Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 1 mediates episome persistence through cis-acting terminal repeat (TR) sequence and specifically binds TR DNA.

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) (also known as human herpesvirus 8) latently infects KS tumors, primary effusion lymphomas (PELs), and PEL cell lines. In latently infected cells, KSHV DNA is maintained as circularized, extrachromosomal episomes. To persist in proliferating cells, KSHV episomes must replicate and efficiently segregate to progeny nuclei. In uninfected B-lymphoblastoid cells, KSHV latency-associated nuclear antigen (LANA1) is necessary and sufficient for persistence of artificial episomes containing specific KSHV DNA. In previous work, the cis-acting sequence required for episome persistence contained KSHV terminal-repeat (TR) DNA and unique KSHV sequence. We now show that cis-acting KSHV TR DNA is necessary and sufficient for LANA1-mediated episome persistence. Furthermore, LANA1 binds TR DNA in mobility shift assays and a 20-nucleotide LANA1 binding sequence has been identified. Since LANA1 colocalizes with KSHV episomes along metaphase chromosomes, these results are consistent with a model in which LANA1 may bridge TR DNA to chromosomes during mitosis to efficiently segregate KSHV episomes to progeny nuclei.

The Kaposi' s sarcoma-associated herpesvirus latency-associated nuclear antigen.

Kaposi's sarcoma-associated herpesvirus (KSHV), also called human herpesvirus 8 (HHV-8), is associated with Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease (6,7,10). While the vast majority of tumor cells from these malignancies are latently infected, only a small subset of viral genes are actually expressed (5,40,47). Of these genes, the latency-associated nuclear antigen (LANA-1, LNA, or LNA1) is the only protein consistently shown to be highly expressed by in situ hybridization and immunohistochemistry (11,20,21,32). Moreover, within the past few years LANA-1 has proven to be a quite versatile protein, playing not only a pivotal role in KSHV episome persistence, but also in interacting with and influencing several cellular genes.

Efficient persistence of extrachromosomal KSHV DNA mediated by latency-associated nuclear antigen.

Primary effusion lymphoma (PEL) cells harbor Kaposi's sarcoma-associated herpesvirus (KSHV) episomes and express a KSHV-encoded latency-associated nuclear antigen (LANA). In PEL cells, LANA and KSHV DNA colocalized in dots in interphase nuclei and along mitotic chromosomes. In the absence of KSHV DNA, LANA was diffusely distributed in the nucleus or on mitotic chromosomes. In lymphoblasts, LANA was necessary and sufficient for the persistence of episomes containing a specific KSHV DNA fragment. Furthermore, LANA colocalized with the artificial KSHV DNA episomes in nuclei and along mitotic chromosomes. These results support a model in which LANA tethers KSHV DNA to chromosomes during mitosis to enable the efficient segregation of KSHV episomes to progeny cells.

Elevation of cyclic AMP levels in astrocytes antagonizes cytokine-induced adhesion molecule expression.

We have examined the effect of elevating cyclic AMP levels on cytokine-mediated enhancement of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) gene expression by astrocytes. Treatment of astrocytes with the cyclic AMP mimetic dibutyryl-cyclic AMP, or the agonists norepinephrine, forskolin, prostaglandin E2, and cholera toxin alone had no effect on ICAM-1 or VCAM-1 mRNA gene expression. However, elevating cyclic AMP levels within the cells by these agents suppressed interleukin-1beta- and tumor necrosis factor-alpha-induced adhesion molecule expression at both the mRNA and protein levels. The phosphodiesterase type IV inhibitor, rolipram, was able to potentiate the inhibitory effect of forskolin on ICAM-1 and VCAM-1 gene expression. Inhibition of tumor necrosis factor-alpha-induced VCAM-1 mRNA levels by forskolin was partially due to enhanced degradation of VCAM-1 message, whereas the decay rates of tumor necrosis factor-alpha-induced ICAM-1 message and interleukin-1beta-induced ICAM-1/VCAM-1 message were not affected by forskolin treatment. These results demonstrate that the pathways used by interleukin-1beta and tumor necrosis factor-alpha to induce adhesion molecule expression are antagonized by cyclic AMP-dependent protein kinase-mediated signaling pathways.

Second messenger systems in the regulation of cytokines and adhesion molecules in the central nervous system.

Cytokines are a group of secreted proteins that exhibit diverse biological activity and are especially important in immune and inflammatory responses. The inappropriate production of cytokines in the central nervous system (CNS) has been implicated in a number of disease states such as Alzheimer's disease, multiple sclerosis, and AIDS dementia complex. This article focuses on the biological role of three cytokines in the CNS, interleukin-6 (IL-6), tumor necrosis factor alpha, and nerve growth factor, with an emphasis on production by glial cells. We will discuss the diverse intracellular signaling pathways that regulate expression of these cytokines by glial cells and then describe the second messenger systems that mediate cytokine-induced responses in the CNS, with an emphasis on adhesion molecule expression. We conclude by discussing the complexities of signal transduction pathways, particularly "cross-talk" between different intracellular mediators.

Interleukin 1-beta- and tumor necrosis factor-alpha-mediated regulation of ICAM-1 gene expression in astrocytes requires protein kinase C activity.

Several adhesion molecules including intercellular adhesion molecule-1 (ICAM-1) are expressed by astrocytes, the predominant glial cell of the central nervous system (CNS). Such molecules are important in the trafficking of leukocytes to sites of inflammation, and in lymphocyte activation. ICAM-1 is constitutively expressed by neonatal rat astrocytes, and expression is enhanced by the proinflammatory cytokines interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma), with IL-1 beta and TNF-alpha being the strongest inducers. In this study, we have examined the nature of the second messengers involved in ICAM-1 gene expression induced by the cytokines IL-1 beta and TNF-alpha. Our results indicate that stimuli related to protein kinase C (PKC) such as the phorbol ester phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187 increase ICAM-1 mRNA expression, whereas cyclic nucleotide analogs and PKA agonists have no effect. Pharmacologic inhibitors of PKC such as H7, H8, and calphostin C inhibit ICAM-1 gene expression inducible by IL-1 beta and TNF-alpha. Prolonged treatment of astrocytes with PMA results in a time-dependent downregulation of the PKC isoforms alpha, delta, and epsilon, and a concomitant diminution of ICAM-1 mRNA expression induced by IL-1 beta, TNF-alpha, and PMA itself at specific time points post-PMA treatment. These data, collectively, demonstrate a role for various PKC isoforms in IL-1 beta and TNF-alpha enhancement of ICAM-1 gene expression in rat astrocytes.

Regulation of intercellular adhesion molecule-1 gene expression by tumor necrosis factor-alpha, interleukin-1 beta, and interferon-gamma in astrocytes.

Intercellular adhesion molecule-1 (ICAM-1) is a cell surface glycoprotein which can be induced on astrocytes, the major glial cell of the central nervous system (CNS). In this study, we examined the effect of three proinflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and interferon-gamma (IFN-gamma), on the expression of ICAM-1 by primary rat astrocytes. Astrocytes constitutively express ICAM-1 mRNA and protein, which is enhanced by treatment with TNF-alpha, IL-1 beta and IFN-gamma. TNF-alpha is the most potent inducer of ICAM-1 expression, followed by IL-1 beta, then IFN-gamma. Kinetic analysis demonstrated optimum ICAM-1 mRNA expression after a 1-h exposure to TNF-alpha, 2 h exposure to IL-1 beta, and 4 h exposure to IFN-gamma. Peak ICAM-1 protein expression was detected 12-16 h after treatment with TNF-alpha or IL-1 beta, and after a 24-h exposure to IFN-gamma. Nuclear run-on analysis demonstrated that the ICAM-1 gene is transcribed under basal conditions in astrocytes, and that both TNF-alpha and IL-1 beta enhance transcriptional activation of the ICAM-1 gene. ICAM-1 mRNA stability studies determined that basal ICAM-1 mRNA has a half-life of about 1 h, and that TNF-alpha, IL-1 beta and IFN-gamma have a modest effect on stabilization of basal ICAM-1 mRNA expression. These results indicate that under inflammatory conditions in the CNS, such as multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE), astrocytes can be induced to express the adhesion molecule ICAM-1, which can contribute to inflammatory events within the CNS.