Tsukasa Okiyoneda

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.

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.

Role of calnexin in the ER quality control and productive folding of CFTR; differential effect of calnexin knockout on wild-type and ΔF508 CFTR

Cystic fibrosis (CF) is caused by the mutation in CF transmembrane conductance regulator (CFTR), a cAMP-dependent Cl(-) channel at the plasma membrane of epithelium. The most common mutant, DeltaF508 CFTR, has competent Cl(-) channel function, but fails to express at the plasma membrane since it is retained in the endoplasmic reticulum (ER) by the ER quality control system. Here, we show that calnexin (CNX) is not necessary for the ER retention of DeltaF508 CFTR. Our data show that CNX knockout (KO) does not affect the biosynthetic processing, cellular localization or the Cl(-) channel function of DeltaF508 CFTR. Importantly, cAMP-induced Cl(-) current in colonic epithelium from CNX KO/DeltaF508 CFTR mice was comparable with that of DeltaF508 CFTR mice, indicating that CNX KO failed to rescue the ER retention of DeltaF508 CFTR in vivo. Moreover, we show that CNX assures the efficient expression of WT CFTR, but not DeltaF508 CFTR, by inhibiting the proteasomal degradation, indicating that CNX might stimulate the productive folding of WT CFTR, but not DeltaF508 CFTR, which has folding defects.

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.

Calnexin Δ185–520 partially reverses the misprocessing of the ΔF508 cystic fibrosis transmembrane conductance regulator1

Abnormal retention of ΔF508 CFTR (cystic fibrosis transmembrane conductance regulator) in the endoplasmic reticulum is a major cause of cystic fibrosis (CF). We show that calnexin Δ185–520 but not calnexin can partially reverse the mislocalization of ΔF508 CFTR. This 256‐amino acid protein has neither the transmembrane domain nor the P domain of calnexin. Calnexin Δ185–520 interacted with CFTR directly, and was secreted into the extracellular compartment over time. Forty‐eight hours after transfection into CHO cells, calnexin Δ185–520 increased the conversion of immature ΔF508 CFTR into mature ΔF508 CFTR. In immortalized human CF cell lines expressing ΔF508 CFTR, a halide efflux assay showed that calnexin Δ185–520 partially restored CFTR function. These data indicate that calnexin Δ185–520 may give a clue to develop the therapeutic way of cystic fibrosis with ΔF508 CFTR.

Characterization of CFTR expression in a human pulmonary mucoepidermoid carcinoma cell line, NCI‐H292 cells

The NCI‐H292 cell, a human pulmonary mucoepidermoid carcinoma cell line, is commonly used for studying bacterial and viral infections of airway epithelial cells. Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) is the main cause of fetal lung infection in cystic fibrosis patients. In this study, we examined CFTR expression in NCI‐H292 cells to determine whether NCI‐H292 cells possess sufficient, normally functioning CFTR. The results of RT‐PCR and Northern blotting analysis indicated that the CFTR gene expression level was much lower in NCI‐H292 cells than in T84 cells. However, Western blotting analysis showed that protein expression in NCI‐H292 cells was comparable to that in T84 cells. Furthermore, whole‐cell and cell‐attached patch clamp electrophysiological techniques indicated that the Cl− current induced by intracellular cAMP elevation in NCI‐H292 cells was comparable to that in T84 cells. These findings suggest that NCI‐H292 cells with a low level of CFTR gene expression possess enough functional CFTR to show a physiological response.