| BackgroudCalreticulin (CRT) is an endoplasmic reticulum (ER) calcium binding protein and chaperone molecule. CRT is closely related to the growth of heart and the angiocardiopathy’genesis, development and prognosis. In mice, targeted deficiency of CRT is embryonic lethal due to impaired cardiac development. These defect has been demonstrated as a result of changes in the extracellular matrix(ECM) composition. MMP-2 and MMP-9 are two number of matrix metalloproteinases (MMPs) family which are proteinases that participate in ECM degradation. Previous study has shown that compared with wide type (wt) cells, the expression and activity of MMP-2 and MMP-9 have changed in the calreticulin deficient cells, but the specific mechanism is still unknown. We also find that the activation of JNK singnal is defferent between wt and crt-/- cells. So, we presume that the JNK singnal probably participates regulateing the activity of MMP-2/9 in crt-/- cells. Next We want to ascertain this presumption and find out the real mechanism of the singnal in doing this.PurposeTo ascertain whether the JNK singnal participates regulateing the activity of MMP-2/9 in crt-/- cells and what is the mechanism of JNK singnal in doing this.Methods1. Cell cultureMouse embryonic fibroblast cells (MEF) from wild type (wt) and calreticulin deficient (crt-/-) 14-day-old mouse embryos were prepared, and cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM).2. Real-time Quantitative PCRWe use Real-time Quantitative PCR to detect the mRNA level of MMP-2 and MMP-9 of wt and crt-/- cells which are treated with anisomycin.3. Cell survival assays In order to test the cytotoxicity of anisomycin(a JNK activator), MTT assay was used to determine cell viability.4. ZymographyAfter treatment with anisomycin,we examined the activity of MMP-2 and MMP-9 in both the wt and the crt-/- cells by Zymography.Results1. The mRNA level of MMP-2,9, compared to control group, declined when treated with 10ng/ml anisomycin after 6h in both wt and crt-/- cell lines.2. The activities of wt cells’MMP-2 and crt-/- cells’MMP-2/9 are upgraded after being treated with 10ng/ml anisomycin , and they are statistically significant compared to the control group. The activity of wt cells’MMP-9 after being treated with 6h 10ng/ml anisomycin and both wt and crt-/- cells’MMP-2/9 after being treated with selected concentration and time are also changed, but they are not statistically significant compared to the control group. Wt cells’MMP-9 activity and crt-/- cells’MMP-2 activity are all decreased in 50ng/ml after being treated with 12 or 24 hours, and they are statistically significant compared to the control group.3. Crt-/- cells’MMP-2 activity and wt cells’MMP-9 activity are keep increased, while wt MMP-2 and crt-/- MMP-9 activity increase first and then descent in 24 hours. The result showed that it existed statistical significance between 6h time point to 12h and 24h time point but no statistical significance between the 12h and 24 time point.4. The activity of both MMP-9 after 10ng/ml anisomycin in 6h and MMP-2/9 after anisomycin(1, 5, 10ng/ml)in 12h and 24h respectively existed statistical significance between the two cell lines. Wt type displayed with low MMP-2 while high MMP-9, however it displayed oppositely in the crt-/- cell line. The activity of both MMP-2 after 10ng/ml anisomycin in 6h and MMP-2/9 after 50ng/ml anisomycin in 12h and 24h respectively existed no statistical significance between the two cell lines.Conclusion1. It suggested that the JNK pathway played an important role in regulating MMP-2 activity in the CRT deficient cells. 2. The JNK pathway maybe also participated in the regulation of MMP-9 activity in the CRT deficient cells, and there maybe some other pathways take part in this regulation in the wt type cells. |
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