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.