SAG therapy restores bone growth and reduces enchondroma incidence in a model of skeletal chondrodysplasias caused by Ihh deficiency

Abstract

Inactivation mutations in the Indian hedgehog (Ihh) gene in humans cause numerous skeletal chondrodysplasias, including acrocapitofemoral dysplasia, brachydactyly type A1, and human short stature. The lack of an appropriate human-relevant model to accurately represent these chondrodysplasias has hampered the identification of clinically effective treatments. Here, we established a mouse model of human skeletal dysplasia induced by Ihh gene mutations via ablation of Ihh in Aggrecan-positive (Acan+) cells using Aggrecan (Acan)-creERT transgenic mice. Smoothen agonist (SAG) promoted Hh activity and rescued chondrocyte proliferation and differentiation by stimulating smoothened trafficking to the cilium in Ihh-silenced cells. SAG treatment corrected mouse stature and significantly decreased mortality without evidence of toxicity. Moreover, Ihh ablation in Acan+ cells produced enchondroma-like tissues near the growth plates that were significantly reduced by SAG treatment. These results demonstrated that SAG effectively treats skeletal dysplasia caused by Ihh gene mutations in a mouse model, suggesting that SAG may represent a potential drug for the treatment of these diseases and/or enchondromas.

GRK2 Kinases in the Primary Cilium Initiate SMOOTHENED-PKA Signaling in the Hedgehog Cascade

“…Here we observed sparse but detectable ciliary anti-pSMO staining that was dramatically enhanced by treatment of embryos with SAG21k, demonstrating endogenous SMO phosphorylation in these structures and consistent with the ability of this SMO agonist to ectopically activate Hh signaling during embryogenesis (Fig. S10) [95][96][97][98][99] . Thus, SMO undergoes GRK2 phosphorylation during eye and brain development.…”

Role of Primary Cilia in Skeletal Disorders

“…Ho et al [ 30 ] reported that only 13.4% of cells are ciliated in enchondroma tissues, which significantly decreased compared with control articular cartilage ( Figure 1 ). Recently, we found that the cilium incidence and cilium length were comparable between human enchondroma cells and control articular chondrocytes, but the cilium orientation largely alters [ 31 ]. The different sample resources may contribute to variation in different samples, and the cilium features in more human enchondroma samples are needed to be identified in the future.…”

“…Hedgehog (Hh) concentration distribution gradient is essential for normal chondrocyte proliferation and differentiation. However, the Hh gradient is disrupted and showed a homogeneous pattern in enchondroma or osteochondroma [ 31 ]. Activated Hh signaling in the growth plate (Col2 α 1-Gli2 overexpressed mouse) leads to enchondroma in mice.…”

“…Some studies [ 32 , 33 ] suggested that Hh signaling is essential for bone tumor growth and process; loss of primary cilium-disrupted Hh signaling can inhibit tumor growth or process. However, we found that Indian hedgehog (Ihh) ablation in aggrecan-positive progenitors produced enchondroma-like tissues near the growth plates in mice, and smoothen agonists can significantly reduce the enchondroma incidence in Ihh-knockout mouse [ 31 ]. Consistently, the dual and opposing roles of primary cilia and Hh signaling were also found in medulloblastoma development [ 34 ].…”

Type II collagen-positive progenitors are important stem cells in controlling skeletal development and vascular formation

“…The embedded samples were cryosectioned at 6 μm using a cryostat (CM1850, Leica). 52 Safranin O/fast green staining The mouse tibia was sectioned and stained with safranin O/fast green to visualize cartilage and assess the proteoglycan content, as described previously. 53 The samples on the slides were stained with Weigert's iron hematoxylin and fast green and then with 0.1% safranin O solution.…”