Cyclin-dependent kinase 9 is required for tumor necrosis factor-α-stimulated matrix metalloproteinase-9 expression in human lung adenocarcinoma cells

Abstract

The proinflammatory cytokine tumor necrosis factor-α (TNF-α) promotes tumor progression through activation of matrix metalloproteinase (MMP) activity. MMP-9 is a gelatinase secreted by both cancer cells and surrounding stromal cells, and it contributes to TNF-α-stimulated tumor invasion and metastasis. Cyclin-dependent kinase 9 (CDK9), the catalytic component of positive transcription elongation factor-b, phosphorylates serine 2 residues in the C-terminal domain of RNA polymerase II for productive transcription elongation and is up-regulated upon exposure to various stresses. This study investigated roles of CDK9 in TNF-α-stimulated MMP-9 expression in human lung adenocarcinoma cells. CDE9 activity was inhibited using three different strategies, including the CDK9 pharmacological inhibitor 5,6-dichloro-1-β- D-ribofuranosylbenzimidazole (DRB), a dominant-negative CDK9, and a CDK9-specific small interfering RNA, All three approaches reduced TNF-α-mediated accumulation of MMP-9 in the conditioned media as demonstrated by gelatin zymography. In contrast, transforming growth factor-β1-induced accumulation of MMP-2 was unaffected by DRB. Expression of the MMP-9 gene was examined using reverse transcription real time PCR and using a transient transfection assay to evaluate MMP-9 promoter activity. DRB reduced the TNF-α-induced increase in MMP-9 mRNA levels but did not effect transforming growth factor-β1-induced MMP-2 mRNA expression. Consistently DRB and dominant-negative CDK9 completely abrogated TNF-α-stimulated human MMP-9 promoter activity. TNF-α did not regulate expression or localization of CDK9 or its regulatory partner Cyclin T. However, TNF-α stimulated CDK9 binding to Cyclin T and MMP-9 gene occupancy by both CDK9 and the serine 2-phosphorylated form of RNA polymerase II. Our findings indicate that CDK9 mediates TNF-α-induced MMP-9 transcription. Disruption of TNF-α signaling using CDK9 inhibitors could serve as a potential therapeutic strategy against tumor invasion and metastasis.