Introduction: Preeclampsia (PE) is a pregnancy disorder where trophoblast (TB) syncytialization is disrupted. One factor hindering syncytialization and increasing TB apoptosis is TNF-α. In endothelial cells, TNF-α mediates apoptosis through a bioactive sphingolipid, sphingosine 1-phosphate (S1P). Like TNF-α, S1P induces TB apoptosis and inhibits syncytialization. However, it is unknown if S1P mediates TNF-α effects in TBs. We propose that sphingosine kinase 1 (SK1), a S1P synthesizing enzyme, is a feasible target downstream of TNF-α to improve syncytialization. Hypothesis: SK1 expression will be increased in term placental chorionic explants and primary human trophoblast cultures treated with TNF-α. TNF-α will decrease syncytialization by activating SK1.
Methods: After 3-4 days of culture to allow original syncytium sloughing, explants were treated with TNF-α (0-10 ng/mL) and/or PF-543 (SK1 antagonist, 1 μM) up to 48 hrs. Re-syncytialization was assessed using E-cadherin immunofluorescence staining (n=3). SK1 mRNA expression in explants (qRT-PCR, n=4) was quantified after 0-48hrs. Primary term TBs were cultured with TNF-α (0-20 ng/mL) for 24 hrs. Syncytialization (hCG ELISA; n=3), cell viability (LDH release assay; n=3), and SK1 expression (immunofluorescence; n=5, qRT-PCR; n=5) were measured. Analysis was by one-way ANOVA.
Results: The greatest reduction in re-syncytialization in explants by TNF-α treatment occurred at 10ng/mL after 48 hrs. 1ng/mL TNF-α, the physiological level in PE, decreased re-syncytialization by 40.9±5.2% (p=0.05). Inhibiting SK1 alone decreased syncytialization by 24.69±1.29%. Blocking SK1 in the presence of TNF-α did not rescue re-syncytialization. No change in SK1 expression was observed. In primary TBs, TNF-α (1-20ng/mL) decreased hCG levels (p<0.01) without affecting LDH levels. SK1 expression did not change in TNF-α treated primary TBs.
Conclusion: TNF-α decreases syncytialization independently of SK1 activity without affecting viability or SK1 mRNA expression. However, endogenous SK1 activity is important. Thus, TNF-α and S1P independently impact syncytialization without synergistic effects.
Funding: CIHR, U of A: FOMD, FGSR, MatCH Program
Kirsten Webster– Undergraduate trainee, Department of Medical Microbiology and Immunology, University of Alberta
Saba Saadat– Undergraduate trainee, University of Alberta, Department of Obstetrics & Gynecology
Denise Hemmings– Associate Professor, Department of Obstetrics and Gynecology, Department of Medical Microbiology and Immunology, University of Alberta