Sequential Epiphyseal Cartilage Changes of Femoral Heads in C57BL/6 Female Mice Treated with Excessive Glucocorticoids

Cartilage ◽  
2020 ◽  
pp. 194760352097857
Author(s):  
Shengyang Jin ◽  
Liang Yang ◽  
Chunqing Meng ◽  
Yu He ◽  
Kaige Ma ◽  
...  
Keyword(s):  
Western Blotting ◽  
Vascular Invasion ◽  
Epiphyseal Cartilage ◽  
Chondrocyte Apoptosis ◽  
Control Group ◽  
Tunel Staining ◽  
Micro Computed Tomography ◽  
Femoral Heads ◽  
Chondrocyte Metabolism

Objective Excessive use of glucocorticoids (GCs) may cause adverse effects on the skeletal system in children. However, only a few studies have reported the effects of GCs on the epiphyseal cartilage. This study aimed to uncover the subsequent epiphyseal cartilage changes of immature femoral heads after excessive GC treatment in a mouse model and explain the pathological changes preliminarily. Design Female C57BL/6 mice were divided into control and model (excessive GC treatment) groups. The structure of the femoral heads was evaluated by using micro-computed tomography, hematoxylin-eosin staining, and safranin staining analyses. Immunohistochemistry was used to detect angiogenesis and cartilage metabolism. Western blotting and TUNEL staining were used to examine epiphyseal cartilage chondrocyte apoptosis. Primary chondrocytes were isolated from the femoral heads of healthy mice for in vitro studies. The effects of GCs on chondrocyte apoptosis and metabolism were determined by flow cytometry and Western blotting. Results The epiphyseal cartilage ossification had started at 4 weeks posttreatment in a portion of mice; the ossification presented as a sequential process in the model group, while the epiphyseal cartilage maintained an unossified state in the control group. Vascular invasion into the epiphyseal cartilage of the model mice was observed at 4 weeks posttreatment. GCs induced chondrocyte apoptosis and altered chondrocyte metabolism in the epiphyseal cartilage. Conclusions The epiphyseal cartilage ossification accelerated in the femoral heads of female C57BL/6 mice after excessive GC treatment. Increased chondrocyte apoptosis, altered chondrocyte metabolism, as well as increased vascular invasion, are the potential factors influencing epiphyseal cartilage ossification.

PSX-B-13 Effect of epidermal growth factor and prolactin on the quality of bovine oocytes aging in vitro

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 327-327
Author(s):  
Ekaterina Shedova ◽  
Galina Singina ◽  
Irina Y Lebedeva ◽  
Aleksandr Lopukhov
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Control Group ◽  
Tunel Staining ◽  
Apoptosis Resistance ◽  
Prolonged Culture ◽  
Epidermal Growth ◽  
Bovine Oocytes

Abstract The evaluation of factors responsible for the protection of the oocytes attained the metaphase-II stage from aging is importance for successful in vitro embryo reproduction. The aim of the present research was to study dose-dependent effects of epidermal growth factor (EGF) and prolactin (PRL) on the quality of bovine oocytes after their aging in vitro. Bovine cumulus-enclosed oocytes (CEOs) were matured in vitro for 20 h in TCM 199 containing 0.2 mM sodium pyruvate, 10% fetal calf serum (FCS), 10 μg/ml FSH and LH. At the end of in vitro maturation, oocytes were transferred to TCM 199 supplemented with 10% FCS (aging medium) and cultured for additional 24 h in the absence (Control) and in presence of EGF (10 and 50 ng/ml) and PRL (20 and 50 ng/ml). After prolonged culture oocytes were used for apoptosis detection (TUNEL staining, n=251) and the state of chromosomes evaluation (Tarkowski’s cytogenetic method, n=359). The data from 3–4 replicates were analyzed by ANOVA. At the end of prolonged culture (24 h) the rate of apoptotic oocytes in the Control group was 47.4±8.5%. EGF at concentration of 10 ng/ml and PRL at both doses decreased this rate to 15.0–22.1% (p < 0.05). Furthermore, PRL (not EGF) reduced the frequency of abnormal chromosome modifications (decondensation, adherence, clumping) at concentrations of 20–50 ng/ml from 58.7±2.1% (Control) to 41.2±1.9 and 45.6±2.7% respectively (p < 0.01). Thus, EGF and PRL is able to maintain the apoptosis resistance of bovine oocytes during their prolonged in vitro culture as well as PRL have the decelerating effect on abnormal modifications of M-II chromosomes. The research was supported by RFBR (17-29-08035) and the Ministry of Science and Higher Education of Russia.

75 EFFECT OF PHENAZINE ETHOSULFATE ON PORCINE BLASTOCYST DEVELOPMENT, APOPTOSIS, AND CRYOTOLERANCE AFTER OPEN PULLED STRAW VITRIFICATION

2008 ◽  
Vol 20 (1) ◽  
pp. 118
Author(s):  
B. Gajda ◽  
Z. Smorag ◽  
M. Bryla
Keyword(s):  
Cell Number ◽  
Developmental Competence ◽  
Control Group ◽  
Tunel Staining ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
Morula Stage ◽  
Phenazine Ethosulfate ◽  
And Control

It is possible to improve the success of cryopreservation of in vitro-produced bovine embryos by modifying the embryos with the metabolic regulator phenazine ethosulfate (PES) (Seidel 2006 Theriogenology 65, 228–235). The PES treatment increased glucose matabolism, tended to increase the pentose phosphate pathway flux of glucose, and clearly reduced accumulation of lipids in cultured bovine embryos (De La Torre-Sanchez et al. 2006 Reprod. Fertil. Dev. 18, 597–607). It is known that porcine embryos have a considerably high content of lipids, and the success rates of their cryopreservation appear to be highly correlated with cytoplasmic lipid content. In our preliminary study, we observed that supplementation of NCSU-23 medium with PES has a positive effect on efficiency of pig blastocysts of good quality (Gajda et al.. 2007 Acta Biochim. Pol. 54(Suppl 1), 52 abst). In the present study, the effects of PES on pig blastocyst development, apoptosis, and survival after vitrification were investigated. In Exp. 1, porcine zygotes obtained from superovulated gilts were cultured in NCSU-23 medium supplemented with 0 (control), 0.025, 0.05, or 0.075 µm PES. The culture was performed at 39�C, with 5% CO2 in air, for 96–120 h. Embryo quality criteria were developmental competence (cleavage, morula stage, and blastocyst stage), cell number per blastocyst, and the degree of apoptosis as assessed by TUNEL staining. In Exp. 2, expanded blastocysts cultured with 0.025 µm PES were vitrified in a ethylene glycol and dimethyl sulfoxide mixture using open pulled straw (OPS) technology (Vajta et al. 1997 Acta Vet. Scand. 38, 349–352). After thawing, the blastocysts were cultured in vitro for re-expansion or transferred to synchronized recipients. Data were analyzed by chi-square test. There was a difference between the 0.025 µm PES-treated and the control group in percentage of cleaved embryos (99.0 and 91.4%, respectively; P < 0.05), between all experimental groups and control in percentage of morula stage (90.7, 87.8, 83.8, and 80.0%, respectively), and between 0.025 and 0.05 µm PES-treated and control in percentage of blastocyst rates (70.0, 75.5, and 65.7%, respectively). The number of cells and percentage of TUNEL-positive nuclei per blastocyst were lower in the PES-treated than in the control group. The survival rate of blastocysts after vitrification and thawing was enhanced in the presence of PES compared to that in the PES-free group (45.2 and 38.9%, respectively; P < 0.05). After transfer of 56 expanded blastocysts cultured with PES and vitrified into 3 recipients, two gilts were confirmed pregnant at 35 days of gestation. In conclusion, a higher blastocyst percentage with a low incidence of apoptosis was obtained in the presence of PES compared to control. These blastocysts also had an increased ability to survive cryopreservation.

Effect of lenalidomide on the response of bladder cancer to BCG immunotherapy in an in vivo murine model.

2012 ◽  
Vol 30 (5_suppl) ◽  
pp. 288-288
Author(s):  
Eugene K. Lee ◽  
Jinesh Gerald ◽  
Ashish M. Kamat
Keyword(s):  
Cell Cycle ◽  
Bladder Cancer ◽  
Western Blotting ◽  
Murine Model ◽  
Statistical Significance ◽  
Combination Group ◽  
Control Group ◽  
Immunomodulatory Agent

288 Background: Intravesical BCG is the gold standard for non-muscle-invasive bladder cancer (NMIBC). However, many patients do not respond to therapy while others relapse and/or progress. As a result, there remains a need for therapies that can enhance the efficacy of BCG. We explore the efficacy of lenalidomide, an immunomodulatory agent used in multiple myeloma and myelodysplastic syndrome, in combination with BCG in vitro and in an in vivo bladder cancer model. Methods: We studied the effects of lenalidomide in combination with BCG induced cytokines in MBT-2 cells using PI-FACS. For in vitro studies, we used 10 and 100 nM of lenalidomide in combination with TNF-a and FasL. We then performed Western blotting for cell cycle and apoptosis regulatory proteins. Subsequently, we tested the efficacy of this combination in an immunocompetent murine model of bladder cancer with MBT-2 cells in C3H mice using the flank injection method. Drug dosages were 30 mg/kg for lenalidomide and 105 CFU of BCG. Tumor growth curves were created for the control, lenalidomide, BCG and combination treatment mice groups. Immunohistochemistry (IHC) was then performed using antibodies against proteins related to cell cycle, apoptosis, angiogenesis and immune response. Results: PI-FACS identified increased DNA fragmentation in the combinations of lenalidomide and TNF-a and FasL compared to control and each agent alone. Using Western blotting, we demonstrated that the combination resulted in apoptosis via caspase-3 activation. In the murine model, combination therapy resulted in a statistically significant decreased tumor size compared to the control group. While the BCG alone and lenalidomide alone groups did show a trend toward smaller tumor, they did not reach statistical significance. Furthermore, the TUNEL assay showed a substantial increase in apoptosis only in the combination group. Immunohistochemistry demonstrated decreased angiogenesis in all treatment groups compared to control, as well as, decreased T-cell infiltration. Conclusions: Our study demonstrates a potential role for the immunomodulatory agent, lenalidomide, in combination with BCG for NMIBC. This in vivo model serves as a template for future clinical trials.

scholarly journals Hypoxia and its possible relationship with endometrial receptivity in adenomyosis: a preliminary study

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Song Guo ◽  
Di Zhang ◽  
Xiaowei Lu ◽  
Qian Zhang ◽  
Ruihuan Gu ◽  
...  
Keyword(s):  
Real Time ◽  
Western Blotting ◽  
Real Time Pcr ◽  
Underlying Mechanism ◽  
Endometrial Receptivity ◽  
Control Group ◽  
Expression Levels ◽  
Preliminary Study

Abstract Background Adenomyosis (AM) is an important cause of female infertility. However, the underlying mechanism remains unclear. This report describes a preliminary study of hypoxia and its possible association with endometrial receptivity in AM. Methods The study was divided into in vitro and in vivo experiments. In vitro, expression levels of the endometrial receptivity markers HOXA10 and HOXA11 in the implantation period were examined using real-time PCR and western blotting. Endometrial expression of hypoxia-inducible factor (HIF)-1α, HIF-2α, and HIF-3α was determined using immunohistochemistry. In vivo, using an AM mouse model established by oral administration of tamoxifen, we inhibited expression of HIF-2α using an HIF-2α antagonist (PT2399; 30 mg/kg body weight, twice daily by oral gavage for 2 days) and then examined expression levels of Hoxa10 and Hoxa11 using real-time PCR and western blotting. Results Endometrial mRNA and protein expression levels of HOXA10 and HOXA11 were significantly lower in patients with AM than in control patients. Expression of HIF-2α was significantly higher in the AM group than in the control group, whereas that of HIF-1α and HIF-3α was equivalent in both groups. In vivo analysis showed that administration of the HIF-2α antagonist resulted in increased expression of Hoxa10 and Hoxa11 at both the mRNA and protein levels in AM model mice. Conclusions HIF-2α overexpression may be one reason for decreased endometrial receptivity in AM. The current findings provide insight into HIF-2α-mediated AM-related infertility and suggest that PT2399 has potential as a treatment for AM. Trial registration This trial was retrospectively registered.

scholarly journals The Expression and Significance of mTORC1 in Diabetic Retinopathy

2019 ◽  
Author(s):  
Yanli Liu ◽  
Yarong Zheng ◽  
Yekai Zhou ◽  
Yi Liu ◽  
Mengjuan Xie ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Diabetic Retinopathy ◽  
Western Blotting ◽  
Rat Model ◽  
High Glucose ◽  
Control Group ◽  
Diabetes Group ◽  
And Migration ◽  
Proliferation And Migration

Abstract Background: To investigate the expression and significance of mechanistic target of rapamycin complex 1(mTORC1) in diabetic retinopathy(DR), and to find new targets and new methods for the treatment of DR.Methods: A DR rat model was prepared by general feeding combined with intraperitoneal injection of 10% streptozotocin (60 mg/kg). The rats were randomly divided into a control group (NDM group) and diabetes group (DM group).Three months later,the degrees of retinopathy were determined using hematoxylin and eosin staining,and the levels of p-S6, VEGF, and PEDF proteins were detected by immunohistochemistry and western blotting. Human retinal capillary endothelial cells (HRCECs) were cultured in high glucose conditions,then treated with rapamycin or transfected with siTSC1.The protein levels of p-S6 were assessed by western blotting. The 5-ethynyl-2´-deoxyuridine assay was used to detect cell proliferation, and the Transwell assay was used to detect cell migration.Results: A DM rat model was successfully developed. The expressions of p-S6 and VEGF proteins were significantly increased in the DM group (p < 0.05), and the expression of PEDF protein was significantly decreased compared with the control group (p < 0.05). In vitro,the p-S6 protein in high glucose(HG) induced HRCECs was increased compared with the normal control (p < 0.05), and cell proliferation and migration were increased compared with the normal glucose(NG) group (p < 0.05). After transfection with siTSC1 to activate mTORC1,the expression of p-S6 was increased,as well as cell proliferation and migration.In contrast rapamycin decreased p-S6 expression in HG induced HRCECs, as well as decrased proliferation and migration (p < 0.05).Conclusion: The mTORC1 played an important role in DR. After activation, mTORC1 induced expression of the p-S6 protein, regulated the expressions of VEGF and PEDF proteins, and changed the proliferation and migration of endothelial cells.The mTORC1 can therefore be used as a new target,as well as in the treatment of DR.

scholarly journals Assortment of Stauntonia hexaphylla and Cornus officinalis protect against testosterone-induced benign prostatic hyperplasia through anti-inflammatory and anti-proliferative activity

2019 ◽  
Author(s):  
Shanika Karunasagara ◽  
Geum-Lan Hong ◽  
Da-Young Jung ◽  
Kyung-Hyun Kim ◽  
Eun-Jeong Koh ◽  
...  
Keyword(s):  
Benign Prostatic Hyperplasia ◽  
Western Blotting ◽  
Specific Antigen ◽  
Prostatic Hyperplasia ◽  
Nuclear Antigen ◽  
Control Group ◽  
Cornus Officinalis

AbstractBenign prostatic hyperplasia (BPH) is a progressive pathological condition associated with proliferation of prostatic tissues, prostate enlargement, and lower-urinary tract symptoms. However, the mechanism underlying the pathogenesis of BPH is not clear. The aim of this study was to investigate the protective effects of Stauntonia hexaphylla and Cornus officinalis (SC extract) on a testosterone propionate (TP)-induced BPH model. For in vitro experiments, a human prostate adenocarcinoma cell line was used to perform western blotting for androgen receptor (AR), prostate specific antigen (PSA), and 5α-reductase type 2. Male Sprague-Dawley rats were randomly divided into 8 groups as follows for the in vivo experiments: control, BPH, Fina, Saw, SC25, SC50, SC100, and SC200. To induce BPH, all rats, except those in the control group, were daily administered with subcutaneous injections of TP (5 mg/kg), and orally treated with appropriate PBS/drugs for 4 consecutive weeks. Our findings indicated that the SC treatment significantly reduced the prostate size and downregulated the serum testosterone and DHT levels in BPH rats. The histological examination revealed that SC treatment markedly recovered the TP-induced abnormalities and reduced the prostatic hyperplasia. In addition, in vitro and in vivo western blotting indicated that SC treatment significantly downregulated the AR, PSA, and 5α-reductase type 2 expression, while an immunohistochemistry examination revealed that the SC extract significantly reduced the expression of type 2 5α-reductase and proliferating cell nuclear antigen positive cell count. Collectively, our findings demonstrated that SC extract attenuates BPH through anti-proliferative and anti-inflammation activities and might be useful in the clinical treatment of BPH.

scholarly journals Debris Removal by Activation of Endodontic Irrigants in Complex Root Canal Systems: A Standardized In-Vitro-Study

2021 ◽  
Vol 11 (16) ◽  
pp. 7331
Author(s):  
Matthias Widbiller ◽  
Lukas Keim ◽  
Ralf Schlichting ◽  
Birgit Striegl ◽  
Karl-Anton Hiller ◽  
...  
Keyword(s):  
Root Canal ◽  
Ethylenediaminetetraacetic Acid ◽  
In Vitro Study ◽  
Model System ◽  
Control Group ◽  
Complex Root ◽  
Micro Computed Tomography ◽  
Canal Systems ◽  
Endodontic Irrigation

Aim of the study was to develop a standardized model system to investigate endodontic irrigation techniques and assess the efficiency of different activation methods on the removal of hard tissue debris in complex root canal systems. Mesial roots of mandibular molars were firstly scanned by micro-computed tomography (µCT) and allocated to three groups of irrigant activation: sonic activation (EDDY, VDW, Munich, Germany), laser activation (AutoSWEEPS, FOTONA, Ljubljana, Slovenia) and conventional needle irrigation (control). Roots were fixed in individual 3D-printed holders to facilitate root canal enlargement under constant irrigation with NaOCl (5%). To enable standardized quantification of remaining debris, BaSO4-enriched dentine powder was compacted into the canals, followed by another µCT-scan. The final irrigation was performed using 17% ethylenediaminetetraacetic acid (EDTA) and 5% sodium hypochlorite (NaOCl) with the respective activation method, and the volume of remaining artificial debris was quantified after a final µCT-scan. The newly developed model system allowed for reliable, reproducible and standardized assessment of irrigation methods. Activation of the irrigant proved to be significantly more effective than conventional needle irrigation regarding the removal of debris, which persisted particularly in the apical third of the root canal in the control group. The efficiency of irrigation was significantly enhanced with laser- and sonic-based activation, especially in the apical third.

scholarly journals Yu-Ping-Feng Formula Exerts Antilung Cancer Effects by Remodeling the Tumor Microenvironment through Regulating Myeloid-Derived Suppressor Cells

2021 ◽  
Vol 2021 ◽  
pp. 1-16
Author(s):  
Yuli Wang ◽  
Ningyang Sun ◽  
Yingbin Luo ◽  
Zhihong Fang ◽  
Yuan Fang ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Tumor Microenvironment ◽  
Western Blotting ◽  
Suppressor Cells ◽  
T Cell Subsets ◽  
Control Group ◽  
Spleen Weight ◽  
Myeloid Derived Suppressor Cells ◽  
Rt Pcr

Yu-Ping-Feng (YPF) formula is a classical prescription used for enhancing the body’s immunity function in traditional Chinese medicine (TCM). In clinical practice, the YPF formula has been reported to exhibit antilung cancer and immunomodulatory effect. However, the relationship between them remains unclear. The present study aimed to investigate the antilung cancer effect of the YPF formula and its immune-related mechanisms. The C57BL/6 tumor-bearing mice model was established and randomly divided into the YPF group and the control group. Tumor volume, spleen weight, and survival in both groups were measured and evaluated during 28 days of consecutive intervention. Flow cytometry was used to detect the proportion of immune cell subsets. Myeloid-derived suppressor cells (MDSCs) were induced in vitro from bone marrow cells. After intervention by the YPF formula, CCK-8 and flow cytometry analyses were performed to detect proliferation and apoptosis of MDSCs. A coculture system containing T cells and MDSCs was established to further study the role of MDSCs in the regulation of T-cell subsets proportion by the YPF formula. The expressions of MDSCs-related genes and proteins were detected by RT-PCR and Western blotting. The results showed the YPF formula inhibited tumor growth, reduced spleen weight, and prolonged the survival of mice. Besides, the proportions of MDSCs subsets and Regulatory T (Treg) in the YPF group decreased, whereas those of CD4+T and CD8+T increased both in vitro and in vivo. CCK-8 and flow cytometry demonstrated that the YPF formula could inhibit proliferation and promote apoptosis of MDSCs. The coculture experiments further confirmed that MDSCs served a critical role in regulating the tumor microenvironment by the YPF formula. RT-PCR and Western blotting indicated that the levels of MDSCs’ activation and proliferation-related proteins and genes were downregulated in the YPF group. Therefore, our results demonstrated that the YPF formula could promote apoptosis and inhibit the proliferation of MDSCs. As a result, the negative regulatory effect on the positive immune cells induced by MDSCs was weakened, thus achieving the antilung cancer effect by remodeling the tumor microenvironment.

scholarly journals Transient Receptor Potential Channel, Vanilloid 5, Induces Chondrocyte Apoptosis in a Rat Osteoarthritis Model Through the Mediation of Ca2+ Influx

2018 ◽  
Vol 46 (2) ◽  
pp. 687-698 ◽  
Author(s):  
Yingliang Wei ◽  
Dianbin Zheng ◽  
Xiaocheng Guo ◽  
Min Zhao ◽  
Linlin Gao ◽  
...  
Keyword(s):  
Articular Cartilage ◽  
Western Blotting ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Transient Receptor Potential Channel ◽  
Chondrocyte Apoptosis ◽  
Caspase 8 ◽  
Pathological Feature ◽  
Transient Receptor

Background/Aims: Chondrocyte apoptosis is the most common pathological feature in cartilage in osteoarthritis (OA). Transient receptor potential channel vanilloid 5 (TRPV5) is important in regulating calcium ion (Ca2+) influx. Accumulating evidences suggest that Ca2+ is a major intracellular second messenger that can trigger cell apoptosis. Therefore, we investigate the potential role of TRPV5 in mediating Ca2+ influx to promote chondrocyte apoptosis in OA. Methods: The monoiodoacetic acid (MIA)-induced rat OA model was assessed by macroscopic and radiographic analyses. Calmodulin protein immunolocalization was detected by immunohistochemistry. The mRNA and protein level of TRPV5, calmodulin and cleaved caspase-8 in articular cartilage were assessed by real time polymerase chain reaction and western blotting. Primary chondrocytes were isolated and cultured in vitro. TRPV5 small interfering RNA was used to silence TRPV5 in chondrocytes. Then, calmodulin and cleaved caspase-8 were immunolocalized by immunofluorescence in chondrocyte. Fluo-4AM staining was used to assess intracellular Ca2+ to reflect TRPV5 function of mediation Ca2+ influx. Annexin V-fluorescein isothiocyanatepropidium iodide flow cytometric analysis was performed to determine chondrocytes apoptosis. Western blotting techniques were used to measure the apoptosis-related proteins in chondrocyte level. Results: Here, we reported TRPV5 was up-regulated in MIA-induced OA articular cartilage. Ruthenium red (a TRPV5 inhibitor) can relieve progression of joint destruction in vivo which promoted us to demonstrate the effect of TRPV5 in OA. We found that TRPV5 had a specific role in mediating extracellular Ca2+ influx leading to chondrocytes apoptosis in vitro. The apoptotic effect was inhibited even reversed by silencing TRPV5. Furthermore, we found that the increase Ca2+ influx triggered apoptosis by up-regulating the protein of death-associated protein, FAS-associated death domain, cleaved caspase-8, cleaved caspase-3, cleaved caspase-6, and cleaved caspase-7, and the up-regulated proteins were abolished by silencing TRPV5 or 1, 2-bis-(o-Aminophenoxy)-ethane-N,N,N’,N’-tetraacetic acid, tetraacetoxymethyl ester (a Ca2+ chelating agent). Conclusion: The up-regulated TRPV5 could used be as an initiating factor that induces extrinsic chondrocyte apoptosis via the mediation of Ca2+ influx. These findings suggested TRPV5 could be an intriguing mediator for drug target in OA.

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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