Ets-1-dependent expression of vascular endothelial growth factor receptors is activated by latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus through interaction with Daxx

Vascular endothelial growth factor (VEGF) and its receptors are highly expressed in Kaposi's sarcoma (KS) lesion and play a key role in angiogenesis. Latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8) has multiple functions related to viral latency and KSHV-induced oncogenesis. In this report, we have identified Daxx as a LANA-binding protein by co-immunoprecipitation analysis of HeLa cells stably expressing LANA. LANA associated with Daxx in a PEL cell line infected with KSHV. LANA and Daxx also bound in vitro, suggesting direct interaction. From the results of binding assays, a region containing the Glu/Asp-rich domain within LANA, and a central region including the second paired amphipathic helix within Daxx contributed to the interaction. To address the physiological significance of this interaction, we focused on a Daxx-mediated VEGF receptor gene regulation. We found that Daxx repressed Ets-1-dependent Flt-1/VEGF receptor-1 gene expression, and that LANA inhibited the repression by Daxx in a reporter assay. Analyses of flow cytometry and real-time PCR revealed that expression of VEGF receptor-1 and -2 in LANA-expressing human umbilical vein endothelial cells (HUVECs) significantly increased. Co-immunoprecipitation and immunoblotting experiments suggested that LANA-bound Daxx to inhibit the interaction between Daxx and Ets-1. Chromatin immunoprecipitation assays showed that Daxx associated with VEGF receptor-1 promoter in HUVECs, and that LANA expression reduced this association. These results suggested that LANA contributes to a high expression of VEGF receptors in KS lesion by interfering with the interaction between Daxx and Ets-1.     

The nfkb1 promoter is controlled by proteins of the Ets family.

The gene encoding NFKB1 is autoregulated, responding to NF-kappa B/Rel activation through NF-kappa B binding sites in its promoter, which also contains putative sites for Ets proteins. One of the Ets sites, which we refer to as EBS4, is located next to an NF-kappa B/Rel binding site, kB3, which is absolutely required for activity of the promoter in Jurkat T cells in response to activation by phorbol 12-myristate 13-acetate (PMA), PMA/ionomycin, or the Tax protein from human T cell leukemia virus type I. We show that EBS4 is, required for the full response of the nfkb1 promoter to PMA or PMA/ionomycin in Jurkat cells. EBS4 is bound by Ets-1, Elf-1, and other species. Overexpression of Ets-1 augments the response to PMA/ionomycin and this is reduced by mutation of EBS4. Elf-1 has less effect in conjunction with PMA/ionomycin, but by itself activates the promoter 12-fold. This activation is only partly affected by mutation of EBS4, and a mutant promoter that binds Ets-1, but not Elf-1, at the EBS4 site responds to PMA/ionomycin as efficiently as the wild-type. Ets proteins may be responsible for fine-tuning the activity of the nfkb1 gene in a cell-type-specific manner.