Background
Nodal, a member of the transforming growth factor-beta (TGF-β) superfamily, regulates the processes of pattern formation and differentiation that occur during early embryo development [
1]. In particular, Nodal signalling is essential for mesoderm and endoderm induction, neural patterning and the specification of the primary body axes [
1]. Nodal signals through activin type I (ALK4) and type II (ActRII or ActRIIB) serine-threonine kinase receptors [
2]. However, unlike activin, Nodal lacks intrinsic affinity for ALK4 and ActRII/IIB, suggesting the requirement for a co-receptor to potentiate its actions [
3]. Indeed, recent studies have shown that Nodal effects are dependent upon interactions with Cripto, a small cysteine-rich extracellular protein that is attached to the plasma membrane through a glycosyl phosphatidyl inositol linkage [
1]. Cripto interacts with Nodal and ALK4, independently, and promotes the formation of a stable high affinity complex with activin type II receptors [
4]. Phosphorylation of ALK4 within this complex initiates signaling via Smad2/Smad3 signal transducers [
3]. The TGF-β signaling antagonist, Lefty, blocks Nodal actions by competing for access to the ligand binding site of Cripto [
5].
Consistent with its crucial developmental function,
nodal is first expressed throughout the embryonic ectoderm shortly after implantation (5.25 days post-coitum). Expression continues during the initial stages of primitive streak formation and is then rapidly down regulated as the streak elongates. Subsequently,
nodal expression is detected in a small subset of node progenitors, and following the formation of the morphologically distinct node becomes restricted to the edges of the notochordal plate [
1,
6,
7]. Until recently, Nodal expression was widely thought to be embryonically restricted [
8]. However, several studies have shown that Nodal and its signalling partners are expressed at defined stages in a variety of adult tissues, including the lactating mammary gland and regenerating islet cells in the pancreas [
9,
10]. In addition, there is increasing evidence that Nodal pathway activity is upregulated in many human cancers. Hendrix and colleagues [
11,
12] have shown that expression of Nodal in metastatic melanomas and breast carcinomas is correlated with cancer progression, whereas pathway inhibition decreases cell invasiveness, colony formation and tumourigenicity.
Components of the Nodal signalling pathway have also been detected in human endometrium. Lefty A, which was originally designated endometrial bleeding associated factor (
ebaf), is highly expressed in endometrium during the late secretory and menstrual phases, but is significantly reduced in proliferative, early and mid-secretory endometria [
13,
14]. Lefty A stimulates the production of several matrix metalloproteinases and may be a key local regulator of focal extracellular matrix breakdown in the cycling human endometrium [
15]. Furthermore, dysregulated endometrial expression of Lefty is associated with infertility [
14], and
in vivo gene transfer of Lefty leads to implantation failure in mice [
16]. Curiously, given Lefty's well-documented mechanism of action during vertebrate embryogenesis [
5,
17], the presence of Nodal and Cripto mRNA in human endometrium has only recently been established [
18].
In the current study, RT-PCR and immunohistochemistry were utilised to examine the site- and menstrual cycle stage-specific expression of Nodal and Cripto in the human endometrium. As recent studies have suggested that increased Nodal signalling has a key role in melanoma cell plasticity and tumourgenicity, the expression profiles of Nodal, Cripto and Lefty were also examined in endometrial carcinomas. The expression of Nodal and Cripto in normal and malignant endometrial cells that lack Lefty strongly supports an important role for this embryonic morphogen in the endometrial remodelling events that occur across the menstrual cycle and in tumourogenesis.
Discussion
The human endometrium is divided into two layers: the functionalis, which comprises the upper two-thirds, and the basalis, which remains during and following menstruation and is thought to be the origin of a new functionalis in the subsequent cycle [
31]. The restructuring of the functional layer is critical to the development of a tissue ready for implantation or, in the absence of a conceptus, for menstruation [
32]. Endometrial repair and regeneration involves re-epithelialization (which occurs very rapidly even while menstruation is still in progress), angiogenesis and vessel remodeling, stromal and glandular epithelial cell proliferation, and extracellular matrix (ECM) deposition ([
33]. Adult stem/progenitor cells likely underpin most aspects of this endometrial regeneration [
31], however, the molecular and cellular mechanisms that mediate the regeneration process are still not well understood.
Given their established roles in wound healing, cell growth, ECM production and the maintenance of stem cell pluripotency [
34‐
36], members of the TGF-β superfamily that signal via Smad2/3 (e.g. TGF-β1, TGF-β2, TGF-β3, activin A, activin B and Nodal) are likely to be important mediators of endometrial repair and regeneration. Indeed, endometrial repair is retarded in the absence of activin A [
37]. In this study, we showed that Nodal, as well as its co-receptor, Cripto, are co-expressed in human endometrial tissue throughout the menstrual cycle. Immunohistochemistry localized Nodal and Cripto primarily to stromal and epithelial cells, although moderate staining was also observed in endothelial cells associated with spiral arterioles. Nodal protein levels were maintained in the glandular and luminal epithelium at relatively steady-state levels across the cycle, however, stromal localization was primarily restricted to the proliferative and early secretory phases. Cripto displayed a similar spatiotemporal localization to Nodal within the endometrium, although the amount of Cripto detected in glandular epithelium increased significantly during the late secretory phase of the cycle.
To be responsive to Nodal, a cell must express activin type I and type II receptors, in addition to Cripto. Jones
et al. [
21] have shown that endometrial stromal cells express each of the activin receptor subtypes (ALK4, ActRII and ActRIIB) with highest expression during the early secretory phase. Potential functions of the Nodal signalling pathway within the endometrial stromal compartment can be extrapolated from its roles in embryogenesis [
1]. In early development, Nodal acts as a graded morphogen, instructing stem cells to adopt specific cell fates concurrent with proliferation and migration [
8]. Similar actions during the proliferative phase of the menstrual cycle would ensure Nodal plays a key role in endometrial restoration.
Interestingly, activin receptors are not present in either surface or glandular epithelium at any stage of the cycle [
21], suggesting that Nodal expressed in these cells cannot be functioning in a paracrine/autocrine manner. Indeed, as the majority of glandular derived products are secreted apically, it seemed likely that the endometrium must secrete Nodal into the uterine cavity. In support, we identified Nodal precursor in the uterine lavage fluid of both normal cycling women and women with endometrial carcinoma. Roles for endometrially derived Nodal could be similar to those proposed for activins, i.e. embryogenesis [
38], steroidogenesis [
39] and trophoblast differentiation [
40]. In contrast to Nodal, Cripto within the epithelial compartment may be functional due to its ability to directly activate mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/Akt pathways via c-Src [
9,
41]. These actions of Cripto have led to its designation as a tumour growth factor and may be particularly relevant in endometrial carcinomas.
During embryogenesis, members of the Lefty subclass of TGF-β proteins act as extracellular antagonists of the Nodal signalling pathway [
17]. Lefty blocks the formation of the Nodal receptor complex by binding to Nodal directly [
17] or by interacting with Cripto [
5]. Lefty expression is absolutely dependent upon Nodal function and within the embryo and developing tissues, such as the pancreas [
10], the juxtaposition of these two factors limits their respective range of influence. It is interesting, therefore, that Nodal and Lefty have spatially and temporally distinct patterns of expression within the endometrium. The lack of Lefty expression in the proliferative phase of the menstrual cycle would ensure that the morphogenic actions of Nodal were not restricted at this time. In contrast, co-localization of Nodal, Cripto and Lefty within glandular and luminal epithelial cells during the late secretory and menstrual phases suggests that, at these times, Nodal signalling requires strict control. Indeed, the recently identified role for Lefty as a key local regulator of focal ECM breakdown in the cycling human endometrium [
15] may derive from its ability to antagonise Nodal signalling.
Finally, recent studies have shown that expression of Nodal in metastatic melanomas and breast carcinomas is correlated with cancer progression, whereas pathway inhibition decreases cell invasiveness, colony formation and tumourigenicity [
11,
12]. These findings are consistent with the upregulation of Cripto that is observed in many epithelial cancers [
41,
42], and with the ability of Cripto to initiate several aspects of tumour progression, including increased proliferation, migration, invasion, angiogenesis, and epithelial-to-mesenchymal transition [
29]. In the current study, we showed that Nodal and Cripto are expressed in normal endometrium and that their expression is dramatically upregulated in endometrial carcinomas.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
IP participated in the immunohistochemistry and PCR studies and helped draft the manuscript. PN participated in the immunohistochemistry studies. FW participated in the PCR studies. ML produced the Cripto monoclonal antibodies. YM helped draft the manuscript and performed the statistical analysis. LS provided endometrial tissues and intellectual input. DR participated in the studies design. CH conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.