Crosstalk between JNK and SUMO Signaling Pathways: deSUMOylation Is Protective against H2O2-Induced Cell Injury

Background: Oxidative stress is a key feature in the pathogenesis of several neurological disorders. Following oxidative stress stimuli a wide range of pathways are activated and contribute to cellular death. The mechanism that couples c-Jun N-terminal kinase (JNK) signaling, a key pathway in stress conditions, to the small ubiquitin-related modifier (SUMO), an emerging protein in the field, is largely unknown. Methodology/Principal Findings: With this study we investigated if SUMOylation participates in the regulation of JNK activation as well as cellular death in a model of H 2O 2 induced-oxidative stress. Our data show that H 2O 2 modulates JNK activation and induces cellular death in neuroblastoma SH-SY5Y cells. Inhibition of JNK’s action with the D-JNKI1 peptide rescued cells from death. Following H2O2, SUMO-1 over-expression increased phosphorylation of JNK and exacerbated cell death, although only in conditions of mild oxidative stress. Furthermore inhibition of SUMOylation, following transfection with SENP1, interfered with JNK activation and rescued cells from H 2O 2 induced death. Importantly, in our model, direct interaction between these proteins can occur. Conclusions/Significance: Taken together our results show that SUMOylation may significantly contribute to modulation o

P-JNK and SUMO-1 interaction following stimulation with H₂O₂ for 3 h.

<p><b>A</b>) Immunoprecipitation (IP) experiment. Untransfected (Control cells) or transfected (SUMO-1+UBC₉ or SUMO-1DGG+UBC₉) cells were stimulated with H₂O₂ (50 mM) and P-JNK was immunoprecipitated usind P-JNK antibody. Representative blot showing immunoprecipetated SUMOylated protein blotted with an anti-GFP antibody since SUMO constructs used were YFP tagged. Membranes were stripped and blotted with an anti-P-JNK antibody as control for the IP. Rabbit-IgG negative control antibody was used in the first lane (Ctr Ab). Bands below 50 kDa are IgG bands (50 and 25 kDa) and the others are unspecific signal of GFP antibody. <b>B</b>) Stimulation of untransfected SH-SY5Y cells with 50 or 75 mM of H₂O₂ did not affect the SUMOylated protein profile (from 250 to 37 kDa). <b>C</b>) Immunostaining shows that SUMO-1 (green) and p-JNK (red) colocalize in the nucleus following stimulation with H₂O₂ (50 mM) for 3 hours. Nuclei were stained with the nuclear marker Hoechst.</p

Effect of SENP1 over-expression on JNK activation and cell viability after overnight or 3 h stimulation with H₂O₂.

<p><b>A</b>) Representative Western blot showing decrease in P-JNK protein levels in cells overexpressing SENP1 following overnight stimulation with H₂O₂. <b>B</b>) Densitometric analysis reveals that P-JNK/JNK ratio is significantly decreased in transfected (strapped lines bars = SENP1) cells compared to untransfected cells (white bars = Control). Data are from four experiments and values are expressed as mean±SEM. <b>C</b>) Transfected cells (strapped lines bars = SENP1) are more resistant to H₂O₂-induced death compared to untransfected (white bars = Control) ones, as assessed by MTT colorimetric assaying cell viability. Data are presented as fold increase and are from five experiments. **<i>P</i><0.01 vs control ^#<i>P</i><0.05 vs cells overexpressing SENP1. <b>D</b>) Representative Western blot showing decrease in P-JNK protein levels in cells overexpressing SENP1 and following stimulation with H₂O₂ for 3 h. <b>E</b>) Densitometric analysis reveals a significant decrease of P-JNK/JNK ratio in transfected cells (strapped lines bars = SENP1) compared to untransfected (white bars = Control). <b>F</b>) Transfected cells (strapped lines bars = SENP1) are more resistant to H₂O₂-induced death compared to untransfected (white bars = Control) ones, as assessed by MTT colorimetric assaying cell viability. Data are presented as fold increase and values are from five experiments. *<i>P</i><0.05, **<i>P</i><0.01 vs control, ^#<i>P</i><0.05 control vs SENP1.</p

Successful transfection of SH-SY5Y cells with SUMO-1+UBC₉ and SENP plasmids.

<p>Representative Western Blot showing transfection, in triplicate per condition, of SH-SY5Y cells with SUMO-1+UBC₉ which leads to an increase in SUMOylation, while transfection of SH-SY5Y cells with SENP1 leads to complete abolishment of SUMOylation.</p

Transcription factor c-Jun activation and cell viability in SH-SY5Y cell line following stimulation with H₂O₂ for 3 h.

<p><b>A</b>) Representative Western blot showing increase of P-c-Jun protein levels following H₂O₂ (50 and 75 mM) stimulation. Application of D-JNKI1, 30 min prior to H₂O₂ stimulation decreased P-c-Jun activation. Densitometric analysis revealed a fold increase ∼1.60 for 50 and ∼1.80 for 75 mM of P-c-Jun/c-Jun ratio in cells stimulated with H₂O₂ compared to control cells. D-JNKI1 inhibited activation of c-Jun by 0.57 fold for 50 and ∼1.00 for 75 mM. Data are from five experiments and values are expressed as mean±SEM. *<i>P</i><0.05, **<i>P</i><0.01 vs control and ^#<i>P</i><0.05 vs H₂O₂ alone <b>B</b>) Cell viability assay was performed employing MTT colorimetric method. Application of D-JNKI1 rescued cells from H₂O₂ induced cell death. Data are presented as fold increase and are from five experiments. *<i>P</i><0.05 vs control, ^#<i>P</i><0.05 vs H₂O₂ alone.</p

Effect of SUMO-1 over-expression on JNK activation and cell viability after overnight or 3 h stimulation with H₂O₂.

<p><b>A</b>) Representative Western blot showing increase in P-JNK protein levels in cells overexpressing SUMO-1+UBC₉ following overnight stimulation with H₂O₂. <b>B</b>) Densitometric analysis reveals that P-JNK/JNK ratio is not significantly increased in untransfected (white bars = Control) cells versus transfected (black bars = SUMO-1+UBC₉) ones. Data are from four experiments and values are expressed as mean±SEM. **<i>P</i><0.01 vs own control. <b>C</b>) Overnight H₂O₂ stimulation of transfected (black bars = SUMO-1+UBC₉) cells did not lead to significant changes in cell death in comparison to untransfected (white bars = Control) ones, as assessed by MTT colorimetric assaying cell viability. Data are presented as fold increase and values are from five experiments. *<i>P</i><0.05, **<i>P</i><0.01 vs Control or H₂O₂ alone. <b>D</b>) Representative Western blot showing increase in P-JNK protein levels in cells overexpressing SUMO-1+UBC₉ following stimulation with H₂O₂ for 3 h. <b>E</b>) Densitometric analysis reveals a significant increase of P-JNK/JNK ratio in transfected cells (black bars = SUMO-1+UBC9) compared to untransfected (white bars = Control) ones. Data are from five experiments and values are expressed as mean±SEM. *<i>P</i><0.05 **<i>P</i><0.01 vs Control or H₂O₂ alone and ^#<i>P</i><0,05 Control versus SUMO-1+UBC₉. <b>F</b>) Transfected cells (black bars = SUMO-1+UBC9) are more sensitive to H₂O₂-induced death compared to untransfected (white bars = Control) ones, as assessed by MTT colorimetric assaying cell viability. 20 mM of D-JNKI was applied 30 minutes before oxidative stress. D-JNKI1 rescued cells from H₂O₂- induced death. Data are presented as fold increase and values are from five experiments. **<i>P</i><0.01 versus control, ^#<i>P</i><0.05 H₂O₂ vs H₂O₂+D-JNKI1.</p