Besides cell death, caspase-9 participates in non-apoptotic events, including cell differentiation. To evaluate a possible impact on the expression of chondrogenic/osteogenic factors, a caspase-9 inhibitor was tested in vitro. For this purpose, mouse forelimb-derived micromass cultures, the most common chondrogenic in vitro model, were used. The following analyses were performed based on polymerase chain reaction (PCR) arrays and real-time PCR. The expression of several chondrogenesis-related genes was shown to be altered, some of which may impact chondrogenic differentiation (Bmp4, Bmp7, Sp7, Gli1), mineral deposition (Alp, Itgam) or the remodelling of the extracellular matrix (Col1a2, Mmp9) related to endochondral ossification. From the cluster of genes with altered expression, Mmp9 showed the most significant decrease in expression, of more than 50-fold. Additionally, we determined the possible impact of caspase-9 downregulation on the expression of other Mmp genes. A mild increase in Mmp14 was observed, but there was no change in the expression of other studied Mmp genes (-2, -3, -8, -10, -12, -13). Interestingly, inhibition of Mmp9 in micromasses led to decreased expression of some chondrogenic markers related to caspase-9. These samples also showed a decreased expression of caspase-9 itself, suggesting a bidirectional regulation of these two enzymes. These results indicate a specific impact of caspase-9 inhibition on the expression of Mmp9. The localisation of these two enzymes overlaps in resting, proliferative and pre-hypertrophic chondrocytes during in vivo development, which supports their multiple functions, either apoptotic or non-apoptotic. Notably, a coincidental expression pattern was identified in Pik3cg, a possible candidate for Mmp9 regulation.

• Keywords
• Caspase-9, Chondrogenic differentiation, Micromasses, Mmp-9, Non-apoptotic functions,
• MeSH
• Cell Differentiation MeSH
• Chondrocytes * MeSH
• Chondrogenesis * physiology   MeSH
• Caspase Inhibitors metabolism   pharmacology   MeSH
• Caspase 9 genetics   metabolism   MeSH
• Cells, Cultured MeSH
• Mice MeSH
• Osteogenesis MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Caspase Inhibitors MeSH
• Caspase 9 MeSH

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,
• Publication type
• Journal Article MeSH

The term apoptosis, as a way of programmed cell death, was coined a half century ago and since its discovery the process has been extensively investigated. The anatomy and physiology of the head are complex and thus apoptosis has mostly been followed in separate structures, tissues or cell types. This review aims to provide a comprehensive overview of recent knowledge concerning apoptosis-related molecules involved in the development of structures of head with a particular focus on caspases, cysteine proteases having a key position in apoptotic pathways. Since many classical apoptosis-related molecules, including caspases, are emerging in several non-apoptotic processes, these were also considered. The largest organ of the head region is the brain and its development has been extensively investigated, including the roles of apoptosis and related molecules. Neurogenesis research also includes sensory organs such as the eye and ear, efferent nervous system and associated muscles and glands. Caspases have been also associated with normal function of the skin and hair follicles. Regarding mineralised tissues within craniofacial morphogenesis, apoptosis in bones has been of interest along with palate fusion and tooth development. Finally, the role of apoptosis and caspases in angiogenesis, necessary for any tissue/organ development and maintenance/homeostasis, are discussed. Additionally, this review points to abnormalities of development resulting from improper expression/activation of apoptosis-related molecules.

• Keywords
• apoptotic, caspases, development, head, non-apoptotic,
• Publication type
• Journal Article MeSH
• Review MeSH

Objective. Caspases, cysteine proteases traditionally associated with apoptosis and inflammation, have recently been identified as important regulators of autophagy and reported within the growth plate, a cartilaginous part of the developing bone. The aim of this research was to identify novel autophagy-related molecules affected by inhibition of pro-apoptotic caspases in chondrocytes. Design. Chondrocyte micromasses derived from mouse limb buds were treated with pharmacological inhibitors of caspases. Autophagy-related gene expression was examined and possible novel molecules were confirmed by real-time polymerase chain reaction and immunocytofluorescence. Individual caspases inhibitors were used to identify the effect of specific caspases. Results. Chondrogenesis accompanied by caspase activation and autophagy progression was confirmed in micromass cultures. Expression of several autophagy-associated genes was significantly altered in the caspases inhibitors treated groups with the most prominent decrease for Pik3cg and increase of Tnfsf10. The results showed the specific pro-apoptotic caspases that play a role in these effects. Importantly, use of caspase inhibitors mimicked changes triggered by an autophagy stimulator, rapamycin, linking loss of caspase activity to an increase in autophagy. Conclusion. Caspase inhibition significantly affects regulation of autophagy-related genes in chondrocytes cultures. Detected markers are of importance in diagnostics and thus the data presented here open new perspectives in the field of cartilage development and degradation.

• Keywords
• autophagy, caspase, gene expression, inhibition, limb,
• MeSH
• Autophagy MeSH
• Chondrocytes * metabolism   MeSH
• Chondrogenesis MeSH
• Caspase Inhibitors metabolism   pharmacology   MeSH
• Caspases * metabolism   pharmacology   MeSH
• Mice MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Caspase Inhibitors MeSH
• Caspases * MeSH

The protein heterogeneity at the single-cell level has been recognized to be vital for an understanding of various life processes during animal development. In addition, the knowledge of accurate quantity of relevant proteins at cellular level is essential for appropriate interpretation of diagnostic and therapeutic results. Some low-copy-number proteins are known to play a crucial role during cell proliferation, differentiation, and also in apoptosis. The fate decision is often based on the concentration of these proteins in the individual cells. This is likely to apply also for caspases, cysteine proteases traditionally associated with cell death via apoptosis but recently being discovered also as important factors in cell proliferation and differentiation. The hypothesis was tested in bone-related cells, where modulation of fate from apoptosis to proliferation/differentiation and vice versa is particularly challenging, e.g., towards anti-osteoporotic treatments and anti-cancer strategies. An ultrasensitive and highly selective method based on bioluminescence photon counting was used to quantify activated caspase-3/7 in order to demonstrate protein-level heterogeneity in individual cells within one population and to associate quantitative measurements with different cell fates (proliferation, differentiation, apoptosis). The results indicate a gradual increase of caspase-3/7 activation from the proliferative status to differentiation (more than three times) and towards apoptosis (more than six times). The findings clearly support one of the putative key mechanisms of non-apoptotic functions of pro-apoptotic caspases based on fine-tuning of their activation levels.

• Keywords
• Apoptosis, Bioluminescence detection, Caspase-3/7, Cell death and differentiation, Single-cell detection and quantification,
• MeSH
• Enzyme Activation MeSH
• Apoptosis MeSH
• Cell Differentiation MeSH
• Cell Line MeSH
• Caspase 3 chemistry   genetics   metabolism   MeSH
• Caspase 7 chemistry   genetics   metabolism   MeSH
• Mice MeSH
• Osteoblasts cytology   physiology   MeSH
• Cell Proliferation MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Caspase 3 MeSH
• Caspase 7 MeSH

The development of a tooth germ in a precise size, shape, and position in the jaw, involves meticulous regulation of cell proliferation and cell death. Apoptosis, as the most common type of programmed cell death during embryonic development, plays a number of key roles during odontogenesis, ranging from the budding of the oral epithelium during tooth initiation, to later tooth germ morphogenesis and removal of enamel knot signaling center. Here, we summarize recent knowledge about the distribution and function of apoptotic cells during odontogenesis in several vertebrate lineages, with a special focus on amniotes (mammals and reptiles). We discuss the regulatory roles that apoptosis plays on various cellular processes during odontogenesis. We also review apoptosis-associated molecular signaling during tooth development, including its relationship with the autophagic pathway. Lastly, we cover apoptotic pathway disruption, and alterations in apoptotic cell distribution in transgenic mouse models. These studies foster a deeper understanding how apoptotic cells affect cellular processes during normal odontogenesis, and how they contribute to dental disorders, which could lead to new avenues of treatment in the future.

• Keywords
• apoptosis, dental lamina, morphogenesis, odontogenesis, teeth,
• Publication type
• Journal Article MeSH
• Review MeSH

Caspases are evolutionary conserved proteases traditionally known as participating in apoptosis and inflammation but recently discovered also in association with other processes such as proliferation or differentiation. This investigation focuses on caspase-12, ranked among inflammatory caspases but displaying other, not yet defined functions. A screening analysis pointed to statistically significant (P < 0.001) increase in expression of caspase-12 in a decisive period of mandibular bone formation when the original mesenchymal condensation turns into vascularized bone tissue. Immunofluorescence analysis confirmed the presence of caspase-12 protein in osteoblasts. Therefore, the osteoblastic cell line MC3T3-E1 was challenged to investigate any impact of caspase-12 on the osteogenic pathways. Pharmacological inhibition of caspase-12 in MC3T3-E1 cells caused a statistically significant decrease in expression of some major osteogenic genes, including those for alkaline phosphatase, osteocalcin and Phex. This downregulation was further confirmed by an alkaline phosphatase activity assay and by a siRNA inhibition approach. Altogether, this study demonstrates caspase-12 expression and points to its unknown physiological engagement in bone cells during the course of craniofacial development.

• Keywords
• alkaline phosphatase, bone, caspase-12, differentiation, osteoblast,
• Publication type
• Journal Article MeSH

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.

• MeSH
• Cell Line MeSH
• Caspase Inhibitors pharmacology   MeSH
• Caspases metabolism   MeSH
• Mice MeSH
• PHEX Phosphate Regulating Neutral Endopeptidase metabolism   MeSH
• Osteoblasts cytology   metabolism   MeSH
• Osteogenesis drug effects   MeSH
• Osteocalcin metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Caspase Inhibitors MeSH
• Caspases MeSH
• PHEX Phosphate Regulating Neutral Endopeptidase MeSH
• Osteocalcin MeSH
• Phex protein, mouse MeSH Browser

FasL is a well-known actor in the apoptotic pathways but recent reports have pointed to its important novel roles beyond cell death, as observed also for bone cells. This is supported by non-apoptotic appearance of FasL during osteogenesis and by significant bone alterations unrelated to apoptosis in FasL deficient (gld) mice. The molecular mechanism behind this novel role has not yet been revealed. In this report, intramembranous bone, where osteoblasts differentiate directly from mesenchymal precursors without intermediary chondrogenic step, was investigated. Mouse mandibular bone surrounding the first lower molar was used as a model. The stage where a complex set of bone cells (osteoblasts, osteocytes, osteoclasts) is first present during development was selected for an initial examination. Immunohistochemical staining detected FasL in non-apoptotic cells at this stage. Further, FasL deficient vs. wild type samples subjected to osteogenic PCR Array analysis displayed a significantly decreased expression of Mmp2 in gld bone. To examine the possibility of this novel FasL-Mmp2 relationship, intramembranous bone-derived osteoblastic cells (MC3T3-E1) were treated with anti-FasL antibody or rmFasL. Indeed, the FasL neutralization caused a decreased expression of Mmp2 and rmFasL added to the cells resulted in the opposite effect. Since Mmp2 -/- mice display age-dependent alterations in the intramembranous bone, early stages of gld mandibular bone were examined and age-dependent phenotype was confirmed also in gld mice. Taken together, the present in vivo and in vitro findings point to a new non-apoptotic function of FasL in bone development associated with Mmp2 expression.

• Keywords
• Fas ligand, Mmp2, intramembranous bone, non-apoptotic, osteogenesis,
• Publication type
• Journal Article MeSH