Delta-like 1/fetal antigen-1 (DLK1/FA1) is a negative regulator of bone mass in vivo as it inhibits osteoblast (OB) and stimulates osteoclast differentiation. However, the molecular mechanisms underlying these effects are not known. Thus, we studied the effect of DLK1/FA1 on different signaling pathways known to regulate OB differentiation. We identified DLK1/FA1 as an inhibitor of BMP2-induced OB differentiation. Stable overexpression of DLK1/FA1 in C2C12 cells or the addition of its soluble form protein significantly inhibited BMP2-induced OB differentiation evidenced by reduced Alp activity and osteoblastic gene expression. We observed that DLK1/FA1 inhibited BMP signaling by reduced expression of BMP-responsive genes, reduced BMP2-induced luciferase activity in BMP luciferase reporter cells (C2C12BRA), and reduced BMP2-induced Smad1/5/8 phosphorylation. Further studies revealed that DLK1/FA1 affected the expression of both positive (Bmpr1a, Bmpr1b, Bmpr2 and Smad4) and negative (Smad6, Smad7, Smur1 and Smurf2) regulators of BMP pathway. Besides, we observed that DLK1/FA1 induced robust NFκB activity evidenced by NFκB-luciferase reporter assay and real-time RT-PCR analysis of NFκB target genes. Activation of NFκB signaling was found to inhibit BMP signaling in C2C12BRA cells by luciferase assay. In conclusion, we propose a novel mechanism where DLK1/FA1 inhibits BMP2-induced OB differentiation by inhibiting BMP signaling directly through regulation of BMP signaling molecules and indirectly through modulation of NFκB signaling. Our results provide new insight into molecular control of DLK1 on OB differentiation and on bone formation.
14 May 2016 - 17 May 2016