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.
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.
- MeSH
- adaptorové proteiny signální transdukční analýza genetika MeSH
- buněčná diferenciace MeSH
- imunohistochemie MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- mandibula růst a vývoj MeSH
- messenger RNA analýza MeSH
- myši MeSH
- osteoblasty fyziologie MeSH
- osteocyty fyziologie MeSH
- osteogeneze fyziologie MeSH
- osteokalcin analýza genetika MeSH
- osteoklasty fyziologie MeSH
- receptory kalcitoninu metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Mammalian Meckel´s cartilage is a temporary structure associated with mandible development. Notably, its elimination is not executed by apoptosis, and autophagy was suggested as the major mechanism. Simultaneous reports point to pro-apoptotic caspases as novel participants in autophagic pathways in general. The aim of this research was to find out whether activation of pro-apoptotic caspases (-2, -3, -6, -7, -8 and -9) was associated with autophagy of the Meckel´s cartilage chondrocytes. Active caspases were examined in serial histological sections of mouse mandible using immunodetection and were correlated with incidence of autophagy based on Beclin-1 expression. Caspase-2 and caspase-8 were found in Beclin-1 positive regions, whereas caspase-3, -6, -7 and -9 were not present. Caspase-8 was further correlated with Fas/FasL and HIF-1alpha, potential triggers for its activation. Some Fas and FasL positivity was observed in the chondrocytes but caspase-8 activation was found also in FasL deficient cartilage. HIF-1alpha was abundantly present in the hypertrophic chondrocytes. Taken together, caspase-8 activation in the Meckel´s cartilage was demonstrated for the first time. Caspase-8 and caspase-2 were the only pro-apoptotic caspases detected in the Beclin-1 positive segment of the cartilage. Activation of caspase-8 appears FasL/Fas independent but may be switched on by HIF-1alpha.
- MeSH
- apoptóza fyziologie MeSH
- autofagie fyziologie MeSH
- kaspasy metabolismus MeSH
- kloubní chrupavka cytologie metabolismus MeSH
- lidé MeSH
- mandibula cytologie metabolismus MeSH
- myši MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
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.
- MeSH
- buněčná diferenciace MeSH
- cytoplazma metabolismus MeSH
- faktor 1 indukovatelný hypoxií - podjednotka alfa metabolismus MeSH
- ligand RANK metabolismus MeSH
- membránové proteiny genetika fyziologie MeSH
- myši inbrední ICR MeSH
- myši knockoutované MeSH
- myši MeSH
- osteoblasty cytologie metabolismus MeSH
- osteocyty cytologie metabolismus MeSH
- osteogeneze * MeSH
- osteoklasty cytologie metabolismus MeSH
- osteoprotegerin metabolismus MeSH
- proliferace buněk MeSH
- protein-serin-threoninkinasy genetika fyziologie MeSH
- vývoj kostí MeSH
- vývojová regulace genové exprese * MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Caspases have functions particularly in apoptosis and inflammation. Increasing evidence indicates novel roles of these proteases in cell differentiation, including those involved in osteogenesis. This investigation provides a complex screening of osteogenic markers affected by pan caspase inhibition in micromass cultures derived from mouse forelimbs. PCR Array analysis showed significant alterations in expression of 49 osteogenic genes after 7 days of inhibition. The largest change was a decrease in CD36 expression, which was confirmed at organ level by caspase inhibition in cultured mouse ulnae followed by CD36 immunohistochemical analysis. So far, available data point to osteogenic potential of pro-apoptotic caspases. Therefore, the expression of pro-apoptotic caspases (-3, -6, -7, -8, -9) within the growth plate of mouse forelimbs at the stage where the individual zones are clearly apparent was studied. Caspase-9 was reported in the growth plate for the first time as well as caspase-6 and -7 in the resting zone, caspase-7 in the proliferation, and caspase-6 and -8 in the ossification zone. For all caspases, there was a gradient increase in activation toward the ossification zone. The distribution of staining varied significantly from that of apoptotic cells, and thus, the results further support non-apoptotic participation of caspases in osteogenesis.
- MeSH
- antigeny CD36 analýza genetika MeSH
- imunohistochemie MeSH
- inhibitory kaspas farmakologie MeSH
- kaspasy metabolismus MeSH
- kultivované buňky MeSH
- myši MeSH
- orgánové kultury - kultivační techniky MeSH
- osteogeneze * účinky léků MeSH
- přední končetina růst a vývoj metabolismus MeSH
- vývojová regulace genové exprese účinky léků MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Haemosporidians and trypanosomes of the northern goshawk (Accipiter gentilis) population in the Czech Republic were studied by morphological and molecular methods. Despite the wide distribution of these medium-large birds of prey, virtually nothing is known about their blood parasites. During a 5-year period, altogether 88 nestlings and 15 adults were screened for haemosporidians and trypanosomes by microscopic examination of blood smears and by nested PCR. Both methods revealed consistently higher prevalence of blood protists in adults, Leucocytozoon (80.0 % in adults vs. 13.6 % in nestlings), Haemoproteus (60.0 vs. 2.3 %), Plasmodium (6.7 vs. 0 %), and Trypanosoma (60.0 vs. 2.3 %). Altogether, five haemosporidian lineages were detected by cytochrome b sequencing. Two broadly distributed and host nonspecific lineages, Plasmodium (TURDUS1) and Leucocytozoon (BT2), were detected only sporadically, while three newly described northern goshawk host-specific Leucocytozoon lineages (ACGE01-03) represent the absolute majority of the haemosporidians identified by molecular methods. Our findings support evidences that in falconiform birds the Leucocytozoon toddi group is formed by several host-specific clusters, with Leucocytozoon buteonis in buzzards and Leucocytozoon mathisi in hawks. Between-year comparisons revealed that the infection status of adults remained predominantly unchanged and individuals stayed uninfected or possessed the same parasite lineages; however, two gains and one loss of blood parasite taxa were also recorded.
- MeSH
- cytochromy b genetika MeSH
- Falconiformes parazitologie MeSH
- fylogeneze MeSH
- Haemosporida klasifikace genetika izolace a purifikace MeSH
- hostitelská specificita MeSH
- malárie ptačí epidemiologie parazitologie MeSH
- nemoci ptáků epidemiologie parazitologie MeSH
- parazitemie epidemiologie parazitologie veterinární MeSH
- Plasmodium klasifikace genetika izolace a purifikace MeSH
- polymerázová řetězová reakce veterinární MeSH
- prevalence MeSH
- protozoální DNA chemie izolace a purifikace MeSH
- protozoální infekce zvířat epidemiologie parazitologie MeSH
- sekvenční seřazení veterinární MeSH
- Trypanosoma klasifikace genetika izolace a purifikace MeSH
- trypanozomiáza epidemiologie parazitologie veterinární MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
- Publikační typ
- abstrakt z konference 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
The Myb transcription factors are involved in essential cellular processes, such as cell proliferation, differentiation and cell death. Biological functions carried out by specific Myb proteins are distinct. Hair follicles are ectodermal-derived organs with cycling character of the growth resulting from the presence of somatic stem cells. In this study, we followed the expression of the Myb proteins in developing hair follicles and in the hair follicle stem cell niche by immunofluorescence staining. During hair follicle development, B-Myb was present in a few cells located in the area of cell division; c-Myb was abundant postanally in dividing cells but also in keratinizing zone. In addition, c-Myb was also detected in cells under the hair follicle bulge. These findings indicate possible involvement of c-Myb in regulation of activated stem cells leaving the niche.
Tooth absence and defects caused by various reasons are frequent events in humans. They are not life threatening but may bring about social consequences. Recent dentistry provides solutions in the form of prosthetics or dental implants; however, several complications and distinct limitations favour bioengineering of dental and periodontal structures. At least two types of cells (epithelial and mesenchymal) have to be recombined to produce a new functional tooth. Moreover, the tooth must be vascularized, innervated and properly anchored in the bone. To study these issues, different approaches have been established in both basic and applied research. In this review, recent strategies and techniques of tooth engineering are comprehensively summarized and discussed, particularly regarding manipulation using stem cells.
- MeSH
- implantace protézy MeSH
- kmenové buňky cytologie MeSH
- lidé MeSH
- tkáňové inženýrství metody trendy MeSH
- tkáňové podpůrné struktury MeSH
- výzkum * MeSH
- zuby fyziologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
