Identification and characterization of TGF-β1-responsive Runx2 acetylation sites for matrix Metalloproteinase-13 expression in osteoblastic cells

In skeletal tissues, transforming growth factor-beta 1 (TGF-β1) serves a number of activities. For example, in osteoblastic cells, TGF-β1 stimulates the expression of matrix metalloproteinase-13 (MMP-13, a bone remodeling gene), which requires the bone transcription factor Runx2. Although TGF-β1 is known to stimulate Runx2 acetylation, the sites involved in MMP-13 gene activation remain unknown. Mass spectrometry analysis revealed that Runx2 was acetylated at one site (K134) and three sites (K24, K134, and K169) following control and TGF-β1-treatment, respectively, in osteoblastic cells. In addition, we mutated the lysine residues in the Runx2 construct into arginine and transfected the construct into mouse mesenchymal stem cells (C3H10T1/2). Wild-type Runx2 expression and acetylation were significantly increased by TGF-β1-treatment, whereas this effect was decreased in the presence of the Runx2 double mutant construct (K24 + K169) in C3H10T1/2 cells. TGF-β1 enhanced MMP-13 promoter activity in cells transfected with the wild-type Runx2 construct, but this effect was considerably reduced in cells transfected with the Runx2 double mutant construct (K24 + K169), according to a luciferase reporter test. Hence, the stability of Runx2 may be mediated by TGF-β1-induced acetylation at K24 and K169 and is required for MMP-13 expression in osteoblastic cells. These findings add to our knowledge of TGF-β1, Runx2, and MMP-13's physiological roles in bone metabolism.