ROS-induced store-operated Ca²⁺ entry coupled to PARP-1 hyperactivation is independent of PARG activity in necrotic cell death

2,3,5-tris(Glutathion-S-yl)hydroquinone, a potent nephrotoxic and nephrocarcinogenic metabolite of benzene and hydroquinone, generates reactive oxygen species (ROS) causing DNA strand breaks and the subsequent activation of DNA repair enzymes, including poly(ADP-ribose) polymerase (PARP)-1. Under robust oxidative DNA damage, PARP-1 is hyperactivated, resulting in the depletion of NAD⁺ and ATP with accompanying elevations in intracellular calcium concentrations (iCa²⁺), and ultimately necrotic cell death. The role of Ca²⁺ during PARP-dependent necrotic cell death remains unclear. We therefore sought to determine the relationship between Ca²⁺ and PARP-1 during ROS-induced necrotic cell death in human renal proximal tubule epithelial cells (HK-2). Our experiments suggest that store-operated Ca²⁺ channel (SOC) entry contributes to the coupling of PARP-1 activation to increases in iCa²⁺ during ROS-induced cell death. Poly(ADP-ribose)glycohydrolase (PARG), which catalyzes the degradation of PARs to yield free ADP-ribose (ADPR), is known to activate Ca²⁺ channels such as TRPM2. However, siRNA knockdown of PARG did not restore cell viability, indicating that free ADPR is not responsible for SOC activation in HK-2 cells. The data indicate that PARP-1 and iCa²⁺ are coupled through activation of SOC mediated Ca²⁺ entry in an apparently ADPR-independent fashion; alternative PAR-mediated signaling likely contributes to PARP-dependent necrotic cell death, perhaps via PAR-mediated signaling proteins that regulate iCa²⁺ homeostasis.