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