SUMO-4: A novel functional candidate in the human placental protein SUMOylation machinery

Abstract

Background: Small ubiquitin-like modifiers (SUMOs) conjugate to proteins post-translationally, thereby affecting target localization, activity and stability. Functional SUMO family members identified in the human placenta include SUMO-1 to SUMO-3, which are elevated in pre-eclampsia. Whether the fourth isoform, SUMO-4, plays a role in placental development and function remains unknown.

Objectives: We tested the hypothesis that SUMO-4 is expressed in the human placenta and demonstrates altered SUMOylation in pre-eclamptic pregnancies.

Methods: SUMO-4 mRNA (qRT-PCR) and protein (Western blot and immunohistochemistry) were measured in Jar cells, BeWo cells, first trimester placental villous explants and placental tissues across normal gestation and in pre-eclampsia. SUMO-4 expression in response to oxidative stress (H2O2: 0, 0.1, 1 and 5mM), as well as, hypoxia-reperfusion (O2: 1%, 8% and 20%) was measured. Lastly, SUMO-4 binding (covalently vs. non-covalently) to target proteins was investigated.

Results: SUMO-4 mRNA and protein were unchanged across gestation. SUMO-4 was present in the villous trophoblast layer throughout gestation. SUMO-4 mRNA expression and protein levels were increased ~2.2-fold and ~1.8-fold in pre-eclamptic placentas compared to age-matched controls, respectively (p<0.01). SUMO-4 mRNA and protein expression increased in Jars, BeWos and first trimester placental explants with 5mM H2O2 treatment, as well as with exposure to hypoxia-reperfusion. SUMO-1 to SUMO-3 did not show consistent trends across models. SUMO-4 hyper-SUMOylation was predominantly covalent in nature.

Conclusions: SUMO-4 is expressed in normal placental development. SUMO-4 expression was increased in pre-eclamptic placentas and in models of oxidative stress and hypoxic injury. These data suggests that SUMO-4 hyper-SUMOylation may be a potential post-translational mechanism in the stressed pre-eclamptic placenta.

Fig 1. SUMO-4 mRNA and protein levels across gestation and in pre-eclampsia (PE).

SUMO-4 (A) mRNA (n = 8–15) and (C) protein expression was unchanged across gestation (n = 3–6). Conversely, PE placentas showed elevated SUMO-4 (B) mRNA (**p<0.01, n = 4–6) and (D) protein (*p<0.05, n = 11–13) relative to pre-term age matched controls (PTC; set as 1). 1^st = first trimester, 2^nd = second trimester. Values represented as mean+SEM.

Fig 2. SUMO-4 immuno-staining in placentas from: (A) First-trimester (T1), (B) Second trimester (T2), (C) Term, (D) Preterm age-matched controls (PTC) and (E) Pre-eclampsia (PE).

SUMO-4 was present in the villous trophoblast layer across gestation. PE placentas showed strong immuno-staining throughout the trophoblast and stroma compared to PTC. Images in left panels are presented at 40x magnification and boxed areas demonstrate images in right panels presented at 100x magnification.

Fig 3. H₂O₂ treatment of placental (A & B) Jar and (C & D) BeWo cells induces SUMOylation at the (A & C) mRNA and (B & D) protein level (representative Western blots).

Treatment of Jar cells with 5mM of H₂O₂ (24h) induced SUMO-4 and UBC9 mRNA, as well as SUMO-4 protein expression. Treatment of BeWo cells also induced SUMO-4, UBC9 and SENP2 mRNA levels and SUMO-4 protein expression. Values represented as mean+SEM, n = 3–4, Significance ***p<0.001, **p<0.01, *p<0.05. CON = control.

Fig 4. H₂O₂ treatment of first trimester placental explants induces SUMOylation at the (A) mRNA and (B) protein level.

H₂O₂ treatment for 24h induced SUMO-2, SUMO-3, SUMO-4 and SENP mRNA levels (compared to vehicle control). (B) Representative Western blots shown of SUMO-2/3 and SUMO-4. SUMO-2/3 protein remains unchanged with treatment, however SUMO-4 protein expression is increased. Values represented as mean+SEM; n = 5, Significance ***p<0.001. CON = control.

Fig 5. Hypoxia-reperfusion stress in first trimester placental explants induced SUMOylation of SUMO-4 at the (A) mRNA and (B) protein level.

Explants were treated with hypoxia (1%), hyperoxia (20%) or hypoxia-reperfusion (cycled between 1% and 20% for 1h intervals) for a total of 8h. (A) Hypoxia-reperfusion induced SUMO-4 mRNA expression relative to vehicle control (normoxic 8% O₂). (B) Representative images of SUMO-4 Western blotting. SUMO-4 protein conjugation is increased with oxygen stress. Values represented as mean+SEM; n = 5, Significance *p<0.05.

Fig 6. SUMO-4 covalently interacts with its targets in BeWO cells.

BeWo cells were stressed by fetal bovine serum (FBS) depravation (24h) to induce global SUMOylation. Elevated levels of SUMO-4 conjugated proteins were observed in high SDS buffer, but not in the low SDS buffer preparations, suggesting that SUMO-4 protein interactions are covalent in nature.