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

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.

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.

Figure 2

Mechanical loading parameter. (a) Scanning electron microscopy exposure of collagen scaffolds. (b) Schematic representation of mechanical loading setup. Cells were seeded on cylindrical collagen scaffolds and load was applied along the symmetry axis of the scaffold (= pore direction). (c) Mechanical loading was performed in a custom-made bioreactor system. Scaffolds were compressed by 10% at a frequency of 1 Hz.

Figure 3

Bone morphogenetic protein 2 and mechanical loading synergistically regulate bone morphogenetic protein-induced Smad phosphorylation events. (a) hFOBs were seeded on collagen scaffolds and subjected to BMP2 stimulation, mechanical loading or a combination of both. Protein lysates were analysed by western blot using specific antibodies. (b) Quantification of western blot analysis depicting phosphoprotein levels normalized to GAPDH. Bar charts represent means ± standard error of the mean (SEM) from three independent experiments; *P < 0.05; **P < 0.01. (c) hFOBs were seeded on collagen scaffolds and subjected for 90 minutes to BMP2 stimulation, mechanical loading or a combination of both. Gene expression was analysed by qRT-PCR. Bar charts summarize three independent experiments and depict means ± SEM. (d) hFOBs were seeded on collagen scaffolds and subjected for 30 min to BMP2 stimulation, mechanical loading or a combination of both. Nuclear and cytosolic protein lysates were fractionated and analysed by western blot. BMP: bone morphogenetic protein; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; hFOB: human fetal osteoblast; qRT-PCR: quantitative reverse transcriptase polymerase chain reaction; SEM: standard error of the mean.

Figure 4

Bone morphogenetic protein 2 and mechanical loading both regulate early bone morphogenetic protein signalling events. (a) hFOB were seeded on collagen scaffolds and subjected to BMP2 stimulation, mechanical loading or a combination of both. Protein lysates were analysed by western blot using specific antibodies. (b) Quantification of western blot analysis depicting phosphoprotein levels normalized to GAPDH. Bar charts represent means ± SEM from three independent experiments; *P < 0.05. (c) hFOBs were seeded on collagen scaffolds and subjected for 90 minutes to BMP2 stimulation, mechanical loading or a combination of both. Gene expression was analysed by qRT-PCR. Bar charts summarize four independent experiments and depict means ± SEM; *P < 0.05; **P < 0.01. BMP: bone morphogenetic protein; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; hFOB: human fetal osteoblast; qRT-PCR: quantitative reverse transcriptase polymerase chain reaction; SEM: standard error of the mean.

Figure 5

Bone morphogenetic protein target gene expression in differentially regulated by bone morphogenetic protein 2 and mechanical loading. (a) hFOBs were seeded on collagen scaffolds and subjected for 24 hours to BMP2 stimulation, mechanical loading or a combination of both. Cell morphology was visualized by actin staining (red), cell nuclei were counterstained by DAPI (blue) and collagen matrix is shown in green. Scale bars, 100 μm. (b and c) hFOBs were seeded on collagen scaffolds and subjected for 24 hours to BMP2 stimulation, mechanical loading or a combination of both. Gene expression was analysed by qRT-PCR. Bar charts summarize three independent experiments and depict means ± SEM; ^#P ≤0.1, *P < 0.05, **P < 0.01, ***P < 0.005. BMP: bone morphogenetic protein; DAPI: 4'-6-diamidino-2-phenylindole; hFOB: human fetal osteoblast; qRT-PCR: quantitative reverse transcriptase polymerase chain reaction; SEM: standard error of the mean.

Figure 6

Crosstalk between bone morphogenetic protein and mechanotransduction pathways might occur on several signalling levels. Schematic model of possible crosstalk levels between mechanical triggers and the BMP signalling cascade as indicated by arrows. BMP: bone morphogenetic protein; BRI-II: bone morphogenetic protein receptor I or II.