Waf1/Cip1 and PUMA in human cervical cancer cell lines. Interestingly, TSC-22 did not affect the interaction between p53 and HDM2. Knock-down of TSC-22 by small interfering RNA clearly enhanced the poly-ubiquitination of p53, leading to the degradation of p53. These results suggest that TSC-22 acts as a tumor suppressor by safeguarding p53 from poly-ubiquitination mediated-degradation.The p53 tumor suppressor function can be compromised in many tumors by the cellular antagonist HDM2 and human papillomavirus oncogene E6 that induce p53 degradation. Restoration of p53 activity has strong therapeutic potential. Here, we identified TSC-22 as a novel p53-interacting protein and show its novel function as a positive regulator of p53. We found that TSC-22 level was significantly down-regulated in cervical cancer tissues. Moreover, over-expression of TSC-22 was sufficient to inhibit cell proliferation, promote cellular apoptosis in cervical cancer cells and suppress growth of xenograft tumors in mice. Expression of also TSC-22 enhanced the protein level of p53 by protecting it from poly-ubiquitination. When bound to the motif between amino acids 100 and 200 of p53, TSC-22 inhibited the HDM2- and E6-mediated p53 poly-ubiquitination and degradation. Consequently, ectopic over-expression of TSC-22 activated the function of p53, followed by increased expression of p21 TSC-22 expression is induced in a variety of cell lines by TGF-β, phorbol ester, serum, and progestin and positively regulates the TGF-β signaling TSC-22 homologous gene-1 (THG-1), and has transcriptional repressor activity TGF-Xenopus laevisDrosophilaβ-dependent signaling Some researchers have identified the physiological roles of TSC-22 in the developmental process. TSC-22 is required for gastrulation during early embryogenesis in Gadd45b and Lzts2Additionally, several studies have focused on the tumor suppression functions of TSC22. TSC22 is thought to be a potent tumor suppressor in salivary cancer cells p53 is a well-known tumor suppressor gene that acts by activating the transcription of its targeted genes such as p21, PUMA, , PKR and BAX Since the mechanism underlying the network of tumor suppressor genes has not yet been explicitly elucidated, we conducted a cDNA microarray analysis of the gene expression profile in cancer tissue to find novel tumor-related genes. We found that TSC-22 expression was significantly decreased in cervical cancer tissues compared to normal tissues. Subsequently, we explored the novel function of TSC-22 during tumorigenesis. We therefore performed a yeast two-hybrid assay to screen for novel TSC-22-binding proteins. From this, p53 was identified as a TSC-22-binding protein. We also found that TSC-22 could enhance the activities of p53 through the inhibition of HDM2 and E6-mediated ubiquitination by directly binding to p53. On the other hand, TSC-22 over-expression stabilized p53 protein level, leading to increased cell death and inhibition of cell proliferation. Finally, tumor growth rate was strongly reduced by the expression of TSC-22 in a xenograft tumor model. Taken together, these results indicate that TSC-22 plays a crucial role in the inhibition of tumor growth through the regulation of p53 ubiquitination.TSC-22 gene expression was noticeably decreased in all cancer samples (data not shown). We then conducted real-time PCR (RT-PCR) analysis to confirm the microarray results. As shown in TSC-22 mRNA expression levels in the patients’ cancer tissues were significantly reduced compared to those in normal tissue.In order to analyze the specific gene expression profile of cervical cancer, we conducted cDNA microarray analysis with cDNA prepared from patients’ cervical cancer tissues. Interestingly, our microarray data showed that HeLa (HPV-18) and Caski (HPV-16) cells with adenovirus expressing TSC-22 or LacZ (infection control). As shown in TSC-22. We next examined whether TSC-22 could induce cell cycle arrest and apoptosis in HeLa cells. We found that the G0/G1 population was dramatically increased among Ad-TSC-22-infected cells compared to that in Ad-LacZ-infected cells within 48 h after infection. Furthermore, most Ad-TSC-22-infected cells underwent apoptosis in the late stage  and PUMA (apoptosis inducer) expression levels were markedly enhanced in Ad-TSC-22 infected HeLa and Caski cell (HeLa cell. p53 and TSC22 level in HeLa cells were barely detected compare(d) to caski cells -driven promoter activity with TSC-22 expression and knock-down of TSC-22 in HeLa cells. A promoter harboring a p53RE was activated by TSC-22 expression in a dose-dependent manner. On the other hand, the promoter activity was decreased by TSC-22 knock-down. These data suggest that p53-mediated transcriptional activity is regulated by TSC-22  were monitored for tumor growth. Tumor growth and morphology were analyzed over 30 days. LacZ or that were untreated. We next investigated the effect of TSC-22 on p53 stabilization in tumor tissues harvested from control and TSC-22 treated mice. TSC-22 transfection was observed to significantly increase the expression level of p53 protein  fetal bovine serum (FBS), TSC22 knock-down HeLa cell was established by previous established method shRNA-expressing recombinant plasmid (sh-TSC) was constructed by manipulation of the pSilencer3.1 vector (Ambion) with the ds-oligonucleotide prepared by annealing the following synthetic oligonucleotides: forward; GATCCGTGGATCTAGGAGTTTACCATTCAAGAGA TGGTAAACTCCTAGATCCATCTTTTTTGGAAA-3′ and reverse; 5′-AGCTTTTCCAA AAAAGATGGATCTAGGAGTTTACCATCTCTTGAATGGTAAACTCCTAGATCCACG-3′. After transfection of recombinant plasmid into HeLa cell, TSC22 knock-down HeLa cells were isolated by culture supplemented with 1 ug/ml of puromycin. DNA transfections were carried out using lipofectamine2000 (invitrogene), or Fugene HD (Roche), each according to the manufacturer’s instructions.Immune-deficient BALB/c nude mice were purchased from Orient Bio . Human 5′-GTAGACCAGTGGCGATGGAT and 3′-GCTACCACACTTGCACCAGA.Total RNA were obtained by extracting tissues in TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. First strand cDNA was prepared from total RNA and oligo dT using I Script cDNA synthesis kit . Real-time PCR was conducted with a Mini Opticon System (Bio-Rad) and SYBR Green (Bio-Rad). Specific primers used for Real-time PCR were R RT-PCR kit . Specific primers used for quantitative RT-PCR were: human TSC22; forward 5′-GCTGCCGTTTTCTGTTTCTC-3′ reverse 5′-ATCCATCGCCACTGGTCTAC-3′, human p53; forward AAGTCCAAAAAGGGTCAG TCT reverse CCCAAACATCCCTCACAGTAA, human PUMA; forward 5′-GGAGGGTCCTGTACAATCTC-3′ reverse 5′-GCTACATGGTGCAGAGAAAG-3′: HPV-18 E6; forward 5′-ATGCTGCATGCCATAAATGT-3′ reverse 5′-TGCCCAGCTATGTTGTGAAA-3′, HPV-16 E6; forward 5′-TTGCTTTTCGGGATTTATGC-3′ reverse 5′-TCAGGACACAGT GGCTTTTG-3′, GAPDH; forward 5′- GACATCAAGAAGGTGGTGAA-3′, reverse 5′-TGTCATACCAGGAAATGAGC - 3′. Real-Time quantitative RT-PCR data was normalized to GAPDH mRNA, and analyzed by fold induction in comparison with a standard control for each experiment, and are represented as mean ± SEM of three independent experiments. Semi-quantitative RT-PCR data was shown by electrophoresis in agarose gel.Semi-quantitative RT-PCR was performed with ONE-STEPAll animal studies were approved by the Animal Care and Use Committee of Samsung Medical Center.Flag-TSC22 plasmid was co-transfected with 200 ng of p53RE-conjugated luciferase reporter plasmid  together with control pCMV-lacZ plasmid into the HeLa cells for 48 h. The luciferase activity was measured and normalized  using Bright-Glo Luciferase Assay system (Promega) and the Genios Luminometer . Data are represented by error bar ± SEM.Luciferase assay were performed as described previously with minor modification 5′-GGGTGTTTTTGGCTGCAAT and 3′-TTCAGTTCACACGCAGCAG. The cloning product was confirmed for sequence from both directions. The pCR-TOPO-TSC22 was digested with EcoRI and cloned into the pΔACMVp(A) EcoRI site. The pΔACMV-TSC22 and adenovirus backbone vector, pJM17 were cotransfected into a packaging cell line, 293 using Fugene HD transfection reagent (Boehringer Mannheim). A replication competent virus (RCV) negative clone was propagated in 293 cells and purified through two rounds of CsCl density gradient centrifugation. The Ad-TSC22 construct was used at 100 M.O.I. for all transfection experiments. AdCMV-lacZ was used as an internal control. Full and partial length cDNAs of human TSC22 and p53 were introduced into pcDNA3-Flag or pcDNA3 (Invitrogen), respectively. Human papillomavirus E6 (type 16) were cloned into pcDNA3-HA vector. pcDNA3-HDM2 vector and HA-Ubiquitin expression plasmids were pleasant gifted from J.W.Song (SKKU). DNA transfections were carried out using lipofectamine2000 (Invitrogen), or Fugene HD (Roche), each according to the manufacturer’s instructions.PCR-amplified, full-length human TSC22 fragment (500 bp) was cloned into pCRII-TOPO vector. The primers used to generate the full-length were 5 cells/well in 6-well plates. Twenty-four hours later, the cells were infected with Ad-TSC22 or Ad-LacZ. Beginning 24 h after infection, cells were counted each day by MTT assay for up to 3 days.Cells were plated in triplicate at a density of 2×10Flow cytometry analysis was carried out as described previously HeLa and Caski cells were plated onto six-chamber slides and infected with the indicated Ad-LacZ or Ad-TSC22. For the observation of nuclear apoptotic body, the nuclei were fixed in methanol and stained with 40, 60-diamidino-2-phenylindole  for 15 min and rinsed twice with PBS, then examined with the fluorescence microscope.Samples were separated on 10% polyacrylamide-SDS gels. The gel was transferred overnight to nitrocellulose membrane and blocked in TBS-Tween-20 (0.1% v/v) with dry skimmed milk (5% w/v) (PBSTM) for 1 h at RT. And then western blotting was carried out by using respective antibody and ECL (Amersham). Antibodies against the following proteins were purchased; p53 mAb (DO-1), p53 pAb (FL393), HDM2 pAb, HA pAb (H9), HRP-conjugated Myc Ab, mouse IgG and rabbit IgG were obtained from Santa Cruz Biotechnology. p21waf/cip mAb (DCS60), PUMA pAb, was purchased from Cell signaling technology. Flag (or HRP conjugated) mAb (M2) and β-actin mAb were purchased from Sigma–Aldrich.In vivo ubiqutination assay were performed as described H1299 cells were transfected with plasmids as described in the legend to figure. The cells were treated with 20 µM of MG132 for 5 h and then harvested at 48 h post-transfection. Ubiquitinated proteins were immunoprecipitated by HA poly clonal antibody. And then ubiquitination of p53 was detected by western blotting by using p53 Antibody (DO-1).420−1.75×A550)]/(time×volume×A600).LexA-human TSC22 fusion protein was constructed and used to screen binding proteins from a human ovary cDNA library . The binding proteins were expressed as pB42 fusion proteins. cDNA encoding full length human p53 were PCR amplified and cloned separately into the EcoRI/XhoI sites of the B42. Experiment of yeast two hybrid assay was previously described in vivo, H1299 cell were transiently transfected with expression plasmids of Flag-tagged TSC22 and Flag-tagged (or non-tagged) p53 or HDM2 if needed using lipofectamin 2000 reagent (invitrogen). The p53 was immunoprecipitated with anti-p53 antibody (DO-1 or FL393) and coimmunoprecipitated TSC22 or HDM2 were detected by Western blot with anti-Flag antibody (for TSC22) and HDM2 antibody respectively. For semi endogenous coimmunoprecipitation with p53 and TSC22, HEK293 cells were transiently transfected with Flag-tagged TSC22 using lipofectamin 2000 reagent. Immunoprecipitation of p53 and Flag-TSC22 were carried out with anti-p53 (DO-1 or FL393) and Flag antibody, respectively. The co-immunoprecipitated Flag-TSC22 and p53 was detected by Western hybridization.To perform co-immunoprecipitation 6HeLa cells were injected s.c. in the mid-dorsal region of nude mouse. An intra-tumoral injection of 1×109 pfu/40uL of Ad-TSC22 was done thrice. Tumor size was evaluated by caliper measurements every 3 days. Mice were sacrificed on day 27 after final virus injection. Tumors were then excised.To establish tumors in mice, 1×10Figure S1Quantification analysis of p53, E6 and TSC-22 genes. The mRNA expression of p53, E6 and TSC-22 in cervical cancer cell lines (A) and patients’ cancer specimens (B) was performed by quantitative RT- PCR by using GAPDH protein as reference gene.(TIF)Click here for additional data file.


One of Stat3 downstream genes products, chitinase 3-like 1 (CHI3L1) protein, showed increased concentration in both bronchioalveolar lavage fluid (BALF) and blood of doxycycline-treated CCSP-rtTA/(tetO)7-CMV-Stat3C bitransgenic mice. When tested in other inflammation-induced lung cancer mouse models, the CHI3L1 protein concentration was also highly increased in BALF and blood of these models with tumors. Immunohistochemical staining showed strong staining of CHI3L1 protein around tumor areas in these mouse models. Analysis of normal objects and lung cancer patients revealed a significant elevation of CHI3L1 protein concentration in human serum samples from all categories of lung cancers. Furthermore, recombinant CHI3L protein stimulated proliferation and growth of Lewis lung cancer cells. Therefore, secretory CHI3L1 plays an important role in inflammation-induced lung cancer formation and potentially serve as a biomarker for lung cancer prediction. Based on our previous publication and this work, this is the first animal study linking overexpression of CHI3L1 to various lung tumor mouse models. These models will facilitate identification of additional biomarkers to predict and verify lung cancer under various pathogenic conditions, which normally cannot be done in humans.Over-activation of the signal transducers and activators of the transcription 3 (Stat3) pathway in lung alveolar type II (AT II) epithelial cells induces chronic inflammation and adenocarcinoma in the lung of CCSP-rtTA/(tetO) The five year survival rate is only about 15%. There is an urgent need for identifying biomarkers that can be used to predict lung cancer occurrence. Recently, we demonstrated that both lung epithelial cell-initiated regional inflammation and myeloid cell-initiated systemic inflammation can induce spontaneous lung tumorigenesis in multiple lung tumor animal models 7Stat3C bitransgenic mice as we reported previously Based on these observations, we hypothesize that Stat3 downstream genes serve as potential biomarkers for inflammation-induced lung cancer prediction. Affymetrix GeneChip microarray analysis reveals around 800 Stat3 downstream genes in the lung of CCSP-rtTA/(tetO)All scientific protocols involving the use of animals have been approved by the Institutional Animal Care and Use Committee (IACUC) of Indiana University School of Medicine and followed guidelines established by the Panel on Euthanasia of the American Veterinary Medical Association. Protocols involving the use of recombinant DNA or biohazardous materials have been approved by the Biosafety Committee of Indiana University School of Medicine and followed guidelines established by the National Institutes of Health. Animals were housed under (IACUC)–approved conditions in a secure animal facility at Indiana University School of Medicine. All the cell specific transgenic mice have been previously described 7-CMV-Stat3, CCSP-rtTA/(TetO)7-CMV-MMP12, CCSP-rtTA/(TetO)7-CMV-Api6, c-fms-rtTA/(TetO)7-CMV-Api6 and c-fms-rtTA/(TetO)7-CMV-MMP12 bitransgenic mice were opened after anesthetizing with triple sedative by intraperitoneal (IP) injection. The mouse blood samples were collected from interior vena cava (IVC). Sera were separated by centrifugation at 1,500 rpm for 10 minutes at 4°C. For bronchioalveolar lavage fluid (BALF) collection, the trachea was isolated and cannulated with a 20 gauge luer stub adapter. Using a 1 cc syringe, bronchioalveolar lavage fluid (BALF) was collected by perfusing the lung with 1 ml aliquot of 0.9% sodium chloride and withdrawing back fluids. BAL fluids were centrifuged for 5 minutes at 1,000 rpm and 4°C to remove cell pellets. CHI3L1 concentrations in mouse and human serum (50–100 µl) were determined by Mouse Chitinase 3-like 1 Quantikine ELISA Kit and Human Chitinase 3-like 1 Quantikine ELISA Kits according to the manufacturer’s instruction . The human serum samples of normal objects and lung cancer patients were obtained from the Biosample Repository Core Facility (BRCF) of Fox Chase Cancer Center in Philadelphia (supported by NCI). The human serum samples were diluted to 1∶100 before assay.For mouse serum collection, the abdominal cavities of doxycycline-treated or untreated CCSP-rtTA/(TetO)7-CMV-Stat3 bitransgenic mice were anesthetized and lungs were removed and homogenized in radioimmunoprecipitation assay (RIPA) buffer. Lung lysates (10 µg) were fractionated on a Novex® 4–20% Tris-Glycine Mini Gel . After transferring to the polyvinylidene difluoride membrane , the membrane was blotted with 5% nonfat dry milk PBS and hybridized with rabbit anti-Flag antibody . Following incubation with secondary antibody, the proteins were detected with chemiluminescent substrate . For CHI3L1 protein detection, BALFs from doxycycline-treated or untreated CCSP-rtTA/(TetO)7-CMV-Stat3 bitransgenic mice were collected as described above and diluted with Laemmli sample buffer  at 1∶1, then heated in boiling water for 5 min. Gel electrophoresis and antibody hybridization were performed following the same procedure as above except that rat anti-mouse ChI3L1 primary antibody  and goat anti rat IgG secondary antibody  were used. The protein bands were visualized with a Vectastain Elite ABC kit  following a procedure recommended by the manufacturer.For Stat3C-Flag fusion protein detection, CCSP-rtTA/(TetO)7-CMV-Stat3, CCSP-rtTA/(TetO)7-CMV-MMP12, CCSP-rtTA/(TetO)7-CMV-Api6, c-fms-rtTA/(TetO)7-CMV-Api6 and c-fms-rtTA/(TetO)7-CMV-MMP12 bitransgenic mice were inflated with a fixative solution , dissected out, and stored in fixative at 4°C for 24 hours as previously described For immunohistochemical staining, the lungs from doxycycline-treated or untreated CCSP-rtTA/(TetO)For immunofluorescence staining, sections were hybridized with rat anti-mouse ChI3L1 antibody (R&D System) and rabbit anti-mouse F4/80  antibody as primary antibodies. A FITC-conjugated donkey anti-rat IgG  and a Cy3-conjugated donkey anti-rabbit IgG  at a dilution of 1/200 were used as the secondary antibodies. Sections were co-stained with DAPI. Slides were examined under a Nikon fluorescent microscope.Chi3l1 coding region was amplified by PCR, and subcloned into the pEGX 4T-1 vector  at the NotI/BamHI restriction enzyme sites. CHI3L1 fusion protein was expressed in BL21 E. coli by 50 µM IPTG induction and purified using Glutathione Sepharose 4 Fast Flow kit (GE life science) according to the manufacture’s instruction.The full-length murine 3/well) were seeded in 96-wells plate with culture medium . GST or GST CHI3L1 fusion protein  was added to the culture medium. After 72 hours, cell numbers of proliferation were counted. For apoptotic assay, LLC cells (2x105/well) were seeded in 6-wells plate with culture medium . GST or GST CHI3L1 fusion protein  was added to the culture medium. After 72 hours, the apoptotic cell population was determined by fluorescein isothiocyanate-labeled-Annexin V (FITC-annexinV) . LLC cells treated with GST or GST-CHI3L1 fusion protein were harvested and washed twice with PBS. After resuspension of the cells in Annexin V-binding buffer containing FITC-conjugated Annexin V (1∶20 dilution) and incubation at room temperature for 15 minutes, stained cells were analyzed by flow cytometry as soon as possible (within 1 hour).Lewis Lung Carcinoma (LLC) cells  curve analysis was used to evaluate the diagnostic ability of CHI3L1, which is quantified by the area under the ROC curve (AUC). Analysis was performed using the pROC package in the R language 7-CMV-Stat3C mice is a spontaneous lung tumor animal model. In this model, Stat3C-Flag fusion protein was highly inducible by doxycycline treatment .As we reported previously, CCSP-rtTA/(TetO)reatment , which c7-CMV-Stat3C mice. The average CHI3L1 concentration was 8.3±6.4 ng/ml in wild type mice, 8.8±10 ng/ml in doxycycline-untreated CCSP-rtTA/(TetO)7-CMV-Stat3C mice. In comparison, the average CHI3L1 concentration was increased more than 11-fold  in doxycycline-treated mice without tumor, further supporting that CHI3L1 expression was elevated prior to tumor formation, and was increased more than 17-fold  in doxycycline-treated mice with tumor 7-CMV-Stat3C mouse model, therefore potentially serves as biomarkers.We further tested the CHI3L1 concentration in the blood. When compared with doxycycline-untreated mice (6.2±3.7 ng/ml), the average CHI3L1 concentration was increased more than 5-fold  in doxycycline-treated mice without tumor (prior tumor formation), and was increased more than 26-fold  in doxycycline-treated mice with tumor  compared with that in BALF of doxycycline-untreated mice  is a smoking-induced matrix metalloproteinase. Over-expression of MMP12 in lung alveolar type II epithelial cells induced local inflammation, emphysema and lung tumor 7-CMV-Api6 spontaneous lung tumor mouse model, the average CHI3L1 concentration in BALF of doxycycline-treated mice with tumor was 409.8±49.2 ng/ml, compared with that in BALF of doxycycline-untreated mice  is another pro-inflammatory molecule that induces inflammation and lung tumor when over-expressed in lung alveolar type II epithelial cells 7-CMV-MMP12 spontaneous lung tumor mouse model, the average CHI3L1 concentration in BALF of doxycycline-treated mice with tumor (70.9±36.5 ng/ml) was 4-fold increase in comparison with that in BALF of doxycycline-untreated mice  was ∼3-fold higher than that in BALF of doxycycline-untreated mice 7-CMV-Stat3C mice, CHI3L1 was mainly expressed in epithelial cells along the conducting airways  airways  A a, dis airways  b, and a airways  A c , the CHI3L1 protein concentration was significantly elevated in human serum samples from all categories of lung cancer patients 7Stat3C bitransgenic mice 7Stat3C bitransgenic mouse model CHI3L1 was first identified as a downstream gene of Stat3 in the CCSP-rtTA/(tetO)nic mice . This isnic mice  g-i. Evenic mice . This ofnic mice  f. Both 7-CMV-Stat3C bitransgenic mice, the increased CHI3L1 protein concentration was observed in BALF and serum of doxycycline-treated CCSP-rtTA/(TetO)7-CMV-MMP12 and CCSP-rtTA/(TetO)7-CMV-Api6 bitransgenic mice with tumor. Unlike that observed in CCSP-rtTA/(TetO)7-CMV-Stat3C bitransgenic mice, no increase of the CHI3L1 protein concentration was observed in non-tumor mice even after doxycycline treatment (7-CMV-MMP12 and c-fms-rtTA/(TetO)7-CMV-Api6 bitransgenic mice In other regional inflammation-induced lung spontaneous tumor mouse models, overexpression of MMP12 and Api6 in alveolar type II epithelial cells results in Stat3 activation (increased phosphorylation at Y705) in lung epithelial cells reatment . The stureatment , in whic7Stat3C bitransgenic mice (Indeed, the CHI3L1 protein concentration in the serum of human patients with several types of lung cancer was significantly elevated . A repornic mice . COPD isnic mice .In summary, this report shows that CHI3L1 is a potential biomarker for inflammation-induced lung cancer prediction in animal models, especially in Stat3-induced cancer. Although CHI3L1 is a useful biomarker, it is necessary to use multiple biomarkers as a panel for more accurate prediction and verification of lung cancer, as increase of CHI3L1 expression has been shown in other inflammation-related mouse models


Pulmonary surfactant is required for lung function at birth and throughout life. Lung lipid and surfactant homeostasis requires regulation among multi-tiered processes, coordinating the synthesis of surfactant proteins and lipids, their assembly, trafficking, and storage in type II cells of the lung. The mechanisms regulating these interrelated processes are largely unknown.We integrated mRNA microarray data with array independent knowledge using Gene Ontology (GO) similarity analysis, promoter motif searching, protein interaction and literature mining to elucidate genetic networks regulating lipid related biological processes in lung. A Transcription factor (TF) - target gene (TG) similarity matrix was generated by integrating data from different analytic methods. A scoring function was built to rank the likely TF-TG pairs. Using this strategy, we identified and verified critical components of a transcriptional network directing lipogenesis, lipid trafficking and surfactant homeostasis in the mouse lung.Within the transcriptional network, SREBP, CEBPA, FOXA2, ETSF, GATA6 and IRF1 were identified as regulatory hubs displaying high connectivity. SREBP, FOXA2 and CEBPA together form a common core regulatory module that controls surfactant lipid homeostasis. The core module cooperates with other factors to regulate lipid metabolism and transport, cell growth and development, cell death and cell mediated immune response. Coordinated interactions of the TFs influence surfactant homeostasis and regulate lung function at birth. Pulmonary surfactant is a lipid-protein complex that is synthesized by type II epithelial cells in the alveoli. Surfactant is stored in intracellular organelles known as lamellar bodies and is secreted into airspace by exocytosis. Surfactant lipids form monolayer and multilayer that line the alveolar surface, reducing surface tension created at the air-liquid interface. Pulmonary surfactant is essential for the proper inflation and function of the lung . Surfactin vitro and in vivo data. In adipocytes, C/EBPα, SREBP-1c and PPARγ induce fatty acid biosynthesis, but only C/EBPα is essential . I(Ti) is normalized relative TF importance ranging from . m is the size of Ct, n is the size of Cg, Ti ∈ Ct and Gj ∈ Cg, and Ct and Cg are in a cluster pair passed Support cutoff. All factors are equally weighted in the equation.where Confidence describes the possibility of a true positive TF-TG relationship according to the integrated information. The first factor of Confidence  denotes the connectivity between a Ti from a cluster Ct and all genes in a cluster Cg, the second factor measures the connectivity between a Gj from cluster Cg and all TFs in cluster Ct, the fourth factor implies the connectivity between Ti and Gj, and the fifth factor I(Ti) denotes the relative importance of Ti in our analysis. We rank TFBS-TG pairs based on the normalized Confidence score for each TF-TG pair. The TFBS-TG pairs with the highest Confidence scores will be selected for experimental validation. For each cluster, we generated a TF-TG association table ranked according to the confidence score. A network graph linking TFs and their TGs was generated using Cytoscape 2.6 http://www.cytoscape.org/.Elovl1, Slc34a2 and Zdhhc3 promoters. Approximately 1 Kb of the 5'-upstream regulatory regions comprising the proximal promoter were PCR amplified, including the transcription start site (TSS) and a part of the 5'-untranslated (5'-UT) region as depicted in Figure n = 6) and depicted as means ± S.D. unless stated otherwise.The MLE-15 cell is an immortalized mouse lung epithelial cell line that maintains some of the morphological and functional characteristics of type II epithelial cells. MLE-15 cells were cultured in HITES medium  for funchttp://research.cchmc.org/pbge/jsp/links_v2.jspAll published microarrays and mouse models we used in this study are listed in Table ABCA3: ATP-binding cassette sub-family A member 3; ACLY: ATP citrate lyase; ACOXL: acyl-Coenzyme A oxidase-like; ACSS2: acyl-CoA synthetase short-chain family member 2; ADORA2B: adenosine A2b receptor; AHR: aryl hydrocarbon receptor; APP: approximation steps; AQP5: aquaporin 5; BARBIE: barbiturate-inducible element; BCL6B: B-cell CLL/lymphoma 6, member B; BEX2: brain expressed X-linked 2; BIOGRID: Biological General Repository for Interaction Datasets; BTG3: B-cell translocation gene 3; CEBPA: CCAAT/enhancer-binding protein alpha; CFTR: cystic fibrosis transmembrane conductance regulator; CHIP: Chromatin immunoprecipitation; CIZ: Cas-associated zinc finger protein; CLOVER: cis-element over-representation; CMV: Cytomegalovirus; DAVID: Database for Annotation, Visualization and Integrated Discovery; DIRE: Distant Regulatory Elements of co-regulated genes; DLK1: delta-like 1 homolog; ECR: Evolutionarily Conserved Regions; EGF: epidermal growth factor; EGR: Early growth response; ELF5: Ef1alpha-like factor-5; ELOVL1: elongation of very long chain fatty acids-like 1; EMP2: epithelial membrane protein 2; ENAC: epithelial sodium channel; ENPP2: ectonucleotide pyrophosphatase/phosphodiesterase 2; ER: Endoplasmic Reticulum; ERM: ets-related molecule; ERR1: estrogen receptor related 1; ETS: erythroblastosis virus E26 oncogene homolog; ETV5: ETS variant gene 5; FABP5: fatty acid binding protein 5; FLI1: Friend leukemia integration 1; FOXA2: forkhead box A2; GATA6: GATA binding protein 6; GO: Gene Ontology; GPAM: glycerol-3-phosphate acyltransferase, mitochondrial; HES1: hairy and enhancer of split 1; HITES: hydrocortisone, insulin, transferrin, estrogen, and selenium; HNF3: Hepatocyte Nuclear Factor 3; HPRD: Human Protein Reference Database; ID2: inhibitor of DNA binding 2; IRF1: interferon regulatory factor 1; IRFF: Interferon regulatory factors; JUN: v-jun sarcoma virus 17 oncogene homolog; KDR: kinase insert domain protein receptor; KNN: k-Nearest-neighbours; LEF1: lymphoid enhancer binding factor 1; LIPG: lipase, endothelial; LMO2COM: LIM domain only 2 complex; LPCAT1: lysophosphatidylcholine acyltransferase 1; MEF2C: myocyte enhancer factor 2C; MLE-15: Murine lung epithelial cells; MTCH2: mitochondrial carrier homolog 2; NF1: nuclear factor I; NFAT: Nuclear factor of activated T-cells; NFATC3: nuclear factor of activated T-cells, calcineurin-dependent 3; NFE2: nuclear factor, erythroid derived 2; NFKB: nuclear factor of kappa light polypeptide gene enhancer in B-cells; NKX2-1: NK2 homeobox 1; NLP: Natural Language Processing; NOTCH1: neurogenic locus notch homolog protein 1; NPT2B: Na(+)/Pi co-transporter 2B; NR1H2/3: nuclear receptor subfamily 1, group H, member 2/3; OCT1: organic cation transporter 1; PDIA5: protein disulfide isomerase associated 5; POU2F1: POU domain, class 2, transcription factor 1; PPAR: peroxisome proliferator-activated receptor; PRDX6: peroxiredoxin 6; PREMOD: predicted transcriptional regulatory modules; QT: Quality Threshold; RAR: retinoic acid receptor; RUNX1T1: runt-related transcription factor 1; translocated to, 1; RVISTA: Rank Vista; S.D.: Standard Deviation; SCAP: SREBP cleavage-activating protein; SCD: stearoyl-Coenzyme A desaturase; SCGB1A1: secretoglobin, family 1A, member 1; SLC34A2: solute carrier family 34 (sodium phosphate), member 2; SOAT1: sterol O-acyltransferase 1; SOX9: Sex determining region Y-Box 9; SP1: Sp1 transcription factor (specificity protein 1); SPP1: secreted phosphoprotein 1; SREBP: Sterol Regulatory Element Binding Proteins; SREPINB9: serpin peptidase inhibitor, clade B , member 9; STAT6: signal transducer and activator of transcription 6; STFPA: surfactant, pulmonary-associated protein A; STFPB: surfactant, pulmonary-associated protein B; STFPC: surfactant, pulmonary-associated protein C; STFPD: surfactant, pulmonary-associated protein D; TCF4: transcription factor 4; TF: Transcription Factor; TFBS: Transcription Factor Binding Site; TG: Target Gene; TRANSFAC: Transcriptional Factor Database; TSS: Transcription start site; TTF-1: thyroid transcription factor 1; VEGFA: vascular endothelial growth factor A; WARS: tryptophanyl-tRNA synthetase; WWTR1: WW domain containing transcription regulator 1; ZDHHC3: zinc finger, DHHC domain containing 3.YX designed and coordinated the overall project, participated in the statistical analysis and drafted the manuscript. MZ and LJL participated in the design; drafting and computational analysis of the data integration section. YW carried out multiple data analysis and assisted manuscript preparation. PK assisted the data analysis and manuscript preparation. VD carried out promoter reporter assays and wrote that part of the manuscript. JAW provided mRNA data, contributed to study design and to the writing and revising of the manuscript. All authors read and approved the final manuscript.Data collecting and Clustering.Click here for fileSupport & Confidence Calculation For C1 Genes.Click here for fileSupport & Confidence Calculation For C2 Genes.Click here for fileSupport & Confidence Calculation For C28 Genes.Click here for fileTop Ranked CEBP Targets According To The Integrative Score.Click here for fileTop Ranked SREBP Targets According To The Integrative Score.Click here for fileTop Ranked HNF3 Targets According To The Integrative Score.Click here for file


HTLV-I proviral load (pVL) is proposed as a biomarker of disease progression. HTLV-I pVLs are higher in symptomatic compared to asymptomatic carriers. The aim of this study was to evaluate HTLV-I pVL in patients with HTLV-I associated diseases (HAM/TSP and ATLL) and other disorders (uveitis and pyoderma gangrenous–PG).6 PBMCs (0.04%). Differences among groups where assessed by Mann-Whitney test.HTLV-I pVL was evaluated in 62 subjects; 52 asymptomatic and 10 symptomatic . HTLV-I pVL in PBMCs was estimated by a quantitative real-time PCR assay with SYBR Green, where HTLV-I pol gene is amplified in parallel with albumin gene as a normalizer. The limit of detection of the assay was of 400 copies of HTLV-I/106 PBMCs, IQR=4.70-5.30) had significantly higher pVLs than asymptomatic carriers . HAM/TSP patients had pVLs between 5.00-5.73 log10 copies/106 PBMCs (10-54%), ATLL patients had pVLs of 4.83 and 5.50 log10 copies/106 PBMCs ; patients with uveitis had pVLs of ~4.00 log10 copies/106 PBMCs (~1%) and PG patient had pVL of 4.56 log10 copies/106 PBMCs (4%). Patients with HAM/TSP and ATLL had significantly higher pVL (median=5.25 log10 copies/106 PBMCs) than those with other disorders .Symptomatic subjects (median=5.02 log10 copies/10HTLV-I pVL seems to be associated with pathogenic phenotype, being higher in subjects with HTLV-I associated diseases compared to those with other disorders.


Variations within the gene locus encoding protein tyrosine phosphatase non-receptor type 22 (PTPN22) are associated with the risk to develop inflammatory bowel disease (IBD). PTPN22 is involved in the regulation of T- and B-cell receptor signaling, but although it is highly expressed in innate immune cells, its function in other signaling pathways is less clear. Here, we study whether loss of PTPN22 controls muramyl-dipeptide (MDP)-induced signaling and effects in immune cells.Stable knockdown of PTPN22 was induced in THP-1 cells by shRNA transduction prior to stimulation with the NOD2 ligand MDP. Cells were analyzed for signaling protein activation and mRNA expression by Western blot and quantitative PCR; cytokine secretion was assessed by ELISA, autophagosome induction by Western blot and immunofluorescence staining. Bone marrow derived dendritic cells (BMDC) were obtained from PTPN22 knockout mice or wild-type animals.MDP-treatment induced PTPN22 expression and activity in human and mouse cells. Knockdown of PTPN22 enhanced MDP-induced activation of mitogen-activated protein kinase (MAPK)-isoforms p38 and c-Jun N-terminal kinase as well as canonical NF-κB signaling molecules in THP-1 cells and BMDC derived from PTPN22 knockout mice. Loss of PTPN22 enhanced mRNA levels and secretion of interleukin (IL)-6, IL-8 and TNF in THP-1 cells and PTPN22 knockout BMDC. Additionally, loss of PTPN22 resulted in increased, MDP-mediated autophagy in human and mouse cells.Our data demonstrate that PTPN22 controls NOD2 signaling, and loss of PTPN22 renders monocytes more reactive towards bacterial products, what might explain the association of PTPN22 variants with IBD pathogenesis. In the healthy gut, commensal bacteria populate our intestine without provoking a significant immune reaction. However, there is an intense and effective immune response as soon as pathogenic bacteria penetrate the epithelial surface. Usually, monocytes and macrophages initiate and orchestrate effective immune responses by the secretion of pro-inflammatory cytokines, as soon pathogenic bacterial components are present. When recruited to the intestine however, peripheral blood monocytes (PBMC) differentiate into intestinal macrophages (iMAC) that are characterized by reduced reactivity and immune tolerance towards commensal bacteria Monocytes, like other innate immune cells, sense bacteria via conserved pattern recognition receptors (PRR), including nucleotide-binding and oligomerization domain containing type 2 (NOD2) Upon activation, NOD2 induces the phosphorylation of proteins of the nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways, resulting in enhanced expression of adhesions molecules and the secretion of pro-inflammatory cytokines Genome-wide association studies revealed that variants within the gene locus encoding for protein tyrosine phosphatase non-receptor type 22 (PTPN22) are linked with the risk to develop autoimmune disorders, including rheumatoid arthritis, type 1 diabetes, UC and CD Here, we address the question whether PTPN22 affects NOD2-induced signaling pathways, cytokine secretion, and autophagy in myeloid cells. We found that loss of PTPN22 leads to aberrant MDP/NOD2-induced MAPK and NF-κB) activation, resulting in changed gene expression and cytokine secretion. Additionally, MDP-induced autophagy was enhanced when PTPN22 was missing. This influence of PTPN22 on IBD associated intracellular pathways could provide an explanation of how PTPN22 variants might functionally be linked with IBD.180/Tyr182), rabbit anti-p38, rabbit anti-phospho-extracellular signal-regulated kinase (ERK)1/2 (Tyr42/Tyr44), rabbit anti-ERK1/2, mouse anti-phospho c-Jun N-terminal kinase , rabbit anti-JNK, rabbit anti-phospho-NF-κB-p65 (Ser536), rabbit anti-NF-κB-p65 (ser276), rabbit anti-phospho-NF-κB p105 (Ser933), rabbit anti-NF-κB p105/p50, rabbit anti-phospho-NF-κB p100/p52 (Ser866/Ser870), rabbit anti-NF-κB p100/p52, and mouse anti-microtubule-associated proteins 1A/1B light chain 3B (LC3B) were obtained from Cell Signaling Technologies .All reagents were of analytical grade and purchased commercially. Monoclonal goat anti-PTPN22 antibody was obtained from Santa Cruz Biotechnologies  and mouse anti-β-actin antibody from Millipore . Mouse anti-phospho-p38  and pLKO.1-shPTPN22 , pMD2.G (packaging plasmid) and pCMV (envelop plasmid). Transductions were performed as described elsewhere THP-1 monocytes were cultured in RPMI 1640  supplemented with 10% FCS at a density between 0.2 and 1×106 cells/well seeded in 6 well plates and left to adhere to the plates for 24 h before performing experiments.Animal experiments were carried out according to Swiss animal welfare laws and were approved by the veterinary authorities of Zurich, Switzerland . Due to the approval of the veterinary authorities of Zürich, no further approval by an Institutional Animal Care and Use Committee (IACUC) or ethics committee was necessary. PTPN22 knockout mice were kindly provided by Andrew C. Chan, Genentech, Inc., South San Francisco, CA Cells were washed twice with ice-cold phosphate buffered saline (PBS) and lysed in M-Per Mammalian protein extraction reagent  supplemented with protease inhibitors  for 45 min. Cells were centrifuged for 10 min at 13,000 g and supernatants assayed for protein content by absorbance measurement .An aliquot of each lysate was mixed with loading buffer (NuPAGE® 4×LDS Sample Buffer (Invitrogen), 50 mM dithiothreitol) and boiled for 5 min at 96°C. Proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes . Membranes were blocked with 1% blocking solution and primary antibody (concentrations according to manufactureŕs instructions) was added in blocking buffer . Membranes were washed for 30 min, HRP-labelled secondary anti-mouse-, anti-goat- or anti-rabbit-IgG-antibody  in blocking buffer was added for 30 min and membranes were washed for 30 min. with 1% TBST. Immunoreactive proteins were detected using an enhanced chemiluminescence detection kit . Densitometric analysis of Western blots was performed by NIH Image software.THP-1 cells were disrupted in RLT buffer (Qiagen) using a 26 G needle. Total RNA was isolated using RNeasy Plus Mini Kit (Qiagen), and DNA removed by TURBO DNA-free Kit  according to manufacturer's instructions. RNA concentration was assessed by absorbance at 260 and 280 nm. Complementary DNA (cDNA) synthesis was performed using a High-Capacity cDNA Reverse Transcription Kit  following the manufacturer’s instructions.Real-time polymerase chain reaction (PCR) was performed using FAST qPCR MasterMix for Taqman Assays (Applied Biosystems) on a Fast HT7900 Real-Time PCR system using SDS Software (Applied Biosystems). Measurements were performed in triplicate, human β-actin was used as endogenous control, and results were analyzed by ΔΔCT method. The real-time PCR contained an initial enzyme activation step  followed by 45 cycles consisting of a denaturing  and an annealing/extending  step. The used gene expression assays were all obtained from Applied Biosystems.Phosphatase activity was assessed using the EnzChek Phosphatase Assay Kit (Molecular Probes) according to manufacturer's instructions and as described previously Cells were collected and washed three times in ice cold PBS before transferring to glass slides by cytospin . Afterwards, cells were fixed with 4% paraformaldehyde followed by incubation with Methanol at −20°C for 20 min. After 3 wash steps in ice-cold PBS, cells were incubated overnight with anti-LC3 antibody . Texas-Red conjugated anti-mouse IgG secondary antibody was added for 2 h, and cell nuclei stained with DAPI for 10 min. Cells were washed 3 times and slides mounted with Fluorescence Mounting Medium . Cells were imaged immediately using a fluorescence microscope  equipped with an AxioCam HRc (Zeiss) and ApoTome.2 (Zeiss) with AxioVision Release 4.8.2 software (Zeiss).Data are presented as means ± S.E.M. for a series of n experiments. Data are expressed as relative values of the respective control. Statistical analysis was performed by analysis of variance (ANOVA) followed by Student–Newman–Keuls post hoc test. P values <0.05 were considered significant.First, we addressed whether PTPN22 expression in THP-1 cells might be regulated by MDP-induced NOD2 activation. Cells were treated with different concentrations of MDP and a concentration of 500 ng/ml MDP seemed to exert the highest effect on PTPN22 mRNA expression p<0.001; . TherefoTo address if activation of other PRRs beside NOD2, might also influence expression of PTPN22, THP-1 cells were treated for increasing time with lipopolysaccharide  4 ligand), the TLR1/2 ligand PamCys or the Nod1 ligand C12-iE-DAP. While stimulation with LPS also led to a significant increase in PTPN22 expression after 24 h, TLR1/2 activation by PamCys reduced PTPN22 expression and C12-iE-DAP-mediated NOD1 activation did not change PTPN22 expression at all . This inAs PTPN22 is induced by MDP-treatment, we addressed if PTPN22 is involved in the regulation of MDP-induced signaling pathways, such as MAPK or NF-κB activation. For this purpose, THP-1 cells were transduced with lentiviral particles either containing non-targeting control, or PTPN22 silencing shRNA. After selection of cells stably expressing shRNA, PTPN22 expression was reduced for about 60–80% . This ceIn cell treated with LPS we could find similar effects of PTPN22 knockdown on p38 and ERK activation as in MDP-treated cells . InteresTo address the effect of PTPN22 knockdown on NF-κB activation, lysates from MDP-treated cells, expressing either non-targeting control or PTPN22 targeting shRNA, were analyzed for IκB-α and NF-κB p65, p105/50 and p100/52 phosphorylation. MDP induced IκB-α phosphorylation in control-transduced cells, and this induction was enhanced in cells lacking PTPN22 . MDP indWe next investigated functional consequences arising from the observed alterations in MDP-induced signaling. Consistent with enhanced MAPK and NF-κB p65 activation, we detected increased IL-6  and IL-8These findings could be fully confirmed using BMDC derived from PTPN22 knockout mice. Loss of PTPN22 resulted in further increased mRNA levels of IL-6  and tumoPTPN22 deficiency finally resulted in enhanced secretion of the pro-inflammatory cytokines IL-6, IL-8 and TNF in human THP-1 monocytes  as well As NOD2 activation leads to an increase in autophagy To further address autophagy induction, THP-1 cells were treated for 24 h with MDP or the autophagy activator rapamycine and whole cells fluorescently stained for LC3B. In untreated, PTPN22 competent cells, LC3B staining was diffuse and rather weak . When ceHere, we demonstrate that PTPN22 is activated by MDP-treatment, and its loss interferes with signaling events downstream of NOD2 receptor in human THP-1 cells and mouse BMDC. By controlling MAPK and NF-κB activation, loss of PTPN22 influences MDP-induced gene expression, cytokine release and autophagy. We show that loss of PTPN22 results in enhanced MDP-mediated p38, JNK and NF-κB p65 phosphorylation, all involved in monocyte and macrophage differentiation and activation In IBD, hyper-activated intestinal macrophages are important drivers of intestinal inflammation et al found decreased levels of ERK phosphorylation and enhanced levels of p38 phosphorylation in patients carrying a gain of function variant of PTPN22, indicating that PTPN22 would regulate ERK signaling and facilitate p38 activation Cao p38-MAPK signaling is important for shaping the monocyte/dendritic cell-induced adaptive immune reaction and its presence in antigen presenting cells is crucial for Th17 cell development In line with our previous findings in IFN-γ-treated cells We further observed that loss of PTPN22 results in enhanced autophagosome formation in response to MDP. Autophagy plays a crucial role in the clearance of intracellular bacteria It has been further demonstrated that p38 and JNK activation play a role in autophagy induction in monocytic cells In summary, our data indicate that PTPN22, beyond its important role in regulating receptor signaling in the adaptive arm of the immune system Figure S1Different effect of distinct bacterial products on PTPN22 mRNA expression. THP-1 cells were treated for increasing time with (A) 100 ng/ml LPS, (B) 100 ng/ml PamCys, or (C) 100 ng/ml C12-iE-DAP. Graphs show PTPN22 mRNA expression relative to non-treated control and normalized to β-actin. Asterisks denote significant differences from the non-treated control .(TIF)Click here for additional data file.Figure S2PTPN22 controls p38 MAPK.(A+B) THP-1 cells were treated for the indicated time with 500 ng/ml MDP. Representative Western blots show levels of (A) phospho-p38 (Thr180/Tyr182) and total p38; and (B) phospho-ERK (Tyr42/Tyr44) and total ERK. (C–E) THP-1 cells were treated for 30 min with (C) LPS, (D) PamCys, or (E) C12-iE-DAP. Representative Western blots show levels of phospho-p38 (Thr180/Tyr182) and total p38 and phospho-ERK (Tyr42/Tyr44) and total ERK. Asterisks denote significant differences from the non-treated control ; # = p<0.05 vs. MDP treatment of control-transduced THP-1 cells.(TIF)Click here for additional data file.Figure S3Loss of PTPN22 affects NF-κB in a stimulus dependent manner.(A+B) THP-1 cells were treated for the indicated time with 500 ng/ml MDP. Representative Western blots show levels of (A) phospho-NF-κB p65 (Ser536) and total NK-κB p65; and (B) phospho-NF-κB p105 (Ser933) and total NF-κB p105. (C+D) THP-1 cells were treated for 30 min with 500 ng/ml MDP. Representative Western blots and densitometric analysis show levels of (C) phospho-NF-κB p100 (Ser866/Ser870) and total NF-κB p100; and (D) phospho-NF-κB p52 (Ser933) and total NF-κB p52. (E–G) THP-1 cells were treated for 30 min with (E) LPS, (F) PamCys or (G) C12-iE-DAP. Representative Western blots show levels of phospho-NF-κB p65 (Ser536) and total NK-κB p65 and of phospho-NF-κB p105 (Ser933) and total NF-κB p105. Asterisks denote significant differences from the non-treated control .(TIF)Click here for additional data file.


Lnk plays a non-redundant role by negatively regulating cytokine signaling of TPO, SCF or EPO. Retroviral expression of Lnk has been shown to suppress hematopoietic leukemic cell proliferation indicating its therapeutic value in cancer therapy. However, retroviral gene delivery carries risks of insertional mutagenesis. To circumvent this undesired consequence, we fused a cell permeable peptide octa-arginine to Lnk and evaluated the efficacy of inhibition of leukemic cell proliferation in vitro.In this study, proliferation assays, flow cytometry, Western Blot analyses were performed on wild-type (WT), mutant Lnk R8 or BSA treated M-MOK cells. We found that delivered WT, but not mutant Lnk R8 blocked TPO-induced M-MOK megakaryoblastic leukemic cell proliferation. In contrast, WT Lnk R8 showed no growth inhibitive effect on non-hematopoietic HELA or COS-7 cell. Moreover, we demonstrated that TPO-induced M-MOK cell growth inhibition by WT Lnk R8 was dose-dependent. Penetrated WT Lnk R8 induced cell cycle arrest and apoptosis. Immunoprecipitation and Western blots data indicated WT Lnk R8 interacted with endogeneous Jak2 and downregulated Jak-Stat and MAPK phosphorylation level in M-MOK cells after TPO stimulation. Treatment with specific inhibitors (TG101348 and PD98059) indicated Jak-Stat and MAPK pathways were crucial for TPO-induced proliferation of M-MOK cells. Further analyses using TF-1 and HEL leukemic cell-lines showed that WT Lnk R8 inhibited Jak2-dependent cell proliferation. Using cord blood-derived CD34+ stem cells, we found that delivered WT Lnk R8 blocked TPO-induced megakaryopoiesis in vitro.Intracellular delivery of WT Lnk R8 fusion protein efficiently inhibited TPO-induced M-MOK leukemic cell growth by promoting apoptosis. WT Lnk R8 protein delivery may provide a safer and more practical approach to inhibit leukemic cell growth worthy of further development. Cytokines play an important role in hematopoiesis. Myeloproliferative leukemia virus proto-oncogene (c-mpl) gene encodes a receptor for thrombopoietin (TPO) Lnk is an intracellular adaptor protein which expressed mainly in hematopoietic cells. Lnk plays a non-redundant role in negative regulation of several cytokine signalings by stem cell factor (SCF), TPO and erythropoietin (EPO) New therapeutic interventions have been made possible through the development of membrane-permeable carrier peptides in vitro.A model suggested that dysregulation of negative regulator such as Lnk may lead to development of cancer in human Umbilical cord blood was obtained from Miyagi Cord Blood Bank. All donors gave informed consent and the study was approved by Tohoku University Hospital Ethnic Committee.2.Human megakaryoblastic cell-line, M-MOK was maintained in RPMI medium with GM-CSF (20 ng/ml). Human erythroleukemic cell-line, TF-1 (ATCC: CRL-2003) was maintained in RPMI medium with IL-3 (10 ng/ml). HEL, human erythroleukemic cell-line with Jak2 V617F gain-of-function mutation (ATCC: TIB-180) was cultured in RPMI. HELA, human epithelial carcinoma cell-line (ATCC: CCL-2) and COS-7 fibroblast cell-line (ATCC: CRL-1651) were cultured in DMEM medium. All culture medium were supplemented with 10% Fetal Calf Serum (FCS), 100 units/ml penicillin, 100 µg/ml streptomycin and cells were grown in humidified 37°C with 5% COMononuclear cells were isolated using Ficoll Hypaque  density centrifugation. Cells were washed and resuspended in PBS with 2% FCS. Cells were incubated for 20 minutes at 4°C with biotin-conjugated anti-CD34 antibody . After washing, cells were incubated for 20 minutes at 4°C with anti-biotin microbeads . Cells were washed twice and CD34+ stem cells were sorted with autoMACS cell separator (Miltenyi). More than 96% CD34+ stem cells were obtained in all preparations.Human pcDNA3 Lnk cDNA was kindly provided by Dr. I. Nobuhisa . A mutant human Lnk (R392E mutation in SH2 domain) was generated by polymerase chain reaction (PCR)-based site-directed mutagenesis WT or mutant Lnk R8 proteins were expressed in BL21 . Cells were resuspended into extraction buffer  with protease inhibitor cocktail  before lysed by sonication for 1 minute in 4 cycles. Bacterial debris were pelleted, supernatant were subjected to affinity chromatography using His-bind affinity column . Eluted protein was further purified with Heparin and ion exchange FPLC . Purified samples were desalted and buffers were exchanged into phosphate-buffered saline PBS (7.5) using PD-10 Sephadex G-25M column  before application.To measure exogenous recombinant proteins uptake efficiency, M-MOK, TF-1, HEL, HELA or COS-7 cells were treated with 5 µM of fluorescein isothiocyanate (FITC)-labeled WT or mutant Lnk R8. After designated time, cells were collected, washed three times with PBS and trypsinized to remove membrane-bound proteins before subjected to flow cytometry analyses . Dead cells were gated out using Side Scatter and Propidium Iodide (PI) stain.4 M-MOK cells were seeded in 24-well plates in 0.5 ml of RPMI before treated with designated concentration of WT, mutant Lnk R8 or BSA  for 6 hours. Treated cells were supplemented with 10 ng/ml of TPO  and further cultured. Viable cell count was performed every day.2×104 M-MOK, TF-1 or HEL cells were seeded in 96-well plates in RPMI prior to protein treatment. HELA or COS-7 cells were seeded at a density of 5×103 cells in DMEM. Cells were treated with 5 µM of WT or mutant Lnk R8 or BSA. After 6 hours, 10% FCS  were supplemented and samples were further cultured for 2 days. Cell viability was measured using Cell-Quanti MTT assays kit according to Manufacturer's instructions . Absorbance was measured at 570 nm using Microplate Reader (Bio-Rad). All samples were done in triplicate. Cell viability was showed as ratio of absorbance (A570nm) in WT or mutant Lnk R8-treated cells relative to absorbance in BSA-treated cells.1×105 cells per ml were seeded in 6-well plates in RPMI with 10% FCS. Cells were treated with 25 µM of TG101348, PD98059 or DMSO (inhibitor solvent) for 1 hour before TPO addition. Cell growth was determined by counting the number of viable cells for 3 days.To study the effect of different inhibitors on TPO-induced M-MOK cell growth, 1×104 M-MOK cells were treated with of WT, mutant Lnk R8 or BSA as described above. At designated time, M-MOK cells were harvested and stained with FITC- annexin V and PI (BD Biosciences) in binding buffer for 15 minutes. Stained cells were immediately subjected to flow cytometry analyses.2×1024 hours after TPO addition, protein-treated M-MOK cells were collected and washed three times with PBS .The supernatant was removed and 70% cold ethanol was added. After fixation, cells were treated with RNase for 1 hour in 37°C. Finally, cells were incubated in PI staining buffer and cell cycle was analyzed with flow cytometry. The percentages of cells in different cell cycle phases  were determined.5 human CD34+ stem cells purified from cord blood were resuspended in X-VIVO 15 medium  and treated with 5 µM of WT or mutant Lnk R8 or BSA. After 6 hours, X-VIVO 15 supplemented with 1% HSA (Human Serum Albumin) and TPO (100 ng/ml) were added to the medium. At day 0 and day 4, cells were collected, washed with PBS and labeled with mouse Phycoerythrin (PE)-CD41 antibody  or mouse PE-isotype control antibody . Cells were washed twice before subjected to flow cytometry analysis.1×10GM-CSF deprived M-MOK cells were treated with WT, mutant Lnk R8 or BSA prior to TPO stimulation. After designated time, all cells were harvested and lysed in lysis buffer  containing protease inhibitors and 1.5 mM PMSF. The following antibodies were used: Lnk , Jak2, pJak2 (Tyr1007/1008), Stat5, pMAPK (Thr202/Tyr204), cleaved PARP, β-actin, c-mpl , MAPK, pStat5 (Y694) , pentaHis (Qiagen). For immunoprecipitation, lysates were precleared with protein G sepharose before adding anti-Jak2 or control IgG antibody (2 µg). After 2 hours rotation, the immunoprecipitates were collected by addition of protein G sepharose. Samples were resolved on 10% SDS-PAGE gel and blotted onto PVDF membrane (Milipore). Membranes were probed with above mentioned primary and corresponding secondary antibodies IgG TrueBlot conjugated to horseradish peroxidase . Membranes were developed using ECL enhanced chemiluminescence reagents (Amersham Biosciences).WT or mutant Lnk R8 treated-M-MOK cells were stimulated with TPO. After 30 minutes, cells were collected, washed and briefly spun onto poly-L-lysine coated cover glass. Immunofluorescence staining was carried out according to previously described method with slight modification t-test. Statistical significance was presumed when *p<0.05, **p<0.01.Significance of differences between two groups was determined by Student's unpaired To investigate the negative regulatory ability of WT Lnk, we included a Lnk mutant construct (R392E) which showed no inhibitive effect on TPO-induced cell growth in a previous report as control To determine the uptake efficiency of the R8 fusion proteins, M-MOK cells were co-cultured with 5 µM of fluorescein isothiocyanate (FITC)-labeled WT or mutant Lnk R8 at 37°C for 2 hours. Cells were harvested and trysinized before flow cytometry analyses. The uptake rate of WT or mutant Lnk R8 was 96.7% and 98.4% respectively . Moreovep<0.01). To confirm the results of cell proliferation, we performed MTT assays to check cell viability after protein treatment. Cell viability of WT Lnk R8-treated cells was 46.6±5.6%, significantly lower compared to those of control groups, mutant Lnk R8 or BSA-treated cells, 93.3±9.3% and 100.0±8.5% respectively  to analyze percentage of apoptotic or dead cells through flow cytometry. On day one, the number of apoptotic cells in WT Lnk R8-treated M-MOK cells was 26.5±7.5%, significantly higher than those of control groups, mutant Lnk R8 or BSA, 17±1.4% or 16.5±2.1% respectively . On day To further elucidate the mechanism of TPO-induced cell growth inhibition, we investigated the effect of WT Lnk R8 on cell cycle distribution 24 hours after TPO addition. WT Lnk R8-treated cell population in the G2 phase was three-fold lower compared to mutant Lnk R8 or BSA–treated cells . In contTo investigate whether penetrated WT Lnk R8 interacted with Jak2, we did immunoprecipitation with Jak2 or control IgG antibodies using lysates from Lnk R8-treated M-MOK cells stimulated with or without TPO. WT, but not mutant Lnk R8 strongly bound with Jak2 after TPO stimulation . ImmunofTo determine whether these pathways involved in TPO-induced cell growth, M-MOK cells were pretreated with recently developed specific Jak2 inhibitor, TG101348 Next, we would like to know whether WT Lnk R8 is effective in other Jak2-dependent signaling such as GM-CSF or IL-3. M-MOK cells also proliferate in response to GM-CSF in vitro  not restricted to leukemic cell growth, thus, it should be cautioned that WT Lnk R8 treatment could carry risk such as thrombocytopenia although further studies are needed to elucidate this.Lnk has been shown to interact with major binding site phosphorylated (pY813) and minor pY613 in Jak2 through its SH2 domain after TPO stimulation Recently, Lnk mutations have been discovered in two Jak2 V617F-negative MPN patients resulting in aberrant Jak-Stat activation due to loss of Lnk negative feedback regulation in vitro data shown here, the potential of WT Lnk R8 can be further investigated in MPN animal model to evaluate its efficacy as a method of cancer therapy. Moreover, the function of WT Lnk R8 can be enhanced by fusing with Hemagglutinin-2 subunit peptides (HA2), where previous study showed that HA2 fusion proteins remarkably reduced protein entrapment in the micropinosomes and enhanced the function of delivered protein at a lower dose In this study, we have demonstrated the efficiency and inhibition mechanism of TPO activation by WT Lnk R8 in M-MOK leukemic cells. In addition, WT Lnk R8 is also effective against other Jak2-dependent cytokine cell growth. Based on our Figure S1(A) GM-CSF deprived M-MOK cells were cultured with or without TPO. Cells were counted for 3 consecutive days. (Data represent mean ± SD and results are representative of three independent experiments). (B) Lysates from unstimulated or TPO-stimulated M-MOK cells were immunoprecipitated with c-mpl antibody. Western blots showing immunoprecipitated c-mpl and total tyrosine phosphorylation detected with c-mpl and 4G10 anti-tyrosine phosphorylation antibodies, respectively.(PDF)Click here for additional data file.


C). To examine the consequence of skeletal muscle-restricted JNK-1 overactivation in the development of insulin resistance and glucose metabolism, JNKC mice were crossed to Mck-Cre mice yielding JNKSM-C mice. However, despite increased muscle-specific JNK activation, energy homeostasis and glucose metabolism in JNKSM-C mice remained largely unaltered compared to controls. In line with these findings, obese mice with skeletal muscle specific disruption of JNK-1, did not affect energy and glucose homeostasis. These experiments indicate that JNK-1 activation in skeletal muscle does not account for the major effects on diet-induced, JNK-1-mediated deterioration of insulin action and points towards a so far underappreciated role of JNK-1 in other tissues than skeletal muscle during the development of obesity-associated insulin resistance.Obesity and associated metabolic disturbances, such as increased circulating fatty acids cause prolonged low grade activation of inflammatory signaling pathways in liver, skeletal muscle, adipose tissue and even in the CNS. Activation of inflammatory pathways in turn impairs insulin signaling, ultimately leading to obesity-associated type 2 diabetes mellitus. Conventional JNK-1 knock out mice are protected from high fat diet-induced insulin resistance, characterizing JNK-1-inhibition as a potential approach to improve glucose metabolism in obese patients. However, the cell type-specific role of elevated JNK-1 signaling as present during the course of obesity has not been fully elucidated yet. To investigate the functional contribution of altered JNK-1 activation in skeletal muscle, we have generated a ROSA26 insertion mouse strain allowing for Cre-activatable expression of a JNK-1 constitutive active construct (JNK The increasing prevalence of obesity in the population of developed countries represents a serious health problem, since obesity is a major risk factor for the development of insulin resistance, hyperglycemia, and metabolic syndrome. It is therefore important to understand the molecular mechanism that accounts for obesity-induced insulin resistance. Weight gain during the course of obesity induces low grade chronic inflammation in white adipose tissue (WAT) and liver leading to the release of pro-inflammatory cytokines such as TNF-α into circulation in vivo is poorly understood. While adipose-tissue-specific JNK-1-deficiency confers some protection to obesity-associated insulin resistance JNKs are expressed by three different genes, JNK-1, -2 and -3, all of which generate various JNK-isoforms by alternative splicing mechanisms However, none of these mouse mutants completely phenocopied the effect observed in conventional JNK-1 knock out mice indicating that other cell types or combinations thereof are involved in mediating JNK-1′s effect on insulin sensitivity. Despite the current cell type-specific knowledge about JNK-1 signaling in the development of obesity-induced insulin resistance, mimicry of JNK-1 overactivation as present in the course of obesity has not been elucidated yet. Here, we investigated mice with isolated transgenic overactivation of JNK-1 in skeletal muscle for alterations in glucose metabolism and energy homeostasis. However, besides cell type-specific aggravation of JNK-1 signaling in these organs, the impact on insulin sensitivity and glucose homeostasis was minor. To further investigate the cell type specific function of JNK-1 in obesity, we have generated conditional JNK-1 knock out mice in skeletal muscle. Strikingly, when exposed to high fat diet, obesity developed to a similar extent in JNK-1 mutants and controls and obesity induced a similar inhibition of insulin action in controls and mutants despite successful reduction of JNK-1 expression in these tissues. Taken together our data reveal, that JNK-1 action in skeletal muscle fails to affect the development of obesity and obesity-associated insulin resistance.2.Care of all animals was within institutional animal care committee guidelines. All animal procedures were conducted in compliance with protocols and approved by local government authorities . Animals were fed either normal chow diet (NCD)  containing 53.5% carbohydrates, 18.5% protein, and 5.5% fat , or a high-fat diet (HFD)  containing 32.7% carbohydrates, 20% protein, and 35.5% fat  starting from 3 weeks of age. All animals had access to water and food ad libitum. Body weight was measured once per week. Food intake was quantified daily over a period of 7 days. Mice were sacrificed using COC construct consisting of a JNKK-2D(MKK-7D)-JNK-1 fusion protein was created by conventional cloning techniques. Sequences encoding the JNKK2(MKK7)-JNK1 fusion protein C) sequences were then inserted into the AscI site of the STOP-EGFP-ROSA targeting vector that additionally contained a CAG promoter. Sequences are available upon request. 107 Bruce4 ES cells were transfected with the targeting vector and positive clones were identified using the ROSA probe C mouse strain, which was further crossed to Mck-Cre mice to generate JNKSM-C mice. JNKSM-C mice were genotyped for the presence of the JNKC construct by performing PCR analysis using primers 5_MKK7_JNK1CA (5′-AAG-AAC-TGC-AAG-ACG-GAC-TT-3′) and 3_MKK7_JNK1CA (5′- TTC-ATA-AGA-ACT-AGC-TCT-CTG-T-3′).The JNKSM-KO male mice Mck-Cre mice FL/FL mice FL/FL mice were crossed to deleter Cre mice to yield JNK-1KO mice. In all experiments, littermates carrying the loxP-flanked JNK-1 allele were used as controls. Genotyping for the Mck-Cre trangene was performed by PCR by using primers mck5 (5′-GTT-CTT-AAG-TCT-GAA-CCC-GG-3′), mck3 (5′-GTC-TGG-ATG-ACA-TCG-TCC-AG-3′) and Cre_intern_rev3 (5′-ATG-TTT-AGC-TGG-CCC-AAA-TGT-3′). For identifying the JNK-1FL/FL and the JNK-1KO alleles, the primers 5J1loxNT (5′-ACA-TGT-ACC-ATG-TAC-TGA-CCT-AAG-3′), 3J1loxNT (5′-CAT-TAC-TCT-ACT-CAC-TAT-AGT-AAC-3′) and 3J1deltaNT (5′-GAT-ATC-AGT-ATA-TGT-CCT-TAT-AG-3′) were used.To generate JNK-1Lean mass and body fat content of live animals was determined using the NMR Analyser Minispec (Bruker Optik).Indirect calorimetry was measured in a PhenoMaster System (TSE Systems) as previously described Glucose-tolerance tests were performed on animals fasted for 16 h. Animals were injected with either 2 g/kg body weight of glucose or 0.75 units/kg body weight of insulin  into the peritoneal cavity. Blood glucose concentrations were determined by using an automated glucose monitor (GlucoMen) for the indicated time points.Plasma insulin and leptin concentrations were examined by ELISA  according to the manufacturer's protocol.[α32P]dATP (Amersham) using the Ladderman™ Labeling Kit (Takara) as previously described For Southern blot, 15 µg of DNA isolated from embryonic stem cells was digested over night with 100 U of EcoRI. The DNA fragments were separated by agarose gel electrophoresis and transferred onto Hybond-XL™- membranes (Amersham) by alkaline capillary transfer. 80 ng DNA of the ROSA26 probe was radioactively labeled with 2.5 mCiC mRNA  was determined by using whole-muscle RNA isolated by TRIzol (Invitrogen). RNA was reversely transcribed with High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and amplified by using TaqMan Gene Expression Master Mix (Applied Biosystems). Relative expression of mRNAs was determined by using standard curves based on cDNA derived from the respective tissues, and samples were adjusted for total RNA content by Hprt1 , Gusb  and Tfrc  quantitative PCR. Calculations were performed by a comparative cycle threshold (Ct) method: starting copy number of test samples was determined in comparison with the known copy number of the calibrator sample (ddCt). The relative gene copy number was calculated as 2-ddCt. Quantitative PCR was performed on an ABI PRISM 7900 Sequence Detector (Applied Bioscience). Assays were linear >4 orders of magnitudes.Quantitative expression for JNKvena cava. Skeletal muscle and liver were collected 2, 5 and 7 min after injection and processed as previously described Mice were anesthetized by intraperitoneal injection of ketamin . 5 mU of human regular insulin  were injected into the inferior Muscle and liver tissues were homogenized with a polytron homogenizer (IKA Werke) in protein lysis buffer and centrifuged at 13,000 g for 1 h at 4°C. Proteins in the supernatant were electrophoresed by SDS–polyacrylamide gel electrophoresis (10%) and transferred to PVDF membranes (Bio-Rad). Membranes were probed with the following antibodies: JNK 1/3 (Santa Cruz Biotechnology Inc.), phospho-JNK , AKT (pan) , phospho-AKT , Calnexin  and goat anti-rabbit conjugated to HRP .SM-C mice were incubated overnight with the GST-c-Jun fusion beads. Kinase reaction was induced by adding 200 µM [γ-32 P]-ATP (3000 Ci/mmol) . After SDS-PAGE, phosphorylation of c-Jun was detected by using a Typhoon Trio Imager .JNK activity assays were performed as previously described t test and one-or two-way analysis of variance (ANOVA) (PASW Statistics 18.0). P values <0.05 were considered significant. For visual clarity, instead of standard deviations, means +/− standard errors of the mean (SEM) are depicted in all figures.Datasets were analyzed for statistical significance by using a two-tailed, unpaired Student's Obesity-induced JNK activation has been described to result in the development of insulin resistance in various organs by inhibitory serine phosphorylation of IRS proteins C) in the ROSA26 locus (ROSA JNKC), by targeted insertion of a MKK-7D-JNK-1 fusion-gene, whose expression is prevented by a loxP-flanked transcriptional stop cassette . Indeed, assessment of JNK expression in skeletal muscle revealed not only successful overexpression of the JNKC construct on the RNA level  causes overall alteration in whole body glucose homeostasis and (ii.) can impair insulin signaling in this organ ce tests . Additioce tests . Moreovece tests . Consistin liver . Taken tob/ob genetic obesity model SM-KO mice) SM-KO mice to normal chow (NCD) and high fat diet (HFD) conditions starting from 3 weeks of age and monitored parameters of adiposity as well as energy homeostasis in these animals. Though this analysis revealed that HFD-exposure significantly increased body weight, body length, body fat content, epididymal fat pad mass as well as circulating plasma leptin concentrations in control mice exposed to HFD compared to those exposed to NCD  as well as on the background of the d to NCD , parametnditions . In summSM-KO mice both under lean and obese conditions . To assess whether this discrepancy might be a consequence of an alternative physiological characterization, we investigated control C57/BL6 mice in the different phenotyping protocols. To this end, cohorts of C57/BL6 mice were exposed to NCD and HFD starting with 3 weeks of age (WTS3), and compared to cohorts of C57/BL6 mice receiving the different diets with 8 weeks of age (WTS8), respectively. This analysis demonstrated that HFD exposure increased bodyweight gain, body length as well as adiposity in both protocols to a similar extent when compared to the NCD exposed control mice , B, C, Duishable , G. NeveFL/FL mice to deleter Cre mice (JNK-1KO mice). Consistent with previously published results, JNK-1KO mice on a NCD show not only unaltered bodyweight but also similar naso-anal lengths at 17 weeks of age when compared to controls , the c-Jun N-terminal kinases (JNK), atypical protein kinase C (PKC) and it has been recently recognized that altered diacylglycerol (DAG) content in skeletal muscle can induce insulin resistance via diacylglycerol kinase (DGK) activation We and others could demonstrate that in liver, IKK activation plays an important function in the development of obesity-associated insulin resistance and that both, liver-specific IKK-2 or NEMO deficiency can protect from obesity-associated impairment of glucose tolerance In light of the dramatic protection from the occurrence of obesity-associated insulin resistance in conventional JNK-1-, but not JNK-2-deficient animals, JNK-1 was predicted to play an important role - particularly in the insulin-sensitive target tissue skeletal muscle - to cause obesity-associated insulin resistance per se had no effect on the development of obesity-associated insulin resistance whereas other reports on bone marrow transplantation chimeras with JNK-1-deficient hematopoetic stem cells have yielded controversial findings Thus, what remains a striking observation requiring further investigation is the profound difference of the metabolic phenotype of conventional JNK-1-deficient mice and the reported phenotypes for mice with cell type-specific disruption of JNK-1 activity. While our report clearly demonstrates that skeletal muscle-specific JNK-1 deficiency is not sufficient to protect against obesity-associated insulin resistance, a recent report indicates that inactivation of JNK-1 in the adipose tissue causes some degree of protection from obesity-associated insulin resistance due to the inactivation of JNK-1 in adipose tissue Figure S1C expression in skeletal muscle enhances phosphorylation of cJun.Transgenic JNK (A) JNK-kinase assay (KA) of protein lysates isolated from muscles of WT and JNKSM-C mice fed a NCD. Calnexin antibodies were used for input control of the lysates. (B) Quantitation of radioactively labeled cJun peptide in skeletal muscle of NCD fed WT and JNKSM-C mice. The data were adjusted to the WT NCD data shown in (TIF)Click here for additional data file.Figure S2Physiological comparison of diet-induced obesity protocols. (A) The average bodyweight of WTS3 NCD fed (open squares) and WTS3 HFD fed (grey circles) mice was compared with WTS8 mice fed a NCD (open squares) or a HFD (grey circles) from 0 to 17 weeks on both diets, respectively (n = 10). (B) Body length of WTS3 (white bar) and WTS8 (grey bar) mice upon NCD and HFD feeding (n = 10). (C) Body composition of WTS3 (white bar) and WTS8 (grey bar) mice when exposed to NCD or HFD was determined by using a Brucker minispec in week 17 of feeding the diets (n = 10). (D) Weight of epigonadal fat pads from NCD and HFD WTS3 (white bar) and WTS8 (grey bar) mice in week 17 of feeding the diets (n = 10). (E) Serum leptin levels of WTS3 (white bar) and WTS8 (grey bar) mice upon NCD and HFD feeding after 17 weeks on both the diets (n = 10). (F) Energy expenditure revealed by the daily and nightly volume of O2 consumption and CO2 release of WTS3 (white bar) and WTS8 (grey bar) mice upon NCD and HFD feeding (n = 10). (G) Respiratory exchange rate (RER) of WTS3 (white bar) and WTS8 (grey bar) mice upon NCD and HFD feeding (n = 10). (H) Glucose tolerance tests of WTS3 NCD fed (open squares) and HFD fed (open circles) mice were performed after 17 weeks on either diet (n = 10). (I) Glucose tolerance tests of WTS8 NCD fed (grey squares) and HFD fed (grey circles) mice were performed after 17 weeks on either diet (n = 10). (J) Insulin tolerance tests of WTS3 NCD fed (open squares) and HFD fed (open circles) mice were performed after 17 weeks on either diet (n = 10). (K) Insulin tolerance tests of WTS8 fed a NCD (grey squares) or a HFD (grey circles) were performed after 17 weeks on either diet (n = 10). (L) Insulin levels from sera isolated after 17 weeks on either of the diets from mice with the indicated genotypes determined by ELISA (n = 10). Values are means ± SEM. **, p≤0.01; ***, p≤0.001.(TIF)Click here for additional data file.Figure S3Phenotypical analysis of bodywide JNK-1 deficiency under normal conditions. (A) The average body weight of WT (white bars) and JNK-1KO (black bars) mice fed a NCD at 17 weeks of age (n = 5). (B) Body length of WT and JNK-1KO mice upon NCD feeding at 17 weeks age (n = 5). (C) Body composition of WT (white bar) and JNK-1KO (black bar) mice when exposed to NCD was determined by using a Brucker minispec in week 17 (n = 5). (D) Weight of epigonadal fat pads from NCD fed WT (white bar) and JNK-1KO (black bar) mice in week 17 (n = 5). (E) Serum leptin levels of WT (white bar) and JNK-1KO (black bar) mice at the age of 17 weeks (n = 5). (F) Daily food intake of WT (white bar) and JNK-1KO (black bar) mice upon NCD feeding at the age of 14 weeks (n = 5). (G) Energy expenditure revealed by the daily and nightly volume of O2 consumption and CO2 release of WT (white bar) and JNK-1KO (black bar) mice upon NCD feeding (n = 5). (H) Respiratory exchange rate (RER) of control (white bar) and JNK-1KO(grey bar) mice upon NCD feeding (n = 5). (I) Glucose tolerance tests of WT (white diamonds) and JNK-1KO (black diamonds) mice when feeding a NCD were performed at 11 weeks of age (n = 5). (J) Insulin tolerance tests of WT (white diamonds) and JNK-1KO (black diamonds) mice upon NCD feeding were performed at 12 weeks of age (n = 5) (K) Insulin levels from sera isolated at week 17 from mice with the indicated genotypes upon NCD feeding determined by ELISA (n = 5). Values are means ± SEM, *, p<0.05.(TIF)Click here for additional data file.


Replicating viral vectors are promising HIV vaccine candidates that may enhance immunogenicity through prolonged antigen expression. VSV is the first replicating viral vector after vaccinia to be tested clinically as an HIV vaccine; here we present preliminary safety and immunogenicity data from a phase 1a trial.3 to 3.4 x 107 PFU) (N=10/group) or placebo (N=2/group), delivered intramuscularly at 0 and 2 months. Reactogenicity over 7 days, adverse events (AEs), and viral cultures from whole blood, urine, saliva and swabs of oral lesions were collected. HIV-1-specific CD4+ and CD8+ T-cell responses to Gag peptides were measured 1 and 2 weeks post-boost by intracellular cytokine staining.HVTN 090 enrolled sixty healthy, HIV-1-uninfected adults in a randomized, double-blinded, placebo-controlled dose escalation study. Groups of 12 participants received rVSV Indiana HIV Gag vaccine at 5 dose levels . At the highest dose, 92% reported systemic symptoms, including flu-like syndrome (41%), fever (41%), and moderate chills (33%). Lymphadenopathy, decreased neutrophil count, oral ulceration, and presyncope were each seen in > 1 participant. No severe reactogenicity, encephalitis, or product-related SAEs were reported, and all VSV cultures were negative at all doses tested. Low frequency HIV-specific CD4+ (9%) and CD8+ (3%) T-cell responses were detected post-boost at the first 3 dose levels.Immunization with an attenuated, replicating rVSV Indiana HIV-1 vaccine has an acceptable reactogencity and safety profile to date. Preliminary data reveal few T-cell responses at lower doses. Immunogenicity of the vaccine at the highest doses and in heterologous prime-boost regimens will guide future vector development.


In Table 2, the baseline data for High-Sensitivity C-Reactive Protein (ng/mL) was incorrect. The correct data is "1430 (264-3478)." Please see the corrected Table 2 here: