Department of Molecular and Integrative Physiology, The University of Michigan Medical School, Ann Arbor, Michigan 48109-0622
Submitted 1 December 2003 ; accepted in final form 22 March 2004
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ABSTRACT |
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exocrine pancreas; cholecystokinin; translation initiation factors; protein phosphatase 2B; immunosuppressants
Because of the importance of increased intracellular calcium as a signaling mechanism in pancreatic acinar cells, the potential role of calcineurin in acinar cell signaling has been studied (21). Using CsA and FK506 as inhibitors, our laboratory previously identified a novel calcineurin substrate of unknown function named calcium-regulated heat-stable protein of 24 kDa (CRHSP-24), based on its calcium regulation, heat stability, and apparent molecular weight of 24 kDa (22). Whereas high concentrations of CsA were found to inhibit amylase secretion (21, 56), it is not clear whether FK506 inhibits pancreatic exocrine secretion (13, 56) or not (unpublished observations). More recently, both FK506 and CsA were found to block pancreatic growth in response to chronic elevation of CCK induced by feeding trypsin inhibitor to mice (55), indicating a possible role for calcineurin in pancreatic growth. An obligatory requirement for cell growth in all cells is the activation of protein synthesis (41). Associated with growth is an increase in protein translation, initially of regulatory and later structural proteins (36) that could be regulated by calcineurin.
Translational control of protein synthesis in the pancreas is important in regulating growth and also in the synthesis of digestive enzymes (51). Regulation of translation is primarily directed at initiation and elongation steps and involves reversible phosphorylation of initiation [eukaryotic initiation factors (eIFs)] and elongation [eukaryotic elongation factors (eEFs)] and ribosomal proteins. The assembly of the eIF4F mRNA cap binding complex, the activity of guanine nucleotide exchange factor eIF2B, the activity of ribosomal S6 kinase (S6K), and the activity of eEF2 are some of the potential regulatory sites (Fig. 1) (45, 51). Stimulation of protein synthesis in pancreatic acinar cells is primarily mediated by the phosphatidylinositol 3-kinase (PI3K)-mammalian target of rapamycin (mTOR) pathway and involves both release of eIF4E from its binding protein and activation of the S6K (2, 3). Inhibition of acinar protein synthesis can be mediated by inhibition of eIF2B (Fig. 1) following phosphorylation of eIF2 (50).
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EXPERIMENTAL PROCEDURES |
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Preparation of pancreatic acini. Pancreatic acini were prepared by collagenase digestion of pancreas from 125- to 150-g male Sprague-Dawley rats (2). Acini were suspended in incubation buffer, consisting of an N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered Ringer solution supplemented with 11.1 mM glucose, Eagle's minimal essential amino acids, 0.1 mg/ml soybean trypsin inhibitor, and 1 mg/ml BSA and was equilibrated with 100% O2. In the assays where the immunosuppressants FK506 or CsA were used, acini were preincubated 1 h and then incubated with FK506 or CsA in the buffer at the specified concentrations.
Incorporation of amino acid into protein. To measure total net protein synthesis in acinar cells, L-[35S]methionine incorporation into protein was evaluated as described previously (3). Following 1 h preincubation in supplemented N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered Ringer solution, aliquots of isolated acini (1 ml) were incubated with agonists for 60 min at 37°C with gentle shaking. During the last 15 min of incubation, 2 µCi/ml of [35S]methionine was added to the medium. The incubation was terminated by dilution with 2 ml of 154 mmol/l NaCl at 4°C. After centrifugation at 300 g for 3 min, acinar pellets were resuspended in 0.5 ml water and sonicated. Samples were precipitated with 10% TCA at 4°C. The precipitates were washed twice with ice-cold 10% TCA and dissolved in 200 µl of 0.1 N NaOH, and radioactivity in the insoluble material was measured in Bio-Safe II scintillation medium. All samples contained an equal amount of water and NaOH to ensure equal quenching. Background samples contained NaOH to control for chemiluminescence. L-Methionine in the TCA-soluble fraction was measured by HPLC on a C18 reverse-phase column after precolumn derivatization with Waters AccQ-Fluor Reagent kit to produce a stable fluorescent derivative of L-methionine. Neither the concentration of total methionine nor its specific activity in the acinar TCA-soluble fraction was altered after stimulation with CCK or exposure to FK506 or CsA (data not shown).
Isoelectric focusing gel analysis of CRHSP-24 and eIF4E phosphorylation.
To determine the phosphorylation state of CRHSP-24 and of eIF4E, pancreatic acini were prepared and incubated as described above. After the incubation with agonists, acini were centrifuged, washed with ice-cold phosphate-buffered saline (pH 7.4), resuspended in 9 M urea buffer (containing 4% Nonidet P-40 and 1% -mercaptoethanol), sonicated, and kept at 70°C. Preparation and running of isoelectric focusing (IEF) gels, containing broad-range (pH 310) ampholytes, were carried out by using Bio-Rad model 111 mini IEF cell apparatus. IEF and Western blotting were performed as described previously (4, 22) by using anti-CRHSP-24 (1:3,000) and anti-eIF4E (1:1,500).
Formation of the eIF4F complex. To quantify the formation of the eIF4F complex, we analyzed the association of its components eIF4E and eIF4G by co-immunoprecipitation, as previously described (4). Briefly, pancreatic samples were homogenized in 2 ml of lysis buffer and centrifuged at 10,000 g for 10 min at 4°C, and the supernatant containing microsomes and soluble protein was used to analyze translation factors. The association of eIF4G and eIF4E was assessed by analyzing the amount of eIF4G bound to eIF4E immunoprecipitated by using specific anti-eIF4E antibody, following the protocol described by Kimball et al. (29). The immunoprecipitates were resolved on 420% gradient gel SDS-PAGE, followed by Western analysis with the use of anti-eIF4G antibody (1:2,000), and each band density was calculated as percentage of control. To ensure equal loading, the same membranes were stripped and reproved for the total amount of eIF4E.
Evaluation of the phosphorylation state of 4E-BP1.
The phosphorylation state of the eIF4E-binding protein (4E-BP1) was determined by protein immunoblot analysis by using an antibody that recognizes all forms of 4E-BP1. 4E-BP1 resolves into multiple electrophoretic forms during SDS-PAGE, depending on which, and how many, sites are phosphorylated (38). Unlike the more rapidly migrating forms ( and
), the slowly migrating
-form does not bind to eIF4E. For this analysis, aliquots of pancreas lysates were boiled for 10 min, cooled to room temperature, and centrifuged at 10,000 g for 30 min at 4°C. Supernatant proteins were resolved in a 15% SDS-PAGE gel, transferred to nitrocellulose, and analyzed by Western blotting by using anti-4E-BP1 (1:7,500) and ECL detection kit. The amount in the
-band was calculated as the percentage of total 4E-BP1 in all bands.
Evaluation of the phosphorylation state of ribosomal protein S6, eEF2, eEF2 kinase, eIF2, and mTOR.
The phosphorylation state of these proteins was determined by the relative amount of protein in the phosphorylated form, quantified by protein immunoblot analysis by using affinity-purified antibodies that specifically recognize the phosphorylated forms of mTOR at Ser-2448, S6 at Ser-240/244, eEF2 at Thr-56, eEF2K at Ser-366, and eIF2
at Ser-51. To ensure equal loading, the same membranes were stripped and reproved for the total amount of the proteins by using polyclonal antibodies diluted 1:1,000 for mTOR, eEF2, and eEF2K, and 1:500 for the ribosomal protein S6. For total eIF2
, a monoclonal antibody to eIF2
(1:500) was used.
Measurement of eIF2B activity. Determination of eIF2B activity in pancreatic tissue samples was performed, as described previously, by measuring the rate of exchange of [3H]GDP present in an exogenous eIF2·[3H]GDP complex for free nonradiolabeled GDP (28, 50). The guanine nucleotide exchange activity was measured as a decrease in the eIF2·[3H]GDP complex bound to nitrocellulose filters and expressed as nanomoles of GDP exchanged per minute per milligram of acinar protein or as a percentage of the control group (50).
Statistical analysis. Data are represented as means ± SE and were obtained from at least four separate experiments. Statistical analysis was carried out by one-way ANOVA and post-hoc Fisher's protected least significant differences test on the Stat View program (SAS Institute, Cary, NC). Differences with P < 0.05 were considered significant.
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RESULTS |
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Next, we analyzed the phosphorylation state of 4E-BP1, which is usually phosphorylated when protein synthesis is stimulated, releasing eIF4E that binds the 5'-cap mRNA and joins with eIF4G to form the eIF4F complex (Fig. 1) (17, 18). Stimulation of acini by CCK increased 4E-BP1 phosphorylation in a biphasic manner. CCK at 100 pM maximally stimulated the phosphorylation of 4E-BP1, expressed as the percentage of total 4E-BP1 in its -form (the more slowly migrating form) from 6.1 ± 2.9 to 21.0 ± 5.2% of total, with this effect being decreased at higher concentrations of CCK (Fig. 6A). Incubation of acini with 100 nM FK506 had no effect on basal 4E-BP1 phosphorylation but strongly inhibited CCK-stimulated 4E-BP1 phosphorylation from 20.8 ± 2.5 to 12.3 ± 2.2% of total compared with FK506 alone (Fig. 6B).
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FK506 does not affect the phosphorylation state of the initiation factor 4E.
Phosphorylation of the initiation factor eIF4E on Ser-209 often occurs when protein translation is stimulated (15, 18), and our laboratory has previously shown that CCK stimulation in vivo increases eIF4E phosphorylation (4). In the present study, we first analyzed its phosphorylation state in isolated acini, in vitro, after stimulation with different concentrations of CCK. Our results showed that 60% of the total eIF4E was phosphorylated in nonstimulated (basal) acini (Fig. 8A) (as indicated by the stronger band in the acidic pole), and this fraction increased with different increasing doses of CCK, reaching a plateau of 95% phosphorylation of the total protein from 100 pM to 10 nM CCK (Fig. 9A). Treatment with 100 nM FK506 had no effect on basal or 100 pM CCK-stimulated eIF4E phosphorylation, as indicated in the pooled data and in the representative IEF gel image (Fig. 9B). Thus calcineurin is not likely to be involved in the regulation of the phosphorylation/dephosphorylation events on eIF4E stimulated by CCK.
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DISCUSSION |
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Pancreatic protein synthesis is mainly stimulated through the activation of the PI3K/Akt/mTOR pathway (51, 57), but it can likely be modulated through other pathways. The involvement of calcineurin in protein synthesis has been reported in other tissues as a result of the side effects of the use of immunosuppressants to block organ transplant rejection (710). In addition to their effect inhibiting the production of interleukin 2 and other lymphokines by T-helper cells (10), CsA and FK506 induce a series of secondary effects in different tissues that have been the object of study. The blockade of renal, cardiac, or hepatic protein synthesis has been described (8, 9), but the activation of S6K and the induction of a hypertrophic response after chronic treatments have also been observed (35). The effects of CsA and FK506 on protein synthesis appear to be organ and immunosuppressant dependent. Thus CsA inhibits translation in microsomes from rat kidney and heart, stimulates it in microsomes from rat liver (8, 9), and has no effect on vascular smooth muscle protein synthesis (49).
In this study, using pancreatic acinar cells in suspension, we established that the immunosuppressants FK506 and CsA inhibited calcineurin activity and the concentration at which this occurred. For that, we used CRHSP-24, the only known substrate for calcineurin in pancreatic acinar cells (21, 22), as an intracellular indicator. CRHSP-24 function is unknown, and preliminary experiments have shown no effect on basal or serum-stimulated protein synthesis when transfected into HEK293 cells (S.-H. Lee and J. A. Williams, unpublished observations). Our results showed that both FK506 and CsA inhibited CRHSP-24 dephosphorylation in a dose-dependent manner, with FK506 being at least 10-fold more potent.
Both FK506 and CsA inhibited protein synthesis at concentrations that inhibited calcineurin, but there were several major differences in their inhibitory profile. FK506 significantly inhibited CCK-stimulated protein synthesis while having only minimal effects on basal protein synthesis. This suggests a role for calcineurin in the stimulatory mechanism rather than the basal translational machinery. CsA at 100 nM partially blocked CCK-stimulated protein synthesis with minimal (but significant) effect on basal synthesis, consistent with partial inhibition of calcineurin at this concentration. At higher concentrations, 10 nM to 1 µM, CsA significantly inhibited basal protein synthesis and blocked CCK stimulation. This suggests the possibility of an action of CsA distinct from that of FK506; effects of CsA on LDH release, K+ channels, and the mitochondrial permeability transition pore have been reported (1, 20, 59). Thus FK506 appears to be a more specific tool to selectively inhibit calcineurin in acinar cells.
We confirmed that FK506 was not inhibiting protein synthesis through blockade of the CCK receptor by using other pancreatic secretagogues, known to stimulate secretion and protein synthesis in acini in vitro through distinct membrane receptors (16, 51). Both CCh, which acts via m3 muscarinic receptors, and BBS, which acts through a neuromedin C receptor, generally activate similar intracellular mechanisms, as does CCK in pancreatic acinar cells, increasing cytoplasmic Ca2+ and diacylglycerol (16, 58). Because their stimulation of protein synthesis was blocked by FK506, similar to the blockage of CCK, FK506 then seems to block distinct intracellular events related to calcineurin activation rather than initial steps in transmembrane signaling by the CCK receptor. This is consistent with an earlier report, where it was concluded that FK506 did not inhibit the CCK receptor in rat pancreas (13). Moreover, the fact that FK506 does not block signaling pathway intermediates, such as mTOR, S6K, and the eIF4E kinase, indicates that much of the intracellular signaling pathways are unaffected.
After establishing that acinar protein synthesis was dependent on calcineurin activity, we analyzed which key regulatory translation factors were affected by calcineurin. Surprisingly, only a few studies have evaluated the regulation of the translational machinery by calcineurin (9, 37), despite the fact that calcineurin has been involved in several processes of cell growth (19, 36) and activation of protein synthesis is required for growth. It has been demonstrated that the PI3K/Akt/mTOR pathway is a major stimulatory pathway for protein synthesis, and that it stimulates the phosphorylation and activation of the ribosomal protein S6K (46), the phosphorylation of the eIF4E binding protein 4E-BP1 (17), and the formation of the eIF4F complex (Fig. 1) (2, 3, 47). Our results indicated no effect of FK506 on the mTOR phosphorylation status (downstream of Akt) or on the ribosomal protein S6 phosphorylation (Fig. 7). This is different from the rabbit heart, in which chronic FK506 treatment increases S6K activity, (35), but similar to other cell types, in which no effect on S6K was seen (40, 44). The lack of effect of FK506 on acinar mTOR and ribosomal protein S6 phosphorylation indicates that this pathway is independent of calcineurin action in pancreatic acinar cells.
On the other hand, the phosphorylation of 4E-BP1, which is also downstream of mTOR (Fig. 1), was inhibited by FK506. This result differs from studies in T cells in which this immunosuppressant was reported not to have any effect on 4E-BP1 (37). The role of calcineurin in regulating the phosphorylation of 4E-BP1 in acinar cells is not obvious. It has been described that mTOR may stimulate phosphorylation of 4E-BP1 indirectly by inactivating some phosphatases (PP2A, PP4, PP6) that would lead to the phosphorylation of 4E-BP1 (47), but it is not known how PP2A or these other phosphatases are regulated. It has also been described that 4E-BP1 phosphorylation can be dependent on calcium and calmodulin activation, independently of the PI3K/Akt/mTOR pathway activation (47), which could account for calcineurin activation in this process. Alternatively, FK506 could inhibit protein synthesis and translation effectors through an increase of intracellular Ca2+. FK506 binding protein and calcineurin interact under physiological conditions to modulate Ca2+ flux in Ca2+ release channels [the ryanodine and inositol 1,4,5-triphosphate receptors (25)]. FK506, by inhibiting calcineurin and preventing receptor dephosphorylation, would increase Ca2+ transport through these channels by blocking Ca2+ oscillations and progressively increase intracellular Ca2+ concentration to a high-plateau level (43). However, the lack of a strong effect on amylase secretion makes this mechanism unlikely.
Further downstream in the mTOR pathway, the reduction in the formation of the eIF4F complex (Fig. 8B) can be readily explained as a consequence of the FK506 effect on 4E-BP1 phosphorylation. Thus calcineurin can be involved in the stimulation of protein synthesis through increasing the phosphorylation of the 4E-BP1 and the formation of eIF4F complex by a yet-unknown mechanism. The lack of effect of FK506 on eIF4E phosphorylation indicates that the phosphorylation status of this initiation factor is not regulated by calcineurin and does not affect the formation of the eIF4F complex.
Because modulation of eIF2B activity is known to be one of the most important regulatory points in translation initiation (27), and intracellular calcium levels are important in regulating its activity through the phosphorylation of eIF2 in pancreatic acini (Fig. 1) (50), we also studied the possible involvement of calcineurin in the regulation of these parameters. FK506 in acini appeared to inhibit the basal activity of the guanine nucleotide exchange factor eIF2B (Fig. 10A), but this modification had only small effects on basal protein synthesis. Also, at the same time, FK506 increased eIF2
phosphorylation in nonstimulated acini (Fig. 10B). This differs from results in PC12 and Neuro2A cells, where FK506 at the same dose had no effect on the phosphorylation status of the eIF2
(31). Because it is known that the proportion of eIF2 and eIF2B in pancreatic acinar cells is 3:1 (50), and because eIF2
(P) inhibits eIF2B activity on an approximately equimolar basis (27), a small increase in eIF2
phosphorylation would be sufficient to reduce eIF2B activity. Thus the increase on basal eIF2
phosphorylation after blockade of calcineurin by FK506 can explain the decrease in basal eIF2B activity. However, this was without effect on basal protein synthesis (Fig. 3A).
An efficient increase in translation requires an increase in translation elongation, which is primarily regulated by changes in eEF2 phosphorylation (5, 45). eEF2 is phosphorylated and inactivated by the Ca2+/calmodulin-dependent protein kinase 3 or eEF2K (Fig. 1) (5, 45). It has been described that phosphorylated eEF2 is a substrate for PP2A (45), but whether calcineurin (PP2B) has an effect on eEF2 phosphorylation has not been fully addressed. The blockade of calcineurin with FK506 significantly reversed CCK-induced eEF2 dephosphorylation in acini, although the reversion was not complete. Because FK506 did not affect the phosphorylation levels of the eEF2K, it is not likely to have any effect on the kinase activity. Similar results were obtained by Buss and coworkers (9) on eEF2 and kidney protein synthesis by blocking calcineurin with CsA administration to rats. Although Buss et al. concluded that "phosphorylated eEF2 is not a substrate for PP2B," our results show that calcineurin (PP2B) is involved in eEF2 dephosphorylation when protein synthesis is activated in pancreatic acinar cells. Whether calcineurin directly regulates eEF2 phosphorylation or is required for the activation of PP2A remains to be determined.
In summary, we conclude that calcineurin is necessary for secretagogue stimulation of pancreatic acinar cell protein synthesis, with its major effect at the level of the eIF4F complex formation and at the elongation step. It will be important to determine whether calcineurin is also required for stimulation of translation by other hormones, such as insulin, and by amino acids and whether this requirement for stimulated protein synthesis is related to the calcineurin dependence of the pancreatic growth response.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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