Exploring the impact of the PNPLA3 I148M variant on primary human hepatic stellate cells using 3D extracellular matrix models

Background and aims The PNPLA3 rs738409 C>G (encoding for I148M) variant is a risk locus for the fibrogenic progression of chronic liver diseases, a process driven by hepatic stellate cells (HSCs). We investigated how the PNPLA3 I148M variant affects HSCs biology using transcriptomic data and validated findings in 3D-culture models. Methods RNA sequencing was performed on 2D-cultured primary human HSCs and liver biopsies of obese individuals, genotyped for the PNPLA3 I148M variant. Data were validated in HSCs wild type (WT) or carrying I148M-PNPLA3 cultured on 3D extracellular matrix (ECM) scaffolds from human healthy and cirrhotic liver with/without TGFB1 or Cytosporone-B (Csn-B) treatment. Results Comparison between transcriptomic analysis of liver biopsies and HSCs highlighted shared PNPLA3 I148M-driven dysregulated pathways related to mitochondrial function, antioxidant response, ECM remodelling and TGFB1 signalling. Analogous pathways were dysregulated in HSCs WT/I148M-PNPLA3 cultured in 3D liver scaffolds. Mitochondrial dysfunction in I148M-PNPLA3 cells was linked to respiratory chain complex IV insufficiency. Antioxidant capacity was lower in I148M-PNPLA3 HSCs, while ROS secretion was increased in I148M-PNPLA3 HSCs and higher in bioengineered cirrhotic versus healthy scaffolds. TGFB1 signalling pathway followed the same trend. In I148M-PNPLA3 cells, TGFB1-endogenous inhibitor NR4A1 expression and activation were decreased: treatment with the Csn-B agonist increased total NR4A1 in HSCs cultured in healthy but not in cirrhotic 3D scaffolds. NR4A1 regulation by TGFB1/Csn-B was linked to Akt signalling in WT-PNPLA3 HSCs and to Erk signalling in I148M-PNPLA3 HSCs. Conclusion HSCs carrying the PNPLA3 I148M variant have impaired mitochondrial function, antioxidant response, and increased TGFB1 signalling dampening the antifibrotic NR4A1 activity. These features are exacerbated by cirrhotic ECM, highlighting the dual impact of the PNPLA3 I148M variant and the fibrotic microenvironment in progressive chronic liver diseases. Impact and implications Hepatic stellate cells (HSCs) play a key role in the fibrogenic process associated with chronic liver disease. The PNPLA3 genetic mutation has been linked with increased risk of fibrogenesis, but its role in HSCs needs further investigation. Here, by using comparative transcriptomic and a novel 3D in vitro model, we demonstrate the impact of the PNPLA3 genetic mutation on primary human HSCs cell behaviour, and we show that it affects the cell's mitochondrial function and antioxidant response, as well as the antifibrotic gene NR4A1. Our publicly available transcriptomic data, 3D platform and our findings on NR4A1 could facilitate the discovery of targets to develop more effective treatments for chronic liver diseases.