MMP9 regulates osteogenesis and MMP2 expression through the TGF-β1/SMAD2/3 signaling pathway in...

Matrix metalloproteinases (MMP) act as effectors and regulators in normal growth and development as well as in pathological processes. The gelatinases MMP2 and MMP9 exhibit similar structures and biological reactions. The purpose of this study was to clarify the relationship between MMP9 and MMP2 in lipopolysaccharide (LPS)-induced osteoblasts. MC3T3-E1 cells were pretreated with or without TGF-β1 inhibitor (20 nM) and SMAD2/3 inhibitor (20 nM) for 1 h, and then with pcDNA3.1-mMMP9 (0.8 μg/mL) for 48 h. Quantitative real-time PCR, western blot, and immunocytochemistry were performed to detect MMP2, TGF-β1, and/or SMAD2/3 expression. Luciferase reporter assay and electrophoretic mobility shift assay (EMSA) were performed to examine the regulatory effect of SMAD2/3 on MMP2 gene transcription. RUNX2, OSX, ALP, type I collagen, and OCN expression was detected in LPS (20 μg/mL)-stimulated MC3T3-E1 cells after MMP9 treatment. MMP9 activated the expression of TGF-β1 and phosphorylation of SMAD2/3. Phosphorylated SMAD2/3 translocalized into nuclei to bind to SMAD-binding elements in the promoter of the MMP2 gene, inhibiting MMP2 gene transcription. Additionally, MMP9 increased RUNX2, OSX, ALP, COL I, and OCN expression in LPS-induced MC3T3 cells. MMP9 may regulate osteogenesis through TGF-β1-SMAD2/3/-MMP2 signaling during the inflammation process.