Matrix metalloproteinases (MMPs) and their specific tissue inhibitors (TIMPs) in mature human odontoblasts and pulp tissue

The regulation of expressions of fibrillar collagens, MMPs and TIMPs by growth factors, transforming growth factor-β 1 (TGF-β 1) and bone morphogenetic protein-2 (BMP-2).

Heidi Palosaari

Institute of Dentistry, University of Oulu

Abstract

Dentin formation in physiological and pathological conditions has been widely studied, but the events and regulation are still not completely understood. Odontoblasts, terminally differentiated post-mitotic cells located in a single cell layer around pulp tissue, synthesize and mineralize dentin organic matrix. Growth factors, such as TGF-β 1 and BMP-2, have been implicated in the regulation of the responses of odontoblasts and pulp tissue to external irritation. Matrix metalloproteinases (MMPs), a family of 28 endopeptidases collectively capable of degrading virtually all extracellular matrix components, and their specific tissue inhibitors (TIMPs) participate in the organo- and morphogenesis, physiological tissue turnover and pathological tissue destruction in many tissues, but very little is known about their presence, function, and regulation in the dentin-pulp complex cells and tissues. The aim of the work presented in this thesis was to analyze the expression and regulation of collagens, MMPs and TIMPs by TGF-β 1 and BMP-2 in mature human odontoblasts and pulp tissue. Odontoblasts synthesize and secrete type I and type III collagens, with no clear effect of TGF-β 1 on their expression levels. MMP-1, -2, -8, -9, -10, -11, -14, -15, -16, -19 and TIMP-1, -2, -3 and -4 were expressed by both odontoblasts and pulp tissue. MMP-3 and MMP-12 were not expressed in native odontoblasts or pulp tissue, and MMP-7, -24, and -25 were expressed only in odontoblasts. MMP-2, -10, -14, -20 and -23 were expressed more abundantly in odontoblasts, whereas pulp tissue expressed more MMP-13 and MMP-17. Growth factors differentially regulated the expression of different MMPs and TIMPs within and among the cells and tissues studied. In odontoblasts, MMP-1, -8 and -14 were down-regulated, but MMP-7, MMP-9, TIMP-1 and TIMP-3 up-regulated, by either TGF-β 1 or BMP-2, alone or in combination. In pulp tissue, growth factors up-regulated the expression of MMP-1, -2, -10, -13, -17 and TIMP-3, but down-regulated TIMP-4. The widespread of expression of MMPs and TIMPs by mature human odontoblasts and pulp tissue suggests that they may participate in dentin matrix organization prior to mineralization, and that growth factors may further control dentin matrix modeling, not by regulating the synthesis of type I or III collagens as previously believed, but rather by differentially regulating each MMPs and TIMPs.

Dedication

To my mother Marja-Leena and my fiancé Timo

Table of Contents
Acknowledgements
Abbreviations
List of original articles
1. Introduction
2. Review of the literature
2.1. Tooth structure
2.2. Dentin-pulp complex
2.2.1. Odontoblasts
2.2.2. Pulp tissue
2.3. Dentin formation and mineralization
2.3.1. Inter- and intratubular dentin
2.3.2. Tertiary dentin
2.4. Organic matrix of dentin
2.5. Growth factors in dentin-pulp complex
2.5.1. Transforming growth factor-β 1, TGF-β 1
2.5.2. Bone morphogenetic protein-2, BMP-2
2.6. Dentin demineralization and destruction
2.7. Metalloproteinase superfamily
2.8. Matrix metalloproteinases (MMPs)
2.8.1. Simple hemopexin domain-containing MMPs
2.8.2. Gelatin-binding MMPs
2.8.3. Furin-activated secreted MMPs
2.8.4. Vitronectin-like insert MMPs
2.8.5. Minimal-domain MMPs
2.8.6. Type I transmembrane MMPs
2.8.7. GPI-linked MMPs
2.8.8. Type II transmembrane MMPs
2.9. Transcriptional regulation of MMPs
2.9.1. Signal transduction by TGF-β s and BMPs
2.9.2. Regulation of MMPs by TGF-β 1 or BMP-2
2.10. Activation of proMMPs
2.11. Inhibition of MMP activities
2.11.1. Tissue inhibitor of matrix metalloproteinase-1, -2, -3 and -4 (TIMP-1, TIMP-2, TIMP-3 and TIMP-4)
2.11.2. Inhibition of MMPs by TIMPs
2.12. Suggested roles for MMPs and TIMPs in dentin-pulp complex
2.12.1. MMPs and TIMPs in dentinogenesis
2.12.2. MMPs and caries pathobiology
2.12.3. MMPs and pulpitis
3. Aims of the present study
4. Materials and methods
4.1. Collection of the samples
4.2. Odontoblasts and pulp tissue (I–IV)
4.2.1. Native samples (I–IV)
4.2.2. Odontoblast and pulp tissue cultures (I–IV)
4.3. Other cells (I, II)
4.3.1. Pulp and gingival fibroblasts (I, II)
4.3.2. Osteoblasts (I)
4.4. Analysis of mRNA expression
4.4.1. RT-PCR procedure (I–IV)
4.4.2. Quantitative analysis of mRNA (I)
4.4.3. Real-Time PCR (IV, Figures 5–14)
4.4.4. Ribonuclease protection assay (RPA) (III)
4.4.5. Verifying amplification products (II, III)
4.5. Analysis of protein synthesis and secretion
4.5.1. Western blot (II–IV, Figures 5–7, 14)
4.5.2. Enzymography (IV)
4.5.3. Immunofluorescence assay (II)
4.5.4. Immunohistochemical staining (II)
4.5.5. Immunoassays for type I and III collagen domains (I)
4.6. ProMMP activation assay (III)
4.7. Statistical analysis (III, IV, Figures 5–14)
5. Results
5.1. Expression of proα1(I) collagen in mature human odontoblasts and pulp tissue, and in pulpal and gingival fibroblasts and bone osteoblasts (I)
5.2. Regulation of type I and III collagens by TGF-β 1 in mature human odontoblasts and pulp tissue, and in pulpal and gingival fibroblasts (I)
5.3. Expression of MMPs and TIMPs, and regulation by TGF-β 1 and BMP-2 in mature human odontoblasts and pulp tissue (II–IV, Figures 5–14)
5.3.1. Simple hemopexin-domain containing MMPs
5.3.2. Gelatin-binding MMPs (IV, Figure 9)
5.3.3. Furin-activated secreted MMPs (IV)
5.3.4. Minimal-domain MMPs (IV, Figure 10)
5.3.5. Type I transmembrane MMPs (III, IV, Figure 11)
5.3.6. GPI-linked MMPs (IV, Figure 12)
5.3.7. Type II transmebrane MMPs (IV, Figure 13)
5.3.8. TIMPs (IV, Figure 14)
5.4. Activation of proMMP-20 and proMMP-2 by MT1-MMP (III)
6. Discussion
6.1. The expression of and effects of TGF-β 1 on type I and type III collagens in human dentin-pulp complex (I)
6.2. Expression of MMPs and regulation by TGF-β 1 and BMP-2 in human dentin-pulp complex (II–IV, Figures 5–14)
6.2.1. Collagenases (II, IV, Figure 5, 6)
6.2.2. Stromelysins (IV, Figure 7)
6.2.3. Other MMPs (IV, Figure 8)
6.2.4. Gelatin-binding MMPs (IV, Figure 9)
6.2.5. Furin-activated MMPs (IV)
6.2.6. Minimal-domain MMPs (IV, Figure 10)
6.2.7. Type I transmembrane MMPs (III, IV, Figure 11)
6.2.8. GPI-linked MMPs (IV, Figure 12)
6.2.9. Type II transmembrane MMPs (IV, Figure 13)
6.3. Expression and regulation of TIMPs in human dentin-pulp complex (IV, Figure 14)
6.4. ProMMP-20 activation (III)
6.5. Possible roles of MMPs and TIMPs in human dentin-pulp complex in health and disease
6.5.1. MMPs and dental matrix synthesis and mineralization
6.5.2. MMPs and dental injury
6.5.3. TIMPs in physiological and pathological conditions of teeth
6.5.4. Other possible roles for MMPs and TIMPs in the dentin-pulp complex
7. Future prospects
References
List of Tables
1. Growth factors detected in human dentin-pulp complex.
2. Overview of the expression levels of MMPs and TIMPs in mature human odontoblasts and pulp tissue, and the effect of growth factors (GFs), TGF-β 1 (T) and BMP-2 (B), on their expression.
List of Figures
1. A schematic picture of tooth structure.
2. Domain structure of MMPs and their classification. MMPs are illustrated as latent zymogens with a catalytic site zinc (Zn) binding to cysteine (C) of cysteine-switch of the prodomain. (Modified from Egeblad & Werb 2002)
3. Signalling cascades of TGF-β and BMP. (Modified from Miyazono et al. 2001).
4. Some activation routes of latent MMPs.
5. A) The effects of 1 ng/ml TGF-β 1 and 100 ng/ml BMP-2 on MMP-1 mRNA expression were analyzed with Real-Time PCR. Graphic presentations show mean and standard error of the mean (SEM) of the relative MMP-1 mRNA expression in odontoblasts (n = 2–3 in all groups) and pulp tissue (n = 3–5 in all groups) after tissue stimulation with either TGF-β 1 (T), BMP-2 (B), TGF-β 1 combined with BMP-2 (T+B) or without mediator (Ctr). One-way ANOVA with LSD test was used for statistical analysis (*: P = 0.05, **: P = 0.01, ***: P = 0.001). B) MMP-1 protein secretion into conditioned culture media of odontoblasts and pulp tissue was analyzed by Western blot procedure, which show the latent (proMMP-1) and active MMP-1 (aMMP-1) immunoreactive bands in all samples.
6. A) The effects of 1 ng/ml TGF-β 1 and 100 ng/ml BMP-2 on MMP-13 mRNA expression were analyzed with Real-Time PCR. Graphic presentations show mean and standard error of the mean (SEM) of the relative MMP-13 mRNA expression in pulp tissue (n = 4–5 in all groups) after tissue stimulation with either TGF-β 1 (T), BMP-2 (B), TGF-β 1 combined with BMP-2 (T+B) or without mediator (Ctr). One-way ANOVA with LSD test was used for statistical analysis (*: P = 0.05, **: P = 0.01, ***: P = 0.001). B) MMP-13 protein secretion into conditioned culture media of pulp tissue was analyzed by Western blot procedure, which shows the active MMP-13 (aMMP-13) immunoreactive bands in all samples.
7. A, B) The effects of 1 ng/ml TGF-β 1 and 100 ng/ml BMP-2 on MMP-10 mRNA expression were analyzed with Real-Time PCR. Graphic presentations show mean and standard error of the mean (SEM) of the relative MMP-10 mRNA expression in odontoblasts (A) (n = 3–4 in all groups) and pulp tissue (B) (n = 4–5 in all groups) after tissue stimulation with either TGF-β 1 (T), BMP-2 (B), TGF-β 1 combined with BMP-2 (T+B) or without mediator (Ctr). One-way ANOVA with LSD test was used for statistical analysis (*: P = 0.05, **: P = 0.01, ***: P = 0.001). C) MMP-10 protein secretion into conditioned culture media of odontoblasts was analyzed by Western blot procedure, which shows the latent (proMMP-10) and active MMP-10 (aMMP-10) immunoreactive bands in all samples.
8. A, B) The effects of 1 ng/ml TGF-β 1 and 100 ng/ml BMP-2 on MMP-19 mRNA and MMP-27 mRNA expression were analyzed with Real-Time PCR. Graphic presentations show mean and standard error of the mean (SEM) of the relative MMP-19 mRNA (A) and MMP-27 mRNA (B) expression in odontoblasts (n = 3–5 in all groups) and pulp tissue (n = 4–5 in all groups) after tissue stimulation with either TGF-β 1 (T), BMP-2 (B), TGF-β 1 combined with BMP-2 (T+B) or without mediator (Ctr). One-way ANOVA with LSD test was used for statistical analysis (*: P = 0.05, **: P = 0.01, ***: P = 0.001).
9. The effects of 1 ng/ml TGF-β 1 and 100 ng/ml BMP-2 on MMP-2 mRNA expression were analyzed with Real-Time PCR. Graphic presentations show mean and standard error of the mean (SEM) of the relative MMP-2 mRNA expression in odontoblasts (n = 3–4 in all groups) and pulp tissue (n = 4 in all groups) after tissue stimulation with either TGF-β 1 (T), BMP-2 (B), TGF-β 1 combined with BMP-2 (T+B) or without mediator (Ctr). One-way ANOVA with LSD test was used for statistical analysis (*: P = 0.05, **: P = 0.01, ***: P = 0.001).
10. A, B) The effects of 1 ng/ml TGF-β 1 and 100 ng/ml BMP-2 on MMP-7 mRNA and MMP-26 mRNA expression were analyzed with Real-Time PCR. Graphic presentations show mean and standard error of the mean (SEM) of the relative MMP-7 mRNA expression in odontoblasts (n = 3–4 in all groups) (A) and MMP-26 mRNA expression in odontoblasts (n = 3–4 in all groups) and pulp tissue (B) (n = 5 in all groups) after tissue stimulation with either TGF-β 1 (T), BMP-2 (B), TGF-β 1 combined with BMP-2 (T+B) or without mediator (Ctr). One-way ANOVA with LSD test was used for statistical analysis (*: P = 0.05, **: P = 0.01, ***: P = 0.001).
11. A, B) The effects of 1 ng/ml TGF-β 1 and 100 ng/ml BMP-2 on MMP-15 mRNA and MMP-16 mRNA expression were analyzed with Real-Time PCR. Graphic presentations show mean and standard error of the mean (SEM) of the relative MMP-15 mRNA (A) and MMP-16 mRNA (B) expression in odontoblasts (n = 3–5 in all groups) and pulp tissue (n = 4–5 in all groups) after tissue stimulation with either TGF-β 1 (T), BMP-2 (B), TGF-β 1 combined with BMP-2 (T+B) or without mediator (Ctr). One-way ANOVA with LSD test was used for statistical analysis (*: P = 0.05, **: P = 0.01, ***: P = 0.001).
12. A, B) The effects of 1 ng/ml TGF-β 1 and 100 ng/ml BMP-2 on MMP-17 mRNA and MMP-25 mRNA expression were analyzed with Real-Time PCR. Graphic presentations show mean and standard error of the mean (SEM) of the relative MMP-17 mRNA expression in pulp tissue (A) (n = 3–4 in all groups) and MMP-25 mRNA expression in odontoblasts (n = 3 in all groups) and pulp tissue (B) (n = 5 in all groups) after tissue stimulation with either TGF-β 1 (T), BMP-2 (B), TGF-β 1 combined with BMP-2 (T+B) or without mediator (Ctr). One-way ANOVA with LSD test was used for statistical analysis (*: P = 0.05, **: P = 0.01, ***: P = 0.001).
13. The effects of 1 ng/ml TGF-β 1 and 100 ng/ml BMP-2 on MMP-23 mRNA expression were analyzed with Real-Time PCR. Graphic presentation shows mean and standard error of the mean (SEM) of the relative MMP-23 mRNA expression in odontoblasts (n = 3–4 in all groups) and pulp tissue (n = 3–4 in all groups) after tissue stimulation with either TGF-β 1 (T), BMP-2 (B), TGF-β 1 combined with BMP-2 (T+B) or without mediator (Ctr). One-way ANOVA with LSD test was used for statistical analysis (*: P = 0.05, **: P = 0.01, ***: P = 0.001).
14. A, C, E, G) The effects of 1 ng/ml TGF-β 1 and 100 ng/ml BMP-2 on TIMP-1 (A), TIMP-2 (C), TIMP-3 (E) and TIMP-4 (G) mRNA expression levels were analyzed with Real-Time PCR. Graphic presentations show mean and standard error of the mean (SEM) of the relative TIMPs expression levels in odontoblasts (n = 3–5 in all groups) and pulp tissue (n = 3–5 in all groups) after tissue stimulation with either TGF-β 1 (T), BMP-2 (B), TGF-β 1 combined with BMP-2 (T+B) or without mediator (Ctr). One-way ANOVA with LSD test was used for statistical analysis (*: P = 0.05, **: P = 0.01, ***: P = 0.001). B, D, F) TIMP protein secretion into conditioned culture media of odontoblasts and pulp tissue was analyzed by Western blot, which show the immunoreactive bands of the respective size of 28.5 kDa TIMP-1 (B), 21 kDa TIMP-2 (D) and 27 kDa TIMP-3 (F) in all samples.
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