Abstract
Marinobufagenin (MBG) is an endogenous mammalian cardiotonic steroid involved in the inhibition of Na(+)/K(+)-ATPase. Increased plasma levels have been reported in patients with volume expansion-related hypertension. We have recently demonstrated that MBG impairs first trimester cytotrophoblast (CTB) cell proliferation, migration, and invasion, which may play a role in the development of preeclampsia. However, whether apoptosis contributes to altered CTB cell function by MBG remains unknown. Using the human extravillous CTB cell line SGHPL-4, we examined the effect of MBG and a similar Na(+)/K(+)-ATPase inhibitor, ouabain, on the phosphorylation status of Jnk, p38, and Src. Additionally, we measured apoptosis by caspase 9 and 3/7 activity and by annexin-V staining. We also investigated interleukin-6 (IL-6) secretion with or without p38 and Jnk inhibition. MBG significantly increased the phosphorylation of Jnk, p38, and Src and increased the expression of caspase 9 and 3/7 indicating the activation of apoptosis. MBG treatment also stimulated the expression of the early apoptosis marker, annexin-V, which was prevented by Jnk and p38 inhibition. MBG also stimulated the secretion of IL-6, which was attenuated by p38 inhibition. Ouabain had similar effects to those of MBG, suggesting that the apoptotic effects on CTB cells may be mediated by inhibition of Na(+)/K(+)-ATPase. In conclusion, the MBG-induced impairment of CTB function occurs via activation of Jnk, p38, and Src leading to increased apoptosis and IL-6 secretion. These observations may have clinical applicability with respect to the therapy of preeclampsia.
Highlights
Drome that occurs in 3–10% of pregnancies [9, 10] and is a leading cause of maternal and fetal morbidity and mortality [9]
We examined the effects of MBG on Jnk, p38, and Src phosphorylation, the early apoptosis markers caspases 9 and 3/7, and annexin-V staining to determine whether MBG causes apoptotic signaling in CTB cells
MBG and Ouabain Increased Phosphorylation of Jnk, p38, and Src—Both 10 and 100 nM MBG and ouabain caused a significant increase in the ratio of phosphorylated Jnk to total Jnk in CTB cells compared with controls (50, 48, and 45% increase at 10, 30, and 60 min, respectively, after MBG treatment and 38, 40, and 40% increase at 10, 30, and 60 min, respectively, after ouabain treatment; p Ͻ 0.001 for each) (Fig. 1, A and B)
Summary
MBG Effects on Cell Signaling in CTB Cells associated with a down-regulation of the cell cycle and antiapoptotic proteins Bcl-2 and Bcl-XL and the concomitant activation of Bax and caspase 9 [33]. It has been postulated that IL-6-induced signal transduction pathways lead to apoptosis of 1A-9-M cells [39] and that the Src family of kinases likely regulate p38-mediated IL-6 production following hypoxia in Kupffer cells [40]. Cardiotonic steroids, including MBG and ouabain, another Naϩ/Kϩ-ATPase inhibitor, can increase intracellular Ca2ϩ, increase reactive oxygen species production, and activate Src, phosphatidylinositol 3-kinase/Akt, and NFB pathways [31]. We examined IL-6 secretion in response to MBG and determined whether apoptosis and IL-6 secretion could be prevented by the inhibition of p38 and Jnk. We determined that the pro-apoptotic effect of MBG and ouabain on CTB cell function was mediated by Jnk, p38, and Src pathways and by the stimulation of IL-6 production. The similarity of the effects of MBG and ouabain on CTB apoptosis suggests a common pathway and that Naϩ/Kϩ-ATPase inhibition may play a role
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