Unaltered Secretion of β-Amyloid Precursor Protein in Gelatinase A (Matrix Metalloproteinase 2)-deficient Mice

Abstract

The ␤-amyloid peptide, which forms extracellular cerebral deposits in Alzheimer's disease, is derived from a large membrane-spanning glycoprotein referred to as the ␤-amyloid precursor protein (APP). The APP is normally cleaved within the ␤-amyloid region by a putative proteinase (␣-secretase) to generate large soluble amino-terminal derivatives of APP, and this event prevents the ␤-amyloid peptide formation. It has been suggested that the gelatinase A (matrix metalloproteinase 2, a 72-kDa type IV collagenase) may act either as ␣-secretase or as ␤-secretase. Mice devoid of gelatinase A generated by gene targeting develop normally, except for a subtle delay in their growth, thus providing a useful system to examine the role of gelatinase A in the cleavage and secretion of APP in vivo. We show here that APP is cleaved within the ␤-amyloid region and secreted into the extracellular milieu of brain and cultured fibroblasts without gelatinase A activity. The data suggest that gelatinase A does not play an essential role in the generation and release of soluble derivatives of APP at physiological conditions. Amyloid precursor protein (APP) 1 is an integral membrane protein that is produced by most cells (1). Several isoforms ranging from 365 to 770 amino acids are generated by alternative splicing of transcripts from the APP gene on the long arm of chromosome 21 (2). Proteolytic cleavage of APP by enzymes termed ␣-, ␤-, and ␥-secretases generates various APP fragments that are released from APP expressing cells (3). ␤-Amyloid peptides (A␤) of 40 -43 amino acids are released by the action of ␤-and ␥-secretases cleaving at or near residues 671 and 713 (numbers refer to APP 770 ), respectively (3). Cleavage of APP at a membrane proximal site by an ␣-secretase releases larger APP fragments (sAPP) and prevents generation of A␤. The absolute and relative amounts of various APP fragments that are released in the brain are thought to be of importance in the formation of first amorphous (diffuse) and then filamentous (amyloid) plaques that are characteristic of Alzheimer's disease. Two larger forms sAPP also known as protease nexin II have a Kunitz type serine protease inhibitor domain (4, 5). In addition all forms of sAPP have in the carboxyl-terminal region a domain that inhibits gelatinase A (matrix metalloproteinase 2, a 72-kDa type IV collagenase) activity (6). On the other hand, gelatinase A has been suggested to act on APP either as ␣-secretase (6) or as ␤-secretase (7), although the hypothesis is controversial (8, 9). To clarify the putative role of this enzyme, we studied APP fragmentation and release in gelatinase A knockout mice. MATERIALS AND METHODSGeneration of Gelatinase A Gene-deficient Mice-A genomic DNA clone of mouse gelatinase A (Clg4a) was isolated from a 129/Sv genomic library. The fragments used for constructing the targeting vector were a 2.0-kb HindIII fragment of the distal region of the promoter and a 4.5-kb XbaI-SacI fragment. In the resulting targeting construct, 5.9 kb containing the exon 1 were r...

The Role of Matrix Metalloproteinase-2 and Matrix Metalloproteinase-9 in Antibody-Induced Arthritis

“…Embryonic fibroblasts from the wild-type mice were injected into the ankle joint on days 0, 3, and 6. These cells secrete MMP-2 in vitro (14). Recovery from the exacerbated arthritis in the MMP-2 KO mice occurred following the injection of wild-type fibroblasts (Fig.…”

“…Gelatin zymography was conducted as described previously (14). Briefly, whole mouse paws were removed proximal to the carpus or tarsus and homogenized in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 1% Nonidet P-40 and centrifuged at 15,000 rpm for 10 min.…”

“…MMP-2 ϩ/Ϫ mice (14) and MMP-9 ϩ/Ϫ mice (16) were crossed five times with BALB/c mice and were crossed with each other to obtain MMP-2 ϩ/Ϫ MMP-9 ϩ/Ϫ mice. We then crossed these mice with each other to obtain MMP-2 Ϫ/Ϫ MMP-9 ϩ/ϩ (MMP-2 KO), MMP-2 ϩ/ϩ MMP-9 Ϫ/Ϫ (MMP-9 KO), MMP-2 Ϫ/Ϫ MMP-9 Ϫ/Ϫ (double knockout), and control wild-type mice.…”

“…Thus, the role of these enzymes in RA is controversial. To more directly examine the major roles of MMP-2 and MMP-9 in RA, we have used MMP-2 KO mice (14,15) and MMP-9 KO mice (16). We investigated the development of Ab-induced arthritis (AbIA), one of the animal models of RA (17,18), in these KO mice.…”

The Collagen Binding Domain of Gelatinase A Modulates Degradation of Collagen IV by Gelatinase B

“…[7][8][9][10] Gene knockout studies have suggested that the gelatinases may cooperate in vivo. [11][12][13] In this respect, an example may be represented by the process of inflammation, wherein the degranulation of extravasated neutrophils allows MMP2 and MMP9 to share the same extracellular space, thus enhancing the probability to interact with the same substrates. 14 Neutrophil degranulation is associated with the ability of neutrophils to cross basement membranes, composed mainly of a type IV collagen network; 15,16 therefore, the enzymatic activities of MMP2 and MMP9 on type IV collagen are likely relevant for this process.…”