ESPE Abstracts (2018) 89 FC6.3

Role of PTEN in the Proliferation and Differentiation of Preadipocytes

Anna Kirsteina, Franziska Kässnerb, Gordian L. Schmidc, Norman Händeld, Melanie Penkea,e, Wieland Kiessa,e & Antje Gartena,f


aCenter for Pediatric Research, Hospital for Children and Adolescent, University of Leipzig, Leipzig, Germany; bDivision of Rheumatology, Department of Internal Medicine, University of Leipzig, Leipzig, Germany; cDepartment of General Practice and Family Medicine, Medical Faculty of the University of Leipzig, Leipzig, Germany; dAcademic Teaching Hospital St. Georg, Leipzig, Germany; eLIFE Leipzig Research Centre for Civilization Diseases, University of Leipzig, Leipzig, Germany; fInstitute for Metabolism and Systems Research, University of Birmingham, Birmingham, UK


Background/aim: The PTEN hamartoma tumor syndrome (PHTS) is an overgrowth syndrome caused mainly by germline mutations in the tumor suppressor PTEN. Patients are predisposed for the development of malignant and benign tumors. Children and adolescents with PHTS frequently develop single or multiple lipomas. PTEN antagonizes the phosphatidylinositide3-kinase/AKT/mechanistic target of rapamycin (PI3K/AKT/mTOR) pathway, which promotes proliferation and differentiation in adipocytes. The aim of this study was to investigate the mechanisms leading to lipoma development in patients with PHTS using isolated lipoma cells from a PHTS patient and a PTEN knock-down cell model.

Methods: PTEN haploinsufficient cells from a lipoma of a PHTS patient (LipPD1) were compared to SVF cells from healthy individuals regarding differentiation capacity during long-term culturing and proliferation. Primary cells of the stromal vascular fraction (SVF) from human fat biopsies (over 20 population doublings) were transfected with siRNA against PTEN and compared to scramble siRNA transfected cells. To analyze their differentiation capacity cells were grown in adipocyte differentiation medium for 12 days after transfection. Lipid droplets were stained with Oil Red O. Proliferation was measured after 7 days incubation in growth medium via counting of cell nuclei.

Results: LipPD1 cells retained their differentiation capacity over a prolonged period of 25 population doublings (60% lipid accumulation) compared to control preadipocytes (20% lipid accumulation after 10 population doublings). The proliferation was 1.3±0.1 fold higher in LipPD1 cells compared to low passage SVF cells. To evaluate the effect of reduced PTEN in direct comparison to controls, PTEN was transiently down regulated in SVF cells via siRNA with a knock-down efficiency of 54%±11% that was stable during 12 days of adipocyte differentiation. PTEN knock-down cells showed an elevated AKT phosphorylation compared to control cells. Lipid accumulation was 1.4±0.2 fold higher in PTEN knock-down cells compared to controls after adipocyte differentiation for 12 days. Cell count was increased 2.7±1.0 fold in PTEN knock-down cells after 7 days.

Conclusion: Primary human preadipocytes lose their ability to differentiate into adipocytes after several weeks in culture. Their differentiation capacity could be partly recovered with reduction in PTEN levels. An enhanced proliferation of these cells corresponds with the enhanced activation of AKT. This resembles the phenotype found in lipoma cells of pediatric patients with PTEN defects.

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