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

<div><p>Background</p><p>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.</p><p>Objectives</p><p>We tested the hypothesis that SUMO-4 is expressed in the human placenta and demonstrates altered SUMOylation in pre-eclamptic pregnancies.</p><p>Methods</p><p>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.</p><p>Results</p><p>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.</p><p>Conclusions</p><p>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.</p></div

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).

<p>Treatment of Jar cells with 5mM of H₂O₂ (24h) induced <i>SUMO-4</i> and <i>UBC9</i> mRNA, as well as SUMO-4 protein expression. Treatment of BeWo cells also induced <i>SUMO-4</i>, <i>UBC9</i> and <i>SENP2</i> 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.</p

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

<p>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.</p

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

<p>H₂O₂ treatment for 24h induced <i>SUMO-2</i>, <i>SUMO-3</i>, <i>SUMO-4</i> and <i>SENP</i> 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.</p

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).

<p>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.</p

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

<p>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 <i>SUMO-4</i> 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.</p

Maternal IL-6 plasma levels and spleen weight in mice exposed to chronic LPS treatments.

<p>Chronic LPS (5 µg/Kg) treatment was performed daily from E11.5 until E15.5. (A) Maternal plasma was extracted 4 h after last LPS treatment on E15.5 (Vehicle n = 9; LPS n = 10). (B) Maternal splenic weight from animals exposed to chronic LPS treatment (Vehicle n = 9; LPS n = 10). Values are means±SEM. *P<0.05, **<i>p<</i>0.001 (one-way ANOVA followed by the Tukey’s post-hoc test).</p

Placental size and P-gp transport efficiency.

<p>Small, mid-range and larger placentas from each litter were grouped and averaged for placental (A) and fetal (B) weight and fetal to placental (F:P) weight ratio (C). (D) shows the relationship between individual placental weights and [³H]digoxin fetal accumulation at E15.5, for vehicle (r = −0.6657, n = 31 fetuses from 5 litters, <i>p</i><0.0001) and chronic LPS (r = −0.5099, n = 39 fetuses from 6 litters, <i>p</i><0.001) groups. (E) [³H]digoxin fetal accumulation from small, mid-range and larger placentas at E15.5 (vehicle n = 5; LPS n = 6). (A,B,C and E, repeated measures two-way ANOVA followed by Bonferonni’s test, <i>p</i><0.05; Pearson’s correlation test).</p

Rate of fetal death/reabsorption after acute LPS exposure.

<p>Rate of fetal death/reabsorption after acute LPS exposure.</p

Placental and maternal myocardial P-gp activity after acute sub-lethal LPS exposure:

<p>Fetal Units [³H]digoxin accumulation (4 fetal units/dam were randomly harvested and assayed) on E15.5 (A) (n = 5 dams/gp) and E17.5 (B) (4 h n = 5 dams/gp; 24 h n = 4 dams/gp); 4 or 24 h after acute LPS treatment. Maternal myocardial [³H]digoxin accumulation on E15.5 (C) and E17.5 (D) 4 or 24 h after acute LPS treatment. Values are means±SEM. *<i>p<</i>0.05 (one-way ANOVA followed by the Tukey’s post-hoc test on E15.5 and Kruskal-Wallis analysis of variance followed by Dunn’s test on E17.5).</p