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

Abstract

Matrix metalloproteinase-9 (MMP-9) plays a critical role in tissue remodeling under both physiological and pathological conditions. 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 ␣ subunit of AMP-activated protein kinase (AMPK), which is a key regulator of energy homeostasis, to identify AMPK as a suppressor of MMP-9 expression. Total AMPK␣ deletion significantly elevated MMP-9 expression compared with wild-type (WT) MEFs, whereas single knock-out of the isoforms AMPK␣1 and AMPK␣2 caused minimal change in the level of MMP-9 expression. The suppressive role of AMPK on MMP-9 expression was mediated through both its activity and presence. Matrix metalloproteinase-9 (MMP-9, 2 gelatinase B) degrades denatured collagens and native collagen type IV, which is a major component of the extracellular matrix (ECM) and basement membranes (1). Under normal circumstances, the degradation of the ECM by MMP-9 is a tightly controlled process involved in physiological wound healing and embryo development (1, 2). Conversely, aberrant degradation of ECM by excess MMP-9 expression results in the pathologic destruction of connective tissue seen in cancer, arterial sclerosis, and rheumatoid arthritis (1, 3). Therefore, under physiological conditions, regulated MMP-9 expression is low (1), but the mechanisms behind this are obscure.AMP-activated protein kinase (AMPK) is a serine/threonine kinase, which regulates energy homeostasis and metabolic stress (4). AMPK acts as a sensor of cellular energy status and maintains the balance between ATP production and consumption. In mammals, AMPK exists as a heterotrimer with ␣, ␤, and ␥ subunits, each of which is encoded by two or three genes (␣1, ␣2, ␤1, ␤2, ␥1, ␥2, and ␥3). The ␣ subunit possesses catalytic activity, whereas the ␤ and ␥ subunits are regulatory and maintain the stability of the heterotrimer complex. The importance of AMPK␣ is illustrated by the fact that dual deficiency of AMPK␣1 and AMPK␣2 is embryonic lethal (5).Recent evidence suggests that AMPK has a much wider range of functions, including the regulation of cell growth, cell proliferation, cell polarity, and autophagy (6, 7). Because these functions are closely linked to the pathology of MMP-9-related diseases, including cancer, arterial sclerosis, and rheumatoid arthritis, we hypothesized that AMPK regulates MMP-9 expression. To address this, in the present study, we utilized AMPK␣-deficient mouse embryonic fibroblasts (MEFs) to investigate the effect of the genetic deletion and activation of AMPK on MMP-9 expression. EXPERIMENTAL PROCEDURESAntibodies, Recombinant Proteins, and Reagents-All antibodies, except for MMP-9 (Abcam, Cambridge, MA) and AMPK␣2 (Santa Cruz Biotechnology, Santa Cruz, CA), were purchased from Cell Signaling (Beverly, MA). Recombinant mouse TNF-␣, MMP-9, and MMP-2 proteins were obtained from R&D Systems (

Insulin sensitive and resistant obesity in humans: AMPK activity, oxidative stress, and depot-specific changes in gene expression in adipose tissue

“…Such a fi nding is puzzling because most cells types found in adipose tissue (i.e., adipocytes, macrophages, endothelial cells) predominantly express the AMPK ␣ 1 isoform (45)(46)(47). To the best of our knowledge, the only cell type in adipose tissue that expresses AMPK ␣ 2 is the fi broblast ( 48 ). Future studies involving the separation of adipocytes from various stromal fractions may help discern the source of the elevated AMPK ␣ 2.…”

AMP-dependent Kinase Inhibits Oxidative Stress-induced Caveolin-1 Phosphorylation and Endocytosis by Suppressing the Dissociation between c-Abl and Prdx1 Proteins in Endothelial Cells

“…AMPK␣1 and -␣2) that show differential tissue-specific expression (8,9,15). To determine the role of both isoforms in the inhibitory effect of AMPK on caveolin-1 phosphorylation under oxidative stress, we used RNA interference technology to knock down AMPK␣1 or -␣2 in HUVEC.…”

“…Protein Extraction and Western Blotting-Protein extraction and Western blotting were carried out as described previously (15). Densitometric analysis of bands was performed using ImageJ software.…”

“…We have recently demonstrated that activation of AMPK inhibits retinoblastoma cell proliferation, tumor growth, angiogenesis, ocular inflammation, and MMP-9 expression (11)(12)(13)(14)(15). Because these functions of AMPK are closely linked to the vascular hyper-permeability induced by oxidative stress, we hypothesized that AMPK has a protective role for vascular permeability.…”

Expression of adipocyte/macrophage fatty acid binding protein promotes tumor growth and metastasis.

“…Because AMPK has been shown to suppress various proteins involved in metastasis, including MMP-9 [167,169], it may also serve as an additional mechanism by which A-FABP deficiency suppresses tumor growth and metastasis in both lean and obese mice. Furthermore, both A-FABP deficiency and AMPK activation improves insulin sensitivity and reduces circulating levels of insulin, which may also contribute to tumor regression, particularly in diet-induced obese mice.…”