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Keywords:

  • ameloblast;
  • apoptosis;
  • caspase;
  • cell differentiation;
  • odontoblast

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of interest
  9. References

Apoptosis during tooth development appears dependent on the apoptotic executioner caspase-3, but not caspase-7. Instead, activated caspase-7 has been found in differentiated odontoblasts and ameloblasts, where it does not correlate with apoptosis. To further investigate these findings, the mouse incisor was used as a model. Analysis of caspase-7-deficient mice revealed a significant thinner layer of hard tissue in the adult incisor. Micro computed tomography scan confirmed this decrease in mineralized tissues. These data strongly suggest that caspase-7 might be directly involved in functional cell differentiation and regulation of the mineralization of dental matrices.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of interest
  9. References

Caspase-7 is a cystein protease involved in apoptosis and inflammation. Caspase-7 has often been considered to be redundant to caspase-3, another member of the death trio of executive caspases. Both of these enzymes become activated by cleavage, mediated by a cascade initiated by a death ligand–receptor interaction or by apoptosome formation after cytochrome-c release from the mitochondria (Lamkanfi & Kanneganti 2010). In addition, particularly caspase-7 has been linked to inflammatory responses (Martinon & Tschopp 2007). Recently, non-apoptotic and non-inflammatory functions of caspases have been suggested, such as a role in tissue regeneration, cell fate determination, neural activation and cell differentiation (Kuranaga 2012). Caspase-3, the most studied caspase, was demonstrated to be a putative gatekeeper in stem cells, playing a role in non-apoptotic events in several stem cell derived lineages (Fujita et al. 2008). Lack of caspase-3 causes disorders also in bone turnover due to decreased osteogenic differentiation of bone marrow stem cells (Miura et al. 2004).

Morphogenesis of tooth germs during odontogenesis is accompanied by proliferation, differentiation, adhesion, migration, and also apoptosis (Peterkova et al. 2003; Matalova et al. 2004). Caspases become activated during the course of tooth development (Matalova et al. 2006; Setkova et al. 2007), particularly in relation to apoptotic events (Matalova et al. 2012a). Notably, non-apoptotic cleavage of caspase-3 was reported in normal and neoplastic odontogenic epithelial cells in human patients (Kumamoto et al. 2001). Moreover, cleaved caspase-7 was found in mouse molars in the primary enamel knot undergoing apoptosis but also in non-apoptotic areas (Matalova et al. 2012b). Attempts were thus made to further investigate the possible non-apoptotic role of caspase-7, by moving to the mouse incisor as a model.

The mouse incisor grows throughout the animal's life with the population of hard tissue producing cells (odontoblasts and ameloblasts) renewed by differentiation of cells derived from populations of stem cells located at the apical base of the tooth. The epithelial stem cells are housed in a bulge at the end of the labial cervical loop, while the mesenchymal stem cells have been suggested to reside distally near from the cervical loop (Harada et al. 1999; Lapthanasupkul et al. 2012). The mouse incisor forms a tooth bud around embryonic day (E) 13, enters the cap stage (E14–15) and forms a bell structure by E16 (Kieffer-Combeau et al. 2001). During embryonic development, the tooth germ elongates in a posterior direction due to cell proliferation. The elongation then proceeds anteriorly and allows tooth eruption and lifelong compensation for the progressive abrasion at the incisor tips. The labial and lingual parts of the incisor differ in histology, as only the labial side is covered by enamel, allowing the upper and lower incisors to self-sharpen on contact. The mouse incisor, therefore, represents a unique model for addressing questions of growth and differentiation, allowing several stages of cell differentiation to be visualized as gradients on a single sagittal section.

This work aims to investigate the possible non-apoptotic functions of caspase-7 in cell differentiation during odontogenesis by evaluation of the cleavage (activation) pattern of caspase-7, and analysis of the phenotype of loss of caspase-7 on formation of the continuously growing mouse incisor.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of interest
  9. References

Samples

CD1 mice were killed according to the experimental protocol approved by the Laboratory Animal Science Committee of the IAPG CAS, v.v.i., Brno, Czech Republic. Mouse heads or mandible quadrants (at postnatal stages) were immediately fixed in 4% buffered paraformaldehyde and decalcified in buffered ethylenediaminetetraacetic acid (EDTA). Samples were dehydrated through a graded series of ethanols, treated with xylene, paraffin embedded and cut in 4-μm serial sections. Prenatal (embryonic) stage E17.5, postnatal (P) stages P0, P5 and P10 were used in the study. Caspase-7−/− mice that had been backcrossed on the C57BL/6 genetic background over at least 10 generations were provided by Professor P. Vandenabeele (Duprez et al. 2011) and samples processed as for wild type animals.

Immunohistochemistry

Immunohistochemistry was performed as described in Matalova et al. (2012b). Briefly, deparaffinized and rehydrated histological sections were pre-treated (only anti-caspase-7 samples) in citrate buffer (pH = 6.0) 10 min/98°C and in 3% hydrogen peroxide in PBS (5 min/RT). The primary antibody (anti-caspase-7: Cell Signaling 9491, anti-amelogenin: Abcam ab59705, anti-PCNA: Santa Cruz sc-7907) was diluted (anti-caspase-7: ready-to-use solution 50x, anti-amelogenin: ready-to-use solution 400x, anti-PCNA: 100×), and applied overnight/4°C. Peroxidase-conjugated streptavidin-biotin system (Vectastain, S7100) and chromogen substrate diaminobenzidine (DAB, Dako, K3466) reactions were used to visualize positive cells as brown. Slides were counterstained by hematoxylin to clearly distinguish the cell nuclei. Negative control was performed by omitting the primary antibody. Specificity of the selected anti-caspase-7 antibodies was verified using caspase-7 deficient tissues.

MicroCT

Specimens for microCT (computed tomography) were scanned using a GE Locus SP microCT scanner. The specimens were immobilized using cotton gauze and scanned to produce 14 μm voxel size volumes, using an X-ray tube voltage of 80 kVp and a tube current of 80 μA. An aluminum filter (0.05 mm) was used to adjust the energy distribution of the X-ray source. The specimens were characterized further by making three-dimensional isosurfaces, generated and measured using Microview software (GE). Mineral measurements were carried out using the Advanced Bone Module of Microview. Scan data was reconstructed and rendering carried out using the Microview 2.2 software package. Dentin and enamel was segmented using Microview's auto-thresholding facility and isosurfaces were reconstructed separately and loaded into the software using the Geometry overlay feature. Statistical significance was determined using a t-test and F-test (P > 0.05). Eight adult specimens were analyzed, four wild type (WT) mice and four knock-out (KO) mice.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of interest
  9. References

Cleaved caspase-7 during incisor development

At E17.5, the incisor tooth germ has reached the bell stage. At this stage the differentiation of functional odontoblasts proceeds as a gradient from the apex (cervical loops), while the ameloblasts are just starting to differentiate (Fig. 1A). Positive staining for cleaved caspase-7 was apparent particularly in the cytoplasm of the differentiating ameloblasts, cells of the stratum intermedium and also the polarized odontoblasts (Fig. 1B). Staining was detected on the inner portion of the epithelium on the labial part of the cervical loop (Fig. 1C). In contrast, no expression was observed on the lingual part of the cervical loop (data not shown).

image

Figure 1. Cleaved caspase-7 in incisor development. (A,D,G) Hematoxylin/Eosin stained tissue. (B,C, E, F, H, I) Immunohistochemistry of cleaved caspase-7. (A–C) Embryonic day (E) 17.5. (D–F) Postnatal day (P) 0. (G–I) P10. (A): Sagittal section, with clearly formed cervical loop. Scale bar = 100 μm. (B) Caspase-7 positive ameloblasts, odontoblasts and stratum intermedium cells. Scale bar = 100 μm. (C) Cervical loop at apex of tooth with positive label in the inner epithelium on the labial side. Scale bar = 50 μm. (D1) Sagittal section. Anterior region with differentiated ameloblasts and odontoblasts. (D2) Apical part of the tooth with the cervical loop. Hematoxylin/Eosin. Scale bar = 100 μm. (E) Caspase-7 positive ameloblasts and odontoblasts. Scale bar = 100 μm. (F) Caspase-7 localized to the inner epithelium of the labial cervical loop. Scale bar = 100 μm. (G) Frontal section. Enamel covers the labial part of the tooth (top) while the lingual side (bottom) remains enamel free. Scale bar = 100 μm. (H) Detailed look at the localization of cleaved caspase-7 at P10 with positive ameloblasts and negative stratum intermedium cells. Scale bar = 50 μm. (I) A border is evident between the caspase-7 positive ameloblasts and odontoblasts and negative periodontal mesenchyme and dental pulp. Scale bar = 50 μm. (J) Detailed look at the localization of cleaved caspase-7 at P10 with positive ameloblasts and negative stratum intermedium cells. Scale bar = 50 μm. (K) Detailed look at the localization of cleaved caspase-7 at P10 with positive odontoblasts. Scale bar = 50 μm. (L) A border is evident between the caspase-7 positive ameloblasts and odontoblasts and negative periodontal mesenchyme and dental pulp. Scale bar = 50 μm. AM, ameloblasts; b, bone; CL, cervical loop; dn, dentin; DP, dental papilla; en, enamel; OD, odontoblasts; pAM, preameloblasts; PDM, peridental mesenchyme; pdn, predentin; SI, stratum intermedium; lin, lingual side; lab, labial side.

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At P0 (Fig. 1D1,2), functional ameloblasts and enamel were covering the labial part of the tooth germ, although only in its anterior portion (Fig. 1D1). Staining for cleaved caspase-7 was found in the odontoblasts and ameloblasts (Fig. 1E), whereas the neighbor cells of stratum intermedium were caspase-7 negative at this stage (Fig. 1E). In the labial part of the cervical loop, caspase-7 positive cells were detected on the inner portion of the epithelium (Fig. 1F), while the outer epithelium in contact with the peridental mesenchyme remained unstained (Fig. 1C,F).

Later on, at P10 (Fig. 1G), the active form of caspase-7 was detected in the cytoplasm of ameloblasts (Fig. 1H,J) and odontoblasts (Fig. 1I, K) while nuclei in both cell types were negative. A clear border was observed between the cleaved caspase-7 positive odontoblasts/ameloblasts and the cells of the peridental mesenchyme, next to these ameloblasts, which was negative (Fig. 1L). Caspase-7 positive odontoblasts were detected also in the root-analog of the incisor (data not shown), whereas the stratum intermedium cells remained caspase-7 negative (Fig. 1H). Dental pulp was in general caspase-7 negative at all investigated stages. Few scattered, slightly positive cells were found in the marginal parts beneath the forming odontoblast layer.

Analysis of caspase-7 knock-out incisor phenotype

In the adult caspase-7 deficient mice, the incisors erupted as in wild type littermates (Fig. 2A,D). Despite normal amelogenin expression in the WT and KO (Fig. 2B,E), the histological analysis suggested some differences in the thickness of the hard tissue with less mineralized matrix laid down in the knock-out compared to the wild type (Fig. 2C,F). There were no histologically apparent differences in the stratum intermedium cells of the wild type and deficient mice (Fig. 2G1,H1). Based on PCNA expression in the adult mice, in both, the wt and deficient mice, the lines of differentiated odontoblasts and ameloblasts were negative; few scattered positive cells were detected in the mesenchymal tissue surrounding the labial part of the incisor (Fig. 2G2,H2) and the mesenchyme around the interface of the enamel and enamel-free region of the tooth (Fig. 2G3,H3). MicroCT (Fig. 2I) and statistical analyses (Fig. 2J) confirmed a significant decrease in the amount of mineralized tissue in caspase-7 KO mice.

image

Figure 2. Analysis of caspase-7 deficient incisors. (A–C) Wild type adult incisor. (D–F) Caspase-7 knock-out mouse. (A) Macroscopic view of fully erupted adult wild type incisor. Scale bar = 3 mm. (B) Immunohistochemistry of amelogenin in the adult wild type incisor. Scale bar = 100 μm. (C) Detailed view at dentin (and odontoblasts) and enamel (and ameloblasts) structure and deposition in the adult wild type mouse. Scale bar = 50 μm. (D) Macroscopic view of fully erupted adult incisor of the caspase-7 deficient mouse. Scale bar = 3 mm. (E) Immunohistochemistry of amelogenin in the adult type incisor of the caspase-7 deficient mouse. Scale bar = 100 μm. (F) Detailed view at dentin (and odontoblasts) and enamel (and ameloblasts) structure and deposition in the adult caspase-7 deficient mouse. Scale bar = 50 μm. (G) Hematoxylin/Eosin stained sagittal section of the wild type incisor, indicating regions magnified in G1,2,3. (G1) Detailed view at the stratum intermedium cells of the wild type incisor. (G2) Proliferation (PCNA staining) in the mesenchymal cells surrounding the labial part of the wild type incisor. (G3) Proliferation (PCNA staining) in the mesenchymal cells surrounding the enamel and enamel-free region interface of the wild type incisor. (H) Hematoxylin/Eosin stained sagittal section of the caspase-7 deficient incisor, indicating regions magnified in H1,2,3. (H1) Detailed view at the stratum intermedium cells of the caspase-7 deficient incisor. (H2) Proliferation (PCNA staining) in the mesenchymal cells surrounding the labial part of the caspase-7 deficient incisor. (H3) Proliferation (PCNA staining) in the mesenchymal cells surrounding the enamel and enamel-free region interface of the caspase-7 deficient incisor. (I) MicroCT of the adult incisor in the wild type (I1) and caspase-7 deficient mice (I2) confirmed decreased mineralization in the caspase-7 incisors. Scale bar = 2 mm. (J) Statistical evaluation of the microCT results. Both, the enamel area and volume display significantly lower value in the knock-out than in the wild type. AM, ameloblasts; dn, dentin; DP, dental papilla; en, enamel; OD, odontoblasts; pAM, preameloblasts; PDM, peridental mesenchyme; SI, stratum intermedium.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of interest
  9. References

Proteins known for their specific roles in apoptosis have recently been reported as associated with a wide range of non-apoptotic functions such as cell cycle progression, differentiation, self-renewal, metabolism or autophagy (Galluzzi et al. 2012). Caspases, as the key proteins/enzymes in the apoptotic machinery (Feinstein-Rotkopf & Arama 2009), are the hottest candidates for such diverse roles.

Dental apoptosis is caspase dependent (Matalova et al. 2012a); however, so far only caspase-3 (Matalova et al. 2006) and caspase-9 (Setkova et al. 2007) have been shown to be essential in molar development as their deficiency leads to absence of apoptotic cells. Caspase-7 was found expressed in the apoptotic regions of the molar tooth signaling centers, the enamel knots, but its absence (knock-out) did not cause any inhibition or prevention of apoptosis (Matalova et al. 2012a). Notably, in the molars cleaved caspase-7 was present in non-apoptotic, differentiated cells, particularly odontoblasts and ameloblasts. Despite this expression, deficiency of caspase-7 did not alter molar development in a distinct way (Matalova et al. 2012a). Therefore, we turned to the incisor, which is permanently growing in the mouse, and differentiation of cells producing dental hard tissues occurs continuously.

Immunohistochemistry confirmed the presence of cleaved caspase-7 in incisor ameloblasts and odontoblasts, in a similar pattern to the molars (Matalova et al. 2012a). In the incisor a difference in expression was shown between the labial and lingual parts of the cervical loop. The epithelial cells in contact with the basement membrane on the lingual side of the incisor were caspase-7 negative. Since these cells do not produce enamel, the absence of caspase-7 cleavage on the lingual side and strong cleavage on the labial side may suggest a role of caspase-7 in formation of hard tissues, unrelated to apoptosis. The expression indicates that caspase-7 may play a role in the fate of dental cells that are producing extracellular matrix and undergoing mineralization. Speculatively, the scattered slightly positive cells in the marginal part of the dental pulp mesenchyme at early stages of development may mirror different fate of pulp located cells as described in molar teeth (Diep et al. 2009; Rothova et al. 2012).

Furthermore, the caspase-7 deficient incisors displayed a significant decrease in the amount of mineralized matrix. However, the loss of caspase-7 did not cause an absence of enamel in the knock-out but rather delayed mineralization and/or hypomineralization. Similar incomplete impairment in enamel formation was reported in other transgenic animals (Yoshioka et al. 2011). There was no obvious difference between localization of amelogenin in the wild type and caspase-7 knock-outs, but it is possible that the levels of such enamel-associated proteins were reduced but this was not quantified in our analysis. MicroCT and histology showed variations in the amount of mineralized matrix. All together, the set of observations reported here supports the hypothesis that active caspase-7 could be involved in the modulation of ameloblast functional differentiation/maturation and not in an on/off regulation. Very complex mechanisms regulate dentinogenesis and amelogenesis (Caton & Tucker 2009; Simmer et al. 2010) and the data presented here suggest that caspase-7 may take part in these networks. The hypothesis that caspase-7 is likely to play a role in the differentiation/mineralization of hard tissue/cells is supported by the fact that cleaved caspase-7 can be observed not only in odontoblasts and ameloblasts but also in osteoblasts forming the bone hard tissue (Matalova et al. 2012a).

Although it has been shown that some caspases, such as caspase-3, are required for differentiation of a variety of cell types (Larsen et al. 2010), the mechanism allowing caspases to influence cell-fate decision and regulate differentiation, survival or proliferation remains unclear.

Subcellular localization of cleaved caspase-7 seems to correspond with its particular function. During apoptosis, activated caspases becomes translocated into the nucleus (Lamkanfi & Kanneganti 2010), whereas cleaved caspase-7 in non-apoptotic areas of the incisor was found exclusively in the cytoplasm. This observation is supported by the finding that in developing molars caspase-3 is localized in the nucleus of apoptotic cells (Matalova et al. 2006); while it is localized to the cytoplasm of non-apoptotic epithelial cells (Kumamoto et al. 2001). The cytoplasmic localization of caspase-7 appears similar in molars and incisors in non-apoptotic regions of the tooth (Matalova et al. 2012b), indicating that the localization of cleaved protein regulates its function.

One hypothesis suggesting the possible mechanisms of caspase-7 function based on its localization within the tooth germ would be engagement in differentiation/maturation of cells by cleavage of proteins typical for the undifferentiated status of cells. In such cases, the non-apoptotic mechanism of caspase function may include the depletion of stem cell markers, OCT4 and NANOG, as demonstrated in cancer cells after drug treatment (Musch et al. 2010). In the study, caspase-3 cleaved NANOG, whereas caspase-7 was able to degrade both of these markers. Oct3/4 was reported to be strongly expressed in the nucleus of the incisor cervical loop cells starting at E18, persisting up to late postnatal stages (Li et al. 2011). As there is no universal marker of the stem cell niche in the incisors so far, Oct3/4 is one of the molecules expected in dental stem cells (Li et al. 2011). Caspase-7 was located in the inner part of the labial cervical loop, while the outer region was caspase-7 negative. This caspase-7 negative domain correlates with the region of the cervical loop where Oct3/4 is present and to the putative location of stem cells as indicated by Sox2 (Li et al. 2011). In contrast to the cervical loop, Oct3/4 is expressed in the cytoplasm in ameloblasts and odontoblasts (Li et al. 2011) similar to the active form of caspase-7. These findings could suggest some putative participation of caspase-7 in the cleavage of Oct4 before/during cell differentiation. Depletion of stem cell factors was shown to be mediated by cleaved caspases and OCT4 is a direct in vitro target of active caspase-7 (Musch et al. 2010).

In this context, the expression of Oct4 was previously reported in relation to different cell populations during tooth root formation (Nakagawa et al. 2012). A similar difference was detected between the regions we show that are caspase-7 positive (ameloblasts) and negative (stratum intermedium and peridental mesenchyme) in the incisors. Therefore, the cleaved form of caspase-7, connected with Oct4, could be a further marker to distinguish these different cell populations.

Alternatively, caspase-7 might participate in the activation of nuclear factor-κB (NF-κB). A link between these pathways has been demonstrated by the effect of caspase-3 on osteoclasts (Szymczyk et al. 2006) or lens (Basu et al. 2012) differentiation. A similar role in teeth is supported by the importance of tumor necrosis factor (TNF) signaling in development and evolution of tooth number, size and shape (Ohazama & Sharpe 2004). Moreover, Fas receptor, as a member of the TNF family, was suggested to have a modulatory function in dental apoptosis (Matalova et al. 2005) and might play a role in a switch in cell fate, as has been reported in apoptotic versus non-apoptotic decisions in other systems (Lavrik & Krammer 2012). Recent data indicate that non-apoptotic functions of caspases involve proteolysis exerted by their catalytic domains as well as non-proteolytic functions exerted by their prodomains (Lamkanfi et al. 2007).

Confirmation of such hypotheses, as well as the final matching of caspase-7 into other molecular networks governing differentiation, maturation and function of hard tissue cells, is an important area for further analysis.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of interest
  9. References

The research was funded by the Grant Agency of the Czech Republic, P502/12/1285 at the UVPS and P304/11/1418 at the IAPG. International cooperation was supported by the Academy of Sciences of the Czech Republic (project M200451201). The Brno lab runs under RVO: 67985904.

Conflict of interest

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of interest
  9. References

The authors declare that they have no conflict of interest.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of interest
  9. References