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Osteopontin: Roles in Implantation and Placentation¹

Center for Animal Biotechnology and Genomics,³ Department of Veterinary Anatomy and Public Health,⁴ College of Veterinary Medicine, Department of Animal Science,⁵ College of Agriculture and Life Sciences, Texas A&M University, College Station, Texas 77843

	ABSTRACT

TOP ABSTRACT INTRODUCTION OSTEOPONTIN STRUCTURE AND... OPN IS A MAJOR... OPN IS A PROGESTERONE-REGULATED... OPN BINDS AND ACTIVATES... OPN IS A MARKER... CONCEPTUS EXPRESSION OF OPN OPN EXPRESSION BY ENDOMETRIAL... SUMMARY REFERENCES

 
Osteopontin (OPN) is an acidic member of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family of extracellular matrix proteins/cytokines that undergoes extensive posttranslational modification, including phosphorylation, glycosylation, and cleavage, yielding molecular mass variants ranging in size from 25 to 75 kDa. The result is a versatile protein(s) with multiple functions arising from its role as a mediator of cell-cell and cell-extracellular matrix (ECM) communication that encompass both normal and tumorigenic developmental processes, immunological responses during inflammation and wound healing, and biomineralization. Studies in primates, pigs, sheep, and rodents have revealed that OPN is a major constituent of the uterine-placental microenvironment with influence as 1) a component of histotroph required for adhesion and signal transduction at the uterine-placental interface throughout pregnancy, 2) a gene product expressed by uterine stroma contributing to a decidualization-like transformation that correlates with the degree of conceptus invasiveness, and 3) a product of resident uterine and placental immune cells that may regulate their behavior and cytokine production. This minireview summarizes information regarding uterine and placental expression of OPN that has accumulated over the past 15 yr, and we briefly describe structural/functional properties of this protein that are likely relevant to its role(s) during pregnancy. Comparative studies have offered insights into the potential hormonal/cytokine, cellular, and molecular mechanisms underlying OPN-mediated adhesion, remodeling, and cell-cell/cell-ECM communication within the uterus and placenta. OPN has the potential to profoundly impact pregnancy, and investigators are now challenged to focus on the mechanistic nature of the functions of this multifaceted and major component of the uterine-placental microenvironment.

conceptus, implantation, placenta, pregnancy, uterus

	INTRODUCTION

TOP ABSTRACT INTRODUCTION OSTEOPONTIN STRUCTURE AND... OPN IS A MAJOR... OPN IS A PROGESTERONE-REGULATED... OPN BINDS AND ACTIVATES... OPN IS A MARKER... CONCEPTUS EXPRESSION OF OPN OPN EXPRESSION BY ENDOMETRIAL... SUMMARY REFERENCES

 
The greatest limitation to reproductive efficiency across mammalian species is embryonic mortality, estimated to be 25%–60% [1]. Most losses are characterized by asynchrony between conceptus (embryo/fetus and associated membranes) signals and uterine receptivity, resulting in defective implantation and/or placentation. Women suffer high rates of unexplained infertility and early embryo loss [2], and several studies have documented a correlation between defects in uterine receptivity and unexplained infertility [3], endometriosis [4], and luteal phase defects [5, 6]. It is widely accepted that asynchronous endometrial development during in vitro fertilization/embryo transfer procedures contributes to low pregnancy rates. In all eutherian mammal species, several physiological events are required for development of a receptive uterus that will support pregnancy. The first event is synthesis and secretion of histotroph by the endometrial glandular epithelium (GE), which supports conceptus development for at least the first one-third of pregnancy in humans [7] and for the duration of pregnancy in domestic animals by influencing fetal-placental development. The second event is reorganization of the endometrial luminal epithelium (LE) to allow its intimate association with conceptus trophectoderm (Tr). Receptors on LE become exposed or induced during the receptive period, and adhesive molecules then function sequentially or in parallel to stabilize attachment of Tr to LE. Once Tr has adhered to the uterine LE, the inherently invasive nature of the conceptus must be inhibited (domestic animals) or restricted to a circumscribed region of the endometrium. Implantation and placentation then take place in an environment enriched in immune cells that protect against infectious pathogens and promote inflammatory cytokines for tissue remodeling while protecting the fetal-placental semiallograft from attack by the maternal immune system. Osteopontin (OPN) is an extracellular matrix (ECM) molecule and cytokine hypothesized to influence the uterine environment as 1) a component of histotroph required for adhesion and signal transduction at the uterine-placental interface, 2) a gene product expressed by uterine stroma as it decidualizes in response to conceptus invasion, and 3) a product of resident placental and uterine immune cells that regulates their behavior and cytokine production. The regulation and immediate functional implications of events involving OPN, although independent, probably are temporally and spatially orchestrated to ensure establishment and maintenance of a successful pregnancy.

	OSTEOPONTIN STRUCTURE AND FUNCTION

TOP ABSTRACT INTRODUCTION OSTEOPONTIN STRUCTURE AND... OPN IS A MAJOR... OPN IS A PROGESTERONE-REGULATED... OPN BINDS AND ACTIVATES... OPN IS A MARKER... CONCEPTUS EXPRESSION OF OPN OPN EXPRESSION BY ENDOMETRIAL... SUMMARY REFERENCES

 
OPN is an acidic member of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family of proteins that include bone sialoprotein, dentin matrix protein I, dentin sialophosphoprotein, and matrix extracellular phosphoglycoprotein, which are the products of five genes clustered along human chromosome 4 [8]. OPN has been described as a transformation-specific phosphoprotein [9] named 2ar [10], as an ECM protein of bone [11–14] with potential to bridge between cells and hydroxyapatite [15], and as a cytokine produced by activated lymphocytes and macrophages called early T-cell activation factor 1 (Eta-1) [16]. Because of the breadth of the scientific literature pertaining to OPN, this minireview focuses exclusively on the multiple roles of OPN within the mammalian uterus. For excellent descriptions of the general structure and function of OPN please see reviews by Denhardt and Guo [17], Butler et al. [18], and Sodek et al. [19].

In general, OPN is a monomer ranging in length from 264 to 301 amino acids that undergoes extensive posttranslational modification, including phosphorylation, glycosylation, and cleavage, resulting in molecular mass variants ranging from 25 to 75 kDa (see Fig. 1). OPN contains a hydrophobic leader sequence characteristic of a secreted protein, a potential calcium phosphate apatite binding region of consecutive asparagine residues, a cell attachment GRGDS sequence, a thrombin cleavage site, and two glutamines that are recognized substrates for transglutaminase-supported multimer formation [18]. Genes encoding OPN have been cloned from eight species, including rat [15], mouse [10], human [20, 21], cow [22], chicken [23], rabbit [24], and sheep [25]. The cDNA sequences from these species reveal only moderate conservation [25], except in the NH₂-terminal region, around the Arg-Gly-Asp (RGD) integrin-binding sequence, and the COOH-terminus [18]. Sequences that are highly conserved in all these species include 1) 4 of the 9 or 10 residues in the poly-Asp region, 2) the GRGDS sequence, 3) 15 serine residues that include an SSEEK sequence (residues 26–30), 4) three threonines (residues 131, 140, and 145), 5) an NES sequence (residues 79–81), and 6) glutamines at positions 50 and 52. Two features shared by ovine and bovine OPN but not other species are deletion of 22 amino acids that would otherwise be inserted between residues 196 and 197 and replacement of the RS with a KS thrombin cleavage site [18].

View larger version (29K): [in this window] [in a new window]   FIG. 1. OPN structure, integrin-binding sequences and cognate receptors, and sites for proteolytic cleavage. In the lower half of the figure are a hypothetical structure for OPN based upon the amino acid sequence and secondary structure prediction of functional domains of the ECM protein/cytokine and positions of posttranslational modifications. The amino acid sequences of several conserved regions that include the GRGD integrin binding site, the polyaspartate sequence believed to be important in binding to bone mineral, the transglutaminase cross-linking site, and thrombin-cleavage site also are shown. The capital letters indicate amino acids that are identical in seven species in which OPN has been sequenced, and the lowercase letters indicate conserved amino acids (adapted from Sodek et al. [19] with permission). In the upper half of the figure is a portion of the human OPN amino acid sequence (residues 185–217) that contains integrin recognition sites and specific integrin receptors that bind these sites. Not listed are the leukocyte integrins mß2 and xß2, which also bind the RGD sequence. The amino acid sequence also shows cleavage sites for thrombin, MMP-3 (stromelysin 1), and MMP-7 (matrilysin), which yield OPN fragments with different bioactivities (adapted from Agnihotri et al. [28] with permission)

OPN undergoes extensive posttranslational modifications believed to be important to its function. These modifications include proteolytic cleavage [26–28], phosphorylation on serine and threonine residues [13, 28], glycosylation with primarily O-linked oligosaccharides [29, 30], cross-linking with self and other macromolecules through transglutaminase [31, 32], and sulfation [33]. Originally isolated from bone [11], OPN has been found on epithelial cells and in secretions of the gastrointestinal tract, kidneys, thyroid, breast, uterus, placenta, and testes [25, 34–40]. OPN is also expressed by leukocytes, smooth muscle cells, and highly metastatic cancer cells [41–43]. OPN has been reported to 1) stimulate cell-cell adhesion, 2) increase cell-ECM communication, 3) promote migration of immune cells, osteocytes, and tumor cells, 4) decrease cell death by reducing reactive oxygen species and nitric oxide production by injured tissues, 5) stimulate immunoglobulin production by B cells, 6) induce changes in the phosphorylation state of focal adhesion kinase and paxillin, 7) stimulate phosphotidylinositol 3'-kinase activity, 8) alter intracellular calcium levels, and 9) affect tissue mineralization and promote calcium phosphate deposition in bone [44–54]. Increases in transcription of the OPN gene are induced by interleukin (IL) 1 and ß, transforming growth factor ß-1, fibroblast growth factor, tumor necrosis factor , interferon (IFN) , estrogen, progesterone, glucocorticoids, and 1,25-dihydroxy vitamin D3 [55–61].

The RGD amino acid sequence of OPN interacts with cell surface integrin receptors to mediate cell adhesion, migration, differentiation, survival, and immune function [44, 62–69]. Integrins are dominant glycoproteins in adhesion cascades. They belong to a ubiquitous family of cation-dependent, heterodimeric intrinsic transmembrane glycoprotein receptors composed of noncovalently bound and ß subunits that participate in cell-cell and cell-ECM adhesion, cause cytoskeletal reorganization to stabilize adhesion, and transduce signals through numerous signaling intermediates [70–72]. Although generally it has been accepted that OPN binds primarily to vß3 integrin heterodimers via its RGD sequence, recent identification of other receptors and alternative binding sequences has expanded this signaling repertoire (Fig. 1). The vß1, vß5, and 8ß1 integrins have affinities for the RGD motif of OPN that are similar to that of vß3 [73, 74], and OPN has binding affinity for the leukocyte integrins mß2 and xß2 [75]. Evidence for non-RGD-mediated integrin binding to OPN is illustrated by the discovery of dual 4ß1 binding sites through which OPN promotes leukocyte adhesion [76, 77]. There is also a cryptic binding sequence recognized by 9ß1 [78]. OPN is also an extracellular ligand for certain splice variants of the cell surface proteoglycan CD44 (CD44v3-v6), where it serves as a proadhesion cytokine immobilized on the luminal surface of endothelium to recruit leukocytes and trigger conversion of integrin expression to an active adherence configuration [27, 49, 79]. CD44 variants may require cooperation with ß1 to bind multiple yet unidentified domains in OPN and stimulate cell motility and chemotaxis [80]. Additionally, intracellular OPN is part of a hyaluronan-CD44-ERM (ezrin/radixin/moesin) attachment complex involved in fibroblast, macrophage, and tumor cell migration, suggesting that OPN stimulates or participates in motogenic activity of cells [81, 82].

The different signals that OPN elicits are attributed to its multiple receptors and binding sites and to its various molecular/structural forms. Upon freezing and thawing or treatment with serine proteases such as thrombin, the 70-kDa native protein gives rise to 24-kDa and 45-kDa fragments that were initially shown to bind antigen and to nonspecifically suppress T-helper lymphocytes, respectively [26, 27]. The 45-kDa amino-terminal OPN fragment retaining the RGD sequence was subsequently shown to improve cell attachment and spreading properties through increased accessibility of the RGD to integrin receptors [83]. Only the 45-kDa OPN supports 9ß1 and 4ß1-mediated cell migration and adhesion [76, 84] and attachment through several other non-RGD sites [85]. OPN is also a substrate for cleavage by matrix metalloproteinases (MMPs) through one novel site cleaved by both MMP-3 (stromelysin 1) and MMP-7 (matrilysin) and another site cleaved by MMP-3 alone (Fig. 1). Fragments formed by these cleavage events bind cell surface integrins to initiate adhesion and migration [28]. OPN exists in a polymerized form cross-linked by tissue transglutaminase (Fig. 1), which alters OPN conformation to increase binding to collagen. In bone, polymerized OPN is hypothesized to participate in cell adhesion, matrix assembly and maturation, and calcification by simultaneously binding multiple receptors on different cells [32]. Homotypic OPN-OPN bonds have high tensile strength in vitro, supporting the idea that self-assembly is involved in cell-cell and cell-matrix adhesion [86].

	OPN IS A MAJOR COMPONENT OF HISTOTROPH

TOP ABSTRACT INTRODUCTION OSTEOPONTIN STRUCTURE AND... OPN IS A MAJOR... OPN IS A PROGESTERONE-REGULATED... OPN BINDS AND ACTIVATES... OPN IS A MARKER... CONCEPTUS EXPRESSION OF OPN OPN EXPRESSION BY ENDOMETRIAL... SUMMARY REFERENCES

 
All mammalian uteri contain endometrial glands that secrete histotroph, which includes enzymes, growth factors, cytokines, lymphokines, hormones, transport proteins, and other substances [87–90]. Histotroph plays a role in conceptus nourishment, production of pregnancy recognition signals, immunoprotection, attachment, implantation, and placentation [91–94]. GE secretions support conceptus development in humans [7] and influence conceptus development and growth of the fetus and placenta in sheep [95]. Recent studies with ewes, in which uterine glands were epigenetically ablated (uterine gland knockout [UGKO] ewes) by neonatal progestin exposure, confirm that histotroph is required to maintain pregnancy through the peri-implantation period when conceptus Tr must adhere to the epithelial lining of the uterine lumen [96–99]. The LE is a simple polarized layer that is normally nonadhesive; however, this characteristic is lost during an adhesion cascade that allows two apical surfaces, i.e., Tr and uterine LE, to make contact with and attach to each other [100, 101]. OPN, a key component of histotroph that is distinctly absent from uterine flushings from UGKO ewes [102], is an adhesion molecule likely involved in the implantation cascade [4, 25, 38, 102, 103].

The original observation that OPN is expressed by uterine epithelia arose from studies to determine possible roles of 2ar/OPN in the cell cycle and in cell proliferation during mouse embryogenesis [104]. In situ hybridization indicated high levels of OPN mRNA in a number of epithelial tissues, including kidney tubules, sensory epithelium of the embryonic ear, and uterine endometrium [104]. Although uterine LE and GE adjacent to deciduoma expressed OPN whereas epithelia in the contralateral horn were negative, the authors did not focus on OPN expression [104]. Subsequent immunocytochemical studies by Young and colleagues [35] localized OPN protein to secretory phase endometrial GE of cyclic women. Because OPN was absent from GE during the proliferative phase of the menstrual cycle, it was suggested that changes in expression in GE of normal cycling endometrium were the result of hormonal regulation and that the function(s) of OPN in the endometrium was likely associated with its ability to enhance cell attachment [35]. A significant advance in thinking about the function of epithelium-derived OPN was made in 1992, when OPN mRNA and protein were localized to the epithelia of a variety of organs, including the hypersecretory endometrial GE associated with pregnancy in women [36]. The investigators hypothesized that OPN secreted by epithelium binds integrins on luminal surfaces to effect communication between LE and the external environment [36].

A substantial body of work in the 1990s established that transient endometrial expression of vß3 and 4ß1 integrins is dependent on the stage of the sexual cycle and defines the window of implantation in women [4, 105, 106] and that altered expression of these integrins is correlated with several conditions associated with human infertility [3, 5]. In mice, null mutations of v, 5, ß1, or ß5 integrin genes result in peri-implantation lethality and failure of chorioallantoic membrane fusion [107], and a functional blockade of v and ß3 integrins in mice and rabbits reduces the number of implantation sites [108, 109]. Noting that the vß3 and 4ß1 integrin heterodimers present during the implantation window bind the RGD amino acid sequence in OPN, Lessey and coworkers [4] suggested that involvement of OPN and integrins in trophoblast-endometrial interactions during the initial attachment phase of implantation is plausible and merits closer examination. This suggestion was reinforced by observations that CD44, another OPN receptor, is present through the blastocyst stage of human preimplantation development [110] and that uterine epithelium expresses CD44 and its larger variant CD44 isoforms [111].

A comprehensive examination of temporal and spatial expression of uterine OPN mRNA and protein in sheep provided the first clear evidence that OPN is a secretory product of endometrial glands (histotroph) that binds integrins on endometrial LE and Tr. Comparison of expression patterns between in situ hybridization and immunofluorescence analyses of cyclic and pregnant ovine uterine sections revealed a significant divergence. OPN mRNA increased in the endometrial GE during the peri-implantation period; however, it was not present in LE or conceptus Tr [25]. In contrast, immunoreactive OPN protein was present at the apical surfaces of endometrial LE and GE and on Tr [38]. A 45-kDa proteolytic fragment of OPN, known to be more stimulatory to cell attachment and migration than native OPN [83], was detected exclusively on LE and Tr (Fig. 2). A wide array of OPN proteins representing the native 70-kDa form, 45- and 25-kDa cleavage fragments, and probable high-molecular-weight transglutaminase-stabilized multimers (Fig. 2) were detected by Western blot analysis of endometrium from pregnant ewes [38]. In addition, the integrin subunits v, 4, 5, ß1, ß3, and ß5, which could contribute to the assembly of several OPN receptors including vß3, vß1, vß5, 4ß1, and 5ß1 heterodimers, were constitutively expressed on Tr and the apical surface of endometrial LE [38, 103]. A key finding was an increase in the amount of 45-kDa OPN in uterine flushings from pregnant ewes during the attachment phase of implantation [38]. Pregnant Day 14 UGKO ewes, which lack uterine glands, exhibit an absence of OPN in uterine flushings compared with normal bred ewes [102]. These results indicated that OPN is a component of histotroph secreted from GE into the uterine lumen of pregnant ewes and that OPN binds integrin receptors expressed on endometrial LE and conceptus Tr, where it can stimulate changes in morphology of Tr and mediate adhesion between LE and Tr essential for implantation and placentation [38].

View larger version (110K): [in this window] [in a new window]   FIG. 2. Detection of OPN protein in ovine uterus and placenta by Western blotting and immunofluorescence staining of frozen sections. Immunoreactive OPN proteins were detected using IgG LF-124 [197] against the amino half of recombinant human OPN (upper left) ending at the thrombin (RS) cleavage site and containing all integrin binding sites and using IgG LF-123 [197] against the carboxyl half of the molecule. When a cocktail of both LF-123 and LF-124 antibodies is used to detect OPN in ovine endometrial extracts by Western blotting (upper right, two different animals), 70-, 45-, 25-, and 140-kDa fragments (presumably OPN multimers stabilized by transglutaminase) are detected. Immunofluorescent staining of frozen pregnant ovine uterus from Days 14 and 120 of pregnancy using LF-123 and LF-124 antibodies separately is shown in the lower panel. The white arrow denotes the uterine-placental interface. The 45-kDa OPN cleavage fragment is exclusively, continuously, and abundantly present along the apical surface of LE, on Tr, and along the entire uterine-placental interface (white arrow) through Day 120 of pregnancy. There is a dramatic increase in OPN at the uterine-placental interface and within the endometrial stromal compartment between Day 14 and Day 120. PL, Placenta; ST, uterine stroma. Width of microscopic fields is 940 µm. Modified from Johnson et al. [123] with permission

Uterine OPN expression has been detected during the peri-implantation period in humans [112, 113], pigs [114], and rabbits [115], confirming that OPN is an important epithelial protein involved in implantation in several mammalian species with diverse types of implantation and placentation. Results from two studies in the human endometrium were highly similar to results from sheep regarding the temporal/spatial deposition of OPN. In both studies, OPN mRNA and protein increased in the GE during the mid to late secretory phase [112, 113], and Apparao et al. [112] localized OPN protein, but not mRNA, to the apical LE surface. OPN protein was also present in GE cells showing evidence of secretory activity [112], and increasing levels of OPN were detected in uterine secretions from the secretory phase in women [113]. Hence, it is probable that in humans OPN is synthesized in the GE and secreted into the uterine lumen and then binds to integrins on the LE. In rabbits, OPN mRNA and protein are also expressed by the endometrial GE during the peri-implantation period. OPN expression in GE increases at the time of initial conceptus attachment but is not detectable in the nonpregnant endometrium [115]. Pigs have a novel expression pattern for OPN mRNA. Unlike in other species, in pigs OPN is transcribed directly in endometrial LE, and GE mRNA expression does not begin until Day 35 of pregnancy [114]. This unique expression of OPN mRNA in LE begins in discrete regions of the endometrial LE associated with the presence of the conceptus prior to conceptus attachment and expands to the entire LE by Day 20, when firm adhesion of conceptus to uterus has occurred [114]. The pig has an epitheliochorial type of placentation in which the LE remains intact throughout pregnancy, whereas other mammals undergo differing degrees of LE degeneration and fusion with Tr during placentation. Therefore, the LE of pigs provides an uninterrupted supply of OPN protein at the interface between LE and conceptus during implantation, whereas other species use OPN synthesized and secreted from GE to ensure continued expression at these sites. The expression of OPN mRNA in porcine LE offers strong evidence that OPN at the maternal-fetal interface is critical and emphasizes that functional properties of LE and interactions between LE and Tr are essential to the processes of implantation and placentation. In support of this hypothesis, the SPP1 gene (OPN) has recently been reported as one of several putative quantitative trait loci on porcine chromosome 8 associated with reproductive performance [116]. Specifically, SPP1 is a candidate for the litter size and prenatal survival traits based on positional data and growing physiological evidence for the involvement of OPN in implantation and placentation.

	OPN IS A PROGESTERONE-REGULATED ENDOMETRIAL SECRETORY PROTEIN

TOP ABSTRACT INTRODUCTION OSTEOPONTIN STRUCTURE AND... OPN IS A MAJOR... OPN IS A PROGESTERONE-REGULATED... OPN BINDS AND ACTIVATES... OPN IS A MARKER... CONCEPTUS EXPRESSION OF OPN OPN EXPRESSION BY ENDOMETRIAL... SUMMARY REFERENCES

 
Progesterone plays pivotal roles during gestation, including preparation of the endometrium for implantation, maintenance of pregnancy, and uterine quiescence. Studies of endometrial OPN have focused on its regulation by progesterone because 1) the OPN gene contains a progesterone-response element in its 5'-flanking region and is induced by progesterone in vivo in mice [58], 2) OPN is upregulated in human cytotrophoblasts by progesterone [60], and 3) the temporal pattern of OPN expression in endometrium suggests an influence of progesterone [25, 35, 36, 104, 117]. Three recent studies have confirmed that endometrial epithelial expression of OPN is influenced by progesterone. In the first, a steroid-treated uterine-infused ovariectomized ewe model was utilized to determine whether regulation of OPN gene expression by endometrial GE is controlled by progesterone and/or IFN, the pregnancy recognition signal in sheep [118]. Although intrauterine infusion of IFN had no effect on OPN gene expression, exogenous progesterone induced OPN mRNA in GE and increased secretion of the 45-kDa OPN cleavage product. This upregulation of OPN in GE is correlated with silencing of expression of progesterone receptors (PRs) in uterine LE and superficial GE, although PR continues to be expressed in stroma and myometrium. It has been suggested that effects of progesterone on OPN expression in ovine GE are to downregulate epithelial PR to allow for upregulation of OPN and/or to regulate a growth factor(s) produced by the stroma or myometrium (progestamedin) that mediates progesterone effects on GE [118]. Administration of ZK137.316, a PR antagonist, ablated progesterone effects in these ewes [118]. In the second study, injection of progesterone alone or in combination with ZK137.316 convincingly created a stimulatory effect of progesterone on endometrial OPN mRNA expression in rabbits [115]. Progesterone induction of OPN mRNA in unmated rabbits was prevented by addition of the PR antagonist, and estrogen priming did not influence effects of progesterone. Although there is no direct evidence for progesterone regulation of OPN in the endometrium of women, in a third study Apparao and coworkers [112] demonstrated effects of progesterone on OPN expression in transformed human endometrial epithelial (Ishikawa) cells. Expression of OPN was increased by in vitro treatment of cells with estrogen plus progesterone, whereas cotreatment with progesterone and RU-486 reduced the stimulatory effect of progesterone on OPN [112]. In women, where PR-A and -B isoforms are differentially regulated in the endometrium during implantation, it is likely that endometrial OPN is modulated by PR-B alone. Progesterone treatment of cells transfected with PR-A or -B resulted in an increase in OPN in cells containing PR-B but not in those containing PR-A [119].

Global gene profiling using high-density microarray technology has identified genes that either increase or decrease in endometrium as it transitions to an implantation receptive state. About 20% of the changes are attributed to genes encoding cell surface receptors, adhesion and ECM proteins, and growth factors [120]. Two studies of human endometrium have confirmed that OPN expression is upregulated during the receptive phase for implantation. A comparison of gene expression in endometrium from Days 8–10 of the cycle and that from Days 8–10 after midluteal LH surge showed an 8.1-fold increase in OPN mRNA [121]. Similarly, microarray comparison of endometrium from Days 2–4 and Days 7–9 post-LH surge showed a 12.3-fold upregulation [120]. OPN is the most highly up-regulated ECM-adhesion molecule in the human uterus as it becomes receptive to implantation. Previous reports indicated that this upregulation occurs in the endometrial GE and results in increased secretion of OPN into the uterine lumen [35, 36, 112], suggesting a role in uterine-conceptus interactions during early adhesion of the conceptus to LE.

Recently, another microarray study extended uterine gene expression profiling throughout pregnancy in rats. OPN gene expression increased 60-fold between Day 0 of the estrous cycle and Day 20 of pregnancy, the second greatest increase of all genes measured [122]. It is unclear what cell type is responsible for the increase in endometrial OPN expression in rats, but both endometrial OPN mRNA and protein increase in pigs and sheep throughout gestation. In pigs, total endometrial OPN mRNA increases 20-fold between Day 25 and Day 85 of pregnancy. Although OPN mRNA continues to be present in endometrial LE, GE is responsible for the significant increase in OPN expression during gestation. OPN mRNA in GE was first detected on Day 35 and increased markedly between Days 40 and 85 [114] of gestation. Progesterone is likely responsible for this expression, because it regulates endometrial secretory activity in the pig, and secretory activity increases after Day 35 of pregnancy to maximal levels between Day 60 and Day 75 [92], a temporal pattern that mirrors transcriptional activity of the OPN gene in GE [114].

In sheep, the increase in OPN mRNA during pregnancy is even greater than that in pigs. Steady-state levels of OPN mRNA in total ovine endometrium remain constant between Day 20 and Day 40, increase 40-fold between Day 40 and Day 100, and remain maximal thereafter [123]. The major source of this OPN is GE, which undergoes hyperplasia through Day 50, followed by hypertrophy and maximal production of histotroph after Day 60 [124]. Endometrial gland morphogenesis and increases in the OPN from GE are likely influenced by uterine exposure to estrogen, progesterone, IFN, placental lactogen, and placental growth hormone. Sequential treatment of ovariectomized ewes with progesterone, IFN, placental lactogen, and growth hormone results in GE development similar to that observed during normal pregnancy. Administration of progesterone alone in these experiments induced expression of OPN in GE, and intrauterine infusion of IFN and placental lactogen to progesterone-treated ovariectomized ewes increased OPN mRNA levels above those for ewes treated with progesterone alone. Intrauterine infusion of recombinant ovine placental growth hormone did not affect OPN expression, although it did increase levels of uterine milk protein (serine protease inhibitor), another GE secretory product in sheep [125, 126]. Immunofluorescence microscopy revealed that secreted 45-kDa OPN cleavage fragment is exclusively, continuously, and abundantly present along the apical surface of LE, on Tr, and along the entire uterine-placental interface of both interplacentomal and placentomal regions through Day 120 of pregnancy [123] (Fig. 2). These findings are significant because they definitively localize a secretory product of the GE to regions of intimate contact between conceptus and uterus where OPN may influence fetal/placental development and growth and mediate communication between placental and uterine tissues to support pregnancy.

	OPN BINDS AND ACTIVATES INTEGRINS IN ENDOMETRIAL LE AND TR

TOP ABSTRACT INTRODUCTION OSTEOPONTIN STRUCTURE AND... OPN IS A MAJOR... OPN IS A PROGESTERONE-REGULATED... OPN BINDS AND ACTIVATES... OPN IS A MARKER... CONCEPTUS EXPRESSION OF OPN OPN EXPRESSION BY ENDOMETRIAL... SUMMARY REFERENCES

 
Functional evidence for a role for OPN in adhesion and communication between conceptus Tr and uterine LE during implantation and placentation is limited and indirect. OPN-, fibronectin-, and vitronectin-null mouse embryos implant normally as do OPN/vitronectin double mutants [127–129]. However, the abundance of potentially compensating integrins and matrix proteins at the maternal-fetal interface makes these data difficult to interpret. Illera et al. [108] functionally blocked the vß3 integrin heterodimer by intrauterine injection of neutralizing antibodies against the respective integrin subunits, by masking ligand binding sites via intrauterine injection of RGD containing peptides, and by intrauterine injection of the disintegrin echistatin and reported that each procedure reduced numbers of implantation sites in mice. A similar study in rabbits demonstrated reduction in the number of implantation sites after intrauterine injection of neutralizing antibody or GRGDSP hexapeptides, whereas GRGESP hexapeptides had no effect [109]. Although these experiments do not address the role of OPN directly, they illustrate a critical role for vß3 and, by inference, one or more of its ligands, including OPN, during implantation. Immortalized and primary ovine and porcine endometrial LE and conceptus Tr cells, which abundantly express v and ß3 heterodimers at focal adhesion sites of cell anchorage to the basal substrate [103], show evidence of integrin activation via OPN binding. Transmembrane accumulation of talin at the apical surface of these cells in contact with OPN-coated beads revealed functional integrin activation and cytoskeletal reorganization to form in vitro focal adhesions in response to OPN binding. However, OPN-coated beads in which the RGD sequence was mutated to RGE or RAD failed to induce focal adhesions [103, 114].

Functional evidence for integrin-mediated signal transduction in vivo at ovine implantation sites is presented in Figure 3. When serial frozen sections of implantation sites from Day 45 of pregnancy were stained with antibodies to v, ß3, ß5, the ECM proteins OPN and fibronectin, and antibodies to detect focal adhesion formation to cytoskeletal proteins talin, vinculin, or -actinin, nearly identical intense punctate staining for v, ß5, and -actinin was observed. Colocalization of the vß5 integrin receptor with -actinin indicates the presence of specific ligand-activated integrins at implantation sites to form focal adhesions in sheep. In vivo focal adhesions have rarely been described [130]. OPN protein is present along the entire maternal-placental interface in sheep through Day 120 of pregnancy (Fig. 2) and may be an ECM ligand bound to these integrin receptors that is responsible for induction of the focal adhesions observed in vivo.

View larger version (117K): [in this window] [in a new window]   FIG. 3. Functional evidence for the presence of integrin-mediated signal transduction in vivo at implantation sites in the ovine uterus. In serial 8-µm-thick frozen sections of a Day 45 ovine placentation site, the conceptus is centrally located and endometrial tissues are located in the top and bottom regions of each panel. The depth of each section (µm) from the original section in the upper left panel is indicated. The upper five panels are stained with antibodies directed against v, ß5, -actinin, OPN, and fibronectin (Fib). The -actinin is a cytoskeletal protein that aggregates at focal adhesions following integrin-ligand interaction and integrin aggregation. Localization provides a sensitive functional index of integrin activation and "outside-in" signaling. Note that v, ß5, -actinin are colocalized and OPN is present along the entire maternal-placental interface. Fibronectin does not appear to be involved in formation of these stable focal adhesions because it is not detected at this maternal-placental interface even though it is abundantly expressed in conceptus tissues underlying Tr. The lower three panels are serial sections from the same implantation site that show additional v, ß5, and -actinin colocalization. By utilizing this approach, focal adhesions were measured from these serial sections and cytoskeletal reorganization were determined to occur in discrete 30- to 80-µm-diameter aggregates. The 30- to 80-µm size also offers the opportunity to identify assemblies of relevant signaling intermediates in these structures and to decipher integrin-mediated signals in LE and Tr. Therefore, vß5 appears to be a major receptor expressed at implantation sites, and this receptor is capable of binding OPN. This is the first demonstration of functional activation of integrins at implantation sites in vivo, which confirms that focal adhesion formation and other integrin-mediated signaling events can be identified in situ with available imaging tools

	OPN IS A MARKER FOR STROMAL DECIDUALIZATION

TOP ABSTRACT INTRODUCTION OSTEOPONTIN STRUCTURE AND... OPN IS A MAJOR... OPN IS A PROGESTERONE-REGULATED... OPN BINDS AND ACTIVATES... OPN IS A MARKER... CONCEPTUS EXPRESSION OF OPN OPN EXPRESSION BY ENDOMETRIAL... SUMMARY REFERENCES

 
Conceptuses are inherently invasive and can attach to and invade a diverse array of artificial ectopic sites and biological matrices without discrimination or need for hormonal priming [131, 132]. Highly invasive Tr shares many phenotypic properties with carcinomas, including altered expression of adhesion molecules [133, 134] and elevated expression of matrix degrading proteinases [135, 136]. The endometrial LE serves as a barrier to Tr invasion until it is cyclically transformed to a receptive state that responds to embryonic signals and permits adhesive contact with conceptus Tr [100, 101]. Invasive implantation, as observed in rodents and primates, is characterized by adhesion of Tr to LE via apical adhesive glycoproteins, local ECM degradation by MMPs, and conceptus cell migration into stroma [137–139]. Penetration of the LE by invasive Tr triggers a series of stromal responses collectively termed decidualization [140]. During decidualization, hyperplasia and hypertrophy transforms small spindlelike fibroblast stroma cells into enlarged polygonal epitheliumlike cells [141–143]. Functionally, decidualized stroma synthesizes and secretes prolactin [144, 145], insulin-like growth factor binding protein 1 [146–149], and a rapidly growing list of other endocrine and paracrine factors [150, 151]. Decidua also exhibits marked accumulation of desmin [152, 153], -smooth muscle actin [154], and ECM components such as OPN [155]. The end result is the formation of a morphologically and functionally distinct tissue that is a source of hormones, promotes conceptus nutrition, prevents fetal allograft rejection, and regulates placentation by limiting Tr invasion through generation of a local cytokine environment that promotes Tr attachment over invasion [156–158].

The earliest reports of OPN mRNA in uteri of mammals noted high expression in a population of periodic acid-Schiff-positive cells corresponding in size and distribution to granulated metrial gland cells in mice [104]. These OPN-positive cells were localized in the central zone and metrial triangle, extending to more antimesometrial regions adjacent to trophoblast giant cells. Both the pseudopregnant deciduoma resulting from intraluminal injection of oil and the endometrium in the uninjected horn of these mice contained OPN-positive cells. A similar pattern of OPN mRNA expression was reported by Waterhouse and coworkers [155] in 1992, who defined OPN as a marker gene for decidualization. In humans, OPN mRNA and protein have been localized in human decidual cells by reverse transcription polymerase chain reaction (RT-PCR), Northern blot, and in situ hybridization procedures. These decidual cells also exhibited intense cytoplasmic immunoreactivity to antiserum against human bone OPN [35]. Eleven years later, Apparao et al. [112] reported identical immunostaining in the decidua of human pregnancy and stressed the striking similarity in the temporal and spatial patterns of expression of the ligand OPN and its integrin receptor vß3. These results confirmed previous results showing coexpression of OPN and vß3 integrins in decidualizing stromal cells of baboons. Fazleabas et al. [117] concluded that increased stromal expression of specific integrins during pregnancy, along with changes in ECM such as OPN, may be essential for stromal cell proliferation and differentiation. Much of the 60-fold increase in OPN gene expression in the rat uterus during pregnancy probably occurs in decidua, because there is a paucity of endometrial GE in rats [122].

OPN mRNA and protein have also been detected in the stroma of pregnant sheep [159]. Although Mossman [160] and Kellas [161] described decidual cell characteristics in syncytial tissues of placentomal crypts of sheep and antelope, these reports have been largely ignored, and decidualization is not thought to occur in species with central and noninvasive implantation, including domestic animals. However, endometrial stromal cells increase in size and become polyhedral in shape in pregnant ewes following conceptus attachment. The classical decidualization markers desmin and -smooth muscle actin are expressed in these cells, suggesting that OPN expression in this stromal compartment is part of a decidualization-like differentiation during ovine pregnancy [159]. In contrast, no mophological changes or induction of OPN mRNA or protein have been detected in uterine stromal cells during porcine pregnancy [159]. One of the primary roles of decidua in invasive implanting species is to restrain conceptus trophoblast invasion to a circumscribed region of the endometrium. Both pigs and sheep have noninvasive implantation, but the extent of Tr invasion into the endometrium differs between these species. Pig conceptuses undergo true epitheliochorial placentation in which LE remains morphologically intact throughout pregnancy and the conceptus Tr simply attaches to the apical LE surface without contacting uterine stromal cells [162, 163]. Synepitheliochorial placentation in sheep involves extensive erosion of the LE due to syncytium formation with Tr binucleate cells. After Day 19 of pregnancy, conceptus tissue is apposed to but does not penetrate ovine uterine stroma [164]. Although speculative, differences in stromal expression of OPN between these species suggest that the extent of decidualization is correlated positively with degree of conceptus invasiveness. OPN expression increases in ovine stratum compactum stroma that has undergone considerable loss of ECM by Day 35 of pregnancy [159]. A possible explanation for this concomitant increase in OPN and decrease in overall ECM might be that stromal OPN is intracellular. OPN has generally been characterized as a protein secreted into biological fluids and luminal spaces, such as the uterine lumen, or into the ECM where it binds receptors to mediate various cell-cell and cell-ECM interactions. However, an intracellular form of OPN associated with a hyaluronan-CD44-ERM attachment complex has been implicated in migration of embryonic fibroblasts, activated macrophages, and invasive cancer cells. As such, intracellular OPN may modulate CD44-mediated changes in cytoskeletal architecture involved in cell differentiation and migration [81, 82]. The nature of the potential intracellular and extracellular OPN interactions with stromal cells will be critical to understanding the function of OPN during decidualization and placentation.

	CONCEPTUS EXPRESSION OF OPN

TOP ABSTRACT INTRODUCTION OSTEOPONTIN STRUCTURE AND... OPN IS A MAJOR... OPN IS A PROGESTERONE-REGULATED... OPN BINDS AND ACTIVATES... OPN IS A MARKER... CONCEPTUS EXPRESSION OF OPN OPN EXPRESSION BY ENDOMETRIAL... SUMMARY REFERENCES

 
Decidua in invasive implanting species, and apical LE in superficial implanting species, constitute the maternal side of the maternal-fetal interface. The fetal side of this interface is the placental trophoblast. Information is exchanged between these intimately apposed tissues to maintain the intricate balance that ensures a successful pregnancy. OPN is consistently present at the maternal surface of this interface, and in most cases, high levels of OPN protein are also present at the surface of Tr that contacts maternal tissues. However, the specific pattern of OPN expression in Tr is highly variable among species. In mice, OPN is expressed in the inner cell mass of early blastocysts, where it may be regulated by the transcription factors Oct-4 and Sox-2, but becomes undetectable in the embryo by Day 5.5 of gestation [165]. Although increased levels of OPN mRNA have been reported in late gestation mouse placenta [104], this expression is confined to the granulated metrial gland cells [165]. OPN mRNA has also been detected in the human placenta [35]. Differential display RT-PCR, based on differences in total RNA between trophoblast-enriched primary cell cultures and peripheral mononuclear blood cells, showed high and exclusive expression of OPN in placental cells [166]. A significant series of early studies from Coutifaris and colleagues firmly established synthesis of OPN by conceptus-derived components of the human placenta [39, 60]. In vivo, OPN mRNA is expressed by invading cytotrophoblast but not syncytial trophoblast [39]. In vitro, expression of OPN mRNA declined during cytotrophoblast aggregation but subsequently increased when syncytia developed [60]. Placental expression of OPN was regulated by progesterone. The temporal and spatial patterns of expression suggested progesterone secretion by syncytiotrophoblast increases expression of OPN by the underlying cytotrophoblast to mediate adhesion and/or signaling events between these trophoblast cell types via v integrin [60]. In hydatidiform mole, the most common type of gestational trophoblastic disease, there is a downregulation of OPN mRNA and protein, compared with normal placentae, concomitant with decreased levels of progesterone and PR [167]. In contrast to mice and humans, OPN mRNA is not expressed by sheep, pig, or rabbit conceptus tissues. However, in sheep and pigs very high levels of immunoreactive OPN protein, presumably supplied in histotroph and bound to placental integrin receptors, are detected on Tr beginning just prior to implantation and maintained throughout gestation [38, 112, 114, 123]. Although OPN is not synthesized by Tr in these species, Tr exhibits greater immunostaining for OPN than does any other cell type in the pregnant uterine environment. This high affinity may indicate essential roles for OPN in mediating embryonic placental changes in morphology and adhesion to the maternal uterus during implantation and placentation.

	OPN EXPRESSION BY ENDOMETRIAL AND PLACENTAL IMMUNE CELLS

TOP ABSTRACT INTRODUCTION OSTEOPONTIN STRUCTURE AND... OPN IS A MAJOR... OPN IS A PROGESTERONE-REGULATED... OPN BINDS AND ACTIVATES... OPN IS A MARKER... CONCEPTUS EXPRESSION OF OPN OPN EXPRESSION BY ENDOMETRIAL... SUMMARY REFERENCES

 
Although OPN is not synthesized by ovine Tr, OPN mRNA and protein are present in subpopulations of both placental and endometrial immune cells [25, 117, 168]. OPN, also known as Eta-1, is an established component of the immune system. Although reports of OPN activity in the immune system preceded its identification in malignant transformed cells and bone, OPN was later isolated independently from T cells [11, 16, 169–172]. Early studies indicated that a 45-kDa thrombin cleavage fragment secreted by T-helper cells stimulated immunoglobulin production by B lymphocytes and that a 70-kDa protein secreted by CD8⁺ T lymphocytes bound antigen and inhibited T-helper cell function [26]. As such, OPN was the most abundant early transcript to increase expression in activated T-cells in response to treatment with conconavalin A [16, 173]. OPN is secreted by activated T lymphocytes [16], induced in monocytes and macrophages by tissue injury [174], and present in CD8^-/CD4^- granulated metrial gland cells [175].

Johnson and coworkers [25] first described OPN mRNA in a small population of cells scattered within the stratum compactum stroma immediately beneath the endometiral LE during the early stages of the estrous cycle and pregnancy in sheep. Unlike OPN mRNA in endometrial GE, expression of OPN by these cells and/or migration of this cell population through the endometrium was not regulated by administration of exogenous progesterone to ovariectomized ewes [118]. Similar results were observed in pigs in which both OPN mRNA and protein were detected in a small population of cells in the endometrium on Day 9 of the estrous cycle and early pregnancy exclusively [114]. Immunostaining for OPN and various immune cell surface antigens in ovine serial uterine cross sections indicated that OPN-producing cells in the uterus may be CD8-bearing T cytotoxic or natural killer cells or CD172a-positive macrophages [168]. Both macrophages and natural killer cells are believed to influence the pregnant endometrium of humans and mice [176, 177]. In primates, OPN mRNA was detected in a population of CD45-positive leukocytes isolated from mid and late secretory phase human endometrium [113] and has been detected in scattered endometrial stromal cells, believed to be of immune origin, during the menstrual phase of cynomolgus monkeys [178]. OPN-producing immune cells are also present in the ovine placenta beginning on Day 25 of pregnancy, and the number of these cells increases as gestation progresses (Fig. 4). In contrast, OPN mRNA was never detected in the placenta of pigs at any stage of gestation studied, and differences in placental immune cell expression of OPN between sheep and pigs may relate to type of placentation [168].

View larger version (145K): [in this window] [in a new window]   FIG. 4. OPN mRNA and protein in immune cells of ovine placenta. Upper panels show in situ hybridization analysis of OPN mRNA in ovine placenta comparing brightfield and darkfield images from Day 35 of pregnancy (width of each field is approximately 450 µm). Lower right panel shows a corresponding immunofluorescence image of ovine placenta from Day 35 of pregnancy stained for OPN, revealing scattered OPN-positive cells, several of which are indicated by arrows (width of the field is approximately 940 µm)

Immune cell OPN is of particular interest because of its role in maintenance and remodeling of tissue during inflammation, where it mediates cell-cell and cell-matrix communication resulting in targeted chemotaxis of immune cells [27]. In vitro, OPN can induce migration of both T cells and macrophages [179, 180], and in vivo macrophages migrate toward OPN injected into rat dermis [181]. OPN induces chemotactic migration of dendritic cells from the skin to draining lymph nodes, a process required for many inflammatory responses [182]. Movement of leukocytes into tissues involves a cascade in which adhesion to endothelium is critical. OPN becomes immobilized on endothelium by CD44 to recruit leukocytes to the endothelial surface and triggers activation of integrins to an active adherence configuration [49, 79]. An intracellular form of OPN associates with the CD44 complex in cell processes and is believed to function in the migration of osteoclasts, which are specialized bone cells of macrophage lineage [183].

OPN is a key component of type 1 (Th-1) immunity characterized by increases in IL-12 and decreases in IL-10 expression by macrophages and monocytes [41, 83, 184]. Macrophage production of IL-12 promotes a cell-mediated or Th-1 immune response that effectively protects against the growth of viral and bacterial pathogens and transformed cells, whereas IL-10 inhibits this response and favors humoral (Th-2) immunity [185, 186]. Although conclusions of recent studies are being debated, OPN appears to regulate Th-1-mediated demyelinating disease in rats paralyzed by experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis. OPN-deficient rats are resistant to progressive EAE and have higher levels of IL-10 than do OPN-positive mice [187–189]. A similar model in mice revealed that OPN-deficient mice have less severe clinical symptoms and faster recovery without spontaneous relapses than do OPN-positive mice. Deficient mice display decreased inflammatory cell infiltration and demyelination of spinal cords, suggesting that OPN sustains autoimmune responses by increasing and/or maintaining Th-1 immunity [190].

Trophoblasts and many tumors use OPN to selectively escape immune surveillance [191]. As a key component of innate immunity, the alternative complement system is directly activated by foreign epitopes on cell surfaces. Molecules such as the plasma protein Factor-H bind and inactivate inappropriately high levels of complement pathway components in serum [192]. OPN and several related SIBLINGS sequester Factor-H to the cell surface and inhibit complement-mediated cell lysis [191, 193]. Human decay-accelerating factor appears to play a similar protective role for endometrial LE during the secretory phase of the menstrual cycle [194].

	SUMMARY

TOP ABSTRACT INTRODUCTION OSTEOPONTIN STRUCTURE AND... OPN IS A MAJOR... OPN IS A PROGESTERONE-REGULATED... OPN BINDS AND ACTIVATES... OPN IS A MARKER... CONCEPTUS EXPRESSION OF OPN OPN EXPRESSION BY ENDOMETRIAL... SUMMARY REFERENCES

 
Comprehensive analyses of temporal and spatial patterns of OPN mRNA and protein expression within the uteri of multiple species suggests that OPN has diverse biological functions in the uterus and placenta throughout the estrous cycle and early pregnancy, during the peri-implantation period, and extending to the end of gestation. Based upon its identified roles as 1) an ECM molecule that mediates cell-cell and cell-matrix adhesion and communication, 2) a cytokine that can exhibit proinflammatory properties (e.g., macrophage/monocyte and T-cell recruitment and stimulation of cytokine production), anti-inflammatory actions (e.g., inhibition of inducible nitric oxide production and MMP-2 expression), and effects on tissue repair at sites of inflammatory responses, and 3) an intracellular protein associated with CD44 and ERM proteins in migrating cells, OPN has potential to profoundly influence the events of implantation and subsequent placental development.

Figure 5 illustrates a working model for the role of OPN in the pregnant ovine uterus, although data from other species indicate remarkably similar patterns of OPN expression. OPN functions within the uterus appear to be complex and omnifarious, encompassing 1) a role as a component of histotroph involved in adhesion and signal transduction at the uterine-placental interface, 2) expression in uterine stroma, suggesting a decidualizationlike transformation that is correlated with the degree of conceptus invasiveness, and 3) expression by resident uterine and placental immune cells that may regulate immune cell behavior and cytokine production. The regulation and immediate functional implications of these expression events, although independent, are temporally and spatially orchestrated to maintain successful pregnancy. Insights have been gained into the potential hormonal/cytokine, cellular, and molecular mechanisms underlying OPN-mediated adhesion, remodeling, and cell-cell/cell-matrix communication within the uterine-placental environment through comparisons among humans, monkeys, sheep, pigs, and rodents. Each animal model offers unique advantages, and investigations must continue to exploit the comparative aspects of OPN expression and function among species. Abundant and continuous immunoreactive OPN protein is present along all areas of intimate contact between maternal and fetal components of the placenta throughout pregnancy [38, 112, 114, 115, 123].

View larger version (27K): [in this window] [in a new window]   FIG. 5. Working model for the complex distribution of OPN mRNA and protein within the ovine uterus and placenta. In the left panel (Day 15), OPN is snythesized and secreted from the middle to deep GE beginning between Day 12 and Day 20 of pregnancy. A population of immune cells within the stratum compactum stroma of the endometrium synthesizes OPN. In the middle panel (Day 45), secreted OPN is cleaved to produce a highly bioactive integrin-binding 45-kDa fragment that binds to apical surfaces of conceptus Tr and endometrial LE and GE. OPN may serve as a bridging ligand for attachment or migration and/or as a conduit for cell-cell communication between fetal and maternal tissues along the entire uterine-placental interface during pregnancy. OPN is also present in stromal cells, where it may limit trophoblast invasiveness. Immune cells within the placenta begin to sythesize OPN after Day 20. In the right panel (Day 80), as placentation progresses, large quantities of secreted 45-kDa OPN fragment are bound along the entire uterine-placental interface of both placentomal and interplacentomal regions of the ovine uterus. Uterine stroma, including interdigitations with chorionic villi in placentomes, continues to synthesize OPN. Immune cells on both sides of the uterine-placental interface express OPN

Future investigations must focus on the mechanistic nature of the functions of this multifaceted component of the uterine/placental environment. For example, studies are underway to identify specific signals that are transduced into LE and Tr, respectively, following binding of OPN to integrin and/or CD44 receptors. The specific signals elicited by OPN result not only from its multiple receptors and binding sites but also from the various molecular forms that arise from glycosylation, phosphorylation, and protease cleavage. Therefore investigators are challenged to focus on understanding the regulation and distribution of the various OPN forms within the uterus and placenta. Investigations should include a determination of whether intracellular OPN-CD44-ERM complex is involved in uterine and placental function. The identity of the molecular forms and functional significance of the induction of ovine stromal OPN accompanying the decidualizationlike differentiation of this tissue during implantation should also be informative. Similarly, the identity of OPN-producing immune cells and the determination of intracellular or secreted molecular forms generated is an important question. Specifically, the production of secreted OPN by immune cells would suggest cell-cell or cell-matrix communication resulting in upregulation of Th-1 inflammatory cytokines and/or chemotaxis of immune cells, whereas intracellular OPN would suggest the presence of a population of immune cells that are themselves migrating through the tissue. Other obvious functions to be addressed include potential roles of OPN in placental angiogenesis and in uterine tissue remodeling that occurs during and following pregnancy. Because nitric oxide and polyamines (products of arginine catabolism) are essential for placental angiogenesis and placental and fetal growth [195, 196], the role and mechanisms for OPN in regulating these biosynthetic processes should be defined. Collectively, clear understanding of these and other functions involving uterine OPN should be useful for the rational design of therapies for the prevention of pregnancy failure related to abnormalities in uterine/placental factors as opposed to current empirical therapies that have low efficiency.

	FOOTNOTES

 
¹ This work was supported in part by USDA-NRICGP grants 98-35203-6223 and 95-37203-2185 and NIH grant P30 ES09106 to F.W.B. and R.C.B. and by NIH 1-F32-HD08501-01A1 and a University of Idaho Seed Grant to G.A.J.

² Correspondence: Greg A. Johnson, Department of Veterinary Anatomy and Public Health, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843-4458. FAX: 979 847 8981; gjohnson{at}cvm.tamu.edu

Received: 25 June 2003.

First decision: 8 July 2003.

Accepted: 16 July 2003.

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TOP ABSTRACT INTRODUCTION OSTEOPONTIN STRUCTURE AND... OPN IS A MAJOR... OPN IS A PROGESTERONE-REGULATED... OPN BINDS AND ACTIVATES... OPN IS A MARKER... CONCEPTUS EXPRESSION OF OPN OPN EXPRESSION BY ENDOMETRIAL... SUMMARY REFERENCES

 

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