MMP-2 downregulates expression of Src Family kinase inhibitors
To elucidate the mechanism underlying the lack of Src phosphorylation at Tyr-416 in MMP-2 KO cells, we analyzed the expression and activity of upstream endogenous SFK inhibitors at both the RNA and protein levels. Based on current literature, we examined three important regulators of the Src Family Kinases: CHK/MATK, Csk and CDC2 (13,29,30). The qPCR showed a significant upregulation of the CHK/MATK gene expression in the MMP-2 KO cells compared to WT cells (110-fold increase) (Figure 4a). Both CDC2 and Csk transcripts were also slightly upregulated in the MMP-2 KO cells (7.0-fold and 2.1-fold, respectively). Nonetheless, our attention was drawn to the substantial increase in CHK/MATK expression within the MMP-2 KO cells (Figure 4a).
Due to the significance of both SFK inhibitors, CSK and CHK/MATK, protein levels were measured, as shown in the western blot in Figure 4b. CSK protein levels were not significantly different between the WT and MMP-2 KO conditions in either untreated or 0.4 µM doxorubicin-treated cells. CHK/MATK protein levels, however, were only detected in the MMP-2 KO cells and were significantly upregulated compared to the WT cells in both doxorubicin-treated and untreated cells (Figure 4b). The increased expression of CHK/MATK, a SFK inhibitor, in MMP-2 KO cells may explain the lack of Src phosphorylation/activation by doxorubicin in these cells. This also suggests that MMP-2 regulates the expression of CHK/MATK gene in osteosarcoma.