Placental production of progestins is fully effective in villous cytotrophoblasts and increases with the syncytiotrophoblast formation.

Placental syncytiotrophoblast (ST) is considered as the main placental endocrine tissue secreting progesterone, a steroid essential for maintenance of pregnancy. However, each step of progestins production has been poorly investigated in villous cytotrophoblast (VCT) regarding ST formation. We aimed to characterize progestins production during human differentiation of VCT into ST. VCTs were isolated from term placenta and cultivated, with or without forskolin (FSK), to stimulate trophoblast differentiation. Secreted progestins concentrations were determined by immuno-assay and Gas Chromatography-tandem mass spectrometry. Intracellular expression of cholesterol transporter and enzymes involved in steroidogenesis were studied by immunofluorescence, western-blot, and RT-qPCR. Progesterone and pregnenolone are produced by VCT and their secretion increases with VCT differentiation while 17-hydroxyprogesterone concentration remains undetectable. HSD3B1 enzyme expression increases whereas MLN64, the cholesterol placental mitochondrial transporter and P450SCC expressions do not. FSK induces progestins production. Progestins placental synthesis is effective since VCT and increases with ST formation thanks to mitochondria.

Retinoic acid-induced rapid loss of nuclear cyclic AMP-dependent protein kinase in teratocarcinoma cells.

To determine what effect retinoic acid might have in modulating cyclic AMP-mediated events at the nucleus of teratocarcinoma cells, we have investigated the effect of retinoic acid treatment of F9 and PC13 cells on cyclic AMP-dependent protein kinase activity and the amounts of the RI and RII cyclic AMP binding proteins present in the nuclear fraction. Exposure of F9 cells to retinoic acid (0.1 microM) induces differentiation to parietal endoderm, while retinoic acid treatment (3 microM) of PC13 cells induces differentiation to visceral endoderm. In both cell types retinoic acid treatment causes a rapid (within 4 h) and pronounced (by 2-fold) decrease in nuclear cyclic AMP-dependent protein kinase activity. Conversely, as measured by cyclic [8-azido-32P]AMP photoaffinity labeling a similar rapid and pronounced decrease in the RI and RII regulatory subunits is observed at the nucleus. This decrease in nuclear cyclic AMP-dependent protein kinases in at least two cell types may be an early event of retinoid action important in the initiation of differentiation.

Effect of phorbol ester on cyclic adenosine 3':5'-monophosphate-dependent protein kinases in PYS teratocarcinoma-derived cells and counteraction with retinoic acid.

Treatment of PYS cells with the tumor promoter (TPA) has been previously shown to enhance calcium- and phospholipid-dependent protein kinase (PK.C) in the membranes and to decrease its activity in the cytosol. Evidence is presented that 0.1 microM TPA treatment of PYS cells causes an opposite effect on the cyclic AMP-dependent protein kinases (PK.A). Within 10 min TPA led to an increase in PK.A in the cytosol and a concomitant decrease in the membranes, as measured by both the kemptide phosphorylation activity and photoaffinity labeling of RI and RH regulatory subunits of PK.A, with 8-azido-cyclic [32P]AMP. Moreover, the antitumor promoter retinoic acid (RA, 0.1 microM), when added simultaneously with TPA to the PYS cells, completely abolished the TPA effects on PK.A. When RA was added 25 min before TPA, the counteraction was not observed, indicating that RA was counteracting the TPA effect directly. These results suggest that TPA induces a rapid change in the compartmentalization of PK.A between the membrane and the soluble fraction. This possible translocation of PK.A seems to be blocked by RA, suggesting that the early antagonistic effects of RA toward TPA-mediated events occur at the plasma membranes.

Expression of HERV-W Env glycoprotein (syncytin) in the extravillous trophoblast of first trimester human placenta.

Although the extravillous trophoblastic invasion has a critical role in human placental development, nothing is known about HERV-W expression in the extravillous phenotype. The aim of the present study was to localize in first trimester placenta the expression of HERV-W Env glycoprotein and its receptor all along the differentiation pathway of the extravillous phenotype. In addition using an in vitro model of extravillous cytotrophoblastic cell isolation and invasion we investigated the presence of HERV-W transcripts and envelope glycoprotein in cultured extravillous trophoblastic cells. Using monoclonal and polyclonal antibodies, the glycoprotein was immunolocalized in all the cell types of the extravillous phenotype lineage: cytotrophoblastic cells of the column, interstitial extravillous trophoblastic cells, multinucleated giant cells and endovascular trophoblast. Furthermore, using a polyclonal antibody, the D mammalian virus receptor was also localized in the various extravillous trophoblastic phenotypes. In addition, the presence of HERV-W transcripts and protein was demonstrated in cultured extravillous trophoblastic cells. HERV-W Env glycoprotein expressed in villous and extravillous trophoblast can be considered as a specific marker of the human trophoblast.

Review: An overview of molecular events occurring in human trophoblast fusion.

During human placentation, mononuclear cytotrophoblasts fuse to form a multinucleated syncytia ensuring hormonal production and nutrient exchanges between the maternal and fetal circulation. Syncytia formation is essential for the maintenance of pregnancy and for fetal growth. The trophoblast cell fusion process first requires the acquisition of cell fusion properties, then cells set up plasma membrane protein macrocomplexes and fusogen machinery that trigger cell-cell fusion. Numerous proteins have been shown to be directly involved in the initiation of trophoblast cell fusion. These proteins must expressed at the right time and in the right place to trigger cell-cell fusion. In this review, we describe the role of certain fusogenic protein macrocomplexes that form the scaffold for the fusogen machinery underlying human trophoblastic-lipid mixing and merging of cell contents that lead to cell fusion in physiological conditions.

Rapid effect of treatment of psoriatic erythrocytes with the synthetic retinoid acitretin to increase 8-azido cyclic AMP binding to the RI regulatory subunit.

We have recently demonstrated a deficiency in the cyclic adenosine monophosphate (cAMP)-dependent protein kinases (PKA), the intracellular mediator of AMP, in psoriasis. This enzyme defect is expressed in fibroblasts and in red blood cells isolated from psoriatic patients. In these cells, the abnormality noted in cAMP binding to PKA correlates well with the severity of the disease and is corrected by long-term treatment with etretinate. In this study, we determined the effect of oral administration of acitretin in four psoriatic patients on the altered cAMP binding observed with the RI regulatory subunit of PKA in erythrocytes prepared from these patients. Acitretin (30 mg/day) induced a rapid (within 1 h) increase in the ability of the RI regulatory subunit of erythrocytes to bind the 8-azido[32P]cAMP photoaffinity analogue of cAMP. The maximal plateau for this effect of acitretin was observed within 24 h of treatment and preceded the clinical improvement of the disease. The effect of acitretin was dose-dependent, with the maximal response observed at 40 mg acitretin/d. In addition, the rapid exposure (15 min) of erythrocytes isolated from untreated patients exhibiting severe psoriasis to acitretin also promoted an increase in binding of 8-azido[32P]cAMP to the RI cAMP binding protein. Retinoic acid and 13-cis-retinoic acid were as efficient as acitretin in inducing the increase in binding of 8-azido[32P]cAMP to the RI regulatory subunit, whereas arotinoid was without effect. These results suggest that acitretin may act to modify PKA (the RI regulatory subunit) at the post-transcriptional level, and this may reflect, in part, on the mechanism of action of this synthetic retinoid. Further, monitoring this biochemical event may be helpful in determining the choice of retinoid therapy and in the management of its pharmacology.

Antioxidant enzymes in psoriatic fibroblasts and erythrocytes.

Antioxidant enzyme activities in fibroblasts and erythrocytes prepared from normal and psoriatic patients were measured and compared. The most significant differences were noted in superoxide dismutase (SOD) activities. A dramatic (5.2-fold) increase in Mn-SOD activity along with a lesser (1.8-fold) increase in CuZn-SOD activity was observed in fibroblasts from lesional and nonlesional psoriatic skin. The increase of Mn-SOD activity was correlated with an increase of both protein and mRNA. A slight (1.2-fold) increase in CuZn-SOD activity was also found in psoriatic as compared to normal red blood cells, while Mn-SOD activity was not present in these cells. In contrast, both glutathione peroxidase and catalase activities were only slightly (1.3-fold) increased in psoriatic fibroblasts, with no appreciable change noted in psoriatic erythrocytes. Likewise, glutathione levels were observed to be similar in normal and psoriatic cells. The increases in SOD activities did not appear to correlate with the severity of the disease as expressed by the Psoriatic Area Severity Index score or with plasma inflammatory markers. These results demonstrate that antioxidant enzyme activities, particularly Mn-SOD in fibroblasts and CuZn-SOD in erythrocytes, are significantly elevated in cells from psoriatic patients.

Effect of retinoic acid on platelet-derived growth factor (PDGF) bioactivity and type-B PDGF receptors in normal and psoriatic human fibroblasts.

Psoriasis is a common skin disease in which retinoids have beneficial effects. It offers a model for the study of benign hyperproliferation with abnormal differentiation. The dermis has a prominent role in the appearance of epidermal lesions. It is therefore of interest to study the factors that modulate dermal cell proliferation. In this study, the role of retinoids in modulating platelet-derived growth factor (PDGF) bioactivity was studied in normal (six subjects) and psoriatic fibroblasts from involved and uninvolved tissues (six patients). Retinoic acid treatment (for 4 d at 10(-6) M) of psoriatic fibroblasts significantly increased the chemotactic effect of PDGF in these cells (p less than 0.01 and p less than 0.05, respectively, in involved and uninvolved skin at 20 ng/ml of platelet-derived growth factor as measured in a modified Boyden Chamber Assay). In the same way, retinoic acid treatment of psoriatic fibroblasts increased the mitogenicity of platelet-derived growth factor in these cells. Retinoic acid treatment has no significant effect on the mitogenic and chemotactic activity of PDGF in normal fibroblasts. The binding of the homodimer BB PDGF to its type-B receptor, which mediates the mitogenic and chemotactic effect of PDGF, was not modified by retinoic acid treatment either in psoriatic and/or normal fibroblasts. These results suggest that retinoic acid may modulate the PDGF bioactivity in psoriatic fibroblasts not by affecting the binding of this ligand to these cells but by influencing a post-receptor event.

Increased oxidative damage to fibroblasts in skin with and without lesions in psoriasis.

Differences in oxidative damage, as measured by an increase in the carbonylation of macromolecules, were determined in situ with skin biopsies from psoriatic patients and controls. High levels of carbonyl residues were consistently detected in the dermis and never in the epidermis of sections of these skin biopsy samples. The dermis of psoriatic skin without lesions had a higher level of carbonylation than the dermis of normal skin. In this study, we found that there was more oxidative damage in cultured fibroblasts prepared from skin with and without lesions from psoriasis patients than in normal fibroblasts from the skin of age-matched controls. The extent of protein carbonylation in cell extracts was determined by immunoblotting, using an antidinitrophenylhydrazone antibody, and in intact cells was determined by immunocytochemical analysis with the same antibody. The higher level of carbonylation detected was used here as a measure of oxidative stress, and showed that some oxidative damage occurred before the appearance of typical psoriatic plaques. These results suggest that fibroblasts are affected before the onset of psoriasis and that this damage is independent of any inflammatory infiltrate.

Calcitonin and calcitonin gene-related peptide are chemotactic for F9 embryonal carcinoma cells.

The chemotactic effect of calcitonin (CT) gene products was tested on F9 teratocarcinoma cells, which are an in vitro model of early embryonic development. CT and CT gene-related peptide (CGRP) induce a significant chemotactic response (chemotactic index, 40-50). The order of potency is: chicken CGRP greater than or equal to salmon CT greater than or equal to human CGRP. Human CT is a less potent chemotactic agent (chemotactic index, 15). Compared to other well known peptides with chemotactic activity, such as platelet-derived growth factor (no activity) and transforming growth factor-beta (chemotactic index, 5), CGRP and CT appear to be very active in attracting F9 cells in the Boyden chamber assay. Interestingly, CT and CGRP exhibit little chemotactic effect toward differentiated teratocarcinoma cells (i.e. retinoic acid-treated F9 cells or parietal endodermal PYS cells). While salmon CT and chicken CGRP activate adenylate cyclase activity in F9 cell membranes by 7- to 8-fold, higher concentrations (greater than 10(-10) M) of these peptides are required to stimulate cAMP formation than are required to mediate the chemotactic effect of these peptides. These data imply the possible involvement of CT gene products in regulating cell migration during early embryonic development.

Oxidative modulation of cyclic AMP-dependent protein kinase in human fibroblasts: possible role in psoriasis.

Previous studies have established that cyclic AMP-dependent protein kinase (PKA) activity, as well as 8-azido-[32P]-cAMP binding to the RI and RII regulatory subunits, are decreased in cells from psoriatic patients compared to cells from normal patients. Here we show that the exposure of normal human dermal fibroblasts in culture to hydrogen peroxide and to oxygen free-radical generating systems decreased PKA activity, as well as cyclic AMP binding to the RI and RII regulatory subunits, to levels similar to those observed with psoriatic fibroblasts. Likewise, treatment of normal cytosolic preparations of PKA, as well as purified bovine PKA II, in vitro with free radical generating systems also resulted in decreased PKA activity and 8-azido [32P]-cAMP binding to the RI and RII regulatory subunits. Further, treatment of psoriatic fibroblasts with free radical scavenging agents such as vitamins E and C, and mannitol, and also with superoxide dismutase, restored the ability of RI and RII to bind 8-azido-[32P]-cAMP toward normal levels. Western blot analysis showed that the protein levels of the RI and RII subunits are similar in normal and psoriatic fibroblasts, and that the amounts of RI and RII are not altered by treatment of the cells with free radical-generating systems. These results suggest that oxidative modification may serve as a mechanism to alter PKA activity in human cells, and that an altered oxidative state may be involved in mediating the decrease in PKA activity and cyclic AMP binding noted in cells from psoriatic patients.

Impact of trisomy 21 on human trophoblast behaviour and hormonal function.

Although trisomy 21 (T21) is the most frequent genetic abnormality and some maternal serum markers for this fetoplacental aneuploidy are of placental origin, little is known of its impact on placental development. We therefore studied the influence of T21 on trophoblast behaviour. Using cultured cells from 46 human T21 pregnancies, we confirmed the defective morphological and functional differentiation of the villous cytotrophoblast in this setting; indeed, villous cytotrophoblast cells aggregate normally but fuse inefficiently to form the syncytiotrophoblast. This is in part related to the abnormal oxidative status of the T21 cytotrophoblast, characterized by a gene dosage-related increase in SOD-1 (copper-zinc superoxide dismutase) expression and activity. This was associated with a significant (P < 0.01) increase in catalase activity but no significant change in glutathione peroxidase activity. On the basis of these in vitro findings and studies of large panels of maternal serum, we propose a pathophysiological explanation for trisomy 21 maternal serum markers of placental origin.

Large variability of trophoblast gene expression within and between human normal term placentae.

Human placenta extracts are widely used in clinical and fundamental research, particularly to study the hormonal and exchange functions of the placenta. However, very little is known about the distribution of the main hormone mRNAs in the placenta as a whole. Total placenta extracts are heterogeneous in their cellular components, as they contain material of both fetal and maternal origin, and in their structure. Results vary greatly depending upon the location of the biopsy and the number of biopsies performed. We used real-time quantitative RT-PCR to determine whether transcripts corresponding to the main hormones secreted by the human placenta (e.g. hCG, HPL and PGH) are equally distributed within and between term placentae. We also measured cytokeratin 7 transcripts, which are specifically expressed in the trophoblast, and transcripts corresponding to nuclear receptors PPARgamma and RXRalpha. A comparison of the results obtained with 12 different samples from each of four normal term placentae revealed that the amounts of transcripts differ considerably within and between each placenta. This emphasizes the need to study large numbers of samples when looking for significant differences in gene expression.

[Spontaneous retroperitoneal hematoma: a rare cause of hemorrhagic shock]. / Hématome rétropéritonéal spontané: cause rare de choc hémorragique.

A spontaneous retroperitoneal haematoma is an uncommon cause of haemorrhagic shock. We report a case of spontaneous rupture of a renal angiomyolipoma resulting in haemorrhagic shock in a 52-year-old woman. The renal tumor was recognized by sonography and diagnosed by CT-scan. Renal angiography was performed, but embolization was not successful. During the surgical procedure, nephrectomy was required because of persistent bleeding, related to disseminated intravascular coagulation. Outcome was uneventful. Diagnosis and treatment of renal angiomyolipoma are discussed. The Lenk's triad, consisting of acute lumbar pain, symptoms of internal bleeding and lumbar tumefaction, is the usual clinical picture of retroperitoneal haemorrhage. The kidney is the most frequent cause and renal angiomyolipoma is the most frequent benign tumor. Renal angiomyolipoma is either isolated or associated with tuberous sclerosis in up to 20 per cent of patients. Diagnosis is suggested by sonography and confirmed by CT-scan. Renal angiography, performed in haemodynamically stable patients, shows the origin of bleeding and allows embolization. Considering the frequent bilaterality of angiomyolipoma, surgery should be as conservative as possible in order to preserve renal function.

Review: Human trophoblast fusion and differentiation: lessons from trisomy 21 placenta.

The syncytiotrophoblast layer plays a major role throughout pregnancy, since it is the site of numerous placental functions, including ion and nutrient exchange and the synthesis of steroid and peptide hormones required for fetal growth and development. Inadequate formation and regeneration of this tissue contributes to several pathologies of pregnancy such as intrauterine growth restriction and preeclampsia, which may lead to iatrogenic preterm delivery in order to prevent fetal death and maternal complications. Syncytiotrophoblast formation can be reproduced in vitro using different models. For the last ten years we have routinely purified villous cytotrophoblastic cells (CT) from normal first, second and third trimester placentas and from gestational age-matched Trisomy 21 placentas. We cultured villous CT on plastic dishes to follow the molecular and biochemical aspects of their morphological and functional differentiation. Taking advantage of this unique collection of samples, we here discuss the concept that trophoblast fusion and functional differentiation may be two differentially regulated processes, which are linked but quite distinct. We highlight the major role of mesenchymal-trophoblast cross talk in regulating trophoblast cell fusion. We suggest that the oxidative status of the trophoblast may regulate glycosylation of proteins, including hCG, and thereby modulate major trophoblast cell functions.

Hyperglycosylated hCG is a marker of early human trophoblast invasion.

CONTEXT: Human chorionic gonadotropin (hCG) is the major pregnancy glycoprotein hormone whose maternal concentration and glycan structure change all along pregnancy. hCG is mainly secreted by the syncytiotrophoblast covering the chorionic villi, but little is known about the source of hyperglycosylated hCG (hCG-H) production. OBJECTIVE: The objective of the study was to analyze expression and secretion of hCG and hCG-H in vitro during human trophoblastic cell differentiation, in situ in first-trimester placentas, and in maternal sera during early pregnancy. DESIGN: hCG and hCG-H were measured in cell supernatants from primary cultures of first-trimester placenta trophoblastic cells, which differentiate in vitro into syncytiotrophoblast or invasive extravillous cytotrophoblasts (evct). hCG-H immunodetection were performed on 9 wk gestation (WG) placental tissue sections. Total hCG and hCG-H were quantified by chemiluminometric assay in 539 maternal sera collected between 9 and 19 WG during normal pregnancies. RESULTS: In vitro, hCG secretion reached 37 ng/ml per µg DNA during syncytiotrophoblast formation but contained few hCG-H (2-5% of total hCG). In contrast, hCG secretion (20 ng/ml per µg DNA) in evct supernatants contained 10-20% hCG-H. In situ, hCG-H immunostaining was strong in invasive and endovascular evct, weaker in mononucleated villous cytotrophoblasts, but negative in the syncytiotrophoblast. In maternal sera, hCG-H concentrations continuously decreased during pregnancy from 406 ± 222 ng/ml at 9 WG to 8 ± 6 ng/ml at 19 WG, whereas total hCG picked up at 11 WG and then decreased. CONCLUSIONS: This study suggests that the high levels of hCG-H observed in first-trimester maternal sera are mainly from invasive evct origin, reflecting the early trophoblast invasion process.

Characterization of specific proteases associated with the surface of human skin fibroblasts, and their modulation in pathology.

Human skin fibroblasts were probed for cell surface protease activity. One activity removing dipeptides from the NH2-terminal end of Gly-Pro-pNA was specifically inhibited by di-isopropyl-fluorophosphate (DFP), phenylmethanesulphony fluoride (PMSF), and diprotin A, and thus was identified as dipeptidyl peptidase IV (DPP IV). A group of bestatin-sensitive N-exoaminopeptidase activities was also characterized when Ala-, Leu-, and Arg-pNA were used as chromogenic substrates. Using human monoclonal antibodies anti-CD 13 and anti-CD 26 that recognized, respectively, an N-Ala-aminopeptidase and DPP IV, it was found that human dermal fibroblasts expressed the CD 13 and CD 26 antigen on their surface. In addition, both peptidases were specifically immunoprecipitated by monoclonal antibodies anti-CD 13 and anti-CD 26 from plasma membranes. Cell surface proteolytic activities were also investigated in human fibroblasts derived from dermatological and rheumatic diseases (i.e., psoriasis, rheumatoid arthritis, and lichen planus). It was found that these fibroblasts also expressed both types of proteinases initially identified on normal skin fibroblasts and that the levels of Ala-aminopeptidase activities were similar in all cases. In contrast, the levels of Arg-, Leu-exoaminopeptidase, and DPP IV activities were significantly higher (up to 6.6-fold) in the three pathological fibroblast populations than in their normal counterparts. These proteolytic enzymes, therefore, can potentially serve as markers in dermatological diseases. Taken together, our results suggest that skin fibroblast-derived proteinases associated with both serine and N-aminopeptidase activities may play an important role by participating in the extracellular events associated with fibroblast behaviour.

Association of type II cAMP-dependent protein kinase with p34cdc2 protein kinase in human fibroblasts.

Previous independent studies suggested that type II cAMP-dependent protein kinase and the p34cdc2 protein kinase cell cycle regulator co-localize at centrosomes. In order to investigate whether there is an association of type II cAMP-dependent protein kinase with p34cdc2 in human fibroblasts, we used three different approaches. First, the regulatory subunits RI and RII were photoaffinity-labeled with 8-N3-[32P]cAMP, and anti-p34cdc2 immunoprecipitates were screened for the presence of either RI or RII regulatory subunits by one- or two-dimensional gel electrophoresis. Second, anti-RII alpha immunoprecipitates were screened for the presence of p34cdc2 by Western blot using three different affinity-purified antibodies recognizing different domains of human p34cdc2. Conversely, anti-p34cdc2 immunoprecipitates (three different antibodies), as well as the material retained on p13suc1-Sepharose Bio-Beads, which binds specifically p34cdc2, were screened for the presence of RII alpha. Finally, we have looked for cAMP-dependent protein kinase activity specifically inhibited by PKI in immunoprecipitates obtained from extracts treated with different anti-p34cdc2 antibodies. All these experiments gave concordant results and demonstrate that at least at G0/G1, human fibroblasts contain a complex of active type II cAMP-dependent protein kinase associated through its RII alpha subunit with p34cdc2.

Altered hormonal responses: markers for embryonal and embryonic carcinoma stem cells and their differentiated derivatives.

Teratocarcinoma cells in culture offer an in vitro system for studying certain aspects of embryonic differentiation. To gain some insight into regulatory systems that might be operative during early development, we have characterized the alterations that occur in the hormonal responsiveness of the membrane adenylate cyclase of different embryonal carcinoma cell lines with differentiation. Each undifferentiated embryonal carcinoma stem cell studied (F9, PCC4, PC13, P19) has an adenylate cyclase system predominantly activated by calcitonin. Of great interest is the fact that cAMP production is also enhanced specifically by calcitonin in an embryo-derived stem cell line. Differentiation of the embryonal carcinoma stem cell population toward parietal endoderm results in a decrease in calcitonin activation with a concomitant appearance of sensitivity to parathyroid hormone. Differentiation toward visceral endoderm is characterized by a lack of response of the adenylate cyclase system to both calcitonin and parathyroid hormone. These results indicate that the changes noted in adenylate cyclase hormonal responsiveness might serve as useful markers during early stages of differentiation.