Category Archives: Human Placenta

Expression of the Erythropoietin Receptor: DISCUSSION(7)

In this regard, the placentas associated with EPO-/- and EPO-R-/- knockout mice were reported to be of normal size at gestational Days 13-15 when the embryos died in utero apparently of severe anemia. However, it was not reported whether the morphology and distribution of the various populations of trophoblast cells were normal. An autocrine role for EPO, whereby the hormone contributes to the survival, proliferation, and differentiation of tro-phoblast cells, may be analogous to its role in early colony-forming [8] and erythroleukemic cells, as well as human hepatocellular carcinoma cells.

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Expression of the Erythropoietin Receptor: DISCUSSION(6)

DISCUSSION(6)

In the present investigation, we provide evidence that the EPO-R on trophoblast cells and trophoblast-derived Jar cells is functional, insofar as it is phosphorylated on tyrosine residues in response to exogenous EPO (Fig. 11). Interestingly, some basal tyrosine phosphorylation was also evident, perhaps reflecting the autocrine action of endogenously produced EPO, since we have previously reported that Jar trophoblast-derived choriocarcinoma cells secrete EPO.

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Expression of the Erythropoietin Receptor: DISCUSSION(5)

The elucidation of potential binding sites and transport of EPO across the placental barrier has clinical significance in terms of assessing safety to the fetus with rhEPO administration to anemic pregnant women (refer to ). In addition, measurement of circulating EPO may serve as a marker of fetal distress since increased nucleated red blood cell counts can be measured in the neonate from complicated pregnancies. Previous studies have resulted in contrasting results with no demonstrable binding of radiolabeled EPO by the human placenta and no transport between the maternal and fetal compartments in a model of perfused cotyledons. Interestingly, in the latter study the authors measured a 50% loss of EPO from the perfusate after 5 h, suggesting that the administered rhEPO was being bound without placental transfer. Our laboratory has also attempted to identify specific EPO binding sites on isolated trophoblast cells using 125I-labeled ligand without success (data not shown). However, since these cells coexpress EPO, prior occupancy of the receptors may mask binding.

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Expression of the Erythropoietin Receptor: DISCUSSION(4)

DISCUSSION(4)

In addition to identification of EPO-R protein, we also detected EPO-R mRNA in placental tissues and trophoblast cells using RT-PCR. Levels of EPO-R message do not seem to be very abundant, as others have reported its detection by Northern analysis in positive control TF-1 cells when 10 ^g of poly(A)+ RNA was used. We noted only faint hybridization signals in placental villous tissues and isolated first- and third-trimester trophoblast cells.

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Expression of the Erythropoietin Receptor: DISCUSSION(3)

The 66-kDa form of the EPO-R reportedly represents the Golgi form of the EPO-R and is the predominant one found in other tissues, including the placenta (current study). In immunoblots of placental homogenates, a less intense band of —76-78 kDa was also observed. This protein is presumably the plasma membrane form of the receptor, which has a reported size range of 70-78 kDa. The TF-1 cells, an erythroleukemic cell line known to express high levels of EPO-R, also demonstrated a prominent band of corresponding size, thus serving as our positive control.

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Expression of the Erythropoietin Receptor: DISCUSSION(2)

DISCUSSION(2)

The explanation for these partly discrepant results may relate to different procedures of tissue fixation and processing, as well as to the generally lower intensity of staining in the syncytiotrophoblast as compared to the fetoplacental endothelium. There was no evaluation of placentas of earlier gestational ages or of basal plate from the term placenta in the previous report, so the observation of EPO-R expression associated with extravillous cytotrophoblast, villous cytotrophoblast, and cytotrophoblast cell columns as reported herein is also new (Figs. 1-3). Further, the present results obtained with the m2her 16.5.1 antibody were observed with a sheep polyclonal antibody. Consistent with our observation of EPO-R expression by various populations of trophoblast cells in the human placenta is the finding of EPO-R immunoreactivity associated with the tro-phoblast giant cells in the mouse placenta.

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Expression of the Erythropoietin Receptor: DISCUSSION(1)

The recent report of EPO expression by trophoblast cells in the human placenta [11] prompted us to test whether the same cells also express the EPO-R. If so, then by analogy to erythroid precursors, important functions for this hormone related to survival, proliferation, and/or differentiation of placental trophoblast cells are possible. The findings of the present work provide evidence for the expression of EPO-R by trophoblast cells: 1) villous and extravillous cy-totrophoblast cells, as well as syncytiotrophoblast at all gestational stages, expressed immunoreactive EPO-R identified by immunohistochemistry; 2) placental tissues and isolated, immunopurified trophoblast cells of various gestational ages, as well as Jar trophoblast-derived choriocarcinoma cells, also expressed immunoreactive EPO-R by Western blot; 3) EPO-R mRNA was detected in the same placental tissues and trophoblast cells by RT-PCR; and 4) the EPO-R was shown to be functional, insofar as tyrosine phosphorylation of the receptor increased in response to exogenously administered EPO.

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Expression of the Erythropoietin Receptor: RESULTS(5)

RESULTS(5)

Protein Tyrosine Phosphorylation

Figure 11A is representative of three different experiments showing that the EPO-R of Jar trophoblast-derived choriocarcinoma cells undergoes phosphorylation on tyrosine residues in response to treatment with exogenous rhuEPO for 2 and 5 min (40 U/ml). After immunoprecipi-tation of cell lysates with monoclonal anti-EPO-R antibody clone mh2er 7.9.2, and probing of the immunoblot with anti-phosphotyrosine antibody, a prominent band of > 80 kDa was observed. Low amounts of basal tyrosine phosphorylation of the receptor were also detectable in cells prior to EPO treatment. Analysis of tyrosine phosphorylation of the EPO-R in immunopurified third-trimester placental cells was also performed (Fig. 11B).

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Expression of the Erythropoietin Receptor: RESULTS(4)

Detection of EPO-R mRNA

By loading 20 ^g of total RNA or poly(A)+ RNA from the villous placenta and from isolated first- and third-trimester trophoblast cells, we were able to detect a faint hybridization signal by Northern analysis at approximately 1.8 kilobases (data not shown). However, we used RT-PCR to verify the expression of placental EPO-R mRNA (Figs. 9 and 10). A 197-bp PCR product was amplified from reverse-transcribed RNA extracted from first- and third-trimester placentas, Jar trophoblast-derived choriocarcinoma cells, isolated and immunopurified first- and third-trimester trophoblast cells, and the positive control fetal mouse liver cells (Figs. 9A and 10A).

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Expression of the Erythropoietin Receptor: RESULTS(3)

RESULTS(3)

Negative control blots (Fig. 5B and Fig. 6, right panel) generated by substituting mouse IgG1K isotype for the primary antibody were virtually devoid of any bands. As a further control in Western blotting experiments, the 7.9.2 monoclonal antibody was also preabsorbed with soluble EPO-R (Fig. 7). The intensity of the 66-kDa band in TF-1 cells and purified trophoblast cells was considerably reduced compared to that obtained after incubation with 7.9.2 antibody alone, and the EPO-R band was eliminated altogether in placental homogenates. Band intensity was not affected when the EPO-R antibody was preabsorbed with a nonspecific antigen (rhEPO, data not shown).

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