The cAMP Effectors Epac and PKA are Involved in the Hepatic Cystogenesis of an Animal Model of ARPKD

doi:10.1002/hep.22636

Hepatology. Author manuscript; available in PMC 2011 March 25.

Published in final edited form as:

Hepatology. 2009 January; 49(1): 160–174.

doi: 10.1002/hep.22636

PMCID: PMC3063896

NIHMSID: NIHMS76793

The cAMP Effectors Epac and PKA are Involved in the Hepatic Cystogenesis of an Animal Model of ARPKD

Jesús M. Banales,^1,² Tatyana V. Masyuk,¹ Sergio A. Gradilone,¹ Anatoliy I. Masyuk,¹ Juan F. Medina,² and Nicholas F. LaRusso¹

¹Miles and Shirley Fiterman Center for Digestive Diseases, Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, Rochester, MN, USA

²Laboratory of Molecular Genetics, Division of Gene Therapy and Hepatology, School of Medicine and CIMA of the University of Navarra, and Ciberehd, Pamplona, Spain

Corresponding author: Nicholas F. LaRusso, MD, Miles and Shirley Fiterman Center for Basic Research in Digestive Diseases, Mayo Clinic College of Medicine, 200 First Street, SW, Rochester, Minnesota 55905, USA, Phone: (507) 284-1006, Fax: (507) 284-0762, Email: larusso.nicholas/at/mayo.edu

The publisher's final edited version of this article is available at Hepatology

See other articles in PMC that cite the published article.

Abstract

PCK rats, an animal model of autosomal recessive polycystic kidney disease (ARPKD), develop cholangiocyte-derived liver cysts associated with increased intracellular adenosine 3′,5′-cyclic monophosphate (cAMP), the inhibition of which suppresses cyst growth. We hypothesized that elevated cAMP stimulates cholangiocyte proliferation via two downstream effectors, exchange proteins activated by cAMP (Epac1 and Epac2 isoforms) and protein kinase A (PKA), and that intracellular calcium is also involved in this process. Assessment of Epac isoforms and PKA regulatory subunits (PKA-Rs) at the mRNA and protein level showed that cultured normal rat cholangiocytes express Epac1, Epac2 and all regulatory PKA subunits. Epac isoforms and the PKA RIβ subunit were over-expressed in cultured PCK cholangiocytes. Proliferation analysis in response to Epac and PKA activation indicated that both normal and PCK cholangiocytes increase their growth upon Epac-specific stimulation, while PKA-specific stimulation results in differential effects, suppressing proliferation in normal cholangiocytes but accelerating this process in PCK cholangiocytes. On the other hand, both PKA and Epac activation of cystic structures generated by normal and PCK cholangiocytes when cultured under 3-D conditions resulted in increased cyst growth, particularly in PCK-cholangiocyte derived cysts. Pharmacological inhibitors and siRNA-mediated gene silencing demonstrated the specificity of each effector activation, as well as the involvement of MEK-ERK1/2 signaling in all the observed effector-associated proliferation changes. Hyperproliferation of PCK cholangiocytes in response to PKA stimulation, but not to Epac stimulation, was found to be associated with decreased intracellular calcium, and restoration of calcium levels blocked the PKA-dependent proliferation via PI3K/AKT pathway.

Conclusion

our data provide strong evidence that both cAMP effectors, Epac and PKA, and the levels of intracellular calcium are involved in the hepatic cystogenesis of ARPKD.

Keywords: cAMP, calcium, cholangiocyte, proliferation, ARPKD

Autosomal recessive polycystic kidney disease (ARPKD) is a genetic disorder in which affected infants often die at birth or shortly thereafter, primarily as the result of markedly enlarged kidney cysts and impaired lung function. In surviving patients, hepatic fibrosis, bile duct dilatation (Caroli’s disease) and/or cyst development becomes progressively more severe and may be the major cause of morbidity and mortality.¹ ARPKD is linked to mutations in the PKHD1 gene which encodes fibrocystin, a large trans-membrane protein with unknown function,²^,³ located on cholangiocyte primary cilia;⁴ these non-motile long tubular organelles extend from the cholangiocyte apical membrane and function as mechano-,⁵ chemo-⁶ and osmo-sensors,⁷ detecting and transmitting luminal stimuli into intracellular signals.

Although the pathophysiology of hepatic cystogenesis in ARPKD is unclear, abnormalities in cholangiocyte fluid secretion and proliferation likely contribute.¹^,⁸ Indeed, in the PCK rat, a well-characterized animal model of ARPKD,⁹ liver cysts originate from intrahepatic bile ducts and their growth is associated with increased proliferative activity of cystic cholangiocytes.¹⁰ However, the mechanism underlying this benign hyperproliferative process is not fully understood.

Accumulated evidence suggests that two intracellular signaling mediators, cAMP and Ca²⁺, regulate proliferation in different cell types,¹¹^,¹² including cholangiocytes.¹³ We recently demonstrated that cAMP levels are increased in cholangiocytes of the PCK rat and that octreotide, a somatostatin analog known to inhibit cAMP, decreases hepatic cyst volume, hepatic fibrotic scores and mitotic indices.¹⁰

cAMP has two downstream targets, cAMP-GEF/Epac (Epac) and protein kinase A (PKA).¹⁴ Epac proteins, a family of Rap guanine nucleotide exchange factors, regulate many cellular processes via PKA-independent mechanisms.¹⁵ There are two different Epac isoforms (Epac1 and Epac2, also known as RapGEF3 and RapGEF4, respectively) which are expressed from different genes in a variety of tissues.¹⁵^–¹⁷

PKA is a heterotetrameric holoenzyme with two regulatory and two catalytic subunits, that responds to intracellular changes of cAMP regulating a wide range of intracellular processes.¹⁴ There exist several members of PKA regulatory (RIα, RIβ, RIIα and RIIβ) and catalytic (Cα, Cβ, Cγ and PrKX) subunits. The biochemical and functional features of PKA are largely determined by the structure and properties of the regulatory subunits, which are differentially expressed depending on the tissue and the cellular state.¹⁴ cAMP binds to the PKA regulatory subunits, leading to dissociation and activation of the catalytic subunits that may regulate the phosphorylation of a number of proteins and the expression of different genes.¹⁴

Both cAMP downstream effectors, Epac and PKA, may be involved in regulation of proliferation in different cell types. In polycystic diseases, however, only PKA has been shown to be responsible for hyperproliferation of epithelial cells lining renal cysts from patients with autosomal dominant polycystic kidney disease (ADPKD).¹⁸ But no data exist regarding the expression, intracellular localization and function of PKA regulatory subunits and Epac isoforms in cholangiocytes, and the involvement of these effectors in cystic cholangiocyte proliferation remains unknown.

Intracellular Ca²⁺ [Ca²⁺]_i may also participate in the control of cell growth.¹¹^,¹² In fact, renal cystic cells of patients with ARPKD have low calcium levels,¹¹ and silencing of Pkhd1 in cultured normal kidney cells results in decreased intracellular calcium and hyperproliferation.¹⁹

The interconnection between cAMP and [Ca²⁺]_i signaling pathways under normal and pathological conditions is currently attracting considerable attention. In particular, it has been shown that in renal cells from ADPKD patients, intracellular calcium modulates cAMP-dependent proliferation. Moreover, these cells were characterized by decreased [Ca²⁺]_i and exhibited higher rates of cell proliferation in response to cAMP, while experimental restoration of [Ca²⁺]_i inhibited their cAMP-stimulated growth.¹¹

We recently demonstrated in normal rat cholangiocytes that elevated [Ca²⁺]_i is able to terminate cAMP signaling (i.e., turn off regulation) initially activated by choleretic stimuli.⁵ However, it is unclear whether intracellular Ca²⁺ levels are altered in cystic cholangiocytes and what role, if any, intracellular Ca²⁺ plays in hepatic cystogenesis. Thus, we examined the: i) expression and intracellular localization of two cAMP downstream effectors, PKA and Epac, in cultured cholangiocytes from normal and PCK rats; ii) role of PKA and Epac activation in proliferation of normal and PCK cholangiocytes; iii) intracellular calcium levels in PCK cholangiocytes; and iv) relationships between cAMP-stimulated proliferation and calcium levels in PCK cholangiocytes.

Material and Methods

Animals and models

Wild-type Sprague–Dawley and PCK rats were maintained on a standard laboratory diet. Animals were anesthetized with pentobarbital prior to in vivo procedures. Livers were harvested, fixed in 10% formaldehyde, and embedded in paraffin for immunohistochemisty. For all experiments with cells we used cholangiocytes that have been cultured for several passages after their isolation from normal and PCK rats.²⁰^,²¹ Studies were approved by the Mayo Institutional Animal Care and Use Committee.

Determination of mRNA expression

Total RNA was obtained from cultured normal and PCK rat cholangiocytes with TRIZOL reagent (Invitrogen, Carlsbad, CA). Detection and quantification of mRNAs for Epac isoforms and PKA regulatory subunits were carried out by RT-PCR and real-time qPCR (see Table 1 for specific primers) using 18S rRNA as normalizing control.

Table 1

Primers employed for RT-PCR analyses.

Proliferation assay

Cultured normal and PCK cholangiocytes were maintained overnight in quiescent media (DMEM-Ham’s-F12 with 2% fetal bovine serum and 1% penicillin/streptomycin) prior to the addition of any of the following activators/inhibitors: 100 µM 8-pCPT-2’-O-Me-cAMP (Epac-specific activator; Biolog, Bremen, Germany), 100 µM 6-Phe-cAMP (PKA-specific activator; Biolog), 100 µM Rp-cAMP (PKA-specific inhibitor; Biolog), 10 µM U0126 (MEK-specific inhibitor; Cell Signaling Technology, Danvers, MA); 50 µM LY294002 (PI3K inhibitor; Cell Signaling Technology), and 40 µM 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (AKT inhibitor; Calbiochem, San Diego, CA). Cholangiocytes were pre-incubated in the absence or presence of an inhibitor, and/or the Ca²⁺ ionophore A23187 (0.1 µM, Sigma-Aldrich, St. Louis, MO) for 1 hour and then with either an Epac or PKA activator during 6 hours. Proliferation rates were assessed by flow cytometry using Click-iT™ EdU Alexa Fluor 488 Cell Proliferation Assay Kit (Invitrogen), data being given as percentage of proliferation rates of non-treated cholangiocytes (100%). However, when showing the combined effects of PKA activation and A23187 in the presence of inhibitors, data are given as a percentage of PKA-associated proliferation rate in the presence of corresponding inhibitors, relative to PKA-associated proliferation without inhibitors. The effect of each inhibitor over the basal state was subtracted from the effect of the inhibitor together with PKA stimulation to eliminate any effect of inhibitors on the basal state.

3-D culture and cyst formation

Cultured normal and PCK cholangiocytes were included in 3-D as described.⁸ Briefly, clusters of normal and PCK cholangiocytes were grown between two layers of collagen (1.5 mg/mL) plus 10% Matrigel (both BD Biosciences, San Jose, CA).²¹ Under these conditions, both normal and PCK cholangiocytes form cystic structures that expand over time.¹⁰ Cyst expansion in quiescent media was then monitored following 24- or 48-hours treatment with PKA and Epac activators, with and without inhibitors. For gene-silencing experiments, Epac1 siRNA: s133230 and Epac2 siRNA: s141111 (Applied Biosystems, Austin, TX) were added to the medium (50 nM each) one day before (day -1) the cyst monitoring (day 0) and maintained until day 2 (changed every day).

Measurement of intracellular Ca²⁺

Cultured normal and PCK cholangiocytes were loaded for 30 minutes with 10 µM fura-2/AM (Invitrogen) in HCO₃⁻-Ringer’s solution (with 2 mM CaCl₂) at 37°C, washed twice and measured for fluorescence intensities (340/380 nm excitation ratios). Alternatively, intracellular Ca²⁺ was measured with fluo-4-AM (Invitrogen) fluorescent dye.⁷ Cultured cells were incubated for 7 hours with 0.1 µM of A23187 or vehicle, washed twice and incubated for 30 minutes with 3 µM fluo-4-AM in HCO₃⁻-Ringer’s solution. Fluorescence was read at 480 and 540 nm wavelengths for excitation and emission, respectively.

Western blot

Cultured normal and PCK cholangiocytes were resuspended in RIPA Buffer (Santa Cruz, CA), 40 µg of proteins being run in 7.5% (for Epac) or 12% (for PKA and ERK1/2) SDS-PAGE, and electrotransferred to a nitrocellulose membrane (BioRad, Hercules, CA). Once blocked, membranes were incubated overnight at 4°C with antibodies against Epac1 and Epac2 (Santa Cruz; 1:200), PKA regulatory subunits (PKA RIβ – Santa Cruz; 1:200; PKA RIα, PKA RIIα and PKA RIIβ - BD Biosciences; 1:200), pERK1/2 (Abcam, Cambridge, MA; 1:1000) and ERK1/2 (BD Bioscience; 1:2000). Respective secondary antibodies (Invitrogen; 1:5000) were applied for 45 minutes at room temperature. Bands were visualized with ECL Plus Western Blotting Detection Kit (BD Biosciences). β-actin staining was employed to normalize for protein loading (Abcam: 1:1000). Protein extracts from either normal rat hepatocytes or rat brain were used as positive controls (cf. Fig. 1). ERK1/2 phosphorylation was analyzed using protein extracts of Epac- or PKA-stimulated cholangiocytes (for 30 minutes).

Figure 1

Expression of Epac isoforms and PKA regulatory subunits in normal rat cholangiocytes. (A) RT-PCR shows that the mRNAs for Epac1, Epac2 and all four PKA regulatory subunits are expressed in cultured normal rat cholangiocytes (NRC). Negative controls with (more ...)

Confocal microscopy

Immunofluorescence microscopy was performed as described.⁵ Briefly, liver samples were incubated overnight at 4°C with antibodies against Epac1 and Epac2 (Santa Cruz; 1:50) and PKA regulatory subunits (PKA RIα, RIIα and RIIβ - Santa Cruz; PKA RIβ - Calbiochem; 1:50) followed by secondary antibodies (Molecular Probes, Eugene, OR; 1:100). Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich).

Statistics

Data are expressed as mean ± SE. Statistical analyses were performed by One-way ANOVA with Bonferroni posthoc test to compare more than two groups and by the Student t test to compare two groups. Results were considered statistically different at p<0.05.

Results

Epac isoforms and PKA regulatory subunits are expressed in normal rat cholangiocytes

First we examined the presence of the cAMP downstream effectors – Epac1 and Epac2 and PKA regulatory subunits (RIα, RIβ, RIIα and RIIβ) – in cultured cholangiocytes from normal rat. RT-PCR revealed that both Epac isoforms and all four PKA regulatory subunits are expressed at mRNA level in these cells (Fig. 1A). The expression of Epac isoforms and PKA regulatory subunits was also confirmed at protein level by western blot (Fig. 1B). By immunofluorescent confocal microscopy, all these proteins were localized to the cytoplasm of cholangiocytes in normal rat liver tissue (Fig. 2C).

Figure 2

Over-expression of Epac1, Epac2 and PKA RIβ in PCK cholangiocytes. (A) Real-time qPCR indicates that the mRNAs for Epac1, Epac2 and PKA RIβ are over-expressed (approx. 2.6-, 1.6-, 4.2- fold, respectively) in PCK cholangiocytes compared (more ...)

Epac1, Epac2 and PKA RIβ are over-expressed in PCK cholangiocytes

Real-time qPCR revealed that mRNAs for Epac isoforms and PKA RIβ were over-expressed in PCK cholangiocytes compared to normal (Fig. 2A), while no differences in the remaining PKA regulatory subunits were observed between normal and PCK cells (data not shown). Over-expression of Epac1, Epac2 and PKA RIβ proteins in PCK cholangiocytes was further confirmed by western blot (Fig. 2B). Moreover, by immunofluorescent confocal microscopy these over-expressed proteins were localized to the cytoplasm of PCK cholangiocytes (Fig. 2C).

Epac activation increases proliferation in both normal and PCK cholangiocytes

The application of 8-pCPT-2’-O-Me-cAMP, a cAMP analogue that specifically activates Epac proteins¹⁵^,²² for 6 hours increased proliferation rates in normal (by 6.7%) and PCK (by 15.7%) cholangiocytes as compared to non-treated cells (Fig. 3A, p<0.05 and p<0.001, respectively). Increased proliferation was significantly higher in PCK cholangiocytes compared to normal cholangiocytes (p<0.05), which is consistent with the aforementioned over-expression of Epac proteins in PCK cholangiocytes. Epac-activated proliferation of both normal and PCK cholangiocytes was specifically mediated by Epac isoforms, as it was not reversed by pre-incubation with the PKA inhibitor Rp-cAMP (Fig. 3B).

Figure 3

Effects of Epac and PKA activation on cell proliferation of normal and PCK rat cholangiocytes. (A) Specific activation of Epac with 8-pCPT-2’-O-Me-cAMP increases cell proliferation in normal (p<0.05) and PCK cholangiocytes (p<0.001). (more ...)

PKA activation decreases proliferation in normal cholangiocytes but accelerates proliferation in PCK cholangiocytes

We also analyzed the effect of PKA stimulation on the proliferation rate of cultured normal and PCK cholangiocytes by using 6-Phe-cAMP, a cAMP analogue that specifically activates PKA.²³ Exposure of normal cholangiocytes to this activator during 6 hours decreased their rate of cell proliferation by 27% (Fig. 3C, p<0.01). By contrast, PKA activation accelerated proliferation of PCK cholangiocytes by 18% (Fig. 3C, p<0.001). These differential proliferation effects in normal and PCK cells were PKA-specific, as each of them was reversed in the presence of the PKA inhibitor Rp-cAMP (Fig. 3D, p<0.001 and p<0.05, respectively).

Role of MEK and ERK1/2 in the proliferation changes induced by activation of cAMP effectors in normal and PCK cholangiocytes

cAMP is known to regulate cell proliferation via stimulation of MEK that subsequently activates ERK1/2 kinases. Thus, we analyzed the effect of Epac and PKA stimulation on MEK and ERK1/2 in cultured normal and PCK cholangiocytes. In normal cholangiocytes, both basal and Epac-stimulated proliferation was blocked by the MEK-specific inhibitor U0126 (Fig. 4A, p<0.001). The phosphorylation status of ERK1/2 was increased upon Epac stimulation (4.7 times, Fig. 4B, p<0.05). This effect was specific, as pre-incubation of the normal cholangiocytes with the PKA inhibitor Rp-cAMP did not reverse Epac-stimulated phosphorylation (Fig. 4B). ERK1/2 phosphorylation was found to be always dependent on MEK activity, as both basal and Epac-stimulated ERK1/2 phosphorylation was blocked by the MEK inhibitor in normal cholangiocytes (Fig. 4B, p<0.01 and p<0.05, respectively).

Figure 4

Role of MEK and ERK1/2 in the proliferation changes induced by Epac and PKA activation in cultured normal and PCK cholangiocytes. (A) MEK-specific inhibitor U0126 blocked Epac-activated proliferation in both normal and PCK cholangiocytes. (B–C) (more ...)

Likewise, Epac-associated effects in PCK cholangiocytes were also MEK-dependent. Thus, pre-incubation of PCK cholangiocytes with the MEK inhibitor decreased the rate of Epac-stimulated proliferation (Fig. 4A, p<0.001). Similarly to normal cholangiocytes, Epac stimulation increased ERK1/2 phosphorylation also in PCK cholangiocytes (3.2 times, Fig. 4C, p<0.05). This effect was again Epac-specific, as pre-incubation with the PKA inhibitor Rp-cAMP did not block Epac-stimulated phosphorylation (Fig. 4C). Basal and Epac-stimulated ERK1/2 phosphorylation in PCK cholangiocytes were both MEK-dependent like in normal cholangiocytes, being also blocked in the presence of the MEK inhibitor (Fig. 4C).

We further analyzed the effect of PKA activation on MEK and ERK1/2. In normal cholangiocytes we observed a PKA-dependent reduction of 37.9% (p<0.05) in the phosphorylated/non-phosphorylated ERK1/2 ratio (Fig. 4E). This finding is consistent with our observation that PKA activation, in contrast to Epac activation, slows the proliferation rate in normal cholangiocytes (cf. Fig. 3C). Moreover, pre-incubation with the PKA inhibitor Rp-cAMP reversed the reduced ERK1/2 phosphorylation associated to PKA-activation (Fig. 4E). Thus, in normal cholangiocytes it may be assumed that PKA stimulation results in decreased MEK activity.

In PCK cholangiocytes, PKA-stimulated proliferation was blocked by the MEK-specific inhibitor (Fig. 4D, p<0.001). Moreover, the phosphorylation status of ERK1/2 was also changed. In contrast to normal cholangiocytes, and in agreement with the aforementioned hyperproliferation of PCK cholangiocytes following PKA activation (cf. Figs. 3C), we observed an increased ERK1/2 phosphorylation (2.5 times, Fig. 4F, p<0.05) in response to PKA activation. This effect was specific, since pre-incubation of PCK cholangiocytes with the PKA inhibitor Rp-cAMP halted the PKA-stimulated ERK1/2 phosphorylation (Fig, 4F, p<0.05). On the other hand, both basal and PKA-stimulated ERK1/2 phosphorylation was inhibited in PCK cholangiocytes by the presence of the MEK inhibitor (Fig. 4F p<0.01). Altogether, our data indicate that Epac- and PKA-associated effects on the proliferation of both normal and PCK cholangiocytes are MEK and ERK1/2-dependent.

Both Epac and PKA activation results in increased growth rate of 3-D cysts derived from normal and PCK cholangiocytes

We analyzed the effect of Epac stimulation on the growth of cysts that normal and PCK cholangiocytes form when cultured in 3-D.¹⁰ Once obtained, their stimulation with 8-pCPT-2’-O-Me-cAMP for 1 and 2 days resulted in increased growth of normal and PCK cysts (Figs. 5A,B). Epac specificity was supported by two inhibition experiments: pre-incubation with the PKA inhibitor Rp-cAMP did not block Epac-stimulated cyst growth (Figs. 5A,B), while this was halted in the presence of siRNAs against Epac1 and Epac2 mRNAs (Figs. 5C,D). The capability of these siRNAs for Epac silencing was ascertained by decreased protein levels of Epac isoforms in their presence (Fig. 5E). Importantly, Epac silencing also inhibited the basal growth of PCK cystic structures (Fig. 5D, p<0.001), further suggesting the involvement of Epac proteins in hepatic cystogenesis. Finally, both baseline and Epac-stimulated growth of normal and PCK cysts were MEK dependent as shown by the blocking effect of the MEK inhibitor U0126 (Figs. 5F,G, p<0.01).

Figure 5

Epac activation increases expansion of 3-D cystic structures derived from normal and PCK cholangiocytes. (A) Representative images of normal and PCK cystic structures grown in 3-D culture for 24 and 48 hours in the presence of the Epac activator 8-pCPT-2’-O-Me-cAMP (more ...)

Similarly to the effect observed with Epac activation, PKA activation during 1 and 2 days also increased the cyst growth rate of both normal and PCK 3-D cysts (Fig. 6A,B). This PKA-stimulated expansion, which was greater in PCK cysts (p<0.001), was always halted by pre-incubation with the PKA inhibitor Rp-cAMP (Figs. 6A,B). Likewise, the MEK inhibitor U0126 blocked both baseline and PKA-stimulated growth of normal and PCK cysts (Figs. 6C,D).

Figure 6

PKA activation increases expansion of 3-D cystic structures derived from normal and PCK cholangiocytes. (A) Representative images of normal and PCK cystic structures grown in 3-D culture for 24 and 48 hours in the presence of the PKA activator 6-Phe-cAMP (more ...)

Decreased intracellular Ca²⁺ in PCK cholangiocyte is responsible for the PKA-associated but not Epac-associated proliferation

Recent evidence indicates that levels of intracellular Ca²⁺ are decreased in renal cystic cells, a finding associated with accelerated cell proliferation.¹¹ Because there is no information on the levels of intracellular calcium in cystic cholangiocytes, we loaded cultured normal and PCK rat cholangiocytes with either fura-2/AM or fluo-4-AM, and found lower intracellular Ca²⁺ levels in PCK compared to normal cholangiocytes (Fig. 7A, p<0.01). We examined the role of the decreased intracellular Ca²⁺ levels in PCK cholangiocyte proliferation by raising intracellular Ca²⁺ with the Ca²⁺ ionophore A23187. As shown in Fig. 7B, pre-incubation of cultured PCK cholangiocytes with 0.1 µM of A23187 increased the fluo-4-AM fluorescence by 22.5% (p<0.01). This A23187-mediated restoration of intracellular Ca²⁺ was able to suppress both baseline and PKA-associated cholangiocyte proliferation (Fig. 7C, p<0.01). Consistent with this, PKA-stimulated ERK1/2 phosphorylation was decreased in response to elevated intracellular Ca²⁺ (Fig. 7D, p<0.05). These observations suggest that intracellular Ca²⁺ has a relevant role in the modulation of the PKA-associated proliferation of PCK cholangiocytes. On the other hand, restoration of intracellular Ca²⁺ did not affect the Epac-associated proliferation of PCK cholangiocytes (Fig. 7E), indicating that Epac-stimulated proliferation of cholangiocytes is not calcium-dependent

Figure 7

Intracellular Ca²⁺ is decreased in PCK cholangiocytes being involved in their PKA-associated (but not their Epac-associated) proliferation. (A) Intracellular Ca²⁺ determination after loading cells with 10 µM of fura-2/AM or 3 µM of fluo-4-AM (more ...)

The inhibition of PKA-associated proliferation by elevation of intracellular Ca²⁺ in PCK cholangiocytes is PI3K and AKT-dependent

It has been previously shown that the PI3K/AKT pathway regulates cAMP-associated cell proliferation.¹¹ Thus, we assessed the involvement of PI3K and AKT in PCK cholangiocyte proliferation in response to intracellular Ca²⁺ restoration by treating cultured PCK cholangiocytes with inhibitors of PI3K (LY294002) and AKT (1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate. The effect of A23187-mediated restoration of intracellular Ca²⁺ slowing PKA-associated proliferation was abolished by pre-incubation of PCK cholangiocytes with PI3K and AKT inhibitors (Fig. 8A). Likewise, the decreased ERK1/2 phosphorylation observed after elevation of intracellular Ca²⁺ was reversed by pre-incubation of PCK cholangiocytes with PI3K and AKT inhibitors (Fig. 8B). Importantly, none of these inhibitors modified the intracellular Ca²⁺ levels elevated with calcium ionophore A23187 (data not shown).

Figure 8

Restoration of intracellular Ca²⁺ levels in PCK cholangiocytes inhibits PKA-associated proliferation via PI3K/AKT pathway. Suppression of either PI3K activity with the LY294002 inhibitor, or AKT activity with the 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (more ...)

Discussion

The key findings reported here indicate that: i) Epac1, Epac2 and all four PKA regulatory subunits are expressed in normal rat cholangiocytes; ii) Epac1 and Epac2 isoforms and the PKA subunit RIβ are over-expressed in PCK cholangiocytes; iii) Epac activation increases proliferation of both normal and PCK cholangiocytes – but mainly in the latter – whereas PKA activation exhibits differential effects decreasing proliferation in normal rat cholangiocytes and accelerating proliferation in PCK cholangiocytes; iv) in 3-D cysts derived from normal and PCK cholangiocytes, however, both Epac and PKA activation results in increased expansion, which is more pronounced in PCK-cholangiocyte derived cysts; v) Epac and PKA associated effects occur via the MEK and ERK1/2 pathway; vi) levels of intracellular Ca²⁺ are lower in PCK than in normal cholangiocytes; vii) restoration of intracellular Ca²⁺ levels in PCK cholangiocytes inhibits both baseline and PKA-associated proliferation via the PI3K/AKT pathway; and viii) Epac-associated proliferation of PCK cholangiocytes is calcium-independent. Altogether, our data suggest that accelerated proliferation of cholangiocytes in the PCK rat is related to abnormalities in two important intracellular signaling pathways: the cAMP pathway, which is up-regulated, and the intracellular Ca²⁺ pathway, which is down-regulated in PCK cholangiocytes. Moreover, in contrast to the previous reports on renal cystic epithelia (in which only PKA seems to modulate accelerated cell proliferation while Epac has no role),¹⁸ both downstream effectors of cAMP, i.e. Epac and PKA, are clearly involved in proliferation of cystic cholangiocytes. Thus, our present observations expand our understanding of the pathogenesis of cystic liver diseases and suggest new therapeutic approaches.

Epac has two isoforms, Epac1 and Epac2, which are cAMP-activated RAP guanine-nucleotide-exchange proteins.¹⁵^–¹⁷ Epac1 is ubiquitously expressed,¹⁴ while Epac2 has been reported mainly in the brain,¹⁴ though a short form of Epac2 was also detected in whole liver.¹⁷ Here we demonstrate that both Epac1 and Epac2 proteins are expressed in normal and PCK cholangiocytes. Moreover, we demonstrate that the two Epac isoforms are over-expressed in PCK rat cholangiocytes, suggesting that they might be involved in the accelerated cholangiocyte proliferation subsequently leading to hepatic cystogenesis. Indeed, the Epac-specific activator, 8-pCPT-2’-O-Me-cAMP, which does not affect PKA,¹⁵^,²² promoted proliferation of cultured cholangiocytes and 3-D cysts derived from cholangiocytes, in a MEK and ERK1/2-dependent manner. This proliferative effect was more pronounced in PCK cholangiocytes and PCK-derived cysts compared to normal. Administration of siRNAs against both Epac isoforms inhibited Epac-activated cyst growth in normal and PCK cysts, confirming the specificity of Epac activation. Importantly, siRNA-mediated Epac silencing also blocked the basal growth of PCK cystic structures, further suggesting that Epac proteins play an important role in hepatic cystogenesis.

PKA is another downstream target of cAMP.¹⁴ While PKA RIα and RIIα regulatory subunits are ubiquitously expressed, PKA RIβ and RIIβ are mainly tissue-specific.¹⁴ We found that all four regulatory subunits are expressed in normal and PCK rat cholangiocytes at mRNA and protein levels, with over-expression of RIβ in PCK cells. RIβ possesses high affinity to cAMP and has been implicated in cell growth.²⁴ RIβ over-expression in PCK cholangiocytes suggests that it might contribute to their hyperproliferation. Indeed, PKA activation for 6 hours accelerated proliferation of cultured PCK cholangiocytes, while it displayed a hypoproliferative effect in cultured normal cholangiocytes. Surprisingly, when we changed to cystic structures – obtained from 3-D culture of normal and PCK cholangiocytes – to test longer PKA activation (for 24 and 48 hours), expansion occurred in both normal and PCK cysts (though with a greater extent in PCK-derived cysts). Expansion of normal-cholangiocyte derived cysts in spite of a putative PKA-mediated weakening in their proliferation rate, points to a counterbalance of PKA-dependent apical fluid secretion that results in increased cystic cavities. This view is in agreement with the well known PKA-mediated hypersecretory effect of secretin.²⁵ Indeed, we have recently shown that cystic structures from normal cholangiocytes respond to secretin stimulation by increasing cyst expansion.⁸ Moreover, PCK cystic structures exhibit increased secretin-stimulated fluid accumulation compared to normal cystic structures, which is associated with over-expression and abnormal location of AQP1, CFTR and AE2.⁸

Similarly to the hypoproliferative effect of PKA activation observed in our cultured normal cholangiocytes and the hyperproliferation in PCK cholangiocytes, PKA activation was reported to inhibit proliferation of normal human renal cells while stimulating this process in cells isolated from ADPKD or ARPKD renal cysts.¹¹^,¹⁸

The opposite effects of PKA reported in renal cells between normal and diseased epithelial cells were associated with decreased [Ca²⁺]_i in cystic cells.¹¹ Also, we found that intracellular Ca²⁺ levels are lower in PCK cholangiocytes compared to normal. The mechanisms leading to decreased intracellular Ca²⁺ and cAMP-associated hyperproliferation may be investigated taking into account both cholangiocyte and renal perspectives in polycystic diseases. Fibrocystin, the protein mutated in ARPKD, is known to form functional complexes within primary cilia with proteins involved in intracellular Ca²⁺ signaling, such as polycystin 2 and CAML (Ca²⁺-modulating cyclophilin1 ligand), suggesting a possible role for fibrocystin in Ca²⁺ signaling.²⁶^–²⁸ In fact, pre-incubation of kidney cells with antibodies against fibrocystin was reported to abolish a flow-induced increase in [Ca²⁺]_i.²⁷ Disappearance of fibrocystin from primary cilia was observed in cholangiocytes from the PCK rat and also in renal cysts of ARPKD patients. ⁴^,²⁹ Thus, the absence of fibrocystin from cilia may lead to its inability to form the aforementioned functional complexes and, subsequently, to maintain baseline [Ca²⁺]_i in cystic cells. Furthermore, other calcium channels present in cholangiocyte cilia (such as TRPV4) may also affect the influx of extracellular Ca²⁺ into the cells.⁷

Here we found that restoration of intracellular Ca²⁺ levels inhibits both baseline and PKA-associated proliferation in PCK cholangiocytes through the PI3K/AKT pathway and ERK1/2 phosphorylation. These data provide further support for a cross-talk between the two intracellular signaling mediators, cAMP and Ca²⁺, in modulating the response to PKA in cholangiocytes. Importantly, restoration of intracellular Ca²⁺ levels had no effect on Epac-associated proliferation of PCK cholangiocytes. These findings may explain in part why Epac stimulates the proliferation of both normal and PCK cholangiocytes and suggest a differential interaction between intracellular Ca²⁺ signaling and the two downstream effectors of cAMP signaling.

In summary (see the working model in Fig. 9), our data suggest that in normal cholangiocytes, activation of two downstream effectors of cAMP have opposite effects on cholangiocyte proliferation – PKA inhibits while Epac stimulates this process. In PCK cholangiocytes, activation of both cAMP downstream targets, Epac and PKA, leads to cholangiocyte hyperproliferation. This is different from the findings in the epithelial cells lining renal cysts since only PKA appears to be involved in their proliferation while Epac proteins play no role.¹⁸ Secondly, restoration of reduced intracellular Ca²⁺ in PCK cholangiocytes suppresses PKA-associated (but not Epac-related) proliferation. Our results are consistent with the notion that disturbances in both pathways contribute to the benign hyperproliferation of PCK cholangiocytes that result in hepatic cystogenesis. Our observations also suggest that therapeutic targeting of either of these signaling pathways could lead to a reduction in the progressive hepatic cystogenesis seen in ARPKD and other cystic liver diseases.

Figure 9

Working model. Downstream effectors of the cAMP, Epac and PKA, differentially affect cell proliferation in cultured normal and PCK rat cholangiocytes. In normal cholangiocytes, Epac activation stimulates proliferation, while PKA activation suppresses (more ...)

Acknowledgments

This work was supported by the National Institutes of Health (N.F. LaRusso: grant DK 24031), by the PKD Foundation (T.V. Masyuk and S.A. Gradilone), by the Mayo Foundation, and by the Spanish Ramón Areces Foundation (J.M. Banales). Ciberehd is funded by the Spanish Instituto de Salud Carlos III.

Abbreviations


ADPKD	Autosomal Dominant Polycystic Kidney Disease

AKT/PKB	protein kinase B

ARPKD	Autosomal Recessive Polycystic Kidney Disease

CAML	Ca²⁺-modulating cyclophilin1 ligand

cAMP	cyclic adenosine monophosphate

cAMP-GEF/Epac	cAMP-dependent guanine nucleotide exchange factor/exchange proteins activated by cAMP

DAPI	4',6-diamidino-2-phenylindole

ERK	extracellular signal-regulated kinase

MEK/MAPKK	mitogen-activated protein kinase kinase

mRNA	messenger RNA

PCR	polymerase chain reaction

PI3K	phosphoinositide Kinase-3

PKA R	protein kinase A regulatory subunit

RT-PCR	reverse transcriptase-PCR

qPCR	real time quantitative PCR

siRNA	small interfering RNA

Footnotes

No conflicts of interest exist.

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