Regucalcin was discovered in 1978 as a calcium (Ca2+) binding protein in the liver of rats (Yamaguchi and Yamamoto, 1978) and is known as the senescence marker protein-30 (SMP30) that decreases expression with ageing in rat livers (Fujita et al., 1992). Being a calcium-binding protein without EF-hand motif, several functions of regucalcin have been reported in mammals including intracellular calcium homeostasis, modulation of Ca2+- or Ca2+/calmodulin-dependent enzymes, participation of the biosynthesis process of ascorbic acid, and transcriptional regulation for several hormones (e.g., insulin and estrogen) (Fujita et al., 1998; Kondo et al., 2006; Yamaguchi, 2011; Yamaguchi, 2013). Many vertebrates have the ability to synthesize ascorbic acid, however this does not include teleosts due to their loss of an important key enzyme, gluconolactone oxidase (Ching et al., 2015). As a calcium binding protein, regucalcin was found to have inhibitory effects on several Ca2+-dependent kinase and enzymes (i.e., cAMP phosphodiesterase, caspase-3, and nitric oxide synthase) which inhibited apoptosis under stress (Matsutama et al., 2004; Izumi and Yamaguchi, 2004; Yamaguchi and Kurota, 1997). Regucalcin knockout in mice has been reported to elevate oxidative stress, antioxidant dysfunction, and hepatocyte apoptosis, leading to liver fibrosis and even death (Park et al., 2010; Kondo et al., 2006). Therefore, regucalcin has critical functions in calcium homeostasis for several physiological responses. Related studies on teleosts, however, are limited.
Hypothermal stress was found to change cell membrane fluidity and protein structure that may lead to rearrangement of cytoskeleton, activation of calcium channels, or ER stress-induced imbalance of cytosolic Ca2+ levels, which then affect several physiological responses (Bayley et al., 2018; Wang et al., 2019). Meanwhile, hypothermal stress may induce reactive oxygen species (ROS) due to the immune response or mitochondria dysfunction (Donaldson et al., 2008). Elevation of cytoplasmic Ca2+ levels was found in pufferfish upon low-temperature challenge, leading to oxidative stress and apoptosis (Cheng et al., 2018). Several calcium binding proteins were reported to be involved in mechanisms of cold tolerance in fish. The livers of the Antarctic notothenioid fish (Dissostichus mawsoni; Dm) contained very high levels of calmodulin. Overexpression of Dm-calmodulin further demonstrated increased cold tolerance in tobacco (Na et al., 2013). In addition, feed supplemented with Dm-calmodulin recombinant protein has enhanced cold tolerance in juvenile, orange-spotted grouper (Epinephelus coioides), showing increased antioxidant enzyme activity and reduced oxidative stress responses upon low-temperature challenge (Luo et al., 2015). In the fruit fly, high expression of the Drosophila-cold-acclimation (Dca) gene, a regucalcin-like protein with the function of maintaining calcium homeostasis, was found to help the fly tolerate cold treatments (Arboleda-Bustos and Segarra, 2011). A proteomic analysis revealed that the protein spot corresponding to regucalcin in seawater (SW) milkfish livers disappeared after one-week acclimation to 18°C (Chang et al., 2016b). Hence, it is suggested that regucalcin may play roles in maintaining calcium homeostasis in milkfish. Furthermore, the disappearance of the regucalcin protein spot in livers of hypothermal milkfish suggested functional deterioration of regucalcin, together with elevation of oxidative stress and dysfunction of antioxidant mechanisms (Chang et al., 2016b). In zebrafish, regucalcin was found to express mainly in the liver, corresponding to liver injury. Meanwhile, mRNA expression of zebrafish regucalcin (rgn) decreased with aging (Fujisawa et al., 2011). In rainbow trout, the TCO and BORN strains revealed different patterns of rgn expression in several tissues including the liver, head kidney, and muscle. After 21-days infection with Aeromonas salmonicida, the rgn was up-regulated in the liver of only the TCO strain. In addition, lower and higher temperature challenges revealed significant differences in expression of rgn between the TCO and BORN strain. There was no comparison, however, in the liver, head kidney, trunk kidney, and muscle between different temperature groups (Verleih et al., 2011). Although plasma calcium in milkfish was not changed under hypothermal challenge, calcium imbalance may happen on cellular levels according to the proteomics analysis (Kang et al., 2015; Chang et al., 2016b). Therefore, regucalcin could be considered a novel indicator of milkfish for hypothermal acclimation.
Milkfish is a tropical species with high mortality during cold snaps in winter in Southeast Asia (Fachry et al., 2018; Liao, 1991; Martinez et al., 2006). Being a euryhaline aquaculture species, milkfish have the ability to survive in environments with a broad range of salinities and thus have been cultured in water with different salinities (Jana et al., 2006). In previous studies, SW-acclimated milkfish showed better low-temperature tolerance than fresh water (FW)-acclimated milkfish (Kang et al., 2015). When acclimated to low-temperature environments, different strategies in energy metabolism and antioxidant mechanisms were found in the livers of FW- and SW-acclimated milkfish (Chang et al., 2016a, 2017, 2018, 2019). Since regucalcin, a calcium binding protein, has regulatory functions for energy metabolism, antioxidant mechanisms, and apoptosis in mammals (Fukaya and Yamaguchi, 2004; Yamaguchi, 2013; Vaz et al., 2016), the regucalcin in the liver of milkfish was thought to be an upstream regulator for differential physiological responses under salinity and hypothermal acclimation. Hence, this study reported the molecular characteristics, gene expression, and relative protein abundance of regucalcin in livers of FW- and SW-acclimated milkfish to illustrate a potential mechanism of modulating cellular calcium for hypothermal acclimation.