Effect of hypoxia on stimulatory effect of TGF-β1 on MMP-2 and MMP-9 activities in mouse fibroblasts

Ghassan M. Saed, Wendy Zhang, Michael Peter Diamond

Research output: Contribution to journalArticle

52 Citations (Scopus)

Abstract

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-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 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 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 activities, 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. Copyright (C) 2000 Society for Gynecologic Investigation.

Original languageEnglish (US)
Pages (from-to)348-354
Number of pages7
JournalJournal of the Society for Gynecologic Investigation
Volume7
Issue number6
DOIs
StatePublished - Dec 6 2000

Fingerprint

Matrix Metalloproteinase 2
Matrix Metalloproteinase 9
Transforming Growth Factors
Fibroblasts
Messenger RNA
Fibrosis
Hypoxia
Ethidium
Densitometry
Multiplex Polymerase Chain Reaction
Reverse Transcriptase Polymerase Chain Reaction
Sepharose
Actins
Gels
RNA
Staining and Labeling
Polymerase Chain Reaction

Keywords

  • Fibroblasts
  • Hypoxia
  • MMP-2
  • MMP-9
  • Multiplex RT-PCR
  • Zymography

ASJC Scopus subject areas

  • Obstetrics and Gynecology

Cite this

Effect of hypoxia on stimulatory effect of TGF-β1 on MMP-2 and MMP-9 activities in mouse fibroblasts. / Saed, Ghassan M.; Zhang, Wendy; Diamond, Michael Peter.

In: Journal of the Society for Gynecologic Investigation, Vol. 7, No. 6, 06.12.2000, p. 348-354.

Research output: Contribution to journalArticle

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abstract = "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-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 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 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 activities, 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. Copyright (C) 2000 Society for Gynecologic Investigation.",
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N2 - 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-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 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 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 activities, 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. Copyright (C) 2000 Society for Gynecologic Investigation.

AB - 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-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 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 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 activities, 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. Copyright (C) 2000 Society for Gynecologic Investigation.

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KW - Multiplex RT-PCR

KW - Zymography

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