Analysis of the heat shock response in mouse liver reveals transcriptional dependence on the nuclear receptor peroxisome proliferator-activated receptor alpha (PPARalpha)

Abstract

Background: The nuclear receptor peroxisome proliferator-activated receptor alpha (PPARalpha) regulates responses to chemical or physical stress in part by altering expression of genes involved in proteome maintenance. Many of these genes are also transcriptionally regulated by heat shock (HS) through activation by HS factor-1 (HSF1). We hypothesized that there are interactions on a genetic level between PPARalpha and the HS response mediated by HSF1.

Results: Wild-type and PPARalpha-null mice were exposed to HS, the PPARalpha agonist WY-14,643 (WY), or both; gene and protein expression was examined in the livers of the mice 4 or 24 hrs after HS. Gene expression profiling identified a number of Hsp family members that were altered similarly in both mouse strains. However, most of the targets of HS did not overlap between strains. A subset of genes was shown by microarray and RT-PCR to be regulated by HS in a PPARalpha-dependent manner. HS also down-regulated a large set of mitochondrial genes specifically in PPARalpha-null mice that are known targets of PPARgamma co-activator-1 (PGC-1) family members. Pretreatment of PPARalpha-null mice with WY increased expression of PGC-1beta and target genes and prevented the down-regulation of the mitochondrial genes by HS. A comparison of HS genes regulated in our dataset with those identified in wild-type and HSF1-null mouse embryonic fibroblasts indicated that although many HS genes are regulated independently of both PPARalpha and HSF1, a number require both factors for HS responsiveness.

Conclusions: These findings demonstrate that the PPARalpha genotype has a dramatic effect on the transcriptional targets of HS and support an expanded role for PPARalpha in the regulation of proteome maintenance genes after exposure to diverse forms of environmental stress including HS.

Figure 1

Altered gene expression by heat shock and WY in wild-type and PPARα-null mice. Mice were fed a control diet or a diet containing WY for 7 days. Groups of mice were subjected to a 42°C HS for 40 min or kept at room temperature. Mice were sacrificed 4 hrs after HS and hepatic mRNA levels were assessed in the livers. A. Principle component analysis. B. Heat map of altered gene expression. Genes were subjected to one-dimensional hierarchical clustering. Red, up-regulation; green, down-regulation; black, no change. The intensity scale indicates fold-change due to chemical exposure relative to controls. Abbreviations: H, heat shock; W, WY-14,643.

Figure 2

Heat shock alters the expression of different sets of genes in wild-type and PPARα-null mice. A. Direct comparison of HS genes in wild-type and PPARα-null mice. The relatively small number of genes which exhibited altered expression in both strains are shown in detail. B. Different classes of HS genes. The genes altered by HS were divided into six classes (I - VI) based on expression in wild-type and PPARα-null mice and comparison with WY. The genes whose expression was confirmed by TaqMan are underlined. Red, up-regulation; green, down-regulation; black, no change. The intensity scales indicates fold-change due to exposure relative to controls. C. Altered expression of genes assessed by TaqMan. There were four animals per treatment group, and each sample was analyzed in duplicate. Variability is expressed as standard error of the mean. Means and S.E. (n = 4) for RT-PCR data were calculated by Student's t test. The level of significance was set at p ≤ 0.05.

Figure 3

PPARα-independent changes in triglyceride levels and fatty acid metabolism genes by heat shock. A. Serum levels of alkaline phosphatase, glucose and triglycerides 4 and 24 hrs after HS. B. Decreases in fatty acid metabolism genes by HS in wild-type and PPARα-null mice. Genes called as significantly altered between control and HS groups were extracted from GSEA gene sets HSA03320_PPAR_SIGNALING_PATHWAY (wild-type mice) and HSA00071_FATTY_ACID_METABOLISM (PPARα-null mice).

Figure 4

Expression of Acox1 and Cyp4a proteins after WY and heat shock in livers and kidneys. A. Expression of ACO and Cyp4a in livers of wild-type or PPARα-null mice on control or WY diet, 24 hrs after HS or mock HS. Expression was assessed by Western blot using primary antibodies against the indicated proteins. B. Quantitation of the western blots in C. C. Altered protein expression in the kidneys of wild-type or PPARα-null mice on control or WY diet, 24 hrs after HS or mock HS. Expression was assessed by Western blot using primary antibodies against the indicated proteins.

Figure 5

Expression of chaperone proteins after heat shock or PPC treatment. A. Expression of Hsp in the livers of mice. Proteins were extracted from the livers of wild-type or PPARα-null mice on control or WY diet, 24 hrs after HS or mock HS. Expression was assessed by Western blot using primary antibodies against the indicated proteins. B and C. Quantitation of the western blots in A. D. Altered protein expression in the kidneys of wild-type and PPARα-null mice exposed to HS or WY. Protein expression was assessed in the kidneys of the mice described in A.

Figure 6

Global decreases in mitochondrial gene expression in PPARα-null mice after heat shock are prevented by WY pretreatment. A. Down-regulation of genes regulated by the co-activator PGC-1. Gene Set Enrichment Analysis (GSEA) was used to identify gene sets that exhibited significant overlaps with those gene differences between control and HS in PPARα-null mice. Left, enrichment plot for genes regulated by PGC-1. Black bars illustrate the position of probe sets belonging to the PGC-1 gene set in the context of all probes on the U74Av2 array. The running enrichment score (RES) plotted as a function of the position within the ranked list of array probes is shown as a green line. The ranked list metric shown in gray illustrates the correlation between the signal to noise values of all individually ranked genes according to the class labels (experimental conditions). Right, individual expression profiles for leading edge probe sets contributing to the normalized enrichment score are shown. Signal intensities are illustrated by varying shades of red (up-regulation) and blue (down-regulation). B. Prevention of down-regulation of electron transporter gene expression by pretreatment with WY. Left, enrichment plot for genes with electron transporter activity as described in A. Right, individual expression profiles for probe sets contributing to the normalized enrichment score are shown. C. Increased expression of PGC1β and regulated genes involved in lipid homeostasis by WY in PPARα-null mice. GSEA-derived heat map of the top 100 differentially expressed probe sets enriched in the control or WY-treated groups from PPARα-null mice. Location of PGC1β (Ppargc1b) and regulated genes are indicated. D. Increased expression of genes involved in fatty acid metabolism after exposure to WY for 5 days in PPARα-null mice. Genes significantly altered in the GSEA set "fatty acid metabolism" are shown. These include a number that overlap with those that are involved in mitochondrial fatty acid metabolism regulated by PGC-1.

Figure 7

Regulation of heat shock genes by HSF1 and PPARα. Genes which exhibited significant changes in expression due to HS from the dataset of Trinklein et al. (2004) and from the present study were identified. A. Heat map of gene expression changes by HS in wild-type (W) and PPARα-null (N) mice compared to the Trinklein et al. (2004) dataset. In the Trinklein et al. (2004) study, mouse embryonic fibroblasts were subjected to HS followed by recovery for the indicated times in hrs. Genes were subjected to one-dimensional hierarchical clustering. Red, up-regulation; green, down-regulation; grey, no data; black, no change. B. Classification of genes based on regulation by PPARα and HSF1.