title: Primary cilium is required for the stimulating effect of icaritin on osteogenic differentiation and mineralization of osteoblasts in vitro.
abstract: OBJECTIVE
  Icaritin, one effective metabolite of Herba Epimedii-derived flavonoid icariin, has a strong osteogenic activity. However, its action mechanism remains unclear. Since primary cilia have been shown to play a pivotal role in regulating the osteogenesis, we hypothesized primary cilia are indispensable in mediating icaritin osteogenic effect.
  
  
  METHODS
  Primary rat calvarial osteoblasts were transfected with siRNA1 targeting intraflagellar transport protein 88 (IFT88), a protein required for ciliogenesis, to prevent formation of primary cilium and were treated with 10-6M icaritin.
  
  
  RESULTS
  Alkaline phosphatase (ALP) activity was significantly increased after 3 days in cells transfected with scrambled siRNA control and treated by icaritin (SC+I group) compared to cells transfected with scrambled siRNA control only (SC group). ALP activity after IFT88 siRNA1 transfection and icaritin treatment (siRNA1+I group) was significantly lower than that of SC+I group. Formation of ALP positively stained colonies after 6 days, osteocalcin secretion after 9 days and formation of calcified nodules after 12 days displayed a similar tendency among the three groups. mRNA expression of osteogenesis-related genes ALP, BMP-2, COL1α, RUNX-2 and OSX after 24 h was significantly increased in SC+I group, but was not different with SC group in siRNA1+I group. Protein levels of BMP-2, COL1α, RUNX-2 and OSX after 48 h showed the similar tendency with gene expression.
  
  
  CONCLUSIONS
  Primary cilia are important in mediating icaritin-stimulated osteogenic differentiation and may be a novel target for pharmacological therapies for bone loss.
authors: Institute of Orthopaedics, Lanzhou General Hospital, Lanzhou Command of CPLA, Lanzhou, 730050, People's Republic of China.|Department of Endocrinology, The First Hospital of Lanzhou University, Lanzhou, 730000, People's Republic of China.
authors: X-N
authors: XN
authors: Ma
authors: Department of Laboratory, Affiliated Hospital of Gansu University of Chinese Medicine, Lanzhou, 730000, People's Republic of China.
authors: C-X
authors: CX
authors: Ma
authors: Institute of Orthopaedics, Lanzhou General Hospital, Lanzhou Command of CPLA, Lanzhou, 730050, People's Republic of China.
authors: W-G
authors: WG
authors: Shi
authors: Institute of Orthopaedics, Lanzhou General Hospital, Lanzhou Command of CPLA, Lanzhou, 730050, People's Republic of China.
authors: J
authors: J
authors: Zhou
authors: Department of Pharmacy, Lanzhou General Hospital of CPLA, Lanzhou, 730050, People's Republic of China.
authors: H-P
authors: HP
authors: Ma
authors: Institute of Orthopaedics, Lanzhou General Hospital, Lanzhou Command of CPLA, Lanzhou, 730050, People's Republic of China.
authors: Y-H
authors: YH
authors: Gao
authors: Sansom Institute for Health Research, School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, SA, 5001, Australia.
authors: C J
authors: CJ
authors: Xian
authors: Institute of Orthopaedics, Lanzhou General Hospital, Lanzhou Command of CPLA, Lanzhou, 730050, People's Republic of China. chenkm@lut.cn.
authors: K-M
authors: KM
authors: Chen
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D055396:Bone Morphogenetic Protein 2 / Q000378:metabolism; D002113:Calcification, Physiologic / Q000187:drug effects*; D002454:Cell Differentiation / Q000187:drug effects*; D002478:Cells, Cultured; D002923:Cilia / Q000502:physiology*; D005260:Female; D005419:Flavonoids / Q000494:pharmacology*; D066298:In Vitro Techniques; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D010012:Osteogenesis / Q000187:drug effects / Q000502:physiology*; D051381:Rats; D017208:Rats, Wistar
chemical_list: D055396:Bone Morphogenetic Protein 2; D005419:Flavonoids; D000469:Alkaline Phosphatase; C499403:icaritin
keywords: Differentiation; Icaritin; Mineralization; Osteoblasts; Primary cilia; Small interfering RNA