Phosphorylation of NG2 Proteoglycan by Protein Kinase C- Regulates Polarized Membrane Distribution and Cell Motility

Cancer Research Center, The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 01/2005; 279(53):55262-70. DOI: 10.1074/jbc.M411045200
Source: PubMed


Protein kinase C (PKC)-alpha phosphorylation of recombinant NG2 cytoplasmic domain and phorbol ester-induced PKC-dependent phosphorylation of full-length NG2 expressed in U251 cells are both blocked by mutation of Thr(2256), identifying this residue as a primary phosphorylation site. In untreated U251/NG2 cells, NG2 is present along with ezrin and alpha(3)beta(1) integrin in apical cell surface protrusions. Phorbol ester treatment causes redistribution of all three components to lamellipodia, accompanied by increased cell motility. U251 cells expressing NG2 with a valine substitution at position 2256 are resistant to phorbol ester treatment: NG2 remains in membrane protrusions and cell motility is unchanged. In contrast, NG2 with a glutamic acid substitution at position 2256 redistributes to lamellipodia even without phorbol ester treatment, rendering transfected U251 cells spontaneously motile. PKC-alpha-mediated NG2 phosphorylation at Thr(2256) is therefore a key step for initiating cell polarization and motility.

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    • "Furthermore, we see a difference in NG2 band distribution by western blot when comparing pericyte cells to meningeal explant cells. This may be due to differential expression of NG2 isoforms in either cell type or due to phosphorylation events which play a role in polarity and migration [53,54]. For the above reasons, although we have operationally defined these cells as pericytes (based on αSMA, PDGFRβ and NG2 staining), it is possible that they represent a mixed cell population that may additionally contain mesenchymal stem cells and fibroblast-like cells. "
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    ABSTRACT: Background Brain inflammation plays a key role in neurological disease. Although much research has been conducted investigating inflammatory events in animal models, potential differences in human brain versus rodent models makes it imperative that we also study these phenomena in human cells and tissue. Methods Primary human brain cell cultures were generated from biopsy tissue of patients undergoing surgery for drug-resistant epilepsy. Cells were treated with pro-inflammatory compounds IFNγ, TNFα, IL-1β, and LPS, and chemokines IP-10 and MCP-1 were measured by immunocytochemistry, western blot, and qRT-PCR. Microarray analysis was also performed on late passage cultures treated with vehicle or IFNγ and IL-1β. Results Early passage human brain cell cultures were a mixture of microglia, astrocytes, fibroblasts and pericytes. Later passage cultures contained proliferating fibroblasts and pericytes only. Under basal culture conditions all cell types showed cytoplasmic NFκB indicating that they were in a non-activated state. Expression of IP-10 and MCP-1 were significantly increased in response to pro-inflammatory stimuli. The two chemokines were expressed in mixed cultures as well as cultures of fibroblasts and pericytes only. The expression of IP-10 and MCP-1 were regulated at the mRNA and protein level, and both were secreted into cell culture media. NFκB nuclear translocation was also detected in response to pro-inflammatory cues (except IFNγ) in all cell types. Microarray analysis of brain pericytes also revealed widespread changes in gene expression in response to the combination of IFNγ and IL-1β treatment including interleukins, chemokines, cellular adhesion molecules and much more. Conclusions Adult human brain cells are sensitive to cytokine challenge. As expected ‘classical’ brain immune cells, such as microglia and astrocytes, responded to cytokine challenge but of even more interest, brain pericytes also responded to such challenge with a rich repertoire of gene expression. Immune activation of brain pericytes may play an important role in communicating inflammatory signals to and within the brain interior and may also be involved in blood brain barrier (BBB) disruption . Targeting brain pericytes, as well as microglia and astrocytes, may provide novel opportunities for reducing brain inflammation and maintaining BBB function and brain homeostasis in human brain disease.
    Full-text · Article · Jun 2014 · Journal of Neuroinflammation
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    • "Experiments with these cellular models consistently delineated the differential activation of multiple signal transduction pathways in NG2-expressing versus NG2-deficient cells upon selective confrontation with Col VI. We identified diverse phosphorylation patterns in both components linked to cell survival pathways of the PCK–PI-3K–Akt-1– mTOR-type and molecules associated with growth-promoting pathways of the ERK–MAPK-type; the latter already known to be associated with the phosphorylation of the cytoplasmic NG2 threonine residues (Makagiansar et al., 2004, 2007). Modulation of the phosphorylation state of molecules affecting the actomyosin-regulated cell contractility and focal adhesion formation was in partial agreement with previous Col VI-unrelated findings and provided additional evidence for a tight NG2 exerted control of the actin microfilament dynamics in critical cytoplasmic domains of motile cells. "
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    ABSTRACT: In soft-tissue sarcoma patients, enhanced expression of NG2/CSPG4 proteoglycan in pre-surgical primary tumours predicts post-surgical metastasis formation and thereby stratifies patients into disease-free survivors and patients destined to succumb from the disease. Both primary and secondary sarcoma lesions also up-regulate collagen type VI, a putative extracellular matrix ligand of NG2, and this matrix alteration potentiates the prognostic impact of NG2. Enhanced constitutive levels of the proteoglycan in isolated sarcoma cells tightly correlates with a superior engraftment capability and local growth in xenogenic settings. This apparent NG2-associated malignancy was also corroborated by the diverse tumorigenic behaviour in vitro and in vivo of immunoselected NG2-expressing and NG2-deficient cell subsets, by RNAi-mediated knock down of endogenous NG2, and by ectopic transduction of full-length or deletion constructs of NG2. Cells with modified expression of NG2 diverged in their interaction with purified Col VI, matrices supplemented with Col VI, and cell-free matrices isolated from wild type and Col VI null fibroblasts. The combined use of dominant-negative NG2 mutant cells and purified domain fragments of the collagen allowed us to pinpoint the reciprocal binding sites within the two molecules and to assert the importance of this molecular interaction in the control of sarcoma cell adhesion and motility. The NG2-mediated binding to Col VI triggered activation of convergent cell survival- and cell adhesion/migration-promoting signal transduction pathways, implicating PI-3 K as a common denominator. Thus, the findings point to an NG2-Col VI interplay as putatively involved in the regulation of the cancer cell-host microenvironment interactions sustaining sarcoma progression.
    Full-text · Article · Apr 2013 · Journal of Molecular Cell Biology
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    • "However, no evidence currently exists for involvement of the proteoglycan in adrenergic signaling. More realistic possibilities are based on our findings that NG2 promotes activation of signaling through β1 integrins [41], [42], [43], [44] and through growth factor receptors for FGF and PDGF [17], [29], [45]. While neither integrins nor growth factor receptors represent traditional G protein-coupled receptors, there are nevertheless numerous reports of G protein-dependent and G protein-independent activation of PKA by β1 integrin [46], [47], [48] and receptors for FGF or PDGF [47], [49], [50], [51]. "
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    ABSTRACT: Obesity is a major health problem worldwide. We are studying the causes and effects of obesity in C57Bl/6 mice following genetic ablation of NG2, a chondroitin sulfate proteoglycan widely expressed in progenitor cells and also in adipocytes. Although global NG2 ablation delays early postnatal adipogenesis in mouse skin, adult NG2 null mice are paradoxically heavier than wild-type mice, exhibiting larger white fat deposits. This adult onset obesity is not due to NG2-dependent effects on CNS function, since specific ablation of NG2 in oligodendrocyte progenitors yields the opposite phenotype; i.e. abnormally lean mice. Metabolic analysis reveals that, while activity and food intake are unchanged in global NG2 null mice, O(2) consumption and CO(2) production are decreased, suggesting a decrease in energy expenditure. Since brown fat plays important roles in regulating energy expenditure, we have investigated brown fat function via cold challenge and high fat diet feeding, both of which induce the adaptive thermogenesis that normally occurs in brown fat. In both tests, body temperatures in NG2 null mice are reduced compared to wild-type mice, indicating a deficit in brown fat function in the absence of NG2. In addition, adipogenesis in NG2 null brown pre-adipocytes is dramatically impaired compared to wild-type counterparts. Moreover, mRNA levels for PR domain containing 16 (PRDM16) and peroxisome proliferator-activated receptor γ coactivator (PGC)1-α, proteins important for brown adipocyte differentiation, are decreased in NG2 null brown fat deposits in vivo and NG2 null brown pre-adipocytes in vitro. Altogether, these results indicate that brown fat dysfunction in NG2 null mice results from deficits in the recruitment and/or development of brown pre-adipocytes. As a consequence, obesity in NG2 null mice may occur due to disruptions in brown fat-dependent energy homeostasis, with resulting effects on lipid storage in white adipocytes.
    Full-text · Article · Jan 2012 · PLoS ONE
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