Translational and transcriptional responses in human primary hepatocytes under hypoxia

Abstract

The liver is the primary source of a large number of plasma proteins and plays a critical role in multiple biological processes. Inadequate oxygen supply characterizing various clinical settings such as liver transplantation exposes the liver to hypoxic conditions. Studies assessing hypoxia-induced global translational changes in liver are lacking. Here, we employed a recently developed ribosome-profiling technique to assess global translational responses of human primary hepatocytes exposed to acute hypoxic stress (1% O2) for the short term. In parallel, transcriptome profiling was performed to assess mRNA expression changes. We found that translational responses appeared earlier and were predominant over transcriptional responses. A significant decrease in translational efficiency of several ribosome genes indicated translational inhibition of new ribosome protein synthesis in hypoxia. Pathway enrichment analysis highlighted altered translational regulation of MAPK signaling, drug metabolism, oxidative phosphorylation, and nonalcoholic fatty liver disease pathways. Gene Ontology enrichment analysis revealed terms related to translation, metabolism, angiogenesis, apoptosis, and response to stress. Transcriptional induction of genes encoding heat shock proteins was observed within 30 min of hypoxia. Induction of genes encoding stress response mediators, metabolism regulators, and proangiogenic proteins was observed at 240 min. Despite the liver being the primary source of coagulation proteins and the implicated role of hypoxia in thrombosis, limited differences were observed in genes encoding coagulation-associated proteins. Overall, our study demonstrates the predominance of translational regulation over transcription and highlights differentially regulated pathways or biological processes in short-term hypoxic stress responses of human primary hepatocytes.NEW & NOTEWORTHY The novelty of this study lies in applying parallel ribosome- and transcriptome-profiling analyses to human primary hepatocytes in hypoxia. To our knowledge, this is the first study to assess global translational responses using ribosome profiling in hypoxic hepatocytes. Our results demonstrate the predominance of translational responses over transcriptional responses in early hepatic hypoxic stress responses. Furthermore, our study reveals multiple pathways and specific genes showing altered regulation in hypoxic hepatocytes.

Hypoxic gene expression in chronic hepatitis B virus infected patients is not observed in state-of-the-art in vitro and mouse infection models

“…In this study we identified increased hypoxia gene signatures in a CHB cohort in the absence of cirrhosis or HCC. We confirmed hypoxic gene expression using MSigDB derived gene sets and recently reported signatures from HepG2 44 and PHH data sets 45 . We noted an increase in HIF-1α mRNA levels, consistent with their transcriptional regulation by inflammatory mediators such as TNFα.…”

“…1b). To further validate these results, we analysed the acute transcriptional response of primary human hepatocytes (PHHs) 45 cultured under 1% oxygen for 4 h and identified 113 upregulated genes (FC > 2; FDR < 0.05) and GSEA showed an enrichment in CHB ( Supplementary Fig. 2a).…”

“…GSEA was performed with GSEA_4.0.3 71 using publicly available Affymetrix microarray datasets 41 , 49 from Gene Expression Omnibus (GEO) (accessions: GSE83148, GSE14323). Gene sets were derived from HepG2 RNA-seq data 44 downloaded from GEO (GSE120886) or PHH RNA-Seq accessed from the published Supplementary Material 45 . To determine whether HBV infection induces hypoxia-responsive genes, we interrogated the mRNA expression patterns of the liver chimeric mice RNA-Seq dataset for the top 80 hypoxia-induced genes as identified in HepG2 hepatic cells 44 .…”

Ribosome profiling of HEK293T cells overexpressing codon optimized coagulation factor IX

“…Ribosome profiling was conducted as described previously 7 using the Illumina TruSeq Ribo Profile (Mammalian) Kit according to manufacturer's instructions with modifications in harvest, RNA isolation/purification (isopropanol isolation used to improve the yield) and ribosome protected fragments size selection (~20-32 nt). During harvest, media was carefully removed, and cells were immediately flash-frozen.…”

Ribosome profiling of HEK293T cells overexpressing codon optimized coagulation factor IX

“…Since there are several other HEK293T ribosomal profiling datasets available, these could be used to examine the reproducibility of the results 6 . Furthermore, by looking into ribosome profiling datasets from other cell types, such as other human cells 7 and/or across species, it would be valuable to examine whether a given gene maintains the same translation kinetics or if there are significant differences that could reflect on the conformation of the protein. Clearly, since a rather large inter-experiment variation is expected, the accumulation of several ribosome profiling databases would be very useful for this type of analysis.…”