Insulin Receptor Signaling Has Dichotomous Roles in Hepatic Stellate Cell Activation: Crosstalk with Insulin and TGFβ Pathways in Liver Fibrosis

The insulin receptor (INSR) has been recently shown to be hyperactive in hepatic stellate cells (HSCs) in human and rodent liver fibrosis. To explore HSC cellular mechanisms that the INSR regulates during pro-fibrotic stimulation, we used CRISPR-Cas9 technology. We knocked out a portion of the INSR in the human LX2 HSC cell line (INSRe5-8 KO) that regulates insulin responsiveness but not the insulin-like growth factor (IGF) or transforming growth factor-β (TGFβ) signaling. The INSRe5-8 KO HSCs had increased cell growth and migration compared to scramble control. We treated the scramble control and INSRe5-8 KO HSCs with insulin or TGFβ for kinase activity profiling using the PamGene PamStation kinome technology. Our analysis showed that serine/threonine kinase (STK) activities were reduced, and most of the protein-tyrosine kinase (PTK) activities were increased in the INSRe5-8 KO compared to scramble control HSCs. To study gene transcripts altered in activated scramble control and INSRe5-8 KO HSCs, we treated with TGFβ for 24 hours. We isolated the RNA for sequencing and found that the INSRe5-8 KO cells, compared to control HSCs, had reduced transcriptional responsiveness to TGFβ stimulation, collagen-activated signaling, smooth muscle cell differentiation pathways, SMAD protein signaling, collagen metabolic process, integrin-mediated cell adhesion, and notch signaling were all altered. This study demonstrates that INSR regulates HSC growth and migration and is involved in TGFβ-induced HSC activation. These findings provide new insights into the development of more effective treatments for liver fibrosis.