Analysis of ORP2‐knockout hepatocytes uncovers a novel function in actin cytoskeletal regulation

Abstract

ORP2 is implicated in cholesterol transport, triglyceride metabolism, and adrenocortical steroid hormone production. We addressed ORP2 function in hepatocytes by generating ORP2-knockout (KO) HuH7 cells by CRISPR-Cas9 gene editing, followed by analyses of transcriptome, F-actin morphology, migration, adhesion, and proliferation. RNA sequencing of ORP2-KO cells revealed >2-fold changes in 579 mRNAs. The Ingenuity Pathway Analysis (IPA) uncovered alterations in the following functional categories: cellular movement, cell-cell signaling and interaction, cellular development, cellular function and maintenance, cellular growth and proliferation, and cell morphology. Many pathways in these categories involved actin cytoskeleton, cell migration, adhesion, or proliferation. Analysis of the ORP2 interactome uncovered 109 putative new partners. Their IPA analysis revealed Ras homolog A (RhoA) signaling as the most significant pathway. Interactions of ORP2 with SEPT9, MLC12, and ARHGAP12 were validated by independent assays. ORP2-KO resulted in abnormal F-actin morphology characterized by impaired capacity to form lamellipodia, migration defect, and impaired adhesion and proliferation. Rescue of the migration phenotype and generation of typical cell surface morphology required an intact ORP2 phosphoinositide binding site, suggesting that ORP2 function involves phosphoinositide binding and transport. The results point at a novel function of ORP2 as a lipid-sensing regulator of the actin cytoskeleton, with impacts on hepatocellular migration, adhesion, and proliferation.-Kentala, H., Koponen, A., Kivelä, A. M., Andrews, R., Li, C., Zhou, Y., Olkkonen, V. M. Analysis of ORP2-knockout hepatocytes uncovers a novel function in actin cytoskeletal regulation.

OSBP-related protein-2 (ORP2): a novel Akt effector that controls cellular energy metabolism

“…ORP2-KO did not reduce but rather increased ER-LD contacts under basal culture conditions and interfered with their expansion upon fatty acid loading. Together with our recently published work (Kentala et al in FASEB J 32:1281-1295, 2018, this study identifies ORP2 as a new regulatory nexus of Akt signaling, cellular energy metabolism, actin cytoskeletal function, cell migration, and proliferation.…”

“…The HuH7 human hepatoma cell line [35] was cultured as described in [21,22]. ORP2-KO lines were derived therefrom by CRISPR-Cas9-mediated gene editing by employing vectors described in [42,44] as reported in [21]. For the generation of ORP2-KO1 guide, RNAs targeting exon 4 of OSBPL2 were used, and for ORP2-KO2 guide RNAs targeting both exon 4 and 5 were used.…”

“…Generation of recombinant lentiviruses expressing wildtype (wt) ORP2 or its mutants ORP2 mPIP (H178-179A, K423A) and mFFAT (F7-8V, D9V), and a GFP-encoding vector control, as well as cell infections of ORP2-KO HuH7 cells are described in [21]. Knockdown of ORP2 in HUVECs was carried out by transducing the cells for 48 h at a multiplicity of infection 10, with an lentivirus expressing the ORP2-specific shRNA, TRCN0000154381 (Sigma-Aldrich), using non-targeting SHC002 (Sigma-Aldrich) as a control.…”

“…Analysis of ORP2 knockout (ORP2-KO) HuH7 cells indicated a role for ORP2, in addition to lipid metabolism, in actin cytoskeletal regulation as well as in the control of cell adhesion, migration, and proliferation [21]. Moreover, analysis of ORP2 protein interactome in HuH7 cells identified ORP2-binding partners involved in RhoA signaling [21], a central actin regulatory pathway. These findings prompted the hypothesis that ORP2 could mediate a crosstalk between lipid metabolism and actin cytoskeletal functions.…”

ORP2 Delivers Cholesterol to the Plasma Membrane in Exchange for Phosphatidylinositol 4, 5-Bisphosphate (PI(4,5)P2)

“…Consistently, the FFAT motif of ORP2 is dispensable for the lipid transfer function of ORP2. Notably, the FFAT motif is not required for ORP2 to induce morphological changes in mammalian cells (Kentala et al, 2018). The FFAT motif may instead be required for ORP2 to Figure 6.…”