Induction of a subgroup of acute phase protein genes in mouse liver by hyperthermia

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Abstract

We have demonstrated that two members of the acute phase reactant family of positively regulated genes, α1-acid glycoprotein (AGP-1 and AGP-2) and C-reactive protein (CRP) are induced by hyperthermia, while two others, the serum amyloid A (SAA) and α1-antitrypsin (AT) genes, are not. Albumin (ALB), a negative acute phase reactant gene, is also induced by hyperthermia. The AGP-1, AGP-2, and CRP genes require glucocorticoids, but not IL-6, IL-1β or TNFα in response to hyperthermia. As with LPS, the C/EBPβ mRNA levels increased, while the C/EBPα mRNA levels decreased in response to LPS. In contrast to the LPS response, C/EBPδ was unchanged. Protein pool levels and DNA-binding activities of the 35 and 20 kDa C/EBPβ isoforms increase, whereas protein pool levels of the 42 kDa C/EBPα decrease and the 30 kDa remained high. These studies suggest that the synthesis of specific C/EBPα and C/EBPβ isoforms is induced by hyperthermia, and that the regulation of the AGP-1 and AGP-2 genes during heat stress may involve one of these isoforms. The difference between the responses to hyperthermia and LPS is that the former, may not involve the participation of cytokines. Furthermore, since cis-acting heat shock elements (HSE) are located in the promoter regions of the ALB, CRP, and C/EBPβ genes, these regulatory sequences may be involved in the in vivo activation of these genes by hyperthermia.

Introduction

α1-Acid glycoprotein (AGP), serum amyloid A (SAA), and C-reactive protein (CRP) are major acute phase reactants whose synthesis is activated primarily in the liver by such inflammatory agents as bacterial lipopolysaccharide (LPS) and heavy metals 1, 2, 3, 4. The AGP gene(s) are transcriptionally activated by glucocorticoids and cytokines through the interaction of cis-acting DNA sequences, i.e. the acute phase responsive element (APRE), with C/EBPβ and glucocorticoid receptor (GR). The APRE is a compound binding site composed of overlapping glucocorticoid (GRE) and IL-6-responsive elements and is located in the promoter region from −104 to −130 bp 5, 6, 7, 8. Transient expression analyses using site-specific mutated expression vectors have suggested that the interaction of GR and C/EBPβ with the APRE is essential for maximal gene expression [7]. The complexity of the DNA–protein interactions at the APRE site in response to stress signals was further indicated by our studies suggesting that this site may be essential for the activation of AGP by heavy metals, and that this activation does not require GRE or cytokines (Yiangou, et al., unpublished data).

Heat shock proteins (HSP) are the products of a family of genes that are transcriptionally activated by hyperthermia and other stress factors such as LPS, heavy metals, and amino acid starvation 9, 10, 11, 12. Transcription of the HSP genes in response to hyperthermia is mediated by the interaction of heat shock trans-acting factors (HSFs) with heat shock elements (HSE) located in the promoters of responsive genes 13, 14. These HSFs are sequestered in the cytoplasm and, upon challenge by various stress factors, are translocated to the nucleus where they interact with cis-acting DNA sequences, i.e. the HSE. The sequences of these HSEs consist of alternating GAA or TTC blocks arranged at two-nucleotide intervals [15]. However, in higher eukaryotes only the G residue remains highly conserved and there are variant sequences, such as nGGGn, nGACn or nGGCn that also serve as contact sites for HSFs 13, 14. Sequence analysis of the 5′ flanking regions of mouse C/EBPβ, CRP, and albumin genes revealed the presence of HSE-like consensus sequences 16, 17, 18. On the basis of these sequence analyses, we conducted experiments to determine if hyperthermia can affect the expression of these specific members of the acute phase reactant gene family, as well as the C/EBP trans-acting factors that regulate their expression, and whether this response requires the interaction of such stress mediators as cytokines.

Section snippets

Animals

Normal and adrenalectomized Balb/c mice (4 month old, 25–30 g), were purchased from Charles River Breeding Laboratory and maintained under 12 h light–dark cycles. The adrenalectomized mice were provided free access to food and 0.9% NaCl for at least one week before experimental use. Exposure to elevated temperatures was achieved by placing animals in a positive forced air incubator at 40°C for varying times [10]. All experiments applying heat stress to mice were done from 07:00 to 08:30 A.M. The

Effects of hyperthermia on acute phase protein and heat shock protein gene expression in normal and adrenalectomized mice

Male mice (25–30 g) were exposed to 40–42.5°C for varying times, and 6 h post-treatment total hepatic RNA was analyzed for changes in the levels of AGP, SAA and CRP mRNA pools. Mice exposed to these stress conditions have shown no incidence of failed or abnormal thermoregulation, since the core body temperature taken from 30 mice was found to be elevated to 40–42.5°C with an average of 41.22±0.24. This is well within the range known to occur with physiological stress conditions such as exercise,

Discussion

We have demonstrated the induction of mouse hepatic AGP-1, AGP-2, CRP and albumin gene expression by heat stress, and that this response occurs in the absence of elevated cytokine levels that induce these genes during the LPS-mediated APR. These observations suggest that the responses of these genes to hyperthermia do not require IL-1β, IL-6 or TNFα. However, there may still be a requirement for both glucocorticoids and C/EBP trans-acting factors, thus, suggesting that hyperthermia may regulate

Acknowledgements

This investigation was supported by a grant from the Shriners Burns Institute, Galveston Unit (awarded to J.P.) and a grant from the Hellenic General Secretary of Research and Technology (awarded to M.Y.) We acknowledge the assistance of M.A. Ridgeway in the preparation of the manuscript and of Dr. David Konkel for his review of the manuscript.

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