Background: We have demonstrated that Kupffer cell-derived cytokines induce liver injury during acute pancreatitis; however, these activated Kupffer cells undergo accelerated apoptosis via Toll-like receptor-4 (TLR4) and NF-κB. Additionally, we showed that deletion of TLR4 downregulates Protein Kinase C-zeta (PKC-ζ), decreases nuclear translocation of NF-κB, and attenuates Kupffer cell apoptosis during acute pancreatitis. Since PKC-ζ plays a pivotal role in cell signaling, we aimed to determine its action mechanism in Kupffer cells apoptosis during acute pancreatitis.
Methods: Cultures of mouse Kupffer cell line (MKCL3-2) were transfected with PKC-ζ siRNA and treated with pancreatic elastase (1U/ml; 2hr) to mimic acute pancreatitis. Cell extracts were assayed for PKC-ζ (protein and activity), TLR4, nuclear translocation of NF-κB, phosphorylated ERK1/2, activated Caspase-3 and DNA fragmentation. All n≥3; data: mean±SD; means were compared by t-test; p<0.05 was considered significant.
Results: Elastase upregulated TLR4 protein (23±1 vs. 14±1), TLR4 mRNA (130±9 vs. 76±4), PKC-ζ protein (15±1 vs. 4±1), PKC-ζ activity (5±0.1 vs. 1±0.1), NF-κB (12±1 vs. 5±0.1), ERK1/2 (67±3 vs. 35±2), caspase-3 (5±0.1 vs. 1±0.1) and DNA fragmentation (29±1 vs. 6±0.3%), all p<0.01 vs. control. Transfection with PKC-ζ siRNA attenuated the elastase-induced upregulation of PKC-ζ (mRNA: 17±1 vs. 68±1; protein: 3±0.1 vs. 8±0.1), PKC-ζ activity (0.3±0.2 vs. 1±0.1), NF-κB (4±0.1 vs. 17±1), ERK1/2 (8±1 vs. 26±2), Caspase-3 (1±0.1 vs. 3±0.1) and DNA fragmentation (8±1 vs. 32±1%), all p<0.001 vs. control siRNA+elastase. We assessed the physical interaction of activated PKC-ζ with other cell signaling systems by co-immunoprecipitation; PKC-ζ did not interact directly with NF-κB or p38-MAPK. However, interaction of PKC-ζ with ERK1/2 increased in elastase-treated cells (67±3 vs. 35±3; p<0.01 vs. control) and was diminshed by PKC-ζ siRNA (8±1 vs. 69±2; p<0.01 vs. control siRNA+elastase). We further confirmed that PKC-ζ and ERK1/2 interacted directly by double immunofluorescent staining; elastase increased soluble staining compare to control and PKC-ζ siRNA decreased staining compared to control siRNA+elastase.
Conclusion: Activation of Kupffer cells upregulates PKC-ζ activity, increases apoptosis and induces nuclear translocation of NF-κB via ERK1/2 dependent mechanisms. Inhibiting the activity of PKC-ζ significantly attenuates Kupffer cell apoptosis, NF-κB and ERK1/2. Targeting the interaction of PKC-ζ and ERK1/2 may have therapeutic implications for Kupffer cell apoptosis and liver injury.