Folliculin Controls Kidney Tumor Development Of BHD Syndrome Through MTORC1

Background:Birt-Hogg-Dube’(BHD) syndrome is an autosomal dominant disease, where patients are predisposed to incident of hamartomas and cysts in multiple organs, including skin, lung, and kidney. Genotype analysis of BHD patients family, the incidence of BHD syndrome is mostly caused by deletion or mutation of BHD gene. The gene coded the protein named folliculin and approved with the symbol FLCN. The function of FLCN protein is not completely understood yet. But the recent research in rodents in animal models recognized the protein is a tumor suppressor.The BHD gene encodes a 70 kD protein with 579 amino acid. FLCN is an ancient and highly conserved protein, with multiple orthologs present in fungi and animals. It was demonstrated that FLCN mRNA was predominantly expressed in various tissues. The von Hippel-Lindau (VHL) and Tuberous Sclerosis Complex syndromes predisposing to malignant and benign tumors. Research on the pathogenesis found that the impaired cilia function due to tumor or cyst formation. BHD syndrome patients appeared cysts and benign and malignant tumors in the kidney, liver and lung and other organs. The clinical similarity suggests shared elements of pathogenesis, which led us to hypothesize that ciliary dysfunction could contribute to the BHD phenotype. And we hypothesized that FLCN might similarly have an important functional role in primary cilia.Cilia is elongate protruding microtubule-based structure outwardly extended to cell surface, which contain three parts:the cilia membrane, cilia axoneme and basal body. In humans, primary cilia are found on nearly every cell in the body. Inside cilia and flagella is a microtubule-based cytoskeleton called the axoneme. The axoneme of primary cilia typically has a ring of nine outer microtubule doublets (called a 9+0 axoneme), and the axoneme of a motile cilium has two central microtubule singlets in addition to the nine outer doublets (called a 9+2 axoneme). Surrounded by the ciliary membrane, a highly specialized domain extension of the cell membrane with many receptor related to cilia signal pathway. At the base of the cilium where it attaches to the cell body is the microtubule organizing center, the basal body. Cilia formation is restricted to cells that have exited the cell cycle, allowing the centrosome to differentiate into a basal body that forms the base of the cilium. A variety of receptors, ion channels and transporter proteins, as well as some of their downstream effector molecules, localize to the basal body.primary cilia, which typically serve as sensory organelles. It can sense mechanical stimulation like bending of the cilium and chemosensation such as growth factor, hormone. It can also respond to light, temperature, osmolality or gravity. The different kinds of external signals transfect into the cells by cilia, which modify the gene expression and physiological responses. Ciliary defects can lead to a number of human diseases. Ciliopathy which are characterized by pathophysiology associated with changes in the morphology and/or number of cilia. This kind of disease can be represented as polycystic kidney disease, blindness, mental retardation, obesity and diabetes. The large group of disorders classified as ciliopathies is particularly associated with cancers in several organs, including kidney cancer, melanoma, pancreatic ductal adenocarcinoma and ovarian cancer.Mammalian target of rapamycin (mTOR) is evolutionarily conserved serine /threonine protein kinase, belongs to the phosphatidylinositol 3-kinase kinase (PIKK) protein family. mTOR could senses cellular nutrient, oxygen, and energy levels. mTOR signaling pathway plays a critical role in cell growth, cell proliferation cell motility, cell survival, protein synthesis and transcription. It can integrate multiple extracellular signals, activate many factors involved in gene transcription, protein translation initiation, ribosome synthesis and cell apoptosis of many biological processes.The mTOR pathway has a biological activity to maintain normal physiological metabolism in the body in normal condition. But the mTOR pathway is dysregulated in human diseases, such as diabetes, obesity, depression and certain cancers. It’s abnormal activation in tumorigenesis due to tumor formation. A new therapeutic approaches to cancer could be developed once we uncovered the mechanism clearly. It is reported, at the cancer tissues separated from BHD patients, detects the abnormal activation of mTOR signaling pathway. While the mechanism between mTOR signal pathway and BHD syndrome of tumor generation still need to be further studied.Objective:This study is to explore the role of FLCN protein plays a key role in the pathogenesis of BHD syndrome in Kidney Tumor. Discuss the function of this protein in primary cilia and signaling pathways are implicated in. We then seek to confirm the necessity of primary cilia to maintain normal kidney function.Method:A series of experiments were accomplished in different cells by using cell culture, Western Blot, shRNA, co-immunoprecipitation and immunofluorescence staining. Human proximal tubular epithelial cell 8 (HKC8), FLCN-null UOK257 cell and UOK257-2 cell restored with ectopic expression of FLCN are used in our experments. The human cell line UOK257, derived from the renal cell carcinoma of a BHD patient with a germline mutation in the FLCN gene, harbors a truncated version of the FLCN protein. The ibidi pump system could culture the cells in flow culture medium which cause a shear stress to the cells. Compare the cells in stationary liquid culture conditions and culture fluid FLOW condition with a fluid shear stress stimulation effect of FLCN protein function in mTOR signaling pathway regulation. Design pcDNA3.1-FLCN-HA, pcDNA3.1-FLCN-GFP and pLKO-Tet-On-FLCN shRNA plasmids to change the level of FLCN protein. The phosphorylation of S6, 4E-BP1 and AMPK, Akt was determined by immunoblotting in FLOW or no-FLOW condition and detect which one is important in the mechanism. At the same time, knock down AMPK protein kinase LKB1 using pLKO-Tet-On-LKB1 shRNA to observe whether the protein kinase involved into the regulation process. Cells extracts were immunoprecipitated with anti-FLCN antibody and anti-LKB1 antibody to determine whether FLCN is required for recruiting LKB1 to AMPK under flow condition. The primary cilia-dependency was detected in the absence of IFT88 protein. Transfect pLKO-Tet-On-IFT88 shRNA to HKC8 cells. Immunofluorescence was used to observe the morphology of primary cilia and the translocation of phospho-AMPK in HKC8s.Results:1. FLCN protein play a role in the primary cilia(1) The growth of cell density reached a very high level, there will be contacted inhibition between the HKC8 cells. They entered the stationary phase from the mitotic phase. Maintain the cells cultured for another 72 hours to form the primary cilia. We observed a clear and specific staining of green anti-acetylated alpha tubulin of the cilia in the immunofluorescence picture. Each cell only has one primary cilia.(2) Using co-immunoprecipitation, we confirmed the association of FLCN with KIF3A with endogenously expressed proteins in cultured cells. Importantly, the association of FLCN with KIF3A was detected only in ciliated resting stationary phase cells but not cycling cells which do not contain primary cilia. KIF3A is one subunit of the heterotrimeric motor protein, This motor consists of two kinesin-related subunits (KIF3A and KIF3B or 3C in vertebrates) and an associated protein KAP. It transports protein complexes, nucleic acids towards the end of cilia. It is suggested that FLCN is a cilia protein.2. Overexpression of FLCN does not affect mTORCl activity under normal growth conditionOverexpress either HA-FLCN or GFP-FLCN fusion protein in HEK293 cells and then analyzed for the protein quantity. We noted exogenous fusion protein exceed endogenous FLCN protein level by 10 times or more. Phosphorylation levels of AKT, AMPK and S6 and 4EBP1 were also assessed by Western Blotting in FLOW or no-FLOW condition. Increase the FLCN protein level had no effect on mTORCl activity.3.FLCN regulates mTORCl activity under FLOW condition(1) Doxycycline with concentrations of 0,0.1,0.5,1.0 μg/ml were applied, respectively, to HKC8 those stably expressing pLKO-Tet-On-FLCN-shRNA plasmid. Doxycycline is an antibiotic. Upon the addition of doxycycline to the growth media, shRNA express is triggered resulting in target gene knock-down. FLCN shRNA efficiently inhibited protein expressions of FLCN in the concentration 0.1μg/ml, but had no effect on P-actin expression and phospho-S6, phospho-4E-BP1.(2) Under no-FLOW condition, two FLCN inducible knockdown cell lines, Western Blotting data suggests knocking down FLCN have no effect on the AKT, AMPK, S6 and 4E-BP1. Whereas in FLOW condition, a decrease in Akt activation was observed in HKC-8 cells as measured by the phosphorylation at T308 and S473. And a strong increase in AMPK activation with increased phosphorylation level of AMPK at T172. This increase correlated with the decrease in mTOR signaling pathway activity. However, when FLCN was depleted by shRNA in HKC-8 cells flow shear stress was unable to activate AMPK, and correlatively, mTOR pathway remained largely active. The density of the phosphorylation levels of AMPK and S6 ware analyzed with Image J and were significantly different between no-FLOW and FLOW conditions using SPSS 13.0 (P-AMPK:F=28.274, P=0.001; P-S6:F=16.449, P<0.001).FLCN-null UOK257 cells also showed actived mTOR pathway under FLOW condition. UOK257-2 cells had the same performance with wild type HKC8 cells. Phosphorylation levels of AMPK and S6 were significantly different using SPSS 13.0 (P-AMPK:F=8.944, P=0.007; P-S6:F=19.636, P=0.002).We provide evidence that FLCN could inhibit mTOR signaling pathway activity through phosphorylate AMPK under FLOW condition.4. LKB1 is involved in the mTOR regulation by FLCN(1) HKC8 cell extracts were immunoprecipitated with anti-FLCN antibody to pull-down AMPK. While We found that the amounts of AMPK Co-IP with FLCN were similar, the amount of phospho-AMPK was more under flow condition. This observation indicates that AMPK recruited by FLCN easier to be phosphorylated.(2) Whether the regulation mechanism of FLCN on AMPK requires AMPK kinase LKB1 proteins. HKC8 cells stably expressing pLKO-Tet-On-FLCN-shRNA plasmid treated with glycolysis inhibitor 2-deoxy-D-glucose(2DG) 20mM for 2hrs. The results suggest that activation of AMPK and phosphorylation of S6,4E-BP1 have no differences with or without FLCN.(3) Design pLKO-Tet-On-LKB1 shRNA plasmids to change the level of LKB1 protein. Reducing LKB1 levels due to attenuated AMPK activation. The experiments prove that the inhibited mTORCl activity is induced through the LKB1 activing AMPK protein.5. FLCN recruits LKB1 through the primary cilia, LKB1 active AMPK at the basal body(1) We examined the association of LKB1 with FLCN and AMPK. We found that LKB1 was not in complex with FLCN-AMPK in the absence of flow but became associated with it under flow condition. We confirmed LKB1, AMPK and FLCN could form a complex playing an inhibitory effect on mTOR pathway, but the complex is formed in the presence of FLOW.(2) Immunofluorescence was used to observe the morphology of primary cilia and the translocation of phospho-AMPK in HKC8. We found that in HKC-8 cells actived AMPK presents the sand like structure, scattered in the cytoplasm in no-FLOW condition. Phospho-AMPK accumulated at the basal body after subjected the cells to flow stress.6. Primary cilia is essential organ in inhibiting mTOR signaling pathway under FLOW conditions(1) Design pLKO-Tet-On-IFT88 shRNA plasmids to change the level of IFT88 protein. Removal of the primary cilia by shRNA-mediated depletion of IFT88, a gene required for formation of primary cilia. The interaction of FLCN and KIF-3A protein became weak or even disappear in impaired primary cilia formation cells, indicating that the association took place in the primary cilia.(2) Downregulation of IFT88 abolishes the inhibitory effect of flow stress on mTORC1 activity. This result demonstrated that the inhibitory effect was mediated through primary cilia. Primary cilia acts as a flow sensor, when the cilia is bent, many messenger molecules transduct the signal to all the cell to inhibit mTOR signal pathway activation.(3)When the formation of primary cilia was blocked by shRNA mediated downregulation of IFT88, association of LKB1 with FCLN and AMPK also decreased, indicating that the association was mediated through the primary cilia. It is very important for a cell primary cilia existed and functional maintain integrity.7. FLCN could regulates Wnt signaling pathwayHKC8 cells and UOK257, UOK257-2 cells, we detect WNT signaling pathway. The results show CD44 and Cyclin D1 protein expression level increased in FLCN knockdown and knockout cells under FLOW condition. The CD44 protein involved in cell proliferation, differentiation and tumor invasion and migration process; cyclin D1 overexpression can lead to uncontrolled cell growth and canceration. These results show that FLCN protein deficiency or dysfunction have close relationship with tumongenesis.Conclusion:1. The FLCN protein is a cilia protein exert their function in the cell.2. Overexpression of FLCN protein has no effect on mTOR signaling pathway activity, but knockdown of FLCN protein will weak the inhibitory effect on mTOR signal pathway under FLOW conditions3. FLCN located in primary cilia and basal body plays an important role on primary cilia related signal transduction pathway. Inhibitory effect of FLCN on mTOR signaling pathway is through the recruitment of LKB1 and AMPK protein to primary cilia basal body and active it.4. Primary cilia as an important shear stress sensor, could regulate the intracellular signal transduction pathway in the presence of shear stress of flow.5. Absence of FLCN protein will upregulate CD44 and cyclin D1 protein level. The overexpression of the proteins lead to abnormal cell growth and division and tumor formation.