Caspases are proteases traditionally associated with inflammation and cell death. Recently, they have also been shown to modulate cell proliferation and differentiation. The aim of the current research was to search for osteogenic molecules affected by caspase inhibition and to specify the individual caspases critical for these effects with a focus on proapoptotic caspases: caspase-2, -3, -6, -7, -8 and -9. Along with osteocalcin (Ocn), general caspase inhibition significantly decreased the expression of the Phex gene in differentiated MC3T3-E1 cells. The inhibition of individual caspases indicated that caspase-8 is a major contributor to the modification of Ocn and Phex expression. Caspase-2 and-6 had effects on Ocn and caspase-6 had an effect on Phex. These data confirm and expand the current knowledge about the nonapoptotic roles of caspases and the effect of their pharmacological inhibition on the osteogenic potential of osteoblastic cells.

Department of Physiology University of Veterinary and Pharmaceutical Sciences Brno Czech Republic

Faculty of Science Masaryk University Brno Czech Republic

Institute of Analytical Chemistry of the Czech Academy of Sciences Brno Czech Republic

Institute of Animal Physiology and Genetics Academy of Sciences Brno Czech Republic

International Clinical Research Center St Anne's University Hospital Brno Czech Republic

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Kudelova J, Fleischmannova J, Adamova E, Matalova E. Pharmacological caspase inhibitors: research towards therapeutic perspectives. J. Physiol. Pharmacol. 2015;66:473–82. PubMed

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

Miura M, et al. A crucial role of caspase-3 in osteogenic differentiation of bone marrow stromal stem cells. J. Clin. Invest. 2004;114:1704–1713. doi: 10.1172/JCI20427. PubMed DOI PMC

Svandova E, Vesela B, Tucker AS, Matalova E. Activation of Pro-apoptotic Caspases in Non-apoptotic Cells During Odontogenesis and Related Osteogenesis. Front. Physiol. 2018;9:174. doi: 10.3389/fphys.2018.00174. PubMed DOI PMC

Mogi M, Togari A. Activation of Caspases Is Required for Osteoblastic Differentiation. J. Biol. Chem. 2003;278:47477–47482. doi: 10.1074/jbc.M307055200. PubMed DOI

Ivaska KK, et al. Urinary osteocalcin as a marker of bone metabolism. Clin. Chem. 2005;51:618–28. doi: 10.1373/clinchem.2004.043901. PubMed DOI

Beck GR, Zerler B, Moran E. Gene Array Analysis of Osteoblast Differentiation. Cell Growth Differ. 2001;12:61–83. PubMed

Raouf A, Seth A. Discovery of osteoblast-associated genes using cDNA microarrays. Bone. 2002;30:463–471. doi: 10.1016/S8756-3282(01)00699-8. PubMed DOI

Choi J-Y, et al. Expression patterns of bone-related proteins during osteoblastic differentiation in MC3T3-E1 cells. J. Cell. Biochem. 1996;61:609–618. doi: 10.1002/(SICI)1097-4644(19960616)61:4<609::AID-JCB15>3.0.CO;2-A. PubMed DOI

Sudo H. In vitro differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria. J. Cell Biol. 1983;96:191–198. doi: 10.1083/jcb.96.1.191. PubMed DOI PMC

Czekanska E, Stoddart M, Richards R, Hayes J. In search of an osteoblast cell model for in vitro research. Eur. Cells Mater. 2012;24:1–17. doi: 10.22203/eCM.v024a01. PubMed DOI

Pérez-Garijo A. When dying is not the end: Apoptotic caspases as drivers of proliferation. Semin. Cell Dev. Biol. 2018;82:86–95. doi: 10.1016/j.semcdb.2017.11.036. PubMed DOI

Thrailkill KM, Siddhanti SR, Fowlkes JL, Quarles LD. Differentiation of MC3T3-E1 Osteoblasts is associated with temporal changes in the expression of IGF-I and IGFBPs. Bone. 1995;17:307–313. doi: 10.1016/8756-3282(95)00223-Z. PubMed DOI

Yan X-Z, et al. Effects of Continuous Passaging on Mineralization of MC3T3-E1 Cells with Improved Osteogenic Culture Protocol. Tissue Eng. Part C Methods. 2014;20:198–204. doi: 10.1089/ten.tec.2012.0412. PubMed DOI

Addison WN, et al. Extracellular matrix mineralization in murine MC3T3-E1 osteoblast cultures: An ultrastructural, compositional and comparative analysis with mouse bone. Bone. 2015;71:244–256. doi: 10.1016/j.bone.2014.11.003. PubMed DOI PMC

Miron RJ, et al. Influence of Enamel Matrix Derivative on Cells at Different Maturation Stages of Differentiation. PLoS One. 2013;8:e71008. doi: 10.1371/journal.pone.0071008. PubMed DOI PMC

Quarles LD, Yohay DA, Lever LW, Caton R, Wenstrup RJ. Distinct proliferative and differentiated stages of murine MC3T3-E1 cells in culture: An in vitro model of osteoblast development. J. Bone Miner. Res. 1992;7:683–692. doi: 10.1002/jbmr.5650070613. PubMed DOI

Wohlrab C, et al. The Association Between Ascorbate and the Hypoxia-Inducible Factors in Human Renal Cell Carcinoma Requires a Functional Von Hippel-Lindau Protein. Front. Oncol. 2018;8:574. doi: 10.3389/fonc.2018.00574. PubMed DOI PMC

Wohlrab C, et al. Ascorbate modulates the hypoxic pathway by increasing intracellular activity of the HIF hydroxylases in renal cell carcinoma cells. Hypoxia. 2019;7:17–31. doi: 10.2147/HP.S201643. PubMed DOI PMC

Atkins GJ, et al. RANKL Expression Is Related to the Differentiation State of Human Osteoblasts. J. Bone Miner. Res. 2003;18:1088–1098. doi: 10.1359/jbmr.2003.18.6.1088. PubMed DOI

Hojo H, Ohba S, He X, Lai LP, McMahon AP. Sp7/Osterix Is Restricted to Bone-Forming Vertebrates where It Acts as a Dlx Co-factor in Osteoblast Specification. Dev. Cell. 2016;37:238–253. doi: 10.1016/j.devcel.2016.04.002. PubMed DOI PMC

Tsapras P, Nezis IP. Caspase involvement in autophagy. Cell Death Differ. 2017;24:1369–1379. doi: 10.1038/cdd.2017.43. PubMed DOI PMC

Watanabe C, Shu GL, Zheng TS, Flavell RA, Clark EA. Caspase 6 regulates B cell activation and differentiation into plasma cells. J. Immunol. 2008;181:6810–9. doi: 10.4049/jimmunol.181.10.6810. PubMed DOI PMC

Ladha S, et al. Constitutive ablation of caspase-6 reduces the inflammatory response and behavioural changes caused by peripheral pro-inflammatory stimuli. Cell Death Discov. 2018;4:40. doi: 10.1038/s41420-018-0043-8. PubMed DOI PMC

Galande S, Dickinson LA, Mian IS, Sikorska M, Kohwi-Shigematsu T. SATB1 Cleavage by Caspase 6 Disrupts PDZ Domain-Mediated Dimerization, Causing Detachment from Chromatin Early in T-Cell Apoptosis. Mol. Cell. Biol. 2001;21:5591–5604. doi: 10.1128/MCB.21.16.5591-5604.2001. PubMed DOI PMC

Dobreva G, et al. SATB2 Is a Multifunctional Determinant of Craniofacial Patterning and Osteoblast Differentiation. Cell. 2006;125:971–986. doi: 10.1016/j.cell.2006.05.012. PubMed DOI

Zhang J, et al. Roles of SATB2 in Osteogenic Differentiation and Bone Regeneration. Tissue Eng. Part A. 2011;17:1767–1776. doi: 10.1089/ten.tea.2010.0503. PubMed DOI PMC

Bell, R. A. V. et al. Chromatin reorganization during myoblast differentiation involves the caspase-dependent removal of SATB2. bioRxiv 2019.12.19.883579 10.1101/2019.12.19.883579 (2019). PubMed PMC

Brentnall M, Rodriguez-Menocal L, De Guevara R, Cepero E, Boise LH. Caspase-9, caspase-3 and caspase-7 have distinct roles during intrinsic apoptosis. BMC Cell Biol. 2013;14:32. doi: 10.1186/1471-2121-14-32. PubMed DOI PMC

Fuentes-Prior P, Salvesen GS. The protein structures that shape caspase activity, specificity, activation and inhibition. Biochem. J. 2004;384:201–232. doi: 10.1042/BJ20041142. PubMed DOI PMC

Ruchon AF, et al. Pex mRNA is localized in developing mouse osteoblasts and odontoblasts. J. Histochem. Cytochem. 1998;46:459–68. doi: 10.1177/002215549804600405. PubMed DOI

Yuan B, et al. Aberrant Phex function in osteoblasts and osteocytes alone underlies murine X-linked hypophosphatemia. J. Clin. Invest. 2008;118:722–734. PubMed PMC

Alos N, Ecarot B. Downregulation of osteoblast Phex expression by PTH. Bone. 2005;37:589–598. doi: 10.1016/j.bone.2005.05.006. PubMed DOI

Tian Y, Xu Y, Fu Q, He M. Parathyroid hormone regulates osteoblast differentiation in a Wnt/β-catenin-dependent manner. Mol. Cell. Biochem. 2011;355:211–216. doi: 10.1007/s11010-011-0856-8. PubMed DOI

Van de Craen M, et al. Proteolytic cleavage of β-catenin by caspases: an in vitro analysis. FEBS Lett. 1999;458:167–170. doi: 10.1016/S0014-5793(99)01153-9. PubMed DOI

Sabbagh Y, Carpenter TO, Demay MB. Hypophosphatemia leads to rickets by impairing caspase-mediated apoptosis of hypertrophic chondrocytes. Proc. Natl. Acad. Sci. 2005;102:9637–9642. doi: 10.1073/pnas.0502249102. PubMed DOI PMC

Diaz-Franco M, Franco-Diaz de Leon R, Villafan-Bernal J. Osteocalcin-GPRC6A: An update of its clinical and biological multi-organic interactions (Review) Mol. Med. Rep. 2019;19:15–22. PubMed PMC

Chlastakova I, et al. Dynamics of caspase-3 activation and inhibition in embryonic micromasses evaluated by a photon-counting chemiluminescence approach. Vitr. Cell. Dev. Biol. - Anim. 2012;48:545–549. doi: 10.1007/s11626-012-9542-8. PubMed DOI

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

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