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Alan Engelman - One of the best experts on this subject based on the ideXlab platform.

  • a peptide derived from Lens Epithelium derived growth factor stimulates hiv 1 dna integration and facilitates intasome structural studies
    Journal of Molecular Biology, 2020
    Co-Authors: Xuemin Chen, Alan Engelman, Huaibin Wang, Kellie A Jurado, Robert Craigie
    Abstract:

    Abstract The low solubility and aggregation properties of HIV-1 integrase (IN) are major obstacles for biochemical and structural studies. The Lens Epithelium–derived growth factor (LEDGF) is a cellular factor that binds IN and tethers preintegration complexes to chromatin before integration. The LEDGF also stimulates HIV-1 IN DNA strand transfer activity and improves its solubility in vitro. We show that these properties are conferred by a short peptide spanning residues 178 to 197 of the LEDGF that encompasses its AT-hook DNA-binding elements. The peptide stimulates HIV-1 IN activity both in trans and in cis. Fusion of the peptide to either the N- or C-terminus of IN results in maximal stimulation of concerted integration activity and greatly improves the solubility of the protein and nucleoprotein complexes of IN with viral DNA ends (intasomes). High-resolution structures of HIV-1 intasomes are required to understand the mechanism of IN strand transfer inhibitors (INSTIs), which are front-line drugs for the treatment of HIV-1, and how the virus can develop resistance to INSTIs. We have previously determined the structure of the HIV-1 strand transfer complex intasome. The improved biophysical properties of intasomes assembled with LEDGF peptide fusion IN have enabled us to determine the structure of the cleaved synaptic complex intasome, which is the direct target of INSTIs.

  • Lens Epithelium-derived growth factor fusion proteins redirect HIV-1 DNA integration
    Proceedings of the National Academy of Sciences of the United States of America, 2010
    Co-Authors: Andrea L. Ferris, Christina M. Hughes, Claudia Stewart, Steven J. Smith, Thomas A. Milne, Gang Greg Wang, Ming Chieh Shun, C. David Allis, Alan Engelman
    Abstract:

    Lens Epithelium-derived growth factor (LEDGF) fusion proteins can direct HIV-1 DNA integration to novel sites in the host genome. The C terminus of LEDGF contains an integrase binding domain (IBD), and the N terminus binds chromatin. LEDGF normally directs integrations to the bodies of expressed genes. Replacing the N terminus of LEDGF with chromatin binding domains (CBDs) from other proteins changes the specificity of HIV-1 DNA integration. We chose two well-characterized CBDs: the plant homeodomain (PHD) finger from ING2 and the chromodomain from heterochromatin binding protein 1α (HP1α). The ING2 PHD finger binds H3K4me3, a histone mark that is associated with the transcriptional start sites of expressed genes. The HP1α chromodomain binds H3K9me2,3, histone marks that are widely distributed throughout the genome. A fusion protein in which the ING2 PHD finger was linked to the LEDGF IBD directed integrations near the start sites of expressed genes. A similar fusion protein in which the HP1α chromodomain was linked to the LEDGF IBD directed integrations to sites that differed from both the PHD finger fusion–directed and LEDGF-directed integration sites. The ability to redirect HIV-1 DNA integration may help solve the problems associated with the activation of oncogenes when retroviruses are used in gene therapy.

  • biochemical and genetic analyses of integrase interacting proteins Lens Epithelium derived growth factor ledgf p75 and hepatoma derived growth factor related protein 2 hrp2 in preintegration complex function and hiv 1 replication
    Virology, 2006
    Co-Authors: Nick Vandegraaff, Pamela A Silver, Eric Devroe, Fanny Turlure, Alan Engelman
    Abstract:

    Human immunodeficiency virus type 1 (HIV-1) integrase (IN) functions in cells within the context of high molecular weight preintegration complexes (PICs). Lens Epithelium-derived growth factor (LEDGF) transcriptional coactivator/p75 and hepatoma-derived growth factor related protein 2 (HRP2) tightly bind to HIV-1 IN and stimulate its integration activity in vitro. Here, we show that each recombinant host cell factor efficiently reconstitutes the in vitro activity of HIV-1 PICs disrupted for functional integration by pre-treatment with high concentrations of salt. Mutational analysis reveals that both the IN-binding and DNA-binding activities of LEDGF/p75 contribute to functional PIC reconstitution. We also investigate a role(s) for these proteins in HIV-1 infection by using short-interfering RNA. HIV-1 infection was essentially unaffected in HeLa-P4 cells depleted for LEDGF/p75, HRP2, or both proteins. We conclude that cells knocked-out for LEDGF/p75 and/or HRP2 will be useful genetic tools to address the roles of these host cell factors in HIV-1 replication.

  • identification of an evolutionarily conserved domain in human Lens Epithelium derived growth factor transcriptional co activator p75 ledgf p75 that binds hiv 1 integrase
    Journal of Biological Chemistry, 2004
    Co-Authors: Peter Cherepanov, Eric Devroe, Pamela A Silver, Alan Engelman
    Abstract:

    Human Lens Epithelium-derived growth factor/transcriptional co-activator p75 (LEDGF/p75) protein was recently identified as a binding partner for HIV-1 integrase (IN) in human cells. In this work, we used biochemical and bioinformatic approaches to define the domain organization of LEDGF/p75. Using limited proteolysis and deletion mutagenesis we show that the protein contains a pair of evolutionarily conserved domains, assuming about 35% of its sequence. Whereas the N-terminal PWWP domain had been recognized previously, the second domain is novel. It is comprised of ∼80 amino acid residues and is both necessary and sufficient for binding to HIV-1 IN. Strikingly, the integrase binding domain (IBD) is not unique to LEDGF/p75, as a second human protein, hepatoma-derived growth factor-related protein 2 (HRP2), contains a homologous sequence. LEDGF/p75 and HRP2 IBDs avidly bound HIV-1 IN in an in vitro GST pull-down assay and each full-length protein potently stimulated HIV-1 IN activity in vitro. LEDGF/p75 and HRP2 are predicted to share a similar domain organization and have an evident evolutionary and likely functional relationship.

Zeger Debyser - One of the best experts on this subject based on the ideXlab platform.

  • fragment based discovery of 8 hydroxyquinoline inhibitors of the hiv 1 integrase Lens Epithelium derived growth factor p75 in ledgf p75 interaction
    Journal of Medicinal Chemistry, 2013
    Co-Authors: Erik Serrao, Bikash Debnath, Hiroyuki Otake, Yuting Kuang, Frauke Christ, Zeger Debyser, Nouri Neamati
    Abstract:

    On the basis of an initial molecular modeling study suggesting the favorable binding of the "privileged" fragment 8-hydroxyquinoline with HIV-1 integrase (IN) at the IN-Lens Epithelium-derived growth factor/p75 (LEDGF/p75) interface , we developed a set of modified 8-hydroxyquinoline fragments demonstrating micromolar IC50 values for inhibition of the IN-LEDGF/p75 interaction, but significant cytotoxicity was associated with these initial compounds. Diverse modifications at the C5 and C7 carbons of the 8-hydroxyquinoline core improved potency, but reduction of diversity to only modifications at the C5 position ultimately yielded potent inhibitors with low cytotoxicity. Two of these particular compounds, 5-((p-tolylamino)methyl)quinolin-8-ol and 5-(((3,4-dimethylphenyl)amino)methyl)quinolin-8-ol, inhibited viral replication in MT-4 cells with low micromolar EC50. This is the first study providing evidence for 8-hydroxyquinolines as novel inhibitors of the IN-LEDGF/p75 interaction. Our lead compounds are druglike, have low molecular weights, and are amenable to various substitutions suitable for enhancing their potency and selectivity.

  • characterization of rare Lens Epithelium derived growth factor p75 genetic variants identified in hiv 1 long term nonprogressors
    AIDS, 2013
    Co-Authors: Rik Schrijvers, Frauke Christ, Zeger Debyser, Jan De Rijck, Jonas Demeulemeester, Melanie Gerard, Rik Gijsbers
    Abstract:

    Objective Lens Epithelium-derived growth factor (LEDGF)/p75 is a cellular binding partner of HIV-1 integrase and a crucial cofactor for HIV-1 replication. Here, we study two LEDGF/p75 exonic variants I436S and T473I, identified in HIV-1 long-term nonprogressors, together with Q472L. Methods In-vitro binding assays, cell culture complementation, and functional rescue. Results Binding affinities of wild-type, I436S, T473I, and Q472L LEDGF/p75 for HIV-1 integrase were comparable. All LEDGF/p75 variants bound equally well to LEDGF/p75 interacting partners JPO2 and PogZ. In addition, HIV-1 replication was evaluated in human somatic LEDGF/p75-knockout cells and LEDGF/p75-knockdown cells complemented with either wild-type LEDGF/p75 or the respective LEDGF/p75 variants. All variants rescued HIV-1 replication to wild-type levels, whereas LEDGF/p75 D366N, defective for interaction with HIV-1 integrase, did not. Conclusion Although identified in a cohort of long-term nonprogressors, our study did not indicate that the I436S or T473I mutation in LEDGF/p75 affects the interaction with HIV-1 integrase.

  • impairing mll fusion gene mediated transformation by dissecting critical interactions with the Lens Epithelium derived growth factor ledgf p75
    Leukemia, 2013
    Co-Authors: Helene Mereau, Frauke Christ, Zeger Debyser, Jan De Rijck, Rik Gijsbers, Jonas Demeulemeester, Kateřina Cermakova, A Kutz, Sabine Juge, Juerg Schwaller
    Abstract:

    The Lens Epithelium-derived growth factor (LEDGF/p75) tethers the mixed-lineage leukemia (MLL1) protein complex to chromatin. Likewise, LEDGF/p75 tethers the HIV-1 pre-integration complex to chromatin. We previously demonstrated that expression of the C-terminal fragment fused to enhanced green fluorescent protein (eGFP) (eGFP-LEDGF(325-530)) impaired HIV-1 replication. Here, we explored this strategy to selectively interfere with the leukemogenic activity of MLL-fusion proteins. We found that expression of LEDGF(325-530) impaired the clonogenic growth of MLL-fusion gene transformed human and mouse hematopoietic cells, without affecting the growth of control cells immortalized by the FLT3-ITD mutant or normal lineage-marker-depleted murine bone marrow cells. Expression of LEDGF(325-530) was associated with downregulation of the MLL target Hoxa9 and impaired cell cycle progression. Structure-function analysis revealed two small eGFP-fused LEDGF/p75 peptides, LEDGF(424-435) and LEDGF(375-386) phenocopying these effects. Both LEDGF(325-530) and the smaller active peptides were able to disrupt the LEDGF/p75-MLL interaction. Expression of LEDGF(325-530) or LEDGF(375-386) fragments increased the latency period to disease development in vivo in a mouse bone marrow transplant model of MLL-AF9-induced AML. We conclude that small peptides disrupting the LEDGF/p75-MLL interface have selective anti-leukemic activity providing a direct rationale for the design of small molecule inhibitors targeting this interaction.

  • Lens Epithelium derived growth factor p75 qualifies as a target for hiv gene therapy in the nsg mouse model
    Molecular Therapy, 2012
    Co-Authors: Sofie Vets, Janine Kimpel, Andreas Volk, Jan De Rijck, Rik Schrijvers, Bert Verbinnen, Wim Maes, Dorothee Von Laer, Zeger Debyser
    Abstract:

    Lens Epithelium-derived growth factor (LEDGF/p75) is an essential cofactor of HIV integration. Both stable overexpression of the C-terminal part of LEDGF/p75 (LEDGF325–530) containing the integrase (IN)-binding domain (IBD) and stable knockdown (KD) of LEDGF/p75 are known to inhibit HIV infection in laboratory cell lines. Here, primary human CD4+ T-cells were transduced with lentiviral vectors encoding LEDGF325–530, the interaction-deficient mutant LEDGF325–530D366N, or a hairpin depleting LEDGF/p75 and challenged with HIV. Maximal protection of primary T-cells from HIV infection was obtained after LEDGF325–530 overexpression reducing HIV replication 40-fold without evidence of cellular toxicity. This strategy was subsequently evaluated in the NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mouse model. Threefold reduction in mean plasma viral load was obtained in mice engrafted with CD4+ T-cells expressing LEDGF325–530 in comparison with engraftment with LEDGF325–530D366N cells. Four weeks after transplantation with LEDGF325–530D366N cells, 70% of the CD4+ cells were lost due to ongoing HIV replication. However, in mice transplanted with LEDGF325–530 cells only a 20% decrease in CD4+ cells was measured. Liver and spleen sections of LEDGF325–530 mice contained less HIV than LEDGF325–530D366N mice as measured by p24 antigen detection. LEDGF325–530 overexpression potently inhibits HIV replication in vivo and protects against HIV mediated cell killing of primary CD4+ T-cells.

  • Lens Epithelium derived growth factor p75 interacts with the transposase derived dde domain of pogz
    Journal of Biological Chemistry, 2009
    Co-Authors: Koen Bartholomeeusen, Frauke Christ, Zeger Debyser, Jelle Hendrix, Jeanchristophe Rain, Stephane Emiliani, Richard Benarous, Rik Gijsbers, Jan De Rijck
    Abstract:

    Lens Epithelium-derived growth factor/p75 (LEDGF/p75) is a prominent cellular interaction partner of human immunodeficiency virus-1 (HIV-1) integrase, tethering the preintegration complex to the host chromosome. In light of the development of LEDGF/p75-integrase interaction inhibitors, it is essential to understand the cell biology of LEDGF/p75. We identified pogZ as new cellular interaction partner of LEDGF/p75. Analogous to lentiviral integrase, pogZ, a domesticated transposase, carries a DDE domain, the major determinant for LEDGF/p75 interaction. Using different in vitro and in vivo approaches, we corroborated the interaction between the C terminus of LEDGF/p75 and the DDE domain of pogZ, revealing an overlap in the binding of pogZ and HIV-1 integrase. Competition experiments showed that integrase is efficient in displacing pogZ from LEDGF/p75. Moreover, pogZ does not seem to play a role as a restriction factor of HIV. The finding that LEDGF/p75 is capable of interacting with a DDE domain protein that is not a lentiviral integrase points to a profound role of LEDGF/p75 in DDE domain protein function.

Shizuya Saika - One of the best experts on this subject based on the ideXlab platform.

  • suppression of injury induced epithelial mesenchymal transition in a mouse Lens Epithelium lacking tenascin c
    Molecular Vision, 2010
    Co-Authors: Saiichi Tanaka, Kathleen C Flanders, Yuka Okada, Osamu Yamanaka, Ai Kitano, Takayoshi Sumioka, Norihito Fujita, Masayasu Miyajima, Shizuya Saika
    Abstract:

    Purpose: To investigate the role of tenascin-C in epithelial-mesenchymal transition (EMT) of the Lens Epithelium during wound healing in mice. Tenascin-C is a component of the extracellular matrix in patients having post-operative capsular opacification. Methods: The crystalline Lens was injured by needle puncture in tenascin-C null (KO, n=56) and wild-type (WT, n=56) mice in a C57BL/6 background. The animals were killed at day 2, 5, or 10 post-injury. Immunohistochemistry was employed to detect α-smooth muscle actin (αSMA), a marker of EMT, collagen type I, transforming growth factor β1 (TGFβ1), phospho-Smad2, phospho-adducin, and phospho-myosin light chain 9 (MLC9). The expression levels of phospho-adducin and phospho-MLC9 were used as markers for the activation of protein kinase C and Rho kinase, respectively. Results: The expression of tenascin-C was upregulated in WT Lens epithelial cells adjacent to the capsular break at day 5. The results showed that injury-induced EMT of the mouse Lens Epithelium, as evaluated by histology and the expression patterns of αSMA and fibronectin, was attenuated in the absence of tenascin-C. Upregulation of TGFβ1 expression in the Epithelium was also inhibited, and loss of tenascin-C attenuated the phosphorylation of Smad2 and adducin in epithelial cells adjacent to the capsular break. The expression of phospho-adducin was suppressed, while the expression level of phospho-MLC9 was unchanged, in the healing Epithelium in the absence of tenascin C. Conclusions: Tenascin-C is required for injury-induced EMT in the mouse Lens Epithelium. The mechanism behind this might involve impaired activation of cytoplasmic signaling cascades; i.e., TGFβ/Smad and protein kinase C-adducing signaling, in the absence of tenascin-C.

  • loss of osteopontin perturbs the epithelial mesenchymal transition in an injured mouse Lens Epithelium
    Laboratory Investigation, 2007
    Co-Authors: Shizuya Saika, Kathleen C Flanders, Kumi Shirai, Yuka Okada, Osamu Yamanaka, Kenichi Miyazaki, Ai Kitano, Shigeyuki Kon, Toshimitsu Uede, W W Kao
    Abstract:

    We previously reported that osteopontin (OPN), a matrix structural glycophosphoprotein, is upregulated in the injured mouse Lens prior to the epithelial-mesenchymal transition (EMT). Here, we investigated the role of this protein in EMT of the Lens Epithelium during wound healing. The crystalline Lens was injured by needle puncture in OPN-null (KO, n=40) and wild-type (WT, n=40) mice. The animals were killed at day 1, 2, 5, and 10 postinjury. Immunohistochemistry was employed to detect alpha-smooth muscle action (alphaSMA), a marker of EMT, collagen type I, transforming growth factor beta1 (TGFbeta1), TGFbeta2, and phospho-Smad2/3. Cell proliferation was assayed by examining uptake of bromodeoxyuridine (BrdU). The results showed that injury-induced EMT of mouse Lens Epithelium, as evaluated by histology, expression pattern of alphaSMA and collagen I, was altered in the absence of OPN with reduced phospho-Smad2/3 signaling. Upregulation of TGFbeta1 and TGFbeta2 in the Epithelium was also inhibited. Cell proliferation was more active in KO mice as compared with WT mice at day 1 and 2, but not at day 5 and 10. An in vitro experiment shows OPN facilitates cell adhesion of Lens epithelial cell line. OPN is required for activation of Smad2/3 signal in an injured Lens Epithelium and Lens cell EMT.

  • a new model of anterior subcapsular cataract involvement of tgfbeta smad signaling
    Molecular Vision, 2006
    Co-Authors: Kumi Shirai, Kathleen C Flanders, Akira Ooshima, Takeshi Tanaka, Yuka Okada, Shizuya Saika, Yoshitaka Ohnishi
    Abstract:

    PURPOSE: To develop a new animal model of anterior subcapsular cataract formation by topical application of alkali to the eye and to examine the role of Transforming growth factorbeta/Smad3 (TGFbeta/Smad3) signaling in the formation of this cataract model. METHODS: Under anesthesia, one eye of adult Wistar rats (n=142) was subjected to alkali burn by topical application of 1 N NaOH. The eye was then histologically examined at specific time intervals. Immunohistochemistry with a battery of antibodies was carried out to examine the epithelial-mesenchymal transition (EMT) in Lens Epithelium. Enzyme immunoassay was employed to determine the level of growth factors in aqueous humor and Lens tissue. Smad3-null mice were also used to examine the role of Smad3 signaling in cataractogenesis in this model. RESULTS: Two days post-burn of the ocular surface, Lens Epithelium underwent EMT as evidenced by the upregulation of Snail and alpha-smooth muscle actin and formed a multilayer of cells beneath the capsule. Smad signaling was found to be activated in EMT-type Lens cells. The majority of myofibroblast-type Lens cells expressed proliferative cell nuclear antigen (PCNA). The total amount of active TGFbeta2, total TGFbeta2, and Fibroblast growth factor 2 (FGF2) increased in the aqueous humor and Lens. Loss of Smad3 attenuated, but did not completely abolish, EMT in the Lens Epithelium. CONCLUSIONS: Topical alkali treatment of the ocular surface readily induces an EMT-type anterior subcapsular cataract. Smad3 signaling is involved, but not required, for achievement of EMT in the Lens Epithelium in this cataract model.

  • a new model of anterior subcapsular cataract involvement of tgfbeta smad signaling
    Molecular Vision, 2006
    Co-Authors: Kumi Shirai, Kathleen C Flanders, Akira Ooshima, Takeshi Tanaka, Yuka Okada, Shizuya Saika, Yoshitaka Ohnishi
    Abstract:

    PURPOSE: To develop a new animal model of anterior subcapsular cataract formation by topical application of alkali to the eye and to examine the role of Transforming growth factorbeta/Smad3 (TGFbeta/Smad3) signaling in the formation of this cataract model. METHODS: Under anesthesia, one eye of adult Wistar rats (n=142) was subjected to alkali burn by topical application of 1 N NaOH. The eye was then histologically examined at specific time intervals. Immunohistochemistry with a battery of antibodies was carried out to examine the epithelial-mesenchymal transition (EMT) in Lens Epithelium. Enzyme immunoassay was employed to determine the level of growth factors in aqueous humor and Lens tissue. Smad3-null mice were also used to examine the role of Smad3 signaling in cataractogenesis in this model. RESULTS: Two days post-burn of the ocular surface, Lens Epithelium underwent EMT as evidenced by the upregulation of Snail and alpha-smooth muscle actin and formed a multilayer of cells beneath the capsule. Smad signaling was found to be activated in EMT-type Lens cells. The majority of myofibroblast-type Lens cells expressed proliferative cell nuclear antigen (PCNA). The total amount of active TGFbeta2, total TGFbeta2, and Fibroblast growth factor 2 (FGF2) increased in the aqueous humor and Lens. Loss of Smad3 attenuated, but did not completely abolish, EMT in the Lens Epithelium. CONCLUSIONS: Topical alkali treatment of the ocular surface readily induces an EMT-type anterior subcapsular cataract. Smad3 signaling is involved, but not required, for achievement of EMT in the Lens Epithelium in this cataract model.

  • transient adenoviral gene transfer of smad7 prevents injury induced epithelial mesenchymal transition of Lens Epithelium in mice
    Laboratory Investigation, 2004
    Co-Authors: Shizuya Saika, Yoshitaka Ohnishi, Akira Ooshima, Osamu Yamanaka, Misako Sato, Yasuteru Muragaki, Kazuo Ikeda, Yuji Nakajima, Kazuhiko Namikawa, Hiroshi Kiyama
    Abstract:

    We examined the effect of adenovirus-mediated transient expression of Smad7, an inhibitory Smad in TGFbeta/activin signaling, on injury-induced epithelial-mesenchymal transition (EMT) of Lens Epithelium in mice. A volume of 3 microl of adenoviral solution was injected into the right Lens of adult male C57BL/6 mice (n=56) at the time of capsular injury made using a hypodermic needle under general anesthesia. A mixture of recombinant adenovirus carrying CAG promoter-driven Cre (Cre adv) and mouse Smad7 complementary DNA (Smad7 adv) was administered to induce Smad7 expression, while control Lenses were treated with Cre adv alone. After healing intervals of 2, 3, 5, and 10 days, animals were killed 2 h after labeling with bromodeoxyuridine (BrdU) and eyes were processed for histology. During healing, marked expression of Smad7 was observed in Lens epithelial cells in the Smad7 adv group with loss of nuclear translocation of Smads2/3, while little Smad7 and abundant nuclear Smads2/3 were seen in cells in the Cre adv group. Lens epithelial cells in the Cre adv control group exhibited a fibroblastic appearance at days 5 and 10 and the capsular break was sealed with fibrous tissue, while Smad7 adv-treated cells around the capsular break retained their epithelial morphology and the break was not sealed. Expression of snail mRNA, and alpha-smooth muscle actin, lumican, and collagen VI proteins, markers of EMT, was observed in control-treated eyes, but not in cells of the Smad7 adv group at day 5 with minimal expression at day 10. Additionally, cell proliferation increased in Epithelium infected with Smad7 adv consistent with suppression of injury-induced upregulation of TGFbeta1 in Epithelium. We conclude that gene transfer of Smad7 in mice prevents injury-induced EMT of Lens epithelial cells and sealing of the capsular break with fibrous tissue.

Yoshitaka Ohnishi - One of the best experts on this subject based on the ideXlab platform.

  • a new model of anterior subcapsular cataract involvement of tgfbeta smad signaling
    Molecular Vision, 2006
    Co-Authors: Kumi Shirai, Kathleen C Flanders, Akira Ooshima, Takeshi Tanaka, Yuka Okada, Shizuya Saika, Yoshitaka Ohnishi
    Abstract:

    PURPOSE: To develop a new animal model of anterior subcapsular cataract formation by topical application of alkali to the eye and to examine the role of Transforming growth factorbeta/Smad3 (TGFbeta/Smad3) signaling in the formation of this cataract model. METHODS: Under anesthesia, one eye of adult Wistar rats (n=142) was subjected to alkali burn by topical application of 1 N NaOH. The eye was then histologically examined at specific time intervals. Immunohistochemistry with a battery of antibodies was carried out to examine the epithelial-mesenchymal transition (EMT) in Lens Epithelium. Enzyme immunoassay was employed to determine the level of growth factors in aqueous humor and Lens tissue. Smad3-null mice were also used to examine the role of Smad3 signaling in cataractogenesis in this model. RESULTS: Two days post-burn of the ocular surface, Lens Epithelium underwent EMT as evidenced by the upregulation of Snail and alpha-smooth muscle actin and formed a multilayer of cells beneath the capsule. Smad signaling was found to be activated in EMT-type Lens cells. The majority of myofibroblast-type Lens cells expressed proliferative cell nuclear antigen (PCNA). The total amount of active TGFbeta2, total TGFbeta2, and Fibroblast growth factor 2 (FGF2) increased in the aqueous humor and Lens. Loss of Smad3 attenuated, but did not completely abolish, EMT in the Lens Epithelium. CONCLUSIONS: Topical alkali treatment of the ocular surface readily induces an EMT-type anterior subcapsular cataract. Smad3 signaling is involved, but not required, for achievement of EMT in the Lens Epithelium in this cataract model.

  • a new model of anterior subcapsular cataract involvement of tgfbeta smad signaling
    Molecular Vision, 2006
    Co-Authors: Kumi Shirai, Kathleen C Flanders, Akira Ooshima, Takeshi Tanaka, Yuka Okada, Shizuya Saika, Yoshitaka Ohnishi
    Abstract:

    PURPOSE: To develop a new animal model of anterior subcapsular cataract formation by topical application of alkali to the eye and to examine the role of Transforming growth factorbeta/Smad3 (TGFbeta/Smad3) signaling in the formation of this cataract model. METHODS: Under anesthesia, one eye of adult Wistar rats (n=142) was subjected to alkali burn by topical application of 1 N NaOH. The eye was then histologically examined at specific time intervals. Immunohistochemistry with a battery of antibodies was carried out to examine the epithelial-mesenchymal transition (EMT) in Lens Epithelium. Enzyme immunoassay was employed to determine the level of growth factors in aqueous humor and Lens tissue. Smad3-null mice were also used to examine the role of Smad3 signaling in cataractogenesis in this model. RESULTS: Two days post-burn of the ocular surface, Lens Epithelium underwent EMT as evidenced by the upregulation of Snail and alpha-smooth muscle actin and formed a multilayer of cells beneath the capsule. Smad signaling was found to be activated in EMT-type Lens cells. The majority of myofibroblast-type Lens cells expressed proliferative cell nuclear antigen (PCNA). The total amount of active TGFbeta2, total TGFbeta2, and Fibroblast growth factor 2 (FGF2) increased in the aqueous humor and Lens. Loss of Smad3 attenuated, but did not completely abolish, EMT in the Lens Epithelium. CONCLUSIONS: Topical alkali treatment of the ocular surface readily induces an EMT-type anterior subcapsular cataract. Smad3 signaling is involved, but not required, for achievement of EMT in the Lens Epithelium in this cataract model.

  • transient adenoviral gene transfer of smad7 prevents injury induced epithelial mesenchymal transition of Lens Epithelium in mice
    Laboratory Investigation, 2004
    Co-Authors: Shizuya Saika, Yoshitaka Ohnishi, Akira Ooshima, Osamu Yamanaka, Misako Sato, Yasuteru Muragaki, Kazuo Ikeda, Yuji Nakajima, Kazuhiko Namikawa, Hiroshi Kiyama
    Abstract:

    We examined the effect of adenovirus-mediated transient expression of Smad7, an inhibitory Smad in TGFbeta/activin signaling, on injury-induced epithelial-mesenchymal transition (EMT) of Lens Epithelium in mice. A volume of 3 microl of adenoviral solution was injected into the right Lens of adult male C57BL/6 mice (n=56) at the time of capsular injury made using a hypodermic needle under general anesthesia. A mixture of recombinant adenovirus carrying CAG promoter-driven Cre (Cre adv) and mouse Smad7 complementary DNA (Smad7 adv) was administered to induce Smad7 expression, while control Lenses were treated with Cre adv alone. After healing intervals of 2, 3, 5, and 10 days, animals were killed 2 h after labeling with bromodeoxyuridine (BrdU) and eyes were processed for histology. During healing, marked expression of Smad7 was observed in Lens epithelial cells in the Smad7 adv group with loss of nuclear translocation of Smads2/3, while little Smad7 and abundant nuclear Smads2/3 were seen in cells in the Cre adv group. Lens epithelial cells in the Cre adv control group exhibited a fibroblastic appearance at days 5 and 10 and the capsular break was sealed with fibrous tissue, while Smad7 adv-treated cells around the capsular break retained their epithelial morphology and the break was not sealed. Expression of snail mRNA, and alpha-smooth muscle actin, lumican, and collagen VI proteins, markers of EMT, was observed in control-treated eyes, but not in cells of the Smad7 adv group at day 5 with minimal expression at day 10. Additionally, cell proliferation increased in Epithelium infected with Smad7 adv consistent with suppression of injury-induced upregulation of TGFbeta1 in Epithelium. We conclude that gene transfer of Smad7 in mice prevents injury-induced EMT of Lens epithelial cells and sealing of the capsular break with fibrous tissue.

  • smad3 signaling is required for epithelial mesenchymal transition of Lens Epithelium after injury
    American Journal of Pathology, 2004
    Co-Authors: Shizuya Saika, Kathleen C Flanders, Yoshitaka Ohnishi, Akira Ooshima, Satoko Konosaika, Misako Sato, Yasuteru Muragaki, Jiyun Yoo, Mario A Anzano, Chiayang Liu
    Abstract:

    Lens epithelial cells undergo epithelial-mesenchymal transition (EMT) after injury as in cataract extraction, leading to fibrosis of the Lens capsule. Fibrosis of the anterior capsule can be modeled in the mouse by capsular injury in the Lens, which results in EMT of the Lens Epithelium and subsequent deposition of extracellular matrix without contamination of other cell types from outside the Lens. We have previously shown that signaling via Smad3, a key signal-transducing element downstream of transforming growth factor (TGF)-β and activin receptors, is activated in Lens epithelial cells by 12 hours after injury and that this Smad3 activation is blocked by administration of a TGF-β2-neutralizing antibody in mice. We now show that EMT of primary Lens epithelial cells in vitro depends on TGF-β expression and that injury-induced EMT in vivo depends, more specifically, on signaling via Smad3. Loss of Smad3 in mice blocks both morphological changes of Lens Epithelium to a mesenchymal phenotype and expression of the EMT markers snail, α-smooth muscle actin, lumican, and type I collagen in response to injury in vivo or to exposure to exogenous TGF-β in organ culture. The results suggest that blocking the Smad3 pathway might be beneficial in inhibiting capsular fibrosis after injury and/or surgery.

  • osteopontin a component of matrix in capsular opacification and subcapsular cataract
    Investigative Ophthalmology & Visual Science, 2003
    Co-Authors: Shizuya Saika, Iku Ishida, Yoshitaka Ohnishi, Takeshi Miyamoto, Akira Ooshima
    Abstract:

    PURPOSE. To examine whether tissues of human capsular opacification and subcapsular cataract contain osteopontin, an adhesive matrix protein, and whether mouse Lens Epithelium expresses osteopontin after injury. METHODS. An immunohistochemical examination was conducted to determine whether matrices in human postoperative capsular specimens and anterior subcapsular cataract contain osteopontin. The spatial and temporal protein expression patterns of osteopontin were then determined in Epithelium of a healing mouse Lens after a capsular incision. RESULTS. Human Lens epithelial cells in the specimens extracted at the time of vitrectomy 10 days after cataract surgery and also after longer healing intervals were labeled with an anti-osteopontin antibody, whereas uninjured Lens Epithelium was not. In the later healing phase, matrix of capsular opacification was positive for osteopontin. Lens cells amid anterior subcapsular cataract tissue were also positive. Osteopontin was detected in the cell surface and membrane and the cytoplasm of Lens cells, as well as in the matrix. Unlike normal uninjured specimens, anterior Lens capsule of some of the healing postoperative specimens and anterior subcapsular cataract specimens also faintly or weakly stained for osteopontin. Mouse Lens Epithelium started to express osteopontin protein at 8 hours after injury, before the cells changed their shape from epithelial cell type to fibroblast type. Expression of osteopontin lasted during the healing interval, even after the cells transformed into fibroblast-like cells. CONCLUSIONS. Extracellular matrix in human postoperative capsular opacification and anterior subcapsular cataract contains osteopontin. Epithelial cells of a mouse Lens also ectopically express osteopontin in response to capsular injury.

Jan De Rijck - One of the best experts on this subject based on the ideXlab platform.

  • characterization of rare Lens Epithelium derived growth factor p75 genetic variants identified in hiv 1 long term nonprogressors
    AIDS, 2013
    Co-Authors: Rik Schrijvers, Frauke Christ, Zeger Debyser, Jan De Rijck, Jonas Demeulemeester, Melanie Gerard, Rik Gijsbers
    Abstract:

    Objective Lens Epithelium-derived growth factor (LEDGF)/p75 is a cellular binding partner of HIV-1 integrase and a crucial cofactor for HIV-1 replication. Here, we study two LEDGF/p75 exonic variants I436S and T473I, identified in HIV-1 long-term nonprogressors, together with Q472L. Methods In-vitro binding assays, cell culture complementation, and functional rescue. Results Binding affinities of wild-type, I436S, T473I, and Q472L LEDGF/p75 for HIV-1 integrase were comparable. All LEDGF/p75 variants bound equally well to LEDGF/p75 interacting partners JPO2 and PogZ. In addition, HIV-1 replication was evaluated in human somatic LEDGF/p75-knockout cells and LEDGF/p75-knockdown cells complemented with either wild-type LEDGF/p75 or the respective LEDGF/p75 variants. All variants rescued HIV-1 replication to wild-type levels, whereas LEDGF/p75 D366N, defective for interaction with HIV-1 integrase, did not. Conclusion Although identified in a cohort of long-term nonprogressors, our study did not indicate that the I436S or T473I mutation in LEDGF/p75 affects the interaction with HIV-1 integrase.

  • impairing mll fusion gene mediated transformation by dissecting critical interactions with the Lens Epithelium derived growth factor ledgf p75
    Leukemia, 2013
    Co-Authors: Helene Mereau, Frauke Christ, Zeger Debyser, Jan De Rijck, Rik Gijsbers, Jonas Demeulemeester, Kateřina Cermakova, A Kutz, Sabine Juge, Juerg Schwaller
    Abstract:

    The Lens Epithelium-derived growth factor (LEDGF/p75) tethers the mixed-lineage leukemia (MLL1) protein complex to chromatin. Likewise, LEDGF/p75 tethers the HIV-1 pre-integration complex to chromatin. We previously demonstrated that expression of the C-terminal fragment fused to enhanced green fluorescent protein (eGFP) (eGFP-LEDGF(325-530)) impaired HIV-1 replication. Here, we explored this strategy to selectively interfere with the leukemogenic activity of MLL-fusion proteins. We found that expression of LEDGF(325-530) impaired the clonogenic growth of MLL-fusion gene transformed human and mouse hematopoietic cells, without affecting the growth of control cells immortalized by the FLT3-ITD mutant or normal lineage-marker-depleted murine bone marrow cells. Expression of LEDGF(325-530) was associated with downregulation of the MLL target Hoxa9 and impaired cell cycle progression. Structure-function analysis revealed two small eGFP-fused LEDGF/p75 peptides, LEDGF(424-435) and LEDGF(375-386) phenocopying these effects. Both LEDGF(325-530) and the smaller active peptides were able to disrupt the LEDGF/p75-MLL interaction. Expression of LEDGF(325-530) or LEDGF(375-386) fragments increased the latency period to disease development in vivo in a mouse bone marrow transplant model of MLL-AF9-induced AML. We conclude that small peptides disrupting the LEDGF/p75-MLL interface have selective anti-leukemic activity providing a direct rationale for the design of small molecule inhibitors targeting this interaction.

  • Lens Epithelium derived growth factor p75 qualifies as a target for hiv gene therapy in the nsg mouse model
    Molecular Therapy, 2012
    Co-Authors: Sofie Vets, Janine Kimpel, Andreas Volk, Jan De Rijck, Rik Schrijvers, Bert Verbinnen, Wim Maes, Dorothee Von Laer, Zeger Debyser
    Abstract:

    Lens Epithelium-derived growth factor (LEDGF/p75) is an essential cofactor of HIV integration. Both stable overexpression of the C-terminal part of LEDGF/p75 (LEDGF325–530) containing the integrase (IN)-binding domain (IBD) and stable knockdown (KD) of LEDGF/p75 are known to inhibit HIV infection in laboratory cell lines. Here, primary human CD4+ T-cells were transduced with lentiviral vectors encoding LEDGF325–530, the interaction-deficient mutant LEDGF325–530D366N, or a hairpin depleting LEDGF/p75 and challenged with HIV. Maximal protection of primary T-cells from HIV infection was obtained after LEDGF325–530 overexpression reducing HIV replication 40-fold without evidence of cellular toxicity. This strategy was subsequently evaluated in the NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mouse model. Threefold reduction in mean plasma viral load was obtained in mice engrafted with CD4+ T-cells expressing LEDGF325–530 in comparison with engraftment with LEDGF325–530D366N cells. Four weeks after transplantation with LEDGF325–530D366N cells, 70% of the CD4+ cells were lost due to ongoing HIV replication. However, in mice transplanted with LEDGF325–530 cells only a 20% decrease in CD4+ cells was measured. Liver and spleen sections of LEDGF325–530 mice contained less HIV than LEDGF325–530D366N mice as measured by p24 antigen detection. LEDGF325–530 overexpression potently inhibits HIV replication in vivo and protects against HIV mediated cell killing of primary CD4+ T-cells.

  • Lens Epithelium derived growth factor p75 interacts with the transposase derived dde domain of pogz
    Journal of Biological Chemistry, 2009
    Co-Authors: Koen Bartholomeeusen, Frauke Christ, Zeger Debyser, Jelle Hendrix, Jeanchristophe Rain, Stephane Emiliani, Richard Benarous, Rik Gijsbers, Jan De Rijck
    Abstract:

    Lens Epithelium-derived growth factor/p75 (LEDGF/p75) is a prominent cellular interaction partner of human immunodeficiency virus-1 (HIV-1) integrase, tethering the preintegration complex to the host chromosome. In light of the development of LEDGF/p75-integrase interaction inhibitors, it is essential to understand the cell biology of LEDGF/p75. We identified pogZ as new cellular interaction partner of LEDGF/p75. Analogous to lentiviral integrase, pogZ, a domesticated transposase, carries a DDE domain, the major determinant for LEDGF/p75 interaction. Using different in vitro and in vivo approaches, we corroborated the interaction between the C terminus of LEDGF/p75 and the DDE domain of pogZ, revealing an overlap in the binding of pogZ and HIV-1 integrase. Competition experiments showed that integrase is efficient in displacing pogZ from LEDGF/p75. Moreover, pogZ does not seem to play a role as a restriction factor of HIV. The finding that LEDGF/p75 is capable of interacting with a DDE domain protein that is not a lentiviral integrase points to a profound role of LEDGF/p75 in DDE domain protein function.

  • overexpression of the Lens Epithelium derived growth factor p75 integrase binding domain inhibits human immunodeficiency virus replication
    Journal of Virology, 2006
    Co-Authors: Jan De Rijck, Frauke Christ, Jelle Hendrix, Rik Gijsbers, Linos Vandekerckhove, Anneleen Hombrouck, J Vercammen, Yves Engelborghs, Zeger Debyser
    Abstract:

    We initially identified Lens Epithelium-derived growth factor/p75 (LEDGF/p75) as a binding partner of human immunodeficiency virus type 1 (HIV-1) integrase. To investigate the role of LEDGF/p75 in HIV replication and its potential as a new antiviral target, we stably overexpressed two different fragments containing the integrase binding domain (IBD) of LEDGF/p75 fused to enhanced green fluorescent protein (eGFP). HIV-1 replication was severely inhibited by overexpression of the eGFP-IBD fusion proteins, while no inhibition was observed in cell lines overexpressing the interaction-deficient D366A mutant. Quantitative PCR pinpointed the block to the integration step, whereas nuclear import was not affected. Competition of the IBD fusion proteins with endogenous LEDGF/p75 for binding to integrase led to a potent defect in HIV-1 replication in both HeLaP4- and MT-4-derived cell lines. A previously described diketo acid-resistant HIV-1 strain remained fully susceptible to inhibition, suggesting that this strategy will also work in patients who harbor strains resistant to the current experimental integrase inhibitors. These data support LEDGF/p75 as an important cofactor for HIV replication and provide proof of concept for the LEDGF/p75-integrase interaction as a novel target for treating HIV-1 infection.