Studies on the role of estradiol 17 B in the regulation of synthesis of proteins by human placenta

Abstract

"STUDIES ON THE ROLE OF ESTRADIOL 17? IN THE REGULATION OF SYNTHESIS OF PROTEINS BY HUMAN PLACENTA" Submitted by Mrs. Usha P. Shetty for the award of Ph.D. Degree of the Indian Institute of Science, Bangalore, India.

The primary objective of the present study is to investigate the possible role of estradiol 17? (E2) in the regulation of total protein synthesis as well as with reference to certain specific proteins in the human placenta.

A brief account of the structure and development of the placenta and the role of estrogen in various reproductive tissues is presented in Chapter I. In addition, this chapter also deals with the salient features of the placenta as an immunological barrier and with various proteins and hormones synthesized and secreted by the human placenta.

The experimental design in the present study basically consisted of incubation of placental villi prepared from either first-trimester human placenta (FTHP, collected after 8–10 weeks of gestation from cases of medical termination of pregnancy) or from term placenta collected after caesarean section. Although the use of purified cytotrophoblasts or syncytiotrophoblasts would have been ideal for this kind of study, in view of the practical problems and due to very low yield of cells with FTHP, all studies were carried out using a suspension of placental villi. Also, the fact that FTHP is metabolically much more active than term placenta, particularly with respect to protein synthesis, prompted the use of FTHP rather than term placenta, though the yield of cells would not have been a problem. Furthermore, most of the proteins synthesized by term placenta are also synthesized by FTHP.

The first series of experiments in the study consisted of examining the effect of addition or deprivation of E2 in FTHP; results of which are presented in Chapter II. It was observed that addition of E2 resulted in a significant stimulation of several proteins as judged by increased [^35S]-methionine incorporation as well as SDS-PAGE analysis followed by autofluorography. Addition of aromatase inhibitor (AI, a competitive inhibitor of aromatase, a key enzyme in estrogen biosynthesis) at a concentration of 4 ?M resulted in a significant decrease in protein synthesis as judged by SDS-PAGE analysis. However, in contrast, the use of TMX (a non-steroidal antagonist of estrogen) to block estrogen action resulted in stimulation of protein synthesis. This is believed to be due to agonistic activity of TMX, as it is known to act as either agonist, antagonist, or partial agonist depending upon the tissue and concentration used.

The use of cycloheximide, a protein synthesis inhibitor, and actinomycin-D, a transcription inhibitor, along with or without E2 showed that while cycloheximide inhibited both basal and E2-stimulated protein synthesis, actinomycin-D specifically inhibited only E2-stimulated protein synthesis. These results suggest a possible control of protein synthesis and secretion in the placenta by E2 at the transcriptional level.

Stimulation of specific proteins by E2 was also established by double-isotope labeling experiments where E2-treated samples were incubated with [^3H]-leucine and control samples with [^14C]-leucine. The increase in the [^3H]/[^14C] ratio of tissue and secretory proteins was monitored after SDS-PAGE analysis and a distinct increase in the ratio indicated stimulation of specific proteins by E2 in human placenta.

After demonstrating a role for E2 in the regulation of protein synthesis and secretion, attempts were made to study its role with respect to specific proteins as this would support the hypothesis of estrogen having a role in the regulation of protein synthesis and secretion. During the studies, it was observed that following addition of E2 there was a significant stimulation in the synthesis and secretion of a 45 kDa protein. The facts that only a few of the placental proteins are identified and characterized functionally and also very little is known about the possible factors involved in their regulation prompted the purification and characterization of this estrogen-stimulated protein. The results of this study are presented in Chapter III.

Purified 45 kDa protein is a monomeric protein with an isoelectric point of 5 ± 0.5 and a blocked N-terminus. It has a high content of acidic amino acids, but cysteine residues are completely absent. Similar protein has been identified in the placentae of rat, guinea pig, and monkey by tryptic map analysis. However, the tryptic map of the 45 kDa protein from bovine placenta (syndesmochorial) did not show any homology with the tryptic map of human or monkey placental protein. Though there was 60–70% similarity in the tryptic peptide distribution of rat and guinea pig placental protein with human and monkey protein, immunological cross-reaction was observed only with the monkey placental protein. This is consistent with similar observations made earlier with other proteins such as retinol-binding protein. The presence of a structurally similar protein in hemochorial placentae across species (human, monkey, rat, and guinea pig) suggests that this protein may have an important function during pregnancy.

The failure to raise high-titer antiserum to the protein in several animals of different species suggested a possibility that either this is a constitutive protein or it may have immunosuppressive activity. Moreover, tryptic map analysis of proteins in the region corresponding to a molecular weight of 45 kDa on SDS-PAGE after differential ammonium sulfate fractionation of either monkey or human liver extract did not reveal any similarity with the placental protein, suggesting that this protein may be pregnancy-specific. The possibility of the protein being actin because of the similarity in molecular weight was ruled out because of the dissimilar tryptic maps and also the polyclonal antiserum against actin did not show any significant cross-reaction with the purified protein.

The possibility of the 45 kDa protein having immunosuppressive activity was examined in vitro by monitoring inhibition of [^3H]-thymidine incorporation by Con-A-stimulated monkey lymphocytes in the presence of different concentrations of the protein. Incubation of lymphocytes in the presence of purified protein resulted in a dose-dependent inhibition of [^3H]-thymidine incorporation. This was also confirmed by in vivo studies, where rabbits previously injected with 45 kDa protein followed by immunization with riboflavin carrier protein (RCP), a potent immunogen, failed to elicit any RCP-specific antibodies as checked by binding of ^125I-labeled RCP. In view of the immunosuppressive activity of the 45 kDa protein, studies were also carried out to rule out the possibility of this protein being placental protein 14 (pp14), which has also been shown to have immunosuppressive activity. However, this purified protein was found to be a completely different entity as determined by tryptic map analysis and by the use of monoclonal antibodies.

To substantiate the role of E2 in the regulation of protein synthesis, it was necessary to study its role with respect to a known estrogen-modulated protein, and for this purpose RCP was chosen. RCP is known to be involved in the transplacental transfer of riboflavin, and the modulation of RCP in various tissues by estrogen is well documented. Before studying modulation of RCP in placenta, it was necessary to demonstrate its presence in placenta. The results of studies on isolation and characterization of RCP from human placenta and its modulation in placenta by various steroids are presented in Chapter IV.

RCP was purified from human placenta to homogeneity using DEAE-Sephacel and riboflavin affinity chromatography. The purified protein was found to be physicochemically and immunologically homologous to its chicken counterpart as determined by the use of various monoclonal antibodies to RCP.

The ability of human placenta to synthesize RCP was studied by employing two approaches. Firstly, by demonstration of RCP-specific message in placenta by dot-blot and Northern blot hybridization using [^32P]-labeled RCP-specific cDNA. A specific message for RCP of 1 Kb was detected by Northern blot analysis using RNA isolated from FTHP or term placenta, which correlated with the reported one. The second approach used to study de novo RCP synthesis consisted of labeling placental proteins in vitro using [^35S]-methionine and immunoprecipitation of labeled RCP using RCP-specific IgG followed by SDS-PAGE and autofluorography. SDS-PAGE analysis of the immunoprecipitate revealed a band corresponding to a molecular weight of 43 kDa. The observation of higher molecular weight species instead of 37 kDa (molecular weight of purified RCP) may represent the unprocessed form of RCP, since it is a phosphoglycoprotein.

Having demonstrated the presence of RCP message in placenta, the role of steroids, mainly that of E2, in the modulation of RCP message was studied by dot-blot and Northern blot analysis. It was observed that addition of different concentrations of E2 to the incubation medium of term placental villi stimulated RCP-specific message in a dose-dependent manner. Addition of TMX, an antiestrogen, resulted in a dose-dependent decrease in the levels of RCP message in placenta and also prevented the E2-induced increase of RCP mRNA level when added along with E2 to the incubation medium.

It is known from earlier studies that the levels of RCP in chicken liver cannot be stimulated by progesterone when injected alone or after estrogen priming. However, it was capable of stimulating RCP levels in the chicken oviduct primed with estrogen. In view of this, it was of interest to examine the effect of progesterone on RCP message in placenta, which is already exposed to high concentrations of estrogen in vivo. Interestingly, progesterone, when added to placental incubation medium, stimulated RCP message in a dose-dependent manner, and this stimulation was not inhibited by TMX. Similar results were obtained with FTHP, though much lower concentrations of steroids were adequate. Thus, the present study involving the identification of one of the proteins with functional importance from placenta and its modulation by estradiol is of considerable significance and provides additional evidence for a role of estrogen in the regulation of protein synthesis either by paracrine and/or autocrine pathway.

It is well known that estrogen regulates progesterone synthesis in the ovary by the induction of receptors for gonadotrophins, which in turn stimulate steroid biosynthesis. In placenta, progesterone synthesis is mainly dependent on the supply of maternal cholesterol in the form of low-density lipoprotein (LDL) cholesterol, which is sequestered by the placenta through specific LDL-receptors. Thus, the number of LDL-receptors in the placenta is rate-limiting in progesterone biosynthesis, and very little is known about the factors involved in the regulation of these receptors.

It is pertinent to note that regulation of ^125I-labeled LDL uptake in placenta by E2 during pregnancy in baboons and in human trophoblast cultures has been demonstrated. Therefore, it was of interest to study the role of estrogen in the regulation of LDL-receptor message levels in human placenta, and the results of these studies are presented in Chapter V. Dot-blot and Northern blot analysis of RNA isolated from FTHP and term placenta using 1.7 Kb LDL-R cDNA probe revealed the presence of 5.3 Kb message. The level of the message was higher in FTHP compared to term placenta.

It was also observed that the expression of LDL-receptor message in human placenta is under negative control of E2. This conclusion was supported by the results obtained with the use of TMX and AI, which resulted in an increase in the level of message. Interestingly, addition of progesterone to term placental minces resulted in a significant increase in the level of LDL-R message. As it is known that the level of LDL-R is induced by hCG in human granulosa cells and luteal cells, the effect of addition of hCG to term placental minces on LDL-R message was examined, and a significant increase was observed. Further studies also revealed that the expression of LDL-R in term placenta appears to be under inhibitory control of a cycloheximide-sensitive factor. While it is evident that more work needs to be done, these preliminary results indicate that placental steroids do have a role in the regulation of the expression of LDL-R message.

In the last chapter, the significance of the results obtained in the present study is discussed.