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. 2017 May;187(5):963-972.
doi: 10.1016/j.ajpath.2016.12.026. Epub 2017 Mar 14.

Neural EGFL-Like 1 Is a Downstream Regulator of Runt-Related Transcription Factor 2 in Chondrogenic Differentiation and Maturation

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Neural EGFL-Like 1 Is a Downstream Regulator of Runt-Related Transcription Factor 2 in Chondrogenic Differentiation and Maturation

Chenshuang Li et al. Am J Pathol. .
Free PMC article

Abstract

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.

Figures

Supplemental Figure S1
Supplemental Figure S1
Proposed model for the compensation of chondrogenic deficit by Nell-1 in Runx2 deficiency. Transgenic overexpression of Nell-1 results in a partial rescue of the Runx2−/− phenotype by presence of hypertrophic chondrocytes and mineralized cartilage in the mid-shaft of the femur. The region of calcified cartilage is shown in yellow. WT, wild-type.
Supplemental Figure S2
Supplemental Figure S2
Nell-1 promotes the proliferation of Runx2−/− chondrocytes in vitro. Proliferation analysis of primary Runx2−/− mesenchymal progenitor cell pellets at day 7 by 5-bromo-2′-deoxyuridine (BrdU) quantification. The cell number in each pellet was counted on BrdU/DAPI-stained sections and expressed as a ratio of BrdU-positive cells/total counted cells. The raw data were presented with box plot. Minimum and maximum values are depicted by short black lines, the box signifies the upper and lower quartiles, and the median is represented by a black line within the box for each group. Nell-1 stimulation significantly increases the proliferation of primary Runx2−/− mesenchymal progenitor cells. P < 0.05. Scale bar = 50 μm. PBS, phosphate-buffered saline.
Supplemental Figure S3
Supplemental Figure S3
Nell-1 promotes chondrogenic differentiation and maturation of ex vivo cultured Runx2−/− E14.5 hind limbs. Hind limb explants were isolated from WT and Runx2−/− E14.5 mouse embryos, cultured in media with or without 2.0 μg/mL Nell-1 protein for 5 days, fixed in 4% paraformaldehyde, divided into sections, and stained with hematoxylin and eosin (H&E) for histological evaluation and immunohistochemistry of collagen II and collagen X to assess chondrogenic differentiation and maturation. Boxed areas are shown at higher magnification below. Scale bars = 100 μm. PBS, phosphate-buffered saline; WT, wild-type.
Figure 1
Figure 1
Spatiotemporal expression of Nell-1 and Runx2 in the femurs of mouse embryos during chondrogenesis. Nell-1 and Runx2 expression in the femoral bone of WT (A) and Runx2−/− (B) embryos at embryonic day (E)14.5, E16.5, E18.5, and neonatal stage as examined by immunohistochemical staining. Boxed areas are shown below at higher magnification. Scale bars = 100 μm (A and B).
Figure 2
Figure 2
Nell-1 is a downstream target of Runx2 in mouse chondrocytes. A: mRNA levels of Nell-1 in mouse chondrocytes with different genotypes of Runx2. B: Transcriptional levels of Nell-1 and Runx2 in AdRunx2-transduced Runx2+/+ mouse chondrocytes. C: mRNA levels of Runx2 in mouse chondrocytes with different genotypes of Nell-1. D: Transcriptional levels of Runx2 in Nell-1 protein stimulated Nell-1+/+ mouse chondrocytes. E: Transcriptional levels of Nell-1 and Runx2 in AdNell-1–transduced Nell-1+/+ chondrocytes. Two sample t-tests were used to compare the data from two groups. Data are expressed as means + SD of three independent experiments performed in triplicate (AE). P < 0.05 versus Runx2 mRNA expression in Runx2+/+ cells; P < 0.05 versus Nell-1 mRNA expression in Runx2+/+ cells; P < 0.05 versus Runx2 mRNA expression in AdLacz-transduced chondrocytes for 24 hours; §P < 0.05 versus Nell-1 mRNA expression in AdLacz-transduced chondrocytes for 24 hours; P < 0.05 versus Nell-1 expression in Nell-1+/+ cells; P < 0.05 versus Nell-1 expression in AdLacz-transduced Nell-1+/+ chondrocytes for 24 hours. PFU, plaque-forming unit.
Figure 3
Figure 3
Exogenous Nell-1 protein promotes cartilaginous nodule formation and maturation of primary Runx2−/− mesenchymal progenitor cells. A: Nell-1 treatment promotes the chondrogenic differentiation of Runx2−/− cells at day 7 shown by hematoxylin and eosin (H&E), Alcian Blue, and Safranin O staining. The cartilage nodules are circled by the dotted yellow lines. B: Nell-1 stimulation rescues chondrogenic marker expression in Runx2−/− limb bud pellets at day 7. C: Nell-1 treatment promotes the terminal chondrogenic maturation markers of Runx2−/− cells at day 21 by gene expression. D: Nell-1 treatment promotes the chondrogenic maturation of Runx2−/− cells at day 21 shown by H&E, Alcian Blue, and osteocalcin (Ocn) staining. The cartilage nodules are circled by the dashed yellow lines. Two sample t-tests were used to compare the data from two groups. Data are expressed as means + SD (B and C). n = 6 (B and C). P < 0.05. Scale bars = 100 μm (A and D). PBS, phosphate-buffered saline; WT, wild-type.
Figure 4
Figure 4
Partial rescue of chondrogenic differentiation in neonatal Runx2−/− by Nell-1 overexpression. A: Alcian Blue/Alizarin Red skeletal staining of neonatal Runx2−/− and Runx2−/−/CMV-Nell-1 mice. The mid-shaft of the femur and lower thoracic vertebrae in neonatal Runx2−/−/CMV-Nell-1 mice clearly stain positively with Alizarin Red (arrows). Endochondral ossification is clearly absent in the vertebrae and proximal limbs of Runx2−/− mice. Calcified cartilage, which stains positively with Alizarin Red, is apparent in Runx2−/−/CMV-Nell-1 femurs. B: Hematoxylin and eosin (H&E) staining shows the cartilage morphology of Runx2−/− and Runx2−/−/CMV-Nell-1 femurs. C: Restoration of a distinct hypertrophic chondrocyte zone is clearly shown by immunostaining of collagen X in restricted regions of the limbs in Runx2−/−/CMV-Nell-1 mice. Boxed areas are shown at higher magnification to the right (AC). Scale bars: 500 μm (A, left column); 50 μm (A, right column); 200 μm (B and C). IHC, immunohistochemistry.

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