Cytological distribution of chorionic gonadotropin subunit and placental lactogen messenger RNA in neoplasms derived from human placenta

Normal trophoblast of the human placenta elaborates at least two major protein hormones, chorionic gonadotropin (hCG), and placental lactogen (hPL). There are several gestational trophoblastic diseases of the placenta called hydatidiform mole, invasive mole, and choriocarcinoma. Molar and choriocarcinoma tissues characteristically synthesize large amounts of hCG and small quantities of hPL. To examine the role of trophoblast differentiation in the expression of the hCG and hPL genes, we studied the cytological distribution of their messenger RNA (mRNA) in tissue sections of human hydatidiform mole and choriocarcinoma by in situ hybridization. Histologically, these tissues are in different stages of cellular differentiation. In normal placenta, hCG alpha and - beta mRNA can be localized to some cytotrophoblasts and primarily to the syncytium, whereas hPL mRNA appears only in the syncytial layer. In hydatidiform mole, which still retains placental villous morphology, the hPL gene and hCG alpha and -beta genes are expressed but are poorly localized because of the admixture of cyto- and syncytiotrophoblasts. By contrast, choriocarcinoma, which is devoid of placental villous pattern but in which the cyto- and syncytiotrophoblast-like components are distinguishable, expresses hCG alpha and -beta in the syncytial- like areas but little, if any, hPL. These results suggest that a certain level of trophoblast differentiation, such as villous formation, is associated with hPL expression, while the hCG alpha gene and the hCG beta gene can be expressed in more disorganized tissues that contain cytotrophoblastic elements.     

Effect of morphine on hCG release by first trimester human trophoblast in vitro

Opiate synthesis by human placental cells and the presence of kappa-type opiate binding sites in the syncytiotrophoblast brush border membrane may indicate the possible role of morphine-like substances in the autocrine regulation of trophoblast cell metabolism. This study was undertaken to examine the in vitro effect of morphine on hCG (human chorionic gonadotrophin) and hPL (human placental lactogen) release by 1st and 3rd trimester placental tissue explants. The results have shown that morphine (100 nM) significantly stimulated hCG secretion by 6-8 weeks old trophoblast and was without effect on hPL. Hormone secretion by term placental tissue explants was unaffected by morphine treatment. Based on these results we assume that opiates may have a role in the local (autocrine and/or paracrine) regulation of hCG secretion in early gestation.     

Modification of human placental lactogen with plasmin. Preparation and characterization of a modified hormone with increased biologic activity.

To determine the structure needed for the biologic activity of human placental lactogen (hPL), we have cleaved hPL with the proteolytic enzyme plasmin. Plasmin modified hPL (PL-hPL) was purified by gel chromatography. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis before and after reduction showed that cleavage had occurred within the Cys53-Cys165 loop and tryptic peptide maps revealed that a single peptide consisting of residues 135 to 140 had been removed. 5-Dimethylaminonaphthalene-1-sulfonyl end group analysis and digestion with carboxypeptidase B confirmed that cleavage was complete and only the single hexapeptide was removed. In a membrane binding assay for lactogenic activity PL-hPL was 2- to 3-fold more potent than hPL. Using growth hormone receptors from rabbit liver membranes, PL-hPL was also more potent than hPL, but still much less potent than growth hormone. The lactogenic activity of PL-hPL in an in vitro bioassay was 75% above that of unmodified hormone. It is concluded that plasmin cleaves homologous peptides from hPL and growth hormone and that removal of the hexapeptide from hPL results in enhanced biologic activity.     

Preservation of human chorionic gonadotropin and placental lactogen secretion and normal morphology following long-term culture of normal human placental cells

In order to study the regulation of hCG and hPL secretion during gestation, a system for the preservation of the functional integrity of normal placental cells in long-term culture was established. Normal term placental cells were dispersed with 0.25% trypsin-500 units DNAse I and cultured in a monolayer in Dulbecco's modified Eagle medium with 10% fetal bovine serum. Normal cell morphology, basal hCG and hPL production and hCG responses to dibutyryl cAMP were preserved till 54 days of culture. This model may be useful for the study of long-term regulation of normal placental hCG and hPL synthesis and secretion.     

Transcriptional products of the human placental lactogen gene

Poly(A+)RNA from human term placenta was translated in a mouse-derived cell-free system. A major band corresponding to preplacental lactogen (pre-hPL) and a minor band co-migrating with mature hPL represent approximately 15% of the total radioactively labeled proteins. Analysis of the poly(A+)RNA by agarose gel electrophoresis showed a prominent band at approximately 860 nucleotides. A corresponding band was observed in Northern blots of total RNA, hybridized with 32P-labeled recombinant plasmid containing a portion of hPL cDNA. Similar analyses of nuclear RNA showed at least four additional bands at 990, 1200, 1460, and 1760 nucleotides, respectively, which are likely precursors of hPL mRNA. Poly(A+)RNA was also used to construct a cDNA library. Approximately 5% of the clones were found to hybridize to hPL DNA sequences, indicating that hPL mRNA is indeed very abundant in term placental tissue. One recombinant plasmid containing an insert of approximately 815 base pairs was isolated and characterized by restriction enzyme mapping and electron microscopy. Heteroduplexes constructed between the cDNA and the DNA isolated from an hPL genomic clone revealed four small intervening sequences which can account for the lengths observed for the hnRNA molecules.     

Biochemical evidence that human placental lactogen and human chorionic gonadotropin are not stored in cytoplasmic secretion granules

The intracellular storage sites for the human placental hormones placental lactogen (hPL) and chorionic gonadotropin (hCG) are unknown. To determine whether hPL and hCG are stored in cytoplasmic secretion granules, we have compared the localization of hPL and hCG in placental homogenates following differential and density-gradient centrifugations to those of prolactin (PRL) and luteinizing hormone (LH) in human and rat pituitary homogenates. In the differential centrifugation studies, 93.1 +/- 4.1% (mean +/- SE) of the hPL and 79.4 +/- 6.0% of the hCG were detected in the postmicrosomal supernatant of placental homogenates. In contrast, 95-98% of the hPRL and hLH in the pituitary homogenates were detected in particulate fractions. Following centrifugation on sucrose-density gradients, particulate hPL and hCG were distributed diffusely throughout the gradients, while greater than 90% of the pituitary hormones sedimented as single peaks with densities of 1.22 g/cm3. When human placental and rat pituitary tissues were homogenized together prior to differential and density-gradient centrifugations, similar marked differences were observed between the distribution of the placental and pituitary hormones. These results strongly suggest that the placental hormones hPL and hCG, unlike pituitary PRL and LH, are not stored in large secretory granules. Differences in the intracellular storage sites of the hormones may explain, in part, differences in the regulation of peptide hormone secretion by placental and pituitary tissues.     

Properties of biologically active messenger RNA from human placenta. Cell-free synthesis of two immunoreactive forms of placental lactogen.

In order to understand better the regulation of human placental proteins the activity of placental lactogen messenger RNA has been examined. Total RNA was extracted from normal term placentas and purified by chromatography on oligo(dT)-cellulose. The poly(A)-containing fraction stimulated amino acid incorporation 5- to 10-fold in wheat germ cell-free extracts, and immunoprecipitation of the translation products with antiserum directed against human placental lactogen (hPL) suggests that about 2% of the peptides contain hPL determinants. Analysis of the material precipitated with hPL antiserum by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels revealed two major species, one co-migrating with hPL and the other migrating slightly slower than hPL. On DEAE-cellulose chromatography the former material eluted close to authentic hPL while the latter material eluted at higher ionic strength than hPL, indicating a difference in net charge of these two species. Tryptic peptide analysis of the large material and authentic hPL shows marked similarities in the primary structure of these two proteins. The slower migrating peptide has an apparent molecular weight about 3000 larger than hPL and thus may represent a precursor molecule. Both cell-free products could be competed out of immunoprecipitates by a large excess of authentic hPL, confirming their immunologic similarities. Centrifugation of the placental poly(A)-containing RNA through aqueous glycerol gradients indicates that the hPL mRNA sediments at about 14 S.     

Synthesis of human placental lactogen messenger RNA as a function of gestation.

It was shown previously that 4 to 5 times more human placental lactogen (hPL) was synthesized in cell-free extracts from term placentae than in comparable extracts prepared from first trimester tissue. In an attempt to define what accounts for this differential rate of synthesis RNA was prepared from first trimester and term placentae. Following purification through an oligo(dT)-cellulose column, these RNA preparations were tested for their ability to direct the synthesis of the hPL precursor in the wheat germ cell-free system. With similar amounts of first trimester and term mRNA, the overall efficiency as defined by the stimulation of total amino acid incorporation was comparable. However, there was approximately 4 times more hPL synthesized in the presence of term RNA. This was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tryptic fingerprinting. The peak of the hPL precursor messenger activity sedimented at 12 to 13 S on sucrose gradient. Analysis of the RNA by formamide-polyacrylamide gel electrophoresis further supported this value. The data indicate that the increased synthesis of hPL at term reflects greater levels of hPL mRNA in term tissue than in first trimester tissue. The data also show that the overall in vivo levels of hPL can be correlated not only with the increase in placental syncytial mass during pregnancy but also in the greater proportion of hPL synthesized per g of tissue. The latter results from the continual differentiation of the placenta occurring throughout gestation.     

The effects of insulin-like growth factor-I and insulin on placental lactogen production by human term placental explants

The effects of insulin-like growth factor (IGF)-I and insulin on placental lactogen production (hPL) by term human placental explants were studied. The hPL content in medium and explant decreased rapidly after first 24 hours of culture. The decrease thereafter was gradual and reached a plateau by day 4 of culture. The decrease of HPL content in placental culture has previously been suggested being due to the depletion of a rapidly secreting preformed pool of hPL. Addition of IGF-I (0.1-10 micrograms/ml) and insulin (1-20 micrograms/ml) stimulated the decreased level of hPL in tissue and medium after 24 hours in culture. IGF-I was 10 times more potent than insulin in stimulating hPL. These findings suggest that IGF-I and insulin effects the production of hPL by placenta. The lower potency of insulin may indicate that the effect of insulin on hPL production is via IGF-I receptor.     

Mutational analysis and protein engineering of receptor-binding determinants in human placental lactogen

Human placental lactogen (hPL) shares 85% sequence identity to human growth hormone (hGH) yet has some very different receptor-binding properties. For example, hPL binds 2300-fold weaker than hGH to the hGH receptor, yet these two hormones have similar affinities for prolactin receptors. We have expressed hPL in Escherichia coli, and we show that, like hGH, hPL requires zinc for tight binding to the extracellular domain of the human prolactin receptor (hPRLbp). In fact, hPL contains virtually the same receptor-binding determinants and zinc ligands (His-18, His-21, and Glu-174) that hGH uses for coordinating zinc in the hGH.hPRLbp complex. As with hGH, mutation of Glu-174 to Ala in hPL reduces the affinity for the hPRLbp by 1400-fold. We can increase the affinity of hPL by over 200-fold for the hGHbp by installing four hGH receptor determinants that are not conserved in hPL. By simultaneously introducing E174A, we produced a pentamutant whose binding affinity for the hGHbp is only 1.6-fold weaker than hGH, but whose binding affinity for the hPRLbp is weaker by greater than 1000-fold relative to wild-type hPL. Thus, we have identified an hPRLbp epitope in hPL, "recruited" an hGHbp epitope into hPL, and produced receptor selective analogs of hPL that are designed to bind tightly to either, neither, or both receptors. Such variants should be important molecular probes to link specific receptor-binding, activation, and biological events.     

Adenosine 3':5'-cyclic monophosphate (cAMP) is not the mediator of kappa opiate effect on human placental lactogen release.

We previously reported that kappa opiates stimulated the release of human placental lactogen (hPL) from human placental cells. In this study, we investigated the role of adenylate cyclase as a potential cellular mediator of such an effect. Incubations with ethylketocyclazocine (EKC) led to a time- and dose-dependent inhibition of adenylate cyclase activity. The maximal inhibition was 45 +/- 5% of control value after 15 min exposure to 10(-7)M EKC. This inhibition was reversed by opiate antagonist naloxone and was specific to kappa opiate type. Preincubation of human trophoblastic cells with 0.1 microgram/ml Islet-Activating-Protein (IAP; also called pertussis toxin) did not modify basal adenylate cyclase activity but abolished the inhibition of adenylate cyclase activity by EKC, indicating that the effect of opiates on cAMP production was mediated by an IAP-sensitive GTP binding protein. Also, IAP stimulated basal hPL release; the control levels were 22.4 ng/ml and 46.5 ng/ml without and with IAP respectively. However, the EKC-stimulated hPL levels were unchanged by preincubation with IAP. This difference in cAMP and hPL response in IAP-treated cells suggested that the opiate receptors are not directly coupled to adenylate cyclase. This hypothesis was confirmed by 1) experiments on placental membranes showing that in absence of the cytoplasmic elements (membranes only), EKC had no effect on membrane adenylate cyclase and 2) experiments on placental cells showing that dibutyryl-cAMP (dbcAMP) stimulated hPL release.     

Placental Lactogen Is Expressed but Is Not Translated into Protein in Breast Cancer

Introduction

Several studies reported that the pregnancy-specific hormone placental lactogen (hPL) is expressed at both mRNA and protein levels in breast cancer. The overall objective was to establish hPL, the product of the CSH1 and CSH2 genes, as a biomarker for breast cancer.

Methods

CSH expression was determined at the mRNA level in breast cancer cell lines (BCC) and primary carcinomas by real-time and conventional PCR and the products verified as CSH1 by sequencing. Expression of hPL protein was examined by western blots and immuno-histochemistry, using commercial and custom-made polyclonal and monoclonal antibodies.

Results

Variable levels of CSH mRNA were detected in several BCC, and in some primary tumors. We detected a protein, slightly larger than recombinant hPL by western blotting using several antibodies, leading us to postulate that it represents an hPL variant (‘hPL’). Furthermore, some monoclonal antibodies detected ‘hPL’ by immunohistochemistry in breast carcinomas but not in normal breast. However, further examination revealed that these antibodies were non-specific, as efficient suppression of CSH mRNA by shRNA did not abolish the ‘hPL’ band. Custom-made monoclonal antibodies against recombinant hPL detected hPL of the correct size in placental lysate and hPL-overexpressing BCC, but not in unmodified cells or primary carcinomas. hPL protein was detected only when mRNA was increased several thousand fold.

Conclusions

We call into question previous reports of hPL expression in breast cancer which relied on mRNA levels as surrogates for protein and/or used improperly validated antibodies to measure hPL protein levels. Our data suggests that an inhibitory mechanism(s) prevents translation of CSH mRNA in breast cancer when not highly expressed. The mechanism by which translation of CSH mRNA is inhibited is intriguing and should be further investigated.     

Synthetic amphipathic peptides resembling apolipoproteins stimulate the release of human placental lactogen

Previous studies from our laboratory demonstrated native high density lipoproteins and apolipoproteins AI, AII, and CI, stimulate the release of human placental lactogen (hPL) from trophoblast cells in culture. To examine the mechanisms by which these apolipoproteins stimulate hPL release, we have studied hPL secretion in response to several synthetic peptide analogs of the amphipathic helical structure of the apolipoproteins. The magnitude of the stimulation of hPL release in response to the analog peptides correlated with the ability to displace apolipoproteins from high density lipoprotein and with other measures of phospholipid binding affinity such as the increase in alpha-helicity and the size of complexes formed between the peptide and phospholipid. The correlation of stimulatory ability and lipid affinity suggests that the action of the apolipoproteins on hPL release may be mediated through an interaction with plasma membrane phospholipids.     

Crystal structure and site 1 binding energetics of human placental lactogen

In primates, placental lactogen (PL) is a pituitary hormone with fundamental roles during pregnancy involving fetal growth, metabolism, and stimulating lactation in the mother. Human placental lactogen (hPL) is highly conserved with human growth hormone (hGH) and both hormones bind to the hPRLR extracellular domain (ECD), the first step in receptor homodimerization, in a Zn2+-dependent manner. A modified surface plasmon resonance method was developed to measure the kinetics for hPL and hGH binding to the hPRLR ECD, with and without Zn2+ and showed that hPL has about a tenfold higher affinity for the hPRLR ECD1 than hGH. The crystal structure of the free state of hPL has been determined to 2.0 A resolution showing the molecule possesses an overall structure similar to other long chain four-helix bundle cytokines. Comparison of the free hPL structure with the 1:1 complex structure of hGH bound to the hPRLR ECD1 suggests that two surface loops undergo conformational changes >10 A upon binding. An 18 residue Ala-scan was used to characterize the binding energy epitope for the site 1 interface of hPL. Individual alanine substitutions at five positions reduced binding affinity by a DeltaDeltaG > or = 3 kcal mol(-1). A comparison of the hPL site 1 epitope with that previously determined for hGH indicates contributions of individual residues track reasonably well between hPL and hGH. In particular, residues involved in the zinc-binding site and Lys172 constitute the principal binding determinants for both hormones. However, several residues that are identical between hPL and hGH contribute quite differently to the binding of the hPRLR ECD1. Additionally, the overall magnitudes of the DeltaDeltaG changes observed from the Ala-scan of hPL were markedly larger than those determined in the comparative scan of hGH to the hPRLR ECD1. The structural and biophysical data presented here show that subtle changes in the structural context of an interaction can lead to significantly different effects at the individual residue level.