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THE ROLE OF P35/CDK5-MEDIATED KINASE CASCADE IN INTRAHEPATIC BILIARY MORPHOGENESIS IN ZEBRAFISH, AND ITS APPLICATION IN HUMAN BILIARY RESEARCH
Maitham A. Moslim*2, Manali Dimri1, Gareth Morris-Stiff2, R Matthew Walsh2, Takuya F. Sakaguchi1
1Department of Stem Cell Biology and Regenerative Medicine, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH; 2Departments of HPB Surgery, Cleveland Clinic Foundation, Cleveland, OH
BACKGROUND: Biliary atresia is associated with branching alterations in the intrahepatic biliary system, and accounts for almost 50% of all liver transplants performed in children. However the availability of donated livers in children is extremely limited. Hence, the development of pharmacological treatment to ameliorate the biliary atresia phenotype would provide a novel and much needed therapeutic option. To our knowledge such medical therapy does not exist.
Cyclin-dependent kinase 5 (Cdk5) is known to regulate a variety of biological processes including cell migration, maturation, cell-cell connection and cytoskeletal organization in the nervous system; however, its role in liver development has not yet been established. We hypothesized that the Cdk5-mediated kinase cascade might play a role in intrahepatic biliary network branching morphogenesis.
METHODS: Zebrafish has been emerging as an excellent model to study human liver diseases. A transgenic line of zebrafish is generated to express an enhanced green fluorescent protein in the intrahepatic biliary system. Using this zebrafish model and with a National Institute of Health (NIH) grant, a new computer-based algorithm was created to quantitatively compute the structural differences of the three-dimensional biliary network and to examine for a Cdk5-mediated kinase cascade.
RESULTS: With inhibition of Cdk5, biliary epithelial cells could properly differentiate and extend projections, although these projections could not efficiently form connections and thus lead to a biliary atresia-like branching defect.
Our experiment showed that the administration of small molecule inhibitors against known Cdk5 targets including p21-activated kinase 1 (Pak1), demonstrated that Pak1 activity in biliary epithelial cells is negatively correlated with the frequency of branch formation in the liver. Pak1 is known to increase the activity of LIM kinases, which in turn, phosphorylate and thereby suppress the activity of cofilin. Since cofilin is an actin depolymerizing factor that influences actin assembly and dynamics, it was demonstrated that the Cdk5-Pak1-LimK-cofilin kinase cascade regulated actin dynamics within biliary epithelial cells.
Generating larvae carrying a mutation in the cdk5 activator 1a (cdk5r1a), an essential activator of Cdk5, the mutant larvae exhibited similar branching defects as those observed in Cdk5 inhibitor-treated larvae. However, inhibition of LIM kinase rescued the functionality of the biliary system in the mutant larvae.
CONCLUSIONS: Our results provide some of the first insights into branching morphogenesis of the intrahepatic biliary network, demonstrating the importance of the Cdk5-Pak1-LimK-Cofilin kinase cascade, and suggesting that LIM kinase inhibition may be a potential target to improve hepatic function in biliary atresia.
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