Objective: To study the effect of signal-selective parathyroid hormone (PTH) analogue peptide on Wnt signaling factors in osteoblasts isolated from neonatal mouse, and provide theoretical basis for the mechanism of PTH's function in bone metabolism.
Methods: Osteoblasts were isolated from calvaria of 2-3-day-old C57BL neonatal mouse and identified by alkaline phosphatase (ALP) staining, and Alizarin red staining. The cells at passage 1 were divided into 4 groups: control group, PTH (1-34) group, G1R19 (1-34) group, and G1R19 (1-28) group. Then the medium was changed to a-MEM supplemented with 1% FBS. After 12 hours, trifluoroacetic acid or three peptides [(10 nmol/L PTH (1-34), 10 nmol/L G1R19 (1-34), and 100 nmol/L G1R19 (1-28)] were added into the culture medium. After 4 hours, the cells were washed gently with cold PBS 3 times before total RNA was isolated. The expressions of Wnt related genes were measured by quantitative real-time PCR.
Results: Most of the cells were polygonal and triangular; the cells were positive for ALP staining with blue cytoplasm at 14 days and the Alizarin red staining showed the formation of red mineralized nodules in the special mineralization induction medium at 28 days. The expressions of osteocalcin mRNA and Wnt5b mRNA in PTH (1-34) group, G1R19 (1-34) group, and G1R19 (1-28) group were significantly higher than those in control group (P < 0.05); the expression of Wnt2 mRNA was significantly lower than that in control group (P < 0.05); the expression of beta-catenin mRNA in PTH (1-34) group was significantly higher than that in control group (P < 0.05); the expression of Wnt7b mRNA in PTH (1-34) group and G1R19 (1-34) group was higher than that in control group, and the G1R19 (1-34) group was higher than PTH (1-34) group and G1R19 (1-28) group (P < 0.05).
Conclusion: In the Wnt-related factors, PTH (1-34) and G1R19 (1-34) affect mainly canonical Wnt signal factors, but the G1R19 (1-28) chiefly acts on non-canonical Wnt signal factors.