Murine CMV-induced hearing loss is associated with inner ear inflammation and loss of spiral ganglia neurons.

Congenital human cytomegalovirus (HCMV) occurs in 0.5-1% of live births and approximately 10% of infected infants develop hearing loss. The mechanism(s) of hearing loss remain unknown. We developed a murine model of CMV induced hearing loss in which murine cytomegalovirus (MCMV) infection of newborn mice leads to hematogenous spread of virus to the inner ear, induction of inflammatory responses, and hearing loss. Characteristics of the hearing loss described in infants with congenital HCMV infection were observed including, delayed onset, progressive hearing loss, and unilateral hearing loss in this model and, these characteristics were viral inoculum dependent. Viral antigens were present in the inner ear as were CD(3+) mononuclear cells in the spiral ganglion and stria vascularis. Spiral ganglion neuron density was decreased after infection, thus providing a mechanism for hearing loss. The lack of significant inner ear histopathology and persistence of inflammation in cochlea of mice with hearing loss raised the possibility that inflammation was a major component of the mechanism(s) of hearing loss in MCMV infected mice.

Requirement of evading apoptosis for HIF-1α-induced malignant progression in mouse cells.

Tumor hypoxia is correlated with genetic alteration and malignant progression. Our previous studies indicated that the hypoxia-inducible transcription factor, HIF-1α, is responsible for hypoxic suppression of DNA repair in tumor cells by a non-canonical mode of action that requires the HIF-1α PAS-B subdomain. The involvement of HIF-1α in genetic alteration has raised an intriguing question as to whether normal cells would respond to hypoxic stress differently to avert genetic alteration. In this study, we chose several mouse cell types ranging from benign to malignant, apoptosis-proficient to apoptosis-deficient, and determined their responses to HIF-1α expression. In agreement with our previous findings, transient hypoxia and HIF-1α expression inhibited DNA repair and induced DNA damage in all cell types examined; however, cumulative DNA damage only occurred in apoptosis-deficient, malignant cells transduced for sustained expression of HIF-1α or HIF-1α PAS-B itself. In keeping with the theory of apoptosis as a cancer barrier, only these apoptosis-deficient cells acquired anchorage-independent growth and epithelial-mesenchymal transition. Furthermore, these cells exhibited increased Akt activity and resistance to etoposide by inhibiting autophagy. Altogether, our results define an essential role for apoptosis to prevent HIF-1α-induced genetic alteration and thereby malignant progression.

HIF-1α mediates tumor hypoxia to confer a perpetual mesenchymal phenotype for malignant progression.

Although tumor progression involves genetic and epigenetic alterations to normal cellular biology, the underlying mechanisms of these changes remain obscure. Numerous studies have shown that hypoxia-inducible factor 1α (HIF-1α) is overexpressed in many human cancers and up-regulates a host of hypoxia-responsive genes for cancer growth and survival. We recently identified an alternative mechanism of HIF-1α function that induces genetic alterations by suppressing DNA repair. Here, we show that long-term hypoxia, which mimics the tumor microenvironment, drives a perpetual epithelial-mesenchymal transition (EMT) through up-regulation of the zinc finger E-box binding homeobox protein ZEB2, whereas short-term hypoxia induces a reversible EMT that requires the transcription factor Twist1. Moreover, we show that the perpetual EMT driven by chronic hypoxia depends on HIF-1α induction of genetic alterations rather than its canonical transcriptional activator function. These mesenchymal tumor cells not only acquire tumorigenicity but also display characteristics of advanced cancers, including necrosis, aggressive invasion, and metastasis. Hence, these results reveal a mechanism by which HIF-1α promotes a perpetual mesenchymal phenotype, thereby advancing tumor progression.

HIF-1α confers aggressive malignant traits on human tumor cells independent of its canonical transcriptional function.

Hypoxia is known to favor tumor survival and progression. Numerous studies have shown that hypoxia-inducible factor 1α (HIF-1α), an oxygen-sensitive transcription factor, is overexpressed in various types of human cancers and upregulates a battery of hypoxia-responsive genes for the growth and survival of cancer cells. Although tumor progression involves the acquisition of genetic and/or epigenetic changes that confer additional malignant traits, the underlying mechanisms of these changes remain obscure. We recently identified an alternative mechanism of HIF-1α function by which HIF-1α suppresses DNA repair by counteracting c-Myc transcriptional activity that maintains gene expression. Here, we show that this HIF-α-c-Myc pathway plays an essential role in mediating hypoxic effects on malignant progression via genetic alterations, resulting in the formation of malignant tumors with aggressive local invasion and epithelial-mesenchymal transition. We show an absolute requirement of the HIF-α-c-Myc pathway for malignant progression, whereas the canonical transcription function of HIF-1α alone is insufficient and seemingly dispensable. This study indicates that HIF-1α induction of genetic alteration is the underlying cause of tumor progression, especially by the hypoxic microenvironment.

An essential role of the HIF-1alpha-c-Myc axis in malignant progression.

Cancer is a disease of genomic aberration. The hypoxic microenvironment is believed to promote tumor progression via the induction of genetic instability. To understand how hypoxia drives tumor progression, we have shown recently that the hypoxia-inducible transcription factor, HIF-1alpha, is critical for transcriptional repression of DNA repair genes by a noncanonical mode of action referred to as the "HIF-1alpha-c-Myc axis." HIF-1alpha action via the HIF-1alpha-c-Myc axis is independent of its DNA-binding and transactivation domains; instead it requires the PAS-B domain to displace the transcription activator c-Myc from the target gene promoter for gene repression. Owing to the functional compromise on DNA repair, tumor cells with activated HIF-1alpha-c-Myc axis display persistent DNA damage, genetic alterations, and malignant progression. However, apoptosis-proficient cells are resistant to such changes. These findings argue that the hypoxic microenvironment plays a critical role in driving genetic alterations especially in apoptosis-deficient cells for malignant progression.

Transcriptional activation of HIF-1 by RORalpha and its role in hypoxia signaling.

OBJECTIVE: Hypoxia-inducible factor 1alpha (HIF-1alpha) is primarily involved in the adapting of cells to changes in oxygen levels, which is essential for normal vascular function. Recently, physiological roles for retinoic acid-related orphan receptor alpha (RORalpha) have been implicated in cardiovascular diseases such as atherosclerosis. In this study, we have investigated the potential roles of RORalpha in the hypoxia signaling pathway in connection with activation of HIF-1alpha. METHODS AND RESULTS: Under hypoxic conditions, expression of RORalpha was induced. When RORalpha was introduced exogenously, protein level as well as transcriptional activity of HIF-1alpha was enhanced. Putative ligands of RORalpha, such as melatonin and cholesterol sulfate, induced transcriptional activity for HIF-1alpha, which was abolished by RNA interference against RORalpha. RORalpha was physically associated with HIF-1alpha through DNA binding domain, which was required to the RORalpha-induced stabilization and transcriptional activation of HIF-1alpha. Finally, either infection with adenovirus encoding RORalpha or treatment with ROR ligands enhanced the formation of capillary tubes by human umbilical vascular endothelial cells. CONCLUSIONS: Our results provide a new insight for the function of RORalpha in amplification of hypoxia signaling and suggest a potential application of RORalpha ligands for the therapy of hypoxia-associated vascular diseases.

Hypoxic suppression of the cell cycle gene CDC25A in tumor cells.

Hypoxia, a key microenvironmental factor for tumor development, not only stimulates angiogenesis and glycolysis for tumor expansion, but also induces cell cycle arrest and genetic instability for tumor progression. Several independent studies have shown hypoxic blockade of cell cycle progression at the G1/S transition, arising from the inactivation of S-phase-promoting cyclin E-CDK2 kinase complex. Despite these findings, the biochemical pathways leading to the cell cycle arrest remain poorly defined. We recently showed that hypoxic activates the expression of CDNK1A encoding the CDK2 inhibitor p21Cip1, through a novel HIF-1alpha-Myc pathway that involves Myc displacement from the CDNK1A promoter by the hypoxia-inducible transcription factor HIF-1alpha. In pursuit of further understanding of the hypoxic effects on cell cycle in tumor cells, here we report that hypoxia inhibits the expression of CDC25A, another cell cycle gene encoding a tyrosine phosphatase that maintains CDK2 activity. In accordance with the HIF-1alpha-Myc pathway, hypoxia requires HIF-1alpha for CDC25A repression, resulting in a selective displacement of an activating Myc from the CDC25A promoter without affecting a canonical Myc binding in the intron. Intriguingly, HIF-1alpha alone fails to recapitulate the hypoxic effect, indicating that HIF-1alpha is necessary but insufficient for the hypoxic repression. Taken together, our studies indicate that hypoxia inhibits cell cycle progression by controlling the expression of various cell cycle genes.

Inhibitor of DNA binding 1 activates vascular endothelial growth factor through enhancing the stability and activity of hypoxia-inducible factor-1alpha.

Inhibitor of DNA binding 1 (Id-1) has been implicated in tumor angiogenesis by regulating the expression of vascular endothelial growth factor (VEGF), but its molecular mechanism has not been fully understood. Here, we show the cross talk between Id-1 and hypoxia-inducible factor-1alpha (HIF-1alpha), that Id-1 induces VEGF by enhancing the stability and activity of HIF-1alpha in human endothelial and breast cancer cells. Although both the transcript and proteins levels of VEGF were induced by Id-1, only the protein expression of HIF-1alpha was induced without transcriptional changes in both human umbilical endothelial cells and MCF7 breast cancer cells. Such induction of the HIF-1alpha protein did not require de novo protein synthesis but was dependent on the active extracellular response kinase (ERK) pathway. In addition, stability of the HIF-1alpha protein was enhanced in part by the reduced association of the HIF-1alpha protein with von Hippel-Lindau protein in the presence of Id-1. Furthermore, Id-1 enhanced nuclear translocation and the transcriptional activity of HIF-1alpha. Transcriptional activation of HIF-1-dependent promoters was dependent on the active ERK pathway, and the association of HIF-1alpha protein with cyclic AMP-responsive element binding protein was enhanced by Id-1. Finally, Id-1 induced tube formation in human umbilical endothelial cells, which also required active ERK signaling. In conclusion, we provide the molecular mechanism of the cross talk between HIF-1alpha and Id-1, which may play a critical role in tumor angiogenesis.

Metastasis-associated protein 1 enhances stability of hypoxia-inducible factor-1alpha protein by recruiting histone deacetylase 1.

The expression of metastasis-associated protein 1 (MTA1) correlates well with tumor metastases; however, the associated molecular mechanism is not fully understood. Here, we explored the possibility of cross-talk between MTA1 and hypoxia-inducible factor-1alpha (HIF-1alpha), a key regulator of angiogenic factors. We observed that the expression of MTA1 was strongly induced under hypoxia in breast cancer cell lines such as MCF-7 and MDA-MB-231. When MTA1 was overexpressed, the transcriptional activity and stability of HIF-1alpha protein were enhanced. MTA1 and HIF-1alpha are physically associated in vivo and they were localized completely in the nucleus when coexpressed. MTA1 induced the deacetylation of HIF-1alpha by increasing the expression of histone deacetylase 1 (HDAC1). MTA1 counteracted to the action of acetyltransferase, ARD1, and it did not stabilize the HIF-1alpha mutant that lacks the acetylation site, K532R. These results indicate that acetylation is the major target of MTA1/HDAC1 function. Collectively, our data provide evidence of a positive cross-talk between HIF-1alpha and MTA1, which is mediated by HDAC1 recruitment, and indicate a close connection between MTA1-associated metastasis and HIF-1-induced tumor angiogenesis.

Negative cross-talk between Nur77 and small heterodimer partner and its role in apoptotic cell death of hepatoma cells.

Nur77, an orphan nuclear receptor, has been implicated in apoptosis of a variety of cell types, including hepatocytes. The small heterodimer partner (SHP) binds and inhibits the function of many nuclear receptors. Here, we investigated cross-talk between Nur77 and SHP during anti-Fas antibody (CH11)-mediated apoptosis of hepatic cells. Expression of SHP decreased, whereas antisense SHP enhanced, the transcriptional activity of Nur77 in HepG2 cells. SHP and Nur77 were physically associated in vivo and colocalized in the nucleus. SHP decreased the transactivation function of the N-terminal domain of Nur77 that recruits coactivators. Nur77 and SHP competitively bound to cAMP response element-binding protein-binding protein and the expression of coactivators, such as cAMP response element-binding protein-binding protein and activating signal cointegrator-2, recovered the decreased function of Nur77 caused by SHP. Finally, SHP was differentially expressed in hepatoma cell lines in that it was not detected in the interferon-gamma (IFNgamma)/CH11-sensitive SNU354, whereas it was significantly expressed in the IFNgamma/CH11-resistant HepG2. Interestingly, a stable SNU354 cell line that expressed SHP became resistant to the IFNgamma/CH11-induced apoptosis. Together, our results suggest that SHP plays a key role in the regulation of Nur77 activation and thereby in Nur77-mediated apoptosis in the liver.

The carboxy-terminus of the hepatitis B virus X protein is necessary and sufficient for the activation of hypoxia-inducible factor-1alpha.

Hepatitis B virus X protein (HBx) of the hepatitis B virus is strongly implicated in angiogenesis and metastasis during hepatocarcinogenesis. Previously, we reported that HBx enhances activity of hypoxia-inducible factor-1alpha (HIF-1alpha), a potent transactivator that induces angiogenic factors. Here, we delineate the structural region of HBx that potentiates HIF-1alpha. The carboxy-terminus of HBx increased the stability of HIF-1alpha protein, probably through inhibiting interaction with von Hippel-Lindau protein. Further, the carboxy-terminus of HBx enhanced the transactivation function of HIF-1alpha by enhancing its association with CREB binding protein (CBP). Finally, we demonstrated the physical association of HBx with the basic helix-loop-helix/PER-ARNT-SIM domain, the inhibitory domain, and the carboxy-terminal transactivation domain of HIF-1alpha in vivo.

Novel function of orphan nuclear receptor Nur77 in stabilizing hypoxia-inducible factor-1alpha.

Hypoxia-inducible factor-1alpha (HIF-1alpha) plays a central role in oxygen homeostasis by inducing the expression of a broad range of genes in a hypoxia-dependent manner. Here, we show that the orphan nuclear receptor Nur77 is an important regulator of HIF-1alpha. Under hypoxic conditions, Nur77 protein and transcripts were induced in a time-dependent manner. When Nur77 was exogenously introduced, it enhanced the transcriptional activity of HIF-1, whereas the dominant negative Nur77 mutant abolished the function of HIF-1. The HIF-1alpha protein was greatly increased and completely localized in the nucleus when coexpressed with Nur77. The N-terminal transactivation domain of Nur77 was required and sufficient for the activation of HIF-1alpha. The association of HIF-1alpha with von Hippel-Lindau protein was not affected, whereas that with mouse double minute 2 (MDM2) was greatly reduced in the presence of Nur77. Further we found that the expression of MDM2 was repressed at transcription level in the presence of Nur77 as well as under hypoxic conditions. Finally, PD98059 decreased Nur77-induced HIF-1alpha stability and recovered MDM2 expression, indicating that the extracellular signal-regulated kinase pathway is critical in the Nur77-induced activation of HIF-1alpha. Together, our results demonstrate a novel function for Nur77 in the stabilization of HIF-1alpha and suggest a potential role for Nur77 in tumor progression and metastasis.

Hepatitis B virus X protein induces expression of Fas ligand gene through enhancing transcriptional activity of early growth response factor.

FasL expressed in tumor cells plays an important role in the escape from immune surveillance by inducing apoptosis in T-cells bearing Fas. Since the Fas/FasL signaling pathway requires transcriptional induction of the FasL gene, elucidation of the precise mechanisms underlying regulation of FasL gene expression may provide useful molecular insights on tumor progression. We and others (Shin, E. C., Shin, J. S., Park, J. H., Kim, H., and Kim, S. J. (1999) Int. J. Cancer 82, 587-591; Lee, M. O., Kang, H. J., Cho, H., Shin, E. C., Park, J. H., and Kim, S. J. (2001) Biochem. Biophys. Res. Commun. 288, 1162-1168) have previously reported that hepatitis B virus X protein (HBx) plays a role in the induction of FasL expression in hepatitis B virus-associated hepatoma. In the present study, we analyzed the potential cis- and trans-acting factors that regulate FasL promoter. We found that HBx induced activity of the reporter containing FasL promoter through binding site for Egr but not through NFAT or SP-1, which are known as strong activators of the FasL promoter in T-cells. Transient expression of antisense Egr-2 and antisense Egr-3 abolished expression of FasL, which further confirmed the role of Egr in the HBx-mediated FasL expression. Also we observed that HBx increased the transcriptional activity of Egr-2 and Egr-3 by enhancing expression as well as the transactivation function of these proteins. HBx interacted with Egr-2 and Egr-3 in vivo and enhanced binding of Egr to the co-activator, cAMP-response element-binding protein-binding protein, which may explain the molecular mechanism by which HBx induced the transactivation function of Egr. Finally, we found that the carboxyl terminus of HBx was necessary and sufficient for FasL induction as well as activation of Egr. Taken together, our results show a novel mechanism by which HBx induces FasL gene expression that is mediated by enhancing transcriptional activity of Egr-2 and Egr-3.

Hepatitis B virus X protein enhances transcriptional activity of hypoxia-inducible factor-1alpha through activation of mitogen-activated protein kinase pathway.

Hepatitis B virus X protein (HBx) of the hepatitis B virus was strongly implicated in angiogenesis and metastasis during hepatocarcinogenesis. Here, we explored the possibility of cross-talk between HBx and hypoxia-inducible factor-1alpha (HIF-1alpha), a potent transcriptional inducer of angiogenic factors. First, we showed that stability of HIF-1alpha protein was increased by HBx in HBx-inducible Chang liver cells as well as in transient HBx expression system of non-hepatic cells. Immunofluorescence studies revealed that the HBx-induced HIF-1alpha was partially translocated into the nucleus in majority of cells while additional CoCl2-induced hypoxic condition caused complete nuclear translocation. Second, HBx induced both phosphorylation of HIF-1alpha and activation of p42/p44 mitogen-activated protein kinases (MAPKs), which were synergistically enhanced in the presence of CoCl2. Furthermore, HBx enhanced transcriptional activity of HIF-1alpha in the reporter genes encoding hypoxia response element or VEGF promoter. Either treatment of MEK inhibitor PD98059 or coexpression of dominant-negative MAPK mutants abolished the HBx-induced transcriptional activity and protein stability as well as nuclear translocation of HIF-1alpha, suggesting that HBx activates HIF-1alpha through MAPK pathway. Third, the association of HIF-1alpha with von Hippel-Lindau was decreased but the association with CREB-binding protein was enhanced in the presence of HBx, suggesting the molecular mechanism by which HBx enhances the protein stability and transactivation function of HIF-1alpha. Finally, we demonstrated that expression of HIF-1alpha and vascular endothelial growth factor was increased in the liver of HBx-transgenic mice, suggesting that the cross-talk between HIF-1alpha and HBx may lead to transcriptional activation of HIF-1alpha target genes, which play a critical role in hepatocarcinogenesis.