Saed G M, Zhang W, Diamond M P
Department of Obstetrics and Gynecology, Wayne State Medical School, Wayne State University, Michigan, Detroit 48201, USA.
J Soc Gynecol Investig. 2000 Nov-Dec;7(6):348-54.
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.
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.
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.
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.
由于慢性低度缺氧与纤维化及术后粘连形成的发病机制有关，我们推测缺氧可能在转录和翻译水平上调节转化生长因子-β1（TGF-β1）对基质金属蛋白酶（MMP）-2和MMP-9的作用。
将小鼠成纤维细胞置于有或无1 ng/mL TGF-β1的缺氧环境中不同时间。对每次处理后收集的细胞上清液进行酶谱分析。通过光密度法对与MMP-2和MMP-9对应的明胶酶解带进行定量。还对每次处理后从细胞中提取的总RNA进行MMP-2和MMP-9的多重逆转录聚合酶链反应（RT-PCR）。PCR扩增产物通过2%琼脂糖凝胶分析，随后对DNA条带进行溴化乙锭染色。使用扫描光密度法确定每条带的强度与β-肌动蛋白的强度比。
缺氧导致MMP-9活性降低64%，MMP-9 mRNA水平降低80%，但不影响MMP-2 mRNA水平或活性。TGF-β1处理分别导致MMP-2和MMP-9活性增加180%和50%。MMP-2和MMP-9 mRNA水平分别增加37.5%和40%。然而，在缺氧条件下，TGF-β1分别导致MMP-2和MMP-9活性增加160%和降低45%，MMP-2和MMP-9 mRNA水平分别增加37.5%和降低71%。
缺氧抑制TGF-β1对MMP-9活性的刺激作用，但不抑制MMP-2。这可能表明MMP-9在缺氧条件下在组织纤维化和术后粘连形成的发病机制中起重要作用。
