 |
INTRODUCTION |
Sodium balance is important for the maintenance of body
electrolyte, extracellular volume, and blood pressure. The
amiloride-sensitive epithelial sodium channel
(ENaC)1 is expressed in
airway epithelium, distal segments of kidney tubule, skin, bladder,
colon, and sweat and salivary glands. In addition, ENaC is a member of
the expanded degenerin/ENaC superfamily (1) and consists of at least
three homologous subunits, 
, 
, and 
(2-5).
Compelling functional and biochemical evidence suggests that all three
subunits form a heteromultimeric complex and contribute to the optimal
epithelial sodium reabsorption activity of the amiloride-sensitive
sodium channel (3, 6, 7). However, when expressed individually in a
Xenopus oocyte system, only the -subunit can produce an
amiloride-sensitive current. Both - and -subunits are not
functional on their own, but augment the channel activity of -ENaC
(3, 8). Thus, the expression of a functionally active sodium channel is
dependent upon the presence of the -ENaC subunit. Indeed, different
phenotypes were observed in -ENaC(
/) and -ENaC(/)
transgenic mice that were generated by homologous gene targeting. For
example, 50% of -ENaC(/) mice develop respiratory distress and
die within 24 h after birth from an inability to clear the lung
fluid (9), whereas none of the -ENaC(/) mice die within the same
time period (10).
Although the composition, structure, and activity of ENaC have been
well studied, the molecular basis for the regulatory mechanisms underlying the gene expression of ENaC subunits remains
unclear. Results from studies by us and others have suggested that the transcriptional control of ENaC gene expression occurs in a
subunit- and tissue-specific manner (Ref. 11; reviewed in Refs. 12 and
13). For instance, the transcription of - and
-ENaC genes is up-regulated by the steroid hormone
aldosterone in the colon, whereas the -ENaC mRNA is
constitutively expressed. In contrast, dexamethasone treatment is
reported to up-regulate the steady-state mRNA levels of all three
subunits in the colon and fetal lung. Moreover, vasopressin increases
only - and -ENaC mRNA levels without
altering -ENaC mRNA level in RCCD1 rat
cortical collecting duct cell line (14). We have previously
demonstrated that TPA treatment represses the -ENaC
mRNA level (11). Taken together, these studies also outline a
putative pathway(s) by which extracellular signals regulate
cell-specific ENaC expression. Therefore, studies that more
precisely define the interaction between the cellular transcriptional
machinery and signaling pathways are needed to provide a framework for
understanding the mechanisms underlying the regulation of
ENaC expression and the resultant electrolyte homeostasis.
Nuclear hormone receptors modulate gene transcription, upon binding of
their cognate ligands, by activation as well as repression (15). One of
the best studied members of the nuclear hormone receptor superfamily is
the glucocorticoid receptor (GR), which plays an important role in
physiology and developmental biology. Transactivation by GR requires
binding of receptor homodimers to specific partial palindromic
sequences in the cis-regulatory region of target genes,
namely glucocorticoid response elements (GREs). In recent years, it has
become apparent that a variety of other factors may have a profound
effect on the transactivation potential of GR. For example, the
phosphorylation status of GR (16) and the formation of composite or
complex GREs (17, 18) can affect classical GR transactivation activity.
Thus, although the GR is expressed in virtually all mammalian cell
types, it is possible that the expression of a distinct set of
GR-responsive genes is cell context-specific and may be modulated by
other signaling pathway(s).
Ras proteins are important signaling intermediates that convey signals
initiated at the cell surface to various effector pathways in the
cytoplasm. Ras exerts effects on cell transformation and proliferation,
cytoskeletal structure, differentiation, and apoptosis. These changes
may be mediated by multiple effectors, including Raf-1, Ral-GDS,
phosphatidylinositol (PI) 3-kinase, and other Ras-binding proteins
(reviewed in Ref. 19), often at the transcriptional level. Even though
both Ras effector pathways and gene regulation are characterized in
molecular detail, the interface between canonical Ras signaling and
transcriptional repression remains poorly described. Clearly, there are
mechanisms, when appropriately cued, whereby signal transduction
cascades operating via Ras activation would negatively modulate
transcriptional events in the nucleus, ultimately leading to
transcriptional repression.
The human -ENaC gene has recently been cloned and
characterized (20). A rat genomic DNA fragment harboring the
-ENaC promoter, first exon, partial first intron, and
5'-flanking region has been isolated and used very recently to
investigate the -ENaC gene regulation by us (11). Since
more than one transcription initiation site has been identified in both
human and rat -ENaC genes accounting for the polymorphism
of -ENaC transcript size
(21),2 we have adopted a
numbering convention that assigns the number +1 to the first base of
the translation start codon. Previously, we have shown that the
regulatory elements in a 1.4-kb (1573 to 154 bp relative to
translation initiator ATG) -ENaC DNA fragment are
sufficient to mediate the basal and the extracellular signal-regulated protein kinase (ERK) pathway-modulated expression of -ENaC/reporter construct in transient transfection assays (11). In this study, we
identified a 508-bp DNA fragment in the 5'-flanking region of the rat
-ENaC gene that is required for transcriptional
activation of the -ENaC promoter and which also confers
Ras-mediated repression. By using heterologous promoter constructs and
deletion analyses, we demonstrate that a functional GRE and GR are
required for both transcriptional activation and Ras-mediated
repression. This is the first report of an -ENaC enhancer
with a dual effect, suggesting that its involvement in the
transcriptional regulation of -ENaC gene expression in
salivary epithelial cells is via the cross-talk between Ras- and
GR-mediated signaling pathways. This mechanism may also be responsible
for the expression of the -ENaC gene in different tissues.
| |
MATERIALS AND METHODS |
Cell Culture and DNA Constructs--
The rat parotid epithelial
cell line Pa-4, also known as parotid C5 cell line (22), was maintained
as described previously (23). Dexamethasone was obtained from Sigma,
resuspended to 1 mM in ethanol, and stored in 80 °C.
The anti-glucocorticoid, ZK98.299 (24, 25), a generous gift from Dr. M. Stallcup (University of Southern California, Los Angeles, CA), was
stored at 80 °C as a 1 mM stock solution in ethanol.
A series of reporter constructs encoding the chloramphenicol
acetyltransferase (CAT) reporter gene were derived from
-ENaC/CATs, as described previously (11). The
1.0-ENaC/CAT, 0.7-ENaC/CAT, and 0.5-ENaC/CAT (Fig. 2)
were made by a double digest of -ENaC/CATs with
SphI (vector)/HindIII, KpnI, and
PstI, respectively. Vector-containing fragments were
isolated, blunt-ended by T4 DNA polymerase (Promega, WI), and
self-ligated. In addition, a 0.5-kb HindIII fragment of
-ENaC (1573 to 1066 bp) was excised from
-ENaC/CATs and ligated into a
HindIII-linearized 0.7-ENaC/CAT plasmid DNA in both
sense and antisense orientations to generate
(1573/1066)-0.7-ENaC/CAT and (1066/1573)-0.7-ENaC/CAT
reporter constructs, respectively. A 0.2-kb DNA fragment extending from
internal PstI (from vector via previous engineering) to
PstI (internal) was deleted from (1066/1573)-0.7-ENaC/CAT to construct the
(1066/1573)-0.5-ENaC/CAT plasmid.
A series of unidirectional deletion mutants harboring the luciferase
reporter gene (Fig. 3) were constructed as follows. The 0.5-kb
HindIII -ENaC DNA fragment (1573 to 1066
bp) was PCR-amplified using primer pairs designed according to the
-ENaC DNA sequence and cloned into pCR2.1 vector with the
Original TA Cloning® kit (Invitrogen) in either sense or
antisense orientation. Subsequently, each insert was excised and
unidirectionally cloned into pGL2-P reporter plasmid (Promega) to
generate p(1573/1066)GL2-P and p(1066/1573)GL2-P,
respectively. These two constructs were then double-digested on the
adjacent KpnI (3'-overhang) and SpeI
(5'-overhang) sites to create a single exonuclease III-sensitive end.
Unidirectional deletions were carried out by digesting DNA fragments
for various periods of time with exonuclease III according to the
instruction of Erase-a-Base® system (Promega). The extent
of deletion in each individual clone was determined by DNA sequence
analyses. Site-specific mutations within or adjacent to the GRE in
p(1414/1066)GL2-P were introduced by TransformerTM
site-directed mutagenesis kit (CLONTECH) to
generate Mt A and Mt B (Fig. 6A). A -ENaC/CATs
plasmid with point mutations on GRE site (GRE Mt; Fig. 7) was
constructed by replacing a SacI/HindIII fragment
containing GRE site with the same fragment from Mt B described above
that carries mutated GRE.
Reporter constructs, p(1334/1306)GL2-P and
p(1334/1306)2 GL2-P, containing one and two copies of
-ENaC GRE, respectively, were generated as follows: a
pair of complementary and 5'-phosphorylated oligomers, which correspond
to 1334 to 1306 bp of -ENaC, were annealed by
incubating 200 pmol of each oligomer in a buffer containing 50 mM Tris-HCl (pH 7.6) and 10 mM
MgCl2, followed by heating to 85 °C for 2 min and step
wise cooling at 65 °C (15 min), 37 °C (15 min), 25 °C (15 min), and 4 °C (15 min). The annealed oligos, which have a built-in
5'-phosphorylated XhoI overhang, were ligated to
XhoI-linearized pGL2-P plasmid DNA.
The DNA sequences and mutations introduced in all constructs were
verified by sequence analyses utilizing T7 Sequenase 7-deaza-GTP Sequencing Kit (Amersham Pharmacia Biotech) as instructed by the manufacturer. Expression plasmids harboring Ras mutants of Ras V12, Ras
S35, Ras G37, and Ras C40 were generous gifts from Dr. D. Johnson
(University of Southern California, Los Angeles, CA), while
glucocorticoid receptor (GR) expression construct and a tyrosine
aminotransferase GRE containing reporter construct,
TAT(GRE)3TK/Luc, were kindly provided by Dr. M. Stallcup
(University of Southern California).
Transient Transfection--
Plasmid DNAs were transiently
transfected into Pa-4 cells using a LipofectAMINETM-mediated method
(Life Technologies, Inc.) as described previously (23). In all
experiments, 0.1 µg of Renilla luciferase plasmid, pRL-TK
(Promega, WI), was included as an indicator plasmid to normalize for
transfection efficiency. Total amount of DNA in each transfection of
cells grown in a 35-mm dish was kept constant at 2 µg by
supplementing with pCMV vector, as needed. To maximize the response of
cells to dexamethasone treatment, the cells were serum-starved 24 h after the start of transfection by replacing growth medium with
0.05% serum-containing medium for 8 h, followed by adding
10
7 M dexamethasone in the same medium. All
plates were harvested 16 h thereafter. The CAT reporter gene
analysis was carried out as described previously (23), whereas firefly
and Renilla luciferase activities were measured using
Dual-LuciferaseTM reporter assay kit (Promega) according to the
manufacturer's instruction. All transient transfection assays were
carried out at least three times independently.
Northern Blot Analysis--
Total RNA (RNA) was isolated from
Pa-4 cells using Trizol® reagent (Life Technologies, Inc.)
as instructed by the manufacturer. The quality and quantity of RNA were
determined by both spectrophotometric analysis and fractionating RNA on
an agarose/formaldehyde gel (1.3% (w/v)/6% (v/v)), followed by
staining with ethidium bromide to compare the ratio of 28 S to 18 S
ribosomal RNAs. For Northern analyses, equal amounts of RNA (18 µg/sample) were loaded onto an agarose/formaldehyde gel, fractionated
by size, transferred to a 0.2-µm nylon membrane (ICN Biomedicals,
Inc.), and UV-cross-linked. All blots were prehybridized for 1 h
in QuikHyb® (Stratagene). The hybridization was carried
out according to manufacturer's instructions with
32P-labeled -ENaC probes that were prepared
from an isolated rat -ENaC cDNA fragment (bases
1-905) using a Random Primed DNA labeling kit (Roche Molecular
Biochemicals). All blots were also reprobed with rat -actin to
ensure that the quality and quantity of mRNA between lanes were
comparable. After hybridization, these blots were washed in a 0.1× SSC
(0.15 M NaCl and 0.015 M sodium citrate) solution containing 0.5% SDS at 60 °C for 2 h with three
changes of washing solution. The radioactive signal was visualized by autoradiography, where blots were exposed to films at 80 °C
overnight, using BioMax TranScreen-LE (Eastman Kodak Corp.) to improve
sensitivity. Blots were also quantitated through the use of electronic
autoradiography with an Instantimager 228 (Packard Instrument Co). The
amount of -ENaC message was quantitatively compared
between lanes after normalizing against that of -actin.
| |
RESULTS |
Ras Inhibits -ENaC/Reporter Activity via Multiple Effector
Pathways--
The Ras pathway has been recently reported to modulate
epithelial sodium channel activity and expression in Xenopus
oocytes (26). Moreover, we have shown that a downstream effector
pathway of Ras, Raf/ERK, can down-regulate the expression of both the endogenous -ENaC gene and transiently transfected
reporter constructs harboring 1.4 kb (1573/154 bp relative to ATG)
of the rat -ENaC 5'-flanking region (11). In order to
more precisely evaluate the ability of Ras and its different effector
pathways to modulate -ENaC expression in mammalian cells,
salivary epithelial Pa-4 cells were cotransfected with an
-ENaC/CATs reporter construct and an expression plasmid
encoding a constitutively activated form of Ras (Ras V12) or a
"single-effector" mutant, i.e. Ras S35, Ras G37, and Ras
C40. The ability of each Ras mutant to modulate -ENaC/CATs reporter activity was assessed.
Among the three Ras "single-effector" mutants, S35 is able to
activate Raf-1 but not Ral-GDS or PI 3-kinase, while G37 activates Ral-GDS but not Raf-1 or PI 3-kinase, and C40 activates PI 3-kinase but
not Raf-1 or Ral-GDS (27). As shown in Fig.
1, Ras V12 markedly repressed
-ENaC/CATs activities when cotransfected into Pa-4 cells
(lanes 3 and 4 versus
lanes 1 and 2). While the C40 mutant had no effect on -ENaC/CATs activities, mutants S35 and
G37 appeared less effective than Ras V12 in our system (Fig. 1).
Although it is possible that the known differences in the activity and
expression level of these mutant proteins (28) may have contributed to these results, it is more likely that both Ral-GDS and Raf/ERK signaling pathways are involved in down-regulating -ENaC
expression.
View larger version (36K):
[in this window]
[in a new window]
|
Fig. 1.
Ras effector mutants modulate
-ENaC/CATs activity in Pa-4
cells. Pa-4 cells were transiently cotransfected with 1.2 µg of
-ENaC/CATs reporter construct and 0.35 µg of expression
plasmid encoding Ras V12, and GTPase deficient (V12) forms of Ras S35,
Ras G37, and Ras C40, respectively. pRL-TK plasmid (0.1 µg) was
cotransfected as an indicator to normalize for transfection efficiency.
The total amount of plasmid transfected was kept constant at 2.0 µg
by supplementing with pCMV vector. CAT assay was performed after
normalizing against Renilla luciferase activity. The percent
conversion shown was calculated as described previously (23).
Transfection with each Ras mutant expression plasmid was performed in
duplicate. Similar results were obtained from three independent
experiments, where one representative CAT assay is shown.
|
|
Previously, we have shown that MEK inhibitor, PD 98059, was able to
block the inhibition of -ENaC/CATs activity by Raf/ERK activation (11). However, the MEK inhibitor had a limited effect on Ras
V12-mediated inhibition,3
suggesting that an effector pathway(s) other than Raf/ERK may also have
been involved in modulating -ENaC expression. Since Ras
V12 elicited the maximum repression on -ENaC/CATs
reporter activity, we used Ras V12 to elucidate the mechanism(s) that
down-regulates -ENaC expression.
Identification of a Ras-repressible Enhancer Required for -ENaC
Expression--
The results shown above and our previously published
data (11) suggest that elements confined within the
-ENaC/CATs construct are essential for recapitulating the
basal and Ras-mediated regulation of -ENaC expression. To
elucidate the cis-element(s) conferring Ras-mediated
repression of the -ENaC/CATs, we constructed a series of
reporter plasmids in which truncated fragments of the
-ENaC promoter/enhancer were placed upstream of the
promoterless CAT reporter gene. As shown in Fig.
2, removal of the first 508 bp from the
parental construct, -ENaC/CATs, reduced the level of CAT
activity approximately 10-fold in Pa-4 cells transiently transfected with 1.0-ENaC/CAT. Since basal activity was so low, the ability of
Ras V12 to repress reporter activity cannot be accurately assessed.
View larger version (41K):
[in this window]
[in a new window]
|
Fig. 2.
Effect of progressive 5'-deletions and
internal deletions on basal activity and Ras-mediated repression of
-ENaC promoter. Salivary Pa-4 cells were
transiently transfected with 1.2 µg of DNA of each
-ENaC/CATs deletion constructs (as depicted in the
left panel) in the presence and absence of 0.7 µg of cotransfected expression plasmid harboring Ras V12 as
indicated. Transfections and CAT assays were performed as described in
Fig. 1. The region between 1573 and 1066 bp in either sense
(construct E) or antisense (constructs
F and G) orientation apparently acts as an
enhancer, of which effect is repressed by Ras pathway activation. A
representative CAT assay from three independent experiments is shown
(right panel).
|
|
Subsequent deletions of 310 and 586 bp from 1.0-ENaC/CAT,
designated as 0.7-ENaC/CAT and 0.5-ENaC/CAT, respectively, resulted in minor variations of the basal CAT activity when compared with those from 1.0-ENaC/CAT-transfected cells (Fig. 2). The reason for these variations is not clear, since they were not always
reproducible. This dramatic decrease in basal activity indicated that a
Ras-repressible enhancer(s) was located within the first 508 bp
(1573/1066) of the 1.4-kb -ENaC DNA fragment. To
confirm this, we inserted the (1573/1066) fragment 5' to the
0.7-ENaC/CAT in both sense and antisense orientations and in the
antisense orientation upstream of the 0.5-ENaC/CAT construct (Fig.
2). High levels of basal activity were observed in cells transiently
transfected with any of the three constructs (Fig. 2). Moreover, the
Ras-mediated repression of reporter gene activity was quantitatively
and qualitatively similar to that observed in
-ENaC/CATs-transfected cells. Thus, the DNA fragment
1573 to 1066 bp of -ENaC enhanced the
-ENaC basal promoter activity in a way that could be
repressed by Ras activation. The DNA sequences between 1066 and 480
bp, on the other hand, were dispensable for modulating
-ENaC expression.
Delineation of the Activating Sequence(s) within the 1573/1066
-ENaC 5'-Flanking Region--
To determine whether the first 508-bp
fragment of the parental -ENaC/CATs construct alone could
modulate -ENaC expression, the (1573/1066) fragment
was inserted in both sense (Fig.
3A, construct
A) and antisense (Fig. 3A, construct
E) orientations upstream of a heterologous minimal SV40
promoter in pGL2-P vector. As shown in Fig. 3B, the
(1573/1066) fragment (construct A) conferred an approximately
4-fold higher basal activity than that of vector alone (construct I).
Moreover, the activity was stimulated in an orientation-independent
manner, a hallmark of an enhancer element. Although it is unclear, the
508-bp fragment when in the antisense orientation (construct E)
elicited a higher activity than the sense counterpart (construct A; see
Fig. 3B). Moreover, coexpression of Ras V12 had little
effect on attenuating reporter activity of vector alone, but markedly
down-regulated the enhancer activity conferred by the 1573/1066
fragment.
View larger version (40K):
[in this window]
[in a new window]
|
Fig. 3.
Functional analyses of the
-ENaC enhancer element by transient transfection
assays with heterologous promoter constructs in Pa-4 cells.
A, schematic diagram of a series of p(1573/1066)GL2-P
and p(1066/1573)GL2-P deletion mutants. The end points of truncated
DNA fragments in p(1573/1066)GL2-P and p(1066/1573)GL2-P
constructs and their deletion mutants are shown. Construct
I represents the vector only, pGL2-P. S and
AS depict the sense and antisense orientations of the
insert. B, Ras pathway activation down-regulates the
enhancer activity in transiently transfected Pa-4 cells cultured in
serum-containing growth media. Pa-4 cells were transiently transfected
with 0.9 µg each of the constructs shown in diagram (A)
with or without 0.5 µg of Ras V12 expression plasmid where indicated.
The transient transfection was carried out as described in Fig. 1. Both
firefly and Renilla luciferase activities were measured
simultaneously using Dual-LuciferaseTM assay system (Promega). The
relative luciferase activity from firefly luciferase reporter gene
shown in each group of transfected cells was determined and normalized
with the indicator Renilla luciferase activity. Each value
shown is the mean ± S.E. based on three independent transfection
experiments. C, GR/dexamethasone (Dex)
transactivates the -ENaC distal enhancer in cells
cultured under serum-free conditions. Pa-4 cells were cotransfected
with 0.9 µg of each construct shown in diagram (A) and/or
0.5 µg of GR expression plasmid as described in B. Sixteen
hours after the start of transfection, the cells were serum-starved for
8 h, followed by the addition of 107 M
dexamethasone to the culture medium, and incubated overnight. The
reporter and indicator luciferase activities were determined as
described in B. The level of induction, expressed as -fold
induction, for each construct is calculated by dividing the normalized
reporter luciferase activity in extracts from
GR-transfected/dexamethasone-treated cells over that of
corresponding GR-transfected/vehicle-treated cells. Error
bars were calculated as in Fig. 3B. D,
Ras pathway activation represses GR transactivation activity. Pa-4
cells were cotransfected with 0.9 µg of each construct shown in
diagram (A) and 0.5 µg of GR expression plasmid in
combination with or without 0.5 µg of Ras V12 expression plasmid as
indicated. The transfection, treatment protocol, and dual luciferase
assay were carried out as described in C. The relative
luciferase activity for each reporter construct in extracts, after
normalization, from GR-transfected/dexamethasone-treated cells and Ras-
plus GR-cotransfected/dexamethasone-treated cells, respectively, is
shown. Error bars represent the standard error of
the mean for each construct based on three independent transfection
experiments.
|
|
Since the expression pattern of the parental, -ENaC/CATs,
construct was functionally reproduced in heterologous promoter constructs containing the (1573/1066) fragment, we focused our attention on characterizing this 508-bp distal enhancer. Serial 5'- and
3'-deletion mutants of the 1573/1066 fragment were constructed to
locate the DNA motif(s) involved in Ras-repressible enhancer activity.
As shown in Fig. 3B, deletions outside the 1355 to 1269
bp sequence had little effect on either enhancer activity or Ras
repressibility (constructs B, C, F, and G). In contrast, constructs
excluding this region (constructs D and H) exhibited a dramatic
reduction in enhancer activity. In addition, Ras repression was
virtually abolished (Fig. 3B, constructs
D and H). These data suggest that the DNA
fragment extending from 1355 to 1269 bp confers both enhancer and
Ras-mediated repressor activity. Thus, the 87-bp fragment (1355 to
1269 bp) in the 5'-flanking region appears to function as both an
enhancer and a Ras-mediated repressor of -ENaC expression
in parotid epithelial cells.
Glucocorticoid Hormone Transactivates the Reporter Expression from
the 1573/1066 DNA Fragment--
Analysis of the 87-bp fragment by
Find Patterns Algorithm of the GCG software package and transcription
factor sites data base (29) identified several putative transcription
factor-binding sites within this region. A DNA sequence around position
1316 bp (1323 to 1309 bp; AGAACANNNTGTCCT) with close
resemblance to the consensus site for the glucocorticoid hormone
receptor (GRE; AGAACANNNTGTTCT) was chosen for further
investigation. To investigate whether the (1323/1309) DNA sequence
could mediate functional induction and was necessary for the
Ras-repressible enhancer activity, Pa-4 cells were cotransfected with a
glucocorticoid receptor (GR) expression plasmid and a reporter
construct shown in Fig. 3A. Addition of 107
M dexamethasone enhanced the reporter activity of reporter
constructs A and E by approximately 7-fold over those from
corresponding vehicle-treated transfected cells (Fig. 3C).
Moreover, despite that a high expression level was induced by exogenous
GR/dexamethasone treatment, the expression of Ras V12 plasmid was still
able to reduce GR/dexamethasone-mediated enhancement in constructs A
and E up to 5-fold (Fig. 3D). In general, shortening the
-ENaC DNA fragment 5' to 1355 bp and 3' to 1269 bp
neither altered dexamethasone induction nor Ras-mediated repression of
reporter activity (Fig. 3, C and D,
constructs B, C, F, and
G). However, deletion of the region containing the
identified GRE virtually abolished GR/dexamethasone-mediated enhancement as well as Ras-mediated repression of the minimal SV40
promoter (Fig. 3, C and D, constructs
D and H). These data are consistent with the
results shown in Fig. 3B, suggesting that deletions outside
the GRE have no effect on reporter activity, whereas deletions of the
GRE abolish both enhancer and repressor properties of the 508-bp fragment.
Based on these results, it appears that in Pa-4 cells
p(1066/1573)GL2-P reporter construct expression can be modulated by two signaling pathways; whereas GR activation stimulates reporter expression, Ras activation antagonizes GR-mediated enhancement. To
examine this hypothesis, Pa-4 cells were transiently cotransfected with
the reporter construct p(1066/1573)GL2-P and a GR expression plasmid or an empty vector. The luciferase activity was increased approximately 1.5-fold by treatment with the GR agonist, dexamethasone (Fig. 4, lane 5 versus lane 4), suggesting that
endogenous GR activation was able to increase -ENaC
enhancer activity modestly via a GRE located between 1573 and 1066
bp. No transcriptional stimulation by dexamethasone was observed in
pGL2-P (Fig. 4, lane 3), which lacks the cloned
-ENaC enhancer fragment. Cotransfection with a GR
expression plasmid in the absence of exogenous dexamethasone failed to
elicit a robust enhancement on the heterologous promoter activity of
p(1066/1573)GL2-P (Fig. 4, lane 6 versus lane 4). However,
dexamethasone substantially induced reporter activity in the same
cotransfection (Fig. 4, lane 7 versus
lane 4). In addition, Ras activation markedly
antagonized exogenous GR/dexamethasone-stimulated expression of
p(1066/1573)GL2-P (Fig. 4, lane 9 versus lane 7) but had little effect
on p(1066/1573)GL2-P alone (lane 8 versus lane 4). A similar result was
also observed with sense-oriented counterpart p(1573/1066)GL2-P
(data not shown). These results not only support an antagonistic
regulatory hypothesis, but also suggest that a direct association
exists between Ras-mediated repression and GR-associated enhancement of
-ENaC expression. Hence, it appears that the maximal
stimulating activity via the 508-bp enhancer requires the presence of a
GRE in the reporter construct and the presence of glucocorticoid
hormone to activate its expression. In addition, its Ras-mediated
repression on -ENaC promoter activity is likely to be
associated with its ability to inhibit GR transactivation.
View larger version (19K):
[in this window]
[in a new window]
|
Fig. 4.
Hormone-dependent activation of
-ENaC enhancer activity by GR. Salivary Pa-4
cells were transiently transfected with 0.9 µg of vector pGL2-P
plasmid or p(1066/1573)GL2-P reporter construct with or without 0.5 µg of Ras V12 expression plasmid in combination with 0.5 µg of GR
expression plasmids, as indicated. The amount of transfected DNA was
kept constant at 2 µg by supplementing with varying amount of pCMV
plasmid DNA. After transfection, the cells were treated as described in
Fig. 3C with 0.1 µM dexamethasone or vehicle
as indicated. The level of induction, expressed as -fold induction, is
calculated by dividing the normalized reporter luciferase activity in
each extract (lanes 2-9, respectively) over that
of pGL2-P-transfected/vehicle-treated cells, which is arbitrarily
designated as 1 (lane 1) and indicated by a
horizontal line. This experiment has been
repeated three times, and one representative plot is shown.
|
|
GR Antagonist and TPA Block Dexamethasone-induced Endogenous
Expression of -ENaC mRNA--
To investigate whether the
identified enhancer DNA fragment functions in its native configuration,
endogenous -ENaC expression was examined in response to
GR and Ras signaling pathway activation. To do this, Pa-4 cells were
treated with dexamethasone in the presence and absence of GR
antagonist, ZK98.299. Our preliminary results indicated that the
dexamethasone EC50 value for inducing -ENaC
expression was approximately 3 × 109 M
in Pa-4 cells (data not shown). This allowed us to reduce concentration of dexamethasone used in this study to 108 M
in order to accommodate the usage of 100-fold excess of ZK98.299 without obvious cytotoxicity. Cells treated with 108
M dexamethasone alone for 16 h exhibited an
approximately 6-fold increase in -ENaC mRNA level,
which was completely blocked by the cotreatment with 1 µM
ZK98.299 (Fig. 5A). -Actin
mRNA expression was used to normalize -ENaC mRNA
level in control and treated Pa-4 cells, and to assure the viability of
the treated cells (Fig. 5, A and B).
View larger version (27K):
[in this window]
[in a new window]
|
Fig. 5.
Glucocorticoid hormone up-regulation of
endogenous -ENaC expression in Pa-4 cells is
antagonized by TPA. A, GR/dexamethasone induces
-ENaC mRNA level in Pa-4 cells. Northern analysis
(upper panel) was performed to analyze the
changes in -ENaC mRNA expression between control and
Pa-4 cells treated with dexamethasone for 16 h in the
presence (+) or absence () of the glucocorticoid hormone
antagonist, ZK98. 299. Cells were cultured in 0.05% serum-stripped
medium for 8 h prior to treatment with either vehicle,
106 M ZK98.299, or 108
M dexamethasone alone (lanes 1,
2, and 3, respectively), or treated with ZK98.299
and dexamethasone concomitantly (lane 4). Total
RNA was isolated 16 h thereafter. RNA blots were prepared and
probed with 32P-labeled -ENaC and -actin
DNA fragments as described under "Materials and Methods."
Lower panel is a summary of experiments where
mRNA levels were quantitated directly by electronic
autoradiography, and normalized with -actin mRNA levels. Results
are depicted as the mean ± S.E. B, TPA antagonizes
GR-mediated transactivation of -ENaC expression. Northern
analysis (upper panel) of -ENaC
mRNA level in control and treated Pa-4 cells was performed as
described in A. Dexamethasone was used at 107
M (lanes 3 and 4) to
maximize -ENaC induction and TPA was used at 100 ng/ml
(lanes 2 and 4). Control cells
(lane 1) were treated with vehicle alone. The
quantitative analyses (lower panel) were
performed as described in A.
|
|
A modest decrease in the basal -ENaC mRNA level by
ZK98.299 treatment was also observed (Fig. 5A),
demonstrating the ability of the remaining glucocorticoid hormone,
after switching to serum-free conditions, to modulate
-ENaC expression. Since TPA has been shown to induce the
Raf/ERK, Ras effector pathway, in Pa-4 cells (11), we investigated
whether TPA could block dexamethasone-mediated induction of the
endogenous -ENaC expression. Cells treated with 107 M dexamethasone for 16 h exhibited a
marked increase in the steady-state -ENaC mRNA level
by approximately 7-fold (Fig. 5B, lane
3). Consistent with the effect of ZK98.299, TPA treatment
attenuated both basal and dexamethasone-induced -ENaC
mRNA levels in Pa-4 cells (Fig. 5B, lanes
2 and 4). These data are consistent with the
results shown above on the regulatory effects on -ENaC/reporter
activity by GR and Ras V12 expression plasmids, and establish the
physiological relevance of this study. Together, these data suggest
that glucocorticoid hormone induces -ENaC expression
through the putative GRE and that the GR-mediated transactivation can
be antagonized by GR antagonist as well as TPA-mediated Raf/ERK activation.
Point Mutations in GRE Abolish Antagonistic Modulation by
Glucocorticoid Hormone/Ras Pathways--
Our data indicate that the
Ras-mediated repressibility and dexamethasone-stimulatory effect are
not operated exclusively via distinct positive and negative regulatory
elements. Based on sequence analysis of the 1334 to 1306 bp DNA
fragment, a second putative transcription factor binding site AP-1 was
identified 15 bases upstream of the GRE (Fig.
6, from the center of putative AP-1 site
to the center of putative GRE). AP-1 is activated by the Ras signaling
pathway (19), and is a well established antagonist of GR-mediated
transcription (17, 18). Therefore, we postulated that Ras activation
may repress GR/GRE association by activating AP-1 and thereby
stimulating a mutual competition between AP-1 and GR for their
respective response element. Second, although we showed that
GR-mediated transcription is necessary for endogenous -ENaC gene expression (Fig. 5A),
whether or not this GR transactivation is mediated via the identified
GRE is unknown. To test these hypotheses directly, Pa-4 cells were
transiently transfected with one of three distinct reporter constructs:
wild type, Mt A, and Mt B. Mt A differs from wild type by three
nucleotide substitutions at positions 1330, 1328, and 1327,
abolishing the putative AP-1 element, whereas Mt B leaves the AP-1 site
unaltered but replaces five nucleotides within the putative GRE (Fig.
6A, upper panel).
View larger version (21K):
[in this window]
[in a new window]
|
Fig. 6.
Functional analyses of putative GRE as a
regulatory element in GR-mediated transactivation and Ras-mediated
repression. A, function of the putative GRE in the
context of heterologous promoter. DNA sequences of the rat
-ENaC distal enhancer region extending from 1334 to
1306 bp are shown. The GRE- and AP-1-like recognition motifs are
boxed. Mt A and Mt B represent mutant
variants used in transfection assays. Sequences that are identical to
the wild type are shown as dashes, whereas the mutated DNA
sequences are shown in letters. Reporter construct (0.9 µg) was cotransfected with 0.4 µg of Ras V12 and/or 0.4 µg of GR
expression plasmids as indicated. Luciferase activities of the wild
type and mutant variants were assayed and analyzed as described in Fig.
3. B, the putative GRE in -ENaC confers
glucocorticoid response in the context of heterologous promoter. Three
different reporter constructs were tested. Two constructs carry one and
two copies of -ENaC GRE encompassing sequences, 1334 to
1306 bp, upstream of the minimal SV40 promoter-containing luciferase
reporter gene and the third construct, TAT(GRE)3TK/Luc
plasmid, harbors three copies of simple GRE from tyrosine
aminotransferase gene upstream of a minimal TK promoter. Pa-4 cells
were cotransfected with 0.9 µg each of the above three con structs in combination with Ras V12 and GR expression constructs
as indicated. Transfection with the empty vector pGL2-P is included as
a control. The transfection, treatment protocol, and dual luciferase
assays were carried out as described in Fig. 3. The luciferase activity
in each extract of p(1334/1306)GL2-P-,
p(1334/1306)2GL2-P-, TAT (GRE)3TK/Luc-,
and pGL2-P-transfected cells, respectively, is arbitrarily designated
as 1 (lanes 1, 5, 9, and
13). The level of induction for each construct, expressed as
-fold induction, is calculated by dividing the normalized reporter
luciferase activity in extracts from Ras-cotransfected cells
(lanes 2, 6, 10, and
14), GR-cotransfected/dexamethasone (Dex)-treated
cells (lanes 3, 7, 11, and
15), and Ras- plus GR-cotransfected/dexamethasone-treated
cells (lanes 4, 8, 12, and
16), respectively, over that of corresponding
vehicle-treated transfected cells (lanes 1,
5, 9, and 13). One representative data
set out of three independent experiments is shown.
|
|
As shown in Fig. 6A, mutations on the putative AP-1 site (Mt
A) had no obvious effect on the heterologous promoter activity when Mt
A was transfected alone or cotransfected with GR and/or Ras V12
expression plasmids. Thus, the putative AP-1 site was not involved in
Ras-mediated GR transrepression in Pa-4 cells. The activity of Mt B in
unstimulated conditions was not affected when compared with that of the
wild type or Mt A (Fig. 6A). Therefore, an intact GRE site
was not required for the identified enhancer to modulate
-ENaC expression in the absence of glucocorticoid hormone. Its presence, however, was indispensable for
dexamethasone-induced -ENaC expression and for the Ras
pathway to block the induction, since altering the nucleotide sequences
crucial to GRE motif drastically reduced the GR/dexamethasone-induced
reporter activity and the magnitude of Ras-mediated,
GR-dependent repression (Fig. 6A). Moreover,
AP-1 site elicited no enhancing effect on the expression of Mt B, which
lacks the functional GRE site, confirming that Ras activation had no
effect on the identified -ENaC enhancer activity in the
absence of GR/dexamethasone transactivation.
The data in Fig. 3 suggest that -ENaC DNA sequences
between 1355 and 1269 bp confer high GR/dexamethasone-induced
activity and Ras-mediated repression of
GR/dexamethasone-dependent activity. Furthermore, the data
in Fig. 6A illustrate that this Ras-repressible enhancer
activity is largely abolished when the half-palindrome (nucleotides
1322 to 1318) of the GRE site is mutated, but not by mutations on
the adjacent AP-1 sequence. To establish that the identified GRE
sequence alone can serve as a GR/dexamethasone-dependent enhancer and confer Ras-repressibility over the
GR/dexamethasone-mediated transactivation on a heterologous promoter,
-ENaC DNA sequences of 1334 to 1306 bp were cloned in
both sense and antisense orientations into a luciferase reporter
vector, pGL2-P, containing a minimal SV40 promoter. In transiently
GR-cotransfected Pa-4 cells, the p(1334/1306)GL2-P reporter
construct exhibited at least a 10-fold increase in luciferase activity
in the presence of dexamethasone; a -ENaC GRE
(1334/1306) copy number-dependent dexamethasone enhancement was also observed (Fig. 6B). A similar result
was also observed from the antisense construct, p(1306/1334)GL2-P (data not shown). As illustrated in Fig. 6B, the
transcriptional enhancement by GR/dexamethasone on reporter constructs
harboring either one copy or two copies of -ENaC GRE was
inhibited by the cotransfected Ras V12 expression plasmid. Similarly,
GR/dexamethasone-dependent enhancement and Ras-mediated
repression were also observed in Pa-4 cells by a tyrosine
aminotransferase (TAT) simple GRE sequence on the minimal TK promoter
(Fig. 6B). These data clearly establish that
-ENaC sequences of 1323 to 1309 bp are capable of
functioning as a canonical GRE and its enhancer activity is inhibited
by Ras pathway activation in salivary Pa-4 cells.
To further ascertain the contribution from the identified GRE in
modulating -ENaC expression, we investigated the consequences of GRE
mutation in its native context. Point mutations were made on the GRE of
-ENaC/CATs and the effects of GR/dexamethasone and Ras
V12 on the wild type and GRE-mutated -ENaC/CATs reporter constructs were assessed. As shown in left panels
of Fig. 7,
GR/dexamethasone-dependent induction of wild type
-ENaC reporter activity was unequivocally abolished by
Ras V12, although not entirely, in either serum-free condition
(A) or serum-containing growth medium (B).
GR/dexamethasone treatment failed to confer a robust induction on
reporter activity of GRE-mutated -ENaC/CATs
(right panels, Fig. 7) when compared with that of
the wild type in both culture conditions. Thus, point mutations on the
identified GRE not only reduced the basal reporter expression but also
abrogated GR/dexamethasone-mediated induction substantially.
Nevertheless, the residual reporter activity from the GRE mutant was
slightly enhanced by GR/dexamethasone treatment and repressed by Ras
V12 (Fig. 7, right panel). Taken together, results from Fig. 7 confirm the observation that the identified GRE
plays an essential role in modulating -ENaC expression. However, this fails to rule out the possibility that a second, albeit much weaker, GRE exists in the 1.4 kb of -ENaC 5'-flanking
region that could also be inhibited by Ras pathway activation.
View larger version (83K):
[in this window]
[in a new window]
|
Fig. 7.
The -ENaC GRE confers
glucocorticoid response in the context of its own promoter.
Salivary Pa-4 cells were transiently transfected with 0.9 µg of
-ENaC/CATs (Wt) or its GRE mutant (GRE
Mt) with point mutations, as described in Fig. 6A, in
the presence or absence of 0.5 µg of Ras V12 and 0.5 µg of GR
expression plasmids, as indicated. Following transfection, the cells
were either serum-starved for 8 h and treated with
107 M dexamethasone (Dex)
overnight (A), or cultured in the growth medium and treated
with dexamethasone overnight (B). Transfection,
normalization, and treatment protocols were as described in Fig. 3,
whereas CAT assay was as described in Fig. 1.
|
|
| |
DISCUSSION |
The results presented herein demonstrate that the transcription of
the rat -ENaC gene in salivary epithelial cells depends upon an enhancer located between 1.5 and 1.0 kb upstream from the
translation initiation site. Elements within this region are required
for Ras pathway-mediated repression of -ENaC expression (Fig. 2). We have mapped a Ras-repressible enhancer activity of this
region to a DNA sequence between 1334 and 1306 bp, which encompasses a GRE site. Our data provide evidence that the overall activity of the -ENaC promoter/enhancer represents
an integrated response to GR/dexamethasone-dependent
activation and Ras-mediated repression.
A number of physiological, pathological, and pharmacological stimuli
are known to regulate ENaC function (reviewed in Refs. 12 and 13). In
order to establish how these stimuli work in concert, it is necessary
to define the signal transduction pathways and transcriptional control
paradigms that are activated by various stimuli and to assess the
integrated response to these stimuli. Previously, we have reported that
TPA-mediated ERK activation appears to be required for regulating
several biological responses in salivary epithelial cells including the
modulation of -ENaC mRNA level (11, 30). In this
context, a partial aim of the present study was to identify the
regulatory elements of the rat -ENaC gene responsible for
Raf/ERK-mediated down-regulation of -ENaC expression in
salivary epithelial cells. The exact steps that follow ERK activation
to attenuate -ENaC mRNA steady-state level are
unclear at present. The activity of a number of transcription factors,
including AP-1, Ets, and SRF (31-34) are known to be modulated by the
Raf/ERK pathway. However, whether or not their activation is associated
with ERK-mediated repression of -ENaC expression has not
been established.
To delineate the mode of -ENaC gene regulation, we have
examined the ability of different Ras effector pathways, essential cis-elements, and their cognate trans-factors to
regulate -ENaC expression. A 508-bp fragment located
between 1573 and 1066 bp of the rat -ENaC gene was
found to activate transcription of -ENaC promoter (Fig.
2). Moreover, the observed enhancing activity was promoter- and
orientation-independent, based on its ability to increase the
transcription via either the SV40 minimal promoter or the proximal
-ENaC promoter in both sense and antisense orientations,
the hallmark of a classical enhancer. One novel aspect of the
identified -ENaC enhancer is that both the activation and
repression elements reside within the same region, 1355 to 1269 bp,
i.e. deletion of the 1355 to 1269 bp region
substantially decreases both enhancement and Ras-mediated repression
(Fig. 3), implying that elements located within this region enhances
the homologous/heterologous promoter activity and that Ras pathway activation antagonizes its enhancing effect. Moreover, site-specific mutations of nucleotides 1322 to 1318 abrogate both enhancing and
repressing effects (Fig. 6A), strongly suggesting that the enhancement and repression are mediated via the same element. In this
regard, we further demonstrate that a single copy of this element alone
is sufficient to recapitulate the dexamethasone-stimulated and
Ras-inhibitable effects on the heterologous promoter (Fig. 6B). In addition, specific mutation on the identified GRE in
its native context drastically attenuated the Ras-repressible and GR/dexamethasone-inducible -ENaC/CATs reporter activity
(Fig. 7). We conclude that Ras pathway activation represses GR-mediated transactivation of -ENaC via the identified GRE located
in nucleotides 1334 to 1306 of the -ENaC 5'-flanking region.
This novel finding extends the potential role of Ras pathway(s) in
modulating various biological responses. The exact mechanism(s) by
which Ras blocks GR/dexamethasone-mediated transactivation used in
modulating -ENaC expression is unknown. Currently, we are
investigating whether Ras pathway activation down-regulates the GR
protein level or attenuates its ability to interact with the
-ENaC GRE in Pa-4 cells. A similar functional antagonism between the activation of JNK and ERK kinases and
dexamethasone-stimulated GRE/reporter activities has recently been
reported (35). These authors showed that phosphorylation of GR at
Ser-246 by JNK led to the inhibition of GR-mediated transcriptional
activation, whereas ERK did so indirectly. Our present work extends the
conclusion by Rogatsky et al. (35) by demonstrating for the
first time that the transcription of the endogenous -ENaC
gene as well as -ENaC/reporter construct(s) is regulated via the
opposing effects of GR- and Ras-mediated signaling pathways.
Furthermore, the findings that endogenous -ENaC
expression is induced by glucocorticoid hormone and attenuated by TPA
treatment (Fig. 5) support the physiological and pharmacological
relevance of our data. Moreover, it rules out the possibility that the
observed transrepression is a result of transcriptional squelching
caused by overexpression of Ras V12 and/or GR expression plasmids.
Substantial progress has been made in understanding the mechanisms
through which Ras exerts its biological effects. For example, there are
multiple effector pathways stimulated by Ras activation, which vary in
different cells. Recent studies have shown that the activation of Ras
signaling pathway regulates total RNA and protein synthesis (36-38). A
wide array of promoters have been reported to be up-regulated by Ras
activation as a result of a stimulatory effect on the basal
transcriptional apparatus of the cells as well as on specific
transcription factors (reviewed in Ref. 39). Using the attenuated
transcription of -ENaC by the Raf/ERK pathway as a
paradigm, we have uncovered a mammalian signal transduction mechanism
underlying Ras-mediated gene repression in epithelial cells. These
results emphasize a fundamental difference between activation and
inhibition of gene expression mediated by the same pathway, and
reinforce the importance of studying Ras action in both the global and
the specific enhancement/repression of gene expression.
Based on our data, a putative model on the regulation of
-ENaC expression in the salivary epithelial cells is
depicted as an integrated response to both Ras- and GR-mediated
signaling pathways (Fig. 8). We propose
that Ras activation leads to the activation of ERK kinases as well as a
less well defined Ral-GDS effector pathway. Subsequently, these two
effector pathways may work in concert or individually to inhibit
GR-stimulated -ENaC transcription in a direct and/or
indirect fashion. This model is supported by the failure of MEK
inhibitor, PD 98059, to fully block Ras-mediated repression, indicating
that other effector pathway(s) may participate in parallel with the
elucidated Raf/ERK pathway to mediate GR-dependent
transrepression of -ENaC expression. The results
obtained from transient transfection studies employing mutant
constructs with homologous (Fig. 2) and heterologous (Fig. 3) promoters
support the hypothesis that GR activation is necessary for Ras-mediated
repression of -ENaC promoter. In addition, part of the
Ras-mediated GR transrepression is independent of the presence of the
adjacent putative AP-1 site (Fig. 6A, Mt A).
Moreover, the expression of Ras V12 inhibits the luciferase activity in cells transfected with reporter constructs containing either
-ENaC- or TAT-GRE (Fig. 6B). These data
suggest that Ras-mediated GR/dexamethasone-dependent repression may be a common mechanism modulating the expression of
GR-target genes in epithelial cells. It is possible, however, that
there is other yet unidentified transcription factor(s) "X" interacting with DNA motif(s) within the cloned 1.4-kb
-ENaC 5'-flanking region that may likewise be modified by
Ras pathway activation to inhibit -ENaC expression (Fig.
8).
View larger version (26K):
[in this window]
[in a new window]
|
Fig. 8.
A model for the regulation of
-ENaC expression in salivary epithelial cells by
glucocorticoid hormone and Ras signaling pathways. Transcription
of -ENaC gene in parotid salivary epithelial cells is
activated by glucocorticoid hormone treatment and repressed by GR
antagonist, ZK98.299. GR/dexamethasone-mediated induction is also
antagonized by Ras activation of at least two distinct effector
pathways, Ral-GDS and Raf/ERK. In addition, treatment of cells with TPA
represses -ENaC transcription possibly through Raf/MEK
pathway. Previously, we have shown that TPA-mediated down-regulation of
-ENaC is attenuated by the MEK inhibitor, PD 98059 (11),
which is consistent with our current results, indicating that the
Ras/Raf/MEK/ERK pathway activation antagonizes -ENaC
expression. Whether Ral-GDS down-regulates -ENaC
expression via another downstream effector(s) or MEK-independent ERK is
unclear. It is possible that the Ras/ERK pathway not only suppresses
GR/dexamethasone-mediated induction, but also simultaneously induces
and/or activates an unidentified protein "X" to repress
-ENaC transcription. This scheme depicts an integrated
molecular model of -ENaC gene regulation, illustrating
the antagonistic effects between glucocorticoid hormone and Ras
pathways. By the same mechanism, Ras pathway activation by cytokine or
growth factor may also influence the expression of other GR-targeted
genes in salivary epithelia.
|
|
-Subunit Is Regulated by Antagonistic Effects between
Glucocorticoid Hormone and Ras Pathways in Salivary Epithelial
Cells*
§,
**
TOP
ABSTRACT
INTRODUCTION
