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R Pine

Publications and source records attributed to R Pine.

At least 19 recordsLinked to original sources
PubMed2000

Differentiation of monocytes to macrophages switches the Mycobacterium tuberculosis effect on HIV-1 replication from stimulation to inhibition: modulation of interferon response and CCAAT/enhancer binding protein beta expression.

HIV-1 replication is inhibited in uninflamed lung macrophages and is stimulated during tuberculosis. Attempts to recapitulate activation of HIV-1 replication in primary monocytes and macrophages ex vivo and in the untreated and PMA-treated THP-1 cell line model in vitro have produced opposite results depending on the state of differentiation of the cells. After infection with Mycobacterium tuberculosis, monocytes enhanced HIV-1 replication and produced a stimulatory 37-kDa CCAAT/enhancer binding protein beta (C/EBPbeta) transcription factor, whereas macrophages suppressed HIV-1 replication and produced an inhibitory 16-kDa C/EBPbeta transcription factor. IFN-beta induced inhibitory 16-kDa C/EBPbeta in macrophages, but had no effect on C/EBPbeta expression in monocytes. Macrophages, but not monocytes, were able to activate IFN-stimulated gene factor-3 (ISGF-3), a transcription factor composed of STAT-1, STAT-2, and IFN regulatory factor (IRF)-9, after infection with M. tuberculosis or stimulation with type I IFN. Macrophages expressed IRF-9 DNA-binding activity, but monocytes did not, and addition of the IRF-9 component reconstituted ISGF-3 in extracts of IFN-treated monocytes. Modulation of IFN responsiveness upon differentiation occurred at least in part through a post-transcriptionally regulated increase in IRF-9 expression. Both monocytes and macrophages maintained IFN responsiveness, activating STAT-1 homodimer formation and transcription of the STAT-1 gene after IFN stimulation. In addition, both monocytes and macrophages were able to activate NF-kappaB upon infection with M. tuberculosis. These results show that induction of ISGF-3, expression of the inhibitory 16-kDa C/EBPbeta, and suppression of HIV-1 replication via a transcriptional mechanism are macrophage-specific responses to infection with M. tuberculosis.

CCAAT-Enhancer-Binding Proteins↗
PubMed1999

Activation of the p38 mitogen-activated protein kinase by type I interferons.

The p38 mitogen-activated protein (Map) kinase plays a critical role in the generation of signals in response to stress stimuli, but its role in interferon (IFN) signaling and its potential regulatory role in the activation of Jak-signal transducer and activator of transcription (Stat) pathway are not known. In the present study, we provide evidence that the p38 Map kinase is rapidly phosphorylated and activated during treatment of cells with Type I interferons (IFNalpha and IFNbeta). Furthermore, the Type I IFN-dependent activation of p38 regulates induction of the catalytic domains of MapKap kinase-2 and MapKap kinase-3, strongly suggesting the existence of an IFNalpha signaling cascade activated downstream of the p38 kinase. The engagement of this pathway in interferon signaling plays a critical role in interferon-dependent transcriptional regulation, as evidenced by the fact that inhibition of p38 activation results in abrogation of interferon-dependent gene transcription via interferon-stimulated response elements. Interestingly, inhibition of the kinase activity of the p38 blocks IFNalpha-induced gene transcription without inhibiting DNA binding or tyrosine phosphorylation of Stat proteins, suggesting that the p38 pathway acts in cooperation with the Stat pathway. Thus, the p38 kinase signaling cascade is activated by the Type I interferon receptor and plays a critical role in interferon signaling and interferon-dependent transcriptional regulation.

Base Sequence↗
PubMed1999

Fluoroquinolone action against clinical isolates of Mycobacterium tuberculosis: effects of a C-8 methoxyl group on survival in liquid media and in human macrophages.

When the lethal action of a C-8 methoxyl fluoroquinolone against clinical isolates of Mycobacterium tuberculosis in liquid medium was measured, the compound was found to be three to four times more effective (as determined by measuring the 90% lethal dose) than a C-8-H control fluoroquinolone or ciprofloxacin against cells having a wild-type gyrA (gyrase) gene. Against ciprofloxacin-resistant strains, the C-8 methoxyl group enhanced lethality when alanine was replaced by valine at position 90 of the GyrA protein or when aspartic acid 94 was replaced by glycine, histidine, or tyrosine. During infection of a human macrophage model by wild-type Mycobacterium bovis BCG, the C-8 methoxyl group lowered survival 20- to 100-fold compared with the same concentration of a C-8-H fluoroquinolone. The C-8 methoxyl fluoroquinolone was also more effective than ciprofloxacin against a gyrA Asn94 mutant of M. bovis BCG. In an M. tuberculosis-macrophage system the C-8 methoxyl group improved fluoroquinolone action against both quinolone-susceptible and quinolone-resistant clinical isolates. Thus, a C-8 methoxyl group enhances the bactericidal activity of quinolones with N1-cyclopropyl substitutions; these data encourage further refinement of fluoroquinolones as antituberculosis agents.

Anti-Infective Agents↗
PubMed1998

Type I interferon induces inhibitory 16-kD CCAAT/ enhancer binding protein (C/EBP)beta, repressing the HIV-1 long terminal repeat in macrophages: pulmonary tuberculosis alters C/EBP expression, enhancing HIV-1 replication.

We have previously observed that HIV-1 replication is suppressed in uninflamed lung and increased during tuberculosis. In vitro THP-1 cell-derived macrophages inhibited HIV-1 replication after infection with Mycobacterium tuberculosis. Suppression of HIV-1 replication was associated with inhibition of the HIV-1 long terminal repeat (LTR) and induction of ISGF-3, a type I interferon (IFN)-specific transcription factor. Repression of the HIV-1 LTR required intact CCAAT/enhancer binding protein (C/EBP) sites. THP-1 cell-derived macrophages infected with M. tuberculosis, lipopolysaccharide, or IFN-beta induced the 16-kD inhibitory C/EBPbeta isoform and coincidentally repressed HIV-1 LTR transcription. C/EBPbeta was the predominant C/EBP family member produced in THP-1 macrophages during HIV-1 LTR repression. In vivo, alveolar macrophages from uninflamed lung strongly expressed inhibitory 16-kD C/EBPbeta, but pulmonary tuberculosis abolished inhibitory C/EBPbeta expression and induced a novel C/EBP DNA binding protein. Therefore, in vitro, proinflammatory stimulation produces an IFN response inhibiting viral replication by induction of a C/EBPbeta transcriptional repressor. THP-1 cell-derived macrophages stimulated with type I IFN are similar to alveolar macrophages in the uninflamed lung in vivo. In contrast, the cellular immune response in active pulmonary tuberculosis disrupts this innate immunity, switching C/EBP expression and allowing high level viral replication.

Base Sequence↗
PubMed1998

Definition of the interferon-alpha receptor-binding domain on the TYK2 kinase.

Interferons and cytokines modulate gene expression via a simple, direct signaling pathway containing receptors, JAK tyrosine kinases, and STAT transcription factors. The interferon-alpha pathway is a model for these cascades. Two receptors, IFNaR1 and IFNaR2, associate exclusively in a constitutive manner with two JAK proteins, TYK2 and JAK1, respectively. Defining the molecular interface between JAK proteins and their receptors is critical to understanding the signaling pathway and may contribute to the development of novel therapeutics. This report defines the IFNaR1 interaction domain on TYK2. In vitro binding studies demonstrate that the amino-terminal half of TYK2, which is approximately 600 amino acids long and contains JAK homology (JH) domains 3-7, comprises the maximal binding domain for IFNaR1. A fragment containing amino acids 171-601 (JH3-6) also binds IFNaR1, but with reduced affinity. Glutathione S-transferase-TYK2 fusion proteins approximating either the JH6 or JH3 domain affinity-precipitate IFNaR1, suggesting that these are major sites of interaction within the larger binding domain. TYK2 amino acids 1-601 act in a dominant manner to inhibit the transcription of an interferon-alpha-dependent reporter gene, presumably by displacing endogenous TYK2 from the receptor. This same fragment inhibits interferon-alpha-dependent tyrosine phosphorylation of TYK2, STAT1, and STAT2.

Binding Sites↗
PubMed1997

Convergence of TNFalpha and IFNgamma signalling pathways through synergistic induction of IRF-1/ISGF-2 is mediated by a composite GAS/kappaB promoter element.

The molecular basis for the well known synergistic biological effects of tumor necrosis factor alpha (TNFalpha) and interferon gamma (IFNgamma) is still poorly understood. This report demonstrates that expression of interferon-regulatory factor 1 (IRF-1), also known as interferon-stimulated-gene factor 2 (ISGF-2), is synergistically induced by these cytokines. The induction is a primary transcriptional response that occurs rapidly without a requirement for new protein synthesis. Synergism is mediated by a novel composite element in the IRF-1 promoter that includes an IFNgamma-activation site (GAS) overlapped by a non-consensus site for nuclear factor kappa B (NFkappaB). These sequences are bound strongly by signal transducer and activator of transcription 1 (STAT-1) and weakly by the p50/p65 heterodimer form of NFkappaB, respectively. However, the binding of STAT-1 and NFkappaB to the GAS/kappaB element in vitro seems to be mutually exclusive and independent. Synergistic induction of IRF-1 is likely to be an important early step in regulatory networks critical to the synergism of TNFalpha and IFNgamma. The GAS/kappaB element may mediate synergistic transcriptional induction of IRF-1 by other pairs of ligands that together activate NFkappaB and STAT family members. Other genes are likely to contain this motif and be regulated similarly.

Binding, Competitive↗
PubMed1997

Kinase-deficient forms of Jak1 and Tyk2 inhibit interferon alpha signaling in a dominant manner.

Signaling by interferon alpha (IFN alpha), an extracellular factor that mediates a number of anti-viral and growth-suppressive effects, requires two members of the Janus family of tyrosine kinases (JAK family): Jak1 and Tyk2. IFN alpha treatment of cells induces the rapid tyrosine phosphorylation of these two kinases, two subunits of the IFN alpha receptor, and two members of the signal transducer and activator of transcription (STAT) family of latent transcription factors. These proteins are believed to be direct substrates of one or both JAKs. Though the requirement for both Jak1 and Tyk2 in the IFN alpha-signaling cascade is well established, the order of activation and the relative contribution of the two kinases has not been elucidated completely. To address these questions, we have employed kinase-deficient mutants of both enzymes. Both mutant kinases suppress transcriptional activation as measured by an IFN alpha-dependent reporter-gene assay. Furthermore, in transient-transfection assays, the kinase-deficient versions of Tyk2 and Jak1 can act independently to inhibit STAT phosphorylation. Thus, kinase-deficient versions of JAK can act in a dominant-negative fashion to suppress IFN alpha signaling. The effects of the overexpressed mutant kinases on the phosphorylation of the kinases themselves, however, are unequal, suggesting that Jak1 functions upstream of Tyk2.

Cells, Cultured↗
PubMed1997

Proteasome- and p53-dependent masking of signal transducer and activator of transcription (STAT) factors.

Hepatoma Hep3B cell lines stably expressing a temperature-sensitive p53 species (p53-Val-135) displayed a reduced response to interleukin-6 (IL-6) when cultured at the wild-type (wt) p53 temperature (Wang, L., Rayanade, R., Garcia, D., Patel, K., Pan, H., and Sehgal, P. B. (1995) J. Biol. Chem. 270, 23159-23165). We now report that in such cultures IL-6 caused a rapid (20-30 min) and marked loss of cellular immunostaining for STAT3 and STAT5, but not for STAT1. The loss of STAT3 and STAT5 immunostaining was transient (lasted 120 min) and tyrosine kinase-dependent, and even though the loss was blocked by the proteasome inhibitors MG132 and lactacystin it was not accompanied by changes in cellular levels of STAT3 and STAT5 proteins suggesting that IL-6 triggered a rapid masking but not degradation of these transcription factors. STAT3 and STAT5 masking was accompanied by a reduction in IL-6-induced nuclear DNA-binding activity. The data suggest that p53 may influence Jak-STAT signaling through a novel indirect mechanism involving a wt p53-dependent gene product which upon cytokine addition is activated into a "STAT-masking factor" in a proteasome-dependent step.

Carcinoma, Hepatocellular↗
PubMed1997

Susceptibility to virus infection is determined by a Stat-mediated response to the autocrine effect of virus-induced type I interferon.

Interferon (IFN) signalling appears to be the predominant pathway that leads to the development of the host defence against virus. However, other mechanisms that are IFN independent may also be involved. The Stat1 transcription factor is specific for the IFN pathway and plays a central role in mediating many, if not all, IFN-dependent biological responses. Viral infection of cells in the presence or absence of Stat1 was studied to examine the role of IFN-dependent and independent antiviral mechanisms. A lower virus yield measured by plaque assays was observed following challenge with VSV, EMCV and NDV of cells in which either Stat1a or Stat1b was present compared to those lacking both forms of the Stat protein. The more efficient antiviral response was abolished when cells were infected with virus in the presence of IFN-neutralizing antibodies, suggesting that a Stat-dependent pathway is activated following virus infection by endogenously produced IFN. Virus-induced Stat protein translocation from the cytoplasmic compartment, detected within 3 h of infection, and Stat-dependent transcriptional activation of ISGF2 in response to virus challenge, were also abolished by neutralizing type I IFN. Thus, susceptibility to virus infection can be determined by the cell's ability to respond to autocrine IFN through the Stat-mediated pathway of gene induction.

Animals↗
PubMed1996

Activation of STAT4 by IL-12 and IFN-alpha: evidence for the involvement of ligand-induced tyrosine and serine phosphorylation.

The immunoregulatory cytokine IL-12 plays a central role in cell-mediated immune responses through its effects on NK cells and T lymphocytes. While IL-12 is known to share some functions with other cytokines, such as IFN-alpha, it also maintains distinct roles, such as its ability to induce Th1 differentiation. The molecular basis for these unique and overlapping functions is not well understood. IL-12 has previously been shown to induce tyrosine phosphorylation and DNA-binding of STAT3 and STAT4, members of the signal transducers and activators of transcription (STAT) family. Because STAT4 has only been shown to be activated in response to IL-12, this specificity has been suggested to be a basis for the unique actions of IL-12. In this study, we demonstrated that STAT4 activation by IL-12 is not unique; IL-12 and IFN-alpha, but not IFN-gamma, induced tyrosine phosphorylation and DNA binding of STAT4. Since tyrosine and serine phosphorylation of STAT1 have previously been shown to be important in IFN-alpha-mediated signaling, we also investigated IL-12- and IFN-alpha-induced serine phosphorylation of STAT4. We demonstrated that both cytokines induced serine phosphorylation. This modification was not required for DNA binding, but may be important in STAT-mediated transcription. Thus, STAT4 activation was not IL-12 specific, and IL-12 and IFN-alpha activated STAT4 through both tyrosine and serine phosphorylation. These findings have significant implications for understanding the role of STAT4 in mediating biologic functions; specifically, the data argue that the unique effects of IL-12 cannot be solely explained by STAT4 activation.

Animals↗
PubMed1996

The SH2 domain-containing tyrosine phosphatase PTP1D is required for interferon alpha/beta-induced gene expression.

Interferons (IFNs) induce early response genes by stimulating Janus family (Jak) tyrosine kinases, leading to tyrosine phosphorylation of Stat (signal transducer and activator of transcription) proteins. Previous studies demonstrated that a protein-tyrosine phosphatase (PTP) is required for activation of the ISGF3 transcription complex by IFNalpha/beta, but the specific PTP responsible remained unidentified. We now show that the SH2 domain containing tyrosine phosphatase PTP1D (also designated as SHPTP2, SHPTP3, PTP2C, or Syp) is constitutively associated with the IFNalpha/beta receptor and becomes tyrosine-phosphorylated in response to ligand. Furthermore, transient expression of a phosphatase-inactive mutant or the COOH-terminal SH2 domain of PTP1D causes a dominant negative effect on IFNalpha/beta-induced early response gene expression. These results provide strong evidence that PTP1D functions as a positive regulator of the IFNalpha/beta-induced Jak/Stat signal transduction pathway.

Base Sequence↗
PubMed1996

Retinoic acid activates interferon regulatory factor-1 gene expression in myeloid cells.

All-trans-retinoic acid (ATRA) is the drug of choice in the treatment of acute promyelocytic leukemia (APL). ATRA induces both in vitro and in vivo differentiation of APL cells into mature granulocytes. However, the molecular mechanisms involved in ATRA-dependent growth inhibition and cellular differentiation are not presently understood. The NB4 cell line, which is derived from the bone marrow of a patient with APL during relapse, can be used as a model system to study the growth and differentiation of APL cells. Because interferon (IFN) regulatory factors (IRF-1 and IRF-2) and other IFN-inducible gene products regulate cell growth, we analyzed the effects of ATRA on the expression of these genes. We show that ATRA directly activates IRF-1 gene expression, followed by activation of IRF-2 and 2'-5' oligoadenylate synthetase (OAS) gene expression with slower kinetics. In addition to NB4 cells, ATRA also activated IRF-1 gene expression in HL-60, U937, and THP-1 cells, which all respond to ATRA by growth inhibition. A more than additive increase in IRF-1 gene expression was seen with ATRA and IFN-gamma in NB4 cells. ATRA did not activate nuclear factor kappa B or signal transducer and activator of transcription (STAT) activation pathways, suggesting that an alternate mechanism is involved in IRF-1 gene activation. The ATRA-induced expression of IRF-1, an activator of transcription and repressor of transformation, may be one of the molecular mechanisms of ATRA-induced growth inhibition, and the basis for the synergistic actions of ATRA and IFNs in myeloid leukemia cells.

2',5'-Oligoadenylate Synthetase↗
PubMed1996

Regulation of LMP2 and TAP1 genes by IRF-1 explains the paucity of CD8+ T cells in IRF-1-/- mice.

The TAP1 and LMP2 genes are central for class I MHC function and share a common promoter. Here, we analyze the molecular mechanism of IFN gamma up-regulation of TAP1 and LMP2. In vivo footprinting indicates IFN gamma up-regulates protein-DNA contacts at an IRF-E that is essential for the up-regulation of TAP1 and LMP2 by IFN gamma. Gel shift analysis indicates that this site binds IRF-1. The expression of TAP1 and LMP2 are both greatly reduced in IRF-1-deficient mice. Surface class I MHC as well as CD8+ T cells are reduced in IRF-1-/- mice. The role of IRF-1 in the regulation of TAP1 and LMP2 suggests a mechanism for the antiviral properties of IRF-1 and the unexpected deficiency of CD8+ T cells observed in IRF-1-/- mice.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Interleukin 2 activates STAT5 transcription factor (mammary gland factor) and specific gene expression in T lymphocytes.

Although prolactin and interleukin 2 (IL-2) can elicit distinct physiological responses, we have found that their signal pathways share a common signal transducer and activator of transcription, STAT5. STAT5 was originally identified as a mammary gland factor induced by prolactin in lactating breast cells. Here we demonstrate that STAT5 is activated after IL-2 stimulation of two responsive lymphocyte cell lines, Nb2 and YT. Activation of STAT5 is measured both by IL-2-induced tyrosine phosphorylation and by IL-2-induced DNA binding. The STAT5 DNA recognition site is the same as the interferon gamma-activated site (GAS) in the interferon regulatory factor 1 gene. We demonstrate that the GAS element is necessary and sufficient for transcriptional induction by both IL-2 and prolactin in T lymphocytes. These results indicate that the role of STAT5 in the regulation of gene expression is not restricted to mammary cells or to prolactin, but is an integral part of the signal pathway of a critical immunomodulatory cytokine, IL-2.

Animals↗
PubMed1995

Requirement for MAP kinase (ERK2) activity in interferon alpha- and interferon beta-stimulated gene expression through STAT proteins.

Activation of early response genes by interferons (IFNs) requires tyrosine phosphorylation of STAT (signal transducers and activators of transcription) proteins. It was found that the serine-threonine kinase mitogen-activated protein kinase (MAPK) [specifically, the 42-kilodalton MAPK or extracellular signal-regulated kinase 2 (ERK2)] interacted with the alpha subunit of IFN-alpha/beta receptor in vitro and in vivo. Treatment of cells with IFN-beta induced tyrosine phosphorylation and activation of MAPK and caused MAPK and Stat1 alpha to coimmunoprecipitate. Furthermore, expression of dominant negative MAPK inhibited IFN-beta-induced transcription. Therefore, MAPK appears to regulate IFN-alpha and IFN-beta activation of early response genes by modifying the Jak-STAT signaling cascade.

Binding Sites↗
PubMed1995

Involvement of two regulatory elements in interferon-gamma-regulated expression of human indoleamine 2,3-dioxygenase gene.

Interferon (IFN)-gamma-induced expression of indoleamine 2,3-dioxygenase (IDO) gene is implicated in the antimicrobial and antiproliferative effects of IFN-gamma in cell cultures. Earlier studies identified a 96 base pair (bp) regulatory region upstream of the IDO gene that conferred IFN-gamma response to the chloroamphenicol acetyltransferase (CAT) gene linked to herpesvirus thymidine kinase promoter. The IFN-gamma-responsive region was further narrowed to a 67 bp fragment by 3' deletion. This 67 bp fragment contains several sequence elements of potential interest, including a 14 bp sequence homologous to the ISRE sequence found in IFN-alpha-inducible genes and two palindromic sequences (PE I and PE II) homologous to the GAS sequence identified in IFN-gamma-inducible genes. Site-directed mutagenesis studies showed that IFN-gamma-induced expression of IDO-CAT constructs involved cooperation between two elements: the ISRE homolog and the PE II (but not PE I). Either element alone with its flanking sequence was inadequate in conferring an IFN-gamma response to CAT reporter gene. Two IFN-gamma-regulated protein factors interacting with these two elements were identified. The factor binding to the ISRE region was induced with a slower kinetics, required new protein synthesis, and reacted with antibodies to IRF-1. The factor interacting with the PE II region appeared rapidly after treatment with IFN-gamma independently of new protein synthesis, and its binding to DNA probe was blocked by antibodies to p91 factor, reported to bind to GAS element.(ABSTRACT TRUNCATED AT 250 WORDS)

Antisense Elements (Genetics)↗
PubMed1995

Endothelial interferon regulatory factor 1 cooperates with NF-kappa B as a transcriptional activator of vascular cell adhesion molecule 1.

Transcription of the vascular cell adhesion molecule 1 (VCAM-1) gene in endothelial cells is induced by lipopolysaccharide and the inflammatory cytokines interleukin-1 beta and tumor necrosis factor alpha (TNF-alpha). Previous studies have demonstrated that tandem binding sites for the inducible transcription factor NF-kappa B are necessary but not sufficient for full cytokine-mediated transcriptional activation. Herein, we demonstrate that full cytokine-induced accumulation of VCAM1 transcript requires protein synthesis. We report the definition of a functional regulatory element in the VCAM1 promoter interacting with the transcriptional activator interferon regulatory factor 1 (IRF-1). DNA-protein binding studies with endothelial nuclear extracts revealed that IRF-1 is cytokine inducible and binds specifically to a consensus sequence motif located 3' of the TATA element. We have identified heterodimeric p65 and p50 as the NF-kappa B species binding to the VCAM1 promoter in TNF-alpha-activated endothelial cells. Experiments with recombinant proteins showed that p50/p65 and high-mobility-group I(Y) protein cooperatively facilitated the binding of IRF-1 to the VCAM1 IRF binding site and that IRF-1 physically interacted with p50 and with high-mobility-group I(Y) protein. Transient transfection assay in endothelial cells showed that overexpressed IRF-1 resulted in superinduction of TNF-alpha-stimulated transcription. Site-directed mutations in the IRF binding element decreased TNF-alpha-induced activity and totally abolished superinduction. Cotransfection assays in P19 embryonal carcinoma cells revealed that IRF-1 synergized with p50/p65 NF-kappa B to activate the VCAM1 promoter or heterologous promoter constructs bearing isolated VCAM1 NF-kappa B and IRF binding motifs. Cytokine inducibility of VCAM1 in endothelial cells utilizes the interaction of heterodimeric p50/p65 proteins with IRF-1.

Base Sequence↗