α5β1 integrins act as sensors for tauroursodeoxycholic acid in hepatocytes

Introduction:

Ursodeoxycholic acid, which in vivo is converted to its taurine conjugate tauroursodeoxycholic acid (TUDC), is a mainstay for the treatment of cholestatic liver disease. Earlier work showed that TUDC exerts its choleretic properties in the perfused rat liver in a α5β1 integrin-mediated way (Häussinger et al., Gastroenterology, 2003). However, the molecular basis of TUDC-sensing in the liver is unknown.

Methods:

Rat livers were perfused and analysed by immunohistochemical staining or Western blot technique thereafter. Cell culture experiments were performed in either primary rat hepatocytes or in a stably expressing FLAG-Ntcp-EGFP HepG2 cell line. Molecular dynamics (MD) simulations of complex structures of the ectodomain of α5ß1 integrin bound to either TUDC, taurocholate (TC), or the integrin-antagonistic peptide GRGDSP were performed and analysed with respect to conformational changes in the head region.

Results:

In perfused rat liver as well as in human FLAG-Ntcp-EGFP-transfected HepG2 cells, TUDC (20µmol/L) induces the rapid appearance of the active conformation of the β1 subunit of α5β1 integrins, followed by an activating phosphorylation of tyrosine residues Y845 and Y1173 of the epidermal growth factor receptor (EGFR) and of extracellular signal-regulated kinases (Erk-1/-2). The TUDC-induced receptor and kinase activation was no longer observed in the presence of the integrin-antagonistic hexapeptide GRGDSP in perfused rat liver or after β1 integrin siRNA knockdown in FLAG-Ntcp-EGFP-transfected HepG2 cells. The TUDC-induced β1 integrin activation occurred predominantly inside the hepatocyte and required TUDC uptake by way of the Na+/taurocholate cotransporting peptide. This requirement for concentrative TUDC uptake and the liver-specificity of Ntcp-expression may explain why TUDC acts primarily in the liver. Molecular dynamics simulations of a 3D model of α5β1 integrin with TUDC bound revealed significant conformational changes within the head region that have been linked to integrin activation before.

Discussion/Conclusion:

In summary, TUDC has the unique property to directly interact with α5ß1 integrins inside the hepatocyte. The resulting conformational change triggers ß1 integrin activation and initiates integrin-dependent signaling, which explains not only the choleretic and cytoprotective effects of this therapeutically used bile acid but also its hepatocyte-specificity.