Elsevier

Brain Research

Volume 983, Issues 1–2, 5 September 2003, Pages 74-83
Brain Research

Research report
Role of glypican-1 in the trophic activity on PC12 cells induced by cultured sciatic nerve conditioned medium: identification of a glypican-1–neuregulin complex

https://doi.org/10.1016/S0006-8993(03)03031-2Get rights and content

Abstract

Glypican-1 is an extracellular matrix component found by microsequencing in a medium conditioned by cultured rat-sciatic nerves (CM). This CM was concentrated by ultrafiltration and fractionated by quaternary ammonium chromatography, followed by Hi-Trap blue affinity chromatography to obtain the active fraction B1.2. Previously, we have reported a 54 kDa neuregulin (NRG) in the same B1.2 fraction [Villegas et al., Brain Res. 852 (2001) 304]. The effect of Glypican-1 on the neuron-like differentiation of PC12 cells was investigated by immunoprecipitation, Western blot and cellular image analysis. Removal of glypican-1 by immunoprecipitation with increasing concentrations of specific antibodies revealed a gradual decrease of the differentiation activity of fraction B1.2, which paralleled the results obtained by removal of the 54 kDa NRG protein. Colorless native electrophoresis and Western blot analysis was used to identify a glypican-1-NRG protein complex, which could be afterwards separated by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis into its individual components. Additionally, it was demonstrated that glypican-1, in cooperation with the 54 kDa NRG, is involved in the neuronal-like differentiation of PC12 cells and could play an important role on the regeneration responses of peripheral nerves.

Introduction

The development, maintenance and repair of vertebrate nervous systems depend on the activity of a variety of neurotrophic factors and other molecules, such as those of the extracellular matrix, which modulate neurite outgrowth.

A remarkable neurotrophic activity has been reported in the sciatic nerve by several investigators [1], [2], [3], [22], [37], [39], [40], [46]. Different sciatic nerve preparations, such as nerve explants [11], [20], [38], [41], [48], [50], nerve extracts [35], [48], nerve conditioned media [13], [45], [46], [47], [50], Schwann cell conditioned media [5], [8] and nerve secreted fluid collected in regeneration chambers [6], [28] have been used. Some of these works, including our own [46], [47], have allowed us to identify the presence of neurotrophic factors underlying the neurotrophic activity of these nerves.

We have immunodetected a 54 kDa NRG in the most active fraction (B1.2) of a medium conditioned by cultured rat sciatic nerves (CM), which was capable of inducing neuron-like differentiation of PC12 cells [47], [48]. In the same fraction, we have also identified by microsequencing other components, such as collagen, fibronectin and glypican-1. Extracellular matrix components, in addition to cell surface adhesion molecules and neurotrophic factors, play an important role in cell-to-cell adhesion and may regulate axon behavior [23]. Glypican is one of the mayor constituents of the extracellular matrix and has been found widely distributed in plasma membranes of nervous tissue [18], [25]. Different studies have indicated that glypican interacts with a variety of heparin binding proteins such as other extracellular matrix components and growth factors [27], [33], [34], [44]. This issue has been particularly well studied for fibroblast growth factors (FGFs) [7] and more recently for neuregulin (NRG) at the neuromuscular junction [24].

In the present work, we found that glypican-1 is involved in the neuronal differentiation activity of PC12 cells exhibited by fraction B1.2. By using a combination of native electrophoresis and Western blot analysis, we identified a glypican-1–NRG protein complex that could be further separated by polyacrylamide gel electrophoresis (SDS–PAGE) into its individual components. The possible role of glypican-1, as modulator of the interaction between NRG proteins and their ErbB receptors is also discussed.

Section snippets

Preparation of cultured sciatic nerve conditioned medium

The conditioned medium (CM) was prepared from cultured adult rat sciatic nerves as indicated in previous works [47], [48]. In summary, sciatic nerves from 60 Sprague–Dawley adult rats, 200–250 g weight, were cultivated within groups of eight in T-.25 culture flasks containing 6 ml of serum-free Dulbecco’s modified Eagle’s medium (DMEM) for 8 days. At the beginning of day 9, nerves were transferred to new flasks with fresh serum-free DMEM. CM was collected every 24 h from the end of day 9 until

Identification of glypican-1 and NRG proteins in fraction B1.2 by SDS–PAGE, microsequencing and Western blotting

According to previous results [48], when fraction B1.2 was analyzed by HPLC (Protein Pak 300 column), the most active peak in the neuron-like differentiation assay contained proteins in the molecular range of 50–70 kDa. Microsequencing analysis of the electrophoretic polypeptide bands obtained from the B1.2 fraction led to the identification of four polypeptides migrating within the 53–54 kDa range, two of them (DEIQISTGNALFIEK and MQQVEASLQPETLR) having 100% homology with known sequences from

Discussion

In previous works [47], [48], we have reported that CM produced neuronal-like differentiation of PC12 cells. Further attempts to identify the components of the active fraction B1.2 revealed the presence of a 54 kDa NRG that seem to play a key role in the differentiation of these cells [48]. Our results obtained here by microsequencing and Western blotting also provided evidences of the presence of glypican-1 in the active fractions separated from the CM. The anti-glypican-1 antibody recognized

Uncited reference

[10]

Acknowledgements

This work was supported by CONICIT Venezuela Grants S1-95000709 (to R.V.) and S1-2000000641 (to R.V), the Vollmer Research Fund of the Centro de Estudios Cientı́ficos de Caracas (CEC-Caracas), and donations from the Vollmer Foundation and the Fundación Pro Ciencia. We thank Dr A.D. Lander for kindly providing the anti-Glypican-1 polyclonal antibody; Professor J. Bubis for critical reading of the manuscript and J. Núñez, E. Majul, F. Castillo and G. Dı́az for technical assistance.

References (50)

  • A.J. Aguayo

    Axonal regeneration from injured neurons in the adult mammalian central nervous system

  • A.J. Aguayo et al.

    A potential for axonal regeneration in neurons of the adult mammalian nervous system

  • A.J. Aguayo et al.

    Axon-sheath cell interactions in peripheral and central nervous system transplants

    Soc. Neurosci. Symp.

    (1979)
  • J.L. Andres et al.

    Membrane-anchored and soluble forms of betaglycan, a polymorphic proteoglycan that binds transforming growth factor-beta

    J. Cell Biol.

    (1989)
  • J.G. Assouline et al.

    Rat astrocytes and Schwann cells in culture synthesize nerve growth factor-like neurite-promoting factors

    Dev. Brain Res.

    (1987)
  • D.J. Bates et al.

    Multiple neurotrophic factors including NGF-like activity in nerve regeneration chamber fluids

    Neurochem. Int.

    (1995)
  • D. Bonneh-Barkay et al.

    Identification of glypican as dual modulator of the biological activity of fibroblast growth factor

    J. Biol. Chem.

    (1997)
  • L.M. Bolin et al.

    Characterization of a Schwann cell neurite-promoting activity that directs motoneuron axon outgrowth

    J. Neurosci. Res.

    (1994)
  • D.J. Carey et al.

    Processing and subcellular distribution of the Schwann cell lipid-anchored heparan slfate proteoglycan and identification as glypican

    Exp. Cell Res.

    (1993)
  • S.L. Carroll et al.

    Expression of neuregulins and their putative receptors, ErbB2 and ErbB3, is induced during Wallerian degeneration

    J. Neurosci.

    (1997)
  • T.J. Collie et al.

    Diffusible factor(s) from adult rat sciatic nerve increases cell number and neurite outgrowth of cultured embryonic ventral mesencephalic tyrosine hydroxylase-positive neurons

    J. Neurosci. Res.

    (1990)
  • G. David

    Integral membrane heparan sulfate proteoglycan

    FASEB J.

    (1993)
  • M. Dobretsov et al.

    Influence of factors released from sciatic nerve on adult dorsal root ganglion neurons

    J. Neurobiol.

    (1992)
  • J. Dong et al.

    Genomic organization and chromosome localization of the newly identified human heparanase gene

    Gene

    (2000)
  • A. Heremans et al.

    The core protein of the matrix-associated heparan sulfate proteoglycan binds to fibronectin

    J. Biol. Chem.

    (1990)
  • R.E. Hill et al.

    Accelerated evolution in the reactive centre regions of serine protease inhibitors

    Nature

    (1987)
  • W.E. Holmes et al.

    Identification of heregulin, a specific activator of p185erbB2

    Science

    (1992)
  • L. Karthikeyan et al.

    Immunocytochemical and in situ hybridization studies of the heparan sulfate proteoglycan, glypican, in nervous tissue

    J. Cell Sci.

    (1994)
  • L. Karthikeyan et al.

    Cloning of a major heparan sulfate proteoglycan from brain and identification as the rat form of glypican

    Biochem. Biophys. Res. Commun.

    (1992)
  • D.P. Kuffler et al.

    Neurotrophic influence of denervated sciatic nerve on adult dorsal root ganglion neurons

    J. Neurobiol.

    (1994)
  • U.K. Laemmli

    Cleavage of structural proteins during the assembly of bacteriophage T4

    Nature

    (1970)
  • G. Leoz-Ortı́n et al.

    Procesos regenerativos del nervio óptico y retina con ocasión de injertos nerviosos

    Trab. Lab. Invest. Biol. Madrid

    (1913)
  • P.C. Letourneau et al.

    Interactions of developing neurons with the extracellular matrix

    J. Neurosci.

    (1994)
  • Q. Li et al.

    Neuregulin-heparan-sulfate proteoglycan interactions produce sustained erbB receptor activation required for the induction of acetylcholine receptors in muscle

    J. Biol. Chem.

    (2001)
  • E.D. Litwack et al.

    Neuronal expresion of glypican, a cell-surface glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan in the adult rat nervous system

    J. Neurosci.

    (1994)
  • Cited by (20)

    • Extracellular Application of CRMP2 Increases Cytoplasmic Calcium through NMDA Receptors

      2018, Neuroscience
      Citation Excerpt :

      The HPLC system consisted of a 1525 binary pump and 2475 multi λ fluorescence detector (Waters, Millford, MA), set at 340-nm excitation and 450-nm emission wavelengths. Samples were prepared from band excised in the range of 58–60 kDa as described before (Malavé et al., 2003). These samples were sequenced (microsequencing) at Harvard Microchemistry Facility, Harvard University (Cambridge, MA).

    • ErbB receptors and PKC regulate PC12 neuronal-like differentiation and sodium current elicitation

      2013, Neuroscience
      Citation Excerpt :

      Other remarkable capabilities of sciatic nerves are described in the findings reported by Villegas et al. (1995) who demonstrated that a conditioned media from rat sciatic nerves (CM) was able to differentiate a pro-neuronal cell model (PC12 cells) into cells with a neuronal-like phenotype, showing neurites as a distinctive feature. In agreement with this idea, it was previously reported that this CM has factors like neuregulin-1 and glypican-1 (Villegas et al., 2000; Malavé et al., 2003) which were implicated in the neurotrophic activity of the above mentioned CM. However, additional studies showed that the CM was incapable of inducing sodium currents in PC12 cells (Castillo et al., 2001).

    • Sciatic nerve conditioned medium depleted of pro-NGF modulates sodium currents and neurite outgrowth in PC12 cells

      2009, Neuroscience
      Citation Excerpt :

      These results are in agreement with findings where the involvement of these receptors in neurite elongation has been established and extensively discussed (Huang and Reichardt, 2001; Segal, 2003; Diolaiti et al., 2007). Our results, together with the findings from experiments where CM was depleted of neuregulin-1 or glypican-1 (long neurites were inhibited but short neurites remained) (Villegas et al., 2000; Malave et al., 2003), suggested that other molecules in the CM might act through p75NTR and TrkA receptors to induce the development of short neurites. As a further characterization of the CM effect, an important aspect to establish was if molecules present in the CM were able to induce neuronal polarization, therefore the unknown nature of the neurites generated either with CM or CM+k252a, was studied.

    View all citing articles on Scopus
    View full text