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Am J Physiol Endocrinol Metab 292: E1110-E1121, 2007. First published December 12, 2006; doi:10.1152/ajpendo.00373.2006
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Progesterone-induced sphingosine kinase-1 expression in the rat uterus during pregnancy and signaling consequences

Yow-Jiun Jeng, Victor R. Suarez, Michael G. Izban, Hui-Qun Wang, and Melvyn S. Soloff

Departments of Obstetrics and Gynecology and Pathology, University of Texas Medical Branch, Galveston, Texas

Submitted 26 July 2006 ; accepted in final form 6 December 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Sphingosine 1-phosphate (Sph-1-P), a product of sphingomyelin metabolism, can act via a family of cognate G protein-coupled receptors or as an intracellular second messenger for agonists acting through their membrane receptors. In view of the general growth promoting and developmental effects of Sph-1-P on target cells, we hypothesized that it plays a role in adaptation of the uterus to pregnancy. We analyzed its potential role and that of the related lysophospholipid lysophosphatidic acid in the pregnant rat uterus by examining changes in mRNA levels of cognate receptors and enzymes involved in their turnover. Of these, only sphingosine kinase-1 (SphK1) was markedly changed (~30-fold increase), being localized in the glandular epithelium, vasculature, and the myometrium. Uterine SphK1 mRNA and protein levels paralleled those of serum progesterone, and treatment with progesterone or an antagonist elevated or reduced SphK1 mRNA expression, respectively. Progesterone also increased SphK1 mRNA steady-state levels in a rat myometrial/leiomyoma cell line (ELT3). Overexpressing human SphK1 in these cells resulted in increased levels of the cell cycle regulator cyclin D1 and increased myosin light-chain phosphorylation. Ectopic expression of SphK1 also resulted in increased proliferation rates, possibly in conjunction with increased cyclin D1 expression. These studies suggest that the uterine expression of SphK1 mediates processes involved in growth and differentiation of uterine tissues during pregnancy.

ELT3 cells; myosin light-chain phosphorylation; cyclin D1; lysophospholipids; sphingosine 1-phosphate; sphingosine-1-phosphate lyase

THE BIOACTIVE LYSOPHOSPHOLIPIDS sphingosine 1-phosphate (Sph-1-P) and lysophosphatidic acid (LPA) are growth factors that act through a family of G protein-coupled receptors (GPCRs), S1P1 through S1P5 and LPA1 through LPA4, respectively (25), causing a broad array of effects on target cells, including cell proliferation, survival, migration, adhesion molecule expression, and morphogenesis (26, 31, 44, 47, 52). Sph-1-P also plays a role in vasculogenesis in the mouse embryo (4, 27). Both lysophospholipids increase myosin light-chain phosphorylation in platelets and endothelial cells (10, 37) by inhibiting myosin light-chain phosphatase (43). They also stimulate DNA synthesis in human myometrial cells in primary culture (20, 32). Platelets are the major source of circulating Sph-1-P and LPA, releasing the lysophospholipids in response to prothrombotic stimuli (47, 54). Circulating Sph-1-P also arises by constitutive secretion by cells of hemangioblastic lineage, such as monocytes, and by mast, endothelial, and red blood cells (54).

Intracellular Sph-1-P is synthesized by a variety of cell types and acts as a second messenger for a growing list of activated membrane receptors, such as platelet-derived growth factor (34), muscarinergic acetylcholine (29), purinergic P2Y2 (2), tumor necrosis factor-{alpha} (53), epidermal growth factor (30), formyl peptide (1), bradykinin B2 (5), and specific antigen (9, 28) receptors. Agonist-induced activation of these receptors results in the activation of sphingosine kinase (SphK), which catalyzes the rapid and transient phosphorylation of sphingosine to Sph-1-P. Two isoforms of SphK, which are encoded by separate genes, have been characterized (44). Comparison of the amino acid sequences of the two indicates that human SphK1 lacks hydrophobic transmembrane regions, whereas human SphK2 appears to be a membrane protein with four transmembrane regions. The two enzymes differ in size, tissue distribution, developmental expression, substrate specificity, specific activity, and likely cellular roles (see Ref. 46 for references). The turnover of Sph-1-P is mediated either by dephosphorylation by specific Sph-1-P phosphohydrolases or by cleavage to ethanolamine phosphate and hexadecenal by a pyridoxal-dependent Sph-1-P lyase.

Extracellular LPA is synthesized by platelets and other cell types by the phospholipase A-catalyzed deacylation of phosphatic acid or from lysophosphatidylcholine in a reaction catalyzed by a specific phosphodiesterase, lysophospholipase D (autotaxin) (35, 49, 50). LPA is degraded through the actions of intracellular phosphatases such as phosphohydrolase-I (LPP1) and lysophospholipase-2 (LYP2) (7, 35).

In considering the broad functions of Sph-1-P, it is surprising that disruption of the SphK1 gene in mice was found to cause no obvious abnormalities in fertility (3). There was a significant decrease in serum Sph-1-P levels in these mice, but Sph-1-P concentrations in most tissues were not markedly reduced. These findings indicate that additional SphKs, such as SphK2, might compensate for the ablation of SphK1 expression. It is clear that Sph-1-P is vital for developmental processes, as disruption of the S1P1 gene in mice caused embryonic hemorrhage leading to intrauterine death between embryonic days 12.5 and 14.5 (27). Vascular maturation was incomplete in these embryos due to a deficiency in vascular smooth muscle cells and pericytes that normally would support endothelial tube structures (27). Disruption of S1P2 and S1P3 together also results in marked perinatal lethality (19).

The rat myometrium undergoes increased cellular division during the first half of pregnancy, followed by myocyte hypertrophy (39). Extensive endometrial gland hyperplasia and hypertrophy (14) and uterine vasculogenesis (56) also occur during gestation. In view of the growth- and development-promoting effects of lysophospholipids (20, 32) and their role in myosin light-chain phosphorylation (10, 37), we investigated changes in the expression of receptors and enzymes involved in Sph-1-P and LPA metabolism and signaling in the rat uterus to begin to elucidate their role in the adaptation of the rat uterus to pregnancy.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Progesterone was purchased from Schein Pharmaceutical (Florham Park, NJ). Onapristone (Schering, Germany) was a gift from Dr. Robert Garfield (Dept. of Obstetrics and Gynecology, Univ. of Texas Medical Branch, Galveston, TX). 17beta-Estradiol, RU-486, and other chemicals were purchased from Sigma Chemical (St. Louis, MO). Steroids were dissolved in sesame oil and injected subcutaneously in a total volume of 0.2 ml, except onapristone, which was dissolved in benzyl benzoate and castor oil.

Animals. Experimental protocols were approved by the Animal Care and Use Committee of the University of Texas Medical Branch at Galveston. Uterine samples were taken from timed-pregnant rats (Sprague-Dawley; Charles River Laboratory, Wilmington, MA) that were randomly selected. Preterm delivery was induced by the administration of single dose of onapristone (3 mg sc) on day 17. The control group received vehicle alone. Pregnancy was prolonged by administration of progesterone (2 mg sc) twice daily, starting on day 19. Nonpregnant rats (9 wk old, 200–225 g) from the same vendor were treated 1 wk after bilateral ovariectomy with either 17beta-estradiol (0.2 µg), progesterone (1 mg), or both for 4 days. Uteri were removed on the 5th day after initiation of treatment. Another group of rats received a regimen of estrogen and progesterone simulating the estrous cycle and first 5 days of pregnancy, as previously described (45). Uteri were removed on the equivalent of day 5 of pregnancy.

Ribonuclease protection assays. Uterine horns were obtained by laparotomy immediately after rats were killed, and the products of conception, including the surface layer of decidual tissue, were removed by gentle scraping. RNA was extracted using TRIzol reagent (GIBCO, Carlsbad, CA) according the manufacturer's protocol. DNA templates used for protection assays were produced by RT-PCR, cloned into pCRII (Invitrogen, Carlsbad, CA), and the sequence was confirmed by DNA sequence analysis. Primer sets used to generate the templates are as follows (forward and reverse): SphK1 (GenBank acc. no. NM_133386) 5'-CGCCTGGGCAACACCGATAA-3' and 5'-GGCTACATAGGGGTTTCTGG-3'; SphK2 (GenBank acc. no. BC079120) 5'-GAGTGAGTGGGAAGGCATTG-3' and 5'-TGGTAGCAGGGAGGTAGGAG-3'; Sph-1-P lyase (GenBank acc. no. NM_173116) 5'-CCAGCATTTCAGCAGATACT-3' and 5'-ATTCCACCCCTTAGCAGACA-3'; Sph-1-P phosphatase (GenBank acc. no. AF329638) 5'-TCATCTTCTTCCCCTTCTGG-3' and 5'-AATGCCCAAAATCAAGTGAA-3'; S1P1 (EDG1, GenBank acc. no. NM_017301) 5'-CCCCTCTCTTCATCCTACTA-3' and 5'-TTGTCCCCATCATCCTTCTG-3'; LPA1 (EDG2; GenBank acc. no. NM_133386) 5'-TGTGGTGGTGGTGATTGTAG-3' and 5'-GCGGTAGGAGTAGATGATGG-3'; S1P2 (EDG5; GenBank acc. no. NM_017192) 5'-CTGCTCAACCCTGTCATCTA-3' and 5'-TTCCTCTCTGCTCCCCAATA-3'; S1P3 (EDG3; GenBank acc. no. XM_225216) 5'-AGCACCCTCATCACCACCAT-3' and 5'-CAGACAGCCGCACACCAACC-3'; cytosolic phospholipase A2 (cPLA2; GenBank acc. no. NM_133551) 5'-TATGAAGGTGGGAGAGAAGA-3' and 5'-CAGTAAAGGTGACAGGAAGG-3'; LYPII (GenBank acc. no. NM_031342) 5'-GCTGACGCCCTCTCCACCAT-3' and 5'-GAAAAGCCACCCAGGACGAT-3'; LPP1 (GenBank acc. no. NM_022538) 5'-CGATGTGATTTGCGTGTTGC-3' and 5'-ATGAGGAGTGCCCCGAGTAG-3'; autotaxin (GenBank acc. no. NM_057104) 5'-TGACCACGGCTTTGATAACA-3' and 5'-TTGACTCCATTCCTTTCTGA-3'.

Radioactive labeled RNA probes for the ribonuclease protection assays were generated by transcription of antisense strands using [32P]CTP (800 Ci/mmol) and the Maxiscript kit from Ambion (Austin, TX). The probes were purified by denaturing polyacrylamide gel electrophoresis (5% polyacrylamide, 8 M urea). Solution hybridization of the RNA probe with 20 µg of total RNA and subsequent RNase digestion were performed using an RPA II kit according to manufacturer's instructions (Ambion). A probe for the ribosomal protein L19 was used as internal control. RNase-protected probes were isolated by denaturing polyacrylamide gel electrophoresis and quantified using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The results are expressed relative to the signal generated by the protected L19 mRNA.

Immunoblotting. Protein extracts from tissues and ELT3 cells were obtained by homogenization in lysis buffer (50 mM Tris, pH 7.4, 10% glycerol, 0.05% Triton X-100, 150 mM NaCl, 1 mM dithiothreitol, 2 mM Na3VO4, 10 mM NaF, 1 mM EDTA), supplemented with complete protease inhibitor mixture (Roche Molecular Biochemicals, Indianapolis, IN). Proteins were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA). The membranes were probed with 1:1,000 dilutions of either anti-SphK1, anti-lyase (Exalpha Biologicals, Maynard, MA), anti-cyclin D1 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-BclXL (Cell Signaling Technology, Danvers, MA), or anti-phosphorylated myosin light-chain 20 (MLC20; a gift from Dr. James M. Staddon, Eisai London Research Laboratories, University College, London, UK). Following rinsing, the blots were incubated with a 1:5,000 dilution of horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse IgG (Amersham Pharmacia Biotech, Piscataway, NJ), depending on the first antibody. Analysis was performed using the Enhanced Chemiluminescence Detection Kit (ECL, Amersham). Images were analyzed by densitometry using a FluorChem Imager (Alpha Innotech, San Leandro, CA). The membranes were stripped and reprobed with antibody to either ERK2 (Santa Cruz Biotechnology) or MLC20 (Sigma Chemical).

Immunohistochemical analysis. Whole uteri, obtained from rats on days 1 and 17 of pregnancy, were fixed in buffered formalin and paraffin embedded. Tissue sections (5 µm) were deparaffinized and rehydrated by passage through xylene and graded ethanol solutions. Slides were then treated with 1% hydrogen peroxide in phosphate-buffered saline (PBS) for 15 min, followed by microwave antigen retrieval at 100°C for 10 min in Target Retrieval Solution (DAKO, Carpinteria, CA) in an H2800 Microwave Processor (Energy Beam Sciences, Agawam, MA). Following sequential 15-min incubations with 0.1% avidin and 0.01% biotin (Vector Laboratories, Burlingame, CA) to block endogenous avidin- and biotin-binding sites, slides were incubated in 0.05% casein (Sigma)-0.05% Tween-20-PBS for 30 min to block nonspecific protein binding. Sections were treated with a 1:150 dilution of either rabbit anti-SphK1 (Exalpha Biologicals, Watertown, MA) or rabbit IgG negative control (DAKO) for 60 min. The biotinylated F(ab')2 fragment of swine anti-rabbit IgG (DAKO) was used to bind to the primary antibody, biotin was detected with streptavidin-HRP, and the complex was visualized using 3,3-diaminobenzidine (DAKO). Slides were counterstained with Mayer's modified hematoxylin (Poly Science, Bay Shore, NY), mounted, and viewed using an Olympus BX51 microscope equipped with a digital camera.

Expression of human SphK1 in ELT3 cells. ELT3 cells were cultured using phenol red-free MEM medium containing 2 mM L-glutamine, 50 IU/ml penicillin, 50 mg/ml streptomycin (Mediatech, Herndon, VA), and 5% fetal bovine serum (Gemini BioProducts, Woodland, CA). The human SphK1 and the dominant negative SphKG82D (36) plasmids were a gift from Dr. Lina M. Obeid (Medical University of South Carolina, Charleston, SC). The SphK1 coding sequence lacking the start codon was inserted into the pRAV-Flag retroviral vector, obtained from Dr. X. Liu (University of Colorado, Boulder, CO) (23), which we modified for selection purposes to encode an enhanced green fluorescent (EGFP)/neomycin-resistance (NEO) fusion protein (pRAV-FLAG EGFP/NEO). The pRAV-FLAG EGFP/NEO construct was engineered in two steps. First, the BamHI/SapI fragment from pRAV-FLAG, containing the EGFP coding sequence, was inserted into BamHI/SapI-linearized pUC18 (pEGFP-pUC18). The NEO, gene derived from pCDNA3, was amplified using BD TITANIUM Taq DNA polymerase (BD Biosciences Clontech, Mountain View, CA) and primers (5'-AGTGTTGTACATTAGAAGAACTCGT-3' and 5'-CGAGCTGTACAAGATGATTGAACAA-3'). The PCR product, containing BsrGI sites near the ends, was digested with BsrGI and inserted into BsrGI-linearized pEGFP-pUC18. The BamHI/SapI fragment from a clone bearing the NEO coding sequence in the proper orientation was then reintroduced into pRAV-FLAG. BamHI (end-filled)/Not1 fragments from pcDNA3-SphK1 and pcDNA3-SphKG82D were inserted into a SalI (end-filled)/NotI-digested pRAV-FLAG EGFP/NEO. All constructs were sequence verified.

The constructs were transfected into RetroPack PT67 packaging cells (BD Biosciences Clontech), using Fugene (Roche Diagnostics, Indianapolis, IN). Retroviruses were harvested and used to infect ELT3 cells, which were subjected to antibiotic selection using G-418 (200 µg/ml, Invitrogen). Expression levels of the Flag-tagged fusion proteins were determined by immunoblot analysis using an anti-Flag antibody (Sigma). ELT3 cells infected with human SphK1, SphKG82D, or empty vector were plated at a density of 200,000 cells/30-mm plate. Cell lysates were subjected to immunoblot analyses.

MTT cell proliferation assay. ELT3 cells, expressing empty vector, SphK1, or SphKG82D, were plated at density of 5,000 cells/well in a 96-well plate in medium described in the preceding section. The next day, the medium was replaced with fresh medium, and the cells were incubated for an additional 24 h. Then the medium was removed from each well, and the cultured cells were rinsed with serum-free medium followed by the addition of 0.1 ml of 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) solution (0.5 mg/ml medium, Sigma) to each well. After incubation at 37°C for 2 h, the MTT solution was removed, and insoluble formazan reaction product was solubilized with 0.1 ml of detergent reagent (10% sodium dodecyl sulfate, 50% N,N-dimethyl formamide, and 40% water containing 0.25% acetic acid and 0.02% hydrochloric acid). Absorbance of the converted dye was measured at a wavelength of 570 nm, with background subtraction at 650 nm, using a microtiter plate reader (Molecular Devices, Sunnyvale, CA).

Statistical analysis. For the animal studies, the results are expressed as means ± SE of uteri from three rats. Experiments using ELT3 cells involved means ± SE of triplicate dishes. Comparison among groups was made by one-way ANOVA and the Holm-Sidak posttest or Student's t-test when only two groups were compared, using SigmaStat software (Systat, Point Richmond, CA). A value of P < 0.05 was considered significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Lysophospholipid receptors. We previously identified three Sph-1-P receptor mRNAs (S1P1, S1P2, S1P3) and one LPA receptor (LPA1) in cultured myometrial cells from pregnant women by use of reverse transcription-polymerase chain reaction (RT-PCR) and DNA sequence analysis (20). Examination of the expression of these receptor subtypes in rat uterus, enriched in myometrium by scraping away the luminal surface, showed that S1P1 and LPA1 mRNA levels were elevated about twofold (relative to L19 mRNA) at the end of gestation and in the postpartum period compared with their levels on day 1 of pregnancy (Fig. 1A). In contrast, S1P3 mRNA levels were reduced by almost 50% on days 15, 17, and 19 compared with day 1. There were no significant changes in the expression of S1P2.


Figure 1
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  		Fig. 1. Steady-state mRNA levels in rat uterus during pregnancy and postpartum (pp) day 1. mRNA levels, determined by ribonuclease protection assay, are expressed relative to those of L19 mRNA, which encodes a ribosomal protein whose level of expression remains constant during pregnancy. Samples on day 22 were taken before labor (N) and after the delivery of 1 pup (L). Each value is mean ± SE of samples from 3 rats. Data were analyzed via 1-way ANOVA followed by the Holm-Sidak multiple comparison test. A: lysophospholipid receptors. The symbols indicate P < 0.05 relative to day 1 of pregnancy: *S1P1, +LPA1, #S1P3. B: enzymes involved in lysophosphatidic acid (LPA) metabolism. Autotaxin and phospholipase A2 (cPLA2) are involved in synthesis of LPA, whereas phosphohydrolase-1 (LPP1) and lysophospholipase-2 (LYP2) are phosphatases that degrade LPA. *P < 0.05 relative to day 1 of pregnancy. C: enzymes involved in sphingosine 1-phosphate (Sph-1-P) metabolism. SphK1 and SphK2, sphingosine kinases involved in synthesis of Sph-1-P, whereas Sph-1-P lyase and phosphatase are degradative enzymes. *P < 0.05 relative to day 1 of pregnancy.

 
LPA and Sph-1-P metabolic enzymes. Because little is known about key steps in the regulation of uterine lysophospholipid production, we examined steady-state mRNA levels of enzymes directly involved in the synthesis and degradation of LPA and Sph-1-P. Of the mRNA pool sizes of the LPA synthesis enzymes lysophospholipase D (autotaxin) and cPLA2, and the degradative enzymes LPP1 and LYP2, respectively, only that of LPP1 was elevated, about twofold, on days 15 and 17 of pregnancy compared with day 1 (Fig. 1B). Of the enzymes directly involved in the synthesis and degradation of Sph-1-P, SphK1, SphK2, Sph-1-P lyase, and Sph-1-P phosphatase, only expression of SphK1 mRNA was elevated significantly (Fig. 1C). The maximal rise in SphK1 mRNA levels was about 30-fold on days 15 and 17 and decreased to about eightfold on days 21 and 22 relative to day 1. SphK1 expression on the first postpartum day was not statistically different from that on day 1 (Fig. 1C). Expression of Sph-1-P lyase was decreased on days 5 and 9 relative to other days of pregnancy, but the decline was not statistically significant by ANOVA.

Immunological analysis of SphK1 protein levels in the uterus during pregnancy. Of the steady-state mRNA species examined, that of SphK1 was elevated to the greatest extent during pregnancy. SphK1 protein levels generally followed SphK1 mRNA levels, as shown by immunoblot analysis (Fig. 2). Uterine SphK1 was not detectable on day 1 of pregnancy but increased by day 5 and was maximal around days 17–19 (Fig. 2). SphK1 protein levels then decreased to barely detectable levels on day 22 during labor and were undetectable 1 day after delivery. There were no significant changes in Sph-1-P lyase levels during pregnancy. Immunoreactive levels of ERK2, which remain constant throughout pregnancy, were used to normalize SphK1 and Sph-1-P lyase levels per sample (Fig. 2).


Figure 2
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  		Fig. 2. Immunoblot analysis of SphK1 levels in uterus during pregnancy and in postpartum period. After being probed with SphK1 antibody, membranes were stripped and reprobed with antibody to ERK2 and to Sph-1-P lyase. Days of pregnancy are denoted as in Fig. 1. Each bar represents the ratio of either SphK1 or Sph-1-P lyase to ERK2 in arbitrary densitometric units and is the mean ± SE of replicate samples from 3 rats. Blots are single representations of each time point. *P < 0.05 relative to day 1 of pregnancy.

 
Immunohistochemical localization of SphK1 in the rat uterus. Cytosolic SphK1 immunoreactivity was seen in the uterine luminal epithelium and myometrium of day 1 pregnant rat (Fig. 3, C and D), but nuclear localization was also apparent in the luminal epithelium and in some cells in the stroma and myometrium. On day 17 of pregnancy, cytosolic staining was markedly increased in the luminal and glandular epithelium, vasculature, and myometrium (Fig. 3, G and H). SphK1 has been shown to be distributed in both cytosolic and nuclear compartments in cultured cells (18, 22), but our findings indicate that the increase in staining is primarily cytosolic in late pregnancy.


Figure 3
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  		Fig. 3. Immunohistochemical localization of SphK1 in day 1 pregnant (A–D) and 17-day pregnant (E–H) rat uterus (original magnification x200). Sections were incubated with control, nonimmune rabbit IgG (A, B, E, F), or rabbit IgG directed against SphK1 (C, D, G, H) and counterstained with hematoxylin (blue staining). The presence of immunoreactive SphK1 is indicated by red to brown staining. The luminal side (A, C, E, G) and serosal side (B, D, F, H) of the uterine horns are shown. LE, luminal epithelium; GE, glandular epithelium; S, serosa; M, myometrium; V, blood vessel.

 
Progesterone as an effector of SphK1 expression. As the temporal profile of uterine SphK1 expression parallels that of serum progesterone levels during pregnancy (41), we examined the effects of progesterone on SphK1 mRNA concentrations in two ways. First, we administered progesterone or a progesterone antagonist to pregnant rats. Progesterone administration (2 mg twice daily) starting on day 19 of pregnancy circumvented endogenous progesterone withdrawal, prolonged gestation, and maintained elevated SphK1 mRNA expression through day 24 (Fig. 4A). Vehicle-injected control rats delivered on day 22, and SphK1 mRNA levels were significantly lower than those in the progesterone-treated group on the equivalent of day 24 (2 days postpartum; Fig. 4A). There were no significant effects of progesterone administration on SphK2, Sph-1-P lyase, or Sph-1-P phosphatase mRNA levels (Fig. 4A). Administration of the progesterone antagonist onapristone on day 17 of pregnancy resulted in preterm delivery on day 19 and reduced uterine SphK1 mRNA levels relative to those of the vehicle-injected control group (Fig. 4B). Sph-1-P phosphatase and SphK2 mRNA levels were not significantly affected by onapristone administration, but there was a significant, almost threefold rise in Sph-1-P lyase mRNA levels (Fig. 4B).


Figure 4
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  		Fig. 4. Effect of progesterone (P4) on SphK1, SphK2, Sph-1-P lyase, and Sph-1-P phosphatase mRNA levels. Mean ± SE values (n = 3) of control and P4-treated groups were analyzed by Student's t-test. A: twice-daily P4 treatment, beginning on day 19, was continued for 5 consecutive days, and mRNA levels were determined 24 days after onset of pregnancy. Control rats (Control) gave birth on day 22, whereas P4-treated rats failed to deliver. *P < 0.05 vs. Control. B: uteri were removed 2 days after treatment of pregnant rats on day 17 of pregnancy with P4 antagonist onapristone. Treated rats delivered prematurely on day 19 and uterine samples were taken after delivery of the first pup, whereas vehicle control rats (Control) were still pregnant and killed on day 19. P < 0.05 for SphK1 (*) and Sph-1-P lyase (+) groups vs. their respective control groups. C: ovariectomized rats were treated for 5 days with either oil vehicle (CON), P4, 17beta-estradiol (E2), P4 + E2 together (E2/P4), or a regimen of sequential E2 and P4 mimicking the steroid milieu over the first 5 days of pregnancy (M5). Data are expressed as %control group. Mean ± SE values were analyzed by 1-way ANOVA followed by Holm-Sidak multiple comparison test between control and steroid-treated animals. P < 0.05 for SphK1 (*) and Sph-1-P lyase (+) groups vs. their respective control groups.

 
We also examined the effects of exogenous progesterone on nonpregnant, ovariectomized rats. Treatment with progesterone or 17beta-estradiol for 4 days or the two simultaneously had no effect on the expression of SphK1, SphK2, Sph-1-P lysase, or Sph-1-P phosphatase mRNA concentrations compared with control animals (Fig. 4C). These results were not surprising, as progesterone receptors are induced by estrogen priming (38). When ovariectomized rats were treated with 17beta-estradiol first, followed by progesterone (in a regimen that simulates the hormonal milieu of the first 5 days of pregnancy), there was a more than fourfold (P < 0.05) rise in steady-state SphK1 mRNA expression in the uterus (Fig. 4C). There were no significant changes in SphK2 or Sph-1-P phosphatase mRNA levels at this time, but Sph-1-P lyase expression was significantly reduced (Fig. 4C). These findings are in agreement with the increase in uterine SphK1 and declining trend in Sph-1-P lyase mRNA levels on day 5 of pregnancy compared with day 1 (Fig. 1C).

Endogenous SphK1 expression in ELT3 cells. As the immunohistochemical results showed that a major site of SphK1 expression during late pregnancy was the myometrium, we utilized a rat uterine smooth muscle cell line to further investigate the effects of progesterone on SphK1 mRNA levels. The Eker rat myometrial cell line (ELT3), derived from a spontaneous leiomyoma, expresses both estrogen and progesterone receptors (16). Under normal culture conditions, ELT3 cells expressed low, but detectable, levels of SphK1 mRNA. Addition of 10 nM progesterone to the culture medium for 4 h caused an approximately sevenfold (P < 0.05) rise in SphK1 mRNA concentration (Fig. 5A). The effects of progesterone were transient, diminishing by ~67% by 24 h and 100% by 48 h. 17beta-Estradiol, at 1 nM, had no effect on SphK1 mRNA levels after 4 h either alone or combined with progesterone (Fig. 5B). The concentrations of estrogen and progestin used are within the range of those shown to be active in ELT3 cells with respect to both estrogen-induced cell proliferation and its inhibition by progestins (16). Incubation of the cells with the progesterone antagonists onapristone (ZK-98299) or RU-486, at effective doses (21) of 1 µM, did not affect basal SphK1 levels but completely blocked the stimulatory effects of progesterone (Fig. 5B).


Figure 5
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  		Fig. 5. A: time course of the effect of P4 on SphK1 mRNA steady-state levels in ELT3 cells. B: effect of treatment of ELT3 cells (4 h) with E2 and/or P4, or P4 antagonists RU-486 (RU) or onapristone (ZK) on SphK1 mRNA levels, expressed as %control group, and each value is mean ± SE of triplicate determinations. One-way ANOVA followed by the Holm-Sidak multiple comparison test was used to determine statistical significance (P < 0.05), indicated by different letters above each column.

 
Concomitant expression of uterine SphK1 expression: cyclin D1 and BclXL. The actions of SphK1 include increased cell proliferation and inhibition of apoptosis in 3T3 and HEK 293 cells (33, 51). To estimate whether these processes are associated with elevated uterine SphK1 expression during pregnancy, we began by investigating whether markers of cell proliferation and antiapoptosis, such as cyclin D1 and BclXL, respectively, are expressed concomitantly with SphK1. BclXL, rather than other members of the Bcl family, was examined, because the pattern of expression in the rat myometrium (39) appears to be similar to that of SphK1. Indeed, the temporal profiles of the relative protein levels of both cyclin D1 and BclXL (Fig. 6) were similar to that exhibited by SphK1 (Fig. 2).


Figure 6
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  		Fig. 6. Immunoblot analysis showing relative levels of cyclin D1 and BclXL proteins in rat uterus during pregnancy. Blot is representation of replicate samples from 3 rats. Blots were stripped and reprobed using antibody to ERK2 to demonstrate equal loading of polyacrylamide gels and transfer onto membranes. Days of pregnancy are denoted as in Fig. 1.

 
Overexpression of SphK1 and the dominant negative mutant Sphk1G82D in ELT3 cells. To examine whether elevated SphK1 expression affects cyclin D1 and BclXL levels, we stably expressed either wild-type SphK1 or affinity protein-tagged variant of wild-type SphK1 into ELT3 cells. Similar results were obtained with both SphK1 wild-type constructs, indicating that the tag does not interfere with activity, and data obtained using the tagged form are shown. Cells overexpressing SphK1 exhibited increased basal levels of cyclin D1 expression (Fig. 7A). However, cells expressing the equivalent amount of the affinity-tagged dominant negative SphKG82D mutant had about one-half and one-third (P < 0.05) the expression of cyclin D1 compared with cells expressing the vector alone or SphK1, respectively (Fig. 7A). There was no statistically significant difference in BclXL protein levels in cells expressing either empty vector, SphK1, or SphKG82D (Fig. 7B).


Figure 7
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  		Fig. 7. Effects of overexpression of SphK1 and the SphK1 dominant negative mutant (SphKG82D) on expression of basal levels of cyclin D1 (A), BclXL (B), and MLC20 phosphorylation (C). Control cells were infected with retroviral vector alone (empty vector). Each value shown is mean ± SE of triplicate dishes. Letters above columns indicate differences (P < 0.05) from empty vector controls. Representative immunoblots are shown above each column.

 
Myosin light-chain phosphorylation. Because we found that the myometrium is a major site of SphK1 expression and extracellularly applied Sph-1-P increases MLC20 phosphorylation in other cell types (10, 37), we examined the effects of overexpression of SphK1 on MLC20 phosphorylation in ELT3 cells. Basal MLC20 phosphorylation was about 4 and 10 times greater (P < 0.05) in cells overexpressing SphK1 compared with cells expressing vector alone or the dominant negative SphK1 mutant, respectively (Fig. 7C).

Effect of SphK1 expression on ELT3 cell proliferation. Among other cellular functions, cyclin D1 is involved in control of the mitotic cell cycle. In line with increased cyclin D1 levels, there was an ~28% increase (P < 0.05) in the proliferation rate of cells ectopically expressing SphK1 (Fig. 8). In contrast, there was an ~20% decrease (P < 0.05) in the proliferation rate in cells expressing SphKG82D, consistent with the decreased levels of cyclin D1 expression seen.


Figure 8
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  		Fig. 8. Effect of expression of SphK1 and SphKG82D on ELT3 cell proliferation using the MTT assay. OD, optical density. Each value shown is mean ± SE of triplicate dishes. Letters a, b, and c indicate that each group is statistically different (P < 0.05) from the others.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Lysophospholipids have been shown to be involved in reproductive processes, such as implantation (55), angiogenesis during decidualization (40), and regulation of placental vascular tone (15). There is evidence to suggest a role for LPA in the induction of labor in that autotaxin activity in serum increases during the third trimester of pregnancy, reaching higher levels in patients with threatened preterm delivery (49). Consistent with the idea that LPA might play a role in the initiation of labor, there was also more than a twofold (P < 0.05) rise in LPA1 receptor expression in the uterus on days 21 and 22. LPA might stimulate myometrial contractions, as indicated by its ability to increase myosin light-chain phosphorylation in cultured human myometrial cells (42). It is not known whether reproductive tissues contribute significantly to the production of LPA. Our findings show that the levels of LPA synthetic/degradative enzymes do not change appreciably in myometrial-enriched uterine tissue during the course of pregnancy except in the case of LPP1 mRNA levels, which were elevated (P < 0.05) on days 15 and 17 of pregnancy. However, it is possible that other enzyme(s) in the LPA synthesis pathway might be key regulators of LPA production or that the enzymes involved are regulated posttranslationally.

There was a statistically significant increase in S1P1 mRNA levels in the rat uterus at the end of gestation and a decrease in S1P3 mRNA between days 15 and 19 of pregnancy. Among the lysophospholipid receptor subtypes and synthetic/degradative enzymes expressed in the uterus, SphK1 steady-state mRNA and protein levels were upregulated to the greatest extent (~30-fold) during pregnancy. SphK1 mRNA and protein expression first became apparent around day 5, coincident with the time of implantation of the blastocyst. Uterine expression levels then rose to maximum around days 15–17 and declined thereafter to near-initial levels in the postpartum period. Serum progesterone levels increase from implantation in the rat until around days 15–17 and then fall off from day 19 to the end of gestation (see Ref. 41 for references). Because changes in SphK1 expression appeared to parallel the changes in serum progesterone levels, we considered the possibility that progesterone regulates SphK1. Indeed, administration of progesterone and the resulting prolongation of pregnancy blunted the fall in SphK1 mRNA expression beyond day 19. In addition, induction of preterm labor by administration of the progesterone receptor antagonist onapristone on day 17 resulted in a premature labor and a fall in SphK1 mRNA expression. Nonpregnant ovariectomized rats were administered progesterone, 17beta-estradiol, or the two together. None of these treatments caused a change in SphK1 mRNA levels. However, it is likely that we did not observe an effect of progesterone treatment alone, as prior estrogen exposure is required to upregulate progesterone receptors (38). Indeed, pretreatment of rats with 17beta-estradiol before progesterone, in a regimen that mimics the ovarian steroid milieu during the first 5 days of pregnancy, resulted in a significant fourfold rise in SphK1 mRNA expression. Parenthetically, there appeared to be a reciprocal relationship with respect to the effects of progesterone and progesterone antagonists on SphK1 and Sph-1-P lyase mRNA levels. Thus, progesterone might elevate Sph-1-P levels by increasing expression of SphK1 and decreasing that of Sph-1-P lyase.

We determined, by incubating ELT3 cells with progesterone, that the effects of progesterone on the uterus were likely direct and not mediated by agents released from other tissue sites. These cells, which were derived from rat myometrium, express both estrogen and progesterone receptors (16). Progesterone increased SphK1 mRNA expression seven- to eightfold by 4 h, but the level of expression declined thereafter. The mechanistic basis for this transient effect was not examined, but the reduced effects of progesterone at the later time points might be due to progesterone-stimulated downregulation of its own receptors in cultured cells (38). Regardless of these results, SphK1 expression is clearly upregulated by progesterone.

The immunohistochemical analysis showed that SphK1 was expressed by several cell types in the pregnant uterus. Expression by the luminal epithelium appears to be constitutive, as approximately equal staining was seen in uteri from day 1 and day 17 pregnant rats. In contrast, there was strikingly greater staining in the glandular epithelium, vasculature, and myometrium on day 17. As not all cells in these tissues were stained, the antibody appears to be specific for SphK1. Although SphK1 was localized predominantly in the cytoplasm of uterine cells in late pregnancy, nuclear staining was observed in the day 1 pregnant uterus. Two functional nuclear export signal sequences in human SphK1 appear to allow shuttling between the nucleus and cytoplasm (18). Platelet-derived growth factor, in addition to rapidly stimulating cytosolic SphK1 in Swiss 3T3 cells, also induced a large increase in nucleoplasm-associated activity after 12–24 h (22). The progression of cells to the S-phase of the cell cycle was correlated with the translocation of a tagged SphK1 fusion protein to the nuclear envelope (22). The apparent shift in primary location of SphK1 in uterine cells from the nucleus to cytoplasm in late pregnancy is a unique observation that could help elucidate the complex actions of SphK1 in future investigations. The glandular epithelium, vasculature, and myometrium undergo growth during pregnancy. To gain a better understanding of the significance of increased SphK1 synthesis in the myometrium during pregnancy, we generated rat myometrium-derived ELT3 cell lines constitutively expressing human SphK1 or the SphK1 dominant negative mutant (36).

Despite the fact that BclXL expression paralleled that of SphK1 in the rat uterus during pregnancy, the levels of BclXL expression were not different in cells expressing either SphK1, the SphK1 dominant negative mutant, or vector. These results indicate that there is probably not a causal relationship between the uterine expression of SphK1 and BclXL; rather, both might be independently regulated by a common agent, such as progesterone.

Uterine SphK1 expression paralleled that of cyclin D1 during pregnancy. In this case, there appears to be a causal relationship between SphK1 and cyclin D1, as ELT3 cells expressing human SphK1 also exhibited elevated basal levels of cyclin D1 protein relative to cells infected with the empty vector or mutant SphK1. Previous studies showed that the ectopic expression of SphK1 in NIH 3T3 cells resulted in expedited G1/S transition, increased DNA synthesis, and increased cell numbers (33). In view of the role of cyclin D1 as a regulatory subunit for cyclin-dependent kinases (CDK) in G1/S transition, it is likely that the stimulatory effect of ectopic expression of SphK1 on ELT3 cell proliferation is mediated by increased cyclin D1 levels. Indeed, suppression of cyclin D1 levels in cells expressing the SphK1 mutant resulted in a reduced rate of proliferation. Cyclin D1 is also involved in CDK-independent functions, such as interacting with transcriptional factors, coactivators, and corepressors that govern epigenetic modifications (12). It is therefore likely that a subset of pleiotropic actions of SphK1 can be accounted for by the induction of cyclin D1 expression by uterine cells during pregnancy.

Lysophospholipids increase MLC20 phosphorylation through their respective receptors activating G protein {alpha}12/13-subunits, which stimulate guanine nucleotide exchange factors, allowing Rho A to bind GTP (10, 37, 43). Thus activated, Rho stimulates Rho kinase, which catalyzes the phosphorylation of the myosin-binding subunit of myosin phosphatase, inhibiting myosin phosphatase activity. Rho kinase also phosphorylates MLC20 directly. Aside from the extracellular effects of Sph-1-P, we show that basal levels of MLC20 phosphorylation were greater in the cells expressing SphK1 relative to cells expressing the empty vector or SphKG82D. Overexpression of SphK1 in smooth muscle cells of isolated resistance arteries results in increased MLC20 phosphorylation, resting tone, and myogenic responses (6). By analogy, the rise in myometrial SphK1 expression during pregnancy might play a role in increasing uterine smooth muscle resting tone and sensitizing the myometrium to contractile agents. Indeed, lysophospholipids are capable of inducing contractions of isolated rat myometrium (48). However, there has been no clear demonstration of a role for Sph-1-P in the initiation of labor. Instead, phosphorylation of MLC20 might be an important factor in the maintenance of stress fiber morphology (24). These fibers are associated with mechanical stretch (11), and it is possible that increased myometrial SphK1 during pregnancy is a response, in part, to uterine stretch associated with growing fetuses. Indeed, lysophospholipid alone can induce stress fiber formation in cultured myometrial cells (13).

The present studies point to an important role for the physiological upregulation of SphK1 expression in the uterus during pregnancy. Whereas basal levels of SphK1 likely clear the cell of sphingomyelin derivatives (17), elevation and/or activation of SphK1 by various agonists confers a signaling role through the production of elevated Sph-1-P. On the basis of the progesterone-induced increase in SphK1 expression, any one of a number of agents, or a combination of them, might activate the enzyme and generate increased amounts of intracellular Sph-1-P and extracellular Sph-1-P transported from intracellular sites during pregnancy. The downregulation of SphK1 at the end of pregnancy is consistent with the findings of Charpigny et al. (8). Those workers, using microarray analysis, showed that parturition is associated with a massive downregulation of developmental and proliferation-related genes in the human myometrium. Insofar as Sph-1-P stimulates development and proliferation, the downregulation of SphK1 at the end of pregnancy might contribute significantly to the reduced expression of these regulatory genes.

In summary, our findings indicate that the capacity of the rat uterus to produce Sph-1-P is increased during pregnancy. Although our understanding of the significance of this finding is still cursory, increased SphK1 expression might result in increased cyclin D1 expression and MLC20 phosphorylation in vivo as well as serving a number of key functions in adaptation of the uterus to pregnancy.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by funds from the Department of Obstetrics and Gynecology and from the Center of Interdisciplinary Research in Women's Health at the University of Texas Medical Branch, Galveston, TX.


   ACKNOWLEDGMENTS
 
We thank Dr. Cheryl Walker, University of Texas M. D. Anderson Cancer Center, for making ELT3 cells available; Dr. Lina M. Obeid, Medical University of South Carolina, for the SphK1 and SphKG82D DNA constructs; Dr. X. Liu, University of Colorado, for the pRAV-Flag retrovirus; and Dr. James M. Staddon, Eisai London Research Laboratories, for the antibody to phosphorylated MLC20.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Soloff, Dept. of Obstetrics and Gynecology, Univ. of Texas Medical Branch, Galveston, TX 77555-1062 (e-mail: msoloff{at}utmb.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 MATERIALS AND METHODS
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 DISCUSSION
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