Yosuke Mai

Autoantibody Profile Differentiates between Inflammatory and Noninflammatory Bullous Pemphigoid

Bullous pemphigoid (BP) is a major autoimmune blistering skin disorder, in which a majority of the autoantibodies (autoAbs) target the juxtamembranous extracellular noncollagenous 16A domain (NC16A) domain of hemidesmosomal collagen XVII. BP-autoAbs may target regions of collagen XVII other than the NC16A domain; however, correlations between epitopes of BP-autoAbs and clinical features have not been fully elucidated. To address correlations between the clinical features and specific epitopes of BP-autoAbs, we evaluated the epitope profiles of BP-autoAbs in 121 patients. A total of 87 patients showed a typical inflammatory phenotype with erythema and autoAbs targeting the anti-NC16A domain, whereas 14 patients showed a distinct noninflammatory phenotype, in which autoAbs specifically targeted the midportion of collagen XVII, but not NC16A. Interestingly, this group clinically showed significantly reduced erythema associated with scant lesional infiltration of eosinophils. Surprisingly, 7 of the 14 cases (50.0%) received dipeptidyl peptidase-IV inhibitors for the treatment of diabetes. Dipeptidyl peptidase-IV inhibitors were used in 3 of 76 (3.9%) typical cases of BP with autoAbs targeting NC16A; thus, dipeptidyl peptidase-IV inhibitors are thought to be involved in the development of atypical noninflammatory BP. This study shows that the autoAb profile differentiates between inflammatory and noninflammatory BP, and that noninflammatory BP may be associated with dipeptidyl peptidase-IV inhibitors.

Different binding property of STIM1 and its novel splice variant STIM1L to Orai1, TRPC3, and TRPC6 channels

Stromal interaction molecule 1 (STIM1) is the endoplasmic reticulum (ER) Ca(2+) sensor to control ER Ca(2+) levels. A recent study has shown that STIM1L, a new splice variant of STIM1, is expressed in various tissues of rodent and in human skeletal muscle, and that the interaction of STIM1L with actin filament allows rapid activation of store-operated Ca(2+) entry (SOCE) mediated through Orai1 channels. Here, we characterize mRNA expression and function of human STIM1 and STIM1L, and compare their binding property to Orai1 functioning as store-operated Ca(2+) channels (SOCCs), and TRPC3 (transient receptor potential canonical 3) and TRPC6 channels functioning as endothelin type A receptor (ET(A)R)-operated Ca(2+) channels (ROCCs). Although mRNA for STIM1 was ubiquitously expressed in human tissues, STIM1L was detected only in skeletal muscle. STIM1L augmented thapsigargin- and endothelin-1-induced SOCE more strongly than STIM1 in human embryonic kidney 293 cells stably expressing ET(A)R, whereas, it tends to suppress ET(A)R-operated Ca(2+) entry (ROCE) via TRPC3 and TRPC6 more strongly than STIM1. Coimmunoprecipitation experiments have revealed that when compared with STIM1, STIM1L binds more abundantly to Orai1 and also to TRPC3 and TRPC6. These results suggest that the higher binding capacity of STIM1L to SOCCs and ROCCs plays an important role in the regulation of Ca(2+) signaling such as the augmentation of SOCE via Orai1 and the inhibition of ROCE via TRPC3 and TRPC6.

Identification of stable cytotoxic factors in the gas phase extract of cigarette smoke and pharmacological characterization of their cytotoxicity.

Smoking is a major risk factor for atherosclerotic vascular diseases, but the mechanism for its genesis is unknown. We have recently shown that the gas phase of cigarette smoke (nicotine- and tar-free cigarette smoke extract; CSE) likely to reach the systemic circulation contains stable substances which cause cytotoxicity like plasma membrane damage and cell death in cultured cells, and also that the plasma membrane damage is caused through sequential activation of protein kinase C (PKC) and NADPH oxidase (NOX) and the resulting generation of reactive oxygen species (PKC/NOX-dependent mechanism), whereas cell death is caused through PKC/NOX-dependent and -independent mechanisms. To identify these stable substances, the CSE was prepared by passing the main-stream smoke of 10 cigarettes through a Cambridge glass fiber filter, trapping of the smoke in a vessel cooled at -80°C, and subsequent dissolution in 10ml of water. The CSE was fractionated into nine fractions using reversed-phase HPLC, and each fraction was screened for cytotoxicity in cultured cells, using propidium iodide uptake assay for cell membrane damage and MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] reduction assay for cell viability. The cytotoxicity was positive in two of the nine fractions (Fr2 and Fr5). After extraction of the active fractions into dichloromethane, GC/MS analysis identified 2-cyclopenten-1-one (CPO) in Fr5 but none in Fr2. After derivatization of the active fractions with O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine hydrochloride, GC/MS analysis identified acrolein, acetone and propionaldehyde in Fr2, and methyl vinyl ketone (MVK) in Fr5. After 4-h incubation, authentic acrolein and MVK induced concentration-dependent cytotoxicity with EC50 values of 75.9±8.2 and 47.0±8.0μM (mean±SEM; n=3), respectively, whereas acetone, propionaldehyde and CPO were without effect. However, after 24-h incubation, CPO induced concentration-dependent cytotoxicity with an EC50 value of 264.0±16.9μM (n=3). The concentrations of acrolein, MVK and CPO in the CSE were 3368±334, 2429±123 and 392.9±31.8μM (n=4), respectively, which were higher than the cytotoxic concentrations. The cytotoxicity of acrolein and MVK consisted of plasma membrane damage and decreased cell viability: the plasma membrane damage was totally prevented by treatment with an inhibitor of PKC or NOX, whereas the decreased cell viability was only partially prevented by these inhibitors. The cytotoxicity of CPO consisted only of decreased cell viability, which was totally resistant to these inhibitors. These results show that acrolein and MVK are responsible for the acute cytotoxicity of the CSE through PKC/NOX-dependent and -independent mechanisms, whereas CPO is responsible for the delayed cytotoxicity of the CSE through a PKC/NOX-independent mechanism.

A Simple and Rapid Method for Standard Preparation of Gas Phase Extract of Cigarette Smoke

Cigarette smoke consists of tar and gas phase: the latter is toxicologically important because it can pass through lung alveolar epithelium to enter the circulation. Here we attempt to establish a standard method for preparation of gas phase extract of cigarette smoke (CSE). CSE was prepared by continuously sucking cigarette smoke through a Cambridge filter to remove tar, followed by bubbling it into phosphate-buffered saline (PBS). An increase in dry weight of the filter was defined as tar weight. Characteristically, concentrations of CSEs were represented as virtual tar concentrations, assuming that tar on the filter was dissolved in PBS. CSEs prepared from smaller numbers of cigarettes (original tar concentrations ≤15 mg/ml) showed similar concentration-response curves for cytotoxicity versus virtual tar concentrations, but with CSEs from larger numbers (tar ≥20 mg/ml), the curves were shifted rightward. Accordingly, the cytotoxic activity was detected in PBS of the second reservoir downstream of the first one with larger numbers of cigarettes. CSEs prepared from various cigarette brands showed comparable concentration-response curves for cytotoxicity. Two types of CSEs prepared by continuous and puff smoking protocols were similar regarding concentration-response curves for cytotoxicity, pharmacology of their cytotoxicity, and concentrations of cytotoxic compounds. These data show that concentrations of CSEs expressed by virtual tar concentrations can be a reference value to normalize their cytotoxicity, irrespective of numbers of combusted cigarettes, cigarette brands and smoking protocols, if original tar concentrations are ≤15 mg/ml.

Endothelin-1 activates extracellular signal-regulated kinases 1/2 via transactivation of platelet-derived growth factor receptor in rat L6 myoblasts

AIMS Endothelin (ET) system plays a critical role in the development of insulin resistance and type 2 diabetes. In skeletal muscle, differentiation of myoblasts to myotubes is accompanied by the development of insulin sensitivity. Activation of extracellular signal-regulated kinase (ERK) 1/2 inhibits the differentiation of myoblasts, leading to insulin resistance. Although ET receptor (ETR) stimulation generally activates ERK1/2, the mechanism for ETR-mediated ERK1/2 activation in skeletal muscle is unknown. The purpose of this study was to determine the signal transduction pathway involved in ET-1-stimulated ERK1/2 phosphorylation in L6 myoblasts derived from rat skeletal muscle. MAIN METHODS Changes in phosphorylation levels of ERK1/2 following stimulation with ET-1 were analyzed by Western blot in L6 myoblasts. To inhibit receptor internalization, dominant-negative dynamin (K44A) was overexpressed in L6 myoblasts using adenovirus-mediated gene transfer. KEY FINDINGS ET-1 induced phosphorylation of ERK1/2 in L6 myoblasts. The ERK1/2 phosphorylation was abolished by BQ123 (a selective ET type A receptor (ETAR) antagonist), YM-254890 (a Gαq/11 protein inhibitor), and AG370 (a platelet-derived growth factor receptor (PDGFR) kinase inhibitor), while U-73122 (a phospholipase C (PLC) inhibitor) was less potent. The ERK1/2 phosphorylation was inhibited by overexpression of dominant-negative dynamin (K44A). These results suggest that ETAR stimulation induces ERK1/2 phosphorylation in L6 myoblasts through Gq/11 protein-dependent, PLC-independent PDGFR transactivation which requires dynamin-dependent ETAR internalization. SIGNIFICANCE Because activation of ERK1/2 is considered to inhibit differentiation of myoblasts with the development of insulin sensitivity, the ETAR-mediated PDGFR transactivation and subsequent ERK1/2 activation play an important role in ET-1-induced insulin resistance.

Agonist-promoted ubiquitination differentially regulates receptor trafficking of endothelin type A and type B receptors.

Background: Agonist stimulation induces different intracellular trafficking of endothelin receptors (ETA/BR) after internalization. The mechanism is unclear. Results: Stimulation induces ubiquitination, lysosomal targeting, and decreased cell surface ETBR levels. Non-ubiquitinated ETAR and ETBR mutant recycled to plasma membrane with smaller changes in the levels. Conclusion: Ubiquitination determines intracellular trafficking of endothelin receptors. Significance: ETBR ubiquitination fine tunes cellular responses to agonist by regulating cellular receptor levels. Two types of G protein-coupled receptors for endothelin-1 (ET-1), ET type A receptor (ETAR) and ETBR, closely resemble each other, but upon ET-1 stimulation, they follow totally different intracellular trafficking pathways; ETAR is recycled back to plasma membrane, whereas ETBR is targeted to lysosome for degradation. However, the mechanisms for such different fates are unknown. Here we demonstrated that ETBR but not ETAR was ubiquitinated on the cell surface following ET-1 stimulation and that ETBR was internalized and degraded in lysosome more rapidly than ETAR. The mutant ETBR (designated “5KR mutant”) in which 5 lysine residues in the C-tail were substituted to arginine was not ubiquitinated, and its rates of internalization and degradation after ET-1 stimulation became slower, being comparable with those of ETAR. Confocal microscopic study showed that following ET-1 stimulation, ETAR and 5KR mutant of ETBR were co-localized mainly with Rab11, a marker of recycling endosome, whereas ETBR was co-localized with Rab7, a marker of late endosome/lysosome. In the 5KR mutant, ET-1-induced ERK phosphorylation and an increase in the intracellular Ca2+ concentration upon repetitive ET-1 stimulation were larger. A series of ETBR mutants (designated “4KR mutant”), in which either one of 5 arginine residues of the 5KR mutant was reverted to lysine, were normally ubiquitinated, internalized, and degraded, with ERK phosphorylation being normalized. These results demonstrate that agonist-induced ubiquitination at either lysine residue in the C-tail of ETBR but not ETAR switches intracellular trafficking from recycling to plasma membrane to targeting to lysosome, causing decreases in the cell surface level of ETBR and intracellular signaling.

Endothelin‐1 suppresses insulin‐stimulated Akt phosphorylation and glucose uptake via GPCR kinase 2 in skeletal muscle cells

Endothelin‐1 (ET‐1) reduces insulin‐stimulated glucose uptake in skeletal muscle, inducing insulin resistance. Here, we have determined the molecular mechanisms underlying negative regulation by ET‐1 of insulin signalling.

Endothelin‐1 suppresses insulin‐stimulated Akt phosphorylation and glucose uptake via G protein‐coupled receptor kinase 2 in skeletal muscle cells

Endothelin‐1 (ET‐1) reduces insulin‐stimulated glucose uptake in skeletal muscle, inducing insulin resistance. Here, we have determined the molecular mechanisms underlying negative regulation by ET‐1 of insulin signalling.

A Standardized Method for the Preparation of a Gas Phase Extract of Cigarette Smoke.

The gas phase of cigarette smoke is important from the viewpoint of human health, because it can pass through alveolar epithelium and enter the circulation. There is no standard method for the preparation of a gas phase extract of cigarette smoke (CSE), although CSE is widely used for research instead of whole cigarette smoke. We have established a standard method for the preparation of CSE. One cigarette per trial is continuously combusted under a reduced pressure generated by an aspiration pump with a velocity of 1.050 L/min: the main stream of the smoke is passed through a Cambridge filter to remove tar, and subsequently, bubbled through a glass ball filter (pore size, 20-30 µm) into 15 mL of phosphate-buffered saline (PBS). To express the concentration of CSE, a virtual tar concentration is introduced, which is calculated assuming that tar trapped on the Cambridge filter is dissolved in the PBS. CSEs prepared from smaller numbers of cigarettes (original virtual tar concentration≤15 mg/mL) show similar concentration-response curves for cytotoxicity versus virtual tar concentrations. CSEs prepared from various brands of cigarettes and by different smoking regimes (continuous and puff smoking) show similar cytotoxic potency if the virtual tar concentrations are the same. In conclusion, using the standardized method for CSE preparation in combination with the virtual tar concentration, it becomes possible to simply and rapidly prepare standard CSEs with defined concentrations from any brand of cigarettes, which are toxicologically equivalent to CSE prepared by puff smoking.

Bullous Pemphigoid Triggered by Thermal Burn Under Medication With a Dipeptidyl Peptidase-IV Inhibitor: A Case Report and Review of the Literature

Bullous pemphigoid (BP) is a common autoimmune blistering disease in which autoantibodies mainly target the hemidesmosomal component BP180 (also known as type XVII collagen) in basal keratinocytes. Various triggering factors are known to induce BP onset, including radiotherapy, burns, ultraviolet exposure, surgery, and the use of dipeptidyl peptidase-IV inhibitors (DPP4i), which are widely used antihyperglycemic drugs. Here, we present a case of BP triggered by a thermal burn under medication with DPP4i. A 60-year-old man with type II diabetes had been treated with the DPP4i linagliptin for 1 year. After the right forearm experienced a thermal burn, blisters developed around the burned area and gradually spread over the whole body with the production of autoantibodies targeting the non-NC16A domain of BP180. The diagnosis of BP was confirmed by immunohistopathological examination. Upon withdrawal of linagliptin and treatment with topical steroid and minocycline, complete remission was achieved after 4 months. Previously, 13 cases of BP that developed after thermal burns have been reported, and our case shared some of the clinical features of these thermal burn-induced BP cases. Interestingly, the present case also showed the typical clinical, histopathological, and immunological features of the non-inflammatory type of DPP4i-associated BP (DPP4i-BP). Although the pathogenesis of BP remains uncertain, the present case suggests that DPP4i may trigger the onset of BP similarly to a thermal burn. In addition, the clinical and histopathological features of DPP4i-BP may be distinct from other types of BP.