Expression Profiling and Functional Analysis of Candidate Col10a1 Regulators Identified by the TRAP Program

Abstract

Hypertrophic chondrocytes and their specific marker, the type X collagen gene (Col10a1), are critical components of endochondral bone formation during skeletal development. We previously found that Runx2 is an indispensable mouse Col10a1 gene regulator and identified many other transcription factors (TFs) that potentially interact with the 150-bp Col10a1 cis-enhancer. However, the roles of these candidate TFs in Col10a1 expression and chondrocyte hypertrophy have not been elucidated. Here, we focus on 32 candidate TFs recently identified by analyzing the 150-bp Col10a1 enhancer using the transcription factor affinity prediction (TRAP) program. We found that 12 TFs (Hoxa3, Lsx, Evx2, Dlx5, S8, Pax2, Egr2, Mef2a, Barhl2, GKlf, Sox17, and Crx) were significantly upregulated and four TFs (Lhx4, Tbx5, Mef2c, and Hb9) were significantly downregulated in hypertrophic MCT cells, which show upregulation of Col10a1 expression. Most of the differential expression pattern of these TFs conformed with the results obtained from ATDC5 cell model and primary mouse chondrocytes. Notably, Tbx5 was downregulated upon Col10a1 upregulation, overexpression of Tbx5 decreased Col10a1 expression, and knock-down of Tbx5 increased Col10a1 expression in hypertrophic chondrocytes, suggesting that Tbx5 is a negative regulator of Col10a1. We further generated a stable Tbx5-overexpressing ATDC5 cell line and ColX-Tbx5 transgenic mice driven by Col10a1-specific enhancers and promoters. Tbx5 overexpression decreased Col10a1 expression in ATDC5 cells cultured as early as day 7 and in limb tissue on post-natal day 1. Slightly weaker alkaline phosphatase staining was also observed in cell culture on day 7 and in limb digits on embryonic day 17.5, indicating mildly delayed ossification. Further characterization of these candidate Col10a1 transcriptional regulators could help identify novel therapeutic targets for skeletal diseases associated with abnormal chondrocyte hypertrophy.

Keywords: Col10a1 regulators; Runx2; TRAP program; Tbx-5; chondrocyte hypertrophy; skeletal disease.

Figure 1

The 150-bp Col10a1 cis-enhancer and its binding sites for selected candidate TFs. (A) Schematic illustration of the cis-enhancer derived from the Col10a1 distal promoter region (−4,296 to −4,147 bp). Positions of putative binding sites for Pax2, Nrf2, Nfat, Tbx5, Gklf, Gli, CACD, and Runx2 are shown. (B–E) Sequences of predicted binding sites for Tbx5, Gklf, Gli, and CACD are highlighted in red. These TFBSs are the same as or overlap with the previously described Runx2 binding site TGTGGGTGTGGC (−4,187 to −4,176 bp) (Li et al., 2011).

Figure 2

Relative Col10a1 and candidate TFs mRNA levels in hypertrophic vs. proliferative chondrocytes. (A) Col10a1 mRNA levels were upregulated in hypertrophic compared with proliferative MCT cells at 1, 2, and 3 days. (B) Col10a1 mRNA level in ATDC5 cells was upregulated on day 14 compared with day 0. (C) Col10a1 mRNA was barely detectable in proliferative primary chondrocytes but was abundantly expressed in hypertrophic primary chondrocytes. (D,E) Hoxa3, lsx, Evx2, Dlx5, S8, Pax2, Egr2, Mef2a, Barhl2, GKlf, Sox17, and Crx were upregulated and Lhx4, Tbx5, Mef2c, and Hb9 were downregulated in hypertrophic MCT cells. (F) Hoxa3, lsx, Lhx4, Evx2, Dlx5, Egr2, Mef2a, Mef2c, Barhl2, GKlf, Sox17, and Crx were upregulated and S8, Pax2, Tbx5, and Hb9 were downregulated in hypertrophic ATDC5 cells. (G) Except for Tbx5, all other genes examined were significantly upregulated in hypertrophic primary chondrocytes. *p < 0.05, **p < 0.01.

Figure 3

Col10a1 and candidate TFs protein expression in hypertrophic chondrocytes. (A) Protein levels of Col10a1, Dlx5, Egr2, Klf4, and Sox17 were increased and that of Tbx5 was decreased in hypertrophic compared with proliferative MCT cells. (B) Relative protein levels of Col10a1, Dlx5, Egr2, Gklf, Sox17, and Tbx5 normalized to β-actin in hypertrophic vs. proliferative MCT cells. (C) Strong green fluorescence signal indicating Col10a1 protein expression was observed throughout the hypertrophic zone. Dlx5, Egr2, Gklf, and Sox17 protein were also abundantly expressed in hypertrophic chondrocytes, with some Sox17 signal also seen in resting or proliferative chondrocytes. Tbx5 signal was much weaker in hypertrophic chondrocytes compared with that of other TFs. (D) Immunohistochemistry analysis detected strong Col10a1 expression only in the extracellular matrix of hypertrophic chondrocytes (top row), whereas no obvious Tbx5 expression was detected in either proliferative or hypertrophic chondrocytes. *p < 0.05, **p < 0.01.

Figure 4

Transfection of Tbx5 in MCT cells. (A) Schema graph of Tbx5 expression plasmid driven by Col10a1-specific enhancer and promoter. (B) The Col10a1 gene (top) and Col10a1–Tbx5 transgenic construct (bottom). The ~300-bp hypertrophic chondrocyte-specific Col10a1 cis-element illustrated previously locate in the distal promoter (−4,296 to −4,009 bp) and four copies of the ~300-bp cis-elements and a short basal promoter (265bp) element were used to drive the Tbx5 gene with a Flag-tag. ATG: start codon; TAG: stop codon; ShXBP: Short Col10a1 basal promoter. (C,D) Transient transfection of Tbx5 increased the mRNA level of Tbx5 and downregulated Col10a1 expression. (E,F) Compared with scrambled RNA, Tbx5 siRNA downregulated Tbx5 expression and increased Col10a1 expression. *p < 0.05, **p < 0.01.

Figure 5

Tbx5 overexpression inhibited Col10a1 expression in ATDC5 cells. (A) Tbx5 mRNA was upregulated in ATDC5 cells stably transfected with Tbx5 expression plasmid compared with vector and blank controls. (B) Col10a1 expression was inhibited in Tbx5-overexpressing cells compared with vector and blank controls. (C) Protein levels of Tbx5 and Col10a1 were similar to their mRNA levels. *p < 0.05, **p < 0.01.

Figure 6

Effects of Tbx5 on chondrogenic differentiation in ATDC5 cells. (A) Tbx5-overexpressing cells showed slightly weaker Alizarin red staining than control cells on day 21 of culture. Tbx5-overexpressing cells showed slightly weaker ALP staining on days 4 and 7. (B) Quantification of mean integrated optical density of Alizarin red and ALP staining in Tbx5-overexpressing and control cells using Image-Pro Plus 6.0 image analysis software. Compared with control cells (blank, pCMV6), Tbx5-overexpressing cells (Tbx5, ColX-Tbx5) showed slightly weaker Alizarin red staining (left). Tbx5-overexpressing cells also showed slightly weaker ALP staining on days 4 and 7 (right). *p < 0.05, **p < 0.01.

Figure 7

Expression of transgene and Col10a1 mRNA in Col10a1-Tbx5 TG mice. (A) Col10a1-Tbx5 transgenic mouse lines. PCR genotyping using mouse skin genome DNA and Col10a1-Tbx5 fragment specific primers indicated that we have successfully generated transgenic founders with ~10% positive rate (lanes 10, 11). (B) Genotype of the offspring of the transgenic founders breeding with wild-type mice was confirmed by PCR either and showed the establishment of Col10a1–Tbx5 transgenic mouse lines. (C) RT-PCR confirmed transgene expression in Col10a1-Tbx5 TG mice (red arrows). (D) Immunohistochemistry staining was used to analyze Flag expression in TG mouse hind limb sections. Dark brown staining shows Flag expression in hypertrophic chondrocytes of the proximal tibia in a TG mouse (right panel; control with no antibody, left panel). (E–G) Tbx5 expression was upregulated in TG mice compared with WT mice at each age, whereas Col10a1 expression was downregulated in limb tissue on P1 and the hypertrophic zone on P7. *p < 0.05, **p < 0.01.

Figure 8

Skeletal phenotype of Col10a1-Tbx5 TG mice. (A) Alcian blue and Alizarin red staining of the mouse skeleton on E17.5. A1: whole skeleton, no difference in Alizarin red staining between TG and WT mice. A2/A3 (zoomed-in pictures of A2): forelimb, no Alizarin red staining in the last phalange of a TG mouse (red arrow), but slight staining in a WT mouse (black arrow). A4/A5 (zoomed-in pictures of A4): hind limb, less Alizarin red staining in the phalanx of a TG mouse (red arrow) compared with a WT mouse (black arrow). (B) Alcian blue and Alizarin red staining of the mouse skeleton on P1. B1: whole skeleton, no difference in Alizarin red staining between TG and WT mice. B2/B3 (zoomed-in pictures of B2): forelimb, no difference between TG and WT mice. B4/B5 (zoomed-in pictures of B4): hind limb, less Alizarin red staining in the metatarsal bones and terminal digits of a TG mouse (red arrows) compared with a WT mouse (black arrows).