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Cardiac transcription factor Nkx2.5 is downregulated under excessive O-GlcNAcylation condition.

Kim HS, Woo JS, Joo HJ, Moon WK - PLoS ONE (2012)

Bottom Line: We investigated whether Nkx2.5 protein, a cardiac transcription factor, is regulated by O-GlcNAc.Recombinant Nkx2.5 (myc-Nkx2.5) proteins were reduced by treatment with the O-GlcNAcase inhibitors STZ and O-(2-acetamido-2-deoxy-D-glucopyroanosylidene)-amino-N-phenylcarbamate; PUGNAC) as well as the overexpression of recombinant O-GlcNAc transferase (OGT-flag).In addition, Nkx2.5 proteins were reduced in the heart tissue of streptozotocin (STZ)-induced diabetic mice and O-GlcNAc modification of Nkx2.5 protein increased in diabetic heart tissue compared with non-diabetic heart.

View Article: PubMed Central - PubMed

Affiliation: The Institute of Radiation Medicine, Medical Research Center, Seoul National University, Jongno-gu, Seoul, Korea.

ABSTRACT
Post-translational modification of proteins with O-linked N-acetylglucosamine (O-GlcNAc) is linked the development of diabetic cardiomyopathy. We investigated whether Nkx2.5 protein, a cardiac transcription factor, is regulated by O-GlcNAc. Recombinant Nkx2.5 (myc-Nkx2.5) proteins were reduced by treatment with the O-GlcNAcase inhibitors STZ and O-(2-acetamido-2-deoxy-D-glucopyroanosylidene)-amino-N-phenylcarbamate; PUGNAC) as well as the overexpression of recombinant O-GlcNAc transferase (OGT-flag). Co-immunoprecipitation analysis revealed that myc-Nkx2.5 and OGT-flag proteins interacted and myc-Nkx2.5 proteins were modified by O-GlcNAc. In addition, Nkx2.5 proteins were reduced in the heart tissue of streptozotocin (STZ)-induced diabetic mice and O-GlcNAc modification of Nkx2.5 protein increased in diabetic heart tissue compared with non-diabetic heart. Thus, excessive O-GlcNAcylation causes downregulation of Nkx2.5, which may be an underlying contributing factor for the development of diabetic cardiomyopathy.

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Related in: MedlinePlus

Reduction of myc-Nkx2.5 protein in cells treated with O-GlcNAcase inhibotirs.(A) Immunoblotting of myc-Nkx2.5 and O-GlycNAcylated proteins in myc-Nkx2.5-transfected HEK293 cells in the presence or absence of 3 mM streptozotocine (STZ). (B) Graphs showed relative O-GlcNAc (left) and myc-Nkx2.5 (right) levels normalized to the tubulin levels in STZ-treated and untreated cells. O-GlcNAcylation levels were compared in untreated cells vs STZ-treated cells and untreated myc-Nkx2.5 cells vs STZ-treated myc-Nkx2.5 cells. The myc-Nkx2.5 protein levels were compared in untreated myc-Nkx2.5 cells vs STZ-treated myc-Nkx2.5 cells. All values are presented as the mean±standard error of three independent experiments. *p<0.05. (C) Immunoblotting analysis of myc-Nkx2.5 and O-GlycNAcylated proteins in myc-Nkx2.5-transduced HEK293 cells in the presence or absence of O-(2-acetamido-2-deoxy-D-glucopyroanosylidene)-amino-N-phenylcarbamate (10 or 100 µM PUGNAC). (D) Graphs showed relative O-GlcNAc (left) and myc-Nkx2.5 (right) levels normalized to the tubulin levels in PUGNAC-treated and untreated cells. The treatment with STZ and PUGNAC significantly increased the O-GlcNAcylation of intracellular proteins, but decreased the myc-Nkx2.5 protein significantly. O-GlcNAcylation levels were compared in untreated myc-Nkx2.5 cells vs PUGNAC-treated myc-Nkx2.5 cells. The myc-Nkx2.5 protein levels were compared in untreated myc-Nkx2.5 cells vs PUGNAC treated myc-Nkx2.5 cells. All values are presented as the mean±standard error of three independent experiments. *p<0.05.
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pone-0038053-g002: Reduction of myc-Nkx2.5 protein in cells treated with O-GlcNAcase inhibotirs.(A) Immunoblotting of myc-Nkx2.5 and O-GlycNAcylated proteins in myc-Nkx2.5-transfected HEK293 cells in the presence or absence of 3 mM streptozotocine (STZ). (B) Graphs showed relative O-GlcNAc (left) and myc-Nkx2.5 (right) levels normalized to the tubulin levels in STZ-treated and untreated cells. O-GlcNAcylation levels were compared in untreated cells vs STZ-treated cells and untreated myc-Nkx2.5 cells vs STZ-treated myc-Nkx2.5 cells. The myc-Nkx2.5 protein levels were compared in untreated myc-Nkx2.5 cells vs STZ-treated myc-Nkx2.5 cells. All values are presented as the mean±standard error of three independent experiments. *p<0.05. (C) Immunoblotting analysis of myc-Nkx2.5 and O-GlycNAcylated proteins in myc-Nkx2.5-transduced HEK293 cells in the presence or absence of O-(2-acetamido-2-deoxy-D-glucopyroanosylidene)-amino-N-phenylcarbamate (10 or 100 µM PUGNAC). (D) Graphs showed relative O-GlcNAc (left) and myc-Nkx2.5 (right) levels normalized to the tubulin levels in PUGNAC-treated and untreated cells. The treatment with STZ and PUGNAC significantly increased the O-GlcNAcylation of intracellular proteins, but decreased the myc-Nkx2.5 protein significantly. O-GlcNAcylation levels were compared in untreated myc-Nkx2.5 cells vs PUGNAC-treated myc-Nkx2.5 cells. The myc-Nkx2.5 protein levels were compared in untreated myc-Nkx2.5 cells vs PUGNAC treated myc-Nkx2.5 cells. All values are presented as the mean±standard error of three independent experiments. *p<0.05.

Mentions: We investigated the effect of the O-GlcNAcase inhibitors, STZ and PUGNAC, on myc-Nkx2.5 protein levels in HEK293 cells. Immunoblotting analysis revealed that treatment with STZ (3 mM) for 24 hours remarkably increased the level of general protein O-GlcNAcylation, but significantly decreased the expression level of myc-Nkx2.5 in cells (Fig. 2A and B, p<0.05). We assessed the expression level of myc-Nkx2.5 in the presence of STZ by immunofluorescence. Myc-Nkx2.5 was detected in the nuclei of cells and at reduced levels in STZ-treated cells (data not shown). The other O-GlcNAcase inhibitor, PUGNAc increased the O-GlcNAcylation dose-dependently but also significantly decreased the expression of myc-Nkx2.5 (Fig. 2C and D, p<0.05).


Cardiac transcription factor Nkx2.5 is downregulated under excessive O-GlcNAcylation condition.

Kim HS, Woo JS, Joo HJ, Moon WK - PLoS ONE (2012)

Reduction of myc-Nkx2.5 protein in cells treated with O-GlcNAcase inhibotirs.(A) Immunoblotting of myc-Nkx2.5 and O-GlycNAcylated proteins in myc-Nkx2.5-transfected HEK293 cells in the presence or absence of 3 mM streptozotocine (STZ). (B) Graphs showed relative O-GlcNAc (left) and myc-Nkx2.5 (right) levels normalized to the tubulin levels in STZ-treated and untreated cells. O-GlcNAcylation levels were compared in untreated cells vs STZ-treated cells and untreated myc-Nkx2.5 cells vs STZ-treated myc-Nkx2.5 cells. The myc-Nkx2.5 protein levels were compared in untreated myc-Nkx2.5 cells vs STZ-treated myc-Nkx2.5 cells. All values are presented as the mean±standard error of three independent experiments. *p<0.05. (C) Immunoblotting analysis of myc-Nkx2.5 and O-GlycNAcylated proteins in myc-Nkx2.5-transduced HEK293 cells in the presence or absence of O-(2-acetamido-2-deoxy-D-glucopyroanosylidene)-amino-N-phenylcarbamate (10 or 100 µM PUGNAC). (D) Graphs showed relative O-GlcNAc (left) and myc-Nkx2.5 (right) levels normalized to the tubulin levels in PUGNAC-treated and untreated cells. The treatment with STZ and PUGNAC significantly increased the O-GlcNAcylation of intracellular proteins, but decreased the myc-Nkx2.5 protein significantly. O-GlcNAcylation levels were compared in untreated myc-Nkx2.5 cells vs PUGNAC-treated myc-Nkx2.5 cells. The myc-Nkx2.5 protein levels were compared in untreated myc-Nkx2.5 cells vs PUGNAC treated myc-Nkx2.5 cells. All values are presented as the mean±standard error of three independent experiments. *p<0.05.
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Related In: Results  -  Collection

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pone-0038053-g002: Reduction of myc-Nkx2.5 protein in cells treated with O-GlcNAcase inhibotirs.(A) Immunoblotting of myc-Nkx2.5 and O-GlycNAcylated proteins in myc-Nkx2.5-transfected HEK293 cells in the presence or absence of 3 mM streptozotocine (STZ). (B) Graphs showed relative O-GlcNAc (left) and myc-Nkx2.5 (right) levels normalized to the tubulin levels in STZ-treated and untreated cells. O-GlcNAcylation levels were compared in untreated cells vs STZ-treated cells and untreated myc-Nkx2.5 cells vs STZ-treated myc-Nkx2.5 cells. The myc-Nkx2.5 protein levels were compared in untreated myc-Nkx2.5 cells vs STZ-treated myc-Nkx2.5 cells. All values are presented as the mean±standard error of three independent experiments. *p<0.05. (C) Immunoblotting analysis of myc-Nkx2.5 and O-GlycNAcylated proteins in myc-Nkx2.5-transduced HEK293 cells in the presence or absence of O-(2-acetamido-2-deoxy-D-glucopyroanosylidene)-amino-N-phenylcarbamate (10 or 100 µM PUGNAC). (D) Graphs showed relative O-GlcNAc (left) and myc-Nkx2.5 (right) levels normalized to the tubulin levels in PUGNAC-treated and untreated cells. The treatment with STZ and PUGNAC significantly increased the O-GlcNAcylation of intracellular proteins, but decreased the myc-Nkx2.5 protein significantly. O-GlcNAcylation levels were compared in untreated myc-Nkx2.5 cells vs PUGNAC-treated myc-Nkx2.5 cells. The myc-Nkx2.5 protein levels were compared in untreated myc-Nkx2.5 cells vs PUGNAC treated myc-Nkx2.5 cells. All values are presented as the mean±standard error of three independent experiments. *p<0.05.
Mentions: We investigated the effect of the O-GlcNAcase inhibitors, STZ and PUGNAC, on myc-Nkx2.5 protein levels in HEK293 cells. Immunoblotting analysis revealed that treatment with STZ (3 mM) for 24 hours remarkably increased the level of general protein O-GlcNAcylation, but significantly decreased the expression level of myc-Nkx2.5 in cells (Fig. 2A and B, p<0.05). We assessed the expression level of myc-Nkx2.5 in the presence of STZ by immunofluorescence. Myc-Nkx2.5 was detected in the nuclei of cells and at reduced levels in STZ-treated cells (data not shown). The other O-GlcNAcase inhibitor, PUGNAc increased the O-GlcNAcylation dose-dependently but also significantly decreased the expression of myc-Nkx2.5 (Fig. 2C and D, p<0.05).

Bottom Line: We investigated whether Nkx2.5 protein, a cardiac transcription factor, is regulated by O-GlcNAc.Recombinant Nkx2.5 (myc-Nkx2.5) proteins were reduced by treatment with the O-GlcNAcase inhibitors STZ and O-(2-acetamido-2-deoxy-D-glucopyroanosylidene)-amino-N-phenylcarbamate; PUGNAC) as well as the overexpression of recombinant O-GlcNAc transferase (OGT-flag).In addition, Nkx2.5 proteins were reduced in the heart tissue of streptozotocin (STZ)-induced diabetic mice and O-GlcNAc modification of Nkx2.5 protein increased in diabetic heart tissue compared with non-diabetic heart.

View Article: PubMed Central - PubMed

Affiliation: The Institute of Radiation Medicine, Medical Research Center, Seoul National University, Jongno-gu, Seoul, Korea.

ABSTRACT
Post-translational modification of proteins with O-linked N-acetylglucosamine (O-GlcNAc) is linked the development of diabetic cardiomyopathy. We investigated whether Nkx2.5 protein, a cardiac transcription factor, is regulated by O-GlcNAc. Recombinant Nkx2.5 (myc-Nkx2.5) proteins were reduced by treatment with the O-GlcNAcase inhibitors STZ and O-(2-acetamido-2-deoxy-D-glucopyroanosylidene)-amino-N-phenylcarbamate; PUGNAC) as well as the overexpression of recombinant O-GlcNAc transferase (OGT-flag). Co-immunoprecipitation analysis revealed that myc-Nkx2.5 and OGT-flag proteins interacted and myc-Nkx2.5 proteins were modified by O-GlcNAc. In addition, Nkx2.5 proteins were reduced in the heart tissue of streptozotocin (STZ)-induced diabetic mice and O-GlcNAc modification of Nkx2.5 protein increased in diabetic heart tissue compared with non-diabetic heart. Thus, excessive O-GlcNAcylation causes downregulation of Nkx2.5, which may be an underlying contributing factor for the development of diabetic cardiomyopathy.

Show MeSH
Related in: MedlinePlus