The expression of the Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame 50 (ORF50) protein, Lyta (lytic transactivator), marks the switch from latent KSHV infection to the lytic phase. ORF50/ Lyta upregulates several target KSHV genes, such as K8 (K-bZip), K9 (vIRF1), and ORF57, finally leading to the production of mature viruses. The auto-upregulation of ORF50/Lyta is thought to be an important mechanism for efficient lytic viral replication. In this study, we focused on this autoregulation and identified the promoter element required for it. An electrophoretic mobility shift assay indicated that the octamerbinding protein 1 (Oct-1) bound to this element. Mutations in the octamer-binding motif resulted in refractoriness of the ORF50/Lyta promoter to transactivation by ORF50/Lyta, and Oct-1 expression enhanced this transactivation. These results suggest that the autoregulation of ORF50/Lyta is mediated by Oct-1.

Kaposi's sarcoma-associated herpesvirus (KSHV) is strongly linked to Kaposi's sarcoma, primary effusion lymphomas, and a subset of multicentric Castleman's disease. The mechanism by which this virus establishes latency and reactivation is unknown. KSHV Lyta (lytic transactivator, also named KSHV͞Rta), mainly encoded by the ORF 50 gene, is a lytic switch gene for viral reactivation from latency, inasmuch as it is both essential and sufficient to drive the entire viral lytic cycle. Here we show that the Lyta promoter region was heavily methylated in latently infected cells. Treatment of primary effusion lymphoma-delivered cell lines with tetradecanoylphorbol acetate caused demethylation of the Lyta promoter and induced KSHV lytic phase in vitro. Methylation cassette assay shows demethylation of the Lyta promoter region was essential for the expression of Lyta. In vivo, biopsy samples obtained from patients with KSHV-related diseases show the most demethylation in the Lyta promoter region, whereas samples from a latently infected KSHV carrier remained in a methylated status. These results suggest a relationship among a demethylation status in the Lyta promoter, the reactivation of KSHV, and the development of KSHV-associated diseases.

EphrinB transmembrane ligands and their cognate EphB receptor tyrosine kinases regulate vascular development through bidirectional cell-to-cell signaling, but little is known about the role of EphrinB during postnatal vascular remodeling. We report that EphrinB is a critical mediator of postnatal pericyte-to-endothelial cell assembly into vascular structures. This function is dependent upon extracellular matrixsupported cell-to-cell contact, engagement of EphrinB by EphB receptors expressed on another cell, and Srcdependent phosphorylation of the intracytoplasmic domain of EphrinB. Phosphorylated EphrinB marks angiogenic blood vessels in the developing and hypoxic retina, the wounded skin, and tumor tissue, and is detected at contact points between endothelial cells and pericytes. Furthermore, inhibition of EphrinB activity prevents proper assembly of pericytes and endothelial cells into vascular structures. These results reveal a role for EphrinB signaling in orchestrating pericyte/endothelial cell assembly, and suggest that therapeutic targeting of EphrinB may prove useful for disrupting angiogenesis when it contributes to disease. (Blood. 2009;114:1707-1716) IntroductionBlood vessels are composed of endothelial cells that form the inner lining of vessels and pericytes, also known as mural cells, that envelope the endothelium. Pericytes are embedded into the vascular basement membrane and make direct contact with endothelial cells through extensions that reach deeply into the endothelial cells, without an intervening basal membrane. 1,2 Endothelial cells are better characterized, but pericytes are emerging as critical regulators of vascular function because they are thought to stabilize the vessel wall and to regulate endothelial cell survival, growth, maturation, and permeability. 2 Pericytes have multilineage progenitor potential and are developmentally close to mesenchymal stem cells (MSCs). 3 Genetic mouse models have implicated several receptor-ligand systems as mediators of endothelial-pericyte interactions, including Ang-1/Tie2, transforming growth factor-␤ and its receptors, plateletderived growth factor (PDGF)/PDGF receptor-1, the sphingosine-1-phosphate (S1P)/S1P 1 , and Dll4/Notch. 2 The family of EphB (erythropoietin-producing hepatoma) receptors tyrosine kinases and their surface-bound EphrinB (erythropoietin-producing hepatoma interactor B) ligands have recently emerged as critical regulators of cardiovascular development and pathologic angiogenesis, modulating both endothelial cells and pericyte function. 4 Upon cell-to-cell contact, EphrinB ligands both activate the cognate EphB receptors (forward signaling) and undergo phosphorylation at the C terminus initiating signaling (reverse signaling). 5,6 Genetic experiments have shown that the global knockout of EphB4, EphrinB2, or the endothelial-specific inactivation of EphrinB2 produced a very similar phenotype of embryonic death with prominent remodeling defects of the vascular system. [7][8][9] The role of EphrinB2 reverse signaling in vascular ...