Potential osmoprotective roles of branchial heat shock proteins towards Na+, K+-ATPase in milkfish (Chanos chanos) exposed to hypotonic stress

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Highlights

  • CcHSPs in milkfish gills were rapidly induced after acute hypotonic exposure.

  • Hypotonicity-induced CcHSPs recovered gradually within 7 days.

  • Co-IP was first used to show the interaction of CcHSPs with NKA in milkfish gills.

  • Increased binding of CcHSPs to NKA protected branchial NKA from hypotonic stress.

Abstract

In euryhaline teleosts, osmoregulatory mechanisms vary with osmotic stresses, and heat shock proteins (HSPs) play a central role in maintaining cellular homeostasis. The present study aimed to investigate the expression and potential roles of HSP70 and HSP90 in the gills of seawater (SW)- and freshwater (FW)-acclimated milkfish (Chanos chanos). Four HSP genes, including Cchsc70 (heat shock cognate 70), Cchsp70, Cchsp90α, and Cchsp90β, were analyzed in milkfish gills. Among these genes, only the mRNA abundance of branchial Cchsp90α was significantly lower in the FW-acclimated than in the SW-acclimated milkfish. Immunoblotting showed no significant difference in the relative protein abundance of branchial HSP70 and HSP90 between the two groups. The time-course experiments (from SW to FW) showed that the protein abundance of HSP70 and HSP90 at the 3 h and 6 h post-transfer and then declined gradually. To further illustrate the potential osmoregulatory roles of HSP70 and HSP90, their interaction with Na+, K+-ATPase (NKA, the primary driving force for osmoregulation) was analyzed using co-immunoprecipitation. The results showed the interaction between HSP70, HSP90 and NKA after acclimation to SW or FW increased within 3 h; and then returned to normal levels within 7 days. To our knowledge, the present study was the first to demonstrate that the interaction between HSP70, HSP90 and NKA changes with hypotonic stress in euryhaline teleosts. Before the transfer, no interaction was detected. When transferred to FW from SW, the interaction of HSP70 and HSP90 with NKA were detected. The results suggested that HSP70 and HSP90 participated in the acute responses of osmoregulatory mechanisms to protect branchial NKA from hypotonic stress in milkfish.

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

References (92)

  • L.E. Hightower

    Heat shock, stress proteins, chaperones, and proteotoxicity

    Cell

    (1991)
  • J. Hiroi et al.

    New insights into gill ionocyte and ion transporter function in euryhaline and diadromous fish

    Respir. Physiol. Neurobiol.

    (2012)
  • Y.C. Hu et al.

    Cortisol regulation of Na+, K+-ATPase β1 subunit transcription via the pre-receptor 11β-hydroxysteroid dehydrogenase 1-like (11β-Hsd1L) in gills of hypothermal freshwater milkfish, Chanos chanos

    J. Steroid Biochem. Mol. Biol.

    (2019)
  • P.P. Hwang et al.

    New insights into fish ion regulation and mitochondrion-rich cells

    Comp. Biochem. Physiol. A Mol. Integr. Physiol.

    (2007)
  • C.K. Kang et al.

    Salinity-dependent expression of a Na+, K+, 2Cl− cotransporter in gills of the brackish medaka Oryzias dancena: amolecular correlate for hyposmoregulatory endurance

    Comp. Biochem. Physiol. A

    (2010)
  • C.K. Kang et al.

    Seawater-acclimation abates cold effects on Na+, K+-ATPase activity in gills of the juvenile milkfish, Chanos chanos

    Aquaculture

    (2015)
  • C.K. Kang et al.

    The expression of VILL protein is hypoosmotic-dependent in the lamellar gill ionocytes of Otocephala teleost fish, Chanos chanos

    Comp. Biochem. Physiol. A Mol. Integr. Physiol.

    (2017)
  • H. Koll et al.

    Antifolding activity of hsp60 couples protein import into the mitochondrial matrix with export to the intermembrane space

    Cell.

    (1992)
  • Y.M. Lin et al.

    The expression of gill Na, K-ATPase in milkfish, Chanos chanos, acclimated to seawater, brackish water and fresh water

    Comp. Biochem. Physiol. B

    (2003)
  • Y.M. Lin et al.

    Short-term effects of hypoosmotic shock on Na+/K+-ATPase expression in gills of the euryhaline milkfish, Chanos chanos

    Comp. Biochem. Physiol. A

    (2006)
  • K.J. Livak et al.

    Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method

    Methods

    (2001)
  • W.S. Marshall

    Osmoregulation in estuarine and intertidal fishes

    Fish Physiol. Vol.

    (2012)
  • A. Molina et al.

    Cloning and expression analysis of an inducible HSP70 gene from tilapia fish

    FEBS Lett.

    (2000)
  • E. Padmini et al.

    Impact of seasonal variation on HSP70 expression quantitated in stressed fish hepatocytes

    Comp. Biochem. Physiol. B

    (2008)
  • E. Padmini et al.

    Evaluation of oxidative stress biomarkers in hepatocytes of grey mullet inhabiting natural and polluted estuaries. Sci

    Total Environ

    (2009)
  • M.C. Ruete et al.

    Na+/K+-ATPase stabilization by Hsp70 in the outer stripe of the outer medulla in rats during recovery from a low-protein diet

    Cell Stress Chaperones

    (2008)
  • N. Valkova et al.

    Constitutive and inducible stress proteins dominate the proteome of the murine inner medullary collecting duct-3 (mIMCD3) cell line

    Biochim. Biophys. Prot. Proteom.

    (2006)
  • M. Yamashita et al.

    Characterization of multiple members of the HSP70 family in platyfish culture cells: molecular evolution of stress protein HSP70 in vertebrates

    Gene

    (2004)
  • W.K. Yang et al.

    Na+/K+-ATPase expression in gills of the euryhaline sailfin molly, Poecilia latipinna, is altered in response to salinity challenge

    J. Exp. Mar. Biol. Ecol.

    (2009)
  • W.K. Yang et al.

    Gene expression of Na+/K+-ATPase α-isoforms and FXYD proteins and potential modulatory mechanisms in euryhaline milkfish kidneys upon hypoosmotic challenges

    Aquaculture

    (2019)
  • A. Ali et al.

    Evaluation of stress-inducible hsp90 gene expression as a potential molecular biomarker in Xenopus laevis

    Cell Stress Chaperones

    (1996)
  • T. Bagarinao

    Systematics, distribution, genetics and life history of milkfish, Chanos chanos

    Environ. Biol. Fish

    (1994)
  • B.A. Barton

    Stress in fishes: a diversity of responses with particular reference to changes in circulating corticosteroids

    Integr. Comp. Biol.

    (2002)
  • J. Buchner

    Supervising the fold: functional principles of molecular chaperones

    J. FASEB

    (1996)
  • C.H. Chang et al.

    The antioxidant peroxiredoxin 6 (Prdx6) exhibits different profiles in the livers of seawater-and fresh water-acclimated milkfish, Chanos chanos, upon hypothermal challenge

    Front. Physiol.

    (2016)
  • E.E. Deane et al.

    Differential gene expression associated with euryhalinity in sea bream (Sparus sarba)

    Am. J. Phys. Regul. Integr. Comp. Phys.

    (2004)
  • E.E. Deane et al.

    Advances and perspectives on the regulation and expression of piscine heat shock proteins

    Rev. Fish Biol. Fish.

    (2011)
  • E.E. Deane et al.

    Chronic salinity adaptation modulates hepatic heat shock protein and insulin-like growth factor I expression in black sea bream

    Mar. Biotechnol.

    (2002)
  • S.L. Edwards et al.

    Principles and patterns of osmoregulation and euryhalinity in fishes

  • R.J. Ellis et al.

    Protein aggregation in crowded environments

    Biol. Chem.

    (2006)
  • D.H. Evans et al.

    The multifunctional fish gill: dominant site of gas exchange, osmoregulation, acid-base regulation, and excretion of nitrogenous waste

    Physiol. Rev.

    (2005)
  • D.F. Fiol et al.

    Osmotic stress sensing and signaling in fishes

    FEBS J.

    (2007)
  • Z. Fishelson et al.

    Contribution of heat shock proteins to cell protection from complement-mediated lysis

    Int. Immunol.

    (2001)
  • B.C. Freeman et al.

    The human cytosolic molecular chaperones hsp90, hsp70 (hsc70) and hdj-1 have distinct roles in recognition of a non-native protein and protein refolding

    EMBO J.

    (1996)
  • F. Gagnon et al.

    Na+, K+ pump and Na+-coupled ion carriers in isolated mammalian kidney epithelial cells: regulation by protein kinase C

    Can. J. Physiol. Pharmacol.

    (1999)
  • M.J. Gething et al.

    Protein folding in the cell

    Nature

    (1992)
  • Cited by (0)

    1

    K. Umam and H.J. Chuang contributed equally.

    2

    Present address: Department of Life Sciences, National Taiwan University, Taipei, 104 Tiwan

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