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Endocrinology, doi:10.1210/en.2007-0548
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Endocrinology Vol. 149, No. 1 131-138
Copyright © 2008 by The Endocrine Society

Oxytocin-Induced Activation of Eukaryotic Elongation Factor 2 in Myometrial Cells Is Mediated by Protein Kinase C

Dominic Devost, Marie-Eve Carrier and Hans H. Zingg

Departments of Pharmacology and Therapeutics (D.D., M.-E.C., H.H.Z.), of Medicine (H.H.Z.), and of Obstetrics and Gynecology (H.H.Z.), McGill University, Montreal, Québec, Canada H3G 1Y6

Address all correspondence and requests for reprints to: Hans H. Zingg, M.D., Ph.D., Department of Pharmacology & Therapeutics, McGill University, 3655 Promenade Sir-William-Osler, Montreal, Quebec, Canada H3G 1Y6. E-mail: hans.zingg{at}mcgill.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The nonapeptide oxytocin (OT) mediates a wide spectrum of biological action, many of them related to reproduction. Recently, we have shown that OT exerts a trophic effect on uterine smooth muscle cells and induces dephosphorylation, and thus activation, of the translation elongation factor eukaryotic elongation factor 2 (eEF2). The present study was designed to elucidate the mechanisms underlying this novel action of OT in the well-characterized human myometrial cell line hTERT-C3. Pathways known to induce eEF2 dephosphorylation are mammalian target of rapamycin (mTOR), and the MAPKs ERK1/2 and p38. Using a panel of chemical inhibitors of specific signaling pathways, we determined that none of these pathways played a role in OT-mediated eEF2 dephosphorylation. Because the OT receptor is a G protein-coupled receptor linked to G{alpha}q, we tested the possibility that this OT action was mediated via protein kinase C (PKC). PKC activity was blocked by application of the general PKC chemical inhibitor Go6983 or by incubation with the cell-permeable PKC inhibitor peptide myr-psi PKC. With either approach, the effect of OT on eEF2 dephosphorylation was suppressed, indicating that the PKC pathway is essential for this OT action. Consistent with this idea, we also found that direct stimulation of PKC with the phorbol ester phorbol 12-myristate 13-acetate induced eEF2 dephosphorylation. Moreover, we observed that the stimulatory effect of OT on [35S]methionine incorporation into nascent proteins was blocked by PKC inhibition. Overall, these results define a novel hormonal signaling pathway that leads to eEF2 dephosphorylation and activation of protein synthesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
AMONG THE MANIFOLD actions of the nonapeptide oxytocin (OT), its effects on behavior, uterine contractions, and prostaglandin biosynthesis have been most intensively studied (1, 2). In addition, cell-specific mitogenic effects have been described (3, 4, 5). Specifically, OT has exerted a growth-promoting effect on uterine smooth muscle cells, and it has been suggested that OT thus contributes to the process of hyperplasia that occurs in the uterus throughout pregnancy (5). Recently, we have discovered through a phosphoproteomics approach that OT induces dephosphorylation of eukaryotic elongation factor 2 (eEF2) and directly activates protein synthesis in human myometrial cells (6). eEF2 is a key regulator of cellular protein synthesis (7, 8). Upon dephosphorylation, it activates the ribosomal translocation step and increases peptide chain elongation rates. The phosphatase involved in eEF2 dephosphorylation is most likely PP2A (7, 8). On the other hand, eEF2 is phosphorylated, and thus inactivated, by eEF2 kinase, formerly known as calmodulin kinase III.

Several hormonally mediated pathways are known to be involved in modulating eEF2 dephosphorylation, either by blocking eEF2 kinase or by stimulating PP2A phosphatase. For example, the trophic effects of the hormones IGF-I and insulin involve eEF2 dephosphorylation, and this effect is mediated via the serine/threonine protein kinase "mammalian target of rapamycin" (mTOR) (9). This pathway is specifically inhibited by the bacterial product rapamycin (7, 10, 11). eEF2 dephosphorylation can also be induced by activation of the MAPKs ERK1 and ERK2 (12, 13). This pathway mediates the trophic effects of endothelin, angiotensin II, and {alpha}1 adrenergic receptor agonists. A third hormonal pathway that has been shown to elicit eEF2 dephosphorylation involves stress-activated protein kinase 2 (or p38MAPK{alpha}/β) (8, 14). By contrast, elevations of the levels of intracellular AMP or calcium ions stimulate eEF2 kinase, leading to phosphorylation-induced inhibition of eEF2 activity (7, 15).

In an attempt to delineate the pathway(s) by which OT exerts its effect on eEF2 dephosphorylation, we systematically explored each of the aforementioned pathways known to stimulate eEF2 activity. Surprisingly, we found that the pathway involved in OT-induced eEF2 dephosphorylation corresponds to none of the previously characterized pathways.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
DMEM/F12 tissue culture medium was purchased from Life Technologies, Inc. (Grand Island, NY), and fetal bovine serum (FBS) from Hyclone (Logan, UT). Insulin and OT were purchased from Sigma-Aldrich (St. Louis, MO) and phorbol 12-myristate 13-acetate (PMA) from Cederlane (Burlington, Ontario, Canada). The inactive PMA variant 4{alpha}-PMA was obtained from Sigma-Aldrich. The specific OT antagonist (OTA) (d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH29]OVT) (16) was obtained from Bachem (King of Prussia, PA). The inhibitors SB203580, rapamycin, Go6983, and the protein kinase C (PKC) inhibitory peptide myr-{psi} PKC were all purchased from Calbiochem (La Jolla, CA). UO126 was purchased from Promega (Madison, WI). All antibodies were purchased from Cell Signaling Technology (Beverly, MA), except the pan anti-ERK1/2 antibody, which was obtained from Stressgen (Ann Arbor, MI). All other analytical grade chemicals were obtained from Sigma-Aldrich, Fisher Scientific (Waltham, MA), and VWR (West Chester, PA).

Tissue culture
Myometrial hTERT-HM cells were obtained from W. E. Rainey (Medical College of Georgia, Augusta, GA) (17). hTERT-HM cells represent a cell population of human myometrial cells that were immortalized by stable transfection with a human telomerase reverse transcriptase (hTERT) expression vector. hTERT-C3 cells represent a selected subclone that we obtained by serial clonal dilution of hTERT-HM cells (18). hTERT-C3 cells were maintained in DMEM/F12 medium supplemented with 10% FBS (Hyclone) and cultured at 37 C in 5% CO2. Cells close to confluency were passaged by trypsinization and plated in T175 flask at a one-quarter dilution every 4–5 d.

Myometrial M11 cells were obtained from John A. Copland (Mayo Clinic College of Medicine, Jacksonville, FL) (18). These cells were derived from dispersed primary human myometrial cells by repeated passage without the use of any immortalizing or transforming agent. M11 cells were maintained in DMEM high glucose (Invitrogen, Carlsbad, CA), supplemented with 10% FBS.

Immunoblotting
Immunoblotting was performed as described previously (6). Briefly, hTERT-C3 cells were plated onto culture dishes and grown in DMEM/F12 medium supplemented with 10% FBS to near confluency. Cells were starved for 18 h in DMEM/F12 supplemented with only 0.5% FBS. Cells were stimulated by 1 nM OT for 5 min at 37 C. Stimulation was stopped by two ice-cold PBS washes, and plates were flash frozen in liquid nitrogen. For inhibitor studies, cells were preincubated with the different inhibitors for 30 min before OT stimulation, except for the PKC inhibitory peptide myr-{psi} PKC, which was left for 3 h in the medium. Cells were lysed on ice with 250 µl lysate buffer [25 mM HEPES/NaOH (pH 7.4), 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 10% glycerol, and 1% Triton X-100], and a mix of protease inhibitors (1 µg/ml each aprotinin, pepstatin A, and leupeptin and 0.5 mM phenylmethylsulfonylfluoride) and phosphatase inhibitors (50 mM NaF, 30 mM sodium pyrophosphate, and 1 mM sodium orthovanadate). Lysates were clarified by centrifugation at 15, 000 x g for 10 min at 4 C in a microcentrifuge. Total protein concentration was determined by a colorimetric bicinchoninic acid assay using BSA as a standard (BCA Protein Assay Reagent Kit; Pierce, Rockford, IL). Proteins were denatured by boiling in Laemmli buffer [50 mM Tris-HCl (pH 6.8), 2% sodium dodecyl sulfate, 10% glycerol, and 0.1 M β-mercaptoethanol] for 5 min, and subjected to SDS-PAGE and Western blotting. Immunodetection involved different primary antibodies in conjunction with a second horseradish peroxidase C-conjugated antibody and a chemiluminescence detection system (Supersignal; Pierce). Quantization of band intensities was performed using ImageQuant 5.1 (Amersham Biosciences Inc., Piscataway, NJ).

[35S]Methionine incorporation into nascent proteins
The level of cellular protein synthesis was assessed as previously described (6). In brief, hTERT-C3 cells were seeded in 60-mm plates. Once confluent, cells were first incubated with DMEM low-glucose medium (Specialty Media, Phillipsburg, NJ) containing 0.5% serum for 12 h, and then for 2 h in L-methionine-free DMEM labeling media (Specialty Media) without serum. Next, 1 µCi [35S]methionine (PerkinElmer, Boston, MA) was directly added to the medium, and incubation was continued for an additional 1 h in the absence or presence of 100 nM OT. At the end of the incubation, cells were washed twice with PBS, flash frozen in liquid nitrogen, and kept at –80 C until use. Cells were lysed in 250 µl lysate buffer, and protein concentration was determined by a protein quantization assay, as described previously. To denature proteins, 50 µl protein lysate was mixed with a same volume of 1 M NaOH/2% H2O2 and incubated for 10 min at 37 C. Proteins were precipitated on ice for 30 min after addition of 1 ml 25% trichloroacetic acid/2% Casamino acids. The precipitates were recovered by filtration through G4 fiberglass filters (Fisher Scientific Co., Pittsburgh, PA). Filters were washed three times with 1 ml ice-cold 5% trichloroacetic acid and once with 3 ml acetone, dried at room temperature, and radioactivity was measured by scintillation counting. The [35S]L-methionine incorporation into protein was evaluated in duplicate for each sample.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
OT-induced dephosphorylation in hTERT-C3 cells
The present studies were undertaken using the human myometrial cell line hTERT-C3 (18). This cell line represents a clonal derivative of the nonclonal myometrial cell line hTERT HM that was rendered immortal by stable transfection with an expression vector containing the hTERT gene (17). hTERT-C3 cells show many features of differentiated myometrial cells, such as the presence of OT receptors (OTRs) at the cell surface and the capacity to contract in response to OT application (18). In these cells, OT induced a dose-dependent dephosphorylation of eEF2 (Fig. 1AGo). These results were comparable to the ones obtained earlier in M11 cells, a cell line derived from primary human myometrial cells by continued passages (6). A significant effect was already observed at 1 nM, a concentration close to the Kd of the OTR. A maximal effect was observed at 10 nM, but at 100 nM, eEF2 phosphorylation tended to increase again. Because it had been shown, in myometrial cells, that increasing the concentration of applied OT from the low to the high nanomolar range leads to a several-fold increase in intracellular calcium (19), high nanomolar concentrations of OT may lead to increased eEF2 phosphorylation via activation of the calcium/calmodulin-dependent eEF2 kinase (7, 8). The effect was observable after 5 min and lasted for at least 2 h (Fig. 1BGo). The effect was specific and mediated by the cognate OTR because the effect was abrogated by addition of a 100-fold excess of the specific OTR antagonist OTA (16) (Fig. 1CGo).


Figure 1
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  		FIG. 1. A, Dose-response curve of OT-induced dephosphorylation of eEF2 (thin line) and OT-induced protein synthesis (thick line). For measurement of phospho-eEF2 levels, myometrial hTERT-C3 cells were treated with buffer (control) or OT at the indicated concentrations for 5 min at 37 C. Cells were lysed, and levels of phosphorylated eEF2 (peEF2) were assessed by immunoblotting with a phospho-eEF2 antibody. Blots were stripped and reprobed with a pan eEF2 antibody. Autoradiograms from eight independent experiments were analyzed densitometrically. Values for phosphorylated eEF2 were normalized with respect to total eEF2 levels and plotted as means ± SEM as a percentage of control (lower panel; thin line; n = 8). A representative blot is shown in the top panel. For the determination of OT-induced protein synthesis, cells were incubated in presence of 1 µCi [35S]methionine for 1 h in the absence (control) or presence of OT at the concentrations indicated. Level of radiolabeled methionine incorporation into protein was assessed as described in Materials and Methods. Data are expressed as counts per min per µg protein as a percentage of control (thick line). Each point represents the mean ± SEM of three independent experiments done in duplicates or triplicates. B, Time course of OT-induced eEF2 dephosphorylation. Phosphorylation was assessed as in A, but cells were treated with a fixed concentration of OT (1 nM) for different times as indicated (lower panel; n = 3). C, Specificity of OT effect. Cells were treated for 5 min with 1 nM OT after pretreatment with 100 nM of the OTR antagonist OTA (OT + OTA), with 1 nM OT without pretreatment (OT) or with buffer alone (control) (lower panel; n = 4). A representative blot is shown in the top panel. *, P < 0.05 vs. control. **, P < 0.01 vs. control.

 
As previously demonstrated for M11 cells (6), OT-induced eEF2 dephosphorylation was accompanied by a concomitant increase in total protein synthesis, as assessed by the incorporation of [35S]methionine into nascent proteins (Fig 1AGo).

Effect of inhibiting established pathways leading to eEF2 dephosphorylation
In an attempt to delineate the pathway(s) by which OT exerts its effect on eEF2 dephosphorylation, we systematically explored each of the pathways known to stimulate eEF2 activity, including: 1) the serine/threonine protein kinase, mTOR, a pathway that is specifically inhibited by the bacterial natural product rapamycin (9); 2) the MAPKs ERK1 and ERK2, a pathway mediating the trophic effects of several G protein-coupled receptor (GPCR) agonists (12, 13); and 3) the p38MAPK pathway (8, 14).

The mTOR pathway.
First, we analyzed the role of the mTOR pathway by using the selective mTOR inhibitor rapamycin. As a positive control, we assessed the established effect of rapamycin to block insulin-induced eEF2 dephosphorylation (10, 11). As shown in Fig. 2Go, insulin was able to induce eEF2 dephosphorylation in hTERT-C3 cells, and pretreatment with rapamycin abrogated this effect. However, the same rapamycin pretreatment was ineffective in blocking the action of OT on eEF2 dephosphorylation. These data indicate that, although both insulin and OT are able to induce eEF2 dephosphorylation in myometrial cells, they do so by different pathways. The finding that the action of OT is rapamycin-insensitive indicates that the mTOR pathway is not involved in the OT-induced eEF2 dephosphorylation in hTERT-C3 cells.


Figure 2
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  		FIG. 2. OT-induced eEF2 dephosphorylation is rapamycin insensitive. hTERT-C3 cells were pretreated with 100 nM rapamycin or vehicle DMSO (control) for 30 min and then treated with control buffer (c), 1 nM OT, or 100 µg/ml insulin for 5 min at 37 C. Cells were lysed, and phospho-eEF2 levels were assessed by immunodetection and expressed as a percentage of the corresponding control (c) as in Fig. 1Go (n = 3) (lower panel). A representative blot is shown in the top panel. *, P < 0.05 vs. the corresponding control (c). peEF2, Phosphorylated eEF2.

 
MAPKs ERK1/2.
A number of GPCRs has activated the MAPKs ERK1/2, and there is evidence that ERK1/2 activation can lead to eEF2 dephosphorylation (12). Consistent with previous reports (4, 18, 20), we found that OT rapidly induced ERK1/2 phosphorylation, and this effect was blocked by pretreatment of hTERT-C3 cells with 1 µM of the specific MAPK kinase-1 inhibitor U0126 (Fig. 3AGo). Although ERK1/2 activation was efficiently inhibited by application of this MAPK kinase-1 inhibitor, the action of OT on eEF2 dephosphorylation remained unaffected (Fig 3BGo). Thus, we conclude that activation of ERK1/2 is not a prerequisite for OT-induced eEF2 dephosphorylation.


Figure 3
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  		FIG. 3. OT-induced eEF2 dephosphorylation is insensitive to blockage of the MAPK ERK1/2 pathway. A, hTERT-C3 cells were pretreated with 1 µM U0126 or its vehicle DMSO (control) for 30 min and then treated with control buffer (C) or 1 nM OT for 5 min at 37 C. Cells were lysed, and levels of phosphorylated ERK1/2 (p-ERK1/2) (A) or phospho-eEF2 (B) were assessed by immunodetection. Levels were normalized with respect to total levels of ERK1/2 (A) or eEF2 (B) and expressed as a percentage of control (C) (leftmost bar; n = 3) (lower panel). Representative blots are shown in the top panels of A and B. *, P < 0.05 vs. the DMSO pretreated, nonstimulated condition (C). peEF2, Phosphorylated eEF2.

 
The p38MAPK pathway.
The p38MAPK pathway has been implicated in eEF2 activation (8, 14, 21). Hoare et al. (22) have shown that OT is able to activate the p38MAPK pathway in OTR-expressing CHO cells, but the downstream effects of this activation have not been investigated. In the present study, we have used an antibody specific for threonine 180 and tyrosine 182-phosphorylated p38. Phosphorylation on both these residues is important for p38 activation. Using this antibody, we found that OT treatment of hTERT-C3 cells led to a more than 2-fold increase in the intensity of the p38 band, indicating that OT induced p38 phosphorylation on both these residues in our hTERT-C3 cells (Fig. 4AGo). To determine to what extent p38 activation was involved in eEF2 dephosphorylation, we used the selective p38MAPK{alpha}/β-kinase inhibitor SB203580. This inhibitor has repressed p38 kinase autoactivation induced by TGFβ (23). When applied to our model, we found that it blocked efficiently OT-induced p38 activation as well (Fig. 4AGo). However, blockage of this pathway had no effect on the ability of OT to induce eEF2 dephosphorylation (Fig. 4BGo). These data indicate that the p38 pathway is not involved in OT-mediated eEF2 dephosphorylation.


Figure 4
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  		FIG. 4. OT-induced eEF2 dephosphorylation is independent of the p38MAPK pathway. hTERT-C3 cells were pretreated with 50 nM SB203580 or its vehicle DMSO (control) for 30 min and then treated with control buffer (C) or 1 nM OT for 5 min at 37 C. Cells were lysed, and levels of phosphorylated p38 (A) or phosphorylated eEF2 (peEF2) (B) were assessed by immunoblotting. Levels were normalized with respect to total levels of p38 (A) or eEF2 (B) and expressed as a percentage of the corresponding control value (C) (n = 3) (lower panel). Representative blots are shown in the top panels of A and B. *, P < 0.05 vs. the corresponding control (C).

 
PKC involvement in eEF2 activation
Effect of PKC inhibition.
The results obtained so far indicated that OT-induced eEF2 dephosphorylation was not mediated by any of the classical pathways tested previously. Due to its coupling to G{alpha}q/11, the OTR is able to activate PKC (1). Therefore, we tested the hypothesis that the process might be mediated by PKC, although a link between PKC and eEF2 dephosphorylation has never been formally demonstrated. We first applied the general PKC inhibitor Go6983. As shown in Fig. 5AGo, in cells pretreated with 1 µM Go6983, the effect of OT on eEF2 dephosphorylation was abolished, suggesting that the PKC pathway was indeed involved in this process. This effect was not specific for the clonal hTERT-C3 cell line and was also observed in the nonclonal M11 cell line. To confirm the involvement of PKC, we applied an alternative strategy to block PKC action. To this end, we pretreated cells with the peptide "myr-{psi} PKC," an N-myristoylated peptide corresponding to the pseudosubstrate domain of PKC with specificity for the PKC{alpha} and PKCβ isotypes (24). As demonstrated in Fig. 5BGo, treatment with myr-{psi} PKC completely blocked the effect of OT on eEF2 dephosphorylation.


Figure 5
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  		FIG. 5. OT-induced eEF2 dephosphorylation is repressed by inhibition of the PKC pathway. A, M11 or hTERT-C3 cells were pretreated with 1 µM of the general PKC inhibitor Go6983 or its vehicle DMSO (control) for 30 min and then treated with control buffer (C) or 1 nM OT for 5 min at 37 C. Cells were lysed, and phospho-eEF2 levels were assessed by immunodetection. Values for phosphorylated eEF2 (p-eEF2) in hTERT-C3 cells were normalized with respect to total eEF2 levels and plotted as means ± SEM as a percentage of control (C) (leftmost bar; n = 3). B, as in A, but instead of Go6983, cells were pretreated with 20 µM"myr-{psi} PKC," an N-myristoylated pseudosubstrate of PKC or its vehicle (control) for 3 h. Data are expressed as a percentage of the corresponding control value (C) (n = 3; lower panel). Representative blots are shown in the top panels of A and B. *, P < 0.05 vs. the corresponding control (C).

 
Effect of PKC activation.
To confirm further the involvement of PKC, we tested whether phorbol ester mediated activation of PKC was able to induce eEF2 dephosphorylation in hTERT-C3 myometrial cells. As shown in Fig. 6Go, this was indeed the case. Application of the phorbol ester PMA, but not of the inactive analog 4{alpha}-PMA, significantly decreased eEF2 phosphorylation.


Figure 6
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  		FIG. 6. Effect of the phorbol ester PMA on eEF2 dephosphorylation. hTERT-C3 cells were treated with vehicle DMSO (control), 100 nM PMA, or the inactive phorbol ester 4{alpha}-PMA for 5 min at 37 C. Cells were lysed, and phospho-eEF2 levels were assessed by immunodetection. Values for phosphorylated eEF2 (p-eEF2) were normalized with respect to total eEF2 levels and plotted as means ± SEM as a percentage of control (n ≥ 4) (lower panel). A representative blot is shown in the top panel. *, P < 0.05 vs. control.

 
Effect of blocking PKC on OT-induced protein synthesis
Because protein chain elongation contributes significantly to total protein synthesis, OT-induced eEF2 dephosphorylation and activation in hTERT-C3 cells should lead to a concomitant increase in protein synthesis. We previously reported that protein synthesis is stimulated by OT in the human myometrial cell line M11 (6). To assess the physiological significance of our present findings, we determined whether protein synthesis was similarly stimulated by OT in hTERT-C3 cells and whether this effect was abrogated by inhibition of PKC. As depicted in Fig. 7Go, we observed that OT led to a 38% increase in [35S]methionine incorporation into nascent proteins in hTERT-C3 cells. This effect was abrogated by preincubation of cells with PKC inhibitor Go6983, indicating that PKC activation represents an obligatory link connecting OTR activation to increased protein synthesis.


Figure 7
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  		FIG. 7. Effect of blockage of the PKC pathway on OT-induced protein synthesis. hTERT-C3 cells were incubated in the presence of 1 µCi [35S]methionine for 1 h in the absence (C) or presence (OT) of 100 nM OT after being pretreated with 1 µM Go6983 or its vehicle DMSO (control). Level of radiolabeled methionine incorporation into protein was assessed as described in Materials and Methods. Data are expressed as counts per minute per microgram of protein as a percentage of control (C) (leftmost bar). Each bar represents the mean ± SEM of four independent experiments done in duplicates or triplicates. *, P < 0.05 vs. the DMSO pretreated, nonstimulated condition (C).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We had previously demonstrated that the hormone OT stimulates protein synthesis in myometrial smooth muscle cells (6). Concomitantly, we had shown that OT induces dephosphorylation of eEF2. However, the causal relations between these two events and the possible signaling mechanisms linking them have remained unclear. We now show that, in contrast to other hormonal systems analyzed previously, both the trophic effect of OT and its ability to induce dephosphorylation of eEF2 depend on the activation of PKC (Fig. 8Go). This connection is novel and has not been described for any other hormonal system.


Figure 8
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  		FIG. 8. OT mediates the dephosphorylation of eEF2 through a novel pathway involving PKC. Starting from the left are represented the main three signal transduction pathways, namely the mTOR, and the MAPKs ERK1/2 and p38, that have been reported to mediate hormonally induced eEF2 dephosphorylation (8 ). eEF2 is actively dephosphorylated either by inhibition of eEF2 kinase or activation of an eEF2 phosphatase, likely PP2A (not shown). As shown here, activation of the OTR by the hormone OT leads to the dephosphorylation of eEF2 through a PKC-dependent pathway. Sites of action of the inhibitors rapamycin, U0126, SB203580, Go6983, and myr-{psi} PKC are also shown.

 
Several hormones acting on cardiomyocyte contraction have a trophic effect and can potentially mediate cardiac hypertrophy. Angiotensin II-induced increase in cardiac protein synthesis has involved eEF2 dephosphorylation via a mechanism that encompasses activation of the MAPKs ERK1/2, but not mTOR (12). On the other hand, phenylephrine and endothelin-1 exert eEF2-mediated trophic effects on cardiomyocytes via ERK1/2 as well as mTOR (13). In rat pancreatic acini, cholecystokinin has had an eEF2-mediated trophic effect mediated by mTOR, ERK1/2, as well as MAPKp38 (25). Finally, in Aplysia neurons, serotonin induces eEF2 dephosphorylation as part of a process that may be related to memory consolidation (26). This effect is specifically blocked by rapamycin and, thus, involves mTOR. It is clear from the aforementioned results that hormonally induced stimulation of protein synthesis appears to evolve along several distinct pathways in a receptor- and cell-specific fashion. The present report now adds PKC as an additional pathway.

The precise PKC subtype(s) involved remain to be determined. PKC subtypes are divided into three subfamilies: classical (isoforms {alpha}, βI, βII, and {gamma}); novel ({delta}, {epsilon}, {eta}, and {theta}); and atypical ({zeta} and {iota}/{lambda}), based on their second messenger requirements (27). Whereas Go6983, our first PKC inhibitor used, is a general PKC inhibitor, our second strategy involved the peptide myr-{psi} PKC, an N-myristoylated pseudosubstrate of PKC with specificity for the "classical" PKC{alpha} and PKCβ isotypes (24). This suggests that the PKC involved belongs to the classical rather than the novel or atypical subfamily.

The pathways by which PKC activates eEF2 remain to be specified as well. The activity of eEF2 is inhibited by phosphorylation at threonine residue 56, and dephosphorylation at this residue leads to its activation (7, 8). In turn, eEF2 dephosphorylation can be induced either by inhibiting eEF2 kinase or by activating the phosphatase(s) targeting eEF2.

eEF2 kinase is activated via phosphorylation on several specific target residues, but there is no evidence so far that any of them is a PKC target. We performed a sequence analysis of the human eEF2 kinase for putative PKC phosphorylation sites using the protein analysis tool "PROSITE" (available through the ExPASy proteomics server at http://ca.expasy.org/prosite). This analysis yielded a total of 13 possible PKC phosphorylation motifs. One of them, Ser359, is known to be the target of the mTOR and MAPKp38 pathways (7). This site could represent a central regulatory switch for the control of the eEF2 kinase activity by mTOR, MAPK p38, as well as PKC.

An alternative pathway for eEF2 stimulation is via activation of an eEF2 phosphatase, most likely PP2A (28, 29). Evidence for the physiological relevance of this pathway stems from the fact that transgenic mice overexpressing PP2A exhibit increased eEF2 dephosphorylation combined with cardiac hypertrophy (30). Moreover, Everett et al. (12) showed that inhibitors of PP2A, but not of PP2B, attenuated the effect of angiotensin II on eEF2 dephosphorylation. We have obtained preliminary evidence that PP2A is rapidly activated by OT in hTERT-C3 cells (our unpublished results). Moreover, there is published evidence that PKC activation leads to increased PP2A activity (31, 32). In view of our preliminary findings, data in the literature, and in view of the fast onset of OT action (5 min), it appears likely that the OT action on eEF2 dephosphorylation involves a PKC-mediated activation of PP2A. Experiments to test this hypothesis are in progress.

It is clear from the aforementioned results that trophic effects of hormones are initiated via several distinct pathways that are cell and receptor specific. It is also clear that, in myometrial cells, OT is not the only factor mediating a trophic effect during pregnancy. Thus, mice deficient in either OT or the OTR undergo normal labor (33, 34, 35, 36). Other important trophic factors include the estrogens (37) and the IGF system (38). We have also recently demonstrated concomitant activation of a set of angiogenesis-related genes in the myometrium at the end of gestation (39). With the present study, a potentially important additional pathway involving PKC has been defined on the example of OT and its trophic actions on myometrial smooth muscle cells. It remains to be determined to what extent the pathway defined in the present study for the myometrial cell may apply to other sites of OT action and to other hormonal systems.


   Footnotes
 
This work was supported by a grant from the Canadian Institutes for Health Research.

Disclosure Statement: The authors have nothing to disclose.

First Published Online October 18, 2007

Abbreviations: eEF2, Eukaryotic elongation factor 2; FBS, fetal bovine serum; GPCR, G protein-coupled receptor; hTERT, human telomerase reverse transcriptase; mTOR, mammalian target of rapamycin; OT, oxytocin; OTA, oxytocin antagonist; OTR, oxytocin receptor; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate.

Received April 27, 2007.

Accepted for publication October 9, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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