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(Chest. 1999;116:87S-88S.)
© 1999 American College of Chest Physicians

Hyperoxia Upregulated Na,K-Adenosine Triphosphatase ß1 Gene Transcription*

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

* 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{at}gold.tc.umn.edu

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.1 Na,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.2 3 4 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 {alpha}1 and ß1 subunit messenger RNA (mRNA), respectively.2 3 6 In 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), {alpha}1 subunit complementary DNA, and ß1 subunit complementary DNA. NRAs revealed a 1.3-fold and 3.0-fold increase in {alpha}1 and ß1 transcription with hyperoxia compared with normoxia. To identify hyperoxia regulatory regions within the promoter of the ß1 subunit, transient transfection experiments using the 5'-flanking region of the Na,K-ATPase ß1 subunit linked to the reporter gene, luciferase, were performed in MDCK cells under hyperoxic and normoxic conditions (Table 1) . The wild-type construct (ß1-817) 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 ß1 subunit transcription. To localize the region(s) necessary for the hyperoxia induction, MDCK cells were transfected with four different 5' deletion constructs of the ß1 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 ß1-102 through ß1-62 constructs; however, hyperoxia did not induce promoter activity in the ß1-41 deletion construct. This localized a 21 bp regulatory region on the ß1 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.


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  		Table 1. Effect of Hyperoxia on Na,K-ATPase ß1 Subunit Promoter Activity*

 
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 {alpha}1 and ß1 subunits. Further, we determined that hyperoxia induced the transcription of the ß1 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.

References

  1. Skou, JC (1988) Overview: the NA, K-pump. Methods Enzymol 156,1-25[ISI][Medline]
  2. Carter, EP, Wangensteen, OD, O'Grady, SM, et al (1997) Effects of hyperoxia on type II cell Na-K-ATPase function and expression. Am J Physiol 272,L542-L551[Abstract/Free Full Text]
  3. Carter, EP, Duvick, SE, Wendt, CH, et al (1994) Hyperoxia increases active alveolar Na resorption in vivo and type II cell Na,K-ATPase in vitro. Chest 105,75S-78S
  4. Harris, ZL, Ridge, KM, Gonzalez-Flecha, B, et al (1996) Hyperbaric oxygenation upregulates rat lung Na,K-ATPase. Eur Respir J 9,472-477[Abstract]
  5. Olivera, WG, Ridge, KM, Wood, LDH, et al (1994) Active sodium transport and alveolar epithelial Na-K-ATPase increase during subacute hyperoxia in rats. Am J Physiol 266,L577-L584[Abstract/Free Full Text]
  6. Nici, L, Dowin, R, Jamieson, JD, et al (1991) Up-regulation of rat type II pneumocyte Na,K-ATPase during hyperoxic lung injury. Am J Physiol 5L,307-314
  7. Wendt, CH, Towle, H, Sharma, R, et al (1998) Regulation of Na-K-ATPase gene expression by hyperoxia in MDCK cells. Am J Physiol 274,C356-C364[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
C. H. Wendt, G. Gick, R. Sharma, Y. Zhuang, W. Deng, and D. H. Ingbar
Up-regulation of Na,K-ATPase beta 1 Transcription by Hyperoxia Is Mediated by SP1/SP3 Binding
J. Biol. Chem., December 22, 2000; 275(52): 41396 - 41404.
[Abstract] [Full Text] [PDF]

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