Tsunehito Higashi

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.

Carbonyl Compounds in the Gas Phase of Cigarette Mainstream Smoke and Their Pharmacological Properties

Cigarette mainstream smoke is composed of gas and tar phases and contains >4000 chemical constituents, including nicotine and tar. The substances in the gas phase but not in the tar phase can pass through the airway epithelial barrier, enter the systemic circulation via the pulmonary circulation, and increase systemic oxidative damage, leading to the development of cigarette smoking-related diseases such as atherosclerosis. Recently, we identified some stable carbonyl compounds, including acrolein (ACR) and methyl vinyl ketone (MVK), as major cytotoxic factors in nicotine- and tar-free cigarette smoke extract (CSE) of the gas phase. CSE, ACR, and MVK induce protein kinase C (PKC)-dependent activation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) and subsequent generation of reactive oxygen species (ROS) via NOX, causing plasma membrane damage and cell apoptosis. CSE, ACR, and MVK also trigger carbonylation of PKC, which is an irreversible oxidative modification. Cell damage and PKC carbonylation in response to treatment with CSE, ACR, or MVK are abolished by thiol-containing antioxidants such as N-acetyl-L-cysteine and reduced glutathione. Thus pharmacological modulation of PKC and NOX activities and the trapping of ROS are potential strategies for the prevention of diseases related to cigarette smoking.

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.

Decreased proteasomal function accelerates cigarette smoke-induced pulmonary emphysema in mice

Chronic obstructive pulmonary disease (COPD) is a disease common in elderly people, characterized by progressive destruction of lung parenchyma and chronic inflammation of the airways. The pathogenesis of COPD remains unclear, but recent studies suggest that oxidative stress-induced apoptosis in alveolar cells contributes to emphysematous lung destruction. The proteasome is a multicatalytic enzyme complex that plays a critical role in proteostasis by rapidly destroying misfolded and modified proteins generated by oxidative and other stresses. Proteasome activity decreases with aging in many organs including lungs, and an age-related decline in proteasomal function has been implicated in various age-related pathologies. However, the role of the proteasome system in the pathogenesis of COPD has not been investigated. Recently, we have established a transgenic (Tg) mouse model with decreased proteasomal chymotrypsin-like activity, showing age-related phenotypes. Using this model, we demonstrate here that decreased proteasomal function accelerates cigarette smoke (CS)-induced pulmonary emphysema. CS-exposed Tg mice showed remarkable airspace enlargement and increased foci of inflammation compared with wild-type controls. Importantly, apoptotic cells were found in the alveolar walls of the affected lungs. Impaired proteasomal activity also enhanced apoptosis in cigarette smoke extract (CSE)-exposed fibroblastic cells derived from mice and humans in vitro. Notably, aggresome formation and prominent nuclear translocation of apoptosis-inducing factor were observed in CSE-exposed fibroblastic cells isolated from Tg mice. Collective evidence suggests that CS exposure and impaired proteasomal activity coordinately enhance apoptotic cell death in the alveolar walls that may be involved in the development and progression of emphysema in susceptible individuals such as the elderly.

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.

Using Phos-Tag in Western Blotting Analysis to Evaluate Protein Phosphorylation

Protein phosphorylation has traditionally been detected by radioisotope phosphate labeling of proteins with radioactive ATP. Several nonradioactive assays with phosphorylation site-specific antibodies are now available for the analysis of phosphorylation status at target sites. However, due to their high specificity, these antibodies they cannot be used to detect unidentified phosphorylation sites. Recently, Phos-tag technology has been developed to overcome the disadvantages and limitations of phosphospecific antibodies. Phos-tag and its derivatives conjugated to biotin, acrylamide, or agarose, form alkoxide-bridged dinuclear metal complexes, which can capture phosphate monoester dianions bound to serine, threonine, and tyrosine residues, in an amino acid sequence-independent manner. Here, we describe our method, which is based on in vitro kinase assay and Western blotting analysis using biotinylated Phos-tag and horseradish peroxidase-conjugated streptavidin, to determine the sites of TRPC6 (transient receptor potential canonical 6) channel phosphorylated by protein kinase A.