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.

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.

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.

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.

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.

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.

Osteogenic impact of pro-apoptotic caspase inhibitors in MC3T3-E1 cells.

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.

Onset of calciotropic receptors during the initiation of mandibular/alveolar bone formation.

Mandibular/alveolar (m/a) bone, as a component of the periodontal apparatus, allows for the proper tooth anchorage and function of dentition. Bone formation around the tooth germs starts prenatally and, in the mouse model, the mesenchymal condensation turns into a complex vascularized bone (containing osteo-blasts, -cytes, -clasts) within only two days. This very short but critical period is characterized by synchronized cellular and molecular events. The m/a bone, as others, is subjected to endocrine regulations. This not only requires vasculature to allow the circulation of active molecules (ligands), but also the expression of corresponding cell receptors to define target tissues. This contribution aimed at following the dynamics of calciotropic receptors´ expression during morphological transformation of a mesenchymal condensation into the initial m/a bone structure. Receptors for all three calciotropic systemic regulators: parathormone, calcitonin and activated vitamin D (calcitriol), were localized on serial histological sections using immunochemistry and their relative expression was quantified by q-PCR. The onset of calciotropic receptors was followed along with bone cell differentiation (as checked using osteocalcin, sclerostin, RANK and TRAP) and vascularization (CD31) during mouse prenatal/embryonic (E) days 13-15 and 18. Additionally, the timing of calciotropic receptor appearance was compared with that of estrogen receptors (ESR1, ESR2). PTH receptor (PTH1r) appeared in the bone already at E13, when the first osteocalcin-positive cells were detected within the mesenchymal condensation forming the bone anlage. At this stage, blood vessels were only lining the condensation. At E14, the osteoblasts started to express the receptor for activated vitamin D (VDR). At this stage, the vasculature just penetrated the forming bone. On the same day, the first TRAP-positive (but not yet multinucleated) osteoclastic cells were identified. However, calcitonin receptor was detected only one day later. The first Sost-positive osteocytes, present at E15, were PTH1r and VDR positive. ESR1 almost copied the expression pattern of PTH1r, and ESR2 appearance was similar with VDR with a significant increase between E15 and E18. This report focuses on the in vivo situation and links morphological transformation of the mesenchymal cell condensation into a bone structure with dynamics of cell differentiation/maturation, vascularization and onset of receptors for calciotropic endocrine signalling in developing m/a bone.

Tuftelin and HIFs expression in osteogenesis.

Tuftelin was originally discovered and mostly studied in the tooth, but later found also in other organs. Despite its wide distribution among tissues, tuftelin's function has so far been specified only in the formation of enamel crystals. Nevertheless, in many cases, tuftelin was suggested to be associated with cellular adaptation to hypoxia and recently even with cell differentiation. Therefore, we aimed to investigate tuftelin expression along with hypoxia-inducible factors (HIFs) during the early development of the mandibular/alveolar (m/a) bone, when osteoblasts started to differentiate in vivo and to compare their expression levels in undifferentiated versus differentiated osteoblastic cells in vitro. Immunohistochemistry demonstrated the presence of tuftelin already in osteoblastic precursors which were also HIF1-positive, but HIF2-negative. Nevertheless, HIF2 protein appeared when osteoblasts differentiated, one day later. This is in agreement with observations made with MC3T3-E1 cells, where there was no significant difference in tuftelin and Hif1 expression in undifferentiated vs. differentiated cells, although Hif2 increased upon differentiation induction. In differentiated osteoblasts of the m/a bone, all three proteins accumulated, first, prenatally, in the cytoplasm and later, particularly at postnatal stages, they displayed also peri/nuclear localization. Such a dynamic time-space pattern of tuftelin expression has recently been reported in neurons, which, as the m/a bone, differentiate under less hypoxic conditions as indicated also by a prevalent cytoplasmic expression of HIF1 in osteoblasts. However, unlike what was shown in cultured neurons, tuftelin does not seem to participate in final osteoblastic differentiation and its functions, thus, appears to be tissue specific.

Specification of Sprouty2 functions in osteogenesis in in vivo context.

Sprouty proteins are modulators of the MAPK/ERK pathway. Amongst these, Sprouty2 (SPRY2) has been investigated as a possible factor that takes part in the initial phases of osteogenesis. However, the in vivo context has not yet been investigated and the underlying mechanisms taking place in vitro remain unknown. Therefore, in this study, the impact of Spry2 deficiency was examined in the developing tibias of Spry2 deficient (-/-) mouse. The investigation was performed when the osteogenic zone became clearly visible and when all three basic bone cells types were present. The main markers of osteoblasts, osteocytes and osteoclasts were evaluated by immunohistochemistry and RT-PCR. RT-PCR showed that the expression of Sost was 3.5 times higher in Spry2-/- than in the wild-type bone, which pointed to a still unknown mechanism of action of SPRY2 on the differentiation of osteocytes. The up-regulation of Sost was independent of Hif-1α expression and could not be related to its positive regulator, Runx2, since none of these factors showed an increased expression in the bone of Spry2-/- mice. Regarding the RANK/RANKL/OPG pathway, the Spry2-/- showed an increased expression of Rank, but no significant change in the expression of Rankl and Opg. Thanks to these results, the impact of Spry2 deletion is shown for the first time in the developing bone as a complex organ including, particularly, an effect on osteoblasts (Runx2) and osteocytes (Sost). This might explain the previously reported decrease in bone formation in postnatal Spry2-/- mice.

p73 expression in medulloblastoma: TAp73/DeltaNp73 transcript detection and possible association of p73alpha/DeltaNp73 immunoreactivity with survival.

The human p73 protein is essential for normal morphogenesis and maintenance of neural tissue. Recently, several TP73 transcripts have been revealed in medulloblastoma (MB), the most common malignant brain tumor in children. Here, we performed immunohistochemical analysis on 29 MB specimens using anti-p73alpha and anti-DeltaNp73 antibodies. Real-time PCR quantification was performed to assess TAp73 and DeltaNp73 transcripts in a subset of 13 MB samples. Normal cerebellar tissues and RNA were used for comparison. Pilot clinical-pathological correlations were also provided. We report significant differences for TAp73 and DeltaNp73 mRNA expression between tumor tissues and reference (P = 0.013, P = 0.028). Immunohistochemically, 52 and 29% MB samples were positive for p73alpha and DeltaNp73, respectively. p73alpha expression was found to be in both the nucleus and cytoplasm, whereas DeltaNp73 was localized predominantly in the cytoplasm. In normal cerebellum, positive staining for p73alpha and DeltaNp73 was observed in the Purkinje cells of newborns, not adult samples, which supports the developmental role of TP73 during organogenesis of the human cerebellum. Survival analysis has shown negative relationship of DeltaNp73-immunoreactivity with overall survival (OS) and event free survival (EFS) (P = 0.026 and P = 0.127, respectively). For p73alpha-positive cases, the negative trend in OS (P = 0.149) and EFS (P = 0.216) was also apparent. Our results indicate the involvement of p73 protein in MB tumorigenesis and define TP73 as a potential prognostic and therapeutic target for medulloblastoma.