Effect of hypoxia on gene expression by human hepatocytes (HepG2)

Abstract

The full extent to which hypoxia produces gene expression changes in human cells is unknown. We used late-generation oligonucleotide arrays to catalog hypoxia-induced changes in gene expression in HepG2 cells. Five paired sets of cultures were subjected to either control (room air-5% CO(2)) or hypoxic (1% O(2)-5% CO(2)) conditions for 24 h, and RNA was analyzed on an Affymetrix cDNA array containing approximately 12,600 sequences. A statistically significant change in expression was shown by 2,908 sequences (1,255 increased and 1,653 decreased). The observed changes were highly concordant with published literature on hypoxic stress but showed relatively little overlap (12-22%) with changes in gene expression that have been reported to occur after heat stress in other systems. Of note, of these 2,908 sequences, only 387 (213 increased and 174 decreased) both exhibited changes in expression of twofold or greater and were highly expressed in at least three of the five experiments. We conclude that the effect of hypoxia on gene expression by HepG2 cells is broad, has a significant component of downregulation, and includes a relatively small number of genes whose response is truly independent of cell and stress type.

Global Gene Expression Analysis of ERK5 and ERK1/2 Signaling Reveals a Role for HIF-1 in ERK5-mediated Responses

“…Significantly, of the 17 genes controlled by ERK5 alone, 14 were found by literature search to be regulated in response to hypoxia ( Table 1). Most of these were previously validated by experiments demonstrating a link to transcription by HIF-1, a basic helix-loop-helix factor that promotes transcription under conditions of low oxygen tension (55,(62)(63)(64)(65). For example, of the set of 14 ERK5-responsive genes, 9 were induced or blocked upon wild-type HIF-1␣ or dominant-negative HIF-1␣ overexpression, respectively, in cell lines (bHLHB3, PFKFB3, PFKFB4, ADM, CA9, PPP1R3C, SLC2A3, HIG2, and CCNG2, see Table 1), and 4 were downregulated upon homologous recombination to delete expression of HIF-1␣ or -␤ (CCNG2, ADM, DDIT4, and SLC2A3; see Table 1).…”

A microarray analysis of the effects of moderate hypothermia and rewarming on gene expression by human hepatocytes (HepG2)

“…In previous experiments comparing a single stress condition to a control, we used T-distribution-derived 95% confidence intervals on natural log-transformed expression ratios to identify statistically significant changes in expression, followed by aggressive post-hoc filtering to reduce the number of false-positive reports (Sonna et al 2003(Sonna et al , 2002bWood et al 2004). While this approach is highly efficient at generating findings that can be reproduced by reverse transcription PCR, it cannot easily be applied to multifactorial experimental designs such as the one reported here.…”

“…The heat shock conditions were experimentally conventional (6°C over normal culture temperatures for 30 min) and have been shown to produce a vigorous heat shock response in HepG2 cells (Sonna et al 2003).…”

“…Supplemental Table 2 details the effect of moderate hypothermia and heat shock on a list of such control sequences, including beta-actin, GAPDH, cyclophilin A, a number of other sequences found in previous gene chip array experiments to demonstrate stable expression after heat shock (Sonna et al 2002b) and hypoxia (Sonna et al 2003), and several of the ribosomal proteins most highly expressed in this cell line as determined by signal intensity on the microarrays. Of the 19 genes represented by the 25 sequences in this table, only three (beta-actin, GAPDH, and ribosomal protein L30)…”

“…HepG2 cells were selected because this cell line is derived from a solid organ that is involved in a wide variety of metabolic processes (and as such, expresses a large number of genes that are potentially inducible or repressible) and in our hands has demonstrated robust changes in gene expression in response to environmental stressors such as hypoxia and heat shock (Sonna et al 2003). Furthermore, unlike our previous microarray study in THP-1 cells, we chose to look at the gene expression changes that occur both during hypothermia itself and after a period of recovery at 37°C.…”