Effect of hypoxia on stimulatory effect of TGF-beta 1 on MMP-2 and MMP-9 activities in mouse fibroblasts.
OBJECTIVE
Because chronic low-grade hypoxia has been implicated in the pathogenesis of fibrosis and postoperative adhesion formation, we hypothesized that hypoxia may modulate the effect of transforming growth factor-beta 1 (TGF-beta 1) on matrix metalloproteinase (MMP)-2 and MMP-9 at both transcriptional and translational levels.


METHODS
Mouse fibroblasts were placed in a hypoxic environment with or without 1 ng/mL TGF-beta 1 for varying periods of time. Zymography was performed on cell supernatants collected after each treatment. Gelatinolytic bands corresponding to MMP-2 and MMP-9 were quantiated by densitometry. Multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) was also performed for MMP-2 and MMP-9 on total RNA extracted from cells after each treatment. Analysis of PCR-amplified products was performed by 2% agarose gel followed by ethidium bromide staining of DNA bands. Scanning densometry was used to determine the ratio of intensity of each band relative to beta-actin.


RESULTS
Hypoxia resulted in a 64% decrease in MMP-9 activity and 80% decrease in MMP-9 mRNA level but did not affect MMP-2 mRNA level or activity. TGF-beta 1 treatment resulted in 180% and 50% increases in MMP-2 and MMP-9 activities, respectively. Increases of 37.5% and 40% in MMP-2 and MMP-9 mRNA levels, respectively, were seen. However, under hypoxic conditions, TGF-beta1 resulted in a 160% increase and 45% decrease in MMP-2 and MMP-9 activities and a 37.5% increase and 71% decrease in MMP-2 and MMP-9 mRNA levels, respectively.


CONCLUSIONS
Hypoxia suppresses the stimulatory effect of TGF-beta1 on the activity of MMP-9 but not MMP-2. This may suggest an important role for MMP-9 under hypoxic conditions in the pathogenesis of tissue fibrosis and postoperative adhesion formation.
Saed
G M
GM
Department of Obstetrics and Gynecology, Wayne State Medical School, Wayne State University, Michigan, Detroit 48201, USA. g.saed@wayne.edu
Zhang
W
W
Diamond
M P
MP
D016475:3T3 Cells; D000818:Animals; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology*; D000860:Hypoxia; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism*; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism*; D051379:Mice; D014176:Protein Biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1
C508961:Tgfb1 protein, mouse; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9