Potential osmoprotective roles of branchial heat shock proteins towards Na+, K+-ATPase in milkfish (Chanos chanos) exposed to hypotonic stress
Introduction
Living organisms interact with their environments throughout their life spans. Some unfavorable environments containing biotic or abiotic stressors threaten or disturb the dynamic equilibrium (homeostasis) of individuals (Wendelaar Bonga, 1997). Environmental stresses are caused by a combination of abiotic factors, such as salinity, temperature extremes, pollutants, and anoxia, and biotic factors, such as parasitism and predation (Fishelson et al., 2001; Iwama et al., 2004; Padmini and Rani, 2009; Roberts et al., 2010; Taleb et al., 2008; Tine et al., 2010). Besides, some compensatory mechanisms might allow an individual to deal with environmental stresses by producing various metabolic or structural components that could maintain basic cellular functions (Palmisano et al., 2000). Among these mechanisms, stress proteins play a central role in maintaining cellular homeostasis and, thus, minimize acute stress damage (Welch, 1993). Moreover, previous studies have demonstrated that stressors induce the production of chaperone proteins, especially heat shock proteins (HSPs), which are involved in protein biogenesis and prevent protein misfolding (Roberts et al., 2010).
The HSP family is an important group of chaperone proteins that were originally identified as proteins whose expression was induced by heat stress (Basu et al., 2002; Poompoung et al., 2014; Roberts et al., 2010). An environmental stressor could disrupt the three-dimensional structure of a protein (Ellis and Minton, 2006; Goldberg, 2003); thus, HSPs are indispensable for maintaining normal cell function under stressful conditions. They are highly conserved in diverse organisms (Iwama et al., 1998, Iwama et al., 1999; Lindquist and Craig, 1988; Metzger et al., 2016; Srivastava, 2002). Moreover, some HSP family members, which play a role in various aspects of protein metabolism, are expressed under normal conditions (Poompoung, 2014) to maintain cellular integrity (Iwama et al., 1998). Previous studies have demonstrated that the HSP family proteins are rapidly induced by various stressors and exhibit cytoprotective functions (Ali et al., 1996; Gething and Sambrook, 1992; Hartl et al., 2011; Hightower, 1991). So far, HSPs have been classified into several distinct groups according to their molecular weights, amino acid sequences, and functions (Freeman and Morimoto, 1996; Lindquist, 1986). Among them, three major HSP families are HSP90 (85–90 kDa), HSP70 (68–73 kDa), and HSP60 (58–62 kDa) (Buchner, 1996). HSP90 genes including hsp90α (i.e., Hsp90AA or inducible form) and hsp90β (i.e., Hsp90AB or constitutive form) have been identified in vertebrates. Both of them were majorly cytosolic HSP90 and are important chaperone proteins that suppress intracellular aggregation in general (Buchner, 1999). In the cellular context, hsp90α emerges as a fast-response isoform, while hsp90β seems to be associated with long-term cellular adaptation and is more specifically responsible for germ cell maturation, cytoskeletal stabilization, cellular transformation and signal transduction (Sreedhar et al., 2004). Furthermore, HSP90s can influence the function of glucocorticoid receptor to regulate the transcription induced by a steroid hormone (Iwama and Didier, 2007). On the other hand, the HSP70 family which is the primary group of HSPs is composed of both environmentally induced (HSP70) and constitutively expressed members (HSC70). By consuming ATP, HSP70 tightly binds to hydrophobic amino acids in order to prevent protein aggregation that renders proteins non-functional (Mashaghi et al., 2016). When facing stressful situation, HSP70 is highly induced from low basal levels, with transcriptional regulation via heat shock factor 1 (Hsf 1) (Deane and Woo, 2005; Westwood et al., 1991), while HSC70 is often considered to be part of constitutive cell functions in “non-stress” situation (Yeh and Hsu, 2002; Yamashita et al., 2004; López-Maury et al., 2008). HSP60 is a mitochondrial chaperone responsible for protein refolding and transportation from the cytoplasm into the mitochondrial matrix (Koll et al., 1992). Among these HSPs, HSP70 and HSP90 have been shown to be an integral part of the cellular stress response pathways in several fishes (Ali et al., 2003; Basu et al., 2002; Boone and Vijayan, 2002; Deane and Woo, 2006, Deane and Woo, 2011; Jesus et al., 2013; Liu et al., 2012; Molina et al., 2000; Padmini and Rani, 2008; Padmini and Tharani, 2015; Wang et al., 2014; Wu et al., 2013; Zhang et al., 2014).
Fish are aquatic organisms that dwell in various habitats and undergo long-term exposure to various stressors (Barton, 2002). To acclimate to external environments, it is important for fish to maintain their internal homeostasis. An exposure to environmental stressors might reestablish the system of behavior, physiology, ecology, and even evolution in fishes (Iwama et al., 1999; Padmini, 2010; Sørensen and Loeschcke, 2007). Among teleosts, approximately 5% are euryhaline and can survive in fresh water (FW), brackish water (BW), and seawater (SW). These groups of fishes are commonly found in habitats, such as estuaries and tide pools, where salinity usually changes dramatically (Evans et al., 2005), making euryhaline fishes an excellent model to study osmotic stress in vivo.
In euryhaline teleosts, Na+, K+-ATPase (NKA) is an indispensable protein that plays an important role in osmoregulation. It provides an ion gradient to promote the activity of other transporters in gill ionocytes by consuming energy (Hwang et al., 2011; Lee et al., 2000; Yang et al., 2019). In the mammalian kidney, NKA plays similar roles as in teleost gills (Gagnon et al., 1999). Furthermore, it was demonstrated that under renal injury, the regulated HSP70 was bound to NKA in porcine kidney epithelial cell line LLC-PK1 (Riordan et al., 2005), and the interaction between HSP70 and NKA increased in rats recovering from a low-protein diet (Ruete et al., 2008). These studies indicated that when mammals were exposed to nutritional stress, HSP70 was activated to maintain the function of NKA. However, it is unknown whether HSPs interact with NKA in euryhaline teleosts, and if milkfish respond to osmotic stress through an NKA-HSP interaction to avoid unfolding. The roles of HSP70 and HSP90 in the osmoregulatory processes of milkfish also remain undetermined. In this study, we aimed to (1) investigate the mRNA and protein expression of branchial HSP70 and HSP90; (2) demonstrate the interaction between HSP70 or HSP90 and NKA by Co-immunoprecipitation (Co-IP) to identify their potential osmoprotective roles in the osmoregulatory mechanisms of euryhaline milkfish reared in SW and FW.
Section snippets
Experimental fish and environments
Juvenile milkfish (C. chanos) with a weight of 20.0 ± 7.0 g and a standard length of 13.5 ± 1.3 cm were obtained from a local fish farm (Tainan, Taiwan). After acclimation to BW (15‰) prepared from dechlorinated local tap water (FW) with proper amounts of Blue Treasure Sea Salts (New South Wales, Australia) for at least 2 weeks, the milkfish were transferred directly to either FW or SW (35‰) with 28 ± 1 °C for at least 4 weeks before sampling for the long-term FW and SW acclimation groups and
mRNA and protein expression of CcHSPs in seawater (SW)- and fresh water (FW)-acclimated milkfish gills
Branchial Cchsp90α was significantly decreased in the FW-acclimated milkfish (approximately 0.5-fold) compared to the SW-acclimated individuals (Fig. 1C). Meanwhile, the quantitative PCR results showed no significant difference in the expressions of Cchsc70, Cchsp70, and Cchsp90β between SW- and FW-acclimated milkfish gills (Fig. 1A, B, D). On the other hand, at the protein level, the representative immunoblots showed single bands at approximately 70 and 90 kDa for HSP70 and HSP90, respectively
Discussion
Euryhaline teleosts have the capacity to maintain individual homeostasis under changing environmental salinities (Burnett et al., 2007; Choi and An, 2008; Deane et al., 2002; Deane and Woo, 2004; Fiol and Kültz, 2007; Hiroi and McCormick, 2007; Kang et al., 2010; Marshall et al., 1999; McCormick et al., 2003; Sangiao-Alvarellos et al., 2003; Tang and Lee, 2013a, Tang and Lee, 2013b; Yang et al., 2009). In SW, the internal environments of fish are hypotonic compared to the external media so that
Funding
This study was financially supported in part by the Taiwan Ministry of Science and Technology (MOST) Research Project grant (MOST-106-2313-B-005-038- MY3) to T.H.L. and the Taiwan-France ORCHID (MOST-109-2911-I-005-501) grants to T.H.L. and C.L.N.. This work was also financially supported in part by the iEGG and Animal Biotechnology Center from The Feature Area Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE), Taiwan (
Author contributions
K.U., H.J.C., W.K.Y., and T.H.L. conceived and designed the experiments. K.U., H.J.C., and L.C. performed the experiments. K.U., H.J.C., W.K.Y., and Y.C.W., and W.Y.W. analyzed the data. K.U. and H.J.C. wrote the manuscript. T.H.L. and C.L.N. organized the whole project and manuscript. All authors have read and approved the final manuscript.
Declaration of Competing Interest
The authors declare that there is no conflict of interests regarding the publication of this paper. The authors alone are responsible for the content and writing of the paper.
Acknowledgements
The authors appreciate Dr. Catherine Lorin-Nebel (MARBEC, Université de Montpellier, UM-CNRS-IRD-IFREMER, Montpellier, France) for her constructive comments and suggestions for this article. This study was dedicated to the memory of Dr. Cheng-Hao Tang (Department of Oceanography, National Sun Yat-Sen University, Kaohsiung, Taiwan), a colleague and friend, who passed away on February 8, 2019, for his suggestion of experimental design. The monoclonal antibody α5 was purchased from the
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K. Umam and H.J. Chuang contributed equally.
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†Present address: Department of Life Sciences, National Taiwan University, Taipei, 104 Tiwan