Mi-Ae Yoo

Regulation and Destabilization of HIF-1α by ARD1-Mediated Acetylation

Abstract Hypoxia-inducible factor 1 (HIF-1) plays a central role in cellular adaptation to changes in oxygen availability. Recently, prolyl hydroxylation was identified as a key regulatory event that targets the HIF-1α subunit for proteasomal degradation via the pVHL ubiquitination complex. In this report, we reveal an important function for ARD1 in mammalian cells as a protein acetyltransferase by direct binding to HIF-1α to regulate its stability. We present further evidence showing that ARD1-mediated acetylation enhances interaction of HIF-1α with pVHL and HIF-1α ubiquitination, suggesting that the acetylation of HIF-1α by ARD1 is critical to proteasomal degradation. Therefore, we have concluded that the role of ARD1 in the acetylation of HIF-1α provides a key regulatory mechanism underlying HIF-1α stability.

Determination of hypoxic region by hypoxia marker in developing mouse embryos in vivo: A possible signal for vessel development

Hypoxia is a well‐known signal for angiogenesis, but the recent proposal that hypoxia exists in developing embryonic tissues and that it induces vascular development remains to be proven. In the present study, we demonstrate the presence of hypoxia in normal developing embryos by means of a hypoxia marker, pimonidazole, and its associated antibody. Our data clearly show that hypoxia marker immunoreactivity was highly detected in developing neural tubes, heart, and intersomitic mesenchyme at an early stage of organogenesis, suggesting that hypoxia may exist in the early stages of embryo development. We also found that hypoxia inducible factor‐1α (HIF‐1α) and vascular endothelial growth factor (VEGF) were spatiotemporally co‐localized with possible hypoxic regions in embryos. Investigation of platelet endothelial cell adhesion molecule (PECAM) expression provides evidence that endothelial cells proliferate and form the vessels in the hypoxic region in developing organs. Furthermore, we found that hypoxia induced both HIF‐1α and VEGF in F9 embryonic stem and differentiated cells. Thus, we suggest that hypoxia may exist widely in developing embryonic tissues and that it may act as a signal for embryonic blood vessel formation in vivo.

Visfatin enhances ICAM-1 and VCAM-1 expression through ROS-dependent NF-κB activation in endothelial cells

Visfatin has recently been identified as a novel visceral adipokine which may be involved in obesity-related vascular disorders. However, it is not known whether visfatin directly contributes to endothelial dysfunction. Here, we investigated the effect of visfatin on vascular inflammation, a key step in a variety of vascular diseases. Visfatin induced leukocyte adhesion to endothelial cells and the aortic endothelium by induction of the cell adhesion molecules, ICAM-1 and VCAM-1. Promoter analysis revealed that visfatin-mediated induction of CAMs is mainly regulated by nuclear factor-kappaB (NF-kappaB). Visfatin stimulated IkappaBalpha phosphorylation, nuclear translocation of the p65 subunit of NF-kappaB, and NF-kappaB DNA binding activity in HMECs. Furthermore, visfatin increased ROS generation, and visfatin-induced CAMs expression and NF-kappaB activation were abrogated in the presence of the direct scavenger of ROS. Taken together, our results demonstrate that visfatin is a vascular inflammatory molecule that increases expression of the inflammatory CAMs, ICAM-1 and VCAM-1, through ROS-dependent NF-kappaB activation in endothelial cells.

A protein kinase similar to MAP kinase activator acts downstream of the raf kinase in Drosophila

D-raf, a Drosophila homolog of Raf-1, plays key roles in multiple signal transduction pathways. Dsor1, a putative factor downstream of D-raf, was genetically identified by screening of dominant suppressors of D-raf. Dsor1Su1 mapped on X chromosome significantly suppressed the D-raf mutant phenotypes, and the loss-of-function mutations of Dsor1 showed phenotypes similar to those of the D-raf null mutations. Dsor1Su1 also significantly suppressed the mutations of other terminal class genes acting further upstream of D-raf. Molecular cloning of Dsor1 revealed its product with striking similarity to the microtubule-associated protein (MAP) kinase activator and yeast PBS2, STE7, and byr1. Our genetic results demonstrate the connection between raf and the highly conserved protein kinase cascade involving MAP kinase in vivo.

Age-related changes in Drosophila midgut are associated with PVF2, a PDGF/VEGF-like growth factor

Age‐associated changes in stem cell populations have been implicated in age‐related diseases, including cancer. However, little is known about the underlying molecular mechanisms that link aging to the modulation of adult stem cell populations. Drosophila midgut is an excellent model system for the study of stem cell renewal and aging. Here we describe an age‐related increase in the number and activity of intestinal stem cells (ISCs) and progenitor cells in Drosophila midgut. We determined that oxidative stress, induced by paraquat treatment or loss of catalase function, mimicked the changes associated with aging in the midgut. Furthermore, we discovered an age‐related increase in the expression of PVF2, a Drosophila homologue of human PDGF/VEGF, which was associated with and required for the age‐related changes in midgut ISCs and progenitor cell populations. Taken together, our findings suggest that PDGF/VEGF may play a central role in age‐related changes in ISCs and progenitor cell populations, which may contribute to aging and the development of cancer stem cells.

Hypoxic induction of human visfatin gene is directly mediated by hypoxia-inducible factor-1

Visfatin has been originally identified as a growth factor for early stage B cells and recently known as an adipokine. Here, we report that hypoxia induces the visfatin mRNA and protein levels in MCF7 breast cancer cells. We also demonstrate that induction of visfatin gene is regulated by hypoxia‐inducible factor‐1α (HIF‐1α). Moreover, 5′‐flanking promoter region of human visfatin gene contains two functional HIF responsive elements (HREs), activating the expression of visfatin. Mutation of these HREs in the visfatin promoter abrogates activation of a luciferase reporter gene driven by visfatin promoter under hypoxia. Taken together, our results demonstrate that visfatin is a new hypoxia‐inducible gene of which expression is stimulated through the interaction of HIF‐1 with HRE sites in its promoter region.

APOPTOTIC ACTIVITY OF URSOLIC ACID MAY CORRELATE WITH THE INHIBITION OF INITIATION OF DNA REPLICATION

Ursolic acid (UA), a pentacyclic triterpene acid, has been reported to exhibit anti‐tumor activity. In this study, we investigated the pro‐apoptotic effect of UA on HepG2 human hepatoblastoma cells. Treatment with UA decreased the viability of HepG2 cells in a concentration‐ and time‐dependent manner. Furthermore, 30 μM of UA induced DNA fragmentation and subdiploid cells and enhanced the release of cytochrome c and the activation of caspase‐3. These results suggest that UA induces cell death through apoptosis, which may be mediated by cytochrome c‐dependent caspase‐3 activation. In addition, cell‐cycle analysis revealed that UA‐treated cells were arrested predominantly in the G0 and G1 phases with a concomitant decrease in the cell population of S phase. Moreover, expression of p21WAF1, a cell‐cycle regulator, was increased by UA, indicating that p21WAF1 might mediate UA‐induced cell‐cycle arrest. However, UA markedly inhibited SV40 DNA replication in the initiation stage in vitro and significantly reduced the DNA cleaving of topoisomerase I and the ssDNA binding activity of replication protein A. These results indicate that the inhibition of DNA replication by UA may result from blockade of the establishment of the replication fork during initiation stage, consequently contributing to UA‐induced cell‐cycle arrest. Taken together, we suggest that UA‐induced cell‐cycle arrest may be mediated by inhibition of DNA replication and the increase of p21WAF1 expression, which induces the release of cytochrome c and the activation of caspase‐3, leading to apoptosis of HepG2 cells. Int. J. Cancer 87:629–636, 2000.

Regional difference of ROS generation, lipid peroxidaton, and antioxidant enzyme activity in rat brain and their dietary modulation

One of the potential causes of age-related neuronal damage can be reactive oxygen species (ROS), as the brain is particularly sensitive to oxidative damage. In the present study, we investigated the effects of aging and dietary restriction (DR) on ROS generation, lipid peroxidation, and antioxidant enzymes in cerebrum, hippocampus, and cerebellum of 6-, 12-, 18-, and 24-month-old rats. ROS generation significantly increased with age in cerebrum ofad libitum (AL) rats. However, no significant age-difference was observed in hippocampus and cerebellum. DR significantly decreased ROS generation in cerebrum and cerebellum at 24-months. On the other hand, the increased lipid peroxidation of AL rats during aging was significantly reduced by DR in all regions. Our results further showed that catalase activity decreased with age in cerebellum of AL rats, which was reversed by DR, although SOD activity had little change by aging and DR in all regions. In a similar way, glutathione (GSH) peroxidase activity increased with age in cerebrum of AL rats, while DR suppressed it at 24-months. These data further support the evidence that the vulnerability to oxidative stress in the brain is region-specific.

The Homeobox Gene Caudal Regulates Constitutive Local Expression of Antimicrobial Peptide Genes in Drosophila Epithelia

ABSTRACT In Drosophila melanogaster, although the NF-κB transcription factors play a pivotal role in the inducible expression of innate immune genes, such as antimicrobial peptide genes, the exact regulatory mechanism of the tissue-specific constitutive expression of these genes in barrier epithelia is largely unknown. Here, we show that the Drosophila homeobox gene product Caudal functions as the innate immune transcription modulator that is responsible for the constitutive local expression of antimicrobial peptides cecropin and drosomycin in a tissue-specific manner. These results suggest that certain epithelial tissues have evolved a unique constitutive innate immune strategy by recruiting a developmental “master control” gene.

The inflammatory process in aging

Aging processes are time-dependent, deteriorative functional changes. These functional changes lead to a progressive loss of the organism’s ability to withstand both internal and environmental stresses, causing the failure of cellular homeostasis. Among the modern hypotheses, the ‘Oxidative Stress Hypothesis’ offers the best mechanistic elucidation of aging phenomena. Based on the ‘Free Radical Theory of Aging’, this hypothesis has gained popularity among researchers in the field of gerontology as well as other biomedical fields. Its primary premise proposes that aging and its related disease processes are the net result of free radical-induced damage, asserting further that an organism’s inability to produce counterbalancing antioxidative defences, i.e. defences that offset disturbances in the redox state, underlies its cause.