Steroids modulate corticotropin-releasing hormone production in human fetal membranes and placenta

The levels of immunoreactive CRH are elevated in both maternal and fetal plasma in late gestation and labor, but fall precipitously after parturition. The major source of this peptide is thought to be the placenta. We determined if the placenta and also the amnion, chorion, and decidua produce CRH, whether this material has biological activity, and whether CRH output is modulated by glucocorticoids and progesterone. In an in vitro monolayer culture system CRH was produced by the fetal membranes and decidua. Media immunoreactive CRH concentrations averaged 625 +/- 45 (SE) pg/10(5) cells in amnion, 701 +/- 56 pg/10(5) cells in chorion, and 580 +/- 60 pg/10(5) cells in decidual tissue obtained at cesarean section. This output was similar to that by the placenta (906 +/- 121 pg/10(5) cells). These values increased in tissue obtained after spontaneous labor. A single peak of CRH immunoreactivity eluting at the same position as synthetic human CRH, and possessing biological activity, was found in all tissues. There was a dose-dependent increase in CRH output by all tissue types when cells were maintained in the presence of increasing concentrations of cortisol and dexamethasone. In contrast, increasing concentrations of progesterone decreased CRH output by all tissue types. We conclude that immuno- and biologically active CRH is produced not only in the human placenta, but also in the fetal membranes. CRH output by the placenta/fetal membranes is moderated by steroids, and changes with labor. These findings raise the possibility of a regulatory system similar to that of the hypothalamic pituitary axis, but residing within the placenta and fetal membranes.     

Human follistatin-related protein: a structural homologue of follistatin with nuclear localization.

Follistatin-related protein is a recently discovered glycoprotein that is highly homologous in both primary sequence and exon/intron domain structure to the activin-binding protein, follistatin. We explored their potential for functional redundancy by investigating the relative affinities and kinetics of their interactions with activin, bone morphogenic protein-6, and bone morphogenic protein-7 and by exploring their expression and distribution in human tissues and cells. Follistatin and follistatin-related protein mRNA were ubiquitous by Northern analyses, although their sites of peak distribution differed, with follistatin-related protein and follistatin predominating in the placenta and ovary, respectively. Follistatin-related protein, like follistatin, preferentially bound activin with high affinity and in an essentially irreversible fashion. Although follistatin-related protein, like follistatin, possesses a signal sequence and no known nuclear localization signals, its secretion was undetectable in most cell lines by RIA. Intriguingly, follistatin-related protein was identified as a nuclear protein in human granulosa cells and all human cell lines tested. Furthermore, Western analyses of CHO cells transfected with human follistatin-related protein revealed this protein to reside within the insoluble nuclear protein fraction. We conclude that despite its remarkably high level of similarity to follistatin with regard to structure and activin binding kinetics, follistatin-related protein is a nuclear as well as a secretory protein that may perform distinct intracellular actions.     

Human pancreatic adenocarcinomas express parathyroid hormone-related protein

PTH-related protein (PTHrP) is expressed in many common malignancies such as breast and prostate cancer and can regulate their growth. Little is known, however, about the role of PTHrP in pancreatic adenocarcinoma. To study PTHrP in pancreatic exocrine cancer, we studied its expression in pancreatic cancer cell lines and surgical specimens. Eight human pancreatic adenocarcinoma cell lines were evaluated: AsPC-1, BxPC-3, Capan-1, CFPAC-1, MIA PaCa-2, PANC-1, PANC-28, and PANC-48. Murine monoclonal antibodies to the amino-terminal (1-34), mid-region (38-64), and carboxyl-terminal peptides (109-141) of PTHrP were used to identify cellular PTHrP and secreted PTHrP, including Western blotting and immunocytochemical staining for PTHrP from each cell line. Cellular PTHrP was detected in all cell line extracts by both Western blotting and immunoassay. CFPAC-1, derived from a pancreatic liver metastasis, had the highest concentration of PTHrP, and MIA PaCa-2, derived from primary pancreatic adenocarcinoma, had the lowest. PTHrP was localized by immunocytochemical staining in the cytoplasm in all but one cell line, and both nuclear and cytoplasmic immunostaining were observed in the MIA PaCa-2 and PANC-1 cells. Secretion of PTHrP into cell medium was also observed for each cell line and paralleled intracellular PTHrP levels. Evidence for differential processing of PTHrP expression was provided by studies demonstrating different patterns of PTHrP among the cell lines when assessed by PTHrP immunoassays directed against different PTHrP peptides. In specific, PTHrP secretion measured by a PTHrP-(38-64) assay was highest for BxPC-3, whereas the highest levels of secreted PTHrP-(109-141) occurred in CFPAC-1 and PANC-1. Growth of AsPC-1 cells was stimulated in a dose-dependent manner by PTHrP-(1-34). Immunostaining from archival tissue of patients with pancreatic adenocarcinoma revealed strong PTHrP expression in all 14 specimens. All patients were eucalcemic preoperatively. These results demonstrate that PTHrP is commonly expressed in pancreatic cancer. Our data suggest that PTHrP may have growth-regulating properties in pancreatic adenocarcinoma cells, but further studies are required.     

Oocytes of baboon fetal primordial ovarian follicles express estrogen receptor beta mRNA

In fetal ovaries of estrogen-suppressed baboons, we have previously shown that follicle numbers were 50% lower than in estrogen-replete animals and contained oocytes with a reduced number of microvilli. In the baboon fetal ovary, although estrogen receptor (ER)α and β have been detected by immunocytochemistry in granulosa cells, it is not known whether oocytes express ER. Because the actions of estrogen are mediated by interaction with cell-specific receptors, the current study determined whether ERα/β mRNA were expressed in oocytes of baboon fetal ovaries obtained on day 165 (term = day 184) of gestation. Oocyte nuclei and cytoplasm from primordial follicles were isolated by laser capture microdissection and ERα, ERβ, GATA-4 (granulosa cell specific marker) mRNAs, and 18S rRNA determined by RT-PCR and products verified by sequencing. ERβ mRNA was expressed in oocytes of 5 of 5 fetuses. In contrast, fetal oocytes did not express ERα mRNA. Although 18S rRNA was expressed in all oocytes, GATA-4 mRNA was not detected in oocytes and only detected in granulosa cells confirming purity of oocytes sampled. We conclude that oocytes of the fetal baboon ovary express ERβ mRNA, thereby providing a mechanism by which estrogen regulates oocyte function, e.g. microvillus development.     

Identification of proliferin mRNA and protein in mouse placenta.

Proliferin is a recently described, prolactin-related protein whose mRNA appears in several murine cell lines during active growth. We have surveyed a number of mouse organs or tissues for the presence of mRNAs that hybridize to cloned proliferin cDNA. Of the tissues tested, only the placenta yielded proliferin-related mRNA. This placental RNA is about 1 kilobase in length, increases sharply between days 8 and 10 of pregnancy, and then gradually declines through day 18. It is more abundant in RNA extracted from the fetal, compared to the maternal, part of the placenta. From a cDNA plasmid library prepared from poly(A)+ placental RNA, two types of proliferin-related clones were isolated, differing in intensity of hybridization to proliferin cDNA. By nucleotide sequence analysis, a strongly hybridizing clone was found to be nearly identical to the proliferin cDNA clone isolated from a library prepared from mRNA of a growing mouse fibroblastic cell line. Using an antiserum prepared against a synthetic proliferin fusion protein, we show that proliferin is secreted as a glycoprotein by minced placental tissue and that it differs from mouse placental lactogen. We conclude that proliferin is a placental hormone that is synthesized in certain mouse cell lines during active growth. Its function during pregnancy and during the growth of cultured cells is presently unknown.     

Leukotriene release by human fetal membranes, placenta and decidua in relation to parturition

Complete placentas and membranes were obtained from women after uncomplicated singleton pregnancies. Six were collected after labour at term, six after preterm labour and six after elective Caesarean section at term. Leukotriene C4 (LTC4), leukotriene D4 (LTD4) and leukotriene E4 (LTE4) release was examined using a short-term incubation technique. Release by amnion was significantly higher than that by the other tissues for the three modes of delivery. Comparison of LTC4, LTD4 and LTE4 release by individual tissues with regard to the type of delivery showed no significant difference.     

Expression of extracellular matrix metalloproteinase inducer in human placenta and fetal membranes at term labor

Matrix metalloproteinases (MMPs) are the main mediators of extracellular matrix (ECM) degradation during human parturition. However, the mechanisms involved in regulation of MMP production during parturition remain poorly understood. Recently, an extracellular matrix metalloproteinase inducer (EMMPRIN) has been shown to play a key role, as a local regulator, in stimulating MMP production in cancer systems. Whether EMMPRIN is expressed and stimulates MMP production in human placenta and fetal membranes is presently unknown. In this study, we investigated the expression of EMMPRIN at the levels of mRNA and protein in human term placenta and fetal membranes with or without labor. Western blot analysis showed that EMMPRIN protein was detected in term placenta and fetal membranes at two molecular masses of 40 and 65 kDa (glycosylated protein) and one of approximately 30 kDa (nonglycosylated protein). The ratio of 65 kDa EMMPRIN to total EMMPRIN significantly increased (P < 0.05) in term labor chorio-decidua and amnion compared with nonlabor chorio-decidua and amnion. Immunohistochemical analysis revealed that EMMPRIN was expressed in placental syncytiotrophoblast, amniotic epithelial cells, trophoblast cells of chorion laeve, and decidua parietalis. EMMPRIN was also detected at the mRNA level using RT-PCR in cultured placental syncytiotrophoblast, amniotic epithelial cells, and chorionic trophoblast cells. We conclude that human placenta and fetal membranes express EMMPRIN, with the potential to stimulate MMP production, thereby facilitating fetal membrane rupture and leading to detachment of the placenta and fetal membranes from the maternal uterus at the time of parturition.     

Expression of membrane prostaglandin E synthase in human placenta and fetal membranes and effect of labor

Initiation and maintenance of labor in humans is associated with an increase in prostaglandin synthesis by intrauterine tissues. The objective of the present study was to characterize the distribution of membrane-bound PGES (mPGES) protein and mPGES mRNA in human placenta, fetal membranes, and decidua at term and to determine whether any changes occurred with labor. Immunoreactive mPGES was found to be highly concentrated in amnion epithelial cells and the chorion laeve trophoblasts, with lower levels in the mesenchymal layers. The enzyme was at very low levels or undetectable in the decidual tissue. Much lower levels of mPGES protein and mRNA were found in placenta than in fetal membranes. mPGES was associated with the syncytiotrophoblast and in cells surrounding blood vessels. The expression of mPGES mRNA did not change with labor in full membranes or placenta, but Western analysis showed an increase in mPGES protein in chorion laeve and a decrease in mPGES protein in placenta during labor, with no change in the amnion. The differences in expression found among placenta, chorion, and amnion before and after labor would indicate that this enzyme is differentially regulated in these tissues at this time.     

Characterization of a novel prolactin-related protein from bovine fetal placenta.

The bovine fetal placenta transcribes a family of PRL-related genes that are distinct from the bovine placental lactogen gene. To demonstrate that one of these cDNAs, bovine PRL-related cDNA-I (bPRCI), is expressed at the protein level, a recombinant form of the bPRCI product was overexpressed in Escherichia coli. An antiserum raised against this recombinant product did not cross-react with the pituitary members of the PRL-GH family or with bovine placental lactogen, although cross-reactivity within the bPRC subfamily cannot be ruled out. The antiserum detected a doublet with apparent mol wt of 34,000 and 35,000 in bovine fetal placenta, but not in other fetal tissues. Treatment with several glycosidases revealed a glycoprotein with asparagine-linked carbohydrates of the biantennary complex or hybrid type. We conclude that the bovine fetal placenta expresses at least one of the novel members of this gene family at the protein level.     

Human platelets express hemochromatosis protein (HFE) and transferrin receptor 2

OBJECTIVES: While body iron status may influence platelets, little information is available about platelet expression of proteins regulating iron homeostasis. HFE, the protein defective in hereditary hemochromatosis, and transferrin receptor 2 (TfR2) are two novel protein candidates that could be involved in mechanisms of iron transport across the platelet plasma membrane. METHODS: The expression and localization of HFE, TfR1 and TfR2 proteins in human platelets were examined using Western blotting and immunocytochemistry. RESULTS: Human platelets expressed HFE and TfR2, whereas no signal for TfR1 was found. The positive reactions for HFE and TfR2 were mainly confined to the platelet plasma membrane. CONCLUSIONS: Expression of HFE and TfR2 proteins in human platelets may indicate that the mutations in the corresponding genes could influence platelet count, size and/or activation. The presence of TfR2 and absence of TfR1 suggests that HFE may serve a different function in platelets compared with the other HFE-positive cell types, e.g. enterocytes, macrophages and syncytiotrophoblasts.     

Human platelets express heat shock protein receptors and regulate dendritic cell maturation

Immunizations using the endoplasmic reticulum-resident heat shock protein Gp96 induce specific immune responses. Specificity is based on the major histocompatibility complex class I-restricted cross-presentation of Gp96-associated peptides derived from endogenous proteins. Initiation of the immune response depends on the ability of Gp96 to induce the production of proinflammatory cytokines by macrophages and dendritic cells (DCs) and of their maturation in a fashion presumably independent of associated peptide. Both events are mediated by Gp96 receptors on antigen-presenting cells. It is known that Gp96 is released from cells at necrosis induced, for example, by virus infection. Although this event supports the efficient induction of immune responses, it might also interfere with processes that are susceptible to chronic inflammation, such as wound healing after tissue damage. Therefore, Gp96-mediated stimulation of the immune system requires tight regulation. Here we show that human thrombocytes specifically interact with Gp96 and that binding of Gp96 to platelets is enhanced more than 10-fold on activation by thrombin. Gp96 interferes with neither thrombin-induced platelet activation nor platelet aggregation. However, the presence of platelets during Gp96-mediated DC activation reduces the secretion of proinflammatory cytokines and the activation of DCs. This effect is independent of soluble platelet factors and cell-to-cell contact between DCs and thrombocytes. Thus, we provide evidence for a regulatory mechanism that neutralizes Gp96 molecules systemically, especially in the blood. This effect might be of significance in wounds in which chronic inflammation and immune responses against autoantigens have to be prevented.     

Genomic organization and promoter analysis of mouse follistatin-related gene (FLRG)

Follistatin (FS) is well characterized as an activin-binding protein. Recently, a novel follistatin-like protein called follistatin-related gene (FLRG) that has a similar domain organization to that of follistatin has been identified. Like follistatins, FLRG binds activins and bone morphogenetic proteins (BMPs). To study the regulation of FLRG expression, we have analyzed the genomic organization and promoter of the mouse FLRG gene. The mouse FLRG gene consists of five exons, and each encodes discrete functional regions. The overall genomic structure of FLRG is similar to that of FS except that the FLRG gene is missing one exon that codes a third FS domain found in FS. The promoter that covers 2.5 kbp and is linked to a luciferase reporter construct is active in human cervical carcinoma HeLa cells as well as in human embryonic kidney (HEK293) cells. Deletion analysis of the promoter regions indicates that a proximal 550 base pairs are enough for basal FLRG promoter activity in the cell lines. FLRG promoter activity is significantly augmented by phorbol 12-myristate 13-acetate (PMA) treatment, but not by cAMP stimulation. By contrast, FS promoter is activatable either by cAMP or PMA. Thus, although FS and FLRG are structurally and functionally related, their modes of regulation by external stimuli are different.     

Human CD34+ cells differentiate into microglia and express recombinant therapeutic protein

In rodents, bone marrow-derived cells enter the brain during adult life. Allogeneic bone marrow transplantation is used to treat genetic CNS diseases, but the fate of human bone marrow and CD34⁺ cells within the brain remains to be elucidated. The present study demonstrates that cells derived from human CD34⁺ cells, isolated from either cord blood or peripheral blood, migrate into the brain after infusion into nonobese diabetic/severe combined immunodeficient mice. Both types of CD34⁺-derived cells differentiate into perivascular and ramified microglia. The lentiviral transfer of genes into CD34⁺ cells before infusion does not modify the differentiation of human CD34⁺ cells into microglia, allowing new transgenic proteins to be expressed in these cells. The transplantation of CD34⁺ cells could thus be used for the treatment of CNS diseases.