Biomedical subjects

W B Stallcup

Publications and source records attributed to W B Stallcup.

At least 19 recordsLinked to original sources

PubMed2004

NG2 proteoglycan expression in the peripheral nervous system: upregulation following injury and comparison with CNS lesions.

The chondroitin sulphate proteoglycan NG2 blocks neurite outgrowth in vitro and thus may be able to inhibit axonal regeneration in the CNS. We have used immunohistochemistry to compare the expression of NG2 in the PNS, where axons regenerate, and the spinal cord, where regeneration fails. NG2 is expressed by satellite cells in dorsal root ganglia (DRG) and in the perineurium and endoneurium of intact sciatic nerves of adult rats. Endoneurial NG2-positive cells were S100-negative. Injury to dorsal roots, ventral rami or sciatic nerves had no effect on NG2 expression in DRG but sciatic nerve section or crush caused an upregulation of NG2 in the damaged nerve. Strongly NG2-positive cells in damaged nerves were S100-negative. The proximal stump of severed nerves was capped by dense NG2, which surrounded bundles of regenerating axons. The distal stump, into which axons regenerated, also contained many NG2-positive/S100-negative cells. Immunoelectron microscopy revealed that most NG2-positive cells in distal stumps had perineurial or fibroblast-like morphologies, with NG2 being concentrated at the poles of the cells in regions exhibiting microvillus-like protrusions or caveolae. Compression and partial transection injuries to the spinal cord also caused an upregulation of NG2, and NG2-positive cells and processes invaded the lesion sites. Transganglionically labelled ascending dorsal column fibres, stimulated to sprout by a conditioning sciatic nerve injury, ended in the borders of lesions among many NG2-positive processes. Thus, NG2 upregulation is a feature of the response to injury in peripheral nerves and in the spinal cord, but it does not appear to limit regeneration in the sciatic nerve.

Animals↗

PubMed2001

An anti-angiogenic state in mice and humans with retinal photoreceptor cell degeneration.

Abnormal angiogenesis accompanies many pathological conditions including cancer, inflammation, and eye diseases. Proliferative retinopathy because of retinal neovascularization is a leading cause of blindness in developed countries. Another major cause of irreversible vision loss is retinitis pigmentosa, a group of diseases characterized by progressive photoreceptor cell degeneration. Interestingly, anecdotal evidence has long suggested that proliferative diabetic retinopathy is rarely associated clinically with retinitis pigmentosa. Here we show that neonatal mice with classic inherited retinal degeneration (Pdeb(rd1)/Pdeb(rd1)) fail to mount reactive retinal neovascularization in a mouse model of oxygen-induced proliferative retinopathy. We also present a comparable human paradigm: spontaneous regression of retinal neovascularization associated with long-standing diabetes mellitus occurs when retinitis pigmentosa becomes clinically evident. Both mouse and human data indicate that reactive retinal neovascularization either fails to develop or regresses when the number of photoreceptor cells is markedly reduced. Our findings support the hypothesis that a functional mechanism underlying this anti-angiogenic state is failure of the predicted up-regulation of vascular endothelial growth factor, although other growth factors may also be involved. Preventive and therapeutic strategies against both proliferative and degenerative retinopathies may emerge from this work.

Animals↗

PubMed2001

NG2 proteoglycan is expressed exclusively by mural cells during vascular morphogenesis.

Immunofluorescence mapping demonstrates that the NG2 proteoglycan is invariably expressed by the mural cell component of mouse neovascular structures. This pattern is independent of the developmental mechanism responsible for formation of the vasculature (vasculogenesis or angiogenesis). Thus, NG2 is expressed in the embryonic heart by cardiomyocytes, in developing macrovasculature by smooth muscle cells, and in nascent microvessels by vascular pericytes. Due to the scarcity of proven markers for developing pericytes, NG2 is especially useful for identification of this cell type. The utility of NG2 as a pericyte marker is illustrated by two observations. First, pericytes are associated with endothelial tubes at an early point in microvessel development. This early interaction between pericytes and endothelial cells has important implications for the role of pericytes in the development and stabilization of microvascular tubes. Second, the pericyte to endothelial cell ratio in developing capillaries varies from tissue to tissue. Because the extent of pericyte investment is likely to affect the physical properties of the vessel in question, it is important to understand the mechanisms that control this process. Additional insight into these and other aspects of vascular morphogenesis should be possible through use of NG2 as a mural cell marker.

Animals↗

PubMed2001

Chondroitin sulfate and cytoplasmic domain-dependent membrane targeting of the NG2 proteoglycan promotes retraction fiber formation and cell polarization.

Targeting of the NG2 proteoglycan to cellular retraction fibers was studied by expressing mutant NG2 molecules lacking specific structural elements of the proteoglycan. Both the cytoplasmic domain and the chondroitin sulfate chain of NG2 appear to have roles in sorting NG2 to subcellular microdomains destined to become retraction fibers. Neither of these structural features alone is sufficient to allow optimal targeting of NG2 to retraction fibers, but together they promote efficient localization of the proteoglycan to these sites. This pattern of NG2 sorting seems to be necessary for optimal retraction fiber formation, as cells expressing poorly targeted NG2 mutants are noticeably deficient in their ability to extend retraction fibers. Furthermore, retraction fiber formation correlates strongly with the tendency of cells to assume a polarized morphology with NG2-positive retraction fibers at one pole of the cell and actin-rich lamellipodia at the other. This polarization can be triggered either through engagement of NG2 by the substratum or by exposure to lysophosphatidic acid, a potent activator of the rho GTPase. These results suggest a possible role for NG2 in regulating rho-dependent mechanisms in the trailing processes of motile cells.

Animals↗

PubMed2000

Binding of the NG2 proteoglycan to kringle domains modulates the functional properties of angiostatin and plasmin(ogen).

Interactions of the developmentally regulated chondroitin sulfate proteoglycan NG2 with human plasminogen and kringle domain-containing plasminogen fragments have been analyzed by solid-phase immunoassays and by surface plasmon resonance. In immunoassays, the core protein of NG2 binds specifically and saturably to plasminogen, which consists of five kringle domains and a serine protease domain, and to angiostatin, which contains plasminogen kringle domains 1-3. Apparent dissociation constants for these interactions range from 12 to 75 nm. Additional evidence for NG2 interaction with kringle domains comes from its binding to plasminogen kringle domain 4 and to miniplasminogen (kringle domain 5 plus the protease domain) with apparent dissociation constants in the 18-71 nm range. Inhibition of plasminogen and angiostatin binding to NG2 by 6-aminohexanoic acid suggests that lysine binding sites are involved in kringle interaction with NG2. The interaction of NG2 with plasminogen and angiostatin has very interesting functional consequences. 1) Soluble NG2 significantly enhances the activation of plasminogen by urokinase type plasminogen activator. 2) The antagonistic effect of angiostatin on endothelial cell proliferation is inhibited by soluble NG2. Both of these effects of NG2 should make the proteoglycan a positive regulator of the cell migration and proliferation required for angiogenesis.

Angiostatins↗

PubMed2000

The multi-PDZ domain protein MUPP1 is a cytoplasmic ligand for the membrane-spanning proteoglycan NG2.

A yeast two-hybrid screen was employed to identify ligands for the cytoplasmic domain of the NG2 chondroitin sulfate proteoglycan. Two overlapping cDNA clones selected in the screen are identical in sequence to a DNA segment coding for the most amino-terminal of the 13 PDZ domains found in the multi-PDZ-protein MUPP1. Antibodies made against recombinant polypeptides representing these two clones (NIP-2 and NIP-7) are reactive with the same 250-kDa molecule recognized by anti-MUPP1 antibodies, confirming the presence of the NIP-2 and NIP-7 sequences in the MUPP1 protein. NIP-2 and NIP-7 GST fusion proteins effectively recognize NG2 in pull-down assays, demonstrating the ability of these polypeptide segments to interact with the intact proteoglycan. The fusion proteins fail to bind NG2 missing the C-terminal half of the cytoplasmic domain, emphasizing the role of the NG2 C-terminus in the interaction with MUPP1. The existence of an NG2/MUPP1 interaction in situ is demonstrated by the ability of NG2 antibodies to co-immunoprecipitate both NG2 and MUPP1 from detergent extracts of cells expressing the two molecules. MUPP1 may serve as a multivalent scaffold that provides a means of linking NG2 with key structural and/or signaling components in the cytoplasm.

Amino Acid Sequence↗

PubMed2000

The third fibronectin type III repeat is required for L1 to serve as an optimal substratum for neurite extension.

As a means of defining functionally important regions of the L1 neuronal cell adhesion molecule, neurite outgrowth from cerebellar neurons was compared on monolayers of L1-negative B28 glioma cells, B28 cells transfected with wild-type human L1, and B28 cells transfected with variant forms of L1. Neurite outgrowth on L1-positive B28 cells is greatly enhanced over that seen on parental B28 cells. Neurite outgrowth on B28 cells expressing L1 variants that lack either the first or the fifth fibronectin type III repeat is comparable to that seen on monolayers expressing wild-type L1. In contrast, B28 cells expressing L1 without the third fibronectin type III repeat do not support neurite outgrowth above the background level seen on parental B28 cells. This suggests that the third fibronectin type III repeat plays a key role in the ability of L1 to promote neurite extension. This is consistent with reports that the third fibronectin type III repeat mediates L1 homomultimerization and integrin binding and that plasmin cleavage within this domain interferes with L1 function by abolishing these molecular interactions.

Amino Acid Sequence↗

PubMed1999

NG2 proteoglycan-binding peptides target tumor neovasculature.

NG2 is the rat homologue of the human melanoma proteoglycan, also known as the high molecular weight melanoma-associated antigen. This developmentally regulated membrane-spanning chondroitin sulfate proteoglycan is expressed primarily by glial, muscle, and cartilage progenitor cells. Upon maturation, these cell types down-regulate NG2 expression. In adult animals, the expression of NG2 is restricted to tumor cells and angiogenic tumor vasculature, making this proteoglycan a potential target for directing therapeutic agents to relevant sites of action. To this end, we have identified specific NG2-binding peptides by screening a phage-displayed random peptide library on purified NG2. Several rounds of biopanning on NG2 resulted in the specific enrichment of two phage-displayed decapeptides, TAASGVRSMH and LTLRWVGLMS. The binding of these phages to NG2 was inhibitable both by soluble NG2 and by glutathione S-transferase (GST) fusion proteins containing the cognate peptide sequences. In addition, direct binding between GST-TAASGVRSMH and GST-LTLRWVGLMS fusion proteins and NG2 was demonstrated in solid-phase binding assays. Interestingly, these NG2-binding fusion proteins cross-inhibited each other's binding to NG2, suggesting that the two sequences bind to the same or overlapping sites on the proteoglycan. Upon injection into tumor-bearing mice, NG2-binding phages specifically homed to tumor vasculature in wild-type mice but did not localize to the tumor vasculature in NG2 knockout mice. The in vivo targeting capability of these sequences suggests that they can be used for tumor targeting.

Animals↗

PubMed1999

High-affinity binding of basic fibroblast growth factor and platelet-derived growth factor-AA to the core protein of the NG2 proteoglycan.

NG2 is a transmembrane chondroitin sulfate proteoglycan that is expressed by immature progenitor cells in several developmental lineages and by some types of malignant cells. In vitro studies have suggested that NG2 participates in growth factor activation of the platelet-derived growth factor-alpha receptor. In this study the ability of recombinant NG2 core protein to interact with several different growth factors (epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF)-AA, PDGF-BB, vascular endothelial growth factor (VEGF)165 and transforming growth factor (TGF)-beta1) was investigated using two different assay systems: enzyme-linked immunosorbent assay-type solid-phase binding and an optical biosensor (BIAcore) system. High-affinity binding of bFGF and PDGF-AA to the core protein of NG2 could be demonstrated with both types of assays. Using both the BIAcore software analysis program and nonlinear regression analysis of the solid phase binding data, KD values in the low nanomolar range were obtained for binding of each of these growth factors to NG2. The results further indicate that NG2 contains at least two binding sites for each of these two growth factors. PDGF-BB, TGF-beta1, VEGF, and EGF exhibited little or no binding to NG2 in either type of assay. These data suggest that NG2 can have an important role in organizing and presenting some types of mitogenic growth factors at the cell surface.

Antigens↗

PubMed1999

Cytoskeletal reorganization induced by engagement of the NG2 proteoglycan leads to cell spreading and migration.

Cells expressing the NG2 proteoglycan can attach, spread, and migrate on surfaces coated with NG2 mAbs, demonstrating that engagement of NG2 can trigger the cytoskeletal rearrangements necessary for changes in cell morphology and motility. Engagement of different epitopes of the proteoglycan results in distinct forms of actin reorganization. On mAb D120, the cells contain radial actin spikes characteristic of filopodial extension, whereas on mAb N143, the cells contain cortical actin bundles characteristic of lamellipodia. Cells that express NG2 variants lacking the transmembrane and cytoplasmic domains are unable to spread or migrate on NG2 mAb-coated surfaces, indicating that these portions of the molecule are essential for NG2-mediated signal transduction. Cells expressing an NG2 variant lacking the C-terminal half of the cytoplasmic domain can still spread normally on mAbs D120 and N143, suggesting that the membrane-proximal cytoplasmic segment is responsible for this process. In contrast, this variant migrates poorly on mAb D120 and exhibits abnormal arrays of radial actin filaments decorated with fascin during spreading on this mAb. The C-terminal portion of the NG2 cytoplasmic domain, therefore, may be involved in regulating molecular events that are crucial for cell motility.

Actins↗

PubMed1999

A potential role for the plasmin(ogen) system in the posttranslational cleavage of the neural cell adhesion molecule L1.

L1 is a neural recognition molecule that promotes neural developmental and regenerative processes. Posttranslational cleavage of L1 is believed to be important for regulating its function in vivo, but little is known of the proteolytic systems responsible. In this study we present evidence that plasmin can regulate both L1 expression and function. The addition of plasmin to cell lines results in a dose-dependent loss of surface L1 expression, with the simultaneous appearance of soluble L1 species. The addition of plasminogen to primary neurons and melanoma cells also resulted in the generation of plasmin and the concomitant release of L1. One product of plasmin-mediated cleavage is an amino-terminal fragment of approximately 140 kDa that has been previously described as a natural posttranslational cleavage product in vivo. This fragment was confirmed to result from cleavage at two sites in the middle of the third fibronectin-like domain of L1. Cleavage at a further site, proximal to the transmembrane domain of L1, was also observed at higher plasmin concentrations. Plasmin was further confirmed to abrogate homophilic L1 interactions required for cellular aggregation. Based on these findings we propose that plasmin is likely to be an important regulator of L1-mediated processes including those documented in the nervous system.

Base Sequence↗

PubMed1999

PDGF (alpha)-receptor is unresponsive to PDGF-AA in aortic smooth muscle cells from the NG2 knockout mouse.

A line of null mice has been produced which fails to express the transmembrane chondroitin sulfate proteoglycan NG2. Homozygous NG2 null mice do not exhibit gross phenotypic differences from wild-type mice, suggesting that detailed analyses are required to detect subtle alterations caused by the absence of NG2. Accordingly, dissociated cultures of aortic smooth muscle cells from null mice were compared to parallel cultures from wild-type mice for their ability to proliferate and migrate in response to specific growth factors. Both null and wild-type smooth muscle cells exhibited identical abilities to proliferate and migrate in response to PDGF-BB. In contrast, only the wild-type cells responded to PDGF-AA in both types of assays. NG2 null cells failed to proliferate or migrate in response to PDGF-AA, implying a defect in the signaling cascade normally initiated by activation of the PDGF (alpha)-receptor. In agreement with this idea, activation of the extracellular signal-regulated kinase (ERK) in response to PDGF-AA treatment occured only in wild-type cells. Failure to observe autophosphorylation of the PDGF (alpha)-receptor in PDGF-AA-treated null cells indicates that the absence of NG2 causes a defect in signal transduction at the level of (alpha)-receptor activation.

Animals↗

PubMed1998

Expression of the NG2 proteoglycan enhances the growth and metastatic properties of melanoma cells.

The human homologue of NG2, the human melanoma proteoglycan (HMP), is expressed on most human melanomas. To investigate the role of this proteoglycan in melanoma progression, we have attempted to identify functionally important molecular ligands for NG2. Immunohistochemical analysis of cell lines that endogenously express NG2/HMP suggests that NG2/HMP associates with CD44 and alpha4beta1 integrin, two molecules previously implicated in melanoma progression. Transfection of rat NG2 into the NG2-negative B16 mouse melanoma cell line also resulted in a highly colocalized pattern of expression between the transfected rat NG2 and the endogenously expressed mouse CD44 and alpha4beta1 integrin molecules. In functional assays, expression of NG2 decreased the adhesion of B16 melanoma cells to CD44 monoclonal antibodies, hyaluronic acid, the C-terminal 40-kDa fibronectin fragment, and the CS1 fibronectin peptide, suggesting that NG2 may negatively modulate CD44- and alpha4beta1-mediated binding events. Expression of NG2 increased the proliferation of melanoma cells in culture and increased tumorigenicity in vivo. Moreover, NG2 expression led to increased lung metastasis of B16F1 and B16F10 melanoma cells in experimental metastasis studies. Together, these studies demonstrate that NG2 is capable of modulating the adhesion, proliferation, and metastatic potential of melanoma cells.

Animals↗

PubMed1997

The brain chondroitin sulfate proteoglycan brevican associates with astrocytes ensheathing cerebellar glomeruli and inhibits neurite outgrowth from granule neurons.

Brevican is a nervous system-specific chondroitin sulfate proteoglycan that belongs to the aggrecan family and is one of the most abundant chondroitin sulfate proteoglycans in adult brain. To gain insights into the role of brevican in brain development, we investigated its spatiotemporal expression, cell surface binding, and effects on neurite outgrowth, using rat cerebellar cortex as a model system. Immunoreactivity of brevican occurs predominantly in the protoplasmic islet in the internal granular layer after the third postnatal week. Immunoelectron microscopy revealed that brevican is localized in close association with the surface of astrocytes that form neuroglial sheaths of cerebellar glomeruli where incoming mossy fibers interact with dendrites and axons from resident neurons. In situ hybridization showed that brevican is synthesized by these astrocytes themselves. In primary cultures of cerebellar astrocytes, brevican is detected on the surface of these cells. Binding assays with exogenously added brevican revealed that primary astrocytes and several immortalized neural cell lines have cell surface binding sites for brevican core protein. These cell surface brevican binding sites recognize the C-terminal portion of the core protein and are independent of cell surface hyaluronan. These results indicate that brevican is synthesized by astrocytes and retained on their surface by an interaction involving its core protein. Purified brevican inhibits neurite outgrowth from cerebellar granule neurons in vitro, an activity that requires chondroitin sulfate chains. We suggest that brevican presented on the surface of neuroglial sheaths may be controlling the infiltration of axons and dendrites into maturing glomeruli.

Animals↗

PubMed1997

A central segment of the NG2 proteoglycan is critical for the ability of glioma cells to bind and migrate toward type VI collagen.

Previous studies have established that the NG2 proteoglycan binds directly to type VI collagen. To further our understanding of the biochemical and functional significance of this interaction we have used NG2 cDNA to construct a series of NG2 mutants with deletions spaced throughout the entire length of the 260-kDa NG2 core protein. Following transfection of these mutant cDNAs into B28 glioma cells, we determined the ability of mutant NG2 molecules to anchor type VI collagen on the cell surface. Eight of 11 transfectant populations were able to anchor type VI collagen. The three NG2 variants incapable of anchoring type VI collagen have deletions clustered within the central one-third of the NG2 ectodomain. These deletions identify a 469-amino-acid domain of NG2 responsible for binding of type VI collagen. Functional consequences of the NG2-type VI collagen interaction were explored by testing the relative ability of NG2-transfected and untransfected glioma cells to migrate toward type VI collagen. NG2-expressing cells exhibited a greater migratory response toward type VI collagen than their NG2-negative counterparts. This enhanced migration could be specifically inhibited with NG2 antibodies. Furthermore, glioma cells expressing NG2 in which the collagen-binding domain was deleted failed to exhibit this enhanced migration, whereas NG2 mutants in which non-collagen-binding regions were deleted continued to exhibit increased chemotaxis toward the type VI collagen. These comparisons confirm the importance of the central collagen-binding domain in mediating functionally important interactions between NG2 and type VI collagen.

Animals↗

PubMed1997

The membrane-spanning proteoglycan NG2 binds to collagens V and VI through the central nonglobular domain of its core protein.

NG2 is a membrane-spanning proteoglycan with a primary structure unique among cell surface or extracellular matrix proteins. To characterize the interaction between NG2 and extracellular matrix proteins, we have used a eukaryotic expression system to produce and purify several recombinant fragments covering not only the entire ectodomain of NG2 but also distinct subdomains of the molecule. Using a solid phase binding assay with various extracellular matrix proteins, we have identified two main ligands for NG2, namely, collagens V and VI. Consistent with previous models of glycosaminoglycan attachment, roughly 50% of the recombinant NG2 fragments containing the central domain have chondroitin sulfate chains attached to the protein core. These glycosaminoglycan chains are not directly involved in collagen binding, since chondroitinase-treated fragments exhibit an unimpaired ability to bind to both collagens. Using more restricted recombinant fragments of NG2, we mapped the binding site for both collagens to the central domain of NG2. Electron microscopy after rotary shadowing of native NG2 molecules indicates that this extended nonglobular domain provides a flexible connection joining the two N- and C-terminal globular regions of NG2. Rotary shadowing of mixtures of NG2 and collagen V or VI confirms a direct interaction between the molecules and indicates that the collagens align with the central region of NG2, giving the appearance of a rod between the N- and C-terminal globules.

Antigens↗

PubMed1997

The characterization, molecular cloning, and expression of a novel hematopoietic cell antigen from CD34+ human bone marrow cells.

The adhesion molecule BEN/SC1/DM-GRASP (BEN) is a marker in the developing chicken nervous system that is also expressed on the surface of embryonic and adult hematopoietic cells such as immature thymocytes, myeloid progenitors, and erythroid progenitors. F84.1 and KG-CAM, two monoclonal antibodies to rat neuronal glycoproteins with similarity to BEN, cross-react with an antigen on rat hematopoietic progenitors, but F84.1 only also recognizes human blood cell progenitors. We have defined the antigen recognized by F84.1 as the hematopoietic cell antigen (HCA). HCA expression was detected on 40% to 70% of CD34+ fetal and adult bone marrow cells and mobilized peripheral blood cells. Precursor cell activity for long-term in vitro bone marrow cell culture was confined to the subset of CD34+ cells that coexpress HCA. HCA is expressed by the most primitive subsets of CD34+ cells, including all rhodamine 123(lo), Thy-1+, and CD38(-/lo) CD34+ adult bone marrow cells. HCA was also detected on myeloid progenitors but not on early B-cell progenitors. We also describe here the cloning and characterization of cDNAs encoding two variants of the human HCA antigen (huHCA-1 and huHCA-2) and of a cDNA clone encoding rat HCA (raHCA). The deduced amino acid sequences of huHCA and raHCA are homologous to that of chicken BEN. Recombinant proteins produced from either human or rat HCA cDNAs were recognized by F84.1, whereas rat HCA but not human HCA was recognized by antirat KG-CAM. Expression of either form of huHCA in CHO cells conferred homophilic adhesion that could be competed with soluble recombinant huHCA-Fc. The molecular cloning of HCA and the availability of recombinant HCA should permit further evaluation of its role in human and rodent hematopoiesis.

Adult↗

PubMed1997

Expression cloning and characterization of a cDNA encoding a novel membrane protein required for the formation of O-acetylated ganglioside: a putative acetyl-CoA transporter.

By expression cloning using COS-1 cells stably transfected with GD3-synthase (COS-1/GD3+) as a recipient cell line, we have isolated a cDNA, termed AT-1, encoding a novel protein required for the formation of O-acetylated (Ac) gangliosides. The cDNA encodes a protein with multitransmembrane spanning domains with a leucine zipper motif. It consists of 549 amino acids and has a molecular mass of 60.9 kDa. Although both O-Ac-GD3 and O-Ac-GT3 were barely detectable in recipient cells or cells transfected with the vector alone, their amount increased significantly in transfectants containing AT-1. When semi-intact cells prepared by treatment with streptolysin O were incubated with [Ac-14C]-Ac-CoA, increased incorporation of radioactivity was found in those cells transfected with AT-1 when compared with the mock transfectants. Northern blot analysis showed two major transcripts of 3.3 and 4.3 kb in all tissues examined. Immunohistochemical study with an antibody specific to the AT-1 protein suggested that it is most probably expressed in the endoplasmic reticulum membrane. Based on these results, the protein encoded by AT-1 is suggested to be an Ac-CoA transporter that is involved in the process of O-acetylation.

Acetylation↗