Enhanced vulnerability to oxidative stress and induction of inflammatory gene expression in 3-phosphoglycerate dehydrogenase-deficient fibroblasts

Abstract

l-Serine (l-Ser) is a necessary precursor for the synthesis of proteins, lipids, glycine, cysteine, d-serine, and tetrahydrofolate metabolites. Low l-Ser availability activates stress responses and cell death; however, the underlying molecular mechanisms remain unclear. l-Ser is synthesized de novo from 3-phosphoglycerate with 3-phosphoglycerate dehydrogenase (Phgdh) catalyzing the first reaction step. Here, we show that l-Ser depletion raises intracellular H₂O₂ levels and enhances vulnerability to oxidative stress in Phgdh-deficient mouse embryonic fibroblasts. These changes were associated with reduced total glutathione levels. Moreover, levels of the inflammatory markers thioredoxin-interacting protein and prostaglandin-endoperoxide synthase 2 were upregulated under l-Ser-depleted conditions; this was suppressed by the addition of N-acetyl-l-cysteine. Thus, intracellular l-Ser deficiency triggers an inflammatory response via increased oxidative stress, and de novo l-Ser synthesis suppresses oxidative stress damage and inflammation when the external l-Ser supply is restricted.

Keywords: Phgdh; Ptgs2; Txnip; l‐serine deficiency; oxidative stress.

Figure 1

l‐Ser deficiency induces the reduction in the intracellular GSH level and resistance to oxidative stress in Phgdh KO‐MEFs. (A) KO‐MEFs were cultured under l‐Ser‐supplemented (+Ser) or l‐Ser‐depleted (–Ser) conditions for 24 h, and intracellular total GSH levels were measured by a GSH assay kit (n = 3; Student's t‐test, *P < 0.05). (B) KO‐MEFs and KO‐MEFs transduced with Phgdh (KO‐MEFs^+Phgdh) were cultured under l‐Ser‐supplemented or l‐Ser‐depleted conditions for 6 h, and the production of intracellular H₂O₂ was analyzed using a fluorescent probe of H₂O₂ with In Cell Analyzer 1000 (n = 3; Student's t‐test, *P < 0.05). (C) WT‐MEFs and KO‐MEFs were cultured in complete DMEM for 16 h, and cells were cultured in EMEM containing 10% FBS supplemented with 0.01, 0.1, and 1 μm H₂O₂ for 6 h. Cell viability (WT‐MEFs: solid line with closed circles, KO‐MEFs: dotted line with open squares) was determined by counting the number of live cells using the MTT assay kit (WT‐MEFs, n = 3; KO‐MEFs, n = 3; Student's t‐test, *P < 0.05, ***P < 0.0005).

Figure 2

Phgdh deletion induced Txnip and Ptgs2 expression caused by l‐Ser deficiency. (A,E) WT‐MEFs and KO‐MEFs were cultured under l‐Ser‐supplemented or l‐Ser‐depleted conditions for 6 h, and Txnip (A) and Ptgs2 (E) mRNA levels were measured (WT‐MEFs, n = 3; KO‐MEFs, n = 3; Student's t‐test, **P < 0.005, ***P < 0.0005). (B,F) KO‐MEFs were cultured under l‐Ser‐supplemented or l‐Ser‐depleted conditions for 6 h, and Txnip (B) and Cox2 (F) protein levels were measured by western blotting and normalized to the Gapdh protein level (KO‐MEFs, n = 3, Student's t‐test, *P < 0.05, **P < 0.005). (C,H) KO‐MEFs, KO‐MEFs transduced with Phgdh (KO‐MEFs^+Phgdh), and KO‐MEFs transduced with Gfp (KO‐MEFs^+Gfp) were cultured under l‐Ser‐supplemented or l‐Ser‐depleted conditions for 6 h, and Txnip (C) and Ptgs2 (H) mRNA levels were measured (KO‐MEFs, n = 3; KO‐MEFs^+Phgdh, n = 3; KO‐MEFs^+Gfp, n = 3; Student's t‐test, **P < 0.005, ***P < 0.0005). (D,G) KO‐MEFs were cultured under l‐Ser‐supplemented or l‐Ser‐depleted conditions for 2 h, 6 h, and 24 h, and Txnip (D) and Ptgs2 (G) mRNA levels were measured by qRT‐PCR and normalized to the Gapdh mRNA level (KO‐MEFs, n = 3, Student's t‐test, *P < 0.05, ***P < 0.0005).

Figure 3

Txnip and Ptgs2 induction is not associated with the ISR pathway activated by amino acid deficiency in l‐Ser‐depleted KO‐MEFs. (A,B) Mock‐ and shAtf4‐transduced KO‐MEFs were cultured under l‐Ser‐supplemented or l‐Ser‐depleted conditions for 6 h, and Txnip (A) and Ptgs2 (B) mRNA levels were measured (mock‐transduced KO‐MEFs, n = 3; shAtf4‐transduced KO‐MEFs, n = 3; Student's t‐test, *P < 0.05, **P < 0.005). (C,D) KO‐MEFs were cultured under l‐Leu‐supplemented or l‐Leu‐depleted conditions for 6 h, and Txnip (C) and Ptgs2 (D) mRNA levels were measured (KO‐MEFs, n = 3, Student's t‐test, *P < 0.05, **P < 0.005, ***P < 0.0005).

Figure 4

Antioxidant NAC treatment suppresses Txnip and Ptgs2 induction caused by l‐Ser deficiency. (A,B) KO‐MEFs were cultured under l‐Ser‐supplemented or l‐Ser‐depleted conditions in the presence of 1 mm or 5 mm NAC for 6 h, and Txnip (A) and Ptgs2 (B) mRNA levels were measured (KO‐MEFs, n = 3; Dunnett's post hoc test, ***P < 0.0005).