Manabu Taura

p53 Regulates Toll-Like Receptor 3 Expression and Function in Human Epithelial Cell Lines

ABSTRACT Toll-like receptors (TLRs) are important sensors of microbial pathogens and mediators of innate immune responses. Although the signal transduction of TLRs is well elucidated, their basal regulation is largely unexplored. Here we show that the tumor suppressor p53 positively regulates the transcription of TLR3, a receptor for viral double-stranded RNA and poly(I-C), by binding to the p53 site in the TLR3 promoter. TLR3 expression was lower in HCT116 p53−/− cells than in HCT116 p53+/+ cells. Activation of p53 by 5-fluorouracil increased the TLR3 mRNA in epithelial cell lines with wild-type p53 but not in cell lines harboring mutant p53. Knockdown of p53 by small interfering RNA decreased the TLR3 expression. TLR3 mRNA was also lower in liver and intestine of p53−/− mice than in p53+/+ mice. Furthermore, the poly(I-C)-induced phosphorylation of IκB-α, nuclear translocation of NF-κB, and phosphorylation of interferon regulatory transcription factor 3, were drastically reduced in HCT116 p53−/− cells, indicating a dysregulation of the two signaling pathways governed by TLR3. Consequently, induction of interleukin-8 and beta interferon after poly(I-C) stimulation was impaired in HCT116 p53−/− cells. These results suggest that p53 influences TLR3 expression and function and highlight a role of p53 in innate immune response in epithelial cells.

TLR3 induction by anticancer drugs potentiates poly I:C-induced tumor cell apoptosis

Toll‐like receptor 3 (TLR3) has gained recognition as a novel molecular target for cancer therapy because TLR3 activation by its synthetic ligand poly I:C directly causes tumor cell death. Recently, we reported that tumor suppressor p53 increases the expression of TLR3 in several tumor cell lines. Another study also showed that interferon‐α (IFN‐α) up‐regulates TLR3 expression. We thus hypothesized that various anticancer drugs such as p53‐activating reagents and IFNs may potentiate poly I:C‐induced tumor cell death through the up‐regulation of TLR3 expression. Here, we screened several anticancer drugs that, together with poly I:C, effectively cause tumor cell death in colon carcinoma HCT116 cells. We found that the DNA‐damaging reagent 5‐fluorouracil (5‐FU) increased TLR3 mRNA expression and potentiated poly I:C‐induced apoptosis in HCT116 p53+/+ cells but had only minimal effect in p53−/− cells, indicating a p53‐dependent pathway. On the other hand, IFN‐α increased poly I:C‐induced apoptosis and the TLR3 mRNA level in HCT116 p53+/+ and p53−/− cell lines. Furthermore, the combination of poly I:C, 5‐FU and IFN‐α induced the highest apoptosis in HCT116 p53+/+ and p53−/− cells. Taken together, these data suggest that the anticancer drugs increased TLR3 expression and subsequently potentiated poly I:C‐induced apoptosis likely via p53‐dependent and ‐independent pathways. Considering that the p53 status in malignant cells is heterogeneous, this combination approach may provide a highly effective tumor therapy. (Cancer Sci 2010)

Antitumor effect of berberine against primary effusion lymphoma via inhibition of NF-κB pathway.

Primary effusion lymphoma (PEL) is an infrequent and distinct entity among the aggressive non‐Hodgkin B cell lymphomas that occurs predominantly in patients with advanced AIDS. It shows serous lymphomatous effusion in body cavities, and is resistant to conventional chemotherapy with a poor prognosis. Thus, the optimal treatment for PEL is not well defined and there is a need for novel agents. PEL has been recognized as the tumor caused by Kaposi sarcoma‐associated herpes virus/human herpes virus‐8 (KSHV/HHV‐8), and nuclear factor (NF)‐κB activation plays a critical role in the survival and growth of PEL cells. In this study, we assessed the antitumor effect of berberine, a naturally occurring isoquinoline alkaloid, on this pathway. The methylthiotetrazole assay showed that cell proliferation in the PEL cell lines was inhibited by berberine. Berberine also induced caspase‐dependent apoptosis and suppressed NF‐κB activity by inhibiting IκB kinase (IKK) phosphorylation, IκB phosphorylation and IκB degradation, upstream targets of the NF‐κB pathway, in PEL cells. In a xenograft mouse model that showed ascites and diffuse organ invasion of PEL cells, treatment with berberine inhibited the growth and invasion of PEL cells significantly compared with untreated mice. These results show that the suppression of NF‐κB is a molecular target for treating PEL, and berberine is a potential antitumor agent for PEL. (Cancer Sci 2012; 103: 775–781)

STT3B-Dependent Posttranslational N-Glycosylation as a Surveillance System for Secretory Protein

Nascent secretory proteins are extensively scrutinized at the endoplasmic reticulum (ER). Various signatures of client proteins, including exposure of hydrophobic patches or unpaired sulfhydryls, are coordinately utilized to reduce nonnative proteins in the ER. We report here the cryptic N-glycosylation site as a recognition signal for unfolding of a natively nonglycosylated protein, transthyretin (TTR), involved in familial amyloidosis. Folding and ER-associated degradation (ERAD) perturbation analyses revealed that prolonged TTR unfolding induces externalization of cryptic N-glycosylation site and triggers STT3B-dependent posttranslational N-glycosylation. Inhibition of posttranslational N-glycosylation increases detergent-insoluble TTR aggregates and decreases cell proliferation of mutant TTR-expressing cells. Moreover, this modification provides an alternative pathway for degradation, which is EDEM3-mediated N-glycan-dependent ERAD, distinct from the major pathway of Herp-mediated N-glycan-independent ERAD. Hence we postulate that STT3B-dependent posttranslational N-glycosylation is part of a triage-salvage system recognizing cryptic N-glycosylation sites of secretory proteins to preserve protein homeostasis.

Comparative Study of Human Hematopoietic Cell Engraftment into Balb/c and C57BL/6 Strain of Rag-2/Jak3 Double-Deficient Mice

Immunodeficient mice are becoming invaluable tools in human stem cell and tumor research. In this study, we generated Rag-2/Jak3 double-deficient (Rag-2−/−Jak3−/−) mice with a C57/BL6 and Balb/c genetic background and compared the human lymphohematopoietic cell engraftment rate. Human cord blood-derived CD34+ hematopoietic stem cells were successfully engrafted into Balb/c Rag-2−/−Jak3−/− mice; however, the engraftment rate was far lower in C57/BL6 Rag-2−/−Jak3−/− mice. Transplantation of human peripheral blood mononuclear cells resulted in the same tendency. Thus, a Balb/c background offers superior engraftment capacity than a C57/BL6 background and provides an attractive model for human hematopoietic cell engraftment.

Sp1-dependent regulation of Myeloid Elf-1 like factor in human epithelial cells

Myeloid Elf‐1 like factor (MEF) is an ETS protein, which activates the promoters of granulocyte macrophage colony‐stimulating factor, interleukin‐3, lysozyme, human beta defensin‐2 and perforin. In spite of its many known functions, little is known about MEF transcriptional regulation. Here, we cloned the 5′‐flanking region of human MEF gene and identified a TATA‐less promoter region at −204/−54 which contains 4 putative binding sites for Sp1, two of which are essential in up‐regulating MEF activity. These were proven by EMSA and blocking Sp1 using RNAi or mithramycin A treatment of HEK293 cells. Our results suggest that Sp1 constitutively regulates the MEF gene.

Endoplasmic reticulum stress increases the expression and function of toll-like receptor-2 in epithelial cells

Endoplasmic reticulum (ER) stress is involved in a wide range of pathological conditions including neurodegenerative disorders, diabetes mellitus, atherosclerosis, inflammation, and infection. The ability of ER stress to induce an inflammatory response is considered to play a role in the pathogenesis of these diseases. However, its role in regulating the gene expression and function of toll-like receptors (TLRs), host defense receptors that recognize invading pathogens, remains unknown. Here we showed that several well-characterized ER stress inducers (thapsigargin, tunicamycin, and dithiothreitol) increase the expression of TLR2 in epithelial cells. Ligand-responsiveness of TLR2 was also enhanced by ER stress inducers, implying a contributory role of ER stress for the regulation of TLR2-dependent inflammatory responses. Furthermore, there was significant increase of TLR2 mRNA level in the livers of tunicamycin-treated mice and high-fat diet-fed mice, suggesting an impact of ER stress in vivo on the expression of TLR2. Overexpression and knockdown experiments showed the importance of activating transcription factor 4 (ATF4), an ER stress-induced transcription factor, in the induction of TLR2 expression during ER stress. This was confirmed by the increased expression and function of TLR2 during treatment with salubrinal, an activator of ATF4 pathway. Taken together, our study provides further insights into the role of ER stress in enhancing host bacterial response or in exaggerating the inflammatory condition via up-regulating TLR2 expression.

Comparative analysis of ER stress response into HIV protease inhibitors: Lopinavir but not darunavir induces potent ER stress response via ROS/JNK pathway

HIV protease inhibitor (PI)-induced ER stress has been associated with adverse effects. Although it is a serious clinical problem for HIV/AIDS patients, comparative analyses of ER stress induction by clinically used PIs have rarely been done. Especially, there is no report on the differential ER stress response between lopinavir (LPV) and darunavir (DRV), although these PIs are the most clinically used PIs. We show here that LPV induces the most potent CHOP expression, ER stress marker, among the 9 Food and Drug Administration (FDA)-approved PIs in human peripheral blood mononuclear cells, several human epithelial cells, and mouse embryonic fibroblasts. LPV induced the most potent ROS production and JNK activation in 9 PIs. A comparison among the most clinically used PIs, ritonavir (RTV), LPV, and DRV, revealed that LPV potently and RTV moderately but not DRV induced ER stress via ROS-dependent JNK activation rather than proteasome inhibition. Finally, we analyzed ER stress induction in tissues of mice intraperitoneally injected with RTV, LPV, and DRV. RTV and LPV but not DRV showed ER stress induction in several mice tissues. In conclusion, we first identify LPV as the most potent ER stress inducing PI among 9 FDA-approved PIs in human cells, and although clinical verification is necessary, we show here that DRV has the advantage of less ROS and ER stress induction potential compared with LPV in vitro and in vivo.

Perturbation of proteasome function by bortezomib leading to ER stress-induced apoptotic cell death in cholangiocarcinoma

PurposeCholangiocarcinoma (CCA) or cancer of the biliary tract is heterogeneous; however, chronic inflammatory-related features are unique in CCA. Moreover, the genes involved in proteasome functions are evidently increased in CCA. Hence, CCA might be vulnerable to endoplasmic reticulum (ER) stressors, particularly a proteasome inhibitor. Therefore, bortezomib (BTZ), a specific 26S proteasome inhibitor, was selected, and its antitumor effects against CCA were investigated.MethodsLiver fluke-associated CCA cell lines were used. Cell proliferation and apoptosis detection were determined by a tetrazolium-based assay, caspase detection and annexin V binding assay. The accumulations of proteasome substrates, the inductions of ER stress and unfolded protein response (UPR) proteins were demonstrated by western blot and reporter systems. The in vivo anti-proliferative effect was accessed in a subcutaneous transplantation mouse model.ResultsBTZ inhibited CCA proliferation and induced caspase-dependent apoptosis, independently of the NF-κB pathway. Inhibition of protein degradation by BTZ led to the induction of UPR; induction of XBP1 splicing, ATF6 proteolysis and nuclear ATF4 as well as BiP and CHOP expressions were evident. Nevertheless, ER stress-induced UPR was overwhelming, leading to the activation of apoptosis demonstrated by proteolytic cleavages of ER-related caspase 4 and 12 as well as classical caspase 8, 9 and 3. The growth inhibitory effect of BTZ was supported by an in vivo model.ConclusionBTZ treatment could be a promising therapeutic approach for CCA treatment.

Inhibition of HIV-1 replication by a tricyclic coumarin GUT-70 in acutely and chronically infected cells

The anti-HIV-1 activity of GUT-70, a natural product derived from the stem bark of Chlophyllum brasiliense, was evaluated. GUT-70 inhibited HIV-1 replication in both acutely and chronically infected cells through suppression of NF-κB. Our results strengthen the idea that NF-κB pathway is one of the potential targets to control HIV-1 replication and that GUT-70 could serve as a lead compound to develop novel therapeutic agents against HIV-1 infection.