Caspases are well known proteases in the context of inflammation and apoptosis. Recently, novel roles of pro-apoptotic caspases have been reported, including findings related to the development of hard tissues. To further investigate these emerging functions of pro-apoptotic caspases, the in vivo localisation of key pro-apoptotic caspases (-3,-6,-7,-8, and -9) was assessed, concentrating on the development of two neighbouring hard tissues, cells participating in odontogenesis (represented by the first mouse molar) and intramembranous osteogenesis (mandibular/alveolar bone). The expression of the different caspases within the developing tissues was correlated with the apoptotic status of the cells, to produce a picture of whether different caspases have potentially distinct, or overlapping non-apoptotic functions. The in vivo investigation was additionally supported by examination of caspases in an osteoblast-like cell line in vitro. Caspases-3,-7, and -9 were activated in apoptotic cells of the primary enamel knot of the first molar; however, caspase-7 and -8 activation was also associated with the non-apoptotic enamel epithelium at the same stage and later with differentiating/differentiated odontoblasts and ameloblasts. In the adjacent bone, active caspases-7 and -8 were present abundantly in the prenatal period, while the appearance of caspases-3,-6, and -9 was marginal. Perinatally, caspases-3 and -7 were evident in some osteoclasts and osteoblastic cells, and caspase-8 was abundant mostly in osteoclasts. In addition, postnatal activation of caspases-7 and -8 was retained in osteocytes. The results provide a comprehensive temporo-spatial pattern of pro-apoptotic caspase activation, and demonstrate both unique and overlapping activation in non-apoptotic cells during development of the molar tooth and mandibular/alveolar bone. The importance of caspases in osteogenic pathways is highlighted by caspase inhibition in osteoblast-like cells, which led to a significant decrease in osteocalcin expression, supporting a role in hard tissue cell differentiation.

Department of Craniofacial Development and Stem Cell Research King's College London London United Kingdom

Department of Physiology University of Veterinary and Pharmaceutical Sciences Brno Brno Czechia

Laboratory of Molecular Morphogenesis Institute of Animal Physiology and Genetics Czech Academy of Sciences Brno Czechia

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Alfaqeeh S. A., Gaete M., Tucker A. S. (2013). Interactions of the tooth and bone during development. J. Dent. Res. 92, 1129–1135. 10.1177/0022034513510321 PubMed DOI

Aoyama I., Calenic B., Imai T., Ii H., Yaegak K. (2012). Oral malodorous compound causes caspase-8 and -9 mediated programmed cell death in osteoblasts. J. Periodont. Res. 47, 365–373. 10.1111/j.1600-0765.2011.01442.x PubMed DOI

Boatright K. M., Salvesen G. S. (2003). Mechanisms of caspase activation. Curr. Opin. Cell Biol. 15, 725–731. 10.1016/j.ceb.2003.10.009 PubMed DOI

Chandler J. M., Cohen G. M., MacFarlane M. (1998). Different subcellular distribution of caspase-3 and caspase-7 following Fas-induced apoptosis in mouse liver. J. Biol. Chem. 273, 10815–10818. 10.1074/jbc.273.18.10815 PubMed DOI

Cohen G. M. (1997). Caspases: the executioners of apoptosis. Biochem. J. 15, 1–16. 10.1042/bj3260001 PubMed DOI PMC

Cohn S. A. (1957). Development of the molar teeth in the albino mouse. Am. J. Anat. 101, 295–319. 10.1002/aja.1001010205 PubMed DOI

Connolly P. F., Jager R., Fearnhead H. O. (2014). New roles for old enzymes: killer caspases as the engine of cell behavior changes. Front. Physiol. 16:149 10.3389/fphys.2014.00149 PubMed DOI PMC

Diekwisch T. G. (2002). Pathways and fate of migratory cells during late tooth organogenesis. Connect. Tissue Res. 43, 245–256. 10.1080/03008200290001221 PubMed DOI

Diep L., Matalova E., Mitsiadis T. A., Tucker A. S. (2009). Contribution of the tooth bud mesenchyme to alveolar bone. J. Exp. Zool. B Mol. Dev. Evol. 312B, 510–517. 10.1002/jez.b.21269 PubMed DOI

Faleiro L., Lazebnik Y. (2000). Caspases disrupt the nuclear-cytoplasmic barrier. J. Cell Biol. 151, 951–959. 10.1083/jcb.151.5.951 PubMed DOI PMC

Fava L. L., Bock F. J., Geley S., Villunger A. (2012). Caspase-2 at a glance. J. Cell Sci. 125, 5911–5915. 10.1242/jcs.115105 PubMed DOI

Fischer U., Jänicke R. U., Schulze-Osthoff K. (2003). Many cuts to ruin: a comprehensive update of caspase substrates. Cell Death Differ. 10, 76–100. 10.1038/sj.cdd.4401160 PubMed DOI PMC

Fuentes-Prior P., Salvesen G. S. (2004). The protein structures that shape caspase activity, specificity, activation and inhibition. Biochem. J. 384, 201–232. 10.1042/BJ20041142 PubMed DOI PMC

Gaunt W. A. (1966). The disposition of the developing cheek teeth in the albino mouse. Acta Anat. 64, 572–585. 10.1159/000142856 PubMed DOI

Godefroy N., Foveau B., Albrecht S., Goodyer C. G., LeBlanc A. C. (2013). Expression and activation of caspase-6 in human fetal and adult tissues. PLoS ONE 8:e79313. 10.1371/journal.pone.0079313 PubMed DOI PMC

Goga Y., Chiba M., Shimizu Y., Mitani H. (2006). Compressive force induces osteoblast apoptosis via caspase-8. J. Dent. Res. 85, 240–244. 10.1177/154405910608500307 PubMed DOI

Haÿ E., Lemonnier J., Fromigué O., Marie P. J. (2001). Bone morphogenetic protein-2 promotes osteoblast apoptosis through a Smad independent, protein kinase C-dependent signaling pathway. J. Biol. Chem. 276, 29028–29036. 10.1074/jbc.M011265200 PubMed DOI

Hock J. M., Krishnan V., Onyia J. E., Bidwell J. P., Milas J., Stanislaus D. (2001). Osteoblast apoptosis and bone turnover. J. Bone Miner. Res. 16, 975–984. 10.1359/jbmr.2001.16.6.975 PubMed DOI

Jernvall J., Aberg T., Kettunen P., Keranen S., Thesleff I. (1998). The life history of an embryonic signalling center: BMP-4 induces p-21 and is associated with apoptosis in the mouse tooth enamel knot. Development 125, 161–169. PubMed

Kamada S., Kikkawa U., Tsujimoto Y., Hunter T. (2005). Nuclear translocation of caspase-3 is dependent on its proteolytic activation and recognition of a substrate-like protein(s). J. Biol. Chem. 280, 857–860. 10.1074/jbc.C400538200 PubMed DOI

Kang T. B., Ben-Moshe T., Varfolomeev E. E., Pewzner-Jung Y., Yogev N., Jurewicz A., et al. . (2004). Caspase-8 serves both apoptotic and nonapoptotic roles. J. Immunol. 173, 2976–2984. 10.4049/jimmunol.173.5.2976 PubMed DOI

Katao Y., Sawai H., Shishido M., Omae A., Liao W., Inami K., et al. (2013). Augmentation of RANKL-induced osteoclast differentiation by Z-VAD-fmk, a pan-caspase inhibitor. J. Osaka Dent. Univ. 47, 41–46. 10.18905/jodu.47.1_41 DOI

Katao Y., Shishido M., Inami K., Matsumoto N., Sawai H. (2014). An inhibitory role for caspase-3 at the late stage of RANKL-induced osteoclast differentiation in RAW264 cells and mouse bone marrow macrophages. Cell Biol. Int. 38, 723–728. 10.1002/cbin.10263 PubMed DOI

Kogianni G., Mann V., Ebetino F., Nuttall M., Nijweide P., Simpson H., et al. . (2004). Fas/CD95 is associated with glucocorticoid-induced osteocyte apoptosis. Life Sci. 75, 2879–2895. 10.1016/j.lfs.2004.04.048 PubMed DOI

Krajewska M., Wang H. G., Krajewski S., Zapata J. M., Shabaik A., Gascoyne R., et al. . (1997). Immunohistochemical analysis of in vivo patterns of expression of CPP32 (Caspase-3), a cell death protease. Cancer Res. 57, 1605–1613. PubMed

Kuida K., Haydar T. F., Kuan C. Y., Gu Y., Taya C., Karasuyama H., et al. . (1998). Reduced apoptosis and cytochrome-c mediated caspase activation in mice lacking caspase-9. Cell 94, 325–337. 10.1016/S0092-8674(00)81476-2 PubMed DOI

Lamkanfi M., Festjens N., Declercq W., Vanden Berghe T., Vandenabeele P. (2007). Caspases in cell survival, proliferation and differentiation. Cell Death Differ. 14, 44–55. 10.1038/sj.cdd.4402047 PubMed DOI

Ledvina V., Janecková E., Matalová E., Klepárník K. (2017). Parallel single-cell analysis of active caspase-3/7 in apoptotic and non-apoptotic cells. Anal. Bioanal. Chem. 409, 269–274. 10.1007/s00216-016-9998-6 PubMed DOI

Lesot H., Hovorakova M., Peterka M., Peterkova R. (2014). Three-dimensional analysis of molar development in the mouse from the cap to bell stage. Aust. Dent. J. 59, 81–100. 10.1111/adj.12132 PubMed DOI

Lisi S., Peterkova R., Peterka M., Vonesch J. L., Ruch J. V., Lesot H. (2003). Tooth morphogenesis and pattern of odontoblast differentiation. Connect. Tissue Res. 44 (Suppl. 1), 167–170. 10.1080/03008200390152278 PubMed DOI

Matalova E., Lesot H., Svandova E., Vanden Berghe T., Sharpe P. T., Healy C., et al. . (2013). Caspase-7 participates in differentiation of cells forming dental hard tissues. Dev. Growth Differ. 55, 615–621. 10.1111/dgd.12066 PubMed DOI

Matalova E., Sharpe P. T., Lakhani S. A., Roth K. A., Flavell R. A., Setkova J., et al. . (2006). Molar tooth development in caspase-3 deficient mice. Int. J. Dev. Biol. 50, 491–497. 10.1387/ijdb.052117em PubMed DOI

Matalova E., Svandova E., Tucker A. S. (2012a). Apoptotic signaling in mouse odontogenesis. OMICS 16, 60–70. 10.1089/omi.2011.0039 PubMed DOI PMC

Matalova E., Vanden Berghe T., Svandova E., Vandenabeele P., Healy C., Sharpe P. T., et al. . (2012b). Caspase-7 in molar tooth development. Arch. Oral Biol. 57, 1474–1481. 10.1016/j.archoralbio.2012.06.009 PubMed DOI

Miura M., Chen X. D., Allen M. R., Bi Y., Gronthos S., Seo B. M., et al. . (2004). A crucial role of caspase-3 in osteogenic differentiation of bone marrow stromal stem cells. J. Clin. Invest. 114, 1704–1713. 10.1172/JCI20427 PubMed DOI PMC

Mogi M., Togari A. (2003). Activation of caspases is required for osteoblastic differentiation. J. Biol. Chem. 278, 47477–47482. 10.1074/jbc.M307055200 PubMed DOI

Nakatsumi H., Yonehara S. (2010). Identification of functional regions defining different activity in caspase-3 and caspase-7 within cells. J. Biol. Chem. 285, 25418–25425. 10.1074/jbc.M110.126573 PubMed DOI PMC

Nhan T. Q., Liles W. C., Schwartz S. M. (2006). Physiological functions of caspases beyond cell death. Am. J. Pathol. 169, 729–737. 10.2353/ajpath.2006.060105 PubMed DOI PMC

Nicholson D. W. (1999). Caspase structure, proteolytic substrates, and function during apoptotic cell death. Cell Death Differ. 6, 1028–1042. 10.1038/sj.cdd.4400598 PubMed DOI

Peterková R., Lesot H., Vonesch J. L., Peterka M., Ruch J. V. (1996). Mouse molar morphogenesis revisited by three dimensional reconstruction. I. Analysis of initial stages of the first upper molar development revealed two transient buds. Int. J. Dev. Biol. 40, 1009–1016. PubMed

Pop C., Oberst A., Drag M., Van Raam B. J., Riedl S. J., Green D. R., et al. . (2011). FLIPL induces caspase 8 activity in the absence of interdomain caspase 8 cleavage and alters substrate specificity. Biochem. J. 433, 447–457. 10.1042/BJ20101738 PubMed DOI PMC

Schmitt R., Ruch J. V. (2000). In vitro synchronization of embryonic mouse incisor preodontoblasts and preameloblasts: repercussions on terminal differentiation. Eur. J. Oral Sci. 108, 311–319. 10.1034/j.1600-0722.2000.108004318.x PubMed DOI

Schwerk C., Schulze-Osthoff K. (2003). Non-apoptotic functions of caspases in cellular proliferation and differentiation. Biochem. Pharmacol. 66, 1453–1458. 10.1016/S0006-2952(03)00497-0 PubMed DOI

Setkova J., Matalova E., Sharpe P. T., Misek I., Tucker A. S. (2007). Primary enamel knot cell death in Apaf-1 and caspase-9 deficient mice. Arch. Oral Biol. 52, 15–19. 10.1016/j.archoralbio.2006.07.006 PubMed DOI

Shalini S., Dorstyn L., Dawar S., Kumar S. (2015). Old, new and emerging functions of caspases. Cell Death Differ. 22, 526–539. 10.1038/cdd.2014.216 PubMed DOI PMC

Shigemura N., Kiyoshima T., Sakai T., Matsuo K., Momoi T., Yamaza H., et al. . (2001). Localization of activated caspase-3-positive and apoptotic cells in the developing tooth germ of the mouse lower first molar. Histochem. J. 33, 253–258. 10.1023/A:1017900305661 PubMed DOI

Solier S., Fontenay M., Vainchenker W., Droin N., Solary E. (2017). Non-apoptotic functions of caspases in myeloid cell differentiation. Cell Death Differ. 24, 1337–1347. 10.1038/cdd.2017.19 PubMed DOI PMC

Sordet O., Rébé C., Plenchette S., Zermati Y., Hermine O., Vainchenker W., et al. . (2002). Specific involvement of caspases in the differentiation of monocytes into macrophages. Blood 100, 4446–4453. 10.1182/blood-2002-06-1778 PubMed DOI

Svandova E., Lesot H., Vanden Berghe T., Tucker A. S., Sharpe P. T., Vandenabeele P., et al. . (2014). Non-apoptotic functions of caspase-7 during osteogenesis. Cell Death Dis. 5:e1366. 10.1038/cddis.2014.330 PubMed DOI PMC

Szymczyk K. H., Freeman T. A., Adams C. S., Srinivas V., Steinbeck M. J. (2006). Active caspase-3 is required for osteoclast differentiation. J. Cell. Physiol. 209, 836–844. 10.1002/jcp.20770 PubMed DOI

Thaler R., Maurizi A., Roschger P., Sturmlechner I., Khani F., Spitzer S., et al. . (2016). Anabolic and antiresorptive modulation of bone homeostasis by the epigenetic modulator sulforaphane, a naturally occurring isothiocyanate. J. Biol. Chem. 291, 6754–6771. 10.1074/jbc.M115.678235 PubMed DOI PMC

Tucker A., Sharpe P. (2004). The cutting-edge of mammalian development; how the embryo makes teeth. Nat. Rev. Genet. 5, 499–508. 10.1038/nrg1380 PubMed DOI

Vaahtokari A., Aberg T., Thesleff I. (1996). Apoptosis in the developing tooth: association with an embryonic signaling center and suppression by EGF and FGF-4. Development 122, 121–129. PubMed

Walsh J. G., Cullen S. P., Sheridan C., Lüthi A. U., Gerne C., Martin S. J. (2008). Executioner caspase-3 and caspase-7 are functionally distinct proteases. Proc. Natl. Acad. Sci. U.S.A. 105, 12815–12819. 10.1073/pnas.0707715105 PubMed DOI PMC

Yazid M. D., Ariffin S. H. Z., Senafi S., Razak M. A., Wahab R. M. A. (2010). Determination of the differentiation capacities of murines' primary mononucleated cells and MC3T3-E1 cells. Cancer Cell Int. 10:42. 10.1186/1475-2867-10-42 PubMed DOI PMC

Zermati Y., Garrido C., Amsellem S., Fishelson S., Bouscary D., Valensi F., et al. . (2001). Caspase activation is required for terminal erythroid differentiation. J. Exp. Med. 193, 247–254. 10.1084/jem.193.2.247 PubMed DOI PMC

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