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EDITORIAL FOCUS

-Adrenergic agonists regulate Na-K-ATPase via p70^S6k

Division of Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611

Submitted 6 August 2002 ; accepted in final form 16 April 2003

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
We have recently reported that the -adrenergic agonist isoproterenol regulates the alveolar epithelial cell Na-K-ATPase via MAPK/extracellular signal-regulated kinase and rapamycin-sensitive pathways. Here we report that isoproterenol phosphorylated the protein S6 kinase (p70^S6k) in alveolar epithelial cells, which was inhibited by both rapamycin and the MEK1/2 inhibitor U-0126. In alveolar epithelial cells transfected with a p70^S6k dominant negative construct, isoproterenol did not increase Na-K-ATPase total protein expression, whereas in cells transfected with a rapamycin-resistant mutant, the isoproterenol-mediated increase in Na-K-ATPase was not prevented by rapamycin. Accordingly, we provide here first evidence that isoproterenol regulates Na-K-ATPase via p70^S6k in alveolar epithelial cells.

isoproterenol; rapamycin; mitogen-activated protein kinase; phosphatidylinositol 3-kinase; lung

-Adrenergic agonists have been shown to increase active Na transport in isolated rat lungs by increasing Na channels and Na-K-ATPase function (28). Isoproterenol increases (short-term) Na-K-ATPase activity by recruiting Na pumps from intracellular pools to the AEC basolateral membranes within 15 min (3). However, the long-term mechanisms of Na-K-ATPase regulation by -adrenergic agonists have not been fully elucidated. Recently, the enhancement of Na-K-ATPase gene expression by -adrenergic agonists in alveolar epithelial cells has been reported (19). We have reported that isoproterenol posttranscriptionally (long-term) increased Na-K-ATPase protein abundance via MAPK/extracellular signal-regulated kinase (ERK) and rapamycin-sensitive pathways in AEC (23).

The mammalian target of rapamycin (mTOR) has been involved in the regulation of translation initiation by being upstream of the protein S6 kinase (p70^S6k) and key proteins that regulate the initial ribosomal recruitment to the mRNA. The p70^S6k is a complex enzyme (25) that targets the S6 protein, part of the 40S ribosomal complex, to regulate the translation of 5'-terminal oligopyrimidine tract (5'-TOP) mRNAs, mostly ribosomal proteins and elongation factors (11, 13, 26). However, it has been described that p70^S6k phosphorylates eIF4B, which may be important in regulating the translation of mRNAs with extensive 5'-untranslated region (UTR) secondary structure (1, 11).

It has been reported that MAPKs regulate the p70^S6k (9, 12, 40) and also that -adrenergic agonists stimulate the MAPK/ERK and the S6 kinase in different cell lines (23, 24). Thus we set out to determine whether the -adrenergic agonist isoproterenol regulates the alveolar epithelial Na-K-ATPase via the p70^S6k.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
Materials. Rapamycin was purchased from Calbiochem (La Jolla, CA), isoproterenol was purchased from Sigma (St. Louis, MO), U-0126 and anti-ERK antibody were purchased from Promega (Madison, WI), Elk-1 fusion protein and antibodies against phospho (P)-(Thr²⁰²/Tyr²⁰⁴)-p44/42, P-Elk-1, P-(Thr³⁸⁹)p70^S6k, and total p70^S6k were purchased from New England Biolabs (Beverly, MA). Antibody against Na-K-ATPase ₁-subunits was purchased from Upstate Biotechnologies (Lake Placid, NY). The p70^S6k constructs were a generous gift of Dr. G. Thomas.

Cell isolation and culture. Animals were handled according to National Institutes of Health guidelines, and experimental protocols were approved by our institutional Animal Care and Use Committee. AEC were isolated from pathogen-free male Sprague-Dawley rats (200-225 g) as previously described (3, 23) according to the method of Dobbs et al. (7a). Briefly, the lungs were digested with elastase (3 U/ml; Worthington Biochemical, Freehold, NJ), AEC were purified by differential adherence to IgG-pretreated dishes, and cell viability was assessed by trypan blue exclusion (>95%). Cells were resuspended in DMEM (Cellgro) containing 10% fetal bovine serum with 2 mM glutamine, 200 U/ml penicillin, 200 µg/ml streptomycin, and 0.5 µg/ml amphotericin B. Cells were seeded in 6-cm plates at 8 million per plate or 5 million per plate (for transfection experiments) and incubated in a humidified atmosphere of 5% CO₂-95% air at 37°C. The day of isolation was designated cultured day 0. Most experimental conditions were conducted in day 3 serum-starved cells. They were treated for 3 days with isoproterenol and processed on day 6. Experiments utilizing transient transfections were performed on day 4, treated for 3 days with isoproterenol, and processed on day 7. Medium was changed every 2 or 3 days. Each experiment corresponds to a different isolation.

Recombinant plasmids. These constructs were tagged by the insertion of the myc 9E10 epitope immediately after the p70^S6k isoform initiator ATG codon as previously described (20).

Transient transfections. Day 2 alveolar type II cells were transiently transfected with either pRK5-myc-p70^S6k (wild-type p70^S6k), myc-d/ED3E-pRK5 (rapamycin-resistant mutant), or myc-2BQ pRK5 (dominant negative) recombinant plasmids. These plasmids have been described elsewhere (20, 22). AEC were transfected with Lipofectin (Invitrogen Life Technologies, Carlsbad, CA) as described by the manufacturer. Expression of the plasmid was allowed to occur for 48 h, after which cells were serum starved for 8 h and then incubated in the presence or absence of isoproterenol with or without rapamycin. A limitation of our experiments is that we did not determine the transfection efficiency in these cells.

Western blot analysis. AEC were treated for the desired times, medium was aspirated, cells were washed twice with cold PBS, and total protein was isolated with 0.5 ml of lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM -glycerolphosphate, 1 mM Na₃VO₄, 1 µg/ml leupeptin, 1 mM PMSF; New England Biolabs) per 60-mm plate and centrifuged at 14,000 g. Total cell lysate protein concentration was measured by the Bradford assay (Bio-Rad, Hercules, CA), resolved in 10 or 12% SDS-PAGE, and transferred to nitrocellulose membranes (Optitran; Schleicher and Schuell, Keene, NH). Incubation with specific antibodies was performed overnight at 4°C. Blots were developed with Western Lightning Chemiluminescence Reagent Plus (Perkin Elmer Life Sciences, Boston, MA) used as recommended by the manufacturer. Bands were analyzed by densitometric scan (Stratagene EagleSight, La Jolla, CA).

Determination of ERK activity. AEC seeded in 6-cm plates were serum starved for 18-24 h and treated for the desired times. Cells lysates were obtained as described, and the ERK activity was determined as described in the p44/p42 MAPK assay kit manual by New England Biolabs, which is based on the immunoprecipitation of the active ERK with a phosphospecific p44/p42 MAPK (Thr²⁰²/Tyr²⁰⁴) monoclonal antibody using an in vitro kinase assay with Elk-1 as substrate. Samples (25 µl) were fractionated in a 12.5% SDS-PAGE and analyzed by immunoblotting with a phospho-specific Elk-1 antibody (New England Biolabs) as a probe.

Statistical analysis. Data are reported as means ± SE. Statistical analysis was done by one-way ANOVA and Tukey's correction. Results were considered significant when P < 0.05.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
Isoproterenol regulates Na-K-ATPase via MAPK/ERKs and rapamycin-sensitive mechanisms. As shown in Fig. 1, AEC incubated in the presence of isoproterenol for 3 days had increased Na-K-ATPase ₁-subunit total protein abundance. This effect was inhibited by the MEK1/2 inhibitor U-0126 and by the mTOR inhibitor rapamycin. No changes in cell morphology or viability were noticed in the cells incubated with these inhibitors. Also, no changes were observed in actin abundance, which was used as an internal control (data not shown). Measurements of protein concentration between samples were not different when measured by the Bradford assay.

View larger version (28K): [in this window] [in a new window]   Fig. 1. Isoproterenol (ISO) regulates Na-K-ATPase via a MAPK/extracellular signal-regulated kinase (ERK) and rapamycin (Rapa)-sensitive mechanism in alveolar epithelial cells (AEC). Serum-starved AEC were incubated in the presence or absence of 1 µM ISO for 3 days. When indicated, Rapa (50 ng/ml) or U-0126 (U0; 10 µM) was added 30 min before ISO. Total cell lysates were analyzed by Western blotting against the Na-K-ATPase 1-subunit. Representative blot (A) and composite graph of 3 independent experiments (B) are shown. Data are expressed as means ± SE; *P < 0.05 compared with controls (CT).

Isoproterenol activates the MAPK/ERKs via phosphatidylinositol 3-kinase, epidermal growth factor receptor-dependent, and mTOR-independent mechanisms. It has been described that p70^S6k is downstream of the phosphatidylinositol 3-kinase (PI 3-kinase) pathway (30, 36) and that -adrenergic agonists activate the MAPK pathway by using epidermal growth factor receptor (EGFR) as a scaffold protein (18). As shown in Fig. 2A, both wortmannin and PD-153035 inhibited the isoproterenol-mediated MAPK/ERK phosphorylation, suggesting that both the PI 3-kinase and the EGFR are upstream of the MAPK/ERKs in AEC. To determine whether the MAPK/ERK activation was rapamycin sensitive, AEC were preincubated in the presence or absence of rapamycin for 30 min before isoproterenol. As shown in Fig. 2B, the ERK activation was not inhibited by rapamycin, suggesting that mTOR is downstream of the MAPK/ERKs.

View larger version (22K): [in this window] [in a new window]   Fig. 2. ISO-mediated ERK activation is phosphatidylinositol 3-kinase (PI 3-kinase) and epidermal growth factor receptor (EGFR)-dependent and -independent of mammalian target of rapamycin (mTOR). A: serum-starved AEC were incubated in the presence or absence of 1 µM ISO for 10 min. When indicated, 100 nM wortmannin (Wort) or 0.25 nM PD-153035 (PD) were added 30 min before ISO. Total cell lysates were analyzed by Western blotting against phospho (P)-(Thr202/Tyr204)-ERK1/2 and membranes were stripped and reprobed against total ERK. Composite graph of 3 independent experiments and representative blots are shown. Data are expressed as means ± SE with values normalized to total ERK. ***P < 0.001 compared with controls. B: serum-starved AEC were incubated in the presence or absence of 1 µM ISO for 10 min. Rapa (50 ng/ml) was added 30 min before ISO when indicated. ERK activity was determined as phosphorylation of the transcription factor Elk-1, as indicated in MATERIALS AND METHODS. Representative blot and composite graph of 3 independent experiments is shown. **P < 0.01 compared with controls. ns, Not significant.

Isoproterenol phosphorylates p70^S6k via a MAPK/ERK-dependent pathway. As shown in Fig. 3A, isoproterenol phosphorylated p70^S6k within 10 min of incubation and remained phosphorylated in cells incubated with isoproterenol for up to 4 days (Fig. 3B). Both the MEK inhibitor U-0126 and rapamycin prevented isoproterenol-mediated p70^S6k phosphorylation in AEC, indicating that isoproterenol phosphorylates p70^S6k via a MAPK/ERK- and mTOR-dependent pathway.

View larger version (19K): [in this window] [in a new window]   Fig. 3. ISO phosphorylates the protein S6 kinase (p70S6k) via a MAPK/ERK-dependent pathway in AEC. A: serum-starved AEC were incubated in the presence of 1 µM ISO for the indicated times, and total cell lysates were analyzed by Western blotting against P-(Thr389)-p70S6k. Membranes were stripped and reblotted against total p70S6k. Representative blots and composite graph of 4 independent experiments are shown. Results were normalized to P-p70S6k/total p70S6k and are expressed as means ± SE. *P < 0.05 compared with controls. B: serum-starved AEC were incubated in the presence of 1 µM ISO for 4 days. When indicated, Rapa (50 ng/ml) or U-0126 (10 µM) were added 30 min before ISO. Total cell lysates were analyzed by Western blotting against P-(Thr389)-p70S6k. Membranes were stripped and reblotted against total p70S6k. Representative blots and composite graph of 4 independent experiments are shown. Results were normalized to P-p70S6k/total p70S6k and are expressed as means ± SE. **P < 0.01 compared with controls.

Isoproterenol regulates Na-K-ATPase via p70^S6k. As shown on Fig. 4A, when AEC were transfected with either the vector (PRK5) or the wild-type p70^S6k, incubation with isoproterenol increased the total Na-K-ATPase ₁-subunit protein abundance. This increase was prevented in cells transfected with the dominant negative form of p70^S6k. Figure 4B shows that rapamycin did not inhibit the isoproterenol-mediated increase in the Na-K-ATPase protein abundance when the cells were transfected with a construct expressing the rapamycin-resistant p70^S6k.

View larger version (29K): [in this window] [in a new window]   Fig. 4. p70S6k is involved in the ISO-mediated Na-K-ATPase regulation. A: AEC were transfected with PRK5 (vector), pRK5-mycp70S6k [wild-type (WT) p70S6k], or myc-2BQ pRK5 [dominant negative (DN) p70S6k] recombinant plasmids or with Lipofectin alone and incubated for 48 h to allow expression of plasmid. Cells were serum starved and incubated with or without ISO (1 µM) for 72 h. Total cell lysates were analyzed against the Na-K-ATPase 1-subunit. Representative blots and composite graph of 3 independent experiments are shown. Data are expressed as means ± SE. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with controls. B: AEC were transfected with either PRK5 (vector) or myc-d/ED3E-pRK5 (Rapa-resistant mutant) and incubated for 48 h to allow expression of plasmid. Cells were then serum starved and incubated with or without ISO (1 µM) for 72 h in the presence or absence of 50 ng/ml of Rapa added 30 min before ISO. Total cell lysates were analyzed against the Na-K-ATPase 1-subunit. Representative blots and composite graph of 3 independent experiments are shown. Data are expressed as means ± SE. **P < 0.01, ***P < 0.001 compared with controls.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
Recently, we reported that the -adrenergic agonist isoproterenol increases (long-term) the Na-K-ATPase total protein abundance via cAMP-dependent protein kinase, MAPK/ERK, and rapamycin-sensitive pathways (23). Rapamycin binds with high affinity to the immunophilin FK binding protein, and this complex inhibits the TOR proteins (6, 33). The TOR proteins have been shown to regulate initiation of translation by regulating the eIF4E binding proteins and p70^S6k (4, 10, 14), which can be activated by -adrenergic agonists and cAMP (5, 32).

The p70^S6k is a complex enzyme that contains an acidic NH₂ terminus, followed by a serine-threonine catalytic domain and a regulatory COOH-terminal tail. It is phosphorylated in up to eight residues. In the initial steps, p70^S6k is phosphorylated at the COOH terminus with subsequent conformational changes in the catalytic domain of the enzyme. The conformational change reveals additional phosphorylation sites such as Thr²²⁹, Thr³⁸⁹, and Ser³⁷¹ in the catalytic domain. Phosphorylation of these residues has been shown to be wortmannin- and rapamycin-sensitive and important in regulating the enzyme activation, being MAPK involved in the initial phosphorylation steps (8, 36).

mTOR and p70^S6k are known to be downstream of the PI 3-kinase pathway (30, 36), and -adrenergic agonists have been shown to activate the MAPK pathway using EGFR as a scaffold protein (18). Here, we provide evidence that in AEC, PI 3-kinase and EGFR are involved in the signaling pathway that regulates Na-K-ATPase because both wortmannin and PD-153035 inhibited the isoproterenol-mediated MAPK/ERK phosphorylation. The ERK activation was not blocked by rapamycin, suggesting mTOR to be downstream of the MAPK/ERKs.

We determined that isoproterenol activates the p70^S6k in AEC by assessing phosphorylation of the enzyme at Thr³⁸⁹, which is a critical phosphorylation site to activate the enzyme (8). Dephosphorylation of this site by rapamycin results in loss of the kinase activity (22). As shown on Fig. 3, isoproterenol phosphorylated p70^S6k in AEC at Thr³⁸⁹ after 10 min of incubation. The isoproterenol-mediated p70^S6k phosphorylation was prevented by both the MEK inhibitor U-0126 and by rapamycin, suggesting that the MAPK/ERK and the mTOR pathways are involved in the isoproterenol-mediated regulation of p70^S6k in AEC.

In cells transfected with a dominant negative form of p70^S6k, isoproterenol did not increase the Na-K-ATPase protein abundance, implicating p70^S6k in the isoproterenol-mediated regulation of Na-K-ATPase in AEC. Furthermore, we provide evidence that the rapamycin-sensitive step regulated by isoproterenol is p70^S6k. In cells transfected with a rapamycin-resistant form of the enzyme, rapamycin was unable to inhibit the isoproterenol-mediated increase in the Na-K-ATPase protein abundance (see Fig. 4).

The p70^S6k has been proposed to regulate mRNAs that have an oligopyrimidine tract in their 5'-UTR (5'-TOP mRNAs), generally recognized as mRNAs from the ribosomal biosynthetic machinery (13); however, some non-TOP mRNAs appear to also be rapamycin sensitive (29, 35, 37, 38). Some of these RNAs have long and structured 5'-UTR that are presumed to require high amounts of eIF4A helicase activity to be translated. Recently, it has been described that p70^S6k phosphorylates the eIF4B, a protein that increases the helicase activity of the EIF4A (11).

Because the Na-K-ATPase subunit mRNAs have 5'-UTR that are rich in G/C sequences that predict complex and stable mRNA secondary structure (7, 15, 31, 39), we speculate that p70^S6k regulates the translation of Na-K-ATPase by phosphorylating the eIF4B with unwinding of the Na-K-ATPase ₁ mRNA 5'-UTR, facilitating its translation. Another possibility is that mTOR and p70^S6k decrease the proteolysis of Na-K-ATPase as a role for mTOR in decreasing protein degradation (6).

On the basis of our current data and previous publications, we propose (as depicted in Fig. 5) that isoproterenol activates the PI 3-kinase using EGFR as a scaffold protein. PI 3-kinase, in turn, activates the MAPK/ERK pathway, with downstream activation of mTOR and p70^S6k, which may facilitate Na-K-ATPase translation, resulting in increased total ₁ Na-K-ATPase protein abundance. To our knowledge, this is the first study reporting that p70^S6k is important in the regulation of Na-K-ATPase.

View larger version (22K): [in this window] [in a new window]   Fig. 5. Proposed pathway for the ISO-mediated (long-term) regulation of Na-K-ATPase. On the basis of our data, we propose that ISO regulates (long-term) the 1 Na-K-ATPase protein expression in AEC. -AR, -adrenergic.

	DISCLOSURES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
This research was supported in part by National Heart, Lung, and Blood Institute Grant HL-48129.

	ACKNOWLEDGMENTS

 
The authors are grateful to Dr. G. Thomas for generously providing the p70^S6k constructs and to Drs. N. Sonenberg, E. Lecuona, L. Dada, and K. M. Ridge for valuable discussions.

	FOOTNOTES

 

Address for reprint requests and other correspondence: J. I. Sznajder, Pulmonary and Critical Care Medicine, Northwestern Univ., 303 E. Chicago Ave., Tarry Bldg. 14-707, Chicago, IL 60611 (E-mail: j-sznajder{at}northwestern.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.

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