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American Journal of Physiology - Cell Physiology logoLink to American Journal of Physiology - Cell Physiology
. 2014 Mar 19;306(10):C961–C971. doi: 10.1152/ajpcell.00048.2014

Protein kinase D1 mediates class IIa histone deacetylase phosphorylation and nuclear extrusion in intestinal epithelial cells: role in mitogenic signaling

James Sinnett-Smith 1,*, Yang Ni 1,2,*, Jia Wang 1, Ming Ming 1, Steven H Young 1, Enrique Rozengurt 1,✉
PMCID: PMC4024715  PMID: 24647541

Abstract

We examined whether class IIa histone deacetylases (HDACs) play a role in mitogenic signaling mediated by protein kinase D1 (PKD1) in IEC-18 intestinal epithelial cells. Our results show that class IIa HDAC4, HDAC5, and HDAC7 are prominently expressed in these cells. Stimulation with ANG II, a potent mitogen for IEC-18 cells, induced a striking increase in phosphorylation of HDAC4 at Ser246 and Ser632, HDAC5 at Ser259 and Ser498, and HDAC7 at Ser155. Treatment with the PKD family inhibitors kb NB 142-70 and CRT0066101 or small interfering RNA-mediated knockdown of PKD1 prevented ANG II-induced phosphorylation of HDAC4, HDAC5, and HDAC7. A variety of PKD1 activators in IEC-18 cells, including vasopressin, lysophosphatidic acid, and phorbol esters, also induced HDAC4, HDAC5, and HDAC7 phosphorylation. Using endogenously and ectopically expressed HDAC5, we show that PKD1-mediated phosphorylation of HDAC5 induces its nuclear extrusion into the cytoplasm. In contrast, HDAC5 with Ser259 and Ser498 mutated to Ala was localized to the nucleus in unstimulated and stimulated cells. Treatment of IEC-18 cells with specific inhibitors of class IIa HDACs, including MC1568 and TMP269, prevented cell cycle progression, DNA synthesis, and proliferation induced in response to G protein-coupled receptor/PKD1 activation. The PKD1-class IIa HDAC axis also functions in intestinal epithelial cells in vivo, since an increase in phosphorylation of HDAC4/5 and HDAC7 was demonstrated in lysates of crypt cells from PKD1 transgenic mice compared with matched nontransgenic littermates. Collectively, our results reveal a PKD1-class IIa HDAC axis in intestinal epithelial cells leading to mitogenic signaling.

Keywords: angiotensin II, vasopressin, IEC-18 cells, PKD family inhibitors, MC1568, TMP269, PKD1 transgenic mice

the mammalian intestine is covered by a single layer of epithelial cells that is renewed every 4–6 days throughout adult life. This high rate of turnover plays an essential role in the organization, maintenance, and restoration of intestinal tissue integrity. The sequential proliferation, differentiation, crypt-villus migration, and death of the epithelial cells of the intestinal mucosa are tightly regulated by a variety of factors (1, 11, 19). The intracellular signal transduction pathways involved, however, are incompletely understood.

Recent studies with crypt-derived intestinal epithelial cells indicate that protein kinase D1 (PKD1), the founding and best-characterized member of the PKD family (40, 41), plays a critical role in mediating migration, proliferation, and signal transduction in these cells (34, 44, 52). Accordingly, multiple growth-promoting stimuli rapidly activate PKD1 catalytic activity in intestinal epithelial cells (2, 5, 39, 44, 52) through activation loop phosphorylation (16, 43, 44, 48). Furthermore, transgenic mice that express elevated PKD1 protein in intestinal epithelial cells display a marked increase in DNA-synthesizing cells in their intestinal crypts and a significant increase in the length and total number of cells per crypt (44). Collectively, these results support the notion that PKD1 signaling is a novel element in the pathway leading to proliferation of intestinal epithelial cells in vitro and in vivo. The mechanism(s) downstream of PKD1, however, remains to be identified.

Class IIa histone deacetylases (HDACs), including HDAC4, HDAC5, HDAC7, and HDAC9, are thought to regulate gene expression by interacting with various transcription factors to repress their transcriptional activity (14, 36), but the precise mechanism of action of these proteins in signal transduction remains incompletely understood (30, 31). Class IIa HDACs are unique among the HDAC family, in that they shuttle between the nucleus and the cytoplasm in response to extracellular signals. Phosphorylation of specific conserved residues in HDAC4, HDAC5, HDAC7, and HDAC9 leads to their nuclear extrusion and sequestration in the cytoplasm, thereby facilitating gene expression (14). PKD1 has been identified as one of the upstream kinases that mediate the phosphorylation and subcellular localization of class IIa HDACs in mesenchymal cells (17, 28, 32, 47), thereby promoting cardiac hypertrophy (10, 47), angiogenesis (13, 49), skeletal muscle gene expression (23), T and B cell receptor function (7, 28, 35), and differentiation into the osteogenic lineage (17). In contrast to mesenchymal cells, little is known about the phosphorylation, dynamic localization, and function of class IIa HDACs in epithelial cells. In fact, phosphorylation-dependent nuclear-cytoplasmic shuttling of endogenous class II HDACs has not been demonstrated in intestinal epithelial cells, nor has any protein kinase that mediates class IIa HDAC phosphorylation been identified in these cells.

In our search for substrates that mediate PKD1 signaling in intestinal cells, we examine the hypothesis that PKD1 participates in a signal transduction pathway triggered by G protein-coupled receptors (GPCRs) through direct phosphorylation of class IIa HDACs, leading to their nuclear export, cytoplasmic localization, and mitogenic signaling. Our results demonstrate that HDAC4, HDAC5, and HDAC7 are strongly phosphorylated in intestinal epithelial cells in response to GPCR agonists that induce PKD1 activation and mitogenic signaling in these cells. Using endogenously and ectopically expressed HDAC5, we show that PKD1-mediated phosphorylation of HDAC5 induces its nuclear extrusion into the cytoplasm. Structurally unrelated pharmacological inhibitors of class IIa HDACs prevented cell cycle progression and entry into DNA synthesis induced in response to GPCR/PKD1 activation. Our results reveal a PKD1-class IIa HDAC axis in intestinal epithelial cells that mediates mitogenic signaling.

MATERIALS AND METHODS

Cell culture.

The nontransformed rat intestinal epithelial cell line IEC-18 (37, 38), originated from intestinal crypt cells, was purchased from American Type Culture Collection. These cells express Gq-coupled receptors for ANG II and vasopressin (2–5, 39, 50, 51) and have been extensively used as a model system to examine signal transduction pathways in response to GPCR activation (2–5, 44, 46, 50, 52). Cultures of IEC-18 cells were maintained as described previously (34, 44). Briefly, cells were cultured in DMEM supplemented with 5% FBS and penicillin-streptomycin and kept at 37°C in a humidified atmosphere containing 10% CO2-90% air. Stock cultures were subcultured every 3–4 days. For experimental purposes, IEC-18 cells were seeded in 35-mm dishes at a density of 2 × 105 cells/dish.

Immunoblotting and detection of HDAC and PKD1 phosphorylation.

Serum-starved, confluent intestinal epithelial IEC-18 cells were lysed in 2× SDS-PAGE sample buffer (20 mM Tris·HCl, pH 6.8, 6% SDS, 2 mM EDTA, 4% 2-mercaptoethanol, and 10% glycerol) and boiled for 10 min. After SDS-PAGE, a Bio-Rad apparatus at 100 V and 0.4 A at 4°C for 4 h was used to transfer proteins to Immobilon-P membranes. The transfer buffer consisted of 200 mM glycine, 25 mM Tris, 0.01% SDS, and 20% CH3OH. For detection of proteins, membranes were blocked using 5% nonfat dry milk in PBS (pH 7.2) and then incubated for ≥2 h with the desired antibodies diluted in PBS containing 3% nonfat dry milk. Primary antibodies bound to immunoreactive bands were visualized by enhanced chemiluminescence detection with horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibody and a FUJI LAS-4000 Mini luminescent image analyzer.

Immunofluorescence.

For immunofluorescence analysis of IEC-18 cells, the cultures were fixed with ice-cold 100% methanol and then permeabilized with 0.2% Triton X-100. Fixed cells were washed extensively in PBS, incubated for 2 h at 25°C in blocking buffer [PBS-5% FBS-2% BSA (BB)], and then stained at 4°C overnight with a rabbit anti-HDAC5 polyclonal antibody (1:200 dilution in BB). Subsequently, the cells were washed with PBS at 25°C, stained at 25°C for 60 min with Alexa Fluor 546-conjugated goat anti-rabbit antibody (1:100 dilution in BB), and washed again with PBS. Nuclei were stained using Hoechst 33342 (1:10,000 dilution). The samples were imaged with an epifluorescence Zeiss Axioskop and a Zeiss water objective (Achroplan 40/0.75 W, Carl Zeiss). Images were captured as uncompressed 24-bit TIFF files with a cooled (−12°C) single charge-coupled device color digital camera (Pursuit, Diagnostic Instruments) driven by SPOT version 4.7 software. Alexa Fluor 546 signals were observed with a high-Q filter set for rhodamine/tetramethylrhodamine isothiocyanate (Chroma Technology). The selected cells are representative of 90% of the population.

Knockdown of PKD1 levels via small interfering RNA transfection.

The pooled small interfering RNA (siRNA) duplexes were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). PKD1 siRNA pools were designed to target the mRNA of mouse PKD1 (GenBank accession no. NM_008858) and consists of three different duplexes: oligo1 (CUCUCUUCGUUCAUUCAUAt), oligo2 (GUGAGCAUUUCCGUUUCAAtt), and oligo3 (GAAGCCAUUGAUCUUAUCAtt). For siRNA transfection, the reverse transfection method was used. The siRNA pool was mixed with Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol and added to 35-mm dishes. IEC-18 cells were then plated on top of the siRNA-Lipofectamine RNAiMAX complex at a density of 1 × 105 cells/35-mm dish. Control transfections were carried out with Stealth siRNA negative control (Invitrogen). At 4 days after transfection, cells were used for experiments and subsequent Western blot analysis.

Cell transfection.

IEC-18 cells were transfected with the plasmid containing a cDNA encoding an epitope (FLAG)-tagged HDAC5 wild-type or an identical construct in which Ser259 and Ser498 were mutated to nonphosphorylatable Ala (catalog nos. 13822 and 32216, respectively, Addgene) with use of Lipofectamine 2000 (Invitrogen), as suggested by the manufacturer. Transiently transfected cells were analyzed 48 h posttransfection.

Immunoprecipitation of HDAC4, HDAC5, and HDAC7.

Confluent IEC-18 cells were lysed in buffer A containing 50 mM Tris·HCl, pH 7.6, 2 mM EGTA, 2 mM EDTA, 1 mM dithiothreitol, 100 μg/ml leupeptin, 10 mM sodium fluoride, 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (Pefabloc), and 1% Triton X-100. HDACs were immunoprecipitated from the cell extracts with antibodies from Cell Signaling Technology. The immune complexes were recovered using protein A coupled to agarose.

Assay of DNA synthesis.

Confluent cultures of IEC-18 cells were washed twice with DMEM and incubated with 1:1 (vol/vol) DMEM-Waymouth's medium containing ANG II and increasing concentrations of the specific class IIa HDAC inhibitors MC1568 and TMP269. After 18 h of incubation at 37°C, [3H]thymidine (0.2 μCi/ml, 1 μM) was added to the cultures for 6 h, and the cultures were washed twice with PBS and incubated in 5% trichloroacetic acid at 4°C for 20 min to remove acid-soluble radioactivity, washed with ethanol, and solubilized in 1 ml of 2% Na2CO3-0.1 M NaOH. The acid-insoluble radioactivity was determined by scintillation counting in 6 ml of Beckman ReadySafe.

Flow cytometric analysis.

The proportion of cells in the G0/G1, S, G2, and M phases of the cell cycle was determined by flow cytometric analysis. Cells were seeded at a density of 1 × 105 cells in 35-mm dishes in DMEM containing 10% FBS for 4 days. The cells were then washed twice with DMEM and incubated with DMEM containing various additions (see Fig. 5 legend) for 6 h before the addition of 1 μM colchicine and incubation for another 24 h. After treatment, the cells were harvested by trypsinization, washed in PBS, and resuspended in a final concentration of 1 × 106 cells/ml in hypotonic propidium iodide (PI) solution containing 0.1% sodium citrate, 0.3% Triton X-100, 0.01% PI, and 0.002% ribonuclease A. Cells were incubated in 4°C for 30 min before acquisition on the flow cytometer (Becton-Dickinson) using CellQuest. One hundred thousand cells were collected for each sample. Excitation occurred at 488 nm, and data were collected in the FL2 channel and analyzed using FCS Express version 3.

Fig. 5.

Fig. 5.

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Mutations of Ser259 and Ser498 to Ala in HDAC5 prevent its nuclear extrusion. IEC-18 cells were transiently transfected with a plasmid encoding FLAG-tagged HDAC5 or FLAG-tagged HDAC5S259A/S498A. Cultures were incubated in the absence (−) or presence of 3.5 μM kb NB 142-70 (kb) for 1 h prior to stimulation with 50 nM ANG II. Cultures were then washed and fixed with 4% paraformaldehyde and stained with an antibody that detects the FLAG tag and Hoechst 33342 stain to visualize the nuclei.

Class IIa HDAC phosphorylation in intestinal epithelial cells in vivo.

To assess the effect of PKD1 on class IIa HDAC phosphorylation in vivo, we used transgenic mice that express elevated PKD1 protein in the ileal epithelium and control nontransgenic littermates. The generation of PKD1 transgenic mice is described elsewhere (44). For anatomic dissection and tissue collection, mice were euthanized in a CO2 chamber. Overexpression of PKD1 in the ileum was verified using epithelial cells isolated sequentially along the crypt-villus axis by timed incubations in EDTA-PBS solutions. For measurement of PKD1 expression and HDAC phosphorylation, lysates of intestinal cells isolated from gender- and age-matched mice were subjected to immunoblotting, as described above. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Animal Research Committee of the University of California, Los Angeles (protocol no. 2001-142-23).

Materials.

DMEM was obtained from Invitrogen (Carlsbad, CA); ANG II, vasopressin, and lysophosphatic acid (LPA) from Sigma Chemical (St. Louis, MO); kb NB 142-70 from R & D Systems (Minneapolis, MN); CRT0066101 from Cancer Research Technology Discovery Laboratories (London, UK); the specific class IIa HDAC inhibitor MC1568 from Selleck Chemicals (Houston, TX); TMP269 from Xcessbio (San Diego, CA); and all antibodies, including the antibody that detects the phosphorylated state of HDAC4 at Ser246, HDAC5 at Ser259, and HDAC7 at Ser155 and the antibody that recognizes the phosphorylated state of HDAC4 at Ser632, HDAC5 at Ser498, and HDAC7 at Ser486, from Cell Signaling Technology (Danvers, MA). All other reagents were of the highest grade available.

RESULTS

Expression and GPCR-induced phosphorylation of class II HDACs in IEC-18 cells.

To determine whether class IIa HDACs are expressed in IEC-18 intestinal epithelial cells, lysates of these cells were analyzed by Western blotting using antibodies that recognize class IIa HDAC4, HDAC5, HDAC7, and HDAC9. As shown in Fig. 1A, robust inmunoreactive signals for HDAC4, HDAC5, and HDAC7 were detected in IEC-18 extracts, whereas only a faint band was seen with the antibody directed against HDAC9 (results not shown). Immunoprecipitation with specific anti-HDAC antibodies followed by Western blotting verified that IEC-18 cells express multiple class IIa HDACs, i.e., HDAC4, HDAC5, and HDAC7 (Fig. 1B).

Fig. 1.

Fig. 1.

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Expression and phosphorylation of class IIa histone deacetylases (HDACs) in IEC-18 intestinal epithelial cells. A: confluent cultures of IEC-18 cells were lysed with 2× SDS-PAGE sample buffer. Lysates were analyzed by SDS-PAGE and by immunoblotting with antibodies that detect HDAC4, HDAC5, and HDAC7. WB, Western blot. B: confluent cultures of IEC-18 cells were lysed with lysis buffer A, and extracts were immunoprecipitated with specific antibodies to HDAC4, HDAC5, and HDAC7. Immunoprecipitates (IP) were analyzed by immunoblotting using antibodies that detect total HDAC4, HDAC5, and HDAC7. C: confluent cultures of IEC-18 cells were incubated without (−) or with 50 nM ANG II for 1 h. Cells were then lysed, and immunoprecipitates were analyzed by immunoblotting with an antibody that detects the phosphorylated state (p) of HDAC4 at Ser246, HDAC5 at Ser259, and HDAC7 at Ser155 or an antibody that recognizes the phosphorylated state of HDAC4 at Ser632 and HDAC5 at Ser498.

Next, we determined whether stimulation of IEC-18 cells with a GPCR agonist enhances the phosphorylation of class II HDACs in these cells. We used an antibody that detects the phosphorylated state of HDAC4 at Ser246, HDAC5 at Ser259, and HDAC7 at Ser155 and a second antibody that recognizes the phosphorylated state of HDAC4 at Ser632, HDAC5 at Ser498, and HDAC7 at Ser486 (Fig. 1C). Stimulation of IEC-18 cells with the Gq-coupled receptor agonist ANG II, a potent mitogen for IEC-18 cells (3, 4, 44), induced striking phosphorylation of class IIa HDACs in these cells, as detected with both antibodies in whole lysates of these cells (Fig. 1C). The first antibody revealed two bands: the upper band corresponded to phosphorylated HDAC4 at Ser246 and/or HDAC5 at Ser259, whereas the lower band corresponded to phosphorylated HDAC7 at Ser155. Western blotting with the second phospho-specific HDAC antibody showed phosphorylation of HDAC4 at Ser632 and/or HDAC5 at Ser498 (Fig. 1C).

To confirm the identity of the phosphorylated bands detected in the cell lysates, endogenous HDAC4, HDAC5, and HDAC7 were recovered from IEC-18 cell extracts by immunoprecipitation, and the resulting immunoprecipitates were analyzed by Western blotting with the phospho-specific HDAC antibodies. As shown in Fig. 1C, cell stimulation with ANG II induced a striking increase in the phosphorylation of HDAC4 at Ser246 and Ser632, HDAC5 at Ser259 and Ser498, and HDAC7 at Ser155. These results demonstrated, for the first time, stimulus-dependent robust phosphorylation of class IIa HDACs in intestinal epithelial cells.

PKD family inhibitors kb NB 142-70 and CRT0066101 or knockdown of PKD1 prevented class IIa HDAC phosphorylation in IEC-18 cells.

To determine the role of the PKD family in mediating ANG II-induced class IIa HDAC phosphorylation in IEC-18 cells, we used the recently identified preferential PKD family inhibitors kb NB 142-70 and CRT0066101 (15, 22), which act as potent PKD1 inhibitors in intact IEC-18 cells (34, 52). Cultures of IEC-18 cells were treated with increasing concentrations of kb NB 142-70 (Fig. 2A) or CRT0066101 (Fig. 2B) for 1 h and then stimulated with ANG II. Prior exposure to kb NB 142-70 or CRT0066101 prevented ANG II-induced phosphorylation of HDAC4 at Ser246 and Ser632, HDAC5 at Ser259 and Ser498, and HDAC7 at Ser155 in a concentration-dependent manner. The concentrations of kb NB 142-70 and CRT0066101 that blunted ANG II-induced phosphorylation of HDAC were virtually identical to those required to suppress PKD1 activity within IEC-18 cells, as shown in our previous studies (34, 52) by monitoring autophosphorylation at Ser916 in the COOH-terminal region (29) and at Ser748 in the activation loop of the kinase catalytic domain (16, 43).

Fig. 2.

Fig. 2.

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PKD family inhibition by kb NB 142-70 or CRT0066101 or small interfering RNA (siRNA) knockdown of PKD1 prevents HDAC4, HDAC5, and HDAC7 phosphorylation in IEC-18 cells. A and B: confluent cultures of IEC-18 cells were incubated in the absence (−) or presence of increasing concentrations of kb NB 142-70 or CRT0066101 for 1 h prior to stimulation without (−) or with 50 nM ANG II for 1 h. All cultures were then lysed with 2× SDS-PAGE sample buffer. C: cultures of IEC-18 cells were transfected with nontargeting siRNA (N Targ) or siRNAs targeting PKD1 (siPKD1). Other cultures were not subjected to transfection (Control). Then the cultures were stimulated with 50 nM ANG II for 1 h and lysed with 2× SDS-PAGE sample buffer. All samples were analyzed by SDS-PAGE and immunoblotting with an antibody that detects the phosphorylated state of HDAC4 at Ser246, HDAC5 at Ser259, and HDAC7 at Ser155 or an antibody that recognizes the phosphorylated state of HDAC4 at Ser632 and HDAC5 at Ser498. Antibodies directed against total HDAC5 and HDAC7 were used to verify equal gel loading. In C, total PKD1 and PKD2 were detected with the PKD C-20 antibody to evaluate siRNA-mediated knockdown of PKD1 expression. Similar results were obtained in ≥3 independent experiments in each case.

To prove that PKD1 mediates phosphorylation of class IIa HDACs in response to GPCR activation in IEC-18 cells, we determined whether knockdown of PKD1 expression prevents ANG II-induced HDAC phosphorylation in these cells. The PKD1 protein level in IEC-18 cells transfected with siRNA targeting PKD1 was dramatically reduced (>90%) compared with cells transfected with nontargeted negative control duplex (Fig. 2C). In contrast, the level of PKD2 protein, the lower band of the doublet detected with the PKD1 C-20 antibody (44), was not affected. PKD1 knockdown strikingly prevented class IIa HDAC phosphorylation in response to ANG II stimulation in IEC-18 cells (Fig. 2C). Collectively, the results with PKD family inhibitors, multiple PKD1 activators, and siRNA-mediated knockdown of PKD1 indicate that this member of the PKD family is the predominant PKD isoform mediating class IIa HDAC phosphorylation in response to GPCR agonists in intestinal epithelial cells.

It was possible that phosphorylation of HDAC4, HDAC5, and HDAC7 could be mediated by different protein kinases at different times after GPCR stimulation. Consequently, we examined the time course of ANG II-induced class IIa HDAC phosphorylation in IEC-18 cells preincubated with or without kb NB 142-70. As shown in Fig. 3A, striking phosphorylation of HDAC4 at Ser246 and Ser632, HDAC5 at Ser259 and Ser498, and HDAC7 at Ser155 was already nearly maximal after 10 min of ANG II stimulation and persisted for ≥4 h. Prior exposure to the PKD family inhibitor kb NB 142-70 drastically reduced class IIa HDAC phosphorylation at all times.

Fig. 3.

Fig. 3.

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PKD1 inhibition by kb NB 142-70 prevents HDAC4, HDAC5, and HDAC7 phosphorylation in IEC-18 cells stimulated with ANG II for 0–240 min or with vasopressin, lysophosphatidic acid (LPA), or phorbol 12,13-dibutyrate (PDBu). A: confluent cultures of IEC-18 cells were incubated in the absence (−) or presence 3.5 μM kb NB 142-70 (kb) for 1 h prior to stimulation with 50 nM ANG II for 0–240 min. B: confluent cultures of IEC-18 cells were incubated in the absence (−) or presence of 3.5 μM kb NB 142-70 (kb) for 1 h prior to stimulation with 50 nM ANG II, 50 nM vasopressin (VP), 10 μM LPA, or 100 ng/ml PDBu. All samples were analyzed for HDAC4, HDAC5, and HDAC7 phosphorylation with an antibody that detects the phosphorylated state of HDAC4 at Ser246, HDAC5 at Ser259, and HDAC7 at Ser155 or a different antibody that recognizes the phosphorylated state of HDAC4 at Ser632 and HDAC5 at Ser498. Antibodies directed against total HDAC5 and HDAC7 were used to verify equal gel loading. Similar results were obtained in ≥3 independent experiments in each case.

We next determined whether stimulation of IEC-18 cells with other GPCR agonists that induce PKD1 activation in these cells also elicits class IIa HDAC phosphorylation through PKD1. As shown in Fig. 3B, stimulation of IEC-18 cells with vasopressin (5, 39, 44), LPA (2), or phorbol 12,13-dibutyrate enhanced phosphorylation of HDAC4 at Ser246 and Ser632, HDAC5 at Ser259 and Ser498, and HDAC7 at Ser155. In each case, prior exposure to kb NB 142-70 markedly attenuated the phosphorylation of these HDACs in IEC-18 cells. These results imply that PKD1 mediates class IIa HDAC phosphorylation in response to multiple GPCR agonists and phorbol esters in IEC-18 intestinal epithelial cells.

PKD1 regulates class IIa HDAC nuclear-cytoplasmic shuttling in response to GPCR activation in IEC-18 cells.

To determine the functional significance of class IIa HDAC phosphorylation in GPCR-mediated signaling in epithelial cells, we examined the effect of ANG II on endogenous HDAC5 nuclear-cytoplasmic shuttling. Immunofluorescence analysis showed that endogenous HDAC5 localizes predominantly to the nucleus in unstimulated IEC-18 cells (Fig. 4). Stimulation with ANG II for 1 h induced a striking intracellular redistribution of HDAC5, from the nucleus to the cytosol (Fig. 4), that persisted for ≥4 h (results not shown). Prior exposure to kb NB 142-70 did not cause a detectable change in the cellular distribution of HDAC5 in unstimulated IEC-18 cells but prevented its nuclear extrusion and cytoplasmic localization induced by ANG II (Fig. 4). These results indicate that HDAC5, a typical class IIa HDAC, shuttles between the nucleus and the cytoplasm in intestinal epithelial cells in response to GPCR activation and that PKD1 plays a critical role in regulating this process.

Fig. 4.

Fig. 4.

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PKD1 regulates endogenous HDAC5 nuclear-cytoplasmic shuttling in response to ANG II stimulation in IEC-18 cells. Confluent cultures of IEC-18 cells were incubated in the absence (−) or presence of 3.5 μM kb NB 142-70 (kb) for 1 h prior to stimulation without (−) or with 50 nM ANG II for 1 h. Cultures were then washed, fixed in ice-cold methanol, and stained with an antibody that detects HDAC5 and with Hoechst 33342 stain to visualize nuclei.

We next determined whether the nuclear extrusion of HDAC5 is mediated by phosphorylation of the residues strongly phosphorylated in response to GPCR/PKD1 activation. Cultures of IEC-18 cells were transfected with epitope (FLAG)-tagged HDAC5 or an identical construct in which Ser259 and Ser498 were mutated to nonphosphorylatable Ala. After 48 h, the cells were treated with or without kb NB 142-70 and subsequently challenged with ANG II. As shown in Fig. 5, FLAG-tagged HDAC5 is present in the nucleus of unstimulated cells and strikingly translocates to the cytosol in response to ANG II stimulation. Treatment with kb NB 142-70 prevented the nuclear extrusion of FLAG-tagged HDAC5, consistent with the results shown for endogenous HDAC5 (Fig. 4). In contrast, FLAG-tagged HDAC5 with Ser259 and Ser498 mutated to Ala were localized to the nucleus in unstimulated and GPCR-stimulated cells (Fig. 5), indicating that the phosphorylation of these residues plays a critical role in mediating nuclear-cytoplasmic HDAC5 shuttling in intestinal epithelial cells.

Role of class IIa HDAC activity in GPCR-induced mitogenesis in IEC-18 cells.

Previous results demonstrated that PKD1 activation plays a key role in mediating GPCR-induced cell proliferation in intestinal epithelial cells (44), but the mechanism(s) downstream of PKD1 remains to be identified. Having established that phosphorylation, nuclear export, and cytoplasmic localization of class IIa HDACs through PKD1 are early events in GPCR-induced signaling in intestinal epithelial cells, we next determined whether class IIa HDAC activity plays a role in promoting the mitogenic response induced through the GPCR/PKD1 pathway. Cultures of IEC-18 cells in serum-free medium were stimulated with ANG II in the absence or presence of increasing concentrations of the specific class IIa HDAC inhibitor MC1568 (8, 25, 33), and DNA synthesis was assessed by measurement of [3H]thymidine incorporation into acid-precipitable material. As shown in Fig. 6A, addition of MC1568 prevented ANG II-induced DNA synthesis in IEC-18 cells in a dose-dependent manner. Half-maximal inhibitory effect was elicited at ∼3 μM. To substantiate the results obtained with MC1568, we also tested TMP269, a recently developed specific inhibitor of class IIa HDAC that contains a trifluoromethyloxadiazolyl moiety and is therefore structurally unrelated to MC1568 (24). Exposure of IEC-18 cells to increasing concentrations of TMP269 potently inhibited [3H]thymidine incorporation induced by ANG II in these cells. Half-maximal inhibitory effect was elicited at ∼1.5 μM (Fig. 6B). These results indicate that class IIa HDAC catalytic domain activity is necessary for mitogenic signaling induced via the GPCR/PKD1 pathway in intestinal epithelial cells.

Fig. 6.

Fig. 6.

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Class IIa HDAC catalytic domain activity is necessary for mitogenic signaling induced by ANG II in intestinal epithelial cells. Confluent cultures of IEC-18 cells were incubated with increasing concentrations of MC1568 (A) or TMP269 (B) for 1 h prior to stimulation with 50 nM ANG II. After 16 h of incubation at 37°C, [3H]thymidine (0.2 μCi/ml, 1 μM) was added, and cultures were incubated for 6 h at 37°C. DNA synthesis was assessed by measurement of [3H]thymidine incorporated into acid-precipitable material. Values (means ± SE) are expressed as percentage of maximum cpm/culture × 10−3 (n = 6). C: confluent IEC-18 cells were incubated in the absence (−) or presence of MC1568 (5 μM) or TMP269 (3 μM) and 50 nM ANG II for 6 h before addition of 1 μM colchicine and incubation for another 24 h. Colchicine was added to arrest cells that progressed through the cell cycle at the G2/M phase. Proportions of cells in the G0/G1 and G2/M phases, determined by flow cytometric analysis, were 71 ± 0.7 and 16 ± 0.4 in control cells, 38 ± 0.8 and 54 ± 0.6 in cells stimulated with ANG II, 92 ± 0.1 and 5 ± 0.3 in cells stimulated with ANG II and treated with MC1568, and 83 ± 0.8 and 11 ± 0.2 in cells stimulated with ANG II and treated with TMP269. Proportions of cells in the G0/G1 and G2/M phases of the cell cycle that were treated with MC1568 or TMP269 but without ANG II were virtually identical to those treated with ANG II. Shift from the G0/G1 to G2/M phase induced by 50 nM ANG II was equivalent to that produced by addition of 10% FBS to parallel cultures, used as a positive control (result not shown). D: suspended IEC-18 cells (5 × 104) were plated onto 35-mm Nunc petri dishes with 2 ml of DMEM containing 1% FBS. At day 0 (24 h after plating), cultures were washed twice with DMEM to remove residual serum and transferred to DMEM in the absence (open bar) or presence of increasing concentrations of MC1568 or TMP269. Cultures represented by the solid bars also received 50 nM ANG II. Cell number was determined by counting trypsinized cells with a Coulter counter. Cell counts were obtained 48 h after addition of agonists. *P < 0.05.

To substantiate that the inhibitory effects elicited by exposure to MC1568 or TMP269 on [3H]thymidine incorporation reflect blockade of cell cycle progression, rather than alteration(s) of [3H]thymidine uptake (e.g., transport and/or phosphorylation), we used flow cytometric analysis to determine the proportion of cells in the various phases (G0/G1, S, and G2/M) of the cell cycle. Confluent and serum-starved cultures of IEC-18 cells were stimulated with ANG II in the absence or presence of 5 μM MC1568 or 4 μM TMP269. Cells that traversed the cell cycle were accumulated in the G2/M phase by addition of colchicine. As shown in Fig. 6C, stimulation of IEC-18 cells with ANG II induced a striking shift from the G0/G1 to the G2/M phase, an effect completely prevented by exposure to MC1568 or TMP269. Furthermore, the inhibitors of class IIa HDACs also abolished the increase in cell number induced by ANG II in IEC-18 cells (Fig. 6D). Collectively, the results demonstrate, for the first time, that pharmacological inhibition of class IIa HDAC activity completely prevented GPCR/PKD1-induced progression of the cell cycle, DNA synthesis, and proliferation in IEC-18 cells.

As controls, we verified that exposure to MC1568 (1–5 μM) or TMP269 (1–5 μM) prevented neither PKD1 activation in response to ANG II in IEC-18 cells, scored by autophosphorylation at Ser916, nor PKD1-mediated phosphorylation of HDAC4 at Ser246 and HDAC5 at Ser259 in these cells (Fig. 7A). Furthermore, treatment with MC1568 or TMP269 did not interfere with GPCR/PKD1-induced nuclear extrusion and cytoplasmic localization of endogenous HDAC5 in IEC-18 cells (Fig. 7B). These results imply that class IIa HDAC's catalytic pocket plays a role in mitogenic signaling when these proteins are localized in the cytoplasm of intestinal epithelial cells.

Fig. 7.

Fig. 7.

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Class IIa HDAC inhibition by MC1568 or TMP269 does not prevent PKD1 activation, HDAC4 and HDAC5 phosphorylation, and HDAC5 nuclear extrusion in IEC-18 cells stimulated with ANG II. A: confluent cultures of IEC-18 cells were incubated in the absence (−) or presence MC1568 (5 and 2.5 μM) or TMP269 (5 and 2.5 μM) for 1 h prior to stimulation without (0) or with 50 nM ANG II for 10 or 60 min. Samples were analyzed for HDAC4/5 phosphorylation with an antibody that detects the phosphorylated state of HDAC4 at Ser246 and HDAC5 at Ser259. Similar results were obtained in ≥3 independent experiments in each case. B: confluent cultures of IEC-18 cells were incubated in the absence (−) or presence of 5 μM MC1568 or 3 μM TMP269 for 1 h prior to stimulation of the cells without (0) or with 50 nM ANG II for 1 h. Cultures were then washed, fixed in ice-cold methanol, and stained with an antibody that detects HDAC5 and with Hoechst stain to visualize nuclei.

Overexpression of PKD1 enhances class II HDAC phosphorylation in intestinal epithelial cells “in vivo.”

Our preceding results indicating that PKD1 is required for class II HDAC phosphorylation and redistribution in IEC-18 cells prompted us to determine whether PKD1 promotes HDAC phosphorylation of intestinal epithelial cells in vivo. To examine this possibility, we used transgenic mice that express elevated PKD1 protein in the small intestine epithelium and display a marked increase in DNA-synthesizing cells in their intestinal crypts and a significant increase in the length and total number of cells per crypt (44). As shown in Fig. 8A, overexpression of PKD1 protein in the ileum was verified by Western blot analysis of total PKD1 in lysates of epithelial cells isolated sequentially along the crypt-villus axis by timed incubations in EDTA-PBS solutions (9, 27). Using these lysates, we also demonstrated that HDAC5 is prominently expressed in fractions 6 and 8 of epithelial cells, corresponding to the proliferative compartment of the crypt, as shown by the detection of proliferating cell nuclear antigen in these fractions. Furthermore, the results in Fig. 8B demonstrate that class IIa HDAC phosphorylation is markedly increased in epithelial cells isolated from PKD1 transgenic mice compared with epithelial cells isolated from their nontransgenic littermates. The results support the notion that PKD1 promotes the phosphorylation of class IIa HDACs in intestinal epithelial cells in vivo.

Fig. 8.

Fig. 8.

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Overexpression of PKD1 enhances class II HDAC phosphorylation in intestinal epithelial cells “in vivo.” A: PKD1 is overexpressed along the crypt-villus axis in PKD1 transgenic mice (Tg) compared with nontransgenic (NTg) littermates. Epithelial cells from the ileum of Tg and NTg mice were isolated sequentially and collected as fractions 2, 4, 6, and 8 along the crypt-villus axis by timed incubations in EDTA-PBS solution. Western blot analysis of lysates of these cells was used to determine total PKD1, HDAC5, and the proliferation marker proliferating cell nuclear antigen (PCNA). Sequential elution along the crypt-villus axis was verified by the gradient of PCNA expression. B: eluted cells from 2 separate fraction 6 preparations (I and II) were lysed, and the extracts were used for analysis by SDS-PAGE and immunoblotting with antibodies that detect total PKD and PKD1 autophosphorylated on Ser916. Lysates were also analyzed with the antibody that recognizes the phosphorylated state of HDAC4 at Ser632, HDAC5 at Ser498, and HDAC7 at Ser486 or an antibody that recognizes the phosphorylated state of HDAC4 at Ser246, HDAC5 at Ser259, and HDAC7 at Ser155 and with antibodies directed against total HDAC4, HDAC5, and HDAC 7. Results are shown for 2 PKD1 transgenic mice and 2 nontransgenic littermates. Values (means ± SE) represent the level of phosphorylation [expressed in optical density (OD) units] of HDAC4 at Ser632 and HDAC5 at Ser498 (n = 5) and HDAC4 at Ser246, HDAC5 at Ser259, and HDAC7 at Ser155 (n = 6). *P < 0.05.

DISCUSSION

PKD1 has been identified as one of the upstream kinases that mediate the phosphorylation and subcellular localization of class IIa HDACs in mesenchymal cells (17, 28, 32, 47), thereby promoting cardiac hypertrophy (10, 47), angiogenesis (13, 49), skeletal muscle gene expression (23), T and B cell receptor function (7, 28, 35), and differentiation into the osteogenic lineage (17). In contrast to mesenchymal cells, little is known about the phosphorylation, dynamic localization, and function of class IIa HDACs in epithelial cells. Consequently, the experiments presented here were designed to determine whether PKD1 regulates the phosphorylation and redistribution of class IIa HDACs in intestinal epithelial cells and explore the role of class IIa HDACs in mitogenic signaling induced through the GPCR/PKD1 pathway in these cells.

Using IEC-18 cells as a model system of intestinal epithelial crypt cells, we produced several lines of evidence indicating that PKD1 is a major upstream kinase that mediates class IIa HDAC phosphorylation and nuclear extrusion. 1) Stimulation of IEC-18 cells with ANG II, a potent mitogen for these cells, induced a striking increase in the phosphorylation of HDAC4 at Ser246 and Ser632, HDAC5 at Ser259 and Ser498, and HDAC7 at Ser155. These results demonstrated, for the first time, robust class IIa HDAC phosphorylation in response to GPCR activation in intestinal epithelial cells. 2) Treatment of IEC-18 cells with the structurally unrelated PKD family inhibitors kb NB 142-70 and CRT0066101 markedly reduced ANG II-induced phosphorylation of HDAC4, HDAC5, and HDAC7 in a dose-dependent manner. 3) A variety of PKD1 activators in IEC-18 cells, including vasopressin (5, 39), LPA (50), and phorbol esters (2), also induced HDAC4, HDAC5, and HDAC7 phosphorylation. 4) siRNA-mediated selective knockdown of PKD1 protein expression prevented ANG II-induced class IIa HDAC phosphorylation. 5) Immunofluorescence analysis showed that stimulation with ANG II induced a striking intracellular redistribution of endogenous HDAC5, from the nucleus to the cytosol. 6) Using HDAC5 as a model of class IIa HDAC, we demonstrated that residues targeted by PKD1 play a critical role in mediating nuclear extrusion and cytoplasmic localization in response to GPCR activation. 7) The PKD1-class IIa HDAC axis also occurs in intestinal epithelial cells in vivo, since an increase in the phosphorylation of HDAC4/5 and HDAC7 was demonstrated in lysates of crypt cells from mice that express elevated PKD1 protein in intestinal epithelial cells. Collectively, these results demonstrate, for the first time, that PKD1 mediates endogenous class IIa phosphorylation, nuclear extrusion, and cytoplasmic localization in response to GPCR activation in intestinal epithelial cells.

ANG II induces DNA synthesis and proliferation in IEC-18 cells (3, 4, 44) via its cognate Gq-coupled receptor endogenously expressed by these intestinal epithelial cells (3, 51). Recent studies revealed that PKD1 activation plays a critical role in mediating ANG II-induced proliferative responses in these cells (44). PKD1 is also rapidly activated by mitogenic stimuli and mediates growth-promoting signaling in Swiss 3T3 fibroblasts (43, 45, 53, 54) and human pancreatic cancer cells (12, 15, 20), but the mechanism(s) downstream of PKD1 remains incompletely understood in all model systems. In our search for substrates that mediate PKD1 signaling in intestinal cells, we tested the hypothesis that PKD1-mediated class IIa HDAC phosphorylation and intracellular relocalization participate in a signal transduction pathway triggered by GPCRs leading to mitogenesis. To explore this hypothesis, we examined whether pharmacological inhibition of class IIa HDAC activity, using MC1568 (8, 25, 26, 33) and TMP269 (24), interferes with ANG II mitogenic signaling. We show here that exposure of IEC-18 cells to MC1568 or the more recently identified class IIa HDAC inhibitor TMP269 prevented the stimulation of DNA synthesis induced by the GPCR agonist ANG II. More evidence for a functional role of class IIa HDAC activity in the regulation of the proliferative response was derived from cell cycle analysis by flow cytometry, demonstrating that MC1568 or TMP269 blocks cell cycle progression in the G0/G1 phase. The results imply that class IIa HDACs are critical players in GPCR/PKD1 mitogenic signaling in intestinal epithelial cells.

In theory, class IIa HDACs could contribute to mitogenic signaling in IEC-18 cells through at least two mechanisms. Since GPCR/PKD1 signaling induces potent nuclear extrusion of class IIa HDACs in these cells, a first mechanism could be the removal of their transcriptional repressive effects in the nucleus mediated by the scaffolding function of the NH2-terminal region of these proteins. If this was the only mechanism or the predominant mechanism, pharmacological inhibition of class IIa HDAC catalytic activity should not interfere with mitogenic signaling. On the other hand, the results presented here with recently developed specific inhibitors support an unexpected second mechanism in which class IIa HDACs function in mitogenic signaling via their HDAC catalytic domain in the cytoplasm (21, 42). In this context, characterization of lysine acetylation sites revealed that many proteins involved in cytoskeleton organization, cell cycle, and signal transduction are acetylated and reside in the cytoplasm (6). Because the deacetylase activity of class IIa HDACs is ∼1,000-fold lower than that of class I HDACs for acetylated histones (21, 42), it is conceivable that class IIa HDACs act on cytoplasmic substrates that remain to be identified. Alternatively, the catalytic domain may mediate binding to acetyl lysine residues of proteins to promote protein-protein interactions (24), thereby regulating the assembly of multiprotein signaling complexes in the cytoplasm (6). Our results imply that the occupation of the catalytic domain of class IIa HDACs in the cytoplasm by MC1568 or TMP269 blocks the stimulation of cell cycle progression in intestinal epithelial cell proliferation. Our findings identify, for the first time, a novel function in mitogenic signaling for cytoplasmic class IIa HDACs in intestinal epithelial cells.

GRANTS

This work was supported by National Institutes of Health Grants R01 DK-55003, P30 DK-41301, and P01 CA-163200 and in part by Department of Veterans Affairs Grant 1I01BX001473 (to E. Rozengurt).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

J.S.-S. and E.R. are responsible for conception and design of the research; J.S.-S., Y.N., J.W., M.M., and S.H.Y. performed the experiments; J.S.-S., Y.N., J.W., M.M., S.H.Y., and E.R. analyzed the data; J.S.-S. and E.R. interpreted the results of the experiments; J.S.-S. prepared the figures; J.S.-S., S.H.Y., and E.R. edited and revised the manuscript; E.R. drafted the manuscript; E.R. approved the final version of the manuscript.

ACKNOWLEDGMENTS

We are grateful to the Morphology and Cell Imaging Core of the CURE: Digestive Diseases Research Center (supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant P30 DK-41301) for their help.

E. Rozengurt is the Ronald S. Hirshberg Professor of Pancreatic Cancer Research.

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