Along with the invasion of trophoblasts into the maternal endometrium, sequential decidual reactions occur and lead to the formation of two distinct zones: the differentiated zone proximal to an implanting embryo termed the primary decidual zone, and the distal peripheral zone consisting of immature mitotic decidual cells, termed the secondary decidual zone [22
]. In our present study, we have demonstrated that ChM-I is expressed in the primary decidual zone during early pregnancy in mice. In contrast to Prl8a2
, which is broadly expressed in decidual tissue and strongly induced during decidualization [10
transcripts were localized to well-differentiated decidual cells in the primary decidual zone and were not detectable in the secondary decidual zone. Consistent with this, ChM-I
was induced in cultured endometrial stromal cells that were undergoing decidualization in the presence of estrogen and progesterone in vitro
. Both ChM-I protein and Prl8a2
mRNA were evident in the enlarged multi-nucleated well-differentiated cells in primary cultures of decidual cells [24
The invasion of trophoblasts and maternal blood vessels are coordinately regulated in the primary decidual zone during early pregnancy. It has been known that the primary decidual zone remains to be vascularized prior to the invasion of trophoblasts [23
]. Interestingly, ChM-I
expression is predominantly detected at the mesometrial side of primary decidua facing the zone of vascularization, whereas the intense signals for TIMP-3
expression, another marker for well-differentiated decidual cells [25
], were found at the antimesometrial side of primary decidua. When the trophoblasts were extensively detected along the margin of the implantation chamber at 7.5 days p.c., ChM-I was expressed at the entire zone of the primary decidua surrounding the invading trophoblasts, suggesting that ChM-I may participate in the invasion processes of trophoblasts.
The capacity for ECM degradation is one of the critical components required for the invasive behavior of cells [26
]. Trophoblasts indeed express various kinds of proteinases, and proteinase inhibitors are localized in the decidua. For instance, it has been shown that TIMPs, cystatins, and plasminogen activator inhibitors are expressed in the distinct domains of the decidua [25
]. In particular, MMP-9 and TIMP-3 are expressed at considerably high levels in the periphery of the implantation chamber and are thought to be pivotal regulators of ECM remodeling during the invasion of trophoblasts. However, trophoblast invasion proceeded relatively unhindered by the administration of a synthetic MMP inhibitor GM6001 into pregnant mice or the overexpression of TIMP-1 [14
], while these treatments had led to a reduced size of the decidua and mesometrial displacement of an embryo. These observations are compatible with our present results showing that GM6001 had only a marginal inhibitory effect on the invasion of Rcho-1 trophoblast cells. TIMP-3 had also little impact on the Matrigel invasion of Rcho-1 trophoblast cells (data not shown). In contrast, ChM-I exhibited remarkable anti-invasive activity in the Matrigel invasion assay of Rcho-1 trophoblast cells, even though it did not interfere with the production and activation of gelatinases in the cells. The anti-invasive activity of rhChM-I was also evident in the Matrigel invasion of HUVECs [8
], in which GM6001 was ineffective to block the invasion of cells (data not shown).
We have previously demonstrated that ChM-I inhibits the migration of vascular endothelial cells induced by angiogenic growth factors including VEGF-A, FGF-2, and IGF-I [8
]. As suggested by the present RT-PCR study, EPCs are also capable of responding to these growth factors. Indeed, it has been reported also that IGF-I is a potent stimulator of mouse EPC cells and human trophoblast migration [13
]. Similarly, IGF-I and FGF-2, but not VEGF-A (data not shown), stimulated the migration of Rcho-1 trophoblast cells in culture. Using a modified Boyden chamber assay, we found that rhChM-I completely abolishes the IGF-I-induced migration of Rcho-1 trophoblast cells. Morphologies of ChM-I-treated Rcho-1 trophoblast cells are also indicative of a critical reduction in cellular motility. This mode of ChM-I action is analogous to that on HUVECs [8
]. Thus, ChM-I is likely to inhibit the invasion of Rcho-1 trophoblast cells and vascular endothelial cells by suppressing their cellular motility stimulated by IGF-I.