Biochimica et Biophysica Acta 1803 (2010) 343–351

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Biochimica et Biophysica Acta

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PTH inactivates the AKT survival pathway in the colonic cell line Caco-2

Natalia Calvo, Ana Russo de Boland, Claudia Gentili ⁎Dept. Biología, Bioquímica y Farmacia, Universidad Nacional del Sur, San Juan 670, (8000) Bahía Blanca, Argentina

⁎ Corresponding author. Tel.: +54 291 4595100x243E-mail address: cgentili@criba.edu.ar (C. Gentili).

0167-4889/$ – see front matter © 2009 Elsevier B.V. Adoi:10.1016/j.bbamcr.2009.11.011

a b s t r a c t

a r t i c l e i n f o

Article history:Received 7 August 2009Received in revised form 25 November 2009Accepted 30 November 2009Available online 18 December 2009

Keywords:PTHCaco-2 cellApoptosisPP2AAKT

In previous works, we found that PTH promotes the apoptosis of human Caco-2 intestinal cells, through themitochondrial pathway. This study was conducted to investigate the modulation of different playersimplicated in the AKT survival pathway in PTH-induced intestinal cell apoptosis. We demonstrate, for thefirst time, that PTH modulates AKT phosphorylation in response to apoptosis via the serine/threoninephosphatase PP2A. PTH treatment induces an association of AKT with the catalytic subunit of PP2A andincreases its phosphatase activity. PTH also promotes the translocation of PP2Ac from the cytosol to themitochondria. Furthermore, our results suggest that PP2A plays a role in hormone-dependent Caco-2 cellsviability and in the cleavage of caspase-3 and its substrate PARP. The cAMP pathway also contributes to PTH-mediated AKT dephosphorylation while PKC and p38 MAPK do not participate in this event. Finally, we showthat PTH induces the dissociation between 14-3-3 and AKT, but the significance of this response remainsunknown. In correlation with PTH-induced Bad dephosphorylation, the hormone also decreases the basalassociation of 14-3-3 and Bad. Overall, our data suggest that in Caco-2 cells, PP2A and the cAMP pathway actin concert to inactivate the AKT survival pathway in PTH-induced intestinal cell apoptosis.

© 2009 Elsevier B.V. All rights reserved.

1. Introduction

Apoptosis is a form of programmed cell death that is character-ized by specific morphological and biochemical features, and istightly regulated by extracellular stimuli and intracellular signalingpathways [1]. There are two distinct signaling pathways of apoptosisin mammals. One is initiated by death receptors (death receptorpathways) [2], and the other is regulated by anti- and pro-apoptoticBcl-2 family members and involves release of cytochrome c frommitochondria into the cytoplasm (mitochondrial pathways) [3,4]. Ithas been suggested that the balance of pro- and anti-apoptoticmembers of the Bcl-2 family determines life or death in a cell [5].Indeed, the regulation of mitochondrial cytochrome c release andmitochondrial function by several Bcl-2 family members plays alarge part in controlling cell death [3]. Bad, one of the pro-apoptoticfamily members, is the first target of AKT, a major inhibitor ofapoptosis [6]. By phosphorylating Bad, AKT precludes its binding tothe Bcl-xL and Bcl-2 anti-apoptotic family members leading toincreased cell survival. AKT, a serine/threonine protein kinase, is akey component of cell survival pathways and is a major downstreamtarget of phosphoinositide 3-kinase (PI3K) [7]. Activation of PI3Kleads to AKT activation by both phosphoinositide-dependent proteinkinase-1 (PDK1), which phosphorylates AKT at Thr-308, and PDK1-independent mechanisms, which result in AKT phosphorylation atSer-473 [7].

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AKT phosphorylation is tightly regulated, representing a balancebetween kinase-activating and phosphatase-inactivating events.Several protein phosphatases, including the dual-action phosphatasePTEN, which transforms PtdIns(3,4,5)P3 into PtdIns(4,5)P2 [8],canonical PP1 [9,10] and PP2A [11–13] as well as newly identifiedAKT phosphatases [14,15] were recently reported to bind anddephosphorylate AKT in an agonist-dependent manner.

Mammalian PTH is an 84-amino acid single-chain polypeptide,although only the first 34 amino acids are required for most biologicaleffects [16,17]. The PTH receptor (PTH1R) is found in a variety oftissues not regarded as classical PTH target tissues, includingintestinal cells [18,19]. In intestinal cells, PTH initiates its effects byinteracting with its PTHR1 that, like other members of the class IIfamily of GPCRs, is capable of coupling to several different G proteins,thereby activating multiple signaling pathways, including adenylylcyclase/cAMP [20], PLC [21], cytoplasmic Ca2+ [22] and the MAPkinases ERK1/2 [23] and p38 MAPK [24].

Apoptosis is especially relevant in the gastrointestinal tract, as themammalian intestinal mucosa undergoes a process of continual cellturnover that is essential for maintenance of normal function [25].Defective apoptosis may allow the progression of disease andmaintain the resistance of colon cancer cells to cytotoxic therapy[26], again illustrating the importance of apoptosis in the gastroin-testinal tract. Thus, it is of interest to know the mechanism as well asthe various physiological inducers of apoptosis in the intestinalepithelium. Since PTH, depending on the cell type involved, inhibits orpromotes the apoptosis [27–29], we therefore used Caco-2 cells, a cellline derived from human colorectal adenocarcinoma, as an in vitromodel for studying the effects of PTH on gut cell apoptosis. In a

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previous work we found that PTH promotes the apoptosis of theseintestinal cells [30], which is mediated by translocation of mitochon-dria to the perinuclear region, dephosphorylation of AKT, dephos-phorylation of Bad and its movement to the mitochondria andsubsequent release of cytochrome c and Smac/Diablo which result inactivation of downstream caspase-3 and degradation of its substratePARP [30,31].

However, relatively little is known about how specific proteintyrosine and serine/threonine phosphatases function to regulate PTH-mediated apoptosis.

This study was conducted to investigate the modulation ofdifferent players implicated in the AKT survival pathway in PTH-induced intestinal cell apoptosis.

2. Materials and methods

2.1. Materials

Human PTH (1–34) was obtained from Calbiochem (San Diego, CA,USA). High glucose Dubelcco's modified Eagle's medium (DMEM) wasfrom Sigma Chemical Co. (St. Louis, MO, USA). Fetal bovine serum(FBS) was from Natocord (Córdoba, Argentina).

Antibodies were from the following sources: anti-Bad, anti-phospho-AKT (S473) anti-PP2Ac and anti-PARP were from CellSignaling Technology (Beverly, MA, USA). Anti-AKT1/2/3, anti-σ14-3-3, anti-caspase-3, anti-cytochrome c, anti-lamin B and goat anti-rabbit peroxidase-conjugated secondary antibodies were from SantaCruz Biotechnology (Santa Cruz, CA, USA). Alexa Fluor 488 conjugat-ed-anti-rabbit antibody was from Molecular Probes. Anti-actinantibody, okadaic acid sodium and p-nitrophenyl phosphate (p-NPP) were from Sigma (Sigma Chemical Co. St. Louis, MO, USA). Ro-31-8220, Rp-cAMP (cyclic adenosine 3′,5′-mono-phosphorothioate,Rp diastereomer) and SB 203580 were from Calbiochem (San Diego,CA, USA).

Protein size markers were from Amersham Biosciences (Piscat-away, NJ, USA), and PVDF (Immobilon polyvinylidene difluoride)membranes and ECL chemiluminescence detection kit were fromAmersham (Little Chalfont, Buckinghamshire, England). All otherreagents used were of analytical grade.

2.2. Cell culture and treatment

The human colon cell line Caco-2 (from the American TissueCulture Bank (Bethesda, USA)) was cultured at 37 °C in DMEMcontaining 10% FBS, 1% non-essential acids, 100 UI/ml penicillin,100 mg/ml streptomycin and 50 mg/ml gentamycin in a humidatmosphere of 5% CO2 in air. Cultures were passaged every 2 days withfresh medium. The treatments were performed with 70% confluentcultures in serum free medium by adding PTH (10−8 M) for 48 h.Where indicated, cells were pretreated for 30 min with one of thefollowing inhibitors: okadaic acid sodium, LY 294002, Ro-31-8220,Rp-cAMP or SB 203580. The inhibitors were also present duringsubsequent exposure to the hormone.

2.3. Western blot analysis

Caco-2 cells were washed with PBS buffer plus 25 mM NaF and1 mM Na3VO4, and lysed in buffer containing 50 mM Tris–HCl (pH7.4), 150 mM NaCl, 3 mM KCl, 1 mM EDTA, 1% Tween-20, 1% NonidetP-40, 20 μg/ml aprotinin, 20 μg/ml leupeptin, 1 mM phenylmethyl-sulfonyl fluoride (PMSF), 25 mM NaF and 1 mM Na3VO4. The lysateswere incubated on ice for 10min, vortexed for 45 s andmaintained onice for another 10 min. After centrifugation at 14,000×g and 4 °Cduring 15 min the supernatant was collected and proteins werequantified by the Bradford method [32]. Lysate proteins dissolved in6× Laemmli sample buffer were separated (25 μg/lane) using SDS–

polyacrylamide gels (10% or 15% acrylamide) and electrotransferredto PVDF membranes. After blocking with 5% non-fat milk in TBSTbuffer (50 mM Tris pH 7.2–7.4, 200 mM NaCl, 0.1% Tween-20), themembranes were incubated overnight with the appropriate dilutionof primary antibody in TBST with 1% non-fat milk. After washing,membranes were incubated with the appropriate dilution of horse-radish peroxidase-conjugated secondary antibody in TBST with 1%non-fat milk. Finally, the blots were developed by ECL with the use ofKodak BioMax Light film and digitalized with a GS-700 ImagingDensitomer (Bio-Rad, Hercules, CA, USA).

2.4. Stripping and reprobing membranes

The complete removal of primary and secondary antibodies fromthe membranes was achieved by incubating the membranes instripping buffer (62.5 mM Tris–HCl pH 6.8, 2% SDS and 50 mM β-mercaptoethanol) at 55 °C for 30 min with agitation. Then,membranes were washed for 10 min in TBST (1% Tween-20) andblocked, as indicated above, for 1 h at room temperature. After that,membranes were ready to reprobewith the corresponding antibodies.

2.5. Co-immunoprecipitation

Co-immunoprecipitation assays were performed under nativeconditions in order to preserve protein–protein associations. To thatend, after hormone treatment, the cells were lysed in buffer A (50mMTris–HCl, pH 7.4, 150 mM NaCl, 3 mM KCl, 0.5 mM EDTA, 0.2 mMNa3VO4, 1 mMNaF, 1 mM PMSF, 6 μg/ml leupeptin, 8 μg/ml aprotinin,and 1% Tween-20). After centrifugation at 14,000×g (4 °C, 15 min)the supernatant was collected and proteins were quantified by theBradford method [32]. Lysate aliquots (200–500 μg protein) wereincubated overnight at 4 °C with anti-Bad, anti-14-3-3, anti-AKT oranti-PP2Ac antibodies, followed by precipitation of the complexeswith protein A conjugated with Sepharose. The immune complexeswere washed three times with buffer A (14,000×g, 4 °C, 5 min) andthen subjected to Western blot analysis. To confirm the association ofboth proteins, immunoprecipitation and immunoblotting wereperformed with the same antibodies used in reverse order.

2.6. Protein phosphatase 2A (PP2A) activity assay

Cell lysates (200 μg protein) were incubated overnight at 4 °C withanti-PP2Ac antibody, followed by precipitation of the complexes withprotein A conjugated with Sepharose. After three washes with lysisbuffer and two washes with a serine/threonine assay buffer (50 mMTris–HCl, pH 7.0, 100 μM CaCl2), the immune complexes wereincubated at 37 °C for 10 min in Ser/Thr assay buffer (45 μl/sample)containing p-nitrophenyl phosphate as an exogenous substrate forPP2A (5 μl/sample). The reaction was terminated by the addition ofNaOH 1N and the color developed was quantified by measuring theabsorbance with a spectrophotometer at 405 nm against a reagentblank. The enzyme activity was expressed as percentage of the activityrespect of control.

2.7. Cell proliferation assay

The MTS assay (Promega) is based on the ability of viable cells tobioreduce the tetrazolium compound (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, innersalt; MTS) into a colored formazan product that is soluble in tissue-culture medium. The intensity of the product color is directlyproportional to the number of living cells in the culture. Cells wereseeded in 96-well plates at a density of 2×103 cells per well. After eachexperiment, 20 μlMTSwas added toeachwell, followedby incubation for45min at 37 °C. Absorbance was then measured with a spectrophotom-eter at 490 nm. The results were expressed as a percentage of the mean

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respect to control cultures. To determine background absorbance, wellswithout cells were used as negative controls.

2.8. Immunocytochemistry

Caco-2 cells grown onto glass cover slips were fixed inmethanol at−20 °C for 15 min. After washing with PBS, non-specific sites wereblocked with 5% BSA in PBS. Samples were then incubated overnightwith anti-PP2Ac antibody prepared in 2% BSA in PBS (1:50). Afterwashing with PBS, the samples were incubated for 1 h at roomtemperature with secondary Alexa Fluor 488 conjugated antibody(1:200). Cells were washed with PBS andmounted. Cells were stainedwith MitoTracker Red CMXRos, (Molecular Probes) before fixation tovisualize mitochondria [33]. It can be used to stain active mitochon-dria and is retained after fixation. The samples were examined using aLeica TCS SP2 AOBS confocal laser microscope.

2.9. Subcellular fractionation

Cells were washed with PBS, resuspended in ice-cold TES buffer(50 mM Tris–HCl (pH 7.4), 1 mM EDTA, 250 mM sucrose containingprotease inhibitors) homogenized in a Teflon-glass hand homogeniz-er (30 stokes), and then centrifuged at 1200×g for 20 min at 4 °C topellet the nuclei. The supernatant was centrifuged at 12,000×g for20 min at 4 °C to pellet mitochondria. The resulting supernatant is thecytosolic fraction. Nuclear and mitochondrial fractions were washedtwice in TES buffer. The purity of each isolated fraction was assessedby assaying for proteins known to be associated with cellularcomponents. Routinely we included anti-lamin B and anti-cyto-chrome c antibodies to check the purity of nuclear, mitochondrial andcytosolic fractions. Proteins from each fraction were quantified by theBradford method [32]. Equal proteins from each fraction weresubjected to SDS–PAGE.

2.10. Statistical analysis

The statistical significance of the data was evaluated usingStudent's t test, and probability values below 0.050 (pb0.050) were

Fig. 1. (A) Okadaic acid revert PTH-mediated AKT dephosphorylation. Caco-2 cells were pretreafor 48 h. Proteins from cell lysates were immunoblottedwith anti-AKT phospho-Ser 473 antibA representative immunoblot and bar graph from three independent experiments are shInteraction of PP2A with AKT in Caco-2 cells stimulated with PTH. Caco-2 cells were eximmunoprecipitated (IP) with anti-AKT antibody under native conditions, resolved onto 10%Materials and methods. Right side: The antibodies were used in reverse order. The mRepresentative immunoblots from three independent experiments are given. (C) PTH increasand assay of PP2A activity using p-nitrophenyl phosphate as substrate is detailed in Materialsrespect to the control.

considered significant. Quantitative data are expressed as means±SDfrom the indicated set of experiments.

3. Results

3.1. PTH increases PP2A activity, induces the association between PP2Aand AKT with subsequent dephosphorylation of AKT

Wehave recentlydemonstrated that the treatmentwithPTHcausedadecrease in AKT basal phosphorylation at Ser-473 [31]. The contributionof activation of protein phosphatases to PTH-induced dephosphorylationof AKT was examined by pre-treating cells with the phosphataseinhibitor okadaic acid (OA). OA inhibits the serine/threonine phospha-tases to differing extents: has a 100-fold higher specificity for PP2A(IC50_0.1 nM) than for PP1 (IC50_10nM) [34], PP2B is even less sensitive(Ki 10 μM) and PP2C is not inhibited at all [35]. Initially, Caco-2 cells werepre-incubated with different doses of OA (0.5–10 nM) followed by PTHtreatment (10−8 M, 48 h) and AKT phosphorylation was determined byWestern blot using a phospho-specific antibody against the Ser-473residue. Maximal inhibition was observed at 1 nM okadaic acid, whichinhibited PP2A activity (not shown). OA at concentrations that inhibitPP2A activity (1 nM) reverted PTH-mediated reduction of AKTphosphorylation at Ser-473 (Fig. 1A). These results provided evidencesupporting a role for PP2A, rather than the related PP1, in regulatingcellular levels of phospho-AKT in intestinal cells exposed to PTH.

The previous observations demonstrated that PP2A activityreduced phospho-AKT levels in cells exposed to PTH, but did notdemonstrate a direct interaction between the proteins. In order toaddress whether PP2A exerted direct effects on AKT, we performedco-immunoprecipitation assays under native conditions to preserveprotein–protein interactions. We found that PP2Ac could be detectedin AKT immunoprecipitates (Fig. 1B, left side), and in addition, AKTwas detected in equivalent PP2Ac immunoprecipitates (Fig. 1B, rightside). These results indicated that the catalytic subunit of PP2Aassociates with AKT when Caco-2 cells are stimulated with PTH. Tostudy the effects of PTH on PP2A activity, the enzyme from lysates ofCaco-2 cells exposed to PTH for 48 h was immunoprecipitated with ananti-PP2A catalytic subunit antibody and PP2A activity was then

ted for 30 min with okadaic acid (OA) (1 nM) followed by exposition to PTH (10−8 M)ody and the same immunoblot was reprobedwith anti-AKT antibody as loading control.own; means±SD are given. ⁎pb0.05 with respect to the corresponding control. (B)posed to PTH (10−8 M) for 48 h. Left side: Cell lysates were obtained, AKT wasSDS–PAGE gels and then immunoblotted (IB) with anti-PP2Ac antibody as described inembranes were dyed using Coomassie brilliant blue to demonstrate equal loading.es PP2A activity in Caco-2 cells. Cells were incubated in presence of PTH (10–8M) for 48 hand methods. Results are the average of three independent experiments. ⁎pb0.01 with

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measured using p-nitrophenyl phosphate as an exogenous substrate.As shown in Fig. 1C, PTH increased by 182% the activity of PP2Acompared with untreated cells.

3.2. PTH induces PP2A translocation to the mitochondria

Since the regulation of subcellular localization is important for theability of PP2A and other Ser/Thr phosphatases to dephosphorylateparticular substrates, we investigated the subcellular distribution ofthe PP2A catalytic subunit in cells exposed to PTH (10−8 M, 48 h). Asshown in Fig. 2A, under basal conditions, PP2Ac localized to both

Fig. 2. PTH promotes the translocation of PP2Ac from the cytosol to the mitochondria. (A) Cacthe mitochondrial and cytosolic fractions were isolated and the proteins were analyzedCoomassie brilliant blue to demonstrate equal loading. A representative immunoblot and themeans±SD are given. ⁎pb0.05 with respect to the corresponding control. (B) Caco-2 cells wmethanol and incubated with a rabbit polyclonal antibody against PP2Ac. Fluorescence-conj(green) and mitochondria (red) localization patterns under a confocal microscope. Red- an

cytosolic and mitochondrial compartments. However, after PTHtreatment, the amount of the enzyme increased in the mitochondrialfraction and decreased in cytosol, indicating that the hormone inducesthe translocation of PP2Ac from the cytosol to themitochondria. Theseresults were confirmed by confocal microscope (Fig. 2B).

3.3. PP2A mediates PTH decrease of live cells and the cleavage ofcaspase-3 and its substrate PARP in Caco-2 cells

AKT is a key component of cell survival pathways and our resultsshow a role of PP2A in the dephosphorylation and inactivation of AKT

o-2 cells were treated with or without PTH (10−8 M) during 48 h. Upon PTH treatment,by Western blotting with an anti-PP2Ac antibody. The membranes were dyed usingquantification by scanning densitometry of three independent experiments are shown;ere treated with or without PTH (10−8 M) during 48 h. The cells were then fixed withugated secondary antibody and MitoTracker Red CMX Ros were used to visualize PP2Acd green-stained images were merged. Areas of co-localization appear yellow.

Fig. 4. PP2A is involved in PTH-dependent caspase-3 activation and PARP degradation.(A) Western blot analysis with an anti-caspase-3 antibody, specific for active caspase-3(that recognizes p11 subunit), was performed to evaluate caspase-3 activation. (B)Cleavage of poly (ADP-ribose) polymerase was analyzed by Western blotting with ananti-PARP antibody that recognizes cleaved-PARP (89 kDa). In order to evaluate theequivalence of protein content among the different experimental conditions, blottedmembranes were reprobed with anti-β-actin antibody. A representative immunoblotand the quantification by scanning densitometry of three independent experiments areshown; means±SD are given. ⁎Pb0.05 with respect to the corresponding control.

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in Caco-2 cells exposed to PTH. Therefore, to explore the possibilitythat PP2A affects cell viability, PTH treated cells (5 days) wereincubated with 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) and,upon treatment, the metabolic capacity of these intestinal cells wasmeasured as an indicator of cell viability. We found that PTHdiminished the bioreduction of the reagent, being the productionof formazan 56% lower respect to control (Fig. 3). Moreover, okadaicacid (1 nM) reversed hormone-mediated decrease of live cells,suggesting that PP2A activation mediates PTH-dependent apoptosisin Caco-2 cells.

Our previous study showed that PTH activation of caspase-3 inCaco-2 cells was followed by cleavage of its substrate poly (ADP-ribose) polymerase PARP [31]. Therefore, we analyzed the cleavagestatus of caspase-3 and PARP in cells exposed to PTH, and in thepresence or absence of OA (1 nM). Western blot analysis with ananti-caspase-3 antibody specific for active caspase-3 (p11 subunit)and with an anti-PARP antibody specific for PARP inactive fragmentof 89 kDa reveal the caspase-3 activation and the PARPdegradation in Caco-2 cells stimulated with PTH (48 h), effectsthat were almost completely suppressed by OA, suggesting theinvolvement of PP2A in the regulation of caspase-3 activity (Fig. 4Aand B). Actin immunoblotting was performed as an internal controlof equal protein loading.

3.4. Role of PKC, cAMP and p38 MAPK on PTH-dependentAKT dephosphorylation

Since increased PP2A activity is an important component of PKC/PKCα induced cell cycle arrest in intestinal epithelial cells [36], wealso investigated if PKC plays a role in PTH-mediated AKT dephos-phorylation. To that end, the cells were treated with PTH (10−8 M,48 h) in the presence of the PKC inhibitor Ro-31-8220 (200 nM)followed by immunoblot analysis using a phospho-specific antibodyagainst the Ser-473 residue. We found that inhibition of PKC activitydid not reverse hormone-mediated reduction of AKT phosphorylation,suggesting that PP2A activity on AKT is independent of PKC signaling(not shown).

AKT is also phosphorylated at Ser-473 by the p38 MAPK-activatedprotein kinase-2 (MAPKAPK-2) pathway [37,38]. Therefore, we alsohave investigated the possible regulation of the PI3K/AKT survivalpathway by p38 MAPK in Caco-2 cells. To that end, cells were treatedwith PTH (10−8 M, 48 h) in the presence of p38 MAPK specificinhibitor, SB203580 (20 μM). Inhibition of the p38 MAPK did notreverse hormone-mediated reduction of AKT phosphorylation, sug-gesting that PP2A activity on AKT is independent of the p38 MAPKpathway (not shown).

Fig. 3. PP2A is involved in PTH-mediated decrease of live cells. Caco-2 cells werepretreated for 30 min with okadaic acid (OA) (1 nM) followed by treatment with PTH(10−8M) for 5 days. The first control is in the absence of the agonist PTH and the secondcontrol is in the presence of OA, without PTH. The number of viable cells wasdetermined by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) assay as described in Materials andmethods. Results are expressed as percentage relative to control of three independentmeasurements performed in quadruplicate. ⁎pb0.05 with respect to the correspondingcontrol.

cAMP can either stimulate [39–41] or inhibit [42–44] AKT activity.Furthermore, there are reports describing cAMP-dependent PP2Aactivation [45,46]. In an attempt to investigate whether the cAMPpathway is involved in PTH-mediated AKT dephosphorylation, Caco-2cells were treated with PTH (10−8 M, 48 h) in the presence of Rp-cAMP (50 μM), a highly specific competitive antagonist for allactivators of the cAMP signal pathway, followed by immunoblotanalysis using a phospho-Ser-473-AKT antibody. As shown in Fig. 5A,Rp-cAMP upregulated the levels of AKT phosphorylated on Ser473.Moreover, PP2A activity significantly decreased when Caco-2 cellswere incubated with Rp-cAMP followed by PTH treatment (Fig. 5B).Taken together, these results indicate that the cAMP pathwaycontributes to PP2A activation leading to the dephosphorylation andinactivation of AKT.

3.5. PTH induces the dissociation of 14-3-3 with Bad and AKT inCaco-2 cells

The 14-3-3 proteins are a family of adaptor and scaffoldingproteins that interact with more than 60 proteins and are involvedin cell cycle regulation, transcriptional control of gene expression, orin apoptosis [47]. These proteins form homo- and heterodimers thatinteract with phosphoserine binding domains [48]. There isevidence that AKT interacts with and phosphorylates 14-3-3 Zeta,leading to modulation of its dimerization [49]. To evaluate thepotential interaction between AKT and 14-3-3 in Caco-2 cells, we

Fig. 5. (A) Effect of Rp-cAMP on PTH-induced AKT dephosphorylation. Caco-2 cells were pretreated for 30 min with Rp-cAMP (50 μM) and were exposed to PTH (10−8 M) for 48 hfollowed byWestern blot analysis of cell lysates with anti-AKT phospho-Ser 473 antibody. Blotted membranes were reprobed with anti-AKT antibody. A representative immunoblotand the quantification by scanning densitometry of three independent experiments are shown; means±SD are given. ⁎Pb0.05 with respect to the control. (B) cAMP is involved inPTH-dependent PP2A activity. Caco-2 cells were pre-incubated for 30 min with Rp-cAMP (50 μM) and then exposed to PTH (10−8 M) for 48 h. PP2A activity assay using p-nitrophenylphosphate as substrate was performed as detailed in Materials and methods.

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performed co-immunoprecipitation assays under native conditionsto preserve protein–protein interactions. As shown in the Fig. 6A(left side), the association of both proteins was detectable underbasal conditions and the treatment with PTH (10−8M, 48 h)diminished the formation of the complex. This result was confirmedwhen the antibodies were used in reverse order (Fig. 6A, right side).These data indicate that, in Caco-2 cells, the dephosphorylation andinactivation of AKT by PTH lead to its dissociation from 14-3-3protein, suggesting that the phosphorylation status of 14-3-3 andperhaps its function might be affected. We have recently shownthat stimulation of Caco-2 cells with PTH results in the dephos-phorylation of Bad, the first target of AKT, and its movement fromthe cytosol to mitochondria [31]. The phosphorylation of Bad resultsin its binding to 14-3-3 protein in the cytosol and precludes itsbinding to membrane-based Bcl-Xl, an event that enhances survival[50]. However, with an apoptotic stimulus, Bad is dephosphorylated,dissociates from 14-3-3 proteins, and translocates to the outermembrane of mitochondria, where it subsequently dimerizes withBcl-Xl to neutralize their anti-apoptotic function [50]. To determinewhether Bad interacted with 14-3-3 in Caco-2 cells, we performedco-immunoprecipitation experiments under native conditions fol-lowed by Western blot analysis on immunoprecipitated 14-3-3 withanti-Bad antibody. As shown in Fig. 6B (left side), PTH (10−8 M,48 h) decreased the basal association of 14-3-3 and Bad, in

Fig. 6. (A) PTH induces the dissociation of 14-3-3 with AKT in Caco-2 cells. Caco-2 ceimmunoprecipitated (IP) with anti-14-3-3 antibody under native conditions, resolved onto 1Materials and methods. Right side: The antibodies were used in reverse order. The mRepresentative immunoblots from three independent experiments are given. (B) PTH induces thfor 48 h. Left side: In cell lysates 14-3-3was immunoprecipitated (IP)with anti-14-3-3 antibodwith anti-Bad antibody as described in Materials and methods. Right side: The antibodies wdemonstrate equal loading. Representative immunoblots from three independent experiment

correlation with PTH-induced Bad dephosphorylation. Similarresults were obtained when the antibodies were used in reverseorder (Fig. 6B, right side). These results suggest that Baddephosphorylation [31] followed by its dissociation from 14-3-3proteins is part of PTH-induced mitochondrial dysfunction leadingto Caco-2 cell apoptosis.

4. Discussion

PTH binding to its receptor is known to trigger initial tyrosinephosphorylation events in intestinal cells, such as the activation ofMAP kinases and Src family kinases, that result in the activation ofdownstream signaling pathways, a number of which involve theactivity of various serine/threonine kinases [21,23,51,52]. However,relatively little is known about how specific protein tyrosine andserine/threonine phosphatases function to regulate PTH-mediatedapoptosis. Like many physiological processes, commitment to apo-ptosis is posttranslationally regulated by reversible phosphorylationof apoptotic signaling proteins and therefore controlled by a balancein protein kinase and protein phosphatase activity. We have recentlyreported that PTH inhibits AKT phosphorylation in the humanintestinal cell line Caco-2 [31]. We now demonstrate that PTHmodulates AKT phosphorylation in response to apoptosis by activat-ing the serine/threonine phosphatase PP2A.

lls were exposed to PTH (10−8 M) for 48 h. Left side: In cell lysates 14-3-3 was0% SDS–PAGE gels and then immunoblotted (IB) with anti-AKT antibody as described inembranes were dyed using Coomassie brilliant blue to demonstrate equal loading.e dissociation of 14-3-3with Bad in Caco-2 cells.Caco-2 cellswere exposed to PTH (10−8M)y under native conditions, resolved onto 15% SDS–PAGE gels and then immunoblotted (IB)ere used in reverse order. The membranes were dyed using Coomassie brilliant blue tos are given.

Fig. 7. Pro-apoptotic events from PTH to PARP cleavage in Caco-2 cells. PTH induces theactivation of cAMP-PP2A pathway and the subsequent dephosphorylation andinactivation of AKT leading to the activation of caspase-3 and degradation of itssubstrate PARP.

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Currently, the PP1 and PP2/4/6 subclasses of human phosphataseshave been reported to contribute most of the Ser/Thr phosphataseactivity in cells [53]. PP2A is by far the best-characterized memberwithin the PP2/4/6 subclass, given that its catalytic, structural andregulatory subunits have been sequenced and identified, and itssusceptibility to inhibition by compounds such as okadaic acid (OA)and fostriecin is well known [54]. PP2A is an abundant and ubiquitousenzyme with pleiotropic functions [55]. The predominant form ofPP2A in cells contains a heterotrimeric subunit structure, consisting ofa 36 kDa catalytic C subunit (PP2Ac), a 65 kDa structural A subunit(PP2AA) and one of a variety of structurally distinct regulatory Bsubunit (PP2AB). In addition to the heterotrimeric form of PP2A, coredimeric form of PP2A consisting of A and C subunit also exists in cells.The different B subunits are believed to change substrate specificityand subcellular localization of PP2A [56]. This family of protein serine/threonine phosphatases can interact with a substantial number ofproteins and contribute to the regulation of numerous signalingpathways [54]. Active PP2A can inhibit the cell cycle, predominantly atthe G2/M checkpoint [57], induce apoptosis [58,59] and act as a tumorsuppressor [60]. The assumption that PP2A plays a key role in theregulation of AKT, was supported by the finding that PTH increasedPP2A activity and induced its physical association with AKT.

Previous results in PC12 neuronal cells [61] hypothesize thatPP2A/Bβ2 holoenzyme exists in equilibrium between cytosol andmitochondrial surface to promote dephosphorylation of unidentifiedproteins associated with outer mitochondrial membrane, thusinhibiting fusion and/or stimulating fission activities which in turnsensitize neurons to pro-apoptotic insults [62]. Other pro-apoptoticPP2A regulatory subunits appear to substitute for Bβ2 in non-neuronal cells. Ruvolo et al. [63,64] reported that ceramide-induceddephosphorylation of Bcl-2 in a lymphocyte cell line involvesmitochondrial redistribution of PP2A containing the B′ α regulatorysubunit. In line with these observations, our results show that, inCaco-2 cells, PTH induced the translocation of PP2Ac from the cytosolto the mitochondria.

Previous findings show that PP2A is a downstream target of p38MAPK in TNF-induced endothelial apoptosis [65]. Furthermore, p38MAPK has been shown to regulate PP2A in neutrophils and cardiacmyocytes [66,67]. However, PP2A has also been reported todephosphorylate the threonine residue of p38 MAPK and therebyimpair the activity of this protein [68]. In addition, PP2A activity is animportant component of PKC/PKCα induced cell cycle arrest inintestinal epithelial cells [36]. Therefore, we further characterize themechanisms involved in PTH-mediated AKT dephosphorylation inCaco-2 cells and our results rule out the participation of PKC and p38MAPK in the hormone regulation of AKT but provide, for the first time,evidence that AKT is dephosphorylated by the phosphatase PP2A,which in turn is activated by cAMP. In agreement with our findings,the activation of cAMP-PP2A pathway and the subsequent inactiva-tion of AKT also plays a key role in the induction of apoptosis inChinese hamster ovarian (CHO) cells [69]. The mechanistic details forcAMP stimulation of PP2A are still unknown. In this regard, Hong et al.[70] showed that cAMP-dependent inhibition of AKT in PCCL3 thyroidcells is mediated by PP2A. They identified a novel and stable Epac–PP2A signaling complex, and observed the modulation of itsphosphatase activity by Rap-GTP and PKA. Also, it has been recentlyshown that cAMP could activate PP2A via PKA-dependent phosphor-ylation of a B regulatory subunit [71,72].

There are studies showing that cAMP-inducing agents could beused in the treatment of human colon cancer. The cAMP activatorforskolin with the cAMP phosphodiesterase-4 (PDE4) inhibitorrolipram cause growth arrest and apoptosis of KM12C colon cancercells [73]. In DLD-1 cells, another colon cancer cell line, PDE4 plays acritical role in regulating intracellular cAMP levels and is involved incancer invasion. Thus, PDE4 can be a novel target of anti-invasion drug[74]. Furthermore, the human colon cancer cells LS174T are unable to

proliferate upon sustained accumulation of intracellular cyclic AMPfollowing forskolin or dibutyryl cyclic AMP treatment [75].

Downstream target of AKT include caspase-9 [76] and phosphor-ylation of this protein by AKT inhibits its pro-apoptotic function,thereby favoring the survival-promoting effects of AKT [77]. In Caco-2cells we hypothesize that after PTH treatment, dephosphorylation andinactivation of AKT by PP2A revert caspase-9 inhibition, therebypromoting its pro-apoptotic functions and thus activating caspase-3.

The AGC family of serine/threonine kinases includes AKT and PKC.Interaction with various 14-3-3 isoforms has been reported tostimulate [78] or to inhibit [79,80] PKC activity. Three phosphoryla-tion sites have been identified on 14-3-3 proteins, Ser-58, Ser-184,and Thr-232 [47] and some kinases have been found to phosphorylatethese residues, although the role of these phosphorylations in theregulation of 14-3-3 function is unknown [81,82]. Ser-58 wasrecognized as a low stringency AKT phosphorylation (2.703%) site[83], suggesting that AKT might phosphorylate 14-3-3 proteins andregulate a function common to both proteins. In Caco-2 cells, wehypothesize that the dissociation between AKT and 14-3-3 induced byPTH could affect the phosphorylation status of 14-3-3 and perhaps itsfunction, but additional studies are required to confirm this idea.

Finally, we observed that, in correlation with PTH-induced Baddephosphorylation [31], PTH decreased the basal association of 14-3-3and Bad. The defined role for Bad is pro-apoptotic, in which it isthe nonphosphorylated species that binds Bcl-Xl to reverse deathprotection [50]. The phosphorylation status of Bad appears to dictateits subcellular localization as well as its capacity to associate with14-3-3.

As summarized in Fig. 7, our results demonstrate that, in thehuman intestinal cell line Caco-2, PTH increases the serine/threoninephosphatase PP2A activity and that PP2A and the cAMP pathway actin concert to inactivate the AKT survival pathway in hormone-inducedintestinal cell apoptosis.

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Acknowledgements

This work was supported by grants from the Agencia Nacional dePromoción Científica y Tecnológica (ANPCYT), Consejo Nacional deInvestigaciones Científicas y Técnicas (CONICET) and UniversidadNacional del Sur, Argentina.

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