Role of ERK5 in liver pathophysiology

Metabolic dysfunction-associated steatotic liver disease (MASLD) comprises a heterogeneous condition in the presence of steatotic liver, and affects 38% of all people worldwide. It’s frequently associated with metabolic conditions, including insulin resistance which is an early event in MASLD. MASLD may lead to its active inflammatory form (referred to metabolic dysfunction-associated steatohepatitis as MASH) and, in some cases, to cirrhosis, liver failure and liver cancer. The extracellular signal-regulated kinase 5 (ERK5), a member of the Mitogen-Activated Protein Kinases (MAPK) family, is highly expressed in hepatocytes and regulates the development and growth of HCC. To investigate the role of Extracellular signal-regulated kinase 5 in the pathogenesis of metabolic dysfunction-associated steatotic liver disease, MASLD, my group has generated mice with hepatocyte-specific ERK5 knock out (ERK5ΔHep) that were fed with a high-fat diet for 16 weeks, which predisposes mice to insulin resistance. ERK5ΔHep mice exhibited impaired glucose tolerance and showed reduced insulin sensitivity. To dissect the molecular mechanisms in vitro, I used a murine immortalized hepatocyte cell line (MMH) to focus on insulin receptor (IR) signaling. MMH was stably silenced for ERK5, using specific shRNAs. In ERK5-silenced cells exposed to lipotoxic stress (i.e. palmitic acid), insulin-induced activation of a well-known downstream mediator of IR signalling, Akt, was abrogated, and the expression of the IR was reduced compare to control cells. Additionally, ERK5 silencing induced an increase in mitochondrial ROS, together with activation of JNK, which, in turn, supported the phosphorylation of IRS-1 on inhibitory residues. In ERK5-silenced cells, the expression of heme oxygenase-1, HMOX1, and NAD(P)H Quinone Dehydrogenase 1, NQO1, which mediate the antioxidant response via Nuclear factor erythroid 2-related factor 2, NRF2, was reduced. To establish whether increased ROS is involved in the reduced insulin signalling observed in ERK5-depleted cells subjected to lipotoxic stress, MMH exposed to palmitic acid were treated with a well-established antioxidant, N-acetyl-cysteine. Treatment with NAC prevented the downregulation of IR and the increase of IRS1 phosphorylation. Next, the impact of ERK5 on mitochondrial functions was investigated. Measurement of mitochondrial membrane potential indicated a strong depolarization in ERK5-silenced cells, together with impaired mitochondrial oxidative phosphorylation. These effects were associated with the up-regulated expression of Peroxisome Proliferator-Activated Receptor γ Coactivator 1α, PGC-1α, and Tribbles Pseudokinase 3, TRIB3, a negative regulator of insulin signaling through inhibition of Akt. Reduced expression of IR, and increased expression of PGC-1α and TRIB3 were also observed in ERK5ΔHep mice. In patients with MASLD and insulin resistance, ERK5 expression correlated inversely with LDL levels, and directly with HMOX1 mRNA in the liver. In conclusion, ERK5 contributes to maintaining hepatocyte sensitivity to insulin, via an antioxidant response involving IR, IRS1, PGC-1α, and TRIB3, that collectively impair IR signalling as witnessed by Akt inactivation. Another study was directed to further investigate ERK5-dependent signals in Hepatocellular carcinoma (HCC). To this end, I used human hepatocellular carcinoma cell lines Huh-7 and HepG2 in vitro, and found that ERK5-silenced cells show increased EGFR mRNA and protein levels, and downstream PI3K/AKT activation. Furthermore, the amount of EGFR protein was also increased in cells treated with the ERK5 inhibitor, JWG-071, compared to control cells. The combination of JWG-071, and an EGFR inhibitor, Gefitinib, resulted to be more effective than single treatments in reducing cell viability in both cell lines. Nuclear accumulation of EGFR has been reposrted to support carcinogenesis and resistance to therapy. Intriguingly, nuclear EGFR levels were higher in ERK5-silenced cells compared to control cells as determined by subcellular fractionation. Immunofluorescence analysis in confocal microscopy confirmed the increased accumulation of EGFR protein in the nuclei, upon ERK5 pharmacologic inhibition. Since EGFR shuttles to the nucleus via the α/β importin system, I performed combined treatment of JWG-071 and the α/β importin inhibitor, Ivermectin. This cotreatment showed a higher antiproliferative effect than single inhibitors on HCC cell lines. In conclusion, these results revealed new potential therapeutic strategies to be explored for the treatment of HCC.