Background: Liver injury is a prognostic indicator during sepsis. Activated Kupffer cells produce cytokines that induce liver injury, yet undergo accelerated apoptosis via Fas/FasL. The balance between activation and apoptosis of Kupffer cells may alter the severity of liver injury. As Protein Kinase C-zeta regulates cell death via NF-κB and Fas/FasL, we aimed to determine the role of PKC-ζ in apoptosis of Kupffer cells during sepsis.
Methods: Sepsis was induced in male rats by cecal ligation and puncture (CLP); livers were assayed for PKC-ζ, NF-κB, Fas/FasL, Caspase-3 and DNA fragmentation. In other experiments, Kupffer cells from un-operated rats were infected with PKC-ζ domain-negative adenovirus (AdPKCζ-DN) to inhibit PKC-ζ activity or transfected with CMV promoter-driven PKC-ζ gene expression plasmid (pCMVPKC-ζ) to over-express PKC-ζ, and treated with lipopolysaccharide (LPS: 0.5μg/ml) to simulate sepsis. Cell extracts were assayed for PKC-ζ, NF-κB, Fas/FasL, Caspase-3, and DNA fragmentation. All n≥3; data: mean±SD.
Results: In rat livers CLP upregulated PKC-ζ protein (3675±40 vs. 1338±21), PKC-ζ activity (3±0.1 vs.1±0.1), NF-κB nuclear translocation (15±0.2 vs. 5±0.1), Fas/FasL protein (Fas: 3671±30 vs. 1655±34; FasL: 3851±37 vs. 1765±28), Fas/FasL mRNA (Fas:3789±35 vs. 1245±25, FasL: 3657±30 vs. 1435±26), activated Caspase-3 (2453±40 vs. 1143±32) and DNA fragmentation (29±0.2 vs. 8 ±0.1); all p<0.001 vs. sham. In liver sections from CLP rats, immunostaining for PKC-ζ increased and predominantly localized to Kupffer cells (but not hepatocytes). We therefore proceeded by treating Kupffer cells from un-operated rats with LPS; in-vivo, LPS upregulated PKC-ζ activity, NF-κB, Fas/FasL, Caspase-3, and DNA fragmentation (data not shown, all p<0.001). AdPKCζ-DN attenuated the LPS-induced upregulation of PKC-ζ activity (2±0.1 vs. 4±0.1), NF-κB (4±0.1 vs. 7±0.1), Fas/FasL protein (Fas: 1248 ±19 vs. 2567±29, FasL: 1326±10 vs. 2682±22), Fas/FasL mRNA (Fas: 891±29 vs. 1742±23, FasL: 821±25 vs. 1892±27), Caspase-3 (987±24 vs. 1750±12) and DNA fragmentation (8±0.1 vs. 13±0.1); all p<0.001 vs. control. Over-expression of PKC-ζ by pCMVPKC-ζ augmented the LPS-induced upregulation of PKC-ζ activity, NF-κB, Fas/FasL, Caspase-3 and DNA fragmentation (data not shown, all p<0.001).
Conclusion: CLP or LPS upregulate PKC-ζ, NF-κB, Fas/FasL, Caspase-3 and DNA fragmentation within Kupffer cells. Manipulating the expression of PKC-ζ alters the activation of proapoptotic pathways and apoptosis of Kupffer cells. The ability of Kupffer cells to auto-regulate their stress response by upregulating death receptor/ligand and key proapoptotic signaling warrants further studies.