Neural EGFL-Like 1 Is a Downstream Regulator of Runt-Related Transcription Factor 2 in Chondrogenic Differentiation and Maturation
2017; Elsevier BV; Volume: 187; Issue: 5 Linguagem: Inglês
10.1016/j.ajpath.2016.12.026
ISSN1525-2191
AutoresChenshuang Li, Jie Jiang, Zhong Zheng, Kevin M. Lee, Yan-Heng Zhou, Eric Chen, Cymbeline T. Culiat, Yiqiang Qiao, Xuepeng Chen, Kang Ting, Xinli Zhang, Chia Soo,
Tópico(s)Osteoarthritis Treatment and Mechanisms
ResumoRecent studies indicate that neural EGFL-like 1 (Nell-1), a secretive extracellular matrix molecule, is involved in chondrogenic differentiation. Herein, we demonstrated that Nell-1 serves as a key downstream target of runt-related transcription factor 2 (Runx2), a central regulator of chondrogenesis. Unlike in osteoblast lineage cells where Nell-1 and Runx2 demonstrate mutual regulation, further studies in chondrocytes revealed that Runx2 tightly regulates the expression of Nell-1; however, Nell-1 does not alter the expression of Runx2. More important, Nell-1 administration partially restored Runx2 deficiency–induced impairment of chondrocyte differentiation and maturation in vitro, ex vivo, and in vivo. Mechanistically, although the expression of Nell-1 is highly reliant on Runx2, the prochondrogenic function of Nell-1 persisted in Runx2−/− scenarios. The biopotency of Nell-1 is independent of the nuclear import and DNA binding functions of Runx2 during chondrogenesis. Nell-1 is a key functional mediator of chondrogenesis, thus opening up new possibilities for the application of Nell-1 in cartilage regeneration. Recent studies indicate that neural EGFL-like 1 (Nell-1), a secretive extracellular matrix molecule, is involved in chondrogenic differentiation. Herein, we demonstrated that Nell-1 serves as a key downstream target of runt-related transcription factor 2 (Runx2), a central regulator of chondrogenesis. Unlike in osteoblast lineage cells where Nell-1 and Runx2 demonstrate mutual regulation, further studies in chondrocytes revealed that Runx2 tightly regulates the expression of Nell-1; however, Nell-1 does not alter the expression of Runx2. More important, Nell-1 administration partially restored Runx2 deficiency–induced impairment of chondrocyte differentiation and maturation in vitro, ex vivo, and in vivo. Mechanistically, although the expression of Nell-1 is highly reliant on Runx2, the prochondrogenic function of Nell-1 persisted in Runx2−/− scenarios. The biopotency of Nell-1 is independent of the nuclear import and DNA binding functions of Runx2 during chondrogenesis. Nell-1 is a key functional mediator of chondrogenesis, thus opening up new possibilities for the application of Nell-1 in cartilage regeneration. Chondrogenesis is an obligatory step for skeletogenesis, which results in the construction of the primary skeleton of the vertebrate embryo.1Lefebvre V. Bhattaram P. Vertebrate skeletogenesis.Curr Top Dev Biol. 2010; 90: 291-317Crossref PubMed Scopus (141) Google Scholar Chondrogenesis begins as the mesenchymal cells migrate and tightly pack at the presumptive skeletogenic sites to form cell mass condensations.1Lefebvre V. Bhattaram P. Vertebrate skeletogenesis.Curr Top Dev Biol. 2010; 90: 291-317Crossref PubMed Scopus (141) Google Scholar, 2Lefebvre P. Martin P.J. 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Chondrogenesis, chondrocyte differentiation, and articular cartilage metabolism in health and osteoarthritis.Ther Adv Musculoskelet Dis. 2012; 4: 269-285Crossref PubMed Scopus (283) Google Scholar, 7Otto F. Thornell A.P. Crompton T. Denzel A. Gilmour K.C. Rosewell I.R. Stamp G.W. Beddington R.S. Mundlos S. Olsen B.R. Selby P.B. Owen M.J. Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development.Cell. 1997; 89: 765-771Abstract Full Text Full Text PDF PubMed Scopus (2403) Google Scholar, 8Inada M. Yasui T. Nomura S. Miyake S. Deguchi K. Himeno M. Sato M. Yamagiwa H. Kimura T. Yasui N. Ochi T. Endo N. Kitamura Y. Kishimoto T. Komori T. Maturational disturbance of chondrocytes in Cbfa1-deficient mice.Dev Dyn. 1999; 214: 279-290Crossref PubMed Scopus (505) Google Scholar, 9Kim I.S. Otto F. Zabel B. Mundlos S. Regulation of chondrocyte differentiation by Cbfa1.Mech Dev. 1999; 80: 159-170Crossref PubMed Scopus (393) Google Scholar Our previous study showed that neural EGFL-like 1 (Nell-1), an extracellular matrix molecule distributed in human uncalcified articular cartilage,12Li C.S. Zhang X. Peault B. Jiang J. Ting K. Soo C. Zhou Y.H. Accelerated chondrogenic differentiation of human perivascular stem cells with NELL-1.Tissue Eng Part A. 2016; 22: 272-285Crossref PubMed Scopus (27) Google Scholar enhances chondrogenic marker expression and cartilage nodule formation of rabbit chondrocytes.13Lee M. Siu R.K. Ting K. Wu B.M. Effect of Nell-1 delivery on chondrocyte proliferation and cartilaginous extracellular matrix deposition.Tissue Eng Part A. 2010; 16: 1791-1800Crossref PubMed Scopus (43) Google Scholar In addition, application of Nell-1 induces hyaline cartilage regeneration, as demonstrated in a rabbit knee subchondral defect model,14Siu R.K. Zara J.N. Hou Y. James A.W. Kwak J. Zhang X. Ting K. Wu B.M. Soo C. Lee M. NELL-1 promotes cartilage regeneration in an in vivo rabbit model.Tissue Eng Part A. 2012; 18: 252-261Crossref PubMed Scopus (42) Google Scholar and administration of Nell-1–overexpressed bone marrow mesenchymal stem cells promotes articular cartilage reestablishment in critical-sized goat mandibular condyle osteochondral defects.15Zhu S. Zhang B. Man C. Ma Y. Hu J. NEL-like molecule-1-modified bone marrow mesenchymal stem cells/poly lactic-co-glycolic acid composite improves repair of large osteochondral defects in mandibular condyle.Osteoarthritis Cartilage. 2011; 19: 743-750Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar Developmentally, in comparison with wild-type (WT) littermates, newborn Nell-1 overexpression transgenic mice exhibit premature hypertrophy and apoptosis in the chondrocranium region,16Zhang X. Cowan C.M. Jiang X. Soo C. Miao S. Carpenter D. Wu B. Kuroda S. Ting K. Nell-1 induces acrania-like cranioskeletal deformities during mouse embryonic development.Lab Invest. 2006; 86: 633-644Crossref PubMed Scopus (24) Google Scholar whereas neonatal Nell-1–deficient mice have shorter and deformed rib cages and vertebral bodies with compressed intervertebral spaces accompanied with reduced cartilage extracellular matrix.17Desai J. Shannon M.E. Johnson M.D. Ruff D.W. Hughes L.A. Kerley M.K. Carpenter D.A. Johnson D.K. Rinchik E.M. Culiat C.T. Nell1-deficient mice have reduced expression of extracellular matrix proteins causing cranial and vertebral defects.Hum Mol Genet. 2006; 15: 1329-1341Crossref PubMed Scopus (89) Google Scholar Moreover, Nell-1 deficiency also results in reduced expression of multiple cartilage-related genes.17Desai J. Shannon M.E. Johnson M.D. Ruff D.W. Hughes L.A. Kerley M.K. Carpenter D.A. Johnson D.K. Rinchik E.M. Culiat C.T. Nell1-deficient mice have reduced expression of extracellular matrix proteins causing cranial and vertebral defects.Hum Mol Genet. 2006; 15: 1329-1341Crossref PubMed Scopus (89) Google Scholar Taken together, these findings indicate that Nell-1 may also play a role in chondrogenic differentiation and maturation. To better understand the potential regulatory roles of Nell-1 in chondrogenesis, the current study focuses on Runx2−/− mice to eliminate the pivotal contribution of Runx2. Mice were bred and maintained, as previously described,7Otto F. Thornell A.P. Crompton T. Denzel A. Gilmour K.C. Rosewell I.R. Stamp G.W. Beddington R.S. Mundlos S. Olsen B.R. Selby P.B. Owen M.J. Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development.Cell. 1997; 89: 765-771Abstract Full Text Full Text PDF PubMed Scopus (2403) Google Scholar, 17Desai J. Shannon M.E. Johnson M.D. Ruff D.W. Hughes L.A. Kerley M.K. Carpenter D.A. Johnson D.K. Rinchik E.M. Culiat C.T. Nell1-deficient mice have reduced expression of extracellular matrix proteins causing cranial and vertebral defects.Hum Mol Genet. 2006; 15: 1329-1341Crossref PubMed Scopus (89) Google Scholar, 18Zhang X. Ting K. Bessette C.M. Culiat C.T. Sung S.J. Lee H. Chen F. Shen J. Wang J.J. Kuroda S. Soo C. Nell-1, a key functional mediator of Runx2, partially rescues calvarial defects in Runx2(+/−) mice.J Bone Miner Res. 2011; 26: 777-791Crossref PubMed Scopus (78) Google Scholar, 19Zhang X. Kuroda S. Carpenter D. Nishimura I. Soo C. Moats R. Iida K. Wisner E. Hu F.Y. Miao S. Beanes S. Dang C. Vastardis H. Longaker M. Tanizawa K. Kanayama N. Saito N. Ting K. Craniosynostosis in transgenic mice overexpressing Nell-1.J Clin Invest. 2002; 110: 861-870Crossref PubMed Scopus (131) Google Scholar under institutional approval by the Chancellor's Animal Research Committee at UCLA (protocol number 2012-041). Runx2 heterozygous deficient mice (Runx2+/−; generated by embryonic stem cells derived from the 129 mouse strain backcrossed with the C57BL/6 strain7Otto F. Thornell A.P. Crompton T. Denzel A. Gilmour K.C. Rosewell I.R. Stamp G.W. Beddington R.S. Mundlos S. Olsen B.R. Selby P.B. Owen M.J. Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development.Cell. 1997; 89: 765-771Abstract Full Text Full Text PDF PubMed Scopus (2403) Google Scholar) were mated with Nell-1–overexpressing mice (CMV-Nell-1; derivative of the B6C3 strain19Zhang X. Kuroda S. Carpenter D. Nishimura I. Soo C. Moats R. Iida K. Wisner E. Hu F.Y. Miao S. Beanes S. Dang C. Vastardis H. Longaker M. Tanizawa K. Kanayama N. Saito N. Ting K. Craniosynostosis in transgenic mice overexpressing Nell-1.J Clin Invest. 2002; 110: 861-870Crossref PubMed Scopus (131) Google Scholar) to obtain Runx2−/−/CMV-Nell-1 mice.18Zhang X. Ting K. Bessette C.M. Culiat C.T. Sung S.J. Lee H. Chen F. Shen J. Wang J.J. Kuroda S. Soo C. Nell-1, a key functional mediator of Runx2, partially rescues calvarial defects in Runx2(+/−) mice.J Bone Miner Res. 2011; 26: 777-791Crossref PubMed Scopus (78) Google Scholar Because severe reduction of Nell-1 expression in homozygotes [Nell-16R/6R; Nell-16R: an N-ethyl-N-nitrosourea–induced point mutation that truncates an 810 amino acid Nell-1 protein at residue 50220Rinchik E.M. Carpenter D.A. Selby P.B. A strategy for fine-structure functional analysis of a 6- to 11-centimorgan region of mouse chromosome 7 by high-efficiency mutagenesis.Proc Natl Acad Sci U S A. 1990; 87: 896-900Crossref PubMed Scopus (116) Google Scholar, 21Rinchik E.M. Carpenter D.A. Johnson D.K. Functional annotation of mammalian genomic DNA sequence by chemical mutagenesis: a fine-structure genetic mutation map of a 1- to 2-cM segment of mouse chromosome 7 corresponding to human chromosome 11p14-p15.Proc Natl Acad Sci U S A. 2002; 99: 844-849Crossref PubMed Scopus (23) Google Scholar] induces neonatal death,17Desai J. Shannon M.E. Johnson M.D. Ruff D.W. Hughes L.A. Kerley M.K. Carpenter D.A. Johnson D.K. Rinchik E.M. Culiat C.T. Nell1-deficient mice have reduced expression of extracellular matrix proteins causing cranial and vertebral defects.Hum Mol Genet. 2006; 15: 1329-1341Crossref PubMed Scopus (89) Google Scholar Nell-16R heterozygous mice (Nell-1+/6R) were used to produce Nell-16R/6R fetuses. Mouse genotypes were determined by PCR, and mRNA expression levels of Nell-1 and Runx2 were monitored using quantitative real-time PCR. The animals in this study were euthanized with an overdose of phenobarbital (Piramal Healthcare, Maharashtra, India). After euthanization, three neonatal Runx2−/−/CMV-Nell-1 mice and five Runx2−/− littermates were skinned, dissected, and fixed in 95% ethanol for 16 hours before standard skeletal staining with Alcian Blue and Alizarin Red to provide gross distinction between cartilage and mineralized tissues. For paraffin embedding, hind limbs isolated from mouse embryos at different stages and from neonatal mice were fixed in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO) at 4°C overnight. For Runx2-deficient mice and N-ethyl-N-nitrosourea–induced Nell-1–deficient mice, embryo pairs from six litters were used for each utero stage. Four neonatal Runx2−/−/CMV-Nell-1 mice and three Runx2−/− littermates were also used for histological analysis. Hematoxylin and eosin staining was performed on paraffin sections (5 μm thick) for histological analysis, whereas Safranin O staining, Alcian Blue staining, and immunohistochemical and immunofluorescence staining were performed following standard protocols.13Lee M. Siu R.K. Ting K. Wu B.M. Effect of Nell-1 delivery on chondrocyte proliferation and cartilaginous extracellular matrix deposition.Tissue Eng Part A. 2010; 16: 1791-1800Crossref PubMed Scopus (43) Google Scholar, 14Siu R.K. Zara J.N. Hou Y. James A.W. Kwak J. Zhang X. Ting K. Wu B.M. Soo C. Lee M. NELL-1 promotes cartilage regeneration in an in vivo rabbit model.Tissue Eng Part A. 2012; 18: 252-261Crossref PubMed Scopus (42) Google Scholar Primary antibodies against collagen II (Developmental Studies Hybdridoma Bank, Iowa City, IA), collagen X (Developmental Studies Hybdridoma Bank), Nell-1 (Allele Biotechnology, San Diego, CA), osteocalcin (Abcam, Cambridge, UK), and Runx2 (Santa Cruz Biotechnology, Santa Cruz, CA) were used for immunohistochemical and/or immunofluorescence staining, respectively. DAPI (Sigma-Aldrich) was used for nuclear counterstaining in immunofluorescence staining. After removing soft tissues by 2 mg/mL protease (Roche, Nutley, NJ) and 3 mg/mL collagenase II (Roche), the rib cages of neonatal mouse embryos were digested in 1 mg/mL collagenase II for 3 hours to achieve single-cell suspension. Chondrocytes were cultured in a basal culture medium (Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin). Medium was changed every 3 days and cells were passaged at 70% to 90% confluence. All cell culture reagents were purchased from Invitrogen (Carlsbad, CA). A total of 5 × 104 cells/well passage 2 (P2) chondrocytes were seeded in 6-well plates with basal culture medium for 6 hours. Recombinant human Nell-1 protein was synthesized by Aragen Bioscience Inc. (Morgan Hill, CA) with a purity of 92%. Before treatment, cells were synchronized by culture in starvation medium [Dulbecco's modified Eagle's medium + 1% insulin–transferrin-sodium selenite media supplement (BD Biosciences, San Jose, CA)] for 18 hours. For adenoviral infection, P2 mouse chondrocytes at 80% confluence were infected with AdLacZ, AdNell-1, or AdRunx2 at a multiplicity of infection of 50, 250, or 500 plaque-forming units per cell, as described previously.22Zhang X. Carpenter D. Bokui N. Soo C. Miao S. Truong T. Wu B. Chen I. Vastardis H. Tanizawa K. Kuroda S. Ting K. Overexpression of Nell-1, a craniosynostosis-associated gene, induces apoptosis in osteoblasts during craniofacial development.J Bone Miner Res. 2003; 18: 2126-2134Crossref PubMed Scopus (57) Google Scholar Primary embryonic mesenchymal progenitor cells were isolated from limb buds of E11.5 embryonic mice, as previously described.23Aydelotte M.B. Kochhar D.M. Development of mouse limb buds in organ culture: chondrogenesis in the presence of a proline analog, L-azetidine-2-carboxylic acid.Dev Biol. 1972; 28: 191-201Crossref PubMed Scopus (66) Google Scholar Briefly, limbs were dissected from the embryos and then digested with 1 mg/mL Dispase (Roche) for 1.5 hours at 37°C. Cells were filtered through a prewashed 40-μm cell strainer to generate single-cell suspension. After wash with Puck's Saline A solution, cells were suspended in culture medium [Dulbecco's modified Eagle's medium:F12 = 2:3 (Invitrogen) containing 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, 0.5 mmol/L glutamine, 50 μg/mL vitamin C (Sigma-Aldrich), 10 nmol/L β-glycerophosphate (Sigma-Aldrich)] with or without 2 μg/mL recombinant human Nell-1 protein, and were plated at the density of 2 × 105 cells per tube, centrifuged at 500 × g for 5 minutes, then incubated at 37°C in a 5% CO2 humidified incubator. Medium was changed every 48 hours. Pellets were cultured at 7 or 21 days for further analysis. Mouse hind limbs were isolated from embryonic day (E)14.5 mouse embryos. Isolated limbs were cultured in BGJb medium (Invitrogen) for 24 hours on polycarbonate tissue culture inserts (pore size, 0.1 μm; EMD Millipore, Billerica, MA). For the treatment, α-modified Eagle's medium containing 5% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.5 mmol/L glutamine (Invitrogen) with or without 2 μg/mL recombinant human Nell-1 protein was changed every 48 hours for 5 days to simulate the environment in neonatal mice (term, 19.5 days). Limb explants were fixed in 4% paraformaldehyde at 4°C overnight, embedded in paraffin, and divided into sections (5 μm thick). 5-Bromo-2′-deoxyuridine (Invitrogen) was added 6 hours before sample collection, and pellets were fixed in 4% paraformaldehyde at 4°C overnight, embedded in paraffin, and divided into sections (5 μm thick). DAPI was used for nuclear counterstaining. Total RNA was isolated by TRIzol Reagent (Invitrogen) followed by DNase (Invitrogen) treatment. RNA (1 μg) was injected for reverse transcription (RT) with the SuperScript II Reverse Transcriptase Kit (Invitrogen), as per the manufacturer's instructions. Real-time PCR was performed on the 7300 Real-Time PCR system with SYBR Green Mastermix (Invitrogen). The primer pair sequences were as follows: Acan, 5′-CCAGGCTCCACCAGATACTC-3′ (forward) and 5′-TGCTCATAGCCTGCCTCATA-3′ (reverse); Adamts4, 5′-ATGGCCTCAATCCATCCCAG-3′ (forward) and 5′-AAGCAGGGTTGGAATCTTTGC-3′ (reverse); Col2α1, 5′-GTCCTGAAGGTGCTCAAGGT-3′ (forward) and 5′-TTTGGCTCCAGGAATACCAT-3′ (reverse); Mmp13, 5′-TGTTTGCAGAGCACTACTTGAA-3′ (forward) and 5′-CAGTCACCTCTAAGCCAAAGAAA-3′ (reverse); Nell-1, 5′-TCCTGGGTAGATGGTGACAA-3′ (forward) and 5′-CATTGGCCAGAAATATGCAC-3′ (reverse); Runx2, 5′-AACGATCTGAGATTTGTGGGC-3′ (forward) and 5′-CCTGCGTGGGATTTCTTGGTT-3′ (reverse); and SRY-Box 9, 5′-ACGGCTCCAGCAAGAACAAG-3′ (forward) and 5′-TTGTGCAGATGCGGGTACTG-3′ (reverse). Concomitant glyceraldehyde 3-phosphate dehydrogenase was also evaluated in separate tubes for each RT reaction as a housekeeping standard [5′-ATTCAACGGCACAGTCAAGG-3′ (forward) and 5′-GATGTTAGTGGGGTCTCGCTC-3′ (reverse)]. Relative gene expression was analyzed by the ΔΔCT method.24Livak K.J. Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.Methods. 2001; 25: 402-408Crossref PubMed Scopus (123392) Google Scholar Images were acquired at room temperature with the CellSens Standard 1.9 software (Olympus, America Inc., Center Valley, PA) on a microscope (Olympus) using 4× (dry HC Plan Apochromat; numerical aperture, 0.13), 10× (dry HC Plan Apochromat; numerical aperture, 0.30), and 20× (dry HC Plan Apochromat; numerical aperture, 0.17) objective lenses. Images were processed in Photoshop CS2 and Illustrator CS4 (Adobe Systems Computer Software Company, San Jose, CA) for merging. Statistical analysis was performed by OriginPro 8 (Origin Lab Corp., Northampton, MA) and included the one-way analysis of variance and two-sample t-test. The Mann-Whitney test was used for nonparametric data. Statistical significance was determined at the P < 0.05 level. To reveal the spatiotemporal expression pattern of Nell-1 and Runx2 during long bone development, the hind limbs of fetal and neonatal WT (Runx2+/+) and Runx2−/− mice were harvested for immunohistochemical examination. In WT animals, from E16.5 onward, both Nell-1 and Runx2 staining were observed in the superficial osteoblasts of the newly formed trabeculae (Figure 1A). Runx2 and Nell-1 were also coexpressed in the perichondrium and prehypertrophic chondrocytes of the WT E14.5 femurs (Figure 1A). Beginning at E16.5, Runx2 was preferentially expressed in hypertrophic chondrocytes of WT mice, where intense Nell-1 expression was also observed (Figure 1A and Supplemental Figure S1). This spatiotemporal overlap in endogenous Nell-1 and Runx2 expression at different chondrogenic differentiation zones during long bone development suggested a possible regulatory relationship between Runx2 and Nell-1 in chondrogenesis, at least during the late stages of chondrogenic differentiation. Unlike WT mice with strong Nell-1 staining in the resting, proliferating, and prehypertrophic chondrocytes of femurs from E16.5 to newborn (Figure 1A), Runx2−/− mice barely exhibited Nell-1 staining throughout the whole femurs during the entire examining period, accompanied with impaired endochondral ossification (Figure 1B and Supplemental Figure S1). Therefore, at least during late limb development, Nell-1 expression in chondrocytes is highly reliant on Runx2. To examine the regulatory relationship between Nell-1 and Runx2 in chondrocytes, we next evaluated the expression of Runx2 mRNA and Nell-1 mRNA in chondrocytes of neonatal mice with different genotypes. The expression level of Nell-1 mRNA in Runx2−/− mouse chondrocytes was only half of that in WT control (Figure 2A). In addition, forcing Runx2 overexpression in WT mouse chondrocytes by adenovirus AdRunx2 significantly stimulated Nell-1 mRNA expression in a viral dose-dependent manner (Figure 2B). In contrast, expression of Runx2 mRNA was retained at similar levels in mouse chondrocytes with different Nell-1 genotypes (Figure 2C). Moreover, neither Nell-1 protein nor AdNell-1 adenovirus had a significant effect on Runx2 mRNA expression in the Nell-1+/+ chondrocytes (Figure 2, D and E). These results clearly demonstrated that, in chondrocytes, Runx2 regulated Nell-1 expression but that Nell-1 did not alter Runx2 expression. After 7 days of chondrogenic differentiation, in vitro pellet cultures of primary mesenchymal progenitor cells isolated from Runx2−/− mouse embryonic limb buds exhibited a larger undifferentiated area with less intense Alcian Blue and Safranin O staining in cartilaginous components in comparison to the pellet controls formed by cells isolated from WT littermates (Figure 3A). Interestingly, administration of Nell-1 protein not only promoted cell proliferation in Runx2−/− pellets (Supplemental Figure S2), but also increased cartilaginous nodule formation in the Runx2−/− pellets, which contained rich cartilage matrix characterized by high staining intensity of Alcian Blue and Safranin O (Figure 3A). Transcriptionally, expression of genes encoding chondrogenic markers, such as Col2α1 (encoding collagen II, α 1, an abundant and specific protein in cartilage), Acan (encoding aggrecan core protein, one of the major structural components in cartilage matrix), and SRY-Box 9,1Lefebvre V. Bhattaram P. Vertebrate skeletogenesis.Curr Top Dev Biol. 2010; 90: 291-317Crossref PubMed Scopus (141) Google Scholar, 2Lefebvre P. Martin P.J. Flajollet S. Dedieu S. Billaut X. Lefebvre B. Transcriptional activities of retinoic acid receptors.Vitam Horm. 2005; 70: 199-264Crossref PubMed Scopus (111) Google Scholar, 3Wuelling M. Vortkamp A. Chondrocyte proliferation and differentiation.Endocr Dev. 2011; 21: 1-11Crossref PubMed Scopus (81) Google Scholar, 4Goldring M.B. Chondrogenesis, chondrocyte differentiation, and articular cartilage metabolism in health and osteoarthritis.Ther Adv Musculoskelet Dis. 2012; 4: 269-285Crossref PubMed Scopus (283) Google Scholar in Runx2−/− pellets was significantly lower than that in WT pellets (Figure 3B). However, disruption of chondrogenic differentiation in Runx2−/− pellets was rescued by Nell-1 protein administration (Figure 3, A and B). Meanwhile, after 21 days of cultivation, Runx2−/− pellets exhibited significantly decreased expression of terminal chondrogenic differentiation markers [Adamts4 (encoding aggrecanase-1)25Djouad F. Delorme B. Maurice M. Bony C. Apparailly F. Louis-Plence P. Canovas F. Charbord P. Noel D. Jorgensen C. Microenvironmental changes during differentiation of mesenchymal stem cells towards chondrocytes.Arthritis Res Ther. 2007; 9: R33Crossref PubMed Scopus (142) Google Scholar and Mmp13 (encoding matrix metallopeptidase 13)1Lefebvre V. Bhattaram P. Vertebrate skeletogenesis.Curr Top Dev Biol. 2010; 90: 291-317Crossref PubMed Scopus (141) Google Scholar, 2Lefebvre P. Martin P.J. Flajollet S. Dedieu S. Billaut X. Lefebvre B. Transcriptional activities of retinoic acid receptors.Vitam Horm. 2005; 70: 199-264Crossref PubMed Scopus (111) Google Scholar, 3Wuelling M. Vortkamp A. Chondrocyte proliferation and differentiation.Endocr Dev. 2011; 21: 1-11Crossref PubMed Scopus (81) Google Scholar, 4Goldring M.B. Chondrogenesis, chondrocyte differentiation, and articular cartilage metabolism in health and osteoarthritis.Ther Adv Musculoskelet Dis. 2012; 4: 269-285Crossref PubMed Sco
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