Abstract
GPR56 is required for the adipogenesis of preadipocytes, and the role of one of its ligands, type III collagen (ColIII), was investigated here. ColIII expression was examined by reverse transcription quantitative polymerase chain reaction, immunoblotting and immunostaining, and its function investigated by knockdown and genome editing in 3T3-L1 cells. Adipogenesis was assessed by oil red O staining of neutral cell lipids and production of established marker and regulator proteins. siRNA-mediated knockdown significantly reduced Col3a1 transcripts, ColIII protein and lipid accumulation in 3T3-L1 differentiating cells. Col3a1−/− 3T3-L1 genome-edited cell lines abolished adipogenesis, demonstrated by a dramatic reduction in adipogenic moderators: Pparγ2 (88%) and C/ebpα (96%) as well as markers aP2 (93%) and oil red O staining (80%). Col3a1−/− 3T3-L1 cells displayed reduced cell adhesion, sustained active β-catenin and deregulation of fibronectin (Fn) and collagen (Col4a1, Col6a1) extracellular matrix gene transcripts. Col3a1−/− 3T3-L1 cells also had dramatically reduced actin stress fibres. We conclude that ColIII is required for 3T3-L1 preadipocyte adipogenesis as well as the formation of actin stress fibres. The phenotype of Col3a1−/− 3T3-L1 cells is very similar to that of Gpr56−/− 3T3-L1 cells, suggesting a functional relationship between ColIII and Gpr56 in preadipocytes.
Highlights
Recent studies have shown that the G protein-coupled receptor 56 (GPR56 or ADGRG1) is required for adipogenesis
Col3a1‐knockout (KO) mice (Col3a1) have previously shown that loss of Gpr56 in 3T3‐L1 cells results in changes in expression of genepreviously transcripts encoding in preadipocytes as well as We have shown other that extracellular matrix (ECM)
(5) changes in ECM Col4a1, Col6a1 and Fn1 gene expression in both preadipocytes as well as in cells 10 days post-differentiation treatment. These observations suggest that a functional relationship exists between ColIII and Gpr56 in 3T3-L1 cells, which contributes to the successful execution of adipogenesis
Summary
Recent studies have shown that the G protein-coupled receptor 56 (GPR56 or ADGRG1) is required for adipogenesis. Gpr (AdgrgI) gene knockdown (KD) and genome editing in 3T3-L1 cells (Gpr56−/− ) inhibit adipogenesis [1]. Genes, loss of actin stress fibres and sustained levels of active β-catenin, all of which may contribute to the inhibition of the adipocyte differentiation programme observed [1]. Type III collagen (COLlIII) binds GPR56 to regulate a range of biological activities, including brain development [2] and pancreatic islet β-cell function [3]. The interaction of COLIII with GPR56 stimulates RHOA activation via G-proteins Gα12/Gα13 [2]. The association of these two molecules raises the possibility that ColIII, like Gpr, might regulate adipogenesis
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