Insulin-induced hepatocyte proliferation involves swelling-dependent activation of the epidermal growth factor receptor

Introduction: The aim of the study was to analyze whether the proliferative properties of insulin in rat liver do involve cross-signaling toward the epidermal growth factor receptor (EGFR), and whether this is mediated via the insulin-induced hepatocyte swelling. Methods: Perfused rat liver and primary rat hepatocytes. EGFR, c-Src and Erk phosphorylation were detected by WB, c-Src/EGFR association by EGFR IP and cell proliferation by BrdU assay. Results: In perfused rat liver, insulin (35 nmol/L) induced phosphorylation of the EGFR (Y⁸⁴⁵ and Y¹¹⁷³, but not at Y¹⁰⁴⁵). Insulin-induced EGFR as well as Erk phosphorylation were sensitive to bumetanide indicating a dependence on hepatocyte swelling. As a proof of principle, also hypoosmolarity (225 mosmol/L)-induced hepatocyte swelling led to an activation of EGFR and Erk. Since hypoosmolarity-induced cell signaling is sensed by the integrin system and mediated via c-Src, it was tested whether those are involved in insulin-induced EGFR activation. Both, inhibition of the integrin system using RGD peptide or integrin β₁-blocking antibody as well as inhibition of c-Src by PP-2 were able to inhibit insulin- and hypoosmotic induced EGFR activation, whereas integrin β₃- or α_Vβ₅-blocking antibodies were ineffective. IP studies revealed that c-Src binds to EGFR in a RGD-peptide-, integrin β₁-blocking AB- and PP-2-sensitive fashion indicating that swelling-induced EGFR activation is c-Src-mediated. Proliferation induced by insulin (100 nmol/L) or hypoosmolarity (205 mosmol/L) was analyzed by BrdU assay in primary rat hepatocytes: all maneuvers which prevented insulin- or hypoosmolarity-induced EGFR activation also inhibited hepatocyte proliferation (RGD, integrin β₁-blocking AB, PP-2, AG1478 and PD098059). Conclusions: Hepatocyte cell swelling by either insulin or hypoosmolarity does lead to an activation of the EGFR and subsequent cell proliferation which is sensed by the integrin β₁-subtype and mediated via the c-Src kinase.