Signalling pathways involved in the chemotactic activity of CXCL12 in cultured rat cerebellar neurons and CHP100 neuroepithelioma cells

Abstract

We compared the signal transduction pathways activated by stromal cell-derived factor-1 (CXCL12) chemokine in two different cell systems: primary cultures of rat cerebellar granule neurons (CGN) and human neuroepithelioma CHP100 cells. Both cell types express functional CXC chemokine receptor 4 (CXCR4), which is coupled both to extracellular signal-regulated kinase (ERK) and Akt phosphorylation pathways. The activation of ERK shows different dependency on the phosphatidylinositol 3-kinase (PI3-K) pathway and different sensitivity to pertussis toxin (PTX) treatment, indicative of coupling to different G proteins in the two cell systems considered. We demonstrate that the inhibition of either the ERK kinase or the PI3-K pathways blocks the CXCL12 induced-chemotaxis in CHP100 cells; while only PI3-K activity is stringently necessary for CGN migration.

Introduction

Chemokines and chemokine receptors are predominantly expressed in the immune system, where they regulate many different cellular functions (reviewed by Murphy et al., 2000). In the last few years, accumulating evidence indicate that chemokines and their receptors are widely expressed in several CNS regions, both under physiological and pathological conditions (Glabinski and Ransohoff, 1999). The role of chemokines in the CNS spans from the recruitment of inflammatory cells in affected regions during host defence to the regulation of many biological functions (Asensio and Campbell, 1999). Specifically, chemokines regulate cell proliferation, survival, and chemotaxis; and modulate synaptic transmission Araujo and Cotman, 1993, Ragozzino et al., 1998, Ragozzino et al., 2002, Robinson et al., 1998, Giovannelli et al., 1998, Limatola et al., 2000a, Limatola et al., 2000b, Limatola et al., 2002, Zheng et al., 1999b, Bezzi et al., 2001, Meucci et al., 1998, Puma et al., 2001, Lax et al., 2002. The CXC chemokine receptor 4 (CXCR4), the sole receptor known for the chemokine stromal cell-derived factor 1α (CXCL12) (Bleul et al., 1996) is expressed in central neurons, glial and microglial cells (Hesselgesser and Horuk, 1999), where its function is essential during development for the proper neuronal migration and hence for ensuring the correct cytoarchitecture of cerebellum and hippocampus Zou et al., 1998, Ma et al., 1998, Lu et al., 2001. In the CNS cells, CXCR4 is coupled to both pertussis toxin (PTX) -sensitive and -insensitive G proteins that lead to the activation of several signalling pathways. These pathways consist in: the activation of phosphoinositide hydrolysis, yielding the generation of Ca²⁺ transients; the phosphorylation of extracellular signal-regulated kinase (ERK), Akt and Pyk2; and the inhibition of adenylate cyclase Tanabe et al., 1997, Meucci et al., 1998, Zheng et al., 1999b, Limatola et al., 2000a, Lazarini et al., 2000, Bezzi et al., 2001, Oh et al., 2001, Gillard et al., 2002, Bajetto et al., 2001. Some correlations between the biological activities of CXCL12 and its intracellular signalling have been traced: specific inhibitors of the ERK kinase and phosphatidylinositol 3-kinase (PI3-K) pathways abolish CXCL12-induced astrocytes proliferation (Bajetto et al., 2001); and embryonic neuron chemotaxis is dependent on ERK signalling (Lazarini et al., 2000).

Even if CXCR4 expressed on neurons are functional, some CXCL12 effects on neurons are mediated through glial-derived factors: upon CXCL12 treatment of hippocampal slices, both astrocytes and activated microglial cells release TNFα that induces glutamate release by astrocytes (Bezzi et al., 2001). In addition, in cerebellar slices, CXCL12 induces a glutamate release mediating both Ca²⁺ entry and current responses in Purkinje neurons (Limatola et al., 2000a), and reducing the evoked excitatory synaptic currents at the parallel fibres synapses (Ragozzino et al., 2002).

During cerebellar maturation, the correct migration of cerebellar granule neurons (CGN) from the external to the inner layer is controlled by multiple and opposite chemotactic stimuli; one of them is represented by CXCL12, whose migratory signalling is impaired by the EphB receptor that affects CXCR4 coupling to G proteins (Lu et al., 2001). It is, therefore, interesting to study the signalling pathways activated by CXCR4 on CGN and their possible involvement in mediating cellular movement. The aim of this study was to analyse the signal transduction pathways involved in CXCL12-induced chemotaxis of CGN to improve the knowledge of the molecular mechanisms that regulate the timing of CGN migration during cerebellar development. The signalling pathways activated were compared with a different cell system of neural origin, the CHP100 neuroepithelioma cells, which has been elsewhere characterised with respect to CXCR4 expression (Catani et al., 2000). Neuroepithelioma and neuroblastoma are tumours with a common neuroectodermal origin; and CXCR4 expression is reported in a variety of neuroepithelioma and neuroblastoma cell lines Banisadr et al., 2000, Catani et al., 2000, Geminder et al., 2001, where they may be involved in directing tumour cell migration during metastases formation (Geminder et al., 2001). We demonstrated that CXCL12 activates ERK1/2 and Akt phosphorylation in both CGN and CHP100 cells, with different PTX dependency; and that while the ERK kinase pathway is involved in CXCL12-mediated chemotaxis of CHP100 cells, the activation of the PI3-K pathway mediates both CGN and CHP100 cell migration.