Figure 4. ZBRK1 represses MMP9 promoter activation.

(A) Left panel: ZBRK1 inhibited the MMP9 transcripts in xenogenic tumor. Expression levels of multiple genes of tumor sample from mice resulting after subcutaneously injected with EGFP (TS-C) or EGFP-ZBRK1#6 (TS-#6) HeLa cells were analyzed by RT-PCR. The transcripts of human GAPDH served as a control. Right panel: The loss-of-function ZBRK1 enhanced MMP9 transcripts. Stable ZBRK1-expressing cells were incubated with lentiviral shRNA of ZBRK1 or the control. The lysates and total RNA of infected cells were harvested for Western blot and RT-PCR analyses, respectively. (B) ZBRK1 inhibited the MMP9 reporter. EGFP-ZBRK1-expressing HeLa cells transfected with the −1940/+113 and −940/+113 MMP9 reporters contained wild-type or loss-of-function ZBRK1 responsive element, respectively. Lysates of the transfectants were harvested after 12 h of transfection for luciferase assay. (C) ZBRK1 regulates MMP9 transcription through binding to ZBRK1 motifs. The heterologous reporters bearing the wild-type or mutant ZBRK1 motifs of the MMP9 promoters were transfected in EGFP-ZBRK1-expressing HeLa cells. The results shown are averages from three independent transfection assays and are plotted as relative activities to cells transfected by the backbone reporter, pGL2-promoter (pGL2-p). The latter activity was considered to be 100. Data are shown as the mean ± SEM. * p < 0.05, by Student’s t-test. (D) Left panel: ZBRK1 suppresses TNFα- and EGF-induced MMP9 transcription. HeLa cells transfected the region of −1940/+113 of the MMP9 promoter and stimulated with or without TNF-α or EGF. Right panel: An increase in ZBRK1 attenuated EGF-induced enzyme activity of MMP9. The concentrated lysates for gelatin zymography were performed by the supernatants harvested from the cells stably expressing EGF or EGF-ZBRK1 upon EGF treatment or not.