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Project description:Chondrocyte hypertrophy is the terminal step in chondrocyte differentiation and is crucial for endochondral bone formation. How signaling pathways regulate chondrocyte hypertrophic differentiation remains incompletely understood. In this study, using a Tbx18:Cre (Tbx18Cre/+) gene-deletion approach, we selectively deleted the gene for the signaling protein SMAD family member 4 (Smad4f/f ) in the limbs of mice. We found that the Smad4-deficient mice develop a prominent shortened limb, with decreased expression of chondrocyte differentiation markers, including Col2a1 and Acan, in the humerus at mid-to-late gestation. The most striking defects in these mice were the absence of stylopod elements and failure of chondrocyte hypertrophy in the humerus. Moreover, expression levels of the chondrocyte hypertrophy-related markers Col10a1 and Panx3 were significantly decreased. Of note, we also observed that the expression of runt-related transcription factor 2 (Runx2), a critical mediator of chondrocyte hypertrophy, was also down-regulated in Smad4-deficient limbs. To determine how the skeletal defects arose in the mouse mutants, we performed RNA-Seq with ChIP-Seq analyses and found that Smad4 directly binds to regulatory elements in the Runx2 promoter. Our results suggest a new mechanism whereby Smad4 controls chondrocyte hypertrophy by up-regulating Runx2 expression during skeletal development. The regulatory mechanism involving Smad4-mediated Runx2 activation uncovered here provides critical insights into bone development and pathogenesis of chondrodysplasia.
| S-EPMC6005452 | biostudies-literature
Project description:Smad4 is a central mediator of canonical TGF/BMP signaling and plays important roles in mesenchymal cell aggregation, chondrocyte differentiation, osteoblast differentiation and maturation. However, the regulatory mechanism of Smad4 underlying chondrocyte hypertrophy during skeletal development is unknown. To elucidate the molecular mechanism by which Smad4 deficiency impairs chondrocyte hypertrophy, we performed high-throughput Chip-seq to identify target genes involved in Smad4-regulated chondrocyte hypertrophy. Our results suggest that Smad4 controls chondrocyte hypertrophy through regulating Runx2 expression during skeletal development.
2018-05-07 | GSE114081 | GEO
Project description:Smad4 is a central mediator of canonical TGF/BMP signaling and plays important roles in mesenchymal cell aggregation, chondrocyte differentiation, osteoblast differentiation and maturation. However, the regulatory mechanism of Smad4 underlying chondrocyte hypertrophy during skeletal development is unknown. To elucidate the molecular mechanism by which Smad4 deficiency impairs chondrocyte hypertrophy, we performed high-throughput RNA-seq to identify target genes involved in Smad4-regulated chondrocyte hypertrophy. Our results suggest that Smad4 controls chondrocyte hypertrophy through regulating Runx2 expression during skeletal development.
2018-05-07 | GSE114079 | GEO
Project description:Smad4 deficiency impairs chondrocyte hypertrophy via the Runx2 transcription factor in mouse skeletal development
| PRJNA454484 | ENA
Project description:Smad4 deficiency impairs chondrocyte hypertrophy via the Runx2 transcription factor in mouse skeletal development [RNA-seq]
| PRJNA464242 | ENA
Project description:Smad4 deficiency impairs chondrocyte hypertrophy via the Runx2 transcription factor in mouse skeletal development [ChIP-seq]
| PRJNA464244 | ENA
Project description:SH2-containing inositol-5'-phosphatase-1 (SHIP-1) controls the phosphatidylinositol-3'-kinase (PI3K) initiated signaling pathway by limiting cell membrane recruitment and activation of Akt. Despite the fact that many of the growth factors important to cartilage development and functions are able to activate the PI3K signal transduction pathway, little is known about the role of PI3K signaling in chondrocyte biology and its contribution to mammalian skeletogenesis. Here, we report that the lipid phosphatase SHIP-1 regulates chondrocyte hypertrophy and skeletal development through its expression in osteochondroprogenitor cells. Global SHIP-1 knockout led to accelerated chondrocyte hypertrophy and premature formation of the secondary ossification center in the bones of postnatal mice. Drastically higher vascularization and greater number of c-kit + progenitors associated with sinusoids in the bone marrow also indicated more advanced chondrocyte hypertrophic differentiation in SHIP-1 knockout mice than in wild-type mice. In corroboration with the in vivo phenotype, SHIP-1 deficient PDGFRα + Sca-1 + osteochondroprogenitor cells exhibited rapid differentiation into hypertrophic chondrocytes under chondrogenic culture conditions in vitro. Furthermore, SHIP-1 deficiency inhibited hypoxia-induced cellular activation of Akt and extracellular-signal-regulated kinase (Erk) and suppressed hypoxia-induced cell proliferation. These results suggest that SHIP-1 is required for hypoxia-induced growth signaling under physiological hypoxia in the bone marrow. In conclusion, the lipid phosphatase SHIP-1 regulates skeletal development by modulating chondrogenesis and the hypoxia response of the osteochondroprogenitors during endochondral bone formation.
| S-EPMC6879780 | biostudies-literature
Project description:FILAMIN B, which encodes a cytoplasmic actin binding protein, is mutated in several skeletal dysplasias. To further investigate how an actin binding protein influences skeletogenesis, we generated mice lacking intact Filamin B. As observed in spondylocarpotarsal synostosis syndrome patients, Filamin B mutant mice display ectopic mineralization in many cartilaginous elements. This aberrant mineralization is due to ectopic chondrocyte hypertrophy similar to that seen in mice expressing Runx2 in chondrocytes. Accordingly, removing one copy of Runx2 rescues the Filamin B mutant phenotype, indicating that Filamin B is a regulator of Runx2 function during chondrocyte differentiation. Filamin B binds Smad3, which is known to interact with Runx2. Smad3 phosphorylation is increased in the mutant mice. Thus, Filamin B inhibits Runx2 activity, at least in part, through the Smad3 pathway. Our results uncover the involvement of actin binding proteins during chondrogenesis and provide a molecular basis to a human genetic disease.
| S-EPMC2064428 | biostudies-literature
Project description:Runx2 is a known master transcription factor for osteoblast differentiation, as well as an essential regulator for chondrocyte maturation. Recently, more and more data has shown that Runx2 regulates hypertrophic chondrocyte-specific type X collagen gene (Col10a1) expression in different species. However, how Runx2 regulation of Col10a1 expression impacts chondrocyte maturation, an essential step of endochondral bone formation, remains unknown. We have recently generated transgenic mice in which Flag-tagged Runx2 was driven by a cell-specific Col10a1 control element. Significantly increased level of Runx2 and Col10a1 mRNA transcripts were detected in transgenic mouse limbs at both E17.5 (embryonic day 17.5) and P1 (post-natal day1) stages, suggesting an in vivo correlation of Runx2 and Col10a1 expression. Surprisingly, skeletal staining suggested delayed ossification in both the axial and the appendicular skeleton of transgenic mice from E14.5 until P6. Histological analysis showed elongated hypertrophic zones in transgenic mice, with less von Kossa and TUNEL staining in long bone sections at both E17.5 and P1 stages, suggesting defective mineralization due to delayed chondrocyte maturation or apoptosis. Indeed, we detected increased level of anti-apoptotic genes B-cell leukemia/lymphoma 2, Osteopontin, and Sox9 in transgenic mice by real-time RT-PCR. Moreover, immunohistochemistry and Western blotting analysis also suggested increased Sox9 expression in hypertrophic chondrocytes of transgenic mice. Together, our data suggest that targeting Runx2 in hypertrophic chondrocytes upregulates expression of Col10a1 and other marker genes (such as Sox9). This will change the local matrix environment, delay chondrocyte maturation, reduce apoptosis and matrix mineralization, and eventually, lead to impaired endochondral ossification.
| S-EPMC3359397 | biostudies-literature
Project description:BackgroundIK is a splicing factor that promotes spliceosome activation and contributes to pre-mRNA splicing. Although the molecular mechanism of IK has been previously reported in vitro, the physiological role of IK has not been fully understood in any animal model. Here, we generate an ik knock-out (KO) zebrafish using the CRISPR/Cas9 system to investigate the physiological roles of IK in vivo.ResultsThe ik KO embryos display severe pleiotropic phenotypes, implying an essential role of IK in embryonic development in vertebrates. RNA-seq analysis reveals downregulation of genes involved in skeletal muscle differentiation in ik KO embryos, and there exist genes having improper pre-mRNA splicing among downregulated genes. The ik KO embryos display impaired neuromuscular junction (NMJ) and fast-twitch muscle development. Depletion of ik reduces myod1 expression and upregulates pax7a, preventing normal fast muscle development in a non-cell-autonomous manner. Moreover, when differentiation is induced in IK-depleted C2C12 myoblasts, myoblasts show a reduced ability to form myotubes. However, inhibition of IK does not influence either muscle cell proliferation or apoptosis in zebrafish and C2C12 cells.ConclusionThis study provides that the splicing factor IK contributes to normal skeletal muscle development in vivo and myogenic differentiation in vitro.
| S-EPMC8015194 | biostudies-literature