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Endocrinology 147(8):3761–3768
Copyright © 2006 by The Endocrine Society
doi: 10.1210/en.2005-1672
Inhibition of the Proteasomal Function in Chondrocytes
Down-Regulates Growth Plate Chondrogenesis and
Longitudinal Bone Growth
Shufang Wu and Francesco De Luca
Section of Endocrinology and Diabetes, St. Christopher’s Hospital for Children, Department of Pediatrics, Drexel University
College of Medicine, Philadelphia, Pennsylvania 19134
The proteasome is a large multiprotein complex that processes intracellular proteins functioning as cell cycle regulators and transcription factors. It has been shown that the
chymotryptic component of the proteasome is an important
regulator of osteoblast differentiation and bone formation,
with inhibitors of the proteasome increasing osteoblast differentiation and bone formation. Yet, little is known about the
effects of the proteasomal activity in the growth plate. In the
present study, we cultured rat metatarsal bones in the presence of proteasome inhibitor I (PSI), a known inhibitor of the
chymotrypsin-like activity of the 20S proteasome. PSI sup-
P
ROTEASOMES ARE LARGE multisubunit protease
complexes that selectively degrade intracellular proteins (1–3). The ubiquitin-proteasomal pathway is recognized as the major intracellular mechanism for degradation
of many short-lived proteins, such as cell cycle regulators
and transcription factors. Thus, protein degradation or processing by the ubiquitin-proteasome system has a decisive
impact on cell survival and death.
The unique and distinguishing feature of the proteasome
is the presence of multiple peptidase activities that include
chymotrypsin-like activity, postglutamyl peptidase activity,
and trypsin-like activity. It has been shown that the chymotryptic component of the proteasome is an important regulator of osteoblast differentiation and bone formation, with
inhibitors of the proteasome increasing osteoblast differentiation and bone formation (4, 5).
Inhibitors of the proteasome have also been used to evaluate the role of proteolytic degradation in the activity of
transcription factors involved in growth plate chondrogenesis and longitudinal bone growth. Nuclear factor ␬B (NF␬B) (6 –11) is a family of transcription factors that exist in a
latent form in the cytoplasm bound to inhibitory proteins,
known as inhibitory NF-␬Bs (I␬Bs) (12–14). Proteasome-mediated degradation of I␬Bs leads to the release of the previously bound NF-␬B, which then translocates to the nucleus
and subsequently modulates the expression of important
First Published Online May 4, 2006
Abbreviations: dpc, Days post conception; I␬B, inhibitory nuclear
factor ␬B; NF-␬B, nuclear factor ␬B; PSI, proteasome inhibitor I; TdT,
terminal deoxynucleotidyl transferase.
Endocrinology is published monthly by The Endocrine Society (http://
www.endo-society.org), the foremost professional society serving the
endocrine community.
pressed growth plate chondrocyte proliferation and hypertrophy/differentiation, and induced chondrocyte apoptosis.
All these cellular effects led to reduced metatarsal linear
growth. In cultured chondrocytes, PSI increased the expression of ␤-catenin (a negative regulator of chondrogenesis) and
reduced the DNA binding of nuclear factor ␬B, a transcription
factor that stimulates growth plate chondrogenesis. In conclusion, our findings suggest that the proteasomal activity
facilitates growth plate chondrogenesis and, in turn, longitudinal bone growth. (Endocrinology 147: 3761–3768, 2006)
target genes involved in cell growth, survival, adhesion, and
death. These target genes include anti-apoptotic (15) as well
as proapoptotic ones (16), suggesting that the effects of
NF-␬B on cell growth and survival may depend on the cell
type and on the nature of the extracellular stimuli. In chick
embryo, overexpression of I␬B-␣, which blocks NF-␬B activation, results in abnormal limb development (17). Mice
deficient in both the p50 and p52 subunits of NF-␬B have
retarded growth and shortened long bones, suggesting that
NF-␬B may promote growth plate chondrogenesis and longitudinal bone growth (18).
␤-Catenin is another important regulator of chondrogenesis. In the absence of extracellular stimuli, ␤-catenin is degraded intracellularly by the proteasome (19). Once a cell is
stimulated by Wnt proteins (a family of secreted signaling
factors involved in a number of developmental processes)
(20), proteasomal degradation is inactivated and ␤-catenin
accumulates in the cytoplasm. ␤-Catenin is required at an
early stage of development to repress chondrocytic differentiation (21). In addition, inhibition of ␤-catenin degradation by proteasome inhibitor causes de-differentiation of
chondrocytes (22).
In the present study, we cultured whole rat metatarsal
bones in the presence of proteasome inhibitor I (PSI), a
known cell-permeable peptide aldehyde which inhibits the
chymotrypsin-like activity of the 20S proteasome. PSI is one
of the most specific proteasome inhibitors currently available
(8). This compound blocks the proteasomal function without
being toxic either in vivo or in vitro (5, 19, 23–24). We demonstrate that the inhibition of the proteasomal function in
growth plate chondrocytes causes reduced longitudinal bone
growth. Such growth inhibition is mediated by decreased
chondrocyte proliferation and hypertrophy/differentiation,
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Endocrinology, August 2006, 147(8):3761–3768
and by increased chondrocyte apoptosis. We also show that
PSI stabilizes the expression of ␤-catenin and reduces the
NF-␬B binding to DNA, suggesting that the proteasome may
facilitate longitudinal bone growth by promoting NF-␬B and
preventing ␤-catenin activities in growth plate chondrocytes.
Materials and Methods
Organ culture
The second, third, and fourth metatarsal bone rudiments were isolated from Sprague Dawley rat fetuses at 20 d post conception (dpc) and
cultured individually in 24-well plates (25, 26). Each well contained 0.5
ml MEM (Invitrogen, Carlsbad, CA) supplemented with 0.05 mg/ml
ascorbic acid (Sigma, St. Louis, MO), 1 mm sodium glycerophosphate
(Sigma), 0.2% BSA (Sigma), 100 U/ml penicillin, and 100 ␮g/ml streptomycin (Invitrogen), and graded concentrations of PSI (Z-lle-Glu(OtBu)-Ala-Leu-CHO, 0 –100 nm) (Calbiochem, San Diego, CA) (5, 8, 23,
24, 27). Bone rudiments were cultured for 3 d in a humidified incubator
with 5% CO2 in air at 37 C. The medium was changed on d 2.
Measurement of longitudinal growth
The length of each bone rudiment was measured under a dissecting
microscope, using an eyepiece micrometer. The eyepiece micrometer
was calibrated every day by using a 1-mm stage micrometer. To calculate
the metatarsal growth rate, bone length was measured at the beginning
and at the end of the culture period using an eyepiece micrometer in a
dissecting microscope.
[3H]Thymidine incorporation
To assess cell proliferation, we measured [3H]thymidine incorporation into newly synthesized DNA (25). After 3 d of culture, [3H]thymidine was added to the metatarsal culture medium at a concentration of
5 ␮Ci/ml (25 Ci/mmol; Amersham, Piscataway, NJ). Bone rudiments
were incubated for an additional 5 h. At the end of the incubation, all
bones were fixed in 4% phosphate-buffered paraformaldehyde, embedded in paraffin, and cut in 5- to 7-␮m-thick longitudinal sections.
Autoradiography was performed by dipping the slides in Hypercoat
emulsion, exposing them for 4 wk, and then developing them with a
Kodak-D19 developer. Sections were counterstained with hematoxylin.
The labeling index was calculated as the number of [3H]thymidinelabeled cells per grid divided by the total number of cells per grid. The
grid circumscribed a portion of the growth plate zone as viewed through
a ⫻20 objective, and generally contained an average of 80 cells. For each
growth plate zone, the fraction of labeled cells in three distinct grid
locations was calculated and averaged. The labeling index (number of
labeled cells divided by number of total cells) was determined separately
for the epiphyseal zone and for the proliferative zone. For each treatment
group, we sampled five bones and analyzed both growth plates of each
of three longitudinal sections per bone (15 bone sections per group). All
determinations were made by the same observer blinded to the treatment category.
To assess proliferation in cultured chondrocytes, subconfluent cell
cultures in 24-well plates were treated for 24 h with DMEM containing
10% FCS and indicated concentration of PSI. Then, 2.5 ␮Ci/well of
[3H]thymidine (Amersham) was added to the culture medium for an
additional 3 h. Cells were released by trypsin and collected onto glass
fiber filters. Incorporation of [3H]thymidine was measured by liquid
scintillation counting.
Quantitative histology
At the end of the culture period, metatarsals were fixed overnight at
4 C in 4% paraformaldehyde in PBS, then dehydrated through a series
of ethanol washes, cleared in xylene, and finally embedded in paraffin.
Three longitudinal, 5- to 7-␮m-thick sections were obtained from each
metatarsal bone and stained with toluidine blue. Briefly, the sections
were dipped into 0.1% toluidine blue for 30 sec and then rinsed thoroughly with tap water for 1 min. From each of the three sections, we
measured the height of the epiphyseal, proliferative, and hypertrophic
zones, and of the primary ossification center, and calculated the average
Wu and De Luca • Proteasomal Function in the Growth Plate
value. The height of the epiphyseal zone was measured from the distal
edge of the metatarsal bone to the upper margin of the first row of
flattened cells. The height of the proliferative zone was measured from
the upper margin of the first row of flattened cells to the upper margin
of the first row of hypertrophic cells. The height of the hypertrophic zone
was measured from the upper margin of the first row of hypertrophic
cells to the edge of the primary ossification center.
All quantitative histology was performed by a single observer
blinded to the treatment category.
In situ cell death
At the end of the culture period, metatarsal bones were fixed in 4%
phosphate-buffered paraformaldehyde, embedded in paraffin, and cut
in 5- to 7-␮m-thick longitudinal sections. From each bone, three sections
parallel to the long axis of the bone were obtained. Apoptotic cells in the
growth plate were identified by terminal deoxynucleotidyl transferase
(TdT)-mediated deoxyuridine triphosphate nick end labeling, according
to the manufacturer’s instructions (TdT-FragEL kit; Oncogene Research
Products, Boston, MA) with slight modifications (deparaffinized and
rehydrated sections were treated with proteinase K for 10 min instead
of 20 min) (28). A positive control was generated by covering the entire
tissue section with 1 ␮g/␮l DNase I in 1⫻ TBS/1 mm MgSO4 for 20 min
after proteinase K treatment, while a negative control was generated by
substituting dH2O for the TdT in the reaction mixture. All other steps
were performed as described above (data not shown).
Apoptosis was quantitated by determining the apoptotic index (calculated as the number of apoptotic cells per grid divided by the total
number of cells per grid). In each growth plate, the apoptotic index was
calculated separately in three distinct grid locations of the growth plate,
and then averaged. For each treatment group, we sampled five to six
bones and analyzed both growth plates of each of three longitudinal
sections per bone (15–18 bone sections per group). All determinations
were made by the same observer blinded to the treatment category.
In situ hybridization
Metatarsals were fixed overnight in 4% paraformaldehyde at 4 C, then
dehydrated in ethanol and embedded in paraffin. Sections (5 ␮m-thick)
were hybridized to 35S-labeled Col10a1 antisense riboprobes. Slides
were exposed to photographic emulsion at 4 C for 4 d, then developed,
fixed, and cleared. Sections were counterstained with hematoxylin and
viewed using a light microscope. Sections hybridized with a labeledsense Col10a1 riboprobe were used as negative controls. The mouse type
X collagen (Col10a1) probe [a gift from Dr. Bjorn Olsen (Harvard Medical
School, Boston, MA] was a 650-bp HindIII fragment containing 400 bp
of noncollagenous (NC1) domain and 250 bp of 3⬘-untranslated sequence of the mouse Col10a1 gene in pBluescript (29).
RT-PCR
At the end of the culture period, total RNA was extracted from fifteen
rat metatarsal bones per group or from cultured chondrocytes treated
without or with PSI (10 and 100 nm), using the QIAGEN RNeasy Mini
kit (QIAGEN Inc., Valencia, CA). Primers specific for rat collagen X (5⬘
primer, 5⬘-ATATCCTGGGGATCCAGGTC-3⬘; 3⬘ primer, 5⬘-TGGGTCACCCTTAGATCCAG-3⬘; product size 241 bp), rat CHOP (5⬘ primer,
5⬘-AGCTGAGTCTCTGCCTTTCG-3⬘; 3⬘ primer, 5⬘-AGGTGCTTGTGACCTCTGCT-3⬘; product size 221 bp), and rat ␤-catenin (5⬘ primer,
5⬘-GATCATAGACAATGACATGGAGGAC-3⬘; 3⬘ primer, 5⬘-GCCATACAACTGTAAAAATGGTTTC-3⬘; product size 351 bp) were used. The
housekeeping gene ␤-actin (5⬘ primer, 5⬘-CTGACAGACTACCTCATGAAGATCC-3⬘; 3⬘ primer, 5⬘-CATAGAGGTCTTTACGGATGTCAAC3⬘; product size 330 bp) was used as normalization control. The recovered RNA was further processed using 1st Strand cDNA Synthesis kit
for RT-PCR (AMV) (Roche Diagnostics Corp., Indianapolis, IN) to produce cDNA. One microgram of total RNA and 1.6 ␮g of oligo-p(dT)15
primer were incubated for 10 min at 25 C, followed by incubation for 60
min at 42 C in the presence of 20 U AMV Reverse Transcriptase and 50
U RNase inhibitor in a total 20-␮l reaction. The cDNA products were
directly used for PCR or stored at ⫺80 C for later analysis. The reaction
(100 ␮l total volume) was performed using a PerkinElmer GeneAmp
PCR system 9600 in the presence of 20 pmol primers, 20 nmol dNTP, 150
Wu and De Luca • Proteasomal Function in the Growth Plate
Endocrinology, August 2006, 147(8):3761–3768
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nmol MgCl2, and 1⫻ PCR buffer (Expand High Fidelity PCR buffer;
Roche Molecular Biochemicals, Mannheim, Germany) and 2.5 U Expand
High Fidelity DNA polymerase (Roche Molecular Biochemicals). The
conditions for amplification were 2 min 30 sec at 96 C, followed by 35
cycles of denaturation for 45 sec at 96 C, annealing for 1 min at 55 C,
elongation for 1 min 30 sec at 72 C, and finally, extension for 10 min at
72 C. PCR products were separated by electrophoresis in a 2% agarose
gel with ethidium bromide (1.5 ␮g/ml).
Chondrocyte culture
The primary ossification center of the metatarsal bone, along with the
adherent soft tissues, was carefully dissected off before digestion under
a dissecting microscope. The cartilaginous ends of the metatarsal rudiments were rinsed in PBS and then incubated in 0.2% trypsin at 37 C for
1 h and 0.2% collagenase for 3 h. Cell suspension was aspirated repeatedly and filtered through a 70-␮m cell strainer, rinsed first in PBS then
in serum-free DMEM, and counted.
Chondrocytes were seeded in 100-mm dishes at a density of 4 ⫻ 106
cells/10 ml in DMEM with antibiotics, 50 ␮g/ml ascorbic acid, and 10%
FBS. The culture medium was changed at 72-h intervals. Once cells
reached 70 – 80% confluence, graded concentrations of PSI (0 –100 nm)
were added to the medium and cells were incubated up to 24 h before
cytoplasmic and nuclear protein were extracted. To confirm the chondrogenic phenotype, we studied the expression of type I and type II
collagen (both by immunocytochemistry and Western blot, data not
shown) in a subset of cells isolated from the cartilaginous portion of the
metatarsal bone. Cells were cultured only if at least 95% of the cells
studied were type II collagen positive and type I collagen negative.
Western blot
FIG. 2. Effect of PSI on collagen X expression in metatarsal bones. A,
Representative normal growth plate. EZ, Epiphyseal zone; PZ, proliferative zone; HZ, hypertrophic zone. B and C, Collagen X mRNA
expression was detected in growth plate chondrocytes of control (A)
and 10 nM PSI-treated (B) metatarsal bones by in situ hybridization.
Black punctate staining (arrow) indicates collagen X mRNA expression in the growth plate hypertrophic zone (HZ). D, RT-PCR analysis
of collagen X mRNA expression in metatarsal bones treated without
or with PSI (10 nM). Total RNA was extracted from the cartilaginous
portions of 15 metatarsal bones treated without or with PSI and then
reverse-transcribed to cDNA. The housekeeping gene ␤-actin was
used as normalization control. PCR products were separated by electrophoresis in a 2% agarose gel with ethidium bromide.
Whole cell lysates prepared from cultured chondrocytes treated with
PSI (0 –100 nm) for 24 h were solubilized with 1% SDS sample buffer and
electrophoresed on a 4 –15% SDS-PAGE gel (Bio-Rad, Richmond, CA).
Proteins were transferred onto a nitrocellulose membrane and were
probed with the following primary antibodies: rabbit polyclonal anti-
bodies against I␬B-␣, I␬B-␤, and caspase III respectively (Santa Cruz
Biotechnology Inc., Santa Cruz, CA). The blots were developed using a
horseradish-peroxidase-conjugated polyclonal goat antirabbit IgG antibody and enhanced chemiluminescence system (Amersham). The protein size was confirmed by molecular weight standards (Invitrogen).
Apoptosis assay
Apoptotic cell death was quantified by a flow cytometric assay based
on the number of cells with fragmented DNA. Cultured chondrocytes
were treated with PSI (0 –100 nm) for 24 h before being harvested by
centrifugation and fixed in 80% ethanol that had been precooled to ⫺20
C. The cells were resuspended in PBS containing 50 ␮g/ml propidium
iodide, 0.1% Nonidet P-40, and 100 ␮g/ml RNase (Sigma), and incubated for 1 h. The number of cells with fragmented DNA was then
quantified using 1–2 ⫻ 104 cells on a FACSort flow cytometer with the
CellQuest analysis program (BD Biosciences).
FIG. 1. Effects of PSI on metatarsal longitudinal growth and growth plate histology. A, Fetal rat metatarsals (20 dpc) were cultured for 3 d
in serum-free MEM containing PSI (0 –100 nM, n ⫽ 34 –38 per group). Bone length was measured at the beginning and at the end of the culture
period using an eyepiece micrometer in a dissecting microscope. B, Fetal rat metatarsals (20 dpc, n ⫽ 8 –9 per group) were cultured for 3 d in
serum-free MEM without or with 10 nm PSI. After routine histological processing, the bones were embedded in paraffin, and 5- to 7-␮m-thick
longitudinal sections were obtained. The heights of the growth plate epiphyseal, proliferative, and hypertrophic zones, and the length of the
primary ossification center (OC) were measured by a single observer blinded to the treatment regimen.
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Wu and De Luca • Proteasomal Function in the Growth Plate
FIG. 3. [3H]Thymidine incorporation and in situ cell death in the metatarsal growth plate. Fetal rat metatarsals (20 dpc) were cultured for 3 d
in serum-free MEM without (A) or with 10 nM PSI (B). A and B, After 3 d in culture, [3H]thymidine was added to the culture medium at a final
concentration of 5 ␮Ci/ml. Bone rudiments were incubated for an additional 5 h. At the end of the incubation, all bones were fixed in 4%
phosphate-buffered paraformaldehyde, embedded in paraffin, and cut in 5- to 7-␮m-thick longitudinal sections. Autoradiography was performed
by dipping the slides in Hypercoat emulsions (Amersham) exposing them for 4 wk, and developing with a Kodak-D19 developer. Sections were
counterstained with hematoxylin. A representative [3H]thymidine-labeled cell is indicated by the arrow. EZ, Epiphyseal zone; PZ, proliferative
zone. C, The labeling index was calculated as the number of [3H]thymidine-labeled cells per grid divided by the total number of cells per grid.
The grid circumscribed a portion of the growth plate zone as viewed through a ⫻20 objective, and generally contained an average of 80 cells.
For each growth plate zone, the fraction of labeled cells in three distinct grid locations was calculated and averaged. The labeling index was
determined separately for the epiphyseal zone and for the proliferative zone. For each treatment group, we sampled five bones and analyzed
both growth plates of each of three longitudinal sections per bone (15 bone sections per group). All determinations were made by the same
observer blinded to the treatment category. D and E, At the end of the culture period, metatarsal bones were fixed in 4% phosphate-buffered
paraformaldehyde and embedded in paraffin. Longitudinal sections 5–7 ␮m thick were obtained and treated with TdT-mediated deoxy-UTP
nick end labeling assay. Representative photomicrographs showing apoptosis in the metatarsal growth plate. Arrow indicates an apoptotic
chondrocyte. F, The apoptotic index was calculated as the number of apoptotic cells per grid divided by the total number of cells per grid. In
each growth plate, the apoptotic index was calculated separately in three distinct grid locations of the growth plate, and then averaged. For
each treatment group, we sampled five to six bones and analyzed both growth plates of each of three longitudinal sections per bone (15–18 bone
sections per group). All determinations were made by the same observer blinded to the treatment category.
EMSA
Nuclear protein extract was prepared from cultured chondrocytes.
Briefly, chondrocytes were washed and scraped in PBS, resuspended in
buffer (10 mm HEPES, pH 7.9, 0.1 mm EDTA, 0.1 mm EGTA, 10 mm KCl,
2 mm MgCl2, 1 mm phenylmethylsulfonyl fluoride, 10 ␮g/ml leupeptin,
10 ␮g/ml aprotinin, 0.15 mm spermine, 0.5 m spermidine, 1 mm dithiothreitol) for 15 min on ice and lysed with 0.5% Nonidet P-40. The nuclei
were pelleted by centrifugation, resuspended in extraction buffer (20 mm
HEPES, pH 7.9, 0.5 m NaCl, 1 mm EDTA, 1 mm EGTA, 0.5% Nonidet
P-40, 1 mm phenylmethylsulfonyl fluoride, 10 ␮g/ml leupeptin, 10
␮g/ml aprotinin, 0.15 mm spermine, 0.5 mm spermidine, 1 mm dithiothreitol), and rotated at 4 C for 40 min. After centrifugation, the supernatant containing nuclear proteins was collected, analyzed by Bradford,
and stored at ⫺80 C.
NF-␬B binding activity was studied by using double-stranded oligonucleotides (5⬘-AGT TGA GGG GAC TTT CCC AGG C-3⬘; Promega,
Madison, WI), corresponding to the consensus NF-␬B binding site). The
oligonucleotide probe was prepared by phosphorylation with T4
polynucleotide kinase (Promega) in the presence of [␥-32P]ATP (Amersham), followed by inactivation of the kinase by adding 1 ␮l of 0.5 m
EDTA. Nuclear proteins (10 ␮g) were preincubated for 10 min in NF-␬B
binding buffer (Promega). Radioactively labeled oligonucleotide was
added and incubated for 30 min at room temperature. The complexes
were then subjected to 6% nondenaturing acrylamide gel, electrophoresed, and analyzed by autoradiography. To assess the specificity of the
NF-␬B-DNA binding, competition experiments were performed by using excess (10⫻) of unlabeled NF-␬B oligonucleotides and nonspecific
competitor DNA sequence (SP1).
Statistics
All data are expressed as the mean ⫾ se. Statistical significance was
determined by t test or by ANOVA.
Results
Effects of PSI on longitudinal bone growth and growth
plate chondrogenesis
During the 3 d of the culture period, higher concentrations
of PSI (10 and 100 nm) induced a significant suppression of
the metatarsal longitudinal growth (n ⫽ 34 –38 per group,
P ⬍ 0.001; Fig. 1A). To rule out any toxic effect of PSI, two
subsets of metatarsal bones (control and 10-nm PSI groups)
were cultured for 7 d, with the addition of PSI to the medium
Wu and De Luca • Proteasomal Function in the Growth Plate
in the latter group being discontinued after d 3 of culture.
Both control and previously PSI-treated bones continued to
grow during the additional 4 d in culture, with no significant
difference between the two groups with respect to the cumulative growth achieved between d 3–7 (n ⫽ 32 per group;
121.6 ⫾ 9.0 vs. 114.8 ⫾ 12.0 ␮m, control vs. PSI, P ⬍ 0.538).
Because the rate of longitudinal bone growth depends
primarily on the rate of growth plate chondrogenesis, we
evaluated the effects of PSI on chondrocyte hypertrophy/
differentiation and chondrocyte proliferation. To assess
chondrocyte hypertrophy, we examined the bone rudiments
histologically. After 3 d in culture, treatment with 10 nm PSI
(the lowest growth-inhibiting concentration) reduced the
height of the growth plate hypertrophic zone (n ⫽ 8 –9 per
group, P ⬍ 0.001; Fig. 1B). In the metatarsal growth plate, the
epiphyseal zone is characterized by small and rounded cells,
irregularly arranged in the cartilage matrix. The proliferative
zone comprises cells with a flattened shape arranged in columns parallel to the longitudinal axis of the bone. In the
hypertrophic zone, large cells (defined by a height ⱖ9 ␮m)
form a layer adjacent to the calcified region of the metatarsal
bone, the primary ossification center (Fig. 2A). We then evaluated the effects of PSI on chondrocyte differentiation by
assessing the expression of collagen X (a marker of chondrocyte differentiation) in the growth plate by in situ hybridization. In the control metatarsal bones, type X collagen
mRNA expression was primarily observed in the hypertrophic zone of the growth plate (Fig. 2B). Treatment with PSI
resulted in a dramatic decrease of such expression (Fig. 2C).
Consistent with the morphological findings, PSI (10 nm)
caused a marked decrease of type X collagen mRNA level by
RT-PCR (Fig. 2D).
To determine the effects of PSI on chondrocyte proliferation, we examined the in situ [3H]thymidine incorporation
into the bone rudiments at the end of the culture period. PSI
(10 nm) significantly decreased 3H thymidine incorporation
into the growth plate epiphyseal and proliferative zones
(representative sections of control and PSI-treated bones, Fig.
3, A and B; labeling index, n ⫽ 15 bone sections per group,
P ⬍ 0.01; Fig. 3C). Consistent with these findings, treatment
with PSI (10 nm) significantly reduced the height of the
growth plate proliferative zone (n ⫽ 8 –9 per group, P ⬍
0.001; Fig. 1B).
To further confirm the effect of PSI on chondrocyte proliferation and differentiation, we evaluated [3H]thymidine
incorporation and collagen X mRNA expression in cultured
chondrocytes isolated from metatarsal bones. Ten and 100
nm PSI reduced [3H]thymidine incorporation (P ⬍ 0.01; Fig.
4A) as well as collagen X expression (Fig. 4B), both findings
consistent with those observed in the whole metatarsal bone.
Effects of PSI on chondrocyte apoptosis
In light of the regulatory role of the proteasome on apoptosis in other cell types, we evaluated the effects of PSI on
growth plate chondrocyte apoptosis. PSI (10 nm) significantly induced apoptosis in the metatarsal chondrocytes
(representative sections; Fig. 3, D and E. Apoptotic index, n ⫽
15–18 bone sections per group, P ⬍ 0.01; Fig. 3F). We then
investigated the effects of PSI on cultured chondrocyte ap-
Endocrinology, August 2006, 147(8):3761–3768
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FIG. 4. Effects of PSI on cultured chondrocyte proliferation and differentiation. A, Cultured chondrocytes were treated without or with
PSI (10 and 100 nM) for 24 h. Then, 2.5 ␮Ci/well of [3H]thymidine
(Amersham) was added for an additional 3 h. Cells were released by
trypsin and collected onto glass fiber filters. Incorporation of [3H]thymidine was measured by liquid scintillation counting. Data were
expressed as percentage of control. B, RT-PCR analysis of collagen X
mRNA expression in cultured chondrocytes treated without or with
PSI (10 and 100 nM). Total RNA was extracted from cultured chondrocytes treated without or with PSI and then reverse-transcribed to
cDNA. The housekeeping gene ␤-actin was used as normalization
control. PCR products were separated by electrophoresis in a 2%
agarose gel with ethidium bromide.
optosis. Ten and 100 nm PSI significantly induced chondrocyte apoptosis in a dose-dependent manner (P ⬍ 0.05; Fig.
5A). We then analyzed the effects of PSI on caspase III, an
intracellular mediator of apoptosis. Consistent with the morphological findings aforementioned, PSI induced caspase III
protein expression in cultured chondrocytes (Fig. 5B).
To determine whether the inhibition of chondrocyte proliferation and differentiation and the induction of apoptosis
resulted from a nonspecific cell oxidative injury, we analyzed
the effects of PSI on the mRNA expression of CHOP (a known
marker of endoplasmic reticulum stress) in cultured chondrocytes. Neither 10 nm PSI nor 100 nm PSI up-regulated
CHOP mRNA expression, assessed by RT-PCR (Fig. 5C).
Effects of PSI on I␬B-␣ and I␬B-␤ degradation
Because the proteasome facilitates the activation of NF-␬B
by inducing the degradation of I␬Bs, we studied the effects
of PSI on I␬B-␣ and I␬B-␤ (two cytoplasmic proteins that
inhibit NF␬B translocation to the nucleus) protein levels in
chondrocytes isolated from metatarsal growth plates. After
24 h of culture, 10 and 100 nm PSI both increased I␬B-␤ level,
whereas no significant changes were found in I␬B-␣ protein
level (Fig. 6A).
Effects of PSI on NF-␬B activation
To determine whether the PSI-mediated increased expression of I␬B-␤ led to a decreased NF-␬B activation, we studied
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Wu and De Luca • Proteasomal Function in the Growth Plate
FIG. 5. Effects of PSI on cultured chondrocyte apoptosis, caspase III activation, and CHOP mRNA expression. A, Cultured chondrocytes were
treated without or with PSI (10 and 100 nM) for 24 h before they were harvested by centrifugation and fixed in 80% ethanol that had been
precooled to ⫺20 C. Apoptotic cells were determined by flow cytometric analysis. The data represent means ⫾ SE from four independent
experiments. B, Cultured chondrocytes were treated without or with PSI (10 and 100 nM) for 24 h, harvested, lysed, electrophoresed, and
immunoblotted for caspase III and the loading control, actin. A typical experiment result was presented. More than five independent experiments
were performed with similar results. C, Total RNA was extracted from cultured chondrocytes treated without or with PSI (10 and 100 nM) for
24 h, and then reverse-transcribed to cDNA. The housekeeping gene ␤-actin was used as normalization control. PCR products were separated
by electrophoresis in a 2% agarose gel with ethidium bromide.
the binding of NF-␬B to DNA by performing EMSA. Chondrocytes isolated from metatarsal growth plates were cultured up to 24 h with or without PSI, and nuclear extracts
were then prepared. Labeled oligonucleotides containing a
NF-␬B consensus sequence were incubated with chondrocyte nuclear extracts, leading to the formation of a proteinDNA complex. Although 100 nm PSI inhibited formation of
the NF-␬B-DNA complex both at 6 and 24 h of culture, 10 nm
PSI did so only at 24 h. To confirm specificity, NF-␬B-DNA
binding was competed out with a NF-␬B cold probe but not
with the SP1 cold probe. (Fig. 6B).
Effects of PSI on ␤-catenin expression
To analyze the effects of PSI on ␤-catenin expression, we
isolated total RNA from chondrocytes cultured without or
with PSI (10 and 100 nm). After 24 h in culture, PSI caused
a concentration-dependent increase of ␤-catenin mRNA expression, assessed by RT-PCR (Fig. 6C).
Discussion
In cultured fetal rat metatarsal bones, PSI caused a concentration-dependent suppression of longitudinal bone
growth. Because the rate of longitudinal bone growth depends primarily on the rate of growth plate chondrogenesis,
we evaluated the effects of PSI on the two main processes
responsible for growth plate formation, chondrocyte proliferation, and chondrocyte hypertrophy/differentiation. PSI
suppressed both growth plate chondrocyte proliferation (assessed by [3H]thymidine incorporation and histology) and
chondrocyte hypertrophy/differentiation (assessed by collagen X expression and histology). Moreover, PSI induced
chondrocyte apoptosis both in the metatarsal growth plate
and in cultured growth plate chondrocytes. In a recent study,
it has been shown that a variety of signals may induce
endoplasmic reticulum stress in both primary and immortalized chondrocytes, resulting in loss of differentiation, impaired cell growth, and apoptosis (30). The lack of any PSImediated effect on the expression of CHOP (a marker of
endoplasmic reticulum stress) in chondrocytes suggests that
proteasomal inhibition leads to a reduced growth plate chondrogenesis through the impaired activation/expression of
growth-promoting transcription factors rather than by inducing a nonspecific cell oxidative response.
The ubiquitin-proteasomal pathway is recognized as the
major intracellular mechanism for degradation of many proteins (1–3). Several of the substrates of the ubiquitin-dependent proteasomal pathway are critical for cell proliferation
and programmed cell death. Examples of proteasomal involvement in proliferative processes are the regulation of the
cell cycle through degradation of cyclins, cyclin-dependent
kinases, and their inhibitors (31, 32), the regulation of oocyte
maturation (33) and of embryonic cell cycle progression (34).
In addition, the proteasome degrades proapoptotic as well as
antiapoptotic proteins (35, 36), with its effects on cell survival
depending on the proliferative state of a cell and on the cell
type. Increasing evidence indicates that altered proteasomal
function results in human diseases, such as cancer and neurodegenerative and myodegenerative disorders characterized by an imbalance between proliferation and apoptosis
(37, 38).
The proteasome appears to play an important regulatory
role in bone biology. ␤-Catenin, a negative regulator of chon-
Wu and De Luca • Proteasomal Function in the Growth Plate
Endocrinology, August 2006, 147(8):3761–3768
3767
recently demonstrated that two specific inhibitors of NF-␬B
activation (pyrrolidinethiocarbamate and BAY11-7082) suppress metatarsal longitudinal growth and growth plate chondrogenesis (manuscript in preparation). In our study, the
evidence of a PSI-mediated increased I␬B-␤ expression and
decreased NF-␬B nuclear translocation implicates the reduced NF-␬B activity as a mechanism underlying the inhibition of growth plate chondrogenesis. Taken together, our
findings indicate that the proteasomal-dependent protein
degradation in growth plate chondrocytes results in increased proliferation and hypertrophy/differentiation, and
in reduced apoptosis. These effects on chondrocyte function
facilitate growth plate chondrogenesis and longitudinal bone
growth. Lastly, the PSI-mediated stabilization of ␤-catenin
and decreased NF-␬B nuclear translocation in growth plate
chondrocytes suggest that the regulatory role of the proteasome in the growth plate is exerted, at least in part, through
␤-catenin degradation and NF-␬B activation.
Acknowledgments
FIG. 6. A, Effects of PSI on I␬B expression in growth plate chondrocytes. Chondrocytes isolated from metatarsal bone were cultured
without or with PSI (1–100 nM) for 24 h. Cell lysates were analyzed
by Western blotting for I␬B-␣ and I␬B-␤ with antirabbit polyclonal
antibodies. Representative results of three experiments are depicted.
B, Effects of PSI on NF-␬B DNA-binding activity in growth plate
chondrocytes. Chondrocytes isolated from metatarsal bone were cultured with graded concentrations of PSI (0, 10, and 100 nM) at the time
points indicated. A labeled oligonucleotide of NF-␬B consensus element was incubated with the growth plate chondrocyte nuclear extract. DNA binding was analyzed by EMSA. The arrow indicates the
NF-␬B/DNA complex. Representative results of three experiments
are depicted. For specificity, NF-␬B DNA binding was competed out
with a NF-␬B cold probe and with the SP1 cold probe. C, Effects of PSI
on ␤-catenin mRNA expression in growth plate chondrocytes. Total
RNA was extracted from cultured chondrocytes treated without or
with PSI (10 and 100 nM) and then reverse-transcribed to cDNA. The
housekeeping gene ␤-actin was used as normalization control. PCR
products were separated by electrophoresis in a 2% agarose gel with
ethidium bromide.
drogenesis, is degraded by the proteasome (19). Previous
evidence indicates that inhibition of ␤-catenin degradation
with proteasome inhibitor causes stabilization of ␤-catenin
and de-differentiation of chondrocytes (22). In addition,
transgenic mice overexpressing ␤-catenin exhibit several
skeletal defects, with their growth plates completely disorganized and failing to undergo endochondral ossification
(39). Consistent with these findings, in our study, we have
demonstrated an increased expression of ␤-catenin in chondrocytes treated with PSI.
NF-␬B is one of the most intensely studied eukaryotic
transcription factors. In the cytoplasm, NF-␬B is bound to
proteins called I␬Bs. Upon cellular stimulation by inflammatory cytokines, viral proteins, and growth factors, the I␬Bs
are degraded by the proteasome, and NF-␬B translocates to
the nucleus, where it activates the expression of multiple
target genes. Proteasome inhibitors have been shown to prevent the activation of NF-␬B in several cell types (6 –11, 40,
41). Interestingly, NF-␬B knockout mice exhibit retarded
growth, shortened long bones, and a significant decrease in
growth plate chondrocyte proliferation (18). We have also
Received December 30, 2005. Accepted April 27, 2006.
Address all correspondence and requests for reprints to: Francesco De
Luca, M.D., St. Christopher’s Hospital for Children, Erie Avenue at Front
Street, Philadelphia, Pennsylvania 19134. E-mail: francesco.deluca@
drexel.edu.
S.W. has nothing to declare. F.D.L. has received lecture fees from
Pfizer Inc.
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