PTH(1-34) Affects Osteoprotegerin Production in Human PDL Cells in vitro

Abstract

Since periodontal ligament (PDL) cells exhibit several osteoblastic traits, we hypothesized that human PDL cells will respond to hormonal stimulation in an osteoblast-like manner. Confluent and pre-confluent PDL cells from six patients were challenged with PTH(1-34). Cell number, ALP, osteocalcin, osteoprotegerin, and RANKL expression were determined. Intermittent PTH(1-34) treatment of confluent PDL cells caused a significant increase in proliferation, whereas differentiation and osteoprotegerin production decreased significantly. In pre-confluent PDL cells, this treatment regimen induced a biphasic decrease in proliferation, but a biphasic increase in differentiation and osteoprotegerin production. Continuous PTH(1-34) exposure enhanced proliferation but inhibited osteocalcin production in confluent cells and stimulated osteoprotegerin production in pre-confluent PDL cells. RANKL was hardly detectable and unaffected by PTH(1-34) treatment. These results indicate that human PDL cells respond to PTH(1-34) in an osteoblast-like manner, and that the PTH(1-34) effect depends on the maturation state of the cells and on the mode of administration.

INTRODUCTION

Periodontal ligament (PDL) cells were demonstrated to exhibit several osteoblastic traits (Chou et al., 2002), suggesting that these cells have the potential to differentiate into osteoblasts (Basdra and Komposch, 1997). Whether PDL cells also respond to hormonal stimulation in an osteoblast-like manner has not been extensively investigated so far. However, a better understanding of PDL cell response to hormonal stimulation would enable us to influence regenerative periodontal processes (Skripitz and Aspenberg, 2001; Barros et al., 2003) and the formation of reparative cementum occurring after tooth-root resorption induced by orthodontic tooth movement. The hormone of interest in this study was parathyroid hormone (PTH). PTH has been demonstrated, in several in vivo and in vitro studies, to modulate the responses of cells in terms of catabolic or anabolic actions (Ishizuya et al., 1997), depending on the mode of its administration (Morley et al., 2001). Catabolic effects have been assigned to continuous PTH exposure (Lee and Lorenzo, 1999), as opposed to anabolic effects in response to intermittent PTH challenge (Iida-Klein et al., 2002). Although the exact molecular mechanisms that mediate the effects of PTH remain to be elucidated, these responses have partially been attributed to modulation of osteoblast precursor proliferation and osteoblast apoptosis (Jilka et al., 1999), mature osteoblast function (Canalis et al., 1989), and also to changes in the production of local factors which have been discovered to be key regulatory molecules in bone remodeling, such as osteoprotegerin (OPG) and receptor activator of nuclear factor kappaB ligand (RANKL) (Huang et al., 2004). Since studies implicating OPG and RANKL expressed in PDL cells (Kanzaki et al., 2001) provided evidence for a potential role for PDL cells in hard-tissue repair, we wished to analyze whether PTH(1-34) might alter the responses of PDL cells with respect to proliferation, differentiation, and OPG and RANKL production. We hypothesized that an intermittent PTH treatment would result in a modulation of the OPG/RANKL ratio, favoring osteogenesis over bone resorption, whereas continuous PTH(1-34) challenge would induce a decrease in differentiation parameters and OPG production. Furthermore, we speculated that the PTH(1-34) effect would be time- and maturation-state-dependent.

MATERIALS & METHODS

Cell Culture and PTH Administration

PDL cells were scraped from the middle third of the roots of premolars from six different healthy human donors, both male and female, aged between 12 and 14 yrs, showing no clinical signs of periodontitis. The teeth had been extracted for orthodontic reasons, with informed parental consent and following a protocol approved by the ethics committee of the University of Bonn. Fourth- to eighth-passage cells were characterized with the use of markers described by Basdra and Komposch (1997) and Chou et al.(2002) (data not shown), prior to being plated at a seeding density of 9300 cells/cm² in 24-well plates (n = 6). Cells were cultured in DMEM containing 10% fetal bovine serum and 0.5% antibiotics in an atmosphere of 5% CO₂. For experiments with pre-confluent cells, cells were treated with PTH(1-34) following a culture period of 24 hrs. For evaluation of the responses of more mature cells, PDL cells were cultured to confluence for 4–5 days prior to PTH(1-34) exposure.

Preliminary experiments in our laboratory did not show any significant dose-dependence of the PTH effect in PDL cells. Thus, to assess whether PTH acts differently at different stages of maturation, as noted by Isogai et al.(1996), we cultured pre-confluent and confluent cells in the presence of 10⁻¹² M PTH(1-34) (Sigma Aldrich, Seelze, Germany) for 0, 1, 3, 6, or 24 hrs within 3 incubation cycles of 48 hrs each. For the remaining time, experimental media were replaced by media without PTH(1-34). Additionally, we subjected pre-confluent and confluent cells to continuous PTH(1-34) challenge, to investigate whether the mode of PTH administration affects the cellular response. Untreated cells cultured in the presence of the PTH-vehicle ethanol served as controls.

RNA Isolation, Reverse Transcription, and Polymerase Chain-reaction

Total RNA was isolated with use of the RNeasy mini kit (Qiagen, Hilden, Germany). Afterwards, 1 μg of RNA was reverse-transcribed with 200 ng of the respective GAPDH, OPG, and RANKL antisense primer in a 15-μL volume, by means of the Amersham-Pharmacia-Biotech RT kit (Amersham Biosciences, Piscataway, NJ, USA). The primers used to amplify human cDNA (Invitrogen, Karlsruhe, Germany) were synthesized according to the sequences specified in the Table. The PCR reaction mix contained 15 μL of the reverse-transcription reaction as the cDNA template in a 100-μL total reaction volume. The amplification was performed including 29 cycles of denaturation, annealing, and extension. The annealing temperatures were 55°C for OPG and RANKL and 60°C for GAPDH. Afterward, each PCR product was run in a 1.5% agarose gel and analyzed in a UV-transilluminator. Serving as an example, the PCR products for OPG, RANKL, and GAPDH following PTH exposure of confluent PDL cells are depicted in Fig. 3A . We quantified the levels of amplified DNAs by averaging 3 separate measurements, normalized to the endogenous reference gene GAPDH. Samples without reverse-transcriptase treatment served as controls.

PTH Effect on Cell Number, Differentiation, and Local Factor Production

At harvest, cells were released from the culture surface by trypsinization. Thereafter, the cell suspension was centrifuged, and the cell pellet re-suspended in 0.9% NaCl. Finally, the cell number was determined by the use of a cell counter (Moelab, Hilgen, Germany). Cells harvested in this manner exhibited > 95% viability based on Trypan blue exclusion.

Alkaline-phosphatase-specific activity was measured in lysates of isolated cells, as described previously (Bretaudiere and Spillman, 1984). The levels of osteocalcin, osteoprotegerin, and sRANKL in the conditioned media were assayed with the use of commercially available enzyme-linked immunoassay kits (IBL GmbH, Hamburg, Germany; Immundiagnostik AG, Bensheim, Germany).

Statistical Analysis

For any given experiment, each datapoint represents the mean ± SEM of 6 independent cultures. Variance and statistical significance of data were analyzed by Bonferroni’s modification of Student’s t test. P-values < 0.05 were considered to be significant. Each set of experiments was repeated twice and analyzed separately. Both sets of experiments had comparable results.

RESULTS

In confluent PDL cells, the cell number was 1.283 ± 0.094 x 10⁵ cells/well at the beginning of the PTH(1-34) exposure. Intermittent PTH(1-34) treatment caused a significant time-dependent increase in cell number, with a maximum increase of 54.4% following 10⁻¹² M PTH(1-34) application for 6 hrs/cycle. Compared with the untreated controls, significantly higher cell numbers were observed after intermittent treatment for 1 hr, 3 hrs, and 6 hrs/cycle (p < 0.05), whereas the increase in proliferation following incubation of the cells in the presence of PTH(1-34) for 24 hrs/cycle displayed only a trend without any statistical significance. Continuous PTH(1-34) challenge of confluent cells resulted in an increase in cell numbers comparable with the intermittent administration for 6 hrs/cycle. In pre-confluent PDL cells, the cell number was 0.342 ± 0.015 x 10⁵ cells/well at the onset of the PTH(1-34) challenge. Intermittent challenge with 10⁻¹² M PTH(1-34) exerted a biphasic effect on cell number, with a significant decrease in response to a 1 hr/cycle exposure. Longer PTH treatments resulted in a gradual increase of cell numbers back to control levels. When PTH(1-34) was administered continuously, cell numbers did not change significantly compared with the untreated control (Fig. 1).

Although alkaline-phosphatase-specific activity showed large standard errors of the mean due to variation in the cultures of the different donors, there seemed to be a slight decrease in ALP activity in confluent PDL cells as opposed to an increase in pre-confluent PDL cells. However, these findings did not prove to be statistically significant (data not shown).

In contrast to the PTH effect on cell number, the same treatment regimen resulted in a gradual time-dependent reduction in osteocalcin protein levels. There were no statistically significant differences observed with respect to the mode of administration, since continuous PTH exposure also resulted in a decrease in osteocalcin production. In pre-confluent PDL cells, differentiation was enhanced, as shown by the significant increase in osteocalcin production following intermittent PTH(1-34) exposure for 1 hr/cycle. A longer presence of PTH(1-34) in the cultures resulted in a decline to control levels, as was also observed for a continuous presence of PTH(1-34) (Fig. 2 ).

OPG mRNA expression levels were reduced in a biphasic manner compared with the untreated control cultures in confluent PDL cells, with the least reduction after PTH(1-34) treatment for 6 hrs/cycle. Continuous PTH administration did not alter OPG expression significantly. In pre-confluent PDL cells, OPG mRNA expression was not affected by intermittent PTH exposure. Similarly, continuous administration did not exert a significant effect compared with the untreated control, but OPG expression was significantly higher than in the experimental groups treated intermittently for 1 hr/cycle and 6 hrs/cycle, respectively (Fig. 3B ). At the translational level, OPG production was reduced in a time-dependent manner in confluent PDL cells, as opposed to the PTH(1-34) effect in pre-confluent cells, in which protein levels were not affected, except for the 6 hrs/cycle treatment group, where OPG protein production was elevated by 49.2% compared with the untreated control. Continuous PTH(1-34) challenge did not significantly alter OPG levels in confluent cells, but caused a distinct increase in less mature cells (Fig. 3C ).

We were unable to detect RANKL in any of the cultures, at either the transcriptional or the translational level.

DISCUSSION

The results of this study show, for the first time, that PTH modulates the ability of PDL cells to proliferate, differentiate, and produce OPG, dependent on the maturation state of the cells and on the mode of PTH administration.

Previous studies have demonstrated that low-dose PTH exposure results in the increased proliferation of osteoprogenitor cells (Nakajima et al., 2002). This is in line with our observations of enhanced cell numbers in PTH-challenged cultures. It is evident that changes in the cell number might result from altered proliferation and from modulation of apoptosis. Unpublished data from our laboratory, from measurements of BrdU incorporation into PTH-treated PDL cells, mirror the changes in cell number we observed. However, this does not exclude the possibility of prolonged cell survival. Analysis of data presented on osteoblasts indicated that the PTH effect on apoptosis strongly depends on the cell status (Chen et al., 2002). In their study, PTH promoted cell viability in pre-confluent cells, while reducing viability in confluent cells.

In our investigation, differentiation parameters were also affected by PTH treatment. Our data are supported by the work of Isogai et al.(1996), who found that PTH preferentially stimulates osteoblast differentiation in immature cells, while inhibiting it in mature cells, as indicated by decreased alkaline-phosphatase-specific activity and osteocalcin production in the latter. Similar observations were made in our study with PDL cells. These findings give further support to the idea that, although the mixed population of PDL cells is comprised mainly of fibroblasts, these cells display traits typical of osteoblasts. This holds true not only for marker gene expression, but also for their response to hormonal stimulation. Apparently, there is a stronger PTH influence on events occurring later in the cascade of differentiation than there is on early events, as outlined by the stronger effect of PTH on osteocalcin levels than on alkaline phosphatase activity. Contrasting reports postulate that mature osteoblastic cells respond to PTH in an anabolic fashion (Ishizuya et al., 1997; Schiller et al., 1999). Different culture conditions, treatment modalities, cell densities, and the sources of the cells used might account for these apparent discrepancies. Finally, we observed a reduction in osteocalcin protein levels in all groups except for the cultures exposed to PTH(1-34) 24 hrs/cycle in confluent cells, which might be a statistical phenomenon. If we had pooled the PTH(1-34)-treated groups, there still might have been a significant reduction in osteocalcin levels.

In contrast to the marked increase in OPG production following intermittent and continuous PTH exposure of pre-confluent cells, neither RANKL nor sRANKL was sensitive to PTH. The physiological relevance of these findings should be evaluated in the light of the OPG/RANKL ratio, which ultimately determines the ability of osteoblasts to coordinate the sequence of osteoclast differentiation during the bone remodeling cycle (Gori et al., 2000). Thus, the net effect was a micro-environment conducive to periodontal repair. In contrast, we detected a transient decrease in OPG in mature PDL cells, suggesting that less OPG would be available at potential sites of osteoclast formation in vivo. Both factors have been detected in PDL cells (Kanzaki et al., 2001). The absence of RANKL in our samples might be explained by different PCR protocols. Alternatively, any changes that may have occurred at the protein level might have been below the threshold of detection of the immunoassay kit we used. The different patterns of OPG mRNA expression and OPG protein production observed in confluent PDL cells might result from the fact that both were determined at the end of the third cycle of PTH(1-34) treatment. Since changes in mRNA expression usually precede alterations in protein production, it is reasonable that the changes in OPG protein levels do not correlate with those in OPG transcription.

As for continuous PTH challenge, our data are not in accordance with an increasing bulk of literature linking continuous PTH exposure to a rapid and sustained decrease in OPG and reciprocal increase in RANKL (Horwood et al., 1998; Lee and Lorenzo, 1999; Kanzawa et al., 2000; Ma et al., 2001; Halladay et al., 2002). Ma and co-workers (2001) observed the continuous PTH effects as early as after 1 hr in a rat model, with a peak after 6 hrs. Our measurements were obtained after a culture period of 7 days in pre-confluent and 10 days in confluent PDL cells. Although PTH receptor levels and responsiveness of the receptor to stimulation appear to increase during osteoblastic phenotypic maturation in vitro (Bos et al., 1996; McCauley et al., 1996; Kondo et al., 1997), sustained PTH stimulation for a culture period of 10 days might lead to a down-regulation of the number or sensitivity of PTH receptors in PDL cells, as part of a feedback mechanism. This might also explain, at least in part, why we did not observe adverse effects of continuous PTH(1-34) treatment as compared with intermittent exposure.

In conclusion, human PDL cells respond to PTH stimulation in an osteoblast-like manner and, therefore, bear the potential to be regulatorily involved in hard-tissue repair. Further research is needed to clarify whether the observed changes in OPG production are sufficient to result in a modification of the reparative properties of the PDL.

Table.

Primer Sequences Used for Polymerase Chain-reaction

Gene	Sequence	Product Size	Accession Number
Osteoprotegerin	(5′-3′): GCT AAC CTC ACC TTC GAG (sense)	324 bp	U94332
	(5′-3′): TGA TTG GAC CTG GTT ACC (antisense)
RANKL	(5′-3′): GCC AGT GGG AGA TGT TAG (sense)	486 bp	AF019047
	(5′-3′): TTA GCT GCA AGT TTT CCC (antisense)
GAPDH	(5′-3′): CGT CTT CAC CAC CAT GGA GA (sense)	300 bp	BC083511
	(5′-3′): CGG CCA TCA CGC CAC AGT TT (antisense)

Figure 1.

Effect of PTH(1-34) treatment on regulation of cell number in confluent and pre-confluent PDL cells. Cells were either treated intermittently with 10⁻¹² M PTH(1-34) for 0, 1, 3, 6, or 24 hrs during 3 cycles of 48 hrs each, or were exposed continuously (cont). Data were acquired from one of two separate experiments, both yielding comparable results. Each value is the mean ± SEM for 6 independent cultures. *P < 0.05, experimental group vs. untreated control. #P < 0.05, experimental group vs. PTH(1-34) for 1 hr/cycle; •P < 0.05, experimental group vs. PTH(1-34) for 3 hrs/cycle.

Figure 2.

Decrease in osteocalcin production in confluent PDL cells following intermittent and continuous exposure to PTH(1-34) as opposed to a significant increase in response to 1 hr/cycle challenge in pre-confluent cells. Cells were either treated intermittently with 10⁻¹² M PTH(1-34) for 0, 1, 3, 6, or 24 hrs during 3 cycles of 48 hrs each, or were challenged continuously (cont). Data were acquired from one of two separate experiments, both yielding comparable results. Each value is the mean ± SEM for 6 independent cultures. *P < 0.05, experimental group vs. untreated control; #P < 0.05, experimental group vs. PTH(1-34) for 1 hr, 3 hrs, 6 hrs/cycle.

Figure 3.

PTH (1-34) effect on OPG expression and production. (A) Serving as an example, the RT-PCR for OPG, RANKL, and GAPDH following PTH exposure of confluent PDL cells from one out of six donors is depicted. (B) Biphasic reduction of OPG mRNA expression following intermittent PTH(1-34) challenge of confluent PDL cells, as compared with no effect in pre-confluent PDL cells. PDL cells were treated intermittently with 10⁻¹² M PTH(1-34) for 0, 1, 3, 6, or 24 hrs during 3 cycles of 48 hrs each, or were exposed continuously (cont). Data were taken from one of two separate experiments, both yielding comparable results. Each value is the mean ± SEM for 6 independent cultures. *P < 0.05, experimental group vs. untreated control; #P < 0.05, experimental group vs. PTH(1-34) for 1 hr/cycle; •P < 0.05, experimental group vs. PTH(1-34) for 6 hrs/cycle. (C) Time-dependent reduction in OPG protein levels in response to intermittent PTH(1-34) challenge of confluent PDL cells. In contrast, there was an increase in OPG protein production following intermittent PTH(1-34) exposure for 6 hrs/cycle, and also after continuous treatment in pre-confluent cells. PDL cells were treated intermittently with 10⁻¹² M PTH(1-34) for 0, 1, 3, 6, or 24 hrs during 3 cycles of 48 hrs each, or were exposed continuously (cont). *P < 0.05, experimental group vs. untreated control; #P < 0.05, experimental group vs. intermittent PTH(1-34) exposure.

Footnotes

Notes

Acknowledgements

The authors thank K. Hoffmann for technical assistance and C. Maelicke for her help in preparing the manuscript. This research was supported by a research grant from the BONFOR program (O-135.0006) of the University of Bonn, Germany.

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