C. Yang1,2, M. Deng2, M. Scott2, T. Billiar2 1Tsinghua University,Medical School,Beijing, BEIJING, China 2University Of Pittsburg,Pittsburgh, PA, USA
Introduction:
Notch is a highly conserved transmembrane receptor well-known to regulate cell to cell communication as well as cell fate decisions. In recent years, Notch signaling has been implicated in inflammation, yet the regulation and roles of Notch in sepsis are unknown. The sheddase, TNFα-converting enzyme (TACE/ADAM17) participates in cleavage and activation of Notch and we have previously shown that TACE is activated via the TLR4-MyD88-iNOS-NO pathway in hepatocytes during endotoxemia. Thus, we hypothesized that Notch signaling pathway is regulated by TLR4/Myd88/iNOS/NO/TACE pathway in spleen during endotoxemia.
Methods:
To test our hypothesis, WT (C57BL/6), Myd88-/-, TLR4-/-, iNOS-/-mice were injected saline or 5mg/kg LPS i.p, spleen and serum were harvested 8 hours after injection. In vitro, primary mice splenocytes were exposed to LPS and treated with or without iNOS inhibitors and NO donors. Activation of Notch and cell death in splenocytes was assessed.
Results:
The Notch signaling pathway was activated in the spleen and in splenocytes after LPS challenge in a time-dependent manner, indicated by increased cleaved intracellular notch measure as notch intracellular domain (NICD) by western. The NICD levels peaked at 8 hours and 4 hours after LPS in the spleen and in the splenocytes, respectively. Inhibition of Notch activation using DAPT (5 mg/kg), 3 hours prior to LPS injection, resulted in a significantly increased level of cleaved-caspase-3, indicating more apoptosis in the absence of Notch signaling. LPS-induced Notch activation was significantly lower in Myd88-/- mice and TLR4-/- mice as well as iNOS-/- after LPS challenge and this we associated with increased levels of cleaved-caspase3. Similar results were also obtained in vitro in splenocytes treated with an iNOS inhibitor 1400w (2 uM). Moreover, Notch activation was induced in splenocytes by using SNAP (200 uM) as an NO donor or directly by the cGMP analog 8-pCPT-cGMP(400 uM). Inhibition of TACE with TAPI-II (800 nM) greatly reduced LPS-induced Notch activation in splenocytes and resulting in increased cleaved-caspase3.
Conclusion:
Our data suggests that Notch signaling pathway is activated during endotoxemia under the regulation of iNOS/NO and TACE and plays a critical role in regulating splenic cell survival and homeostasis.