| The primary cilium,an organelle with special structure and complex function,senses and transduces extracellular mechanical and chemical stimuli into the cell through a variety of molecular signaling pathways.The primary cilia can be found on the surface of many organs in the human body.Murine 3T3-L1 fibroblast cells are the good cell models for ciliopathy study since they allow the formation of primary cilia after contact inhibition.Cells show high dependence on appropriate cell-matrix interactions for their survival.The interaction between cells and extracellular matrix influenced cell attachment and signal transduction.Collagen I(Col I),the major component of extracellular matrix,is abundant in dermal tissue where fibroblasts are located.The membrane of primary cilia contains collagen receptor integrins,however,little is known about the effect of collagen on primary cilia.We cultured 3T3-L1 cells on collagen molecule-coated dishes,which simulated the cellular microenvironment,to investigate the influence of collagen I on the cell morphology and the structure of primary cilia.Cell morphology after phalloidine staining was observed under a fluorescence microscope.The 3T3-L1 cells on Col I-coated dishes exhibited a fibroblast-like shape,which was different from the irregular round-shape of the cells on non-coated dishes.But there was no significant difference on the proliferation of 3T3-L1 cells between the two groups.Moreover,Col I promoted the ciliogenesis in confluent 3T3-L1 cells.The extracellular matrix affects many intracellular signaling pathways,including autophagy pathway,which regulates the materials recycle and the life activities in cells,and the YAP pathway,which is responsible for sensing extracellular mechanical pressure.Consequently,we focus on the two pathways respectively.Col I promotes primary cilia growth through down-regulating HDAC6-mediated autophagy.Transfection of the siRNA targeting microtubule-associated protein light chain 3(LC3)further enhanced Col I-induced growth of primary cilia.In the study of the YAP pathway,we found that YAP localization in the nucleus was dramatically increased when cells were cultured on Col I.Inhibition of YAP by Verteporfin or small interfering RNA(siRNA)against YAP reversed collagen-induced cilia elongation.The effects of extracellular matrix are widely present in tumor cell invasion and migration.We examined the effect of Col I and the enhanced ciliary length on cell migration.The results of cell-scraped wound model assay and transwell assay showed that Col I treatment promoted the migration of confluent 3T3-L1 cells while knockdown of primary cilia associated protein IFT88 or YAP attenuated the migration.The results showe that Col I promotes primary cilia growth and contributes to cell migration.During the process of Col I induced-ciliogenesis,HDAC6-mediated autophagy is inhibited while the YAP pathway is activated.As an organelle sensing changes in the external environment,primary cilia can be influenced by a variety of drug ingredients except for the regulation of extracellular matrix.Abnormal structure of the primary cilia is suggested to be closely associated with a series of congenital human diseases such as Alzheimer's disease,cardiopathy and diabetes.Silibinin,a flavanone from the milk thistle,reversed the learning and memory impairment induced with LPS and A?.Silibinin protects cardiacmyocytes against isoproterenol-induced injury and reverses the death of pancreatic islet ? cells.Previous study reported that silibinin attenuates adipogenesis in 3T3-L1 cells.It attracted our attention whether these functions of silibinin are relevant to primary cilia.Therefore,we foucused on the regulation mechanism of silibinin in primary cilia.The results showed that cultured with 50 ?M silibinin changed confluent cells into fibrous morphology accompanying shorter primary cilia,while in 100 ?M silibinin-treated cases,the cells shrank and primary cilia disappeared.Silibinin has been reported to affect cell autophagy level,and autophagy is closely related to the cilia length.So we investigate whether the autophagic pathway involved in the regulation of the primary cilia.The cells were treated with 50 ?M silibinin for 24 h,contrary to the results of Col I,the autophagy-associated proteins increased,while cilia-associated proteins decreased.Transfection of the siRNA targeting microtubule-associated protein light chain 3(LC3)reversed the silibinin-induced shortening of primary cilia.Moreover,we found that the regulation of autophagy by silibinin was mediated through histone deacetylase 6(HDAC6).Subsequently 3T3-L1 cells treated with siRNA against HDAC6 had reduced silibinin-induced autophagy and promoted primary cilia growth.Therefore silibinin negatively contributes to primary cilia length via up-regulating HDAC6-mediated autophagy.According to above results,we found that Col I promotes primary cilia growth and cell migraton through down-regulating HDAC6-mediated autophagy and up-regulating YAP nuclear expression,while silibinin,a polyphenolic flavonoid,decreased primary cilia growth through up-regulating HDAC6-mediated autophagy. |
PDF Full Text Request