ESPE Abstracts

Background and aim: PTEN hamartoma tumor syndrome (PHTS) is caused by germline mutation in the phosphatase and tensin homolog (PTEN) gene. PTEN is a tumor suppressor gene and antagonist of the growth and survival signalling Phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of Rapamycin (mTOR)- cascade. Patients with PHTS, amongst other symptoms, develop lipomas, for which the underlying mechanism is not completely understood. To investigate the role of PTEN in lipoma formation, we previously performed a PTEN knockdown (KD) in adipose progenitor cells (APCs) and observed enhanced proliferation and adipocyte differentiation. RNA sequencing showed downregulation of phospholipid scramblase 4 (PLSCR4) in PTEN KD APCs. While mice with knockout of the closely related PLSCR3 exhibit an adipose tissue expansion, the role of PLSCR4 in adipose tissue is unknown. We asked whether PLSCR4 downregulation is involved in adipose tissue expansion by modulating PI3K/AKT signalling.

Methods: We compared PLSCR4 expression in lipoma tissue from lipoma Pten/Retinoblastoma (Rb) double knockout (DKO) mice in comparison to epididymal WAT (epiWAT) and inguinal WAT (ingWAT) from control mice. PLSCR4 expression during adipogenesis was analysed in human LipPD1 cells. Further we established siRNA mediated knockdown and plasmid mediated overexpression of PLSCR4 in APCs. After transfection we analysed proliferation and adipocyte differentiation. For influence of PLSCR4 on PI3K/AKT/m-TOR signalling we analysed phosphatidylinositol-(3,4,5)-trisphosphate (PIP3) levels and activation of AKT.

Results: We could observe that PLSCR4 expression in lipoma tissue from Pten/ Rb DKO mice was diminished in comparison to epiWAT by 67.7% ± 15.4% (n = 7, P< 0.05) and ingWAT by 58.4% ± 23.5% (n = 7, P< 0.05). During adipogenesis PLSCR4 was significantly downregulated in LipPD1 cells P< 0.001 (n = 4). We saw an influence of PLSCR4 on adipogenesis but not on proliferation. The differentiated cell fraction in PLSCR4 knockdown cells was increased by 44.1% ± 10.5% (n = 7, P< 0.05). These results could be verified by PLSCR4 overexpression, which led to a decrease of differentiated adipocytes by 25.3% ± 7.3% (n = 3, P< 0.05). PLSCR4 KD APCs showed increased PIP3 levels by 94.4% ± 24.5% in PLSCR4 KD compared to control APCs (n = 3, P=0.0611). In line with this, AKT phosphorylation was increased in PLSCR4 KD (n = 5, P< 0.05).

Conclusion: PLSCR4 downregulation enhances adipogenesis in APCs via activation of AKT. This might contribute to adipose tissue overgrowth as observed in PHTS patients.