Identification of BARD1 splice-isoforms involved in human trophoblast invasion

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Abstract

The tumor suppressor protein BARD1, originally discovered as BRCA1-binding protein, acts in conjunction with BRCA1 as ubiquitin ligase. BARD1 and BRCA1 form a stable heterodimer and dimerization, which is required for most tumor suppressor functions attributed to BRCA1. In addition, BARD1 has BRCA1-independent functions in apoptosis, and a role in control of tissue homeostasis was suggested. However, cancer-associated mutations of BARD1 are rare; on the contrary, overexpression of truncated BARD1 was found in breast and ovarian cancer and correlated with poor prognosis. Here we report that human cytotrophoblasts, which show a strong similarity with cancer cells in respect of their invasive behavior and capacity of matrix metalloprotease production, overexpress isoforms of BARD1 derived from differential splicing. We demonstrate that expression of BARD1 and its isoforms is temporally and spatially regulated by human chorionic gonadotropin and by hypoxia, both factors known to regulate the invasive phase and proliferation of cytotrophoblasts. Interestingly, we found a subset of BARD1 isoforms secreted by cytotrophoblasts. BARD1 repression by siRNAs, mitigates the interference of cytotrophoblasts with cell adhesion of collagen matrix-dependent epithelial cells, suggesting a role of BARD1 isoforms in extracellular matrix remodelling and in cytotrophoblasts invasion.

Introduction

The BRCA1-associated RING-domain protein (BARD1) was identified in a two-hybrid screen as a protein interacting with BRCA1 (Wu et al., 1996). BARD1 is structurally related to BRCA1 in that it harbours an amino-terminal RING domain and two carboxy-terminal BRCT motifs. BARD1 and BRCA1 stabilize each other through interaction via their respective RING domains (Brzovic, Rajagopal, Hoyt, King, & Klevit, 2001). The BRCA1-BARD1 heterodimer participates in DNA repair, transcription regulation, RNA processing and ubiquitination (Irminger-Finger & Jefford, 2006). BARD1 also has a BRCA1-independent function in apoptosis (Irminger-Finger & Leung, 2002; Irminger-Finger et al., 2001). It binds to p53 facilitating its phosphorylation and stabilization required for induction of apoptosis (Feki et al., 2005). In vitro experiments showed that elevated expression of BARD1 leads to apoptosis and repression of BARD1 reduced apoptotic response to genotoxic stress (Irminger-Finger et al., 2001).

BARD1 is highly expressed in spleen and testis and in proliferative cells in normal tissues where it locates to the nuclei (Ayi, Tsan, Hwang, Bowcock, & Baer, 1998; Irminger-Finger, Soriano, Vaudan, Montesano, & Sappino, 1998). However, BARD1 also appears to be expressed independently of BRCA1 in tissues responding to hormonal control, especially in breast, uterus, testis and ovary (Feki et al., 2004, Irminger-Finger et al., 1998). BARD1 mutations were found in breast, ovarian and uterine tumors (Ishitobi et al., 2003; Karppinen, Heikkinen, Rapakko, & Winqvist, 2004; Thai et al., 1998), consistent with its tumor suppressor function. BARD1 protein expression was aberrantly elevated and localized to the cytoplasm in sporadic breast and ovarian cancers in both tumors with wild type and tumors with mutated BARD1 (Wu et al., 2006). Since the level of expression of truncated BARD1 was correlated with poor prognosis for breast and ovarian cancers (Wu et al., 2006), BARD1 isoforms in cancer might have oncogenic potential.

We hypothesized that tumors might reproduce a developmental pattern of gene expression, thus expressing BARD1 isoforms that are functional during a specific developmental process. One such process that shows high similarity with cancer cell behavior is the proliferation and invasion process of the human cytotrophoblasts (CTBs) (Bischof & Irminger-Finger, 2005). CTBs are specialized placental cells that play a pivotal role during the early stage of placental development and during embryo implantation. Implantation in the endometrium allows the CTBs to invade the decidua and myometrium, which is important for gas exchange, nutrition, endocrine function, and immunological support for foetal growth.

At present, many factors have been found involved in CTB invasion, such as serine proteases, cathepsin, matrix metalloproteinases (MMPs) (Westermark et al., 2003), extracellular matrix proteins (Bischof & Irminger-Finger, 2005; Xu et al., 2001), hypoxia (James, Stone, & Chamley, 2006a), human chorionic gonadotropin (hCG) (Zygmunt, Hahn, Munstedt, Bischof, & Lang, 1998) and VEGF (Lash, Warren, Underwood, & Baker, 2003). While hCG might be a temporal regulator of CTB invasion, a local modulator is the oxygen supply. During the first 10–12 weeks of gestation, extravillous trophoblast plugs the spiral arteries to prevent maternal blood flow from entering the intervillous space, thereby creating an environment of physiological hypoxia. Before the ninth week of gestation, placental oxygen is as low as ∼20 mm Hg, but after 10–12 weeks of gestation, it increases to ∼55 mm Hg. It was found that oxygen can regulate CTB gene expression and phenotype (James et al., 2006a; James, Stone, & Chamley, 2006b) and may increase the invasiveness of CTBs.

Since BARD1 expression is aberrantly upregulated in cancer and associated with an aberrant form and intracellular localization, it was interesting to evaluate whether BARD1 expression in CTBs resembled its expression in cancer. In this manuscript, we describe the temporal and spatial expression pattern of BARD1 in the human placenta and address the question of the regulation of its expression and the functions of its gene products.

Section snippets

CTB purification and cell culture

Placental tissues were obtained from patients who voluntarily and legally chose to terminate pregnancy during the first trimester (7–12 weeks of gestation). Informed written consent was obtained from all patients before their inclusion in the study, for which approval was obtained from the local ethic committee.

Cytotrophoblast cells were isolated from first trimester placenta as described (Bischof, Haenggeli, & Campana, 1995). In brief, fresh tissue specimen were isolated and washed several

Expression of BARD1 in CTBs of early pregnancy

Since the human cytotrophoblast has invasive properties reminiscent of cancer cells, we were interested in elucidating the function of BARD1 in CTBs of early pregnancy. In a first approach RT-PCR was performed by amplifying full length (FL) BARD1 to determine its expression in purified CTBs at different weeks of pregnancy (Fig. 1A). When investigating expression levels in CTBs from 7 to 12 weeks of pregnancy, we found that the expression level of FL BARD1 was variable. It increased from 7 to 9

Discussion

We were interested in studying the function and expression of BARD1 in human CTBs that share invasive properties with cancer cells. Surprisingly, BARD1 expression is highly elevated in CTBs, as compared to other tissues, suggesting that it plays an important role in this cell type. The heterodimeric protein binding partner of BARD1, BRCA1, however, is only weakly expressed in CTBs, indicating that the role of BARD1 in CTBs is independent of BRCA1.

In the first trimester of human pregnancy, the

Acknowledgments

We are grateful to Aurélie Caillon, Chantal Genet, and Christine Wuillemin for excellent technical help. We are indebted to Dr. Stephan Ryser and Dr. Charles Edward Jefford for advice and helpful discussions and Dr. Marie-Francoise Pelte for expert opinion. This work was supported by Swiss National Science grants 3100AO-110038 to IIF and 3200BO-101526 to PB.

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