Claudin family proteins play an important role in the formation of tight junctions in several tissues. Individual claudins display organ- and tissue-specific expression. Claudin domain containing 1 (CLDND1), also known as claudin 25 (CLDN25), is a homolog of the claudin family, and its expression was reported to be downregulated in a mouse model of cerebellar hemorrhage. We have also reported that the retinoic acid receptor-related orphan receptor α (RORα) is involved in the transcriptional activation of CLDND1 by binding to the RORα responsive element (RORE) in the CLDND1 promoter region. Cholesterol and its derivative oxysterol reportedly serve as ligands for the nuclear receptor RORα. However, the effect of cholesterols on CLDND1 expression is unclear. The present study aimed to evaluate the effect of inhibiting steroid synthesis via lovastatin on RORα-mediated CLDND1 transcriptional regulation. Chromatin immunoprecipitation and luciferase reporter assays revealed that RORα-mediated transcriptional regulation of CLDND1 was suppressed upon lovastatin treatment of HepG2 cells; however, this inhibitory effect was attenuated by supplementation with cholesterol. Furthermore, quantitative reverse transcription-PCR and immunoblotting analyses revealed the downregulated expression of CLDND1 mRNA and protein in HepG2 cells upon lovastatin treatment with no parallel changes in RORα mRNA and protein levels. These results confirm that cholesterol serve as ligands for RORα and are, therefore, involved in the activation of CLDND1 transcriptional regulation by RORα.

Thiazolidinediones, such as troglitazone and rosiglitazone, are anti-diabetic insulin-sensitizing agents that bind to the peroxisome proliferator-activated receptor γ (PPARγ) and have potent adipogenic effects on 3T3-L1 preadipocytes. During 3T3-L1 preadipocyte differentiation, which was induced by isobutyl methylxanthine, dexamethasone, and insulin, troglitazone treatment increased lipid content and decreased PPARγ protein levels compared with DMSO-treated control cells. However, the level of CCAAT/enhancer binding protein α (C/EBPα) and C/EBPβ proteins did not decrease in troglitazone-treated cells compared with DMSO-treated cells. Real-time PCR analysis showed that PPARγ mRNA but not C/EBPα mRNA was downregulated in troglitazone-treated adipocytes, suggesting that PPARγ protein reduction occurred due to the decrease in its transcription level. Rosiglitazone treatment also increased lipid content but decreased PPARγ expression during 3T3-L1 preadipocyte differentiation. Both thiazolidinediones significantly increased the levels of adipokines such as adipocyte protein 2 (aP2) and adiponectin in 3T3-L1 adipocytes compared with that in DMSO-treated cells. We propose that thiazolidinediones are involved in adipogenic homeostasis rather than act as agonists of PPARγ during 3T3-L1 adipocyte differentiation.

Melanocytes increase melanin production upon exposure to ultraviolet rays (UV) as a defense mechanism. Melanin synthesis in melanocytes is regulated by bioactive factors produced and secreted by keratinocytes. In addition, melanocytes transfer biosynthesized melanin to keratinocytes, causing skin pigmentation. During the pigmentation process, melanocytes communicate with surrounding cells. However, the molecules involved in this cell-to-cell communication, particularly in melanocytes, are poorly understood. In this study, we focused on changes in cell membrane protein expression following exposure of melanocytes to UV irradiation. After mouse melanoma B16 cells were exposed to UV irradiation, alterations in the expression of cell membrane proteins were analyzed using peptide mass fingerprinting. We identified the membrane protein vinculin from proteins with enhanced expression. Western blot results confirmed UV exposure increased vinculin in cell membranes. In contrast, there was no change in vinculin levels in whole cell extracts. Furthermore, we observed no variation in mRNA expression levels using real time PCR. Melanocytes exposed to UV enhanced vinculin migration to cell membranes without altering expression levels. We suggest vinculin involved in the cellular responses of melanocytes and keratinocytes.

Spinal muscular atrophy (SMA) is a progressive neuromuscular disease, associated with motoneuron loss and muscle wasting. Numerous SMA-causative mutations have been reported in survival motor neuron (SMN) gene(s); however, the pathogenic mechanism underlying SMA remains unclear. In the present study, we showed that SMN modulates the expression of transcription factor EB (TFEB), a master regulator of lysosomal genes that plays a key role in lysosome function, autophagy, and the mammalian target of rapamycin (mTOR) signaling pathway. The transfection of small interfering RNA (siRNA) targeting SMN caused a reduction in TFEB expression in the motoneuron-like NSC-34 cell line. In differentiated NSC-34 cells, either SMN or TFEB knockdown resulted in reduced lysosomes at neurites and the atypical accumulation of swollen and enlarged lysosomes in cell bodies. SMN knockdown caused the reduced expression of lysosome-related genes, resulting in the decline of lysosomal degradation and increased autophagic flux. These SMN-depletion-induced aberrations in lysosomes and autophagy could be rescued by the exogenous expression of TFEB. Furthermore, SMN depletion in NSC-34 cells resulted in the decreased phosphorylation of mTOR and its downstream signals. Finally, SMA transgenic mice exhibited reduced TFEB and lysosomal protein expression and the inactivation of mTOR signaling in the lumbar spinal cord at postnatal day 11, compared with their counterparts. These findings indicated that SMN regulates lysosomal gene expression and functions by altering TFEB expression in motoneurons. The targeting of lysosomes might represent a new strategy for the treatment of SMA.

Polymethoxyflavones (PMFs) are flavone compounds that contain more than two methoxyl groups and are almost exclusively found in citrus peel. In this study, we examined and compared the effects of the PMFs nobiletin, heptamethoxyflavone and natsudaidain on antigen-specific T cell activation in vitro and in vivo. The three PMFs suppressed proliferation responses in an in vitro study. The PMFs also suppressed IFN-γ, IL-2 and IL-10 production from splenocytes in a dose-dependent manner. Interestingly, nobiletin and heptamethoxyflavone, but not natsudaidain, enhanced IL-4 production from splenocytes. In an in vivo model, antigen-specific T cell proliferation responses were reduced in mice treated with nobiletin, heptamethoxyflavone and natsudaidain compared with responses in control mice. The results suggest that citrus PMFs suppress T cell activation and that nobiletin and heptamethoxyflavone enhance IL-4 production.

Exposure to cadmium, an environmental pollutant, has been associated with adverse health effects such as atherosclerosis. Nucleolin is a multifunctional protein present in the nucleus, cytoplasm, and cell surface and involved in the angiogenesis and repair of the vascular endothelium following injury. Nucleolin dysfunction has been suggested to cause the development of vascular lesions. In the present study, we assessed the effect of cadmium on nucleolin expression in cultured bovine aortic vascular endothelial cells. After incubation with cadmium (0–5 µM), cellular NCL (nucleolin) mRNA levels and nucleolin protein levels were determined by real-time RT-PCR and western blotting, respectively. Cadmium did not alter nucleolin levels in vascular endothelial cells. We then evaluated whether nucleolin modulates endothelial cadmium-induced cytotoxicity by lactate dehydrogenase leakage. In nucleolin-knockdown vascular endothelial cells, cadmium-induced cytotoxicity was significantly higher than in control cells. Furthermore, nucleolin knockdown did not decrease the mRNA levels of metallothionein 1A and 2A, suggesting that nucleolin protects vascular endothelial cells against cadmium-induced cytotoxicity via metallothionein-independent pathways.

Clostridium perfringens iota-toxin consists of an enzymatic Ia component and binding Ib component. Ib binds to membrane receptors, forms heptamers in lipid rafts, and associates with Ia. The toxin complex is internalized into cells. In this study, we evaluated the inhibitory effects of cholera toxin B subunit (CTB), which binds to lipid rafts, on iota-toxin-mediated cytotoxicity. We examined the effect of CTB on iota-toxin-induced cell rounding activity against MDCK (Madin-Darby canine kidney) cells and Ib-induced cell death in A431 (human epithelial) cells. The presence of CTB inhibited both the cell rounding activity of iota-toxin (MDCK cells) and Ib-induced cell death (A431 cells). Moreover, CTB blocked binding of the Ib monomer to both cells at 4°C. We found by immunofluorescence microscopy that, after loading with Ib and CTB at 4°C, Ib and CTB bound to distinct regions in the plasma membranes. In MDCK cells at 37°C, Ib internalized and partially coexisted with CTB in cytoplasmic vesicles. These findings demonstrated that CTB blocks the binding of iota-toxin to target cells. CTB may provide a protective effect against infection.