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Endocrinology Vol. 145, No. 8 3850-3857
Copyright © 2004 by The Endocrine Society

Leptin-Induced Increase in Leukemia Inhibitory Factor and Its Receptor by Human Endometrium Is Partially Mediated by Interleukin 1 Receptor Signaling

R. R. Gonzalez, B. R. Rueda, M. P. Ramos, R. D. Littell, S. Glasser and P. C. Leavis

Boston Biomedical Research Institute (R.R.G., M.P.R., P.C.L.), Watertown, Massachusetts 02472; Vincent Center for Reproductive Biology (R.R.G., B.R.R., M.P.R., R.D.L.), Massachusetts General Hospital, Boston, Massachusetts 02114; Harvard Medical School (B.R.R., R.D.L.), Boston, Massachusetts 02115; Department of Molecular and Cellular Biology (S.G.), Baylor College of Medicine, Texas 77030; and Department of Physiology (P.C.L.), Tufts University School of Medicine, Boston, Massachusetts 02111

Address all correspondence and requests for reprints to: R. R. Gonzalez, Boston Biomedical Research Institute, 64 Grove Street, Watertown, Massachusetts 02472. E-mail: gonzalezr{at}bbri.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Leptin and leukemia inhibitory factor (LIF) have been implicated as important mediators of implantation. The present study was designed to investigate whether leptin can directly regulate the expression of LIF and its receptor (LIF-R) in human endometrial cells and/or whether leptin-induced effects are linked to, or regulated in part by IL-1 signaling. Primary endometrial cells and endometrial epithelial cell lines (HES and Ishikawa cells) were cultured for 24–48 h in a medium containing insulin (5 µg/ml) and leptin (3, 10, and 62 nM) or IL-1ß (0.6, 3, and 10 nM) in the presence or absence of cytokines and/or receptor antagonists. The endpoints included phosphorylation of signal transducer and activator of transcription 3 (STAT3) and the relative levels of LIF, LIF-R, IL-1ß, IL-1 receptor antagonist (IL-1Ra) and IL-1 receptor type I (IL-1R tI) as determined by ELISA or Western blotting techniques. Leptin treatment increases the level of phosphorylated STAT3, LIF-R, and LIF. Leptin also increases the levels of IL-1 ligand, receptor, and antagonist as was previously reported. Blockade of OB-R with antibodies or with a specific OB-R inhibitor (leptin peptide antagonist-2) abrogated leptin-induced effects, suggesting that leptin binding to its receptor activates Janus kinase 2/STAT3 signaling. Treatment of endometrial cells with IL-1ß also results in elevated levels of LIF-R. Interestingly, the inhibition of IL-1R tI with a specific antibody or with IL-1Ra negatively affects both leptin-induced and IL-1-induced effects on LIF-R levels. Abnormal endometrial LIF expression has been associated with human infertility and leptin has profound effects on the levels of LIF, IL-1, and their cognate receptors in vitro. Thus, it is tempting to speculate that leptin’s role in vivo could include the regulation of other key cytokines to be fundamental to endometrial receptivity during implantation (i.e. LIF and IL-1).


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE IMPLANTATION PROCESS is a rate-limiting step in mammalian reproduction that depends on complex and precisely controlled interactions between the preimplantation embryo and the receptive endometrium. Several growth factors and cytokines have been implicated as regulators of implantation. Among these factors, leukemia inhibitory factor (LIF) (1) and leptin (2) play essential roles.

LIF, a pleiotropic and ubiquitous cytokine, promotes myeloid leukemia cell differentiation and is considered essential for reproduction in mice. Evidence to support this conviction continues to accumulate. LIF has the ability to preserve the totipotentiality of murine embryonic stem cells (3). Mouse preimplantation embryos and endometrium express mRNA for LIF and its receptor (LIF-R). Female mice lacking LIF produce viable blastocysts, but they fail to implant (1, 4). Administration of LIF rescues fertility in LIF-deficient mice (1). The importance of LIF during early implantation is primarily confined to its effects on the endometrium because LIF-R-deficient-embryos implant normally (5, 6).

The importance of LIF to reproduction is not limited to the murine model. LIF sequences are highly conserved among species, including humans (7). It has also been suggested that LIF plays an important role in rabbit (8, 9) and human implantation (10). The human blastocyst expresses the functional LIF-R, and the addition of the ligand increases the quality and number of human blastocysts in vitro (11, 12). In contrast to the mouse (13), the mRNA encoding LIF is not expressed by the human preimplantation embryo (11). LIF mRNA is, however, expressed in human endometrial cells. The levels of LIF mRNA and protein vary in accordance to the stage of the menstrual cycle (14, 15, 16, 17, 18, 19). In humans, maximal LIF secretion by the endometrium coincides with the window of implantation (17). Similar to that observed in the mouse, infertility in humans has also been related to abnormal expression of LIF by the endometrium (10, 20, 21). The diminution of immunoreactive LIF with specific antibodies leads to significant reduction in pregnancy rates in rhesus monkeys (22), suggesting that LIF does play a significant role in the primate.

Similar to LIF, leptin is also a pleiotropic and ubiquitous cytokine that plays a critical role in reproductive function (2, 23). Leptin promotes preimplantation embryo development (24) and leptin (25, 26) and leptin receptor (OB-R) are expressed by endometrium (25, 26, 27, 28), suggesting a role for leptin in embryo-maternal cross talk at the time of implantation. This idea is supported with evidence that human blastocysts cocultured with endometrial epithelial cells (EECs) modulate leptin secretion (25). More importantly, leptin (ob/ob)- or OB-R (db/db)-deficient mice cannot reproduce and fertility can be restored by exogenous administration of leptin (29) in the ob/ob mouse. Pregnancies from leptin-treated ob/ob females fail if leptin is withdrawn shortly after conception (30).

Leptin and LIF signal via specific transmembrane receptors that are activated upon dimerization after binding to their respective ligands. Activated OB-R is a homodimer that belongs to the class I cytokine superfamily of receptors (31, 32). In contrast, the activated LIF-R is composed of LIF-R{alpha} (also called LIF-Rß) and gp130 protein (7). In addition to LIF, other cytokines (i.e. IL-6 and IL-11) can transmit intracellular signals through LIF-R. When activated, both OB-R (33, 34) and LIF-R (35) signal through phosphorylation of Janus kinase (JAK) and signal transducer and activator of transcription 3 (STAT3), which, in turn, activate several downstream signaling pathways. In addition to the JAK-STAT signaling pathway, other pathways, including the MAPK, protein kinase C, and phosphoinositol 3-kinase pathways, are also activated by leptin (34, 36) and LIF (37).

IL-1 and leptin have synergistic functions on endometrial receptivity (37, 38). Leptin up-regulates IL-1 receptor antagonist (IL-1Ra), IL-1ß, and IL-1 receptor type I (IL-1R tI) levels in primary human EEC and endometrial stromal cells (ESCs) cell cultures (38). IL-1 (39) and leptin (40) induce the expression of ß3 integrin, an adhesion molecule and a marker of endometrial receptivity, which likely increases the possibility of successful implantation (40, 41). The blockade of functional OB-R impairs leptin and IL-1ß effects on EEC and ESC cultures (38). In contrast to LIF, leptin and IL-1 induce the expression of markers of endometrial receptivity and of the invasive trophoblast (42, 43).

To further assess the leptin-induced signaling in the endometrium, we examined leptin-induced effects on LIF and LIF-R protein levels in human endometrial cells. We also investigated whether leptin-induced changes are related to IL-1 function.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and antibodies
Goat polyclonal antihuman LIF-R{alpha}, anti-OB-R (anti-NH2 terminal end of human OB-R), and monoclonal anti-IL-1R type I (IL-1R tI) antibodies, human recombinant leptin, IL-1ß, and IL-1Ra were obtained from R&D Systems Inc. (Minneapolis, MN) Antibodies for STAT3 (F-2) and phosphorylated STAT3 (p-STAT3, B-7) and nonspecific mouse and goat IgGs were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antivimentin, anticytokeratin, and anti-CD45 antibodies were from Dako Corp. (Carpinteria, CA). Human recombinant insulin was obtained from Sigma Chemical Co. (St. Louis, MO). Fetal bovine serum (FBS) was obtained from Gemini Bioproducts (Woodland, CA), and DMEM/F-12 and antibiotic-antimycotic mixture were from Gibco BRL Products (Gaithersburg, MD). Other chemicals were obtained from Sigma Chemical Co.

Endometrial tissues
Endometrial tissues were obtained from hysterectomies of nonmalignant etiologies under an institutional review board at the Vincent Center for Reproductive Biology [Massachusetts General Hospital, Boston, MA (B.R.R.)]. Tissues were digested with proteases for endometrial cell isolation as described elsewhere (41). Briefly, endometrial biopsies were minced and treated with collagenase I (0.1%)-deoxyribonuclease I (0.005%) for 1 h at 37 C. After gland sedimentation, ESC were separated from supernatants. EEC were purified of ESC and macrophage contaminants by repeated incubation at 37 C in a Falcon flask. Stromal and epithelial cell dispersions were counted in a hemocytometer, and cell viability (≥ 90%) was assessed by optical microscopy using the Trypan Blue exclusion method. To assess homogeneity of cell preparations several specific monoclonal antibodies were used in cell smears (41), i.e. antivimentin (ESC+), cytokeratin (EEC+), and CD45 (leukocyte +). Homogeneity of cell preparations was higher than 98%.

Cell cultures and treatments
LIF/LIF-R are mainly expressed in the endometrial epithelium; thus, we investigated leptin-induced effects in a variety of EEC types in an attempt to recognize any cell type-specific responses that could be misinterpreted as a global response. The cell types we used included: Ishikawa (a endometrial cancer cell line widely used in reproductive biology research), HES (a cell line derived from a benign proliferative endometrium), and primary EECs. LIF/LIF-R are also expressed in ESCs, and therefore their response to leptin were analyzed for general comparison with the epithelial response.

Primary human endometrial cells (ESC and EEC) were cultured for 5–9 d in DMEM-F12 containing 5% FBS, 5 µg/ml insulin, 1% amphopthericin B, 100 µg/ml streptomycin, and 100 U/ml penicillin until they were 80% confluent. Two types of endometrial epithelial cell lines, HES (originally derived from a benign proliferative endometrium, provided by Dr. Douglas Kniss, Ohio State University, Columbus, OH) and Ishikawa (human endometrial epithelial adenocarcinoma, ECACC 9832301, Wiltshire, UK) were cultured under the same conditions described above. The cells were washed twice with 100 mM PBS, pH 7.2 and cultured for additional 2 d in the same medium but without FBS (basal medium). Cells were washed as described before and cultured in basal medium containing leptin (0, 3, 10, or 62 nM) or IL-1ß (0, 0.6, 3, or 10 nM).

The anti-OB-R antibody (10 or 20 µg/ml) and leptin peptide antagonist-2 (LPA-2), a specific inhibitor of OB-R (3, 120, or 300 nM or 30 µM) were used to assess whether leptin-induced effects were regulated primarily by the OB-R. Nonspecific species-matched IgGs to the OB-R antibody and a scrambled version of LPA-2 (LPA-2Sc) served as negative controls (44). Inhibitors of IL-1R were used (monoclonal antibody anti-IL-1R tI and IL-1Ra) to assess whether leptin activated IL-1-induced signaling pathways or just activated similar components responsible for LIF/LIF-R expression. Treatment with the cytokine, inhibitor or the two combined was implemented for 5, 15, 30, and 60 min, and 24 and 48 h. The cells were prepared for Western blot analysis. The conditioned media was collected, lyophilized and stored at –80 C until analysis for LIF, IL-1ß, and IL-1Ra by ELISA could be performed. Duplicate wells were run for each treatment and the experiments repeated at least three times with different cell preparations.

Cell lysates
Endometrial cells were washed with ice-cold PBS and lysed by homogenization on ice with lysis buffer A [20 nM Tris (pH 7.4) containing 137 nM NaCl, 2 mM EDTA, 10% glycerol, 50 mM ß-glycerophosphate, 1% Nonidet P-40, and a mixture of proteases and phosphatase inhibitors composed of 100 µM antipain, 0.1 mg/ml trypsin inhibitor, protease inhibitor cocktail 1:50 (Sigma), 50 nM NaF, 2 mM phenyl-methylsulfonyl fluoride, and 2 mM sodium orthovanadate]. Cellular lysates were centrifuged at 2400 x g at 4 C for 10 min. For nuclear lysates, the cells were scraped from the culture plates and homogenized with lysis buffer B [10 nM Tris (pH 7.4), 0.25 M sucrose, 0.1 mM EDTA containing the same mixture of protease and phosphatase inhibitors as buffer A]. Protein concentrations were determined using the Bradford protein assay (Bio-Rad Laboratories Inc., Hercules, CA).

Immunoprecipitation
Thirty micrograms of protein from nuclear or cellular lysates were incubated in Eppendorf tubes containing 0.5 µg of primary antibodies (anti-LIF-R, OB-R, STAT3, and p-STAT3 diluted in buffer A) for 2 h at 4 C under constant stirring. The immunocomplexes were incubated with 20 µl of Protein G-Agarose (Amersham Pharmacia Biotech, Piscataway, NJ) diluted 1:1 with buffer A for an additional 2 h under constant stirring. The beads were centrifuged for 1 min at 1000 x g and washed with buffer A containing 0.5 M NaCl followed by a final wash with buffer A.

Western blot
Cellular lysates and their immunoprecipitates containing 10–30 µg of protein plus Laemmli buffer (1:1) were incubated at 95 C for 5 min. Electrophoresis was performed at 220 V for 5 min followed by 130 V for 45 min (Bio-Rad, electrophoresis apparatus) on 7.5% (for STAT3, p-STAT3, LIF-R, and IL-1R tI) and 10% (for OB-R) SDS-PAGE gels. Nuclear lysates were also used to detect p-STAT3. Electroblotting onto 0.2 µm nitrocellulose membranes was performed at 22 V overnight at 4 C in 48 nM Tris-39 nM glycine buffer containing 0.037% sodium dodecyl sulfate, and 20% methanol. Membranes were washed with 20 mM Tris, 137 mM NaCl (pH 7.4) buffer containing 0.5% Tween 20 (vol/vol) (wash buffer) and incubated for 1 h at room temperature in blocking buffer containing low-fat dried milk (5%, wt/vol) in wash buffer. The membranes were subsequently incubated at room temperature for 1 h with 2 µg/ml of anti-OB-R, LIF-R, and 1 µg/ml of IL-1R tI, STAT3, and p-STAT3 antibodies in blocking buffer. After washing, the membranes were incubated for 30 min in wash buffer containing 2.5% normal horse or rabbit serum (Vector Laboratories, Burlingame, CA). Immune complexes were detected with biotinylated horse antimouse or rabbit antigoat antibodies (Vector Laboratories) followed by incubation with streptavidin-horseradish peroxidase-conjugate (Amersham) for 30 min at room temperature. Specific bands in the blots were visualized using an ECL chemiluminescent assay (Amersham) and Imagetek-B film (American X-Ray & Medical Supply, Rancho Cordoba, CA). Nonspecific mouse and goat IgGs (Santa Cruz Biotechnology) were used instead of primary antibodies to produce negative control blots.

To quantitatively assess the effects of cytokines and inhibitors of OB-R and IL-1R tI on the antigen expression, the blots were scanned and analyzed (TotalLab version 2003.02, NonLinear Dynamics Ltd., Durham, NC).

Determination of LIF, IL-1ß, and IL-1Ra secretion by human endometrial cells
Conditioned media from cells cultured under the experimental conditions described above were used to quantify the secretion of LIF, IL-1ß, and IL-1Ra by ELISA (Quantikine, R&D Systems). The experiments were replicated three times and standards, controls, and samples were assayed in duplicate. Cytokine concentrations as determined by ELISA were within the range of the standard curve and were expressed in picograms per milliliter per milligram of protein. The intra- and interassay coefficients of variation were between 5 and 8% and 8.5 and 10%, respectively. The lowest detectable concentration of each cytokine by ELISA was LIF (9 pg/ml), IL-1 (0.11 pg/ml), and IL-1Ra (15.6 pg/ml). These concentrations are similar to those reported by the manufacturer. LIF-ELISA is reported by the manufacturer to have a sensitivity of less than 8 pg/ml for both natural and recombinant human LIF and 100% specificity (no significant cross-reactivity or interference with a diversity of human and mouse cytokines and growth factors was observed). The performance characteristics for human IL-1ß and IL-1Ra ELISAs reported are: 100% specificity; sensitivity 0.1 pg/ml and 14 pg/ml, respectively.

Statistical analysis
A one-way ANOVA test with Dunnett error protection and a confidence interval of 95% was used from the Analyze-it for Microsoft Excel (Leeds, UK; http//www.analyze-it.com) for data analysis. Data are expressed as mean ± SEM. Values for P < 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Leptin effects on p-STAT3, LIF-R, and LIF levels in human endometrial cell cultures
After confirming via Western blot that the primary EEC and ESC and endometrial cell lines used herein had OB-R, we evaluated leptin-induced effects on the levels of p-STAT3. Leptin induced a concentration-dependent increase in the levels of p-STAT3 in EEC at 5 min (Fig. 1Go, A and B). Similar significant effects were observed in the ESC and the Ishikawa cell line. However, the response in HES cells was not significant (data not shown).



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  		FIG. 1. Leptin induced changes in the levels of p-STAT3, LIF-R, and LIF by human endometrial cells. A, Representative Western blot of p-STAT3 obtained from nuclear extracts of human EEC (h-EEC) after 5 min of stimulation with leptin at increasing concentrations. B, Quantitative analysis of normalized p-STAT3 levels in h-EEC (5 min). p-STAT3 levels were normalized against non-p-STAT3. C, Qualitative changes in LIF-R levels found in cellular extracts after a 24-h treatment of h-EEC. D, Quantification of leptin-induced changes in LIF-R levels after densitometric analysis. E, LIF levels in h-EEC in response to treatment with or without leptin for 24 h. Quantitative analyses of Western blot results were performed with the program TotalLab, and the levels of LIF were determined by ELISA. All data were derived from a minimum of three independent experiments using different cell preparations. The results presented in the graphs are the mean and SE. *, P ≤ 0.05 when comparing levels in response to treatments to that of the nontreated controls.

 
Leptin also induced a concentration-dependent increase in the levels of LIF-R (Fig. 1Go, C and D) and LIF (Fig. 1EGo) in primary EEC at 24 h. Leptin (3–62 nM) induced a more moderate expression of LIF-R in HES (1.3- to 2-fold) than in Ishikawa cells (1.5- to 3-fold) or primary EEC (1.7- to 8-fold) and ESC (1.4- to 3.2-fold) (data not shown). Leptin induced a significant increase in LIF levels in ESC and Ishikawa cells but had no effect on LIF levels in HES cells (data not shown).

Inhibition of leptin-induced effects by OB-R antagonists
Leptin (62 nM) increased the levels of p-STAT3 in Ishikawa cells (Fig. 2AGo). The antagonist, LPA-2 (300 nM) inhibited the leptin-induced increase, whereas LPA-2Sc had no effect (Fig. 2Go, A and B). Interestingly LPA-2 treatment decreased the level of p-STAT3 when compared with the nontreated control Ishikawa cells (Fig. 2BGo). In a similar pattern, the levels of LIF-R were elevated over control after treatment with leptin (62 nM). Cotreatment with LPA-2 (300 nM) reduced the leptin-induced increase. As before, treatment with LPA-2Sc had no effect on LIF-R levels (Fig. 2Go, C and D) when added in combination with leptin.



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  		FIG. 2. Leptin-induced changes in the levels of p-STAT3, LIF-R, and LIF are blocked by OB-R inhibitors. A, Qualitative representation of a Western blot evaluating levels of p-STAT3 in Ishikawa cells after treatment (5 min) with leptin and/or leptin and LPA-2. B, Quantitative analysis of normalized p-STAT3 levels. p-STAT3 levels were normalized against non-p-STAT3. C, Representative Western blot depicting changes in LIF-R levels in Ishikawa cells after leptin and/or leptin and LPA-2 treatments for 24 h. D, Quantitative analysis of LIF-R levels. E, LIF levels in medium of cultured Ishikawa cells after 24-h treatments with leptin in the presence or absence of OB-R antagonists/inhibitors. Controls included Ishikawa cells cultured with leptin plus LPA-2Sc or nonspecific species-matched IgG. Quantitative analyses of Western blot results were performed with the program TotalLab, and the data were calculated from results of three or more experiments (n ≥ 3) using different cell preparations. The results presented in the graphs are the mean and SE. *, P ≤ 0.05 when comparing levels in response to treatments to that of the nontreated controls.

 
Figure 2EGo illustrates a quantitative assessment of the effects of inhibitors on leptin-induced effects on LIF levels found in the medium of cultured Ishikawa cells. Treatment with leptin (3 nM) increased the levels of LIF in the conditioned medium of cultured Ishikawa cells. However, treatment of Ishikawa cells with leptin signaling inhibitors (either antibody to OB-R or LPA-2) resulted in reduced LIF levels, whereas treatment with leptin plus control IgG or scrambled peptide did not inhibit leptin (Fig. 2EGo).

Treatment of EEC with leptin resulted in elevated levels of IL-1ß and IL-1Ra (Fig. 3Go, A and B). The leptin-induced increase in IL-1ß and IL-1Ra levels was abrogated by cotreatment with LPA-2 and not affected by LPA-2Sc (Fig. 3BGo). Leptin also increased the levels of IL-1R tI and again LPA-2 inhibited the leptin-induced effects (Fig. 3Go, C and D). An analogous effect was observed in ESC and Ishikawa cells. The leptin-induced effect on IL-1ß and IL-1Ra levels was inconsistent in the HES cells (data not shown).



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  		FIG. 3. Leptin-mediated increase of IL-1ß and IL-1Ra and IL-1R tI levels by human endometrial cells are blocked by OB-R inhibitors. Leptin (10 nM) induced effects on IL-1ß (A) and IL-1Ra (B) levels in the conditioned medium (24 h) of human EECs. Leptin induced changes in the levels of IL-1R tI (C) in EEC cultures. D, Quantitative analysis of IL-1R tI levels as determined by Western blot. Interestingly, LPA-2 (30 µM) inhibited the leptin-induced changes in IL-1ß (A), IL-1Ra (B), and LIF-R (C) levels. Controls included EEC cultured with leptin plus LPA-2Sc. Analyses of Western blot results were performed with the program TotalLab. All determinations were made in duplicate and the experiments were repeated at least three times. The results presented in the graphs are the mean and SE. *, P ≤ 0.05 when comparing levels in response to treatments to that of the nontreated controls.

 
The blockade of IL-1R tI inhibits leptin’s effects on LIF/LIF-R levels
Treatment with IL-1ß increased the levels of LIF-R found in Ishikawa cells in a concentration-dependent (3 and 10 nM) manner (Fig. 4Go, A and B). Moreover, when Ishikawa cells were treated with a combination of IL-1ß and leptin (10 nM) a further increase of LIF-R levels was observed in comparison to the cells treated with IL-1ß (10 nM) alone (see Fig. 4BGo). The leptin-induced increase in LIF-R levels in Ishikawa cells was partially blocked by the addition of IL-1Ra (1000 pg/ml) or anti-IL-1R tI antibody (20 µg/ml). No effects were found when the antibody was substituted by nonspecific species-matched IgGs (see Fig. 4Go, C and D). Similar results on the induction of LIF-R levels by leptin and IL-1ß were found in primary EEC. However, IL-1R tI inhibitors (antibody and antagonist) at the concentrations described above were unable to totally inhibit the leptin-induced increase of LIF-R or LIF levels by primary EEC cultures.



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  		FIG. 4. Leptin and IL-1-induced LIF-R expression by human endometrial cells are partially blocked by IL-1R tI inhibitors. A, Representative Western blot of IL-1ß effects on LIF-R levels in Ishikawa cells in the presence or absence of leptin. B, Quantitative analysis of IL-1 effects on LIF-R levels in Ishikawa cells. C, Representative Western blot of LIF-R levels after treatment with or without leptin in the presence or absence of IL-1Ra (1000 pg/ml), monoclonal antibody anti IL-1R tI (20 µg/ml), or mouse IgG by Ishikawa cells. D, Quantitative analysis of the impact of IL-1R tI inhibitors on leptin-mediated increase of LIF-R levels in Ishikawa cells. All data were derived from a minimum of three independent experiments using different cell preparations. The results presented in the graphs are the mean and SE. *, P ≤ 0.05 when comparing levels in response to treatments to that of the nontreated controls.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
LIF and leptin have several similar features. Both signal through JAK/STAT3 (33, 34, 35) and are down-regulated by the suppressor of cytokine signaling-3 (SOCS-3) (45, 46). Preimplantation mouse embryos express LIF-R and OB-R simultaneously. From a functional standpoint, LIF is presumed to modify the effect of leptin during preimplantation and embryo development in mice. LIF is thought to interfere with activation of STAT3 signaling (47). We investigated whether leptin treatment affects the levels of members of the LIF system (ligand and/or receptor) by human endometrial cells (primary and cell lines). The primary EEC and ESC, HES and Ishikawa cells (benign and neoplastic epithelial cell lines, respectively), constitutively express OB-R. These findings corroborate our previous observations on OB-R expression by human (37, 38) and rabbit endometrial cells (44). In these cells, leptin effectively up-regulated phosphorylation of STAT3, suggesting that leptin interaction with OB-R activated JAK/STAT3 pathways (36, 48).

Our data are not in total agreement with others. It was recently reported human endometrial stromal cells do not secrete LIF and leptin had no effect on LIF or IL-6 secretion (49). In a similar study, it was reported that human endometrial stromal cells did secrete LIF, and leptin increased IL-6 secretion but had no effects on LIF secretion by the endometrial stromal cells and in a human endometrial epithelial cell line (50). The exact reasons for the differences between these studies and our own are not clear and could be attributed to differences in experimental conditions.

Our data suggest significant qualitative and quantitative differences between established cell lines and primary endometrial cells. In addition, the different cell types evaluated displayed differential sensitivity to leptin-induced effects with the EEC most responsive. Moreover, an impressive decrease in basal LIF levels was seen in epithelial cell lines compared with primary cells (EEC). The basal levels of LIF in the primary epithelial cell cultures were significantly higher (71-fold) than in Ishikawa cells. HES cells showed inconsistent response to leptin induction of LIF and IL-1 components. However, leptin at low concentration (3 nM) was able to significantly induce p-STAT3, LIF, and LIF-R levels in Ishikawa cells compared with primary EEC. Reasons for these differences could be due to the metabolic differences between normal and immortalized endometrial cells.

Endometrial stromal cells express both LIF and LIF-R (51, 52). Immunoreactive LIF protein is observed in the glandular EEC and LIF-R is primarily detected in luminal EEC (51). Interestingly, decidualized ESC are more responsive to treatment with LIF (52). EEC constitutively express LIF mRNA, whereas the levels of LIF mRNA in ESC vary (16). Our present findings corroborate the findings by others (51) that LIF and LIF-R are expressed principally by primary EEC when compared with ESC or cell lines as determined by ELISA and Western blot, respectively. Moreover, leptin induced an increase in LIF levels in the conditioned medium of EEC, ESC, or Ishikawa cells.

Previously we reported that leptin increased IL-1ß, IL-1Ra, and IL-1R tI in primary human endometrial cells (38). Leptin also induced the expression of these factors in HES and Ishikawa cell lines. It has been reported that IL-1ß induces the secretion of LIF (16). In the present study, treatment of endometrial cells with IL-1ß resulted in elevated LIF-R levels. Moreover, treatment with IL-1R tI antagonists (anti-IL-1R tI antibody or IL-1Ra) partially blocked the IL-1-induced effect in these cells. Interestingly, leptin regulation of LIF-R expression involves, to some extent, the normal function of IL-1R tI in endometrial cells. The addition of IL-1R tI inhibitors partially blocked the leptin-induced changes of LIF/LIF-R levels in endometrial cells.

Leptin and IL-1 functions appear to be closely related in the endometrium. IL-1 up-regulates leptin and OB-R expression and both leptin (37) and IL-1 (39) up-regulate ß3 integrin expression (a marker of endometrial receptivity) by endometrial cells. Leptin also up-regulates the expression of the IL-1 system (ligand, receptor, and receptor antagonist) (38). Interestingly, the blockade of IL-1R with specific antibodies decreases the expression of the IL-1 system, but leptin neutralizes this effect. Moreover, the blockade of OB-R negatively affects both leptin and IL-1ß effects on IL-1 system expression (38). The present data also suggest that leptin and IL-1ß have similar if not synergistic effects on LIF-R expression by endometrial cells. These findings further suggest that leptin is a molecular mediator for the endometrial actions of the IL-1 system and that the blockade of OB-R impairs leptin and IL-1ß functions.

Redundancy in the mechanism of action of several cytokines during implantation is well known. In fact, leptin appears to substitute for several IL-1 functions in the endometrium and trophoblast (38, 42). Despite the initial findings that IL-1Ra reduces implantation rates in mice (53), IL-1 has not been proven indispensable for implantation and reproduction in mice (13, 54). Therefore, the leptin mediated induction of LIF expression could also occur by one or more alternative mechanisms that are not obligatory to IL-1 mediating action.

Interestingly, IL-1, as well as TNF{alpha}, platelet-derived growth factor, epidermal growth factor, and TGF-ß are potent inducers of LIF in endometrium (16). Leptin is also a potent inducer of IL-1 expression (38). The data presented in the current manuscript suggest that leptin regulation of implantation could involve IL-1. Leptins effects on cytokine induction and endometrial receptivity might involve other cytokines and growth factors, i.e. TGF-ß, TNF-{alpha} (55), or vascular epithelial growth factor (VEGF) (56). IL-1 induces VEGF (57) and colony stimulator factor-1 (CSF-1) (58) expression in nonendometrial cell types. Therefore, we hypothesize leptin could potentially increase the expression of VEGF and CSF-1 in the endometrium through the up-regulation of IL-1. A scheme depicting how leptin could be one of the primary signals that initiate a wave of expression of several important molecules during implantation is presented in Fig. 5Go.



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  		FIG. 5. A schematic representation of leptin interactions with important factors for implantation. At the time of implantation, leptin secreted by the embryo and/or endometrium (25 ) induces the expression of IL-1 ligand, receptor and receptor antagonist (38 ). Leptin directly or indirectly via IL-1 induces the expression of LIF ligand and receptor and ß3-integrin (a marker for endometrial receptivity) (37 ). IL-1 could also induce the expression of VEGF (57 ) and CSF-1 (58 ). Leptin could be one of the primary factors that initiate and regulate the cascade system of molecules that promote the development of endometrial receptivity and successful implantation.

 
LIF and leptin-deficient mice cannot reproduce, but pregnancies can be achieved by injection of LIF and leptin respectively (1, 29, 30). LIF is indispensable for implantation, but LIF receptor-deficient-mouse embryos implant normally. LIF requirement in implantation is related to the absence of estrogen-induced LIF synthesis in the uterine wall at the time of blastocyst implantation (59). Interestingly, the function of LIF-R expressed by mouse luminal EEC is temporally regulated independently of LIF secretion. Although the molecular basis of the change of LIF-R activity is unknown, the negative regulation of p-STAT3 by SOCS-3 has been suggested (35). Further studies are needed to evaluate the contribution of leptin and LIF to the induction of SOCS-3 synthesis for the regulation of LIF intracellular signals at the time of implantation.

In conclusion, we found that leptin increases p-STAT3, LIF, LIF-R, and IL-1 ligand, receptor, and receptor antagonist levels in cultured human endometrial cells and in human endometrial cell lines. IL-1ß similarly increases LIF-R expression. Inhibition of IL-1R tI partially prevents leptin-induced effects on LIF/LIF-R, suggesting that IL-1 may mediate in part some of leptin’s effects on endometrial cells. The timing of the expression of LIF (16) and the tight regulation of LIF-R function at the time of implantation (35) by the endometrium could be related to leptin actions. Leptin effects on LIF/LIF-R levels in endometrial stromal and epithelial cells are closely associated to IL-R tI-regulated events. These data, together with the absolute requirement of leptin for mouse reproduction (29, 30), the leptin-induced expression of an established marker of endometrial receptivity (ß3-integrin) (37), and IL-1 (38) by endometrial cells suggest that leptin plays an important role in implantation. Leptin could be one of the primary factors that initiate and regulate the cascade system of molecules that promote the development of endometrial receptivity and successful implantation.


   Acknowledgments
 
We thank Ms. E. Gowell for helping in the purification and characterization of peptides and Dr. M. Lynch for her careful revision and helpful comments on this paper.


   Footnotes
 
This work was supported in part by Consortium for Industrial Collaboration in Contraceptive Research (CICCR), a program of Contraceptive Research and Development Program (CONRAD), Eastern Virginia Medical School Grant CIG-02-87 (to R.R.G.), the Vincent Memorial Hospital Research Funds (to B.R.R., R.D.L.), and by Analytical Biotechnology Inc.

The views expressed by the authors do not necessarily reflect the views of CONRAD or CICCR.

Abbreviations: CSF-1, Colony stimulatory factor-1; EECs, endometrial epithelial cells; ESCs, endometrial stromal cells; FBS, fetal bovine serum; IL-1Ra, IL-1 receptor antagonist; IL-1R tI, IL-1 receptor type I; JAK, Janus kinase; LIF, leukemia inhibitory factor; LIF-R, LIF receptor; LPA-2, leptin peptide antagonist-2; OB-R, leptin receptor; p-STAT3, phosphorylated STAT3; SOCS-3, suppressor of cytokine signaling-3; STAT3, signal transducer and activator of transcription 3; VEGF, vascular epithelial growth factor.

Received March 25, 2004.

Accepted for publication May 4, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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