scholarly journals Site-1 protease is essential for endochondral bone formation in mice

2007 ◽  
Vol 179 (4) ◽  
pp. 687-700 ◽  
Author(s):  
Debabrata Patra ◽  
Xiaoyun Xing ◽  
Sherri Davies ◽  
Jennifer Bryan ◽  
Carl Franz ◽  
...  
Keyword(s):  
Er Stress ◽  
Hedgehog Signaling ◽  
Endochondral Ossification ◽  
Bone Development ◽  
Active Form ◽  
Type Ii Collagen ◽  
Chondrocyte Apoptosis ◽  
Endochondral Bone Formation ◽  
Endochondral Bone ◽  
Er Stress Response

Site-1 protease (S1P) has an essential function in the conversion of latent, membrane-bound transcription factors to their free, active form. In mammals, abundant expression of S1P in chondrocytes suggests an involvement in chondrocyte function. To determine the requirement of S1P in cartilage and bone development, we have created cartilage-specific S1P knockout mice (S1Pcko). S1Pcko mice exhibit chondrodysplasia and a complete lack of endochondral ossification even though Runx2 expression, Indian hedgehog signaling, and osteoblastogenesis is intact. However, there is a substantial increase in chondrocyte apoptosis in the cartilage of S1Pcko mice. Extraction of type II collagen is substantially lower from S1Pcko cartilage. In S1Pcko mice, the collagen network is disorganized and collagen becomes entrapped in chondrocytes. Ultrastructural analysis reveals that the endoplasmic reticulum (ER) in S1Pcko chondrocytes is engorged and fragmented in a manner characteristic of severe ER stress. These data suggest that S1P activity is necessary for a specialized ER stress response required by chondrocytes for the genesis of normal cartilage and thus endochondral ossification.

scholarly journals Constitutive E2F1 Overexpression Delays Endochondral Bone Formation by Inhibiting Chondrocyte Differentiation

2003 ◽  
Vol 23 (10) ◽  
pp. 3656-3668 ◽  
Author(s):  
Blanca Scheijen ◽  
Marieke Bronk ◽  
Tiffany van der Meer ◽  
René Bernards
Keyword(s):  
Bone Formation ◽  
Endochondral Ossification ◽  
Bone Growth ◽  
Late Phase ◽  
Type Ii Collagen ◽  
Endochondral Bone Formation ◽  
Nodule Formation ◽  
Chondrocyte Differentiation ◽  
Endochondral Bone ◽  
Chondrocyte Maturation

ABSTRACT Longitudinal bone growth results from endochondral ossification, a process that requires proliferation and differentiation of chondrocytes. It has been shown that proper endochondral bone formation is critically dependent on the retinoblastoma family members p107 and p130. However, the precise functional roles played by individual E2F proteins remain poorly understood. Using both constitutive and conditional E2F1 transgenic mice, we show that ubiquitous transgene-driven expression of E2F1 during embryonic development results in a dwarf phenotype and significantly reduced postnatal viability. Overexpression of E2F1 disturbs chondrocyte maturation, resulting in delayed endochondral ossification, which is characterized by reduced hypertrophic zones and disorganized growth plates. Employing the chondrogenic cell line ATDC5, we investigated the effects of enforced E2F expression on the different phases of chondrocyte maturation that are normally required for endochondral ossification. Ectopic E2F1 expression strongly inhibits early- and late-phase differentiation of ATDC5 cells, accompanied by diminished cartilage nodule formation as well as decreased type II collagen, type X collagen, and aggrecan gene expression. In contrast, overexpression of E2F2 or E2F3a results in only a marginal delay of chondrocyte maturation, and increased E2F4 levels have no effect. These data are consistent with the notion that E2F1 is a regulator of chondrocyte differentiation.

scholarly journals Ablation of Ephrin B2 in Col2 Expressing Cells Delays Fracture Repair

Endocrinology ◽  
2020 ◽  
Vol 161 (12) ◽  
Author(s):  
Yongmei Wang ◽  
Lin Ling ◽  
Faming Tian ◽  
Sun Hee Won Kim ◽  
Sunita Ho ◽  
...  
Keyword(s):  
Bone Formation ◽  
Cell Activation ◽  
Bone Development ◽  
Type Ii Collagen ◽  
Fracture Site ◽  
Fracture Repair ◽  
Endochondral Bone Formation ◽  
Endochondral Bone ◽  
Intramembranous Bone ◽  
Endothelial Growth Factor

Abstract Ephrin B2 is critical for endochondral bone development. In this study, we investigated its role in fracture repair by deleting ephrin B2 in type II collagen (Col.2) expressing cells. We used a nonstable tibia fracture model to evaluate fracture repair at 3 sites: intramembranous bone formation, endochondral bone formation, and intramedullary bone formation. We observed that during fracture repair, deletion of ephrin B2 impaired periosteal stem cell activation, inhibited their proliferation, decreased their survival, and blocked their differentiation into osteoblasts and chondrocytes. In addition, deletion of ephrin B2 decreased vascular endothelial growth factor production as well as vascular invasion into the fracture site. These changes led to reduced cartilage to bone conversion in the callus with decreased new bone formation, resulting in impaired fracture repair. Our data indicate that ephrin B2 in Col2-expressing cells is a critical regulator of fracture repair, pointing to a new and potentially targetable mechanism to enhance fracture repair.

scholarly journals Prolactin inhibits the apoptosis of chondrocytes induced by serum starvation

2006 ◽  
Vol 189 (2) ◽  
pp. R1-R8 ◽  
Author(s):  
C Zermeño ◽  
J Guzmán-Morales ◽  
Y Macotela ◽  
G Nava ◽  
F López-Barrera ◽  
...  
Keyword(s):  
Articular Chondrocytes ◽  
Cell Types ◽  
Cartilage Degradation ◽  
Chondrocyte Apoptosis ◽  
Heat Inactivation ◽  
Serum Starvation ◽  
Endochondral Bone Formation ◽  
Endochondral Bone ◽  
Therapeutic Value ◽  
Apoptotic Effect

The apoptosis of chondrocytes plays an important role in endochondral bone formation and in cartilage degradation during aging and disease. Prolactin (PRL) is produced in chondrocytes and is known to promote the survival of various cell types. Here we show that articular chondrocytes from rat postpubescent and adult cartilage express the long form of the PRL receptor as revealed by immunohistochemistry of cartilage sections and by RT-PCR and Western blot analyses of the isolated chondrocytes. Furthermore, we demonstrate that PRL inhibits the apoptosis of these same chondrocytes cultured in low-serum. Chondrocyte apoptosis was measured by hypodiploid DNA content determined by flow cytometry and by DNA fragmentation evaluated by the ELISA and the TUNEL methods. The anti-apoptotic effect of PRL was dose-dependent and was prevented by heat inactivation. These data demonstrate that PRL can act as a survival factor for chondrocytes and that it has potential preventive and therapeutic value in arthropathies characterized by cartilage degradation.

scholarly journals Exogenous PTH 1-34 Attenuates Impaired Fracture Healing in Endogenous PTH Deficiency Mice via Activating Indian Hedgehog Signaling Pathway and Accelerating Endochondral Ossification

2022 ◽  
Vol 9 ◽  
Author(s):  
Cheng Ma ◽  
Huan Liu ◽  
Yifan Wei ◽  
He Li ◽  
Dengshun Miao ◽  
...  
Keyword(s):  
Fracture Healing ◽  
Signaling Pathway ◽  
Endochondral Ossification ◽  
Healing Process ◽  
Fracture Repair ◽  
Luciferase Reporter ◽  
Endochondral Bone Formation ◽  
Fracture Callus ◽  
Endochondral Bone ◽  
Bony Callus

Fracture healing is a complicated, long-term, and multistage repair process. Intermittent administration of parathyroid hormone (PTH) has been proven effective on intramembranous and endochondral bone formation during the fracture healing process, however, the mechanism is unclear. In this study, we investigated the role of exogenous PTH and endogenous PTH deficiency in bone fracture healing and explored the mechanism by using PTH knockout (PTH-/-) mice and ATDC5 cells. In a mouse femur fracture model, endogenous PTH deficiency could delay endochondral ossification whereas exogenous PTH promotes accumulation of endochondral bone, accelerates cartilaginous callus conversion to bony callus, enhances maturity of bony callus, and attenuates impaired fracture healing resulting from endogenous PTH deficiency. In fracture callus tissue, endogenous PTH deficiency could inhibit chondrocyte proliferation and differentiation whereas exogenous PTH could activate the IHH signaling pathway to accelerate endochondral ossification and rescue impaired fracture healing resulting from endogenous PTH deficiency. In vitro, exogenous PTH promotes cell proliferation by activating IHH signaling pathway on ATDC5 cells. In mechanistic studies, by using ChIP and luciferase reporter assays, we showed that PTH could phosphorylate CREB, and subsequently bind to the promoter of IHH, causing the activation of IHH gene expression. Therefore, results from this study support the concept that exogenous PTH 1-34 attenuates impaired fracture healing in endogenous PTH deficiency mice via activating the IHH pathway and accelerating endochondral ossification. Hence, the investigation of the mechanism underlying the effects of PTH treatment on fracture repair might guide the exploration of effective therapeutic targets for fracture.

Identifying Candidate Mechanoregulators of Skeletal Development

2009 ◽  
Author(s):  
Niamh C. Nowlan ◽  
Patrick J. Prendergast ◽  
Shahragim Tajbakhsh ◽  
Paula Murphy
Keyword(s):  
Skeletal Muscle ◽  
Endochondral Ossification ◽  
Muscle Development ◽  
Skeletal Development ◽  
Endochondral Bone Formation ◽  
Mechanical Forces ◽  
Endochondral Bone ◽  
Mutant Strains ◽  
The Relationship

Studying the relationship between mechanical forces and skeletal development can provide vital clues to the mechanoregulation of skeletogenesis, providing important information to tissue engineers hoping to create functional cartilage or bone in vitro. Many studies of the mechanoregulation of skeletal development have focused on the chick embryo e.g., [1, 2]. However, as no endochondral ossification takes place in the embryonic chick long bones [1], mammalian systems must be used to examine the effect of mechanical forces on endochondral bone formation. Mouse mutant strains exist in which muscle development is affected, providing models with which to examine skeletogenesis in the absence of skeletal muscle contractions. One such strain is Pax3sp/sp [3], also known as splotch. The splotch mutant lacks the transcription factor Pax3, which prevents the migration of muscle pre-cursor cells into the limb buds, resulting in a complete absence of skeletal muscle.

scholarly journals Missense mutations in IHH impair Indian Hedgehog signaling in C3H10T1/2 cells: Implications for brachydactyly type A1, and new targets for Hedgehog signaling

2010 ◽  
Vol 15 (1) ◽  
Author(s):  
Shengzhen Guo ◽  
Jian Zhou ◽  
Bo Gao ◽  
Jianxin Hu ◽  
Hongsheng Wang ◽  
...  
Keyword(s):  
Hedgehog Signaling ◽  
Bmp Signaling ◽  
Indian Hedgehog ◽  
Endochondral Bone Formation ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Endochondral Bone ◽  
Proliferation And Differentiation ◽  
Microarray Expression ◽  
Bmp Antagonist

AbstractHeterozygous missense mutations in IHH result in Brachydactyly type A1 (BDA1; OMIM 112500), a condition characterized by the shortening of digits due to hypoplasia/aplasia of the middle phalanx. Indian Hedgehog signaling regulates the proliferation and differentiation of chondrocytes and is essential for endochondral bone formation. Analyses of activated IHH signaling in C3H10T1/2 cells showed that three BDA1-associated mutations (p.E95K, p.D100E and p.E131K) severely impaired the induction of targets such as Ptch1 and Gli1. However, this was not a complete loss of function, suggesting that these mutations may affect the interaction with the receptor PTCH1 or its partners, with an impact on the induction potency. From comparative microarray expression analyses and quantitative real-time PCR, we identified three additional targets, Sostdc1, Penk1 and Igfbp5, which were also severely affected. Penk1 and Igfbp5 were confirmed to be regulated by GLI1, while the induction of Sostdc1 by IHH is independent of GLI1. SOSTDC1 is a BMP antagonist, and altered BMP signaling is known to affect digit formation. The role of Penk1 and Igfbp5 in skeletogenesis is not known. However, we have shown that both Penk1 and Igfbp5 are expressed in the interzone region of the developing joint of mouse digits, providing another link for a role for IHH signaling in the formation of the distal digits.

scholarly journals Parathyroid hormone-related peptide-depleted mice show abnormal epiphyseal cartilage development and altered endochondral bone formation.

1994 ◽  
Vol 126 (6) ◽  
pp. 1611-1623 ◽  
Author(s):  
N Amizuka ◽  
H Warshawsky ◽  
J E Henderson ◽  
D Goltzman ◽  
A C Karaplis
Keyword(s):  
Bone Formation ◽  
Embryonic Stem ◽  
Type Ii Collagen ◽  
Epiphyseal Plate ◽  
Epiphyseal Cartilage ◽  
Endochondral Bone Formation ◽  
Type Ii ◽  
Endochondral Bone ◽  
Cartilage Development ◽  
Calcified Cartilage

To elucidate the role of PTHrP in skeletal development, we examined the proximal tibial epiphysis and metaphysis of wild-type (PTHrP-normal) 18-19-d-old fetal mice and of chondrodystrophic litter mates homozygous for a disrupted PTHrP allele generated via homologous recombination in embryonic stem cells (PTHrP-depleted). In the PTHrP-normal epiphysis, immunocytochemistry showed PTHrP to be localized in chondrocytes within the resting zone and at the junction between proliferative and hypertrophic zones. In PTHrP-depleted epiphyses, a diminished [3H]thymidine-labeling index was observed in the resting and proliferative zones accounting for reduced numbers of epiphyseal chondrocytes and for a thinner epiphyseal plate. In the mutant hypertrophic zone, enlarged chondrocytes were interspersed with clusters of cells that did not hypertrophy, but resembled resting or proliferative chondrocytes. Although the overall content of type II collagen in the epiphyseal plate was diminished, the lacunae of these non-hypertrophic chondrocytes did react for type II collagen. Moreover, cell membrane-associated chondroitin sulfate immunoreactivity was evident on these cells. Despite the presence of alkaline phosphatase activity on these nonhypertrophic chondrocytes, the adjacent cartilage matrix did not calcify and their persistence accounted for distorted chondrocyte columns and sporadic distribution of calcified cartilage. Consequently, in the metaphysis, bone deposited on the irregular and sparse scaffold of calcified cartilage and resulted in mixed spicules that did not parallel the longitudinal axis of the tibia and were, therefore, inappropriate for bone elongation. Thus, PTHrP appears to modulate both the proliferation and differentiation of chondrocytes and its absence alters the temporal and spatial sequence of epiphyseal cartilage development and of subsequent endochondral bone formation necessary for normal elongation of long bones.

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

2017 ◽  
Vol 292 (8) ◽  
pp. 3164-3171 ◽  
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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