SLRPs in the Endometrium
Our data show for the first time the distribution of the SLRPs decorin, lumican, biglycan, and fibromodulin in the uterus of mice in each stage of the estrous cycle. Moreover, the organization and distribution of these molecules in the uterine tissues were found to be estrous cycle-stage dependent, suggesting that these molecules undergo ovarian hormonal control and probably participate in the preparation of the uterus for decidualization and embryo implantation.
We found that decorin and lumican were expressed in the whole stroma in estrus and metaestrus, stages of estradiol predominance. Interestingly, in proestrus and diestrus—high progesterone levels—decorin was restricted to the deep stroma, and lumican was diminished in the superficial stroma. Similar distribution was observed by San Martin et al. (2003a) in the preimplantation period in the mouse. This result supports our hypothesis that the deposition of these proteoglycans appears to be related with both hormonal profile and the region of the endometrial stroma.
We have previously documented that, in mice, remodeling of the extracellular matrix begins early in pregnancy, characterized by synthesis, degradation, and alteration of collagen fibrillogenesis, accompanied by pregnancy-stage dependent expression of collagen, glycosaminoglycans and proteoglycans. Interestingly, decorin is abolished from the superficial stroma after decidualization and lumican is maintained, although in very low levels (Zorn et al., 1995; San Martin et al., 2003a, b; Spiess and Zorn, 2007). In fact, studies from Vogel and Trotter (1987) showed that decorin and lumican link to fibrilar collagens in vitro, stabilizing thin fibrils. During pregnancy, the loss of decorin and reduction of lumican are related to the appearance of thick collagen fibrils in the mature decidua. We do not know yet whether these molecules participate in collagen fibrillogenesis during the estrous cycle.
Decorin is also known to control cell division and to stop cell growth of neoplastic cells of different origins by its ability to bind TGF-β (Hildebrand et al, 1994; Santra et al., 1997). We speculate that the absence of decorin in the superficial stroma in diestrus may be favorable to the cellular proliferation in this region. Furthermore, overexpression of decorin regulates the distribution of several matrix metalloproteinases (MMPs) and cytokines by gingival fibroblasts, evidencing its role in tissue metabolism (Al Haj Zen et al., 2003).
Biglycan was found exclusively in the deep stroma in all stages of the estrous cycle, except in estrus, where it is present in both superficial and deep stroma. Similarly, biglycan is distributed preferentially in the deep stroma of non decidualized interimplantation sites. However, in the implantation sites, biglycan appears in the decidualized stroma on day 4 of pregnancy, remaining around decidual cells until day 8 (San Martin et al., 2003a). These data may suggest that the extracellular matrix architecture is spatial, temporal, and functionally modulated in the uterus of mice.
Among the studied SLRPs, fibromodulin was the only one to be absent in the endometrial stroma. Fibromodulin was absent in the mouse antimesometrial pregnant stroma until the beginning of decidualization when it is weakly expressed in the deep stroma (San Martin et al., 2003a). Fibromodulin has been found mainly in dense connective tissues. Interestingly, in the connective tissue of papillary gingiva, fibromodulin was detected, along with biglycan and lumican, mainly in the deep region (Alimohamad et al., 2005). These results highlight differences between distinct types of connective tissues, showing that the deposition of these molecules is tissue especific as previously shown by our group in developing embryonic tissues (Miqueloto and Zorn, 2007). Moreover, our results on the distribution of proteoglycans reinforce the regional differences between superficial and deep stroma.
Similar distinct regional distribution was found for other ECM molecules in the endometrial stroma of mice during the preimplantation period. Grecca et al. (1998) showed differences in safrannin O positive network, when superficial stroma is compared with deep endometrial stroma; Stumm and Zorn (2007) showed that fibrilin-1 declines in the superficial stroma only in diestrus; Spiess and Zorn (2007) studied the distribution of collagens I, III, and V in the mouse pregnant endometrium, showing that collagen III was the only one present at the maternal-fetal interface, suggesting that this interface needs a specific molecular composition, favorable to embryo implantation and development. Those and the present results suggest a predefined regionalization of the mouse endometrium into superficial and deep stroma that may be related to the centrifugal development of the decidua (reviewed by Oliveira et al., 1998).
Studies by Lee and Jeung (2007) showed the distribution of TRPV6, a calcium-regulating protein, in the mouse uterus during the estrous cycle and pregnancy. TRPV6 was highly expressed in estrus, an E2-dominant stage. On the contrary, CAPB-9K, another calcium-regulating protein, was found to increase in diestrus and to be induced after progesterone replacement therapy (Kim et al., 2006). All these data together reinforce the idea of a dynamic organization of uterine tissues, including their extracellular matrices. They also demonstrate that ECM molecules are under control of the ovarian steroid hormones, which alternate periodically during the estrous cycle, regulating uterine structure and function. High resolution autoradioautography demonstrated a highly differential binding of 3H-estradiol to luminal and glandular epithelia with region- and time-specific changes of related effects on cell proliferation, differentiation, and secretion, probably involving involution and remodeling (Zorn et al., 2003). Similar region-and time specific changes are observed in the endometrial compartments (manuscript in preparation). These regional differences on estradiol binding may be related with the differential synthesis of proteoglycans exhibited by cells of the superficial and deep stroma.
SLRPs in the Myometrium
We found that all four SLRPs were present in the myometrium. However, we observed notable changes in their expression and degradation, according to the estrous cycle stage. Fascinatingly, we also observed that each muscle layer of the myometrium has a particular behavior concerning the expression of proteoglycans and the estrous cycle stage.
Decorin had an intriguing distribution in the myometrium. In all stages of the estrous cycle, it was present inside muscle cells of the internal layer, whereas immunoreaction was never present inside cells of the external layer, suggesting that only the cells of the internal layer are committed with synthesis and secretion of decorin. Interestingly, the immunoreaction for decorin around bundles of cells of the external layer seems to be stronger in metaestrus than in proestrus and estrus. However, in diestrus, the immunoreaction was weakly detected. These results indicate an intense remodeling of decorin in the myometrium that is probably modulated by the hormonal levels. Differently from decorin, biglycan was immunolocalized inside of cells from the internal or the external layer only in proestrus and diestrus, respectively. This is a very intriguing behavior that suggests the existence of a fine control specific for each one of the myometrium layers. Both decorin and biglycan are known as TGF-β binding molecules modulating the proliferative capability of this growth factor (Hildebrand et al., 1994). It is possible that synthesis and degradation of these molecules may be related with proliferation of muscle cells. In fact, the morphological observation shows intense modifications in the thickness of the myometrium layer along the estrous cycle (data not shown).
An interesting and dynamic relationship between fibromodulin and lumican was observed. These two molecules were alternately expressed inside and outside smooth muscle cells according to the stage of the estrous cycle, showing a dynamic synchrony between synthesis, secretion, and degradation of both molecules. In fact, Svensson et al. (1999) studied the ratio between lumican and fibromodulin and estimated it to be 1:3, in normal mice tendons. In absence of fibromodulin, the amount of lumican was multiplied by four. This suggests that lumican and fibromodulin compete for the same binding site on collagen fibrils and that these proteoglycans may exert important roles in smooth muscle biological processes, such as cell proliferation and differentiation, possibly under control of ovarian hormones.
A previous study conducted by Levens et al. (2005) showed that gonadotropin-releasing hormone analogue (GnRHa) influences the differential expression of fibromodulin in the myometrium at the proliferative and secretory phases of the menstrual cycle, suggesting again that this proteoglycan is under the control of ovarian hormones. Notwithstanding, the functional relevance of fibromodulin and the other proteoglycans in the myometrium needs to be better understood.
SLRPs in Glandular and Luminal Epithelia
Curiously, only biglycan and fibromodulin were immunodetected in the epithelial tissues. Proteoglycans have been previously observed in epithelial cells in other models. Schaefer et al. (2000) evidenced the presence of biglycan in glomerular endothelial cells and in distal tubular cells of the kidney, whereas Qian et al. (2003) showed that fibromodulin was strongly expressed in gingival epithelia. These data suggest that these molecules might be synthesized or internalized by some epithelial cells via specific receptors.
In addition to playing structural roles, SLRPs have been reported to interact with molecular regulators, such as EGF, TGF-β, and TNFα. Their glycosaminoglycan chains enable these proteoglycans to provide a sink for growth factor accumulation, thus modulating cell metabolism. By binding to growth factors, SLRPs influence and regulate cell functions, such as adhesion, migration, proliferation, differentiation, and apoptosis. Therefore, they may induce intracellular signaling cascades through cell-ECM interactions (Yamaguchi and Ruoslahti, 1988; Hildebrand et al., 1994; Roughley, 2006). Fibromodulin is known to be responsible for TGF-β retention in the ECM. In fact, Levens et al. (2005) demonstrated that TGF-β increased five-fold the expression of fibromodulin in the myometrium of human uteri. Burton-Wurster et al. (2003) reported that TGF-β modulates the synthesis and accumulation of decorin, biglycan, and fibromodulin in cartilage. Moreover, there are indications that the interaction between decorin and TGF-β is competitively inhibited by biglycan (Hocking et al., 1998). Decorin binding is thought to neutralize TGF-β biological activity. Nevertheless, due to the reversibility of this interaction, decorin also acts as a local reservoir for TGF-β in tissues (Ruoslahti and Yamaguchi, 1991).