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