Biol Reprod. 2014 Sep;91(3):65.

Adrenomedullin promotes rat trophoblast stem cell differentiation.

Gao H, Liebenthal DA, Yallampalli U, Yallampalli C.

Department of Obstetrics & Gynecology, Baylor College of Medicine; Texas Children’s Hospital, Houston TX 77030, USA



Accumulating data suggest that adrenomedullin (ADM) regulates the trophoblast cell growth, migration, and invasion. However, the effect of ADM on trophoblast differentiation is poorly understood. In this study, we hypothesized that ADM promotes the differentiation of trophoblast stem cells (TSCs) into trophoblast giant cells (TGCs). Using rat TSCs, Rcho-1 cells, we investigated the effect of ADM on TSC differentiation into TGCs in differentiation or stem cell media, respectively, and explored the effect of ADM on the mechanistic target of rapamycin (MTOR) signaling in trophoblast cell differentiation. The results include: 1) in the presence of differentiation medium, 10⁻⁷ M ADM, but not lower doses, elevated (P < 0.05) Prl3b1/Esrrb (i.e., the ratio of mRNA levels) by 1.7-fold compared to that in control; 2) the supplementation of ADM antagonist, regardless of the concentration of ADM, reduced (P < 0.05) Prl3b1/Esrrb by 2-fold, compared to control group, while the supplementation of CGRP antagonist, regardless of the concentration of ADM, did not change Prl3b1/Esrrb; 3) in the presence of stem cell medium, ADM did not alter the expression of TSC and TGC marker genes, however, the ratio of Prl3b1/Esrrb was reduced (P < 0.05) by ADM antagonist compared to that in control; and 4) ADM increased (P < 0.05) phosphorylated MTOR proteins and the ratio of phosphorylated to total MTOR proteins by 2.0- and 1.7-fold, respectively. The results indicate that ADM promotes but does not induce the differentiation of TSCs to TGCs in a dose-dependent manner and MTOR signaling may play a role in this process.

PMID: 25061099



Pregnancy is associated with complicated maternal adaptations to maintain pregnancy and also to secure nutrient supply for placental and fetal growth. One of the mechanisms responsible for maternal adaptions is driven by hormones of placental origin, for instance, placental lactogens. Trophoblast giant cells (TGC) play versatile functions as pregnancy advances, with secretion of a variety of agents including steroid hormones and placental lactogens at different stages [1], and thus are critical for implantation and placentation. Together with other types of trophoblasts, TGC are derived from the differentiation of trophoblast stem cells (TSC).

Adrenomedullin (ADM), a member of calcitonin/calcitonin gene-related peptides (CALCA/CGRP) family. In both humans and rats, maternal plasma ADM levels progressively increase with pregnancy [1;2] and feto-placental tissue is considered the major source of ADM during pregnancy[3]. On the other hand, Adm null mice are embryonic lethal [4] and decrease in maternal expression of Adm results in impaired fertility, placentation and fetal growth [5], supporting the importance of ADM in implantation and placentation. The physiological or cellular effects of ADM are dependent on the type of tissues or organs, especially the distribution of its receptors consisting of CALCRL (calcitonin receptor-like) and receptor activity modifying proteins (RAMP) [9]. Recently, PI3K (phosphatidylinositol-4,5-bisphosphate 3-kinase)-AKT

(thymoma viral proto-oncogene) signaling pathway has been demonstrated to play a crucial role in rodent trophoblast cell differentiation, and enhanced ADM expression is associated with the differentiation of TSC [6]. It is well-known that PI3K is one of the upstream regulators of MTOR (mechanistic target of rapamycin) signaling pathway in trophoblast cells in humans [7], mice [8], rats [9], and sheep [10]. However, to date, the role of MTOR signaling has not been linked to trophoblast stem cell differentiation.

In this study, we hypothesized that ADM enhances the differentiation of trophoblast stem cell and MTOR signaling pathway is involved in this process. This hypothesis was tested by a series of experiments, primarily using cell culture of rat Rcho-1 rat trophoblast stem cells which were derived from rat blastocyst, a well characterized in vitro model for studying trophoblast [11].

First, we investigated the effect of ADM on TSC differentiation into TGC in Differentiation Medium. Rat TSCs were cultured in Differentiation Medium only or Differentiation Medium with ADM at 3 doses, ADM antagonist (ADM22-52), CGRP antagonist (CGRP8-37), and/or their combinations for 6 days. As a result, in the presence of Differentiation Medium, ADM at high dose promoted TSC differentiation into TGC, but ADM at lower doses did not; the treatment with ADM antagonist, regardless of the dose of ADM, reduced the TSC differentiation, compared to that in the control group. However the CGRP antagonist (CGRP8-37) treatment, regardless of the dose of ADM, did not change TSC differentiation. These results suggest that ADM promotes TSC differentiation in a dose-dependent manner, and high dose is required for this process. In addition, ADM promoting TSC differentiation is mediated by RAMP2, instead of RAMP3, since ADM actions involving RAMP2 are reduced by ADM antagonist, whereas RAMP3 related ADM actions are blocked by CGRP antagonist[12].

Second, we investigated the effect of ADM on TSC differentiation into TGC in Stem Cell Medium. Rat TSCs were cultured in Stem Cell Medium only or Stem Cell Medium with ADM and/or ADM antagonist for 4 days. As a result, in the presence of Stem Cell Medium the supplementation of ADM, ADM antagonist and their combination did not alter TSC differentiation. These results clearly showed that ADM alone cannot initiate TSC differentiation into TGC, and therefore, some components in the Differentiation Medium are required for the process of initiation.

Third, we explored the role of MTOR protein in ADM promoted TSC differentiation into TGC. Rat TSCs were cultured in Differentiation Medium only or Differentiation Medium with other treatments for 6 days. As a result, ADM treatment caused increases in both the abundance of phosphorylated MTOR (Ser2448) proteins and the ratio to total MTOR proteins. These indicate that ADM can stimulate MTOR phosphorylation in the process of TSC differentiation. Interestingly, treatment of ADM antagonist itself or the combination of ADM and ADM antagonist also resulted in increases in the abundance of phosphorylated MTOR proteins and the ratio to total MTOR proteins. This unexpected result may be due to the extended culture time for 4 days, instead of few minutes which is routinely used in most signaling studies. In addition, it is known that some of these antagonists may exert agonistic functions under certain conditions [13].

The importance of this study is two-fold. First, this study for the first time demonstrates that ADM promotes but does not initiate trophoblast stem cell differentiation to trophoblast giant cells. The effect of ADM appears to be through the ADM receptor consisting of CALCRL and RAMP2. Second, we show for the first time that ADM can stimulate MTOR phosphorylation in the process of TSC differentiation. Moreover, activation of MTOR signaling in response to ADM may help elucidate the underlying mechanisms responsible for TSC differentiation during gestation in that MTOR signaling integrates various nutritional and hormonal stimuli, and plays a critical role in protein synthesis.

In summary, according to data from this study and others, we propose the role of ADM in regulating TSC differentiation as follows. During pregnancy, ADM binding to its receptor CALCRL and co-receptor RAMP2 in TSC, activates PI3K-AKT-MTOR signaling, which stimulates differentiation of TSC into TGC. TGC synthesize and secret various steroid hormones and placental lactogens, which cause maternal adaptations to pregnancy status. Meanwhile, fetal and placental tissues together with maternal organs or tissues produce increasing amount of ADM with the progress of gestation, which further stimulates the differentiation of TSC to TGC (Figure 1).

sd fig1

Fig.1 Schematic illustration of role of ADM in TSC differentiation to TGC during pregnancy. TSC: trophoblast stem cells; TGC: trophoblast giant cells; ADM: adrenomedulin; CALCRL: calcitonin receptor; RAMP2: receptor activity modifying protein 2; PI3K: phosphatidylinositol-4,5-bisphosphate 3-kinase; AKT: thymoma viral proto-oncogene; MTOR: mechanistic target of rapamycin.



This study was supported by National Institutes of Health grants R01HL102866and R01HL58144.



Chandra Yallampalli, Ph.D

Professor and Director, Basic Sciences Perinatology Research Laboratories, Department of Obstetrics and Gynecology, Baylor College of Medicine, 1102 Bates Avenue, Room #1850.34, Houston, Texas 77030. Office: 832-824 4188; Fax: 832-825 7946; Email:


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