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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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Overexpression of catalytically active GFP–OGT induces apoptosis. a CMV-driven expression of the mitochondrial form of OGT (pGFP–OGT) induces apoptosis as shown by the TUNEL assay. b A catalytically inactive form of OGT (pGFP–OGT-G) does not induce apoptosis. EcR-293 cells were used for transfections in a and b. c and d An insulinoma cell line, INS-1, was transfected with active OGT (pGFP–OGT). The TUNEL assay was used to reveal apoptotic cells. Representative focal planes near the bottom (c) and top (d) of INS-1 cell clusters are shown. Note the apoptotic cells on the outer layer of cells in the clusters. Black and red arrows denote cells scored as positive by the TUNEL assay. e–g EcR-293 cells were transfected with GFP–OGT (pIND/GFP–OGT) and induced with Ponasterone A (PoA) at indicated concentrations. Note the increasing amount of TUNEL-positive cells (black arrows) in g and h compared with the two negative controls e and h pIND/GFP–OGT, no PoA and no plasmid, 15 µM PoA, respectively. i–l Annexin V staining was used to identify apoptotic cells. i EcR-293 cells were treated with 3.0 μM of Ponasterone A to induce expression of pIND/GFP–OGT (green). j Same field stained with an anti-annexin V antibody to reveal apoptotic cells (red). Note near-complete colocalization of GFP–OGT expression and Annexin V staining (compare i and j). k Untreated cells did not stain positive for Annexin V. l As a positive control for apoptosis, 4 μ/ml camptothecin was added to uninduced cells. m DNA ladder assay. Genomic DNA from pIND/GFP–OGT transfected cells (lanes 1–5) was separated on an agarose gel to reveal DNA laddering. Uninduced cells are shown in lane 1. Increasing amounts of Ponasterone A were added at the following concentrations: 0.1 µM (lane 2), 0.5 µM (lane 3), 3.0 µM (lane 4), 15.0 (lane 5). The DNA laddering from CMV-driven expression of pGFP–OGT (lane 6) is comparable to the commercially available positive control from Calf thymus (lane 7). DNA size markers of 100 bp (lane 8) and 1 kb DNA (lane 9) are shown
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Fig4: Overexpression of catalytically active GFP–OGT induces apoptosis. a CMV-driven expression of the mitochondrial form of OGT (pGFP–OGT) induces apoptosis as shown by the TUNEL assay. b A catalytically inactive form of OGT (pGFP–OGT-G) does not induce apoptosis. EcR-293 cells were used for transfections in a and b. c and d An insulinoma cell line, INS-1, was transfected with active OGT (pGFP–OGT). The TUNEL assay was used to reveal apoptotic cells. Representative focal planes near the bottom (c) and top (d) of INS-1 cell clusters are shown. Note the apoptotic cells on the outer layer of cells in the clusters. Black and red arrows denote cells scored as positive by the TUNEL assay. e–g EcR-293 cells were transfected with GFP–OGT (pIND/GFP–OGT) and induced with Ponasterone A (PoA) at indicated concentrations. Note the increasing amount of TUNEL-positive cells (black arrows) in g and h compared with the two negative controls e and h pIND/GFP–OGT, no PoA and no plasmid, 15 µM PoA, respectively. i–l Annexin V staining was used to identify apoptotic cells. i EcR-293 cells were treated with 3.0 μM of Ponasterone A to induce expression of pIND/GFP–OGT (green). j Same field stained with an anti-annexin V antibody to reveal apoptotic cells (red). Note near-complete colocalization of GFP–OGT expression and Annexin V staining (compare i and j). k Untreated cells did not stain positive for Annexin V. l As a positive control for apoptosis, 4 μ/ml camptothecin was added to uninduced cells. m DNA ladder assay. Genomic DNA from pIND/GFP–OGT transfected cells (lanes 1–5) was separated on an agarose gel to reveal DNA laddering. Uninduced cells are shown in lane 1. Increasing amounts of Ponasterone A were added at the following concentrations: 0.1 µM (lane 2), 0.5 µM (lane 3), 3.0 µM (lane 4), 15.0 (lane 5). The DNA laddering from CMV-driven expression of pGFP–OGT (lane 6) is comparable to the commercially available positive control from Calf thymus (lane 7). DNA size markers of 100 bp (lane 8) and 1 kb DNA (lane 9) are shown

Mentions: We then examined the effects of elevated O-GlcNAc levels on cell viability. The cell line EcR-293 (a kidney line) was chosen because it contains low levels of OGT (Lubas et al. 1997; Kreppel et al. 1997) and stably expresses ecdysone receptor which facilitates the ecdysone induction. Transient overexpression of catalytically active OGT was found to induce apoptosis as measured by the TUNEL assay (Fig. 4a, pGFP–OGT). The apoptosis was dependent upon catalytic activity since the transfection with the C-terminus deleted, catalytically defective, mutation of OGT did not result in TUNEL-positive cells (Fig. 4b, pGFP–OGT-G).Fig. 4


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

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

Overexpression of catalytically active GFP–OGT induces apoptosis. a CMV-driven expression of the mitochondrial form of OGT (pGFP–OGT) induces apoptosis as shown by the TUNEL assay. b A catalytically inactive form of OGT (pGFP–OGT-G) does not induce apoptosis. EcR-293 cells were used for transfections in a and b. c and d An insulinoma cell line, INS-1, was transfected with active OGT (pGFP–OGT). The TUNEL assay was used to reveal apoptotic cells. Representative focal planes near the bottom (c) and top (d) of INS-1 cell clusters are shown. Note the apoptotic cells on the outer layer of cells in the clusters. Black and red arrows denote cells scored as positive by the TUNEL assay. e–g EcR-293 cells were transfected with GFP–OGT (pIND/GFP–OGT) and induced with Ponasterone A (PoA) at indicated concentrations. Note the increasing amount of TUNEL-positive cells (black arrows) in g and h compared with the two negative controls e and h pIND/GFP–OGT, no PoA and no plasmid, 15 µM PoA, respectively. i–l Annexin V staining was used to identify apoptotic cells. i EcR-293 cells were treated with 3.0 μM of Ponasterone A to induce expression of pIND/GFP–OGT (green). j Same field stained with an anti-annexin V antibody to reveal apoptotic cells (red). Note near-complete colocalization of GFP–OGT expression and Annexin V staining (compare i and j). k Untreated cells did not stain positive for Annexin V. l As a positive control for apoptosis, 4 μ/ml camptothecin was added to uninduced cells. m DNA ladder assay. Genomic DNA from pIND/GFP–OGT transfected cells (lanes 1–5) was separated on an agarose gel to reveal DNA laddering. Uninduced cells are shown in lane 1. Increasing amounts of Ponasterone A were added at the following concentrations: 0.1 µM (lane 2), 0.5 µM (lane 3), 3.0 µM (lane 4), 15.0 (lane 5). The DNA laddering from CMV-driven expression of pGFP–OGT (lane 6) is comparable to the commercially available positive control from Calf thymus (lane 7). DNA size markers of 100 bp (lane 8) and 1 kb DNA (lane 9) are shown
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Fig4: Overexpression of catalytically active GFP–OGT induces apoptosis. a CMV-driven expression of the mitochondrial form of OGT (pGFP–OGT) induces apoptosis as shown by the TUNEL assay. b A catalytically inactive form of OGT (pGFP–OGT-G) does not induce apoptosis. EcR-293 cells were used for transfections in a and b. c and d An insulinoma cell line, INS-1, was transfected with active OGT (pGFP–OGT). The TUNEL assay was used to reveal apoptotic cells. Representative focal planes near the bottom (c) and top (d) of INS-1 cell clusters are shown. Note the apoptotic cells on the outer layer of cells in the clusters. Black and red arrows denote cells scored as positive by the TUNEL assay. e–g EcR-293 cells were transfected with GFP–OGT (pIND/GFP–OGT) and induced with Ponasterone A (PoA) at indicated concentrations. Note the increasing amount of TUNEL-positive cells (black arrows) in g and h compared with the two negative controls e and h pIND/GFP–OGT, no PoA and no plasmid, 15 µM PoA, respectively. i–l Annexin V staining was used to identify apoptotic cells. i EcR-293 cells were treated with 3.0 μM of Ponasterone A to induce expression of pIND/GFP–OGT (green). j Same field stained with an anti-annexin V antibody to reveal apoptotic cells (red). Note near-complete colocalization of GFP–OGT expression and Annexin V staining (compare i and j). k Untreated cells did not stain positive for Annexin V. l As a positive control for apoptosis, 4 μ/ml camptothecin was added to uninduced cells. m DNA ladder assay. Genomic DNA from pIND/GFP–OGT transfected cells (lanes 1–5) was separated on an agarose gel to reveal DNA laddering. Uninduced cells are shown in lane 1. Increasing amounts of Ponasterone A were added at the following concentrations: 0.1 µM (lane 2), 0.5 µM (lane 3), 3.0 µM (lane 4), 15.0 (lane 5). The DNA laddering from CMV-driven expression of pGFP–OGT (lane 6) is comparable to the commercially available positive control from Calf thymus (lane 7). DNA size markers of 100 bp (lane 8) and 1 kb DNA (lane 9) are shown
Mentions: We then examined the effects of elevated O-GlcNAc levels on cell viability. The cell line EcR-293 (a kidney line) was chosen because it contains low levels of OGT (Lubas et al. 1997; Kreppel et al. 1997) and stably expresses ecdysone receptor which facilitates the ecdysone induction. Transient overexpression of catalytically active OGT was found to induce apoptosis as measured by the TUNEL assay (Fig. 4a, pGFP–OGT). The apoptosis was dependent upon catalytic activity since the transfection with the C-terminus deleted, catalytically defective, mutation of OGT did not result in TUNEL-positive cells (Fig. 4b, pGFP–OGT-G).Fig. 4

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