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Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114
Address all correspondence and requests for reprints to: Jun Guo, M.D., Ph.D., Endocrine Unit, Massachusetts General Hospital, 50 Blossom Street, Boston, Massachusetts 02114. E-mail: guo{at}helix.mgh.harvard.edu
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Abstract |
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In hC1–5 cells, PTH and cAMP analog, but not phorbol ester, inhibited both ALP and mineralization. PTH1R-null hC1–5 subclones were isolated by in vitro selection and then reconstituted by stable transfection with wild-type PTH1Rs or mutant (DSEL) PTH1Rs defective in PLC activation. ALP and mineralization were inhibited similarly via both forms of the receptor. These results indicate that PLC activation is not required for PTH1R regulation of mineralization or ALP in hypertrophic chondrocytes and are consistent with a major role for cAMP in regulating differentiation of hypertrophic chondrocytes.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Chondrocytes have long been recognized as skeletal target cells for PTH (16, 17) and PTHrP (9, 11). The importance of the PTH1R in cartilage and bone development was highlighted by the demonstration that ablation of the gene for either PTHrP or the PTH1R in mice leads to a neonatal-lethal phenotype that includes profound abnormalities in endochondral growth plates, most notably an acceleration of chondrocytic differentiation with accompanying shortening of the columns of proliferating chondrocytes, premature mineralization, and early formation of primary spongiosa (9, 10). Overexpression of PTHrP in chondrocytes, in contrast, delays their differentiation and slows endochondral bone formation (13). Similarly, transgenic expression of a constitutively active PTH1R in chondrocytes slows their differentiation, culminating in delayed cell death and chondrocyte persistence in the marrow space of long bones (14). Expression of the PTH1R messenger RNA (mRNA) varies markedly among chondrocytic cells that populate the various strata of the growth plate in rodents. The receptor mRNA is expressed at low levels through most of the proliferative layers, and expression is most intense in the region between the proliferating and hypertrophic layers. There, the area of expression overlaps, but is noncongruent with, areas of intense collagen X and Indian hedgehog (Ihh) mRNA expression (10, 11, 12, 18). In organ-cultured fetal hindlimbs, PTHrP mediates the action of Ihh to slow chondrocyte differentiation and participates in a local feedback loop in which it delays differentiation of chondrocytes into cells that produce Ihh (12, 15).
Interaction of PTH or PTHrP with the PTH1R activates multiple intracellular effectors, including adenylyl cyclase and phospholipase C (PLC), which then generate various second messengers [i.e. cAMP, inositol trisphosphate (IP3), diacylglycerol, and cytosolic Ca2+ transients] that mediate specific cellular responses in bone and cartilage (19, 20, 21). The roles of these various PTH1R second messengers in regulating the differentiation and function of chondrocytes are poorly understood. We previously reported identification and characterization of a signal-defective form of the PTH1R (DSEL), incapable of activating PLC (22). If expressed in PTH1R-null chondrocytes, this receptor mutant could be used to define the role of PTH1R-dependent PLC signaling in such cells.
To better understand the function(s) of the PTH1R in controlling the successive steps of chondrocyte differentiation and to complement studies in vivo and of intact tissue or primary cultures of normal chondrocytes in vitro, it would be advantageous if nontransformed cell lines were available that corresponded to specific PTH1R-expressing chondrocytic phenotypes. In the work reported here, we have isolated a series of phenotypically diverse, conditionally transformed chondrocyte cell lines from the growth plates of neonatal mice produced by matings of H-2Kb-tsA58 transgenic mice expressing a temperature-sensitive SV40 T antigen with mice heterozygous for ablation of the PTH1R gene (10, 23). These cell lines were chosen for: 1) expression of the tsA58 transgenic immortalizing transgene; 2) the presence of one PTH1R knockout allele and one normal PTH1R allele (i.e. PTH1R(±); and 3) expression of functional PTH1Rs (transcribed from the remaining normal allele of the PTH1R gene). Such cells were expected to proliferate indefinitely when maintained in permissive conditions (33 C) but to become more differentiated upon inactivation of the temperature-sensitive SV40 T-antigen at restrictive conditions (39 C) (16, 24, 25, 26, 27, 28). Further, we anticipated that, by using drug selection to isolate rare subclones that had undergone spontaneous homologous recombination at the PTH1R gene locus, we could obtain PTH1R-null subclones of these PTH1R(±) cells. The resulting subclones, now homozygous for PTH1R ablation, lacking endogenous PTH1R expression and yet originally PTH-responsive, then could be used to analyze the function of stably transfected wild-type or mutant PTH1Rs (29, 30, 31, 32).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Isolation of chondrocytes
Conditionally immortalized chondrocyte cell lines were
established from the growth plates of 2-day-old mice derived from
matings of H-2Kb-tsA58 transgenic mice (Immortomouse; Charles River Laboratories, Inc., Wilmington, MA) with mice heterozygous
for ablation of the PTH/PTHrP receptor gene (10, 23).
Animals were maintained in facilities operated by the Center for
Comparative Research of the Massachusetts General Hospital in
accordance with the NIH Guide for the Care and Use of Laboratory
Animals and were employed using protocols approved by the
institution’s Subcommittee on Animal Care.
Under sterile conditions, distal femoral and proximal tibial growth plates were isolated, the epiphyses were carefully removed, and chondrocytes then were isolated by digestion for 6 h with 1 mg/ml of mixed collagenases (Worthington types I and II; ratio 1:3) on a rocking platform at room temperature (RT). Fractions of dispersed cells were collected at 2-h intervals during the digestion, centrifuged, resuspended in growth medium, and incubated for 2 days at 33 C, under 5% CO2 in air, before trypsinization and replating at high dilution in 100-mm culture dishes. After 1–3 weeks, individual colonies could be harvested for further expansion, subcloning, cryopreservation, and analysis.
Radioligand binding analysis
Measurement of specific PTH/PTHrP receptor binding was performed
as previously described (20). Briefly, confluent
chondrocyte monolayers in 24-well plates were washed twice with 0.5 ml
binding buffer before incubation with
125I-labeled [Tyr36]human
PTHrP(1–36) amide or [NIe8,
18,Tyr34]bovine PTH(1–34) (100,000
cpm/well), with or without nonradioactive competing peptide, in 0.5 ml
binding buffer for 4 h at 15 C. Receptor number was ascertained by
Scatchard analysis, as described previously (20).
cAMP accumulation
Confluent monolyers of cells in 24-well plates were treated with
appropriate agonists, for 20 min at 37 C, in the presence of
isobutylmethylxanthine (1 mM), after which the
concentration of cellular cAMP in the acid extracts (50 mM
HCl) was measured by RIA, as previously described
(20).
Alkaline phosphatase (ALP) activity and mineralization
Cells in 6-well plates were grown to confluence at 33 C and then
incubated in 37 C with DMEM medium (2 ml/well) containing 10
mM 
-glycerophosphate. Various treatments were added, and
the medium was replaced every 2 days. For cytochemical staining of ALP,
the cells were washed twice with PBS and then stained with a commercial
ALP kit (Sigma Kit 86-C), following the procedure
recommended by the manufacturer. For enzymatic measurement of ALP
activity, cells were plated in 12-well plates and incubated at 37 C in
the presence of the indicated treatments, after which they were
harvested with Tris-Triton buffer (100 mM Tris, pH 7.6;
0.1% Triton X-100), scraped, and sonicated. ALP activity was
determined in cell sonicates as the hydrolysis of p-nitrophenol from
p-nitrophenyl phosphate over 10 min at 30 C (23).
Mineralization was visualized by von Kossa staining. Briefly, cells were fixed with 95% ethanol for 20 min at RT and then serially rehydrated by immersion in 80%, 50%, 20%, and 0% ethanol. After staining with 5% silver nitrate for 1 h at RT, the wells were exposed to natural light for 30 min. For quantitative determination of the calcium content of the cultures, aliquots of cell sonicates, prepared as above, were incubated with 0.6 N HCl for 24 h. The extracted calcium then was measured spectrophotometrically at 612 nm, after reaction with methylthymol blue (23).
Plasmid transfections
Chondrocytes were stably cotransfected with plasmids encoding
hygromycin resistance and either the wild-type or a PLC-defective
mutant form (DSEL) of the rat PTH1R complementary DNA, using the
calcium-phosphate precipitation technique, as previously described
(22, 31, 32).
RT-PCR
Reverse transcription was carried out by direct addition of BSA
(1 µg), Ribonuclease inhibitor (10 µg), specific antisense
primer (100 ng), NP-40 (2.5%), deoxynucleotide triphosphate (500
µM), and 25 U reverse transcriptase and its buffer, in a
total vol of 10 µl, to the cell monolayers in 96-well dishes and
incubation at 37 C for 90 min. Specific sets of nested primers used for
each mRNA species are shown in Table 1
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The PCR amplification procedure was conducted using 36 cycles of
denaturation at 94 C for 1 min, annealing at 56 C for 1 min, and
polymerization at 72 C for 2 min. Three microliters of the initial RT
reaction then were used as template for primary PCR amplification with
external primers, after which 2% of the product of this primary PCR
amplification was withdrawn and used as a template for secondary
amplification with internal primers. Products were analyzed by
electrophoresis in 1.5% agarose gels and visualized by ethdium bromide
staining.
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Statistical analysis
All experiments were repeated at least three times (unless
specified otherwise), and results were expressed as the mean and
SEM of replicate determinations. Significance of
differences was assessed by ANOVA, followed by the Bonferroni-corrected
Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) and others that
appeared smaller, flattened, and elongated, i.e.
hypertrophic (nhC2–27) (Fig. 1B). Clonal populations initially were
screened by RT-PCR for expression of mRNA for the PTH1R, type II
collagen and type X collagen (
1 subunits), which were detected in
28%, 90%, and 29%, respectively, of the cell lines. Expression of
PTH1R and type X collagen mRNAs were not concordant. Thus, expression
of both genes was observed in 8 clones, of the PTH1R only in 21 clones,
and of collagen X only in 22 clones. All clones that expressed PTH1Rs
also expressed collagen II. cAMP responses to rPTH(1–34), ranging from
20- to 50-fold over basal levels, were detected in each of the
PTH1R-expressing clones. No consistent differences in this response
were observed among cells that retained only 1 vs. both
normal PTH1R alleles (maximal response to 100 nM
rPTH(1–34), as fold over basal, = 34.0 ± 2.7 vs.
35.8 ± 3.3, respectively, for 10 subclones of each type).
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), the presence of both the tsTAg
transgene and a single copy of the neo-containing PTH1R knockout
allele, and evidence of similar levels of functional PTH1R expression,
as demonstrated by specific PTH1R radioligand binding and cAMP
responsiveness to rPTH(1–34). hC1–5 cells were morphologically larger
in size and expressed genes characteristic of the hypertrophic
chondrocyte phenotype, including collagens X and II as well as
osteopontin, osteocalcin, and matrix Gla protein (Fig. 2). The other cells, designated nhC2–27,
were smaller in size and expressed collagen II but not collagen X, ALP,
osteopontin, osteocalcin, or matrix Gla protein mRNAs (Fig. 2). mRNA
for Ihh was detected in nh2–27 cells but not in hC1–5 cells (Fig. 2),
whereas both cell lines expressed PTH1R mRNA, as expected. Cytochemical
staining for ALP was positive for hC1–5 cells but not for nhC2–27
cells (Fig. 1, C and D).
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, when cultured in
medium containing -glycerophosphate, hC1–5 cells displayed focal
mineralization demonstrable by von Kossa staining. Mineralization of
these cultures was observed at 33 C within 1 week (Fig. 3A) but was
more extensive in cultures maintained for 1 or 2 weeks at 37 C (Fig. 3, B and C). No mineralization was observed in nhC2–27 cultures
maintained for 1 week at 33 C or 37 C (Fig. 3, D and E), although low
amounts were detected after 2 weeks at 37 C (Fig. 3F).
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, cells cultured under permissive
conditions (33 C) proliferated continuously, whereas cells cultured
under fully restrictive conditions (39 C) ceased proliferating
completely within 48 h.
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, C and D). Specific
125I-[Tyr36]hPTHrP(1–36)amide
radioligand binding also was evident and was competed by the
nonradioactive peptide, with IC50s of
approximately 1 nM (Fig. 5, A and B). In both cell lines,
competition binding and cAMP responses for rPTH(1–34) and
[Tyr36]hPTHrP(1–36)amide were identical (Fig. 5). No PTH-stimulated
[3H]IP3 formation could
be detected in these cells (not shown), each of which expressed fewer
than the level of 50,000 PTH1Rs/cell (i.e. 30,000 and 45,000
for nhC2–27 and hC1–5, respectively), previously shown to be required
for detection of PLC activation in kidney cells stably transfected with
the rat PTH1R (20). Increased
[3H]IP3 formation was
observed in response to PGF2 and ADP, however (not shown).
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).
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). Thus, no functional evidence for
alternate species of Gs-linked PTH or PTHrP receptors was found in
these cells.
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). cAMP responses to rPTH(1–34),
however, were identical in the 2 cell lines (Fig. 7B).
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, inhibitory effects
of rPTH(1–34) on ALP activity and mineralization (by approximately
70% and 50%, respectively) were observed not only in wild-type PTH1R
(SPR31) but also in the DSEL mutant PTH1R (SPD90). These results, which
are consistent with the finding that the inhibitory actions of PTH in
hC1–5 cells could be mimicked by 8-BrcAMP, but not by active phorbol
ester (Table 2), suggest that PTH1R-mediated regulation of growth plate
chondrocyte differentiation does not require PLC activation but may be
cAMP-dependent.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The normal chondrocytic phenotypes to which the clonal hC1–5 and nhC2–27 cells most closely correspond cannot be determined unequivocally, although their morphologies and profiles of gene expression suggest that they may represent late hypertrophic and prehypertrophic chondrocytes, respectively. Both hC1–5 and nhC2–27 cells were selected from among the minority (28%) of clones initially isolated from the growth plate that expressed PTH1Rs. Chondrocytic expression of the PTH1R varies dramatically along the axis of the growth plate (11, 12). The highest levels of PTH1R mRNA expression, as demonstrated by in situ hybridization, occur in a region at the transition between prehypertrophic and hypertrophic chondrocytes that is distinct from that featuring abundant type X collagen expression, although some overlap occurs between these two zones (11). Thus, our finding that 28% of the clonal, conditionally transformed cell lines randomly isolated from the growth plate of 2-day-old mice expressed the PTH1R is consistent with the results of previous in situ hybridization analysis, as was the observation that mRNAs for the PTH1R and for collagen X were sometimes, but not always, expressed in the same cell (11). We observed Ihh expression in nhC2–27 cells but not in hC1–5 cells, whereas the opposite was true for matrix GLA protein mRNA. In vivo, Ihh mRNA expression is observed mainly in cells of the murine growth plate that are less differentiated than most hypertrophic chondrocytes but more differentiated than the prehypertrophic chondrocytes that express the highest levels of PTH1R mRNA (although, again, significant overlap exists) (10, 12, 34, 35). On the other hand, matrix GLA protein is highly expressed in proliferative chondrocytes and late hypertrophic chondrocytes but not in early hypertrophic chondrocytes (36). Thus, nhC2–27 cells, which exhibit a nonhypertrophic morphology and express mRNAs for type II collagen, Ihh and PTH1R but not type X collagen or matrix Gla protein, could be representative of cells in the late prehypertrophic zone of the growth plate. hC1–5 cells are much larger, more readily mineralize their matrix, and express PTH1Rs, type X collagen, and matrix GLA protein (as well as osteocalcin and osteopontin, also features of hypertrophic chondrocytes) (37) but not Ihh, and thus possess characteristics more typical of the mid-late portion of the hypertrophic zone.
To address the role of PLC vs. adenylyl cyclase (or possibly other effectors), in mediating PTH1R-dependent regulation of differentiation in hypertrophic chondrocytes, we produced subclones of hypertrophic hC1–5 cells that expressed only wild-type or PLC-defective mutant PTH1Rs. In vivo, overexpression of PTHrP in murine chondrocytes delays their differentiation (13), whereas ablation of the gene for either PTHrP or the PTH1R exerts an opposite effect (9, 10). Our findings that both ALP activity and mineralization, markers of chondrocyte differentiation, were inhibited by PTH1R activation in hC1–5 cells are consistent with these observations and suggest that PTHrP may regulate chondrocyte differentiation, at least in part, by acting directly upon differentiating chondrocytes. Moreover, these inhibitory effects on chondrocyte differentiation were observed also in cells expressing PLC-defective mutant PTH1Rs, which indicates that they are mediated by mechanisms independent of PLC. This conclusion is consistent with the effects of pharmacologic agonists of protein kinases A and C (i.e. 8-BrcAMP and phorbol ester) in these cells and is concordant also with the delay in endochondral bone formation caused by targeted expression of constitutively active PTH1Rs to chondrocytes in vivo (the Jansen’s mutation causes preferential constitutive activation of adenylyl cyclase vs. PLC) (14, 33, 38). Collectively, these observations suggest that the adenylyl cyclase/cAMP/protein kinase A pathway is the principal mediator of the inhibitory effects of PTHrP on hypertrophic chondrocyte differentiation, although involvement of other possible PLC-independent effectors cannot yet be rigorously excluded.
Finally, the availability of clonal chondrocytes expressing both, one,
or none of the two normal endogenous PTH1R alleles provided
opportunities to address several other issues. First, with respect to
cAMP stimulation by PTH, no consistent differences in PTH1R function
were observed between cells that harbored one vs. two
functional alleles of the endogenous PTH1R gene. These data suggest
that there is no substantial gene-dose effect for PTH1R function in
growth-plate chondrocytes, at least at the level of adenylyl cyclase
signaling, and are consistent with observations that fetal PTH1R(±)
mice exhibit an apparently normal growth plate (10).
Second, it has been shown that the midregion of the PTH(1–84) molecule
exerts a mitogenic action on chick embryo chondrocytes
(29) and that the carboxylterminal region of PTH(1–84),
not known to activate the PTH1R, can induce histological changes in
murine cartilage and can regulate collagen gene expression in bovine
and human chondrocytes (39, 40). Such evidence indicates
that chondrocytes may express other species of PTH receptors, distinct
from the PTH1R, that recognize the middle or carboxylterminal region(s)
of PTH or PTHrP. It has been impossible to formally exclude involvement
of the PTH1R in these responses, however, when primary chondrocytes or
cartilage explants are studied. In this regard, we observed no evidence
of PTH(1–34) or PTHrP(1–36) radioligand binding to any of our
PTH1R(-/-) chondrocytes. Moreover, these cells showed no detectable
cAMP response to even high concentrations of these peptides, even
though they do express functional Gs and adenylyl cyclase proteins,
as indicated by cAMP responses to isoproterenol (not shown), to
forskolin, and (after subsequent transfection with PTH1Rs) to PTH.
These findings indicate that murine growth plate chondrocytes, at least
those with prehypertrophic or hypertrophic phenotypes, probably do not
express alternate species of receptors, particularly not Gs-linked
receptors, with specificity for the aminoterminal portion(s) of PTH or
PTHrP. In studies to be reported elsewhere, however, we have identified
binding sites on the PTH1R(-/-) hC5m8 cells with specificity for the
mid- and carboxylterminal regions of PTH(1–84). Thus, the PTH1R(-/-)
cell lines we have isolated here, in which potentially confounding
effects of PTH1R expression can be confidently excluded or controlled,
should be extremely useful in further studies of the potential roles of
alternate species of PTH or PTHrP receptors in chondrocyte biology.
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Footnotes |
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Received July 18, 2000.
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