Heparan Sulfate Proteoglycans and Their Binding Proteins in Embryo Implantation and Placentation

Abstract

Complex interactions occur among embryonic, placental and maternal tissues during embryo implantation. Many of these interactions are controlled by growth factors, extracellular matrix and cell surface components that share the ability to bind heparan sulfate (HS) polysaccharides. HS is carried by several classes of cell surface and secreted proteins called HS proteoglycan that are expressed in restricted patterns during implantation and placentation. This review will discuss the expression of HS proteoglycans and various HS binding growth factors as well as extracellular matrix components and HS-modifying enzymes that can release HS-bound proteins in the context of implantation and placentation.

I. Introduction

The processes of embryo implantation and placentation involve a complex coordination among multiple cell types of the embryo/fetus and mother [1]. Control of gene expression in these cell types involves the actions of transcription factors regulated, in many cases, by activation of various intracellular signal transduction pathways. Activation of many of these pathways involves interaction of cell surface receptors with extracellular matrix (ECM) components and growth factors which are often deposited in ECM. Binding to heparan sulfate proteoglycans (HSPGs) sequesters and concentrates growth factors in species ranging from Drosophila melanogaster and Caenorhabditis elegans to mammals [2]. HSPGs are proteins to which one or more heparan sulfate (HS) polysaccharides are covalently attached. In this review, we will focus on the role HSPGs play in modulating key events in embryo implantation and placentation as well as recent work on functions of novel HS binding proteins and extracellular HS modifying enzymes. The expression pattern and proposed functions of molecules involved in HS-dependent events in the fetal-maternal interface is summarized in Table I. Detailed descriptions of the cellular and morphological events that occur during embryo implantation and placentation are available [1,3,4].

Table I.

Expression of HS proteoglycans and HS-binding proteinsin the uteroplacental unit (partial list)¹

Molecule	Trophectoderm	Uterine Epithelia	Decidua	Trophoblast	Villus fibroblasts	Proposed function(s)
HSPGs						Tissue integrity, cell adhesion, growth factor binding
• Syndecans	+	-	+ (m,h)	+ (h; Cyto, Syn)	+ (h)
• Glypicans	?	?	+ (h)	+ (h; Syn)	?
• Perlecan	+	+	+ (m,h)	+ (m, h; Cyto, Syn)	- (h)
• Collagen XVIII	-	-	+ (h)	- (h)	+ (h)
Heparanase	?	+ (m)	+ (m)	+ (m & h)	?	Trophoblast invasion, angiogenesis, decidualization, remodeling of extracellular matrix, release of growth factors
Sulfs	?	?	+ (m,h)	+ (h, but location unknown)	?	HS 6-O-desulfation, modulation of growth factor binding and signaling
HS-binding growth factors
• HBEGF	+	+	+ (h)	+ cyto(h) I-III	?	Blastocyst attachment, trophoblast invasion, and maintenance of pregnancy [73,74]
• HGF	+	-	+ (h)	+ syn+cyto (h)	+ (h)	Trophoblast invasion [75]
• FGF-2			+ (m)	+ syn+cyto(h) I	+ (h)	Placental growth and angiogenesis [76]
• FGF-10			+ (h)(I-III)	+ syn+cyto (II-III)		Sprouting and branching of placental villi [77]
• VEGF				+ (h)	+ (h)	Placental angiogenesis and vasculogenesis [78]
• BMPs			+ (m)			Decidualization, embryo spacing [79,80]
• Wnts			+ (m)			Labyrinth morphogenesis [79,81]
• MK			-	Giant troph.(m)		Regulation of endometrial growth [82]
• PTN			+(m)	-		Decidualization [82]
HS-binding ECM components
• LAMA-111	+BM	+BM				Post-implantation cyto invasion direction [83,84]
• FN			+(h)			Decidual cell differentiation and invasion [85-88]
• Selectins	+(h)	-		+syn+cyto (h)		Attachment and establishment of pregnancy in human [89,90]
• CNN1 (Cyr61)			-	-syn(I-III)+cyto(III)	+(h)I-III	Vessel bifurcation in developing placenta[91]
Other HS-binding molecules
• HIP/RPL29	+ (m)	+ (m)	+ (m)	+cyto(h)	+(h)	Global protein synthesis and putative role in embryo attachment and invasion in human

¹The symbols “+” or “- “ are used to indicate whether the corresponding molecule has been detected by any method with no attempt made to indicate relative levels of expression among these compartments. For trophoblast, the abbreviations “Cyto” and “Syn” are used to indicate whether expression has been observed in cytotrophoblasts or syncytiotrophoblast, respectively. The symbols “h” and “m” are used in parentheses to indicate whether the observations were made in human or mouse tissues, respectively. If differential expression is observed in human placental tissue during pregnancy, then Roman numerals I-III are used to discriminate among the first, second and third trimesters. For further details, see text. BM, basement membrane.

II. Heparan sulfate proteoglycans

Core proteins

There are three major classes of HSPG core proteins. The first is the syndecans, a family of four type I transmembrane proteins (reviewed in Beauvais and Rapraeger [5]). Only syndecans-1, -3 and -4 have been reported in the uteroplacental units of humans and rodents [6-10]. The characteristic short cytoplasmic tails of syndecans interact with cytoskeletal components and, in the case of syndecan-4, the signaling molecules PKCα and PIP2 (reviewed in Beauvais and Raprager, 2004 [5]). In the preimplantation mouse embryo, syndecan is detected at the surfaces of blastomeres at the morula stage, but becomes restricted to the inner surface of blastocoel cavity by the blastocyst stage [11]. Syndecans also are found in uterine stroma, syncytiotrophoblast and fetal vessels in the labyrinthine zone [6,8,12]. In the human fetal-maternal interface, syndecan-1 is restricted to the syncytiotrophoblast. Syndecan-4 is expressed in villous and extravillous cytotrophoblast early in pregnancy, but is lost from these regions later [10].

The glypicans are a family of six HSPGs linked to the cell surface via glycosylphosphatidyl inositol anchors. Very little information is available on glypican expression in the utero-placental unit, but one report indicates that glypican-3, like syndecans, accumulates in syncytiotrophoblast [13] while another tentatively identified glypican-1 in human decidual tissues [9,10].

The third proteoglycan class is that of the ECM, namely agrin, collagen XVIII and perlecan. These proteoglycans all have relatively large protein cores and, in the cases of collagen type XVIII and perlecan, functions have been identified for proteolytic fragments derived from them which modulate angiogenesis [14]. There are no reports of agrin expression in the utero-placental unit. In contrast, collagen XVIII is expressed by decidua and villous fibroblasts, but not in trophoblastic tissue or cell lines [15,16]. Perlecan is more broadly expressed in basal lamina underlying uterine epithelia, trophoblast, maternal and placental vasculature and in decidua. In humans, perlecan also accumulates in the region of cytotrophoblast invasion into the decidua [17]. In the preimplantation mouse embryo, perlecan is transiently expressed before the morula stage [18]. During blastocyst development, perlecan expression is enhanced and is deposited on the external surface of the trophectoderm [19]. In delayed implantation blastocysts, perlecan mRNA accumulates, but the protein is not expressed until the blastocyst is activated indicating translational control [20]. The distribution of perlecan relative to syndecans and glypicans during the post-implantation and placentation periods in murine implantation site and the human fetal-maternal interface is illustrated in Figure 1.

Figure 1. Heparan sulfate proteoglycan distribution at implantation sites.

Panel A shows the pattern of perlecan (black areas) and syndecan-1 (green shaded area) in a cross section of a day 8 implantation site in the mouse. Perlecan is widely distributed in decidua as well as in both embryonic and maternal basal lamina. In contrast, syndecan-1 is restricted to the deciduas as well as embryonic tissue elements. Symbols: e, embryo; mm, mesometrial aspect of uterus. Panel B is a drawing representing main features of the human fetal-maternal interface showing the blood supply (red, arteries; blue, veins) leading to and from the umbilical cord and into the chorion as well as the maternal decidua. In panel C, an anchoring villus has been expanded from panel B to highlight the presence of invading cytotrophoblast (red cells), villar fibroblasts (purple shaded area), syncytiotrophoblast (yellow area), deciduas (gray area) and decidual vascular elements (V) surrounded by vascular smooth muscle cells (green). Perlecan is heavily deposited in areas indicated by the thick black lines. Syndecan-1 and -3 are found in the yellow region.

Gene knockouts have been generated for a variety of proteoglycan core proteins as well as HS biosynthetic enzymes (reviewed in Forsberg and Kjellen, 2001 [21]). None of these animals display an obvious implantation phenotype suggesting that HSPG-dependent functions are either dispensable or redundant at this early stage of development. A defect in placental development was noted in syndecan-4 nulls involving defects in fetal blood vessels [12]. Perlecan null mice display embryonic lethality with defects in formation of multiple fetal tissues [22]. These authors reported that the placental morphology through mid-gestation was similar to that of wild type and heterozygous animals. It should be noted that maternal RNA may support early murine embryonic development in certain cases [23] and it is possible that this occurs in the case of some proteoglycan nulls. If this is the case, then treatment of null embryos with the corresponding proteoglycan directed siRNA or ribozymes might reveal additional implantation/placentation phenotypes.

HS polysaccharides

The steps and genes involved in HS assembly have been reviewed in detail elsewhere [24]. An ordered series of reactions involving N-deacetylation/N-sulfation, uronic acid epimerization and O-sulfation occur that produce sequences within these large (20-50,00 MW) polysaccharides which provide binding sites for growth factors and ECM proteins. The particular balance in expression of these distinct activities gives rise to cell-type specific differences in expression of HS structures. Moreover, the size, charge and linearity of HS polysaccharides provide a capacity to bind more than one protein per polysaccharide chain [25,26]. Interestingly, several extracellular enzymes occur that can modify HS chains at the cell surface or ECM and modulate protein binding (see below). Thus, HS-dependent interactions with proteins can be dynamically modulated both at the synthetic and post-synthetic levels.

III. HS Binding Proteins

HS chains interact with a variety of proteins such as growth and differentiation factors, morphogens, ECM proteins and others (reviewed in Bernfield et al., 1999 [27]). There are many examples of HS-binding protein expression in uteroplacental tissues and these are summarized in Table 1. These interactions have been shown to participate in both normal and pathological events and to require HS chains with specific oligosaccharide sequences and sulfation patterns that selectively bind to domains in interacting protein ligands. For example, recent studies using HS oligoaccharide libraries established the importance of either 2-O-sulfate and/or 6-O-sulfate, and N-sulfate groups in particular HS-binding protein-HS interactions [28,29]. Such specific HS-protein interactions serve to regulate the signal output of growth factor receptors as well as the storage and diffusion of extracellular protein effectors. Thus, HS chains of proteoglycans are believed to act as co-receptors which are capable of influencing cell fate by integrating cellular signals. Binding HS provides a means to both restrict the diffusion and concentrate of soluble proteins like growth factors. In addition, HS-binding by ECM components enhances the structural integrity of the tissue. In other cases, HS-binding may serve more complex functions as in the case of HIP/RPL29.

HIP/RPL29

Heparin/heparan sulfate interacting protein (HIP) is a highly conserved basic protein identical to ribosomal protein L29 (RPL29) found associated with perinuclear membranes and translationally active ribosomes after polysomal fractionation [30,31]. In human uterine epithelial all lines, HIP/RPL29 is found at the cell surface where it is believed to participate in HS-binding [32]. HIP RL29 also can modulate blood coagulation processes [33] and inhibits heparanase activity (see below). HIP/RPL29 is prominently expressed in uterine luminal and glandular epithelia in both humans and mice at all stages of the cycle including during the window of receptivity [32,34]. During the post-implantation period in mice, HIP/RPL29 accumulates in decidualizing stroma and actively dividing embryonic structures [34]. These expression patterns suggest an important role for HIP/RPL29 in the establishment and maintenance of pregnancy. In human placenta, HIP/RPL29 is expressed at high level by invasive cytotrophoblasts and to a lesser extent in syncytiotrophoblasts [17]. Hip/Rpl29 null mice display low birth weight followed by decreased fertility in adults [30]. Reduced rates of cellular proliferation and global protein synthesis appear to cause this runting phenotype. In utero, the growth defect of null animals becomes evident at 12.5 days of development and is systematically associated with decreased placental weight suggesting that the small phenotype might be the result of placental insufficiency.

IV. Heparanase

Growth factors and cytokines must be released from ECM sites of deposition, cell contacts with HS-bearing substrates must be broken and extracellular matrices must be remodeled during implantation and placentation. Heparanase (HPSE) is an endo-β-D-glucuronidase that cleaves HS at specific sites to generate oligosaccharides that are 10 - 20 sugar units in length [35]. Heparanase has been identified in a number of cell types and tissues, including the placenta [36-41]. The inactive 65 kD pro-enzyme subsequently is proteolytically cleaved to generate 50 kD and 8 kD polypeptides [35,42] that remain associated to provide catalytic activity [42]. HPSE displays optimal activity at pH 5 with an 80-90% activity reduction at neutral pH [35,43]. In this regard, HPSE binds HS and may function as a cell adhesion molecule at pH 7.0 [44]. Recently, an mRNA splice variant of human HPSE lacking exon 5 was identified that lacks HS hydrolytic activity, but may retain adhesion-promoting function [45].

Another member of the HPSE family, heparanase-2 (HPSE2), encodes for three different proteins due to alternative mRNA splicing [46]. Hpse2 mRNA is present in a number of human tissues, including uterus [46]. No information is available on HPSE2 protein expression, but we recently found that Hpse2 transcripts in human and baboon decidual tissue (S. D’Souza, A.T. Fazleabas and D.D. Carson, submitted).

HPSE can promote angiogenesis, a crucial process during placentation, by: 1) releasing HS:angiogenic growth factor complexes, allowing optimal binding between HS:growth factor and receptor; 2) promoting endothelial cells migration and; 3) degradation of the subendothelial basement membrane [47-49]. Little progress has been made in determining HPSE expression and function in implantation and placentation. HPSE is present in human and bovine placenta [50-53] as well as endometrium of cycling human uteri [54,55]. In transgenic mice, HPSE overexpression resulted in a significant increase in the number of implanted embryos [56]. Furthermore, exogenous HPSE supplementation increased the number of implantation sites and promoting embryo attachment and outgrowth in vitro [57]. Recently, we demonstrated large increases in HPSE expression and activity in decidua of mouse endometrium, in areas where the potential substrate, perlecan, also accumulates [58]. These studies also demonstrated that injection of the heparanase inhibitor, PI-88, severely inhibited embryo implantation and caused malformations of the implantation sites, supporting the notion that maintenance of HPSE activity is critical during this process. In baboon and human receptive phase uteri, HPSE is localized to the glandular epithelia and glandular secretions and at the maternal-fetal interface HPSE is localized in villi and decidua (manuscript submitted). Collectively, these studies indicate that HPSE plays a key role in early implantation as well as placentation in various species.

Two naturally occurring inhibitors of HPSE have been identified: eosinophil major basic protein [59] and HIP/RPL29 [34]. Human placenta express eosinophil basic protein mRNA [60] and a precursor form of eosinophil major basic protein is secreted constitutively by human trophoblast cells isolated from the first to third trimester as well as decidualizing human uterine stroma cells [61]. HIP/RPL29 accumulates in mouse decidua and is expressed in a complementary pattern to the HSPG, perlecan, at the human fetal-maternal interface [17]. Thus, these proteins are expressed in patterns consistent with a role in modulating HPSE activity during early pregnancy and placentation.

V. Sulf1 and Sulf2

Sulf1 and Sulf2 are secreted endosulfatases that remove 6-O-sulfate residues from HS [62]. They have been identified in a many species, including quail, zebrafish, C. elegans, Drosophila, mouse, rat and human [62-65]. Human SULFs have been shown to modulate binding of FGF-1 and VEGF to HS [66]. Sulf overexpression impacts signaling pathways including those activated by FGF-2, FGF-4, hepatocyte growth factor, Wnt, bone morphogenetic protein and HB-EGF [62,67-71]. The Sulf genes are expressed in various tissues during mouse embryogenesis including the nervous system, skeleton and heart [72]. In the pregnant mouse uterus, Sulf transcripts were detected in the decidual region [72]. In the same study, Sulf2 mRNA was detected in the spongiotrophoblast region of placenta while Sulf1 was not detected except in a subset of placental vasculature. Sulf transcripts have also been identified in human uterus and placenta [63]. Given their intriguing functions and expression patterns in the developing mouse embryo, SULF proteins may play a role in modulating HS-dependent processes.

Summary and Future Directions

HSPG expression changes dynamically during embryo implantation and placentation. Nonetheless, data is still incomplete on the expression of HSPG core proteins, particularly that of the glypicans and little is known about how the patterns of HS structures or the expression of key enzymes involved in HS assembly and extracellular modification change during these key biological events. HSPGs bind a wide array of growth factors and ECM components. In the former case, this binding serves to concentrate and restrict the diffusion of these growth factors to specific regions within the uteroplacental unit. Null mutations in genes encoding some proteoglycan core proteins have subtle effects suggesting they are not required or have redundant function in implantation and placentation; however, mutations in other HSPG-encoding genes have such massive developmental defects that it will be necessary to use more sophisticated methods, e.g., tissue-specific, inducible knockouts in the uterus or placenta, to dissect the role these gene products play in this process. Given the profound biological roles HS-binding growth factors and HSPGs are known to play in other biological events, expanding our knowledge of the expression and function of HSPGs, HS structures and HS modifying enzymes is likely to add significantly to our understanding of the biology of embryo implantation and placentation and pathologies associated with these processes, e.g., pre-eclampsia and intrauterine growth retardation.