FcRL4hi B cells exhibit an activated TLR-signaling pathway identified by elevated phosphorylation levels of: pERK (p = 0.0373), p38 (p = 0.0337), p65 (p = 0.1097), and cJUN (p = 0.0239), concomitant with significantly elevated expression of the TLR-signaling modulator hematopoietic cell kinase . Compared to FcRL4neg B cells from healthy controls, TLR9-stimulated FcRL4pos B cells express significantly higher levels of lL-6 (p = 0.0179). Further, TLR9-stimulated B cells also upregulate the expression of FcRL4 (p = 0.0415) and HcK (p = 0.0386). In B-cell lines, siRNA-mediated HcK knockdown downmodulates TLR9-induced FcRL4-mediated activation quantified by CD23 upregulation (p = 0.0553). We present data suggesting that, in viremic HIV-infected individuals, FcRL4 expression identifies unique IL-6 producing pro-inflammatory B-cell subsets. Further, TLR stimulation likely modulates FcRL4 expression and FcRL4 expression is associated with Hck, potentially enhancing the activation of TLR-signaling associated transcription factors. Pathogenic B-cells have been identified in other disease settings, and this study represents a novel report describing a pro-inflammatory B cell subset in HIV-infected patients.In autoimmune diseases, toll-like receptor (TLR)-stimulated pro-inflammatory IL-6-secreting B cells exert pathogenic roles. Similarly, B cell Fc receptor-like 4 (FcRL4) expression amplifies TLR stimulation, and in rheumatoid arthritis patients, FcRL4 expression identifies a pro-inflammatory B cell subset. B cells from HIV-infected subjects also express heightened levels of FcRL4 and secrete high levels of IL-6: a critical mediator of HIV disease progression. In this study, we sought to determine if FcRL4 identifies a pro-inflammatory B cell subset in HIV-infected subjects and further elucidate the mechanisms underlying FcRL4 amplification of TLR stimulation. We determine that tissue-like memory B cells express the highest endogenous levels of FcRL4 positively correlating with IL-6 expression ( Our data indicate that FcRL4hi B-cell subsets are high producers of IL-6, and TLR-signaling modulates FcRL4 expression. Finally, FcRL4 mediates amplification of TLR-signaling likely by recruiting Src Kinase proteins.In this study, we investigated: (1) if in untreated HIV infection, FcRL4VIR), naïve subjects had a median CD4 count of 466 cells/μl , and median viral load of 20,000 copies/ml  .Ramos (a human Burkitt lymphoma cell line) FcRL4 stable transfectants were a generous gift from Dr. Susan Pierce (NIH) and previously described . The FcRCells were stained with the following antibodies: FcRL4-APC (Biolegend), IL-6-PE, CD23-PE-Cy7, CD19-PE-Texas Red, CD10-Pe-Cy5, CD21-V450, CD27-AF700, phospho-p38-PE, phospho-Erk-AF647, phospho-p65-PE, phospho-C-Jun-FITC, (BD Biosciences), Sheep anti-rabbit IgG-DyLight 488 (Biolegend), purified hematopoietic cell kinase (Hck), and phospho-Hck (Abcam).® Cell Separation Media, Mediatech) gradient centrifugation. Cryopreserved PBMC from HIV-infected subjects were used in the immunophenotyping experiments. The cells were stained with FcRL4-APC and CD19-PE-Texas Red and CD19+FcRL4pos and CD19+FcRL4neg B cells were FACS purified and cultured overnight in the presence of 10 µg/ml CpG-B ODN2006 (TLR9L), 2 µg/ml PAM3CSK4 (TLR2L), or 2 µg/ml Imiquimod (InvivoGen). B cells (CD19+) from healthy controls were purified from PBMC using the B Cell Isolation Kit II (Miltenyi Biotec) and AUTOmacs (Miltenyi Biotec). After 4H, the cultures of CD19+ B cells were supplemented with Brefeldin A . After overnight incubation, the cells were surface stained , fixed/permeabilized (Fix/Perm Kit BD Biosciences), and stained for intracellular IL-6 . All samples were acquired on an LRSII (BD Biosciences) flow cytometer and the data analyzed using FlowJo software (Tree Star Inc.). Florescence parameters were normalized using Rainbow Calibration Particles (Spherotech) and antibody bound CompBead (BD Biosciences). Gating was determined by unstained controls.PBMCs were isolated from whole blood using Ficoll  were acquired on an LRSII (BD Biosciences) flow cytometer and the data analyzed using FlowJo software (Tree Star Inc.).Chemical inhibition of Hck was achieved using PP2 (Millipore). Cells were incubated overnight with indicated concentrations of the inhibitor, supplemented with TLR9-L, and further cultured overnight. Only events corresponding to living cells (determined by Live/DeadGAPDH expression was used as an endogenous control.RNA was extracted using the RNeasy Kit (QIAGEN) according to the manufacturer’s instructions. The extracted RNA was measured by spectrophotometer and equimolar concentrations used for cDNA synthesis according to the manufacturer’s instructions . The following primers were used for the qPCR reaction: HcK-Forward 5′-CGGATCCCACATCCACCATCA-3′, Reverse 5′-ACCACGATGATGTCCTCAGAGC-3′, FcRL4-Forward 5′-TCAGCTGGGAGAAGAAGAGGAA-3′, Reverse 5′-GAGTTATCTGGGTGTTGTGTCTTTACC-3′, GAPDH-Forward 5′-CTTCAACGACCACTTTGT-3′ and reverse 5′-TGGTCCAGGGGTCTTACT-3′. Real-time RT-PCR was performed using a Quantitect SYBR Green PCR kit (Qiagen) in a 7900HT Fast Real-Time PCR system (Applied Biosystems). Melting curve analysis was performed to ensure that the primers amplified the desired amplicon and that primer-dimers were absent. Fold change in mRNA expression was calculated by relative quantification using the comparative cycle threshold method. siRNA targeting HcK were purchased from Santa Cruz Biotechnology and Dharmacon, and cells were transfected using the Lipofectamine RNAiMax kit (Life Technologies) according to the manufacturer’s instructions. Knockdown was confirmed by qPCR 48H post-transfection.p-value between group parameters was determined using either unpaired or paired Student’s t-test (with a confidence level of 95%). The statistical dependence between variables was calculated using the Spearman rank correlation analysis. p-Values of <0.05 were considered statistically significant. Pair and multiple comparisons were done using the Wilcoxon-matched-pairs signed rank test.Results are expressed as mean ± SEM or as indicated. GraphPad Prism software, version 5.03 was used for all statistical analysis. The statistical significance VIR) subjects  expressed the highest levels of FcRL4 among different B cell subsets , but significantly higher than naïve B cells  and resting memory B cells . TLM B cells also expressed the highest endogenous levels of IL-6  and RM B cells (p = 0.0204). Likewise, in AM B cells  and RM B cells (p = 0.0241). Moreover, AM cells expressed the highest frequency of FcRL4+IL-6+ cells , N (p < 0.0001) and RM (p < 0.0001) B cells. Taken together, TLM and AM B cells express the highest levels of FcRL4 and IL-6 as well as the frequency of FcRL4+IL-6+ cells. Finally, in the TLM B cells, we observed a significant positive correlation between the FcRL4 and IL-6 expression  as well as FcRL4 and HIV viral load .In rheumatoid arthritis patients, FcRL4 expression identifies a pro-inflammatory B-cell subset . Differesubjects ; howeversubjects . We inveVIR individuals exhibit enhanced FcRL4 expression . Although the level of phosphorylated p65 was higher in FcRL4pos B cells than FcRL4neg B cells, the difference did not attain statistical significance .HIV-infection is associated with an increase in serum concentration of several TLR ligands –9, and Bpression . As FcRLpression , we nextNEG) subjects upregulate expression of the pro-inflammatory cytokine IL-6 (neg) B cells, TLR stimulation of purified FcRL4pos B cells significantly upregulated IL-6 expression .We previously demonstrated that TLR stimulated B cells from healthy controls . We confirmed that while TLR stimulation induces FcRL4 upregulation, the anti-FcRL4 flow cytometry antibody did not lead to FcRL4 upregulation. In human secondary lymphoid tissue, elevated FcRL4 expression is associated with heightened levels of the Src kinase family member HcK   and phosphorylated HcK .Elevated FcRL4 expression on blood B cells has been identified in malaria and HIV-infected viremic patients , 19, conmber HcK , which imber HcK . We, theHcK expression in FcRL4.WT transfectants was reduced using siRNA and confirmed by qPCR . Finally, TLR9 activation was quantified by change in CD23 expression, a readout of TLR9 activity (p = 0.0553) following TLR9 stimulation. To confirm these results, we chemically inhibited HcK using 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolopyrimidine (PP2) as described elsewhere .The effect of HcK on TLR-signaling in B cells was further investigated using a B cell line stably expressing FcRL4 (FcRL4.WT) and a loss-of-function FcRL4 mutant cell line, incapable of amplifying TLR-signaling (FcRL4.FFF) . We deteactivity . HcK knolsewhere , 26. HcKhi TLM and AM blood B cells express high endogenous levels of IL-6, strongly indicating that high FcRL4 expression identifies pro-inflammatory B cells. We demonstrate that frequency of FcRL4+ B-cells correlates strongly with IL-6+ B-cell frequency in the TLM subset. However, AM B cells exhibit the highest frequency of FcRL4+IL-6+ double-positive cells suggesting the possibility that divergent mechanisms drive IL-6 production in AM and TLM B cells. This concept of divergent mechanisms is further supported by the distinct characteristics of these subsets, with TLM displaying elevated expression of inhibitory receptors and increased frequency of HIV-specific B cells, while the AM subset show greater specificity for other pathogens  subjects exhibit elevated serum levels of TLR-ligands  ligands (not shown). Sohn et al. elegantly demonstrated that FcRL4 expression switches B-cell responsiveness from adaptive to innate stimulus (hi B cells concurrently express high levels of the Src-kinase family member HcK, and Smolinska et al. (NEG subjects upregulate HcK and FcRL4hi B cells from HIVVIR subjects exhibit elevated endogenous levels of HcK. Further, HcK downmodulation resulted in a reduction of TLR-signaling in FcRL4 B-cell transfectants. These data confirm recent reports by Liu et al. (While FcRL4 expression has been well documented in HIV, its function remains only partly elucidated. During HIV-1 infection, FcRL4 is elevated on TLM of non-treated individuals, but expression is greatly reduced following treatment ; this sustimulus ; howeverstimulus  reporteda et al.  determinu et al.  and suggB cells have been well established as a critical source of pro-inflammatory IL-6 in autoimmune diseases , and som+ B cells, a population of B cells previously identified as exhausted, in viremic HIV infection. Finally, we present data elucidating the mechanisms of FcRL4-mediated amplification of TLR-signaling in B cells. We provide data indicating that increased expression of FcRL4 coincides with upregulation of the Src kinase HcK, and HcK is necessary for FcRL4’s amplification of TLR signaling.The data from this study indicate that in viremic HIV infected subjects, high expression of FcRL4 identifies pro-inflammatory B cell subsets. In autoimmune conditions, B cells have been established as critical IL-6 expressing cells . Our datAll studies were performed after signed informed written research consent by each study subject. The study was reviewed and approved by the Institutional Review Board of the Rush University Medical Center, and the University of Iowa City VAMC and University of Iowa.BS and AL conceived/designed study and wrote manuscript. BS and AN performed experiments. BS, AN, AL, HS, and JS analyzed data and edited manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer CS and handling editor declared their shared affiliation.


Treatment with dexamethasone in human osteoblasts leads to oxidative stress and cell injures. NF-E2-related factor 2 (Nrf2) is a key anti-oxidant signaling. We want to induce Nrf2 activation via microRNA-mediated silencing its suppressor Keap1. Our results show that microRNA-200a (“miR-200a”) expression depleted Keap1, causing Nrf2 protein stabilization in OB-6 osteoblastic cells. Reversely, the miR-200a anti-sense led to Keap1 upregulation and Nrf2 degradation. miR-200a expression activated Nrf2 signaling, which inhibited dexamethasone-induced reactive oxygen species production and OB-6 cell death/apoptosis. Keap1 shRNA also activated Nrf2 and protected OB-6 cells from dexamethasone. Importantly, miR-200a was in-effective in Keap1-silenced (by shRNA) OB-6 cells. In the primary human osteoblasts, Keap1 silence by targeted-shRNA or miR-200a protected cells from dexamethasone. Significantly, miR-200a level was decreased in necrotic femoral head tissues, which was correlated with Keap1 mRNA upregulation. Together, miR-200a expression activates Nrf2 signaling and protects human osteoblasts from dexamethasone. Existing literatures have demonstrated that dexamethasone (Dex) and other glucocorticoids shall exert direct injuries to human osteoblasts –3. OsteoWe –9, 11 hamiRNAs (miRs) inhibit targeted-gene expression via binding to 3′-untranslated region (UTR) of the mRNAs –25. Our First, we demonstrate that miR-200a (“-3p”) putatively targets the 3′-UTR of Keap1 mRNA (at position 131-138) Figure , as repoNext, the miR-200a anti-sense (“antamiR-200a”) was introduced to OB-6 cells. AntamiR-200a indeed decreased miR-200a-3p expression Figure . As a reIn the resting condition, Keap1 binds to Nrf2 to cause Nrf2 ubiquitination and degradation . Above rIf Keap1 is the primary target of miR-200a in OB-6 cells, then direct silence of Keap1 should also protect cells from Dex. Thus, shRNA method was utilized to knockdown Keap1. A set of two distinct lentiviral Keap1 shRNAs , with non-overlapping sequence, were applied. qRT-PCR assay results in Figure Since Keap1 shRNA induced Nrf2 stabilization in OB-6 cells, expressions of Nrf2-regulaed genes were also tested. qRT-PCR assay results in Figure If Keap1 is the primary target of miR-200a, then miR-200a should be ineffective in Keap1-silenced cells. To test this hypothesis, miR-200a vector was introduced to Keap1 shRNA (“Sequ1”)-expressing OB-6 cells Figure . MiR-200Next, we focused the potential effect of miR-200a in the primary human osteoblasts. The primary cells were also transfected with miR-200a expression vector or Keap1-shRNA (“Sequ1”). qRT-PCR assay results showed that miR-200a (−3p) level was increased significantly only after introduction of the miR-200a construct Figure . Keap1 mAt last, we tested expression of miR-200a in human necrotic femoral head tissues. A total of 10 different necrotic femoral head tissues (“Necrosis”) from Dex-taking patients were tested. As compared to the surrounding normal femoral head tissues , expression of miR-200a (−3p) was significantly decreased in the “Necrosis” tissues Figure . ReverseRecent studies have suggested that Dex-induced ROS production and subsequent oxidative stress are the major reason of osteoblast injuries , 13, 34.The transcription factor Nrf2 dictates a genetic program which protects cells from oxidative stress and maintains cellular redox homeostasis , 36, 37.et al., showed that miR-200a activates Nrf2 by targeting Keap1 in hepatic stellate cells [et al., showed that miR-141 activates Nrf2-dependent antioxidant pathway also via depleting Keap1 [et al., showed that miRNA-141 attenuated UV-induced oxidative stress by activating Keap1-Nrf2 signaling [Very recent studies have identified potential miRNAs that could possibly activate Nrf2 via depleting Keap1. For example, Yang te cells . Shi et ng Keap1 . Similarignaling . Therefoin vivo.In the current study, we demonstrate that miR-200a activated Nrf2 signaling in OB-6 cells and primary human osteoblasts. First, forced-expression of miR-200a induced Keap1 degradation, causing Nrf2 stabilization in human osteoblasts/osteoblastic cells. Reversely, miR-200a depletion by the antamiR-200a led to Keap1 upregulation and Nrf2 degradation. Second, stabilized Nrf2 induced transcription of several Nrf2-regulated genes  in miR-200a-expressing cells. Significantly, expression of miR-200a, by inducing Nrf2 activation, largely inhibited Dex-induced oxidative stress and subsequent osteoblasts/osteoblastic cell death and apoptosis. Third, Keap1 shRNA, mimicking miR-200a, activated Nrf2 and protected OB-6 cells from Dex. More importantly, miR-200a was unable to further protect OB-6 cells from Dex when Keap1 was silenced by shRNA. We therefore conclude that Keap1 is the primary target of miR-200a in osteoblasts in mediating its anti-Dex functions. Significantly, we found that miR-200a level was decreased in necrotic femoral head tissues, which was correlated with Keap1 mRNA upregulation. It will be interesting to further test the potential function of miR-200a on Dex-damaged human osteoblasts Dex and puromycin were purchased from Sigma Aldrich . Cell culture reagents were from Gibco . Antibodies of this study were purchased from Cell Signaling Technology .The OB-6 human osteoblastic cells , obtaineThe primary human osteoblasts were achieved from the redundant trabecular bone fragments of healthy donors. The trabecular bone fragments were minced into small pieces and washed. The bone pieces were then digested with 2 mg/mL collagenase type II  for 2 hours, which were placed in culture flasks with the described medium . Medium A total of 10 Dex-taking patients undergoing femoral head surgery were included. The necrotic femoral head tissues along with paired surrounding normal femoral head tissues were obtained. Tissues were subjected to tissues lysis buffer  incubation. Samples were stored in liquid nitrogen for further analysis. Written-informed consent was obtained from each participant. The protocols were approved by Ethics Review Board of Nanjing Medical University.The pSuper-GPF-puro-miR-200a expression vector, encoding miR-200a, was designed by Genepharm . The miR-200a anti-sense (“antamiR-200a”) was provide by Ambion . Human osteoblasts/osteoblastic cells were transfected with miR-200a expression vector, antamiR-200a, scramble miRNA control (“miR-C”) or scramble antagomiR control (antagomiR-C) via the Lipofectamine 2000 reagent . Stable cells were selected by puromycin  for 72 hours.As previously described , Keap1 mAs described in our previous studies –11, the The histone-DNA ELISA cell apoptosis plus kit  was applied to quantify cell apoptosis in human osteoblasts/osteoblastic cells –9.As described –9, cell The detailed protocol of Western blotting assay was descibred previously, and blot results were quantified via ImageJ software –9.The two lentiviral Keap1 shRNAs  were designed and synthesized by Genepharm Co. . The shRNA was directly to cultured osteoblasts/osteoblastic cells  for 24 hours. Puromycin  was added afterwards to select stable cells for additional 72 hours. Keap1 knockdown was verified via qRT-PCR assay and Western blotting assay. Control cells were infected with lentiviral scramble control shRNA [Nrf2, HO-1, GCLC and NQO1, as well as Keap1 and GAPDH were previously described [RNA from human osteoblasts/osteoblastic cells or fresh femoral head tissues was extracted via RNeasy Midi Kit . For each sample, 500 ng of total RNA was reverse-transcribed via the RT-PCR kit . Quantitative real-time PCR (“qRT-PCR”) was performed using the SYBR green kit , 43 throescribed –47. In aescribed . miR-200As previously described , 11, 12,p < 0.05 was considered significant.All values were expressed as means ± standard deviation (SD). The statistical significance of differences among groups were determined by one-way analysis of variance (ANOVA) followed by the Tukey's post hoc multiple comparison tests. MiR-200a expression activates Nrf2 signaling and protects human osteoblasts/osteoblastic cells from Dex. Keap1 is the primary target of miR-200a in mediating its actions.


Staphylococcus aureus JH4899, a community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) isolate collected from a patient with systematically disseminated infection, is classified as sequence type 8 and carries the staphylococcal cassette chromosome mec type IVl (SCCmecIVl). It produces TSST-1, SEC, a newly discovered enterotoxin (SE1), and epidermal cell differentiation inhibitor A (EDIN-A). Here, we present the complete genome sequence of the chromosome and a plasmid harboring the se1 and ednA genes. Staphylococcus aureus (CA-MRSA) is genetically heterogeneous, and several genotypes are spreading worldwide. Recently, emergence of the Japanese CA-MRSA strain (CA-MRSA/J), which is sequence type 8 (ST8) of multiple locus sequencing typing (MLST), has been reported (Community-acquired methicillin-resistant reported . Genotypreported .mec type IVl (SCCmecIVl) isolated in 2012 from the expectorated sputum of the patient.We previously reported a patient with systematically disseminated CA-MRSA/J infection and described the genotypic analysis . Here, wde novo using CLC Genomics Workbench version 7 . These analyses yielded 5 scaffolds and 38 contigs, and gaps were closed by sequencing PCR products spanning the gaps. The completed genome sequence was automatically annotated using the Microbial Genome Annotation Pipeline (MiGAP)  (The genomic DNA was isolated from JH4899 grown on tryptic soy broth (TSB) at 37°C using the QIAamp DNA minikit  with the addition of lysostaphin (10 µg/mL) and incubation at 37°C for 1 h. The DNA library was prepared using the Nextera XT DNA library prep kit  and sequenced as paired-end reads using an Illumina MiSeq platform and MiSeq reagent kit v2 500 cycles) (Illumina). The run generated 1,552,210 reads (approximately 123-fold genome coverage). Illumina reads were assembled 00 cyclesThe JH4899 genome comprised a 2,808,921-bp chromosome and 32,580-bp plasmid (pJSA01), with average G+C contents of 32.8% and 28.7%, respectively. The chromosome contained 2,619 predicted protein-coding sequences (CDSs), 59 tRNA genes, and 6 rRNA operons. pJSA01 encoded 45 predicted CDSs.mecIVl of JH4899 contained a 3,939-bp sasL gene encoding a cell wall-anchored surface protein, SasL (tst-1), enterotoxin C (sec), and enterotoxin L (sel) genes, and phage ϕSa3j, carrying sak, scn, and sep, encoding staphylokinase, staphylococcal complement inhibitor, and enterotoxin P, respectively (aac(6′)/aph(2′′)] resistance transposon Tn4001, and transposon Tn552, carrying the β-lactam resistance gene (blaZ). Like NN50, the first CA-MRSA/J isolated in Japan, the JH4899 genome did not carry genes for Panton-Valentine leukocidin (PVL) or an arginine catabolic mobile element (ACME), which are present in the prophage and SCCmecIVa on the USA300 genome, respectively /aph′′] resisSasL tst-, enterot257s embedded in the plasmid. Furthermore, this plasmid possessed an antiseptic efflux protein gene, qacB.pJSA01 carried two virulence genes encoding a newly discovered enterotoxin (SE1) and ADP-ribosyltransferase and epidermal cell differentiation inhibitor A (EDIN-A) . These gThe complete genome sequence of JH4899 will contribute to further understanding of the virulence mechanism of CA-MRSA/J infection.S. aureus JH4899 and pJSA01 have been deposited in the DDBJ/EMBL/GenBank under the accession numbers AP014921 and AP014922 for the chromosome and plasmid, respectively. The version described in this paper is the first version.Whole-genome sequences of


Phd2 knockout (Phd2-cKO) mice and their littermate control mice (Control) were created and fed HFD or LFD. Control mice on HFD (Control HFD) exhibited renal damages and renal ischemia with impaired hypoxic response compared with those on LFD. After tamoxifen treatment, HFD-fed knockout mice (Phd2-cKO HFD) had increased peritubular capillaries and the increased expressions of hypoxia responsive genes compared to Control HFD mice. Phd2-cKO HFD also exhibited the mitigation of tubular damages, albuminuria and glomerulomegaly. In human PT cells, the increased expressions of hypoxia-inducible genes in hypoxic condition were attenuated by free fatty acids. Thus, aberrant hypoxic responses due to dysfunction of PHD2 caused both glomerular and tubular damages in HFD-induced obese mice. Phd2-inactivation provides a novel strategy against obesity-induced kidney injury.The involvement of tissue ischemia in obesity-induced kidney injury remains to be elucidated. Compared with low fat diet (LFD)-mice, high fat diet (HFD)-fed mice became obese with tubular enlargement, glomerulomegaly and peritubular capillary rarefaction, and exhibited both tubular and glomerular damages. In HFD-fed mice, despite the increase in renal pimonidazole-positive areas, the expressions of the hypoxia-responsive genes such as Prolyl-hydroxylase PHD2, a dominant oxygen sensor, and VEGFA were unchanged indicating impaired hypoxic response. Tamoxifen inducible proximal tubules (PT)-specific  Obesity is worldwide health problem with a variety of complications including hypertension, dyslipidemia, and abnormal glucose metabolism. Obesity is also considered as a major risk factor for chronic kidney disease (CKD)26Although previous studies reported that diabetes induced a hypoxic condition in the kidney, it has not been determined whether this condition is also evident in obesity8prolyl hydroxylation of HIFα by PHDs enables its association with the von Hippel-Lindau tumor suppressor E3 ubiquitin ligase complex, leading to the degradation of HIFα via the ubiquitin-proteasome pathway12When there is an imbalance between oxygen demand and supply, cells activate hypoxic responses to protect themselves against the hypoxic environment. Among the molecules responsible for tissue hypoxic response, the transcription factor hypoxia-inducible factor (HIF), which is composed of HIF-1, HIF-2 and HIF-3, is the master regulator of the hypoxic responses. Although little is known about the cell type-specific functions of HIFs in response to ischemic kidney injury, many cellular adaptive processes are regulated by HIF-111Vegfa, Slc2a1, Pgk1, and Ldha were not upregulated in response to hypoxia, indicating a lack of HIF-1α activation. By using tamoxifen (Tam)-inducible PT-specific Phd2 knockout mice, we demonstrated that deletion of Phd2 in PT cells mitigated the renal damage, by ameliorating the peritubular capillary rarefaction and tissue ischemia. Our data suggest that the inhibition of PHD2 in PT cells is a potential therapeutic strategy against obesity-induced kidney disease.In this study, we used high-fat-diet (HFD)-fed C57BL/6J background mice as an obese mice model. Though this model is characterized by increased plasma free fatty acids (FFA) and insulin levels with impaired glucose tolerance, they did not show frank hyperglycemia. This model is relevant to study obesity-related nephropathyIn mice fed HFD, body weight increased but blood pressure did not change . Though Vegfa (VEGF-A), Pgk1 (PGK1), Slc2a1 (Glut-1), and Ldha (LDHA)   were notPhd2 knockout mice (Phd2-cKO) by crossing Phd2-floxed mice (Phd2F/F)Cre-recombinase gene driven by a PT-specific N-myc downstream regulated gene 1 (Ndrg1) promoterPhd2-cKO mice on either diet group, confirming that Phd2-inactivation by the Cre-loxP system was effective in Phd2-cKO mice (Control mice (Phd2-cKO mice (Vegfa (VEGF-A), Pgk1 (PGK1), Slc2a1 (Glut-1), and Ldha (LDHA), were increased in Phd2-cKO mice, suggesting that hypoxia responsible gene downstream of PHD2 was subsequently induced by inactivating PHD2  condition to mimic kidney environment in HFD-induced obese mice. The expressions of hypoxia-responsible genes downstream of PHD2-HIF pathway including glucose transporter 1 (GLUT1) and VEGFA were increased under hypoxia  or HFD  during the 12-wk experimental protocol as described beforeEight-wk-old male C57BL/6J mice  weighing 20 ± 1 g were housed in a temperature- and light -controlled room with Phd2 knockout (Phd2-cKO) mice by crossing the Phd2F/F mice from Dr. Yoji Andrew Minamishima (Keio University)Ndrg1-CreERT2/+ mice with a PT-specific Ndrg1 gene promoter from Prof. Motoko Yanagita (Kyoto University)Phd2-cKO mice, Phd2F/F mice were crossed with Ndrg1-Cre mice to generate Phd2/F+; Ndrg1-Cre mice. Phd2/F+; Ndrg1-Cre mice were then crossed with Phd2/F+ mice to generate Phd2F/F; Ndrg1-Cre (Phd2-cKO) mice and Phd2+/+; Ndrg1-Cre (Control) as littermate controls. Genomic DNA was isolated from tail biopsies at 4 week of age using a DNeasy kit  and genomic DNA samples were screened by polymerase chain reaction using the transgene-specific oligonucleotide primers shown in Phd2F/F allele are also shown in Cre gene was designed to be specifically activated by the estrogen receptor (ER) agonist, tamoxifen (Tam). PT-specific conditional Phd2 knockout mice (Phd2-cKO) and their control littermates (Control) were maintained on LFD (n = 8) or HFD (n = 8) from 8 to 20 wk of age. Mice were divided into 8 groups; (1) LFD-fed Control mice without Tam injection (sunflower oil vehicle injected) (Control LFD/Tam−); (2) LFD-fed Phd2-cKO mice without Tam injection (Phd2-cKO LFD/Tam−); (3) LFD-fed Control mice with Tam injection (Control LFD/Tam+); (4) LFD-fed Phd2-cKO mice with Tam injection (Phd2-cKO LFD/Tam+); (5) HFD-fed Control mice without Tam injection (Control HFD/Tam−); (6) HFD-fed Phd2-cKO mice without Tam injection (Phd2-cKO HFD/Tam−); (7) HFD-fed Control mice with Tam injection (Control HFD/Tam+); and (8) HFD-fed Phd2-cKO mice with Tam injection (Phd2-cKO HFD/Tam+). At 8 wk of age, mice received 1 mg of tamoxifen  by intraperitoneal injection for 5 consecutive days as described previouslyWe generated PT-specific conditional ® . Urinary albumin was measured by Albuwell M  and neutrophil-associated lipocalin (NGAL) and cystatin C were measured by ELISA kits .Serum concentrations of insulin and free fatty acids (FFA) were measured with LabAssayKidney sections (5 μm) were stained with periodic acid-Schiff’s stainTEM analysis was performed as previously reportedμg/mL streptomycin in a CO2 incubator .The human renal proximal tubular cell line, HK-2 cells  were expanded in Dulbecco’s modified Eagle’s medium nutrient mixture F-12  with 10% bovine serum (FBS) supplemented with 50 U/mL penicillin and 50 2, 5% CO2, 94% nitrogen gas) for 24 h in a hypoxia workstation  or remained in the CO2 incubator used for routine culture (normoxia). An oxygen sensor was used to ensure that the oxygen concentration inside the workstation was maintained at 1% throughout the experiments . Cells were further supplemented with 1 μM human insulin (Sigma Aldrich) or 0.3 mM FFA. The FFA stock solution consisted of 6.35 mM sodium palmitate (Sigma Aldrich) and 12.7 mM sodium oleate (Sigma Aldrich) in FFA-free bovine serum albumin (BSA) (1.8 mM) as previously described482 concentration of 20%), (2) cells without insulin or FFA in hypoxia, (3) cells with insulin in normoxia, (4) cells with insulin in hypoxia, (5) cells with FFA in normoxia and (6) cells with FFA in hypoxia. After incubation for 24 h, cells were collected using TRIzol reagent  to perform real-time RT-PCR.Early passage (passages 3–4) HK-2 cells were grown to 80% confluence and made quiescent by serum starvation for 24 h. Cells were then exposed to hypoxia (1% ORPLP0 (36B4) genes. The sequences of primers used are shown in Equal amount (1 μg) of total RNA from each sample was subjected to reverse transcription in a 20 μL reaction mixture containing random primers and Superscript II enzyme (Invitrogen). Real-time PCR was performed using an ABI Step One Plus sequence detector P < 0.05 was considered statistically significant.Data are expressed as means ± standard error of mean. One-way analysis of variance was used to determine significant differences among groups. In the overall analysis of variance, the Kruskal–Wallis test for multiple comparisons was used to assess individual group differences. This study was performed in accordance with the Institutional Guidelines on Animal Experimentation at Keio University. All methods were carried out in accordance with the guidelines for animal experiments of the Ministry of Education, Culture, Sports, Science and Technology, Japan. The experimental protocols were approved by the Animal Care and Experimentation Committee in Keio University ). All surgery was performed under sodium pentobarbital anesthesia by intraperitoneal injection, and all efforts were made to minimize suffering. At the end of the experiments, the mice were euthanized by intraperitoneal injection of sodium pentobarbital.How to cite this article: Futatsugi, K. et al. Obesity-induced kidney injury is attenuated by amelioration of aberrant PHD2 activation in proximal tubules. Sci. Rep.6, 36533; doi: 10.1038/srep36533 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Human immunodeficiency virus type 1 (HIV-1) Tat is a virus-encoded trans-activator that plays a central role in viral transcription. We used our recently developed parallel analysis of in vitro translated open reading frames (ORFs) (PLATO) approach to identify host proteins that associate with HIV-1 Tat. From this proteomic assay, we identify 89 Tat-associated proteins (TAPs). We combine our results with other datasets of Tat or long terminal repeat (LTR)-associated proteins. For some of these proteins , we confirm their strong association with Tat. These TAPs also suppress Tat-mediated HIV-1 transcription. Removing suppression of HIV-1 transcription benefits the reversal of post-integrated, latent HIV-1 proviruses. We demonstrate that these transcriptionally suppressing TAPs contribute to HIV-1 latency in Jurkat latency (J-LAT) cells. Therefore, our proteomic analysis highlights the previously unappreciated TAPs that play a role in maintaining HIV-1 latency and can be further studied as potential pharmacological targets for the “shock and kill” HIV-1 cure strategy.  Tat is a virus-encoded regulatory protein that is essential for human immunodeficiency virus type 1 (HIV-1) replication. Although Tat possesses multiple functions, it is primarily involved in the transcription of integrated HIV-1 proviruses. Once the host cellular transcription factors nuclear factor kappa B (NF-κB) and Nuclear factor of activated T-cells 1 (NFAT1) bind to the enhancer region of the HIV-1 5’ long terminal repeat (LTR) promoter, they recruit histone acetyltransferase (HAT) to acetylate histones and stimulate viral gene expression. Tat protein is therefore produced, which induces a positive feedback loop to amplify HIV-1 transcription. Tat associates with the RNA polymerase II (Pol II) pre-initiation complex (PIC), and plays a role in both initiation and elongation . Trans-aTat is a small protein (86–101 amino acids), containing the N-terminal, proline-rich, cysteine-rich core, basic, and the C-terminal glutamine-rich regions. The structural studies indicate that Tat alone lacks a defined three-dimensional folding with a stable conformation and is intrinsically a disordered protein . However®attL-containing entry clone and an attR-containing destination vector (LR) cloning using pDONR223-TAP entry vector and pcDNA-DEST40 destination vector .HEK293 and HEK293T cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS). J-LAT A2 was obtained from the National Institutes of Health (NIH) AIDS reagent repository, and cultured in Roswell Park Memorial Institute (RPMI) 1640 medium with 10% FBS. pQCXIP-FLAG-Tat and HIV-LTR-luciferase vectors were described previously . pcDNA-VProstratin, suberoylanilide hydroxamic acid (SAHA), and JQ1 were purchased . Compounds were used at the following concentrations: prostratin (1 μM), SAHA (0.5 μM), JQ1 (1 μM). Drug-treated cells were cultured in the presence of compounds for 24 h and subjected to flow cytometry assays on a FACS Calibur flow cytometer , and results acquired using BD CellQuest software and analyzed using FlowJo vx.0.7 program. Puromycin for selecting stably-transduced cells was purchased . Lentviruses were produced by transfecting pAPM-TAP-short hairpin RNA (shRNA) plasmids in HEK293T cells using TransIT®-293 Transfection Reagent . Cell supernatants containing lentiviruses were harvested and filtered through 0.45 μm filter . Viruses were stored in aliquots at −80 °C for later use. To generate cell lines for stable expression of shRNAs, lentiviruses were transduced in cells . At 72 h of post transduction, puromycin (1 μg/mL) was added to the medium for stable selection. The following antibodies were used in this study: rabbit anti-KA5/Tip60 ; rabbit anti-SIN3A ; mouse anti-JMJD1A ; mouse anti-V5 ; rabbit anti-FLAG ; mouse anti-FLAG ; unlabeled rabbit and mouse IgGs .shRNAs targeting TAPs were cloned into the pAPM lentiviral vector following the reported protocol . The tarPLATO for this study followed previously described protocol ,16, exce® Luciferase Assay System  and normalized to Renilla signal. All results were collected on a Luminoskan Ascent Microplate Luminometer . HEK293 cells were transiently transfected with pcDNA-V5-TAP for the gain-of-function luciferase assays. To measure the effect of TAPs on HIV-1 LTR promoter activity, HEK293 cells stably expressing pAPM-TAP-shRNA were co-transfected with pcDNA-Tat, HIV-LTR-luciferase, and pRL-TK-renilla vectors using turbofect reagent . At 48 h post-transfection, luciferase unit (LU) was measured using Dual Glo® LDS Sample Buffer , and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blots for FLAG-Tat. To investigate the interaction of FLAG-Tat and endogenous TAP, pQCXIP-FLAG-Tat was transfected in HEK293 cells. Cell lysate was immunoprecipitated using mouse/rabbit endogenous anti-TAP or mouse/rabbit IgG antibody. Protein samples were analyzed by SDS-PAGE and western blots for FLAG-Tat. Co-IP assays followed the previously described protocol  with min® Green Supermix . The qPCR reaction was performed on the CFX Connect™ Real-Time PCR Detection System (Bio-Rad), in a 20 μL volume using the following program: 95 °C for 1 m, 40 cycles of 95 °C for 15 s and 60 °C for 30 s. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. The primer sequences for TAP qPCRs: NAT10 forward, 5′-GCT CAG TCG GCT CTT CTC TT-3′, NAT10 reverse, 5′-TTC ACA ACT TTG CGG ATG AT-3′; TINP1 forward, 5′-GGA AGT CCC TCT GCC TAA AG-3′; TINP1 reverse, 5′-CCA TCT CCA ACA AAG CAC AC-3′; XRCC5 forward, 5′-GTT GCA CTT TCC TCC CTG AT-3′, XRCC5 reverse, 5′-GCC GAC TTG AGG ATT AGC TC-3′; SIN3A forward, 5′-CAT TGC TGT TCC AAT TGT CC-3′, SIN3A reverse, 5′-CTG GTG GTC CAG AGA CTT CA-3′; TH1L forward, 5′-TTG TGT TGC AAC GAG AAC AA-3′, TH1L reverse, 5′-TGC AGC TGA AAG TAC CTT GG-3′; NME1 forward, 5′-GAG ACC AAC CCT GCA GAC TC-3′, NME1 reverse, 5′-CCT TCT CTG CAC TCT CCA CA-3′; IFI6 forward, 5′-GGT CTG CGA TCC TGA ATG GG-3′, IFI6 reverse, 5′-TCA CTA TCG AGA TAC TTG TGG GT-3′  ≥4)  B. p < 0.001) are mostly related to transcription factor activity (p < 0.0001) for 566 and 34 proteins ” minigenome. J-LAT A2 cells stably expressing TAP-shRNAs were treated with dimethyl sulfoxide (DMSO), JQ1 (a BETi), or SAHA (a HDACi), and subjected to flow cytometry analysis for the quantification of GFP-positive cells. The depletion of TH1L (NELFCD) or NME1 by shRNA served as the positive or negative control, respectively. Relative fold of GFP-positive cells as compared to shNT control was used to represent the data.At the basal level (DMSO), the percentage of the GFP-positive cell population for shNT-transduced J-LAT A2 cells was 2.06%, and all TAPs except for SIN3A showed a two-fold induction in GFP-positive cells as compared to control shNT A. In theIt is generally believed that HIV-1 trans-activator Tat recruits host proteins required for promoting viral transcription . Howeverp < 0.001) are relevant to transcription factor activity . From our studies, we successfully identified a set of TAPs that strongly associate with Tat  and suppIndeed, the depletion of transcription-suppressing TAPs enhances the reversal of latently infected HIV-1 proviruses in the J-LAT–A2 cell model, albeit at a moderate effect . It geneOverall, our proteomic studies of TAPs have identified new and previously described host factors that associate with Tat. Moreover, we have shown that some of these TAPs can impose an inhibitory effect on HIV-1 transcription and may contribute to the maintenance of HIV-1 latency. Our initial results in the simple but well-defined J-LAT–A2 cell model of HIV-1 latency support this idea. Nevertheless, further examination of these TAPs’ effects on HIV-1 latency is warranted and will be the subject of future studies. On the other hand, this present study also establishes the PLATO method as a great tool to study host-virus protein interactions. Here, we used HIV Tat as a platform to study such interactions; however, it is conceivable that this technique can be used for viral proteins from other viruses. The fact that we identified both new and previously described TAPs argues that our assay is highly reliable. In addition, we have demonstrated that our hits also had a functional role in HIV-1 infection. Consequently, in the ever-growing concern for emerging pathogens such as Zika or Ebola, our technique will be a great additional tool to elucidate important virus-host interactions to help tailor proper drug development.


MicroRNAs (miRs) are mRNA-regulatory molecules that fine-tune gene expression and modulate both processes of development and tumorigenesis. Our previous studies identified progranulin A (GrnA) as a growth factor which induces zebrafish hepatic outgrowth through MET signaling. We also found that miR-145 is one of potential fine-tuning regulators of GrnA involved in embryonic hepatic outgrowth. The low level of miR-145 seen in hepatocarinogenesis has been shown to promote pathological liver growth. However, little is known about the regulatory mechanism of miR-145 in embryonic liver development. In this study, we demonstrate a significant decrease in miR-145 expression during hepatogenesis. We modulate miR-145 expression in zebrafish embryos by injection with a miR-145 mimic or a miR-145 hairpin inhibitor. Altered embryonic liver outgrowth is observed in response to miR-145 expression modulation. We also confirm a critical role of miR-145 in hepatic outgrowth by using whole-mount in situ hybridization. Loss of miR-145 expression in embryos results in hepatic cell proliferation, and vice versa. Furthermore, we demonstrate that GrnA is a target of miR-145 and GrnA-induced MET signaling is also regulated by miR-145 as determined by luciferase reporter assay and gene expression analysis, respectively. In addition, co-injection of GrnA mRNA with miR-145 mimic or MO-GrnA with miR-145 inhibitor restores the liver defects caused by dysregulation of miR-145 expression. In conclusion, our findings suggest an important role of miR-145 in regulating GrnA-dependent hepatic outgrowth in zebrafish embryonic development. The liver is the largest metabolic organ that maintains numerous vital functions in the body. The liver is composed of approximately 80% hepatocytes and other cell types, including cholangiocytes, Kupffer cells, stellate cells and sinusoidal endothelial cells . Liver dProgranulin (PGRN), a pleiotropic autocrine growth factor, contributes to wound healing , frontotMicroRNAs (miRNAs) are approximately 22-nucleotide noncoding RNAs that regulates gene expression by directly targeting complementary messenger RNAs and affect mRNA degradation or translational inhibition . Many stZebrafish are a suitable animal model for liver development studies because the stages and mechanisms of liver development are conserved between zebrafish and mammals. In the present study, we reveal that miR-145 regulates embryonic liver size by controlling hepatocyte proliferation. In this process, miR-145 directly targets the mRNA of GrnA, and GrnA rescues the miR-145-induced liver defect. Taken together, our findings indicate that miR-145 regulates GrnA-dependent hepatic outgrowth in zebrafish development.Tg (fabp10:EGFP) (> 3 months) were used for egg production. The approximately 30 fish of mixed sex were kept in recirculation systems  in 2-litre aquaria under a 14h:10h light-dark cycle and fed twice daily. The embryos were collected using natural mating and transferred to Petri dishes filled with E3 medium  methylene blue, in dH2O). The embryos and larvae were kept in an incubator set at 28°C under a 14h:10h light-dark period [The wild-type (AB) zebrafish (Danio rerio) and the transgenic line k period . To prevk period . In all GrnA, miR-9, miR-206, miR-731, and miR-217 on 3’ UTR were predicted by TargetScan (http://www.targetscan.org/). The miR-145 on GrnA CDS region was predicted by MicroInspector [Computer-based programs were used to predict potential miRNAs targeting zebrafish GrnA. From miRNAs prediction of nspector  and RNAhnspector .5’-UCACAACCUCCUAGAAAGAGUAGA-3’), 1.5 and 3 ng/embryo miR-145 mimic (5’-GUCCAGUUUUCCCAGGAAUCCCU-3’), 3 ng/embryo miR-9 mimic (5’-UCUUUGGUUAUCUAGCUGUAUGA-3’), 0.03ng/embryo miR-206 mimic (5′-UGGAAUGUAAGGAAGUGUGUGG-3’), 3 ng/embryo miR-731 mimic (5’-AAUGACACGUUUUCUCCCGGAUCG-3’), 3 ng/embryo miR-217 mimic (5’-UACUGCAUCAGGAACUGAUUGG-3’) and 1.5 and 3 ng/embryo miR-145 inhibitor  were injected into one-cell stage embryos. The miR-145 inhibitor is 2′-O-methyl antisense oligonucleotides that against miR-145 were synthesized by Dharmacon RNA Technologies. Zebrafish GrnA  mRNA was synthesized using the mMESSAGE mMACHINE kit  and grnA morpholino  . These were co-injected with miR-145 mimic or inhibitor for the rescue assay.The 3 ng/embryo of control mimic (ZFL cells (ATCC Number: CRL-2643) from zebrafish normal liver tissue were cultured in Leibovitz’s L-15 medium containing 50% 2 mM L-glutamine (Vitacell 30–2008), 35% Dulbecco's Modified Eagle's medium (GIBCO12100), and 15% Ham’s F12 (GIBCO 21700), which was supplemented with 5% fetal bovine serum, 0.15 g/L sodium bicarbonate, 15 mM HEPES, 0.01 mg/ml insulin, 50 ng/ml EGF and penicillin/streptomycin (Invitrogen). miR-145 mimic, miR-145 hairpin inhibitor and negative-control mimic were purchased from Dharmacon (Thermo). The day before transfection, the cells were seeded in antibiotic-free medium. Transfections of plasmids and RNA duplexes were carried out using LipofectamineTM 2000 in accordance with the manufacturer's guidelines (Invitrogen).TCGAGTCAGCCGGAGACTTGGACTGGAGCAACTGGGTCAACTGGAAGC, reverse CAGTCGGCCTCTGAACCTGACCTCGTTGACCCAGTTGACCTTCGCCGG. ZFL cells (~5×105) were co-transfected with miR-145 mimic (50 nM) or control mimic (50 nM) and target reporter plasmid (1.6 μg) using LipofectamineTM 2000 (Invitrogen). The transfections and luciferase activity measurements were carried out using the Invitrogen LipofectamineTM 2000 / Promega Dual-luciferase kit according to the manufacturer’s instructions. The relative renilla activity was assessed as renilla/firefly luciferase ratios.The predicted target sequences of GrnA were cloned downstream of the renilla luciferase gene (XhoI/NotI sites) in the psiCheck-2 plasmid (Promega) and designated the psi-Grna vector. The predicted target sequences of grnA were (5’ to 3’): forward For immunohistochemistry, fixed and paraffin-embedded embryos were sectioned and hybridized with a PCNA antibody  and using an In situ Cell Death Detection Kit, POD . For whole-mount in situ hybridization (WISH), digoxigenin (DIG)-labeled or fluorescein-labeled antisense RNA probes for grna (NM_001001949), hhex (NM_130934), sePb (XM_001923882), prox1 (NM_131405) and fabp10 (NM_152960) were generated by in vitro transcription using T7 or SP6 RNA polymerase. miR-145 5’ and 3’ DIG-labeled LNA probes (Exiqon) were used for miRNA in situ hybridization. Confocal images were obtained using a Leica SP5 confocal microscope and were analyzed using Imaris software.The expression levels of miR-145, GrnA and MET signaling were measured in zebrafish embryos treated with control, miR-145 mimic or miR-145 hairpin inhibitor using a High Capacity cDNA Reverse Transcription kit and Power SYBR Green PCR master Mix (Applied Biosystems). Total RNAs were extracted from each sample with TRIzol reagent (Invitrogen). The reverse transcription reaction was performed with 1 μg of total RNA. Real-time PCR was performed using a LightCycler 480 Real-time PCR system (Roche) according to the manufacturer’s instructions. The levels of U6 RNA and ef1a mRNA were used to normalize the relative miRNA and mRNA abundance.EWEDHKQKKPETQRTTTRPTG (corresponding to residues 244–264 of GrnA) to immunize BALB/c mice ; Met antibody ; and GAPDH antibody .ZFL cells were transfected in six-well plates with miR-145 mimic , miR-145 hairpin inhibitor  or miR-control . After transfection, cells were cultured for 48 hours. Intermediate samples were collected and analyzed by western blot to assess GrnA and MET expression. The lysates were hybridized with the following primary antibodies: the polyclonal anti-GrnA antibody (1:1000), which was produced using the 4MAPS peptide All 2-dimensional images were captured using Zeiss SV-11 APO Microscope and SPOT RT3 color digital camera and analyzed by SPOT image software. The liver size was quantified by measuring the fluorescence intensity in 3-dimensional images using Leica SP5 confocal microscope and analyzed by Imaris software.t-test was used to determine the significance of differences between groups .Twenty, 30 or 100 embryos (from one clutch) per experimental group were used. Three independent replicates were performed. Data were presented as mean ± SEM (standard error of mean). Two-tailed unpaired Student’s Tg(fabp10:EGFP) embryos. The liver size was examined by measuring the volume of EGFP expression in 3-dimension confocal images. The EGFP expression of Tg (fabp10:EGFP) fish showed no significantly changes in liver size in miR-9 mimic-, miR-206 mimic-, miR-731 mimic- and miR-217 mimic-injected fish, but the miR-145 mimic-injected fish has a smaller liver size (-3mm3 and reduced to 25% liver size compared to those seen in WT (2 ± 0.38×10-3mm3)-, control mimic (1.95 ± 0.53×10-3mm3)-, miR-9 mimic (1.93 ± 0.13×10-3mm3)-, miR-206 mimic (2.05 ± 0.18×10-3mm3)-, miR-731 mimic (2.16 ± 0.19×10-3mm3)- and miR-217 mimic (1.94 ± 0.42×10-3mm3)-injected fish  from the miRNA predictions of GrnA using bioinformatics software. The miRNA predictions of GrnA included miR-9, miR-206, miR-731, miR-217 on 3’UTR and miR-145 on CDS region . Liver mver size . The livted fish . These fGrnA expression patterns specifically in liver development. Fluorescence-labeled RNA probes of the hepatoblast-specific marker prospero-related homeobox 1 (prox1) and DIG-labeled miR-145 or GrnA RNA probes were detected at 30, 50, 72 and 96 hpf in wild-type (AB) zebrafish embryos. miR-145 was expressed in hepatoblasts at 30 and 50 hpf but decreased at 72 and 96 hpf  was used to determine the miR-145 and d 96 hpf . In contd 96 hpf . To analt 72 hpf . These rC. elegans microRNAs that have minimal sequence identity to zebrafish. A total of 3 ng of control, miR-145 inhibitor or miR-145 mimic was injected into wild-type zebrafish embryos. In situ hybridization using an LNA antisense probe against mature miR-145 indicated that the miR-145 inhibitor reduced the level of miR-145  zebrafish. The Tg(fabp10:EGFP) is a transgenic fish that has liver-specific expression of enhanced green fluorescent protein (EGFP) at 36 hpf [Tg(fabp10:EGFP) embryos was 2.0 ± 0.5×10-3mm3. The liver size of miR-145 inhibitor-injected Tg(fabp10:EGFP) embryos increased by approximately 170% (3.4 ± 0.6×10-3mm3); however, the liver size of miR-145 mimic-injected Tg(fabp10:EGFP) embryos reduced to 25% of the liver size of the control group (0.5± 0.1×10-3mm3)  . These rhhex). The miR-145 inhibitor- and miR-145 mimic-injected embryos exhibited a normal expression pattern of hhex at 30 hpf (sePb) was detected at 50 hpf and also exhibited a normal expression pattern in miR-145 inhibitor- and miR-145 mimic-injected embryos as compared to those in controls (prox1) and fatty acid-binding protein 10 (fabp10) were expressed in mature hepatocytes after 72 hpf. miR-145 inhibitor-injected embryos had increased prox1 expression and miR-145 mimic-injected embryos had reduced prox1 expression at 72 hpf  was used to clarify the stages of liver development affected by miR-145. We detected the gene expression of the liver specification marker hematopoietically expressed homeobox  to observe cell proliferation in liver sections at 4 dpf in control mimic-, miR-145 mimic- and miR-145 inhibitor-injected fish. The percentage of PCNA-positive hepatocytes was increased by 2.4-fold in response to miR-145 inhibition in embryos at 4 dpf: 36.7 ± 6.7% in the control versus 90 ± 10% in the miR-145 inhibitor-injected fish . In contpsi-grna vector, psi-grna / psi-grna mutant vector into the zebrafish liver cell line (ZFL cell line) for the luciferase assay. Luciferase activity was suppressed approximately 45% when miR-145 was co-expressed with the psi-grna vector, but there was no significant effect on the mutant control  fish (Tg(fabp10: EGFP) embryos restored normal liver size (Tg(fabp10: EGFP) fish (Tg(fabp10: EGFP) embryos and observed that the liver size was restored to normal (-3mm3) in miR-145 inhibitor-injected fish and smaller liver (0.5± 0.1×10-3mm3) in miR-145 mimic-injected fish as compared with normal livers in WT and control mimic-injected fish . Co-injection of miR-145 inhibitor with GrnA morpholino restored normal liver size (1.8 ± 0.2×10-3mm3); similarly, co-injection of miR-145 mimic and GrnA mRNA also restored normal liver size (2.2 ± 0.2×10-3mm3)  fish . Co-injever size . SmallerFP) fish . We co-io normal  comparedo normal . The qua10-3mm3) . In addit manner . These rThe regulatory mechanisms of liver development are complex, and involve in many growth factors and transcription factors across three stages-hepatic specification, differentiation and outgrowth. The miRNAs are known to be important genetic regulators in development. A liver specific miRNA, miR-122a, has been reported to control hepatocyte differentiation . Besideshhex, a marker for liver specification, is affected neither by the loss of miR-145 nor by miR-145 overexpression. At liver budding stages, sePb expression is also not significantly altered. In contrast, at hepatic outgrowth stages, loss of miR-145 increases prox1 and fabp10 expression, while overexpression of miR-145 decreases prox1 and fabp10 expression. We show that manipulation of miR-145 has minor effects on the expression of specification- and differentiation-related genes. However, we cannot exclude the possibilities of miR-145 involvement in hepatic specification and/or differentiation. We hypothesize that hepatocyte outgrowth is the major target of miR-145 during liver development and that the process of hepatic proliferation is activated in the outgrowth stages. We reason that loss or attenuation of miR-145 expression promotes hepatic proliferation and liver outgrowth, and inversely, miR-145 overexpression suppresses hepatic proliferation and liver growth. Interestingly, miR-145 expression is downregulated in liver cancer, and restoration of miR-145 expression in liver tumor cells results in inhibition of proliferation in these cells [Numerous reports have indicated that miR-145 is a suppressor of tumor cell proliferation in various types of cancers –39, inclse cells , 22. OurGrnA. In a previous study, GrnA is shown to be essential for hepatic outgrowth through transcriptional regulation of MET during liver development [In this study, we decide to determine whether the direct target gene of the miRNA is useful for studying the biological function of miR-145 during liver development. The seed sequence between the 5’ end of the microRNA and the mRNA is a critical determinant of the potential binding site. Usually, a perfect or near perfect match of the seed sequence between miRNA and mRNA targets will lead to mRNA degradation . We haveelopment . Our finIn addition, we find an inverse correlation between miR-145 downregulation and GrnA overexpression in zebrafish liver outgrowth. Similar gene expression patterns of miR-145 and PGRN also occur in pathological liver growth. miR-145 downregulation and PGRN overexpression are required for liver tumor cell proliferation. PGRN is a growth factor and has been reported to be associated with the tumor growth of many cancers, such as breast , ovary , liver  and bileTaken together, we identify a new role of miR-145 that is required for hepatic outgrowth through regulating GrnA expression. Moreover, we provide a zebrafish model that could be used to study the regulatory mechanisms of miRNAs and their target mRNAs during liver development. The regulatory mechanism of miR-145 and GrnA is important not only in physiological liver growth but also in pathological liver growth. This mechanism may be a new therapeutic target for liver cancer.S1 Figgata6 expression is regulated by altered expression of miR-145 (H). The ef1a and U6 expression was measured as a loading control. .The miR-145 expression pattern at 72 hpf was examined using WISH and qPCR in control-, miR-145 inhibitor- and miR-145 mimic-injected fish. At 72 hpf, WISH indicates normal miR-145 expression in control-injected fish (A and B), reduced miR-145 expression in miR-145 inhibitor-injected fish (C and D) and overexpression of miR-145 in miR-145 mimic-injected fish (E and F). . The qPCR analysis reveals that the miR-145 mimic and inhibitor modulate miR-145 expression (G). In addition, the qPCR analysis reveals that (TIFF)Click here for additional data file.S2 FigTg(fabp10:EGFP) embryos. .Liver morphology at 4 dpf after 1.5 ng and 3 ng control mimic, miR-145 inhibitor, miR-145 mimic injection in (TIFF)Click here for additional data file.S3 Fighand2) at 30 hpf and myogenic differentiation 1(myod) at 54 hpf was examined using WISH. The result reveals mesoderm development was not significantly affected by manipulation of miR-145 expression (A). The body length were measured at 96 hpf after control mimic, miR-145 inhibitor, miR-145 mimic injection in Tg(fabp10:EGFP) embryos (B). .Two mesodermal genes, heart and neural crest derivatives expressed transcript 2 ((TIFF)Click here for additional data file.S4 FigTg(fabp10:EGFP) embryos at 4 dpf (A) or in embryos injected with control mimic (B); miR-145 inhibitor (C); miR-145 mimic (D); miR-145 inhibitor with three doses of grnA MO 0.0625 ng/embryo (E), 0.125 ng/embryo (F), or 0.25 ng/embryo (G); and miR-145 mimic with three doses of grnA mRNA 0.1 ng/embryo (H), 0.2 ng/embryo (I), or 0.4 ng/embryo (J). .Liver morphology was determined by EGFP expression in (TIFF)Click here for additional data file.S5 Fig(TIFF)Click here for additional data file.


Hippo (Hpo) signaling pathway controls tissue growth by regulating the subcellular localization of Yorkie (Yki)/Yap via a cytoplasmic kinase cassette containing an upstream kinase Hpo/MST1/2 and a downstream kinase Warts (Wts)/Lats1/2. Here we show that PRP4K, a kinase involved in mRNA splicing, phosphorylates Yki/Yap in the nucleus to prevent its nuclear accumulation and restrict Hpo pathway target gene expression. PRP4K inactivation accelerates whereas excessive PRP4K inhibits Yki-driven tissue overgrowth. PRP4K phosphorylates a subset of Wts/Lats1/2 sites on Yki/Yap to inhibit the binding of Yki/Yap to the Scalloped (Sd)/TEAD transcription factor and exclude Yki/Yap nuclear localization depending on nuclear export. Furthermore, PRP4K inhibits proliferation and invasiveness of cultured breast cancer cells and its high expression correlates with good prognosis in breast cancer patients. Our study unravels an unanticipated layer of Hpo pathway regulation and suggests that PRP4K-mediated Yki/Yap phosphorylation in the nucleus provides a fail-safe mechanism to restrict aberrant pathway activation. The Hippo signaling pathway controls tissue growth by regulating the subcellular localization of Yorkie /Yap. Here the authors show that PRP4K, a kinase involved in mRNA splicing, phosphorylates Yki/Yap in the nucleus, which prevents its nuclear accumulation and inhibits Hippo signaling. Drosophila to mammals4, and deregulation of Hpo pathway activity has been implicated in many human cancers5.How organs know when they reach final size and stop growth during development is an important yet still poorly understood problem in modern biology. The regulation of tissue growth and organ size depends on a delicate balance between cell proliferation and cell death, which is tightly controlled by both global and local cues. Indeed, tissue growth is not only influenced by environmental factors, such as hormonal signals and nutrients, but also by organ-intrinsic mechanisms. The Hippo (Hpo) signaling pathway has emerged as an evolutionarily conserved developmental pathway that controls tissue growth and organ size in species ranging from 10, and a downstream kinase Warts (Wts)/Lats1/2, members of the Nuclear Dbf-2-related (NDR) kinase family12. Hpo/MST1/2 phosphorylates and activates Wts/Lats1/2, which in turn phosphorylates and inactivates Yorkie (Yki)/YAP14. Recent studies indicate that MAP4K family members act in parallel with Hpo/MST1/2 to regulate Wts/Lats1/2 and Yki/YAP in both Drosophila and mammalian cells18. Phosphorylation of Yki/YAP restricts its nuclear localization in part through recruiting 14-3-322. When the activity of the kinase cascade is compromised, unphosphorylated Yki/YAP enters the nucleus and interacts with the TEAD family transcription factors Scalloped (Sd) in Drosophila and TEAD1-4 in mammals25, leading to activation of Hpo pathway target genes that regulate cell growth, proliferation, and survival.The core of the Hpo pathway is a conserved kinase cassette consisting of an upstream kinase Hpo/MST1/2, members of the Ste20 kinase family29; however, it has not been implicated in Hpo signaling and organ size control. We provided both genetic and biochemical evidence that PRP4K regulated Hpo signaling by phosphorylating Yki/Yap. We found that PRP4K acted in parallel with Wts/Lats1/2 to phosphorylate a subset of Wts/Lats1/2 sites on Yki/Yap, and that PRP4K-mediated phosphorylation restricted Yki/Yap activity by attenuating its binding to the Scalloped (Sd)/TEAD transcription factor and excluding its nuclear localization. Furthermore, PRP4K inhibited proliferation and invasiveness of cultured breast cancer cells and its high expression correlated with good prognosis in triple-negative breast cancer patients.How Yki/YAP is regulated in the nucleus is still poorly understood. Here, we conducted a kinome screen and identified PRP4K as a genetic modifier of the eye overgrowth phenotype caused by Yki overexpression. PRP4K is a nuclear kinase implicated in the regulation of mRNA splicing and spindle assembly checkpointDrosophila eyes (GMR>Yki)30. By screening through a collection of transgenic RNAi lines targeting the Drosophila kinome, we identified several kinases including Hpo, aPKC, Tao-1, Misshapen (Msn), Happy hour (Hppy), and PRP4 kinase  whose inactivation enhanced GMR>Yki induced eye overgrowth  that target different regions of PRP4K coding sequence and found that they enhanced GMR>Yki induced eye overgrowth in a similar fashion, although expression of these RNAi transgenes with GMR in otherwise wild-type eyes did not cause a discernible change in eye size  grew larger than GMR-Yki eyes carrying control clones  failed to suppress the GMR>Yki phenotype  increased the expression of multiple Hpo pathway target genes including expanded (ex-lacZ), diap1 (diap1-GFP3.5), bantam (ban-lacZ), and wingless (wg)  Fig. . Co-exprlls Fig. . On the nes Fig. , consistnes Fig. .Fig. 2PRKR with Myc-Yki in S2 cells induced a mobility shift of Myc-Yki on a phospho-tag gel that reduces the mobility of phosphorylated proteins  did not affect PRP4K-induced mobility shift of Yki, whereas mutating S111 and S250 (Yki2SA) or mutating all three sites (Yki3SA) abolished PRP4K-induced mobility shift in S2 cells  were immunopurified from S2 cells and incubated with purified GST fusion proteins containing Yki fragments that harbor either wild type or mutated S111, S168, or S250  at the +2 position, S168 contains Pro at this position, which confers binding to 14-3-3 after S168 is phosphorylated by Wts. Interestingly, conversion of Pro to Asp (GST-S168PD) allowed this site to be phosphorylated by PRP4K  and its S168A mutant (Myc-YkiS168A); however, PRP4K did not affect the subcellular localization of Myc-Yki2SA and Myc-Yki3SA  reporter activity stimulated by Yki and YkiS168A but failed to inhibit Yki2SA- or Yki3SA-induced 3XSd2-luc expression , into HEK293 cells induced phosphorylation of co-transfected Yap 17 abolished Mst1-induced but not PRP4K-induced Yap phosphorylation  are similar to the S111/S250 site of Yki in that they have acid residues (D/E) or T at this position  or all five sites (Yap5SA) abolished PRP4K-mediated phosphorylation of Yap 35 abolished PRP4K-mediated phosphorylation of Taz , leading to its nuclear exclusion in a manner depending on nuclear export.40, which promotes its association with 14-3-3 and cytoplasmic sequestration. Yki/Yap/Taz phosphorylation is likely to be dynamic due to the action of Yki/Yap/Taz phosphatase42. As a consequence, dephosphorylated Yki/Yap/Taz may enter the nucleus to cause leaky activation of Hpo pathway target genes. However, we found that Yki/Yap/Taz was phosphorylated by PRP4K in the nucleus, which promoted its nuclear export  is a P-element insertional allele of PRP4K/CG7028 (Flybase). It is a lethal mutation caused by an insertion of P{EPgy2} at the 5' region of the CG7028 coding sequence. P{EPgy2} was inserted in the opposite orientation relative to CG7028 transcription. wtsX1 is strong allele of wts12. Transgenic RNAi lines: UAS-PRP4K-RNAi-1 (VDRC#107042), UAS-PRP4K-RNAi-2 (VDRC#27698), UAS-PRP4K-RNAi-3 (BL#55640), and UAS-Wts-RNAi (VDRC#106174). Transgenes: UAS-Yki, UAS-YkiS168A, UAS-Sd-GA, and diap-GFP3.521, UAS-Yki2SA and UAS-Yki3SA22, UAS-InRAC (BL#8263), bam-lacZ43, and ex-lacZ44. Gal4 drivers: GMR-Gal445 and hh-Gal421. Drosophila eyes containing control or PRP4KEY11156 mutant clones with or without Yki overexpression were generated using the following genotypes:Mutant stocks: y w ey-Flp; FRT80A/RpS14[4] w+FRT80A, y w ey-Flp; EY11156 FRT80A/RpS14[4] w+FRT80A, y w ey-Flp; GMR-Gal4 UAS-Yki; FRT80A/RpS14[4] w+FRT80A, y w ey-Flp; GMR-Gal4 UAS-Yki; EY11156 FRT80A/RpS14[4] w+FRT80A.wts clones expressing UAS-GFP or UAS-PRP4K-GFP: y w UAS-GFP hs-Flp; tubulin-GaL4/+; FRT82B tubulin-Gal80/FRT82B wtsX1,y w hs-Flp; tubulin-GaL4/UAS-PPR4K-GFP; FRT82B tubulin-Gal80/FRT82B wtsX1.pUAST constructs: UAS-Myc-Yki, UAS-Myc-YkiS168A, UAS-Myc-Yki2SA, UAS-Myc-Yki3SA, UAS-Flag-Hpo, UAS-Sav, and UAS-HA-Sd were previously described 22. Myr-Yki-GFP contained a myristoylation signal MGNKCCSKRQ and GFP tag at the N- and C-terminus, respectively. Drosophila PRP4K cDNA clone was obtained from DGRC (Flybase). PRP4KKR (K617R) variant was generated by PCR-mediated site-directed mutagenesis. GFP or HA tag was inserted at the C-terminus of PRP4K or PRP4KKR. The constructs were subcloned into the pUAST vector digested with NotI and XbaI. pcDNA3.1 constructs expressing Flag-Yap, Flag-YAPS127A, Flag-YAP5SA, HA-Taz, and HA-Taz4SA are a gift from Dr. Yingzi Yang . Flag-YAP4SA was derived from Flag-YAP5SA by PCR-mediated site-directed mutagenesis to convert A127 back to S127. Human PRP4K was obtained from Dharmacon. PRP4KKR (K718R) was generated by PCR-mediated site direct mutagenesis. GFP tag was inserted at the C-terminus of PRP4K or PRP4KKR and the constructs were subcloned into pcDNA3.1 for transient transfection experiments or FUXW for lentiviral infection experiments. To make GST-Yki fusion constructs, synthetic DNA oligos corresponding to the coding sequences for Yki aa105-122, aa160-181, aa 237-260, and their SA or PD mutants  were annealed and subcloned into pGEX 4T-1 vector digested with EcoRI and XhoI.Drosophila Medium (Life Technologies) with 10% fetal bovine serum , penicillin , and streptomycin  at 24 °C. Transfection of S2 cells was performed using Calcium Phosphate Transfection Kit  following manufacturer’s instruction. A ubiquitin-Gal4 construct was co-transfected with pUAST constructs to drive UAS transgene expression in S2 cells. HEK293A and MDA-MB-231 cells were obtained from the American Type Culture Collection (ATCC). Cell line authentication utilizing Short Tandem Repeat (STR) profiling were performed with PowerPlex® 21 System , which allowed for detection of 21 loci. The PCR amplification of DNA templates were analyzed by capillary electrophoresis with genetic analyzer 3130  and allele call was performed by GeneMapper software V3.2.1. The markers were used to compare with the accessible database archived by ATCC. The cells were cultured in DMEM (Sigma-Aldrich) containing 10% BCS (ATCC), and transfected using GenJet Plus in vitro DNA transfection kit (SignaGen). For starvation experiments, HEK293A cells were cultured in DMEM (Sigma-Aldrich) without serum for 6 h two days after transfection. For lentivirus production, HEK293T-17 cells were seeded and transfected with psPAX2, VSVG, and FUXW expressing GFP, PRP4K-GFP, or PRP4KK718R-GFP. The recombinant viruses were infected into MDA-MB-231 cells using the standard method.S2 cells were cultured in Schneider’s For RNAi experiments in S2 cells, dsRNAs were generated using the MEGAscript High Yield Transcription Kit (Ambion). The following primers were used for generating the dsRNA targeting Wts: TAATACGACTCACTATAGGGCACCCAGTTATTGTCG (F) and TAAACGACTCACTATAGGGGTTCTTCATGGAGCAGCACATAG (R). dsRNA targeting the coding sequence of Luciferase was used as a control. For RNAi in mammalian cells, siRNA was transfected into cells using Lipofectamine RNAi-MAX (Life Technologies) in antibiotics-free medium according to manufacturer’s instruction. Oligonucleotides of siRNA duplexes were purchased from Dharmacon as follow: siPRP4K#1 (J-004074-11), siPRP4K#2 (J-004074-12), and siYap1 (J-012200-07 and J-012200-08), and siControl (D-001810-03-05).3VO4, 1% NP-40, 10% Glycerol, and 0.5 M EDTA (pH8.0). Cell lysates were incubated with protein A–Sepharose beads (Thermo scientific) for 1 h at 4 C° to eliminate non-specific binding proteins. After removal of the protein-A beads by centrifugation, the cleared lysates were incubated with Myc (HA or Flag) antibody for 2 h or overnight. The complexes were collected by incubation with protein A–Sepharose beads for 1 h at 4 C°, followed by centrifugation. The immunoprecipitates were then washed three times for 5 min each with lysis buffer and were separated on SDS-PAGE. West blot was carried out using standard protocol. Uncropped blots are shown in Supplementary Fig. 46. Immunostaining of imaginal discs was carried out using standard protocal47. For immunostaining of cultured cells, cells were seeded on LAB-TEK chamber slides, transfected with indicated constructs and stained using standard protocols. Antibodies used: rabbit anti-sed: , mouse anti-Wg , rat anti-Ci , rabbit anti-Yki 16, Yki S168 phospho-specific antibody 19, mouse anti-GFP , rabbit anti-GFP , mouse anti-Myc , mouse anti-Flag , mouse anti-HA , rabbit anti-HA , rabbit anti-Lats1 , mouse anti-Yap , rabbit anti-Yap , rabbit anti-PRP4K , mouse anti-Histon-H3 , and mouse anti-tubulin .For immunoprecipitation assay, cells were harvested and washed twice with PBS after transfection for 48 h and then lysed on ice for 30 min with lysis buffer containing 1 M Tris pH8.0, 5 M NaCl, 1 M NaF, 0.1 M NaTotal RNA was extracted using RNeasy Plus Mini Kit (Qiagen #74134), and cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad). RT-qPCR was performed using iQ SYBR Green System (Bio-Rad) and a Bio-rad CFX96 real-time PCR system.36B4 expression level was used a normalization control. Primer sequences used are:CTGF, CATCTTCGGTGGTACGGTGT (F) and TTCCAGTCGGTAAGCCGC (R); CYR61, CGGGTTTCTTTCACAAGGCG (F) and TGAAGCGGCTCCCTGTTTTT (R); ANKRD1, GCCATGCCTTCAAAATGCCA (F) and AGAACTGTGCTGGGAAGACG (R) AJUBA, TACCAGGACGAGCTAACAGC (F) and TACAGGTGCCGAAGTAGTCC (R) 36B4, GGCGACCTGGAAGTCCAACT (F) and CCATCAGCACCACAGCCTTC (R).32p-ATP, and immune-purified HA-PRP4K or HA-PRP4KKR expressed in S2 cells. After incubation at 30 °C for 1.5 h in the reaction buffer, the reactions were stopped by adding 4 × SDS loading buffer and boiled at 100 °C for 5 min, the phosphorylation of GST-fusion proteins were analyzed by autoradiography after SDS-PAGE.GST-fusion proteins were expressed in bacteria, purified with glutathione bead, and mixed with 0.1 mM ATP, 10 mCi γ-To measure the activity of Yki/YAP/TAZ, cells were seeded in 24-well plates and co-transfected with a Sd -dependent luciferase reporter construct (3XSd2-luc) with pTK-Renilla and effector plasmids. The luciferase activities were analyzed using a dual-luciferase reporter assay kit (Promega) according to manufacturer’s instruction.MDAMB231 cells were seeded and transfected with 50 μM PRP4K siRNA or control siRNA. Twenty-four hours after transfection, cells were seeded into 96-well plates. Cell numbers were determined using WST-1 cell proliferation reagent  at indicated time points.t = 0)] × 100%MDAMB231 cells were seeded and transfected with 50 μM PRP4K siRNA or control siRNA. Twenty-four hours after transfection, the cells were seeded into 6-well plates with 1% FBS with 100% confluence. One yellow pipette tip was used to make a straight scratch. The wound distance was measured at indicated time points and normalized with starting time point. Percentage wound recovery was expressed as: [1-. For the transwell assay, 1 × 10http://kmplot.com/analysis/index.php?p=service&cancer=breast). The gene affy IDs are 224894_at and 202127_at for YAP and PRP4K, respectively. 'Auto selected best cutoff' and 'basal type' were selected for triple-negative breast cancer analysis.The PFS survival data of PRP4K and YAP was generated from KMPLOT online analysis database (t-test to determine significance when only 2 groups were compared. P-values of less than 0.05 and 0.01 were considered significant. Error bars on all graphs are presented as the s.d.All experiments were performed for at least two independent times unless noted otherwise. Statistical analysis between groups was performed by two-tailed Student’s All relevant data are available from the authors.Supplementary InformationPeer Review File


Mutations in clr-1/RPTP result in reduced NLG-1 GRASP fluorescence and impaired behavioral output of the PHB circuit. Temperature-shift experiments demonstrate that clr-1/RPTP acts early in development, consistent with a role in SPR. Expression and cell-specific rescue experiments indicate that clr-1/RPTP functions in postsynaptic AVA neurons, and overexpression of clr-1/RPTP in AVA neurons is sufficient to direct additional PHB-AVA synaptogenesis. Genetic analysis reveals that clr-1/RPTP acts in the same pathway as the unc-6/Netrin ligand and the unc-40/DCC receptor, which act in AVA and PHB neurons, respectively. This study defines a new mechanism by which SPR is governed, and demonstrates that these three conserved families of molecules, with roles in neurological disorders and cancer, can act together to regulate communication between cells.During neural circuit formation, most axons are guided to complex environments, coming into contact with multiple potential synaptic partners. However, it is critical that they recognize specific neurons with which to form synapses. Here, we utilize the split GFP-based marker Neuroligin-1 GFP Reconstitution Across Synaptic Partners (NLG-1 GRASP) to visualize specific synapses in live animals, and a circuit-specific behavioral assay to probe circuit function. We demonstrate that the receptor protein tyrosine phosphatase (RPTP)  The nervous system is required for many body functions including perception, behavior and thought. Cells in the nervous system called neurons function in interconnected groups called circuits to carry out these basic functions. While we have learned a great deal about how circuits function, we know much less about how they are set up during development. Discovering the mechanisms that organize these neural circuits could help us to understand neurological disorders that may result from defects in this process. Our work has identified a key role for the cell surface molecule CLR-1 in a critical step in the formation of neural circuits: the recognition between neurons that must link together. We find that CLR-1 acts with the ligand Netrin and its receptor Deleted in Colorectal Cancer (DCC) to mediate communication between adjacent cells. Interestingly, all three of these molecules have been linked to schizophrenia and to cancer, indicating that our discovery may help inform our understanding of these diseases. Perception, thought, and behavior all rely on the faithful transfer of information between neurons. During development, neurons form circuits through a series of well-characterized steps, including neuronal migration, guidance of axons and dendrites into target regions, and finally the formation of synapses between presumptive neuronal partners. However, within a target region, most neurites contact many potential partners. To form functional circuits, neurons must faithfully recognize and form synapses only with the correct neuronal partners , 2. RelaCaenorhabditis elegans hermaphrodites, which mediates avoidance of toxin-producing Streptomyces bacteria [C. elegans  with extracellular domains similar to those in the Leukocyte common Antigen-Related protein (LAR) family of RPTPs [clr-1/RPTP is necessary for formation of PHB-AVA synapses, and overexpression promotes increased synapse formation between the two neurons. CLR-1/RPTP acts in postsynaptic AVA neurons to direct SPR, and is enriched in AVA neurites in the region of synapse formation with PHB neurons. Finally, we find that clr-1/RPTP acts in the same pathway as unc-6/Netrin and unc-40/DCC to mediate SPR. Our findings demonstrate a new role for clr-1/RPTP in SPR, and indicate that these three conserved families of proteins can act together to mediate communication between cells, which may provide insight into their roles in neurological disorders [Here, we show that the of RPTPs . We demoisorders , 14 and isorders –17.clr-1 mutants, cell migration, axon guidance, and contact between PHB and AVA neurites appear normal  with immunoglobulin-like and fibronectin III domains, similar to the Leukocyte common Antigen-Related protein (LAR) family (type IIa) RPTPs [clr-1/RPTP(e1745) mutants, PHB axon length is slightly reduced, but other mutants with similarly reduced axon length, such as ced-10/Rac1(n1993), have normal NLG-1 GRASP intensity, indicating that a slight reduction in PHB-AVA contact is unlikely to result in decreased synapse formation  RPTPs , 18. In ormation . clr-1/R-10/Rac1n93, have  mutants .clr-1/RPTP mutants have a severe defect in SDS avoidance, indicating that PHB circuit function is disrupted  . Wild-tyisrupted . This deher cell . We prevnormally , thus imclr-1/RPTP mutants are due to a defect in trafficking synaptic components to the distal synaptic region of PHB and AVA neurites, or if these defects might be due to a later recognition defect in SPR, we examined levels of synaptic components in the distal region of the neurites. Levels of the presynaptic vesicle marker RAB-3::mCherry and the presynaptic active zone marker GFP::ELKS-1 in PHB, and the postsynaptic marker NLG-1::YFP in AVA, are not reduced in clr-1/RPTP mutants  and introduced it into clr-1/RPTP mutants. NLG-1 GRASP intensity and the behavioral output of the PHB circuit were rescued to wild-type levels in these transgenic animals, indicating that clr-1/RPTP can function in AVA neurons to direct SPR. Expression of the clr-1 cDNA in PHB neurons did not rescue NLG-1 GRASP intensity or the behavioral output of the PHB circuit in clr-1/RPTP mutants, consistent with a role for clr-1/RPTP in AVA neurons , and introduced it into clr-1/RPTP mutants. Unlike the full-length clr-1/RPTP cDNA under the direction of the AVA promoter, pAVA::clr-1/RPTPΔxcd did not rescue either NLG-1 GRASP fluorescence or the behavioral output of the circuit to wild-type levels; NLG-1 GRASP fluorescence was 51% of wild-type levels, and the relative response index was 206% of wild-type levels . Again, the NLG-1 GRASP intensity and behavioral output of the PHB circuit were not rescued to wild-type levels; NLG-1 GRASP fluorescence was 41% of wild-type levels, and the relative response index was 221% of wild-type levels (clr-1/RPTP phosphatase activity is required for full SPR function as well.To test if the extracellular domain of e levels . This ine domain  (pAVA::ce levels . This inclr-1/RPTP cDNA to YFP under the direction of an AVA-selective promoter (pAVA::clr-1/RPTP::YFP). We found that CLR-1/RPTP was localized throughout the AVA axon, but was brightest in the preanal ganglion, where AVA neurons contact PHB neurons. Interestingly, within the region of the preanal ganglion, it was usually concentrated in the anterior half, where the majority of synapses between PHB and AVA normally form (clr-1/RPTP cDNA to mCherry (pAVA::clr-1/RPTP::mCherry) and introduced it into animals expressing PHB-AVA NLG-1 GRASP. We similarly found that the NLG-1 GRASP signal was usually confined to the anterior half of the preanal ganglion where CLR-1/RPTP localized  allele [clr-1(e1745) animals placed at the restrictive temperature only during embryogenesis, or placed at the restrictive temperature only after embryogenesis, indicating that clr-1/RPTP function is required both during and after embryogenesis  and after the first larval stage (L1). Animals placed at the restrictive temperature only during the 3-fold embryo and L1 stages exhibited impaired behavior, while animals placed at the permissive temperature during the same stages exhibited normal behavior  allele , 20. Intogenesis . To testbehavior . This inclr-1/RPTP animals shifted from the restrictive to the permissive temperature at the 3-fold embryo stage, and shifted from the permissive to the restrictive temperature at the end of the L1 stage, possibly due to stress on the mutant animals from multiple temperature shifts. Even so, NLG-1 GRASP intensity was severely and significantly reduced from this level in animals moved to the restrictive temperature for the 3-fold embryo and L1 stages, compared with animals kept at the permissive temperature during the same periods  of pAVA::clr-1/RPTP was sufficient to drive a significant increase in PHB-AVA synaptogenesis  mutation into animals overexpressing unc-6/Netrin to determine if the high levels of synaptogenesis were suppressed. Indeed, PHB-AVA synapses were dramatically reduced  II, clr-1(e2530) II, clr-1(n1992) II, [unc-6(ev400) X [unc-40(e271) I [ced-10(n1993) IV [wyEx1364, wyEx1402, iyEx323, and iyIs8 (see below), all strains contain the integrated PHB-AVA NLG-1 GRASP marker wyIs157 IV (pSM::pgpa-6::nlg-1::spGFP1-10 (60 ng/μl), pSM::pflp-18::nlg-1::spGFP11 (30 ng/μl), pSM::pnlp-1::mCherry (10 ng/μl), pSM::pflp-18::mCherry (5 ng/μl) and podr-1::DsRed2 (20 ng/μl)) [All worms were maintained according to standard protocols  and were992) II, , 20, uncev400) X , unc-40(1993) IV . Except pAVA::clr-1/RPTP for both cell-specific rescue of clr-1(e1745) II (iyEx97 (pSM::prig-3::clr-1 (40 ng/μl), punc-122::RFP (20 ng/μl)) and iyEx101 (pSM::prig-3::clr-1 (60 ng/μl), punc-122::RFP (20 ng/μl)). Transgenic lines carrying pPHB::clr-1/RPTP include iyEx364, iyEx365, and iyEx366 (pSM::pnlp-1::clr-1 (1–5 ng/μl), punc-122::RFP (22 ng/μl)) , punc-122::RFP (20 ng/μl)) , punc-122::RFP (21 ng/μl)) (iyEx121 (pSM::prig-3::clr-1::YFP (85 ng/μl), punc-122::RFP (20 ng/μl)). The transgenic line generated to visualize CLR-1/RPTP co-localization with NLG-1 GRASP contained iyEx175 (pSM::prig-3::clr-1::mCherry (60 ng/μl), punc-122::RFP (20 ng/μl)) and iyIs2 (pSM::pgpa-6::nlg-1::GFP1-10 (30 ng/μl), pSM::pflp-18::nlg-1::GFP11 (15 ng/μl), podr-1::DsRed2 (50 ng/μl)), in a clr-1(e1745) background  II , and ove1745) II : iyEx97  ng/μl)) . Transge ng/μl)) . Transge ng/μl)) . The trackground . For ovewyEx1364 (pSM::pgpa-6::GFP (50ng/μl), pSM::pflp-18::mCherry (10 ng/μl) and punc-122::RFP (20 ng/μl)) ) .wyEx1402 (pSM::pgpa-6::GFP (50 ng/μl) and podr-1::DsRed2 (20 ng/ μl)) and wyIs157 , punc-122::RFP (20 ng/μl)) with wyIs157, and in the head was iyEx323 (pSM::pclr-1::GFP (20 ng/μl), pSM::prig-3::mCherry (17 ng/μl), punc-122::RFP (22.5 ng/μl)) (The transgenic lines generated to determine the expression pattern of  ng/μl)) .clr-1/RPTP is required for AVA-VA synapses, we stably integrated the wyEx1845 AVA-VA/DA NLG-1 GRASP marker (pSM::punc-4::nlg-1::spGFP1-10 (20 ng/μl), pSM::pflp-18::nlg-1::spGFP11 (30 ng/μl), pSM::punc-4::mCherry (5 ng/μl), podr-1::DsRed2 (50 ng/μl)) [iyIs8 (To test if ) [iyIs8 .pSM::prig-3::clr-1 (referred to as pAVA::clr-1/RPTP in the text), an N2 C. elegans cDNA library was generated by isolating C. elegans mRNA using Sigma Tri Reagent (TRIzol) to break the worm cuticle, chloroform to isolate RNA, and the Qiagen RNeasy Mini kit to purify the RNA. The Invitrogen SuperScript II Reverse Transcriptase kit was used to generate cDNA. clr-1 cDNA was amplified using clr-1-specific primers (MVP281: AGACGTCGACATGCGAATAAATCGATGGATC and MVP282: TATTTGGTACCCTACCTATATGTCTTAGAGATA) that introduced the SalI and Acc65I restriction sites. The clr-1 cDNA was subcloned into the SalI-Acc65I fragment from pSM::prig-3, which was made by subcloning the rig-3 promoter from pSM::prig-3::MT::unc-6 [pSMΔ (a gift from S. McCarroll).Constructs were generated using standard molecular techniques. To generate T::unc-6  using SppSM::pclr-1::GFP construct, (referred to as pclr-1::GFP in the text) the clr-1 promoter (4480 bp upstream of the clr-1 start site) was amplified from genomic DNA using pclr-1 specific primers (MVP227: TAACGGCGCGCCGAGAATGAGGTTACGATCTAC and MVP228: ACATACCCGGGGTTTCCGCGTTAATTTAAAAGCC) that introduced AscI and SmaI restriction sites. Then, the pclr-1 fragment was subcloned into the AscI-SmaI fragment from pSM::GFP (a gift from S. McCarroll).To generate the pSM::prig-3::clr-1::YFP (referred to as pAVA::clr-1/RPTP::YFP in the text), the clr-1 cDNA (without its stop codon) was amplified from pSM::prig-3::clr-1 using clr-1 specific primers that introduced the SalI and Acc65I restriction sites (MVP344: TATTGGTACCCCTATATGTCTTAGAGATATAG and MVP281: AGACGTCGACATGCGAATAAATCGATGGATC). The clr-1 cDNA was subcloned into the SalI-Acc65I fragment from pSM::prig-3::YFP. pSM::prig-3::YFP was made by amplifying the YFP fragment from pSM::prig-3::unc-6::YFP [pSM::prig-3.To generate c-6::YFP  using prpSM::prig-3::clr-1::mCherry (referred to as pAVA::clr-1/RPTP::mCherry in the text), the clr-1 cDNA (without its stop codon) was amplified from pSM::prig-3::clr-1 using clr-1 specific primers that introduced the SalI and Acc65I restriction sites (MVP344: TATTGGTACCCCTATATGTCTTAGAGATATAG and MVP281: AGACGTCGACATGCGAATAAATCGATGGATC). The clr-1 cDNA was subcloned into the SalI-Acc65I fragment from pSM::prig-3::mCherry. pSM::prig-3::mCherry, was made by amplifying mCherry from pttx-3::mCherry [10GSlinker::mCherry was then subcloned into the Acc65I-SacI fragment of pSM::prig-3 to generate pSM::prig-3::mCherry.To generate :mCherry  using prpSM::prig-3::clr-1Δxcd::mCherry (referred to as pAVA::clr-1/RPTPΔxcd in the text), the transmembrane and intracellular domains of the clr-1 cDNA were amplified from pSM::prig-3::clr-1, adding SalI and Acc65I sites (MVP342: AGACGTCGACGCGTATGGATATTCTGCATACT and MVP344: TATTGGTACCCCTATATGTCTTAGAGATATAG) and subcloned into pSM::prig-3::mCherry using the SalI and Acc65I sites.To generate pSM::prig-3::clr-1pd::mCherry (referred to as pAVA::clr-1/RPTPpd in the text) was generated using the Stratagene QuikChange site-directed mutagenesis kit to make a point mutation in the catalytic sequence of the D1 intracellular phosphatase domain of pSM::prig-3::clr-1, the domain predicted to be active. The cysteine at position 1013 was replaced with a serine (C1013S). Specifically, we changed G at 3038 to C (G3038C) [(G3038C) .pSM::pnlp-1::clr-1 (referred to as pPHB::clr-1/RPTP in the text) was generated by subcloning the nlp-1 promoter, flanked by the SphI and SmaI restriction sites, from pSM::pnlp-1 into the SphI-SmaI fragment of pSM::prig-3::clr-1.C. elegans under 630X magnification. Worms were anesthetized on 2% agarose pads using a 2:1 ratio of 0.3 M 2,3-butanedione monoxime (BDM) and 10 mM levamisole in M9 buffer. All micrographs taken were of larval stage 4 (L4) animals, except micrographs in A Zeiss Axio Imager.A1 compound fluorescent microscope and a Zeiss LSM710 confocal microscope were used to capture images of live All data from micrographs were quantified using NIH ImageJ software , as prevclr-1(e2530) and (n1992) did not back sufficiently for behavioral testing.To test the PHB-AVA circuit function, we utilized a high-throughput SDS avoidance assay based on the previously published SDS dry drop test , 10. BriP-values . P-values provide accurate information about whether two samples differ significantly. P-values are generated by a procedure that incorporates both the sample sizes and variability in samples, so that the reader is not required to multiply each standard error of the mean (obtained by assessing the length of error bars) by a factor that depends on each sample size. Error bars  are included for the behavioral measurements throughout the manuscript. However, the NLG-1 GRASP intensity data is not normally distributed, as previously described [In the figures, the results are reported in the form of escribed , and theescribed ) for theP-value less than 0.05, or when comparing only two NLG-1 GRASP relative median intensity values, pair-wise comparisons were made using the Mann-Whitney U-test, which is a non-parametric significance test that compares medians of two independent groups. If more than one independent test was performed, P-values were adjusted for multiple comparisons using the Hochberg procedure. If pair-wise comparisons are not independent, the Hochberg procedure is no longer appropriate, as in P-values.For comparing more than two NLG-1 GRASP relative median intensity values, a Kruskal-Wallis test, a nonparametric alternative to ANOVA that does not rely on a normality assumption, was first used. If a Kruskal-Wallis test yielded a P-values were adjusted for multiple comparisons using the Hochberg method. Statistical significance was confirmed by conducting a multi-way ANOVA model with appropriate interaction terms using the linear model procedure . For axon length, mean values were compared using an ANOVA F-test, and pair-wise comparisons were made using the t-test, and adjusted for multiple comparisons using the Hochberg method. Microsoft Excel and R statistical computing software [For behavior analysis, relative SDS response indices were compared using the t-test, and software  were useS1 Figclr-1/RPTP(e1745), and ced-10/Rac1(n1993) animals (n>40). ***P<0.001, t-test, comparison with wild-type. P-values were adjusted for multiple comparisons using the Hochberg method. (B) Quantification of a reduction in NLG-1 GRASP fluorescence in clr-1/RPTP(e1745) animals and no significant difference in NLG-1 GRASP fluorescence in ced-10/Rac1(n1993) animals, indicating that a reduction in length is not sufficient to cause a reduction in PHB-AVA synapses (n≥40). ***P<0.001, NS, not significant, U-test, comparison with wild-type. P-values were adjusted for multiple comparisons using the Hochberg method. 95% confidence intervals for the medians are included in clr-1/RPTP(e1745) mutants. AVA was labeled with mCherry (pAVA::mCherry). Note that the shorter distance in clr-1/RPTP mutants indicates slightly increased AVA neurite length.(A) Quantification of PHB axon length in wild-type, (TIF)Click here for additional data file.S2 Figclr-1/RPTP animals using the same promoters that drive the PHB-AVA NLG-1 GRASP marker in PHB and AVA neurons. NS, not significant, U-test, comparison with wild-type. P-values were adjusted for multiple comparisons using the Hochberg method. (B to D) Representative schematics and micrographs of wild-type (B and C) and clr-1/RPTP(e1745) (D and E) labeled with the presynaptic vesicle marker mCherry::rab-3 expressed in PHB (pPHB::mCherry::rab-3). (E to H) Representative schematics and micrographs of wild-type (F and G) and clr-1/RPTP(e1745) (H and I) labeled with the presynaptic active zone marker GFP::elks-1 expressed in PHB (pPHB:::GFP::elks-1). Representative schematics and micrographs of wild-type (J and K) and clr-1/RPTP(e1745) (L and M) labeled with the postsynaptic marker nlg-1::YFP expressed in AVA (pAVA::nlg-1::YFP). (N) Quantification of no significant difference in NLG-1 GRASP fluorescence in the region of the preanal ganglion in pPHB::mCherry::rab-3, pPHB:::GFP::elks-1, or pAVA::nlg-1::YFP in clr-1/RPTP(e1745) mutants compared with wild-type animals (n≥24). NS, not significant, U-test. P-values were adjusted for multiple comparisons using the Hochberg method.(A) Quantification of similar median neurite fluorescence intensity in wild-type and (TIF)Click here for additional data file.S3 Figclr-1 promoter and mCherry under the direction of the rig-3 promoter, which drives expression in AVA neurons and a few other cells in the head [pclr-1::GFP transcriptional fusion was expressed in AVA neurons (arrows), which were identified based on expression of prig-3::mCherry, their cell body position and axon morphology. (B) Wild-type animals expressing pclr-1::GFP and mCherry under the direction of a promoter that, in the posterior of the worm, is specifically expressed in PHB neurons (pnlp-1::mCherry). The pclr-1::GFP transcriptional fusion was not expressed in PHB neurons (arrow indicates placement of PHB neurons).(A) Wild-type animals expressing GFP under the direction of the the head . The pcl(TIF)Click here for additional data file.S4 Figclr-1(e1745) mutants. (B) Quantification of a severe reduction in median NLG-1 GRASP fluorescence in clr-1(e1745) animals in comparison to wild-type animals in the region between the VA10 and DA7 neurons, where synapses between AVA and VA10 are observed in wild-type animals. ***P<0.001, U-test, comparison with wild type.(A) Representative diagrams and micrographs of NLG-1 GRASP fluorescence labeling synapses between AVA and VA and DA motorneurons in wild-type and (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(XLSX)Click here for additional data file.