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Am J Physiol Lung Cell Mol Physiol 292: L6-L14, 2007. First published September 1, 2006; doi:10.1152/ajplung.00078.2006
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EDITORIAL FOCUS

Developmental acquisition of T3-sensitive Na-K-ATPase stimulation by rat alveolar epithelial cells

Jianxun Lei, Christine H. Wendt, Daosheng Fan, Cary N. Mariash, and David H. Ingbar

Department of Medicine, University of Minnesota Medical School, Minneapolis, Minnesota

Submitted 6 March 2006 ; accepted in final form 14 July 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Late in gestation, the developing air space epithelium switches from chloride and fluid secretion to sodium and fluid absorption. Absorption requires Na-K-ATPase acting in combination with apical sodium entry mechanisms. Hypothyroidism inhibits perinatal fluid resorption, and thyroid hormone [triiodothyronine (T3)] stimulates adult alveolar epithelial cell (AEC) Na-K-ATPase. This study explored the developmental regulation of Na-K-ATPase by T3 in fetal rat distal lung epithelial (FDLE) cells. T3 increased Na-K-ATPase activity in primary FDLE cells from gestational day 19 [both primary FDLE cells at embryonic day 19 (E19) and the cell line FD19 derived from FDLE cells at E19]. However, T3 did not increase the Na-K-ATPase activity in less mature FDLE cells, including primary E17 and E18 FDLE cells and the cell line FD18 (derived from FDLE cells at E18). Subsequent experiments assessed the T3 signal pathway to define whether it was similar in the late FDLE and adult AEC and to determine the site of the switch in responsiveness to T3. As in adult AEC, in the FD19 cell line, the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin blocked the T3-induced increase in Na-K-ATPase activity and plasma membrane quantity. T3 caused a parallel increase in phosphorylation of Akt at Ser473 in FDLE cells from E19, but not from E17 or E18. In the FD18 cell line, transient expression of a constitutively active mutant of the PI3K catalytic p110 subunit significantly augmented the Na-K-ATPase activity and the cell surface expression of Na-K-ATPase {alpha}1 protein. In conclusion, FDLE cells from E17 and E18 lacked T3-sensitive Na-K-ATPase activity but acquired this response at E19. The developmental stimulation of Na-K-ATPase by T3 in rat FDLE cells requires activation of PI3K, and the acquisition of T3 responsiveness may be at PI3K or upstream in the signaling pathway.

triiodothyronine

DURING MOST OF FETAL LIFE the distal lung epithelia secretes chloride and fluid into the developing air spaces, establishing a distending pressure that is crucial for lung morphogenesis (36). However, by the time of birth, this fluid must be removed from the incipient air spaces to permit normal gas exchange (4, 33, 36). The developmental shift from alveolar epithelial fluid secretion to fluid absorption is a very important prenatal physiological change. Changes in oxygen partial pressure and the levels of hormones such as catecholamines, glucocorticoid, and thyroid hormones all may be involved in this physiological switch of fetal lung (2, 4, 33). However, the triggers and mechanisms of the developmental shift of the fetal alveolar epithelium from chloride and fluid secretion to fluid absorption are incompletely defined (17).

In the adult lung, active Na+ absorption drives liquid movement from the air spaces into the interstitium (26). Similar to the adult lung, active Na+ absorption across fetal distal lung epithelium is the primary mechanism of the removal of fetal lung fluid and occurs mainly by an increased apical Na+ conductance and Na-K-ATPase activity (4, 7, 25). Na+ enters the alveolar epithelial cells (AEC) through apical membrane amiloride-sensitive or -insensitive Na+-permeable channels (ENaCs) and is extruded actively through ouabain-sensitive Na-K-ATPase in the basolateral plasma membrane (25, 30). Mice lacking the {alpha}-subunit of ENaC die shortly after birth with fluid-filled alveoli (16). Adenovirus-mediated overexpression of Na-K-ATPase beta1-subunit increases the active Na+ transport across monolayers of fetal day 19 and fetal day 20 primary fetal rat distal epithelial (FDLE) cells, indicating that Na-K-ATPase activity can be a limiting factor for the amount of transcellular sodium transport in rat FDLE cells in late gestation (40).

The perinatal upregulation of Na-K-ATPase in lung AEC plays an important role in removing fluid from the lung during labor and after birth (for review, see Refs. 4, 7, 25, 26, 33). The quantities of fetal lung Na-K-ATPase {alpha}1- and beta1-subunit mRNA (17, 32) and protein (17) and the enzyme activity increase from gestational day 17 to gestational day 20 (17). Both ENaC (31) protein and Na-K-ATPase protein (17) also rise in the fetal lung epithelium several days before birth. Just before birth, AEC Na-K-ATPase and ENaC quantities are upregulated in parallel with increases in concentrations of catecholamine, glucocorticoid, and thyroid hormone in fetal circulation (13, 17, 29, 30, 32, 33). During normal development, fetal lung fluid clearance is partially induced by an increase in catecholamines at birth, including the surge accompanying labor (9, 13, 33); both triiodothyronine (T3) and glucocorticoid hormone are essential for the epinephrine-induced fetal lung fluid clearance at birth (3, 4, 6, 37). The fetal serum T3 concentration increases perinatally in parallel with the increases in fluid absorption and Na-K-ATPase activity (19, 29), indicating that T3 may involve the developmental regulation of function of Na-K-ATPase in fetal lung epithelium. In mature rat FDLE cells at gestational day 22, beta-adrenergic agonists, thyroid hormone (T3), glucocorticoid, and increased oxygen pressure (PO2) in culture condition increase the Na-K-ATPase activity (1, 4, 36). However, when and how the thyroid hormone functions on developmental regulation of Na-K-ATPase is not clear.

Although T3 acts primarily through receptors that control the transcription of target genes (10, 12), increasing recent evidence shows that T3 also regulates some functions via nongenomic actions that are independent of transcription (11, 12, 14). Previously, we demonstrated that T3 upregulates Na-K-ATPase activity in adult rat AEC via stimulating increases in the quantity of plasma membrane sodium pump protein in a transcription-independent manner (21) and that the T3-stimulated increase in Na-K-ATPase activity requires activation of Src family kinases and phosphatidylinositol 3-kinase (PI3K) (20).

In this study, we sought to assess the impact of T3 on Na-K-ATPase in developing FDLE cells. We assessed the timing of acquisition of the T3 stimulatory effect on Na-K-ATPase activity in rat FDLE cells in late gestation. We also explored whether the effect of T3 is transcriptional or is a more direct induction via a signaling pathway similar to that seen in the adult AEC. We found that the Na-K-ATPase enzyme became sensitive to T3 stimulation at gestational day 19 and that T3 stimulation of Na-K-ATPase activity in FDLE cells was transcription independent and required activation of PI3K.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
T3, protease inhibitor cocktail, elastase, and DNase were purchased from Sigma. Biotin-X-NHS (water soluble) was purchased from Calbiochem. Polyclonal anti-PKB/Akt and anti-phospho-PKB/Akt (Ser473) antibodies and 10x cell lysis buffer were obtained from Cell Signaling Technology. Polyclonal anti-thyroid hormone receptor {alpha}1 and monoclonal anti-thyroid hormone receptor beta1 were purchased from Santa Cruz Biotechnology. Monoclonal antibodies against {alpha}1- and beta1-subunits of Na-K-ATPase and monoclonal anti-PI3K p85 subunit were purchased from Upstate Biotechnology. Cell culture reagents, including Waymouth's MB752/1 medium, DMEM/F-12 medium, HEPES, FBS, and antibiotics, were purchased from GIBCO/BRL Life Technologies. Nitex mesh (120 and 40 µm) was purchased from Tetko. Bicinchoninic acid (BCA) protein assay kit was purchased from Pierce.

Isolation and culture of rat AEC. Adult primary alveolar type II (ATII) cells were isolated from pathogen-free adult male Sprague-Dawley rats (190–220 g) as described by Jiang et al. (18) and maintained in DMEM/F-12 medium with 10% FBS and antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin). Identification of ATII cells was based on the presence of lamellar bodies in cells by electron microscopy and tannic acid-osmium staining.

Rat primary FDLE cells were isolated using the protocol described by Mallampalli et al. (23) with modification. Lungs were resected from rat fetuses and were inflated twice with solution (in nM: 140 NaCl, 5 KCl, 2.5 NaH2PO4, 1.3 MgSO4, 2.0 CaCl2, 6 glucose, and 10 HEPES) and were then minced into ~1-mm fragments in the above solution. Cells were dispersed using 4.3 U/ml elastase and 150 µg/ml DNase. The resulting cell suspension was filtered through 120- and 40-µm Nitex mesh. The cell filtrate was then centrifuged to obtain a pellet and resuspended in Waymouth's MB752/1 medium with 10% FBS. The harvested cells were incubated in 100-mm tissue culture dishes for 1 h at 37°C to remove the fibroblasts. The nonadherent cells (i.e., primary FDLE) were then replated on tissue culture dishes for experimental use. Primary FDLE cells were maintained in Waymouth's MB752/1 medium with 10% FBS and antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin). Both male and female fetuses at 17-, 18-, 19-, and 20 days of gestation from timed-pregnant Sprague-Dawley rats (term = 22 days) were used. All studies on rats were approved by the University of Minnesota Committee on Animal Research.

Cell culture. Fetal rat ATII-like cell lines FD18 and FD19 that were derived from primary rat FDLE cells at gestational days 18 and 19, respectively, were generous gifts of R. Mallampalli and G. Hunninghake at University of Iowa (23). The adult rat ATII-like cell line MP48 was kindly provided by G. Hunninghake at University of Iowa (35). The FD18, FD19, and MP48 cell lines all exhibit characteristics of ATII cells, such as phospholipid synthesis profile and lipid-containing inclusion bodies. All lines were maintained in Waymouth's MB752/1 media supplemented with 10% FBS and antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin).

To assess the effect of T3 on the hydrolytic activity of Na-K-ATPase, FD18, FD19, and MP48 cells were grown to ~90% confluence in Waymouth's MB752/1 with 10% FBS. Cells were then cultured overnight in Waymouth's MB752/1 medium plus hormone-stripped 1% FBS in which endogenous T3 and glucocorticoids previously had been removed by mixing the serum with a resin slurry (5 g Bio-Rad AG 1-X8 resin/100 ml serum) as described by Samuels et al. (38), along with antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin). The cells were then incubated with indicated concentrations of T3 in Waymouth's MB752/1 medium with 1% hormone-stripped FBS. For the primary adult ATII and FDLE cells, in contrast, the cells initially were cultured in maintenance medium with 10% FBS and antibiotics 24 h after isolation, and were then cultured in maintenance medium with 5% hormone-stripped FBS and antibiotics overnight. The cells were then incubated with indicated concentrations of T3 for indicated periods in maintenance medium with 1% hormone-stripped FBS. The T3 concentrations used in these experiments range across the physiological to pharmacological range. The lowest concentration of T3 used (10–8 M) in the presence of 1% serum has a free hormone concentration similar to the normal serum concentration of 10–9 M.

In experiments performed to detect phosphorylation of PKB/Akt, the primary and cell line cells were starved in serum-free medium for 24 or 48 h before T3 treatment, respectively. Then, cells were treated with 10–5 M T3 for 30 min in serum-free medium.

Transient transfection of AEC lines. FD18 and MP48 cells were transfected with LipofectAMINE PLUS Reagent (Life Technologies, GIBCO BRL) in six-well plates using 2 µg of plasmid DNA per well according to the manufacturer's instructions. The transfected cells were cultured in medium with 10% FBS for 48 h. After cells were incubated for an additional 24 h in either serum-free medium (for protein phosphorylation) or 1% stripped FBS (for Na-K-ATPase activity assay and biotinylation), the cells were used for Na-K-ATPase activity assay, protein phosphorylation, and biotinylation experiments. The constitutively active PI3K p110 subunit mutant in vector pSG5 was used for transfection as previously described (42).

Na-K-ATPase activity assay. The hydrolytic activity of Na-K-ATPase was determined based on the ouabain-sensitive ATP hydrolysis under Vmax conditions by measuring the release of inorganic phosphate (Pi) from ATP as previously described (21). Na-K-ATPase specific activity was calculated as the difference in Pi concentration per milligram of protein per minute in the absence and presence of 10 mM ouabain. Results were expressed as the percentage change from the control Na-K-ATPase activity from the same experiment.

Cell lysis and immunoprecipitation. The cells were lysed in lysis buffer containing 20 mM Tris·HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% (vol/vol) Triton X-100 with protease inhibitors (1 mM PMSF, 2 µg/ml pepstatin, and 10 µg/ml each of aprotinin and leupeptin), and inhibitors of phosphatases (2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, and 1 mM Na3VO4). The lysate was drawn 10 times through a 25-gauge needle on ice for further lysis and was then centrifuged at 13,000 rpm for 15 min at 4°C. The supernatant was collected, and the protein concentrations were determined using the BCA protein assay kit. Immediately after this step, equal amounts of protein were subjected to Western blotting.

Immunoprecipitation of the p85 subunit of PI3K was performed as previously described (20) with slight modification. Briefly, the cells were lysed in immunoprecipitation buffer. The immunoprecipitation buffer differs from the above lysis buffer only in the concentration of Tris·HCl (50 mM for the immunoprecipitation buffer). Equal amounts of protein from each treatment were incubated with 5 µg of anti-p85 antibody and 20 µl of protein A agarose beads in 1x immunoprecipitation buffer at 4°C overnight with gentle rocking. The immunoprecipitates were washed three times with 1x immunoprecipitation buffer. Proteins were eluted from the beads by boiling for 10 min in Western blot loading buffer and were then subjected to Western blotting.

Western blot. Western blotting was performed as previously described (20, 21). For reuse of membranes that had been Western blotted, the antibodies adherent to the membrane were removed by incubating the membrane with Restore Western Blot Stripping Buffer (Pierce) at 45°C for 1 h and subsequently washing five times in 1x TBST buffer (20 mM Tris·HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20). We assessed Akt phosphorylation with Western blotting as an indirect measure of the PI3K activity. In the adult ATII cells and the MP48 cell line, the amount of T3-induced Akt phosphorylation correlated directly with the PI3K activity as assessed by either phosphatidylinositol 3,4,5-triphosphate production or p85 phosphorylation (20).

Measurement of plasma membrane Na-K-ATPase protein. To determine the amount of Na-K-ATPase in plasma membrane, the cell surface proteins were biotinylated as previously described (21). The biotinylated proteins were eluted from the beads by incubation of the biotinylated protein-streptavidin agarose beads for 10 min in 50 µl of SDS-containing buffer (5.6% SDS, 240 mM Tris·HCl, pH7.5, 6% 2-mercaptoethanol, 16% glycerol, and 0.008% bromphenol blue) and analyzed by Western blotting as described above. The densitometric amount of protein was expressed in arbitrary units. To insure that our biotinylation method was specific for plasma membrane proteins, we probed samples for the presence of biotinylated actin that had been pulled down with avidin beads, but none was detected.

Statistics. All data are expressed as means ± SD of a minimum of three independent experiments, unless otherwise noted. In most experiments, individual data points within an experiment represent the mean of at least two replicates. Comparisons involving three or more groups were analyzed by ANOVA and post hoc pairwise comparisons. Differences between means were considered significant at P < 0.05, adjusted for the number of comparisons by Bonferroni correction.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
T3 stimulated Na-K-ATPase activity in 19-day gestation rat FDLE cells but not at earlier ages. We previously reported that T3 upregulated Na-K-ATPase activity in adult rat AEC from physiological (10–9 M) to pharmacological concentrations (10–5 M) and that the increased Na-K-ATPase activity was detected within 30 min and reached a maximal activity at 6 h (21). We hypothesized that T3 also would augment Na-K-ATPase in FDLE cells, at least once they reach the period of prebirth predominant sodium absorption. Primary cultures of FDLE cells derived from fetal rats of timed-pregnant mothers at gestational day 17 (E17), 18 (E18), 19 (E19), and (as control) adult rat ATII cells were exposed to either 10–8 M or 10–5 M of T3 for 6 h. At both concentrations, T3 had no discernible effect on Na-K-ATPase activity in E17 and E18 cells. However, Na-K-ATPase activity increased significantly in both E19 and adult AEC exposed to either 10–8 or 10–5 M T3 (Fig. 1A). These data indicated that the rat AEC acquired T3-sensitive Na-K-ATPase stimulation at fetal day 19.


Figure 1
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  		Fig. 1. Triiodothyronine (T3)-stimulated the Na-K-ATPase activity in alveolar epithelial cells (AEC) starting at gestational day 19. Ouabain-sensitive hydrolytic activity of Na-K-ATPase was measured after permeabilization of crude cell membrane preparations. The results are expressed as a percentage of controls, with means ± SD from 3 or more independent experiments. *P < 0.05, **P < 0.01. For the activity assay, the individual data points within an experiment represent the mean of at least 2 replicates. A: primary fetal distal lung epithelial (FDLE) cells and adult alveolar type II (ATII) cells were T3 starved for 24 h by being cultured in Waymouth's MB752/1 medium with 5% stripped FBS. Cells were then incubated with 10–8 or 10–5 M T3 for 6 h in Waymouth's MB752/1 medium with 1% stripped FBS. B: cells of rat AEC lines FD18, FD19, and MP48 were T3 starved for 24 h by being cultured in Waymouth's MB752/1 medium with 1% stripped FBS after cells were grown to ~90% confluence. Cells were then incubated with 10–8 or 10–5 M T3 for 6 h in Waymouth's MB752/1 medium with 1% stripped FBS.

 
To facilitate subsequent mechanistic studies, we also assessed the effect of T3 on Na-K-ATPase activity in two immortalized rat FDLE cell lines, FD18 and FD19, and the similarly derived adult rat AEC line MP48. The FD18 and FD19 cell lines were derived from rat fetal lungs at gestational days 18 and 19, respectively. Both the FD18 and FD19 cell lines exhibit many characteristics of FDLE cells, including abundant stores of glycogen, and surface binding of the lectin Maclura pomifera (23). These cell lines served as excellent models since they behaved similarly to primary FDLE of the same age and permitted greater manipulation (transfection, etc.). We found that the FD18 cell line Na-K-ATPase activity was not significantly altered by either 10–8 or 10–5 M of T3 at the 6-h time point, similar to primary E18 FDLE. However, T3 augmented significantly the Na-K-ATPase activity in either FD19 or MP48 cell lines (Fig. 1B), as in their primary cell counterparts. Thus the FD18, FD19, and MP48 cell lines each exhibited age-dependent responses to T3 similar to those of their primary cell analogs.

T3 increased PI3K activity in FDLE cells starting at 19-day gestation fetuses. In adult AEC, T3 stimulation of Na-K-ATPase activity required the activation of PI3K (20). Because PI3K activates Akt with phosphorylation of Akt at Ser473, PI3K activity is indirectly measured in AEC by the PI3K phosphorylation of Akt at Ser473 (20). We sought to determine whether the FDLE cells use a similar or different signaling pathway for T3 to stimulate Na-K-ATPase. We assessed the effect of T3 on Akt phosphorylation at Ser473 in primary cultures of E17, E18, E19, and adult AEC. Consistent with our previous results (20), T3 increased significantly the Akt phosphorylation at Ser473 in adult rat primary ATII cells and in the immortalized adult rat AEC line MP48. T3 also significantly enhanced Akt phosphorylation at Ser473 in fetal rat primary E19 FDLE cells and the FD19 cell line, but not in fetal rat primary E17 or E18 FDLE cells or in the FD18 cell line (Fig. 2). In addition, in the FD19 cells, wortmannin (50 nM) completely blocked the increase in Na-K-ATPase activity induced by 6 h of exposure to 10–5 M T3 (Fig. 3). Thus T3 stimulation of the sodium pump required PI3K activation in FDLE cells starting from gestational day 19 and suggested that the reason that T3 does not stimulate Na-K-ATPase at the earlier ages might be due to failure of activation of PI3K or an earlier point in the signaling pathway.


Figure 2
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  		Fig. 2. T3 increased phosphorylation of Akt at Ser473 in FDLE cells beginning at gestational day 19. Cells were cultured for 24 h in media with 1% FBS stripped of thyroid hormone before treatment with 10–5 M T3 for 30 min in Waymouth's MB752/1 medium with 1% stripped FBS. In A and B, the top blot shows a representative Western blot for phosphorylated Akt at Ser473, and the bottom blot shows the same Western blot reprobed (after stripping) with an antibody against total Akt. This lane represents protein loading control. The corresponding densitometric data are shown as a histogram plotting the results expressed as a percentage of controls, showing the means ± SD of 3 or more independent experiments (*P < 0.05; the open and solid bars represent the control and T3-treated groups, respectively). A: Akt phosphorylated at Ser473 by T3 in primary FDLE cells is increased at gestational day 19 (E19) and adult AEC, but not E17 or E18. B: T3 increased Akt phosphorylation at Ser473 in FDLE cells immortalized at E19 (FD19) and adult AEC (MP48), but not the FD18 cell line.

 

Figure 3
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  		Fig. 3. The effects of inhibitors on T3-induced increase in Na-K-ATPase activity in FD19 cell line. The cells were prepared as described in Fig. 1B. The cells then were incubated with inhibitor for 1 h before exposure to 10–5 M T3. The ouabain-sensitive Vmax hydrolytic activity of Na-K-ATPase was measured after cells were incubated for 6 h in 1% stripped FBS medium containing 10–5 M of T3 in the presence or absence of inhibitor. The results are expressed as a percentage of controls and are means ± SD of 3 or more independent experiments. *P < 0.05. Act-D, 10 µg/ml actinomycin D; Wort, 20 nM wortmannin; BA, 10 µg/ml brefeldin A; NS, not significant.

 
Brefeldin A, but not actinomycin D, blocked the T3-induced increase in Na-K-ATPase activity in FD19 cells. In adult ATII cells, T3 stimulation of Na-K-ATPase was nontranscriptional and was accompanied by an increase in plasma membrane Na-K-ATPase. The T3-stimulated increase in Na-K-ATPase activity was sensitive to the protein trafficking inhibitor brefeldin A (20) but not to actinomycin D (21). To explore whether the FDLE cells employ the same mechanisms of T3 response, we investigated the effects of brefeldin A and actinomycin D on T3-induced increase in Na-K-ATPase activity in the FD19 cell line. T3 (10–5 M) increased Na-K-ATPase activity significantly at the 6-h time point. This increase was completely blocked by 10 µg/ml brefeldin A but was unaffected significantly by actinomycin D (10 µg/ml) (Fig. 3). Neither brefeldin A nor actinomycin D impacted the baseline Na-K-ATPase activity compared with the control. In prior experiments with primary rat ATII cells, the half-life of the Na-K-ATPase protein subunits was relatively long, ~8–12 h (data not shown), thus the maintained basal enzyme activity is not very surprising. Thus, based on two parameters assessed, the mechanism of the T3 stimulation in FD19 cells is similar to that in the adult AEC; the T3 effect required insertion of Na-K-ATPase protein into the plasma membrane, but not transcription of Na-K-ATPase.

T3 increased the amount of plasma membrane Na-K-ATPase {alpha}1-subunit protein without altering the total amount of Na-K-ATPase subunit proteins in FD19 cells. Both Na-K-ATPase activity and the quantity of lung Na-K-ATPase {alpha}1- and beta1-isoform proteins increase gradually towards term (17, 32, 34), paralleling the increases in concentrations of epinephrine, glucocorticoids, and T3 (13, 19, 29, 33). In adult rat ATII cells, short-term exposure to T3 induced an increase in the Na-K-ATPase activity and plasma membrane protein expression but did not alter the total cellular amount of {alpha}1- or beta1-isoform proteins (21). We evaluated whether T3 altered the amount of Na-K-ATPase protein expression in the FD19 cell line, comparing the total cell amount of Na-K-ATPase {alpha}1- and beta1-isoform proteins between the T3-treated cells and control cells. T3 did not increase the amount of total cellular Na-K-ATPase {alpha}1- or beta1-subunit protein in the FD19 cell line (Fig. 4A) but did increase the plasma membrane quantity of {alpha}1-subunit (Fig. 4B). The T3-induced increase in steady-state cell surface expression of {alpha}1-subunit was blocked by either wortmannin or brefeldin A. These data indicated that T3 stimulation of Na-K-ATPase activity was due to increased plasma membrane distribution of Na-K-ATPase protein in FD19 cells, not an increase in the total amount of protein; this is similar to the observations in the adult ATII cells (21).


Figure 4
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  		Fig. 4. T3 increased the plasma membrane Na-K-ATPase {alpha}1-subunit protein but did not alter the total cellular Na-K-ATPase {alpha}1- and beta1-subunit proteins in the FD19 cell line. The cells were starved for 24 h in 1% stripped FBS medium after the cells were grown to ~90% confluence. The cells were then incubated with 10–5 M T3 for 6 h in 1% stripped FBS medium and lysed. Western blots were performed, and the densitometric results are expressed as percentage of control and are means ± SD of 3 different experiments; **P < 0.01. A: total cellular Na-K-ATPase subunit protein levels are unchanged by T3. Tubulin is shown as a loading control, and the densitometric intensities of Na-K-ATPase subunits were normalized using the corresponding tubulin signal. B: plasma membrane Na-K-ATPase {alpha}1-subunit protein amount is increased by T3, and this response is inhibited by either wortmannin or brefeldin A.

 
Increased PI3K activity is sufficient to augment the Na-K-ATPase activity and cell surface expression of Na-K-ATPase {alpha}1 protein in FD18 cells. The constitutively active mutant of PI3K catalytic subunit p110 increases Na-K-ATPase activity and cell surface expression of this enzyme in adult AEC (20). Because the PI3K inhibitor wortmannin prevented T3 stimulation of Na-K-ATPase activity and cell surface expression in FD19 cells (Figs. 3 and 4B), we hypothesized that the lack of T3 stimulation before gestational day 19 was due to the failure of T3 to activate the PI3K activity. To test whether the stimulatory pathway of PI3K was competent to increase Na-K-ATPase activity, we transiently expressed a constitutively active mutant of p110 in FD18 cells and determined whether this mutant enhanced the activity and cell surface expression of Na-K-ATPase as it did in adult AEC.

In the FD18 cell line that was transiently transfected by the active p110 mutant, but not the empty vector, Akt phosphorylation increased significantly (Fig. 5A), indicating that the active mutant of p110 was expressed and functional in FD18 cells using this experimental protocol. Transient expression of active mutant of p110 in the FD18 cells also enhanced significantly both the Na-K-ATPase activity (Fig. 5B) and the amount of Na-K-ATPase {alpha}1-subunit protein at the cell surface (Fig. 5C). Active mutant p110 also had the same effects in MP48 cells (Fig. 5, B and C). Together, these data suggested that the absence of T3 stimulation of Na-K-ATPase activity in E17 and E18 FDLE cells is due to a block in the signaling pathway at or before the PI3K step.


Figure 5
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  		Fig. 5. A constitutively active mutant of phosphatidylinositol 3-kinase (PI3K) increased the activity and cell surface expression of Na-K-ATPase in both MP48 and FD18 cell lines. FD18 and MP48 cells were incubated in 10% FBS medium without antibiotics for 48 h after cells were transfected with vector or constitutively active mutant p110. The cells then were starved overnight in medium with 1% stripped FBS. The results in B and C are expressed as percentage of controls in which cells were concurrently transfected with empty vector with bars showing the means ± SD of 3 or more independent experiments; *P < 0.05, **P < 0.01. A: constitutively active mutant p110 increased phosphorylation of Akt at Ser473. This experiment was performed twice with virtually identical results, and the findings recapitulate those in adult ATII cells. B: constitutively active mutant p110 enhanced the Na-K-ATPase activity. C: constitutively active p110 increased the amounts of Na-K-ATPase {alpha}1-subunit protein at cell surface measured by biotinylation assay with immunoprecipitation and Western blotting. The top shows representative Western blots, and the bottom shows the quantitative densitometric data and the statistical test results. Bars represent means ± SD of 3 different experiments.

 
FDLE cells from E17 onward express Na-K-ATPase, PI3K, and thyroid hormone receptors. A possible alternate explanation for the lack of T3 response in the E17 or E18 FDLE cells is that they lack thyroid hormone receptors, PI3K, or Na-K-ATPase subunit proteins. We used Western blots to qualitatively assess the expression of Na-K-ATPase, PI3K, and thyroid hormone receptor protein in FDLE cells from gestation days 17–19 and adult rat AEC. All the FDLE cells expressed the proteins of Na-K-ATPase, {alpha}1- and beta1-isoforms, PI3K p85 subunit, and thyroid hormone receptor {alpha}- and beta-isoforms (Fig. 6). Thus the lack of T3 response in E17, E18 FDLE cells, and the FD18 cell line was not due to lack of PI3K, thyroid hormone receptor, or Na-K-ATPase proteins.


Figure 6
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  		Fig. 6. Expression of Na-K-ATPase, thyroid receptors (TR), and PI3K regulatory subunit p85 proteins did not account for the lack of T3 response at E17 and E18. All proteins except p85 were detected by Western blotting. The p85 protein was detected by Western blotting after immunoprecipitation was performed using p85 antibody. The results shown are representative Western blots. Because the blots were performed on multiple different samples with different amounts of protein per lane in each experiment, the findings for qualitative and quantitative data are not presented, although each finding was confirmed at least 3 times.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present research found that the primary rat FDLE cells undergo a developmental switch with acquisition of T3-sensitive Na-K-ATPase activity late in gestation. FDLE cells at gestational days 17 and 18 did not respond to T3 with stimulation of Na-K-ATPase, but they acquire this response at gestational day 19. As in adult AEC, the E19 FDLE Na-K-ATPase response to T3 is nontranscriptional, requires PI3K, and involves protein trafficking that results in an increase in the plasma membrane amount of sodium pump protein. The nonresponsive E17 and E18 primary FDLE cells and the FD18 cell line also do not show increased Akt phosphorylation in response to T3. Overexpression of active PI3K in the FD18 cell line increased Na-K-ATPase activity and the amount of plasma membrane protein. These data suggest that in addition to there being a late gestational increase in thyroid hormone levels, there also is a time-sensitive switch in FDLE cell response to T3. This switch may play a role in the late gestational activation of Na-K-ATPase and in the shift from ion secretion to sodium and fluid absorption in preparation for birth.

FDLE cells acquired T3-sensitive Na-K-ATPase stimulation at gestational day 19. T3 increased Na-K-ATPase activity in primary cultures of both FDLE cells at gestational day 19 and adult rat ATII cells, but not in primary cultures of FDLE cells younger than gestational day 19, indicating that Na-K-ATPase in FDLE cells acquired T3 stimulatory capability at gestational day 19 (Fig. 1).

FDLE cells from all gestational day 19 litters responded to T3 stimulation. However, in one of the five experiments performed using FDLE cells isolated from E18 fetal rats, Na-K-ATPase activity was sensitive to T3 stimulation. The single outlier litter result may reflect differences in the exact mating time of the timed-pregnant mother or some biological variability in the exact timing of acquisition of the response.

In our experiments, we measured the Na-K-ATPase Vmax activity of cell membranes. The rationale for this measurement is that it best reflects the peak transport capacity of the cell. Measurement of the steady-state Na-K-ATPase activity in intact cells is difficult to interpret outside of the setting of monolayers because the sodium pump activity is highly regulated to avoid overconsumption of ATP and at the same time maintain essential cellular ion gradients. For example, with hyperoxic exposure of rat ATII cells, the Na-K-ATPase Vmax activity is decreased, whereas the steady-state activity is unchanged.

T3 increased Na-K-ATPase activity in FDLE cells via mechanism(s) similar to that of adult AEC. In adult rat ATII cells, the T3-induced increase in Na-K-ATPase activity was transcription independent and was accompanied by the increased quantities of this enzyme at the plasma membrane. T3-increased activity and cell surface insertion of Na-K-ATPase required the activation of PI3K by T3 (20, 21). As in the adult AEC, in the FD19 cell line, T3 increased plasma membrane sodium pump protein expression but did not alter the total cell content of Na-K-ATPase protein (Fig. 4). The T3-induced increase in Na-K-ATPase activity was not sensitive to the general transcriptional inhibitor actinomycin D (Fig. 3), indicating that T3 increases Na-K-ATPase activity in FDLE cells via a transcription-independent manner. T3 augmented Akt phosphorylation at Ser473 in FDLE cells at gestation day 19 and the FD19 cell line (Fig. 2). The PI3K inhibitor wortmannin abolished the T3-induced increase in Na-K-ATPase activity and plasma membrane {alpha}1-subunit protein in FD19 cells (Figs. 3 and 4B), suggesting that T3-activated PI3K activity in FDLE cells is also required for stimulation of Na-K-ATPase activity by T3 in FDLE cells. Together, both FDLE cells and adult AEC employ similar mechanisms to respond to T3 stimulation.

PI3K activation was necessary for T3 stimulation of Na-K-ATPase activity in FD19 cells. Phosphorylation of Akt at Ser473 was augmented in FDLE cells at gestational day 19 but not at gestational days 17 or 18 (Fig. 2). In parallel with the responses of Akt to T3 stimulation, Na-K-ATPase activity was increased by T3 in FDLE cells at gestational day 19 but not at gestational days 17 or 18 (Fig. 1). This parallel increase in Akt phosphorylation and Na-K-ATPase activity by T3 implied that PI3K likely was involved in the response of Na-K-ATPase activity to T3 stimulation during this developmental stage. Deficiency of activation of PI3K by T3 may account for the lack of effect of T3 on Na-K-ATPase activity in FDLE cells younger than gestational day 19. Moreover, transient expression of a constitutively active mutant of PI3K (active p110) increased significantly the activity and cell surface expression of Na-K-ATPase in FD18 cells that originally were not sensitive to T3 stimulation (Figs. 1 and 2 and Fig. 5, B and C), consistent with the view that activation of PI3K by T3 controlled the response of T3 stimulation of Na-K-ATPase activity in FDLE cells. These findings are consistent with a model in which the switch to T3 responsiveness in late gestation occurs at the PI3K step or upstream from PI3K in the signaling pathway.

T3 plays important roles during normal development. Thyroid hormone is essential for normal human development. For example, rat enterocyte T3 responsiveness is developmentally regulated (15). The role of thyroid hormone in lung development is exerted predominantly in the late phase of lung development, with effects on alveolar cell growth and surfactant production. Administration of T3 accelerates the process of septation, resulting in a greater alveolar surface area in rats (24).

The effects of thyroid hormone on gene expression depend on the developmental period in many organs. For example, thyroid hormone differentially regulated the expression of the transcription factor NGFI-A in the developing rat brain depending on age. In the piriform cortex, the expression of NGFI-A is thyroid hormone dependent on postnatal day 0 and 5, but not on postnatal day 15 (27). There are few or no effects of T3 on intestinal lactase and 3.0-kb intestinal phosphatase gene expression during the suckling period (postnatal day 10), whereas with postweaning (postnatal day 25), T3 has great effects on the expression of these two genes (15).

The sensitivity of Na-K-ATPase gene expression to T3 changes during development. The postnatal increases in Na-K-ATPase expression in kidney, brain, and lung depend on normal thyroid hormone status (28). The expression of Na-K-ATPase {alpha}3-isoform in ferret myocardium is responsive to T3 in newborns younger than 3 wk of age but not in mature ferrets older than 6 wk of age (8). In the adult ferret heart, expression of {alpha}3 mRNA is not responsive to T3 (8). In the developing rat brain, thyroid hormone increases Na-K-ATPase activity and {alpha}-subunit protein in newborn, but not in adult, rats (22, 39). Brain Na-K-ATPase activity and proteins of {alpha}-isoforms are sensitive to T3 as late as postnatal day 15, but after postnatal day 22, they no longer respond to T3 (39). Neonatal, but not adult, brain Na-K-ATPase protein levels are thyroid hormone (T3) responsive (39). Thus there are many examples of developmental windows of sensitivity of sodium pump gene expression to thyroid hormones. Interestingly, most of these models show disappearance of T3 sensitivity after a developmental period.

Although in many systems T3 exerts developmental effects on Na-K-ATPase gene transcription, there is increasing recognition that thyroid hormones can act through other nontranscriptional cellular mechanisms (11, 12, 22). For example, thyroxine linked to sepharose beads (so that it remains extracellular) can alter cell signaling pathways. Our present data suggested that FDLE acquire T3-increased Na-K-ATPase activity developmentally, after a period of nonresponsiveness. As in the adult ATII cell response to T3, this effect is nongenomic. The acquisition of T3 responsiveness is accompanied by enzyme insertion at plasma membrane and requires the T3 activation of PI3K pathway. It suggests that there is a cooperative interaction between increasing late gestational levels of thyroid hormone and "turn on" of a developmental switch in the FDLE cells; together, these effects help promote the late gestational increase in Na-K-ATPase activity and alveolar fluid clearance prebirth. The precise mechanism of the developmental switch needs further definition, but the signaling switch is likely at or upstream of PI3K in the T3 response pathway. Defining the mechanistic steps involved and how this switch works will add to the understanding of developmental regulation by hormones, particularly for this novel nongenomic T3 effect.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. H. Ingbar, Pulmonary, Allergy, & Critical Care Division, MMC 276, 420 Delaware St. SE, Minneapolis, MN 55455 (e-mail: ingba001{at}umn.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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