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

Fibroblast growth factors (FGFs) deliver extracellular signals that govern many developmental and regenerative processes, but the mechanisms regulating FGF signaling remain incompletely understood. Here, we explored the relationship between intrinsic stability of FGF proteins and their biological activity for all 18 members of the FGF family. We report that FGF1, FGF3, FGF4, FGF6, FGF8, FGF9, FGF10, FGF16, FGF17, FGF18, FGF20, and FGF22 exist as unstable proteins, which are rapidly degraded in cell cultivation media. Biological activity of FGF1, FGF3, FGF4, FGF6, FGF8, FGF10, FGF16, FGF17, and FGF20 is limited by their instability, manifesting as failure to activate FGF receptor signal transduction over long periods of time, and influence specific cell behavior in vitro and in vivo. Stabilization via exogenous heparin binding, introduction of stabilizing mutations or lowering the cell cultivation temperature rescues signaling of unstable FGFs. Thus, the intrinsic ligand instability is an important elementary level of regulation in the FGF signaling system.

• MeSH
• chondrosarkom genetika   metabolismus   patologie   MeSH
• cirkulární dichroismus MeSH
• fibroblastové růstové faktory chemie   klasifikace   genetika   metabolismus   MeSH
• krysa rodu rattus MeSH
• lidé MeSH
• mutace genetika   MeSH
• mutantní proteiny chemie   metabolismus   MeSH
• nádorové buňky kultivované MeSH
• nádory kostí genetika   metabolismus   patologie   MeSH
• nádory prsu genetika   metabolismus   patologie   MeSH
• proliferace buněk * MeSH
• signální transdukce * MeSH
• stabilita proteinů MeSH
• teplota MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Aberrant fibroblast growth factor (FGF) signaling disturbs chondrocyte differentiation in skeletal dysplasia, but the mechanisms underlying this process remain unclear. Recently, FGF was found to activate canonical WNT/β-catenin pathway in chondrocytes via Erk MAP kinase-mediated phosphorylation of WNT co-receptor Lrp6. Here, we explore the cellular consequences of such a signaling interaction. WNT enhanced the FGF-mediated suppression of chondrocyte differentiation in mouse limb bud micromass and limb organ cultures, leading to inhibition of cartilage nodule formation in micromass cultures, and suppression of growth in cultured limbs. Simultaneous activation of the FGF and WNT/β-catenin pathways resulted in loss of chondrocyte extracellular matrix, expression of genes typical for mineralized tissues and alteration of cellular shape. WNT enhanced the FGF-mediated downregulation of chondrocyte proteoglycan and collagen extracellular matrix via inhibition of matrix synthesis and induction of proteinases involved in matrix degradation. Expression of genes regulating RhoA GTPase pathway was induced by FGF in cooperation with WNT, and inhibition of the RhoA signaling rescued the FGF/WNT-mediated changes in chondrocyte cellular shape. Our results suggest that aberrant FGF signaling cooperates with WNT/β-catenin in suppression of chondrocyte differentiation.

• MeSH
• beta-katenin genetika   metabolismus   MeSH
• biologické modely MeSH
• buněčná diferenciace účinky léků   genetika   MeSH
• chondrocyty účinky léků   metabolismus   MeSH
• chrupavka cytologie   účinky léků   metabolismus   MeSH
• fibroblastové růstové faktory farmakologie   MeSH
• fibroblastový růstový faktor 2 farmakologie   MeSH
• HEK293 buňky MeSH
• končetinové pupeny účinky léků   embryologie   metabolismus   MeSH
• konfokální mikroskopie MeSH
• krysa rodu rattus MeSH
• kultivované buňky MeSH
• LDL receptor related protein 6 genetika   metabolismus   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• protein Wnt3A farmakologie   MeSH
• proteiny Wnt genetika   metabolismus   farmakologie   MeSH
• receptory fibroblastových růstových faktorů genetika   metabolismus   MeSH
• signální transdukce účinky léků   genetika   MeSH
• synergismus léků MeSH
• transkriptom účinky léků   genetika   MeSH
• western blotting MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH