Parathyroid Hormone (1-34) Receptor-Binding and ... - Springer Link

Calcif Tissue Int (1998) 62:255–259

© 1998 Springer-Verlag New York Inc.

Parathyroid Hormone (1-34) Receptor-Binding and Second-Messenger Response in Rat Incisor Odontoblasts T. Lundgren, V. Stenport, A. Wetter, A. Linde Department of Oral Biochemistry, Faculty of Odontology, Go¨teborg University, Medicinaregatan 7B, S-413 90 Go¨teborg, Sweden

Received: 12 March 1997 / Accepted: 26 June 1997

Abstract. Even though indirect evidence indicates that PTH exerts an anabolic effect on dentinogenesis, the existence of PTH receptors and any second-messenger response in odontoblasts have not been demonstrated. The aim of this study was to investigate whether rat incisor odontoblasts express PTH receptors, and to identify which second messenger pathway the hormone may activate. Odontoblasts were dissected from rat incisors. Amino-terminal (1-34) fragment rat PTH [rPTH(1-34)] conjugated to fluorescein isothiocyanate visualized receptor sites on the cell surface. Upon incubation of odontoblasts with rPTH(1-34), cAMP formation was increased. However, no fluctuations in intracellular calcium activity were observed upon rPTH(1-34) stimulation when using Fura-2 as a Ca2+ probe. In long-time incubations, stimulation with PTH(1-34) upregulated APase activity. The results demonstrate that rPTH(1-34) evokes an anabolic response in dentinogenically active odontoblasts, and that this may be mediated through the protein kinase A/cAMP pathway, whereas no indications for Ca2+ as a second messenger were evident. Key words: Alkaline phosphatase — Dentinogenesis — Odontoblasts — Parathyroid hormones — Rats — Second messenger systems.

Several studies have been performed to elucidate the effects of parathyroid hormone (PTH) on mineralized tissues. It has been demonstrated that osteoblasts, osteocytes, osteosarcoma cells, cementoblasts, and dental pulp fibroblasts respond to PTH [1–6], and that cyclic adenosine monophosphate (cAMP) [7–12] and possibly Ca2+ [13–19] act as second messengers, resulting in liberation of both Ca2+ and phosphate ions from bone into the circulation. The presence of several second messengers might explain that PTH, except for enhancing bone resorption, also has a documented anabolic effect on bone [20–24]. Alkaline phosphatase (APase) activity is expressed by several cell types, both mineralizing and nonmineralizing cells. Due to its high expression in hard tissue-forming cells, the enzyme is regarded as a feature of a mineralizing cell phenotype and is often used as a marker of calcified tissue formation. In cultured osteoblast-like cells, APase has been co-localized with PTH receptors [25], and stimulation with the amino-terminal human PTH(1-34) upregulates the activity of APase in such cell lines [26]. APase is, however,

Correspondence to: T. Lundgren

not specific for the anabolic action of all cells. In osteosarcoma cells, e.g., the effect of amino-terminal PTH was reported to be the opposite, a decrease in activity of APase [27, 28]. Osteoblasts and odontoblasts, bone- and dentin-forming cells, respectively, are different cells types, but with many similarities. They synthesize and secrete the organic matrices of the respective tissue, which to a large extent comprise the same components and which are mineralized by the cells; mature bone and dentin display many common features. Considering the similarities between the two cell types, there are reasons to believe that the dentin-forming cells also express PTH receptors. The functional importance of PTH binding during dentinogenesis is not obvious, but there are data that imply a role for this hormone. As an effect of feeding rats a calcium-deficient and vitamin D-free diet, the levels of serum PTH were considerably increased. This occurred simultaneously as the width of the predentin in their incisors increased [29–31], shown to be due to an overall increased metabolic activity in the odontoblasts with a rise in energy metabolism, APase activity and protein synthesis [32–35]. This increase in metabolic activity might thus be due to an induction of a secondary hyperparathyroidism as a result of the dietary restraint [31], indicating that PTH may have an effect during dentinogenesis. The aim of this study was to investigate whether dissected rat incisor odontoblasts express PTH(1-34) receptors, identify which second-messenger pathway the hormone activates in these cells and, as an indication of a putative anabolic effect, to examine the influence of PTH(1-34) stimulation on APase activity. Materials and Methods Fura-2/AM and pluronic F-127 were purchased from Calbiochem (La Jolla, CA, USA). Bovine serum albumin, the amino-terminal fraction (1-34) of rat PTH (rPTH(1-34)), collagenase (1.8 U/mg), and APase substrate (p-nitro-phenyl phosphate (p-NPP)) were obtained from Sigma (St. Louis, MO, USA). The cAMP 125I radioimmunoassay (RIA) kit was from New England Nuclear-DuPont (Boston, MA, USA). Quick Tag™ FITC Conjugation Kit was from Boehringer (Mannheim, Germany), and the BCA (bicinchoninic acid) Micro Protein Assay Reagent from Pierce (Rockford, IL, USA). Dulbecco’s Modified Eagle Medium/Mix F12 (DMEM) was from Gibco BRL Life Technologies (Gaithersburg, MD, USA). Spectra/Port dialysis tubing (nominal Mr cutoff 3,500) was from Spectrum Medical Industries (Houston, TX, USA). DMSObased stock solutions of Fura-2/AM (Fura-2 acetoxymethyl ester) (5 mM) and pluronic F-127 (10%) were prepared. The rPTH(1-34) was dissolved in 5 mM acetic acid. The Sprague-Dawley rats, body weight 250–350 g, were bred by B & K Universal (Sollentuna, Sweden). The medium referred to as the incubation medium contained (in mM) NaCl 120, Hepes 20, KCl 4.7, KH2PO4 1.2,

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MgSO4, 1.2, CaCl2 1.25, glucose 10, and was adjusted to pH 7.4. The APase assay buffer contained 20 mM Hepes, 5 mM MgCl2, and 1 mM p-NPP; the pH was adjusted to 8.8.

FITC-rPTH(1-34) Conjugation and Fluorescence Microscopy The conjugation procedure was performed according to the manufacturer’s instructions. In each conjugation batch, 250 mg rPTH(134) was coupled to an excess of FITC. Separation of conjugated from nonconjugated PTH was performed in a miniaturized Sephadext G-25 column during centrifugation. Odontoblasts were dissected from Sprague-Dawley albino rat incisors [36], each cell sample being derived from two animals. Cells suspended in the incubation medium were collected as a pellet at 4000 × g for 10 minutes and were resuspended in 400 ml of the incubation medium. Fifty microliters of the FITC-rPTH(1-34) conjugate, giving a final concentration of 1.5 mM, was added to the resuspended pellet, and the sample was incubated at room temperature. After 20 minutes, the tube was filled with ice cold incubation medium in order to stop the reaction. The pellet was washed twice at 4°C, 2000 × g for 10 minutes. Incubation medium (200 ml) was added, and the pellet was briefly vortexed. The sample was placed on a microscope slide, and individual cells were photographed in a fluorescence microscope, equipped with 546 nm excitation and 590 nm emission filters. The fluorescent cells were also photographed in phase contrast. For each test sample, a negative sample was made by incubating the cells with 125 mg (200 ml) nonconjugated hormone for 10 minutes.

Fura-2 Measurement of [Ca2+]i Odontoblasts were dissected from rat incisors. Each sample consisted of cells from six incisors in 1 ml of the incubation medium. In the presence of Pluronic F-127 (0.025%) and bovine serum albumin (2%), the cells were loaded with Fura-2 (5 ml of the stock solution) at 37°C with slow magnetic stirring for 30 minutes, after which the cells were spun down and washed twice (4000 × g, 10 minutes). rPTH(1-34) (100 nM) was added to the cuvette after initiation of a [Ca2+]i measurement. After assays of hormonal effects, calibrations were made by additions of Triton X-100 followed by EGTA. Every single addition of liquid to the cuvette corresponded to 1% (15 ml) of the total sample volume (1.5 ml). Intracellular calcium activity ([Ca2+]i) was measured with a Perkin-Elmer LS 50B double-beam fluorescence spectrophotometer at 340 and 380 nm excitation wavelengths, and a 510 nm emission wavelength, using a quartz cuvette at 37°C with slow magnetic stirring. [Ca2+]i was calculated on a computerized measuring device, using Kd 4 220 for Fura-2. In order to quantitate the Ca2+ fluxes, the area between the graphs and the x-axis was measured during 100-second intervals. The total protein content in the samples was measured using a BCA protein assay kit.

RIA Measurement of cAMP Odontoblasts were dissected from rat incisors. Each sample contained cells from six incisors in 1 ml of the incubation medium. To control samples, only carrier (100 ml 5 mM acetic acid) was added. To the other cell samples 100 nM rPTH(1-34) was added. The samples were incubated for either 5 or 15 minutes. After incubation, the cells were collected by centrifugation at 4000 × g for 10 minutes. The pellets were resuspended in 1 ml ice-cold 90% propanol/10% water and the cells were homogenized with 10 firm strokes in an all-glass homogenizer. The samples were then left for cAMP extraction at 4°C for 24 hours. After extraction, the homogenates were dried by evaporation in a water bath at 60°C. For detection of cAMP in the samples, a RIA (125I) kit was used, the radioactivity being detected by a g-scintillation measuring device. The radioactivity was related to the amount of protein in each sample (CPM/mg), assayed using BCA protein assay.

T. Lundgren et al.: PTH Receptors in Odontoblasts

APase Activity Odontoblasts were dissected from rat incisors. Each sample consisted of cells from six to eight incisors in 1 ml of the incubation medium. The cells were collagenase treated in the presence of 1 mM CaCl2 for 20 minutes at 37°C. The odontoblasts were washed twice (500 × g, 10 minutes) in the same medium in the presence of 0.1 mM EGTA. The pellets were suspended in 250 ml of DMEM/ F12. To the test samples, 200 nM of rPTH(1-34) was added, controls receiving carrier only. The cell suspensions were incubated for 24 hours at 37°C in a tissue incubator at 5% CO2 in air. After addition to 500 ml butanol, the cells were homogenized with 10 firm strokes in an all glass-homogenizer. After 2 hours of shaking, the homogenates were transferred to tubings for dialysis overnight against 1 mM MgCl2 in 20 mM Hepes buffer, pH 8.2. APase activity was assayed in the samples using assay buffer as substrate. After 30 minutes, color intensity of the samples was measured using an E-maxt microplate reader (Molecular Devices, San Francisco, CA, USA) at 405 nm. The APase activity values were normalized against protein content of the samples as measured with a BCA protein assay kit. Statistical analyses were performed using a computerized, unpaired Students t-test or the Spearman rank correlation test (StatView 4.01). The experiments were approved by the Ethical Committee for Animal Experiments at Go¨teborg University. Results Visualization of PTH Receptors

The interaction of FITC-conjugated rPTH(1-34) with plasma membrane-associated PTH receptors on isolated odontoblasts was assayed in vivo by means of fluorescence microscopy. Odontoblasts incubated with this probe revealed a bright fluorescence (Fig. 1A, C). When including unconjugated rPTH(1-34) in excess during incubation, the fluorescence was damped, demonstrating a competitive inhibition (Fig. 1B). Though odontoblasts exhibited binding of the FITC-rPTH(1-34) conjugate, erythrocytes included in the samples did not (Fig. 1C). Measurement of [Ca2+]i

To measure possible alterations in [Ca2+]i induced by rPTH(1-34) receptor binding, fluorescence from dissected odontoblasts in suspension was recorded using Fura-2 as a Ca2+ probe. The cell content in each measurement corresponded to a protein content of 8.0 ± 1.2 mg (mean ± SD, n 4 18; range 5.8–9.2). The baseline value for [Ca2+]i was normally at or somewhat below pCa 7. No fluctuations were recorded with addition of rPTH(1-34) to the incubations (data not shown). cAMP Formation

Effects of rPTH(1-34) on cAMP contents in odontoblasts is shown in Figure 2. Without adding any drugs, the odontoblasts contained a small amount of cAMP. When incubated for 5 or 15 minutes in the presence of rPTH(1-34), the odontoblast cAMP amount increased 2- to 5-fold compared with controls (both P < 0.01). APase Activity

Odontoblast APase activities, assayed as p-NPPase activity,


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Fig. 1. Interaction of FITC-conjugated rPTH(1-34) with plasma membrane-associated PTH-receptors was demonstrated in vivo by fluorescence microscopy. (A) Receptors on the odontoblasts were visualized as a fluorescent plasma membrane. (B) When unconjugated rPTH(1-34) as well was present in excess during incubation, the fluorescence was damped in a competitive mode. Bar 4 20 mm. (C) The specificity of binding between hormone and receptor is demonstrated by a red blood cell (arrow), adjacent to an odontoblast. In contrast to the odontoblast, the erythrocyte reveals no fluorescence. (D) The same odontoblast and erythrocyte in phase contrast mode. Bar equals 10 mm.

Fig. 2. Odontoblast cAMP contents. When hormone was not added, the odontoblasts expressed a low baseline value of cAMP. After incubation with rPTH(1-34) for 5 or 15 minutes, the amount of cAMP was increased (P < 0.01; n 4 3).

are shown in Figure 3. When treated with rPTH(1-34) in 24-hour incubations, odontoblasts expressed a 2.8-fold increase in APase activity compared with control cells incubated for the same period with carrier only (P < 0.05, n 4 6). Discussion

Little is known about the hormonal regulation of dentinogenesis. In this study, the presence of PTH(1-34) receptors was demonstrated in dentinogenically active odontoblasts. Furthermore, the second-messenger response to this recep-

Fig. 3. Odontoblast APase activity. When treated with rPTH(134) in 24-hour incubations, odontoblasts expressed a 2.8-fold increased (P < 0.05, n 4 6) APase activity. Untreated cells, receiving carrier only, expressed some APase activity as well, but at low levels. APase assayed with p-NPP as a substrate. Mean values ± SEM.

tor binding was examined, revealing a synthesis of cAMP. In contrast, no fluctuations in [Ca2+]i were observed as a result of rPTH(1-34) binding, thus suggesting that cAMP is the sole PTH second messenger in odontoblasts. An upregulation of APase was demonstrated upon PTH stimulation. The odontoblasts line the pulpal aspect of dentin (really the unmineralized predentin) and are responsible for the formation of dentin and the production of its constituents. Odontoblasts thus line the surface of the tissue they have formed in a manner similar to the osteoblasts in bone. During active dentinogenesis, the odontoblasts are columnar, highly polarized, secretory cells. After formation of dentin, the cells decrease in height and density of organelles [37, 38]. Isolation of dentinogenically active odontoblasts is easily performed by a micro-dissection technique [36]. A va-

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riety of morphological and biochemical characterizations of this noncycling cell type have been performed [29, 31–33, 36–38]. The amino-terminal (1-34) portion of PTH, containing the first 34 amino acid residues, has been demonstrated to exhibit characteristics similar to the native hormone comprising 84 amino acids. It binds to the same receptor and stimulates adenylate cyclase equally. Hence, the aminoterminal (1-34) of rat PTH was used in these experiments. Localization of PTH receptors on bone cells has been made by autoradiography, using 125I-labeled PTH [35, 39]. In the present study, rPTH(1-34) was conjugated instead to fluorescein isothiocyanate (FITC). Conjugation of FITC directly to PTH has not been previously used. The competitive decrease in fluorescence, demonstrated in the control experiments where unconjugated rPTH(1-34) in excess was added together with the FITC-rPTH(1-34) conjugate (Fig. 1B), gives evidence for the specificity in interaction. Some authors have suggested a differentiated response to the hormone, where expression of PTH receptors might be dependent on the cellular stage of differentiation. In the studies of Rouleau et al. [40, 41], for example, PTH bound to bone cells other than the fully differentiated, mature osteoblasts. Freshly isolated odontoblasts from rat incisor, however, comprise primarily postmitotic, terminally differentiated cells. APase activity is regarded as a characteristic of a calcified tissue cell phenotype and is often used as a marker of hard tissue formation. In cultured osteoblast-like cells, APase has been co-localized with PTH-receptors [25]. Stimulation with the amino-terminal hPTH(1-34) elevated the activity of APase in osteoblast-like cells [26], being in accordance with the results of this study. In some osteosarcoma cells, however, the effect of amino-terminal PTH has been reported to be the opposite: a decrease in APase activity. In contrast, the carboxy-terminal portion increased the activity in those cells [27, 28]. In one report, the stimulatory effect of carboxy-terminal human PTH(53-84) was dependent on whether the osteosarcoma cells were dexamethasone treated or not [42]. It might be that the aminoterminal portion of PTH is responsible for anabolic effects of the hormone, as the expression of the mineralization marker APase increases in odontoblasts and some osteoblast-like cell lines. It has been shown that functional domains for anabolic activity lie within the 1-34 fragment. The residues 1-7, e.g., is the activation domain for adenylate cyclase/PKA [43, 44]. Receptor binding of the carboxylterminal to other types of mineralizing cells induces a decreased enzyme activity, which might be related to downregulation of anabolic activities. The role of PTH in the regulation of dentin formation is not clear, nor is the role of endocrine regulation of dentinogenesis in general, but earlier data imply that the hormone might have an anabolic effect. Secondary hyperparathyroidism in adult rats, as a result of lowered serum Ca2+ levels, resulted in an increased synthesis of the organic components of dentin, with an increased appositional rate of dentin formation, as well as a rise in odontoblast APase, cytochrome oxidase, and lactate dehydrogenase activities [29–34]. In cultured murine embryonic molars, however, Sakakura et al. [45, 46] noted an inhibitory effect of PTH on predentin formation, which was attributed to an inhibitory effect on collagen synthesis. An alternative explanation is that those authors, in fact, observed the effects of PTH on the differentiation of the odontogenic tissues, rather than an effect on dentin production as such.


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