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

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.In the canonical Wnt (Wnt/β-catenin (β-cat)) signaling cascade, Wnt binds to Frizzled (Frz) receptors and the low-density lipoprotein receptor-related protein (LRP) 5 or 6, thereby activating dishevelled, suppressing the glycogen synthase kinase 3β (GSK3β) activity and inhibiting phosphorylation of β-cat at Thr41, Ser37, and Ser33 sites. The stabilized cytosolic β-cat enters the nucleus and consequently activates its downstream target genes via lymphoid enhancer-binding factor-1 (Lef-1) and T-cell factors.^{1, 2} This signaling is fine-tuned in part via a negative feedback mechanism involving secreted and transmembrane Wnt inhibitors and activators, secreted Frz-related proteins (SFRPs), and Dickkopf-1 (Dkk1).^{3, 4}Canonical Wnt signaling is critical not only to bone development in embryogenesis but also to the maintenance of bone mass during adult life.⁵ The initial evidence came from the discoveries that in humans loss- or gain-of-function mutations in LRP5 were linked with the osteoporosis-pseudoglioma syndrome and a high-bone-density syndrome, respectively.^{6, 7, 8} Subsequent studies in mice showed that Wnt signaling might promote ossification by inducing the differentiation of bone-forming osteoblasts, suppressing the development of bone-resorbing osteoclasts, and driving the differentiation of multi-potent stem cells toward an osteoblast cell fate.⁹Non-canonical Wnt signaling is β-cat independent and consists of two main pathways: the Rho small GTPases-mediated planar cell polarity pathway and the Wnt/Ca²⁺ pathway,¹⁰ involved in various aspects of cell fate differentiation and cell movement. Non-canonical Wnt signaling has profound effects on tissue morphogenesis in a variety of vertebrate species.¹⁰ The potential role for non-canonical Wnt signaling in bone formation has been investigated recently in limited studies, which have shown that the non-canonical Wnt-Gαq/11-PKC pathway operates in mammalian osteoprogenitors to promote osteoblast development, and that Wnt16 exhibits a stimulatory effect on bone metabolism.^{11, 12, 13} Nevertheless, the molecular events in the non-canonical Wnt signaling regulation of bone development and homeostasis have yet to be further elucidated.Phosphodiesterases (PDEs) are a large family of enzymes that cleave cyclic nucleotides. To date, 11 PDE subtypes have been identified, among which PDE5 has been most extensively studied. PDE5, cyclic guanosine monophosphate (cGMP), and cGMP-dependent protein kinase (PKG) are among the major components of the non-canonical Wnt signaling pathway and are involved in the regulation of intracellular Ca²⁺ concentration.^{14, 15} It is now well established that PDE5 degrades 3''-5′- cGMP and its inhibition leads to an increase in intracellular cGMP levels and activation of protein kinase G (PKG), resulting in a decrease in Ca²⁺ influx and consequent relaxation of smooth muscles, which produces the therapeutic effects in clinical erectile dysfunction (ED) and pulmonary hypertension (PH).¹⁴ Currently, little is known regarding the involvement of PDE5 in Wnt signaling regulation of bone formation and homeostasis. The objective of this study was to determine the effect of PDE5 inhibition on canonical Wnt signaling and bone mass.