Two transactivation domains of hypoxia-inducible factor-1alpha regulated by the MEK-1/p42/p44 MAPK pathway

At a low-oxygen tension, cells increase the expression of several genes (such as erythropoietin, the vascular endothelial growth factor, and glycolytic enzymes) in order to adapt to hypoxic stress. A common transactivator, named the hypoxia-inducible factor 1 (HIF-1) activates these genes. HIF-1 is a heterodimeric transactivator that is composed of alpha and beta subunits. HIF-1 activity is primarily determined by the hypoxia-induced stabilization of the alpha subunit, whereas the HIF-1beta subunit is expressed constitutively. Our previous observation implied that the MEK-1/p42/p44 MAPK pathway is involved in the hypoxia-induced transactivation ability, but not in the stabilization and DNA binding of HIF-1alpha. In this paper, we dissected the transactivation domain of HIF-1alpha in more detail, and tested the correlation between specific domains of HIF-1alpha and specific signaling pathways. We designed several fusion proteins that contain deletion mutants of HIF-1alpha that is linked to the DNA binding domain of the yeast protein Gal4. By using the Gal4-driven reporter system, we tested the transactivation activities of the Gal4/HIF-1alpha fusion proteins in Hep3B cells. Our findings suggest that tyrosine kinases, the MEK-1/p42/p44 MAPK pathway, but not the PI-3 kinase/Akt pathway, are involved in the hypoxia-induced transactivation of HIF-1alpha. We have shown that the functional transactivation activities are located at both 522-649 and 650-822 amino acids of HIF-1alpha. Treatment of PD98059, a MEK-1 inhibitor, blocked the hypoxia-induced transactivation abilities of both the 522-649 and 650-822 amino acids of the C-terminal half of HIF-1alpha. This implies that the MEK-1/p42/p44 MAPK signaling pathway cannot distinguish between the two hypoxia-induced transactivation domains.     

Hepatitis B virus X protein modulates peroxisome proliferator-activated receptor gamma through protein-protein interaction

Ligand activation of peroxisome proliferator-activated receptor gamma (PPARgamma) has been reported to induce growth inhibition and apoptosis in various cancers including hepatocellular carcinoma (HCC). However, the effect of hepatitis B virus X protein (HBx) on PPARgamma activation has not been characterized in hepatitis B virus (HBV)-associated HCC. Herein, we demonstrated that HBx counteracted growth inhibition caused by PPARgamma ligand in HBx-associated HCC cells. We found that HBx bound to DNA binding domain of PPARgamma and HBx/PPARgamma interaction blocked nuclear localization and binding to recognition site of PPARgamma. HBx significantly suppressed a PPARgamma-mediated transactivation. These results suggest that HBx modulates PPARgamma function through protein-protein interaction.     

Mechanism of regulation of the hypoxia-inducible factor-1 alpha by the von Hippel-Lindau tumor suppressor protein

In normoxic cells the hypoxia-inducible factor-1 alpha (HIF-1 alpha) is rapidly degraded by the ubiquitin-proteasome pathway, and activation of HIF-1 alpha to a functional form requires protein stabilization. Here we show that the product of the von Hippel-Lindau (VHL) tumor suppressor gene mediated ubiquitylation and proteasomal degradation of HIF-1 alpha under normoxic conditions via interaction with the core of the oxygen-dependent degradation domain of HIF-1 alpha. The region of VHL mediating interaction with HIF-1 alpha overlapped with a putative macromolecular binding site observed within the crystal structure of VHL. This motif of VHL also represents a mutational hotspot in tumors, and one of these mutations impaired interaction with HIF-1 alpha and subsequent degradation. Interestingly, the VHL binding site within HIF-1 alpha overlapped with one of the minimal transactivation domains. Protection of HIF-1 alpha against degradation by VHL was a multistep mechanism, including hypoxia-induced nuclear translocation of HIF-1 alpha and an intranuclear hypoxia-dependent signal. VHL was not released from HIF-1 alpha during this process. Finally, stabilization of HIF-1 alpha protein levels per se did not totally bypass the need of the hypoxic signal for generating the transactivation response.     

Hepatitis B virus X protein induces cell death by causing loss of mitochondrial membrane potential

The hepatitis B virus X protein (HBx) has been implicated in the carcinogenicity of this virus as a causative factor by means of its transactivation function in development of hepatocellular carcinoma. However, we and others have recently reported that HBx is located in mitochondria and causes subsequent cell death (Takada, S., Shirakata, Y., Kaneniwa, N., and Koike, K. (1999) Oncogene 18, 6965-6973; Rahmani, Z., Huh, K. W., Lasher, R., and Siddiqui, A. (2000) J. Virol. 74, 2840-2846). In this study, we, therefore, examined the mechanism of HBx-related cell death. Using enhanced green fluorescent protein (EGFP) fusion constructs of HBx, the region required for its mitochondrial localization was mapped to amino acids (aa) 68-117, which is essential for cell death but inactive for transactivation function. In vitro binding analysis supported the notion that the recombinant HBx associates with isolated mitochondria through the region of aa 68-117 without causing redistribution of cytochrome c and apoptosis-inducing factor (AIF). A cytochemical analysis revealed that mitochondrial membrane potential was decreased by HBx association with mitochondria, suggesting that HBx induces dysfunction of permeability transition pore (PTP) complex. Furthermore, PTP inhibitors, reactive oxygen species (ROS) scavengers and Bcl-xL, which are known to stabilize mitochondrial membrane potential, prevented HBx-induced cell death. Collectively, the present results suggest that location of HBx in mitochondria of hepatitis B virus-infected cells causes loss of mitochondrial membrane potential and subsequently induces mitochondria-dependent cell death.