Runx2 is essential for the transdifferentiation of chondrocytes into osteoblasts

Chondrocytes proliferate and mature into hypertrophic chondrocytes. Vascular invasion into the cartilage occurs in the terminal hypertrophic chondrocyte layer, and terminal hypertrophic chondrocytes die by apoptosis or transdifferentiate into osteoblasts. Runx2 is essential for osteoblast differentiation and chondrocyte maturation. Runx2-deficient mice are composed of cartilaginous skeletons and lack the vascular invasion into the cartilage. However, the requirement of Runx2 in the vascular invasion into the cartilage, mechanism of chondrocyte transdifferentiation to osteoblasts, and its significance in bone development remain to be elucidated. To investigate these points, we generated Runx2fl/flCre mice, in which Runx2 was deleted in hypertrophic chondrocytes using Col10a1 Cre. Vascular invasion into the cartilage was similarly observed in Runx2fl/fl and Runx2fl/flCre mice. Vegfa expression was reduced in the terminal hypertrophic chondrocytes in Runx2fl/flCre mice, but Vegfa was strongly expressed in osteoblasts in the bone collar, suggesting that Vegfa expression in bone collar osteoblasts is sufficient for vascular invasion into the cartilage. The apoptosis of terminal hypertrophic chondrocytes was increased and their transdifferentiation was interrupted in Runx2fl/flCre mice, leading to lack of primary spongiosa and osteoblasts in the region at E16.5. The osteoblasts appeared in this region at E17.5 in the absence of transdifferentiation, and the number of osteoblasts and the formation of primary spongiosa, but not secondary spongiosa, reached to levels similar those in Runx2fl/fl mice at birth. The bone structure and volume and all bone histomophometric parameters were similar between Runx2fl/fl and Runx2fl/flCre mice after 6 weeks of age. These findings indicate that Runx2 expression in terminal hypertrophic chondrocytes is not required for vascular invasion into the cartilage, but is for their survival and transdifferentiation into osteoblasts, and that the transdifferentiation is necessary for trabecular bone formation in embryonic and neonatal stages, but not for acquiring normal bone structure and volume in young and adult mice.

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Raf Kinases Are Essential for Phosphate Induction of ERK1/2 Phosphorylation in Hypertrophic Chondrocytes and Normal Endochondral Bone Development

Journal of Biological Chemistry ◽

10.1074/jbc.m116.763342 ◽

2017 ◽

Vol 292 (8) ◽

pp. 3164-3171 ◽

Cited By ~ 8

Author(s):

Garyfallia Papaioannou ◽

Elizabeth T. Petit ◽

Eva S. Liu ◽

Manuela Baccarini ◽

Catrin Pritchard ◽

...

Keyword(s):

Growth Plate ◽

Vascular Invasion ◽

Bone Development ◽

Skeletal Development ◽

Hypertrophic Chondrocytes ◽

Chondrocyte Apoptosis ◽

Caspase 9 ◽

Endochondral Bone ◽

Hypertrophic Chondrocyte

Hypophosphatemia causes rickets by impairing hypertrophic chondrocyte apoptosis. Phosphate induction of MEK1/2-ERK1/2 phosphorylation in hypertrophic chondrocytes is required for phosphate-mediated apoptosis and growth plate maturation. MEK1/2 can be activated by numerous molecules including Raf isoforms. A- and B-Raf ablation in chondrocytes does not alter skeletal development, whereas ablation of C-Raf decreases hypertrophic chondrocyte apoptosis and impairs vascularization of the growth plate. However, ablation of C-Raf does not impair phosphate-induced ERK1/2 phosphorylation in vitro, but leads to rickets by decreasing VEGF protein stability. To determine whether Raf isoforms are required for phosphate-induced hypertrophic chondrocyte apoptosis, mice lacking all three Raf isoforms in chondrocytes were generated. Raf deletion caused neonatal death and a significant expansion of the hypertrophic chondrocyte layer of the growth plate, accompanied by decreased cleaved caspase-9. This was associated with decreased phospho-ERK1/2 immunoreactivity in the hypertrophic chondrocyte layer and impaired vascular invasion. These data further demonstrated that Raf kinases are required for phosphate-induced ERK1/2 phosphorylation in cultured hypertrophic chondrocytes and perform essential, but partially redundant roles in growth plate maturation.

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A-Raf and B-Raf Are Dispensable for Normal Endochondral Bone Development, and Parathyroid Hormone-Related Peptide Suppresses Extracellular Signal-Regulated Kinase Activation in Hypertrophic Chondrocytes

Molecular and Cellular Biology ◽

10.1128/mcb.00617-07 ◽

2007 ◽

Vol 28 (1) ◽

pp. 344-357 ◽

Cited By ~ 35

Author(s):

Sylvain Provot ◽

Gregory Nachtrab ◽

Jennifer Paruch ◽

Adele Pin Chen ◽

Alcino Silva ◽

...

Keyword(s):

Cell Proliferation ◽

Parathyroid Hormone ◽

Bone Development ◽

Hypertrophic Chondrocytes ◽

Chondrocyte Proliferation ◽

Endochondral Bone ◽

Extracellular Signal Regulated Kinase ◽

Chondrocyte Maturation ◽

Extracellular Signal ◽

Parathyroid Hormone Related Peptide

ABSTRACT Parathyroid hormone-related peptide (PTHrP) and the parathyroid hormone-PTHrP receptor increase chondrocyte proliferation and delay chondrocyte maturation in endochondral bone development at least partly through cyclic AMP (cAMP)-dependent signaling pathways. Because data suggest that the ability of cAMP to stimulate cell proliferation involves the mitogen-activated protein kinase kinase kinase B-Raf, we hypothesized that B-Raf might mediate the proliferative action of PTHrP in chondrocytes. Though B-Raf is expressed in proliferative chondrocytes, its conditional removal from cartilage did not affect chondrocyte proliferation and maturation or PTHrP-induced chondrocyte proliferation and PTHrP-delayed maturation. Similar results were obtained by conditionally removing B-Raf from osteoblasts. Because A-raf and B-raf are expressed similarly in cartilage, we speculated that they may fulfill redundant functions in this tissue. Surprisingly, mice with chondrocytes deficient in both A-Raf and B-Raf exhibited normal endochondral bone development. Activated extracellular signal-regulated kinase (ERK) was detected primarily in hypertrophic chondrocytes, where C-raf is expressed, and the suppression of ERK activation in these cells by PTHrP or a MEK inhibitor coincided with a delay in chondrocyte maturation. Taken together, these results demonstrate that B-Raf and A-Raf are dispensable for endochondral bone development and they indicate that the main role of ERK in cartilage is to stimulate not cell proliferation, but rather chondrocyte maturation.

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BONE DEVELOPMENT AND FRACTURE HEALING IS NORMAL IN MICE THAT HAVE A DEFECT IN THE DEVELOPMENT OF THE LYMPHATIC SYSTEM

Lymphology ◽

10.2458/lymph.4669 ◽

2021 ◽

Vol 53 (4) ◽

Author(s):

AL McCarter ◽

A Khalid ◽

Y Yi ◽

M Monroy ◽

H Zhao ◽

...

Keyword(s):

Fracture Healing ◽

Lymphatic System ◽

Bone Development ◽

Bone Destruction ◽

Wild Type ◽

Normal Bone ◽

Adult Mice ◽

Generalized Lymphatic Anomaly ◽

Kaposiform Lymphangiomatosis ◽

Lymphatic Vasculature

Ectopic lymphatics form in bone and promote bone destruction in diseases such as Gorham-Stout disease, generalized lymphatic anomaly, and kaposiform lymphangiomatosis. However, the role lymphatics serve in normal bone development and repair is poorly understood. The objective of this study was to characterize bone development and fracture healing in mice that have a defect in the development of the lymphatic vasculature. We found that bones in wild-type adult mice and mouse embryos did not have lymphatics. We also found that bone development was normal in Vegfr3Chy/Chy embryos. These mice do not have lymphatics and die shortly after birth. To determine whether lymphatics serve a role in postnatal bone development and fracture healing, we analyzed bones from Vegfr3wt/Chy mice. These mice are viable and have fewer lymphatics than wild-type mice. We found that postnatal bone development and fracture healing was normal in Vegfr3wt/Chy mice. Taken together, our results suggest that lymphatics do not play a major role in normal bone development or repair.

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Delta-1 negatively regulates the transition from prehypertrophic to hypertrophic chondrocytes during cartilage formation

Development ◽

10.1242/dev.126.5.987 ◽

1999 ◽

Vol 126 (5) ◽

pp. 987-998 ◽

Cited By ~ 6

Author(s):

R. Crowe ◽

J. Zikherman ◽

L. Niswander

Keyword(s):

Notch Signaling ◽

Cell Fate ◽

Bone Development ◽

Hypertrophic Chondrocytes ◽

Signaling System ◽

Endochondral Bone ◽

Cell Fate Decisions ◽

Cartilage Formation ◽

Chondrocyte Maturation

Endochondral bone development begins with the formation of a cartilage template. Chondrocytes within this template undergo a progressive program of maturation from proliferative to prehypertrophic chondrocytes to hypertrophic chondrocytes. The progression of cells through these steps of differentiation must be carefully controlled to ensure coordinated growth. Because the Delta/Notch signaling system is known to regulate cell fate choices, we sought to determine if these molecules might be involved in the progressive cell fate decisions that chondocytes undergo. Here we demonstrate in the chick that Delta/Notch signaling negatively regulates progression from the prehypertrophic to hypertrophic state of differentiation. Delta-1 is expressed specifically in the hypertrophic chondrocytes while Notch-2 is expressed in chondrocytes at all stages. Misexpression of Delta-1 using a replication-competent retrovirus blocks chondrocyte maturation. Prehypertrophic cells form normally but do not undergo differentiation to hypertrophic cells, resulting in shortened skeletal elements that lack ossification. We conclude that Delta-1 acts during chondrogenesis to inhibit the transition from prehypertrophic chondrocytes to hypertrophic chondrocytes, thus defining a novel mechanism for the regulation of the chondrocyte maturation program. In addition, these results reveal a new role for Delta/Notch signaling in regulating the progression to a terminally differentiated state.

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Expression of bone-specific genes by hypertrophic chondrocytes: Implications of the complex functions of the hypertrophic chondrocyte during endochondral bone development

Journal of Cellular Biochemistry ◽

10.1002/(sici)1097-4644(199607)62:1<1::aid-jcb1>3.0.co;2-x ◽

1996 ◽

Vol 62 (1) ◽

pp. 1-9 ◽

Cited By ~ 136

Author(s):

L.C. Gerstenfeld ◽

F.D. Shapiro

Keyword(s):

Bone Development ◽

Hypertrophic Chondrocytes ◽

Endochondral Bone ◽

Hypertrophic Chondrocyte ◽

Complex Functions

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Abstract 280: Deficiency of FKBP12 in the Heart Leads to Impaired Contractility and Pregnancy Induced Cardiomyopathy

Circulation Research ◽

10.1161/res.113.suppl_1.a280 ◽

2013 ◽

Vol 113 (suppl_1) ◽

Author(s):

Joshua Oakes ◽

Susan Hamilton

Keyword(s):

Ejection Fraction ◽

Ryanodine Receptors ◽

Left Ventricular ◽

Heart Weight ◽

Mild Phenotype ◽

Adult Mice ◽

Significant Difference ◽

Fk506 Binding Proteins ◽

Deficient Mice ◽

Fractional Shortening

FK506 Binding Proteins (FKBPs) are a family of cis-trans prolyl isomerases that bind rapamycin and FK506. FKBP12 and 12.6 interact with ryanodine receptors (RyR), homotetrameric transmembrane ion channels that regulate Ca2+ release from the sarcoplasmic reticulum (SR). FKBP12 interacts with RyR1 in skeletal muscle and FKBP12.6 interacts with RyR2 in cardiac muscle to regulate the Ca2+ leak properties of these channels. Recently it has been suggested that FKBP12 also plays a role in regulating RyR2 activity. Using mice with a cardiac specific deficiency in FKBP12, we analyzed the role of FKBP12 in cardiac function. We found that both male and female mice with a α-MyHC Cre/Lox mediated deficiency in FKBP12 in the heart (FKBP12 KD) developed a mild dilated cardiomyopathy, with enlarged left ventricular diameter both during systole and diastole, decreased ejection fraction and decreased fractional shortening. To elucidate the mechanism for these effects we assessed Ca2+ sparks in isolated cardiomyocytes. We found an increase in both Ca2+ spark frequency and spark amplitude in FKBP12 cardiac deficient mice without a change in spark duration. Despite a mild phenotype in adult mice, we found that approximately 25% of all pregnancies (26/106) in the FKBP12 deficient mice resulted in the mothers dying following the birth. Autopsies show that these cardiac specific FKBP12 deficient mice had increased heart weight and significantly dilated ventricles compared to female Cre mice. Our data suggest that a cardiac specific deficiency in FKBP12 leads to the development of pregnancy induced cardiomyopathy. Echocardiography on FKBP12 deficient mice one day after giving birth found that there was no significant difference in ejection fraction or fractional shortening compared to α-MyHC Cre control mice. FKBP12 deficient females, however, had larger hearts and 50% (2/4) displayed heart failure and died. In conclusion, we show that FKBP12 does indeed alter Ca2+ handling in the heart and that a loss of FKBP12 leads to the development of pregnancy induced cardiomyopathies in females.

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Bone and muscle development in three inbred female mouse strains

Osteologie/Osteology ◽

10.1055/a-1287-6016 ◽

2021 ◽

Author(s):

Ursula Föger-Samwald ◽

Maria Papageorgiou ◽

Katharina Wahl-Figlash ◽

Katharina Kerschan-Schindl ◽

Peter Pietschmann

Keyword(s):

Trabecular Bone ◽

Muscle Development ◽

Bone Growth ◽

Volume Fraction ◽

Bone Structure ◽

Bone Development ◽

Mouse Strains ◽

Structural Reorganization ◽

Bone Volume Fraction ◽

Trabecular Thickness

AbstractMuscle force is thought to be one of the main determinants of bone development. Hence, peak muscle growth is expected to precede peak bone growth. In this study, we investigated muscle and bone development in female C57BL/6 J, DBA/2JRj, and C3H/HeOuJ mice. Femoral cortical and trabecular bone structure and the weights of selected muscles were assessed at the ages of 8, 16, and 24 weeks. Muscle mass increased from 8 to 24 weeks in all 3 strains, suggesting peak muscle development at 24 weeks or later. Bone volume fraction, trabecular number, and connectivity density of the femur decreased or remained unchanged, whereas trabecular density and trabecular thickness largely increased. These results suggest a peak in trabecular bone accrual at 8 weeks or earlier followed by further increases in density and structural reorganization of trabeculae. Cortical density, cortical thickness, and cortical cross sectional area increased over time, suggesting a peak in cortical bone accrual at 24 weeks or later. In conclusion, our data provide evidence that growth of muscle lags behind trabecular bone accrual.

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Increased Fetal and Extramedullary Hematopoiesis in Fas-Deficient C57BL/6-lpr/lpr Mice

Blood ◽

10.1182/blood.v94.8.2613.420k33_2613_2621 ◽

1999 ◽

Vol 94 (8) ◽

pp. 2613-2621 ◽

Cited By ~ 41

Author(s):

Elke Schneider ◽

Géraldine Moreau ◽

Anne Arnould ◽

Florence Vasseur ◽

Naushad Khodabaccus ◽

...

Keyword(s):

Progenitor Cell ◽

Bone Marrow Cells ◽

Extramedullary Hematopoiesis ◽

Interferon Γ ◽

Regulatory Function ◽

Fetal Life ◽

Adult Mice ◽

Colony Forming Units ◽

Immature Myeloid Cells ◽

Deficient Mice

In this study, we examined the consequences of Fas deficiency on hematopoiesis in C57BL/6-lpr/lpr mice. We found a striking extramedullary increase in hematopoietic progenitor cells, comprising erythroid and nonerythroid lineages alike. These modifications preceded the lymphadenopathy, because early progenitors (colony-forming units-spleen [CFU-S] day 8) were already augmented in day-18 fetal livers of the lpr phenotype. Three weeks after birth, CFU-S increased in peripheral blood and spleen and colony-forming cells (CFU-C) began to accumulate 1 to 3 weeks later. Extramedullary myelopoiesis augmented progressively in Fas-deficient mice, reaching a maximum within 6 months. By then, mature and immature myeloid cells had infiltrated the spleen, the liver, and the peritoneal cavity. Similar changes occurred in C57BL/6-gld/gld mice, indicating that they resulted from Fas/FasL interactions. Medullary hematopoiesis was not significantly modified in adult mice of either strain. Yet, the incidence of CFU-S decreased after Fas cross-linking on normal bone marrow cells in the presence of interferon γ, consistent with a regulatory function of Fas/FasL interactions in early progenitor cell development. These data provide evidence that Fas deficiency can affect hematopoiesis both during adult and fetal life and that these modifications occur independently from other pathologies associated with the lpr phenotype.

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