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Endocrinology, doi:10.1210/en.2002-221039
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Endocrinology Vol. 144, No. 7 2947-2956
Copyright © 2003 by The Endocrine Society

Extracellular Signal-Regulated Kinase 1/2 Activation by Myometrial Oxytocin Receptor Involves G{alpha}qGß{gamma} and Epidermal Growth Factor Receptor Tyrosine Kinase Activation

Miao Zhong, Ming Yang and Barbara M. Sanborn

Department of Biochemistry and Molecular Biology, University of Texas Medical School Houston, Houston, Texas 77030

Address all correspondence and requests for reprints to: Barbara M. Sanborn, Ph.D., Department of Biomedical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado 80523. E-mail: Barbara.Sanborn{at}colostate.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The mechanisms by which oxytocin (OT) stimulates extracellular signal-regulated kinase 1/2 (ERK1/2) are only partially understood. OT receptor (OTR) signals predominantly through G{alpha}q, but ERK1/2 phosphorylation (ERK1/2-P) in PHM1 myometrial cells was not eliminated by inhibition of downstream effectors such as phospholipase C or protein kinase C. Inconsistent with a G{alpha}i-coupled response, pertussis toxin inhibition of OT-induced ERK1/2-P was reversed by the protein kinase A inhibitors Rp-cAMPS and KT5720. Consistent with an inhibitory role for protein kinase A, pertussis toxin pretreatment raised cellular cAMP and 8-(4-chlorophenylthio)-cAMP inhibited OT-induced ERK1/2-P. Attenuation of the OT response by the Gß{gamma} scavenger carboxyl terminus of the ß-adrenergic receptor kinase implicated a Gß{gamma}-mediated pathway. In both COSM6 cells overexpressing OTR (OTR-COSM6) and in PHM1 cells, the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor AG1478 markedly reduced OT-induced ERK1/2-P, whereas the platelet-derived growth factor receptor tyrosine kinase inhibitor AG1296 had no effect. Furthermore, OT increased EGFR tyrosine phosphorylation in OTR-COSM6 cells, which was inhibited by AG1478 or EGTA plus thapsigargin pretreatment. AG1478 did not affect inositol 1,4,5-triphosphate production by OT or protein kinase C-stimulated ERK1/2-P but completely blocked ionomycin-induced ERK1/2-P and EGFR tyrosine phosphorylation. In both OTR-COSM6 and PHM1 cells, EGTA reduced OT-stimulated ERK1/2-P; no ERK1/2-P was observed when intracellular calcium increases were blocked by pretreatment with thapsigargin plus EGTA. These data are consistent with activation of a Gß{gamma}-mediated pathway as a consequence of G{alpha}q activation in myometrium and OTR-COSM6 cells that results in increased ERK1/2-P. This pathway involves both EGFR activation and an influence of calcium.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
EXTRACELLULAR SIGNAL-REGULATED kinase 1/2 (ERK1/2) are part of one of the most ubiquitous signal transduction cascades (1). Gene transcription is initiated through ERK1/2 activation by signals from both growth factor (2) and G protein-coupled membrane receptors (GPCRs) (3). The mechanism underlying ERK1/2 activation by growth factor receptor is well defined and includes tyrosine phosphorylation of the receptor itself, formation of the Grb2, Shc, and SOS adapter protein complex, activation of Ras and then Raf kinase, activation of MAPK kinase and finally dual phosphorylation of ERK1/2 (ERK1/2-P) on tyrosine and threonine by MAPK kinase (1, 4).

In contrast, the mechanisms for ERK1/2 activation as a consequence of GPCR activation are less well understood and are determined in part by the cell type and the receptor involved (5, 6). In some cells, transactivation of receptor tyrosine kinases like epidermal growth factor receptor (EGFR) (7) and platelet-derived growth factor receptor (PDGFR) (8) appear to be a critical convergent point for GPCR-mediated ERK1/2 activation, but there is not general consensus on the mechanism involved (9). Protein kinase C (PKC) was essential for M1 muscarinic receptor-mediated EGFR transactivation in HEK293 cells (10), but in GN4 rat liver epithelial cells, EGFR transactivation by angiotensin II was suppressed by PKC and was activated when PKC activity was blocked (11). The calcium/calmodulin pathway was implicated in angiotensin II-mediated EGFR transactivation in cardiac fibroblasts (12) but was not observed in PC12 cells treated with bradykinin (13).

Oxytocin (OT) regulates a wide spectrum of physiological functions through its G protein-coupled receptor. Immunoneutralization of interaction with G{alpha}q inhibited OT-stimulated GTPase activity, phospholipase C (PLC) activation, and intracellular calcium elevation in myometrium (14, 15). OT receptor (OTR) was associated with and activated G{alpha}i in Chinese hamster ovary (CHO) cells overexpressing OTR and was associated with G{alpha}i in pregnant rat myometrium (16, 17). OT stimulated G{alpha}h in human myometrium (18). OT-activated myometrial tyrosine kinase (19, 20) and ERK-2 (21) were implicated in the regulation by OT of cyclooxygenase-2 gene expression and prostaglandin synthesis in myometrium at term (17, 19), but the specific signaling linkage remains to be clarified. In CHO cells overexpressing OTR, OT stimulated ERK-2 phosphorylation predominantly through the G{alpha}q-PLC-PKC pathway (17). On the other hand, OT-stimulated ERK-2 phosphorylation was completely inhibited by pertussis toxin in cultured myometrial cells, suggesting the possible involvement of G{alpha}i (21).

Truncated OTR constructs lacking part of the fourth intracellular domain appeared to differentiate between pathways stimulated by G{alpha}q and G{alpha}i in CHO cells and implicated part of the fourth intracellular domain of OTR as important for OTR/G{alpha}q coupling (22). In contrast, we determined that mutation of a single residue, lysine-270 to valine, in the third intracellular domain abolished OT-stimulated activation of both PLC and ERK1/2 in OTR-COSM6 cells, thus indicating the need for more detailed analysis of specificity determinants (23). To establish whether PLC and ERK signaling pathways might be used to distinguish coupling of OTR to different G proteins in structure/function studies, we attempted to define the pathways used by OTR overexpressed in COSM6 cells and endogenously expressed in PHM1 myometrial cells to activate ERK1/2.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals and plasmids
AG1478, AG1296, KT5720, and Go6983 were obtained from Calbiochem Corp. (San Diego, CA). [3H]Myoinositol (22.3 Ci/mmol) and [tyrosyl-2, 6-3H]oxytocin (33 Ci/mmol) were obtained from NEN Life Science Products (Boston, MA) and BSA (fatty acid free, fraction V) from ICN Biomedicals, Inc. (Irvine, CA). Human oxytocin, ionomycin, pertussis toxin, phorbol 12-myristate 13-acetate (PMA), CPT-cAMP [8-(4-chlorophenylthio)-cAMP], 8-bromoadenosine-cAMP (Rp-isomer), chelerythrine chloride and thapsigargin were obtained from Sigma (St. Louis, MO). Cell culture reagents and lipofectamine were obtained from Life Technologies, Inc. (Gaithersburg, MD). ECL Western blotting detection reagents was obtained from Amersham Pharmacia Biotech (Piscataway, NJ). cAMP Direct Correlate-enzyme immunoassay kit was purchased from Assay Designs Inc. (Ann Arbor, MI). Anti-phospho-p44/42 MAPK monoclonal antibody was purchased from Cell Signaling (Beverly, MA). Polyclonal anti-p44/42 MAPK antibody and anti-phospho-tyrosine monoclonal antibody PY99 were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-EGFR monoclonal antibody was obtained from Signal Transduction Laboratory (Lexington, KY). The AG 1-X8 resin was obtained from Bio-Rad Laboratories, Inc. (Hercules, CA) and nitrocellulose membranes from Schleicher \|[amp ]\| Schuell (Keene, NH). Poly-Lys-linked adenovirus for transfection was purchased from N. Weigel (Baylor College of Medicine, Houston, TX).

The cDNA clone of human OTR was obtained from Dr. M. J. Brownstein (NIH, Bethesda, MD). The clone was removed from the original plasmid by digestion with BamH1 and BstE2 (Promega Corp., Madison, WI) and inserted into these sites in pCDNA6 (Invitrogen, Carlsbad, CA). The cDNA clone for G{alpha}q was obtained from Dr. M. I. Simon (California Institute of Technology, Pasadena, CA). DNA encoding the carboxyl terminus of the ß-adrenergic receptor kinase (ßARK-ct) peptide subcloned into pRK5 was obtained from Dr. R. J. Lefkowitz (Duke University, Durham, NC).

Cell culture and transfection
COSM6 cells and PHM1 myometrial cells were cultured in DMEM containing 8% fetal calf serum, 2 mM L-glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin. For transient transfection, COSM6 cells were plated into 35-mm culture dishes (Corning, Inc., Corning, NY) at 1.5 x 105/dish. The next day, plasmid DNA (0.5 µg of OTR plasmid/dish) was mixed with 6 µl of lipofectamine in 200 µl of DMEM and incubated for 1 h before addition to cells washed with PBS in a final volume of 0.95 ml. In phosphatidylinositide turnover experiments, 0.08 µg of G{alpha}q plasmid was also cotransfected. The cells were incubated at 37 C for 5 h before they were returned to growth medium. Where indicated, 1 µg of ß-ARK-ct plus 0.1 µg OTR plasmid/dish was transfected.

PHM1 cells were plated in 35-mm dishes at 1.5 x 105 cells/dish and were subjected to transfection the next day. Plasmid containing the ßARK-ct minigene or empty vector (0.2 µg) was incubated with 2.25 x 108 poly-Lys-adenovirus particles (24) in 35.5 µl of sterilized HBS (0.15 M NaCl and 0.02 M HEPES, pH 7.3) for 30 min. Polylysine (0.3 µg) in 15.1 µl of HBS was added and incubation was continued for an additional 30 min. PHM1 cells were washed twice with PBS and replaced in 1ml of DMEM; 50 µl of the virus mixture was added in the dish and incubated for 2 h. The transfected cells were returned to growth medium and incubated overnight at 37 C. Transfection efficiency was assessed by examining fluorescence at 485 nm following transfection under similar conditions with a plasmid expressing green fluorescent protein. The dishes were washed twice with PBS, incubated in DMEM for 4 h, and incubated for an additional hour in DMEM (minus glucose and phenol red) containing 0.2% BSA before they were stimulated.

Phosphatidylinositide turnover
Twenty-four hours after transfection, COSM6 cells were washed twice with PBS, placed in 1 ml DMEM containing 0.5% fetal calf serum, and 0.4 µM [3H]myoinositol, and incubated at 37 C overnight. The labeled cells were washed twice with PBS and were incubated with Hanks’ balanced salt solution (pH 7.4) containing 0.2% BSA and 10 mM LiCl at 37 C for 30 min. The cells were stimulated for another 30 min with 40 nM of OT. 3H-inositol phosphates were isolated by ion exchange chromatography (23).

Protein phosphorylation assay and Western blots
Twenty-four hours after transfection, COSM6 cells were washed twice with PBS and incubated in DMEM with 0.5% fetal bovine serum overnight. Cells were washed once with PBS and placed in DMEM (minus phenol red and glucose) with 0.2% BSA, and incubated at 37 C for 1 h to reduce background phosphorylation. Inhibitors were added as indicated in the figure legends and incubation continued for 1 h. The cells were then exposed to OT at 37 C for 5 min before stopping the reaction with 200 µl of sample buffer (62.5 mM Tris, pH 6.8; 2% sodium dodecyl sulfate; 10% glycerol; 50 mM dithiothreitol; 0.01% bromphenol blue). The samples were separated by SDS-PAGE as described previously (23). Antibody bands were detected by chemiluminescence and density was analyzed with the imaging system from Bio-Rad Laboratories, Inc. (Hercules, CA).

Phospho-ERK1/2 and total ERK1/2 were detected with anti-phospho-p44/42 MAPK and anti-p44/42 MAPK antibody on the same blot, respectively. Tyrosine phosphorylation of EGFR was detected with anti-phospho-tyrosine antibody PY99 and the total amount of EGFR was detected with anti-EGFR antibody on the same blot.

cAMP assay
PHM1 cells grown in 35-mm dishes were treated with 100 ng/ml pertussis toxin overnight in DMEM containing 0.5% FBS. In the next day, cells were stimulated with 10-7 M OT for 5 min before the reaction was terminated with 1 ml 0.1 N HCl. cAMP was measured in the cell lysates by immunoassay as recommended by the kit manufacturer. Data were normalized to protein, determined with the Bradford reagent (Bio-Rad Laboratories, Inc.).

Ligand binding assay
PHM1 cells grown in six-well plates were washed once with PBS after pertussis toxin treatment as described above. Cells were incubated in 0.9 ml/well Ca2+-free HBSS containing 5 mM MgCl2, 0.1% BSA, and 10-8 M [3H]oxytocin in the absence (total binding) or presence (nonspecific binding) of 10-5 M unlabeled oxytocin for 2.5 h at room temperature. The reaction was stopped by aspiration, followed by two washes with cold HBSS (Ca2+, Mg2+ free) containing 0.1% BSA. The cells were lysed with 0.5 ml 1 N NaOH and neutralized with 0.5 ml 1 N HCl, and aliquots were counted in Scintisafe Econo 1 (Fisher Scientific, Fairlawn, NJ).

Data analysis
Data are presented as mean ± SE and were analyzed by ANOVA and Duncan’s modified multiple range tests where appropriate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
OT-induced ERK1/2-P involves a pathway independent of the action of PLC or PKC in human myometrial PHM1 cells
Binding of OT to OTR activates G{alpha}q, resulting in increased PLC activity in myometrium (15). This increases both diacylglycerol and inositol 1,4,5-triphosphate formation, resulting in an increase in intracellular calcium and favoring PKC activation. In turn, activated PKC can potentially stimulate ERK1/2. To test whether this pathway plays a major role in OT-induced ERK1/2-P, PHM1 cells were preincubated with the PLC inhibitor U73122, or the PKC inhibitors Go6983 or chelerythrine, for 1 h before they were challenged with OT. U73122 pretreatment eliminated OT-induced PLC activity (Fig. 1AGo), but did not affect ERK1/2-P in PHM1 cells (Fig. 1BGo). Figure 2AGo shows that Go6983 did not reduce OT-induced ERK1/2-P significantly under conditions where it eliminated the effect of the PKC activator PMA (Fig. 2BGo). As shown in Fig. 2CGo, the broad spectrum PKC inhibitor chelerythrine also did not affect OT-induced ERK1/2-P, but inhibited the effect of PMA (Fig. 2DGo). Taken together, these data provide little support for significant involvement of the G{alpha}q/PLC or PKC pathways in stimulation of ERK1/2-P in PHM1 cells.



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  		Figure 1. Inhibition of PLC does not eliminate OT-induced ERK1/2-P in PHM1 cells. PHM1 cells were incubated with 4 x 10-5 M PLC inhibitor U73122 for 1 h before stimulation with 10-7 M OT for 30 min to determine inositol 1,4,5-triphosphate production (A) or for 5 min to determine ERK1/2-P (B) in the whole cell lysates. A, Results are expressed as mean ± SEM, n = 3. Data were analyzed by ANOVA and Duncan’s modified range tests. Significant differences (P < 0.05) between groups are indicated by different lowercase letters. Data in (B) represent the mean and range of two determinations.

 


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  		Figure 2. Inhibition of PKC does not eliminate OT-induced ERK1/2-P in PHM1 cells. PHM1 cells were incubated with 10-6 M PKC inhibitor Go6983 (A and B) or 10-5 M PKC inhibitor chelerythrine (C and D) for 1 h before stimulation with 10-7 M OT (A and C) or 10-6 M PKC activator PMA (B and D) for 5 min. Results are expressed as mean ± SEM (A, n = 4; C, n = 3), or represent one of two similar experiments (B and D). Data in (A) and (C) were analyzed by ANOVA and Duncan’s modified range tests. Significant differences between groups (P < 0.05) are indicated by different lowercase letters.

 
G{alpha}i is not required for OT-induced ERK1/2-P in PHM1 cells, but Gß{gamma}-mediated pathways are implicated
Because pertussis toxin ADP-ribosylates and inactivates G{alpha}i, pertussis toxin sensitivity is often viewed as evidence of the role of G{alpha}i in GPCR-mediated signaling. However, pertussis toxin can raise cytosolic cAMP levels and activate PKA, probably as a consequence of G{alpha}i ADP-ribosylation (25) and inhibits ERK1/2 activation as a consequence of cAMP-mediated effects on other components of the signaling pathway (26). As shown in Fig. 3AGo, pretreatment of PHM1 cells with 100 ng/ml pertussis toxin overnight partially inhibited OT-induced ERK1/2-P (P < 0.01). Importantly, this inhibitory effect of pertussis toxin was completely reversed by pretreatment with the specific PKA inhibitor Rp-cAMPS. Neither pertussis toxin nor Rp-cAMPS affected basal ERK1/2-P status. KT5720, another specific PKA inhibitor, attenuated the effect of pertussis toxin, similar to the effect of Rp-cAMPS (Fig. 3BGo). Pertussis toxin treatment did not affect OTR expression in PHM1 cells as determined by intact cell binding assay (71 vs. 80 fmol/mg protein for control and pertussis toxin treated groups, respectively). Consistent with an indirect effect of pertussis toxin via cAMP generation, pretreatment of PHM1 cells with CPT-cAMP, a cell permeable cAMP analog, inhibited OT-induced ERK1/2-P (Fig. 3CGo). Similar to our previous finding in rat myometrium (25), pertussis toxin treatment raised PHM1 cell intracellular cAMP and this was not affected by OT (Fig. 3DGo). Taken together, these data are not consistent with a requirement for G{alpha}i activation in the OT-stimulated ERK1/2-P in these cells.



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  		Figure 3. Pertussis toxin inhibits OT-stimulated ERK1/2-P through activation of PKA and this is reversible by pretreatment with PKA inhibitors. PHM1 cells were pretreated with or without 100 ng/ml pertussis toxin overnight and then with or without 10-5 M Rp-cAMPS (A) or 10-6 M KT5720 (B) for 1 h, with or without 10-3 M CPT-cAMP (C) for 30 min, or with no additions (D) before stimulation with 10-7 M OT for 5 min. Whole cell lysates were collected for ERK1/2-P (A–C) or cAMP (D) assays. Results are expressed as mean ± SEM (A, n = 6; B, n = 3; D, n = 5). Data were analyzed by ANOVA and Duncan’s modified range tests. Significant differences between groups (P < 0.05) are indicated by different lowercase letters. Data in panel C are from one of two similar experiments.

 
Overexpression of Gß{gamma} subunits activates ERK1/2. To determine the role of Gß{gamma} in OT-stimulated ERK1/2-P, we transiently transfected a plasmid expressing the Gß{gamma} scavenger ßARK-ct into PHM1 cells. As shown in Fig. 4AGo, this treatment partially attenuated OT-induced ERK1/2-P (P < 0.01). Because transfection efficiency under these conditions is approximately 30%, we would not expect complete inhibition. We also observed a significant inhibitory effect of ßARK-ct on OT-induced ERK1/2-P in OTR-COSM6 cells (P < 0.01; Fig. 4BGo). These data are consistent with a role for a Gß{gamma}-mediated pathway in OT-stimulated ERK1/2 activation, presumably as a consequence of G{alpha}qGß{gamma} activation by OTR.



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  		Figure 4. The Gß{gamma} scavenger ßARK-ct reduces OT-induced ERK1/2-P in both PHM1 and OTR-COSM6 cells. PHM1 cells (A) and OTR-COSM6 cells (B) were stimulated with 10-7 M OT for 5 min. ßARK-ct plasmid was transfected into PHM1 cells 24 h before assay and into COSM6 cells along with OTR plasmid 48 h before assay. Results are expressed as mean ± SEM (A, n = 3; B, n = 4). Data were analyzed by ANOVA and Duncan’s modified range tests. Significant differences between groups (P < 0.05) are indicated by different lowercase letters.

 
Transactivation of EGFR tyrosine kinase is required for OT-stimulated ERK1/2-P
Both G{alpha}i- and G{alpha}q-coupled receptor activation of ERK1/2 can involve cross-talk and activation of receptor tyrosine kinases, but the mechanisms employed depend on the specific receptor and the cell type in which these receptors are expressed. Therefore, the involvement of receptor tyrosine kinases in OT-stimulated ERK1/2-P was investigated in PHM1 and OTR-COSM6 cells. Preincubation with the EGFR tyrosine kinase inhibitor AG1478 significantly decreased OT-stimulated ERK1/2-P by 83 ± 4% in OTR-COSM6 cells (P < 0.01; Fig. 5AGo) and by 56 ± 5% in PHM1 cells (P < 0.01; Fig 5BGo). Because the effect of OT on ERK1/2-P was more pronounced in OTR-COSM6 cells, this system was used to investigate the effect of OT on tyrosine kinase signaling further. In contrast to the effect of AG1478, pretreatment of OTR-COSM6 cells with PDGFR tyrosine kinase inhibitor AG1296 had no effect on OT-stimulated ERK1/2-P (Fig. 5CGo), indicating a measure of specificity in the response. An increase in EGFR tyrosine phosphorylation was elicited in these cells by OT, and this increase was also blocked by AG1478 (Fig. 5DGo). The effect of OT was weaker than that elicited by EGF (Fig. 5DGo). Consistent with the lack of involvement of the PLC pathway in OT-stimulated ERK1/2-P, AG1478 did not affect PLC activation by OT in OTR-COSM6 cells (Fig. 6Go).



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  		Figure 5. Activation of EGFR but not PDGFR tyrosine kinase is implicated in OT-stimulated ERK1/2-P. OTR-COSM6 cells and PHM1 cells were incubated with or without 10-7 M AG1478 or 10-6 M AG1296 for 1 h before stimulation with 10-7 M OT or 1 ng/ml recombinant EGF for 5 min. ERK1/2-P (A–C) or EGFR tyrosine phosphorylation (D) were determined in cell lysates. Results are expressed as mean ± SEM (A, n = 3; B, n = 5; C, n = 3). Data were analyzed by ANOVA and Duncan’s modified range tests. Significant differences between groups (P < 0.05) are indicated by different lowercase letters. Data in (D) are from one of two similar experiments.

 


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  		Figure 6. EGFR tyrosine kinase inhibitor AG1478 did not block OT-stimulated PLC in OTR-COSM6 cells. OTR-COSM6 cells were incubated with or without 10-7 M AG1478 for 1 h before stimulation with 40 nM OT for 30 min. Results are expressed as mean ± SEM (n = 3). Data were analyzed by ANOVA and Duncan’s modified range tests. Significant differences between groups (P < 0.05) are indicated by different lowercase letters.

 
Requirement for calcium in OT- and ionomycin-stimulated ERK1/2-P
Both calcium and PKC activation have been reported to stimulate ERK1/2 activation. Figure 7AGo shows that in OTR-COSM6 cells, the PKC activator PMA and the calcium ionophore ionomycin increased ERK1/2-P by 2.7 ± 0.3 (P < 0.05) and 3.6 ± 0.2 (P < 0.01) fold over control, respectively. The EGFR tyrosine kinase inhibitor AG1478 selectively inhibited the ionomycin-induced response, suggesting that the involvement of EGFR lies downstream of a calcium-dependent step. To investigate the role of calcium in OT signaling further, cells were treated with 0.2 mM EGTA to eliminate extracellular calcium and with EGTA plus 0.1 µM thapsigargin, an endoplasmic reticulum Ca2+-ATPase inhibitor that depletes intracellular calcium stores. As shown in Fig. 7BGo, the OT-stimulated increase in ERK1/2-P in OTR-COSM6 cells was blocked 58 ± 8% (P < 0.01) by removing extracellular calcium with EGTA and essentially the entire response was blocked by EGTA plus thapsigargin. In PHM1 cells, both treatments completely blocked OT-stimulated ERK1/2-P (P < 0.01).



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  		Figure 7. Stimulation of ERK1/2-P by ionomycin, but not by PMA is sensitive to AG1478, and by OT shows a requirement for calcium. A, OTR-COSM6 cells were incubated with or without 10-7 M AG1478 for 1 h before stimulation with 1 µM of PMA (n = 3) or ionomycin (n = 6) for 5 min. B, OTR-COSM6 (n = 3) or PHM1 (n = 6) cells were incubated with 0.2 mM EGTA or EGTA plus 10-7 M thapsigargin in the absence of extracellular calcium for 1 h before stimulation with 10-7 M OT for 5 min. Results are expressed as mean ± SEM. Data were analyzed by ANOVA and Duncan’s modified range tests. Significant differences between groups (P < 0.05) are indicated by different lowercase letters.

 
To verify the involvement of calcium in EGFR tyrosine phosphorylation and investigate the mechanism underlying the role of calcium in OT-stimulated ERK1/2-P, we examined the effect of ionomycin on EGFR phosphorylation and of EGTA plus thapsigargin on OT-induced EGFR phosphorylation in OTR-COSM6 cells. As shown in Fig. 8Go, ionomycin increased tyrosine phosphorylation of EGFR; this was eliminated by pretreatment with AG1478. Preincubation with EGTA plus thapsigargin blocked OT-induced EGFR tyrosine phosphorylation.



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  		Figure 8. Calcium transient in OTR-COSM6 cells is critical for ionomycin- and OT-induced EGFR tyrosine phosphorylation. OTR-COSM6 cells were preincubated with or without 10-7 M AG1478 or 0.2 mM EGTA plus 10-7 M thapsigargin for 1 h before stimulation with 10-6 M ionomycin or 10-7 M OT for 5 min. Data are from one of two similar experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Because OTR couples predominantly to G{alpha}q, the PLC/PKC pathway is one candidate for mediating ERK1/2 activation. However, OT-induced cyclooxygenase-2 gene up-regulation and prostaglandin production were reported to be independent of PKC activation in human myometrial cells (19). Consistent with these observations, we found that neither the PLC inhibitor U73122 nor the PKC inhibitors Go6983 or chelerythrine eliminated OT-induced ERK1/2 activation in the human myometrium PHM1 cell line under conditions where they abolished PLC and PKC activities, respectively. In contrast, in CHO cells stably expressing OTR, PGE2 synthesis and ERK-2 activation by OT were significantly attenuated by both PLC and PKC inhibitors (17). Thus, the relative importance of the PLC/PKC pathway in OT signaling appears to depend on the specific cell type.

The Gß{gamma} subunits released as a result of G{alpha}i activation have been widely implicated in ERK1/2 activation (3), and some evidence has been presented for stimulation of G{alpha}i by OTR (16, 21, 27). Pertussis toxin ADP-ribosylates and irreversibly inhibits G{alpha}i activation, and hence pertussis toxin sensitivity is considered a potential indication of coupling to G{alpha}i. Such inhibitory effects of pertussis toxin have been observed for OT-stimulated intracellular calcium elevation and phosphatidylinositide turnover in myometrium (27, 28), OT-stimulated cyclooxygenase gene transcription in myometrium (19) and ERK-2 phosphorylation in both myometrium and CHO cells stably expressing OTR (17, 21). Nonetheless, pertussis toxin treatment can also result in an increase in cAMP and activation of PKA, which can result in inhibition of PLC (25, 29) and c-Raf (26). Therefore, inhibition of ERK1/2-P by pertussis toxin does not constitute a priori proof of direct G{alpha}i involvement in a pathway. In rat myometrium and PHM1 cells, the inhibitory effect of pertussis toxin on OT-stimulated PLC activity was reversed by preincubation with PKA inhibitors (25, 29). Similarly, in the present study we found that the inhibitory effect of pertussis toxin on OT-induced ERK1/2-P was reversed by preincubation with the PKA inhibitors Rp-cAMPS or KT5720. These observations are not consistent with activation of either PLC or ERK1/2 as a result of G{alpha}i activation by OTR in myometrial cells because the inhibitory effect of pertussis toxin should be irreversible in this case. In fact, pertussis toxin treatment raised basal cytosolic cAMP and CPT-cAMP treatment effectively inhibited OT-induced ERK1/2-P in PHM1 cells. These studies indicate minimal involvement of G{alpha}i in OT-induced ERK1/2-P. These findings, coupled with the observation that the PLC/PKC pathway is not involved, is more consistent with the concept that Gß{gamma} liberated from a heterotrimeric complex with G{alpha}q as a result of OTR activation is responsible for stimulation of the ERK1/2 activation pathway in these cells. Consistent with our hypothesis, the Gß{gamma} scavenger ßARK-ct (30) significantly attenuated OT-induced ERK1/2-P in both PHM1 and OTR-COSM6 cells. Recently, Gß{gamma} subunits released as a result of G{alpha}q activation has been reported to be responsible for G protein-coupled proteinase activated receptor-1 regulation of NF-{kappa}B activity in endothelial cells (31). A similar effect of Gß{gamma} subunits on angiotensin II-mediated activation of ERK has also been observed in cardiac fibroblasts, but not in cardiac myocytes (32).

Activation of receptor tyrosine kinases is an obligatory step in the pathway resulting in ERK1/2 activation by some GPCRs (9). Overexpressing Gß{gamma} subunits in COS-7 cells led to an increase in basal EGFR phosphorylation (9, 33), which could trigger Ras-dependent activation of ERK1/2 independent of PKC activation (3, 34, 35). Both PDGFR (8) and EGFR (12) participated in angiotensin II-induced ERK1/2 activation. Several lines of evidence have indicated the involvement of tyrosine kinase in OT-stimulated pathways. Genestein, a relatively wide spectrum tyrosine kinase inhibitor, blocked the Gß{gamma}-mediated calcium transient elicited by a truncated OTR in CHO cells (22) and attenuated OT-stimulated RGS2 mRNA expression in human myometrial cells (20). Herbimycin, another tyrosine kinase inhibitor, suppressed OT-induced cyclooxygenase-2 expression and prostacyclin production in human myometrial cells (19). In the present study, participation of EGFR but not PDGFR tyrosine kinase was implicated in OT-induced ERK1/2 activation in both myometrial and OTR-COSM6 cells, indicating some specificity for EGFR involvement.

Calcium appears to play a role in receptor tyrosine kinase stimulation by some GPCRs. Both calcium-dependent (12, 36, 37) and calcium-independent (38, 39) pathways have been implicated in EGFR activation by angiotensin II, bradykinin and endothelin-1 receptors. In CHO cells overexpressing OTR, removal of calcium inhibited OT-stimulated ERK-2 (17). In the present study, OT-mediated ERK1/2-P in both myometrium and OTR-COSM6 cells demonstrated a requirement for calcium. Because the EGFR tyrosine kinase inhibitor AG1478 blocked ionomycin- as well as OT-induced ERK1/2-P, this may reflect a calcium requirement for EGFR activation. Consistent with this postulation, we observed that ionomycin triggered EGFR tyrosine phosphorylation and thapsigargin plus EGTA pretreatment inhibited OT-induced EGFR tyrosine phosphorylation. Calcium-mediated activation of tyrosine kinases that phosphorylate EGFR has also been reported (40, 41). A role for PKC, also activated in some cases by calcium, in EGFR activation in myometrium is ruled out by the finding that AG1478 did not block ERK1/2-P by the PKC activator PMA.

OTR-mediated stimulation of PLC was not affected by pretreatment of OTR-COSM6 cells with AG1478, consistent with involvement of the EGFR tyrosine kinase in a pathway independent of PLC. OTR activation has been shown to elicit mitogenic effects and induce cell proliferation in human trophoblast cells and a choriocarcinoma cell line that express endogenous OTR, as well as in Madin-Darby canine kidney cells stably expressing human OTR (42, 43). OTR activation also induced P19 embryonic stem cell differentiation into cardiomyocytes (44). Because EGFR plays a pivotal role in the regulation of cell differentiation and proliferation (2), it is possible that these actions of OT involve EGFR activation.

In summary, our data are consistent with a mechanism whereby OT-mediated ERK1/2 activation in myometrial cells involves a PLC-independent pathway mediated through the action of Gß{gamma} subunits released from activated G{alpha}q. The pathway involves activation of EGFR tyrosine kinase and has a calcium requirement. The effects of OTR on a number of pathways are apparently different in different cell types (45). It is possible that overexpression of receptors or receptor derivatives may enhance coupling to pathways that would ordinarily not be used, but because both COSM6 cells overexpressing OTR and PHM1 cells expressing endogenous OTR behaved similarly, this does not provide an explanation for differences between the results reported here and other data in the literature. More likely, these differences may reflect the ability of the OTR to associate with other membrane and membrane-associated signal components in a cell-specific manner (43, 46).


   Acknowledgments
 
The authors thank Drs. Brownstein, Lefkowitz, and Simon for providing plasmids used in this work.


   Footnotes
 
This work was supported in part by NIH Grant HD-9618.

Abbreviations: ßARK-ct, Carboxyl terminus of the ß-adrenergic receptor kinase; CHO, Chinese hamster ovary; CPT-cAMP, 8-(4-chlorophenylthio)-cAMP; EGFR, epidermal growth factor receptor; ERK1/2, extracellular signal-regulated kinase 1/2; ERK1/2-P, ERK1/2 phosphorylation; GPCRs, G protein-coupled receptors; OT, oxytocin; OTR-COSM6; COSM6 cells overexpressing OTR; PDGFR, platelet-derived growth factor receptor; PKC, protein kinase C; PLC, phospholipase C; PMA, phorbol 12-myristate 13-acetate.

Received October 8, 2002.

Accepted for publication March 20, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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