Tubby-like protein superfamily member PLSCR3 functions as a negative regulator of adipogenesis in mouse 3T3-L1 preadipocytes by suppressing induction of late differentiation stage transcription factors.

PLSCR3 (phospholipid scramblase 3, Scr3) belongs to the superfamily of membrane-associated transcription regulators named Tubby-like proteins (TULPs). Physiological phospholipid scrambling activities of PLSCRs in vivo have been skeptically argued, and knowledge of the biological functions of Scr3 is limited. We investigated the expression of Scr3 during differentiation of mouse 3T3-L1 preadipocytes by Western blotting (WB) and by reverse-transcription and real-time quantitative PCR (RT-qPCR). The Scr3 protein decreased during 3T3-L1 differentiation accompanied by a reduction in the mRNA level, and there was a significant increase in the amount of Scr3 protein secreted into the culture medium in the form of extracellular microvesicles (exosomes). On the other hand, Scr3 expression did not significantly decrease, and the secretion of Scr3 in 3T3 Swiss-albino fibroblasts (a parental cell-line of 3T3-L1) was not increased by differentiation treatment. Overexpression of human Scr3 during 3T3-L1 differentiation suppressed triacylglycerol accumulation and inhibited induction of the mRNAs of late stage pro-adipogenic transcription factors [CCAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ)] and X-box-binding protein 1 (XBP1). Expression of early stage pro-adipogenic transcription factors (C/EBPß and C/EBPδ) was not significantly affected. These results suggest that Scr3 functions as a negative regulator of adipogenesis in 3T3-L1 cells at a specific differentiation stage and that decrease in the intracellular amount of Scr3 protein caused by reduction in Scr3 mRNA expression and enhanced secretion of Scr3 protein appears to be important for appropriate adipocyte differentiation.

ALG-2-interacting Tubby-like protein superfamily member PLSCR3 is secreted by an exosomal pathway and taken up by recipient cultured cells.

PLSCRs (phospholipid scramblases) are palmitoylated membrane-associating proteins. Regardless of the given names, their physiological functions are not clear and thought to be unrelated to phospholipid scrambling activities observed in vitro. Using a previously established cell line of HEK-293 (human embryonic kidney-293) cells constitutively expressing human Scr3 (PLSCR3) that interacts with ALG-2 (apoptosis-linked gene 2) Ca²âº-dependently, we found that Scr3 was secreted into the culture medium. Secretion of Scr3 was suppressed by 2-BP (2-bromopalmitate, a palmitoylation inhibitor) and by GW4869 (an inhibitor of ceramide synthesis). Secreted Scr3 was recovered in exosomal fractions by sucrose density gradient centrifugation. Palmitoylation sites and the N-terminal Pro-rich region were necessary for efficient secretion, but ABSs (ALG-2-binding sites) were dispensable. Overexpression of GFP (green fluorescent protein)-fused VPS4B(E235Q), a dominant negative mutant of an AAA (ATPase associated with various cellular activities) ATPase with a defect in disassembling ESCRT (endosomal sorting complex required for transport)-III subunits, significantly reduced secretion of Scr3. Immunofluorescence microscopic analyses showed that Scr3 was largely localized to enlarged endosomes induced by overexpression of a GFP-fused constitutive active mutant of Rab5A (GFP-Rab5A(Q79L)). Secreted Scr3 was taken up by HeLa cells, suggesting that Scr3 functions as a cell-to-cell transferable modulator carried by exosomes in a paracrine manner.