Focal adhesion kinase and osmotic responses in ionocytes of Fundulus heteroclitus, a euryhaline teleost fish

Comparative Biochemistry and Physiology, Part A 241 (2020) 110639

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Focal adhesion kinase and osmotic responses in ionocytes of Fundulus heteroclitus, a euryhaline teleost fish

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Breton Fougere, Katelyn R. Barnes, Magen E. Francis, Lauren N. Claus, Regina R.F. Cozzi, ⁎ William S. Marshall Department of Biology, St. Francis Xavier University, P.O. Box 5000, Antigonish, Nova Scotia B2G 2W5, Canada

A R T I C LE I N FO

A B S T R A C T

Keywords: Cystic fibrosis transmembrane conductance regulator (CFTR) Tyrosine phosphorylation Epithelial transport Teleost osmoregulation Ion channel regulation Hypotonic Hypertonic Focal adhesion kinase (FAK) Saracatinib Y15

Cystic Fibrosis Transmembrane conductance Regulator (CFTR) anion channels are the regulated exit pathway in Cl− secretion by teleost salt secreting ionocytes of the gill and opercular epithelia of euryhaline teleosts. By confocal light immunocytochemistry using regular and phospho-antibodies directed against conserved sites, we found that killifish CFTR (kfCFTR) and the tyrosine kinase Focal Adhesion Kinase (FAK) phosphorylated at Y407 (FAKpY407) and FAKpY397 are colocalized at the apical membrane and in subjacent membrane vesicles of ionocytes. Hypotonic shock and the α-2 adrenergic agonist clonidine rapidly and reversibly inhibit Cl− secretion by isolated opercular epithelia, simultaneous with dephosphorylation of FAKpY407 and increased FAKpY397, located in the apical membrane of ionocytes in the opercular epithelium. FAKpY407 is re-phosphorylated at the apical membrane of ionocytes and Cl− secretion rapidly restored by hypertonic shock, detectable at 2 min., maximum at 5 min and still elevated at 30 min. In isolated opercular epithelia, the FAK phosphorylation inhibitor Y15 and p38MAP kinase inhibitor SB203580 significantly blunted the recovery of short-circuit current (Isc, equal to Cl− secretion rate) after hypertonic shock. The cSRC inhibitor saracatinib dephosphorylated FAKpY861 seen near tight junctions of pavement cells, and reduced the increase in epithelial resistance normally seen with clonidine inhibition of ion transport, while FAKpY397 was unaffected. The results show rapid osmosensitive responses in teleost fish ionocytes involve phosphorylation of CFTR by FAKpY407, an opposing role for FAKpY397 and a possible role for FAKpY861 in tight junction dynamics.

1. Introduction Cystic Fibrosis Transmembrane conductance Regulator (CFTR), the anion channel and regulatory protein that causes cystic fibrosis, is activated by cyclic AMP (cAMP) activated protein kinase A (PKA) and by protein kinase C (PKC), pathways that terminate with serine and threonine residue phosphorylation in the regulatory (R) domain of CFTR protein, exon 13, nominally amino acid residues 590–831 (reviews: Aleksandrov et al., 2007; Dahan et al., 2001; Hwang et al., 2018). In human and mummichog CFTR sequences there are multiple, approximately 20, PKA and PKC candidate sites in the R domain. The disease Cystic Fibrosis (CF) often arises from mutations that interfere with the trafficking of CFTR product into the plasma membrane (Class II type mutations), of which the ΔF508 deletion is the most common (Aleksandrov et al., 2007; Bush et al. 2006). Without functional CFTR in the plasma membrane, the lack of the channel produces a suite of CF symptoms, to include highly viscous mucus overlaying the airway epithelium of the lung, the insufficiency of pancreatic acinar ⁎

bicarbonate secretion, suppressed anion transport in the colon and inadequate salt recovery along sweat duct, yielding salty perspiration, among other symptoms. The disease progresses to chronic lung infection, cystic lesions and ultimately death. Class III type of CF occurs with defects in the regulation of CFTR, particularly its failure to be activated by the cAMP-PKA pathway; example mutations are G551D and Y569D, both in the first nucleotide binding domain (NBD1), that result in inadequate phosphorylation of the regulatory domain and sub-optimal activation of the channel (review: Farinha et al., 2016). In class III type CF, alternate pathways to activate CFTR may be important. CFTR is also known to be activated by tyrosine kinases. We reported that the tyrosine kinase FAK activates CFTR-mediated transport in euryhaline teleost fish (Marshall et al., 2008), followed by recognition that tyrosine kinases, notably cSRC and PYK2, also activate CFTR in mammalian cell systems (Liang et al., 2011; Billet et al., 2016a, 2016b) with potential tyrosine target residues at positions 625 and 627 in the regulatory domain of CFTR (Billet et al., 2016a, 2016b). Interestingly, the tyrosine kinase inhibitors genistein and dephostatin also activate

Corresponding author. E-mail address: [email protected] (W.S. Marshall).

https://doi.org/10.1016/j.cbpa.2019.110639 Received 21 October 2019; Received in revised form 12 December 2019; Accepted 13 December 2019 Available online 19 December 2019 1095-6433/ © 2019 Elsevier Inc. All rights reserved.

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saracatinib and responses to phosphatase inhibition by okadaic acid, to propose roles for FAKpY397 and 407 in CFTR activation and a possible role for FAKpY861 in maintaining tight intercellular junctions.

CFTR channels and can stimulate ion secretion by the shark rectal gland epithelium (Lehrich and Forrest, 1995) and in previously-inhibited mummichog opercular epithelium (Marshall et al., 2000), but apparently these effects involve direct binding to CFTR protein, rather than by inhibition of tyrosine kinases (Hwang et al., 1997; Wang et al., 1998). Because of the potential for tyrosine kinases to activate CFTR with class III mutations (mutations that result in misregulation of CFTR), the relationships between tyrosine kinases and CFTR warrants further examination. Teleost fish possess CFTR in the apical membrane of mitochondrion rich salt transporting ionocytes in the gills (e.g. Wilson et al., 2000) and opercular epithelia (e.g. Marshall et al., 2002) that are responsible for salt secretion and successful acclimation of marine fish to seawater and for hardy euryhaline species, such as Fundulus heteroclitus, also to survive in hypersaline conditions (Griffith, 1974; Marshall, 2013; Cozzi et al., 2015). CFTR has been cloned and sequenced from mummichog gill and is a divergent homolog of the mammalian version of the gene (Singer et al., 1998). The channel is activated by cAMP and PKA (Marshall et al., 1995; Singer et al., 1998), as is true for mammalian systems (e.g. Farinha et al., 2016) and is trafficked into the apical membrane of seawater ionocytes during seawater acclimation (Marshall et al., 2002; Scott et al., 2008). Most phosphorylation sites of human and teleost CFTR are conserved, such that the regulation and activation of CFTR in teleost fish is in many ways similar to that in mammals. Euryhaline teleost fish are distinct from mammals in that CFTR that is usually expressed in a static “housekeeping” fashion in mammalian tissues, whereas in teleost fish, cftr expression can be induced to increase expression by simple transfer of the animal from dilute salinity to seawater or higher salinities (Marshall et al., 1999; Scott et al., 2008; Marshall, 2013). Thus, regulation of CFTR expression and its plasticity is easily studied in the teleost model system. Furthermore, teleostean salt secretion can be rapidly deactivated and activated through manipulation of neurotransmitters (Degnan et al., 1977; May and Degnan, 1985), hormones (Marshall et al., 1999; Marshall et al., 2005) and medium osmolality (Zadunaisky et al., 1995; Marshall et al., 2000; Marshall et al., 2005; Marshall et al., 2008). In this way, the euryhaline teleost Fundulus heteroclitus, that is a well-known and intensely studied physiological and genomic model of salt transport (Burnett et al., 2007), provides unique opportunities to study the regulation of this clinically important regulatory ion channel. FAK is a nonreceptor tyrosine kinase that generally resides in focal adhesions (Tani et al., 1996), is associated with cell motility (Parsons, 2003) and invasive cancer (Owens et al., 1995), possesses a “FERM” (Four pt. one, Ezrin, Radixin, Moesin) domain and exists in unphosphorylated (FAK-Related NonKinase, FRNK) and activated phosphorylated forms (Parsons, 2003). FAK can be phosphorylated at tyrosines Y397, Y407, Y576, Y577, Y861 and Y925 (Parsons, 2003) and in teleost fish, FAKpY407, FAKpY576, FAKpY577 and FAKpY861 have been detected in seawater opercular epithelium and gill, but FAKpY397 has not yet been detected (Marshall et al., 2005, 2009). We previously connected osmosensing to integrin and FAK activation of CFTR and ion secretion (Hoffmann et al., 2007; Marshall et al., 2008, 2009); we observed that hypotonic shock inhibits Cl− secretion by the chloride cells, simultaneously dephosphorylates FAKpY407 and restoration of osmolality rephosphorylates FAK and restores Cl− secretion. Thus, there is a potential functional relationship between CFTR and FAK in the apical membrane of chloride cells. In mammalian cells, the autophosphorylation site Y397 activates FAK functions, whereas phosphorylation at Y407 occurs with contact inhibition, cell cycle arrest and reduces FAK activity (Lim et al., 2007). This reciprocal relationship has not yet been examined in teleost epithelial systems. The present study expands on these initial findings to start to reveal the relationship between FAK phosphorylation and CFTR activation. Here we establish the time course of rephosphorylation of FAKpY407, osmosensitive phosphorylation of FAKpY397, its sensitivity to the small molecule FAK inhibitor Y15, dephosphorylation of FAKpY861 by

2. Materials and methods 2.1. Animals Adult common killifish or mummichog (Fundulus heteroclitus L.) of both sexes were obtained from the Antigonish estuary (Nova Scotia, Canada), transferred to indoor holding facilities and kept in full strength seawater with salinity 32 g.L−1 at 17-21 °C and ambient photoperiod under artificial light. Fish were fed marine fish food blend (Nutrafin flakes; R.C. Hagen, Montreal, Canada) twice daily at a rate of 1.0 g.100 g−1 body mass day−1, supplemented three times weekly with mealworms (Tenebrio molitor). Fish were anaesthetized in 1.0 g.L−1 MS222 (2-aminobenzoic acid ethyl ester, Sigma) in isotonic saline (9.0 g NaCl per liter distilled water), adjusted to pH 7.0 and killed by pithing prior to the experiments. Animal care was performed under animal care protocol 16-003R2, approved by St Francis Xavier University Animal Care Committee and followed Canadian Council on Animal Care guidelines. 2.2. Bathing solutions Opercular epithelia were incubated in modified Cortland's isotonic (ISO) saline (composition in mmol.L−1: NaCl 160, KCl 2.55, CaCl2 1.56, MgSO4 0.93, NaHCO3 17.85, NaH2PO4 2.97 and glucose 5.55, pH 7.8, 315 mOsm.kg−1, when equilibrated with a 99% O2/ 1% CO2 gas mixture). Test membranes that received hypotonic shock treatment (HYPO) were flushed with a diluted 80% Cortland's saline/20% high quality (HQ) 18 MΩ ion exchange organopure water and continuously aerated with a 99% O2/1% CO2 gas mixture to maintain pH balance of the solutions. Test membranes that received hypertonic post-treatments (HYPER) were incubated with a higher osmolality Cortland's saline (375 mOsm.kg− 1) where NaCl content was increased by 30 mmol.L−1. 2.3. Antibodies The primary phosphor-antibodies used to detect FAK phosphorylated at tyrosine sites were rat monoclonal anti-hFAKpY397 (clone 820755, R&D Systems, Minneapolis, MN), rabbit polyclonal anti-human FAKpY407 and rabbit polyclonal anti-human FAKpY861 (BioSource Int., Camarillo, CA, USA); all are immunopurified against the epitope. The phosphorylated tyrosine epitope regions of FAK corresponding to this antibody are known to be highly conserved (TDDYAEI for Y397, EDTYTMP for Y407 and QHIYQPV for Y861) between human (GenBank accession no. NP_72560.1) and Fundulus heteroclitus (accession no. XP_021167183.1). The anti FAK pY397, pY407 and pY861 antibodies have been used previously in immunocytochemistry and were confirmed by immunoblot, 140 kDa (Marshall et al., 2008) and antiFAKpY407 has been confirmed by immuno-TEM (Marshall et al., 2008). The primary antibody used for detection of F-actin was mouse monoclonal anti-chicken F-actin (JLA-20, Developmental Studies Hybridoma Bank) with broad species specificity. Primary antibody anti-Na+, K+ATPase antibody used for detection of ionocytes was mouse monoclonal anti-chicken (α5, Developmental Studies Hybridoma Bank, University of Iowa). The polyclonal secondary antibodies used for immunofluorescence microscopy were goat anti-rat IgG-AlexaFluor 488 (Life technologies, Eugene OR, USA), donkey anti-mouse IgG-NorthernLights 557 (R&D Systems) or goat anti-rabbit IgG Alexa Fluor 594 (Molecular Probes, Eugene, OR, USA). 2.4. Pharmaceuticals The p38 MAPK inhibitor SB203580 (MilliporeSigma, Etobicoke, ON, 2

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saline (80:20 Cortland's saline:HQ water). The control and test epithelia were immediately fixed and rinsed (see above) and incubated in primary phosphoantibodies (rabbit anti-hFAKpY397 and mouse antiNa+,K+-ATPase) and secondary antibodies (goat anti-rat IgGAlexaFluor and donkey anti-mouse IgG-NorthernLights, as above).

Canada), the phosphatase inhibitor okadaic acid potassium salt (Calbiochem EMD Biosciences, La Jolla Ca, USA) and the cSRC kinase inhibitor saracatinib (Cayman Chemical, Ann Arbor MI, USA) were dissolved in DMSO and added to both sides of the isolated epithelium bathing solutions. The focal adhesion tyrosine kinase inhibitor Y15 (MedChem Express, Monmouth Junction, NJ, USA, HY-12444 lot13853) was dissolved in saline (9 g.L−1 NaCl) and added to both sides of the preparations in aerated Cortland's saline at a concentration of 50 μM or 200 μM. The α2-adrenoreceptor agonist clonidine hydrochloride (MilliporeSigma, Etobicoke, ON, Canada) was also dissolved in saline but added only to the serosal side. If the solutions were exchanged to change osmolality, then the drug was replaced immediately after the exchange was complete.

2.5.4. Y15 dephosphorylation of FAKpY397 protocol Paired opercular membranes were exposed to control incubations in well-aerated Cortland's saline or test conditions of an additional hour exposure with inhibitor Y15, 50 μM or 200 μM, in well-aerated Cortland's saline. The control and test epithelia were immediately fixed and rinsed (see above) and incubated in primary phosphoantibodies (rabbit anti-hFAKpY397 and mouse anti-Na+,K+-ATPase) and secondary antibodies (goat anti-rat IgG-AlexaFluor and donkey anti-mouse IgG-NorthernLights, as above).

2.5. Immunocytochemistry The opercular epithelia were dissected without the dermal chromatophore layer and pinned to modeler's sheet wax. The membranes received each treatment on the wax and were then fixed for 2 h at -20 °C in 80% methanol/20% dimethyl sulfoxide (DMSO). Following fixation, they were rinsed three times in rinsing buffer comprising 0.1% bovine serum albumin (BSA) in 0.05% Tween 20 in phosphate-buffered saline (TPBS) (composition in mmol.L−1: NaCl 137, KCl 2.7, Na2HPO4 4.3, and KH2PO4 1.4 at pH 7.4), then immersed in a blocking solution with 5% normal goat serum (NGS), 0.1% BSA, 0.2% NaN3 in TPBS, pH 7.4 for 30 min at room temperature in the dark and finally incubated in each primary antibody (8 μg.mL−1 in blocking solution), singly and in combination, at 4 °C overnight. Control and test membranes were then rinsed three times and exposed to the secondary antibodies (8 μg.mL−1 in blocking solution), singly and in combination, for 4 h at room temperature. After three final rinses the membranes were mounted in mounting medium (Fluoroshield®; Sigma-Aldrich F6182). Slides were viewed and images were collected with a laser scanning confocal microscope (Olympus, Markham, ON, Canada; model FV300). In each opercular membrane, randomly selected Z-stack series were collected using a 40× water objective (N.A. 1.15 W), zoom of 3.0 and with optical sections of 1.0 ± 0.05 μm. An average of 25 sections was collected for each image.

2.6. Electrophysiology The opercular epithelia were removed and mounted in two paired Ussing-style chambers as described previously (Marshall et al., 1999). The epithelium was supported by a nylon mesh and pinned out over the circular aperture (0.125 cm2) with the rim area lightly greased and beveled to minimize edge damage. In the Ussing chambers, the following epithelial electrophysiological variables were monitored: transepithelial potential Vt (mV), transepithelial resistance Rt (Ω.cm2) and short-circuit current Isc (μA.cm−2). Isc is expressed as positive for secretion of anions and is equivalent to the net secretion of Cl− (Degnan et al., 1977). A current-voltage clamp (D. Lee Co., Sunnyvale, CA, USA) was used to measure the electrophysiological variables. Temperature was controlled by recirculating 20 °C water in the water jackets of the epithelial chambers. To test the effects of SB203580 the drug was added (10 μM both sides final concentration) and allowed to take effect (45 min), then the bathing solutions were made hypertonic by addition of dry mannitol to add 60 mM (60 mOsm). To test Y15 (10 μM both sides) for effects on the Cl− secretion by ionocytes in opercular epithelia, Isc of the test OE was monitored, in parallel to the paired control OE from the same animal. After a period of at least 30 min to establish the resting Isc of the membrane in isotonic saline on the both serosal and mucosal sides, both sides received drug addition, while the paired control epithelium received a similar volume of the drug vehicle. The drug was allowed to take effect for 30–45 min, then the ion transport was osmotically inhibited by flow-through exchange of the fluids with 80:20 Cortland's saline:HQ water and the Isc allowed to come to a new steady-state (hypotonic shock). We showed previously that this hypotonic treatment dephosphorylates FAKpY407 (Marshall et al., 2008, 2009). To test for possible effects of the drug on FAK-mediated stimulation of ion transport, NaCl was added to both sides of the chamber to bring the osmolality back to isotonicity; this treatment rephosphorylates FAKpY407 and restores Isc in these preparations (Marshall et al., 2009). A modified sequence was used with okadaic acid, where inhibition of Isc was produced by clonidine addition, followed by hypertonic stimulation of Isc by mannitol addition (60 mM final concentration, both sides). A further modification was used with saracatinib (0.5 or 1.0 μM final concentration), where osmotic stimulation of Isc was effected by addition of 60 mOsm (30 mM) NaCl to both side bathing solutions, then Isc was inhibited by addition of clonidine (10 μM final concentration, serosal side). In each case, the ability of the drug to affect stimulation of transport was tested and, for Y15, okadaic acid and saracatinib, possible effects on ion transport inhibition were also tested.

2.5.1. FAKpY407 rephosphorylation protocol Paired tissues were exposed to control incubations in well-aerated Cortland's saline or test conditions of an additional hour exposure to well-aerated hypotonic Cortland's saline (80:20 Cortland's saline:HQ water), followed by return to isotonic conditions for several time points: 1, 2, 5 and 30 min. The control and test epithelia were immediately fixed and rinsed (see above) and incubated in primary phosphoantibody (rabbit anti-hFAKpY407) and secondary antibody (goat anti-rabbit IgG with AlexaFluor, as above). 2.5.2. Saracatinib dephosphorylation of FAKpY861 protocol Paired opercular epithelia were incubated for two hours in wellaerated Cortland's saline with addition of saracatinib or drug vehicle, then placed in fixative and processed for immunocytochemistry for Factin (to localize actin bands near intercellular junctions) and FAKpY861, which also localizes to intercellular junctions in these tissues (Marshall et al., 2008). To measure level of FAKpY861, control tissues were observed first and photomultiplier tube (PMT) voltage, gain and offset were adjusted for optimal FAKpY861 fluorescence, then the paired test tissues were observed with no adjustments of PMT settings. Only one primary/secondary antibody set was used in these comparisons, to also eliminate the possibility of channel bleed-through.

2.7. Statistical analysis 2.5.3. Hypotonicity effect protocol Paired opercular membranes were exposed to control incubations in well-aerated Cortland's saline or test conditions of an additional five minutes or one hour exposure to well-aerated hypotonic Cortland's

Data are expressed as means ± 1 S. E. M. Comparisons between treatments were performed using paired t-tests and single classification analysis of variance (ANOVA) followed by Tukey's a posteriori 3

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Fig. 1. Time course for the rephosphorylation of FAK-pY407 in opercular epithelium of seawater acclimated mummichogs. A) Positive control preparation exposed to isotonic conditions only, B) negative control after one hour of hypotonic shock with FAK-pY407 dephosphorylated. The epithelia were then returned to isotonic conditions for one minute (C), 2 min (D), 5 min (E) or 30 min (F). N = 7–10 preparations per treatment. Phosphorylation is principally in the apical crypts that appear in optical section as rings (arrowheads) 2–3 μm in diameter. Scale bars are 20 μm.

comparisons test using GraphPad Prism® 7.0. Statistical significance was ascribed if P < .05. 3. Results 3.1. FAKpY407 distribution and rephosphorylation We previously showed that FAKpY407, distributed across the cytosol of ionocytes and at high levels in the apical crypts (colocalized with CFTR) was rapidly dephosphorylated by hypotonic shock (Marshall et al., 2009) and that return to isotonic conditions or application of hypertonic conditions caused rephosphorylation of FAKpY407 mostly near the apical crypts of ionocytes (Marshall et al., 2009). Thus, the ionocytes are osmosensitive and FAK responds to the level of osmolality. Immunocytochemistry revealed positive staining for FAKpY407 in apical crypts of ionocytes in opercular epithelia in control conditions and low level staining across the cytosol of ionocytes (Fig. 1A). The negative control was one hour exposure to hypotonic media before fixation and immunofluorescence for FAKpY407 was not detectable (Fig. 1B). Return of the epithelia to isotonic conditions restored FAKpY407 immunofluorescence, detectable at 1 min (Fig. 1C), higher at 2 min (Fig. 1D), maximal at 5 min (Fig. 1E) and still detectable at 30 min (Fig. 1F).

Fig. 2. The P38-MAPK inhibitor SB203580 (10 μM) had no effect on Isc by itself (control vs. SB203580), but blunted the stimulatory effect of hypertonic shock by addition of mannitol (P < .05, paired t-test, one tailed). Dissimilar letters indicate P < .05 Tukey's test, two tailed after one way ANOVA, N = 4 paired preparations.

3.4. FAK inhibitor Y15 3.2. p38 MAP kinase inhibitor SB203580

The small molecule FAK phosphorylation inhibitor Y15 was added to both sides of the test epithelium and vehicle to both sides of the paired control tissue; after 45 min there was no significant change in Isc (Initial, Fig. 4) and hypotonic flow through exchange inhibited Isc equally in test and control tissues (Hypo, Fig. 4). Return to isotonic osmolality stimulated Isc significantly in both test and control tissues in the first 20 min (Iso initial, Fig. 4) but in the Y15-treated tissues the effect was not sustained, whereas the control tissues after one hour of isotonic conditions continued to increase Isc (Iso final, Fig. 4). Thus, FAK phosphorylation seems essential to the sustained increase in Isc produced by an increase in osmolality.

In isolated opercular epithelia in Ussing-style chambers, the p38 MAP kinase inhibitor SB203580 (10 μM) had little effect on the resting ion transport rate, as Isc after one hour exposure to SB203580 was not significantly different from vehicle-treated controls (Fig. 2). When both sides of both epithelia were made hypertonic by addition of 60 mM mannitol, Isc increased significantly in the control tissues, as expected, but the increase was smaller and not significantly higher than the initial period in the SB203580-treated epithelia. 3.3. Phosphatase inhibitor okadaic acid In contrast, the protein phosphatase inhibitor okadaic acid (10 μM, both sides), while not having apparent effect after one hour exposure (OK, Fig. 3) and not apparently affecting the inhibitory effect of clonidine,10 μM, serosal side, (OK + Clon, Fig. 3), severely restricted the effect of hypertonic shock (OK + Clon + Hyper, Fig. 3). Apparently, phosphatases are essential to reset the stimulatory osmosensing pathway and p38MAP kinase may be part of that pathway.

3.5. SRC inhibitor saracatinib The cSRC inhibitor saracatinib, added to both sides of the test tissue (10 μM), had little effect on resting Isc or R after 45 min exposure (+sara, Fig. 5) and had little effect on the increase in Isc with hypertonicity (sara+hyper, Fig. 5). With the subsequent addition of clonidine (shown previously to dephosphorylate FAKpY407 in these 4

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Fig. 3. Okadaic acid (10 μM, both sides) had no significant effect on Isc (control vs OK) and did not block the inhibitory effect of clonidine (10 μM, serosal side) (P = .952, Clon vs OK + Clon), but significantly blunted the stimulatory effect of hypertonic shock by addition of mannitol without drug removal (P < .0001, Clon+Hyper vs OK + Clon+Hyper). Dissimilar letters indicate P < .05, two tailed Tukey's tests, following one way ANOVA, N = 5 paired preparations.

Fig. 5. Effect of saracatinib on Isc and epithelial resistance (R), compared to parallel-running vehicle-treated control epithelia (N = 10 pairs). Inhibition of cSRC by saracatinib had little effect on resting Isc and, with hypertonic shock, saracatinib treated and control epithelia both had a higher trend in Isc. Clonidine addition profoundly inhibited Isc in control and saracatinib-treated tissues and the normal increase in epithelial resistance after clonidine was blunted by saracatinib. Dissimilar letters indicate P < .05, two-tailed Tukey tests after one way ANOVA.

that cSRC may be involved in this pathway. Because of the prior recognized distribution of FAKpY861 in intercellular junctions in this tissue and, as cSRC is known to activate FAK in some tissues (Calalb et al., 1995), we examined the possible indirect role of cSRC in disrupting FAK-pY861 using immunocytochemistry.

Fig. 4. FAK phosphorylation inhibitor Y15 (50 μM, both sides) effect on recovery from hypotonic shock. Initially, Isc was not significantly different between control and test paired epithelial preparations. Inhibition of Isc was similar after hypotonic shock (Hypo). Addition of NaCl to restore osmolality to isotonic conditions increased Isc more in control epithelia than in Y15 treated preparations and finally, the Y15 epithelia were significantly lower than the controls.

3.6. Dephosphorylation of FAKpY861 by saracatinib Epithelia were dissected and incubated in vitro with saracatinib (0.5 or 1.0 μM for two hours) or control (DMSO). In controls (N = 5 different animals), JLA20 stained F-actin near intercellular junctions and in microridges of pavement cells (Fig. 6A) and the apical crypt rings (arrowheads in Fig. 6A), whereas FAKpY861 was present only in intercellular junctions and apical crypt rings (Fig. 6B); the merged image demonstrated considerable colocalization (Fig. 6C). The ionocytes underlying the apical crypt rings can be seen in the bright field image as fine granulated large round cells (Fig. 6 D). Saracatinib 0.5 μM for 2 h (N = 5) diminished the intercellular tight junction FAKpY861 and reduced tight junctions to interrupted lines (arrowheads, Fig. 6 F), compared to F-actin staining (Fig. 6 E, G), while underlying granular leucocytes (g in Fig. 6H) were unstained. At a higher dose, 1.0 μM, saracatinib greatly decreased FAKpY861 fluorescence (three different animals, Fig. 6 J, K, L) in preparations without JLA20 (to eliminate any possibility of channel-to-channel bleed through), compared to controls that had clear FAKpY861 immunofluorescence (Fig. 6 I).

epithelia, Marshall et al., 2009) both preparations decreased Isc greatly (−89% for controls and − 92.3% for test epithelia, for both, P < .0001, unpaired t-test, two tailed, N = 10), but the test tissues had a smaller increase in R (+12%, sara vs sara+hyper+clon, P < .01, N = 11, paired t-test, two tailed) than did the controls (+56%, DMSO vs DMSO+clon+hyper, P < .05, paired t-test, two tailed, N = 11, Fig. 5). The dominant conductance in this epithelium is the paracellular pathway that is the Na+ secretion pathway (Cozzi et al., 2015) and comprises salinity-responsive claudin-10 isoforms that compose cationpermeable pores (Marshall et al., 2018) between ionocytes and accessory cells in single-stranded tight junctions (Cozzi et al., 2015). The ability of the fish to close this cation diffusive pathway when the threat of freshwater dilute environment occurs is modeled here by addition of the alpha agonist clonidine. Although we are unsure exactly how the pathway is closed, as it could be selective removal of claudin-10 from the junctions or it could also be the mechanical withdrawal of ionocytes and their closing-over by pavement cells. The latter mechanism has been recognized previously and can occur within minutes (Daborn et al., 2001). The fact that saracatinib impeded this process suggests

3.7. Effects of hypotonicity and Y15 on FAKpY397 Under control conditions of isotonic saline incubation, opercular epithelia from SW acclimated mummichogs had immunofluorescence 5

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Fig. 6. Effect of saracatinib (AZD0530) on phosphorylation rate of FAKpY861 with JLA20 staining for F-actin (green) as contrast agent. Control opercular epithelia at 2 h incubation (N = 5, A-D) had clearly stained pavement cell margins, microridges and apical crypts (arrowheads in A) for F-actin, whereas FAKpY861 immunofluorescence (red) was restricted to cell margins and apical crypts (B); merged channels indicate extensive colocalization (yellow in C) and bright field (BF) in D indicates underlying ionocytes (i). Saracatinib 0.5 μM 2 h (N = 5, E-H) had little effect on F-actin immunofluorescence (E, G), but FAKpY861 fluorescence in intercellular margins was diminished (arrowhead in F). Granular leucocytes in the epithelium (g in H) were negative for both fluorophores. Immunofluorescence for FAKpY861 alone was similar to two channel controls (B,I) and saracatinib 1.0 μM 2 h (N = 3) diminished FAKpY861 immunofluorescence (J,K,L). Scale bars are 10 μm.

expected time course of rephosphorylation, that we also measured using the rephosphorylation of FAK upon introduction of hypertonic conditions, seen as increases in FAKpY407 immunofluorescence. We confirmed that p38MAPK inhibition by SB203580 also blunts the response to increases in osmolality, supporting the idea that p38MAPK is involved with hypertonic augmentation of ion transport. Whereas saracatinib, a cSRC inhibitor, had little effect on the osmotic responses and little effect on FAKpY407 immunofluorescence, we happened to observe that FAK phosphorylation at Y861, which in immunocytochemistry is localized to pavement cell borders, was inhibited and in isolated epithelia, the normal increase in transepithelial resistance after clonidine inhibition was reduced, thus opening up the possibility that FAKpY861 may be involved in tight junction responses.

for FAKpY397 in the margins between pavement cells with concentrations in a ring-shaped locus at the apical crypts of ionocytes, highlighted by immunofluorescence for Na+,K+-ATPase alpha subunit (Fig. 7A). Perinuclear cytosolic colocalization of immunofluorescence of FAKpY397 and Na+,K+-ATPase was observed (Fig. 7B). Hypotonic shock for 5 min consistently enhanced the FAKpY397 immunofluorescence and initiated perinuclear cytosolic fluorescence in pavement cells and increased the marginal immunofluorescence between pavement cells (Fig. 7C). However, the effect was transient and at 60 min of hypotonic exposure the immunofluorescence was less obvious in apical crypts and cell margins and absent from the perinuclear areas of pavement cells (Fig. 7E and F) (N = 6 for experimental and 12 for control). As in previous control membranes, FAKpY397 was detected in the pavement cell margins, apical crypt and perinuclear cytosolic region of ionocytes (Fig. 8A, B). Sixty minute treatments with Y15, 50 μM (Fig. 8C, D, N = 8) and Y15, 200 μM (Fig. 8E, F, N = 6) prompted the dephosphorylation of FAKpY397 resulting in undetectable immunofluorescence in the apical crypt, in the pavement cell margins and in the perinuclear cytosolic area of ionocytes, whereas Y15 treatment had no effect on cytosolic Na+,K+-ATPase immunofluorescence (N = 6 for experimental and 14 for control).

4.2. Congruence with mammalian systems Our results are in accord with previous human cell findings: 1) dasatinib, another cSRC inhibitor, dephosphorylates FAKpY576, FAKpY577 and FAKpY861, but does not dephosphorylate FAKpY397, the mammalian autophosphorylation site (Caccia et al., 2010; Roseweir et al., 2016), 2) cSRC expression is associated with tyrosine phosphorylation of FAKpY407, FAKpY576, FAKpY577 and FAKpY861 in tumor cell line KM12c (Brunton et al., 2005), 3) saracatinib inhibits growth and invasion of thyroid cancer cell lines, associated with dephosphorylation of FAKpY861 (Schweppe et al., 2009), 4) squamous cell carcinomas also are inhibited by saracatinib apparently via dephosphorylation of FAKpY861 (Ammer et al., 2009) and 5) Our observation that phosphatase inhibition by okadaic acid impedes the hypertonic recovery of Isc is consistent with the previously-established close complex of CFTR and protein phosphatase 2A in human airway epithelial cells (Thelin et al., 2005). Mammalian systems involving FAK

4. Discussion 4.1. Support for FAK, p38MAPK, cSRC model The most important finding of the present study was that FAK inhibition by Y15, a small molecule inhibitor of FAK, disrupts the regulation of ion transport by osmotic stimuli, specifically a partial block of the stimulation of ion transport by increases in osmolality of bathing solutions in the isolated opercular epithelium. The effect follows the 6

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Fig. 8. Effects of FAK inhibitor Y15 on FAKpY397. Images A,C and E are at the plane of the ionocyte tight junctions and apical crypts, while images B,D and F are the same frame but 5.0 μm deeper into the epithelium. A) Control opercular epithelia at 60 min showed presence of Na+,K+-ATPase (red) in ionocytes and FAKpY397 (green) in pavement cell margins and apical crypts (white arrowheads). B) Merged image showed colocalization of Na+,K+-ATPase and FAKpY397 in ionocytes (yellow) 5 μm below the surface (N = 14). Granular leucocytes in the epithelium (white asterisk, B) were positive for FAKpY397. C,D) Dephosphorylation of FAKpY397 occurred following a 60 min incubation period with 50 μM Y15. There was an absence of green immunofluorescence in the apical crypts (C), pavement cell margins (C) and ionocytes (D, N = 8). E,F) Similar results were observed following a 60 min incubation period with 200 μM Y15. There was no FAKpY397 immunofluorescence in the apical crypts and pavement cell margins with only Na+,K+-ATPase detected in the perinuclear cytosolic area of the ionocytes (E,F, N = 6). Scale bars are 10 μm.

Fig. 7. Effects of hypotonicity on FAK pY397 phosphorylation. A) Control opercular epithelia showed presence of Na+,K+-ATPase (red) in ionocytes and FAKpY397 (green) in pavement cell margins and apical crypts (white arrowheads). B) Merged channels showed colocalization of Na+,K+-ATPase and FAKpY397 in ionocytes (yellow) evident 5 μm below the surface (N = 12, A-B). C) Following a 5 min hypotonic shock, apical crypts revealed strong FAKpY397 immunofluorescence, but with diffuse fluorescence in pavement cells, yet stronger fluorescence in ionocytes (yellow in D) in comparison to the controls (N = 6). E) Following a 60 min hypotonic shock FAKpY397 remained present in apical crypts and fluorescence was diffuse in the pavement cells. However there was no apparent FAKpY397 immunofluorescence in the ionocytes; Na+,K+ATPase red fluorescence only in F (N = 6). Granular leucocytes in the epithelium (white asterisks, A-D) were positive for FAKpY397. Scale bars are 10 μm.

generally go through initial autophosphorylation at Y397 often followed by SRC family kinase phosphorylation of FAK at Y576 and Y577 (Calalb et al., 1995; Brunton et al., 2005; Lie et al., 2012).

epithelium, FAKpY407, present in apical crypts of ionocytes, is associated with increased ion transport and is dephosphorylated by cell swelling (hypotonic shock) simultaneously with inhibition of ion transport and is rapidly rephosphorylated in the apical crypts simultaneously with restoration of ion transport (Marshall et al., 2009). In the present work, FAKpY397, also present in the apical crypts of ionocytes, transiently increases phosphorylation after hypotonic shock in pavement cells and apical crypts (opposite to the response of FAKpY407), followed by a regressive dephosphorylation. Hence the opercular epithelium suggests a biphasic response of FAKpY397 operating opposite to FAKpY407 which implies that FAK responds to changes in osmolality in two distinct and opposing ways. Our results are similar to the two previous studies (Lim et al., 2007; Lie et al., 2012) in that the responses are opposite to each other. Because of the strong FAKpY397 and FAKpY861 immunofluorescence in the pavement cell margins of (but not FAKpY407 or FAKpY576) and the decrease in transepithelial electrical resistance by saracatinib, coincident with dephosphorylation of FAKpY861, our results suggest that FAK may have an additional regulatory role in maintenance of tight junctions in ion transporting epithelia. In the apical crypts of ionocytes, the location of ion transport regulation, there is osmosensitive FAKpY407 (present work and

4.3. Relationship between FAKpY397 and FAKpY407 FAKpY407, a cSRC phosphorylation target site (Ciccimaro et al., 2006), has been examined in mammalian cell systems along with the more well-described FAKpY397 autophosphorylation site. In NIH3T3 (mouse embryonic fibroblast) cells, FAKpY407 is associated with contact inhibition, cell cycle arrest and is invoked by serum starvation, whereas FAKpY397 is associated with cell adhesion, cell proliferation and cell migration, implying opposite effects of these two tyrosine phosphorylation sites (Lim et al., 2007). In the seminiferous epithelium of the rat testis, FAKpY407 negatively regulates FAKpY397 such that a nonphosphorylatable mutant (Y407F) increased FAKpY397, whereas the phosphomimetic mutant (Y407E) downregulated FAKpY397, increased transepithelial resistance and increased expression of the tight junctional protein occludin, consistent with a role for FAKpY407 in maintaining integrity of the blood-testis barrier junctions (Lie et al., 2012). Previously, we demonstrated that in an ion-transporting 7

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FAK at Y407 and leaves Y861 unchanged (Marshall et al., 2008, 2009, present study). Return to isotonic conditions (or application of hypertonic shock) rebounds Isc, rephosphorylates FAK at Y407 (Marshall et al., 2008, present study) while phosphorylation at Y397 decreases and Y861 is unchanged (present study). In contrast, at the basolateral membrane under resting conditions FAK is phosphorylated at Y397 and Y407 (Y861 is absent), colocalized with basolateral NKCC1 (Marshall et al., 2008, present study). Hypotonic shock dephosphorylates FAK at Y407 (Marshall et al., 2008), while Y397 increased (present study). Return to isotonic conditions or application of hypertonic shock rephosphorylates Y407 while Y397 returns to control levels (Marshall et al., 2008, present study). Thus, FAK is involved at both apical and basolateral locations, is osmosensitive and the responses of FAKpY407 and FAKpY397 at the apical membrane are distinct and suggest opposing actions.

Marshall et al., 2009), as well as osmotically unresponsive FAKpY576 and FAKpY861 (Marshall et al., 2009), reinforcing the notion that FAKpY407 is involved with transporter regulation. Our results do not clearly indicate which, if any, of the forms of FAK control the special cation-selective pore-junctions that are essential for Na+ secretion to accompany transcellular Cl− secretion by the marine teleost gill and opercular epithelia (Marshall et al., 2018). 4.4. FAK and cell volume regulation In mammalian heart, FAK has been associated with volume activated, outward rectifying anion channel (VSOAC) activation and FAK/ SRC inhibition paradoxically enhances this Cl− current (Walsh and Zhang, 2005), whereas a similar stimulus in the opercular epithelium dephosphorylates FAK specifically at Y407 and deactivates CFTR anion channel (Marshall et al., 2009). In mammalian nerves, hypotonic shock instead increases overall phosphorylation of FAK coincident with taurine efflux (Lezama et al., 2005), characteristic of regulatory volume decrease response. In contrast, FAKpY397 dephosphorylates in hypotonic shock of Ehrlich ascites tumor cells but FAK is not connected to activation of acid-sensitive K+ channels of regulatory volume decrease (Kirkegaard et al., 2010). A closer examination of site-specific FAK phosphorylation in different tissue types may reveal a common osmosensitive phosphorylation site on FAK.

Acknowledgements Supported by NSERC Canada through discovery grant to W.S.M., USRA scholarship to K.R.B. and L.P. Chiasson scholarships to B.F. and M.E.F. The JLA20, R26.4c and α5 antibodies (developed by Lin, J.J.-C., Goodenough, D.A. and Fambrough, D.M., respectively) were obtained from the Developmental Studies Hybridoma Bank, created by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) of the National Institutes of Health (NIH) and maintained at The University of Iowa.

4.5. CFTR and FAK

Declaration of Competing Interest

CFTR in human and killifish have a PDZ binding domain in the carboxy terminus (Singer et al., 1998) and via this domain appears to interact with the regulatory protein Na+/H+ exchanger regulatory factor (NHERF) (Li et al., 2005; Naren et al., 2003) and ablation of this domain enhances surface expression by slowing endocytic recycling of CFTR (Peter et al., 2002; Swiatecka-Urban et al., 2002). The two NHERF subunits in turn interact via a FERM-binding domain at the carboxy terminus with ezrin-radixin-moesin via a FERM domain on ezrin. Thus the concept of a regulatory complex involving FERMbinding domains preexists with an apparent stoichiometry of CFTR:NHERF:ezrin in a ratio of 2:1:1 (Li et al., 2005). As with NHERF, FAK possesses the FERM-binding domain at the carboxy terminus (Parsons, 2003) and thus may form an alternate complex with CFTR and ezrin. This is reasonable in the context of seawater fish, as apical membranes of seawater ionocytes are known to contain apical Na+/H+ exchanger, and presumably NHERF, in context of acid-base balance (review Hwang et al., 2011). Because rephosphorylation of FAKpY407 is a common feature of cAMP-dependent (forskolin, isoproterenol, IBMX) and cAMP-independent hyperosmotic stimulation of CFTRmediated Cl− secretion, we believe the activation pathways converge on FAK, thus implying a close relationship between FAK and CFTR. CFTR structure has recently been resolved by cryo-EM and phosphorylation of the regulatory (R) domain is essential to remove its inhibition of dimer formation of the two nucleotide binding domains (NBDI and II) that allow channel opening (Zhang and Chen, 2016). CFTR is activated by R domain phosphorylation by ser/thr kinases, classically by PKA and PKC, as well as by serum and glucocorticoid activated kinase (SGK1) in mammalian systems and in mummichog opercular epithelium (Sato et al., 2007; Shaw et al., 2007, 2008). The phosphorylation of CFTR by tyrosine kinases (FAK and PYK2) is still not completely understood, but the conserved potential FAK/PYK2 target sites in the R domain are Y625 and Y627 (Billet et al., 2016a).

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