Primary cilia control cell alignment and patterning in bone development via ceramide-PKCζ-β-catenin signaling

Abstract

Intraflagellar transport (IFT) proteins are essential for cilia assembly and function. IFT protein mutations lead to ciliopathies, which manifest as variable skeletal abnormalities. However, how IFT proteins regulate cell alignment during bone development is unknown. Here, we show that the deletion of IFT20 in osteoblast lineage using Osterix-Cre and inducible type I Collagen-CreERT cause a compromised cell alignment and a reduced bone mass. This finding was validated by the disorganized collagen fibrils and decreased bone strength and stiffness in IFT20-deficient femurs. IFT20 maintains cilia and cell alignment in osteoblasts, as the concentric organization of three-dimensional spheroids was disrupted by IFT20 deletion. Mechanistically, IFT20 interacts with the ceramide-PKCζ complex to promote PKCζ phosphorylation in cilia and induce the apical localization of β-catenin in osteoblasts, both of which were disrupted in the absence of IFT20. These results reveal that IFT20 regulates polarity and cell alignment via ceramide-pPKCζ-β-catenin signaling during bone development.

Characterization of primary cilia features reveal cell-type specific variability in in vitro models of osteogenic and chondrogenic differentiation

“…5). Frequencies of ciliated non-starved day 0 uninduced C3H10T1/2 (85.3 ± 2.6%) and MC3T3-E1 (81.4 ± 3.9%) cells were greater than in mouse primary osteoblasts (~70%) (Lim et al, 2020b). Notably the preponderance of ciliated cells significantly decreased in 21 day OS differentiated MC3T3-E1 cells compared to controls (72.5 ± 0.6%).…”

“…Congruently, we observed cilia lengthening, although in a cell-type specific manner, during OS differentiation. The mean cilia length in non-starved day 0 uninduced MC3T3-E1 preosteoblasts (2.2 ± 0.03 µm) and C3H10T1/2 mesenchymal stem cells (2.3 ± 0.03 µm) were shorter compared to mouse primary osteoblasts (~2.9 µm) (Lim et al, 2020b). Cilia length in MLO-Y4 osteocytes have been reported to range from 2 µm to 4 µm (Xiao et al, 2006).…”

“…In this context mechanical stimulation serves as a crucial anabolic signal; it promotes osteogenic (OS) differentiation in MSCs and mineralization of the chondrocyte matrix (Chen et al, 2015;Hoey, Kelly & Jacobs, 2011;O'Conor et al, 2014). Primary cilia are also required for osteoblast, osteocyte polarity, alignment in bone development and regulate chondrocyte cell polarity at the growth plate (Lim et al, 2020a;Song et al, 2007). They are chemosensitive and serve as a nexus of signaling activity important for OS and chondrogenic (CH) differentiation, such as Hh, TGFβ, Fibroblast growth factor (FGF), Wnt, platelet derived growth factor and parathyroid hormone related peptide (Cai et al, 2012;Caplan & Correa, 2011;Chen, Deng & Li, 2012;Jiang et al, 2016;Kozhemyakina, Lassar & Zelzer, 2015).…”

Type II collagen-positive progenitors are major stem cells to control skeleton development and vascular formation

“…To date, much progress has been made in defining the molecular and cellular properties of the osteoblast phenotype through characterization of primary calvarial-derived osteoblast cultures from chicks, rats, and mice 24,38 . We report that Col2+ cells were present in calvarial-derived cells when we isolated POBs from mouse calvaria according to a previously described protocol 39 . Interestingly, calvarial-derived cells from Col2-cre control mice can differentiate into osteoblasts, chondrocytes, and adipocytes.…”

“…Bone marrow stem cells (BMSCs) were isolated as previously described 39 . Briefly, femurs and tibias were dissected from 4-week old Col2-cre; tdTomato mice.…”