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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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Related in: MedlinePlus

Tadalafil reduced bone mass in the adult SFRP1−/− mice.(a) Tadalafil reduced bone mass of the distal femur in the adultSFRP1−/− mice. H&E staining of paraffin sectionsand μCT analyses of the distal femur fromSFRP1+/− or SFRP1−/− miceintragastrically administrated with normal saline or indicated dosage of tadalafildaily for 2 months. (b and c) Quantification of bone parameters fromthree-dimensional reconstruction μCT. (d and e)Formation of mineralized nodules in BMSCs from the above mice. BMSCs were isolatedfrom the indicated mice and were cultured in the media containing ascorbic acid,β-glycerophosphate, and dexamethasone in the presence or absenceof indicated concentration of tadalafil. After incubation for 21 days, cells weredetected for bone nodules by alizarin-red staining and quantitative determination.(f–i) Tadalafil treatment reduced the mRNA levels ofosteoblast marker genes (AP and RunX2) and target genes ofcanonical Wnt signaling (Lef1 and Dkk1) in BMSCs from the femurand tibia of above mice. (h) Immunohistochemistry analyses of AP, Runx2,Lef1, and DKK1 expression in the distal femur ofSFRP1+/− or SFRP1−/− micetreated with vehicle or indicated dosages of tadalafil. *P<0.05,**P<0.01 versus vehicle treatment.
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fig6: Tadalafil reduced bone mass in the adult SFRP1−/− mice.(a) Tadalafil reduced bone mass of the distal femur in the adultSFRP1−/− mice. H&E staining of paraffin sectionsand μCT analyses of the distal femur fromSFRP1+/− or SFRP1−/− miceintragastrically administrated with normal saline or indicated dosage of tadalafildaily for 2 months. (b and c) Quantification of bone parameters fromthree-dimensional reconstruction μCT. (d and e)Formation of mineralized nodules in BMSCs from the above mice. BMSCs were isolatedfrom the indicated mice and were cultured in the media containing ascorbic acid,β-glycerophosphate, and dexamethasone in the presence or absenceof indicated concentration of tadalafil. After incubation for 21 days, cells weredetected for bone nodules by alizarin-red staining and quantitative determination.(f–i) Tadalafil treatment reduced the mRNA levels ofosteoblast marker genes (AP and RunX2) and target genes ofcanonical Wnt signaling (Lef1 and Dkk1) in BMSCs from the femurand tibia of above mice. (h) Immunohistochemistry analyses of AP, Runx2,Lef1, and DKK1 expression in the distal femur ofSFRP1+/− or SFRP1−/− micetreated with vehicle or indicated dosages of tadalafil. *P<0.05,**P<0.01 versus vehicle treatment.

Mentions: We next assessed the effect of tadalafil on osteoblastogenesis and Wnt signalingin 2-month-old SFRP1 knockout (SFRP1−/−) mice. At thebaseline level, SFRP1−/− mice had a significantlyhigher mass of cancellous bone but not cortical bone as compared withSFRP1+/− mice, as revealed by histological analysisof the longitudinal sections of the distal femur. At an oral dose of75 mg/kg daily for 2 months, tadalafil robustly decreased the mass ofcancellous bone but not in cortical bone in SFRP1−/−mice (Figure 6a). Three-dimensional reconstruction ofthe distal femur using μCT further confirmed that there weresignificant increases in BMD (2.1-fold), BMTV (2.3-fold), and TbN (1.5-fold) butnot in TbTh and TbSp in SFRP1−/− mice as compared withSFRP1+/− mice, and that tadalafil treatment resultedin significant decreases in BMD (38%), BMTV (28%), and TbN(47%), but not in TbTh and TbSp as compared with the vehicle treatment(Figures 6a–c, and Supplementary Figure 4c and d). Likewise, BMSCs fromSFRP1−/− mice formed much more mineralized nodulesthan those from SFRP1+/− mice (1.4-fold), and tadalafilattenuated SFRP1 knockout-associated excessive formation of mineralized nodules by~25% over vehicle (Figures 6d and e). Moreover,BMSCs from SFRP1−/− mice exhibited higher transcriptionactivities of Lef1 and Dkk1 (13- and 14-fold, respectively) than thosefrom SFRP1+/− mice, whereas systemic treatment withtadalafil in SFRP1−/− mice robustly attenuated thetranscription of these genes (39 and 54%, respectively; Figures 6h and i). Similarly, BMSCs fromSFRP1−/− mice also exhibited higher transcriptionalactivities of osteoblast differential markers including AP and Runx2 (24- and26-fold, respectively) than those from SFRP1+/− mice,and systemic treatment with tadalafil in SFRP1−/− micerobustly attenuated the transcription of these markers (21 and 16%,respectively; Figures 6f and g). As demonstrated byimmunohistochemistry staining of the sections of the distal femur, deletion ofSFRP1 in mice led to a robust increase in the numbers of Lef1- and Dkk1-positivecells, as well as AP- and Runx2-positive cells, and that systemic treatment withtadalafil in SFRP1−/− mice robustly attenuated theincreases in the number of not only Lef1- and Dkk1-positive cells but also AP- andRunx2-positive cells (Figure 6j). As indicated bytartrate-resistant acid phosphatase (TRAP) staining, neither deletion of SFRP1 norsystemic treatment with tadalafil had any significant effect on the number ofTRAP-positive cells in SFRP1−/− mice (Supplementary Figure 3a and 3b). Together, these resultsfurther confirm that systemic inhibition of PDE5 specifically reduces theexcessive bone growth derived from forced activation of canonical Wnt signaling inadult SFRP1−/− 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 SFRP1−/− mice.(a) Tadalafil reduced bone mass of the distal femur in the adultSFRP1−/− mice. H&E staining of paraffin sectionsand μCT analyses of the distal femur fromSFRP1+/− or SFRP1−/− miceintragastrically administrated with normal saline or indicated dosage of tadalafildaily for 2 months. (b and c) Quantification of bone parameters fromthree-dimensional reconstruction μCT. (d and e)Formation of mineralized nodules in BMSCs from the above mice. BMSCs were isolatedfrom the indicated mice and were cultured in the media containing ascorbic acid,β-glycerophosphate, and dexamethasone in the presence or absenceof indicated concentration of tadalafil. After incubation for 21 days, cells weredetected for bone nodules by alizarin-red staining and quantitative determination.(f–i) Tadalafil treatment reduced the mRNA levels ofosteoblast marker genes (AP and RunX2) and target genes ofcanonical Wnt signaling (Lef1 and Dkk1) in BMSCs from the femurand tibia of above mice. (h) Immunohistochemistry analyses of AP, Runx2,Lef1, and DKK1 expression in the distal femur ofSFRP1+/− or SFRP1−/− micetreated with vehicle or indicated dosages of tadalafil. *P<0.05,**P<0.01 versus vehicle treatment.
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fig6: Tadalafil reduced bone mass in the adult SFRP1−/− mice.(a) Tadalafil reduced bone mass of the distal femur in the adultSFRP1−/− mice. H&E staining of paraffin sectionsand μCT analyses of the distal femur fromSFRP1+/− or SFRP1−/− miceintragastrically administrated with normal saline or indicated dosage of tadalafildaily for 2 months. (b and c) Quantification of bone parameters fromthree-dimensional reconstruction μCT. (d and e)Formation of mineralized nodules in BMSCs from the above mice. BMSCs were isolatedfrom the indicated mice and were cultured in the media containing ascorbic acid,β-glycerophosphate, and dexamethasone in the presence or absenceof indicated concentration of tadalafil. After incubation for 21 days, cells weredetected for bone nodules by alizarin-red staining and quantitative determination.(f–i) Tadalafil treatment reduced the mRNA levels ofosteoblast marker genes (AP and RunX2) and target genes ofcanonical Wnt signaling (Lef1 and Dkk1) in BMSCs from the femurand tibia of above mice. (h) Immunohistochemistry analyses of AP, Runx2,Lef1, and DKK1 expression in the distal femur ofSFRP1+/− or SFRP1−/− micetreated with vehicle or indicated dosages of tadalafil. *P<0.05,**P<0.01 versus vehicle treatment.
Mentions: We next assessed the effect of tadalafil on osteoblastogenesis and Wnt signalingin 2-month-old SFRP1 knockout (SFRP1−/−) mice. At thebaseline level, SFRP1−/− mice had a significantlyhigher mass of cancellous bone but not cortical bone as compared withSFRP1+/− mice, as revealed by histological analysisof the longitudinal sections of the distal femur. At an oral dose of75 mg/kg daily for 2 months, tadalafil robustly decreased the mass ofcancellous bone but not in cortical bone in SFRP1−/−mice (Figure 6a). Three-dimensional reconstruction ofthe distal femur using μCT further confirmed that there weresignificant increases in BMD (2.1-fold), BMTV (2.3-fold), and TbN (1.5-fold) butnot in TbTh and TbSp in SFRP1−/− mice as compared withSFRP1+/− mice, and that tadalafil treatment resultedin significant decreases in BMD (38%), BMTV (28%), and TbN(47%), but not in TbTh and TbSp as compared with the vehicle treatment(Figures 6a–c, and Supplementary Figure 4c and d). Likewise, BMSCs fromSFRP1−/− mice formed much more mineralized nodulesthan those from SFRP1+/− mice (1.4-fold), and tadalafilattenuated SFRP1 knockout-associated excessive formation of mineralized nodules by~25% over vehicle (Figures 6d and e). Moreover,BMSCs from SFRP1−/− mice exhibited higher transcriptionactivities of Lef1 and Dkk1 (13- and 14-fold, respectively) than thosefrom SFRP1+/− mice, whereas systemic treatment withtadalafil in SFRP1−/− mice robustly attenuated thetranscription of these genes (39 and 54%, respectively; Figures 6h and i). Similarly, BMSCs fromSFRP1−/− mice also exhibited higher transcriptionalactivities of osteoblast differential markers including AP and Runx2 (24- and26-fold, respectively) than those from SFRP1+/− mice,and systemic treatment with tadalafil in SFRP1−/− micerobustly attenuated the transcription of these markers (21 and 16%,respectively; Figures 6f and g). As demonstrated byimmunohistochemistry staining of the sections of the distal femur, deletion ofSFRP1 in mice led to a robust increase in the numbers of Lef1- and Dkk1-positivecells, as well as AP- and Runx2-positive cells, and that systemic treatment withtadalafil in SFRP1−/− mice robustly attenuated theincreases in the number of not only Lef1- and Dkk1-positive cells but also AP- andRunx2-positive cells (Figure 6j). As indicated bytartrate-resistant acid phosphatase (TRAP) staining, neither deletion of SFRP1 norsystemic treatment with tadalafil had any significant effect on the number ofTRAP-positive cells in SFRP1−/− mice (Supplementary Figure 3a and 3b). Together, these resultsfurther confirm that systemic inhibition of PDE5 specifically reduces theexcessive bone growth derived from forced activation of canonical Wnt signaling inadult SFRP1−/− 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