Sweat Protects against Contact Hypersensitivity: Transient Sweat Suppression Compromises Skin Barrier Function in Mice.

Although subtle barrier defects may facilitate allergen penetration, thereby enabling allergic sensitization, the relationship between sweating disturbance and skin barrier function is unknown. However, many studies on contact hypersensitivity in mice examined ear skin, which does not sweat, instead of the footpad, where sweating is uniquely present. In this study, we assessed whether sweat suppression in the footpad before hapten application provoked a skin barrier abnormality and reduced inflammatory thresholds to topical haptens. Mice without any genetic skin barrier dysfunction displayed markedly reduced inflammatory thresholds to haptens under transient sweat suppression before hapten application. Epicutaneously applied haptens penetrated the skin more robustly in the presence of sweat suppression compared with that in its absence, although this increase was abolished by exposure to high-humidity conditions. These mice displayed a subtle atopic dermatitis-like inflammation mediated by type 2 response-dominant inflammation and increased IgE responses, mimicking some events occurring in nonlesional atopic dermatitis skin in humans and in murine models. These lesions were dramatically attenuated by exposure to high-humidity conditions. In our model, hapten sensitization does not require mechanical injury, explaining why sensitization occurs through nonlesional atopic dermatitis skin. Awareness of the importance of preserving sweating responses is essential to prevent occupational contact dermatitis and atopic dermatitis.

GDE7 produces cyclic phosphatidic acid in the ER lumen functioning as a lysophospholipid mediator.

Cyclic phosphatidic acid (cPA) is a lipid mediator, which regulates adipogenic differentiation and glucose homeostasis by suppressing nuclear peroxisome proliferator-activated receptor γ (PPARγ). Glycerophosphodiesterase 7 (GDE7) is a Ca2+-dependent lysophospholipase D that localizes in the endoplasmic reticulum. Although mouse GDE7 catalyzes cPA production in a cell-free system, it is unknown whether GDE7 generates cPA in living cells. Here, we demonstrate that human GDE7 possesses cPA-producing activity in living cells as well as in a cell-free system. Furthermore, the active site of human GDE7 is directed towards the luminal side of the endoplasmic reticulum. Mutagenesis revealed that amino acid residues F227 and Y238 are important for catalytic activity. GDE7 suppresses the PPARγ pathway in human mammary MCF-7 and mouse preadipocyte 3T3-L1 cells, suggesting that cPA functions as an intracellular lipid mediator. These findings lead to a better understanding of the biological role of GDE7 and its product, cPA.

Coculture in vitro with endothelial cells induces cytarabine resistance of acute myeloid leukemia cells in a VEGF-A/VEGFR-2 signaling-independent manner.

An interaction between acute myeloid leukemia (AML) cells and endothelial cells in the bone marrow seems to play a critical role in chemosensitivity on leukemia treatment. The endothelial niche reportedly enhances the paracrine action of the soluble secretory proteins responsible for chemoresistance in a vascular endothelial growth factor A (VEGF-A)/VEGF receptor 2 (VEGFR-2) signaling pathway-dependent manner. To further investigate the contribution of VEGF-A/VEGFR-2 signaling to the chemoresistance of AML cells, a biochemical assay system in which the AML cells were cocultured with human endothelial EA.hy926 cells in a monolayer was developed. By coculture with EA.hy926 cells, this study revealed that the AML cells resisted apoptosis induced by the anticancer drug cytarabine. SU4312, a VEGFR-2 inhibitor, attenuated VEGFR-2 phosphorylation and VEGF-A/VEGFR-2 signaling-dependent endothelial cell migration; thus, this inhibitor was observed to block VEGF-A/VEGFR-2 signaling. Interestingly, this inhibitor did not reverse the chemoresistance. When VEGFR-2 was knocked out in EA.hy926 cells using the CRISPR-Cas9 system, the cytarabine-induced apoptosis of AML cells did not significantly change compared with that of wild-type cells. Thus, coculture-induced chemoresistance appears to be independent of VEGF-A/VEGFR-2 signaling. When the transwell, a coculturing device, separated the AML cells from the EA.hy926 cells in a monolayer, the coculture-induced chemoresistance was inhibited. Given that the migration of VEGF-A/VEGFR-2 signaling-dependent endothelial cells is necessary for the endothelial niche formation in the bone marrow, VEGF-A/VEGFR-2 signaling contributes to chemoresistance by mediating the niche formation process, but not to the chemoresistance of AML cells in the niche.

Development of a selective fluorescence-based enzyme assay for glycerophosphodiesterase family members GDE4 and GDE7.

Lysophosphatidic acid (LPA) is a lipid mediator that regulates various processes, including cell migration and cancer progression. Autotaxin (ATX) is a lysophospholipase D-type exoenzyme that produces extracellular LPA. In contrast, glycerophosphodiesterase (GDE) family members GDE4 and GDE7 are intracellular lysophospholipases D that form LPA, depending on Mg2+ and Ca2+, respectively. Since no fluorescent substrate for these GDEs has been reported, in the present study, we examined whether a fluorescent ATX substrate, FS-3, could be applied to study GDE activity. We found that the membrane fractions of human GDE4- and GDE7-overexpressing human embryonic kidney 293T cells hydrolyzed FS-3 in a manner almost exclusively dependent on Mg2+ and Ca2+, respectively. Using these assay systems, we found that several ATX inhibitors, including α-bromomethylene phosphonate analog of LPA and 3-carbacyclic phosphatidic acid, also potently inhibited GDE4 and GDE7 activities. In contrast, the ATX inhibitor S32826 hardly inhibited these activities. Furthermore, FS-3 was hydrolyzed in a Mg2+-dependent manner by the membrane fraction of human prostate cancer LNCaP cells that express GDE4 endogenously but not by those of GDE4-deficient LNCaP cells. Similar Ca2+-dependent GDE7 activity was observed in human breast cancer MCF-7 cells but not in GDE7-deficient MCF-7 cells. Finally, our assay system could selectively measure GDE4 and GDE7 activities in a mixture of the membrane fractions of GDE4- and GDE7-overexpressing human embryonic kidney 293T cells in the presence of S32826. These findings allow high-throughput assays of GDE4 and GDE7 activities, which could lead to the development of selective inhibitors and stimulators as well as a better understanding of the biological roles of these enzymes.

Sphingosine 1-phosphate receptor type 2 positively regulates interleukin (IL)-4/IL-13-induced STAT6 phosphorylation.

Previous reports have demonstrated that sphingosine 1-phosphate receptor type 2 (S1P2) is involved in the activation of signal transducer and activator of transcription (STAT) 6. Additionally, the major signaling pathway of S1P2 is the Rho-Rho kinase pathway. In this study, we examined the role of S1P2 in STAT6 activation in a macrophage (Mφ) model using THP-1 cells differentiated with phorbol 12-myristate 13-acetate (PMA). We established S1P2knockout THP-1 cells using the CRISPR-Cas9 gene editing system. The PMA-treated S1P2knockout THP-1 Mφs showed decreases in IL-4/IL-13-induced phosphorylation of Janus-activated kinase (JAK) 1, JAK2, and STAT6 as well as mRNA expression of the M2 marker ARG1 compared with wild-type THP-1 Mφs. Pretreatment of PMA-treated THP-1 Mφs with the S1P2 antagonist JTE-013, the Rho inhibitor Rhosin or the Rho kinase inhibitor Y27632 inhibited the IL-4/IL-13-induced increase in STAT6 phosphorylation. The expressions of suppressor of cytokine signaling 3 in the S1P2knockout THP-1 Mφs were higher than those in wild-type THP-1 Mφs. In addition, the protein tyrosine phosphatase inhibitor vanadate enhanced IL-4-induced STAT6 phosphorylation in the S1P2knockout THP-1 Mφs, suggesting that S1P2-Rho-Rho kinase inhibited the negative regulation of STAT6. These results suggest that the S1P2-Rho-Rho kinase pathway is necessary for full activation of STAT6 by IL-4/IL-13 in Mφs.

ATF4-mediated transcriptional regulation protects against ß-cell loss during endoplasmic reticulum stress in a mouse model.

OBJECTIVE: Activating transcription factor 4 (ATF4) is a transcriptional regulator of the unfolded protein response and integrated stress response (ISR) that promote the restoration of normal endoplasmic reticulum (ER) function. Previous reports demonstrated that dysregulation of the ISR led to development of severe diabetes. However, the contribution of ATF4 to pancreatic ß-cells remains poorly understood. In this study, we aimed to analyze the effect of ISR enhancer Sephin1 and ATF4-deficient ß-cells to clarify the role of ATF4 in ß-cells under ER stress conditions. METHODS: To examine the role of ATF4 in vivo, ISR enhancer Sephin1 (5 mg/kg body weight, p.o.) was administered daily for 21 days to Akita mice. We also established ß-cell-specific Atf4 knockout (ßAtf4-KO) mice that were further crossed with Akita mice. These mice were analyzed for characteristics of diabetes, ß-cell function, and morphology of the islets. To identify the downstream factors of ATF4 in ß-cells, the islets of ßAtf4-KO mice were subjected to cDNA microarray analyses. To examine the transcriptional regulation by ATF4, we also performed in situ PCR analysis of pancreatic sections from mice and ChIP-qPCR analysis of CT215 ß-cells. RESULTS: Administration of the ISR enhancer Sephin1 improved glucose metabolism in Akita mice. Sephin1 also increased the insulin-immunopositive area and ATF4 expression in the pancreatic islets. Akita/ßAtf4-KO mice exhibited dramatically exacerbated diabetes, shown by hyperglycemia at an early age, as well as a remarkably short lifespan owing to diabetic ketoacidosis. Moreover, the islets of Akita/ßAtf4-KO mice presented increased numbers of cells stained for glucagon, somatostatin, and pancreatic polypeptide and increased expression of aldehyde dehydrogenase 1 family member 3, a marker of dedifferentiation. Using microarray analysis, we identified atonal BHLH transcription factor 8 (ATOH8) as a downstream factor of ATF4. Deletion of ATF4 in ß-cells showed reduced Atoh8 expression and increased expression of undifferentiated markers, Nanog and Pou5f1. Atoh8 expression was also abolished in the islets of Akita/ßAtf4-KO mice. CONCLUSIONS: We conclude that transcriptional regulation by ATF4 maintains ß-cell identity via ISR modulation. This mechanism provides a promising target for the treatment of diabetes.

Involvement of acid ceramidase in the degradation of bioactive N-acylethanolamines.

Bioactive N-acylethanolamines (NAEs) include palmitoylethanolamide, oleoylethanolamide, and anandamide, which exert anti-inflammatory, anorexic, and cannabimimetic actions, respectively. The degradation of NAEs has been attributed to two hydrolases, fatty acid amide hydrolase and NAE acid amidase (NAAA). Acid ceramidase (AC) is a lysosomal enzyme that hydrolyzes ceramide (N-acylsphingosine), which resembles NAAA in structure and function. In the present study, we examined the role of AC in the degradation of NAEs. First, we demonstrated that purified recombinant human AC can hydrolyze various NAEs with lauroylethanolamide (C12:0-NAE) as the most reactive NAE substrate. We then used HEK293 cells metabolically labeled with [14C]ethanolamine, and revealed that overexpressed AC lowered the levels of 14C-labeled NAE. As analyzed with liquid chromatography-tandem mass spectrometry, AC overexpression decreased the amounts of different NAE species. Furthermore, suppression of endogenous AC in LNCaP prostate cells by siRNA increased the levels of various NAEs. Lastly, tissue homogenates from mice genetically lacking saposin D, a presumable activator protein of AC, showed much lower hydrolyzing activity for NAE as well as ceramide than the homogenates from wild-type mice. These results demonstrate the ability of AC to hydrolyze NAEs and suggest its physiological role as a third NAE hydrolase.

Growth differentiation factor 15 facilitates lung fibrosis by activating macrophages and fibroblasts.

Lung fibrosis is a devastating disease characterized by fibroblast accumulation and extracellular matrix deposition in lungs. However, its molecular and cellular pathogenesis is not fully understood and the current therapeutic strategies are ineffective. Bleomycin-induced lung fibrosis is the most widely used experimental model for research aimed at in-depth analysis of lung fibrosis mechanisms. The present study aimed to analyse the effects of growth differentiation factor 15 (GDF15), which is associated with many diseases, in lung fibrosis. GDF15 mRNA expression was elevated in the lungs of bleomycin-treated mice, revealed by comprehensive gene analysis. Its protein levels were also increased in the lungs, bronchoalveolar lavage fluid, and plasma obtained from bleomycin-treated mice as compared to those in saline-treated mice. Bleomycin administration in mice resulted in a marked increase in senescence-associated ß-galactosidase-positive and p16INK4a-positive lung structural cells including alveolar epithelial cells and macrophages. Immunohistochemical staining using anti-GDF15 antibody and increased mRNA expression of GDF15 in bleomycin-induced senescent A549 cells indicated that GDF15 is produced from alveolar epithelial cells undergoing bleomycin-induced cellular senescence. GDF15 was also implicated in the augmentation of interleukin-4/interleukin-13-induced mRNA expression of M2 markers including arginase 1 and chitinase-3-like protein and was also responsible for increased α-smooth muscle actin expression through the ALK5-Smad2/3 pathway in WI-38 lung fibroblasts. Therefore, GDF15 secreted from senescent alveolar epithelial cells might act as a profibrotic factor through activation of M2 macrophages and fibroblasts. This implies that GDF15 could be a potential therapeutic target and a predictor of lung fibrosis progression.

Cell-based HTS identifies a chemical chaperone for preventing ER protein aggregation and proteotoxicity.

The endoplasmic reticulum (ER) is responsible for folding secretory and membrane proteins, but disturbed ER proteostasis may lead to protein aggregation and subsequent cellular and clinical pathologies. Chemical chaperones have recently emerged as a potential therapeutic approach for ER stress-related diseases. Here, we identified 2-phenylimidazo[2,1-b]benzothiazole derivatives (IBTs) as chemical chaperones in a cell-based high-throughput screen. Biochemical and chemical biology approaches revealed that IBT21 directly binds to unfolded or misfolded proteins and inhibits protein aggregation. Finally, IBT21 prevented cell death caused by chemically induced ER stress and by a proteotoxin, an aggression-prone prion protein. Taken together, our data show the promise of IBTs as potent chemical chaperones that can ameliorate diseases resulting from protein aggregation under ER stress.

Tailored Synthesis of 162-Residue S-Monoglycosylated GM2-Activator Protein (GM2AP) Analogues that Allows Facile Access to a Protein Library.

A synthetic protocol for the preparation of 162-residue S-monoglycosylated GM2-activator protein (GM2AP) analogues bearing various amino acid substitutions for Thr69 has been developed. The facile incorporation of the replacements into the protein was achieved by means of a one-pot/N-to-C-directed sequential ligation strategy using readily accessible middle N-sulfanylethylanilide (SEAlide) peptides each consisting of seven amino acid residues. A kinetically controlled ligation protocol was successfully applied to the assembly of three peptide segments covering the GM2AP. The native chemical ligation (NCL) reactivities of the SEAlide peptides can be tuned by the presence or absence of phosphate salts. Furthermore, NCL of the alkyl thioester fragment [GM2AP (1-31)] with the N-terminal cysteinyl prolyl thioester [GM2AP (32-67)] proceeded smoothly to yield the 67-residue prolyl thioester, with the prolyl thioester moiety remaining intact. This newly developed strategy enabled the facile synthesis of GM2AP analogues. Thus, we refer to this synthetic protocol as "tailored synthesis" for the construction of a GM2AP library.

Protease-resistant modified human ß-hexosaminidase B ameliorates symptoms in GM2 gangliosidosis model.

GM2 gangliosidoses, including Tay-Sachs and Sandhoff diseases, are neurodegenerative lysosomal storage diseases that are caused by deficiency of ß-hexosaminidase A, which comprises an αß heterodimer. There are no effective treatments for these diseases; however, various strategies aimed at restoring ß-hexosaminidase A have been explored. Here, we produced a modified human hexosaminidase subunit ß (HexB), which we have termed mod2B, composed of homodimeric ß subunits that contain amino acid sequences from the α subunit that confer GM2 ganglioside-degrading activity and protease resistance. We also developed fluorescent probes that allow visualization of endocytosis of mod2B via mannose 6-phosphate receptors and delivery of mod2B to lysosomes in GM2 gangliosidosis models. In addition, we applied imaging mass spectrometry to monitor efficacy of this approach in Sandhoff disease model mice. Following i.c.v. administration, mod2B was widely distributed and reduced accumulation of GM2, asialo-GM2, and bis(monoacylglycero)phosphate in brain regions including the hypothalamus, hippocampus, and cerebellum. Moreover, mod2B administration markedly improved motor dysfunction and a prolonged lifespan in Sandhoff disease mice. Together, the results of our study indicate that mod2B has potential for intracerebrospinal fluid enzyme replacement therapy and should be further explored as a gene therapy for GM2 gangliosidoses.

Combined replacement effects of human modified ß-hexosaminidase B and GM2 activator protein on GM2 gangliosidoses fibroblasts.

GM2 gangliosidoses are autosomal recessive lysosomal storage diseases (LSDs) caused by mutations in the HEXA, HEXB and GM2A genes, which encode the human lysosomal ß-hexosaminidase (Hex) α- and ß-subunits, and GM2 activator protein (GM2A), respectively. These diseases are associated with excessive accumulation of GM2 ganglioside (GM2) in the brains of patients with neurological symptoms. Here we established a CHO cell line overexpressing human GM2A, and purified GM2A from the conditioned medium, which was taken up by fibroblasts derived from a patient with GM2A deficiency, and had the therapeutic effects of reducing the GM2 accumulated in fibroblasts when added to the culture medium. We also demonstrated for the first time that recombinant GM2A could enhance the replacement effect of human modified HexB (modB) with GM2-degrading activity, which is composed of homodimeric altered ß-subunits containing a partial amino acid sequence of the α-subunit, including the GSEP loop necessary for binding to GM2A, on reduction of the GM2 accumulated in fibroblasts derived from a patient with Tay-Sachs disease, a HexA (αß heterodimer) deficiency, caused by HEXA mutations. We predicted the same manner of binding of GM2A to the GSEP loop located in the modified HexB ß-subunit to that in the native HexA α-subunit on the basis of the x-ray crystal structures. These findings suggest the effectiveness of combinational replacement therapy involving the human modified HexB and GM2A for GM2 gangliosidoses.

Development of a Chemical Methodology for the Preparation of Peptide Thioesters Applicable to Naturally Occurring Peptides Using a Sequential Quadruple Acyl Transfer System.

Peptide thioesters are very useful in protein chemistry, and chemistry- and biochemistry-based protocols are used for the preparation of thioesters. Among such protocols, only a few biochemistry-based approaches have been use for naturally occurring peptide sequences. The development of chemistry-based protocols applicable to natural sequences remains a challenge, and the development of such methods would be a major contribution to protein science. Here, we describe the preparation of peptide thioesters using innovative methodology that features nickel(II)-mediated alcoholysis of a naturally occurring peptide sequence, followed by O-N and N-S acyl transfers. This protocol involves sequential quadruple acyl transfer, termed SQAT. Notably, the SQAT system consists of sequential chemical reactions that allow naturally occurring peptide sequences to be converted to thioesters without requiring an artificial chemical unit.

The total chemical synthesis of the monoglycosylated GM2 ganglioside activator using a novel cysteine surrogate.

We describe a novel peptide ligation/desulfurization strategy using a ß-mercapto-N-glycosylated asparagine derivative. The newly developed procedure was successfully applied to the total chemical synthesis of the GM2 ganglioside activator protein bearing a monosaccharide on the native glycosylation site.

Chemical synthesis of biologically active monoglycosylated GM2-activator protein analogue using N-sulfanylethylanilide peptide.

Going to SEA(lide): Total chemical synthesis of a 162-residue glycoprotein analogue of the monoglycosylated human GM2-activator protein (GM2AP) was achieved. Key steps were the use of N-sulfanylethylanilide (SEAlide) peptides in the kinetic chemical ligation synthesis of a large peptide fragment, and a convergent native chemical ligation for final fragment assembly.

Lyso-GM2 ganglioside: a possible biomarker of Tay-Sachs disease and Sandhoff disease.

To find a new biomarker of Tay-Sachs disease and Sandhoff disease. The lyso-GM2 ganglioside (lyso-GM2) levels in the brain and plasma in Sandhoff mice were measured by means of high performance liquid chromatography and the effect of a modified hexosaminidase (Hex) B exhibiting Hex A-like activity was examined. Then, the lyso-GM2 concentrations in human plasma samples were determined. The lyso-GM2 levels in the brain and plasma in Sandhoff mice were apparently increased compared with those in wild-type mice, and they decreased on intracerebroventricular administration of the modified Hex B. The lyso-GM2 levels in plasma of patients with Tay-Sachs disease and Sandhoff disease were increased, and the increase in lyso-GM2 was associated with a decrease in Hex A activity. Lyso-GM2 is expected to be a potential biomarker of Tay-Sachs disease and Sandhoff disease.

Therapeutic potential of intracerebroventricular replacement of modified human ß-hexosaminidase B for GM2 gangliosidosis.

To develop a novel enzyme replacement therapy for neurodegenerative Tay-Sachs disease (TSD) and Sandhoff disease (SD), which are caused by deficiency of ß-hexosaminidase (Hex) A, we designed a genetically engineered HEXB encoding the chimeric human ß-subunit containing partial amino acid sequence of the α-subunit by structure-based homology modeling. We succeeded in producing the modified HexB by a Chinese hamster ovary (CHO) cell line stably expressing the chimeric HEXB, which can degrade artificial anionic substrates and GM2 ganglioside in vitro, and also retain the wild-type (WT) HexB-like thermostability in the presence of plasma. The modified HexB was efficiently incorporated via cation-independent mannose 6-phosphate receptor into fibroblasts derived from Tay-Sachs patients, and reduced the GM2 ganglioside accumulated in the cultured cells. Furthermore, intracerebroventricular administration of the modified HexB to Sandhoff mode mice restored the Hex activity in the brains, and reduced the GM2 ganglioside storage in the parenchyma. These results suggest that the intracerebroventricular enzyme replacement therapy involving the modified HexB should be more effective for Tay-Sachs and Sandhoff than that utilizing the HexA, especially as a low-antigenic enzyme replacement therapy for Tay-Sachs patients who have endogenous WT HexB.