Coordinate upregulation of guanylin and uroguanylin
expression by hypertonicity in HT29-18-N2 cells
Kris A.
Steinbrecher,
Jeffrey A.
Rudolph,
Guangju
Luo, and
Mitchell B.
Cohen
Division of Pediatric Gastroenterology, Hepatology, and
Nutrition and Graduate Program in Molecular and Developmental Biology,
Children's Hospital Research Foundation, Children's Hospital Medical
Center and University of Cincinnati, Cincinnati, Ohio 45229
 |
ABSTRACT |
Guanylin and uroguanylin are
particulate guanylate cyclase-activating peptides that are secreted
from the epithelia of the intestine, kidney, pancreas, and salivary
gland. These peptides elicit chloride and bicarbonate secretion via the
cystic fibrosis transmembrane conductance regulator. To test the
hypothesis that hypertonicity mediates an increase in guanylin and
uroguanylin mRNA, we subjected HT29-18-N2 to osmotic stress. Guanylin
and uroguanylin RNA were increased substantially in the presence of hypertonicity but only with solutes that were relatively impermeable to
the cell membrane. This hypertonicity-mediated increase was transcriptional and did not require protein synthesis. Herbimycin A and
mitogen-activated protein kinase inhibitors SB-203580 and PD-98059 had
no effect on basal or induced levels of guanylin or uroguanylin. Both
staurosporine and prolonged exposure to phorbol ester reduced basal
levels and completely blocked hypertonicity-related increases in
guanylin or uroguanylin RNA. These data suggest that serine/theonine
protein kinases, possibly protein kinase C (PKC), mediate the
hypertonicity-associated increase in guanylin and uroguanylin RNA. We
conclude that guanylin and uroguanylin are released in response to
hypertonic stress and that regulation of these genes may be mediated by
PKC isoforms.
protein kinase C; osmotic stress; guanylate kinase C
 |
INTRODUCTION |
GUANYLIN AND UROGUANYLIN
are produced and secreted by intestinal epithelia. One or both
ligands are released by kidney, pancreatic, and salivary epithelia, as
well. They are produced as prohormones, and the carboxy terminal region
of each is cleaved to release the active, receptor-binding ligand,
which then binds the transmembrane receptor guanylate cyclase C (GC-C).
Specifically, activation of GC-C by guanylin or uroguanylin results in
elevated cGMP and increased cystic fibrosis transmembrane conductance
regulator (CFTR) activity (6, 10). This epithelial
signaling system is thought to mediate release of intestinal chloride
and probably bicarbonate but may also have distant effects on other
epithelial cells (14, 15). Specifically, guanylin and in
particular uroguanylin may serve a hormonal function and form an
endocrine axis between the intestine and the kidneys. These peptides
may regulate renal electrolyte and water transport during periods of
salt absorption (5, 13, 16, 22). We have previously shown
that guanylin and uroguanylin are increased in a whole animal model of
osmotic diarrhea and speculated that these genes are responsive to the increased intraluminal hypertonicity inherent to this model
(29).
To determine more fully the factors that result in increased
guanylin and uroguanylin expression, we identified a novel in vitro
intestinal epithelial model system that expresses both guanylin and
uroguanylin RNA. The HT29-18-N2 intestinal cell line represents a
reductionist system that lends itself to addressing questions concerning guanylin and uroguanylin production during isotonic and
hypertonic exposure. Here, we show that guanylin and uroguanylin RNA
demonstrate a transcription-dependent increase in response to
hypertonicity and that proguanylin protein secretion is also increased
during hypertonic exposure. Similar to other genes responsive to
hypertonicity, the compatible osmolyte betaine blocks the induction of
guanylin or uroguanylin RNA. Studies to determine which signaling pathways influence both basal and induced RNA levels of these ligands
suggest that serine/threonine protein kinase cascades are involved and
that, specifically, PKC isoforms may mediate increases in levels of
guanylin and uroguanylin RNA during osmotic stress.
 |
EXPERIMENTAL PROCEDURES |
Cell culture and reagents.
HT29-18-N2 cells were a gift from Dr. Cynthia Sears of Johns Hopkins
University. These cells are mucin-secreting human intestinal epithelial
cells and have been previously shown to express guanylin RNA
(18). The experiments described here were performed by
using cells from passages 5 through 15 that were
<2 days past confluence because the effects of hypertonicity were lost
at subsequent time points. This cell line does not form resistive
monolayers with tight junctions. HT29-18-N2 cells were grown at 37°C
and 10% CO2 in Dulbecco's modified Eagle's medium (DMEM)
plus 10% fetal calf serum supplemented with 10 µg/ml penicillin, 50 IU/ml streptomycin, and 5 mg of human transferrin (Sigma-Aldrich, St.
Louis, MO) per liter. Cells were seeded into 12-well plates at equal
amounts. Sodium chloride, urea, and mannitol were all supplied by
Fisher Scientific (St. Louis, MO). Lactose was obtained from ICN
Biochemicals (Aurora, OH). All other reagents and pharmacologicals used
in experimental protocols, unless otherwise stated, were supplied by
Sigma-Aldrich.
Experiments using hypertonic medium were performed as follows. Medium
was removed from confluent wells of HT29-18-N2 cells, and the cells
were washed twice with warm phosphate-buffered saline (PBS). Serum-free
DMEM medium, with or without hypertonic agents, was placed on the
monolayers, and the plates were then incubated at 37°C/10%
CO2 for 24 h. Concentrated solutions of NaCl, urea, lactose, or mannitol were added to bring the experimental medium to the
indicated osmolality. Unless otherwise stated, experiments were
performed using +50 mM NaCl (+100 mosmol/kgH2O),
resulting in hypertonic medium of a calculated 400 mosmol/kgH2O. Aliquots of medium containing each
hypertonic agent and control medium were measured for pH and did not
differ from the accepted norm for HT29-18-N2 culture medium (~pH
7.4). After the 24-h incubation period, the medium was removed, placed
in microcentrifuge tubes, and stored at
80°C until use as described
below. Cells within each well were quickly washed with ice-cold PBS and
then processed in one of two ways. Cells were either placed in Trizol
Reagent (Life Technologies, Gaithersburg, MD) for RNA extraction or in Tris-mannitol lysis buffer for protein extraction. RNA and protein extraction methods are described below. Several activators and inhibitors of cellular proteins were used. RNA transcription and protein translation were inhibited by using actinomycin D and cycloheximide, respectively. PKC was activated and depleted in these
studies by using differing exposure times to phorbol 12-myristate 13-acetate (PMA). A broad range of kinases was blocked by using herbimycin A, a tyrosine phosphorylation inhibitor, or staurosporine, a
serine/threonine kinase inhibitor. The mitogen-activated protein kinases p38 and p42/p44 (ERK) were blocked with SB-203580 and PD-98059,
respectively. All drugs were placed on the cells 15 min before the
introduction of hypertonic medium unless otherwise stated, and
concentrations of each can be found in the text. All pharmacologicals
were dissolved in the minimum amount of vehicle (water, methanol, or
dimethysulfoxide) as recommended by the supplier. Within each study,
control medium contained the same amount of vehicle as was used in
drug-containing medium, which, in all cases, was <0.2%.
Trypan blue assay.
HT29-18-N2 cells that had been incubated with either serum-free control
or serum-free hypertonic mannitol medium (~300
mosmol/kgH2O water control vs. ~400 or ~500
mosmol/kgH2O hypertonic medium) for 24 h were
trypsinized, briefly centrifuged, and resuspended. Cells were then
incubated with 0.4% trypan blue for 5 min. Two wells each of control
and hypertonic medium were used to determine cell viability by counting
total cells, as well as stained cells in duplicate for each well using
a hemocytometer.
Northern analysis.
Total RNA was extracted from cells by using Trizol reagent. Briefly,
cells were washed with PBS and ice-cold Trizol reagent was placed in
each well. The cell monolayer was scraped free, and well contents were
moved to a microcentrifuge tube and thoroughly mixed. Total RNA was
then extracted according to the manufacturer's protocol and stored at
80°C. RNA (20 µg/lane for each sample) was separated by using
1.5% agarose/7% formaldehyde gels. After confirmation of equal
amounts of RNA in each well by ultraviolet visualization of 18S
ribosomal RNA, gels were blotted onto Magnacharge nylon membrane
(Osmonics, Westborough, MA) by using standard capillary transfer
methods. Human guanylin and uroguanylin cDNAs were kindly provided by
Dr. Mark Currie (Monsanto, St. Louis, MO). An actin cDNA probe was a
gift from James Lessard (Children's Hospital Research Foundation,
Cincinnati, OH). Guanylin, uroguanylin, and actin cDNAs were labeled
with 32P-dCTP by using a commercially available random
primer labeling system (Life Technologies). Probes were hybridized to
filters as described previously (7, 34). Hybridization
signals were visualized by using the Molecular Dynamics PhosphorImager
system (Molecular Dynamics, Cambridge, MA).
Western analysis.
Proguanylin-specific antiserum (Ab4696) was generated by
using standard protocols. Briefly, a portion of the human guanylin gene
that encodes prohormone residues 18-115 was placed into the expression vector pET21B, and a recombinant antigen (guanylin prohormone with polyhistidine tag) was produced according to the manufacturer's suggested protocol (Novagen, Madison, WI). This antigen
was injected into New Zealand White rabbits, and human proguanylin-specific antiserum was extracted as per standard
techniques. Antiserum 4696 recognizes the correctly sized band during
Western analysis only in human tissues, e.g., jejunum, ileum, and
colon, that are expected to produce guanylin prohormone.
Protein extraction from HT29-18-N2 cells was performed at the indicated
time points as follows. Medium was removed from each well, and this
"spent" medium was stored at
80°C until analysis. Cells were
quickly washed with ice-cold PBS twice before collection of cell
monolayer. Ice-cold 2 mM Tris · HCl/50 mM
mannitol buffer containing a protease inhibitor cocktail (Protease
Inhibitor Cocktail III; Calbiochem, San Diego, CA) was placed on each
well (0.5 ml), and the cell monolayer was scraped free and placed in a
glass dounce. After complete homogenization with the dounce, each
sample was placed in a 1.5-ml centrifuge tube and spun at 16,000 g at 4°C for 20 min. The supernatant was then moved to a
new tube and stored at
80°C. Cell supernatant (40 µg) or spent
medium (5 µl) from each sample was electrophoresed by using NuPAGE
4-12% gradient polyacrylamide gels and electroblotted onto
nitrocellulose membranes according to the manufacturer's protocol
(Invitrogen, Carlsbad, CA). Membranes were immunoblotted by using
proguanylin-specific antibody 4696-4 at a 1:1,000 dilution. After
incubation with a secondary antibody conjugated to horseradish
peroxidase, signal was visualized on Kodak X-OMAT AR film by using a
commercially available chemiluminescence kit (NEN Life Science
Products, Boston, MA).
Statistical analysis.
All values are presented as means ± SE. Unless otherwise stated,
all comparisons are made between cells in control isotonic DMEM for
24 h and cells in hypertonic medium using the unpaired t-test or analysis of variance (ANOVA) where appropriate.
Differences were considered statistically significant at
P < 0.05.
 |
RESULTS |
Hypertonicity increases guanylin and uroguanylin RNA.
To determine the effect of hypertonicity on guanylin and uroguanylin
RNA levels in HT29-18-N2 cells, hypertonic medium was placed on cells
for 24 h and RNA was extracted. Medium was made hypertonic with
mannitol or sodium chloride to levels approximately +50 or +100
mosmol/kgH2O above isotonicity (350 or 400 mosmol/kgH2O vs. 300 mosmol/kgH2O
control). Guanylin and uroguanylin RNA were increased substantially in
cells exposed to hypertonic medium of both +50 and +100
mosmol/kgH2O for 24 h (Fig. 1,
A-D). These increases were dose dependent with a maximal increase seen at +100
mosmol/kgH2O using either mannitol or sodium chloride
as the extracellular osmolyte. Use of more substantial hypertonicity (up to +300 mosmol/kgH2O mannitol) did not further
increase guanylin or uroguanylin RNA past that seen with +100
mosmol/kgH2O (data not shown). In a parallel set of
experiments, HT29 cells were exposed to +100
mosmol/kgH2O mannitol for 24 h (Fig.
1E). RNA was prepared and probed for guanylin, uroguanylin,
actin, and GAPDH. When normalized for GAPDH expression (which was
unchanged) in the presence of mannitol, guanylin mRNA was increased
50% (P < 0.0001), uroguanylin mRNA was increased 52%
(P < 0.0001), and actin mRNA was decreased 33%
(P = 0.03).

View larger version (48K):
[in this window]
[in a new window]
|
Fig. 1.
Dose-dependent induction of guanylin and uroguanylin RNA during
hypertonic stress. Guanylin (A, B) and
uroguanylin (C, D) RNA are elevated after a 24-h
incubation with medium made hypertonic with 50 mosmol/kgH2O or 100 mosmol/kgH2O of
mannitol or sodium chloride. Insets represent typical
Northern blots at top and 18S ribosomal RNA at
bottom to demonstrate equal loading. All values are
expressed as means ± SE. Values for control (isotonic) medium
were set to 100 for each experiment, and all others were adjusted
accordingly, n = 4 per group. ANOVA for A:
P < 0.0001; for B: P = 0.02; for C: P < 0.0001; for D:
P = 0.003. Asterisks indicate significance vs. control
(P < 0.05). E: 50% increased expression of
guanylin and uroguanylin (P < 0.0001) and 33%
decreased expression of actin after incubation of HT29 cells with 100 mosmol/kgH2O of mannitol for 24 h.
|
|
Guanylin and uroguanylin RNA levels were first elevated at 8 h and
remained elevated for 24-48 h after exposure to hypertonic DMEM
(Fig. 2, A and B).
In the experiment depicted in Fig. 2, basal levels of guanylin and
uroguanylin decreased after 16 h. Levels of guanylin/uroguanylin
consistently declined postconfluence. When preconfluent cells
(70-80% confluent) were cultured for 24-48 h to achieve
confluence, the decline in guanylin and uroguanylin levels was not seen
between time zero and 16 h.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 2.
Time course of guanylin and uroguanylin RNA increase
during hypertonic stress. Guanylin (A) and uroguanylin
(B) RNA are elevated beginning 8 h after exposure to
medium made hypertonic with 100 mosmol/kgH2O of
mannitol. All values are expressed as means ± SE. Values for
control (isotonic) medium were set to 100 for each experiment, and all
others were adjusted accordingly. In this experiment, cells were plated
at confluence and levels of guanylin and uroguanylin declined in
postconfluent culture; n = 3 per group. ANOVA for
A: P < 0.008; for B:
P = 0.03, with 8 h being different by Bonferroni
correction in both groups. Asterisks indicate P < 0.05 vs. control without Bonferroni correction.
|
|
Cell death due to osmotic stress would influence guanylin and
uroguanylin levels. Several studies have determined that HT29-18-N2 cells are viable at hypertonicity levels of ~500
mosmol/kgH2O and higher (1, 21). To
confirm this finding, cells exposed to +100
mosmol/kgH2O or +200 mosmol/kgH2O
mannitol for 24 h were stained with 0.4% trypan blue to determine
cell viability. No significant difference in cell viability between
control and hypertonic medium was seen (95.3 ± 2.1% living
control cells vs. 93.3 ± 2.7% +100 mosmol/kgH2O
mannitol and 93.1 ± 1.2% +200 mosmol/kgH2O mannitol).
Having established that RNA of both genes is elevated after exposure to
an increase of 50 mosmol/kgH2O above isotonic levels, we next sought to identify whether these genes respond in a
solute-specific manner. We incubated HT29-18-N2 cells for 24 h
with +100 mosmol/kgH2O of NaCl, mannitol, lactose, or
urea. Guanylin and uroguanylin RNA induction was dependent on a
transmembrane osmotic gradient (Fig. 3, A and
B). Both ionic (NaCl) and
non-ionic (mannitol and lactose) solutes that are not readily membrane
permeable caused increases in guanylin and uroguanylin levels. However,
urea, a reagent that moves across the cell membrane, does not increase RNA for either gene and elicits a decrease in guanylin RNA. Of note,
urea has been shown to negatively regulate the osmotically responsive
gene aldose reductase, as well (30). Thus guanylin and
uroguanylin are increased in response to the osmotic gradient created
by impermeable solutes and not simply to hyperosmolality.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 3.
Hypertonicity, not hyperosmolality, causes increased
guanylin and uroguanylin RNA. Guanylin (A) and uroguanylin
(B) respond to extracellular solutes that are relatively
non-membrane permeable. NaCl, mannitol, and lactose cause elevated
guanylin and uroguanylin levels, but the membrane-permeable urea does
not. This implies the need for a transmembrane osmotic gradient for the
guanylin and uroguanylin RNA response. All values are expressed as
means ± SE. Values for control (isotonic) medium were set to 100 for each experiment, and all others were adjusted accordingly;
n = 4 to 6 per group. ANOVA for A:
P < 0.0001; for B: P < 0.0001. Asterisks indicate significance vs. control (P < 0.05).
|
|
Proguanylin production and secretion are elevated after exposure to
hypertonicity.
We next sought to determine whether increases in guanylin and
uroguanylin RNA during osmotic stress were reflected in increased prohormone production and secretion. Toward this end, the availability of a human proguanylin-specific antibody, Ab4696, allowed for the
determination of cellular and secreted proguanylin levels in response
to hypertonic conditions. Initial studies to assess the feasibility of
the HT29-18-N2 cell line for use in determining proguanylin protein
levels were performed. Proguanylin levels could be easily detected in
newly confluent cells and in medium that had bathed confluent cells for
as little as 4 h (data not shown). Hypertonic medium (+100
mosmol/kgH2O NaCl) was placed on HT29-18-N2 cells for
24 h, and levels of proguanylin in cell homogenates and in medium
were determined. Proguanylin levels found in HT29-18-N2 cells were
increased 23% in response to hypertonic medium (Fig.
4A; P = 0.01).
In addition, 41% more proguanylin hormone was released from HT29-18-N2
cells after 24 h in hypertonic medium compared with cells in
control isotonic DMEM (Fig. 4B; P = 0.0005).
Elevated levels of proguanylin in cell supernatant and medium that
resulted from exposure to hypertonic conditions were of similar
magnitude to changes in RNA levels (Fig. 1, A and
B). This suggests that RNA levels of guanylin are increased to support elevated production and export of guanylin prohormone. Similar studies to assess the response of human prouroguanylin to
osmotic stress will begin upon availability of prouroguanylin-specific antiserum.

View larger version (70K):
[in this window]
[in a new window]
|
Fig. 4.
Cellular and secreted proguanylin protein is increased
after exposure to hypertonicity. Proguanylin-specific antibody 4696 (Ab4696), which recognizes the ~12-kDa prohormone, was used to
determine both cellular and secreted levels of proguanylin. Cellular
levels of proguanylin (A) and secreted proguanylin found in
cell culture medium (B) are significantly higher after
exposure to hypertonicity (+100 mosmol/kgH2O NaCl).
Typical blots from 4 to 6 experiments per group are shown.
|
|
Guanylin and uroguanylin RNA transcription is increased during
osmotic shock, and protein synthesis is not required for induction.
Next, we addressed the mechanism of the increase in RNA levels of
guanylin and uroguanylin. To determine whether elevations in guanylin
and uroguanylin RNA were due to increased transcription vs. increased
RNA transcript stability, HT29-18-N2 cells were exposed to
hypertonicity for 24 h. Medium was then replaced with hypertonic
DMEM containing 5 µg/ml actinomycin D (1). Samples were
taken at 6 h after exposure to actinomycin D. This approach allowed for the assessment of sustained increases in uroguanylin or
guanylin after impairment of new transcript generation. A decrease was
noted in the levels of guanylin after 6 h with actinomycin D,
suggesting that RNA stability was not sufficient to result in the
hypertonicity-induced elevation. Similarly, hypertonicity-induced uroguanylin expression was also decreased in the presence of
actinomycin, albeit not to basal levels. (Fig.
5). RNA from control wells containing hypertonic medium and methanol (actinomycin D vehicle) did not change during the 6-h time course for guanylin but was lower for uroguanylin. Although further characterization is necessary, this study
suggests that guanylin and uroguanylin RNA levels are increased in
response to hypertonicity through transcriptional means.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 5.
Effects of actinomycin D on basal and
hypertonicity-induced guanylin and uroguanylin RNA levels. Elevated
levels of guanylin and uroguanylin RNA are not maintained after 6 h of 5.0 µg/ml actinomycin D (Act. D). All values are expressed as
means ± SE. Values for control (isotonic) medium were set to 100 for each experiment, and all others were adjusted accordingly;
n = 4 per group. a indicates significance
vs. isotonic untreated control (P < 0.05);
b indicates significance vs. hypertonic untreated
(P < 0.05).
|
|
To determine whether protein translation was necessary to mediate
hypertonic induction of guanylin and uroguanylin, we used cycloheximide
to block protein synthesis in cells exposed to hypertonic medium.
Cycloheximide (6.0 µM) in isotonic medium was placed on control and
experimental cells 2 h before study initiation. At the start of
the study, the preincubation medium was removed and fresh control and
hypertonic medium containing 6.0 µm cycloheximide was placed on the
cells for 24 h. Control and hypertonic medium samples were
increased 2.5- to 5-fold compared with samples not exposed to
cycloheximide (Fig. 6, A and
B). Even in the presence of
cycloheximide, however, a consistent increase in guanylin and uroguanylin levels was seen during osmotic stress. This finding suggests that the transcriptional response of these genes to
hypertonicity is not dependent on protein synthesis. The increases in
guanylin and uroguanylin mRNA seen after cycloheximide treatment, in
the presence or absence of hypertonicity, could be consistent with the
loss of a normally present inhibitor that represses
guanylin/uroguanylin transcription.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 6.
Cycloheximide treatment does not block osmotic
stress-mediated RNA increases in guanylin and uroguanylin. The presence
of cycloheximide has no effect on guanylin (A) and
uroguanylin (B) induction during exposure to hypertonicity.
Basal levels of expression are significantly increased during exposure
to cycloheximide. All values are expressed as means ± SE. Values
for control (isotonic) medium were set to 100 for each experiment, and
all others were adjusted accordingly; n = 5 to 6 per
group. a indicates significance vs. isotonic untreated
control (P < 0.05); b indicates
significance vs. cycloheximide-treated control (P < 0.05).
|
|
The compatible osmolyte, betaine, blocks guanylin and uroguanylin
RNA induction during hypertonic stress.
To establish the induction of guanylin and uroguanylin as a response to
hypertonicity and its effects on cellular function, betaine was added
to both control and hypertonic medium. This provides the HT29-18-N2
cell layer with a compatible osmolyte with which to counter the
hypertonic effects of the NaCl gradient on the cell exterior. Although
addition of 5.0 mM betaine to control medium did not significantly
change the level of guanylin or uroguanylin RNA, addition of betaine
blocked the expected increase in guanylin or uroguanylin after 24 h in hypertonic medium (Fig. 7, A and B). The lack of guanylin or
uroguanylin RNA elevation in hypertonic medium containing betaine is
similar to that seen in other osmotically responsive genes (12,
28).

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 7.
The compatible osmolyte betaine blocks
hypertonicity-mediated increases in guanylin and uroguanylin RNA.
Guanylin (A) and uroguanylin (B) are not induced
by hypertonicity (+100 mosmol/kgH2O NaCl) when
HT29-18-N2 cells are coincubated for 24 h with 5.0 mM betaine. All
values are expressed as means ± SE. Values for control (isotonic)
medium were set to 100 for each experiment, and all others were
adjusted accordingly; n = 4 to 6 per group. Asterisks
indicate significance vs. control (P < 0.05).
|
|
Serine/threonine protein kinase pathways regulate guanylin and
uroguanylin responses to hypertonic shock.
To identify the intracellular signaling pathways that regulated the
induction of guanylin and uroguanylin during osmotic stress, we
determined the response of these genes to hypertonic medium in the
presence of broad-spectrum kinase inhibitors (Fig.
8). First, HT29-18-N2 cells were exposed
to 1.0 µM herbimycin A (1) for 15 min before and during
the 24-h exposure to +100 mosmol/kgH2O medium made
hypertonic with additional NaCl. This tyrosine protein kinase inhibitor
did not significantly alter either basal and hypertonicity-induced
guanylin or uroguanylin levels (Fig. 8, A and B).
Therefore, tyrosine protein kinase activity does not appear to have a
significant role in regulating guanylin or uroguanylin RNA abundance.
Second, staurosporine was used to block signal transduction through
serine/threonine phosphorylation-dependent pathways. Addition of
staurosporine (0.1 µM) resulted in a striking decrease in both basal
and tonicity-activated guanylin and uroguanylin RNA transcripts (Fig.
8, A and B). These data implicate
serine/threonine protein kinase pathways in regulation of both basal
and induced levels of guanylin and uroguanylin RNA.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 8.
Serine/threonine protein kinase pathways modulate
guanylin and uroguanylin RNA levels in isotonic and hypertonic
conditions. Both basal and hypertonicity-induced levels of guanylin
(A) and uroguanylin (B) RNA are unaffected by
exposure to herbimycin A, indicating little or no role for tyrosine
kinase-dependent pathways in regulating this process. However,
staurosporine significantly decreased both isotonic and
hypertonicity-induced RNA levels of both genes, suggesting that
serine/threonine protein kinase pathways are relevant to guanylin and
uroguanylin transcript regulation and stress response. All values are
expressed as means ± SE. Values for control (isotonic) medium
were set to 100 for each experiment, and all others were adjusted
accordingly; n = 6 per group. a indicates
significance vs. isotonic untreated control (P < 0.05); b indicates significance vs. herbimycin A-treated
control (P < 0.05); c indicates
significance vs. hypertonic untreated group (P < 0.05).
|
|
PKC is inhibited by staurosporine and represented a candidate-signaling
kinase for further investigation. The phorbol ester PMA activates some
PKC isoforms in the short term (minutes to a few hours) but eventually
results in depletion of PKC activity through exhaustion of PKC stores
(2, 24, 27, 31). PMA was used to deplete PKC levels in
HT29-18-N2 cells bathed in isotonic or hypertonic NaCl-containing
medium, and this resulted in a substantial decrease in guanylin and
uroguanylin RNA (Fig. 9). We next asked whether guanylin and uroguanylin RNA would increase if PKC were stimulated. A short, 2-h exposure to 0.1 µM PMA in isotonic medium resulted in large increases in guanylin and uroguanylin RNA and suggests that PKC activity influences basal guanylin and uroguanylin RNA transcript levels (Fig. 10).

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 9.
Long-term treatment of HT29-18-N2 cells with phorbol
ester results in loss of hypertonicity-mediated increases in guanylin
and uroguanylin RNA. Similar to staurosporine treatment, 24-h exposure
to phorbol 12-myristate 13-acetate (PMA) resulted in substantial
decreases in basal levels of guanylin and uroguanylin RNA. PMA
treatment also eliminates the inducing effect of osmotic shock. All
values are expressed as means ± SE. Values for control (isotonic)
medium were set to 100 for each experiment, and all others were
adjusted accordingly; n = 6 to 12 per group;
a indicates significance vs. isotonic untreated control
(P < 0.05); b indicates significance vs.
hypertonic untreated group (P < 0.05).
|
|

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 10.
Activation of protein kinase C (PKC) by PMA results in
elevation of guanylin and uroguanylin RNA. A 2-h exposure to PMA under
isotonic conditions induces guanylin and uroguanylin RNA levels to a
degree that is very similar to that seen during hypertonic shock. All
values are expressed as means ± SE. Values for control (isotonic)
medium were set to 100 for each experiment, and all others were
adjusted accordingly; n = 4 to 6 per group. Asterisks
indicate significance vs. control (P < 0.05).
|
|
Because mitogen-activated protein kinases (MAPK) mediate some cellular
responses to osmotic shock (23, 26, 33), we next determined the effect of inhibition of certain p38 isoforms and p42/p44
(ERK) signaling using the specific inhibitors SB-203580 and PD-98059
(9, 11). Inhibition of p38
and p38
activity with 10 µM SB-203580 or ERK activity with 10 µM PD-98059 had no effect on
basal expression of guanylin or uroguanylin RNA and also did not change
the magnitude of the guanylin and uroguanylin RNA increase after 24-h
exposure to medium made hypertonic (+100 mosmol/kgH2O) with NaCl (Table
1). Although these data suggest no role
for these extensively characterized MAPKs, the potential importance of
other members of this family (e.g., p38
) will need to be
investigated (19, 32, 35).
 |
DISCUSSION |
Guanylin and uroguanylin are both expressed in the epithelia of
the mammalian intestine. Guanylin is present primarily in the distal
small intestine and colon, and uroguanylin is largely produced in the
proximal small intestine. However, some overlap exists and it is
presumed that both ligands have similar function(s). Very little is
known about the physiological conditions that control guanylin and
uroguanylin expression and the signal transduction pathways that
mediate this regulation. Identification of an in vitro model of
guanylin and uroguanylin expression would therefore be desirable. Of
the many human and rodent intestinal cell lines that have been
surveyed, most do not express either guanylin or uroguanylin (7,
18, 34). The human Caco-2 cell line expresses guanylin after
postconfluence differentiation but does not express uroguanylin.
Several subclones of the HT29 tissue culture line exhibit low levels of
guanylin RNA, but few express uroguanylin at levels that are
readily detectable by Northern analysis. Only the goblet cell-like
HT29-18-N2 tissue culture line (a subclone of the human HT29
adenocarcinoma clone) expresses both guanylin and uroguanylin RNA. Both
genes are robustly expressed before and at confluence. Here, we
describe the coordinate transcriptional induction of guanylin and
uroguanylin in response to extracellular hypertonicity. Overall, the
response of guanylin and uroguanylin was qualitatively and
quantitatively similar. Furthermore, we present the first demonstration
that serine/threonine protein kinase pathways may modulate both basal
and osmotic stress-mediated increases in guanylin and uroguanylin.
We have previously reported induction of guanylin and uroguanylin in a
mouse model of osmotic diarrhea (29). After 48 h of
lactose-rich chow or 40 mM polyethylene glycol-containing drinking water, guanylin and uroguanylin RNA and prohormone levels were increased substantially in intestinal epithelia, leading us to speculate that persistent hypertonic intestinal contents mediated this
increase. Consistent with these animal studies are the data presented
here. Increases in guanylin and uroguanylin RNA are similar in
magnitude to that seen in vivo. These data are also consistent with the physiological observations that salt loading increases guanylin levels (20) and that uroguanylin may
serve as a mechanism for the intestine to alert the kidney to high salt intake (14, 15, 22). However, we have demonstrated that hypertonic conditions other than salt can increase guanylin and uroguanylin to a similar degree. The physiological significance of
these stimuli remains to be investigated.
Treatment with either ionic or non-ionic solutes that did not easily
cross the cell membrane resulted in increases in guanylin and
uroguanylin RNA despite the broadly negative effects of cell shrinkage
associated with hypertonicity (17). Urea, however, is
relatively membrane permeable and the hyperosmolality that it generates
did not cause increases in guanylin and uroguanylin RNA levels. These
differences suggest that the presence of an osmotic gradient across the
cell membrane is necessary for guanylin and uroguanylin mRNA induction.
Moreover, the regulation of these genes by tonicity and not simply
osmolality suggests that guanylin and uroguanylin are regulated by
changes in intracellular ion concentration, cytoskeletal
reorganization, and/or other membrane perturbations. Further
experimentation involving cytoskeletal stabilizing agents and ion
channel inhibitors is planned to distinguish among these possibilities.
In Madin-Darby canine kidney (MDCK) epithelial cells, external
hypertonicity may be rectified by transport of betaine to levels 1,000-fold above that found in medium (36). The
supplementation of compatible osmolytes, i.e., betaine, into hypertonic
medium greatly diminishes the transcriptional response of two important osmotically controlled genes, aldose reductase and the betaine transporter (12, 28). Similarly, we found that including
betaine in the medium of HT29-18-N2 cells that are exposed to tonicity of +100 mosmol/kgH2O eliminates the increase in
guanylin and uroguanylin RNA. This provides additional evidence that
these genes are tightly controlled by intestinal epithelial cell osmoregulation.
Several osmotic stress-activated kinase cascades are known to
facilitate transcriptional increases, stabilization or degradation of
RNA, increased protein translation, and activation or inhibition of
functional proteins (3, 4, 17). The serine/threonine protein kinase inhibitor staurosporine caused a decrease in both basal
level of guanylin and uroguanylin RNA, as well as a diminished response
to hypertonicity. Extended PMA treatment had a similar effect on
guanylin and uroguanylin RNA. PKC activity is regulated by
hypertonicity but is blocked by both staurosporine and long-term exposure to phorbol esters (1, 37). Initial studies
suggest that by using highly specific inhibitors of PKC such as
bisindolylmaleimide, basal and induced RNA levels of guanylin and
uroguanylin are almost totally blocked (data not shown). We also report
here that activation of PKC through short-term exposure to PMA
increases guanylin and uroguanylin RNA. Although the MAPKs p38 and ERK
are activated in many cell types by changes in hypertonicity, the use
of specific inhibitors of these kinases did not affect guanylin and
uroguanylin basal expression or tonicity-induced elevation in
HT29-18-N2 cells.
Collectively, these data support several conclusions. First, the
identification of serine/threonine protein kinase pathways in the
regulation of guanylin and uroguanylin levels represents a novel
association between specific intracellular signaling networks and the
RNA abundance of these genes. Second, PKC is a likely candidate for the
regulation of guanylin/uroguanylin RNA levels at both basal and
hypertonicity-stimulated levels in HT29-18-N2 cells. Because PMA
results in a relatively rapid increase in
guanylin/uroguanylin mRNA and the induction of guanylin/uroguanylin by
hypertonicity requires a longer incubation, it is possible that other
serine/threonine kinases are involved or that other signal transduction
mechanisms subsequent to PKC activation are required for
hypertonicity-mediated changes. It was recently reported that PKC
positively influences transcription of the guanylin/uroguanylin
receptor, GC-C, and that the activity of this receptor is upregulated
by PKC-mediated phosphorylation (8, 25, 31). Taken
together, these data suggest that PKC may act at several points in the
guanylin/uroguanylin-GC-C-signaling pathway, perhaps in response to
hypertonicity caused by high levels of extracellular solutes. Further
work to determine which specific PKC isoforms are involved, what
transcription factors are activated, whether cytoskeletal
reorganization is a constituent factor in this process, and what other
serine/threonine protein kinases may influence these genes will provide
a more complete picture of how intracellular signaling networks control
guanylin and uroguanylin expression.
 |
ACKNOWLEDGEMENTS |
This work was supported by a grant from National Institute of
Diabetes and Digestive and Kidney Diseases DK-47318.
 |
FOOTNOTES |
Address for reprint requests and other correspondence:
M. B. Cohen, Div. of Pediatric Gastroenterology, Hepatology,
and Nutrition, MLC 2010, Children's Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229 (E-mail:
mitchell.cohen{at}chmcc.org).
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.
August 22, 2002;10.1152/ajpcell.00010.2002
Received 8 January 2002; accepted in final form 20 August 2002.
 |
REFERENCES |
1.
Baudouin-Legros, M,
Brouillard F,
Cougnon M,
Tondelier D,
Leclerc T,
and
Edelman A.
Modulation of CFTR gene expression in HT-29 cells by extracellular hyperosmolarity.
Am J Physiol Cell Physiol
278:
C49-C56,
2000[Abstract/Free Full Text].
2.
Breuer, W,
Glickstein H,
Kartner N,
Riordan JR,
Ausiello DA,
and
Cabantchik IZ.
Protein kinase C mediates down-regulation of cystic fibrosis transmembrane conductance regulator levels in epithelial cells.
J Biol Chem
268:
13935-13939,
1993[Abstract/Free Full Text].
3.
Burg, MB,
Kwon ED,
and
Kultz D.
Osmotic regulation of gene expression.
FASEB J
10:
1598-1606,
1996[Abstract/Free Full Text].
4.
Burg, MB,
Kwon ED,
and
Kultz D.
Regulation of gene expression by hypertonicity.
Annu Rev Physiol
59:
437-455,
1997[ISI][Medline].
5.
Carrithers, SL,
Hill MJ,
Johnson BR,
O'Hara SM,
Jackson BA,
Ott CE,
Lorenz J,
Mann EA,
Giannella RA,
Forte LR,
and
Greenberg RN.
Renal effects of uroguanylin and guanylin in vivo.
Braz J Med Biol Res
32:
1337-1344,
1999[ISI][Medline].
6.
Chao, AC,
de Sauvage FJ,
Dong YJ,
Wagner JA,
Goeddel DV,
and
Gardner P.
Activation of intestinal CFTR Cl
channel by heat-stable enterotoxin and guanylin via cAMP-dependent protein kinase.
EMBO J
13:
1065-1072,
1994[Abstract].
7.
Cohen, MB,
Hawkins JA,
and
Witte DP.
Guanylin mRNA expression in human intestine and colorectal adenocarcinoma.
Lab Invest
78:
101-108,
1998[ISI][Medline].
8.
Crane, JK,
and
Shanks KL.
Phosphorylation and activation of the intestinal guanylyl cyclase receptor for Escherichia coli heat-stable toxin by protein kinase C.
Mol Cell Biochem
165:
111-120,
1996[ISI][Medline].
9.
Cuenda, A,
Rouse J,
Doza YN,
Meier R,
Cohen P,
Gallagher TF,
Young PR,
and
Lee JC.
SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin-1.
FEBS Lett
364:
229-233,
1995[ISI][Medline].
10.
Cuthbert, AW,
Hickman ME,
MacVinish LJ,
Evans MJ,
Colledge WH,
Ratcliff R,
Seale PW,
and
Humphrey PP.
Chloride secretion in response to guanylin in colonic epithelial from normal and transgenic cystic fibrosis mice.
Br J Pharmacol
112:
31-36,
1994[Abstract].
11.
Dudley, DT,
Pang L,
Decker SJ,
Bridges AJ,
and
Saltiel AR.
A synthetic inhibitor of the mitogen-activated protein kinase cascade.
Proc Natl Acad Sci USA
92:
7686-7689,
1995[Abstract].
12.
Ferraris, JD,
Burg MB,
Williams CK,
Peters EM,
and
Garcia-Perez A.
Betaine transporter cDNA cloning and effect of osmolytes on its mRNA induction.
Am J Physiol Cell Physiol
270:
C650-C654,
1996[Abstract/Free Full Text].
13.
Fonteles, MC,
Greenberg RN,
Monteiro HS,
Currie MG,
and
Forte LR.
Natriuretic and kaliuretic activities of guanylin and uroguanylin in the isolated perfused rat kidney.
Am J Physiol Renal Physiol
275:
F191-F197,
1998[Abstract/Free Full Text].
14.
Forte, LR,
and
Currie MG.
Guanylin: a peptide regulator of epithelial transport.
FASEB J
9:
643-650,
1995[Abstract/Free Full Text].
15.
Forte, LR,
Fan X,
and
Hamra FK.
Salt and water homeostasis: uroguanylin is a circulating peptide hormone with natriuretic activity.
Am J Kidney Dis
28:
296-304,
1996[ISI][Medline].
16.
Greenberg, RN,
Hill M,
Crytzer J,
Krause WJ,
Eber SL,
Hamra FK,
and
Forte LR.
Comparison of effects of uroguanylin, guanylin, and Escherichia coli heat-stable enterotoxin STa in mouse intestine and kidney: evidence that uroguanylin is an intestinal natriuretic hormone.
J Investig Med
45:
276-282,
1997[ISI][Medline].
17.
Haussinger, D.
The role of cellular hydration in the regulation of cell function.
Biochem J
313:
697-710,
1996[ISI][Medline].
18.
Hochman, JA,
Sciaky D,
Whitaker TL,
Hawkins JA,
and
Cohen MB.
Hepatocyte nuclear factor-1
regulates transcription of the guanylin gene.
Am J Physiol Gastrointest Liver Physiol
273:
G833-G841,
1997[Abstract/Free Full Text].
19.
Hu, MC,
Wang YP,
Mikhail A,
Qiu WR,
and
Tan TH.
Murine p38-delta mitogen-activated protein kinase, a developmentally regulated protein kinase that is activated by stress and proinflammatory cytokines.
J Biol Chem
274:
7095-7102,
1999[Abstract/Free Full Text].
20.
Kita, T,
Kitamura K,
Sakata J,
and
Eto T.
Marked increase of guanylin secretion in response to salt loading in the rat small intestine.
Am J Physiol Gastrointest Liver Physiol
277:
G960-G966,
1999[Abstract/Free Full Text].
21.
Ludeking, A,
Fegert P,
Blin N,
and
Gott P.
Osmotic changes and ethanol modify TFF gene expression in gastrointestinal cell lines.
FEBS Lett
439:
180-184,
1998[ISI][Medline].
22.
Potthast, R,
Ehler E,
Scheving LA,
Sindic A,
Schlatter E,
and
Kuhn M.
High salt intake increases uroguanylin expression in mouse kidney.
Endocrinology
142:
3087-3097,
2001[Abstract/Free Full Text].
23.
Roger, F,
Martin PY,
Rousselot M,
Favre H,
and
Feraille E.
Cell shrinkage triggers the activation of mitogen-activated protein kinases by hypertonicity in the rat kidney medullary thick ascending limb of the Henle's loop. Requirement of p38 kinase for the regulatory volume increase response.
J Biol Chem
274:
34103-34110,
1999[Abstract/Free Full Text].
24.
Roman, RM,
Bodily KO,
Wang Y,
Raymond JR,
and
Fitz JG.
Activation of protein kinase C alpha couples cell volume to membrane Cl
permeability in HTC hepatoma and Mz-ChA-1 cholangiocarcinoma cells.
Hepatology
28:
1073-1080,
1998[ISI][Medline].
25.
Roy, N,
Guruprasad MR,
Kondaiah P,
Mann EA,
Giannella RA,
and
Visweswariah SS.
Protein kinase C regulates transcription of the human guanylate cyclase C gene.
Eur J Biochem
268:
2160-2171,
2001[Abstract/Free Full Text].
26.
Sheikh-Hamad, D,
Di Mari J,
Suki WN,
Safirstein R,
Watts BA, 3rd,
and
Rouse D.
p38 kinase activity is essential for osmotic induction of mRNAs for HSP70 and transporter for organic solute betaine in Madin-Darby canine kidney cells.
J Biol Chem
273:
1832-1837,
1998[Abstract/Free Full Text].
27.
Shen, BQ,
Barthelson RA,
Skach W,
Gruenert DC,
Sigal E,
Mrsny RJ,
and
Widdicombe JH.
Mechanism of inhibition of cAMP-dependent epithelial chloride secretion by phorbol esters.
J Biol Chem
268:
19070-19075,
1993[Abstract/Free Full Text].
28.
Smardo, FL, Jr,
Burg MB,
and
Garcia-Perez A.
Kidney aldose reductase gene transcription is osmotically regulated.
Am J Physiol Cell Physiol
262:
C776-C782,
1992[Abstract/Free Full Text].
29.
Steinbrecher, KA,
Mann EA,
Giannella RA,
and
Cohen MB.
Increases in guanylin and uroguanylin in a mouse model of osmotic diarrhea are guanylate cyclase C-independent.
Gastroenterology
121:
1191-1202,
2001[ISI][Medline].
30.
Tian, W,
and
Cohen DM.
Urea inhibits hypertonicity-inducible TonEBP expression and action.
Am J Physiol Renal Physiol
280:
F904-F912,
2001[Abstract/Free Full Text].
31.
Wada, A,
Hasegawa M,
Matsumoto K,
Niidome T,
Kawano Y,
Hidaka Y,
Padilla PI,
Kurazono H,
Shimonishi Y,
and
Hirayama T.
The significance of Ser1029 of the heat-stable enterotoxin receptor (STaR): relation of STa-mediated guanylyl cyclase activation and signaling by phorbol myristate acetate.
FEBS Lett
384:
75-77,
1996[ISI][Medline].
32.
Wang, Z,
Canagarajah BJ,
Boehm JC,
Kassisa S,
Cobb MH,
Young PR,
Abdel-Meguid S,
Adams JL,
and
Goldsmith EJ.
Structural basis of inhibitor selectivity in MAP kinases.
Structure
6:
1117-1128,
1998[ISI][Medline].
33.
Watts, BA, 3rd,
Di Mari JF,
Davis RJ,
and
Good DW.
Hypertonicity activates MAP kinases and inhibits HCO-3 absorption via distinct pathways in thick ascending limb.
Am J Physiol Renal Physiol
275:
F478-F486,
1998[Abstract/Free Full Text].
34.
Whitaker, TL,
Witte DP,
Scott MC,
and
Cohen MB.
Uroguanylin and guanylin: distinct but overlapping patterns of messenger RNA expression in mouse intestine.
Gastroenterology
113:
1000-1006,
1997[ISI][Medline].
35.
Wilson, KP,
McCaffrey PG,
Hsiao K,
Pazhanisamy S,
Galullo V,
Bemis GW,
Fitzgibbon MJ,
Caron PR,
Murcko MA,
and
Su MS.
The structural basis for the specificity of pyridinylimidazole inhibitors of p38 MAP kinase.
Chem Biol
4:
423-431,
1997[ISI][Medline].
36.
Yamauchi, A,
Uchida S,
Kwon HM,
Preston AS,
Robey RB,
Garcia-Perez A,
Burg MB,
and
Handler JS.
Cloning of a Na+- and Cl
-dependent betaine transporter that is regulated by hypertonicity.
J Biol Chem
267:
649-652,
1992[Abstract/Free Full Text].
37.
Zhuang, S,
Hirai SI,
and
Ohno S.
Hyperosmolality induces activation of cPKC and nPKC, a requirement for ERK1/2 activation in NIH/3T3 cells.
Am J Physiol Cell Physiol
278:
C102-C109,
2000[Abstract/Free Full Text].
Am J Physiol Cell Physiol 283(6):C1729-C1737
0363-6143/02 $5.00
Copyright © 2002 the American Physiological Society