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Hyperoxia Upregulated Na,K-Adenosine Triphosphatase β1 Gene Transcription* FREE TO VIEW

Christine H. Wendt, MD, FCCP; Renuka Sharma, MS; Howard Towle, PhD; Gregory Gick, PhD; David H. Ingbar, MD, FCCP
Author and Funding Information

*From the University of Minnesota Medical School (Drs. Wendt, Towle, and Ingbar, and Ms. Sharma), Minneapolis, MN; and State University of New York (Dr. Gick), Brooklyn, NY.

Correspondence to: Christine H. Wendt, MD, FCCP, Assistant Professor of Medicine, Pulmonary and Critical Care, University of Minnesota, FUMC Box 276, 420 Delaware St SE, Minneapolis, MN 55455; e-mail: wendt005@gold.tc.umn.edu



Chest. 1999;116(suppl_1):87S-88S. doi:10.1378/chest.116.suppl_1.87S
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Alveolar sodium and fluid transport occur via type II cell apical sodium channels and basolateral Na,K-adenosine triphosphatases (ATPases), both of which are fundamental in resorbing edema fluid and restoring gas exchange following lung injury.1Na,K-ATPase gene expression is upregulated in the whole lung and type II cells in both in vitro and in vivo models of hyperoxic lung injury.5 This increase in Na,K-ATPase may serve as an homeostatic protective mechanism against alveolar flooding. Using a type II cell in vitro model of hyperoxic injury (≥ 95% O2 for 48 h), we demonstrated a threefold and fivefold increase in steady-state levels of Na,K-ATPase α1 and β1 subunit messenger RNA (mRNA), respectively.,23,6In addition, hyperoxia did not alter messenger RNA stability of either subunit.7 To study the mechanism of Na,K-ATPase gene upregulation by hyperoxia, we developed an in vitro model using MDCK cells exposed to hyperoxia (95% O2/5% CO2 for 24 to 48 h).,7

To measure transcription rates of the Na,K-ATPase subunits, nuclear run-on assays (NRAs) were performed with nuclei isolated from MDCK cells incubated in either normoxia or hyperoxia for 24 h. Slot blots containing the following plasmids were used for the NRAs: pGEM plasmid (control plasmid), actin (control), α1 subunit complementary DNA, and β1 subunit complementary DNA. NRAs revealed a 1.3-fold and 3.0-fold increase in α1 and β1 transcription with hyperoxia compared with normoxia. To identify hyperoxia regulatory regions within the promoter of the β1 subunit, transient transfection experiments using the 5′-flanking region of the Na,K-ATPase β1 subunit linked to the reporter gene, luciferase, were performed in MDCK cells under hyperoxic and normoxic conditions (Table 1). The wild-type construct (β1-817) contained 817 basepairs (bp) of the 5′ promoter region upstream from the transcription start site plus 151 bp of the first exon linked to a promoterless firefly luciferase expression vector (pXP1-luc). This construct was transfected via lipofection and revealed a 1.9-fold increase in promoter activity in hyperoxia compared with normoxia, confirming that hyperoxia induced Na,K-ATPase β1 subunit transcription. To localize the region(s) necessary for the hyperoxia induction, MDCK cells were transfected with four different 5′ deletion constructs of the β1 promoter (Table 1). Transfection of the deletion constructs in MDCK under normoxic conditions demonstrated a decrease in basal promoter activity with decreasing size of the deletion construct. The induction by hyperoxia was present in the β1-102 through β1-62 constructs; however, hyperoxia did not induce promoter activity in the β1-41 deletion construct. This localized a 21 bp regulatory region on the β1 promoter between bp-41 and -62 that was necessary for the twofold induction by hyperoxia. Since the full induction by hyperoxia was not seen with transfection of the wild-type or deletion constructs, other regions outside of our constructs may be necessary for further hyperoxia induction.

To identify proteins that bind to this putative regulatory region, electromobility shift assays (EMSAs) were performed using whole cell extracts from MDCK cells under normoxic and hyperoxic conditions on an oligonucleotide spanning the 21 bp regulatory region identified from the transfection experiments. EMSAs revealed two bands that had increased binding in extracts obtained from hyperoxic cells compared with normoxic cells. In addition, cells treated with the thiol oxidizer, diamide, manifested mobility shift patterns identical to extracts from cells exposed to hyperoxia. This suggested that hyperoxia induced an increased protein binding within the regulatory region identified in the transfection experiments and that thiol oxidation played a role in the protein binding and therefore, the hyperoxia induction.

The Na,K-ATPase is an important protein for maintaining vectoral ion and fluid transport, along with normal cellular homeostasis.1 This is especially important in the lung, where ion and fluid transport is necessary to maintain normal gas exchange, especially in the setting of lung injury. In our model system, we demonstrated that hyperoxia increased the gene expression of the Na,K-ATPase α1 and β1 subunits. Further, we determined that hyperoxia induced the transcription of the β1 subunit and identified a 21 bp region within its promoter that is necessary for this induction. Further analysis with EMSA suggested that thiol oxidation may be playing a role in the upregulation by hyperoxia. This upregulation of the Na,K-ATPase by hyperoxia may help to maintain gas exchange in the injured lung that is undergoing alveolar flooding.

Table Graphic Jump Location
Table 1. Effect of Hyperoxia on Na,K-ATPase β1 Subunit Promoter Activity*
* 

Luciferase activity was normalized to either cytomegalovirus-β-galactosidase activity or protein concentration, then reported as a percent activity over control. Control is designated as the β1-817 promoter construct in normoxic conditions.

References

Skou, JC (1988) Overview: the NA, K-pump.Methods Enzymol156,1-25
 
Carter, EP, Wangensteen, OD, O’Grady, SM, et al Effects of hyperoxia on type II cell Na-K-ATPase function and expression.Am J Physiol1997;272,L542-L551
 
Carter, EP, Duvick, SE, Wendt, CH, et al Hyperoxia increases active alveolar Na resorptionin vivoand type II cell Na,K-ATPasein vitro.Chest1994;105,75S-78S
 
Harris, ZL, Ridge, KM, Gonzalez-Flecha, B, et al Hyperbaric oxygenation upregulates rat lung Na,K-ATPase.Eur Respir J1996;9,472-477
 
Olivera, WG, Ridge, KM, Wood, LDH, et al Active sodium transport and alveolar epithelial Na-K-ATPase increase during subacute hyperoxia in rats.Am J Physiol1994;266,L577-L584
 
Nici, L, Dowin, R, Jamieson, JD, et al Up-regulation of rat type II pneumocyte Na,K-ATPase during hyperoxic lung injury.Am J Physiol1991;5L,307-314
 
Wendt, CH, Towle, H, Sharma, R, et al Regulation of Na-K-ATPase gene expression by hyperoxia in MDCK cells.Am J Physiol1998;274,C356-C364
 

Figures

Tables

Table Graphic Jump Location
Table 1. Effect of Hyperoxia on Na,K-ATPase β1 Subunit Promoter Activity*
* 

Luciferase activity was normalized to either cytomegalovirus-β-galactosidase activity or protein concentration, then reported as a percent activity over control. Control is designated as the β1-817 promoter construct in normoxic conditions.

References

Skou, JC (1988) Overview: the NA, K-pump.Methods Enzymol156,1-25
 
Carter, EP, Wangensteen, OD, O’Grady, SM, et al Effects of hyperoxia on type II cell Na-K-ATPase function and expression.Am J Physiol1997;272,L542-L551
 
Carter, EP, Duvick, SE, Wendt, CH, et al Hyperoxia increases active alveolar Na resorptionin vivoand type II cell Na,K-ATPasein vitro.Chest1994;105,75S-78S
 
Harris, ZL, Ridge, KM, Gonzalez-Flecha, B, et al Hyperbaric oxygenation upregulates rat lung Na,K-ATPase.Eur Respir J1996;9,472-477
 
Olivera, WG, Ridge, KM, Wood, LDH, et al Active sodium transport and alveolar epithelial Na-K-ATPase increase during subacute hyperoxia in rats.Am J Physiol1994;266,L577-L584
 
Nici, L, Dowin, R, Jamieson, JD, et al Up-regulation of rat type II pneumocyte Na,K-ATPase during hyperoxic lung injury.Am J Physiol1991;5L,307-314
 
Wendt, CH, Towle, H, Sharma, R, et al Regulation of Na-K-ATPase gene expression by hyperoxia in MDCK cells.Am J Physiol1998;274,C356-C364
 
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