title: Cyclin-dependent kinase 9 is required for tumor necrosis factor-alpha-stimulated matrix metalloproteinase-9 expression in human lung adenocarcinoma cells.
abstract: The proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) 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-alpha-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-alpha-stimulated MMP-9 expression in human lung adenocarcinoma cells. CDK9 activity was inhibited using three different strategies, including the CDK9 pharmacological inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), a dominant-negative CDK9, and a CDK9-specific small interfering RNA. All three approaches reduced TNF-alpha-mediated accumulation of MMP-9 in the conditioned media as demonstrated by gelatin zymography. In contrast, transforming growth factor-beta1-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-alpha-induced increase in MMP-9 mRNA levels but did not effect transforming growth factor-beta1-induced MMP-2 mRNA expression. Consistently DRB and dominant-negative CDK9 completely abrogated TNF-alpha-stimulated human MMP-9 promoter activity. TNF-alpha did not regulate expression or localization of CDK9 or its regulatory partner Cyclin T. However, TNF-alpha 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-alpha-induced MMP-9 transcription. Disruption of TNF-alpha signaling using CDK9 inhibitors could serve as a potential therapeutic strategy against tumor invasion and metastasis.
authors.affiliation: Pulmonary and Critical Care Section, Department of Medicine, Tulane University Health Sciences Center, New Orleans, Louisiana 70112, USA.
authors.forename: Bin
authors.initials: B
authors.lastname: Shan
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhuo
authors.forename: Dawn
authors.initials: D
authors.lastname: Chin
authors.forename: Cindy A
authors.initials: CA
authors.lastname: Morris
authors.forename: Gilbert F
authors.initials: GF
authors.lastname: Morris
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Lasky
mesh_terms: D000230:Adenocarcinoma / Q000201:enzymology* / Q000235:genetics; D045744:Cell Line, Tumor; D017077:Culture Media, Conditioned / Q000378:metabolism; D056906:Cyclin T; D042863:Cyclin-Dependent Kinase 9 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism; D004004:Dichlororibofuranosylbenzimidazole / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D008175:Lung Neoplasms / Q000201:enzymology* / Q000235:genetics; D020778:Matrix Metalloproteinase 2 / Q000235:genetics; D020780:Matrix Metalloproteinase 9 / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D014158:Transcription, Genetic / Q000187:drug effects / Q000235:genetics; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D015854:Up-Regulation / Q000187:drug effects
chemical_list: C503684:CCNT1 protein, human; D017077:Culture Media, Conditioned; D056906:Cyclin T; D016213:Cyclins; D014409:Tumor Necrosis Factor-alpha; D004004:Dichlororibofuranosylbenzimidazole; D042863:Cyclin-Dependent Kinase 9; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9