Thyroid hormone (T3) increases Na-K-ATPase activity in rat adult alveolar type II cells via a PI3K-dependent pathway. In these cells, dopamine and β-adrenergic agonists can stimulate Na-K-ATPase activity through either PI3K or MAPK pathways. We assessed the role of the MAPK pathway in the stimulation of Na-K-ATPase by T3. In the adult rat alveolar type II-like cell line MP48, T3 enhanced MAPK/ERK1/2 activity in a dose-dependent manner. Increased ERK1/2 phosphorylation was observed within 5 min, peaked at 20 min, and then decreased. Two MEK1/2 inhibitors, U0126 and PD-98059, each abolished the T3-induced increase in the quantity of Na-K-ATPase α1-subunit plasma membrane protein and Na-K-ATPase activity. T3 also increased the phosphorylation of MAPK/p38; however, SB-203580, a specific inhibitor of MAPK/p38 activity, did not prevent the T3-induced Na-K-ATPase activity. SP-600125, a specific inhibitor of the MAPK/JNK pathway, also did not block the T3-induced Na-K-ATPase activity. Phorbol 12-myristate 13-acetate (PMA) significantly increased ERK1/2 phosphorylation and Na-K-ATPase activity. The PMA-induced Na-K-ATPase activity was inhibited by U0126. These data indicate that activation of MAPK-ERK1/2 was required for the T3-induced increase in Na-K-ATPase activity in addition to the requirement for the PI3K pathway.
- thyroid hormone
na-k-atpase (sodium pump) is an ubiquitous transmembrane heterodimer composed of a catalytic α-subunit and a glycosylated β-subunit (10). The ouabain-inhibitable α-subunit exchanges intracellular Na+ for extracellular K+ (9). The Na-K-ATPase plays a crucial role in maintenance of cellular ion homeostasis and is of particular importance in transporting epithelia (44). In lung tissue, alveolar Na-K-ATPase is essential for active transepithelial resorption of Na+ ions and alveolar fluid to keep the alveolar space free from the excessive fluid caused by pathological reasons such as acute lung injury or acute respiratory distress syndrome (43). The accumulation of fluid in the alveolar space may be life threatening because it impairs alveolar oxygen exchange. Active Na+ absorption across lung epithelium is the primary mechanism for the removal of lung fluid (31) and is driven by increases of both apical Na+ conductance and Na-K-ATPase activity in alveolar epithelial cells (AECs). Na+ enters the AECs primarily through apical membrane amiloride-sensitive or -insensitive Na+ channels and is extruded actively through the ouabain-sensitive Na-K-ATPase in the basolateral plasma membrane (30, 35). Adenovirus-mediated overexpression of Na-K-ATPase β1-subunit increases the active Na+ transport across monolayers of AECs (9) and augments lung liquid clearance in normal and injured rat lungs (1, 8). In contrast, decreased expression of both the α1- and α2-subunits of the Na-K-ATPase reduces maximal alveolar epithelial fluid clearance (29).
Mitogen-activated protein kinases (MAPKs), a family of kinases, regulate essential cellular activities in the response to extracellular stimuli. Three MAPK pathways have been characterized, including the extracellular signal-regulated kinases (ERK1 and 2), the Jun-NH2-terminal kinases (JNK1, 2, and 3), and the p38 isoforms (α, β, γ, and δ) (5, 40). All MAPKs are highly conserved serine/threonine kinases that are activated through phosphorylation of a T-X-Y motif. Activation is performed by upstream MAPK kinases, a family of conserved dual specificity kinases that are, in turn, activated by MAPK kinase (MKK) kinases. In general, each group of MAPKs is activated by two homologous MKKs: MEK1 and 2 activate the ERKs, JNK kinases 1 and 2 (JNKK1 and 2 or MKK4 and 7) activate the JNKs, and MKK3 and 6 activate the p38s (5, 19, 23). Upstream activators of the MAPK pathways include small GTPases of the Ras family, and downstream effectors include transcription factors and other kinases (21, 25, 33).
Activation of MAPK-ERK by hormones and other stimuli is involved in regulation of Na-K-ATPase activity in AECs and other cell types. In vascular smooth muscle cells, angiotensin II increases Na-K-ATPase activity and gene transcription of α1-subunit with either acute (10 min) or prolonged (24 h) treatment via activation of phosphatidylinositol 3-kinase (PI3K) and ERK1/2 pathways (25). Inhibition of ERK1/2 blocked the insulin-promoted stimulation of Na-K-ATPase in skeletal muscle and renal cells (2, 38, 49). In adult AECs, fibroblast growth factor-10 enhances Na-K-ATPase activity via the Grb2-sos/Ras/MAPK/ERK pathway (45), β-adrenergic agonists upregulate Na-K-ATPase activity via an adenosine 3′,5′-cyclic monophosphate (cAMP)-PKA pathway and MAPK/ERK pathway (37), and dopamine enhances Na-K-ATPase activity and its cell surface insertion through the PKC (39) and MAPK/ERK pathways (17, 18).
In Hela and CV-1 cells, thyroid hormone l-thyroxine (T4) rapidly increases tyrosine phosphorylation and nuclear translocation (activation) of MAPK-ERK1/2 (28). Hypothyroidism decreases phosphorylated, but not total ERK1/2 in the CA1 region of the hippocampus in rats; this decrease may contribute to the hypothyroidism-induced impairment of late long-term potentiation in CA1 (16). However, the effects of thyroid hormone on activation of MAPK-ERK1/2 pathway in AECs are unknown. Previously, we demonstrated that T3 upregulates Na-K-ATPase activity in a transcription-independent manner in adult rat AECs, acting through a pathway that involved PI3K and Src kinase and accompanied by increases in the quantity of plasma membrane Na-K-ATPase protein (26). Since stimuli such as β-adrenergic agonists and dopamine regulate Na-K-ATPase in AEC via MAPK-dependent mechanisms, we hypothesized that T3 activation of MAPK also may be involved in T3-induced increases in activity and plasma membrane quantity of Na-K-ATPase in AECs. In this study, we sought to assess the impact of T3 on MAPK and whether MAPK were involved in the T3 effect on Na-K-ATPase. Our results demonstrate that two distinct kinase pathways are needed for activation of Na-K-ATPase by T3: both PI3K and MAPK-ERK1/2.
MATERIALS AND METHODS
3,3′,5-Triiodo-l-thyronine (T3) and protease inhibitor cocktail were purchased from Sigma. Phorbol 12-myristate 13-acetate (PMA), biotin-X-NHS (water soluble), and MAPK inhibitors U0126, PD-98059, SB-203580, and SP-600125 were purchased from Calbiochem and were used at concentrations of 20 μM in most experiments, as previously used by other investigators. Polyclonal anti-ERK1/2 and anti-phospho-ERK1/2 (P-Thr202/Tyr204) antibodies and 10× cell lysis buffer were obtained from Cell Signaling Technology. Monoclonal antibody against α1-subunit of Na-K-ATPase was 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. Bicinchoninic acid (BCA) protein assay kit was purchased from Pierce.
The adult rat alveolar type II (ATII)-like cell line MP48 was kindly provided by G. Hunninghake (Univ. of Iowa) (36). This cell line exhibits characteristics of ATII cells, such as their phospholipid synthesis profile and lipid-containing inclusion bodies. MP48 cells 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, cells were grown to ∼85% 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 of Bio-Rad AG 1-X8 resin/100 ml serum) as described by Samuels et al. (41), along with antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin). The cells then were incubated with indicated concentrations of T3 for a variety of time periods in Waymouth's MB752/1 medium with 1% hormone-stripped FBS.
In experiments performed to detect phosphorylation of ERK1/2, the cells were incubated in medium with 1% stripped FBS for 24 h before T3 treatment. Then cells were treated with 10−5 M T3 for the indicated times in medium with 1% stripped FBS.
For inhibitory experiments, cells were incubated with inhibitors for 1 h, and then cells were treated with T3 in the continuous presence of the inhibitors.
Cell lysis and Western blot.
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 β-glycerophosphate, and 1 mM Na3VO4). The lysate was drawn 10 times through a 25-gauge needle on ice for further lysis and then was 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.
Western blotting was performed as previously described (26). To reuse membranes, any 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 1× TBST buffer (20 mM Tris·HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 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 (27). The biotinylated proteins were eluted from the streptavidin agarose beads by incubation of the beads for 10 min at ∼100°C in 50 μl of SDS-containing buffer (5.6% SDS, 240 mM Tris·HCl, pH7.5, 6% 2-mercaptoethanol, 16% glycerol, and 0.008% bromophenol 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 streptavidin beads, but none was detected.
Na-K-ATPase activity assay.
The hydrolytic activity of Na-K-ATPase was determined based on the ouabain-sensitive ATP hydrolysis under maximal reaction velocity (Vmax) conditions by measuring the release of inorganic phosphate (Pi) from ATP as previously described (27). 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.
All data are expressed as means ± SD of a minimum of three or more 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.
T3-induced Na-K-ATPase stimulation is blocked by the MEK1/2 inhibitor, but not by inhibitors for p38 or JNK.
Previously, we demonstrated that T3 increased Na-K-ATPase activity in a transcription-independent manner in MP48 cells (27). Others have shown that T3 also activates MAPK/ERK1/2 in CV-1 and Hela cells (28). Activation of MAPK/ERK1/2 in AECs is required for increase in Na-K-ATPase activity stimulated by the hormone dopamine or β-adrenergic agonists (17, 18). We predicted that T3-induced Na-K-ATPase activity in AECs requires activation of MAPK. To test this hypothesis in MP48 cells, the effects of specific inhibitors for each of the three MAPK pathways on T3-induced Na-K-ATPase activity were assessed. The experimental combination of 10−5 M of T3 and a 6-h time point was used because T3 increased Na-K-ATPase activity in AECs with maximum effects at this pharmacological combination (27). T3 significantly enhanced the Na-K-ATPase activity (Fig. 1) as we reported before; however, this increase was completely blocked by the MEK1/2 inhibitor U0126 at 20 μM (Fig. 1). These data indicated that T3 activation of MAPK/ERK1/2 pathway was required for T3-induced Na-K-ATPase activity in MP48 cells. In contrast, neither the inhibitors SB-203580 of MAPK/p38 nor SP-600125 of MAPK/JNK blocked T3-induced Na-K-ATPase activity (Fig. 1). This suggested that neither MAPK/p38 nor MAPK/JNK were involved in the effect of T3 on Na-K-ATPase activity.
T3 increased ERK1/2 phosphorylation in a dose-dependent fashion in MP48 cells.
Tyrosine phosphorylation of ERK1/2 is an indicator of MAPK-ERK1/2 activity. Thyroid hormones (T3 and T4) increased MAPK/ERK phosphorylation in CV-1 and Hela cells, respectively (28). To identify T3 activation of MAPK/ERK1/2 in AECs, the tyrosine phosphorylation level of ERK1/2 was measured in MP48 cells. T3 increased the tyrosine phosphorylation of ERK1/2 in a dose-dependent fashion from physiological concentrations of 10−8 M to pharmacological concentrations of 10−5 M (Fig. 2A). The T3-stimulated increase in ERK1/2 phosphorylation was observed within 5 min, peaked at 20 min, and then decreased over the first hour. T3 increased ERK1/2 phosphorylation by approximately fourfold at 20 min (Fig. 2B). The time course of stimulation of ERK1/2 phosphorylation by physiological concentrations of T3 (10−8 M) was the same as at 10−5 M (data not shown). The MEK1/2 inhibitor U0126 suppressed the T3-activated tyrosine phosphorylation of ERK1/2 (Fig. 2C), indicating that ERK1/2 likely was activated by T3 stimulation of MEK1/2 activity.
U0126 and PD-98059 prevented T3-stimulated cell surface expression of Na-K-ATPase.
Previously, we found that T3 increased the quantity of Na-K-ATPase subunit proteins on the plasma membrane, and this increase accounted for the T3-stimulated Na-K-ATPase activity (27). MEK1/2 is the direct upstream stimulator of ERK1/2, so inhibition of MEK1/2 activity prevents MAPK/ERK1/2 pathway activity. U0126 and PD-98059 block MEK1/2 activity specifically and are widely used to assess the involvement of MAPK/ERK1/2. To further confirm that activation of MAPK/ERK1/2 is required for T3-induced Na-K-ATPase activity in MP48 cells, the effects of the MEK1/2 inhibitors U0126 and PD-98059 on the T3-induced increase in amounts of Na-K-ATPase α1-subunit proteins at the plasma membrane were assessed. T3 (10−5 M) significantly augmented the amount of plasma membrane α1 protein at the 6-h time point; however, this increase was completely blocked by U0126 or PD-98059 (Fig. 3).
PMA increased MAPK/ERK activity and Na-K-ATPase activity.
PMA is a powerful stimulus of the MAPK/ERK1/2 pathway in CHO cells (13). PMA (100 nM) activates PKC efficiently in alveolar type II cells (12). To further confirm the role of the MAPK/ERK1/2 pathway on regulation of Na-K-ATPase activity in AECs, the effects of PMA on MAPK/ERK1/2 activation and Na-K-ATPase activity were assessed. PMA (100 nM) dramatically augmented tyrosine phosphorylation of ERK1/2 at 20 min (Fig. 4A). In the absence of T3, PMA at 50 and 100 nM also significantly increased Na-K-ATPase activity; these increases were detectable within 3 h and lasted for 24 h (Fig. 4B). Moreover, the PMA-stimulated increase in Na-K-ATPase activity was also blocked by the MEK1/2 inhibitor U0126 (Fig. 4C). Together, these data further supported the role of MAPK/ERK1/2 activation in T3-induced stimulation of Na-K-ATPase activity in AECs.
T3 activation of MAPK/ERK1/2 was required for T3-induced increase in Na-K-ATPase activity in AECs.
Previously, we demonstrated that T3 stimulated alveolar epithelial Na-K-ATPase in a posttranscriptional mechanism that increases the quantity of Na-K-ATPase subunit proteins in the plasma membrane (21, 22). T3 activated MAPK/ERK1/2 activity in MP48 cells in a dose- and time-dependent manner (Fig. 2). Moreover, two MAPK/ERK1/2 pathway inhibitors, U0126 and PD-98059, completely blocked T3-induced Na-K-ATPase activity (Fig. 1) and increased protein quantities of Na-K-ATPase α1-subunit at the plasma membrane (Fig. 3). In addition, PMA increased MAPK/ERK1/2 phosphorylation in AECs, and PMA-activated ERK1/2 was also required for the PMA-induced Na-K-ATPase activity (Fig. 4). These data all indicate that activation of MAPK/ERK1/2 has a direct role in T3-stimulated Na-K-ATPase activity in AECs.
Activation of ERK by a variety of stimuli increases the Na-K-ATPase activity in various cell types. In adult AECs, β-adrenergic agonists upregulate Na-K-ATPase activity via an adenosine 3′,5′-cyclic monophosphate (cAMP)-PKA pathway and MAPK/ERK pathway (37), and dopamine enhances Na-K-ATPase activity and its cell surface insertion through the PKC (39) and MAPK/ERK pathways (17). In human skeletal muscle cells, activation of MAPK/ERK1/2 is required for insulin-induced Na-K-ATPase activity (2). Angiotensin II activates the Na-K-ATPase in vascular smooth muscle cells through MAPK/ERK signaling pathways (11, 25, 46). Na-K-ATPase activity and transmembrane Na-K-ATPase current in renal cells were blocked by inhibition of ERK1/2 (32). Inhibition of ERK1/2 blocked the insulin-promoted stimulation of Na-K-ATPase and phosphorylation of the α-subunit of Na-K-ATPase in both skeletal muscle and renal cells (2, 49). Inhibition of ERK1/2 reduces Na-K-ATPase activity in parotid gland epithelial cells (38). With prolonged treatment, ERK1/2 also regulates the expression of the α- and β-subunit proteins of Na-K-ATPase (17, 25, 37). Thus, there is a consistent pattern in which activation of the ERK1/2 pathway upregulates Na-K-ATPase function across many tissues.
Although the inhibitor of JNK also did not inhibit the T3-induced Na-K-ATPase activity (Fig. 1), the possibility of involvement of JNK cannot be completely excluded, due to the lack of the measurement of the dose response effects of this inhibitor.
In fact, different members of the MAPK family have distinct roles depending on the cell type. In osteoblast-like MC3T3-Ei cells (20), IGF-I induced phosphorylation of ERK1/2 and p38, but not SAPK/JNK (stress-activated protein kinase/c-Jun NH2-terminal kinase). PD-98059 and U0126, specific inhibitors of the upstream kinase that activates ERK1/2, significantly suppress the IGF-I-induced alkaline phosphatase activity. In contrast, SB-203580 and PD-169316, specific inhibitors of p38, fail to affect the activity induced by IGF-I. Although T3 markedly induces the phosphorylation of p38 and ERK1/2 in osteoblasts, only MAPK/p38, but not ERK1/2, participates in T3-stimulated osteocalcin synthesis (24). Similarly, p38 but not p44/42 mitogen-activated protein kinase is required for nitric oxide synthase induction mediated by lipopolysaccharide in RAW 264.7 macrophages (6).
Interaction of T3-stimulated MAPK/ERK and PI3K pathway in regulation of Na-K-ATPase activity.
We demonstrated previously that in AECs, T3-activated PI3K was necessary for T3-induced Na-K-ATPase activity and cell surface expression of Na-K-ATPase (26). We now indentified that T3-activated ERK1/2 is also required for T3-increased activity and cell surface expression of Na-K-ATPase. How the Src kinase/PI3K/Akt and MAPK/ERK1/2 pathways interact in response to T3 and how these two pathways cooperatively regulate the stimulatory effects of T3 on Na-K-ATPase are not yet defined in AECs. However, it is reasonable to speculate that the PI3K/Akt pathway may play a different role from the MAKP/ERK pathway, possibly related to effects on the cellular machinery for translocation of Na-K-ATPase into the plasma membrane. This speculation is based on several observations. First, in human skeletal muscle cells (2), Na-K-ATPase is a good substrate for the ERK1/2; insulin-activated ERK1/2 mediated phosphorylation of the α-subunit is linked to the translocation of Na-K-ATPase protein into the cell plasma membrane. Second, the PI3K/Akt pathway is involved in the dopamine-induced endocytosis of Na-K-ATPase through the clathrin-coated vesicles in kidney epithelial cells (34, 48). Third, activation of the PI3K/Akt pathway also is involved in the clathrin vesicle-related exocytosis of glucose transporter-4 in adipocytes (22, 47).
Role of thyroid hormone receptors in T3 stimulation.
The mechanism by which thyroid hormone initiates its signal transduction is incompletely understood. In monkey fibroblasts, binding of thyroxine (T4) to the cell surface integrin αVβ3 stimulates the ERK1/2 MAPK pathway and activating thyroid hormone receptor (TR), estrogen receptor, and p53 (3, 7). In rat pituitary cells, T3 binds to TRβ on the cell surface, activating the PI3K pathway (42). In two systems, liganded TR binds to PI3K subunits, activating the PI3K pathway (4, 14, 15). Whether TR is involved in the activation of MAPK or PI3K by T3 in AECs remains to be determined.
In summary, T3 stimulated MAPK/ERK1/2 pathway in a dose- and time-dependent fashion in AECs. Activation of ERK1/2 was required for T3-induced increases in activity and plasma membrane content of Na-K-ATPase in AECs. T3-stimulated MAPK/ERK1/2 plays a necessary role in the regulation of Na-K-ATPase activity in the alveolar epithelium. This pathway may have important effects in determining the clearance of alveolar fluid during lung injury and recovery.
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