Induction of Tissue Plasminogen Activator Gene Expression by Proinflammatory Cytokines in Human Pulp and Gingival Fibroblasts

JOURNAL OF ENDODONTICS Copyright © 2003 by The American Association of Endodontists

Printed in U.S.A. VOL. 29, NO. 2, FEBRUARY 2003

Induction of Tissue Plasminogen Activator Gene Expression by Proinflammatory Cytokines in Human Pulp and Gingival Fibroblasts Yu-Chao Chang, DDS, MMS, Shun-Fa Yang, MS, Fu-Mei Huang, DDS, MMS, Kuo-Wei Tai, DDS, MSD, and Yih-Shou Hsieh, PhD

ized tissue, dentin. The inflammatory process in the pulp is essentially the same as that in connective tissue elsewhere in the body. In infectious diseases, the invasion of the tissue by microorganisms frequently induces a wide variety of inflammatory and immunopathological reactions. Proinflammatory cytokines will initiate and augment subsequent inflammatory cascades leading to tissue destruction. These cytokines are produced by a variety of cell types, such as macrophages, monocytes, and polymorphonuclear leukocytes, and are potent inducers of mineralized tissue resorption in vitro (1, 2). An immunohistochemistry study has shown that interleukin (IL)-1 and tumor necrosis factor-␣ (TNF-␣)-positive cells were found to be present in rat pulp and periapical lesions after surgical pulp exposure (3). Recently, studies reported that IL-1 and TNF-␣ stimulated the production of elevated levels of matrix metalloproteinases (MMPs) in human pulp cells by gelatin zymography (4, 5). These findings suggested the significance of proinflammatory cytokines in pulpal injury. However, few studies reported on the mechanisms of tissue destruction in pulpal and periapical diseases. The subsequent reactions leading to pulpal and periapical injury after the induction of IL-1␣ and TNF-␣ remains to be elucidated. Plasmin can be formed locally at sites of inflammation and is produced by limited proteolysis of its inactive precursor, plasminogen, which circulates in plasma and interstitial fluids (6). Plasminogen activators (PA) are serine proteases that form part of the complex enzyme cascade involved in fibrinolysis. These enzymes convert plasminogen into plasmin, which is not only responsible for the degradation of fibrin but also contributes directly and indirectly, via conversion of latent collagenase into active collagenase, to the degradation and turnover of the extracellular matrix (7). Plasminogen is activated by either urokinase type plasminogen activator (u-PA) or tissue-type plasminogen activator (t-PA). These catalytic reactions generally take place at the plasma membrane (u-PA) or on a fibrin surface (t-PA). These activating enzymes are produced by a wide range of mesenchymal, epithelial, and endoepithelial cells in response to a variety of cytokines and growth factors. Thus, at sites of inflammation, the potential for up-regulation of the plasminogen-activating system is high. The resultant activated plasmin can degrade a wide range of substrates, including extracellular matrix macromolecules and fibrin.

Plasminogen activator converts plasminogen to plasmin, and plasmin activates the latent matrix metalloproteinases. Tissue plasminogen activator (t-PA) is one of the important proteolysis factors present in human inflamed tissues. However, few studies reported on the mechanisms of tissue destruction via a t-PA proteolysis pathway in pulpal and periapical diseases. The subsequent reactions leading to pulpal and periapical injury after the induction of proinflammatory cytokines remains to be elucidated. The aim of this study was to investigate the effects of interleukin-1␣ and tumor necrosis factor-␣ on the expression of t-PA mRNA gene in cultured human pulp and gingival fibroblasts. The mRNAs for t-PA were measured by reverse transcription-polymerase chain reaction at 2, 6, and 24 h. The results show that both cytokines induced significantly high levels of t-PA mRNA gene expression in human pulp fibroblasts. The peak of t-PA mRNA levels induced by both proinflammatory cytokines was at the 6-h incubation period. Interleukin-1␣ was found to be more effective in induction of t-PA gene expression than tumor necrosis factor-␣. In addition, a similar induction pattern was also found in human gingival fibroblasts. These results indicate that proinflammatory cytokines can induce t-PA gene expression and such an effect may partially contribute to the destruction of pulpal and periapical tissues through dysregulated pericellular proteolysis. An understanding of the mechanism could not only further define the role of immune events in pulpal and periapical diseases but also have important implication for pharmacological intervention.

Dental pulp is a loose, mesenchymal tissue characterized by its particular location and almost entirely enclosed within a mineral114

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The PA/plasmin proteolytic system has received considerable attention because of its participation in a wide variety of connective tissue matrix alterations, including those secondary to inflammation (8). Surprisingly, there have been relatively few studies addressing the presence and activity of the PA/plasmin system in pulpal disease. The fibroblast is a prominent cellular component of pulpal and periapical tissues. The aim of this study was to examine the effects of proinflammatory cytokines on the PA/plasmin system in cultured human pulp and gingival fibroblasts. MATERIALS AND METHODS All tissue culture biologicals, TRIzol, RNase A, and Moloney murine leukemia virus reverse transcriptase were purchased from Gibco Laboratories (Grand Island, NY). IL-1␣ and TNF-␣ were purchased from Sigma Chemical Co. (St. Louis, MO). Cytokines were directly dissolved in the culture medium. The final concentration of IL-1␣ and TNF-␣ used in this study was 10 ng/ml. Cell Culture Human pulp and gingival fibroblasts were cultured using an explant technique as described previously (5, 9, 10). Briefly, impacted third molars were obtained from healthy patients of the Department of Oral and Maxillofacial Surgery, Chung Shan Medical University Hospital, Taichung, Taiwan. For culture of gingival fibroblasts, gingival connective tissues were minced into small fragments. For culture of pulp fibroblasts, teeth were sectioned horizontally below the CEJ with a #330 high-speed bur with water spray. The pulp tissue was removed aseptically, rinsed with Hanks’ buffered saline solution, and placed in a 60-mm Petri dish. Pulp tissue was also minced with a blade into small fragments and grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS) and antibiotics (100 U/ml of penicillin, 100 ␮g/ml of streptomycin, and 0.25 ␮g/ml of Fungizone). Cultures were maintained at 37°C in a humidified atmosphere of 5% CO2 and 95% air. Confluent cells were detached with 0.25% trypsin and 0.05% EDTA for 5 min, and aliquots of separated cells were subcultured. Cell cultures between the third and eighth passages were used in this study. Cytokines Treatments Confluent fibroblasts were trypsinized, counted, and plated at a concentration of 1 ⫻ 105 cells in 60-mm culture dish and allowed to achieve confluence. Cells arrested in G0 by serum deprivation (0.5% FCS for 48 h) were generally used in these experiments. Before treatment, the cells were washed with serum-free DMEM and immediately exposed for the indicated incubation times (2, 6, and 24 h) to both cytokines. Cultures with 10% FCS were used as positive control.

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reverse transcriptase. The reaction mixture was diluted with 20 ␮l of water and 3 ␮l of the diluted reaction mixture was used for the polymerase chain reaction (PCR). PCR reaction mixture contains 10 pmol of forward and reverse primers and 2 units of Tag DNA polymerase. Amplification was performed at 25 cycles for GAPDH and 35 cycles for t-PA in a thermal cycle. Each cycle consisted of 1 min of denaturation at 94°C, 1 min of annealing at 57°C, and 1 min of extension at 72°C. The sequences of primers used are listed in Table 1 (11). The PCR products were analyzed by agarose gel electrophoresis. The intensity of each band after normalization with GAPDH mRNA was quantified by the photographed gels with a densitometer (AlphaImager 2000). Each densitometric value, expressed as the mean ⫾ SD, was obtained from three independent experiments. RESULTS The effects of proinflammatory cytokines on the expression of t-PA gene expression in human pulp and gingival fibroblasts are shown in Figs. 1 and 2. As visualized, PCR products corresponding to GAPDH are almost equivalent in RNA preparations, the amount of PCR product should be a sufficiently sensitive indicator of mRNA expression levels. After exposure to cytokines for 2, 6, and 24 h, total RNA in pulp fibroblasts was isolated and evaluated by RT-PCR. Densitometric analysis of the t-PA mRNA gene expression, after normalization by GAPDH, demonstrated that exposure to IL-1␣ and TNF-␣ resulted in induction of t-PA mRNA (Fig. 1). The values of induction by IL-1␣ were 2.35-, 6.5-, and 2.9-fold at 2 h, 6 h, and 24 h, respectively. Moreover, the value of induction by TNF-␣ was 1.5-, 4.3-, and 4.1-fold at 2 h, 6 h, and 24 h, respectively. The peak of t-PA mRNA levels induced by both proinflammatory cytokines was at the 6-h incubation period. IL-1␣ was found to be more effective in induction of t-PA gene expression than TNF-␣. The expression of t-PA mRNA gene in human gingival fibroblast by proinflammatory cytokines is shown in Fig. 2. It reveals TABLE 1. Nucleotide sequence and size of the expected PCR products for oligonucleotide primers used for RT-PCR Gene

Sequence

GAPDH 5⬘-TCCTCTGACTTCAACAGCGACACC-3⬘ 5⬘-TCTCTCTTCCTCTTGTGCTCTTGG-3⬘ t-PA 5⬘-AGGCTCATGTCAGACTGTACC-3⬘ 5⬘-CCTGAAATCAGACCAAGTCC-3⬘

PCR product (bp) 207 500

Total RNA Preparation and Reverse-Transcriptase Polymerase Chain Reaction Total RNA was prepared using TRIzol reagent following the manufacturer instructions. Single-stranded DNA was synthesized from RNA in a 15-␮l reaction mixture containing 100 mg of random hexamer and 200 units of Moloney murine leukemia virus

FIG 1. Expression of t-PA gene in proinflammatory cytokine-treated human pulp fibroblasts by RT-PCR assays. M ⫽ DNA molecular size marker. Lanes 1 and 2 are control: 0.5% and 10% FCS. Lanes 3 to 5 are IL-1␣: 2, 6, and 24 h, respectively. Lanes 6 to 8 are TNF-␣: 1, 3, and 6 h, respectively.

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FIG 2. Expression of t-PA gene in proinflammatory cytokine-treated human gingival fibroblasts by RT-PCR assays. M ⫽ DNA molecular size marker. Lanes 1 and 2 are control: 0.5% and 10% FCS. Lanes 3 to 5 are IL-1␣: 2, 6, and 24 h, respectively. Lanes 6 to 8 are TNF-␣: 1, 3, and 6 h, respectively.

induction of t-PA after treatment of cell cultures with IL-1␣ and TNF-␣. Similar induction patterns were also found in human gingival fibroblasts. From the AlphaImager 2000, the value of induction by IL-1␣ was approximately 1.9-, 6.0-, and 3.1-fold at 2 h, 6 h, and 24 h, respectively. The values of induction by TNF-␣ were 1.7-, 5.0-, and 3.4-fold at 2 h, 6 h, and 24 h, respectively. DISCUSSION At sites of inflammation and tissue destruction, cells communicate with one another through the interaction of cytokines and other related molecules. Thus, their importance in the complex cascade of events associated with inflammation mediated tissue destruction and repair cannot be discounted. In many inflammatory-mediated conditions, the PA/plasmin proteolytic system has received considerable attention because of its participation in a wide variety of biological activities and in pathological conditions involving tissue destruction. Regulation of PA is a key element in controlling proteolytic events in the extracellular matrix, and this regulation is achieved through the action of specific PA inhibitors. At sites of inflammation, the PAs are involved in cell migration and tissue remodeling (7). Plasmin is generated from its precursor plasminogen by the action of PA, of which there are two types, t-PA and u-PA. u-PA is thought to be involved in more generalized proteolysis and has increasingly been implicated in tumor invasion (12). t-PA, which is activated by fibrin, is thought to be a key enzyme involved in fibrinolysis (13). The degradation of matrix is thought to play a major role in tissue destruction during pulpal and periapical lesions. Thus, we focused on the effects of proinflammatory cytokines on t-PA expression in human pulp and gingival fibroblasts in this study. IL-1 and IL-1-producing cells have been demonstrated in inflamed pulp cells (4, 14) as well as periapical lesions (4). This proinflammatory cytokine may, in turn, initiate and augment inflammatory processes in pulpal and periapical lesions. The experiments reported here demonstrated that human IL-1␣ induced normal human pulp fibroblasts express t-PA mRNA gene. To the best of our knowledge, this is the first study to report such an effect of IL-1␣ on t-PA gene expression in pulp cells. In addition, the same pattern was found in human gingival fibroblasts. Our results are in agreement with Mochan et al. (15), who reported that addition of human IL-1 stimulated the production of PA activity. These results suggest that IL-1␣ induced pulpal and periapical lesions may be via the t-PA pathway. TNF-␣ is a protein of approximately 17,000 KDa and is one of several cytokines secreted by macrophages in response to bacterial

lipopolysaccharide (16). TNF-␣ has been demonstrated in pulp and periapical lesions by immunohistochemistry (4). To our knowledge, no information is available concerning the effect of TNF-␣ on gene expression of t-PA. In this study, we first found that TNF-␣ induced t-PA mRNA gene expression in human gingival fibroblasts. Moreover, the same pattern was also found in human pulp fibroblasts. This result was in agreement with Ueda and Matsushima (17), who reported that a significant level of t-PA activation occurred in a time- and dose-dependent manner in pulp cells treated with TNF-␣. Therefore, persistent induction of the t-PA expression by TNF-␣ may be one of the mechanisms in the pathogenesis of pulpal and periapical lesions. In this study, IL-1␣ and TNF-␣ have been shown to induce proteolytic enzyme t-PA mRNA gene expression in human pulp and gingival fibroblasts. Plasmin degrades fibrin and several extracellular matrix and adhesion proteinases and, by activation of procollagenases, may contribute to collagen degradation (9). Indeed, the plasmin-dependent pathway is understood to be a significant alternative pathway for the initiation of extracellular matrix degradation by MMPs (18). Previous studies have shown IL-1 and TNF-␣ can stimulate the synthesis and secretion of MMPs in human pulp cell cultures (5, 6, 19, 20). Recently, a study has shown that TNF-␣ stimulates t-PA gene expression, and t-PA-activated TNF-␣ stimulated MMP-9 activity in human pulp cells (17). The interaction between MMPs and t-PA is worthy of further investigation. In summary, the results of our study demonstrate that proinflammatory cytokines induce human pulp and gingival fibroblast t-PA mRNA gene expression. These results suggest that t-PA gene expression could be important in initiating inflammatory events that could lead to tissue destruction associated with pulp and periapical diseases. An understanding of this mechanism could not only further define the role of immune events pulpal and periapical diseases but also have important implications for pharmacological intervention. However, the detailed mechanism of activation of t-PA by proinflammatory cytokines in vivo remains to be further defined. Drs. Chang, Huang, and Tai are specialists, Dental Department, Chung Shan Medical University Hospital. Dr. Chang is associate professor, Institute of Stomatology, Mr. Yang is a PhD student, and Dr. Hsieh is associate professor, Institute of Biochemistry, Chung Shan Medical University, Taichung, Taiwan. Address requests for reprints to Dr. Yih-Shou Hsieh, Institute of Biochemistry, Chung Shan Medical and University, 110, Sec. 1, ChienKuo N. Road, Taichung, Taiwan.

References 1. Gowen M, Wood DD, Mundy GR, Russell RGG. Studies on the actions of interleukin-1 on bone metabolism: IL-1 stimulation of bone cell proliferation and inhibition of IL-1-induced bone resorption by interferon-␥. Br J Rheum 1985;24:147–9. 2. Thomson BM, Mundy GR, Chambers TJ. Tumor necrosis factor ␣ and ␤ induce osteoblastic cells to stimulate osteoclastic bone resorption. J Immunol 1987;138:775–9. 3. Tani-Ishii N, Wang CY, Stashenko P. Immunolocalization of bone-resorptive cytokines in rat pulp and periapical lesions following surgical pulp exposure. Oral Microbiol Immunol 1995;10:213–9. 4. O’Boskey FJ Jr, Panagakos FS. Cytokines stimulate matrix metalloproteinase production by human pulp cells during long-term culture. J Endodon 1998;24:9 –11. 5. Chang YC, Yang SF, Hsieh YS. Regulation of matrix metalloproteinase-2 production by cytokines and pharmacological agents in human pulp cell cultures. J Endodon 2001;27:679 – 82. 6. Deutsch DG, Mertz ET. Plasminogen: purification from human plasma by affinity chromatography. Science 1970;170:1095– 6. 7. Vassall JD, Sappino AP, Belin D. The plasminogen activator/plasmin system. J Clin Invest 1991;88:1067–72.

Vol. 29, No. 2, February 2003 8. Kruithof EKO, Baker MS, Bunn CL. Biological and clinical aspects of plasminogen activator type 2. Blood 1995;86:4007–24. 9. Chang YC, Chou MY. Cytotoxicity of halothane on human gingival fibroblast cultures in vitro. J Endodon 2001;27:82– 4. 10. Chang YC, Lai CC, Yang SF, Chan Y, Hsieh YS. Stimulation of matrix metalloproteinases by black-pigmented Bacteroides in human pulp and periodontal ligament cell cultures. J Endodon (in press). 11. Xiao Y, Bunn CL, Bartold PM. Effect of lipopolysaccharide from periodontal pathogens on the production of tissue plasminogen activator and plasminogen activator inhibitor 2 by human gingival fibroblasts. J Periodontal Res 2001;36:25–31. 12. Mignatti P, Robbins E, Rifkin DB. Tumor invasion through the human amniotic membrane requirement for a proteinase cascade. Cell 1986;47:487–98. 13. Saksela O. Plasminogen activation and regulation of pericellular proteolysis. Biochim Biophys Acta 1985;823:35– 65. 14. D’Souze R, Brown LR, Newland JR, Lachman LB. Detection and characterization of interleukin-1 in human dental pulps. Arch Oral Biol 1989; 34:307–13.

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15. Mochan E, Armor L, Sporer R. Interleukin-1 stimulation of plasminogen activator production in cultured gingival fibroblasts. J Periodontal Res 1988;23:28 –32. 16. Beutler B, Ceramia A. Cachectin and tumor necrosis factor as two sides of the same biological coin. Nature 1986;32:584 – 8. 17. Ueda L, Matsushima K. Stimulate of plasminogen activation activity and matrix metalloproteinases of human dental pulp-derived cells by tumor necrosis factor-␣. J Endodon 2001;27:175–9. 18. Birkedal-Hansen H, More WG, Bodden MK, et al. Matrix metalloproteinases: a review. Crit Rev Oral Biol Med 1993;4:197–250. 19. Tamura M, Nagaoka S, Kawagoe M. Interleukin-1␣ stimulates interstitial collagenase gene expression in human dental pulp fibroblasts. J Endodon 1996;22:240 –3. 20. Lin SK, Wang CC, Huang S, et al. Induction of dental pulp fibroblast matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 gene expression by Interleukin-1␣ and tumor necrosis factor-␣ through a prostaglandin-dependent pathway. J Endodon 2001;27:185–9.