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From the Institute of Pathology*
and Institute of
Experimental Internal Medicine,
Otto-von-Guericke-University of Magdeburg, Magdeburg, Germany; the
Biotechnology Center of Oslo,
University of
Oslo, Oslo, Norway; and the Department of Infectious and Tropical
Diseases,
Immunology Unit, London School of
Hygiene and Tropical Medicine, London, England
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Introduction |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant human MMP-1, -2, and -3, and fluorogenic MMP substrate (DNP-Pro-Leu-Gly-Trp-Ala-D-Arg-NH2) were purchased from Calbiochem-Novabiochem GmbH (Bad Soden, Germany). Human THP-1 cells were purchased from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany), and the RPMI cell culture medium was obtained from PAA (Martinsried, Germany). Fetal calf serum, dextran sulfate (DS), lipopolysaccharide (LPS), o-phenanthroline, the protease inhibitor set, p-aminophenylmercuric acetate, and NaN3 were all from Sigma (Deisenhofen, Germany). The cathepsin/subtilisin inhibitor and pepstatin A were purchased from Calbiochem-Novabiochem, and E64 was from Bachem (Heidelberg, Germany). Monoclonal antibodies against MMP-1 (clone 41-1E5, corresponding to residues 332 to 350 of human MMP-1), MMP-2 (clone 42-5D11, corresponding to residues 524 to 539 of human MMP-2), MMP-3 (clone 55-2A4 or clone B) were purchased from Oncogene (Cambridge, England) and amyloid A (clone mc1) was obtained from DAKO (Hamburg, Germany). The secondary biotinylated rabbit anti-mouse antibody, streptavidin conjugated with alkaline phosphatase, and the chromogen 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium were purchased from BioTrend Chemikalien GmbH (Cologne, Germany).
Methods
Preparation of Serum Amyloid A
Plasma was obtained by plasmapheresis and measured for SAA by enzyme-linked immunosorbent assay (ELISA).13 Plasma with a minimum SAA concentration of 100 µg/ml was used for SAA purification using hydrophobic interaction chromatography, gel filtration, and ion-exchange chromatography as described,13 with the modification that 10 mmol/L ethylenediaminetetraacetic acid (EDTA) was added to plasma before it was run through the hydrophobic interaction column. SAA purity was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 4% stacking gel and 16% (acrylamide:bisacrylamide 19:1 v/v; Severn Biotech, Ltd., Worcestershire, Great Britain) resolving gel.
Preparation of Amyloid Fibril Proteins
Amyloid fibril proteins were prepared as described by Skinner and
colleagues.14
Briefly, this was as follows: 
6 g of
amyloidotic tissue from a spleen (10% of the splenic tissue was
replaced by amyloid), which had been stored at -80°C, was
homogenized in 60 ml of aqueous 0.9% NaCl solution for 30 seconds
using an Ultra-Turrax T8 (IKA-Labortechnik, Staufen, Germany). The
homogenate was centrifuged at 10,000 rpm for 30 minutes at 4°C. The
supernatant was discarded and the pellet was homogenized in 60 ml of
0.9% NaCl for 30 seconds, centrifuged, resuspended for 15 seconds in
60 ml sodium citrate buffer (0.05 mol/L sodium citrate, 0.01 mol/L
Tris, pH 8.0), and centrifuged again. Subsequently the supernatant was
discarded and the pellet was resuspended in 60 ml of 0.9% NaCl
solution for 15 seconds and centrifuged. The last washing step was
repeated until the optical density (OD 280) of the supernatant was
below 0.1. Thereafter the pellet was resuspended in 60 ml of deionized
water and centrifuged for 200 minutes at 10,000 rpm (4°C); this step
was repeated three times. The final pellet had a whitish top layer and
contained AA fibril proteins (AFPs) as demonstrated by SDS-PAGE,
Western blotting, amino acid sequencing, and mass spectrometry. This
was stored at -20°C until further use.
Degradation Experiments with Recombinant MMP-1, -2, and -3
Before the degradation experiment, MMP-1 was activated with 1 mmol/L p-aminophenylmercuric acetate for 60 minutes at 37°C. MMP-2 and MMP-3 did not necessitate activation before the degradation experiments. Degradation experiments with recombinant MMPs were performed as follows: samples from the pellet containing AFP were dissolved in water by heating for 30 minutes at 100°C before the supernatant was sterile filtrated (Millex HV, 0.45 µm; Millipore, Eschborn, Germany) and concentrated to 6 µg/µl (whole protein). Ten µl of the concentrated sample were mixed with 5 µl of protease and 20 µl of reaction buffer (containing 20 mmol/L Tris, 150 mmol/L NaCl, 5 mmol/L CaCl2, and 1 mmol/L ZnCl2, pH 7.6), and then made up to a volume of 40 µl with deionized water. The MMPs were used in a concentration of 0.15 µmol/L or 0.30 µmol/L and the degradation reaction was performed at 37°C for 18 hours before being stopped by the addition of EDTA (2.86 mmol/L). Incubation in the absence of MMPs or in the presence of 2.86 mmol/L EDTA served as controls. For the degradation experiments with SAA, SAA was dissolved in 4 mol/L of urea and subsequently dialyzed against deionized water. SAA was diluted with reaction buffer to a final concentration of 20 µmol/L. For mass spectrometry, aliquots were removed after 0 minutes, 90 minutes, and 18 hours. All degradation experiments were performed in triplicate. The activity of MMPs (30 µmol/L/200 µl of either MMP-1, MMP-2, or MMP-3) was tested by fluorospectroscopy (excitation 280 nm, emission 360 nm; Spectramax Gemini dual-scanning microplate spectrofluorometer; Molecular Devices Corp., Sunnyvale, CA) using a 80 µmol/L of DNP-PLGWAR (200 µl) fluorogenic substrate. All MMPs were active and degraded the substrate.
Degradation Experiments with THP-1 Cells
Four days before the degradation experiments, a THP-1 cell suspension was seeded in a 5-ml culture chamber (Falcon, Becton-Dickinson, Heidelberg, Germany) with an average cell density of 80,000 cells/ml of RPMI medium containing 10% fetal calf serum. Three days later, the cells were transferred into serum-free medium and stimulated with 10 µmol/L DS or 5 µg/ml LPS for 24 hours. On the day of the degradation experiments, the cell medium was changed and AFP (final concentration, 150 µg/ml) was added. After 6, 24, and 48 hours, 500-µl samples were removed from the THP-1 cell suspension and homogenized by sonication in the presence of 2 mg/ml of a protease inhibitor set, before centrifugation at 14,000 rpm for 10 minutes at room temperature. The resulting supernatant was stored at -20°C until further use (the pellet did not contain any AA immunoreactive material as tested by Western blotting and ELISA). Incubations without prestimulation and in the presence of o-phenanthroline (10 mmol/L), pepstatin A, E64, or cathepsin/subtilisin inhibitor served as controls. Incubation without AFP served as further negative control. All experiments were performed in triplicate.
SDS-PAGE and Western Blotting
Proteins were resolved in 16.5% polyacrylamide gels according to Schägger and Jagow15 and visualized by staining with Coomassie blue. For Western blotting, proteins on unstained polyacrylamide gels were transferred onto a polyvinylidene difluoride membrane [Immobilon-PSQ (polyvinylidene difluoride), pore size 0.1 µm; Millipore, Bedford, MA] using the tank-blotting system from Bio-Rad Laboratories (München, Germany) according to the manufacturer’s instructions. Transferred proteins were visualized by Coomassie blue staining. Immunostaining of the proteins was performed using anti-amyloid A antibodies (dilution 1:200). Free binding sites of the membrane were blocked overnight at room temperature with phosphate-buffered saline (PBS) containing 3% bovine serum albumin and 0.05% Tween-20. Incubation with the primary antibody was performed at 37°C for 90 minutes. After extensive washing in Tris-buffered saline supplemented with 0.05% Tween-20, the secondary biotinylated rabbit anti-mouse antibody was administered for 30 minutes at 37°C (dilution, 1:3000). Immunostaining was visualized with 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium.
Solid Phase Binding Assay
An ELISA was used to determine the concentration of AFP during degradation experiments with THP-1 cells. The assay protocol followed that used for antibody capture assays.16 The wells of polystyrene microtiter plates (96 well; Costar, Cambridge, MA) were each coated with 50 µl of the supernatant from the homogenized cell suspensions. After 2 hours of incubation at room temperature, the plates were washed three times with PBS (10 minutes each time), then incubated with 3% bovine serum albumin in PBS containing 0.1% Tween-20 and 0.2% NaN3 for 2 hours (room temperature) to block residual hydrophobic surfaces. Incubation with anti-amyloid A antibody (dilution 1:400) was performed overnight at 4°C. Plates were washed thoroughly with PBS, incubated with alkaline phosphatase-conjugated secondary antibody (dilution 1:5000, 2 hours at room temperature, rabbit anti-mouse immunoglobulin G; Bio-Rad), and than washed again in the same way. Bound immunoglobulin G was detected by the EIA Kit (Bio-Rad) according to the manufacturer’s instructions. Absorbance was measured at 405 nm using an ELISA plate reader and the amount of bound ligand was determined by subtracting the optical density of the blank wells, in which the ligand was omitted. The data were analyzed using the graphic program Sigma Plot (Jandel Scientific, Chicago, IL). For direct quantitation, AFP standards (1 to 8 µg/ml, whole protein content), which had been isolated from human spleen, were precoated onto wells of the same plates as the test solutions to generate a standard curve. Duplicate assays were performed throughout.
Amino Acid Sequencing
For amino acid sequencing the polyvinylidene difluoride-electroblotted bands were cut out and placed into a 477A protein sequencer connected to a 120A PTH amino acid analyzer (Applied Biosystems, Foster City, CA). The samples were analyzed for 7 to 11 cycles.
Mass Spectrometry
Cleavage sites were determined using a matrix-assisted laser-desorption/ionization time-of-flight mass spectrometer (Reflex III; Bruker Daltonics, Germany). Human SAA or AFP were incubated with either MMP-1, MMP-2, or MMP-3 as described above and 0.5-µl aliquots were removed at the time points indicated. The samples were subsequently co-crystallized with 0.5 µl of 3,5-dimethoxy-4-hydroxycinnamic acid (20 mg/ml) in 70% acetonitrile on a SCOUT 384 matrix-assisted laser-desorption/ionization target. The mass spectrometry was performed on an matrix-assisted laser-desorption/ionization time-of-flight mass spectrometer in linear mode with internal calibration. The BioTools 1.0 software (Bruker Daltonics) was used for the annotation of the SAA fragments; the accepted mass tolerance was 100 ppm.
Immunocytochemistry
Immunocytochemistry was performed using cell pellets obtained by cytocentrifugation onto precoated glass slides (5 minutes, 1500 rpm) (Cytospin 3; Shandon GmbH, Frankfurt/Main, Germany). The morphology and structural integrity of the cells was investigated using May-Grünwald-Giemsa stain. MMPs were detected with monoclonal antibodies directed against MMP-1 (dilution 1:100), MMP-2 (1:20), and MMP-3 (1:20). AFPs were localized with anti-amyloid A antibody (1:600). THP-1 cells were identified as macrophages using an antibody directed against CD68 (monoclonal, dilution 1:40; DAKO, Glostrup, Denmark). Before immunostaining the slides were pretreated with 10 mmol/L EDTA (twice for 10 minutes in a 450 W microwave oven). Immunoreactions were visualized with the avidin-biotin-peroxidase complex method applying a Vectastain avidin-biotin complex-alkaline phosphatase kit (distributed by Camon, Wiesbaden, Germany). Neufuchsin or 3,3'-diaminobenzidine tetrahydrochloride served as chromogens. The cells were counterstained with hematoxylin.
Electron Microscopy
For electron microscopy cells were fixed in a mixture of 2% formalin/2.5% glutaraldehyde (pH 7.2, overnight at 4°C) and then in 3.125% glutaraldehyde (7 hours at 4°C). After standard procedures of tissue processing for electron microscopy, the cells were finally embedded in Lowicryl using the K4M kit (Plano, Wetzlar, Germany). Samples for postembedding immunoelectron microscopy were not postfixed with OsO4. Polymerization took place throughout 24 hours at -30°C and was initiated by UV light. Semithin sections (1 µm) were stained with toluidine blue. Ultrathin sections (80 to 120 nm) were mounted on copper grids and counterstained with 3% aqueous uranyl acetate (30 minutes at room temperature) and contrasted with 1% aqueous lead citrate (15 minutes at room temperature).
For postembedding immunoelectron microscopy, ultrathin sections (120 nm) were mounted on formvar-coated nickel grids (200 mesh; Plano). Immunoelectron microscopy was performed in triplicate as described previously.17 The specificity of immunostaining was controlled by omitting the primary antibody. The sections were air-dried and examined using a Zeiss EM900 electron microscope.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Degradation of SAA by MMP-1, -2, and -3
The first set of experiments was performed to determine whether
the precursor protein of AA amyloidosis is susceptible to proteolytic
cleavage by MMPs. Purified, delipidated human acute-phase SAA (20
µmol/L) was incubated with 0.30 µmol/L of either recombinant MMP-1,
-2, or -3 at 37°C overnight. SDS-PAGE, Western blotting, and mass
spectrometry were applied to identify proteolytic fragments and
cleavage sites. After SDS-PAGE bands of 4 to 5 kd were detected
(Figure 1A)
. Fragments and cleavage sites
were identified using mass spectrometry. Three fragments were annotated
after degradation with MMP-1: fragment 1 to 57, 58 to 104, and a
fragment of 2726.5 Da corresponding to either 7 to 29, 8 to 30, or 9 to
31 (Figure 2)
. The latter fragment could
not be further specified. The fragment spanning 58 to 104 most likely
correlates with the band identified by Coomassie blue staining (Figure 1A)
. The amount of the N-terminal fragment spanning 1 to 57 (Figure 2)
was probably too low to be detected either by Coomassie blue or silver
staining (Figure 1A)
. Three fragments were annotated after degradation
with MMP-2: 1 to 51, 52 to 104, and 8 to 55. Four fragments were
annotated after degradation with MMP-3: 8 to 55, 57 to 104, 58 to 104,
and 74 to 104 (Figure 2)
. The fragments generated by MMP-2 or -3
probably are not separated by SDS-PAGE or the amount was too low to be
detected either by Coomassie blue or silver staining (Figure 1A)
. After
degradation with MMP-1, MMP-2, or MMP-3 the amount of SAA was reduced
accordingly. However, SAA was still detectable after 18 hours of
incubation (Figures 1 and 2)
.
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In almost all patients with AA amyloidosis the fibril protein is
an N-terminal cleavage product of the precursor protein that is
generated by proteolysis.5,18
Thus, the isolated fibril
protein differs from the precursor protein in that it lacks between 18
and 60 amino acid residues at the C-terminus and, therefore, precursor
and fibril proteins may not be accessible to the same proteases. With
the second set of experiments we tested whether recombinant MMP-1, -2,
and -3 are able to cleave AFP. AFP was prepared from the spleen of a
patient who had suffered from generalized AA amyloidosis. After
SDS-PAGE and immunoblotting, three AA-immunoreactive bands of 6, 7,
and 9 kd were detected (Figure 1, B and C)
. Amino acid sequencing for
seven and eight cycles showed that all three bands were homologous with
the N-terminus of serum amyloid A (SAA) starting with serine in
position 2. Using mass spectrometry the following eight N-terminally
intact fragments were annotated: 2 to 21, 2 to 64, 2 to 65, 2 to 66, 2
to 67, 2 to 68, 2 to 76, and 2 to 86 (Figure 3)
. The fragment spanning residues 2 to
86 corresponds to the 9-kd band (Figure 1B)
, 2 to 76 corresponds to the
7-kd band, and 2 to 64, 2 to 65, 2 to 66, 2 to 67, and 2 to 68 probably
are not separated by SDS-PAGE and correspond to the 6-kd band (Figure 1B)
. It was interesting to note that the purified AFP contained a
fragment corresponding to residues 2 to 21, which was probably too
small to be retained in the gel.
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. The same banding pattern was
generated using either 0.15 µmol/L or 0.30 µmol/L samples of MMPs
(data not shown). Whereas MMP-1 and MMP-3 generated fragments of
approximately the same size, a slightly smaller polypeptide was found
after proteolysis with MMP-2. In each case the degradation was
incomplete and after overnight incubation fibril proteins were still
detectable by immunoblotting (Figure 1C)
. However, the amount of
detectable AFP was reduced accordingly. No degradation was observed in
the absence of MMPs or in the presence of an inhibitor (EDTA). The
fragments generated were immunoreactive for AA amyloid (Figure 1C)
.
Amino acid sequencing for seven and eight cycles showed that all
additional bands occurring after proteolysis revealed a sequence of SAA
starting with serine in position 2. Cleavage sites were identified
using mass spectrometry. MMP-1 generated a fragment spanning position 2
to 57 (Figure 3)
. MMP-2 generated a fragment spanning 2 to 51 (Figure 3)
. Five fragments were found after degradation with MMP-3: 2 to 16, 2
to 23, 2 to 51, 2 to 56, and 2 to 57 (Figure 3)
. After degradation with
all three MMPs, the amount of the seven largest fibril proteins was
reduced accordingly. However, degradation was not complete and all
fibril proteins were detectable after 18 hours of incubation
(Figure 3)
. None of the MMPs tested here cleaved the peptide spanning 2
to 21 (Figure 3)
. Degradation of AFPs by THP-1
In vivo an individual protease is embedded in a complex
network of activating and inactivating factors, such as specific
compartments, pH, salt concentrations, presence of metal ions, other
proteases, and protease inhibitors. The activity of a particular
protease depends on the specific constellation of these activating and
inactivating factors as well as accessibility of the substrate.
Therefore, a third set of experiments was performed, using cell culture
to test whether metalloproteinases, both in general and in a more
complex system, may participate in the degradation of AFP. A
monocyte/macrophage cell line (THP-1) was chosen because macrophages
are commonly found in many different amyloid diseases, including AA
amyloidosis, and it has been proposed that they are involved in
amyloidogenesis. They synthesize a broad range of proteases that may
process the precursor protein to generate the fibril protein, and they
may also be involved in the degradation of the
deposits.19-22
Before the degradation experiments, MMPs
were up-regulated by stimulating THP-1 cells with either 10 µmol/L of
DS or 5 µg/ml of LPS. Unstimulated cells served as controls. An
increased amount of MMP-1, -2, and -3 protein was detected
immunocytochemically in DS-stimulated cells (Figure 4)
. After 24 hours of stimulation, AFP
(150 µg/ml) was added to the culture medium and its subsequent
degradation was assessed by ELISA using an antibody directed against
amyloid A.
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24, 39, and 55% after 6, 24, and 48 hours,
respectively. After similar time intervals, LPS-stimulated THP-1 cells
degraded AFP by 36, 43, and 64%, and DS-stimulated cells degraded AFP
by 16, 50, and 74%. These differences were not statistically
significant (Figure 5)
. However, after
the addition of o-phenanthroline, a general MMP inhibitor,
the degradation of AFP was significantly delayed in DS-stimulated THP-1
cells as compared to unstimulated cells (P =
0.043) and DS-stimulated cells without the inhibitor
(P = 0.006) (Figure 5)
. All other protease
inhibitors tested, ie, pepstatin A, E64, and a cathepsin/subtilisin
inhibitor (alone or in combination), had no effect on the degradation
(data not shown).
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and
immunoelectron microscopy (Figure 6B)
were applied to identify the
compartment where degradation took place. Immunostaining for AA amyloid
was confined to THP-1 cells at 6, 24, and 48 hours and localized at the
cell surface or cell membrane (Figure 6A
, arrows). In addition, patchy
immunoreactive deposits were identified that were located in close
proximity to or enclosed by THP-1 cells (Figure 6A
, asterisk). However,
immunostaining gradually diminished throughout time. No immunostaining
was found after incubation in the absence of AFP (Figure 6B)
.
Immunoelectron microscopy with gold-labeled secondary antibodies helped
to identify the patchy immunoreactive deposits as AA immunoreactive
fibrillar material (Figure 6C)
. Occasionally gold particles were found
at the cell surface, but no immunolabeling was found intracellularly.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) are potent
inducers of some MMPs including MMP-1, -3, and -9, and they may
increase transcription by as much as 100-fold.27-29
The
production and secretion of MMP-2 and -3 can be stimulated by the
acute-phase protein SAA.27
Thus, AA amyloidosis occurs
under conditions that are known to generate significantly increased
serum and tissue levels of MMPs, as compared with healthy control
individuals, and it is tempting to speculate that these MMPs may
influence the pathogenesis of AA amyloidosis.
Previously we have shown that MMP-1, -2, and -3 are present within AA
deposits,12
and we hypothesize that MMPs may be involved
in the processing of the precursor (SAA) or fibril proteins (AFP). In
the present study we believe that we are the first to show that human
SAA and AFP are indeed susceptible to proteolytic cleavage by MMPs.
Both, SAA and AFPs were degraded by MMP-1, -2, and -3 generating
fragments of different sizes. Proteolysis was neither uniform nor
complete, and although each protease had its own pattern of cleavage
sites some were generated by more than one of the MMPs (Figures 2 and 3)
. For instance redundancy was observed for MMP-1 and MMP-3 both
cleaving between position 57 and 58, and for MMP-2 and MMP-3 both
cleaving between residues 7 and 8, 51 and 52 as well as between 55 and
56. The cleavage sites for SAA and AFP also showed similarities:
between residue 57 and 58 for MMP-1, between 51 and 52 for MMP-2, and
between 56 and 57, and 57 and 58 for MMP-3. All MMPs share the
propensity to cleave SAA and AFP within the region spanning residues 51
to 57. Similar results have been reported by Mitchell and
colleagues30
who proposed that MMP-1 and -3 are able to
degrade rabbit acute-phase SAA3 within the region spanning residues 50
to 57.
However, despite similarities degradation of SAA and AFP also showed differences. MMP-2 and MMP-3 generated fragments from SAA, which we were unable to find after degradation of AFP, whereas MMP-3 generated fragments from AFP, which we could not detect after degradation of SAA. These differences may be related to differences in the secondary or tertiary structure. AFP lacked the Arg1 and the cleavage within the N-terminal half of the protein such as between residues 7 and 8, 15 and 16, and 23 and 24 may require an intact N-terminus. However, future investigations will have to clarify this issue.
The evaluation of the degradation results by SDS-PAGE, immunoblotting,
and mass spectrometry was not strictly comparable, because the size of
the peaks obtained by mass spectrometry do not necessarily correlate
directly with the amount of protein or peptide present on the target
(see Figures 1, 2, and 3
).
The results of our study show that 1) SAA and AFP seem to have
overlapping but probably not completely identical substrate
characteristics; 2) whereas all three MMPs have partly similar but not
completely identical proteolytic profiles; and 3) MMP-1, -2, and -3 do
not lead to complete degradation of SAA or AFP. The latter finding is
supported by our third set of experiments; cell culture experiments
revealed that degradation of AFP by DS-stimulated THP-1 cells was
delayed in the presence of a general metalloproteinase inhibitor.
Degradation was not entirely blocked by o-phenanthroline,
which indicated that other proteases, besides metalloproteinases, are
involved in the degradation process. However, as yet, we are unable to
identify any additional specific protease group by the use of protease
inhibitors such as pepstatin A (aspartate proteases such as cathepsin
D), E64 (cysteine proteases such as cathepsin B and K), and
cathepsin/subtilisin inhibitor (cysteine and serine proteases). Based
on this observation one may speculate that different proteases are able
to degrade AFP sufficiently. We have already identified four proteases
[ie, MMP-1, -2, -3 (present investigation) and cathepsin
K31
] that are able to degrade AFP. However, if redundancy
is present for the degradation of SAA and AFP, then why does AA occur
or persist? It is possible that the degrading system becomes imbalanced
during the course of AA deposition or that the presence of protease
inhibitors such as 2-macroglobulin, which have been detected in AA
deposits,12
may prevent sufficient degradation. To the
best of our knowledge, AFP ending at position 51, 56, 57, or 58 (as
generated here by degradation with MMPs) have not been described, which
would have indicated in vivo activity of MMPs. This may be
interpreted in three ways: 1) MMP-1, -2, and -3 are inactive in
vivo; 2) MMP-1, -2, and -3 cleave at different sites in
vivo to those in vitro; 3) or a fibril protein, that
has been cleaved by MMPs is rapidly degraded by other proteases and
escapes detection. Further investigations are needed to clarify this
issue.
The cell culture experiments presented here indicate that degradation
of AFP may take place initially in the extracellular or at least
pericellular compartment. AA immunoreactive material was not found
within THP-1 cells, and immunostaining was confined to the cell surface
or to large extracellular patches of fibrillar material (Figure 6)
. The
main site of activity of the MMPs is the peri- or extracellular
compartment.28,29
In addition, it was recently shown that
murine acute phase SAA and murine AA fibril proteins bind to a cell
surface receptor of advanced glycation end products (RAGE), which is a
signal transduction receptor and does not lead to the internalization
of SAA/AA fibril proteins.32
However, after binding to
RAGE, signal transduction is mediated by p21ras,
mitogen-activated kinases, ERK1 and ERK232,33
that are
known to be involved in the regulation of MMP genes.34
Blocking the binding of acute phase SAA or AA fibril protein to RAGE
delays the onset of AA amyloidosis.32
Whether this effect
is mediated through regulation of MMP genes and hence protein
expression and activity needs to be determined.
In summary, the putative interaction between SAA/AFP and MMPs seems to be complex. SAA can induce synthesis and secretion of MMPs27 at concentrations that are equivalent to those observed in serum and synovial fluid samples of patients with inflammatory arthritis.27,35 MMPs, in turn, are able to cleave SAA and AFP. Therefore, MMPs may be involved in the pathology of AA amyloidosis by partially degrading SAA or AA deposits. MMPs also degrade basement membranes and connective tissue and play an essential role in the homeostasis of the extracellular matrix. The deposition of amyloid disrupts the integrity of the vascular and interstitial matrix, which again, may modify the activity of MMPs. Thus, investigation of the putative role of MMPs in AA amyloidosis merits further attention.
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Acknowledgements |
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Footnotes |
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Supported by grants from the Deutsche Forschungsgemeinschaft (grant no. RO 1173/3-1 and SFB 387), Bonn Bad-Godesberg, Germany; and the Wilhelm Vaillant-Stiftung, München, Germany.
Accepted for publication April 19, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-macroglobulin in cases of generalized AA and AL amyloidosis. Virchows Arch 2000, 437:521-527[Medline]
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 S.M.P.M. Ruwanpura, K. Noguchi, and I. Ishikawa Prostaglandin E2 Regulates Interleukin-1{beta}-induced Matrix Metalloproteinase-3 Production in Human Gingival Fibroblasts Journal of Dental Research, March 1, 2004; 83(3): 260 - 265. [Abstract] [Full Text] [PDF]
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 G. Merlini and V. Bellotti Molecular Mechanisms of Amyloidosis N. Engl. J. Med., August 7, 2003; 349(6): 583 - 596. [Full Text] [PDF]
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