Stable expression of α1-antitrypsin Portland in MDA-MB-231 cells increased MT1-MMP and MMP-9 levels, but reduced tumour progression.

C L Evered,M A Cepeda,S Damjanovski,J A Willson,C A Muir

doi:10.1007/s12079-017-0407-5

Abstract

The membrane bound matrix metalloproteinase MT1-MMP plays roles in modulating cell movement, independent of its abilities to remodel the extracellular matrix. Unlike many MMPs, MT1-MMP is activated in the Golgi prior to secretion by a pro-protein convertase, primarily furin. Regulation of the activation of pro-MT1-MMP has been methodically investigated, as altering the level of the active protein has broad implications in both activating other pro-MMPs, including pro-MMP-2, and many subsequent remodelling events. Our previous work in MCF-7 cells has demonstrated that modest, and not extremely high, levels of active MT1-MMP manifests into altered cell morphology and movement. At this low but optimal amount of MT1-MMP protein, changes to MT1-MMP levels are always mirrored by MMP-9 and pERK levels, and always opposite to MMP-2 levels. In this study, stable expression of the furin inhibitor α1-antitrypsin Portland (α1-PDX) in MDA-MB-231 cells increased overall MT1-MMP levels, but cells maintained a 21% proportion of pro-MT1-MMP. The increase in MT1-MMP was mirrored by increases in MMP-9 and pERK, but a decrease in MMP-2. These changes were associated with increased NF-κB transcription. In vitro analysis showed that α1-PDX decreased cell protrusions and migration, and this manifested as decreased tumourigenesis when examined in vivo using a chick CAM assay.

Highlights

Matrix metalloproteinases (MMPs) are a family of zincdependent endopeptidases which function collectively to cleave all components of the extracellular matrix (ECM) (Bourboulia and Stetler-Stevenson 2010)
While the level of pro-MT1MMP did increase in 231-PDX cells (Fig. 2a and data not shown), densitometric quantification showed that the percentage of pro-MT1-MMP compared to total MT1-MMP protein levels did not differ between 231-PDX cells (21%), and MDA-MB-231 parental cells (21%) (Fig. 2b)
These results suggest that stable expression of α1-PDX in MDA-MB-231 cells increased MT1-MMP, both at the transcript and total protein level, while the percentage of proMT1-MMP protein remained at about 21% of total MT1MMP

Summary

Introduction

Matrix metalloproteinases (MMPs) are a family of zincdependent endopeptidases which function collectively to cleave all components of the extracellular matrix (ECM) (Bourboulia and Stetler-Stevenson 2010). The correct MMP family member must be present in its appropriate form (inactive, inhibited, or active) at the proper time and location (Egeblad and Werb 2002; Ellis and Crawford 2016; Nieuwesteeg et al 2012, Walsh et al 2012). Retaining proper MMP activity in tissues is achieved through regulation of MMP expression, pro-enzyme activation, and enzymatic activity through catalytic inhibition by their endogenous inhibitors, TIMPs and RECK (Visse and Nagase 2003). The pro-domain of MT1-MMP contains a furin cleavage motif, with trans-Golgi localized furin being a key component in its activation (Scamuffa et al 2006; Seidah et al 2008)

Methods

Results

Conclusion