§
* Department of Cell Biology, Department of Orthopaedics, ¶ Department of Internal Medicine, ** Department of Cellular and
Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut, 06510; § Department of Bone Pathology,
Hamburg University School of Medicine, 20246 Hamburg, Germany; and
Tokyo Metropolitan Institute of Medical Science,
Tokyo 113, Japan
Parathyroid hormone-related peptide (PTHrP) appears to play a major role in skeletal development. Targeted disruption of the PTHrP gene in mice causes skeletal dysplasia with accelerated chondrocyte maturation (Amizuka, N., H. Warshawsky, J.E. Henderson, D. Goltzman, and A.C. Karaplis. 1994. J. Cell Biol. 126:1611-1623; Karaplis, A.C., A. Luz, J. Glowacki, R.T. Bronson, V.L.J. Tybulewicz, H.M. Kronenberg, and R.C. Mulligan. 1994. Genes Dev. 8: 277-289). A constitutively active mutant PTH/PTHrP receptor has been found in Jansen-type human metaphyseal chondrodysplasia, a disease characterized by delayed skeletal maturation (Schipani, E., K. Kruse, and H. Jüppner. 1995. Science (Wash. DC). 268:98- 100). The molecular mechanisms by which PTHrP affects this developmental program remain, however, poorly understood. We report here that PTHrP increases the expression of Bcl-2, a protein that controls programmed cell death in several cell types, in growth plate chondrocytes both in vitro and in vivo, leading to delays in their maturation towards hypertrophy and apoptotic cell death. Consequently, overexpression of PTHrP under the control of the collagen II promoter in transgenic mice resulted in marked delays in skeletal development. As anticipated from these results, deletion of the gene encoding Bcl-2 leads to accelerated maturation of chondrocytes and shortening of long bones. Thus, Bcl-2 lies downstream of PTHrP in a pathway that controls chondrocyte maturation and skeletal development.
Parathyroid hormone-related peptide (PTHrP)1
was initially isolated from human carcinomas
(Strewler et al., 1987 The critical role played by PTHrP and its receptor in
skeletal development has recently been emphasized by
gene deletion experiments in mice and by a natural mutation in humans. Mice homozygous for PTHrP gene ablation exhibit skeletal deformities that are due to a decrease
in proliferation and the accelerated differentiation of
chondrocytes in the developing skeleton (Amizuka et al.,
1994 During endochondral ossification, the chondrocytes present
in the early cartilaginous model, and later in the growth
plate, first proliferate and then progressively differentiate
into mature hypertrophic chondrocytes. Once fully differentiated, these hypertrophic cells participate in the mineralization of the cartilaginous matrix and undergo cell
death. In normal bone development, this is followed by, and
may be the necessary signal for, the local recruitment of
blood vessels and osteoclasts into the zone of provisional mineralization. This leads to the progressive replacement
of cartilage by bone, the homing of the hematopoietic
bone marrow, and ultimately, longitudinal bone growth. In
bone, osteoblasts and growth plate chondrocytes express
PTH/PTHrP receptors and secrete PTHrP, suggesting the
existence of autocrine/paracrine regulatory loops (Jüppner et al., 1988 Apoptosis has been proposed as the mechanism responsible for the death of chondrocytes during endochondral
bone formation (Farnum and Wilsman, 1989 The temporal-spatial distribution of Bcl-2 suggests that
it serves to regulate apoptotic cell death during embryonic
development and in adulthood. Bcl-2 is widely expressed
among fetal tissues, with substantial levels present in the
developing limb bud (Veis Novack and Korsmeyer, 1994),
and apoptosis is now recognized as an important process
in organogenesis and development. In the adult, Bcl-2 expression is limited to renewing stem cell populations, such
as those found in hematopoietic lineages, complex differentiating epithelia, and glandular epithelia, and to longlived postmitotic cell populations (Hockenbery et al.,
1991 We report here that Bcl-2 and Bax are expressed in
chondrocytes in vivo and show a characteristic distribution
within the developing growth plate, which is consistent
with their role in regulating chondrocyte programmed cell
death. Furthermore, we provide evidence that Bcl-2 is a
direct player and not just a bystander in skeletal development, since accelerated endochondral bone maturation occurs in bcl-2 knockout mice, leading to a phenotype paralleling that of the PTHrP knockout mice (Amizuka et al.,
1994 Confocal Immunofluorescence Analysis
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.
In situ Analysis of Apoptotic Cells
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 Cell Cultures
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 Western Blot Analysis
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 Generation and Analysis of col II-PTHrP
Transgenic Mice
Transgenic mice were generated as previously described (Weir et al., 1996 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 and Histology of bcl-2 Knockout Mice
Generation of germ line mutant mice carrying ablated bcl-2 coding regions was previously described (Nakayama et al., 1993 Localization of Bcl-2 and Bax in Normal Growth Plates
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
PTHrP Increases Bcl-2 Expression in Chondrocytes
In Vitro
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 In
Vivo Leads to Increased Bcl-2 Expression and Delayed
Skeletal Development
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
Accelerated Endochondral Bone Formation in bcl-2
Knockout Mice
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 presence of short ears and short and deformed
limbs in the bcl-2 knockout mice (Nakayama et al., 1993 Since absence of PTHrP in the null mutant leads to accelerated chondrocyte differentiation in the growth plate
(Amizuka et al., 1994 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 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 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 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 Most recently, Indian hedgehog protein was found to be
upstream of PTHrP in the control of chondrocyte differentiation (Lanske et al., 1996; Suva et al., 1987
; Mangin et
al., 1988
) and is responsible for the humoral hypercalcemia associated with various malignancies. PTHrP is structurally related to parathyroid hormone (PTH), a hormone
of major importance in calcium metabolism. Both peptides
share 8 of 13 NH2-terminal residues and bind to and activate the same G-protein-coupled PTH/PTHrP receptor
(Jüppner et al., 1991
). Unlike PTH, however, PTHrP does not circulate in appreciable amounts in normal subjects
but is instead, widely expressed in fetal and adult tissues,
where it is thought to regulate cell differentiation, cell proliferation, and organogenesis as a paracrine or autocrinesoluble factor (Goltzman et al., 1989
; Broadus and Stewart, 1994
; Wysolmerski et al., 1994
). In this context, PTHrP
is a mediator of cellular growth and differentiation (Amizuka et al., 1994
; Karaplis et al., 1994
) and is involved in
mesenchymal-epithelial interactions in several tissues
(Hardy, 1992
; Van de Stolpe et al., 1993; Wysolmerski et
al., 1994
).
; Karaplis et al., 1994
). At the other end of the spectrum, striking skeletal deformities are observed in Jansen's
metaphyseal chondrodysplasia, a human form of shortlimbed dwarfism with delayed endochondral maturation
(Jansen, 1934
). This has been recently attributed to a single heterozygous nucleotide exchange in exon M2 of the
gene encoding for the PTH/PTHrP receptor, resulting in a
constitutively active mutant PTH/PTHrP receptor (Schipani et al., 1995
). Although these findings clearly establish
that PTHrP plays a regulatory role in the process of endochondral ossification, the precise mechanism by which
PTHrP affects skeletal development is not known.
; Amizuka et al., 1994
). The latter appear to
be essential for normal chondrocyte maturation and/or endochondral bone formation, as shown by the various transgenic models and natural mutation discussed above (Amizuka et al., 1994
; Karaplis et al., 1994
; Schipani et al.,
1995
).
; Lewinson
and Silbermann, 1992
). In several cell types, apoptosis is
regulated by the ratio of expression of the cell death inhibitor, Bcl-2, and the cell death inducer, Bax. Bcl-2 is the
founding member of an emerging family of proteins whose
function involves the regulation of programmed cell death (Vaux et al., 1988
; Korsmeyer, 1992
). Bax is another Bcl-2
family member which forms heterodimers with Bcl-2 and,
when overexpressed, counters the anti-apoptotic effect of
Bcl-2, causing accelerated cell death. Within a cell, it is the
ratio of Bcl-2 to Bax that determines whether a cell dies or
not. It has been shown that apoptosis is repressed when
half or more of the endogenous Bax is heterodimerized
with Bcl-2 (Oltvai et al., 1993
; Miyashita et al., 1994
; Yin et
al., 1994
; Sedlak et al., 1995
).
). Several observations led us to hypothesize that
PTHrP and Bcl-2 might participate in the same regulatory
pathways during skeletal development: (a) the temporal-
spatial similarities of the expression patterns of PTHrP
(Lee et al., 1995
) and Bcl-2 (Veis Novack and Korsmeyer, 1994); and the characteristic changes in endochondral
bone formation due to either the absence of PTHrP (Amizuka et al., 1994
) or the constitutive activation of its receptor (Schipani et al., 1995
).
). Finally, we present in vitro and in vivo evidence, using transgenic mice with overexpression of PTHrP targeted to chondrocytes, that Bcl-2 is downstream of PTHrP
in a signaling pathway that is required for normal skeletal
development.
Materials and Methods
). 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.
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
MEM 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.
-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.
).
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.
; 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.
Results
) (Fig. 1 E).
Fig. 1.
Expression of Bcl-2 and Bax in endochondral bone formation. (A) The normal growth plate shows a typical zonal structure of differentiating chondrocytes. Proliferating chondrocytes (P) progressively enlarge to prehypertrophic chondrocytes (PH), and further differentiate into hypertrophic chondrocytes (H). (B) Age-matched growthplates were labeled by immunofluorescence for collagen type X,
which serves as a specific marker in the chondrocyte differentiation process. Intracellular expression of collagen X is characteristic of prehypertrophic chondrocytes, whereas the dense, pericellular matrix labeling shown in red is found solely in the hypertrophic zone. (C)
Highest levels of Bcl-2 protein are detected in late proliferative and prehypertrophic chondrocytes, whereas, (D), hypertrophic chondrocytes exhibit an increased Bax protein expression. Age-matched frozen sections were labeled for DNA fragmentation by TUNEL. (E)
Nick end labeling of DNA fragments was observed only in fully differentiated hypertrophic chondrocytes, at the level where resorption
of cartilage and replacement by bone is taking place.
[View Larger Version of this Image (53K GIF file)]
Fig. 2.
PTHrP increases Bcl-2
expression in chondrocytes in
vitro. (A) Murine chondrocytes,
cultured for 24 d as high-density
monolayers, differentiated in
vitro into hypertrophic chondrocytes with the typical extracellular matrix pattern of collagen X
(compare to Fig. 1 B). (B) In
contrast, treatment of these cultures with PTHrP 1-37 resulted
in an inhibition of terminal differentiation and an accumulation of prehypertrophic chondrocytes, as confirmed by the
pathognomonic intracellular collagen X expression. After 12 d in
culture, a time at which treated
and untreated cultures show no
apparent morphological differences, (C) the untreated chondrocytes show only low cytoplasmic Bcl-2 protein expression, whereas, (D), a marked increase
in Bcl-2 expression is visible after PTHrP treatment. In contrast, the same treatment had no detectable effect on the level of
Bax protein expression (E, control; F, treated). Bars, 10 µm.
[View Larger Version of this Image (154K GIF file)]
Fig. 3.
PTHrP effect on Bcl-2 is restricted to chondrocytes.
Western blotting analysis revealed that treatment of murine
chondrocytes in vitro (cultured for 12 d) with PTHrP 1-37 resulted in a marked increase in Bcl-2 expression. In contrast, the
same treatment had no detectable effect on Bcl-2 levels in primary osteoblasts, obtained from 3-d-old mouse calvaria, and a
kidney cell line (CAKI), both of which express the PTH/PTHrP
receptor. The blots were then stripped of antibody and reprobed
with anti-actin antibody to confirm that equal amounts of protein
were loaded in each corresponding treated and untreated lane.
[View Larger Version of this Image (56K GIF file)]
). 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).
Fig. 4.
Targeted overexpression of PTHrP to chondrocytes in transgenic animals leads to increased Bcl-2 expression and delayed
chondrocyte maturation. (A) Metatarsals of a normal 6-d-old mouse, showing the zonal structures of a growth plate, trabecular and cortical bone, and the presence of a marrow cavity. In contrast (B), the metatarsal of the col II-PTHrP transgenic littermate demonstrates a delay in chondrocyte differentiation, an accumulation of prehypertrophic chondrocytes (as confirmed by intracellular collagen X expression, data not shown), and the complete absence of bone formation. At a higher magnification of the insets in A and B, the progression of chondrocyte differentiation is visible in the normal growth plate: proliferating cells differentiate first into prehypertrophic cells,
and after further enlargement, they finally die at the border to bone formation (compare to Fig. 1) (C). In contrast, the same region in
the metatarsal of the col II-PTHrP transgenic mouse shows that the cells accumulate at the prehypertrophic stage (D). Frozen sections
of the corresponding growth plate regions labeled for Bcl-2 and Bax by immunofluorescence demonstrate the normal pattern of Bcl-2
expression in the growth plate, with highest levels in the zone of prehypertrophic chondrocytes, (E). In contrast, in the col II-PTHrP
transgenic animals, not only the number of chondrocytes expressing Bcl-2 but also the level of Bcl-2 expression are markedly increased
(F); panels G and H demonstrate that Bax expression is not affected in the col II-transgenic animals (H) and is comparable to normal
levels in prehypertrophic chondrocytes (G).
[View Larger Version of this Image (101K GIF file)]
Fig. 5.
Bcl-2 directly affects skeletal development and endochondral bone formation. bcl-2 knockout mice exhibit accelerated chondrocyte differentiation. (A) While under normal conditions at day 1, the foot middle phalanx is still a cartilaginous
model. (B) In bcl-2 knockout littermates, vascular invasion, progressive replacement of cartilage by bone, and homing of bone
marrow cells has already taken place. (D) This acceleration of
chondrocyte differentiation leads to a reduction in the growth
plate thickness, mostly in the proliferative zone (P), shown here
for the distal metatarsal bone of a 6-d-old bcl-2 knockout mouse
as compared to a 6-d-old normal control (C). Even more striking
is the almost complete loss of cartilage at the proximal end of the
metatarsal bones (day 6) in the bcl-2 /
(F), while in the normal
mice, the present zone of chondrocytes is still a resource of bone
formation (E). Consequently, bcl-2 knockout mice are markedly
smaller than control littermates (G) (contact x-ray of 60-d-old
mice; on the right bcl-2
/
, on the left normal control littermate), and the absence of Bcl-2 leads to a significant decrease
(15%-20%) in overall bone length (H). Significant levels of P < 0.05, Student's t test, are indicated by asterisks.
[View Larger Versions of these Images (117 + 48 + 18K GIF file)]
Discussion
). 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
).
;
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.
; 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.
; 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
).
). 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.
;
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
).
) 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.
; 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.
Received for publication 21 August 1996 and in revised form 30 October 1996.
Please address all correspondence to Roland Baron, Yale University School of Medicine, PO Box 208044, 333 Cedar Street, New Haven, CT 06520-8044. Tel.: (203) 785-4150; Fax: (203) 785-2744.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.