Kenneth Roebuck | National Institutes of Health (original) (raw)
Papers by Kenneth Roebuck
American Journal of Physiology-cell Physiology, Apr 1, 2001
Reactive oxygen species (ROS) are generated at sites of inflammation and injury, and at low level... more Reactive oxygen species (ROS) are generated at sites of inflammation and injury, and at low levels, ROS can function as signaling molecules participating as signaling intermediates in regulation of fundamental cell activities such as cell growth and cell adaptation responses, whereas at higher concentrations, ROS can cause cellular injury and death. The vascular endothelium, which regulates the passage of macromolecules and circulating cells from blood to tissues, is a major target of oxidant stress, playing a critical role in the pathophysiology of several vascular diseases and disorders. Specifically, oxidant stress increases vascular endothelial permeability and promotes leukocyte adhesion, which are coupled with alterations in endothelial signal transduction and redox-regulated transcription factors such as activator protein-1 and nuclear factor-B. This review discusses recent findings on the cellular and molecular mechanisms by which ROS signal events leading to impairment of endothelial barrier function and promotion of leukocyte adhesion. Particular emphasis is placed on the regulation of cell-cell and cell-surface adhesion molecules, the actin cytoskeleton, key protein kinases, and signal transduction events.
BMC Infectious Diseases, Mar 28, 2002
Background: Respiratory syncytial virus (RSV) infection of airway epithelial cells stimulates the... more Background: Respiratory syncytial virus (RSV) infection of airway epithelial cells stimulates the expression and secretion of a variety of cytokines including the chemotactic cytokines interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), and RANTES (regulated upon activation, normal T cell expressed and secreted). Chemokines are important chemoattractants for the recruitment of distinct sets of leukocytes to airway sites of inflammation. Results: We have shown previously that chemokine expression is regulated in airway epithelial cells (A549) in a stimulus-specific manner in part through the redox-responsive transcription factors AP-1 and NF-κB. In this study, we examined the NF-κB-mediated effects of RSV and the proinflammatory cytokine TNFα on the induction of IL-8, MCP-1 and RANTES chemokine gene expression in A549 epithelial cells. The results demonstrate that RSV induces chemokine expression with distinct kinetics that is associated with a specific pattern of NF-κB binding activity. This distinction was further demonstrated by the differential effects of the NF-κB inhibitors dexamethasone (DEX) and N-acetyl-L-cysteine (NAC). NAC preferentially inhibited RSV induced chemokine expression, whereas DEX preferentially inhibited TNFα induced chemokine expression. DNA binding studies using NF-κB subunit specific binding ELISA demonstrated that RSV and TNFα induced different NF-κB binding complexes containing Rel A (p65) and NF-κB1 (p50). Both TNFα and RSV strongly induced Rel A the activation subunit of NF-κB, whereas only TNFα was able to substantially induce the p50 subunit. Consistent with the expression studies, RSV but not TNFα induction of Rel A and p50 were markedly inhibited by NAC, providing a mechanism by which TNFα and RSV can differentially activate chemokine gene expression via NF-κB. Conclusions: These data suggest that RSV induction of chemokine gene expression, in contrast to TNFα, involves redox-sensitive NF-κB complexes containing predominantly Rel A. Background Respiratory syncytial virus (RSV) belongs to the Pneumovirinae subfamily of the Paramyxovirodae family of enveloped single-stranded negative sense RNA viruses. RSV infection of the lower respiratory tract cells results in cell death and sloughing into the lumen of the respiratory tree. Worldwide, RSV is the leading cause of infant mortality from respiratory infections and is so highly contagious
Journal of Immunology, Apr 15, 2000
IFN-γ up-regulates expression of the MHC class II transactivator (CIITA) type IV promoter in mult... more IFN-γ up-regulates expression of the MHC class II transactivator (CIITA) type IV promoter in multiple myeloma cells (35.20)
PubMed, 1999
Human immunodeficiency virus type-1 (HIV-1) is a highly pathogenic lentivirus that requires trans... more Human immunodeficiency virus type-1 (HIV-1) is a highly pathogenic lentivirus that requires transcription of its provirus genome for completion of the viral life cycle and the production of progeny virions. Since the first genetic analysis of HIV-1 in 1985, much has been learned about the transcriptional regulation of the HIV-1 genome in infected cells. It has been demonstrated that HIV-1 transcription depends on a varied and complex interaction of host cell transcription factors with the viral long terminal repeat (LTR) promoter. The regulatory elements within the LTR interact with constitutive and inducible transcription factors to direct the assembly of a stable transcription complex that stimulates multiple rounds of transcription by RNA polymerase II (RNAPII). However, the majority of these transcripts terminate prematurely in the absence of the virally encoded trans-activator protein Tat, which stimulates HIV-1 transcription elongation by interacting with a stem-loop RNA element (TAR) formed at the extreme 5' end of all viral transcripts. The Tat-TAR interaction recruits a cellular kinase into the initiation-elongation complex that alters the elongation properties of RNAPII during its transit through TAR. This review summarizes our current knowledge and understanding of the regulation of HIV-1 transcription in infected cells and highlights the important contributions human lentivirus gene regulation has made to our general understanding of the transcription process.
Virology, Jun 1, 1998
The HIV-1 long terminal repeat (LTR) responds to a variety of cellular signal transduction pathwa... more The HIV-1 long terminal repeat (LTR) responds to a variety of cellular signal transduction pathways. We demonstrate that the cAMP-dependent protein kinase A (PKA) and protein kinase C (PKC) signaling pathways synergize to increase HIV-1 LTR-mediated transcription and viral replication in a latently infected promonocytic cell line (U1). The LTR-mediated synergy induced by cholera toxin (Ctx), a potent activator of the cAMP-dependent PKA pathway, and the PKC activator phorbol 12-myristate 13-acetate (PMA) was abrogated by a PKC--specific inhibitor (LY333531). In contrast, the LTR-mediated synergy induced by Ctx and TNF␣ was not affected by LY333531. The synergy induced by Ctx and TNF␣ was also abrogated by mutation of the cAMP-responsive downstream sequence elements (DSE) in the 5Ј untranslated leader region, whereas the DSE mutations did not affect the synergy induced by Ctx and PMA. These distinctions indicate that Ctx cooperates differently with TNF␣ and PMA to activate the HIV-1 LTR. Ctx and PMA synergistically activated AP-1-and NF-B-dependent transcription, even though no cooperative binding of AP-1 or NF-B was observed in gel shift assays. An extensive mutational analysis of the HIV-1 LTR that included the NF-B and AP-1 binding sites revealed no distinct cis-acting element or region within the HIV-1 LTR that was required for the transcriptional synergy. Ctx and PMA also synergistically interact to activate the HTLV-1 LTR. These results indicate that the transcriptional synergy elicited by Ctx and PMA targets multiple functional elements and promoters, requires a cooperative interaction between the PKA and PKC- pathways, and differs mechanistically from the transcriptional synergy induced by Ctx and TNF␣.
The Journal of Allergy and Clinical Immunology, 2000
Journal of Clinical Investigation, Sep 1, 1993
Activation of HIV-1 requires the binding of host cell transcription factors to cis elements in th... more Activation of HIV-1 requires the binding of host cell transcription factors to cis elements in the proviral long terminal repeat (LTR). This study identifies c-fos-responsive sequence motifs in the U5 transcribed noncoding leader sequences downstream of the viral transactivator responsive (TAR) element. These DNA sequence motifs are the most downstream regulatory elements described thus far in the HIV-1 LTR. Functional studies, using human colon epithelial cell lines, demonstrate that the downstream elements are transactivated by expression of the c-fos protooncogene and can transmit PMA and TNFa activation signals to the viral LTR. Moreover, the c-fos-responsive elements mediate HIV-1 LTR transcription independent of Tat and the NFiB-binding enhancer element. Nuclear extracts of colon epithelial cells form distinct gel mobility shift complexes with the c-fos-responsive elements. These complexes comigrate with a gel shift complex formed on a classical CRE oligonucleotide and are competed by CRE oligonucleotides. These data indicate that the HIV-1 LTR contains previously unrecognized functional DNA cis-regulatory elements downstream of TAR in the transcribed noncoding 5' leader sequence and suggest that early response genes such as c-fos play a role in the
Biochemical and Biophysical Research Communications, Oct 1, 1990
The anomalous electrophoretic behavior of a 686 base pair restriction fragment containing an in v... more The anomalous electrophoretic behavior of a 686 base pair restriction fragment containing an in vitro-generated inversion mutation within the enhancer region of a chicken U1 RNA gene was investigated. This DNA fragment migrated with an abnormally slow mobility in polyacrylamide gels but migrated normally in agarose gels relative to the wild type fragment of identical size and base composition. In polyacrylamide gels, the degree of retardation was enhanced at low temperature, a phenomenon associated with bent DNA. A putative site of bending was localized at or near one end of the inverted region. These data suggest that the altered DNA conformation results from the juxtaposition of two normally remote DNA sequences.
Journal of Bone and Mineral Research, Mar 1, 2001
Previously, we showed that exposure of human osteoblasts to titanium particles stimulates protein... more Previously, we showed that exposure of human osteoblasts to titanium particles stimulates protein tyrosine phosphorylation (PTP), activates the transcription factor nuclear factor κB (NF‐κB), and causes an approximately 50% decrease in the steady‐state messenger RNA (mRNA) level of procollagen α1[I]. In this study, we identify three NF‐κB binding sites within the human procollagen α1[I] gene promoter, show that titanium particles stimulate their binding of the NF‐κB subunits Rel A (p65) and NF‐κB1 (p50), and find NF‐κB activation correlates with collagen gene suppression by titanium particles in osteoblasts. Protein tyrosine kinase (PTK) inhibitors, which significantly reduce the suppressive effect of titanium particles on collagen gene expression, inhibited NF‐κB binding activity showing that titanium particle stimulation of PTK signals in osteoblasts are critical for both NF‐κB activation and collagen gene expression. The antioxidant pyrrolidine dithiocarbamate (PDTC), which also inhibits the titanium particle suppression of collagen, abrogated the titanium particle activation of NF‐κB, suggesting the involvement of redox signals in NF‐κB‐mediated collagen gene expression. The RNA polymerase II inhibitor actinomycin D (Act D) decreased procollagen α1[I] mRNA expression and effectively blocked the titanium‐induced suppressive effect, suggesting that titanium particles activate a cascade of signals in osteoblasts, which result in a suppression of procollagen α1[I] mRNA. Collectively, these results show that titanium particles can activate NF‐κB signaling in osteoblasts and suggest that NF‐κB binding to the collagen gene promoter has a functional role in the down‐regulation of procollagen α1[I] gene transcription.
Nucleic Acids Research, 1987
The signals controlling the expression of a chicken U4 small nuclear RNA (snRNA) gene have been s... more The signals controlling the expression of a chicken U4 small nuclear RNA (snRNA) gene have been studied by microinjection into Xenopus oocytes. At least two distinct regions in the 5'-flanking DNA contribute to U4B RNA gene expression. The proximal regulatory element, which is inactivated by a 5'-flanking DNA deletion to position-38, provides a basal level of U4B RNA synthesis. The distal regulatory region, centered near position-200, acts as a transcriptional enhancer. It provides a 4-5 fold stimulation of U4B RNA gene expression above the basal level, and, like mRNA enhancers, is composed of multiple functional motifs. One of these, the octamer sequence ATGCAAAG, has previously been recognized as an important element of Ul and U2 snRNA gene enhancers, as well as being involved in the expression of a number of uRNA genes. However, the octamer sequence is not sufficient for U4B enhancer activity. An additional element, an "Sph motif," is located 12 base pairs downstream of the octaxer and is an essential component of the U4B enhancer. Transcriptional competition studies indicate that the U4B and Ul snRNA genes utilize a common set of transcription factors.
Journal of Biomedical Materials Research Part A, 2006
Particulate wear debris induces the expression of pro-inflammatory cytokine and chemokine genes i... more Particulate wear debris induces the expression of pro-inflammatory cytokine and chemokine genes in various cell types of the periprosthetic region. We have previously reported that titanium particles stimulate the selective induction of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) chemokines in human osteoblast-like osteosarcoma cells. In this study, we characterize the human bone marrow-derived osteoblast chemokine response to titanium particles. We demonstrate that titanium particles result in enhanced IL-8 and MCP-1 protein secretion as well as differential chemokine gene activation. Osteoblast chemokine expression was regulated at the level of gene transcription, with a time-dependent induction of NF-B activation. Inhibition studies with N-acetyl-l-cysteine (Nac) and MG-132 suggest that titanium particle activation of NF-B activity and IL-8 chemokine exression involves oxidant signaling and IB␣-proteasomal degradation. Activation of the NF-B transcription factor, as well as the IL-8 gene, are redoxregulated. We also demonstrate that while cytochalasin D, a potent inhibitor of phagocytosis, suppressed the titanium particle effect on IL-8 protein release in human bone marrow-derived osteoblasts, the inhibitor had no effect on IL-8 expression in MG-63 osteoblast-like cells. Collectively, these results provide insight into the potential mechanisms responsible for the particulate activation of osteoblast chemokine expression and suggest an important role for the osteoblast in the pathogenesis of periprosthetic osteolysis.
Journal of Bone and Mineral Research, Sep 1, 2000
Particulate wear debris generated mechanically from prosthetic materials is phagocytosed by a var... more Particulate wear debris generated mechanically from prosthetic materials is phagocytosed by a variety of cell types within the periprosthetic space including osteoblasts, which cells with an altered function may contribute to periprosthetic osteolysis. Exposure of osteoblast‐like osteosarcoma cells or bone marrow‐derived primary osteoblasts to either metallic or polymeric particles of phagocytosable sizes resulted in a marked decrease in the steady‐state messenger RNA (mRNA) levels of procollagen α1[I] and procollagen α1[III]. In contrast, no significant effect was observed for the osteoblast‐specific genes, such as osteonectin and osteocalcin (OC). In kinetic studies, particles once phagocytosed, maintained a significant suppressive effect on collagen gene expression and type I collagen synthesis for up to five passages. Large particles of a size that cannot be phagocytosed also down‐regulated collagen gene expression suggesting that an initial contact between cells and particles can generate gene responsive signals independently of the phagocytosis process. Concerning such signaling, titanium particles rapidly increased protein tyrosine phosphorylation and nuclear transcription factor κB (NF‐κB) binding activity before the phagocytosis of particles. Protein tyrosine kinase (PTK) inhibitors such as genistein and the NF‐κB inhibitor pyrrolidine dithiocarbamate (PDTC) significantly reduced the suppressive effect of titanium on collagen gene expression suggesting particles suppress collagen gene expression through the NF‐κB signaling pathway. These results provide a mechanism by which particulate wear debris can antagonize the transcription of the procollagen α1[I] gene in osteoblasts, which may contribute to reduced bone formation and progressive periprosthetic osteolysis.
International Journal of Molecular Medicine, May 1, 1998
Human immunodeficiency virus type-1 (HIV-1) transcription is dependent on the interaction of host... more Human immunodeficiency virus type-1 (HIV-1) transcription is dependent on the interaction of host-cell transcription factors with cis-regulatory DNA elements within the viral long terminal repeat (LTR). Much attention has focused on the series of sequence elements upstream of the transcriptional initiation site in the U3 region of the LTR including the Sp1 and NF-kappaB binding sites. Recent studies, however, demonstrate that the transcribed 5'-untranslated leader region (5'-UTR) also contains important transcriptional elements. These regulatory elements situated downstream of transcription interact with constitutive and inducible transcription factors, mediate transmission of cellular activation signals, and are important for efficient HIV-1 transcription and replication. The 5'-UTR contains binding sites for the transcription factors AP-1, NF-kappaB, NF-AT, IRF, and Sp1. Mutations in these binding sites can interfere with the viral response to cell activation signals, decrease LTR transcription, and inhibit viral replication. The 5'-UTR also interacts with a specific nucleosome that is rapidly displaced during transcriptional activation of the latent provirus. We propose that the inducible transcription factor binding sites in the 5'-UTR comprise a downstream enhancer domain that can function independent of, or in concert with, the LTR promoter to rapidly increase latent proviral transcription in response to cell activation signals. In this review, we describe the host-cell transcription factors that interact with the 5'-UTR and discuss their role in the transcriptional regulation of HIV-1 gene expression.
Clinical Orthopaedics and Related Research, Dec 1, 2001
Since the recognition of aseptic loosening by Charnley in the early 1960s, much information has b... more Since the recognition of aseptic loosening by Charnley in the early 1960s, much information has been gained on the basic science of periprosthetic bone loss. Initially termed cement disease, it now generally is accepted that, in most instances, osteolysis is a manifestation of an adverse cellular response to phagocytosable particulate wear and corrosion debris, possibly facilitated by local hydrodynamic effects. Tissue explant, animal, and cell culture studies have allowed us to compile an appreciation of the complexity of cellular interactions and chemical mediators involved in osteolysis. Cellular participants have been shown to include the macrophage, osteoblast, fibroblast, and osteoclast. The plethora of chemical mediators that are responsible for the cellular responses and effects on bone include prostaglandin E2, tumor necrosis factor-alpha, interleukin-1, and interleukin 6. However, an increasing number of other proinflammatory and antiinflammatory cytokines, prostenoids, and enzymes have been shown to play important roles in this process. The ultimate goal of basic research is to develop novel strategies for evaluation and treatment of patients with osteolysis. Although initial animal studies are promising for possible pharmacologic treatment and prevention of osteolysis, well-controlled human trials are required before agents such as bisphosphonates can be recommended for general clinical use.
Clinical and Experimental Immunology, Aug 1, 2000
HIV-1 replicates in activated T cells at significantly higher levels than in resting cells. Thus,... more HIV-1 replicates in activated T cells at significantly higher levels than in resting cells. Thus, certain molecules up-regulated during T cell activation appear to be important for HIV-1 replication. In this study, we present evidence suggesting that expression of MHC class II (class II) molecules on CD4 1 T cells facilitate HIV-1 replication. T cells that expressed class II supported greater virus replication than T cells lacking class II. The class II 1 cells, when either infected with HIV-1 or transfected with an envminus HIV-1 provirus plasmid, produced 10±20-fold greater virus expression than class II 2 cells. Anticlass II antibody markedly inhibited virus expression in class II 1 cells (but not class II 2 cells) and also decreased the nuclear binding activity of AP-1, an inducible transcription factor important in T cell activation and HIV-1 expression. Most importantly, the induction of class II expression by transfection of the MHC class II transactivator (CIITA) stimulated HIV-1 replication in Jurkat T cells. Taken together, these data suggest that expression of MHC class II molecules and/or CIITA in T cells enhances HIV-1 transcription.
Journal of Biological Chemistry, May 1, 2002
Journal of Orthopaedic Research, May 1, 2002
Exposure of human osteoblasts to ultrafine titanium (Ti) particles has been shown to alter osteob... more Exposure of human osteoblasts to ultrafine titanium (Ti) particles has been shown to alter osteoblast gene expression. We previously reported that Ti particles can increase IL‐6 release and suppress the gene expression of procollagens α1[I] and α1[III] in human osteoblasts. In this study, we now demonstrate that Ti particles can rapidly induce the chemotactic cytokines interleukin‐8 (IL‐8) and monocyte chemoattractant protein‐1 (MCP‐1), two immediate early stress responsive chemokines important for the activation and chemotaxis of neutrophils and macrophages, respectively. In MG‐63 osteosarcoma cells and bone marrow derived primary osteoblasts Ti particles selectively increased the steady state levels of IL‐8 and MCP‐1 mRNA in a time and concentration dependent manner. The increased chemokine mRNA correlated with increased secretion of IL‐8 and MCP‐1 protein. Actinomycin D, a potent RNA polymerase II inhibitor, blocked the Ti particle induction of IL‐8 and MCP‐1 mRNA expression, whereas cycloheximide, which inhibits protein synthesis, failed to inhibit chemokine gene expression suggesting Ti particles directly target activation of chemokine gene transcription. Consistent with a transcriptional mechanism not involving new protein synthesis, we demonstrate that Ti particles induce the binding of the p65 and p50 subunits of the latent transcription factor NF‐κB to the IL‐8 gene promoter. Taken together, these data demonstrate that Ti particles can activate transcription of the stress responsive chemokine genes IL‐8 and MCP‐1 in human osteoblasts. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Journal of Interferon and Cytokine Research, May 1, 1999
Interleukin-8 (IL-8), a m em ber of the C XC chem okine fam ily, is an important activator and ch... more Interleukin-8 (IL-8), a m em ber of the C XC chem okine fam ily, is an important activator and chemoattractant for neutrophils and has been im plicated in a variety of inflamm atory diseases. IL-8 is secreted in a stimulusspecific m anner by a wide variety of cell types and is regulated prim arily at the level of gene transcription. Functional studies indicate that IL-8 transcriptional responses to proinflam matory m ediators are rapid and require only 100 nucleotides of 59-flanking DNA upstream of the TATA box. W ithin the IL-8 promoter sequence are DNA binding sites for the indu cible transcription factors AP-1, NF-IL-6, and NF-j B. Transcription factors in these fam ilies bind the IL-8 promoter as dim ers, and several distinct subunit combinations have been identified as important for IL-8 transcription. In addition, these factors can act in concert to synergistically activate the IL-8 promoter. AP-1 and NF-IL-6 physically interact with NF-j B, and functional cooperativity am ong the factors appears to be critical for optim al IL-8 prom oter activity in different cell types. IL-8 transcription appears to be activated by a promoter recruitm ent mechanism where inducible transcription factor binding to the IL-8 prom oter is required for binding of constitutively active TATA box-binding proteins and form ation of a stable preinitiation complex. This review discusses the regulatory role these higherorder synergistic interactions play in IL-8 transcription and in generation of the stimulus-specific and cell type-specific patterns of IL-8 expression. 429 INTRODUC TIO N REVIEW RO EBUC K 430 FIG. 1. Structure of the IL-8 gene. The IL-8 gene contains four exons, indicated by rectangles, and three introns of varying lengths. The coding regions are indicated by solid rectangles. The number of amino acids (aa) encoded by each exon is indicated above each rectangle. The IL-8 gene contains a TATA box at the 59-end and a polyadenylation signal at the 39-end. The amino acid sequence of the IL-8 protein is shown below the gene. The four conserved cysteine residues, the characteristic CXC sequence, and the 20 amino acid signal peptide are underlined. The extracellular processing required to generate the major 72 amino acid form is indicated by dashes.
American Journal of Physiology-cell Physiology, Apr 1, 2001
Reactive oxygen species (ROS) are generated at sites of inflammation and injury, and at low level... more Reactive oxygen species (ROS) are generated at sites of inflammation and injury, and at low levels, ROS can function as signaling molecules participating as signaling intermediates in regulation of fundamental cell activities such as cell growth and cell adaptation responses, whereas at higher concentrations, ROS can cause cellular injury and death. The vascular endothelium, which regulates the passage of macromolecules and circulating cells from blood to tissues, is a major target of oxidant stress, playing a critical role in the pathophysiology of several vascular diseases and disorders. Specifically, oxidant stress increases vascular endothelial permeability and promotes leukocyte adhesion, which are coupled with alterations in endothelial signal transduction and redox-regulated transcription factors such as activator protein-1 and nuclear factor-B. This review discusses recent findings on the cellular and molecular mechanisms by which ROS signal events leading to impairment of endothelial barrier function and promotion of leukocyte adhesion. Particular emphasis is placed on the regulation of cell-cell and cell-surface adhesion molecules, the actin cytoskeleton, key protein kinases, and signal transduction events.
BMC Infectious Diseases, Mar 28, 2002
Background: Respiratory syncytial virus (RSV) infection of airway epithelial cells stimulates the... more Background: Respiratory syncytial virus (RSV) infection of airway epithelial cells stimulates the expression and secretion of a variety of cytokines including the chemotactic cytokines interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), and RANTES (regulated upon activation, normal T cell expressed and secreted). Chemokines are important chemoattractants for the recruitment of distinct sets of leukocytes to airway sites of inflammation. Results: We have shown previously that chemokine expression is regulated in airway epithelial cells (A549) in a stimulus-specific manner in part through the redox-responsive transcription factors AP-1 and NF-κB. In this study, we examined the NF-κB-mediated effects of RSV and the proinflammatory cytokine TNFα on the induction of IL-8, MCP-1 and RANTES chemokine gene expression in A549 epithelial cells. The results demonstrate that RSV induces chemokine expression with distinct kinetics that is associated with a specific pattern of NF-κB binding activity. This distinction was further demonstrated by the differential effects of the NF-κB inhibitors dexamethasone (DEX) and N-acetyl-L-cysteine (NAC). NAC preferentially inhibited RSV induced chemokine expression, whereas DEX preferentially inhibited TNFα induced chemokine expression. DNA binding studies using NF-κB subunit specific binding ELISA demonstrated that RSV and TNFα induced different NF-κB binding complexes containing Rel A (p65) and NF-κB1 (p50). Both TNFα and RSV strongly induced Rel A the activation subunit of NF-κB, whereas only TNFα was able to substantially induce the p50 subunit. Consistent with the expression studies, RSV but not TNFα induction of Rel A and p50 were markedly inhibited by NAC, providing a mechanism by which TNFα and RSV can differentially activate chemokine gene expression via NF-κB. Conclusions: These data suggest that RSV induction of chemokine gene expression, in contrast to TNFα, involves redox-sensitive NF-κB complexes containing predominantly Rel A. Background Respiratory syncytial virus (RSV) belongs to the Pneumovirinae subfamily of the Paramyxovirodae family of enveloped single-stranded negative sense RNA viruses. RSV infection of the lower respiratory tract cells results in cell death and sloughing into the lumen of the respiratory tree. Worldwide, RSV is the leading cause of infant mortality from respiratory infections and is so highly contagious
Journal of Immunology, Apr 15, 2000
IFN-γ up-regulates expression of the MHC class II transactivator (CIITA) type IV promoter in mult... more IFN-γ up-regulates expression of the MHC class II transactivator (CIITA) type IV promoter in multiple myeloma cells (35.20)
PubMed, 1999
Human immunodeficiency virus type-1 (HIV-1) is a highly pathogenic lentivirus that requires trans... more Human immunodeficiency virus type-1 (HIV-1) is a highly pathogenic lentivirus that requires transcription of its provirus genome for completion of the viral life cycle and the production of progeny virions. Since the first genetic analysis of HIV-1 in 1985, much has been learned about the transcriptional regulation of the HIV-1 genome in infected cells. It has been demonstrated that HIV-1 transcription depends on a varied and complex interaction of host cell transcription factors with the viral long terminal repeat (LTR) promoter. The regulatory elements within the LTR interact with constitutive and inducible transcription factors to direct the assembly of a stable transcription complex that stimulates multiple rounds of transcription by RNA polymerase II (RNAPII). However, the majority of these transcripts terminate prematurely in the absence of the virally encoded trans-activator protein Tat, which stimulates HIV-1 transcription elongation by interacting with a stem-loop RNA element (TAR) formed at the extreme 5' end of all viral transcripts. The Tat-TAR interaction recruits a cellular kinase into the initiation-elongation complex that alters the elongation properties of RNAPII during its transit through TAR. This review summarizes our current knowledge and understanding of the regulation of HIV-1 transcription in infected cells and highlights the important contributions human lentivirus gene regulation has made to our general understanding of the transcription process.
Virology, Jun 1, 1998
The HIV-1 long terminal repeat (LTR) responds to a variety of cellular signal transduction pathwa... more The HIV-1 long terminal repeat (LTR) responds to a variety of cellular signal transduction pathways. We demonstrate that the cAMP-dependent protein kinase A (PKA) and protein kinase C (PKC) signaling pathways synergize to increase HIV-1 LTR-mediated transcription and viral replication in a latently infected promonocytic cell line (U1). The LTR-mediated synergy induced by cholera toxin (Ctx), a potent activator of the cAMP-dependent PKA pathway, and the PKC activator phorbol 12-myristate 13-acetate (PMA) was abrogated by a PKC--specific inhibitor (LY333531). In contrast, the LTR-mediated synergy induced by Ctx and TNF␣ was not affected by LY333531. The synergy induced by Ctx and TNF␣ was also abrogated by mutation of the cAMP-responsive downstream sequence elements (DSE) in the 5Ј untranslated leader region, whereas the DSE mutations did not affect the synergy induced by Ctx and PMA. These distinctions indicate that Ctx cooperates differently with TNF␣ and PMA to activate the HIV-1 LTR. Ctx and PMA synergistically activated AP-1-and NF-B-dependent transcription, even though no cooperative binding of AP-1 or NF-B was observed in gel shift assays. An extensive mutational analysis of the HIV-1 LTR that included the NF-B and AP-1 binding sites revealed no distinct cis-acting element or region within the HIV-1 LTR that was required for the transcriptional synergy. Ctx and PMA also synergistically interact to activate the HTLV-1 LTR. These results indicate that the transcriptional synergy elicited by Ctx and PMA targets multiple functional elements and promoters, requires a cooperative interaction between the PKA and PKC- pathways, and differs mechanistically from the transcriptional synergy induced by Ctx and TNF␣.
The Journal of Allergy and Clinical Immunology, 2000
Journal of Clinical Investigation, Sep 1, 1993
Activation of HIV-1 requires the binding of host cell transcription factors to cis elements in th... more Activation of HIV-1 requires the binding of host cell transcription factors to cis elements in the proviral long terminal repeat (LTR). This study identifies c-fos-responsive sequence motifs in the U5 transcribed noncoding leader sequences downstream of the viral transactivator responsive (TAR) element. These DNA sequence motifs are the most downstream regulatory elements described thus far in the HIV-1 LTR. Functional studies, using human colon epithelial cell lines, demonstrate that the downstream elements are transactivated by expression of the c-fos protooncogene and can transmit PMA and TNFa activation signals to the viral LTR. Moreover, the c-fos-responsive elements mediate HIV-1 LTR transcription independent of Tat and the NFiB-binding enhancer element. Nuclear extracts of colon epithelial cells form distinct gel mobility shift complexes with the c-fos-responsive elements. These complexes comigrate with a gel shift complex formed on a classical CRE oligonucleotide and are competed by CRE oligonucleotides. These data indicate that the HIV-1 LTR contains previously unrecognized functional DNA cis-regulatory elements downstream of TAR in the transcribed noncoding 5' leader sequence and suggest that early response genes such as c-fos play a role in the
Biochemical and Biophysical Research Communications, Oct 1, 1990
The anomalous electrophoretic behavior of a 686 base pair restriction fragment containing an in v... more The anomalous electrophoretic behavior of a 686 base pair restriction fragment containing an in vitro-generated inversion mutation within the enhancer region of a chicken U1 RNA gene was investigated. This DNA fragment migrated with an abnormally slow mobility in polyacrylamide gels but migrated normally in agarose gels relative to the wild type fragment of identical size and base composition. In polyacrylamide gels, the degree of retardation was enhanced at low temperature, a phenomenon associated with bent DNA. A putative site of bending was localized at or near one end of the inverted region. These data suggest that the altered DNA conformation results from the juxtaposition of two normally remote DNA sequences.
Journal of Bone and Mineral Research, Mar 1, 2001
Previously, we showed that exposure of human osteoblasts to titanium particles stimulates protein... more Previously, we showed that exposure of human osteoblasts to titanium particles stimulates protein tyrosine phosphorylation (PTP), activates the transcription factor nuclear factor κB (NF‐κB), and causes an approximately 50% decrease in the steady‐state messenger RNA (mRNA) level of procollagen α1[I]. In this study, we identify three NF‐κB binding sites within the human procollagen α1[I] gene promoter, show that titanium particles stimulate their binding of the NF‐κB subunits Rel A (p65) and NF‐κB1 (p50), and find NF‐κB activation correlates with collagen gene suppression by titanium particles in osteoblasts. Protein tyrosine kinase (PTK) inhibitors, which significantly reduce the suppressive effect of titanium particles on collagen gene expression, inhibited NF‐κB binding activity showing that titanium particle stimulation of PTK signals in osteoblasts are critical for both NF‐κB activation and collagen gene expression. The antioxidant pyrrolidine dithiocarbamate (PDTC), which also inhibits the titanium particle suppression of collagen, abrogated the titanium particle activation of NF‐κB, suggesting the involvement of redox signals in NF‐κB‐mediated collagen gene expression. The RNA polymerase II inhibitor actinomycin D (Act D) decreased procollagen α1[I] mRNA expression and effectively blocked the titanium‐induced suppressive effect, suggesting that titanium particles activate a cascade of signals in osteoblasts, which result in a suppression of procollagen α1[I] mRNA. Collectively, these results show that titanium particles can activate NF‐κB signaling in osteoblasts and suggest that NF‐κB binding to the collagen gene promoter has a functional role in the down‐regulation of procollagen α1[I] gene transcription.
Nucleic Acids Research, 1987
The signals controlling the expression of a chicken U4 small nuclear RNA (snRNA) gene have been s... more The signals controlling the expression of a chicken U4 small nuclear RNA (snRNA) gene have been studied by microinjection into Xenopus oocytes. At least two distinct regions in the 5'-flanking DNA contribute to U4B RNA gene expression. The proximal regulatory element, which is inactivated by a 5'-flanking DNA deletion to position-38, provides a basal level of U4B RNA synthesis. The distal regulatory region, centered near position-200, acts as a transcriptional enhancer. It provides a 4-5 fold stimulation of U4B RNA gene expression above the basal level, and, like mRNA enhancers, is composed of multiple functional motifs. One of these, the octamer sequence ATGCAAAG, has previously been recognized as an important element of Ul and U2 snRNA gene enhancers, as well as being involved in the expression of a number of uRNA genes. However, the octamer sequence is not sufficient for U4B enhancer activity. An additional element, an "Sph motif," is located 12 base pairs downstream of the octaxer and is an essential component of the U4B enhancer. Transcriptional competition studies indicate that the U4B and Ul snRNA genes utilize a common set of transcription factors.
Journal of Biomedical Materials Research Part A, 2006
Particulate wear debris induces the expression of pro-inflammatory cytokine and chemokine genes i... more Particulate wear debris induces the expression of pro-inflammatory cytokine and chemokine genes in various cell types of the periprosthetic region. We have previously reported that titanium particles stimulate the selective induction of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) chemokines in human osteoblast-like osteosarcoma cells. In this study, we characterize the human bone marrow-derived osteoblast chemokine response to titanium particles. We demonstrate that titanium particles result in enhanced IL-8 and MCP-1 protein secretion as well as differential chemokine gene activation. Osteoblast chemokine expression was regulated at the level of gene transcription, with a time-dependent induction of NF-B activation. Inhibition studies with N-acetyl-l-cysteine (Nac) and MG-132 suggest that titanium particle activation of NF-B activity and IL-8 chemokine exression involves oxidant signaling and IB␣-proteasomal degradation. Activation of the NF-B transcription factor, as well as the IL-8 gene, are redoxregulated. We also demonstrate that while cytochalasin D, a potent inhibitor of phagocytosis, suppressed the titanium particle effect on IL-8 protein release in human bone marrow-derived osteoblasts, the inhibitor had no effect on IL-8 expression in MG-63 osteoblast-like cells. Collectively, these results provide insight into the potential mechanisms responsible for the particulate activation of osteoblast chemokine expression and suggest an important role for the osteoblast in the pathogenesis of periprosthetic osteolysis.
Journal of Bone and Mineral Research, Sep 1, 2000
Particulate wear debris generated mechanically from prosthetic materials is phagocytosed by a var... more Particulate wear debris generated mechanically from prosthetic materials is phagocytosed by a variety of cell types within the periprosthetic space including osteoblasts, which cells with an altered function may contribute to periprosthetic osteolysis. Exposure of osteoblast‐like osteosarcoma cells or bone marrow‐derived primary osteoblasts to either metallic or polymeric particles of phagocytosable sizes resulted in a marked decrease in the steady‐state messenger RNA (mRNA) levels of procollagen α1[I] and procollagen α1[III]. In contrast, no significant effect was observed for the osteoblast‐specific genes, such as osteonectin and osteocalcin (OC). In kinetic studies, particles once phagocytosed, maintained a significant suppressive effect on collagen gene expression and type I collagen synthesis for up to five passages. Large particles of a size that cannot be phagocytosed also down‐regulated collagen gene expression suggesting that an initial contact between cells and particles can generate gene responsive signals independently of the phagocytosis process. Concerning such signaling, titanium particles rapidly increased protein tyrosine phosphorylation and nuclear transcription factor κB (NF‐κB) binding activity before the phagocytosis of particles. Protein tyrosine kinase (PTK) inhibitors such as genistein and the NF‐κB inhibitor pyrrolidine dithiocarbamate (PDTC) significantly reduced the suppressive effect of titanium on collagen gene expression suggesting particles suppress collagen gene expression through the NF‐κB signaling pathway. These results provide a mechanism by which particulate wear debris can antagonize the transcription of the procollagen α1[I] gene in osteoblasts, which may contribute to reduced bone formation and progressive periprosthetic osteolysis.
International Journal of Molecular Medicine, May 1, 1998
Human immunodeficiency virus type-1 (HIV-1) transcription is dependent on the interaction of host... more Human immunodeficiency virus type-1 (HIV-1) transcription is dependent on the interaction of host-cell transcription factors with cis-regulatory DNA elements within the viral long terminal repeat (LTR). Much attention has focused on the series of sequence elements upstream of the transcriptional initiation site in the U3 region of the LTR including the Sp1 and NF-kappaB binding sites. Recent studies, however, demonstrate that the transcribed 5'-untranslated leader region (5'-UTR) also contains important transcriptional elements. These regulatory elements situated downstream of transcription interact with constitutive and inducible transcription factors, mediate transmission of cellular activation signals, and are important for efficient HIV-1 transcription and replication. The 5'-UTR contains binding sites for the transcription factors AP-1, NF-kappaB, NF-AT, IRF, and Sp1. Mutations in these binding sites can interfere with the viral response to cell activation signals, decrease LTR transcription, and inhibit viral replication. The 5'-UTR also interacts with a specific nucleosome that is rapidly displaced during transcriptional activation of the latent provirus. We propose that the inducible transcription factor binding sites in the 5'-UTR comprise a downstream enhancer domain that can function independent of, or in concert with, the LTR promoter to rapidly increase latent proviral transcription in response to cell activation signals. In this review, we describe the host-cell transcription factors that interact with the 5'-UTR and discuss their role in the transcriptional regulation of HIV-1 gene expression.
Clinical Orthopaedics and Related Research, Dec 1, 2001
Since the recognition of aseptic loosening by Charnley in the early 1960s, much information has b... more Since the recognition of aseptic loosening by Charnley in the early 1960s, much information has been gained on the basic science of periprosthetic bone loss. Initially termed cement disease, it now generally is accepted that, in most instances, osteolysis is a manifestation of an adverse cellular response to phagocytosable particulate wear and corrosion debris, possibly facilitated by local hydrodynamic effects. Tissue explant, animal, and cell culture studies have allowed us to compile an appreciation of the complexity of cellular interactions and chemical mediators involved in osteolysis. Cellular participants have been shown to include the macrophage, osteoblast, fibroblast, and osteoclast. The plethora of chemical mediators that are responsible for the cellular responses and effects on bone include prostaglandin E2, tumor necrosis factor-alpha, interleukin-1, and interleukin 6. However, an increasing number of other proinflammatory and antiinflammatory cytokines, prostenoids, and enzymes have been shown to play important roles in this process. The ultimate goal of basic research is to develop novel strategies for evaluation and treatment of patients with osteolysis. Although initial animal studies are promising for possible pharmacologic treatment and prevention of osteolysis, well-controlled human trials are required before agents such as bisphosphonates can be recommended for general clinical use.
Clinical and Experimental Immunology, Aug 1, 2000
HIV-1 replicates in activated T cells at significantly higher levels than in resting cells. Thus,... more HIV-1 replicates in activated T cells at significantly higher levels than in resting cells. Thus, certain molecules up-regulated during T cell activation appear to be important for HIV-1 replication. In this study, we present evidence suggesting that expression of MHC class II (class II) molecules on CD4 1 T cells facilitate HIV-1 replication. T cells that expressed class II supported greater virus replication than T cells lacking class II. The class II 1 cells, when either infected with HIV-1 or transfected with an envminus HIV-1 provirus plasmid, produced 10±20-fold greater virus expression than class II 2 cells. Anticlass II antibody markedly inhibited virus expression in class II 1 cells (but not class II 2 cells) and also decreased the nuclear binding activity of AP-1, an inducible transcription factor important in T cell activation and HIV-1 expression. Most importantly, the induction of class II expression by transfection of the MHC class II transactivator (CIITA) stimulated HIV-1 replication in Jurkat T cells. Taken together, these data suggest that expression of MHC class II molecules and/or CIITA in T cells enhances HIV-1 transcription.
Journal of Biological Chemistry, May 1, 2002
Journal of Orthopaedic Research, May 1, 2002
Exposure of human osteoblasts to ultrafine titanium (Ti) particles has been shown to alter osteob... more Exposure of human osteoblasts to ultrafine titanium (Ti) particles has been shown to alter osteoblast gene expression. We previously reported that Ti particles can increase IL‐6 release and suppress the gene expression of procollagens α1[I] and α1[III] in human osteoblasts. In this study, we now demonstrate that Ti particles can rapidly induce the chemotactic cytokines interleukin‐8 (IL‐8) and monocyte chemoattractant protein‐1 (MCP‐1), two immediate early stress responsive chemokines important for the activation and chemotaxis of neutrophils and macrophages, respectively. In MG‐63 osteosarcoma cells and bone marrow derived primary osteoblasts Ti particles selectively increased the steady state levels of IL‐8 and MCP‐1 mRNA in a time and concentration dependent manner. The increased chemokine mRNA correlated with increased secretion of IL‐8 and MCP‐1 protein. Actinomycin D, a potent RNA polymerase II inhibitor, blocked the Ti particle induction of IL‐8 and MCP‐1 mRNA expression, whereas cycloheximide, which inhibits protein synthesis, failed to inhibit chemokine gene expression suggesting Ti particles directly target activation of chemokine gene transcription. Consistent with a transcriptional mechanism not involving new protein synthesis, we demonstrate that Ti particles induce the binding of the p65 and p50 subunits of the latent transcription factor NF‐κB to the IL‐8 gene promoter. Taken together, these data demonstrate that Ti particles can activate transcription of the stress responsive chemokine genes IL‐8 and MCP‐1 in human osteoblasts. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Journal of Interferon and Cytokine Research, May 1, 1999
Interleukin-8 (IL-8), a m em ber of the C XC chem okine fam ily, is an important activator and ch... more Interleukin-8 (IL-8), a m em ber of the C XC chem okine fam ily, is an important activator and chemoattractant for neutrophils and has been im plicated in a variety of inflamm atory diseases. IL-8 is secreted in a stimulusspecific m anner by a wide variety of cell types and is regulated prim arily at the level of gene transcription. Functional studies indicate that IL-8 transcriptional responses to proinflam matory m ediators are rapid and require only 100 nucleotides of 59-flanking DNA upstream of the TATA box. W ithin the IL-8 promoter sequence are DNA binding sites for the indu cible transcription factors AP-1, NF-IL-6, and NF-j B. Transcription factors in these fam ilies bind the IL-8 promoter as dim ers, and several distinct subunit combinations have been identified as important for IL-8 transcription. In addition, these factors can act in concert to synergistically activate the IL-8 promoter. AP-1 and NF-IL-6 physically interact with NF-j B, and functional cooperativity am ong the factors appears to be critical for optim al IL-8 prom oter activity in different cell types. IL-8 transcription appears to be activated by a promoter recruitm ent mechanism where inducible transcription factor binding to the IL-8 prom oter is required for binding of constitutively active TATA box-binding proteins and form ation of a stable preinitiation complex. This review discusses the regulatory role these higherorder synergistic interactions play in IL-8 transcription and in generation of the stimulus-specific and cell type-specific patterns of IL-8 expression. 429 INTRODUC TIO N REVIEW RO EBUC K 430 FIG. 1. Structure of the IL-8 gene. The IL-8 gene contains four exons, indicated by rectangles, and three introns of varying lengths. The coding regions are indicated by solid rectangles. The number of amino acids (aa) encoded by each exon is indicated above each rectangle. The IL-8 gene contains a TATA box at the 59-end and a polyadenylation signal at the 39-end. The amino acid sequence of the IL-8 protein is shown below the gene. The four conserved cysteine residues, the characteristic CXC sequence, and the 20 amino acid signal peptide are underlined. The extracellular processing required to generate the major 72 amino acid form is indicated by dashes.