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  • 1
    Publication Date: 2018-12-08
    Description: The temperature-sensitive and calcium-permeable transient receptor potential vanilloid 3 (TRPV3) channel abundantly expressed in keratinocytes plays important functions in skin physiology. Dysfunctional gain-of-function TRPV3 gene mutations cause genetic Olmsted syndrome characterized by periorificial keratoderma, palmoplantar keratoderma, inflammation, and severe itching, which suggests that pharmacological inhibition of overactive TRPV3 function may be beneficial in treating pruritus or skin disorders. To test this hypothesis, we identified natural compound forsythoside B as a TRPV3 inhibitor through screening of human embryonic kidney 293 (HEK293) cells expressing human TRPV3 channels in a calcium fluorescent assay. Whole-cell patch-clamp recordings of HEK293 cells expressing TRPV3 confirmed that forsythoside B selectively inhibited the channel current activated by agonist 2-aminoethoxydiphenyl borate (50 µ M) in a dose-dependent fashion, with an IC 50 value of 6.7 ± 0.7 μ M. In vivo evaluation of scratching behavior demonstrated that pharmacological inhibition of TRPV3 by forsythoside B significantly attenuated acute itch induced by either the TRPV3 agonist carvacrol or the pruritogen histamine, as well as chronic itch induced by acetone-ether-water in a mouse model of dry skin. Furthermore, forsythoside B was able to prevent the death of HEK293 cells or native human immortalized nontumorigenic keratinocyte cells from human keratinocytes expressing a gain-of-function TRPV3 G573S mutant or in the presence of the TRPV3 agonist carvacrol. Taken together, our findings demonstrate the crucial role of TRPV3 in pruritus and keratinocyte toxicity; thus, specific inhibition of overactive TRPV3 by natural forsythoside B may possess therapeutic potential for treatment of chronic pruritus, skin allergy, or inflammation-related skin diseases.
    Print ISSN: 0022-3565
    Electronic ISSN: 1521-0103
    Topics: Medicine
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  • 2
    Keywords: RECEPTOR ; APOPTOSIS ; ENDOTHELIAL-CELLS ; Germany ; IN-VIVO ; THERAPY ; ACTIVATION ; LIGAND ; T-CELLS ; MOUSE ; transactivation ; SMOOTH-MUSCLE-CELLS ; hematology ; ACUTE KIDNEY INJURY ; PAR1 ; REDUCED ANTICOAGULANT ACTIVITY ; THROMBIN
    Abstract: The cytoprotective effects of activated protein C (aPC) are well established. In contrast, the receptors and signaling mechanism through which aPC conveys cytoprotection in various cell types remain incompletely defined. Thus, within the renal glomeruli, aPC preserves endothelial cells via a protease-activated receptor-1 (PAR-1) and endothelial protein C receptor-dependent mechanism. Conversely, the signaling mechanism through which aPC protects podocytes remains unknown. While exploring the latter, we identified a novel aPC/PAR-dependent cytoprotective signaling mechanism. In podocytes, aPC inhibits apoptosis through proteolytic activation of PAR-3 independent of endothelial protein C receptor. PAR-3 is not signaling competent itself as it requires aPC-induced heterodimerization with PAR-2 (human podocytes) or PAR-1 (mouse podocytes). This cytoprotective signaling mechanism depends on caveolin-1 dephosphorylation. In vivo aPC protects against lipopolysaccharide-induced podocyte injury and proteinuria. Genetic deletion of PAR-3 impairs the nephroprotective effect of aPC, demonstrating the crucial role of PAR-3 for aPC-dependent podocyte protection. This novel, aPC-mediated interaction of PARs demonstrates the plasticity and cell-specificity of cytoprotective aPC signaling. The evidence of specific, dynamic signaling complexes underlying aPC-mediated cytoprotection may allow the design of cell type specific targeted therapies.
    Type of Publication: Journal article published
    PubMed ID: 22117049
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  • 3
    Keywords: CELL-LINE ; BLOOD-PRESSURE ; KNOCKOUT MICE ; STEROID-HORMONE RECEPTORS ; aldosterone ; HISTONE METHYLATION ; EPITHELIAL SODIUM-CHANNEL ; MEDULLARY COLLECTING DUCT ; NA+ CHANNEL ; FUSION PARTNER
    Abstract: Aldosterone is a major regulator of Na(+) absorption and acts by activating the mineralocorticoid receptor (MR) to stimulate the epithelial Na(+) channel (ENaC). MR(-/-) mice exhibited pseudohypoaldosteronism type 1 (hyponatremia, hyperkalemia, salt wasting, and high levels of aldosterone) and died around postnatal day 10. However, if and how MR regulates ENaC transcription remain incompletely understood. Our earlier work demonstrated that aldosterone activates alphaENaC transcription by reducing expression of Dot1a and Af9 and by impairing Dot1a-Af9 interaction. Most recently, we reported identification of a major Af9 binding site in the alphaENaC promoter and upregulation of alphaENaC mRNA expression in mouse kidneys lacking Dot1a. Despite these findings, the putative antagonism between the MR/aldosterone and Dot1a-Af9 complexes has never been addressed. The molecular defects leading to PHA-1 in MR(-/-) mice remain elusive. Here, we report that MR competes with Dot1a to bind Af9. MR/aldosterone and Dot1a-Af9 complexes mutually counterbalance ENaC mRNA expression in inner medullary collecting duct 3 (IMCD3) cells. Real-time RT-quantitative PCR revealed that 5-day-old MR(-/-) vs. MR(+/+) mice had significantly lower alphaENaC mRNA levels. This change was associated with an increased Af9 binding and H3 K79 hypermethylation in the alphaENaC promoter. Therefore, this study identified MR as a novel binding partner and regulator of Af9 and a novel mechanism coupling MR-mediated activation with relief of Dot1a-Af9-mediated repression via MR-Af9 interaction. Impaired ENaC expression due to failure to inhibit Dot1a-Af9 may play an important role in the early stages of PHA-1 (before postnatal day 8) in MR(-/-) mice.
    Type of Publication: Journal article published
    PubMed ID: 24026182
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  • 4
    Keywords: RECEPTOR ; ENDOTHELIAL-CELLS ; EXPRESSION ; PROTEIN ; LYMPHOCYTES ; B-CELLS ; STOMATITIS-VIRUS GLYCOPROTEIN ; EFFICIENT GENE-TRANSFER ; LYSE TARGET-CELLS ; IMMUNORECEPTOR
    Abstract: Playing a central role in both innate and adaptive immunity, CD4(+) T cells are a key target for genetic modifications in basic research and immunotherapy. In this article, we describe novel lentiviral vectors (CD4-LV) that have been rendered selective for human or simian CD4(+) cells by surface engineering. When applied to PBMCs, CD4-LV transduced CD4(+) but not CD4(-) cells. Notably, also unstimulated T cells were stably genetically modified. Upon systemic or intrasplenic administration into mice reconstituted with human PBMCs or hematopoietic stem cells, reporter gene expression was predominantly detected in lymphoid organs. Evaluation of GFP expression in organ-derived cells and blood by flow cytometry demonstrated exclusive gene transfer into CD4(+) human lymphocytes. In bone marrow and spleen, memory T cells were preferentially hit. Toward therapeutic applications, we also show that CD4-LV can be used for HIV gene therapy, as well as for tumor therapy, by delivering chimeric Ag receptors. The potential for in vivo delivery of the FOXP3 gene was also demonstrated, making CD4-LV a powerful tool for inducible regulatory T cell generation. In summary, our work demonstrates the exclusive gene transfer into a T cell subset upon systemic vector administration opening an avenue toward novel strategies in immunotherapy.
    Type of Publication: Journal article published
    PubMed ID: 26232436
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  • 5
    Keywords: ACTIVATION, ADULT, ANGIOGENESIS, BINDING, BLOOD, BMPER, BONE, bone morphogenetic proteins, cardiovas
    Abstract: Bone morphogenetic proteins (BMPs) are involved in embryonic and adult blood vessel formation in health and disease. BMPER (BMP endothelial cell precursor -derived regulator) is a differentially expressed protein in embryonic endothelial precursor cells. In earlier work, we found that BMPER interacts with BMPs and when overexpressed antagonizes their function in embryonic axis formation. In contrast, in a BMPER-deficient zebrafish model, BMPER behaves as a BMP agonist. Furthermore, lack of BMPER induces a vascular phenotype in zebrafish that is driven by disarray of the intersomitic vasculature. Here, we investigate the impact of BMPER on endothelial cell function and signaling and elucidate its role in BMP-4 function in gain-and loss-of-function models. As shown by Western blotting and immunocytochemistry, BMPER is an extracellular matrix protein expressed by endothelial cells in skin, heart, and lung. We show that BMPER is a downstream target of FoxO3a and consistently exerts activating effects on endothelial cell sprouting and migration in vitro and in vivo. Accordingly, when BMPER is depleted from endothelial cells, sprouting is impaired. In terms of BMPER related intracellular signaling, we show that BMPER is permissive and necessary for Smad 1/5 phosphorylation and induces Erk1/2 activation. Most interestingly, BMPER is necessary for BMP-4 to exert its activating role in endothelial function and to induce Smad 1/5 activation. Vice versa, BMP-4 is necessary for BMPER activity. Taken together, BMPER is a dose-dependent endothelial cell activator that plays a unique and pivotal role in fine-tuning BMP activity in angiogenesis
    Type of Publication: Journal article published
    PubMed ID: 18787191
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  • 6
    Keywords: EXPRESSION ; MICE ; DOWN-REGULATION ; METHYLATION ; METHYLTRANSFERASE ; NEPHROGENIC DIABETES-INSIPIDUS ; AQUAPORIN-2 TRAFFICKING ; WATER CHANNELS ; ENAC-ALPHA ; SET DOMAIN
    Abstract: Dot1l encodes histone H3 K79 methyltransferase Dot1a. Mice with Dot1l deficiency in renal Aqp2-expressing cells (Dot1l(AC)) develop polyuria by unknown mechanisms. Here, we report that Aqp5 links Dot1l deletion to polyuria through Aqp2. cDNA array analysis revealed and real-time RT-qPCR validated Aqp5 as the most upregulated gene in Dot1l(AC) vs. control mice. Aqp5 protein is barely detectable in controls, but robustly expressed in the Dot1l(AC) kidneys, where it colocalizes with Aqp2. The upregulation of Aqp5 is coupled with reduced association of Dot1a and H3 dimethyl K79 with specific subregions in Aqp5 5' flanking region in Dot1l(AC) vs. control mice. In vitro studies in IMCD3, MLE-15 and 293Tcells using multiple approaches including real-time RT-qPCR, luciferase reporter assay, cell surface biotinylation assay, colocalization, and co-immunoprecipitation uncovered that Dot1a represses Aqp5. Human AQP5 interacts with AQP2 and impairs its cell surface localization. The AQP5/AQP2 complex partially resides in the ER/Golgi. Consistently, AQP5 is expressed in none of 15 normal controls, but in all of 17 kidney biopsies from patients with diabetic nephropathy. In the patients with diabetic nephropathy, AQP5 colocalizes with AQP2 in the perinuclear region and AQP5 expression is associated with impaired cellular H3 dimethyl K79. Taken together, these data for the first time identify Aqp5 as a Dot1a potential transcriptional target, and an Aqp2 binding partner and regulator, and suggest that the upregulated Aqp5 may contribute to polyuria, possibly by impairing Aqp2 membrane localization, in Dot1l(AC) mice and in patients with diabetic nephropathy.
    Type of Publication: Journal article published
    PubMed ID: 23326416
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  • 7
    Keywords: kidney ; METHYLATION ; H+-ATPASE ; V-ATPASE ; ENAC-ALPHA ; DUCT-SPECIFIC KNOCKOUT ; COLLECTING DUCT ; EPITHELIAL POLARITY ; CARBONIC-ANHYDRASE ; TUBULAR-ACIDOSIS
    Abstract: The mammalian collecting duct comprises principal and intercalated cells, which maintain sodium/water and acid/base balance, respectively, but the epigenetic contributors to the differentiation of these cell types remain unknown. Here, we investigated whether the histone H3 K79 methyltransferase Dot1l, which is highly expressed in principal cells, participates in this process. Taking advantage of the distribution of aquaporin 2 (Aqp2), which localizes to principal cells of the collecting duct, we developed mice lacking Dot1l in Aqp2-expressing cells (Dot1l(AC)) and found that these mice had approximately 20% fewer principal cells and 13%-16% more intercalated cells than control mice. This deletion of Dot1l in principal cells abolished histone H3 K79 methylation in these cells, but unexpectedly, most intercalated cells also had undetectable di-methyl K79, suggesting that Aqp2(+) cells give rise to intercalated cells. These Aqp2(+) cell-derived intercalated cells were present in both developing and mature kidneys. Furthermore, compared with control mice, Dot1l(AC) mice had 40% higher urine volume and 18% lower urine osmolarity with relatively normal electrolyte and acid-base homeostasis. In conclusion, these data suggest that Dot1l deletion facilitates the differentiation of some alpha- and beta-intercalated cells from Aqp2-expressing progenitor cells or mature principal cells.
    Type of Publication: Journal article published
    PubMed ID: 23308014
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  • 8
    Abstract: Comprehensively investigate the association of CT morphology and clinical findings of adenocarcinoma with EGFR mutation status. Retrospectively included 282 patients who was pathologically proved as lung adenocarcinoma with known EGFR mutation status (mutations: 138 patients, female: 86, median age: 66 years; wildtype: 144 patients, female: 67, median age: 62 years) and their pre-treatment CT scans were analyzed. CT findings and clinical information were collected. Univariate and multivariable logistic regression analysis were performed. Adjusted for age, gender and smoking history of two groups, significantly more patients with pleural tags, pleural and liver metastases were found in the EGFR mutated group (P = 0.007, 0.004, and 0.043, respectively). Multivariable logistic regression analysis found that the model included age, gender, smoking history, air bronchogram, pleural tags, pleural and liver metastasis had a moderate predictive value for EGFR mutation status (AUC = 0.741, P 〈 .0001). Exon-19 deletion was associated with air bronchogram which adjusted for age, gender and smoking history (P = 0.007, OR: 2.91, 95%CI: 1.25-7.79). The evidence of pleural tags, pleural and liver metastases go along with a higher probability of EGFR mutation in adenocarcinoma patients and air bronchogram is positively associated with Exon-19 deletion mutation.
    Type of Publication: Journal article published
    PubMed ID: 28949965
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  • 9
    Abstract: The discovery of RNAs (for example, messenger RNAs, non-coding RNAs) in sperm has opened the possibility that sperm may function by delivering additional paternal information aside from solely providing the DNA (1) . Increasing evidence now suggests that sperm small non-coding RNAs (sncRNAs) can mediate intergenerational transmission of paternally acquired phenotypes, including mental stress(2,3) and metabolic disorders(4-6). How sperm sncRNAs encode paternal information remains unclear, but the mechanism may involve RNA modifications. Here we show that deletion of a mouse tRNA methyltransferase, DNMT2, abolished sperm sncRNA-mediated transmission of high-fat-diet-induced metabolic disorders to offspring. Dnmt2 deletion prevented the elevation of RNA modifications (m(5)C, m(2)G) in sperm 30-40 nt RNA fractions that are induced by a high-fat diet. Also, Dnmt2 deletion altered the sperm small RNA expression profile, including levels of tRNA-derived small RNAs and rRNA-derived small RNAs, which might be essential in composing a sperm RNA 'coding signature' that is needed for paternal epigenetic memory. Finally, we show that Dnmt2-mediated m(5)C contributes to the secondary structure and biological properties of sncRNAs, implicating sperm RNA modifications as an additional layer of paternal hereditary information.
    Type of Publication: Journal article published
    PubMed ID: 29695786
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  • 10
    Keywords: CELLS ; EXPRESSION ; CELL ; human ; NETWORK ; NETWORKS ; SUPPORT ; DISEASE ; DISEASES ; CDNA ; EPITHELIA ; TISSUE ; MICE ; DOMAIN ; TISSUES ; SKIN ; PHOSPHORYLATION ; TRANSGENIC MICE ; IDENTIFICATION ; PATTERNS ; CARE ; MUTATION ; transgenic ; REGION ; WILD-TYPE ; REGIONS ; MONOCLONAL-ANTIBODIES ; cytoskeleton ; keratin ; STOMACH ; ABNORMALITIES ; ORGANIZATION ; DOMAINS ; OVEREXPRESSES ; CELL-DIFFERENTIATION ; I CYTOKERATIN ; intermediate filament
    Abstract: Of the 〉20 epithelial keratins, keratin 20 (K20) has an unusual distribution and is poorly studied. We began to address K20 function, by expressing human wild-type and Arg80--〉His (R80H) genomic (18 kb) and cDNA K20 in cells and mice. Arg80 of K20 is conserved in most keratins, and its mutation in epidermal keratins causes several skin diseases. R80H but not wild-type K20 generates disrupted keratin filaments in transfected cells. Transgenic mice that overexpress K20 R80H have collapsed filaments in small intestinal villus regions, when expressed at moderate levels, whereas wild-type K20-overexpressing mice have normal keratin networks. Overexpressed K20 maintains its normal distribution in several tissues, but not in the pancreas and stomach, without causing any tissue abnormalities. Hence, K20 pancreatic and gastric expression is regulated outside the 18-kb region. Cross-breeding of wild-type or R80H K20 mice with mice that overexpress wild-type K18 or K18 that is mutated at the conserved K20 Arg80-equivalent residue show that K20 plays an additive and compensatory role with K18 in maintaining keratin filament organization in the intestine. Our data suggest, the presence of unique regulatory domains for pancreatic and gastric K20 expression and support a significant role for K20 in maintaining keratin filaments in intestinal epithelia
    Type of Publication: Journal article published
    PubMed ID: 12857878
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