Acute hyperthermic responses of heat shock protein and estrogen receptor mRNAs in rainbow trout hepatocytes

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Abstract

Heat shock proteins (HSPs) are induced upon elevated temperature in fishes. HSPs also function as molecular chaperones for cellular proteins, including steroid hormone receptors. Estrogen receptors (ERs) are critical for the hormone signaling necessary during the liver production of the yolk precursor protein vitellogenin in oviparous vertebrates. Considering the possible regulatory role of HSPs on the ER signaling pathway, the present study characterized the mRNA expression of all known isoforms of HSP70 (hsp70a, hsp70b), HSP90 (hsp90a1a, hsp90a1b, hsp90a2a, hsp90a2b, hsp90b1, hsp90b2), and ERs (erα1, erα2, erβ1, erβ2) in Rainbow Trout hepatocytes following an acute heat shock (1 h at 25 °C) compared to a control treatment (12 °C). The results showed that the mRNA levels of hsp70a, hsp70b, hsp90a1b, hsp90a2a, and hsp90b2 were significantly increased after heat shock, while erα1 mRNA levels were significantly reduced by this treatment. hsp90a1a, hsp90a2b, hsp90b1, erα2, erβ1 and erβ2 were unaffected by this acute hyperthermic treatment. Comparatively, the responses of the two hsp70 isoforms were much greater than the hsp90 isoforms. Acute heat shock treatment of hepatocytes followed by a 24 h exposure to 17β-estradiol (E2) exposure also resulted in decreased expression of erα1 mRNA, but not vitellogenin (vtg) mRNA. This study showed that some hsp70 and hsp90 isoforms display a robust response to an acute hyperthermic treatment in Rainbow Trout hepatocytes. Among the transcripts measured here, the erα1 isoform uniquely showed significantly decreased mRNA levels upon acute heat treatment.

Introduction

Heat shock proteins (HSPs) have been demonstrated to be induced in all organisms by a variety of stressors such as heat, osmotic shock, heavy metals, starvation or oxidative stress (Kregel, 2002, Basu et al., 2002, Cara et al., 2005, Roberts et al., 2010). Based on their molecular weights, HSPs are traditionally classified into several families and each family plays various roles at the cellular level. The high molecular weight HSPs, HSP70 and HSP90, are particularly well known for their interaction with other intracellular proteins that are involved in many cell functions (Ellis, 1987, Welch, 1993, Hartl, 1996, Wegele et al., 2004, Pratt et al., 2010, Richter et al., 2010, Mashaghi et al., 2014). It has emerged through DNA sequencing efforts that most animals have multiple HSP70 and HSP90 isoforms (Gupta, 1995, Kregel, 2002, Daugaard et al., 2007). This is very complex in fishes, due to a past genome duplication event in this lineage that has resulted in many HSP isoforms (Iwama et al., 1998, Iwama et al., 1999, Manchado et al., 2008, Deane and Woo, 2011, Garcia de la serrana and Johnston, 2013). In most cases it is not known which isoforms are stress responsive. Additionally, amongst HSPs that demonstrate a stress response there are differences in the magnitude of those responses. This has been shown by the work of Boone and Vijayan (2002) and Ojima et al. (2005) that found differences in HSP70 mRNA isoform expression after heat shock in Rainbow Trout hepatocytes and RTG-2 cells, respectively. Because of the numerous HSP70 and HSP90 isoforms existing within a species it becomes important to know which exhibit a stress response in order to interpret how HSP induction might affect their cellular function.

HSPs are well known to be necessary for the normal functioning of steroid hormone receptors (Picard, 2006). The HSP90 family work as chaperone proteins for steroid hormone receptors (Chambraud et al., 1990, Pratt and Toft, 1997) and the HSP70 family is necessary for translocation and protein folding (Gething and Sambrook, 1992, Kohno et al., 2010). Estrogen receptors (ERs) are a steroid hormone group that have interactions with HSPs (Cheung and Smith, 2000, Pratt and Toft, 1997, Pratt and Toft, 2003, Pratt et al., 2004). Briefly, unliganded ERs are assembled into hetero-complexes with HSP90, HSP70, and other proteins by a multi-protein chaperone machinery. The ligand binding of ERs cause the dissociation of the receptor from these hetero-complexes, allowing the receptor-ligand complex to bind estrogen response elements, which can trigger an ER-mediated signal cascade reaction (Pratt et al., 2004). By forming hetero-complexes with HSPs, the chaperone machinery stabilizes the ER for proper folding, trafficking, nuclear cycling and turnover. Estrogens are important endocrine hormones and involved in the regulation of numerous physiological processes. The free steroid binds to ERs in order to initiate the transcription of estrogen responsive genes (Berg et al., 2004, Menuet et al., 2004, Guiguen et al., 2010, Hao et al., 2013, Bondesson et al., 2015). In most vertebrates, the ER family consists of two ERs, ERα and ERβ, (Eick and Thornton, 2011). However, in fishes due to the genome duplication described above there are additional ER isoforms similar to the situation for HSPs. How these numerous HSP and ER isoforms interact within the cells of fishes is not known.

Fishes, as aquatic ectotherms, typically experience variable water temperatures throughout their lives. Temperature has a modifying role, particularly in cueing the precise timing of gamete maturation and spawning, providing the capacity for reproductive cycles to be locally tuned to shorter term and less predictable variations in thermal condition (Pankhurst and Porter, 2003). Increasing temperatures will at some level have adverse effects on reproduction in fishes (Van Der Kraak and Pankhurst, 1997). Studies on salmonids indicated that elevated temperature retards final oocyte maturation and ovulation, and impairs reproductive success (Pankhurst and Thomas, 1998, Pankhurst et al., 1996, King et al., 2007, Pankhurst and King, 2010). Most fishes exhibit a robust heat shock response by increasing the transcription of HSP mRNA and protein (Dietz and Somero, 1993, Palmisano et al., 1999, Boone and Vijayan, 2002, Yamashita et al., 2004, Ojima et al., 2005, Manchado et al., 2008). Induction of HSPs is presumably adaptive but few physiological responses have been investigated in detail and a direct connection between heat responsive proteins and reproduction has yet to be made.

It has been reported that elevated temperature affects reproduction in Rainbow Trout (Oncorhynchus mykiss) by impairing estrogen synthesis (Pankhurst and King, 2010). One mechanism by which temperature could impact estrogen function would be through the role HSPs have as molecular chaperones of ERs. The goal of this study was to measure the mRNA expression patterns of all known hsp70, hsp90, and er isoforms in Rainbow Trout (Oncorhynchus mykiss) hepatocytes following an acute hyperthermic shock to identify which isoforms are temperature responsive. In addition, an experiment was conducted to examine the effect of elevated temperature of the induction pathway of an estrogen-responsive gene, vitellogenin (vtg). This information will be important to guide future studies to establish the validity of the mechanism described above.

Section snippets

Animals

Mixed sexes of juvenile rainbow trout (Oncorhynchus mykiss), body weight = 117 ± 8.5 g, were obtained from the University of Idaho Aquaculture Research Institute Moscow Facility. Fish were maintained in re-circulating tanks with a photoperiod of 12 h Light: 12 h Dark, a water temperature of 12 °C, and were fed a commercial fish food (Rangen, Inc., Buhl, ID). All live fish procedures were conducted according to the guidelines established by the Institutional Animal Care and Use Committee at the

Results

All HSP, ER, and vitellogenin primers amplified the expected amplicons in the RT-PCR (Table 1). Because the Rainbow Trout HSP90 isoforms (i.e., hsp90a1a, hsp90a1b, hsp90a2a, hsp90a2b, hsp90b1, hsp90b2) have not been previously described, RT-PCR was initially based on Atlantic Salmon primers sequences reported in Table 1. Primer sets that did amplify as expected were redesigned based on Rainbow Trout genomic sequence in NCBI or found within the Rainbow Trout genome database (Berthelot et al.,

Discussion

This study represents the first time that the mRNAs for all known hsp70 and hsp90 isoforms have been measured in juvenile rainbow trout hepatocytes and quantified in response to an acute hyperthermic shock. It was demonstrated that amongst the putative HSP mRNAs that could be measured by RT-PCR that many, but not all, showed a significant increase in isolated hepatocytes upon exposure for 1 h to 25 °C. The hsp70 group is comprised of two isoforms, hsp70a and hsp70b, and both show a robust

Acknowledgements

This study was supported by a Chinese Scholarship Council award (201403260021) to Yudong Jia and EPA-STAR Grant #R835167.

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