Kif3a deficiency reverses the skeletal abnormalities in Pkd1 deficient mice by restoring the balance between osteogenesis and adipogenesis

Pkd1 localizes to primary cilia in osteoblasts and osteocytes. Targeted deletion of Pkd1 in osteoblasts results in osteopenia and abnormalities in Runx2-mediated osteoblast development. Kif3a, an intraflagellar transport protein required for cilia function, is also expressed in osteoblasts. To assess the relationship between Pkd1 and primary cilia function on bone development, we crossed heterozygous Pkd1- and Kif3a-deficient mice to create compound Pkd1 and Kif3a-deficient mice. Pkd1 haploinsufficiency (Pkd1(+/Δ)) resulted in osteopenia, characterized by decreased bone mineral density, trabecular bone volume, and cortical thickness. In addition, deficiency of Pkd1 resulted in impaired osteoblastic differentiation and enhanced adipogenesis in both primary osteoblasts and/or bone marrow stromal cell cultures. These changes were associated with decreased Runx2 expression, increased PPARγ expression, and impaired hedgehog signaling as evidenced by decreased Gli2 expression in bone and osteoblast cultures. In contrast, heterozygous Kif3a(+/Δ) mice display no abnormalities in skeletal development or osteoblast function, but exhibited decreased adipogenic markers in bone and impaired adipogenesis in vitro in association with decreased PPARγ expression and upregulation of Gli2. Superimposed Kif3a deficiency onto Pkd1(+/Δ) mice paradoxically corrected the effects of Pkd1 deficiency on bone mass, osteoblastic differentiation, and adipogenesis. In addition, Runx2, PPARγ and Gli2 expression in bone and osteoblasts were normalized in compound double Pkd1(+/Δ) and Kif3a(+/Δ) heterozygous mice. The administration of sonic hedgehog, overexpression of Gli2, and the PC1 C-tail construct all increased Gli2 and Runx2-II expression, but decreased PPARγ2 gene expression in C3H10T1/2 cells. Our findings suggest a role for Pkd1 and Kif3a to counterbalance the regulation of osteogenesis and adipogenesis through differential regulation of Runx2 and PPARγ by Gli2.     

Twist genes regulate Runx2 and bone formation

Runx2, a master regulator of osteoblast differentiation, is expressed several days before osteoblast genes in bone anlage. In this issue of Developmental Cell, show that Twist-1 and Twist-2 suppress the activity of Runx2 and thereby regulate bone formation.     

Selective Runx2-II deficiency leads to low-turnover osteopenia in adult mice

Runx2 transcribes Runx2-II and Runx2-I isoforms with distinct N-termini. Deletion of both isoforms results in complete arrest of bone development, whereas selective loss of Runx2-II is sufficient to form a grossly intact skeleton with impaired endochondral bone development. To elucidate the role of Runx2-II in osteoblast function in adult mice, we examined heterozygous Runx2-II (Runx2-II(+/-)) and homozygous Runx2-II (Runx2-II(-/-))-deficient mice, which, respectively, lack one or both copies of Runx2-II but intact Runx2-I expression. Compared to wild-type mice, 6-week-old Runx2-II(+/-) had reduced trabecular bone volume (BV/TV%), cortical thickness (Ct.Th), and bone mineral density (BMD), decreased osteoblastic and osteoclastic markers, lower bone formation rates, impaired osteoblast maturation of BMSCs in vitro, and significant reductions in mechanical properties. Homozygous Runx2-II(-/-) mice had a more severe reduction in BMD, BV/TV%, and Ct.Th, and greater suppression of osteoblastic and osteoclastic markers than Runx2-II(+/-) mice. Non-selective Runx2(+/-) mice, which have an equivalent reduction in Runx2 expression due to the lack one copy of Runx2-I and II, however, had an intermediate reduction in BMD. Thus, selective Runx2-II mutation causes diminished osteoblastic function in an adult mouse leading to low-turnover osteopenia and suggest that Runx2-I and II have distinct functions imparted by their different N-termini.     

Twist plays an essential role in FGF and SHH signal transduction during mouse limb development

Loss of Twist gene function arrests the growth of the limb bud shortly after its formation. In the Twist(-/-) forelimb bud, Fgf10 expression is reduced, Fgf4 is not expressed, and the domain of Fgf8 and Fgfr2 expression is altered. This is accompanied by disruption of the expression of genes (Shh, Gli1, Gli2, Gli3, and Ptch) associated with SHH signalling in the limb bud mesenchyme, the down-regulation of Bmp4 in the apical ectoderm, the absence of Alx3, Alx4, Pax1, and Pax3 activity in the mesenchyme, and a reduced potency of the limb bud tissues to differentiate into osteogenic and myogenic tissues. Development of the hindlimb buds in Twist(-/-) embryos is also retarded. The overall activity of genes involved in SHH signalling is reduced.Fgf4 and Fgf8 expression is lost or reduced in the apical ectoderm, but other genes (Fgf10, Fgfr2) involved with FGF signalling are expressed in normal patterns. Twist(+/-);Gli3(+/XtJ) mice display more severe polydactyly than that seen in either Twist(+/-) or Gli3(+/XtJ) mice, suggesting that there is genetic interaction between Twist and Gli3 activity. Twist activity is therefore essential for the growth and differentiation of the limb bud tissues as well as regulation of tissue patterning via the modulation of SHH and FGF signal transduction.     

Haploinsufficiency of Runx2 results in bone formation decrease and different BSP expression pattern changes in two transgenic mouse models

Runx2 has been identified as "a master gene" for the differentiation of osteoblasts and Runx2-deficient mice has demonstrated a complete absence of mature osteoblast and ossification. To further characterize the Runx2 responsive elements within the bone sialoprotein (BSP) promoter and further investigate into the role of Runx2 haploinsufficiency in osteoblast differentiation, mBSP9.0Luc mice and mBSP4.8Luc mice were crossed with Runx2-deficient mice respectively. Luciferase assay, micro CT scan, and histological analysis were performed using tissues isolated from mBSP9.0luc/Runx2+/- mice, mBSP4.8luc/Runx2+/- mice and their corresponding Runx2+/+ littermates. Alkaline phosphatase activity, mineralization assays and RT-PCR analysis using calvarial osteoblasts isolated from these transgenic mice were also performed. Luciferase assay demonstrated an early increase in luciferase expression in mBSP9.0luc/Runx2+/- mice before the expression level of luciferase dramatically decreased and turned lower than that in their control littermates in later stages. In contrast, luciferase expression in mBSP4.8luc/Runx2+/- failed to show such an early increase. Micro CT scan and histological analysis showed that BMD and trabecular bone volume were decreased and bone formation was delayed in Runx2+/- mice. Furthermore, mineralization assay and semi-quantitative RT-PCR assay demonstrated a gene-dose-dependent decrease in bone nodule formation and bone marker genes expression levels in cultured calvarial osteoblasts derived from Runx2 knockout mice. Reconstitution of Runx2-null cells with Runx2 vector partially rescued the osteoblast function defects. In conclusion, the 9.0 kb BSP promoter demonstrated a higher tissue-specific regulation of the BSP gene by Runx2 in vivo and full Runx2 gene dose is essential for osteoblast differentiation and normal bone formation.