PNPLA3 downregulation exacerbates the fibrotic response in human hepatic stellate cells.

Brian Rady,David A Brenner,Takahiro Nishio,Xiao Liu,Alessandro Pocai,Debanjan Dhar,Mark Erion,Tatiana Kisseleva,Matias A Avila

doi:10.1371/journal.pone.0260721

Abstract

Non-alcoholic steatohepatitis (NASH) results, in part, from the interaction of metabolic derangements with predisposing genetic variants, leading to liver-related complications and mortality. The strongest genetic determinant is a highly prevalent missense variant in patatin-like phospholipase domain-containing protein 3 (PNPLA3 p.I148M). In human liver hepatocytes PNPLA3 localizes to the surface of lipid droplets where the mutant form is believed to enhance lipid accumulation and release of pro-inflammatory cytokines. Less is known about the role of PNPLA3 in hepatic stellate cells (HSCs). Here we characterized HSC obtained from patients carrying the wild type (n = 8 C/C) and the heterozygous (n = 6, C/G) or homozygous (n = 6, G/G) PNPLA3 I148M and investigated the effect of genotype and PNPLA3 downregulation on baseline and TGF-β-stimulated fibrotic gene expression. HSCs from all genotypes showed comparable baseline levels of PNPLA3 and expression of the fibrotic genes α-SMA, COL1A1, TIMP1 and SMAD7. Treatment with TGF-β increased PNPLA3 expression in all 3 genotypes (~2-fold) and resulted in similar stimulation of the expression of several fibrogenic genes. In primary human HSCs carrying wild-type (WT) PNPLA3, siRNA treatment reduced PNPLA3 mRNA by 79% resulting in increased expression of α-SMA, Col1a1, TIMP1, and SMAD7 in cells stimulated with TGF-β. Similarly, knock-down of PNPLA3 in HSCs carrying either C/G or G/G genotypes resulted in potentiation of TGF-β induced expression of fibrotic genes. Knockdown of PNPLA3 did not impact fibrotic gene expression in the absence of TGF-β treatment. Together, these data indicate that the presence of the I148M PNPLA3 mutation in HSC has no effect on baseline activation and that downregulation of PNPLA3 exacerbates the fibrotic response irrespective of the genotype.

Highlights

With the increased prevalence of obesity and insulin resistance worldwide, non-alcoholic fatty liver disease (NAFLD) is becoming the leading cause of chronic liver disease and liver transplantation
Baseline expression of the hepatic stellate cells (HSCs) activation markers of α-SMA, collagen 1, TIMP1, and SMAD7, as well as expression of patatin-like phospholipase domain-containing 3 (PNPLA3) were similar between all genotypes (p = ns vs WT genotype for all) (Fig 1A–1E) suggesting that the presence of the I148M mutation in HSCs does not impact their level of baseline activation
Knockdown of PNPLA3 had no impact on α-SMA, collagen 1, TIMP1, or SMAD7 expression in the absence of TGF-β (Fig 2A; p = ns vs scramble siRNA for all)

Summary

Introduction

With the increased prevalence of obesity and insulin resistance worldwide, non-alcoholic fatty liver disease (NAFLD) is becoming the leading cause of chronic liver disease and liver transplantation. Romeo et al first reported that the rs738409 non-synonymous SNP in PNPLA3 was significantly associated with liver steatosis [2]. This finding has since been robustly replicated in different populations across the full spectrum of NAFLD, including the identification of associations with NASH histological severity and hepatic fibrosis, with confirmation of these findings in pediatric patients as well [3,4,5]. Rs738409 is highly prevalent among those with NASH, with up to 34% of patients homozygous carriers of the mutant alleles [8] Population-based studies have shown that compared to the normal variant homozygosity for this PNPLA3 risk allele is associated with 2 to 4-fold greater risk for NASH and cirrhosis, with up to a 12-fold increased risk for hepatocellular carcinoma and, most recently, an 18-fold increase in liverrelated mortality [6, 7]. rs738409 is highly prevalent among those with NASH, with up to 34% of patients homozygous carriers of the mutant alleles [8]

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Conclusion