SUN-109 PTEN Regulates Differentiation and Proliferation of Aging Preadipocytes

Abstract

Background: The tumor suppressor gene phosphatase and tensin homologe (PTEN) is one of the most frequently altered genes in human cancer. It antagonizes the PI3K/AKT/mTOR pathway, which is activated by various factors like IGF-1 or insulin and promotes cell growth and proliferation. Germline PTEN mutations cause the PTEN hamartoma tumor syndrome, characterized by an overgrowth of different soft tissues. This includes the formation of lipomas. To understand these adipose tissue malformations we aim to assess the function of PTEN in the context of preadipocyte proliferation, differentiation and senescence. Methods: We used preadipocytes obtained from healthy individuals and reduced the PTEN levels either transiently by siRNA mediated PTEN knock-down or stably using the CRISPR/Cas9 system. Results: Reduced PTEN levels (0.59 ± 0.03 fold) led to an increased proliferation of the preadipocytes as measured by nuclei counting (1.5 ± 0.2 fold after 7 days) and staining of the proliferation marker Ki-67 (1,09 ± 0.01 fold). Moreover, the adipocyte differentiation capacity of low passage preadipocytes was increased after PTEN knock-down (1.14 ± 0.04 fold lipid accumulation) compared to control transfected cells. Differentiation capacity could even be restored in high passage presenescent cells that had already lost their ability to differentiate (1.41 ± 0.14 fold lipid accumulation). This corresponded with an increase in the adipocyte differentiation marker PPARy, as analyzed via qPCR (1.8 ± 0.4 fold). We not only observed an activation of AKT signaling (enhanced AKT and ribosomal protein S6 phosphorylation) in PTEN knock-down cells, but noticed a decrease of the senescence marker p21 (0.4 ± 0.09 fold) and an increase in the NAD-synthesis enzyme NAMPT (visfatin) (1.28 ± 0.06 fold) suggesting a role of PTEN in regulating cellular lifespan. To address the question, if PTEN plays a role not only in disease but also in normal preadipocyte function, we measured PTEN protein levels in cells of different passages. During long-term culture PTEN levels increased (3.7 ± 0.7 fold after 25 days), resulting in a decreased AKT phosphorylation (0.4 ± 0.3 fold). Levels of NAMPT decreased during long-term culture (0.04 ± 0.02 fold). Conclusion: Considering the observations in normal preadipocytes during cellular aging and the findings in PTEN knock-down cells, our results provide strong evidence that PTEN is involved in regulation of preadipocyte aging.