Ochratoxin A, citrinin and deoxynivalenol decrease claudin-2 expression in mouse rectum CMT93-II cells.

Intestinal epithelial cells are the first targets of ingested mycotoxins, such as ochratoxin A, citrinin and deoxynivalenol. It has been reported that paracellular permeability regulated by tight junctions is modulated by several mycotoxins by reducing the expression of specific claudins and integral membrane proteins in cell-cell contacts, accompanied by increase in phosphorylation of mitogen-activated protein kinases, including extracellular signal-related kinase (ERK) 1/2, p38 and c-Jun NH2-terminal protein kinase. Claudin-2 is expressed in the deep crypt cells, but not in the villus/surface cells in vivo. While Caco-2, T84 and IPEC-J2 cells, which are widely used intestinal epithelial cell lines to assess the influence of mycotoxins, do not express claudin-2, CMT93-II cells express claudin-2. We previously reported that inhibition of the ERK pathway reduced claudin-2 levels in cell-cell contacts in CMT93-II cells. In this study, we examined whether ochratoxin A, citrinin and deoxynivalenol affect claudin-2 expression and ERK1/2 phosphorylation in CMT93-II cells. We found that all mycotoxins reduced claudin-2 expression in cell-cell contacts, with reduction (by citrinin and deoxynivalenol) or no change (by ochratoxin A) in phosphorylated ERK1/2. All mycotoxins increased transepithelial electrical resistance, but did not affect flux of fluorescein. While ochratoxin A and citrinin are known to be nephrotoxic, only deoxynivalenol reduced claudin-2 expression in MDCK II cells derived from the renal tubule. These results suggest that claudin-2 expression is regulated not only by the ERK pathway, but also by other pathways in an organ-specific manner.福岡歯科大学2017年

Anisomycin, a JNK and p38 activator, suppresses cell-cell junction formation in 2D cultures of K38 mouse keratinocyte cells and reduces claudin-7 expression, with an increase of paracellular permeability in 3D cultures.

Keratinocytes in the oral mucosal epithelium, which is a non-keratinized stratified epithelium, are exposed to various stimuli from the oral cavity. JNK and p38 are stress-activated mitogen-activated protein kinases (MAPKs) that are phosphorylated by various stimuli and are involved in the assembly and disassembly of tight junctions (TJs) in keratinocytes. Therefore, we investigated the effects of stress-activated MAPKs on TJs in a mouse keratinocyte cell line during cell-cell junction formation in two-dimensional (2D) cultures or stratification to form non-keratinized epithelium in 3D cultures. In 2D cultures, calcium induced zipper-like staining for ZO-1 at 2 h and string-like staining for ZO-1 at 12 h, which indicated immature and mature cell-cell junctions, respectively. Anisomycin (AM), a JNK and p38 activator, inhibited formation of string-like staining for ZO-1, whereas inhibition of JNK, but not p38, after AM treatment restored string-like staining for ZO-1, although claudins (CLDNs) 4, 6, and 7 did not completely colocalize to ZO-1-positive sites. In 3D cultures, AM treatment for 2 weeks activated only p38, suppressed flattening of the superficial cells, removed CLDN7 from ZO-1-positive spots on the surface of 3D cultures, which represent TJs, and decreased transepithelial electrical resistance. Thus, short-term AM treatment inhibited maturation of cell-cell junctions by JNK, but not p38, activation. p38 activation by long-term AM treatment affected morphology of stratified structures and paracellular permeability, which was increased by CLDN7 removal from TJs. Various chronic stimuli that activate stress-activated MAPKs may weaken the keratinocyte barrier and be involved in TJ-related diseases.福岡歯科大学2018年

Serum affects keratinization and tight junctions in three-dimensional cultures of the mouse keratinocyte cell line COCA through retinoic acid receptor-mediated signaling.

Vitamin A, which is found in serum, is known to affect keratinocyte proliferation, epidermal differentiation, and keratinization. In mice, stratified epithelia in the oral cavity, esophagus, and forestomach are keratinized; however, these epithelia are not keratinized in humans. Several studies have reported that three-dimensional (3D) cultures of human keratinocytes in serum-containing medium could form keratinized epithelia. Here, we evaluated the effects of serum on the morphology, expression, and localization of differentiation markers and tight junction proteins, and paracellular permeability in 3D cultures of mouse keratinocytes. We found that only 0.1% calcium-depleted serum inhibited keratinization and induced a change in the expression of differentiation marker proteins from loricrin to keratin 4; the inhibition of retinoic acid receptor-mediated signaling reversed these changes. Furthermore, the serum reduced claudin-1 protein expression and prevented its localization at occludin-positive spots on the surface of 3D cultures. On the other hand, the serum increased the protein expression of claudin-4, occludin, zonula occludens-1, and E-cadherin. These changes may contribute to the reduction of the transepithelial electrical resistance by approximately half. In conclusion, mouse keratinocytes derived from the epidermis formed non-keratinized structures in 3D cultures in response to vitamin A in serum. The results suggest that retinoic acid receptor-mediated signaling may be inhibited in the mouse epithelia in the oral cavity, esophagus, and forestomach as well as the epidermis, leading to the keratinization of these epithelia.福岡歯科大学2018年

Multivariate analysis of risk factors for QT prolongation following subarachnoid hemorrhage

BACKGROUND: Subarachnoid hemorrhage (SAH) often causes a prolongation of the corrected QT (QTc) interval during the acute phase. The aim of the present study was to examine independent risk factors for QTc prolongation in patients with SAH by means of multivariate analysis. METHOD: We studied 100 patients who were admitted within 24 hours after onset of SAH. Standard 12-lead electrocardiography (ECG) was performed immediately after admission. QT intervals were measured from the ECG and were corrected for heart rate using the Bazett formula. We measured serum levels of sodium, potassium, calcium, adrenaline (epinephrine), noradrenaline (norepinephrine), dopamine, antidiuretic hormone, and glucose. RESULTS: The average QTc interval was 466 ± 46 ms. Patients were categorized into two groups based on the QTc interval, with a cutoff line of 470 ms. Univariate analyses showed significant relations between categories of QTc interval, and sex and serum concentrations of potassium, calcium, or glucose. Multivariate analyses showed that female sex and hypokalemia were independent risk factors for severe QTc prolongation. Hypokalemia (<3.5 mmol/l) was associated with a relative risk of 4.53 for severe QTc prolongation as compared with normokalemia, while the relative risk associated with female sex was 4.45 as compared with male sex. There was a significant inverse correlation between serum potassium levels and QTc intervals among female patients. CONCLUSION: These findings suggest that female sex and hypokalemia are independent risk factors for severe QTc prolongation in patients with SAH

Inhibition of retinoid X receptor improved the morphology, localization of desmosomal proteins and paracellular permeability in three-dimensional cultures of mouse keratinocytes.

Retinoic acid (RA) plays an important role in epithelial homeostasis and influences the morphology, proliferation, differentiation and permeability of epithelial cells. Mouse keratinocytes, K38, reconstituted non-keratinized stratified epithelium in three-dimensional (3D) cultures with serum, which contains retinol (a source of RA), but the morphology was different from in vivo epithelium. The formed epithelium was thick, with loosened cell-cell contacts. Here, we investigated whether the inhibition of RA receptor (RAR)/retinoid X receptor (RXR)-mediated signaling by an RXR antagonist, HX 531, improved K38 3D cultures in terms of morphology and intercellular junctions. The epithelium formed by 0.5 μM HX531 was thin, and the intercellular space was narrowed because of the restoration of the layer-specific distribution of desmoglein (DSG)-1, DSG3 and plakoglobin (PG). Moreover, the levels of desmosomal proteins and tight junction proteins, including DSG1, DSG2, DSG3, PG, claudin (CLDN)-1 and CLDN4 increased, but the adherens junction protein, E-cadherin, did not show any change. Furthermore, CLDN1 was recruited to occludin-positive cell-cell contacts in the superficial cells and transepithelial electrical resistance was increased. Therefore, K38 3D cultures treated with 0.5 μM HX531 provides a useful in vitro model to study intercellular junctions in the non-keratinized epithelium.福岡歯科大学2021年

The reduced susceptibility of mouse keratinocytes to retinoic acid may be involved in the keratinization of oral and esophageal mucosal epithelium.

Keratinocytes take up serum-derived retinol (vitamin A) and metabolize it to all-trans-retinoic acid (atRA), which binds to the nuclear retinoic acid receptor (RAR). We previously reported that serum-affected keratinocyte differentiation and function; namely, it inhibited keratinization, decreased loricrin (LOR) and claudin (CLDN) 1 expression, increased keratin (K) 4 and CLDN4 levels, and reduced paracellular permeability in three-dimensional (3D) cultures of mouse keratinocytes (COCA). Contrarily, RAR inhibition reversed these changes. Here, we aimed to examine whether atRA exerted the same effects as serum, and whether it was involved in the differential oral mucosa keratinization among animal species. Porcine oral mucosal keratinocytes, which form non-keratinized epithelium in vivo, established keratinized epithelium in 3D cultures. Both mouse and porcine sera induced non-keratinized epithelium at 0.1% in COCA 3D cultures. Although atRA caused the same changes as serum, its effective concentration differed. atRA inhibited keratinization at 0.1 nM and 1 nM in porcine or human keratinocytes and COCA, respectively. Furthermore, atRA upregulated CLDN7 in the cytoplasm but not in cell-cell contacts. These atRA-induced changes were reverted by RAR inhibition. The results indicate that serum-induced changes are probably due to the effect of serum-derived atRA, and that mouse keratinocytes require higher atRA concentrations to suppress keratinization than porcine and human keratinocytes. We propose that the lower susceptibility of mouse keratinocytes to atRA, rather than a lower retinol concentration, is a possible reason for the keratinization of mouse oral mucosal epithelium.福岡歯科大学2019年