Phorbol esters, including phorbol myristate acetate (PMA), are analogues of diacylglycerol (DAG) and can therefore activate conventional and novel PKC isoforms (Zalewski et al., 1990; Obeid et al., 1992; Quest and Bell, 1994; Newton, 1997; Medkova and Cho, 1999). Unlike DAG, phorbol esters are not readily metabolised following binding to PKC and induce chronic PKC activation (Newton, 1997). This prolonged activation accounts for the tumorigenic effects of phorbol esters and led to the identification of PKC as the intracellular effector molecule (Marquez et al., 1992; Szallasi et al., 1996; Ventura and Maioli, 2001). Phorbol esters have consequently been used
extensively in research on PKC biochemistry (Zalewski et al., 1990; James and Olson, 1992; Obeid et al., 1992; Quest and Bell, 1994; Newton, 1997; Medkova and Cho, 1999), gene expression (Ventura and Maioli, 2001; Suh et al., 2004; Brettingham-Moore et al., 2005a) tumorigenesis (Correale et al., 1997; Gopalakrishna et al., 1997; Chen et al., 2000; Esteve et al., 2002), inflammation (Genot et al., 1995; Han et al., 2003; Abramov et al., 2005; Hull et al., 2006; Hashioka et al., 2007), cytoskeletal regulation (Wilms et al., 1997; Abe and Saito, 1999; Cheng et al., 2000; Kobayashi et al., 2001) and other effects (Kermorgant et al., 2003; Michel et al., 2005; Kheifets et al., 2006) mediated by PKC. The phorbol ester research includes numerous studies on the intracellular targets of PKC, including NF-κB (Baldwin, 1996; Schwaninger et al., 1999; Huang et al., 2003; Brettingham-Moore et al., 2005a), in a variety of cell types including astrocytes (Abe and Saito, 2000; Suh et al., 2004; Amos et al., 2005; Ludwig et al., 2005; Vermeiren et al., 2005; Hull et al., 2006) and microglia (Han et al., 2003; Hashioka et al., 2007; Moon- Sook et al., 2008; Woo et al., 2008).
3.03.1 Ionophores induce intracellular calcium ion influx
As described above, calcium ions are important second messengers in intracellular signalling pathways and increases in intracellular calcium ion concentration regulate many of the cellular responses to CNS injury. Ionophores, such as ionomycin or calcium ionophore (also known as calcimycin or A23187), can transport divalent cations across cell membranes down electrochemical gradients (Kolber and Haynes, 1981; Fasolato and Pozzan, 1989; Bergling et al., 1998). Since intracellular calcium ion (Ca2+) concentration is actively maintained by cells at much lower levels than extracellular levels (Carafoli, 2002; Clapham, 2007), ionophores induce Ca2+ influx that can initiate calcium transients under normal physiological and cell culture conditions (Venance et al., 1997; Calegari et al., 1999). The increased intracellular Ca2+ can promote activation of NF-κB through conventional PKC isoform activation (Nishizuka, 1988; Ahmed et al., 1991; Li et al., 2005a) and other calcium signalling pathways (Tsuboi et al., 1994; Baldwin, 1996; Abramov et al., 2005; Belcheva et al., 2005; Brettingham-Moore et al., 2005a; Silberman et al., 2005). Smooth endoplasmic reticulum calcium (SERCA) pumps and other Ca2+- ATPases actively transport calcium into intracellular calcium stores that are
predominantly located in the endoplasmic reticulum and mitochondria (Kannurpatti et al., 2000; Guerini et al., 2002; Beech et al., 2003; Paschen and Mengesdorf, 2003). Blocking these Ca2+-ATPase transporters with thapsigargin, can also elevate intracellular calcium ion concentrations (Lampe et al., 1995; Bergling et al., 1998; Brunig et al., 2004) leading to PKC and NF-κB activation (Pahl and Baeuerle, 1996; Paria et al., 2003).
3.03.2 Combined PMA and ionophore activation of NF-κB
Full activation of inflammatory genes requires interactions between NF-κB and other transcription factors (Chan et al., 2001; Grassl et al., 2003; Hayden and Ghosh, 2004; Chen et al., 2007; Wolter et al., 2008). Consequently, combined treatment with PMA and calcium ionophore has commonly been used to stimulate expression of NF-κB-regulated inflammatory genes in lymphocytes (Tsuboi et al., 1994; Baldwin, 1996; Schwaninger et al., 1999; Brettingham-Moore et al., 2005a; Silberman et al., 2005). Combined PMA and calcium ionophore also stimulated PKC-dependent production of IL-6 (Norris et al., 1994) and ICAM-1 expression (Ballestas and Benveniste, 1995) in astrocytes in a similar manner to the inflammatory cytokines, IL-1β and TNF-α. Furthermore, PMA and ionomycin-induced calcium influx promoted ROS generation by NADPH oxidase in astrocytes (Abramov et al., 2005) and activated NF-κB to induce COX-2 and subsequent prostaglandin production in an astroglial cell line (Rael et al., 2004a). However, the effects of these reagents are not always synergistic, with nerve growth factor production in astrocytes being promoted by PMA and inhibited by calcium ionophore- or thapsigargin-induced intracellular Ca2+ elevation (Jehan et al., 1995). As described above, both PKC and calcium signalling are involved in activating NF-κB to promote production of inflammatory mediators in astrocytes. Therefore, since PMA stimulates PKC and ionophore stimulates calcium signalling, NF-κB translocation to astrocyte nuclei in response to combined PMA and ionophore treatment was investigated in this Chapter as
an in vitro model of astrocyte inflammatory activation.
3.04 Research rationale
The aim of this Chapter was to establish a model of astrocyte inflammatory activation relevant to CNS injury, as required for the following research on the possible moderation
of this activation by OECs. To investigate activation, NF-κB translocation to astrocyte nuclei in vitro was measured by immunocytochemistry following stimulation with PMA and calcium ionophore. Translocation of NF-κB to astrocyte nuclei was chosen as a suitable relevant measure of astrocyte activation, since NF-κB is a key transcriptional regulator of inflammatory genes including many of the inflammatory genes involved in astrogliosis. Translocation of the p65 (or RelA) subunit was measured since it is the most potent transcriptional activator of the NF-κB family (Baeuerle and Henkel, 1994; Baldwin, 1996; Wang and Baldwin, 1998; Madrid et al., 2001; Ridder and Schwaninger, 2009) and most NF-κB activity in response to CNS injury involves the p50/p65 dimers (Schneider et al., 1999; Hang et al., 2006; Ridder and Schwaninger, 2009). The combined treatment with PMA and calcium ionophore is an established, well-characterised treatment that induces NF-κB translocation via activation of the PKC and calcium signalling pathways, both of which regulate astrocyte responses to CNS injury. GFAP expression was measured in response to stimulation with PMA and calcium ionophore since it is upregulated by reactive astrocytes following CNS injury.
3.05 Specific aims
1. Investigate PMA and calcium ionophore stimulation of p65 NF-κB translocation to astrocyte nuclei as a measure of inflammatory activation.
2. Investigate GFAP expression in astrocytes following stimulation with PMA and calcium ionophore.