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FASEB J. Sep 2011; 25(9): 3057–3067.
PMCID: PMC3157684

Deletion of Zfp521 rescues the growth plate phenotype in a mouse model of Jansen metaphyseal chondrodysplasia


Jansen metaphyseal chondrodysplasia (JMC) is caused by a constitutively activating mutation of the parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptor (PTHR1) and is characterized by widening of the metaphyses, reduction of long bone length, and short stature. A transgenic mouse expressing this mutation under the collagen α1(II) promoter has been generated to investigate the mechanisms responsible for this chondrodysplasia. We recently identified zinc finger protein 521 (Zfp521) as a downstream target gene of PTHrP signaling. Interestingly, loss of Zfp521 from chondrocytes leads to reduced cell proliferation and increased differentiation in the growth plate. Thus, we hypothesized that specifically ablating Zfp521 from Jansen chondrocytes could sufficiently rescue the chondrodysplasia phenotype. Our results show that Zfp521 expression is up-regulated in Jansen mouse growth plate chondrocytes and that PTHR1 is required for Zfp521 expression. Its ablation from Jansen chondrocytes restored normal cell differentiation, thus initiating chondrocyte apoptosis at the chondro-osseous junction, leading to partial rescue of endochondral bone formation shown by proper bone length. This study provides the first genetic evidence that Zfp521 is required downstream of PTHR1 signaling to act on chondrocyte proliferation, differentiation, and cell death.—Seriwatanachai, D., Densmore, M. J., Sato, T., Correa, D., Neff, L., Baron, R., Lanske, B. Deletion of Zfp521 rescues the growth plate phenotype in a mouse model of Jansen metaphyseal chondrodysplasia.

Keywords: chondrocyte, endochondral bone formation, PTHrP, JMC, zinc finger protein

Endochondral ossification is a well-studied process, whereby a cartilaginous framework is replaced by true bone to create the axial and appendicular skeleton (1, 2). Parathyroid hormone-related protein (PTHrP) is one of the regulators of endochondral bone formation (3), and loss of its function from either lack of its expression or loss of its receptor (PTHR1) results in severe skeletal malformations and perinatal lethality (4, 5). Moreover, an H223R-residue mutation in human PTHR1 was found to be responsible for a rare autosomal dominant disorder known as Jansen metaphyseal chondrodysplasia (JMC; refs. 6, 7). This mutation leads to hypercalcemia and hypophosphatemia with disturbed longitudinal bone formation characterized by short stature. Interestingly, serum parathyroid hormone (PTH) levels are normal to low, distinguishing it from hyperparathyroidism (8, 9). To study this disease in more detail, a transgenic mouse model expressing the H223R mutation under the control of the collagen α1(II) promoter, the Jansen mouse, has been generated (7, 10, 11). In both mice and humans, the mutant PTHR1 leads to cAMP accumulation, and thereby constitutive signaling, despite the absence of a ligand. It is obvious that such uncontrolled receptor activation must disturb normal growth plate morphogenesis and longitudinal growth. Nevertheless, no attempt had been made to rescue the anomalies found in this type of chondrodysplasia prior to this study.

We recently identified zinc finger protein 521 (Zfp521) as a downstream target gene of PTHrP, PTHR1, and cAMP signaling. Zfp521 is expressed in the embryonic limb bud and in limbs throughout postnatal life. Loss of Zfp521, specifically in chondrocytes, leads to a reduction in proliferation and an increase in differentiation in growth plate development (12). Since Zfp521 is regulated by PTHrP via PKC and PKA, we hypothesized that specifically ablating Zfp521 expression in Jansen mice chondrocytes could rescue the Jansen metaphyseal chondrodysplasia phenotype. We tested this hypothesis by crossing the Jansen mouse with a Col2Cre Zfp521d/d mouse.


Cell culture

Primary chondrocytes were isolated from rib cages of postnatal day (P)2 mice, as described previously (13); seeded at a density of 1 × 105 cells/cm2 in DMEM/F-12 medium supplemented with 5% FBS, penicillin (100 U/ml), streptomycin (100 μg/ml), and 1% l-glutamine (maintenance medium); and cultured until confluent (~3 d). Cells were cultured in chondrogenic differentiation medium (maintenance medium plus 10 μg/ml insulin, 10 μg/ml transferrin, and 3×10−8 M sodium selenite), as described previously (14). The medium was changed every other day.

Adenovirus-transfecting chondrocytes

Primary chondrocytes from rib cages of P2 mice were isolated from PTHR1 homozygous floxed mice (15). Cells were plated at a density of 1 × 106 cells/well in 6-well-plates and grown as monolayers in cultured medium (as described earlier). At 1 d postplating, adherent chondrocytes were infected with adenovirus (MOI 10) containing either β-galactosidase or cre recombinase to generate control chondrocytes (PTHR1fl/fl) or PTHR1-deleted chondrocytes (PTHR1d/d), respectively. After incubation for 24 h, the efficiency of the PTHR1 gene deletion was determined by quantitative RT-PCR (see Supplemental Fig. S1).

Growth plate histology and tissue preparation

Paraffin sections of sternebrae and tibia were cut for histological analyses, and ulna and radius were collected for size measurements from newborn, P7, and P14 mice. Tissues were fixed in 10% buffered formalin, decalcified in 20% EDTA, dehydrated at room temperature through an ethanol series, cleared in xylene, embedded in paraffin, and sectioned at 5-μm thickness. Growth plate length was directly measured from the chondro-osseous junction at 100-μm intervals on low-power images (×4 objective lens). Higher-power images (×10 objective lens) were used to measure lengths of the two proliferative zones, randomly selected at the middle of the growth plate and at 50-μm intervals. For routine morphological analyses, sections were stained with hematoxylin-and-eosin. At least 4 stained sections from each group were randomly selected and photographed. Bone length of ulna and radius of each group was photographed and randomly measured by calibrator. At a representative field of ×40, width and total number of hypertrophic chondrocytes from Jansen and double-mutant sternebrae were analyzed with Scion Image software (Scion Corp., Frederick, MD, USA) using ≥4 sections/group.

Generation of Col2Cre Zfp521d/d, Col2 Jansen transgenic mice, and Jansen/Col2Cre Zfp521d/d mice

The Col2 Jansen hemizygous transgenic mouse line (Tg-A) expressing a constitutively active form of the human PTHR1 receptor under the control of the rat α1(II) collagen promoter was kindly provided by Dr. Ernestina Schipani (Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA: ref. 7). The constitutively active PTHR1 in chondrocytes causes a delay in chondrocyte differentiation. However, no other gross abnormalities were detected when compared to wild type. Zfp521fl/fl animals (12) were crossed with mice expressing Cre recombinase under the control of the collagen type II α1 promoter (16) to obtain Col2Cre-Zfp521fl/+ offspring. Subsequently, Col2Cre-Zfp521fl/+ mice were interbred with Jansen transgenic mice to eventually obtain a double mutant. All animal studies performed were approved by the institutional animal care and use committee at Harvard Medical School.

PCR analysis for genotyping

Jansen mouse genotyping was performed using the following specific primers: Jansen forward, 5′-TAGTTGGCCCACGTCCTGT-3′; Jansen reverse, 5′-TAACCATGTTCATGCCTTCTTC-3′. After an initial denaturation step for 5 min at 95°C, amplification cycles consisted of denaturation at 95°C for 1 min, annealing at 58°C for 45 s, and 1 min extension at 72°C for 35 cycles, followed by a final extension for 10 min at 72°C. The expected amplicon for the Jansen transgene is 560 bp.

Cre recombinase mice were genotyped using primers Cre(275), 5′-CGCGGTCTGGCAGTAAAAACTATC-3′, and Cre(603), 5′-CCCACCGTCAGTACGTGAATATC-3′. After an initial denaturation step for 8 min at 94°C, amplification cycles consisted of denaturation at 94°C for 30 s, annealing at 68°C for 30 s, and 45 s extension at 72°C for 35 cycles, followed by a final extension for 10 min at 72°C. The expected amplicon for the Cre allele is 328 bp.

PTHR1fl/fl mice genotyping was performed using primers PTHR1 forward, 5′-ATGAGGTCTGAGGTACATGGCTCTGA-3′, and PTHR1 reverse, 5′-CCTGCTGACCTCTCTGAAAGAATGT-3′. After an initial denaturation step for 8 min at 95°C, amplification cycles consisted of denaturation at 94°C for 25 s, annealing at 55°C for 15 s, and 20-s extension at 72°C for 34 cycles, followed by a final extension for 2 min at 72°C. The expected amplicons are 210 bp for the wild-type PTHR1 allele and 280 bp for the floxed PTHR1 allele.

Quantitative PCR (qPCR) using mRNA extracted from the growth plate

To analyze gene expression in growth plate chondrocytes, 60-μm frozen sections were prepared from tibiae of P14 control, Jansen transgenic, Col2Cre-Zfp521d/d, and Jansen/Col2Cre-Zfp521d/d mice. Growth plates were microdissected and collected in lysis buffer (Agilent Technologies, Santa Clara, CA, USA), and total RNA was extracted with TRIzol (Invitrogen, Carlsbad, CA, USA), purified, reverse transcribed, and quantified by qRT-PCR. RNA from primary rib chondrocytes was extracted with TRIzol, followed by DNaseI digestion and purification with the RNeasy mini kit (Qiagen, Valencia, CA, USA). High-quality RNA (1 μg) was reverse transcribed with SuperScript III (Invitrogen), producing 20 ng of cDNA. All cDNA samples were amplified by qRT-PCR using the primers listed in Table 1 in an iCycler real-time PCR thermocycler (Bio-Rad Laboratories, Hercules, CA, USA) using SYBR-green. Results were normalized to the endogenous expression of GAPDH, and fold overexpression was calculated with the 2−ΔΔCT method. This analysis was performed using ≥4–10 mice of each genotype, using duplicates for each sample.

Table 1.
Primers for quantitative PCR using mRNA extracted from growth plates

In situ hybridization

35S-UTP-labeled riboprobes were synthesized from linearized plasmids using an in vitro transcription kit (Promega, Madison. WI, USA) and 35S-UTP (Amersham, Piscataway, NJ, USA). In situ hybridization using antisense riboprobes for collagen type II and collagen type X was performed on 3.7% buffered formaldehyde-fixed, decalcified, paraffin-embedded proximal tibiae of 2-wk-old mice, as described previously (17). For nonradioactive in situ hybridization, Zfp521 (NM_145492.3) riboprobes incorporating digoxigenin (DIG)-labeled nucleotides (18) were synthesized from linearized plasmid templates with T7 or SP6 polymerase (Roche Diagnostic, Indianapolis, IN, USA). The labeled probes were resuspended in 0.1 M DTT in diethylpyrocarbonate (DEPC) water containing RNase inhibitor, and integrity was assessed by gel electrophoresis. Sections were deparaffinized and rehydrated in serial dilution of ethanol, then incubated overnight at 60°C with the hybridization probe in hybridization buffer. To increase stringency, sections were sequentially washed with 5×, 2×, and 0.2× sodium chloride, sodium citrate (SSC) at 60°C for 1 h each. Unhybridized probe was removed by treating sections with RNase A (10 mg/ml) in TNE (1 M Tris-HCl, 5 M NaCl, and 1 mM EDTA) at 37°C for 10 min, followed by a wash with TNE at 37°C for 10 min. Detection was achieved by treatment with DIG buffer 1 (1 M Tris, pH 7.5; and 5 M NaCl) for 5 min, followed by treatment with DIG buffer 2 (1% blocking reagent in buffer 1) for 60 min. Sections were treated with the anti-DIG antibody (1:5000) overnight at 4°C. Subsequently, samples were washed 3 times with DIG buffer 1 for 15 min and then washed with DIG buffer 3 (1 M Tris, pH 9.5; 5 M NaCl; 2 M MgCl2; and 2 mM levamisole). To detect the hybridized probe, sections were incubated with nitro blue tetrazolium (NBT; 450 mg/ml) and 5-bromo-4-chloro-3-indoyl phosphate (BCIP; 175 mg/ml) in DIG buffer 3 at 37°C. As internal controls, in situ hybridizations using sense riboprobes were performed.


Chondrocytes extracted from wild-type or Jansen transgenic rib cages at P2 were cultured on coverslips for 3 d in maintaining medium before harvesting. Slides were washed and fixed in cold 4% paraformaldehyde/PBS. Rabbit polyclonal anti-Zfp521 (19) was diluted 1:200 and incubated overnight at 4°C. For detection, goat anti-rabbit conjugated fluorescence (rhodamine) was used for visualization under an inverted microscope.

TUNEL assay

The number of apoptotic chondrocytes in the growth plate of the proximal tibia was assessed by TUNEL staining of paraffin sections from 2-wk-old mice using a kit from Roche. Apoptotic cells at the chondro-osseous junction were counted and normalized by the width of the growth plate.

Statistical analysis

All values are given as means ± se. Analyses were performed using Excel (Microsoft, Redmond, WA). Comparison between groups was performed by 1-way ANOVA using Graph Prism 4.0 (GraphPad, San Diego, CA, USA). Values of P < 0.05 were considered statistically significant.


Zfp521 expression is up-regulated in the growth plate of Jansen mice

PTHrP has been shown to regulate chondrocyte maturation, and Zfp521 is a key downstream target of PTHrP in this process (12). In vitro data suggest that PTHrP stimulates Zfp521 expression in chondrocytes via the Gsα/PKA pathway. In the current study, we investigated the expression and regulation of Zfp521 in the growth plate of Jansen mice, a mouse model that expresses a constitutively active PTHR1 in chondrocytes. These mice thereby exhibit a metaphyseal chondrodysplasia phenotype that mimics the human disorder (6). We performed in situ hybridization for Zfp521 on tibial sections from 7-d-old Jansen mice and compared the expression pattern to that of wild-type littermates. Zfp521 expression was detected in all zones of the growth plate, with the strongest signals in the prehypertrophic cells, where the Jansen receptor is mostly expressed (Fig. 1A). We isolated chondrocytes from growth plates of 14-d-old wild-type, Jansen, double-mutant, and Col2Cre-Zfp521d/d mice by microdissection (Fig. 1B) and performed qRT-PCR analyses to quantify our histological data. We also performed immunocytochemistry in order to confirm up-regulation of Zfp521 protein in Jansen chondrocytes compared to that in wild-type chondrocytes (Fig. 1C). These data clearly confirmed that expression of Zfp521 in chondrocytes of the Jansen mice is significantly increased. To show that Zfp521 expression is activated directly via PTHR1 signaling, we specifically ablated PTHR1 in prehypertrophic chondrocytes (Supplemental Fig. S1). We cultured chondrocytes from PTHR1fl/fl mice for 15 d to enrich for prehypertrophic chondrocytes as confirmed by the spatial and temporal patterns of chondrocyte markers such as PTHR1, Col II, and Col X (data not shown). We found that after ablation of PTHR1 with adenovirus Cre-recombinase in these prehypertrophic enriched chondrocytes (Supplemental Fig. S1B), endogenous Zfp521 expression was reduced ~50% compared to cells transfected with a lacZ-cre control virus (Fig. 1D). This suggests that PTHR1 signaling is necessary for basal expression of Zfp521 expression in prehypertrophic chondrocytes.

Figure 1.
Expression of Zfp521. A) In situ hybridization of P7 control and Jansen growth plates using a DIG-labeled Zfp521 probe. Zfp521 expression is increased in the Jansen growth plate when compared to control. Negative control was performed on a Jansen sample ...

Ablation of Zfp521 from Jansen chondrocytes partially rescues the delayed endochondral bone formation

We generated double-mutant Jansen/Col2Cre-Zfp521d/d mice by crossing Jansen mice with Col2Cre-Zfp521fl/fl mice. The phenotype of Col2Cre-Zfp521d/d mice has been recently described (12). Briefly, Col2Cre-Zfp521d/d mice are morphologically indistinguishable from wild-type mice at birth but start to show growth retardation by 1–2 wk postnatal with shorter long bones. No other gross anatomical alterations have been observed.

Histological analyses from paraffin sections of tibia from P14 wild-type, Jansen, Col2Cre-Zfp521d/d, and Jansen/Col2Cre-Zfp521d/d double-mutant mice were performed. We could confirm the previously reported delay in chondrocyte differentiation in Jansen mice characterized by an extended growth plate length and the absence of a clear secondary ossification center when compared to wild-type littermates (Fig. 2E, F). These changes in chondrogenesis were also obvious in other skeletal elements at earlier time points, as can be seen in the sternum of newborn Jansen mice (Fig. 2A). In these bones, chondrocytes have just started to differentiate into hypertrophy, whereas in wild-type mice, full replacement of cartilage by bone has already occurred. In contrast, chondrocyte differentiation in Col2Cre-Zfp521d/d mice was more advanced, with a narrowing of the tibial growth plate (Fig. 2E) and an advanced ossification center in the sternebrae (Fig. 2A). Interestingly, the tibial growth plate of Jansen/Col2Cre-Zfp521d/d double mutants resembled that of wild-type mice, suggesting that deletion of Zfp521 from Jansen chondrocytes improved the delay in chondrocyte differentiation (Fig. 2E, F). A representative quantification of hypertrophic chondrocytes was determined from ≥4 stained sections from each sample using Scion Image software. The sternebrae of newborn Jansen/Col2Cre-Zfp521d/d double mutants showed a significant increase in the number of hypertrophic chondrocytes when compared to Jansen mice (Fig. 2B, D). We then performed in situ hybridization in order to confirm our histological observations and to examine the differentiation stage of cells in the growth plate of all four genotypes (Fig. 3). Hybridization of tibial sections with probes specific for collagen type II (Fig. 3A–D) and type X (Fig. 3E–H), markers for proliferative and hypertrophic chondrocytes, respectively, demonstrated that the delay in chondrocyte differentiation in Jansen mice was rescued in Jansen/Col2Cre-Zfp521d/d double mutants. Interestingly, the expression of these markers, which is decreased in the growth plate of Col2Cre-Zfp521d/d mice, was altered as well, as shown by the increase in collagen type II and type X expression in the chondrocytes of double-mutant growth plates. Furthermore, the slightly reduced secondary ossification center in these mice indicates that additional signaling of PTHR1 can occur in the absence of Zfp521 (Fig. 3C, D, G, H). The growth plate of Jansen mice lacked a clear secondary ossification center and collagen type II-positive cells were seen throughout the growth plate. In contrast, the double mutants had an expression pattern resembling that of wild-type controls (Fig. 3C, G). These data suggest that ablation of Zfp521 from Jansen chondrocytes can restore proper chondrocyte differentiation in Jansen mice.

Figure 2.
Histological and quantitative analyses of the growth plate. A) H&E staining of decalcified sternebrae of P0 control, Jansen transgenic, Jansen/Col2Cre-Zfp521d/d, and Col2Cre-Zfp521d/d mice; black arrows indicate primary ossification center. B–D ...
Figure 3.
In situ hybridization of the growth plate of the proximal tibia of P14 mice. Mislocalization of cells expressing collagen type II and type X was rescued in Jansen/Col2Cre-Zfp521d/d double mutants. In situ hybridization using an S35-labeled type II collagen ...

Increased expression of hypertrophic markers demonstrates enhanced differentiation of chondrocytes in double mutants

The impaired endochondral ossification process in Jansen mice is clearly due to a reduction in chondrocyte differentiation (11, 20). In contrast, ablation of Zfp521 from chondrocytes was shown to result in reduced chondrocyte proliferation, accelerated chondrocyte differentiation, and increased cell death (12). We examined the expression of several key genes in Jansen/Col2Cre-Zfp521d/d double mutants to further analyze these observations. We found that genes expressed during chondrocyte proliferation, such as CyclinD1 and collagen type II are significantly up-regulated in Jansen mice, while their expression in Zfp521-depleted chondrocytes is decreased when compared to wild type. Interestingly, expression of CyclinD1, collagen type II, and Bcl2 in chondrocytes of double mutants was normal and resembled the levels in controls (Fig. 4). No significant difference between Jansen and double mutants was observed in collagen type X mRNA levels (Fig. 4). Notably, the decreased collagen type X expression in Col2Cre-Zfp521d/d mutants could be normalized by the Jansen transgene (Fig. 4), confirming the data obtained by in situ hybridization (Fig. 3G, H). Furthermore, we could demonstrate that Zfp521-deficient Jansen chondrocytes exhibit a significant increase in Alp, Runx2, and Mmp13, supporting our earlier data that chondrocyte differentiation is advanced in double-mutant mice and resembles the levels seen in Col2Cre-Zfp521d/d mice. Notably, we found that P53, a cell arrest mediator, is significantly increased in both Col2Cre-Zfp521d/d and double-mutant mice (Fig. 4). We therefore examined double-mutant chondrocytes in order to determine whether they undergo terminal transition from hypertrophic chondrocytes into bone formation via apoptosis.

Figure 4.
qRT-PCR analyses from chondrocytes isolated from the tibial growth plate of P14 mice of all four genotypes. CyclinD1 (Ccnd1), type II collagen (Col II), type X collagen (Col X), P53, Alp, Runx2, Mmp13, and Bcl2 expression. Values are expressed as means ...

Chondrocyte apoptosis is partially rescued in Jansen/Col2Cre-Zfp521d/d double-mutant mice

Endochondral bone is formed on replacement of a cartilage mold. This process involves controlled cell death of chondrocytes (3). It has previously been shown that PTHrP not only delays chondrocyte hypertrophy but also protects cells from programmed cell death by up-regulation of Bcl2 (20). Our previous work has demonstrated that Zfp521 plays an important role in the control of cell differentiation and apoptosis by mediating the actions of PTHrP on chondrocytes (12). We performed TUNEL staining on sections of the tibial growth plate of wild-type, Jansen, Col2Cre-Zfp521d/d, and Jansen/Col2Cre-Zfp521d/d double-mutant mice. The results show a decrease in apoptosis in Jansen mice, whereas there was a significant increase in cell death in Col2Cre-Zfp521d/d mice. The growth plate of Jansen/Col2Cre-Zfp521d/d mice, however, had a significantly higher rate of chondrocyte apoptosis than Jansen mice and levels were equivalent to those in control mice (Fig. 5A, B).

Figure 5.
Quantification of chondrocyte apoptosis. A) TUNEL staining (green dots) reveals an accumulation of apoptotic cells in tibial growth plate of P14 control (a), Jansen transgenic (b), Jansen/Col2Cre-Zfp521d/d (c) and Col2Cre-Zfp521d/d (d) mice; red arrows ...

Increased bone length in double-mutant mice indicates a clear improvement in endochondral bone formation

We investigated whether the increased chondrocyte differentiation and chondrocyte apoptosis at the chondro-osseous junction in double-mutant mice will result in an improvement of bone length. We dissected representative long bones (left radius and ulna) from random P14 mice from each group. As expected, Jansen mice and Col2Cre-Zfp521d/d mice had significantly shorter bones, confirming our previous observations (12). In contrast, the gross appearance, bone length (Fig. 6), and body weight (Supplemental Fig. S2) of the double-mutant mice were rescued and similar to that of control mice. These results corroborate our previous data demonstrating a significant improvement in proper endochondral bone formation in Jansen/Col2Cre-Zfp521d/d double-mutant mice. Taken together, our data indicate that ablation of Zfp521 from Jansen chondrocytes partially rescues the defect in hypertrophic chondrocyte differentiation and thereby endochondral bone formation.

Figure 6.
Macroscopic appearance and bone length of all four genotypes. A) Top view of P2 control (Ctrl), Jansen transgenic (Jansen), double-mutant, and Col2Cre-Zfp521d/d littermates revealing that the short stature of Jansen transgenic was visibly rescued in double ...


JMC is a rare autosomal dominant disorder characterized by progressive widening of the metaphyses and distal clavicles, a reduction in the length of the long bones, and a short stature (8, 21). Affected patients show high bone turnover, as indicated by high levels of serum bone alkaline phosphatase, intact osteocalcin, and tartrate-resistant acid phosphatase (TRAP). Relatively elevated urine cAMP excretion indicated a potential cause of this phenotype. In vitro and in vivo studies have demonstrated that the H223R conversion in PTHR1 causes a constitutive activation of the receptor, resulting in the accumulation of intracellular cAMP, which is correlated to the biochemical phenotype observed in these patients (6, 7). As expected from the increased PTHrP signaling in Jansen chondrocytes, we have found that endochondral bone formation in the Jansen mouse model is characterized by a delay in bone ossification and blood vessel invasion. A consistent delay of tibial growth plate development in postnatal life was observed, as indicated by the absence of a secondary ossification and lengthening of the total growth plate (7). Zfp521 has recently been identified as a target gene and key effector of PTHrP in the regulation of chondrocyte maturation (12). This finding led us to explore a possible role for Zfp521 in the JMC disorder. Interestingly, we discovered that Zfp521 expression is up-regulated in the growth plate of Jansen mice, indicating the possible significance of this factor as a contributor to the Jansen phenotype. This finding is consistent with our previous finding that Zfp521 is down-regulated in chondrocytes extracted from PTHrP-knockout mice (12). In addition, we could demonstrate that removal of PTHR1 from prehypertrophic enriched primary chondrocytes leads to a significant decrease in endogenous Zfp521 expression, indicating that Zfp521 is a crucial mediator of PTHrP signaling (Fig. 1D). These observations, along with our previous findings, suggest that Zfp521 is required to maintain the balance chondrocyte proliferation and differentiation, which is important for proper growth plate development. Further studies are required, however, to determine how or whether other chondrogenic signaling factors mediate Zfp521 expression at different stages of chondrogenesis.

Runx2 expression is localized in perichondrial cells and hypertrophic chondrocytes surrounding the columnar proliferating and hypertrophic chondrocytes, with limited expression in prehypertrophic chondrocytes (22). Several factors have been reported to inhibit or enhance Runx2 activity to coordinate chondrocyte and osteoblast differentiation (23). Among these, Zfp521 has recently been identified as a corepressor of Runx2 in both chondrocytes and osteoblasts (12, 19). Thus, one explanation for the improvement in chondrocyte differentiation in Jansen/Col2Cre-Zfp521 double-mutant mice could be increased Runx2 activity in chondrocytes depleted for Zfp521, leading to accelerated chondrocyte differentiation. The role of PTHrP in decreasing Runx2 mRNA and protein levels has been extensively studied (24, 25), raising the possibility that the PTHrP-induced decline in Runx2 may be responsible for the abnormal growth plate development that we observed. However, the delay in chondrocyte hypertrophy by PTH/PTHrP signaling is reported to involve two different mechanisms. Administration of PTH to endochondral bone cultures clearly demonstrates reduction of Runx2 expression in chondrocytes expressing collagen type X, but not in perichondrial zones where osteoblast precursors are located. This suggests that regulation of Runx2 by PTHrP specifically occurs in hypertrophic chondrocytes. Alternatively, an in vivo study of Runx2 and PTHrP interactions revealed that PTHR1 stimulation prevented hypertrophy in Runx2-null mice, indicating that PTH/PTHrP actions on chondrocyte hypertrophy can occur in a Runx2-independent manner (22). Moreover, we previously reported in ex vivo studies that ablation of Zfp521 is able to prevent PTHrP actions, indicating a role for Zfp521 in blocking Runx2 expression and activity. Here, we report that Runx2 expression is enhanced on Zfp521 ablation in contrast to the slightly decreased levels found in Jansen mice (Fig. 4) where PTHrP signaling is solely mediated by cAMP accumulation. Notably, we show in the double-mutant mice that constitutively activation of PTHrP signaling could no longer inhibit Runx2 expression in the absence of Zfp521. Our studies, therefore, demonstrate for the first time in vivo that Zfp521 is a crucial mediator of the actions of PTHrP to control chondrocyte differentiation.

We could demonstrate that ablation of Zfp521 improves endochondral bone formation in Jansen mice by significantly restoring the number of hypertrophic chondrocytes. Collagen type II expression was increased and collagen type X mRNA level was not significantly different in Jansen and double-mutant mice. However, the misexpression of these markers was rescued in double mutants (Fig. 3), indicating a normal transition from proliferative to prehypertrophic and eventually to hypertrophic chondrocytes during growth plate formation. Interestingly, we could confirm the previously reported decrease in collagen type X expression in the Col2Cre-Zfp521 growth plate (12). The observation that Zfp521 deletion increases differentiation of chondrocytes, but also represses collagen type X expression, is still unexplained. Notably, in the current study, we could demonstrate that double mutants exhibit a complete restoration of the collagen type X expression pattern to resemble the one seen in wild-type mice. This observation requires further attention and needs to be addressed in a separate study. Consistent with the role of PTHrP in preventing apoptosis and promoting chondrocyte proliferation, we observed an increase of Bcl2 and CyclinD1 levels in growth plate chondrocytes extracted from Jansen mice, respectively (Fig. 4). In a previous in vitro study, we found that PTHrP failed to up-regulate CyclinD1 and Bcl2 when Zfp521 was absent (12). As expected, we found that expression of CyclinD1 and Bcl2 was normalized in double-mutant mice. In addition, our in vivo data demonstrate a clear reduction in the length of the proliferative zone in double mutants when compared to the one in Jansen mice. Taken together, these data imply that chondrocyte proliferation in these mutants was recovered. In addition, we observed that the number of hypertrophic chondrocytes and markers such as Alp, Runx2, and Mmp13 were increased in double mutants when compared to Jansen mice. This would indicate that the population of premineralizing chondrocytes was recouped in these mice. These changes in double-mutant mice lead to an improvement of endochondral bone length, which suggests proper endochondral bone formation. One of the hallmark phenotypes in Jansen metaphyseal chondrodysplasia is markedly retarded ossification in endochondral bones, i.e., cervical and lumbar vertebrae (radiography detected at 12 mo in patient, distorted ossification; refs. 8, 26). Here, we also found that chondrocyte differentiation was improved in newborn sternebrae of double mutants, as indicated by the accelerated chondrocyte differentiation and the appearance of a secondary ossification center in the tibia.

Interestingly, we found a significant increase in P53 levels in the Zfp521-deficient growth plate, correlated with an increase in chondrocyte apoptosis at the chondro-osseous junction. However, we found that accumulation of cAMP in chondrocytes in vivo did not alter P53 mRNA levels. This finding is consistent with a report showing lack of nodule formation in hypertrophic chondrocyte-like cells derived from P53-knockout mice. Furthermore, blocking PTHrP function using either an antagonist or neutralizing antibodies failed to rescue this phenotype (27). This raises the possibility that Zfp521 could directly interact with P53, but further studies would be needed to test this hypothesis.

This study provides the first genetic proof that Zfp521 is a crucial mediator of PTH/PTHrP signaling in chondrocytes. We could demonstrate that ablation of Zfp521 significantly improves the delay in normal growth plate development in the Jansen metaphyseal chondrodysplasia mouse model by controlling cell proliferation, differentiation, and apoptosis. Future studies will be required to investigate whether Zfp521 also acts downstream of other growth factors such as Ihh and BMPs and whether or not Zfp521 can mediate the function of factors that are independent of PTHrP signaling. Taken together, this study highlights the in vivo role of Zfp521 as an important factor in PTH/PTHrP signaling in chondrocytes. In conclusion, this study provides first genetic evidence that Zfp521 acts downstream of PTHR1 signaling to control chondrocyte proliferation, differentiation, and cell death. This finding suggests a potential role for Zfp521 as a candidate for therapeutic application in JMC.

Supplementary Material

Supplemental Data:


The authors thank Drs. Neal G. Copeland, Sǿren Warming, Riku Kiviranta, and Yukiko Maeda for helpful suggestions.

This work was supported by U.S. National Institutes of Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases grants AR050560 (B.L.) and AR057769 (R.B.).


This article includes supplemental data. Please visit http://www.fasebj.org to obtain this information.


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