Gadd45beta promotes hepatocyte survival during liver regeneration in mice by modulating JNK signaling - PubMed (original) (raw)

Gadd45beta promotes hepatocyte survival during liver regeneration in mice by modulating JNK signaling

Salvatore Papa et al. J Clin Invest. 2008 May.

Abstract

In the liver, the JNK cascade is induced downstream of TNF receptors (TNFRs) in response to inflammatory, microbial, and toxic challenges. Sustained activation of JNK triggers programmed cell death (PCD), and hepatocyte survival during these challenges requires induction of the NF-kappaB pathway, which antagonizes this activation by upregulating target genes. Thus, modulation of JNK activity is crucial to the liver response to TNFR-mediated challenge. The basis for this modulation, however, is unknown. Here, we investigated the role of the NF-kappaB target Gadd45b in the regulation of hepatocyte fate during liver regeneration after partial hepatectomy. We generated Gadd45b(-/-) mice and found that they exhibited decreased hepatocyte proliferation and increased PCD during liver regeneration. Notably, JNK activity was markedly increased and sustained in livers of Gadd45b(-/-) mice compared with control animals after partial hepatectomy. Furthermore, imposition of a Jnk2-null mutation, attenuating JNK activity, completely rescued the regenerative response in Gadd45b(-/-) mice. Interestingly, Gadd45beta ablation did not affect hepatotoxic JNK signaling after a TNFR-mediated immune challenge, suggesting specificity in the inducible hepatic program for JNK restraint activated during distinct TNFR-mediated challenges. These data provide a basis for JNK suppression during liver regeneration and identify Gadd45beta as a potential therapeutic target in liver diseases.

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Figures

Figure 1

Figure 1. Impaired liver regeneration in Gadd45b–/– mice.

(A) Percent survival in Gadd45b+/+ (+/+) and Gadd45b–/– (–/–) mice (n = 12) at the indicated times after PH. *P < 0.05, log-rank test. (B–K) Images of H&E-stained liver sections from representative _Gadd45b+/+_and Gadd45b–/– mice at the indicated times after PH. Original magnification: ×20 (B–I); ×40 (J and K); ×40 (J, inset). 0 h, untreated; black arrowheads, necrosis; white arrowheads, inflammatory infiltrates; black arrows (J), mitotic figures. (L) Necrosis grade in livers of Gadd45b+/+ and Gadd45b–/– mice (n = 10) 48 hours after PH. Values are mean ± SEM. *P < 0.05. (M) Inflammation grade in the same livers used in L. Values are mean ± SEM. *P < 0.05. (N) Percent mitosis in livers of Gadd45b+/+ and Gadd45b–/– mice (n = 27) 48 hours after PH. Values are mean ± SEM. *P < 0.05. (O) Steatosis grade in the same livers used in L. Values are mean ± SEM. Alanine transaminase (ALT) (P) and aspartate transaminase (AST) (Q) activities in sera of Gadd45b+/+ and Gadd45b–/– mice prior to (0; n = 3) or 48 hours after (n = 14) PH. Values are mean ± SEM. **P < 0.01.

Figure 2

Figure 2. Impaired hepatocyte proliferation in Gadd45b–/– mice after PH, despite normal cytokine induction.

(A) Histomorphometric quantification of BrdU+ nuclei in livers of _Gadd45b+/+_and Gadd45b–/– mice prior to (n = 3) or 24 (n = 4), 36 (n = 6), and 48 hours (n = 20) after PH. Values are mean ± SEM. **P < 0.01. (B) Images of liver sections depicting representative clusters of BrdU+ cells from the mice shown in A. Original magnification, ×40. ELISA showing concentrations of TNF-α (C) and IL-6 (D) in livers of Gadd45b+/+ and Gadd45b–/– mice (n = 3) at the indicated times after PH. Values are mean ± SEM. (E) RT-PCR showing Tgfa mRNA levels in livers of Gadd45b+/+ and Gadd45b–/– mice at the indicated times after PH. (F–H) Western blots showing levels of TGF-β, HGFα, and cell cycle–regulating proteins in livers of Gadd45b+/+ and Gadd45b–/– mice at the indicated times after PH. n.s., nonspecific.

Figure 3

Figure 3. Increased apoptosis in livers of Gadd45b–/– mice after PH.

(A) Histomorphometric quantification of TUNEL+ nuclei in livers of Gadd45b+/+ and Gadd45b–/– mice prior to (0; n = 3) or 48 hours after (n = 16) PH. Values are mean ± SEM. **P < 0.01. (B) Images of liver sections depicting representative clusters of TUNEL+ cells from the mice shown in A. Original magnification, ×40. Open arrowheads, TUNEL+ hepatocytes with evident chromatin condensation. (C–F) TEM images showing hepatocytes from representative Gadd45b+/+ and Gadd45b–/– mice prior to (0 hours) and 48 hours after PH. (C and D) Hepatocytes from untreated animals exhibiting a thin nuclear envelop (NE), distinct nucleoli (N), euchromatin (E) associated with the nuclear pores (P), ER, and mitochondria (M). (E) Hepatocyte from a hepatectomized wild-type animal displaying many of the same features of the cells depicted in C and D. H, heterochromatin. (F) Hepatocyte from a hepatectomized Gadd45b–/– animal displaying signs of necrosis, including cytoplasmic disorganization (CD) with the presence of poorly defined organelles and ER, swollen organelles (S), and only few intact mitochondria. The nucleus shows dense marginated chromatin (MC) and few nuclear pores. F, intracytoplasmic fat. Scale bars: 2 μm. (G) Western blots showing procaspase-3 and PARP-1 cleavage products and c-FLIPL levels in livers of Gadd45b+/+ and Gadd45b–/– mice at the indicated times after PH.

Figure 4

Figure 4. Exacerbated JNK activation in livers of Gadd45b–/– mice after PH.

(A and B) Western blots with antibodies against phosphorylated or total kinases and c-Jun, and kinase assays (K.A.) showing MKK7, MAPK, and c-Jun activation in livers of Gadd45b+/+ and Gadd45b–/– mice at the indicated times after PH.

Figure 5

Figure 5. Deletion of JNK2 completely corrects the defective regenerative response of Gadd45b–/– mice.

ALT (A) and AST (B) activities in sera of Gadd45b+/+ (n = 5), Gadd45b–/– (n = 14), and Gadd45b–/–Jnk2–/–(dKO; n = 7) mice 48 hours after PH. Values are mean ± SEM. *P < 0.05. (C) Images of liver sections depicting representative clusters of TUNEL+ cells from the mice shown in D. Original magnification, ×40. White arrowheads, TUNEL+ hepatocytes with evident chromatin condensation. (D) Histomorphometric quantification of TUNEL+ nuclei in livers of Gadd45b+/+ (n = 5), Gadd45b–/– (n = 14), and Gadd45b–/–Jnk2–/– (n = 7) mice 48 hours after PH. Values are mean ± SEM. *P < 0.05, **P < 0.01. (E) Images of liver sections depicting representative clusters of BrdU+ cells from the mice shown in F. Original magnification, ×40. (F) Histomorphometric quantification of BrdU+ nuclei in livers of Gadd45b+/+(n = 5), Gadd45b–/– (n = 14), and Gadd45b–/–Jnk2–/– (n = 7) mice 48 hours after PH. Values are mean ± SEM. *P < 0.05. (G) Western blots with antibodies against phosphorylated or total JNK1/2 or c-Jun showing JNK and c-Jun activation in livers of Gadd45b+/+, Gadd45b–/–, and Gadd45b–/–Jnk2–/– mice prior to (0 hours) or 48 hours after PH. Each lane represents a different mouse.

Figure 6

Figure 6. Normal susceptibility of Gadd45b–/– mice to ConA-induced hepatotoxicity.

(A) Kinase assays showing JNK activation in livers of Gadd45b+/+ and Gadd45b–/– mice at the indicates times after ConA injection (25 mg/kg), in the presence or absence (–) of BHA treatment. Total JNK is shown (Western blots). (B) Kinase assay showing IKK activity in livers of Gadd45b+/+ and Gadd45b–/– mice treated with ConA as in A. Total IKKβ is shown (Western blots). (C) Histomorphometric quantification of TUNEL+ nuclei in livers of Gadd45b+/+ and Gadd45b–/– mice (n = 4) 8 hours after injection of saline (0 hours) or ConA (8 hours; 25 mg/kg). Values are mean ± SEM. (D) Images of TUNEL-stained liver sections from representative mice shown in C. Original magnification, ×40. ALT (E) and AST (F) activities in sera of Gadd45b+/+ and Gadd45b–/– mice (n = 4) 8 hours after injection of saline (0 hours) or ConA (8 hours; 25 mg/kg). Values are mean ± SEM.

Figure 7

Figure 7. ROSs are not involved in PH-induced, JNK overactivation in Gadd45b–/– mice.

(A) Kinase assays and Western blots with antibodies against phosphorylated or total kinases showing MAPK activation in livers of Gadd45b+/+ and Gadd45b–/– mice 48 hours after PH, in the presence or absence of BHA treatment. Each lane represents a different mouse. (B) Images of liver sections depicting representative clusters of TUNEL+ cells from the mice shown in C. Original magnification, ×40. White arrowheads, TUNEL+ hepatocytes with evident chromatin condensation. (C) Histomorphometric quantification of TUNEL+ nuclei in livers of Gadd45b+/+(n = 3) and normally (–; n = 6) or BHA-fed (n = 6) Gadd45b–/– mice 48 hours after PH. Values are mean ± SEM. *P < 0.05. ALT (D) and AST (E) activities in sera of Gadd45b+/+ (n = 3) and normally (–; n = 6) or BHA-fed (n = 6) Gadd45b–/– mice 48 hours after PH. Values are mean ± SEM. *P < 0.05, **P < 0.01. (F) Images of liver sections depicting representative clusters of BrdU+ cells from the mice shown in G. Original magnification, ×40. (G) Histomorphometric quantification of BrdU+ nuclei in livers of Gadd45b+/+(n = 3) and normally (–; n = 6) or BHA-fed (n = 6) Gadd45b–/– mice 48 hours after PH. Values are mean ± SEM. *P < 0.05, **P < 0.01.

References

    1. Wajant H., Pfizenmaier K., Scheurich P. Tumor necrosis factor signaling. Cell Death Differ. 2003;10:45–65. doi: 10.1038/sj.cdd.4401189. - DOI - PubMed
    1. Papa S., et al. The NF-κB-mediated control of the JNK cascade in the antagonism of programmed cell death in health and disease. Cell Death Differ. 2006;13:712–729. doi: 10.1038/sj.cdd.4401865. - DOI - PubMed
    1. Basseres D.S., Baldwin A.S. Nuclear factor-κB and inhibitor of κB kinase pathways in oncogenic initiation and progression. Oncogene. 2006;25:6817–6830. doi: 10.1038/sj.onc.1209942. - DOI - PubMed
    1. Karin M. Nuclear factor-κB in cancer development and progression. Nature. 2006;441:431–436. doi: 10.1038/nature04870. - DOI - PubMed
    1. De Smaele E., et al. Induction of gadd45β by NF-κB downregulates proapoptotic JNK signalling. Nature. 2001;414:308–313. doi: 10.1038/35104560. - DOI - PubMed

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