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calmodulin-dependent protein kinase or epidermal growth factor receptor tyrosine kinase abolishes lysophosphatidic acid-mediated DNA-synthesis in human myometrial smooth muscle cells
Cell Biology International
Cell Biology International 27 (2003) 341–347
www.elsevier.com/locate/cellbi
Inhibition of Ca2+/calmodulin-dependent protein kinase or epidermal growth factor receptor tyrosine kinase abolishes lysophosphatidic acid-mediated DNA-synthesis in human myometrial smooth muscle cells Ulrika K. Nilsson *, Samuel P.S. Svensson Department of Medicine and Care, Division of Pharmacology, Faculty of Health Sciences, Linko¨pings Universitet, SE-581 85 Linko¨ping, Sweden Received 24 June 2002; revised 8 October 2002; accepted 3 December 2002
Abstract Human myometrial smooth muscle cells (SMCs) were used to evaluate the proliferative activity of lysophosphatidic acid (LPA). This study specifically focuses on the role of Ca2+/calmodulin-dependent protein (CaM) kinase and epidermal growth factor (EGF) receptor tyrosine kinase. Myometrial SMCs were cultured from biopsies taken at Cesarean sections. The expression of LPA receptors was determined by reverse transcriptase polymerase chain reaction (RT-PCR), and DNA-synthesis was measured by [3H]thymidine incorporation. LPA1, LPA2, and LPA3 receptor subtypes were detected in the SMCs using RT-PCR. KN-62, an inhibitor of CaM kinase, and Tyrphostin AG 1478, an inhibitor of EGF receptor tyrosine kinase, dose-dependently decreased LPA-stimulated [3H]thymidine incorporation. Furthermore, BB-3103, an inhibitor of matrix metalloproteinases (MMPs), also reduced DNA-synthesis induced by LPA in these cells. The results show, for the first time, that human myometrial SMCs express all three known LPA receptor subtypes. Growth stimulatory effects of LPA on myometrial SMCs seems to be mediated by several pathways, where transactivation of EGF receptors through MMPs appears to be of importance. Furthermore, CaM kinase activity may be critical for LPA signaling since inhibition of CaM kinase totally abolish the proliferative effect of LPA. 2003 Elsevier Science Ltd. All rights reserved. Keywords: Calcium; Calmodulin; DNA-synthesis; Epidermal growth factor; Lysophosphatidic acid; Smooth muscle cells
1. Introduction During pregnancy, the human myometrium undergoes considerable enlargement both through hypertrophy and hyperplasia of smooth muscle cells (SMCs), and increases in weight from approximately 50 to about 950 g (Llewellyn-Jones, 1994). This increase depends on differentiation of new SMCs from undifferentiated cells in the connective tissue and involves mitosis of differentiated SMCs (Bacon and Niles, 1983). Earlier studies concentrated on the growth-promoting effects of sex steroids and peptide growth factors. The precise mechanism of how female sex steroids regulate growth and differentiation of the myometrium is unknown. One * Corresponding author. Tel.: +46-13-223456; fax: +46-13-149106. E-mail address: [email protected] (U.K. Nilsson).
possibility is that estrogen controls growth indirectly by regulating the synthesis of polypeptide growth factors and their receptors. Uterine insulin-like growth factor-I production is increased after estrogen administration to rats (Murphy et al., 1987). Furthermore, estrogen treatment of rats and mice increase the levels of epidermal growth factor (EGF) receptor tyrosine kinases (Stancel et al., 1987). Lysophosphatidic acid (LPA) is an intercellular lipid messenger produced and released from membrane microvesicles of activated cells (Fourcade et al., 1995). In microvesicles, phospholipase (PL) C and diacylglycerol kinase or PLD hydrolyze phospholipids to generate high concentrations of phosphatidic acid which is then converted to LPA by PLA2 (Goetzl and An, 1998) before being released into extracellular fluids. The concentration of LPA in serum has been estimated to be in the micromolar range (Eichholtz et al., 1993).
1065-6995/03/$ - see front matter 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1065-6995(02)00352-9
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Abbreviations [Ca2+]i cytosolic free Ca2+ concentration CaM Ca2+/calmodulin-dependent protein cDNA complementary DNA Edg endothelial differentiation gene EDTA ethylenediamine-tetraacetic acid EGF epidermal growth factor LPA lysophosphatidic acid MAP mitogen-activated protein MMP matrix metalloproteinase PBS phosphate-buffered saline PLA2/C/D phospholipase A2/C/D RT-PCR reverse transcriptase polymerase chain reaction SEM standard error of the mean SMC smooth muscle cell TCA trichloroacetic acid vzg-1 ventricular zone gene-1
LPA activates at least three types of G-protein-coupled receptors called endothelial differentiation gene (Edg)-2, Edg-4, and Edg-7 (An et al., 1997, 1998; Bandoh et al., 1999). In 1996, Hecht et al. (1996) isolated mouse cDNA, termed ventricular zone gene-1 (vzg-1), encoding a receptor for LPA. Thereafter the human homologue of vzg-1, called Edg-2, was identified and later on another subtype called Edg-4 was discovered (An et al., 1997, 1998). Recently another novel human subtype, Edg-7, has been characterized (Bandoh et al., 1999). However, according to Tigyi (2001) a new nomenclature for these receptors has been proposed. The new names are LPA1 (Edg-2), LPA2 (Edg-4), and LPA3 (Edg-7). These LPA receptors differ with respect to cell distribution and intracellular signal transduction mechanisms (Bandoh et al., 1999). LPA can exert such diverse effects as smooth muscle contraction (Vogt, 1963), platelet aggregation (Tokumura et al., 1981), and neurite retraction in PC12 cells (Tigyi et al., 1996). LPA also induces growth of fibroblasts (Van Corven et al., 1989) and SMCs (Tokumura et al., 1994). The effects of LPA on intracellular signaling transduction pathways and cellular growth are far from clear. Most studies have been performed in transfected cell lines. Conversely, in the present study we have examined the expression of LPA receptor subtypes and how LPA induces DNA-synthesis in primary cultures of human myometrial SMCs from biopsies taken at Cesarean sections. It has previously been shown that LPA has the ability to raise the cytosolic free Ca2+ concentration ([Ca2+]i) and increase synthesis of DNA in this cell type (Nilsson et al., 1998). Since the exact role of Ca2+ in the regulation of cellular growth is unclear, we have used a pharmacological approach to investigate Ca2+/calmodulin-dependent
protein kinase (CaM kinase) and EGF receptor tyrosine kinase, with regard to the proliferative activity of LPA in myometrial SMCs.
2. Materials and methods 2.1. Materials All cell culture reagents were obtained from GIBCO (Gaithersburg, MD, USA). EGF, --lysophosphatidic acid (oleoyl-sn-glycero-3-phosphate), and the monoclonal antibody against anti--smooth muscle actin were purchased from Sigma Chemicals (St. Louis, MO, USA); KN-62 was from Biomol Research Laboratories (Plymouth Meeting, PA, USA); anti-mouse IgG heavy and light chain (goat) Texas Red conjugated antibodies, Tyrphostin AG 1478 and Tyrphostin A1 were from Calbiochem (La Jolla, CA, USA); the primers were synthesized by Invitrogen (Paisley, UK); the kilo base molecular ruler was from Bio-Rad Laboratories (Hercules, CA, USA); BB-3103 was a generous gift from British Biotech Pharmaceuticals Ltd (Oxford, UK); and [3H]thymidine (5 Ci/mmol) was from Amersham Life Science (Buckinghamshire, UK). 2.2. Myometrial SMC culture All experiments were performed using cultured SMCs from human myometrial tissue taken at Cesarean sections at term. The Ethical Committee at the University of Linko¨ping approved the study and the delivering mothers were informed about the purpose of the study before surgery and gave their consent to the biopsy. Single cells were obtained by using the explant method;
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i.e. cells grow out from small tissue pieces placed in culture dishes. Cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 5% fetal bovine serum, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin, and non-essential amino acids. The culture flasks were stored in a humidified incubator at 37 (C in an atmosphere of 95% air and 5% CO2. The culture medium was changed every 72 h. For subcultures, cells were detached using a 0.05% trypsin, 0.02% ethylenediamine-tetraacetic acid (EDTA) solution. The identity of SMCs was confirmed by immunofluorescent staining against -smooth muscle actin (Skalli et al., 1986). 2.3. Amplification of LPA receptor subtype cDNA Confluent SMCs were washed with phosphatebuffered saline (PBS; 137 mM NaCl, 8 mM Na2HPO4, 2.7 mM KCl, and 1.5 mM KH2PO4; pH 7.4) and trypsinated. The suspension of cells was diluted in PBS and centrifuged at 300g for 5 min. The pellet was thereafter used to isolate total RNA using StrataPrep Total RNA Microprep Kit (Stratagene, La Jolla, CA, USA) according to the manufacturer’s protocol. RNase-free DNase was used to digest contaminated DNA. Reverse transcriptase polymerase chain reaction (RT-PCR) was carried out using Ready-to-go RT-PCR Beads (Amersham Pharmacia Biotech, Uppsala, Sweden). A final volume of 50 µl contained 2.0 U taq DNA polymerase, M-MuLV reverse transcriptase, 60 mM KCl, 10 mM tris(hydroxymethyl)-aminomethane (Tris)–HCl (pH 9.0), 1.5 mM MgCl2, 200 µM dNTP mix, 1 µM of each primer and 0.5–1.0 µg RNA template. Complementary DNA (cDNA) was synthesized by using random hexamer primers during incubation at 42 (C for 30 min. The reverse transcriptase was thereafter denaturated at 95 (C for 5 min. A negative control was made by denaturating reverse transcriptase before adding primers and template. After the incubation, aliquots of cDNA were transferred to Ready-to-go PCR beads (Amersham Pharmacia Biotech). A final volume of 25 µl contained 1.5 U taq DNA polymerase, 50 mM KCl, 10 mM Tris– HCl (pH 9.0), 1.5 mM MgCl2, 200 µM dNTP mix, 1 µM of each primer and 6 µl DNA template. Sense and antisense primers used for the PCR-step were the same as used by Motohashi et al. (2000). The reactions were run for 40 cycles consisting of 95 (C for 30 s, 55 (C for 30 s, and 72 (C for 30 s. At the first cycle, the samples had an extended denaturation period and were heated to 95 (C for 2 min. The products were electrophoresed in a 2% agarose gel and visualized by staining with ethidium bromide. To verify the identity of the PCR products, three different restriction enzymes were used to cut the products. LPA1 was cut by the enzyme Ban I, LPA2 was cut by Stu I, and LPA3 was cut by Pst I.
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2.4. [3H]thymidine incorporation DNA-synthesis was quantified by measuring [3H]thymidine incorporation into DNA according to a modified version of Bornfeldt et al. (1991). The SMCs were seeded at a density of 10,000 cells per well in 96 multi-well plates. After culturing for 48 h, cells were deprived of serum for 24 h. The cells were incubated with drugs in serum-free medium containing [3H]thymidine (2 µCi/ml). After 24 h of incubation at 37 (C, cells were rinsed with medium and the DNA was precipitated with ice-cold 5% trichloroacetic acid (TCA) at 4 (C for 15 min. Thereafter, the wells were rinsed once with TCA and the DNA was solubilized in 200 µl of 0.1 M KOH for at least 30 min at room temperature. The solution was agitated and 170 µl aliquots were transferred to 5 ml scintillation liquid and quantified by liquid scintillation counting. The decrease in [3H]thymidine incorporation induced by BB-3103, KN-62, or Tyrphostin AG 1478 was compared to control cells incubated with LPA alone. 2.5. Cytotoxic test of BB-3103, KN-62, and Tyrphostin AG 1478 Eventual toxic effects of incubation with the different drugs were analyzed with the dye trypan blue. Cells were deprived of serum for 24 h and thereafter incubated with drugs in serum-free medium for another 24 h. Subsequently, cells were detached by using trypsin and diluted once with 0.4% trypan blue. Cells were then incubated for 6 min before the total and viable cell counts, and the percentage of dead cells present were counted in a Bu¨rker chamber. 2.6. Statistics Results are expressed as meansstandard error of the means (SEM). Statistical analyses of the data were performed by one-way ANOVA followed by Bonferroni’s multiple comparison test as post hoc test for comparisons between groups. A difference was considered statistically significant when P<0.05. Data were analyzed using GraphPad Prisme (GraphPad Software, San Diego, CA, USA). 3. Results 3.1. Cell morphology Myometrial cells did not seem to exhibit contact inhibition and grew in a so-called hill-and-valley pattern, which is known to be morphologically characteristic for SMCs (Chamley-Campbell et al., 1979). Characterization of the phenotype of the cells, with a monoclonal antibody against -smooth muscle actin, indicated that the cultures consisted of a homogenous population of SMCs (data not shown).
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Fig. 2. Inhibitory effects of KN-62, a CaM kinase inhibitor, on the incorporation of [3H]thymidine into human myometrial SMCs stimulated with 10 µM LPA. Cells stimulated with 10 µM LPA represent 100%. The bar marked with basal represents cells incubated without serum and drugs. Data points represent meansSEM of five separate experiments with six samples in each. ***P<0.001 compared to cells treated only with LPA. Fig. 1. Expression of LPA receptor mRNA in human myometrial SMCs. RT-PCR analysis of LPA receptors was carried out with mRNA from cultured cells. Lane 1, contains negative control; lane 2, LPA1; lane 3, LPA2; lane 4, PCR molecular ruler indicated on the right in base pair (bp); and lane 5, LPA3. This is representative of three independent experiments.
3.2. PCR amplification of LPA receptor cDNA To investigate which of the LPA receptor subtypes LPA1, LPA2, or LPA3 are expressed in myometrial SMCs, we isolated total RNA and synthesized cDNA from cultured cells. Amplification of the cDNA product was carried out using PCR with primer homologues for the three different receptor subtypes (Motohashi et al., 2000). All three receptor subtypes were detected in three independent experiments (Fig. 1). In the negative control, where the reverse transcriptase was denaturated before adding primers and template, no PCR product was obtained, confirming that the product originated from mRNA. A comparison of the sizes of the PCR products with that of Motohashi et al. (2000), and by using restriction enzyme analysis, show that myometrial SMCs express LPA1, LPA2, and LPA3 receptor subtypes. 3.3. KN-62 inhibited LPA-induced DNA-synthesis in myometrial SMCs LPA at a concentration of 10 µM, without the addition of other growth factors or serum, significantly
increased DNA-synthesis in SMCs. To determine whether a CaM kinase inhibitor could reduce the growth-promoting effect of LPA, cells were treated either with or without KN-62 for 24 h. The inhibitor was used at 10 nM and 100 nM. Addition of KN-62 reduced the [3H]thymidine incorporation down to the level of basal incorporation (Fig. 2). Both 10 nM and 100 nM KN-62 significantly (P<0.001) inhibited DNA-synthesis in myometrial SMCs. 3.4. Tyrphostin AG 1478 and BB-3103 inhibited DNA-synthesis induced by LPA Treatment with the EGF receptor tyrosine kinase inhibitor Tyrphostin AG 1478 inhibited DNA-synthesis induced by 10 µM LPA (Fig. 3). Tyrphostin AG 1478 was used over the range 10 nM–10 µM. The dosedependent inhibition was statistically significant at concentrations of 1 µM (P<0.01) and 10 µM (P<0.001) of Tyrphostin AG 1478. The inactive inhibitor, Tyrphostin A1, used as a negative control at 10 µM, did not significantly affect the response by LPA (P>0.05). To determine whether matrix metalloproteinases (MMPs) could be involved in the LPA-induced stimulation of DNA-synthesis, a synthetic inhibitor of MMPs, BB-3103, was also used over the range 1 nM–10 µM. Addition of BB-3103 reduced the [3H]thymidine incorporation (Fig. 4). The inhibition was statistically significant at concentrations of 0.1 µM (P<0.01) and 10 µM (P<0.001). However, 1 µM BB-3103 did not
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4. Discussion
Fig. 3. Effects of Tyrphostin AG 1478, an EGF receptor tyrosine kinase inhibitor, on the incorporation of [3H]thymidine into human myometrial SMCs stimulated with 10 µM LPA. The bar to the right (A1) shows cells incubated with LPA together with 10 µM of the negative control Tyrphostin A1. Cells stimulated with 10 µM LPA represent 100%. The bar marked with basal represents cells incubated without serum and drugs. Data points represent meansSEM of eight separate experiments with six samples in each. **P<0.01 and ***P<0.001 compared to cells treated only with LPA.
Fig. 4. Effects of BB-3103, an MMP inhibitor, on the incorporation of [3H]thymidine into human myometrial SMCs stimulated with 10 µM LPA. Cells stimulated with 10 µM LPA represent 100%. The bar marked with basal represent cells incubated without serum and drugs. Data points represent meansSEM of three separate experiments with six samples in each. **P<0.01 and ***P<0.001 compared to cells treated only with LPA.
significantly reduce LPA-induced stimulation. The biological significance of this is unknown. 3.5. BB-3103, KN-62, or Tyrphostin AG 1478 and cell death Trypan blue was used to evaluate the eventual toxic effects of incubation with the different drugs. When studying cells in microscope, trypan blue colored dead cells blue but left viable cells uncolored. No correlation were found between lower cell viability and increasing concentrations of BB-3103, KN-62, or Tyrphostin AG 1478 (data not shown).
LPA seems to play multiple roles in both female and male reproductive physiology and pathology. LPA receptor transcripts have, for example, been detected in prostate and testis (Bandoh et al., 1999; Im et al., 2000). SMCs from benign prostate hyperplasia proliferate when stimulated with LPA (Adolfsson et al., 2002). Furthermore, LPA has been purified from ascites, and detected at higher levels in plasma from ovarian cancer patients compared with a control group (Xu et al., 1995, 1998). According to this, LPA has been proposed to be used as a biomarker for gynecologic cancers (Xu et al., 1998). LPA can also induce stress fiber formation in myometrial SMCs via a pathway involving Rho-kinase (Gogarten et al., 2001). Nevertheless, almost nothing is known about LPA and its role in normal myometrial tissue. RT-PCR was used here to characterize which LPA receptor subtypes are expressed in human myometrial SMCs. Our results show that myometrial SMCs express all three known LPA receptor subtypes (An et al., 1997, 1998; Bandoh et al., 1999). Hooks et al. (2001) suggest that mitogenic and platelet aggregation responses to LPA are independent of LPA receptors. Instead, there is an unknown low affinity LPA-signaling pathway, which mediates these responses regulated by lipid phosphoric acid phosphatase enzymes. This hypothesis was, however, not explored here. LPA can stimulate a rapid and transient rise in [Ca2+]i, and DNA-synthesis in myometrial SMCs (Nilsson et al., 1998). Furthermore, Seewald et al. (1997) reported that LPA can induce an increase in [Ca2+]i in vascular SMCs. The role of Ca2+ in regulation of cellular growth is unclear, and seems to be cell specific. Increasing evidence suggests that progression of the cell cycle is dependent on transient rises of [Ca2+]i (Santella, 1998). Huang et al. (1995) have shown that extracellular Ca2+ can stimulate sustained growth of fibroblasts and mitogen-activated protein (MAP) kinase in the absence of growth factors. On the other hand, LPA stimulates the activity of MAP kinase in rat fibroblasts which does not depend on rises in [Ca2+]i. However, Ca2+ is necessary for expression of MAP kinase phosphatase and to fully reconstitute the response to LPA (Cook et al., 1997). Since we noticed that KN-62 potently inhibited DNA-synthesis induced by LPA, we suggest that CaM kinase plays an important role in LPA-mediated cell growth in myometrial SMCs. Minami et al. (1994) showed that cell growth is inhibited by KN-62 in leukemia cells. Similarly, incubation of lung carcinoma cells with KN-62 potently inhibits DNA-synthesis and slows progression through S phase (Williams et al., 1996). Stimulated G-protein-coupled receptors can use receptor tyrosine kinases as signal transduction elements. EGF receptors can be transactivated by stimulation with LPA in rat fibroblasts and neuroepithelioma
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cells (Buist et al., 1998; Daub et al., 1996). It has not been clearly defined how G-protein-coupled receptors induce receptor tyrosine kinase transactivation, although several mechanisms have been suggested. Interestingly, results suggest that transactivation of the EGF receptors is a result of MMP-mediated release of membrane-bound EGF, i.e. an autocrine activation of the receptor and not a ligand-independent transactivation (Dong et al., 1999; Prenzel et al., 1999). MMPs are naturally occurring enzymes that degrade components of extracellular matrix in both physiologic and pathologic states (Woessner, 1991). Eguchi et al. (1998) suggest that Ca2+ is necessary for transactivation of EGF receptors which thereby leads to MAP kinase activation in vascular SMCs. Furthermore, Murasawa et al. (1998) have shown that Ca2+/calmodulin plays a role in the transactivation of EGF receptors. The presence of EGF receptors in human myometrial cells has been reported by Chegini et al. (1986). Based on the fact that treatment with Tyrphostin AG 1478 reduced the LPA-induced DNA-synthesis, our results suggest that EGF receptor tyrosine kinase activity is also important for LPAmediated DNA-synthesis in myometrial SMCs. In agreement with earlier studies (Dong et al., 1999; Prenzel et al., 1999), LPA-induced activation of EGF receptors might be mediated by MMPs in myometrial SMCs. This is based on the fact that treatment with the MMP inhibitor BB-3103 reduced the LPA-induced DNAsynthesis. This is interesting since the transactivation phenomenon has mainly been studied in overexpressed systems. It is important to study signal transduction pathways in primary cell cultures and not only in transfected and immortalized cell lines (Della Rocca et al., 1999). Although transactivation of EGF receptors seems to be of importance in other SMCs our results suggest that CaM kinases also mediate proliferative signals of LPA. How CaM kinases might be involved in the transactivating cascade remains unclear. Taken together, LPA seems to be an important growth stimulatory signal in myometrial SMCs. How different sex steroids influence this signaling system, and whether it changes during different phases of the pregnancy, are other interesting questions to be resolved. However, LPA can inhibit ligand binding to estrogen and progesterone receptors, and LPA might thereby regulate pregnancy maintenance (Pulkkinen and Ha¨ma¨la¨inen, 1995).
Acknowledgements We acknowledge Sitti Hultgren for excellent work with RT-PCR. We wish to thank the staff at the Division of Obstetrics and Gynecology, Department of Health and Environment for help in collecting biopsies. We also thank British Biotech Pharmaceuticals Ltd for providing the MMP inhibitor BB-3103. This work was supported
by grants from the Swedish Society for Medical Research (K1999-14X-013046-01A, K1999-14P-013050¨ stergo¨tland. 01A) and the County Council of O
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Cell Biology International 27 (2003) 341–347
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Inhibition of Ca2+/calmodulin-dependent protein kinase or epidermal growth factor receptor tyrosine kinase abolishes lysophosphatidic acid-mediated DNA-synthesis in human myometrial smooth muscle cells Ulrika K. Nilsson *, Samuel P.S. Svensson Department of Medicine and Care, Division of Pharmacology, Faculty of Health Sciences, Linko¨pings Universitet, SE-581 85 Linko¨ping, Sweden Received 24 June 2002; revised 8 October 2002; accepted 3 December 2002
Abstract Human myometrial smooth muscle cells (SMCs) were used to evaluate the proliferative activity of lysophosphatidic acid (LPA). This study specifically focuses on the role of Ca2+/calmodulin-dependent protein (CaM) kinase and epidermal growth factor (EGF) receptor tyrosine kinase. Myometrial SMCs were cultured from biopsies taken at Cesarean sections. The expression of LPA receptors was determined by reverse transcriptase polymerase chain reaction (RT-PCR), and DNA-synthesis was measured by [3H]thymidine incorporation. LPA1, LPA2, and LPA3 receptor subtypes were detected in the SMCs using RT-PCR. KN-62, an inhibitor of CaM kinase, and Tyrphostin AG 1478, an inhibitor of EGF receptor tyrosine kinase, dose-dependently decreased LPA-stimulated [3H]thymidine incorporation. Furthermore, BB-3103, an inhibitor of matrix metalloproteinases (MMPs), also reduced DNA-synthesis induced by LPA in these cells. The results show, for the first time, that human myometrial SMCs express all three known LPA receptor subtypes. Growth stimulatory effects of LPA on myometrial SMCs seems to be mediated by several pathways, where transactivation of EGF receptors through MMPs appears to be of importance. Furthermore, CaM kinase activity may be critical for LPA signaling since inhibition of CaM kinase totally abolish the proliferative effect of LPA. 2003 Elsevier Science Ltd. All rights reserved. Keywords: Calcium; Calmodulin; DNA-synthesis; Epidermal growth factor; Lysophosphatidic acid; Smooth muscle cells
1. Introduction During pregnancy, the human myometrium undergoes considerable enlargement both through hypertrophy and hyperplasia of smooth muscle cells (SMCs), and increases in weight from approximately 50 to about 950 g (Llewellyn-Jones, 1994). This increase depends on differentiation of new SMCs from undifferentiated cells in the connective tissue and involves mitosis of differentiated SMCs (Bacon and Niles, 1983). Earlier studies concentrated on the growth-promoting effects of sex steroids and peptide growth factors. The precise mechanism of how female sex steroids regulate growth and differentiation of the myometrium is unknown. One * Corresponding author. Tel.: +46-13-223456; fax: +46-13-149106. E-mail address: [email protected] (U.K. Nilsson).
possibility is that estrogen controls growth indirectly by regulating the synthesis of polypeptide growth factors and their receptors. Uterine insulin-like growth factor-I production is increased after estrogen administration to rats (Murphy et al., 1987). Furthermore, estrogen treatment of rats and mice increase the levels of epidermal growth factor (EGF) receptor tyrosine kinases (Stancel et al., 1987). Lysophosphatidic acid (LPA) is an intercellular lipid messenger produced and released from membrane microvesicles of activated cells (Fourcade et al., 1995). In microvesicles, phospholipase (PL) C and diacylglycerol kinase or PLD hydrolyze phospholipids to generate high concentrations of phosphatidic acid which is then converted to LPA by PLA2 (Goetzl and An, 1998) before being released into extracellular fluids. The concentration of LPA in serum has been estimated to be in the micromolar range (Eichholtz et al., 1993).
1065-6995/03/$ - see front matter 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1065-6995(02)00352-9
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Abbreviations [Ca2+]i cytosolic free Ca2+ concentration CaM Ca2+/calmodulin-dependent protein cDNA complementary DNA Edg endothelial differentiation gene EDTA ethylenediamine-tetraacetic acid EGF epidermal growth factor LPA lysophosphatidic acid MAP mitogen-activated protein MMP matrix metalloproteinase PBS phosphate-buffered saline PLA2/C/D phospholipase A2/C/D RT-PCR reverse transcriptase polymerase chain reaction SEM standard error of the mean SMC smooth muscle cell TCA trichloroacetic acid vzg-1 ventricular zone gene-1
LPA activates at least three types of G-protein-coupled receptors called endothelial differentiation gene (Edg)-2, Edg-4, and Edg-7 (An et al., 1997, 1998; Bandoh et al., 1999). In 1996, Hecht et al. (1996) isolated mouse cDNA, termed ventricular zone gene-1 (vzg-1), encoding a receptor for LPA. Thereafter the human homologue of vzg-1, called Edg-2, was identified and later on another subtype called Edg-4 was discovered (An et al., 1997, 1998). Recently another novel human subtype, Edg-7, has been characterized (Bandoh et al., 1999). However, according to Tigyi (2001) a new nomenclature for these receptors has been proposed. The new names are LPA1 (Edg-2), LPA2 (Edg-4), and LPA3 (Edg-7). These LPA receptors differ with respect to cell distribution and intracellular signal transduction mechanisms (Bandoh et al., 1999). LPA can exert such diverse effects as smooth muscle contraction (Vogt, 1963), platelet aggregation (Tokumura et al., 1981), and neurite retraction in PC12 cells (Tigyi et al., 1996). LPA also induces growth of fibroblasts (Van Corven et al., 1989) and SMCs (Tokumura et al., 1994). The effects of LPA on intracellular signaling transduction pathways and cellular growth are far from clear. Most studies have been performed in transfected cell lines. Conversely, in the present study we have examined the expression of LPA receptor subtypes and how LPA induces DNA-synthesis in primary cultures of human myometrial SMCs from biopsies taken at Cesarean sections. It has previously been shown that LPA has the ability to raise the cytosolic free Ca2+ concentration ([Ca2+]i) and increase synthesis of DNA in this cell type (Nilsson et al., 1998). Since the exact role of Ca2+ in the regulation of cellular growth is unclear, we have used a pharmacological approach to investigate Ca2+/calmodulin-dependent
protein kinase (CaM kinase) and EGF receptor tyrosine kinase, with regard to the proliferative activity of LPA in myometrial SMCs.
2. Materials and methods 2.1. Materials All cell culture reagents were obtained from GIBCO (Gaithersburg, MD, USA). EGF, --lysophosphatidic acid (oleoyl-sn-glycero-3-phosphate), and the monoclonal antibody against anti--smooth muscle actin were purchased from Sigma Chemicals (St. Louis, MO, USA); KN-62 was from Biomol Research Laboratories (Plymouth Meeting, PA, USA); anti-mouse IgG heavy and light chain (goat) Texas Red conjugated antibodies, Tyrphostin AG 1478 and Tyrphostin A1 were from Calbiochem (La Jolla, CA, USA); the primers were synthesized by Invitrogen (Paisley, UK); the kilo base molecular ruler was from Bio-Rad Laboratories (Hercules, CA, USA); BB-3103 was a generous gift from British Biotech Pharmaceuticals Ltd (Oxford, UK); and [3H]thymidine (5 Ci/mmol) was from Amersham Life Science (Buckinghamshire, UK). 2.2. Myometrial SMC culture All experiments were performed using cultured SMCs from human myometrial tissue taken at Cesarean sections at term. The Ethical Committee at the University of Linko¨ping approved the study and the delivering mothers were informed about the purpose of the study before surgery and gave their consent to the biopsy. Single cells were obtained by using the explant method;
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i.e. cells grow out from small tissue pieces placed in culture dishes. Cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 5% fetal bovine serum, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin, and non-essential amino acids. The culture flasks were stored in a humidified incubator at 37 (C in an atmosphere of 95% air and 5% CO2. The culture medium was changed every 72 h. For subcultures, cells were detached using a 0.05% trypsin, 0.02% ethylenediamine-tetraacetic acid (EDTA) solution. The identity of SMCs was confirmed by immunofluorescent staining against -smooth muscle actin (Skalli et al., 1986). 2.3. Amplification of LPA receptor subtype cDNA Confluent SMCs were washed with phosphatebuffered saline (PBS; 137 mM NaCl, 8 mM Na2HPO4, 2.7 mM KCl, and 1.5 mM KH2PO4; pH 7.4) and trypsinated. The suspension of cells was diluted in PBS and centrifuged at 300g for 5 min. The pellet was thereafter used to isolate total RNA using StrataPrep Total RNA Microprep Kit (Stratagene, La Jolla, CA, USA) according to the manufacturer’s protocol. RNase-free DNase was used to digest contaminated DNA. Reverse transcriptase polymerase chain reaction (RT-PCR) was carried out using Ready-to-go RT-PCR Beads (Amersham Pharmacia Biotech, Uppsala, Sweden). A final volume of 50 µl contained 2.0 U taq DNA polymerase, M-MuLV reverse transcriptase, 60 mM KCl, 10 mM tris(hydroxymethyl)-aminomethane (Tris)–HCl (pH 9.0), 1.5 mM MgCl2, 200 µM dNTP mix, 1 µM of each primer and 0.5–1.0 µg RNA template. Complementary DNA (cDNA) was synthesized by using random hexamer primers during incubation at 42 (C for 30 min. The reverse transcriptase was thereafter denaturated at 95 (C for 5 min. A negative control was made by denaturating reverse transcriptase before adding primers and template. After the incubation, aliquots of cDNA were transferred to Ready-to-go PCR beads (Amersham Pharmacia Biotech). A final volume of 25 µl contained 1.5 U taq DNA polymerase, 50 mM KCl, 10 mM Tris– HCl (pH 9.0), 1.5 mM MgCl2, 200 µM dNTP mix, 1 µM of each primer and 6 µl DNA template. Sense and antisense primers used for the PCR-step were the same as used by Motohashi et al. (2000). The reactions were run for 40 cycles consisting of 95 (C for 30 s, 55 (C for 30 s, and 72 (C for 30 s. At the first cycle, the samples had an extended denaturation period and were heated to 95 (C for 2 min. The products were electrophoresed in a 2% agarose gel and visualized by staining with ethidium bromide. To verify the identity of the PCR products, three different restriction enzymes were used to cut the products. LPA1 was cut by the enzyme Ban I, LPA2 was cut by Stu I, and LPA3 was cut by Pst I.
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2.4. [3H]thymidine incorporation DNA-synthesis was quantified by measuring [3H]thymidine incorporation into DNA according to a modified version of Bornfeldt et al. (1991). The SMCs were seeded at a density of 10,000 cells per well in 96 multi-well plates. After culturing for 48 h, cells were deprived of serum for 24 h. The cells were incubated with drugs in serum-free medium containing [3H]thymidine (2 µCi/ml). After 24 h of incubation at 37 (C, cells were rinsed with medium and the DNA was precipitated with ice-cold 5% trichloroacetic acid (TCA) at 4 (C for 15 min. Thereafter, the wells were rinsed once with TCA and the DNA was solubilized in 200 µl of 0.1 M KOH for at least 30 min at room temperature. The solution was agitated and 170 µl aliquots were transferred to 5 ml scintillation liquid and quantified by liquid scintillation counting. The decrease in [3H]thymidine incorporation induced by BB-3103, KN-62, or Tyrphostin AG 1478 was compared to control cells incubated with LPA alone. 2.5. Cytotoxic test of BB-3103, KN-62, and Tyrphostin AG 1478 Eventual toxic effects of incubation with the different drugs were analyzed with the dye trypan blue. Cells were deprived of serum for 24 h and thereafter incubated with drugs in serum-free medium for another 24 h. Subsequently, cells were detached by using trypsin and diluted once with 0.4% trypan blue. Cells were then incubated for 6 min before the total and viable cell counts, and the percentage of dead cells present were counted in a Bu¨rker chamber. 2.6. Statistics Results are expressed as meansstandard error of the means (SEM). Statistical analyses of the data were performed by one-way ANOVA followed by Bonferroni’s multiple comparison test as post hoc test for comparisons between groups. A difference was considered statistically significant when P<0.05. Data were analyzed using GraphPad Prisme (GraphPad Software, San Diego, CA, USA). 3. Results 3.1. Cell morphology Myometrial cells did not seem to exhibit contact inhibition and grew in a so-called hill-and-valley pattern, which is known to be morphologically characteristic for SMCs (Chamley-Campbell et al., 1979). Characterization of the phenotype of the cells, with a monoclonal antibody against -smooth muscle actin, indicated that the cultures consisted of a homogenous population of SMCs (data not shown).
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Fig. 2. Inhibitory effects of KN-62, a CaM kinase inhibitor, on the incorporation of [3H]thymidine into human myometrial SMCs stimulated with 10 µM LPA. Cells stimulated with 10 µM LPA represent 100%. The bar marked with basal represents cells incubated without serum and drugs. Data points represent meansSEM of five separate experiments with six samples in each. ***P<0.001 compared to cells treated only with LPA. Fig. 1. Expression of LPA receptor mRNA in human myometrial SMCs. RT-PCR analysis of LPA receptors was carried out with mRNA from cultured cells. Lane 1, contains negative control; lane 2, LPA1; lane 3, LPA2; lane 4, PCR molecular ruler indicated on the right in base pair (bp); and lane 5, LPA3. This is representative of three independent experiments.
3.2. PCR amplification of LPA receptor cDNA To investigate which of the LPA receptor subtypes LPA1, LPA2, or LPA3 are expressed in myometrial SMCs, we isolated total RNA and synthesized cDNA from cultured cells. Amplification of the cDNA product was carried out using PCR with primer homologues for the three different receptor subtypes (Motohashi et al., 2000). All three receptor subtypes were detected in three independent experiments (Fig. 1). In the negative control, where the reverse transcriptase was denaturated before adding primers and template, no PCR product was obtained, confirming that the product originated from mRNA. A comparison of the sizes of the PCR products with that of Motohashi et al. (2000), and by using restriction enzyme analysis, show that myometrial SMCs express LPA1, LPA2, and LPA3 receptor subtypes. 3.3. KN-62 inhibited LPA-induced DNA-synthesis in myometrial SMCs LPA at a concentration of 10 µM, without the addition of other growth factors or serum, significantly
increased DNA-synthesis in SMCs. To determine whether a CaM kinase inhibitor could reduce the growth-promoting effect of LPA, cells were treated either with or without KN-62 for 24 h. The inhibitor was used at 10 nM and 100 nM. Addition of KN-62 reduced the [3H]thymidine incorporation down to the level of basal incorporation (Fig. 2). Both 10 nM and 100 nM KN-62 significantly (P<0.001) inhibited DNA-synthesis in myometrial SMCs. 3.4. Tyrphostin AG 1478 and BB-3103 inhibited DNA-synthesis induced by LPA Treatment with the EGF receptor tyrosine kinase inhibitor Tyrphostin AG 1478 inhibited DNA-synthesis induced by 10 µM LPA (Fig. 3). Tyrphostin AG 1478 was used over the range 10 nM–10 µM. The dosedependent inhibition was statistically significant at concentrations of 1 µM (P<0.01) and 10 µM (P<0.001) of Tyrphostin AG 1478. The inactive inhibitor, Tyrphostin A1, used as a negative control at 10 µM, did not significantly affect the response by LPA (P>0.05). To determine whether matrix metalloproteinases (MMPs) could be involved in the LPA-induced stimulation of DNA-synthesis, a synthetic inhibitor of MMPs, BB-3103, was also used over the range 1 nM–10 µM. Addition of BB-3103 reduced the [3H]thymidine incorporation (Fig. 4). The inhibition was statistically significant at concentrations of 0.1 µM (P<0.01) and 10 µM (P<0.001). However, 1 µM BB-3103 did not
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4. Discussion
Fig. 3. Effects of Tyrphostin AG 1478, an EGF receptor tyrosine kinase inhibitor, on the incorporation of [3H]thymidine into human myometrial SMCs stimulated with 10 µM LPA. The bar to the right (A1) shows cells incubated with LPA together with 10 µM of the negative control Tyrphostin A1. Cells stimulated with 10 µM LPA represent 100%. The bar marked with basal represents cells incubated without serum and drugs. Data points represent meansSEM of eight separate experiments with six samples in each. **P<0.01 and ***P<0.001 compared to cells treated only with LPA.
Fig. 4. Effects of BB-3103, an MMP inhibitor, on the incorporation of [3H]thymidine into human myometrial SMCs stimulated with 10 µM LPA. Cells stimulated with 10 µM LPA represent 100%. The bar marked with basal represent cells incubated without serum and drugs. Data points represent meansSEM of three separate experiments with six samples in each. **P<0.01 and ***P<0.001 compared to cells treated only with LPA.
significantly reduce LPA-induced stimulation. The biological significance of this is unknown. 3.5. BB-3103, KN-62, or Tyrphostin AG 1478 and cell death Trypan blue was used to evaluate the eventual toxic effects of incubation with the different drugs. When studying cells in microscope, trypan blue colored dead cells blue but left viable cells uncolored. No correlation were found between lower cell viability and increasing concentrations of BB-3103, KN-62, or Tyrphostin AG 1478 (data not shown).
LPA seems to play multiple roles in both female and male reproductive physiology and pathology. LPA receptor transcripts have, for example, been detected in prostate and testis (Bandoh et al., 1999; Im et al., 2000). SMCs from benign prostate hyperplasia proliferate when stimulated with LPA (Adolfsson et al., 2002). Furthermore, LPA has been purified from ascites, and detected at higher levels in plasma from ovarian cancer patients compared with a control group (Xu et al., 1995, 1998). According to this, LPA has been proposed to be used as a biomarker for gynecologic cancers (Xu et al., 1998). LPA can also induce stress fiber formation in myometrial SMCs via a pathway involving Rho-kinase (Gogarten et al., 2001). Nevertheless, almost nothing is known about LPA and its role in normal myometrial tissue. RT-PCR was used here to characterize which LPA receptor subtypes are expressed in human myometrial SMCs. Our results show that myometrial SMCs express all three known LPA receptor subtypes (An et al., 1997, 1998; Bandoh et al., 1999). Hooks et al. (2001) suggest that mitogenic and platelet aggregation responses to LPA are independent of LPA receptors. Instead, there is an unknown low affinity LPA-signaling pathway, which mediates these responses regulated by lipid phosphoric acid phosphatase enzymes. This hypothesis was, however, not explored here. LPA can stimulate a rapid and transient rise in [Ca2+]i, and DNA-synthesis in myometrial SMCs (Nilsson et al., 1998). Furthermore, Seewald et al. (1997) reported that LPA can induce an increase in [Ca2+]i in vascular SMCs. The role of Ca2+ in regulation of cellular growth is unclear, and seems to be cell specific. Increasing evidence suggests that progression of the cell cycle is dependent on transient rises of [Ca2+]i (Santella, 1998). Huang et al. (1995) have shown that extracellular Ca2+ can stimulate sustained growth of fibroblasts and mitogen-activated protein (MAP) kinase in the absence of growth factors. On the other hand, LPA stimulates the activity of MAP kinase in rat fibroblasts which does not depend on rises in [Ca2+]i. However, Ca2+ is necessary for expression of MAP kinase phosphatase and to fully reconstitute the response to LPA (Cook et al., 1997). Since we noticed that KN-62 potently inhibited DNA-synthesis induced by LPA, we suggest that CaM kinase plays an important role in LPA-mediated cell growth in myometrial SMCs. Minami et al. (1994) showed that cell growth is inhibited by KN-62 in leukemia cells. Similarly, incubation of lung carcinoma cells with KN-62 potently inhibits DNA-synthesis and slows progression through S phase (Williams et al., 1996). Stimulated G-protein-coupled receptors can use receptor tyrosine kinases as signal transduction elements. EGF receptors can be transactivated by stimulation with LPA in rat fibroblasts and neuroepithelioma
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cells (Buist et al., 1998; Daub et al., 1996). It has not been clearly defined how G-protein-coupled receptors induce receptor tyrosine kinase transactivation, although several mechanisms have been suggested. Interestingly, results suggest that transactivation of the EGF receptors is a result of MMP-mediated release of membrane-bound EGF, i.e. an autocrine activation of the receptor and not a ligand-independent transactivation (Dong et al., 1999; Prenzel et al., 1999). MMPs are naturally occurring enzymes that degrade components of extracellular matrix in both physiologic and pathologic states (Woessner, 1991). Eguchi et al. (1998) suggest that Ca2+ is necessary for transactivation of EGF receptors which thereby leads to MAP kinase activation in vascular SMCs. Furthermore, Murasawa et al. (1998) have shown that Ca2+/calmodulin plays a role in the transactivation of EGF receptors. The presence of EGF receptors in human myometrial cells has been reported by Chegini et al. (1986). Based on the fact that treatment with Tyrphostin AG 1478 reduced the LPA-induced DNA-synthesis, our results suggest that EGF receptor tyrosine kinase activity is also important for LPAmediated DNA-synthesis in myometrial SMCs. In agreement with earlier studies (Dong et al., 1999; Prenzel et al., 1999), LPA-induced activation of EGF receptors might be mediated by MMPs in myometrial SMCs. This is based on the fact that treatment with the MMP inhibitor BB-3103 reduced the LPA-induced DNAsynthesis. This is interesting since the transactivation phenomenon has mainly been studied in overexpressed systems. It is important to study signal transduction pathways in primary cell cultures and not only in transfected and immortalized cell lines (Della Rocca et al., 1999). Although transactivation of EGF receptors seems to be of importance in other SMCs our results suggest that CaM kinases also mediate proliferative signals of LPA. How CaM kinases might be involved in the transactivating cascade remains unclear. Taken together, LPA seems to be an important growth stimulatory signal in myometrial SMCs. How different sex steroids influence this signaling system, and whether it changes during different phases of the pregnancy, are other interesting questions to be resolved. However, LPA can inhibit ligand binding to estrogen and progesterone receptors, and LPA might thereby regulate pregnancy maintenance (Pulkkinen and Ha¨ma¨la¨inen, 1995).
Acknowledgements We acknowledge Sitti Hultgren for excellent work with RT-PCR. We wish to thank the staff at the Division of Obstetrics and Gynecology, Department of Health and Environment for help in collecting biopsies. We also thank British Biotech Pharmaceuticals Ltd for providing the MMP inhibitor BB-3103. This work was supported
by grants from the Swedish Society for Medical Research (K1999-14X-013046-01A, K1999-14P-013050¨ stergo¨tland. 01A) and the County Council of O
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