Identification and characterization of Runx2 phosphorylation sites involved in matrix metalloproteinase-13 promoter activation

Abstract 

Matrix metalloproteinase-13 (MMP-13) plays a critical role in parathyroid hormone (PTH)-induced bone resorption. PTH acts via protein kinase A (PKA) to phosphorylate and stimulate the transactivation of Runx2 for MMP-13 promoter activation. We show here that PTH stimulated Runx2 phosphorylation in rat osteoblastic cells. Runx2 was phosphorylated on serine 28 and threonine 340 after 8-bromo cyclic adenosine mono phosphate (8-Br-cAMP) treatment. We further demonstrate that in the presence of 8-Br-cAMP, the wild-type Runx2 construct stimulated MMP-13 promoter activity, while the Runx2 construct having mutations at three phosphorylation sites (S28, S347 and T340) was unable to stimulate MMP-13 promoter activity. Thus, we have identified the Runx2 phosphorylation sites necessary for PKA stimulated MMP-13 promoter activation and this event may be critical for bone remodeling.

Abbreviations: PTH, parathyroid hormone, PKA, protein kinase A, 8-Br-cAMP, 8-bromo cyclic adenosine mono phosphate, MMP-13, matrix metalloproteinase-13, MALDI, matrix-assisted laser desorption/ionization, TLC, thin layer chromatography, CAT, chloramphenicol acetyl transferase, GAPDH, glyceraldehyde 3-phosphate dehydrogenase

Keywords: Runx2, Matrix metalloproteinase-13, Collagenase-3, Cyclic adenosine mono phosphate, Parathyroid hormone