Original Research
A Functional Role of GAS6/TAM in Nonalcoholic Steatohepatitis Progression Implicates AXL as Therapeutic Target

https://doi.org/10.1016/j.jcmgh.2019.10.010Get rights and content
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Background and Aims

GAS6 signaling, through the TAM receptor tyrosine kinases AXL and MERTK, participates in chronic liver pathologies. Here, we addressed GAS6/TAM involvement in Non-Alcoholic SteatoHepatitis (NASH) development.

Methods

GAS6/TAM signaling was analyzed in cultured primary hepatocytes, hepatic stellate cells (HSC) and Kupffer cells (KCs). Axl-/-, Mertk-/- and wild-type C57BL/6 mice were fed with Chow, High Fat Choline-Deficient Methionine-Restricted (HFD) or methionine-choline-deficient (MCD) diet. HSC activation, liver inflammation and cytokine/chemokine production were measured by qPCR, mRNA Array analysis, western blotting and ELISA. GAS6, soluble AXL (sAXL) and MERTK (sMERTK) levels were analyzed in control individuals, steatotic and NASH patients.

Results

In primary mouse cultures, GAS6 or MERTK activation protected primary hepatocytes against lipid toxicity via AKT/STAT-3 signaling, while bemcentinib (small molecule AXL inhibitor BGB324) blocked AXL-induced fibrogenesis in primary HSCs and cytokine production in LPS-treated KCs. Accordingly; bemcentinib diminished liver inflammation and fibrosis in MCD- and HFD-fed mice. Upregulation of AXL and ADAM10/ADAM17 metalloproteinases increased sAXL in HFD-fed mice. Transcriptome profiling revealed major reduction in fibrotic- and inflammatory-related genes in HFD-fed mice after bemcentinib administration. HFD-fed Mertk-/- mice exhibited enhanced NASH, while Axl-/- mice were partially protected. In human serum, sAXL levels augmented even at initial stages, whereas GAS6 and sMERTK increased only in cirrhotic NASH patients. In agreement, sAXL increased in HFD-fed mice before fibrosis establishment, while bemcentinib prevented liver fibrosis/inflammation in early NASH.

Conclusion

AXL signaling, increased in NASH patients, promotes fibrosis in HSCs and inflammation in KCs, while GAS6 protects cultured hepatocytes against lipotoxicity via MERTK. Bemcentinib, by blocking AXL signaling and increasing GAS6 levels, reduces experimental NASH, revealing AXL as an effective therapeutic target for clinical practice.

Keywords

Liver Fibrosis
Hepatic Stellate Cells
Bemcentinib (BGB324)
GAS6/TAM Signaling
Liver Inflammation

Abbreviations used in this paper

ADAM10
a disintegrin and metalloproteinase 10
ADAM17
a disintegrin and metalloproteinase 17
cDNA
complementary DNA
ECM
extracellular matrix
ELISA
enzyme-linked immunosorbent assay
GAS6
Growth arrest-specific gene 6
H&E
hematoxylin and eosin
HCC
hepatocellular carcinoma
HFD
high-fat choline-deficient methionine-restricted diet
HSC
hepatic stellate cell
IL
interleukin
KC
Kupffer cell
KO
knockout
LPS
lipopolysaccharide
MCD
methionine-choline-deficient diet
MCP-1
monocyte chemoattractant protein-1
MMP9
matrix metalloproteinase-9
MPO
myeloperoxidase
mRNA
messenger RNA
NAFLD
nonalcoholic fatty liver disease
NAS
NAFLD activity score
NASH
nonalcoholic steatohepatitis
PA
palmitic acid
PBS
phosphate-buffered saline
PMH
primary mouse hepatocyte
sAXL
soluble AXL
sMERTK
soluble MERTK
TAM
Tyro3-Axl-Mertk
TNF
tumor necrosis factor
WT
wild-type

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Conflicts of interest These authors disclose the following: James B. Lorens is a co-founder of BerGenBio. Gro Gausdal is employed by BerGenBio. Pablo García de Frutos, Montserrat Marí, and Albert Morales received research funding from BerGenBio. The remaining authors disclose no conflicts.

Funding This study was funded by grants from Instituto de Salud Carlos III (PI16/00930 and PI19/01410 to Montserrat Marí) and CIBEREHD; Ministerio de Economía y Competitividad (SAF2015-66515-R and RTI2018-095672-B-I00 to Albert Morales and Pablo García de Frutos, and RTI2018-095572-B-100 to Anna Colell), and co-funded by FEDER (Fondo Europeo de Desarrollo Regional, Unión Europea), Fundació Marató de TV3 (to Pablo García de Frutos), AGAUR (2017_SGR_177 to Albert Morales), and CERCA Programme/Generalitat de Catalunya.