The Study Of Foxc2 In Adjusting MMPs In The Calreticulin Deficient Cells

BackgroudCalreticulin(CRT) is one of calcium binding proteins in endoplasmic reticulum (ER) and sarcoplasmic reticulum(SR), many unique functions have been reported for CRT, including calcium homeostasis, protein folding, apoptosis, integrin-dependent cell adherence, steroid-dependent gene expression, autoimmune reaction and so on. CRT is closely related in heart development, and the occurrence, development and prognosis of angiocardiopathy. Cardiovascular system depends on series of coordinations in cardiocytes occurrence, development, angiogenesis, extracellular matrix(ECM) and so on. Targeted deletion of CRT in mice cause embryonic lethal as a result of impaired heart development. These defects had been demonstrated as a result of changes in the extracellular matrix composition. Matrix metalloproteinases (MMP)-2 and MMP-9 are two of MMPs which are a family of zinc-dependent proteinases that participate in ECM and collagen degradation. Previous study has shown that the expression and activity of MMP-2 and MMP-9 changes in the calreticulin deficient cells, calreticulin deficient cells have high expression of MMP-2 mRNA, while MMP-9’s mRNA is low, which run directly counter to wt cells’MMPs expression, with significant difference between the two kind of cells. This result exactly the same as MMP-2 and MMP-9’s expression in protein level. These experimental results also demonstrate that the changes of MMP-2 and MMP-9 may possibly be one important reason in mice heart development. However, the mechanism of how MMP-2 and MMP-9 turn to be activated is still unknown, also, less known in MMP’s activity of calreticulin deficient cells. Our gene chip results suggest that Foxc2 in wt and calreticulin deficient cells display a marked difference expression, that is Foxc2 mRNA high express in wt cells, while low in calreticulin deficient cells. We speculate that Foxc2 may be involved in the MMP-2/9’s regulation.Naoyuki Miura fist separated Foxc2 gene from mesoderm of mice in 1933, which strongly express in the developing cartilaginous tissues, kidney and dorsal aortas. Tsutomu Kume etc found Foxc2 expressed in aortic branch, outflow tract, heart mesenchyme and endothelial cells. Foxc2-defcient mice has almost the same performance in cardiovascular development. Both of them exhibited cardiovascular defects. Because of the different expression of Foxc2 in two kind of cells, we consider Foxc2 may participate in regulating MMP-2 and MMP-9’s activity and expression. Basing on the original founds before, we can make clear whether transcription factors Foxc2 regulating MMP-2/9’s activity, adjusting by this process, we can know more about the intracellular mechanisms of MMP-2/9’s changes, which can clarify the mechanisms of cardiogenesis, and provides relevant pathological process of it, moreover it will show a new theory basis for clinical disease’s treatment.CRT and Foxc2 deficient mice have the same exhibition in cardiovascular system, our research is to find whether and how Foxc2 involved in regulating MMP-2 and MMP-9’s activity and expression. PurposeCulture wt and crt-/- cells in high glucose Dulbecco’s modified Eagle’s medium (DMEM). We aims to prove Foxc2 adjusting MMP-2/9 in the calreticulin deficient cells, and to ascertain Foxc2 plays a capital role in the regulation of MMP-2/9. In this way, we can learn more about the regulation mechanism of MMP-2/9.Methods1. Cell cultureMouse embryonic fibroblast cells (MEFs) from wild type (wt) and calreticulin deficient (crt-/-) 14-day-old mouse embryos were prepared, and cultured in DMEM.2. Gene chip analysisIn order to find differentially expressed transcription factors in wild type (wt) and calreticulin deficient (crt-/-) cells, we use gene chip analysis.3. RT-PCRRT-PCR-based assay is the method for confirming gene expression patterns and comparing mRNA levels in different sample populations. Through this way, we can know the different mRNA level expression of MMP-2/9 between wt and crt-/- cells. We also can know changes of Foxc2’s mRNA expression after we block down this taget gene.4. Small interfering RNA (siRNA)We use this way to block down Foxc2 mRNA’s expression. 5. Zymography analysisWe examined the activity of MMP-2 and MMP-9 in the wt cells by zymography analysis.Results1. Gene chip analysis suggest that Foxc2 in wt and calreticulin deficient cells display a marked difference expression, which gray-scale value>3.2. We annlyzed the expression of MMP-2 and MMP-9 by RT-PCR. In wild type cells, the expression of MMP-2 is low, while MMP-9 is high; however, in calreticulin deficient cells, MMP-2 and MMP-9’s expression are directly counter to wild type cells’. Compared to the wild type cells, the differences were considered statistically significant (P<0.05).3. After transfection in wild type cells, RT-PCR proved Foxc2 mRNA expression was lower than transfected before, the difference was considered statistically significant (P<0.05).4. We examined the expression of MMP-2 and MMP-9 by zymography. It shows that MMP-2’s activity highten, when MMP-9’s activity was declined. In contrast with blank control group, the changes of MMPs activity were considered statistically significant (P<0.05). When MMP-2’s changes are contrasted with the negative control(NC) group, MMP-2’s difference is very apparent (P<0.05).5. Both NC and si-Foxc2 groups’have the same change in MMP-9 activity, changes were considered no statistically significant (P>0.05).ConclusionFoxc2 maybe also participated in the regulation process of MMP-2 and MMP-9.