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Elevated O-GlcNAc-dependent signaling through inducible mOGT expression selectively triggers apoptosis.

Shin SH, Love DC, Hanover JA - Amino Acids (2010)

Bottom Line: One differentially spliced isoform of OGT is normally targeted to mitochondria (mOGT) but is quite cytotoxic when expressed in cells compared with the ncOGT isoform.Apoptosis was dependent upon the unique N-terminus of mOGT, and its catalytic activity.Induction of mOGT expression triggered programmed cell death in every cell type tested including INS-1, an insulin-secreting cell line.

View Article: PubMed Central - PubMed

Affiliation: Gyeong-Gi Bio Center, Suwon, Korea.

ABSTRACT
O-linked N-acetylglucosamine transferase (OGT) catalyzes O-GlcNAc addition to numerous cellular proteins including transcription and nuclear pore complexes and plays a key role in cellular signaling. One differentially spliced isoform of OGT is normally targeted to mitochondria (mOGT) but is quite cytotoxic when expressed in cells compared with the ncOGT isoform. To understand the basis of this selective cytotoxicity, we constructed a fully functional ecdysone-inducible GFP-OGT. Elevated GFP-OGT expression induced a dramatic increase in intracellular O-GlcNAcylated proteins. Furthermore, enhanced OGT expression efficiently triggered programmed cell death. Apoptosis was dependent upon the unique N-terminus of mOGT, and its catalytic activity. Induction of mOGT expression triggered programmed cell death in every cell type tested including INS-1, an insulin-secreting cell line. These studies suggest that deregulated activity of the mitochondrially targeted mOGT may play a role in triggering the programmed cell death observed with diseases such as diabetes mellitus and neurodegeneration.

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Inducible expression of mOGT in EcR293 cells. a Diagram of the GFP–mOGT construct under the control of an inducible promoter (pIND/GFP–OGT). b Western blot analysis using an anti-OGT antibody to compare expression levels of pIND/GFP–OGT with increasing amounts of Ponasterone A (PoA) to a CMV-driven GFP–OGT fusion (pGFP–OGT). c Western blot analysis of same samples using an anti-GFP antibody. For b and c EcR-293 cells were transfected with pIND/GFP–OGT and incubated without Ponasterone A(PoA) (lane 1), or with the following concentration of PoA: 0.1 µM (lane 2), 0.5 µM (lane 3), 3.0 µM (lane 4), 15.0 µM (lane 5). Cells were also transfected with the CMV-driven plasmid pGFP–OGT (lane 6). On the right side are molecular weight markers. d Longer exposure of the Western blot shown in c between lanes2 and 4 at 140 kDa range
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Fig2: Inducible expression of mOGT in EcR293 cells. a Diagram of the GFP–mOGT construct under the control of an inducible promoter (pIND/GFP–OGT). b Western blot analysis using an anti-OGT antibody to compare expression levels of pIND/GFP–OGT with increasing amounts of Ponasterone A (PoA) to a CMV-driven GFP–OGT fusion (pGFP–OGT). c Western blot analysis of same samples using an anti-GFP antibody. For b and c EcR-293 cells were transfected with pIND/GFP–OGT and incubated without Ponasterone A(PoA) (lane 1), or with the following concentration of PoA: 0.1 µM (lane 2), 0.5 µM (lane 3), 3.0 µM (lane 4), 15.0 µM (lane 5). Cells were also transfected with the CMV-driven plasmid pGFP–OGT (lane 6). On the right side are molecular weight markers. d Longer exposure of the Western blot shown in c between lanes2 and 4 at 140 kDa range

Mentions: In another series of experiments, GFP was directly appended to the amino terminus of the 103-kDa isoform of mOGT and placed under the control of the ecdysone promoter, pIND/GFP–OGT (Fig. 2a). This allowed direct visualization and detection of the mOGT transgene. Expression of this fusion protein was monitored by immunoblotting (Fig. 2b–d). Increasing concentrations of Ponasterone A induced expression of the fusion protein. This expression was detected by an anti-OGT antibody (Fig. 2b) or an anti-GFP antibody (Fig. 2c, d). The level of expression observed when fully induced compared favorably with that seen using a CMV promoter, pGFP–OGT (Fig. 2b, c; compare lanes 5 and 6). Longer exposures of the immunoblots revealed that even at low levels of Ponasterone A, induction of OGT occurred (Fig. 2d). These data suggest that the ecdysone system allowed exquisite control over the levels of OGT expression.Fig. 2


Elevated O-GlcNAc-dependent signaling through inducible mOGT expression selectively triggers apoptosis.

Shin SH, Love DC, Hanover JA - Amino Acids (2010)

Inducible expression of mOGT in EcR293 cells. a Diagram of the GFP–mOGT construct under the control of an inducible promoter (pIND/GFP–OGT). b Western blot analysis using an anti-OGT antibody to compare expression levels of pIND/GFP–OGT with increasing amounts of Ponasterone A (PoA) to a CMV-driven GFP–OGT fusion (pGFP–OGT). c Western blot analysis of same samples using an anti-GFP antibody. For b and c EcR-293 cells were transfected with pIND/GFP–OGT and incubated without Ponasterone A(PoA) (lane 1), or with the following concentration of PoA: 0.1 µM (lane 2), 0.5 µM (lane 3), 3.0 µM (lane 4), 15.0 µM (lane 5). Cells were also transfected with the CMV-driven plasmid pGFP–OGT (lane 6). On the right side are molecular weight markers. d Longer exposure of the Western blot shown in c between lanes2 and 4 at 140 kDa range
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040817&req=5

Fig2: Inducible expression of mOGT in EcR293 cells. a Diagram of the GFP–mOGT construct under the control of an inducible promoter (pIND/GFP–OGT). b Western blot analysis using an anti-OGT antibody to compare expression levels of pIND/GFP–OGT with increasing amounts of Ponasterone A (PoA) to a CMV-driven GFP–OGT fusion (pGFP–OGT). c Western blot analysis of same samples using an anti-GFP antibody. For b and c EcR-293 cells were transfected with pIND/GFP–OGT and incubated without Ponasterone A(PoA) (lane 1), or with the following concentration of PoA: 0.1 µM (lane 2), 0.5 µM (lane 3), 3.0 µM (lane 4), 15.0 µM (lane 5). Cells were also transfected with the CMV-driven plasmid pGFP–OGT (lane 6). On the right side are molecular weight markers. d Longer exposure of the Western blot shown in c between lanes2 and 4 at 140 kDa range
Mentions: In another series of experiments, GFP was directly appended to the amino terminus of the 103-kDa isoform of mOGT and placed under the control of the ecdysone promoter, pIND/GFP–OGT (Fig. 2a). This allowed direct visualization and detection of the mOGT transgene. Expression of this fusion protein was monitored by immunoblotting (Fig. 2b–d). Increasing concentrations of Ponasterone A induced expression of the fusion protein. This expression was detected by an anti-OGT antibody (Fig. 2b) or an anti-GFP antibody (Fig. 2c, d). The level of expression observed when fully induced compared favorably with that seen using a CMV promoter, pGFP–OGT (Fig. 2b, c; compare lanes 5 and 6). Longer exposures of the immunoblots revealed that even at low levels of Ponasterone A, induction of OGT occurred (Fig. 2d). These data suggest that the ecdysone system allowed exquisite control over the levels of OGT expression.Fig. 2

Bottom Line: One differentially spliced isoform of OGT is normally targeted to mitochondria (mOGT) but is quite cytotoxic when expressed in cells compared with the ncOGT isoform.Apoptosis was dependent upon the unique N-terminus of mOGT, and its catalytic activity.Induction of mOGT expression triggered programmed cell death in every cell type tested including INS-1, an insulin-secreting cell line.

View Article: PubMed Central - PubMed

Affiliation: Gyeong-Gi Bio Center, Suwon, Korea.

ABSTRACT
O-linked N-acetylglucosamine transferase (OGT) catalyzes O-GlcNAc addition to numerous cellular proteins including transcription and nuclear pore complexes and plays a key role in cellular signaling. One differentially spliced isoform of OGT is normally targeted to mitochondria (mOGT) but is quite cytotoxic when expressed in cells compared with the ncOGT isoform. To understand the basis of this selective cytotoxicity, we constructed a fully functional ecdysone-inducible GFP-OGT. Elevated GFP-OGT expression induced a dramatic increase in intracellular O-GlcNAcylated proteins. Furthermore, enhanced OGT expression efficiently triggered programmed cell death. Apoptosis was dependent upon the unique N-terminus of mOGT, and its catalytic activity. Induction of mOGT expression triggered programmed cell death in every cell type tested including INS-1, an insulin-secreting cell line. These studies suggest that deregulated activity of the mitochondrially targeted mOGT may play a role in triggering the programmed cell death observed with diseases such as diabetes mellitus and neurodegeneration.

Show MeSH
Related in: MedlinePlus