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Decrease in membrane phospholipid unsaturation induces unfolded protein response.

Ariyama H, Kono N, Matsuda S, Inoue T, Arai H - J. Biol. Chem. (2010)

Bottom Line: In this study we showed that stearoyl-CoA desaturase 1 (SCD1) knockdown increased the amount of saturated fatty acids and decreased that of monounsaturated fatty acids in phospholipids without affecting the amount or the composition of free fatty acid and induced unfolded protein response (UPR), evidenced by increased expression of C/EBP homologous protein (CHOP) and glucose-regulated protein 78 (GRP78) mRNAs and splicing of Xbox-binding protein 1 (XBP1) mRNA.Finally we showed that palmitic acid-induced UPR was significantly enhanced by LPCAT3 knockdown as well as SCD1 knockdown.These results suggest that a decrease in membrane phospholipid unsaturation induces UPR.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033, Japan.

ABSTRACT
Various kinds of fatty acids are distributed in membrane phospholipids in mammalian cells and tissues. The degree of fatty acid unsaturation in membrane phospholipids affects many membrane-associated functions and can be influenced by diet and by altered activities of lipid-metabolizing enzymes such as fatty acid desaturases. However, little is known about how mammalian cells respond to changes in phospholipid fatty acid composition. In this study we showed that stearoyl-CoA desaturase 1 (SCD1) knockdown increased the amount of saturated fatty acids and decreased that of monounsaturated fatty acids in phospholipids without affecting the amount or the composition of free fatty acid and induced unfolded protein response (UPR), evidenced by increased expression of C/EBP homologous protein (CHOP) and glucose-regulated protein 78 (GRP78) mRNAs and splicing of Xbox-binding protein 1 (XBP1) mRNA. SCD1 knockdown-induced UPR was rescued by various unsaturated fatty acids and was enhanced by saturated fatty acid. Lysophosphatidylcholine acyltransferase 3 (LPCAT3), which incorporates preferentially polyunsaturated fatty acids into phosphatidylcholine, was up-regulated in SCD1 knockdown cells. Knockdown of LPCAT3 synergistically enhanced UPR with SCD1 knockdown. Finally we showed that palmitic acid-induced UPR was significantly enhanced by LPCAT3 knockdown as well as SCD1 knockdown. These results suggest that a decrease in membrane phospholipid unsaturation induces UPR.

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SCD1 knockdown induces UPR. HeLa cells were transfected with the indicated siRNA. At 72 h after transfection, total RNA and cell lysates were prepared. A and B, expression of CHOP (A) and GRP78 (B) mRNAs were detected by quantitative real-time PCR. The expression level of each gene was normalized to the GAPDH gene and is represented as -fold induction over siControl. Thapsigargin-treated cells (Tg) were used as a positive control. C, semiquantitative reverse transcription-PCR analysis of XBP1 spliced and unspliced mRNA is shown. The positions of the unspliced form (u) and spliced form (s) are indicated. Thapsigargin-treated cells were used as a positive control. D, shown is an immunoblot analysis of PERK. Thapsigargin-treated cells were used as a positive control, and α-tubulin expression was used as a loading control. The data represent the mean ± S.E. of three experiments. The asterisks indicate significant differences compared with siControl-transfected cells (p < 0.01).
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Figure 2: SCD1 knockdown induces UPR. HeLa cells were transfected with the indicated siRNA. At 72 h after transfection, total RNA and cell lysates were prepared. A and B, expression of CHOP (A) and GRP78 (B) mRNAs were detected by quantitative real-time PCR. The expression level of each gene was normalized to the GAPDH gene and is represented as -fold induction over siControl. Thapsigargin-treated cells (Tg) were used as a positive control. C, semiquantitative reverse transcription-PCR analysis of XBP1 spliced and unspliced mRNA is shown. The positions of the unspliced form (u) and spliced form (s) are indicated. Thapsigargin-treated cells were used as a positive control. D, shown is an immunoblot analysis of PERK. Thapsigargin-treated cells were used as a positive control, and α-tubulin expression was used as a loading control. The data represent the mean ± S.E. of three experiments. The asterisks indicate significant differences compared with siControl-transfected cells (p < 0.01).

Mentions: UPR is initiated by three endoplasmic reticulum (ER) transmembrane proteins, inositol-requiring 1, PERK, and activating transcription factor 6 (ATF6). UPR induces transcription of a set of genes whose protein products increase the capacity for protein folding and ER-associated degradation and induces apoptosis when the ER function is severely impaired (30). Of these genes, CHOP and glucose-regulated protein 78 (GRP78) were often used as UPR markers. Quantitative real-time PCR analysis showed that SCD1 knockdown strikingly induced the expression of CHOP and GRP78 mRNAs, which is comparable with that induced by 0.5 μm thapsigargin treatment for 12 h (Fig. 2, A and B). We also examined activations of the UPR sensor proteins IRE1 and PERK. Activation of IRE1 leads to alternative splicing of transcription factor XBP1, and PERK is activated by autophosphorylation, a modification that slows its electrophoretic mobility (31, 32). SCD1 knockdown induced XBP1 splicing to the same extent as thapsigargin treatment induced XBP1 splicing (Fig. 2C). Western blotting using anti-PERK revealed that SCD1 knockdown induced a shift in PERK protein mobility that was similar to the shift induced by thapsigargin treatment (Fig. 2D). These results indicate that SCD1 knockdown induces UPR activation.


Decrease in membrane phospholipid unsaturation induces unfolded protein response.

Ariyama H, Kono N, Matsuda S, Inoue T, Arai H - J. Biol. Chem. (2010)

SCD1 knockdown induces UPR. HeLa cells were transfected with the indicated siRNA. At 72 h after transfection, total RNA and cell lysates were prepared. A and B, expression of CHOP (A) and GRP78 (B) mRNAs were detected by quantitative real-time PCR. The expression level of each gene was normalized to the GAPDH gene and is represented as -fold induction over siControl. Thapsigargin-treated cells (Tg) were used as a positive control. C, semiquantitative reverse transcription-PCR analysis of XBP1 spliced and unspliced mRNA is shown. The positions of the unspliced form (u) and spliced form (s) are indicated. Thapsigargin-treated cells were used as a positive control. D, shown is an immunoblot analysis of PERK. Thapsigargin-treated cells were used as a positive control, and α-tubulin expression was used as a loading control. The data represent the mean ± S.E. of three experiments. The asterisks indicate significant differences compared with siControl-transfected cells (p < 0.01).
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Related In: Results  -  Collection

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Figure 2: SCD1 knockdown induces UPR. HeLa cells were transfected with the indicated siRNA. At 72 h after transfection, total RNA and cell lysates were prepared. A and B, expression of CHOP (A) and GRP78 (B) mRNAs were detected by quantitative real-time PCR. The expression level of each gene was normalized to the GAPDH gene and is represented as -fold induction over siControl. Thapsigargin-treated cells (Tg) were used as a positive control. C, semiquantitative reverse transcription-PCR analysis of XBP1 spliced and unspliced mRNA is shown. The positions of the unspliced form (u) and spliced form (s) are indicated. Thapsigargin-treated cells were used as a positive control. D, shown is an immunoblot analysis of PERK. Thapsigargin-treated cells were used as a positive control, and α-tubulin expression was used as a loading control. The data represent the mean ± S.E. of three experiments. The asterisks indicate significant differences compared with siControl-transfected cells (p < 0.01).
Mentions: UPR is initiated by three endoplasmic reticulum (ER) transmembrane proteins, inositol-requiring 1, PERK, and activating transcription factor 6 (ATF6). UPR induces transcription of a set of genes whose protein products increase the capacity for protein folding and ER-associated degradation and induces apoptosis when the ER function is severely impaired (30). Of these genes, CHOP and glucose-regulated protein 78 (GRP78) were often used as UPR markers. Quantitative real-time PCR analysis showed that SCD1 knockdown strikingly induced the expression of CHOP and GRP78 mRNAs, which is comparable with that induced by 0.5 μm thapsigargin treatment for 12 h (Fig. 2, A and B). We also examined activations of the UPR sensor proteins IRE1 and PERK. Activation of IRE1 leads to alternative splicing of transcription factor XBP1, and PERK is activated by autophosphorylation, a modification that slows its electrophoretic mobility (31, 32). SCD1 knockdown induced XBP1 splicing to the same extent as thapsigargin treatment induced XBP1 splicing (Fig. 2C). Western blotting using anti-PERK revealed that SCD1 knockdown induced a shift in PERK protein mobility that was similar to the shift induced by thapsigargin treatment (Fig. 2D). These results indicate that SCD1 knockdown induces UPR activation.

Bottom Line: In this study we showed that stearoyl-CoA desaturase 1 (SCD1) knockdown increased the amount of saturated fatty acids and decreased that of monounsaturated fatty acids in phospholipids without affecting the amount or the composition of free fatty acid and induced unfolded protein response (UPR), evidenced by increased expression of C/EBP homologous protein (CHOP) and glucose-regulated protein 78 (GRP78) mRNAs and splicing of Xbox-binding protein 1 (XBP1) mRNA.Finally we showed that palmitic acid-induced UPR was significantly enhanced by LPCAT3 knockdown as well as SCD1 knockdown.These results suggest that a decrease in membrane phospholipid unsaturation induces UPR.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033, Japan.

ABSTRACT
Various kinds of fatty acids are distributed in membrane phospholipids in mammalian cells and tissues. The degree of fatty acid unsaturation in membrane phospholipids affects many membrane-associated functions and can be influenced by diet and by altered activities of lipid-metabolizing enzymes such as fatty acid desaturases. However, little is known about how mammalian cells respond to changes in phospholipid fatty acid composition. In this study we showed that stearoyl-CoA desaturase 1 (SCD1) knockdown increased the amount of saturated fatty acids and decreased that of monounsaturated fatty acids in phospholipids without affecting the amount or the composition of free fatty acid and induced unfolded protein response (UPR), evidenced by increased expression of C/EBP homologous protein (CHOP) and glucose-regulated protein 78 (GRP78) mRNAs and splicing of Xbox-binding protein 1 (XBP1) mRNA. SCD1 knockdown-induced UPR was rescued by various unsaturated fatty acids and was enhanced by saturated fatty acid. Lysophosphatidylcholine acyltransferase 3 (LPCAT3), which incorporates preferentially polyunsaturated fatty acids into phosphatidylcholine, was up-regulated in SCD1 knockdown cells. Knockdown of LPCAT3 synergistically enhanced UPR with SCD1 knockdown. Finally we showed that palmitic acid-induced UPR was significantly enhanced by LPCAT3 knockdown as well as SCD1 knockdown. These results suggest that a decrease in membrane phospholipid unsaturation induces UPR.

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