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. Author manuscript; available in PMC: 2016 Jul 19.
Published in final edited form as: J Dent Res. 1998 Oct;77(10):1779–1790. doi: 10.1177/00220345980770100501

Autoregulation of Periodontal Ligament Cell Phenotype and Functions by Transforming Growth Factor-β1

TA Brady 1, NP Piesco 2, MJ Buckley 3, HH Langkamp 2, LL Bowen 3, S Agarwal 2,✉
PMCID: PMC4950996  NIHMSID: NIHMS802055  PMID: 9786634

Abstract

During orthodontic tooth movement, mechanical forces acting on periodontal ligament (PDL) cells induce the synthesis of mediators which alter the growth, differentiation, and secretory functions of cells of the PDL. Since the cells of the PDL represent a heterogeneous population, we examined mechanically stress-induced cytokine profiles in three separate clones of human osteoblast-like PDL cells. Of the four pro-inflammatory cytokines investigated, only IL-6 and TGF-β1 were up-regulated in response to mechanical stress. However, the expression of other pro-inflammatory cytokines such as IL-1β, TNF-α, or IL-8 was not observed. To understand the consequences of the increase in TGF-β1 expression following mechanical stress, we examined the effect of TGF-β1 on PDL cell phenotype and functions. TGF-β1 was mitogenic to PDL cells at concentrations between 0.4 and 10 ng/mL. Furthermore, TGF-β1 down-regulated the osteoblast-like phenotype of PDL cells, i.e., alkaline phosphatase activity, calcium phosphate nodule formation, expression of osteocalcin, and TGF-β1, in a dose-dependent manner. Although initially TGF-β1 induced expression of type I collagen mRNA, prolonged exposure to TGF-β1 down-regulated the ability of PDL cells to express type I collagen mRNA. Our results further show that, within 4 hrs, exogenously applied TGF-β1 down-regulated IL-6 expression in a dose-dependent manner, and this inhibition was sustained over a six-day period. In summary, the data suggest that mechanically stress-induced TGF-β1 expression may be a physiological mechanism to induce mitogenesis in PDL cells while down-regulating its osteoblast-like features and simultaneously reducing the IL-6-induced bone resorption.

Keywords: periodontal ligament cells, mechanical stress, transforming growth factor-β1, cytokines

Introduction

Mechanical forces play a major role during tooth movement by inducing synthesis and secretion of different mediators involved in bone remodeling (Davidovitch, 1991; Davidovitch et al., 1992; Lanyon, 1992; Shimizu et al., 1994; Yamaguchi and Shimizu, 1994; Lowney et al., 1995). These mediators alter the growth, differentiation, and secretory functions of cells of the periodontal ligament (PDL) (Saito et al., 1990, 1991). Cells of PDL lineage have been shown to elaborate TGF-β1 as well as pro-inflammatory mediators (interleukin-1, IL-1; tumor necrosis factor-α, TNF-α; prostaglandin E2, PGE2), whereas osteoblasts and osteoblast-like cells exhibit TGF-β1 expression and phenotypic changes, but not pro-inflammatory cytokine induction, in response to mechanical stress in vivo and in vitro. For example, mixed populations of PDL cells in response to tensile forces in vitro exhibit IL-1β and PGE2 synthesis, type I collagen synthesis, and decreased alkaline phosphatase activity (Nakagawa et al., 1994; Yamaguchi and Shimizu, 1994; Yamaguchi et al., 1994; Shimizu et al., 1995). Additionally, IL-1β, TGF-β1, and TNF-α have been found in gingival crevicular fluid around teeth subjected to mechanical stress (Lowney et al., 1995; Uematsu et al., 1996a). These observations suggest that mechanical stress induces an inflammatory response in the cells of the PDL; however, the exact nature of this response still needs to be analyzed. Several studies performed on rat calvarial cell cultures grown in vitro have shown that mechanical stress induced increased synthesis of PGE2 (Binderman et al., 1988; Rawlinson et al., 1995; Klein-Nulend et al., 1996; Zhuang et al., 1996), decreased alkaline phosphatase activity, and reduced the synthesis of osteocalcin (Roelofsen et al., 1995; Stanford et al., 1995; Mikuni-Takagaki et al., 1996), while not affecting collagen synthesis (Hillsley and Frangos, 1997). Mechanical stress in human osteoblasts has been shown to induce increased expression of TGF-β1 without significantly affecting alkaline phosphatase activity (Neidlinger-Wilke et al., 1995). Additionally, human osteosarcoma cells and osteoblasts have been shown to exhibit increased mitogenesis as well as increased RNA and protein synthesis in response to mechanical stress in vitro (Buckley et al., 1988).

The above observations suggest that the tension-induced cellular responses entail phenotypic changes in osteoblasts and osteoblast-like cells. Recently, TGF-β1, an osteotropic growth factor, has been shown to affect growth and differentiation of osteoblast-like cells in a fashion similar to those observed following exposure to mechanical stress. For example, in rat calvarial cells, TGF-β1 has been shown to inhibit osteogenic differentiation (Breen et al., 1994), expression of alkaline phosphatase activity, as well as expression of osteocalcin, osteopontin, and collagenase (Breen et al., 1994; Harris et al., 1994). TGF-β1 induces osteopontin and chondrocytic differentiation in rat calvarial osteoprogenitor cells (Basic et al., 1995; Cheifetz et al., 1996; Rydziel et al., 1997). In these cells, TGF-β1 has been shown to inhibit mineralization without affecting cell growth, alkaline phosphatase activity, or collagen type I synthesis (Tally-Ronsholdt et al., 1995). In human osteosarcoma cells, TGF-β1 autostimulates expression of TGF-β1 (Liu et al., 1996), whereas responses of human PDL cells to TGF-β1 include increased RNA and protein synthesis (Mailhot et al., 1995) and mitogenesis in vitro (Oats et al., 1993; Dennison et al., 1994).

Since a variety of cells constitutes the PDL (Pitaru et al., 1994; Lekic and McCulloch, 1996), we speculated that the different populations of cells in the PDL may secrete different cytokines in response to mechanical stress. Hence, this study was initiated to examine the effect of mechanical stress on well-defined PDL cell lines exhibiting osteoblast-like phenotype. We show that PDL cells with osteoblast-like features, when subjected to intermittent stress, exhibit increased expression of TGF-β1 and IL-6 mRNA and proteins. Subsequent examination of the consequences of TGF-β1 augmentation shows that TGF-β1 autoregulates its expression. It also regulates growth, alkaline phosphatase activity, expression of type I collagen, osteocalcin, and IL-6 synthesis in osteoblast-like PDL cells. TGF-β1-mediated regulation of the osteoblast-like PDL cell phenotype and functions is similar, in most cases, to that observed following exposure of osteoblasts or osteoblast-like cells to mechanical stress. Furthermore, we show that osteoblast-like PDL cells do not elaborate pro-inflammatory cytokines such as IL-1β or TNF-α in response to mechanical stress.

Materials and methods

Isolation, cloning, and characterization of periodontal ligament cells

Healthy human premolars extracted for orthodontic reasons (two females, one male, ages between 12 and 16 yrs) were obtained according to the protocols approved by the Internal Review Board of the University of Pittsburgh. The middle third of a root surface of the tooth was scraped and the cells cultured in a tissue culture medium (TCM) containing RPMI-1640 (Life Technologies, Grand Island, NY), 10% defined fetal calf serum (Hyclone Laboratories, Logan, UT), 2 mM glutamine, penicillin (100 U/mL), streptomycin (100 µg/mL), and tylosin (80 µg/mL). The semiconfluent cultures were grown further in a tylosin-free medium, cloned by the limitation of dilutions, and used for experimentation between the 6th and 20th generations. PDL cell clones (PL-1, PL-4, and PL-124) were characterized as described earlier (Somerman et al., 1988; Quintero et al., 1995). The osteoblast-like phenotypic characteristics of PDL cells—such as alkaline phosphatase activity, expression of TGF-β1, osteocalcin, and type I-α1 collagen mRNA expression—were assessed as described below. Additionally, we assessed cAMP generation in response to PGE2 to confirm the osteoblast-like phenotype of PDL cells. Briefly, PL-1, PL-4, or PL-124 cells (105 cells/well of six-well plate) were incubated for 48 hrs. Subsequently, cells were rinsed and re-suspended in 400 µL DMEM containing 0.5% BSA and 0.5 mM 3-isobutyl-1-methyl xanthine. Based on earlier studies (Somerman et al., 1990), PDL cells were activated for 10 min with 25 µg/mL PGE2 in 100 µL DMEM (5 µg/mL final concentration) or, for controls, 100 µL DMEM alone. Thereafter, the cells were rinsed, lysed, and extracted for analysis of total cAMP contents by competitive binding assay (Amersham Life Science Inc., Arlington Heights, IL). In parallel cultures, we counted cells to assess the total cAMP contents per 105 cells. All of the above experiments were done in triplicate, and mean and standard error of the mean were calculated for each point.

Application of mechanical stress to PDL cells

Monolayers of PL-1 or PL-124 cells (5 × 104/well) were grown on Flex I cell plates, having collagen-coated flexible Teflon bottoms. The monolayers were subjected to mechanical stress in a Flexercell® strain instrument (Flexercell® Corp., McKeesport, PA) at a rate of 3 cycles per min (0.5 Hz), i.e., 10 sec of a maximum of 24% elongation followed by 10 sec of relaxation (180 cycles/hr) at a vacuum level equivalent to 5 inches of mercury. Short-term mechanical stress was defined as 180 cycles of stress, and long-term mechanical stress as 24 hrs of stress regimen, in which 180 cycles of stress/hr were applied for 24 consecutive hrs. At a vacuum level equivalent to 5 inches of mercury, the empirically measured deformation on the surface of a Flex I plate follows an exponential curve from 0 to 24% elongation along the radius from the center of the well to the edge (Banes et al., 1985). The mRNA from stressed and unstressed (control) cells was extracted 4 hrs following completion of stress cycles, whereas the presence of cytokines in the culture supernatants was assessed 6 hrs after the completion of stress cycle by enzyme-linked immunoassays (Medgenix, Belgium).

Effect of TGF-β1 on PDL cell phenotype

Alkaline phosphatase activity

To examine the effect of TGF-β1 on the alkaline phosphatase activity in PDL cells, we grew PL-4, PL-1, or PL-124 cells (104/100 µL) in 96-well microtiter plates with 0, 0.016, 0.08, 0.4, 2.0, and 10.0 ng/mL concentrations of natural human TGF-β1 (Genzyme, Cambridge, MA) in a final volume of 150 µL of TCM, at 37°C in 5% CO2 and 95% humidity. The cultures were replenished with 50 µL of fresh TCM containing identical concentrations of TGF-β1 on day 6. PDL cells in long-term cultures were replenished with 150 µL of TCM on day 14. The alkaline phosphatase activity in PDL cells was measured spectrophotometrically. The cells were first permeabilized with digitonin (20 µg/mL in 50 mM Tris, pH 9.4; Sigma Chemical Co., St. Louis, MO) so that the substrate could enter. Subsequently, p-nitrophenyl phosphate (1 mg/mL; Sigma Chemical Co., St. Louis, MO) was added in the presence of 5 mM MgCl2 in 50 mM Tris, pH 9.4. The cells were incubated for 20 min at 37°C, supernatant was removed to a new 96-well plate, and the optical density of supernatants was measured at 450 nm in a Dynatech Microtiterplate Reader. In some experiments, alkaline phosphatase activity was assessed by histochemical analysis on 10% buffered formalin-fixed PDL cells in the presence of fast-red TR (1 mg/mL) and naphthol ASMX phosphate (0.2 mg/mL in 50 mM Tris HCl buffer, pH 9.4).

Mitogenesis

We evaluated the effect of TGF-β1 on the mitogenesis in PDL cells by treating PL-1, PL-4, and PL-124 cells with various concentrations of TGF-β1 as described above. Thereafter, the mitogenesis in cells was assessed by spectrophotometric analysis of crystal-violet-stained PDL cells as described earlier (Quintero et al., 1995).

Expression of TGF-β1 and osteocalcin

The expression of osteocalcin- and TGF-β1-specific mRNA was assessed by reverse transcriptase/polymerase chain-reaction (RT/PCR) as described below.

Expression of mRNA by RT/PCR

To examine the relative expressions of IL-1β, IL-6, IL-8, TNF-α, TGF-β1, osteocalcin, and type I-α1 collagen, we exposed PL-4 or PL-124 cells (5 × 104/well), grown in six-well plates, to various concentrations of TGF-β1 for predetermined time intervals. Subsequently, the expression of mRNA was assessed by RT/PCR.

The total RNA from control and activated PDL cells was extracted according to a method described earlier (Chomczynski and Sacchi, 1987). A total of 1 µg RNA was denatured at 65°C for 15 min. The RNA was then mixed with 4 µL of 25 mM MgCl2, 2 µL of 10 × PCR buffer (Promega, Madison, WI), 1 µL of 10 mM oligo dT15, 1 µL Murine Maloney Leukemia Virus reverse transcriptase (RT), 5 µL of RNasin, 2 µL of 10 mM deoxynucleoside triphosphates (dNTPs), and distilled water to make the final volume of 20 µL. The mixture was incubated at room temperature for 10 min and then placed in a Perkin-Elmer Gene Amp PCR system 9600. The oligonucleotide primers used were synthesized by the University of Pittsburgh DNA Synthesis Facility. The size of amplified DNA and the oligonucleotide sequences of primers used were as follows: human osteocalcin (294 bp), 5'-ATGAGAGCCCTCAGACTCCTC-3' and 5'CGGGCCGTAGAAGCGCCGATA-3' (Fleet and Hock, 1994), human TGF-β1 (161 bp), 5'-GCCCTGGACACCAACTATTGCT-3' and 5'-AGGCTCCAAATGTAGGGGCAGG-3' (Huo et al., 1995); human β-actin (548 bp), 5'-GTGGGGCGCCCCAGGCACCA-3' and 3'-CTCCTTAATGTCACGCACGATTTC-5'; human IL-1β (388 bp), 5'-AAACGAATGAAGTGCTCCTTC AGC-3'; human IL-6 (628 bp), 5'-ATGAACTCCTTCTCCACAAGCGC-3' and 5'-GAAGAGCCCTCAGGCTGGACTG-3'; interleukin 8 (302 bp), 5'-ATGACTTCCAAGCTGGCCGTG 3' and 3'-TTATGAATTCTCAGC-CCT-CTTCAAAAACTT-5'; TNF-α (325 bp), 5'-ATGAGCACTGAAAGCATGATC 3' and 3'-TCACAGGGC-AATGATCCCAAAGTAGACCTGCCC 5' (Zhong et al., 1993); Human Collagen Iα1 (685 bp), 5'-AGCAAGTTGCTAACATCAGC-3' and 5' TCACCGGTGAATTCCCAAGCTCTCATA-CCAG-3' (Rose et al., 1991). The reverse transcription was carried out at 42°C for 15 min. After an initial denaturation, the DNA was amplified by 35 cycles of PCR. One PCR cycle run was made under the following conditions: DNA denaturation at 94°C for 20 sec, primer annealing at 60°C for 30 sec, and DNA extension at 72°C for 45 sec. The reaction was terminated at 72°C for 10 min. The PCR products were separated on 2% agarose gels and photographed. We determined quantitatively the relative differences between the PCR products by assessing luminescence values of each DNA band by a Dage CCD camera and Optimas software (Bioscan, Seattle, WA).

Statistical analysis

All experiments for the assessment of alkaline phosphatase activity and mitogenesis were performed in quadruplicate. Cytokine synthesis in culture supernatants was determined in triplicate by ELISA. The mean of the absorbance and standard error of the mean were calculated. The levels of significance were calculated by Student’s t test. Using two different cell lines, we performed each experiment 4 times to assess the expression of mRNA by RT/PCR. The luminescence values of PCR products were measured 3 times on each gel. The mean and standard error of the mean of luminescence values were calculated, and data were expressed as % of untreated controls.

Results

Characteristics of PDL cell lines

The PDL cell lines PL-1, PL-4, and PL-124, obtained from healthy premolars, used in this study exhibited similar osteoblast-like characteristics. As shown in Fig. 1A, in passage 9, PL-1 and PL-4 cells exhibited 22% higher alkaline phosphatase activity than PL-124 cells. All of these cell lines also exhibited increased generation of cAMP in response to PGE2 (Fig. 1B; Somerman et al., 1990). The ability of all 3 cell lines to form calcium phosphate nodules over a period of 4 wks was similar (Fig. 1C). PL-1, PL-4, and PL-124 cells exhibited the presence of TGF-β1, osteocalcin, and type I collagen-specific mRNA expression (Fig. 1D). All PDL cells were negative for cytokeratins and Factor VIII by immunofluorescence with anti-human cytokeratin and anti-Factor VIII-related antigen monoclonal IgG1 (clone MNF116 or clone F8/86, Dako Corp, Carpinteria, CA), respectively.

Figure 1.

Figure 1

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Characterization of various PDL cell lines. (A) Alkaline phosphatase activity in PDL cells, PL-1, PL-4, and PL-124 (104 cells/well), cultured for 7 days in vitro. (B) Generation of cAMP in PDL cells activated with PGE2, for 10 min. (C) Calcium phosphate nodule formation in PDL cells grown for 28 days in vitro. (D) Expression of mRNA for TGF-β1, osteocalcin, and type I-α1 collagen in PL-1, PL-4, and PL-124 cells as assessed by RT/PCR. The data in (A), (B), and (C) represent the mean and standard error of the mean (± SEM) of quadruplicate values. Bands of PCR products in (D) represent one out of three separate experiments on PL-1, PL-4, and PL-124 cells.

The effect of mechanical stress on PDL cells

Unstressed PDL cells grown on Flexercell plates constitutively expressed TGF-β1-specific mRNA. Exposure of PL-1 or PL-124 cells to mechanical stress for 1 hr augmented the expression of TGF-β1 and IL-6 mRNA. However, the expression of mRNA for pro-inflammatory cytokines IL-1β, TNF-α, or IL-8 was not observed following the above mechanical stress regimen (Fig. 2A). The pronounced TGF-β1 and IL-6 mRNA expressions after short-term mechanical stress were paralleled by the synthesis of their proteins in both PL-1 and PL-124 cells, as analyzed by ELISA. Similarly, following exposure to mechanical stress, the lack of the induction of mRNA synthesis for IL-1β, IL-8, and TNF-α in PDL cells was also confirmed by the absence of their proteins in culture supernatants.

Figure 2.

Figure 2

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Effect of mechanical stress on PDL cells. (A) PL-1 or PL-124 cells were subjected to either 0 hr (lanes 1, 3, 5, and 7), 1 hr (lanes 2 and 4) or 24 hrs (lanes 6 and 8) of mechanical stress. Subsequently, cells were incubated for 4 hrs; RNA was extracted and analyzed by RT/PCR for the presence of mRNA for cytokines. Gingival fibroblasts activated with recombinant human IL-1β for 4 hrs were used as positive controls in these experiments (lane 9). Alternatively, PL-1 or PL-124 unstressed control cells (PL-1c, PL-124c) or cells subjected to mechanically induced tension (PL-1ms, PL-124ms) were incubated for 6 hrs at 37°C, after a one-hour stress regimen (B) or a 24-hour stress regimen (C). Thereafter, the supernatants were harvested and analyzed by ELISA for the presence of cytokines. The data in (B) and (C) represent the mean ± SEM of triplicate values. The data in all panels represent one out of three separate experiments.

To examine the effects of repeated cycles of mechanical stress over a 24-hour time interval, we subjected PL-1 and PL-124 cells to 180 cycles 2 of MS per hr for 24 hrs, followed by 4 hr of rest before the extraction of RNA. As shown in Fig. 2A, TGF-β1 mRNA expression in mechanically stressed cells increased as compared with untreated control PL-1 as well as PL-124 cells. Additionally, the expression of IL-6 mRNA was not sustained following long-term mechanical stress, but the presence of measurable quantities of IL-6 in the supernates could be detected. It is of interest that the expression of IL-8, IL-1β, or TGF-α mRNA was not observed even after cells were subjected to 24 hrs of mechanical stress.

The effect of TGF-β1 on PDL cell mitogenesis

Since mechanical stress caused sustained induction of TGF-β1, we examined the consequences of the increased TGF-β1 production on PDL cells. To analyze the effect of TGF-β1 on PDL cell growth, we exposed PL-4 cells to various concentrations of TGF-β1 for 1, 3, 6, 9, 12, or 28 days, following which cell growth was analyzed in quadruplicate wells. As shown in Fig. 3, incubation of PL-4 cells for 3 days with TGF-β1 at concentrations of 0.4, 2, and 10 ng/mL resulted in 137%, 167%, and 192% increases in mitogenesis, respectively. This represented a significant (p < 0.05) increase in mitogenesis, as compared with untreated controls, which exhibited 0% mitogenesis over a three-day period. PL-4 cells exposed to 0.4, 2, and 10 ng/mL TGF-β1 exhibited 181%, 192%, and 209% increases in cell growth, respectively, during the first 12 days. Thereafter, cell growth, slowed. PDL cells in TCM alone exhibited a 14% increase in growth over the same time period. Similar results were observed when PL-124 cells were used (data not shown).

Figure 3.

Figure 3

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Effects of various concentrations of TGF-β1 on PDL cell growth. PL-4 cells (5 × 103/well) grown in 96-well plates were incubated in the absence or presence of various concentrations of TGF-β1 for 3, 6, 9, 12, or 28 days. The cell growth was assessed spectrophotometrically following crystal violet staining and solubilization of cells. The data represent the mean ± SEM of quadruplicate values from one of three separate experiments.

The effect of TGF-β1 on PDL cell alkaline phosphatase activity

Examination of PL-4 cells undergoing TGF-β1-induced mitogenesis demonstrated that these cells exhibited a dose-dependent decrease in alkaline phosphatase activity. As shown in Fig. 4A, total alkaline phosphatase activity, when calculated per 10,000 PDL cells, decreased rapidly within 24 hrs in the presence of 0.4 ng/mL, or higher, concentrations of TGF-β1. For example, the presence of 0.4, 2, and 10 ng/mL TGF-β1 decreased alkaline phosphatase activity by 29.3%, 34.4%, and 46.2%, respectively, within the first 24 hrs. The down-regulation of alkaline phosphatase activity continued during the first 12 days of TGF-β1 treatment. Thereafter, no significant decrease in alkaline phosphatase activity was observed during the next 16 days (Fig. 4A). In these experiments, PL-4 cells with 0 ng/mL TGF-β1 did not exhibit down-regulation of alkaline phosphatase activity during the first 9 days; thereafter, a 12.7% decrease was observed. PL-124 cells also exhibited similar results (data not shown).

Figure 4.

Figure 4

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Effects of various concentrations of TGF-β1 on the alkaline phosphatase activity and calcium phosphate nodule formation in PDL cells. (A) PL-4 cells (5 × 103/well) grown in 96-well plates were incubated in the absence or presence of various concentrations of TGF-β1 for 3, 6, 9, 12, or 28 days. Alkaline phosphatase activity was assessed spectrophotometrically in the presence of p-nitrophenyl phosphate in digitonin-permeabilized cells. All experiments were done in quadruplicate. The SEM at each point was less than 2% of the mean. (B) Localization of alkaline phosphatase activity in PL-4 cells following TGF-β1 treatment. PL-4 cells were incubated in the continuous presence of 0 ng/mL (a) or 10 ng/mL (b) TGF-β1 for 28 days. Alternatively, PL-4 cells were incubated in the presence of TGF-β1 for 14 days, washed, and replenished with medium devoid of TGF-β1 and examined on day 28 (c). All cells were examined for the presence of alkaline phosphatase activity by histochemical staining as described in “ Methods”. The photographs represent one of three separate experiments performed in duplicates. (C) PL-4 cells were incubated with various concentrations of TGF-β1 for 9 days (solid bars). To examine the effect of removal of TGF-β1, we grew PL-4 cells in 10 ng/mL TGF-β1 for 14 days, then washed them and grew them again in TCM devoid of TGF-β1 for an additional 14 days (hatched bars). The numbers of nodules were counted after von Kossa staining of cells. Each point represents the means ± SEM of triplicate values.

We also investigated the presence of alkaline phosphatase activity in individual cells by the histochemical staining of PDL cells. PL-4 cells initially showed the presence of alkaline phosphatase activity. It was of interest that, in the presence of 0.4 ng/mL (or higher) concentrations of TGF-β1, PDL cell monolayers undergoing mitosis did not exhibit high levels of alkaline phosphatase activity, whereas high alkaline phosphatase activity remained localized in the nodule-forming cells. It is important to note, however, that a reduction in the number of nodules was observed with increasing concentrations of TGF-β1 treatment. Furthermore, as shown in Fig. 4B, the removal of TGF-β1 from PDL cells for 14 days resulted in the re-expression of alkaline phosphatase activity in cells incubated with the highest concentration of TGF-β1 (10 ng/mL).

As compared with untreated PL-4 cells, the down-regulation of alkaline phosphatase activity in PDL cells by TGF-β1 (0.4, 2, and 10 ng/mL) was accompanied by a significant decrease (p < 0.05) in the number of nodules formed in each well over a period of 9 days. Furthermore, removal of TGF-β1 (10 ng/mL) from cultures resulted in the resumption of nodule formation (Fig. 4C).

The effect of TGF-β1 on the expression of mRNA for TGF-β1 in PDL cells

The expression of TGF-β1 is associated with the osteoblast-like characteristics of PDL cells. Since TGF-β1 induced a reduction in alkaline phosphatase activity, an osteogenic marker for the PDL cell phenotype, we examined the regulation of TGF-β1 mRNA expression by TGF-β1 itself. As shown in Fig. 5, TGF-β1, at all concentrations tested, induced expression of TGF-β1 mRNA within the first 4 hrs of PL-4 cells treatment. This increase persisted for the next 24 hrs, except at 0.032 ng/mL TGF-β1. However, the continuous presence of TGF-β1 for 3 or 6 days inhibited the expression of TGF-β1 in PDL cells in a dose-dependent manner. PL-1 as well as PL-124 cells exhibited similar effects of TGF-β1 on the induction of TGF-β1 (data not shown).

Figure 5.

Figure 5

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Effect of TGF-β1 on the expression of TGF-β1 mRNA in PDL cells (5 × 104/well) were incubated in the absence or presence of various concentrations of TGF-β1 for 0.16, 1, 3, or 6 days. Subsequently, RNA was extracted, and the expression of TGF-β1 mRNA was examined by RT/PCR. The histograms represent the mean of triplicate luminescence values of PCR products on agarose gels in one of three separate experiments performed on PL-4 or PL-124 cells with similar results. The SEM were within 5% of the mean in Figs. 5 to 8.

The effect of TGF-β1 on the expression of mRNA for osteocalcin in PDL cells

An examination of the effects of co-incubation of PL-4 cells and TGF-β1 revealed that TGF-β1 down-regulated osteocalcin mRNA expression within 4 hrs. The continuous presence of TGF-β1 at concentrations as low as 32 pg/mL was sufficient to inhibit the expression of osteocalcin mRNA. The extent of TGF-β1-induced inhibition of osteocalcin mRNA expression was time- and dose-dependent, i.e., the decline in osteocalcin mRNA expression was observed with increasing concentrations of TGF-β1 as well as with increasing incubation time (Fig. 6). Similar results were observed when PL-124 cells were used (data not shown).

Figure 6.

Figure 6

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Effect of TGF-β1 on the expression of osteocalcin mRNA in PDL cells. PL-4 cells (5 × 104/well) were incubated in the absence or presence of various concentrations of TGF-β1 for 0.16, 1, 3, or 6 days. Subsequentlv, RNA was extracted, and the expression of osteocalcin-specific mRNA was assessed by RT/PCR. The histograms depict the mean of triplicate luminescence values of PCR products on agarose gels shown in the photographs. The data represent one of three separate experiments performed on PL-4 or PL-124 cells with similar results.

The effect of TGF-β1 on the expression of mRNA for collagen-I in PDL cells

The effect of TGF-β1 on the ability of PDL cells to synthesize extracellular matrix was assessed by their expression of mRNA for type I-α1 collagen. As shown in Fig. 7, following exposure to TGF-β1, PL-4 cells exhibited no significant change in collagen-I mRNA expression during the first 4 hrs. However, at 24 hrs, the expression of mRNA for collagen-I increased in a dose-dependent manner, showing a 6% increase in the presence of 32 pg/mL TGF-β1 and a 23% increase in the presence of 10 ng/mL TGF-β1. In the continuous presence of TGF-β1, a significant decrease (p < 0.05) in collagen-I mRNA expression was observed over periods of 3 and 6 days, i.e., during a six-day period, reductions of 33% and 47% type I-α1 collagen mRNA expression were observed in the presence of 2 and 10 ng/mL TGF-β1, respectively.

Figure 7.

Figure 7

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Effect of TGF-β1 on type I-α1 collagen mRNA expression in PDL cells. PL-4 cells (5 × 104/well) were incubated in the absence or presence of various concentrations of TGF-β1 for 0.16, 1, 3, or 6 days. Subsequently, RNA was extracted, and the expression of type I-α1 collagen mRNA was examined by RT/PCR. The histograms represent the mean of triplicate luminescence values of PCR products in one of three separate experiments performed on PL-4 or PL-124 cells with similar results.

The effect of TGF-β1 on the expression of mRNA for IL-6 and IL-1 in PDL cells

We had observed an increased production of IL-6 in PDL cells subjected to mechanical stress (Fig. 2). Furthermore, gingival crevicular fluids obtained from sulci of teeth subjected to mechanical stress have been shown to contain IL-1β and TNF-α (Saito et al., 1991; Grieve et al., 1994; Uematsu et al., 1996a). Therefore, we determined whether TGF-β1 can induce or inhibit the expression of pro-inflammatory cytokines such as IL-6, IL-1β, or TNF-α mRNA in PDL cells.

PL-4 or PL-124 cells did not constitutively produce mRNA for IL-1β or TNF-α. Furthermore, TGF-β1 did not induce the expression of mRNA for IL-1β or TNF-α at all concentrations tested over a period of 6 days (data not shown). As indicated in Fig. 8, PL-4 cells produced IL-6 constitutively, and incubation of PL-4 cells with TGF-β1 down-regulated the expression of IL-6 mRNA. In comparison with untreated PL-4 control cells, the presence of 0.4, 2, or 10 ng/mL exogenous TGF-β1 caused a significant decrease (p < 0.01) in IL-6 mRNA expression in PL-4 cells within 4 hrs. This down-regulation of IL-6 mRNA expression was persistent during the next 6 days, when PL-4 and PL-124 cells were co-incubated with various concentrations of TGF-β1.

Figure 8.

Figure 8

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Effect of TGF-β1 on IL-6 mRNA expression in PDL cells. PL-4 cells (5 × 104/well) were incubated in the absence or presence of various concentrations of TGF-β1 for 0.16,1, 3, or 6 days. Subsequently, RNA was extracted, and the expression of IL-6 mRNA was examined by RT/PCR. The histograms represent the mean of the triplicate luminescence values of PCR products in one of three separate experiments performed on PL-4 or PL-124 cells with similar results.

Discussion

PDL cells with an osteoblast-like phenotype exhibit characteristics such as constitutive presence of high levels of alkaline phosphatase, ability to synthesize calcium phosphate nodules, as well as the presence of mRNA for TGF-β1 and osteocalcin (Somerman et al., 1988; Nojima et al., 1990; Arceo et al., 1991; Quintero et al., 1995). Our data demonstrate that these osteoblast-like PDL cells, when subjected to mechanical stress for a period as short as one hour, exhibit increased expression of TGF-β1 mRNA. This observation is not unexpected, because the application of mechanical stress has previously been shown to increase the levels of mRNA for TGF-β in osteoblasts in vitro (Neidlinger-Wilke et al., 1995). Furthermore, gingival crevicular fluids around teeth subjected to stress also exhibit high concentrations of TGF-β1 (Uematsu et al., 1996b). Therefore, we examined the consequences of the increased TGF-β1 synthesis on the osteogenic characteristics of PDL cells. Our data suggest that TGF-β1 exerts strong mitogenic effects on PDL cells and that its presence results in rapid PDL cell proliferation. A similar increase in cell growth during the first two days of TGF-β1 exposure to PDL cells and osteoblasts has been reported earlier (Oates et al., 1993; Dennison et al., 1994; Harris et al., 1994; Mailhot et al., 1995). However, in some cases, TGF-β1 has been shown to inhibit mitogenesis in osteoblasts (Bonewald and Mundy, 1990).

The mitogenic effects of TGF-β1 were paralleled by a rapid decrease in alkaline phosphatase activity in PDL cells. The extent of the down-regulation of alkaline phosphatase activity was directly proportional to the concentration of exogenously applied TGF-β1, and inversely proportional to the mitogenic activity observed in response to TGF-β1. Since the half-life of TGF-β1 in culture is only few days and not weeks, it is tempting to speculate that the sustained decrease in alkaline phosphatase activity and the increase in mitogenesis in TGF-β1-treated cells may be due to the TGF-β1 synthesized by PDL cells in response to exogenous TGF-β1. It is also important to note that the effect of TGF-β1 on down-regulation of alkaline phosphatase activity appears to be disparate, i.e., highly differentiated nodule-forming PDL cells retain their high alkaline phosphatase activity, whereas the dividing PDL cells in monolayers exhibit minimal alkaline phosphatase activity. In this respect, PDL cells mimic osteoblasts in that the only cells affected by TGF-β1 are the younger dividing cells and not the well-differentiated osteoblasts (Harris et al., 1994). It has also been shown that concentrations of TGF-β1 at or above 2 ng/mL completely inhibit nodule formation in fetal rat calvarial cells (Breen et al., 1994). Likewise, PDL cells in our studies exhibited inhibition of nodule formation, albeit concentrations of TGF-β1 greater than 10 ng/mL were required for a complete inhibition. It is of interest that the effects of TGF-β1 on alkaline phosphatase activity are transient, since removal of TGF-β1 from cultures results in the restoration of complete alkaline phosphatase activity.

TGF-β1 regulates its own expression in PDL cells. This is evident through the observations that, during initial exposure of PDL cells to TGF-β1, low doses of TGF-β1 induced higher expression of TGF-β1-specific mRNA than higher doses of TGF-β1. Yet within 3 days, even low doses of TGF-β1 inhibited TGF-β1 mRNA expression. These results suggest that endogenous TGF-β1 produced by PDL cells in response to TGF-β1 may play a critical role in synergy with exogenously applied TGF-β1 in down-regulating TGF-β1 expression. Furthermore, following initial up-regulation, TGF-β1 down-regulates PDL cell TGF-β1 synthesis in a dose- and time-dependent manner, i.e., TGF-β1 is down-regulated with time and increasing concentrations of TGF-β1. In this respect, these cells behave similarly to osteoblasts, where TGF-β1 has been shown to autoregulate its expression (Liu et al., 1996).

Besides TGF-β1, osteocalcin is also known to be associated with the osteoblast-like characteristics of PDL cells. Therefore, it is not surprising that, as with alkaline phosphatase activity, osteocalcin mRNA expression was also inhibited with time and increasing concentrations of TGF-β1. Thus, TGF-β1 regulates multiple genes associated with the osteoblast-like phenotype in PDL cells.

The major extracellular matrix produced by osteoblast-like cells is type I collagen. We have observed augmentation of type I-α1 collagen mRNA by TGF-β1 in the initial phase of activation of PDL cells with TGF-β1, followed by a reduction in the sustained presence of TGF-β1 for a three- to six-day period. It is quite likely that, with these longer exposures, the reduction in collagen-I mRNA expression may be associated with the major cell population, which also exhibits lower alkaline phosphatase activity in the sustained presence of TGF-β1. Recently, TGF-β1 has been shown to down-regulate nodule formation, alkaline phosphatase activity, osteocalcin, and type I collagen in fetal rat calvarial cells (Harris et al., 1994). Our results suggest that although PDL cells are not fully differentiated osteoblasts or cementoblasts, their responses to TGF-β1 are quite similar to responses in osteoblasts.

In addition to TGF-β1, mechanical stress augmented the expression of IL-6 in PDL cells. The observations that IL-6 was detected in the supernate of PDL cells subjected to both one-hour and 24-hour regimens of mechanical stress, and that TGF-β1 inhibited IL-6 mRNA expression, suggest that in vivo IL-6 induction following MS may be short-lived due to the inhibition of IL-6 expression by TGF-β1. The observed suppression of IL-6 synthesis is not surprising, since TGF-β1 is known to be an anti-inflammatory cytokine (Tsunawaki et al., 1988). Furthermore, these results suggest that IL-6 and TGF-β1 expressions are regulated by distinct pathways.

Recently, heterogeneous populations of fibroblasts from periodontal ligament have been shown to synthesize IL-1β in response to mechanical stress (Shimizu et al., 1994). However, in our studies, IL-1β mRNA expression was not observed in cloned PDL cells in response to either mechanical stress or TGF-β1 application. Since in our experiments a homogeneous population of PDL cells with an osteoblast-like phenotype was used, it is conceivable that mechanical stress may induce IL-6 only in a distinct population of PDL cells and IL-1β in other types of fibroblasts. Although a battery of pro-inflammatory cytokines has been shown to be present in the sulcus of teeth subjected to mechanical stress (Saito et al., 1991; Grieve et al., 1994; Uematsu et al., 1996a), our observations indicate that, in mechanically stress-induced inflammation, the primary source of pro-inflammatory cytokines may be immune cells and not PDL cells. Furthermore, PDL cells may synthesize IL-1β in response to other pro-inflammatory cytokines (Quintero et al., 1995). Our observations also indicate that the profiles of cytokine expression by PDL cells in response to mechanical stress are discrete from those induced by a microbial challenge.

Taken together, these results demonstrate that the cloned PDL cells with osteoblast-like characteristics express increased levels of TGF-β1 following exposure to mechanical stress. TGF-β1 serves primarily as a mitogenic signal, while inhibiting the osteoblast-like phenotype as a whole in PDL cells. Down-regulation of the osteoblast-like phenotype appears to be a transient process, since removal of TGF-β1 results in up-regulation of alkaline phosphatase activity. TGF-β1 also down-regulates expression of IL-6, which, in turn, may be an important factor in the mechanically stress-induced inflammatory response. Additional work is needed to elucidate the consequences of stress-mediated IL-6 induction, as well as the effects of this cytokine in initiating an inflammatory response in the absence of infection.

Acknowledgments

The authors express their sincere appreciation to Drs. Herbert L. Langdon and Mark P. Mooney for their critical comments on the manuscript. This work was supported by PHS grants DE09830 and DE11010. It was based on a thesis submitted by Thomas A. Brady to the graduate faculty, University of Pittsburgh School of Dental Medicine, in partial fulfillment of the requirements for the Master in Dental Science degree.

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