Gene Depletion Research Articles

Presence of an amino acid residue at position 619 required for the function of YidC in Rhodobacter sphaeroides

YidC, the bacterial homologous protein of Oxa1 and Alb3, could insert membrane proteins into the membrane. Rhodobacter sphaeroides is a kind of photobacteria with abundant intracytoplasmic membranes. In this study, the functions of R. sphaeroides YidC and its C-terminus were investigated in the Escherichia coli YidC gene depletion strain FTL10. The results showed that RS_YidC could complement the growth of the strain FTL10, but the RS_YidC last 5 residues (619–623, KKRKP) deletion mutant could not. Interestingly, the site-directed RS_YidC mutants of any one or all of these 5 residues were still active. The deletion mutant of the last 4 residues and even the last 4 residues deletion mutant with substitution of the Ala or Glu for Lys619 still had sufficient activity to complement the growth of the strain FTL10. These results indicated that the length of the C-terminus of Rs_YidC is more important for its function than the amino acid composition or the charges of it, and the presence of an amino acid residue at position 619 is required for Rs_YidC function in E. coli. Our result also suggests that Rs_YidC may function differently as compared to its homologs.

Class I Arfs (Arf1 and Arf3) and Arf6 are localized to the Flemming body and play important roles in cytokinesis

Small GTPases play important roles in various aspects of cell division as well as membrane trafficking. We and others previously showed that ADP-ribosylation factor 6 (Arf6) is locally activated around the ingressing cleavage furrow and recruited to the Flemming body in late cytokinesis phases, and involved in faithful completion of cytokinesis. However, knockout of the Arf6 gene or Arf6 depletion by siRNAs did not drastically influence cytokinesis. We here show that, in addition to Arf6, Class I Arfs (Arf1 and Arf3) are localized to the Flemming body, and that double knockdown of Arf1 and Arf3 moderately increases the proportion of multinucleate cells and simultaneous knockdown of Arf1, Arf3 and Arf6 leads to severe cytokinesis defects. These observations indicate that Arf1 and Arf3 as well as Arf6 play important roles in cytokinesis. We further show that EFA6 (exchange factor for Arf6) activates not only Arf6 but also Arf1 in the cell. Taken together with our previous data, these Arf GTPases are likely to be locally activated by EFA6 and in turn targeted to the Flemming body to complete cytokinesis.

ShRNA Depletion of cIAP1 Sensitizes Human Ovarian Cancer Cells to Anticancer Agent-Induced Apoptosis.

Emerging evidence suggests a potential role of cellular inhibitor of apoptosis protein 1 (cIAP1) in the development of human ovarian cancer. However, its function in the progression of ovarian cancer has not been clearly determined. Our study aimed to investigate the effect of cIAP1 gene depletion on the chemosensitivity of ovarian cancer cells. We developed a novel short hairpin RNA (shRNA) plasmid specifically targeting cIAP1. Cell proliferation, invasion, and apoptosis of the shRNA-transfected cells were evaluated using MTT, Transwell chamber, and flow cytometric assays, respectively. The concentration of MMP-9 in the supernatant was detected by ELISA. Targeted depletion of cIAP1 by shRNA significantly reduced expression levels of cIAP1 mRNA and protein, leading to inhibition of cell proliferation and invasion capability in SKOV3 cells. At the same time, cIAP1 downregulation decreased the secretion of MMP-9. shRNA depletion of cIAP1 enhanced chemosensitivity of ovarian cancer cells to Taxol and carboplatin-induced apoptosis. cIAP1 is associated with tumor progression in human ovarian cancer. Therefore, cIAP1 might be a potential target for therapeutic anticancer drugs.

Nanoparticle-mediated RNA interference of angiotensinogen decreases blood pressure and improves myocardial remodeling in spontaneously hypertensive rats.

Angiotensinogen (AGT) has been shown to have a role in cardiac hypertrophy, while depletion of the AGT gene in spontaneously hypertensive rats (SHR) has not been investigated. The present study investigated the effect of AGT knockdown on cardiac hypertrophy in SHR. For this, small hairpin (sh)RNAs were intravenously injected into SHRs, using a nanoparticle‑mediated transfection system. The experimental rats were divided into the following groups: a) Blank control with water treatment only, b) negative control with biscarbamate‑crosslinked Gal‑polyethylene glycol polyethylenimine nanoparticles (GPE)/negative shRNA, c) AGT‑RNA interference (RNAi) group with GPE/AGT‑shRNA, and 4) normotensive control using Wistar‑Kyoto rats (WKY) with water treatment. Three and five days following the first injection, the levels of hepatic AGT mRNA and AGT protein as well as plasma levels of AGT were markedly decreased in the AGT‑RNAi group (P<0.05). Furthermore, a significant decrease in systolic blood pressure (SBP), left ventricular weight to body weight ratio and heart weight to body weight ratio were observed in the AGT‑RNAi group compared with those in the control groups. The depletion of AGT in SHR led to a reduction in SBP by 30±4 mmHg, which was retained for >10 days. Cardiac hypertrophy was also significantly improved in AGT‑knockdown rats. In conclusion, the present study showed that AGT‑silencing had a significant inhibitory effect on hypertension and hypertensive‑induced cardiac hypertrophy in SHRs.

FOXO1/3 and PTEN Depletion in Granulosa Cells Promotes Ovarian Granulosa Cell Tumor Development.

The forkhead box (FOX), FOXO1 and FOXO3, transcription factors regulate multiple functions in mammalian cells. Selective inactivation of the Foxo1 and Foxo3 genes in murine ovarian granulosa cells severely impairs follicular development and apoptosis causing infertility, and as shown here, granulosa cell tumor (GCT) formation. Coordinate depletion of the tumor suppressor Pten gene in the Foxo1/3 strain enhanced the penetrance and onset of GCT formation. Immunostaining and Western blot analyses confirmed FOXO1 and phosphatase and tensin homolog (PTEN) depletion, maintenance of globin transcription factor (GATA) 4 and nuclear localization of FOXL2 and phosphorylated small mothers against decapentaplegic (SMAD) 2/3 in the tumor cells, recapitulating results we observed in human adult GCTs. Microarray and quantitative PCR analyses of mouse GCTs further confirmed expression of specific genes (Foxl2, Gata4, and Wnt4) controlling granulosa cell fate specification and proliferation, whereas others (Emx2, Nr0b1, Rspo1, and Wt1) were suppressed. Key genes (Amh, Bmp2, and Fshr) controlling follicle growth, apoptosis, and differentiation were also suppressed. Inhbb and Grem1 were selectively elevated, whereas reduction of Inha provided additional evidence that activin signaling and small mothers against decapentaplegic (SMAD) 2/3 phosphorylation impact GCT formation. Unexpectedly, markers of Sertoli/epithelial cells (SRY [sex determining region Y]-box 9/keratin 8) and alternatively activated macrophages (chitinase 3-like 3) were elevated in discrete subpopulations within the mouse GCTs, indicating that Foxo1/3/Pten depletion not only leads to GCTs but also to altered granulosa cell fate decisions and immune responses. Thus, analyses of the Foxo1/3/Pten mouse GCTs and human adult GCTs provide strong evidence that impaired functions of the FOXO1/3/PTEN pathways lead to dramatic changes in the molecular program within granulosa cells, chronic activin signaling in the presence of FOXL2 and GATA4, and tumor formation.

Adenovirus siMDM2 and NDRG2 Gene Therapy Inhibits Cell Proliferation and Induces Apoptosis of Squamous Cell Carcinoma.

Squamous cell carcinoma (SCC) is one of the most common skin cancers. In the present study, we explored the effects of depletion of murine double minute gene 2 (MDM2) together with overexpression of N-myc downstream-regulated gene 2 (NDRG2) on cutaneous SCC. In order to achieve high efficiency of gene knockdown and overexpression in SCC-13 cells, recombinant adenovirus carrying siMDM2 and NDRG2 expression construct was produced. We found Ad-siMDM2, Ad-NDRG2, and Ad-siMDM2-NDRG2 infections inhibit the growth of SCC-13 cells in vitro, and Ad-siMDM2-NDRG2 infection has the highest inhibitory effect. Subcutaneous injections of Ad-siMDM2, Ad-NDRG2, and Ad-siMDM2-NDRG2 into SCC-13 xenograft nude mice resulted in the reduction of tumor volume. Moreover, we found that apoptosis protein caspase 3 was up-regulated in the Ad-siMDM2-, Ad-NDRG2-, and Ad-siMDM2-NDRG2-treated groups. Our data indicate that the adenovirus-mediated MDM2 silencing and NDRG2 overexpression can synergistically inhibit local cancer cell proliferation, induce apoptosis, and further prevent metastases of SCC. Our study provides a promising method that can be further developed as a new therapeutic approach against SCC.

Cellular Cholesterol Accumulation Facilitates Ubiquitination and Lysosomal Degradation of Cell Surface-Resident ABCA1.

By excreting cellular cholesterol to apolipoprotein A-I, ATP-binding cassette transporter A1 (ABCA1) mediates the biogenesis of high-density lipoprotein in hepatocytes and prevents foam cell formation from macrophages. We recently showed that cell surface-resident ABCA1 (csABCA1) undergoes ubiquitination and later lysosomal degradation through the endosomal sorting complex required for transport system. Herein, we investigated the relevance of this degradation pathway to the turnover of csABCA1 in hypercholesterolemia. Immunoprecipitation and cell surface-biotinylation studies with HepG2 cells and mouse peritoneal macrophages showed that the ubiquitination level and degradation of csABCA1 were facilitated by treatment with a liver X receptor (LXR) agonist and acetylated low-density lipoprotein. The effects of an LXR agonist and acetylated low-density lipoprotein on the degradation of csABCA1 were repressed completely by treatment with bafilomycin, an inhibitor of lysosomal degradation, and by depletion of tumor susceptibility gene 101, a major component of endosomal sorting complex required for transport-I. RNAi analysis indicated that LXRβ inhibited the accelerated lysosomal degradation of csABCA1 by the LXR agonist, regardless of its transcriptional activity. Cell surface coimmunoprecipitation with COS1 cells expressing extracellularly hemagglutinin-tagged ABCA1 showed that LXRβ interacted with csABCA1 and inhibited the ubiquitination of csABCA1. Immunoprecipitates with anti-ABCA1 antibodies from the liver plasma membranes showed less LXRβ and a higher ubiquitination level of ABCA1 in high-fat diet-fed mice than in normal chow-fed mice. Under conditions of high cellular cholesterol content, csABCA1 became susceptible to ubiquitination by dissociation of LXRβ from csABCA1, which facilitated the lysosomal degradation of csABCA1 through the endosomal sorting complex required for transport system.

MP29-04 THE LOSS OF PTEN CONSEQUENT TO URETERAL OBSTRUCTION CONTRIBUTES TO RENAL FIBROSIS

INTRODUCTION AND OBJECTIVES: Phosphate Tensin Homologue on Chromosome 10 (PTEN), the principle negative regulator of PI3K pathway, is a known tumor suppressor that is mutated or lost in many cancer types. Recent studies highlight the emerging role of tumor suppressor PTEN in organ fibrosis including lung and skin where loss of PTEN expression is linked to progression of fibrosis via promoting epithelial plasticity in the lung and induction of myofibroblasts in the skin. However, potential involvement of this molecule in obstructive uropathies is not defined. METHODS: Obstruction induced renal fibrosis was modeled in vivo by unilateral ureteral obstruction (UUO) in mice and in vitro by gene silencing and pharmacological inhibition of PTEN in HK-2 human renal tubular epithelial cells and NRK-49F rat renal fibroblasts. RESULTS: PTEN expression is dramatically decreased in the obstructed (ligated) kidney, while profibrotic TGF-b1/SMAD3 and p53 signal transduction is increased compared to contralateral control or sham kidney. PTEN silencing induced phenotypic modifications from typical cuboidal to an elongated fibroblast-like shape (4-fold increase) and a 40% decrease in the relative cell number compared to control shRNA expressing HK-2 cultures (p<0.01). Stable gene depletion of PTEN in HK-2 cells long term (3-5 days) resulted in increased expression of the profibrotic markers CTGF, PAI-1 and fibronectin compared to control shRNA expressing cultures. Loss of PTEN resulted in activation of SMAD2/3 transcription factor. SMAD3 knockdown or pharmacological inhibition of SMAD3 in PTEN depleted HK-2 cells reversed growth inhibition and suppressed PAI-1 induction, confirming a critical role of SMAD3 downstream of PTEN in orchestrating fibrotic responses. CONCLUSIONS: Unilateral ureteral obstruction induces loss of PTEN expression, resulting in the increased expression of profibrotic cytokines in renal tubular epithelial cells that is dependent on the SMAD3 signaling pathway. PTEN is a potential therapeutic target in the treatment of ureteral obstruction and renal fibrosis. This model paves the way for further clinical studies in humans.

T cell lipid peroxidation induces ferroptosis and prevents immunity to infection.

The selenoenzyme glutathione peroxidase 4 (Gpx4) is a major scavenger of phospholipid hydroperoxides. Although Gpx4 represents a key component of the reactive oxygen species-scavenging network, its relevance in the immune system is yet to be defined. Here, we investigated the importance of Gpx4 for physiological T cell responses by using T cell-specific Gpx4-deficient mice. Our results revealed that, despite normal thymic T cell development, CD8(+) T cells from T(ΔGpx4/ΔGpx4) mice had an intrinsic defect in maintaining homeostatic balance in the periphery. Moreover, both antigen-specific CD8(+) and CD4(+) T cells lacking Gpx4 failed to expand and to protect from acute lymphocytic choriomeningitis virus and Leishmania major parasite infections, which were rescued with diet supplementation of high dosage of vitamin E. Notably, depletion of the Gpx4 gene in the memory phase of viral infection did not affect T cell recall responses upon secondary infection. Ex vivo, Gpx4-deficient T cells rapidly accumulated membrane lipid peroxides and concomitantly underwent cell death driven by ferroptosis but not necroptosis. These studies unveil an essential role of Gpx4 for T cell immunity.

Depletion of the cereblon gene activates the unfolded protein response and protects cells from ER stress-induced cell death

Previous studies showed that cereblon (CRBN) binds to various cellular target proteins, implying that CRBN regulates a wide range of cell responses. In this study, we found that deletion of the Crbn gene desensitized mouse embryonic fibroblast cells to various cell death-promoting stimuli, including endoplasmic reticulum stress inducers. Mechanistically, deletion of Crbn activates pathways involved in the unfolded protein response prior to ER stress induction. Loss of Crbn activated PKR-like ER kinase (PERK) with enhanced phosphorylation of eIF2α. Following ER stress induction, loss of Crbn delayed dephosphorylation of eIF2α, while reconstitution of Crbn reversed enhanced phosphorylation of PERK and eIF2α. Lastly, we found that activation of the PERK/eIF2α pathway following Crbn deletion is caused by activation of AMP-activated protein kinase (AMPK). We propose that CRBN plays a role in cellular stress signaling, including the unfolded protein response, by controlling the activity of AMPK.

Proteomic analysis of mitochondria in respiratory epithelial cells infected with human respiratory syncytial virus and functional implications for virus and cell biology.

The aim of this study was to quantitatively characterise the mitochondrial proteome of airway epithelial cells infected with human respiratory syncytial virus (HRSV), a major cause of paediatric illness. Quantitative proteomics, underpinned by stable isotope labelling with amino acids in cell culture, coupled to LC-MS/MS, was applied to mitochondrial fractions prepared from HRSV-infected and mock-infected cells 12 and 24 h post-infection. Datasets were analysed using ingenuity pathway analysis, and the results were validated and characterised using bioimaging, targeted inhibition and gene depletion. The data quantitatively indicated that antiviral signalling proteins converged on mitochondria during HRSV infection. The mitochondrial receptor protein Tom70 was found to act in an antiviral manner, while its chaperone, Hsp90, was confirmed to be a positive viral factor. Proteins associated with different organelles were also co-enriched in the mitochondrial fractions from HRSV-infected cells, suggesting that alterations in organelle dynamics and membrane associations occur during virus infection. Protein and pathway-specific alterations occur to the mitochondrial proteome in a spatial and temporal manner during HRSV infection, suggesting that this organelle may have altered functions. These could be targeted as part of potential therapeutic strategies to disrupt virus biology.

Synthetic vanillate-regulated promoter for graded gene expression in Sphingomonas.

Regulated promoters are an important basic genetic tool allowing, for example, gene-dosage and gene depletion studies. We have previously described a cumate-inducible promoter (PQ5) that is functional in diverse Alphaproteobacteria. This promoter has been engineered by combining a synthetic minimal promoter, Psyn2, and operator sites and the repressor of the Pseudomonas putida F1 cym/cmt system. In the present study, we engineered a vanillate-regulated promoter using Psyn2 and the regulatory elements of the Caulobacter crescentus vanR-vanAB system. We show that the resulting promoter, which we called PV10, responds rapidly to the inducer vanillate with an induction ratio of about two orders of magnitude in Sphingomonas melonis Fr1. In contrast to the switch-like behavior of PQ5, PV10 shows a linear dose-response curve at intermediate vanillate concentrations, allowing graded gene expression. PV10 is functionally compatible with and independent of PQ5 and cumate, and vice versa, suggesting that both systems can be used simultaneously.

ZNF750 interacts with KLF4 and RCOR1, KDM1A, and CTBP1/2 chromatin regulators to repress epidermal progenitor genes and induce differentiation genes.

ZNF750 controls epithelial homeostasis by inhibiting progenitor genes while inducing differentiation genes, a role underscored by pathogenic ZNF750 mutations in cancer and psoriasis. How ZNF750 accomplishes these dual gene regulatory impacts is unknown. Here, we characterized ZNF750 as a transcription factor that binds both the progenitor and differentiation genes that it controls at a CCNNAGGC DNA motif. ZNF750 interacts with the pluripotency transcription factor KLF4 and chromatin regulators RCOR1, KDM1A, and CTBP1/2 through conserved PLNLS sequences. ChIP-seq (chromatin immunoprecipitation [ChIP] followed by high-throughput sequencing) and gene depletion revealed that KLF4 colocalizes ∼ 10 base pairs from ZNF750 at differentiation target genes to facilitate their activation but is unnecessary for ZNF750-mediated progenitor gene repression. In contrast, KDM1A colocalizes with ZNF750 at progenitor genes and facilitates their repression but is unnecessary for ZNF750-driven differentiation. ZNF750 thus controls differentiation in concert with RCOR1 and CTBP1/2 by acting with either KDM1A to repress progenitor genes or KLF4 to induce differentiation genes.

How cells explore shape space: a quantitative statistical perspective of cellular morphogenesis.

Through statistical analysis of datasets describing single cell shape following systematic gene depletion, we have found that the morphological landscapes explored by cells are composed of a small number of attractor states. We propose that the topology of these landscapes is in large part determined by cell-intrinsic factors, such as biophysical constraints on cytoskeletal organization, and reflects different stable signaling and/or transcriptional states. Cell-extrinsic factors act to determine how cells explore these landscapes, and the topology of the landscapes themselves. Informational stimuli primarily drive transitions between stable states by engaging signaling networks, while mechanical stimuli tune, or even radically alter, the topology of these landscapes. As environments fluctuate, the topology of morphological landscapes explored by cells dynamically adapts to these fluctuations. Finally we hypothesize how complex cellular and tissue morphologies can be generated from a limited number of simple cell shapes.

Negative regulation of NF-κB activity by brain-specific TRIpartite Motif protein 9.

The TRIpartite Motif (TRIM) family of RING-domain-containing proteins participate in a variety of cellular functions. The β-transducin repeat-containing protein (β-TrCP), a component of the Skp-Cullin-F-box-containing (SCF) E3 ubiquitin ligase complex, recognizes the NF-κB inhibitor IκBα and precursor p100 for proteasomal degradation and processing respectively. β-TrCP thus plays a critical role in both canonical and non-canonical NF-κB activation. Here, we report TRIM9 is a negative regulator of NF-κBactivation. Interaction between the phosphorylated degron motif of TRIM9 and the WD40 repeat region of β-TrCP prevented β-TrCP from binding its substrates, stabilizing IκBα and p100 and thereby blocking NF-κB activation. Consequently, expression or depletion of the TRIM9 gene significantly affected NF-κB-induced inflammatory cytokine production. This study not only elucidates a mechanism for TRIM9-mediated regulation of the β-TrCP SCF complex activity, but also identifies TRIM9 as a brain-specific negative regulator of the NF-κB pro-inflammatory signaling pathway.

Regulation of nasal airway homeostasis and inflammation in mice by SHP-1 and Th2/Th1 signaling pathways.

Allergic rhinitis is a chronic inflammatory disease orchestrated by Th2 lymphocytes. Src homology 2 domain-containing protein tyrosine phosphatase (SHP)-1 is known to be a negative regulator in the IL-4α/STAT-6 signaling pathway of the lung. However, the role of SHP-1 enzyme and its functional relationship with Th2 and Th1 cytokines are not known in the nasal airway. In this study, we aimed to study the nasal inflammation as a result of SHP-1 deficiency in viable motheaten (mev) mice and to investigate the molecular mechanisms involved. Cytology, histology, and expression of cytokines and chemokines were analyzed to define the nature of the nasal inflammation. Targeted gene depletion of Th1 (IFN-γ) and Th2 (IL-4 and IL-13) cytokines was used to identify the critical pathways involved. Matrix metalloproteinases (MMPs) were studied to demonstrate the clearance mechanism of recruited inflammatory cells into the nasal airway. We showed here that mev mice had a spontaneous allergic rhinitis-like inflammation with eosinophilia, mucus metaplasia, up-regulation of Th2 cytokines (IL-4 and IL-13), chemokines (eotaxin), and MMPs. All of these inflammatory mediators were clearly counter-regulated by Th2 and Th1 cytokines. Deletion of IFN-γ gene induced a strong Th2-skewed inflammation with transepithelial migration of the inflammatory cells. These findings suggest that SHP-1 enzyme and Th2/Th1 paradigm may play a critical role in the maintenance of nasal immune homeostasis and in the regulation of allergic rhinitis.

Stromal cell-derived factor-1α/C-X-C chemokine receptor type 4 axis promotes endothelial cell barrier integrity via phosphoinositide 3-kinase and Rac1 activation.

Although stromal cell-derived factor (SDF)-1αis well known to modulate the mobilization of hematopoietic stem cells and endothelial progenitor cells, its effects on some pre-existing vascular functions remain unknown. We have investigated here the role of SDF-1αsignaling in endothelial barrier function. Treatment with SDF-1α elevated transendothelial electrical resistance and inhibited the dextran hyperpermeability elicited by thrombin in bovine aortic endothelial cells, both indicating an increase in endothelial barrier function. SDF-1α binds to 2 receptors, C-X-C chemokine receptor types 4 and 7 (CXCR4 and CXCR7). Pretreatment with a CXCR4 antagonist or CXCR4 gene depletion by small interfering RNA (siRNA) eliminated SDF-1α-induced endothelial barrier enhancement. In contrast, CXCR7 antagonist or CXCR7 gene depletion by siRNA did not influence SDF-1α-induced barrier enhancement. Pretreatment with a Gi-protein inhibitor, a phosphoinositide 3-kinase (PI3K) inhibitor, or PI3K p110γsubunit gene depletion by siRNA also inhibited SDF-1α-induced barrier enhancement significantly. Western blot analysis revealed that SDF-1α phosphorylated Akt(Ser473) in endothelial cells, suggesting PI3K activation. Immunostaining showed that treatment with SDF-1αformed a cortical actin rim, which was accompanied by Rac1 activation. In vivo, SDF-1αinhibited croton oil-induced vascular leakage indexed by dye extravasation, which is attenuated by a pretreatment with a CXCR4 antagonist. We have identified SDF-1α as a novel suppressor of endothelial permeability. Specifically, SDF-1α stimulates the CXCR4/PI3K/Rac1 signaling pathway and the subsequent cytoskeletal rearrangement.

Alcohol dehydrogenase III exacerbates liver fibrosis by enhancing stellate cell activation and suppressing natural killer cells in mice

The important roles of retinols and their metabolites have recently been emphasized in the interactions between hepatic stellate cells (HSCs) and natural killer (NK) cells. Nevertheless, the expression and role of retinol metabolizing enzyme in both cell types have yet to be clarified. Thus, we investigated the expression of retinol metabolizing enzyme and its role in liver fibrosis. Among several retinol metabolizing enzymes, only alcohol dehydrogenase (ADH) 3 expression was detected in isolated HSCs and NK cells, whereas hepatocytes express all of them. In vitro treatment with 4-methylpyrazole (4-MP), a broad ADH inhibitor, or depletion of the ADH3 gene down-regulated collagen and transforming growth factor-β1 (TGF-β1) gene expression, but did not affect α-smooth muscle actin gene expression in cultured HSCs. Additionally, in vitro, treatments with retinol suppressed NK cell activities, whereas inhibition of ADH3 enhanced interferon-γ (IFN-γ) production and cytotoxicity of NK cells against HSCs. In vivo, genetic depletion of the ADH3 gene ameliorated bile duct ligation- and carbon tetrachloride-induced liver fibrosis, in which a higher number of apoptotic HSCs and an enhanced activation of NK cells were detected. Freshly isolated HSCs from ADH3-deficient mice showed reduced expression of collagen and TGF-β1, but enhanced expression of IFN-γ was detected in NK cells from these mice compared with those of control mice. Using reciprocal bone marrow transplantation of wild-type and ADH3-deficient mice, we demonstrated that ADH3 deficiency in both HSCs and NK cells contributed to the suppressed liver fibrosis. ADH3 plays important roles in promoting liver fibrosis by enhancing HSC activation and inhibiting NK cell activity, and could be used as a potential therapeutic target for the treatment of liver fibrosis.

Uncovering novel roles of tandem BRCA1 C‐terminal (BRCT) domains in DNA repair, genome stability and breast cancer (LB236)

Pax transactivation domain‐interacting protein (PTIP) has an important role in preserving genomic stability and is essential for cell survival following DNA damage, although the precise mechanism is undefined. PTIP consists of three pairs of tandem BRCA1 C‐terminal (tBRCT) domains required for assembly into multiple protein complexes. Mutations that disrupt BRCT domain binding correlate with increased cancer rates. To determine its precise role in DNA repair, we profiled the expression of PTIP in breast cancer cell lines. Surprisingly, we’ve discovered a novel truncated isoform of PTIP, containing the last four C‐terminal BRCT domains, (PTIP‐delta). RNAi gene depletion studies reveal a post‐transcriptional origin of PTIP‐delta. Kinetic profiling of PTIP synthesis indicates that a significant portion of PTIP is processed to PTIP‐delta within hours of translation. Recombinant forms of this PTIP‐delta retain the ability to form nuclear repair foci. Biophysical characterization uncovers new oligomerization interfaces of higher order structures composed exclusively of PTIP‐delta. These studies indicate that PTIP‐delta participates in the formation of multiple structurally and stoichiometrically diverse complexes during DNA repair. These studies implicate new and unusual functional roles of PTIP in DNA repair, genome stability and breast cancer.Grant Funding Source: NCI K 22

Involvement of adrenomedullin in spinal glial activation following chronic administration of morphine in rats

Adrenomedullin (AM) belongs to the calcitonin gene-related peptide (CGRP) family. Our previous studies show that chronic exposure to morphine increases spinal AM bioactivity, contributing to the development and maintenance of morphine tolerance. This study investigated the possible involvement of AM in morphine-evoked gliosis. Real-time polymerase chain reaction was performed to determine interleukin-1β (IL-1β), IL-6 and tumour necrosis factor-α (TNF-α) mRNAs in the spinal dorsal horn and cultured sensory ganglion explants. Immunohistochemistry was performed to identify spinal microglia and astrocytes. Repetitive intrathecal (i.t.) injection of morphine (20 μg) increased the expression of IL-1β, IL-6 and TNF-α mRNAs in the spinal dorsal horn. The co-administration of the selective AM receptor antagonist AM22-52 (36 μg) markedly attenuated chronic morphine-evoked increase in IL-1β and IL-6, but not TNF-α, mRNA levels. Exposure of cultured dorsal root ganglion (DRG) explants to morphine (3.3 μmol/L) for 6 days up-regulated IL-1β and IL-6 mRNA expressions. The depletion of AM gene using small interfering RNA (siRNA) approach abolished morphine-evoked increase in IL-1β and IL-6 syntheses in the cultured DRG. The blockade of AM receptors by i.t. AM22-52 also inhibited chronic morphine-evoked cell hypertrophy of microglia and astrocytes as well as an increase in OX-42 and GFAP (glial fibrillary acidic protein) immunoreactivities. Furthermore, the 6-day treatment with AM (10 μg, i.t.) induced morphological changes of microglia and astrocytes as well as an increase in IL-1β, IL-6 and TNF-α mRNA levels in the spinal dorsal horn. The present study supports the idea that up-regulation of the pronociceptive mediator AM can recruit spinal glial cells, resulting in an increase in cytokines during chronic use of morphine.