chondrogenesis
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Caspase-12 is a molecule whose functions are still not well understood. Although its expression has been found in various tissues, specific roles have been described in only a few cases. These include the effect of caspase-12 on murine bone cell differentiation during craniofacial development. This work focused on the development of the limbs taking place through endochondral ossification, which precedes the formation of the cartilaginous growth plate. Caspase-12 was described here for the first time in growth plate chondrocytes during physiological development. Using pharmacological inhibition, caspase-12 was found to affect chondrogenesis. Limb-derived micromass cultures showed a significantly increased area of chondrogenic nodules after caspase-12 inhibition and there were changes in gene expression, the most significant of which was the reduction of Mmp9. These data point to potential new functions of caspase-12 in chondrogenesis.
- MeSH
- buněčná diferenciace MeSH
- chondrocyty * MeSH
- chondrogeneze * fyziologie MeSH
- inhibitory kaspas farmakologie MeSH
- kaspasa 12 * metabolismus genetika MeSH
- kultivované buňky MeSH
- matrixová metaloproteinasa 9 metabolismus genetika MeSH
- myši MeSH
- růstová ploténka růst a vývoj MeSH
- vývojová regulace genové exprese MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The Myb locus encodes the c-Myb transcription factor involved in controlling a broad variety of cellular processes. Recently, it has been shown that c-Myb may play a specific role in hard tissue formation; however, all of these results were gathered from an analysis of intramembranous ossification. To investigate a possible role of c-Myb in endochondral ossification, we carried out our study on the long bones of mouse limbs during embryonic development. Firstly, the c-myb expression pattern was analyzed by in situ hybridization during endochondral ossification of long bones. c-myb positive areas were found in proliferating as well as hypertrophic zones of the growth plate. At early embryonic stages, localized expression was also observed in the perichondrium and interdigital areas. The c-Myb protein was found in proliferating chondrocytes and in the perichondrium of the forelimb bones (E14.5-E17.5). Furthermore, protein was detected in pre-hypertrophic as well as hypertrophic chondrocytes. Gain-of-function and loss-of-function approaches were used to test the effect of altered c-myb expression on chondrogenesis in micromass cultures established from forelimb buds of mouse embryos. A loss-of-function approach using c-myb specific siRNA decreased nodule formation, as well as downregulated the level of Sox9 expression, a major marker of chondrogenesis. Transient c-myb overexpression markedly increased the formation of cartilage nodules and the production of extracellular matrix as detected by intense staining with Alcian blue. Moreover, the expression of early chondrogenic genes such as Sox9, Col2a1 and activity of a Col2-LUC reporter were increased in the cells overexpressing c-myb while late chondrogenic markers such as Col10a1 and Mmp13 were not significantly changed or were downregulated. Taken together, the results of this study demonstrate that the c-Myb transcription factor is involved in the regulation and promotion of endochondral bone formation.
- MeSH
- biologické markery metabolismus MeSH
- buněčná diferenciace MeSH
- chondrogeneze fyziologie MeSH
- hybridizace in situ MeSH
- končetiny embryologie MeSH
- myši MeSH
- protoonkogenní proteiny c-myb genetika fyziologie MeSH
- umlčování genů MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- buněčná diferenciace MeSH
- chondrocyty * MeSH
- chondrogeneze * fyziologie MeSH
- inhibitory kaspas metabolismus farmakologie MeSH
- kaspasa 9 genetika metabolismus MeSH
- kultivované buňky MeSH
- myši MeSH
- osteogeneze MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The degeneration of articular cartilage results from osteoarthritis and many other forms of severe arthritis. Current treatments for cartilage repair are less than satisfactory, and rarely restore a full function or return the tissue to its natural state. The leading strategies in the treatment are aimed at the transplantation of cells and/or the use of various biological grafts, bioactive agents, or biologically compatible implant matrices. The insertion of a crushed autologous bone graft has been reported as a possible therapy. However, the regenerative quality of the tissue was less than 70% of healthy cartilage for fragments and controls. The implantation of cycloolefin-blend 3D-cylinders with hydrogel scaffolds on their proximal parts and with the applications of type I collagen films is one of several surgical therapies. The replacement and continuous biomechanical properties of the subchondral bone play an important role in the morphology and the quality of chondrogenesis. The initial biomechanical stability of COC-blend polymer replacements in the subchondral bone contributes to the formation of a new cartilage tissue. The initial bearing capacities of the implanted tissue/replacements and vertical positions of the replacements have a principal influence upon both the quality and the quantity of new articular cartilage.
- MeSH
- analýza selhání vybavení MeSH
- chondrogeneze fyziologie MeSH
- kloubní chrupavka cytologie růst a vývoj chirurgie MeSH
- kostní náhrady terapeutické užití MeSH
- prasata MeSH
- protézy - design MeSH
- výsledek terapie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The FGF signaling pathway plays an important role in the regulation of limb development, controlling cell migration, proliferation, differentiation, and apoptosis. Sprouty proteins act as antagonists of the FGF pathway and control the extent of FGF signaling as part of a negative feedback loop. Sprouty2/4 deficient mice evince defects in endochondral bone formation and digit patterning in their forelimbs, with pathogenesis recently related to ciliopathies. To understand the mechanisms behind these pathologies, the limb defects in Sprouty2+/-;Sprouty4-/- male and female mice were characterized and correlated to the dynamic expression patterns of Sprouty2 and Sprouty4, and the impact on the main signaling centers of the limb bud was assessed. Sprouty2 and Sprouty4 exhibited dynamic expressions during limb development. Interestingly, despite similar expression patterns in all limbs, the hindlimbs did not evince any obvious alterations in development, while the forelimbs showed consistent phenotypes of variable severity. Prenatally as well as postnatally, the left forelimb was significantly more severely affected than the right one. A broad variety of pathologies was present in the autopodium of the forelimb, including changes in digit number, size, shape, and number of bones, hand clefts, and digit fusions. Ectopic ossification of bones and abnormal bone fusions detected in micro-CT scans were frequently observed in the digital as well as in the carpal and metacarpal areas. Sprouty2+/-;Sprouty4-/- limb buds showed patchy loss of Fgf8 expression in the apical ectodermal ridge, and a loss of tissue underlying these regions. The zone of polarizing activity was also impacted, with lineage analysis highlighting a change in the contribution of Sonic hedgehog expressing cells. These findings support the link between Sproutys and Hedgehog signaling during limb development and highlight the importance of Sprouty2 and Sprouty4 in controlling early signaling centers in the limb.
- Publikační typ
- časopisecké články MeSH
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.
- MeSH
- buněčná diferenciace * MeSH
- chondrocyty metabolismus cytologie MeSH
- chondrogeneze * MeSH
- chrupavka metabolismus MeSH
- cytokiny metabolismus MeSH
- iniciační kaspasy * metabolismus MeSH
- kaspasy metabolismus MeSH
- myši MeSH
- zánět * patologie metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články 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.
