Hepatocytic c-Jun N-terminal kinases (JNK) protect against cystogenesis in NEMO-deficient mice

Background:

Jnk1 and Jnk2 are the two major Jnk genes ubiquitously expressed in all the mammalian cells, including hepatocytes. Inhibition of nuclear factor kappa B (NF-κB) activity via deletion of NF-κB essential modulator (IKKγ/Nemo) triggers cell apoptosis, which subsequently promotes compensatory hepatocyte proliferation triggering c-Jun N-terminal kinases (JNK)-mediated hepatocarcinogenesis (HCC) via prolonged activation. Thus, JNK/NF-κB interplay is a major determinant for hepatocyte fate and carcinogenesis. In this study, we sought to investigate the relevance of NF-kB/JNK crosstalk in the NEMO model of hepatocarcinogenesis (HCC).

Methods:

JNK signaling in hepatocytes was inhibited by crossing AlbCre-Jnk1LoxP/LoxP mice with Jnk2-deficient mice to generate Jnk1LoxP/LoxP/Jnk2–/– mice. Jnk1Δhepa/Jnk2-/-micewere further interbred with hepatocyte-specific Nemo-knockout mice (NemoΔhepa), a model of chronic liver inflammation and spontaneous HCC, to generate NemoΔhepa/JnkΔhepa mice. The phenotype was characterized during disease progression. The impact of JNK deletion on liver damage, cell death, compensatory proliferation, fibrogenesis, and tumor development in NemoΔhepa was investigated. Moreover, regulation of essential genes was assessed by RT-PCR, immunoblotting and immunostaining. Additionally, Jnk2 inhibition specifically in hepatocytes of NemoΔhepa/Jnk1Δhepa mice was performed using siRNA (siJnk2) nanodelivery. Finally, active signaling pathways were blocked using specific inhibitors.

Results:

Compound deletion of Jnk1 and Jnk2 in hepatocytes diminished hepatocarcinogenesis, but, in contrast, exacerbated liver injury caused by Nemo ablation as demonstrated by significant higher levels of ALT, AST, AP and GLDH. Interestingly, microscopic analysis of liver sections showed strong-ductular proliferation and cyst formation in NemoΔhepa/JnkΔhepa livers. Indeed, JNK deficiency in hepatocytes of NemoΔhepa (NemoΔhepa/JnkΔhepa) caused elevated fibrosis, increased apoptosis, increased compensatory proliferation, and elevated inflammatory cytokines expression. Furthermore, siJnk2 treatment in NemoΔhepa/Jnk1Δhepa mice resembled the phenotype of NemoΔhepa/JnkΔhepa mice.

Next, we sought to investigate the impact of molecular pathways in response to compound JNK deficiency in NemoΔhepa mice. We found NemoΔhepa/JnkΔhepa livers exhibited overexpression of the IL-6/Stat3 pathway in addition to EGFR-Raf-MEK-ERK cascade. To test the functional relevance of this finding we administered lapatinib – a dual tyrosine kinase inhibitor interrupting ErbB2 and EGFR signaling – to NemoΔhepa/JnkΔhepa mice. Lapatinib effectively inhibited cyst formation, improved transaminases and effectively blocked EGFR-Raf-MEK-ERK signaling.

Conclusion:

The regulatory crosstalk between NF-κB and JNK in hepatocytes is indispensable for hepatocytes homeostasis. JNK1 together with JNK2 play an essential role during carcinogenesis of NEMO deleted hepatocytes, but combined deletion in hepatocytes triggers ductular proliferation with cyst formation via the EGFR-Raf-MEK-ERK pathway. Lapatinib therapy successfully prevented cyst formation via inhibition of EGFR/ErbB2 phosphorylation.