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Hyperglycemia: GDNF-EGR1 pathway target renal epithelial cell migration and apoptosis in diabetic renal embryopathy.

Lin CY, Lin TY, Lee MC, Chen SC, Chang JS - PLoS ONE (2013)

Bottom Line: Preliminary study found EGR-1 persistently expressed with GDNF in hyperglycemic environment.EGR-1 siRNA also reduced GDNF/EGR-1-induced cRaf/MEK/ERK phosphorylation by 80%.Our findings reveal a novel mechanism of GDNF/MAPK/EGR-1 activation playing a critical role in HRPTE cell migration, apoptosis and fetal hyperglycemic nephropathy.

View Article: PubMed Central - PubMed

Affiliation: Clinical Immunology Center, China Medical University Hospital, Taichung, Taiwan. cylin@mail.cmuh.org.tw

ABSTRACT
Maternal hyperglycemia can inhibit morphogenesis of ureteric bud branching, Glial cell line-derived neurotrophilic factor (GDNF) is a key regulator of the initiation of ureteric branching. Early growth response gene-1 (EGR-1) is an immediate early gene. Preliminary study found EGR-1 persistently expressed with GDNF in hyperglycemic environment. To evaluate the potential relationship of hyperglycemia-GDNF-EGR-1 pathway, in vitro human renal proximal tubular epithelial (HRPTE) cells as target and in vivo streptozotocin-induced mice model were used. Our in vivo microarray, real time-PCR and confocal morphological observation confirmed apoptosis in hyperglycemia-induced fetal nephropathy via activation of the GDNF/MAPK/EGR-1 pathway at E12-E15. Detachment between ureteric branch and metanephrons, coupled with decreasing number and collapse of nephrons on Day 1 newborn mice indicate hyperglycemic environment suppress ureteric bud to invade metanephric rudiment. In vitro evidence proved that high glucose suppressed HRPTE cell migration and enhanced GDNF-EGR-1 pathway, inducing HRPTE cell apoptosis. Knockdown of EGR-1 by siRNA negated hyperglycemic suppressed GDNF-induced HRPTE cells. EGR-1 siRNA also reduced GDNF/EGR-1-induced cRaf/MEK/ERK phosphorylation by 80%. Our findings reveal a novel mechanism of GDNF/MAPK/EGR-1 activation playing a critical role in HRPTE cell migration, apoptosis and fetal hyperglycemic nephropathy.

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Effects of high glucose on GDNF-dependent phosphorylation of cRaf, MEK and ERK protein expression.HRPTE cells were treated with or without 20 mM glucose and/or GDNF (100 ng/ml) for 30 minutes, protein measured by Western blot. Phosphorylated cRaf, MEK and ERK significantly increased after co-treatment with high glucose and GDNF, the latter downregulated by pretreated with ERK inhibitor, PD98059 for 1 hour. Bands were quantified by densitometry, values normalized to that of β-actin protein in the same sample. Similar results emerged from three independent experiments. * vs lane 1 p<0.05, □ vs lane 2 p<0.05, # vs lane 4 p<0.05 (Fig 3a). EGR-1 mRNA expression was significantly increased in a time-dependent manner. Real-time quantitative polymerase chain reaction of EGR-1 mRNA expression in HRPTE cells, with time-dependent manner was noted in high glucose concentration (20 nM) with GDNF (100 ng/ml) for 12, 24 and 48 hours. Relative amount was compared with their own β-actin mRNA. Each point represents mean ± SD of three independent experiments. *p<0.05 vs control, #p<0.05 vs GDNF 12 hrs, □p<0.05 vs GDNF 24 hours, ★p<0.05 vs. high glucose 24 hrs, ⊚p<0.05 vs. GDNF 48 hrs, ☆p<0.05 vs. high glucose 48 hr.(Fig.3b). siRNA of EGR-1 downregulated high glucose and GDNF mediated cRaf/MEK/ERK phosphorylation.(Fig.3c). High glucose enhanced GDNF-induced DNA damage was determined by the comet assay. Higher concentration of glucose led to more damaged cells being stained (Fig.3d)
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pone-0056731-g003: Effects of high glucose on GDNF-dependent phosphorylation of cRaf, MEK and ERK protein expression.HRPTE cells were treated with or without 20 mM glucose and/or GDNF (100 ng/ml) for 30 minutes, protein measured by Western blot. Phosphorylated cRaf, MEK and ERK significantly increased after co-treatment with high glucose and GDNF, the latter downregulated by pretreated with ERK inhibitor, PD98059 for 1 hour. Bands were quantified by densitometry, values normalized to that of β-actin protein in the same sample. Similar results emerged from three independent experiments. * vs lane 1 p<0.05, □ vs lane 2 p<0.05, # vs lane 4 p<0.05 (Fig 3a). EGR-1 mRNA expression was significantly increased in a time-dependent manner. Real-time quantitative polymerase chain reaction of EGR-1 mRNA expression in HRPTE cells, with time-dependent manner was noted in high glucose concentration (20 nM) with GDNF (100 ng/ml) for 12, 24 and 48 hours. Relative amount was compared with their own β-actin mRNA. Each point represents mean ± SD of three independent experiments. *p<0.05 vs control, #p<0.05 vs GDNF 12 hrs, □p<0.05 vs GDNF 24 hours, ★p<0.05 vs. high glucose 24 hrs, ⊚p<0.05 vs. GDNF 48 hrs, ☆p<0.05 vs. high glucose 48 hr.(Fig.3b). siRNA of EGR-1 downregulated high glucose and GDNF mediated cRaf/MEK/ERK phosphorylation.(Fig.3c). High glucose enhanced GDNF-induced DNA damage was determined by the comet assay. Higher concentration of glucose led to more damaged cells being stained (Fig.3d)

Mentions: First we proved HRPTE cells can express EGR-1 by GDNF stimulation (data not shown). To ascertain the signal-transduction pathway induced by GDNF in HRPTE cells, we detected GDNF-dependent phosphorylation of MAPK signaling components by Western blot. Within the MAPK signaling cascade, MAPK kinase (MEK)-1 is a key protein that mediates signaling cascade from MAPK kinase kinase (MAPKKK) to both components of MAPK, ERK-1 and -2. Activation of ERK facilitates its translocation into nuclei, where it phosphorylates transcription activators like cRaf, MEK and ERK, all activated by GDNF (Fig. 3a, third line). Serine phosphorylation of cRaf, MEK, as well as tyrosine-threonine phosphorylation of ERK, was induced within 30 minutes, indicating the GDNF signal in cultured HRPTE cells as mediated by GDNF-MAPK signal pathways.


Hyperglycemia: GDNF-EGR1 pathway target renal epithelial cell migration and apoptosis in diabetic renal embryopathy.

Lin CY, Lin TY, Lee MC, Chen SC, Chang JS - PLoS ONE (2013)

Effects of high glucose on GDNF-dependent phosphorylation of cRaf, MEK and ERK protein expression.HRPTE cells were treated with or without 20 mM glucose and/or GDNF (100 ng/ml) for 30 minutes, protein measured by Western blot. Phosphorylated cRaf, MEK and ERK significantly increased after co-treatment with high glucose and GDNF, the latter downregulated by pretreated with ERK inhibitor, PD98059 for 1 hour. Bands were quantified by densitometry, values normalized to that of β-actin protein in the same sample. Similar results emerged from three independent experiments. * vs lane 1 p<0.05, □ vs lane 2 p<0.05, # vs lane 4 p<0.05 (Fig 3a). EGR-1 mRNA expression was significantly increased in a time-dependent manner. Real-time quantitative polymerase chain reaction of EGR-1 mRNA expression in HRPTE cells, with time-dependent manner was noted in high glucose concentration (20 nM) with GDNF (100 ng/ml) for 12, 24 and 48 hours. Relative amount was compared with their own β-actin mRNA. Each point represents mean ± SD of three independent experiments. *p<0.05 vs control, #p<0.05 vs GDNF 12 hrs, □p<0.05 vs GDNF 24 hours, ★p<0.05 vs. high glucose 24 hrs, ⊚p<0.05 vs. GDNF 48 hrs, ☆p<0.05 vs. high glucose 48 hr.(Fig.3b). siRNA of EGR-1 downregulated high glucose and GDNF mediated cRaf/MEK/ERK phosphorylation.(Fig.3c). High glucose enhanced GDNF-induced DNA damage was determined by the comet assay. Higher concentration of glucose led to more damaged cells being stained (Fig.3d)
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pone-0056731-g003: Effects of high glucose on GDNF-dependent phosphorylation of cRaf, MEK and ERK protein expression.HRPTE cells were treated with or without 20 mM glucose and/or GDNF (100 ng/ml) for 30 minutes, protein measured by Western blot. Phosphorylated cRaf, MEK and ERK significantly increased after co-treatment with high glucose and GDNF, the latter downregulated by pretreated with ERK inhibitor, PD98059 for 1 hour. Bands were quantified by densitometry, values normalized to that of β-actin protein in the same sample. Similar results emerged from three independent experiments. * vs lane 1 p<0.05, □ vs lane 2 p<0.05, # vs lane 4 p<0.05 (Fig 3a). EGR-1 mRNA expression was significantly increased in a time-dependent manner. Real-time quantitative polymerase chain reaction of EGR-1 mRNA expression in HRPTE cells, with time-dependent manner was noted in high glucose concentration (20 nM) with GDNF (100 ng/ml) for 12, 24 and 48 hours. Relative amount was compared with their own β-actin mRNA. Each point represents mean ± SD of three independent experiments. *p<0.05 vs control, #p<0.05 vs GDNF 12 hrs, □p<0.05 vs GDNF 24 hours, ★p<0.05 vs. high glucose 24 hrs, ⊚p<0.05 vs. GDNF 48 hrs, ☆p<0.05 vs. high glucose 48 hr.(Fig.3b). siRNA of EGR-1 downregulated high glucose and GDNF mediated cRaf/MEK/ERK phosphorylation.(Fig.3c). High glucose enhanced GDNF-induced DNA damage was determined by the comet assay. Higher concentration of glucose led to more damaged cells being stained (Fig.3d)
Mentions: First we proved HRPTE cells can express EGR-1 by GDNF stimulation (data not shown). To ascertain the signal-transduction pathway induced by GDNF in HRPTE cells, we detected GDNF-dependent phosphorylation of MAPK signaling components by Western blot. Within the MAPK signaling cascade, MAPK kinase (MEK)-1 is a key protein that mediates signaling cascade from MAPK kinase kinase (MAPKKK) to both components of MAPK, ERK-1 and -2. Activation of ERK facilitates its translocation into nuclei, where it phosphorylates transcription activators like cRaf, MEK and ERK, all activated by GDNF (Fig. 3a, third line). Serine phosphorylation of cRaf, MEK, as well as tyrosine-threonine phosphorylation of ERK, was induced within 30 minutes, indicating the GDNF signal in cultured HRPTE cells as mediated by GDNF-MAPK signal pathways.

Bottom Line: Preliminary study found EGR-1 persistently expressed with GDNF in hyperglycemic environment.EGR-1 siRNA also reduced GDNF/EGR-1-induced cRaf/MEK/ERK phosphorylation by 80%.Our findings reveal a novel mechanism of GDNF/MAPK/EGR-1 activation playing a critical role in HRPTE cell migration, apoptosis and fetal hyperglycemic nephropathy.

View Article: PubMed Central - PubMed

Affiliation: Clinical Immunology Center, China Medical University Hospital, Taichung, Taiwan. cylin@mail.cmuh.org.tw

ABSTRACT
Maternal hyperglycemia can inhibit morphogenesis of ureteric bud branching, Glial cell line-derived neurotrophilic factor (GDNF) is a key regulator of the initiation of ureteric branching. Early growth response gene-1 (EGR-1) is an immediate early gene. Preliminary study found EGR-1 persistently expressed with GDNF in hyperglycemic environment. To evaluate the potential relationship of hyperglycemia-GDNF-EGR-1 pathway, in vitro human renal proximal tubular epithelial (HRPTE) cells as target and in vivo streptozotocin-induced mice model were used. Our in vivo microarray, real time-PCR and confocal morphological observation confirmed apoptosis in hyperglycemia-induced fetal nephropathy via activation of the GDNF/MAPK/EGR-1 pathway at E12-E15. Detachment between ureteric branch and metanephrons, coupled with decreasing number and collapse of nephrons on Day 1 newborn mice indicate hyperglycemic environment suppress ureteric bud to invade metanephric rudiment. In vitro evidence proved that high glucose suppressed HRPTE cell migration and enhanced GDNF-EGR-1 pathway, inducing HRPTE cell apoptosis. Knockdown of EGR-1 by siRNA negated hyperglycemic suppressed GDNF-induced HRPTE cells. EGR-1 siRNA also reduced GDNF/EGR-1-induced cRaf/MEK/ERK phosphorylation by 80%. Our findings reveal a novel mechanism of GDNF/MAPK/EGR-1 activation playing a critical role in HRPTE cell migration, apoptosis and fetal hyperglycemic nephropathy.

Show MeSH
Related in: MedlinePlus