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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References

Partial hepatectomy leads to an orchestrated regenerative response, activating a cascade of cell signaling events necessary for cell cycle progression and proliferation of hepatocytes. However, the identity of the humoral factors that trigger the activation of these pathways in the concerted regenerative response in hepatocytes remains elusive. In recent years, extracellular ATP has emerged as a rapidly acting signaling molecule that influences a variety of liver functions, but its role in hepatocyte growth and regeneration is unknown. In this study, we sought to determine if purinergic signaling can lead to the activation of c-jun N-terminal kinase (JNK), a known central player in hepatocyte proliferation and liver regeneration. Hepatocyte treatment with ATPγS, a nonhydrolyzable ATP analog, recapitulated early signaling events associated with liver regeneration—that is, rapid and transient activation of JNK signaling, induction of immediate early genes c-fos and c-jun, and activator protein-1 (AP-1) DNA-binding activity. The rank order of agonist preference, UTP>ATP>ATPγS, suggests that the effects of extracellular ATP is mediated through the activation of P2Y2 receptors in hepatocytes. ATPγS treatment alone and in combination with epidermal growth factor (EGF) substantially increased cyclin D1 and proliferating cell nuclear antigen (PCNA) protein expression and hepatocyte proliferation in vitro. Extracellular ATP as low as 10 nM was sufficient to potentiate EGF-induced cyclin D1 expression. Infusion of ATP by way of the portal vein directly activated hepatic JNK signaling, while infusion of a P2 purinergic receptor antagonist prior to partial hepatectomy inhibited JNK activation. In conclusion, extracellular ATP is a hepatic mitogen that can activate JNK signaling and hepatocyte proliferation in vitro and initiate JNK signaling in regenerating liver in vivo. These findings have implications for enhancing our understanding of novel factors involved in the initiation of regeneration, liver growth, and development. (HEPATOLOGY 2004;39:393–402.)

Based on investigations performed over the last two decades, it is now recognized that the hepatic regenerative response involves a well-orchestrated series of events culminating in hepatocyte cell cycle entry and liver morphogenesis.1–4 Although many growth factors and cytokines appear to play important roles in the regenerative response, none is individually sufficient to trigger liver regeneration.3, 5–8

Data from several laboratories suggest that signaling by way of c-jun N-terminal kinase (JNK), a member of the stress-activated protein kinase family, plays a key role in hepatocyte proliferation and hepatogenesis.9–13 Activation of a cascade of events upstream of JNK results in its phosphorylation at two residues, Thr 183/Tyr 185, which enhances its kinase activity. Activated JNK phosphorylates its main substrate c-jun (Ser 63/Ser 73), which positively regulates c-jun function. c-jun is an immediate early gene product and a component of the activator protein-1 (AP-1) transcription factor complex.14 Activation of AP-1, in turn, influences the expression of several genes essential for the proliferation of hepatocytes, including cyclin D1.14, 15

The importance of JNK signaling and AP-1 activation in hepatocyte proliferation is apparent from analyses of regenerating liver and gene knock-out studies in mice.9–11 Studies of regenerating livers show increased JNK activity within 15 minutes of partial hepatectomy.1, 16 The deletion of either mitogen-activated protein kinase activating kinase 4 (MKK4), a potent upstream activator of JNK, or c-jun genes is embryonic lethal, with marked impairments in murine hepatocyte proliferation.17, 18 It has been shown recently that the perinatal, liver-specific deletion of the JNK target c-jun leads to decreased hepatocyte proliferation and liver regeneration after partial hepatectomy11. The findings of the above gene deletion studies concur with recent observations that a small molecule JNK inhibitor (SP600125) blocks cyclin D1 expression and proliferation during regeneration.13 Collectively, these studies suggest that JNK signaling is essential for normal liver development and liver regeneration after partial hepatectomy. However, the upstream molecular mechanisms that lead to the activation of JNK signaling in regenerating liver remains unknown.

In recent years, extracellular ATP, acting through cell surface purinergic receptors, has emerged as an important signaling molecule influencing a variety of epithelial cell functions, including hepatocytes and cholangiocytes.19–23 Cellular stress induced by changes in osmosis or mechanical perturbations such as shear stress induce discrete release of ATP into the extracellular milieu. In fact, cell swelling is a potent stimulus for ATP release from hepatocytes, and recent studies in both hepatocytes and cholangiocytes suggest that extracellular ATP plays a key role in cell volume recovery from swelling in both cell types.21, 24, 25 Hepatocytes are known to swell soon after partial hepatectomy.26, 27 Although ATP is a highly labile molecule, concentrations of 50 to 1,000 nM ATP have been detected in portal blood and bile.28–30 Additionally, ATP released from hepatocytes has been shown to influence intracellular calcium signaling in neighboring cells.31 Multiple metabolic processes such as hepatic protein synthesis, gluconeogenesis, and ureagenesis in the liver are under the autocrine and paracrine control of extracellular ATP.19–21, 29

Extracellular ATP is mitogenic for multiple cell types, including vascular smooth muscle cells, endothelial cells, renal mesangial cells, several types of fibroblasts, and neuroblastoma- and osteoblast-like cell lines.32–34 However, the potential role of purinergic signaling on hepatocyte proliferation remains unknown.

Because JNK activation takes place within minutes of partial hepatectomy and plays a key role in hepatocyte proliferation, the purpose of this study was to determine if extracellular ATP influences JNK signaling and cell cycle progression in hepatocytes. The findings of this study provide the first evidence that extracellular ATP and P2 purinergic receptors play a key role in the early induction of JNK signaling response during liver regeneration. Interestingly, the temporal profile of ATP-induced JNK activation in hepatocytes in vitro is similar to the activation of JNK signaling associated with partial hepatectomy and treatment with known hepatic mitogens (e.g., hepatocyte growth factor).16, 35 Moreover, the present study provides evidence that extracellular ATP activates multiple components of hepatocyte cell cycle progression and proliferation (c-fos and c-jun mRNA expression, c-jun protein, AP-1 activity, cyclin D1, and PCNA expression), thus identifying a new role for extracellular ATP as a hepatic mitogen.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References

Hepatocyte Isolation and ATPγS Treatment of Cultured Hepatocytes.

Hepatocytes were isolated by the two-step perfusion procedure using 0.025% collagenase as previously described.36, 37 They were plated at a density of 105 to 5 × 105 cells/well in 35-mm-diameter Primaria dishes (Becton and Dickenson, San Diego, CA) or rat type 1 collagen (Sigma, St. Louis, MO) coated glass cover slips in Williams E medium (Sigma) containing 10% fetal bovine serum, glutamine (2 mM), dexamethasone (400 ng/mL), insulin (2.5 μg/mL), glucagon (4 ng/mL), transferrin (2.5 μg/mL), and sodium selenite (2.5 ng/mL), penicillin (10,000 U/mL), streptomycin (10,000 μg/mL) and gentamycin (50 μg/mL). After 3 hours the medium was replaced with serum- and mitogen-free Williams E medium supplemented with glutamine, penicillin, streptomycin, and gentamicin; the medium was renewed every day.

Primary rat hepatocytes maintained under serum- and mitogen-free conditions for 42 hours were replenished with fresh media 3 to 6 hours before treatment.38 Treatment was initiated by the addition of a nonhydrolyzable analog of ATP, ATPγS [adenosine 5′-O-(3-thiotriphosphate)] to the culture media and was terminated by placing culture dishes on ice and washing twice with ice-cold phosphate buffered saline (PBS). Cells were rapidly processed for the isolation of total RNA or proteins from the nuclei or total cell lysates. All experiments described have been performed at least three times.

Assay of JNK Activity.

Primary rat hepatocytes were treated with 100 μM ATPγS for 5 to 120 minutes and washed with ice-cold PBS; total cell extracts were obtained in lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), 1.0% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 mM phenylmethanesulfonyl fluoride (PMSF), 1 mM leupeptin). After centrifuging at 12,000 rpm for 10 minutes at 4° C, the supernatants containing 250 μg proteins were incubated with 2 μg of c-jun fusion protein beads overnight with gentle rocking at 4° C. The beads were washed twice with 500 μL lysis buffer and 500 μL kinase buffer (25 mM Tris, pH 7.4, 5 mM β-glycerophosphate 2 mM DTT, 0.1 mM Na3VO4, 10 mM MgCl2). The pellets were suspended in 50 μL kinase buffer supplemented with 100 μM ATP and incubated at 30° C for 30 minutes. The kinase reaction was terminated by the addition of 100 μL of 2 × Laemmli buffer and boiling at 95° C for 5 minutes. Laemmli extracts were removed by centrifugation at 12,000 rpm for 5 minutes for analysis by Western blotting with rabbit polyclonal antibody raised against c-jun peptides phosphorylated at Serine 63.39

Immunoblotting.

At the end of treatment period cells were washed twice with ice-cold PBS and total cell lysates were obtained in the lysis buffer. The amount of total protein was determined using the BCA protein assay method (Pierce, Rockford, IL). Proteins were analyzed by SDS-polyacrylamide gel electrophoresis with antibodies specific for phospho-JNK (Thr 183/Tyr185), phospho-c-jun (Ser 63/Ser 73), JNK (p54/46) and c-jun (New England Biolabs, Beverly, MA), and cyclin D1 and PCNA (Santa Cruz Biotechnology, Santa Cruz, CA). The protein bands were detected by enhanced chemiluminescent reaction according to the manufacturer's instructions (NEN Life Sciences, Boston, MA) as described previously.40

RNA Isolation and Northern Blotting.

Total RNA was isolated by acid guanidinium thiocyanate-phenol-chloroform extraction with subsequent centrifugation in cesium chloride solution.36 Total RNA (20 μg) was denatured, electrophoresed on a 1.2% agarose/formaldehyde gel, transferred to a nylon membrane (Genescreen; DuPont-NEN, Boston, MA) by overnight capillary blotting, and UV cross-linked (UV Stratalinker 1800; Strategene, La Jolla, CA). Membrane prehybridization, hybridization, and washing procedures were performed as described previously.36 RNA levels were detected by exposure of the membrane to Hyperfilm (Amersham Corporation, Piscataway, NJ) for 1 to 3 days. Equal loading of mRNA was determined by reprobing the stripped blots for GAPDH. The size of mRNA was estimated by a 0.24- to 9.5-kb RNA ladder (Gibco-BRL, Life Technologies, Gaithersburg, MD). The specific cDNA probes used were c-fos (2.1-kb EcoR1 fragment), c-jun (2.0-kb EcoR1/BamH1 fragment), and GAPDH cDNA (1.25-kb Pst1 fragment).41

Nuclear Protein Extraction.

Preparation of crude nuclear extract was basically as described previously.42 Briefly, after treatment with ATP for the times indicated, hepatocytes were washed twice with ice-cold PBS and gently lysed in 10-mM Tris buffer, pH 7.9 containing 1 mM EDTA, 150 mM NaCl, 0.6% NP-40, 1 mM DTT, and protease inhibitors for 5 minutes. Hepatocyte nuclei were isolated by centrifuging the lysates at 4,000 rpm for 5 minutes in the cold room. Nuclear proteins were extracted with 10 mM Hepes buffer, pH 7.6 containing 0.1 mM EDTA, 1.5 mM MgCl2, 420 mM NaCl, 25% glycerol, 1 mM DTT, and protease inhibitors by incubating at 4° C for 30 minutes and centrifuging at 12,000 rpm for 5 minutes. Aliquots of the supernatant that contained nuclear proteins were flash-frozen on powdered ice and stored at −70° C. Protein was determined using a Bio-Rad kit (Bio-Rad, Hercules, CA) according to the manufacturer's instructions.

Electrophoretic Mobility Shift Assays.

AP-1 oligonucleotides were obtained from Santa Cruz Biotechnology. Double-stranded oligonucleotide probes were end-labeled and purified according to standard procedures.43 Ten micrograms of nuclear extracts were incubated with poly-dI:dC (μg/ml) in binding buffer for 10 minutes at 4° C prior to the incubation with 2 × 104 cpm of 32P end-labeled oligonucleotide as described previously.42 In competition assays, 100-fold molar excess of the specific unlabeled oligonucleotide was added to the binding mixtures along with the labeled oligonucleotide. Gels were dried and autoradiographed using Hyperfilm (Amersham Corporation) at −70° C for 1 to 3 days.

Infusion of ATP by Way of Portal Vein.

Adult male Sprague-Dawley rats (250 g) were anesthetized with 1.0% phentobarbitol and were infused with ATP (7.0 μmoles) or vehicle (0.9 % saline, 0.3 mL) via the portal vein over a period of 1 minutes. Thirty minutes after infusion, liver tissues were harvested and total homogenates obtained in the lysis buffer were analyzed by Western blotting with antibodies specific for phosphorylated JNK (Thr183/Tyr185), total JNK, and phosphorylated c-jun (Ser63).40

Infusion of Pyridoxal Phosphate-6-azo(benzene-2,4-disulfonic acid) (PPADS) by Way of Portal Vein Prior to Partial Hepatectomy of Adult Rat Liver.

Adult male Sprague-Dawley rats (250 g) were anesthetized with 1.0% phentobarbital and infused with 10 μmoles of PPADS (Sigma) or vehicle (0.9 % saline, 0.3 mL) by way of the portal vein 5 minutes prior to 70% partial hepatectomy. Partial hepatectomy was performed according to the methods previously described.44 The resected lobes (0 minutes) and the remnant right lateral lobes (60 minutes) were homogenized in the lysis buffer and analyzed by SDS-polyacrylamide gel electrophoresis with antibodies specific for phosphorylated JNK (Thr183/Tyr185), total JNK, and phosphorylated c-jun (Ser63).40 All animal study protocols were approved by the animal care and use committee of the Baylor College of Medicine.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References

Extracellular ATP Activates JNK Signaling in Hepatocytes.

Considering the importance of JNK signaling in liver regeneration, we first sought to determine if extracellular ATP leads to the activation of JNK signaling in primary rat hepatocytes in culture. Hepatocytes in culture exhibit a prolonged G1 phase and remain growth arrested in the absence of growth stimuli. As shown by Loyer and colleagues, this mitogen-dependent restriction point occurs at the end of the first two thirds of the G1 phase, after approximately 42 to 48 hours in culture.45 Therefore, we tested the role of extracellular ATP on JNK signaling using the culture conditions optimized by Loyer's group and used by several investigators.38, 45–47

Primary rat hepatocytes were maintained under serum-free conditions for 42 hours and treated with 0.01 to 100 μM ATPγS, a nonhydrolyzable analog of ATP. ATPγS treatment for 15 minutes induced dual phosphorylation of JNK at Threonine 183 and Tyrosine 185 in a dose-dependent manner starting at 10 μM, maximally at 100 μM (Fig. 1A). JNK activation was detectable within 5 minutes of ATPγS incubation, with maximal levels noted between 15 and 60 minutes and returning to near baseline levels within 2 hours (Fig. 1B, upper panel). However, ATPγS treatment did not influence total cellular JNK protein levels (Fig. 1B, lower panel).

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Figure 1. Extracellular ATP activates JNK in primary rat hepatocytes. (A) Dose-response of ATPγS leading to JNK phosphorylation. Hepatocytes were treated with a series of ATPγS concentrations for 15 minutes and 10 μg total cell lysates were analyzed by Western blotting with antibodies specific for phospho-JNK. (B) Time-course of ATPγS-dependent phosphorylation of JNK. Hepatocytes were treated with 100 μM ATPγS for 5 to 120 minutes and total cell lysates were analyzed by Western blotting with antibodies specific for phospho-JNK and total JNK protein. (C) Time course of ATPγS-dependent JNK activity. Hepatocytes were treated with 100 μM ATPγS for 5 to 120 minutes, and an increase in JNK activity was confirmed by the phosphorylation of GST-c-jun at Serine 63. Western blotting for total JNK confirms the precipitation of equivalent quantities of JNK. (D) Dose-response of ATPγS on c-jun phosphorylation. Hepatocytes were treated with 100 μM ATPγS for 60 minutes, and total cell lysates (10 μg) were analyzed by Western blotting for phosphorylated c-jun at Serine 73. (E) Time course of ATPγS-dependent phosphorylation of c-jun. Hepatocytes were treated with 100 μM ATPγS for 5 to 120 minutes and total cell lysates (10 μg) were analyzed by Western blotting with antibodies specific for phosphorylated c-jun at Serine 63 and Serine 73 and total c-jun protein.

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To confirm that changes in phosphorylation of JNK truly reflects its activation of kinase activity in hepatocytes, we performed the in vitro kinase assay for JNK using a glutathione-S-transferase-c-jun (GST-c-jun) fusion protein target. ATPγS treatment induced a robust and rapid stimulation of GST-c-jun phosphorylation in hepatocytes commensurate with the timeline of JNK phosphorylation (Fig. 1C, upper panel). Thus ATP-induced phosphorylation of JNK temporally correlated with its kinase activity. The kinase reaction mixture contained equal amounts of JNK protein at each time point (Fig. 1C, lower panel) confirming that the increase in GST-c-jun phosphorylation resulted from JNK activation, not changes in JNK protein content.

Prompted by our observations that extracellular ATP activates JNK activity, we next tested if the activation of JNK indeed leads to the phosphorylation of the endogenous JNK target, c-jun in primary rat hepatocytes. ATPγS treatment of hepatocytes led to a dose-dependent increase in c-jun phosphorylation (Fig. 1D). Endogenous c-jun was phosphorylated at both Ser 73 and Ser 63 within 5 minutes of ATPγS treatment, with maximal phosphorylation apparent at 1 hour (Fig. 1E, upper panel). In addition to increased phosphorylation, the total c-jun protein level increased, with peak levels observed after 60 to 120 minutes of ATPγS treatment (Fig. 1E, lower panel). Although an increase in total c-jun protein levels can potentially result from multiple mechanisms, our data suggest a role for ATP-mediated increase in c-jun mRNA playing a role in the observed increase of c-jun protein in primary rat hepatocytes (Fig. 2).

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Figure 2. Extracellular ATP activates c-fos and c-jun mRNA expression and AP-1 activity. (A) Northern blotting for the expression of immediate early genes c-fos and c-jun. Hepatocytes were treated with 100 μM ATPγS for 5 to 240 minutes and total RNA was analyzed by Northern blotting for the expression of c-fos and c-jun. The blot was probed with GAPDH to ensure equal loading of RNA between lanes. (B) Electrophoretic mobility shift assay for AP-1 DNA-binding activity. Hepatocytes were treated with 100 μM ATPγS for 5 to 240 minutes and nuclear proteins (10 μg) were analyzed for AP-1 activity by electrophoretic mobility shift assay.

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Extracellular ATP Induces the Expression of c-fos and c-jun RNA and AP-1 DNA-Binding Activity.

Immediate early genes c-fos and c-jun are well-known targets of JNK activation in hepatocytes.14 To test the effects of extracellular ATP on the expression of these genes, primary rat hepatocytes were treated with ATPγS for 5 to 240 minutes, and total RNA was analyzed by Northern blotting. Both c-fos and c-jun mRNA levels increased within 15 minutes of treatment, reaching their maximum by 30 minutes and returning to baseline levels within 2 hours (Fig. 2). Correspondingly, ATPγS treatment increased c-jun protein level (Fig. 1E).

Immediate early gene products c-fos and c-jun are key components of the AP-1 transcription factor, which controls a variety of cellular response genes, including genes involved in cell cycle progression.14 AP-1 activation is a key downstream target event of JNK signaling in hepatocytes. Prompted by the observations that ατPγS treatment induced c-fos mRNA and c-jun mRNA and c-jun protein expression in hepatocytes, we next tested if extracellular ATP influences AP-1 DNA-binding activity in primary rat hepatocytes. As expected, ATPγS treatment of primary rat hepatocytes enhanced AP-1 DNA-binding activity within 30 minutes of treatment, reaching its maximum within 2 hours (Fig. 2B). The temporal profile of AP-1 activation parallels ATP-induced c-jun expression in hepatocytes (Fig. 1E).

c-jun Phosphorylation Is Mediated by P2 Receptors.

To test the hypothesis that extracellular ATP activates JNK signaling cascade by activation of P2 cell-surface purinergic receptors, hepatocytes were pretreated with P2 receptor antagonists (100 μM suramin, oATP [periodate oxidized ATP], or reactive blue-2) 30 minutes prior to the addition of 100 μM ATPγS.23 Pretreatment with each P2 receptor antagonist markedly inhibited ATPγS-induced c-jun phosphorylation at serine 73, confirming the importance of cell-surface P2 purinergic receptors receptors in the extracellular ATP-mediated activation of JNK signaling in hepatocytes (Fig. 3).

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Figure 3. Purinergic P2 receptor blockade prevents ATP-mediated phosphorylation of c-jun. Hepatocytes were incubated with P2 receptor antagonists suramin (100 μM), oATP (100 μM), and reactive blue-2 (100 μM) for 30 minutes prior to the treatment with ATPγS (100 μM) for 60 minutes. Total cell lysates (10 μg) were analyzed by Western blotting with antibodies specific for phosphorylated c-jun at Serine 73.

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Extracellular ATP-Mediated Activation of JNK Signaling Profile Exhibits P2Y2 Receptor Specificity.

Hepatocytes express multiple receptor subtypes of the P2 purinergic receptor family coupled with diverse downstream signaling events.48 To identify the purinergic receptor subtype predominantly responsible for JNK activation, primary rat hepatocytes were treated with analogs of ATP with known selectivity toward multiple purinergic receptor subtypes.23 Based on densitometric analysis of Western blots, UTP was found to be the most potent activator of JNK phosphorylation, with an apparent rank order potency of UTP>ATP>ATPγS> 2MeSATP>ADPβS>UDP, the characteristic agonist preference of Y2 subtype of the P2 purinergic receptor family (P2Y2) (Table 1).23

Table 1. Extracellular ATP-Mediated JNK Signaling Profile Exhibits P2-Y2 Receptor Specificity
Conc.ATPγSATPUTPUDPADPβS2MeSATP
  1. Hepatocytes were treated with multiple nucleotide analogs (10 nM–100 μM) for 15 minutes, and total cell lysates (10 μg) were analyzed by Western blotting for the phosphorylation of JNK. Fold activation was calculated with reference to vehicle treatment. The data is representative of two independent experiments.

Veh1.01.01.01.01.01.0
10 nM0.61.22.21.20.82.0
100 nM1.01.42.31.41.13.3
1 μM1.12.23.61.30.64.0
10 μM4.66.411.51.92.47.5
100 μM6.89.816.02.34.58.1

Extracellular ATP Induces Cell Cycle Progression.

AP-1 activity influences a variety of cell functions, including the modulation of expression of several genes involved in cell cycle progression. Cyclin D1 is a key regulatory protein that controls cell cycle progression in the late G1 phase and is a well-known AP-1 target gene in fibroblasts and epithelial cells. Moreover, it has been suggested that cyclin D1 expression alone is sufficient to promote hepatocyte progression through the G1 restriction point.38 Therefore, we tested the role of extracellular ATP on cyclin D1 expression using the culture conditions optimized by Loyer and colleagues.45 After maintaining in serum- and mitogen-free conditions for 42 hours, hepatocytes were treated with ATPγS and harvested 30 hours later. ATPγS treatment led to a 3.7-fold increase in cyclin D1 expression in primary rat hepatocytes; EGF, a known hepatic mitogen, led to a 26-fold induction of cyclin D1. Combining ATPγS and EGF together led to a 41-fold enhancement of cyclin D1 expression (Fig. 4A).

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Figure 4. Extracellular ATP activates cell-cycle progression. Hepatocytes were maintained in serum- and mitogen-free conditions for 42 hours, then treated with ATPγS (100 μM) or EGF (20 ng/mL) alone or a combination thereof for 30 hours; the total cell lysates (10 μg) were analyzed by Western blotting with antibodies specific for (A) cyclin D1, (B) PCNA, and (C) β-actin. (D) Dose-response of ATP on EGF-induced cyclin D1 expression. Numbers below each band represent the fold activation in comparison to vehicle treatment, based on the densitometric analysis of band intensity.

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ATP treatment induced cyclin D1 expression in a dose-dependent manner. EGF (20 ng/mL) alone induced a 6.9-fold increase. Further increases were apparent at ATP concentrations as low as 10 nM (10-fold) (Fig. 4D). It should be noted that ATP is a more potent inducer of JNK than ATPγS and the ATP concentrations in the lower nM range appear to be sufficient to potentiate EGF-induced cell cycle progression in hepatocytes. The current observation underscores the physiologic significance of ATP as a potential comitogen, enhancing the effects of growth factors in hepatocytes.

Extracellular ATP Induces Hepatocyte Proliferation.

We next tested if ATPγS treatment induces hepatocyte proliferation in vitro.49–53 PCNA is a protein that functions as an auxiliary protein of DNA polymerase-δ and widely used in the detection of proliferating cells by immunocytochemistry and western blotting. ατPγS treatment induced a 2.7-fold increase in PCNA expression in primary rat hepatocytes. Parallel to our observations with cyclins D1, ατPγS treatment enhanced EGF-induced PCNA expression from 12-fold to 19-fold (Fig. 4B).

Hepatocytes maintained in serum and growth factor-free conditions rarely divide and remain growth arrested during the late G1 phase. Consistent with its role in the induction of cell cycle progression, hepatocyte proliferation in vitro is induced by ATPγS treatment. After 4 days of treatment, there was a 2.2-fold increase in hepatocyte number in the ATPγS treated group compared with controls (Fig. 5). EGF treatment alone led to a 5.2-fold increase in hepatocyte number, whereas a combination of EGF and ATPγS led to even higher increase in hepatocytes (7.1-fold) (Fig. 5).

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Figure 5. Extracellular ATP activates proliferation of hepatocytes in vitro. Hepatocytes were treated with ATPγS (100 μM) or EGF (20 ng/mL) alone or a combination thereof for 4 days. Cells were replenished with fresh media and treatment components every 24 hours. Cells were fixed with cold acidified ethanol and analyzed under the ×10 objective of a phase-contrast light microscope. Cells were counted from 10 randomly selected fields of view for each experimental group and represented as mean ± SEM (n = 6). *P < .001 with reference to vehicle-treated control. #P < .001 with reference to EGF alone.

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Extracellular ATP-Mediated Cell Cycle Progression Is Dependent on Functional P2 Purinergic Receptors and the Induction of JNK Signaling.

Extracellular ATPγS led to a robust and transient activation of JNK signaling in hepatocytes, and P2 receptor antagonists blocked ATPγS-induced JNK signaling (Figs. 1 and 3). To determine if activation of JNK plays a role in extracellular ATPγS-mediated proliferation, primary rat hepatocytes were treated with P2 receptor antagonists (10 μM PPADS or 10 μM oATP) and a small molecule inhibitor of JNK signaling (10 μM SP600125) 30 minutes prior to ATPγS treatment. JNK inhibition and P2 purinergic receptor blockade diminished extracellular ATP-mediated PCNA expression in vitro, suggesting a role for P2 purinergic receptor–mediated early activation of JNK signaling in hepatocyte proliferation (Fig. 6).

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Figure 6. Extracellular ATP mediated cell-cycle progression is dependent on P2 purinergic receptors and activation of JNK signaling. Hepatocytes were pretreated with the (A) JNK inhibitor SP600125 (10 μM), (B) P2 receptor antagonist PPADS (10 μM), or (C) oATP (10 μM) for 30 minutes prior to the treatment with ATPγS (100 μM) for 4 days. The total cell lysates (10 μg) were analyzed by Western blotting with antibodies specific for PCNA.

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ATP Infusion Activates Hepatic JNK Signaling In Vivo.

Our observations on the robust activation of extracellular ATP mediated JNK signaling in primary rat hepatocytes in vitro led us to explore if extracellular ATP alone is sufficient to activate hepatic JNK signaling in vivo. Male Sprague-Dawley rats were anesthetized and infused with a single dose of ATP (7 μmoles in 0.9% saline) or 0.9% saline vehicle alone, by way of the portal vein 30 minutes prior to the excision of liver for analysis for JNK activation. Mirroring our observations in vitro, ATP infusion alone led to a robust activation of hepatic JNK signaling, as evidenced by the increased phosphorylation of JNK and c-jun in the adult rat liver (Fig. 7A). To our knowledge, this observation provides the first evidence that extracellular ATP directly activates intracellular signaling in liver.

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Figure 7. Extracellular ATP and P2 purinergic receptors activate hepatic JNK signaling in vivo. Male Sprague-Dawley rats were infused with ATP (7.0 μmoles) or vehicle (0.9 % saline, 0.3 mL) by way of the portal vein over a period of 1 minute. Thirty minutes after infusion, liver tissues were harvested and total homogenates were analyzed by Western blotting with antibodies specific for phosphorylated JNK (Thr183/Tyr185), total JNK, and phosphorylated c-jun at Serine 63 (A). P2 receptor blockade inhibits partial hepatectomy–induced JNK signaling in vivo. Male Sprague-Dawley rats were infused with 10 μmoles of PPADS or vehicle (0.9 % saline, 0.3 mL) by way of the portal vein 5 minutes prior to 70% partial hepatectomy and total homogenates of liver tissues harvested before (0 minutes) and after (60 minutes) 70% partial hepatectomy were analyzed by Western blotting with antibodies specific for phosphorylated JNK (Thr183/Tyr185), total JNK, and phosphorylated c-jun at Serine 63 (B). PH, 70% partial hepatectomy.

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P2 Purinergic Receptor Blockade Inhibits Partial Hepatectomy-Induced JNK Signaling In Vivo.

Our current finding that extracellular ATP can induce hepatic JNK signaling in vivo prompted us to test the functional role of ATP and its cognate P2 receptors in a well-established model for the analysis of hepatocyte proliferation and liver growth in vivo (e.g., partial hepatectomy of adult rats). Partial hepatectomy (70%) is known to induce a robust activation of JNK signaling in the remnant liver within minutes, with maximal activation at 30 to 60 minutes.1, 16 Pretreatment with P2 purinergic receptor antagonist PPADS (10 μmoles) inhibited the activation of JNK signaling at 60 minutes after partial hepatectomy (Fig. 7B). The present findings implicate for the first time that the extracellular ATP and P2 purinergic receptors play a key role in the early induction of JNK signaling in vivo in a regenerating liver.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References

The principal findings of the present study indicate that extracellular ATP is a novel and potent hepatic mitogen. Extracellular ATP activates hepatocyte cell cycle progression and proliferation in vitro and plays a key role in the initiation of JNK signaling soon after partial hepatectomy. ATP infusion by way of the portal vein activates hepatic JNK signaling in adult rats, and infusion of PPADS—a P2 purinergic receptor antagonist—prior to partial hepatectomy inhibits endogenous JNK activation, one of the earliest signaling events in the remnant liver. JNK phosphorylation, c-fos and c-jun mRNA expression, AP-1 DNA binding activity, markers of cell cycle progression (e.g., cyclin D1), and hepatocyte number are all increased by extracellular ATP treatment in vitro. We believe the above findings suggest a role for extracellular ATP as a rapid activator and initiator of hepatocyte cell cycle progression and proliferation (Fig. 8).

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Figure 8. Extracellular ATP activates hepatocyte cell cycle progression by multiple mechanisms. Schematic representation of the effects of extracellular ATP on JNK signaling, immediate early gene expression, AP-1 activity, and cyclin D1 protein levels collectively leading to cell cycle progression in hepatocytes. Solid arrows indicate the experimental findings in this report. Arrows with dotted lines indicate potential mechanisms based on other studies.

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Supportive of its role in proliferation, we show that ατPγS treatment of hepatocytes leads to a transient but robust increase in JNK phosphorylation and activation of its kinase activity. Extracellular ATP has been shown to induce JNK signaling in nonepithelial cells such as macrophages and smooth muscle cells.54, 55 However, this study provides the first evidence that extracellular ATP transiently activates JNK signals in an epithelial cell type (e.g., hepatocytes). The temporal profile of ATP-induced JNK activation in hepatocytes is similar to hepatocyte growth factor, an established hepatic mitogen.6

The in vitro study design and the concentration range of ATPγS (10 nM–100 μM) employed in this study were adapted from previous reports from multiple laboratories exploring purinergic signaling in hepatocytes in culture.21, 31, 48, 56, 57 Although robust activation of JNK and c-jun phosphorylation have been observed with 100 μM ATPγS, the dose-response analysis reveals that ATPγS as low as 1 μM and ATP as low as 10 nM concentrations can activate JNK phosphorylation (Table 1) that falls within the physiologic range reported in blood and bile.29 Moreover, only 10 nM of ATP activates cyclin D1 expression. However, the intracelluar concentration of ATP is in the mM range, and considering the well-documented potential of hepatocytes to discretely secrete ATP into the extracellular millieu under stress—as well as the highly labile nature of ATP—it is not difficult to envisage local cell surface ATP concentrations to be much higher than those reported in blood or bile.29

Our results obtained from electrophoretic mobility shift assays suggest purinergic signaling potently activates DNA binding activity of AP-1 transcription factors in hepatocytes (Fig. 2B) and AP-1 target genes, cyclin D1 and PCNA (Fig. 4). Differential expression of AP-1 proteins in response to extracellular stimuli has been suggested as one of the major mechanisms that modulate AP-1 activity.14 The present study provides evidence that extracellular ATP increases immediate early gene expression, c-jun and c-fos mRNA levels, leading to increased AP-1 DNA binding activity.

AP-1 activity plays a key role in the control of cellular proliferation in multiple cell systems. Growth-promoting signals activate AP-1 activity, which in turn causes the activation of genes whose products stimulate cell cycle progression. We provide evidence that extracellular ATP enhances the expression of cyclin D1. Cyclin D1 is an AP-1 target gene and is one of the earliest expressed cyclins that controls hepatocyte cell cycle progression through G1 and G1/S transition.38, 58 The promoter for cyclin D1 contains an AP-1 site, and ectopic expression of either c-fos or c-jun induces cyclin D1 mRNA expression. Overexpression of cyclin D1 alone is sufficient for mitogen-independent cell cycle progression in hepatocytes beyond the mitogenic restriction point and into S phase of the cell cycle.59 Transgenic overexpression of cyclin D1 in mice leads to marked hepatomegaly.60

Our findings suggest that extracellular ATP alone is sufficient to induce cell cycle progression beyond G1 phase of the cell cycle and potentiate the effects of EGF on cell cycle progression and proliferation. As low as 10 nM ATP seems to be sufficient to amplify the effects of EGF-induced cyclin D1 expression in hepatocytes in culture. The current observations underscore the physiological significance of the potential cross-talk between the signals emenating from extracellular ATP and the known hepatocyte growth factors such as EGF and hepatocyte growth factor. The modulation of EGF receptor sensitivity at the cell surface and amplification of downstream signals such as JNK and p44/42MAPK pathways induced by EGF remain attractive candidates potentially influenced by extracellular ATP-mediated JNK signaling.

The search for the “humoral agent” that serves as the initial trigger for cell cycle progression and proliferation in hepatocytes in vivo has been an area of intense study for the past few decades.61–63 The known characteristics of ATP and its ability to be released discretely into the extracellular milieu in response to cellular stress fits the profile of the humoral agent speculated to induce hepatocyte proliferation after partial hepatectomy.21 Although not reported, we believe that partial hepatectomy has the potential to increase extracellular ATP levels in the remnant parenchyma. One of the immediate consequences of partial hepatectomy is the reduction of liver mass and the consequent increase in shear stress associated with the passage of total blood flow, typically through the remaining one third of the original liver mass.6 Shear stress has been shown to induce ATP release and induction of JNK signaling in endothelial cells.55 The short half-life of extracellular ATP in vivo and the technical limitations associated with accurate in vivo measurement of ATP have been the major limitations in testing this hypothesis in animal models. However, in vivo experimental models employed in this study provide the first evidence that extracellular ATP and purinergic receptor activation are involved in liver regeneration. The portal venous perfusion of ATP and PPADS, the respective agonist and antagonist specific for P2 purineregic receptors, facilitates the analysis of the physiological significance of the in vitro findings based on the isolated hepatocytes in culture.

In summary, extracellular ATP is an important autocrine and paracrine signaling molecule influencing liver function. The present study provides the first evidence that extracellular ATP can rapidly activate multiple components of the JNK signaling cascade both in vitro and in vivo and induce hepatocyte proliferation alone and in concert with known hepatic mitogens (e.g., EGF) and that purinergic receptor blockade blunts early JNK activation in regenerating liver. These findings identify extracellular ATP as a hepatic mitogen and comitogen with implications in the regulation of liver growth and repair.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References