Adaptation to hypoxia is regulated by hypoxia‐inducible factor 1 (HIF‐1), a heterodimeric transcription factor consisting of an oxygen‐regulated α subunit and a constitutively expressed β subunit. Although HIF‐1 is regulated mainly by oxygen tension through the oxygen‐dependent degradation of its α subunit, in vitro it can also be modulated by cytokines, hormones and genetic alterations. To investigate HIF‐1 activation in vivo, we determined the spatial and temporal distribution of HIF‐1 in healthy mice subjected to varying fractions of inspiratory oxygen. Immunohisto‐chemical examination of brain, kidney, liver, heart, and skeletal muscle revealed that HIF‐1α is present in mice kept under normoxic conditions and is further in‐creased in response to systemic hypoxia. Moreover, immunoblot analysis showed that the kinetics of HIF‐1 α expression varies among different organs. In liver and kidney, HIF‐1 α reaches maximal levels after1hand gradually decreases to baseline levels after4hof continuous hypoxia. In the brain, however, HIF‐1α is maximally expressed after 5 h and declines to basal levels by 12 h. Whereas HIF‐1 β is constitutively expressed in brain and kidney nuclear extracts, its hepatic expression increases concomitantly with HIF‐1 α. Overall, HIF‐1 α expression in normoxic mice suggests that HIF‐1 has an important role in tissue homeostasis.—Stroka, D. M., Burkhardt, T., Desbaillets, I., Wenger, R. H., Neil, D. A. H., Bauer, C., Gassmann, M., Candinas, D. HIF‐1 is expressed in normoxic tissue and displays an organ specific regulation under systemic hypoxia. FASEB J . 15, 2445–2453 (2001)

The liver forms a firewall that protects against vascular-borne gut microbes and is commonly impaired in liver disease.

Hypermutations in hepatitis B virus (HBV) DNA by APOBEC3 cytidine deaminases have been detected in vitro and in vivo, and APOBEC3G (A3G) and APOBEC3F (A3F) have been shown to inhibit the replication of HBV in vitro, but the presumably low or even absent hepatic expression of these enzymes has raised the question as to their physiological impact on HBV replication. We show that normal human liver expresses the mRNAs of APOBEC3B (A3B), APOBEC3C (A3C), A3F, and A3G. In primary human hepatocytes, interferon alpha (IFN‐α) stimulated the expression of these cytidine deaminases up to 14‐fold, and the mRNAs of A3G, A3F, and A3B reached expression levels of 10%, 3%, and 3%, respectively, relative to GAPDH mRNA abundance. On transfection, the full‐length protein A3B_L inhibited HBV replication in vitro as efficiently as A3G or A3F, whereas the truncated splice variant A3B_S and A3C had no effect. A3B_L and A3B_S were detected predominantly in the nucleus of uninfected cells; however, in HBV‐expressing cells both proteins were found also in the cytoplasm and were associated with HBV viral particles, similarly to A3G and A3F. Moreover, A3G, A3F, and A3B_L, but not A3B_S, induced extensive G‐to‐A hypermutations in a fraction of the replicated HBV genomes. In conclusion, the editing enzymes A3B_L, A3F, and most markedly A3G, which are expressed in liver and up‐regulated by IFN‐α in hepatocytes, are candidates to contribute to the noncytolytic clearance of HBV. (HEPATOLOGY 2006;43:1364–1374.)

The A20 gene product is a novel zinc finger protein originally described as a tumor necrosis factor alpha (TNF)-inducible early response gene in human umbilical vein endothelial cells (HUVEC). Its described function is to block TNF-induced apoptosis in fibroblasts and B lymphocytes, but more recently it has also been shown to play a role in lymphoid cell maturation. The mechanism of action of A20 is unknown. The aim of our study was to assess the effect of A20 upon endothelial cell activation. By transfecting bovine aortic endothelial cells (BAEC) with A20 as well as reporter constructs consisting of the promoters of genes known to be up-regulated during endothelial cell activation, i.e. E-selectin, interleukin (IL)-8, tissue factor (TF), and inhibitor of nuclear factor kappaBalpha (IkappaBalpha), we demonstrate that A20 expression inhibits gene up-regulation associated with TNF, lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA), and hydrogen peroxide (H2O2)-induced endothelial cell (EC) activation. The mechanism of action of A20 is in part, or totally, due to the blockade of nuclear factor kappaB (NF-kappaB), as shown by its ability to suppress the activity of a NF-kappaB reporter. This effect is specific, as A20 does not block a noninducible, constitutively expressed reporter, Rous sarcoma virus-luciferase (RSV-LUC); nor does it block the c-Tat-inducible, NF-kappaB-independent reporter, human immunodeficiency virus-chloramphenicol acetyltransferase (HIV-CAT). How A20 blocks NF-kappaB is unclear, although we demonstrate that it does not affect p65 (RelA)-mediated gene transactivation. The inhibition of endothelial cell activation by A20 is a novel function for A20.

To become insulin independent, patients with type 1 diabetes mellitus require transplantation of at least two donor pancreata because of massive β–cell loss in the early post‐transplantation period. Many studies describing the introduction of new immunosuppressive protocols have shown that this loss is due to not only immunological events but also nonimmunological factors. To test to what extent hypoxia may contribute to early graft loss, we analyzed the occurrence of apoptotic events and the expression of hypoxia‐inducible factor 1 (HIF‐1), a heterodimeric transcription factor consisting of an oxygen‐dependent α subunit and a constitutive β subunit. Histological analysis of human and rat islets revealed nuclear pyknosis as early as 6 h after hypoxic exposure (1% O ₂ ). Moreover, immunoreactivity to activated caspase‐3 was observed in the core region of isolated human islets. Of note, both of these markers of apoptosis topographically overlap with HIF‐1α immunoreactivity. HIF‐1α mRNA was detected in islets from human and rat as well as in several murine β–cell lines. When exposed to hypoxia, mouse insulinoma cells (MIN6) had an increased HIF‐1α protein level, whereas its mRNA level did not alter. In conclusion, our data provide convincing evidence that reduced oxygenation is an important cause of β–cell loss and suggest that HIF‐1α protein level is an indicator for hypoxic regions undergoing apoptotic cell death. These observations suggest that gene expression under the control of HIF‐1 represents a potential therapeutic tool for improving engraftment of transplanted islets.

Repression of NFκB-dependent gene expression is one of the major elements of immunosuppression by glucocorticoids. Protein-protein interactions between the glucocorticoid receptor and NFκB have been characterized and shown to be a possible mechanism of mutual inhibition of transactivation properties. More recently, glucocorticoid-mediated induction of IκBα, an inhibitor of NFκB, has been described in monocytes and lymphocytes; an increase in IκBα mRNA and protein resulted in inactivation and cytosolic retention of NFκB. Thus, rather than the physical interaction between the glucocorticoid receptor and NFκB, the up-regulation of IκBα was presented as the key element in immunosuppression by glucocorticoids. In contrast, we show that the IκBα pathway is not involved in glucocorticoid-mediated inhibition of NFκB activity in endothelial cells. Although transcriptional activation by NFκB was significantly reduced in the presence of glucocorticoids, we did not detect induction of IκBα protein that could prevent nuclear translocation of NFκB upon stimulation with lipopolysaccharide or tumor necrosis factor α. Furthermore, treatment with glucocorticoids did not seem to affect the transcription rate or mRNA stability of IκBα. We therefore conclude that, although induction of IκBα expression by glucocorticoids seems to be of importance in monocytes and lymphocytes, it cannot explain inhibition of NFκB-dependent gene expression in endothelial cells. Our results emphasize the relevance of physical interaction between the glucocorticoid receptor and NFκB in endothelial cells and thus in suppression of inflammation by glucocorticoids.