Extracellular Purines Via The Activation Of P2 Purinergic Receptors Induce Cell Cycle Progression And Proliferation Ofhepatocytes

Adult liver has a remarkable ability to regenerate after the loss of hepatic tissue. After surgical removal of 70% of the mass of liver (partial hepatectomy), the remaining tissue undergoes rapid regeneration and the original mass is restored in its entirely, typically within a week or two after surgery. Interestingly, hepatocytes are the first to enter the cell cycle in a concerted manner in response to regenerative stimulus. Despite intense investigations for the past several decades, the identity of the key humoral factors which trigger cell cycle progression in adult hepatocytes remains elusive.C-Jun N-Termianl Kinase (JNK) is a family of serine/threonine kinase that transducer signals from the cell membrane to the nucleus in response to a wide range of stimuli which control a spectrum of cellular processes, including cell growth, differentiation, transformation and apoptosis. C-Jun N-Teminal Kinase signaling cascade plays a key role in hepatocyte proliferation and liver regeneration. However, the factors responsible for the activation of JNK early on during regeneration remain unknown.In recent years, extracellular ATP which is discretely released from cells under stress, has proven to be a bona-fide signaling molecule influencing a variety of cellular functions via activation of cell surface receptors, but its role in hepatocyte growth and regeneration is unknown, In this study, we sought to determine if purinergic signaling can lead to the activation of c-Jun N-terminal kinase. (JNK), a known central player in hepatocyte proliferation and liver regeneration. Moreover, if P2 purinergic receptor mediated activation of Ca²⁺ and protein kinase C signaling networks play a key role in the induction of JNK signaling in hepatocyte also remain unclear. Hepatocyte treatment with ATPγS, a non-hydrolyzable ATP analog, recapitulated early signaling events associated with liver regeneration, i.e. rapid and transient activation of JNK signaling, induction of immediate early genes c-fos, and c-jun, and Activator Protein-1 (AP-1) DNA-binding activity. The rank order of agonist preference, UTP>ATP>ATPγS, suggests that the effects ofextracellular ATP is mediated via the activation of P2Y2 receptors in hepatocytes. ATPyStreatment alone, and in combination with epidermal growth factor (EGF), substantiallyincreased cyclin Dl, and PCNA protein expression and hepatocyte proliferation in vitro.Extracellular ATP as low as 10 nM was sufficient to potentiate EGF-induced cyclin Dlexpression. Partial hepatectomy can induce a rapid and robust activation of JNK signalingand potent proliferation response in the remnant liver, based on the immunoblotting ofphosphor-JNK, phosphor-c-jun, Cyclin Dl and PCNA expression andimmunohistochemical analysis for Ki67 and PCNA. Infusion of ATP via the portal veindirectly activated hepatic JNK signaling, while infusion of a P2 purinergic receptorantagonist prior to partial hepatectomy inhibited JNK activation and cell cycle proliferationand liver regeneration in vivo. In order to test the role of PKC as a potential upstreammediator of JNK activation, hepatocytes were treated with a battery of small molecule PKCinhibitors, which exhibit differential specificity towards multiple PKC isoforms—prior tothe treatment with ATPyS. Our results suggests that extracellular ATP via the activation ofP2 purinergic receptors activates multiple Ca-dependent, Ca-independent and atypical PKCisoforms which includes PKCu. Furthermore, extracellular ATP can rapidly induce PKCactivation and translocation to membranes. Based on the inhibition of JNK by the PKCinhibitior G66976 and the apparent lack of inhibition by PKC inhibitors G66983 andG66850, we believe PKCu is a potential key upsteam mediator of ATP-mediated JNKsignaling in hepatocytes. We also find immunohistochemical evidence that extracellularATP leads to the activation of PKCu in hepatocytes. Within 10 min of treatment of ATPyS,PKCu is translocated into the nucleus from the cytoplasma. We also find that ATPySinduced JNK activation in not dependent on Ca2+ or the activation of PLC. In conclusion,extracellular ATP is a hepatic mitogen that can activate JNK signaling and hepatocyteproliferation in vitro and initiate JNK signaling in regenerating liver in vivo. ExtracellularATP also activates multiple PKC isoforms as determined by the translocation andphosphorylation of PKC in hepatocytes. Duo to JNK and PKC activation are early eventsin hepatocyte proliferation and liver regeneration, these findings implicate that extracellularATP as a potential humoral mediator responsible for the induction of a cascade of signalingevents which collectively induce hepatocyte proliferation.