt and PDK1, and thus promoted ROS-mediated apoptosis. This was further supported by the protection of sub-G1 accumulation, MMP loss, cleavage of caspase-3 and PARP in the presence of N-acetylcysteine (NAC). Additionally, MB induced cell death via down-regulation of ubiquitin-like with PHD and ringfinger domains 1 (UHRF1) and Bcl-xL. Taken together, this study provides a new insight into the apoptosis- inducing potential of MB, and its molecular mechanisms are associated with an increase in oxidative stress and inhibition of the PDK1/Akt pathway.

The anti-lipidemic drug simvastatin modifies epigenetic biomarkers in the amphipod Gammarus locusta.

The adverse effects of certain environmental chemicals have been recently associated with the modulation of the epigenome. Although changes in the epigenetic signature have yet to be integrated into hazard and risk assessment, they are interesting candidates to link environmental exposures and altered phenotypes, since these changes may be passed across multiple non-exposed generations. Here, we addressed the effects of simvastatin (SIM), one of the most prescribed pharmaceuticals in the world, on epigenetic regulation using the amphipod Gammarus locusta as a proxy, to support its integration into hazard and environmental risk assessment. SIM is a known modulator of the epigenome in mammalian cell lines and has been reported to impact G. locusta ecological endpoints at environmentally relevant levels. G. locusta juveniles were exposed to three SIM environmentally relevant concentrations (0.32, 1.6 and 8 µg L-1) for 15 days. Gene transcription levels of selected epigenetic regulators, i.e., dnmt1, dmap1, usp7, kat5 and uhrf1 were assessed, along with the quantification of DNA methylation levels and evaluation of key ecological endpoints: survival and growth. Exposure to 0.32 and 8 µg L-1 SIM induced significant downregulation of DNA methyltransferase 1 (dnmt1), concomitant with global DNA hypomethylation and growth impacts. Overall, this work is the first to validate the basal expression of key epigenetic regulators in a keystone marine crustacean, supporting the integration of epigenetic biomarkers into hazard assessment frameworks.

USP7 inhibitors suppress tumour neoangiogenesis and promote synergy with immune checkpoint inhibitors by downregulating fibroblast VEGF.

BACKGROUND: Understanding how to modulate the microenvironment of tumors that are resistant to immune checkpoint inhibitors represents a major challenge in oncology.Here we investigate the ability of USP7 inhibitors to reprogram the tumor microenvironment (TME) by inhibiting secretion of vascular endothelial growth factor (VEGF) from fibroblasts. METHODS: To understand the role played by USP7 in the TME, we systematically evaluated the effects of potent, selective USP7 inhibitors on co-cultures comprising components of the TME, using human primary cells. We also evaluated the effects of USP7 inhibition on tumor growth inhibition in syngeneic models when dosed in combination with immune checkpoint inhibitors (ICIs). RESULTS: Abrogation of VEGF secretion from fibroblasts in response to USP7 inhibition resulted in inhibition of tumor neoangiogenesis and increased tumor recruitment of CD8-positive T-lymphocytes, leading to significantly improved sensitivity to immune checkpoint inhibitors. In syngeneic models, treatment with USP7 inhibitors led to striking tumor responses resulting in significantly improved survival. CONCLUSIONS: USP7-mediated reprograming of the TME is not linked to its previously characterized role in modulating MDM2 but does require p53 and UHRF1 in addition to the well-characterized VEGF transcription factor, HIF-1α. This represents a function of USP7 that is unique to fibroblasts, and which is not observed in cancer cells or other components of the TME. Given the potential for USP7 inhibitors to transform "immune desert" tumors into "immune responsive" tumors, this paves the way for a novel therapeutic strategy combining USP7 inhibitors with immune checkpoint inhibitors (ICIs).

Molecular Mechanisms Responsible for In Vitro Cytotoxic Attributes of Conyza bonariensis Extract against Lymphoblastic Leukaemia Jurkat Cells.

BACKGROUND: Conyza bonariensis is known to have anti-cancer properties. OBJECTIVE: The current study investigated the in vitro pro-apoptotic properties of Conyza bonariensis (C. bonariensis) towards human lymphoblastic leukemia Jurkat cells. METHODS: Ariel parts of C. bonariensis were macerated in a non-polar (n-Hexane) solvent. MTS cell viability assay was employed to determine the cytotoxic activity of the extract towards human leukemia Jurket cells and normal Peripheral Blood Mononuclear Cells (PBMCs). The phytochemical composition of the extract was screened using HPLC method. Flow cytometric studies (FACS) were conducted to explore the pro-apoptotic potential of the extract. Western blot studies were employed to identify the molecular targets involved in the induction of apoptosis. RESULTS: The n-hexane extract showed selective cytotoxic activity towards Jurkat cells. FACS analysis indicated that the extract induced early and late apoptosis in Jurkat cells. Western blot studies revealed that the extract downregulated the expression of DNMT1, SIRT1, and UHRF1 with a simultaneous up-regulation of p73 and caspases-3 proteins expression. HPLC characterization of the extract revealed the presence of phenolic compounds. CONCLUSION: Overall, these findings demonstrate that the anti-cancer effects of a Conyza bonariensis extract towards human lymphoblastic leukemia Jurkat cells are due to the modulation of the activity of multiple oncogenic and tumor suppressor proteins. Phenolic contents of the extract are proposed to be responsible for these activities.

Selective deletion of adipocytes, but not preadipocytes, by TNF-alpha through C/EBP- and PPARgamma-mediated suppression of NF-kappaB.

Tumor necrosis factor-alpha (TNF-alpha) is a key regulator of adipose tissue mass, but mechanisms underlying this effect have not been fully elucidated. We found that exposure to TNF-alpha caused a significant decrease in the number of adipocytes, but not preadipocytes. Subsequent experiments revealed that TNF-alpha selectively deleted adipocytes through induction of apoptosis. Following exposure to TNF-alpha, rapid activation of nuclear factor-kappaB (NF-kappaB) was observed only in preadipocytes, but not in adipocytes. Inhibition of NF-kappaB rendered preadipocytes susceptible to TNF-alpha-induced apoptosis, suggesting that different activity of NF-kappaB is the determinant for the distinct apoptotic responses. During adipocyte differentiation, expression and activity of peroxisome proliferator-activated receptor-gamma (PPARgamma) were upregulated. Treatment of preadipocytes with a PPARgamma agonist attenuated NF-kappaB activation and rendered the cells vulnerable to TNF-alpha-induced apoptosis. Conversely, treatment of adipocytes with a PPARgamma antagonist enhanced NF-kappaB activation and conferred resistance to TNF-alpha-induced apoptosis, suggesting involvement of PPARgamma in the suppression of NF-kappaB in adipocytes. We also found that, following differentiation, expression and activity of CCAAT/enhancer binding protein (C/EBP), especially C/EBPalpha and C/EBPbeta, were upregulated in adipocytes. Overexpression of individual C/EBPs significantly inhibited activation of NF-kappaB in preadipocytes. Furthermore, transfection with siRNA for C/EBPalpha or C/EBPbeta enhanced activity of NF-kappaB in adipocytes, suggesting that C/EBP is also involved in the repression of NF-kappaB in adipocytes. These results suggested novel mechanisms by which TNF-alpha selectively deletes adipocytes in the adipose tissue. The C/EBP- and PPARgamma-mediated suppression of NF-kappaB may contribute to TNF-alpha-related loss of adipose tissue mass under certain pathological situations, such as cachexia.

Regulation of gene expression by SREBP and SCAP.

Sterol regulatory element binding proteins (SREBPs) function as transcription factors that activate specific genes involved in cholesterol synthesis, endocytosis of low density lipoproteins, the synthesis of both saturated and unsaturated fatty acids and glucose metabolism. As such, these proteins provide a link between lipid and carbohydrate metabolism. There are three SREBPs, SREBP-1a, SREBP-1c and SREBP-2, that are encoded by two genes. SREBPs are synthesized as 125 kDa precursor proteins that are localized to the endoplasmic reticulum. The precursor is transported to the Golgi by a chaperone protein (SREBP-cleavage activating protein) and then cleaved by two proteases to release the mature, transcriptionally active 68 kDa amino terminal domain. Recent studies have shown that formation of mature SREBP is controlled at multiple levels in response to changes in the levels of oxysterols, insulin/glucose and polyunsaturated fatty acids. These recent findings have important clinical implications relevant to hyperlipidemia and diabetes and are the topic of this review.

Structure and regulation of mammalian squalene synthase.

Mammalian squalene synthase (SQS) catalyzes the first reaction of the branch of the isoprenoid metabolic pathway committed specifically to sterol biosynthesis. SQS produces squalene in an unusual two-step reaction in which two molecules of farnesyl diphosphate are condensed head-to-head. Recent studies have advanced understanding of the reaction mechanism, the functional domains of the enzyme, and transcriptional regulation of the gene. Site-directed mutagenesis has identified conserved Asp, Tyr, and Phe residues that are essential for SQS activity. The Asp residues are hypothesized to be required for substrate binding; the Tyr and Phe residues may stabilize carbocation reaction intermediates. The elucidation of SQS crystal structure will most likely direct future research on the relationship between enzyme structure and function. SQS activity, protein, and mRNA levels are regulated by cholesterol status and by the cytokines TNF-alpha and IL-1beta. Activation of the SQS promoter in response to cholesterol deficit is mediated by sterol regulatory element binding proteins SREBP-1a and SREBP-2. The precise contributions made by individual SREBPs and accessory transcription factors to SQS transcriptional control, and the mechanisms underlying cytokine regulation of SQS are major foci of current research.

Cholesterol and hepatic lipoprotein assembly and secretion.

The assembly and secretion of apo B100 containing lipoproteins (i.e., VLDL) by the liver and cholesterol metabolism are interrelated on several different levels and for several different physiologic reasons. Firstly, hepatic VLDL is the major precursor for LDL, which in the human is the major vehicle responsible for transporting cholesterol to peripheral tissues. Secondly, cholesterol is supplied to many tissues by a specific uptake of LDL via LDL receptor, which is expressed in a regulated manner by most mammalian tissues. Thirdly, the rate of hepatic cholesterol biosynthesis and metabolism to bile acids correlates with production of VLDL. This apparent coordinate expression of cholesterol biosynthetic/catabolic enzymes and hepatic VLDL assembly/secretion are mediated at least in part through the sterol response element binding protein (SREBP) transcription factor family. Their gene targets include a plethora of enzymes that regulate glycolysis, energy production, lipogenesis and cholesterol catabolism. Studies of hepatoma cells overexpressing CYP7A1, the rate-limiting enzyme controlling bile acid synthesis, show that as a result of increased mature SREBP1, there is a coordinate induction of lipogenesis and the assembly and secretion of VLDL. These and additional studies show that the bile acid synthetic pathway and the VLDL assembly/secretion pathway are coordinately linked through SREBP-dependent transcription. Based on studies showing that within the liver acinus, the expression of CYP7A1 is mainly in the pericentral region while HMG-CoA reductase is mainly periportal, we propose that a 'metabolic zonal segregation' plays an important role in coordinate regulation of cholesterol and VLDL metabolism. This putative 'metabolic zonal segregation' may provide segregation of metabolic functions which may be mutually antagonistic. For example, there may be physiologic states in which the bile acid synthetic pathway may compete with the VLDL assembly/secretion pathway for a limited amount of cholesterol. Metabolic antagonism (e.g., competition for cholesterol) may be avoided via inducing SREBP-mediated transcription. Adaptation of catabolic hepatocytes to accommodate the expression of VLDL assembly/secretion may occur in response to activation of SREBP-mediated transcription. Support for these is discussed.

Sterols and gene expression: control of affluence.

Intracellular and extracellular cholesterol levels are tightly maintained within a narrow concentration range by an intricate transcriptional control mechanism. Excess cholesterol can be converted into oxysterols, signaling molecules, which modulate the activity of a number of transcription factors, as to limit accumulation of excess of cholesterol. Two key regulatory pathways are affected by oxysterols. The first pathway involves the uptake and de novo synthesis of cholesterol and is controlled by the family of sterol response element binding proteins, whose activity is regulated by a sterol-dependent feedback mechanism. The second pathway, which only recently has become a topic of interest, involves the activation by a feedforward mechanism of cholesterol utilization for either bile acid or steroid hormone synthesis by oxysterol-activated nuclear receptors, such as liver X receptor and steroidogenic factor-1. Furthermore, biosynthesis and enterohepatic reabsorption of bile acids are regulated by the farnesol X receptor, a receptor activated by bile acids. Both the feedback inhibition of cholesterol uptake and production and the stimulation of cholesterol utilization will ultimately result in a lowering of the intracellular cholesterol concentration and allow for a fine-tuned regulation of the cholesterol concentration.

Adenovirus-mediated overexpression of sterol regulatory element binding protein-1c mimics insulin effects on hepatic gene expression and glucose homeostasis in diabetic mice.

In vitro, the transcription factor sterol regulatory element binding protein-1c (SREBP-1c) mimics the positive effects of insulin on hepatic genes involved in glucose utilization, such as glucokinase (GK) and enzymes of the lipogenic pathway, suggesting that it is a key factor in the control of hepatic glucose metabolism. Decreased glucose utilization and increased glucose production by the liver play an important role in the development of the hyperglycemia in diabetic states. We thus reasoned that if SREBP-1c is indeed a mediator of hepatic insulin action, a hepatic targeted overexpression of SREBP-1c should greatly improve glucose homeostasis in diabetic mice. This was achieved by injecting streptozotocin-induced diabetic mice with a recombinant adenovirus containing the cDNA of the mature, transcriptionally active form of SREBP-1c. We show here that overexpressing SREBP-1c specifically in the liver of diabetic mice induces GK and lipogenic enzyme gene expression and represses the expression of phosphoenolpyruvate carboxykinase, a key enzyme of the gluconeogenic pathway. This in turn increases glycogen and triglyceride hepatic content and leads to a marked decrease in hyperglycemia in diabetic mice. We conclude that SREBP-1c has a major role in vivo in the long-term control of glucose homeostasis by insulin.

Sterol regulatory element binding protein-1c expression and action in rat muscles: insulin-like effects on the control of glycolytic and lipogenic enzymes and UCP3 gene expression.

Sterol regulatory element binding protein-1c (SREBP-1c) is a transcription factor that mediates insulin effects on hepatic gene expression. It is itself transcriptionally stimulated by insulin in hepatocytes. Here we show that SREBP-1c mRNA is expressed in adult rat skeletal muscles and that this expression is decreased by diabetes. The regulation of SREBP-1c expression was then assessed in cultures of adult muscle satellite cells. These cells form spontaneously contracting multinucleated myotubes within 7 days of culture. SREBP-1c mRNA is expressed in contracting myotubes. A 4-h treatment with 100 nmol/l insulin increases SREBP-1c expression and nuclear abundance by two- to threefold in myotubes. In cultured myotubes, insulin increases the expression of glycolytic and lipogenic enzyme genes and inhibits the 9-cis retinoic acid-induced UCP3 expression. These effects of insulin are mimicked by adenovirus-mediated expression of a transcriptionally active form of SREBP-1c. We conclude that in skeletal muscles, SREBP-1c expression is sensitive to insulin and can transduce the positive and negative actions of the hormone on specific genes and thus has a pivotal role in long-term muscle insulin sensitivity.

Protein kinase A-dependent stimulation of rat type II secreted phospholipase A(2) gene transcription involves C/EBP-beta and -delta in vascular smooth muscle cells.

Type II secreted phospholipase A(2) (sPLA(2)) releases precursors of important inflammatory lipid mediators from phospholipids. Some observations have indicated that the sPLA(2), which has been implicated in chronic inflammatory conditions such as arthritis, contributes to atherosclerosis in the arterial wall. sPLA(2) was not detected in control vascular smooth muscle cells (VSMC). Treatment of VSMC with agents that increase intracellular cAMP (eg, forskolin, dibutyryl [db]-cAMP) resulted in a time- and concentration-dependent increase in sPLA(2) gene expression. Semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) showed a marked dose-dependent inhibition of forskolin-induced mRNA by protein kinase A inhibitor. Electrophoretic mobility shift analysis of nuclear proteins from forskolin-treated and db-cAMP-treated VSMC with C/EBP consensus oligonucleotides and C/EBP oligonucleotides from the rat promoter revealed greater binding than in control VSMC. Incubation of VSMC with H89, a specific protein kinase inhibitor, also blocked the binding of nuclear C/EBP to the C/EBP site of the rat promoter induced by db-cAMP and forskolin. Binding was unchanged with the use of CRE consensus oligonucleotides. Antibodies revealed the specific formation of C/EBP/DNA complexes, the majority of which were supershifted by C/EBP-ss and -delta antibodies. Functional activation of C/EBP was confirmed by a luciferase reporter gene assay. A construct comprising 4 tandem repeat copies of the C/EBP element from the rat sPLA(2) promoter linked to luciferase was transcriptionally activated in VSMC by cotransfection with expression vector for the protein kinase A catalytic subunit. It was also significantly activated in transfected VSMC treated by forskolin or db-cAMP. H89 inhibited this activations. We therefore conclude that the increases in sPLA(2) mRNA and enzyme activity produced by cAMP-elevating agents is controlled by a mechanism involving nuclear C/EBP-ss and -delta acting through a protein kinase A signaling pathway.

CCAAT/enhancer binding protein homologous protein (DDIT3) induces osteoblastic cell differentiation.

CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP/DDIT3), a member of the C/EBP family of transcription factors, plays a role in cell survival and differentiation. CHOP/DDIT3 binds to C/EBPs to form heterodimers that do not bind to consensus Cebp sequences, acting as a dominant-negative inhibitor. CHOP/DDIT3 blocks adipogenesis, and we postulated it could induce osteoblastogenesis. We investigated the effects of constitutive CHOP/DDIT3 overexpression in murine ST-2 stromal cells transduced with retroviral vectors. ST-2 cells differentiated toward osteoblasts, and CHOP/DDIT3 accelerated and enhanced the appearance of mineralized nodules, and the expression of osteocalcin and alkaline phosphatase mRNAs, particularly in the presence of bone morphogenetic protein-2. CHOP/DDIT3 overexpression opposed adipogenesis, and did not cause substantial changes in cell number. CHOP/DDIT3 overexpression did not modify C/EBPalpha or -beta mRNA levels but decreased C/EBPdelta after 24 d of culture. Electrophoretic mobility shift and supershift assays demonstrated that overexpression of CHOP/DDIT3 decreased the binding of C/EBPs to their consensus sequence by interacting with C/EBPalpha and -beta, confirming its dominant-negative role. In addition, CHOP/DDIT3 enhanced bone morphogenetic protein-2/Smad signaling. In conclusion, CHOP/DDIT3 enhances osteoblastic differentiation of stromal cells, in part by interacting with C/EBPalpha and -beta and also by enhancing Smad signaling.

Sox21 is a repressor of neuronal differentiation and is antagonized by YB-1.

Sox21, a high-mobility group box transcription factor, is expressed throughout the immature neural stem/progenitor population in ventricular zone but not in the cortical plate in embryonic mouse brain and its expression is restricted to the subventricular zone in the adult brain. In undifferentiated PC12 cells, endogenous Sox21 expression is detected but its expression ceases during the nerve growth factor (NGF)-induced neuronal differentiation. Overexpression of Sox21 results in a substantial repression of NGF-induced neurite outgrowth in PC12 cells. Further, we biochemically identified a Sox21-associating protein, Y-box binding protein 1 which not only binds to Sox21 but also partially restores NGF-induced neurite outgrowth of PC12 cells inhibited by Sox21. These results suggest that Sox21 is a repressor of neuronal differentiation in the developing nervous system.

Bacterial lipopolysaccharide induces type 2 deiodinase in cultured rat astrocytes.

In the brain, 3,5,3'-triiodothyronine, which binds to the thyroid hormone receptor with high affinity, is locally generated from thyroxine by type 2 iodothyronine deiodinase (D2) expressed mainly in astrocytes and tanycytes. We have investigated the effects of bacterial lipopolysaccharide (LPS) on D2 in cultured rat astrocytes. LPS induced D2 activity with a lag-time of 4-8 h and a maximum at 24 h. LPS also promoted D2 mRNA accumulation. Glucocorticoids enhanced both the basal and LPS-stimulated D2 activity and mRNA accumulation. These glucocorticoid effects were blocked by the glucocorticoid receptor antagonist RU486. Our results obtained with different specific signaling pathway inhibitors indicated that D2 induction by LPS required ERK and p38-MAPK signaling pathways. NF-κB inhibitor sulfasalazine blocked the effects of LPS on both D2 activity and mRNA accumulation. Hence, D2 induction by LPS appeared to implicate NF-κB pathway in astrocytes. NF-κB responsiveness of the rat dio2 gene was studied in astrocytes with dio2 5'-flanking region promoter assays. The long form of the dio2 promoter was transactivated by NF-κB. CCAAT/enhancer-binding protein ß, which is upregulated by LPS in astrocytes, increased the transcriptional activity of the dio2 promoter in its long or truncated forms containing CCAATs. Our observations, which demonstrate D2 induction by LPS in astrocytes and specify some characteristics of D2 induction mechanism, support the possible implication of brain D2 in adaptative responses to an infectious stress.

CCAAT/enhancer binding protein homologous protein-dependent death receptor 5 induction and ubiquitin/proteasome-mediated cellular FLICE-inhibitory protein down-regulation contribute to enhancement of tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by dimethyl-celecoxib in human non small-cell lung cancer cells.

2,5-Dimethyl-celecoxib (DMC) is a derivative of celecoxib, a cyclooxygenase-2 (COX-2) inhibitor with anticancer activity in both preclinical studies and clinical practice, and lacks COX-2-inhibitory activity. Several preclinical studies have demonstrated that DMC has better apoptosis-inducing activity than celecoxib, albeit with undefined mechanisms, and exhibits anticancer activity in animal models. In this study, we primarily investigated DMC's cooperative effect with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on the induction of apoptosis and the underlying mechanisms in human non-small-cell lung cancer (NSCLC) cells. We found that DMC was more potent than celecoxib in decreasing the survival and inducing apoptosis of NSCLC cells. When combined with TRAIL, DMC exerted enhanced or synergistic effects on the induction of apoptosis, indicating that DMC cooperates with TRAIL to augment the induction of apoptosis. To determine the underlying mechanism of the synergy between DMC and TRAIL, we have demonstrated that DMC induces a CCAAT/enhancer binding protein homologous protein-dependent expression of DR5, a major TRAIL receptor, and reduces the levels of cellular FLICE-inhibitory protein (c-FLIP) (both the long and short forms), key inhibitors of death receptor-mediated apoptosis, by facilitating c-FLIP degradation through a ubiquitin/proteasome-dependent mechanism. It is noteworthy that enforced expression of c-FLIP or silencing of DR5 expression using DR5 small interfering RNA abrogated the enhanced effects on induction of apoptosis by the combination of DMC and TRAIL, indicating that both DR5 up-regulation and c-FLIP reduction contribute to cooperative induction of apoptosis by the combination of DMC and TRAIL. Together, we conclude that DMC sensitizes human NSCLC cells to TRAIL-induced apoptosis via induction of DR5 and down-regulation of c-FLIP.

Norepinephrine stimulates calprotectin expression in human monocytic cells.

BACKGROUND AND OBJECTIVE: Calprotectin is composed of two proteins, S100A8 and S100A9, which are S100 family members, and is detected in gingival crevicular fluid and gingival tissue with inflammation. The release and production of calprotectin are regulated by lipopolysaccharides of periodontopathic bacteria and cytokines. Emotional or psychological stress, a risk factor of periodontal disease, is transmitted by stress modulators including norepinephrine and cortisol. The aim of the present study was to investigate the effect of stress on calprotectin expression using norepinephrine and cortisol. METHODS: U-937 cells, a human monocytic cell line, were incubated with norepinephrine in the presence or absence of beta- or alpha-adrenergic receptor antagonists, or with cortisol. The expression of S100A8/S100A9 mRNAs was examined by northern blotting and the amount of calprotectin was measured by enzyme-linked immunosorbent assay (ELISA). The DNA binding activity of C/EBPalpha (CCAAT enhancing binding protein), a transcription factor, was examined by electrophoretic mobility shift assay. RESULTS: Norepinephrine stimulated the expression of S100A8/S100A9 mRNAs via beta-adrenergic receptors in U-937 cells and significantly increased calprotectin production to about 3.6-fold that of the control. However, cortisol had no effect on calprotectin expression at the mRNA and protein levels. Norepinephrine elevated C/EBPalpha DNA binding activity, but cortisol did not increase the activity. CONCLUSION: Norepinephrine, a stress modulator, stimulated calprotectin expression in human monocytic cells. Calprotectin expression may be regulated by stress in addition to inflammatory factors.

Representational difference analysis using myeloid cells from C/EBP epsilon deletional mice.

C/EBP epsilon is a recently cloned member of the C/EBP family of transcriptional factors. Previous studies demonstrated that the expression of this gene is tightly regulated in a tissue specific manner; it is expressed exclusively in myeloid cells. C/EBP epsilon-deficient mice developed normally but failed to generate functional neutrophils and eosinophils, and these mice died of opportunistic infections suggesting that C/EBP epsilon may play a central role in myeloid differentiation. To identify myelomonocytic genes regulated by the C/EBP epsilon gene, we performed representational difference analysis (RDA), a polymerase chain reaction (PCR)-based subtractive hybridization using neutrophils and macrophages from wild-type and C/EBP epsilon knockout mice. We identified a set of differentially expressed genes, including chemokines specific to myelomonocytic cells. Several novel genes were identified that were differentially expressed in normal myelomonocytic cells. Taken together, we have found several genes whose expression might be enhanced by C/EBP epsilon. (Blood. 2000;96:3953-3957)

Differential effects on human immunodeficiency virus type 1 replication by alpha-defensins with comparable bactericidal activities.

In addition to their antibacterial activities, certain antimicrobial peptides inactivate enveloped viruses, including the human immunodeficiency virus (HIV). To determine whether peptide bactericidal activities are predictive of antiviral activity, the anti-HIV properties of recombinant human alpha-defensin 5, mouse alpha-defensins, cryptdins (Crp) 3 and 4, and rhesus macaque myeloid alpha-defensins (RMADs) 3 and 4 were determined in vitro. The peptides, purified to homogeneity, had equivalent bactericidal activities that were similar to those of the native molecules. Nuclear magnetic resonance spectroscopy showed RMAD-4 and Crp3 had characteristic alpha-defensin tridisulfide arrays. Of the peptides analyzed, only RMAD-4 inhibited HIV infectivity at 150 microg/ml, and Crp3 unexpectedly increased HIV replication. Quantitative real-time PCRs for minus-strand strong stop DNA and complete viral cDNA synthesis were used to distinguish between preentry and postentry anti-HIV effects by RMAD-4. Viral exposure to RMAD-4 for 1 h prior to infection reduced HIV minus-strand strong stop DNA and HIV cDNA by 4- to 20-fold during the first round of replication, showing that RMAD-4-exposed virions were not entering cells during the first 24 h. On the other hand, when RMAD-4 was added coincident with HIV inoculation, no anti-HIV activity was detected. Viral exposure to Crp3 resulted in a threefold increase in both HIV minus-strand strong stop DNA and HIV cDNA over the first round of replication. Therefore, two alpha-defensins, RMAD-4 and Crp3, inhibit or augment HIV replication, respectively, by mechanisms that precede reverse transcription.