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Endocrinology Vol. 147, No. 8 3761-3768
Copyright © 2006 by The Endocrine Society

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

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{at}drexel.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 suppressed 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 ß-catenin (a negative regulator of chondrogenesis) and reduced the DNA binding of nuclear factor {kappa}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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROTEASOMES ARE LARGE multisubunit protease complexes that selectively degrade intracellular proteins (1, 2, 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 {kappa}B (NF-{kappa}B) (6, 7, 8, 9, 10, 11) is a family of transcription factors that exist in a latent form in the cytoplasm bound to inhibitory proteins, known as inhibitory NF-{kappa}Bs (I{kappa}Bs) (12, 13, 14). Proteasome-mediated degradation of I{kappa}Bs leads to the release of the previously bound NF-{kappa}B, which then translocates to the nucleus and subsequently modulates the expression of important 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-{kappa}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{kappa}B-{alpha}, which blocks NF-{kappa}B activation, results in abnormal limb development (17). Mice deficient in both the p50 and p52 subunits of NF-{kappa}B have retarded growth and shortened long bones, suggesting that NF-{kappa}B may promote growth plate chondrogenesis and longitudinal bone growth (18).

ß-Catenin is another important regulator of chondrogenesis. In the absence of extracellular stimuli, ß-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 ß-catenin accumulates in the cytoplasm. ß-Catenin is required at an early stage of development to repress chondrocytic differentiation (21). In addition, inhibition of ß-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, and by increased chondrocyte apoptosis. We also show that PSI stabilizes the expression of ß-catenin and reduces the NF-{kappa}B binding to DNA, suggesting that the proteasome may facilitate longitudinal bone growth by promoting NF-{kappa}B and preventing ß-catenin activities in growth plate chondrocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
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 µg/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 µCi/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-µm-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]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 x20 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 µCi/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-µm-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 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-µm-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 µg/µl DNase I in 1x 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 µm-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 labeled-sense 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 ß-catenin (5' primer, 5'-GATCATAGACAATGACATGGAGGAC-3'; 3' primer, 5'-GCCATACAACTGTAAAAATGGTTTC-3'; product size 351 bp) were used. The housekeeping gene ß-actin (5' primer, 5'-CTGACAGACTACCTCATGAAGATCC-3'; 3' primer, 5'-CATAGAGGTCTTTACGGATGTCAAC-3'; 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 µg 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-µl reaction. The cDNA products were directly used for PCR or stored at –80 C for later analysis. The reaction (100 µl total volume) was performed using a PerkinElmer GeneAmp PCR system 9600 in the presence of 20 pmol primers, 20 nmol dNTP, 150 nmol MgCl2, and 1x 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 µg/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-µm 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 x 106 cells/10 ml in DMEM with antibiotics, 50 µg/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.

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 µg/ml propidium iodide, 0.1% Nonidet P-40, and 100 µg/ml RNase (Sigma), and incubated for 1 h. The number of cells with fragmented DNA was then quantified using 1–2 x 104 cells on a FACSort flow cytometer with the CellQuest analysis program (BD Biosciences).

Western blot
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 antibodies against I{kappa}B-{alpha}, I{kappa}B-ß, 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).

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 µg/ml leupeptin, 10 µg/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 µg/ml leupeptin, 10 µg/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-{kappa}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-{kappa}B binding site). The oligonucleotide probe was prepared by phosphorylation with T4 polynucleotide kinase (Promega) in the presence of [{gamma}-32P]ATP (Amersham), followed by inactivation of the kinase by adding 1 µl of 0.5 M EDTA. Nuclear proteins (10 µg) were preincubated for 10 min in NF-{kappa}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-{kappa}B-DNA binding, competition experiments were performed by using excess (10x) of unlabeled NF-{kappa}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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
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. 1AGo). 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 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 µm, control vs. PSI, P < 0.538).


Figure 1
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  		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-µm-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.

 
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. 1BGo). 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 ≥9 µm) form a layer adjacent to the calcified region of the metatarsal bone, the primary ossification center (Fig. 2AGo). 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. 2BGo). Treatment with PSI resulted in a dramatic decrease of such expression (Fig. 2CGo). Consistent with the morphological findings, PSI (10 nM) caused a marked decrease of type X collagen mRNA level by RT-PCR (Fig. 2DGo).


Figure 2
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  		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 ß-actin was used as normalization control. PCR products were separated by electrophoresis in a 2% agarose gel with ethidium bromide.

 
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. 3Go, A and B; labeling index, n = 15 bone sections per group, P < 0.01; Fig. 3CGo). 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. 1BGo).


Figure 3
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  		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 µCi/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-µm-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 x20 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 µm 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.

 
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. 4AGo) as well as collagen X expression (Fig. 4BGo), both findings consistent with those observed in the whole metatarsal bone.


Figure 4
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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 µCi/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 ß-actin was used as normalization control. PCR products were separated by electrophoresis in a 2% agarose gel with ethidium bromide.

 
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. 3Go, D and E. Apoptotic index, n = 15–18 bone sections per group, P < 0.01; Fig. 3FGo). We then investigated the effects of PSI on cultured chondrocyte apoptosis. Ten and 100 nM PSI significantly induced chondrocyte apoptosis in a dose-dependent manner (P < 0.05; Fig. 5AGo). 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. 5BGo).


Figure 5
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  		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 ß-actin was used as normalization control. PCR products were separated by electrophoresis in a 2% agarose gel with ethidium bromide.

 
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. 5CGo).

Effects of PSI on I{kappa}B-{alpha} and I{kappa}B-ß degradation
Because the proteasome facilitates the activation of NF-{kappa}B by inducing the degradation of I{kappa}Bs, we studied the effects of PSI on I{kappa}B-{alpha} and I{kappa}B-ß (two cytoplasmic proteins that inhibit NF{kappa}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{kappa}B-ß level, whereas no significant changes were found in I{kappa}B-{alpha} protein level (Fig. 6AGo).


Figure 6
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  		FIG. 6. A, Effects of PSI on I{kappa}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{kappa}B-{alpha} and I{kappa}B-ß with antirabbit polyclonal antibodies. Representative results of three experiments are depicted. B, Effects of PSI on NF-{kappa}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-{kappa}B consensus element was incubated with the growth plate chondrocyte nuclear extract. DNA binding was analyzed by EMSA. The arrow indicates the NF-{kappa}B/DNA complex. Representative results of three experiments are depicted. For specificity, NF-{kappa}B DNA binding was competed out with a NF-{kappa}B cold probe and with the SP1 cold probe. C, Effects of PSI on ß-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 ß-actin was used as normalization control. PCR products were separated by electrophoresis in a 2% agarose gel with ethidium bromide.

 
Effects of PSI on NF-{kappa}B activation
To determine whether the PSI-mediated increased expression of I{kappa}B-ß led to a decreased NF-{kappa}B activation, we studied the binding of NF-{kappa}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-{kappa}B consensus sequence were incubated with chondrocyte nuclear extracts, leading to the formation of a protein-DNA complex. Although 100 nM PSI inhibited formation of the NF-{kappa}B-DNA complex both at 6 and 24 h of culture, 10 nM PSI did so only at 24 h. To confirm specificity, NF-{kappa}B-DNA binding was competed out with a NF-{kappa}B cold probe but not with the SP1 cold probe. (Fig. 6BGo).

Effects of PSI on ß-catenin expression
To analyze the effects of PSI on ß-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 ß-catenin mRNA expression, assessed by RT-PCR (Fig. 6CGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
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 PSI-mediated 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, 2, 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. ß-Catenin, a negative regulator of chondrogenesis, is degraded by the proteasome (19). Previous evidence indicates that inhibition of ß-catenin degradation with proteasome inhibitor causes stabilization of ß-catenin and de-differentiation of chondrocytes (22). In addition, transgenic mice overexpressing ß-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 ß-catenin in chondrocytes treated with PSI.

NF-{kappa}B is one of the most intensely studied eukaryotic transcription factors. In the cytoplasm, NF-{kappa}B is bound to proteins called I{kappa}Bs. Upon cellular stimulation by inflammatory cytokines, viral proteins, and growth factors, the I{kappa}Bs are degraded by the proteasome, and NF-{kappa}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-{kappa}B in several cell types (6, 7, 8, 9, 10, 11, 40, 41). Interestingly, NF-{kappa}B knockout mice exhibit retarded growth, shortened long bones, and a significant decrease in growth plate chondrocyte proliferation (18). We have also recently demonstrated that two specific inhibitors of NF-{kappa}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{kappa}B-ß expression and decreased NF-{kappa}B nuclear translocation implicates the reduced NF-{kappa}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 ß-catenin and decreased NF-{kappa}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 ß-catenin degradation and NF-{kappa}B activation.


   Footnotes
 
S.W. has nothing to declare. F.D.L. has received lecture fees from Pfizer Inc.

First Published Online May 4, 2006

Abbreviations: dpc, Days post conception; I{kappa}B, inhibitory nuclear factor {kappa}B; NF-{kappa}B, nuclear factor {kappa}B; PSI, proteasome inhibitor I; TdT, terminal deoxynucleotidyl transferase.

Received December 30, 2005.

Accepted for publication April 27, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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