*4.3. MAPK Signaling*

A number of groups have implicated MAPK signaling in propagating bystander effects in non-irradiated cells [146,147,153]. Using human B-lymphoblastoid cell lines, one group reported phosphorylation of ERK1/2, JNK and p38 in bystander cells exposed to CM from X-ray irradiated cells [153]. Additionally, bystander-induced caspase 3/7 activation could be diminished by treatment with ERK inhibitor U0126, JNK inhibitor SP600125 or p38 inhibitor SB203580. Human keratinocytes treated with conditioned medium (CM) from irradiated keratinocytes showed elevated ERK and JNK signaling [146]. CM-mediated apoptosis in bystander keratinocytes could be blocked by JNK inhibition, although ERK inhibition seemed to increase bystander-induced apoptosis in these cells [146].

Gq-coupled P2Y receptors (P2Y1,2,4,6,11Rs) activate phospholipase C (PLC) resulting in the production of inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG), in turn mobilizing intracellular Ca2<sup>+</sup> and activating protein kinase C (PKC) and subsequent downstream signaling pathways [144], including ERK1/2 activation, that have been implicated in mediating the bystander effect [146,154]. Further, through its C-terminal Src homology 3 (SH3) domain, the P2Y2R is involved in Src-dependent activation of growth factor receptors (e.g., EGFR, VEGFR-2), ERK1/2, and JNK [144]. P2Y2R is also capable of transactivation of EGFRs by activating metalloproteases (i.e., ADAM10 and ADAM17) [155]. In turn, EGFR activation initiates MAPK signaling cascades. In this way, P2Y2Rs are capable of mediating MAPK signaling through canonical Gq-coupled signaling or through activation of growth factor receptors such as EGFR.

Another potential role for P2Rs in mediating bystander effects involves PKC. We discussed above that knocking down PKCδ expression inhibits IR-induced apoptosis in irradiated murine parotid glands [63], while blocking PKCδ activity by administration of nanoparticles containing siRNA targeting PKCδ reduces apoptosis in irradiated mouse SMG [3]. Directly blocking c-Src with imatinib or dasatinib had a similar anti-apoptotic effect and enhanced saliva production [78]. P2R antagonists (DIDS, suramin and Cibacron Blue 3GA) increase phosphorylation of PKCδ in rat parotid acinar

cells, confirming that P2 activity promotes PKCδ activation [156]. Together these data suggest that ATP-induced activation of P2Rs promotes apoptosis in bystander cells via PKCδ phosphorylation.
