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RECEPTORS AND SIGNAL TRANSDUCTION
and TNF-
regulate human NHE3 gene expression by modulating the Sp family transcription factors in human intestinal epithelial cell line C2BBe1
1Section of Digestive Diseases and Nutrition, Department of Medicine, University of Illinois at Chicago; and 2Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois
Submitted 15 December 2005 ; accepted in final form 26 May 2006
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ABSTRACT |
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 TOP ABSTRACT EXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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and TNF-
, which have been shown to downregulate the expression of the sodium-hydrogen exchanger-3 (NHE3) gene. In this study, we have investigated the mechanism of NHE3 gene regulation by IFN- and TNF- in C2BBe1 cells. In response to both IFN- (30 ng/ml) and TNF- (20 ng/ml), the construct containing the bp –95 to +5 region of the human NHE3 promoter, which harbors a number of cis-elements including four potential Sp1 binding sites, showed a maximum repression of 60%. Knockdown of Sp1 and Sp3 expression using small interfering RNA resulted in a significant inhibition of the NHE3 promoter activity and resistance to cytokines effects. These cytokines showed no effects on the expression of Sp1 and Sp3 mRNA and protein levels as assessed by RT-PCR and Western blot analyses, respectively. After treatment with cytokines, the binding of Sp1 and Sp3 proteins to NHE3 promoter decreased significantly, as seen by gel mobility shift assays and chromatin immunoprecipitation assays. The inhibitory effects of both cytokines on the NHE3 promoter were completely blocked by the broad-range kinase inhibitor staurosporine and the selective protein kinase A (PKA) inhibitor 8-bromoadenosine-3',5'-cyclic monophosphorothioate, Rp-isomer. The binding affinity of Sp1 and Sp3 proteins for NHE3 Sp1 probe was significantly decreased after in vitro phosphorylation of nuclear proteins by the -catalytic subunit of PKA. Our data indicate that IFN- and TNF- may repress the NHE3 promoter activity in C2BBe1 cells by PKA-mediated phosphorylation of Sp1 and Sp3 transcription factors.
Na+/H+ exchanger-3; Sp1; Sp3; protein kinase A; phosphorylation; interferon-; tumor necrosis factor-
Diarrhea associated with inflammatory bowel disease (IBD) has traditionally been attributed to stimulated secretion of proinflammatory cytokines, such as interferon- (IFN-) and tumor necrosis factor- (TNF-), which have been shown to repress the NHE3 activity (29). The IBDs are complex disorders with multiple interacting components, including the microbiota, the immune system, and genetic factors among others (12). After failure of the innate immune system to deal with the microbiota in a noninflammatory manner, the acquired immune system of the gut becomes activated, leading to increased expression of inflammatory cytokines such as IFN-, TNF-, and IL-12 by type 1 helper T cell (Th1) and IL-4 and IL-13 by type 2 helper T cell (Th2). These elevated cytokines in the gut are involved in the pathogenesis of chronic intestinal inflammation, causing malabsorption and diarrhea due to intestinal barrier dysfunction and inhibition of NHE3-mediated transport (9, 12, 29).
We have previously reported that the NHE3 promoter region from bp –95 to +5, which harbors a number of cis-elements including four potential Sp1 binding sites, is sufficient for the basal expression of the NHE3 promoter in C2BBe1 cells (22, 23). In the present study, we found that the construct containing the bp –95 to +5 region of the NHE3 promoter was repressed by a combination of IFN- and TNF-.
Promoter-specific factor binding protein-1, also called specificity protein-1 (Sp1), is a member of the C2-H2 zinc finger family (11) and has been implicated in the regulation of the human and rat NHE3 genes (15, 23). Sp1 is also involved in the regulation of a wide variety of genes, including housekeeping genes (17), those involved in proliferation (19), extracellular matrix proteins (33), the early promoter of SV40 (5), and a number of growth factors (8). The mechanisms of Sp1-mediated transcription appear to be promoter specific and in response to specific extracellular signals. Promoter activity could be affected either through a change in the absolute quantity of Sp1 molecules in the cell or by the posttranslational modification of the transcription factor (10). Sp1 protein is known to be phosphorylated by a number of kinases such as casein kinase II, DNA-dependent protein kinase, cAMP-dependent protein kinase (PKA), and protein kinase C (PKC) (1, 18, 28, 30). The DNA binding activity of nuclear factors can be modulated either positively or negatively by phosphorylation (1, 4, 18, 20, 28, 30, 37).
In the present study, we have investigated the cis-elements, the transcription factors, and the signal transduction pathway that mediate the repressive effects of IFN- and TNF- on the NHE3 promoter. Our data suggest that IFN- and TNF- repress the NHE3 promoter activity in C2BBe1 cells via PKA-mediated phosphorylation of Sp1 and Sp3 transcription factors.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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, 8-bromoadenosine-3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-8-Br-cAMP), PKA, catalytic subunit, and fostriecin were from Sigma-Aldrich (St. Louis, MO). Gel shift assay core system, recombinant human TNF-, and calyculin A were from Promega (Madison, WI). The small interfering RNAs (siRNAs) against Sp1 and Sp3, goat polyclonal anti-NHE3, polyclonal anti-rabbit Sp1 and Sp3, and mouse monoclonal actin and tubulin antibodies were purchased from Santa Cruz Biotechnology, (Santa Cruz, CA). Staurosporine, streptomyces Sp, and okadaic acid were obtained from Calbiochem (La Jolla, CA). Promoter reporter plasmids. Plasmids used for functional analysis of the NHE3 promoter activity were generated using pGL2-basic (Promega) containing a promoterless luciferase reporter gene, previously described (22).
Cell culture and transfection.
C2BBe1 cells obtained from the American Type Culture Collection (Manassas, VA) were maintained in culture as previously described (21). This cell line is a subclone derived from a heterogeneous population of the Caco-2 cells and has been shown to undergo spontaneous differentiation as determined by the appearance of the domes characteristics of micovilli and the presence of the markers of differentiation (27). For all the experiments in this study, differentiated (14 days postplating) C2BBe1 cells were used except for transfection studies, for which undifferentiated cells were utilized. These cells were used between the passage numbers 3 and 20. Cells (5 x 104 cells/cm2) were seeded onto collagen-coated 100-mm plastic plates in 10 ml of media [DMEM containing 10% FBS, transferrin (10 µg/ml), 50 units/ml penicillin G-sodium, and 50 µg/ml streptomycin sulfate] for 11 days. Before treatment with IFN- (30 ng/ml) and TNF- (20 ng/ml) for various time periods, C2BBe1 cells were incubated in serum-reduced media (0.5% FBS) for 24 h. For transient transfection studies, cells (1.8 x 105) were seeded onto collagen-coated 12-well plastic plates and cotransfected on the next day (
90% confluent) with one of the NHE3-luc reporter constructs along with pSV-
gal as an internal control using Lipofectamine-2000 (Invitrogen). A total of 2.0 µg DNA/well at a ratio of 4:1 for experimental vs. pSV-gal was used for each transfection. Cells were incubated for 4 h with the DNA/transfection mixture, and then media were replaced with 0.5% FBS-supplemented DMEM for 24 h. Cells were then treated with a cocktail of IFN- (30 ng /ml) and TNF- (20 ng /ml) (referred to as IFN-/TNF- hereafter) for the next 24 h. At the conditions used, exposure of these cells to IFN-/TNF- did not affect the cellular viability in proliferating or differentiated cells (16). For signal transduction experiments, 24 h posttransfection, cells were incubated with staurosporine (10 nM) or Rp-8-Br-cAMP (0.5 mM) for 1 h before addition of cytokines, and incubation was continued for 24 h. As a control, the transfected cells were incubated with 0.5% FBS-supplemented DMEM. Forty-eight hours posttransfection, cells were harvested and lysed, and luciferase and -galactosidase activities were determined. Luciferase activity was normalized to -galactosidase activity. Reporter gene activities of both the treated and untreated samples were normalized to the activity of the promoterless pGL2-basic vector and presented as a percentage. Values shown are means ± SE for triplicate assays from three different experiments.
Sp1 and Sp3 siRNAs and transfections.
Transfections of cells with siRNA were performed using Santa Cruz reagents as described by the manufacturer. Briefly, cells (1.0 x 105) were seeded onto collagen-coated 12-well plastic plates and transfected with siRNA on the next day (60–75% confluent). After 24 h, siRNA-treated cells were transfected again with NHE3 core promoter plasmid p–95/+5 and pSV--gal plasmid as an internal control. Next, these transfected cells were treated with IFN-/TNF- for 24 h and harvested at 48 or 72 h post-siRNA transfection. Reporter gene activities were determined according to the procedure described above. Cells treated with specific siRNA for 48 h were used for Sp1 and Sp3 Western blotting and NHE3 mRNA expression studies.
RNA extraction and RT-PCR analysis.
Differentiated (14 days postplating) and proliferating C2BBe1 (95% confluent) cells were serum starved in DMEM containing 0.5% FBS for 24 h and treated with IFN-/TNF- for the indicated time period. Total RNA was isolated with RNA STAT-60 (TEL-TEST, Friendswood, TX) according to the manufacturer’s instructions. For cDNA synthesis, 5 µg of total RNA were reverse transcribed in the presence of oligo(dT), RNase Out, deoxyribonucleotide triphosphates (dNTPs), 5x first-strand buffer, and Superscript II RT according to the protocol of Invitrogen. Polymerase chain reactions (PCR) were performed using Perkin-Elmer DNA thermal cycler and Platinum PCR SuperMix kit (Invitrogen). The following primers were used: NHE3, sense 5'-GCAGACCTGGCTTCTGAACC-3' and anti-sense 5'-CTCAGCCACGTAGCTGATGGCATC-3'; GAPDH, sense 5'-ATGGCACCGTCAAGGCTGAGA-3' and anti-sense 5'-GGCATGGACTGTGGTCATGAG-3'. PCR conditions for NHE3 were as follows: denaturation at 95°C for 2 min, then the first four cycles of 94°C for 30 s, 57°C for 30 s, 72°C for 45 s, followed by 26 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 45 s. PCR conditions for GAPDH were as follows: 20 cycles of 94°C for 30 s, 57°C for 30 s, and 72°C for 45 s. Final extension was at 72°C for 5 min. cDNA were analyzed by 1.5% agarose gel electrophoresis and visualized by ethidium bromide staining. The amplification reactions yielded the expected cDNA sizes: NHE3, 484 bp; GAPDH, 371 bp.
Western blot analysis.
Total cell lysates and nuclear proteins (NP) were used for NHE3, Sp1, and Sp3 Western immunoblottings. Differentiated C2BBe1 cells were serum starved in DMEM containing 0.5% FBS for 24 h and treated with IFN-/TNF- for various time periods as indicated (see 
Fig. 4). For NHE3, 100 µg of total cell lysate, and for Sp1 and Sp3, 20 µg of NP were separated by 10% SDS-PAGE and electroblotted onto polyvinylidene difluoride (PVDF) membrane (Immobilon-P Millipore). The NHE3, Sp1, and Sp3 proteins were detected using anti-NHE3 goat polyclonal and anti-Sp1 and anti-Sp3 rabbit polyclonal antibodies, respectively. A peroxidase-linked donkey anti-goat IgG (NHE3 protein) and goat anti-rabbit IgG (Sp1 and Sp3 proteins) were used as a secondary antibody, respectively. The complexes were detected using an enhanced chemiluminescence system (ECL plus; Amersham Pharmacia Biotech, Chicago, IL). The blots were reprobed for actin and tubulin mouse monoclonal antibodies as loading control for Sp1, Sp3, and NHE3, respectively. To ensure reproducibility, these experiments were repeated at least three times.
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-32P]ATP (Amersham, Arlington Heights, IL). Binding reactions were initiated by incubating 5 µg of NP and 2 µl of binding buffer [50 mM Tris·HCl, pH 7.5, 5 mM MgCl2, 2.5 mM EDTA, 2.5 mM DTT, 250 mM NaCl, 0.25 µg/µl poly(dI-dC), 20% glycerol (Promega)] for 10 min at room temperature in a volume of 10 µl, and then 40,000 counts/min (cpm) labeled probes were added and incubated for 25 min at room temperature. In competition assays, the unlabeled probes were added to the reaction for 10 min before addition of the labeled probe. For supershift analysis, anti-Sp1 and anti-Sp3 rabbit polyclonal gel shift antibodies were incubated with the binding reaction mixtures for 30 min at room temperature before electrophoresis on 5% polyacrylamide gel in 0.25x TBE running buffer (0.5x TBE is 45 mM Tris base, 45 mM boric acid, 1 mM EDTA, pH 8.0). Gels were dried and visualized by autoradiography.
Chromatin immunoprecipitation assay.
Chromatin immunoprecipitation (ChIP) assays were performed using ChIP-IT kits according to the protocol of ACTIVE MOTIF (Carlsbad, CA). Before treatment with IFN-/TNF- for 24 h, differentiated cells were incubated in serum-reduced media (0.5% FBS) for 24 h. Cells were formaldehyde cross-linked by addition of cell culture medium containing 1% formaldehyde to cells and incubated at room temperature for 10 min, and cross-linking was then stopped by the addition of glycine to a final concentration of 0.125 M. After a washing with ice-cold PBS, cold cell scraping solution was added, and cells were scraped with a rubber policeman. Cells were then pelleted by centrifugation for 10 min at 2,500 rpm at 4°C, resuspended in ice-cold lysis buffer, and incubated on ice for 30 min. Cells were homogenized by a dounce homogenizer, nuclei were pelleted by centrifugation at 5,000 rpm for 10 min at 4°C, and pelleted nuclei were then resuspended in shearing buffer. The resulting chromatin solution was sonicated on ice to an average length of 200–800 bp and then centrifuged at 14,000 rpm for 12 min at 4°C. The supernatant was precleared with protein G beads for 1.5 h at 4°C and was aliquoted and incubated with Sp1 and Sp3 antibodies overnight at 4°C on a rotating wheel. Immunoprecipitated material was washed several times with different wash buffers as described in protocol. DNA-protein cross-links were reversed by incubating samples for 4 h at 65°C in 200 mM NaCl and 10 µg of RNase A to eliminate RNA. Recovered material was treated with proteinase K, and DNA purification was performed by using mini-column. DNA was eluted by adding 50 µl of dH2O to the resin at the bottom of each mini-column and centrifuging for 1 min at 10,000 rpm. PCR analyses were performed with the use of NHE3 promoter-specific primers (forward, 5'-CTGCGTTTATGAAGGAGGGTTGG-3'; reverse, 5-'GCCGGGGTGGCCTTTAATCC-3') and ChIP-IT kit negative control primers (forward, 5'-ATGGTTGCCACTGGGGATCT-3'; reverse, 5'-TGCCAAAGCCTAGGGGAAGA-3'). PCR conditions for NHE3 were as follows: denaturation at 95°C for 3 min and 35 cycles at 94°C for 30 s, 62°C for 30 s, and 72°C for 45 s. PCR products were analyzed by running on a 2% agarose gel.
In vitro phosphorylation of NP by PKA, -catalytic subunit, and DNA binding activity.
In vitro phosphorylation reactions of NP present in the nuclear extracts of C2BBe1 cells were performed as described previously (30). Briefly, 5 µg of NP were incubated with 250 ng of cAMP-dependent protein kinase, -catalytic subunit (Promega), in a 10-µl reaction volume in the presence of 25 mM HEPES, pH 7.5, 34 mM KCl, and 50 mM MgCl2 at 30°C for 45 min. Binding reactions and supershift analysis of in vitro phosphorylated NP were carried out as described above.
Statistical analyses. The difference between two groups was evaluated using Student’s t-test. P < 0.05 was used to indicate statistical significance.
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RESULTS |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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and TNF- on the NHE3 promoter in C2BBe1 cells and identification of the responsive region.
Functional analyses of a series of the 5'-deletion NHE3 promoter reporter constructs were carried out in C2BBe1 cells by transient transfection in the presence and absence of IFN- and TNF-. Before treatment, transiently transfected C2BBe1 cells were serum starved in DMEM containing 0.5% FBS for 24 h. Treatment with IFN- alone (10, 20, 30 ng/ml) (Fig. 1A) or TNF- alone (5, 10, 20 ng /ml) (Fig. 1B) for 24 h resulted in a dose-dependent repression of NHE3 promoter activity. At the highest concentrations, IFN- or TNF- showed 30% repression of the NHE3 promoter activity. However, simultaneous addition of both the cytokines led to enhanced reduction of the NHE3 promoter activity. Among the series of 5'-deletion NHE3 promoter reporter constructs, p–95/+5 showed a maximum repression of 60% in response to co-stimulation with IFN-/TNF- for 24 h (Fig. 1C). We have previously reported that the NHE3 promoter region from bp –95 to +5 was sufficient for the basal expression of the NHE3 promoter, and transcription factors Sp1 and Sp3 are involved in this process (2). Plasmid p–95/+5, which showed maximum repression in response to both cytokines, harbors a number of cis-elements including four potential Sp1 binding sites (Fig. 1D). As shown in Fig. 1C, 5'-deletion construct p–75/+5 showed decreased basal promoter activity as well as decreased responsiveness to IFN-/TNF-. These results indicate that response elements mediating the effects of IFN-/TNF- are present in the bp –95 to +5 region of the NHE3 promoter.
Role of Sp1/Sp3 in cytokine-induced regulation of the NHE3 promoter.
Because the NHE3 core promoter contains several Sp1/ Sp3 binding sites (Fig. 1D) and cytokine treatment showed maximum repression at this region, we hypothesized that IFN-/TNF- response may be mediated by the Sp1 and Sp3 transcription factors. The role of Sp1 and Sp3 on NHE3 promoter activity was examined by using Sp1 and Sp3 siRNAs. In transient transfection studies, individual Sp1- and Sp3-specific siRNAs caused 60 and 30% repression of the NHE3 promoter activity at 48 h, respectivity (Fig. 2A). Simultaneous transfection of C2BBe1 cells with Sp1 and Sp3 siRNAs resulted in 85 and 75% inhibition of the promoter activity at 48 and 72 h, respectivity (Fig. 2A).
As an extension of these observations, we examined whether that inhibitory effect of IFN-/TNF- on the NHE3 promoter activity could still be detected after transfection of the cells with the Sp1/Sp3 siRNAs. C2BBe1 cells were transfected with Sp1 and Sp3 siRNAs individually (Fig. 2, B and C, respectively) or simultaneously (Fig. 2D), along with p–95/+5 and treated with the cytokines as described in EXPERIMENTAL PROCEDURES. The results of these studies revealed that, under these experimental conditions, the NHE3 core promoter showed much less responsiveness to these cytokines. Silencing of Sp1 and Sp3 caused decreased production of Sp1 and Sp3 proteins (Fig. 2E), causing decreased expression of NHE3 gene (Fig. 2E). The sensitivity of NHE3 core promoter activity to IFN-/TNF- decreased from 60 to 20% after simultaneous treatment with Sp1 and Sp3 siRNAs (Fig. 2C).
Time-course effects of IFN-/TNF- on the expression of the NHE3 mRNA in C2BBe1 cells.
To examine the time-dependent effects of the simultaneous presence of IFN- and TNF- on the NHE3 mRNA expression in differentiated and proliferating C2BBe1 cells, we performed RT-PCR experiments. Total RNA was obtained from cytokine-treated and -untreated cells and subjected to reverse transcription and subsequent PCR amplification using NHE3 and GAPDH gene-specific primers. Treatment with both cytokines decreased the NHE3 mRNA levels in a time-dependent manner. In differentiated cells, IFN-/TNF- treatment caused 70% repression of the NHE3 mRNA expression (Fig. 3A), whereas treatment of proliferating cells with IFN-/TNF- caused only 25% repression of the NHE3 mRNA expression (Fig. 3C). So, proliferating C2BBe1 cells respond to the cytokines to a much lesser extent than the differentiated cells.
Effect of IFN-/TNF- on NHE3 mRNA stability.
To examine the effect of IFN-/TNF- on NHE3 mRNA stability, differentiated cells were serum starved in DMEM containing 0.5% FBS for 24 h and then treated with the transcriptional inhibitor actinomycin D (5 µg/ml) alone or in combination with IFN-/TNF- for the indicated time periods (Fig. 3B). Total RNA was prepared, and subsequently RT-PCR analysis was performed using NHE3- and GAPDH-specific primers. A similar amount of NHE3 mRNA was present after treatment of cells with IFN-/TNF- individually or simultaneous treatment with IFN-/TNF- and actinomycin D (5 µg/ml) for the respective time period (Fig. 3, A and B). So, NHE3 mRNA stability is not altered by IFN-/TNF- treatment.
Time-course effects of IFN-/TNF- on the expression of the NHE3, Sp1, and Sp3 proteins in C2BBe1 cells.
Differentiated cells were serum starved in DMEM containing 0.5% FBS for 24 h and treated with IFN-/TNF- for the indicated time periods; total cell lysates were prepared. Anti-NHE3 goat polyclonal antibody was used for Western blot analyses. Treatment with both cytokines decreased the NHE3 protein levels in a time-dependent manner (Fig. 4A). Maximum repressions of NHE3 protein expression were found at 36–48 h. The tubulin expression used as loading control for NHE3 blot. To examine the effects of IFN-/TNF- on the Sp1 and Sp3 protein expression in these cells, Western blot experiments were performed with NP prepared from cells treated as indicated (see Fig. 4, B and C), and blots were probed with anti-Sp1, -Sp3, and -actin antibodies. Sp1 (Fig. 4B) and Sp3 (Fig. 4C) proteins levels remained unaffected at all time periods after treatment of C2BBe1 cells with both cytokines. Actin expression was used as loading control for Sp1 and Sp3 blots.
Analysis of DNA binding activities of the NP from C2BBe1 cells treated with IFN-/TNF- with Sp1 sites.
Using GMSA and supershift analyses with NP from proliferating C2BBe1 cells and a Sp1 probe (bp –89 to –67), we have previously shown that Sp1 and Sp3 transcription factors bind to the NHE3 core promoter region and are involved in the basal promoter activity (23). Figure 5 shows the results of a GMSA. Using the Sp1 probe (bp –89 to –67) and NP from cytokine-treated and untreated differentiated C2BBe1 cells, three DNA-protein complexes were detected in both nuclear extracts (Fig. 5A, lanes 1 and 6). However, an 50% reduction in the intensity of Sp1/Sp3 DNA-protein complexes was observed in NP from IFN-/TNF--treated C2BBe1 cells (Fig. 5A, lanes 1 and 6). The binding specificity of these complexes was examined by competition assays where excess unlabeled NHE3 Sp1-specific probe or nonspecific probe was used in these assays (Fig. 5A, lanes 5 and 10). Unlabeled Sp1-specific probe completely competed out Sp1/Sp3 DNA-protein complexes, suggesting that the protein components of these complexes were Sp1 related. The identities of the proteins present in these complexes were established by supershift assays. Incubation of anti-Sp1 rabbit polyclonal antibody with NP-probe mixtures resulted in the formation of a slow migrating supershift band (Fig. 5A, lanes 2, 4, 7, and 9). Anti-Sp3 rabbit polyclonal antibody completely removed Sp3 protein-probe complex, indicating that one of the protein components of this complex was Sp3 (Fig. 5A, lanes 3, 4, 8, and 9). These results confirm that the Sp1 binding sites present on the human NHE3 gene promoter bind to Sp1 and Sp3 transcription factors, and IFN-/TNF- decreases the binding activity of Sp1 and Sp3 proteins for the NHE3 promoter. The other Sp1 sites that are present on the bp –95 to +5 region of the NHE3 gene showed results similar to the probe (bp –89 to –67) with NP from C2BBe1 cells treated with both IFN- and TNF- (data not shown).
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/TNF- decreases in vivo binding of Sp1 and Sp3 proteins to NHE3 promoter.
Differentiated C2BBe1 cells were incubated in serum-reduced media (0.5% FBS) for 24 h before treatment with IFN-/TNF-. Formaldehyde cross-linked chromatin was prepared from both untreated and cytokine-treated cells. Chromatin was then immunoprecipitated with Sp1 and Sp3 antibodies. Immunoprecipitated DNA was analyzed by PCR using NHE3 promoter-specific primers. As shown in Fig. 5B, in vivo binding of Sp1 and Sp3 transcription factors to the NHE3 promoter decreased significantly compared with untreated cells after treatment with IFN-/TNF-. The amplification reactions with the NHE3 promoter-specific primers and negative control primers yielded the expected cDNA size, 337 bp and 174 bp, respectively.
IFN-/TNF--induced repression of NHE3 promoter is mediated via activation of PKA signaling pathway.
C2BBe1 cells were transiently transfected with the p–95/+5 or p–1507/+131 promoter reporter constructs, serum starved in DMEM containing 0.5% FBS for 24 h, and then treated with IFN-/TNF- in the presence and absence of staurosporine (10 nM) or Rp-8-Br-cAMP (0.1 and 0.5 mM) for 24 h. Cells were preincubated with the inhibitors for 1 h. As a control, the transfected cells were incubated with 0.5% FBS-supplemented DMEM. Staurosporine (Fig. 6, A and C) or Rp-8-Br-cAMP (Fig. 6, B and D) completely blocked the repressive activities of IFN-/TNF- on the NHE3 promoter in transient transfection assays. However, the plasmid containing the NHE3 core promoter region exhibited a higher level of sensitivity to the inhibitory effect of these cytokines. Rp-8-Br-cAMP is an analog of cAMP, and it occupies cAMP binding sites on the regulatory subunit and prevents dissociation and thus activation of the kinase holoenzyme. It is already established that PKA is involved in the phosphorylation of Sp1 protein (30). Therefore, we speculate that IFN-/TNF--induced repression of the NHE3 promoter activity may be mediated via PKA phosphorylation of Sp family transcription factors.
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-catalytic subunit of PKA or untreated NP. Nuclear extract treated with PKA, -catalytic subunit, showed significantly decreased Sp1 binding activity compared with untreated nuclear extract (Fig. 7A, lanes 1 and 6). The supershift experiments clearly showed that nuclear extract contains Sp1 and Sp3 transcription factors (Fig. 7B, lanes 2–4). These results suggest that the -catalytic subunit of PKA causes phosphorylation of Sp1 and Sp3 proteins, and phosphorylated Sp1 and Sp3 proteins may have decreased binding affinity for the NHE3 promoter.
Nuclear extract treated with PKA, -catalytic subunit, did not change the Sp1 protein level compared with untreated nuclear extract as determined by Western blot analysis.
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DISCUSSION |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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/TNF- has not been explored. For the first time, in the present study, we demonstrate that IFN-/TNF- represses the NHE3 gene via cAMP-dependent, protein kinase-mediated phosphorylation of Sp1 and Sp3 transcription factors in the human intestinal epithelial cell line C2BBe1. Functional analysis of a series of the 5'-deletion NHE3 promoter reporter constructs in C2BBe1 cells by transient transfection indicates that the IFN-/TNF- response elements are located in the bp –95 to +5 region of the NHE3 promoter. Plasmid p–95/+5 showed 60 repression of promoter activity in response to IFN-/TNF- treatment.
In differentiated cells, cytokine treatment caused 70% repression of the NHE3 mRNA expression. On the other hand, in proliferating cells, it caused only 25% repression of the NHE3 mRNA. In transient transfection studies, we used proliferating C2BBe1 cells, which are less sensitive to the cytokines than differentiated cells. This might be the basis for the relatively lower NHE3 promoter activity subsequent to IFN-/TNF- treatment in proliferating cells compared with the endogenous NHE3 mRNA level in differentiated cells. Because the NHE3 core promoter contains several potential Sp1/Sp3 binding sites and IFN-/TNF- treatment showed maximum repression of the NHE3 promoter at this region, we hypothesized that IFN- and TNF- might repress NHE3 promoter activity via regulating the activity of the Sp1 family of transcription factors.
We have used siRNAs specific for Sp1 and Sp3 to knock down the expression of these factors to confirm the involvement of the Sp1 and Sp3 transcription factors in the basal and IFN-/TNF- response in C2BBe1 cells. We have recently reported that transcription factors Sp1 and Sp3 are involved in the basal transcriptional activity of the NHE3 core promoter in C2BBe1 cells (23). Here we have shown that the knockdown of both Sp1 and Sp3 by specific siRNA, individually or simultaneously, leads to a significant reduction in the NHE3 basal promoter activity and a minor decrease in response to the cytokine cocktail. This small reduction, in an already reduced promoter activity, may be attributed to the residual Sp1 and Sp3 remaining subsequent to siRNA treatment. However, we could not rule out the contribution of other potential factors to the inhibitory effect displayed by these cytokines. Gel mobility shift assay using a probe spanning the Sp1 binding site from bp –89 to –67 and the nuclear proteins from differentiated C2BBe1 cells treated with IFN-/TNF- showed 50% reduction in the intensity of both Sp1/Sp3 DNA-protein complexes compared with nuclear proteins from untreated cells. Chromatin immunoprecipitation assays revealed that, after treatment with IFN-/TNF-, the in vivo binding of Sp1 and Sp3 transcription factors to the NHE3 promoter decreased significantly. By RT-PCR and Western blot analyses, we showed that cytokines treatment had no effect on the Sp1 and Sp3 mRNA or protein expression. Therefore, we hypothesized that the reduced binding activity of these transcription factors with the NHE3 promoter may be mediated by posttranslational modification of Sp1/Sp3 transcription factors.
Sp family transcription factors are subjected to different forms of posttranslational modifications, including phosphorylation. It is known that kinases and phosphatases maintain the phosphorylation/dephosphorylation state of cellular proteins. When phosphatase activity is inhibited, the activities of cellular kinases appear indirectly to become upregulated. Gel mobility shift assay with the NHE3 Sp1 probe (–89 to –67) and nuclear proteins from cells treated with different phosphatase inhibitors showed decreased DNA binding activity of Sp1 protein compared with untreated cells. These results support the idea that activated kinase activity in cells may lead to phosphorylation of Sp1 transcription factor, resulting in its decreased binding affinity for the NHE3 promoter. The broad-spectrum kinase inhibitor staurosporine and the selective PKA inhibitor Rp-8-Br-cAMP completely blocked the repressive effects of IFN-/TNF- on the NHE3 promoter in transient transfection assays, indicating that these cytokines repress the NHE3 gene via activation of the PKA signaling pathway. Each PKA molecule is composed of two catalytic subunits and two regulatory subunits. The PKA catalytic subunit, complexed with the PKA regulatory subunit in the inactive state, has been reported to be associated with cytoskeleton and different cytoplasmic membrane structures like Golgi stacks through anchoring proteins, e.g., AKAP79 and AKAP75 (25, 26). This localization has been assumed to be essential for the PKA-induced transcriptional regulation of several genes. In response to different cytokines, catalytic subunits dissociate from regulatory subunits and translocate to the nucleus, where they phosphorylate different transcription factors and modulate expression of different genes (7). Our results can be summarized in the working model presented in Fig. 8. After activation of PKA by cytokines, the catalytic subunit of the enzyme translocates to the nucleus, where it phosphorylates Sp1 and Sp3 proteins. Phosphorylated Sp1 and Sp3 show reduced DNA binding affinity to the NHE3 promoter region, leading to repression of NHE3 gene transcription. To examine the effect of PKA-induced phosphorylation on Sp1 and Sp3 protein binding to the NHE3 promoter, we performed DNA binding assays using Sp1 probe and in vitro phosphorylated nuclear proteins of C2BBe1. These results showed reduced binding of Sp1 and Sp3 proteins to the NHE3 promoter region, confirming our hypothesis that phosphorylation of Sp family members leads to their decreased DNA binding affinity and consequently reduces NHE3 promoter activity. There are several reports relating to PKA activation, phosphorylation of NHE3 protein, and inhibition of NHE3 transport activity (14, 24, 38). In this study, for the first time we show that NHE3 gene transcription is negatively regulated by PKA-mediated phosphorylation of Sp1 and Sp3 transcription factors. It has been shown that gene transcription can be modulated positively or negatively by Sp family transcription factor phosphorylation (1, 4, 18, 20, 28, 30, 37). The varied mechanisms of Sp1-mediated transcription appear to be promoter specific and in response to specific extracellular signals.
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, IFN-, and IL-1, -2, -6, -12, and -18 (9, 31, 32). The elevated levels of these cytokines in the human intestine cause intestinal epithelial barrier dysfunction and the inhibition of NHE3, resulting in malabsorption and diarrhea (6, 29, 34). Further studies addressing the role of other cytokines on NHE2 and NHE3 genes and exploring the mechanisms of gene regulation would be of great interest. |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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FOOTNOTES |
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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.
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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X174RF DNA/HaeIII fragments, Invitrogen Life Technologies). Results shown are representative of at least 3 separate experiments.
