Caspase-8 Deficient Osteoblastic Cells Display Alterations in Non-Apoptotic Pathways

Barbora Vesela; Michael Killinger; Kamila Rihova; Petr Benes; Eva Svandová; Adela Kratochvilová; Filip Trcka; Karel Kleparnik; Eva Matalova

doi:10.3389/fcell.2022.794407

Caspase-8 Deficient Osteoblastic Cells Display Alterations in Non-Apoptotic Pathways

Front Cell Dev Biol. 2022 Mar 15:10:794407. doi: 10.3389/fcell.2022.794407. eCollection 2022.

Authors

Barbora Vesela¹, Michael Killinger^{2

3}, Kamila Rihova², Petr Benes^{2

4}, Eva Svandová¹, Adela Kratochvilová^{1

2}, Filip Trcka², Karel Kleparnik³, Eva Matalova^{1

5}

Affiliations

¹ Institute of Animal Physiology and Genetics, Czech Academy of Sciences, Brno, Czechia.
² Faculty of Science, Masaryk University, Brno, Czechia.
³ Institute of Analytical Chemistry, Czech Academy of Sciences, Brno, Czechia.
⁴ International Clinical Research Center, St. Anne's University Hospital, Brno, Czechia.
⁵ Department of Physiology, University of Veterinary Sciences Brno, Brno, Czechia.

Abstract

Caspase-8 is the key component of the receptor-mediated (extrinsic) apoptotic pathway. Immunological localization of active caspase-8 showed its presence in osteoblasts, including non-apoptotic ones. Further in vivo exploration of caspase-8 functions in the bone is hindered by the fact that the caspase-8 knock-out is lethal prenatally. Examinations were thus performed using individual cell populations in vitro. In this study, caspase-8 was eliminated by the CRISPR/cas9 technology in MC3T3-E1 cells, the most common in vitro model of osteoblastic populations. The aim of the work was to specify the consequences of caspase-8 deficiency on non-apoptotic pathways. The impact on the osteogenic gene expression of the osteoblastic cells along with alterations in proliferation, caspase cascades and rapamycin induced autophagy response were evaluated. Osteogenic differentiation of caspase-8 deficient cells was inhibited as these cells displayed a decreased level of mineralization and lower activity of alkaline phosphatase. Among affected osteogenic genes, based on the PCR Array, major changes were observed for Ctsk, as down-regulated, and Gdf10, as up-regulated. Other significantly down-regulated genes included those coding osteocalcin, bone morphogenetic proteins (-3, -4 and -7), collagens (-1a1, -14a1) or Phex. The formation of autophagosomes was not altered in rapamycin-treated caspase-8 deficient cells, but expression of some autophagy-related genes, including Tnfsf10, Cxcr4, Dapk1 and Igf1, was significantly downregulated. These data provide new insight into the effects of caspase-8 on non-apoptotic osteogenic pathways.

Keywords: CRISPR/cas9; MC3T3-E1; apoptosis; autophagy; bone; osteogenesis.