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Endocrinology Vol. 142, No. 7 2752-2759
Copyright © 2001 by The Endocrine Society

ARTICLES

C-Terminal Parathyroid Hormone-Related Protein (PTHrP) (107–139) Stimulates Intracellular Ca2+ through a Receptor Different from the Type 1 PTH/PTHrP Receptor in Osteoblastic Osteosarcoma UMR 106 Cells1

Alvaro ValÍn, Carlos Guillén and Pedro Esbrit

Bone and Mineral Metabolism Laboratory, Research Unit, Fundación Jiménez Díaz, 28040 Madrid, Spain

Address all correspondence and requests for reprints to: P. Esbrit, Ph.D., Bone and Mineral Metabolism Laboratory, Research Unit, Fundación Jiménez Díaz, Avda. Reyes Católicos 2, 28040 Madrid, Spain. E-mail: pesbrit{at}fjd.es


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Studies were undertaken to determine whether PTH-related protein (PTHrP) (107–139) mobilizes [Ca2+]i in osteoblastic osteosarcoma UMR 106 cells. PTHrP (107–139), in a manner similar to PTHrP (107–111), induced a rapid [Ca2+]i response in these cells that was dose dependent (EC50 of {approx}0.1 pM) and more efficient than that of PTHrP (1–36) (EC50 of {approx} 1 nM). This effect of PTHrP (107–139) was abrogated by micromolar doses of verapamil or nifedipine. However, it was unaffected by 10 µM U73122 (a phospholipase C inhibitor), 100 µg/ml heparin (an inositol 1,4,5-trisphosphate receptor inhibitor), or 400 ng/ml pertussis toxin (a Gi inhibitor), which inhibited the [Ca2+]i response to PTHrP (1–36), or by either 25 nM bisindolylmaleimide I (BIM), a protein kinase (PK) C inhibitor, or 1 µM phorbol-12-myristate-13-acetate preincubation (22 h). PTHrP (107–139) and PTHrP (1–36), at 100 nM, desensitized the [Ca2+]i response to a second challenge with the same peptide, but not with the other peptide in these cells. PTHrP (7–34), a type 1 PTH/PTHrP receptor (PTH1R) antagonist, decreased the effect of PTHrP (1–36) on [Ca2+]i. In contrast, PTHrP (107–111), but neither PTHrP (109–138) nor PTHrP (7–34), abolished this effect of PTHrP (107–139). Both PTHrP (107–139) and PTHrP (1–36), added together at submaximal doses, induced a higher [Ca2+]i response. Moreover, PTHrP (107–139) increased the efficacy of PTHrP (1–36) on [Ca2+]i, but decreased its induced increase in PKA activity in these cells. Verapamil or nifedipine (at 50 µM) or 25 nM BIM, but not 25 µM adenosine 3',5'-cyclic monophosphorothioate, Rp-isomer, a PKA inhibitor, abolished the PTHrP (107–139)-induced increase in interleukin 6 messenger RNA (assessed by RT, followed by PCR) in UMR 106 cells. This peptide also increased c-fos messenger RNA in these cells; an effect inhibited by BIM, but unaffected by either verapamil or EGTA. These findings support the existence of high-affinity receptors for PTHrP (107–139), associated with an induced Ca2+ influx, different from the PTH1R in UMR 106 cells. The present results suggest that PTHrP could affect bone turnover by interacting with the PTH1R and other yet unknown receptors in bone cells through complex mechanisms.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
BONE CELLS FUNCTION in a complex and dynamic microenvironment in which they must constantly sense and respond to the ambient Ca2+ concentration ([Ca2+]). Regulation of Ca2+ entry via activation of various plasma membrane Ca2+ channels is an important signaling pathway in osteoblasts (1). A variety of agents develop intracellular [Ca2+] ([Ca2+]i) signals in these cells, which occurs as an early event in processes associated with osteoblastic differentiation and bone remodeling (1, 2). Both mechanosensitive and voltage-sensitive Ca2+ channels are present in osteoblasts (1). In addition, many stimuli (e.g. PTH) induce not only a Ca2+ influx through these channels but also a Ca2+ release from intracellular stores in these cells (3, 4, 5, 6, 7, 8).

PTH-related protein (PTHrP) was initially isolated from hypercalcemia-associated tumors, but is now known to be produced by many normal tissues, including bone (9). The N-terminal region of both PTH and PTHrP interacts with the type 1 PTH/PTHrP receptor (PTH1R) cloned in osteoblastic cells, thereby activating both adenylate cyclase and phospholipase (PL) C/[Ca2+]i (10). This receptor is also widely distributed, suggesting that PTHrP acts in an autocrine/paracrine manner in normal tissues (9). Multiple PTHrP fragments, including the 1–36 domain, and other poorly characterized C-terminal fragments, seem to be secreted by various cell types (11, 12). One of these putative fragments, PTHrP (107–139) has recently been shown, both in vitro and in vivo, to affect osteoblastic growth and differentiation (13, 14, 15, 16, 17, 18). Furthermore, these effects of this fragment are likely to be mediated by its interaction with an unidentified, specific receptor, different from the PTH1R, which might also be present in other bone and nonbone cell types (18, 19, 20, 21, 22). In the wide majority of these cells, including osteoblastic cells, activation of this putative receptor seems to interact with protein kinase (PK) C activation (13, 14, 15, 16, 19, 20, 21). In addition, a recent report has shown that PTHrP (107–139) induces [Ca2+]i transients, but in contrast to either PTHrP (1–34) or PTH (1–34), it does not activate cAMP in rat hippocampal neurons (22). Interestingly, different bone regulators seem to modulate the induction of various osteoblastic genes through both PKC activation and [Ca2+]i signaling (23, 24, 25). However, the putative role of the latter mechanism associated with the effects of PTHrP (107–139) in osteoblastic cells has not yet been assessed.

The present study was carried out to evaluate the effect of PTHrP (107–139) on [Ca2+]i in rat osteoblastic osteosarcoma UMR 106 cells, which respond to this peptide (13, 16). We have compared this effect of PTHrP (107–139) with that induced by PTHrP (1–36) acting through the PTH1R in these cells (26). We also assessed whether the effect of PTHrP (107–139) on [Ca2+]i might be related to the induction of interleukin 6 (IL-6) and c-fos gene expression in these cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Human PTHrP (107–139), pertussis toxin (PTX), fura-2 acetoxymethylester, and 3-isobutyl-1-methylxanthine were from Sigma (St. Louis, MO). Human PTHrP (1–36), human [109Tyr]PTHrP (109–138) ([PTHrP (109–138)]), and human PTHrP (38–64) amide ([PTHrP (38–64)]) were kindly donated by Dr. A. F. Stewart (Division of Endocrinology, University of Pittsburgh, Pittsburgh, PA). Human PTHrP (107–111) amide ([PTHrP (107–111)]) was synthesized in an Abimed multiple peptide synthesizer (Lagenfeld, Germany), using Fmoc chemistry. (Asn10,Leu11,D-Trp12)PTHrP (7–34) amide ([PTHrP (7–34)]) was from Bachem (Bubendorf, Switzerland). Kemptide was from Roche Molecular Biochemicals (Mannheim, Germany). Verapamil was obtained from Knoll Pharmaceutical Co. (Manidón; Madrid, Spain). Nifedipine, U73122, bisindolylmaleimide I (BIM), and phorbol-12-myristate-13- acetate (PMA) were from Calbiochem (San Diego, CA). Sodium heparin was from Rovi (Madrid, Spain). Adenosine 3',5'-cyclic monophosphorothioate, Rp-isomer (RpcAMPS) was from Biolog Life Science Institute (Bremen, Germany).

Cell culture
UMR 106 cells (ATCC CRL 1661) were seeded on round glass coverslips (12-mm diameter; Menzel, Braunschweig, Germany) at a density of 30,000 cells/coverslip and grown in DMEM with 1 g/liter glucose, 4 mM L-glutamine, 10% FBS, and antibiotics (13). The cells were incubated in a humidified atmosphere containing 5% CO2 in air at 37 C for 2 days.

Measurement of cytosolic calcium
We used subconfluent cells, at days 2–4 after seeding, because these cells have been previously shown to exhibit a higher [Ca2+]i in the proliferative phase (8, 16). Cells were plated onto coverslips, and 48 h later adherent cells were loaded with the acetoxymethyl ester of fura-2 (5 µM) for 1 h at room temperature in a loading buffer containing 10 mM HEPES, 145 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM KH2PO4, 1 mM MgCl2, 5 mM glucose, and 0.1% BSA, adjusted to pH 7.4. After loading, cells were washed three times and resuspended in loading buffer. Fluorescence emission was monitored in a LS-50B spectrofluorimeter (Perkin-Elmer Corp., Norwalk, CT), as described previously (27). Maximal and minimal fluorescence values were determined by adding 1% Triton X-100 and 10 mM EGTA, respectively. [Ca2+]i (nM) was calculated, assuming 225 nM as the dissociation constant (Kd) for Ca2+-fura-2. All experiments were performed at 37 C.

Measurement of PKA
UMR 106 cells were stimulated with the different agonists in FBS-depleted medium for variable time periods. PKA activity was measured in cell extracts, as described previously (13). PKA activity was expressed as the ratio of activity in the absence and in the presence of 6.25 µM cAMP.

RNA extraction and RT-PCR
Cell total RNA was isolated using guanidinium thiocyanate-phenol-chloroform extraction (Tri Reagent; Molecular Research Center, Inc., Cincinnati, OH). Changes in IL-6 levels were assessed by semiquantitative RT-PCR. Total RNA was added to a reaction mixture containing 1.5 mM MgSO4, 0.2 mM dNTP, 1 U avian myeloblastosis virus reverse transcriptase, 1 U thermostable DNA polymerase from Thermus flavus (Access RT-PCR System; Promega Corp., Madison, WI), and 1 µM of the specific primers: 5'-TTCCAGCCAGTTGCCTTCT-3' (sense) and 5'-TGCTCTGAATGACTCTGGC-3' (antisense), corresponding to bases 89–498 of the rat IL-6 complementary DNA (accession number M26744); or 5'-GGGAATTC-GGAGAATCCGAAGGGAAAGG-3' (sense), and 5'-CCGGATCC-GTGAAGGCCTCCTCAGACTC-3' (antisense), corresponding to bases 553–853 in rat-c-fos complementary DNA (accession number X06769). PCR amplification yields 409-bp (IL-6) and 316-bp (c-fos) products. The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase was amplified as a constitutive control (15).

Preliminary experiments established the linear amplification range for each PCR product. Then, total RNA [200 ng (IL-6); 10 ng (c-fos); 1 ng (glyceraldehyde-3-phosphate dehydrogenase)] was incubated in a reaction mixture (10 µl) for 45 min at 48 C, followed by 32–35 cycles of 1 min at 95 C, 1 min at 58–60 C, and 2 min at 68 C, with a final extension of 7 min at 68 C. A negative control without RNA was usually included in the PCR reaction. Abundance of PCR products was assessed on 2% agarose gels stained with ethidium bromide using DNA markers. To confirm the identity of the PCR products, they were extracted with Wizard PCR prep columns (Promega Corp.) following agarose gel electrophoresis. The extracted PCR products were analyzed by automatic sequencing (373 DNA sequencer; PE Applied Biosystems, Branchburg, NJ).

Statistical analysis
Data are expressed as mean ± SEM throughout the text. Statistical significance was determined by either Mann-Whitney test or ANOVA, when appropriate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PTHrP (107–139) stimulates [Ca2+]i dose dependently in UMR 106 cells
We found that PTHrP (107–139) induced an acute and transient [Ca2+]i rise, followed by a variable sustained [Ca2+]i response, in UMR 106 cells (Fig. 1AGo). This effect was dose dependent, with an EC50 of ~0.1 pM (Fig. 2AGo). The pattern and magnitude of the [Ca2+]i response was similar to that produced by PTHrP (107–111) within the same dose range in these cells (Figs. 1BGo and 2AGo). PTHrP (1–36) induced a transient [Ca2+]i signal that was less efficient (EC50 of ~1 nM) than that induced by PTHrP (107–139) in UMR 106 cells (Fig. 2BGo). PTHrP (38–64) was without effect on [Ca2+]i in these cells (Fig. 1CGo).



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  		Figure 1. The C-terminal PTHrP region stimulates [Ca2+]i in UMR 106 cells. The tracings represent the response of these cells after stimulation with either PTHrP (107–139) (A) or PTHrP (107–111) (B), at 0.1 nM. The lack of response of the cells following the addition of 100 nM PTHrP (38–64) is also shown (C).

 


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  		Figure 2. The stimulatory effect of the C-terminal PTHrP peptides on [Ca2+]i is dose dependent in UMR 106 cells. A, Dose-response curve for the [Ca2+]i signal induced by either PTHrP (107–139) (•) or PTHrP (107–111) ({triangleup}). P < 0.01, for all the values corresponding to peptide doses in the range 1 pM to 1 µM, compared with the nonstimulated [Ca2+]i value; *, P < 0.05, compared with the [Ca2+]i value corresponding to 1 pM PTHrP (107–139). B, Dose dependence of [Ca2+]i stimulation by PTHrP (1–36). *, P < 0.05, compared with nonstimulated [Ca2+]i value; **, P <0.01, compared with nonstimulated [Ca2+]i value. The different peptide doses are indicated as log. Data are mean ± SEM of at least four measurements at each dose.

 
Mechanism by which PTHrP (107–139) stimulates [Ca2+]i in UMR 106 cells
The effect of PTHrP (107–139) on [Ca2+]i in UMR 106 cells seems to depend on Ca2+ influx from the extracellular compartment, because the addition of either EGTA (data not shown), verapamil or nifedipine, two L-type calcium channel blockers at micromolar concentrations (28), abolished this maximal effect triggered by PTHrP (107–139) in these cells (Table 1Go). However, treatment with these channel blockers, at 10 nM, did not inhibit the stimulatory effect of PTHrP (107–139) on [Ca2+]i in UMR 106 cells (Table 1Go). On the contrary, 10 nM of these agents increased [Ca2+]i in these cells (Table 1Go), consistent with previous findings (29, 30). On the other hand, 10 µM U73122, a PLC inhibitor (31), or 100 µg/ml heparin, which inhibits inositol 1,4,5-trisphosphate receptors (32), did not change the effect on [Ca2+]i rise triggered by PTHrP (107–139), but abolished this effect induced by PTHrP (1–36) in these cells (Table 2Go). In various cell types, receptor coupling to voltage-dependent Ca2+ channels seems to depend on a PTX-sensitive G protein activation (33). Thus, we assessed whether such a mechanism operates for PTHrP (107–139)-stimulated Ca2+ influx in UMR 106 cells. Preincubation with 400 ng/ml PTX failed to affect the [Ca2+]i response to PTHrP (107–139), although this pretreatment inhibited this effect induced by PTHrP (1–36), in these cells (Table 2Go).


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  		Table 1. Effect of different calcium channel blockers on [Ca2+]i stimulation by PTHrP (107–139) in UMR 106 cells

 

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  		Table 2. Effect of different inhibitors on [Ca2+]i stimulation by each PTHrP peptide in UMR 106 cells

 
Interaction between the effects on [Ca2+]i induced by PTHrP (107–139) and PTHP (1–36)
We next sought to determine whether the stimulated [Ca2+]i signals induced by each PTHrP peptide were affected by the other peptide in UMR 106 cells. Pretreatment of these cells for at least 5 min with either PTHrP (107–139) or PTHrP (1–36), at 100 nM, desensitized the changes in [Ca2+]i to a second challenge with the same peptide (Fig. 3Go, A and C). On the other hand, the addition of one of these peptides after initial exposure to the other peptide failed to decrease the corresponding induced change in [Ca2+]i in UMR 106 cells (Fig. 3Go, B and D). Pretreatment with 100 nM PTHrP (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34), a PTH1R antagonist (34), decreased the effect triggered by PTHrP (1–36), although it did not affect that of PTHrP (107–139), each at 100 nM, on [Ca2+]i in these cells (Fig. 4Go). Moreover, preincubation with 100 nM PTHrP (107–111), in contrast to PTHrP (109–138), abolished the maximal effect on [Ca2+]i induced by PTHrP (107–139) in UMR 106 cells (Fig. 5Go).



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  		Figure 3. Preincubation of UMR 106 cells with either PTHrP (107–139) or PTHrP (1–36) induces homologous but not heterologous down-regulation of the respective [Ca2+]i response in UMR 106 cells. Tracings A and C show that addition of a second dose of the same PTHrP peptide after initial stimulation with 100 nM of either PTHrP (107–139) or PTHrP (1–36) blocks the [Ca2+]i response in UMR 106 cells. B and D show tracings corresponding to similar experiments but adding the other peptide after initial stimulation with each PTHrP peptide.

 


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  		Figure 4. Pretreatment with PTHrP (7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ) inhibited the [Ca2+]i response to PTHrP (1–36) but failed to affect this effect of PTHrP (107–139) in UMR 106 cells. All peptides were added at 100 nM.

 


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  		Figure 5. Pretreatment with PTHrP (109–138) failed to affect the PTHrP (107–139) effect on [Ca2+]i in UMR 106 cells (A). However, PTHrP (107–111) abolished the [Ca2+]i response triggered by PTHrP (107–139) in these cells (B). These peptides were added at 100 nM.

 
Cell stimulation with both PTHrP (107–139) and PTHrP (1–36) together at doses inducing a maximal [Ca2+]i response failed to produce a higher [Ca2+]i signal. However, cell stimulation with these peptides at 0.1 pM and 0.1 nM, respectively, which trigger a submaximal [Ca2+]i signal, elevated [Ca2+]i to 158 ± 16 nM, a higher value than that corresponding to the effect of each peptide alone (P < 0.01; n = 4) (Fig. 2Go, A and B). Moreover, we found that pretreatment with PTHrP (107–139) for at least 5 min, at a concentration inducing its maximal effect on [Ca2+]i, increased the efficacy of PTHrP (1–36) in stimulating [Ca2+]i (Fig. 6Go). In contrast, 100 nM PTHrP (38–64) was ineffective (data not shown). Both the stimulatory effect of PTHrP (107–139) on [Ca2+]i and the synergistic effect of this factor on PTHrP (1–36)-induced [Ca2+]i were unchanged by 25 nM BIM, a PKC inhibitor (35) (data not shown), or by prolonged (22 h) preincubation with 1 µM PMA, which down regulates PKC (36) (Fig. 7Go).



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  		Figure 6. PTHrP (107–139) pretreatment increases the efficacy of the [Ca2+]i response induced by PTHrP (1–36) in UMR 106 cells. Cells were preincubated for at least 5 min with 100 pM PTHrP (107–139), and then different doses of PTHrP (1–36) were added ({triangleup}). The different peptide doses are indicated as log. Each value represents the mean ± SEM of at least four measurements. *, P < 0.05, compared with PTHrP (1–36) alone (•).

 


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  		Figure 7. Prolonged preincubation with 1 µM PMA failed to affect the enhancing effect of 100 nM PTHrP (107–139) on the PTHrP (1–36)-induced [Ca2+]i signal in UMR 106 cells. The latter peptide was added at 0.1 nM.

 
Effect of PTHrP (107–139) on PTHrP (1–36)-stimulated PKA activity in UMR 106 cells
We next determined whether the PKA response to PTHrP (1–36) was also modified by PTHrP (107–139) pretreatment in UMR 106 cells. In agreement with a previous report (13), we found that the latter peptide, at 1 µM, had no significant effect on basal PKA activity in these cells, in the absence (data not shown) or presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (Fig. 8Go). Nevertheless, preincubation with this peptide for 15 min decreased the maximal increase in PKA activity induced by PTHrP (1–36) in these cells (Fig. 8Go).



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  		Figure 8. PTHrP (107–139) interacts with PTHrP (1–36)-stimulated PKA activity in UMR 106 cells. Cells were preincubated or not with 1 µM PTHrP (107–139) for 15 min, and then either PTHrP (1–36) or PTHrP (107–139), at 1 µM, were added for an additional 10 min. Data are mean ± SEM of four experiments in duplicate. *, P < 0.01, compared with nonstimulated control; a, P < 0.01, compared with PTHrP (1–36) stimulation without PTHrP (107–139) preincubation.

 
Role of [Ca2+]i on IL-6 and c-fos induction by PTHrP (107–139)
The possible involvement of [Ca2+]i in IL-6 and c-fos gene induction by PTHrP (107–139) was also investigated in UMR 106 cells. Consistent with our previous findings in human osteoblastic cells (15), this C-terminal PTHrP peptide (at 10 nM) increases IL-6 messenger RNA (mRNA) at 1 h in UMR 106 cells, an effect that was abolished by either verapamil or nifedipine (at 50 µM) or 25 nM BIM, but not by 25 µM RpcAMPS, a PKA inhibitor (37) (Fig. 9Go).



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  		Figure 9. The increase in IL-6 mRNA induced by PTHrP (107–139) in UMR 106 cells is abolished by calcium channel blockers. Cells were stimulated for 1 h in serum-free medium with this peptide, at 10 nM, in the presence or absence of verapamil or nifedipine (at 50 µM), 25 nM BIM, or 25 µM RpcAMPS. Inhibitors were added 1 h before the addition of PTHrP (107–139). Cell total RNA was then isolated, and RT-PCR was performed with IL-6-specific primers.

 
Considering the inhibitory effects of PTHrP (107–139) on both cell growth and alkaline phosphatase in these cells (13, 16), we hypothesized that this peptide would affect the expression of c-fos, a gene involved in the differentiation of these cells (38). We found that PTHrP (107–139), at 10 nM, stimulates c-fos mRNA in UMR 106 cells within 1 h, but this effect was unaffected by 3 mM EGTA or 50 µM verapamil, although it was inhibited by 6 nM BIM (Fig. 10Go).



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  		Figure 10. PTHrP (107–139) stimulates c-fos mRNA in UMR 106 cells, and this effect is not affected by calcium channel blockers. Cells were stimulated for 1 h in serum-free medium with this peptide, at 10 nM, in the presence or absence of verapamil or nifedipine (at 50 µM), 3 mM EGTA, or 6 nM BIM. Cell total RNA was then isolated, and RT-PCR was performed with c-fos-specific primers.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
PTHrP is thought to be an important factor in fetal and adult bone metabolism (9, 39). A variety of studies indicate that the cAMP/PKA pathway coupled to PTH1R activation acts as a main regulator of osteoblastic differentiation (25, 40, 41). In addition, the [Ca2+]i/PKC signaling pathway, which is also associated with PTH1R activation, seems to modulate osteoblastic function either directly or interacting with cAMP synthesis in osteoblasts (1, 8, 25, 30, 41, 42). Also, recent data suggest that the effects of PTHrP on bone metabolism might not only be mediated by interaction of its N-terminal domain with the PTH1R, but that other PTHrP regions also seem to be involved (13, 14, 15, 16, 17, 18, 19, 43, 44).

In the present study, PTHrP (1–36) triggered a dose- dependent [Ca2+]i response in cycling UMR 106 cells, consistent with previous findings using PTH (1–34) or PTHrP (1–34) in these cells (5, 6, 7, 8, 26). We found herein that PTHrP (107–139) also induced a [Ca2+]i signal, characterized by both transient and sustained phases, which was more efficient than that observed with PTHrP (1–36) in these cells. Preincubation with the latter peptide or PTHrP (107–139) induced homologous, but not heterologous, [Ca2+]i desensitization in these cells. Moreover, we found a synergistic effect of both peptides together at submaximal concentrations on Ca2+ transients in UMR 106 cells. These findings, taken together with previous findings (13, 16), support the existence of two different receptors for each PTHrP peptide in UMR 106 cells, which associated with PTHrP (107–139)-induced Ca2+ influx showing an apparent higher affinity. Our results also show that this effect of PTHrP (107–139) in these cells, in contrast to that of PTHrP (1–36), depends on Ca2+ influx through a voltage-sensitive Ca2+ channel. In this regard, the three major isoforms of L-type Ca2+ channels have been identified in osteoblastic cells, including UMR 106 cells (45).

Our data indicate that the [Ca2+]i response to PTHrP (107–139) in UMR 106 cells did not require PLC activation. Recent studies in renal cells expressing the PTH1R have shown that the low abundance of this receptor is associated with less efficient or even absent InsP3-dependent [Ca2+]i spikes after its activation (46, 47, 48). The present results do not allow us to raise any conclusion on whether a similar mechanism would also be associated with the putative PTHrP (107–139) receptor(s) in UMR 106 cells. PTX pretreatment was found to reduce the [Ca2+]i response to PTHrP (1–36) in these cells. In contrast, this pretreatment did not affect this effect triggered by PTHrP (107–139) in UMR 106 cells. Thus, a Gi protein seems to be involved in the mechanism of [Ca2+]i signaling through the PTH1R activation, as suggested by previous studies (6, 8), but not in that associated with the induced Ca2+ influx by PTHrP (107–139) in these cells.

Interestingly, a previous report has shown that PTHrP (107–138) failed to increase [Ca2+]i in UMR 106 cells in suspension after trypsinization (26). In this study, and in other studies using a similar approach, the [Ca2+]i response to either PTH (1–34) or PTHrP (1–34) was weak in these cells (5, 26). Thus, the [Ca2+]i response to either N- or C-terminal PTHrP in UMR 106 cells seems to require intercellular communication, as occurs when attached to coverslips (49, 50).

The pattern of the [Ca2+]i response to PTHrP (107–139) was found to be similar to that induced by PTHrP (107–111) in UMR 106 cells. In fact, similar effects of both peptides on cell proliferation and/or on several osteoblastic markers have previously been reported in these cells and in human osteoblastic cells (13, 14). In this regard, the 107–111 epitope has been shown to mimic the inhibitory effect of PTHrP (107–139) on osteoblastic-mediated bone resorption, and also the effects of this peptide on keratinocyte proliferation and PKC activity (18, 19, 20, 21, 36, 44). Furthermore, a recent study on the molecular structure of PTHrP (107–139), using nuclear magnetic resonance spectroscopy, has given credit to the hypothesis, based on current biological data, that the region 107–111 of this peptide would be responsible for its binding to a putative receptor (51).

In the present study, we found that PTHrP (107–139) pretreatment enhanced the efficacy of the [Ca2+]i response to PTHrP (1–36) in UMR 106 cells, an effect that seems to be related to PTHrP (107–139)-stimulated Ca2+ influx. Interestingly, a previous study has demonstrated that PTHrP (1–141) was more effective than [Tyr36]PTHrP (1–36) amide in stimulating [Ca2+]i, and also prostaglandin E2 release, in human osteoblastic osteosarcoma Saos-2 cells (52). Moreover, the former but not the latter PTHrP peptide was shown to increase Ca2+ influx even at 10 pM, the lowest dose tested (52). Our present results are consistent with, and establish a rationale to better understand, these previous findings. Previously, both endothelin and several prostaglandins have been shown to increase the [Ca2+]i response to PTH in UMR 106 cells (5, 53). These combined findings support the hypothesis that PTH1R activation of the [Ca2+]i pathway might be susceptible to homologous and heterologous modulation in the bone microenvironment. On the other hand, PTHrP (107–139) decreased PTHrP (1–36)-induced PKA activity in UMR 106 cells. This was also consistent with the lower effect of PTHrP (1–141) on cAMP production, compared with that of [Tyr36]PTHrP (1–36) amide, in Saos-2 cells (52). Because increased Ca2+ influx has proven to induce desensitization of PTH1R-mediated cAMP production in UMR 106 cells (42), we cannot rule out that the potentiating effect of PTHrP (107–139) on PTHrP (1–36)-stimulated [Ca2+]i might occur through an increased binding of the latter peptide to the PTH1R in these cells.

[Ca2+]i signals can affect gene transcription by interaction with specific promoters that contain Ca2+-responsive elements. In addition, elevation of [Ca2+]i may increase PKC activity because this is a calcium-dependent enzyme (54). In fact, activation of the [Ca2+]i/PKC pathway has been shown to modulate the response of IL-6 expression to various agonists in osteoblastic cells (23, 24). PTHrP (107–139), within the same concentration range that maximally increases [Ca2+]i in UMR 106 cells, stimulates IL-6 secretion in human osteoblastic cells, and this effect was abrogated by a PKC inhibitor (15). These previous results and the present findings support a putative role of Ca2+ influx in modulating IL-6 expression in osteoblastic cells. On the other hand, we found that PTHrP (107–139) rapidly induces c-fos mRNA, apparently dependent on PKC activation but not on [Ca2+]i changes, in UMR 106 cells. This is consistent with the previously reported effects of this peptide on growth and differentiation (13, 16), and the putative regulatory role of c-fos (38), in these cells.

The effects of PTHrP (107–139) in osteoblastic cells reported herein might have pathophysiological significance. Thus, the actions of the N-terminal region of PTHrP could be modulated not only by other bone cytokines but also by the putative C-terminal fragment PTHrP (107–139) in the bone microenvironment. The present findings also support the notion that PTHrP, by interacting with the PTH1R and other yet unidentified receptors in bone cells, can regulate bone turnover through complex mechanisms.


   Footnotes
 
1 Supported in part by a grant from the Spanish Ministry of Health (FIS 00/0125). Portions of this study were presented at the XXVIth and XXVIIth European Symposia on Calcified Tissues, in Maastricht, The Netherlands, and Tampere, Finland, respectively. Back

Received December 28, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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