In diabetic mice, CCR2 inhibitor decreased plasma and hepatic triglycerides levels and improved insulin sensitivity. Moreover, CCR2 inhibitor treatment decreased ER stress markers  and inflammatory cytokines  while increasing markers of mitochondrial biogenesis  in the liver. We suggest that CCR2 inhibitor may ameliorate hepatic steatosis by reducing ER stress and inflammation in type 2 diabetes mellitus.Hepatic steatosis is the accumulation of excess fat in the liver. Recently, hepatic steatosis has become more important because it occurs in the patients with obesity, type 2 diabetes, and hyperlipidemia and is associated with endoplasmic reticulum (ER) stress and insulin resistance. C-C chemokine receptor 2 (CCR2) inhibitor has been reported to improve inflammation and glucose intolerance in diabetes, but its mechanisms remained unknown in hepatic steatosis. We examined whether CCR2 inhibitor improves ER stress-induced hepatic steatosis in type 2 diabetic mice. In this study,  The liver is a vital organ for energy homeostasis and glucose metabolism. It absorbs and stores fatty acids from the blood and releases neutral fats into the blood as very-low-density lipoproteins when needed . AccordiAccumulated TGs exacerbate insulin resistance in the liver. Furthermore, hepatic TG accumulation and cytokines released from adipose tissue damage the liver, causing inflammation and endoplasmic reticulum (ER) stress . ER streMonocyte chemoattractant proteins (MCPs) and their receptors play pivotal roles in the development of inflammatory disorders, such as in hepatic steatosis, by recruiting immune cells to the area of inflammation . MCP-1 bdb/db and db/m mice were purchased from Japan Shizuoka Laboratory Center ; db/m mice were used as controls in all experiments. The mice were divided into two groups: CCR2 inhibitor-treated mice and untreated controls. CCR2 inhibitor (RS102895) was purchased from Sigma-Aldrich . Eight-week-old mice were fed either normal chow diet (NCD) or chow mixed with 2 mg/kg/day of RS102895 for 9 weeks. The amount of RS102895 added to NCD was adjusted according to the body weight of each mouse. Food and water intake, urine volume, body weight, and blood pressure were measured monthly. Blood glucose concentration was measured with SureStep . The animals were sacrificed 10 weeks after beginning treatment. All extracted tissues were immediately frozen in liquid nitrogen and stored at −80°C until analysis. All experiments were conducted in accordance with the National Institutes of Health guidelines and with the approval of the Yonsei University Institutional Animal Care and Use Committee .Six-week-old C57BLKS/J AML12 hepatocytes  were grown at 37°C in 5% CO₂ in Dulbecco’s modified Eagle’s medium/F12  containing 10% fetal bovine serum, 10 ml/L penicillin streptomycin . The medium was then replaced with DMEM/F12 containing 10% FBS and 100X Insulin-Transferrin-Selenium (ITS) , and was changed every 2 days. Free fatty acids  were dissolved in ethanol containing bovine serum albumin  and conjugated with BSA at a 10:1 molar ratio before use.2. All tissue TG values were converted to glycerol content and corrected for liver weight.Hepatic triglyceride (TG) content was assayed by saponification in ethanolic KOH, and glycerol content was measured with an FG0100 (Sigma-Aldrich) after neutralization with MgClTissue RNA was extracted using TRIzol (Invitrogen), and total RNA (0.5 μg) was reverse-transcribed into cDNA according to the manufacturer’s instructions. For the quantitative, real-time, reverse transcriptase polymerase chain reaction (PCR) assays, the linearity of the amplification of SREBP1c, FASN, HSL, L-FABP, SCD1, mTOR, GLUT4, PGC-1α, COX1, Tfam, and GAPDH cDNAs was established in preliminary experiments. All signal mRNAs were normalized to GAPDH mRNA. cDNAs were amplified by real-time PCR in duplicate with a SYBR premix Ex Taq kit (Invitrogen) using Thermal Cycler Dice . Primers and optimal cycling conditions were as follows: mSREBP1c: sense 5’-GGA-GAC-ATC-GCA-AAC-AAG-CTG-A-3’, antisense 5’-AGA-CTG-CAG-GCC-AGA-TCC-A-3’; mFasN: sense 5’-AGC-ACT-GCC-TTC-GGT-TCA-GTC-3’, antisense 5’-AAG-AGC-TGT-GGA-GGC-CAC-TTG-3’; mHSL: sense 5’-TCC-TGG-AAC-TAA-GTG-GAC-GCA-AG-3’, antisense 5’-CAG-ACA-CAC-TCC-TGC-GCA-TAG-AC-3’; mL-FABP: sense 5’-AAG-TAC-CAA-TTG-CAG-AGC-CAG-GA -3’, antisense 5’-GGT-GAA-CTC-ATT-GCG-GAC-CA-3’; mSCD-1: sense 5’-TCG-CCC-CTA-CGA-CAA-GAA-CA-3’, antisense 5’-GTA-AGC-CAG-GCC-CA-3’; GAPDH: sense 5’-CTG-GAG-AAA-CCT-GCC-AAG-TA-3’, antisense 5’-AGT-GGG-AGT-TGC-TGT-TGA-AG-3’. All reactions were performed in the same manner: 95°C for 10 seconds, followed by 45 cycles of 95°C for 15 seconds and 60°C for 1 minute. The results were analyzed with real-time system AB 7900HT software (Life Technologies), and all values were normalized to the levels of GAPDH.g for 20 minutes. Fifty micrograms of total protein was subjected to Western blot analysis using a polyclonal antibody to phosphorylated Akt (Thr308), IRS-1 (Ser307), JNK (Thr183/Tyr185), or IKKβ (Ser176/180) or to non-phosphorylated Akt, IRS-1, JNK, IKKβ, CHOP, BiP , CD68, IL-10, XBP-1, ATF4, SOD2, 4HNE , CD16, or β-actin . The band intensities were measured with an Image J Analyzer .The liver was dissected and immediately frozen in liquid nitrogen. Three hundred milligrams of each liver was homogenized with 500 μL of lysis buffer (pH 7.4) supplemented with protease and phosphatase inhibitors. Lipids were removed by centrifugation at 10,000 The cytokines in tissue homogenates were assayed by enzyme-linked immunosorbent assays (ELISA). The assays were conducted using the OptEIA mouse TNF-α, murine MCP-1 set , and murine IL-6 antibody sets . Cytokine levels were quantified from standard curves using the BioTek curve-fitting program . Serum adiponectin levels were measured with a mouse adiponectin antibody set (R&D Systems).Liver tissues were fixed overnight at room temperature in 4% formaldehyde and embedded in paraffin. Sections (8 μm thick) were stained with hematoxylin-eosin and were mounted on glass slides. The stained sections were viewed with an Axio-Star Plus light microscope . Liver sections were stained with CD68 and XBP-1 antibodies (Santa Cruz).The mice were sacrificed after an 8-hour fast, and blood was collected through cardiac puncture. Serum triglyceride concentrations were determined using commercially available enzymatic assay kits . Serum insulin levels were measured with an ultrasensitive mouse insulin ELISA kit .P < 0.05.All data are presented as mean ± SEM. Statistical analysis was performed using one-way analysis of variance (ANOVA) and Tukey’s test for multiple comparisons using SPSS version 17.0 . The differences were considered to be statistically significant at vs. 401.5 ± 43.5 mg/dl) and insulin (15.3 ± 2.0 vs. 9.1±1.5 ng/ml) levels of db/db mice treated with CCR2 inhibitor were significantly lower than those of control db/db mice (db/m mice (vs. 100.2 ± 4.9 mg/dl) and ALT (74.6 ± 7.1 vs. 54.7 ± 4.6 IU/L) levels were significantly lower in treated db/db mice (db/db mice (The fasting serum glucose (584.7 ± 31.1 /db mice . There wb/m mice . Serum T/db mice . No sign/db mice .db/db mice (db/db mice compared with control db/db mice (db/db mice (db/db mice were significantly lower than those of control db/db mice (21.1 ± 2.7 vs 14.7 ± 2.2 mg/g liver weight) (db/db mice (db/db mice.Body weights did not differ between control and treated /db mice . However/db mice . This di/db mice . The liv weight) . The exp/db mice . This fidb/db mice  . Western/db mice , which w/db mice .db/db mice had lower levels of inflammatory cytokines  in the liver compared with control db/db mice  inhibits insulin signaling (16). mTOR and AKT play roles in GLUT4 translocation. Akt phosphorylation was significantly higher (307) phosphorylation was significantly lower in the livers of treated db/db mice  was significantly lower, and Akt phosphorylation was significantly higher in CCR2 inhibitor-treated AML12 hepatocytes (Hepatocytes (AML12) were treated with palmitate (250 μM) for 24 hours. CCR2 inhibitor reduced lipid accumulation by 70% compared with palmitate alone . We treaatocytes . We examatocytes . Westernatocytes . This fiWestern blot analysis showed increased levels of an antioxidative marker (SOD-2) and decreased levels of an oxidative stress marker (4-HNE) in CCR2 inhibitor-treated AML12 hepatocytes . We confdb/db and treated db/db mice, but the liver weight decreased significantly in treated db/db mice. Moreover, hepatic TG levels decreased in treated db/db mice. SREBP-1c and FasN play important roles in controlling fatty acid synthesis in the liver [in vivo and in vitro. This study suggests that CCR2 inhibitor plays an important role in regulating hepatic lipid homeostasis.In this study, we investigated the protective effect of CCR2 inhibitor on hepatic steatosis in a diabetic mouse model. Hepatic steatosis is the most common chronic liver disease, and it is characterized by the accumulation of TGs in the liver . The pathe liver , 20. SREdb/db mice may activate an anti-inflammatory response in the liver.Recent studies have investigated the molecular mechanism of inflammatory signal activation and the role of Kupffer cells in hepatic insulin resistance . Kupfferdb/db mice had improved glucose tolerance in the liver, which resulted from decreased recruitment of hepatic M1 macrophages, leading to reduced cytokine levels. These findings imply that CCR2 inhibitor reduces inflammatory responses in fatty liver by reducing the recruitment of specific macrophages, thereby ameliorating hepatic ER stress.Kupffer cell activation releases pro-inflammatory cytokines and chemokines. Inflammatory cytokines  worsen insulin resistance and inflammatory signaling in mice . TNFα, Iin vivo and in vitro. The expression of mitochondrial biogenesis genes  increased significantly in the livers of treated db/db mice compared with control db/db mice. The decreased ER stress markers and increased mitochondrial biogenesis markers in the liver might indicate a pathway by which hepatic steatosis improved in treated db/db mice.Chronic ER stress affects risk factors associated with dyslipidemia and diabetes . Recent db/db mice compared with control db/db mice [db/m mice. However, CCR2 inhibitor did significantly reduce those parameters in db/db mice. Insulin is a key hormone regulating carbohydrate and lipid metabolism. Phosphorylated Akt induces mTOR and GLUT4 in the mitochondria [Recent reports have shown that accumulated hepatic TGs result in insulin resistance by inhibiting insulin receptor signaling. Insulin resistance is a crucial pathological factor in the progression and development of NAFLD . Our pre/db mice . In thischondria . Serine chondria . We founNo other studies so far have described a detailed mechanism involving CCR2 inhibitor and NAFLD, except for its inhibition of adipose tissue-induced hepatic steatosis. This study is the first to describe the mechanism of CCR2 inhibitor in hepatic steatosis, and supports the involvement cross talk between MCP-1 and CCR2, which could activate inflammatory responses and blunt insulin signaling in the fatty liver. Therefore, CCR2 inhibitor may be a useful therapeutic agent in ameliorating fatty liver in type 2 diabetes.Despites, there are still several limitations in our study. First, we could not elucidate the exact mechanism of inflammatory signaling regulation by CCR2 inhibitor. Further study should be done to reveal a clear relationship of inflammation through the treatment of NF-κB or MAPK inhibitor. Secondly, we used AML12 mouse hepatocytes cell line in vitro. Primary hepatocyte is more close to the character of mouse liver. Although, AML12 hepatocytes cell line is also normal hepatocytes, it is different with primary hepatocytes. Finally, we could not confirm how CCR2 inhibitor implicated CCR2 binding with MCP-1 and only suggested that it competed against MCP-1 binding to CCR2.S1 FigHepatocytes (AML12) were treated with a fatty liver-related factor . Palmitate was prepared in ethanol containing bovine serum albumin . AML12 cells were stained with Oil red O. Original magnification is x200 .(TIF)Click here for additional data file.S2 FigWestern blots of BiP, SREBP-1, p-IRS1 and IRS1 in AML12 cells. Bands intensities were measured with an Image J Analyzer. Results shown are means ± SEM. *P < 0.05 compared with control. #P < 0.05 compared with palmitate-treated control.(TIF)Click here for additional data file.


Drosophila melanogaster are specified by long-range BMP signaling and are especially sensitive to loss of extracellular modulators of BMP signaling such as the Chordin homolog Short gastrulation (Sog). However, the role of the extracellular matrix in BMP signaling and Sog activity in the crossveins has been poorly explored. Using a genetic mosaic screen for mutations that disrupt BMP signaling and posterior crossvein development, we identify Gyc76C, a member of the receptor guanylyl cyclase family that includes mammalian natriuretic peptide receptors. We show that Gyc76C and the soluble cGMP-dependent kinase Foraging, likely linked by cGMP, are necessary for normal refinement and maintenance of long-range BMP signaling in the posterior crossvein. This does not occur through cell-autonomous crosstalk between cGMP and BMP signal transduction, but likely through altered extracellular activity of Sog. We identify a novel pathway leading from Gyc76C to the organization of the wing extracellular matrix by matrix metalloproteinases, and show that both the extracellular matrix and BMP signaling effects are largely mediated by changes in the activity of matrix metalloproteinases. We discuss parallels and differences between this pathway and other examples of cGMP activity in both Drosophila melanogaster and mammalian cells and tissues.The developing crossveins of the wing of  Drosophila melanogaster to identify mutations that reduce or eliminate signals carried by Bone Morphogenetic Proteins (BMPs), and show that BMP signaling is sensitive Gyc76C, a peptide receptor that stimulates the production of cGMP in cells. We identify downstream intracellular effectors of this cGMP activity, but provide evidence that the effects on the BMP pathway are not mediated at the intracellular level, but rather through cGMP’s effects upon the extracellular matrix and matrix-remodeling proteinases, which in turn affects the activity of extracellular BMP-binding proteins. We discuss differences and parallels with other examples of cGMP activity in Drosophila melanogaster and mammals.Signaling between cells regulates many processes, including the choices cells make between different fates during development and regeneration, and misregulation of such signaling underlies many human pathologies. To understand how such signals control developmental decisions, it is necessary to elucidate both how cells regulate and respond to different levels of signaling, and how different types of signals combine and regulate each other. We have used genetic screening in the fruitfly  Drosophila melanogaster wing are positioned, elaborated and maintained by a series of well-characterized intercellular signaling pathways. The wing is easily visualized, and specific mutant vein phenotypes have been linked to changes in specific signals, making the wing an ideal tissue for examining signaling mechanisms, for identifying intracellular and extracellular crosstalk between different pathways, and for isolating new pathway components )D. melanogaster development, we screened for novel BMP regulators in the PCV by creating large mitotic recombinant clones homozygous for mutagenized third chromosomes in the posterior, PCV territory of the developing wing. We utilized posteriorly-expressed engrailed (en)-Gal4 and UAS- FLPase to induce mitotic recombination between mutagenized FRT-bearing chromosome . 24 . 24 for the PCV . Removalossveins , but did8 cases) . We willX1) maps to and is allelic to the ade5 gene  after extraction from S2 cells )There is also precedent for cGMP activity specifically affecting BMP and TGFβ signaling in mammals. cGMP-dependent kinase activity increases BMP signaling in C2C12 cells, and this effect has been suggested to underlie some of the effects of nitric oxide-induced cGMP on BMP-dependent pulmonary arterial hypertension , 52. Congyc76C from embryonic muscles affects the distribution and vesicular accumulation of the βintegrin Mys  FRT2A/TM3, Sb (kindly provided by G. Struhl)82By w; FRTy w, FRT82B, RpS3Plac92[also known as M(3)w or M(3)95A] ubi-GFP/TM6B, TbFRT82BdysEP3397/TM6,Tb (FRT recombinant kindly provided by D. Olson)gyc76CKG03723ex33/TM3,Sb, UAS-myc-gyc76C and UAS-myc-gyc76D945A, kindly provided by A. Kolodkin [gyc76CKG03723ex33FRT2A, en-Gal4 UAS-myc-gyc76C and hh-Gal4 UAS-myc-gyc76C recombinants were lab-generated. Gyc76C overexpression experiments used a second chromosome UAS-myc-gyc76C, except for those of for02; hh-Gal4 UAS-myc-gyc76C /CyO-TM6,Tb stock.Kolodkin . gyc76Cy cv1v ade51fl/FM6, kindly provided by D. Clark [Df(1)ED7165/FM7h; the deficiency covers ade5 but not cv.D. Clark . CrossveEY11089y w; Nplp1for02/CyO,Tb, forK04703/CyO,Tbfor02FRT40A/CyO, recombinant generated in lab.UAS-PDE6-RNAi, UAS-PDE6 and UAS-PDE6C1128S kindly provided by Dr. S. Davies [. Davies , 71.cv43 [cv43 UAS-dpp-GFP, UAS-tlr-HA, UAS-tldA53, UAS-sog-HA, cv70, kindly provided by M. O’Connor [O’Connor , 13.cv-2P(EP)1103 (Szeged Stock Center) [ Center) cvP(EP)1349; cv-2P(EP)1103UAS-sog-HA stock generated in lab.1cv-cP(EP)1349cvy w; P(UASp-YFP.Rab9) [FP.Rab9) MB10509CG4839UAS-gyc76C-RNAi, UAS-dys-RNAi and UAS-Pkg24-RNAi lines were from the VDRC. UAS-gyc76C RNAi; hh-Gal4/CyO-TM6,Tb and en-Gal4 UAS-dys-RNAi stocks generated in lab.trol-GFP (P{PTT-un1}trolG00022) [G00022)  from KyoMmp2::GFP/CyO, kindly provided by J. Sun [y J. Sun UAS-TIMP, UAS-Mmp1-RNAi, UAS-Mmp2-RNAi and Mmp1-lacZ kindly provided by D. Bohmann [ Bohmann .P(GSV2)trolGS7407 (UAS-trol) kindly provided by J. Pastor-Pareja [r-Pareja .w+ insertions for mapping.Bloomington deficiency kits and molecularly defined deletions and P element 50 or more males were transferred to empty vials, allowed to dehydrate for 30 minutes, and then transferred overnight to new bottles contained filter paper soaked in a solution of 24mM EMS, 10Mm Tris pH 7.5 and 1% sucrose. The males were then transferred to dry tubes containing damp filter paper for 30 minutes before being crossed to 50 females for two days.ade5X1 was initially mapped using meiotic recombination relative to molecularly mapped w+ P element insertions [gyc76CL635H was identified by Sanger sequencing (University of Wisconsin-Madison Biotechnology Center) using primers 5’ ATGGATTGTTTGCCACCAACAG Fwd, and 5’ TCAAACAATCGGAATGAAGCTG Rev.Mapping was as described in the Results, except that 3L043 lethality was initially mapped using Bloomington deficiency kits DK3,3L and DK3,3R (Bloomington), while sertions . The 3L0Wing disc and pupal wing dissection, fixation and staining were as described previously , 8; idenConcentrations and sources of primary antibodies were: 1:2000 rabbit anti-phosphoSmad3 (Epitomics); 1:500–1000 mouse anti-DSRF (Cold Spring Harbor Laboratory Antibody Facility); 1:50 mouse anti-Engrailed 4D9, mouse anti-FasIII or mouse anti-Mmp1 ; 1:500 rabbit anti-MTYamide  kindly pS1 Figen-Gal4 UAS-Flp and the Minute method.Adult phenotypes caused by large homozygous posterior clones generated using (PDF)Click here for additional data file.S2 Figiso-1 strain used for BDGP sequencing, and was likely caused by the mobilization of genomic DNA around an original Doc insertion [CG14101 (termed CG42529 in one duplicate) and the coding exons, but not promoter or 5’ UTR exons, of gyc76C . The duplication is very unlikely to be present in the 3L043 chromosome: the duplication is not even present in all iso-1 flies, and Canton-S and Oregon-R wild type strains lack both the Doc insertions and the duplication [gyc76CL635H mutation likely causes the 3L043 genotype, it is doubtful that the 3L043 chromosome contains a second, functioning duplication of gyc76C coding exons.Predicted duplicate gene names have the same color. The duplication has only been observed in a subset of the nsertion . When prlication . Since t(PDF)Click here for additional data file.S3 FigX1 mutation to the ade5 region. X1 does not lie at the other end of the deficiencies, since the proximal end of the BSC545 deficiency extends further than the proximal end of ED7165. (B) PCV disruption in adeX1 wing. (C) PCV loss in ade51 wing. (D) PCV disruption in ade5X1/ade51 wing.  anti-pMad staining is present in the PCV region of ade51 wings at 21 hours AP (E) but is largely lost by 25 hours AP (F). (G) en-Gal4-driven expression of UAS-tlr rescues the PCV in an ade5X1/Y wing.(A) Mapping the (PDF)Click here for additional data file.S4 FigNplp1 locus. The P element insertion Nplp1EY11089 lies in the second coding exon of Nplp1, placing stop codons between the region coding the N-terminal secretion signal peptide and the region coding neuropeptide precursors.  Larval CNS stained with antiserum against the Nplp1 peptide MTYamide. The strong staining in dorsal and ventral segmentally repeated neuronal cell bodies and axons in wild type (B) is missing in an Nplp1EY11089 homozygote (C).  Two adult wings homozygous for Nplp1EY11089. Unlike after gyc76C knockdown, the PCVs are present and complete, and occasionally show a small ectopic branch (D).(A) (PDF)Click here for additional data file.S5 Figgyc76C knockdown in 20 hour AP ap-Gal4 UAS-GFP UAS-gyc76C-RNAi wing. Top panels show that ap-driven GFP expression is limited to the dorsal epithelium. Middle panels show that anti-pMad staining is broader in the dorsal epithelium, both around the PCV and the LVs. Lower left panel shows overlay of dorsal (green) and ventral (red) pMad, with overlap in yellow. (B) In situ hybridization to 24 hour AP wing with gyc76C antisense probe. (C) Retention of anti-pMad in PCV of hh-Gal4 UAS-gyc76C-RNAi wing at 20 hours AP.  24 hour AP en-Gal4 UAS-gyc76C-RNAi wings showing loss of pMad (D) but suppression of DSRF (D’) in PCV.(A) Effects of dorsal (PDF)Click here for additional data file.S6 Figfor02 homozygous (B-B”‘) pupal wings at 20 hours AP, with anti-pMad staining in red and anti-FasIII-stained cell membranes in green.  Low magnification image of single apical (nuclear) focal plane showing normal (A) and partially disrupted  pMad in PCV.  Low magnification image of basal focal plane, showing green membranes where dorsal and ventral epithelia have attached, and the dark basal lumen where epithelia have not yet attached. The lumen does not yet define physical veins in the region of the PCV.  Cross-sections (x) reconstructed from high magnification z-series images, along x lines in A’ or B’. Widths of normal (A”) or abnormal (B”) PCV pMad (red) regions are much narrower than the basal lumens.  High magnification projections of z-series images from all the pMad-containing focal planes on one epithelium, again showing PCV disruption (arrow) in the for mutant.Comparison of wild type (A-A”‘) and (PDF)Click here for additional data file.S7 FigL5-Gal4 (control) adult wing. Arrows demarcate the region of L5 posterior to the ACV, used in measurements of vein width. (C) L5 width in hh-Gal4 UAS-GFP/+ flies is similar to wild-type and L5-Gal4. (D) Increased width of L5 in hh-Gal4 UAS-GFP/ UAS-gyc76C-RNAi (VDRC 6552) flies. (E) Comparison of L5 widths between wild type (wt), hh-Gal4 UAS-GFP/+ and hh-Gal4 UAS-GFP/UAS-gyc76C-RNAi wings. (F and G) Normal L5 width in L5-Gal4 UAS-gyc76C-RNAi wing. (H) Increased width of L5 in L5-Gal4 UAS-dpp-GFP/+ wing. (I) Partial rescue of L5 width increase by knockdown of Gyc76C in L5-Gal4 UAS-dpp-GFP/ UAS-gyc76C-RNAi wing. (J) Comparison of L5 widths between L5-Gal4, L5-Gal4 UAS-dpp-GFP and L5-Gal4 UAS-dpp-GFP/UAS-gyc76C-RNAi wings. n = 10 for all experimental groups in E and J. * = p<0.001. L5-Gal4 UAS-gyc76C-RNAi was not significantly different from L5-Gal4 . (PDF)Click here for additional data file.S8 Figgyc76C3L043 clones in 28 hour AP hs-FLP/+; gyc76C3L043FRT2A/hs-GFP RpS174FRT2A wings.  Large clones overlapping the PCV on both surfaces, indicated by the absence of GFP  resulting in a complete loss of pMad from the PCV region (A’). (B-B’) Smaller clones, indicated by the absence of GFP (green) on the dorsal and ventral epithelium of two individual pupal wings. pMad often persists within homozygous gyc76C3L043 clones encompassing the parts of the PCV, even when clones overlap on the dorsal and ventral epithelia (arrows).  Anti-pMad staining (red) in homozygous gyc76CKG0372ex33 clones in 28 hour AP hs-FLP/+; gyc76CKG0372ex33FRT2A/hs-GFP RpS174FRT2A wings. Large clones overlap on the dorsal and ventral surfaces, resulting in the loss of most, but not all (arrows), pMad from the PCV.  Anti-DSRF staining  in homozygous for02 clones in hsFlp; for02FRT40A/ubi-RFP FRT40A 28 hour AP wings. (D) DSRF is still decreased in clone (arrow) on the PCV. (E) Increased DSRF increased in part of PCV adjacent to for clone (arrow).(A-B’) Anti-pMad staining (red) in homozygous (PDF)Click here for additional data file.S9 Fighh-Gal4 UAS-gyc76C-RNAi wings containing either trol-GFP  or UAS-rab9.YFP (B).  Broad Trol-GFP (green) in L5 and pockets in PCV area extend outside vein regions defined by reduction in DSRF (blue) or heightened pMad (red).  High magnification detail showing that anti-Trol staining does not significantly overlap Rab9.YFP vesicles. (C-D’) High magnification details showing Trol-GFP-containing vesicles (green) do not significantly overlap anti-Rab5  or anti-Rab11  staining .  Moderate magnification detail showing only rare overlap (arrow) between Trol-GFP-containing vesicles (green) and anti-Rab7 staining . Anti-FasIII staining (blue) shows cell outlines. (F) Apical focus on intervein region of trol-GFP; for02/ for02 wing showing accumulation of Trol-GFP vesicles. (G-J) Comparison of 6G7 anti-CgIV and anti-Trol staining in for02 / CyO  and for02/ for02  wings at 24 (G-H’) and 27–28 (I-J’) hours AP. Diffuse, broad vein accumulation of CgIV and Trol, and alterations in intervein cells, become increasingly apparent from 24 to 27 hours AP, but loss from intervein pockets is more apparent at later stages  No defects in 6G7 anti-CgIV staining are apparent in cv43 (L) or cv-c1 (M) wings. (N) No defects in anti-Trol staining are apparent in en-Gal4 UAS-dys-RNAi wings.  28 hour AP hh-Gal4 UAS-Mmp2-RNAi wings have slight posterior increases in CgIV aggregates.  No obvious difference in anti-βGal staining between Mmp1-lacZ (P) and Mmp1-LacZ hh-Gal4 UAS-gyc76C-RNAi (Q) wings, except for those that correlate with altered venation. (S-U) Accumulation of Mmp1 in posterior intervein pockets in hh-Gal4 UAS-gyc76C-RNAi wings at 26 hours AP (S) and 28–30 hours AP (T), but loss at 32 hours AP (U). (V) Loss of Mmp1 from intervein pockets, but diffuse increase in veins, in 32 hour AP for02 homozygote.  Anti-Mmp1 staining in control wild type wings at 26 (W) and 30 (X) hours AP.  DIC optic detail of region posterior to L5 in hh-Gal4 UAS-gyc76C-RNAi wings at 30 hours AP, without (Y) and with (Z) UAS-Mmp2-RNAi. The abnormally large vesicles normally seen after gyc76C knockdown (Y) are much less frequent after knockdown of Mmp2 (Z).(A-E’). (PDF)Click here for additional data file.S10 Fighh-Gal4 UAS-gyc76C-RNAi, except  which show wild type control. In some, limits of posterior knockdown is marked with UAS-GFP (green), while in others it is shown by approximate position just anterior to L4 (lines). High magnification figures are 2.4x low magnification figures. (A-E) Focus on basal region of maximal integrin concentration, stained with anti-Mys (A-B), anti-Mew (C-C”) or anti-If (D-E).  Apico-lateral (nuclear) focus on anti-DSRF and anti-Dei.  Basolateral focus and cross-section of anti-Dlp.  Basolateral focus and cross-section of anti-Dlp in wild type control. (I) Basal focus on anti-Dg. (J.J’) Basolateral focus on anti-Notch.  Basolateral focus on anti-Tkv.Candidates  do not change, except in regions of altered venation. All show (PDF)Click here for additional data file.


The HTLV-1 bZIP factor (HBZ) gene, which is encoded in the minus strand of HTLV-1, plays critical roles in pathogenesis. In this study, RNA-seq and ChIP-seq analyses using HBZ transduced T cells revealed that HBZ upregulates the expression and promoter acetylation levels of a co-inhibitory molecule, T cell immunoglobulin and ITIM domain (TIGIT), in addition to those of regulatory T cells related genes, Foxp3 and Ccr4. TIGIT was expressed on CD4+ T cells from HBZ-transgenic (HBZ-Tg) mice, and on ATL cells and HTLV-1 infected CD4+ T cells of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in vivo. Expression of Blimp1 and IL-10 was upregulated in TIGIT+CD4+ cells of HBZ-Tg mice compared with TIGIT-CD4+ T cells, suggesting the correlation between TIGIT expression and IL-10 production. When CD4+ T cells from HBZ-Tg mice were stimulated with TIGIT’s ligand, CD155, their production of the inhibitory cytokine IL-10 was enhanced. Furthermore, dendritic cells from HBZ-Tg mice produced high levels of IL-10 after stimulation. These data suggest that HBZ alters immune system to suppressive state via TIGIT and IL-10. Importantly, TIGIT suppressed T-cell responses to another HTLV-1 virus protein, Tax, in vitro. Blocking of TIGIT and PD-1 slightly increased anti-Tax T-cell activity in some HAM/TSP patients. These results suggest that HBZ-induced TIGIT on HTLV-1 infected cells impairs T-cell responses to viral antigens. This study shows that HBZ-induced TIGIT plays a pivotal role in attenuating host immune responses and shaping a microenvironment favorable to HTLV-1.Human T-cell leukemia virus type 1 (HTLV-1) infects CD4 HTLV-1 is a T-cell-tropic, latently infectious virus that causes a T-cell malignancy, ATL, and inflammatory diseases. The mechanisms by which HTLV-1 evades the immune response and establishes chronic infection are not yet understood. Recent studies have demonstrated that TIGIT, a co-inhibitory molecule, is expressed on tumor infiltrating T cells and T cells during viral infection, which suppresses the anti-tumor and anti-viral immune responses. Furthermore, blockade of co-inhibitory molecules of TIGIT and programmed cell death-1 (PD-1) disrupts immune checkpoints and enhances anti-tumor activity. We found that TIGIT is upregulated by HBZ, and TIGIT impairs anti-virus immune responses through an immunosuppressive cytokine, IL-10. These findings show that HTLV-1 utilizes a co-inhibitory molecule on infected cells to evade the host immune responses. We also found that blocking of TIGIT and PD-1 on peripheral blood mononuclear cells in HTLV-1 infected patients enhances immune responses to virus. These findings suggest a mechanism by which HTLV-1 shapes a microenvironment favorable to its persistence using induced TIGIT. TIGIT is a potential therapeutic target for ATL and HTLV-1 infected patients. Oncogenic viruses, including Epstein-Barr virus (EBV), Kaposi’s sarcoma-associated herpesvirus (KSHV), human papilloma virus (HPV), hepatitis B virus (HBV), hepatitis C virus (HCV), Merkel cell polyomavirus and human T-cell leukemia virus type 1 (HTLV-1), cause approximately 12% of human cancers. In these virus-induced cancers, a limited number of viral proteins play critical roles in oncogenesis—proteins that include HBx for HBV, E6 and E7 for HPV, and Tax and HTLV-1 bZIP factor (HBZ) for HTLV-1 . Al. Al+ T cIP-qPCR) . Taken tTIGIT associated genome, we performed ChIP-seq analysis with ectopic 3xFLAG-HBZ fusion protein expressing mouse CD4+ T cells using FLAG antibody. ChIP-seq tag peaks were analyzed when they were located around TSS. 201 peaks were identified in HBZ-FLAG expressing CD4+ T cells, 37 (18.4%) of which were associated with genes  or mutant HBZ and GFP was transduced into mouse activated CD4+ T cells. In the TTG mutant, the start codon ATG is replaced by TTG. Therefore, TTG mutant does not generate its protein. In the SM mutant, the entire coding region of HBZ is mutated with silent mutations, which indicates that the SM encodes the same protein while its RNA sequences and secondary structure are altered. Both TTG and SM mutant HBZ induced TIGIT expression on CD4+ T cells like wtHBZ (HBZ RNA and protein could induce TIGIT expression. Enhanced TIGIT expression was more potent by HBZ protein (SM) compared with HBZ RNA (TTG).We have reported that both ar genes , 24. To ke wtHBZ , indicatTIGIT. wtHBZ and mutant HBZ slightly enhanced transcription from TIGIT promoter  since mouse retrovirus can infect only dividing cells. These findings suggest that HBZ activates the TIGIT gene transcription along with cell activation or Tax.Next we examined the effect of HBZ on the promoter activity of promoter . In the promoter  althoughd by HBZ . Transcrd by HBZ , which s+ Treg cells exhibit a more potent suppressive function than TIGIT- Treg cells [Blimp1, a transcription factor that transactivates IL-10, is upregulated in TIGIT+ Treg cells [Blimp1 and Il-10. When HBZ was expressed in mouse primary CD4+ T cells, both Blimp1and Il-10 gene transcriptions were enhanced  production of T cells stimulated by Tax peptides using enzyme-linked immunosorbent spot (ELISPOT) assay. C57BL/6J mice were immunized twice with recombinant Tax protein and CpG adjuvant  of HAM/TSP patients, which suggests that TIGIT and PD-1 are targets of treatment for HTLV-1 associated diseases.Antigen recognition by the T-cell receptor and a second signal mediated by the CD28-CD80/86 co-stimulatory pathway are critical for T-cell activation. In addition, a multitude of other co-stimulatory and co-inhibitory pathways are also involved in T-cell activation . In part T cells . Here we+ and CD8+ T cells. Viral loads were reduced and cytokine productions were increased in LCMV infected mice in which CD4+ T cells lack TIGIT expression. Furthermore, blockade of PD-1/PD-L1 and TIGIT by the antibodies synergistically enhanced viral clearance and CD8+ T cell effector functions. These data clearly indicates that TIGIT on T cells is implicated in anti-viral immunity. PD-1 and TIGIT of tumor infiltrating CD8+ T cells or exhausted CD8+ T cells in chronic infection have been extensively studied [+ effector T cells is implicated in evasion of the host defense.It has been reported that TIGIT plays critical roles to control viral infection . In mice studied , 31. Rec studied . In cons+ T cells, illustrating the significance of PD-1 and TIGIT on CD8+ T cells and the interactions of these receptors with their ligands on APCs and cancer cells [As shown in this study, ATL cells and HTLV-1 infected cells express not only TIGIT but also PD-1 on their surfaces. HBZ can induce expression of both molecules. It has been reported that reverse signaling via PD-L1 and PD-L2 into DCs reduces DC maturation . Furtherer cells . The preHost immune responses play pivotal roles in controlling viruses. Accordingly, viruses acquire ways of counteracting host immune responses. For example, HCV NS3/4A cleaves IFN-β promoter stimulator-1, which blocks signaling via RIG-I/MDA5 . Thus, H+ T cell activation [IL-10, a pleiotropic cytokine, exerts immunosuppressive functions on cytokine and chemokine production, suppresses MHC expression, and suppresses the maturation and function of DCs. It has been reported that KSHV microRNAs target C/EBPβ p20, thus inducing basal secretion of IL-6 and IL-10 by macrophages . Viral htivation . Thus, I+CD4+ T cells that convert to Foxp3-CD4+ T cells produce excess amount of interferon-γ (IFN-γ), which leads to inflammation [HAM/TSP is an inflammatory disease of the central nervous system . The immammation . On the ammation . Thus, bFoxp3, TIGIT, and CCR4 genes [This study also illustrates epigenetic modulation by HBZ using genome-wide analysis. HBZ has been shown to interact with KIX domain of p300 . We prevR4 genes , 46. It In this study, we demonstrate that HBZ induced TIGIT expression likely impairs T-cell response to viral antigens through enhanced IL-10 production by T cells and DCs. This study suggests a new therapeutic strategy for ATL patients: the co-blockade of TIGIT and PD-1 to restore anti-tumor and anti-virus immune responses.HBZ gene in CD4+ T cells (HBZ-Tg) were described previously [C57BL/6J mice were purchased from CLEA Japan. Transgenic mice expressing the eviously . All micAnimal experiments were performed in strict accordance with the Japanese animal welfare bodies (Law No. 105 dated 19 October 1973 modified on 2 June 2006), and the Regulation on Animal Experimentation at Kyoto University. The protocol was approved by the Institutional Animal Research Committee of Kyoto University . Experiments using clinical samples were conducted according to the principles expressed in the Declaration of Helsinki, and approved by the Institutional Review Board of Kyoto University (Permit numbers are G310 and G204). ATL and HAM/TSP patients provided written informed consent for the collection of samples and subsequent analysis.TIGIT and FGL2 were amplified from human genomic DNA by PCR using the primer sets described in Expression vectors for HBZ (wild-type and mutants) and C/EBPα were described previously , 47. FLA+ cells were enriched by a CD4 enrichment kit (BD Pharmingen) and were activated by 0.5 μg/ml anti-CD3 Ab and 50 U/ml rIL-2 in the presence of T-cell-depleted and x-irradiated (20Gy) C57BL/6J splenocytes as APCs in 12 well plates. After 16 hours, activated T cells were transduced with viral supernatant and 4 μg/ml polybrene, and centrifuged at 3,000 rpm for 60 min. Cells were cultured in medium supplemented with 50 U/ml rIL-2 [CD4Cells were transduced and sorted as described above, then crosslinked in 1% 0.5% for  formaldeWe prepared RNA-seq libraries from the RNA described above and ChIP-seq libraries from chromatin-immunoprecipitated or input DNA samples, and we performed sequencing using the HiSeq2500 (Illumina) according to the manufacturer’s protocol. For HBZ-FLAG ChIP-seq analysis, library preparation and high-throughput sequencing were performed at BGI  using the Hiseq2000 (Illumina).The obtained RNA-seq and ChIP-seq data were mapped to the murine reference genome using Bowtie . For RNA5 cells/ml and transfected with 300 ng of luciferase reporter plasmid, 10 ng of pGL4-TK (Promega), and indicated amount of HBZ-expressing plasmid with or without Tax-expressing or C/EBPα-expressing plasmid using LTX (Invitrogen) according to the manufacturer’s protocol. After 24 hours, cells were harvested and luciferase activities were measured using the Dual Luciferase Reporter Assay Kit (Promega). Relative luciferase activities of Firefly to Renilla were then calculated. For PMA /ionomycin stimulation, cells were stimulated for 4 hours before being harvested. To compare the expression levels of HBZ mutants, transcripts in SRα region were quantified by realtime PCR.Jurkat cells were seeded at 2 x 10Human PBMCs and murine splenocytes were stained with the antibodies indicated according to the manufacturer’s protocol and analyzed using FACS Verse with Suite software (BD). Data was analyzed by FlowJo software (Treestar).http://www.activemotif.com/catalog/details/39755/histone-h3-acetyl-lys18-antibody-pab-3). Anti-FLAG (M2) was purchased from Sigma. Normal rabbit and mouse IgG (Santa Cruz Biotechnology) were used as a control.ChIP-seq for histone modifications was performed using the following antibodies: anti-H3ac  , anti-H3For flow cytometric analysis of murine samples, PE/Cy7 anti-CD3 (145-2C11), PerCP-Cy5.5 anti-CD4 (RM4-5), APC/Cy7 or FITC anti-CD8a (53–6.7), APC anti-TIGIT (1G9) and PE anti-CD226 (10E5) antibodies were purchased from BioLegend; PE anti-CD45R/B220 (RA3-6B2), FITC anti-CD11c (HL3), PE anti-PD-1 (J43), PE anti-IFN-γ #554412) and PE anti-PD-1 (#551892) antibodies were purchased from BD Bioscience; and eFluor450 anti-Foxp3 (FJK-16s), PE anti-IL-10 (JES5-16E3), Biotin anti-CD90.1 (HIS51) and PE anti-Blimp1 antibodies were purchased from eBioscience. For flow cytometric analysis of human samples, PerCP-Cy5.5 anti-CD4 (OKT4) was purchased from eBioscience, and APC anti-TIGIT (741182) antibodies was purchased from R&D systems. Brilliant Violet421 anti-IL-10 and FITC-labeled goat anti-mouse IgG were purchased from BioLegend. Anti-HTLV I gp46 (67/5.5.13.1) was purchased from Abcam. Biotin conjugated anti-CADM1 (3E1) antibody was generated as described [4412 and + T cells from non-Tg and HBZ-Tg mice were enriched by CD4 magnetic particles (BD) and then seeded into a 96-well plate at 1 or 2x105 cells and cultured for the indicated time. Supernatants from cultured cells were centrifuged to remove debris and IL-10 was then measured using QuantikineELISA from R&D systems according to the manufacturer’s protocol.CD4Mice were immunized with bacterially generated recombinant Tax protein and CpG by subcutaneous inoculation at days 0 and 14, and splenocytes were collected at day 21. Splenocytes of immunized mice and human PBMCs were subjected to ELISPOT assay as described . Recombi6/ml and cultured in the presence of LPS for 24 hours. Supernatants were centrifuged to remove debris and IL-10 was then measured using IL-10 Enhanced Sensitivity Flex Set from BD according to the manufacturer’s protocol.Splenocytes from non-Tg and HBZ-Tg mice were stimulated with LPS overnight, followed by sorting of dendritic cells using FACS AriaII (BD). Sorted cells were seeded into a 96-well plate at 1 x 10+ cells were collected using Human CD4 T Lymphocyte Enrichment Set (BD) and then stimulated with PHA (3 μg/ml) in the presence of IL-2 (150 U/ml) for 3 days, followed by co-cultured with irradiated MT-2 for 3 days. Infected primary CD4+ T cells were detected by anti-HTLV-1 gp46 (Env) followed by FITC-labeled anti-mouse IgG.Human CD4ATL- 43T(-) cells were transfected with short interfering RNA (siRNA) twice at 0 and 48 hours using Lipofectamin2000 as described . Over 90P < 0.05 and **P < 0.01.The Mann-Whitney test using GraphPad Prism software or the Student’s t test was used to determine significance where appropriate. All statistical analyses are shown as *All raw sequence data were deposited in the DNA Data Bank of Japan (DDBJ) under the accession number DRA003229 and DRA003744.S1 Fig+ T cells were stimulated with PHA (3μg/ml) for 3 days and then co-cultured with irradiated HTLV-1 infected cell line MT-2 for 3 days. Expression of TIGIT was analyzed by FCM in HTLV-1 infected Env+CD4+ T cells.Human primary CD4(PPTX)Click here for additional data file.S2 FigP < 0.05.HBZ was knock-down by siRNA in an ATL cell line, 43T(-). Expression levels of TIGIT were analyzed by realtime PCR. Results shown are the mean ± SD in triplicate. *(PPTX)Click here for additional data file.S3 FigExpression levels of HBZ-deletion mutants in luciferase assays  were ana(PPTX)Click here for additional data file.S4 Fig+ T cells from non-Tg and HBZ-Tg mouse. Splenocytes were stimulated with plate-coated anti-CD3 (1 μg/ml) and soluble anti-CD28 (1 μg/ml).Flow cytometry analysis of Blimp1 on CD4(PPTX)Click here for additional data file.S5 FigPBMCs from HAM/TSP patients (n = 5) and healthy donors (n = 3) were stimulated with PMA/ionomycin for 4 hours in the presence of brefeldin A. Expression of IL-10 were analyzed by FCM levels. IL-10 MFI of stained with anti-IL10 and isotype control were shown in the upper right.(PPTX)Click here for additional data file.S6 FigTIGIT, Fgl2, Blimp1 and IL-10 were analyzed by realtime PCR in PBMCs from ATL patients (n = 10–13) and in CD4+ T cells from HD (n = 4).Expression levels of (PPTX)Click here for additional data file.S7 FigExpression level of C/EBPα in luciferase assays  were ana(PPTX)Click here for additional data file.S8 FigCd226 were analyzed by realtime PCR in CD4+ T cells from non-Tg (n = 4), TIGIT+CD4+ and TIGIT-CD4+ T cells from HBZ-Tg (n = 3). Expression levels of CD226 were analyzed by realtime PCR in CD4+ T cells from HD (n = 4) and ATL patients (n = 10).Expression levels of (PPTX)Click here for additional data file.S9 Fig+ T cells from non-Tg (n = 4) and HBZ-Tg (n = 4) mice. Representative histograms were shown. *P < 0.05.Expression levels of PD-1 were analyzed by FCM in CD4(PPTX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table+ T cells. The mean percentages (± SD) from 4 donors are shown for healthy donor (HD).The numbers indicate the percentage of CD8(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.


Neutrophils have an important role in the rapid innate immune response,and the release or active secretion of elastase from neutrophils is linked to variousinflammatory responses. Purpose of this study was to determine how the humanneutrophil elastase affects the interleukin-10 (IL-10) response in peripheral bloodmononuclear cells (PBMC). In this prospective study, changes in IL-10 messenger RNA(mRNA) and protein expression levels in monocytes derived from human PBMCswere investigated after stimulation with human neutrophil elastase (HNE). A set ofinhibitors was used for examining the pathways for IL-10 production induced by HNE.IL-10 mRNA expression by monocytes, while theenzyme-linked immunosorbent assay (ELISA) revealed an increase of IL-10 proteinlevel in the culture medium. A phospholipase C inhibitor (U73122) partially blunt-ed the induction of IL-10 mRNA expression by HNE, while IL-10 mRNA expressionwas significantly reduced by a protein kinase C (PKC) inhibitor (Rottlerin). A calciumchelator octyl ester: TMB-8) inhibitedthe response of IL-10 mRNA to stimulation by HNE. In addition, pretreatment witha broad-spectrum PKC inhibitor (Ro-318425) partly blocked the response to HNE.Finally, an inhibitor of PKC theta/delta abolished the increased level of IL-10 mRNAexpression. Reverse transcription polymerase chain reaction (RT-PCR) showed thatstimulation with HNE upregulated IL-10 mRNA ex-pression and protein production in moncytes via a novel PKC theta/delta, althoughpartially via the conventional PKC pathway.These results indicate that HNE mainly upregulates  Neutrophils are an important part of the rapidinnate immune response that accompanies acuteinflammation. Neutrophils release the contentsof various granules while migrating to sites ofinflammation, and these granule proteins play acentral role in the early inflammatory response.The serine proteases cathepsin G, human leukocyte elastase, and proteinase 3 are abundantin neutrophil granules . These p2+ mobilization,a transient increase of the inositol 1, 4,5-trisphosphate (IP3) level, and translocation ofprotein kinase C (PKC)  induce Ca C (PKC) . Binding C (PKC) . It was  C (PKC) .HoweverAll human materials such as the peripheral bloodcells used in this study were collected from male(n=15) and female (n=15) non-smoking healthyvolunteers aged 26-33 after their informed consentwas obtained. The protocol of this study was approvedby Institutional Review Board of KumamotoHealth Science University, and the study wasconducted in accordance with the Declaration ofHelsinki.HNE with 81 U/mg of activity was purchasedfrom SERVA Electrophoresis .3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester , A23187 , Rottlerin Tumor necrosis factor-alpha(TNF-α)protease inhibitor I (TAPI-1), U73122,R59022 , and pyrrolidinedithiocarbamatewere employed to investigate the intracellularsignal transduction pathways involved in IL-10mRNA expression. The actions of these reagentsare summarized in table 1.In addition, PKC inhibitors including Ro-318425 (Merck Millipore), Go 6976, Go 6983,and CGP 41251 , aswell as a PKC theta/delta inhibitor (Merck Millipore)were utilized to investigate the roles ofPKC isoforms in IL-10 production. The PKCisoform-specific inhibition profile of these reagentsis summarized in table 2.Xestospongin C, which antagonizes the calciumreleasingaction of IP3 at the receptor level, wasobtained from Sigma-Aldrich, USA. Each reagentsolution was negative for endotoxin according tothe Endospecy test , 23.6 cells per wellwere distributed into 12-well plates  and allowed to adhere in a 5% CO2 incubator at 37˚C for 2 hours. Monocytes were furtherenriched by virtue of their attachment to a cultureplate for 2 hours and washed 3 times with warmphosphate-buffered saline to remove nonadherent cells. Then, monocytes werecultured in complete medium consisting of RPMI1640 supplemented with 10% heat-inactivatedfetal calf serum (FCS), and 10 μg/mL gentamicin at 37˚C in 5% CO2 humidifiedair. The adherent monocytes were recovered with acell scraper. The purity of monocytes was evaluatedby fluorescent staining with CD14-phycoerythrin(PE) mouse anti-human monoclonal antibody  and fluorescence-activated cellsorting (FACS) analysis. The recovery of monocyteswas also evaluated by trypan blue staining andcounted using a microscope . Onlyisolated CD14+ monocytes of >85% purity wereused for each experiment. After monocytes wereresuspended in RPMI-1640 medium supplementedwith 25 mM HEPES , 100 mmol/L L-glutamine ,100 U/mL penicillin100 μg/mL streptomycin, and 10% FCS, the cells werestimulated with HNE for 6 hours.Lymphocyte medium for thawing (BBLYMPH1)was obtained from Zen-Bio, Inc. . PBMCs were isolated as describedpreviously . BrieflyIL-10 mRNA expression wasanalysed by RT-PCR. The primer sequences usedwere as follows:Extraction was done with 500 μL of TRIzol™reagent , and total RNA wasisolated and precipitated according to the manufacturer’sinstructions. Then 1 μg of total RNAwas subjected to reverse transcription using randomheptamer primers with Moloney murineleukemia virus . Next, 1 μl ofreverse-transcribed RNA was amplified by polymerasechain reaction (PCR) on an ABI PRISM7000 thermal cycler using the Taqman™ Master Mix Kit. Quantificationof target mRNA was performed by comparingthe number of cycles required to reach the referenceand target threshold values . MonocytIL-10F: 5´-ATGCCCCAAGCTGAGAACCAAGAC-3´R: 5´-TCTCAAGGGGCTGGGTCAGCTATCCCA-3´β-actinF: 5´-GTGGGGCGCCCCAGGCACCA-3´R: 5´-CTCCTTAATGTCACGCACGATTTC-3´The PCR conditions were as follows: for IL-10,35 cycles  and for β-actin, 40cycles  were used. The PCRproducts were analyzed on agarose gels.After monocytes were stimulated with HNE for6 hours, the level of IL-10 protein in the supernatantwas measured by enzyme-linked immunosorbentassay (ELISA) with an anti-IL-10 monoclonalantibody .PKC kinase activity assay kit was obtained fromAbcam Inc. (USA). This kit is based on a solidphase ELISA that utilizes a specific synthetic peptideas a substrate for PKC and a polyclonal antibodythat recognizes the phosphorylated formof the substrate. Monocytes were incubated for 6hours with or without HNE (5 μg/mL). Then, cellswere lysed in 1 mL of lysis buffer, and 30 μL weretested for PKC activity.Data are expressed as the mean ± SD. Analysisof variance and the t test of independent meanswere used to determine differences between multiplegroups and differences between two groups,respectively. When the F ratio was significant,mean values were compared using a post hoc Bonferroni’stest. A P value<0.05 was considered toindicate a significant difference in all analyses.IL-10 mRNA expression tostimulation with HNE. In contrast, R59022 (a diacylglycerolkinase inhibitor) had no effect on IL-10mRNA levels. Similarly, neither a TNF-α convertingenzyme (TACE) inhibitor, TAPI-1, nor an inhibitorof nuclear factor-kappa B (NF-kB), PDTC, reducedIL-10 mRNA expression by HNE-stimulatedmonocytes  did not augmentIL-10 mRNA expression. Interestingly, the PKCinhibitor Rottlerin blunted the increase of IL-10mRNA expression after stimulation of monocyteswith HNE . Consistent with thedata revealing PBMCs increase IL-10 expressionafter HNE treatment , secretionocytes . Howeverwith HNE . Monocytwith HNE .IL-10 mRNA expression was examined byRT-PCR. Ro-318425 (1 μM/mL) partially inhibitedthe increase of IL-10 mRNA expression in monocytes exposed to HNE and more effectivelyinhibited the response of IL-10 mRNA at a higherconcentration (5 μM/mL). Go 6976 (1 μM/mL)had no influence on the increase of IL-10 mRNAin monocytes exposed to HNE, but partially inhibitedthe response of IL-10 mRNA at a higher concentration(5 μM/mL). Similarly, Go 6983 had noeffect at a concentration of 1 μM/mL, but partiallyinhibited IL-10 mRNA expression at a concentrationof 5 μM/mL. Interestingly, addition of a PKCtheta/delta inhibitor (5 μM/mL) completely abolishedIL-10 expression  family of G protein-coupled receptorsis activated by a unique mechanism that involvesproteolytic unmasking of an N-terminal self-activatingtethered ligand. Proteinases can either activatePAR signaling by unmasking the tethered ligandsequence or disarm the receptor for subsequent enzymeactivation by cleavage downstream from thissequence ,the role of PKC isoforms from each subgroup inIL-10 production was investigated. The broad-spectrumPKC inhibitor bisindolylmaleimide Ro-318425 partiallyblocked upregulation of IL-10 mRNA expression byHNE in a concentration-dependent manner. However,the Ca2+-dependent PKC inhibitor Go 6976  hadless effect on the response of IL-10 mRNA to HNE.Similarly, the broad-spectrum PKC inhibitor Go6983 (a PKCβII inhibitor) partially blocked the increaseof IL-10 mRNA expression induced by HNE.Interestingly, a PKC theta/delta inhibitor (which inhibitsnovel PKC isoforms) strongly suppressed IL-10 mRNA expression after HNE stimulation. Thesefindings suggest that HNE upregulates IL-10 expressionin monocytes by promoting intracellular Ca2+influx, activating phospholipase C, and preferentiallyactivating the novel PKC signaling pathway over theconventional pathway.Tumor necrosis factor-alpha converting enzyme(TACE) cleaves TNF at the Ala-76–Val-77 site andTACE expression has been detected on alveolar macrophagesby flow cytometry. It is known that activationof the epidermal growth factor receptor and itsdownstream signaling cascade are involved in theproduction of mucin. TACE cleaves pro-transforminggrowth factor-α in airway epithelial cells to releaseits mature soluble form, which subsequently binds toand activates the epidermal growth factor receptor.Shao and Nadel  previous studies , 32. In  studies  reported2+ and activation of thephospholipase C pathway. IL-10 production alsodepends on activation of the novel PKC theta/delta,rather than conventional PKC isoforms.Monocytes produce anti-inflammatory IL-10after stimulation with HNE. IL-10 productioninvolves intracellular Ca


Caenorhabditis elegans, LON-2/glypican, a heparan sulfate proteoglycan, modulates UNC-6/netrin signaling and may do this through interactions with the UNC-40/DCC receptor. We show that developing axons misorient in the absence of LON-2/glypican when the SLT-1/slit guidance pathway is compromised and that LON-2/glypican functions in both the attractive and repulsive UNC-6/netrin pathways. We find that the core LON-2/glypican protein, lacking its heparan sulfate chains, and secreted forms of LON-2/glypican are functional in axon guidance. We also find that LON-2/glypican functions from the epidermal substrate cells to guide axons, and we provide evidence that LON-2/glypican associates with UNC-40/DCC receptor–expressing cells. We propose that LON-2/glypican acts as a modulator of UNC-40/DCC-mediated guidance to fine-tune axonal responses to UNC-6/netrin signals during migration.Netrin is a key axon guidance cue that orients axon growth during neural circuit formation. However, the mechanisms regulating netrin and its receptors in the extracellular milieu are largely unknown. Here we demonstrate that in  Work in nematodes shows that the proteoglycan glypican acts as an extracellular modulator of netrin signals during nervous system development, interacting with cells that express the netrin receptor. Caenorhabditis elegans. We identify that the response of migrating neurons to a key guidance cue, UNC-6/netrin, is modulated by a specific proteoglycan, LON-2/glypican. We show that LON-2/glypican may carry out this regulation by interacting with the UNC-40/DCC netrin receptor on the cell surface. We propose that LON-2/glypican acts as an extracellular modulator of UNC-40/DCC-mediated guidance to fine-tune axonal responses to UNC-6/netrin signals during migration.During the development of the nervous system, migrating axons are guided as they navigate through complex environments to reach their target destinations. These directed migrations are essential to ensure the proper wiring and function of the nervous system and are instructed by guidance cues and receptors. There is a remarkably small set of guidance cues and receptors relative to the large number of neuronal migrations, suggesting that the actions of these guidance cues might be diversified by regulatory mechanisms. We have addressed this question in the genetically tractable nematode  Directed migrations of developing axons are essential for the proper wiring of the nervous system. A host of guidance cues and their receptors instruct axon guidance decisions. However, how these cues and the growth cone’s responses to them are spatially and temporally regulated in vivo remains largely unknown. Answering this question is central to our understanding of how growing axons navigate in complex environments to reach their targets during development and regeneration.Caenorhabditis elegans [UNC-6/netrin is a highly conserved secreted guidance cue with structural similarity to the extracellular matrix protein laminin –3. UNC-6 elegans  and bioc elegans . A secon elegans ,8, and a elegans ,10. WhilC. elegans, which has been instrumental for discovering major conserved axon guidance pathways. During larval development, the axon of the mechanosensory neuron AVM migrates ventrally as its growth cone integrates signals from two complementary guidance cues , the two glypicans (lon-2 and gpn-1), perlecan (unc-52), and agrin (agr-1). We found that the mild AVM axon guidance defects of sdn-1/syndecan mutants, including a null, [lon-2/glypican in double mutants lon-2 sdn-1  and misguides the axon of AVM [lon-2/glypican enhanced the defects caused by slt-1/Slit misexpression  and of the GABAergic motorneuron axons [lon-2/glypican single null mutants are defective in dorsal DTC migrations . In wild-type animals, PVM, like AVM, expresses the receptor unc-40/DCC, and its axon grows ventrally towards unc-6/netrin  in PVM ependent . In contgrations , highliglon-2/glypican functions in the same genetic pathway as known downstream mediators of unc-6/netrin signaling, we tested for genetic interactions between lon-2/glypican and unc-34/enabled. unc-34/enabled is a regulator of actin polymerization for axonal filopodia outgrowth [unc-6/netrin and slt-1/Slit guidance pathways renders the analysis of genetic interactions in the context of normal AVM axon guidance challenging. Therefore, we used the unc-6/netrin-specific gain-of-function approach as above, in which the dorsal migration of the PVM axon upon ectopic expression of unc-5/UNC5 is unc-34/enabled dependent  transgenes expressed under the heterologous epidermal promoters Pdpy-7 and Pelt-3  rescued lon-2 slt-1 double mutants back to slt-1 single mutant levels, as efficiently as when expressed under the endogenous promoter Plon-2 , in the migrating neuron itself (Pmec-7), in the intestine (Pelt-2), or in body wall muscles (Pmyo-3)  . Our ressdn-1(+) in the AVM neuron (using the heterologous promoter Pmec-7) rescued axon defects of lon-2 sdn-1 double mutants  cannot replace the function of lon-2/glypican; expressing sdn-1/syndecan in either the cells that normally express lon-2/glypican (using Plon-2::sdn-1) or the migrating neuron itself (Pmec-7::sdn-1) did not rescue the loss of lon-2/glypican (lon-2/glypican and sdn-1/syndecan have specific roles in axon guidance.We found that expressing wild-type copies of  mutants . Accordin-1::gfp ) revealeM neuron , at the glypican , supportGlypicans are composed of a core protein moiety with covalently linked HS chains attached via a tetrasaccharide linker at specific Serine residues , superfolder-GFP (SfGFP), and FLAG, respectively , sax-3/Robo, and sqv-5, suggesting that lon-2/glypican functions specifically in the unc-6/netrin pathway. Third, sdn-1/syndecan, cannot replace lon-2/glypican function, highlighting a requirement for lon-2/glypican that cannot be achieved by any HSPG. Given that the core protein of LON-2/glypican, devoid of its HS chains, is fully functional in guidance, the specificity of action of LON-2/glypican in netrin-mediated guidance appears to reside in the core protein itself. As a note, whereas lon-2/glypican mutants are defective in DTC migration, the lon-2/glypican mutant by itself does not show drastic alterations in AVM axon guidance as is observed with other modulators [lon-2/glypican, another HSPG may provide compensation or that our scoring of strong alterations in pathfinding did not include more subtle phenotypes, as could be expected from a modulator of the signal [The impact of dulators . It is pe signal .unc-6/netrin-mediated guidance. Thus, the core protein is the critical region of LON-2/glypican for netrin-mediated axon guidance. This is in line with previous studies showing a contextual dependence of HS chains for glypican function. For instance, the core protein of C. elegans LON-2/glypican and of Drosophila glypican Dally do not require HS chains to function in the transforming growth factor beta (TGFβ) pathway [Drosophila glypican Dally-like interacts with Wg and Hh through their protein core in a HS-independent manner [We show that the LON-2/glypican core protein, devoid of HS attachment sites, is able to associate with UNC-40-expressing cells and is functional in  pathway ,39. Simit manner ,40,41, at manner –45. Whilt manner ,39,41,42LON-2/glypican is predicted to localize at the cell surface via its GPI anchor . HoweverDrosophila glypican Dally-like protein is cleaved at the GPI anchor by the lipase Notum, to negatively regulate Wnts [Xenopus embryo [Glypican cleavage by lipases and proteases has been demonstrated to occur and to be functionally important in other contexts, such as in regulating fibroblast growth factor (FGF) and Wnt signaling during morphogenesis ,46. For ate Wnts . Similarate Wnts . The funate Wnts . In addis embryo  and incrs embryo , respectWe demonstrate that LON-2/glypican is secreted into the extracellular medium and decorates the outline of UNC-40/DCC-expressing cells. Deleting the extracellular domain of UNC-40 (UNC-40ΔNt) abrogated the association of LON-2/glypican with UNC-40/DCC-expressing cells, indicating that LON-2/glypican may interact with UNC-40/DCC at the cell surface. The association of LON-2/glypican with UNC-40/DCC may be direct or indirect through interactions with other molecules . Our explon-2/glypican functions in the same signaling pathway as the UNC-40/DCC downstream mediator unc-34/enabled during axon guidance.We found that LON-2/glypican leads to increased irregular morphology of UNC-40/DCC-expressing cells. Ectopic expression of DCC in mammalian cells activates downstream signaling via Cdc42 and Rac1, producing cytoskeletal rearrangements that lead to filopodia outgrowth and cell surface extensions . Our finOur results suggest a possible regulatory mechanism in the extracellular space in which secreted LON-2/glypican modulates the activity of the receptor UNC-40/DCC. LON-2/glypican may directly bind UNC-40/DCC, or alternatively, LON-2/glypican may instead interact with other molecules to impact UNC-40/DCC to modulate its stability, distribution, or activity. Alternatively, LON-2/glypican could potentially function as a co-receptor for UNC-6/netrin, where it may facilitate the formation of UNC-6/netrin-UNC-40/DCC-LON-2/glypican signaling complexes, similar to the situation in FGF signaling . It is aIn summary, our studies uncover a novel mechanism by which UNC-6/netrin signaling through its UNC-40/DCC receptor is modulated by the HSPG LON-2/glypican during axon pathfinding. Given the evolutionary conservation of the UNC-6/netrin pathway components (UNC-6/netrin and its receptors UNC-40/DCC and UNC-5/UNC5) and of glypicans  and that synthesis of HS chains is required for mammalian axons to respond to netrin-1 in vitro ,10, glypNematode cultures were maintained at 20°C on NGM plates seeded with OP50 bacteria as described . Strainsgpn-1 cDNA amplification, and primers oCB992 (TCGCTTCAAATCAGTTCAGC) and oCB993 (GCGAGCATTGAACAGTGAAG) were used for the control gene Y45F10D.4 [Total RNA was extracted from worm samples using Trizol (Invitrogen) according to the manufacturer’s instructions. 500 ng RNA was used to reverse transcribe using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and random primers. PCR reactions were carried out with first-strand cDNA template, primers oCB834 (ATCAAGACCGAGTGATAGTG) and oCB1321 (TGGCGAGTATTCCCGTTTAG) were used for 45F10D.4  cDNA ampAnimals were mounted on agarose pads, anaesthetized with 100 mM sodium azide, and examined under a Zeiss Axio Scope.A1 or a Zeiss Axioskop 2 Plus.zdIs5, an integrated Pmec-4::gfp reporter [Axons of neurons AVM and PVM were examined in L4 larvae and adult animals using reporter . AnimalsWorms were counted as mutant for AVM ventral axon guidance if (a) AVM failed to send an axon ventrally and instead projected laterally to the anterior or (b) the AVM axon projected laterally, in the anterior or the posterior direction, for at least ~15 μm (>3 AVM cell body lengths) before projecting to the ventral side. The angle between the anterior/posterior axon projection and the ventral axon projection had to be >45° to be counted as mutant, therefore excluding animals with a slight curve in the ventral axon of AVM from the mutant count.zdIs5 labeled neurons were too misplaced to be identified were excluded from analysis.Worms were counted as having their PVM axon misoriented dorsally if the axon of PVM was projecting to the dorsal side of the animal. The vulva was used as a reference for the ventral side. Worms whose The path of migration of the DTCs brings about the shape of the mature gonad arms  and oCB1124 (ATCATGGGGCCCTAAGCTGAATTCCCATAAC) were used to amplify a PCR product out of N2 genomic DNA containing bases 13,104 of cosmid C39E6 to 26,408 of cosmid F55D10 of the dpy-7 was cut with XmaI and ApaI and ligated with insert of lon-2 cDNA digested out of pCB251 (Pmec-7::lon-2) with XmaI and ApaI.Vector Pdpy-7::lon-2 (pCB268) was digested with HindIII and XmaI to release Pdpy-7 and ligated with insert of Pelt-3 2-kb promoter  amplified out of N2 genomic DNA (modeled after [HindIII and XmaI sites.Vector Ped after ) using pgrd-10::GFP (in pPD95.75 backbone) was digested with KpnI and EcoRI to release GFP and ligated with insert of lon-2 cDNA (yk1346g07) amplified with primers oCB1034 (CATGATGGTACCATGGTCTTCCGGTGGCTCATTC) and oCB1035 (ATCATGGAATTCTCAAAAAAGTTTAATAACTGC) to add on KpnI and EcoRI sites.Vector PXmaI and NheI and ligated with insert of lon-2 cDNA (yk1346g07) amplified by primers oCB963 (CATGATCCCGGGATGGTCTTCCGGTGGCTCATTC) and oCB964 (ATCATGGCTAGCTCAAAAAAGTTTAATAACTGC) to add on XmaI and NheI sites.Vector pPD96.41 was digested with dpy-7::lon-2 (pCB268) was digested with HindIII and XmaI to release Pdpy-7 and ligated with insert of Pelt-2 1-kb promoter (coordinates on cosmid C33D3: 2933–3875) amplified out of N2 genomic DNA (modeled after [HindIII and XmaI sites.Vector Ped after ) using pdpy-7::lon-2) was digested with HindIII and XbaI to release Pdpy-7 and was ligated with insert of Pmyo-3 digested out of vector pPD95.86 with HindIII and XbaI.Vector pCB268 (Pdpy-7::lon-2 (pCB268) was digested with HindIII and XmaI to release Pdpy-7 and ligated with insert of lon-2 3-kb promoter  amplified out of N2 genomic DNA (modeled after [HindIII and XmaI sites.Vector Ped after ) using plon-2::lon-2) was digested with XmaI and EcoRI to release the lon-2 cDNA and ligated with insert of lon-2 cDNA (yk1346g07) amplified with primers oCB963 (CATGATCCCGGGATGGTCTTCCGGTGGCTCATTC) and oCB1074 (ATCATGCTCGAGTCAATCCGGCTGAATTTCTTTTTCC) to add on XmaI and EcoRI sites and generate a truncated form of LON-2 after amino acid 488 to remove the GPI anchor (modeled after [Vector pCB246 (Ped after ).dpy-7::lon-2) was digested with XmaI and EcoRI to release the lon-2 cDNA and was ligated with insert of lon-2 cDNA (yk1346g07) amplified by primers oCB963 (CATGATCCCGGGATGGTCTTCCGGTGGCTCATTC) and oCB1075 (ATCATGGAATTCTCACCTTCCGAGTCGGTCCCACG), to add on XmaI and EcoRI sites and generate a truncated form of LON-2 after amino acid 368, i.e., containing amino acids 1–368 (modeled after [Vector pCB268 (Ped after ).lon-2::lon-2) was digested with XmaI and NheI to release lon-2 cDNA and was ligated with insert of lon-2 cDNA amplified by primers oCB964 (ATCATGGCTAGCTCAAAAAAGTTTAATAACTGC) and (1) oCB1253 (TCCGTCCTACCTGCAGAAGAAGTGAAAATCTGTGATCACTCG), (2) oCB1254 (ATTCTTTTTGTATTGCTCTACCGGTCCGTCCTACCTGCAGAAG), and (3) oCB1255 (CATGATCCCGGGATGGTCTTCCGGTGGCTCATTCTTTTTGTATTGCTCTACC) to add on XmaI and NheI sites, to add on the 22 amino acid lon-2 signal peptide sequence, and to truncate LON-2 to begin at amino acid 369. Thus, this construct codes for 22 amino acid residues of the signal peptide of LON-2, followed by residues 369–508.Vector pCB246 (PXmaI and XhoI and ligated with insert of sdn-1 cDNA (yk139f3) amplified with primers oCB903 (CATGATCCCGGGATGATTCTGAAACTCAATTTC) and oCB904 (ATCATGCTCGAGTTACGCGTAAAATTCTTTTG) to add on XmaI and XhoI sites.Vector pPD96.41 was digested with lon-2::lon-2) was digested with XmaI and EcoRI to release lon-2 and was ligated with an insert of sdn-1 cDNA digested out of pCB242 (Pmec-7::sdn-1) with XmaI and EcoRI.Vector pCB246 (Pttx-3::mCherry (50 ng/μL), Pceh-22::gfp (50–75 ng/μL), and/or Punc-122::rfp (50–75 ng/μL). pBSK+ (90–100 ng/μL) was used to increase total DNA concentration if needed. For details on specific coinjection marker(s) used for each rescued transgenic line, see Transgenic animals were generated by standard microinjection techniques . Each colqIs4), LON-2::GFP (TLG257), and LON-2ΔGAG::GFP (TLG199) worms were collected in buffer and protease inhibitors (Roche). Worm pellets were subjected to repeated freeze-thaw cycles. Protein concentration was measured using the Pierce 660 nm Protein Assay on a Nanodrop. 70 μg of samples mixed with 2x Laemmli sample buffer (Bio-Rad) were boiled, separated by SDS-PAGE on a 4%–20% Mini-Protean TGX gel (Bio-Rad), and transferred to PVDF membrane. Membranes were incubated in 1:3000 anti-GFP primary antibody (Millipore #AB3080) and 1:9000 goat anti-rabbit HRP secondary antibody (Bio-Rad #166-2408EDU). For the loading control, membranes were incubated in 1:5000 anti-HSP90 antibody (CST #4874) and 1:10000 goat anti-rabbit HRP secondary antibody (Bio-Rad #166-2408EDU). Signal was revealed using Clarity Western ECL Substrate (Bio-Rad) and imaged using film (LabScientific).Mixed-stage wild-type (N2), GFP control  was amplified by PCR with primers adding the EcoRI and ApaI restriction enzyme sites and cloned into pBlueScript II (Life Technologies). HA::LON-2 was made by synthesizing a DNA fragment, carrying the restriction sites EcoRI and HincII and containing the 5ʹ end of the lon-2 cDNA with the HA tag added after the signal peptide sequence (at cDNA position 61) and cloned into pBlueScript/lon-2. The HA::LON-2 EcoRI/ApaI fragment was then cloned into the pActin5.1/V5-His vector (Life Technologies).The lon-2 cDNA (bases 739–1527) with the myc tag DNA sequence before the GPI anchor domain sequence (at cDNA position 1375) and an ApaI restriction site at the 3ʹ end. The PmlI/ApaI synthesized fragment was then cloned into the pActin5.1/HA::LON-2 (pCB285) construct cut with the same restriction enzymes.The HA::LON-2::myc construct was made by synthesizing the 3ʹ end of the XhoI/BsmI fragment (bases 803–1340 of the lon-2 cDNA) in the pBlueScript/HA::lon-2 plasmid with synthesized fragment where the lon-2 sequence is mutated at the three predicted heparan sulfate chain attachment sites  modeled after [lon-2, the HA::LON-2ΔGAG EcoRI/ApaI fragment was then cloned into the pActin5.1/V5-His vector (Life Technologies).The LON-2ΔGAG construct was made by replacing the ed after . SimilarBsmI/ApaI fragment (bases 1340–1527 of the lon-2 cDNA) in the pBlueScript/HA::LON-2ΔGAG plasmid with a synthesized fragment containing the myc tag DNA sequence before the GPI anchor domain sequence (at cDNA position 1375) and an ApaI restriction site at the 3ʹ end. The HA::LON-2ΔGAG::myc EcoRI/ApaI fragment was then cloned into the pActin5.1/V5-His vector (Life Technologies).The double tagged HA::LON-2ΔGAG::myc construct was made by replacing the C. elegans unc-6 full-length cDNA (yk603d12) was amplified by PCR with primers adding the EcoRI and ApaI restriction enzyme sites and cloned into pBlueScript II. The superfolder-GFP::UNC-6 construct was made by synthesizing a DNA fragment, carrying the restriction sites EcoRI and HincII, and containing the 5ʹ end of the unc-6 cDNA with the superfolder-GFP sequence after the signal peptide sequence (cDNA position 70) and cloned into pBlueScript/unc-6. The superfolder-GFP::UNC-6 EcoRI/ApaI fragment was then cloned into the pActin5.1/V5-His vector.The C. elegans full length unc-40 cDNA (yk449d8) was amplified by two sequential PCR reactions that added the XhoI and ApaI restriction enzyme sites, as well as the FLAG tag at the 3ʹ end before the stop codon (adding the FLAG tag at the C-terminus of UNC-40 after the intracellular domain). This UNC-40::FLAG cDNA was then cloned into the pActin5.1/V5-His vector.The unc-40 intracellular fragment (cDNA bases 3022–4245 that include the coding sequence for the transmembrane and intracellular domains of UNC-40), along with the FLAG tag, was amplified by PCR using the UNC-40::FLAG cDNA as template and primers carrying the EcoRI and ApaI sites. A start codon (ATG) was added to the forward primer. The UNC-40ΔNt::FLAG fragment was then cloned into the pActin5.1/V5-His vector.The qyIs155, which contains a deletion of fibronectin III domains 4 and 5 of the unc-40 cDNA , was amplified by two sequential PCR reactions adding the EcoRI and ApaI restriction sites and the myc tag sequence at the 3ʹ end before the stop codon. The sdn-1::myc EcoRI/ApaI fragment was then cloned into the pActin5.1/V5-His vector (Life Technologies).The The pAc/Evi::EGFP  and pAc/S2 cells were transfected with HA::LON-2::myc (pCB313) and HA::LON-2ΔGAG::myc (pCB330) constructs. Cells were washed once with 1X Phosphate Buffered Saline and lysed for 30 min at 4°C in 1X Phosphate Buffered Saline, 0.5% Triton X-100, and 1X Protease Inhibitor Cocktail (Roche). Samples of supernatant and cell lysates were each mixed with 2X Laemmli sample buffer (BioRad). Proteins were separated by SDS-PAGE and transferred to PVDF membrane. Membranes were incubated with rabbit anti-HA (Life Technologies #715500) and rabbit anti-myc (Santa Cruz #sc-789) primary antibodies and HRP-linked goat anti-rabbit (Bio-Rad #166-2408EDU) secondary antibody. Signals were revealed by chemiluminescence with Clarity Western ECL Substrate (BioRad) and imaged using the ChemiDoc System (BioRad).S2 cells were independently transfected with HA::LON-2::myc (pCB313), UNC-40::FLAG (pCB301), or SfGFP::UNC-6 (pCB292) constructs. 48 h after transfection, old culture medium was removed, and new medium was added to resuspend the cells. Equal volumes of resuspended cells that had been transfected with individual constructs were mixed and cocultured overnight. Cells were harvested, centrifuged, and combined with their corresponding supernatant from each of these cell mixes. 100 μL of supernatant of each mixture was saved and kept on ice. Cell pellets were washed once with 1X Phosphate Buffered Saline and lysed for 30 min at 4°C in 100 μL of ice-cold RIPA buffer  supplemented with Protease Inhibitor Cocktail (Roche) and PMSF. Cell lysates were combined with their corresponding supernatant and mixed with 2X Laemmli sample buffer (BioRad). Each sample was split into three in order to run three protein gels in parallel. Proteins were separated by SDS-PAGE and transferred to PVDF membrane. Membranes were incubated with rabbit anti-myc (Santa Cruz #sc-789), mouse anti-FLAG (Sigma #F3165), and rabbit anti-GFP (Millipore AB3080) primary antibodies as well as HRP-linked goat anti-rabbit (Bio-Rad #166-2408EDU) and HRP-linked horse anti-mouse (Vector Labs PI-2000) secondary antibodies. Signals were revealed by chemiluminescence with Clarity Western ECL Substrate (BioRad) and imaged using the ChemiDoc System (BioRad).S2 cells were maintained in SFX Insect Media (HyClone) containing 10% Fetal Bovine Serum (HyClone) and Penicillin-Streptomycin (50 units-50 μg/mL) (Sigma). 70%–90% confluent S2 cells were transfected with 500 ng of each construct using Effectene (Qiagen) according to the manufacturer’s protocol. 48 h after transfection, old culture medium was removed and new medium was added to resuspend the cells. Equal volumes of resuspended cells that had been transfected with individual constructs were plated onto coverslips and cocultured overnight. Cells were then fixed with 4% paraformaldehyde and immunostained with rabbit anti-HA (Life Technologies #715500) and mouse anti-FLAG (Sigma #F3165) primary antibodies and Alexa594 donkey anti-rabbit (Life Technologies #R37119) and Alexa647 goat anti-mouse (Life Technologies #A21235) secondary antibodies. Confocal analysis was performed on a Zeiss LSM 5 Pascal confocal microscope. Confocal images were processed using ImageJ. Each experiment was repeated at least three times.For the experiment in which we use HA::LON-2-conditioned medium (supernatant) of cells expressing HA::LON-2, the culture medium was also changed 48 h after transfection, fresh medium was added, and the cells were incubated for another 48 h. This medium was collected and centrifuged at 1,500 rpm to remove cells and debris. This supernatant was added onto cells expressing UNC-40::FLAG, incubated overnight, and as above, fixed, stained, and imaged.Independent populations of S2 cells were transfected with (1) 450 ng of pActin5.1::mCherry alone, (2) 50 ng of the UNC-40::FLAG construct plus 450 ng of the cotransfection marker pActin5.1::mCherry, or (3) 500 ng of HA::LON-2. The medium was changed and cells were mixed 48 h after transfection. Control mCherry-expressing cells were mixed with untransfected cells or with HA::LON-2-expressing cells. Similarly, UNC-40::FLAG/mCherry-expressing cells were mixed with untransfected cells or with HA::LON-2-expressing cells. To maintain the total number of cells constant in our different mixes, one volume of UNC-40::FLAG/mCherry cells was mixed with either (a) one volume of control/untransfected cells or (b) one volume of LON-2-transfected cells. Cell mixes were cocultured overnight. Cells were then fixed with 4% paraformaldehyde and examined under a Zeiss LSM 5 Pascal confocal microscope. Control mCherry-expressing cells or UNC-40::FLAG/mCherry-expressing cells were identified by the cotransfection marker mCherry. 20 fields of ~300 cells each per mix per were photographed for each of three independent experiments. Cells were categorized as having the typical S2 cell round and smooth shape, irregular edges, and/or extensions protruding from the cell.S1 Data(XLSX)Click here for additional data file.S2 Data(XLSX)Click here for additional data file.S1 Figunc-6/netrin and slt-1/slit pathways result in partially redundant defects, as previously established by the Culotti and Bargmann labs [p ≤ 0.001 . ns, not significant.Mutations in ann labs . Mutants(TIF)Click here for additional data file.S2 Figgpn-1(ok377) and gpn-1(tm595) are deletions (brackets) in the gpn-1 locus. (B) RT-PCR using primers oCB834 and oCB1321 yields truncated products in gpn-1(ok377) and gpn-1(tm595). Y45F10D.4 is a housekeeping gene used as an RT-PCR control [gpn-1 RT-PCR products for ok377 (blue) reveals that the transcript lacks most of exon 3 and has several in-frame stop codons (*), as intronic sequence (hatch pattern) gets incorporated into the mature transcript. Sequencing of the gpn-1 RT-PCR product for tm595 (red) reveals that the transcript lacks exons 2 and 3 and has several in-frame stop codons (*), as intronic sequence (hatch pattern) gets incorporated into the mature transcript. No alternatively spliced products were detected in the mutants ok377 and tm595. (C) gpn-1(ok377) and gpn-1(tm595) are strong loss-of-function mutations, likely nulls, in which, at most, small truncated proteins would get produced.(A)  control . Sequenc(TIF)Click here for additional data file.S3 Figsqv-5, the gene coding for the chondroitin sulfate polymerase [lon-2 function. Error bars are standard error of the proportion. Asterisks denote significance: *** p ≤ 0.001 . Click here for additional data file.S4 Figsdn-1::gfp (opIs171), we found that SDN-1::GFP is expressed in hypodermal cells and neurons, as previously reported [sdn-1/syndecan expression in AVM (Pmec-7::sdn-1) rescues the defects of sdn-1 mutants Click here for additional data file.S5 Figlon-2 slt-1 double mutants exhibit enhanced AVM guidance defects as compared to slt-1 single mutants. The defects of the double mutants can be rescued back down to slt-1 single mutant levels with expression of wild-type Plon-2::lon-2(+). In contrast, expression of sdn-1(+) in which lon-2/glypican is normally expressed (using the Plon-2 promoter) or in the AVM neuron (using the heterologous promoter Pmec-7) cannot rescue the axon guidance defects of lon-2 slt-1 double mutants. Data for wild type, lon-2, slt-1, lon-2 slt-1, and Plon-2::lon-2 in lon-2 slt-1 are as in p ≤ 0.001, * p ≤ 0.05.  (see ons) see  Tables.(TIF)Click here for additional data file.S6 Fig(A) Diagram of LON-2/glypican variants expressed in S2 cells, HA::LON-2::myc and HA::LON-2ΔGAG::myc, in which the three HS attachment sites were mutated from Serine to Alanine residues. The core protein of LON-2/glypican is red, and the heparan sulfate chains (HS) are yellow. (B) In the supernatant of cells expressing HA::LON-2::myc, high molecular weight species were detected with the anti-myc antibody, which likely corresponds to full-length HA::LON-2::myc with HS chains attached. In contrast, the species detected in the supernatant of HA::LON-2ΔGAG::myc-expressing cells are smaller and fainter, indicating that HA::LON-2ΔGAG::myc indeed affects the synthesis of HS chains onto the LON-2/glypican core protein. No signal was detected with the anti-HA antibody in the supernatants, likely due to technical limitations. In the cell extracts from HA::LON-2::myc-expressing cells, the main species runs at ~90 kDa, and it is detected with both the anti-myc and anti-HA antibodies, suggesting that it is full length. This signal likely corresponds to the LON-2/glypican core protein devoid of HS chains, as it runs as a tight band. The slight mobility shift in cell extracts of HA::LON-2ΔGAG::myc-expressing cells compared to HA::LON-2::myc might correspond to a difference of mass and isoelectric point between HA::LON-2::myc and HA::LON-2ΔGAG::myc. Anti-actin and anti-HSP90 antibodies were used as loading controls. Representative blots of more than four independent repeats.(TIF)Click here for additional data file.S7 Fig(A) Diagram of constructs used to express these proteins in S2 cells, showing the tags used to detect them. (B) Western blots for detection of HA::LON-2::myc, UNC-40::FLAG, and SfGFP::UNC-6. Constructs were individually and transiently transfected in S2 cells. Two d later, cells from single transfections were mixed and incubated overnight. Cells were harvested and combined with their corresponding supernatant from each of these cell mixes. Samples of each cell mix were split into three in order to run three parallel western blots and detect the proteins. As shown in (TIF)Click here for additional data file.S8 FigIn order to distinguish HA::LON-2-producing cells from HA::LON-2-acquiring cells, two separate populations of cells were transfected. One population of S2 cells was transfected with UNC-40::FLAG. A second population of S2 cells was simultaneously transfected with both GFP and HA::LON-2. Two d later, the two populations of cells were mixed, incubated overnight, and immunostained with anti-HA and anti-FLAG antibodies, as described for (TIF)Click here for additional data file.S9 FigDrosophila type I transmembrane receptor Evi. Evi-expressing cells were mixed with cells expressing LON-2/glypican and/or UNC-40/DCC. As shown in experiment 2 and in p ≤ 0.001 (t-test versus untransfected cells). ns, not significant. (C) Quantification of the association of SDN-1::myc with cells expressing UNC-40::FLAG, HA::LON-2, and untransfected cells. Ten different optical fields containing ~300 cells from three independent experiments were quantified.(A) Experiments 1 and 2 show that HA::LON-2 does not associate with cells expressing the unrelated (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.S9 Table(DOCX)Click here for additional data file.


End Stage Renal Disease (ESRD) is influenced by genetic and epigenetic factors. Few studies have been reported earlier depicting the role of Matrix Metalloproteinase in different populations for ESRD risk, but we have reported few novel and functional genes in North Indian population. The prime objective of the present study was to find out the association of four variants viz. MMP1-519(A/G) and MMP1-1607(1G/2G) and MMP3+5356(A/G) and MMP3 -1161(A/G) with ESRD risk.4 SNPs were genotyped by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) in 200 ESRD patients and 200 healthy controls, that were age and gender matched. To evaluate the SNP effects on ESRD, odds ratio (OR) and confidence interval (CI) 95% were calculated by SPSSver16.0. Haplotype analysis was done for MMP1 by SNPanalyzer1.2 ver.In case of MMP1-519(A/G) the variant genotype GG showed significant reduced risk of ESRD . On combining the heterozygous and variant genotypes marginal reduced risk was observed , although we found significance reduced risk of ESRD  in G allele. In case of MMP1-1607(1G/2G) the variant genotype 2G2G showed significant reduced risk of ESRD  whereas the combination of heterozygous and variant genotypes reduced risk was obtained . At allelic level, 2G allele showed reduced risk  of ESRD. Gene-gene interaction was also performed for above gene variants. We found a significant reduced risk in combination GG+AA  on interacting MMP1-519(A/G) and MMP3-1161(A/G). We also found reduced risk for ESRD for MMP1-519(A/G) and MMP1-1607(1G/2G) while looking risk for ESRD at haplotype level. Bioinformatics analysis (in-silico) showed no association with ESRD.Our results indicated that polymorphism in MMP1-519(A/G) and MMP1-1607(1G/2G) showed reduced risk for ESRD in North-Indian population. More relevant studies with large sample size and diverse ethnicity are required to validate these observations.


A significant proportion of recurrent respiratory papillomatosis (RRP) is caused by human papillomavirus type 6 (HPV-6). The long control region (LCR) contains cis-elements for regulation of transcription. Our aim was to characterize LCR HPV-6 variants in RRP cases, compare promoter activity of these isolates and search for cellular transcription factors (TFs) that could explain the differences observed. The complete LCR from 13 RRP was analyzed. Transcriptional activity of 5 variants was compared using luciferase assays. Differences in putative TFs binding sites among variants were revealed using the TRANSFAC database. Chromatin immunoprecipation (CHIP) and luciferase assays were used to evaluate TF binding and impact upon transcription, respectively. Juvenile-onset RRP cases harbored exclusively HPV-6vc related variants, whereas among adult-onset cases HPV-6a variants were more prevalent. The HPV-6vc reference was more transcriptionally active than the HPV-6a reference. Active FOXA1, ELF1 and GATA1 binding sites overlap variable nucleotide positions among isolates and influenced LCR activity. Furthermore, our results support a crucial role for ELF1 on transcriptional downregulation. We identified TFs implicated in the regulation of HPV-6 early gene expression. Many of these factors are mutated in cancer or are putative cancer biomarkers, and must be further studied. Recurrent Respiratory Papillomatosis (RRP) is characterized by the proliferation of multiple papillomas within the respiratory tract affecting especially the larynx ,2. RRP cL1 gene [The prototype HPV-6b clone was originally isolated from a condyloma acuminatum specimen . BecauseL1 gene . RecentlL1 gene . While tL1 gene .The viral long control region (LCR) comprises approximately 10% of the viral genome and encloses cis-regulatory elements for cellular and viral transcription factors (TFs) that modulate early gene expression and replication . The repHPVs 16 and 18 intratype variability has been epidemiologically correlated to viral persistence and development of clinically relevant lesions –18. ConcWe analyzed 23 laryngeal papillomas biopsy specimens from 13 patients diagnosed with RRP from 2005 to 2010, and treated at the Laryngology Clinic of the School of Medicine, University of São Paulo, Ribeirão Preto, Brazil. Disease severity was accessed using the Derkay system at each surgical intervention . This sthttp://trap.molgen.mpg.de/cgi-bin/home.cgi) using the TRANSFAC database [DNA was extracted using the QIAamp DNA Micro kit (Qiagen Inc.). The complete HPV-6 LCR (nt 7292 to 101) was amplified in two independent reactions. Amplicons were cloned using the pCR XL-TOPO Vector (Invitrogen), and clones were purified using the GeneJET Plasmid Miniprep Kit (Fermentas Life Sciences). Sequencing reactions were performed in an ABI 3130XL sequencer (Applied Biosystems) using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). Variants were identified by alignment of sequences with the references of HPV-6b (GenBank n° X00203), HPV-6a (GenBank n° L41216) and HPV-6vc (GenBank n° AF092932) using CLUSTALW [database .E6/E7 or pLXSN-HPV-16-E6/E7 were maintained in keratinocyte serum free medium (KSFM), supplemented with 5ng/mL epidermal growth factor and 50μg/mL bovine pituitary extract (Invitrogen).C33A (ATCC HTB-31) and C33A cells permanently transfected with pGL3-HPV-6-LCR were grown in Dulbecco’s Modified Eagle medium supplemented with 10% fetal calf serum and antibiotics. Primary human foreskin keratinocytes (PHFK) (Clonetics), and PHFK infected with pLXSN-HPV-6b-6 C33A cells were plated in 10cm diameter dishes. Cells were co-transfected using Lipofectamine (Invitrogen) with 4μg of pGL3-LCR-Luc plasmids and 1μg of pCMV-βGal vector for internal control of transfection efficiency [The complete LCR from the different HPV-6 variants was cloned upstream the luciferase gene in the pGL3-Basic vector (Promega). Accurate construction of all plasmids was confirmed by sequencing. Approximately 24 hours before transfection, 4x10ficiency . ExtractC33A cells were co-transfected in 96-well plates using Fugene (Promega) with 100ng of pGL3-LCR-Luc plasmids of different variants of HPV-6 together with increasing amounts of selected TFs expression plasmids , and 50ng of pCMV-β-Gal vector. Cells were harvested 48 hours after transfection by addition of 100μL of 1X Reporter Lysis Buffer (Promega). Relative luciferase activity was normalized to β-galactosidase measurements. Transcriptional activity was compared to basal activity of the same variant with no TFs superexpressed. Averages and standard deviations are based on the mean of triplicates of at least twelve independent experiments.Fisher´s exact test was used to compare HPV-6 variant distribution between JORRP and AORRP cases. We used T-test to compare Derkay mean values with 95% confidence interval (CI) between HPV-6a and -6vc-related RRP cases. These analyses were conducted using the GraphPad Prism statistical package (version 5.01). Kruskal-Wallis and Tukey’s tests were employed to compare LCR activity among HPV-6 variants. For TFs validation assays, one-way analysis of variance and Tukey’s multiple comparison tests were performed using the SPSS statistical package version 20.0. P< 0.05 was considered significant for all tests.5 cells were incubated with 20μg of anti-GATA1 (Abcam), anti-ELF1 (Santa Cruz Biotechnology), or anti-FOXA1 (Santa Cruz Biotechnology) overnight at 4°C with moderate agitation. Recovered DNA was subjected to PCR using specific primers surrounding putative TFs binding sites.ChIP assays were perfomed using the MAGnify Chromatin Immunoprecipitation System (Invitrogen). Briefly, C33A cells permanently transfected with pGL3-HPV-6-LCR from different variants were cross-linked with methanol-free formaldehyde (Polyscience) and further sonicated using a Sonic Dismembrator Model 100 (Fisher Scientific). Production of DNA fragments of 100-500bp was checked by gel electrophoresis in agarose 2%. Approximately 1x10Cells were washed with ice-cold phosphate-buffered saline (PBS), scraped, and centrifuged. Radio immunoprecipitation assay (RIPA) buffer  containing complete protease inhibitor cocktail (Roche) was used for protein extraction. Eighty micrograms of each lysate were submitted to 12% SDS polyacrylamide gel electrophoresis, and further transferred to nylon membranes (Hybond). Incubations with anti-GATA1 (1:500), anti-ELF1 (1:200), anti-FOXA1 (1:500), or anti-tubulin (1:1.000) (Sigma) were conducted overnight in PBS-T  at 4°C with 5% milk. We followed incubation with anti-HRP-conjugated antirabbit secondary antibody (1:5.000)  for 1 hour at room temperature. Proteins were visualized using the Amersham ECL Western Blotting Detection Reagents  in a ImageQuant LAS4.000 equipment , and quantified using the ImageQuant TL software .We sequenced the LCR of 23 HPV-6 RRP specimens obtained from 13 patients. Individuals with more than one biopsy harbored identical HPV genomes. We detected 7 different HPV-6 variants .Maximum genomic distance was 7 mutations between HPV-6a-ref and -6vc-ref (2 insertions and 5 substitutions). HPV-6a-var1, -6a-var2, -6vc-var1, -6vc-var2 and -6vc-var3 have unique substitutions at nucleotide positions 16 (G→A), 7320 (A→G), 7626 (T→G), 7978 (C→T) and 7681 (A→G), respectively. We detected HPV-6a-related sequences  in 32.5% (4/13) patients. Additionally, 67.5% (9/13) individuals contained HPV-6vc-related sequences , of which HPV-6vc-ref was the most prevalent.Age at RRP diagnosis ranged from newborn to 51 years old. All JORRP cases harbored HPV-6vc-related variants, whereas most AORRP specimens had HPV-6a-related variants  (p = 0.02) . AverageLuciferase assays were used to access the impact of LCR nucleotide sequence heterogeneity on early viral transcription. Unfortunately, we were unable to construct a recombinant plasmid containing the HPV-6a-var2 LCR. We observed an 11.5 fold enhanced promoter activity for HPV-6vc-ref variant as compared to HPV-6a-ref. A transition at nucleotide position 16 detected solely in HPV-6a-var1 led to an increase in transcriptional activity similar to that of the HPV-6vc-ref, whereas the transversion at position 7630 inherent of the HPV-6vc-var1 abolished the increased activity observed for HPV-6-vc-ref. HPV-6vc-var2 and -6vc-var3 showed promoter activities similar to HPV-6vc-ref Search for putative TFs binding sites was restricted to nucleotide positions divergent among HPV-6a-ref, -6a-var1, -6vc-ref, -6vc-var1 because these isolates showed differences in transcription. This approach revealed that nucleotide heterogeneity of HPV-6 variants may impact on binding of different TFs including FOXA1, GATA1 and ELF1 .The substitution in nucleotide position 16 (G→A) detected in HPV-6a-var1 leads to the elimination of putative binding sites for GATA1 and ELF1 as compared to the HPV-6a-ref . We obseDivergence in nucleotide position 7626 (T→G) between HPV-6vc-ref and HPV-6vc-var1 results in the replacement of a putative ELF1 for a FOXA1 binding site as suggested by computational analysis . ELF1 inE6/E7 from both HPV-16 and HPV-6b  ELF1, (B) GATA1 and (C) ELF1 levels in primary human foreskin keratinocytes (PHFK), and PHFK infected with pLXSN-HPV-6b-E6/E7 or pLXSN-HPV-16-E6/E7. One representative experiment from two is shown.(TIFF)Click here for additional data file.