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RESEARCH REPORT |
Division of Periodontology and Endodontology, Tohoku University Graduate School of Dentistry, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan;
* corresponding author, e-nemoto{at}umin.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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disappeared with an increasing dose of elastase, and PDGFR-ß was degraded into several fragments. Elastase degraded both receptors on fixed cells, indicating that the degradation resulted from direct proteolysis on the cell surface. Elastase also then disturbed the phosphorylation of ERK1/2, JNK/SARK, and p38, triggered by PDGF-AA and PDGF-BB, suggesting that elastase inhibited PDGFR-dependent cell activation in PDL cells. These results suggest that elastase may modulate the PDGF-mediated activity of PDL cells during periodontal wound healing.
KEY WORDS: elastase • PDGF receptor • periodontal ligament cells • MAP kinase
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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During the process of periodontal tissue regeneration, periodontal ligament (PDL) cells are regarded to have the capacity to differentiate into cementoblasts or osteoblasts, depending on the need, and to form cementum or alveolar bone (MacNeil and Somerman, 2000). Recently, research has focused on the regeneration of periodontal tissue with the use of growth factors. Among the various growth factors, platelet-derived growth factors (PDGFs) regulate diverse cellular functions in connective tissue cells, and are important for normal embryonic development (Betsholtz et al., 2001). The PDGF family consists of 4 members—PDGF-A, PDGF-B, PDGF-C, and PDGF-D—which form 4 functional homodimers—PDGF-AA, PDGF-BB, PDGF-CC, and PDGF-DD—as well as the heterodimer PDGF-AB (Betsholtz et al., 2001). PDGF-AA and PDGF-BB have been shown to have effects on proliferation, chemotaxis, and matrix synthesis in PDL cells (Oates et al., 1993; Boyan et al., 1994; Giannobile, 1996). These responses of PDL cells to PDGF depend on the expression of 2 types of receptors, the PDGF receptor (PDGFR)- and -ß, both of which are protein tyrosine kinase receptors (Heldin et al., 1998).
These observations led us to investigate whether elastase can affect the PDGFR on PDL cells and regulate the cellular activity triggered by PDGF.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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(extracellular domain) and rPDGFR-ß/Fc chimera (extracellular domain), human rPDGF-AA, and rPDGF-BB were obtained from R&D Systems Inc. (Minneapolis, MN, USA). Cell lysis buffer® was obtained from Cell Signaling Technology (Beverly, MA, USA).
Cells
After receiving informed consent, we obtained human PDL cells from the periodontal ligaments of fully erupted third molar teeth of healthy individuals (aged between 16 and 23 yrs), without clinical signs of inflammation in the periodontal tissues. Periodontal ligaments were dissected from the middle third of the root with a sharp blade, cut into small pieces, and cultured in -Minimum Essential Medium (MEM) (Gibco BRL, Rockville, MD, USA) with 10% heat-inactivated FBS and antibiotics, with a medium change every 3 days until confluent cell monolayers formed. After confluency, the cells were passaged with 0.25% trypsin-0.1% EDTA. PDL cells were used from the fourth and seventh passages in all experiments. The experimental procedures were approved by the Ethical Review Board of Tohoku University Graduate School Dentistry (Sendai, Japan).
Elastase Treatment
PDL cells were collected from confluent monolayer cells with the use of Cell Dissociation Solution®, and the cells (2 x 105 cells) were treated with the indicated concentration of elastase in 500 µL of -MEM containing 0.1% (w/v) BSA at 37°C for the indicated times. Confluent monolayers of cells cultured in PRIMARIATM EASYGRIPTM 35-mm tissue culture dishes (BD Bioscience Discovery Labware, Bedford, MA, USA) were treated with the indicated concentration of elastase in 1 mL of -MEM containing 0.1% (w/v) BSA. Human rPDGFR- (10 ng) and rPDGFR-ß (10 ng) were treated with the indicated molar ratio of elastase (29.5 kDa) for 30 min at 37°C in 20 µL of PBS, and then the reaction was stopped by the addition of 5 µL of PMSF to a final concentration of 1 mmol/L.
Flow Cytometry
In total, 1 x 105 PDL cells, collected with the use of Cell Dissociation Solution®, were stained with phycoerythrin-conjugated monoclonal antibodies (mAbs) for human PDGFR- (R1, mouse IgG2a) and PDGFR-ß (28D4, mouse IgG2a) (BD Biosciences PharMingen, San Diego, CA, USA) or isotype-matched control IgG (BD Biosciences PharMingen). Staining was analyzed on a FACScan® (BD Biosciences Immunocytometry Systems, San Jose, CA, USA). The arithmetic mean was used in the computation of the mean fluorescence intensity (MFI).
PDGF Stimulation
Confluent monolayer cells cultured in 35-mm-diameter culture dishes were cultured without FBS for 24 hrs, and then stimulated with 50 ng/mL PDGF in 1 mL of -MEM for 5 min at 37°C.
Preparation of Cell Lysates
Cell lysates were prepared from cell pellets and confluent monolayer cells. PDL cell pellets (8 x 105 cells), collected with Cell Dissociation Solution®, underwent lysis in 100 µL of cell lysis buffer® [20 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 1 mmol/L Na2 EDTA, 1 mmol/L EGTA, 1% Triton, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L ß-glycerophosphate, 1 mmol/L Na3VO4, 1 µg/mL leupeptin] containing 1 mmol/L PMSF. Confluent monolayer cells on 35-mm-diameter tissue culture dishes were harvested with a cell scraper and subjected to lysis in 50 µL and 100 µL of cell lysis buffer® for the analysis of PDGFR expression and kinase activity, respectively. After lysis, cells were incubated on ice for 30 min, followed by centrifugation at 12,000 x g at 4°C for 10 min, after which the supernatants were collected and stored at –20°C until use.
Western Blotting
Cell lysates (25 µL) and rPDGFR (25 µL) were solubilized with Laemmli sample buffer at 100°C for 5 min, separated by SDS-polyacrylamide gel electrophoresis (PAGE) (7.5% or 10%), and transferred to a polyvinylidene difluoride (PVDF) membrane (ATTO Co., Tokyo, Japan) via a semi-dry transblot system (ATTO). The blot was blocked with 0.5% (w/v) non-fat dried milk and 0.1% (v/v) Tween 20 in PBS at 4°C overnight, followed by incubation for 1 hr at room temperature with goat anti-human PDGFR- and -ß polyclonal Abs (R&D Systems Inc.) at 1 µg/mL or rabbit anti-phospho MAPK polyclonal Abs (Cell Signaling Technology) at 1:1000. The blot was incubated with horseradish-peroxidase-conjugated affiniPure donkey anti-goat IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) at 1:30,000 or goat anti-rabbit IgG (Cell Signaling Technology) at 1:2000 for 1 hr at room temperature. The blot was treated with Western blotting detection reagent ECL Plus® (Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA), as instructed by the manufacturer. The detected blot was exposed to PolaroidTM film with the use of the ECL mini-camera. Phospho-MAPK antibodies on membranes were removed by a Re-Blot Plus Western Blot Recycling Kit® (Chemicon International, Inc., Temecula, CA, USA), according to the manufacturer’s instructions, and the membrane was re-probed with corresponding rabbit anti-MAPK polyclonal Abs (Cell Signaling Technology) at 1:1000. The Mr of the proteins was estimated by comparison with the position of the standard (Bio-Rad Laboratories, Hercules, CA, USA).
Statistical Analysis
We performed all experiments in this study at least three times to test the reproducibility of the results, and the representative findings are shown. In some experiments, experimental values are given as means ± standard errors (SE). The significance of differences between two means was evaluated by one-way ANOVA. P values less than 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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and PDGFR-ß on the cell surface, as assessed by flow cytometry (Figs. 1A
, 1B). PDGFR- expression was significantly reduced by 1 µg/mL elastase treatment for 30 min, compared with elastase-untreated cells (Fig. 1C). However, PDGFR-ß expression was substantially unchanged by elastase treatment (Fig. 1D). The time kinetics experiment with 5 µg/mL elastase revealed that PDGFR- was reduced almost completely after 30 to 60 min of treatment (Fig. 1E), but PDGFR-ß was unchanged (Fig. 1F).
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, expressed as an approximately 175-kDa band on untreated PDL cells, was gradually diminished as the concentration of elastase increased, and disappeared completely at 5 µg/mL of elastase. PDGFR-ß, expressed as an approximately 180-kDa band, was degraded into smaller bands with MWs of approximately 120 and 100 kDa (Fig. 2A). The reduction should have resulted from one of three possibilities: (1) proteolytic cleavage on the cell surface by elastase, or (2) internalization or (3) shedding by endogenous enzymes following cell activation by elastase. Therefore, to clarify this, we fixed PDL cells with paraformaldehyde before elastase treatment. PDGFR- and -ß were degraded in fixed cells as well as unfixed cells by elastase treatment (Fig. 2B). These findings suggested that elastase reduced the PDGFR on the cell surface proteolytically. The degradation was inhibited completely by pre-treatment of elastase with 1 mmol/mL HLE/CMK, an elastase-specific inhibitor (Fig. 2B), indicating that enzymatic activity was required for the degradation. Adherent cells exhibited sensitivity to elastase (Fig. 2C) similar to that of suspended cells (Figs. 2A, 2B), and showed a clearer degradation pattern of PDGFR-ß to proteins of 120, 100, 90, and 80 kDa (Fig. 2C). To confirm that elastase can degrade PDGFR, we treated human rPDGFR- and -ß with elastase. These proteins had a predicted MW of 56 kDa and 84 kDa and migrated at approximately 100 kDa and 150 kDa, respectively, due to glycosylation. As the molar ratio of elastase:PDGFR increased, the 2 receptors were gradually degraded (Fig. 2D). Fragments of PDGFR- exhibited a strong sensitivity for elastase and disappeared completely at a high ratio. However, fragments of PGDFR-ß, which was degraded with 2 steps, appeared relatively resistant to elastase. These characteristics of elastase sensitivity were consistent with those of intact cells.
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). Pre-treatment of cells with elastase almost completely inhibited PDGF-AA- and -BB-induced ERK-1/2 phosphorylation to the level of background, where a slight phosphorylation of ERK-1, but not ERK-2, was induced by elastase treatment itself. Neither PDGF-AA nor -BB induced the phosphorylation of JNK-1 and p38 in elastase-treated cells. Thus, these findings indicate that elastase inhibited the MAPK activation in PDL cells triggered by PDGF-AA and -BB.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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and PDGFR-ß on PDL cells via direct proteolysis on the cell surface, and resulted in the down-regulation of MAPK activation triggered by PDGF-AA and PDGF-BB. In view of a previous report that elastase releases a PDGF lacking the fibroblast chemotactic activity (Senior et al., 1985), the pathway of PDGF/PDGFR in fibroblasts can be exhaustively down-regulated by elastase. In addition, although serum contains abundant naturally occurring protease inhibitors, the pericellular concentration of elastase exceeds that of naturally occurring inhibitors by approximately 2 orders of magnitude (Campbell and Campbell, 1988), suggesting that our finding is likely to occur in pericellular lesions in vivo.
PDGFR- and -ß are structurally similar, consisting of an extracellular ligand-binding domain containing 5 immunoglobulin-like motifs and an intracellular tyrosine kinase domain (Heldin et al., 1998). However, the extracellular ligand-binding domains of the 2 receptors are only 31% identical, whereas they share 85% and 75% identity, respectively, in the N- and C-terminal kinase portions (Matsui et al., 1989). Accordingly, our finding—that the 2 receptors had different sensitivities for elastase (Fig. 2
)—is conceivable. PDGFR- could be degraded into multiple fragments, which were not detected by Western blot analysis; meanwhile, PDGFR-ß was degraded into several fragments, which were relatively resistant for elastase. This different sensitivity was also demonstrated by an experiment with human rPDGFR- and -ß proteins (Fig. 2D). Furthermore, the substantially unchanged expression of PDGFR-ß by elastase treatment, analyzed by flow cytometry (Fig. 1B), indicates the possibility that the mAb might recognize the site within the smaller fragment of PDGFR-ß.
The 2 receptors mediate similar, but not identical, cellular responses, such as mitogenicity, chemotaxis, edge ruffling, and Ca2+ mobilization (Heldin et al., 1998). Increasing evidence suggests that the 2 receptors initiate distinct signaling pathways (Rosenkranz and Kazlauskas, 1999); while the 2 receptors activate ERKs, and PDGFR-, but not PDGFR-ß, activates JNK-1 (Yu et al., 2000). The 2 receptors have different ligand-binding capacities (Heldin et al., 1998); PDGF-BB binds to both of the receptors, while PDGF-AA effectively binds only to PDGFR-. Accordingly, our finding—that both PDGF-AA and PDGF-BB failed to activate members of the MAPK family, ERK1/2, JNK-1, and p38 after elastase treatment (Fig. 3)—suggests that not only was PDGFR- signaling impaired, but also that fragmented PDGFR-ß did not function as a receptor.
MAPK signaling is reported to be important for PDL cell functions. The mitogenic responses of PDL cells to enamel matrix derivative, which was used for the regeneration of functional periodontal tissue (Hammarström, 1997), are associated with ERK1/2 activation (Matsuda et al., 2002). PDGF-BB-induced migration of PDL cells is mediated through the p38 MAPK signaling pathway (Ray et al., 2003). Our finding that MAPK activation, triggered by PDGF, was inhibited by elastase suggested that elastase might induce deleterious effects on the periodontal regenerative responses.
It has been reported that the combination of PDGF-BB and IGF-I stimulates periodontal regeneration in various animals and humans (Lynch et al., 1989; Howell et al., 1997). In contrast, elastase is released at inflammatory sites where neutrophils accumulate. Besides, early-onset periodontitis patients have been suggested to have intrinsic hypergranulopoiesis (Nemoto et al., 1997), and their neutrophils produced much more elastase than the controls (Åsman, 1988). Elastase remaining in periodontal tissue may impair not only the regenerative response initiated by PDL cells, but also the gingival fibroblast-mediated host defense (Nemoto et al., 2000, 2002). Since elastase induces proliferative responses in epithelial cells (Rogalski et al., 2002), elastase may lead to downgrowth of epithelial cells into the periodontal lesion, which would not be advantageous for periodontal regeneration. Consequently, careful control of elastase activity would be required for predictable results to be obtained during the periodontal regenerative process.
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ACKNOWLEDGMENTS |
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Received August 29, 2004; Last revision February 24, 2005; Accepted April 8, 2005
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Bank U, Ansorge S (2001). More than destructive: neutrophil-derived serine proteases in cytokine bioactivity control. J Leukocyte Biol 69:197–206.
Belaaouaj A, McCarthy R, Baumann M, Gao Z, Ley TJ, Abraham SN, et al. (1998). Mice lacking neutrophil elastase reveal impaired host defense against Gram negative bacterial sepsis. Nat Med 4:615–618.[ISI][Medline]
Betsholtz C, Karlsson L, Lindahl P (2001). Developmental roles of platelet-derived growth factors. Bioessays 23:494–507.[ISI][Medline]
Boyan LA, Bhargava G, Nishimura F, Orman R, Price R, Terranova VP (1994). Mitogenic and chemotactic responses of human periodontal ligament cells to the different isoforms of platelet-derived growth factor. J Dent Res 73:1593–1600.
Campbell EJ, Campbell MA (1988). Pericellular proteolysis by neutrophils in the presence of proteinase inhibitors: effects of substrate opsonization. J Cell Biol 106:667–676.
Figueredo CM, Gustafsson A, Asman B, Bergstrom K (1999). Increased release of elastase from in vitro activated peripheral neutrophils in patients with adult periodontitis. J Clin Periodontol 26:206–211.[ISI][Medline]
Giannobile WV (1996). Periodontal tissue engineering by growth factors. Bone 19:23S–37S.[Medline]
Hammarström L (1997). Enamel matrix, cementum development and regeneration. J Clin Periodontol 24:658–668.[ISI][Medline]
Heldin CH, Ostman A, Ronnstrand L (1998). Signal transduction via platelet-derived growth factor receptors. Biochim Biophys Acta 1378:F79–F113.[Medline]
Howell TH, Fiorellini JP, Paquette DW, Offenbacher S, Giannobile WV, Lynch SE (1997). A phase I/II clinical trial to evaluate a combination of recombinant human platelet-derived growth factor-BB and recombinant human insulin-like growth factor-I in patients with periodontal disease. J Periodontol 68:1186–1193.[ISI][Medline]
Lynch SE, Williams RC, Polson AM, Howell TH, Reddy MS, Zappa UE, et al. (1989). A combination of platelet-derived and insulin-like growth factors enhances periodontal regeneration. J Clin Periodontol 16:545–548.[ISI][Medline]
MacNeil RL, Somerman MJ (2000). Development and regeneration of the periodontium: parallels and contrasts. Periodontol 2000 19:8–20.
Matsuda N, Horikawa M, Watanabe M, Kitagawa S, Kudo Y, Takata T (2002). Possible involvement of extracellular signal-regulated kinases 1/2 in mitogenic response of periodontal ligament cells to enamel matrix derivative. Eur J Oral Sci 110:439–444.[ISI][Medline]
Matsui T, Heidaran M, Miki T, Popescu N, La Rochelle W, Kraus M, et al. (1989). Isolation of a novel receptor cDNA establishes the existence of two PDGF receptor genes. Science 243:800–804.
Nemoto E, Nakamura M, Shoji S, Horiuchi H (1997). Circulating promyelocytes and low levels of CD16 expression on polymorphonuclear leukocytes accompany early-onset periodontitis. Infect Immun 65:3906–3912.[Abstract]
Nemoto E, Sugawara S, Tada H, Takada H, Shimauchi H, Horiuchi H (2000). Cleavage of CD14 on human gingival fibroblasts cocultured with activated neutrophils is mediated by human leukocyte elastase resulting in down-regulation of lipopolysaccharide-induced IL-8 production. J Immunol 165:5807–5813.
Nemoto E, Tada H, Shimauchi H (2002). Disruption of CD40/CD40 ligand interaction with cleavage of CD40 on human gingival fibroblasts by human leukocyte elastase resulting in down-regulation of chemokine production. J Leukocyte Biol 72:538–545.
Oates TW, Rouse CA, Cochran DL (1993). Mitogenic effects of growth factors on human periodontal ligament cells in vitro. J Periodontol 64:142–148.[ISI][Medline]
Owen CA, Campbell EJ (1995). Neutrophil proteinases and matrix degradation. The cell biology of pericellular proteolysis. Semin Cell Biol 6:367–376.[ISI][Medline]
Perng DW, Wu YC, Tsai MC, Lin CP, Hsu WH, Perng RP, et al. (2003). Neutrophil elastase stimulates human airway epithelial cells to produce PGE2 through activation of p44/42 MAPK and upregulation of cyclooxygenase-2. Am J Physiol Lung Cell Mol Physiol 285:L925–L930.
Ray AK, Jones AC, Carnes DL, Cochran DL, Mellonig JT, Oates TW Jr (2003). Platelet-derived growth factor-BB stimulated cell migration mediated through p38 signal transduction pathway in periodontal cells. J Periodontol 74:1320–1328.[ISI][Medline]
Rogalski C, Meyer-Hoffert U, Proksch E, Wiedow O (2002). Human leukocyte elastase induces keratinocyte proliferation in vitro and in vivo. J Invest Dermatol 118:49–54.[ISI][Medline]
Rosenkranz S, Kazlauskas A (1999). Evidence for distinct signaling properties and biological responses induced by the PDGF receptor alpha and beta subtypes. Growth Factors 16:201–216.[ISI][Medline]
Senior RM, Huang JS, Griffin GL, Deuel TF (1985). Dissociation of the chemotactic and mitogenic activities of platelet-derived growth factor by human neutrophil elastase. J Cell Biol 100:351–356.
Walsh DE, Greene CM, Carroll TP, Taggart CC, Gallagher PM, O’Neill SJ, et al. (2001). Interleukin-8 up-regulation by neutrophil elastase is mediated by MyD88/IRAK/TRAF-6 in human bronchial epithelium. J Biol Chem 276:35494–35499.
Yu J, Deuel TF, Kim HR (2000). Platelet-derived growth factor (PDGF) receptor-alpha activates c-Jun NH2-terminal kinase-1 and antagonizes PDGF receptor-beta-induced phenotypic transformation. J Biol Chem 275:19076–19082.
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