Characterization of dermatan sulfate proteoglycan 3 (DSPG3) and cartilage oligomeric matrix protein (COMP)

This dissertation describes the identification and characterization of human dermatan sulfate proteoglycan 3 (DSPG3) and the characterization of the transcriptional regulation of human cartilage oligomeric matrix protein (COMP) in cartilage, ligament, and tendon cells. DSPG3 and COMP are two extracellular matrix proteins. The function of these ECM proteins is unknown.^ DSPG3 was cloned, sequenced, and shown to be expressed in cartilage, ligament, and placenta. DSPG3 was mapped to human chromosome 12q21, and the genomic structure was identified. 1.6 kb of the promoter region has been sequenced, and several putative SOX9 sites were identified as well as 3 TATA sites. Furthermore, an evolutionary tree of the SLRP gene family, which includes DSPG3, is presented.^ The promoter region of COMP was cloned and sequenced. Several putative transcription factor binding sites were identified including multiple AP2 and SP1 sites. Three transcription start sites were found to be located directly downstream of one of the SP1 sites. In addition, the expression of COMP was demonstrated to be higher in tendon than in cartilage and ligament by both Northern and Western blot analysis, and several regions of the COMP promoter were shown to contain cell-specific regulatory elements. Analysis of the proximal 370bp region of the COMP promoter has also identified distinct patterns of nuclear protein binding for the three tissues, and two SP1 sites may play a role in the tissue-specific expression of COMP. ^

Involvement of heparin-like glycosaminoglycans and expression of a novel heparan sulfate binding protein (p24) during human placentation and in a model for human implantation

Previous studies in our laboratory have indicated that heparan sulfate proteoglycans (HSPGs) play an important role in murine embryo implantation. To investigate the potential function of HSPGs in human implantation, two human cell lines (RL95 and JAR) were selected to model uterine epithelium and embryonal trophectoderm, respectively. A heterologous cell-cell adhesion assay showed that initial binding between JAR and RL95 cells is mediated by cell surface glycosaminoglycans (GAG) with heparin-like properties, i.e., heparan sulfate and dermatan sulfate. Furthermore, a single class of highly specific, protease-sensitive heparin/heparan sulfate binding sites exist on the surface of RL95 cells. Three heparin binding, tryptic peptide fragments were isolated from RL95 cell surfaces and their amino termini partially sequenced. Reverse transcription-polymerase chain reaction (RT-PCR) generated 1 to 4 PCR products per tryptic peptide. Northern blot analysis of RNA from RL95 cells using one of these RT-PCR products identified a 1.2 Kb mRNA species (p24). The amino acid sequence predicted from the cDNA sequence contains a putative heparin-binding domain. A synthetic peptide representing this putative heparin binding domain was used to generate a rabbit polyclonal antibody (anti-p24). Indirect immunofluorescence studies on RL95 and JAR cells as well as binding studies of anti-p24 to intact RL95 cells demonstrate that p24 is distributed on the cell surface. Western blots of RL95 membrane preparations identify a 24 kDa protein (p24) highly enriched in the 100,000 g pellet plasma membrane-enriched fraction. p24 eluted from membranes with 0.8 M NaCl, but not 0.6 M NaCl, suggesting that it is a peripheral membrane component. Solubilized p24 binds heparin by heparin affinity chromatography and $\sp{125}$I-heparin binding assays. Furthermore, indirect immunofluorescence studies indicate that cytotrophoblast of floating and attached villi of the human fetal-maternal interface are recognized by anti-p24. The study also indicates that the HSPG, perlecan, accumulates where chorionic villi are attached to uterine stroma and where p24-expressing cytotrophoblast penetrate the stroma. Collectively, these data indicate that p24 is a cell surface membrane-associated heparin/heparan sulfate binding protein found in cytotrophoblast, but not many other cell types of the fetal-maternal interface. Furthermore, p24 colocalizes with HSPGs in regions of cytotrophoblast invasion. These observations are consistent with a role for HSPGs and HSPG binding proteins in human trophoblast-uterine cell interactions. ^

cDNA cloning and expression of a novel cell surface-expressed \b heparan sulfate/heparin \b interacting \b protein (HIP) from human cell lines and functional studies of a synthetic HIP peptide

Heparan sulfate proteoglycans and their corresponding binding sites have been suggested to play an important role during the initial attachment of 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, characterized a single class of cell surface heparin/heparan sulfate (HP/HS)-binding sites. Three major HP/HS-binding peptide fragments were isolated from RL95 cell surfaces by tryptic digestion and partial amino-terminal amino acid sequence from each peptide fragment was obtained. In the current study, using the approaches of reverse transcription-polymerase chain reaction and cDNA library screening, a novel cell surface $\rm\underline{H}$P/HS $\rm\underline{i}$nteracting $\rm\underline{p}$rotein (HIP) has been isolated from RL95 cells. The full-length cDNA of HIP encodes a protein of 259 amino acids with a calculated molecular weight of 17,754 Da and pI of 11.75. Transfection of HIP 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 kb in both total RNA and poly(A$\sp+$) RNA. Examination of human cell lines and normal tissues using both Northern blot and Western blot analysis revealed that HIP is differentially expressed in a variety of human cell lines and normal tissues, but absent in some cell lines examined. HIP has about 80% homology, at the level of both mRNA and protein, to a rodent protein, designated as ribosomal protein L29. Thus, members of the L29 family may be displayed on cell surfaces where they participate in HP/HS binding events. Studies on a synthetic peptide derived from HIP demonstrate that HIP peptide binds HS/HP with high selectivity and has high affinity (Kd = 10 nM) for a subset of polysaccharides found in commercial HIP preparations. Moreover, HIP peptide also binds certain forms of cell surface, but not secreted or intracellular. HS expressed by RL95 and JAR cells. This peptide supports the attachment of several human trophoblastic cell lines and a variety of mammalian adherent cell lines in a HS-dependent fashion. Furthermore, studies on the subset of HP specifically recognized by HIP peptide indicate that this high-affinity HP (HA-HP) has a larger median MW and a greater negative charge density than bulk HP. The minimum size of oligosaccharide required to bind to HIP peptide with high affinity is a septa- or octasaccharide. HA-HP also quantitatively binds to antithrombin-III (AT-III) with high affinity, indicating that HIP peptide and AT-III may recognize the same or similar oligosaccharide structure(s). Furthermore, HIP peptide antagonizes HP action and promotes blood coagulation in both factor Xa- and thrombin-dependent assays. Finally, HA-HP recognized by HP peptide is highly enriched with anticoagulant activity relative to bulk HP. Collectively, these results demonstrate that HIP may play a role in the HP/HS-involved cell-cell and cell-matrix interactions and recognizes a motif in HP similar or identical to that recognized by AT-III and therefore, may modulate blood coagulation. ^

The heparan sulfate-fibroblast growth factor signaling system in liver growth and function

The heparan sulfate (HS)-fibroblast growth factor (FGF) signaling system is a ubiquitous regulator that senses local environmental changes and mediates cell-to-cell communication. This system consists of three mutually interactive components. These are regulatory polypeptides (FGF), FGF receptor (FGFR) and heparan sulfate proteoglycans (FGFRHS). All four FGFR genes are expressed in the adult liver. Expression of the FGFR1–3 genes is generally associated with non-parenchymal cells while expression of the FGFR4 gene is associated with parenchymal hepatocytes. We showed that livers of mice lacking FGFR4 exhibited normal morphology and regenerated normally in response to partial hepatectomy. However, the FGFR4 (−/−) mice exhibited depleted gallbladders, an elevated bile acid pool and elevated excretion of bile acids. Cholesterol- and bile acid-controlled liver cholesterol 7α-hydroxylase (Cyp7a), the limiting enzyme for bile acid synthesis, was elevated, unresponsive to dietary cholesterol, but repressed normally by dietary cholate. These results indicated that FGFR4 was not directly involved in liver growth but exerted negative control on liver bile acid synthesis. This was confirmed in transgenic mice overexpressing the constitutively active human FGFR4 in livers. The transgenic mice exhibited decreased fecal bile acid excretion, bile acid pool size, and expression of Cyp7a. Introduction of this constitutively active human FGFR4 into FGFR4 (−/−) mice restored the inhibition of bile acid synthesis. Activation of the c-Jun N-terminal Kinase (JNK) pathway by FGFR4 correlated with the repressive effect on bile acid synthesis. ^ To determine whether FGFR4 played a broader role in liver-specific metabolic function, we examined the impact of both acute and chronic exposure to CCl 4 in FGFR4 (−/−) mice. Following acute CCl4 exposure, the FGFR4 (−/−) mice exhibited accelerated liver injury, a significant increase in liver mass and delayed hepatolobular repair, with no apparent effect on liver cell proliferation and restoration of cellularity. Chronic CCl4 exposure resulted in severe fibrosis in livers of FGFR4 (−/−) mice compared to normal mice. Analysis at both mRNA and protein levels indicated an 8 hr delay in FGFR4-deficient mice in the down-regulation of cytochrome P450 2E1 (CYP2E1) protein, the major enzyme whose products underlie CCl 4-induced injury. These results show that hepatocyte FGFR4 protects against acute and chronic insult to the liver and prevents accompanying fibrosis. ^ Of the 23 FGF polypeptides, FGF1 and FGF2 are present at significant levels in the liver. To determine whether FGF1 and FGF2 played a role in CCl 4-induced liver injury and fibrosis, we examined the impact of both acute and chronic exposure to CCl4 in both wild-type and FGF1-FGF2 double-knockout mice. Following acute CCl4 exposure, FGF1(−/−)FGF2(−/−) mice exhibited accelerated liver injury, overall normal liver growth and repair, and decreased liver collagen α1(I) induction. Liver fibrosis resulting from chronic CCl4 exposure was markedly decreased in livers of FGF1(−/−)FGF2(−/−) mice compared to wild-type mice. This study suggests a role for FGF1 and FGF2 in hepatic fibrogenesis. ^ In summary, our three part study shows that specific components of the ubiquitous HS-FGF signaling family in the liver context interfaces with metabolite- and xenobiotic-controlled networks to regulate liver function, but has no apparent direct effect on liver cell growth. ^

Interactions and structural analysis of the zinc -binding motif in decorin, a small leucine rich proteoglycan in extracellular matrix

Decorin, a dermatan/chondroitin sulfate proteoglycan, is ubiquitously distributed in the extracellular matrix (ECM) of mammals. Decorin belongs to the small leucine rich proteoglycan (SLRP) family, a proteoglycan family characterized by a core protein dominated by Leucine Rich Repeat motifs. The decorin core protein appears to mediate the binding of decorin to ECM molecules, such as collagens and fibronectin. It is believed that the interactions of decorin with these ECM molecules contribute to the regulation of ECM assembly, cell adhesions, and cell proliferation. These basic biological processes play critical roles during embryonic development and wound healing and are altered in pathological conditions such as fibrosis and tumorgenesis. ^ In this dissertation, we discover that decorin core protein can bind to Zn2+ ions with high affinity. Zinc is an essential trace element in mammals. Zn2+ ions play a catalytic role in the activation of many enzymes and a structural role in the stabilization of protein conformation. By examining purified recombinant decorin and its core protein fragments for Zn2+ binding activity using Zn2+-chelating column chromatography and Zn2+-equilibrium dialysis approaches, we have located the Zn2+ binding domain to the N-terminal sequence of the decorin core protein. The decorin N-terminal domain appears to contain two Zn2+ binding sites with similar high binding affinity. The sequence of the decorin N-terminal domain does not resemble any other reported zinc-binding motifs and, therefore, represents a novel Zn 2+ binding motif. By investigating the influence of Zn2+ ions on decorin binding interactions, we found a novel Zn2+ dependent interaction with fibrinogen, the major plasma protein in blood clots. Furthermore, a recombinant peptide (MD4) consisting of a 41 amino acid sequence of mouse decorin N-terminal domain can prolong thrombin induced fibrinogen/fibrin clot formation. This suggests that in the presence of Zn2+ the decorin N-terminal domain has an anticoagulation activity. The changed Zn2+-binding activities of the truncated MD4 peptides and site-directed mutagenesis generated mutant peptides revealed that the functional MD4 peptide might contain both a structural zinc-binding site in the cysteine cluster region and a catalytic zinc site that could be created by the flanking sequences of the cysteine cluster region. A model of a loop-like structure for MD4 peptide is proposed. ^