Folliculin Regulates Hedgehog Signaling Pathway Through Primary Cilia

Background:Birt-Hogg-Dube'(BHD)syndrome is a rare autosomal dominant disorder.BHD patients were also predisposed to develop kidney cancer mostly of the oncocytic,chromophobe,or mixed subtype.The gene responsible for BHD,flcn,was mapped to chromosome 17p11.2 by linkage analysis.flcn encodes a novel cytoplasmic 64 kDa protein Folliculin(FLCN),which is expressed in most epithelial tissues.FLCN protein is highly conserved from unicellular organisms(yeast)through mammalian species(rodents,dog and humans).BHD patients also develop cysts in multiple organs such as kidney,liver and lungs.FLCN localizes to primary cilia,basal bodies and both centrosomes.Therefore,we hypothesized that FLCN might similarly have a functional role in primary cilia and that ciliary dysfunction could contribute to the BHD phenotype,highlighting that BHD might be a member of a larger group of disorders called ciliopathies.Cilia are elongate protruding microtubule-based structure outwardly extended to cell surface.Cilia can either be motile or immotile.Primary cilia are found on nearly every cell in vertebrate,and are found as a single cilium per cell.Primary cilium contains three parts:the cilium membrane,cilium axoneme and basal body.Primary cilia are usually immotile,and they lack this central pair(the 9+0 arrangement).The cilium consists of a microtubule-based axoneme covered by a specialized plasma membrane that extends from the cell surface into the extracellular space.Cilia act as 'antennae' to sense extracellular signals,such as growth factors,hormones,odorants and developmental morphogens.It has been known that cilia play pivotal roles in embryo development,cell and tissue homeostasis,and human diseases.The ciliary membrane(which is continuous with the plasma membrane)contains various cilia-specific receptors,ion channels and signaling molecules.Recent observations indicate that chemosensation,as well as signaling through receptor-dependent pathways such as the sonic hedgehog(SHH)pathway,platelet-derived growth factor receptor(PDGFR)pathway or non-canonical Wnt signaling pathway,is also mediated through cilia.Hedgehog signaling is involved in multiple developmental pathways and several adult homeostatic processes.Hedgehog signaling pathway is highly conservative between species.It generally exits in from lower animals(Drosophila)through mammalian species(humans).Multiple components of the Hedgehog pathway localize to the primary cilium or its basal body,including Sonic Hedgehog(Shh),Patch,Smo,suppressor of Fused(SuFu)(a key negative regulator of Gli activity),and Gli transcription factors.In the mammalian signaling pathway,the binding of a Hh ligand to its receptor Patched(Patch)relieves Patch-mediated inhibition of a second transmembrane protein Smoothened(Smo).Activation of Smo triggers an intracellular signal transduction cascade that culminates in Gli-dependent,transcriptional activities.Hedgehog signaling is a central developmental pathway uniquely tied to primary cilium function.The primary cilium has emerged as a central organelle essential for Hedgehog signal transduction in mammalian systems.Futher studies need to focus on how FLCN protein regulates Hedgehog signaling pathway through primary cilia.Objective:This study is to explore the functions of FLCN protein in cilia of HKC-8 cells,UOK257 cells and UOK257-2 cells.Discuss the regulation of FLCN to Hedgehog signaling pathway in the ciliated resting cells.This study contributes to provide a novel thought and direction of treating BHD syndrome or relative tumors.Method:A series of experiments were accomplished in different cells by using cell culture,western blot,shRNA,co-immunoprecipitation,immunofluorescence,real-time PCR,cell wound healing assay.Human proximal tubular epithelial cell 8(HKC-8),FLCN-null UOK257 cell and UOK257-2 cell restored with ectopic expression of FLCN are used in our experiments.Design pcDNA3.1-FLCN-HA,pcDNA3.1-FLCN-GFP and pLKO-Tet-On-FLCN shRNA plasmids to change the level of FLCN protein.To discuss the regulation of FLCN to Hedgehog signaling pathway.The primary cilia-dependency was detected in the absence of IFT88 protein.Transfect pLKO-Tet-On-IFT88 shRNA to HKC-8 cells.Immunofluorescence was used to observe the morphology of primary cilia.Co-immunoprecipitation was used to detect the interaction between FLCN a KIF3A,KIF3B or GSK3?.Results:1.FLCN localizes to primary cilia and basal bodiesHKC-8 cells,UOK257 cells and UOK257-2 cells were cultured till cell density up to 80%,and then serum started for 48 h.Cells become resting cells.Immunofluorescence was used to observe primary cilia which marked with acetylized-tubulin.One cell had only one primary cilium.Using immunofluorescence,FLCN was observed to localize in primary cilia and basal bodies in ciliated resting cells.2.FLCN interacts with KIF3A/3B in a cilium-dependent mannerCo-immunoprecipitation was used to observe the interaction between FLCN and KIF3 A in resting cells but not cycling cells.Futher,the interaction between FLCN and KIF3A was not observing in IFT88 protein knockdown cells which loss cilia.Similar results were observed in KIF3B protein.3.Truncation domain from 315 to 345 a.a.of FLCN protein is the KIF3 A binding domainProtein expression system was used to express and purify FLCN protein(wildtype and truncations)and KIF3 A protein global domain.Protein Pull-down assay was used to observe the direct binding between FLCN protein(wildtype and truncations)and KIF3 A protein global domain.We found out that the truncation from 315 to 345 a.a.of FLCN was the KIF3A protein binding domain.4.The effect of FLCN on frequency and length of primary ciliaImmunofluorescence was used to observe the frequency and length of primary cilia.We found out that loss of FLCN didn't affect the frequency of primary cilia both in UOK cells and HKC-8 cells under NO-FLOW condition.And loss of FLCN didn't affect the frequency of primary cilia in UOK cells and HKC-8 cells under FLOW condition;But loss of FLCN increased length of primary cilia under FLOW condition.5.Loss of FLCN increases the expression of Gli1 in ciliated cells(1)Primary cilia could form in HKC-8 cells,UOK257 cells and UOK257-2 cells after serum starvation.(2)Western blot was used to observe Glil protein level in cells treated with serum starvation.We found that Gli1 protein level increased in FLCN knockdown cells but not in control cells.It means that FLCN protein inhibit Gli1 protein expression in ciliated cells.6.Loss of FLCN increases the relative protein expressions of Hedgehog pathway in ciliated cells(1)Western blot was used to observe the effect of FLCN on the expressions of Hedgehog pathway relative proteins.We found that loss of FLCN increased the expressions of Hedgehog pathway relative proteins in ciliated cells,but not in cycling cells.(2)Western blot was used to observe the effect of FLCN on the expressions of Hedgehog pathway relative proteins under FLOW condition.We found that loss of FLCN increased the expressions of Hedgehog pathway relative proteins under Non-FLOW condition,but the effect become larger under FLOW condition.7.Loss of FLCN increases the relative gene expressions of Hedgehog pathway in ciliated cellsReal-time PCR was used to observe the effect of FLCN on the expressions of Hedgehog pathway relative genes.We found that loss of FLCN increased the expressions of HIP and Patch mRNA.8.Overexpress FLCN-HA inhibites Hedgehog pathway activationWestern blot was used to observe the effect of FLCN overexpression on the expressions of Hedgehog pathway relative proteins.We found that the expressions of Hedgehog pathway relative proteins was inhibited by FLCN overexpression.It means that FLCN protein inhibit Hedgehog pathway activation.9.Loss of FLCN increases Smo accumulation in cilia(1)Immunofluorescence was used to observe Smo accumulation in primary cilia.We found that Smo accumulated in primary cilia of FLCN knockdown cells,but not in control cells.(2)To find out if the effect of FLCN on Smo accumulation in primary cilia is relative with Hedgehog pathway activation,immunofluorescence was used.We found that Smo accumulated in primary cilia after treated with SAG,whether in FLCN knockdown cells or control cells.This result means that loss of FLCN active Hedgehog pathway,leading to Smo accumulation in primary cilia.10.FLCN interacts with GSK3? in a cilium-dependent manner and regulates GSK3P localization in primary ciliaCo-immunoprecipitation was used to observe the interaction between FLCN and GSK3?.We found that FLCN interacted with GSK3? in ciliated cells,but not in cycling cells or IFT88 knockdown cells.It means that the interaction between FLCN and GSK3? is dependent on primary cilia.Furthermore,using immunofluorescence,GSK3? was observed to localize to primary cilia in control cells but not FLCN deficient cells.It means that FLCN may regulate Hedgehog pathway through collecting GSK3? into primary cilia.11.FLCN inhibits cell migration through Hedgehog pathway(1)Cells wound healing assay was used to observe the effect of FLCN on cell migration.We found that FLCN inhibited migration of cells.(2)Cells wound healing assay was used to observe the effect of FLCN on cell migration after treated with SAG or Cyclopamine.We found that control cells migration increased after treated with SAG.Conversely,FLCN knockdown cells migration decreased after treated with Cyclopamine.This result means that FLCN inhibits cell migration of cells through Hedgehog pathway.12.Loss of FLCN promotes cell survival under serum starvation(1)Cell survival assay was used to observe the effect of FLCN on cell survival under serum starvation.We found that loss of FLCN increased cell survival under serum starvation.(2)Cell survival assay was used to observe the effect of FLCN on cell survival under serum starvation after treated with SAG or Cyclopamine.We found that there was no significant difference in cell survival between UOK257-2 cells and SAG treated cells.Similarly,there was not significant difference in cell survival between UOK257 cells and Cyclopamine treated cells.This result means that loss of FLCN increases cell survival but not through Hedgehog pathway.