Idiopathic pulmonary fibrosis (IPF) is a serious lung disease characterized by excessive collagen matrix deposition and extracellular remodeling. Signaling pathways mediated by fibrotic cytokine transforming growth factor β1 (TGF-β1) make important contributions to pulmonary fibrosis, but it remains unclear how TGF-β1 alters metabolism and modulates the activation and differentiation of pulmonary fibroblasts. We found that TGF-β1 lowers NADH and NADH/NAD levels, possibly due to changes in the TCA cycle, resulting in reductions in the ATP level and oxidative phosphorylation in pulmonary fibroblasts. In addition, we showed that butyrate (C4), a short chain fatty acid (SCFA), exhibits potent antifibrotic activity by inhibiting expression of fibrosis markers. Butyrate treatment inhibited mitochondrial elongation in TGF-β1-treated lung fibroblasts and increased the mitochondrial membrane potential (MMP). Consistent with the mitochondrial observations, butyrate significantly increased ADP, ATP, NADH, and NADH/NAD levels in TGF-β1-treated pulmonary fibroblasts. Collectively, our findings indicate that TGF-β1 induces changes in mitochondrial dynamics and energy metabolism during myofibroblast differentiation, and that these changes can be modulated by butyrate, which enhances mitochondrial function.

Reactive oxygen species (ROS) play a significant role in intracellular signaling and regulation, particularly when they are maintained at physiologic levels. However, excess ROS can cause cell damage and induce cell death. We recently reported that eIF2 alpha phosphorylation protects hepatocytes from oxidative stress and liver fibrosis induced by fructose metabolism. Here, we found that hepatocyte-specific eIF2 alpha phosphorylation-deficient mice have significantly reduced expression of the epidermal growth factor receptor (EGFR) and altered EGFR-mediated signaling pathways. EGFR-mediated signaling pathways are important for cell proliferation, differentiation, and survival in many tissues and cell types. Therefore, we studied whether the reduced amount of EGFR is responsible for the eIF2 alpha phosphorylation-deficient hepatocytes' vulnerability to oxidative stress. ROS such as hydrogen peroxide and superoxides induce both EGFR tyrosine phosphorylation and eIF2 alpha phosphorylation. eIF2 alpha phosphorylation-deficient primary hepatocytes, or EGFR knockdown cells, have decreased ROS scavenging ability compared to normal cells. Therefore, these cells are particularly susceptible to oxidative stress. However, overexpression of EGFR in these eIF2 alpha. phosphorylation-deficient primary hepatocytes increased ROS scavenging ability and alleviated ROS-mediated cell death. Therefore, we hypothesize that the reduced EGFR level in eIF2 alpha phosphorylation-deficient hepatocytes is one of critical factors responsible for their susceptibility to oxidative stress.

EIF2α Phosphorylation helps maintain cellular homeostasis and overcome endoplasmic reticulum (ER) stress through transcriptional and translational reprogramming. This study aims to elucidate the transcriptional regulation of glutathione (GSH) and nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) homeostasis through eIF2α phosphorylation and its impact on cell death during ER stress. eIF2α phosphorylation-deficient (A/A) cells exhibited decreased expression of multiple genes involved in GSH synthesis and NADPH production, leading to an exacerbated depletion of both cellular and mitochondrial GSH, as well as mitochondrial NADPH, during ER stress. Impaired GSH homeostasis resulted from deficient expression of ATF4 and/or its dependent factor, Nrf2, which are key transcription factors in the antioxidant response during ER stress. In contrast, the exacerbation of NADPH depletion may primarily be attributed to the dysregulated expression of mitochondrial serine-driven 1-carbon metabolism pathway genes, which are regulated by an unidentified eIF2α phosphorylation-dependent mechanism during ER stress. Moreover, the eIF2α phosphorylation-ATF4 axis was responsible for upregulation of ferroptosis-inhibiting genes and downregulation of ferroptosis-activating genes upon ER stress. Therefore, ER stress strongly induced ferroptosis of A/A cells, which was significantly inhibited by treatments with cell-permeable GSH and the ferroptosis inhibitor ferrostatin-1. ATF4 overexpression suppressed impairment of GSH homeostasis in A/A cells during ER stress by promoting expression of downstream target genes. Consequently, ATF4 overexpression mitigated ferroptosis as well as apoptosis of A/A cells during ER stress. Our findings underscore the importance of eIF2α phosphorylation in maintaining GSH/NADPH homeostasis and inhibiting ferroptosis through ATF4 and unidentified eIF2α phosphorylation-dependent target(s)-mediated transcriptional reprogramming during ER stress.