Knockdown Of C-fos Gene Implacts The Expression Of MMPs In Mouse Embryonic Fibroblasts

BackgroudCalreticulin （CRT） is a Ca²⁺ buffering chaperone in the endoplasmic reticulum （ER）. It’s criticalfor protein folding, Ca²⁺ homeostasis, and cardiac development. Targeted deletion of CRT inmice induces embryonic lethality due to impaired heart with thinner ventricular wall, which maybe caused by changed extracellular matrix. Matrix metalloproteinases （MMPs） are a family ofproteolytic enzymes dependent on zinc that degrade the extracellular matrix, which are veryimportant for the regulation of extracellular matrix （ECM） and collagen in heart. Previous studyhas shown that the expression and activity of MMP-2 and MMP-9 changes, complicateddecreased expression of c-fos in the calreticulin deficient MEFs, but the specific mechanism isstill unknown.ObjectiveThis study focuses on ascertaining that c-fos plays a capital role in the regulation of MMPs in thecalreticulin deficient cells.Methods1. Cell cultureMouse embryonic myofibroblasts （MEFs） from wild type （wt） and calreticulin deficient （crt-/-）14-day-old mouse embryos were prepared, and cultured in high glucose Dulbecco’s modifiedEagle’s medium （DMEM）.2. Observation with invert microscopeObservate cells with invert microscope before and after transfection with siRNA of c-fos.3. RT-PCRDetect the mRNA expression of c-fos, MFH-1 and HEB in wt and crt-/- MEFs, and the themRNA expression of c-fos, MMP-2 and MMP-9 after transfection with siRNA of c-fos.4. ZymographyExamined the activity of MMP-2 and MMP-9 after transfection with siRNA of c-fos.Results1. In the calreticulin deficient MEFs, the mRNA expression of c-fos decreased significantly （P<0.05）, MFH-1 increased significantly （P<0.05）, while HEB equaled as compared to the wildtype MEFs.2. After transfection with siRNA of c-fos, the mRNA expression of c-fos decreased, complicatedwith decreased mRNA expression and activity of MMP-9, while the activity of MMP-2increased in both treated groups and control groups, even in reagent control group, which issimlar with what we observed in crt-/- MEFs.Conclusion1. C-fos played an important role in regulating MMPs activity in the MEFs.2. In crt-/- MEFSS cells, decreased activity of MMP-9 caused by descreased c-fos might inducedefect heart in mice targeted deletion of crt.