Deletion of IKK2 in hepatocytes does not sensitize these cells to TNF-induced apoptosis but protects from ischemia/reperfusion injury

Abstract

The inhibitor of NF-kappaB (I-kappaB) kinase (IKK) complex consists of 3 subunits, IKK1, IKK2, and NF-kappaB essential modulator (NEMO), and is involved in the activation of NF-kappaB by various stimuli. IKK2 or NEMO constitutive knockout mice die during embryogenesis as a result of massive hepatic apoptosis. Therefore, we examined the role of IKK2 in TNF-induced apoptosis and ischemia/reperfusion (I/R) injury in the liver by using conditional knockout mice. Hepatocyte-specific ablation of IKK2 did not lead to impaired activation of NF-kappaB or increased apoptosis after TNF-alpha stimulation whereas conditional NEMO knockout resulted in complete block of NF-kappaB activation and massive hepatocyte apoptosis. In a model of partial hepatic I/R injury, mice lacking IKK2 in hepatocytes displayed significantly reduced liver necrosis and inflammation than wild-type mice. AS602868, a novel chemical inhibitor of IKK2, protected mice from liver injury due to I/R without sensitizing them toward TNF-induced apoptosis and could therefore emerge as a new pharmacological therapy for liver resection, hemorrhagic shock, or transplantation surgery.

Figure 1

Conditional knockout of Ikk2 and Nemo in the mouse liver. (A) Deletion in the mouse liver is shown at the DNA level by Southern blot using genomic live DNA from mice with genetic status for the floxed (f) allele and positive (+) or negative (–) cre status as indicated. (B) Deletion in cre-positive floxed Ikk2 mice (Ikk2Δhepa) was verified in comparison to cre-negative control mice (Ikk2^f/f) at the RNA level by semiquantitative RT-PCR using 1 μg of liver RNA and primers for IKK2 and GAPDH. (C) Western blot analysis with antibodies against IKK2 or α-tubulin (for loading control) from 100 μg of whole liver protein extracts from Ikk2Δhepa and Ikk2^f/f mice. (D) Immunohistochemical staining of IKK2 on liver slides from Ikk2Δhepa and Ikk2^f/f mice, showing that in Ikk2Δhepa mice, IKK2 expression is restricted to nonparenchymal liver cells such as bile duct cells (arrows). Original magnification, ×40. (E) For inducible knockout of NEMO, poly I/C was injected into both cre-positive (NemoΔ) mice and cre-negative control mice (Nemo^f/f). Efficiency of deletion in the mouse liver is shown at the RNA level by RT-PCR. (F) Western blot analysis with antibodies against NEMO and α-tubulin in NemoΔ and Nemo^f/f mice. (G) Inducible deletion of Ikk2. Deletion was induced by injection with poly I/C into Mx-cre–positive (Ikk2Δ) mice and Mx-cre–negative control mice (Ikk2^f/f). Efficiency of the deletion in the mouse liver is shown at the RNA level by RT-PCR. (H) Western blot for IKK2 or α-tubulin from Ikk2Δ and Ikk2^f/f mice.

Figure 2

Nemo, but not Ikk2, deletion results in apoptotic cell death in the liver upon TNF-α stimulation. (A) Ikk2Δhepa, Ikk2Δ, or NemoΔ mice and their respective control litter mates (Ikk2^f/f or Nemo^f/f) were injected intravenously with 6 μg/kg of recombinant TNF-α or intraperitoneally with LPS (100 μg/kg) and GalN (800 mg/kg) (right top panel). Liver injury was measured by determining ALT levels. Values are mean ± SD for independent animals (n = 6). Single asterisks indicate statistical significance with P < 0.01 versus Nemo^f/f control mice. All NemoΔ mice died between 2 and 3 hours after TNF-α stimulation from hepatic failure. (B) TUNEL staining of liver slides before and 2 hours after TNF-α stimulation showing clear positive staining in NemoΔ, but not in Ikk2Δhepa mice. Results are representative of those obtained in mice (n = 6). Original magnification, × 40. (C) Detection of caspase 3–like activity by an enzymatic, fluorometric assay from whole liver protein lysates at different time points after TNF-α stimulation in Ikk2Δhepa, NemoΔ, and control littermates. Values are mean ± SD for independent animals (n = 6). (D) ALT levels as markers for liver injury at different time points following ConA injection into Ikk2Δhepa and Ikk2^f/f mice. Values are mean ± SD for independent animals (n = 6). (E) ALT levels following Fas-stimulating Jo-2 injection into Ikk2Δhepa and Ikk2^f/f mice. Values are mean ± SD for independent animals (n = 6). U/l, units per liter; AFC, 7-amino-trifluoromethyl coumarin.

Figure 3

NF-κB activation in the liver upon TNF-α stimulation is blocked in mice lacking Nemo but not Ikk2. (A) Liver nuclear protein extracts (5 μg) from the indicated mice and time points after TNF-α stimulation were subjected to a gel-retardation assay with an NF-κB consensus probe. In lanes 10/22/34 and 11/23/35, antibodies for the NF-κB subunits p50 or p65 were added as indicated as supershift control. The figure depicts results from 3 different assays. (B) I-κBα degradation in the different mouse groups was assessed by Western blot analysis with 50 μg of whole cell liver protein extracts before and 10 minutes after TNF-α stimulation using an antibody against I-κBα or α-tubulin (as loading control). (C) I-κBα phosphorylation was detemined after TNF-α stimulation by subjecting 50 μg of proteins to a Western blot analysis with an antibody detecting I-κBα phosphorylated at Ser32. (D) Evaluation of IKK activity. Proteins (300 μg) from mice stimulated with TNF-α were IP with a Nemo-antibody and subjected to a kinase assay using a truncated glutathione-S-transferase–I-κBα(1–54) protein as substrate. (E) Gel-retardation assay with an NF-κB consensus site using 5 μg of nuclear protein extracts from primary hepatocyte cultures. Results are representative of those obtained in mice (n = 4). (F) JNK activity was measured by Western blot using protein from mice stimulated with TNF, LPS, and ConA as indicated. Antibodies detecting c-Jun phosphorylated at Ser63 or JNK phosphorylated at Thr183/Tyr185 as well as nonphosphorylated JNK1 and α-tubulin as loading control were used.

Figure 4

A dominant-negative IKK2 form can block NF-κB activation after TNF-α stimulation. (A) IP in 300 μg of whole cell liver protein extracts from Ikk2^f/f and Ikk2Δhepa mice was performed with a polyclonal antibody against NEMO, followed by Western blot analysis with a monoclonal antibody against IKK1 or IKK2 as indicated. Association between NEMO and IKK1 was assessed before as well as 30 and 60 minutes after stimulation with TNF-α. (B) Western blot of protein extracts that were IP with anti-IKK1 and blotted with a monoclonal antibody against IKK1 or IKK2 as indicated. (C) Primary hepatocyte cultures were prepared from livers of Ikk2^f/f and Ikk2Δhepa mice and either treated with PBS alone, infected with LacZAdv, or infected with dnIKK2Adv in a viral dose of 20 PFUs for 12 hours. Protein extracts (200μg) were subjected to IP with a NEMO antibody, followed by Western blot with an IKK2 antibody. (D) Primary hepatocytes from Ikk2^f/f and Ikk2Δhepa mice were infected with LacZAdv or dnIKK2Adv in a viral dose of 20 PFUs for 12 hours and treated with TNF-α (30 ng/ml). Protein extracts were subjected to an NF-κB gel-retardation assay. (E) Protein extracts (100 μg) from primary hepatocytes infected with LacZAdv or dnIKK2Adv were IP with anti-Nemo and blotted with a monoclonal antibody against IKK1.

Figure 5

Ikk2 deletion in hepatocytes protects against liver injury and inflammation after hepatic I/R. (A) Ikk2Δhepa and Ikk2^f/f control mice underwent a procedure of partial hepatic ischemia lasting for 60 minutes, which was followed by reperfusion. Serum AST and ALT levels were measured at the indicated time points before the procedure and after reperfusion as markers for liver injury. Values are mean ± SD for independent animals (n = 8). Asterisks indicate statistical significance: *P < 0.02 versus Ikk2^f/f control mice; **P < 0.05 versus Ikk2^f/f control mice. (B) H&E staining of liver slides from Ikk2^f/f and Ikk2Δhepa mice at 6 hours and 24 hours after reperfusion. N, necrotic area (arrows indicate margins of necrotic areas; P, portal vein; C, central vein. Results are representative of those obtained in 8 mice. Original magnification, ×20. The area of necrotic parenchymal surface was measured and quantified (right panel). Values are mean ± SD for independent animals (n = 4). Hatch mark indicates statistical significance: P < 0.01 versus Ikk2^f/f control mice. (C) Quantification of PMN leukocytes per high power field (×40) at different time points after reperfusion. Values are mean ± SD for independent animals (n = 4). Double hatch marks indicate statistical significance: P < 0.001 versus Ikk2^f/f control mice.

Figure 6

Activation of NF-κB and induction of NF-κB target genes after I/R is dependent on functional IKK2. (A) Nuclear protein extracts (5 μg) from livers of Ikk2^f/f and Ikk2Δhepa mice at different time points after I/R were subjected to a gel-retardation assay with an NF-κB consensus oligonucleotide. In lanes 9 and 10, antibodies for the NF-κB subunits p50 or p65 were added as supershift control. (B) Immunohistochemical staining for iNOS at 6 hours after I/R in Ikk2^f/f and Ikk2Δhepa mice. Original magnification, ×40. Results are representative of those obtained in mice (n = 4). (C) Immunohistochemical staining for TNF-α at 6 hours after I/R. Original magnification, ×40. The number of TNF-α–positive cells was quantified (right panel). Values are mean ± SD for independent animals (n = 4). The asterisk indicates statistical significance: P < 0.01 versus Ikk2^f/f control mice. (D) Kupffer cells were isolated from livers of Ikk2^f/f and Ikk2Δhepa mice and stained with an antibody against the F4/80 antigen, which is a macrophage-specific marker, to verify the specificity of the preparation procedure. Equal expression of IKK2 was verified by staining with a polyclonal antibody against IKK2. Cells were stimulated with 1 μg/ml LPS for 1 hour and TNF-α expression examined by immunohistochemical staining to prove that Kupffer cells in the livers of Ikk2Δhepa mice are functionally active. Original magnification, ×400. For negative control, no primary antibody was added.

Figure 7

The pharmacological IKK2 inhibitor AS602868 protects mice from liver injury due to hepatic I/R. (A) C57BL6 mice received an oral administration of 150 μl of the IKK2 inhibitor AS602868 (10 μg/g body weight) or the vehicle substance (control) at 20 hours and 2 hours before stimulation with 6 μg/kg recombinant TNF-α. Evaluation of serum ALT levels at the indicated time points after TNF-α stimulation showing no significant difference between treated and untreated animals. (B) NF-κB EMSA using 5 μg of nuclear protein extracts from animals treated with AS602868 or control. (C) Serum AST and ALT levels were measured at the indicated time points after reperfusion in mice that were pretreated with AS602868 or vehicle substance (control) and had undergone partial I/R. Values are mean ± SD for independent animals (n = 4). The asterisk indicates statistical significance with P < 0.05 versus control mice. (D) H&E staining of liver slides at 6 hours after reperfusion from control mice and mice treated with AS602868. Original magnification, ×20. The area of necrotic parenchymal surface was measured and quantified (right panel). Values are mean ± SD for independent animals (n = 4). The double asterisk indicates statistical significance with P < 0.01 versus control mice. (E) Quantification of PMN leukocytes per high power field (×40) at 6 hours after reperfusion. Values are mean ± SD for independent animals (n = 4). The hatch mark indicates statistical significance with P < 0.001 versus control mice.