Mary Ann Suico

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.

Promoter hypomethylation of Toll-like receptor-2 gene is associated with increased proinflammatory response toward bacterial peptidoglycan in cystic fibrosis bronchial epithelial cells.

Cystic fibrosis (CF) is the most common lethal inherited disorder caused by mutation in the gene encoding CF transmembrane regulator (CFTR). The clinical course of CF is characterized by recurrent pulmonary infections and chronic inflammation. Here, we show that toll‐like receptor‐2 (TLR2) expression and response were strongly enhanced in the human CF bronchial epithelial cell line, CFBE41o‐. Treatment of the cells with 5‐azacytidine decreased the promoter methylation within TLR2 proximal promoter and increased endogenous expression of TLR2 in non‐CF 16HBE14o‐ cells, suggesting that TLR2 expression is epigenetically regulated by CpG methylation. Moreover, bisulfite sequence analysis revealed that TLR2 promoters were highly demethylated in CFBE41o‐ cells, implying that decreased methylation of the TLR2 promoter is responsible for CF‐related up‐regulation of TLR2. Finally, stable expression of WT‐CFTR in CFBE41o‐ cells (CFBE41o‐/WT‐CFTR cells) reduced TLR2 expression and the response to its ligand peptidoglycan (PGN), implying a causal relationship between CFTR dysfunction and TLR2 up‐regulation. Consistent with reduced expression of TLR2 in CFBE41o‐/WT‐CFTR cells, CpG methylation was increased in CFBE41o‐/WT‐CFTR cells. Taken together, our results demonstrate that TLR2 expression is epigenetically up‐regulated in CF bronchial epithelial cells and suggest that TLR2 overexpression or prolonged activation of TLR2 signaling might be critical in CF pathogenesis.

The Crystal Structure of the Green Tea Polyphenol (―)-Epigallocatechin Gallate―Transthyretin Complex Reveals a Novel Binding Site Distinct from the Thyroxine Binding Site

Amyloid fibril formation is associated with protein misfolding disorders, including neurodegenerative diseases such as Alzheimers, Parkinsons, and Huntingtons diseases. Familial amyloid polyneuropathy (FAP) is a hereditary disease caused by a point mutation of the human plasma protein, transthyretin (TTR), which binds and transports thyroxine (T(4)). TTR variants contribute to the pathogenesis of amyloidosis by forming amyloid fibrils in the extracellular environment. A recent report showed that epigallocatechin 3-gallate (EGCG), the major polyphenol component of green tea, binds to TTR and suppresses TTR amyloid fibril formation. However, structural analysis of EGCG binding to TTR has not yet been conducted. Here we first investigated the crystal structure of the EGCG-V30M TTR complex and found novel binding sites distinct from the thyroxine binding site, suggesting that EGCG has a mode of action different from those of previous chemical compounds that were shown to bind and stabilize the TTR tetramer structure. Furthermore, EGCG induced the oligomerization and monomer suppression in the cellular system of clinically reported TTR variants. Taken together, these findings suggest the possibility that EGCG may be a candidate compound for FAP therapy.

Mild Electrical Stimulation with Heat Shock Ameliorates Insulin Resistance via Enhanced Insulin Signaling

Low-intensity electrical current (or mild electrical stimulation; MES) influences signal transduction and activates phosphatidylinositol-3 kinase (PI3K)/Akt pathway. Because insulin resistance is characterized by a marked reduction in insulin-stimulated PI3K-mediated activation of Akt, we asked whether MES could increase Akt phosphorylation and ameliorate insulin resistance. In addition, it was also previously reported that heat shock protein 72 (Hsp72) alleviates hyperglycemia. Thus, we applied MES in combination with heat shock (HS) to in vitro and in vivo models of insulin resistance. Here we show that 10-min treatment with MES at 5 V (0.1 ms pulse duration) together with HS at 42°C increased the phosphorylation of insulin signaling molecules such as insulin receptor substrate (IRS) and Akt in HepG2 cells maintained in high-glucose medium. MES (12 V)+mild HS treatment of high fat-fed mice also increased the phosphorylation of insulin receptor β subunit (IRβ) and Akt in mice liver. In high fat-fed mice and db/db mice, MES+HS treatment for 10 min applied twice a week for 12–15 weeks significantly decreased fasting blood glucose and insulin levels and improved insulin sensitivity. The treated mice showed significantly lower weight of visceral and subcutaneous fat, a markedly improved fatty liver and decreased size of adipocytes. Our findings indicated that the combination of MES and HS alleviated insulin resistance and improved fat metabolism in diabetes mouse models, in part, by enhancing the insulin signaling pathway.

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)

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.

DNA demethylation-dependent enhancement of toll-like receptor-2 gene expression in cystic fibrosis epithelial cells involves SP1-activated transcription.

BackgroundThe clinical course of cystic fibrosis (CF) is characterized by recurrent pulmonary infections and chronic inflammation. We have recently shown that decreased methylation of the toll-like receptor-2 (TLR2) promoter leads to an apparent CF-related up-regulation of TLR2. This up-regulation could be responsible, in part, for the CF-associated enhanced proinflammatory responses to various bacterial products in epithelial cells. However, the molecular mechanisms underlying DNA hypomethylation-dependent enhancement of TLR2 expression in CF cells remain unknown.ResultsThe present study indicates that there is a specific CpG region (CpG#18-20), adjacent to the SP1 binding site that is significantly hypomethylated in several CF epithelial cell lines. These CpGs encompass a minimal promoter region required for basal TLR2 expression, and suggests that CpG#18-20 methylation regulates TLR2 expression in epithelial cells. Furthermore, reporter gene analysis indicated that the SP1 binding site is involved in the methylation-dependent regulation of the TLR2 promoter. Inhibition of SP1 with mithramycin A decreased TLR2 expression in both CF and 5-azacytidine-treated non-CF epithelial cells. Moreover, even though SP1 binding was not affected by CpG methylation, SP1-dependent transcription was abolished by CpG methylation.ConclusionThis report implicates SP1 as a critical component of DNA demethylation-dependent up-regulation of TLR2 expression in CF epithelial cells.

Hyperthermia With Mild Electrical Stimulation Protects Pancreatic β-Cells From Cell Stresses and Apoptosis

Induction of heat shock protein (HSP) 72 improves metabolic profiles in diabetic model mice. However, its effect on pancreatic β-cells is not known. The current study investigated whether HSP72 induction can reduce β-cell stress signaling and apoptosis and preserve β-cell mass. MIN6 cells and db/db mice were sham-treated or treated with heat shock (HS) and mild electrical stimulation (MES) (HS+MES) to induce HSP72. Several cellular markers, metabolic parameters, and β-cell mass were evaluated. HS+MES treatment or HSP72 overexpression increased HSP72 protein levels and decreased tumor necrosis factor (TNF)-α–induced Jun NH2-terminal kinase (JNK) phosphorylation, endoplasmic reticulum (ER) stress, and proapoptotic signal in MIN6 cells. In db/db mice, HS+MES treatment for 12 weeks significantly improved insulin sensitivity and glucose homeostasis. Upon glucose challenge, a significant increase in insulin secretion was observed in vivo. Compared with sham treatment, levels of HSP72, insulin, pancreatic duodenal homeobox-1, GLUT2, and insulin receptor substrate-2 were upregulated in the pancreatic islets of HS+MES-treated mice, whereas JNK phosphorylation, nuclear translocation of forkhead box class O-1, and nuclear factor-κB p65 were reduced. Apoptotic signals, ER stress, and oxidative stress markers were attenuated. Thus, HSP72 induction by HS+MES treatment protects β-cells from apoptosis by attenuating JNK activation and cell stresses. HS+MES combination therapy may preserve pancreatic β-cell volume to ameliorate glucose homeostasis in diabetes.

Calreticulin Negatively Regulates the Cell Surface Expression of Cystic Fibrosis Transmembrane Conductance Regulator

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent Cl- channel at the plasma membrane, and its malfunction results in cystic fibrosis, the most common lethal genetic disease in Caucasians. Quality control of CFTR is strictly regulated by several molecular chaperones. Here we show that calreticulin (CRT), which is a lectin-like chaperone in the endoplasmic reticulum (ER), negatively regulates the cell surface CFTR. RNA interference-based CRT knockdown induced the increase of CFTR expression. Consistently, this effect was observed in vivo. CRT heterozygous (CRT+/-) mice had a higher endogenous expression of CFTR than the wild-type mice. Moreover, CRT overexpression induced cell surface expression of CRT, and it significantly decreased the cell surface expression and function of CFTR. CRT overexpression destabilized the cell surface CFTR by enhancing endocytosis, leading to proteasomal degradation. Deletion of the carboxyl domain of CRT, which results in its ER export, increased the negative effect and enhanced the interaction with CFTR. Thus, CRT in the post-ER compartments may act as a negative regulator of the cell surface CFTR.

MEF up-regulates human β-defensin 2 expression in epithelial cells

Human β‐defensin 2 (HBD2), an antimicrobial peptide, is widely expressed in epithelial tissues and displays a potent killing activity in response to the invasiveness of a wide range of microorganisms and the stimulation of various molecules. Myeloid ELF‐1‐like factor (MEF) has been reported to be involved in innate immunity responses, such as activation of perforin and lysozyme transcription. The role of MEF in the transcription regulation of HBD2, however, is unknown. Here, we show that MEF not only activated HBD2 promoter activity, but also increased the endogenous HBD2 transcription level. Moreover, the activated HBD2 promoter activity was attenuated by the antisense MEF RNA input and the loss of the ETS binding site (EBS: GGAA core sequence) in the HBD2 promoter. The interaction between the EBS and MEF protein was further confirmed by electrophoretic mobility shift assay. Thus, our data indicate that MEF may play an important role in regulating HBD2 expression in epithelial cells.