Objective
This study was aimed at assessing the role of leptin on human myometrium, by studying its receptor expression in pregnant myometrium and the interaction of leptin with inflammation-induced apoptosis.
Study Design
Myometrial samples were obtained from women with uncomplicated pregnancies who underwent cesarean delivery at term before labor onset. The effect of leptin on apoptosis was assessed by the incubation of myometrial strips with leptin (10 –10 to 10 –8 mol/L; 48 hours) before lipopolysaccharide treatment (10 μg/mL; 48 hours).
Results
Long and short leptin receptor isoforms were expressed in myometrial cells of pregnant women. Leptin prevented lipopolysaccharide-induced apoptosis, in a concentration-dependent manner, by down-regulating cleaved caspase-3 and BCL2–associated X protein and up-regulating BCL2 expression. This effect was mediated specifically through leptin receptor stimulation, followed by ERK1/2 signaling pathway activation.
Conclusion
These results suggest a new potential pathway that is involved in delivery disorders of obese women and propose a role for the leptin-induced inhibition of myometrial apoptosis in the development of such disorders.
The prevalence of obesity is increasing worldwide, including in women of childbearing age, which is of alarming concern because maternal obesity increases the risk of complications for both mother and fetus. Maternal obesity is associated with a wide spectrum of reproductive, pregnancy, and delivery disorders (such as subfertility, miscarriage, preeclampsia, diabetes mellitus, and delayed onset of labor that lead to higher rates of postdate pregnancies). Higher rates of labor induction, failed induction, cesarean section deliveries, and postpartum hemorrhage have also been reported. This increased rate of cesarean section deliveries in obese women might not be explained solely by cephalopelvic disproportion but also by an altered uterine contractility, as suggested by Zhang et al who reported that obesity impairs the ability of the uterus to contract at delivery. Indeed, in vitro measurements of spontaneous contractions from myometrial strips demonstrated that myometrium from obese women contracted with less force and frequency than that from normal-weight women. This was recently challenged in an article that suggests that the observed implications of obesity on parturition cannot be explained by a direct effect on myometrial contractile mechanisms.
Leptin, the obese gene product, is a 16-kd cytokine-type hormone of 146 amino acid residues mainly produced by adipocytes. Circulating leptin levels correlate closely with both the body mass index (BMI) and the total amount of body fat and appear to be elevated in obese subjects, which leads to a state of leptin resistance.
During pregnancy, the placenta becomes a major source of leptin. Circulating leptin levels increase throughout pregnancy, reach a peak during the second trimester, decline thereafter, and return to normal values after delivery. Leptin levels increase in both obese and normal-weight women during pregnancy, but the levels are higher in women with higher BMIs. Whereas leptin and its receptors are both expressed in the placenta, only leptin receptors have been detected in endometrium and nonpregnant myometrium. Additionally, Moynihan et al demonstrated that leptin inhibits in vitro myometrial contractility. The combination of these data supports a central involvement of leptin in the development of obesity-related delivery disorders. However, little is known about the potential mechanism(s) of leptin action on the myometrium of normal or obese women. Leptin acts through its specific obesity gene receptors (OB-R), which belong to the class I cytokine receptor superfamily. In humans, at least 4 splice variants of OB-R messenger RNA (mRNA) encoding protein have been identified and differ in the length of their intracellular domains. Although the long form, which is referred to as OB-RL, predominates in the hypothalamus, 3 short forms (B219.1-B219.3) are expressed in various tissues. Unlike what has been described in mice, no mRNA for a splice form encoding the soluble leptin receptor has been detected so far in humans, where the soluble leptin receptor is generated by ectodomain shedding of membrane-bound receptor forms and seems to act as a leptin-binding protein in the peripheral blood. Leptin’s binding to OB-RL is known to activate 4 signaling pathways: extracellular signal-regulated kinase (ERK1/2), mitogen-activated protein kinase (p38MAPK), janus kinases (JAK)/signal transducers and activators of transcription (STAT) and phosphatidylinositol-3-kinase (PI3K)/AKT in several tissues. OB-Rs have reduced signaling capabilities and are unable to activate the JAK/STAT pathway.
The molecular mechanisms that lead to the onset of labor, from uterine quiescence to contractile activity, remain poorly understood. But, this switch coincides with an activation of the intrinsic apoptotic pathway. Charpigny et al first suggested that apoptosis might be involved in the delivery process as proapoptotic genes were up-regulated in the myometrium of laboring, compared with nonlaboring women. The implication of apoptosis in parturition was further suggested by Shynlova et al who showed that caspase cascade activation may regulate the transition from myometrial proliferative to contractile status and was confirmed by a study that showed that, before labor, prominent changes in the myometrial fibers reflected apoptosis. We previously reported that inflammation that is induced by chorioamnionitis, which is a major cause of preterm delivery, is associated with myometrial apoptosis and validated in an in vitro lipopolysaccharide model that mimics the effect of chorioamnionitis.
Because several reports have linked leptin to an inhibition of apoptosis, we hypothesized that the antiapoptotic effect of leptin might be a key event in the delivery disorders that are observed in obese women, by inhibiting myometrial differentiation towards a contractile phenotype. The aims of our study in human near-term myometrium were (1) to determine the presence and the localization of leptin receptors, (2) to assess the signaling pathways that are induced by leptin stimulation, and (3) to study the ability of leptin to modulate lipopolysaccharide-induced apoptosis.
Materials and Methods
Drugs and solutions
Recombinant human leptin (L4146) and lipopolysaccharide (Escherichia coli O55:B5, L2880) were purchased from and dissolved according to manufacturer’s instructions. OB-R antibody was purchased from Abcam Inc (Cambridge, UK); cleaved caspase-3 and signaling pathways antibodies were purchased from Cell Signaling Technology Inc (Danvers, MA); BCL2–associated X protein (BAX), BCL2, glyceraldehyde-3-phospate dehydrogenase, and secondary antibodies were purchased from Santa Cruz Biotechnology Inc (Santa Cruz, CA). Leptin receptor antagonist (LPrA; Ki ≈ 6.10 –7 mol/L) was a gift from the Boston Biomedical Research Institute (Watertown, MA). Pharmacologic inhibitor of MEK1/2 (U0126; IC 50 = 72 nmol/L and 58 nmol/L for MEK1 and MEK2, respectively) was purchased from Calbiochem (Nottingham, UK).
Biologic samples
Myometrial biopsy specimens were obtained from women who underwent elective cesarean section delivery, mostly between 38 and 40 weeks of gestation, for cephalopelvic disproportion, before labor onset. Myometrial strips were excised from the subserosal region, at an antiplacental site, as previously described. Maternal prepregnancy BMI was categorized according to the World Health Organization’s definitions as underweight (BMI, <18.5 kg/m 2 ), normal weight (BMI, 18.5-24.9 kg/m 2 ), overweight (BMI, 25-29.9 kg/m 2 ), and obese (BMI, ≥30 kg/m 2 ). Because of tissue sample size limitation, experiments were performed once for each patient. This study was approved by the French Drug Agency (registration no. 2008-A01334-51) and the local ethics committee (CPP-Est1, Dijon, France). A written informed consent was obtained from all donors.
Stimulation of myometrial biopsies
Myometrial biopsy specimens were transferred immediately in sterile DMEM (Invitrogen, Carlsbad, CA) and washed twice in sterile phosphate-buffered saline solution (PBS; Lonza, Basel, Switzerland). Biopsy specimens were cut into small strips and placed in a 24-well plate that contained 2 mL DMEM that was supplemented with antibiotics and antifungal, as previously described. Whatever the consecutive stimulations were to be, strips were incubated at 37°C with 5% CO 2 for 48 hours to allow cytokine levels to return to basal values. Then, depending on the intended investigation, the duration of the subsequent stimulations varied. To investigate leptin signaling pathways in near-term myometrium, we stimulated biopsy specimens with leptin to perform time and concentration course experiments (15, 30, and 60 minutes with leptin concentrations that ranged from 10 –10 to 10 –8 mol/L). Changes in signal phosphorylation of ERK1/2, p38MAPK, STAT3, and AKT expression were analyzed by Western blotting. To assess the ability of leptin to oppose lipopolysaccharide-induced apoptosis, myometrial strips were incubated for 48 hours, either with lipopolysaccharide alone (10 μg/mL) as previously described or with a mixture of lipopolysaccharide plus leptin (range, 10 –10 to 10 –8 mol/L); leptin was added 15-20 minutes before the lipopolysaccharide. Western blotting experiments were performed to investigate changes in proapoptotic (cleaved caspase-3, BAX) and antiapoptotic (BCL2) marker expression. To assess the specificity of leptin anti–apoptotic effect, we used a selective OB-R antagonist (LPrA ; range, 10 –7 to 10 –5 mol/L) that was added with the following sequence: LPrA was first put in the medium; leptin was then added 1 hour later; lipopolysaccharide was added 15-20 minutes later. Myometrial strips were incubated with this mixture for 48 hours. Finally, to investigate signaling pathways that were involved in the observed effect, inhibitors of activated pathways (ie, MEK1/2 [MAPK kinase]; U0126 ; 1.10 –5 and 3.10 –5 mol/L) were added 1 hour before leptin and lipopolysaccharide stimulation.
For each experiment, nonstimulated time-matched controls were used. At the end of the stimulation period, the supernatant samples and tissues were frozen quickly in liquid nitrogen and stored at −80°C.
Immunohistochemical analysis
Myometrial strips were fixed for 1 hour with paraformaldehyde 4%, embedded in paraffin, and cut into 5-μm–thick sections. After deparaffinization and rehydration, antigen retrieval was performed by incubating slides for 10 minutes in warm citric acid. After endogenous peroxidase activity was blocked with hydrogen peroxide 3%, nonspecific binding sites were blocked for 30 minutes with 3% bovine serum albumin in PBS. Slides were incubated overnight with a 1/1000 primary anti–OB-R (ab5593) dilution, washed 3 times with PBS, and incubated with a biotinylated anti-rabbit antibody (1/250) for 1 hour. After a new washing, slides were incubated with peroxidase-labeled streptavidin (1/500) for 30 minutes and finally with 3-amino-9-ethyl-carbazole solution until it was a clearly visible color. The reaction was stopped by extensive washing in water. Subsequently, slides were counterstained with hematoxylin. Negative controls were carried out by omitting the primary antibody.
Reverse transcriptase–polymerase chain reaction (RT-PCR) analysis
Total RNA was extracted from snap-frozen myometrial tissues by the Trizol reagent method and purified by deoxyribonuclease I treatment, according to the manufacturer’s instructions (Invitrogen). The integrity of RNA was verified by ethidium bromide staining of agarose gel analysis and by optical density (OD) absorption ratio OD260 nm/OD280 nm ≥1.8. One microgram of total RNA was then subjected to reverse transcription (SuperScript II Reverse Transcriptase kit; Invitrogen). The first-strand complementary DNA samples were amplified by PCR with specific primers and conditions according to Li et al. Amplification of a housekeeping gene, 18S ribosomal RNA , was performed on each complementary DNA sample as an internal control. A negative control was carried out by omitting complementary DNA during PCR. The PCR products were electrophoresed on 2% agarose gels that were stained with ethidium bromide and analyzed with the Gel Doc 1000 system (Bio-Rad Laboratories, Hercules, CA).
Western blotting analysis
Snap-frozen myometrial tissues were crushed with liquid nitrogen, homogenized in lysis buffer (40 mmol/L Tris-HCl [pH 7.4], 150 mmol/L NaCl, 2 mmol/L EDTA, 1 mmol/L sodium orthovanadate, 50 mmol/L sodium fluoride, 1% Triton X100, 1% protease inhibitor [P8340; Sigma-Aldrich, St. Louis, MO], 1% phosphatase inhibitor [P2850; Sigma-Aldrich]), and kept on ice for 30 minutes. After a centrifugation at 10,000 g for 15 minutes at 4°C, total protein supernatant content was determined by the Bradford assay, with bovine serum albumin as the standard. Samples (30-50 μg of protein by lane) were dissolved (volume/volume) in 2X Laemmli buffer and boiled for 5 minutes. Proteins were subjected to electrophoresis on an 8% or 12% sodium dodecylsulfate-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride membrane. To block nonspecific antibody-binding sites, membranes were incubated for 1 hour in 5% nonfat dried milk powder in Tris-buffered saline solution/Tween 20 (TBST; 10 mmol/L Tris, 150 mmol/L NaCl, 0.1% Tween 20, pH 7.8) at room temperature. Membranes were washed 3 times with TBST. The blots were then incubated overnight at 4°C with a 1/2000 dilution of primary rabbit anti-OB-R antibody (ab5593; Abcam Inc) or a 1/1000 dilution of primary rabbit anti-phosphoSTAT3 (#9131; Cell Signaling Technology Inc), anti-phosphoAKT (#9271; Cell Signaling Technology Inc), anti-phosphoERK1/2 (#9101; Cell Signaling Technology Inc) or anti-phospho-p38MAPK (#9211; Cell Signaling Technology Inc) antibody in 1% non-fat dried milk powder in TBST. For apoptosis assessment, the blots were incubated overnight at 4°C with a 1/400 dilution of primary cleaved caspase-3 antibody (#9661; Cell Signaling Technology Inc) or a 1/1000 dilution of primary BAX (sc-493) or BCL2 antibody (sc-7382) in 1% non-fat dried milk powder in TBST. After 3 washes with TBST, the blots were incubated with horseradish peroxidase-conjugated anti-rabbit antibody (sc-2313) or anti-mouse antibody (sc-2314) at a dilution of 1/10,000 for 1 hour at room temperature and washed 3 times. Immunoreactive proteins were detected by chemiluminescence. The intensities of the bands were analyzed densitometrically with Quantity One software (Bio-Rad Laboratories). Blots were stripped and reprobed with anti-STAT3, anti-AKT, anti-ERK1/2, anti-p38MAPK antibody, or glyceraldehyde-3-phospate dehydrogenase antibody, which was used as the protein loading control. Results are expressed as the mean ± SEM in arbitrary density units.
4-6 diamidino-2-phenylindole (DAPI) staining
To distinguish between apoptotic and necrotic cells, chromatin condensation and fragmentation in apoptotic bodies were assessed by staining of nuclei with DAPI (D9542; Sigma-Aldrich). Optimal cutting temperature-embedded sections of stimulated biopsy specimens were incubated with a 0.1 μg/mL DAPI solution for 15 minutes in the dark. Slides were rinsed with water and mounted with Aquatex (MacDermid Autotype Inc, Rolling Meadows, IL). The stained nuclei were visualized with convert fluorescent microscope at magnification of ×1000 with excitation light at 350 nm.
Statistical analysis
Differences among groups were determined by an analysis of variance, followed by the Bonferroni’s or the Dunn’s multiple comparison tests. Statistical analysis was carried out using GraphPad Instat software (version 3; GraphPad Software, Inc, San Diego, CA). All differences were considered significant at a probability value of < .05.
Results
Patient demographics
Twenty-two women with uncomplicated pregnancies who had undergone cesarean delivery at term were included in the study. Maternal characteristics are summarized in the Table .
| Characteristic | Patients, n | Mean ± SD | Range |
|---|---|---|---|
| Maternal age, y | 22 | 32.6 ± 5.6 | 19–42 |
| Gestational age, wk | 22 | 36.5 ± 3.6 | 30–41 |
| Body mass index, kg/m 2 | 26.2 ± 5.5 | 17.7–37.4 | |
| <18.5 | 1 | ||
| 18.5-24.9 | 11 | ||
| 25-29.9 | 6 | ||
| ≥30 | 4 | ||
| Parity | |||
| Nulliparous | 6 | ||
| Primiparous | 11 | ||
| Multiparous | 5 |
Leptin receptors are present in the human near-term pregnant myometrium
Immunostaining of myometrial samples that were obtained from 3 different women showed an intense OB-R staining ( Figure 1 , A) that was not observed in negative control experiments ( Figure 1 , C). This staining was located specifically in myometrial cells ( Figure 1 , B). This finding was confirmed by RT-PCR and Western blotting experiments that were performed on samples that were obtained from 5 different women. RT-PCR experiments demonstrated the expression of specific mRNA transcripts encoding for the OB-RL isoform and for the 3 OB-RS isoforms ( Figure 1 , D). Western blotting experiments revealed 120-kd and 100-kd protein bands that corresponded to OB-RL and OB-RS, respectively ( Figure 1 , E).
