Posphodiesterase5Inhibitors Reduce Bone Mass By Ression Of Canonical Wnt Signaling

Wnt signaling is critical for normal development of multicellular organisms. Depending on the different Wnt ligands, people divide the signaling into canonical Wnt signaling (also known as Wnt/β-catenin signaling) and non-canonical Wnt signaling. Activation of canonical Wnt signaling will cause the stabilization and accumulation of P-catenin in the cytoplasm. The stabilized β-catenin will enter nucleus to activate transcription of downstream genes via lymphoid enhancer-binding factor-1(Lef-1) and T cell factors (TCF). Thus, the canonical Wnt signaling plays a very important role in development, particularly in skeleton development of embryos and maintaining bone mass of individual. The non-canonical Wnt signaling, also called β-catenin independent Wnt signaling, is mainly regulated by small G proteins to control Wnt/planar cell polarity (planar cell polarity, PCP) pathway and Wnt/Ca2+signaling pathways. The non-canonical Wnt signaling pathway mainly regulates cell fate, cell differentiation and cell migration. However, only a few recent studies have shown that non-canonical Wnt signaling pathway is also involved in the regulation of bone formation.Phosphodiesterase (PDEs) is a large multi-gene family that includes11PDE subtypes about30kinds of phosphodiesterase isoenzymes. PDEs are capable of hydrolyzing intracellular cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), thus the signaling they transduced as second messengers is end. The catalytic domains of PDEs determine the substrate specificity, wherein the phosphodiesterase-5(PDE5) is only capable of hydrolyzing cGMP. Inhibition of PDE5leads to the elevation of intracellular cGMP levels and activation of cGMP-dependent protein kinase G (PKG), resulting in decreases of the influx of calcium concentration and consequent relaxation of smooth muscle. As PDE5is abundantly expressed in the blood vessels supplying the corpus cavernosum of the penis and in the arterial wall of smooth muscle cells within the lungs, therefore PDE5inhibitors are clinically used in the therapy of erectile dysfunction (ED) and pulmonary hypertension (PH) as well. Since the first PDE5inhibitor Sildenafil developed listing, there has been listed other PDE5inhibitors, such as Tadalafil and Vardenafil. Previous studies have shown that PDE5/cGMP/PKG is mainly involved in the regulation of non-canonical Wnt signaling to control the intracellular calcium concentration. However, whether PDE5is involved in the regulation of canonical Wnt signaling to affect bone mass and the possible molecular mechanism is unknown.Objective:By in vitro and animal experiments, we want to figure out whether PDE5inhibitors regulate canonical Wnt signaling to affect bone mass and the possible molecular mechanism.Methods and results:1. The effects of PDE5on canonical Wnt signaling and β-catenin. We transfected293T cells with Lef1reporter plasmid, and then treated them with PDE5inhibitor Tadalafil. We found that tadalafil dose-dependently inhibited Lefl-luciferase activities induced by Wnt3a conditional medium (Wnt3a). Consistent with this result, knocking down PDE5a with PDE5a siRNA also inhibited Lefl-luciferase activities induced by Wnt3a. To further confirm this, we examined the effects of PKG inhibitor KT5823and PKG2siRNA on Lefl-luciferase activity. We found that both KT5823and PKG2siRNA increased Lefl-luciferase activities induced by Wnt3a. Next we examined the effects of Tadalafil on P-catenin levels. Through western blots and immunofluorescence, we found that Tadalafil attenuated Wnt3a-induced increase in the whole, cytosolic, and nuclear β-catenin levels. Meanwhile treatment with Tadalafil did not significantly change β-catenin mRNA levels through quantitative RT-PCR. All these results indicated that PDE5inhibitor Tadalafil negatively regulates canonical Wnt signaling through PDE5/cGMP/PKG signaling. 2. The molecular mechanism of PDE5/cGMP/PKG in regulating canonical Wnt signaling. We first examined the effects of Tadalafil and cGMP analogs8-Br-cGMP on GSK3β (Glycogen synthase kinase3β) that is an important kinase in canonical Wnt signaling and phosphorylates β-catenin at Ser33, Ser37and Thr41. Through western blots we found that Tadalafil and8-Br-cGMP attenuated the Wnt3a-induced GSK3β phosphorylation but increased the phosphorylation of β-catenin at Ser33, Ser37, and Thr41. Consistent with this result, knocking down of PKG2with PKG2siRNA led to increases in GSK3β phosphorylation but decreases in β-catenin phosphorylation. All these data illustrated that Tadalafil possibly regulates canonical Wnt signaling through regulating GSK3β activities. To confirm this, we examined the effects of GSK3β inhibitor SB216763and GSK3β siRNA on Lefl-luciferase activity. We found that Tadalafil did not change the Lefl-luciferase activity that induced by SB216763and GSK3β siRNA. Finally, we found that PKG2and GSK3β interact with each other through co-immunoprecipitation. Taken together, activation of PKG2by Tadalafil suppresses canonical Wnt signaling through activating GSK3β-mediated phosphorylation of P-catenin at Thr41, Ser37and Ser33sites.3. The effects of PDE5inhibitor Tadalafil on osteoblastic differentiation. We used C3H10T1/2cells, a mouse embryonic fibroblasts cell, that has the potential to differentiate into osteoblast or adipocyte. In a similar way to293T cells, we found that Tadalafil attenuated Wnt3a-induced increases of P-catenin levels in the whole, cytosol, and nuclear in C3H10T1/2cells. Treatment with Tadalafil also decreased the activity of alkaline phosphatase (AP), which is a marker gene of osteoblastic. Tadalafil treatment affected not only the osteoblastic marker genes AP, OSX and RunX2, but also the canonical Wnt signaling target genes Lefl and Dkkl. Consistent with these results, the formation of mineralized nodule also decreased after Tadalafil treatment. Thus, in C3H10T1/2cells, Tadalafil suppresses not only the canonical Wnt signaling, but also the osteogenesis.4. The effects of Tadalafil on bone mass in C57BL/6mice. We administrated C57BL/6mice (2-month old) with Tadalafil for2months at dosages of45and75mg/kg daily. Through microCT analyses and HE staining of femurs, we found that Tadalafil treatment led to a robust decrease in bone mass. Moreover, Tadalafil treatment reduced the transcription of osteoblastic marker genes (AP, RunX2) and canonical Wnt signaling target genes (Lef1, Dkk1) in bone marrow derived stromal cells (BMSCs) through quantitative RT-PCR and immunochemistry staining. Taken together, these results demonstrated that systemic inhibition of PDE5leads to robust inhibition of osteoblastogenesis and consequent reduction in bone mass possibly through inhibition of canonical Wnt signaling in adult mice.5. The effects of Tadalafil on bone mass in sFRP1-/-(secreted Frizzled-related protein1) mice. The sFRP1-/-mice have a high bone mass because knockout of the canonical Wnt signaling inhibitor sFRP1will lead to over activation of canonical Wnt signaling. We administrated sFRP1-/-mice (2-month old) with Tadalafil for2months at dosages of75mg/kg daily. Through microCT analyses and HE staining of femurs, we found that Tadalafil treatment led to a robust decrease in bone mass and rescued high bone mass. Moreover, Tadalafil treatment reduced the transcription of osteoblastic marker genes (AP, RunX2) and canonical Wnt signaling target genes (Lefl, Dkkl) in BMSCs through quantitative RT-PCR and immunochemistry staining. Consistent with these results, Tadalafil treatment attenuated excessive formation of mineralized nodules in BMSCs derived from sFRP1-/-mice. Taken together, these results demonstrate that systemic inhibition of PDE5specifically reduces the excessive bone growth derived from canonical Wnt signaling in adult sFRP1-/-mice.Conclusions:Tadalafil activates GSK3β-mediated phosphorylation of β-catenin through PDE5/cGMP/PKG2signaling. Thus, Tadalafil negatively regulates canonical Wnt signaling to suppress osteoblastogenesis and reduce bone mass.