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Abundance of MMPs and Cysteine Cathepsins in Caries-affected Dentin C.M.P. Vidal, L. Tjäderhane, P.M. Scaffa, I.L. Tersariol, D. Pashley, H.B. Nader, F.D. Nascimento and M.R. Carrilho J DENT RES 2014 93: 269 originally published online 19 December 2013 DOI: 10.1177/0022034513516979 The online version of this article can be found at: http://jdr.sagepub.com/content/93/3/269

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RESEARCH REPORTS Biological

C.M.P. Vidal1, L. Tjäderhane2,3, P.M. Scaffa1, I.L. Tersariol4,5, D. Pashley6, H.B. Nader5, F.D. Nascimento7*, and M.R. Carrilho7,8* 1

Department of Restorative Dentistry, Dental Materials Area, Piracicaba Dental School, State University of Campinas, Piracicaba, Brazil; 2Institute of Dentistry, University of Oulu, Oulu, Finland; 3Oulu University Hospital, Oulu, Finland; 4 Centro Interdisciplinar de Investigação Bioquímica, University of Mogi das Cruzes, Mogi das Cruzes, Brazil; 5 Department of Biochemistry, Federal University of São Paulo, Brazil; 6Department of Oral Biology, College of Dental Medicine, Georgia Regents University, Augusta, GA, USA; 7 Biomaterials Research Group and Biotechnology Division, UNIBAN, São Paulo, Brazil; and 8Schulich School of Medicine & Dentistry, Western University, London, ON, Canada; *corresponding author, [email protected], [email protected]

Abundance of MMPs and Cysteine Cathepsins in Cariesaffected Dentin

J Dent Res 93(3):269-274, 2014

Abstract

Degradation of dentin matrix components within caries dentin has been correlated with the activity of host-derived proteases, such as matrix metalloproteases (MMPs) and cysteine cathepsins (CTs). Since this relationship has not been fully established, we hypothesized that the abundance of MMPs and CTs in caries-affected dentin must be higher than in intact dentin. To test this premise, we obtained 5 slices (200 µm) from 5 intact teeth and from 5 cariesaffected teeth (1 slice/tooth) and individually incubated them with primary antibodies for CT-B, CT-K, MMP-2, or MMP-9. Negative controls were incubated with pre-immune serum. Specimens were washed and re-incubated with the respective fluorescent secondary antibody. Collagen identification, attained by the autofluorescence capture technique, and protease localization were evaluated by multi-photon confocal microscopy. The images were analyzed with ZEN software, which also quantitatively measured the percentages of collagen and protease distribution in dentin compartments. The abundance of the test enzymes was markedly higher in caries-affected than in intact dentin. CT-B exhibited the highest percentage of co-localization with collagen, followed by MMP-9, MMP-2, and CT-K. The high expression of CTs and MMPs in caries-affected teeth indicates that those host-derived enzymes are intensely involved with caries progression.

KEY WORDS:

fluorescent immunohistochemistry, dental caries, dentin cysteine proteases, metalloproteases, collagen. DOI: 10.1177/0022034513516979 Received June 18, 2013; Last revision November 19, 2013; Accepted November 23, 2013 © International & American Associations for Dental Research

Introduction

C

ariogenic bacteria have traditionally been thought to be responsible for the destruction of dentin organic matrix. However, in situ and in vitro experiments have questioned the role of bacterial enzymes in demineralized dentin degradation (Katz et al., 1987; van Strijp et al., 1994; Kawasaki and Featherstone, 1997). Almost 3 decades ago, Dayan et al. (1983) reported an enhanced collagenolytic activity in caries-affected dentin, implying a causal relationship between the presence of collagenase activators and enzymatic destruction of an endogenous collagenase inhibitor complex. Later, the presence and activity of some matrix metalloproteases (MMPs), the MMPs-2, -8, and -9, were identified in demineralized dentinal lesions by a combination of Western blot analysis, zymography, and functional gelatinolytic/collagenolytic SDS-PAGE assays (Tjäderhane et al., 1998b). MMPs were the first family of proteases implicated in dentin matrix destruction within caries lesions, because several of its members are known to degrade almost all components of the extracellular matrices, especially highly cross-linked triple-helical collagen (i.e., the most prevalent organic component of mature dentin), in either native or denatured conditions (Visse and Nagase, 2003). Work from our group showed that another class of proteases, the cysteine cathepsins (CTs), is also expressed by mature human odontoblasts and pulp tissue (Tersariol et al., 2010), suggesting that, in terms of gene expression, the variety of CTs present in dentin-pulp complex can be compared with that described for MMPs (Palosaari et al., 2003). Total CTs activity has been demonstrated in intact dentin (Tersariol et al., 2010) and noticeably stronger (approximately 10-fold higher) in carious dentin (Nascimento et al., 2011). Despite these findings suggesting that endogenous proteases may be involved with collagen degradation in caries lesions, the dental community has not yet embraced this concept because its potential mechanisms urge clarification. Cathepsin B (CT-B) is the only member of the CTs that has thus far been shown to be distributed in dentin tissue (Tersariol et al., 2010; Nascimento et al., 2011). Since CT-B has never been described as being able to break

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Figure 1.  Image of intact dentin obtained by confocal scanning laser microscopy. (A) Differential interference contrast (DIC), showing the optical density of dentin. (B) Acquired image in green channel, showing emitted fluorescence of collagen within areas corresponding to inner dentin. (C) Merged images A and B.

down triple-helical collagens, we speculate that abundance of CT-B in carious dentin (Nascimento et al., 2011) may reflect its role in a posteriori events by degrading non-helical telopeptides of collagen that sterically block access of MMP-8 to the isoleucine-glycine peptide bond favored by true collagenases (Perumal et al., 2008). Cathepsin K (CT-K) has been shown to be the only cathepsin capable of truly triple-helical collagenase activity (Garnero et al., 1998; Kafienah et al., 1998). CT-K is highly expressed in osteoclasts, and its secretion at the matrix-osteoclast interface is considered a key event in bone matrix degradation (Wang and McCauley, 2011). Although the gene expression of CT-K in cultured odontoblasts derived from sound teeth has been detected (Tersariol et al., 2010), we have never shown its presence/ distribution in dentin. We hypothesize that the balance between host-derived proteases in dentin may change according to the integrity of this tissue. The aim of this study was to test this hypothesis by comparing the abundance of CTs and MMPs in intact and caries-affected dentin. In addition, the interaction of these proteases with collagen in both tissues was determined.

Materials & Methods The study protocol was approved by the institutional review board (IRB) committee of Piracicaba School of Dentistry/ UNICAMP (#119/2009). Samples were collected with patients’ informed consent and consisted of 10 third molars, 5 intact and 5 with caries lesions, which were extracted for therapeutic reasons from individuals from 25 to 38 yrs of age. Teeth containing cavitated caries lesions compromising no more than 1/3 of the occlusal surface were selected for this study. Reagents were purchased from Sigma Chemical (St. Louis, MO, USA) unless otherwise specified.

Specimen Preparation Third molar crowns (from 5 intact and 5 caries-affected teeth) were cut perpendicular to their longitudinal axis with a slowspeed diamond saw under water irrigation (Isomet, Buehler, Lake Bluff, IL, USA) to produce one 200-µm-thick slice per tooth. The slices were washed with phosphate-buffered saline (PBS), conditioned with 10% phosphoric acid for 5 sec for

J Dent Res 93(3) 2014 smear layer removal, and fixed with 2% formaldehyde for 30 min at room temperature (RT). Specimens were incubated for 2 hrs at RT with one of the respective antibodies: rabbit antihuman CT-B (Calbiochem EMD Millipore Corporation, Billerica, MA, USA), rabbit anti-human CT-K (Merck EMD Millipore Corporation), mouse anti-human MMP-2 (Chemicon International Inc., Temecula, CA, USA), and rabbit anti-human MMP-9 (Calbiochem EMD Millipore Corporation) diluted in PBS at 1:100. Negative controls were incubated with preimmune serum in the same conditions. They were then incubated with the respective fluorescent secondary antibody (mouse anti-rabbit conjugated with Alexa Fluor® 594 diluted in PBS at 1:200 for 1 hr at RT under agitation), protected from light exposure, and washed with PBS.

Specimen Analysis The abundance/distribution of proteases and the autofluorescence of collagen in the different compartments of pulp-dentin complex (pulp tissue/chamber, predentin, dentin – up to 400 μm from predentin) were analyzed by inverted confocal laser scanning microscopy (Zeiss LSM-780; Carl Zeiss, Jena, Germany). When specific excitation and emission wavelengths are used, structured collagen can be observed without labeling (Elbaum et al., 2007; Lin et al., 2010). Dentin autofluorescence was attained under a 488-nm excitation from a CW Argon ion laser, and its emission was detected at 490 to 520 nm. Immunolabeled enzymes were observed with a HeNe2 laser with excitation at 633 nm, and fluorescence emission was detected at 640 to 670 nm. Dentin slices were also imaged by differential interference contrast microscopy (DIC), with a HeNe2 laser at 633 nm. Falsecolor fluorescent images and transmitted light images (512 × 512 pixels) were stored, quantified, and managed with resident ZEN software (Carl Zeiss). This allowed fluorescence intensity line profiles and intensity histograms to be measured, thereby providing mean intensity values for green and red emission channels of each image. The percentages of enzyme/structured collagen co-localization in intact and caries-affected dentin were statistically analyzed by analysis of variance (ANOVA) and Tukey’s test at a significance level of 5%.

Results Collagen Detected by Autofluorescence Collagen autofluorescence is shown in Fig. 1, which represents one of the intact, non-carious teeth. Dentinal collagen fibrillar structure can be visualized at the green channel (Fig. 1B). The fluorescent signal emitted by the molecularly well-structured collagen was notably lost in caries-affected dentin (Figs. 2F, 2N, 3F, 3N), compared with intact dentin (Figs. 1, 2B, 2J, 3B, 3J).

Protease Abundance in Intact and Caries-affected Dentin The abundance of CT-B, CT-K, MMP-2, and MMP-9 in intact and caries-affected specimens is presented in Figs. 2 and 3. The predentin and dentin morphology for intact and caries-affected

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J Dent Res 93(3) 2014  271 MMPs and Cysteine Cathepsins in Caries-affected Dentin dentin can be observed by differential interference contrast (DIC) (Figs. 2A, 2E, 2I, 2M, 3A, 3E, 3I, 3M). Both families of proteases, CTs and MMPs, were more intensely localized in regions that correspond to the pulp chamber, predentin, and/or inner dentin. Immunodetection of proteases was markedly higher in caries than in intact dentin (Figs. 2C, 2G, 2K, 2O, 3C, 3G, 3K, 3O), regardless of the enzyme detected. The intensity of emitted fluorescence relative to the area occupied by proteases (in the red channel – Figs. 2R, 2T, 2W, 2Y and 3R, 3T, 3W, 3Y) and collagen (in the green channel – Figs. 2Q, 2S, 2V, 2X and 3Q, 3S, 3V, 3X) was quantified. The abundance of CT-B and CT-K was six- and seven-fold higher, respectively, in caries than in intact dentin (Figs. 2U, 2Z). With regard to MMPs, the abundance of MMP-2 and MMP-9 was, respectively, five- and 15-fold higher in cariesaffected dentin in comparison with intact dentin (Figs. 3U, 3Z). Immunofluorescence was not detected in negative control specimens, confirming the absence of cross-reaction between the secondary antibodies and the dentin organic matrix and/or enzymes (data not shown).

Percentage of Co-localization between Proteases and Collagen Co-localization between proteases and intact collagen is seen in yellowish/orange (Figs. 2D, 2L, 3D, 3L) when the images from the green (i.e., collagen – Figs. 2B, 2J, 3B, 3J) and red channels (i.e., enzymes – Figs. 2C, 2K, 3C, 3K) were merged. ZEN software quantifies the co-localization between molecular species when they emit fluorescence at the same pixel (i.e., digital area). Thus, the percentages of overlapping signals in different regions of samples provided the rate of enzyme/structured collagen co-localization. These results are summarized in the Table. Regardless of the enzyme, the percentage of their respective co-localization with structured collagen was significantly higher in intact than in caries-affected dentin (p < .05).

Discussion The co-existence of CT and MMP activities has been previously shown in caries-infected dentin (Nascimento et al., 2011). Here we show, for the first time, similar co-existence in cariesaffected dentin, where the collagen matrix is believed to maintain the ability to remineralize. The increase of all examined CTs and MMPs in caries-affected dentin allows the original hypothesis to be accepted. While CT and MMP

Figure 2. Cathepsin abundance obtained by confocal microscopy, quantification of emitted fluorescence, and relative area occupied by collagen and proteases in intact and caries-affected dentin. (A, E, I, and M) Intact and caries-affected dentin morphology observed in differential interference contrast mode (DIC). (B, F, J, and N) Acquired image in green channel, showing autofluorescence of collagen within intact (B, J) and caries-affected (F, N) dentin. (C and G) CT-B immunolabeling shown in the red channel in intact (C) and caries-affected (G) dentin. (K and O) CT-K immunolabeling shown in the red channel in intact (K) and caries-affected (O) dentin. (D, H, L, and P) Merged images of DIC, collagen, and CT panels are observed as yellow/orange for each sample. (D and H) Collagen and CT-B co-localization in intact (D) and caries-affected (H) dentin. (L and P) Collagen and CT-K co-localization in intact (L) and caries-affected (P) dentin. (Q, R, S, T and V, W, X, Y) Emitted fluorescence of collagen (Q, S, V, and X) and CTs (R, T, W, and Y) quantified by relative intensity and distribution. (U and Z) Percentage of the relative area occupied by structured collagen and CTs in intact and caries-affected dentin. D = dentin; PD = predentin; P = pulp chamber/tissue.

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Figure 3. MMP abundance obtained by confocal microscopy, quantification of emitted fluorescence, and relative area occupied by collagen and proteases in intact and cariesaffected dentin. (A, E, I, and M) Intact and caries-affected dentin morphology observed in differential interference contrast mode (DIC). (B, F, J, and N) Acquired image in green channel, showing autofluorescence of collagen within intact (B, J) and caries-affected (F, N) dentin. (C and G) MMP-2 immunolabeling shown in the red channel in intact (C) and cariesaffected (G) dentin. (K and O) MMP-9 immunolabeling shown in the red channel in intact (K) and caries-affected (O) dentin. (D, H, L, and P) Merged images of DIC, collagen, and MMP panels are observed as yellow/orange for each sample. (D and H) Collagen and MMP-2 co-localization in intact (D) and caries-affected (H) dentin. (L and P) Collagen and MMP-9 co-localization in intact (L) and caries-affected (P) dentin. (Q, R, S, T and V, W, X, Y) Emitted fluorescence of collagen (Q, S, V, and X) and MMPs (R, T, W, and Y) quantified by relative intensity and distribution. (U and Z) Percentage of the relative area occupied by structured collagen and MMPs in intact and caries-affected dentin. D = dentin; PD = predentin; P = pulp chamber/tissue.

J Dent Res 93(3) 2014 distribution in dentin and interaction with dentinal collagen does not describe these enzymes’ biochemical pathways, the fact that they were found to be far more abundant in caries-affected than in intact dentin (Figs. 2, 3) supports the notion that CTs and MMPs may work synergistically in pathophysiological processes wherein the dentin organic matrix remodeling takes place. In addition, this is the first time that the presence of CT-K in dentin has been demonstrated. As previously mentioned, CT-K is the only member of the cysteine protease family reported to be a true collagenase (Garnero et al., 1998; Kafienah et al., 1998). Although the detailed interplay among proteolytic enzymes in the turnover of bone is not completely elucidated, it is thought that cysteine proteases, especially CT-K, start degrading the demineralized bone matrix in an acidic environment. Later, when the pH is restored to neutrality, other osteoclast-derived proteases, specifically from the MMP family, are released and continue the degradation of collagen and other matrix components (Everts et al., 1998). Thus, because dentin phylogenetically seems to bear a strong resemblance to bone, it was not surprising to detect the presence of CT-K in intact and, mainly, in caries-affected dentin. The distribution of CT-K in intact dentin was observed mainly in inner mineralized dentin (Fig. 2L), and in comparison with CT-B (Fig. 2D), the percentage of its co-localization with structured collagen was noticeably lower (Table). This suggests that these enzymes may exert a function even in the physiological state, wherein little remodeling of dentin organic matrix takes place. With this assumption, it can be thought that, in normal conditions, CT-B may play a preponderant role in the metabolism of mature dentin if compared with that exerted by CT-K. Less evidently, this assumption could also be extended to MMP distribution in intact dentin, in which MMP-9 was seen to be slightly more abundant than MMP-2 (Figs. 3C, 3K). Collagen detection was dramatically reduced in caries-affected specimens (Figs. 2F, 2N, 3F, 3N). This does not necessarily mean that collagen was completely digested in such conditions. It is more likely that, in cariesaffected dentin, the macromolecular structure of collagen was not sufficiently organized and arranged in planes for accurate detection by autofluorescence (Lin et al., 2010). In cariesaffected dentin, with 10% to 30% (mean, 21%) loss of minerals, ca. 20% decreases in collagen periodicity were already observed when evaluated with spatially resolved small-angle x-ray scattering (SAXS) (Deyhle et al., 2011). The

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J Dent Res 93(3) 2014  273 MMPs and Cysteine Cathepsins in Caries-affected Dentin values are close to those observed in the transparent zone under caries lesions (ca. 14% mineral loss) and well above the values in caries-affected (lightly stainable) and infected zones, with approximately 39% and 71% mineral loss, respectively (Pugach et al., 2009). Therefore, in our caries-affected dentin samples, changes in collagen matrix components may already be sufficient to affect collagen detection. Additionally, because the abundance of proteases in these specimens had significantly increased, the rate of co-localization between those proteases and collagen was also consistently lower (Figs. 2T, 2Y, 3T, 3Y). This combined occurrence – that is, an increase in protease abundance and a loss of collagen observation – sustains the hypothesis of a host-derived proteolytic process leading to the degradation of dentin organic matrix in caries progression. The prominent increase in CT abundance (present outcome) and activity (Nascimento et al., 2011) in carious dentin strongly indicates that CTs derived from either odontoblasts or other pulp cells may be key participants in active caries lesions. Additionally, the high abundance of CTs and MMPs in caries dentin embodies the notion that, during caries progression, milieu acidification not only favors matrix demineralization and activation, but also promotes an odontoblast-derived response to approaching infection. Although the effect of TGF-β on enzyme expression was not evaluated in the present study, it is tempting to speculate that the dramatic increase in CT-K and MMP-9 detection in carious dentin may be related to TGF-β modulation. TGF-β has been shown to increase CT-K gene expression (Tersariol et al., 2010) and MMP-9 protein synthesis (Tjäderhane et al., 1998a) in cultured mature human odontoblasts. Since TGF-β1 released from dentin has been described as an important dentinal growth factor in regulating the dentin-pulp complex response to approaching caries (Charadram et al., 2012), it is possible that at least part of the abundant detection of CT-B and -K in caries-affected dentin is caused by increased protein synthesis by cells of the dentinpulp complex. MMPs and CT-K have been reported to be active in osteoclastic bone resorption. Although osteoclasts can secrete MMP-1 (Bord et al., 1997; Delaissé et al., 2003), MMP-9 (Tezuka et al., 1994), and MMP-12 (Kobori et al., 1998), only MMP-1 was shown to efficiently hydrolyze native type I collagen, a major component of bone and dentin matrices (Shapiro et al., 1992; Tezuka et al., 1994; Husmann et al., 2013). Thus, so far, the main MMP described as participating in osteoclastic bone resorption has not yet been identified in dentin. Conversely, CT-K, a true mammalian collagenase that has been considered the main protease involved in bone resorption, is highly expressed by both osteoclasts (Drake et al., 1996) and odontoclasts (Tsuji et al., 2001). Actually, in the absence of CT-K, osteoclasts are hampered in their capacity to hydrolyze bone collagen (Delaissé et al., 2003; Tjäderhane et al., 2013). Thus, it seems reasonable to speculate that, in dental caries, CT-K is likely leading the hydrolysis of dentin collagen, but this hypothesis needs to be tested. In contrast to the significant extracellular matrix remodeling that occurs in bone, dentin is described classically as a relatively static tissue. However, even if the turnover of dentin is considered to be low after its maturation, the presence of proteolytic

Table.  Percentage of Co-localization between Proteases and Structured Collagen in Intact and Caries-affected Dentin Co-localization (%)   CT-B CT-K MMP-2 MMP-9

Intact 69.3 13.0 26.8 41.5

± ± ± ±

Caries-affected

17.6 4.7a 10.2C 12.4c A

6.7 1.1 6.1 2.1

± ± ± ±

2.8B 0.3b 3.3D 0.6d

Values are means ± standard deviation. Comparisons were performed only in rows. Different superscript values indicate statistically significant differences between intact and caries-affected dentin (p < .05).

enzymes in this matrix suggests that they play pathophysiological roles in tissue homeostasis. A serious limitation in this field is our poor understanding of the identity and functions of proteases in dentin matrix. Because the abundance of proteases increases significantly in dentin caries, it is of paramount importance to determine and characterize the complete proteolytic profile of dentin and to understand the interplay among proteases in the degradation of dentin. Determining which proteases are most important in dentin matrix degradation would, ultimately, help guide future development of therapies to effectively control caries.

Acknowledgments The authors thank the following funding agencies: FAPESP (2007/54618-4 and 2011/12226-8, P.I. Carrilho; 2013/05822-9, P.I. Nascimento; and 2009/13652-0, P.I. Vidal) and CNPq (306100/2010-0, P.I. Carrilho), Brazil. The authors declare no potential conflict of interest with respect to the authorship and/ or publication of this manuscript.

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Abundance of MMPs and cysteine cathepsins in caries-affected dentin.

Degradation of dentin matrix components within caries dentin has been correlated with the activity of host-derived proteases, such as matrix metallopr...
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