Human hepatitis B virus (HBV) is one of the causative agents of hepatocellular carcinoma (HCC). The virus encodes a 17 kDa protein, X, which is known to be a causative agent in the formation of HCC. An insulin-like growth factor-II (IGF-II) is expressed during the formation of HCC. Among the four promoters of the IGF-II gene, promoters 2, 3 and 4 become activated during the formation of HCC. The high frequency of detection of hepatitis B virus X (HBV-X) antigen in liver cells from patients with chronic hepatitis, cirrhosis, and liver cancer suggested that the expressions of HBV-X and IGF-II are associated. Studies were carried out to test the relationship between the HBV-X gene product and the activation of IGF-II promoter 4. We demonstrated that the HBV-X protein increases the endogenous IGF-II expression from promoter 3 and 4 of IGF-II gene. Analysis of the fourth promoter of IGF-II gene showed that the HBV-X gene product positively regulates transcription. Two copies of a motif are responsible for conferring HBV-X regulation on the fourth promoter of IGF-II. These motifs have been identi®ed as Sp1 binding sites. Sp1 binding to IGF-II P4 promoter was identi®ed by gel mobility shift assay using puri®ed Sp1. By using a GAL4-Sp1 fusion protein it was demonstrated that HBV-X positively regulates the Sp1 mediated transcriptional activity of IGF-II in vivo. A protein-anity chromatography experiment showed that HBV-X protein does not bind directly to Sp1, but HBV-X does augment the DNA binding activity of the phosphorylated form of Sp1 in HepG2 cells. Sp1 was phosphorylated by HBV-X and its DNA-binding activity was up-regulated upon HBV-X transfections. Various HBV-X mutant expression vectors were used for the demonstration of speci®c interactions between Sp1 and HBV-X. These results indicate that HBV-X functions as a positive regulator of transcription, and that Sp1 is a direct target for the transcriptional regulation of IGF-II. Increasing the DNA binding ability of the phosphorylated form of Sp1 by HBV-X might be an important mechanism for regulating the IGF-II gene expression and possibly promoting cell division during hepatic carcinogenesis. Our experimental results suggest that expression of HBV-X might induce the expression of IGF-II and the IGF-II might play a role in hepatitis B virus pathogenesis during the formation of HCC.

The Eph (erythropoietin-producing hepatoma) family of receptor tyrosine kinases and their membrane-bound ligands, the ephrins, have been implicated in regulating cell adhesion and migration during development by mediating cell-to-cell signaling events. The transmembrane ephrinB (Eph receptor interactor B) protein is a bidirectional signaling molecule that sends a forward signal through the activation of its cognate receptor tyrosine kinase, residing on another cell. A reverse signal can be transduced into the ephrinBexpressing cell via tyrosine phosphorylation of its conserved Cterminal cytoplasmic domain. Although some insight has been gained regarding how ephrinB may send signals affecting cytoskeletal components, little is known about how ephrinB1 reverse signaling affects transcriptional processes. Here we report that signal transducer and activator of transcription 3 (STAT3) can interact with ephrinB1 in a phosphorylation-dependent manner that leads to enhanced activation of STAT3 transcriptional activity. This activity depends on the tyrosine kinase Jak2, and two tyrosines within the intracellular domain of ephrinB1 are critical for the association with STAT3 and its activation. The recruitment of STAT3 to ephrinB1, and its resulting Jak2-dependent activation and transcription of reporter targets, reveals a signaling pathway from ephrinB1 to the nucleus.FGF receptor ͉ Eph receptor ͉ neuroepithelial cells ͉ tyrosine phosphorylation ͉ Jak2 M embrane-anchored ephrinB (erythropoietin-producing hepatoma interactor B) proteins are ligands for EphB receptors and have been involved in many biological processes such as cell adhesion, neural crest migration, brain segmentation, and vasculogenesis (1-3). Upon cell-to-cell contact, ephrinB1 not only sends a forward signal through the activation of its cognate receptor, but it also rapidly undergoes tyrosine phosphorylation at the C terminus and recruits the adaptor protein growth-factor-receptor-bound protein 4 (Grb4)/Nck adaptor protein 2 (Nck2), resulting in an increase of focal adhesion kinase (FAK) catalytic activity as well as cell rounding (4). In addition, the C-terminal PDZ-binding motif of ephrinB1 is able to mediate reverse signaling by recruitment of PDZ-RGS3, a GTP exchange factor that regulates the migration of cerebellar granule cells (5). Moreover, it has been reported that ephrinB1 signals through its intracellular domain to control retinal progenitor cell movement into the eye field by interacting with the scaffold protein Dishevelled (6). Thus, several signaling molecules have been identified to transmit reverse signaling of ephrinB1 by either phosphorylation-dependent or -independent means. ephrinB1 can be phosphorylated in response to binding a cognate Eph receptor (7,8) or the tight junction protein Claudin (9) or in response to FGF receptor activation (10). Among the six tyrosine residues present in the intracellular domain of ephrinB1, one resides in a well conserved YXXQ motif at the C terminus and is rapidly phosphorylated upon engagement of t...

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