Changes in Tissue Inhibitors of Metalloproteinases (TIMPs) During Inhibition of MMP–2 and –9 by a Proteasome Inhibitor

Abstract

Purpose: : Matrix metalloproteinase (MMP) activities in human lens explants are increased by TGFß₂ treatment and decreased by the proteasome inhibitor, MG132, as reported previously (ARVO 2005). MMPs are regulated in vivo by TIMPs. The aim of this study was to assess activities and transcriptional levels of MMP–2 & –9 and TIMP–1, –2 & –3, after MG132 and TGFß₂ treatments.

Methods: : Capsular bags with intraocular lenses (IOLs) were cultured in serum free DMEM medium (14 days), treated with TGFß₂ (14–24 days), and then with MG132 (26–38 days). The medium was sampled and replaced every 2 days. HLE B–3 cells were treated with MG132 +/– TGFß₂. Samples were analyzed for MMPs & TIMPs by direct & reverse zymography, respectively. The HLE B–3 cells were harvested and RNA was isolated for RT–PCR of MMP–2 and TIMP–1 & –2.

Results: : TGFß₂ treatment transiently increased the pro– and active forms of MMP–2 & –9, while TIMP–1 & –3 continued to decrease. In contrast, TIMP–2 showed high activity, which decreased only slightly. MG132 completely eliminated MMP activities with little effect on TIMP–2. TIMP–1 and –3 increased transiently. TGFß₂ treatment of HLE B–3 cells increased both pro– and active MMP–2 & –9 and slightly decreased TIMP–1 & –3. MG132 treatment diminished MMP–2 & –9 activities. Concurrently, TIMP–1 & –3 activities increased. MG132 treatment of TGFß₂ treated cells resulted in diminished MMP activities, while TIMP–1 & –3 activities increased, then gradually returned to un–treated levels. Again, TIMP–2 activity was not affected by treatment. TGFß₂ treatment of HLE B–3 cells increased MMP–2 mRNA almost 4 fold, whereas MG132 treatment decreased MMP–2 mRNA. TIMP–2 mRNA showed a small decrease after TGFß2 treatment but increased almost 2.5–fold after MG132 treatment.

Conclusions: : In capsular bag/IOL and HLE B–3 cultures, TGFß₂ increased MMP activities, decreased TIMP–1 & –3, but did not change TIMP–2. MG132 eliminated MMP activities, transiently increased TIMP–1 and 3, with no effect on TIMP–2. In HLE B–3 cells, MG132 blocked the TGFß₂–induced increase in MMP activities and mRNA, and increased TIMP–1 and –3 activities and TIMP–2 mRNA. These effects of MG132 support its potential to block PCO.

Keywords: posterior capsular opacification (PCO) • proteolysis • enzymes/enzyme inhibitors