Arsenic inhibits SGK1 activation of CFTR Cl− channels in the gill of killifish, Fundulus heteroclitus
Introduction
Arsenic, a toxic metalloid, is prevalent in the environment, where it occurs both naturally and as result of pollution. Exposure to arsenic via occupational and environmental sources represents a major health concern worldwide according to the World Health Organization (WHO). Chronic exposure to arsenic contaminated water or air causes cancers of the skin, lung, bladder, prostate, liver and kidney (Karagas et al., 1998, Abernathy et al., 1999, Henke, 2009). Arsenic has also been linked to type 2 diabetes, vascular disease, cardiovascular disease, neuropathy and reproductive and developmental disorders (USEPA, 1980, Abernathy et al., 1999, National Research Council, 1999). To protect against the adverse effects of chronic arsenic exposure, the U.S. EPA and the WHO have established a limit for safe drinking water of 10 ppb (USEPA, 1980). In addition, to protect humans consuming seafood from the risk of arsenic associated cancers, the U.S. EPA has established a maximum safe-level of total dissolved inorganic arsenic in seawater of 0.0175 ppb. However, these concentrations are often exceeded even in clean coastal waters (1–3 ppb), and arsenic can be as high as 1000 ppb in polluted seawater (Boyle and Jonasson, 1973, Neff, 1997).
Exposure to arsenic disrupts Cl− balance and blocks seawater acclimation in killifish, Fundulus heteroclitus (Stanton et al., 2006, Shaw et al., 2007b), but the underlying mechanism for this inhibition is unknown. When moving from freshwater to seawater, acclimation is accomplished in large part by increasing CFTR-mediated Cl− secretion across the gill and opercula epithelium to balance Cl− (and Na+) intake in seawater. Acclimation to seawater involves both short-term (h) (Sato et al., 2007, Shaw et al., 2008) and longer term (days) up-regulation of NaCl excretion (Wood and Laurent, 2003, Shaw et al., 2007b). The short-term mechanism is mediated by the translocation of CFTR from an intracellular vesicular pool to the plasma membrane, an effect mediated by SGK1, which in killifish is increased by plasma hypertonicity (Shaw et al., 2008). The long-term increase in Cl− secretion is mediated by cortisol, which via activation of the glucocorticoid receptor (GR), enhances the mRNA and protein abundance of CFTR, Na+-K+-ATPase and NKCC1 (Singer et al., 1998, Marshall et al., 1999, Marshall et al., 2005, Marshall, 2002, Marshall, 2003, Marshall and Singer, 2002, Scott et al., 2004). Although arsenic has no effect on the cortisol-GR mediated increase in CFTR, Na+-K+-ATPase, and NKCC1 mRNA, or on the protein abundance of Na+-K+-ATPase and NKCC1 (Shaw et al., 2007b) arsenic reduces CFTR protein in the gill and rapidly (h) reduces CFTR Cl− currents (Stanton et al., 2006). However, the mechanism for this effect of arsenic is unknown. Thus, the goal of this study is to test the hypothesis that arsenic reduces the seawater induced increase in CFTR abundance by inhibiting the seawater induced up-regulation of SGK1.
SGK1, a 50 kDa, serine/threonine protein kinase, is transcriptionally regulated by a wide variety of environmental and cytotoxic stressors, including hypertonicity, as well as by steroids (including cortisol) and peptide hormones (Loffing et al., 2006). SGK1 regulates the location and abundance of many plasma membrane proteins including ion channels, receptors and peptide hormone receptors. For example, SGK1 increases K+ and Ca++ transport in epithelial cells by enhancing the plasma membrane expression of ROMK1 and TRPV5 channels, respectively (Lang et al., 2006, Tessier and Woodgett, 2006). SGK1 also stimulates sodium reabsorption in the kidney by increasing the number of ENaC sodium channels in the plasma membrane (Lang et al., 2003, Lang et al., 2006, Pearce, 2003, Thomas and Itani, 2004, Vallon et al., 2005, Bhalla et al., 2006). Briefly, aldosterone and glucocorticoids, by binding to the mineralocorticoid (MR) and glucocorticoid receptor (GR), respectively, promote the transcription and subsequent phosphorylation of SGK1 (ppSGK). ppSGK1 phosphorylates, and thereby inhibits Nedd4-2, a E3 ubiquitin ligase, which decreases the Nedd4-2 induced ubiquitination of ENaC channels. Because ubiquitinated ENaC is removed from the membrane by endocytosis and is then degraded in the lysosome, reduced ubiquitination of ENaC leads to the accumulation of ENaC in the plasma membrane, which results in enhanced Na+ transport. SGK1 also enhances CFTR Cl− currents in Xenopus oocytes and in pancreatic cells in culture by increasing the abundance of CFTR in the plasma membrane (Wagner et al., 2002, Sato et al., 2007, Caohuy et al., 2009). In recent studies in killifish we demonstrated that transfer from freshwater to seawater rapidly (h) increased SGK1 mRNA and protein levels, and that the increase in SGK1 preceded the rise in the abundance of CFTR in the apical membrane of the opercula epithelium (which is similar in form and function to the gill) (Shaw et al., 2008). In killifish, the increase in SGK1 is stimulated by plasma hypertonicity rather than steroid hormones (i.e., cortisol activation of the GR) (Shaw et al., 2008).
Since arsenic blocks the acclimation to seawater in killifish by a mechanism that does not disrupt GR-mediated induction of CFTR gene expression, yet acutely decreases CFTR protein abundance (Shaw et al., 2007b), studies in this manuscript were designed to test the hypothesis that arsenic interferes with the ability of killifish to acclimate to seawater by interfering with SGK1 regulated trafficking of CFTR to the apical plasma membrane. The data demonstrate for the first time that arsenic reduces SGK1 mRNA expression and protein abundance, increases the ubiquitination and lysosomal degradation of CFTR, and decreases the abundance of CFTR in the apical membrane of the opercula epithelium in the killifish. These findings demonstrate that environmentally relevant levels of arsenic (10 and 100 ppb), increase the ubiquitination and degradation of CFTR, which will reduce the ability of killifish to acclimate to increased salinity and to maintain NaCl homeostasis.
Section snippets
Animals
Studies were performed in compliance with Institutional animal care and use guidelines approved by MDIBL (#A3562-01) and Dartmouth Medical School (#A3259-01). Killifish, F. heteroclitus, were collected from Northeast Creek (Bar Harbor, ME, USA) and held in glass aquaria containing running seawater (pH 8.1 ± 0.4; salinity 33 ± 0.5‰, 15 °C) at the MDIBL for at least 2 weeks to ensure acclimation to seawater. For all tests fish were maintained outdoors under natural light cycles (photoperiod 15:9-h
Arsenic reduces CFTR Cl− secretion
Previous studies demonstrated that arsenic (12,000 ppb) reduced the ability of freshwater acclimated killifish to survive a seawater challenge (Shaw et al., 2007b). Because acclimation to seawater is mediated by an increase in CFTR Cl− secretion by the gill and the opercular membrane (Hoffmann et al., 2002, Marshall et al., 1999, Shaw et al., 2007b, Stanton et al., 2006, Wood and Laurent, 2003), studies were conducted to determine if arsenic reduced CFTR Cl− secretion. In these studies CFTR Cl−
Discussion
There are four major new findings in this manuscript that extend our understanding of SGK1 regulation of plasma membrane CFTR abundance and implicate SGK1 as a target for arsenic. During acclimation to seawater (i.e., direct transfer from freshwater to seawater), arsenic rapidly (h): (1) inhibits the increase in SGK1 mRNA expression and protein abundance in the gills; (2) increases the amount of ubiquitinated CFTR, leading to the lysosomal degradation of CFTR; (3) decreases the abundance of
Conflict of interest
None disclosed.
Acknowledgements
This study was supported by NIEHS Superfund Basic Research Program Project grant P42 ESO7373 (BAS, JRS), NIEHS Center for Membrane Toxicity Studies at MDIBL P30-ES03828 (BAS and JRS), a Research Development Program grant from the Cystic Fibrosis Foundation (BAS), and a MDIBL New Investigator Award and funds from the DOD SERDP program ER1503 (JRS). We thank Dawoon Jung, J. Denry Sato, Chris Chapline, Kristen Gabor, Caitlin Stanton, Lydia Durant, and Renee Thibodeau for valuable support and
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