hCG stimulates leptin mRNA expression and enhances cAMP levels in placenta.

<p>(A) JEG-3 cells (1×10⁶ cells) were plated in complete DMEM-F12 media supplemented with 1% FBS and incubated during 3 days with different doses of hCG (IU/ml). (B) Placental explants were obtained as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046216#s2" target="_blank">Materials and Methods</a> and treated with increasing hCG concentrations. In (A) and (B), total RNA was extracted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046216#s2" target="_blank">Material and Methods</a>. Leptin mRNA was quantified by real time RT-PCR. Cyclophilin was used as internal standard. (C) BeWo cells (1×10⁵) were seeded in 96-well plate and treated during 24 h with increasing concentrations of hCG, as indicated. cAMP-Glo assay kit was used to measure intracellular cAMP concentration. (D) Cells were transiently transfected with pCre-Luc plasmid construction and treated with hCG, (Bu)₂cAMP or cotransfected with CREB, as indicated, during 72 h in DMEM-F12 media supplemented with 1% FBS. Luciferase activity was measured in cellular extracts and normalized to β-galactosidase activity. Activity obtained with empty vector (PGL-3 basic vector) was set as a control. (E) BeWo cells were transiently transfected with pL1951 and treated with 100 IU/ml hCG and/or cotransfected with CREBM plasmid. Cells were incubated during 72 h in DMEM-F12 1% FBS media. Luciferase activity was measured in cellular extracts and normalized to β-galactosidase activity. Activity obtained with empty vector (PGL-3 basic vector) was set as a control. Results shown are from a representative experiment and are expressed as means ± S.E.M. for three independent experiments performed in duplicates. *p<0.05, **p<0.01, ***p<0.001 vs control; ###p<0.001 vs hCG treatment.</p

PKA blocks hCG stimulation of leptin.

<p>(A) BeWo cells were incubated during 3 days with hCG and/or H89, as indicated. Extracts from cells were prepared as previously described and loaded in a 12% SDS-PAGE. Leptin expression was determined by Western-blot. Loading controls were performed by immunoblotting the same membranes with anti-β-actin. Bands densitometry is shown in lower panels. Molecular weight (kDa) is indicated at the right of the blot. Representative results from three replicates are shown. (B) BeWo cells were transiently transfected with pL1951 and treated with 100 IU/ml hCG, 10 µM H89 and/or 100 µM SQ, or cotransfected with a plasmid expressing the catalytic subunit of PKA (PKA) (1 µg/ml) (C), or with a dominant negative mutant of the regulatory subunit of PKA (PKI) (1 µg/ml). Cells were incubated during 72 h in DMEM-F12 1% FBS media. Luciferase activity was measured in cellular extracts and normalized to β-galactosidase activity. Activity obtained with empty vector (PGL-3 basic vector) was set as a control. Results are expressed as mean ± S.E.M. for three independent experiments. *p<0.05, **p<0.01, ***p<0.001 vs. control; #p<0.05, ###p<0.001 vs. hCG treatment.</p

cAMP induces leptin stimulation by hCG at low hormone concentrations.

<p>(A) Placental explants were processed as previously described and treated with increasing hCG and/or (Bu)₂cAMP concentrations during 4 h. Total RNA was extracted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046216#s2" target="_blank">Material and Methods</a>. Leptin mRNA was quantified with real time RT-PCR. Cyclophilin was used as internal standard. (B) BeWo cells (1×10⁶ cells) were plated in complete DMEM-F12 media supplemented with 1% FBS and incubated during 3 days with different doses of hCG (IU/ml) and/or (Bu)₂cAMP (µM), as indicated. Cell extracts were prepared as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046216#s2" target="_blank">Materials and Methods</a>. Proteins were separated on SDS-PAGE gels and leptin expression was determined by Western-blot. Molecular weights were estimated using standard protein markers. Loading controls were performed by immunoblotting the same membranes with anti-β-actin. Bands densitometry is shown in lower panels. Molecular weight (kDa) is indicated at the right of the blot. Representative results from three replicates are shown. **p<0.01, ***p<0.001.</p

The MAPK and the alternative cAMP/Epac signaling pathways participate in leptin stimulation by hCG in placenta.

<p>Proposed model of the signaling pathways involved in hCG stimulation based on current data and its relation to leptin expression. Pointed arrow: Stimulation; Flat arrow: Inhibition. Dash arrow: possible pathways involved.</p

Leptin enhances BCL-2/BAX relationship in placental cells.

<p>A) Swan-71 cells (1×10⁶ cells) were plated in DMEM-F12 media in the absence of serum and incubated during 72 h with different doses of leptin. DMEM-F12 10% FBS was used as a control. Cell extracts were prepared as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099187#s2" target="_blank">Materials and Methods</a>. Proteins were separated on SDS-PAGE gels and BCL-2 and BAX expression was determined by Western blot analysis. Molecular weights were estimated using standard protein markers. Molecular mass (kDa) is indicated at the right of the blot. Loading controls were performed by immunoblotting the same membranes with anti-α-tubulin. Bands densitometry is shown in lower panels, results are expressed as mean ± SD for three independent experiments. B) Leptin increased BCL-2/BAX relationship. C) BeWo cells were transiently transfected with a plasmid containing a section of BAX promoter (pBax-Luc). After transfection, cells were incubated for 72 h in DMEM-F12 and treated with increasing leptin doses. Cell extracts were prepared as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099187#s2" target="_blank">Materials and Methods</a> and Luciferase activity was normalized to β-galactosidase activity. Activity obtained in the absence of leptin and pBax-Luc was set as control. Statistical analyzes were performed by ANOVA. Asterisks indicate significant differences from the control according to Bonferroni's multiple comparison <i>post hoc</i> test, relative to FBS 10% (#) or FBS 0% (*). ## p<0.01, * p<0.05, *** p<0.001.</p

Leptin diminishes BID level in placenta.

<p>A) Swan-71 cells (1×10⁶ cells) were plated in DMEM-F12 media 10% FBS. After 24 h, cells were incubated during 72 h with increasing doses of leptin in DMEM-F12 0% FBS. Cell extracts were prepared as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099187#s2" target="_blank">Materials and Methods</a> and proteins were separated on SDS-PAGE gels. B) Placental explants were processed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099187#s2" target="_blank">Material and Methods</a> and incubated in DMEM-F12 0% FBS media supplemented with increasing leptin doses during 24 h. Placental extracts were prepared and proteins were separated on SDS-PAGE gels. In both cases (A and B) BID cleaved fragment was determined by Western blot analysis. DMEM-F12 10% FBS was used as control. Molecular weights were estimated using standard protein markers. Molecular mass (kDa) is indicated at the right of the blot. Loading controls were performed by immunoblotting the same membranes with anti-α-tubulin or anti-β-actin. Bands densitometry is shown in lower panels. Results are expressed as mean ± SD for three independent experiments. Statistical analyzes were performed by ANOVA. Asterisks indicate significant differences from the control according to Bonferroni's multiple comparison <i>post hoc</i> test, relative to FBS 10% (#) or FBS 0% (*). ## p<0.01, * p<0.05, ** p<0.01</p

Leptin enhances BCL-2/BAX relationship in human placental explants.

<p>A) Placental explants were processed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099187#s2" target="_blank">Materials and Methods</a> and incubated during 24 h in DMEM-F12 media supplemented with increasing leptin doses. DMEM-F12 10% FBS was used as control. Placental extracts were prepared and proteins were separated on SDS-PAGE gels. BCL-2 and BAX expression were determined by Western blot analysis as indicated in the Figure. Molecular weights were estimated using standard protein markers. Molecular mass (kDa) is indicated at the right of the blot. Loading controls were performed by immunoblotting the same membranes with anti-α-tubulin. Bands densitometry is shown in lower panels, results are expressed as mean ± SD for three independent experiments. B) BCL-2/BAX relationship is shown. Statistical analyzes were performed by ANOVA. Asterisks indicate significant differences from the control according to Bonferroni's multiple comparison <i>post hoc</i> test, relative to FBS 0% (*). * p<0.05, ** p<0.01</p

Leptin reduces p53 levels in placenta.

<p>A) Swan-71 cells (1×10⁶ cells) were plated in DMEM-F12 media in the absence of serum and incubated during 72 h with different leptin concentrations. Cell extracts were prepared as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099187#s2" target="_blank">Materials and Methods</a> and proteins were separated on SDS-PAGE gels. B) Placental explants were processed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099187#s2" target="_blank">Material and Methods</a> and incubated in DMEM-F12 media supplemented with increasing leptin doses during 24 h. Placental extracts were prepared and proteins were separated on SDS-PAGE gels. In both cases (A and B) p53 was determined by Western blot analysis. DMEM-F12 10% FBS was used as a control. Molecular weights were estimated using standard protein markers. Molecular mass (kDa) is indicated at the right of the blot. Loading controls were performed by immunoblotting the same membranes with anti-α-GAPDH. Bands densitometry is shown in lower panels. Results are expressed as mean ± SD for three independent experiments. C) p53 expression in human placental explants was determined by <i>q</i>RT-PCR. RNA was extracted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099187#s2" target="_blank">Materials and Methods</a>. Statistical analyzes were performed by ANOVA. Asterisks indicate significant differences from the control according to Bonferroni's multiple comparison <i>post hoc</i> test, relative to FBS 10% (#) or FBS 0% (*). # p<0.05, ## p<0.01, * p<0.05, ** p<0.01.</p