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Expression of protein kinase C isozymes in nonpregnant and pregnant human myometrium
Expression of protein kinase C isozymes in nonpregnant and pregnant human myometrium William W. Hurd, MD,a Victor P. Fomin, PhD,a Viswanathan Natarajan, PhD,b Haywood L. Brown, MD,a Robert M. Bigsby, PhD,a and Dawn M. Singh, BSa Indianapolis, Indiana OBJECTIVE: The aim of this study was to compare the distributions of protein kinase C isozymes in human nonpregnant and pregnant myometrial tissues and primary cell cultures. STUDY DESIGN: Myometrial tissues were obtained at hysterectomy from nonpregnant women and at cesarean delivery from women both before and during early labor at term. Western immunoblot analysis was performed on homogenates of myometrial tissues and primary cell cultures with monoclonal antibodies specific for protein kinase C isozymes. Redistribution and translocation of protein kinase C were examined by means of immunocytochemical methods. RESULTS: Nonpregnant myometrial tissues contained protein kinase C isozymes α, γ, δ, µ, ι, and ζ but not β1, β2, θ, or ε. Pregnant myometrial tissues both before and during early labor contained the same protein kinase C isozymes and also β1 and β2. The protein kinase C isozyme distribution in primary myometrial cell cultures was identical to that in the myometrial tissues. Protein kinase C redistribution and translocation were demonstrated in these cultured myometrial cells. CONCLUSION: Both human myometrial tissues and primary cell cultures expressed a broad range of protein kinase C isozymes. Protein kinase C isozymes β1 and β2 were absent in nonpregnant myometrium but were induced during pregnancy. Labor at term did not alter protein kinase C isozyme expression. (Am J Obstet Gynecol 2000;183:1525-31.)
Key words: Human, isozymes, myometrium, pregnancy, protein kinase C, uterus
The mammalian uterus is remarkable in that it is composed of individual smooth muscle cells that change dramatically but only temporarily, both morphologically and physiologically, during pregnancy. These changes allow the uterus to adapt to one or more rapidly growing fetuses until maturity, and then a period of coordinated uterine activity begins and culminates in delivery of these fetuses. However, the mechanisms that regulate contractility in the pregnant human uterus are only partially understood. An important aspect of this regulation is selective phosphorylation of intracellular proteins, which leads to their activation or inactivation. This phosphorylation is carried out by a large family of protein kinases, including protein kinase C (PKC). PKC plays an important role in transmembrane signal transduction in numerous mammalian cells. In many receptor and G-protein–mediated path-
From the Department of Obstetrics and Gynecologya and the Department of Internal Medicine,b Indiana University School of Medicine. Supported by National Institutes of Health grant HD36692. Received for publication June 8, 1999; revised February 8, 2000; accepted March 5, 2000. Reprint requests: William W. Hurd, MD, 550 N University Blvd, Room 2440, Indianapolis, IN 46202-5274. Copyright © 2000 by Mosby, Inc. 0002-9378/2000 $12.00 + 0 6/1/107783 doi:10.1067/mob.2000.107783
ways, PKC is activated by second messengers such as diacylglycerol and calcium ion.1 The role of PKC in the uterus remains uncertain. In the rat myometrium activation of PKC inhibits oxytocininduced contractility.2 In human myometrium, however, activation of PKC augments oxytocin-mediated myometrial contractility and has been hypothesized to play a role in the sustained stimulation of myometrial activity during labor.3 Either a stimulatory or an inhibitory effect of PKC on the myometrium could be important in the regulation of contractility. It has been uncertain what PKC isozymes are present in human myometrium. It is well appreciated that the distribution of PKC isozymes differs among tissue types. This may be important, because different PKC isozymes are known to differ in sensitivities to diacylglycerol, calcium ion, and phospholipids and also in substrate specificity.2, 4 In most tissue types both calcium ion– dependent and calcium ion–independent PKC isozymes are present. However, until now the distribution of PKC isozymes in human myometrium has been unknown. Likewise, the distribution of PKC isozymes in primary cultures of pregnant human myometrial cells has not previously been established. Because these cells are commonly used as a model to study mechanisms of signal transduction, the relative distributions in the tissues and cultured cells are important. 1525
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In this study we identified the PKC isozymes in nonpregnant and pregnant human myometrium. Western immunoblot analysis was used to identify the presence of both calcium ion–dependent and calcium ion–independent PKC isozymes in myometrial tissues and primary cell cultures. Redistribution of calcium ion–dependent and calcium ion–independent PKC isozymes was examined after exposure to either 12-O-tetradecanoylphorbol-13acetate (TPA) or oxytocin. Finally, oxytocin-stimulated translocation of PKC-α was examined in cultured myometrial cells by means of immunocytochemical methods. Our results indicate that both nonpregnant and pregnant human myometrial tissues contain a relatively broad spectrum of PKC isozymes and that PKC-β1 and PKC-β2 are induced during human pregnancy. Material and methods Tissue acquisition and preparation. All tissue specimens were obtained in accordance with a protocol approved by the university committee on human use in research. Nonpregnant myometrium was obtained from 4 premenopausal women undergoing hysterectomy; all had a grossly normal uterus and no malignancy. The primary diagnoses were chronic pelvic pain (n = 2) and endometriosis (n = 2). After the uterus was removed, a full-thickness section of the lower segment was removed under sterile conditions. After written consent was given, myometrium was obtained from 9 pregnant women at term undergoing cesarean delivery; 6 were not in labor and 3 were in early labor. Labor was defined as regular firm contractions resulting in progressive cervical dilatation to ≥4 cm. Women were excluded if they had any major complications of pregnancy (including hypertension, diabetes, and premature labor) or if they had been treated with any medications other than prenatal vitamins before delivery. At cesarean delivery a full-thickness strip of uterine tissue was taken from the upper margin of a lower transverse incision. Uterine tissues were transported to the laboratory on ice in a sterile Hanks balanced salt solution. The myometrium was separated from the endometrium and connective tissue with a scalpel with the aid of a dissecting microscope. The myometrium was either used for experiments immediately or processed for cell culture. Tissue samples from each patient were used for 2 different experiments. Tissue preparation. The myometrium was separated from the endometrium and cut into small pieces. Then 2 or 3 pieces (approximately 0.3 g) were put into each well of a 6-well plate containing 5 mL Dulbecco modified Eagle medium without fetal bovine serum. To demonstrate PKC redistribution duplicate wells were exposed to TPA (10–7 mol/L) or oxytocin (10–6 mol/L) in a carbon dioxide incubator at 37°C for 30 minutes. After the tissue specimen was washed twice with Hanks balanced salt so-
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lution, the pieces were blotted semidry and weighed. The pieces were frozen in liquid nitrogen and pulverized to a fine powder, which was resuspended in lysis buffer, homogenized, and centrifuged at 1000g for 20 minutes at 4°C. The supernatants were ultracentrifuged at 105,000g for 90 minutes at 4°C. The soluble (cytosolic) fraction was the resulting supernatant, and the particulate (membrane) fraction was the resulting pellet, which was resuspended in 1.0 mL buffer. Protein concentration was determined by the method of Lowry et al.5 Myometrial cell cultures. Primary myometrial cell cultures were established from pregnant myometrial specimens as previously described elsewhere.6 Briefly, the myometrium was minced into 1-mm3 pieces and digested in a solution of collagenase and deoxyribonuclease for 7 hours with intermittent trituration and filtration. The myometrial cells were separated from fibroblasts and other cells with a discontinuous Percoll density gradient (Amersham Pharmacia Biotech AB, Uppsala, Sweden) and found to have >90% myometrial cell viability by trypan blue exclusion. To verify that our cultured cells were myometrial cells rather than fibroblasts, we evaluated the ability of the myometrial cells to express the smooth muscle–specific protein α-actin by immunostaining with smooth muscle–specific monoclonal anti–α-actin (clone 1A4; Sigma, St Louis, Mo) by a method described by Skalli et al.7 The cells were plated on Petri dishes (35, 60, or 100 mm) at a concentration of 5 × 105 cells/cm2 and maintained at 37°C in a 5% carbon dioxide incubator until they reached 95% confluence. These primary cell cultures were serum deprived for 24 hours before use. Cultured human myometrial cells prepared by the described protocol have been extensively characterized morphologically, biochemically, and physiologically by other investigators.8 In primary culture these cells retain basic morphologic and physiologic properties of myometrial cells in the tissue. The cells also maintain a pattern of actin myofibril distribution that is identical to that of myometrial cells in tissue and is different from that of fibroblasts. The cells also produce approximately the same amount of prostaglandins and have the same activities and amounts of both myosin and myosin light-chain kinase as does myometrial tissue. Moreover, the myosin light-chain kinase in these cultured cells retains calciumcalmodulin dependence. These myometrial cells have been shown to respond to physiologic concentrations of oxytocin and endothelin 1 with increases in intracellular free calcium concentration.9 Myometrial cell culture preparation. Myometrial cell cultures were used 6 to 8 days after plating, after each 100-mm plastic culture dish had reached ≥95% confluence. The cells were placed in Dulbecco modified Eagle medium without fetal bovine serum the night before the experiment. To demonstrate PKC redistribution duplicate wells were exposed to TPA (10–7 mol/L) or oxytocin
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(10–6 mol/L) at 37°C for 30 minutes. The cells were washed twice with 3 mL ice-cold Hanks balanced salt solution, and 0.5 mL lysis buffer was added. The cells were scraped from the dishes, homogenized, and centrifuged at 1000g for 20 minutes at 4°C. The supernatants were transferred to different tubes and then ultracentrifuged at 105,000g for 90 minutes at 4°C. The resulting supernatant (soluble fraction) was transferred to an Eppendorf tube. The resulting pellet (particulate fraction) was resuspended in 0.6 mL lysis buffer. Protein concentration was determined by the Lowry method.5 PKC translocation and isozyme identification. Basal locations of PKC isozymes were determined in the soluble and particulate fractions by means of Western blot analysis.4 PKC activation was determined by translocation of PKC from the soluble fraction to the particulate fraction. Each gel lane was loaded with 20 µg protein from either the soluble or the particulate fraction. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis separation was performed in 4% to 20% gradient gels (Bio-Rad Laboratories Inc, Hercules, Calif). The separated proteins were transferred by electrophoresis onto nitrocellulose sheets and exposed to monoclonal antibodies specific for the classic PKC isozymes PKC-α, PKC-β1, PKC-β2, and PKC-γ; the novel PKC isozymes PKC-δ, PKC-ε, PKC-µ, and PKC-θ; and the atypical PKC isozymes PKC-ι and PKC-ζ. Because of reported cross-reactivity between the PKC-α and PKC-γ antibodies from the original supplier (Transduction Laboratories, Inc, Lexington, Ky), the experiments were repeated with highly specific monoclonal antibodies from a second company (Life Technologies, Inc, Rockville, Md). To verify specificity, binding was evaluated in the presence of the corresponding authentic peptide sequence obtained from the same source. Primary antibody exposure was followed by incubation with secondary antimouse immunoglobulin G (IgG) peroxidaseconjugated antibody (Sigma). The relative amount of immunospecific protein was determined by means of an enhanced chemiluminescence detection kit (Amersham Life Science, Arlington Heights, Ill). The bands were quantified with the Gel Documentation System (Alpha Innotech Corporation, San Leandro, Calif). Antibody specificity was verified by processing the blots without the primary antibodies. Immunocytochemical studies. Primary myometrial cell cultures were plated onto 4-well glass slides and were grown for 2 days or until 15% to 20% confluence. Twentyfour hours before the experiment the cell medium was replaced with Dulbecco modified Eagle medium without fetal bovine serum. The cells were treated with TPA (10–7 mol/L), oxytocin (10–6 mol/L), or bisindolylmaleimide I (10–5 mol/L) for 30 minutes at 37°C. The medium was removed, and the cells were fixed with 100% methanol and washed with phosphate-buffered saline solution plus 5% polysorbate. The slides were incubated in bovine serum
albumin–supplemented phosphate-buffered saline solution plus 5% polysorbate for 10 minutes. The slides were exposed to primary antibodies to PKC-α overnight at 4°C at a dilution of 1:25, as recommended by the manufacturer. The slides were washed with phosphate-buffered saline solution plus 5% polysorbate, and a fluorescent secondary antibody, Cy2–conjugated goat antimouse IgG, was added for 1 hour at room temperature. The secondary antibody was removed, and the slides were washed with phosphate-buffered saline solution plus 5% polysorbate, rinsed with water, and blotted. Glass coverslips were fixed to the slides, which were viewed with a Nikon Optiphot (Nikon Inc, Melville, NY) microscope and photographed with a Nikon FX-35A (Nikon Inc) camera. The specificity of the immunostaining was verified by incubating the slides in the medium that did not contain the primary antibody. The experiment was repeated 3 times with cell cultures derived from different patients. Material. Mouse monoclonal antibodies (IgG fractions) directed against isozyme-specific carboxy terminal peptide sequences of PKC isozymes were purchased from either Transduction Laboratories (PKC-α, PKC-γ, PKC-δ, PKC-ε, PKC-ζ, PKC-θ, PKC-ι, and PKC-µ) or Life Technologies (PKC-α, PKC-γ, PKC-β1, and PKC-β2). The PKC inhibitor, bisindolylmaleimide I, was obtained from Alexis (Alexis Corporation, San Diego, Calif). For immunocytochemical studies a fluorescent secondary antibody, Cy2–conjugated goat antimouse IgG, was obtained from Jackson ImmunoResearch Laboratories (Jackson ImmunoResearch Laboratories, Inc, West Grove, Pa). Antibodies to calponin and actin were obtained from Sigma. Other reagents were from standard commercial sources. Results PKC isozyme profiles. Human myometrial tissues and primary myometrial cell cultures derived from these tissue specimens were found to contain significant amounts of most of the known PKC isozymes (Fig 1). Nonpregnant myometrial tissues contained detectable quantities of 2 calcium ion–dependent classic PKC isozymes, PKC-α and PKC-γ. The other two classic PKC isozymes, PKC-β1 and PKC-β2, were not detected in any of the nonpregnant samples. Of the calcium ion–independent novel PKC isozymes, both PKC-δ and PKC-µ were present but neither PKC-θ nor PKC-ε was detected. Both of the atypical PKC isozymes investigated, PKC-ι and PKC-ζ, were detected in nonpregnant myometrium. In contrast, pregnant myometrial tissues obtained either before or after the onset of labor contained all 4 calcium ion–dependent classic PKC isozymes (PKC-α, PKCβ1, PKC-β2, and PKC-γ). The distribution of the remaining isozymes was identical to that in the nonpregnant tissues, with the presence of PKC-δ, PKC-µ, PKC-ι, and PKC-ζ isozymes and the absence of PKC-θ and PKC-ε
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Fig 1. Western immunoblot analysis of PKC isozymes in human myometrial tissues. Tissue specimens were obtained from nonpregnant women (n = 4) and pregnant women at term both before (n = 4) and after (n = 3) onset of labor. Primary cell cultures were derived from pregnant myometrium obtained from women before labor. Each lane was loaded with 20 µg protein. cPKC, Classic PKC; nPKC, novel PKC; aPKC, atypical PKC; T, myometrial tissue, C, primary cell cultures. Plus sign, Positive rat brain control (exception, Jurkat cell line for PKC-µ and PKC-θ). Numbers to right indicate apparent molecular weight of band of interest as calculated from molecular weight standards. Results are from representative myometrial tissue samples and corresponding primary myometrial cell cultures.
isozymes. Primary cell cultures derived from pregnant myometrium from patients before labor revealed a distribution of isozymes identical to that in the tissues from which they were derived. To verify that both PKC-α and PKC-γ were present, primary myometrial cell cultures were evaluated with highly specific antibodies from two different manufacturers, Transduction Laboratories and Life Technologies. Specificity was confirmed by incubation with the authentic peptide sequence to which the antibodies were raised, which resulted in complete disappearance of the bands (Fig 2). Redistribution. Under the experimental conditions of this study, classic PKC-α and novel PKC-δ were distributed in myometrial cells derived from pregnant myometrium almost equally between the soluble and particulate fractions in the resting state (Fig 3). Stimulation with either TPA or oxytocin for 30 minutes resulted in redistribution of both the calcium ion–dependent (classic PKC-α) and calcium ion–independent (novel PKC-δ) isozymes to the particulate fraction.
Immunocytochemical studies. In resting control myometrial cells, classic PKC-α showed a coarse, granular distribution in the cytosol (Fig 4, A). In approximately 75% of the cells in each well, exposure to oxytocin for 30 minutes resulted in translocation manifested by increased immunofluorescence along the cell membranes but not along the nuclear membrane (Fig 4, B). When the myometrial cells were treated in advance for 30 minutes with the specific PKC inhibitor bisindolylmaleimide I, no translocation was seen (Fig 4, C). Comment PKC represents a family of phospholipid-dependent kinases that were first described by Takai et al10 in brain tissue. Since that original report, PKC isozymes have been found in most tissue types; these isozymes differ in their kinetic properties, substrate specificities, and dependence on cofactors. In this study the human myometrium was found to contain significant amounts of most of the known PKC isozymes. In nonpregnant myometrial tissues 2 of the 4
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Fig 2. Western immunoblot analysis of PKC isozymes PKC-α and PKC-γ in primary myometrial cell cultures. Each lane was loaded with 20 µg protein. Monoclonal antibodies specific for PKC-α and PKC-γ isozymes were obtained from either Transduction Laboratories (Transduction) or Life Technologies (Gibco). Two lanes on right were incubated in advance with corresponding authentic peptide sequences. C, Cell culture membrane preparations. Plus sign, Positive rat brain control. Numbers to right indicate apparent molecular weight of band of interest as calculated from molecular weight standards. Experiments were repeated with cell cultures derived from 3 different patients. Results shown are from representative experiment.
Fig 3. Western immunoblot analysis demonstrating redistribution of PKC-α and PKC-δ in pregnant human myometrial tissue specimens (Tissue) and primary cell cultures (Cells) after exposure to TPA or oxytocin (OT). Each lane was loaded with 20 µg protein. S, Soluble (cytosolic) fraction; P, particulate (membrane) fraction. Plus sign, Positive rat brain control. Numbers to right indicate apparent molecular weight of band of interest as calculated from molecular weight standards. Experiments were repeated in tissues and cell cultures derived from 6 different patients. Results shown are from representative myometrial tissue sample and primary myometrial cell culture.
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Fig 4. Fluorescence microscopy of PKC-α in control myometrial cells (A) and cells that had been exposed for 30 minutes to oxytocin (10–6 mol/L) alone (B) or oxytocin plus PKC inhibitor bisindolylmaleimide I (10–5 mol/L; C). After fixation, cells were exposed overnight to mouse monoclonal primary antibodies to PKC-α, followed by fluorescent goat antimouse IgG secondary antibody. Experiments were repeated in cell cultures derived from 3 different patients. Approximately 75% of cells in each culture well exhibited changes shown in this representative experiment.
calcium ion–dependent classic PKC isozymes (PKC-α and PKC-γ) were found, and in pregnant myometrium and primary cell cultures all 4 (PKC-α, PKC-β1, PKC-β2, and PKC-γ) were found. Of the calcium ion–independent novel PKC isozymes, PKC-δ and PKC-µ were abundant in
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both nonpregnant and pregnant myometrium, whereas PKC-θ and PKC-ε were not detectable. Both of the calcium ion–independent atypical PKC isozymes, PKC-ι and PKC-ζ, were detected in both pregnant and nonpregnant myometrial tissues. The similarity in distribution of PKC isozymes between myometrial tissues and primary cell cultures suggests that these cells may be useful in investigating the role of PKC in vitro. The finding that PKC isozymes PKC-β1 and PKC-β2 could be detected in pregnant myometrial tissues but not in nonpregnant myometrial tissues is especially noteworthy. This apparent selective isozyme induction suggests that PKC may play an important role in the myometrial changes that occur during pregnancy. The mechanism of induction may be analogous to the induction of PKC-β1 and PKC-β2 in human cardiac muscle undergoing hypertrophy related to heart failure.11 Although cardiac and smooth muscle are morphologically distinct, stretch may differentially induce PKC isozymes in myometrial cells, as it does in cardiac myocytes.12 Our study did not evaluate pregnant myometrium before term; therefore it remains uncertain at what point during pregnancy PKC isozymes are induced. Another interesting finding in this study was our demonstration of classic PKC-γ in both myometrial tissues and primary cell cultures. Although PKC-γ has been demonstrated in rat myometrium,4 it has not been previously demonstrated in any human smooth muscle. To verify this finding we repeated our experiments with PKC-α and PKC-γ antibodies from 2 different laboratories (Transduction Laboratories, and Life Technologies) and demonstrated that the bands completely disappeared when the gels were incubated in advance with authentic peptide sequences (Fig 2). An important characteristic of the PKC family is that its activation depends on the association of PKC with the plasma membrane phospholipids and diacylglycerol.13 We thus evaluated redistribution and translocation of a representative calcium ion–dependent isozyme (classic PKC-α) and a representative calcium ion–independent isozyme (novel PKC-δ). Redistribution of PKC from the soluble fraction to the particulate fraction, a reflection of translocation, was demonstrated for both isozymes after stimulation with either TPA or oxytocin (Fig 3). These results verify that both calcium ion–dependent and calcium ion–independent PKC isozymes are activated by stimulation of membrane receptors in cells from pregnant human myometrium. Translocation of PKC-α was also demonstrated visually by means of immunocytochemical methods (Fig 4). However, this translocation appeared to be selective to the cell membrane rather than to the nuclear membrane. Although our experiments were not designed to characterize this further, it could be an example of differential regulation of PKC translocation, such as has been recently demonstrated in vascular smooth muscle cells.14
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The exact role of PKC in the pregnant human myometrium remains to be elucidated. The ability of the myometrium to express both PKC-δ and PKC-ζ suggests that PKC may play a role in regulation of mitogenesis in this system. Inhibition or down-regulation of PKC has been shown to suppress proliferation of cultured myometrial cells.15 In other cells, activation of the PKC-δ isozyme markedly suppresses cell growth,16 whereas activation of the PKC-ζ isozyme is involved in the oncogene stimulation of cell maturation and deoxyribonucleic acid synthesis.17 The role of PKC in regulation of contractility is also complex. PKC appears to have both stimulatory and inhibitory effects on myometrial contractility.18 In rat myometrium, PKC stimulation with phorbol 12,13-dibutyrate augments contractility,18 and down-regulation of PKC by TPA exposure diminishes oxytocin-stimulated contractility.19 In human myometrium the selective PKC inhibitor RO 31-8220 almost completely inhibits oxytocin-induced contractility.2 PKC stimulation is believed to be a secondary effect of phosphorylation of contractile and cytoskeletal proteins, including caldesmon, myosin light chain (MLC20), and “myristoylated, alanine-rich C-kinase substrate.”20 In other studies PKC activation has been shown to inhibit myometrial contractility. In rat myometrium PKC stimulation with phorbol 12,13-dibutyrate significantly suppressed oxytocin-mediated contractions,3 and PKC inhibition potentiated oxytocin-inducted contractility.21 PKC may inhibit contractility either by phosphorylating some element of the G-protein and phospholipase C cascade3 or by phosphorylating myosin light-chain kinase.20 Taken together these dichotomous studies suggest that PKC may play a bimodal role in uterine contractility. In support of this hypothesis is the finding in rat myometrium that PKC stimulation with phorbol 12,13-dibutyrate increases contractility after 10 minutes of exposure but decreases contractility after ≥25 minutes.18 Whether this bimodal effect is related to changes in PKC concentrations or to selective activation of different isozymes remains to be determined. Regardless, the broad spectrum of PKC isozymes present in pregnant human myometrium may suggest a role for PKC in myometrial regulation.
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REFERENCES
1. Nishizuka Y. Protein kinase C and lipid signaling for sustained cellular responses. FASEB J 1995;9:484-96. 2. Phillippe M. Protein kinase C, an inhibitor of oxytocinstimulated phasic myometrial contractions. Biol Reprod 1994; 50:855-9. 3. Morrison JJ, Dearn SR, Smith SK, Ahmed A. Activation of pro-
20.
21.
tein kinase C is required for oxytocin-induced contractility in human pregnant myometrium. Hum Reprod 1996;11:2285-90. Ruzycky AL, Kulick A. Estrogen increases the expression of uterine protein kinase C isozymes in a tissue specific manner. Eur J Pharmacol 1996;313:257-63. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193:265-75. Fomin VP, Singh DM, Brown HL, Natarajan V, Hurd WW. Effect of cocaine on intracellular calcium regulation in myometrium from pregnant women. J Soc Gynecol Investig 1999;6:147-52. Skalli O, Ropraz P, Trzeciak A, Benzonana G, Gillessen D, Gabbiani G. A monoclonal antibody against alpha-smooth muscle actin: a new probe for smooth muscle differentiation. J Cell Biol 1986;103:2787-96. Casey ML, MacDonald PC, Mitchell MD, Snyder JM. Maintenance and characterization of human myometrial smooth muscle cells in monolayer culture. In Vitro 1984;20:396-403. Fomin VP, Cox BE, Word RA. Effect of progesterone on intracellular Ca2+ homeostasis in human myometrial smooth muscle cells. Am J Physiol 1999;276(45):C379-C385. Takai Y, Kishimoto A, Inoue M, Nishizuka Y. Studies on a cyclic nucleotide-independent protein kinase and its proenzyme in mammalian tissues. I. Purification and characterization of an active enzyme from bovine cerebellum. J Biol Chem 1977;252: 7603-9. Bowling N, Walsh RA, Song G, Estridge T, Sandusky GE, Fouts RL, et al. Increased protein kinase C activity and expression of Ca2+-sensitive isoforms in the failing human heart. Circulation 1999;99:384-91. Sadoshima J, Izumo S. Mechanical stretch rapidly activates multiple signal transduction pathways in cardiac myocytes: potential involvement of an autocrine/paracrine mechanism. EMBO J 1993;12:1681-92. Rasmussen H, Isales CM, Calle R, Throckmorton D, Anderson M, Gasalla-Herraiz J, et al. Diacylglycerol production, Ca2+ influx, and protein kinase C activation in sustained cellular responses. Endocr Rev 1995;16:649-81. Haller H, Maasch C, Lindschau C, Brachmann M, Buchner K, Luft FC. Intracellular targeting and protein kinase C in vascular smooth muscle cells: specific effects of different membranebound receptors. Acta Physiol Scand 1998;164:599-609. Hirst SJ, Webb BL, Giembycz MA, Barnes PJ, Twort CH. Inhibition of fetal calf serum–stimulated proliferation of rabbit cultured tracheal smooth muscle cells by selective inhibitors of protein kinase C and protein tyrosine kinase. Am J Respir Cell Mol Biol 1995;12:149-61. Watanabe T, Ono Y, Taniyama Y, Hazama K, Igarashi K, Ogita K, et al. Cell division arrest induced by phorbol ester in CHO cells overexpressing protein kinase C-delta subspecies. Proc Natl Acad Sci U S A 1992;89:10159-63. Berra E, Diaz-Meco MT, Lozano J, Frutos S, Municio MM, Sanchez P, et al. Evidence for a role of MEK and MAPK during signal transduction by protein kinase C zeta. EMBO J 1995; 14:6157-63. Savineau JP, Mironneau J. An analysis of the action of phorbol 12, 13-dibutyrate on mechanical activity in rat uterine smooth muscle. Pharmacol Exp Ther 1990;255:133-9. Karibe H, Oishi K, Uchida MK. Involvement of protein kinase C in Ca(2+)-independent contraction of rat uterine smooth muscle. Biochem Biophys Res Commun 1991;179:487-94. Throckmorton DC, Packer CS, Brophy CM. Protein kinase C activation during Ca2+-independent vascular smooth muscle contraction. J Surg Res 1998;78:48-53. Phaneuf S, Europe-Finner GN, Carrasco MP, Hamilton CH, López Bernal A. Oxytocin signalling in human myometrium. Adv Exp Med Biol 1995;395:453-67.
Key words: Human, isozymes, myometrium, pregnancy, protein kinase C, uterus
The mammalian uterus is remarkable in that it is composed of individual smooth muscle cells that change dramatically but only temporarily, both morphologically and physiologically, during pregnancy. These changes allow the uterus to adapt to one or more rapidly growing fetuses until maturity, and then a period of coordinated uterine activity begins and culminates in delivery of these fetuses. However, the mechanisms that regulate contractility in the pregnant human uterus are only partially understood. An important aspect of this regulation is selective phosphorylation of intracellular proteins, which leads to their activation or inactivation. This phosphorylation is carried out by a large family of protein kinases, including protein kinase C (PKC). PKC plays an important role in transmembrane signal transduction in numerous mammalian cells. In many receptor and G-protein–mediated path-
From the Department of Obstetrics and Gynecologya and the Department of Internal Medicine,b Indiana University School of Medicine. Supported by National Institutes of Health grant HD36692. Received for publication June 8, 1999; revised February 8, 2000; accepted March 5, 2000. Reprint requests: William W. Hurd, MD, 550 N University Blvd, Room 2440, Indianapolis, IN 46202-5274. Copyright © 2000 by Mosby, Inc. 0002-9378/2000 $12.00 + 0 6/1/107783 doi:10.1067/mob.2000.107783
ways, PKC is activated by second messengers such as diacylglycerol and calcium ion.1 The role of PKC in the uterus remains uncertain. In the rat myometrium activation of PKC inhibits oxytocininduced contractility.2 In human myometrium, however, activation of PKC augments oxytocin-mediated myometrial contractility and has been hypothesized to play a role in the sustained stimulation of myometrial activity during labor.3 Either a stimulatory or an inhibitory effect of PKC on the myometrium could be important in the regulation of contractility. It has been uncertain what PKC isozymes are present in human myometrium. It is well appreciated that the distribution of PKC isozymes differs among tissue types. This may be important, because different PKC isozymes are known to differ in sensitivities to diacylglycerol, calcium ion, and phospholipids and also in substrate specificity.2, 4 In most tissue types both calcium ion– dependent and calcium ion–independent PKC isozymes are present. However, until now the distribution of PKC isozymes in human myometrium has been unknown. Likewise, the distribution of PKC isozymes in primary cultures of pregnant human myometrial cells has not previously been established. Because these cells are commonly used as a model to study mechanisms of signal transduction, the relative distributions in the tissues and cultured cells are important. 1525
1526 Hurd et al
In this study we identified the PKC isozymes in nonpregnant and pregnant human myometrium. Western immunoblot analysis was used to identify the presence of both calcium ion–dependent and calcium ion–independent PKC isozymes in myometrial tissues and primary cell cultures. Redistribution of calcium ion–dependent and calcium ion–independent PKC isozymes was examined after exposure to either 12-O-tetradecanoylphorbol-13acetate (TPA) or oxytocin. Finally, oxytocin-stimulated translocation of PKC-α was examined in cultured myometrial cells by means of immunocytochemical methods. Our results indicate that both nonpregnant and pregnant human myometrial tissues contain a relatively broad spectrum of PKC isozymes and that PKC-β1 and PKC-β2 are induced during human pregnancy. Material and methods Tissue acquisition and preparation. All tissue specimens were obtained in accordance with a protocol approved by the university committee on human use in research. Nonpregnant myometrium was obtained from 4 premenopausal women undergoing hysterectomy; all had a grossly normal uterus and no malignancy. The primary diagnoses were chronic pelvic pain (n = 2) and endometriosis (n = 2). After the uterus was removed, a full-thickness section of the lower segment was removed under sterile conditions. After written consent was given, myometrium was obtained from 9 pregnant women at term undergoing cesarean delivery; 6 were not in labor and 3 were in early labor. Labor was defined as regular firm contractions resulting in progressive cervical dilatation to ≥4 cm. Women were excluded if they had any major complications of pregnancy (including hypertension, diabetes, and premature labor) or if they had been treated with any medications other than prenatal vitamins before delivery. At cesarean delivery a full-thickness strip of uterine tissue was taken from the upper margin of a lower transverse incision. Uterine tissues were transported to the laboratory on ice in a sterile Hanks balanced salt solution. The myometrium was separated from the endometrium and connective tissue with a scalpel with the aid of a dissecting microscope. The myometrium was either used for experiments immediately or processed for cell culture. Tissue samples from each patient were used for 2 different experiments. Tissue preparation. The myometrium was separated from the endometrium and cut into small pieces. Then 2 or 3 pieces (approximately 0.3 g) were put into each well of a 6-well plate containing 5 mL Dulbecco modified Eagle medium without fetal bovine serum. To demonstrate PKC redistribution duplicate wells were exposed to TPA (10–7 mol/L) or oxytocin (10–6 mol/L) in a carbon dioxide incubator at 37°C for 30 minutes. After the tissue specimen was washed twice with Hanks balanced salt so-
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lution, the pieces were blotted semidry and weighed. The pieces were frozen in liquid nitrogen and pulverized to a fine powder, which was resuspended in lysis buffer, homogenized, and centrifuged at 1000g for 20 minutes at 4°C. The supernatants were ultracentrifuged at 105,000g for 90 minutes at 4°C. The soluble (cytosolic) fraction was the resulting supernatant, and the particulate (membrane) fraction was the resulting pellet, which was resuspended in 1.0 mL buffer. Protein concentration was determined by the method of Lowry et al.5 Myometrial cell cultures. Primary myometrial cell cultures were established from pregnant myometrial specimens as previously described elsewhere.6 Briefly, the myometrium was minced into 1-mm3 pieces and digested in a solution of collagenase and deoxyribonuclease for 7 hours with intermittent trituration and filtration. The myometrial cells were separated from fibroblasts and other cells with a discontinuous Percoll density gradient (Amersham Pharmacia Biotech AB, Uppsala, Sweden) and found to have >90% myometrial cell viability by trypan blue exclusion. To verify that our cultured cells were myometrial cells rather than fibroblasts, we evaluated the ability of the myometrial cells to express the smooth muscle–specific protein α-actin by immunostaining with smooth muscle–specific monoclonal anti–α-actin (clone 1A4; Sigma, St Louis, Mo) by a method described by Skalli et al.7 The cells were plated on Petri dishes (35, 60, or 100 mm) at a concentration of 5 × 105 cells/cm2 and maintained at 37°C in a 5% carbon dioxide incubator until they reached 95% confluence. These primary cell cultures were serum deprived for 24 hours before use. Cultured human myometrial cells prepared by the described protocol have been extensively characterized morphologically, biochemically, and physiologically by other investigators.8 In primary culture these cells retain basic morphologic and physiologic properties of myometrial cells in the tissue. The cells also maintain a pattern of actin myofibril distribution that is identical to that of myometrial cells in tissue and is different from that of fibroblasts. The cells also produce approximately the same amount of prostaglandins and have the same activities and amounts of both myosin and myosin light-chain kinase as does myometrial tissue. Moreover, the myosin light-chain kinase in these cultured cells retains calciumcalmodulin dependence. These myometrial cells have been shown to respond to physiologic concentrations of oxytocin and endothelin 1 with increases in intracellular free calcium concentration.9 Myometrial cell culture preparation. Myometrial cell cultures were used 6 to 8 days after plating, after each 100-mm plastic culture dish had reached ≥95% confluence. The cells were placed in Dulbecco modified Eagle medium without fetal bovine serum the night before the experiment. To demonstrate PKC redistribution duplicate wells were exposed to TPA (10–7 mol/L) or oxytocin
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(10–6 mol/L) at 37°C for 30 minutes. The cells were washed twice with 3 mL ice-cold Hanks balanced salt solution, and 0.5 mL lysis buffer was added. The cells were scraped from the dishes, homogenized, and centrifuged at 1000g for 20 minutes at 4°C. The supernatants were transferred to different tubes and then ultracentrifuged at 105,000g for 90 minutes at 4°C. The resulting supernatant (soluble fraction) was transferred to an Eppendorf tube. The resulting pellet (particulate fraction) was resuspended in 0.6 mL lysis buffer. Protein concentration was determined by the Lowry method.5 PKC translocation and isozyme identification. Basal locations of PKC isozymes were determined in the soluble and particulate fractions by means of Western blot analysis.4 PKC activation was determined by translocation of PKC from the soluble fraction to the particulate fraction. Each gel lane was loaded with 20 µg protein from either the soluble or the particulate fraction. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis separation was performed in 4% to 20% gradient gels (Bio-Rad Laboratories Inc, Hercules, Calif). The separated proteins were transferred by electrophoresis onto nitrocellulose sheets and exposed to monoclonal antibodies specific for the classic PKC isozymes PKC-α, PKC-β1, PKC-β2, and PKC-γ; the novel PKC isozymes PKC-δ, PKC-ε, PKC-µ, and PKC-θ; and the atypical PKC isozymes PKC-ι and PKC-ζ. Because of reported cross-reactivity between the PKC-α and PKC-γ antibodies from the original supplier (Transduction Laboratories, Inc, Lexington, Ky), the experiments were repeated with highly specific monoclonal antibodies from a second company (Life Technologies, Inc, Rockville, Md). To verify specificity, binding was evaluated in the presence of the corresponding authentic peptide sequence obtained from the same source. Primary antibody exposure was followed by incubation with secondary antimouse immunoglobulin G (IgG) peroxidaseconjugated antibody (Sigma). The relative amount of immunospecific protein was determined by means of an enhanced chemiluminescence detection kit (Amersham Life Science, Arlington Heights, Ill). The bands were quantified with the Gel Documentation System (Alpha Innotech Corporation, San Leandro, Calif). Antibody specificity was verified by processing the blots without the primary antibodies. Immunocytochemical studies. Primary myometrial cell cultures were plated onto 4-well glass slides and were grown for 2 days or until 15% to 20% confluence. Twentyfour hours before the experiment the cell medium was replaced with Dulbecco modified Eagle medium without fetal bovine serum. The cells were treated with TPA (10–7 mol/L), oxytocin (10–6 mol/L), or bisindolylmaleimide I (10–5 mol/L) for 30 minutes at 37°C. The medium was removed, and the cells were fixed with 100% methanol and washed with phosphate-buffered saline solution plus 5% polysorbate. The slides were incubated in bovine serum
albumin–supplemented phosphate-buffered saline solution plus 5% polysorbate for 10 minutes. The slides were exposed to primary antibodies to PKC-α overnight at 4°C at a dilution of 1:25, as recommended by the manufacturer. The slides were washed with phosphate-buffered saline solution plus 5% polysorbate, and a fluorescent secondary antibody, Cy2–conjugated goat antimouse IgG, was added for 1 hour at room temperature. The secondary antibody was removed, and the slides were washed with phosphate-buffered saline solution plus 5% polysorbate, rinsed with water, and blotted. Glass coverslips were fixed to the slides, which were viewed with a Nikon Optiphot (Nikon Inc, Melville, NY) microscope and photographed with a Nikon FX-35A (Nikon Inc) camera. The specificity of the immunostaining was verified by incubating the slides in the medium that did not contain the primary antibody. The experiment was repeated 3 times with cell cultures derived from different patients. Material. Mouse monoclonal antibodies (IgG fractions) directed against isozyme-specific carboxy terminal peptide sequences of PKC isozymes were purchased from either Transduction Laboratories (PKC-α, PKC-γ, PKC-δ, PKC-ε, PKC-ζ, PKC-θ, PKC-ι, and PKC-µ) or Life Technologies (PKC-α, PKC-γ, PKC-β1, and PKC-β2). The PKC inhibitor, bisindolylmaleimide I, was obtained from Alexis (Alexis Corporation, San Diego, Calif). For immunocytochemical studies a fluorescent secondary antibody, Cy2–conjugated goat antimouse IgG, was obtained from Jackson ImmunoResearch Laboratories (Jackson ImmunoResearch Laboratories, Inc, West Grove, Pa). Antibodies to calponin and actin were obtained from Sigma. Other reagents were from standard commercial sources. Results PKC isozyme profiles. Human myometrial tissues and primary myometrial cell cultures derived from these tissue specimens were found to contain significant amounts of most of the known PKC isozymes (Fig 1). Nonpregnant myometrial tissues contained detectable quantities of 2 calcium ion–dependent classic PKC isozymes, PKC-α and PKC-γ. The other two classic PKC isozymes, PKC-β1 and PKC-β2, were not detected in any of the nonpregnant samples. Of the calcium ion–independent novel PKC isozymes, both PKC-δ and PKC-µ were present but neither PKC-θ nor PKC-ε was detected. Both of the atypical PKC isozymes investigated, PKC-ι and PKC-ζ, were detected in nonpregnant myometrium. In contrast, pregnant myometrial tissues obtained either before or after the onset of labor contained all 4 calcium ion–dependent classic PKC isozymes (PKC-α, PKCβ1, PKC-β2, and PKC-γ). The distribution of the remaining isozymes was identical to that in the nonpregnant tissues, with the presence of PKC-δ, PKC-µ, PKC-ι, and PKC-ζ isozymes and the absence of PKC-θ and PKC-ε
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Fig 1. Western immunoblot analysis of PKC isozymes in human myometrial tissues. Tissue specimens were obtained from nonpregnant women (n = 4) and pregnant women at term both before (n = 4) and after (n = 3) onset of labor. Primary cell cultures were derived from pregnant myometrium obtained from women before labor. Each lane was loaded with 20 µg protein. cPKC, Classic PKC; nPKC, novel PKC; aPKC, atypical PKC; T, myometrial tissue, C, primary cell cultures. Plus sign, Positive rat brain control (exception, Jurkat cell line for PKC-µ and PKC-θ). Numbers to right indicate apparent molecular weight of band of interest as calculated from molecular weight standards. Results are from representative myometrial tissue samples and corresponding primary myometrial cell cultures.
isozymes. Primary cell cultures derived from pregnant myometrium from patients before labor revealed a distribution of isozymes identical to that in the tissues from which they were derived. To verify that both PKC-α and PKC-γ were present, primary myometrial cell cultures were evaluated with highly specific antibodies from two different manufacturers, Transduction Laboratories and Life Technologies. Specificity was confirmed by incubation with the authentic peptide sequence to which the antibodies were raised, which resulted in complete disappearance of the bands (Fig 2). Redistribution. Under the experimental conditions of this study, classic PKC-α and novel PKC-δ were distributed in myometrial cells derived from pregnant myometrium almost equally between the soluble and particulate fractions in the resting state (Fig 3). Stimulation with either TPA or oxytocin for 30 minutes resulted in redistribution of both the calcium ion–dependent (classic PKC-α) and calcium ion–independent (novel PKC-δ) isozymes to the particulate fraction.
Immunocytochemical studies. In resting control myometrial cells, classic PKC-α showed a coarse, granular distribution in the cytosol (Fig 4, A). In approximately 75% of the cells in each well, exposure to oxytocin for 30 minutes resulted in translocation manifested by increased immunofluorescence along the cell membranes but not along the nuclear membrane (Fig 4, B). When the myometrial cells were treated in advance for 30 minutes with the specific PKC inhibitor bisindolylmaleimide I, no translocation was seen (Fig 4, C). Comment PKC represents a family of phospholipid-dependent kinases that were first described by Takai et al10 in brain tissue. Since that original report, PKC isozymes have been found in most tissue types; these isozymes differ in their kinetic properties, substrate specificities, and dependence on cofactors. In this study the human myometrium was found to contain significant amounts of most of the known PKC isozymes. In nonpregnant myometrial tissues 2 of the 4
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Fig 2. Western immunoblot analysis of PKC isozymes PKC-α and PKC-γ in primary myometrial cell cultures. Each lane was loaded with 20 µg protein. Monoclonal antibodies specific for PKC-α and PKC-γ isozymes were obtained from either Transduction Laboratories (Transduction) or Life Technologies (Gibco). Two lanes on right were incubated in advance with corresponding authentic peptide sequences. C, Cell culture membrane preparations. Plus sign, Positive rat brain control. Numbers to right indicate apparent molecular weight of band of interest as calculated from molecular weight standards. Experiments were repeated with cell cultures derived from 3 different patients. Results shown are from representative experiment.
Fig 3. Western immunoblot analysis demonstrating redistribution of PKC-α and PKC-δ in pregnant human myometrial tissue specimens (Tissue) and primary cell cultures (Cells) after exposure to TPA or oxytocin (OT). Each lane was loaded with 20 µg protein. S, Soluble (cytosolic) fraction; P, particulate (membrane) fraction. Plus sign, Positive rat brain control. Numbers to right indicate apparent molecular weight of band of interest as calculated from molecular weight standards. Experiments were repeated in tissues and cell cultures derived from 6 different patients. Results shown are from representative myometrial tissue sample and primary myometrial cell culture.
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Fig 4. Fluorescence microscopy of PKC-α in control myometrial cells (A) and cells that had been exposed for 30 minutes to oxytocin (10–6 mol/L) alone (B) or oxytocin plus PKC inhibitor bisindolylmaleimide I (10–5 mol/L; C). After fixation, cells were exposed overnight to mouse monoclonal primary antibodies to PKC-α, followed by fluorescent goat antimouse IgG secondary antibody. Experiments were repeated in cell cultures derived from 3 different patients. Approximately 75% of cells in each culture well exhibited changes shown in this representative experiment.
calcium ion–dependent classic PKC isozymes (PKC-α and PKC-γ) were found, and in pregnant myometrium and primary cell cultures all 4 (PKC-α, PKC-β1, PKC-β2, and PKC-γ) were found. Of the calcium ion–independent novel PKC isozymes, PKC-δ and PKC-µ were abundant in
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both nonpregnant and pregnant myometrium, whereas PKC-θ and PKC-ε were not detectable. Both of the calcium ion–independent atypical PKC isozymes, PKC-ι and PKC-ζ, were detected in both pregnant and nonpregnant myometrial tissues. The similarity in distribution of PKC isozymes between myometrial tissues and primary cell cultures suggests that these cells may be useful in investigating the role of PKC in vitro. The finding that PKC isozymes PKC-β1 and PKC-β2 could be detected in pregnant myometrial tissues but not in nonpregnant myometrial tissues is especially noteworthy. This apparent selective isozyme induction suggests that PKC may play an important role in the myometrial changes that occur during pregnancy. The mechanism of induction may be analogous to the induction of PKC-β1 and PKC-β2 in human cardiac muscle undergoing hypertrophy related to heart failure.11 Although cardiac and smooth muscle are morphologically distinct, stretch may differentially induce PKC isozymes in myometrial cells, as it does in cardiac myocytes.12 Our study did not evaluate pregnant myometrium before term; therefore it remains uncertain at what point during pregnancy PKC isozymes are induced. Another interesting finding in this study was our demonstration of classic PKC-γ in both myometrial tissues and primary cell cultures. Although PKC-γ has been demonstrated in rat myometrium,4 it has not been previously demonstrated in any human smooth muscle. To verify this finding we repeated our experiments with PKC-α and PKC-γ antibodies from 2 different laboratories (Transduction Laboratories, and Life Technologies) and demonstrated that the bands completely disappeared when the gels were incubated in advance with authentic peptide sequences (Fig 2). An important characteristic of the PKC family is that its activation depends on the association of PKC with the plasma membrane phospholipids and diacylglycerol.13 We thus evaluated redistribution and translocation of a representative calcium ion–dependent isozyme (classic PKC-α) and a representative calcium ion–independent isozyme (novel PKC-δ). Redistribution of PKC from the soluble fraction to the particulate fraction, a reflection of translocation, was demonstrated for both isozymes after stimulation with either TPA or oxytocin (Fig 3). These results verify that both calcium ion–dependent and calcium ion–independent PKC isozymes are activated by stimulation of membrane receptors in cells from pregnant human myometrium. Translocation of PKC-α was also demonstrated visually by means of immunocytochemical methods (Fig 4). However, this translocation appeared to be selective to the cell membrane rather than to the nuclear membrane. Although our experiments were not designed to characterize this further, it could be an example of differential regulation of PKC translocation, such as has been recently demonstrated in vascular smooth muscle cells.14
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The exact role of PKC in the pregnant human myometrium remains to be elucidated. The ability of the myometrium to express both PKC-δ and PKC-ζ suggests that PKC may play a role in regulation of mitogenesis in this system. Inhibition or down-regulation of PKC has been shown to suppress proliferation of cultured myometrial cells.15 In other cells, activation of the PKC-δ isozyme markedly suppresses cell growth,16 whereas activation of the PKC-ζ isozyme is involved in the oncogene stimulation of cell maturation and deoxyribonucleic acid synthesis.17 The role of PKC in regulation of contractility is also complex. PKC appears to have both stimulatory and inhibitory effects on myometrial contractility.18 In rat myometrium, PKC stimulation with phorbol 12,13-dibutyrate augments contractility,18 and down-regulation of PKC by TPA exposure diminishes oxytocin-stimulated contractility.19 In human myometrium the selective PKC inhibitor RO 31-8220 almost completely inhibits oxytocin-induced contractility.2 PKC stimulation is believed to be a secondary effect of phosphorylation of contractile and cytoskeletal proteins, including caldesmon, myosin light chain (MLC20), and “myristoylated, alanine-rich C-kinase substrate.”20 In other studies PKC activation has been shown to inhibit myometrial contractility. In rat myometrium PKC stimulation with phorbol 12,13-dibutyrate significantly suppressed oxytocin-mediated contractions,3 and PKC inhibition potentiated oxytocin-inducted contractility.21 PKC may inhibit contractility either by phosphorylating some element of the G-protein and phospholipase C cascade3 or by phosphorylating myosin light-chain kinase.20 Taken together these dichotomous studies suggest that PKC may play a bimodal role in uterine contractility. In support of this hypothesis is the finding in rat myometrium that PKC stimulation with phorbol 12,13-dibutyrate increases contractility after 10 minutes of exposure but decreases contractility after ≥25 minutes.18 Whether this bimodal effect is related to changes in PKC concentrations or to selective activation of different isozymes remains to be determined. Regardless, the broad spectrum of PKC isozymes present in pregnant human myometrium may suggest a role for PKC in myometrial regulation.
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REFERENCES
1. Nishizuka Y. Protein kinase C and lipid signaling for sustained cellular responses. FASEB J 1995;9:484-96. 2. Phillippe M. Protein kinase C, an inhibitor of oxytocinstimulated phasic myometrial contractions. Biol Reprod 1994; 50:855-9. 3. Morrison JJ, Dearn SR, Smith SK, Ahmed A. Activation of pro-
20.
21.
tein kinase C is required for oxytocin-induced contractility in human pregnant myometrium. Hum Reprod 1996;11:2285-90. Ruzycky AL, Kulick A. Estrogen increases the expression of uterine protein kinase C isozymes in a tissue specific manner. Eur J Pharmacol 1996;313:257-63. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193:265-75. Fomin VP, Singh DM, Brown HL, Natarajan V, Hurd WW. Effect of cocaine on intracellular calcium regulation in myometrium from pregnant women. J Soc Gynecol Investig 1999;6:147-52. Skalli O, Ropraz P, Trzeciak A, Benzonana G, Gillessen D, Gabbiani G. A monoclonal antibody against alpha-smooth muscle actin: a new probe for smooth muscle differentiation. J Cell Biol 1986;103:2787-96. Casey ML, MacDonald PC, Mitchell MD, Snyder JM. Maintenance and characterization of human myometrial smooth muscle cells in monolayer culture. In Vitro 1984;20:396-403. Fomin VP, Cox BE, Word RA. Effect of progesterone on intracellular Ca2+ homeostasis in human myometrial smooth muscle cells. Am J Physiol 1999;276(45):C379-C385. Takai Y, Kishimoto A, Inoue M, Nishizuka Y. Studies on a cyclic nucleotide-independent protein kinase and its proenzyme in mammalian tissues. I. Purification and characterization of an active enzyme from bovine cerebellum. J Biol Chem 1977;252: 7603-9. Bowling N, Walsh RA, Song G, Estridge T, Sandusky GE, Fouts RL, et al. Increased protein kinase C activity and expression of Ca2+-sensitive isoforms in the failing human heart. Circulation 1999;99:384-91. Sadoshima J, Izumo S. Mechanical stretch rapidly activates multiple signal transduction pathways in cardiac myocytes: potential involvement of an autocrine/paracrine mechanism. EMBO J 1993;12:1681-92. Rasmussen H, Isales CM, Calle R, Throckmorton D, Anderson M, Gasalla-Herraiz J, et al. Diacylglycerol production, Ca2+ influx, and protein kinase C activation in sustained cellular responses. Endocr Rev 1995;16:649-81. Haller H, Maasch C, Lindschau C, Brachmann M, Buchner K, Luft FC. Intracellular targeting and protein kinase C in vascular smooth muscle cells: specific effects of different membranebound receptors. Acta Physiol Scand 1998;164:599-609. Hirst SJ, Webb BL, Giembycz MA, Barnes PJ, Twort CH. Inhibition of fetal calf serum–stimulated proliferation of rabbit cultured tracheal smooth muscle cells by selective inhibitors of protein kinase C and protein tyrosine kinase. Am J Respir Cell Mol Biol 1995;12:149-61. Watanabe T, Ono Y, Taniyama Y, Hazama K, Igarashi K, Ogita K, et al. Cell division arrest induced by phorbol ester in CHO cells overexpressing protein kinase C-delta subspecies. Proc Natl Acad Sci U S A 1992;89:10159-63. Berra E, Diaz-Meco MT, Lozano J, Frutos S, Municio MM, Sanchez P, et al. Evidence for a role of MEK and MAPK during signal transduction by protein kinase C zeta. EMBO J 1995; 14:6157-63. Savineau JP, Mironneau J. An analysis of the action of phorbol 12, 13-dibutyrate on mechanical activity in rat uterine smooth muscle. Pharmacol Exp Ther 1990;255:133-9. Karibe H, Oishi K, Uchida MK. Involvement of protein kinase C in Ca(2+)-independent contraction of rat uterine smooth muscle. Biochem Biophys Res Commun 1991;179:487-94. Throckmorton DC, Packer CS, Brophy CM. Protein kinase C activation during Ca2+-independent vascular smooth muscle contraction. J Surg Res 1998;78:48-53. Phaneuf S, Europe-Finner GN, Carrasco MP, Hamilton CH, López Bernal A. Oxytocin signalling in human myometrium. Adv Exp Med Biol 1995;395:453-67.