Autoregulation of RUNX1 Activity by Homodimerization.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1940-1940 ◽  
Author(s):  
Kislay K. Sinha ◽  
Donglan Li ◽  
Maher A. Hay ◽  
Giuseppina Nucifora
Keyword(s):  
Transcription Factor ◽  
Critical Role ◽  
Gene Activation ◽  
Point Mutations ◽  
Chromosomal Translocations ◽  
Human Leukemia ◽  
Thymocyte Development ◽  
C Terminus

Abstract RUNX1 also known as AML1 is a transcription factor essential for normal hematopoiesis, platelet production and thymocyte development. RUNX1 is the most frequent target of chromosomal translocations and acquired or inherited point mutations associated with human leukemia. RUNX1 is a DNA-binding transcription factor that can act both as an activator and a repressor of gene expression depending probably on the association of RUNX1 with co-activator or corepressors in large transcription complexes at promoter sites. The C-terminus of RUNX1 contains an inhibitory region, ID, which represses positive regulation of RUNX1-dependent genes. Thus, this region could potentially act as a switch and co-operate with RUNX1-interacting transcription factors in the choice between gene activation or gene repression. Here we have examined the role of ID in gene regulation by RUNX1. We found that this region is a homo-dimerization motif that controls RUNX1-RUNX1 interaction in vitro and in vivo. The association of RUNX1 with itself through this domain appears to reduce the positive transactivating potential of RUNX1 and, if provided in trans, this domain can repress by itself the activity of RUNX1 in reporter gene assays. Our studies suggest that RUNX1 autoregulates itself through its ability to form a homodimer. Data will be shown on the effects of the ID region in hematopoietic differentiation of cell lines. The results indicate that the ID region plays critical role in RUNX1 activity and is essential to control the correct execution of hematopoietic programs. It is of importance that aside from the t(12;21), all chromosomal translocations and virtually all point mutations associated with leukemia profoundly affect the integrity of the C-terminus including the ID region. These studies suggest a novel pathway involved in RUNX1 leukemogenesis and provide new targets for the management of RUNX1-leukemia. Kislay Sinha and Donglan Li contributed equally to this study.

scholarly journals Inhibition of RANKL-Induced Osteoclastogenesis by Novel Mutant RANKL

2021 ◽  
Vol 22 (1) ◽  
pp. 434
Author(s):  
Yuria Jang ◽  
Hong Moon Sohn ◽  
Young Jong Ko ◽  
Hoon Hyun ◽  
Wonbong Lim
Keyword(s):  
Nuclear Translocation ◽  
Critical Role ◽  
Osteoclast Differentiation ◽  
Point Mutations ◽  
Tartrate Resistant Acid Phosphatase ◽  
Mice Model ◽  
Gsk 3Β ◽  
Rank Signaling

Background: Recently, it was reported that leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4, also called GPR48) is another receptor for RANKL and was shown to compete with RANK to bind RANKL and suppress canonical RANK signaling during osteoclast differentiation. The critical role of the protein triad RANK–RANKL in osteoclastogenesis has made their binding an important target for the development of drugs against osteoporosis. In this study, point-mutations were introduced in the RANKL protein based on the crystal structure of the RANKL complex and its counterpart receptor RANK, and we investigated whether LGR4 signaling in the absence of the RANK signal could lead to the inhibition of osteoclastogenesis.; Methods: The effects of point-mutated RANKL (mRANKL-MT) on osteoclastogenesis were assessed by tartrate-resistant acid phosphatase (TRAP), resorption pit formation, quantitative real-time polymerase chain reaction (qPCR), western blot, NFATc1 nuclear translocation, micro-CT and histomorphological assay in wild type RANKL (mRANKL-WT)-induced in vitro and in vivo experimental mice model. Results: As a proof of concept, treatment with the mutant RANKL led to the stimulation of GSK-3β phosphorylation, as well as the inhibition of NFATc1 translocation, mRNA expression of TRAP and OSCAR, TRAP activity, and bone resorption, in RANKL-induced mouse models; and Conclusions: The results of our study demonstrate that the mutant RANKL can be used as a therapeutic agent for osteoporosis by inhibiting RANKL-induced osteoclastogenesis via comparative inhibition of RANKL. Moreover, the mutant RANKL was found to lack the toxic side effects of most osteoporosis treatments.

scholarly journals Functions of the C-Terminal Domain of Varicella-Zoster Virus Glycoprotein E in Viral Replication In Vitro and Skin and T-Cell Tropism In Vivo

2004 ◽  
Vol 78 (22) ◽  
pp. 12406-12415 ◽  
Author(s):  
Jennifer Moffat ◽  
Chengjun Mo ◽  
Jason J. Cheng ◽  
Marvin Sommer ◽  
Leigh Zerboni ◽  
...  
Keyword(s):  
T Cell ◽  
Viral Replication ◽  
Varicella Zoster Virus ◽  
Point Mutations ◽  
Glycoprotein E ◽  
Varicella Zoster ◽  
Terminal Domain ◽  
C Terminus

ABSTRACT Varicella-zoster virus (VZV) glycoprotein E (gE) is essential for VZV replication. To further analyze the functions of gE in VZV replication, a full deletion and point mutations were made in the 62-amino-acid (aa) C-terminal domain. Targeted mutations were introduced in YAGL (aa 582 to 585), which mediates gE endocytosis, AYRV (aa 568 to 571), which targets gE to the trans-Golgi network (TGN), and SSTT, an “acid cluster” comprising a phosphorylation motif (aa 588 to 601). Substitutions Y582G in YAGL, Y569A in AYRV, and S593A, S595A, T596A, and T598A in SSTT were introduced into the viral genome by using VZV cosmids. These experiments demonstrated a hierarchy in the contributions of these C-terminal motifs to VZV replication and virulence. Deletion of the gE C terminus and mutation of YAGL were lethal for VZV replication in vitro. Mutations of AYRV and SSTT were compatible with recovery of VZV, but the AYRV mutation resulted in rapid virus spread in vitro and the SSTT mutation resulted in higher virus titers than were observed for the parental rOka strain. When the rOka-gE-AYRV and rOka-gE-SSTT mutants were evaluated in skin and T-cell xenografts in SCIDhu mice, interference with TGN targeting was associated with substantial attenuation, especially in skin, whereas the SSTT mutation did not alter VZV infectivity in vivo. These results provide the first information about how targeted mutations of this essential VZV glycoprotein affect viral replication in vitro and VZV virulence in dermal and epidermal cells and T cells within intact tissue microenvironments in vivo.

Wnt5a Induces Association of ROR1 with 14-3-3ζ to Enhance Chemotaxis and Proliferation in Chronic Lymphocytic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 349-349 ◽  
Author(s):  
Jian Yu ◽  
Liguang Chen ◽  
Yun Chen ◽  
Ling Zhang ◽  
Laura Z. Rassenti ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Research Funding ◽  
Critical Role ◽  
Leukemia Cell ◽  
Lymphocytic Leukemia ◽  
Orphan Receptor ◽  
Binding Motif ◽  
Human Leukemia

Abstract Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncoembryonic antigen that is expressed on CLL cells, but not on normal postpartum tissues. We found that ROR1 was a receptor for Wnt5a, which could activate Rho GTPases (e.g. RhoA and Rac1) in CLL cells by inducing the recruitment to ROR1 of guanine exchange factors (GEFs), notably ARHGEF2. How ARHGEF2 can complex with ROR1 was not known. We performed mass spectrometry-based proteomics to interrogate immune-precipitates of Wnt5a-activated ROR1 and identified 14-3-3ζ, a highly conserved, cytoplasmic-protein member of the tetratricopeptide repeat-like superfamily. 14-3-3ζ plays a critical role in cell-signaling pathways, which promote proliferation, adhesion, and survival in a variety of human cancers. We validated the Wnt5a-induced interaction of ROR1 with 14-3-3ζ in primary CLL cells using co-immunoprecipitation studies and immunoblot analyses. We found the capacity of Wnt5a to induce ROR1 to associate with 14-3-3ζ could be blocked by cirmtuzumab, a first-in-class humanized mAb specific for a functional epitope in the ROR1-extracellular domain; this mAb is undergoing clinical testing in patients with CLL. Furthermore, we found that 14-3-3ζ could interact with ARHGEF2 in CLL cells. Silencing 14-3-3ζ via RNAi impaired the capacity of Wnt5a to: (1) induce recruitment of ARHGEF2 to ROR1, (2) enhance the in vitro exchange activity of ARHGEF2 for RhoA and Rac1, and (3) induce activation of RhoA and Rac1 in primary CLL cells. Consistent with these findings, we found that Difopein, an inhibitor of 14-3-3ζ, also could inhibit Wnt5a-enhanced chemokine-directed migration and proliferation of primary CLL cells in vitro, at low concentrations that did not result in leukemia-cell apoptosis. To examine structure-function relationships, we employed the MEC1 cell line, which was derived from human CLL. Prior studies found MEC1 cells expressed Wnt5a, which induced activation of RhoA and Rac1 in MEC1 cells made to express ROR1 (MEC1-ROR1), but not in parental MEC1 cells, which lacked ROR1. Similar to work on primary CLL cells, we detected 14-3-3ζ in anti-ROR1 immune precipitates via mass spectrometry and immunoblot analyses. We identified a 14-3-3ζ binding motif (RSPS857SAS) in the cytoplasmic domain of ROR1; site directed mutagenesis and transfection of MEC1 with mutant forms of ROR1 determined that serine-857 was required for the recruitment of 14-3-3ζ and ARHGEF2-dependent activation of RhoA and Rac1, respectively. In addition, we used CRISPR/Cas9 technology to delete 14-3-3ζ (Δ14-3-3ζ) in MEC1 and MEC1-ROR1 cells. We found that MEC1-ROR1 had significantly higher rates of proliferation than MEC1-ROR1-Δ14-3-3ζ cells, which in turn had rates of proliferation comparable to those of MEC1 cells or MEC1-Δ14-3-3ζ lacking expression of ROR1. MEC1-ROR1 also had a significantly greater capacity to migrate in response to chemokine (CCL21) than did MEC1-ROR1-Δ14-3-3ζ cells, which migrated in response to CCL21 as well as MEC1-Δ14-3-3ζ or MEC1 cells lacking expression of ROR1. To examine whether such differences affected leukemia-cell growth in vivo, we engrafted Rag2−/−γc−/− mice each with equal numbers of MEC1, MEC1-Δ14-3-3ζ, MEC1-ROR1, or MEC1-ROR1-Δ14-3-3ζ cells. We found that mice that received MEC1-ROR1 cells had significantly higher levels of engraftment and human leukemia-cell proliferation than did mice that received MEC1, MEC1-Δ14-3-3ζ, or ROR1-Δ14-3-3ζ cells, which had comparable levels of engraftment and proliferation, indicating that 14-3-3ζ was necessary for the engraftment/growth advantage of MEC1-ROR1 over MEC1 cells in vivo. Collectively, this study reveals that 14-3-3ζ plays a critical role in Wnt5a/ROR1-dependent-signaling leading to enhanced migration and proliferation of CLL cells in vitro and in vivo. Disclosures Kipps: Roche: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Speakers Bureau; AbbVie: Consultancy, Honoraria, Research Funding.

scholarly journals S. mansoni SmKI-1 Kunitz-domain: Leucine point mutation at P1 site generates enhanced neutrophil elastase inhibitory activity

2021 ◽  
Vol 15 (1) ◽  
pp. e0009007
Author(s):  
Fábio Mambelli ◽  
Bruno P. O. Santos ◽  
Suellen B. Morais ◽  
Enrico G. T. Gimenez ◽  
Duana C. dos S. Astoni ◽  
...  
Keyword(s):  
Inhibitory Activity ◽  
Neutrophil Elastase ◽  
Point Mutations ◽  
Point Of View ◽  
Mutant Proteins ◽  
Inflammatory Parameters ◽  
C Terminus ◽  
Kunitz Domain

The Schistosoma mansoni SmKI-1 protein is composed of two domains: a Kunitz-type serine protease inhibitor motif (KD) and a C-terminus domain with no similarity outside the genera. Our previous work has demonstrated that KD plays an essential role in neutrophil elastase (NE) binding blockage, in neutrophil influx and as a potential anti-inflammatory molecule. In order to enhance NE blocking capacity, we analyzed the KD sequence from a structure-function point of view and designed specific point mutations in order to enhance NE affinity. We substituted the P1 site residue at the reactive site for a leucine (termed RL-KD), given its central role for KD’s inhibition to NE. We have also substituted a glutamic acid that strongly interacts with the P1 residue for an alanine, to help KD to be buried on NE S1 site (termed EA-KD). KD and the mutant proteins were evaluated in silico by molecular docking to human NE, expressed in Escherichia coli and tested towards its NE inhibitory activity. Both mutated proteins presented enhanced NE inhibitory activity in vitro and RL-KD presented the best performance. We further tested RL-KD in vivo in an experimental model of monosodium urate (MSU)-induced acute arthritis. RL-KD showed reduced numbers of total cells and neutrophils in the mouse knee cavity when compared to KD. Nevertheless, both RL-KD and KD reduced mice hypernociception in a similar fashion. In summary, our results demonstrated that both mutated proteins showed enhanced NE inhibitory activity in vitro. However, RL-KD had a prominent effect in diminishing inflammatory parameters in vivo.

GATA-1 Forms Distinct Activating and Repressive Complexes in Erythroid Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 356-356
Author(s):  
John Strouboulis ◽  
Patrick Rodriguez ◽  
Edgar Bonte ◽  
Jeroen Krijgsveld ◽  
Katarzyna Kolodziej ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Chromatin Remodeling ◽  
Gene Activation ◽  
Erythroid Differentiation ◽  
Specific Gene ◽  
Erythroid Cells ◽  
In Erythroid Cells

Abstract GATA-1 is a key transcription factor essential for the differentiation of the erythroid, megakaryocytic and eosinophilic lineages. GATA-1 functions in erythropoiesis involve lineage-specific gene activation and repression of early hematopoietic transcription programs. GATA-1 is known to interact with other transcription factors, such as FOG-1, TAL-1 and Sp1 and also with CBP/p300 and the SWI/SNF chromatin remodeling complex in vitro. Despite this information the molecular basis of its essential functions in erythropoiesis remains unclear. We show here that GATA-1 is mostly present in a high (> 670kDa) molecular weight complex that appears to be dynamic during erythroid differentiation. In order to characterize the GATA-1 complex(es) from erythroid cells, we employed an in vivo biotinylation tagging approach in mouse erythroleukemic (MEL) cells1. Briefly, this involved the fusion of a small (23aa) peptide tag to GATA-1 and its specific, efficient biotinylation by the bacterial BirA biotin ligase which is co-expressed with tagged GATA-1 in MEL cells. Nuclear extracts expressing biotinylated tagged GATA-1 were bound directly to streptavidin beads and co-purifying proteins were identified by mass spectrometry. In addition to the known GATA-1-interacting transcription factors FOG-1, TAL-1 and Ldb-1, we describe novel interactions with the essential hematopoietic transcription factor Gfi-1b and the chromatin remodeling complexes MeCP1 and ACF/WCRF. Significantly, GATA-1 interaction with the repressive MeCP1 complex requires FOG-1. We also show in erythroid cells that GATA-1, FOG-1 and MeCP1 are stably bound to repressed genes representing early hematopoietic (e.g. GATA-2) or alternative lineage-specific (e.g. eosinophilic) transcription programs, whereas the GATA-1/Gfi1b complex is bound to repressed genes involved in cell proliferation. In contrast, GATA-1 and TAL-1 are bound to the active erythroid-specific EKLF gene. Our findings on GATA-1 complexes provide novel insight as to the critical roles that GATA-1 plays in many aspects of erythropoiesis by revealing the GATA-1 partners in the execution of specific functions.

VWRPY motif–dependent and –independent roles of AML1/Runx1 transcription factor in murine hematopoietic development

Blood ◽  
2004 ◽  
Vol 103 (2) ◽  
pp. 562-570 ◽  
Author(s):  
Motohiro Nishimura ◽  
Yoko Fukushima-Nakase ◽  
Yasuko Fujita ◽  
Mitsushige Nakao ◽  
Shogo Toda ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Es Cells ◽  
Embryonic Stem ◽  
Es Cell ◽  
Thymocyte Development ◽  
Hematopoietic Development ◽  
Cell Clones ◽  
Definitive Hematopoiesis

Abstract AML1/Runx1 is a frequent target of leukemia-associated gene aberration, and it encodes a transcription factor essential for definitive hematopoiesis. We previously reported that the AML1 molecules with trans-activation subdomains retained can rescue in vitro hematopoietic defects of AML1-deficient mouse embryonic stem (ES) cells when expressed by using a knock-in approach. Extending this notion to in vivo conditions, we found that the knock-in ES cell clones with AML1 mutants, which retain trans-activation subdomains but lack C-terminal repression subdomains including the conserved VWRPY motif, contribute to hematopoietic tissues in chimera mice. We also found that germline mice homozygous for the mutated AML1 allele, which lacks the VWRPY motif, exhibit a minimal effect on hematopoietic development, as was observed in control knock-in mice with full-length AML1. On the other hand, reduced cell numbers and deviant CD4 expression were observed during early T-lymphoid ontogeny in the VWRPY-deficient mice, whereas the contribution to the thymus by the corresponding ES cell clones was inadequate. These findings demonstrate that AML1 with its trans-activating subdomains is essential and sufficient for hematopoietic development in the context of the entire mouse. In addition, its trans-repression activity, depending on the C-terminal VWRPY motif, plays a role in early thymocyte development.

scholarly journals The β-cell specific transcription factor Nkx6.1 inhibits glucagon gene transcription by interfering with Pax6

2007 ◽  
Vol 403 (3) ◽  
pp. 593-601 ◽  
Author(s):  
Benoit R. Gauthier ◽  
Yvan Gosmain ◽  
Aline Mamin ◽  
Jacques Philippe
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Gene Transcription ◽  
Promoter Activity ◽  
Islet Cells ◽  
Gene Activation ◽  
Physical Interaction ◽  
Specific Expression

The transcription factor Nkx6.1 is required for the establishment of functional insulin-producing β-cells in the endocrine pancreas. Overexpression of Nkx6.1 has been shown to inhibit glucagon gene expression while favouring insulin gene activation. Down-regulation resulted in the opposite effect, suggesting that absence of Nkx6.1 favours glucagon gene expression. To understand the mechanism by which Nkx6.1 suppresses glucagon gene expression, we studied its effect on the glucagon gene promoter activity in non-islet cells using transient transfections and gel-shift analyses. In glucagonoma cells transfected with an Nkx6.1-encoding vector, the glucagon promoter activity was reduced by 65%. In BHK21 cells, Nkx6.1 inhibited by 93% Pax6-mediated activation of the glucagon promoter, whereas Cdx2/3 and Maf stimulations were unaltered. Although Nkx6.1 could interact with both the G1 and G3 element, only the former displayed specificity for Nkx6.1. Mutagenesis of the three potential AT-rich motifs within the G1 revealed that only the Pax6-binding site preferentially interacted with Nkx6.1. Chromatin immunoprecipitation confirmed interaction of Nkx6.1 with the glucagon promoter and revealed a direct competition for binding between Pax6 and Nkx6.1. A weak physical interaction between Pax6 and Nkx6.1 was detected in vitro and in vivo suggesting that Nkx6.1 predominantly inhibits glucagon gene transcription through G1-binding competition. We suggest that cell-specific expression of the glucagon gene may only proceed when Nkx6.1, in combination with Pdx1 and Pax4, are silenced in early α-cell precursors.

scholarly journals The C-terminus of the P22 tailspike protein acts as an independent oligomerization domain for monomeric proteins

2009 ◽  
Vol 419 (3) ◽  
pp. 595-602 ◽  
Author(s):  
Tawnya Webber ◽  
Sarsati Gurung ◽  
Justin Saul ◽  
Trenton Baker ◽  
Michelle Spatara ◽  
...  
Keyword(s):  
Hydrophobic Interactions ◽  
Critical Role ◽  
Oligomeric Proteins ◽  
C Terminus ◽  
Monomeric Proteins ◽  
P22 Tailspike ◽  
Tailspike Protein ◽  
Oligomerization Domain

TSP (P22 tailspike protein) is a well-established model system for studying the folding and assembly of oligomeric proteins, and previous studies have documented both in vivo and in vitro folding intermediates using this protein. Especially important is the C-terminus of TSP, which plays a critical role in the assembly and maturation of the protrimer intermediate to its final trimeric form. In the present study, we show that by grafting the C-terminus of TSP on to the monomeric MBP (maltose-binding protein), the resulting chimaera (MBP-537) is a trimeric protein. Moreover, Western blot studies (using an anti-TSP antibody) indicate that the TSP C-terminus in the MBP-537 chimaera has the same conformation as the native TSP. The oligomerization of the MBP-537 chimaera appears to involve hydrophobic interactions and a refolding sequence, both of which are analogous to the native TSP. These results underscore the importance of the TSP C-terminus in the assembly of the mature trimer and demonstrate its potential utility as a model to study the folding and assembly of the TSP C-terminus in isolation.

scholarly journals Enhancers with cooperative Notch binding sites are more resistant to regulation by the Hairless co-repressor

PLoS Genetics ◽  
2021 ◽  
Vol 17 (9) ◽  
pp. e1009039
Author(s):  
Yi Kuang ◽  
Anna Pyo ◽  
Natanel Eafergan ◽  
Brittany Cain ◽  
Lisa M. Gutzwiller ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Binding Sites ◽  
Target Gene ◽  
Gene Activation ◽  
Notch Intracellular Domain ◽  
Repressor Complex ◽  
Notch Activation

Notch signaling controls many developmental processes by regulating gene expression. Notch-dependent enhancers recruit activation complexes consisting of the Notch intracellular domain, the Cbf/Su(H)/Lag1 (CSL) transcription factor (TF), and the Mastermind co-factor via two types of DNA sites: monomeric CSL sites and cooperative dimer sites called Su(H) paired sites (SPS). Intriguingly, the CSL TF can also bind co-repressors to negatively regulate transcription via these same sites. Here, we tested how synthetic enhancers with monomeric CSL sites versus dimeric SPSs bind Drosophila Su(H) complexes in vitro and mediate transcriptional outcomes in vivo. Our findings reveal that while the Su(H)/Hairless co-repressor complex similarly binds SPS and CSL sites in an additive manner, the Notch activation complex binds SPSs, but not CSL sites, in a cooperative manner. Moreover, transgenic reporters with SPSs mediate stronger, more consistent transcription and are more resistant to increased Hairless co-repressor expression compared to reporters with the same number of CSL sites. These findings support a model in which SPS containing enhancers preferentially recruit cooperative Notch activation complexes over Hairless repression complexes to ensure consistent target gene activation.

Angiotensin II increases the expression of the transcription factor ETS-1 in mesangial cells

2008 ◽  
Vol 294 (5) ◽  
pp. F1094-F1100 ◽  
Author(s):  
Damien D. Pearse ◽  
Run-Xia Tian ◽  
Jessica Nigro ◽  
Julian B. Iorgulescu ◽  
Leopold Puzis ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Angiotensin Ii ◽  
Nadph Oxidase ◽  
Mesangial Cells ◽  
Critical Role ◽  
Ang Ii ◽  
Specific Sequence ◽  
Cox 2

Maladaptive activation of the renin-angiotensin system (RAS) has been shown to play a critical role in the pathogenesis of chronic kidney disease. Reactive oxygen species (ROS) are critical signals for many of the nonhemodynamic effects of angiotensin II (ANG II). We have demonstrated that ANG II increases mesangial and cortical cyclooxygenase-2 (COX-2) expression and activity via NADPH oxidase-derived ROS. The transcription factor ETS-1 (E26 transformation-specific sequence) has been identified as a critical regulator of growth-related responses and inflammation. The present studies were designed to determine: 1) whether ANG II induces ETS-1 expression in vitro in cultured rat mesangial cells and in vivo in rats infused with ANG II; and 2) whether ROS and COX-2 are mediators of ETS-1 induction in response to ANG II. Mesangial cells stimulated with ANG II (10−7 M) exhibited a significant increase in ETS-1 expression that was prevented by the angiotensin type 1 receptor blocker candesartan. NADPH oxidase inhibition with dyphenilene iodinium or apocynin also prevented ETS-1 induction, establishing the role of ROS as mediators of ETS-1 expression in response to ANG II. COX-2 inhibition prevented ETS-1 expression in response to ANG II, suggesting that COX-2 is required for ETS-1 induction. By utilizing short interfering RNAs against ETS-1, we have also determined that ETS-1 is required to induce the production of fibronectin in response to ANG II. Furthermore, rats infused with ANG II manifested increased glomerular expression of ETS-1. These studies unveil novel pathways that may play an important role in the pathogenesis of renal injury when RAS is activated.

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