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. 2008 Apr 17;17(14):2244–2254. doi: 10.1093/hmg/ddn125

Uncoupling of chondrocyte differentiation and perichondrial mineralization underlies the skeletal dysplasia in tricho-rhino-phalangeal syndrome

Dobrawa Napierala 1, Kathy Sam 1, Roy Morello 1, Qiping Zheng 1,†, Elda Munivez 1, Ramesh A Shivdasani 3, Brendan Lee 1,2,*
PMCID: PMC2710999  NIHMSID: NIHMS101676  PMID: 18424451

Abstract

Tricho-rhino-phalangeal syndrome (TRPS) is an autosomal dominant craniofacial and skeletal dysplasia that is caused by mutations involving the TRPS1 gene. Patients with TRPS have short stature, hip abnormalities, cone-shaped epiphyses and premature closure of growth plates reflecting defects in endochondral ossification. The TRPS1 gene encodes for the transcription factor TRPS1 that has been demonstrated to repress transcription in vitro. To elucidate the molecular mechanisms underlying skeletal abnormalities in TRPS, we analyzed Trps1 mutant mice (Trps1ΔGT mice). Analyses of growth plates demonstrated delayed chondrocyte differentiation and accelerated mineralization of perichondrium in Trps1 mutant mice. These abnormalities were accompanied by increased Runx2 and Ihh expression and increased Indian hedgehog signaling. We demonstrated that Trps1 physically interacts with Runx2 and represses Runx2-mediated trans-activation. Importantly, generation of Trps1ΔGT/+;Runx2+/− double heterozygous mice rescued the opposite growth plate phenotypes of single mutants, demonstrating the genetic interaction between Trps1 and Runx2 transcription factors. Collectively, these data suggest that skeletal dysplasia in TRPS is caused by dysregulation of chondrocyte and perichondrium development partially due to loss of Trps1 repression of Runx2.

INTRODUCTION

The TRPS1 gene (OMIM: 604386) is located on 8q24.12 and encodes for a zinc-finger (ZF) transcriptional repressor, TRPS1. Trps1 binds DNA through a single GATA-type ZF that recognizes a DNA consensus sequence common to all GATA transcription factors. However, the repression activity of Trps1 maps to the C-terminal Ikaros-like double ZF motif; in vitro studies demonstrated that the presence of the DNA-binding domain is indispensable for the Trps1 repression function (1). Mutations involving the TRPS1 gene cause tricho-rhino-phalangeal syndrome (TRPS) (2). Genotype–phenotype correlation analyses revealed that three distinctive clinical types of TRPS are associated with different kinds of the TRPS1 gene mutations (3). The mildest form, TRPS type I (OMIM: 190350), is caused mostly by entire gene deletions and nonsense mutations located before the region coding for the DNA-binding domain. Missense mutations in the DNA-binding domain cause more severe TRPS type III (OMIM: 190351). TRPS type II (Langer-Gideon syndrome) (OMIM: 150230) combines features of TRPS type I and multiple exostoses and is caused by contiguous deletion of the TRPS1 and EXT1 genes. While human mutation data together with in vitro studies indicate that TRPS can result from haploinsufficiency, the more severe phenotype of TRPS type III suggests that the molecular mechanism of TRPS may be more complex.

Characteristic TRPS features have been phenocopied in mice with a heterozygous in frame deletion of the DNA-binding domain of Trps1 (Trps1ΔGT/+ mice) (4). Recently, another mouse Trps1 mutant allele was generated by insertion of an IRES–β-galactosidase–neomycin cassette into ATG-containing exon 3 of the Trps1 gene that produces a null allele (5). The general phenotype of Trps+/− mice is milder than Trps1ΔGT/+ mice. This may be due to differences in genetic background of the respective mice, and it also raises the possibility that Trps1+/− mice are a model of TRPS type I, while Trps1ΔGT/+ mice are a model of more severe TRPS type III that is caused by mutation abolishing only the DNA-binding domain.

Long bones of the vertebrate appendicular skeleton form by the process of endochondral ossification when initial cartilaginous anlagen are replaced by bone (6). This process begins with migration of mesenchymal cells to the site of future skeletogenesis, where they aggregate into compact modules. The mesenchymal cells within the region of condensation differentiate into chondrocytes, proliferate, mature into hypertrophic chondrocytes and, ultimately, undergo apoptosis. Chondrocyte differentiation in the cartilaginous anlagen is accompanied by molecular and morphological changes in the surrounding perichondrial cells. These mesenchymal cells sustain their undifferentiated status until the chondrocytes within the anlagen begin to hypertrophy. Signals from the early hypertrophic chondrocytes induce differentiation of perichondrial cells into osteoblasts which secrete mineralizing matrix to form a bone collar (7,8).

Many transcription factors and signaling pathways have been demonstrated to be involved in the cross-talk between differentiating chondrocytes and perichondrium during endochondral bone formation. The Runx2 transcription factor is, however, the master regulator of osteoblast differentiation and is required for chondrocyte hypertrophy (9,10). Runx2 null mice have no osteoblasts and chondrocyte development is blocked before hypertrophy in a majority of proximal bones, thereby resulting in an entirely cartilaginous skeleton populated mostly by immature chondrocytes (11,12).

Previous studies by our group identified the Trps1 transcription factor as a potentially novel transcriptional repressor of the Runx2 promoter by identifying single nucleotide variation in patients with cleidocranial dysplasia, a human skeletal dysplasia caused by haploinsufficiency of RUNX2 (13). Interestingly, during the development of endochondral bones, Trps1 is highly expressed in regions where Runx2 is downregulated. Trps1 demonstrates a dynamic and specific expression pattern during mouse limb development (Supplementary Material, Fig. S1) (14), which suggests that Trps1 is involved in chondrocyte differentiation both during early development and in established growth plate. At embryonic day 12.5 (E12.5), when the mesenchymal cells have already formed the anlagen of future endochondral bones, Trps1 is highly expressed in the mesenchymal condensations destined to the chondrogenic lineage. Once mesenchymal cells commit to chondrogenic phenotype, Trps1 expression decreases and becomes restricted to joints and cells of the perichondrium. After establishment of endochondral ossification, Trps1 expression is further confined to prehypertrophic chondrocytes and perichondrium. In comparison, Runx2 expression is detected as early as E10 during endochondral bone formation (15). However, Runx2 is downregulated in early mesenchymal condensations committed to chondrogenic lineages. Also later, during chondrocyte maturation, Runx2 is attenuated in prehypertrophic chondrocytes where the balance of cellular proliferation versus terminal hypertrophy is maintained (16,17). Trps1 and Runx2 also demonstrate overlapping expression in the perichondrial cells adjacent to proliferating chondrocytes. Although these cells express Runx2, they do not undergo differentiation into osteoblasts until they receive essential signals from chondrocytes undergoing hypertrophy.

To understand the molecular mechanisms underlying the skeletal dysplasia in TRPS type I patients and to elucidate the TRPS1 function during endochondral bone formation and its potential role in RUNX2 regulation, we studied Trps1ΔGT mice (4). Our analyses of mutant mice together with in vitro data indicate that Trps1 regulates cross-talk between developing chondrocytes and perichondrium to coordinate chondrocyte hypertrophy and perichondrial mineralization during the development of growth plates. This is achieved in part by Trps1’s direct repression of Runx2.

RESULTS

Abnormal chondrocyte differentiation and mineralization of perichondrium in Trps1Δgt/Δgt mice

To elucidate how the loss of Trps1 repression activity affects the development of endochondral bones, we compared long bones of homozygous Trps1ΔGT/ΔGT mice and wild-type (WT) littermates. At embryonic day E14.5, cells in the center of cartilaginous anlagen of long bones of WT mice are enlarged and express collagen type X (Col10a1), a marker of hypertrophy (Fig. 1A). The expression of Indian hedgehog (Ihh), a prehypertrophic chondrocyte marker, is organized into two zones flanking the Col10a1-expressing hypertrophic chondrocytes. In comparison with WT littermates, the development of chondrocytes in Trps1ΔGT/ΔGT mice is delayed as evidenced by the diminished size of cells in the center of long bones. The zone of Col10a1-expressing cells is also smaller, and these cells express Ihh along with Col10a1, indicating that they are in early stage of the transition to hypertrophy. Interestingly, the expression of the Ihh receptor and target gene Patched (Ptc-1) and Runx2 in perichondrium of the Trps1ΔGT/ΔGT mice is dramatically elevated in comparison with WT littermates. Since perichondrial expression of both Ptc-1 and Runx2 depends on Ihh signaling from prehypertrophic chondrocytes (7,8), these results indicate an increase in Ihh signaling in perichondrium in Trps1ΔGT/ΔGT.

Figure 1.

Figure 1.

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Delayed onset of chondrocyte hypertrophy and endochondral ossification in Trps1ΔGT/ΔGT mice. Immunohistochemical (IHC) and RNA in situ hybridization (ISH) analyses of long bones of WT and Trps1ΔGT/ΔGT littermates were performed. ColX–IHC for collagen type X, a marker of hypertrophic chondrocytes, Ihh–ISH for Ihh, Ptc-1–ISH for Patched, Runx2–ISH for Runx2; von Kossa staining demonstrates mineralized tissue in black. (A) E14.5 femur; (B) E15.5 femur; (C) E16.5 femur.

Delayed onset of hypertrophy is followed by slower progression of chondrocyte differentiation in Trps1ΔGT/ΔGT mice. At E15.5, the zone of hypertrophic chondrocytes is reduced in the Trps1ΔGT/ΔGT compared with WT littermates (Fig. 1B). Moreover, cells in the center of Trps1ΔGT/ΔGT long bones express Col10a1, while in the WT mice the chondrocytes expressing Col10a1 are separated into two domains by terminally differentiated chondrocytes, in which expression of Col10a1 has already been turned off. These chondrocytes are embedded in calcifying matrix in WT mice, while there is no sign of mineralization in the Trps1ΔGT/ΔGT long bones. Further evidence of delayed endochondral ossification is demonstrated at E16.5 when, in the WT mice, there are two domains of Col10a1-expressing hypertrophic chondrocytes separated by primary spongiosa (Fig. 1C). In contrast, in the homozygous mutant littermates, the growth plate has not been established yet, the primary spongiosa is not yet present and Col10a1 is expressed only in a narrow region in the center of long bones. The extracellular matrix in the region is mineralized, which indicates that these chondrocytes are at the terminal stage of differentiation.

Although there is no difference in the size of WT and Trps1ΔGT/ΔGT long bones at E18.5, histological analyses revealed dramatic growth plate abnormalities in the Trps1ΔGT/ΔGT mice. The cartilaginous region of the long bones of Trps1ΔGT/ΔGT embryos is significantly elongated, resulting in a decreased marrow cavity size (Fig. 2A, left panel). To determine which chondrocyte populations contribute to the expansion of the growth plate in Trps1ΔGT/ΔGT mice, we measured the length of the respective zones of chondrocyte differentiation. These were defined by cell morphology: flattened cells organized in columnar structures constitute the proliferating zone, small round cells at the ends of bones constitute the resting chondrocyte zone and enlarged cells embedded in less extracellular matrix constitute the pre- and hypertrophic chondrocyte zone. However, the morphological differences between pre- and hypertrophic chondrocytes are not sufficiently apparent for precise distinction between these cells; therefore, we measured the total length of the post-proliferating chondrocyte zone. Histomorphometric analyses demonstrated that the zones of proliferating as well as prehypertrophic and hypertrophic chondrocytes are significantly larger in the Trps1ΔGT/ΔGT mice compared with WT littermates (Fig. 2A, right panel). There is no difference in the size of the resting chondrocyte zone. RNA in situ hybridization (ISH) with probes specific for Ihh and for Col10a1 was performed to further define the developmental status of chondrocytes in the expanded growth plates of Trps1ΔGT/ΔGT mice. These results correlated with our histomorphometric analyses and showed that both prehypertrophic and hypertrophic chondrocyte zones are elongated (Fig. 2B). Moreover, in Trps1ΔGT/ΔGT mice, there is a larger zone of cells co-expressing both Ihh and Col10a1, which indicates an increased number of cells in the transition from prehypertrophic to mature hypertrophic stage.

Figure 2.

Figure 2.

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Elongation of growth plate in Trps1ΔGT/ΔGT mice. (A) Left panel: H&E staining of E18.5 femur shows reduced size of marrow cavity (mc) due to elongation of cartilaginous region (c) in Trps1ΔGT/ΔGT mice. Right panel: comparison of the length of resting (r), proliferating (p) and prehypertrophic and hypertrophic (h) chondrocyte zones in femur of E18.5 WT and Trps1ΔGT/ΔGT mice. The long bones of Trps1ΔGT/ΔGT mice demonstrate significantly elongated zones of proliferating (*P < 0.005) as well as prehypertrophic and hypertrophic chondrocytes (**P ≤ 0.001). (B) Histological comparison of WT and Trps1ΔGT/ΔGT growth plates. ISH with probes specific for Ihh and Col10a1 reveals elongation of both prehypertrophic and hypertrophic chondrocyte zones.

Besides the elongation of the growth plate, Trps1ΔGT/ΔGT mice have abnormal mineralization of perichondrium. During development, mineralization of perichondrium is coupled with differentiation of growth plate chondrocytes and depends on Ihh signals from prehypertrophic chondrocytes to adjacent perichondrial cells (7,8). Hence, the transition of chondrocytes from proliferation to hypertrophy precedes mineralization of perichondrium. At the earlier stages, mineralization of perichondrium in Trps1ΔGT/ΔGT mice is delayed probably due to overall impediment of long bone development (Fig. 1B and C). However, the von Kossa staining of long bones with established growth plate demonstrated that the mineralization of the perichondrium was more advanced in Trps1ΔGT/ΔGT mice than in WT littermates (Fig. 3). To quantify this phenomenon, we measured the onset of perichondrial mineralization relative to the onset of chondrocytes hypertrophy. We assigned the ‘zero’ value to the border between proliferating and prehypertrophic chondrocytes, and then measured the distance between the proliferating–prehypertrophic border and the front of mineralization of perichondrial cells. In cases when the mineralization was more advanced proximally toward the proliferating chondrocytes, we assigned a positive value to the distance, while the negative value was assigned when the mineralization of perichondrium started distally toward the zone of hypertrophy. As expected, our analyses showed that mineralization of perichondrium in the WT mice begins in the cells surrounding prehypertrophic chondrocytes that are already progressing toward hypertrophy. This pattern of perichondrial mineralization following chondrocyte hypertrophy was observed for all WT mice analyzed; therefore, WT samples were assigned the negative values. In contrast, Trps1ΔGT/ΔGT samples were assigned positive values, since the mineralization of perichondrium was more advanced, reaching cells adjacent to late proliferating chondrocytes (Fig. 3).

Figure 3.

Figure 3.

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Increased mineralization of perichondrium in Trps1ΔGT/ΔGT mice. Upper panel: Von Kossa staining shows advanced mineralization of perichondrium in Trps1ΔGT/ΔGT mice. Lower panel: measurement of the relative distance between the junction of proliferating-prehypertrophic chondrocytes versus the front of mineralization of perichondrium. The zero value was assigned to the junction between proliferating and prehypertrophic chondrocytes, negative values indicate the retardation of perichondrial mineralization and positive values indicate advanced mineralization.

It has been previously demonstrated that Runx2 is one of the targets of Ihh signaling in perichondrial cells. Moreover, Runx2 expression in perichondrial cells is required for Ihh-induced mineralization of perichondrium (8). Since the Ihh expression and mineralization of perichondrium are increased in Trps1ΔGT/ΔGT mice, we analyzed the Runx2 expression in the long bones of Trps1ΔGT/ΔGT mice. As demonstrated by RNA ISH, in both WT and Trps1ΔGT/ΔGT littermates, Runx2 is expressed in all perichondrial cells except those cells adjacent to resting chondrocytes (Fig. 4). However, in growth plate chondrocytes and perichondrium of Trps1ΔGT/ΔGT mice, the expression of Runx2 is upregulated in comparison with the WT littermates (Figs 1A and 4). In addition to the higher levels of the Runx2 expression in Trps1ΔGT/ΔGT mice, Runx2 mRNA is found in a broader zone expanding beyond pre- and hypertrophic chondrocytes to proliferating chondrocytes. A similar trend of increased expression was observed for Ptc-1 (Figs 1A and 4). Elevated expression of Ptc-1 in chondrocytes and perichondrial cells indicated increased Ihh signaling to its target cells.

Figure 4.

Figure 4.

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Increased expression of Runx2 and Ptc-1 in growth plate and perichondrium in Trps1ΔGT/ΔGT mice. Left panel: H&E staining of distal femur. Middle panel: ISH with probe specific for Runx2. Right panel: ISH with a probe specific for Ptc-1.

Both Ihh and Runx2 have opposite effects on chondrocyte proliferation. It has been demonstrated that Runx2 expression in perichondrium inhibits chondrocytes proliferation through Fgf18 (18), whereas the Ihh signaling stimulates proliferation of chondrocytes (7). Additionally, Ihh regulates the length of columnar chondrocytes zone. Since the expression of both Runx2 and Ihh is increased in Trps1ΔGT/ΔGT growth plates, we compared the proliferation of chondrocytes in WT and Trps1ΔGT/ΔGT mice. The results of the BrdU incorporation assays demonstrated reduced number of BrdU positive cells in long bones of Trps1ΔGT/ΔGT mice (Fig. 5). However, decreased proliferation of chondrocytes and increased length of columnar chondrocytes zone were also reported for Trps1−/− mice; the spectrum of the growth plate abnormalities is broader in Trps1ΔGT/ΔGT than in Trps1−/− mice (5). Unlike Trps1ΔGT/ΔGT mice, the Trps1−/− show no changes in post-proliferating chondrocytes, including the expression of Ihh and Col10a1. The phenotypic differences between these two mutant mice suggest that the Trps1ΔGT allele may have gain-of-function consequences in addition to loss of the DNA binding and repression activity of Trps1.

Figure 5.

Figure 5.

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Decreased chondrocytes proliferation in Trps1ΔGT/ΔGT mice . Decreased BrdU incorporation in Trps1ΔGT/ΔGT limbs in comparison with WT at E18.5 (*P < 0.05).

In summary, the Trps1ΔGT/ΔGT mutant mouse phenotype suggests that Trps1 has two major functions during endochondral ossification. First, Trps1 regulates the onset and the rate of chondrocyte differentiation since Trps1ΔGT/ΔGT mice demonstrate delayed chondrocyte hypertrophy and slower progression of chondrocytes to terminally differentiated stage. Secondly, Trps1 synchronizes the mineralization of perichondrium with differentiation of growth plate chondrocytes.

Trps1 binds RUNX2 and represses RUNX2-mediated trans-activation in vitro

Previous studies by our group identified the TRPS1 as a potentially novel transcriptional repressor of the RUNX2 promoter by identifying single nucleotide variation in patients with cleidocranial dysplasia, a human skeletal dysplasia caused by haploinsufficiency of RUNX2 (13). Moreover, since both mineralization and chondrocyte hypertrophy are regulated by Runx2, we hypothesized that Trps1 might act as a repressor of Runx2 function. Interestingly, both Trps1 and Runx2 are expressed in perichondrial cells adjacent to proliferating chondrocytes (Supplementary Material, Fig. S1, Fig. 4). However, in spite of the presence of Runx2, the mineralization of perichondrial cells begins only when the Trps1 expression is turned off in the perichondrial cells adjacent to hypertrophic chondrocytes.

To test whether TRPS1 could act as a repressor of RUNX2 activity, we performed co-transfection experiments in COS7 cells that do not express endogenous RUNX2. We used the 6xOSE reporter that responds only to the Runx2 trans-activation; hence, it is not expressed in the absence of Runx2. Co-transfection of the reporter construct with the plasmid expressing Runx2 led to activation of the reporter. When the reporter and the Runx2-expressing plasmids were co-transfected with the Trps1-expressing plasmid, we observed decreased expression of the reporter. This repression effect was dose dependent on Trps1 levels (Fig. 6A). Western blot analysis of the Flag-tagged Runx2 and V5-tagged Trps1 proteins demonstrated that the expression of exogenous Runx2 in the transfected cells does not change upon expression of Trps1. Therefore, these results suggest that Trps1 inhibits the Runx2-mediated trans-activation, but it does not affect the expression of Runx2 or the stability of the Runx2 protein in this heterologous system.

Figure 6.

Figure 6.

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Trps1 represses Runx2 mediated trans-activation in vitro. Co-transfection experiments in COS7 cells transiently transfected with 6xOSE-luc reporter with or without Runx2 and Trps1 expressing vectors. White bars represent the reporter activity in the absence of Runx2; black bars represent Runx2-mediated trans-activation of the reporter in the absence of Trps1; gray bars represent reporter activity in the presence of Runx2 and Trps1. Error bars represent standard deviation of three independent transfections. (A) Runx2-mediated trans-activation is repressed in a presence of Trps1. Lower panel: western blots (WB) with anti-V5, anti-Flag and anti-tubulin antibodies demonstrate that the increased expression of V5-tagged Trps1 corresponds to decreased activity of the Runx2-dependent reporter, without changes in the exogenous Flag-tagged Runx2 expression. (B) Comparison of the Trps1 repression activity on full length Runx2 (FL), Runx2 deletion mutants (ΔNt, ΔQ and R391X) and chimeric protein containing Runt domain fused to activation domain (Runt-AD). Runt domain of Runx2 is required for Trps1 repression activity on Runx2. (C) Comparison of the repression activity of the full length Trps1 (FL) and Trps1 deletion mutants (Δzf2-7 and zf7-9). The full length and C-terminal part of Trps1 containing GATA and Ikaros domains (zf7-9-Trps1) can repress Runx2-mediated trans-activation, whereas the deletion of GATA (Δzf2-7-Trps1) domain abolished the repression activity.

To evaluate whether the Trps1 repression activity is specific for Runx2, we performed a similar co-transfection experiment with Sox9, a transcription factor critical for chondrogenesis. Sox9 is highly expressed in chondrogenic mesenchymal condensates and chondrocytes before terminal differentiation. It has been demonstrated that Sox9 activates the enhancer element from the type II collagen (Col2a1) promoter (19). In co-transfection studies with the Col2a1 reporter, Sox9- and Trps1-expressing plasmids, Trps1 did not affect the Sox9-mediated trans-activation of the Col2a1 reporter (Supplementary Material, Fig. S2). These results indicated that TRPS1 specifically represses the RUNX2 protein without inhibiting the global transcriptional machinery.

Next, we attempted to identify which domain of the RUNX2 protein is necessary for the response to TRPS1 repression. We compared Trps1 repression activity on full length Runx2 and a series of Runx2 deletion mutants (Fig. 6B). In co-transfection experiments, Trps1 repressed the expression of the reporter driven by the full length Runx2 protein as well as all analyzed Runx2 mutants. The common feature of these proteins was the presence of the Runt DNA-binding domain. The Runt domain can both bind DNA and mediate protein–protein interaction, but it cannot activate the transcription; therefore, to test whether the Runt domain is a target of the Trps1 repression, we analyzed the 6xOSE reporter trans-activation by the chimeric protein consisting of Runt DNA-binding domain fused with transcription activation domain from the Vp16 virus (AD). The transcriptional activity of the Runt–AD fusion protein was repressed by Trps1, indicating that the presence of Runt DNA-binding domain is sufficient to mediate Trps1's repression.

Similarly, we analyzed which of the Trps1 domains was necessary to repress the Runx2-mediated trans-activation. The Trps1 protein contains nine ZF domains (Supplementary Material, Fig. S3). Consistent with previous data, the deletion of DNA-binding domain results in loss of the Trps1 repression activity. In our co-transfection experiments with 6xOSE reporter and Runx2-expressing plasmid, the Trps1 mutant carrying the deletion of the central portion of the protein (Δzf2-7–Trps1 mutant) did not affect the Runx2 trans-activation of the reporter (Fig. 6C). In contrast, a truncated Trps1 mutant consisting only of GATA and Ikaros domains (zf7-9–Trps1) strongly repressed the Runx2-mediated trans-activation.

To determine whether Trps1 and Runx2 transcription factors can interact directly, we performed a GST pull-down experiment. Since we identified which domains of TRPS1 and RUNX2 are necessary and sufficient for the TRPS1 repression of the RUNX2 protein, we used a GST-tagged Runt domain expressed in prokaryotic cells and zf7-9–Trps1 truncated protein synthesized in vitro. The C-terminal part of the TRPS1 protein was pulled down with the GST-tagged Runt domain, but not with the GST alone, demonstrating that the TRPS1 and Runx2 transcription factors can directly interact in vitro (Fig. 7A). Furthermore, this interaction was confirmed using co-immunoprecipitation (co-IP) analyses. COS7 cells were transfected with either FLAG-tagged Runx2, or V5-tagged Trps1 expressing vectors, or both vectors together. The immunoprecipitation was performed with either anti-Flag or anti-V5 antibodies and followed by western blot analyses with anti-V5 and anti-FLAG antibodies, respectively. The results of this analysis confirmed direct TRPS1 and RUNX2 interaction in a cellular context, since the V5–Trps1 signal was detected in proteins immunoprecipitated with anti-Flag antibodies, and vice versa, the Flag–Runx2 was detected in proteins immunoprecipitated with anti-V5 antibodies (Fig. 7B).

Figure 7.

Figure 7.

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Trps1 physically interacts with Runx2. (A) GST pull-down experiment. GST-tagged Runt domain from Runx2 and GST tag alone were expressed in E. coli cells. C-terminal part of Trps1-containing GATA and Ikaros domains (zf5-7-Trps1) was synthesized in vitro with 35S-methionine (input). zf7-9-Trps1 was pulled down with GST-tagged Runt domain (Runt), but not with GST alone (GST). The arrowhead indicates the signal from 35S-zf7-9-Trps1. (B) Co-immunoprecipitation of Runx2 and Trps1. COS7 cells were transfected with plasmids expressing either V5-tagged Trps1 (lanes 2 and 6) or Flag-tagged Runx2 (lanes 3 and 7) or both proteins (lanes 4 and 8); lanes 1 and 5 represents mock transfection. Upper panel: immunoprecipitation (IP) performed using anti-Flag antibody followed by WB with anti-V5 antibody. Lower panel: IP with anti-V5 antibody followed by WB with anti-Flag antibody.

Hence, our in vitro experiments demonstrated that TRPS1 and RUNX2 physically interact, and that Trps1 specifically represses the Runx2-mediated trans-activation of target elements via the binding of its C-terminal domain with the Runt domain.

Haploinsufficiency of Trps1 rescues the growth plate phenotype of Runx2+/− mice

To confirm our in vitro biochemical data, we determined whether Trps1 and Runx2 can interact genetically. We generated Runx2+/−;Trps1ΔGT/+ double heterozygous mice (d-het) by crossing Runx2+/− females with Trps1ΔGT/+ males. Trps1ΔGT/ΔGT mice show dramatic elongation of growth plates. Consistent with a gene dose effect, milder elongation of cartilaginous part of long bones is observed in Trps1ΔGT/+ mice. Interestingly, the opposite phenotype, shortening of the hypertrophic chondrocyte zone, has been reported in Runx2+/− mice as well as in human growth plates from patients with cleidocranial dysplasia (20,21). Histological analyses of femurs of P2 mice revealed that hypertrophic chondrocyte zones in d-het mice, defined by cell morphology and Col10a1 expression, have intermediate lengths between that of Runx2+/− and Trps1ΔGT/+ mice. More importantly, there is no statistical difference between WT and d-het mice (Fig. 8A and D). Hence, the loss of one copy of Trps1 rescued the growth plate phenotype attributable to haploinsufficiency of Runx2, and vice versa.

Figure 8.

Figure 8.

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Haploinsufficiency of Trps1 rescues growth plate phenotype of Runx2+/− mice. Hindlimbs were collected from WT, Runx2+/−, Trps1ΔGT/+ and Runx2+/;Trps1ΔG/+ (d-het) mice at P2. Each bar represents a mean value of at least three samples. Error bars represent standard deviation. (A) Type X collagen IHC on distal femur. The black bars on the right of each image represent average length of the hypertrophic chondrocyte zone. (B) Von Kossa staining of distal femur. Arrow heads point to the mineralization front on perichondrium. Dotted lines delineate border between proliferating and prehypertrophic chondrocytes. (C) Higher magnifications of boxed regions in (B) show that advanced mineralization of perichondrium in Trps1ΔGT/+ mice and delayed mineralization of perichondrium in Runx2+/− mice are corrected in double heterozygous mice. (D) the length of the hypertrophic zone measured on distal femurs. Asterisks indicate statistically significant difference between the groups analyzed: P < 0.01 for WT versus Runx2+/−, P < 0.05 for WT versus Trps1ΔGT/+, P < 0.05 for Runx2+/− versus d-het, P < 0.05 for Trps1ΔGT/+ versus d-het. There is no statistical ly significant difference in the length of hypertrophic zone between WT versus d-het mice. (E) Onset of perichondrial mineralization measured as the distance between the junction of proliferating–prehypertrophic chondrocytes and the front of mineralization of perichondrium. Asterisks indicate statistically significant differences between the groups analyzed: P < 0.05 for WT versus Runx2+/−, P < 0.05 for Runx2+/− versus d-het, P < 0.01 for Runx2+/− versus Trps1ΔGT/+. There is no statistically significant difference between the onset of perichondrial mineralization of WT versus Trps1ΔGT/+, and Trps1ΔGT/+ versus d-het mice.

To further investigate the genetic interaction between Trps1 and Runx2, we analyzed the mineralization status of perichondrium of the Runx2+/− and d-het mice. Interestingly, haploinsufficiency of Runx2 alone causes significant delay of perichondrial mineralization in comparison with the WT littermates (Fig. 8B and D). Similar to the length of the hypertrophic zone, the abnormal perichondrial mineralization phenotype is corrected in Runx2+/−;Trps1ΔGT/+ double heterozygous mice. As shown in Figure 8, the opposite phenotypes of Trps1- and Runx2-deficient mice are corrected in double heterozygous mice. These results demonstrate a genetic interaction between the Trps1 and Runx2 transcription factors during endochondral bone formation and support our in vitro data, demonstrating that TRPS1 acts as a repressor of RUNX2.

DISCUSSION

Taken together, analyses of the growth plate development in mutant Trps1 mice, in concert with our in vitro studies and Runx2+/−;Trps1ΔGT/+ double heterozygous mice analyses provide evidence that the Trps1 transcription factor represses Runx2 during chondrocyte differentiation and perichondrial mineralization. This repression activity is necessary for the timely progression of chondrocyte maturation and synchronization of chondrocyte development with perichondrial mineralization.

The Runx2 transcription factor is a crucial regulator of chondrocyte terminal differentiation (10,22,23). Chondrocytes of Runx2-deficient mice do not express Ihh and fail to progress to hypertrophy. It has been demonstrated that Runx2 directly regulates the Ihh promoter in developing chondrocytes (24). In turn, Ihh signaling from prehypertrophic chondrocytes to the perichondrium is necessary for perichondrial Runx2 expression and subsequent mineralization of perichondrium. Therefore, Runx2 and Ihh establish a positive regulatory loop during endochondral bone formation. We postulate that Trps1 acts antagonistically to Ihh in the regulation of Runx2, and that Runx2 transcriptional activity is balanced by the positive regulator, Ihh, and the negative regulator, Trps1. Interestingly, Trps1 and Runx2 are co-expressed in perichondrial cells adjacent to proliferating chondrocytes (Supplementary Material, Fig. S1; Fig. 4). These cells are also a target of the Ihh signaling as demonstrated by expression of Ptc-1. However, in spite of the Runx2 expression and Ihh signaling, these cells do not mineralize. In the Trps1ΔGT/ΔGT mice, the absence of the Trps1 repression of Runx2 shifts the balance toward activating Ihh signaling resulting in premature mineralization of perichondrium. Alternatively, since Runx2 positively regulates its own expression (25), deficiency of Trps1 may cause increased Runx2 trans-activation of its own promoter both in perichondrial cells and growth plate chondrocytes (Figs 1A and 4).

It has been demonstrated that Ihh signaling induces Runx2 expression in the perichondrium. It is likely that Ihh signaling has a potential to activate Runx2 expression in chondrocytes also. However, under physiological conditions, the presence of Trps1 transcriptional repression in chondrocytes that are also a target of Ihh signaling may constrain the feedback activation of Runx2 expression by Ihh signaling. The observed increased expression of both Runx2 and Ihh in the Trps1ΔGT/ΔGT growth plate chondrocytes is a pathological consequence of a loss of this inhibitory mechanism. Increased Ihh expression, in turn, results in increased Ihh signaling and elongation of the zone of proliferating chondrocytes. On the other hand, the increased Runx2 expression in perichondrium represses proliferation of chondrocytes.

In summary, the loss of repression of the Runx2–Ihh positive regulatory loop in Trps1ΔGT/ΔGT mice results in altered endochondral bone formation, which is characterized by dysregulation of chondrocyte differentiation and uncoupling of processes of perichondrial mineralization and chondrocyte maturation. These data help to explain the puzzling clinical observation in TRPS patients, where delayed bone age is often accompanied by increased mineralization radiographically (26). Moreover, since the elongation of pre- and hypertrophic chondrocytes zone phenotype is not present in Trps1 null mice (5), the Trps1ΔGT allele may have gain-of-function consequences in addition to loss of DNA binding and repression activity of Trps1. Thus, Trps1+/− mice are the model of TRPS type I, while Trps1ΔGT/+ mice are the model for more severe TRPS type III.

MATERIALS AND METHODS

Mutant mice

Trps1ΔGT- and Runx2-deficient mice were described previously (4,27). Trps1ΔGT/+ mice were maintained on 129svev background, and Runx2+/− mice were maintained on C57B6 background. Genotyping was performed by PCR analysis of genomic DNA.

Basic histology, RNA ISH, immunohistochemistry (IHC), von Kossa staining and BrdU incorporation assay

Limbs were fixed in 4% paraformaldehyde and embedded in paraffin; 7 µm sections were stained with H&E and von Kossa staining according to standard protocols. ISH was performed as described previously (28). IHC was performed with rabbit anti-Col10a1 antibody (pXNC2, a kind gift from Dr. Greg Lunstrum), and ABC reagent (Vector Laboratories, Burlingame, CA, USA) was used as secondary antibody for detection. BrdU incorporation was detected using a Zymed BrdU staining kit. Sections were counterstained with 4′-6-diamidino-2-phenylindole (DAPI). At least four embryos from each genotype were analyzed. BrdU positive signals were counted using Zeiss AxioVision software.

Histomorphometry

Growth plate measurements were conducted using Zeiss Axioplan II microscope and Zeiss AxioVision software. Five sections, 50 µM apart, of each limb were analyzed for at least three mice of each genotype. Measurements were performed by two independent observers blinded to genotype.

DNA transfection and reporter assays

Transfection, luciferase and β-galactosidase assays were performed as described previously (29). All transfections were performed with pSV2βgal as an internal control for transfection efficiency. All data are presented as fold activation relative to the activity obtained with the pcDNA3.1. The bars represent the average ratios of luciferase to β-galactosidase activity.

GST pull-down and co-IP

GST pull-down was performed as described previously (17). For co-IP experiments, COS7 cells were transiently transfected with expression plasmids using Lipofectamine 2000 (Invitrogen). Thirty-six hours after transfection, cells were collected in a lysis/binding buffer (50 mm Tris–HCl, pH = 7.5, 100 mm NaCl, 0.5% Triton-X). Two micrograms of antibodies were used to pull-down the specific tagged proteins with protein G agarose.

Western blot

Western blots were performed using an anti-V5 (Invitrogen) and anti-Flag (Sigma) monoclonal antibodies, and normalized with anti-α-tubulin monoclonal antibody (Sigma).

Statistical analyses

Data are expressed as mean values ± standard deviation. Statistical significance was computed using the Student's t-test. A P-value of <0.05 was considered statistically significant.

SUPPLEMENTARY MATERIAL

Supplementary Material is available at HMG Online.

FUNDING

This work was supported by NIH grants DE016990 and HD22657, and the Bone Diseases Program of Texas.

Supplementary Material

[Supplementary Data]
ddn125_index.html (835B, html)

ACKNOWLEDGEMENTS

We thank Yuching Chen, Margaretha Guenther, Ming-Ming Jiang and Sujatha Kakuru for technical assistance, and Terry Bertin for critically reading the manuscript.

Conflict of Interest statement. None declared.

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Supplementary Materials

[Supplementary Data]
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