BOK (BCL-2-related ovarian killer) is a member of the pro-apoptotic BCL-2 family that is highly expressed in the human placenta. BOK excess causes increased trophoblast autophagy and apoptosis in pre-eclampsia, a pathological condition of hypoxia and oxidative stress. In the present study, we identified an HRE (hypoxia-response element) at the junction of exon-1 and intron-1 (+229 to +279) in the human BOK gene, as well as an antisense transcript driven by a promoter located in intron-2. The isolated BOK-HRE bound hypoxia-inducible HIF (hypoxia-inducible factor) proteins in vitro as well as in trophoblastic JEG3 cells and was functional in its natural position as well as in front of a heterologous promoter. Being a reverted repeat, the BOK-HRE functioned in both orientations. This directionless feature of the BOK-HRE facilitates hypoxia regulation via HIF of both BOK and its antisense transcript as demonstrated by RNAi knockdown of the HIF system. Although the antisense transcript was expressed in several human carcinoma cell lines, including choriocarcinoma-derived JEG3 cells, no antisense-regulated mechanism for BOK expression was noted. Taken together, these findings indicate that hypoxia-induced expression of BOK in placental cells is regulated via HIF and is not affected by its antisense transcript.

GCM1 (glial cell missing 1) is a short-lived transcription factor essential for placental development. The F-box protein, FBW2 (F-box and WD-repeat domain-containing 2), which contains five WD (tryptophan–aspartate) repeats, recognizes GCM1 and mediates its ubiquitination via the SCF FBW2 E3 ligase complex. Although the interaction between GCM1 and FBW2 is facilitated by GCM1 phosphorylation, it is possible that this interaction might be regulated by additional cellular factors. In the present study, we perform tandem-affinity purification coupled with MS analysis identifying RACK1 (receptor for activated C-kinase 1) as an FBW2-interacting protein. RACK1 is a multifaceted scaffold protein containing seven WD repeats. We demonstrate that the WD repeats in both RACK1 and FBW2 are required for the interaction of RACK1 and FBW2. Furthermore, RACK1 competes with GCM1 for FBW2 and thereby prevents GCM1 ubiquitination, which is also supported by the observation that GCM1 is destabilized in RACK1 -knockdown BeWo placental cells. Importantly, RACK1 knockdown leads to decreased expression of the GCM1 target gene HTRA4 (high-temperature requirement protein A4), which encodes a serine protease crucial for cell migration and invasion. As a result, migration and invasion activities are down-regulated in RACK1 -knockdown BeWo cells. The present study reveals a novel function for RACK1 to regulate GCM1 activity and placental cell migration and invasion.

The human hydroxysteroid SULT (sulphotransferase) 2B1 subfamily consists of two isoforms, SULT2B1a and SULT2B1b. These two isoenzymes are transcribed from the same gene by alternative splicing of their first exons and share 94% amino acid sequence identity. The SULT2B1 isoforms are highly selective for the sulphation of 3β-hydroxysteroids. Immunoblot analysis of SULT2B1 expression in several human tissues indicates the presence of only SULT2B1b protein. Immunoreactive SULT2B1b protein was detected in human prostate, skin, placenta and lung tissue. SULT2B1b mRNA expression was detected in RNA isolated from term placenta, normal prostate, prostate carcinoma, benign prostate hyperplasia, LNCaP prostate cancer cells, breast cancer specimens and MCF-7 breast cancer cells. Immunohistochemical localization of SULT2B1b, in terms placental and prostate tissues, detected it in nuclei of placental syncytiotrophoblasts and cytoplasm of epithelial cells in prostate tissues. Immunoreactive and catalytically active SULT2B1b was identified in nuclei isolated from term human placenta. Also SULT2B1b was capable of translocating to nuclei in BeWo placental cells after stable transfection and differentiation. In contrast, immunohistochemical analysis of human prostate showed only cytosolic localization of SULT2B1b in the basal and luminal prostate epithelial cells. SULT2B1b was not detected in isolated nuclei from LNCaP prostate cancer cells but was present in the cytosolic fraction. Differential subcellular localization of SULT2B1b in prostate and placenta suggests that SULT2B1b may be differentially regulated and have different physiological functions in these two hormonally responsive human tissues.

The mouse genome contains a family of clan C1A proteases that appear to be restricted to rodents within Eutherian (placental) mammals. mRNA analysis has shown that these genes are expressed exclusively in placenta. Sequence analysis predicts that the expressed proteins will be functional and consequently it was proposed that this family of proteases may have evolved to perform subspecialized functions of the closely related protease, cathepsin L, that is expressed in placental tissues of all mammalian species. In the present study, it was shown that cathepsin P can be expressed in Pichia pastoris as an inactive zymogen that can be activated with proteinase K, chymotrypsin or pancreatic elastase at neutral pH. Unlike other mammalian cathepsins, cathepsin P could also be autoactivated at neutral pH, but not at acidic pH. The activated enzyme was capable of hydrolysing peptidyl substrates and the protein substrates azocasein and transferrin, with optimal activity at pH 6.5–7.5. Little activity could be detected at pH 5.0 and below. Salts such as Na 2 SO 4 and hyaluronate stimulated the activity of the protease against peptidyl substrates. The properties of cathepsin P appear to be quite distinct from those of cathepsin L, indicating that the duplication that gave rise to cathepsin P has probably not yielded an enzyme that provides a subfunction of cathepsin L in rodents. It seems probable that cathepsin P has evolved to perform a function that is performed by an evolutionarily unrelated protease in other mammalian species.

Recent functional studies have suggested that, in addition to simple diffusion, carrier-mediated transport may play an important role in foetal unconjugated bilirubin (UCB) uptake by the placenta. We have investigated the role of organic anion-transporting polypeptides (OATPs) in UCB transport by the placenta–maternal liver tandem. RNA was obtained from human liver (hL), human placenta (hPl) at term, and purified (> 80%) cytokeratin-7-positive mononucleated human trophoblast cells (hTCs). By analytical reverse transcription (RT)-PCR, agarose gel electrophoresis separation and sequencing, the mRNA of OATP-A ( SLC21A3 ) and OATP-8 ( SLC21A8 ) was identified in hL, hPl and hTCs, whereas that of OATP-C ( SLC21A6 ) was detectable only in hL. Real-time quantitative RT-PCR revealed that in hL the abundance of mRNA was OATP-8 > OATP-C ≫ OATP-A, whereas in hPl and hTCs this was OATP-8 ≫ OATP-A ≫ OATP-C. Expression levels for these OATPs were hL ≫ hTCs > hPl. Injection of mRNA of OATP-A, OATP-C or OATP-8 or RNA from hL, hPl or hTCs into Xenopus laevis oocytes conferred on them the ability to take up [ 3 H]17β- d -glucuronosyl oestradiol ([ 3 H]E 2 17βG) and [ 3 H]UCB, although in the case of OATP-A mRNA, the induced uptake of [ 3 H]UCB was very low. Cis -inhibition of [ 3 H]E 2 17βG and [ 3 H]UCB uptake by both unlabelled E 2 17βG and UCB was found in all cases. The affinity and efficiency of [ 3 H]UCB transport was OATP-C > OATP-8. Kinetic parameters for [ 3 H]UCB uptake induced by RNA from hTCs resembled most closely those of OATP-8. In conclusion, our results suggest that OATP-8 may play a major role in the carrier-mediated uptake of foetal UCB by the placental trophoblast, whereas both OATP-8 and OATP-C may substantially contribute to UCB uptake by adult hepatocytes.

Maternal iron deficiency during pregnancy induces anaemia in the developing fetus; however, the severity tends to be less than in the mother. The mechanism underlying this resistance has not been determined. We have measured placental expression of proteins involved in iron transfer in pregnant rats given diets with decreasing levels of iron and examined the effect of iron deficiency on iron transfer across BeWo cell layers, a model for placental iron transfer. Transferrin receptor expression was increased at both mRNA and protein levels. Similarly, expression of the iron-responsive element (IRE)-regulated form of the divalent metal transporter 1 (DMT1) was also increased. In contrast, the non-IRE regulated isoform showed no change in mRNA levels. Protein levels of DMT1 increased significantly. Iron efflux is thought to be mediated by the metal transporter protein, IREG1/ferroportin1/MTP1, and oxidation of Fe(II) to Fe(III) prior to incorporation into fetal transferrin is carried out by the placental copper oxidase. Expression of IREG1 was not altered by iron deficiency, whereas copper oxidase activity was increased. In BeWo cells made iron deficient by treatment with desferrioxamine (‘deferioxamine’), iron accumulation from iron-transferrin increased, in parallel with increased expression of the transferrin receptor. At the same time, iron efflux also increased, showing a higher flux of iron from the apical to the basolateral side. The data show that expression of placental proteins of iron transport are up-regulated in maternal iron deficiency, resulting in an increased efficiency of iron flux and a consequent minimization of the severity of fetal anaemia.

Indoleamine 2,3-dioxygenase (IDO)-initiated tryptophan degradation in the placenta has been implicated in the prevention of the allogeneic fetus rejection [Munn, Zhou, Attwood, Bondarev, Conway, Marshall, Brown, and Mellor (1998) Science 281 , 1191-1193]. To determine how IDO is associated with the development of the fetus and placenta, the time course of IDO expression (tryptophan-degrading activity, IDO protein and IDO mRNA) in the embryonic and extra-embryonic tissues as well as maternal tissues of mice was examined. A high tryptophan-degrading activity was detected in early concepti on days 6.5 and 7.5, whereas IDO protein and its mRNA were not expressed during early gestation, but appeared 2-3 days later, lasted for about 3 days and declined rapidly thereafter. The expression of IDO basically coincided with the formation of the placenta. On the contrary, the early tryptophan-degrading activity was due to gene expression of tryptophan 2,3-dioxygenase (TDO), as shown by Northern and Western analysis. These findings indicate that IDO is transiently expressed in the placenta but that the expression does not last until birth, and that the IDO expression is preceded by expression of another tryptophan-degrading enzyme, TDO, in the maternal and/or embryonic tissues in early concepti.

The human growth hormone (GH) gene family consists of five tandemly arranged and highly related genes, including the chorionic somatomammotropins (CSs), at a single locus on chromosome 17. Despite striking homologies in promoter and flanking DNA sequences, the genes within this locus have different tissue-specific patterns of expression: GH-N is expressed almost exclusively in the somatotrophs of the anterior pituitary; the remaining genes, including CS-A, are expressed in placental syncytiotrophoblast. Previously we proposed that active repression of the placental gene promoters in pituitary GC cells is mediated by upstream ‘P’ sequences and, specifically, a 263bp region containing two ‘P’ sequence elements (PSE-A and PSE-B) and corresponding factors (PSF-A and PSF-B). We have now examined the possibility that PSF-A and PSF-B are members of the nuclear factor (NF)-1 family. Transcripts of NF-1A, NF-1C and NF-1X, but not of NF-1B, were readily detected in GC cells. High-affinity binding of NF-1 to PSE-B, but not to PSE-A, was confirmed by competition of DNA–protein interactions by using NF-1 DNA elements and antibodies. Functionally, a NF-1 element was able to substitute for PSE-B as a promoter-specific repressor in GC cells after gene transfer. However, there was a difference in the magnitude of repression exerted by the NF-1 and PSF-B elements on the CS-A promoter and, with the use of mutations, this difference was shown to be consistent with variations in NF-1-binding sequences. These results indicate that PSF-B, but not PSF-A, is a member of the NF-1 family, which participates in the PSF complex and in the repression of the CS-A promoter in pituitary GC cells.

To identify extracellular proteins with epidermal growth factor (EGF) domains that are potentially involved in the control of haemopoiesis, we performed degenerate reverse-transcriptase-mediated PCR on the murine bone-marrow stromal cell line MS-5 and isolated a new partial cDNA encoding EGF-like domains related to those in the Notch proteins. Cloning and sequencing of the full-length cDNA showed that it encoded a new extracellular multi-domain protein that we named polydom. This 387kDa mosaic protein contained a signal peptide followed by a new association of eight different protein domains, including a pentraxin domain and a von Willebrand factor type A domain, ten EGF domains, and 34 complement control protein modules. The human polydom mRNA is strongly expressed in placenta, its expression in the other tissues being weak or undetectable. The particular multidomain structure of the encoded protein suggests an important biological role in cellular adhesion and/or in the immune system.

A mucin-type glycoprotein has been described in murine, rat and canine tissues as a differentiation antigen and influenza-virus receptor. We have cloned a cDNA from human placenta RNA encoding the corresponding human protein, a type-I integral membrane protein of 162 amino acids. Madin-Darby canine kidney cells transfected with the cDNA clone directed the cell-surface expression of a 36-kDa O-glycosylated sialoglycoprotein, gp36, and two minor isoforms of 28 and 70 kDa. gp36 has a broad tissue distribution with strong expression in lung, placenta and skeletal muscle, as shown by PCR screening of different cDNA libraries. Immunohistochemical detection of gp36 in cryo-sections of human placenta, kidney, lung and nasal polyps showed that the glycoprotein is expressed at the apical plasma membrane of vascular endothelial cells. Expression of gp36 was not restricted to endothelial cells, as alveolar epithelial cells were found to express gp36 as well.