Opposite effect of stable transfection of bioactive transforming growth factor-beta 1 (TGF beta 1) versus exogenous TGF beta 1 treatment on expression of 92-kDa type IV collagenase in mouse skin squamous cell carcinoma CH72 cells.
We have previously shown that transforming growth factor-beta 1 (TGF beta 1) mRNA is consistently overexpressed in squamous cell carcinomas relative to normal mouse skin. Here we show that 92-kDa type IV collagenase (matrix metalloproteinase) (MMP-9) mRNA was likewise progressively overexpressed during mouse skin carcinogenesis. To determine if overexpression of MMP-9 and TGF beta 1 are linked, we stably transfected a bioactive TGF beta 1 into a mouse skin squamous cell carcinoma cell line (CH72), which resulted in about twofold to three-fold higher levels of secreted active TGF beta 1. Active TGF beta 1-transfected cells grew only slightly, but not significantly, more slowly in vitro and in vivo than vector-only transfectants. Two clones overexpressing active TGF beta 1 secreted much reduced levels of MMP-9 activity, as determined by zymogram analyses. However, treatment of these clones with 40 pM exogenous TGF beta 1 for 48 h enhanced secretion of MMP-9 activity. Constitutive mRNA expression of MMP-9 was reduced twofold to 70-fold in five untreated active TGF beta 1-transfected clones relative to the other transfectants. In contrast, treatment with 40 pM exogenous TGF beta 1 induced MMP-9 mRNA expression in a time-dependent fashion, from twofold to fourfold after 4 h to a maximum of 12- to 19-fold after 24-48 h. Induction of MMP-9 mRNA was dose dependent at TGF beta 1 concentrations of 4-400 pM. Thus, stable transfection of bioactive TGF beta 1 downregulated whereas exogenous TGF beta 1 treatment upregulated MMP-9 activity and expression. Treatment of transfectants with a neutralizing TGF beta 1 antibody slightly downregulated constitutive MMP-9 mRNA (20-30%) but completely blocked induction by exogenous TGF beta 1. Thus, the effect of TGF beta 1 transfection was not due to secreted TGF beta 1 but may have been a secondary effect.
Rundhaug
J E
JE
University of Texas M. D. Anderson Cancer Center, Science Park-Research Division, Smithville, Texas 78957, USA.
Park
J
J
Pavone
A
A
Opdenakker
G
G
Fischer
S M
SM
D000818:Animals; D019138:Antibodies, Blocking / Q000494:pharmacology; D015152:Blotting, Northern; D002294:Carcinoma, Squamous Cell / Q000201:enzymology*; D017364:Collagenases / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005260:Female; D020780:Matrix Metalloproteinase 9; D051379:Mice; D008819:Mice, Nude; D009154:Mutation; D010212:Papilloma / Q000201:enzymology; D012333:RNA, Messenger / Q000032:analysis; D012878:Skin Neoplasms / Q000201:enzymology*; D013997:Time Factors; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D019138:Antibodies, Blocking; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D017364:Collagenases; D020780:Matrix Metalloproteinase 9