Elsevier

Aquatic Toxicology

Volume 98, Issue 2, 10 June 2010, Pages 157-164
Aquatic Toxicology

Arsenic inhibits SGK1 activation of CFTR Cl channels in the gill of killifish, Fundulus heteroclitus

https://doi.org/10.1016/j.aquatox.2010.02.001Get rights and content

Abstract

Seawater acclimation in killifish, Fundulus heteroclitus, is mediated in part by a rapid (1 h) translocation of CFTR Cl channels from an intracellular pool to the plasma membrane in gill and increased CFTR-mediated Cl secretion. This effect is mediated by serum and glucocorticoid-inducible kinase 1 (SGK1), which is stimulated by plasma hypertonicity rather than cortisol. Since arsenic exposure prevents acclimation to seawater by decreasing CFTR protein levels we tested the hypothesis that arsenic (as sodium arsenite) blocks acclimation to seawater by down regulating SGK1 expression. Freshwater adapted killifish were exposed to arsenic (48 h) and transferred to seawater containing arsenic, and SGK and CFTR expression were measured. Arsenic reduced the seawater induced increase in SGK1 mRNA and protein abundance, and reduced both the total amount of CFTR and the amount of CFTR in the plasma membrane. The decrease in membrane CFTR reduced Cl secretion. Arsenic also increased the amount of ubiquitinated CFTR and its degradation by the lysosome. Thus, we propose a model whereby arsenic reduces the ability of killifish to acclimate to seawater by blocking the seawater induced increase in SGK1, which results in increased ubiquitination and degradation of CFTR.

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

References (49)

  • A. Moeller et al.

    Elements in fish of Malibu Creek and Malibu Lagoon near Los Angeles, California

    Mar. Pollut. Bull.

    (2003)
  • B.D. Moyer et al.

    Membrane trafficking of the cystic fibrosis gene product, cystic fibrosis transmembrane conductance regulator, tagged with green fluorescent protein in madin-darby canine kidney cells

    J. Biol. Chem.

    (1998)
  • A. Swiatecka-Urban et al.

    PDZ domain interaction controls the endocytic recycling of the cystic fibrosis transmembrane conductance regulator

    J. Biol. Chem.

    (2002)
  • A. Swiatecka-Urban et al.

    The short apical membrane half-life of resuced Δ508-cystic fibrosis transmembrane conductance regulator (CFTR) results from accelerated encocytosis of Δ508-CFTR in polarized human airway epithelial cells

    J. Biol. Chem.

    (2005)
  • C.M. Wood et al.

    Na+ versus Cl transport in the intact killifish after rapid salinity transfer

    Biochim. Biophys. Acta

    (2003)
  • X.H. Zheng et al.

    Low-level arsenite induced gene expression in HEK293 cells

    Toxicology

    (2003)
  • C.O. Abernathy et al.

    Arsenic: health effects, mechanisms of actions, and research issues

    Environ. Health Perspect.

    (1999)
  • American Society for Testing and Materials

    Annual Book of ASTM Standards. Water and Environmental Technology. Water (I)

    (1985)
  • V. Bhalla et al.

    Disinhibitory pathways for control of sodium transport: regulation of ENaC by SGK1 and GILZ

    Am. J. Physiol. Renal Physiol.

    (2006)
  • R.W. Boyle et al.
  • S.A. Ernst et al.

    Structural diversity of occluding junctions in the low-resistance chloride-secreting opercular epithelium of seawater-adapted killifish (Fundulus heteroclitus)

    J. Cell Biol.

    (1980)
  • M. Gentzsch et al.

    Endocytic trafficking routes of wild-type and ΔF508 CFTR

    Mol. Biol. Cell

    (2004)
  • K.R. Henke
    (2009)
  • M.R. Karagas et al.

    Design of an epidemiologic study of drinking water arsenic exposure and skin and bladder cancer risk in a U.S. population

    Environ. Health Perspect.

    (1998)
  • Cited by (0)

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