New aspects of redox signaling mediated by supersulfides in health and disease.

Oxygen molecules accept electrons from the respiratory chain in the mitochondria and are responsible for energy production in aerobic organisms. The reactive oxygen species formed via these oxygen reduction processes undergo complicated electron transfer reactions with other biological substances, which leads to alterations in their physiological functions and cause diverse biological and pathophysiological consequences (e.g., oxidative stress). Oxygen accounts for only a small proportion of the redox reactions in organisms, especially under aerobic or hypoxic conditions but not under anaerobic and hypoxic conditions. This article discusses a completely new concept of redox biology, which is governed by redox-active supersulfides, i.e., sulfur-catenated molecular species. These species are present in abundance in all organisms but remain largely unexplored in terms of redox biology and life science research. In fact, accumulating evidence shows that supersulfides have extensive redox chemical properties and that they can be readily ionized or radicalized to participate in energy metabolism, redox signaling, and oxidative stress responses in cells and in vivo. Thus, pharmacological intervention and medicinal modulation of supersulfide activities have been shown to benefit the regulation of disease pathogenesis as well as disease control.

PNPO-PLP axis senses prolonged hypoxia in macrophages by regulating lysosomal activity.

Oxygen is critical for all metazoan organisms on the earth and impacts various biological processes in physiological and pathological conditions. While oxygen-sensing systems inducing acute hypoxic responses, including the hypoxia-inducible factor pathway, have been identified, those operating in prolonged hypoxia remain to be elucidated. Here we show that pyridoxine 5'-phosphate oxidase (PNPO), which catalyses bioactivation of vitamin B6, serves as an oxygen sensor and regulates lysosomal activity in macrophages. Decreased PNPO activity under prolonged hypoxia reduced an active form of vitamin B6, pyridoxal 5'-phosphate (PLP), and inhibited lysosomal acidification, which in macrophages led to iron dysregulation, TET2 protein loss and delayed resolution of the inflammatory response. Among PLP-dependent metabolism, supersulfide synthesis was suppressed in prolonged hypoxia, resulting in the lysosomal inhibition and consequent proinflammatory phenotypes of macrophages. The PNPO-PLP axis creates a distinct layer of oxygen sensing that gradually shuts down PLP-dependent metabolism in response to prolonged oxygen deprivation.

The Complex Roles of Redox and Antioxidant Biology in Cancer.

Redox reactions control fundamental biochemical processes, including energy production, metabolism, respiration, detoxification, and signal transduction. Cancer cells, due to their generally active metabolism for sustained proliferation, produce high levels of reactive oxygen species (ROS) compared to normal cells and are equipped with antioxidant defense systems to counteract the detrimental effects of ROS to maintain redox homeostasis. The KEAP1-NRF2 system plays a major role in sensing and regulating endogenous antioxidant defenses in both normal and cancer cells, creating a bivalent contribution of NRF2 to cancer prevention and therapy. Cancer cells hijack the NRF2-dependent antioxidant program and exploit a very unique metabolism as a trade-off for enhanced antioxidant capacity. This work provides an overview of redox metabolism in cancer cells, highlighting the role of the KEAP1-NRF2 system, selenoproteins, sulfur metabolism, heme/iron metabolism, and antioxidants. Finally, we describe therapeutic approaches that can be leveraged to target redox metabolism in cancer.

Unique and overlapping roles of NRF2 and NRF1 in transcriptional regulation.

Transcription is regulated by specific transcription factors that mediate signaling in response to extrinsic and intrinsic stimuli such as nutrients, hormones, and oxidative stresses. Many transcription factors are grouped based on their highly conserved DNA binding domains. Consequently, transcription factors within the same family often exhibit functional redundancy and compensation. NRF2 (NFE2L2) and NRF1 (NFE2L1) belong to the CNC family transcription factors, which are responsible for various stress responses. Although their DNA binding properties are strikingly similar, NRF2 and NRF1 are recognized to play distinct roles in a cell by mediating responses to oxidative stress and proteotoxic stress, respectively. In this review, we here overview the distinct and shared roles of NRF2 and NRF1 in the transcriptional regulation of target genes, with a particular focus on the nuclear protein binding partners associated with each factor.

Longevity control by supersulfide-mediated mitochondrial respiration and regulation of protein quality.

Supersulfides, which are defined as sulfur species with catenated sulfur atoms, are increasingly being investigated in biology. We recently identified pyridoxal phosphate (PLP)-dependent biosynthesis of cysteine persulfide (CysSSH) and related supersulfides by cysteinyl-tRNA synthetase (CARS). Here, we investigated the physiological role of CysSSH in budding yeast (Saccharomyces cerevisiae) by generating a PLP-binding site mutation K109A in CRS1 (the yeast ortholog of CARS), which decreased the synthesis of CysSSH and related supersulfides and also led to reduced chronological aging, effects that were associated with an increased endoplasmic reticulum stress response and impaired mitochondrial bioenergetics. Reduced chronological aging in the K109A mutant could be rescued by using exogenous supersulfide donors. Our findings indicate important roles for CARS in the production and metabolism of supersulfides-to mediate mitochondrial function and to regulate longevity.

Aging exacerbates murine lung ischemia-reperfusion injury by excessive inflammation and impaired tissue repair response.

Donor shortage is a major problem in lung transplantation (LTx), and the use of lungs from elderly donors is one of the possible solutions in a rapidly aging population. However, the utilization of organs from donors aged >65 years has remained infrequent and may be related to a poor outcome. To investigate the molecular events in grafts from elderly donors early after LTx, the left lungs of young and old mice were subjected to 1 hour of ischemia and subsequent reperfusion. The left lungs were collected at 1 hour, 1 day, and 3 days after reperfusion and subjected to wet-to-dry weight ratio measurement, histological analysis, and molecular biological analysis, including RNA sequencing. The lungs in old mice exhibited more severe and prolonged pulmonary edema than those in young mice after ischemia reperfusion, which was accompanied by upregulation of the genes associated with inflammation and impaired expression of cell cycle-related genes. Apoptotic cells increased and proliferating type 2 alveolar epithelial cells decreased in the lungs of old mice compared with young mice. These factors could become conceptual targets for developing interventions to ameliorate lung ischemia-reperfusion injury after LTx from elderly donors, which may serve to expand the old donor pool.

Clinical, Genomic, and Transcriptomic Featurses of Lung Adenocarcinoma With Uncommon EGFR Mutation.

PURPOSE: The purpose of this study is to identify the clinical, genomic, and transcriptomic features of patients with lung adenocarcinoma (LUAD) harboring uncommon epidermal growth factor receptor (EGFR) mutations (UCM) compared with common EGFR mutations (CM). MATERIALS AND METHODS: In this multicenter retrospective cohort study, clinicopathological data were collected from 1047 consecutive patients who underwent complete surgical resection for LUAD, as well as EGFR mutation analysis, between 2005 and 2012 at 4 institutions. Differences in postoperative overall survival (OS) and recurrence-free survival (RFS) according to EGFR mutation status were evaluated. For the genomic and transcriptomic analyses, 5 cohorts from public databases were evaluated. RESULTS: Of 466 eligible patients, 415 (89.1%) and 51 (10.9%) had CM and UCM, respectively. The 5-year OS and RFS rates in the CM/UCM groups were 86.8%/77.0% and 74.8%/59.0%, respectively. OS and RFS were significantly shorter in the UCM than CM group (both P < .01). Multivariable analysis of OS showed that UCM was an independent prognostic factor (hazard ratio 1.72, 95% confidential interval 1.01-2.93). According to the genomic analysis, tumors with UCM had a significantly higher tumor mutation burden and TP53 mutation frequency. Transcriptomic analysis showed that the T-cell-inflamed gene signature, a biomarker of the treatment for immunotherapy, was significantly associated with tumors with UCM. CONCLUSION: UCM were associated with a poor prognosis in patients with surgically resected EGFR-mutated LUAD. Tumors with UCM had unique genomic and transcriptomic features suggestive of a tumor microenvironment responsive to immunotherapy.

Supersulfide biology and translational medicine for disease control.

For decades, the major focus of redox biology has been oxygen, the most abundant element on Earth. Molecular oxygen functions as the final electron acceptor in the mitochondrial respiratory chain, contributing to energy production in aerobic organisms. In addition, oxygen-derived reactive oxygen species including hydrogen peroxide and nitrogen free radicals, such as superoxide, hydroxyl radical and nitric oxide radical, undergo a complicated sequence of electron transfer reactions with other biomolecules, which lead to their modified physiological functions and diverse biological and pathophysiological consequences (e.g. oxidative stress). What is now evident is that oxygen accounts for only a small number of redox reactions in organisms and knowledge of biological redox reactions is still quite limited. This article reviews a new aspects of redox biology which is governed by redox-active sulfur-containing molecules-supersulfides. We define the term 'supersulfides' as sulfur species with catenated sulfur atoms. Supersulfides were determined to be abundant in all organisms, but their redox biological properties have remained largely unexplored. In fact, the unique chemical properties of supersulfides permit them to be readily ionized or radicalized, thereby allowing supersulfides to actively participate in redox reactions and antioxidant responses in cells. Accumulating evidence has demonstrated that supersulfides are indispensable for fundamental biological processes such as energy production, nucleic acid metabolism, protein translation and others. Moreover, manipulation of supersulfide levels was beneficial for pathogenesis of various diseases. Thus, supersulfide biology has opened a new era of disease control that includes potential applications to clinical diagnosis, prevention and therapeutics of diseases.

NRF2 signalling in cytoprotection and metabolism.

The KEAP1-NRF2 system plays a central role in cytoprotection in defence mechanisms against oxidative stress. The KEAP1-NRF2 system has been regarded as a sulfur-utilizing cytoprotective mechanism, because KEAP1 serves as a biosensor for electrophiles by using its reactive thiols and NRF2 is a transcriptional factor regulating genes involved in sulfur-mediated redox reactions. NRF2 is a key regulator of cytoprotective genes, such as antioxidant and detoxification genes, and also possesses potent anti-inflammatory activity. Recently NRF2 has been the focus of attention as a regulator of cellular metabolism and mitochondrial function. The NRF2-mediated regulatory mechanisms of metabolites and mitochondria have been considered diverse, but have not yet been fully clarified. This review article provides an overview of molecular mechanisms that regulate NRF2 signalling and its cytoprotective roles, and highlights NRF2 contribution to cellular metabolism, particularly in the context of mitochondrial function and newly-found sulfur metabolism.

Sulfur metabolic response in macrophage limits excessive inflammatory response by creating a negative feedback loop.

The excessive inflammatory response of macrophages plays a vital role in the pathogenesis of various diseases. The dynamic metabolic alterations in macrophages, including amino acid metabolism, are known to orchestrate their inflammatory phenotype. To explore a new metabolic pathway that regulates the inflammatory response, we examined metabolome changes in mouse peritoneal macrophages (PMs) in response to lipopolysaccharide (LPS) and found a coordinated increase of cysteine and its related metabolites, suggesting an enhanced demand for cysteine during the inflammatory response. Because Slc7a11, which encodes a cystine transporter xCT, was remarkably upregulated upon the pro-inflammatory challenge and found to serve as a major channel of cysteine supply, we examined the inflammatory behavior of Slc7a11 knockout PMs (xCT-KO PMs) to clarify an impact of the increased cysteine demand on inflammation. The xCT-KO PMs exhibited a prolonged upregulation of pro-inflammatory genes, which was recapitulated by cystine depletion in the culture media of wild-type PMs, suggesting that cysteine facilitates the resolution of inflammation. Detailed analysis of the sulfur metabolome revealed that supersulfides, such as cysteine persulfide, were increased in PMs in response to LPS, which was abolished in xCT-KO PMs. Supplementation of N-acetylcysteine tetrasulfide (NAC-S2), a supersulfide donor, attenuated the pro-inflammatory gene expression in xCT-KO PMs. Thus, activated macrophages increase cystine uptake via xCT and produce supersulfides, creating a negative feedback loop to limit excessive inflammation. Our study highlights the finely tuned regulation of macrophage inflammatory response by sulfur metabolism.

Supersulfide catalysis for nitric oxide and aldehyde metabolism.

Abundant formation of endogenous supersulfides, which include reactive persulfide species and sulfur catenated residues in thiols and proteins (supersulfidation), has been observed. We found here that supersulfides catalyze S-nitrosoglutathione (GSNO) metabolism via glutathione-dependent electron transfer from aldehydes by exploiting alcohol dehydrogenase 5 (ADH5). ADH5 is a highly conserved bifunctional enzyme serving as GSNO reductase (GSNOR) that down-regulates NO signaling and formaldehyde dehydrogenase (FDH) that detoxifies formaldehyde in the form of glutathione hemithioacetal. C174S mutation significantly reduced the supersulfidation of ADH5 and almost abolished GSNOR activity but spared FDH activity. Notably, Adh5C174S/C174S mice manifested improved cardiac functions possibly because of GSNOR elimination and consequent increased NO bioavailability. Therefore, we successfully separated dual functions (GSNOR and FDH) of ADH5 (mediated by the supersulfide catalysis) through the biochemical analysis for supersulfides in vitro and characterizing in vivo phenotypes of the GSNOR-deficient organisms that we established herein. Supersulfides in ADH5 thus constitute a substantial catalytic center for GSNO metabolism mediating electron transfer from aldehydes.

Supersulphides provide airway protection in viral and chronic lung diseases.

Supersulphides are inorganic and organic sulphides with sulphur catenation with diverse physiological functions. Their synthesis is mainly mediated by mitochondrial cysteinyl-tRNA synthetase (CARS2) that functions as a principal cysteine persulphide synthase (CPERS). Here, we identify protective functions of supersulphides in viral airway infections (influenza and COVID-19), in aged lungs and in chronic lung diseases, including chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF). We develop a method for breath supersulphur-omics and demonstrate that levels of exhaled supersulphides increase in people with COVID-19 infection and in a hamster model of SARS-CoV-2 infection. Lung damage and subsequent lethality that result from oxidative stress and inflammation in mouse models of COPD, IPF, and ageing were mitigated by endogenous supersulphides production by CARS2/CPERS or exogenous administration of the supersulphide donor glutathione trisulphide. We revealed a protective role of supersulphides in airways with various viral or chronic insults and demonstrated the potential of targeting supersulphides in lung disease.

Phosphorylation of phase-separated p62 bodies by ULK1 activates a redox-independent stress response.

NRF2 is a transcription factor responsible for antioxidant stress responses that is usually regulated in a redox-dependent manner. p62 bodies formed by liquid-liquid phase separation contain Ser349-phosphorylated p62, which participates in the redox-independent activation of NRF2. However, the regulatory mechanism and physiological significance of p62 phosphorylation remain unclear. Here, we identify ULK1 as a kinase responsible for the phosphorylation of p62. ULK1 colocalizes with p62 bodies, directly interacting with p62. ULK1-dependent phosphorylation of p62 allows KEAP1 to be retained within p62 bodies, thus activating NRF2. p62S351E/+ mice are phosphomimetic knock-in mice in which Ser351, corresponding to human Ser349, is replaced by Glu. These mice, but not their phosphodefective p62S351A/S351A counterparts, exhibit NRF2 hyperactivation and growth retardation. This retardation is caused by malnutrition and dehydration due to obstruction of the esophagus and forestomach secondary to hyperkeratosis, a phenotype also observed in systemic Keap1-knockout mice. Our results expand our understanding of the physiological importance of the redox-independent NRF2 activation pathway and provide new insights into the role of phase separation in this process.

Synthesis of Sulfides and Persulfides Is Not Impeded by Disruption of Three Canonical Enzymes in Sulfur Metabolism.

Reactive sulfur species, or persulfides and polysulfides, such as cysteine hydropersulfide and glutathione persulfide, are endogenously produced in abundance in both prokaryotes and eukaryotes, including mammals. Various forms of reactive persulfides occur in both low-molecular-weight and protein-bound thiols. The chemical properties and great supply of these molecular species suggest a pivotal role for reactive persulfides/polysulfides in different cellular regulatory processes (e.g., energy metabolism and redox signaling). We demonstrated earlier that cysteinyl-tRNA synthetase (CARS) is a new cysteine persulfide synthase (CPERS) and is responsible for the in vivo production of most reactive persulfides (polysulfides). Some researchers continue to suggest that 3-mercaptopyruvate sulfurtransferase (3-MST), cystathionine ß-synthase (CBS), and cystathionine γ-lyase (CSE) may also produce hydrogen sulfide and persulfides that may be generated during the transfer of sulfur from 3-mercaptopyruvate to the cysteine residues of 3-MST or direct synthesis from cysteine by CBS/CSE, respectively. We thus used integrated sulfur metabolome analysis, which we recently developed, with 3-MST knockout (KO) mice and CBS/CSE/3-MST triple-KO mice, to elucidate the possible contribution of 3-MST, CBS, and CSE to the production of reactive persulfides in vivo. We therefore quantified various sulfide metabolites in organs derived from these mutant mice and their wild-type littermates via this sulfur metabolome, which clearly revealed no significant difference between mutant mice and wild-type mice in terms of reactive persulfide production. This result indicates that 3-MST, CBS, and CSE are not major sources of endogenous reactive persulfide production; rather, CARS/CPERS is the principal enzyme that is actually involved in and even primarily responsible for the biosynthesis of reactive persulfides and polysulfides in vivo in mammals.

The anti-inflammatory and anti-oxidative effect of a classical hypnotic bromovalerylurea mediated by the activation of NRF2.

The Kelch-like ECH-associated protein 1-nuclear factor erythroid 2-related factor 2 (KEAP1-NRF2) system plays a central role in redox homeostasis and inflammation control. Oxidative stress or electrophilic compounds promote NRF2 stabilization and transcriptional activity by negatively regulating its inhibitor, KEAP1. We have previously reported that bromovalerylurea (BU), originally developed as a hypnotic, exerts anti-inflammatory effects in various inflammatory disease models. However, the molecular mechanism underlying its effect remains uncertain. Herein, we found that by real-time multicolor luciferase assay using stable luciferase red3 (SLR3) and green-emitting emerald luciferase (ELuc), BU potentiates NRF2-dependent transcription in the human hepatoblastoma cell line HepG2 cells, which lasted for more than 60 h. Further analysis revealed that BU promotes NRF2 accumulation and the transcription of its downstream cytoprotective genes in the HepG2 and the murine microglial cell line BV2. Keap1 knockdown did not further enhance NRF2 activity, suggesting that BU upregulates NRF2 by targeting KEAP1. Knockdown of Nfe2l2 in BV2 cells diminished the suppressive effects of BU on the production of pro-inflammatory mediators, like nitric oxide (NO) and its synthase NOS2, indicating the involvement of NRF2 in the anti-inflammatory effects of BU. These data collectively suggest that BU could be repurposed as a novel NRF2 activator to control inflammation and oxidative stress.

Contribution of NRF2 to sulfur metabolism and mitochondrial activity.

NF-E2-related factor 2 (NRF2) plays a crucial role in the maintenance of cellular homeostasis by regulating various enzymes and proteins that are involved in the redox reactions utilizing sulfur. While substantial impacts of NRF2 on mitochondrial activity have been described, the precise mechanism by which NRF2 regulates mitochondrial function is still not fully understood. Here, we demonstrated that NRF2 increased intracellular persulfides by upregulating the cystine transporter xCT encoded by Slc7a11, a well-known NRF2 target gene. Persulfides have been shown to play an important role in mitochondrial function. Supplementation with glutathione trisulfide (GSSSG), which is a form of persulfide, elevated the mitochondrial membrane potential (MMP), increased the oxygen consumption rate (OCR) and promoted ATP production. Persulfide-mediated mitochondrial activation was shown to require the mitochondrial sulfur oxidation pathway, especially sulfide quinone oxidoreductase (SQOR). Consistently, NRF2-mediated mitochondrial activation was also dependent on SQOR activity. This study clarified that the facilitation of persulfide production and sulfur metabolism in mitochondria by increasing cysteine availability is one of the mechanisms for NRF2-dependent mitochondrial activation.

Structural basis of transcription regulation by CNC family transcription factor, Nrf2.

Several basic leucine zipper (bZIP) transcription factors have accessory motifs in their DNA-binding domains, such as the CNC motif of CNC family or the EHR motif of small Maf (sMaf) proteins. CNC family proteins heterodimerize with sMaf proteins to recognize CNC-sMaf binding DNA elements (CsMBEs) in competition with sMaf homodimers, but the functional role of the CNC motif remains elusive. In this study, we report the crystal structures of Nrf2/NFE2L2, a CNC family protein regulating anti-stress transcriptional responses, in a complex with MafG and CsMBE. The CNC motif restricts the conformations of crucial Arg residues in the basic region, which form extensive contact with the DNA backbone phosphates. Accordingly, the Nrf2-MafG heterodimer has approximately a 200-fold stronger affinity for CsMBE than canonical bZIP proteins, such as AP-1 proteins. The high DNA affinity of the CNC-sMaf heterodimer may allow it to compete with the sMaf homodimer on target genes without being perturbed by other low-affinity bZIP proteins with similar sequence specificity.

Establishment of Neh2-Cre:tdTomato reporter mouse for monitoring the exposure history to electrophilic stress.

Cells are often exposed to exogenous and endogenous redox disturbances and exert their protective mechanisms in response to stimuli. The KEAP1-NRF2 system plays pivotal roles in counteracting oxidative damage. Due to the transient nature of NRF2 activation, the identification of cells in which NRF2 is activated in response to systemic stimuli is sometimes not easy. To examine the electrophilic stress response at a single-cell resolution, we aimed to develop a new reporter mouse in this study. A cell-tracing strategy exploiting Cre recombinase-mediated activation of a reporter gene was chosen for stable detection of reporter expression instead of real-time monitoring of the cellular response. We established a transgenic mouse line expressing the Neh2-Cre recombinase fusion protein. As Neh2 is an amino-terminal domain of NRF2 that serves as a degron and mediates KEAP1-dependent degradation and electrophile-inducible stabilization, Neh2-Cre was expected to be activated in response to electrophiles. The Neh2-Cre transgenic mouse was crossed with the ROSA26-loxP-stop-loxP-tdTomato reporter mouse (ROSA-LSL-tdTomato mouse). The compound mutant reporter mice exhibited accumulation of tdTomato-positive cells in various organs after repeated administration of CDDO-Im, one of the NRF2-inducing electrophiles. The mice were also successfully used for the detection of cells that experienced a cisplatin-induced electrophilic stress response.

8-Nitro-cGMP suppresses mineralization by mouse osteoblasts.

Nitric oxide and reactive oxygen species regulate bone remodeling, which occurs via bone formation and resorption by osteoblasts and osteoclasts, respectively. Recently, we found that 8-nitro-cGMP, a second messenger of nitric oxide and reactive oxygen species, promotes osteoclastogenesis. Here, we investigated the formation and function of 8-nitro-cGMP in osteoblasts. Mouse calvarial osteoblasts were found to produce 8-nitro-cGMP, which was augmented by tumor necrosis factor-α (10 ng/ml) and interleukin-1ß (1 ng/ml). These cytokines suppressed osteoblastic differentiation in a NO synthase activity-dependent manner. Exogenous 8-nitro-cGMP (30 µmol/L) suppressed expression of osteoblastic phenotypes, including mineralization, in clear contrast to the enhancement of mineralization by osteoblasts induced by 8-bromo-cGMP, a cell membrane-permeable analog of cGMP. It is known that reactive sulfur species denitrates and degrades 8-nitro-cGMP. Mitochondrial cysteinyl-tRNA synthetase plays a crucial role in the endogenous production of RSS. The expression of osteoblastic phenotypes was suppressed by not only exogenous 8-nitro-cGMP but also by silencing of the Cars2 gene, indicating a role of endogenous 8-nitro-cGMP in suppressing the expression of osteoblastic phenotypes. These results suggest that 8-nitro-cGMP is a negative regulator of osteoblastic differentiation.

Deficiency of the bZIP transcription factors Mafg and Mafk causes misexpression of genes in distinct pathways and results in lens embryonic developmental defects.

Deficiency of the small Maf proteins Mafg and Mafk cause multiple defects, namely, progressive neuronal degeneration, cataract, thrombocytopenia and mid-gestational/perinatal lethality. Previous data shows Mafg -/-:Mafk +/- compound knockout (KO) mice exhibit cataracts age 4-months onward. Strikingly, Mafg -/-:Mafk -/- double KO mice develop lens defects significantly early in life, during embryogenesis, but the pathobiology of these defects is unknown, and is addressed here. At embryonic day (E)16.5, the epithelium of lens in Mafg -/-:Mafk -/- animals appears abnormally multilayered as demonstrated by E-cadherin and nuclear staining. Additionally, Mafg -/-:Mafk -/- lenses exhibit abnormal distribution of F-actin near the "fulcrum" region where epithelial cells undergo apical constriction prior to elongation and reorientation as early differentiating fiber cells. To identify the underlying molecular changes, we performed high-throughput RNA-sequencing of E16.5 Mafg -/-:Mafk -/- lenses and identified a cohort of differentially expressed genes that were further prioritized using stringent filtering criteria and validated by RT-qPCR. Several key factors associated with the cytoskeleton, cell cycle or extracellular matrix (e.g., Cdk1, Cdkn1c, Camsap1, Col3a1, Map3k12, Sipa1l1) were mis-expressed in Mafg -/-:Mafk -/- lenses. Further, the congenital cataract-linked extracellular matrix peroxidase Pxdn was significantly overexpressed in Mafg -/-:Mafk -/- lenses, which may cause abnormal cell morphology. These data also identified the ephrin signaling receptor Epha5 to be reduced in Mafg -/-:Mafk -/- lenses. This likely contributes to the Mafg -/-:Mafk -/- multilayered lens epithelium pathology, as loss of an ephrin ligand, Efna5 (ephrin-A5), causes similar lens defects. Together, these findings uncover a novel early function of Mafg and Mafk in lens development and identify their new downstream regulatory relationships with key cellular factors.