Targeting Runx2 expression in hypertrophic chondrocytes impairs endochondral ossification during early skeletal development

Abstract

Runx2 is a known master transcription factor for osteoblast differentiation, as well as an essential regulator for chondrocyte maturation. Recently, more and more data has shown that Runx2 regulates hypertrophic chondrocyte-specific type X collagen gene (Col10a1) expression in different species. However, how Runx2 regulation of Col10a1 expression impacts chondrocyte maturation, an essential step of endochondral bone formation, remains unknown. We have recently generated transgenic mice in which Flag-tagged Runx2 was driven by a cell-specific Col10a1 control element. Significantly increased level of Runx2 and Col10a1 mRNA transcripts were detected in transgenic mouse limbs at both E17.5 (embryonic day 17.5) and P1 (post-natal day1) stages, suggesting an in vivo correlation of Runx2 and Col10a1 expression. Surprisingly, skeletal staining suggested delayed ossification in both the axial and the appendicular skeleton of transgenic mice from E14.5 until P6. Histological analysis showed elongated hypertrophic zones in transgenic mice, with less von Kossa and TUNEL staining in long bone sections at both E17.5 and P1 stages, suggesting defective mineralization due to delayed chondrocyte maturation or apoptosis. Indeed, we detected increased level of anti-apoptotic genes B-cell leukemia/lymphoma 2, Osteopontin, and Sox9 in transgenic mice by real-time RT-PCR. Moreover, immunohistochemistry and Western blotting analysis also suggested increased Sox9 expression in hypertrophic chondrocytes of transgenic mice. Together, our data suggest that targeting Runx2 in hypertrophic chondrocytes upregulates expression of Col10a1 and other marker genes (such as Sox9). This will change the local matrix environment, delay chondrocyte maturation, reduce apoptosis and matrix mineralization, and eventually, lead to impaired endochondral ossification.

FIG.1. Generation of Runx2 transgenic mice using cell-specific Col10a1-control element

(A). Positions of the ~300-bp (-4296 to -4009 bp) hypertrophic chondrocyte-specific Col10a1 distal promoter and its 330-bp basal promoter (-220 to +110 bp) elements (top) that have been used to generate transgenic reporter (LacZ) construct were as illustrated (middle) (Zheng et al., 2009). Same Col10a1 distal promoter and a shorter Col10a1 basal promoter (-220 to +45 bp) elements were used to generate Runx2-expressing (with a flag-tag) transgenic construct. The 3’-sequence of this shorter basal promoter ends at exon I allowing transgene expression without additional splicing acceptor site. XBP: Col10a1 basal promoter; ShXBP: Shorter Col10a1 basal promoter. (B). Paraffin embedded sections of tibia and fibula from X-gal-stained Tg-4x300 transgenic mouse embryos at E15.5 were counterstained with nuclear fast red. Blue staining indicating reporter activity was exclusively throughout the hypertrophic zone (left panel). No surrounding tissues showed reporter expression (right panel, lower magnification). Tg: transgenic mice, HZ: hypertrophic zone of growth plate. (C). PCR genotyping using Runx2 and Flag fragment specific primers indicated that we have successfully generated five transgenic founders (lanes 1, 3, 5, 8, and 10).

FIG.2. Runx2 and Col10α1 mRNA is upregulated in transgenic mice at E17.5 and P1 stages

(A). Immunohistochemistry analysis was conducted on TG mouse hind limb sections with or without anti-Flag antibody. Brown staining showing Flag expression was primarily restricted to the pre-hypertrophic and hypertrophic chondrocytes of proximal tibia (right panel, red arrows). Left panel shows no antibody control. TG: Transgenic. HZ: hypertrophic zone. (B). Relative mRNA transcripts of Col10a1 and Runx2 were examined using total RNAs prepared from whole hind limbs at E17.5 stage. Runx2 showed two-fold upregulation in transgenic (TG) mice compared to their wild-type (WT) littermates (WT vs. TG: 1.00 ± 0.20 vs. 2.10 ± 0.36, n = 6, t = 2.69, df = 10, p = 0.02). Meanwhile, Col10a1 expression was also increased in TG mice (WT vs. TG: 1.00 ± 0.32 vs. 2.89 ± 0.77, n = 5, t = 2.27, df = 8, p = 0.05). (C). As illustrated, both Runx2 and Col10a1 showed similar increased level of mRNA transcripts in TG mice at P1 stage (Runx2, WT vs. TG: 1.00 ± 0.15 vs. 2.25 ± 0.43, n = 6, t = 2.78, df = 10, p = 0.02; Col10α1, WT vs. TG: 1.00 ± 0.26 vs. 3.11 ± 0.63, n = 5-6, t = 3.30, df = 9, p = 0.009). (D). Runx2 and Col10a1 also showed upregulation in transgenic mice using total RNAs prepared from hypertrophic zone-enriched tissues at P1 stag (Runx2, WT vs. TG: 1.00 ± 0.13 vs. 1.94 ± 0.28, n = 4-8, t = 2.04, df = 10, p = 0.043; Col10α1, WT vs. TG: 1.00 ± 0.19 vs. 2.59 ± 0.43, n = 4-8, t = 2.50, df = 10, p = 0.031). TG: transgenic; WT: wild-type littermates. Bars denote means ± SE for Runx2 and Col10α1 expression. “*”: p < 0.05; “**”: p < 0.01.

FIG.3. Delayed ossification in Col10α1-Runx2 transgenic mice

(A). Alcian blue and alizarin red staining of mouse embryos at E17.5 stage showed slightly delayed ossification (less alizarin red staining) in TG mice compared to littermate controls in following skeletal tissues. A1: Whole skeletal staining, no ossification signs were observed in the phalanges in both TG and WT embryos (black and red arrows). A2: Skull, sagittal suture and front/posterior fontanels are wider in TG embryos (red arrows). A3: Rib and thoracic vertebrae, less alizarin red can be observed in vertebrae and xiphoid (red arrows). A4/A5: Fore and hind limbs, obvious alizarin red staining can be seen in the last metatarsal bone of WT embryos while TG embryos can barely see the red staining (red arrows). A6: Tails, alizarin red staining showed no difference in the 4^th caudal vertebrae of both WT and TG mouse embryos (black and red arrows). Bottom panel shows zoomed-in pictures of A3, A4, and A5. (B). Less ossification signals in the same panel of skeletons at P1 stage. B1: Whole skeletal staining, detectable alizarin red staining was observed in the phalanges in both TG and WT P1 mice (black and red arrows). B2: Skull, sagittal suture is wider and front/posterior fontanels are more open in TG mice (red arrows) compared to WT ones (black arrows). B3: Rib and thoracic vertebrae, less alizarin red signaling can be observed in vertebrae and the xiphoid is shorter in TG mice (red arrows). B4/B5: Fore and hind limbs, less alizarin red staining show in the metatarsal/phalange bones and calceneus of the TG mice (red arrows) compared to WT controls (black arrows). B6: Tails, alizarin red staining can be seen until the 8^th caudal vertebrae of TG mice (red arrow) compared to WT littermates (up to 11^th caudal vertebrae, black arrow). Bottom panel shows zoomed-in pictures of B3, B4, and B5.

FIG.3. Delayed ossification in Col10α1-Runx2 transgenic mice

(A). Alcian blue and alizarin red staining of mouse embryos at E17.5 stage showed slightly delayed ossification (less alizarin red staining) in TG mice compared to littermate controls in following skeletal tissues. A1: Whole skeletal staining, no ossification signs were observed in the phalanges in both TG and WT embryos (black and red arrows). A2: Skull, sagittal suture and front/posterior fontanels are wider in TG embryos (red arrows). A3: Rib and thoracic vertebrae, less alizarin red can be observed in vertebrae and xiphoid (red arrows). A4/A5: Fore and hind limbs, obvious alizarin red staining can be seen in the last metatarsal bone of WT embryos while TG embryos can barely see the red staining (red arrows). A6: Tails, alizarin red staining showed no difference in the 4^th caudal vertebrae of both WT and TG mouse embryos (black and red arrows). Bottom panel shows zoomed-in pictures of A3, A4, and A5. (B). Less ossification signals in the same panel of skeletons at P1 stage. B1: Whole skeletal staining, detectable alizarin red staining was observed in the phalanges in both TG and WT P1 mice (black and red arrows). B2: Skull, sagittal suture is wider and front/posterior fontanels are more open in TG mice (red arrows) compared to WT ones (black arrows). B3: Rib and thoracic vertebrae, less alizarin red signaling can be observed in vertebrae and the xiphoid is shorter in TG mice (red arrows). B4/B5: Fore and hind limbs, less alizarin red staining show in the metatarsal/phalange bones and calceneus of the TG mice (red arrows) compared to WT controls (black arrows). B6: Tails, alizarin red staining can be seen until the 8^th caudal vertebrae of TG mice (red arrow) compared to WT littermates (up to 11^th caudal vertebrae, black arrow). Bottom panel shows zoomed-in pictures of B3, B4, and B5.

FIG.4. Histology analysis of mouse growth plates

(A). H&E staining of sagittal sections of proximal tibia suggested longer hypertrophic zone in E17.5 TG mouse embryos (red double arrows) compared to WT littermates (black double arrows). Bars represent 50 μm. (B). Safranin O/Fast Green staining of P1 proximal tibia sections also indicated that TG mice (red double arrows) have longer hypertrophic zone than littermate controls (black arrows). (C). More layers of hypertrophic chondrocytes can be seen in sagittal sections of proximal ulna in TG mice (yellow rectangle, red arrow) at P1 stage compared to littermate controls (yellow rectangle, black arrow). (D). von Kossa staining was performed to quantify matrix mineralization on proximal tibia of TG or WT mice at P1 stage. Less von Kossa staining (black dots) suggesting decreased mineralization was shown in the hypertrophic zone of TG mice (red square and arrows) compared to WT littermate controls (black square and arrows).

FIG.5. Differential expression of chondrogenic and apoptotic marker genes

(A). qRT-PCR was performed to examine following chondrogenic and apoptotic marker genes using total RNAs prepared from mouse hind limbs at E17.5 stage. As illustrated, Sox9 (2.30-fold, p=0.0297) is significantly upregulated in TG mice compared to their WT littermates. Meanwhile Bcl-2 (2-fold, p = 0.016), and Opn (1.93-fold, p = 0.0097) were also significantly upregulated in TG mice compared to littermate controls, while Bax (1.42 fold, p=0.1837) is slightly upregulated but without statistical significance. (B). Relevant marker genes were also examined at the P1 stage. As illustrated, Bcl-2 (2.3-fold, p = 0.002), Opn (1.69-fold, p = 0.0361), and Sox9 (17.88-fold, p=0.0484) were also significantly increased in TG mice compared to WT littermates. Similar to the result of E17.5, Bax (1.18 fold, p=0.2892) did not show significant changes between TG and WT controls. Bars denote means ± SE for marker gene expression. TG: transgenic; WT: wild-type. “*”: p<0.05; “**”: p<0.01. (C). Western blotting assay was performed using protein extracts prepared from TG or WT mouse hind limbs at the P1 stage and Runx2 or Sox9 antibodies. Both Runx2 and Sox9 are highly expressed in TG or WT mouse limbs (top). Anti-Lamin B antibody was used as an internal control. Densitometry analysis showed that Sox9 increased ~50%, while Runx2 was slightly decreased in TG mouse limbs (bottom), but not statistically significant. (D). Western blotting was also conducted using protein extracts from primary cultured chondrocytes of TG or WT mice at P1 stage and the antibodies as mentioned above. The results showed that Runx2 is weakly expressed, while Sox9 is highly expressed in all the primary chondrocytes examined (top). Densitometry analysis suggested that both runx2 and Sox9 are slightly increased in TG groups but without statistical significance (bottom).

FIG.6. Runx2, Sox9 expression and apoptosis assay in mouse growth plates

(A). IHC assay was performed on distal tibia sections of TG or WT littermates at P1 stage with anti-Runx2 antibody. Intense brown staining showing Runx2 expression was primarily restricted to pre-hypertrophic and hypertrophic chondrocytes of WT controls (top, black circles). Much less brown staining indicating decreased Runx2 expression was observed in comparable tibia sections of TG mice (red circles, bottom). (B). IHC assay on proximal tibia sections of WT controls with anti-Sox9 antibody showed that Sox9 is weakly expressed in pre-hypertrophic chondrocytes, while it is barely detectable in hypertrophic chondrocytes (top, black arrows). IHC assay on comparable TG tibia sections showed more intense immune-staining signal in pre-hypertrophic and hypertrophic chondrocytes, suggesting ectopic expression of Sox9 in TG mice (bottom, red arrows). (C). Apoptosis was determined by TUNEL staining on comparable proximal tibia sections of TG and WT littermates. The majority of the apoptotic cells were localized in the hypertrophic zone of WT control (top, black rectangles). Less apoptotic cells were observed in comparable tibia section of TG mice (bottom, red rectangles).

FIG.7. Potential mechanisms of Runx2-Col10a1 effects on endochondral bone formation

The putative impacts on bone formation in this Col10a1-Runx2 mouse model are proposed: selectively targeting Runx2 overexpression in hypertrophic chondrocytes results in upregulated Col10a1 expression; this will cause local matrix environment change and the subsequent matrix mineralization. The increased Sox9 expression in hypertrophic chondrocytes possibly mediates Runx2 degradation or dominates over Runx2 and other genes’ function, delays chondrocyte maturation and apoptosis, subsequently affects the process of blood vessel invasion, bone mineralization and eventually, leads to impaired endochondral ossification. Meanwhile, upregulation of Bcl-2 and Opn may also delay chondrocyte apoptosis or terminal chondrocyte maturation and therefore, an expanded hypertrophic zone. Red arrows: promote chondrocyte maturation or bone formation; Blue arrows: inhibitory effect.