Osteopontin: a leading candidate adhesion molecule for implantation in pigs and sheep

Abstract

Osteopontin (OPN; also known as Secreted Phosphoprotein 1, SPP1) is a secreted extra-cellular matrix (ECM) protein that binds to a variety of cell surface integrins to stimulate cell-cell and cell-ECM adhesion and communication. It is generally accepted that OPN interacts with apically expressed integrin receptors on the uterine luminal epithelium (LE) and conceptus trophectoderm to attach the conceptus to the uterus for implantation. Research conducted with pigs and sheep has significantly advanced understanding of the role(s) of OPN during implantation through exploitation of the prolonged peri-implantation period of pregnancy when elongating conceptuses are free within the uterine lumen requiring extensive paracrine signaling between conceptus and endometrium. This is followed by a protracted and incremental attachment cascade of trophectoderm to uterine LE during implantation, and development of a true epitheliochorial or synepitheliochorial placenta exhibited by pigs and sheep, respectively. In pigs, implanting conceptuses secrete estrogens which induce the synthesis and secretion of OPN in adjacent uterine LE. OPN then binds to αvβ6 integrin receptors on trophectoderm, and the αvβ3 integrin receptors on uterine LE to bridge conceptus attachment to uterine LE for implantation. In sheep, implanting conceptuses secrete interferon tau that prolongs the lifespan of CL. Progesterone released by CL then induces OPN synthesis and secretion from the endometrial GE into the uterine lumen where OPN binds integrins expressed on trophectoderm (αvβ3) and uterine LE (identity of specific integrins unknown) to adhere the conceptus to the uterus for implantation. OPN binding to the αvβ3 integrin receptor on ovine trophectoderm cells induces in vitro focal adhesion assembly, a prerequisite for adhesion and migration of trophectoderm, through activation of: 1) P70S6K via crosstalk between FRAP1/MTOR and MAPK pathways; 2) MTOR, PI3K, MAPK3/MAPK1 (Erk1/2) and MAPK14 (p38) signaling to stimulate trohectoderm cell migration; and 3) focal adhesion assembly and myosin II motor activity to induce migration of trophectoderm cells. Further large in vivo focal adhesions assemble at the uterine-placental interface of both pigs and sheep and identify the involvement of sizable mechanical forces at this interface during discrete periods of trophoblast migration, attachment and placentation in both species.

Keywords: Implantation; Integrins; Pigs; Psteoponti; Sheep.

Figure 1

OPN is synthesized and secreted from the luminal epithelium (LE) only at sites of direct attachment of uterus to placenta. A) H&E stained paraffin embedded thin section of the uterine/placental interface of a Day 80 pregnant gilt illustrating an areola containing histotroph (note the intense red eosin protein staining) secreted by the glandular epithelium (GE). B) OPN mRNA (top panels) and protein (bottom panels) is expressed in the uterus of a Day 80 pregnant gilt (expression begins in luminal epithelium (LE) on Day 13, in GE by Day 35, and then in both cell types to term). Note that OPN is not detectable in uterine LE associated with areolae where there is no direct attachment of uterine LE to placental trophectoderm/chorion). This precise spatial distribution for OPN expression strongly suggests that it plays a role for attaching uterus to placenta during epitheliochorial placentation.

Figure 2

Expression, regulation and proposed function of OPN produced by the uterine LE of pregnant pigs. A) As porcine conceptuses (Trophoblast) elongate they secrete estrogens for pregnancy recognition. These estrogens also induce the synthesis and secretion of OPN (osteopontin) from the uterine LE (luminal epithelium) directly adjacent to the conceptus undergoing implantation[58]. The implantation cascade is initiated when progesterone from CL down-regulates Muc 1 on the surface of uterine LE[84]. This exposes integrins on the LE and trophoblast surfaces[84] for interaction with OPN, and likely other ECM proteins, to mediate adhesion of trophoblast to LE for implantation[58, 59, 64]. B) In vitro experiments have identified the αvβ6 integrin receptor on trophoblast, and the αvβ3 integrin receptor on LE as binding partners for OPN[64]. OPN may bind individually to these receptors to act as a bridging ligand between these receptors. Alternatively, OPN may serve as a bridging ligand between one of these receptors and an as yet unidentified integrin receptor expressed on the opposing tissue.

Figure 3

OPN stimulates in vitro activation of integrin receptors to form focal adhesions at the apical surface of oTr1 cells. A) Cartoon illustrating a polystyrene bead coated with recombinant rat OPN containing an intact RGD integrin binding sequence, and allowed to settle onto a cultured oTr1 cell. Note the illustrated representation of aggregated integrins, indicative of focal adhesion assembly, at the interface between the surface of the bead and the apical membrane of the cell[52, 64, 66]. B) Immunofluorescence co-localization (left panels) to detect the aggregation of αv integrin subunit (right panels) and talin middle panels), an intracellular component of focal adhesions, around beads coated with recombinant rat OPN containing an intact RGD integrin binding sequence (RGD) or coated with recombinant OPN containing a mutated RAD sequence that does not bind integrins[66]. Optical slice images from the apical plasma membrane of oTr1 cells are shown. Note the apical focal adhesions represented by immunofluo rescence co-localization (yellow color) of the integrin αv subunit with talin that results from integrin activation in response to binding of intact OPN on the surface of the bead. No apical focal adhesions were induced by beads coated with mutated OPN as evidenced by lack of integrin αv and talin aggregation around the bead.

Figure 4

Expression, regulation and proposed function of OPN produced by the uterine GE of pregnant sheep. A) As the lifespan of the CL is extended as the result of the actions of interferon tau secretion from elongating ovine conceptuses (Trophoblast) they secrete progesterone. Progesterone then induces the synthesis and secretion of OPN (Osteopontin) from the uterine GE (Glandular Epithelium)[51]. The implantation cascade is initiated with down-regulation Muc 1 (the regulatory mechanism remains to be identified) on the LE surface to expose integrins on the LE and trophoblast surfaces for interaction with OPN to mediate adhesion of trophoblast to LE for implantation[29, 51, 52, 66]. B) In vitro experiments have identified the αvβ3 integrin receptor on trophoblast as a binding partner for OPN[66]. OPN then likely acts as a bridging ligand between αvβ3 on trophoblast and as yet unidentified integrin receptor(s) expressed on the opposing uterine LE. Note that the α5 integrin subunit was immunoprecipitated from membrane extracts of biotinylated oTr1 cells that were eluted from an OPN-Sepharose column, but the β1 integrin subunit, the only known binding partner for α5, could not be immunoprecipitated. Therefore, while we cannot definitively state that OPN binds α5β1 integrin on oTr1, we are reticent to exclude this possibility.