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Department of Orthopedics (R.S.G., D.A., T.M.M.), University of California, San Diego School of Medicine, La Jolla, California 92093-0630; and Department of Medicine (K.A.J., D.W.B., A.G., L.J.D., R.T.), Veterans Affairs Medical Center, University of California, San Diego School of Medicine, San Diego, California 92161
Address all correspondence and requests for reprints to: L. J. Deftos, M.D., VAMC, 3350 La Jolla Village Drive, San Diego, California 92161. E-mail: ljdeftos{at}ucsd.edu
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In growth cartilage, the chondrocyte functions that PTHrP can modulate include matrix synthesis and degradation and chondrocyte growth, differentiation, and survival (1, 2, 3, 4, 5, 6). Significantly, foci of chondrocyte proliferation and hypertrophy and an increase in apoptotic chondrocytes are observed in OA cartilage (10). In this context, perichondrial and endochondral chondrocyte expression of PTHrP can promote growth plate chondrocyte proliferation (1, 3) and suppress chondrocyte apoptosis (2). PTHrP also coordinates endochondral chondrocyte differentiation by effects that include suppression of development of chondrocyte hypertrophy (1, 2, 3, 4, 5, 6).
Progressive loss of articular cartilage matrix occurs in OA (10). In this regard, PTHrP seems to modulate cartilage matrix synthesis and degradation. For example, increased accumulation of type II collagen occurs in the hypertrophic zone of the growth plate in PTHrP null mice (1). Suppression of PTHrP expression, using antisense oligonucleotides, induced an increase in type II collagen expression in cultured rat articular chondrocytes (11). Moreover, stable expression of transfected rat PTHrP induced a sustained decrease in type II collagen, aggrecan, and link protein messenger RNA (mRNA) expression in a clonal rat calvarial chondrocytic cell line (3). PTHrP also induced the matrix metalloproteinases (MMPs) MMP-2 and MMP-9 in a PTH/PTHrP receptordependent manner in rat growth plate chondrocytes (6).
Articular chondrocytes, unlike growth plate chondrocytes, do not normally deposit mineral crystals in pericellular matrix (10, 12, 13). However, pathologic mineralization with hydroxyapatite and/or calcium pyrophosphate dihydrate is common in OA, particularly in advanced disease, and could promote disease progression (12, 13). Though endochondral chondrocyte-mediated and osteoblast-mediated mineralization of vascularized bone is not strictly comparable with crystal deposition in avascular articular cartilage, it is noteworthy that PTHrP controls not only the organization but also the extent of endochondral matrix mineralization (1).
Humans express three PTHrP isoforms as a consequence of alternative splicing of exons in the PTHrP gene (1, 14). Human PTHrP isoforms 1–139, 1–141, and 1–173 are differentially regulated at the level of mRNA production (1, 14, 15). Moreover, each isoform polypeptide is endoproteolytically processed into biologically active peptides (1, 14, 15). Importantly, each PTHrP isoform has monobasic and multibasic sites susceptible to proteolysis by certain proprotein-processing proteases, including furin and furin-like subtilisin family proteases, cysteine and aspartyl proteases, and MMPs that act as monobasic- and dibasic-selective endopeptidases (16, 17, 18).
Human PTHrP isoforms each have sizable domains of conservation in comparison with lower mammalian and avian PTHrPs, including the N-terminal PTH/PTHrP receptor-binding domain 1–34 (1, 14). However, the C-terminal 140–173 domain of the PTHrP 1–173 isoform, which is uniquely encoded by a single exon in the human PTHrP gene (14), is comprised of a sequence that may be primate-specific (1, 14).
The PTHrP 1–173 isoform is the least broadly expressed PTHrP isoform by nonmalignant human tissues (14, 15). However, PTHrP 1–173 is selectively expressed by normal human articular chondrocytes in monolayer culture, preferentially employing the GC-rich P2 alternative PTHrP promoter to do so (7). Because the PTHrP 140–173 epitope was abundant in OA cartilage (7), we recently examined the role of the PTHrP 140–173 domain in chondrocyte function. We focused on chondrocyte PPi metabolism (7), because extracellular PPi is constitutively elaborated in substantial amounts by articular chondrocytes, and the PPi production serves as a major physiologic inhibitor of the deposition of hydroxyapatite in articular cartilage (19). Conversely, a marked increase in extracellular PPi in aging and OA cartilage promotes pericellular calcium pyrophosphate dihydrate crystal deposition (12, 19).
Expression of PTHrP 1–173, but neither expression of the C-terminal truncation mutants PTHrP 1–146 and 1–87 nor treatment with a panel of exogenous PTHrP-derived peptides, including PTHrP 140–173, induced a significant decrease in both human immortalized costal chondrocyte and articular chondrocyte extracellular PPi (7). Therefore, PTHrP 1–173, through the 140–173 domain, was previously demonstrated to exert an isoform-specific effect on chondrocytes. In this study, we examined the molecular mechanisms by which the 140–173 domain of PTHrP 1–173 regulates the function of chondrocytes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. For each mutagenesis, we used the
Transformer site-directed mutagenesis kit (CLONTECH Laboratories, Inc., Palo Alto, CA) (20). Resulting sequence
changes were verified by sequencing both complementary DNA (cDNA)
strands. The PTHrP 1–87 and 1–173 constructs and its site-directed
mutants (20) were inserted downstream of a human
promoter/enhancer in a mammalian cytomegalovirus expression vector
(pCMV5) from Dr. J. Habener (Harvard University, Cambridge,
MA), and plasmids were amplified and purified as described
(20).
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Five New Zealand White rabbits (mature, 8–10 months old, with closed epiphysis) were euthanized by a protocol approved by the UCSD Animal Subjects Committee, and femoral condyle and tibial plateau articular cartilage were isolated and primary chondrocytes extracted as described (21). Cells were grown in DMEM/F12 (1:1) supplemented with 10% FBS, 25 µg ascorbic acid, and 50 µg Gentamicin/ml. After filtration through a nylon mesh 70-µm cell strainer, cells were resuspended at 4 x 104 cells/ml media into 10-cm diameter dishes, 6-well plates, or culture slides (Costar, Acton, MA). Only primary cultures (after 5–7 days) were transfected.
Transfection and peptide incorporation
TC28 cells (limited to passages 26–37) were transfected using
LipofectAMINE-PLUS (Life Technologies, Inc., Grand Island,
NY), achieving greater than a 40% transfection efficiency, as
described (19). Primary rabbit articular chondrocytes at
70–80% confluence were transfected using a
DNA/transferrin-poly-L-lysine/liposome technique previously
demonstrated to produce transfection of primary rabbit chondrocytes at
efficiencies more than 70%, as described (22). Purified
wild-type PTHrP peptide 140–173 and mutant PTHrP 140–173, bearing the
missense mutation GQKG at the 147–150 domain (purchased from Genemed
Synthesis (South San Francisco, CA), were introduced into TC28 cells
via permeabilization by a protocol that we first optimized using
radioiodinated human calcitonin (Bachem, Torrance,
CA). In brief, cells were exposed (where indicated), for 30 min, to the
permeabilizing agent lysolecithin (0.003% wt/vol), after which
purified peptides were added for 30 min, followed by replacement with
fresh peptide-free medium. Determination of the extracellular PPi in
the conditioned media from the peptide-treated cells was measured
48 h after peptide incorporation as above, and we confirmed >95%
cell viability using trypan blue exclusion. For initial optimization,
and as an internal control, calcitonin incorporation into the
permeabilized cells was verified by 
-counting of
125I-radiolabeled peptide.
Human articular cartilages and RT-PCR
Specimens of knee hyaline cartilage from patients with advanced
OA were obtained from femoral condyles and tibial plateaus at the time
of joint replacement, under informed consent and with approval of the
protocol by the UCSD Human Subjects Committee, and normal knee
cartilage specimens were obtained at autopsy as described
(19). For RT-PCR, total RNA was isolated from the
cartilages in situ using Trizol (Life Technologies, Inc.) (19), and isoform-specific RT-PCR reactions
for PTHrP and G3PDH, as a control, were performed for 40 cycles as
described (7). Positive RT-PCR controls (also 40 cycles)
for each PTHrP isoform were each from the mRNA of NCI-H727 lung
carcinoid tumor cells.
We also similarly isolated RNA from cells and performed RT-PCR for collagen II expression (vs. G3PDH) in transfected TC28 cells, with semiquantitative densitometric analysis of each specific RT-PCR product (at 30 cycles of amplification) in the photographed agarose gels, using previously described and validated protocols for both the RT-PCR and densitometric analyses (19).
PTHrP immunoassay, quantification of nuclear PTHrP
Cell pellets were extracted in 0.25 M Tris (pH 7.5),
0.25% NP-40, and 0.25 mM EDTA; and immunoassays were
performed using domain-specific antisera to human PTHrP, as previously
described (7).
For nuclear PTHrP quantification, TC28 cells (7.5 x 106) were harvested and washed in PBS. Cell pellets were suspended in 250 µl buffer A [10 mM HEPES (pH 8.0); 1.5 mM MgCl2; 10 mM KCl; 0.5 mM dithiothreitol; 300 mM sucrose; 0.1% NP-40; 1 µg/ml each of pepstatin, antipain, chymostatin, aprotinin; 0.1 µg/ml leupeptin; and 0.5 mM phenylmethylsulfonylfluoride (PMSF)], incubated on ice for 5 min and then centrifuged at 21,000 x g for 15 min to collect nuclear pellets, as previously described (23). The nuclei were washed in 250 µl buffer A, resuspended in 250 µl buffer B [20 mM HEPES (pH 8.0); 20% glycerol; 100 mM KCl; 100 mM NaCl; 0.2 mM EDTA; 0.5 mM PMSF; 0.5 mM dithiothreitol; and 1 µl of 1 µg/ml each of pepstatin, antipain, chymostatin, aprotinin, and 0.1 µg/ml leupeptin] and then sonicated and centrifuged at 21,000 x g for 15 min to collect nuclear protein as previously described (23), which was then studied by immunoassay for PTHrP 38–64, our most sensitive PTHrP immunoassay (7).
Cell proliferation assay
Transfected cells were plated in 96-well cell culture plates
(5,000 cells/well) and incubated at 37 C in their respective complete
media (described above). Cells were allowed to adhere for 18 h,
then washed and incubated in serum-free medium for 6 h, and then
placed in their respective complete media. The fluorogenic
double-stranded DNA binding dye Hoescht H33258 was used to quantify the
cell numbers at 24 h (for TC28 cells) and 72 h (for rabbit
chondrocytes) via scanning in a fluorometric plate reader (excitation
355 nm, emission 460 nm), using a reference standard curve to convert
sample fluorescence values to cell numbers.
PPi metabolism assays
PPi concentrations in conditioned media were determined as
previously, via differential adsorption on activated charcoal of
UDP-D-[6-3H] glucose
(Amersham Pharmacia Biotech, Chicago, IL) from the
PPi-stimulated reaction product 6-phospho
[6-3H] gluconate (7, 19). PPi was
equalized for the DNA concentration in each well, determined
chromogenically after precipitation in perchlorate (7, 19). Measurements of PPi-generating nucleoside triphosphate
pyrophosphohydrolase (NTPPPH) and PPi-degrading alkaline phosphatase
activities were performed as described (7, 19).
Collagen synthesis assay
We incubated transfected cells with 50 µg/ml ascorbate, 25
mM NaPi, and media containing 1% FBS for 24 h at 37
C. Cells were pulsed for 24 h with 5 µg/ml
3H-proline and lysed in 1 M NaCl, 1
mM N-ethylmaleimide, 0.2
mM PMSF, and 0.75 mM EDTA
in water. Lysate was added to collected media, and the extract was
precipitated with 15% trichloroacetic acid and resuspended in
0.05 M Tris-HCl (pH 7.6), 5
mM CaCl2, 2.5
mM N-ethylmaleimide, and 80 U/ml
collagenase. Digestion proceeded for 12 h at 37 C, followed by
reprecipitation with trichloroacetic acid. The percent total protein
was determined before collagenase digestion and compared with percent
collagenase digestible protein and percent incorporated
3H.
Confocal microscopy, immunocytochemistry
TC28 cells were grown on glass coverslips in 6-well cell culture
plates in RPMI 1640 supplemented with 10% FBS and transfected as
above, and incubated further for 48 h, then fixed with 10%
buffered-formalin for 10 min. Cells were then permeabilized for 5 min
with 0.1% Triton X-100 in PBS, then washed in PBS and blocked using
20% FBS, 0.25% gelatin, 0.01% azide in PBS. Mouse anti-PTHrP
109–141 antibody (9H7) (7) was applied (10 µg IgG/ml)
to the wells for 18 h at 4 C. The 9H7 antibody (7)
was employed because of its particularly high signal/noise ratio.
Biotinylated goat antimouse IgG antibody was then applied for 1 h,
and streptavidin-Alexa 488 fluorescent dye conjugate (Molecular Probes, Inc., Eugene, OR.) for 1 h. Coverslips were removed
and mounted on glass slides. Controls were cells incubated with
antibodies preadsorbed with their specific antigen overnight and
irrelevant antibodies. Immunostaining was evaluated using an LSM 510
Inverted Confocal Laser Scanning Microscope with an Argon/Krypton laser
specific for 488-nm fluorescence (Carl Zeiss, New York,
NY).
Fluorescent labeling of PTHrP peptides
PTHrP peptides were labeled with Oregon Green 488 fluorescent
dye (Molecular Probes, Inc.), which has a reactive
succinimidyl ester moiety that reacts efficiently with primary amines
of proteins to form stable dye-protein conjugates. In brief, 100 µg
PTHrP peptide, diluted in 0.1 M sodium bicarbonate buffer
(pH 8.5), was added to 100 µg Oregon Green dye. The mixture was
incubated for 1 h at room temperature. The reaction was stopped by
adding 0.1 ml of 1.5 M hydroxylamine (pH 8.5), which also
was added to promote removal of dye molecules noncovalently attached to
the protein or attached to tyrosine or histidine residues by labile
bonds. The free Oregon Green dye was then removed using a P2
size-exclusion chromatography column (Bio-Rad Laboratories, Inc., Hercules, CA).
Statistical analysis
Unless otherwise indicated, error bars represent
SD. Statistical analysis was performed using the
Student’s t test (paired 2-sample testing for means).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Thus, we directly studied the effects
of PTHrP 1–173 in chondrocyte function. To do so, we expressed PTHrP
in human TC28 cells and primary rabbit articular chondrocytes. Within
each PTHrP isoform, processing motifs facilitate endoproteolytic
cleavage to generate peptides with potentially distinct activities,
subcellular localizations, and secretory patterns (1, 14, 20). Thus, we generated mutants of two potential multibasic
processing sites in the 140–173 domain and four more in the human
PTHrP 1–106 region (Fig. 1
). Using TC28 cells, and immunoassay for
PTHrP 1–34, we confirmed that transfection with each of the expression
plasmids encoding PTHrP 1–173 and the 1–173 site-directed mutants, as
well as the truncation mutant PTHrP 1–87, caused significant increases
in secreted PTHrP, relative to empty plasmid (Fig. 3).
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). Three of the six PTHrP
1–173 site-directed mutants also induced a significant decrease in
extracellular PPi levels in TC28 cells. Importantly, the PTHrP 1–173
construct m5 (bearing mutation of KKKK to GQKG at the 147–150 domain)
induced a marked increase in PPi levels in TC28 cells (Fig. 4). We
tested a more limited number of mutants in rabbit articular
chondrocytes but also observed that the m5 mutant of PTHrP 1–173 had a
unique ability to markedly increase extracellular PPi levels (Fig. 4).
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). In TC28 cells, the ability of
wild-type PTHrP 1–173 to suppress collagen synthesis was shared by the
PTHrP 1–173 mutants, with the exception of the 147–150 mutant m5
(Fig. 5A). In rabbit articular chondrocytes, the m4 and m6 mutants,
unlike wild-type PTHrP 1–173, had no significant effect on collagen
synthesis (Fig. 5B).
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). Using semiquantitative RT-PCR and
densitometric analysis in TC28 cells, we observed a consistent increase
in type II collagen mRNA expression by PTHrP bearing the mutation of
the 147–150 domain, relative to cells transfected with empty plasmid
or wild-type PTHrP 1–173 (Fig. 6).
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). Opposing effects
on rabbit articular chondrocyte proliferation of wild-type PTHrP 1–173
and the 147–150 mutant m5 were observed (Fig. 7), though these effects
were small in comparison with the effects on collagen synthesis and PPi
generation.
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).
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), analogous to
previously observed effects of transfection with wild-type PTHrP
1–173. Conversely, treatment of permeabilized cells with the mutated
140–173 peptide induced a dose-dependent increase in PPi production
(Fig. 8), analogous to effects of transfection of the 147–150 mutant
of PTHrP.
Because the 147–150 motif of PTHrP 1–173 determined intracrine
effects of PTHrP 1–173, we used confocal microscopy, immunocytology,
and immunoassay to determine the ability of wild-type PTHrP 1–173 and
the 147–150 m5 mutant of PTHrP 1–173 to localize in the nucleus,
after plasmid transfection of PTHrP 1–173 constructs in TC28 cells,
which was associated with increased PTHrP production, as demonstrated
in Fig. 3. Control (vector-transfected) cells demonstrated stellate
PTHrP immunostaining in focal regions of the nucleus (Fig. 9). Cells transfected with wild-type
PTHrP 1–173 cDNA demonstrated an increase in diffuse nuclear
localization of PTHrP. No increase in nuclear localization of PTHrP was
detectable by immunocytochemistry in cells after transfection with the
147–150 m5 mutant of PTHrP 1–173 (Fig. 9). Immunoassay for nuclear
PTHrP revealed a significant increase after transfection of wild-type
PTHrP 1–173, relative to transfection of the 147–150 m5 mutant of
PTHrP 1–173 (Fig. 9E). Thus, we tested whether the 140–173 domain of
PTHrP 1–173 (and the tetrabasic 147–150 motif) were directly
modulating nuclear localization. To do so, we fluorescently labeled
wild-type PTHrP 140–173, and PTHrP 140–173 bearing the m5 mutation at
the 147–150 motif, and we then studied nuclear localization of the
peptides in permeabilized TC28 cells. Under these conditions, nuclear
localization of the wild-type 140–173 peptide (but not the m5 mutant
140–173 peptide) was detected in the permeabilized TC28 cells (Fig. 10).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The suppressive effects of wild-type PTHrP 1–173 and the inductive effects of the 147–150 mutant of this PTHrP isoform on collagen synthesis by chondrocytes paralleled respective (and opposing) effects on levels of extracellular PPi. Because expression of wild-type PTHrP 1–173 or the 147–150 mutant did not significantly alter cell-associated NTPPPH (19) or alkaline phosphatase activity, it seems likely that regulation of chondrocyte matrix protein synthesis (24) was a central pathway by which PTHrP 1–173 modulated levels of extracellular PPi. However, it will be of interest to determine whether PTHrP 1–173 also can modulate chondrocyte extracellular PPi levels via effects on plasma membrane PPi efflux (25). Importantly, regulatory effects modulated by the 147–150 domain of PTHrP 1–173 on collagen synthesis and extracellular PPi concentrations (per total cellular DNA) in chondrocytes seemed more striking than effects on proliferation of TC28 cells and rabbit articular chondrocytes modulated by the 147–150 domain of PTHrP 1–173.
In this study, we determined that the 147–150 domain of PTHrP 1–173 functions, in part, by regulating subcellular and nuclear localization of PTHrP 1–173-derived peptides. In this context, biologic effects of PTHrP are mediated both by binding of peptides to membrane receptors (including PTHrP 1–34 binding to the PTH/PTHrP receptor) and by intracrine functions of processed peptides of PTHrP (1, 14). Our results reinforce evidence of 140–173 domain-dependent PTHrP intracrine effects (1, 2, 14, 26). For example, the PTHrP 140–173 domain promotes intracellular retention of PTHrP 1–173, in preference to secretion, in a variety of cells (20, 27), including chondrocytes (7). Here, we observed that the tetrabasic 147–150 motif was required for PTHrP 1–173 nuclear localization. We also demonstrated the nuclear localization in permeabilized TC28 cells of fluorescently labeled wild-type PTHrP 140–173 (but not PTHrP 140–173) mutated at the 147–150 region. Thus, the tetrabasic 147–150 motif of PTHrP 1–173 has the potential to act as a nuclear localization signal (NLS). It will be of interest to determine whether the paired arginines (amino acids 154–155), which also lie in the N-terminal half of the C-terminal PTHrP 140–173 domain, can modulate nuclear localization of PTHrP 1–173 (28, 29, 30).
Studies of nuclear and nucleolar localization of PTHrP isoforms other than 1–173 have revealed that PTHrP nuclear localization occurred at the G1 phase of the cell cycle, mediated by a SV40 large T antigen-like sequence in amino acids 61–94, binding to importin ß, and transport mediated by the GTP-binding protein Ran (28, 29, 30). Nucleolar localization of PTHrP in chondrocytes required the HIV-1 Tat-like 87–107 domain (2). Nuclear export required phosphorylation of Thr (85) by cyclin-dependent kinases CDC2-CDK2, and occurred in a cell-cycle-dependent manner (28, 29, 30).
The function of the highly conserved 87–107 amino acid domain of PTHrP in nuclear localization also has been associated with the ability of human PTHrP 1–139 and rat PTHrP to modulate proliferation and resistance to apoptosis in vascular smooth muscle cells (26) and chondrocytes (2), respectively. Here, the tetrabasic 147–150 motif of the 1–173 isoform imposed a critical mode of regulation of PTHrP 1–173 nuclear localization in chondrocytic cells, suggesting the possibility of concerted regulatory action of more than one NLS in PTHrP 1–173. Synergistic action of more than one NLS has a precedent in FGF3, which, like PTHrP, undergoes nuclear/nucleolar localization and secretion (31).
Our results revealed intracrine functions of the 147–150 GQKG mutant m5 of PTHrP 1–173 that were not simply attributable to a loss of function of wild-type PTHrP 1–173 and that seemed to be modulated, in part, by effects other than nuclear localization. In this context, PTHrP 1–173 mutated at the 147–150 domain induced a marked increase in both collagen synthesis and collagen II mRNA expression. Further investigation will be of interest, to determine the subcellular locus, the signal transduction mechanism, and the full extent of chondrocyte collagens and other secreted matrix constituents involved in matrix synthetic stimulatory activity of the 147–150 mutant of PTHrP 1–173.
The PTHrP 1–173 isoform may be human-specific (or primate-specific) via the exon encoding 140–173 (1, 14). However, existence of a PTHrP 1–173 homologue has not been excluded in lagomorphs such as rabbits. Interestingly, functional effects of wild-type and mutant PTHrP 1–173 expression on collagen synthesis and extracellular PPi in human and rabbit chondrocytic cells were comparable. Thus, it will be of interest to determine whether rabbits and humans comparably recognize the C-terminal 140–173 domain of PTHrP 1–173.
In conclusion, physiologic and pathologic changes in both PTHrP 1–173 isoform-specific expression and processing may have unique regulatory effects on chondrocyte synthetic function, mineralizing capacity and growth. The differential abilities of wild-type PTHrP 1–173, and PTHrP 1–173 mutated at the 147–150 tetrabasic motif to modulate collagen synthesis and the concentration of extracellular PPi, may prove useful for developing new approaches to modulate endochondral growth, cartilage matrix calcification, and articular cartilage repair in vivo.
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Footnotes |
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2 Contributed equally to this work.
Received May 5, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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