1 Institute of Molecular Pathology, University of Copenhagen, Frederik's
vej 11, 2100 Copenhagen, Denmark
2 Copenhagen University Hospital, Blegdamsvej 9, 2100 Copenhagen, Denmark
3 Department of Pathology, University of Ulm, Albert-Einstein-Allee 11, D-89081
Ulm, Germany
4 Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical
School, Boston, MA 02215, USA
Author for correspondence (e-mail:
ullaw{at}pai.ku.dk)
Accepted 5 June 2003
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Summary |
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Key words: ADAM12, ß1 integrin, Actin cytoskeleton, Extracellular matrix, Adipogenesis
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Introduction |
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Adipogenesis is a complex process characterized by the strict temporal
regulation of multiple and interacting signaling events that ultimately lead
to the expression of adipocyte-specific genes
(Gregoire et al., 1998;
Rosen and Spiegelman, 2000
). A
cascade of transcription factors is induced that involves the sequential
activation of the CCAAT/enhancer-binding proteins (C/EBPs) and the peroxisome
proliferator-activated receptor
(PPAR
), which leads to the
activation of several genes, such as those responsible for lipid transport and
metabolism. Initially, fibroblastic preadipocytes stop dividing and acquire a
rounded morphology. The change of shape from fibroblastic preadipocytes to
rounded, mature adipocytes is accompanied by changes in cytoskeletal
organization and contacts with the ECM. The expression of fibronectin,
integrins, actin and several cytoskeletal proteins is downregulated during
adipogenesis (Rodriguez Fernandez and
Ben-Ze'ev, 1989
; Spiegelman
and Farmer, 1982
). In fact, the disruption of contacts with the
ECM is a requirement for adipocyte differentiation
(Spiegelman and Ginty,
1983
).
Our understanding of the molecular events involved in adipogenesis is based
mainly on in vitro, cell culture models, especially 3T3-L1 preadipocytes
(Gregoire et al., 1998;
Rosen and Spiegelman, 2000
).
Several genetically modified mouse models have also contributed to the current
knowledge of adipocyte differentiation
(Moitra et al., 1998
;
Rosen et al., 1999
;
Wang et al., 1995
). We have
recently reported increased adipogenesis in transgenic mice overexpressing the
disintegrin and metalloprotease, ADAM12, driven by the muscle creatine kinase
promoter (Kawaguchi et al.,
2002
). Cells expressing early markers of adipogenesis were
apparent in the perivascular space in the muscle tissue of 1- to 2-week-old
transgenic mice, whereas mature, lipid-laden adipocytes were seen at 3 to 4
weeks of age, suggesting a crucial role for ADAM12 in adipogenesis in
vivo.
ADAM12 belongs to the ADAMs, a family of adhesion proteins and
metalloproteases comprising more than 30 members. The prototype ADAM molecule
is a transmembrane protein composed of several distinct domains including a
prodomain, metalloprotease, disintegrin, cysteine-rich, EGF-like and
transmembrane domains, as well as a short cytoplasmic tail
(Black and White, 1998;
Schlondorff and Blobel, 1999
).
Human ADAM12 exists in two alternatively spliced forms - a long transmembrane
form, ADAM12-L, and a shorter secreted form, ADAM12-S, which lacks the
transmembrane and cytoplasmic domains
(Gilpin et al., 1998
). Only
the long, membrane-anchored form of ADAM12 has been reported in mice
(Yagami-Hiromasa et al.,
1995
). In our transgenic mouse models, increased adipogenesis
occurred in the skeletal muscle of mice expressing human ADAM12-S, ADAM12-L,
or ADAM12-L lacking the cytoplasmic domain (ADAM12-
cyt)
(Kawaguchi et al., 2002
)
(U.M.W., unpublished), indicating that it is primarily the extracellular
domain of ADAM12 that elicits the robust adipogenic response observed in these
mice. The phenotype of ADAM12-deficient mice was recently characterized and it
was noted that the interscapular brown adipose tissue was reduced in some pups
(Kurisaki et al., 2003
).
The present study was undertaken to elucidate the molecular mechanisms underlying the stimulatory effects of ADAM12 on adipocyte differentiation. We present evidence that ADAM12 is transiently expressed at the cell surface just before the onset of adipogenesis, concomitant with the reduced activity of ß1 integrin, when preadipocytes begin to change their morphology from a fibroblastic to a rounded shape. Overexpression and RNAi knockdown experiments show that ADAM12 induces several changes in ß1 integrin-dependent functions in these mesenchymal cells. These changes in integrin function appear to involve lateral associations between ADAM12 and ß1 integrin within the plasma membrane that result in increased detergent solubility of ß1 integrin.
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Materials and Methods |
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Antibodies
Antibodies to ADAM12 included mouse monoclonal antibodies (mAbs) 6E6, 6C10
and 8F8; and polyclonal antibodies rb122 and rb109
(Kronqvist et al., 2002).
Mouse anti-human ß1 integrin (clone 12G10) was obtained from Serotec
(Oxford, UK). Rabbit-antiserum to ß1 integrin was kindly provided by S.
Johansson (Bottger et al.,
1989
) and mouse monoclonal antibody to vinculin was kindly
provided by M. Glukhova. Mouse anti-human ß1 integrin (clone 18) and
phycoerythrin-conjugated CD29 (clone MAR4), anti-mouse ß1 integrin
(9EG7), anti-human fibronectin Ab (A245) and phycoerythrin-conjugated goat
anti-mouse immunoglobulin G (IgG) were all obtained from BD Biosciences
(Brøndby, Denmark). Mouse anti-human ß1 integrin (JB1A) was
obtained from Chemicon Ltd (Hampshire, UK). Mouse anti-human ß1 integrin
(K20), isotype-controls, mouse IgG, goat anti-mouse-horse radish peroxidase
(HRP) Ab, and goat anti-rabbit-HRP Ab were obtained from Dako A/S (Glostrup,
Denmark).
Transient transfections
Cells were transfected using FuGene 6 transfection reagent (Roche
Diagnostic, Mannheim, Germany) or Lipofectamine (Invitrogen) in
serum-containing medium or Opti-MEM I according to the manufacturer's
instructions, and analyzed two days later. To create fusion proteins with
enhanced green fluorescent protein (EGFP), human ADAM12 cDNAs were cloned into
the pEGFP-N1 vector (Clontech Laboratories, Heidelberg, Germany). The
following constructs were used (Hougaard
et al., 2000): human ADAM12-L truncated at nt 2498, resulting in a
membrane-inserted protein lacking the cytoplasmic tail, called
ADAM12-
cyt (#1442); and ADAM12-
cyt with a E351-Q catalytic site
mutation (#1455). As a control, the empty vector, pEGFP-NI (#1422), was used.
Mouse full-length ADAM12 (#1629) (kindly provided by J. Frey) or empty-vector
control pcDNA3 (#1695) (Invitrogen) was transfected into CHO-K1 cells that
were used as positive or negative controls, respectively, in western
blots.
Retroviral transduction
ADAM12-cyt cDNA was subcloned into the SalI site of
pBABEpuro (Morgenstern and Land,
1990
) for retroviral transduction, as described previously
(Porse et al., 2001
). Briefly,
retroviral stocks were obtained by transiently transfecting Phoenix ecotropic
retroviral packaging cells with pBABE-based proviral constructs. Cell culture
supernatants were harvested and used to infect 3T3-L1 and C3H10T1/2 cells.
After infection, cells were cultured in the presence of 1 µg/ml puromycin
(Sigma-Aldrich, Vallensbaek, Denmark) to select stable clones.
Northern blot analysis
TriZol reagent (Invitrogen) was used to isolate total RNA from embryonic
muscle, white and brown adipose tissue, or cultured cells at various stages of
differentiation. RNA (15 µg total RNA per lane) was separated on 1%
formaldehyde gels and blotted onto Hybond N nylon membranes (Amersham
Pharmacia Biotech, Uppsala, Sweden). Hybridization was carried out at 68°C
for 1 hour using [32P]-dCTP-labeled mouse ADAM12,
PPAR
(kindly provided by B. M. Spiegelman), or
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Clontech. cDNAs in
QuickHyb hybridization solution (Stratagene, Amsterdam, The Netherlands).
After washing with 2xsaline sodium citrate (SSC)/1% sodium dodecyl
sulphate (SDS), 2xSSC/0.1% SDS and 0.2xSSC/0.1% SDS twice each at
65°C, the blots were exposed to Kodak X-OMAT AR film at -80°C with
intensifying screens, or analyzed by a PhosphorImager (Storm840, Molecular
Dynamics, Amersham Biosciences, Hørsholm, Denmark).
Protein extraction, immunoprecipitation and western blot
analysis
Proteins from growing 3T3-L1, C3H10T1/2, LiSa-2 cells and CHOK1 cells
transfected with control vector (#1695) or mouse full-length ADAM12 (#1629)
were extracted at 4°C in 20 mM Tris, pH 8.0, 150 mM NaCl, 1 mM
MgCl2, 1 mM CaCl2, 50 mM octylglucoside, and a protease
inhibitor cocktail (Complete, ethylenediaminetetraacetic acid-free protease
inhibitor cocktail tablets; Roche Molecular Biochemical, Hvidovre, Denmark).
Insoluble material was removed by a brief centrifugation (100
g for 1 minute), and the supernatant was further centrifuged
at 14,800 g for 45 minutes. The resulting pellets were
extracted for another 30 minutes in the extraction buffer, incubated overnight
at 4°C with a mixture of ADAM12 mAbs (6E6, 6C10 and 8F8; 7.5 µg total),
or a control antibody (total IgG concentration per sample 7.5 µg) and
protein-G Sepharose beads (Amersham Pharmacia Biotech), rinsed twice with
extraction buffer and eluted by boiling in sample buffer. Samples were
separated on 8% polyacrylamide gels. Western blots were performed, as
described previously (Loechel et al.,
1998), using a polyclonal anti-ADAM12 antibody (rb122), or - for
co-immunoprecipitation studies - monoclonal antibodies against mouse (clone
18) or human (JB1A) ß1 integrin.
Immunostaining and imaging
Tissue specimens were fixed in formalin and embedded in paraffin. For
immunoperoxidase staining, 4 µm tissue sections were deparaffinized,
subjected to microwave treatments in citrate buffer at pH 6.0 and incubated
with the primary antibody for 25 minutes at room temperature. Detection was
performed using the DakoChemMate detection kit (code K 5001), which is based
on an indirect streptavidin-biotin technique with a biotinylated secondary
antibody. Cultured cells were analyzed by immunoperoxidase or
immunofluorescence staining on suspended or adherent cells. To stain suspended
cells, cells were detached with trypsin/EDTA or cell dissociation buffer (both
from Invitrogen), restored for 5 minutes at 37°C in growth medium
supplemented with 10% FBS, then transferred to ice and incubated for 30
minutes with antibodies to ADAM12, fibronectin, vinculin or ß1 integrin.
The cells were rinsed once in PBS then fixed in 4% paraformaldehyde for 2
minutes, and immediately centrifuged for 2 minutes in a Cytospin microfuge
(Shandon, Pittsburgh, PA) onto glass slides and air-dried. The secondary
antibodies were applied as described above, except that fluorescein
isothiocyanate (FITC)- or tetramethyl rhodamine isothiocyanate
(TRITC)-conjugated secondary antibodies were used for immunofluorescence
staining. Adherent cells cultured on plastic dishes were fixed with 4%
paraformaldehyde for 5 minutes, permeabilized with 0.1% Triton X-100 for 5
minutes and incubated with antibodies for 1 hour at room temperature.
Secondary antibodies were applied, and peroxidase staining was performed as
described above. Staining for F-actin was performed with TRITC-phalloidin
(Molecular Probes, Leiden, The Netherlands). Cells were examined using an
inverted Zeiss Axiovert microscope equipped with phase contrast optics and
connected to a PentaMAX chilled charge-coupled device camera. The images were
processed using the Metamorph Software Program (Universal Imaging Corporation,
Brock and Michelsen; Birkerød, Denmark). To estimate the degree of
stress fiber and focal adhesion formation, more than 600 cells in 20 fields
were analyzed and the percentage of cells with fewer than five stress fibers
or focal adhesions was calculated. The experiments were repeated twice and
with two to three dishes per experiment.
Triton X-100 extraction
Two experimental techniques were used to assess the extractability of
ß1 integrin. Adherent cells were treated for 5 minutes on ice with either
0.01% Triton X-100 in DMEM or, as previously described
(Fey et al., 1984), with 0.5%
Triton X-100 in a cytoskeletal (CSK) buffer (100 mM NaCl, 300 mM sucrose, 10
mM Pipes pH 6.8, 3 mM MgCl2, 0.5% Triton X-100 and 1.2 mM
phenylmethylsulfonyl fluoride). Both extraction conditions yielded similar
results. The cells were subsequently rinsed with PBS and incubated with
polyclonal anti-ß1 integrin antibodies for 30 minutes, rinsed again and
fixed with 4% paraformaldehyde for 2 minutes. Secondary antibodies were
applied as described above.
Cell attachment assays
Nunc-Immuno 96-well plates with a MaxiSorp surface (Nunc A/S, Roskilde,
Denmark) were coated with 10 µg/ml 9EG7 rat anti-mouse ß1 integrin
antibody (BD Biosciences) or 10 µg/ml fibronectin in 0.1 M
NaHCO3 buffer overnight at 4°C, rinsed with PBS and blocked
with 1% bovine serum albumin in PBS for 1 hour at 37°C. The plate was
washed once with PBS and 6x104 cells/well were added and
allowed to attach for 1 hour at 37°C in 5% CO2 in a humidified
atmosphere. For cell attachment assays, the plates were centrifuged in an
inverted position at 25 g for 2 minutes. Cells were washed
twice in serum-free DMEM, fixed for 20 minutes at room temperature in 2%
glutaraldehyde in 0.1 M cacodylate buffer and stained with 0.1% crystal violet
in 10% methanol. Absorbance was measured with a Multiscan ELISA reader
(Lab-systems, Helsinki, Finland) at 590 nm. Each assay was carried out in
eight separate wells in each of two repetitions.
Apoptosis assay
Cells were trypsinized and resuspended in growth medium with or without 10
µg/ml of the ß1 integrin-activating antibody, 9EG7. After incubation
for 20 minutes at 37°C in suspension, cells were plated and cultured for
20 hours. Cells were re-incubated with 9EG7 (10 µg/ml) for 40 minutes
immediately before treatment with tumour necrosis factor (TNF)-
(Strathmann Biotec AG, Hamburg, Germany) (100 ng/ml) and cycloheximide
(Sigma-Aldrich) (5 µM). After 5 hours of TNF-
and cycloheximide
treatment, cells were fixed with 4% paraformaldehyde, permeabilized with 0.1%
Triton X-100 and stained with 4',6-diamidino-2-phenylindole
dihydrochloride (DAPI) (10 mM). The number of apoptotic cells were assessed by
fluorescence microscopy (Higuchi et al.,
2003
).
Flow cytometry
Growing and confluent (day 0) LiSa-2 cells were detached with cell
dissociation buffer (Invitrogen) and restored for 5 minutes at 37°C in
growth medium supplemented with 10% FBS, then transferred to ice and washed
twice in cold washing buffer (PBS supplemented with 1% BSA). Mouse anti-human
12G10 and MAR4 were diluted according to the description of the manufacturer
and added to the cell suspension. After 20 minutes at 4°C, cells were
washed twice with washing buffer, incubated with the secondary antibody
(phycoerythrin-conjugated goat anti-mouse IgG) for 20 minutes at 4°C and
rinsed twice in washing buffer. After the second wash, cell pellets were
resuspended in 500 µl washing buffer. Flow cytometric analyses were
performed according to standard settings on a FACStar PLUS flow cytometer with
CELLQUEST software (both from BD Biosciences).
RNA interference and microinjection
Short interfering RNA (siRNA) oligos were purchased from Dharmacon
(Lafayette, CO) in a 2'-deprotected, desalted, single-stranded form
(option B), and annealed according to the manufacturer's protocol. Oligo
sequences were: AAUCCCACGACAAUGCUCAGCdTdT (mouse ADAM12 siRNA), and
AACGACGUCCACUCUACAGCAdTdT (scrambled siRNA). A mixture of siRNA (2 µM) and
EGFP expression vector (0.1 µg/µl) was injected into 3T3-L1 cells using
a FemtoJet microinjector and an Inject Man NI 2 manipulator (Eppendorf,
Hamburg, Germany) at an injection pressure of 150 hPa and an injection time of
0.7 second. Confluent cells were injected, and differentiation was induced the
following day as described above. At day 4 after differentiation, cells were
processed for phalloidin staining or immunostaining with antibodies to ADAM12
and observed under a fluorescence microscope.
Statistical analysis
All data are expressed as means ± s.e. The unpaired Student's t-test
or the Mann Whitney test was used for comparisons. A value of
P0.05 was considered statistically significant.
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Results |
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Cell-surface expression of ADAM12 before the onset of
adipogenesis
To study the subcellular distribution pattern of ADAM12 during different
stages of 3T3-L1 adipogenesis, permeabilized adherent cells were immunostained
(Fig. 3A-C). Growing cells
displayed mostly intracellular staining
(Fig. 3A), whereas day 0
confluent cells showed a distinct cell-surface staining
(Fig. 3B). Most maturing
adipocytes (day 4) exhibited intense cytoplasmic ADAM12 staining, which was
confined to small separate vacuoles that probably represented late endosomes
or lysosomes (Fig. 3C). The
number of adipocytes with intracellular ADAM12 immunostaining decreased in
later stages of differentiation (after 6-8 days, data not shown).
|
To more rigorously establish the cell-surface localization of ADAM12, suspended cells were immunostained for ADAM12 without previous permeabilization (Fig. 3D-F). Growing cells had little or no detectable cell-surface immunostaining (Fig. 3D). By contrast, day 0 cells had distinct, evenly distributed cell-surface staining (Fig. 3E). During the adipogenic stage (the 4 days following induction), maturing adipocytes exhibited cell-surface staining that was clearly fainter than the staining of day 0 cells (Fig. 3F). A similar temporal pattern of ADAM12 expression was observed in mouse C3H10T1/2 and in human LiSa-2 cells (data not shown).
The dynamic subcellular localization of ADAM12 during adipogenesis indicated that the protein is endocytosed after the onset of differentiation. To test this hypothesis, anti-ADAM12 IgG (a mixture of mAbs, 6E6, 6C10, 8F8; 7.5 µg total) or control IgG (7.5 µg) was incubated with growing 3T3-L1 cells. After 5 hours, 2 days or 6 days, the cultures were rinsed, fixed, permeabilized and stained with secondary antibody to detect the ADAM12:IgG complex (Fig. 3G-I). No ADAM12:IgG was detected in growing cell cultures (Fig. 3G); however, cell-surface ADAM12:IgG was detected in confluent preadipocytes (Fig. 3H). In day 4 cultures, ADAM12:IgG was located intracellularly (Fig. 3I). No intracellular staining was observed when control IgG was added (data not shown). These results suggest that ADAM12 is, in fact, endocytosed during adipocyte differentiation.
Together, these results indicate that the subcellular distribution of ADAM12 is highly regulated: ADAM12 is translocated to the cell surface of preadipocytes before the onset of differentiation, and internalized during adipogenesis.
Formation of ADAM12/ß1 integrin complexes and reduced ß1
integrin activity as assessed by 12G10 immunostaining upon ADAM12 cell-surface
localization
Under nonadipogenic conditions, fibronectin in the ECM
(Fig. 4A) and intracellular
actin stress fibers in preadipocytic cells
(Fig. 4B) appear to be arranged
in parallel, most probably with ß1 integrins as coordinating matrix
points at the cell surface (Fig.
4C). Because ADAM12 can interact with ß1 integrins in trans,
i.e. ADAM12 present on one cell may bind to the integrin present on another
cell (Eto et al., 2000), we
hypothesized that ADAM12 itself could be part of a ß1 integrin
cell-adhesion complex and that this might influence the adipogenic
differentiation pathway. To test this hypothesis, we performed
co-immunoprecipitation experiments using antibodies against ADAM12 followed by
western blotting with antibodies against ß1 integrin. As shown in
Fig. 4D, ADAM12 and ß1
integrin formed complexes in LiSa-2, C3H10T1/2 and 3T3-L1 cells.
|
The localization pattern of ß1 integrin at the cell surface was examined by immunostaining and fluorescence-activated cell sorter (FACS) analysis (Fig. 5). Growing LiSa-2 cells showed prominent ß1 integrin cell-surface immunostaining using antibodies to the activated human ß1 integrin (12G10) (Fig. 5C). Less intense ß1 integrin immunostaining was detected on confluent day 0 cells stained in parallel (Fig. 5D), which was confirmed by FACS analysis (Fig. 5G). There was little or no detectable difference in the total amount of ß1 integrin as determined by immunostaining using the K20 antibody (Fig. 5E,F), or by FACS analysis using the MAR4 antibody against ß1 integrin (Fig. 5H).
|
These results indicate that upon ADAM12 cell-surface expression, ß1 integrin/ADAM12 complexes are formed, which appears to result in reduced ß1 integrin activity. To assess the consequences of reduced ß1 integrin activity, potential changes in cell shape, actin cytoskeleton organization, cell adhesion, organization of ECM and cell survival were evaluated.
Cell-surface ADAM12 induces actin cytoskeleton reorganization
Concomitant with the onset of adipocyte differentiation, the
fibronectin-rich ECM becomes degraded, the actin cytoskeleton undergoes a
dramatic reorganization and the cell shape changes
(Cornelius et al., 1994).
Growing preadipocytes have a fibroblastic morphology and exhibit an elaborate
network of stress fibers (Fig.
6A). At day 0 the preadipocytic cells are closely packed, and some
begin to round up and loose their actin stress fibers
(Fig. 6B). Within 4 days after
induction of differentiation, maturing adipocytes are round, the actin stress
fibers have disappeared and have reorganized into a cortical actin network
located just beneath the cell membrane
(Kanzaki and Pessin, 2001
)
(Fig. 6C). To determine whether
ADAM12 influences the early changes in the organization of the actin
cytoskeleton, growing 3T3-L1 preadipocytes were transfected with
ADAM12-
cyt. This construct allows the efficient translocation of ADAM12
to the cell surface in contrast to the full-length ADAM12-L construct, which
results in a predominantly intracellular accumulation of protein
(Hougaard et al., 2000
).
ADAM12-
cyt, therefore, provides a good model to study the effect of
ADAM12 at the cell surface. Two days after transfection with
ADAM12-
cyt, the cell morphology had changed dramatically. Transfected
cells were smaller, and more rounded. They retracted their cytoplasm, and
their actin stress fibers gradually disappeared and were replaced, first by
dot-like actin elements, then by a distinct cortical localization of actin
(Fig. 6D-F). Cells transfected
with a protease-deficient ADAM12-
cyt construct (ADAM12-
cyt cat
mut) had the same morphological appearance as cells transfected by
ADAM12-
cyt, i.e. the actin stress fibers in these cells disappeared and
re-organized into a cortical localization
(Fig. 6G-I). Cells transfected
with a control EGFP vector maintained their stress fibers and did not exhibit
morphological changes (Fig.
6J-L).
|
To test whether endogenous ADAM12 in preadipocytes influences reorganization of the actin cytoskeleton, we used an RNAi strategy to reduce ADAM12 expression. Confluent 3T3-L1 preadipocytes were microinjected with ADAM12 or scrambled siRNA oligos together with EGFP to identify injected cells, and differentiation was induced the following day. On day 4 of differentiation, cell cultures were fixed and examined with phalloidin staining to monitor F-actin. 3T3-L1 cells microinjected with ADAM12 siRNA maintained their stress fibers (Fig. 7B), whereas cells injected with scrambled siRNA were similar in appearance to the surrounding noninjected cells and did not contain stress fibers (Fig. 7D). To confirm that the microinjected ADAM12 siRNA reduced the expression of ADAM12, these cells were immunostained and ADAM12 immunostaining was not seen in any injected cells (Fig. 7E,F), whereas noninjected cells exhibited positive ADAM12 immunostaining (Fig. 7G,H). As a further control, cells injected with scrambled siRNA also exhibited positive ADAM12 immunostaining (data not shown).
|
Cells with increased cell-surface ADAM12 expression are less
adhesive, are more prone to apoptosis and have a reorganized extracellular
fibronectin matrix
We used retroviral transduction to establish a 3T3-L1 cell line that
constitutively expressed ADAM12-cyt at the surface
(Fig. 8A,B), and confirmed
protein expression by western blot (Fig.
8C). These cells, like preadipocytes transiently transfected with
ADAM12-
cyt (see Fig.
6D), showed fewer stress fibers compared with cells infected with
a control vector (Fig. 8D,E).
Compared with the control cells, a significantly higher percentage of the
ADAM12-
cyt-expressing cells had fewer than five stress fibers
(Fig. 8F). As shown in
Fig. 8G-I, the
ADAM12-
cyt-expressing cells also exhibited a marked reduction in
vinculin-positive focal adhesions, suggesting reduced ß1
integrin-mediated adhesion activity when ADAM12-
cyt is expressed at the
cell surface. We therefore tested the capacity of these cells to attach to
9EG7 mAb, to mouse ß1 integrin (data not shown) and to fibronectin
(Fig. 8J) in cell attachment
assays. The 3T3-L1 cells expressing ADAM12-
cyt were significantly less
adhesive than 3T3-L1 cells that did not express ADAM12-
cyt
(Fig. 8J). The same results
were obtained with C3H10T1/2 cells infected with ADAM12-
cyt (data not
shown). These results led us to test whether ADAM12-
cyt might also
influence ß1 integrin-mediated cell survival pathways. As shown in
Fig. 9A,B, 3T3-L1 cells
overexpressing ADAM12-
cyt are more sensitive to TNF-
-induced
apoptosis than are control cells. It should be noted that this effect of
ADAM12-
cyt was also observed in other cell types, including CHO-K1
cells (U.M.W., unpublished). The increased sensitivity to apoptosis was
prevented by the ß1 integrin-activating mAb 9EG7
(Fig. 9C).
|
|
Finally, the organization of the pericellular fibronectin-rich ECM was
analyzed in 3T3-L1/ADAM12-cyt cells. We found that the
fibronectin-matrix of these ADAM12-
cyt-expressing cells no longer
appeared as an extensive, dense network, as in the control fibroblastic
preadipocytes (Fig. 9E), but
instead was distributed as a dense basement membrane-like rim encircling the
cell borders (Fig. 9D).
Together, these results indicate that ADAM12-cyt induces a negative
regulation of typical ß1 integrin functions.
ADAM12 increases the Triton X-100 solubility of ß1 integrin
It is well known that integrins associate with the actin cytoskeleton and
modulate cell shape - for example, during formation of filopodia and
lamellopodia. The findings described above suggest that ADAM12 induces changes
in the association between ß1 integrin and the actin cytoskeleton. To
test this hypothesis, the Triton X-100 extractability of ß1 integrin was
examined. We used two different experimental procedures: one was the standard
0.5% Triton X-100 in CSK buffer and the other was 0.01% Triton X-100 in DMEM.
Under both experimental conditions ADAM12-cyt-expressing 3T3-L1 cells
exhibited a dramatic reduction of ß1 integrin immunostaining
(Fig. 10A,E) compared with
control 3T3-L1 cells, which exhibited intense ß1 integrin immunostaining
(Fig. 10B,F). The ß1
integrin immunostaining in untreated ADAM12-
cyt-expressing 3T3-L1 and
control cells are shown in Fig.
10C,D for comparison. Triton X-100 solubility of ß1 integrin
is another demonstration of how overexpression of ADAM12-
cyt mimics the
dynamic changes that accompany early adipocyte differentiation
(Fig. 10G,H).
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Discussion |
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Several studies have pointed to an important role of ADAMs during
mesenchymal cell differentiation (Gilpin
et al., 1998; Yagami-Hiromasa
et al., 1995
). In the mouse embryo, ADAM12 is most prominently
expressed in condensed mesenchymal cells in regions of muscle and bone
formation, including cranial membranous bones, ribs and limbs, and in the bone
marrow (Kurisaki et al.,
1998
). ADAM12 expression is not detectable in adult skeletal
muscle; however, expression is upregulated during muscle regeneration
(Galliano et al., 2000
).
Interestingly, ADAM12 has been shown to alleviate the pathology of the
dystrophin-deficient mdx mice
(Kronqvist et al., 2002
).
Furthermore, transgenic mice overexpressing ADAM12 show increased adipogenesis
(Kawaguchi et al., 2002
),
whereas ADAM12-deficient mice showed decreased interscapular brown adipose
tissue (Kurisaki et al.,
2003
). In the present study we examined the expression profile of
ADAM12 during adipogenesis. We found that ADAM12 mRNA was detected throughout
most stages of adipogenesis but was present at maximal levels in confluent
preadipocytes just before the onset of differentiation. These committed
preadipocytes were characterized by increased levels of ADAM12 immunostaining
at the cell surface that was present in complexes with ß1 integrin.
ADAM12 mRNA expression decreased in later stages of adipogenesis, and ADAM12
located at the cell-surface disappeared, which was most probably due to
endocytosis.
Adhering fibroblastic cells, including preadipocytes, are characterized by
an elaborated network of stress fibers and focal adhesions and show an intense
cell-surface ß1 integrin immunostaining with 12G10 mAb. As preadipocytes
become confluent and round up, their stress fibers become reorganized into a
cortical network and their adhesion to the ECM is weakened. This is thought to
be crucial for the subsequent induction of adipogenic transcription factors.
Direct evidence that ADAM12 is critically involved in the process of
reorganization of stress fibers was obtained from experiments in which
ADAM12-cyt was overexpressed in 3T3-L1 preadipocytes by either
transient transfection or by retroviral transduction. Thus, fibroblastic cells
overexpressing ADAM12-
cyt attained a rounded morphology, and their
actin stress fibers disappeared and reorganized into a distinct F-actin
cortical localization, even before they reached confluence. Furthermore, we
found that 3T3-L1 preadipocytes microinjected with ADAM12 siRNA oligos
maintained their stress fibers even 4 days after the induction of adipogenic
differentiation.
We hypothesized that the effect of ADAM12-cyt on the actin
cytoskeleton is mediated through ß1 integrin, and therefore asked whether
other typical integrin-mediated functions were also altered. We found that
ADAM12-expressing cells had decreased 12G10 immunostaining at the cell
surface, had fewer vinculin-positive focal adhesions and adhered less
efficiently to ß1 integrin antibodies and fibronectin. Even
integrin-mediated cell-survival pathways appeared to be altered - that is,
cells overexpressing ADAM12-
cyt were more prone to TNF
-induced
apoptosis (Fig. 9) as well as
apoptosis induced by ultraviolet light and thapsigargin (U.M.W., unpublished).
The finding that 9EG7 mAb could prevent TNF-
-induced apoptosis in
3T3-L1 cells presents strong evidence that this effect of ADAM12-
cyt
is, in fact, mediated through ß1 integrin.
In other cell systems, it has been shown that the interaction between ADAMs
and ß1 integrin is mediated through the disintegrin or cysteine-rich
domains. For example, the disintegrin domains of ADAM2 and 9 supports cell
adhesion through 6ß1 integrin
(Chen et al., 1999
;
Nath et al., 2000
), and that
of ADAM12 through
9ß1 integrin
(Eto et al., 2000
). The
recombinant cysteine-rich domain of ADAM12, however, interacts with syndecans
and mediates integrin-dependent cell spreading
(Iba et al., 2000
). The
intriguing role of the cysteine-rich domain was elucidated further in a recent
study on ADAM13 (Smith et al.,
2002
) in which the authors showed that this domain could cooperate
intramolecularly with the metalloprotease domain to regulate its function in
vivo. Further studies are needed to determine which of these adhesion
domain(s) of ADAM12 are involved in mediating ß1 integrin-mediated
changes in cell behavior.
ADAM12 induced a reorganization of the fibronectin-rich ECM - that is,
preadipocytes overexpressing ADAM12-cyt showed reduced fibronectin-rich
ECM that was organized into a basement membrane-like rim encircling the cells
while control preadipocytes maintained an elaborate meshwork of
fibronectin-rich ECM covering the cells. This is an important finding as
reduction of the fibronectin-rich ECM is a crucial determinant for adipose
differentiation. The role of the ECM in adipogenesis was first suggested in
1983 when it was reported that adipogenesis was inhibited in preadipocytes
grown on fibronectin-coated dishes
(Spiegelman and Ginty, 1983
).
Subsequent studies showed that transforming growth factor (TGF)-ß
inhibits adipogenesis by increasing the expression of fibronectin and collagen
(Bortell et al., 1994
). More
recent studies have pointed to an important role for matrix metalloproteases
(MMPs)-2 and -9 and plasmin in promoting adipogenesis by degrading the
fibronectin-rich ECM (Alexander et al.,
2001
). Although, on the basis of the studies presented here, we
suggest that the effect of ADAM12 on the reorganization of the
fibronectin-rich ECM is mediated via the reduced activity of ß1 integrin,
we cannot exclude the contribution of the metalloprotease activity of ADAM12.
Little is known about the physiological substrate for the ADAM12 protease,
although it was recently proposed to cleave heparin-binding epidermal growth
factor-like growth factor (HB-EGF) in the mouse heart
(Asakura et al., 2002
). We have
previously shown that human ADAM12-S can degrade insulin-like growth factor
binding protein (IGFBP)-3 and IGFBP-5
(Loechel et al., 2000
;
Shi et al., 2000
). Transgenic
mice expressing a form of ADAM12-S lacking the prodomain and metalloprotease
domains (called ADAM12-S minigene) did not exhibit increased adipogenesis
(Kawaguchi et al., 2002
),
suggesting a role for the protease activity of ADAM12 in adipogenesis.
The data presented here strongly suggest that ADAM12 may directly or
indirectly influence the function of ß1 integrin. The exact molecular
mechanisms involved, however, warrant further consideration. We found that
ß1 integrin coimmunoprecipitates with ADAM12, suggesting that these two
proteins can be part of the same adhesion complex. This indicates that ADAM12
in-cis could modulate ß1 integrin function through lateral associations,
and this further raises the interesting question as to whether ADAM12 may
induce a redistribution of ß1 integrins in the cell membrane, i.e. out of
the rafts or caveolae microdomains. In this context, the recent demonstration
that an extracellular matrix component, laminin-2, could induce a
colocalization of 6ß1 integrins and platelet derived growth factor
(PDGF)
R into the same lipid rafts during oligodendrocyte
differentiation is of interest (Baron et
al., 2003
). The exact localization of ADAM12 in the cell membrane
is not yet known, but ADAM10 (
-secretase) seems not to be present in
lipid microdomains (Kojro et al.,
2001
). Even if the exact localization of ADAM12 and ß1
integrin in the membranes of these cells is not yet determined, it was
observed that the effect of the association between the two proteins was to
disengage ß1 integrin from the actin cytoskeleton. This conclusion is
based on the striking finding that 0.5% Triton X-100 extracted ß1
integrin from ADAM12-
cyt-expressing 3T3-L1 cells but not from control
cells.
The investigations described in this study focused on the role of ADAM12 in
early adipogenesis before the onset of terminal differentiation. However, the
ADAM family contains at least 33 members, and it is likely that other ADAMs
function in parallel with ADAM12 in a synergistic or antagonistic fashion.
Preliminary RT-PCR data showed that ADAM9, 10, 17 and 19 are also expressed in
the 3T3-L1 cells during adipogenesis, and additional immunochemical data
demonstrated the expression of ADAM15 (U.M.W., unpublished). The finding that
ADAM12 null mice do not exhibit striking defects in the white adipose tissue
(Kurisaki et al., 2003)
further suggests functional overlap among the ADAMs. Additional studies,
including those using double-knockout technology, are needed to definitively
determine the contribution of other ADAMs during adipogenesis.
In conclusion, the findings reported here show that ADAM12 induces reorganization of the actin cytoskeleton, reduced cell adhesion, changes in cell survival pathways and reorganization of the fibronectin-rich ECM in fibroblastic preadipocytes. Furthermore, our observations strongly suggest that ADAM12 at the cell surface mediates its effects via a direct or indirect interaction with ß1 integrin that results in an impairment of ß1 integrin function, possibly by decreasing the association of ß1 integrin with the actin cytoskeleton. These results may have implications, not only for adipocyte differentiation and maturation, but also for mesenchymal cell differentiation in general.
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Acknowledgments |
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Footnotes |
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References |
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