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BOR - Papers in Press, published online ahead of print March 5, 2008.
Biol Reprod 2008, 10.1095/biolreprod.107.064485
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BIOLOGY OF REPRODUCTION 78, 1007–1017 (2008)
DOI: 10.1095/biolreprod.107.064485
© 2008 by the Society for the Study of Reproduction, Inc.

Mechanisms of Hormone Action; 

Multiple Signals Regulate Phospholipase CBeta3 in Human Myometrial Cells1

Miao Zhong  3, Dilyara A. Murtazina  3, Jennifer Phillips , Chun-Ying Ku , and Barbara M. Sanborn  2

Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado 80523

ABSTRACT

Phospholipase CB3 (PLCB3) serine1105 (S1105), a substrate for multiple protein kinases, represents a potential point of convergence of several signaling pathways in the myometrium. To explore this hypothesis, the regulation of PLCB3-S1105 phosphorylation (P-S1105) was studied in immortalized and primary human myometrial cells. 8-[4-chlorophenylthio] (CPT)-cAMP and calcitonin gene-related peptide (CALCA) transiently increased P-S1105. Relaxin also stimulated P-S1105; this effect was partially blocked by the protein kinase A (PRKA) inhibitor, Rp-8-CPT-cAMPS. Oxytocin, which stimulates Galphaq-mediated pathways, also rapidly increased P-S1105, as did prostaglandin F2alpha and ATP. Oxytocin-stimulated phosphorylation was blocked by protein kinase C (PRKC) inhibitor Go6976 and by pretreatment overnight with a phorbol ester. Cypermethrin, a PP2B phosphatase inhibitor, but not okadaic acid, a PP1/PP2A inhibitor, prolonged the effect of CALCA on P-S1105, whereas the reverse was the case for the oxytocin-stimulated increase in P-S1105. PLCB3 was the predominant PLC isoform expressed in the myometrial cells and PLCB3 short hairpin RNA constructs significantly attenuated oxytocin-stimulated increases in intracellular calcium. oxytocin-induced phosphatidylinositol (PI) turnover was inhibited by CPT-cAMP and okadaic acid, but was enhanced by pretreatment with Go6976. CPT-cAMP inhibited oxytocin-stimulated PI turnover in the presence of overexpressed PLCB3, but not overexpressed PLCB3-S1105A. These data demonstrate that both negative crosstalk from the cAMP/PRKA pathway and a negative feedback loop in the oxytocin/G protein/PLCB pathway involving PRKC operate in myometrial cells and suggest that different protein phosphatases predominate in mediating P-S1105 dephosphorylation in these pathways. The integration of multiple signal components at the level of PLCB3 may be important to its function in the myometrium.

kinases, myometrium, phosphatases, phospholipase CB3, phosphorylation, protein kinase A, protein kinase C, protein phosphatase

INTRODUCTION

The myometrium maintains relative quiescence during pregnancy, exhibits sporadic contractures near term and gradually develops the coordinated, rhythmic contractions of labor. The biochemical changes that account for this transition reflect a shift in the relative predominance of relaxant and contractant pathways, but are not completely understood at the molecular level [1–3]. The influence of cAMP, a second messenger linked to myometrial relaxation, diminishes near the end of pregnancy. In contrast, G{alpha}q/phospholipase CB (PLCB) pathways mediating the signaling of a number of uterine contractants, including oxytocin (OXT), prostaglandins E2 and F2{alpha}, and ATP, are enhanced near parturition.

Phosphatidylinositide-specific PLC enzymes catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to produce inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). Six subfamilies of PLCs (B, G, D, E, Z, and H) constitute part of ubiquitous signaling cascades that translate hormonal signals into intracellular events, leading to alternations in cell function [4, 5]. PLCB isoforms 1–4 are stimulated by G protein activation (G{alpha}q/11 and/or Gβ{gamma}) [4, 5].

Several PLCB isoforms can be phosphorylated by protein kinases, and this may contribute to the regulation of their activities [6]. Protein kinase C (PRKC) phosphorylates and negatively regulates PLCB1 activity [7], but PLCB1 is not directly phosphorylated by protein kinase A (PRKA) [8]. PLCB2 is a substrate of PRKA; this phosphorylation accounts for the inhibition by cAMP of Gβ{gamma}-induced PLCB2 activity in vitro [9]. PRKA phosphorylates PLCB3 exclusively on Serine1105 (S1105) and inhibits both G{alpha}q- and Gβ{gamma}-stimulated activity in transfected cells [8, 10]. S1105 is conserved between a number of species in a reasonable consensus PRKA target sequence in PLCB3, but a comparable sequence is not present in the other PLCB isoforms [11]. Interestingly, PLCB3-S1105 is also phosphorylated by activation of PRKC and protein kinase G [10, 12]. In the latter case, phosphorylation at another residue (S26) was required for full inactivation [12]; hence, PLCB3 may be regulated by multiple protein kinases and control hormonal signaling. Regulation of PLCB4 activity by protein kinases has not been reported to date.

In myometrial smooth muscle, the contractant hormone OXT signals through a G protein-coupled receptor that primarily couples to G{alpha}q/11 to activate PLCBs [13, 14]. Cyclic AMP generally induces relaxation in smooth muscles [15]. The inhibition of OXT-induced PI turnover by increasing intracellular cAMP in myometrial cells is both PRKA and A-kinase anchoring protein (AKAP) dependent [16, 17]. Activation of PRKA by 8-[4-chlorophenylthio] (CPT)-cAMP inhibits GTP{gamma}S-stimulated PI turnover in rat myometrial plasma membrane, indicating that the effect of PRKA is distal to OXT receptor (OXTR)/G protein coupling [18].

To examine the functional importance of specific PLCB3 phosphorylation sites in hormone signaling in myometrial cells, we have examined the effect of stimulating PRKA and PRKC pathways on the phosphorylation of endogenous PLCB3-S1105. We demonstrate that PLCB3 is the predominant PLCB isoform in the human myometrial cells used. The data support the presence of negative crosstalk from relaxant-stimulated cAMP pathways, as well as negative feedback from the OXT-induced PLCB activation pathway itself, targeted at PLCB3-S1105. In addition, the data suggest that protein phosphatase (PP) 2B regulates PRKA-mediated P-S1105 by calcitonin gene-related peptide (CALCA), whereas PP1 or PP2A regulate OXT-stimulated, PRKC-dependent P-S1105 in PHM1 cells.

MATERIALS AND METHODS

Materials

[3H]-myoinositol (22.3 Ci/mmol) was obtained from Perkin-Elmer Life Sciences (Boston, MA) and BSA (fatty acid free, fraction V) from ICN Biomedicals Inc. (Irvine, CA). CALCA (rat) was obtained from Tocris Bioscience (Ellisville, MO). Cypermethrin, Go6976, okadaic acid, OXT, and phosphatase inhibitor cocktail set 1 were obtained from Calbiochem (La Jolla, CA). CPT-cAMP, 8-[4-chlorophenylthio]-cAMP-thioate, Rp-isomer (Rp-8-CPT-cAMPS), 3-isobutyl-1-methylxanthine, phorbol 12-myristate 13-acetate (PMA), ATP, prostaglandin F2{alpha} (PGF2{alpha}) Tris salt, Reactive Blue-2, and protease inhibitor cocktail (no. P8340) were obtained from Sigma-Aldrich (St. Louis, MO). PK and PP inhibitors were used at concentrations approximately 1000 times higher than their Ki values. Fura-2/acetoxymethylester was purchased from Invitrogen (Carlsbad, CA). Polyclonal antibodies against human PLCB1, PLCB3, human PRKA catalytic subunit, and horseradish peroxidase-conjugated donkey anti-rabbit secondary antibody (1:2000) were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Polyclonal antibody against phosphorylated PLCB3-S1105 was produced for us by Cell Signaling Technology (Beverly, MA). Enhanced chemiluminescence (ECL) reagent was obtained from Amersham Biosciences (Piscataway, NJ). Primers were purchased from Integrated DNA Technologies, Inc. (Coralville, IA). iScript One-Step RT-PCR Kit with SYBR Green and AG 1-X8 resin were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA). Restriction enzymes were obtained from New England Biolabs Inc. (Beverly, MA) or Promega (Madison, WI). SureSilencing short hairpin RNA (shRNA) plasmids for human PLCB3 (no. KH02817G) were obtained from SuperArray Bioscience Corporation (Frederick, MD). The cAMP ELISA kit was obtained from Assay Designs, Inc. (Ann Arbor, MI). AG 1-X8 resin was obtained from Bio-Rad Laboratories, Inc. Nitrocellulose membranes were purchased from Whatman Schleicher & Schuell (Florham Park, NJ).

Primary uterine smooth muscle cells (UtSMC) were obtained from Cambrex (Walkersville, MD). AD293 cells were obtained from Stratagene (La Jolla, CA). Human myometrial preterm labor samples were obtained at the time of cesarean section under approved institutional review board protocols at the University of Texas Medical School at Houston (B.M. Sanborn and M. Monga) and the University of Texas Southwestern Medical School (B.M. Sanborn and R.A. Word).

PLCB3 Adenoviral and psi-CHECK-2 Reporter Construction

Complementary DNAs encoding human PLCB3 and PLCB3-S1105A were excised from pCR3.1 vectors [8] and subcloned into the NotI/XhoI sites of the pAdTrack-CMV shuttle vector (ATCC, Manassas, VA; Stratagene, LaJolla, CA [pAdeasy]). These vectors were used to produce adenoviral vectors expressing PLCB3 or the S1105A mutant according to the manufacturer's instructions. The multiplicity of infection (MOI) for each adenovirus was determined in AD293 cells, as recommended by the manufacturer.

For use in shRNA silencing experiments, the PLCB3 sequence was subcloned into the SgfI/PmeI sites of the psi-CHECK-2 vector (Promega), located downstream of the reporter gene (Renilla luciferase). The resulting PLCB3-psi-CHECK-2 (psiPLCB3) construct was used in reporter assays.

Cell Culture

Immortalized PHM1–41 cells derived from pregnant human myometrial tissue (passages 19–23) [19], UtSMC, and AD293 cells were cultured in Dulbecco modified Eagle medium (DMEM) containing 10% fetal calf serum (FCS), 2 mM L-glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin. Myometrial cells were trypsinized and plated (2 x 105 cells/well) into 6-well plates for PI turnover and cAMP assays, or into 35-mm dishes (1–2 x 105 cells/dish) for the PLCB3 phosphorylation assay.

Viral Infection and Plasmid Electroporation

In overexpression studies, adenoviral constructs expressing PLCB3 or the S1105A mutant were added at an MOI of 1000 to PHM1 cells at the time of plating, and the cells were used 72 h later. Under these conditions, the infection efficiency was ~100%, estimated by expression of green fluorescent protein (GFP) from a second promoter. Infected PHM1 cells exhibited similar morphology and PI turnover in response to OXT as noninfected cells.

In RNA interference experiments, SureSilencing shRNA plasmid DNA (4 µg) was added to 100 µl of Basic Nucleofector Solution (Basic Nuclefection kit, no. VPI-1004; Amaxa Biosystems, Gaithersburg, MD) and was electroporated into 1 x 106 PHM1 cells. Cells were pulsed at 140 V/35 msec/2 mm cuvette with time-constant protocol by using the Genepulser Xcell (Bio-Rad Laboratories, Inc.). After electroporation, cells were plated into dishes and were cultured for 72 h before measurement of intracellular calcium. Transfection efficiency was estimated by fluorescence of modified GFP (mGFP) expressed in the vector from a second promoter.

In the experiments using psi-CHECK-2 system, psiPLCB3 (0.5 µg) was cotransfected with PLCB3-shRNA plasmids (1.5 µg) into AD293 cells (1 x 105 cells/12-well plate) with GenePORTER 2 transfection reagent (Genlantis, San Diego, CA), as recommended by the manufacturer. Samples were analyzed 72 h after transfection by using the Dual-Luciferase Reporter Assay (Promega, Madison, WI). Firefly luciferase activity, driven by a second promoter, allowed normalization of Renilla luciferase expression.

Measurement of Intracellular Calcium

Cells were loaded at room temperature with 5 µM of Fura-2 for 30 min in fluorescence buffer (145 mM NaCl, 5 mM KCl, 1 mM Na2HPO4, 0.5 mM MgCl2, 1mM CaCl2, 10 mM Hepes, and 5 mM glucose, pH 7.4). After loading, the cells were washed twice in the same buffer and, after 30 min, were placed in Ca2+-free buffer (substitution of 100 µM EGTA for 1 mM CaCl2 in fluorescence buffer). Changes in individual cell intracellular free Ca2+ concentration after the addition of 100 nM OXT were measured at 340- and 380-nm excitation and 510-nm emission (Intracellular Imaging, Inc., Cincinnati, OH). The individual responses of 20–36 cells in 1 dish were aligned and averaged with the CalciumComp program produced for us by an engineering consultant (K.J. Bois, Fort Collins, CO).

Membrane Preparation and Protein Phosphorylation Analysis

PHM1 cells and UtSMC were cultured [20] and harvested either as total cell lysates or used to prepare crude membranes (PHM1 cells). For whole-cell lysates, PHM1 cells were harvested in 200 µl lysis buffer (50 mM NaCl, 0.3% Triton X-100). To prepare membranes, cells were collected in chilled hypotonic buffer (20 mM Hepes, pH 7.4, 10 mM EDTA) containing 1.04 mM 4-(2_aminoethyl) benzenesulfonyl fluoride hydrochloride, 0.8 µM aprotinin, 21 µM leupeptin, 36 µM bestatin, 15 µM pepstatin A, 14 µM N-(trans-epoxysuccinyl)-L-leucine 4-guanidinobutylamide (E-64). The collected cells were lysed by passing through a 0.5 inch needle (27 gauge). Cell debris was removed by centrifugation at 3000 x g for 15 min at 4°C. Crude membrane fractions were prepared by centrifugation at 100 000 x g for 1 h at 4°C, resuspended in PBS (2.68 mM KCl, 1.47 mM KH2PO4, 136.9 mM NaCl, 8.1 mM Na2HPO4, pH 7.2), and stored at –80°C. Protein concentration was quantified by the Bradford method (Bio-Rad Laboratories, Inc.).

Total cell lysates (10–40 µl) or membrane proteins (10 µg) were separated on 6% SDS-PAGE gels and transferred onto nitrocellulose membranes. Phosphorylated PLCB3-S1105 was detected with anti-P-S1105 antibody (1:250-1000) with the ECL reagent. Following stripping, total PLCB3 was probed with anti-PLCB3 (1:1000) antibody on the same blot. In overexpression experiments, PLCB3 and PRKA catalytic subunits (1:2000) were detected sequentially on the same blot with different antibodies. PRKA served as an internal control and did not change with treatment. ECL signals were detected with a Storm imager and quantitated with ImageQuant TL software (Amersham Biosciences).

In Vitro PLCB3 Phosphorylation

Purified recombinant PLCB3(His)6 (1 µg) and S1105/A-PLCB3 (1 µg) [8] were incubated with or without PRKA-cat subunit (69 µM) in PRKA buffer (10 mM Tris-HCl, pH 7.0, 5 mM MgCl2, 0.1 mM ATP) for 30 min at 30°C, and the reactions were terminated by addition of 4x SDS sample buffer (final concentration, 62.5 mM Tris-HCl, pH 6.8, 4 M urea, 10% glycerol, 2% SDS, 0.01% β-mercaptoethanol, 0.002% bromophenol blue) and boiled for 5 min. Proteins were separated on SDS-PAGE (4%–15%) and were transferred to nitrocellulose membranes. The blots were probed with primary (1:500) and secondary antibodies, and the bands visualized by ECL. Dephosphorylation of purified recombinant PLCB3(His)6 (1 µg) was carried out at 37°C for 1 h in 50 mM Tris-HCl, 10 mM MgCl2, 100 mM NaCl, 1 mM dithiothreitol, pH 7.9, with calf intestine alkaline phosphatase (1 U/µg protein) from New England Biolabs Inc.

PLCB Isoform RNA and Protein Determination

RNA was isolated from PHM1 and UtSMC cells with the RNeasy kit (QIAGEN, Valencia, CA) and from human myometrium tissue with TRIZOL reagent (Invitrogen). Primers for PLCB1, PLCB2, PLCB3, PLCB4, and the RNA normalizer, hydroxymethylbilane synthase (HMBS) are presented in Table 1. RT-PCR was performed with the iScript one-step RT-PCR kit with the SYBR Green protocol in a 25-µl reaction volume with 500 ng RNA (denatured at 95°C for 15 min and subjected to 35 cycles of denaturation at 94°C for 15 sec, annealing for 30 sec at 58°C for PLCB1, PLCB2, and PLCB3, and for 60°C for PLCB4, and extension at 72°C for 1 min) in an iCycler (Bio-Rad Laboratories, Inc.). The identities of the products were confirmed by sequencing. Melting curves for PLCB1, PLCB2, PLCB3, PLCB4, and HMBS cDNA exhibited one peak. Abundance was determined by the {Delta}{Delta}Ct method, in which data were normalized to the housekeeping gene, HMBS, in the same sample and expressed relative to PLCB3.


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  		TABLE 1. PCR primers and shRNA constructs.

Quantitation of PLCB1 and PLCB3 protein was determined by Western blot analysis in reference to standard curves generated using known quantities of purified recombinant PLCB1(His)6 and PLCB3(His)6 [8].

PI Turnover

One day prior to the experiments, PHM1 cells were washed twice with PBS and cultured in 1 ml DMEM, 0.5% FCS, and 0.4 µM [3H]-myoinositol at 37°C overnight. The labeled cells were washed twice with PBS and were incubated with Hanks balanced salt solution (HBSS, pH 7.4) containing 0.2% BSA, 10 mM LiCl, and the indicated inhibitors of protein kinases or phosphatases at 37°C for 30 min. The cells were then stimulated for 30 min with 30 nM OXT. Total [3H]-IPs were isolated by ion exchange chromatography and counted as described previously [21].

cAMP Assay

PHM1 cells were washed twice with PBS and preincubated with HBSS for 1 h at 37°C. The cells were stimulated with 10 nM CALCA for the intervals indicated in the figure legends at 37°C. The medium was removed and the reaction stopped by the addition of 0.5 ml of 0.1 N HCl. Cyclic AMP content in the cell lysate was measured by immunoassay following the protocol recommended by the manufacturer (Assay Designs, Inc., Ann Arbor, MI).

Data Analysis

Dose-response curves were analyzed with a four-parameter logistics curve-fitting program (M.L. Jaffe, Silver Spring, MD). Where indicated, data are presented as mean ± SEM and were analyzed by ANOVA and Duncan modified multiple range test.

RESULTS

PLCB3 Is the Predominant Isoform in Human Myometrial Cells

To examine the relative expression of PLCB isoforms, we carried out both RNA (real-time quantitative RT-PCR) and protein (Western blot) analysis. PLCB3 mRNA was expressed at 1.6- and 2-fold greater relative abundance than PLCB1 in PHM1 and UtSMC cells, respectively (Fig. 1, A and B). In contrast, PLCB2 and PLCB4 mRNAs were barely detectable in these samples.


Figure 01
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  		FIG. 1 Comparison of the expression of PLCB isoforms in human myometrial cells. Relative expression of PLC mRNA in PHM1 (A) and UtSMC (B), as analyzed by quantitative RT-PCR. Cycle threshold (Ct) values were normalized to HMBS and expressed relative to PLCB3 using the {Delta}{Delta}Ct method (mean ± SEM; n = 6 for PLCB1, PLCB2, and PLCB3; and n = 3 for PLCB4 in PHM1 cells; n = 4 for all PLC isoforms in UtSMC). C) Expression of PLCB1 (open bars) and PLCB3 (black bars) protein in PHM1 and UtSMC cell lysates. Concentrations of PLCB1 and PLCB3 in PHM1 cells were 1.6 ± 0.4 and 20.2 ± 7.8 ng/µg cell lysate protein, respectively (n = 4), and in UtSMC were 0.8 ± 0.1 and 17.2 ± 4.8 ng/µg of cell lysate protein, respectively (n = 3). Representative standard curves for UtSMC cell lysates are shown on the right. A–C) Data were analyzed by ANOVA; significant differences (P < 0.05) between groups are designated with different lowercase letters.

Previous studies had detected PLCB1 and PLCB3, but not PLCB2, protein in PHM1 cells [22]. Since we had recombinant PLCB1 and PLCB3 available, we were able to quantitate PLCB1 and PLCB3 protein in reference to standard curves. PLCB3 was significantly more abundant than PLCB1 in both PHM1 and UtSMC cells (Fig. 1C). The PLCB3/PLCB1 ratios were 12.6 ± 2.0 and 21.2 ± 4.9 in PHM1 and UtSMC cells, respectively. In three pregnant, late-term human myometrial tissue homogenates, the PLCB3:PLCB1 concentration ratios were 1.3, 2.0, and 16.0 (data not shown), suggesting some patient-to-patient variability.

SureSilencing short hairpin RNA interference was used to probe the functional importance of PLCB3 to OXT signaling in myometrial cells. Prior experience has indicated that efficiencies for conventional transfection are very poor in these cells. Therefore, we used an electroporation protocol. Plasmids expressing a commercial PLCB3 shRNA (PLCB3sh1) or scrambled sequence were expressed at ~40%–50% efficiency in PHM1 cells following electroporation, as judged by mGFP fluorescence. There were no significant differences in calcium responses to OXT between cells electroporated without adding DNA and cells electroporated with plasmid expressing scrambled shRNA (Fig. 2A). Individual PHM1 cells electroporated with PLCB3sh1 shRNA and selected by mGFP fluorescence showed a marked decrease in the ability of OXT to increase intracellular free calcium compared with the scrambled control (43.1% ± 11.5% decrease in the initial rise in intracellular calcium, 39.5% ± 11.2% decrease in the integrated calcium transient) (Fig. 2A). Since the experiment was conducted in the absence of extracellular calcium, primarily the Gq/PLC/IP3-mediated response was being measured. Because of the relatively low electroporation efficiency, it was not possible to accurately assess the amount of suppression of PLCB3 expression by conventional methods in PHM1 cells. However, we constructed a PLCB3 psi-CHECK-2 reporter assay in order to assess the amount of PLCB3 mRNA suppression in AD293 cells. Figure 2B shows that PLCB3sh1 suppressed PLCB3 reporter expression by 49% ± 2.2 % in transfected AD293 cells. We have observed >85% knockdown of other reporters by their corresponding shRNA plasmids in this assay (A. Ulloa and D.M., unpublished observations), suggesting that PLCB3sh1 may not be an optimal sequence. Although the knockdown achieved with this commercial plasmid was only partial, the effect of PLCB3sh1 on OXT-stimulated calcium transients supports the contention that the PLCB3 isoform plays a significant role in OXT-stimulated signal transduction.


Figure 02
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  		FIG. 2. The OXT-stimulated increase in intracellular calcium in PHM1 myometrial cells was significantly suppressed by PLCB3 shRNA. A) PHM1 cells were electroporated with plasmids expressing no DNA, scrambled shRNA, or PLCB3sh1 as described in Materials and Methods. Fura-2 loaded cells were stimulated with OXT (100 nM) in the absence of intracellular calcium. The data represent the mean ± SEM of data from all cells selected for mGFP expression. The OXT response is reported as the peak height of the initial calcium increase and integrated area of the calcium transient, relative to scrambled control, for 185 (no DNA, gray bars), 131 (scrambled sequence, black bars), and 141 (PLCB3 shRNA, open bars) individual cells collected in three separate experiments. B) The effectiveness of PLCB3sh1 to suppress PLCB3 mRNA was assessed in AD293 cells by using a PLCB3 psi-CHECK-2 reporter luminescence. The reporter (0.5 µg) and other plasmids (pUC19 plasmid DNA, plasmid expressing scrambled shRNA sequence [scrambled], or PLCB3sh1, 1.5 µg) were transfected into AD293 cells as described in Materials and Methods. The data represent the mean ± SEM for the ratio of luminescence of Renilla luciferase/Firefly luciferase in one experiment (n = 3). Data in (A) and (B) were analyzed by ANOVA; significant differences (P < 0.05) between groups are designated with different lowercase letters.

Activation of Either Endogenous PRKA or PRKC Results in PLCB3-S1105 Phosphorylation

PLCB3-S1105 represents a potential site for regulation by both G{alpha}q/11-stimulated contractant and cAMP-mediated relaxant pathways. To determine if regulation of PLCB3 phosphorylation by these signaling pathways is operable in intact myometrial cells, we arranged for a rabbit polyclonal antibody against the PLCB3-P-S1105 sequence to be produced. Figure 3A shows that this antibody reacts only weakly with recombinant PLCB3, but detects this protein after it is phosphorylated in vitro with PRKA. In contrast, the antibody does not recognize an epitope in the recombinant S1105A mutant. The weak immunoreactivity observed in the absence of PRKA treatment probably represents residual endogenous S1105 phosphorylation in the isolated protein, as it was reduced by >90% by treatment with alkaline phosphatase (data not shown).


Figure 03
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  		FIG. 3. PRKA activation by CPT-cAMP increases phosphorylation of endogenous PLCB3-S1105 in PHM1 cells. A) The specificity of the anti-P-S1105 antibody for phosphorylated PLCB3-S1105. Recombinant PLCB3 and the S1105A mutant proteins were treated with PRKA catalytic subunit as described in Materials and Methods and examined by immunoblot. The anti-P-S1105 antibody detected increased phosphorylation of PLCB3 but not the S1105A mutant after PRKA treatment. The membrane was reprobed for detection of total PLCB3 (anti-PLCB3) after stripping. B) Dose dependence of phosphorylation of crude membrane PLCB3-S1105 following CPT-cAMP treatment for 10 min (mean ± SEM; n = 4). Data were analyzed by ANOVA. Asterisks indicate significant difference from solvent control (P < 0.05).

Treatment of PHM1 cells with CPT-cAMP for 10 min induced a dose-dependent increase in P-S1105 in crude membranes (Fig. 3B); the EC50 of this response was 0.21 ± 0.02 mM. To determine if an increase in endogenous cAMP would induce a similar P-S1105 response, effects of hormones that are able to increase intracellular myometrial cAMP, such as relaxin and CALCA, were examined. Treatment with 1.6 nM relaxin for 15 min induced P-S1105, and this was partially inhibited by the PRKA inhibitor, Rp-8-CPT-cAMPS (Fig. 4A). Treatment with 10 nM CALCA induced a transient increase in intracellular cAMP and in P-S1105 (Fig. 4B) with an EC50 of 0.33 ± 0.14 nM (Fig. 4C). Pretreatment with the PRKC inhibitor, Go6976, lowered basal phosphorylation, but did not alter the net response to CALCA-induced P-S1105 (Fig. 4C). Stimulation of P-S1105 by 10 nM CALCA was also observed in primary UtSMC cells (Fig. 4D).


Figure 04
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  		FIG. 4. Phosphorylation of endogenous PLCB3-S1105 by hormones that increase endogenous cAMP in PHM1 cells. A) Sensitivity of relaxin (RLN)-induced P-S1105 to Rp-CPT-cAMPS (1 mM). C, Solvent control. Data are expressed as net relaxin-stimulated phosphorylation (n = 3) and were analyzed by paired Student t-test. Significant difference compared to relaxin treatment (P < 0.05) is indicated by asterisks. B) Time-course of CALCA (10 nM)-induced P-S1105 and cAMP accumulation (n = 3). C) Dose dependence and lack of effect of PRKC inhibition by Go6976 (1 µM) on net CALCA-induced P-S1105 (n = 4). Data from (B) and (C) are expressed as the mean ± SEM and were analyzed by ANOVA. Asterisks indicate significant difference from control (P < 0.05). D) Increased P-S1105 in UtSMC in response to 10 nM CALCA and 30 nM OXT. Data represent mean ± SEM of triplicate determinations in one of four (CALCA) or three (OXT) similar experiments and were analyzed by ANOVA. Asterisks indicate significant difference from solvent control (P < 0.05). C, Solvent control.

OXT also rapidly increased P-S1105, with an EC50 of 11.9 ± 6.9 nM (Fig. 5, A and B). Pretreatment with the PRKC inhibitor, Go6976 (1 µM), completely eliminated OXT-induced phosphorylation (Fig. 5C). Consistent with an effect mediated by PRKC activation, treatment overnight with 1 µM PMA to desensitize PRKC eliminated the effect of OXT on P-S1105 (Fig. 5D). OXT also stimulated P-S1105 in UtSMC (Fig. 4D).


Figure 05
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  		FIG. 5. OXTR activation increases endogenous PLCB3-S1105 phosphorylation by a PRKC-dependent pathway in PHM1 cells. A) Time-course of P-S1105 following treatment with OXT (100 nM) (n = 6). B) Dose-dependent increase in P-S1105 following OXT treatment for 5 min (n = 4). Data are expressed as OXT-stimulated phosphorylation relative to solvent control. C) Sensitivity of OXT-induced P-S1105 to Go6976 (1 µM) (n = 3). D) Reduction in OXT-induced P-S1105 after desensitization of PRKC by exposure for 24 h to PMA (1 µM) (n = 3). All data are expressed as mean ± SEM and were analyzed by ANOVA. A and B) Asterisks indicate significant difference from the first point in each graph (P < 0.05); (C and D) significant differences between groups (P < 0.05) are designated with different lowercase letters.

In addition to stimulation by OXT, P-S1105 was increased by stimulation of other G protein-coupled receptors. Figure 6A shows that treatment with PGF2{alpha} and ATP significantly increased P-S1105 in PHM1 cells. The effect of ATP was inhibited >90% by the P2Y receptor antagonist, Reactive Blue-2 (100 µM) (data not shown), confirming that the effect of ATP was G protein-coupled receptor-mediated.


Figure 06
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  		FIG. 6. G protein-coupled receptor activation increases endogenous PLCB3-S1105 in PHM-1 cells. A) Cells were exposed to PGF2{alpha} (100 nM), ATP (100 µM), or OXT (30 nM) for 5 min. Data represent mean ± SEM of triplicate determinations in one experiment. The effects of PGF2{alpha} and ATP were replicated in one and two other experiments, respectively. Significant differences between groups (P < 0.05) are designated with different lowercase letters. B) Diagrammatic representation of negative crosstalk from the cAMP/PRKA pathway and negative feedback from the PLC/PRKC pathway focused on PLCB3-S1105 in myometrial cells. According to this scheme, inhibition of PRKA or PRKC would enhance PLCB3 activity, whereas inhibition of PPs would reduce PLCB3 activity.

CALCA- and OXT-Induced PLCB3-S1105 Phosphorylation Are Sensitive to Different Phosphatase Inhibitors

To determine which type of phosphatase is responsible for reversing CALCA- and OXT-stimulated phosphorylation of PLCB3 (Fig. 6B), the effects of okadaic acid, an inhibitor with high affinity for PP1/PP2A phosphatases and cypermethrin, an inhibitor with high affinity for PP2B, were assessed at concentrations used by others to distinguish the effects of PP1/PP2A and PP2B [23–26]. Since Go6976 pretreatment did not interfere with the net response to CALCA, but reduced basal P-S1105 and, therefore, enhanced detection of stimulated changes in P-S1105, PHM1 cells were pretreated with this inhibitor for 1 h prior to these experiments. As shown in Figure 7A, P-S1105 levels in cells treated with CALCA (10 nM), with or without prior exposure to 2 µM okadaic acid, returned to basal levels after 10 min. In contrast, the response to CALCA was relatively persistent for 15 min in cells pretreated with 4 nM cypermethrin. On the other hand, the decrease in OXT-stimulated PLCB3-S1105 was attenuated more by okadaic acid than it was by cypermethrin (Fig. 7B). These data are consistent with a predominant influence of PP2B on PRKA-mediated S1105 phosphorylation and a predominant influence of PP1/PP2A on OXT-mediated phosphorylation of PLCB3-S1105 in PHM1 cells.


Figure 07
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  		FIG. 7. The effect of PP inhibitors on CALCA- and OXT-induced PLCB3-S1105 phosphorylation. A) PHM1 cells were pretreated with Go6976 (1 µM) for 1 h before treatment with either okadaic acid (2 µM) or cypermethrin (4 nM) for an additional 40 min prior to treatment with 10 nM CALCA. B) PHM1 cells were pretreated with either okadaic acid (2 µM) or cypermethrin (4 nM) for 40 min prior to treatment with 100 nM OXT. Data points within a given experiment were normalized to the maximal response to CALCA (A, closed circles) or OXT (B, closed circles) in that experiment in order to allow pooling of data between experiments. Data are expressed as the mean ± SEM (n = 3~4) and were analyzed by ANOVA. Differences at a given time interval between phosphatase inhibitor treatment and the corresponding CALCA (A, closed circles) or OXT (B, closed circles) control are indicated by an asterisk (P < 0.05). CY, Cypermethrin; OA, okadaic acid.

Roles of PRKA, PRKC, and PPs in the Regulation of OXT-Induced PI Turnover

PLCB3 has several phosphorylation sites. To address the link between specific phosphorylation of S1105 and OXT-stimulated PLC activity, we constructed adenoviruses that express PLCB3 or the S1105A mutant and infected PHM1 cells. As shown in the inset in Figure 8A, both PLCB3 and the S1105A mutant were overexpressed in total cell lysates from infected cells compared with those infected with empty adenovirus, whereas PRKA catalytic subunit was expressed at a similar concentration in all groups. In all groups, basal PLC activity, as reflected in PI turnover, was comparable (data not shown). OXT treatment significantly stimulated PI turnover in cells infected with empty adenovirus and viruses expressing either PLCB3 or the S1105A mutant (Fig. 8A). While enhancement of OXT-stimulated PI turnover was occasionally observed with overexpression of the S1105A mutant, it is not clear why this was neither consistent nor statistically significant overall. CPT-cAMP (1.5 mM) pretreatment reduced OXT-stimulated PI turnover by ~50% in cells infected with either empty adenovirus or the adenovirus expressing PLCB3. Importantly, this inhibitory effect was not observed in cells overexpressing the S1105A mutant (Fig. 8A). These data suggest that a significant proportion of the inhibition by PRKA of OXT-induced PI turnover in these cells can be attributed to effects on PLCB3-S1105 (Fig. 6B).


Figure 08
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  		FIG. 8. Both PRKA-mediated negative crosstalk and PRKC-mediated negative feedback mechanisms regulate OXT-induced PI turnover in PHM1 cells. A) Phosphorylation of PLCB3-S1105 contributes to the inhibition by PRKA of net OXT-induced PI turnover. PHM1 cells were pretreated with 1.5 mM CPT-cAMP for 20 min before stimulation with 30 nM OXT. The inset demonstrates overexpression of PLCB3 and the S1105A mutant in PHM1 cells, detected by an antibody that recognizes both forms. PRKA catalytic subunit (PRKA) was analyzed in the same blot as a loading control. B) Both PRKC and PP1/PP2A influence the amount of OXT-stimulated PI turnover. PHM1 cells labeled with 3H-inositol were exposed to dimethyl sulfoxide, Go6976 (1 µM), okadaic acid (2 µM), or cypermethrin (4 nM) in the presence of 10 mM LiCl for 20 min prior to stimulation with 30 nM OXT (OXT) for 30 min and PI turnover determined. OXT-stimulated PI turnover is expressed as the mean ± SEM (n = 3~4) and data were analyzed by ANOVA. Significant differences between groups (P < 0.05) are designated with different lowercase letters. CY, cypermethrin; OA, okadaic acid.

We next attempted to determine if effects on P-S1105 were reflected in influences on OXT-stimulated PI turnover in PHM1 cells and assessed effects of phosphatase inhibition on this parameter. OXT induced a significant increase in PI turnover (Fig. 8B). Pretreatment with the PRKC inhibitor, Go6976 (1 µM), had no effect on basal PI turnover (data not shown), but significantly enhanced OXT-stimulated PI turnover, as might be expected if negative feedback from OXT-stimulated PRKC activity were inhibited (Fig. 6B). In contrast to effects on CALCA-stimulated P-S1105, okadaic acid (2 µM) significantly reduced OXT-stimulated PI turnover, whereas cypermethrin (4 nM) had no effect (Fig. 8B). These data are consistent with the interpretation that negative feedback by PRKC has an impact on the OXT-PLC-PI turnover pathway in PHM1 cells, and that the reversal of PRKC-mediated P-S1105, primarily by PP1/PP2A, partially attenuates this pathway (Fig. 6B).

DISCUSSION

Control of PLCB3 covalent modification by multiple pathways could play an important role in the regulation of myometrial calcium dynamics. We demonstrate here that PLCB3 is the predominant PLCB isoform in the human myometrial cells used, and that suppression of this protein by shRNA significantly reduces the initial rise in intracellular calcium induced by OXT. We show that site-specific phosphorylation of PLCB3-S1105 occurs in intact myometrial cells in response to diverse hormonal stimuli, and that specific PPs are involved in reversing these effects.

Cyclic AMP is known to promote relaxation in smooth muscle [15]. We observed increases in P-S1105 in PHM1 cells in response to treatment with CPT-cAMP and with two hormones (CALCA and relaxin) that have receptors in myometrium and that increase cAMP [27–30]. The effect of relaxin was partially inhibited by Rp-8-CPT-cAMPS. Taken together, these data are consistent with an effect of G protein-coupled receptor/G{alpha}s pathways on the PRKA-mediated increase in P-S1105 in PHM1 cells. Inhibition by cAMP of OXT-induced PI turnover has been observed in pregnant rat uterus, but not at term, coincident with a partial loss of membrane- and AKAP-associated PRKA [16, 17, 31, 32]. PRKA inhibited GTP{gamma}S-stimulated PI turnover in rat myometrial plasma membrane, indicating that this effect was distal to possible effects on the OXTR itself [18]. These data support the presence of negative crosstalk from the receptor/G{alpha}s/adenylyl cyclase/PRKA pathway that contributes to the inhibition of the OXTR/G{alpha}q/PLCB3 pathway via phosphorylation of PLCB3-S1105 (Fig. 6B).

CPT-cAMP pretreatment significantly reduced OXT-induced PI turnover in PHM1 cells infected with empty adenovirus, as well as in cells overexpressing PLCB3, but had no effect on PI turnover in cells overexpressing the S1105A mutant, consistent with the importance of S1105 as a target for PRKA. Overexpression of either PLCB3 or the S1105A mutant did not enhance basal or OXT-stimulated PI turnover. These data are consistent with the lack of constitutive activity of overexpressed PLCB3 [8], and support the concept that activated G{alpha}q and Gβ{gamma} are probably limiting factors in OXT-stimulated PI turnover. Overexpression of PLCB3 and the S1105A mutant should not have any impact on signal pathways upstream of PLCB coupling and activation, such as the phosphorylation of regulator of G protein signaling 4 and adrenergic beta receptor kinase 1, also known as G protein-coupled receptor kinase 2 proteins [33].

In addition to negative signaling pathway crosstalk targeted at PLCB3-S1105, our data provide evidence for negative feedback by downstream components of the PLCB pathway itself in intact cells (Fig. 6B). OXT stimulated phosphorylation of PLCB3-S1105, and this was inhibited by the PRKC inhibitor, Go6976, or by prior treatment with PMA to desensitize PRKC. Importantly, Go6976 pretreatment also enhanced OXT-induced PI turnover, which would be expected if negative feedback elicited by PRKC were inhibited. This implies that downstream effects of OXT on calcium release and entry may be influenced by the phosphorylation status of PLCB3. Interestingly, the PRKC inhibitor, Go6976, decreased basal P-S1105. These data suggest that PRKC activity also contributes to basal phosphorylation of PLCB3-S1105. Basal PLCB3 phosphorylation was noted in 32P-labeled cells in previous studies, but the specific sites were not defined [8, 12].

Conventional PRKC isozymes (A, B, G) are activated by both DAG and calcium, whereas novel PRKC isozymes (D, E, H, Q, M) are activated by DAG, but are insensitive to calcium, and atypical PRKC isozymes are not regulated by either of these second messengers [34]. Both PRKCA and PRKCD have been identified as downstream components of OXTR activation in pregnant human myometrium [35]. Downregulation of the PRKC effect by overnight treatment with PMA is consistent with involvement of conventional or novel PRKC isozymes. According to data provided by the supplier, PRKCD is not inhibited by the concentration of Go6976 used in this study, suggesting that the effects noted are most likely due to the action of conventional PRKC isozymes.

The dynamics of protein phosphorylation are controlled by both protein kinases and PPs, as well as by multiple protein-protein interactions that result in the formation of localized signaling complexes [36, 37]. The inhibitory effect of cAMP on PI turnover in myometrial cells requires interaction with an AKAP PRKA anchoring protein. AKAP5 (AKAP79) is expressed in myometrial membranes [17]. PLCB3-S1105 is an in vitro substrate for PP2B [16]. PP2B also binds to AKAP5, and has been implicated in local control of PRKA-stimulated phosphorylations [38]. In the present study, cypermethrin, which exhibits preferential specificity for PP2B, but not okadaic acid, which shows preferential specificity for PP1/PP2A, slowed the decay of PRKA-stimulated P-S1105. These data are consistent with the presence of a functional signaling complex involving AKAP, PRKA, and PP2B that exerts an influence on the phosphorylation status of PLCB3-S1105 when cAMP activates the PRKA pathway.

In contrast, okadaic acid, but not cypermethrin, slowed the decay of OXT (PRKC)-stimulated PLCB3-S1105 phosphorylation, implicating a role for PP1/PP2A in dephosphorylation in response to this stimulus. Consistent with these observations, okadaic acid inhibited OXT-induced PI turnover, whereas cypermethrin had no effect. Although PRKCs have multiple effects on the contractile apparatus as well as on the signaling pathways, the fact that okadaic acid does not affect basal contractile activity, but significantly impairs the ability of OXT to induce mouse myometrial contraction [39], is at least consistent with a negative effect of PP1/PP2A on OXT action. PP1 regulates many cell functions, with substrate targeting and specificity determined by its interaction with different regulatory units [40, 41]. PRKCs can also be targeted by interactions with scaffolding proteins [42, 43]. The data presented here suggest that PRKC-mediated PLCB3-S1105 phosphorylation may involve the close physical proximity of PP1 or PP2A with one or more of the other signaling components.

Three PLCB isoforms (1, 3, and 4) are expressed in detectable amounts in PHM1 cells (C.-Y. Ku, unpublished observations). On the basis of data presented here and previously [14, 18], it appears that a significant proportion of OXTR/G{alpha}q is coupled to PLCB3 in PHM1 cells, primary human cells, and rat myometrium. PLCB1 is not a substrate of PRKA, and may account for the PRKA-resistant PLC component of the activity elicited by OXT. PLCB4 has not been shown to be a target of PRKA or PRKC, and lacks a potential target sequence in the homologous region [11]. Although there is some evidence that receptors coupling to G{alpha}q can preferentially couple to a given PLCB subtype [44–46], there is no evidence to date that OXTR exhibits this ability. The OXTR can signal both within and outside membrane subdomains [47, 48], whereas PLCB1 and PLCB3 form complexes with scaffolding proteins or other signal components that may restrict their location in cells [49, 50].

There are numerous implications of these findings for uterine smooth muscle function. CALCA inhibits human myometrial contractility during pregnancy [51, 52]. Because of upregulation of relaxant G protein-coupled receptors, the Gs:Gi ratio, and adenylyl cyclase isoforms, the relaxant effects of cAMP-mediated pathways predominate during mid-pregnancy [16, 53, 54]. The inhibitory effect of CPT-cAMP on OXT-stimulated PI turnover in pregnant rat myometrium decreases shortly before the initiation of labor, in conjunction with a decrease in PRKA binding to AKAP5 (AKAP150) [16]. The PRKA-dependent phosphorylation of PLCB3-S1105 could potentially contribute to the dampening by cAMP of both OXT- and PGF2{alpha}-stimulated and spontaneous contractions in pregnant rat myometrium [55, 56]. While important, this is only one of several effects of PRKA that would favor relaxation [57]. On the other hand, at the end of pregnancy, contractant G protein-coupled receptors and Gq are upregulated, the phosphodiesterases that hydrolyze cAMP are also upregulated, and membrane PRKA diminishes, thus reducing the effectiveness of this relaxant pathway [1, 54, 58, 59]. The data presented here also indicate that PLCB3-S1105 phosphorylation occurs in response to OXT and PGF2{alpha} stimulation. Presumably, this pathway would become more important near the end of pregnancy, when contraction-related signaling components are upregulated [1, 54, 59]. The OXT-generated feedback pathway has effects on PI turnover and, hence, IP3 generation. OXT stimulates intracellular calcium oscillations in near-confluent PHM1 myometrial cells [60], and fluctuations in IP3 have been implicated in calcium oscillations, along with complex feedback loops regulating calcium pump activity [61–63]. As a result of downstream signaling through PLCB, OXT also stimulates calcium entry through receptor-operated pathways [57]. Therefore, regulation of OXT-stimulated PLC activity by PRKA- and PRKC-mediated pathways may have important implications for the nature of uterine contractile activity at different stages of pregnancy.

In summary, endogenous PLCB3 is phosphorylated on S1105 in intact PHM1 cells by both PRKA- and PRKC-dependent pathways, representing negative signaling pathway crosstalk and a negative feedback loop, respectively. Since the relative predominance of these two signaling pathways changes over gestation, their relative influence may be dependent on gestational stage.

ACKNOWLEDGMENTS

We acknowledge the contribution of J. Jamison in developing the PLCB PCR assay, A. Ulloa for advice on construction of the psi-CHECK PLCB3 reporter, and Dr. M. Monga (University of Texas Medical School at Houston) and Dr. R.A. Word (Human Biological Tissues and Fluid Repository, University of Texas Southwestern Medical Center at Dallas) for the human tissue samples.

FOOTNOTES

1Supported in part by National Institutes of Health grant NIH-HD09618. Back

Correspondence: 2Barbara M. Sanborn, Department of Biomedical Sciences, 102 Physiology Campus Delivery 1680, Colorado State University, Fort Collins, CO 80523. FAX: 970 491 7569; e-mail: Barbara.Sanborn{at}colostate.edu

3These authors contributed equally to this work. Back

Received: 20 July 2007.

First decision: 6 September 2007.

Accepted: 27 February 2008.

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K. Heng, R. Ivell, P. Wagaarachchi, and R. Anand-Ivell
Relaxin signalling in primary cultures of human myometrial cells
Mol. Hum. Reprod., October 1, 2008; 14(10): 603 - 611.
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biolreprod.107.064485v1
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