Stimulation of 92-kDa gelatinase B promoter activity by ras is mitogen-activated protein kinase kinase 1-independent and requires multiple transcription factor binding sites including closely spaced PEA3/ets and AP-1 sequences.
The 92-kDa type IV collagenase (92-kDa gelatinase B also referred to as MMP-9), which plays a critical role in extracellular matrix degradation, is regulated by growth factors that mediate their effects through the ras proto-oncogene. The current study was undertaken to determine the transcriptional requirements for the induction of 92-kDa gelatinase B expression by an activated ras oncogene. Transfection of OVCAR-3 cells with an expression vector encoding an activated Ha-ras increased 92-kDa gelatinolytic activity and stimulated (over 10-fold) the activity of a CAT reporter driven by 670 nucleotides of 5' flanking sequence of the 92-kDa gelatinase B gene. Transient assays using a CAT reporter driven by 5' deleted fragments of the 92-kDa gelatinase B promoter indicated that a region spanning -634 to -531 was required for optimal induction of the promoter. The individual deletion, or mutation, of a PEA3/ets (-540) motif, AP-1 sites (-533, -79), a NF-kappa B (-600) consensus sequence, and a GT box (-52) substantially reduced the activation of the promoter by ras. An expression vector encoding the PEA3 transcription factor caused a 3-fold stimulation of the wild type but not the PEA3/ets-deleted 92-kDa gelatinase B promoter. Coexpression of a dominant negative c-jun antagonized the ras-dependent stimulation of the 92-kDa gelatinase B promoter-driven CAT reporter. The signaling pathway mediating the induction of 92-kDa gelatinase B promoter activity by ras was examined. The expression of a phosphatase (CL100) which inactivates multiple mitogen-activate protein kinase members abrogated the stimulation of 92-kDa gelatinase B promoter activity by ras. However, the expression of a kinase-deficient mitogen-activated protein kinase kinase 1 (MEK1) did not prevent activation of the 92-kDa gelatinase B promoter by ras and a constitutively activated c-raf expression vector was insufficient for 92-kDa gelatinase B promoter activation. Thus, the stimulation of the 92-kDa gelatinase B promoter by ras requires multiple elements including closely spaced PEA3/est and AP-1 sites and is MEK1-independent.
Gum
R
R
Department of Tumor Biology, M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
Lengyel
E
E
Juarez
J
J
Chen
J H
JH
Sato
H
H
Seiki
M
M
Boyd
D
D
D001483:Base Sequence; D001665:Binding Sites; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D017364:Collagenases / Q000235:genetics*; D011905:Genes, ras; D006801:Humans; D020780:Matrix Metalloproteinase 9; D020929:Mitogen-Activated Protein Kinase Kinases; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D011494:Protein Kinases / Q000378:metabolism*; D000090063:Proto-Oncogene Mas; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured
C000715470:MAS1 protein, human; D009838:Oligodeoxyribonucleotides; D000090063:Proto-Oncogene Mas; D018808:Transcription Factor AP-1; D014157:Transcription Factors; C065788:transcription factor PEA3; D015500:Chloramphenicol O-Acetyltransferase; D011494:Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D017364:Collagenases; D020780:Matrix Metalloproteinase 9