NLRP7 is increased in human idiopathic fetal growth restriction and plays a critical role in trophoblast differentiation.

Fetal growth restriction (FGR) the leading cause of perinatal mortality and morbidity is highly related to abnormal placental development, and placentas from FGR pregnancies are often characterized by increased inflammation. However, the mechanisms of FGR-associated inflammation are far from being understood. NLRP7, a member of a family of receptors involved in the innate immune responses, has been shown to be associated with gestational trophoblastic diseases. Here, we characterized the expression and the functional role of NLRP7 in the placenta and investigated its involvement in the pathogenesis of FGR. We used primary trophoblasts and placental explants that were collected during early pregnancy, and established trophoblast-derived cell lines, human placental villi, and serum samples from early pregnancy (n = 38) and from FGR (n = 40) and age-matched controls (n = 32). Our results show that NLRP7 (i) is predominantly expressed in the trophoblasts during the hypoxic period of placental development and its expression is upregulated by hypoxia and (ii) increases trophoblast proliferation ([3H]-thymidine) and controls the precocious differentiation of trophoblasts towards syncytium (syncytin 1 and 2 and ß-hCG production and xCELLigence analysis) and towards invasive extravillous trophoblast (2D and 3D cultures). We have also demonstrated that NLRP7 inflammasome activation in trophoblast cells increases IL-1ß, but not IL-18 secretion. In relation to the FGR, we demonstrated that major components of NLRP7 inflammasome machinery are increased and that IL-1ß but not IL-18 circulating levels are increased in FGR. Altogether, our results identified NLRP7 as a critical placental factor and provided evidence for its deregulation in FGR. NLRP7 inflammasome is abundantly expressed by trophoblast cells. It is regulated by a key parameter of placental development, hypoxia. It controls trophoblast proliferation, migration, and invasion and exhibits anti-apoptotic role. NLRP7 machinery is deregulated in FGR pregnancies. KEY MESSAGES: NLRP7 inflammasome is abundantly expressed by trophoblast cells. It is regulated by a key parameter of placental development, hypoxia. It controls trophoblast proliferation, migration, and invasion and exhibits anti-apoptotic role. NLRP7 machinery is deregulated in FGR pregnancies.

Endocrine gland-derived endothelial growth factor (EG-VEGF) is a potential novel regulator of human parturition.

EG-VEGF is an angiogenic factor that we identified as a new placental growth factor during human pregnancy. EG-VEGF is also expressed in the mouse fetal membrane (FM) by the end of gestation, suggesting a local role for this protein in the mechanism of parturition. However, injection of EG-VEGF to gravid mice did not induce labor, suggesting a different role for EG-VEGF in parturition. Here, we searched for its role in the FM in relation to human parturition. Human pregnant sera and total FM, chorion, and amnion were collected during the second and third trimesters from preterm no labor, term no labor, and term labor patients. Primary human chorion trophoblast and FM explants cultures were also used. We demonstrate that circulating EG-VEGF increased toward term and significantly decreased at the time of labor. EG-VEGF production was higher in the FM compared to placentas matched for gestational age. Within the FM, the chorion was the main source of EG-VEGF. EG-VEGF receptors, PROKR1 and PROKR2, were differentially expressed within the FM with increased expression toward term and an abrupt decrease with the onset of labor. In chorion trophoblast and FM explants collected from nonlaboring patients, EG-VEGF decreased metalloproteinase-2 and -9 activities and increased PGDH (prostaglandin-metabolizing enzyme) expression. Altogether these data demonstrate that EG-VEGF is a new cytokine that acts locally to ensure FM protection in late pregnancy. Its fine contribution to the initiation of human labor is exhibited by the abrupt decrease in its levels as well as a reduction in its receptors.

Characterization of the adverse effects of nicotine on placental development: in vivo and in vitro studies.

In utero exposure to nicotine is associated with increased risk of numerous adverse fetal and neonatal outcomes, which suggests that it acts directly to affect placental development and the establishment of the fetomaternal circulation (FC). This study used both in vivo [Wistar rats treated with 1 mg/kg nicotine from 2 wk prior to mating until gestational day (GD) 15] and in vitro (RCHO-1 cell line; treated with 10(-9) to 10(-3)M nicotine) models to examine the effects of nicotine on these pathways. At GD 15, control and treated placentas were examined for the impact of nicotine on 1) trophoblast invasion, proliferation, and degree of hypoxia, 2) labyrinth vascularization, 3) expression of key genes of placental development, and 4) expression of placental angiogenic factors. The RCHO-1 cell line was used to determine the direct effects of nicotine on trophoblast differentiation. Our in vivo experiments show that nicotine inhibits trophoblast interstitial invasion, increases placental hypoxia, downregulates labyrinth vascularization as well as key transcription factors Hand1 and GCM1, and decreases local and circulating EG-VEGF, a key placental angiogenic factor. The in vitro experiments confirmed the inhibitory effects of nicotine on the trophoblast migration, invasion, and differentiation processes and demonstrated that those effects are most likely due to a dysregulation in the expression of nicotine receptors and a decrease in MMP9 activity. Taken together, these data suggest that adverse effects of maternal smoking on pregnancy outcome are due in part to direct and endocrine effects of nicotine on the main processes of placental development and establishment of FC.

[Potential role of the angiogenic factor "EG-VEGF" in gestational trophoblastic diseases]. / Rôle potentiel du facteur angiogénique «EG-VEGF¼ dans les maladies gestationnelles trophoblastiques.

Gestational trophoblastic disease (MGT) includes a wide spectrum of pathologies of the placenta, ranging from benign precancerous lesions, with gestational trophoblastic tumors. Metastases are the leading causes of death as a result of this tumor. They represent a major problem for obstetrics and for the public health system. To date, there is no predictor of the progression of molar pregnancies to gestational trophoblastic tumor (GTT). Only an unfavorable plasma hCG monitoring after evacuation of hydatidiform mole is used to diagnose a TTG. The causes of the development of this cancer are still poorly understood. Increasing data in the literature suggests a close association between the development of this tumor and poor placental vascularization during the first trimester of pregnancy. The development of the human placenta depends on a coordination between the trophoblast and endothelial cells. A disruption in the expression of angiogenic factors could contribute to uterine or extra-uterine tissue invasion by extravillous trophoblast, contributing to the development of TTG. This review sheds lights on the phenomenon of angiogenesis during normal and abnormal placentation, especially during the MGT and reports preliminary finding concerning, the variability of expression of "Endocrine Gland-Derived Vascular Endothelial Growth Factor" (EG-VEGF), a specific placental angiogenic factor, in normal and molar placentas, and the potential role of differentiated expressions of the main placental angiogenic factors in the scalability of hydatidiform moles towards a recovery or towards the development of gestational trophoblastic tumor. Deciphering the mechanisms by which the angiogenic factor influences these processes will help understand the pathophysiology of MGT and to create opportunities for early diagnosis and treatment of the latter.

Human cellular prion protein hPrPC is sorted to the apical membrane of epithelial cells.

Propagation of the scrapie isoform of the prion protein (PrP(Sc)) depends on the expression of endogenous cellular prion (PrP(C)). During oral infection, PrP(Sc) propagates, by conversion of the PrP(C) to PrP(Sc), from the gastrointestinal tract to the nervous system. Intestinal epithelium could serve as the primary site for PrP(C) conversion. To investigate PrP(C) sorting in epithelia cells, we have generated both a green fluorescent protein (EGFP) or hemagglutinin (HA) tagged human PrP(C) (hPrP(C)). Combined molecular, biochemical, and single living polarized cell imaging characterizations suggest that hPrP(C) is selectively targeted to the apical side of Madin-Darby canine kidney (MDCKII) and of intestinal epithelia (Caco2) cells.

COOH-terminal truncations promote proteasome-dependent degradation of mature cystic fibrosis transmembrane conductance regulator from post-Golgi compartments.

Impaired biosynthetic processing of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel, constitutes the most common cause of CF. Recently, we have identified a distinct category of mutation, caused by premature stop codons and frameshift mutations, which manifests in diminished expression of COOH-terminally truncated CFTR at the cell surface. Although the biosynthetic processing and plasma membrane targeting of truncated CFTRs are preserved, the turnover of the complex-glycosylated mutant is sixfold faster than its wild-type (wt) counterpart. Destabilization of the truncated CFTR coincides with its enhanced susceptibility to proteasome-dependent degradation from post-Golgi compartments globally, and the plasma membrane specifically, determined by pulse-chase analysis in conjunction with cell surface biotinylation. Proteolytic cleavage of the full-length complex-glycosylated wt and degradation intermediates derived from both T70 and wt CFTR requires endolysosomal proteases. The enhanced protease sensitivity in vitro and the decreased thermostability of the complex-glycosylated T70 CFTR in vivo suggest that structural destabilization may account for the increased proteasome susceptibility and the short residence time at the cell surface. These in turn are responsible, at least in part, for the phenotypic manifestation of CF. We propose that the proteasome-ubiquitin pathway may be involved in the peripheral quality control of other, partially unfolded membrane proteins as well.

Conformational and temperature-sensitive stability defects of the delta F508 cystic fibrosis transmembrane conductance regulator in post-endoplasmic reticulum compartments.

Deletion of phenylalanine at position 508 (DeltaF508) is the most common cystic fibrosis (CF)-associated mutation in the CF transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel. The consensus notion is that DeltaF508 imposes a temperature-sensitive folding defect and targets newly synthesized CFTR for degradation at endoplasmic reticulum (ER). A limited amount of CFTR activity, however, appears at the cell surface in the epithelia of homozygous DeltaF508 CFTR mice and patients, suggesting that the ER retention is not absolute in native tissues. To further elucidate the reasons behind the inability of DeltaF508 CFTR to accumulate at the plasma membrane, its stability was determined subsequent to escape from the ER, induced by reduced temperature and glycerol. Biochemical and functional measurements show that rescued DeltaF508 CFTR has a temperature-sensitive stability defect in post-ER compartments, including the cell surface. The more than 4-20-fold accelerated degradation rate between 37 and 40 degrees C is, most likely, due to decreased conformational stability of the rescued DeltaF508 CFTR, demonstrated by in situ protease susceptibility and SDS-resistant thermoaggregation assays. We propose that the decreased stability of the spontaneously or pharmacologically rescued mutant may contribute to its inability to accumulate at the cell surface. Thus, therapeutic efforts to correct the folding defect should be combined with stabilization of the native DeltaF508 CFTR.

C-terminal truncations destabilize the cystic fibrosis transmembrane conductance regulator without impairing its biogenesis. A novel class of mutation.

Defective cAMP-stimulated chloride conductance of the plasma membrane of epithelial cell is the hallmark of cystic fibrosis (CF) and results from mutations in the cystic fibrosis transmembrane conductance regulator, CFTR. In the majority of CF patients, mutations in the CFTR lead to its misfolding and premature degradation at the endoplasmic reticulum (ER). Other mutations impair the cAMP-dependent activation or the ion conductance of CFTR chloride channel. In the present work we identify a novel mechanism leading to reduced expression of CFTR at the cell surface, caused by C-terminal truncations. The phenotype of C-terminally truncated CFTR, representing naturally occurring premature termination and frameshift mutations, were examined in transient and stable heterologous expression systems. Whereas the biosynthesis, processing, and macroscopic chloride channel function of truncated CFTRs are essentially normal, the degradation rate of the mature, complex-glycosylated form is 5- to 6-fold faster than the wild type CFTR. These experiments suggest that the C terminus has a central role in maintaining the metabolic stability of the complex-glycosylated CFTR following its exit from the ER and provide a plausible explanation for the severe phenotype of CF patients harboring C-terminal truncations.

Cystic fibrosis phenotype associated with pancreatic insufficiency does not always reflect the cAMP-dependent chloride conductive pathway defect. Analysis of C225R-CFTR and R1066C-CFTR.

We have previously screened the cystic fibrosis transmembrane conductance regulator (CFTR) gene and identified new disease-causing mutations. C225R and R1066C are both associated with pancreatic insufficiency, but the former mutation is associated with mild and unusual lung disease, whereas the latter is associated with severe lung disease. In the present study, we expressed these mutants heterologously in HeLa cells, and we analyzed protein synthesis by immunoprecipitation and chloride channel function by using a halide-sensitive fluorescent dye, 6-methoxy-N-ethylquinolinium. Immunoprecipitation and functional studies showed that cells transfected with C225R-CFTR exhibit cAMP-dependent chloride fluxes; C225R-CFTR protein is poorly expressed but fully glycosylated and can be compared with R117H-CFTR. R1066C-CFTR protein is not correctly processed and, unlike DeltaF508-CFTR, this defect cannot be corrected by reduced temperature or overexpression in butyrate-treated cells; defective processing may occur at a different step in the biosynthetic pathway. These results point to two different mechanisms underlying the same pancreatic status and suggest that it is unwise to use pancreatic sufficiency and insufficiency to define mild and severe cystic fibrosis (CF) disease, respectively. Finally, the experimental model described here may be helpful to predict the pulmonary status of CF patients bearing mutations located in putative membrane-spanning domains of the CFTR protein.

Properties of chloride-conductive pathways in rat kidney cortical and outer-medulla brush-border membranes--inhibition by anti-(cystic fibrosis transmembrane regulator) mAbs.

The activity of the Cl(-)-conductive pathways, their regulation by protein kinase A (PKA) and their relationship to the cystic fibrosis transmembrane regulator (CFTR) protein were assessed in rat kidney cortical brush-border-membrane vesicles (cBBMV) and outer medullary vesicles (OMV) by measuring the rate of valinomycin-induced microsomal swelling by light scattering in the presence of an inward Cl- gradient. Valinomycin increased the rate of swelling of cBBMV and OMV, which is consistent with the presence of a Cl(-)-conductive pathway. PKA further increased these rates. This effect was blocked by the inhibitor of protein kinase A, suggesting that phosphorylation by PKA activates these pathways. Four anion-transport inhibitors were tested ¿N-phenylanthranilic acid (PhNHPhCOOH), 5-nitro-2-(3-phenylpropylamino)benzoic acid [N(PhPrNH2)BzOH], glybenclamide and 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid¿. Ph2COOH and 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid inhibited the basal Cl(-)-conductive pathways, while PKA-treated microsomes were sensitive also to N(PhPrNH2)BzOH and glybenclamide, suggesting that additional Cl- pathways were activated by phosphorylation. The pharmacological properties of these pathways were similar to those of the CFTR Cl- channel. Two anti-CFTR mAbs inhibited PKA-activated valinomycin-induced swelling in cBBMV and OMV, while immunoblot analysis of the corresponding proteins with the same antibodies indicated the presence of a 170-kDa protein. The results thus indicate the presence of a PKA-activated Cl(-)-conductive pathway in cBBMV and OMV, and suggest that CFTR protein is involved in PKA-activated Cl- fluxes in these vesicles.

Partial purification of the pig kidney cystic fibrosis transmembrane regulator protein.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a transmembrane protein that is expressed in several epithelia, including kidney tubules. Mutations in CFTR (a PKA-chloride channel and/or regulator of other epithelial channels) give rise to the clinical manifestation of cystic fibrosis, and result in the synthesis of mutated proteins responsible for altering ion transport across secretory epithelia. The low abundance of endogenous CFTR makes a difficult to purify enough of the native protein to prepare anti-CFTR antibodies. We have used differential centrifugation to prepare cortical brush border membrane vesicles from pig kidney, cBBMV, and developed a method for the partial purification of CFTR. This is the first step in the isolation of native CFTR. The results show that CFTR is present in cBBMV. The purified protein will provide a clearer picture of the biophysical and biochemical properties of native CFTR.

Properties of a Cl(-)-conductive pathway(s) in microsomes from rat kidney inner medulla. Involvement of cystic fibrosis transmembrane regulator protein.

The properties of a protein-kinase-A(PKA)-activated Cl(-)-conductive pathway(s) in alkaline phosphatase-enriched microsomes from the rat inner medulla (IMV) were investigated. Transcripts of cystic fibrosis transmembrane regulator (CFTR) were detected by reverse transcription/polymerase analysis of total RNA from the inner medulla, while immunoblot analysis using anti-CFTR antibodies detected a 170-kDa protein in the IMV. The PKA Cl(-)-conductive pathway(s) was studied by measuring the rate of valinomycin-induced microsomal swelling by light scattering. PKA increased the rate of valinomycin-induced swelling of vesicles consistent with the presence of Cl(-)-conductive pathway(s). The pharmacological properties and anion selectivity of the PKA-activated Cl(-)-conductive pathway(s) were similar to those of the CFTR Cl(-) channel. Our results show that a CFTR Cl(-) channel and possibly another cAMP-activated pathway(s) may participate in Cl(-) secretion in the rat inner medulla.