Mechanism Of N-terminus Of Adenomatous Polyposis Coli Inhibiting Primary Cilia Assembly

Adenomatous polyposis coli (APC) is a tumor suppressor gene, and its truncated mutations commonly cause familial adenomatous polyposis (FAP). Cilium is a microtubule-based organelle projecting from cell surface. Cilia are involved in many important biological functions, such as regulating cell motility and participating in diverse signaling pathways. Defects in cilia structure and function have been associated with a range of diseases, which collectively call ciliopathies. It has been reported that there are many same clinical symptoms between extracolonic manifestations of FAP and ciliopathies, such as fibioma, osteoma, CHRPE and so on, which implied inevitable relationship between APC mutation and cilia disorder. APC-N, a high conserved fragment of APC is be retained in many APC mutation individuals. In the present study, we investigated the effects and mechanism of APC-N on primary cilia assembly. This thesis consists of three parts:Part one:Effect of APC-N on ciliogenesis. First of all, MDCK cells were transfected with plasmids pEGFP-C3 and pEGFP-C3-APC-N and further cultured for 3 days to grow cilia. Western blotting and immunofluorescence staining showed that APC-N successfully expressed in MDCK cells and mainly located in the cytoplasm. Cilia statistical results showed that the overexpression of APC-N significantly inhibited cilia assembly in MDCK cells.Part two:Effect of APC-N on pericentrin, cell cycle progression and acetylated tubulin. In part one, we found that APC-N inhibits cilia formation. This part aim to investigate the mechanism following several aspects:1, Effec of APC-N on pericentrin location; 2, Effect of APC-N on cell cycle progression; 3, Effect of APC-N on acetylated tubulin expression. Results showed that APC-N inhibits cilia formation not through influencing pericentrin location and disrupting cell cycle progression. Moreover, reduced acetylated tubulin level and enhanced HDAC6 expression were observed in MDCK cell containing APC-N, which suggests that the APC-N inhibits cilia formation by upregulating HDAC6 and thereby reducing acetylated tubulin level.Part three:A feedback loop of β-catenin/PI3K/AKT/GSK-3β regulates N-terminal fragments of APC-induced cilia defects. To further research the mechanism of APC-N fragment inhibiting cilia formation, we detected β-catenin expression. Furthermore, we also measured GSK3β, which was reported to be related to cilia formation. The results showed that β-catenin was up-regulated along with inhibited GSK3β activity. To assess whether changes of β-catenin and GSK3β are responsible for cilia damages, we treated MDCK cells containing APC-N with siRNA targeted β-catenin or GSK-3β inhibitor SB216763. The results indicated both β-catenin and GSK-3β are associated with cilia loss in cells expressing APC-N. To elucidate potential mechanism between β-catenin and GSK3β, we detected P-AKT (T308) that is the upstream signaling molecules of GSK3β. We found that APC-N expression resulted in increased P-AKT (T308), which suggested PI3K/AKT signaling was activated. We treated MDCK cells containing APC-N with siRNA targeted β-catenin or PI3K inhibitor LY294002 to access the role of PI3K/AKT signaling between β-catenin and GSK3β. Results suggested a feedback loop of β-catenin/PI3K/AKT/GSK-3β was involved in N-terminal fragments of APC-induced cilia defects. To determine whether PI3K/AKT signaling directly participates in ciliogenesis, cells were treated with both inhibitors LY294002 and SB216763. We found treatment of both LY294002 and SB216763 only decreased the P-AKT (T308) but had no effects on GSK-3β,β-catenin and cilia. These results indicated PI3K/AKT signaling indirectly affected cilia formation through GSK-3β and β-catenin.In summary, APC-N overexpression inhibits cilia formation in MDCK cells, which is not through influencing pericentrin location and disrupting cell progression. This study suggested APC-N overexpression suppresses cilia formation via upregulating HDAC6 and thereby increasing acetylated tubulin level. In addition, we also found a feedback loop of β-catenin/PI3K/AKT/GSK-3β was involved in N-terminal fragments of APC-induced cilia defects.