Expression dynamics of metalloproteinases during mandibular bone formation: association with Myb transcription factor.

As the dentition forms and becomes functional, the alveolar bone is remodelled. Metalloproteinases are known to contribute to this process, but new regulators are emerging and their contextualization is challenging. This applies to Myb, a transcription factor recently reported to be involved in bone development and regeneration. The regulatory effect of Myb on Mmps expression has mostly been investigated in tumorigenesis, where Myb impacted the expression of Mmp1, Mmp2, Mmp7, and Mmp9. The aim of this investigation was to evaluate the regulatory influence of the Myb on Mmps gene expression, impacting osteogenesis and mandibular bone formation. For that purpose, knock-out mouse model was used. Gene expression of bone-related Mmps and the key osteoblastic transcription factors Runx2 and Sp7 was analysed in Myb knock-out mice mandibles at the survival limit. Out of the metalloproteinases under study, Mmp13 was significantly downregulated. The impact of Myb on the expression of Mmp13 was confirmed by the overexpression of Myb in calvarial-derived cells causing upregulation of Mmp13. Expression of Mmp13 in the context of other Mmps during mandibular/alveolar bone development was followed in vivo along with Myb, Sp7 and Runx2. The most significant changes were observed in the expression of Mmp9 and Mmp13. These MMPs and MYB were further localized in situ by immunohistochemistry and were identified in pre/osteoblastic cells as well as in pre/osteocytes. In conclusion, these results provide a comprehensive insight into the expression dynamics of bone related Mmps during mandibular/alveolar bone formation and point to Myb as another potential regulator of Mmp13.

Expression of osteogenic factors in FasL-deficient calvarial cells.

During bone development, FasL acts not only through the traditional apoptotic mechanism regulating the amount of bone-resorbing osteoclasts, but there is also growing evidence about its effect on cell differentiation. Expression of osteoblastic factors was followed in non differentiated and differentiating primary calvarial cells obtained from FasL-deficient (gld) mice. The gld cells showed decreased expression of the key osteoblastic molecules osteocalcin (Ocn), osteopontin (Opn), and alkaline phosphatase (Alpl) in both groups. Notably, receptor activator of nuclear factor kappa-B ligand (Rankl) was unchanged in non-differentiated gld vs. wild type (wt) cells but decreased in differentiating gld cells. Osteoprotegerin (Opg) in the gld samples was increased in both groups. Opg vs. Rankl expression levels favored Opg in the case of non-differentiated cells but Rankl in differentiating ones. These results expand information on the involvement of FasL in non-apoptotic cell pathways related to osteoblastogenesis and consequently also osteoclastogenesis and pathologies such as osteoporosis.

Osteogenic Potential of the Transcription Factor c-MYB.

The transcription factor c-MYB is a well-known marker of undifferentiated cells such as haematopoietic cell precursors, but recently it has also been observed in differentiated cells that produce hard tissues. Our previous findings showed the presence of c-MYB in intramembranous bones and its involvement in the chondrogenic steps of endochondral ossification, where the up-regulation of early chondrogenic markers after c-myb overexpression was observed. Since we previously detected c-MYB in osteoblasts, we aimed to analyse the localisation of c-MYB during later stages of endochondral bone formation and address its function during bone matrix production. c-MYB-positive cells were found in the chondro-osseous junction zone in osteoblasts of trabecular bone as well as deeper in the zone of ossification in cells of spongy bone. To experimentally evaluate the osteogenic potential of c-MYB during endochondral bone formation, micromasses derived from embryonic mouse limb buds were established. Nuclear c-MYB protein expression was observed in long-term micromasses, especially in the areas around nodules. c-myb overexpression induced the expression of osteogenic-related genes such as Bmp2, Comp, Csf2 and Itgb1. Moreover, alizarin red staining and osteocalcin labelling promoted mineralised matrix production in c-myb-overexpressing cultures, whereas downregulation of c-myb by siRNA reduced mineralised matrix production. In conclusion, c-Myb plays a role in the osteogenesis of long bones by inducing osteogenic genes and causing the enhancement of mineral matrix production. This action of the transcription factor c-Myb might be of interest in the future for the establishment of novel approaches to tissue regeneration.

Root and Eruption Defects in c-Fos Mice Are Driven by Loss of Osteoclasts.

c-Fos homozygous mice lack osteoclasts with a failure of the teeth to erupt and with an arrest of root development. Here, we characterize the defects associated with the failure in root development and the loss of the tooth-bone interface, and we investigate the underlying causes. We show that, while homozygous c-Fos mice have no multinucleated osteoclasts, heterozygous mice have a reduction in the number of osteoclasts with a reduction in the tooth-bone interface during development and subtle skeletal defects postnatally. In the homozygous mutants bone is found to penetrate the tooth, particularly at the apical end, physically disrupting the root forming HERS (Hertwig's epithelial root sheath) cells. The cells of the HERS continue to proliferate but cannot extend downward due to the presence of bone, leading to a loss of root formation. Tooth germ culture showed that the developing tooth invaded the static bone in mutant tissue, rather than the bone encroaching on the tooth. Although c-Fos has been shown to be expressed in developing teeth, the defect in maintenance of the tooth-bone interface appears to be driven solely by the lack of osteoclasts, as this defect can be rescued in the presence of donor osteoclasts. The rescue suggests that signals from the tooth recruit osteoclasts to clear the bone from around the tooth, allowing the tooth to grow, form roots, and later erupt.

Role of c-Myb in chondrogenesis.

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.