Caffeine Reduces 11β-Hydroxysteroid Dehydrogenase Type 2 Expression in Human Trophoblast Cells through the Adenosine A2B Receptor

Maternal caffeine consumption is associated with reduced fetal growth, but the underlying molecular mechanisms are unknown. Since there is evidence that decreased placental 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) is linked to fetal growth restriction, we hypothesized that caffeine may inhibit fetal growth partly through down regulating placental 11β-HSD2. As a first step in examining this hypothesis, we studied the effects of caffeine on placental 11β-HSD2 activity and expression using our established primary human trophoblast cells as an in vitro model system. Given that maternal serum concentrations of paraxanthine (the primary metabolite of caffeine) were greater in women who gave birth to small-for-gestational age infants than to appropriately grown infants, we also studied the effects of paraxanthine. Our main findings were: (1) both caffeine and paraxanthine decreased placental 11β-HSD2 activity, protein and mRNA in a concentration-dependent manner; (2) this inhibitory effect was mediated by the adenosine A2B receptor, since siRNA-mediated knockdown of this receptor prevented caffeine- and paraxanthine-induced inhibition of placental 11β-HSD2; and (3) forskolin (an activator of adenyl cyclase and a known stimulator of 11β-HSD2) abrogated the inhibitory effects of both caffeine and paraxanthine, which provides evidence for a functional link between exposure to caffeine and paraxanthine, decreased intracellular levels of cAMP and reduced placental 11β-HSD2. Taken together, these findings reveal that placental 11β-HSD2 is a novel molecular target through which caffeine may adversely affect fetal growth. They also uncover a previously unappreciated role for the adenosine A2B receptor signaling in regulating placental 11β-HSD2, and consequently fetal development

Concentration-dependent effects of caffeine on 11β-HSD2 activity and expression.

<p>Human trophoblast cells were treated with increasing concentration of caffeine (100–500 µM) for 48 h. At the end of treatment, levels of 11β-HSD2 activity in intact cells (<b>A</b>) as well as levels of 11β-HSD2 protein (<b>B</b>) and 11β-HSD2 mRNA (<b>C</b>) were determined by a standard radiometric conversion assay, western blotting, and qRT-PCR, respectively. Data are presented as mean ± SEM of four to five independent experiments (*P<0.05, **P<0.01, ***P<0.001 vs. control).</p

Effects of forskolin on caffeine and paraxanthine inhibition of 11β-HSD2 expression.

<p>Human trophoblast cells were pretreated for 2 h with 20 µM of forskolin, and were then treated with 500 µM of caffeine or paraxanthine for 48 h. At the end of treatment, levels of 11β-HSD2 protein were determined by western blot analysis. Data are presented as mean ± SEM of three independent experiments (**P<0.01, ***P<0.001 vs. control).</p

Effects of siRNA-mediated knockdown of ADORA_2B on caffeine and paraxanthine inhibition of 11β-HSD2 expression.

<p>Human trophoblast cells were transfected with 100 nM of ADORA_2B siRNA or the transfection agent alone to serve as control. Forty-eight hours after transfection, cells were lysed, and total RNA extracted and subjected to a semi-quantitative RT-PCR to determine levels of ADORA_2B mRNA (<b>A</b>). GAPDH was used as a control to show the specificity of siRNA mediated knockdown of ADORA_2B. Alternatively, 12 h after transfection, cells were treated for 48 h with or without 500 µM of caffeine (<b>B</b>) or paraxanthine (<b>C</b>). At the end of treatment, levels of 11β-HSD2 11β-HSD2 protein were determined by western blot analysis. Data are presented as mean ± SEM of four to five independent experiments (***P<0.001 vs. control).</p

Expression of adenosine receptor mRNA in cultured human trophoblast cells.

<p>Total RNA was extracted from cultured trophoblast cells and from placental villi at term (to serve as a positive control for ADORA₁). One microgram of RNA was used in a standard RT-PCR to amplify mRNA for the four human adenosine receptors, ADORA₁, ADORA_2A, ADORA_2B and ADORA₃. A fraction of the RT-PCR products was subjected to electrophoresis on a 1.2% agarose gel. This figure shows the results of one representative experiment.</p

Concentration-dependent effects of paraxanthine on 11β-HSD2 activity and expression.

<p>Human trophoblast cells were treated with increasing concentration of paraxanthine (PX; 100–500 µM) for 48 h. At the end of treatment, levels of 11β-HSD2 activity in intact cells (<b>A</b>) as well as levels of 11β-HSD2 protein (<b>B</b>) and 11β-HSD2 mRNA (<b>C</b>) were determined by a standard radiometric conversion assay, western blotting, and qRT-PCR, respectively. Data are presented as mean ± SEM of four to five independent experiments (*P<0.05, **P<0.01, ***P<0.001 vs. control).</p

PCR primers for the four human adenosine receptors and human GAPDH.

<p>PCR primers for the four human adenosine receptors and human GAPDH.</p