Angiopoietin-like protein 2 promotes chondrogenic differentiation during bone growth as a cartilage matrix factor
Hironori TanoueJun MorinagaTatsuya YoshizawaMasaki YugamiIto HTakayuki NakamuraY. UeharaT. MasudaHaruki OdagiriTaichi SugizakiTsuyoshi KadomatsuKeishi MiyataMotoyoshi EndoKazutoyo TeradaHiroki OchiSatoshi TakedaKazuya YamagataTakaichi FukudaHiroshi MizutaYuichi Oike
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Chondrogenesis
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Introduction: NF-κB/p65 has been reported to be involved in regulation of chondrogenic differentiation.However, its function in relation to key chondrogenic factor Sox9 and onset of chondrogenesis during endochondral ossification is poorly understood.We hypothesized that the early onset of chondrogenic differentiation is initiated by transient NF-κB/p65 signaling. Methodology:The role of NF-κB/p65 in early chondrogenesis was investigated in different in vitro, ex vivo and in vivo endochondral models: ATDC5 cells, hBMSCs, chicken periosteal explants and growth plates of six weeks old mice.NF-κB/p65 activation was manipulated using pharmacological inhibitors, RNAi and activating agents.Gene expression and protein expression analysis, and (immuno)histochemical stainings were employed to determine the role of NF-κB/p65 in the chondrogenic phase of endochondral development.Results: Our data show that chondrogenic differentiation is facilitated by early transient activation of NF-κB/p65.NF-κB/p65-mediated signaling determines early expression of Sox9 and facilitates the subsequent chondrogenic differentiation programming by signaling through key chondrogenic pathways. Conclusions:The presented data demonstrate that NF-ĸB/p65 signaling, as well as its intensity and timing, represents one of the transcriptional regulatory mechanisms of the chondrogenic developmental program of chondroprogenitor cells during endochondral ossification.Importantly, these results provide novel possibilities to improve the success of cartilage and bone regenerative techniques.
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Hypoxia, a common environmental condition, influences cell signals and functions. Here, we compared the effects of hypoxia (1% oxygen) and normoxia (air) on chondrogenic differentiation of human mesenchymal stem cells (MSCs). For in vitro chondrogenic differentiation, MSCs were concentrated to form pellets and subjected to conditions appropriate for chondrogenic differentiation under normoxia and hypoxia, followed by the analysis for the expression of genes and proteins of chondrogenesis and endochondral ossification. MSCs induced for differentiation under hypoxia increased in chondrogenesis, but decreased in endochondral ossification compared to those under normoxia. MSCs induced for differentiation were more resistant to apoptosis under hypoxia compared to those under normoxia. The hypoxia-dependent protection of MSCs from chondrogenesis-induced apoptosis correlated with an increase in the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt/FoxO pathway. These results suggest that the PI3K/Akt/FoxO survival pathway activated by hypoxia in MSCs enhances chondrogenesis and plays an important role in preventing endochondral ossification.
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We previously reported that transcription factor XBP1S is upregulated during chondrocyte differentiation and demonstrates the temporal and spatial expression pattern during skeletal development. Herein, we found that XBP1S stimulates chondrocyte differentiation from mesenchymal stem cells in vitro and endochondral ossification ex vivo. In addition, XBP1S activates granulin-epithelin precursor (GEP), a growth factor known to stimulate chondrogenesis, then enhances GEP-stimulated chondrogenesis and endochondral bone formation. Collectively, these findings demonstrate that XBP1S positively regulates endochondral bone formation by activating GEP chondrogenic growth factor.
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NF-κB/p65 has been reported to be involved in regulation of chondrogenic differentiation. However, its function in relation to key chondrogenic factor Sox9 and onset of chondrogenesis during endochondral ossification is poorly understood. We hypothesized that the early onset of chondrogenic differentiation is initiated by transient NF-κB/p65 signaling.
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Chondrogenesis and endochondral ossification are the cartilage differentiation processes that lead to skeletal formation and growth in the developing vertebrate as well as skeletal repair in the adult. The exquisite regulation of these processes, both in normal development and in pathologic situations, is impacted by a number of different types of stress. These include normal stressors such as mechanical loading and hypoxia as well pathologic stressors such as injury and/or inflammation and environmental toxins. This article provides an overview of the processes of chondrogenesis and endochondral ossification and their control at the molecular level. A summary of the influence of the most well-understood normal and pathologic stressors on the differentiation program is also presented.
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Introduction: Developmental engineering based on endochondral ossification has been proposed as a potential strategy for repairing of critical bone defects. Bone development is driven by growth plate-mediated endochondral ossification. Under physiological conditions, growth plate chondrocytes undergo compressive forces characterized by micro-mechanics, but the regulatory effect of micro-mechanical loading on endochondral bone formation has not been investigated. Methods: In this study, a periodic static compression (PSC) model characterized by micro-strain (with 0.5% strain) was designed to clarify the effects of biochemical/mechanical cues on endochondral bone formation. Hydrogel scaffolds loaded with bone marrow mesenchymal stem cells (BMSCs) were incubated in proliferation medium or chondrogenic medium, and PSC was performed continuously for 14 or 28 days. Subsequently, the scaffold pretreated for 28 days was implanted into rat femoral muscle pouches and femoral condylar defect sites. The chondrogenesis and bone defect repair were evaluated 4 or 10 weeks post-operation. Results: The results showed that PSC stimulation for 14 days significantly increased the number of COL II positive cells in proliferation medium. However, the chondrogenic efficiency of BMSCs was significantly improved in chondrogenic medium, with or without PSC application. The induced chondrocytes (ichondrocytes) spontaneously underwent hypertrophy and maturation, but long-term mechanical stimulation (loading for 28 days) significantly inhibited hypertrophy and mineralization in ichondrocytes. In the heterotopic ossification model, no chondrocytes were found and no significant difference in terms of mineral deposition in each group; However, 4 weeks after implantation into the femoral defect site, all scaffolds that were subjected to biochemical/mechanical cues, either solely or synergistically, showed typical chondrocytes and endochondral bone formation. In addition, simultaneous biochemical induction/mechanical loading significantly accelerated the bone regeneration. Discussion: Our findings suggest that microstrain mechanics, biochemical cues, and in vivo microenvironment synergistically regulate the differentiation fate of BMSCs. Meanwhile, this study shows the potential of micro-strain mechanics in the treatment of critical bone defects.
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Chondrogenesis and endochondral ossification are precisely controlled by cellular interactions with surrounding matrix proteins and growth factors that mediate cellular signaling pathways. Here, we report that extracellular matrix protein 1 (ECM1) is a previously unrecognized regulator of chondrogenesis. ECM1 is induced in the course of chondrogenesis and its expression in chondrocytes strictly depends on parathyroid hormone-related peptide (PTHrP) signaling pathway. Overexpression of ECM1 suppresses, whereas suppression of ECM1 enhances, chondrocyte differentiation and hypertrophy in vitro and ex vivo. In addition, target transgene of ECM1 in chondrocytes or osteoblasts in mice leads to striking defects in cartilage development and endochondral bone formation. Of importance, ECM1 seems to be critical for PTHrP action in chondrogenesis, as blockage of ECM1 nearly abolishes PTHrP regulation of chondrocyte hypertrophy, and overexpression of ECM1 rescues disorganized growth plates of PTHrP-null mice. Furthermore, ECM1 and progranulin chondrogenic growth factor constitute an interaction network and act in concert in the regulation of chondrogenesis.—Kong L., Zhao, Y.-P., Tian, Q.-Y., Feng J.-Q., Kobayashi, T., Merregaert, J., Liu, C.-J. Extracellular matrix protein 1, a direct targeting molecule of parathyroid hormone-related peptide, negatively regulates chondrogenesis and endochondral ossification via associating with progranulin growth factor. FASEB J. 30, 2741-2754 (2016). www.fasebj.org
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Orthodontics deals with the correction of skeletal anomalies of the face occurring in the form of jaw discrepancies. There is an abundance of findings in the literature that the development and growth of the cranial base influence facial shape and jaw discrepancies. Cranial base develops by the mechanism of endochondral ossification taking place at its midline axis, where all the synchondroses are located. Chondrogenesis is the initial and indispensable part of endochondral bone formation. In the light of evidence underlying the need of reactive oxygen species in the regulation of chondrogenesis, this study aimed to investigate the ubiquitously present antioxidant enzyme Gpx1 and its contribution to redox regulation in chondrogenesis.
Provided that the levels of oxidative stress were previously found to fluctuate according to the differentiation stage of chondrocytes, the gene expression of Gpx1 was measured with quantitative RT-PCR during chondrogenic differentiation. For this purpose, the chondrogenic cell line ATDC5 was utilized and cultured for 21 days. The time points of measurements were on days 0, 2, 6, 10, 14, and 21. The chondrogenic differentiation of the utilized cell line was determined with the stains Alcian blue and Alizarin red, and with the gene expression of chondrogenic biological markers Col2a1 and Col10a1. The present results suggest that the expression of Gpx1 is not of constitutive nature during chondrogenic differentiation.
Taking this as a starting point, the next step was to quantitatively assess the distribution pattern of Gpx1 at the different differentiation stages of chondrogenesis. To examine this, the spheno-occipital synchondrosis from eight newborn male Wistar rats was isolated and samples were processed for formaldehyde-fixed paraffin-embedded immunohistochemistry. Photographs of the immunostained sections were analysed by two independent observers and a five-grade semiquantitative scale was used to assess Gpx1 immunoreactivity at the synchondrosis. The present findings show that Gpx1 is expressed the most at the proliferative differentiation stage and the lowest at the hypertrophic differentiation stage. Existing literature reports that an increase in oxidative levels is needed for inhibition of proliferation and initiation of hypertrophy. Further, chondrocytes at the hypertrophic stage have the highest levels of ROS compared to other differentiation stages. In this context, the present results implied that Gpx1 is involved in redox regulation in chondrogenesis.
To pursue this further, the expression of Gpx1 was manipulated in ATDC5 chondrogenic cells and cells were then exposed to exogenous H2O2. The manipulation of Gpx1 expression included overexpression and silencing. A control group was also included. The apoptotic percentage of cells was measured flow cytometrically with FITC-labelled Annexin V in conjunction with PI dye. The highest apoptotic percentage was observed in Gpx1-depleted chondrocytes, followed by the control group. The lowest apoptotic percentage was presented in Gpx1-overexpressing cells. These results indicate that Gpx1 possesses an active role on the cellular enzymatic antioxidant system of chondrocytes and can regulate the cellular redox state by H2O2 scavenging. Furthermore, its presence in chondrocytes can prevent H2O2-induced apoptosis.
The contribution of cranial base growth to craniofacial morphology continues until adulthood, since spheno-occipital synchondrosis is the last of the synchondroses to ossify and is active until then. This study localizes the expression of Gpx1 at the spheno-occipital synchondrosis and documents the role of Gpx1 as a redox regulator in chondrocytes.
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Many studies have reported on the effects of cyclooxygenase-2 (COX-2) inhibition on osteogenesis.However, far less is known about the effects of COX-2 inhibition on chondrogenic differentiation.Previous studies conducted by our group show that COX-2 inhibition influences in vitro chondrogenic differentiation.Importantly, this might have consequences on endochondral ossification processes occurring in vivo, such as bone fracture healing, growth plate development and ectopic generation of cartilage.The goal of our study was to investigate, in vivo, the effect of COX-2 inhibition by celecoxib on the cartilaginous phase of three different endochondral ossification scenarios.10 mg/kg/d celecoxib or placebo were orally administered for 25 d to skeletally-immature New Zealand White rabbits (n = 6 per group).Endochondral ossification during fracture healing of a non-critical size defect in the ulna, femoral growth plate and ectopically-induced cartilaginous tissue were examined by radiography, micro-computed tomography (µ-CT), histology and gene expression analysis.Celecoxib treatment resulted in delayed bone fracture healing, alterations in growth plate development and progression of mineralisation.In addition, chondrogenic differentiation of ectopically-induced cartilaginous tissue was severely impaired by celecoxib.In conclusion, we found that celecoxib impaired the chondrogenic phase of endochondral ossification.
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Abstract Skeletal growth and fracture healing rely on the mineralization of cartilage in a process called endochondral ossification. Chondrocytes firstly synthesize and then modify cartilage by the release of a wide range of particles into their extracellular space. Extracellular vesicles (EVs) are one type of such particles, but their roles in endochondral ossification are yet to be fully understood. It remains a challenge to obtain representative populations of chondrocyte‐derived EVs, owing to difficulties both in preserving the function of primary chondrocytes in culture and in applying the serum‐free conditions required for EV production. Here, we used the ATDC5 cell‐line to recover chondrocyte‐derived EVs from early‐ and late‐differentiation stages, representing chondrocytes before and during cartilage mineralization. After screening different culture conditions, our data indicate that a serum‐free Opti‐MEM‐based culture medium preserves chondrocyte identity and function, matrix mineralization and cell viability. We subsequently scaled‐up production and isolated EVs from conditioned medium by size‐exclusion chromatography. The obtained chondrocyte‐derived EVs had typical ultrastructure and expression of classical EV markers, at quantities suitable for downstream experiments. Importantly, chondrocyte‐derived EVs from late‐differentiation stages had elevated levels of alkaline phosphatase activity. Hence, we established a method to obtain functional chondrocyte‐derived EVs before and during cartilage mineralization that may aid the further understanding of their roles in endochondral bone growth and fracture healing.
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