CRAC channels in the human placenta: expression patterns and functional implications

Steroid hormones play a crucial role in supporting a successful pregnancy and ensuring proper fetal development. The placenta is one of the principal tissues in steroid production and metabolism, expressing a vast range of steroidogenic enzymes. Nevertheless, a comprehensive characterization of steroidogenic pathways in the human placenta and potential developmental changes occurring during gestation are poorly understood. Furthermore, the specific contribution of trophoblast cells in steroid release is largely unknown. Thus, this study aimed to (i) identify gestational age-dependent changes in the gene expression of key steroidogenic enzymes and (ii) explore the role of trophoblast cells in steroid biosynthesis and metabolism. Quantitative and Droplet Digital PCR analysis of 12 selected enzymes was carried out in the first trimester (n = 13) and term (n = 20) human placentas. Primary trophoblast cells (n = 5) isolated from human term placentas and choriocarcinoma-derived cell lines (BeWo, BeWo b30 clone, and JEG-3) were further screened for gene expression of enzymes involved in placental synthesis/metabolism of steroids. Finally, de novo steroid synthesis by primary human trophoblasts was evaluated, highlighting the functional activity of steroidogenic enzymes in these cells. Collectively, we provide insights into the expression patterns of steroidogenic enzymes as a function of gestational age and delineate the cellular origin of steroidogenesis in the human placenta.

Placental Expression of EG-VEGF and its Receptors PKR1 (Prokineticin Receptor-1) and PKR2 Throughout Mouse Gestation

Background: Small non-coding RNAs play important roles in leukemogenesis. The canonical function of small nucleolar RNAs (snoRNAs) is to facilitate 2’-O-methylation of rRNA via a target site-specific sequence and thus promote ribosomal biogenesis and function. Recently, we have shown that a subset of small non-coding RNAs, i.e. C/D box snoRNAs are overexpressed in acute myeloid leukemia (AML) and required for AML1/ETO-driven leukemia initiation and maintenance. Depletion of snoRNP protein components or even single snoRNAs can potently inhibit AML cell growth or decrease clonogenicity, and the levels of many snoRNAs positively correlate with leukemic stem cell frequency in AML patients. Over the past years, it has been shown that snoRNAs can be further processed into shorter snoRNA-derived RNA fragments (sdRNAs), but the expression patterns of sdRNAs in AML, as well as mechanistic and functional implications, remain widely elusive. Aims: In this study, we investigated sdRNA expression patterns, functions and mechanisms in AML and healthy hematopoiesis. Methods: We prospectively characterized snoRNA and sdRNA expression in 96 AML patient samples, as well as in healthy hematopoietic stem and progenitor cells and differentiated white blood cells by small RNA sequencing. mRNA sequencing was performed to investigate connections between snoRNAs, sdRNAs and the transcriptome. Further, we depleted AML cells of different snoRNA loci by CRISPR/Cas9-based knock-out and performed lentiviral rescue overexpression of the respective sdRNA or full-length snoRNA. Results: We discovered that more than 120 snoRNAs are further processed into sdRNA fragments in AML. Single snoRNAs often evolve into multiple sdRNA isoforms. Interestingly, sdRNA expression varied significantly between different AML patients. Correlation of sdRNA and full-length snoRNA expression with patient characteristics, clinical outcome and mutational status revealed numerous implications. Those include e.g. a distinct pattern of enriched sdRNAs in NPM1-mutated patients. Expression of several sdRNAs was associated with poor overall, event-free and relapse-free survival in AML. We identified sdRNAs that were overexpressed in AML as compared to HSCs and found characteristic expression patterns among differentiated white blood cells. Notably, ratios of sdRNAs and their host snoRNA were not stable across different samples, suggesting an active mechanism regulating sdRNA processing and thus their downstream pathways. Gene set enrichment analyses based on mRNA and sdRNA correlations suggested distinct cell type specific properties. Forced expression of several sdRNAs rescued the reduced clonogenic potential observed upon depletion of the respective host snoRNA locus. This effect was often identical or even more pronounced than the rescue with the respective snoRNA precursor. Of note, overexpression of distinct sdRNAs in AML cell lines enhanced clonogenic potential. Summary/Conclusion: SnoRNA-derived sdRNAs are a common feature of AML with characteristic expression patterns determined by mutation status and clinical features. Targeting certain sdRNAs or factors involved in sdRNA processing or function might constitute promising novel therapeutic leverage points in AML.

Protein kinase C (PKC) isozymes are key signal transducers involved in normal physiology and disease and have been widely implicated in cancer progression. Despite our extensive knowledge of the signaling pathways regulated by PKC isozymes and their effectors, there is essentially no information on how individual members of the PKC family regulate gene transcription. Here, we report the first PKC isozyme-specific analysis of global gene expression by microarray using RNAi depletion of diacylglycerol/phorbol ester-regulated PKCs. A thorough analysis of this microarray data revealed unique patterns of gene expression controlled by PKCα, PKCδ, and PKCε, which are remarkably different in cells growing in serum or in response to phorbol ester stimulation. PKCδ is the most relevant isoform in controlling the induction of genes by phorbol ester stimulation, whereas PKCε predominantly regulates gene expression in serum. We also established that two PKCδ-regulated genes, FOSL1 and BCL2A1, mediate the apoptotic effect of phorbol esters or the chemotherapeutic agent etoposide in prostate cancer cells. Our studies offer a unique opportunity for establishing novel transcriptional effectors for PKC isozymes and may have significant functional and therapeutic implications.

The cellular localization of GLUT3 messenger ribonucleic acid (mRNA) and protein was examined in human term placenta using a combination of methodologies. In situ hybridization indicated that GLUT3 mRNA was present in the trophoblast cell layer and in vascular endothelium with a heterogeneous distribution pattern. GLUT3 protein migrating at an apparent molecular mass of 49 kDa was detected by immunoblotting in membranes from whole placenta and endothelial cells derived from intraplacental microvessels, but not in isolated trophoblast cells. This cell-specific pattern of expression was confirmed by immunocytochemical studies showing a prominent localization of GLUT3 protein in vascular endothelium. These findings indicate a differential distribution of GLUT3 mRNA and protein in the human placenta. Based on its cell-specific distribution at the fetal interface, GLUT3 protein could be of cardinal importance in the transport of glucose from the placenta to the fetal circulation.

Pregnancy-associated plasma protein-A and -A2 (PAPPA and PAPPA2) are proteases that cleave IGF binding proteins (IGFBPs) and thereby increase the bioavailability of growth factors. PAPPA has long been recognized as a marker of fetal genetic disorders and adverse pregnancy outcomes. In contrast, although PAPPA2 is also highly expressed in human placenta, its physiological importance is not clear. To establish whether mice will be a useful model for the study of PAPPA2, we compared the patterns of expression of PAPPA2 in the placentae of mouse and human. We show, for the first time, that Pappa2 is highly expressed in mouse placenta, as is the case in humans. Specifically, it is expressed at the interface of the maternal and fetal layers of the mouse placenta at all gestational stages studied (10.5-16.5 days post coitum). Similarly, PAPPA2 is expressed in the syncytiotrophoblast layer of human placental villi and is also detected in some invasive extravillous trophoblasts in the first trimester. These results are consistent with a model whereby PAPPA2 cleaves IGFBPs produced in the maternal decidua to promote feto-placental growth, and indicate that this protein may play analogous roles in human and mouse placenta. PAPPA2 protein is detectable in the circulation of pregnant mice and humans during the first trimester and at term, raising the possibility that PAPPA2 may be a useful biomarker of placental dysfunction. Pappa2 expression also shows specific localization within the mouse embryo and therefore may play roles in fetal development, independent of its action in the placenta.

Background: Preeclampsia remains a leading cause of maternal and perinatal morbidity and mortality, yet its pathophysiology is not fully understood. Recent studies suggest that dysregulated maternal immune responses, particularly involving decidual Natural Killer (dNK) cells and immune checkpoint molecules such as B7-H3, may play a role in the pathogenesis of this heterogeneous syndrome, particularly in the development of early-onset preeclampsia (EOP). Objective: The aim of this study was to investigate the expression patterns of B7-H3 on extravillous trophoblasts (EVTs) and the abundance of dNK cells in preeclamptic versus normotensive pregnancies and to analyze the relationship between these two immune parameters. Methods: A cross-sectional study was conducted using 42 placental samples (21 preeclampsia, 21 controls). Immunohistochemistry (IHC) was performed to detect CD56 (dNK cells) and CD276 (B7-H3) expression. Expression was semi-quantitatively evaluated using the Remmele Immunoreactive Score (IRS). Statistical comparisons and correlation analyses were conducted. Results: Preeclamptic placentas exhibited significantly higher dNK cell expression (IRS 7.19 ± 2.16) and significantly lower B7-H3 expression (IRS 2.63 ± 0.90) compared to controls (p < 0.001 and p = 0.002, respectively). A positive correlation was found between B7-H3 and dNK cell expression in both groups, with a stronger correlation in normotensive pregnancies (r = 0.605; p = 0.004) and preeclampsia (r = 0.465; p = 0.034). Conclusions: The inverse expression pattern and reduction in B7-H3 expression compared to dNK cells in preeclampsia suggest a loss of immune tolerance at the maternal–fetal interface. These findings highlight the potential of B7-H3 as a biomarker and immunoregulatory target for early prediction and prevention of preeclampsia.

Retinoid Binding Proteins—Expression Patterns in the Human Placenta

Physiological oxygen tension rises dramatically in the placenta between 8 and 14 weeks of gestation. Abnormalities in this period can lead to gestational diseases, whose underlying mechanisms remain unclear. We explored the changes at mRNA level by comparing the transcriptomes of human placentas at 8–10 gestational weeks and 12–14 gestational weeks. A total of 20 samples were collected and divided equally into four groups based on sex and age. Cytotrophoblasts were isolated and sequenced using RNAseq. Key genes were identified using two different methods: DESeq2 and weighted gene co-expression network analysis (WGCNA). We also constructed a local database of known targets of hypoxia-inducible factor (HIF) subunits, alpha and beta, to investigate expression patterns likely linked with changes in oxygen. Patterns of gene enrichment in and among the four groups were analyzed based on annotations of gene ontology (GO) and KEGG pathways. We characterized the similarities and differences between the enrichment patterns revealed by the two methods and the two conditions (age and sex), as well as those associated with HIF targets. Our results provide a broad perspective of the processes that are active in cytotrophoblasts during the rise in physiological oxygen, which should benefit efforts to discover possible drug-targeted genes or pathways in the human placenta.

SWAP-70 has been demonstrated as a multiple functional signaling protein involved in formation of membrane ruffling induced by signal cascade of tyrosine kinase growth factor receptors. In the present study, the spatial and temporal expression pattern of SWAP-70 on human fetomaternal interface was investigated using specimens collected from tubal and normal pregnancies by in situ hybridization, immunohistochemistry, and Western blotting. Data showed an intense expression of SWAP-70 in trophoblasts at weeks 3-6 of fallopian implantation and at weeks 6-7 of normal pregnancy. The most intense expression was exhibited by those highly motile and invasive extravillous trophoblasts. From gestational week 8 on, the level of SWAP-70 in trophoblasts decreased significantly, and the signal was restricted in villous cytotrophoblast cells. In the in vitro cultured human trophoblast cell line, B6Tert-1, colocalization of SWAP-70 with F-actin was verified. Data in human placenta were similar to what we recently reported on rhesus monkey fetomaternal interface. Our results suggest that SWAP-70 may be involved in regulating migration and invasion of trophoblast cells during the processes of embryonic implantation and placentation in primates.

350 POSTER Chass I histone deacetylase isoforms in prostate cancer: expression patterns in vivo and functional implications in vitro

Endothelin-1, initially identified as potent vasoconstrictor secreted by vascular endothelial cells, was subsequently found to have many effects on both vascular and nonvascular tissues. We have identified from a human placenta cDNA library a clone (cDNA-2) which corresponds to a novel 5'-extended preproendothelin 1 (preproET-1) mRNA. Comparison with the known preproET-1 mRNA (cDNA-1), showed that the two molecules share the same coding sequence but differ in the 5'-untranslated region. Interestingly, cDNA-2 extends upstream of promoter regions previously shown to be essential for full preproET-1 expression. Primer extension and PCR analysis of human placenta RNA demonstrated the presence of additional transcription initiation sites located upstream of the previously identified preproET-1 CAP site. Moreover, the two mRNAs show different pattern of expression. To elucidate the mechanisms controlling the production of alternative transcripts we transfected COS-1 cells with a series of preproET-1 promoter deletion mutants. This analysis revealed that the human preproET-1 gene can be transcribed from a proximal and a distal promoter element which has hitherto been undetected. In addition, we demonstrate the presence of a region in the down-epithelial specific expression.

HLA-G is a non-classical human leukocyte antigen expressed primarily in fetal tissues at the maternal-fetal interface. This expression pattern is unique among HLA genes and suggests that HLA-G may be involved in interactions that are critical in establishing and/or maintaining pregnancy. To evaluate the role of polymorphisms at this locus in maternal-fetal interactions, 113 couples with unexplained recurrent miscarriage were genotyped for seven polymorphisms that define 12 HLA-G alleles. Logistic regression analysis was used to assess whether HLA-G genotypes were associated with an increased risk for a subsequent miscarriage. The presence of an HLA-G*0104 or HLA-G*0105N allele in either partner was significantly associated with an increased risk for miscarriage, after adjustment for maternal age, number of previous miscarriages, history of a previous liveborn, and treatment with paternal mononuclear cells. The *0104 and *0105N alleles are defined by polymorphisms in the alpha-2 domain and encode protein variants that are present only in the full-length HLA-G1 protein. The significant genotype-specific risk in this population suggests that allelic variation in the alpha-2 domain of the HLA-G1 isoforms contributes to recurrent miscarriage.

Distribution of Glutamate Transporters in the Human Placenta

Gonadotropin-releasing hormone (GnRH) is a decapeptide widely known for its role in regulating vertebrate reproduction by serving as a signal from the hypothalamus to pituitary gonadotropes. The first form of GnRH to be identified was isolated from mammals (mGnRH) and the same form has been reported for all mammals studied, which includes marsupials and placental mammals. Later, another variant, chicken GnRH-II (cGnRH-II) was shown to be expressed together with mGnRH in the brains of all jawed vertebrates, including mammals such as rats, monkeys and humans. Our objective was to characterize a third form of GnRH that was isolated previously as mRNA from guinea pigs (gpGnRH), but has not been reported for any other mammal to date. Furthermore, the gonadotropic activity of gpGnRH has not been fully characterized. Our results, using chromatographical and immunological methods, show for the first time that gpGnRH is expressed together with mGnRH in some rodents (wild guinea pig and capybara), but not in others (mouse and hamster). Also, the gonadotropic activity of gpGnRH and mGnRH was tested in two different rat cell culture systems. Although there have been reports that the salmon(s) form of GnRH is present in mammals, we did not detect sGnRH in capybara, wild guinea pigs, hamsters, rats or mice. Taken together with previous reports, the present results support the idea that the expression of multiple GnRH variants in a single species is a common pattern in most vertebrate groups.