1 Section of Digestive and Liver Diseases, Department of Medicine, University of Illinois at Chicago, and Westside Veterans Affairs Medical Center, Chicago 60612; and 2 Department of Medicine, University of Chicago, Chicago, Illinois 60637
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ABSTRACT |
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Na+/H+ exchange (NHE)
activity has been shown to be regulated by various external signals and
protein kinases in many tissues and cell types. A family of six NHE
isoforms has been identified. Three isoforms, NHE1, NHE2, and NHE3,
have been shown to be expressed in the human intestine. The present
studies were designed to study regulation of these human NHE isoforms
by the -isoform of protein kinase C (PKC) in the Caco-2 cell line.
The mRNA levels of the NHE isoforms in Caco-2 cells were initially
measured by a semiquantitative RT-PCR technique in response to PKC
downregulation by long-term exposure to 1 µM
12-O-tetradecanoylphorbol-13-acetate (TPA) for 24 h.
PKC downregulation resulted in an ~60% increase in the mRNA level
for NHE3, but not for NHE1 or NHE2. Utilizing dichlorobenzimidazole riboside, an agent to block the synthesis of new mRNA, we demonstrated that the increase in the NHE3 mRNA in response to downregulation of PKC
was predominantly due to an increase in the rate of transcription, rather than a decrease in the NHE3 mRNA stability. Consistent with the
mRNA results, our data showed that amiloride-sensitive 22Na+ uptake was increased after incubation of
Caco-2 cells with 1 µM TPA for 24 h. To elucidate the role of
PKC-
, an isoform downregulated by TPA, the relative abundance of NHE
isoform mRNA levels and the apical NHE activity were assessed in Caco-2
cells over- and underexpressing PKC-
. Our results demonstrated that
NHE3, but not NHE1 or NHE2, mRNA was downregulated by PKC-
and that
apical NHE activity was higher in cells underexpressing PKC-
and
lower in cells overexpressing PKC-
than in control cells. In
conclusion, these data demonstrate a differential regulation of NHE3,
but not NHE2 or NHE1, expression by PKC in Caco-2 cells, and this regulation appears to be predominantly due to PKC-
.
intracellular pH; phorbol esters; sodium absorption; mRNA; protein kinase C; human intestine
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INTRODUCTION |
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IN EUKAROYTES, the Na+/H+ exchanger (NHE) is a membrane protein that mediates the electroneutral exchange of extracellular Na+ with intracellular H+. A family of six NHE isoforms has been described, with different tissue distributions and functional properties (9, 12, 29, 30). Three isoforms, NHE1, NHE2, and NHE3, have been described in the human intestine (13). In intestinal epithelial cells, the ubiquitous housekeeping isoform NHE1 has been localized to the basolateral membrane and is suggested to be involved in regulation of cell volume and intracellular pH (pHi) (33, 35, 37). NHE2 and NHE3 isoforms have been localized to the apical membrane and are suggested to be involved in vectorial Na+ transport (18).
Previous studies have revealed that many molecular signals modulate NHE activity in various cells and tissues (17). The three intestinal NHE isoforms have been shown to be differentially regulated with various external signals and second messengers (21). NHE1 and NHE2 isoforms are stimulated by growth factors and hormones, including insulin, thrombin, and phorbol esters (16, 36). In contrast, although the NHE3 isoform has been shown to be stimulated by growth factors, serum, and okadaic acid, it has been shown to be inhibited by acute protein kinase C (PKC) stimulation by phorbol esters. The regulation of NHE1 by these factors has been shown to be through an increase in the affinity of the exchanger for H+, with no changes in the maximal reaction velocity (Vmax) (24). In contrast, NHE2 and NHE3 isoforms have been shown to be regulated with different kinetic characteristics, namely, an alteration of Vmax values of these isoforms with no changes in Michealis-Menten constant (Km) (4, 36, 41).
Previous studies have shown that a number of regulatory factors modulate the activity of the NHE isoforms via protein kinase A (PKA)- and PKC-mediated pathways and that heterotrimeric G proteins play an essential intermediary role in these regulatory pathways (21). Structural analysis suggested that the cytoplasmic domain of the rat and rabbit NHE3 isoform has potential consensus sequences for phosphorylation by various kinases (31, 36). Furthermore, studies have shown that although NHE3 is a phosphoprotein under basal conditions, changes in the phosphorylation of NHE3 are not involved in its regulation by growth factors and PKC (41). One recent study has demonstrated that PKC regulates NHE3 acutely by altering the subcellular distribution of the protein product of NHE3 in the human colonic cell line Caco-2 (20).
PKC comprises a large family of phospholipid-dependent serine/threonine
kinases with multiple isoforms and different individual characteristics
and distinct patterns of tissue distribution (19, 28).
According to their cofactor requirement, these isoforms are classified
as 1) classical isoforms (,
I,
II, and
), which are regulated by diacylglycerol
(DAG) and calcium; 2) novel isoforms (
,
,
, and
), which are activated by DAG but are calcium independent, and
3) atypical isoforms (
,
, and
), which are DAG and
calcium independent. However, the role of individual isoforms of PKC in the regulation of NHE has not been fully defined. In a recent study,
Chen and Wu (7) showed, however, that PKC-
appears to
be involved in the activation of NHE in glioma cells.
On the basis of the above-mentioned considerations, the present studies
were, therefore, undertaken to determine whether the human colonic
adenocarcinoma cell line Caco-2 is a suitable experimental model to
study the modulation of NHE isoforms by PKC by examining the effect of
phorbol esters on the expression and/or function of various human NHE
isoforms in Caco-2 cells and to investigate the role of PKC-, in
particular, on the regulation of human NHE isoforms in Caco-2 cells. To
analyze the role of PKC-
in the regulation of NHE activity in Caco-2
cells, we utilized stably transfected Caco-2 cells with the full-length
cDNA of PKC-
in the sense orientation and the antisense orientation
to over- and underexpress PKC-
, respectively, in these cells
(34).
Our present studies demonstrate that Caco-2 cells express the human
NHE1, NHE2, and NHE3 isoforms and serve as an excellent experimental
model to assess the regulation of NHE isoforms by PKC. Utilizing this
experimental model, we have demonstrated differential regulation of
NHE3, but not NHE2 or NHE1, at the transcriptional level by PKC. This
regulation of NHE3 by PKC appears to be predominantly due to the
PKC- isoform.
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MATERIALS AND METHODS |
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Cell culture.
Caco-2 cells were acquired from American Type Culture Collection
(Manassas, VA) and were cultured at 37°C in an atmosphere of 5%
CO2-95% air and maintained in Dulbecco's modified
Eagle's medium supplemented with 40% F-12 nutrient mixture and 20%
FBS. Cells between passages 20 and 25 were grown
in 24-well plastic plates and incubated for required periods with
various concentrations of phorbol 12-myristate 13-acetate [PMA, i.e.,
12-O-tetradecanoylphorbol-13-acetate (TPA)], or
4-phorbol 12-myristate 13-acetate (4
-TPA) as a control, at
day 7 after confluence. To inhibit RNA synthesis, cells were incubated with 5,6-dichlorobenzimidazole riboside (DRB) for 12 h.
Plasmid constructions and transfection of human PKC- in Caco-2
cells.
The full-length cDNA encoding human PKC-
in pBluescript was obtained
from Dr. Hubert Hug (14). The PKC-
cDNA was subcloned into the eukaryotic expression vector pRc/CMV, in the sense or the
antisense orientation, after digestion with
NotI/ApaI or XbaI/HindIII, respectively. Caco-2 cells were transfected with the constructed plasmids or the pRc/CMV vector alone by calcium phosphate
coprecipitation methods (34). The over- or underexpression
of PKC-
alone, but not the other PKC isoforms, was confirmed by
immunoblotting utilizing specific antipeptide antibodies for PKC-
(Santa Cruz Antibodies, Santa Cruz, CA), as previously described in
detail (34).
Isolation of RNA. Total RNA was extracted from Caco-2 cells by the method of Chomczynski and Sacchi (8) using RNAzol solution supplied by the manufacturer (Tel-Test, Friendswood, TX) and following the manufacturer's protocol.
Designing PCR primer sets for NHE1, NHE2, NHE3, and histone H3.3. The unique PCR primer sets for NHE1, NHE2, and NHE3 were designed from the human sequences that have been retrieved from the Gene-Bank CD-ROM, utilizing GeneWorks software (Intelligenetics, Mountain View, CA). The primer sequences utilized for semiquantitative PCR are as follows: 5'-GACTACACACACGTGCGCACCCC-3' (5' primer) and 5'-TCCAGGATGATGGGCGGCAGCAGGAAGAGGAA-3' (3' primer), with length of amplified region equal to 245 residues (nt 1766-2010 of the human NHE1, M81768) (33), for NHE1; 5'-GAAGATGTTTGTGGACATTGGGG-3' (5'-primer) and 5'-CGTCTGAGCTGCTGCTATTGC-3' (3' primer), with length of amplified region equal to 550 residues (nt 16-565 of the partial human NHE2 clone, S83549, Malakooti et al., unpublished results, representing nt 1744-2293 of the rat NHE2, L11004) (10), for NHE2; 5'-ATCTTCATGTTCCTGGGCATCTCGGC-3' (5' primer) and 5'-GTGCTGAAGTCCACATTGACCAT-3' (3' primer), with length of amplified region equal to 380 residues (nt 1174-1553 of the human NHE3, U28043) (4), for NHE3; and 5'-GCA AGA GTG CGC CCT CTA CTG-3' (5'-primer) and 5'-GGC CTC ACT TGC CTC CTG CAA-3' (3' primer), with length of amplified region equal to 218 residues (nt 454-671 of the human histone H3.3) (40) for histone.
RT-PCR technique. RT-PCR was performed essentially as previously described (13). Briefly, 2-4 µg of total RNA were used for reverse transcription with sequence-specific primers and SuperScript II reverse transcriptase (GIBCO BRL, Gaithersburg, MD). The reaction was carried out in a 20-µl reaction containing 100 mM Tris · HCl (pH 8.3), 2.5 mM MgCl2, 10 mM dithiothreitol, 50 mM KCl, 1 µM dNTPs, 10 µM antisense primer, and 1 µl (200 U) of SuperScript II reverse transcriptase and incubated at 42°C for 1 h. The reaction was terminated by heating at 70°C for 15 min, then 1 µl of RNase H was added and the mixture was incubated for 20 min at 37°C.
Two microliters of the RT reaction mixture were diluted into a final volume of 50 µl of a PCR mixture containing 100 mM Tris · HCl (pH 8.3), 2.5 mM MgCl2, 50 mM KCl, 200 µM dNTPs, antisense and sense primers at 1 µM each, and 0.5 µl (2.5 U) of a 16:1 (unit/unit) mixture of Taq DNA polymerase (GIBCO BRL) and Taq Pfu (Stratagene, La Jolla, CA). PCR was carried out using a Microcycler programmable heating/cooling dry block (Perkin-Elmer, Norfolk, CT) for 30-40 cycles of amplification (94°C for 30 s, 55°C for 1 min, and 72°C for 30 s) followed by 10 min at 72°C. PCR products were separated by electrophoresis on a 1% agarose gel containing ethidium bromide (0.5 µg/ml). Bands of expected sizes were visualized under ultraviolet light utilizing the Eagle Eye II Still Video System (Stratagene).Semiquantitative PCR. Using a modification of the method of Kelley et al. (22), we performed the RT-PCR as described above. Histone H3.3 gene-specific primers were added to the RT and the PCR as an internal control simultaneously with NHE gene-specific primers, and [32P]dCTP (10 µCi at 3,000 Ci/mmol) was added only to the PCR. Aliquots (10 µl) of the PCR were removed from cycles 24, 26, 28, 30, and 32, and the reaction was quenched with 50 mM EDTA and was kept on ice until all the samples were collected. The PCR products were separated on a 5% polyacrylamide gel, which was dried onto Whatman 3MM paper and exposed to a storage phosphor screen overnight, and then analyzed utilizing a PhosphorImager (Molecular Dynamics). The relative abundance of the NHE isoforms was calculated by comparing the number of counts of radioactivity of each isoform product band in various cell types or in response to TPA treatment after normalization with the number of counts in the H3.3 product.
Measurement of pHi. The pHi was measured in Caco-2 cells in suspension using the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-AM (Sigma, St. Louis, MO), as previously described (25, 32). Briefly, cells were detached from the culture plate with trypsin (0.05%)-EDTA (0.53 mM) and washed twice with buffer containing 140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2 5 mM glucose, and 6 mM HEPES-KOH, pH 7.4. Cells were then incubated at 37°C for 30 min with the same buffer containing 10 µM BCECF-AM. Caco-2 cells were washed twice, and the ratio of fluorescence of the intracellularly trapped BCECF dye was determined with excitation at wavelengths of 490 and 440 nm and emission at 530 nm utilizing a luminescence spectrometer (model LS50, Perkin-Elmer, Beaconsfield, UK). To estimate pHi, the BCECF excitation fluorescence ratios were calibrated utilizing the K+/nigericin methods, as previously described (25). The calibration curve demonstrated that the fluorescence ratios were linear as a function of pHi in the range 6-8, as previously reported (25).
22Na uptake. Caco-2 cells were acid loaded by incubation with Na+-free NH4Cl preload buffer (50 mM NH4Cl, 70 mM choline chloride, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM glucose, and 15 mM MOPS, pH 7.0) for 1 h. After the cells were washed with PBS, the uptake buffer (10 mM NaCl, 110 mM choline chloride, 1 mM MgCl2, 2 mM CaCl2, 20 mM HEPES, pH 7.4, and 1 mCi/ml 22Na+) was added to the cells. The uptake rates at various time points were measured in the presence or absence of 0.5 mM amiloride. The reaction was terminated by rapid washes with ice-cold PBS. The cells were then solubilized by incubation with 0.1 N NaOH for 4 h. The protein concentration was measured by the method of Bradford, and the radioactivity was counted by a liquid scintillation analyzer (Tri-CARB 1600-TR, Packard Instrument, Downers Grove, IL). Because the 5-min time point was in the linear range of the uptake (uptake was linear up to 10 min), 22Na+ uptake was measured at 5 min and expressed as nanomoles per milligram per 5 min (counts per minute for 22Na in each sample were in the range 500-2,000 cpm/mg protein).
Statistical analysis. All experiments were performed utilizing at least three or four independent sets of samples on different days. Each independent set represents mean of data from three wells of cells utilized as triplicates. Values are means ± SE. Student's t-tests were used in statistical analysis. P < 0.05 was considered statistically significant.
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RESULTS |
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Effect of PKC downregulation by TPA on NHE isoform mRNA.
To determine whether the Caco-2 cell line represents a good model to
study the role of different PKC isozymes in the regulation of NHE
isoforms, we first examined the expression of NHE1, NHE2, and NHE3
isoforms in Caco-2 cells and their possible regulation in response to
downregulation of PKC by 24 h of exposure to the phorbol ester
TPA. RT-PCR was performed on total RNA extracted from the cells
utilizing gene-specific primers for each individual isoform. The
expected-size bands were detected for each isoform, and the identity of
each isoform was confirmed by sequencing (not shown). To examine the
relative abundance of the NHE isoform mRNA in response to PKC
downregulation, Caco-2 cells were incubated for 24 h with TPA,
4-TPA (inactive form), or vehicle (control). Total RNA was
extracted, and the relative levels of mRNA for NHE isoforms (normalized
to histone H3.3) were compared utilizing the semiquantitative RT-PCR
technique. As shown in Fig. 1, 24 h
of incubation with TPA resulted in an increase in the level of the NHE3
mRNA, but not NHE1 (NHE1-to-H3.3 ratio = 0.64 ± 0.02 for
4
-TPA vs. 0.61 ± 0.04 for TPA, n = 4) or NHE2
(NHE2-to-H3.3 ratio = 0.52 ± 0.06 for 4
-TPA vs. 0.60 ± 0.08 for TPA, n = 3), compared with 4
-TPA and the
untreated control.
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Effect of PKC downregulation by TPA on NHE functional activity.
To analyze whether the increase in the level of NHE3 mRNA in response
to PKC downregulation by TPA has a functional significance, the effect
of 24 h of incubation with TPA on the apical NHE activity in
Caco-2 cells was measured via the assessment of
22Na+ uptake after the cells were acidified by
incubation with the NH4Cl buffer and its subsequent
removal. Under these experimental conditions, total 22Na
uptake in Caco-2 cells was inhibited by ~50% in the presence of 0.5 mM amiloride (7.26 ± 0.31 vs. 14.89 ± 0.51 nmol · mg protein1 · 5 min
1), representing the activity of the apical NHE
isoforms. Therefore, NHE activity in response to PKC downregulation was
evaluated by examining the effect of TPA treatment on amiloride (0.5 mM)-sensitive 22Na uptake in Caco-2 cells. Figure
4 shows that, in parallel to the
increases in the NHE3 mRNA levels, the amiloride-sensitive 22Na+ uptake significantly increased in
response to downregulation of PKC.
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Effect of PKC- on the NHE isoform mRNA levels in Caco-2 cells.
In an attempt to analyze the effect of individual PKC isoforms on NHE3
regulation, we investigated the potential role of PKC-
, one of the
important PKC isoforms, which is downregulated in response to TPA
treatment of Caco-2 cells for 24 h (23), on NHE3 mRNA levels and activity in Caco-2 cells. We used Caco-2 cells under- or
overexpressing PKC-
that had been stably transfected with the
eukaroyte expression vector pRc/CMV containing the full-length cDNA of
PKC-
in the sense or the antisense orientation downstream of the CMV
promoter (34). As shown by immunoblotting in Fig. 5, the Caco-2 transfectants with the
sense-oriented vector demonstrated a nearly threefold increase in the
expression of PKC-
, whereas the antisense-oriented transfectant
cells showed a markedly reduced PKC-
protein expression. We
previously showed by Western blotting that the expression of the other
nontargeted PKC isoforms present in these transfectants remains
unchanged (34). The NHE1, NHE2, and NHE3 isoform mRNA
levels were then analyzed in these transfectants by semiquantitative
RT-PCR. As shown in Fig. 6C,
the relative abundance of the NHE3 mRNA significantly increased
(P < 0.05) in the cells transfected with PKC-
antisense (underexpressing PKC-
) and significantly decreased
(P < 0.05) in the sense transfectant (overexpressing
PKC-
) compared with the transfectants with the empty vector alone or
the wild-type cells. Figure 6, A and B, also
shows no changes in the NHE1 and NHE2 mRNA levels between the
transfectants and the wild-type cells. These findings, taken together
with our previous PKC downregulation studies utilizing 24 h of
incubation with TPA, showing that PKC expression was markedly decreased
under these conditions (23), indicate that the effect of
phorbol ester on NHE3, but not NHE1 or NHE2, mRNA level may be due to
the PKC-
isozyme.
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Effect of PKC- over- and underexpression on NHE activity.
To correlate the mRNA levels with NHE function, the effect of PKC-
on NHE activity in Caco-2 cells was evaluated by measuring the
amiloride-sensitive 22Na+ uptake in the
wild-type cells compared with Caco-2 transfectants over- and
underexpressing PKC-
. As shown in Fig.
7, similar to the mRNA results,
amiloride-sensitive 22Na+ uptake was
significantly increased in the antisense transfectants and decreased in
the sense transfectants compared with the wild-type cells. These data
indicate that, in Caco-2 cells, PKC-
appears to be the predominant
isozyme of PKC that regulates apical membrane NHE activity via
alterations in NHE3, but not NHE1 or NHE2, mRNA levels.
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DISCUSSION |
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NHE activity in various tissues and cell lines has been shown to
be regulated by a variety of external molecular signals (15, 17). These signals appear to regulate the activity of the NHE isoforms predominantly via pathways mediated by protein kinases (16, 31, 36). PKC and PKA, in particular, have been shown to be important regulators of NHE activity (21). However,
the role of individual PKC isoforms in the regulation of the NHE
isoform activities has not been investigated. Additionally, previous
studies have shown that even though the NHEs are phosphoproteins, the regulation by the aforementioned kinases does not appear to be entirely
due to altered phosphorylation of the NHE isoforms (41) but may also involve accessory regulatory factors, such as NHE regulatory factor and NHE3 kinase A regulatory protein (39, 42). To study the mechanisms of regulation of the NHE isoforms via PKC pathways in the human intestine, we initiated the present study
to explore whether the human colonic adenocarcinoma Caco-2 cell line
represents a suitable model to study the effect of individual PKC
isoforms on the activity of NHE isoforms. In the present study, we
showed that the mRNA level of NHE3, but not NHE1 or NHE2, was increased
in response to the downregulation of PKC in Caco-2 cells after 24 h of incubation with TPA. In parallel, the apical NHE activity in
Caco-2 cells was increased in response to 24 h of incubation with
TPA. To further define the role of individual PKC isoforms responsible
in this regulation, we showed that the mRNA level of NHE3, but not NHE2
or NHE1, was increased in Caco-2 cells underexpressing PKC-, whereas
it was decreased in Caco-2 cells overexpressing PKC-
. Consistent
with the above findings, our functional studies in Caco-2 cells
overexpressing PKC-
showed a decrease, while in Caco-2 cells
underexpressing PKC-
an increase in the apical NHE activity was found.
The human colon adenocarcinoma cell line Caco-2 has previously been used as a model to characterize the electrolyte transport processes in the small intestine and colon. In the present studies, 5- to 7-day postconfluent Caco-2 cells were utilized to evaluate NHE3 isoform expression and function in response to PKC downregulation. Previous studies from our laboratory have demonstrated that Caco-2 cells grown under the same conditions have shown the early morphological parameters of differentiation, e.g., formation of domes, development of well-defined brush-border membranes possessing microvilli, and expression of the alkaline phosphatase enzyme, as indicated by the increases in its activity over the indicated time after reaching confluence (34). Watson et al. (38) showed that Caco-2 cells expressed only the basolateral isoform NHE1, but not the apical NHE2 or NHE3 isoforms, whereas Janecki et al. (20) showed that a clone of Caco-2 cells also expressed an endogenous NHE3 at 17-22 days after confluence. These differences in the expression of NHE isoforms have been suggested to be due to a variability in substrains of Caco-2 cells in various laboratories, as well as on the culture conditions, e.g., days after confluence is reached. Our results showed that Caco-2 cells, in the culture conditions described in MATERIALS AND METHODS, express all three intestinal NHE isoforms, NHE1, NHE2, and NHE3, at 5-7 days after confluence.
Among the three isoforms expressed in the intestine, NHE1 has been
shown to be the ubiquitous housekeeping isoform localized on the
basolateral plasma membrane, whereas NHE3 and NHE2 isoforms were shown
to be localized on the apical plasma membrane and suggested to be
responsible for the electroneutral absorption of the NaCl in the
intestine (18). Of the three isoforms, NHE3 has been suggested to be the predominant apical isoform responsible for the
luminal Na+ absorption (12). The results of
our present functional studies demonstrated an increase in the
amiloride-sensitive uptake of 22Na+ by Caco-2
cells (in parallel to the increase in the level of NHE3 mRNA) in
response to downregulation of PKC- after 24 h of incubation
with TPA. Because there were no changes in the mRNA level of NHE2
isoforms, these alterations in activity appear to be mainly contributed
by the changes in NHE3 mRNA.
Although, as noted earlier, PKC and PKA have been shown to be the
important regulators for NHE activities, the role of individual isoforms of PKC has not been elucidated. Chen and Wu (7)
showed that PKC- was involved in NHE activation in glioma cells. In that study, however, it was not clear which isoform(s) of NHE was
expressed in these cells and stimulated by PKC-
. Previous studies
have shown that chronic exposure to TPA caused downregulation in
PKC-
, -
II, and -
in Caco-2 cells
(23). PKC-
is involved in upregulation of phospholipase
D gene expression and cellular differentiation in many cells (3,
23, 27). PKC-
and -
I modulate phospholipase C
and calcium channel activity in mouse pancreatic
-cells
(2). To analyze the role of the PKC-
isoform involved
in the NHE3 mRNA regulation in Caco-2 cells in response to PKC
downregulation by 24 h of exposure to TPA, we utilized Caco-2
cells generated to over- and underexpress the PKC-
isoform, without
any changes in expression of the other nontargeted isoforms of PKC
present in these cells (34). Consistent with the long-term exposure to TPA, NHE3, but not NHE1 or NHE2, mRNA levels were increased
in cells underexpressing PKC-
and were decreased in Caco-2 cells
overexpressing PKC-
. In parallel to the alteration in NHE3 mRNA, the
functional studies have shown an increase in NHE activity in Caco-2
cells that are underexpressing PKC-
and a decrease in the NHE
activity in Caco-2 cells that are overexpressing PKC-
.
We previously showed that over- or underexpressing PKC- in Caco-2
cells altered the proliferative phenotype of Caco-2 cells. Cells that
overexpressed PKC-
(in sense transfectants) demonstrated less
proliferation and a more differentiated phenotype, while underexpression of PKC-
(in antisense transfectants) was associated with greater proliferation and a less differentiated phenotype (1, 34). Therefore, one of the possible explanations of
NHE3 regulation in the transfectants could be that changes in NHE3 mRNA
may be secondary to changes in the proliferation status of these
transfectants. However, we have shown previously (13), utilizing the technique of in situ hybridization, that in the human
ascending colon the mRNA of NHE3, but not NHE2, was strictly localized
to the well-differentiated surface cells, but not to the crypt
proliferative epithelial cells. In light of these previous studies, it
is more likely that the increases in the mRNA levels of the NHE3
isoform in the cells underexpressing PKC-
and the decrease in the
cells overexpressing PKC-
are due to downregulation of the NHE3
isoform at the mRNA level by PKC-
, but not to the phenotypic changes
of the cells under- or overexpressing PKC.
The regulation of NHE1 has previously been shown to be through changes
in the affinity of the exchanger to the H+ concentration,
whereas the regulation of NHE3 and NHE2 was via an alteration in its
Vmax (36). The changes in the
Vmax of the NHE3 isoform in response to TPA
could be a result of decreasing the number of the active molecules on
the membrane or changing its turnover. Our results demonstrated no
changes in the mRNA levels of NHE1 or NHE2, whereas 24 h of
exposure of Caco-2 cells to TPA caused an increase in the mRNA level of
the NHE3 isoform. To investigate whether the increases in the mRNA of
NHE3 in response to downregulating PKC- were due to an increase in
the synthesis of new mRNA or a decrease in the stability of existing
mRNA, we utilized the DRB reagent, which has previously been used to
block de novo RNA synthesis (11). Our results showed that
the increase in the levels of the mRNA of the NHE3 isoform in response
to downregulating PKC-
was predominantly due to an increase in the
transcription rate, rather than a decrease in the stability of the mRNA.
In a recent study, Janecki et al. (20) showed that acute
regulation of the NHE3 isoform by PKC in Caco-2 cells involves subcellular redistribution of the exchanger from the brush-border membrane to the subapical cytoplasmic compartment by a mechanism that
contributes up to 50% of the overall PKC-induced inhibition of the
exchanger. However, in the present study, we investigated the long-term
regulation of the exchanger by PKC in Caco-2 cells by a longer exposure
to TPA (24 h) and in Caco-2 cells that stably over- and underexpressed
PKC-. It is not clear whether the regulation of NHE3 by PKC has two
phases, an acute phase at the level of the protein by internalizing the
exchanger and a chronic phase by altering the rate of its
transcription, or the difference is due to the effect of the different
PKC isoforms involved. These important issues require additional studies.
In conclusion, our results demonstrate for the first time that PKC
downregulates the apical membrane NHE activity and NHE3, but not NHE2
and NHE1, mRNA levels in Caco-2 cells and that the increases in the
mRNA levels are likely to be due to increases in the transcriptional
rate, rather than a decrease in the stability of the NHE3 mRNA. This
regulation of NHE3 appears to be predominantly due to PKC-. Unlike
most of the previous studies of NHE3 regulation by PKC, which have
focused at the acute posttranslational regulation of the exchanger,
e.g., by phosphorylation and recycling, our present studies address the
long-term regulation of the NHE3 isoform by PKC-
most likely at the
transcriptional level. Although the promoter for the human NHE3 has not
been completely cloned, the promoter region for rat and human NHE3 has
consensus sequences for a variety of transcription factors including
Sp-1 and AP-2 (5, 6, 26). In future studies, it will be of
interest to determine the differences between these transcription
factors under basal conditions between the stably transfected Caco-2
cells with PKC-
in the sense and the antisense orientation and to
investigate any correlation between these changes and changes in NHE3
mRNA level.
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ACKNOWLEDGEMENTS |
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These studies were supported by the Department of Veterans Affairs (P. K. Dudeja and K. Ramaswamy) and National Institutes of Health Grants DK-54016 (P. K. Dudeja), DK-33349 (K. Ramaswamy), DK-09930 (W. A. Alrefai), P30-DK-42086 (Digestive Diseases Research Core Center, T. A. Brasitus), 5T32-DK-0704 (B. Scaglione-Sewell), DK-39573 (T. A. Brasitus), and CA-36745 (T. A. Brasitus).
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FOOTNOTES |
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Address for reprint requests and other correspondence: P. K. Dudeja, University of Illinois at Chicago, Medical Research Service (600/151), VA Medical Center, 820 South Damen Ave., Chicago, IL 60612 (E-mail: pkdudeja{at}uic.edu).
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.
Received 30 July 1999; accepted in final form 9 July 2001.
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