Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen Induces Expression of the Helix-Loop-Helix Protein Id-1 in Human Endothelial Cells

Abstract

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) (also known as human herpesvirus 8) is a gamma-2 herpesvirus believed to be the etiologic agent responsible for KS. The pathogenesis of this potentially lifethreatening neoplasm is complex and unclear, and it is currently unknown how KSHV causes KS. Id (named for inhibitor of DNA binding or inhibitor of differentiation) proteins were identified in 1990 and found to be naturally occurring dominant-negative inhibitors of basic helix-loop-helix transcription factors. Id-1, the most well-studied member of this family, has since been shown to play a key role in several biological systems including cellular differentiation, cell cycle regulation, and tumorigenesis. In this report, we demonstrate that Id-1 is expressed at high levels in KS tumor cells both in vitro and in vivo but is expressed at relatively modest levels in endothelial cells (ECs), the likely precursor of the KS tumor cell. Infection of precursor cells with KSHV may be responsible for this enhanced expression, as KSHV infection induced Id-1 27-fold in ECs under our experimental conditions. Furthermore, we demonstrate that the KSHV-encoded latency-associated nuclear antigen (LANA) protein appears to be involved. Expression of LANA in ECs resulted in Id-1 induction that was almost identical to the induction seen with KSHV-infected ECs. These results demonstrate the expression of Id-1 in KS tumor cells and indicate the KSHV LANA protein may be, at least in part, responsible. This may be an important mechanism by which KSHV allows KS tumor cells to escape normal cell cycle regulation and enhances their proliferation.Id proteins are a family of four helix-loop-helix (HLH) proteins (Id-1 to Id-4) initially recognized as growth factor-inducible genes that inhibit cellular differentiation (5, 36). These proteins lack the basic amino acid sequence characteristic of basic HLH (bHLH) transcription factors that is necessary for DNA binding and target gene transcription (5). Instead, Id proteins bind to bHLH transcription factors and form nonfunctional heterodimers, thereby acting as naturally occurring dominant-negative inhibitors (5).Although Id proteins have traditionally been viewed as negative regulators of cell differentiation, recent studies indicate they have wider biological roles, including cell cycle regulation, embryonic development, cell death, and tumorigenesis (36). Several of these functions may be related to recently described interactions of Id proteins with certain non-HLH proteins, including ETS transcription factors, pRb, the pocket proteins p107 and p130, MIDA1, and Pax proteins (22, 51). With respect to normal cell cycle regulation, Id genes are induced in the G 1 phase and expression is down regulated in quiescent, senescent, or terminally differentiated cells. These proteins can promote cell cycle progression through several distinct mechanisms. Id proteins prevent expression of the cyclin-dependent kinase inhibitor (CDKI) p21 by blocking bHLH transcription factor activity (41). Id-2 and I...

Lymphatic reprogramming of blood vascular endothelium by Kaposi sarcoma–associated herpesvirus

“…Efficient KSHV infection was confirmed by high levels of expression of LANA mRNA in infected HDMECs. We found that 3-7% of infected HDMECs were ORF59-positive and 1.5-3% were K8.1 positive, in agreement with previous results [12][13][14] . This indicates that <10% of the cells were undergoing lytic reactivation.…”

Gamma secretase inhibitor blocks Notch activation and induces apoptosis in Kaposi's sarcoma tumor cells

“…Whole-cell extracts were prepared by lysing cells in CHAPS buffer containing a mixture of protease inhibitors (MiniComplete, Roche Molecular Biochemicals), incubating the cells for 20 min on ice and centrifuging to remove insoluble cellular debris (Tang et al, 2003). Protein concentration was determined using a Bradford Assay (Bio-Rad Laboratories, Hercules, CA, USA).…”

Activation of NF-κB by the Latent vFLIP Gene of Kaposi's Sarcoma-Associated Herpesvirus Is Required for the Spindle Shape of Virus-Infected Endothelial Cells and Contributes to Their Proinflammatory Phenotype

“…While many established cells in culture can be latently infected by KSHV, most such cells display no phenotype (2). However, recent studies from several groups show that primary vascular endothelial cells from dermal microvasculature (9) or umbilical vein (15,30,42) can undergo conversion from cuboidal to spindle-like morphology, strongly reminiscent of KS tumor cells, upon infection by KSHV in vitro.Here we show that such morphological changes are due to the cell-autonomous action of latent viral gene products and occur in both lymphatic and vascular endothelium. By expressing the known KSHV latency genes individually in primary endothelial cells, we show that this morphological change can be effected by a single viral gene encoding the vFLIP protein.…”