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Inhibition of phosphodiesterase 5 reduces bone mass by suppression of canonical Wnt signaling.

Gong Y, Xu CY, Wang JR, Hu XH, Hong D, Ji X, Shi W, Chen HX, Wang HB, Wu XM - Cell Death Dis (2014)

Bottom Line: In the in vitro experiments, PDE5 inhibition resulted in activation of cGMP-dependent protein kinase 2 and consequent inhibition of glycogen synthase kinase 3β phosphorylation, destabilization of cytosolic β-catenin and the ultimate suppression of canonical Wnt signaling and reduced osteoblastic differentiation in HEK293T and C3H10T1/2 cells.In animal experiments, systemic inhibition of PDE5 suppressed the activity of canonical Wnt signaling and osteoblastogenesis in bone marrow-derived stromal cells, resulting in the reduction of bone mass in wild-type adult C57B/6 mice, significantly attenuated secreted Frizzled-related protein-1 (SFRP1) deletion-induced activation of canonical Wnt signaling and excessive bone growth in adult SFRP1(-/-) mice.Together, these results uncover a hitherto uncharacterized role of PDE5/cGMP/PKG signaling in bone homeostasis and provide the evidence that long-term treatment with PDE5 inhibitors at a high dosage may potentially cause bone catabolism.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, School of Medicine, Zhejiang University, Hangzhou 310058, China.

ABSTRACT
Inhibitors of phosphodiesterase 5 (PDE5) are widely used to treat erectile dysfunction and pulmonary hypertension in clinics. PDE5, cyclic guanosine monophosphate (cGMP), and protein kinase G (PKG) are important components of the non-canonical Wnt signaling. This study aimed to investigate the effect of PDE5 inhibition on canonical Wnt signaling and osteoblastogenesis, using both in vitro cell culture and in vivo animal models. In the in vitro experiments, PDE5 inhibition resulted in activation of cGMP-dependent protein kinase 2 and consequent inhibition of glycogen synthase kinase 3β phosphorylation, destabilization of cytosolic β-catenin and the ultimate suppression of canonical Wnt signaling and reduced osteoblastic differentiation in HEK293T and C3H10T1/2 cells. In animal experiments, systemic inhibition of PDE5 suppressed the activity of canonical Wnt signaling and osteoblastogenesis in bone marrow-derived stromal cells, resulting in the reduction of bone mass in wild-type adult C57B/6 mice, significantly attenuated secreted Frizzled-related protein-1 (SFRP1) deletion-induced activation of canonical Wnt signaling and excessive bone growth in adult SFRP1(-/-) mice. Together, these results uncover a hitherto uncharacterized role of PDE5/cGMP/PKG signaling in bone homeostasis and provide the evidence that long-term treatment with PDE5 inhibitors at a high dosage may potentially cause bone catabolism.

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Tadalafil reduced bone mass in the adult wild-type mice. (a) Tadalafilreduced bone mass of distal femur in the adult mice. H&E staining of paraffinsections and μCT analyses of the distal femur from miceintragastrically treated with normal saline or indicated dosages of tadalafildaily for 2 months. (b and c) Quantification of bone parameters fromthree-dimensional reconstruction μCT. (d–g)Tadalafil treatment reduced the mRNA levels of osteoblast marker genes(AP and RunX2) and target genes of canonical Wnt signaling(Lef1 and Dkk1) in BMSCs compared with the vehicle treatment.(h) Immunohistochemistry analyses of AP, Runx2, Lef1, and DKK1expression in the distal femur of mice treated with vehicle or indicated dosagesof tadalafil. *P<0.05, **P<0.01versus vehicle treatment.
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fig5: Tadalafil reduced bone mass in the adult wild-type mice. (a) Tadalafilreduced bone mass of distal femur in the adult mice. H&E staining of paraffinsections and μCT analyses of the distal femur from miceintragastrically treated with normal saline or indicated dosages of tadalafildaily for 2 months. (b and c) Quantification of bone parameters fromthree-dimensional reconstruction μCT. (d–g)Tadalafil treatment reduced the mRNA levels of osteoblast marker genes(AP and RunX2) and target genes of canonical Wnt signaling(Lef1 and Dkk1) in BMSCs compared with the vehicle treatment.(h) Immunohistochemistry analyses of AP, Runx2, Lef1, and DKK1expression in the distal femur of mice treated with vehicle or indicated dosagesof tadalafil. *P<0.05, **P<0.01versus vehicle treatment.

Mentions: To assess the potential role of inhibition of PDE5 in bone mass in vivo,we first evaluated the effect of tadalafil on osteoblastogenesis andWnt/β-cat signaling in wild-type adult (2 months) C57BL/6mice. Orally administered for 2 months at 45 or75 mg/kg daily that was20- and 32-fold higher than the clinical dosage for ED (10 mg daily) and 5-and 8-fold higher than the clinical dosage for PH (40 mg daily),17, 18 tadalafil robustly decreased the mass of cancellous bonebut not of cortical bone as revealed by morphological and histological analyses(Figure 5a). Three-dimensional reconstruction ofthe distal femur using micro computed tomography (μCT) confirmed thattadalafil at both 45 and 75 mg/kg resulted in decreases in bone mineraldensity (BMD) by 30 and 35%, in trabecular bone volume (BMTV) by 42 and48%, in trabecular number (TbN) by 35 and 50%, but increases intrabecular separation (TbSp) by 58 and 97%, respectively, while it had noeffect on trabecular thickness (TbTh), when compared with the vehicle treatment(Figures 5a–c, and Supplementary Figure 3a and b). Moreover, tadalafil at 45 and75 mg/kg reduced mRNA levels of Lef1 (34 and 49%, respectively)and Dkk1 (24 and 56%, respectively) in bone marrow-derived stromal cells(BMSCs) (Figures 5f and g). In line with theinhibition of canonical signaling, tadalafil dose dependently reduced mRNA levelsof osteoblastogenic markers including AP (18 and 38%, respectively) andRunx2 (18 and 43%, respectively) (Figures 5d ande). Finally, tadalafil robustly decreased the numbers of not only Lef-and Dkk1-positive cells but also AP- and Runx2-positive cells as revealed byimmunohistochemical staining of longitudinal sections of the distal femur (Figure 5h). Taken together, these results demonstrate thatsystemic inhibition of PDE5 may lead to robust inhibition of osteoblastogenesisand consequent reduction in bone mass possibly through inhibition of canonical Wntsignaling in adult mice.


Inhibition of phosphodiesterase 5 reduces bone mass by suppression of canonical Wnt signaling.

Gong Y, Xu CY, Wang JR, Hu XH, Hong D, Ji X, Shi W, Chen HX, Wang HB, Wu XM - Cell Death Dis (2014)

Tadalafil reduced bone mass in the adult wild-type mice. (a) Tadalafilreduced bone mass of distal femur in the adult mice. H&E staining of paraffinsections and μCT analyses of the distal femur from miceintragastrically treated with normal saline or indicated dosages of tadalafildaily for 2 months. (b and c) Quantification of bone parameters fromthree-dimensional reconstruction μCT. (d–g)Tadalafil treatment reduced the mRNA levels of osteoblast marker genes(AP and RunX2) and target genes of canonical Wnt signaling(Lef1 and Dkk1) in BMSCs compared with the vehicle treatment.(h) Immunohistochemistry analyses of AP, Runx2, Lef1, and DKK1expression in the distal femur of mice treated with vehicle or indicated dosagesof tadalafil. *P<0.05, **P<0.01versus vehicle treatment.
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Related In: Results  -  Collection

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fig5: Tadalafil reduced bone mass in the adult wild-type mice. (a) Tadalafilreduced bone mass of distal femur in the adult mice. H&E staining of paraffinsections and μCT analyses of the distal femur from miceintragastrically treated with normal saline or indicated dosages of tadalafildaily for 2 months. (b and c) Quantification of bone parameters fromthree-dimensional reconstruction μCT. (d–g)Tadalafil treatment reduced the mRNA levels of osteoblast marker genes(AP and RunX2) and target genes of canonical Wnt signaling(Lef1 and Dkk1) in BMSCs compared with the vehicle treatment.(h) Immunohistochemistry analyses of AP, Runx2, Lef1, and DKK1expression in the distal femur of mice treated with vehicle or indicated dosagesof tadalafil. *P<0.05, **P<0.01versus vehicle treatment.
Mentions: To assess the potential role of inhibition of PDE5 in bone mass in vivo,we first evaluated the effect of tadalafil on osteoblastogenesis andWnt/β-cat signaling in wild-type adult (2 months) C57BL/6mice. Orally administered for 2 months at 45 or75 mg/kg daily that was20- and 32-fold higher than the clinical dosage for ED (10 mg daily) and 5-and 8-fold higher than the clinical dosage for PH (40 mg daily),17, 18 tadalafil robustly decreased the mass of cancellous bonebut not of cortical bone as revealed by morphological and histological analyses(Figure 5a). Three-dimensional reconstruction ofthe distal femur using micro computed tomography (μCT) confirmed thattadalafil at both 45 and 75 mg/kg resulted in decreases in bone mineraldensity (BMD) by 30 and 35%, in trabecular bone volume (BMTV) by 42 and48%, in trabecular number (TbN) by 35 and 50%, but increases intrabecular separation (TbSp) by 58 and 97%, respectively, while it had noeffect on trabecular thickness (TbTh), when compared with the vehicle treatment(Figures 5a–c, and Supplementary Figure 3a and b). Moreover, tadalafil at 45 and75 mg/kg reduced mRNA levels of Lef1 (34 and 49%, respectively)and Dkk1 (24 and 56%, respectively) in bone marrow-derived stromal cells(BMSCs) (Figures 5f and g). In line with theinhibition of canonical signaling, tadalafil dose dependently reduced mRNA levelsof osteoblastogenic markers including AP (18 and 38%, respectively) andRunx2 (18 and 43%, respectively) (Figures 5d ande). Finally, tadalafil robustly decreased the numbers of not only Lef-and Dkk1-positive cells but also AP- and Runx2-positive cells as revealed byimmunohistochemical staining of longitudinal sections of the distal femur (Figure 5h). Taken together, these results demonstrate thatsystemic inhibition of PDE5 may lead to robust inhibition of osteoblastogenesisand consequent reduction in bone mass possibly through inhibition of canonical Wntsignaling in adult mice.

Bottom Line: In the in vitro experiments, PDE5 inhibition resulted in activation of cGMP-dependent protein kinase 2 and consequent inhibition of glycogen synthase kinase 3β phosphorylation, destabilization of cytosolic β-catenin and the ultimate suppression of canonical Wnt signaling and reduced osteoblastic differentiation in HEK293T and C3H10T1/2 cells.In animal experiments, systemic inhibition of PDE5 suppressed the activity of canonical Wnt signaling and osteoblastogenesis in bone marrow-derived stromal cells, resulting in the reduction of bone mass in wild-type adult C57B/6 mice, significantly attenuated secreted Frizzled-related protein-1 (SFRP1) deletion-induced activation of canonical Wnt signaling and excessive bone growth in adult SFRP1(-/-) mice.Together, these results uncover a hitherto uncharacterized role of PDE5/cGMP/PKG signaling in bone homeostasis and provide the evidence that long-term treatment with PDE5 inhibitors at a high dosage may potentially cause bone catabolism.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, School of Medicine, Zhejiang University, Hangzhou 310058, China.

ABSTRACT
Inhibitors of phosphodiesterase 5 (PDE5) are widely used to treat erectile dysfunction and pulmonary hypertension in clinics. PDE5, cyclic guanosine monophosphate (cGMP), and protein kinase G (PKG) are important components of the non-canonical Wnt signaling. This study aimed to investigate the effect of PDE5 inhibition on canonical Wnt signaling and osteoblastogenesis, using both in vitro cell culture and in vivo animal models. In the in vitro experiments, PDE5 inhibition resulted in activation of cGMP-dependent protein kinase 2 and consequent inhibition of glycogen synthase kinase 3β phosphorylation, destabilization of cytosolic β-catenin and the ultimate suppression of canonical Wnt signaling and reduced osteoblastic differentiation in HEK293T and C3H10T1/2 cells. In animal experiments, systemic inhibition of PDE5 suppressed the activity of canonical Wnt signaling and osteoblastogenesis in bone marrow-derived stromal cells, resulting in the reduction of bone mass in wild-type adult C57B/6 mice, significantly attenuated secreted Frizzled-related protein-1 (SFRP1) deletion-induced activation of canonical Wnt signaling and excessive bone growth in adult SFRP1(-/-) mice. Together, these results uncover a hitherto uncharacterized role of PDE5/cGMP/PKG signaling in bone homeostasis and provide the evidence that long-term treatment with PDE5 inhibitors at a high dosage may potentially cause bone catabolism.

Show MeSH
Related in: MedlinePlus