Type III Collagen is Required for Adipogenesis and Actin Stress Fibre Formation in 3T3-L1 Preadipocytes.

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 3a1 transcripts, ColIII protein and lipid accumulation in 3T3-L1 differentiating cells. 3a1 3T3-L1 genome-edited cell lines abolished adipogenesis, demonstrated by a dramatic reduction in adipogenic moderators: Pparγ (88%) and C/ebpα (96%) as well as markers aP2 (93%) and oil red O staining (80%). 3a1 3T3-L1 cells displayed reduced cell adhesion, sustained active β-catenin and deregulation of fibronectin () and collagen (4a1, 6a1) extracellular matrix gene transcripts. 3a1 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 3a1 3T3-L1 cells is very similar to that of 56 3T3-L1 cells, suggesting a functional relationship between ColIII and Gpr56 in preadipocytes.