Jayonta Bhattacharjee1, Sruthi Alahari2, Julien Sallais3, Andrea Tagliaferro4, Martin Post5, Isabella Caniggia6. 1. Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada. Electronic address: jayontabhatta@gmail.com. 2. Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada; Department of Physiology, University of Toronto, Toronto, Ontario, Canada. Electronic address: alahari@lunenfeld.ca. 3. Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada; Institute of Medical Sciences, University of Toronto, Toronto, Ontario, Canada. Electronic address: sallais@lunenfeld.ca. 4. Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada. Electronic address: tagliaferro@lunenfeld.ca. 5. Department of Physiology, University of Toronto, Toronto, Ontario, Canada; Institute of Medical Sciences, University of Toronto, Toronto, Ontario, Canada; Department of Pediatrics, University of Toronto, Toronto, Ontario, Canada; The Hospital for Sick Children, Toronto, Ontario, Canada. Electronic address: martin.post@sickkids.ca. 6. Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada; Department of Obstetrics and Gynecology, University of Toronto, Toronto, Ontario, Canada; Department of Physiology, University of Toronto, Toronto, Ontario, Canada; Institute of Medical Sciences, University of Toronto, Toronto, Ontario, Canada. Electronic address: caniggia@lunenfeld.ca.
Abstract
INTRODUCTION: Hypoxia-inducible factor 1A (HIF1A) stability is tightly regulated by hydroxylation and ubiquitination. Emerging evidence indicates that HIF1A is also controlled by the interplay between SUMO-specific ligases, which execute protein SUMOylation, and Sentrin/SUMO-specific proteases that de-SUMOylate target proteins. Given the significance of HIF1A in the human placenta, we investigated whether placental HIF1A is subject to SUMOylation in physiological and pathological conditions. METHODS: Placentae were obtained from normal and pregnancies complicated by preeclampsia. Human choriocarcinoma JEG3 cells were maintained at either 21% or 3% oxygen or exposed to sodium nitroprusside (SNP). Cells were transfected with SUMO2/3 constructs with and without Mg132, a proteasome inhibitor. Expression, distribution and associations of SUMO/SENPs and HIF1A were evaluated by Western blotting, immunohistochemistry and co-immunoprecipitation. RESULTS: HIF1A-SUMO2/3 associations peaked at 9-10 weeks, while its deSUMOylation by SENP3 was greatest at 10-12 weeks. In E-PE, HIF1A deSUMOylation by SENP3 was significantly elevated, while HIF1A-SUMO2/3 associations remained constant. In vitro, overexpression of SUMO2/3 de-stabilized HIF1A in hypoxia, and abrogated HIF1A expression following Mg132 treatment in normoxia. Hypoxia and SNP treatments promoted SENP3 nuclear redistribution from nucleoli to the nucleoplasm where it associates with HIF1A. CONCLUSION: During early placental development, SUMOylation events control HIF1A stability in an oxygen-dependent manner. In E-PE, enhanced deSUMOylation of HIF1A by SENP3 may in part contribute to increased HIF1A activity and stability found in this pathology.
INTRODUCTION:Hypoxia-inducible factor 1A (HIF1A) stability is tightly regulated by hydroxylation and ubiquitination. Emerging evidence indicates that HIF1A is also controlled by the interplay between SUMO-specific ligases, which execute protein SUMOylation, and Sentrin/SUMO-specific proteases that de-SUMOylate target proteins. Given the significance of HIF1A in the human placenta, we investigated whether placental HIF1A is subject to SUMOylation in physiological and pathological conditions. METHODS: Placentae were obtained from normal and pregnancies complicated by preeclampsia. Humanchoriocarcinoma JEG3 cells were maintained at either 21% or 3% oxygen or exposed to sodium nitroprusside (SNP). Cells were transfected with SUMO2/3 constructs with and without Mg132, a proteasome inhibitor. Expression, distribution and associations of SUMO/SENPs and HIF1A were evaluated by Western blotting, immunohistochemistry and co-immunoprecipitation. RESULTS:HIF1A-SUMO2/3 associations peaked at 9-10 weeks, while its deSUMOylation by SENP3 was greatest at 10-12 weeks. In E-PE, HIF1A deSUMOylation by SENP3 was significantly elevated, while HIF1A-SUMO2/3 associations remained constant. In vitro, overexpression of SUMO2/3 de-stabilized HIF1A in hypoxia, and abrogated HIF1A expression following Mg132 treatment in normoxia. Hypoxia and SNP treatments promoted SENP3 nuclear redistribution from nucleoli to the nucleoplasm where it associates with HIF1A. CONCLUSION: During early placental development, SUMOylation events control HIF1A stability in an oxygen-dependent manner. In E-PE, enhanced deSUMOylation of HIF1A by SENP3 may in part contribute to increased HIF1A activity and stability found in this pathology.