Highlights
- •Ninjurin2 is increased in fibrotic livers in both humans and mice.
- •Ninjurin2 deficiency ameliorates MCD-diet-induced liver fibrosis in mice.
- •Hepatocyte-specific overexpression of Ninjurin2 exacerbates liver fibrosis in mice.
- •Hepatocyte Ninjurin2 promotes PDGFRB signaling in HSC via a paracrine manner.
- •Systemic inhibitory Ninjurin2 peptide therapy attenuates liver fibrosis in mice.
Abstract
Background
Liver fibrogenesis is orchestrated by the paracrine signaling interaction between
several resident cell types regulating the activation of hepatic stellate cells (HSCs).
However, the molecular mechanisms underlying paracrine regulation are largely unknown.
The aim of this study is to elucidate the role of Ninjurin2 in the crosstalk between
hepatocytes and HSCs and better understand the implications of Ninjurin2 in liver
fibrosis.
Methods
Ninj2 knockout mice (Ninj2−/−) and hepatocyte-specific Ninj2 overexpression mice (Ninj2Hep-tg) were constructed and followed by the induction of liver fibrosis using methionine-
and choline-deficient (MCD) diet. The relationship between Ninjurin2 and liver fibrosis
phenotype was evaluated in vivo by measurement of fibrotic markers and related genes. We used an in vitro transwell cell co-culture model to examine the impact of Ninjurin2 in hepatocytes
on the crosstalk to HSCs. The interaction of Ninjurin2 and IGF1R and the regulation
of PI3K-AKT-EGR1 were analyzed in vivo and in vitro. Finally, an inhibitory Ninjurin2 peptide was injected intravenously via the tail vein to investigate whether inhibiting of Ninjurin2 cascade can attenuate
MCD diet-induced liver fibrosis in mice.
Results
We found that hepatic Ninjurin2 expression was significantly increased in fibrotic
human liver and MCD diet-induced liver injury mouse models. In the mouse model, hepatocyte-specific
overexpression of Ninj2 exacerbates MCD-induced liver fibrosis, while global Ninj2 knockout reverses the phenotype. To mimic hepatocyte-HSC crosstalk during liver fibrosis,
we used co-culture systems containing hepatocytes and HSCs and determined that Ninjurin2
overexpression in hepatocytes directly activates HSCs in vitro. Mechanistically, Ninjurin2 directly interacts with insulin-like growth factor 1
receptor (IGF1R) and increases the hepatocyte secretion of the fibrogenic cytokine,
platelet-derived growth factor-BB (PDGF-BB) through IGF1R-PI3K-AKT-EGR1 cascade. Inhibition
of PDGFRB signaling in HSCs can abolish the profibrogenic effect of Ninjurin2. In
addition, we demonstrated that a specific inhibitory Ninjurin2 peptide containing
an N-terminal adhesion motif mitigates liver fibrosis and improves hepatic function
in the mouse models by negatively regulating the sensitivity of IGF1R to IGF1 in hepatocytes.
Conclusion
Hepatic Ninjurin2 plays a key role in liver fibrosis through paracrine regulation
of PDGF-BB/PDGFRB signaling in HSCs, and the results suggesting Ninjurin2 may be a
potential therapeutic target.
Abbreviations:
ALT (alanine aminotransferase), AST (aspartate aminotransferase), α-SMA (α-smooth muscle actin), CCL2 (C-C Motif Chemokine Ligand 2), ECM (extracellular matrix), HCV (Hepatitis C virus), HSCs (hepatic stellate cells), H&E (hematoxylin and eosin), IF (Immunofluorescence), IGF1 (insulin-like growth factor 1), IGF1R (insulin-like growth factor 1 receptor), IHC (immunohistochemistry), IL-6 (cytokine interleukin-6), MCD (methionine- and choline-deficient), NASH (nonalcoholic steatohepatitis), N-NAM (N-terminal adhesion motif), NINJ2 (human nerve injury-induced protein 2 gene), Ninj2 (mouse nerve injury-induced protein 2 gene), PDGF (platelet-derived growth factor), RTKs (receptor tyrosine kinases), STD (standard chow diet), TGF-β (transforming growth factor-β), TNFα (tumor necrosis factor-α)Keywords
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Article info
Publication history
Published online: December 19, 2022
Accepted:
December 15,
2022
Received:
August 19,
2022
Identification
Copyright
© 2022 Elsevier Inc. All rights reserved.
