Fresh Complete Medium Research Articles

Effect of Baicalin on the LPS-Induced Production of Inflammatory Mediators in Low-Dose Corticosterone Pretreated RAW 267.4 cells and Hepa1c1c-7 Cells

Low-dose glucocorticoids (GCs) are known to induce inflammation and immunosuppression. However, whether preconditioning with a low dose of GCs induces hepatic inflammation remains unclear. Although baicalin has beneficial therapeutic activity against liver damage, 100 μM baicalin enhances the lipopolysaccharide (LPS)-induced production of inflammatory acute-phase proteins in Hepa1c1c-7 cells. This study was performed to investigate the effect of 100 μM baicalin on the LPS-induced production of inflammatory mediators in liver cells, including Kupffer cells and hepatocytes, pre-conditioned with a low dose of GCs. RAW 267.4 cells and Hepa1c1c-7 cells were pretreated with 50 (cort 50) or 100 ng/mL (cort 100) corticosterone for 24 h and then cultured with 100 μM baicalin in fresh complete medium in the presence of LPS. Our results demonstrated that baicalin markedly decreased the LPS-induced production of TNF-α, IL-6, IL-1β, and nitric oxide in RAW 267.4 cells. Baicalin also strongly attenuated the LPS-induced production of TNF-α in cort 50- and cort 100-pretreated RAW 267.4 cells. Baicalin significantly upregulated the production of IL-6 induced by LPS and the production of VEGF decreased by LPS, but decreased the LPS-induced production of CRP in cort 100- pretreated Hepa1c1c-7 cells compared to untreated Hepa1c1c-7 cells. In conclusion, these findings suggest that 100 μM baicalin may enhance the LPS-induced production of inflammatory acute-phase proteins in hepatocytes, whereas it may downregulate these protein levels in macrophages and Kupffer cells. Moreover, preconditioning with a low dose of corticosterone may prime the effects of 100 μM baicalin on the LPS-induced production of IL-6 in hepatocytes, leading to hepatic insulin resistance or fatty liver disease.

Effect of low-dose corticosterone pretreatment on the production of inflammatory mediators in super-low-dose LPS-primed immune cells.

Pretreatment of super-low-dose lipopolysaccharide (SL-LPS) induces a more hyperresponsive state on the production of proinflammatory mediators to a subsequent secondary challenge with high-dose LPS in innate immune cells. Low-dose glucocorticoids (GCs) are also known to induce inflammation and immunosuppression in the immune cells. However, there is limited knowledge on whether preconditioning of low-dose GCs enhances inflammatory responses and dysregulates T lymphocyte responses to secondary LPS in SL-LPS-primed immune cells. In the present study, RAW 264.7 and EL4 cells were pretreated with SL-LPS (50pg/ml) or low-dose corticosterone (CORT50: 50ng/ml and CORT100: 100ng/ml) in fresh complete medium once a day for 2-3days, consecutively, and then cultured in fresh complete medium for 6 or 24h in the presence or absence of LPS (1-10μg/ml) or concanavalin A (Con A). The results demonstrated that the repeated pretreatment of CORT50 strongly enhanced production of IL-6, IL-10, TNF-α, and nitric oxide (NO) by RAW 264.7 cells in EP (SL-LPS-primed cells: endotoxin priming) in the absence of LPS compared to those in control (vehicle-pretreated cells), whereas CORT100 reduced production of TNF-α and IL-10. Further, the repeated pretreatment of CORT50 markedly enhanced LPS-induced production of IL-6, IL-10, TNF-α, PGE2, and NO by RAW 264.7 cells in EP compared to those in control, whereas CORT100 attenuated LPS-induced production of IL-6, IL-10, and NO. Moreover, the repeated pretreatments of CORT50 and CORT100 greatly attenuated the Con A-stimulated production of IFN-γ and IFN-γ/IL-10 and LPS-stimulated production of IL-10, IFN-γ, and IFN-γ/IL-10 by SL-LPS-primed EL4 cells (EP). These findings suggest that double preconditionings of low grade hypercortisolemia and metabolic endotoxemia may act as important risk factors for metabolic disorder and severe morbidity and mortality in septic shock via upregulated production of inflammatory mediators and immunosuppression of IFN-γ-mediated responses.

Retroviral vector production by transient transfection of 293VEC-GALV cells

Background & Aim Introduction Retroviral vectors can permanently integrate their genetic material into chromosomes in the target cells for long-term, stable expression of the introduced genetic elements. It has been widely used since the 1980s and is the second most commonly employed system for gene transfer. Traditionally, retroviral vector production involves the preparation and characterization of a stable producer cell line from which the vector is produced as supernatant. It usually takes about 12 to 20 months from generation the stable producer cell line clone to the final release of the retroviral vector for clinical use. Due to the recent rapid growth of CAR-T cell therapies, a faster, more efficient method for retroviral vector production is required. We have, therefore, employed a novel manufacturing system using transient transfection for rapid GMP preparation of retroviral vectors. Methods, Results & Conclusion Methods Clinical retroviral vectors are produced from a master cell bank of 293Vec-Galv (BioVec Pharma, Quebec, Canada), a human embryonic kidney HEK293-based packaging cell line that stably expresses the Moloney Murine Leukemia gag-pol and Gibbon Ape Leukemia Virus envelope viral proteins. The cells are transfected with plasmid DNA that carries the gene(s) of interest (purified plasmid DNAs are produced by Puresyn Inc. Malvern PA, USA or Aldevron Inc. Fargo ND, USA). The transfection is carried out at 37°C and is mediated by PEIpro®-HQ (Polyplus Transfection, Illkirch, France), which is a highly qualified grade of polyethylenimine developed for large-scale bioproduction processes. About 24 hours post-transfection the medium is replaced by fresh complete medium. Supernatant containing the retrovirus is harvested at 48, 72 and 96 hours post-transfection and filtered through 0.45µm filter. The filtered supernatant is aliquoted, snap frozen and immediately transferred to storage in -80°C freezers. Results and Conclusion Eight retroviral vectors have been manufactured using this novel system at the CAGT GMP Vector Production Facility. Vector production from a single master cell bank of 293Vec-Galv takes about 4 weeks. Four vectors have been released for clinical trials. The transduction rate, phenotype and cytotoxic effect of T cells transduced by vectors produced by this novel system met release requirements, including absence of replication-competent retrovirus. Retroviral vector production by transient transfection of 293Vec-Galv cells saves time, is cost-efficient and safe ( Table 1 ).

Pretreatment of Low-Dose and Super-Low-Dose LPS on the Production of In Vitro LPS-Induced Inflammatory Mediators

Pretreatment of low-dose lipopolysaccharide (LPS) induces a hyporesponsive state to subsequent secondary challenge with high-dose LPS in innate immune cells, whereas super-low-dose LPS results in augmented expression of pro-inflammatory cytokines. However, little is known about the difference between super-low-dose and low-dose LPS pretreatments on immune cell-mediated inflammatory and hepatic acute-phase responses to secondary LPS. In the present study, RAW 264.7 cells, EL4 cells, and Hepa-1c1c7 cells were pretreated with super-low-dose LPS (SL-LPS: 50 pg/mL) or low-dose LPS (L-LPS: 50 ng/mL) in fresh complete medium once a day for 2~3 days and then cultured in fresh complete medium for 24 hr or 48 hr in the presence or absence of LPS (1∼10 µg/mL) or concanavalin A (Con A). SL-LPS pretreatment strongly enhanced the LPS-induced production of tumor necrosis factor (TNF)-α, interleukin (IL)-6, TNF-α/IL-10, prostaglandin E2 (PGE2), and nitric oxide (NO) by RAW 264.7 cells compared to the control, whereas L-LPS increased IL-6 and NO production only. SL-LPS strongly augmented the Con A-induced ratios of interferon (IFN)-γ/IL-10 in EL4 cells but decreased the LPS-induced ratios of IFN-γ/IL-10 compared to the control, while L-LPS decreased the Con A- and LPS-induced ratios of IFN-γ/IL-10. SL-LPS enhanced the LPS-induced production of IL-6 by Hepa1c1c-7 cells compared to the control, while L-LPS increased IL-6 but decreased IL-1β and C reactive protein (CRP) levels. SL-LPS pretreatment strongly enhanced the LPS-induced production of TNF-α, IL-6, IL-10, PGE2, and NO in RAW 264.7 cells, and the IL-6, IL-1β, and CRP levels in Hepa1c1c-7 cells, as well as the ratios of IFN-γ/IL-10 in LPS- and Con A-stimulated EL4 cells compared to L-LPS. These findings suggest that pre-conditioning of SL-LPS may contribute to the mortality to secondary infection in sepsis rather than pre-conditioning of L-LPS.

Human IL10 Gene Repression by Rev-erbα Ameliorates Mycobacterium tuberculosis Clearance

Nuclear receptors modulate macrophage effector functions, which are imperative for clearance or survival of mycobacterial infection. The adopted orphan nuclear receptor Rev-erbα is a constitutive transcriptional repressor as it lacks AF2 domain and was earlier shown to be present in macrophages. In the present study, we highlight the differences in the relative subcellular localization of Rev-erbα in monocytes and macrophages. The nuclear localization of Rev-erbα in macrophages is subsequent to monocyte differentiation. Expression analysis of Rev-erbα elucidated it to be considerably more expressed in M1 phenotype in comparison with M2. Rev-erbα overexpression augments antimycobacterial properties of macrophage by keeping IL10 in a basal repressed state. Further, promoter analysis revealed that IL10 promoter harbors a Rev-erbα binding site exclusive to humans and higher order primates and not mouse, demonstrating a species barrier in its functionality. This direct gene repression is mediated by recruitment of co-repressors NCoR and HDAC3. In addition, our data elucidate that its overexpression reduced the survival of intracellular pathogen Mycobacterium tuberculosis by enhancing phagosome lysosome maturation, an event resulting from IL10 repression. Thus, these findings suggest that Rev-erbα bestows protection against mycobacterial infection by direct gene repression of IL10 and thus provide a novel target in modulating macrophage microbicidal properties.

Oral cancer-associated fibroblasts inhibit heat-induced apoptosis in Tca8113 cells through upregulated expression of Bcl-2 through the Mig/CXCR3 axis

The aim of this study was to detect oral cancer-associated fibroblast-secreted cytokines and their regulation of heat-induced apoptosis in the human tongue squamous cell carcinoma cell line Tca8113. We isolated cancer-associated fibroblasts (CAFs) from human tongue squamous cell carcinoma and normal fibroblasts (NFs) from normal oral mucosa. The expression profiles of cytokines secreted by CAFs and those secreted by NFs were detected using the RayBio® human cytokine antibody microarray. The conditioned medium was prepared by mixing the CAF or NF supernatant cell culture medium with fresh complete medium. The expression levels of Bax, Bcl-2 and CXCR3 in Tca8113 cells were detected by western blot analysis. The heat-induced apoptosis ratio of the Tca8113 cells was detected by propidium iodide staining combined with flow cytometry. The quantity of the Mig factor, one of the chemokines secreted by CAFs, was clearly increased 19-fold when compared with the level in NFs. The conditioned medium of NFs had no obvious effect on the expression levels of Bax/Bcl-2 and the heat-induced apoptosis ratio in the Tca8113 cells. However, the expression levels of Bcl-2 were significantly upregulated and heat-induced Tca8113 cell apoptosis was inhibited in the CAF-conditioned medium. After adding neutralizing antibodies against Mig or its receptor CXCR3, the enhanced expression of the Bcl-2 protein and the inhibited heat-induced apoptosis of Tca8113 cells in the CAF-conditioned medium were significantly attenuated. CAFs may increase the expression levels of Bcl-2 through the paracrine secretion of Mig and reduce the thermosensitivity of Tca8113 cells.

Natural killer cells can exert a graft-vs-tumor effect in haploidentical stem cell transplantation for pediatric solid tumors

Little progress has been made with regard to the survival of children with metastatic and refractory solid tumors. Preliminary data from haploidentical stem cell transplantation (haplo-SCT) suggested a clinically beneficial allograft-vs-tumor effect associated with natural killer cell (NK) donor-recipient mismatch. We hypothesized that interaction between activatory receptors on NK cells and their ligands on tumor cells could be also important. To evaluate the NK-cell-mediated allograft-vs-tumor effect, we conducted a pilot study of haplo-SCT on six children with refractory solid tumors. Our specific goal for this study was NKG2D-major histocompatibility complex class I-related chain A interaction. Tasks include specific immunoassays that support haplo-SCT in refractory solid tumors. Patients suffered from neuroblastoma (n = 1), Ewing sarcoma (n = 2), a desmoplastic tumor (n = 1), nasopharyngeal carcinoma (n = 1), and embryonal rhabdomyosarcoma (n = 1). Pretransplantation disease status showed progressive disease in 2 patients, partial remission in 2 patients, and complete remission in 2 patients. NK-cell mismatch was present in three donor-recipients. Ligands for NKG2D receptors, major histocompatibility complex class I-related chain A and UL16 binding protein 2 were overexpressed in six of six and four of six tumors, respectively. NK cells led early immune reconstitution. After haplo-SCT, three patients were in complete remission, one patient showed partial remission, and two patients were in stable disease. With a median follow-up of 14 months, three patients were alive and in complete remission, and three patients had died; two due to progressive disease and one of transplant-related toxicity. Blocking NKG2D-major histocompatibility complex class I-related chain A interaction in vitro reduced NK-cell cytotoxicity. Our preliminary results suggest a beneficial effect from haplo-SCT in refractory solid tumors.

Activated Apoptotic Cells Induce Dendritic Cell Maturation via Engagement of Toll-like Receptor 4 (TLR4), Dendritic Cell-specific Intercellular Adhesion Molecule 3 (ICAM-3)-grabbing Nonintegrin (DC-SIGN), and β2 Integrins

Dendritic cells (DCs) are professional antigen-presenting cells playing a central role in connecting innate and adaptive immunity. Maturation signals are, however, required for DCs to undergo phenotypic and functional changes to acquire a fully competent antigen-presenting capacity. We previously reported that activated apoptotic peripheral lymphocytes (ActApo) provide activation/maturation signals to human monocyte-derived DCs. In this paper, we have characterized the signaling pathways and molecules involved in ActApo-mediated DC maturation. We found that both cellular and supernatant fractions from ActApo are required for DC maturation signaling. ActApoSup-induced CD80 and CD86 expression was significantly blocked in the presence of neutralizing antibodies against tumor necrosis factor-α (TNF-α). Cell-cell contact-dependent signaling involved β2 integrins, dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN), and TLR4 because ActApo-induced up-regulation of the maturation markers CD80 and CD86 was significantly inhibited in the presence of neutralizing antibodies against CD18, CD11a, CD11b, and DC-SIGN as well as TLR4. The role of TLR4 was further confirmed by silencing of TLR4 in DCs. In addition, the endogenous adjuvant effect exerted by activated apoptotic splenocytes (ActApoSp) was reduced after immunization with human serum albumin in TLR4(-/-) mice. We detected activation of multiple signaling pathways and transcription factors in DCs upon co-culture with ActApo, including p38, JNK, PI3K-Akt, Src family kinases, NFκB p65, and AP1 transcription factor family members c-Jun and c-Fos, demonstrating the complex interactions occurring between ActApo and DCs. These studies provide important mechanistic insight into the responses of DCs during encounter with cells undergoing immunogenic cell death.

Heparan Sulfate Chains of Syndecan-1 Regulate Ectodomain Shedding

Matrix metalloproteinases release intact syndecan-1 ectodomains from the cell surface giving rise to a soluble, shed form of the proteoglycan. Although it is known that shed syndecan-1 controls diverse pathophysiological responses in cancer, wound healing, inflammation, infection, and immunity, the mechanisms regulating shedding remain unclear. We have discovered that the heparan sulfate chains present on syndecan core proteins suppress shedding of the proteoglycan. Syndecan shedding is dramatically enhanced when the heparan sulfate chains are enzymatically degraded or absent from the core protein. Exogenous heparan sulfate or heparin does not inhibit shedding, indicating that heparan sulfate must be attached to the core protein to suppress shedding. Regulation of shedding by heparan sulfate occurs in multiple cell types, for both syndecan-1 and syndecan-4 and in murine and human syndecans. Mechanistically, the loss of heparan sulfate enhances the susceptibility of the core protein to proteolytic cleavage by matrix metalloproteinases. Enhanced shedding of syndecan-1 following loss of heparan sulfate is accompanied by a dramatic increase in core protein synthesis. This suggests that in response to an increase in the rate of shedding, cells attempt to maintain a significant level of syndecan-1 on the cell surface. Together these data indicate that the amount of heparan sulfate present on syndecan core proteins regulates both the rate of syndecan shedding and core protein synthesis. These findings assign new functions to heparan sulfate chains, thereby broadening our understanding of their physiological importance and implying that therapeutic inhibition of heparan sulfate degradation could impact the progression of some diseases.

Loss of Hypermethylated in Cancer 1 (HIC1) in Breast Cancer Cells Contributes to Stress-induced Migration and Invasion through β-2 Adrenergic Receptor (ADRB2) Misregulation

The transcriptional repressor HIC1 (Hypermethylated in Cancer 1) is a tumor suppressor gene inactivated in many human cancers including breast carcinomas. In this study, we show that HIC1 is a direct transcriptional repressor of β-2 adrenergic receptor (ADRB2). Through promoter luciferase activity, chromatin immunoprecipitation (ChIP) and sequential ChIP experiments, we demonstrate that ADRB2 is a direct target gene of HIC1, endogenously in WI-38 cells and following HIC1 re-expression in breast cancer cells. Agonist-mediated stimulation of ADRB2 increases the migration and invasion of highly malignant MDA-MB-231 breast cancer cells but these effects are abolished following HIC1 re-expression or specific down-regulation of ADRB2 by siRNA treatment. Our results suggest that early inactivation of HIC1 in breast carcinomas could predispose to stress-induced metastasis through up-regulation of the β-2 adrenergic receptor.

Peroxiredoxin II Restrains DNA Damage-induced Death in Cancer Cells by Positively Regulating JNK-dependent DNA Repair

The 2-Cys peroxiredoxins (Prx) belong to a family of antioxidant enzymes that detoxify reactive oxygen and nitrogen species and are distributed throughout the intracellular and extracellular compartments. However, the presence and role of 2-Cys Prxs in the nucleus have not been studied. This study demonstrates that the PrxII located in the nucleus protects cancer cells from DNA damage-induced cell death. Although the two cytosolic 2-Cys Prxs, PrxI and PrxII, were found in the nucleus, only PrxII knockdown selectively and markedly increased cell death in the cancer cells treated with DNA-damaging agents. The increased death was completely reverted by the nuclearly targeted expression of PrxII in an activity-independent manner. Furthermore, the antioxidant butylated hydroxyanisole did not influence the etoposide-induced cell death. Mechanistically, the knockdown of Prx II expression impaired the DNA repair process by reducing the activation of the JNK/c-Jun pathway. These results suggest that PrxII is likely to be attributed to a tumor survival factor positively regulating JNK-dependent DNA repair with its inhibition possibly sensitizing cancer cells to chemotherapeutic agents.

MiR-146a Is Critical for Endotoxin-induced Tolerance

The human toll-like receptor 4 (TLR4) pathway is activated in response to lipopolysaccharide (LPS), and subsequent signal transductions lead to the production of cytokines such as tumor necrosis factor-α (TNF-α) by innate immune cells. Defects in innate immune response may contribute to the overproduction of TNF-α leading to systemic inflammation and diseases. Thus, the innate immune response needs to be tightly regulated by elaborate mechanisms to control its onset and termination. LPS tolerance is a state of hyporesponsiveness to subsequent LPS challenge and is achieved by monocytic cells after prolonged exposure to LPS. In this report, kinetics of endotoxin-responsive microRNAs expression analysis revealed a unique pattern of gradual increase for miR-146a starting 4 h after LPS stimulation in THP-1 cells and continued up to 35-fold over 24 h. Conversely, TNF-α increased up to 4 h and then decreased gradually implicating a negative correlation with miR-146a progression. The characteristic up-regulation of miR-146a toward subsequent LPS challenge in THP-1 cells was studied. Strikingly, microRNA expression analysis during the tolerized state of THP-1 cells showed only miR-146a overexpression suggesting its important role in LPS tolerance. In addition, LPS tolerance was dependent on a LPS-priming dose and associated miR-146a up-regulation. LPS-tolerized cells were observed to regain responsiveness in TNF-α production 22 h after LPS removal correlating with a decrease in miR-146a level. Transfection of miR-146a into THP-1 cells mimicked LPS priming, whereas transfection of miR-146a inhibitor largely abolished LPS tolerance. Thus our studies demonstrated that miR-146a is critical for the in vitro monocytic cell-based endotoxin tolerance.

Formation of “Nonculturable” dormant forms of the phytopathogenic enterobacterium Erwinia carotovora

Reversible transition of the phytopathogenic gram-negative bacterium Erwinia carotovora, subsp. atroseptica, strain SCRI1043, to a dormant state was demonstrated; it was associated with a complete loss of cell ability to form colonies on the standard medium, i.e., with acquiring “non-culturability”. Entering of Erwinia cells to a nonculturable state occurred after long-term incubation (100–150 days) of the stationary-phase cell suspensions in either a fresh complete medium or in the carbon-free mineral medium or treatment with a chemical analogue of microbial anabiosis autoinducers (4 × 10−4 M of C12-alkylhydroxybenzene, AHB). However, confocal laser microscopy of the cells stained with the Live/Dead BacLight kit revealed that the majority of E. carotovora cells (90%) from long-incubated suspensions retained membrane integrity. In these suspensions, round cells of smaller size prevailed, with the envelope, containing an electron-dense outer layer and an underlying layer of lower density; the cytoplasm was coarse-granulated. Detection of “nonculturable” E. carotovora cells by quantitative real-time PCR analysis (Q-PCR) with specific primers by using standard procedures of sample preparation was shown to be inefficient. A special procedure including cell washing from the incubation medium in the absence of growth stimulation was developed, which promoted recovery of the colony-forming ability of the cells (up to 10% of the initial CFU number) and improved cell detection by Q-PCR from the number of genomic copies. The results provided further insight into the ways of long-term survival of phytopathogenic bacteria under environmental changes and carbon starvation.

Scavenger Chemokine (CXC Motif) Receptor 7 (CXCR7) Is a Direct Target Gene of HIC1 (Hypermethylated in Cancer 1)

The tumor suppressor gene HIC1 (Hypermethylated in Cancer 1) that is epigenetically silenced in many human tumors and is essential for mammalian development encodes a sequence-specific transcriptional repressor. The few genes that have been reported to be directly regulated by HIC1 include ATOH1, FGFBP1, SIRT1, and E2F1. HIC1 is thus involved in the complex regulatory loops modulating p53-dependent and E2F1-dependent cell survival and stress responses. We performed genome-wide expression profiling analyses to identify new HIC1 target genes, using HIC1-deficient U2OS human osteosarcoma cells infected with adenoviruses expressing either HIC1 or GFP as a negative control. These studies identified several putative direct target genes, including CXCR7, a G-protein-coupled receptor recently identified as a scavenger receptor for the chemokine SDF-1/CXCL12. CXCR7 is highly expressed in human breast, lung, and prostate cancers. Using quantitative reverse transcription-PCR analyses, we demonstrated that CXCR7 was repressed in U2OS cells overexpressing HIC1. Inversely, inactivation of endogenous HIC1 by RNA interference in normal human WI38 fibroblasts results in up-regulation of CXCR7 and SIRT1. In silico analyses followed by deletion studies and luciferase reporter assays identified a functional and phylogenetically conserved HIC1-responsive element in the human CXCR7 promoter. Moreover, chromatin immunoprecipitation (ChIP) and ChIP upon ChIP experiments demonstrated that endogenous HIC1 proteins are bound together with the C-terminal binding protein corepressor to the CXCR7 and SIRT1 promoters in WI38 cells. Taken together, our results implicate the tumor suppressor HIC1 in the transcriptional regulation of the chemokine receptor CXCR7, a key player in the promotion of tumorigenesis in a wide variety of cell types.

Ribosomal Protein S9 Is a Novel B23/NPM-binding Protein Required for Normal Cell Proliferation

B23 (NPM/nucleophosmin) is a multifunctional nucleolar protein and a member of the nucleoplasmin superfamily of acidic histone chaperones. B23 is essential for normal embryonic development and plays an important role in genomic stability, ribosome biogenesis, and anti-apoptotic signaling. Altered protein expression or genomic mutation of B23 is encountered in many different forms of cancer. Although described as multifunctional, a genuine molecular function of B23 is not fully understood. Here we show that B23 is associated with a protein complex consisting of ribosomal proteins and ribosome-associated RNA helicases. A novel, RNA-independent interaction between ribosomal protein S9 (RPS9) and B23 was further investigated. We found that S9 binding requires an intact B23 oligomerization domain. Depletion of S9 by small interfering RNA resulted in decreased protein synthesis and G(1) cell cycle arrest, in association with induction of p53 target genes. We determined that S9 is a short-lived protein in the absence of ribosome biogenesis, and proteasomal inhibition significantly increased S9 protein level. Overexpression of B23 facilitated nucleolar storage of S9, whereas knockdown of B23 led to diminished levels of nucleolar S9. Our results suggest that B23 selectively stores, and protects ribosomal protein S9 in nucleoli and therefore could facilitate ribosome biogenesis.

α2 but Not α1 AMP-activated Protein Kinase Mediates Oxidative Stress-induced Inhibition of Retinal Pigment Epithelium Cell Phagocytosis of Photoreceptor Outer Segments

Oxidative stress causes retinal pigment epithelium (RPE) cell dysfunction and is a major risk factor leading to the development of dry-type age-related macular degeneration. Taking pharmacological and genetic approaches, we address the mechanisms by which sublethal oxidative stress inhibits RPE cell phagocytosis. Sublethal oxidative stress dose-dependently inhibited RPE cell phagocytosis of photoreceptor outer segments (POS) and activated AMP-activated protein kinase (AMPK) as determined by increased Thr172 and Ser79 phosphorylation of AMPKalpha and its substrate acetyl-CoA carboxylase, respectively. Similar to oxidative stress, 5-aminoimidazole-4-carboxamide riboside (AICAR), a pharmacological activator of AMPK, inhibited RPE cell phagocytosis of POS in a dose-dependent manner. Inhibition of RPE cell phagocytosis by AICAR was fully reversed by blockade of AICAR translocation into cells by dipyridamole or inhibition of AICAR conversion to ZMP by adenosine kinase inhibitor 5-iodotubercidin. In agreement, AICAR-induced activation of AMPK was abolished by preincubation with dipyridamole or 5-iodotubercidin. Knock-out experiments further revealed that alpha2 but not alpha1 AMPK was involved in RPE cell phagocytosis and that activation of alpha2 AMPK contributed to the inhibition of RPE cell phagocytosis by oxidative stress. Inhibition of RPE cell phagocytosis by activation of alpha2 AMPK was associated with a dramatic increase in acetyl-CoA carboxylase phosphorylation. In comparison, AMPK had no role in oxidative stress-induced breakdown of RPE barrier function. Taken together, reduction in POS load under oxidative stress might direct RPE cells to a self-protected status. Thus, activating AMPK could have therapeutic potential in treating dry macular degeneration.

DJ-1 Decreases Bax Expression through Repressing p53 Transcriptional Activity

DJ-1, originally identified as an oncogene product, is a protein with various functions in cellular transformation, oxidative stress response, and transcriptional regulation. Although previous studies suggest that DJ-1 is cytoprotective, the mechanism by which DJ-1 exerts its survival functions remains largely unknown. Here we show that DJ-1 exerts its cytoprotection through inhibiting p53-Bax-caspase pathway. DJ-1 interacts with p53 in vitro and in vivo. Overexpression of DJ-1 decreases the expression of Bax and inhibits caspase activation, whereas knockdown of DJ-1 increases Bax protein levels and accelerates caspase-3 activation and cell death induced by UV exposure. Our data provide evidence that the protective effects of DJ-1 on apoptosis are associated with its ability of decreasing Bax level through inhibiting p53 transcriptional activity.

Pro-apoptotic Bim Induction in Response to Nerve Growth Factor Deprivation Requires Simultaneous Activation of Three Different Death Signaling Pathways

Bim is a pro-apoptotic member of the Bcl-2 family that is induced and contributes to neuron death in response to nerve growth factor (NGF) deprivation. Past work has revealed that Bim is downstream of multiple independent transcriptional pathways in neurons, including those culminating in activation of the c-Jun, FoxO, and Myb transcription factors. This study addresses the issue of whether the three signaling pathways are redundant with respect to Bim induction or whether they act cooperatively. Examination of the proximal Bim promoter reveals binding sites for FoxO, Mybs, and, as shown here, c-Jun. We find that mutation of any one of these types of sites abolishes induction of a Bim promoter-driven reporter in response to NGF deprivation. Moreover, down-regulation of either c-Jun, FoxOs, or Mybs by short hairpin RNAs blocks induction of Bim promoter-reporter activity triggered by withdrawal of NGF. This was the case for reporters driven by either the proximal promoter or a promoter that also includes additional regulatory elements in the first intron of the Bim gene. Such short hairpin RNAs also suppressed the induction of endogenous Bim protein. These findings thus indicate that the Bim promoter acts as a coincidence detector that optimally responds to the simultaneous activation of three different pro-apoptotic transcriptional pathways. Such a mechanism provides a "fail-safe" that prevents neurons from dying by accidental activation of any single pathway. It also permits neurons to utilize individual pathways such as JNK signaling for other purposes without risk of demise.

Selective 5-Lipoxygenase Expression in Langerhans Cells and Impaired Dendritic Cell Migration in 5-LO-Deficient Mice Reveal Leukotriene Action in Skin

5-Lipoxygenase (5-LO) catalyzes the initial steps in the formation of leukotrienes (LTs), which are implicated in immune reactions. Recently, it was shown that FITC-triggered epidermal Langerhans cell (LC) emigration to draining lymph nodes (LNs) is impaired in LTC4 export pump (multidrug resistance-associated protein 1)-deficient mice. Here, we sought genetic evidence for a role of endogenous LTs in dendritic cell function through the study of 5-LO-deficient mice. Though DC numbers in skin, spleen, and peripheral LNs were similar in both 5-LO-deficient and wild-type (WT) mice, DC homing from skin to draining LNs induced by FITC was reduced by 75% in 5-LO-deficient mice. Moreover, in WT mice, all epidermal LCs, dermal langerin+ LCs, and subsets of dermal macrophages and langerin+ LCs in T-cell areas of skin-draining LNs markedly expressed 5-LO. However, the enzyme was noticeably absent in all DC subsets of the dermis, thymus, spleen, Peyer's patches, mesenteric LNs, and mucosal surfaces of lung and intestine. As all epidermal cells other than LCs lacked 5-LO and because differentiation and activation of DCs generated from 5-LO-deficient mice in vitro were normal, these data support a selective role of endogenous LTs in DC homing following skin sensitization.

Mouse Ornithine Decarboxylase-like Gene Encodes an Antizyme Inhibitor Devoid of Ornithine and Arginine Decarboxylating Activity

Ornithine decarboxylase (ODC), a key enzyme in the biosynthesis of polyamines, is a labile protein that is regulated by interacting with antizymes (AZs), a family of polyamine-induced proteins. Recently, a novel human gene highly homologous to ODC, termed ODC-like or ODC-paralogue (ODCp), was cloned, but the studies aimed to determine its function rendered contradictory results. We have cloned the mouse orthologue of human ODCp and studied its expression and possible function. mRNA of mouse Odcp was found in the brain and testes, showing a conserved expression pattern with regard to the human gene. Transfection of mouse Odcp in HEK 293T cells elicited an increase in ODC activity, but no signs of arginine decarboxylase activity were evident. On the other hand, whereas the ODCp protein was mainly localized in the mitochondrial/membrane fraction, ODC activity was found in the cytosolic fraction and was markedly decreased by small interfering RNA against human ODC. Co-transfection experiments with combinations of Odc, Az1, Az2, Az3, antizyme inhibitor (Azi), and Odcp genes showed that ODCp mimics the action of AZI, rescuing ODC from the effects of AZs and prevented ODC degradation by the proteasome. A direct interaction between ODCp and AZs was detected by immunoprecipitation experiments. We conclude that mouse ODCp has no intrinsic decarboxylase activity, but it acts as a novel antizyme inhibitory protein (AZI2).