Transcriptome of the GSH-Depleted Lens Reveals Changes in Detoxification and EMT Signaling Genes, Transport Systems, and Lipid Homeostasis

Abstract

PurposeTo understand the effects of glutathione (GSH)-deficiency on genetic processes that regulate lens homeostasis and prevent cataractogenesis.MethodsThe transcriptome of lens epithelia and fiber cells was obtained from C57BL/6 LEGSKO (lens GSH-synthesis knockout) and buthionine sulfoximine (BSO)-treated LEGSKO mice and compared to C57BL/6 wild-type mice using RNA-Seq. Transcriptomic data were confirmed by qPCR and Western blot/ELISA on a subset of genes.ResultsRNA-Seq results were in excellent agreement with qPCR (correlation coefficients 0.87–0.94 and P < 5E-6 for a subset of 36 mRNAs). Of 24,415 transcripts mapped to the mouse genome, 441 genes showed significantly modulated expression. Pathway analysis indicated major changes in epithelial-mesenchymal transition (EMT) signaling, visual cycle, small molecule biochemistry, and lipid metabolism. GSH-deficient lenses showed upregulation of detoxification genes, including Aldh1a1, Aldh3a1 (aldehyde dehydrogenases), Mt1, Mt2 (metallothioneins), Ces1g (carboxylesterase), and Slc14a1 (urea transporter UT-B). Genes in canonical EMT pathways, including Wnt10a, showed upregulation in lens epithelia samples. Severely GSH-deficient lens epithelia showed downregulation of vision-related genes (including crystallins). The BSO-treated LEGSKO lens epithelia transcriptome has significant correlation (r = 0.63, P < 0.005) to that of lens epithelia undergoing EMT. Protein expression data correlated with transcriptomic data and confirmed EMT signaling activation.ConclusionsThese results show that GSH-deficiency in the lens leads to expression of detoxifying genes and activation of EMT signaling, in addition to changes in transport systems and lipid homeostasis. These data provide insight into the adaptation and consequences of GSH-deficiency in the lens and suggest that GSH plays an important role in lenticular EMT pathology.

Proteomic analysis of the glutathione-deficient LEGSKO mouse lens reveals activation of EMT signaling, loss of lens specific markers, and changes in stress response proteins

“…The trends discussed above fit well with those found in the GSH-deficient lens transcriptome [7], with the exception of major shifts in lipid homeostasis and transport systems, which were seen in the transcriptome but not the proteome of the GSH-deficient lens. Specific overlap in significant gene and protein expression changes was also investigated (Table 2).…”

“…As was found in the GSH-deficient lens transcriptome [7], the GSH-deficient lens proteome showed a broad downregulation of β- and γ-crystallins in both lens epithelia and fiber cells (Fig. 6B–J).…”

“…4R) were all modestly upregulated in BSO-treated LEGSKO lens fiber cells. CAR2, which was also upregulated in the GSH-deficient lens transcriptome (Table 2) [7], regulates acid-base balance through the bicarbonate buffer system and may be upregulated in order to accommodate for the loss of GSH, which is acidic and normally present at millimolar levels within the lens. HERPUD1 is a chaperone involved in the unfolded protein response.…”

“…We were previously surprised to find a very muted oxidative stress response in the GSH-deficient lens transcriptome [7]. Aldh1a1, Aldh3a1, Mt1 , and Hmox1 are all upregulated by NRF2 activation [31] but, while Aldh1a1, Aldh3a1, and Mt1 were upregulated in the GSH-deficient lens transcriptome, Hmox1 was downregulated.…”

“…In order to determine the genetic response to GSH deficiency in the lens, an in-depth RNA-Seq analysis was carried out on the lens epithelia and cortical fiber cells of wild-type (WT) (GSH ~3.8 mM), LEGSKO (GSH ~1.3 mM), and BSO-treated LEGSKO mice (GSH ~0.35 mM) [7]. GSH levels were determined by LC-MS/MS analysis of whole lenses [6].…”