The Mechanism And The Differential Expression Of MMP2 And MMP9 In Calreticulin Deficient Cells

Introduction: Calreticulin is an endoplasmic reticulum luminal resident protein, involving in several of the processes including folding of newly synthesized proteins (chaperone activity), regulating of the cell adhesion, and regulating of Ca2+ homeostasis. The gene targeted deletion of calreticulin is embryonic lethal. The main phenotype of these mice is defect in heart and formation of omphaloceal. These defects could be due to altered extracellular matrix protein. The matrix metalloproteinases (MMPs) are family secreted proteases that are important in many cardiac diseases e.g. aortic aneurysm ventricular remodeling, cardiac injury and restenosis. Activation of MMPs (2 and 9) leads to migration and proliferation of endothelial cells and smooth muscle cells thereby contributing to angiogenesis. But there are no more data about the defects observed in the heart and body wall of the calreticulin null embryos are due to changes in the MMPS activity. Therefore, the embryonic fibroblast cells and embryonic tissue were used to test the mechanism that crt-/- mice defect in heart. Methods: Mouse embryonic fibroblast cells (MEF) from wild type (wt) andcalreticulin knockout (crt-/-) 14-day-old mouse embryos were seeded at a density of 2xlO6 cells per dish were cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco) with 10% fetal bow serum. The experiments were separated into 2 groups: wt and crt-/-. And each of them were added thapargagin or wortmanin, respectively. Calcium inhibitor, thapargagin at l(amol/ml, PI 3 kinase inhibitor, wortmanin at 10 nmol/ml, experiments with chemicals were run for 5 hours and 24 hours before the media or cell lysated were collected, respectively. The activity of MMP-2 and MMP-9 in media was measured by gelatin zymography. To determine if there was any changes in the expression of these enzymes we carried out western blot and RT-PCR for MMP-2, MMP-9, MT1-MMP, TIMP1 and TIMP2. To be able to quantitate the data actin (for western) and GAPDH (for RT-PCR) were used as internal controls. In order to test the MMP2 and MMP9 in cell and embryo tissue, immunofluence and immunohischemical were used.Results: Our findings showed conditioned media collected from the crt-/- cells had less detectable MMP-9 activity and high MMP2 activity. However, in the wt cells this observation was reversed. The changes of activities for MMP2 and MMP9 were corresponding to the changes of their RNA, respectively. Western blot analysis for MMP2 and MMP-9 expression in cells showed no difference in the activity in wt and crt-/- cells cultured in MMP2, but MMP9 expression in crt-/- was lower than that in wt. And MT1-MMP RNA of crt-/- was higher than that of in wt cell. There were no changes in TIMP1 and TIMP2 mRAN. The inhibitor experiment showed both wortmanin (inhibitor of PI3 kinase) and thapsigargin (blocker of SERCA ATPase)were able to significantly reduce the level of MMP-2 and MMP9 mRNA in both of wt and crt-/- cells. However, the mRNA level of MMP-2 after these treatments was still significantly higher in the crt-/- cells than in the treated wt cells. There were no differences in the MMP-9 mRNA levels following similar treatments in the wt and crt-/- cells. Interestingly, blockade of PI3 kinase in the wt cells did not affect the mRNA level of MT1-MMP. There were no changes in the mRNA level of TIMP-2 and TIMP-1 following treatment with wortmanin or thapsigargin. The immunohichemical results of MMP2 and MMP9 were similar to the cells's. MMP2 and MMP9 were expressed on the cell surface by immunocytochemical.Conclusion: (1) Targetted deletion of CRT gene results in up-regulation of MMP-2 and MT1-MMP expression and activity accompanied by decreased MMP-9 expression and activity. (2) calreticulin has chaperone function with MMP9 protein. (3) the activities of MMP2 and MMP9 may participate in the heart deficit in crt-/-mice; (4)These changes were due to both PI3 kinase dependent and independent pathways and not via changes in intracellular calcium. ( Supported by an operating grant from CIHR).