Projects

1. Relaxin signalling in the monocyte cell line, THP-1

The peptide hormone relaxin is structurally related to insulin and IGF1, yet unlike these appears to act through a recently cloned receptor of the 7-transmembrane, G-protein coupled family, called LGR7. Relaxin has recently been shown not only to be a key hormone involved in embryo implantation and pregnancy (Bartsch et al., 2001, 2004), but also is now being seen as an exciting new agent influencing general cardiovascular function, the formation of connective tissue and fibrosis, and having very positive effects in wound-healing (Ivell, 2002; Ivell & Einspanier, 2002).

There are very few cell-lines in which the peptide hormone relaxin has been shown to have similar intracellular effects to what is seen in primary cell cultures, for example, of human endometrial cells. The monocyte cell-line THP-1, while not derived from a reproductive tissue, nevertheless shows many features of natural relaxin receptor signalling. In particular, there is a robust and dose-dependent cAMP response, suggesting that relaxin is using its Gs-coupled receptor LGR7 in a conventional sense to activate adenylate cyclase (Bartsch et al., 2001). However, there are many features of this system, which do not fit this simplistic pattern of hormone action. For example, the response cannot be obtained using cell membranes alone, and it is exquisitely sensitive to certain specific inhibitors of tyrosine kinases (Bartsch et al., 2001). All evidence to date suggests that we are dealing here with a completely new signalling pathway linking the relaxin receptor to intracellular systems maintaining high cAMP levels.

In this project molecular pharmacological approaches will be taken to dissect the relaxin-dependent signaling pathway in THP-1 cells, thereby providing a basis for understanding the molecular mode of action of this increasingly important hormone. It is also proposed to extend this work to examine the role of relaxin in artherosclerosis. Knowledge gained about new cellular signalling pathways also offers new opportunities for devising novel pharmacological intervention paradigms of more general application in health and disease.

2. Mechanisms of differentiation of human primary endometrial cells

As stated in the preceding project, the peptide hormone relaxin is a key player in the initiation of pregnancy, implantation and the establishment of the placenta (Ivell & Einspanier, 2002). We still know very little about how relaxin achieves this positive effect, mostly because few if any of these functions can be modelled using cell-lines. Much of our understanding of these processes has been achieved using human primary cell cultures derived from operation tissues obtained at hysterectomy. Using this approach, we have previously shown that relaxin is possibly the most important factor, even more so than steroid hormones, in inducing the differentiation process known as decidualization in the stromal cell compartment of the endometrium (Bartsch et al., 2001, 2004; Telgmann & Gellersen, 1998), which in turn drives differentiation of the other component cell types. Relaxin, however, is probably also able to influence both epithelial and smooth muscle cells directly, through as yet unknown mechanisms (Ivell et al., 2003).

This project will make use of the established human endometrial stromal cell culture model to elaborate the mode of action of relaxin in this cell type. In particular, attention will be paid both the to the immediate signalling pathways employed, thus working together with Project 1, as well as to more downstream events related to gene expression and cell differentiation. Part of this project will probably involve the application and interpretation of DNA microarrays in order to gain a holistic appraisal of the differentiation events governed by relaxin.

3. Leydig cell differentiation and the regulation of INSL3 function

Insulin-like factor 3 (INSL3) is a new peptide hormone of the relaxin-insulin-IGF family and is produced in large amounts by the Leydig cells of the testis in all mammals (Ivell & Bathgate, 2002). In the embryo the production of this hormone is responsible for the first transabdominal phase of testicular descent. While in the ovary in the female there is evidence to suggest an important role in follicle selection (Irving-Rodgers et al., 2001), we have as yet no hard evidence for a role in the adult testis, although preliminary data suggest an anti-apoptotic function in regard to germ cells (Kawamura et al., 2004). The INSL3 gene appears to be expressed in a differentiation-dependent manner in adult-type Leydig cells, being hardly expressed before puberty, but increasing dramatically as the Leydig cells begin to produce testosterone (Balvers et al., 1998).

In this project we shall be using INSL3 and its gene to investigate the molecular differentiation of Leydig cells in primary culture under conditions, which mimic the changes incurred by adult-type Leydig cells during puberty, and possibly similar to the comparable changes in foetal Leydig cells before birth. In particular, this project will address the signalling mechanisms responsible for this strong pubertal upregulation of INSL3, and ways in which this dynamic differentiation process can be modulated or altered by hormones and substances considered as environmental endocrine disruptors. It is hoped in this way to be able to reproduce in vitro some of the effects shown for in vivo animal models, wherein endocrine disrupting substances, and particularly xenoestrogens, prenatally induce cryptorchidism (a failure of the testes to descend). By elaborating the molecular mechanisms involved, it is intended to gain a better understanding of pathologies, like cryptorchidism, which are caused by defects in Leydig cell differentiation.

This project will involve the establishment and analysis of primary cultures of Leydig cells from young rodents. INSL3 gene expression during in vitro induction of puberty will be assessed by quantitative RT-PCR and by immunological methods. Pharmacological inhibitors and gene transfection will be used to dissect the pathways involved in this process, and the influence on these regulatory pathways of known disrupting substances.

4. Feed forward mechanisms drive the differentiation of ovarian granulosa cells

(Collaborative project with A/Prof. Ray Rodgers and Dr Darryl Russell Dept of Obstetrics & Gynaecology, University of Adelaide)

In all mammals it is the changing differentiation status of the inner cell layers of the ovarian follicles - the co-called granulosa cells - and their interaction with the outer theca cell layers, which determines whether a follicle matures and ovulates to release a fertile oocyte, or enters the apoptotic pathway. The final differentiation step, whereby granulosa cells luteinize to become the major cell type of the ovarian corpus luteum, is considered to be triggered by a surge of luteinizing hormone (LH) from the pituitary, approximately 1 day before ovulation occurs (Luck et al., 1987; Rodgers et al., 1995; Ivell et al., 1999; Irving-Rodgers et al., 2004). However a novel matrix recently discovered by us has been proposed to initiate this process (Irving-Rodgers et al., 2004) and the LH surge to allow it to continue. Upon the success of this differentiation step depends the capacity of the later corpus luteum to produce sufficient amounts of the steroid hormone progesterone and other factors to support the development of the growing embryo in the first trimester of pregnancy. The cow, whose ovary shares many common features with the human ovary, offers an excellent model with which to analyse this process at the molecular level. Sufficient primary cells are easily available from abattoir tissue for extensive cell culture studies, and many years of research have established a wide range of well-characterized marker genes and end-points. Nevertheless, very little is still known about the molecular mechanisms inside the granulosa cells which are responsible for these critical differentiation processes.

Some years ago we were able to establish that the peptide hormone oxytocin offers a very important surrogate marker for the bovine luteinization process, and that this process was regulated by a finely tuned dynamic competition of two transcription factors, steroidogenic factor-1 (SF-1) and COUP-TF for occupancy of a site in the proximal promoter of the oxytocin gene (Wehrenberg et al., 1994). In other tissues and cells, this site can be influenced by more complex indirect mechanisms, including steroids (Stedronsky et al., 2002). In 1997, we made the surprising observation that progesterone, which begins to be made in large amounts by the luteinizing granulosa cells, itself is part of a feed forward mechanism driving the expression of the oxytocin gene, although there is no obvious binding site for the known progesterone receptors (Lioutas et al., 1997). In this project, we want to revisit this model system in order to elaborate the mechanisms by which progesterone is itself able to induce the luteinization process.

The project will make use of primary cultures of bovine granulosa cells, and as end-points we shall be able to measure the endogenous expression of the oxytocin and other genes using real time PCR. We will also measure the oxytocin peptide itself in the culture medium using an ELISA system we have developed. Through a collaboration with industry we will be using a series of different progesterone receptor agonists and (partial) antagonists, as well as a series of pharmacological inhibitors known to intercept critical points of diverse signalling pathways in these cells. Should time allow, then it may also be possible to analyse the interactions with the oxytocin gene promoter itself, DNA constructs for which are already well established in the group. The results from this project will not only deliver important new information on the molecular mechanisms involved in the luteinization process, and hence the formation of an adequate corpus luteum, they will also provide vital information on what could be a very exciting new mode of action for progesterone and its clinically relevant analogues.

Funding Sources

Until recently projects were funded by the German government or by the European Union. Since arriving in Adelaide, the research of the Ivell lab is being supported by a fellowship from BioInnovation SA, and more recently by a large project grant from the NHMRC to support the research on relaxin signaling. With Professor Shemesh, we are developing an ARC linkage project on male germ-line transgenesis and siRNA technology. We expect that more grants from commonwealth agencies and industry will follow.