Hormone-regulated expression and distribution of versican in mouse uterine tissues

Reproductive Biology and Endocrinology Hormone-regulated expression and distribution of versican in mouse uterine tissues Renato M Salgado 1 Luciane P Capelo 1 Rodolfo R Favaro 1 Jocelyn D Glazier 0 John D Aplin 0 Telma MT Zorn 1 0 Maternal and Fetal Health Research Group, School of Clinical and Laboratory Sciences, University of Manchester , Manchester , UK 1 Laboratory of Reproductive and Extracellular Matrix Biology, Department of Cell and Developmental Biology, Institute of Biomedical Sciences, University of Sao Paulo , Sao Paulo , Brazil Background: Remodeling of the extracellular matrix is one of the most striking features observed in the uterus during the estrous cycle and after hormone replacement. Versican (VER) is a hyaluronan-binding proteoglycan that undergoes RNA alternative splicing, generating four distinct isoforms. This study analyzed the synthesis and distribution of VER in mouse uterine tissues during the estrous cycle, in ovariectomized (OVX) animals and after 17beta-estradiol (E2) and medroxyprogesterone (MPA) treatments, either alone or in combination. Methods: Uteri from mice in all phases of the estrous cycle, and animals subjected to ovariectomy and hormone replacement were collected for immunoperoxidase staining for versican, as well as PCR and quantitative Real Time PCR. Results: In diestrus and proestrus, VER was exclusively expressed in the endometrial stroma. In estrus and metaestrus, VER was present in both endometrial stroma and myometrium. In OVX mice, VER immunoreaction was abolished in all uterine tissues. VER expression was restored by E2, MPA and E2+MPA treatments. Real Time PCR analysis showed that VER expression increases considerably in the MPA-treated group. Analysis of mRNA identified isoforms V0, V1 and V3 in the mouse uterus. Conclusion: These results show that the expression of versican in uterine tissues is modulated by ovarian steroid hormones, in a tissue-specific manner. VER is induced in the myometrium exclusively by E2, whereas MPA induces VER deposition only in the endometrial stroma. - Background The estrous cycle is orchestrated by ovarian sex hormones [1]. In the mature mouse, estrogen (E2) produced during estrus stimulates epithelial cell proliferation and synthesis of progesterone receptors (PR). On the other hand, progesterone (P4) inhibits epithelial proliferation while stimulating the multiplication of stromal cells that characterizes the beginning of decidualization [2,3]. The combined action of E2 and P4 prepares uterine tissues for blastocyst implantation. Estrogen receptors (ER and ER) and progesterone receptors (PRA, PRB and PRC) are transcription factors that regulate gene expression by direct binding to DNA regulatory sequences and by specific interactions with coactivators and/or co-repressor proteins [4,5]. In response to the normal changes in the levels of E2 and P4, the endometrium and myometrium undergo extensive cellular and extracellular modification [6]. The ECM is a complex structure of macromolecules capable of self-assembly and is composed predominantly of collagens, non-collagenous multiadhesive glycoproteins, elastin, hyaluronan and proteoglycans [7]. The endometrial ECM plays important roles in endometrial decidualization, embryo implantation, trophoblast cell invasion and the maintenance of gestation [8-10]. Previous reports have documented the remodeling of collagen [11-14], as well as glycosaminoglycans and proteoglycans in the mouse uterus during early pregnancy [15,16]. Salgado et al. [17] have shown the differential distribution of four members of the small leucine-rich proteoglycan (SLRP) family in mouse endometrium and myometrium during the estrous cycle, suggesting that their expression in the uterine ECM is modulated by ovarian hormones. San Martin et al. [18] also detected hyaluronan (HA) and versican (VER) in endometrial stroma during the periimplantation period, when angiogenesis, cell migration, trophoblast invasion and cell proliferation occur. After implantation, HA disappeared from the decidual region immediately surrounding the implantation chamber, whereas VER accumulated in the same region, suggesting this proteoglycan plays a role in proliferation and differentiation of endometrial fibroblasts into decidual cells, and may influence trophoblast invasion. VER is a large chondroitin sulfate proteoglycan that belongs to the family of hyaluronan-binding proteoglycans termed hyalectins, and is found in many soft tissues. The middle region of the core protein is encoded by two large exons that specify the chondroitin sulfate attachment regions [19,20]. RNA splicing occurs in the two GAG attachment domains, encoded by exons 7 (GAG) and 8 (GAG), giving rise to four distinct isoforms termed V0, V1, V2 and V3. V0 possesses both exons 7 and 8, V1 only exon 8, V2 only exon 7 and V3 possesses neither [21]. The interaction between VER and hyaluronan (HA) is mediated by the N-terminal G1 globular domain, while the carboxy-terminal globular domain (G3) consists of a C-type lectin adjacent to two epidermal growth factor domains and a complement regulatory region. VER interacts with other ECM molecules, such as tenascin-R, collagen I, fibronectin and the elastic fiber-associated proteins fibrillin-1 and fibulin-2 [22]. Fibrillin-1 is present in the endometrial stroma in estrus and diestrus [23]. In addition, VER binds to chemokines and cell surface receptors including 1-integrin, CD44, epidermal growth factor receptor and selectins [24]. These interactions facilitate essential biological processes, including cell migration [20]. The expression of different VER isoforms influences the formation of the extracellular matrix network and might modulate cell-matrix and cell-cell interactions [19]. Moreover, RNA splicing patterns suggest distinct functions for different domains of the protein. V0/V1 isoforms show similar properties with respect to cell anti-adhesion, proliferation and growth, and resistance to apoptosis. V2 is known to inhibit cell growth and proliferation, and to be expressed only in the nervous system, as the major isoform in adult brain [25]. The truncated isoform V3, sometimes called "versicant", is thought to possess proadhesive properties, due to the lack of the highly negatively charged chondroitin sulfate side chains [26]. Previous reports describe changes in the distribution of HA and VER in human and murine cervix during late pregnancy and parturition [27,28]. In the female rodent reproductive system, VER distribution and the expression of its isoforms were shown for the first time by Russel et al. [29] in the mouse ovary. However, little is known about VER expression and function in the non-pregnant uterus. The major objectives of the present study were (i) to analyze whether E2 and P4 modulate the expression and distribution of VER in the uterine tissues of mice and (ii) to characterize VER isoforms in the mouse uterus. Methods Tissue collection Forty (...truncated)