NOV/CCN3 promotes maturation of cerebellar granule neuron precursors

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Abstract

A body of evidence points to the matricial CCN proteins as key regulators of organogenesis. NOV/CCN3, a founder CCN member, is expressed in the developing central nervous system but its functions during neural development have not been studied yet. Here we describe the pattern of NOV expression during rat cerebellar postnatal development and show that NOV expression increases during the second postnatal week, a critical period for the maturation of granule neuron precursors (GNP). NOV transcripts are specifically produced by Purkinje neurons and NOV protein localises extracellularly in the molecular layer and the inner part of the external granule layer, at a key position to control GNP proliferation and migration. In vitro, NOV reduces Sonic Hedgehog-induced GNP proliferation through β3 integrins and stimulation of GSK3-β activity whereas NOV stimulates GNP migration through distinct RGD-dependent integrins. These findings identify a new paracrine role of NOV in the development of cerebellar granule neurons.

Introduction

The cerebellum, which is involved in motor coordination and regulation of cognitive processes, is a useful model for studying the complexity of central nervous system development. Indeed, the cerebellum has a well-defined cytoarchitecture and its development continues throughout the whole developmental window, from early neural embryogenesis until postnatal completion of brain maturity (Goldowitz and Hamre, 1998, Sotelo, 2004). In mammals, growth of the cerebellum occurs mainly during postnatal development and involves a dramatic increase in volume (Goldowitz and Hamre, 1998). This event is predominantly due to the expansion of granule neurons, the most abundant neuronal population in the brain (Wang and Zoghbi, 2001). Studies over the last decade have demonstrated that proliferation of cerebellar granule neuron precursors (GNP) lying in the external granule layer (EGL) is driven by homotypic cell–cell contacts through the Notch pathway (Solecki et al., 2001) and by underlying Purkinje neurons through secretion of diffusible factors (Dahmane and Ruiz i Altaba, 1999, Smeyne et al., 1995, Ye et al., 1996). Among those, Sonic Hedgehog (SHH) represents so far the main mitogenic factor for GNP both in vitro and in vivo (Dahmane and Ruiz i Altaba, 1999, Lewis et al., 2004, Wechsler-Reya and Scott, 1999). Medulloblastoma, the most common malignant childhood brain tumour, is thought to derive from dysregulated GNP proliferation (Marino, 2005). This has stimulated research to identify the factors directing GNP cell cycle exit. These factors include the pituitary adenylate cyclase-activating polypeptide (PACAP) (Nicot et al., 2002), several bone morphogenetic proteins (BMPs) (Alvarez-Rodriguez et al., 2007, Rios et al., 2004) and fibroblast growth factors (FGFs). Some evidence also indicate, that migration away from the mitogenic niche may also be required for GNP to exit the cell cycle successfully. Therefore, there is now a great interest in the identification of factors that could coordinate downregulation of GNP proliferation with stimulation of their migration from this mitogenic niche. Extra-cellular matrix proteins present in the molecular layer, just beneath the EGL, are attractive candidates for this role.

NOV/CCN3 is a founder member of the CCN family (Cyr61/ccn1, Ctgf/ccn2, Nov/ccn3) of multitasking matricial proteins which are now emerging as key players during organogenesis (Chen and Lau, 2009). Recent reports evidence the importance of NOV during skeletal and cardiac development (Heath et al., 2008, Rydziel et al., 2007). However, although the central nervous system is a major site of NOV expression during development (Joliot et al., 1992, Kocialkowski et al., 2001), no study has yet tried to identify the putative functions of NOV during development of this tissue. Interestingly, the abundance of Nov mRNA increases throughout the postnatal period of rat brain development (Su et al., 2001). NOV is particularly abundant in the cerebellum of young adult rats and is specifically produced by Purkinje neurons at this stage (Su et al., 2001).

NOV and the other cysteine-rich multimodular CCN proteins can regulate cellular processes as diverse as adhesion, migration, proliferation, differentiation and survival (Leask and Abraham, 2006). In particular, NOV regulates proliferation in various cellular contexts (Calhabeu et al., 2006, Ellis et al., 2000, Liu et al., 1999) and its overexpression leads to the growth arrest of several tumorigenic cell types (Benini et al., 2005, Gupta et al., 2001, McCallum et al., 2006). NOV can also stimulate the migration of several cell types including fibroblasts, endothelial and glioblastoma cells (Lin et al., 2005, Lin et al., 2003, Laurent et al., 2003). Interestingly, Nov overexpression inhibits proliferation and stimulates migration of a Ewing's Sarcoma cell line (Benini et al., 2005). Consistent with their multimodular structure and the diversity of their biochemical properties, NOV and other CCN members can modulate the activity of numerous signalling pathways including TGF-beta, TNF-alpha, VEGF, BMP, Wnt and Notch (Chen and Lau, 2009). In many cases, these modulations seem to result from direct interactions between CCN proteins and ligands, receptors or co-receptors of these pathways (Chen and Lau, 2009). However, it is currently assumed that NOV and other CCN members mainly act through integrin receptors (Chen and Lau, 2009). The specific integrin dimers described as possible NOV receptors include αvβ3 integrins (Ellis et al., 2003, Lin et al., 2005, Lin et al., 2003). Interestingly, two ligands of αvβ3 integrins, vitronectin and fibronectin, partially inhibit the mitogenic activity of SHH on GNP (Pons et al., 2001). NOV can also interact with several integrin dimers containing the β1 subunit which has been involved in the regulation of GNP proliferation in vivo and shown to be crucial for the synergistic effects of laminin on SHH-induced proliferation (Blaess et al., 2004, Graus-Porta et al., 2001).

To study the roles of NOV during central nervous system development, we focused on the potential function of NOV during cerebellar development. We report that in rat cerebellum, NOV is specifically expressed by Purkinje neurons and its expression increases during the postnatal period. Moreover, we observed that NOV preferentially localises in the inner part of the external granule layer and in the molecular layer, at a key position to regulate both proliferation and migration of cerebellar granule neuron precursors. We therefore investigated whether NOV modulated these processes using GNP primary cultures. We found that NOV decreases SHH-induced proliferation by interacting with β3 integrins and triggering a pathway leading to the stimulation of GSK3-β activity and subsequent destabilization of N-Myc. In parallel, we demonstrate that NOV stimulates the migration of GNP through a distinct RGD-dependent integrin receptor.

Section snippets

NOV expression increases during postnatal cerebellar development and specifically derives from Purkinje neurons

Real-time RT-PCR experiments indicated that Nov transcript abundance was unchanged between postnatal days (P) 1 and 7 but then increased substantially between P07 and P14 (Fig. 1A). There were no obvious changes between P14 and P21 when cerebellar development is ending. Overall, the amount of Nov transcripts increased 3-fold between P01 and P21 (Fig. 1A) and it then doubled between P21 and P90. As assayed by western blotting on whole cerebellar extracts, NOV protein was already detected at P01,

Discussion

To investigate the function of NOV during cerebellar development, we first established that NOV is specifically expressed and secreted by Purkinje neurons; we also demonstrated that NOV is mostly localised in the molecular layer and the inner part of the EGL, at the interface between Purkinje cell bodies and proliferating granule neuron precursors (GNP). Moreover, induction of NOV expression during postnatal cerebellar development coincides with the period of granule cell transition from the

Animals

Wistar rats from Janvier were kept under standard housing, feeding and lighting conditions. All animal procedures were performed in accordance with the European Union Guidelines for the Care of Laboratory Animals.

Antibodies and chemicals

NOV immunoreactivity was detected using a commercial goat (R&D Systems) or the home-made affinity-purified rabbit (referred as K19M, (Chevalier et al., 1998) anti-human NOV antibodies. The flowthrough resulting from K19M purification was used as a negative control. For western blotting

Acknowledgments

This work was supported by l'Institut National de la Santé et de la Recherche Médicale (INSERM). G. Le Dréau was recipient of a fellowship from the Association pour la Recherche contre le Cancer (ARC). We are grateful to I. Renault and her team for devoted animal care. We also acknowledge Dr F. Calhabeu (Randall Division of Cell and Molecular Biophysics, King's college, Guy's Hospital, London, UK) for skilful assistance with real-time RT-PCR. We thank Dr C. Dubois (INSERM UMR-S 893, Hôpital

References (65)

  • LinC.G. et al.

    Integrin-dependent functions of the angiogenic inducer NOV (CCN3): implication in wound healing

    J. Biol. Chem.

    (2005)
  • LiuC. et al.

    Nephroblastoma overexpressed gene (NOV) codes for a growth factor that induces protein tyrosine phosphorylation

    Gene

    (1999)
  • MarinoS.

    Medulloblastoma: developmental mechanisms out of control

    Trends Mol. Med.

    (2005)
  • McCallumL. et al.

    A novel mechanism for BCR-ABL action: stimulated secretion of CCN3 is involved in growth and differentiation regulation

    Blood

    (2006)
  • RydzielS. et al.

    Nephroblastoma overexpressed (Nov) inhibits osteoblastogenesis and causes osteopenia

    J. Biol. Chem.

    (2007)
  • SmeyneR.J. et al.

    Local control of granule cell generation by cerebellar Purkinje cells

    Mol. Cell. Neurosci.

    (1995)
  • SoleckiD.J. et al.

    Activated Notch2 signaling inhibits differentiation of cerebellar granule neuron precursors by maintaining proliferation

    Neuron

    (2001)
  • SoteloC.

    Cellular and genetic regulation of the development of the cerebellar system

    Prog. Neurobiol.

    (2004)
  • WallaceV.A.

    Purkinje-cell-derived Sonic hedgehog regulates granule neuron precursor cell proliferation in the developing mouse cerebellum

    Curr. Biol.

    (1999)
  • Wechsler-ReyaR.J. et al.

    Control of neuronal precursor proliferation in the cerebellum by Sonic Hedgehog

    Neuron

    (1999)
  • YeP. et al.

    In vivo actions of insulin-like growth factor-I (IGF-I) on cerebellum development in transgenic mice: evidence that IGF-I increases proliferation of granule cell progenitors

    Brain Res. Dev. Brain Res.

    (1996)
  • AdamsN.C. et al.

    Mice that lack astrotactin have slowed neuronal migration

    Development

    (2002)
  • BeniniS. et al.

    In Ewing's sarcoma CCN3(NOV) inhibits proliferation while promoting migration and invasion of the same cell type

    Oncogene

    (2005)
  • BlaessS. et al.

    Beta1-integrins are critical for cerebellar granule cell precursor proliferation

    J. Neurosci.

    (2004)
  • BorghesaniP.R. et al.

    BDNF stimulates migration of cerebellar granule cells

    Development

    (2002)
  • CarnegieJ.

    Influence of extracellular matrix gradients on the haptotactic migration of F9 embryocarcinoma-derived primitive and parietal endoderm-like cells

    J. Biol. Reprod.

    (1994)
  • CohenP. et al.

    The renaissance of GSK3

    Nat. Rev. Mol. Cell Biol.

    (2001)
  • CreanJ.K. et al.

    Connective tissue growth factor [CTGF]/CCN2 stimulates mesangial cell migration through integrated dissolution of focal adhesion complexes and activation of cell polarization

    FASEB J.

    (2004)
  • CrossD.A. et al.

    Selective small-molecule inhibitors of glycogen synthase kinase-3 activity protect primary neurones from death

    J. Neurochem.

    (2001)
  • ChevalierG. et al.

    novH: differential expression in developing kidney and Wilm's tumors

    Am. J. Pathol.

    (1998)
  • ChoiY. et al.

    Migration from a mitogenic niche promotes cell-cycle exit

    J. Neurosci.

    (2005)
  • ChomezP. et al.

    Increased cell death and delayed development in the cerebellum of mice lacking the rev-erbA(alpha) orphan receptor

    Development

    (2000)
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