Glucocorticoid receptor enhances involucrin expression of keratinocyte in a ligand-independent manner.

In this study, we investigated the role of glucocorticoid receptor (GR) in epidermal keratinocytes. In adult normal human skin, GR was highly expressed in the upper layers of the epidermis. Consistent with normal skin, GR expression was increased after calcium treatment of HaCaT keratinocytes cultured in vitro, suggesting that GR is involved in keratinocyte differentiation process. Overexpression of GR using an adenovirus showed that expression of involucrin, an early differentiation marker of keratinocytes, was markedly increased due to GR overexpression. However, treatment with dexamethasone, a GR agonist, did not increase involucrin expression. Overexpression of GR led to phosphorylation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinases (ERK) in the absence of glucocorticoid, suggesting that the GR effect on involucrin expression is related to activation of intracellular signaling cascades. This idea was supported by the fact that GR-mediated involucrin induction was abolished after treatment with JNK and ERK inhibitors. In addition, GR mutants lacking the ligand-binding domain increased involucrin expression concomitantly with increase of ERK phosphorylation. Together, these results suggest that GR modulates involucrin expression of keratinocytes by regulating the intracellular signaling network in a ligand-independent manner.

An Emerging Role of Micro- and Nanoplastics in Vascular Diseases.

Vascular diseases are the leading causes of death worldwide, and they are attributable to multiple pathologies, such as atherosclerosis, diabetes, and chronic obstructive pulmonary disease. Exposure to various environmental contaminants is associated with the development of various diseases, including vascular diseases. Among environmental contaminants, micro- and nanoplastics have gained attention as global environmental risk factors that threaten human health. Recently, extensive research has been conducted on the effects of micro- and nanoplastics on various human diseases, including vascular diseases. In this review, we highlight the effects of micro- and nanoplastics on vascular diseases.

Intron-derived aberrant splicing of A20 transcript in rheumatoid arthritis.

OBJECTIVE: Aberrant splicing is one of the most significant components generating functional diversity in many pathological conditions. The objective of this study was to analyse the mutations or aberrant splicing of A20 transcript, the region encompassing the ovarian tumour (OTU) domain [which is functionally important as an inhibitor of nuclear factor (NF)-κB activation] in fibroblast-like synoviocytes (FLSs) from RA patients. METHODS: Alterations in A20 transcripts were determined through sequence analysis of 10 clones of A20 cDNA in FLSs from each of the five RA patients. The levels of aberrant A20 transcript were measured by quantitative real-time RT-PCR with primers to specifically recognize the inserted introns. The functional role of A20 and its aberrant variants were examined by analysing NF-κB luciferase reporter activity and NF-κB-dependent target gene expression. RESULTS: In RA FLSs, we discovered four novel aberrant A20 transcripts, most of which resulted from insertion of partial intron 2, intron 4 and/or deletion of exon 4. In each of these FLSs, sequence analysis revealed that these aberrant insertional sequences were flanked by consensus splice donor and acceptor sequences without nucleotide substitution, suggesting alternative splicing as the likely mutational mechanism. These variants elicited a codon frame shift by creating a premature translational stop codon, and eventually, disruption of the OTU domain (which is functionally important as an inhibitor of NF-κB activation) of A20. The expression level of aberrant A20 transcript was correlated well with persisitently enhanced status of NF-κB signalling, as evident by the phosphorylation of inhibitor of NF-κB (IκB)-α and transcription of NF-κB target genes. CONCLUSION: The results suggest that A20 inactivation by the novel aberrant splicing may contribute to RA progression by inducing persistent NF-κB activation.

A novel protease activity assay method based on an engineered autoinhibited protein using an enzyme-linked immunoassay.

Proteases are involved in various biological phenomena, and their aberrant activity can be an important indicator of disease. Thus, various methods have been developed to analyze the activities of proteases, but their wide application has been hampered because each method has drawbacks. In this report, we propose a new protease assay method based on an engineered autoinhibited protein and enzyme-linked immunoassay (ELISA) in which a protease of interest activates the autoinhibited protein and the signal is amplified via ELISA. Using this concept a sensitive assay method for MMP2 and caspase-3 was developed. The limit of detection for the two proteases was as low as 7 pM for MMP2 and 0.1 pM for caspase-3. The autoinhibited protein is designed modularly, and the new platform is general enough for the development of assay methods for other proteases with minimal modification.

Recent development of highly sensitive protease assay methods: Signal amplification through enzyme cascades.

Proteases are involved in almost all biological processes, and therefore, aberrant activity of many of these enzymes is an important indicator of disease. Various methods have been developed to analyze protease activity, among which, protease assays based on resonance energy transfer are currently used most widely. However, quantitative methods with relatively higher sensitivity are needed, especially for disease diagnosis at early stages. One of the strategies to achieve higher sensitivity is to implement signal amplification of the protease activity. In this review, we briefly summarize the protease assay methods based on resonance energy transfer, and then elaborate the efforts to develop sensitive protease assays through signal amplification by using enzyme cascades.

Expression of N-terminal truncated desmoglein 3 (deltaNDg3) in epidermis and its role in keratinocyte differentiation.

During a search for keratinocyte differentiation-related genes, we obtained a cDNA fragment from the 5'-untranslated region of a previously identified splicing variant of desmoglein 3 (Dg3). This transcript encodes a protein of 282 amino acids, which corresponds to the N-terminal truncated intracellular domain of Dg3 (deltaNDg3). Northern blot analysis detected a 4.6-kb transcript matching the predicted size of deltaNDg3 mRNA, and Western blot analysis with an antibody raised against the Dg3 C-terminus (H-145) detected a 31-kDa protein. Increased deltaNDg3 expression was observed in differentiating keratinocytes by RT-PCR and Western blot analysis, suggesting that deltaNDg3 is indeed a differentiation-related gene product. In immunohistochemical studies of normal and pathologic tissues, H-145 antibody detected the protein in the cytoplasm of suprabasal layer cells, whereas an antibody directed against the N-terminal region of Dg3 (AF1720) reacted with a membrane protein in the basal layer. In addition, deltaNDg3 transcript and protein were upregulated in psoriatic epidermis, and protein expression appeared to increase in epidermal tumors including Bowen's disease and squamous cell carcinoma. Moreover, overexpression of deltaNDg3 led to increased migration and weakening of cell adhesion. These results suggest that deltaNDg3 have a role in keratinocyte differentiation, and that may be related with tumorigenesis of epithelial origin.

Prediction and evaluation of protein-protein interaction in keratinocyte differentiation.

Terminal differentiation of skin keratinocytes is a vertically directed multi-step process that is tightly controlled by the sequential expression of a variety of genes. We previously investigated the gene expression profile and found that many of differentiation-related genes expressed in a temporally regulated manner. In this study, we attempted to find the hub-molecules and their intracellular signaling networks during keratinocyte differentiation using in silico analysis of data obtained from previous studies. We used protein-protein interaction prediction software called PSIMAP, and drew a hypothetical signaling network. We chose one candidate hub-molecule SHC1 that were predicted to link EGFR and MAPK signal, and then evaluated the protein-protein interactions experimentally. As predicted, SHC1 bound to the MEK1 in an EGF-regulated manner. Furthermore, SHC1 bound to the MEK1 and p38 MAPK in a keratinocyte differentiation dependent manner. These results demonstrate that in silico protein-protein interaction prediction system can be used to efficiently and cost-effectively select the experimental candidates.

Upregulation of Bcl-2 is associated with cisplatin-resistance via inhibition of Bax translocation in human bladder cancer cells.

The efficacy of cisplatin in cancer chemotherapy is limited by the development of resistance. To elucidate the molecular basis of resistance to cisplatin, we compared cisplatin-induced apoptotic responses of the parental human bladder cancer cell line, T24 and its resistant subclone, T24R2. In T24 cells, cisplatin induce apoptosis and the activation of caspase-8, -9 and -3 and poly(ADP-ribose) polymerase cleavage. The expression levels of Fas, FasL, and FADD were not changed by the treatment with cisplatin. Furthermore, neither Fas-neutralizing antibody nor dominant negative mutant of FADD affected cisplatin-induced apoptosis. Western blot analysis of subcellular fractions showed that cisplatin induced redistribution of Bax and cytochrome c. Thus, cisplatin causes apoptosis in a death receptor-independent and mitochondria-dependent fashion in T24 cells. In contrast, overexpressed Bcl-2 protein inhibited cisplatin-induced Bax translocation and its downstream events in T24R2. Downregulation of Bcl-2 by RNAi potentiated the redistribution of Bax and cytochrome c and reversed cisplatin-resistance. Our results indicate that upregulation of Bcl-2 contributes to the development of cisplatin-resistance and usage of siRNA which targets the Bcl-2 gene may offer a potential tool to reverse the resistance to cisplatin in bladder cancer.

Up-regulation of Bfl-1/A1 via NF-kappaB activation in cisplatin-resistant human bladder cancer cell line.

The potent anti-cancer agent cis-diamminedichloroplatinum (II) (cisplatin) is currently used for treating bladder cancer. However, clinical use of this drug for long periods is often limited because of the appearance of cisplatin-resistant bladder tumor cells. We employed the method of a differential display reverse transcriptase polymerase chain reaction to identify the differentially expressed genes in the parental human bladder cancer cell line, T24 and three cisplatin-resistant cell lines. We report here that cisplatin-resistant cell lines overexpress Bcl-2 family protein Bcl-2-related gene expressed in fetal liver (Bfl-1)/A1 as compared with their parental cell. Cisplatin and gamma-irradiation induced expression of Bfl-1/A1 in T24R2 cells but not in T24 cells. Among Bcl-2 family members, Bfl-1/A1 showed the most significant alteration of the expression level in resistant cells. The nuclear translocation of nuclear factor-kappaB (NF-kappaB) by cisplatin and gamma-irradiation selectively occurred in T24R2 cells. Mitochondrial depolarization and cell death by cisplatin were also prevented in T24R2 cells. Moreover, Bfl-1/A1 inhibited cisplatin- and TNF-alpha-induced apoptosis in BOSC23 cells. Our findings suggest that the induction of Bfl-1/A1 by NF-kappaB may be important in controlling resistance to cisplatin responses in bladder tumor cells.

Engineering ß-lactamase zymogens for use in protease activity assays.

In this study, the concept of autoinhibition, a mechanism of protein activity regulation, was applied to the design and engineering of a ß-lactamase zymogen. Using this zymogen, a sensitive protease assay method was developed in which activation of the zymogen by proteases produces an amplified absorbance signal. The approach reported here can be adapted for engineering of zymogens as biological sensors and components of synthetic signaling pathways.

Water extract of Cynanchi atrati Radix regulates inflammation and apoptotic cell death through suppression of IKK-mediated NF-κB signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Cynanchi atrati Radix has been traditionally used as an anti-inflammatory agent to treat febrile diseases, acute urinary infection or subcutaneous pyogenic infection with invasion of the pathogenic factors. AIM OF STUDY: Nuclear factor (NF)-κB is a pleiotropic transcriptional factor of many genes involved in inflammatory and anti-apoptotic responses. To identify a novel, potent inhibitor of NF-κB signaling pathway, a plant extract library of traditional oriental medicine was screened for the capability to block the NF-κB activity in cells overexpressing toll-like receptor 4 (TLR4), and then evaluated the anti-inflammatory and pro-apoptotic functions of water extract of Cynanchi atrati Radix (WECR) in macrophages and cancer cells, respectively. MATERIALS AND METHODS: The effect of WECR on the proinflammatory mediators (inducible NO synthase [iNOS], cyclooxygenase [COX]-2), IκB-α degradation, RelA/p65 phosphorylation and caspase cleavages were measured by immunblotting. NF-κB transcriptional activity, IκB kinase (IKK) activity and nitric oxide (NO) production was measured using the luciferase assay, in vitro kinase assay and Griess reaction. RESULTS: WECR efficiently inhibited LPS-induced expression of proinflammatory mediators including iNOS and COX-2. IKK kinase activity, IκB-α degradation, nuclear translocation of RelA/p65 and NF-κB transcriptional activity induced by LPS were suppressed by WECR. Furthermore, WECR dramatically enhances the apoptotic response, as evident by the combination with tumor necrosis factor (TNF) was able to induce the cytotoxic action through caspase-dependent pathway. CONCLUSION: These results indicate that WECR has a potential to inhibit IKK-mediated NF-κB activation, and is a valuable compound for modulating inflammatory or cancerous conditions.

Role of plasminogen activator inhibitor-2 (PAI-2) in keratinocyte differentiation.

BACKGROUND: Plasminogen activator inhibitor-2 (PAI-2) is an enzyme inhibitor which is involved in various biological processes including cell differentiation, tissue regrowth and regeneration. Although PAI-2 has been originally isolated as an extracellular inhibitor of urokinase plasminogen activator (uPA), recent studies indicate that PAI-2 has other intracellular effects in keratinocyte, such as the component of cornified envelope. OBJECTIVE: The aim of this study is to investigate the expression and functional role of PAI-2 during the keratinocyte differentiation. METHODS: We transduced keratinocytes with adenovirus harboring the expression cassette for PAI-2, then examined the effect on keratinocytes differentiation. RESULTS: When cultured epidermal keratinocytes were treated with 1.2 mM calcium, PAI-2 expression was increased time-dependently at both mRNA and protein levels. The calcium-induced PAI-2 expression was abolished by treatment with p38 MAPK inhibitor, while overexpression of MKK6 led to the increase of PAI-2 expression. When PAI-2 was overexpressed by adenoviral transduction, the expression of keratinocyte differentiation markers such as involucrin, keratin 10 and loricrin was markedly increased. Concomitantly, overexpression of PAI-2 resulted in the retardation of cell growth, with the increase of Rb and p53. CONCLUSION: These results suggest that PAI-2 has a role for promoting the differentiation of epidermal keratinocytes.