Quinoline compound KM11073 enhances BMP-2-dependent osteogenic differentiation of C2C12 cells via activation of p38 signaling and exhibits in vivo bone forming activity

Abstract

Recombinant human bone morphogenetic protein (rhBMP)-2 has been approved by the FDA for clinical application, but its use is limited due to high cost and a supra-physiological dose for therapeutic efficacy. Therefore, recent studies have focused on the generation of new therapeutic small molecules to induce bone formation or potentiate the osteogenic activity of BMP-2. Here, we show that [4-(7-chloroquinolin-4-yl) piperazino][1-phenyl-5-(trifluoromethyl)-1H-pyrazol-4-yl]methanone (KM11073) strongly enhances the BMP-2-stimulated induction of alkaline phosphatase (ALP), an early phase biomarker of osteoblast differentiation, in bi-potential mesenchymal progenitor C2C12 cells. The KM11073-mediated ALP induction was inhibited by the BMP antagonist noggin, suggesting that its osteogenic activity occurs via BMP signaling. In addition, a pharmacological inhibition study suggested the involvement of p38 activation in the osteogenic action of KM11073 accompanied by enhanced expression of BMP-2, -6, and -7 mRNA. Furthermore, the in vivo osteogenic activity of KM11073 was confirmed in zebrafish and mouse calvarial bone formation models, suggesting the possibility of its single use for bone formation. In conclusion, the combination of rhBMP-2 with osteogenic small molecules could reduce the use of expensive rhBMP-2, mitigating the undesirable side effects of its supra-physiological dose for therapeutic efficacy. Moreover, due to their inherent physical properties, small molecules could represent the next generation of regenerative medicine.

Fig 1. Chemical structure of KM11073.

Fig 2. KM11073 enhanced BMP-2-induced osteoblast differentiation in C2C12 cells.

Cell viability was assayed 1 and 3 days after treatment with KM11073 (A). Effect of KM11073 on BMP-2-stimulated ALP induction. Cells (4 × 10³ cells/well) were cultured in a 96-well plate for 1 day and then the medium replaced with DMEM containing 5% FBS and KM11073 in the presence or absence of rhBMP-2 (100 ng/ml). The medium was changed every 3 days. On differentiation day 6, ALP staining and its activity were assayed (B). Effect of noggin on KM11073-mediated enhancement of BMP-2-stimulated ALP induction. Osteogenesis was enhanced by KM11073 in the presence of BMP-2 on differentiation days 0 and 2, and then noggin was treated on differentiation day 4. On differentiation day 6, ALP staining and its activity were assayed (C). *** p < 0.001 compared to the BMP-2-treated group; ^## p < 0.01, ^### p < 0.001 compared to the group treated with BMP-2 and KM11073.

Fig 3. Involvement of p38 in the KM11073-mediated enhancement of BMP-2-stimulated ALP induction.

In a 96-well plate, cells (4 × 10³ cells/well) were treated with each inhibitor for 2 h and then treated with BMP-2 and KM11073. After 3 days, the cells were treated with each inhibitor. On differentiation day 6, ALP staining (A) and its activity (B) were assayed. ^### p < 0.001 compared to the BMP-2-treated group; * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the group treated with BMP-2 and KM11073.

Fig 4. Effect of KM11073 on the activation of p38.

Cells (1 × 10⁵ cells/well) were cultured in a 6-well plate for 1 day and then incubated with DMEM containing 5% FBS in the presence or absence of BMP-2 and/or KM11073 (A). Inhibitory effects of p38 inhibitors (1, SB202190; 2, PD169316; 3, SB203580) on the activation of p38 by BMP-2 and KM11073. Western blot analysis was performed with protein samples prepared with cells treated with each inhibitor for 30 min and then incubated with BMP-2 and KM11073 for 30 min (B). The relative, normalized ratio between phosphorylated protein and the protein itself was presented.

Fig 5. Evaluation of the in vivo osteogenic activity of KM11073 in zebrafish and mouse calvariae.

Five days after fertilization, zebrafish were treated with KM11073 (1 μM) for 2 days and then fixed and stained with alizarin red S. The parasphenoid (ps), notochord (n), ceratobranchial 5 (cb5), otolith (ot), and vertebrae (vb) are indicated with arrows (A). Collagen sponges soaked in 5 μl of 2.5 or 5 mM KM11073 were placed onto mouse calvarial bones. After 3-week implantation, the mice were sacrificed. Calvarial bones were removed, fixed, decalcified, embedded in paraffin, and sectioned. Sections were stained with H&E and photographed at 200 × magnification. Arrows indicate the thickness of newly formed woven bones (B). The thickness of newly formed woven bones was quantified compared to the scale bar.