Abstract
Differentiation of mesenchymal stem cells into adipocyte requires coordination of external stimuli and depends upon the functionality of the primary cilium. The Rab8 small GTPases are regulators of intracellular transport of membrane-bound structural and signaling cargo. However, the physiological contribution of the intrinsic trafficking network controlled by Rab8 to mesenchymal tissue differentiation has not been fully defined in vivo and in primary tissue cultures. Here, we show that mouse embryonic fibroblasts (MEFs) lacking Rab8 have severely impaired adipocyte differentiation in vivo and ex vivo. Immunofluorescent localization and biochemical analyses of Rab8a-deficient, Rab8b-deficient, and Rab8a and Rab8b double-deficient MEFs revealed that Rab8 controls the Lrp6 vesicular compartment, clearance of basal signalosome, traffic of frizzled two receptor, and thereby a proper attenuation of Wnt signaling in differentiating MEFs. Upon induction of adipogenesis program, Rab8a- and Rab8b-deficient MEFs exhibited severely defective lipid-droplet formation and abnormal cilia morphology, despite overall intact cilia growth and ciliary cargo transport. Our results suggest that intracellular Rab8 traffic regulates induction of adipogenesis via proper positioning of Wnt receptors for signaling control in mesenchymal cells.
Highlights
A hallmark of adipocyte maturation is the formation, trafficking, and fusion of lipid droplets, all processes linked to the Rab family of small GTPases [7]
Quantification of the lipid-droplet size and total area indicated a similar extent of impairment in Rab8a−/−;Rab8bKD mouse embryonic fibroblast (MEF) as Kif3a−/− MEFs (Fig. 5, F and G), which displayed a delayed formation and fewer lipid droplets (Fig. 5E). These data suggested a defective activation of the adipogenic program in Rab8-deficient MEFs, which was further supported by significantly reduced transcripts of adipocyte differentiation, including Fabp4 and Glut4 in these Rab8a−/−;Rab8bKD MEFs compared with WT MEFs (Fig. 5H)
As Rab8 has been implicated in ciliary cargo transport and cilia development [11,12,13, 17, 18], and the primary cilium inhibits Wnt/β-catenin signaling activity [2, 25, 27], we examined if Rab8-deficient MEFs might exhibit defects in primary cilia development and functionality, none of which has been examined during induced MEF adipogenesis
Summary
A hallmark of adipocyte maturation is the formation, trafficking, and fusion of lipid droplets, all processes linked to the Rab family of small GTPases [7]. To examine whether Rab8 regulates Lrp6 vesicular compartment and MEF’s signaling capacity in response to Wnt ligands, we first established stable Rab8b knockdown (KD) MEFs and an MEF line deficient in both Rab8a and Rab8b (Fig. 2A).
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