Mitogenic Signaling via Endogenous κ-Opioid Receptors in C6 Glioma Cells: Evidence for the Involvement of Protein Kinase C and the Mitogen-Activated Protein Kinase Signaling Cascade

The μ, κ, and δ members of the opioid receptor (OR) family are expressed by astroglial cells (Eriksson et al., 1990, 1991; Barg et al., 1991, 1993; Stiene-Martin and Hauser, 1991; Ruzicka et al., 1995; Bohn et al., 1998; Stiene-Martin et al., 1998). Opioid signaling via these G protein-coupled receptors (GPCRs) can either inhibit (Stiene-Martin and Hauser, 1991; Barg et al., 1994; Gurwell et al., 1996; Hauser et al., 1996) or stimulate (Barg et al., 1993; Gorodinsky et al., 1995) glial cell proliferation. Because astrocytes are intimately associated with neurons and can effect neuronal responses and play a role in neuronal development, regulation of astroglial growth during brain ontogeny is critical (Gasser and Hatten, 1990; Lin et al., 1993; Nedergaard, 1994; Parpura et al., 1994). Current evidence suggests that κ-OR-selective agonists activate L-type Ca2+ channels (Eriksson et al., 1993) and increase astroglial proliferation (Barg et al., 1993). C6 cells have been widely used as an astrocytic model system for the study of neurotrophic actions mediated by mitogens such as endothelin (MacCumber et al., 1990; Chuang et al., 1991; Couraud et al., 1991; Lin et al., 1992; Barg et al., 1994), nerve growth factor (Kumar et al., 1990), insulin-like growth factor (Lowe et al., 1992), and basic fibroblast growth factor (Okumura et al., 1989; Luo and Miller, 1996). On treatment with the tricyclic antidepressant desipramine (DMI), both high-affinity β-endorphin and μ-OR-selective agonist binding is induced (Albouz et al., 1982; Reggiani et al., 1987; Barg et al., 1991; Bohn et al., 1998). Recently, κ-OR expression by C6 cells has been documented by RNase protection assays (L.M.B., unpublished data), RT-PCR analysis, and radioligand binding studies, which reveal a Bmax of 75 fmol/mg of protein (Bohn et al., 1998). In addition, these cells bind the κ-selective agonist U69,593 independent of DMI pretreatment. Therefore, C6 cells are an attractive model system for the study of the signal transduction mechanisms used by endogenously expressed κ-ORs. ORs can activate the mitogen-activated protein (MAP) kinase signaling cascade when overexpressed in Chinese hamster ovary or COS cells (Avidor-Reiss et al., 1996; Fukuda et al., 1996; Li and Chang, 1996; Belcheva et al., 1998; Ignatova et al., 1999). In vivo, chronic morphine treatment stimulates MAP kinase activity in the ventral tegmental area of rat brain (Berhow et al., 1996). A correlation between astrocyte proliferation and endothelin GPCR-mediated stimulation of the MAP kinase cascade has been demonstrated (Lazarini et al., 1996). Recently, it has been proposed that GPCR activation of the MAP kinase signaling cascade can be mediated by a putative member of the focal adhesion kinase family, PYK2 (also termed RAFTK and CAKβ), in PC12 cells, which are a neuronal model system (Avraham et al., 1995; Lev et al., 1995; Soltoff, 1998; Soltoff et al., 1998). Here we examine the signaling pathways activated by κ-opioids in C6 cells. Furthermore, we evaluate whether κ-ORs can mediate increased proliferation of C6 cells and whether activation of the MAP kinase signaling cascade is involved. We show that the κ-OR-selective agonist U69,593 stimulates proliferation of C6 cells via a pertussis toxin (PTX)-sensitive activation of phospholipase C (PLC), protein kinase C (PKC), and extracellular signal-regulated kinase (ERK). By use of interfering or dominant-negative mutants we demonstrate that ERK phosphorylation is dependent on functional Gβγ subunits and Ras. In addition, we also present the observation that U69,593 increases phosphorylation of PYK2.