AMP-activated Protein Kinase Suppresses Matrix Metalloproteinase-9 Expression in Mouse Embryonic Fibroblasts

Abstract

Purpose: : Matrix metalloproteinase-9 (MMP-9) plays a critical role in both developmental and pathological ocular tissue remodeling and angiogenesis. Although MMP-9 expression is low in most cells and is tightly controlled, the mechanism of its regulation is poorly understood. We utilized mouse embryonic fibroblasts (MEFs) that were nullizygous for the catalytic α-subunit of AMP-activated protein kinase (AMPK), which is a key regulator of energy homeostasis, to investigate the role of AMPK in the regulation of MMP-9 expression.

Methods: : AMPKα wild type (WT), AMPKα1 or α2 subunit single-knockout (AMPKα1-/- and AMPKα2-/-, respectively), and double knockout of AMPKα1 and AMPKα2 subunit (AMPKα-/-) mouse embryonic fibroblasts (MEFs) were cultured. MEFs were treated with AMPK activators and nuclear factor-ΚB (NF-ΚB) inhibitors, or transduced with adenoviral vector of dominant negative (DN) AMPKα. The level of MMP-9 expression was determined by zymography, ELISA, and quantitative real time PCR.

Results: : Total AMPKα deletion significantly elevated MMP-9 expression compared with WT MEFs (7.8 ± 0.9 times higher in the ELISA, P < 0.01), whereas single knockout of the isoforms AMPKα1 and AMPKα2 caused minimal change. The AMPK activators, 5-amino-4-imidazole carboxamide riboside and A769662, suppressed MMP-9 expression in WT MEFs dose-dependently (P < 0.01) and AMPK inhibition by the over-expression of DN AMPKα elevated MMP-9 expression. In AMPKα-/- MEFs transduced with DN AMPKα, MMP-9 expression was suppressed (P < 0.01). AMPKα-/- MEFs showed constitutive activation of NF-ΚB pathway compared with WT MEFs. Consistently, selective NF-ΚB inhibitors decreased MMP-9 expression in AMPKα-/- MEFs (P < 0.01).

Conclusions: : The energy sensor of the cell, AMPK, suppresses MMP-9 expression by inhibiting the NF-ΚB pathway. AMPK may be a therapeutic target to inhibit pathological tissue remodeling and angiogenesis in the eye.

Keywords: extracellular matrix • metabolism • wound healing