The primary cilium is a cellular organelle that coordinates signaling pathways critical for cell proliferation, differentiation, survival, and homeostasis. Intraflagellar transport (IFT) plays a pivotal role in assembling primary cilia. Disruption and/or dysfunction of IFT components can cause multiple diseases, including skeletal dysplasia. However, the mechanism by which IFT regulates skeletogenesis remains elusive. Here, we show that a neural crest-specific deletion of intraflagellar transport 20 (Ift20) in mice compromises ciliogenesis and intracellular transport of collagen, which leads to osteopenia in the facial region. Whereas platelet-derived growth factor receptor alpha (PDGFRα) was present on the surface of primary cilia in wildtype osteoblasts, disruption of Ift20 down-regulated PDGFRα production, which caused suppression of PDGF-Akt signaling, resulting in decreased osteogenic proliferation and increased cell death. Although osteogenic differentiation in cranial neural crest (CNC)-derived cells occurred normally in Ift20-mutant cells, the process of mineralization was severely attenuated due to delayed secretion of type I collagen. In control osteoblasts, procollagen was easily transported from the endoplasmic reticulum (ER) to the Golgi apparatus. By contrast, despite having similar levels of collagen type 1 alpha 1 (Col1a1) expression, Ift20 mutants did not secrete procollagen because of dysfunctional ER-to-Golgi trafficking. These data suggest that in the multipotent stem cells of CNCs, IFT20 is indispensable for regulating not only ciliogenesis but also collagen intracellular trafficking. Our study introduces a unique perspective on the canonical and noncanonical functions of IFT20 in craniofacial skeletal development.cranial neural crest cells | intracellular trafficking | intraflagellar transport | PDGF signaling | primary cilia

Type I collagen, a major extracellular component of the periodontal ligament (PDL), is post-translationally modified by a series of specific enzymes. Among the collagen-modifying enzymes, lysyl oxidase (LOX) is essential to initiate collagen cross-linking and lysyl hydroxylases (LHs) to regulate the cross-linking pathways that are important for tissue specific mechanical properties. The purpose of this study was to investigate the effects of mechanical loading on the expression of collagen-modifying enzymes and subsequent tissue changes in PDL. Primary human PDL cells were subjected to mechanical loading in a 3D collagen gel, and gene expression and collagen component were analyzed. Wistar rats were subjected to excessive occlusal loading with or without intra-peritoneal injection of a LOX inhibitor, β-aminopropionitrile (BAPN). Upon mechanical loading, gene expression of LH2 and LOX was significantly elevated, while that of COL1A2 was not affected on hPDL-derived cells. The mechanical loading also elevated formation of collagen α-chain dimers in 3D culture. The numbers of LH2 and LOX positive cells in PDL were significantly increased in an excessive occlusal loading model. Notably, an increase of LH2-positive cells was observed only at the bone-side of PDL. Intensity of picrosirius red staining was increased by excessive occlusal loading, but significantly diminished by BAPN treatment. These results demonstrated that mechanical loading induced collagen maturation in PDL by up-regulating collagen-modifying enzymes and subsequent collagen cross-linking which are important for PDL tissue maintenance. J. Cell. Physiol. 231: 926-933, 2016. © 2015 Wiley Periodicals, Inc.

Cranial neural crest cells (CNCCs) are a population of multipotent stem cells that give rise to craniofacial bone and cartilage during development. Bone morphogenetic protein (BMP) signaling and autophagy have been individually implicated in stem cell homeostasis. Mutations that cause constitutive activation of the BMP type I receptor ACVR1 cause the congenital disorder fibrodysplasia ossificans progressiva (FOP), which is characterized by ectopic cartilage and bone in connective tissues in the trunk and sometimes includes ectopic craniofacial bones. Here, we showed that enhanced BMP signaling through the constitutively activated ACVR1 (ca-ACVR1) in CNCCs in mice induced ectopic cartilage formation in the craniofacial region through an autophagy-dependent mechanism. Enhanced BMP signaling suppressed autophagy by activating mTORC1, thus blocking the autophagic degradation of β-catenin, which, in turn, caused CNCCs to adopt a chondrogenic identity. Transient blockade of mTORC1, reactivation of autophagy, or suppression of Wnt–β-catenin signaling reduced ectopic cartilages in ca-Acvr1 mutants. Our results suggest that BMP signaling and autophagy coordinately regulate β-catenin activity to direct the fate of CNCCs during craniofacial development. These findings may also explain why some patients with FOP develop ectopic bones through endochondral ossification in craniofacial regions.

Fibrillar type I collagen, the predominant organic component in bone, is stabilized by lysyl oxidase (LOX)-initiated covalent intermolecular cross-linking, an important determinant of bone quality. However, the impact of collagen cross-linking on the activity of bone cells and subsequent tissue remodeling is not well understood. In this study, we investigated the effect of collagen cross-linking on bone cellular activities employing a loss-of-function approach, using a potent LOX inhibitor, β-aminopropionitrile (BAPN). Osteoblastic cells (MC3T3-E1) were cultured for 2 weeks in the presence of 0–2 mM BAPN to obtain low cross-linked collagen matrices. The addition of BAPN to the cultures diminished collagen cross-links in a dose-dependent manner and, at 1 mM level, none of the major cross-links were detected without affecting collagen production. After the removal of cellular components from these cultures, MC3T3-E1, osteoclasts (RAW264.7), or mouse primary bone marrow-derived stromal cells (BMSCs) were seeded. MC3T3-E1 cells grown on low cross-link matrices showed increased alkaline phosphatase (ALP) activity. The number of multinucleate tartrate-resistant acid phosphatase (TRAP)-positive cells increased in RAW264.7 cells. Initial adhesion, proliferation, and ALP activity of BMSCs also increased. In the animal experiments, 4-week-old C57BL/6 mice were fed with BAPN-containing diet for 8 weeks. At this point, biochemical analysis of bone demonstrated that collagen cross-links decreased without affecting collagen content. Then, the diet was changed to a control diet to minimize the direct effect of BAPN. At 2 and 4 weeks after the change, histological samples were prepared. Histological examination of femur samples at 4 weeks showed a significant increase in the number of bone surface osteoblasts, while the bone volume and surface osteoclast numbers were not significantly affected. These results clearly demonstrated that the extent of collagen cross-linking of bone matrix affected the differentiation of bone cells, underscoring the importance of collagen cross-linking in the regulation of cell behaviors and tissue remodeling in bone. Characterization of collagen cross-linking in bone may be beneficial to obtain insight into not only bone mechanical property, but also bone cellular activities.

Condylar cartilage is a joint cartilage essential for smooth jaw movement. The importance of ciliary proteins in condylar cartilage development has been reported. However, little is known about how ciliary proteins control the homeostasis of condylar cartilage. Here we show that intraflagellar transport 20 (IFT20), a ciliary protein, is required for the maintenance of cartilaginous matrix in condylar cartilage. Utilizing NG2-CreER mice expressed in condylar cartilage, we deleted Ift20 by tamoxifen treatment at juvenile-to-adult stages. In wild-type condylar cartilage, IFT20 was robustly produced in the cis-Golgi, but deletion of Ift20 by tamoxifen induction of NG2-CreER (Ift20:NG2-CreER) resulted in reduced cell proliferation and decreased Golgi size in condylar cartilage. Importantly, while the primary cilia were present in cartilage cells in the condylar layers of wild-type mice, no primary cilia were present in the Ift20:NG2-CreER condylar layers. Consistent with this finding, ciliary-mediated Hedgehog signaling was severely attenuated in Ift20 mutant chondrocytes, and thus the production levels of type X collagen were significantly reduced in Ift20:NG2-CreER mice. These results suggest that IFT20 is required for Golgi size and Hedgehog signaling to maintain cartilaginous matrix in condylar cartilage. Our study highlights the unique function of IFT20 in the homeostasis of condylar cartilage.

Craniofacial abnormalities, including facial skeletal defects, comprise approximately one-third of all birth defects in humans. Since most bones in the face derive from cranial neural crest cells (CNCCs), which are multipotent stem cells, craniofacial bone disorders are largely attributed to defects in CNCCs. However, it remains unclear how the niche of CNCCs is coordinated by multiple gene regulatory networks essential for craniofacial bone development. Here we report that tumor suppressors breast cancer 1 (BRCA1) and breast cancer 2 (BRCA2) are required for craniofacial bone development in mice. Disruption of Brca1 in CNCC-derived mesenchymal cells, but not in epithelial-derived cells, resulted in craniofacial skeletal defects. Whereas osteogenic differentiation was normal, both osteogenic proliferation and survival were severely attenuated in Brca1 mutants. Brca1-deficient craniofacial skeletogenic precursors displayed increased DNA damage and enhanced cell apoptosis. Importantly, the craniofacial skeletal defects were sufficiently rescued by superimposing p53 null alleles in a neural crest-specific manner in vivo, indicating that BRCA1 deficiency induced DNA damage, cell apoptosis, and that the pathogenesis of craniofacial bone defects can be compensated by inactivation of p53. Mice lacking Brca2 in CNCCs, but not in epithelial-derived cells, also displayed abnormalities resembling the craniofacial skeletal malformations observed in Brca1 mutants. Our data shed light on the importance of BRCA1/BRCA2 function in CNCCs during craniofacial skeletal formation.

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