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Endocrinology Vol. 142, No. 2 916-925
Copyright © 2001 by The Endocrine Society


ARTICLES

Receptors for the Carboxyl-Terminal Region of PTH(1–84) Are Highly Expressed in Osteocytic Cells1

P. Divieti, N. Inomata, K. Chapin, R. Singh, H. Jüppner and F. R. Bringhurst

Endocrine Unit (P.D., K.C., R.S., H.J., F.R.B.), Massachusetts General Hospital, and Harvard Medical School, Boston Massachusetts 02114; and Chugai Pharmaceutical Company (N.I.), Ltd., Shizuoka, Japan

Address all correspondence and requests for reprints to: Paola Divieti M.D., Ph.D., Endocrine Unit, Wellman 5, Massachusetts General Hospital, Boston, Massachusetts 02114. E-mail: divieti{at}helix.mgh.harvard.edu


    Abstract
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PTH is a potent systemic regulator of cellular differentiation and function in bone. It acts upon cells of the osteoblastic lineage via the G protein-coupled type-1 PTH/PTH-related peptide receptor (PTH1R). Carboxyl fragments of intact PTH(1–84) (C-PTH fragments) are cosecreted with it by the parathyroid glands in a calcium-dependent manner and also are generated via proteolysis of the hormone in peripheral tissues. Receptors that recognize C-PTH fragments (CPTHRs) have been described previously in osteoblastic and chondrocytic cells. To directly study CPTHRs in bone cells, we isolated clonal, conditionally transformed cell lines from fetal calvarial bone of mice that are homozygous for targeted ablation of the PTH1R gene and transgenically express a temperature-sensitive mutant SV40 T antigen. Cells with the highest specific binding of the CPTHR radioligand 125I-[Tyr34]hPTH(19–84) exhibited a stellate, dendritic appearance suggestive of an osteocytic phenotype and expressed 6- to 10-fold more CPTHR sites/cell than did osteoblastic cells previously isolated from the same bones. In these osteocytic (OC) cells, expression of mRNAs for CD44, connexin 43, and osteocalcin was high, whereas that for alkaline phosphatase and cbfa-1/osf-2 was negligible. The CPTHR radioligand was displaced completely by hPTH(1–84), hPTH(19–84) and hPTH(24–84) (IC50s = 20–50 nM) and by hPTH(39–84) (IC50 = 500 nM) but only minimally (24%) by 10,000 nM hPTH(1–34). CPTHR binding was down-regulated dose dependently by hPTH(1–84), an effect mimicked by ionomycin and active phorbol ester. Human PTH(1–84) and hPTH(39–84) altered connexin 43 expression and increased apoptosis in OC cells. Apoptosis induced by PTH(1–84) was blocked by the caspase inhibitor DEVD. We conclude that osteocytes, the most abundant cells in bone, may be principal target cells for unique actions of intact PTH(1–84) and circulating PTH C-fragments that are mediated by CPTHRs.


    Introduction
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PTH IS THE major physiologic regulator of blood calcium and phosphate, and it exerts potent effects upon cells in bone and cartilage (1). PTH and PTH-related peptide (PTHrP) activate a common G protein-coupled receptor, the type-1 PTH/PTHrP receptor (PTH1R) (2, 3, 4). The PTH1R recognizes the highly conserved amino (N)-terminal domain of PTH (and the homologous N terminus of PTHrP) and thus is fully activated by both PTH(1–34) and the intact hormone, PTH(1–84). Carboxyl(C)-fragments of intact PTH(1–84), such as PTH(39–84) or PTH(53–84), neither bind nor activate the PTH1R (5, 6, 7). On the other hand, a possible physiologic role for this region of the hormone is suggested by observations that the amino acid sequence of the PTH C-terminal domain is highly homologous across species (8); that PTH C-fragments, arising via both secretion from the parathyroid glands and proteolysis of PTH(1–84) in peripheral tissues, circulate in blood at levels much higher than those of the intact hormone; and that parathyroid secretion of PTH C-fragments is strongly regulated by serum calcium (9, 10, 11, 12, 13).

In rat osteoblastic cell lines, fragments from within the sequence PTH(35–84), which cannot activate PTH1Rs, regulate expression of alkaline phosphatase, osteocalcin, collagen {alpha}1(I), and IGF binding protein-5 (14, 15, 16). Direct effects of PTH C-fragments upon osteoclasts and osteoclast progenitors (17) and upon expression of collagen {alpha}1(I) and {alpha}1(X) expression in hypertrophic chondrocytes (18) also have been observed. Several such fragments induce cytosolic free calcium transients in human fetal hypertrophic chondrocytes (19). Direct physical evidence of a putative receptor (CPTHR) with binding specificity for C-terminal PTH sequences was obtained by cross-linking of the peptide 125I-[Tyr34]hPTH(19–84) (which does not bind to PTH1Rs) to 40-kDa and 90-kDa proteins in ROS 17/2.8 rat osteoblastic cells (6).

Collectively, these observations suggest that CPTHRs are expressed normally in bone and cartilage and that they may be involved in physiologic control of cell differentiation and function in these tissues. The problem of renal osteodystrophy is of particular interest in this regard, as PTH C-fragments normally are cleared mainly by the kidneys and thus accumulate to very high levels in blood during renal failure (7, 9, 12, 13). Also, hPTH (7–84) potently reduces the calcemic action of intact PTH(1–84) at concentrations much lower than those required for binding to the PTH1R (20).

To establish a model system in which to address the possible functions of CPTHRs in bone, we isolated, from cultures of enzymatically dispersed primary fetal murine calvarial cells, those that expressed CPTHRs in greatest abundance. To eliminate confounding effects of coexpressed PTH1Rs, these cells were derived from fetuses in which most exons encoding the PTH1R had been completely ablated by gene targeting (21, 22). These homozygous mice also were bred to ubiquitously express a transgene encoding a temperature-sensitive mutant SV40 large-T antigen (tsTAg), which enabled isolation of conditionally transformed clonal cell lines (21, 22). We report here that the subpopulation of calvarial-derived bone cells that expresses the highest levels of CPTHRs exhibits morphologic and molecular features characteristic of osteocytes, the most terminally differentiated cells of the osteoblast lineage (23, 24). Initial findings indicate that activation of CPTHRs may play a role in regulating osteocytic cell survival and intercellular communication.


    Materials and Methods
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Materials
Culture media were obtained from the Media Kitchen (Pediatric Surgery, Massachusetts General Hospital, Boston, MA); other tissue culture reagents were purchased from Life Technologies, Inc. (Grand Island, NY), 1,2-bis (2-aminophenoxy) ethane-tetraacetic acid (BAPTA-AM) and other reagents and chemicals were obtained from Sigma (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA). Radioactive Na[125I] and 32P-dATP were purchased from NEN Life Science Products (Boston, MA). Recombinant human (h)PTH(1–84), [Tyr34]hPTH(19–84), hPTH(24–84), were gifts of Chugai Pharmaceutical Co. (Shizuoka, Japan) and [Asp76]human PTH(39–84) was purchased from Peninsula Laboratories, Inc. (Belmont, CA).

Cell isolation and culture
Cells were isolated by enzymatic digestion from calvarial bones of 18.5 day-old tsA58(+)/PTH1R(-/-) fetuses, as previously described (21). Animals were maintained in facilities operated by the Massachusetts General Hospital Center for Comparative Resources in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals and were employed using protocols approved by the Institutional Animal Care and Use Committee.

The calvarial bones were dissected aseptically and sequentially digested in 0.5 ml of {alpha}-MEM containing 0.1% BSA, 1 mM CaCl2 and 1 mg/ml of collagenase (type I and II; ratio 1:3) (Worthington Biochemical Corp., Freehold, NJ). Bones were sequentially digested six times for 20 min each at 37 C on a rocking platform at 90 oscillations per minute under 5% CO2 in air. Cells were cultured at 33 C in a humidified atmosphere (95% air/5% CO2) using growth medium [{alpha}-MEM containing 10% FBS (lot no. 1011961 Life Technologies, Inc.) and 1% penicillin-streptomycin (PS)].

HeLa and BHK21 cells were obtained from Dr. Joel Habener (Molecular Endocrine Unit, Massachusetts General Hospital, Boston, MA) and NIH-3T3 cells were provided by Dr. Gino Segre (Endocrine Unit, Massachusetts General Hospital, Boston, MA). MS-1 cells are clonal conditionally immortalized murine bone marrow stromal cells previously isolated in our laboratory (25).

Radioligand binding
The [Tyr34]hPTH(19–84) peptide was radioiodinated with Na[125I] (2000 Ci/mmol) by the chloramine-T method and purified by HPLC, as previously described (6). For binding experiments, cells were plated in 24-well dishes at 100,000 cells/ml and cultured at 33 C for 7–14 days. Confluent monolayers then were washed with 0.5 ml binding buffer [100 mM NaCl, 5 mM KCl, 2 mM CaCl2, 50 mM Tris-HCl (pH 7.8) plus 5% heat-inactivated horse serum] before incubation with 125I- [Tyr34]hPTH(19–84) (100,000–200,000 cpm/well) in 0.5 ml binding buffer for 4 h at 15 C. Receptor number was ascertained by Scatchard analysis, using [Tyr34]hPTH(19–84) or hPTH(1–84) as competing ligand. Cellular protein was measured using the BCA protein assay kit (Pierce Chemical Co., Rockford, IL) and was found to average 0.33 mg/106 cells and 0.47 mg/106 cells for F cells and C cells, respectively. In some experiments, cells were washed with acidic buffer (50 mM glycine, 150 mM NaCl, pH 4.0) to remove residual bound ligand, before addition of radioligand.

cAMP accumulation
Cells were rinsed twice with assay buffer (135 mM NaCl, 6 mM KCl, 1 mM MgCl2, 2.8 mM glucose, 1.2 mM CaCl2, and 20 mM HEPES, pH 7.4) and then incubated for 15 min at 37 C with the same buffer containing 0.1% heat inactivated BSA, 1 mM isobutylmethylxanthine (IBMX), and agonist, conditions under which cAMP accumulation was found to be linear with time for at least 15 min. The buffer then was rapidly aspirated, the plates were frozen in liquid nitrogen, and the frozen cells subsequently were thawed directly into 0.5 ml of 50 mM HCl. Cell-associated cAMP in the acid extracts was measured using an RIA kit (NEN Life Science Products). Results were expressed as picomoles of cAMP produced per well over 15 min.

Northern blot and RT-PCR analysis
Cells were plated in 10-cm dishes and cultured at the appropriate temperature in growth medium. Total RNA was extracted using the TRIreagent method (Sigma) and quantified by UV absorbance. Fifteen micrograms of total RNA was separated by 1% agarose-gel electrophoresis in the presence of formaldehyde and transferred to a nylon membrane (Bio-Rad Laboratories, Inc. Hercules, CA) using standard procedures. Membranes were hybridized with 32P-labeled cDNA probes labeled by random primer extension, including the {alpha}1 subunit of human type I collagen, mouse osteopontin, mouse alkaline phosphatase, rat osteocalcin, mouse connexin 43 (Cx43), cbfa-1/osf-2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Rat osteocalcin was a gift of Dr. Marie Demay, mouse Cx43 was a gift of Dr. David Paul and cbfa-1/osf-2 was a gift of Dr. Gerard Karsenty.

RT-PCR was performed on total RNA using the SuperScript Preamplification System from Life Technologies, Inc. with a Peltier Thermal Cycler (MJ Research, Inc., Watertown, MA). The following pairs of primers were used for the cDNA amplification: CD44: 5'-CAAGTTTTGGTGGCACACAGC, 3'-GGTTAAGGAAGCTACCTGGC; mouse cbfa-1/osf-2; 5'-TGGAAGGGATGAAAGGCTGC, 3'-CGGTGTTTACCACAGCAGGT and alkaline phosphatase 5'-CCGTTCTTTCTCTATTC, 3'-ACAGATGGGTCCCACCG as previously reported (24, 25). The annealing temperature for CD44 and cbfa1/osf2 was 58 C, and the reaction was repeated for 30 cycles; for alkaline phosphatase the annealing temperature was 54 C, and the reaction was repeated for 35 cycles. PCR products were electrophoresed on 1.5% agarose gels and visualized using ethidium bromide staining.

Von Kossa staining
Cells, plated in six-well dishes, were maintained at 33 C for 3–4 days, until confluent, before refeeding with fresh medium containing 10 mM ß-glycerophosphate and 50 µg/ml ascorbic acid and transferring to nonpermissive conditions (39 C). Cells were refed with fresh medium twice per week. After 3–4 weeks, the presence of mineralized nodules was assessed by von Kossa staining, as previously described (21). Briefly, after fixation in 95% ethanol for 15 min at 37 C, cells were gradually rehydrated with water and stained with 5% silver-nitrate for 1 h at 37 C. Cells then were exposed to incandescent light (100 W) for 15–30 min.

Western blot analysis and immunocytochemical staining
For Western blot analysis, confluent cell monolayers were washed twice with PBS before lysis by incubation in ice-cold RIPA buffer (50 mM Tris-HCl, pH7.2, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate) for 5 min at 4 C. Lysates were centrifuged at 14,000 rpm for 10 min, supernatants were collected and passed ten times through a 22 gauge needle, and proteins were separated by SDS-PAGE (12% acrylamide gel) and transblotted to Hybond nitrocellulose membranes using standard procedures. Membranes were first blocked for 30 min at room temperature in Tris-buffered saline, pH 8.0, plus 0.05% TWEEN-20 (TBS-T) with 5% powdered milk solution, incubated for 60 min with a 1:1000 dilution of first antibody specific for murine Cx43 (Zymed Laboratories, Inc., San Francisco, CA). The membranes were washed several times in TBS-T and then incubated for 60 min with the second antibody/enzyme conjugate (HRP antimouse, IgG diluted 1:5000). Immunoreactive bands were detected by enhanced chemiluminesence assay (ECL, Amersham Pharmacia Biotech, Arlington Heights, IL) according to the manufacturer’s instructions.

For immunocytochemical analysis, cells were plated on glass chamber slides at 72,000 cells/cm2, cultured for 2 days at 33 C and then incubated for an additional 4–6 days at 39 C. Cells were treated with vehicle alone (0.1% TFA) or with the appropriate hormone for the time indicated. The immunocytochemical staining was performed as previously described (24). Briefly, cells were fixed in 3% paraformaldehyde/2% sucrose in PBS and incubated for 5 min. in 0.05% Triton-X 100 diluted in PBS. The fixed slides were blocked in 5% BSA in TBS-T buffer for 2 h at room temperature. The cells then were incubated with a 1:125 dilution of anti-Cx43 monoclonal antibody for 30 min at room temperature. The bound antibody was detected using a Vectastain ABC kit, followed by staining with VIP substrate according to manufacturer’s instructions (Vector Laboratories, Inc. Burlingame, CA). Counterstaining was performed using 0.5% methyl green. In some cases, anti-Cx43 antibody was omitted to control for nonspecific staining.

Apoptosis
The pyknotic fragmentation of nuclei typical of apoptotic cells was detected using Hoechst-33258 fluorescent dye (Sigma Co., St. Louis, MO). Briefly, cells were cultured on glass coverslips and maintained in culture at nonpermissive conditions for 4–6 days in {alpha}-MEM supplemented with 2.5% of FBS and 1% PS. After exposure to the test hormones for 6 h, culture medium was aspirated and cells were fixed in 4% paraformaldehyde in phosphate buffered solution and then stained with Hoechst-33258 (2.5 mg/ml) for 5 min at room temperature. Cells were washed and mounted with glycerol:PBS (9:1, vol/vol). The stained nuclei were visualized by fluorescence microscopy using a DAPI filter. In some experiments, apoptotic cells were detected by the terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) reaction, using the In situ Cell Death Detection Kit from Roche Molecular Biochemicals (Indianapolis, IN) following the manufacturer’s instructions.

Trypan blue staining was used for routine quantification of cell death, as it previously was shown to correlate well with apoptosis in an osteocytic cell line (26). Briefly, cells were plated at 50,000 cells/well in 24-well dishes at 33 C and shifted to nonpermissive conditions (39 C) after 2 days. Cells were grown at 39 C for an additional 4–6 days in {alpha}-MEM supplemented with 2.5% of FBS and 1% PS and then treated with different hormones for an additional 16–18 h. Nonadherent cells were combined with adherent cells that were released from the cultures with trypsin-EDTA, centrifuged, and resuspended in 0.1% trypan blue solution. The percentage of cells exhibiting both nuclear and cytoplasmic staining was determined using a hemocytometer.

Statistical analysis
All results were expressed as the mean ± SD. Each experiment was repeated at least twice. Significance of differences between treatment and control groups was assessed by Student’s t test using Bonferroni correction.


    Results
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cell isolation and C-PTH radioligand binding
Primary mixed calvarial cells, F1, were isolated by sequential collagenase digestion from a PTH1R(-/-)/tsAg(+) animal, as previously described (21). We reported previously that these cells, and osteoblastic subclones derived from them, did not express PTH1Rs, or any other species of Gs-linked PTH receptors, as indicated by the absence of PTH1R DNA by genomic PCR; detectable cAMP response to hPTH(1–34), hPTH(1–84) or hPTHrP(1–36); or specific binding of the 125I-[Nle8,18, Tyr34]bPTH(1–34)NH2 radioligand. Cells expressing CPTHRs were present in this heterogeneous cell preparation, however, as demonstrated in preliminary experiments by the specific displacement of 125I-[Tyr34]hPTH(19–84) radioligand by intact hPTH(1–84) (data not shown).

To identify cells with high expression of CPTHRs, single colonies were isolated from the mixed F1 population by limiting dilution and then screened for specific binding of 125I-[Tyr34] hPTH(19–84). Among twenty subclones isolated, three clones, initially designated C1, C14, and C59 (i.e. C cells) were selected for further characterization on the basis of their distinctly high specific binding of the 125I-[Tyr34] hPTH(19–84) radioligand. Like the previously described osteoblastic clonal cell lines (21), these three C cells did not express any other species of Gs-linked PTH receptors, as demonstrated by the absence of a detectable cAMP response to either hPTH(1–34) or hPTH(1–84) (not shown). Comparison of the CPTHR binding of these C cells with those of the previously described osteoblastic (F) cells, which had been selected for high alkaline phosphatase activity rather than for CPTHR binding (21), is shown in Fig. 1Go. The amount of total radioactivity bound averaged 14.4 ± 3.2% for the three C cell clones, vs. 2.98 ± 0.51% for the three previously reported F cell lines. Nonspecific binding, assessed in the presence of 10-6 M hPTH(1–84), was 2.8 ± 1.2% and 1.04 ± 0.15% for the C and F cell clones, respectively. As assessed by Scatchard analysis, CPTHR expression by C cells ranged from 1,900,000 to 3,400,000 sites/cell on the C cells but was less than 600,000 sites/cell on the F cells (ranging from 200,000 to 600,000) (Fig. 1Go, inset).



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Figure 1. Competitive ligand binding in C cells (C1, C14, and C59) and F cells (F1–10, F1–14 and F1–20). Different cell lines were tested for expression of CPTHRs by radioligand binding analysis at 15 C using 125I-[Tyr34] hPTH(19–84) as tracer. Cells were plated at 100,000 cells/ml in 24-well plates and maintained in culture at 33 C for 7–10 days. C cells expressed between 1,900,000 and 3,400,000 CPTHRs/cell, whereas the F cells expressed less than 600,000 CPTHRs/cell as determined by Scatchard analysis (insert; ordinate= bound/free, abscissa= specific binding, pmol/mg of protein). Specific binding is expressed as mean ± SD of triplicates in this representative experiment.

 
To identify the region(s) of the PTH molecule required for binding to the CPTHR, competitive displacement analysis was performed using various amino-terminally truncated human PTH fragments. As shown in Fig. 2Go, hPTH(1–84) displaced the 125I-[Tyr34] hPTH(19–84) radioligand as effectively as [Tyr34] hPTH(19–84) (IC50 = 20–50 nM for both). Binding by the fragment hPTH(24–84) also was equivalent to that of hPTH(1–84), whereas the shorter peptide hPTH(39–84) was much less potent, with an IC50 in the range of 500–700 nM. Human PTH(1–34) displaced the radioligand slightly (24 ± 11.5%; n = 3 experiments) at 10,000 nM but was ineffective at 1000 nM.



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Figure 2. Binding of N-terminally truncated human PTH fragments to CPTHRs in C59 cells. The human PTH peptides shown were tested for their ability to displace the 125I-[Tyr34] hPTH(19–84) tracer in C59 cells. Experiments were performed as described in Fig. 1Go. Results are expressed as the percentage of maximal specific binding observed in the absence of competing ligand and are shown as mean ± SD of triplicates in this representative experiment.

 
Regulation of CPTHR expression
Expression of CPTHRs, as assessed by specific 125I-[Tyr34]hPTH(19–84) binding, was down-regulated in a concentration-dependent manner by pretreatment of C59 cells for 16 h with hPTH(1–84) (Fig. 3AGo). Binding was reduced 19 ± 5% and 66 ± 7% by preincubation with hPTH(1–84) at concentrations of 200 nM and 1000 nM, respectively. Pretreatment with hPTH(39–84) reduced CPTHR binding by less than 20% when added at 10,000 nM but not at all at 1000 nM (not shown). To assure that this change was due to a reduction in total binding sites and not simply to persistent receptor occupancy by preadministered hPTH(1–84), cells were rinsed, before radioligand addition, with acidic buffer that was found in preliminary experiments to completely and reversibly remove previously bound radioligand. CPTHR binding also was down-regulated following preincubation for 16 h in the presence of calcium ionophore (ionomycin, 1 µM) or active phorbol ester (TPA, 10 nM), which reduced specific binding by 71 ± 3% and 48 ± 12%, respectively. The effects of these drugs were at least partly reversible, as radioligand binding returned to 100% and 63% of controls, respectively, 24 h after their removal (data not shown). No down-regulation occurred when cells were pretreated with inactive phorbol ester (4-{alpha} phorbol) at concentrations as high as 100 nM (not shown). Also, the effect of ionomycin (1 µM) was not blocked by prior incubation of the cells with the intracellular calcium chelator BAPTA-AM (25 µM) (not shown). Finally, no down-regulation of CPTHR binding occurred following exposure for 24 h to hPTH(1–34) (1 µM), 1,25(OH)2D3 (10 nM), 8-bromo cAMP (1 mM), forskolin (10 µM), insulin (100 ng/ml), IGF-1 (100 nM) or dexamethasone (100 nM) (data not shown).



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Figure 3. Homologous down-regulation of CPTHRs in C59 cells. Cells were treated for 16 h with the agent indicated and then rinsed three times with binding buffer and once with an acidic buffer (see Materials and Methods), to completely remove any previously added PTH peptide from the receptor. Radioligand binding then was conducted using 125I-[Tyr34]hPTH(19–84) as tracer ± hPTH(1–84) as competing ligand in cells pretreated with (A) hPTH(1–84) at the indicated concentrations (nM), (B) ionomycin (µM), and (C) the active phorbol ester TPA (nM). Specific binding is expressed as mean ± SD of triplicates. * P < 0.05, ** P < 0.01.

 
Morphology and gene expression
The C cells, which had been selected for high expression of CPTHRs, exhibited a characteristic morphology, including the presence of numerous elongated dendritic processes reminiscent of those seen in mature osteocytes (Fig. 4Go, A–C). This stellate appearance was seen in all three C cell lines and was distinct from the more cuboidal shape of the osteoblastic (F) cells that previously had been isolated from the same calvarial digest on the basis of high alkaline phosphatase activity (21) (Fig. 4DGo). Because of their osteocytic morphology, the three C cell lines were renamed OC cells.



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Figure 4. Connexin 43 expression at permissive and nonpermissive temperature. C59 cells (A and B), C1 (C), and F1–14 cells (D) were plated on glass coverslips at 72,000 cells/cm2 and cultured at 33 C (A) or at 33 C for 2 days and then at 39 C for 3–5 days (B-D) before staining for Cx43 as described in Materials and Methods. Because of their osteocyte-like appearance, C cells were renamed OC cells.

 
Others have reported that osteocytes, unlike osteoblasts, express little or no alkaline phosphatase, cbfa-1/osf-2, or collagen I but do express high levels of mRNAs for osteocalcin, Cx43, and CD44 (24, 27). To further define the phenotype of the osteocyte-like OC cell lines, expression of specific mRNAs characteristic of osteoblasts or osteocytes was analyzed by Northern blotting and RT-PCR. As shown in Fig. 5AGo, OC cell expression of osteocalcin and Cx43 mRNAs was higher than in F1–14 osteoblastic cells or in early cultures of normal murine calvarial osteoblasts, whereas osteopontin mRNA was barely detectable in the OC cells. Transcripts for alkaline phosphatase or cbfa-1/osf-2 could not be detected by Northern blot analysis in these cells but were present at low levels, as revealed by RT-PCR (Fig. 5BGo). Type 1 collagen was well expressed in all of the cell lines. None of the mRNAs measured by Northern blot analysis increased significantly in cells incubated for 5 days at 39 C vs. 33 C. Cx43 expression, as detected by direct immunostaining of fixed cells, was observed in cytoplasm and along the dendritic processes (Figs. 4Go and 6Go). After incubation for 2 h with hPTH(1–84) (100 nM) or hPTH(39–84) (1000 nM), Cx43 staining was more prominent, especially in a perinuclear location (Fig. 6Go). This represented mainly a redistribution of Cx43, as it was not accompanied by an increase in total cellular Cx43 protein, as assessed by Western blotting (Fig. 6DGo).



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Figure 5. mRNA expression in OC cells. OC14 lanes (1 3 ) and OC59 (lanes 2 and 4) cells were cultured at 33 C for 7 days (lanes 1 and 2) or at 33 C for 2 days and at 39 C for 5 additional days (lanes 3 and 4) before RNA extraction. F1–14 cells (lane 5) also were cultured at 33 C 2 days and at 39 C for 5 additional days whereas primary calvarial osteoblasts (lane 6) were cultured at 37 C. Expression of specific mRNAs was assessed by Northern blotting or by RT-PCR as described in Materials and Methods. A, Northern blot analysis of mRNAs for osteocalcin (Oc), connexin 43 (Cx43), osteopontin (OP), and collagen {alpha}1 (I) (Col) and GAPDH. B, RT-PCR for alkaline phosphatase (Alk), CD44 and cbfa-1/osf-2 (Osf2). M, Molecular weight. H, Reaction with primers in the absence of cDNAs. The expected size for each PCR product is as indicated.

 


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Figure 6. Connexin 43 regulation by CPTHR ligands. OC14 cells were plated on glass coverslips at 72,000 cells/cm2, cultured at 33 C for 2 days and then shifted at 39 C for 3–5 days before stimulation with CPTHR ligands. OC14 cells were treated for 2 h with (A) vehicle alone, (B) 100 nM hPTH(1–84), or (C) 1000 nM hPTH(39–84). Cells were fixed and stained for Cx43 expression as described in Materials and Methods. D, Western blot for Cx43 in OC14 cells cultured at 33 C (lane 1) or at 39 C (lanes 2–5), as described in Materials and Methods and treated for 2 h with vehicle alone (lanes1–3) or with 100 nM hPTH(1–84) (lanes 4 and 5).

 
The OC cells were capable of mineralization, as detected by von Kossa staining after 4 weeks in the presence of 10 mM ß-glycerophosphate and 50 µg/ml of ascorbic acid (Fig. 7Go). Mineralization (Fig. 7BGo vs. A) was increased when cells were maintained at 39 C, conditions under which the transforming T antigen is inactive.



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Figure 7. Mineralization by OC 14. OC 14 cells were grown to confluence at 33 C on 6 well-dishes and then maintained for an additional 4 weeks at 33 C (A) or 39 C (B) in the presence of 10 mM ß-glycerophosphate and 50 µg/ml ascorbic acid before visualizing calcium phosphate deposition by von Kossa staining. Magnification, 40x.

 
CPTHR expression in other cells
To determine if abundant CPTHR expression is a specific characteristic of osteocytic cells or a more generalized feature of bone cells, we performed 125I[Tyr34] hPTH(19–84) radioligand binding with several other bone- and non bone-derived cell lines. As shown in Table 1Go, CPTHR specific binding was much greater in the osteocytic cells than in the unfractionated original calvarial digest (F1) or the previously isolated osteoblastic (F1–14) or bone marrow stromal cells (MS-1) (25). Minimal specific binding was detected on HeLa, BHK1, NIH3T3 cells or, as previously reported, on LLC-PK1 porcine kidney cells (6).


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Table 1. CPTHR binding in various cell lines

 
Apoptosis
It was shown recently that PTH(1–34), presumably acting via PTH1Rs, can reduce the rate at which osteoblasts and osteocytes undergo apoptosis in vivo and in vitro (26, 28). As osteocytes are terminally differentiated osteoblasts, it was of interest to determine if CPTHR activation might play a role in regulating apoptosis in the OC cells. As shown in Fig. 8Go (A and B) when OC cells, which lack functional PTH1R genes, were incubated for 6 h with 100 nM hPTH(1–84), increased nuclear pyknosis and chromatin condensation appeared, as revealed by DNA staining with Hoechst dye 33258. Increased apoptosis also was observed using a TUNEL immunocytochemical assay (Fig. 8Go, C and D). Similarly, when cell death was monitored using trypan blue staining of combined adherent and nonadherent cells from these cultures (26), hPTH(1–84) induced a doubling in trypan blue-stained cells after 16 h (Fig. 9AGo). This effect of hPTH(1–84) upon cell death was concentration dependent and maximal at 10 nM, with an EC50 in the range of 0.1 nM (Fig. 9BGo). The effect was mimicked by 100 nM hPTH (24–84) and 1000 nM hPTH(39–84), which produced, respectively, 1.8- and 1.5-fold increases over basal. (Fig. 9Go, C and D). As expected, hPTH(1–34) (1000 nM) was inactive, consistent with its inability to effectively bind to CPTHR sites on these cells (Fig. 2Go). In these experiments, the percentage of trypan blue-stained cells in controls was substantial (20–30%), perhaps because of prolonged incubation in reduced serum at the nonpermissive temperature. A similar proapoptotic effect of hPTH(1–84) was observed in analogous experiments performed at 37 C, however, where basal rates of cell death were lower (Fig. 9AGo). Cell death triggered by hPTH(1–84) was blocked completely by prior addition of the caspase-3 inhibitor DEVD (50 µg/ml) (Fig. 9EGo).



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Figure 8. Induction of apoptosis by PTH(1–84) in OC14 cells. Cells plated on glass coverslips and maintained at 33 C for 3 days were shifted to 39 C for 5–7 days before addition of 100 nM hPTH(1–84) (B, D) or vehicle alone (A, C) for 6 h. At the end of the incubations, cells were fixed and stained with Hoechst 33258 (A and B) or for TUNEL (C and D), as described in Materials and Methods. Arrows indicate apoptotic cells. Of the total Hoechst 33258 stained cells counted (>200), 20 ± 2.1% and 29 ± 0.7% of the cells were apoptotic in the control and in PTH-treated, respectively. Similarly, in TUNEL assay, there were 5.5 ± 0.4 and 8 ± 0.3 TUNEL positive cells/field in the control and in the PTH-treated, respectively.; 200x magnification.

 


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Figure 9. Cell death induced by in PTH in OC14 cells. OC14 cells were plated, cultured at 33 C for 3 days and then incubated at 37 C (hatched bars in A) or 39 C (solid bars in A; B–E) for 5–7 days before addition of PTH peptides for a further 16 h, followed by trypan blue staining (see Materials and Methods). A, Response to hPTH(1–84), 100 nM; B, Human PTH 1–84 dose response; C, Response to hPTH(1–84), 100 nM and hPTH(39–84),1000 nM; D, Response to hPTH(24–84), 100 nM; E, Caspase dependence (C = controls) The Caspase-3 inhibitor DEVD (D) was added 1 h before addition of 100 nM hPTH(1–84) (P). Results are expressed as mean ± SD of quadruplicate determinations of the percentage of nonvital trypan blue-stained cells. * P < 0.05, ** P < 0.01.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Osteocytes comprise over 90% of bone cells, yet their functions, and the involvement of systemic hormones in regulating them, are incompletely understood. Located in interconnecting lacunae deep within the mineralized matrix of bone, osteocytes are believed to fulfill an important mechanosensory function, whereby they transduce shear and strain forces into chemical signals that communicate this information to other osteocytes, osteoblasts and, ultimately, osteoclasts (28, 29, 30). Osteocytes express PTH1Rs, and their response to mechanical forces in vivo is enhanced by, and may even require, circulating PTH (31).

We report here that clonal murine calvarial-derived cell lines with many characteristics of osteocytes also express unusually large numbers of receptors that specifically recognize the C-terminal portion of intact PTH. In fact, these cells express considerably higher levels of CPTHRs (up to 10-fold more per cell) than do other bone cell subtypes, including mature osteoblasts (21). It is difficult to identify the osteocytic phenotype with complete confidence in vitro, absent the normal surrounding lacunar environment, but the three conditionally immortalized, clonal cell lines that we selected exclusively on the basis of high CPTHR expression share many morphological features and a distinctive profile of gene expression with normal osteocytes (23) and with a previously characterized osteocytic cell line (24). These include a stellate shape, with numerous dendritic processes, and abundant expression of mRNAs for osteocalcin, Cx43, and CD44 but not for alkaline phosphatase or cbfa-1/osf-2, which are more characteristic of early or mature osteoblasts. The osteocytic phenotype of these cells is most intense when they are maintained at a temperature (i.e. 39 C) that is nonpermissive for expression of functional transforming tsTAg. For example, mineralization (Fig. 7Go) was much more intense in cells cultured at 39 C than at 33 C, as we previously observed in other clonal osteoblastic cells isolated from these mice (21).

The OC cells were isolated intentionally from animals that genetically lack functional PTH1Rs to eliminate possible confounding effects of coexpressed PTH1Rs and to allow us to focus directly upon CPTHR-dependent actions of intact PTH. The results of radioreceptor binding assays in OC cells, using the 125I-[Tyr34]hPTH(19–84) radioligand, were remarkably similar to those reported previously with ROS17/2.8 cells (6, 16), which suggests that the CPTHRs on these different cell types probably are similar or identical. Thus, like Inomata et al., we found that hPTH(1–84) and the hPTH(19–84) peptide bound equivalently to CPTHRs and that hPTH(39–84) bound more weakly, with an IC50 roughly 15-fold higher. We have further narrowed the ligand domain responsible for this higher affinity interaction, moreover, by studying hPTH (24–84), which was found to bind at least as well as hPTH(1–84). This indicates that CPTHRs require sequences within the region hPTH(24–38) for high affinity binding, although other, more C-terminal domains also must be involved to account for the residual affinity of hPTH(39–84). The small amount of binding observed at high concentrations of hPTH(1–34) is consistent with the presence of a binding determinant within hPTH(24–38), most of which is contained within hPTH(1–34). It seems that this domain probably cannot function well independently of the cooperative effects of more C-terminal sequences, however. The manner in which of these independent domains contribute to the formation of an active conformation of the ligand remains obscure, especially as available spectroscopic studies do not suggest a highly ordered secondary structure for the C-terminal sequence of PTH (32, 33, 34).

Enumeration of CPTHR sites on the OC cells led to estimates of 1,900,000 to 3,400,000 per cell, which was 6- to 10-fold higher than on the osteoblastic (F) cells obtained from the same bones and at least 5-fold higher than on ROS 17/2.8 cells (6). A survey of other available cell lines indicated that, with the exception of a bone marrow stromal cell line (MS-1) (25), little or no binding was detectable on NIH-3T3, HeLa or BHK21 fibroblast cells, as was reported previously for OK and LLC-PK1 kidney cells, YCC cells, and SaOS-2, MG63 and UMR106–01 osteosarcoma cells (6). Thus, the very high abundance of expressed CPTHRs on osteocytic OC cells is unique, even among other bone cells tested, and suggests that these receptors may play an important role in osteocyte regulation. The possibility that bone is an important C-PTH target tissue in vivo is further supported by our recent observation that 125I-[Tyr34]hPTH(19–84) radioligand binds specifically to frozen sections of 18.5-day normal mouse fetal calvarial bone (unpublished results) and that hPTH (7–84) reduces the calcemic effects of intact PTH(1–84) in vivo at concentrations much lower than those required for effective antagonism of the PTH1R (20).

We observed homologous down-regulation of CPTHR binding following preincubation with hPTH(1–84). This response required relatively high concentrations of ligand (>100 nM), in contrast to the effects upon apoptosis, and was mimicked by addition of active phorbol ester or ionomycin. The failure of BAPTA to block the ionomycin effect suggests mediation by a sustained influx of calcium, presumably from the extracellular compartment, as opposed to a rapid burst of calcium released from intracellular stores. This is consistent also with our previous report of CPTH-stimulated calcium uptake in SaOS-2 human osteoarcoma cells (35). Also, in C-PTH-treated human fetal chondrocytes, others have reported increased cytosolic free calcium due to activation of calcium influx via nickel-insensitive channels (18). Our down-regulation data are most compatible with involvement of a calcium-dependent PKC, although further study will be needed to pursue this possibility and to clarify the role and source of the calcium signal implicated in this response.

Functional studies of CPTHRs on OC cells have pointed so far to involvement in at least two general types of cellular processes: cell survival and intercellular communication. We observed a rapid CPTHR-dependent change in cellular expression of Cx43, a gap-junction protein important in mediating intercellular communication and previously shown to be expressed by both osteoblasts and osteocytes (36, 37). The possible physiologic significance of this action is not yet clear. The response of osteocytes to mechanical stress in vivo is promoted by PTH(1–34) and blocked by parathyroidectomy (38). It is interesting to speculate that activation of CPTHRs by C-PTH fragments, preferentially secreted by the parathyroids during hypercalcemia (9), might trigger communication from osteocytes to other bone cells so as to limit osteoclast formation or activity and thereby exert negative feedback on bone resorption. Further understanding of CPTHR function and ligand activity, facilitated by availability of these osteocytic cell lines, will be needed to pursue such hypotheses.

We also observed that CPTHR-interacting ligands increased the rate of osteocyte cell death in vitro. As this action was accompanied by evidence of increased nuclear pyknosis, chromatin condensation and TUNEL staining and was completely blocked by the caspase-3 inhibitor DEVD, we conclude that apoptosis, mediated by activation of the caspase cascade, is the likely mechanism. Importantly, this proapoptotic effect was observed also with the C-terminal fragment hPTH(39–84) (at an appropriately higher concentration), which indicates that the ligand domain required for CPTHR activation of this response likely is located C-terminal to the above-mentioned hPTH(24–38) region needed for high-affinity binding. In contrast, others (26) have observed an opposite, anti-apoptotic action of hPTH(1–34), via the PTH1R activation, in dexamethasone-stimulated osteoblastic and osteocytic cells. In OC cells, which lack endogenous PTH1Rs, hPTH(1–84) exerts only a pro-apoptotic effect (via CPTHRs), as does hPTH(39–84). This suggests the possibility that PTH1Rs and CPTHRs may exert opposite effects upon cell survival in osteocytes and that these cells may be able to integrate signals arising from these two types of PTH receptors in response to discoordinate changes in circulating intact hormone and C-PTH fragments. In renal insufficiency, for example, excessive CPTHR activation by high levels of circulating CPTH fragments might lead to exaggerated, pathological loss of osteocytes and contribute to some forms of renal osteodystrophy (39).

Expression of the PTH1R by osteocytes has been documented in vivo (40), and it is of interest to consider the potential for interactions between PTH1Rs and CPTHRs in these cells. It is possible that the genetic absence of PTH1R in our cells may have modified in some way the expression or function of CPTHRs. Almost certainly, the response of these PTH1R-null cells to hPTH(1–84) in vitro differs from that of authentic osteocytes in vivo, in which both receptor types presumably can be activated concurrently by the intact hormone. Preliminary evidence, in ROS 17/2.8 cells, indicates that C-PTH fragments may modify the function of hPTH(1–84) [but not hPTH(1–34)] in cells that express both types of PTH receptors (41). The possible involvement of PTH1Rs in modulating CPTHR function, or vice versa, now can be addressed directly in our OC cells by reconstituting them through transfection with cDNA encoding PTH1Rs; efforts in this direction are underway in our laboratory.


    Acknowledgments
 
The authors wish to thank Dr. H. M. Kronenberg for critical review of the manuscript and Dr. B. Lanske for provision of the PTH1R -/- mice.


    Footnotes
 
1 This work was supported by the NIH Grant DK-11794. Back

Received July 7, 2000.


    References
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Clinical Utility of an Immunoradiometric Assay for Parathyroid Hormone (1-84) in Primary Hyperparathyroidism
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