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(Received for publication, April 30, 1997, and in revised form, June 16, 1997)
From the ABSTRACT The INTRODUCTION 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 MATERIALS AND METHODS 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 replaced with the
pgk-neo gene cassette (10). A diphtheria toxin A fragment
gene cassette was used for negative selection of nonhomologous recombinants (11). The mouse embryonic stem (ES) cell line used was E14
(a gift of M. Hooper). Cell culture, targeting experiment, and
microinjection of ES clones into C57BL/6J blastocysts were carried out
as described previously (12). The mutation heterozygous mice were
obtained by crossing the chimeras to C57BL/6J mice. Heterozygotes were
backcrossed to C57BL/6J one to five times and then crossed to obtain
the mutation homozygous mice. The genotypes of ES clones and mice were
determined by Southern blot analysis as schematically presented in Fig.
1.
Gelatin zymography was carried out as
described (13). The tissues from the newborn mice were homogenized in
50 mM Tris-HCl (pH 7.5) and centrifuged at 15,000 rpm for 5 min. 10 µg of supernatant proteins was separated by nonreduced
SDS-polyacrylamide gel electrophoresis using 7.5% gels containing
0.1% gelatin.
Used are monoclonal antibody
22C11 (Anti-N), specific for the amino-terminal domain of APP (14)
(Boehringer Mannheim); rabbit polyclonal antibodies I-56/Affi-28
(Anti-A The mouse brain soluble and membrane
fractions were prepared as described (17). Whole brains were
homogenized in 50 mM Tris-HCl (pH 7.4) containing 0.5 M NaCl, 10 mM MgCl2, 2 mM EDTA, 1 µg/ml leupeptin, and 2 mM
phenylmethylsulfonyl fluoride and centrifuged at 100,000 × g for 30 min at 4 °C. The resulting supernatants were
used as soluble fractions. The remaining pellets were solubilized in 50 mM Tris-HCl (pH 7.4) with 4 M urea and 1% SDS
and used as membrane fractions. 30 µg of supernatant proteins or 50 µg of the membrane proteins per lane were separated by 7%
SDS-polyacrylamide gel electrophoresis and electroblotted onto
polyvinylidene difluoride membrane (Millipore) in a buffer containing
10 mM CAPS (pH 11)/10% methanol. After blocking the
membranes with 5% skim milk in Tris-buffered saline, the blots were
immersed with antibodies mentioned above and peroxidase-conjugated
anti-mouse or anti-rabbit antibodies and developed in ECL Western
blotting detection reagent (Amersham Corp.). Protein concentration was
determined by a protein assay (Bio-Rad).
Embryonic fibroblasts were prepared from embryos of 12.5 days
post-coitus. Upon confluence, cells were washed three times with
serum-free Dulbecco's modified Eagle's medium (DMEM) and then
incubated in serum-free DMEM with or without lipopolysaccaride (LPS)
over night. To detect sAPP, we concentrated the conditioned medium 200 times by Centricon-30 (Millipore), and 20 µg of protein was applied
to the Western blot analysis.
Embryonic fibroblasts (2.5-3 × 106/10-cm
diameter dish) were labeled for 20 min at 37 °C with
[35S]methionine in 2.5 ml of methionine-free and
serum-free DMEM and then chased with methionine-containing serum-free
DMEM for 5, 30, 60, and 120 min at 37 °C. The cells were then lysed
and processed for immunoprecipitation with anti-C antibody as described (16). sAPP in the supernatant was immunoprecipitated with anti-N antibody. Immunoprecipitated samples were separated with 7.5 or 15%
polyacrylamide gels. The radioactivities of specific bands were
quantitated by a BioImage Analyzer BAS2000 (Fuji, Japan).
RESULTS AND DISCUSSION To introduce a null mutation in the mouse gelatinase A gene
(Clg4a), we replaced its promoter and first exon with the
pgk-neo gene by gene targeting (Fig.
1a). Three of four homologous
recombinant ES clones transmitted the mutated allele to the germ line
(Fig. 1b). Gelatin zymography revealed a complete lack of
gelatinase A activity in the mutation homozygous mice (hereafter simply
referred to as mutant mice) (Fig. 1c). Although
gelatinolytic bands with molecular masses of 64 and 57 kDa (probably
the proenzyme and mature form of the gelatinase A, respectively) were
detected in the brain, lung, and kidney of wild-type mice, we detected
neither of these forms in tissues from the mutant mice. Reduced levels were found in tissues from mutation heterozygous mice, indicating that
the mutant allele is nonfunctional. There was no evidence for a
compensatory expression of gelatinase B and/or other gelatinolytic enzymes (Fig. 1c).
The mutant mice developed normally and were fertile. Intercrosses
between the heterozygotes backcrossed five times into C57BL/6 background gave rise to 151 wild-type, 318 heterozygous, and 143 homozygous mice, nearly proportional to the expected frequency of
1:2:1. Although gelatinase A is expressed ubiquitously and has been
thought to be important for the remodeling of tissue architecture
during development, the mutant mice did not show any gross anatomical
abnormalities. However, it is of interest that the mutant mice show
significantly slower growth rate (approximately 15% reduction) as
compared with that of wild-type litter mates (Fig.
2) from postnatal day 3 to adulthood,
suggesting a role of gelatinase A in development. The mutant mice seem
to be smaller even at birth. The reason for slower growth rate is not
known. Cultured fibroblasts from mutant embryos grew and migrated
normally on collagen- or gelatin-coated and uncoated dishes (data not
shown).
To determine whether sAPP is secreted from the mutant brain, we carried
out Western blotting of the brain-derived soluble and membrane
fractions using various anti-APP antibodies: Anti-N, Anti-A
To examine more directly the secretion of APP, we cultured fibroblasts
from mutant and wild-type embryos and analyzed the conditioned medium
for gelatinase A activity by zymography and for APPs by Western
blotting. Wild-type fibroblasts produced and secreted large amounts of
gelatinase A (Fig. 4a,
lane 1). As observed earlier with astrocytes (18) and
macrophages (19), LPS enhanced synthesis and secretion of gelatinase A
by fibroblasts in a dose-dependent manner (Fig.
4a, lanes 1-3). The activated form of gelatinase A was significantly increased in LPS-stimulated cultures. However, no
gelatinase A activity was detected in the medium from mutant fibroblasts (Fig. 4a, lanes 4-6). LPS treatment
also enhanced APP synthesis by fibroblasts from both wild-type and
mutant mice as shown by Western blotting of cell lysates with anti-C
antibody (Fig. 4b). Conditioned medium from wild-type cells
contained 120-130-kDa proteins that reacted with anti-N and anti-A
To analyze the kinetics of maturation and secretion of APP, we carried
out a pulse-chase experiment of cultured fibroblasts. As summarized in
Fig. 5, we observed insignificant
differences between wild-type and mutant cells in the rates of
maturation of APP, secretion of sAPP and generation of the 15-kDa
cytoplasmic tails (cAPP). Thus, we concluded that the gelatinase A
(matrix metalloproteinase 2) does not play a role in the metabolism of APP at physiological conditions. However, we cannot rule out the possibility that during development alternative enzymatic systems replace gelatinase A activity for APP processing, although no abnormal
gelatinolytic activities were detected. The analyses under the acutely
stressed conditions in vivo and in vitro may help
to elucidate the issue. Furthermore, this study does not exclude the
possibility that the enzyme hydrolyzes A
It has been suggested that gelatinase A plays an important role in the
metastasis of malignant tumors and in angiogenesis associated with
tumor growth (23, 24). The mutant mice provide a valuable tool to study
these putative functions of gelatinase A.
FOOTNOTES ACKNOWLEDGEMENTS We thank Drs M. Hooper and T. Kunishita for
the generous gift of E14 cells and I-56/Affi-28 antibodies,
respectively, and Drs. W. Haas and D. Gerber for critical
readings.
REFERENCES
Volume 272, Number 36,
Issue of September 5, 1997
pp. 22389-22392
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
COMMUNICATION:
-Amyloid Precursor Protein in
Gelatinase A (Matrix Metalloproteinase 2)-deficient Mice*
§,,,,
Institute for Virus Research, Kyoto
University, 53 Kawahara, Syogo-in, Sakyo-ku, Kyoto 606-01, Japan and
the ¶ Faculty of Pharmaceutical Sciences, University of Tokyo,
Hongo, Bunkyo-ku, Tokyo 113, Japan
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-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.
-, -, 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 APP770), 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.
Fig. 1.
a, schematic representations of the
targeting vector and wild-type and mutant mouse Clg4a loci.
The targeting vector was designed to replace the promoter and exon 1 of
the Clg4a gene by the pgk-neo gene cassette.
Diphtheria toxin A fragment gene cassette, indicated by the
shaded box, was used for negative selection of nonhomologous recombinants (11). The 5
and 3 probes used for the Southern blots in
b are indicated by thick bars. Sa,
SacI; Sp, SpeI. b, Southern
blot analysis of progeny from a cross of heterozygotes. Genomic DNA was
extracted from tail, digested, and probed as described in the figure
legends. Lanes 1 and 5 represent mutation
homozygotes. Lanes 2, 6, and 7 represent wild-type mice. Others represent heterozygotes. The positions
of the wild-type (wt) and the mutant (m)
fragments are indicated by arrows. c, gelatin
zymography of the proteins of the gelatinase A mutant mice. +/
,
heterozygote; +/+, wild type; /, homozygote. The pro- and mature
gelatinase A (GelA) and gelatinase B (GelB) are
indicated by arrows.
[View Larger Version of this Image (46K GIF file)]
), specific for residues 1-28 of A (15), a gift from Dr.
T. Kunishita; and G-369 (Anti-C), specific to the cytoplasmic domain of
APP (16). These antibodies have been shown not to cross-react with
amyloid precursor-like proteins under the conditions used.
Fig. 2.
Postnatal growth kinetics of wild-type and
mutant mice. 
, wild type (female, n = 8; male,
n = 9). 
, mutant (female, n = 8;
male, n = 9). Mutant mice were significantly smaller
than wild-type mice (p < 0.01 for female and male;
Two-way Repeated-Measures ANOVA).
[View Larger Version of this Image (20K GIF file)]
, and
Anti-C (Fig. 3a). These
antibodies detected 110-125-kDa proteins in the membrane fraction from
the wild-type and mutant mice (Fig. 3, b, c, and
d, lanes 3 and 4). Anti-N and
anti-A antibodies detected 105-110-kDa proteins in the soluble
fractions from the brains of wild-type mice (Fig. 3, b and
c, lane 1). These proteins did not bind anti-C
antibodies (Fig. 3d, lane 1), indicating that
they were carboxyl-terminal truncated forms of APP, presumably sAPP.
The sAPP was also detected in the soluble fraction from mutant mice at
a level similar to wild-type mice (Fig. 3b, lane 2, 97.7 ± 9.1%, average ± S.E. (mutants:
n = 5) of wild types (n = 5); Fig.
3c, lane 2, 99 ± 10% (mutants:
n = 3) of wild types (n = 3)). The
relative intensities of bands obtained with anti-N and anti-A
antibodies were the same for the soluble fractions from wild-type and
mutant mice, suggesting that there is no shift in the choice of -
and -sites in the mutant mice. These results suggest that APP is
normally secreted in the brain of the mutant mice.
Fig. 3.
Synthesis and secretion of APP in the brains
of mutant mice. Schematic shows APP with -, -, and
-secretase sites (a). The region of A is indicated by
the hatched box. The regions recognized by the three
antibodies (see "Materials and Methods") are indicated by
thick bars at the bottom of the figure. Western blot analyses of the brain extracts of the mutant mice used anti-N (22C11) (b), anti-A (I-56/Affi-28) (c), and
anti-C (G-369) (d) antibodies. Lanes 1 and
2 represent brain soluble fractions, and lanes 3 and 4 represent brain membrane fractions. Lanes 1 and 3, wild-type mice; lanes 2 and 4,
mutant mice.
[View Larger Version of this Image (33K GIF file)]
antibodies (Fig. 4, c and d, lanes
1-3) but not anti-C antibodies (Fig. 4e). These results indicated that these APP molecules were processed and secreted
rather than released from dying cells. sAPP secretion was increased by
LPS stimulation (Fig. 4, d and e, lanes
1-3). sAPPs from fibroblasts have slightly larger molecular mass
than those detected in the brain. This difference reflects the isoform distribution in different tissues (20). Western blotting revealed sAPP
in the conditioned medium from the mutant cells (Fig. 4, c,
d, and e, lanes 4-6) as well as from
wild-type cells. The intensity of lane 6 in Fig.
4c was slightly weaker than that of lane 3. However, the difference was inconsistently observed in three
independent experiments. In sum, the level of cumulative sAPP from
mutant cell cultures seemed to be nearly equivalent to that from wild types.
Fig. 4.
Secretion of sAPP and gelatinase A from
cultured embryonic fibroblasts stimulated with and without LPS.
Gelatinase A in the serum-free conditioned medium demonstrated by
gelatin zymography (a). Western blot analysis of APP in the
cell lysates using anti-C (G-369) antibodies (b). Western
blot analyses of sAPP in the conditioned medium (CM) using
anti-N (c), anti-A (d), and anti-C
(e) antibodies, respectively. Lanes 1 and
4, without LPS; lanes 2 and 5, with 1 µg/ml of LPS; lanes 3 and 6, with 10 µg/ml of
LPS. Lanes 1-3, wild-type cells; lanes 4-6,
mutant cells..
[View Larger Version of this Image (40K GIF file)]
in vivo (21,
22). This point may be addressed by crossing the mutant mice with A
high producer transgenic lines. In any case, the mutant mice might help
to elucidate further the relationships between gelatinase A and
APP.
Fig. 5.
Time course of maturation and secretion of
APP in cultured fibroblasts. The fibroblasts from wild-type and
mutant embryos were metabolically labeled with [35S]
methionine for 20 min, and then chased for times indicated in the
figure with serum-free DMEM containing methionine. Immature, mature,
and truncated cytoplasmic fragments of APP were immunoprecipitated with
anti-C (G-369) antibody from cell lysates. sAPP was immunoprecipitated with anti-N (22C11) antibody from culture supernatants. Radioactivity for each given time point is represented as a ratio of the highest activity of each product. Each point represents an average of duplicate
experiments. The variance between the duplicates was less than 15%.
, immature +/+; 
, immature /; 
, mature +/+; 
, mature
/; 
, sAPP +/+; 
, sAPP /; 
, cAPP +/+; 
, cAPP /.
[View Larger Version of this Image (28K GIF file)]
§
Present address: Shionogi Inst. for Medical Science, Shionogi & Co., Ltd. 12-4, 5, Sagisu, Fukushima-ku, Osaka 553, Japan.
To whom correspondence should be addressed. Tel.:
81-75-751-3990; Fax: 81-75-761-5626; E-mail:
sitohara{at}virus.kyoto-u.ac.jp.
1
The abbreviations used are: APP, amyloid
precursor protein; A, -amyloid protein; sAPP, soluble
amino-terminal derivatives of APP; ES, embryonic stem cell; CAPS,
cyclohexylaminopropanesulfonic acid; DMEM, Dulbecco's modified
Eagle's medium; LPS, lipopolysaccaride; kb, kilobase pair(s); cAPP,
15-kDa cytoplasmic tails of APP.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
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H. Matsusaka, M. Ikeuchi, S. Matsushima, T. Ide, T. Kubota, A. M. Feldman, A. Takeshita, K. Sunagawa, and H. Tsutsui Selective disruption of MMP-2 gene exacerbates myocardial inflammation and dysfunction in mice with cytokine-induced cardiomyopathy Am J Physiol Heart Circ Physiol, November 1, 2005; 289(5): H1858 - H1864. [Abstract] [Full Text] [PDF]
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R. E. Nisato, G. Hosseini, C. Sirrenberg, G. S. Butler, T. Crabbe, A. J.P. Docherty, M. Wiesner, G. Murphy, C. M. Overall, S. L. Goodman, et al. Dissecting the Role of Matrix Metalloproteinases (MMP) and Integrin {alpha}v{beta}3 in Angiogenesis In vitro: Absence of Hemopexin C Domain Bioactivity, but Membrane-Type 1-MMP and {alpha}v{beta}3 Are Critical Cancer Res., October 15, 2005; 65(20): 9377 - 9387. [Abstract] [Full Text] [PDF]
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J. Xu, P. W. Park, F. Kheradmand, and D. B. Corry Endogenous Attenuation of Allergic Lung Inflammation by Syndecan-1 J. Immunol., May 1, 2005; 174(9): 5758 - 5765. [Abstract] [Full Text] [PDF]
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 K. Lehti, E. Allen, H. Birkedal-Hansen, K. Holmbeck, Y. Miyake, T.-H. Chun, and S. J. Weiss An MT1-MMP-PDGF receptor-{beta} axis regulates mural cell investment of the microvasculature Genes & Dev., April 15, 2005; 19(8): 979 - 991. [Abstract] [Full Text] [PDF]
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