Exploring caspase functions in mouse models.

Caspases are enzymes with protease activity. Despite being known for more than three decades, caspase investigation still yields surprising and fascinating information. Initially associated with cell death and inflammation, their functions have gradually been revealed to extend beyond, targeting pathways such as cell proliferation, migration, and differentiation. These processes are also associated with disease mechanisms, positioning caspases as potential targets for numerous pathologies including inflammatory, neurological, metabolic, or oncological conditions. While in vitro studies play a crucial role in elucidating molecular pathways, they lack the context of the body's complexity. Therefore, laboratory animals are an indispensable part of successfully understanding and applying caspase networks. This paper aims to summarize and discuss recent knowledge, understanding, and challenges in caspase knock-out mice.

Inhibition of caspase-8 cascade restrains the osteoclastogenic fate of bone marrow cells.

Osteoclasts are multinucleated cells of hematopoietic origin, with a pivotal role in bone development and remodeling. Failure in osteoclast differentiation and activation leads to various bone disorders; thus, attention has focused on a search of molecules involved in osteoclast regulatory pathways. Caspase-8 appears to be an interesting candidate for further exploration, due to its potential function in bone development and homeostasis. Mouse bone marrow cells were differentiated into osteoclasts by RANKL stimulation. Increased activation of caspase-8 and its downstream executioner caspases (caspase-3 and caspase-6) was found during osteoclastogenesis. Subsequent inhibition of caspase-8, caspase-3, or caspase-6, respectively, during osteoclast differentiation showed distinct changes in the formation of TRAP-positive multinucleated cells and reduced expression of osteoclast markers including Acp5, Ctsk, Dcstamp, and Mmp9. Analysis of bone matrix resorption confirmed significantly reduced osteoclast function after caspase inhibition. The results clearly showed the role of caspases in the proper development of osteoclasts and contributed new knowledge about non-apoptotic function of caspases.

Inhibition of caspase-11 under inflammatory conditions suppresses chondrogenic differentiation.

Caspase-11 is the murine homologue of human caspases-4 and -5 and is involved in mediating the inflammatory response. However, its functions are often confused and misinterpreted with the more important and better described caspase-1. Therefore, this study focused exclusively on the specific roles of caspase-11, both in cartilage formation and in the inflammatory environment. The presence of caspase-11 during mouse limb development and in chondrogenic cell cultures was investigated by immunofluorescence detection. Subsequently, the function of caspase-11 was downregulated and the affected molecules investigated. The expression analysis applied for osteo/chondrogenesis associated factors and inflammatory cytokines. Simultaneously, morphological appearance of the micromass cultures was evaluated. The results revealed that caspase-11 is physiologically present during cartilage development, but its inhibition under physiological conditions has no significant effect on chondrogenic differentiation. However, in an inflammatory environment, inhibition and downregulation of caspase-11 leads to reduced differentiation of cartilage nodules. Additionally, reduced expression of several genes including Col2a1 and Sp7 and conversely increased expression of Mmp9 were observed. In the cytokine expression panel, a significant decrease was found in molecules that, along with the inflammatory function, may also be involved in cartilage differentiation. The findings bring new information about caspase-11 in chondrogenesis and show that its downregulation under inflammatory conditions reduces cartilage formation.

FasL impacts Tgfb signaling in osteoblastic cells.

Fas ligand (FasL, CD178) belongs to classical apoptotic molecules, however, recent evidence expands the spectrum of FasL functions into non-apoptotic processes which also applies for the bone. Tgfb subfamily members (Tgfb1, Tgfb2, Tgfb3) represent major components in osteogenic pathways and extracellular matrix. Their possible association with FasL has not yet been investigated but can be postulated. To test such a hypothesis, FasL deficient (gld) calvaria-derived cells were examined with a focus on the expression of Tgfb receptor ligands. The qPCR analysis revealed significantly increased expression of Tgfb1, Tgfb2 and Tgfb3 in gld cells. To check the vice versa effect, the gld cells were stimulated by soluble FasL. As a consequence, a dramatic decrease in expression levels of all three ligands was observed. This phenomenon was also confirmed in IDG-SW3 (osteoblastic cells of endochondral origin). TFLink gateway identified Fosl2 as an exclusive candidate of FasL capable to impact expression of all three Tgfb ligands. However, Fosl2 siRNA did not cause any significant changes in expression of Tgfb ligands. Therefore, the upregulation of the three ligands is likely to occur separately. In this respect, we tested the only exclusive candidate transcription factor for Tgfb3, Prrx1. Additionally, an overlapping candidate for Tgfb1 and Tgfb2, Mef2c capable to modulate expression of sclerostin, was examined. Prrx1 as well as Mef2c were found upregulated in gld samples and their expression decreased after addition of FasL. The same effect of FasL treatment was observed in the IDG-SW3 model. Taken together, FasL deficiency causes an increase in the expression of Tgfb ligands and stimulation by FasL reduces Tgfb expression in osteoblastic cells. The candidates mediating the effect comprise Prrx1 for Tgfb3 and Mef2c for Tgfb1/2. These results indicate FasL as a novel cytokine interfering with Tgfb signaling and thus the complex osteogenic network. The emerging non-apoptotic functions of FasL in bone development and maintenance should also be considered in treatment strategies such as the anti-osteoporotic factor.

Caspase-9 Is a Positive Regulator of Osteoblastic Cell Migration Identified by diaPASEF Proteomics.

Caspase-9 is traditionally considered the initiator caspase of the intrinsic apoptotic pathway. In the past decade, however, other functions beyond initiation/execution of cell death have been described including cell type-dependent regulation of proliferation, differentiation/maturation, mitochondrial, and endosomal/lysosomal homeostasis. As previous studies revealed nonapoptotic functions of caspases in osteogenesis and bone homeostasis, this study was performed to identify proteins and pathways deregulated by knockout of caspase-9 in mouse MC3T3-E1 osteoblasts. Data-independent acquisition-parallel accumulation serial fragmentation (diaPASEF) proteomics was used to compare protein profiles of control and caspase-9 knockout cells. A total of 7669 protein groups were quantified, and 283 upregulated/141 downregulated protein groups were associated with the caspase-9 knockout phenotype. The deregulated proteins were mainly enriched for those associated with cell migration and motility and DNA replication/repair. Altered migration was confirmed in MC3T3-E1 cells with the genetic and pharmacological inhibition of caspase-9. ABHD2, an established regulator of cell migration, was identified as a possible substrate of caspase-9. We conclude that caspase-9 acts as a modulator of osteoblastic MC3T3-E1 cell migration and, therefore, may be involved in bone remodeling and fracture repair.

Microfluidic device for enhancement and analysis of osteoblast differentiation in three-dimensional cell cultures.

Three-dimensional (3D) cell cultures are to date the gold standard in biomedical research fields due to their enhanced biological functions compared to conventional two-dimensional (2D) cultures. 3D cell spheroids, as well as organoids, are better suited to replicate tissue functions, which enables their use both as in vitro models for basic research and toxicology, as well as building blocks used in tissue/organ biofabrication approaches. Culturing 3D spheroids from bone-derived cells is an emerging technology for both disease modelling and drug screening applications. Bone tissue models are mainly limited by the implementation of sophisticated devices and procedures that can foster a tissue-specific 3D cell microenvironment along with a dynamic cultivation regime. In this study, we consequently developed, optimized and characterized an advanced perfused microfluidic platform to improve the reliability of 3D bone cell cultivation and to enhance aspects of bone tissue maturation in vitro. Moreover, biomechanical stimulation generated by fluid flow inside the arrayed chamber, was used to mimic a more dynamic cell environment emulating a highly vascularized bone we expected to improve the osteogenic 3D microenvironment in the developed multifunctional spheroid-array platform. The optimized 3D cell culture protocols in our murine bone-on-a-chip spheroid model exhibited increased mineralization and viability compared to static conditions. As a proof-of-concept, we successfully confirmed on the beneficial effects of a dynamic culture environment on osteogenesis and used our platform for analysis of bone-derived spheroids produced from primary human pre-osteoblasts. To conclude, the newly developed system represents a powerful tool for studying human bone patho/physiology in vitro under more relevant and dynamic culture conditions converging the advantages of microfluidic platforms with multi-spheroid array technologies.

Markers of dental pulp stem cells in in vivo developmental context.

Teeth and their associated tissues contain several populations of mesenchymal stem cells, one of which is represented by dental pulp stem cells (DPSCs). These cells have mainly been characterised in vitro and numerous positive and negati ve markers for these cells have been suggested. To investigate the presence and localization of these molecules during development, forming dental pulp was examined using the mouse first mandibular molar as a model. The stages corresponding to postnatal (P) days 0, 7, 14, and 21 were investigated. The expression was monitored using customised PCR Arrays. Additionally, in situ localization of the key trio of markers (Cd73, Cd90, Cd105 coded by genes Nt5e, Thy1, Eng) was performed at prenatal and postnatal stages using immunohistochemistry. The expression panel of 24 genes assigned as in vitro markers of DPSCs or mesenchymal stem cells (MSCs) revealed their developmental dynamics during formation of dental pulp mesenchyme. Among the positive markers, Vcam1, Fgf2, Nes were identified as increasing and Cd44, Cd59b, Mcam, Alcam as decreasing between perinatal vs. postnatal stages towards adulthood. Within the panel of negative DPSC markers, Cd14, Itgb2, Ptprc displayed increased and Cd24a decreased levels at later stages of pulp formation. Within the key trio of markers, Nt5e did not show any significant expression difference within the investigated period. Thy1 displayed a strong decrease between P0 and P7 while Eng increased between these stages. In situ localization of Cd73, Cd90 and Cd105 showed them overlap in differentiated odontoblasts and in the sub-odontoblastic layer that is speculated to host odontoblast progenitors. The highly prevalent expression of particularly Cd73 and Cd90 opens the question of potential multiple functions of these molecules. The results from this study add to the in vitro based knowledge by showing dynamics in the expression of DPSC/MSC markers during dental pulp formation in an in vivo context and thus with respect to the natural environment important for commitment of stem cells.

DiaPASEF proteotype analysis indicates changes in cell growth and metabolic switch induced by caspase-9 inhibition in chondrogenic cells.

Caspase-9 is the major apical caspase responsible for triggering the intrinsic apoptotic pathway. Our previous study indicated that specific inhibition of caspase-9 caused microscopically evident alterations in appearance of the primary chondrogenic cultures which cannot be explained by decrease in apoptosis. To describe a complex molecular background of this effect, proteomics analysis of control and caspase-9 inhibitor-treated chondrogenic cultures were performed. Proteins were extracted, identified and quantified using LC-MS in both data dependent and data independent acquisition (DIA) mode. While directDIA analysis of diaPASEF data obtained using timsTOF Pro LC-MS system revealed 7849 protein groups (Q-value <0.01), a parallel analysis of iTRAQ-2DLC-MS3 and conventional DIA-MS data identified only 5146 and 4098 protein groups, respectively, showing diaPASEF a superior method for the study. The detailed analysis of diaPASEF data disclosed 236/551 significantly down-/up-regulated protein groups after caspase-9 inhibition, respectively (|log2FC|>0.58, Q value <0.05). Classification of downregulated proteins revealed changes in extracellular matrix organization, collagen metabolism, and muscle system processes. Moreover, deregulations suggest a switch from glycolytic to lipid based metabolism in the inhibited cells. No essential changes were found in the proteins involved in apoptosis. The data indicate new non-apoptotic participation of caspases in chondrocyte homeostasis with potential applications in cartilage pathophysiology.

FasL is a catabolic factor in alveolar bone homeostasis.

AIM: Fas ligand (FasL) belongs to the tumour necrosis factor superfamily regulating bone turnover, inflammation, and apoptosis. The appendicular and axial skeleton phenotype of mature Faslgld mice has been reported. The impact of FasL on the alveolar bone providing support for the teeth at mature stages under healthy and induced inflammatory conditions remains unknown. MATERIALS AND METHODS: We performed a phenotypical analysis of mice carrying the homozygous Faslgld mutation and wild-type (WT) mice (C57BL/6) under healthy conditions and upon ligature-induced periodontitis. After 12 days, micro-computed tomography analysis revealed the distance between the cement enamel junction and the alveolar bone crest. Additional structural parameters, such as the bone volume fraction (BV/TV) and the periodontal ligament space volume, were measured. Histological analyses were performed to visualize the catabolic changes at the defect site. RESULTS: Healthy Faslgld mice were found to have more periodontal bone than their WT littermates. Faslgld had no significant effect on inflammatory osteolysis compared to WT controls with ligatures. Histology revealed eroded surfaces at the root and in the inter-proximal bone in both strains. CONCLUSIONS: Our findings suggest that FasL is a catabolic factor in alveolar bone homeostasis but it does not affect the inflammatory osteolysis.

Johann Gregor Mendel: Born to be a scientist?

Johann Gregor Mendel, born 200 years ago, was supposed to be a farmer, intended to be a teacher, became a priest, turned to being a researcher, and later became a world famous scientist associated with genetics. Here, we look into his life through his own words.

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

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.

Autophagy-related proteases accompany the transition of pre-chondrogenic cells into chondroblasts.

BACKGROUND: Autophagy is classified as a form of programmed cell death. Nevertheless, besides the death-inducing function, autophagy enables removal of damaged organelles, energy savings, and thus cell survival. This applies in particular to cells with poor renewal capabilities, such as chondroblasts. Autophagy is regulated by a complex molecular network, including proteases and their substrates. In autopodium, autophagy-related proteases have been examined particularly within the context of the elimination of the interdigital tissue. However, the death-inducing effects of their expression/activation have not been specified yet. This work focuses on autophagy-associated proteases (cathepsins, matrix metalloproteinases, and caspases) in development of phalangeal cartilage of the mouse autopodium. METHODS: PCR Array, Real-time PCR, and immunohistochemistry were used to follow the expression of autophagy-associated genes in vivo at two developmental stages prenatal/embryonic (E)12 vs. E14. Real-time PCR was then applied to investigate the influence of rapamycin (an inducer of autophagy) on the expression of autophagy-associated proteases in chondroblasts in vitro using micromass culture. RESULTS: Several proteases showed increased expression levels during the transition of pre-chondrogenic cells into chondroblasts in vivo. The most significant increases were observed for Ctsb (fold regulation 2.22), Ctsd (fold regulation 2.37), Ctss (fold regulation 2.92), Mmp9 (up to 445%), and Casp8 (up to 250%). The transition was associated also with the high expression of crucial autophagic inducers, such as Atgs. The in vitro treatment of chondroblasts by rapamycin showed significantly decreased expression of cathepsins, a mild increase in expression of metalloproteinases, and no effect in caspase expression. CONCLUSIONS: The present data provide a screening of autophagy-associated proteases accompanying the formation of cartilage in vivo and specify their expression under rapamycin treatment in vitro. Notably, the selected proteases are assigned to osteoarthritis, therefore their regulation might be used in clinically oriented studies.

Making the head: Caspases in life and death.

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.

General Caspase Inhibition in Primary Chondrogenic Cultures Impacts Their Transcription Profile Including Osteoarthritis-Related Factors.

OBJECTIVE: The knowledge about functions of caspases, usually associated with cell death and inflammation, keeps expanding also regarding cartilage. Active caspases are present in the growth plate, and caspase inhibition in limb-derived chondroblasts altered the expression of osteogenesis-related genes. Caspase inhibitors were reported to reduce the severity of cartilage lesions in osteoarthritis (OA), and caspase-3 might represent a promising biomarker for OA prognosis. The objective of this investigation was to decipher the transcriptomic regulation of caspase inhibition in chondrogenic cells. DESIGN: Limb-derived chondroblasts were cultured in the presence of 2 different inhibitors: Z-VAD-FMK (FMK) and Q-VD-OPH (OPH). A whole transcriptome RNA sequencing was performed as the key analysis. RESULTS: The analysis revealed a statistically significant increase in the expression of 252 genes in the FMK samples and 163 genes in the OPH samples compared with controls. Conversely, there was a significant decrease in the expression of 290 genes in the FMK group and 188 in the OPH group. Among the top up- and downregulated genes (more than 10 times changed), almost half of them were associated with OA. Both inhibitors displayed the highest upregulation of the inflammatory chemokine Ccl5, the most downregulated gene was the one for mannose receptors Mrc1. CONCLUSIONS: The obtained datasets pointed to a significant impact of caspase inhibition on the expression of several chondro-/osteogenesis-related markers in an in vitro model of endochondral ossification. Notably, the list of these genes included some encoding for factors associated with cartilage/bone pathologies such as OA.

Caspase-1 Inhibition Impacts the Formation of Chondrogenic Nodules, and the Expression of Markers Related to Osteogenic Differentiation and Lipid Metabolism.

Caspase-1, as the main pro-inflammatory cysteine protease, was investigated mostly with respect to inflammation-related processes. Interestingly, caspase-1 was identified as being involved in lipid metabolism, which is extremely important for the proper differentiation of chondrocytes. Based on a screening investigation, general caspase inhibition impacts the expression of Cd36 in chondrocytes, the fatty acid translocase with a significant impact on lipid metabolism. However, the engagement of individual caspases in the effect has not yet been identified. Therefore, the hypothesis that caspase-1 might be a candidate here appears challenging. The primary aim of this study thus was to find out whether the inhibition of caspase-1 activity would affect Cd36 expression in a chondrogenic micromass model. The expression of Pparg, a regulator Cd36, was examined as well. In the caspase-1 inhibited samples, both molecules were significantly downregulated. Notably, in the treated group, the formation of the chondrogenic nodules was apparently disrupted, and the subcellular deposition of lipids and polysaccharides showed an abnormal pattern. To further investigate this observation, the samples were subjected to an osteogenic PCR array containing selected markers related to cartilage/bone cell differentiation. Among affected molecules, Bmp7 and Gdf10 showed a significantly increased expression, while Itgam, Mmp9, Vdr, and Rankl decreased. Notably, Rankl is a key marker in bone remodeling/homeostasis and thus is a target in several treatment strategies, including a variety of fatty acids, and is balanced by its decoy receptor Opg (osteoprotegerin). To evaluate the effect of Cd36 downregulation on Rankl and Opg, Cd36 silencing was performed using micromass cultures. After Cd36 silencing, the expression of Rankl was downregulated and Opg upregulated, which was an inverse effect to caspase-1 inhibition (and Cd36 upregulation). These results demonstrate new functions of caspase-1 in chondrocyte differentiation and lipid metabolism-related pathways. The effect on the Rankl/Opg ratio, critical for bone maintenance and pathology, including osteoarthritis, is particularly important here as well.

Impact of FasL Stimulation on Sclerostin Expression and Osteogenic Profile in IDG-SW3 Osteocytes.

The Fas ligand (FasL) is known from programmed cell death, the immune system, and recently also from bone homeostasis. As such, Fas signalling is a potential target of anti-osteoporotic treatment based on the induction of osteoclastic cell death. Less attention has been paid to osteocytes, although they represent the majority of cells within the mature bone and are the key regulators. To determine the impact of FasL stimulation on osteocytes, differentiated IDG-SW3 cells were challenged by FasL, and their osteogenic expression profiles were evaluated by a pre-designed PCR array. Notably, the most downregulated gene was the one for sclerostin, which is the major marker of osteocytes and a negative regulator of bone formation. FasL stimulation also led to significant changes (over 10-fold) in the expression of other osteogenic markers: Gdf10, Gli1, Ihh, Mmp10, and Phex. To determine whether these alterations involved caspase-dependent or caspase-independent mechanisms, the IDG-SW3 cells were stimulated by FasL with and without a caspase inhibitor: Q-VD-OPh. The alterations were also detected in the samples treated by FasL along with Q-VD-OPh, pointing to the caspase-independent impact of FasL stimulation. These results contribute to an understanding of the recently emerging pleiotropic effects of Fas/FasL signalling and specify its functions in bone cells.

A single-cell analytical approach to quantify activated caspase-3/7 during osteoblast proliferation, differentiation, and apoptosis.

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.

FasL Is Required for Osseous Healing in Extraction Sockets in Mice.

Fas ligand (FasL) is a member of the tumor necrosis factor (TNF) superfamily involved in the activation of apoptosis. Assuming that apoptosis is initiated after tooth extraction it is reasonable to suggest that FasL may play a pivotal role in the healing of extraction sockets. Herein, we tested the hypothesis of whether the lack of FasL impairs the healing of extraction sockets. To this end, we extracted upper right incisors of FasL knockout (KO) mice and their wildtype (WT) littermates. After a healing period of two weeks, bone volume over total volume (BV/TV) via µCT and descriptive histological analyses were performed. µCT revealed that BV/TV in the coronal region of the socket amounted to 39.4% in WT and 21.8% in KO, with a significant difference between the groups (p=0.002). Likewise, in the middle region of the socket, BV/TV amounted to 50.3% in WT and 40.8% in KO (p<0.001). In the apical part, however, no difference was noticed. Consistently, WT mice displayed a significantly higher median trabecular thickness and a lower trabecular separation when compared to the KO group at the coronal and central region of the socket. There was the overall tendency that in both, female and male mice, FasL affects bone regeneration. Taken together, these findings suggest that FasL deficiency may reduce bone regeneration during the healing process of extraction sockets.

Caspase Inhibition Affects the Expression of Autophagy-Related Molecules in Chondrocytes.

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.

Caspase-12 Is Present During Craniofacial Development and Participates in Regulation of Osteogenic Markers.

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.