Article

Interferon-gamma-mediated inhibition of cyclin A gene transcription is independent of individual cis-acting elements in the cyclin A promoter

Harvard University, Cambridge, Massachusetts, United States
Journal of Biological Chemistry (Impact Factor: 4.6). 05/1999; 274(17):12139-46. DOI: 10.1074/jbc.274.17.12139
Source: PubMed

ABSTRACT Interferons (IFNs) affect cellular functions by altering gene expression. The eukaryotic cell cycle is governed in part by the periodic transcription of cyclin genes, whose protein products associate with and positively regulate the cyclin-dependent kinases. To understand better the growth inhibitory effect of IFN-gamma on vascular smooth muscle cells (VSMCs), we compared the expression and activity of G1 and S phase cyclins in control and IFN-gamma-treated VSMCs. IFN-gamma treatment did not inhibit the G1 cyclins but did decrease cyclin A protein, mRNA, and associated kinase activity by 85, 90, and 90%, respectively. Nuclear run-on and mRNA stability determinations indicated that this decrease was the result of transcriptional inhibition. To investigate the molecular basis of this inhibition, we examined protein-DNA interactions involving the cyclin A promoter. Electromobility shift assays showed little change with IFN-gamma treatment in the binding of nuclear proteins to isolated ATF, NF-Y, and CDE elements. In vivo genomic footprinting indicated that IFN-gamma treatment changed the occupancy of chromosomal NF-Y and CDE sites slightly and did not affect occupancy of the ATF site. In a previous study of transforming growth factor-beta1-mediated inhibition of the cyclin A promoter, we mapped the inhibitory effect to the ATF site; in the present study of IFN-gamma treatment, functional analysis by transient transfection showed that inhibition of the cyclin A promoter persisted despite mutation of the ATF, NF-Y, or CDE elements. We hypothesize that IFN-gamma inhibits cyclin A transcription by modifying co-activators or general transcription factors within the complex that drives transcription of the cyclin A gene.

0 Bookmarks
 · 
39 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Porcine circovirus type 2 (PCV2) is an important pathogen in swine, and it is assumed that PCV2 replication is cell cycle dependent (especially during S phase). However, the cellular molecules that regulate PCV2 replication have not been fully identified. Here, we cloned the porcine cyclin A (CycA) and CDK2 genes, the major regulators of the S phase, and established CycA or CDK2 overexpression and lower-expression cell lines. The propagation efficiency of strains PCV2a/CL or PCV2b/YJ in these cell lines was investigated using a capture enzyme-linked immunosorbent assay (ELISA) or an immunoperoxidase monolayer assay (IPMA), and the cell cycle was analyzed by flow cytometry. The results showed that CycA overexpression suppressed PCV2 replication. In contrast, CycA down-regulation by shRNA induced increases during the S and G2/M phases and resulted in increased PCV2 propagation. In contrast, overexpression or lower expression of CDK2 exhibited no significant influence on PCV2 replication. Furthermore, the subcellular localization of the PCV2 replicase protein (Rep) and capsid protein (Cap), CycA, and CDK2 in PK-15 cells was analyzed by confocal microscopy. The results showed that overexpression of CycA, rather than CDK2, altered normal nuclear localization of PCV2-Rep, which was transferred to the cytoplasm. In conclusion, PCV2 replication is both S- and G2/M-phase dependent and CycA, is an important regulator of the PCV2 life cycle.
    Archives of Virology 07/2013; 158(12). DOI:10.1007/s00705-013-1785-5 · 2.28 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that play crucial roles in proteolytic degradation of the extracellular matrix. Aberrant expression of the 92-kDa type IV collagenase (MMP-9) is implicated in the invasion and angiogenesis process of malignant tumors and in inflammatory diseases of the CNS. We investigated the effects of IFN-γ and IFN-β, cytokines used for treating some cancers and multiple sclerosis, on MMP-9 expression in human astroglioma and fibrosarcoma cell lines and primary astrocytes. Our results demonstrate that IFN-γ and IFN-β significantly inhibit MMP-9 enzymatic activity and protein expression that is induced by PMA and the cytokine TNF-α. The inhibitory effects of IFN-γ and IFN-β on MMP-9 expression correlate with decreased steady state MMP-9 mRNA levels and suppression of MMP-9 promoter activity. IFN-γ- and IFN-β-mediated inhibition of MMP-9 gene expression is dependent on the transcription factor STAT-1α, since IFN-γ and IFN-β fail to suppress MMP-9 expression in STAT-1α-deficient primary astrocytes and human fibrosarcoma cells. Reconstitution of human STAT-1α successfully restores the inhibitory effects of IFN-γ and IFN-β on MMP-9 gene expression. Thus, these data demonstrate the critical role of STAT-1α in IFN-γ and IFN-β suppression of MMP-9 gene expression.
    The Journal of Immunology 11/2001; 167(9):5150-5159. · 5.36 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Studies have shown that Porcine circovirus (PCV)-2 induces apoptosis in PK15 cells. Here we report that cell death is induced in PCV2b-infected PK15 cells that express Capsid (Cap) protein and this effect is enhanced in interferon gamma (IFN-γ)-treated cells. We further show that transient PCV2a and 2b-Cap protein expression induces cell death in PK15 cells at rate similar to PCV2 infection, regardless of Cap protein localization. These data suggest that Cap protein may have the capacity to trigger different signaling pathways involved in cell death. Although further investigation is needed to gain deeper insights into the nature of the pathways involved in Cap-induced cell death, this study provides evidence that PCV2-induced cell death in kidney epithelial PK15 cells can be mapped to the Cap protein and establishes the need for future research regarding the role of Cap-induced cell death in PCV2 pathogenesis.
    Virology 11/2014; s 468–470:126–132. DOI:10.1016/j.virol.2014.07.051 · 3.28 Impact Factor