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The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 10 3609-3614
Copyright © 1998 by The Endocrine Society

Original Studies

Augmentation of Leptin Synthesis and Secretion Through Activation of Protein Kinases A and C in Cultured Human Trophoblastic Cells1

Shigeo Yura, Norimasa Sagawa, Yoshihiro Ogawa, Hiroaki Masuzaki, Hiroko Mise, Tsunekazu Matsumoto, Ken Ebihara, Shingo Fujii and Kazuwa Nakao

Department of Gynecology and Obstetrics (S.Y., N.S., H.M., T.M., S.F.) and Department of Medicine and Clinical Science (Y.O., H.M. K.E., K.N.), Kyoto University Graduate School of Medicine, Kyoto 606-8507, Japan

Address all correspondence and requests for reprints to: Norimasa Sagawa, Department of Gynecology and Obstetrics, Kyoto University Graduate School of Medicine, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. E-mail: fetus{at}kuhp.kyoto-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Leptin is a fat cell-derived hormone that regulates food intake and energy expenditure. We previously demonstrated that leptin is produced by nonadipose cells, i.e. by placental trophoblasts. We also reported that a human trophoblastic cell line, BeWo cells, expresses leptin gene and secretes leptin into culture media. To elucidate the regulatory mechanisms of leptin production by human trophoblasts, we investigated synthesis and secretion of leptin in BeWo cells and in explant cultures of human placental tissue. Leptin production and gene expression in BeWo cells were increased by treatment with forskolin. The forskolin-induced increase in leptin production was completely suppressed by H89, an inhibitor of protein kinase A. Leptin production and gene expression in BeWo cells were increased by treatment with phorbol myristate acetate (PMA). The PMA-induced increase in leptin production was completely suppressed by H7 and staurosporine, both of which are inhibitors of protein kinase C. Leptin secretion from first trimester chorionic tissue was approximately 50-fold greater than that from term placental tissue. Leptin production and gene expression in explant cultures of placental tissue at both stages of pregnancy were augmented markedly by treatment with forskolin or PMA. The present study demonstrated augmentation of leptin production by protein kinase A and protein kinase C in cultured human trophoblasts, thereby leading to a better understanding of the regulatory mechanisms of leptin production in human trophoblasts in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
HUMAN placental trophoblasts produce various hormones and cytokines such as estradiol, progesterone, CG, human placental lactogen, tumor necrosis factor-{alpha}, and interleukin-6 (1). The production of these substances is regulated by multiple factors. For example, CG production is stimulated by GnRH (2), ß-adrenergic agents (3), and phorbol esters (4), and is suppressed by PRL (5). These placental hormones and cytokines play essential roles in the maintenance of pregnancy and in fetomaternal adaptation to pregnancy (1).

Leptin is a fat cell-derived satiety factor that regulates food intake and energy expenditure in humans and rodents (6, 7, 8). Plasma leptin concentrations are correlated well with body mass index or percent body fat in humans (9), suggesting that leptin is a useful marker for body energy balance. We recently demonstrated production of leptin by nonadipose tissue (10). In pregnant women, leptin is synthesized in and secreted from placental trophoblasts into the maternal and fetoplacental circulation (11). Plasma leptin concentrations are significantly elevated in pregnant women as compared with those in age- and body mass index-matched nonpregnant women (10, 12). Although plasma leptin concentrations are also elevated in mice and rats during pregnancy, leptin is not produced by their placenta (13, 14), suggesting the species specificity of placental production of leptin. Production of leptin in the placenta is markedly augmented in patients with severe preeclampsia (15) or in patients with diabetes mellitus (16). Plasma leptin concentrations are also elevated in patients with gestational trophoblastic diseases, hydatidiform mole, and choriocarcinoma (10, 17). These findings, taken together, suggest that leptin is a novel trophoblast-derived hormone in humans, thereby raising its possible functional significance in pregnancy and in gestational trophoblastic diseases.

The present study was designed to elucidate the regulation of leptin synthesis and secretion in placental trophoblasts. We examined leptin production by a human choriocarcinoma cell line, BeWo cells, that is a useful in vitro model system with which to assess the regulation of leptin production by human placental trophoblasts (10). We also examined the synthesis and secretion of leptin in explant cultures of human chorionic and placental tissues because of the potential differences in the regulation of leptin production between BeWo cells and normal placental trophoblasts (18).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

Forskolin, phorbol myristate acetate (PMA), H89, H7, and staurosporine were purchased from Sigma Chemical Co. (St. Louis, MO). They were dissolved in dimethyl sulfoxide and stored at -20 C until use. Other reagents were obtained from Nakarai Tesque (Kyoto, Japan) and were of analytical grade, unless otherwise indicated. RPMI 1640 (Life Technologies, Inc., Rockville, MD) was used as the culture medium and was supplemented with 10% FCS, penicillin (100 U/mL), and streptomycin (100 µg/mL), unless otherwise indicated.

Culture of BeWo cells

Culture of BeWo cells was performed as previously described (10). Cells were cultured at a density of 2.0 x 106 in 10-cm culture dishes with 10 mL culture medium. At confluency (day 0), culture media were changed, and cells were incubated with or without forskolin (0, 2, and 20 µmol/L) or PMA (0, 1, 10, and 100 nmol/L). Aliquots of 1 mL conditioned media were obtained on days 1, 3, and 5 and replaced with 1 mL fresh medium to correct for the final concentrations of forskolin or PMA.

BeWo cells were also incubated with H89 (10 µmol/L), H7 (20 µmol/L), or staurosporine (10 nmol/L). Thirty minutes after the initial treatment with these agents, forskolin (final concentration of 20 µmol/L) or PMA (final concentration of 100 nmol/L) was added. Culture media were collected on day 3. Collected samples were stored at -20 C until use.

Explant culture of human chorionic and placental tissues

Chorionic villous tissues in the first trimester of pregnancy were obtained under aseptic conditions from women undergoing legal artificial termination of pregnancy for medical or social reasons (6–12 weeks of gestation). Placental tissues at term were obtained from normal pregnant women after delivery (39–41 weeks of gestation). Informed written consent was obtained from each patient. The present study was approved by the ethical committee on human research of Kyoto University Graduate School of Medicine (No. 90).

Explant culture of human chorionic and placental tissues was performed as described previously (2, 3) with slight modifications. Briefly, tissues were rinsed with PBS, dissected free of maternal decidua and the fetal amniotic membranes, and minced into 1- to 2-mm pieces. Approximately 50 mg first trimester chorionic tissue or approximately 200 mg term placental tissue was dispersed per well in 6-well dishes with 0.5 mL culture medium (day 0) and cultured under humidified and 5% CO2 balanced air at 37 C. Twelve hours later, when tissue explants were attached to the dishes, 1.5 mL fresh culture medium was added (total 2.0 mL/well). In the time course study, culture media were collected every 2 days from the beginning of culture until day 8. For examination of the effects of forskolin or PMA, 2 mL fresh medium with or without forskolin (0, 2, and 20 µmol/L) or PMA (0, 3, and 30 nmol/L) was added to each well on day 2. Conditioned media were collected on day 4. Samples were stored at -20 C until assay.

Measurement of human leptin and CG concentrations

Human leptin-like immunoreactivity (leptin-LI) in the culture media was measured by the RIA for human leptin as described previously (19). The limit of detection of the RIA was 0.05 ng/tube. Inter- and intraassay variations were 5.9% and 5.3%, respectively. CG concentrations were determined using a commercially available enzyme immunoassay kit according to manufacturer’s instructions. (Amerlite hCG-60; Johnson & Johnson Clinical Diagnostics, Rochester, NY) (10). The limit of detection was 10 mIU/mL. Inter- and intraassay variations were 7.2% and 5.0%, respectively.

RNA extraction and Northern blot analysis

Total RNA was extracted from BeWo cells’ and chorionic tissues, and placental tissues using TRIzol reagent (Life Technologies) according to the manufacturer’s protocol. Total RNA from adipose tissue obtained with informed consent from a pregnant woman at the time of Cesarean section was used as a control. Northern blot analysis was performed with the 32P-labeled full-length human leptin complementary DNA as a probe (10). Autoradiography was performed at -70 C with intensifying screens.

Gel permeation chromatography (GPC) analysis

GPC analysis of culture media of BeWo cells and chorionic tissue was performed as described previously (19).

Data expression and statistical analysis

Each experiment was conducted in triplicate and repeated at least three times. Representative results of each experiment are shown in the figures. All values are expressed as means ± SEM. Statistical analysis was performed by ANOVA with Fisher’s least significance difference test or Mann-Whitney U test, where applicable. Differences between groups were considered significant when P values were less than 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Leptin-LI concentrations in culture media of BeWo cells stimulated by forskolin

Leptin-LI concentration in the culture medium of BeWo cells was elevated time dependently. Leptin-LI concentration in the medium of BeWo cells treated with forskolin was increased significantly and dose dependently (data not shown), consistent with our previous report (10). Leptin-LI concentration in the medium of BeWo cells treated with 20 µmol/L forskolin (8.1 ± 1.2 ng/mL) was approximately 3-fold higher (P < 0.005) than that of the vehicle-treated control group (3.5 ± 0.7 ng/mL). The forskolin-induced increase in leptin-LI concentration was reduced by H89 (4.5 ± 0.9 ng/mL) to the same level as that of the vehicle-treated group (P < 0.05) (Fig. 1Go). Leptin-LI concentration in the medium of BeWo cells treated with H89 but without forskolin (2.6 ± 0.1 ng/mL) was not different from that of the vehicle-treated control group.



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  		Figure 1. Effects of forskolin and H89 on leptin secretion from BeWo cells. Cells were preincubated with or without H89 (10 µmol/L) for 30 min before addition of forskolin at a final concentration of 20 µmol/L. *, P < 0.05.

 
Leptin-LI in culture media of BeWo cells stimulated by PMA

Leptin-LI concentration in the culture medium of BeWo cells treated with PMA was increased significantly and dose dependently (Fig. 2AGo). Leptin-LI concentration in the medium of BeWo cells treated with 100 nmol/L PMA (7.4 ± 0.2 ng/mL) was 3-fold higher than that of the vehicle-treated group (2.3 ± 0.0 ng/mL, P < 0.005). CG concentration in the culture medium was increased in parallel with leptin-LI concentration (Fig. 2BGo). The PMA-induced increase in leptin-LI concentration (8.9 ± 3.1 ng/mL) was reduced by H7 or staurosporine (1.8 ± 0.5 and 4.4 ± 0.1 ng/mL, respectively) to the same level as that of the vehicle-treated group (P < 0.005 and P < 0.05, respectively) (Fig. 2DGo). Leptin-LI concentrations in the media of BeWo cells treated with H7 or staurosporine but without PMA (1.6 ± 0.9 and 3.0 ± 0.6 ng/mL, respectively) were not different from that of the vehicle-treated group.



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  		Figure 2. Effects of PMA treatment on leptin and CG secretion from BeWo cells. A, Time course of changes in leptin-LI in culture media. Various concentrations of PMA (0, 1, 10, and 100 nmol/L) were added to medium at confluence. B, Time course of changes in CG concentrations in culture media. C, Northern blot analysis of leptin mRNA in BeWo cells treated with PMA for 120 h. RNA from adipose tissue was used as a control. Fifteen micrograms total RNA was used per lane. 28S ribosomal RNA bands visualized with ethidium bromide are shown in bottom panel. D, Effects of H7 or staurosporine on stimulation of leptin secretion by PMA in BeWo cells. Cells were preincubated with H7 (20 µmol/L) or staurosporine (10 nmol/L) for 30 min before PMA addition at a final concentration of 100 nmol/L. *, P < 0.05; **, P < 0.005; ***, P < 0.001 vs. control.

 
Leptin messenger RNA (mRNA) expression in BeWo cells

Northern blot analysis revealed a single leptin mRNA species in BeWo cells of the same size as in mature adipocytes (Fig. 2CGo) (10). Leptin mRNA expression was augmented dose dependently in BeWo cells treated with PMA compared with vehicle-treated cells (Fig. 2CGo).

Leptin-LI concentrations in culture media of human chorionic and placental tissues

Leptin-LI concentrations in the culture media of first trimester chorionic tissue and term placental tissue were 34.0 ± 2.4 and 0.78 ± 0.04 ng/day per 100 mg tissue, respectively, during the first 2 days in culture (Fig. 3Go, A and B). The secretion rate of leptin-LI from first trimester chorionic tissue was 50- to 200-fold higher than that from term placental tissue. Leptin-LI concentrations in the culture media at both stages of pregnancy were slightly decreased after 6 days of culture, although this was not statistically significant.



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  		Figure 3. Time course of changes in leptin-LI in explant cultures of human chorionic and placental tissue. First trimester chorionic tissue fragments (A) or term placental tissue fragments (B) were cultured for 8 days. Culture media were exchanged every 2 days.

 
Leptin-LI concentrations in culture media of human chorionic and placental tissues stimulated by forskolin and PMA

Leptin-LI concentrations in the culture media of first trimester chorionic tissue and term placental tissue treated with forskolin (20 µmol/L) were significantly higher (P < 0.05 and P < 0.001, respectively) than those of the vehicle-treated groups (Fig. 4Go, A and C). Leptin-LI concentrations in the culture media of first trimester chorionic tissue and term placental tissue treated with PMA (30 nmol/L) were also significantly higher (P < 0.005 and P < 0.05, respectively) than those of the vehicle-treated groups (Fig. 4Go, B and D).



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  		Figure 4. Effects of forskolin or PMA on leptin-LI secretion from explant culture of first trimester chorionic tissue (A and B) and term placental tissue (C and D). Culture media were exchanged every 2 days, and forskolin or PMA was added to culture media on day 2. Increases in secretion are indicated as fold increases calculated as ratio of leptin concentration on day 4 to that on day 2. *, P < 0.05; **, P < 0.005; ***, P < 0.001 vs. vehicle-treated tissue.

 
Leptin mRNA expression in human chorionic and placental tissues

In first trimester chorionic tissue, leptin mRNA of the same size as that in mature adipocytes was expressed abundantly, and was up-regulated dose dependently with forskolin or PMA treatment (Fig. 5AGo). In term placental tissue, leptin mRNA of the same size as that in mature adipocytes was expressed at low levels and was also up-regulated dose dependently with forskolin or PMA treatment (Fig. 5BGo).



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  		Figure 5. Northern blot analysis of leptin mRNA in first trimester chorionic tissue and term placental tissue treated with forskolin or PMA for 24 h. RNA from adipose tissue was used as a control. Twenty micrograms total RNA was used per lane. 28S ribosomal RNA bands are shown as described in Fig. 2Go.

 
GPC analysis of culture media of BeWo cells and chorionic tissue

GPC analysis was performed for culture media of BeWo cells treated with forskolin or PMA, because human leptin-LI concentration in the culture medium of nontreated BeWo cells was too low to apply GPC (< 5 ng/mL). Culture media of BeWo cells treated with forskolin and PMA had essentially identical profiles (Fig. 6AGo). Human leptin-LI was eluted in a single peak at the position corresponding to recombinant human leptin, as in culture medium of adipose tissue explants (19).



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  		Figure 6. Gel permeation chromatography profile for culture media of BeWo cells (A) and first trimester chorionic tissue (B). BeWo cells were treated with forskolin (20 µmol/L) or PMA (100 nmol/L). Two hundred microliters conditioned media was applied to a Sephadex G-50 column (1 x 30 cm). Flow rate was 30 mL/h, and fraction volume was 0.5 mL. Column was calibrated with blue dextran (Vo) and 125I (Vt). Open symbols indicate that leptin-LI was below limit of detection of assay. Vo, Void volume; Vt, total volume.

 
Human leptin-LI in the culture medium of first trimester chorionic tissue was eluted as two peaks. The major elution position of leptin-LI was identical to that in BeWo cells, and another small peak eluted in the void volume (Vo). GPC profiles of culture media of first trimester chorionic tissue treated with forskolin or PMA were essentially identical to those of the vehicle-treated group (data not shown). GPC study of the culture medium of term placental tissue was not performed because leptin-LI concentration in the medium was too low (< 5 ng/mL).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrated that leptin synthesis and secretion in BeWo cells are augmented by forskolin, an activator of protein kinase A (PKA), and by PMA, an activator of protein kinase C (PKC), in vitro. Augmentation of leptin production by forskolin is blocked completely by H89, an inhibitor of PKA (20). Augmentation of leptin production by PMA is also blocked completely by H7 or staurosporine, both of which are inhibitors of PKC (21, 22). We also examined synthesis and secretion of leptin in explant cultures of human chorionic and placental tissues, because choriocarcinoma cell lines do not always function similarly to normal placental trophoblasts (18). Leptin secretion from first trimester chorionic tissue was 50-fold greater than that from term placental tissue. This gestational age-related difference in leptin secretion is compatible with the marked difference in leptin gene expression between first trimester chorionic tissue and term placental tissue. Leptin production in explant cultures of first trimester chorionic tissue and term placental tissue was also stimulated by forskolin and PMA treatment. These findings, taken together, suggest that leptin synthesis and secretion are augmented through the activation of PKA and PKC in human trophoblasts in vitro, and also probably in vivo.

Forskolin and PMA stimulate secretion of various hormones in human placental trophoblasts including GnRH, CG, and human placental lactogen (18, 23, 24). Forskolin is also known to facilitate syncytial formation in BeWo cells, and so it was not clear whether the forskolin-induced increase in leptin production by BeWo cells was associated with morphological differentiation. Cytotrophoblasts are markedly reduced in number in term placental tissue (1). In primary cultures of cytotrophoblasts from the term placenta, the rate of syncytial formation is not affected by forskolin (18). Therefore, it is suggested that the augmented leptin production through stimulation of PKA is caused by functional activation of trophoblasts, independent of morphological changes. Although PMA is a potent mitogen, it has no effect on proliferation of BeWo cells (24), suggesting that the augmented leptin production by PKC does not depend on proliferation of trophoblasts.

PKA and PKC play central roles in biological signaling of various hormones and cytokines. For example, PKA is activated by epinephrine, prostanoids, and CG (18), and PKC is activated by angiotensin II, endothelin, and epidermal growth factor (25, 26, 27, 28). Angiotensin I and angiotensin-converting enzyme are expressed in intrauterine tissues, and angiotensin II receptor is present in placental trophoblasts (29, 30, 31). In our preliminary study, leptin secretion was augmented by angiotensin II in explant cultures of chorionic and placental tissues (Yura et al., unpublished data). These findings, taken together, indicate that leptin secretion is regulated at least partly by the renin-angiotensin system in vivo. We also found that placental production of leptin is augmented in severe preeclampsia associated with placental hypoxia (15). It has been reported that hypoxia-induced effects are mediated by PKC in multiple types of cells (32, 33). In addition, several transcription factors including Fos and Jun (components of activator protein-1 complex), which are also activated by PKC, are induced under hypoxic conditions (34). Thus, increased levels of activator protein-1 complex, through the activation of PKC, might be involved in the augmentation of leptin production in the placenta under hypoxic conditions. These findings also suggest that leptin production in the placenta is regulated by various physiological and pathophysiological factors in vivo.

It has been demonstrated that leptin circulates both in bound and free forms in humans and mice (13, 35). On GPC analysis, human leptin-LI in the culture medium of first trimester chorionic tissue is eluted in two different peaks, as in serum from humans and mice (13, 35), whereas it is eluted in a single peak in BeWo cells. The peak in the Vo, which is observed only in chorionic tissue, is likely to represent leptin bound to macromolecule(s), and the other peak corresponding to recombinant human leptin represents free leptin. One possible reason for the lack of a peak in the Vo in BeWo cells is that leptin concentrations in bound form in the media may be below the limit of detection of the RIA. We recently observed that leptin receptor is expressed in human chorionic tissue (Masuzaki et al., unpublished data). The soluble isoform of leptin receptor (Ob-Re) secreted from the placenta acts as a binding protein in pregnant mice (13). It is tempting to speculate that the soluble isoform of leptin receptor is secreted from chorionic tissue and contributes to leptin binding capacity in explant cultures of chorionic tissue.

In contrast to placental trophoblasts, activation of PKA suppresses leptin secretion in explant cultures of mature adipocytes (36). PMA has no significant effect on leptin secretion from mature adipocytes (Ogawa et al., unpublished observation). We and others have demonstrated that DNA sequences in the leptin 5'-flanking region, which are important for transcription of the leptin gene in trophoblastic cells, are different from those in adipocytes (37, 38). It is likely that a specific enhancer for human leptin gene that is not active in adipocytes is present in the trophoblast (38). All these findings indicate that the regulation of leptin production in placental trophoblasts is different from that in adipocytes.

In summary, we demonstrated that synthesis and secretion of leptin are augmented through the activation of PKA and PKC in cultured human trophoblastic cells. Our findings will lead to a better understanding of the regulatory mechanisms of leptin production by placental trophoblasts and of the physiological and pathophysiological roles of leptin during pregnancy.


   Acknowledgments
 
We thank Mr. H. Iwai and Ms. H. Yoshida for technical assistance in RIA and data analysis, and Ms. A. Kishimoto for secretarial assistance.


   Footnotes
 
1 This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan (08457441, 09877319, and 09671675), and a grant from Smoking Research Foundation, Japan. Back

Received April 18, 1998.

Revised June 18, 1998.

Accepted July 8, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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