Yunwon Moon

Hypoxia-inducible Factor-2α-dependent Hypoxic Induction of Wnt10b Expression in Adipogenic Cells

Background: In obesity, enlarged adipocytes become hypoxic, which inhibits adipocyte differentiation. Results: Hypoxia induces the expression of Wnt1 and Wnt10b in both human and mouse adipogenic cells in a hypoxia-inducible factor (HIF)-2α-dependent manner. Conclusion: Hypoxia enhances the secretion of Wnt ligands, which trigger Wnt signaling in the neighboring cells. Significance: Wnt10b is a novel HIF-2α-specific target gene and a paracrine factor in hypoxia. Adipocyte hyperplasia and hypertrophy in obesity can lead to many changes in adipose tissue, such as hypoxia, metabolic dysregulation, and enhanced secretion of cytokines. In this study, hypoxia increased the expression of Wnt10b in both human and mouse adipogenic cells, but not in hypoxia-inducible factor (HIF)-2α-deficient adipogenic cells. Chromatin immunoprecipitation analysis revealed that HIF-2α, but not HIF-1α, bound to the Wnt10b enhancer region as well as upstream of the Wnt1 gene, which is encoded by an antisense strand of the Wnt10b gene. Hypoxia-conditioned medium (H-CM) induced phosphorylation of lipoprotein-receptor-related protein 6 as well as β-catenin-dependent gene expression in normoxic cells, which suggests that H-CM contains canonical Wnt signals. Furthermore, adipogenesis of both human mesenchymal stem cells and mouse preadipocytes was inhibited by H-CM even under normoxic conditions. These results suggest that O2 concentration gradients influence the formation of Wnt ligand gradients, which are involved in the regulation of pluripotency, cell proliferation, and cell differentiation.

Chenodeoxycholic Acid Reduces Hypoxia Inducible Factor-1α Protein and Its Target Genes

This study evaluated HIF-1α inhibitors under different hypoxic conditions, physiological hypoxia (5% O2) and severe hypoxia (0.1% O2). We found that chenodeoxy cholic acid (CDCA) reduced the amount of HIF-1α protein only under physiological hypoxia but not under severe hypoxia without decreasing its mRNA level. By using a proteasome inhibitor MG132 and a translation inhibitor cyclohexamide, we showed that CDCA reduced HIF-1α protein by decreasing its translation but not by enhancing its degradation. The following findings indicated that farnesoid X receptor (FXR), a CDCA receptor and its target gene, Small heterodimer partner (SHP) are not involved in this effect of CDCA. Distinctly from CDCA, MG132 prevented SHP and an exogenous FXR agonist, GW4064 from reducing HIF-1α protein. Furthermore a FXR antagonist, guggulsterone failed to prevent CDCA from decreasing HIF-1α protein. Furthermore, guggulsterone by itself reduced HIF-1α protein even in the presence of MG132. These findings suggested that CDCA and guggulsterone reduced the translation of HIF-1α in a mechanism which FXR and SHP are not involved. This study reveals novel therapeutic functions of traditional nontoxic drugs, CDCA and guggulsterone, as inhibitors of HIF-1α protein.

Hypoxic repression of CYP7A1 through a HIF-1α- and SHP-independent mechanism

Liver cells experience hypoxic stress when drug-metabolizing enzymes excessively consume O2 for hydroxylation. Hypoxic stress changes the transcription of several genes by activating a heterodimeric transcription factor called hypoxia-inducible factor-1α/β (HIF-1α/β). We found that hypoxic stress (0.1% O2) decreased the expression of cytochrome P450 7A1 (CYP7A1), a rate-limiting enzyme involved in bile acid biosynthesis. Chenodeoxycholic acid (CDCA), a major component of bile acids, represses CYP7A1 by activating a transcriptional repressor named small heterodimer partner (SHP). We observed that hypoxia decreased the levels of both CDCA and SHP, suggesting that hypoxia repressed CYP7A1 without inducing SHP. The finding that overexpression of HIF-1α increased the activity of the CYP7A1 promoter suggested that hypoxia decreased the expression of CYP7A1 in a HIF-1-independent manner. Thus, the results of this study suggested that hypoxia decreased the activity of CYP7A1 by limiting its substrate O2, and by decreasing the transcription of CYP7A1. [BMB Reports 2016; 49(3): 173-178]

Abstract 122: Hypoxia-enhanced proliferation and effector function of NK cells

Hypoxia promotes tumor cell survival while providing immune cell shaping and resistance. Although NK cells function within hypoxic lymphatics and inflamed tissues, they were shown to mount impaired anti-tumor effector functions in ex-vivo hypoxic environment. This raises a question that additional cues exist in vivo to allow full NK cell activation. By varying the percentage and time of pO 2 exposure ex-vivo, we were able to fine-tune a condition that reverses hypoxia-induced NK cell suppression to become highly proliferative and enhanced cytolytic phenotypes. We found that exposing NK cells to moderate hypoxia in the presence of IL-2 and feeder cells allowed HIF-1α stabilization and its target gene expression, metabolic changes toward glycolysis, specific upregulation of NKp44 receptor, and increased STAT3 phosphorylation with concomitant reduction of apoptosis and p16 senescence pathways. Therefore, the hypoxic exposure allows HIF-1α-mediated adaptation of mature NK cells toward highly proliferative and cytolytic NK phenotypes, which may occur in vivo at the time of tumor growth and inflammation. [This work was supported by the National Research Foundation of Korea (NRF), grants NRF-2013M3A9D3046248] Citation Format: Yunwon Moon, Seon Ah Lim, Kyung-Mi Lee, Hyunsung Park. Hypoxia-enhanced proliferation and effector function of NK cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 122.

Distinct hypoxic regulation of preadipocyte factor-1 (Pref-1) in preadipocytes and mature adipocytes

Preadipocyte factor-1 (Pref-1) is a secretory soluble protein, which exerts pleiotropic effects on maintenance of cancer stem cell characteristics and commitment of mesenchymal stem cell lineages by inhibiting adipogenesis. Observations that obesity renders the microenvironment of adipose tissues hypoxic and that hypoxia inhibits adipogenesis lead us to investigate whether hypoxia increases the expression of anti-adipogenic Pref-1 in preadipocytes, mature adipocytes, and adipose tissues from obese mouse. In 3T3-L1 preadipocytes, hypoxia induces Pref-1 by a hypoxia-inducible factor 1 (HIF-1)-dependent mechanism accompanied by increase in the levels of the active histone mark, acetylated H3K9/14 (H3K9/14Ac). Adipogenesis increased the levels of the heterochromatin histone mark, trimethylated H3K27 (H3K27me3), whereas it decreased the levels of H3K4me3 and H3K9/14Ac euchromatin marks of the mouse Pref-1 promoter. However, differently from preadipocytes, in mature adipocytes hypoxia failed to reverse the repressive epigenetic changes of Pref-1 promoter and to increase its expression. Short term (8weeks) high fat diet (HFD) increased HIF-1α protein in subcutaneous and epididymal adipose tissues, but did not increase Pref-1 expression. Unlike in 3T3-L1 preadipocytes, HIF-1α did not increase Pref-1 expression in adipose tissues in which mature adipocytes constitute the main population. Interestingly, long term (35weeks) HFD increased Pref-1 in serum but not in obese adipose tissues. This study suggests that Pref-1 is an endocrine factor which is synergistically increased by obesity and age.

Multi-dimensional histone methylations for coordinated regulation of gene expression under hypoxia

Abstract Hypoxia increases both active and repressive histone methylation levels via decreased activity of histone demethylases. However, how such increases coordinately regulate induction or repression of hypoxia-responsive genes is largely unknown. Here, we profiled active and repressive histone tri-methylations (H3K4me3, H3K9me3, and H3K27me3) and analyzed gene expression profiles in human adipocyte-derived stem cells under hypoxia. We identified differentially expressed genes (DEGs) and differentially methylated genes (DMGs) by hypoxia and clustered the DEGs and DMGs into four major groups. We found that each group of DEGs was predominantly associated with alterations in only one type among the three histone tri-methylations. Moreover, the four groups of DEGs were associated with different TFs and localization patterns of their predominant types of H3K4me3, H3K9me3 and H3K27me3. Our results suggest that the association of altered gene expression with prominent single-type histone tri-methylations characterized by different localization patterns and with different sets of TFs contributes to regulation of particular sets of genes, which can serve as a model for coordinated epigenetic regulation of gene expression under hypoxia.