Factors affecting cytotrophoblast cell viability and differentiation: Evidence of a link between syncytialisation and apoptosis.

A relationship between cytotrophoblast differentiation (syncytialisation) and apoptosis is hypothesised to exist, but has not been clearly determined. To address this, we explored the effects of cAMP, an inducer of syncytialisation, on human choriocarcinoma cell differentiation and viability under three different culture conditions related to diverse survival status: no serum, 10% fetal calf serum or 10% charcoal-stripped fetal calf serum. 8-Br-cAMP increased BeWo cell viability in culture media without serum, but viability was decreased in a dose- and time-dependent manner when serum was present. The appearance of apoptotic nuclei fragments were only observed when BeWo cells were cultured in media containing serum combined with 8-Br-cAMP treatment. In addition, the ratio of FasL to Fas expression following treatment with 8-Br-cAMP increased by 20-fold in 10% charcoal-stripped fetal calf serum media and 65-fold 10% fetal calf serum media, and activation of caspase-3 also required media with serum. The markers of syncytialisation (syncytin 1 expression and human chorionic gonadotropin secretion) were induced significantly by 8-Br-cAMP, and were higher in 10% fetal calf serum media than in 10% charcoal-stripped fetal calf serum media, than in the absence of serum. Syncytia formation was stimulated by 8-Br-cAMP and this required serum in the media. We now show that factors contained within serum are necessary for cAMP-stimulated cytotrophoblast differentiation, that syncytialisation involves apoptotic events, and that a lack of serum based factors could switch the cellular program away from differentiation.

Progesterone receptors A and B differentially modulate corticotropin-releasing hormone gene expression through a cAMP regulatory element.

Corticotrophin-releasing hormone (CRH) plays a major role in mechanisms controlling human pregnancy and parturition. Gene regulation by progesterone may be a key point in the control of placental CRH production. Studies in primary placental cells show that antagonism of progesterone activity or production by RU486 or trilostane leads to an increase in CRH promoter activity. This effect can be reversed by the addition of progesterone. Overexpression of progesterone receptor A (PR-A) or glucocorticoid receptor resulted in a decrease in CRH promoter activity following progesterone treatment, whereas an increase in promoter activity was observed with overexpressed PR-B. Studies including mutation of the cAMP regulatory element (CRE) confirm this site to be essential for the progesterone-mediated effects. In summary, our results demonstrate that progesterone regulates CRH gene transcription via a CRE in the CRH promoter and that PR-A and PR-B exhibit different actions in the regulation of CRH gene expression.

The regulation of human corticotrophin-releasing hormone gene expression in the placenta.

Corticotrophin-releasing hormone (CRH) is a 41 amino acid neuropeptide that is expressed in the hypothalamus and the human placenta. Placental CRH production has been linked to the determination of gestational length in the human. Although encoded by a single copy gene, CRH expression in the placenta is regulated differently to the hypothalamus. Glucocorticoids stimulate CRH promoter activity in the placenta but inhibit it's activity in the hypothalamus, via mechanisms involving different regions of the CRH promoter. We discuss how various stimuli alter CRH promoter activity and why these responses are unique to the placenta.

The regulation of human corticotrophin-releasing hormone gene expression in the placenta.

Corticotrophin-releasing hormone (CRH) is a 41 amino acid neuropeptide that is expressed in the hypothalamus and the human placenta. Placental CRH production has been linked to the determination of gestational length in the human. Although encoded by a single copy gene, CRH expression in the placenta is regulated differently to the hypothalamus. Glucocorticoids stimulate CRH promoter activity in the placenta but inhibit it's activity in the hypothalamus, via mechanisms involving different regions of the CRH promoter. We discuss how various stimuli alter CRH promoter activity and why these responses are unique to the placenta.

Placental corticotrophin-releasing hormone, local effects and fetomaternal endocrinology.

The human placenta produces corticotrophin-releasing hormone (CRH) in exponentially increasing amounts during pregnancy with peak levels during labour. CRH in human pregnancy appears to be involved in many aspects of pregnancy including placental bloodflow, placental prostaglandin production, myornetrial function, fetal pituitary and adrenal function and the maternal stress axis. Since fetal cortisol levels are associated with pulmonary development and maturity, placental CRH may have an indirect role in fetal development.Although the precise role of placental CRH in the regulation of gestational length and timing of parturition is unclear it appears to be involved in a placental clock. While glucocorticoids inhibit hypothalamic CRH production they stimulate CRH gene expression in the placenta.This difference may allow the fetal and maternal stress axes to influence this placental clock.Maternal CRH levels are elevated in many pathological conditions of pregnancy where fetal well-being is compromised, and in these situations it may act to maintain a stable intrauterine environment. Therefore, CRH appears to link placental function, maternal well-being, fetal well-being and fetal development to the duration of gestation and the timing of parturition.

Glucocorticoid stimulation of corticotropin-releasing hormone gene expression requires a cyclic adenosine 3',5'-monophosphate regulatory element in human primary placental cytotrophoblast cells.

Production of placental CRH, which is identical to the peptide synthesized and secreted in the hypothalamus, has been linked to human parturition. Glucocorticoids stimulate placental CRH secretion and messenger ribonucleic acid expression, in contrast to their inhibition of CRH synthesis in the hypothalamus. A positive feedforward loop involving glucocorticoid-CRH-ACTH-glucocorticoid is thought to drive the exponential increase in placental CRH leading to delivery. Tissue-specific effects of glucocorticoids on CRH expression are therefore of interest. Using human primary placental cells, we investigated the mechanism by which glucocorticoids stimulate placental CRH gene expression. Nuclear run-on transcription shows that in human placental cells glucocorticoids up-regulate transcription of human CRH (hCRH). Using transient transfection assays we demonstrate that dexamethasone up-regulates both basal and cAMP-stimulated hCRH promoter activity, correlating well with the increase in endogenous CRH peptide levels. Through mutagenesis and deletion analyses we show that dexamethasone stimulation of hCRH gene transcription requires a functional cAMP regulatory element (CRE); this CRE is adequate to confer dexamethasone stimulation upon a heterologous promoter, and electrophoretic mobility shift assay studies show that a placental nuclear protein specifically binds to the hCRH CRE.

Corticotropin-releasing hormone gene expression in primary placental cells is modulated by cyclic adenosine 3',5'-monophosphate.

CRH, the principal neuropeptide regulator of pituitary ACTH secretion, is also expressed in placenta. Placental CRH has been linked to the process of human parturition. However, the mechanisms regulating transcription of the CRH gene in placenta remain unclear. cAMP signaling pathways play important roles in regulating the expression of a diverse range of endocrine genes in the placenta. Therefore, we have explored the effect of cAMP on CRH promoter activity in primary cultures of human placental cells. Both forskolin and 8-bromo-cAMP, activators of protein kinase A, can increase CRH promoter activity 5-fold in transiently transfected human primary placental cells, in a manner that parallels the increase in endogenous CRH peptide. Maximal stimulation of CRH promoter activity occurs at 500 micromol/L 8-bromo-cAMP and 10 micromol/L forskolin. Electrophoretic mobility shift assay and mutation analysis combined with transient transfection demonstrate that in placental cells cAMP stimulates CRH gene expression through a cAMP regulatory element in the proximal CRH promoter region and involves a placental nuclear protein interacting specifically with the cAMP regulatory element.

MIC-1, a novel macrophage inhibitory cytokine, is a divergent member of the TGF-beta superfamily.

Macrophages play a key role in both normal and pathological processes involving immune and inflammatory responses, to a large extent through their capacity to secrete a wide range of biologically active molecules. To identify some of these as yet not characterized molecules, we have used a subtraction cloning approach designed to identify genes expressed in association with macrophage activation. One of these genes, designated macrophage inhibitory cytokine 1 (MIC-1), encodes a protein that bears the structural characteristics of a transforming growth factor beta (TGF-beta) superfamily cytokine. Although it belongs to this superfamily, it has no strong homology to existing families, indicating that it is a divergent member that may represent the first of a new family within this grouping. Expression of MIC-1 mRNA in monocytoid cells is up-regulated by a variety of stimuli associated with activation, including interleukin 1beta, tumor necrosis factor alpha (TNF-alpha), interleukin 2, and macrophage colony-stimulating factor but not interferon gamma, or lipopolysaccharide (LPS). Its expression is also increased by TGF-beta. Expression of MIC-1 in CHO cells results in the proteolytic cleavage of the propeptide and secretion of a cysteine-rich dimeric protein of Mr 25 kDa. Purified recombinant MIC-1 is able to inhibit lipopolysaccharide -induced macrophage TNF-alpha production, suggesting that MIC-1 acts in macrophages as an autocrine regulatory molecule. Its production in response to secreted proinflammatory cytokines and TGF-beta may serve to limit the later phases of macrophage activation.

Short-term diethylnitrosamine-induced oval cell responses in three strains of mice.

The oval cells of the liver have been identified as target cells of chemical carcinogens during rat hepatocarcinogenesis and are believed to act as liver stem cells. In this study mice (strains C3H/EJ (C3H), C57/BL6J (C57) and hybrid B6C3F1 (F1)) were sacrificed at 1, 3 and 7 days after administration of a single dose of the carcinogen diethylnitrosamine (DEN), and histopathological studies of oval cells were evaluated using Haematoxylin and Eosin (H&E), Picro-Mallory (P-M), alpha-fetoprotein (A-FP) and glutathione S-transferase placental form (GST-pi) staining techniques and electron microscopy (EM). Increased oval cell proliferation was observed as soon as one day following exposure of the mice to DEN, in a manner consistent with C3H and C57 mice exhibiting high and low susceptibility to DEN respectively, with hybrid F1 mice being intermediate in DEN sensitivity. This analysis indicates that, in mice, oval cells are target cells at very early stages of liver carcinogenesis and supports the notion that oval cells are potential liver stem cells.

High levels of c-myc gene expression precede point mutational activation of Ki-ras in mouse lung cancer.

A cell culture model of mouse lung alveologenic carcinoma, consisting of type 2 pneumocyte-related cells, known to contain an A to G transition in the second base position of codon 61 of the c-Ki-ras gene has been examined for molecular changes affecting nuclear oncogenes. A small, twofold increase in c-myc mRNA levels and transcription levels was observed in malignant cell lines (C4SE9 and NULB5) compared with non-malignant cells (C4E10). Interestingly, the transcriptional level of the c-myc gene in C4E10 cells was very high relative to any other gene examined. Similar high levels of c-myc gene expression levels were also observed in type 2 pneumocytes obtained from normal mouse lung tissue. No major DNA rearrangements or amplifications were detected between C4E10 cells and either C4SE9 or NULB5 cell lines. These data suggest that high levels of c-myc gene expression occurred prior to the activating point mutation of the c-Ki-ras gene and may predispose the type 2 pneumocyte to transformation.

Sequence of the mouse fibronectin-encoding gene promoter region.

The polymerase chain reaction (PCR) procedure has been utilised to clone, from mouse genomic DNA, a region of the fibronectin (FN)-encoding gene (FN) which includes the 5' flanking nucleotide (nt) sequences. The mouse promoter nt sequence shows high homology to the rat and human FN promoters, including consensus nt sequences for several DNA-binding transcription factors.

Negative regulation of the rat stromelysin gene promoter by retinoic acid is mediated by an AP1 binding site.

Stromelysin is a member of the metalloproteinase family which plays an important role in extracellular matrix remodelling during many normal and disease processes. We show here that in polyomavirus-transformed rat embryo fibroblast cells (PyT21), the transcription from the stromelysin gene is repressed by the vitamin A derivative retinoic acid (RA). Furthermore, expression vectors encoding the human RA receptors hRAR-alpha, hRAR-beta and hRAR-gamma repress chloramphenicol acetyltransferase (CAT) expression from stromelysin promoter-CAT gene expression vectors in RA-treated PyT21 and human HeLa cells, as determined by transient transfection assays. Through mutation and deletion analysis, we show that the RA dependent repression is mediated by a 25 bp region from nucleotide positions -72 to -48 of the rat stromelysin 5'-flanking DNA sequence. Further mutation analysis of this region indicates that the DNA sequence required for RA dependent repression colocalizes with an AP1 binding site which is essential for promoter activity. We show also that RA represses the transcriptional activity of a reporter gene containing a TPA responding AP1 binding site driving the HSV tk promoter. Thus the RAR-RA complex appears to repress transcription of the stromelysin gene by blocking activation by positive regulatory factors. However, we found no evidence supporting the possibility that the RA dependent repression could be due to RAR binding to the AP1 binding site or to the AP1 components c-fos and c-jun.

An essential member of the HSP70 gene family of Saccharomyces cerevisiae is homologous to immunoglobulin heavy chain binding protein.

Immunoglobulin heavy chain binding protein (BiP) is present in the lumen of the mammalian endoplasmic reticulum, where it associates transiently with a variety of newly synthesized secretory and membrane proteins or permanently with mutant proteins that are incorrectly folded. We describe a unique member of the Saccharomyces cerevisiae 70-kDa heat shock protein gene family (HSP70) that encodes a protein homologous to mammalian BiP. The DNA sequence contains a 2046-nucleotide open reading frame devoid of introns, and examination of the predicted amino acid sequence reveals features not found in most other yeast HSP70 proteins but which are present in BiP. Most notable are a 42-residue sequence at the N terminus that exhibits characteristics of a cleavable signal sequence and a C-terminal sequence, -His-Asp-Glu-Leu, that is involved in determining endoplasmic reticulum localization in yeast. The 5' flanking region of this gene contains two overlapping sequences between nucleotides -146 and -169 that closely resemble consensus heat shock elements. The yeast BiP gene is strongly heat shock-inducible, whereas the BiP genes in various other species are either weakly or non-heat-inducible. We demonstrate that a functional BiP gene is essential for vegetative growth. An evolutionary comparison of amino acid sequences of 34 HSP70 proteins from 17 species suggests that BiP genes share a common ancestor, which diverged from other HSP70 genes near the time when eukaryotes first appeared.

Expression of a Drosophila heat-shock gene in cells of the yeast Saccharomyces cerevisiae.

A 3.52-kilobase (kb) segment of Drosophila melanogaster DNA carrying the 2.15-kb transcribed sequence for the 70 000-dalton heat-shock protein (hsp70) and 1.14-kb of the 5' flanking sequence was inserted into an autonomously replicating chimeric plasmid and used to transform the yeast Saccharomyces cerevisiae. The Drosophila gene is efficiently transcribed in the transformed cells, yielding a transcript which is 21 nucleotides shorter than the normal Drosophila mRNA at the 5' end. Significant increases in the amount of Drosophila-specific RNA occur when the transformed cells are subjected to heat shock, indicating that the Drosophila gene is inducible in the yeast cells.

The major heat-shock protein (hsp70) gene family: related sequences in mouse, Drosophila, and yeast.

Heat shock induces the synthesis of a 70-kdalton protein in Escherichia coli, Drosophila, yeast, and mouse. We show that the genes for this heat-shock protein in mouse, yeast, and Drosophila share extensive sequence homology as determined by heteroduplex formation at different stringencies. We calculate that the heat-shock gene homology is 74% between yeast and Drosophila and 85% between mouse and Drosophila. The organization of the six copies of the Drosophila gene for the 70-kdalton heat-shock protein at two separate loci is summarized and evidence is presented that the yeast and mouse genomes each contain multiple copies of sequences related to the Drosophila gene for the 70-kdalton heat-shock protein. These results demonstrate that not only the sequence but also the repetitive organization of the major heat-shock genes is conserved.

Specificity of the cholesterol side-chain cleavage enzyme system of adrenal cortex.

Incubation of lanosta-8, 24-dien-3beta-o1-1,2-3H and lanost-8-en-3beta-o1-1,2-3H with an adrenocortical bovine mitochondrial acetone-dried preparation did not yield any significant (less than 0.01%) 3beta-hydroxy-4, 4, 14-trimethyl-5alpha-pregn-8-en-20-one. Under the same conditions cholesterol-1,2-3H yielded 8.3% pregnenolone. Incubation of (20S)-17alpha, 20-di-hydroxycholesterol-7-3H yielded 0.6 to 1.6% (20S,22R)-17alpha, 20, 22-trihydroxycholesterol, 1.0 to 3.2% of 17alpha-hydroxy-pregnenolone, but no significant (less than 0.02%) (20S,22S)-17alpha,20,22-trihydroxycholesterol. In another experiment incubation of cholesterol-1,2-3H yielded 5% pregnenolone, 0.5% 17alpha-hydroxypregnenolone, 0.2% (20R,22R)-20,22-dihydroxy-cholesterol, but no significant ( less than 0.01%) 17alpha-hydroxy-cholesterol, (20S)-17alpha, 20-dihydroxycholesterol or (20S,22R)-17alpha, 20,22-trihydroxycholesterol.