The biphasic redox sensing of SENP3 accounts for the HIF-1 transcriptional activity shift by oxidative stress.
OBJECTIVE
To investigate the mechanisms underlying the biphasic redox regulation of hypoxia-inducible factor-1 (HIF-1) transcriptional activity under different levels of oxidative stress caused by reactive oxidative species (ROS).


METHODS
HeLa cells were exposed to different concentrations of H(2)O(2) as a simple model for mild and severe oxidative stress. Luciferase reporter assay and/or quantitative real-time PCR were used to investigate the transcriptional activity. Immunoblot was used to detect protein expression. Chromatin immunoprecipitation assay was used to detect HIF-1/DNA binding. The interaction of p300 with HIF-1α or with SENP3, and the SUMO2/3 conjugation states of p300 were examined by coimmunoprecipitation.


RESULTS
HIF-1 transcriptional activity in HeLa cells was enhanced by low doses (0.05-0.5 mmol/L) of H(2)O(2), but suppressed by high doses (0.75-8.0 mmol/L) of H(2)O(2). The amount of co-activator p300 bound to HIF-1α in HeLa cells was increased under mild oxidative stress, but decreased under severe oxidative stress. The ROS levels differentially modified cysteines 243 and 532 in the cysteine protease SENP3, regulating the interaction of SENP3 with p300 to cause different SUMOylation of p300, thus shifting HIF-1 transcriptional activity.


CONCLUSIONS
The shift of HIF-1 transactivation by ROS is correlated with and dependent on the biphasic redox sensing of SENP3 that leads to the differential SENP3/p300 interaction and the consequent fluctuation in the p300 SUMOylation status.
Department of Biochemistry and Molecular Cell Biology, Key Laboratory of the Shanghai Science and Technology Commission for Cancer Microenvironment and Inflammation, Institutes of Medical Sciences, Shanghai Jiao Tong University School of Medicine, China.
Ying
Y
Wang
Jie
J
Yang
Kai
K
Yang
Hui
H
Cang
Xin-zhi
XZ
Huang
Hui
H
Li
Jing
J
Yi
D003545:Cysteine / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D051793:Hypoxia-Inducible Factor 1 / Q000235:genetics* / Q000378:metabolism; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D017382:Reactive Oxygen Species / Q000378:metabolism*; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism; D015533:Transcriptional Activation; D014452:Ubiquitins / Q000378:metabolism; D050880:p300-CBP Transcription Factors / Q000378:metabolism
D051793:Hypoxia-Inducible Factor 1; D017382:Reactive Oxygen Species; C117909:SUMO2 protein, human; C492189:SUMO3 protein, human; D025841:Small Ubiquitin-Related Modifier Proteins; D014452:Ubiquitins; D006861:Hydrogen Peroxide; D050880:p300-CBP Transcription Factors; D003546:Cysteine Endopeptidases; C499718:SENP3 protein, human; D003545:Cysteine