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Deletion of core-binding factor ? (Cbf?) in mesenchymal progenitor cells provides new insights into Cbf?/Runxs complex function in cartilage and bone development.

ABSTRACT: Core-binding factor ? (Cbf?) is a subunit of the Cbf family of heterodimeric transcription factors, which plays a critical role in skeletal development through its interaction with the Cbf? subunits, also known as Runt-related transcription factors (Runxs). However, the mechanism by which Cbf? regulates cartilage and bone development remains unclear. Existing Cbf?-deficient mouse models cannot specify the role of Cbf? in skeletal cell lineage. Herein, we sought to specifically address the role of Cbf? in cartilage and bone development by using a conditional knockout (CKO) approach. A mesenchymal-specific Cbf? CKO mouse model was generated by using the Dermo1-Cre mouse line to specifically delete Cbf? in mesenchymal stem cells, which give rise to osteoblasts and chondrocytes. Surprisingly, the mutant mice had under-developed larynx and tracheal cartilage, causing alveolus defects that led to death shortly after birth from suffocation. Also, the mutant mice exhibited severe skeletal deformities from defective intramembranous and endochondral ossification, owing to delayed chondrocyte maturation and impaired osteoblast differentiation. Almost all bones of the mutant mice, including the calvariae, vertebrae, tibiae, femurs, ribs, limbs and sternums were defective. Importantly, we showed that Cbf? was expressed throughout the skeleton during both embryonic and postnatal development, which explains the multiple-skeletal defects observed in the mutant mice. Consistently, Cbf? deficiency impaired both chondrocyte proliferation and hypertrophy zone hypertrophy during growth-plate development in the long bones of mutant mice. Notably, Cbf?, Runx1 and Runx2 displayed different expression patterns in the growth plates of the wild-type mice, indicating that Cbf?/Runx1 complex and Cbf?/Runx2 complex may regulate chondrocyte proliferation and hypertrophy, respectively, in a spatial and temporal manner. Cbf? deletion in the mesenchymal progenitors affected bone development by dramatically down-regulating Collagen X (Col X) and Osterix (Osx) but had a dispensable effect on osteoclast development. Collectively, the results demonstrate that Cbf? mediates cartilage and bone development by interacting with Runx1 and Runx2 to regulate the expressions of Col X and Osx for chondrocyte and osteoblast development. These findings not only reveal a critical role for Cbf? in cartilage and bone development but also facilitate the design of novel therapeutic approaches for skeletal diseases.

SUBMITTER: Wu M

PROVIDER: S-EPMC4644668 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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Deletion of core-binding factor β (Cbfβ) in mesenchymal progenitor cells provides new insights into Cbfβ/Runxs complex function in cartilage and bone development.

Wu Mengrui M Li Chenguan C Zhu Guochun G Wang Yiping Y Jules Joel J Lu Yun Y McConnell Matthew M Wang Yong-Jun YJ Shao Jian-Zhong JZ Li Yi-Ping YP Chen Wei W

Bone 20140504

Core-binding factor β (Cbfβ) is a subunit of the Cbf family of heterodimeric transcription factors, which plays a critical role in skeletal development through its interaction with the Cbfα subunits, also known as Runt-related transcription factors (Runxs). However, the mechanism by which Cbfβ regulates cartilage and bone development remains unclear. Existing Cbfβ-deficient mouse models cannot specify the role of Cbfβ in skeletal cell lineage. Herein, we sought to specifically address the role o ...[more]

PMID: 24798493

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Project description:Core binding factor beta (Cbfβ) is essential for embryonic bone morphogenesis. Yet the mechanisms by which Cbfβ regulates chondrocyte proliferation and differentiation as well as postnatal cartilage and bone formation remain unclear. Hence, using paired-related homeobox transcription factor 1-Cre (Prx1-Cre) mice, mesenchymal stem cell-specific Cbfβ-deficient (Cbfβ(f/f) Prx1-Cre) mice were generated to study the role of Cbfβ in postnatal cartilage and bone development. These mutant mice survived to adulthood but exhibited severe sternum and limb malformations. Sternum ossification was largely delayed in the Cbfβ(f/f) Prx1-Cre mice and the xiphoid process was noncalcified and enlarged. In newborn and 7-day-old Cbfβ(f/f) Prx1-Cre mice, the resting zone was dramatically elongated, the proliferation zone and hypertrophic zone of the growth plates were drastically shortened and disorganized, and trabecular bone formation was reduced. Moreover, in 1-month-old Cbfβ(f/f) Prx1-Cre mice, the growth plates were severely deformed and trabecular bone was almost absent. In addition, Cbfβ deficiency impaired intramembranous bone formation both in vivo and in vitro. Interestingly, although the expression of Indian hedgehog (Ihh) was largely reduced, the expression of parathyroid hormone-related protein (PTHrP) receptor (PPR) was dramatically increased in the Cbfβ(f/f) Prx1-Cre growth plate, indicating that that Cbfβ deficiency disrupted the Ihh-PTHrP negative regulatory loop. Chromatin immunoprecipitation (ChIP) analysis and promoter luciferase assay demonstrated that the Runx/Cbfβ complex binds putative Runx-binding sites of the Ihh promoter regions, and also the Runx/Cbfβ complex directly upregulates Ihh expression at the transcriptional level. Consistently, the expressions of Ihh target genes, including CyclinD1, Ptc, and Pthlh, were downregulated in Cbfβ-deficient chondrocytes. Taken together, our study reveals not only that Cbfβ is essential for chondrocyte proliferation and differentiation for the growth and maintenance of the skeleton in postnatal mice, but also that it functions in upregulating Ihh expression to promoter chondrocyte proliferation and osteoblast differentiation, and inhibiting PPR expression to enhance chondrocyte differentiation.

Project description:BackgroundAnimal cell-based systems have been critical tools in understanding tissue development and physiology, but they are less successful in more practical tasks, such as predicting human toxicity to pharmacological or environmental factors, in which the congruence between in vitro and clinical outcomes lies on average between 50 and 60%. Emblematic of this problem is the high-density micromass culture of embryonic limb bud mesenchymal cells, derived from chick, mouse, or rat. While estimated predictive value of this model system in toxicological studies is relatively high, important failures prevent its use by international regulatory agencies for toxicity testing and policy development. A likely underlying reason for the poor predictive capacity of animal-based culture models is the small but significant physiological differences between species. This deficiency has inspired investigators to develop more organotypic, 3-dimensional culture system using human cells to model normal tissue development and physiology and assess pharmacological and environmental toxicity.MethodsWe have developed a modified, miniaturized micromass culture model using adult human bone marrow-derived mesenchymal progenitor cells (hBM-MPCs) that is amenable to moderate throughput and high content analysis to study chondrogenesis. The number of cells per culture was reduced, and a methacrylated gelatin (gelMA) overlay was incorporated to normalize the morphology of the cultures.ResultsThese modified human cell-based micromass cultures demonstrated robust chondrogenesis, indicated by increased Alcian blue staining and immunodetectable production of collagen type II and aggrecan, and stage-specific chondrogenic gene expression. In addition, in cultures of hBM-MPCs transduced with a lentiviral collagen type II promoter-driven GFP reporter construct, levels of GFP reporter activity correlated well with changes in endogenous collagen type II transcript levels, indicating the feasibility of non-invasive monitoring of chondrogenesis.ConclusionsThe modified hBM-MPC micromass culture system described here represents a reproducible and controlled model for analyzing mechanisms of human skeletal development that may later be applied to pharmacological and environmental toxicity studies.

Dataset Information

Deletion of core-binding factor ? (Cbf?) in mesenchymal progenitor cells provides new insights into Cbf?/Runxs complex function in cartilage and bone development.

Publications

Deletion of core-binding factor β (Cbfβ) in mesenchymal progenitor cells provides new insights into Cbfβ/Runxs complex function in cartilage and bone development.

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