Abstract: 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［Gly1, Arg19］hPTH(1-28) plus 10 nmol/L RP-cAMP, 10 nmol/L［Gly1, Arg19］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［Gly1, Arg19］hPTH(1-28)+
RP-cAMP,［Gly1, Arg19］hPTH(1-34)+RP-cAMP,［Gly1, Arg19］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［Gly1,
Arg19］hPTH(1-34)+ RP-cAMP group than in both the control group and［Gly1, Arg19］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. Conclusion 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.