Inhibition Of PDE5Lowers The Bone Mass Through Canonical Wnt Signaling

Objective:Wnt signaling pathway includes the canonical pathway and non-canonical pathway. Studies have shown that the canonical Wnt signaling is essential in bone development and bone formation. Phosphodiesterase-5(PDE5) specifically hydrolyzes cyclic guanosine monophosphate (cGMP) and participates in the regulation of non-canonical Wnt signaling pathway. Currently, PDE5inhibitors are used for the treatment of erectile dysfunction (ED). This research studies the effect of PDE5inhibitor Tadalafil in canonical Wnt signaling and explores the impact of PDE5inhibitors on bone.Methods:To determine the role of PDE5in the regulation of canonical Wnt signaling, we measured Lef1-luciferase activity of HEK293cell line stimulated by L conditioned medium (CM) or Wnt3a CM with treatment of different concentration (0,1,5,10μM) of Tadalafil. Inhibition of PDE5results in the accumulation of cGMP in cytoplasm leading to the activation of Protein kinase G (PKG). To determine whether PKG is involved in the regulation of Wnt signaling, we measured Lef1-luciferase activity of HEK293treated with different concentration (0,0.2,1,5μM) of PKG inhibitor KT5823and PKG siRNA transfection.When canonical Wnt signaling is active, β-catenin accumulates in the nucleus and binds to T cell factor (Tcf) and Lymphoid enhancer factor (Lef) family members to activate the transcription of target genes. We measured the protein expression (both in nucleus and cytoplasm) and mRNA level of β-catenin in HEK293with Tadalafil treatment. In C3H10T1/2cell line, the mRNA levels of alkaline phosphatase (AP), Runt-related transcription factor2(Runx2) and Osterix (OSX) were measured by RT-PCR to determine the impact of Tadalafil on the differentiation of mesenchymal stem cells (MSC) to osteoblast. Mineral deposition was measured with Alizarin Red staining.In vivo, Tadalafil (0,3,15,45mg/kg) was intragastrically injected once a day for8weeks to C57female mice. The bone mineral density (BMD) of mice was tested at0w,4w and8w. After administration, the femur of mice was isolated and BMSC was harvested. The mRNA levels of osteoblast marker genes AP, Runx2and OSX and Wnt signaling target genes (Lefl and Dkkl) in bone marrow derived mesenchymal stem cell (BMSC) were measured by RT-PCR.Results:Tadalafil reduced Lefl-luciferase activity. Tadalafil did not affect β-catenin in mRNA level but reduced the protein level of β-catenin both in nucleus and cytoplasm. PKG2knockdown significantly increased Lefl-luciferase activity. Thus, PKG2negatively regulated Wnt/p-catenin signaling. It demonstrated that PDE5inhibitor regulated Wnt/β-catenin signaling. The mRNA levels of AP, Runx2and OSX were significantly reduced by Tadalafil in C3H10T1/2cells. The mineral deposition in C3H10T1/2cell was also reduced. In vivo, the BMD of mice with Tadalafil treatment was significantly lower than vehicle. Tadalafil also reduced the expression of osteoblast marker genes and the expression of Wnt signaling target genes (Lefl and Dkk1).Conclusion:PDE5inhibitor Tadalafil lowered the bone mass through canonical Wnt signaling. NO/cGMP/PKG signaling pathway may be involved in this mechanism. We firstly report the influence of tadalafil in bone. It implies that long-term use of tadalafil may lead to the loss of bone mass. Thus it may be necessary to monitor BMD during the application of tadalafil in clinic. However, further clinical studies are warranted to confirm this conclusion.