Macchi, C. | Search bioRxiv

bioRxiv2020

TM6SF2/PNPLA3/MBOAT7 loss-of-function genetic variants impact on NAFLD development and progression both in patients and in in vitro models

Background and aimsThe I148M PNPLA3, the rs641738 in MBOAT7/TMC4 locus and the E167K TM6SF2 polymorphisms represent the main predisposing factors to non-alcoholic fatty liver disease (NAFLD) development and progression. We previously generated a full knockout of MBOAT7 in HepG2 cells (MBOAT7-/-), homozygous for the I148M PNPLA3. Therefore, we aimed to:1) investigate the synergic impact of the 3 at-risk variants on liver injury and hepatocellular carcinoma (HCC) in a large cohort of NAFLD patients;2) create in vitro models of genetic NAFLD by silencing TM6SF2 in both HepG2 and MBOAT7-/- cells. MethodsNAFLD patients (n=1380) of whom 121 had HCC were stratified with a semi-quantitative score ranging from 0 to 3 according to the number of PNPLA3, TM6SF2 and MBOAT7 at-risk variants. TM6SF2 was silenced in HepG2 (TM6SF2-/-) and MBOAT7-/- (MBOAT7-/-TM6SF2-/-) through CRISPR/Cas9. ResultsIn NAFLD patients, the additive weight of these mutations was associated with liver disease severity and increased risk to develop HCC. In HepG2 cells, TM6SF2 silencing altered lipid composition and induced the accumulation of micro-vesicular LDs, whereas the MBOAT7-/-TM6SF2-/- cells showed a mixed micro/macro pattern of LDs. TM6SF2 deletion strongly affected endoplasmic reticulum (ER) and mitochondria ultrastructures thus increasing ER/oxidative stress. Mitochondrial number raised in both TM6SF2-/- and MBOAT7-/-TM6SF2-/- models, suggesting an unbalancing in mitochondrial dynamics and the silencing of both MBOAT7 and TM6SF2 impaired mitochondrial activity with a shift towards anaerobic glycolysis. MBOAT7-/-TM6SF2-/- cells also showed the highest proliferation rate. ConclusionsThe co-presence of the 3 at-risk variants impacts on NAFLD course, in both patients and experimental models affecting LDs accumulation, mitochondrial functionality and metabolic reprogramming towards HCC.

genetics↗

bioRxiv2020

Cyclase-associated protein 2 (CAP2) controls MRTF-A localization and SRF activity in mouse embryonic fibroblasts

Recent studies identified cyclase-associated proteins (CAPs) as important regulators of actin dynamics that control assembly and disassembly of actin filaments (F-actin). While these studies significantly advanced our knowledge of their molecular functions, the physiological relevance of CAPs largely remained elusive. Gene targeting in mice implicated CAP2 in heart physiology and skeletal muscle development. Heart defects in CAP2 mutant mice were associated with altered activity of serum response factor (SRF), a transcription factor involved in multiple biological processes including heart function, but also skeletal muscle development. By exploiting mouse embryonic fibroblasts (MEFs) from CAP2 mutant mice, we aimed at deciphering the CAP2-dependent mechanism relevant for SRF activity. Reporter assays and mRNA quantification by qPCR revealed reduced SRF-dependent gene expression in mutant MEFs. Reduced SRF activity was associated with moderately increased G-actin levels and reduced nuclear levels of MRTF-A, a transcriptional SRF coactivator that is shuttled out of the nucleus and, hence, inhibited upon G-actin binding. Moreover, pharmacological actin manipulation restored MRTF-A distribution in mutant MEFs. Our data are in line with a model in which CAP2 controls the MRTF-SRF pathway in an actin-dependent manner. While MRTF-A localization and SRF activity was impaired under basal conditions, serum stimulation induced nuclear MRTF-A translocation and SRF activity in mutant MEFs similar to controls. In summary, our data revealed that in MEFs CAP2 controls basal MRTF-A localization and SRF activity, while it was dispensable for serum-induced nuclear MRTF-A translocation and SRF stimulation.

cell biology↗