HREF="#IVANOVSKI-ETAL-2001">Ivanovski et al., 2001). It was suggested that PDLF respond to IGF-1 more strongly than GF, and this differential response to IGF-1 could stem from the tissue specificity of these cells within the periodontium (Haase et al., 1998). However, the role of IGF-1 in preventing apoptosis in PDLF compared with GF still remains unclear. The present study was aimed at testing the hypothesis that the IGF-1 signaling pathway is preferentially engaged in the protection against cell apoptosis in PDLF compared with GF.
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MATERIALS & METHODS
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Materials
Cell culture media and reagents were purchased from Invitrogen (Carlsbad, CA, USA). Recombinant human IGF-1 was purchased from Upstate Biotechnology (Lake Placid, NY, USA). PhosphoPlus Akt (Ser473) antibody kit, PhosphoPlus Bad (Ser112) antibody kit, and PI3K inhibitor LY294002 were from Cell Signaling Technology (Beverly, MA, USA). Rabbit anti-human IGFBP-5 antibody (H-100), HRP-conjugated goat anti-rabbit IgG, and Western blotting luminol reagent were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). DAB substrate was purchased from Roche Diagnostics (Indianapolis, IN, USA).
Cell Culture and Experimental Design
Human PDLF and GF cells were obtained, cultured, and validated as previously described (Han and Amar, 2002). All the procedures were performed with appropriate informed consent and were approved by the Institutional Review Board at the Goldman School of Dental Medicine at Boston University. For some experiments, cells were grown in six-well culture dishes until they reached 60-70% confluence. Cell apoptosis was induced by serum starvation as described previously (Santiago et al., 2001). Briefly, cells were grown to sub-confluence in low serum (0.1% FBS) DMEM and cultured for another 6-8 days. The cells were then washed with PBS and incubated 24 hrs in the absence or presence of IGF-1 (10-8 M) in serum-free DMEM (Parrizas et al., 1997). In some experiments, the PI3K inhibitor LY294002 (10-6 M) was added 6 hrs prior to and during incubation of the cells in the absence or presence of IGF-1 as previously described (Parrizas et al., 1997).
Reverse-transcription/Polymerase Chain-reaction (RT-PCR)
The total cellular RNA fraction was isolated from cultured human PDLF and GF with RNeasy isolation columns (Qiagen, Valencia, CA, USA) according to the standard protocol. Total RNA (0.2 µg each) was used as a template for reverse-transcription, and PCR was performed for 25 cycles within the linear range of amplification. The primer pair for PCR included: IGFBP-5 (sense, 5’-GGCTCCGAATCTAAGTGCTG-3’; antisense, 5’-GCAGCCCTGTCTCACTAACC-3’, 457 bp) and ß-actin (sense, 5’-GCTCGTCGTCGACAACGGCTC-3’; antisense, 5’-CAAACATGATCTGGGTCATCTTCTC-3’, 353 bp).
RNase Protection Assay
Probe synthesis and RNA hybridization were carried out with use of the RiboQuant RPA system (Pharmingen, San Diego, CA, USA). Briefly, labeled riboprobes were hybridized to target RNAs (20 µg per assay in all experiments) at 56°C overnight followed by RNase A digestion at 30°C for 45 min. Protected fragments were resolved on 6% denaturing polyacrylamide gels, and radioactive signals were quantified with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA, USA).
Immunohistochemistry
Formaldehyde-fixed, paraffin-embedded healthy periodontal tissues from Macaca mulatta monkeys were used for the in situ detection of IGFBP-5 and quantification of apoptosis. All animal procedures and manipulations were approved by the Institutional Animal Care and Use Committee (IACUC) at Boston University. Five-micrometer-thick tissue sections were cut for all immunoassays. For each section, two regions were selected representing periodontal ligament (Region A) and deep gingival tissue (Region B). Region A was defined as the mid-third of the periodontal ligament area, with the coronal and apical boundaries set between the alveolar bone crest and apex. The root and alveolar bone surface determined the lateral boundaries. Region B was defined as vertically between the level of the sulcular bottom and the alveolar bone crest, with lateral boundaries set as mid-third between the root surface and the basement membrane of the oral epithelium. The areas containing the cells were analyzed by Image-Pro Plus 4.0 (Media Cybernetics, Silver Spring, MD, USA).
For the detection of IGFBP-5, the sections were first digested with 20 µg/mL proteinase K at 37°C for 10 min. The sections were then incubated with the anti-IGFBP-5 antibody (1:200) for 2 hrs. After that, the sections were treated for 1 hr with HRP-conjugated goat anti-rabbit IgG (1:500) and developed with DAB substrate. Sections incubated with PBS instead of primary antibody were used as negative controls.
We performed the quantification of apoptosis using terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay with an in situ cell death detection kit (Boehringer Mannheim, Germany) according to the manufacturer’s instruction. Tissue sections of 4 molar teeth from 2 monkeys (3 serial sections per tooth, 2 teeth per animal, totaling 12 sections) were analyzed. Only spindle-shaped cells were counted at 400 magnification by two methods: (a) The apoptotic cell percentage was obtained as the percentage of TUNEL-positive cells relative to the total number of counted fibroblasts; and (b) the apoptotic cell density was expressed as TUNEL-positive cells per mm2.
Flow Cytometry
Annexin-V labeling was monitored with an apoptosis detection kit (Clontech, Palo Alto, CA, USA). Briefly, cells were washed twice with cold PBS, and FITC-labeled annexin-V and propidium iodide (PI) were added. After 15 min of incubation, the cell suspensions were analyzed on a FACScan flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA). Cells with Annexin-V+ but PI- were counted. At least 10,000 events were collected per sample in all experiments.
DNA Fragmentation Analysis
After different treatments, we collected both attached cells and detached cells floating in the medium, by scraping and centrifuging. Cellular DNA fragmentation was quantified as described previously with the diphenylamine (DPA) assay (Drexler, 1997).
Western Blot Analysis
After different treatments, cells underwent lysis, and equivalent protein concentrations were resolved on a 10% SDS/PAGE electrophoretically transferred to a poly(vinylidene difluoride) membrane. The membranes were blocked with 5% non-fat dry milk in TBS/Tween-20, incubated with the appropriate antibodies (1:1000 for each antibody) for immunoblotting, and visualized by enhanced chemiluminescence.
Caspase-3 Measurement
Caspase-3 in cytosolic fractions was quantified by an ELISA system (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instruction. For each assay, a standard curve was generated, and the measured absorbance based on replicates demonstrated an average of less than 5% variance.
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RESULTS
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Apoptotic Rate in PDLF vs. GF in situ
We used an in situ TUNEL assay to measure the constitutive level of fibroblast apoptosis in monkey periodontal ligament and deep gingival tissues (Figs. 1A, 1B
). The apoptotic cell percentage was significantly lower in periodontal ligament (0.49% ± 0.16%) than in deep gingiva (1.49% ± 0.29%) (Fig. 1C). A significant difference in apoptotic cell density was also observed between PDLF and GF. Only 1.58 ± 0.51 TUNEL-positive cells per mm2 were observed in periodontal ligament, while the number of TUNEL-positive cells in deep gingival tissue was 12.33 ± 2.42 per mm2 (Fig. 1D).
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Figure 1. In situ detection of apoptosis. (A,B) Formaldehyde-fixed, paraffin-embedded healthy periodontal tissues of 4 molar teeth from 2 monkeys were analyzed by means of a TUNEL detection kit (Boehringer Mannheim). TUNEL-positive cells are arrowed. (C) The apoptotic cell percentage was obtained as the ratio of TUNEL-positive cells relative to the total number of counted fibroblasts. (D) The apoptotic cell density was expressed as TUNEL-positive cells per mm2. Mean ± SD (n = 12). **p < 0.01, t test, difference between PDLF and GF. Ce, cementum; PDL, periodontal ligament; AvB, alveolar bone; DG, deep gingiva.
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In vitro Detection of Cell Apoptosis
Early cell apoptosis in cultured human PDLF and GF was evaluated by Annexin-V staining and analyzed by flow cytometry. There was a clear intensity shift between two cell populations upon IGF-1 stimulation, indicating an apparent decrease in apoptosis in PDLF compared with GF (Fig. 2B). A slight reduction of PDLF apoptosis levels was also observed in the DMEM-only group (Fig. 2A) and in the group in the presence of both IGF-1 and LY294002 (Fig. 2C). The extent of DNA fragmentation was determined by the diphenylamine assay (Fig. 2E). After IGF-1 stimulation, the proportion of soluble DNA fragments decreased in both PDLF and GF, when compared with their unstimulated counterparts (DMEM only). However, the percentage of DNA fragmentation showed a significant decrease in PDLF compared with GF (p < 0.05). This retardation of DNA fragmentation was inhibited by the addition of PI3K inhibitor LY294002, leading to a comparable level of DNA fragmentation between PDLF and GF.
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Figure 2. The role of IGF-1 signaling on PDLF vs. GF. Human PDLF and GF were cultured in six-well plates and were treated with IGF-1 and LY294002 as described in MATERIALS & METHODS. (A-D) We used flow cytometry to detect the annexin V-FITC-generated signal at 488 nm (FL1-H). The shadowed area represents fluorescent intensity distribution of PDLF cells. The area under the bold line represents fluorescent intensity distribution of GF cells. (E) The extent of DNA fragmentation was assessed by the diphenylamine (DPA) assay as described in MATERIALS & METHODS. (F) Cell lysate was analyzed by Western blot. Phosphorylated proteins were detected by the use of antibodies against phospho-PKB (Ser473) and phospho-Bad (Ser112). Antibodies against PKB or Bad were also used for detection of the total amount of PKB or Bad in the lysate. (G) We used an ELISA system (R&D Systems, Minneapolis, MN, USA) to determine the concentration of active caspase 3 by measuring the optical density at 450 nm. All bar graphs represent Mean ± SD, n = 4. *p < 0.05, t test, difference between PDLF and GF. D, serum-free DMEM. F, DMEM containing 10% fetal bovine serum. I, 10-8 M IGF-1. LY, 10-6 M LY294002.
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Phosphorylation of PKB and Bad
Phosphorylation of PKB in cultured human PDLF and GF was investigated by Western blot. Phosphorylated PKB (Ser473) was hardly noticeable in PDLF and GF in the absence of serum, but became detectable in PDLF and GF in the presence of serum (Fig. 2F
, upper two lanes). After stimulation with IGF-1, phosphorylated PKB (Ser473) was observed in both PDLF and GF; however, PDLF levels were substantially higher than GF levels. As expected, the increased level of phosphorylated PKB was antagonized by the PI3K inhibitor LY294002, confirming the direct correlation between PKB activity and PI3K signaling. The phosphorylation of the Bcl-2 family protein, Bad, was also tested by Western blot (Fig. 2F, lower two lanes). Phosphorylated Bad (Ser112) was greatly increased in PDLF upon IGF-1 stimulation, whereas no significant change of Bad phosphorylation was observed in GF. The elevation of Bad phosphorylation by IGF-1 was blocked in PDLF when exposed to LY294002. However, such an effect was not observed in GF, since Bad phosphorylation remained unchanged at all conditions tested.
Caspase-3 Activity
After IGF-1 stimulation, a 70.6% decrease in the active caspase-3 level was observed in PDLF in contrast to a 42.1% decrease in GF, when compared, respectively, to the non-stimulated group (Fig. 2G). This decrease in caspase-3 activity was significantly greater in PDLF than in GF (p < 0.05). The IGF-1-induced suppression of caspase-3 activity was antagonized in PDLF after the addition of LY294002, with an 80% increase relative to the group in the absence of LY294002. However, caspase-3 activity was increased by only 23% in IGF-1-stimulated GF when LY294002 was present. This suggests that IGF-1-induced inhibition of caspase-3 activity is more PI3K-dependent in PDLF than in GF.
Mitochondria-associated Anti-apoptotic Genes
Mitochondria play a central role in mammalian cell apoptosis, in part through the regulation of Bcl-2 family genes, including Bad, Bcl-2, and Bid (Wang, 2001). We observed no significant differences in the expression of Bcl-2 family genes between PDLF and GF under normal culture conditions (data not shown). However, after induction of apoptosis, the anti-apoptotic molecules Bcl-w, Bcl-xL, Bcl-2, and Mcl-1 were significantly up-regulated in PDLF compared with GF (Figs. 3A, 3B). The transcript levels of pro-apoptotic molecules Bax, Bid, Bad, and Bak were also checked, and Bid was observed to be significantly down-regulated in PDLF compared with GF (Figs. 3C, 3D).
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Figure 3. Expression of bcl-2 family genes between PDLF and GF. Human PDLF and GF were cultured, and apoptosis was induced by serum deprivation as described in MATERIALS & METHODS. The mRNA levels of bcl-2 family members were measured by RNase protection assay for anti-apoptotic genes (A,B) and pro-apoptotic genes (C,D). Radioactive signals were quantified with a PhosphorImager equipped with IMAGEQUANT software (Molecular Dynamics). A GAPDH probe was used as an internal control. Mean ± SD, n = 3. *P < 0.05, **P < 0.01, t test, difference between PDLF and GF.
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Up-regulation of IGFBP-5 in PDLF
The expression levels of IGFBP-5 in cultured human PDLF and GF were checked by RT-PCR and Western blot. The result showed a much greater expression of IGFBP-5 in PDLF compared with GF at both the mRNA and protein levels (Figs. 4A, 4B). To verify this observation further in vivo, we used immunohistochemical staining to investigate the presence of IGFBP-5 in monkey periodontal tissues. The reactivity of anti-IGFBP-5 antibody with monkey periodontal tissues was verified by Western blot (data not shown). Many fibroblasts in the periodontal ligament were strongly reactive for IGFBP-5, whereas positive staining for IGFBP-5 was barely detectable from the fibroblasts in gingival tissue, indicating a differential distribution of IGFBP-5 in these two neighboring tissues (Figs. 4C, 4D).
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Figure 4. Up-regulation of IGFBP-5 in PDLF. (A) Human PDLF and GF were cultured in DMEM containing 10% FBS until sub-confluent. Total RNA (0.2 µg each) was extracted from cells of passages 4 and 7, and gene-specific primers (1 µM) were used for amplification of IGFBP-5 by RT-PCR. Human ß-actin control primers (0.2 µM) were used as internal standard. (B) The cell samples from passage 4 were analyzed by 10% SDS-PAGE with Western blotting and human IGFBP-5 antibody. (C,D) Sections of healthy periodontal tissues from Macaca mulatta monkeys were analyzed by immunohistochemistry as described in MATERIALS & METHODS. Cells immunoreactive for IGFBP-5 are indicated by arrows. Ce, cementum; PDL, periodontal ligament; AvB, alveolar bone; DG, deep gingiva.
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DISCUSSION
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It has been known that PDLF have specialized functions for repair, remodeling, and regeneration of the adjacent tissues compared with GF (Nishimura and Terranova, 1996;Lackler et al., 2000). This difference could partially stem from the delayed rate of turnover required for the performance of the specialized functions associated with PDLF. Our results from the use of the in situ TUNEL assay on monkey periodontal tissues corroborated this hypothesis and suggested that apoptosis is constitutively reduced in PDLF when compared with GF.
Our results also indicated that, upon IGF-1 stimulation, the reduction of apoptosis was more pronounced in PDLF compared with GF (Figs. 2A-2E). This suggests that the IGF-1 pathway is preferentially used for the enhancement of cell survival in PDLF. Furthermore, an increased level of PKB activation and Bad phosphorylation in PDLF was observed upon IGF-1 stimulation (Fig. 2F). Since PKB may phosphorylate Bad and protect cells from apoptosis (Datta et al., 1997), inhibition of Bad by the up-regulation of PKB activity may be one of the mechanisms involved in the subsequent enhancement of PDLF cell survival. Interestingly, PKB activation is not seen when cells are cultured in serum-free medium. It is still conceivable that endogenous levels of active PKB exist at the basal state in PDLF and GF yet are not detectable by Western blot analysis. Recent studies on fibroblast differentiation corroborate this observation regarding the basal level of PKB activity (Hansen et al., 2002).
The Bcl-2 family proteins regulate the cellular responses to apoptotic stimuli (Korsmeyer, 1999). Our observations suggest a contribution of mitochondria stabilization to the enhanced cell survival in PDLF via constitutive up-regulation of anti-apoptotic Bcl-2 genes (Bcl-w, Bcl-xL, Bcl-2, and Mcl-1) and down-regulation of pro-apoptotic Bcl-2 genes (Bid). Caspase-3 is the primary activator of apoptotic DNA fragmentation (Hatai et al., 2000), and activation of Bcl-2 prevents the activation of caspase-3 (Cheng et al., 2001). Our results also suggest that the effect of the IGF-PI3K pathway on the enhancement of cell survival in PDLF might be partially through the inhibition of caspase-3 activity, which is associated with the initiation of Bcl-2-dependent mitochondrial stabilization.
Up-regulation of IGFBP-5 is currently viewed as an adaptive cell survival mechanism that helps potentiate the anti-apoptotic effects of IGF-1, in part through activation of the PI3K-PKB signaling pathway (Miyake et al., 2000;Roschier et al., 2001). As observed in the present study, the greater expression of IGFBP-5 in PDLF compared with GF, together with IGF-1-induced reduction of apoptosis in PDLF, suggests a potential role of IGFBP-5 in the up-regulation of the IGF-1 pathway in PDLF compared with GF. Furthermore, an IGF-independent effect of IGFBP-5 has been demonstrated recently (Miyakoshi et al., 2001). The identification of putative IGFBP-5 receptors (Andress, 1998) and the detection of IGFBP-5 in the nucleus (Schedlich et al., 2000) support the idea that IGFBP-5 may also function independently as a growth factor. The exact role of IGFBP-5 in the regulation of cell survival in PDLF compared with GF still remains to be determined.
In summary, this study suggests that PDLF has an enhanced cell survival compared with GF. This may be achieved, at least in part, via preferential up-regulation of the IGF-1 signaling pathway. This up-regulation probably occurs via the activation of signaling molecules, including PI3K/PKB, IGFBP-5, and mitochondria-associated anti-apoptotic events. The differential effect of IGF-1 on the periodontium may certainly translate into distinct outcomes of periodontal treatments. Analysis of the present data supports the concept that clinical application of IGF-1 may enhance periodontal wound healing and regeneration by specifically modifying periodontal ligament cell turnover.
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ACKNOWLEDGMENTS
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We thank Dr. Eraldo Batista, Jr. and Dr. Shenghe Cai for expert technical assistance. This work was supported by the National Institute of Dental and Craniofacial Research Grant DE 12482.
Received November 1, 2002;
Last revision January 28, 2003;
Accepted March 3, 2003
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ABSTRACT
INTRODUCTION
