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BMP2 and mechanical loading cooperatively regulate immediate early signalling events in the BMP pathway.

Kopf J, Petersen A, Duda GN, Knaus P - BMC Biol. (2012)

Bottom Line: 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.Mechanical signals are integrated into the BMP signalling pathway by enhancing immediate early steps within the Smad pathway, independent of autocrine ligand secretion.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Chemistry/Biochemistry, FU Berlin, Berlin, Germany.

ABSTRACT

Background: Efficient 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.

Results: Here, 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.

Conclusions: Mechanical 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.

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Bone morphogenetic protein signalling dynamics in hFOBs under two-dimensional and three-dimensional culture conditions. (a) hFOBs in two-dimensional monolayer cultures were stimulated with 5 nM BMP2 for indicated time points and protein phosphorylation was analysed by western blot using specific antibodies. (b) hFOBs in two-dimensional monolayer cultures were transfected with a BMP responsive reporter construct (BRE-Luc) and stimulated with different ligand concentrations for 24 hours. Bar charts depict means ± standard error of the mean of relative luciferase activity (RLA); n = 3; ***P < 0.001. (c) hFOBs were cultured on collagen scaffolds and cell morphology was assessed by immunofluorescent staining. Cell morphology was visualized by actin staining (red), cell nuclei were counterstained by DAPI (blue) and collagen matrix is depicted in green. (d) hFOBs were cultured on collagen scaffolds, stimulated with 10 nM BMP2 and protein phosphorylation was analysed by western blot using specific antibodies. BMP: bone morphogenetic protein; DAPI: 4'-6-diamidino-2-phenylindole; hFOB: human fetal osteoblast; RLA: relative luciferase activity.
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Figure 1: Bone morphogenetic protein signalling dynamics in hFOBs under two-dimensional and three-dimensional culture conditions. (a) hFOBs in two-dimensional monolayer cultures were stimulated with 5 nM BMP2 for indicated time points and protein phosphorylation was analysed by western blot using specific antibodies. (b) hFOBs in two-dimensional monolayer cultures were transfected with a BMP responsive reporter construct (BRE-Luc) and stimulated with different ligand concentrations for 24 hours. Bar charts depict means ± standard error of the mean of relative luciferase activity (RLA); n = 3; ***P < 0.001. (c) hFOBs were cultured on collagen scaffolds and cell morphology was assessed by immunofluorescent staining. Cell morphology was visualized by actin staining (red), cell nuclei were counterstained by DAPI (blue) and collagen matrix is depicted in green. (d) hFOBs were cultured on collagen scaffolds, stimulated with 10 nM BMP2 and protein phosphorylation was analysed by western blot using specific antibodies. BMP: bone morphogenetic protein; DAPI: 4'-6-diamidino-2-phenylindole; hFOB: human fetal osteoblast; RLA: relative luciferase activity.

Mentions: Mesenchymal precursor cells respond to BMP2 stimulation by inducing Smad and several non-Smad signalling cascades [8]. To investigate the influence of mechanical forces on BMP signalling events, we used the osteoblastic precursor cell line human fetal osteoblasts (hFOBs). Since little is known about BMP signalling in this cell type, we examined the BMP responsiveness of hFOBs under two-dimensional monolayer culture conditions (Figure 1a). BMP2 stimulation strongly induced Smad1/5/8 phosphorylation, which peaked around 30 minutes after stimulation. In addition, non-Smad pathways involving MAPK and Akt/PKB were initiated, as shown by p38, Erk1/2 and Akt phosphorylation. BMP signalling activity was furthermore assessed by a BMP/Smad responsive reporter gene assay (BRE-Luc). Stimulation of BMP2 for 24 hours led to a significant dose-dependent increase in luciferase activity (Figure 1b).


BMP2 and mechanical loading cooperatively regulate immediate early signalling events in the BMP pathway.

Kopf J, Petersen A, Duda GN, Knaus P - BMC Biol. (2012)

Bone morphogenetic protein signalling dynamics in hFOBs under two-dimensional and three-dimensional culture conditions. (a) hFOBs in two-dimensional monolayer cultures were stimulated with 5 nM BMP2 for indicated time points and protein phosphorylation was analysed by western blot using specific antibodies. (b) hFOBs in two-dimensional monolayer cultures were transfected with a BMP responsive reporter construct (BRE-Luc) and stimulated with different ligand concentrations for 24 hours. Bar charts depict means ± standard error of the mean of relative luciferase activity (RLA); n = 3; ***P < 0.001. (c) hFOBs were cultured on collagen scaffolds and cell morphology was assessed by immunofluorescent staining. Cell morphology was visualized by actin staining (red), cell nuclei were counterstained by DAPI (blue) and collagen matrix is depicted in green. (d) hFOBs were cultured on collagen scaffolds, stimulated with 10 nM BMP2 and protein phosphorylation was analysed by western blot using specific antibodies. BMP: bone morphogenetic protein; DAPI: 4'-6-diamidino-2-phenylindole; hFOB: human fetal osteoblast; RLA: relative luciferase activity.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3361481&req=5

Figure 1: Bone morphogenetic protein signalling dynamics in hFOBs under two-dimensional and three-dimensional culture conditions. (a) hFOBs in two-dimensional monolayer cultures were stimulated with 5 nM BMP2 for indicated time points and protein phosphorylation was analysed by western blot using specific antibodies. (b) hFOBs in two-dimensional monolayer cultures were transfected with a BMP responsive reporter construct (BRE-Luc) and stimulated with different ligand concentrations for 24 hours. Bar charts depict means ± standard error of the mean of relative luciferase activity (RLA); n = 3; ***P < 0.001. (c) hFOBs were cultured on collagen scaffolds and cell morphology was assessed by immunofluorescent staining. Cell morphology was visualized by actin staining (red), cell nuclei were counterstained by DAPI (blue) and collagen matrix is depicted in green. (d) hFOBs were cultured on collagen scaffolds, stimulated with 10 nM BMP2 and protein phosphorylation was analysed by western blot using specific antibodies. BMP: bone morphogenetic protein; DAPI: 4'-6-diamidino-2-phenylindole; hFOB: human fetal osteoblast; RLA: relative luciferase activity.
Mentions: Mesenchymal precursor cells respond to BMP2 stimulation by inducing Smad and several non-Smad signalling cascades [8]. To investigate the influence of mechanical forces on BMP signalling events, we used the osteoblastic precursor cell line human fetal osteoblasts (hFOBs). Since little is known about BMP signalling in this cell type, we examined the BMP responsiveness of hFOBs under two-dimensional monolayer culture conditions (Figure 1a). BMP2 stimulation strongly induced Smad1/5/8 phosphorylation, which peaked around 30 minutes after stimulation. In addition, non-Smad pathways involving MAPK and Akt/PKB were initiated, as shown by p38, Erk1/2 and Akt phosphorylation. BMP signalling activity was furthermore assessed by a BMP/Smad responsive reporter gene assay (BRE-Luc). Stimulation of BMP2 for 24 hours led to a significant dose-dependent increase in luciferase activity (Figure 1b).

Bottom Line: 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.Mechanical signals are integrated into the BMP signalling pathway by enhancing immediate early steps within the Smad pathway, independent of autocrine ligand secretion.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Chemistry/Biochemistry, FU Berlin, Berlin, Germany.

ABSTRACT

Background: Efficient 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.

Results: Here, 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.

Conclusions: Mechanical 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.

Show MeSH
Related in: MedlinePlus