Search PubMed⌕ Search

Biomedical subjects

J M Boss

Publications and source records attributed to J M Boss.

At least 37 records · Page 2Linked to original sources
PubMed1998

Human cytomegalovirus inhibits major histocompatibility complex class II expression by disruption of the Jak/Stat pathway.

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that is able to persist for decades in its host. HCMV has evolved protean countermeasures for anti-HCMV cellular immunity that facilitate establishment of persistence. Recently it has been shown that HCMV inhibits interferon gamma (IFN-gamma)-stimulated MHC class II expression, but the mechanism for this effect is unknown. IFN-gamma signal transduction (Jak/Stat pathway) and class II transactivator (CIITA) are required components for IFN-gamma-stimulated MHC class II expression. In this study, we demonstrate that both a clinical isolate and a laboratory strain of HCMV inhibit inducible MHC class II expression at the cell surface and at RNA level in human endothelial cells and fibroblasts. Moreover, reverse transcriptase polymerase chain reaction and Northern blot analyses demonstrate that neither CIITA nor interferon regulatory factor 1 are upregulated in infected cells. Electrophoretic mobility shift assays reveal a defect in IFN-gamma signal transduction, which was shown by immunoprecipitation to be associated with a striking decrease in Janus kinase 1 (Jak1) levels. Proteasome inhibitor studies with carboxybenzyl-leucyl-leucyl-leucine vinyl sulfone suggest an HCMV-associated enhancement of Jak1 protein degradation. This is the first report of a mechanism for the HCMV-mediated disruption of inducible MHC class II expression and a direct virus-associated alteration in Janus kinase levels. These findings are yet another example of the diverse mechanisms by which HCMV avoids immunosurveillance and establishes persistence.

Cells, Cultured↗
PubMed1998

MHC class II gene silencing in trophoblast cells is caused by inhibition of CIITA expression.

PROBLEM: Major histocompatibility complex (MHC) class II molecule expression is specifically suppressed on fetal trophoblasts, even in response to interferon (IFN)-gamma, a potent inducer of MHC class II genes. The suppression of class II induction has been suggested to play a role in preventing rejection of the fetal allograft. The mechanism of this suppression is unknown. METHOD OF STUDY: Human trophoblast cell lines were examined for expression of MHC class II transcription factors and for activity of the IFN-gamma signaling pathway. Additionally, trophoblast cells were transfected with a vector expressing the class II transactivator, CIITA, and assayed for class II expression. RESULTS: The MHC class II transcription factors RFX and X2BP and the IFN-gamma signaling pathway components are expressed constitutively and are functional in trophoblasts. However, CIITA expression was absent in trophoblasts and could not be induced by IFN-gamma. Transfection of CIITA into trophoblast cells resulted in derepression of class II gene expression. CONCLUSIONS: The lack of induction of MHC class II genes in response to IFN-gamma in trophoblast cells is caused neither by the absence of factors that bind class II promoters, nor by a lesion in the IFN-gamma signaling pathway, but results from a specific inhibition of the CIITA gene.

Cell Line↗
PubMed1997

Formation of a regulatory factor X/X2 box-binding protein/nuclear factor-Y multiprotein complex on the conserved regulatory regions of HLA class II genes.

Coordinate regulation of MHC class II genes occurs in a tissue-specific and cytokine-inducible manner. While the upstream regulatory sequences are conserved among all MHC class II genes, multiple base pair changes are found, even within the essential X box region. Analysis of all class II X boxes reveals differential binding between two transcription factors known to interact with the X box region, regulatory factor X and X2 box-binding protein (RFX and X2BP) of the HLA-DRA gene. These data presented a paradox with regard to the coordinate regulation of the class II genes if the factors though to regulate the HLA-DRA gene do not bind to the homologous sequence of all class II genes. Previous results suggested that cooperative interactions between the DNA binding proteins may be the key to understanding this paradox. Here RFX/X2BP/DNA complexes were formed on all class II isotypes regardless of the ability of the X box region to bind either factor individually. To further determine the role of the interactions between the X and Y factors, multiprotein/DNA complexes containing RFX, X2BP, NF-Y, and X-Y box DNA of the DRA and DRB genes were formed. This quaternary complex was extremely stable to competitor DNA, with a half-life > 4 h. These results suggest that the conserved X and Y boxes of class II genes function similarly and define a single multiprotein regulatory complex for class II expression in B cells.

Base Composition↗
PubMed1997

Regulatory factor X, a bare lymphocyte syndrome transcription factor, is a multimeric phosphoprotein complex.

Regulatory factor X (RFX) is a transcription factor that binds the conserved X1 box of MHC class II promoters and is essential for transcription of class II genes. The subunit structure of the native RFX complex was examined by coimmunoprecipitation using polyclonal antisera to the 75-kDa subunit of RFX, RFX5. Two polypeptides with apparent masses of 41 and 36 kDa coimmunoprecipitated with RFX5 and appear to be subunits of the native RFX complex. Metabolic labeling of wild-type and mutant B cells with [32P]orthophosphate demonstrated that each of the RFX subunits was phosphorylated in vivo and that the phosphorylation of the RFX subunits was independent of the essential MHC class II regulatory factor, CIITA. The trimeric RFX complex was also present in fibroblast cells with or without IFN-gamma treatment. Both the p41 and p36 subunits were absent in immunoprecipitations of RFX5 from lysates of independently established B cell lines from bare lymphocyte syndrome complementation groups B and D. Together, these results suggest that RFX complex assembly is required for class II expression and that the mutations in bare lymphocyte syndrome complementation groups B and D result in an inability to assemble the RFX complex.

Amino Acid Sequence↗
PubMed1997

HLA-DMA and HLA-DMB gene expression functions through the conserved S-X-Y region.

The MHC class II homologous proteins HLA-DMA and HLA-DMB function in the loading of peptides onto class II molecules. Like the class II genes, the HLA-DM genes contain upstream regulatory sequences similar to the S-X-Y regulatory region as well as additional putative regulatory sites. To determine whether the DM genes are regulated in a similar manner as class II genes, a series of in vivo and in vitro analyses was performed. Deletion analysis showed that expression from the DM promoters is dependent on the conserved S-X-Y region. The class II-specific transcription factors RFX and CIITA are also required for expression, as cell lines deficient in these factors failed to allow transcription from the DM promoters. In addition, in vivo footprint analysis showed the putative X and Y boxes to be occupied by transcription factors in wild-type B cells, but not in RFX-deficient B cells. In astrocytes, IFN-gamma treatment induced increased occupancy of these sites. None of the other putative regulatory sites was occupied in vivo, indicating that they may not be functional. Finally, gel shift analysis showed synergistic complex formation between proteins that bind to the putative X boxes of the DM genes, as is found for the DRA gene. Therefore, the DM genes share a common mechanism of regulation with the class II genes.

B-Lymphocytes↗
PubMed1997

TGF-beta suppresses IFN-gamma induction of class II MHC gene expression by inhibiting class II transactivator messenger RNA expression.

Recently, a non-DNA binding protein, class II transactivator (CIITA), has been shown to be required for constitutive and IFN-gamma-inducible class II MHC transcription. The cytokine TGF-beta inhibits IFN-gamma-induced class II MHC expression at the transcriptional level. In this study, we provide evidence that TGF-beta blocks IFN-gamma-induced CIITA mRNA accumulation. TGF-beta down-regulates class II MHC and CIITA mRNA accumulation in human astroglioma and fibrosarcoma cell lines, but TGF-beta does not destabilize the CIITA message, suggesting an effect at the transcriptional level. In cells that stably overexpressed CIITA, leading to a constitutive class II MHC-positive phenotype, the inhibitory effect of TGF-beta on class II MHC was abrogated, but the cells remained responsive for expression of TGF-beta-inducible genes. Cell lines that possessed defects in TGF-beta signaling also became refractory to inhibition of IFN-gamma-induced CIITA and class II MHC expression. Our data indicate that TGF-beta suppresses IFN-gamma-induced class II MHC expression by inhibiting accumulation of CIITA mRNA.

Astrocytoma↗
PubMed1997

Changing J774A.1 cells to new medium perturbs multiple signaling pathways, including the modulation of protein kinase C by endogenous sphingoid bases.

Sphingosine, sphinganine, and other long-chain (sphingoid) bases are highly bioactive intermediates of sphingolipid metabolism that have diverse effects when added to cells, including the inhibition of protein kinase C (PKC) as evaluated by both enzymatic activity and [3H]phorbol dibutyrate ([3H]PDBu) binding. Nonetheless, changes in endogenous sphingoid bases have not been proven to affect PKC or other signal transduction pathways. We have discovered recently that changing J774A.1 cells to new medium results in up to 10-fold increases in sphingoid bases (Smith, E. R., and Merrill, A. H., Jr. (1995) J. Biol. Chem. 270, 18749-18758); therefore, this system was used to elevate sphingosine and sphinganine and determine if PKC was affected. Incubation of J774A.1 cells in new medium for 30 min increased the levels of these endogenous sphingoid bases to approximately 0.5 nmol/mg of protein and decreased [3H]PDBu binding by 40-60%. Addition of NH4Cl, which suppresses the change in sphingosine, restored [3H]PDBu binding. Elevation of endogenous sphinganine by a second method (addition of fumonisin B1, an inhibitor of ceramide synthase) also reduced [3H]PDBu binding; therefore, elevations in sphingosine and sphinganine can both affect PKC. The elevation in sphingoid bases was also associated with an increase in the amount of PKC-delta (the major PKC isozyme in J774A. 1 cells) in the cytosol, as determined by activity assays and immunoblot analyses. Changing the culture medium affected other PKC isozymes, increased cellular levels of diacylglycerol, dihydroceramide, and ceramide, and altered the expression of two genes (the expression of JE was increased, and the induction of MnSOD by TNF-alpha was potentiated). Thus, changing the culture medium has numerous effects on J774A.1 cells, including the modulation of PKC by endogenous sphingoid bases.

Ammonium Chloride↗
PubMed1997

Regulation of transcription of MHC class II genes.

Genetic and biochemical analyses have identified multiple DNA-binding and non-DNA-binding proteins that functionally regulate MHC class II genes. These include RFX, X2BP, NF-Y, CIITA, OCT-2 and Bob1. One of the essential non-DNA-binding proteins, CIITA, appears to function as a limiting molecular switch that is responsible for the control of class II expression and the regulation of expression by interferon-gamma.

Gene Expression Regulation↗
PubMed1997

Tumor necrosis factor alpha and interleukin-1beta regulate the murine manganese superoxide dismutase gene through a complex intronic enhancer involving C/EBP-beta and NF-kappaB.

Manganese superoxide dismutase (MnSOD), a tumor necrosis factor (TNF)-inducible reactive oxygen-scavenging enzyme, protects cells from TNF-mediated apoptosis. To understand how MnSOD is regulated, transient transfections of promoter-reporter gene constructions, in vitro DNA binding assays, and in vivo genomic footprint (IVGF) analysis were carried out on the murine MnSOD gene. The results of this analysis identified a 238-bp region of intron 2 that was responsive to TNF and interleukin-1beta (IL-1). This TNF response element (TNFRE) had the properties of a traditional enhancer element that functioned in an orientation- and position-independent manner. IVGF of the TNFRE revealed TNF- and IL-1-induced factor occupancy of sites that could bind NF-kappaB and C/EBP. The 5' portion of the TNFRE bound C/EBP-beta in vitro and was both necessary and sufficient for TNF responsiveness with the MnSOD promoter or with a heterologous promoter when in an upstream position. The 3' end of the TNFRE bound both NF-kappaB and C/EBP but was not necessary for TNF responsiveness with the MnSOD promoter. However, this 3' portion of the TNFRE was required for the TNFRE to function as a downstream enhancer with a heterologous promoter. These data functionally separate the MnSOD TNFRE into a region responsible for TNF activation and one that mediates induction when it is downstream of a promoter.

3T3 Cells↗
PubMed1996

Apoptosis-independent retinoblastoma protein rescue of HLA class II messenger RNA IFN-gamma inducibility in non-small cell lung carcinoma cells. Lack of surface class II expression associated with a specific defect in HLA-DRA induction.

Work from our laboratory indicates that HLA class II induction by IFN- gamma in the retinoblastoma (RB) protein-defective breast carcinoma line MDA-468-S4 (S4) requires reconstitution of functional RB. To determine whether RB is required for HLA class 11 expression in multiple tumor types, the RB-defective non-small cell lung carcinoma line H2009 and its RB-reconstituted subclones were examined for class II inducibility. Surface HLA-DR (DR) was not inducible by IFN-gamma in H2009. However, unlike the RB-reconstituted subclones of S4, DR surface expression was not detected in the H2009 RB-positive subclones. IFN-gamma induction of CIITA, a major regulator of class II transcription, suggested that H2009 retained at least part of the IFN-gamma signaling pathway leading to class II expression. Examination of class II mRNA indicated that IFN-gamma induction of RB was rescued in the RB-positive subclones of H2009, confirming the requirement for RB for HLA class II inducibility and revealing that RB is required for inducibility in developmentally distinct tumor types. However, DRA inducibility was not rescued in the H2009 RB-positive subclones, which explained the lack of surface DR induction in the RB-positive H2009 subclones. DPA and DPB were also only weakly inducible in the RB-reconstituted H2009 subclones, compared with the previously described, S4 RB-positive subclones. Finally, data reported here indicates that RB's ability to inhibit IFN-gamma-induced apoptosis is not a viable explanation for why RB expression rescues DRB inducibility in H2009.

Apoptosis↗
PubMed1996

TNF regulates the in vivo occupancy of both distal and proximal regulatory regions of the MCP-1/JE gene.

In vivo genomic footprinting (IVGF) was used to examine regulatory site occupancy during the activation of the murine inflammatory response gene MCP-1/JE by TNF. In response to TNF, both promoter distal and proximal regulatory regions became occupied in vivo. EMSA analysis showed that while some of the factors involved in expression, including NF-kappa B, were translocated to the nucleus following TNF treatment, others were already present and able to bind DNA in vitro. Protein kinase inhibitor studies showed that protein phosphorylation was required for TNF activation but not factor assembly. These studies provide evidence for a multistep model of TNF-mediated gene regulation involving chromatin accessibility, transcription factor complex assembly, and protein phosphorylation.

3T3 Cells↗
PubMed1996

Complex architecture of major histocompatibility complex class II promoters: reiterated motifs and conserved protein-protein interactions.

The S box (also known as at the H, W, or Z box) is the 5'-most element of the conserved upstream sequences in promoters of major histocompatibility complex class II genes. It is important for their B-cell-specific and interferon gamma-inducible expression. In this study, we demonstrate that the S box represents a duplication of the downstream X box. First, RFX, which is composed of the RFX5-p36 heterodimer that binds to the X box, also binds to the S box and its 5'-flanking sequence. Second, NF-Y, which binds to the Y box and increases interactions between RFX and the X box, also increases the binding of RFX to the S box. Third, RFXs bound to S and X boxes interact with each other in a spatially constrained manner. Finally, we confirmed these protein-protein and protein-DNA interactions by expressing a hybrid RFX5-VP16 protein in cells. We conclude that RFX binds to S and X boxes and that complex interactions between RFX and NF-Y direct B-cell-specific and interferon gamma-inducible expression or major histocompatibility complex class II genes.

Animals↗
PubMed1995

Interferon (IFN) beta acts downstream of IFN-gamma-induced class II transactivator messenger RNA accumulation to block major histocompatibility complex class II gene expression and requires the 48-kD DNA-binding protein, ISGF3-gamma.

Interferon (IFN) gamma, a cardinal proinflammatory cytokine, induces expression of the gene products of the class II locus of the major histocompatibility complex (MHC), whereas IFN-alpha or -beta suppresses MHC class II expression. The mechanism of IFN-beta-mediated MHC class II inhibition has been unclear. Recently, a novel factor termed class II transactivator (CIITA) has been identified as essential for IFN-gamma-induced MHC class II transcription. We studied the status of IFN-gamma-induced CIITA messenger RNA (mRNA) accumulation and CIITA-driven transactivation in IFN-beta-treated cells and used cell lines that had defined defects in the type I IFN response pathway to address the roles of IFN signaling components in the inhibition of MHC class II induction. IFN-beta treatment did not suppress IFN-gamma-induced accumulation of CIITA mRNA. After cells were stably transfected with CIITA, endogenous MHC class II genes were constitutively expressed, and MHC class II promoters, delivered by transfection, were actively transcribed in CIITA-expressing cells. Expression of these promoters was significantly impaired by pretreatment with IFN-beta. These results suggest that IFN-beta acts downstream of CIITA mRNA accumulation, and acts in part by reducing the functional competence of CIITA for transactivating MHC class II promoters. IFN stimulated gene factor 3 (ISGF3) gamma was essential for IFN-beta to mediate inhibition of MHC class II induction, regardless of whether MHC class II transcription was stimulated by IFN-gamma or directly by CIITA expression. Results of these experiments suggest that inhibition of MHC class II in IFN-beta-treated cells requires expression of gene(s) directed by the ISGF3-IFN-stimulated response element pathway, and that these gene product(s) may act by blocking CIITA-driven transcription of MHC class II promoters.

DNA-Binding Proteins↗
PubMed1995

Purified X2 binding protein (X2BP) cooperatively binds the class II MHC X box region in the presence of purified RFX, the X box factor deficient in the bare lymphocyte syndrome.

The conserved X2 box sequence of MHC class II promoters is homologous to TRE/CRE elements, and is required for B cell expression and IFN-gamma induction of MHC class II genes. The X2 binding protein (X2BP) was initially identified as a DNA-binding activity that specifically interacts with the conserved X2 box sequence in both the MHC HLA-DRA and HLA-DRB promoters. To begin to demonstrate that X2BP is the X2 box factor responsible for class II expression in B cells, we have purified X2BP to homogeneity from B cell nuclear extracts using DNA-affinity chromatography. X-box DNA-affinity purification indicates that X2BP is most likely composed of two polypeptides of 120 kDa and 46 kDa. The 120-kDa protein was specifically cross-linked to an X-box probe by exposure to UV irradiation. The 46-kDa subunit of X2BP cross-reacted with anti-rat CREB polyclonal Abs but not to anti-human CREB Abs in Western analysis and supershift assays, indicating that it may be a novel member of the ATF/CREB family. Purified X2BP interacted with purified RFX, a factor that binds to the adjacent X1 box and is absent in some cell lines that are mutant for MHC class II transcription. This interaction increases the DNA-binding half-life of RFX from 5 to at least 60 min, suggesting that X2BP functions in class II MHC gene expression by forming a stable complex with RFX.

Base Sequence↗
PubMed1995

Cloning and characterization of the murine manganous superoxide dismutase-encoding gene.

The murine manganous superoxide dismutase-encoding gene (MnSOD) is highly homologous in both sequence and organization to its rat homolog. MnSOD is encoded by five exons spanning approx. 7 kb of DNA. RNA blot analysis indicated multiple RNA species, with the major RNA corresponding to a 960-bp message. This major transcript is highly inducible by tumor necrosis factor (TNF) in murine fibroblasts. Analysis of other murine tissues demonstrated ubiquitous expression. RNase protection and primer extension assays used to map the 5' end of the gene revealed a series of closely spaced multiple transcription start points. Sequence analysis of the 1.7 kb of 5' flanking DNA showed high homology to the 5' proximal 950 bp of the rat homolog. Within this region, multiple potential regulatory elements are present, including several SP1 sites, two NF kappa B sites and an antioxidant-response element. However, no TATA box was identified, placing MnSOD in the family of inducible genes that lack consensus TATA-box elements and contain G+C-rich promoter regions.

3T3 Cells↗
PubMed1995

Activation of class II MHC genes requires both the X box region and the class II transactivator (CIITA).

CIITA, a gene that can complement a transcriptional mutation of the major histocompatibility complex (MHC) class II genes, was tested for its ability to function as a coactivator, CIITA cDNA clones isolated showed alternative RNA splicing, but only one splice site combination was able to restore class II MHC gene expression. DNA-mediated transfection experiments showed that CIITA directs its activity through the X box element; the presence of CIITA leads to the formation of a higher order complex at the X box region; and CIITA contains a potent activation domain. These findings support the hypothesis that CIITA directly interacts with the MHC class II-specific transcription factors and is required for expression.

Alternative Splicing↗
PubMed1994

Retinoblastoma protein regulation of surface CD74 (invariant chain) expression in breast carcinoma cells.

The HLA class II genes encode heterodimeric cell surface proteins which bind peptide antigen recognized by T-cell receptors on CD4+ T-cells. The class II proteins are inducible by IFN-gamma, and this induction requires, or is strongly enhanced, by retinoblastoma protein (RB) in a series of breast carcinoma cell lines. Loading of peptide onto the class II protein appears to be regulated by CD74, which associates with class II during their transition to the endosomal compartment, where class II binds peptide. Class II proteins and CD74 are largely regulated in concert, provoking the question, is CD74 induction by IFN-gamma affected by RB? Results described here indicate that IFN-gamma induction of CD74 surface expression in a series of breast carcinoma lines is enhanced by RB, while RB has no effect on CD74 mRNA induction. Also, neither the class II nor the CD74 promoter regions are activated by RB in cotransfection experiments where RB activates the SV40 promoter.

Antigens, CD↗