Negative Cross-Talk between TLR2/4-Independent AMPKα1 and TLR2/4-Dependent JNK Regulates S. pneumoniae-Induced Mucosal Innate Immune Response.

Streptococcus pneumoniae is major cause of otitis media (OM) and life-threatening pneumonia. Overproduction of mucin, the major component of mucus, plays a critical role in the pathogenesis of both OM and pneumonia. However, the molecular mechanisms underlying the tight regulation of mucin upregulation in the mucosal epithelium by S. pneumoniae infection remain largely unknown. In this study, we show that S. pneumoniae pneumolysin (PLY) activates AMP-activated protein kinase α1 (AMPKα1), the master regulator of energy homeostasis, which is required for S. pneumoniae-induced mucin MUC5AC upregulation in vitro and in vivo. Moreover, we found that PLY activates AMPKα1 via cholesterol-dependent membrane binding of PLY and subsequent activation of the Ca2+- Ca2+/calmodulin-dependent kinase kinase ß (CaMKKß) and Cdc42-mixed-lineage protein kinase 3 (MLK3) signaling axis in a TLR2/4-independent manner. AMPKα1 positively regulates PLY-induced MUC5AC expression via negative cross-talk with TLR2/4-dependent activation of MAPK JNK, the negative regulator of MUC5AC expression. Moreover, pharmacological inhibition of AMPKα1 suppressed MUC5AC induction in the S. pneumoniae-induced OM mouse model, thereby demonstrating its therapeutic potential in suppressing mucus overproduction in OM. Taken together, our data unveil a novel mechanism by which negative cross-talk between TLR2/4-independent activation of AMPKα1 and TLR2/4-dependent activation of JNK tightly regulates the S. pneumoniae PLY-induced host mucosal innate immune response.

Trimerization profile of type IV collagen COL4A5 exon deletion in X-linked Alport syndrome.

BACKGROUND: Alport syndrome (AS) is a genetic kidney disease caused by a mutation in type IV collagen α3, α4, and α5, which are normally secreted as heterotrimer α345(IV). Nonsense mutation in these genes causes severe AS phenotype. We previously revealed that the exon-skipping approach to remove a nonsense mutation in α5(IV) ameliorated the AS pathology. However, the effect of removing an exon on trimerization is unknown. Here, we assessed the impact of exon deletion on trimerization to evaluate their possible therapeutic applicability and to predict the severity of mutations associated with exon-skipping. METHODS: We produced exon deletion constructs (ΔExon), nonsense, and missense mutants by mutagenesis and evaluated their trimer formation and secretion activities using a nanoluciferase-based assay that we previously developed. RESULTS: Exon-skipping had differential effects on the trimer secretion of α345(IV). Some ΔExons could form and secrete α345(IV) trimers and had higher activity compared with nonsense mutants. Other ΔExons had low secretion activity, especially for those with exon deletion near the C-terminal end although the intracellular trimerization was normal. No difference was noted in the secretion of missense mutants and their ΔExon counterpart. CONCLUSION: Exon skipping is advantageous for nonsense mutants in AS with severe phenotypes and early onset of renal failure but applications may be limited to ΔExons capable of normal trimerization and secretion. This study provides information on α5(IV) exon-skipping for possible therapeutic application and the prediction of the trimer behavior associated with exon-skipping in Alport syndrome.

Combinatorial screening for therapeutics in ATTRv amyloidosis identifies naphthoquinone analogues as TTR-selective amyloid disruptors.

Hereditary ATTR amyloidosis is caused by the point mutation in serum protein transthyretin (TTR) that destabilizes its tetrameric structure to dissociate into monomer. The monomers form amyloid fibrils, which are deposited in peripheral nerves and organs, resulting in dysfunction. Therefore, a drug that dissolves amyloid after it has formed, termed amyloid disruptor, is needed as a new therapeutic drug. Here, we first established a high throughput screening system to find TTR interactors from the LOPAC1280 compound library. Among the hit compounds, thioflavin T-based post-treatment assay determined lead compounds for TTR amyloid disruptors, NSC95397 and Gossypol, designated as B and R, respectively. Because these compounds have naphthoquinone-naphthalene structures, we tested 100 naphthoquinone derivatives, and found 10 candidate compounds that disrupted TTR amyloid. Furthermore, to determine whether these 10 compounds are selective for TTR amyloid, we evaluated them against beta-amyloid (Aß1-42). We found two compounds that were selective for TTR and did not disrupt Aß-derived amyloid. Therefore, we succeeded in identifying TTR-selective amyloid disruptors, and demonstrated that naphthoquinone compounds are useful structures as amyloid disruptors. These findings contribute to the on-going efforts to discover new therapeutic tools for TTR amyloidosis.

Metformin suppresses epithelial sodium channel hyperactivation and its associated phenotypes in a mouse model of obstructive lung diseases.

Chronic obstructive pulmonary disease (COPD) is one of the leading causes of death in the world, and has no radical treatment. Inhibition of amiloride-sensitive epithelial sodium ion channel (ENaC) has now been considered as a potential therapeutic target against COPD. One possible modulator of ENaC is AMP-activated protein kinase (AMPK), a key molecule that controls a wide variety of cellular signals; however, little is known about whether metformin, a clinically available AMPK activator, has a protective role against ENaC-associated chronic pulmonary phenotypes, such as emphysema and pulmonary dysfunction. We first used ENaC-overexpressing human bronchial epithelial cells (ß/γENaC-16HBE14o-) and identified that Metformin significantly reduced ENaC activity. Consistently, in vivo treatment of ENaC-overexpressing COPD mouse model (C57BL/6-ßENaC-Tg mice) showed improvement of emphysema and pulmonary dysfunction, without any detrimental effect on non-pulmonary parameters (blood glucose level etc.). Bronchoalveolar lavage fluid (BALF) and lung tissue analyses revealed significant suppression in the infiltration of neutrophils as well as the expression of inflammatory markers (KC), neutrophil gelatinase (MMP9) and macrophage elastase (MMP12) in metformin-treated C57BL/6-ßENaC-Tg mice. Overall, the present study demonstrates that metformin directly inhibits ENaC activity in vitro and provides the first evidence of therapeutical benefit of Metformin for COPD with higher ENaC activity.

A Splice Switch in SIGIRR Causes a Defect of IL-37-Dependent Anti-Inflammatory Activity in Cystic Fibrosis Airway Epithelial Cells.

Cystic fibrosis (CF) is a hereditary disease typically characterized by infection-associated chronic lung inflammation. The persistent activation of toll-like receptor (TLR) signals is considered one of the mechanisms for the CF hyperinflammatory phenotype; however, how negative regulatory signals of TLRs associate with CF inflammation is still elusive. Here, we showed that the cell surface expression of a single immunoglobulin interleukin-1 receptor (IL-1R)-related molecule (SIGIRR), a membrane protein essential for suppressing TLRs- and IL-1R-dependent signals, was remarkably decreased in CF airway epithelial cells compared to non-CF cells. Notably, CF airway epithelial cells specifically and highly expressed a unique, alternative splice isoform of the SIGIRR that lacks exon 8 (Δ8-SIGIRR), which results in the production of a C-terminal truncated form of the SIGIRR. Δ8-SIGIRR was expressed intracellularly, and its over-expression abolished the cell surface expression and function of the full-length SIGIRR (WT-SIGIRR), indicating its dominant-negative effect leading to the deficiency of anti-inflammatory activity in CF cells. Consistently, IL-37, a ligand for the SIGIRR, failed to suppress viral dsRNA analogue poly(I:C)-dependent JNK activation and IL-8 production, confirming the reduction in the functional WT-SIGIRR expression in the CF cells. Together, our studies reveal that SIGIRR-dependent anti-inflammatory activity is defective in CF airway epithelial cells due to the unique splicing switch of the SIGIRR gene and provides the first evidence of IL-37-SIGIRR signaling as a target of CF airway inflammation.

Intrapopulation analysis of longitudinal lifespan in Caenorhabditis elegans identifies W09D10.4 as a novel AMPK-associated healthspan shortening factor.

Caenorhabditis elegans is a model organism widely used for longevity studies. Current advances have been made in the methods that allow automated monitoring of C. elegans behavior. However, ordinary manual assays as well as automated methods have yet to achieve qualitative whole-life analysis of C. elegans longevity based on intrapopulation variation. Here, we utilized live-cell analysis system to determine the parameters of nematode lifespans. Image-based superposition method enabled to determine not only frailty in worms, but also to measure individual and longitudinal lifespan, healthspan, and frailspan. Notably, k-means clustering via principal component analysis revealed four clusters with distinct longevity patterns in wild-type C. elegans. Physiological relevance of clustering was confirmed by assays with pharmacological and/or genetic manipulation of AMP-activated protein kinase (AMPK), a crucial regulator of healthspan. Finally, we focused on W09D10.4 among the possible regulators extracted by integrative expression analysis with existing data sets. Importantly, W09D10.4 knockdown increased the high-healthspan populations only in the presence of AMPK, suggesting that W09D10.4 is a novel AMPK-associated healthspan shortening factor in C. elegans. Overall, the study establishes a novel platform of longitudinal lifespan in C. elegans, which is user-friendly, and may be a useful pharmacological tool to identify healthspan modulatory factors.

The role of discoidin domain receptor 2 in the renal dysfunction of alport syndrome mouse model.

Alport syndrome (AS) is a hereditary glomerular nephritis caused by mutation in one of the type IV collagen genes α3/α4/α5 that encode the heterotrimer COL4A3/4/5. Failure to form a heterotrimer due to mutation leads to the dysfunction of the glomerular basement membrane, and end-stage renal disease. Previous reports have suggested the involvement of the receptor tyrosine kinase discoidin domain receptor (DDR) 1 in the progression of AS pathology. However, due to the similarity between DDR1 and DDR2, the role of DDR2 in AS pathology is unclear. Here, we investigated the involvement of DDR2 in AS using the X-linked AS mouse model. Mice were treated subcutaneously with saline or antisense oligonucleotide (ASO; 5 mg/kg or 15 mg/kg per week) for 8 weeks. Renal function parameters and renal histology were analyzed, and the gene expressions of inflammatory cytokines were determined in renal tissues. The expression level of DDR2 was highly elevated in kidney tissues of AS mice. Knockdown of Ddr2 using Ddr2-specific ASO decreased the Ddr2 expression. However, the DDR2 ASO treatment did not improve the proteinuria or decrease the BUN level. DDR2 ASO also did not significantly ameliorate the renal injury, inflammation and fibrosis in AS mice. These results showed that Ddr2 knockdown by ASO had no notable effect on the progression of AS indicating that DDR2 may not be critically involved in AS pathology. This finding may provide useful information and further understanding of the role of DDRs in AS.

A cell-based high-throughput screening method to directly examine transthyretin amyloid fibril formation at neutral pH.

Transthyretin (TTR) is a major amyloidogenic protein associated with hereditary (ATTRm) and nonhereditary (ATTRwt) intractable systemic transthyretin amyloidosis. The pathological mechanisms of ATTR-associated amyloid fibril formation are incompletely understood, and there is a need for identifying compounds that target ATTR. C-terminal TTR fragments are often present in amyloid-laden tissues of most patients with ATTR amyloidosis, and on the basis of in vitro studies, these fragments have been proposed to play important roles in amyloid formation. Here, we found that experimentally-formed aggregates of full-length TTR are cleaved into C-terminal fragments, which were also identified in patients' amyloid-laden tissues and in SH-SY5Y neuronal and U87MG glial cells. We observed that a 5-kDa C-terminal fragment of TTR, TTR81-127, is highly amyloidogenic in vitro, even at neutral pH. This fragment formed amyloid deposits and induced apoptosis and inflammatory gene expression also in cultured cells. Using the highly amyloidogenic TTR81-127 fragment, we developed a cell-based high-throughput screening method to discover compounds that disrupt TTR amyloid fibrils. Screening a library of 1280 off-patent drugs, we identified two candidate repositioning drugs, pyrvinium pamoate and apomorphine hydrochloride. Both drugs disrupted patient-derived TTR amyloid fibrils ex vivo, and pyrvinium pamoate also stabilized the tetrameric structure of TTR ex vivo in patient plasma. We conclude that our TTR81-127-based screening method is very useful for discovering therapeutic drugs that directly disrupt amyloid fibrils. We propose that repositioning pyrvinium pamoate and apomorphine hydrochloride as TTR amyloid-disrupting agents may enable evaluation of their clinical utility for managing ATTR amyloidosis.

Intratracheal GLP-1 receptor agonist treatment up-regulates mucin via p38 and exacerbates emphysematous phenotype in mucus hypersecretory obstructive lung diseases.

Glucagon-like peptide-1 (GLP-1) is a gastrointestinal hormone that stimulates glucose-mediated insulin production by pancreatic beta cells. It is also associated with protective effects in multiple tissues. GLP-1 receptor is highly expressed in pulmonary tissue, hinting possible pulmonary delivery of GLP-1 drugs. However, little is known about the role of GLP-1 signaling in the lung, especially in mucus hypersecretory obstructive lung diseases. Here, we showed that treatment with exendin-4, a clinically available GLP-1 receptor agonist, up-regulates mucin expression in normal airway epithelial cells and in the lung of normal mice, indicating mucus stimulatory effect of GLP-1 under physiological condition. Exendin-4 also increased mucin expression in in vitro cellular and in vivo murine models of obstructive lung diseases via the activation of p38 MAP kinase. Notably, mucin induction in vivo exacerbated key pulmonary abnormalities including emphysematous phenotypes, implying that GLP-1 signaling in the lung is detrimental under pulmonary obstructive condition. Another GLP-1 receptor agonist liraglutide had similar induction of mucin. Together, our studies not only demonstrate novel physiological and pathological roles of GLP-1 in the lung but may also caution against the clinical use of inhaled GLP-1 receptor agonists in the patients with obstructive lung diseases.

Azithromycin Inhibits Constitutive Airway Epithelial Sodium Channel Activation in Vitro and Modulates Downstream Pathogenesis in Vivo.

Epithelial sodium channel (ENaC) is an amiloride-sensitive sodium ion channel that is expressed in epithelial tissues. ENaC overexpression and/or hyperactivation in airway epithelial cells cause sodium over-absorption and dysregulated ciliary movement for mucus clearance; however, the agents that suppress constitutive airway ENaC activation are yet to be clinically available. Here, we focused on macrolides, which are widely used antibiotics that have many potential immunomodulatory effects. We examined whether macrolides could modulate constitutive ENaC activity and downstream events that typify cystic fibrosis (CF) and chronic obstructive pulmonary diseases (COPD) in in vitro and in vivo models of ENaC overexpression. Treatment of ENaC-overexpressing human bronchial epithelial cells (ß/γENaC-16HBE14o- cells) with three macrolides (erythromycin, clarithromycin, azithromycin) confirmed dose-dependent suppression of ENaC function. For in vivo studies, mice harboring airway specific ßENaC overexpression (C57BL/6J-ßENaC-transgenic mice) were treated orally with azithromycin, a well-established antimicrobial agent that has been widely prescribed. Azithromycin treatment modulated pulmonary mechanics, emphysematous phenotype and pulmonary dysfunction. Notably, a lower dose (3 mg kg-1) of azithromycin significantly increased forced expiratory volume in 0.1 s (FEV0.1), an inverse indicator of bronchoconstriction. Although not statistically significant, improvement of pulmonary obstructive parameters such as emphysema and lung dysfunction (FEV0.1%) was observed. Our results demonstrate that macrolides directly attenuate constitutive ENaC function in vitro and may be promising for the treatment of obstructive lung diseases with defective mucociliary clearance, possibly by targeting ENaC hyperactivation.

STT3B-dependent posttranslational N-glycosylation as a surveillance system for secretory protein.

Nascent secretory proteins are extensively scrutinized at the endoplasmic reticulum (ER). Various signatures of client proteins, including exposure of hydrophobic patches or unpaired sulfhydryls, are coordinately utilized to reduce nonnative proteins in the ER. We report here the cryptic N-glycosylation site as a recognition signal for unfolding of a natively nonglycosylated protein, transthyretin (TTR), involved in familial amyloidosis. Folding and ER-associated degradation (ERAD) perturbation analyses revealed that prolonged TTR unfolding induces externalization of cryptic N-glycosylation site and triggers STT3B-dependent posttranslational N-glycosylation. Inhibition of posttranslational N-glycosylation increases detergent-insoluble TTR aggregates and decreases cell proliferation of mutant TTR-expressing cells. Moreover, this modification provides an alternative pathway for degradation, which is EDEM3-mediated N-glycan-dependent ERAD, distinct from the major pathway of Herp-mediated N-glycan-independent ERAD. Hence we postulate that STT3B-dependent posttranslational N-glycosylation is part of a triage-salvage system recognizing cryptic N-glycosylation sites of secretory proteins to preserve protein homeostasis.

Impact of hepatic HSP72 on insulin signaling.

Heat shock protein 72 (HSP72) is a major inducible molecule in the heat shock response that enhances intracellular stress tolerance. Decreased expression of HSP72 is observed in type 2 diabetes, which may contribute to the development of insulin resistance and chronic inflammation. We used HSP72 knockout (HSP72-KO) mice to investigate the impact of HSP72 on glucose metabolism and endoplasmic reticulum (ER) stress, particularly in the liver. Under a high-fat diet (HFD) condition, HSP72-KO mice showed glucose intolerance, insulin resistance, impaired insulin secretion, and enhanced hepatic gluconeogenic activity. Furthermore, activity of the c-Jun NH2-terminal kinase (JNK) was increased and insulin signaling suppressed in the liver. Liver-specific expression of HSP72 by lentivirus (lenti) in HFD-fed HSP72-KO mice ameliorated insulin resistance and hepatic gluconeogenic activity. Furthermore, increased adipocyte size and hepatic steatosis induced by the HFD were suppressed in HSP72-KO lenti-HSP72 mice. Increased JNK activity and ER stress upon HFD were suppressed in the liver as well as the white adipose tissue of HSP72-KO lenti-HSP72 mice. Thus, HSP72 KO caused a deterioration in glucose metabolism, hepatic gluconeogenic activity, and ß-cell function. Moreover, liver-specific recovery of HSP72 restored glucose homeostasis. Therefore, hepatic HSP72 may play a critical role in the pathogenesis of type 2 diabetes.

Integrative expression analysis identifies a novel interplay between CFTR and linc-SUMF1-2 that involves CF-associated gene dysregulation.

Cystic fibrosis transmembrane regulator (CFTR) is a cyclic AMP-dependent Cl- channel, and its dysfunction, due to CFTR gene mutations, causes the lethal inherited disorder cystic fibrosis (CF). To date, widespread dysregulation of certain coding genes in CF airway epithelial cells is well studied and considered as the driver of pulmonary abnormality. However, the involvement of non-coding genes, novel classes of functional RNAs with little or no protein-coding capacity, in the regulation of CF-associated gene dysregulation is poorly understood. Here, we utilized integrative analyses of human transcriptome array (HTA) and characterized 99 coding and 91 non-coding RNAs that are dysregulated in CFTR-defective CF bronchial epithelial cell line CFBE41o-. Among these genes, the expression level of linc-SUMF1-2, an intergenic non-coding RNA (lincRNA) whose function is unknown, was inversely correlated with that of WT-CFTR and consistently higher in primary human CF airway epithelial cells (DHBE-CF). Further integrative analyses under linc-SUMF1-knockdown condition determined MXRA5, SEMA5A, CXCL10, AK022877, CTGF, MYC, AREG and LAMB3 as both CFTR- and linc-SUMF1-2-dependent dysregulated gene sets in CF airway epithelial cells. Overall, our analyses reveal linc-SUMF1-2 as a dysregulated non-coding gene in CF as well as CFTR-linc-SUMF1-2 axis as a novel regulatory pathway involved in CF-associated gene dysregulation.

Auto-measure emphysematous parameters and pathophysiological gene expression profiles in experimental mouse models of acute and chronic obstructive pulmonary diseases.

Pulmonary emphysema, inflammation and senescence-like phenotype are pathophysiological characteristics of chronic obstructive pulmonary disease (COPD). Recently, a murine model of COPD has been established by inducing airway-specific overexpression of epithelial Na+ channel ß subunit (ßENaC-Tg mice). However, little is known about the histological and biochemical differences between ßENaC-Tg mice and an existing acute emphysematous mouse model (elastase-induced model). Here, we first utilized whole lung image-based quantification method for histological analysis to determine auto-measure parameters, including alveolar area, alveolar perimeter, (major axis + minor axis)/2 and Feret diameter. Even though the extent of emphysema was similar in both models, the coefficient of variation (CV) of all histological parameters was smaller in ßENaC-Tg mice, indicating that ßENaC-Tg mice show homogeneous emphysema as compared with elastase-induced acute model. Expression analysis of lung tissue RNAs further revealed that elastase-induced model exhibits transient changes of inflammation markers (Kc, Il-6, Lcn2) and senescence-related markers (Sirt1, p21) at emphysema-initiation stage (1 day), which does not last until emphysema-manifestation stage (3 weeks); while the up-regulation is stable at emphysema-manifestation stage in ßENaC-Tg mice (14-week old). Thus, these studies demonstrate that ßENaC-Tg mice exhibit diffuse-type emphysema with stable expression of inflammatory and senescence-like markers.

Curcumin Down-Regulates Toll-Like Receptor-2 Gene Expression and Function in Human Cystic Fibrosis Bronchial Epithelial Cells.

Cystic fibrosis (CF), the most common lethal inherited disorder caused by mutation in the gene encoding the CF transmembrane regulator (CFTR), is characterized by chronic inflammation that ultimately leads to death from respiratory failure. In CF patients, up-regulation of toll-like receptor-2 (TLR2), a pattern recognition receptor that senses CF-pathogenic bacteria Staphylococcus aureus peptidoglycan (PGN), in airway epithelial cells is observed, and enhanced proinflammatory responses towards PGN may result in detrimental effects in CF patients. Here, we showed that curcumin, a well known anti-inflammatory agent derived from the curry spice turmeric, inhibits TLR2 expression in CF bronchial epithelial cell line, CFBE41o- cells. Strong suppression of TLR2 gene and protein expression was observed at more than 40 µM of curcumin treatment in CFBE41o- cells. Consistent with decreased expression of TLR2, PGN-dependent interleukin-8 (IL-8) gene up-regulation was markedly reduced by 40 µM of curcumin treatment. Strong reductions of TLR2 gene expression and function were also observed in primary human CF bronchial epithelial cells, but not in human non-CF primary cells. Interestingly, curcumin treatment decreased nuclear expression of transcription factor specificity protein 1 (SP1), a factor that is critical for increased basal TLR2 expression in CF cell line and primary cells. Finally, curcumin-dependent SP1 reduction was diminished by anti-oxidant N-acetylcystein (NAC) and proteasomal inhibitor MG-132, suggesting the crucial roles of oxidative and proteasomal degradation pathways. Taken together, our study shows that curcumin down-regulates TLR2 gene expression and function in CF bronchial epithelial cells possibly by accelerating SP1 degradation via an oxidative process.

Transthyretin Amyloid Fibril Disrupting Activities of Extracts and Fractions from Juglans mandshurica Maxim. var. cordiformis (Makino) Kitam.

Transthyretin-related amyloidosis is a slowly progressive disorder caused by deposition of insoluble amyloid plaques formed by fibrillization of mutant or defective transthyretin (TTR) monomers that leads to neurodegeneration and organ failure. Thus, any compound exhibiting TTR amyloid formation inhibitory activity or TTR amyloid fibril disrupting activity might be a potential candidate for the development of therapies for these disorders. Our aim in this study was the evaluation of the TTR amyloid fibril disrupting potential of extracts of leaves and immature fruits of two Juglans plants, i.e., Juglans mandshurica var. sachalinensis and Juglans mandshurica var. cordiformis. The TTR amyloid fibril disrupting activity was measured by Thioflavin-T (ThT) assay and PROTEOSTAT® Protein aggregation assay methods. A fifty percent acetone extract of the fruits of Juglans mandshurica var. cordiformis showed strong amyloid fibril disrupting activity, and was further fractionated using different solvents. Ethyl acetate and n-butanol fractions showed significant activity in both assays. Syringic acid was isolated and identified as main compound in both of these fractions; however, it did not show any activity. Furthermore, some of the previously reported compounds from Juglans plants including naphthoquinone derivatives and phenolic compounds were evaluated to identify the potential bioactive compounds. Among them, juglone, a naphthoquinone derivative showed promising activity. However, juglone also showed strong cytotoxicity in HEK293 cells. Thus, future studies should focus on the isolation and identification of naphthoquinone derivatives or other compounds from Juglans plan ts with potent bioactivity and low cytotoxicity.

Mild electrical stimulation with heat shock reduces inflammatory symptoms in the imiquimod-induced psoriasis mouse model.

Psoriasis is a chronic skin disease caused by immune disorder. The chronic skin inflammation involves inflammatory molecules that are released from T lymphocytes and keratinocytes. Therefore, developing an anti-inflammatory therapy that is suitable for long-term treatment is needed. Electrical stimulation induces biological responses by modulating intracellular signaling pathways. Our previous studies showed that the optimized combination treatment of mild electrical stimulation (MES, 0.1-millisecond; ms, 55-pulses per second; pps) and heat shock (HS, 42°C) modulates inflammatory symptoms of metabolic disorders and chronic kidney disease in mice models and clinical trials. Here, we investigated the effect of MES+HS treatment on imiquimod-induced psoriasis mouse model. Topical application of imiquimod cream (15 mg) to mice ear induced keratinocyte hyperproliferation and psoriasis-like inflammation. In MES+HS-treated mice, imiquimod-induced skin hyperplasia was significantly decreased. MES+HS treatment reduced the protein expression of IL-17A and the infiltration of CD3-positive cells in lesioned skin. In addition, MES+HS-treated mice had decreased mRNA expression level of antimicrobial molecules (S100A8 and Reg3γ) which aggravate psoriasis. In IL-17A-stimulated HaCaT cells, MES+HS treatment significantly lowered the mRNA expression of aggravation markers (S100A8, S100A9 and ß-defensin2). Taken together, our study suggested that MES+HS treatment improves the pathology of psoriasis via decreasing the expression of inflammatory molecules.

STAT3 inhibition attenuates the progressive phenotypes of Alport syndrome mouse model.

Background: Alport syndrome (AS) is a hereditary, progressive nephritis caused by mutation of type IV collagen. Previous studies have shown that activation of signal transducer and activator of transcription 3 (STAT3) exacerbates other renal diseases, but whether STAT3 activation exacerbates AS pathology is still unknown. Here we aim to investigate the involvement of STAT3 in the progression of AS. Method: Phosphorylated STAT3 expression was assessed by immunoblotting analysis of kidneys and glomeruli of an AS mouse model (Col4a5 G5X mutant). To determine the effect of blocking STAT3 signaling, we treated AS mice with the STAT3 inhibitor stattic (10 mg/kg i.p., three times per week for 10 weeks; n = 10). We assessed the renal function [proteinuria, blood urea nitrogen (BUN), serum creatinine] and analyzed the glomerular injury score, fibrosis and inflammatory cell invasion by histological staining. Moreover, we analyzed the gene expression of nephritis-associated molecules. Results: Phosphorylated STAT3 was upregulated in AS kidneys and glomeruli. Treatment with stattic ameliorated the progressive renal dysfunction, such as increased levels of proteinuria, BUN and serum creatinine. Stattic also significantly suppressed the gene expression levels of renal injury markers (Lcn2, Kim-1), pro-inflammatory cytokines (Il-6, KC), pro-fibrotic genes (Tgf-ß, Col1a1, α-Sma) and Mmp9. Stattic treatment decreased the renal fibrosis congruently with the decrease of transforming growth factor beta (TGF-ß) protein and increase of antifibrosis-associated markers p-Smad1, 5 and 8, which are negative regulators of TGF-ß signaling. Conclusion: STAT3 inhibition significantly ameliorated the renal dysfunction in AS mice. Our finding identifies STAT3 as an important regulator in AS progression and provides a promising therapeutic target for AS.

Taurine Inhibits TRPV-Dependent Activity to Overcome Oxidative Stress in Caenorhabditis elegans.

Taurine has important physiological roles as well as a wide range of pharmacological effects. Studies have suggested that taurine ameliorates diabetes, hypertension, oxidative stress, and inflammatory diseases. However, its mechanisms of action are still unclear. It has been reported that N-acyl taurine activates transient receptor potential vanilloid-1 (TRPV1), which is related to the pathogenesis of many inflammatory diseases. In this study, we hypothesized that taurine has a regulatory effect on TRPV1 activation via N-acyl taurine. To evaluate this hypothesis, we assessed the calcium influx activated by a TRPV1 agonist in human keratinocyte (HaCaT) cells and paraquat-induced oxidative stress in Caenorhabditis elegans. Our results indicate that taurine inhibits TRPV-dependent activity to overcome oxidative stress in cultured cell lines and in C. elegans.

Characterization of Non-amyloidogenic G101S Transthyretin.

Transthyretin (TTR) is a tetrameric beta-sheet-rich protein that is important in the plasma transport of thyroxine and retinol. Mutations in the TTR gene cause TTR tetramer protein to dissociate to monomer, which is the rate-limiting step in familial amyloid polyneuropathy. Amyloidogenicity of individual TTR variants depends on the types of mutation that induce significant changes in biophysical, biochemical and/or biological properties. G101S TTR variant was previously identified in a Japanese male without amyloidotic symptom, and was considered as a non-amyloidogenic TTR variant. However, little is known about G101S TTR. Here, we found slight but possibly important biophysical differences between wild-type (WT) and G101S TTR. G101S TTR had slower rate of tetramer dissociation and lower propensity for amyloid fibril formation, especially at mild low pH (4.2 and 4.5), and was likely to have strong hydrophobic interaction among TTR monomers, suggesting relatively higher stability of G101S TTR compared with WT TTR. Cycloheximide (CHX)-based assay in HEK293 cells revealed that intracellular G101S TTR expression level was lower, but extracellular expression was higher than WT TTR, implying enhanced secretion efficiency of G101S TTR protein compared with WT TTR. Moreover, we found that STT3B-dependent posttranslational N-glycosylation at N98 residue occurred in G101S TTR but not in other TTR variants, possibly due to amino acid alterations that increase N-glycosylation preference or accelerate rigid structure formation susceptible to N-glycosylation. Taken together, our study characterizes G101S TTR as a stable and N-glycosylable TTR, which may be linked to its non-amyloidogenic characteristic.