When mouse 3T3-L1 preadipocytes are induced to differentiate into adipocytes, they change from an extended fibroblast-like morphology to a rounded one. This change most likely occurs through extracellular matrix remodelling, a process known to be mediated in part by matrix metalloproteinases (MMPs). In this study, we have shown by semi-quantitative reverse transcriptase-PCR, zymographic and immunoblot analysis that MMP-2, MMP-9 and membrane type 1 (MT1)-MMP are regulated during adipose conversion. To assess the importance of MMPs for adipocytic differentiation we have used MMP-specific inhibitors as well as neutralizing antibodies. Treatment of 3T3-L1 preadipocytes with the broad MMP inhibitor Ilomastat or the more restricted MMP-2 Inhibitor I prevented their differentiation into adipocytes in a dose-dependent manner, as evidenced by absence of triglyceride accumulation. Inhibitor treatment prevented the fibronectin-network degradation, as well as the induction of the genes for peroxisome-proliferator-activated receptor γ and adipsin, two adipocyte phenotype markers. Inhibitor treatment was effective when applied during the early stages of adipocytic conversion, whereas inhibitor treatment during later stages had little effect. Inhibitor treatment did not inhibit clonal mitotic expansion; nor did it affect the expression pattern of the adipogenic transcription factor CCAAT/enhancer-binding protein β (C/EBPβ) or its nuclear translocation. It did, however, markedly reduce C/EBPβ DNA-binding capacity. Taken together, these results suggest that MMPs, and notably MMP-2 and MMP-9, may be necessary mediators of adipocytic differentiation of 3T3-L1 cells.
Abbreviations used: ADD1/SREBP1, sterol regulatory element binding protein 1; C/EBP, CCAAT/enhancer-binding protein; Dex, dexamethasone; DMEM, Dulbecco's modified Eagle's medium; ECM, extracellular matrix; EMSA, electrophoretic mobility-shift assay; FBS, fetal bovine serum; IBMX, isobutylmethylxanthine; Ins, insulin; LAP, liver-enriched activating protein; LIP, liver-enriched inhibitory protein; MMP, matrix metalloproteinase; proMMP, precursor MMP; intMMP, intermediate MMP; MT1 MMP, membrane type 1 MMP; PPARγ, peroxisome-proliferator-activated receptor gamma; RT-PCR, reverse transcriptase-PCR; TIMP-2, tissue inhibitor of metalloproteinase-2.