Effect of Hypoxia on Stimulatory Effect of TGF-β1 on MMP-2 and MMP-9 Activities in Mouse Fibroblasts

OBjective: Because chronic low-grade hypoxia has been implicated in the pathogensis of fibrosis and postoperative adhesion formation, we hypothesized that hypoxia may modulate the effect of transforming growth factor-β1 (TGF-β1) 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-β1 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 desitometry. Multiplex reverse transcriptase-polymerase chain rection (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 β-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-β1 treatment resulted in 180% and 50% increases in MMP-2 and MMP-9 acitivites, respectively. Increases of 37.5% and 40% in MMP-2 and MMP-9 mRNA levels, respectively, were seen. However, under hypoxic conditions, TGF-β1 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.

Conclusion: Hypoxia suppresses the stimulatory effect of TGF-β1 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.