α5β1 integrins in hepatocytes act as receptors for bile acids with a (nor)ursodeoxycholane scaffold

Ursodeoxycholic acid (UDCA) is a standard treatment in several cholestatic liver diseases (Figure 1A) [1]. In vivo, conjugation with taurine occurs rapidly and yields tauroursodeoxycholic acid (TUDC), which has been shown to promote choleresis by triggering the insertion of ATP-dependent transport proteins (e.g., the bile salt export pump (Bsep) and the multidrug resistance protein-2 (Mrp2)) into the canalicular membrane [2]. TUDC-induced recruitment of Bsep results from activation of focal adhesion kinase (FAK), phosphatidylinositol 3-kinase (PI3 kinase), and c-Src, which leads to downstream activation of extracellular signal-regulated kinases (Erks) and p38 mitogen-activated protein kinase (p38). Upon hepatocyte swelling, either induced by exposure to a hypoosmotic environment or insulin, a5b1 integrins become activated and trigger similar signaling events towards choleresis. a5b1 Integrins may also become activated by a swelling-independent way as previously shown by exposing hepatocytes to pathophysiological concentrations of urea [3]. Both TUDC-induced and swelling-dependent signaling were abolished in the presence of an antagonistic, RGD-motif containing hexapeptide (GRGDSP). These findings led us to hypothesize that a5b1 integrin will act as a receptor for TUDC in hepatocytes. We tested this hypothesis in a combined experimental and computational study [4]. Immunofluorescence staining on cryosections of isolated perfused rat liver (IPRL) revealed the active conformation of b1 integrin within 1 min after addition of TUDC at a concentration of 20 μM. Furthermore, phosphorylation of Erk-1 and -2 as well as activation of the epidermal growth factor receptor were induced by TUDC within the same time span. These effects were sensitive to inhibition by GRGDSP but insensitive to the presence of an inactive control peptide (GRADSP). As TUDC does not affect hepatocyte volume, which excludes that TUDC triggers integrin activation osmotically, these findings demonstrated that TUDC directly activates a5b1 integrins and triggers signaling events towards choleresis. While swelling-induced b1 integrin activation occurs primarily in the plasma membrane, TUDC-induced b1 integrin activation occurs primarily in the cytosol of hepatocytes. We demonstrated that the presence of the Na/taurocholate cotransporting polypeptide (Ntcp) is required for the latter. The need to uptake and/or concentrate TUDC inside the hepatocyte for b1 integrin activation to occur may explain why TUDC primarily acts in the liver. In order to provide insights at a molecular level as to how TUDC activates a5b1-integrin, a complex structure of a homology model of the ectodomain of a5b1-integrin and TUDC was generated by molecular docking and subsequently subjected to molecular dynamics (MD) simulations of 200 ns length [4]. These simulations revealed pronounced conformational changes in three regions of the bA domain of the integrin (Figure 1B): I) Helix a1 straightens and becomes continuous; II) this leads to a tighter packing between the top of helix a7 and the center of a1, which has been characterized as “T-junction formation” in an X-ray structure of integrin aIIbb3 bound to a ligand as well as in computational studies of agonistbound integrins; III) as a result, helix a7 moves downwards and outwards, which imposes a torque on the hybrid domain. The induced rotational motion of the hybrid domain is a prerequisite for the unbending of the integrin ectodomain, which, in turn, is required for integrin activation according to current models. Neither did MD simulations of the ectodomain of a5b1 integrin bound to GRGDSP nor to taurocholic acid (TC) (Figure 1A) reveal such conformational changes, in line with results * Correspondence: [email protected] Department of Mathematics and Natural Sciences, Institute for Pharmaceutical and Medicinal Chemistry, Heinrich Heine University, 40225 Düsseldorf, Germany Full list of author information is available at the end of the article Bonus et al. European Journal of Medical Research 2014, 19(Suppl 1):S13 http://www.eurjmedres.com/content/19/S1/S13 EUROPEAN JOURNAL OF MEDICAL RESEARCH