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J Dent Res 83(9):703-707, 2004
© 2004 International and American Associations for Dental Research

RESEARCH REPORT
Biomaterials & Bioengineering

Inhibition of Phosphatidylinositol 3-Kinase Amplifies TEGDMA-induced Apoptosis in Primary Human Pulp Cells

G. Spagnuolo1, K. Galler2, G. Schmalz2, C. Cosentino3, S. Rengo1, and H. Schweikl2,*

1 Department of Oral and Maxillo-Facial Sciences and
3 Department of Cellular and Molecular Biology and Pathology, University of Naples "Federico II", via S. Pansini 5, 80131-Naples, Italy; and
2 Department of Operative Dentistry and Periodontology, University of Regensburg, D-93042 Regensburg, Germany;

* corresponding author, helmut.schweikl{at}klinik.uni-regensburg.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cytotoxicity of triethylene glycol dimethacrylate (TEGDMA), a co-monomer of dental resinous restorative materials, is firmly established in vitro, but the molecular mechanisms are unknown. Here we examined apoptosis and necrosis induced by TEGDMA in human primary pulp cells. The levels of apoptotic and necrotic cell populations differentially increased after exposure to increasing concentrations of TEGDMA. A two-fold increase in the percentage of apoptotic cells was induced by 1 mmol/L TEGDMA. However, a population shift among cells in apoptosis and necrosis was detected when cell cultures were exposed to 2 mmol/L TEGDMA. Inhibition of the MAP Kinase/ERK pathway had no influence on cell survival, but inhibition of phosphatidylinositol 3 kinase (PI3-Kinase; Akt/protein kinase B) by LY294002 amplified TEGDMA-induced apoptosis. Moreover, Akt phosphorylation was inhibited in the presence of TEGDMA. These results suggest that depression of PI3K signaling may be a primary target in TEGDMA-induced apoptosis.

KEY WORDS: pulp cells • apoptosis • necrosis • TEGDMA • PI3K


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dental composite materials and adhesives release monomers because of degradation processes or incomplete polymerization. More than 30 different compounds have been extracted from polymerized dental composites and from major monomers, co-monomers, and other substances that have been identified as cytotoxic (Hanks et al., 1991; Spahl et al., 1998; Pelka et al., 1999). Cytotoxicity is usually indicated by decreased cell proliferation, mitochondrial activity, and protein or nucleic acid synthesis (Geurtsen, 2000).

Cells die by two major processes, apoptosis and necrosis. Necrosis is a passive process and the result of severe cellular damage, because of the loss of protein function or plasma membrane integrity. In contrast, apoptosis (programmed cell death), as an active process, is stimulated by developmental signals or environmental factors (Dragovich et al., 1998; Denecker et al., 2001). Cells respond to stress caused by endogenous or exogenous sources by a network of signal transduction pathways. The activation of protein kinases is a key event in most of these pathways investigated so far. Members of the mitogen-activated protein kinase (MAPK) family activate regulatory proteins involved in the active response to environmental stress (Clerk et al., 1998; Boldt et al., 2002; Ramachandiran et al., 2002). The phosphorylation cascade of the extracellular signal-regulated kinase (ERK1/2) is a well-understood MAPK pathway, which is thought to mediate cell proliferation, survival, and apoptosis (Hindley and Kolch, 2002). A survival pathway different from MAP kinases is initiated by the phosphatidylinositol 3-kinase (PI3K), which catalyzes the addition of phosphate groups to phosphoinositides. Protein kinase B (Akt) is activated by phosphoinositides and phosphorylates downstream target proteins, including Bcl-2 family members, thus inhibiting apoptosis (Cantrell, 2001).

Induction of apoptosis in human gingival cells was reported recently (Janke et al., 2003). However, the molecular mechanisms of the cytotoxicity of resinous materials or monomers remain to be analyzed, and the signal transduction pathways leading to apoptosis induced by these compounds are yet to be elucidated. Here, we tested the hypothesis that the cytotoxicity of a major compound of dental resinous materials, triethylene glycol dimethacrylate (TEGDMA), is mediated by apoptosis and might influence major intrinsic cellular survival pathways.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and Reagents
Cell culture media and TEGDMA (CAS-No. 109-16-0) were purchased from Sigma (Deisenhofen, Germany). 4-morpholinyl-8-phenyl-4H-1-benzopyran-4-one (LY294002; CAS-No. 154447-36-6) and 2-amino-3-methoxyflavone (PD98059; CAS-No. 167869-21-8) were obtained from Calbiochem (Bad Soden, Germany). A TACS annexin V-fluorescein labeling kit was purchased from R&D Systems (Wiesbaden, Germany).

Cell Culture
The use of human primary pulp cells conformed to an informed consent protocol reviewed and approved by an appropriate Institutional Review Board. Impacted tooth germs of human third molars, after extraction, were obtained from the Department of Oral and Maxillofacial Surgery. Briefly, the central part of the dental papilla was cut into small pieces, washed, and incubated in phosphate-buffered saline (PBS). The tissues were digested with accutase solution (PAA Laboratories, Coelbe, Germany) for 10 min at 37°C, collected in {alpha}MEM containing 10% fetal bovine serum, penicillin (150 U/mL), streptomycin (150 µg/mL), and amphotericin B (0.125 µg/mL), and then explanted into tissue culture dishes. Cells from these explants were cultivated in culture medium in a humidified atmosphere containing 5% CO2 at 37°C. Cells of the fourth and fifth passages, which were morphologically and histochemically analyzed as fibroblast-like cells, were used in all experiments.

Induction of Apoptosis and Necrosis
Human primary pulp cells (1.5 x 105) were plated in 60-mm culture dishes and incubated at 37°C for 24 hrs. For the detection of a dose-related induction of apoptosis and necrosis, the cell cultures were exposed to increasing concentrations of TEGDMA (from 0 to 3 mmol/L) for 24 hrs at 37°C. For analysis of the time-dependent induction of apoptosis and necrosis by 1 mmol/L TEGDMA, the cell cultures were exposed for 6, 12, and 24 hrs. We also tested the effects of the inhibitors LY294002 (50 µmol/L) and PD98059 (40 µmol/L) on the induction of apoptosis by 1 mmol/L TEGDMA. PD98059 is an inhibitor of MAPK/ERK1/2, and LY294002 inhibits PI3K (Dudley et al., 1995; Baumann and West, 1998). Pulp cell cultures were pre-incubated with each inhibitor, and then continuously exposed to 1 mmol/L TEGDMA for 24 hrs. The exposure was stopped when the exposure media were discarded, and cell cultures were washed with PBS at room temperature. The cell cultures were then trypsinized, washed, and collected by centrifugation.

Detection of Apoptosis and Necrosis by Flow Cytometry
Phosphatidylserine exposure in human pulp cells, as a marker of apoptosis, was measured by the binding of annexin V-FITC. For the differentiation of apoptosis and necrosis, the cells were also stained with propidium iodide (PI) (Vermes et al., 1995). Usually, 1.5 x 105 - 1 x 106 cells per cell culture were incubated in 100 µL binding buffer containing annexin V-FITC and PI as recommended by the manufacturer. The samples were analyzed on a FACSCalibur flow cytometer (Beckton Dickinson, San Jose, CA, USA) equipped with a 15-mW 488-nm argon ion laser. FITC fluorescence (FL-1) was analyzed through a 530/30 band-pass filter, and PI fluorescence (Fl-2) through a 650-nm-long pass filter. Data acquisition (at least 104 events for each sample) was performed with CellQuest software, and the data were stored as list mode files. Data were analyzed by means of WinMDI2.8 software. Numbers of viable (annexin V-; PI-) cells were counted in the lower left quadrant, and the percentages of apoptotic (annexin V+) (lower right quadrant) and necrotic cell populations in the upper left (annexin V-; PI+) and upper right (annexin V+; PI+) quadrants were determined accordingly (Vermes et al., 1995).

All experiments were repeated at least twice. Statistical analysis was performed with the Mann-Whitney U-test for pairwise comparisons among groups at the 0.05 and 0.01 levels of significance.

Western Blotting
Antibodies to the endogenous proteins were the following: P-Akt ser473 (mouse monoclonal, UBI), Akt (rabbit polyclonal, Cell Signaling), P-Erk 1/2 (mouse monoclonal, Santa Cruz), Erk 2 (rabbit polyclonal, Santa Cruz), and tubulin (mouse monoclonal, Sigma). Total extracts were prepared in 50 mM Tris-HCl (pH 7.4), 1% Nonidet NP-40, 100 mM NaCl, 2 mM EDTA, 50 mM NaF, 0.1 mM NaVO3, 1 mM ß-glycerophosphate, 2.5 mM sodium pyrophosphate, and a protease inhibitor cocktail for 30 min. Whole-cell extracts were separated on 10% SDS-PAGE and transferred onto a nitrocellulose filter. Immunoblots were performed according to the manufacturer’s recommendations.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
TEGDMA Induces Apoptosis and Necrosis in Human Pulp Cells
Human primary pulp-derived cells were exposed to increasing concentrations of TEGDMA, and the induction of apoptosis was analyzed by flow cytometry. The onset of apoptosis was indicated when the cells were stained with annexin V-FITC and PI. Cells in control conditions responded uniformly, and only very few cells displayed phosphatidylserine (annexin V+) on the cell surface or were identified as cells with membrane damage (PI+) (Fig. 1Go). These basal levels of apoptosis and necrosis were 6.9 ± 2.3 (annexin V+) and 7.4 ± 2.6 (PI+) %, respectively (Fig. 2Go). A dose-related increase in apoptosis was induced by TEGDMA concentrations higher than 0.5 mmol/L (Fig. 1Go). A two-fold difference between the percentages of apoptotic cells in control cultures and cultures exposed to 1 mmol/L TEGDMA was highly significant (p ≤ 0.01), but no such significant increase was observed with the necrotic cell populations (Fig. 2Go). However, there was a clear population shift among cells in apoptosis and necrosis when the cultures were analyzed after exposure to 2 mmol/L TEGDMA (Fig. 1Go). The percentage of necrotic cells (16.9 ± 3.4) was higher than the percentage of apoptotic cells (14.3 ± 3.5) (Fig. 2Go). Furthermore, a high percentage of the cell population (40.2 ± 5.1) showed necrosis after exposure of the cells to 3 mmol/L TEGDMA, but the apoptotic cell population was decreased to almost basal levels (Figs. 1Go, 2Go). Since the present investigation focused mostly on apoptosis, we have studied the time course of the onset of phosphatidylserine-presenting pulp cells induced by 1 mmol/L TEGDMA (Fig. 3Go). No significant differences between apoptotic cell populations in controls and treated cell cultures were detected after a six-hour exposure. After 12 hrs, a higher percentage of the cell population stained positively with annexin V, but a significant increase of apoptotic cells was observed only after a 24-hour exposure (Fig. 3Go).



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  		Figure 1. The effect of TEGDMA on the induction of apoptosis and necrosis in human primary pulp cells. Cell cultures were incubated with annexin V-FITC (apoptotic cells) and PI (necrotic cells) and analyzed by flow cytometry. The representative dual-parameter fluorescence histograms were derived from cell cultures exposed to various TEGDMA concentrations for 24 hrs. The numbers indicate the percentages of the viable (annexin V-; PI-) cell population (lower left quadrant), the apoptotic (annexin V+) cell population (lower right quadrant), and the necrotic cell populations in the upper right (annexin V+; PI+) and the upper left quadrants (annexin V-; PI +) in one typical experiment.

 


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  		Figure 2. Dose-dependent induction of apoptosis and necrosis in human pulp cells. Cell cultures were treated with various concentrations of TEGDMA in duplicate for 24 hrs. The percentages of apoptotic ({circ}) and necrotic ({square}) cell populations were analyzed by flow cytometry. Values (means ± SD) from 6 independent experiments are presented (n = 6); * indicates significant (p ≤ 0.05), and ** indicates highly significant (p ≤ 0.01) differences from untreated control cultures (Mann-Whitney U-test).

 


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  		Figure 3. Time-dependent induction of apoptosis by TEGDMA. Primary human pulp cell cultures were continuously exposed to 1 mmol/L TEGDMA for 6, 12, and 24 hrs. The percentages of the apoptotic cell population in untreated controls ({circ}) and TEGDMA-treated cultures (•) were analyzed by flow cytometry. Values (means ± SD) from 3 independent experiments in duplicate are presented (n = 3); * indicates a significant (p ≤ 0.05) difference from untreated control cultures (Mann-Whitney U-test).

 
PI3K Signaling in the Induction of Apoptosis
To get insight into the mechanism of induction and the pathways of apoptosis, we treated human primary pulp cells with TEGDMA, and evaluated phosphorylation of ERK1/2 or Akt by immunoblotting with specific antibodies recognizing phosphorylated and active ERK1/2 or Akt. Akt phosphorylation was inhibited in the presence of TEGDMA, but no effects on ERK1/2 were observed (Figs. 4aGo, 4bGo). The Akt inhibitory effect was time-dependent, being maximal at 15 min (data not shown). These results suggest that Akt may be a major target in TEGDMA-induced apoptosis. To investigate this hypothesis further, we treated cells with the MAPK-ERK1/2 inhibitor PD98059 or the PI3K inhibitor LY294002. PD98059 elicited no significant effect on cell survival in the presence or absence of TEGDMA (Fig. 4cGo). In contrast, LY294002 significantly increased the percentage of apoptotic cells in otherwise-untreated cell cultures (p ≤ 0.05) (Fig. 4cGo). Moreover, LY294002 inhibited the phosphorylation of Akt, and it amplified the effect of TEGDMA on the induction of apoptosis in pulp cells about two-fold (p ≤ 0.01) (Figs. 4cGo, 4dGo). Although the percentage of necrotic cells was not affected by LY294002 alone, this population also increased in the presence of TEGDMA, resulting in a significant reduction of cell viability (p ≤ 0.01) (Fig. 4cGo).



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  		Figure 4. The effects of the inhibition of MAPK/ERK and PI3K pathways in human pulp cells. (a) Total extracts derived from human primary pulp cells treated with 1 mmol/L TEGDMA or 3 mmol/L TEGDMA for 15 min were separated on 10% SDS-PAGE, and immunoblotted with anti-P-Akt (ser 473) or anti-P-ERK 1/2. The amounts of total protein present in the extracts were determined by immunoblotting with anti-Akt, anti-tubulin, and anti-ERK 2. Experiments were performed 3 times, and a representative result is shown. (b) The ratio between P-Akt and total Akt in pulp cells after treatment with TEGDMA determined by densitometry. Asterisk indicates significant differences from untreated cell cultures (n = 3). (c) Induction of apoptosis and necrosis in human pulp cells. Cells were pre-treated with 50 µmol/L LY294002 and 40 µmol/L PD98056 for 30 min, and then further exposed to 1 mmol/L TEGDMA for 24 hrs. Apoptotic (annexin V+) and necrotic (PI+) cell populations were analyzed by flow cytometry as described. Viability of a cell population is expressed as the difference between the total cell population and the populations which stained with annexin V-FITC and PI. Values (means ± SD) from at least 4 independent experiments in duplicate are presented (n = 4); * indicates significant (p ≤ 0.05), and ** indicates highly significant (p ≤ 0.01) differences from untreated control cultures (Mann-Whitney U-test). (d) Inhibition of Akt phosphorylation in human pulp cells. The cells were treated with 40 mmol/L LY294002 for 30 min, and then exposed to 1 mmol/L TEGDMA for 15 min. Cell lysates were separated on SDS-PAGE and blotted with anti-P-Akt or total Akt antibodies.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Here, we report on the biological effects of TEGDMA in primary human pulp cells. Analyses by flow cytometry after cells were stained with annexin V and PI allowed for clear discrimination between apoptosis and necrosis (Vermes et al., 1995). The pulp cells appeared to be very resistant to the initiation of apoptosis and necrosis, because only a two-fold increase in apoptotic cells was detected in cultures exposed to 1 mmol/L TEGDMA, and the percentage of necrotic cells increased only slightly. Note that 1 mmol/L TEGDMA was considered cytotoxic in V79 cells (Schweikl et al., 2001). Our results, however, are in agreement with those of Janke et al.(2003), who reported the induction of apoptosis in human gingival cells detected after exposure to TEGDMA concentrations even higher than those used in the present investigation. Here, apoptosis and necrosis were linearly dependent on the concentration of TEGDMA. Thus, we provide experimental evidence that cytotoxicity of TEGDMA in mammalian cells is differentially induced by processes as different as apoptosis (active intrinsic pathway) and necrosis (passive process).

The second goal of the present work was to shed light on the molecular mechanisms leading to signaling pathways affected by cytotoxic effects of monomers like TEGDMA. Hence, we analyzed two pathways important for cell survival and stress response (Dragovich et al., 1998). Among other effects, MAP kinases like ERK1/2 are transducers of cellular stress caused by reactive oxygen species (ROS) to initiate apoptosis (Clerk et al., 1998; Wang et al., 1998; Ramachandiran et al., 2002). It has been reported that TEGDMA decreased the intracellular concentration of glutathione in gingival fibroblasts, and that GSH depletion might lead to an increase in ROS production (Engelmann et al., 2002). Therefore, we hypothesized that ERK1/2 activation might be important for the transduction of TEGDMA effects. However, we found that inhibition of the MAPK/ERK1/2 pathway did not change the percentage of apoptotic cell populations.

The other main signaling pathway involved in cytoprotection is represented by phosphatidylinositol 3-kinase (PI3K). Activation of PI3K leads to the downstream phosphorylation of protein kinase B (Akt/PKB), which inhibits various pro-apoptotic molecules like Bad and caspase 9 (Edwards et al., 2002). The results presented here provide evidence that PI3K signaling is a key component in human pulp cell survival, because blocking this pathway by LY294002 increased the percentage of apoptotic cells, even in untreated cultures. The increase in the percentage of apoptotic cells was similar to the effect of 1 mmol/L TEGDMA in the absence of LY294002. Moreover, inhibition of PI3K signaling by LY294002 amplified the effect of TEGDMA on the induction of apoptosis in pulp cells. Interestingly, Akt phosphorylation was inhibited in the presence of TEGDMA, and the inhibition of PI3K/Akt by TEGDMA may be a primary target in TEGDMA-induced cytotoxicity. Thus, we suggest that PI3K is not only important for the survival of human pulp cells per se, but it may also protect cells by counteracting TEGDMA-induced cytotoxicity. However, the relation of glutathione depletion and ROS production observed in recent investigations and PI3K signaling remains to be elucidated (Engelmann et al., 2002; Stanislawski et al., 2003).

Tissue repair and pulp healing are associated with cell death of damaged cells via apoptosis and reactionary and reparative dentinogenesis (Smith et al., 1995). It has been reported recently that heat stress was initiated through c-Jun N-terminal kinase (JNK) activity, after cavity preparation induced pulp cell apoptosis (Kitamura et al., 2003). PI3K signaling counteracting stress induced by TEGDMA, as presented here, might be of similar clinical relevance. Dental materials and some components of restorative dental materials have been suggested to cause pulp injury, if the remaining dentin thickness is less than 0.5 mm (Murray et al., 2002). Moreover, the TEGDMA concentrations released from dentin adhesives into the pulp might be within the range used in the present investigation (Noda et al., 2002). Thus, the TEGDMA concentrations effective in the induction of apoptosis, as demonstrated here, should be taken into account for the interpretation of cellular events in the pulp after cavity restoration. TEGDMA might also modify the cellular signal transduction pathways initiating dentinogenesis, differentiation of pulp cells, wound healing, and tissue repair (Smith et al., 1995; Smith and Lesot, 2001). Investigation of the cross-talk between PI3K signaling and the JNK pathway will give more insight into the complex regulation of cell survival, apoptosis of pulp cells, and repair of damaged tissues as a response to environmental stress factors.


   ACKNOWLEDGMENTS
 
The skilled technical assistance of Mrs. C. Bolay is gratefully acknowledged. The authors thank Dr. K.-A. Hiller (University of Regensburg) for his help on the statistical analyses. The authors are indebted to Dr. L.J. Nunez (Memphis, TN, USA) and Prof. Dr. E.V. Avvedimento (University of Naples, Italy) for critically reading the manuscript. This project was supported by the Medical Faculty of the University of Regensburg.

Received July 30, 2003; Last revision July 8, 2004; Accepted July 8, 2004


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 MATERIALS & METHODS
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 DISCUSSION
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Denecker G, Vercammen D, Steemans M, Vanden Berghe T, Brouckaert G, Van Loo G, et al. (2001). Death receptor-induced apoptotic and necrotic cell death: differential role of caspases and mitochondria. Cell Death Differ 8:829–840.[ISI][Medline]

Dragovich T, Rudin CM, Thompson CB (1998). Signal transduction pathways that regulate cell survival and cell death. Oncogene 17:3207–3213.[ISI][Medline]

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