title: [Activation of phospholipase C-independent protein kinase C signaling pathway of parathyroid hormone enhances CITED1 expression in mouse osteoblasts].
abstract: OBJECTIVE
  To explore the functions of phospholipase C (PLC)-independent protein kinase C signaling pathway (PTH/nonPLC/PKC) of parathyroid hormone (PTH) and its role in bone metabolism.
  
  
  METHODS
  Osteoblasts isolated from the calvaria of 2- or 3-day-old C57BL mice, identified by alkaline phosphatase staining and Alizarin red staining, were treated for 4 h with 100 nmol/L [Gly(1), Arg(19)]hPTH(1-28) plus 10 nmol/L RP-cAMP, 10 nmol/L [Gly(1), Arg(19)]hPTH(1-34) plus 10 nmol/L RP-cAMP , 10 nmol/L PTH(1-34), or and 0.1% trifluoroacetic acid (TFA). The total RNA was then isolated for screening differentially expressed genes related to PTH/nonPLC/PKC pathway using Affymetrix mouse 12x135K gene expression profile microarray, and the identified genes were confirmed by real-time quantitative PCR. MC3T3-E1 cells treated with [Gly(1), Arg(19)]hPTH(1-28)+RP-cAMP, [Gly(1), Arg(19)]hPTH(1-34)+RP-cAMP, [Gly(1), Arg(19)]hPTH(1-34)+ RP-cAMP +100 nmol/L Go6983, or 0.1% TFA were also examined for GR(1-28)- or GR(1-34)-mediated gene expression changes using real-time quantitative PCR.
  
  
  RESULTS
  Alizarin red staining visualized red mineralized nodules in the osteoblasts at 28 days of culture. According to the genechip results, we selected 56 target genes related to PTH/nonPLC/PKC pathway, among which CITED1 showed higher expressions in [Gly(1), Arg(19)]hPTH(1-34)+ RP-cAMP group than in both the control group and [Gly(1), Arg(19)]hPTH(1-28)+RP-cAMP group (P<0.05), and its expression was the highest in PTH(1-34) group (P<0.05). RT-PCR of MC3T3-E1 cells yielded consist results with those in the primary osteoblasts, and the cells treated with Go6983 (a PKC inhibitor) did not show GR(1-28)- or GR(1-34)-mediated differential expression of CITED1.
  
  
  CONCLUSIONS
  The activation of PLC-independent protein kinase C signaling pathway of PTH enhances the expression of CITED1 in mouse osteoblasts to mediate the effect of PTH on bone metabolism, and this pathway is not dependent on the activation of PLC or PKA signaling.
authors.affiliation: Department of Spinal Surgery, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China. E-mail: htony1988@163.com.
authors.forename: Song
authors.initials: S
authors.lastname: Hao
authors.forename: Yue
authors.initials: Y
authors.lastname: Meng
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.forename: Shao-Yu
authors.initials: SY
authors.lastname: Hu
authors.forename: De-Hong
authors.initials: DH
authors.lastname: Yang
mesh_terms: D000818:Animals; D051017:Apoptosis Regulatory Proteins; D002478:Cells, Cultured; D007211:Indoles; D008301:Maleimides; D051379:Mice; D008810:Mice, Inbred C57BL; D009687:Nuclear Proteins / Q000502:physiology*; D010006:Osteoblasts / Q000502:physiology*; D010281:Parathyroid Hormone / Q000502:physiology*; D011493:Protein Kinase C / Q000502:physiology*; D015398:Signal Transduction; D012886:Skull; D015534:Trans-Activators / Q000502:physiology*; D010738:Type C Phospholipases
chemical_list: C465664:2-(1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl)-3-(1H-indol-3-yl)maleimide; D051017:Apoptosis Regulatory Proteins; C102208:Cited1 protein, mouse; D007211:Indoles; D008301:Maleimides; D009687:Nuclear Proteins; D010281:Parathyroid Hormone; D015534:Trans-Activators; D011493:Protein Kinase C; D010738:Type C Phospholipases