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.

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.

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.

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.

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.

Diverse Fate of an Enigmatic Structure: 200 Years of Meckel's Cartilage.

Meckel's cartilage was first described by the German anatomist Johann Friedrich Meckel the Younger in 1820 from his analysis of human embryos. Two hundred years after its discovery this paper follows the development and largely transient nature of the mammalian Meckel's cartilage, and its role in jaw development. Meckel's cartilage acts as a jaw support during early development, and a template for the later forming jaw bones. In mammals, its anterior domain links the two arms of the dentary together at the symphysis while the posterior domain ossifies to form two of the three ear ossicles of the middle ear. In between, Meckel's cartilage transforms to a ligament or disappears, subsumed by the growing dentary bone. Several human syndromes have been linked, directly or indirectly, to abnormal Meckel's cartilage formation. Herein, the evolution, development and fate of the cartilage and its impact on jaw development is mapped. The review focuses on developmental and cellular processes that shed light on the mechanisms behind the different fates of this cartilage, examining the control of Meckel's cartilage patterning, initiation and maturation. Importantly, human disorders and mouse models with disrupted Meckel's cartilage development are highlighted, in order to understand how changes in this cartilage impact on later development of the dentary and the craniofacial complex as a whole. Finally, the relative roles of tissue interactions, apoptosis, autophagy, macrophages and clast cells in the removal process are discussed. Meckel's cartilage is a unique and enigmatic structure, the development and function of which is starting to be understood but many interesting questions still remain.

Formation and Developmental Specification of the Odontogenic and Osteogenic Mesenchymes.

Within the mandible, the odontogenic and osteogenic mesenchymes develop in a close proximity and form at about the same time. They both originate from the cranial neural crest. These two condensing ecto-mesenchymes are soon separated from each other by a very loose interstitial mesenchyme, whose cells do not express markers suggesting a neural crest origin. The two condensations give rise to mineralized tissues while the loose interstitial mesenchyme, remains as a soft tissue. This is crucial for proper anchorage of mammalian teeth. The situation in all three regions of the mesenchyme was compared with regard to cell heterogeneity. As the development progresses, the early phenotypic differences and the complexity in cell heterogeneity increases. The differences reported here and their evolution during development progressively specifies each of the three compartments. The aim of this review was to discuss the mechanisms underlying condensation in both the odontogenic and osteogenic compartments as well as the progressive differentiation of all three mesenchymes during development. Very early, they show physical and structural differences including cell density, shape and organization as well as the secretion of three distinct matrices, two of which will mineralize. Based on these data, this review highlights the consecutive differences in cell-cell and cell-matrix interactions, which support the cohesion as well as mechanosensing and mechanotransduction. These are involved in the conversion of mechanical energy into biochemical signals, cytoskeletal rearrangements cell differentiation, or collective cell behavior.

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.

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.

Growth-dependent phenotype in FasL-deficient mandibular/alveolar bone.

FASL (CD178) is known for its role in triggering apoptosis, mostly in relation with immune cells but additional functions have been reported more recently, including those in bone development. Examination of postnatal FasL-deficient mice (gld) showed an increased bone deposition in adult mice when compared with wild types. However, a different phenotype was observed prenatally, when the gld bone was underdeveloped. The aim of the following investigation was to evaluate this indication for an growth-dependent bone phenotype of gld mice and to search for the 'switch point'. This study focused on the mandibular/alveolar bone as an important structure for tooth anchorage. In vivo micro-computed tomography (CT) analysis was performed at different stages during the first month (6, 12 and 24 days) of postnatal bone development. In 6-day-old gld mice, a decrease in bone volume/tissue volume (BV/TV), trabecular thickness and trabecular number was revealed. In contrast, the 12-day-old gld mice showed an increased BV/TV and trabecular thickness in the alveolar bone. The same observation applied for bone status in 24-day-old gld mice. Therefore, changes in the bone phenotype occurred between day 6 and 12 of the postnatal development. The switch point is likely related to the changing proportion of bone cells at these stages of development, when the number of osteocytes increases. Indeed, the immunohistochemical analysis of FASL localized this protein in osteoblasts, whereas osteocytes were mostly negative at examined stages. The impact of FASL particularly on osteoblasts would agree with an earlier in vivo observed effect of FASL deficiency on expression of Mmp2, typical for osteoblasts, in the gld mandibular/alveolar bone. Notably, an age-dependent bone phenotype was reported in Mmp2-deficient mice.

Osteogenic and Angiogenic Profiles of Mandibular Bone-Forming Cells.

The mandible is a tooth-bearing structure involving one of the most prominent bones of the facial region. Mesenchymal cell condensation is the first morphological sign of osteogenesis, and several studies have focused on this stage also in the mandible. Little information is available about the early post-condensation period, during which avascular soft condensation turns into vascularized bone, and all three major bone cell types, osteoblasts, osteocytes, and osteoclasts, differentiate. In the mouse first lower molar region, the post-condensation period corresponds to the prenatal days 13-15. If during this critical period, when osteogenesis reaches the point of major bone cell differentiation, vascularization already has to play a critical role, one should be able to show molecular changes which support both types of cellular events. The aim of the present report was to follow in organ context the expression of major osteogenic and angiogenic markers and identify those that are up- or downregulated during this period. To this end, PCR Array was applied covering molecules involved in osteoblastic cell proliferation, commitment or differentiation, extracellular matrix (ECM) deposition, mineralisation, osteocyte maturation, angiogenesis, osteoclastic differentiation, and initial bone remodeling. From 161 analyzed osteogenic and angiogenic factors, the expression of 37 was altered when comparing the condensation stage with the bone stage. The results presented here provide a molecular survey of the early post-condensation stage of mandibular/alveolar bone development which has not yet been investigated in vivo.

FasL Modulates Expression of Mmp2 in Osteoblasts.

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.

Activation of Pro-apoptotic Caspases in Non-apoptotic Cells During Odontogenesis and Related Osteogenesis.

Caspases are well known proteases in the context of inflammation and apoptosis. Recently, novel roles of pro-apoptotic caspases have been reported, including findings related to the development of hard tissues. To further investigate these emerging functions of pro-apoptotic caspases, the in vivo localisation of key pro-apoptotic caspases (-3,-6,-7,-8, and -9) was assessed, concentrating on the development of two neighbouring hard tissues, cells participating in odontogenesis (represented by the first mouse molar) and intramembranous osteogenesis (mandibular/alveolar bone). The expression of the different caspases within the developing tissues was correlated with the apoptotic status of the cells, to produce a picture of whether different caspases have potentially distinct, or overlapping non-apoptotic functions. The in vivo investigation was additionally supported by examination of caspases in an osteoblast-like cell line in vitro. Caspases-3,-7, and -9 were activated in apoptotic cells of the primary enamel knot of the first molar; however, caspase-7 and -8 activation was also associated with the non-apoptotic enamel epithelium at the same stage and later with differentiating/differentiated odontoblasts and ameloblasts. In the adjacent bone, active caspases-7 and -8 were present abundantly in the prenatal period, while the appearance of caspases-3,-6, and -9 was marginal. Perinatally, caspases-3 and -7 were evident in some osteoclasts and osteoblastic cells, and caspase-8 was abundant mostly in osteoclasts. In addition, postnatal activation of caspases-7 and -8 was retained in osteocytes. The results provide a comprehensive temporo-spatial pattern of pro-apoptotic caspase activation, and demonstrate both unique and overlapping activation in non-apoptotic cells during development of the molar tooth and mandibular/alveolar bone. The importance of caspases in osteogenic pathways is highlighted by caspase inhibition in osteoblast-like cells, which led to a significant decrease in osteocalcin expression, supporting a role in hard tissue cell differentiation.