Abstract
Polarization of hepatocytes is manifested by bile canalicular network formation and activation of LKB1 and AMPK, which control cellular energy metabolism. The bile acid, taurocholate, also regulates development of the canalicular network through activation of AMPK. In the present study, we used collagen sandwich hepatocyte cultures from control and liver-specific LKB1 knockout mice to examine the role of LKB1 in trafficking of ABCB11, the canalicular bile acid transporter. In polarized hepatocytes, ABCB11 traffics from Golgi to the apical plasma membrane and endogenously cycles through the rab 11a-myosin Vb recycling endosomal system. LKB1 knockout mice were jaundiced, lost weight and manifested impaired bile canalicular formation and intracellular trafficking of ABCB11, and died within three weeks. Using live cell imaging, fluorescence recovery after photobleaching (FRAP), particle tracking, and biochemistry, we found that LKB1 activity is required for microtubule-dependent trafficking of ABCB11 to the canalicular membrane. In control hepatocytes, ABCB11 trafficking was accelerated by taurocholate and cAMP; however, in LKB1 knockout hepatocytes, ABCB11 trafficking to the apical membrane was greatly reduced and restored only by cAMP, but not taurocholate. cAMP acted through a PKA-mediated pathway which did not activate AMPK. Our studies establish a regulatory role for LKB1 in ABCB11 trafficking to the canalicular membrane, hepatocyte polarization, and canalicular network formation.
Highlights
Structural and functional generation of polarized domains of the plasma membrane of hepatocytes is essential for proper hepatic function
Canalicular network formation is regulated by taurocholate, a major mammalian bile acid, through cAMP-Epac-MEK-mediated activation of AMPK [3]
Control mouse hepatocytes in collagen sandwich cultures are not polarized on day 1, but progressively develop a mature interconnecting canalicular network similar to rat hepatocyte cultures and mammalian liver [2,19]
Summary
Structural and functional generation of polarized domains of the plasma membrane of hepatocytes is essential for proper hepatic function (for a recent comprehensive review see [1]). Hepatocellular canalicular network formation, an important component of hepatocyte polarization, requires activation of LKB1 and AMPK, which control cellular energy metabolism [2]. In 1999, LeCluyse et al described a collagen sandwich technique by which hepatocytes can be maintained for 2–3 weeks with retention of structure and function [11]. In this and subsequent studies, hepatocytes were isolated from liver of rats or humans, and recently from mice [12]. Because genetically modified mice provide a powerful experimental tool to identify regulatory and signaling factors, in the present studies we combined hepatocyte collagen sandwich culture technique with mouse knockout methodology to investigate the role of LKB1 in hepatocyte polarization
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