Cilia-like Structures and Polycystin-1 in Osteoblasts/Osteocytes and Associated Abnormalities in Skeletogenesis and Runx2 Expression

Abstract

We examined the osteoblast/osteocyte expression and function of polycystin-1 (PC1), a transmembrane protein that is a component of the polycystin-2 (PC2)-ciliary mechano-sensor complex in renal epithelial cells. We found that MC3T3-E1 osteoblasts and MLO-Y4 osteocytes express transcripts for PC1, PC2, and the ciliary proteins Tg737 and Kif3a. Immunohistochemical analysis detected cilia-like structures in MC3T3-E1 osteoblastic and MLO-Y4 osteocyte-like cell lines as well as primary osteocytes and osteoblasts from calvaria. Pkd1 m1Bei mice have inactivating missense mutations of Pkd1 gene that encode PC1. Pkd1 m1Bei homozygous mutant mice demonstrated delayed endochondral and intramembranous bone formation, whereas heterozygous Pkd1 m1Bei mutant mice had osteopenia caused by reduced osteoblastic function. Heterozygous and homozygous Pkd1 m1Bei mutant mice displayed a gene dose-dependent decrease in the expression of Runx2 and osteoblastrelated genes. In addition, overexpression of constitutively active PC1 C-terminal constructs in MC3T3-E1 osteoblasts resulted in an increase in Runx2 P1 promoter activity and endogenous Runx2 expression as well as an increase in osteoblast differentiation markers. Conversely, osteoblasts derived from Pkd1 m1Bei homozygous mutant mice had significant reductions in endogenous Runx2 expression, osteoblastic markers, and differentiation capacity ex vivo. Co-expression of constitutively active PC1 C-terminal construct into Pkd1 m1Bei homozygous osteoblasts was sufficient to normalize Runx2 P1 promoter activity. These findings are consistent with a possible functional role of cilia and PC1 in anabolic signaling in osteoblasts/osteocytes.

Repression of osteocyte Wnt/β-catenin signaling is an early event in the progression of renal osteodystrophy

“…(77) Activating Nek8 mutations lead to increased primary cilia length in the kidney, (28,77) whereas loss of function mutations in PKD1 induce PKD, skeletal defects in vivo, and decreased osteoblast differentiation in vitro. (78,79) As primary cilia may be part of the mechanosensing machinery that influences bone remodeling via b-catenin signaling, (80) NEK8 mutations in jck could theoretically influence the onset of HTO bone disease. However, tibia and femur length and total body weight were not different between 6-week-old WT and jck mice before the onset of renal dysfunction indicating that the mutation had no influence on normal growth (data not shown).…”

ConditionalKif3aablation causes abnormal hedgehog signaling topography, growth plate dysfunction, and excessive bone and cartilage formation during mouse skeletogenesis

“…In several but not all respects, these abnormalities are reminiscent of those seen in developing Ihh -/-long bones (St-Jacques et al, 1999), pointing to the pivotal nature and general requirement of hedgehog action in skeletogenesis. Given their roles in hedgehog signaling, primary cilia should be as essential for skeletal development and growth as hedgehog proteins, and previous genetic studies certainly support that premise (Murcia et al, 2000;Olsen et al, 2005;Xiao et al, 2006;Zhang et al, 2003). In particular, limb and cranial skeletal abnormalities were found in conditional mouse mutants deficient in Kif3a or in the ciliaassociated mechanosensory protein polycystin-1 (Pkd1), as well as in mice bearing a missense mutation in the Pkd1 gene (Olsen et al, 2005;Xiao et al, 2006).…”

“…Given their roles in hedgehog signaling, primary cilia should be as essential for skeletal development and growth as hedgehog proteins, and previous genetic studies certainly support that premise (Murcia et al, 2000;Olsen et al, 2005;Xiao et al, 2006;Zhang et al, 2003). In particular, limb and cranial skeletal abnormalities were found in conditional mouse mutants deficient in Kif3a or in the ciliaassociated mechanosensory protein polycystin-1 (Pkd1), as well as in mice bearing a missense mutation in the Pkd1 gene (Olsen et al, 2005;Xiao et al, 2006). A more extensive recent study indicated that long bone anlagen in mouse embryo limbs deficient in Ift88 or Kif3a (prx1cre;Ift88 fl/n or prx1cre;Kif3a fl/n ) are short, exhibit accelerated chondrocyte hypertrophy and lack an intramembranous bone collar (Haycraft et al, 2007).…”

“…2K) and there was an abrupt transition from cartilage to marrow, and a concurrent reduction in primary spongiosa and endochondral bone (Fig. 2L) (Xiao et al, 2006). In line with the absence of flat-shaped cells, mutant growth plates contained few proliferating chondrocytes, as indicated by gene expression of the mitotic marker histone 4C (Fig.…”

The “soft” side of the bone: unveiling its endocrine functions

“…The classically recognized one is the role as mechanosensors, which is accomplished by translating mechanical stimuli into biochemical signals, which are then transferred to the other cells by means of the cellular processes [39]. This function requires the involvement of a protein complex, composed by the cilium and the cilia-associated proteins PolyCystin 1 and 2 [40].…”