Differential regulation of the expression of Na+/H+ exchanger isoform NHE3 by PKC-alpha in Caco-2 cells

W. A. Alrefai1, B. Scaglione-Sewell2, S. Tyagi1, L. Wartman1, T. A. Brasitus2, K. Ramaswamy1, and P. K. Dudeja1

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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-alpha , 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-alpha . Our results demonstrated that NHE3, but not NHE1 or NHE2, mRNA was downregulated by PKC-alpha and that apical NHE activity was higher in cells underexpressing PKC-alpha and lower in cells overexpressing PKC-alpha 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-alpha .

intracellular pH; phorbol esters; sodium absorption; mRNA; protein kinase C; human intestine


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 (alpha , beta I, beta II, and gamma ), which are regulated by diacylglycerol (DAG) and calcium; 2) novel isoforms (delta , epsilon , eta , and theta ), which are activated by DAG but are calcium independent, and 3) atypical isoforms (tau , lambda , and zeta ), 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-delta 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-alpha , in particular, on the regulation of human NHE isoforms in Caco-2 cells. To analyze the role of PKC-alpha in the regulation of NHE activity in Caco-2 cells, we utilized stably transfected Caco-2 cells with the full-length cDNA of PKC-alpha in the sense orientation and the antisense orientation to over- and underexpress PKC-alpha , 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-alpha isoform.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 4alpha -phorbol 12-myristate 13-acetate (4alpha -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-alpha in Caco-2 cells. The full-length cDNA encoding human PKC-alpha in pBluescript was obtained from Dr. Hubert Hug (14). The PKC-alpha 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-alpha alone, but not the other PKC isoforms, was confirmed by immunoblotting utilizing specific antipeptide antibodies for PKC-alpha (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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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, 4alpha -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 4alpha -TPA vs. 0.61 ± 0.04 for TPA, n = 4) or NHE2 (NHE2-to-H3.3 ratio = 0.52 ± 0.06 for 4alpha -TPA vs. 0.60 ± 0.08 for TPA, n = 3), compared with 4alpha -TPA and the untreated control.


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Fig. 1.   Effect of protein kinase C (PKC) downregulation by 12-O-tetradecanoylphorbol-13-acetate (TPA) on Na+/H+ exchanger (NHE) isoform 3 (NHE3) mRNA. Caco-2 cells were incubated for 24 h with 1 µM TPA, 1 µM 4alpha -TPA, or vehicle (control). Cells were harvested, and total RNA was extracted. Relative abundance of NHE3 mRNA is normalized to histone H3.3 as an internal standard. Values are means ± SE (n = 3). *P < 0.005 compared with control and 4alpha -TPA.

To further evaluate whether the effect of TPA on the level of NHE3 mRNA was dose and time dependent, first, the effect of 24 h of exposure to various doses of TPA (1, 10, and 1,000 nM) on NHE3 mRNA level was examined. At 1 and 10 nM TPA, there was no effect on NHE3 mRNA; however, NHE3 mRNA became significantly (P < 0.05) elevated only at 1,000 nM TPA (Fig. 2A). We next examined the time course of 1,000 nM (1 µM) TPA on the level of NHE3 mRNA (Fig. 2B): at 4 h there was no effect on NHE3 mRNA; however, it tended to increase at 12 h and was significantly higher at 24 h. These data agree with dose- and time-dependent downregulation of PKC by TPA in Caco-2 cells, as shown previously (23).


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Fig. 2.   Effect of various concentrations of TPA and its time course on NHE3 mRNA. A: Caco-2 cells incubated for 24 h with various concentrations of TPA (solid bars) or 4alpha -TPA (open bars). B: Caco-2 cells incubated with 1 µM TPA (solid bars) or 1 µM 4alpha -TPA (open bars) at 4, 12, and 24 h. Cells were harvested, and total RNA was extracted. Relative abundance of NHE3 mRNA is normalized to histone H3.3 as an internal standard. Values are means ± SE (n = 3-4). *P < 0.05 compared with control and 4alpha -TPA.

To elucidate whether the increase in the level of NHE3 mRNA was due to new synthesis of NHE3 transcripts or a decrease in their stability, Caco-2 cells were incubated with DRB (an inhibitor of de novo RNA synthesis) along with TPA. As shown in Fig. 3, the decrease in the relative level of NHE3 mRNA after incubation with DRB, a known transcription inhibitor, was similar in the cells incubated with TPA and in the cells incubated with 4alpha -TPA (both showing ~50% decreases), indicating that the increase in NHE3 mRNA in response to TPA as shown above (Fig. 1) was predominantly a result of an increase in the transcription rate.


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Fig. 3.   Effect of dichlorobenzimidazole riboside (DRB) on NHE3 mRNA in TPA-treated cells. Caco-2 cells were incubated for 24 h with 1 µM TPA or 4alpha -TPA (control), and DRB (50 µg/ml) was added 12 h after addition of TPA (DRB + TPA) and to untreated cells (control + DRB). Relative abundance of NHE3 mRNA is normalized to histone H3.3. *P < 0.005 compared with control; **P < 0.005 compared with cells not treated with DRB.

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 protein-1 · 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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Fig. 4.   Effect of PKC downregulation by TPA on NHE activity. Amiloride (0.5 mM)-sensitive 22Na+ uptake was measured in Caco-2 cells after 24 h of incubation with 1 µM TPA or vehicle (control). Values are means ± SE (n = 3). *P < 0.05 compared with control.

To rule out the possibility that the increase in 22Na uptake may be secondary to changes in the driving force for 22Na uptake (i.e., due to alterations in the pHi in control vs. treated cells), rather than functional changes in NHE3 activity in Caco-2 cells, we measured the steady-state pHi in wild-type cells and in cells treated with 1 µM TPA or 4alpha -TPA for 24 h. Our results demonstrated no significant changes in pHi in TPA-treated Caco-2 cells compared with cells treated with 4alpha -TPA or wild-type cells (7.27 ± 0.05, 7.18 ± 0.05, and 7.32 ± 0.07, respectively, n = 4), indicating that the increase in 22Na uptake represents an increase in NHE3 activity in response to PKC downregulation by TPA, but not to possible changes in basal pHi.

Effect of PKC-alpha 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-alpha , 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-alpha that had been stably transfected with the eukaroyte expression vector pRc/CMV containing the full-length cDNA of PKC-alpha 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-alpha , whereas the antisense-oriented transfectant cells showed a markedly reduced PKC-alpha 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-alpha antisense (underexpressing PKC-alpha ) and significantly decreased (P < 0.05) in the sense transfectant (overexpressing PKC-alpha ) 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-alpha isozyme.


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Fig. 5.   Western blot analysis of PKC-alpha expression in parental and transfected Caco-2 cells. Transfectant Caco-2 cells were collected 8 days after they were plated. Proteins were separated by SDS-PAGE, electrotransferred to Immobilon membranes, and probed with PKC-alpha -specific antibodies: wild type (A), empty vector (B), sense transfectants (C), and antisense transfectants (D). Results are representative of 3 independent experiments.



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Fig. 6.   Relative abundance of NHE1, NHE2, and NHE3 in Caco-2 cells over- and underexpressing PKC-alpha . Relative abundance of NHE1 (A), NHE2 (B), and NHE3 (C) mRNA was calculated by normalizing their abundance to histone H3.3 in Caco-2 cells overexpressing (sense) or underexpressing (antisense) PKC-alpha compared with parental Caco-2 cells (wild type) and cells transfected with the empty vector alone (empty vector). Values are means ± SE (n = 3-6). *P < 0.05 compared with empty vector.

Effect of PKC-alpha over- and underexpression on NHE activity. To correlate the mRNA levels with NHE function, the effect of PKC-alpha 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-alpha . 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-alpha 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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Fig. 7.   Effect of PKC-alpha over- and underexpression on NHE activity calculated as amiloride (0.5 mM)-sensitive 22Na+ uptake in Caco-2 overexpressing (sense) and underexpressing (antisense) PKC-alpha compared with wild-type cells. Values are means ± SE (n = 3-6). *P < 0.05 compared with wild type.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha , whereas it was decreased in Caco-2 cells overexpressing PKC-alpha . Consistent with the above findings, our functional studies in Caco-2 cells overexpressing PKC-alpha showed a decrease, while in Caco-2 cells underexpressing PKC-alpha 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-alpha 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-delta 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-delta . Previous studies have shown that chronic exposure to TPA caused downregulation in PKC-alpha , -beta II, and -delta in Caco-2 cells (23). PKC-alpha is involved in upregulation of phospholipase D gene expression and cellular differentiation in many cells (3, 23, 27). PKC-alpha and -beta I modulate phospholipase C and calcium channel activity in mouse pancreatic beta -cells (2). To analyze the role of the PKC-alpha 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-alpha 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-alpha and were decreased in Caco-2 cells overexpressing PKC-alpha . 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-alpha and a decrease in the NHE activity in Caco-2 cells that are overexpressing PKC-alpha .

We previously showed that over- or underexpressing PKC-alpha in Caco-2 cells altered the proliferative phenotype of Caco-2 cells. Cells that overexpressed PKC-alpha (in sense transfectants) demonstrated less proliferation and a more differentiated phenotype, while underexpression of PKC-alpha (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-alpha and the decrease in the cells overexpressing PKC-alpha are due to downregulation of the NHE3 isoform at the mRNA level by PKC-alpha , 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-alpha 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-alpha 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-alpha . 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-alpha . 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-alpha 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-alpha in the sense and the antisense orientation and to investigate any correlation between these changes and changes in NHE3 mRNA level.


    ACKNOWLEDGEMENTS

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).


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Cell Physiol 281(5):C1551-C1558