No abstract

Bone morphogenetic proteins (BMPs) are key regulators of cell fate decisions during embryogenesis and tissue homeostasis. BMPs signal through a coordinated assembly of two types of transmembrane serine/ threonine kinase receptors to induce Smad1/5/8 plus non-Smad pathways, such as MAPK and Akt. The recent discovery of BMP receptor inhibitors opened new avenues to study specific BMP signalling and to delineate this effect from TGF-β and Activin signalling. Here we present comprehensive and quantitative analyses on both canonical and non-Smad mediated BMP signalling under Dorsomorphin (DM) and LDN-193189 (LDN) treatment conditions. We demonstrate for the first time, that both compounds affect not only the Smad but also the non-Smad signalling pathways induced by either BMP2, BMP6 or GDF5. The activation of p38, ERK1/2 and Akt in C2C12 cells was inhibited by DM and LDN. In addition “off-target” effects on all branches of BMP non-Smad signalling are presented. From this we conclude that the inhibition of BMP receptors by DM and more efficiently by LDN-193189 affects all known BMP induced signalling cascades.

BackgroundEfficient osteogenic differentiation is highly dependent on coordinated signals arising from growth factor signalling and mechanical forces. Bone morphogenetic proteins (BMPs) are secreted proteins that trigger Smad and non-Smad pathways and thereby influence transcriptional and non-transcriptional differentiation cues. Crosstalk at multiple levels allows for promotion or attenuation of signalling intensity and specificity. Similar to BMPs, mechanical stimulation enhances bone formation. However, the molecular mechanism by which mechanical forces crosstalk to biochemical signals is still unclear.ResultsHere, we use a three-dimensional bioreactor system to describe how mechanical forces are integrated into the BMP pathway. Time-dependent phosphorylation of Smad, mitogen-activated protein kinases and Akt in human fetal osteoblasts was investigated under loading and/or BMP2 stimulation conditions. The phosphorylation of R-Smads is increased both in intensity and duration under BMP2 stimulation with concurrent mechanical loading. Interestingly, the synergistic effect of both stimuli on immediate early Smad phosphorylation is reflected in the transcription of only a subset of BMP target genes, while others are differently affected. Together this results in a cooperative regulation of osteogenesis that is guided by both signalling pathways.ConclusionsMechanical signals are integrated into the BMP signalling pathway by enhancing immediate early steps within the Smad pathway, independent of autocrine ligand secretion. This suggests a direct crosstalk of both mechanotransduction and BMP signalling, most likely at the level of the cell surface receptors. Furthermore, the crosstalk of both pathways over longer time periods might occur on several signalling levels.

The assumption that mesenchymal stromal cell (MSC)-based-therapies are capable of augmenting physiological regeneration processes has fostered intensive basic and clinical research activities. However, to achieve sustained therapeutic success in vivo, not only the biological, but also the mechanical microenvironment of MSCs during these regeneration processes needs to be taken into account. This is especially important for e.g., bone fracture repair, since MSCs present at the fracture site undergo signifi cant biomechanical stimulation. This study has therefore investigated cellular characteristics and the functional behaviour of MSCs in response to mechanical loading.Our results demonstrated a reduced expression of MSC surface markers CD73 (ecto-5'-nucleotidase) and CD29 (integrin β1) after loading. On the functional level, loading led to a reduced migration of MSCs. Both effects persisted for a week after the removal of the loading stimulus. Specifi c inhibition of CD73/CD29 demonstrated their substrate dependent involvement in MSC migration after loading. These results were supported by scanning electron microscopy images and phalloidin staining of actin fi laments displaying less cell spreading, lamellipodia formation and actin accumulations. Moreover, focal adhesion kinase and Src-family kinases were identifi ed as candidate downstream targets of CD73/CD29 that might contribute to the mechanically induced decrease in MSC migration.These results suggest that MSC migration is controlled by CD73/CD29, which in turn are regulated by mechanical stimulation of cells. We therefore speculate that MSCs migrate into the fracture site, become mechanically entrapped, and thereby accumulate to fulfi l their regenerative functions.

The ability to specifically target a cell-type is important for the development of vectors for in vivo gene therapy. In order to produce retrovirus vectors targeting ovarian cancer cells, which specifically overexpress ␣ folate receptor (␣FR), a single chain antibody was fused as an N-terminal extension of the ecotropic and amphotropic murine leukemia virus (MLV) envelope glycoproteins. Vector particles bearing the modified glycoproteins were produced and analysed. Although conventional FACS studies indicated that viral particles bearing the modified Env could bind to ovarian cancer cells, targeted infection was not achieved. The initial step of viruscell interaction was further studied using an immunofluorescence technique, which allows visualisation of single retrovirus particles. Vectors bearing chimeric or wild-type