JoAnne Julian

Cell Surface Expression of HIP, a Novel Heparin/Heparan Sulfate-binding Protein, of Human Uterine Epithelial Cells and Cell Lines

Previous studies established that uterine epithelial cells and cell lines express cell surface heparin/heparan sulfate (HP/HS)-binding proteins (Wilson, O., Jacobs, A. L., Stewart, S., and Carson, D. D.(1990) J. Cell. Physiol. 143, 60-67; Raboudi, N., Julian, J., Rohde, L. H., and Carson, D. D.(1992) J. Biol. Chem. 267, 11930-11939). The accompanying paper (Liu, S., Smith, S. E., Julian, J., Rohde, L. H., Karin, N. J., and Carson, D. D.(1996) J. Biol. Chem. 271, 11817-11823) describes the cloning of a full-length cDNA corresponding to a candidate cell surface HP/HS interacting protein, HIP, expressed by a variety of human epithelia. A synthetic peptide was synthesized corresponding to an amino acid sequence predicted from the cDNA sequence and used to prepare a rabbit polyclonal antibody. This antibody reacted with a protein with an apparent M of 24,000 by SDS-polyacrylamide gel electrophoresis that was highly enriched in the 100,000 × g particulate fraction of RL95 cells. This molecular weight is similar to that of the protein expressed by 3T3 cells transfected with HIP cDNA. HIP was solubilized from this particulate fraction with NaCl concentrations ≥0.8 M demonstrating a peripheral association consistent with the lack of a membrane spanning domain in the predicted cDNA sequence. HIP was not released by heparinase digestion suggesting that the association is not via membrane-bound HS proteoglycans. NaCl-solubilized HIP bound to heparin-agarose in physiological saline and eluted with NaCl concentrations of 0.75 M and above. Furthermore, incubation of I-HP with transblots of the NaCl-solubilized HIP preparations separated by two-dimensional gel electrophoresis demonstrated direct binding of HP to HIP. Indirect immunofluorescence studies demonstrated that HIP is expressed on the surfaces of intact RL95 cells. Binding of HIP antibodies to RL95 cell surfaces at 4°C was saturable and blocked by preincubation with the peptide antigen. Single cell suspensions of RL95 cells formed large aggregates when incubated with antibodies directed against HIP but not irrelevant antibodies. Finally, indirect immunofluorescence studies demonstrate that HIP is expressed in both lumenal and glandular epithelium of normal human endometrium throughout the menstrual cycle. In addition, HIP expression increases in the predecidual cells of post-ovulatory day 13-15 stroma. Collectively, these data indicate that HIP is a membrane-associated HP-binding protein expressed on the surface of normal human uterine epithelia and uterine epithelial cell lines.

Synthesis and Intracellular Trafficking of Muc-1 and Mucins by Polarized Mouse Uterine Epithelial Cells

Mucins function as a protective layer rendering the apical surface of epithelial cells nonadhesive to a variety of microorganisms and macromolecules. Muc-1 is a transmembrane mucin expressed at the apical cell surface of mouse uterine epithelial cells (UEC) that disappears as UEC become receptive for embryo implantation (Surveyor, G. A., Gendler, S. J., Pemberton, L., Das, S. K., Chakraborty, I., Julian, J., Pimental, R. A., Wegner, C. W., Dey, S. K., and Carson, D. D. (1995) Endocrinology 136, 3639-3647). In the present study, the kinetics of Muc-1 assembly, cell surface expression, release, and degradation were examined in polarized mouse UEC in vitro. Mucins were identified as the predominant glycoconjugates synthesized, apically expressed, and vectorially released in both wild-type and Muc-1 null mice. When mucins were released, greater than 95% were directed to the apical compartment. Approximately half of the cell-associated mucins lost during a 24-h period were found in the apical compartment. Vectorial biotinylation detected apically disposed, cell-surface mucin and indicated that at least 34% of these mucins are released apically within 24 h. This suggests that release of mucin ectodomains is part of the mechanism of mucin removal from the apical cell surface of UEC. The half-lives of total cell-associated mucins and Muc-1 were 19.5 ± 1 and 16.5 ± 0.8 h, respectively. Muc-1 represented approximately 10% of the [3H]glucosamine-labeled, cell-associated mucins. Studies of the kinetics of intracellular transport of Muc-1 indicated transit times of 21 ± 15 min from the rough endoplasmic reticulum to Golgi apparatus and 111 ± 28 min from the Golgi apparatus to the cell surface. Collectively, these studies provide the first comprehensive description of Muc-1 and mucin maturation, metabolism, and release by polarized cells, as well as defining a major metabolic fate for mucins expressed by UEC. Normal metabolic processing appears to be sufficient to account for the removal of Muc-1 protein during the transition of UEC to a receptive state.

cDNA Cloning and Expression of HIP, a Novel Cell Surface Heparan Sulfate/Heparin-binding Protein of Human Uterine Epithelial Cells and Cell Lines

Heparan sulfate proteoglycans and their corresponding binding sites have been suggested to play an important role during the initial attachment of murine blastocysts to uterine epithelium and human trophoblastic cell lines to uterine epithelial cell lines. Previous studies on RL95 cells, a human uterine epithelial cell line, had characterized a single class of cell surface heparin/heparan sulfate (HP/HS)-binding sites. Three major HP/HS-binding peptide fragments were isolated from cell surfaces by tryptic digestion, and partial amino-terminal amino acid sequence for each peptide fragment was obtained (Raboudi, N., Julian, J., Rohde, L. H., and Carson, D. D.(1992) J. Biol. Chem. 267, 11930-11939). In the current study, using approaches of reverse transcription-polymerase chain reaction and cDNA library screening, we have cloned and expressed a novel, cell surface HP/HS-binding protein, named HP/HS interacting protein (HIP), from RL95 cells. The full-length cDNA of HIP encodes a protein of 159 amino acids with a calculated molecular mass of 17,754 Da and pI of 11.75. Transfection of HIP full-length cDNA into NIH-3T3 cells demonstrated cell surface expression and a size similar to that of HIP expressed by human cells. Predicted amino acid sequence indicates that HIP lacks a membrane spanning region and has no consensus sites for glycosylation. Northern blot analysis detected a single transcript of 1.3 kilobases in both total RNA and poly(A) RNA. Examination of human cell lines and normal tissues using both Northern blot and Western blot analyses revealed that HIP is expressed at different levels in a variety of human cell lines and normal tissues but absent in some cell lines and some cell types of normal tissues examined. HIP has relatively high homology (80% both at the levels of nucleotide and protein sequence) to a rodent ribosomal protein L29. Thus, members of the L29 family may be displayed on cell surfaces where they may participate in HP/HS binding events.

HEPARIN/HEPARAN SULFATE (HP/HS) INTERACTING PROTEIN (HIP) SUPPORTS CELL ATTACHMENT AND SELECTIVE, HIGH AFFINITY BINDING OF HP/HS

Heparin/heparan sulfate (HP/HS), HS proteoglycans, and their binding proteins play important roles in a variety of biological processes. Previously, we identified a novel cell surface HP/HS interacting protein (HIP) from human uterine epithelia and a variety of other human epithelial and endothelial cells and cell lines (Liu, S., Smith, S. E., Julian, J., Rohde, L. H., Karin, N. J., and Carson, D. D. (1996) J. Biol. Chem. 271, 11817–11823; Rohde, L. H., Julian, J., Babaknia, A., and Carson, D. D. (1996) J. Biol. Chem. 271, 11824–11830). In the current studies, we have purified and characterized HIP from HEC cells, a human uterine epithelial cell line, as well as recombinant HIP from a bacterial expression system. HIP supports attachment of the human trophoblastic cell line, JAR, in a HS-dependent fashion. Predigestion of JAR cells with a mixture of heparitinases, but not chondroitinase AC, abolished cell attachment to HIP. In addition, JAR cell attachment to HIP is highly sensitive to HP inhibition and much more selective for HP/HS than other glycosaminoglycans. Dermatan sulfate displays partial inhibitory activity as well, consistent with the observation that chondroitinase ABC digestion partially reduces JAR cell attachment to HIP. Solid-phase binding assays indicate HIP binds [3H]HP with high affinity (apparentK D = 8 nm). Furthermore, HIP bound cell surface/extracellular matrix-associated HS, expressed by RL95 cells, a human uterine epithelial cell line. Anti-HIP antibody generated against a synthetic peptide derived from a putative HP/HS-binding motif resident within HIP inhibited about half of [3H]HP binding to HIP, indicating that this domain is a functional HP-binding domain of HIP. Similarly, this same synthetic peptide motif of HIP could block about 50% of [3H]HP binding to HIP; however, this peptide almost completely inhibited cell attachment to HIP, suggesting a critical role, in this regard. Collectively, these results suggest that HIP can function as a HP/HS-binding cell-cell/cell-matrix adhesion molecule.

Uterine stromal cell chondroitin sulfate proteoglycans bind to collagen type I and inhibit embryo outgrowth in vitro

Chondroitin sulfate proteoglycans (CSPGs) are the major class of proteoglycans synthesized by mouse uterine stroma in vitro (Jacobs, A. L., and Carson, D. D. (1991). J. Biol. Chem. 266, 15,464-15,473). In the present study, stromal CSPGs were isolated and examined with regard to their ability to bind to specific extracellular matrix (ECM) components. Of a variety of ECM components tested, only collagen type I formed stable complexes with stromal CSPGs in both solid phase and solution binding assays. Proteolytic digestion of the CSPGs did not affect binding and suggested that the protein cores did not participate directly in binding. Furthermore, free chondroitin sulfate polysaccharides do not compete effectively in the binding assays. Therefore, interactions with multiple CS chains and/or the higher charge density afforded by intact CSPGs appear to be required for retention by collagen type I. Intact CSPGs were examined for their ability to modulate embryo attachment and outgrowth in vitro on fibronectin- or collagen type I-coated surfaces. In both cases, intact CSPGs, but not their constituent protein cores or polysaccharides, inhibited both the rate and the extent of outgrowth formation. In addition, embryo outgrowth on stromal ECM was enhanced by predigestion with chondroitinase. Addition of exogenous CSPG markedly retarded embryo outgrowth on stromal matrix. Collectively, these data indicate that stromal cell-derived CSPGs are retained by collagen type I in the stromal interstitial ECM where these molecules may attenuate trophoblast invasive behavior.

Murine HIP/L29 Is a Heparin-binding Protein with a Restricted Pattern of Expression in Adult Tissues

Heparin/heparan sulfate (Hp/HS)-binding proteins are implicated in a variety of cell biological processes including cell adhesion, modulation of blood coagulation, and cytokine/growth factor action. Hp/HS-interacting protein (HIP) has been identified in various adult tissues in humans. HIP supports high affinity, selective binding to Hp/HS, promotes cell adhesion, and modulates blood coagulation activities via Hp/HS-dependent mechanisms. Herein, a murine ortholog of human HIP is described that is 78.8% identical to human HIP and 99.8% identical at the cDNA level and identical at the amino acid level to a previously described murine ribosomal protein, L29. Western blot analyses and immunohistological staining with affinity-purified antibodies generated against two distinct peptide sequences of murine HIP/L29 indicate that HIP/L29 is differentially expressed in adult murine tissues and cell types. In the normal murine mammary epithelial cell line, NMuMG, HIP/L29 is enriched in the 100,000 × g particulate fraction. HIP/L29 can be solubilized from the 100,000 × g particulate fraction with 0.8 m NaCl, suggesting that it is a peripheral membrane protein. HIP/L29 directly binds 125I-Hp in gel overlay assays and requires 0.75 m NaCl for elution from Hp-agarose. In addition, recombinant murine HIP expressed inEscherichia coli binds Hp in a saturable and highly selective manner, compared with other glycosaminoglycans including dermatan sulfate, chondroitin sulfate, keratan sulfate, and hyaluronic acid. Collectively, these data indicate that murine HIP/L29, like its human ortholog, is a Hp-binding protein expressed in a restricted manner in adult tissues.

Mucins and Proteoglycans as Modulators of Embryo-Uterine Epithelial Cell Attachment

Blastocyst implantation involves the conversion of cell surfaces from a nonadhesive to an adhesive state. The blastocyst progresses to the adhesive state in response to a developmental program driven, at least in part, by internally or externally generated growth factors and cytokines. The uterus progresses from a nonadhesive or nonreceptive state to a receptive state. In the absence of implantation, the uterus further converts to a refractory state in which the uterine environment is, in fact, hostile to embryos (1). The uterine program is driven, directly or indirectly, by ovarian steroid hormones. It seems likely that some of the steroid hormone control of uterine functions involves the action of growth factors and cytokines as well (2, 3).

Mucins Provide a Barrier to Embryo Implantation

The process of embryo implantation is in actuality a multistep event. The initial phase involves the interaction or attachment of the embryonic trophectoderm with the uterine lumenal epithelia. Subsequently, a more intimate contact forms between the embryo and the uterus with ultimate placental formation. The secondary processes of implantation are classified into several categories based on whether the trophectoderm/trophoblast cells invade or fuse with the uterine epithelia (UE). In contrast, although there are differences in these secondary processes, the initial phase of implantation, that is, the attachment stage, is a universal phenomenon among species (reviewed in (1)). On the gross anatomical and histological scale, the process appears identical in all species examined. Yet, it is still unclear at the molecular level whether these different mammalian species utilize common factors to facilitate this initial event in implantation.

Proteoglycans as Modulators of Embryo-Uterine Interactions

The process by which mammalian embryos attach to and invade the uterine endometrium is both fascinating and complex. From early conception until parturition, embryonal and maternal tissues must exist symbiotically without imposing detrimental effects on the other. It remains unclear why the immunologically competent mother fails to reject the embryo in spite of histocompatibility differences. It also is unclear why the highly invasive trophoblast tissue of the embryo normally halts its progress within the endometrium although it clearly has the capacity to invade a wide range of tissues (1, 2). In this regard, the interesting feature of the uterus may not be that it supports embryo implantation, but that it has the unique ability to prevent and limit embryo invasion.