Hypoxia induces transcription of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-4 gene via hypoxia-inducible factor-1α activation

AbstractThe PFKFB4 gene encodes isoenzyme of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB or PFK-2/FBPase-2) which originally was found in the testes. We have studied hypoxic regulation of PFKFB4 gene in prostate cancer cell line, PC-3, and several other cancer cell lines. It was shown that hypoxia significantly induced PFKFB4 mRNA levels in PC-3 as well as in HeLa, Hep3B and HepG2 cell lines. Hypoxia increased PFKFB4 protein levels also. Moreover, desferrioxamine and cobalt chloride, which are known to mimic hypoxia, also had a stimulatory effect on the expression of PFKFB4 mRNA. In order to investigate the mechanisms of hypoxic regulation of PFKFB4 gene expression, we used dimethyloxalylglycine, which has the ability to mimic effect of hypoxia by significant induction of hypoxia-inducible factor (HIF-1α) protein levels. Our studies showed that PFKFB4 mRNA expression in PC-3, HeLa, Hep3B and HepG2 cell lines was highly responsive to dimethyloxalylglycine, an inhibitor of HIF-1α hydroxylase enzymes, suggesting that the hypoxia responsiveness of this gene is regulated by HIF proteins. To better understand the hypoxic regulation of PFKFB4 gene expression, we isolated genomic DNA, which includes the promoter region of PFKFB4. Cell transfection, deletion and site-specific mutagenesis of the PFKFB4 promoter region indicates that hypoxic induction of PFKFB4 gene expression is mediated by the hypoxia-responsive element (HRE). These experiments identified a HRE 422–429 bp upstream from the translation start site. Thus, our results indicate that testis-specific form of PFKFB or PFK-2/FBPase-2 is also expressed in several cancer cell lines and that hypoxia induces transcription of PFKFB4 gene in these cell lines by HIF-1α dependent mechanism. HRE in 5′-promoter region of PFKFB4 gene mediates hypoxic induction of PFKFB4 gene transcription

Identification of hypoxia-inducible factor 1 ancillary sequence and its function in vascular endothelial growth factor gene induction by hypoxia and nitric oxide.

Transcription of hypoxia-inducible genes is regulated by hypoxia response elements (HREs) located in either the promoter or enhancer regions. Analysis of these elements reveals the presence of one or more binding sites for hypoxia-inducible factor 1 (HIF-1). Hypoxia-inducible genes include vascular endothelial growth factor (VEGF), erythropoietin, and glycolytic enzyme genes. Site-directed mutational analysis of the VEGF gene promoter revealed that an HIF-1 binding site (HBS) and its downstream HIF-1 ancillary sequence (HAS) within the HRE are required as cis-elements for the transcriptional activation of VEGF by either hypoxia or nitric oxide (NO). The core sequences of the HBS and the HAS were determined as TACGTG and CAGGT, respectively. These elements form an imperfect inverted repeat, and the spacing between these motifs is crucial for activity of the promoter. Gel shift assays demonstrate that as yet unknown protein complexes constitutively bind to the HAS regardless of the presence of these stimuli in several cell lines, in contrast with hypoxia- or NO-induced activation of HIF-1 binding to the HBS. A common structure of the HRE, which consists of the HBS and the HAS, is seen among several hypoxia-inducible genes, suggesting the presence of a novel mechanism mediated by the HAS for the regulation of these genes

Identification of a novel protein kinase mediating Akt survival signaling to the ATM protein

We identified a novel human AMP-activated protein kinase (AMPK) family member, designated ARK5, encoding 661 amino acids with an estimated molecular mass of 74 kDa. The putative amino acid sequence reveals 47, 45.8, 42.4, and 55% homology to AMPK-alpha1, AMPK-alpha2, MELK and SNARE respectively, suggesting that it is a new member of the AMPK family. It has a putative Akt phosphorylation motif at amino acids 595600, and Ser(600) was found to be phosphorylated by active Akt resulting in the activation of kinase activity toward the SAMS peptide, a consensus AMPK substrate. During nutrient starvation, ARK5 supported the survival of cells in an Akt-dependent manner. In addition, we also demonstrated that ARK5, when activated by Akt, phosphorylated the ATM protein that is mutated in the human genetic disorder ataxia-telangiectasia and also induced the phosphorylation of p53. On the basis of our current findings, we propose that a novel AMPK family member, ARK5, is the tumor cell survival factor activated by Akt and acts as an ATM kinase under the conditions of nutrient starvation

The whole blood transcriptional regulation landscape in 465 COVID-19 infected samples from Japan COVID-19 Task Force

「コロナ制圧タスクフォース」COVID-19患者由来の血液細胞における遺伝子発現の網羅的解析 --重症度に応じた遺伝子発現の変化には、ヒトゲノム配列の個人差が影響する--. 京都大学プレスリリース. 2022-08-23.Coronavirus disease 2019 (COVID-19) is a recently-emerged infectious disease that has caused millions of deaths, where comprehensive understanding of disease mechanisms is still unestablished. In particular, studies of gene expression dynamics and regulation landscape in COVID-19 infected individuals are limited. Here, we report on a thorough analysis of whole blood RNA-seq data from 465 genotyped samples from the Japan COVID-19 Task Force, including 359 severe and 106 non-severe COVID-19 cases. We discover 1169 putative causal expression quantitative trait loci (eQTLs) including 34 possible colocalizations with biobank fine-mapping results of hematopoietic traits in a Japanese population, 1549 putative causal splice QTLs (sQTLs; e.g. two independent sQTLs at TOR1AIP1), as well as biologically interpretable trans-eQTL examples (e.g., REST and STING1), all fine-mapped at single variant resolution. We perform differential gene expression analysis to elucidate 198 genes with increased expression in severe COVID-19 cases and enriched for innate immune-related functions. Finally, we evaluate the limited but non-zero effect of COVID-19 phenotype on eQTL discovery, and highlight the presence of COVID-19 severity-interaction eQTLs (ieQTLs; e.g., CLEC4C and MYBL2). Our study provides a comprehensive catalog of whole blood regulatory variants in Japanese, as well as a reference for transcriptional landscapes in response to COVID-19 infection

DOCK2 is involved in the host genetics and biology of severe COVID-19

「コロナ制圧タスクフォース」COVID-19疾患感受性遺伝子DOCK2の重症化機序を解明 --アジア最大のバイオレポジトリーでCOVID-19の治療標的を発見--. 京都大学プレスリリース. 2022-08-10.Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge. Here we conducted a genome-wide association study (GWAS) involving 2, 393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3, 289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target