Bcl-2 Lies Downstream of Parathyroid Hormone–related Peptide in a Signaling Pathway That Regulates Chondrocyte Maturation during Skeletal Development

The antibodies used in these experiments include a rabbit anti-mouse collagen type X antibody (dilution 1:100) generously provided by Dr. B. Olsen (Harvard University, Cambridge, MA), polyclonal antibodies against mouse Bcl-2 (dilution 1:200) obtained from PharMingen (San Diego, CA), and Bax (dilution 1:200) obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The fluorescein-conjugated secondary antibodies (dilution 1:100) and the normal goat serum were purchased from Boehringer Mannheim Biochemicals (Indianapolis, IN). For immunofluorescence analysis, cultured cells plated on glass coverslips, 6 μm frozen sections, and 6 μm paraffin embedded sections of 6-d-old mice [(Procollagen Ia type II) col II-PTHrP transgenic mice and control littermates] were used. Mice were perfused via the heart with paraformaldehyde (2%), lysine (0.75 M), and sodium periodate (0.01 M) (PLP), for 5 min. The long bones were dissected out and fixed in PLP for an additional 4 h at 4°C, washed in PBS for 1 h, and infiltrated overnight with 40% sucrose in PBS. The tissue was subsequently quick-frozen or embedded in paraffin. The cultured cells were fixed in 3.7% formaldehyde in PBS for 10 min at room temperature and washed in PBS. Further processing of tissue sections and isolated cells was identical. For collagen X staining, the samples were preincubated with bovine hyaluronidase in PBS (1 mg/ml) for 45 min at 37°C. All subsequent incubations were performed at room temperature with PBS containing 0.05% saponin, 0.1% BSA, and 5% normal goat serum (NGS). The samples were incubated in PBS–saponin–BSA–NGS for 30 min to block nonspecific binding, and then for 2 h with the primary antibody. The samples were washed in PBS–saponin–BSA and incubated with secondary antibody for 1 h in the dark. After washing in PBS, the samples were mounted in FluorSave fluorescent mounting media (Calbiochem-Novabiochem Corp., La Jolla, CA). Samples were examined with a scanning laser confocal microscope (Axiovert 10, Zeiss, Inc., Thornwood, NY and MRC 600 confocal imaging system; Bio-Rad Laboratories, Richmond, CA) with a krypton/argon laser using an optical slice thickness of 1–2 μm. Computer images were collected on optical memory discs and were computer enhanced with the Adobe Photoshop program.

Terminal deoxynucleotidyl transferase (TdT)–mediated dUTP nick endlabeling (TUNEL) was performed using a molecular biological–histochemical system, ApopTag kit (Oncor, Gaithersburg, MD) for specific staining of DNA fragmentation and apoptotic bodies (Gavrieli et al., 1992). Cells exhibiting DNA fragmentation and containing apoptotic bodies, thereby morphologically consistent with apoptosis, are referred to as apoptotic cells. TdT, which catalyzes a template-independent addition of deoxyribonucleotide to 3′-OH ends of DNA, was used to incorporate digoxigenin-conjugated dUTP to the ends of DNA fragments. Briefly, after digesting protein in quick-frozen bone sections (control mice and col IIPTHrP transgenic mice) with 10 μg/ml proteinase K at room temperature and then quenching endogenous peroxidase activity with 1% H₂O₂ in PBS, slides were placed in equilibration buffer, and then in terminal deoxynucleotidyl transferase solution, followed by stop/wash buffer. The signal of TUNEL was then detected by an anti-digoxigenin antibody conjugated with peroxidase, a reporter enzyme that catalytically generates a browncolored product from the chromogenic substrate diaminobenzidine. Because processing in paraffin may increase the number of apparent apoptoses, we used cryosections. Controls for specificity of labeling included positive control slides prepared by nicking DNA with DNAse I (Boehringer Mannheim Biochemicals) (Arends et al., 1990), and negative control sections were prepared by substituting distilled water for TdT enzyme. The slides were washed, dried, and mounted in Permount media.

Chondrocytes were isolated from the resting zone of the proximal tibia and distal femur of 10-d–old mice. Growth plates were cleaned of perichondrium; the reserve zone was dissected by a transverse cut through the calcification zone and separated from the epiphysis under a dissecting microscope. Pooled growth plates from 10 to 12 animals were predigested for 30 min in a 0.1% solution of clostridial collagenase Ia (Sigma Chemical Co., St. Louis, MO) in culture media (Ham F-12/DME 1:1, 10% FBS, 1% penicillin/streptomycin [PS], 50 μg/ml L-ascorbic acid, 100 μg/ml sodium pyruvate) in a shaker at room temperature. Cartilage fragments were then washed twice in culture media and subsequently digested for 3 h, in a 0.2% solution of collagenase in culture media, in an incubator shaker at 37°C. The solution containing the isolated cells was filtered (through 50 μm mesh), and cells were recovered by centrifugation (1,500 rpm, 4°C, 5 min). Cells were resuspended in culture medium at a final concentration of 300,000 cells/ml. Cells were cultured as high-density monolayers in 24-well dishes (150,000 cells/well) (Falcon Labware, Oxnar, CA) and in 60-mm dishes (10⁶ cells/dish) for protein extraction. Medium was changed every other day after the third day. PTH 1-34 and PTHrP 1-37 (both at a final concentration of 10⁻⁷ M) or forskolin (final concentration 50 μM) were added with fresh media every other day. Mouse primary osteoblastic cells were obtained from 3-d–old mouse calvaria and cultured in αMEM containing 10% FBS and 1% PS. The human renal carcinoma cell line (CAKI) was cultured in RPMI containing 10% FBS and 1% PS. All cultures were maintained at 37°C, gassed with 95% air/5% CO₂.

All extraction procedures were performed at 4°C. Cells were washed once with ice-cold PBS, and cell lysates were generated with ice-cold RIPA buffer (10% mM Tris-HCl, pH 7.2, 158 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS) containing protease inhibitors (0.1 mM PMSF, 1 μM pepstatin, and 1 μM leupeptin) which disrupt Bcl-2/Bax interactions. RIPA extracts were prepared by sonication and subsequent centrifugation at 12,000 g for 10 min. The protein was eluted by boiling in sample buffer (62.5 mM Tris–HCl, pH 6.9, 2 mM EDTA, 3% SDS, 3.75% glycerol, and 180 mM β-mercaptoethanol) for 2 min. Western blotting was performed using the enhanced chemiluminescence Western blotting detection reagents (Amersham Corp., Arlington Heights, IL) according to the conditions recommended by the supplier. Briefly, samples (chondrocytes and osteoblasts, 200 μg/lane, kidney cells, 100 μg/lane) were analyzed by 12.5% SDS-PAGE followed by electrophoretic transfer of the proteins to nitrocellulose filters. Filters were first blocked in TBST (20 mM Tris, pH 7.6, 134 mM NaCl, 0.1% Tween-20) containing 5% Carnation nonfat dry milk for 2 h and then incubated with the rabbit anti–Bcl-2 N-19 (Santa Cruz Biotech.) polyclonal antibody (1:200 in TBST) for 6 h at 4°C. After three sequential 15 min washes in TBST, the filters were incubated with anti–rabbit peroxidase-conjugated secondary antibody (1:20,000 in TBST) for 1 h at room temperature and then again washed as described above. For human kidney cells, filters were first incubated with 5% BSA in TBST, to block nonspecific binding, and then with monoclonal mouse anti–human Bcl-2 124 (dilution 1:200 in TBST) (DAKO Corp., Carpinteria, CA); anti–mouse peroxidase-conjugated secondary antibody was used as described above. Bound protein was detected by enhanced chemiluminescence reaction. The blots were then stripped of antibody and reprobed with anti-actin monoclonal antibody (Boehringer Mannheim Biochemicals, 1 μg/ml) to confirm that equal amounts of protein were loaded in each corresponding treated and untreated lane.

Transgenic mice were generated as previously described (Weir et al., 1996). Briefly, a 568-bp human PTHrP cDNA fragment encoding the 1-141 isoform was inserted into a 6.5-kb segment of the mouse procollagen a1 type II (col II) promoter region (obtained from S. Garofolo, Shriners Hospital, Portland, OR and B. de Crombrugghe, M.D. Anderson Cancer Center, Houston, TX) at a site within the first intron. A consensus splice acceptor (courtesy of P. Soriano, Fred Hutchinson Cancer Research Center, Seattle, WA) was included in the col II construct at the 5' end of the insertion point to create an artificial exon and thus circumvent alternative splicing. The initiation codon in exon 1 of the procollagen gene was inactivated to allow translation to start within the cDNA sequences. Also, a 2.2-kb segment of the human growth hormone gene was added downstream of the cDNA to provide termination/polyadenylation signals and to increase expression efficiency. The growth hormone coding sequences are not translated.

For histology, col II-PTHrP transgenic mice and control littermates (6 d of age) were perfused via the heart with PLP for 5 min. Metatarsals were dissected out and fixed by 3.7% paraformaldehyde for 12 h at 4°C, embedded in epon, and 2 μm sections were prepared on a ultramicrotome (Reichert Scientific Instruments, Buffalo, NY). Sections were stained with toluidine blue and evaluated on a Reichert microscope (UnivaR, C. Reichert, Austria). Immunohistochemical analysis and TUNEL analysis were done as stated above for confocal immunfluorescence analysis and in situ analysis of apoptotic cells, respectively.

Generation of germ line mutant mice carrying ablated bcl-2 coding regions was previously described (Nakayama et al., 1993; 1994). Offspring from heterozygote intercrosses were genotyped by PCR and by Southern blot analysis, as previously reported (Nakayama et al., 1993; 1994). After radiological analysis of the skeleton, histomorphometric analysis was performed on undecalcified, processed bones of neonate, 6-d, and 60-d–old knockout mice and control littermates (Hahn et al., 1991). Three samples each were analyzed, and statistical analysis was performed using Student's t test.

Bcl-2 protein is expressed in chondrocytes throughout the growth plate, with highest levels in late proliferative and prehypertrophic chondrocytes (characterized by their intracellular expression of collagen type X), and markedly decreased levels in late hypertrophic chondrocytes (characterized by their morphology and pathognomonic, pericellular, ring-like secretion of collagen type X) (Fig. 1, A–C). The opposite pattern was observed for Bax protein expression, with undetectable levels in proliferative cells and a progressive increase towards hypertrophic chondrocytes (Fig. 1,D). Thus, within the growth plate, the ratio of Bcl-2 to Bax progressively decreases in favor of Bax, and fully differentiated (hypertrophic) chondrocytes die in an apoptotic manner, as confirmed by nick end-labeling of DNA fragments by TUNEL (Gavrieli et al., 1992) (Fig. 1 E).

Treatment of cultured murine growth plate chondrocytes with the NH₂-terminal fragments PTHrP 1-37 (Figs. 2 and 3) or PTH 1-34 (data not shown) resulted in an increase in cAMP production and a marked increase in Bcl-2 expression. By comparing pixel intensity of Bcl-2 immunolabeling, differences in Bcl-2 levels in treated and untreated chondrocyte cultures were clearly detectable, with a twofold increase in treated culture, as early as 3 d after starting PTHrP treatment, i.e., before the appearance of hypertrophic cells (data not shown). In parallel, in longer cultures (12 or 24 d), we observed an inhibition of chondrocyte differentiation, as determined by reduced expression of alkaline phosphatase and matrix calcification (data not shown); a marked decrease in the number of hypertrophic cells; and an accumulation of prehypertrophic chondrocytes (Fig. 2, A and B). This increase in Bcl-2 expression was found to be cAMP dependent and was mimicked in these cultures by the addition of Forskolin (data not shown). Bax expression was lower and was not affected by PTH or PTHrP treatment (Fig. 2 E). Thus, in vitro treatment of chondrocytes with PTH or PTHrP results in a shift of the Bcl-2/Bax ratio in favor of Bcl-2, a change that delays terminal differentiation, prolongs chondrocyte survival, and leads to the accumulation of cells in their prehypertrophic stage.

As shown in Fig. 3, the effects of PTHrP on Bcl-2 expression were found to be restricted to chondrocytes, since treating both primary osteoblasts and a kidney cell line, that expresses the PTH/PTHrP receptor, with PTH or PTHrP failed to alter Bcl-2 levels in these other cell types, despite the fact that they responded to PTH/PTHrP with the expected increase in cAMP.

Targeted overexpression of PTHrP to chondrocytes, using the collagen type II promoter in transgenic mice, leads to a profound delay in endochondral bone formation with Jansen's metaphyseal dysplasia–like skeletal abnormalities (Weir et al., 1996). As shown on Fig. 4, this also leads to a marked increase in Bcl-2 expression in prehypertrophic chondrocytes, with no detectable change in Bax levels. At 6 d of age, the metatarsals of col II–PTHrP transgenic animals were still at the cartilaginous model stage with an accumulation of prehypertrophic chondrocytes, thereby mimicking the effects of PTHrP on chondrocytes in culture. These cells contained intracellular collagen type X (data not shown). Hypertrophic or apoptotic chondrocytes were not detectable, as further confirmed by the absence of extracellular collagen type X rings and signal from TUNEL analysis (data not shown). In contrast with control littermates, the formation of bone or bone marrow was not yet apparent in the transgenic animals (Fig. 4).

To determine whether Bcl-2 is directly involved in endochondral bone formation and whether its characteristic expression pattern within the growth plate is related to a functional role in regulating chondrocyte terminal differentiation, we analyzed the skeleton in bcl-2 knockout mice. We found that bcl-2 knockout mice exhibit premature chondrocyte maturation and terminal differentiation. Consequently, the program of endochondral bone formation is significantly accelerated, leading to earlier vascular invasion, replacement of cartilage by bone, and homing of bone marrow cells, as compared to control littermates (Fig. 5). Furthermore, bcl-2 knockout mice exhibit a reduction in the growth plate thickness, mostly due to a decrease in the height of proliferative zone, and ultimately a significant decrease (15%–20%) in overall bone length. These data demonstrate that Bcl-2 is a direct player in the regulation of the endochondral bone formation and that bcl-2 knockout mice display a skeletal phenotype paralleling that of PTHrP knockout mice, though far less pronounced (Fig. 5).

The present study suggests that PTHrP, a major player in chondrocyte maturation and endochondral bone formation, exerts its effect on skeletal development via a pathway involving Bcl-2 expression and alterations in chondrocyte maturation and apoptotic cell death.

Bcl-2 is expressed in chondrocytes throughout the growth plate, with highest levels in late proliferative and prehypertrophic chondrocytes, and markedly decreased levels in late hypertrophic chondrocytes, a pattern of expression similar to that of PTHrP (Amizuka et al., 1994). The opposite pattern was observed for Bax expression, with undetectable levels in proliferative cells and a progressive increase towards hypertrophic chondrocytes. Thus, within the growth plate, the ratio of Bcl-2 to Bax progressively decreases in chondrocytes in favor of Bax. This should result in the apoptotic death (Oltvai et al., 1993) of fully differentiated (hypertrophic) chondrocytes, as confirmed by morphology and nick end-labeling of DNA fragments by TUNEL and previously suggested by others (Farnum and Wilsman, 1989; Lewinson and Silbermann, 1992; Henderson et al., 1995).

The presence of short ears and short and deformed limbs in the bcl-2 knockout mice (Nakayama et al., 1993; Nakayama et al., 1994; Veis et al., 1993) also suggests a functional role for Bcl-2 in chondrocyte maturation. To directly study the role of Bcl-2 in chondrocyte differentiation, we analyzed the skeletal phenotype in bcl-2 knockout. We found that despite the existence of nine family members and efficient redundancies, the pathway of endochondral bone formation is significantly altered in bcl-2 knockout mice. A marked reduction in growth plate thickness, predominantly due to a shortening of the proliferative zone, and a significant decrease in overall bone length, provided evidence for accelerated chondrocyte differentiation. This firmly established that Bcl-2 is directly involved and required for normal skeletal development.

Since absence of PTHrP in the null mutant leads to accelerated chondrocyte differentiation in the growth plate (Amizuka et al., 1994; Karaplis et al., 1994), we and others (Amizuka et al., 1994; Henderson et al., 1995) hypothesized that this could be due to an increase in programmed cell death. To further test this hypothesis and to study the mechanisms by which PTHrP delays chondrocyte differentiation (Kato et al., 1990; Iwamoto et al., 1994; Klaus et al., 1994) and apoptotic cell death (Henderson et al., 1995), we first determined whether PTHrP could alter Bcl-2 expression in chondrocytes. We found that in vitro treatment of chondrocytes with PTH or PTHrP results in a shift of the Bcl-2/Bax ratio in favor of Bcl-2, a change that delays terminal differentiation and apoptosis of hypertrophic chondrocytes and leads to the accumulation of cells in their prehypertrophic stage.

Despite the fact that other cell types are known to express PTH/PTHrP receptors and to respond to PTHrP with an increase in cAMP, the only alterations reported in the PTHrP knockout mice, and in individuals with the receptor mutation, are chondrodysplasias. We consequently determined whether the effects of PTHrP on Bcl-2 expression in chondrocytes were cell-specific, by treating primary osteoblasts and a kidney cell line that expresses the PTH/ PTHrP receptor with PTH or PTHrP. While responding to PTH/PTHrP with the expected increase in cAMP production, this treatment had no detectable effect on Bcl-2 or Bax levels in these two cell types. We therefore concluded that the downstream effect of PTH/PTHrP on Bcl-2 expression is, at least among those cells that are known to express the PTH/PTHrP receptor, specific for chondrocytes. These findings are entirely consistent with the seemingly chondrocyte-specific abnormalities in the PTHrP knockout mice (Amizuka et al., 1994; Karaplis et al., 1994), as well as in individuals with Jansen's-type metaphyseal chondrodysplasia (Schipani et al., 1995; Jüppner, 1996). This specificity may be due to the fact that in chondrocytes, Bcl-2 may alter an antioxidative pathway to repress cell death (Allsopp et al., 1993; Boise et al., 1993; Hockenbery et al., 1993; Kane et al., 1993), since chondrocytes have been shown to be resistant to various stresses, such as the withdrawal of growth factors or serum starvation, as long as antioxidants are also present (Tschan et al., 1990; Ishizaki et al., 1994). These cells literally commit suicide in response to nitric oxide (Blanco et al., 1995).

The most compelling evidence that the regulation of Bcl-2 in chondrocytes is a key mechanism by which PTHrP exerts its control on endochondral ossification during skeletal development, however, comes from our recent observations in vivo. Targeted overexpression of PTHrP to chondrocytes using the collagen type II promoter in transgenic mice leads to a profound delay in endochondral bone formation with Jansen's-like skeletal abnormalities (Weir et al., 1996). We show here that this targeted overexpression of PTHrP also induces a marked increase in Bcl-2 expression (fourfold increase) in prehypertrophic chondrocytes, with no detectable change in Bax levels. The observation that levels of Bcl-2 expression in chondrocytes of the col II-PTHrP transgenic animals were markedly higher than that of growth plate chondrocytes in control animals, further indicates that Bcl-2 lies downstream of PTHrP, rather than PTHrP directly inhibiting differentiation, and thereby keeping the cells at a stage intrinsically programmed to produce Bcl-2.

We therefore conclude that the apoptotic inhibitor, Bcl-2, is involved in the regulation of the programmed cell death of hypertrophic chondrocytes in the growth plate, an event that is critical for endochondral ossification and skeletal development. PTH/PTHrP increases the expression of Bcl-2 in chondrocytes in a cell-specific manner, thereby delaying their terminal differentiation and subsequent apoptosis, and regulating the maturation of the growth plate. We propose that this regulation may be the mechanism underlying the chondrodysplasias observed in the PTHrP knockout mice, in the mice with targeted overexpression of PTHrP in cartilage, and in human Jansen-type metaphyseal chondrodysplasia. These findings are entirely consistent with the alterations in endochondral bone formation reported here after manipulating Bcl-2 levels independently of PTHrP. Thus, as would be expected if one was lying downstream of the other, the changes in the bcl-2 knockout mice are similar, though far less pronounced, to those in the PTHrP knockout mice, i.e., accelerated chondrocyte maturation. The fact that the bcl-2 knockout mice show a phenotype which is less severe than that in the mice lacking PTHrP (Amizuka et al., 1994; Karaplis et al., 1994) is most likely due to the fact that the function of Bcl-2 during endochondral bone formation can be in part compensated for by redundant pathways, possibly involving other Bcl-2 family members. Although we were not able to detect Bcl-x in the normal growth plate, redundancy is a very likely explanation, since compensation of the absence of Bcl-2 expression by the other 9 Bcl-2 family members has been previously described in different cell systems (Reed, 1995; Han et al., 1996).

These observations directly establish the functional role of Bcl-2 in endochondral bone formation. However, it still remains to be determined whether the regulation of Bcl-2 expression by PTHrP is of relevance to tumorigenesis: if the upregulation of PTHrP, which is known to occur in several malignancies, affects the level of Bcl-2 expression, it might thereby increase the malignant potential of a tumor by repressing the death of tumor cells due to this autocrine/paracrine pathway. Preliminary data analyzing the coexpression of PTHrP and Bcl-2 in human chondrosarcomas (Pösl et al., 1996) further confirms the importance of the PTHrP/Bcl-2 pathway, at least in chondrogenic tumors, where the level of coexpression of PTHrP and Bcl-2 seems to be correlated with the degree of malignancy of the tumor. Thus, Bcl-2 is the first in a new category of proto-oncogenes that oppose apoptosis and extend cell survival rather than promote proliferation (Vaux et al., 1988; Korsmeyer, 1992). We show here that it may be involved in both physiological development and tumorigenesis of the skeleton.

Most recently, Indian hedgehog protein was found to be upstream of PTHrP in the control of chondrocyte differentiation (Lanske et al., 1996; Vortkamp et al., 1996). Our observations suggest that Bcl-2 acts downstream of PTHrP in the same pathway, slowing down chondrocyte maturation during normal skeletal development.

The authors are grateful to Dr. B. Olsen for providing the mice collagen X antibody; Dr. D. Loh (Nippon Roche Research Center, Kanagawa, Japan) for providing the bcl-2 knockout mice; Dr. J. Orloff (West Haven VA Hospital, West Haven, CT) for providing the renal cell line; K.J. Ford for maintaining the bcl-2 colony, and to P. Male (Yale University School of Medicine, New Haven, CT) for his help with confocal images. We thank Dr. G. Delling (Hamburg University, Hamburg, Germany) for critical discussion.

This work was supported by National Institutes of Health Grants to R. Baron (DE-04724) and to A.E. Broadus (AR-30102 and DR-48108). M. Amling is a fellow of the German Research Community (DFG Am 103/2-1).

col II

collagen type II

PTH

parathyroid hormone

PTHrP

parathyroid hormone–related peptide

TUNEL

terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling