Inhibition of nuclear export attenuates IkBa phosphorylation

CRITICAL CARE II Cellular stimulation results in IkBa phosphorylation and degradation, followed by resynthesis and transport to the nucleus. Nuclear IkBa binds NF-kB and recycles it to the cytoplasm via the protein exportin-1. We hypothesized that inhibition of exportin-1 would cause nuclear sequestration of IkBa and attenuate IkBa phosphorylation.

Functional toll-like receptor 4 signaling in liver nonparenchymal cells is required for hepatic ischemia/ reperfusion (I/R) injury Allan Tsung MD, Sarah McAshon, Rosemary Hoffman PhD, David Geller MD, Timothy Billiar MD, FACS University of Pittsburgh Medical Center, Pittsburgh, PA

METHODS: Human microvascular endothelial cells were treated with 20 nM Leptomycin B (LMB), a specific exportin-1 inhibitor, or vehicle for 60 minutes. Following IL-1B (100 ng/ml)stimulation, cells were fractionated; nuclear and cytoplasmic extracts were collected. Western blotting was performed to examine total IkBa, phosphorylated IkBa (P-IkBa), phosphorylated IKKa/B, and phosphorylated p38 MAP kinase. The chemokine GRO-a was measured as a readout of NF-kB activation.

INTRODUCTION: Recent evidence suggests that endogenous ligands from damaged cells can activate the innate immune system via Toll-like receptor 4(TLR4) signaling. We have previously shown that the injury and inflammation following hepatic warm I/R requires activation of TLR4 using TLR4-deficient mice. To determine whether the TLR4-dependent injury required TLR4 expression on liver parenchymal (hepatocytes) or non-parenchymal cells (Kupffer cells, endothelial cells), we performed adoptive cell transfer to produce chimeric mice.

RESULTS: Cytoplasmic P-IkBa was upregulated at 5 minutes and was sustained thirty and sixty minutes after IL-1B stimulation. In LMB⫹IL-1B treated cells, cytoplasmic levels of P-IkBa were decreased by 50%. Following IL-1B alone, cytoplasmic IkBa levels were markedly decreased whereas cells exposed to LMB⫹IL-1B demonstrated persistent cytoplasmic IkBa. LMB⫹IL-1B treated cells demonstrated sequestration of IkBa but not P-IkBa in the nuclear fraction. Phosphorylated IKKa/B, the activator of IkBa, and phosphorylated p38 MAPK were unchanged with exportin-1 inhibition. GRO-a production was inhibited in LMB⫹IL-1B treated cells by 70% (p⬍0.05).

METHODS: Chimeric mice were produced by adoptive transfer of donor bone marrow cells into irradiated recipient animals using combinations of TLR4 wild type(HeOuj) and TLR4-deficient(HeJ) mice in the following combinations: WT/WT, WT/Mutant, Mutant/ Mutant, Mutant/WT. Partial warm ischemia was produced in the left and median lobes of mice. RESULTS: Consistent with previous results we found the TLR4deficient mice were protected from hepatic I/R-induced damage in association with markedly lower TNF, IL-6, and ICAM-1 mRNA levels. Further, we report here that hepatic JNK, but not P38 activation was lower in TLR4-deficient mice. Our studies with chimeric mice show that WT/Mutant(recipient/donor) chimeric mice consisting of recipient TLR4 WT parenchymal cells and TLR4-deficient donor non-parenchymal cells were protected from hepatic I/R and exhibited decreased JNK activation compared to WT/WT chimeric mice. In contrast, Mutant/WT chimeric mice were not protected from I/R and were associated with increased JNK activation compared to Mutant/Mutant chimeric mice. Chimeric mice (Recipient/ Donor) ALT

WT/WT

WT/Mutant

Mutant/Mutant

Mutant/WT

1210 ⫾ 138

482 ⫾ 74*

303 ⫾ 36*

801 ⫾ 168**

CONCLUSIONS: Cellular shuttling is essential to NF-kB signaling. Nuclear export inhibition sequesters IkBa in the nucleus and attenuates cytoplasmic IkBa phosphorylation and degradation, leading to diminished NF-kB activation.

A polymerized hemoglobin based oxygen carrier attenuates TLR4 stimulated NF-kB activation by inhibiting phosphorylation of IkB-a Aaron M Cheng MD, Ernest E Moore MD, FACS, Jeffrey Johnson MD, Mark Walsh MD, Christine Hamiel BS, Anirban Banerjee PhD, Tomohiko Masuno MD, Christopher Silliman MD, PhD University of Colorado Health Sciences Center/Denver Health, Denver, CO

Data are mean ⫾ SEM; n⫽6 per group; *indicates p⬍0.05 vs WT/WT; **indicated p⬍0.05 vs Mutant/Mutant.

INTRODUCTION: Previous clinical trials using polymerized hemoglobin (PolyHb) suggested that this hemoglobin-based oxygen carrier (HBOC) also conveys anti-inflammatory effects. In this study we hypothesize that this HBOC inhibits TLR4-stimulated activation of the NF-kB pathway.

CONCLUSIONS: This study demonstrates that TLR4-dependent hepatic I/R injury activates JNK signaling. We also show that TLR4 signaling in non-parenchymal cells and not hepatocytes is required for the I/R induced injury.

METHODS: Cultured human pulmonary endothelial cells were pre-incubated with PolyHb/cell media (50% v/v) mixture for 5 hrs. After pre-incubation, cells were stimulated with LPS in fresh media. NF-kB activation was determined by a p65 DNA binding assay and by p65 nuclear translocation; IkB-a phosphorylation was determined by protein immunoblot.

Inhibition of nuclear export attenuates IkBa phosphorylation Mark Walsh MD, Anirban Banerjee PhD, Christine Hamiel BS, Aaron Cheng MD, Guillermo Escobar MD, Lynn Gries MD, Robert McIntyre MD University of Colorado Health Sciences Center, Denver, CO

RESULTS: In the LPS-treated groups, PolyHb decreased nuclear lysate p65 DNA binding by 79% compared to control (0.26 ⫾ 0.05 vs. 1.23 ⫾ 0.27, p⬍ 0.05). By digital immunofluorescence, PolyHb also decreased LPS-induced p65 intensity in the nucleus (MFI/Cell:

INTRODUCTION: The NF-kB inhibitor, IkBa, is a predominantly cytoplasmic protein that undergoes basal nuclear/cytoplasmic shuttling.

© 2005 by the American College of Surgeons Published by Elsevier Inc.

ISSN 1072-7515/05/$30.00

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