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.

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.

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.

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.

NG2 proteoglycan and the actin-binding protein fascin define separate populations of actin-containing filopodia and lamellipodia during cell spreading and migration.

The transmembrane proteoglycan NG2 is able to interact both with components of the extracellular matrix and with the actin cytoskeleton. An examination of the distribution of NG2 during cell spreading suggests that NG2 can associate with two distinct types of actin-containing cytoskeletal structures, depending on the nature of the stimulus derived from the substratum. On fibronectin-coated dishes, cell surface NG2 associates exclusively with stress fibers developing within the cell. On poly-L-lysine-coated dishes, cell surface NG2 is associated with radial processes extending from the cell periphery. Spreading on fibronectin/poly-L-lysine mixtures, as well as on matrix components such as laminin, tenascin, and type VI collagen, produces cells with mosaic characteristics, i.e., NG2 is associated with both types of structures. NG2-positive radial processes are distinct from a second population of radial structures that contain fascin. NG2-positive extensions appear to be individual self-contained units (filopodia), whereas fascin is associated with actin ribs within sheets of membrane (lamellipodia). NG2- and fascin-positive structures are often localized to opposite poles of spreading cells, suggesting a possible role for the two classes of cellular extensions in the establishment of cell polarity during morphogenesis or migration. Time lapse imaging confirms the presence of lamellipodia on the leading edges of migrating cells, while numerous filopodia are present on trailing edges.

Participation of the NG2 proteoglycan in rat aortic smooth muscle cell responses to platelet-derived growth factor.

Through immunohistochemical studies we have identified the cell-surface proteoglycan, NG2, on blood vessels throughout the rat embryo. The particular cell type expressing this chondroitin sulfate proteoglycan, however, is dependent upon tissue location. Microvessels within the rat CNS express NG2 on endothelial cells, while in blood vessels outside the CNS, NG2 is found on smooth muscle cells. To analyze what role NG2 might play in these blood vessels, an enzymatic dissociation protocol was used to establish primary cultures of vascular smooth muscle cells from Postnatal Day 3 rat aorta. In this study we demonstrate the involvement of NG2 in the mitogenic and chemoattractant responses of smooth muscle cells to PDGF. In assays measuring either DNA synthesis or cell migration, treatment of smooth muscle cells with anti-NG2 immunoglobulins decreased their responses to PDGF-AA but had no effect upon their ability to react to PDGF-BB. These results support a role for NG2 in potentiating signaling through the alpha PDGF receptor in vascular smooth muscle cells. The presence of the proteoglycan on a large subpopulation of these cells could provide an enhanced response to the growth factor in times of active normal growth or in pathological conditions, such as arterial injury or atherosclerosis.

Hybrid formation between endogenous mouse and transfected human tyrosine kinase-deficient (A/K1018) insulin receptors leads to decreased insulin sensitivity in 3T3-L1 adipocytes.

Swiss Mouse 3T3-L1 cells provide a unique model for insulin-sensitive primary fat cells. Under defined conditions this fibroblast cell line can be converted to fully differentiated adipocytes, characterized by increased insulin receptor number and induction of adipogenic-specific proteins. 3T3-L1 cells were therefore transfected with the cDNA for the A/K1018 insulin receptor (alanine substituted for lysine at amino acid 1018 in the ATP binding region of the kinase domain). The cell lines in which the dominant inhibitory effects of the A/K1018 receptor had been previously demonstrated [Rat 1 fibroblasts and Chinese Hamster Ovary cells] have relatively few endogenous insulin receptors; however, the 3T3-L1 cell line has about 200,000 insulin receptors when differentiated. In this study we have used this cell line to explore the molecular mechanisms for the dominant inhibitory effect of a tyrosine kinase-defective insulin receptor on insulin action. The A/K1018 receptor was inhibitory in the 3T3-L1 cells as shown by decreased glucose transport. Further, altered differentiation in the transfected cell implicates the insulin receptor as an important downstream regulator in this process. We were able to demonstrate the presence of numerous hybrid receptors, composed of endogenous mouse heterodimers and human kinase-deficient heterodimers in these cells. Trans phosphorylation did occur within these hybrids as evidenced by autophosphorylation of human beta-subunits; however, these hybrids are unable to phosphorylate substrates. These results establish hybrid formation as an important determinant in the dominant negative nature of the A/K1018 insulin receptor.

Tyrosine kinase-defective insulin receptors undergo decreased endocytosis but do not affect internalization of normal endogenous insulin receptors.

To characterize tyrosine kinase activity in signaling ligand/receptor internalization, metabolic labeling and surface radioligand binding were used to follow the processing of both normal and tyrosine kinase-deficient human insulin receptors. The mutant receptor (A/K1018) has an alanine substituted for lysine 1018 in the ATP-binding domain. Rat 1 fibroblasts, expressing either normal human insulin receptors (HIRc) or A/K1018 receptors, were assayed to determine the insulin receptor half-life as well as internalization and down-regulation. Our results show that insulin greatly reduces the half-life of normal insulin receptors (from 9.9 to 5.7 h). The A/K1018 receptor had a much longer half-life (24 h), which was not reduced by the presence of saturating insulin concentrations. The A/K1018 receptor does not undergo down-regulation after long term insulin exposure, while HIRc cells showed a 34% decrease in insulin receptor number. This down-regulation is accounted for by the accelerated turnover rate of normal receptors in the presence of insulin. To confirm that the kinase activity is necessary for normal endocytosis, we also show that ATP depletion in HIRc cells resulted in significant decreases in receptor internalization and that tyrosine kinase-defective receptors also fail to internalize in a different cell type (rat Fao hepatocytes). Lastly, the complement of normal rat insulin receptors in cells expressing the kinase-defective receptors endocytose normally. We conclude that the defect in endocytosis observed in kinase-defective receptors is intrinsic to this receptor and not due to a dominant inhibitory effect on cellular endocytotic machinery.