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. 2009 Dec;27(8):526-34.
doi: 10.1002/cbf.1615.

Development and optimization of a cell-based assay for the selection of synthetic compounds that potentiate bone morphogenetic protein-2 activity

Motohiro Okada  1 , Sreedhara Sangadala, Yunshan Liu, Munehito Yoshida, Boojala Vijay B Reddy, Louisa Titus, Scott D Boden
Affiliations

Development and optimization of a cell-based assay for the selection of synthetic compounds that potentiate bone morphogenetic protein-2 activity (V体育安卓版)

V体育安卓版 - Motohiro Okada et al. Cell Biochem Funct. 2009 Dec.

Abstract (V体育2025版)

The requirement of large amounts of the recombinant human bone morphogenetic protein-2 (BMP-2) produces a huge translational barrier for its routine clinical use due to high cost. This leads to an urgent need to develop alternative methods to lower costs and/or increase efficacies for using BMP-2. In this study, we describe the development and optimization of a cell-based assay that is sensitive, reproducible, and reliable in identifying reagents that potentiate the effects of BMP-2 in inducing transdifferentiation of C2C12 myoblasts into the osteoblastic phenotype. The assay is based on a BMP-responsive Smad1-driven luciferase reporter gene. LIM mineralization protein-1 (LMP-1) is a novel intracellular LIM domain protein that has been shown by our group to enhance cellular responsiveness to BMP-2. Our previous report elucidated that the binding of LMP-1 with the WW2 domain in Smad ubiquitin regulatory factor-1 (Smurf1) rescues the osteogenic Smads from degradation. Here, using the optimized cell-based assay, we first evaluated the activity of the recombinantly prepared proteins, LMP-1, and its mutant (LMP-1DeltaSmurf1) that lacks the Smurf1-WW2 domain-binding motif. Both the wild type and the mutant proteins were engineered to contain an 11-amino acid HIV-TAT protein derived membrane transduction domain to aid the cellular delivery of recombinant proteins. The cell-based reporter assay confirmed that LMP-1 potentiates the BMP-induced stimulation of C2C12 cells towards the osteoblastic phenotype. The potentiating effect of LMP-1 was significantly reduced when a specific-motif known to interact with Smurf1 was mutated. We validated the results obtained in the reporter assay by also monitoring the expression of mRNA for osteocalcin and alkaline phosphatase (ALP) which is widely accepted osteoblast differentiation marker genes. Finally, we provide further confirmation of our results by measuring the activity of alkaline phosphatase in support of the accuracy and reliability of our cell-based assay. Direct delivery of synthesized protein can be limited by high cost, instability or inadequate post-translational modifications. Thus, there would be a clear benefit for a low cost, cell penetrable chemical compound. We successfully used our gene expression-based assay to choose an active compound from a select group of compounds that were identified by computational screenings as the most likely candidates for mimicking the function of LMP-1 VSports手机版. Among them, we selected SVAK-3, a compound that showed a dose-dependent potentiation of BMP-2 activity in inducing osteoblastic differentiation of C2C12 cells. We show that either the full length LMP-1 protein or its potential mimetic compound consistently exhibit similar potentiation of BMP-2 activity even when multiple markers of the osteoblastic phenotype were parallely monitored. .

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Figures

Figure 1
Figure 1
LMP-1 regulates cellular responsiveness to BMP. Upon binding of BMP ligand to its specific cell surface receptor, intracellular signaling proteins Smad1/5 are phosphorylated. The activated Smad1/5 associate with Smad4. The oligomerized Smad complex then enters the nucleus to induce BMP-responsive genes in concert with other transcription factors. LMP-1 competitively binds to Smurf1 and rescues Smads1/5 from being targeted for Smurf1-dependent ubiquitin-mediated proteasomal degradation. Consequently, the rescued Smads1/5 lead to potentiation of the BMP-pathway by enhancing the expression of BMP-induced genes such as alkaline phosphatase and osteocalcin
Figure 2
Figure 2
Determination of BMP-2 activity in a cell-based reporter assay. A dose-dependent induction of luciferase activity by BMP-2 is shown in C2C12 cells transfected with the BMP-specific and Smad1-driven 9 × GCCG reporter plamid for 24 h. Luciferase activities were determined in triplicates. * Denotes statistical significance, determined as described in methods, between the indicated treatments ( p < 0.001)
Figure 3
Figure 3
The Smurf1-interacting motif in LMP-1 is required for potentiation of BMP-2 induced reporter activity. A comparison of the effect of wild type LMP-1 protein and the mutant LMP-1ΔSmurf1 protein on BMP-2 induced 9 × GCCG reporter activity is shown. The LMP-1 wild type protein enhanced the BMP-induced luciferase activity by 2.7-fold. LMP-1ΔSmurf1 mutant protein treatment resulted in a 45% loss of the activity observed with the wild type LMP-1 protein. Luciferase activities were determined in triplicates. Statistical significance among different treatment groups was (p < 0.001) obtained as detailed in methods
Figure 4
Figure 4
The Smurf1-interacting motif in LMP-1 is required to enhance BMP-2 induced expression of ALP mRNA. Comparison of the effect of wild type LMP-1 protein and the mutant LMP-1ΔSmurf1 protein on the potentiation of the BMP-2- induced increase in the ALP mRNA level is shown at 25 and 50 nM concentration of proteins. The BMP-2 induced increase in the ALP mRNA level was 2.2-fold and 3.0-fold by 25 nM and 50 nM LMP-1, respectively. These increases were reduced by 53% and 34% when the cells were treated with LMP-1ΔSmurf1 mutant, respectively. Quantitation of mRNA was based on duplicate determinations
Figure 5
Figure 5
The Smurf1-interacting motif in LMP-1 is required for enhancing BMP-2 induced expression of osteocalcin mRNA. Comparison of the effect of the wild type LMP-1 protein and the LMP-1ΔSmurf1 protein on enhancement of BMP-2 induced osteocalcin mRNA expression is shown at 25 and 50 nM concentration of proteins, respectively. BMP-2 induced osteocalcin mRNA expression was enhanced 1.7-fold and 2.6-fold by 25 nM and 50 nM LMP-1, respectively. These increases were reduced by 30% and 24% when the cells were treated with 25 and 50 nM LMP-1ΔSmurf1 mutant, respectively. Quantitation of mRNA was based on duplicate determinations
Figure 6
Figure 6
The Smurf1-interacting motif in LMP-1 is required for enhancing BMP-2 induced ALP enzyme activity. Comparison of the effect of the wild type LMP-1 protein and the LMP-1ΔSmurf1 mutant protein on the enhancement of BMP-2 induced ALP enzyme activity is shown. BMP-2 induced ALP activity was enhanced 4.7-fold by 100 nM LMP-1. This enhancement was reduced by 42% when cells were treated with 100 nM LMP-1ΔSmurf1 mutant instead of wild type LMP-1. Data points were determined in triplicates. Statistical significance among different treatment groups was (p < 0.001) obtained as detailed in methods
Figure 7
Figure 7
Determination of the efficacy of various mimetic compounds to enhance BMP-induced luciferase activity. Relative activities of a representative set of candidate LMP-1 mimetic compounds in the luciferase reporter assay are shown. Compounds were tested at a concentration of 1.0 μg ml−1 while BMP-2 was used at 0.25 ng ml−1. Data points were determined in triplicate. Statistical significance among different treatment groups was (p < 0.001) obtained as detailed in methods. In no compound controls, cells were treated with DMSO (0.01%) alone. Compounds showed no activity in the absence of BMP-2 at the concentration (1.0 μg ml−1) tested. The same compounds that were identified as being active were also determined to be active at compound concentrations of 0.1 and 10 μg ml−1 (data not shown)
Figure 8
Figure 8
SVAK-3 potentiates BMP-2 responsiveness in the Smad1-driven reporter assay. A 2.3-fold enhancement of luciferase activity was observed at a compound concentration of 0.5 μg ml−1 when compared to BMP-2 alone (5 ng ml−1). Data points were determined in triplicates. Statistical significance from the BMP-2 control group was ( p < 0.001) obtained as detailed in Methods section and is denoted by (*)
Figure 9
Figure 9
SVAK-3 enhances the BMP-induced increase of the ALP mRNA level in C2C12 cells. The compound dose-dependently enhanced the BMP-2 induced ALP mRNA level. The peak 3.4-fold enhancement of ALP mRNA level was observed at a SVAK-3 compound concentration of 0.25 μg ml−1. LMP-1 alone or compound alone controls showed no significant effect. Quantitation of mRNA was based on duplicate determinations
Figure 10
Figure 10
SVAK-3 enhances the BMP-induced osteocalcin mRNA level in C2C12 cells. The peak 2.6-fold enhancement of the osteocalcin mRNA level was observed at a SVAK-3 compound concentration of 0.25 μg ml−1. LMP-1 alone or compound alone showed no significant effect. Quantitation of mRNA was based on duplicate determinations
Figure 11
Figure 11
Enhancement of BMP-2 induced ALP enzyme activity by LMP-1 and SVAK-3. The compound dose-dependently enhanced the BMP-2 (100 nM)-induced ALP activity at 100 ng ml−1 of BMP-2. The peak 3.6-fold enhancement of ALP activity was observed at a SVAK-3 compound concentration of 0.25 μg ml−1. Similarly, 100 nM LMP-1 protein treatment, when used as a positive control, showed a 2.8-fold enhancement of ALP activity. Data points were determined in triplicates. * Denotes significant difference from BMP alone control. # Denotes significant difference from BMP alone control in the presence of equal amount of DMSO (0.01%) as in compound treatments. Statistical significance among different treatment groups was ( p < 0.001) obtained as detailed in Methods section
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