Increased NFAT5 expression stimulates transcription of Hsp70 in preeclamptic placentas.

OBJECTIVE: We investigated the expression of heat shock protein 70 (Hsp70), nuclear factor of activated T cells 5 (NFAT5), and hypoxia-induced factor-1α (HIF-1α) in the placentas of normal and preeclamptic pregnancies and in human placental hypoxia models in vitro to examine the regulatory mechanisms of placental Hsp70 expression. METHODS: The expression levels of HIF-1α, NFAT5, and Hsp70 were examined in placental samples from 10 females with preeclampsia and 10 normotensive control patients and in human choriocarcinoma trophoblast cells treated with 1 mM CoCl2 by western blotting. Using models of placental hypoxia, pharmacological inhibition of HIF-1α with chetomin and shRNA knockdown and overexpression of NFAT5 were performed to investigate the roles of HIF-1α and NFAT5 in induction of Hsp70 by placental hypoxia. RESULTS: The levels of HIF-1α, NFAT5, and Hsp70 expression were significantly higher in the preeclamptic compared to normal placentas. In the placental hypoxia models, the expression of HIF-1α, NFAT5, and Hsp70 were significantly higher after 3, 6, and 12 h of 1 mM CoCl2 treatment, respectively. Pharmacological inhibition of HIF-1α suppressed the induction of NFAT5 and Hsp70 at the protein level. shRNA knockdown of NFAT5 suppressed the induction of Hsp70 protein and overexpression of NFAT5 stimulated the induction of Hsp70 mRNA and protein in models of human placental hypoxia in vitro. CONCLUSION: HIF-1α positively regulates the induction of NFAT5 and Hsp70 by placental hypoxia and NFAT5 stimulates transcription of Hsp70 in response to placental hypoxia in models of human placental hypoxia in vitro.

Inhibition of the PI3K-Akt pathway suppresses sFlt1 expression in human placental hypoxia models in vitro.

OBJECTIVE: Although elevated expression of soluble fms-like tyrosine kinase 1 (sFlt1) plays a major role in the pathogenesis of pre-eclampsia, it is unclear how hypoxia regulates placental sFlt1 expression. Thus, we investigated sFlt1 expression in placentas from normal and preeclamptic pregnancies and in human placental hypoxia models in vitro to examine the role of the PI3K-Akt pathway in regulating the expression of this molecule. METHODS: We examined the expression of VEGF, PlGF, sFlt1, PI3K, Akt, and HIF-1 in placental samples from ten women with pre-eclampsia and ten normotensive control patients and in human choriocarcinoma trophoblast cells treated with 600muM CoCl(2) by Western blotting. Using models of placental hypoxia, we also determined whether inhibition of the PI3K-Akt pathway plays a direct role in regulating the expression of sFlt1. RESULTS: The VEGF, PlGF, sFlt1, PI3K, Akt, and HIF-1 levels were significantly higher in the preeclamptic placentas than the normal placentas. In the placental hypoxia models, the expression of VEGF and PlGF increased in a time-dependent manner, whereas the expression of sFlt1 plateaued after 3h of CoCl(2) treatment. The expression levels of p-Akt and PI3K were maximal after 6 and 12h of CoCl(2) treatment, respectively. The expression of HIF-1alpha increased in a time-dependent manner with CoCl(2) treatment. Inhibition of the PI3K-Akt pathway with the PI3K-specific inhibitor LY294002 leads to decreased sFlt1 levels and unchanged or increased VEGF and PlGF levels. CONCLUSION: Inhibition of the PI3K-Akt pathway may be a useful therapeutic approach, if it were to decrease sFlt1 secretion without inhibiting VEGF or PlGF secretion. This pathway provides a potential target for a new treatment strategy in patients with pre-eclampsia.

PKC delta in preeclamptic placentas promotes Bax dissociation from 14-3-3 zeta through 14-3-3 zeta phosphorylation.

OBJECTIVE: We investigated placental apoptosis and the expression of and interactions between 14-3-3 and Bcl-2 family proteins during preeclampsia. In addition, we explored the mechanism of Bax dissociation from 14-3-3, hypothesizing that PKC-mediated phosphorylation of 14-3-3 results in dissociation of Bax from 14-3-3 proteins, and leads to apoptosis. METHODS: Placental samples from 10 women with preeclampsia and 10 normotensive control patients were analyzed using M30-specific immunohistochemistry to assess placental apoptosis. Biochemical markers of cellular apoptosis, such as cleaved caspase-3, Bax, Bcl-2, 14-3-3, and PKC were followed by Western blotting. Interaction of 14-3-3 proteins with Bax and with PKC was assessed by immunoprecipitation. RESULTS: M30-positive cells were widespread in the preeclamptic placentas. The levels of cleaved caspase-3, Bax, 14-3-3 zeta, phospho-(Ser)-14-3-3, and PKC delta were significantly higher in the preeclamptic placentas than in normal placentas. Preeclampsia was also associated with weaker interactions between 14-3-3 zeta and Bax and stronger interactions between 14-3-3 zeta and PKC delta. CONCLUSION: Our results suggest that PKC delta in preeclamptic placentas promotes Bax dissociation from 14-3-3 zeta through the phosphorylation of 14-3-3 zeta. This finding may at least in part explain the apoptosis-inducing activity of PKC delta, revealing the important role of PKC delta in the development of apoptosis-related diseases such as preeclampsia.

The localization of dopamine D2 receptor mRNA in the human placenta and the anti-angiogenic effect of apomorphine in the chorioallantoic membrane.

While there are some reports indicating that dopamine (DA) and D2-like receptors (Rc) are detected in human placenta, there is little evidence available regarding the function of DA or the precise localization of its receptors in this organ. In the present study, we confirmed the placental expression of DA D2 Rc transcripts by Northern blot analysis. Using in situ hybridization, we also first revealed that DA D2 Rc mRNA was expressed in cytotrophoblasts, syncytial trophoblasts, vascular endothelial cells, Hafbauer cells, and fibroblasts in the chorionic villi of the human placenta. The expression sites of DA D2 Rc mRNA led us to suspect other functions of DA in the placenta besides the regulation of human placental lactogen. Since the cells expressing DA D2 Rc mRNA are related to proliferation and remodeling of placental tissue, we tried to evaluate a possible involvement of DA in the regulation of placental angiogenesis. To this end, we used the chicken chorioallantoic membrane (CAM) assay. In CAM assay, apomorphine, a potent nonselective agonist of DA, has an anti-angiogenic effect. These results suggest that DA may regulate the vascularization of human placenta through its receptors present in the chorionic villi.

Wortmannin-sensitive and -insensitive steps in calcium-controlled exocytosis in pituitary gonadotrophs: evidence that myosin light chain kinase mediates calcium-dependent and wortmannin-sensitive gonadotropin secretion.

In cultured rat pituitary cells, increases in the cytosolic calcium concentration ([Ca2+]i) and LH release are induced by activation of GnRH receptors as well as by nonreceptor-mediated stimuli. Treatment of pituitary cells with the myosin light chain kinase (MLCK) inhibitor, wortmannin, attenuated GnRH-induced LH release. Wortmannin also reduced the LH responses to nonreceptor-mediated elevation of [Ca2+]i by ionomycin and activation of voltage-sensitive Ca2+ channels by Bay K 8644 or high K+, as well as Ca2+-induced LH release in permeabilized pituitary cells. The [Ca2+]i responses to these stimuli were unaltered in wortmannin-treated pituitary cells, indicating that this compound inhibits a Ca2+-dependent step in exocytosis without affecting Ca2+ signaling. In perifused pituitary cells, the GnRH-induced early spike phase of LH release was not affected by wortmannin, whereas the subsequent plateau phase was almost completely inhibited. No significant changes in GnRH-induced phospholipase D activity and diacylglycerol production were observed in wortmannin-treated pituitary cells during the sustained phase of agonist stimulation. Wortmannin also had no effect on LH responses to the protein kinase C activator, phorbol 12-myristate 13-acetate, further indicating that the attenuation of agonist-induced LH release is not related to inhibition of the diacylglycerol/protein kinase C pathway. In addition, agonist-induced LH release was attenuated by two other MLCK inhibitors, MS-347a and KT5926. These data suggest that MLCK mediates the downstream effects of Ca2+ on exocytosis, an action supported by the finding of wortmannin-sensitive phosphorylation of a 20-kDa protein in pituitary cells and alphaT3-1 gonadotrophs treated with GnRH, K+, and Bay K 8644. This protein was coprecipitated from pituitary extracts with a specific antibody to nonmuscle myosin IIB and comigrated with 20-kDa smooth muscle myosin light chain on SDS-PAGE. These results demonstrate that Ca2+ controls exocytosis through an initial wortmannin-insensitive step and a sustained wortmannin-sensitive step and suggest that the latter event in the cascade of cellular responses is dependent on phosphorylation of nonmuscle myosin IIB light chain by MLCK.

Effects of the phospholipase-C inhibitor, U73122, on signaling and secretion in pituitary gonadotrophs.

The effects of inhibition of phosphoinositide hydrolysis by U73122 [1-(6-[17 beta-3-methoxyestra-1,3,5- (10) triene-17-yl] amino/hexyl) 1H-pyrroledione] and neomycin on agonist-stimulated intracellular signaling and secretory responses were analyzed in cultured pituitary cells and alpha T3-1 gonadotrophs. GnRH (100 nM)- and endothelin-1 (ET-1; 100 nM)-induced inositol (1,4,5)-trisphosphate and diacylglycerol formation in normal cells and immortalized gonadotrophs were reduced by U73122 in a concentration-dependent manner, with IC50 values of about 2 microM and complete inhibition at 10 microM U73122. Neomycin also reduced GnRH- and ET-induced inositol phosphate production in both cell types. Agonist-induced intracellular Ca2+ responses were also inhibited in both cell types by U73122 and neomycin at the same concentrations that inhibited their inositol phosphate responses. In cultured pituitary cells, agonist-induced LH release was inhibited by U73122 and neomycin in a dose-dependent manner. In perifused pituitary cells, U73122 completely inhibited GnRH- and ET-1-induced LH release, but after 10 min caused a progressive and substantial increase in basal LH release. In static cultures, U73122 inhibited agonist-induced LH response at low concentrations (up to 3 microM), but stimulated LH release at higher concentrations due to direct activation of exocytosis by the compound. When added alone, U73122 caused a concentration-dependent increase in LH release with an EC50 of about 7 microM and a maximum response similar that that elicited by GnRH. The stimulatory action of U73122 on LH release was not reduced in the absence of extracellular Ca2+. In contrast to cultured pituitary cells, alpha T3-1 gonadotrophs showed only constitutive exocytosis that was not affected by either neomycin or U73122. These results demonstrate that GnRH and ET(A) receptors are coupled to the phosphoinositide/Ca2+ transduction system in pituitary gonadotrophs, and provide evidence for the dependence of agonist-regulated exocytosis on this signaling pathway. The ability of U73122 to stimulate LH release could reflect an additional action of the compound at late steps in the exocytic pathway.