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THE REGULATORY MECHANISM OF EXTRACELLULAR HSP90α ON MATRIX
METALLOPROTEINASE-2 (MMP-2) PROCESSING AND TUMOR ANGIOGENESIS*
Xiaomin Song1,2,3, Xiaofeng Wang1,2,3, Wei Zhuo1,2,3, Hubing Shi1,2,3, Dan Feng1,2,3, Yi Sun1,2,3,
Yun Liang1,2,3, Yan Fu1,2,3, Daifu Zhou1 and Yongzhang Luo1,2,3
1National Engineering Laboratory for Anti-tumor Protein Therapeutics, 2Beijing Key Laboratory for Protein Therapeutics, 3Cancer Biology Laboratory, School of Life Sciences,
Tsinghua University, Beijing 100084, China. Running Head: Extracellular Hsp90α and MMP-2 in tumor angiogenesis
Address Correspondence to: Yongzhang Luo, Ph.D., School of Life Sciences, Tsinghua University,
Beijing 100084, China. Phone: 86-10-6277-2897; Fax: 86-10-6279-4691; E-mail:
yluo@tsinghua.edu.cn
Heat shock protein 90α (Hsp90α) is a
ubiquitously expressed molecular chaperone,
which is essential for eukaryotic homeostasis.
Hsp90α can also be secreted extracellularly,
where it has been shown to be involved in
tumor metastasis. Extracellular Hsp90α
interacts with and promotes the proteolytic
activity of matrix metalloproteinase-2
(MMP-2). However, the regulatory
mechanism of Hsp90α on MMP-2 activity is
still unknown. Here we show that Hsp90α
stabilizes MMP-2 and protects it from
degradation in tumor cells. Further
investigation reveals that this stabilization
effect is isoform-specific, ATP-independent,
and mediated by the interaction between
Hsp90α middle domain and MMP-2
C-terminal hemopexin domain. Moreover,
this mechanism also applies to endothelial
cells which secrete more Hsp90α in their
proliferating status. Furthermore, endothelial
cell transmigration, Matrigel plug, and tumor
angiogenesis assays demonstrate that
extracellular Hsp90α promotes angiogenesis
in an MMP-2 dependent manner. In sum, this
study provides new insights into the
molecular mechanism of how Hsp90α
regulates its extracellular client proteins and
also reveals for the first time the function of
extracellular Hsp90α in promoting tumor
angiogenesis.
Heat shock protein 90 (Hsp90) is an
ATP-dependent molecular chaperone, which is
ubiquitously expressed and essential for cell
viability (1). Unlike other types of chaperones,
Hsp90 is not required for the biogenesis of most
polypeptides, but instead functions in the
maintenance of the active state of several
conformationally labile signaling proteins (2-4).
Many of Hsp90 client proteins are mutated,
chimeric or overexpressed oncogenic proteins
(3). Therefore, the chaperoning function of
Hsp90 is subverted to a biochemical buffer for
genetic lesions in tumor cells, facilitating the
malignant transformations of the cells (3). Hsp90
has emerged as a promising target for cancer
therapy (5).
There are two isoforms of Hsp90 in the
cytosol, referred to as Hsp90α and Hsp90β (6).
Intriguingly, the Hsp90α isoform also exists
extracellularly (7). Recent studies indicate that
extracellular Hsp90α is significantly correlated
with tumor invasiveness and metastasis (8), and
the antibody or impermeable inhibitor of Hsp90α
can suppress tumor metastasis efficiently in
mouse models (9-11). Furthermore, Hsp90α can
be detected in the blood of cancer patients and
the level of Hsp90α is positively associated with
tumor malignancy (9). In addition to tumor cells,
extracellular Hsp90α has also been identified in
neuron cells, dermal fibroblasts, keratinocyte,
macrophages and epithelial cells, and
participates in neuronal cell migration, wound
1
http://www.jbc.org/cgi/doi/10.1074/jbc.M110.181941The latest version is at JBC Papers in Press. Published on October 11, 2010 as Manuscript M110.181941
Copyright 2010 by The American Society for Biochemistry and Molecular Biology, Inc.
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healing, and viral and bacteria infections (7).
Accumulating evidence indicates that
extracellular Hsp90α plays important roles in
both physiological and pathological processes,
especially in tumor progression (7). However,
the molecular mechanism of how extracellular
Hsp90α functions is still largely unknown (12).
Eustace et al. reported that extracellular Hsp90α
can interact with matrix metalloproteinase 2
(MMP-2) and that the impermeable inhibitor of
Hsp90α (immobilized geldanamycin) inhibits
MMP-2 proteolytic activity (8), but the
regulatory molecular mechanism behind this
phenomenon is still a mystery.
In the present study, in order to further
elucidate the molecular mechanism of
extracellular Hsp90α function, we have
investigated the regulatory mechanism of
extracellular Hsp90α on MMP-2 activity. We
reveal that extracellullar Hsp90α stabilizes
MMP-2 and protects it from processing and
subsequent inactivation in tumor cells. The
regulatory function of Hsp90α on MMP-2
processing is isoform-specific and
ATP-independent. The interaction of Hsp90α
and MMP-2 is mediated by the middle domain
of Hsp90α and the C-terminal hemopexin
domain of MMP-2. Moreover, we further
confirm this mechanism in endothelial cells,
which can secrete increasing amount of Hsp90α
upon the treatment of VEGF. The effects of
recombinant human Hsp90α (rHsp90α) and the
Hsp90α antibody on angiogenesis in vitro and in
vivo were also examined. The result shows that
rHsp90α promotes, whereas the antibody of
Hsp90α suppresses angiogenesis in an MMP-2
dependent manner, suggesting that extracellular
Hsp90α is a potential therapeutic target for not
only tumor metastasis but also tumor
angiogenesis.
Experimental Procedures
Cell lines and transfectants- Human breast
cancer cell lines MDA-MB-231, MCF-7, and
mouse melanoma cell line B16/F10 were from
the American Type Culture Collection. HMEC is
a human dermal microvascular endothelial cell
line (HDMEC, Sciencell) transfected with SV40
large T antigen (13). Human umbilical venous
endothelial cells (HUVECs) were isolated from
human umbilical vein (14).
The stable cell line overexpressing MMP-2
was screened by G418 (200 µg/ml) from MCF-7
cells transfected with pcDNA3.1-MMP-2.
Antibodies- Anti-Myc, anti-His, and
anti-CD31 antibody (PECAM-1, M-20) was
from Santa Cruz Biotechnology (Santa Cruz,
CA). Anti-MMP-2 antibody (Ab-7) for Western
blotting was from Calbiochem (Darmstadt,
Germany). Anti-Hsp90α antibody (9D2) was
from Stressgen Bioreagents (Victoria, Canada).
Anti-Hsp90β antibody (H90-10) was from
Abcam (Cambridge, UK). Anti-flag antibody
was from Sigma. Monoclonal antibody against
Hsp90α (Hsp90α mAb) and Hsp90β (Hsp90β
mAb) for endothelial cell transmigration, tube
formation and tumor growth assay was prepared
by our lab. The specificity and efficiency were
confirmed by ELISA.
MMP-2 processing assay in vitro- This assay
was performed according to the previous report
(15). Purified rMMP-2 was incubated with the
indicated proteins with or without ATP (2 mM)
at 37°C for different periods. The incubation
buffer contains 40 mM HEPES, 10 mM MgCl2,
20 mM KCl, 5 mM CaCl2, 2 mM
p-Aminophenylmercuric acetate (APMA), pH7.4.
The processing was terminated by
electrophoresis sample buffer.
Endothelial cell transmigration assay- This
assay was performed using 6.5-mm millicell
inserts (8 μm pore size, Millipore) coated with
Matrigel (50 μg/insert) (Vigorous, Beijing,
China) on the upper side of the filter membranes.
2×105 HMECs per insert were inoculated. 0.5%
FBS was added in the lower chamber. Then the
cells were cultured for 24 h. Cells at the lower
surface of the filter membranes were stained
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with Hematoxylin & Eosin (H&E). The images
were visualized by microscope (Olympus IX71)
and 5 random fields per insert were captured by
CCD camera (Olympus DP71) for counting.
Tube formation assay- 24-well plates were
coated with 125 μl/well of Matrigel. HMECs
were seeded (1×105 cells/well) and cultured for
12 h in DMEM plus 2% FBS with indicated
treatment. The tubule images were visualized by
microscope (Olympus IX71) and captured by
CCD camera. The tubule length was calculated
in 5 random fields of view per well using
Image-Pro Plus.
In vivo Matrigel plug assay- 400 μl Matrigel
containing VEGF-A165 (50 ng/ml) (PeproTech,
Rocky Hill, NJ), lentivirus with vector
expressing MMP-2 shRNA or empty vector
(5×106 TU) (GenePharma, Shanghai, China),
and recombinant Hsp90α (rHsp90α),
recombinant Hsp90β (rHsp90β), non-immune
mouse IgG, Hsp90α mAb or Hsp90β mAb (50
μg/ml) was injected subcutaneously into the
nude mice. After 7 days, the plug was removed
and the tube formation was examined by
immuno-fluorescence using anti-CD31 antibody.
The target sequence of MMP-2 shRNA is
AAGAACCAGATCACATACAGG.
Tumor growth assay- B16/F10 cells (1×106,
100 µl) were subcutaneously inoculated in the
back of nude mice (6–8 weeks old).
Non-immune mouse IgG (10 mg/kg), Hsp90α
mAb (5, 10 mg/kg, according to the previous
report (10)) or Hsp90β mAb (5, 10 mg/kg) were
injected intraperitoneal every other day from the
day after implantation. The experiment was
performed with 6 mice in each group. After 25
days treatment, the mice were sacrificed, and the
tumors were excised, weighed, fixed, and
applied to immuno-fluorescence. All animal
studies were performed with the approval of the
Scientific Investigation Board of Tsinghua
University.
Immuno-fluorescence assay- Paraffin
embedded tumor tissues or Matrigel plugs were
processed into 5 μm thick sections. Sections
were re-hydrated and blocked with 5% rabbit or
goat serum, then incubated with the indicated
antibodies overnight at 4 °C. The primary
antibodies were detected by TRITC or FITC
conjugated secondary antibodies. The nucleus
was stained with DAPI. Sections were analyzed
by confocal microscopy (Nikon A1).
RESULTS
Hsp90α prevents the processing and
inactivation of MMP-2. To investigate the
regulatory mechanism of Hsp90α on MMP-2
activity, an MCF-7 stable cell line, which
overexpresses human MMP-2 with C-terminal
tandem Myc- and His-tags, was established (Fig.
1A). MMP-2 expression in the conditioned
media (CM) of this cell line treated with
rHsp90α or its antibody was analyzed. As
expected, activated MMP-2 was increased upon
the treatment with increasing amounts of
rHsp90α (Fig. 1B-C), which is consistent with
the previous report (8). Intriguingly, the amount
of ProMMP-2 was also increased, while some
over-processed fragments of MMP-2 (~30 kDa),
which were detected by Western blotting but not
zymography (Fig. 1B-C), were decreased in this
process (Fig. 1B). Furthermore, the antibody of
Hsp90α exhibited the opposite effect on MMP-2
processing (Fig. 1D-E), while rHsp90β and
Anti-Hsp90β antibody showed no such effect
(Supplementary Fig. S1A-B). These results
indicate that Hsp90α can modulate the activity
of MMP-2 via interfering with the processing or
degradation of MMP-2 at a specific cleavage site.
In addition, the densitometry reading result of
the MMP-2 processing upon the treatment of
rHsp90α or anti-Hsp90α antibody also showed
that Hsp90α was mainly involved in the
inactivation processing but not the activation of
MMP-2 (Fig. 1F-G).
We next sequenced the major processed
fragment (~30 kDa) observed above and
identified its N-terminus as LYGAS
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(Supplementary Fig. S2), beginning from the
amino acid residue 444 (Leu444) of MMP-2 (Fig.
1H). Since this fragment can be detected by the
anti-Myc antibody, it should end at the
Myc-tagged C-terminus of MMP-2 (Cys660).
Thus this fragment was identified as
Leu444-Cys660 comprising of both the hemopexin
domain (Pro466-Cys660) and 22 residues of the
linker domain. The theoretical molecular weight
of this fragment (Leu444-Cys660) is 24.5 kDa.
With the additional C-terminal tags, the
molecular weight of the fragment is increased to
27.4 kDa which is consistent with its migratory
position on the SDS-PAGE (Fig. 1B,D).
According to the previous report, this fragment
is a stable and inactive product of MMP-2
autocatalytic degradation (15,16). The cleavage
at Glu443-Leu444 initiates the MMP-2 inactivation
which is completed later by cleavage at the zinc
binding domain (15). Based on our results and
aforementioned literature, we propose that
Hsp90α can stabilize MMP-2 and protect it from
autocatalytic cleavage at Glu443-Leu444 which
subsequently leads to the complete inactivation
of MMP-2.
Hsp90α stabilizes MMP-2 in an
ATP-independent and isoform-specific manner.
To affirm the above hypothesis, we prepared the
purified rHsp90α, rHsp90β (Supplementary Fig.
S3A), and rProMMP-2 (Supplementary Fig. S3B)
and investigated the influence of these
chaperones on MMP-2 autocatalytic processing
in an in vitro non-cell system. Since Hsp90 is an
ATP-dependent molecular chaperone in the
cytosol (17,18), we first examined whether the
stabilization effect of Hsp90α on MMP-2 is ATP
dependent. Purified rProMMP-2 was mixed with
PBS, equal molar of Hsp90α or Hsp90β with or
without ATP, and then incubated at 37°C for 3 h.
The autocatalytic processing products of
ProMMP-2 were assayed by Western blotting. It
was found that Hsp90α but not Hsp90β can
protect MMP-2 from autocatalytic processing
and inactivation, and that ATP exhibited no
effect on the stabilization activity of both
Hsp90α and Hsp90β (Supplementary Fig. S3C),
demonstrating that the stabilization of MMP-2
mediated by Hsp90α is ATP-independent.
Next the effect of Hsp90α and Hsp90β on
MMP-2 processing was compared at different
incubation times. Without the protection of other
factors, only 20% of Pro- or active MMP-2
remained after incubation at 37°C for 1 h (Fig.
2A, top left and Fig. 2B); whereas with equal
molar amounts of rHsp90α, > 90% MMP-2 were
in their Pro- or activated forms after 1 h
incubation at 37°C, and even after 20 h
incubation, more than 30% MMP-2 was still
active (Fig. 2A, top right and Fig. 2B). This
result is indicative that Hsp90α has the ability to
remarkably stabilize MMP-2. Similar to the
result shown in Supplementary Fig. S3C, here
Hsp90β also showed no obvious effect on the
stabilization of MMP-2 (Fig. 2A, bottom and Fig.
2B).
To further confirm this result, the effects of
Hsp90α and Hsp90β on MMP-2 stabilization
were compared in a concentration gradient. The
result showed that after incubation at 37°C for 1
h (without Hsp90α or Hsp90β), 80% of Pro- or
active MMP-2 was processed into small inactive
fragments, while equal molar amounts of
Hsp90α can inhibit the inactivation processing of
MMP-2 completely (Fig. 2C, top and Fig. 2D).
As for Hsp90β, even after increasing its
concentration to 40 μM, which is the eight-fold
of the concentration of MMP-2, the inactivation
processing of MMP-2 could not be inhibited
completely (Fig. 2C, bottom and Fig. 2D). Based
on the above results, we conclude that Hsp90α
can stabilize MMP-2 directly and specifically.
Since Hsp90α and Hsp90β are closely related
isoforms of Hsp90, which are 86% identical and
93% similar in their amino acid sequences
(19,20), it seems contradictory that Hsp90β
shows no effect on the stabilization of MMP-2.
To investigate this mystery, the status of Hsp90α
and Hsp90β during incubation with MMP-2 was
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explored. Interestingly, Hsp90α was found to be
gradually and slowly degraded by equal molar
amounts of MMP-2 within 20 h (Supplementary
Fig. S4A), while Hsp90β was rapidly degraded
within 1 h and was found to be much more
unstable than Hsp90α (Supplementary Fig. S4B).
This result provides an explanation for the
different behaviors of Hsp90α and Hsp90β in the
stabilization of MMP-2, which is not only
determined by the interaction with MMP-2 but
also the individual stabilities of each chaperone.
The hemopexin domain is essential for the
interaction of MMP-2 with Hsp90α. To
investigate the molecular mechanism of the
stabilization of MMP-2 by Hsp90α, the binding
region of MMP-2 to Hsp90α was mapped. The
different functional domains of MMP-2 are
shown in the top panel of Fig. 3A. Mutants
ΔPEX (full length MMP-2 lacking the
hemopexin domain) and PEX (hemopexin
domain with the signal-peptide for secretion)
were constructed and their interactions with
Hsp90α were examined by
co-immunoprecipitation (Co-IP). Hsp90α was
co-precipitated with both the MMP-2 full length
(FL) and PEX but not ΔPEX (Fig. 3A, bottom),
demonstrating that MMP-2 binds to Hsp90α via
the hemopexin domain.
The interaction of Hsp90α with the
hemopexin domain was further confirmed by a
competitive Co-IP assay. Increasing amounts of
GST-fused recombinant hemopexin domain
(GST-PEX, 0, 1, 10 μg/ml) were added to the
concentrated CM of MCF-7 stable cell line
overexpressing MMP-2 (Fig. 3B, top left). The
Co-IP of Hsp90α and MMP-2 was blocked by
GST-PEX in a dose-dependent manner (Fig. 3B,
top right and bottom left), while the Co-IP of
Hsp90α and GST-PEX was increased along with
the concentration gradient of GST-PEX (Fig. 3B,
top right and bottom right). In sum, these results
consistently demonstrate that the hemopexin
domain is essential for the interaction of Hsp90α
and MMP-2.
Hsp90α binds to MMP-2 via its middle
domain. Next the binding site of Hsp90α to
MMP-2 was mapped. We constructed flag
tagged truncation mutants of Hsp90α according
to its functional domains, the N-terminal (N),
middle (M) and C-terminal (C) domains, which
are schematically shown in the top panel of Fig.
3C. Then we transfected these truncations to
MCF-7 cells and used anti-flag antibody to
perform Co-IP. The precipitated proteins were
detected by anti-MMP-2 antibody. The result
showed that MMP-2 was co-precipitated with
Hsp90α FL, M, NM and MC domains but not N
or C domain, demonstrating that Hsp90α
interacts with MMP-2 via its middle domain (Fig.
3C, bottom left). Intriguingly, the MMP-2
proteins co-precipitated with Hsp90α were
mainly in the activated form (Fig. 3C, bottom
left), implicating the specific stabilization effect
of Hsp90α on the activated MMP-2.
It is reported that the middle domain of
Hsp90α is responsible for the binding of several
Hsp90α substrate proteins, and is considered to
discriminate different substrate types and to
regulate the chaperone machinery for proper
substrate activation (21). According to the
reported mutations which were shown to be
important for these substrates binding (21,22),
we constructed point mutations of Hsp90α (Fig.
3C, top) and screened the binding sites of
Hsp90α to MMP-2. Strikingly, we identified that
three clusters of mutations,
F349A/L351A/F352A, R400A/E401K and
V368A/F369A can completely abrogate the
interaction of Hsp90α to MMP-2 (Fig. 3C,
bottom right); while another mutation, W320A,
which disrupts the binding of PKB/AKT to
Hsp90α (23), showed no such effect (Fig. 3C,
bottom right).
Subsequently, we prepared the recombinant
proteins of Hsp90α mutants
F349A/L351A/F352A and R400A/E401K, then
compared their abilities on MMP-2 stabilization
with that of the wild type (WT) Hsp90α. As
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expected, these two non-MMP2-binding Hsp90α
mutants cannot protect MMP-2 from inactivation
processing (Fig. 3D), demonstrating that the
protection of Hsp90α on MMP-2 requires
physical interaction and this further confirms
that Hsp90α can specifically stabilize MMP-2.
Hsp90α promotes the transmigration and tube
formation of endothelial cells. As we
demonstrated that Hsp90α stabilizes MMP-2 and
protects it from inactivation processing via
binding to the hemopexin domain, we further
explored the biological relevance of this
mechanism. In addition to the cancer cells,
endothelial cells also express high level of
MMP-2 (24) which is an important positive
regulator of angiogenesis (12), we thus
considered whether Hsp90α can be secreted by
endothelial cells, which would subsequently be
involved in the modulation of angiogenesis.
To address this question, the secretion of
Hsp90α from two kinds of endothelial cells,
human dermal microvascular endothelial cells
(HMECs) and human umbilical venous
endothelial cells (HUVECs), was examined.
Without any stimulation, both HMECs and
HUVECs secrete low level of Hsp90α compared
to tumor cells (Fig. 4A), while when endothelial
cells were treated with increasing amount of
vascular endothelial growth factor
(VEGF-A165), which can stimulate the
proliferation and induce the angiogenic
responses of endothelial cells, the secretion of
Hsp90α from both HMECs and HUVECs was
increased remarkably (Fig. 4B-C), suggesting
that the secretion of Hsp90α is correlated with
the angiogenic status of endothelial cells.
Subsequently, we examined the influence of
rHsp90α and Hsp90α mAb on MMP-2
processing and angiogenesis using endothelial
cells. rHsp90β and Hsp90β mAb were taken as
the control proteins. As expected, rHsp90α
inhibited, while Hsp90α mAb promoted the
inactivation processing of MMP-2 in HMECs
(Fig. 5A). rHsp90β and Hsp90β mAb exhibited
no obvious effect on MMP-2 processing (Fig.
5A). Coincidently, the transmigration of HMECs
through extracellular matrix (Matrigel) was
increased upon the treatment of rHsp90α, and
decreased with the treatment of Hsp90α mAb
(Fig. 5B; Supplementary Fig. S5B). Moreover,
with the knock down of MMP-2 in HMECs
(Supplementary Fig. S5A), the
pro-transmigration effect of rHsp90α was almost
completely abrogated (Fig. 5B; Supplementary
Fig. S5B). Similar results were also obtained in
the tube formation assay in vitro (Fig. 5C).
To further confirm the direct effect of the
interaction between Hsp90α and MMP-2 in
promoting angiogenesis, a Matrigel plug assay
with lentivirus delivered MMP-2 shRNA was
employed to examine the role of Hsp90α in
angiogenesis in vivo. The knock down and
infectious efficiency of the lentivirus delivered
MMP-2 shRNA was examined and shown in
Supplementary Fig. S6A-B. The tube formation
in Matrigel plug was detected by
immuno-fluorescence staining with anti-CD31
antibody. The result showed that Hsp90α but not
Hsp90β promoted angiogenesis in an MMP-2
dependent manner (Fig. 5D; Supplementary Fig.
S6C), while the antibody of Hsp90α suppressed
angiogenesis remarkably (Fig. 5D;
Supplementary Fig. S6C). Taken together, these
results demonstrate that Hsp90α can promote
angiogenesis via stabilizing MMP-2.
Hsp90α promotes tumor angiogenesis and
growth in vivo. As we found that Hsp90α can be
secreted by endothelia cells and promotes
endothelial cell transmigration and tube
formation in vitro and in vivo, we proposed that
Hsp90α may also be involved in the
angiogenesis of tumors in vivo. So we next
examined the effect of Hsp90α mAb on tumor
growth and angiogenesis using the B16/F10
melanoma mouse model. Hsp90β mAb was
taken as the control protein. The results indicate
that Hsp90α mAb but not Hsp90β mAb can
suppress tumor growth in a dose-dependent
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manner (Fig. 6A-B). Furthermore, the blood
vessel density (BVD) of the different groups was
assessed using CD31 staining, which showed a
significant decrease upon the treatment of
Hsp90α mAb but not Hsp90β mAb (Fig. 6C).
Collectively, the above results suggest that the
antibody of Hsp90α is a potential therapeutic
agent for the inhibition of not only tumor
metastasis (8-10) but also tumor angiogenesis
and growth.
Since Hsp90α mAb remarkably inhibits tumor
angiogenesis, we wondered whether the direct
localization of Hsp90α mAb on tumor blood
vessels can be observed in vivo. Therefore the
localization of the administrated antibodies in
tumor tissues was detected using the
FITC-conjugated anti-mouse IgG antibody. In
the groups treated with control mouse IgG or
Hsp90β mAb, no specific staining was observed
in the tumor tissues; while Hsp90α mAb was
specifically localized on the tumor blood vessels
and the periphery of the tumor cells (Fig. 6D),
reflecting the localization of endogenous
Hsp90α on the same sites and its important role
in modulating tumor angiogenesis.
Next we compared the level of active MMP-2
in tumors treated with Hsp90 monoclonal
antibodies by Western blotting. Consistently, the
antibody of Hsp90α but not Hsp90β promoted
the degradation of MMP-2. The total amounts of
Pro- and activated MMP-2 were decreased in the
tumors treated with Hsp90α mAb but not
Hsp90β mAb (Fig. 6E). These results further
confirm that Hsp90α can actually stabilize and
enhance the activity of MMP-2 in vivo, which
subsequently promotes tumor angiogenesis and
invasiveness.
DISCUSSION
The regulatory mechanism of Hsp90α on
MMP-2 processing. The activity of MMP-2 is
regulated at four levels: gene expression,
compartmentalization (localization), proenzyme
(zymogen) activation, and enzyme inactivation
(inhibition or processing) (25). The mechanism
of ProMMP-2 activation (26) and the inhibition
of activated MMP-2 (27) are relatively clear, but
the regulation of MMP-2 processing is still
poorly understood.
In this study, we demonstrate that the
processing of MMP-2 is regulated by
extracellular Hsp90α (Fig. 1-2). Many substrates
or clients of Hsp90α are structurally labile
signaling proteins (3). In the absence of Hsp90α
binding, these proteins are susceptible to
aggregation or degradation (1,3,4). Structural
variability was also found in MMPs. Almost all
of the MMPs possess two terminal globular
domains (the catalytic domain and the
hemopexin domain) connected by an
unstructured linker. Recent studies have found
that the linker is flexible and facilitates the
conformational change of MMPs from a
compact structure into an elongated one (28-30).
Although not experimentally confirmed, a
similar conformational freedom was also
observed by molecular dynamics simulation in
MMP-2 (31). Upon the conformational change
of MMP-2 from a compact to an elongated one,
its linker domain becomes loose and exposed,
which may facilitate the access and cleavage by
degrading enzymes. In our study, using
N-terminal sequencing, we verified a cleavage
site of MMP-2 processing protected by Hsp90α,
which is Glu443-Leu444 and locates in the linker
domain of MMP-2 (Supplementary Fig. S7A).
Interestingly, a similar cleavage mechanism was
also observed in the hinge domain of MT1-MMP,
which contains a highly exposed loop and is the
prime target for proteolysis (32). The
aforementioned result provides evidence that this
conformational change induces the instability of
MMP-2. In the presence of Hsp90α, which is
demonstrated here to bind with MMP-2 via the
hemopexin domain, the elongated structure
would be stabilized and the cleavage site would
be shielded. A hypothetical model of Hsp90α
mediated the stabilization of MMP-2 is shown in
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Supplementary Fig. S7B.
The interaction of Hsp90α and MMP-2.
Previous studies have shown that Hsp90α
interacts with MMP-2, and that the impermeable
inhibitor of Hsp90α (immobilized geldanamycin)
decreases the activity of MMP-2 (8). In this
study, we demonstrate that Hsp90α interacts
with the C-terminal hemopexin domain of
MMP-2, and this binding protects MMP-2 from
inactivation processing (Fig. 1-2). Furthermore,
Hsp90α antibody promotes the inactivation
processing of MMP-2 (Fig. 1D, 5A and 6E),
which is consistent with the effect of the
impermeable Hsp90 inhibitor reported
previously (8). Therefore, our findings provide a
novel mechanistic explanation for the regulation
of MMP-2 activity by Hsp90α, which is
attributed to the stabilization effect of Hsp90α on
the processing of MMP-2.
In addition, we observed that the stabilization
effect of Hsp90α on MMP-2 is ATP-
independent (Supplementary Fig. S3C). Hsp90α
contains an ATPase domain, and the hydrolysis
of ATP is essential for Hsp90α to bind with its
cochaperones and various client proteins in the
cytosol (18). However, it has also been observed
that ATP is not required for Hsp90α to exert its
holding and stabilization functions on partially
unfolded proteins (33,34), our results are
consistent with this aspect. It is proposed that
ATP binding and hydrolysis may only be
essential for Hsp90α chaperoning machinery that
requires the interplay of Hsp90α with its
cochaperones, regulators and clients (2). On the
other hand, extracellular Hsp90α was identified
to be hyperacetylated, which cannot bind to ATP
but still bind to MMP-2 (35). Very recently, our
group reported that extracellular Hsp90α is
phosphorylated at Thr90 which is located in the
ATP-binding pocket and may also influence the
binding of ATP to Hsp90α (9). These reports all
implicate that the function of extracellular
Hsp90α is ATP-independent.
Besides, though ATP is not essential for the
activity of extracellular Hsp90α, geldanamycin
and its derivatives, the inhibitors of Hsp90α
which act by blocking ATP binding (17,36), are
still able to bind with extracellular Hsp90α (35).
Then their inhibitory effects on MMP-2 activity
and tumor invasiveness (8) may be attributed to
the attenuation of clients binding.
Extracellular Hsp90α is a substrate of MMP-2.
As an exosite, the hemopexin domain determines
the interaction of several substrates to MMP-2
(37). Extracellular Hsp90α, which is identified to
be a hemopexin domain binding protein here
(Fig. 3A-B), is a highly probable substrate of
MMP-2. In fact, the cleavage of Hsp90α by
MMP-2 was detected previously by analyzing
the substrate degradome of MMP-2 (38). In our
work, the degradation of Hsp90α upon the
treatment of MMP-2 was also observed
(Supplementary Fig. S4A), and the major
degradation products (~80 and ~50 kDa) were
consistent with the previous report (38). More
interestingly, Hsp90β, which is quite similar to
Hsp90α (6,39), is actually extremely unstable
upon MMP-2 treatment. It can be degraded
completely within 1 h at 37°C by equal molar
amounts of MMP-2 (Supplementary Fig. S4B),
while Hsp90α is degraded much more slowly
under the same condition (Supplementary Fig.
S4A). These results indicate that the proteolysis
mechanisms of MMP-2 on Hsp90α and Hsp90β
are quite different, which may be determined by
both the amino acid sequences and secondary
structures of these two isoforms. Although we
have detected the cleavage of Hsp90β by
MMP-2 by an in vitro non-cell system, whether
it is a natural substrate of MMP-2 remains to be
determined. Ironically, an unfair game appears to
exist here: on one hand, Hsp90α stabilizes
MMP-2 and prevents it from inactivation
processing, but MMP-2 degrades Hsp90α! This
observation once again illustrates that the
biological system is nothing if not complex.
Extracellular Hsp90α, angiogenesis and
tumor microenvironment. Since intracellular
8
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9
Hsp90α is essential for the stability of diverse
cell signaling pathways of cancer cell (40), the
inhibitors of Hsp90 can suppress tumor
progression by targeting almost all hallmarks of
cancer cells, including angiogenesis (41-43). On
the other side, extracellular Hsp90α was
considered to play a unique role in tumor
metastasis (7). In our study, extracellular Hsp90α
is demonstrated for the first time to be a positive
regulator of tumor angiogenesis (Fig. 5-6),
signifying the potential role of Hsp90α antibody
on the inhibition of tumor angiogenesis and
growth.
In addition, along with the discovery of
additional extracellular Hsp90α client proteins
(44-46), more functions of extracellular Hsp90α
will be revealed. We propose that Hsp90α is not
only a biochemical buffer for the genetic lesions
of tumor cells in the cytosol, but also essential
for the homeostasis of entire tumor
microenvironment. Consequently, the
applications of Hsp90α antibody or impermeable
inhibitors of Hsp90α will be extended and not
only limited to the inhibition of metastasis.
Moreover, it was shown that other molecular
chaperones, such as Hsp70, GRP78, and
GRP94/gp96, also exist extracellularly and are
involved in the regulation of angiogenesis
(47-49) or other components of tumor
microenvironment (7). The applications of these
extracellular chaperones as therapeutic targets in
cancer treatment all merit further investigations.
In summary, our results demonstrate for the
first time that extracellular Hsp90α stabilizes
MMP-2 via the interaction of the middle domain
of Hsp90α and the hemopexin domain of
MMP-2, providing novel mechanistic
explanations for both the function of
extracellular Hsp90α and the regulatory
mechanism of MMP-2 activity. Furthermore, we
reveal that Hsp90α can be secreted by
endothelial cells and promotes angiogenesis in
vitro and in vivo, while the antibody of Hsp90α
is a potential anti-tumor drug targeting not only
metastasis but also angiogenesis.
REFERENCES 1. Pearl, L. H., and Prodromou, C. (2006) Annu Rev Biochem 75, 271-294
2. Picard, D. (2002) Cell Mol Life Sci 59, 1640-1648
3. Whitesell, L., and Lindquist, S. L. (2005) Nat Rev Cancer 5, 761-772
4. Caplan, A. J., Mandal, A. K., and Theodoraki, M. A. (2007) Trends Cell Biol 17, 87-92
5. Kim, Y. S., Alarcon, S. V., Lee, S., Lee, M. J., Giaccone, G., Neckers, L., and Trepel, J. B. (2009) Curr
Top Med Chem 9, 1479-1492
6. Chen, B., Piel, W. H., Gui, L., Bruford, E., and Monteiro, A. (2005) Genomics 86, 627-637
7. Tsutsumi, S., and Neckers, L. (2007) Cancer Science 98, 1536-1539
8. Eustace, B. K., Sakurai, T., Stewart, J. K., Yimlamai, D., Unger, C., Zehetmeier, C., Lain, B., Torella,
C., Henning, S. W., Beste, G., Scroggins, B. T., Neckers, L., Ilag, L. L., and Jay, D. G. (2004) Nat Cell
Biol 6, 507-514
9. Wang, X., Song, X., Zhuo, W., Fu, Y., Shi, H., Liang, Y., Tong, M., Chang, G., and Luo, Y. (2009) Proc
Natl Acad Sci U S A 106, 21288-21293
10. Stellas, D., Karameris, A., and Patsavoudi, E. (2007) Clinical Cancer Research 13, 1831-1838
11. Tsutsumi, S., Scroggins, B., Koga, F., Lee, M. J., Trepel, J., Felts, S., Carreras, C., and Neckers, L.
(2008) Oncogene 27, 2478-2487
12. Stefanidakis, M., and Koivunen, E. (2006) Blood 108, 1441-1450
13. Ades, E. W., Candal, F. J., Swerlick, R. A., George, V. G., Summers, S., Bosse, D. C., and Lawley, T. J.
by guest on August 29, 2018
http://ww
w.jbc.org/
Dow
nloaded from
(1992) J Invest Dermatol 99, 683-690
14. Jaffe, E. A., Nachman, R. L., Becker, C. G., and Minick, C. R. (1973) Journal of Clinical Investigation
52, 2745-2756
15. Bergmann, U., Tuuttila, A., Stetler-Stevenson, W. G., and Tryggvason, K. (1995) Biochemistry 34,
2819-2825
16. Howard, E. W., Bullen, E. C., and Banda, M. J. (1991) J Biol Chem 266, 13064-13069
17. Prodromou, C., Roe, S. M., O'Brien, R., Ladbury, J. E., Piper, P. W., and Pearl, L. H. (1997) Cell 90,
65-75
18. Grenert, J. P., Johnson, B. D., and Toft, D. O. (1999) J Biol Chem 274, 17525-17533
19. Stebbins, C. E., Russo, A. A., Schneider, C., Rosen, N., Hartl, F. U., and Pavletich, N. P. (1997) Cell 89,
239-250
20. Wright, L., Barril, X., Dymock, B., Sheridan, L., Surgenor, A., Beswick, M., Drysdale, M., Collier, A.,
Massey, A., Davies, N., Fink, A., Fromont, C., Aherne, W., Boxall, K., Sharp, S., Workman, P., and
Hubbard, R. E. (2004) Chemistry & Biology 11, 775-785
21. Hawle, P., Siepmann, M., Harst, A., Siderius, M., Reusch, H. P., and Obermann, W. M. (2006) Mol Cell
Biol 26, 8385-8395
22. Meyer, P., Prodromou, C., Hu, B., Vaughan, C., Roe, S. M., Panaretou, B., Piper, P. W., and Pearl, L. H.
(2003) Mol Cell 11, 647-658
23. Sato, S., Fujita, N., and Tsuruo, T. (2000) Proc Natl Acad Sci U S A 97, 10832-10837
24. Egeblad, M., and Werb, Z. (2002) Nat Rev Cancer 2, 161-174
25. Nagase, H., Visse, R., and Murphy, G. (2006) Cardiovasc Res 69, 562-573
26. Ra, H. J., and Parks, W. C. (2007) Matrix Biology 26, 587-596
27. Stetler-Stevenson, W. G., and Seo, D. W. (2005) Trends Mol Med 11, 97-103
28. Bertini, I., Calderone, V., Fragai, M., Jaiswal, R., Luchinat, C., Melikian, M., Mylonas, E., and Svergun,
D. I. (2008) J Am Chem Soc 130, 7011-7021
29. Rosenblum, G., Van den Steen, P. E., Cohen, S. R., Grossmann, J. G., Frenkel, J., Sertchook, R., Slack,
N., Strange, R. W., Opdenakker, G., and Sagi, I. (2007) Structure 15, 1227-1236
30. Bertini, I., Fragai, M., Luchinat, C., Melikian, M., Mylonas, E., Sarti, N., and Svergun, D. I. (2009) J
Biol Chem
31. Diaz, N., Suarez, D., and Valdes, H. (2008) J Am Chem Soc 130, 14070-14071
32. Osenkowski, P., Meroueh, S. O., Pavel, D., Mobashery, S., and Fridman, R. (2005) J Biol Chem 280,
26160-26168
33. Minami, Y., Kawasaki, H., Minami, M., Tanahashi, N., Tanaka, K., and Yahara, I. (2000) J Biol Chem
275, 9055-9061
34. Yonehara, M., Minami, Y., Kawata, Y., Nagai, J., and Yahara, I. (1996) J Biol Chem 271, 2641-2645
35. Yang, Y., Rao, R., Shen, J., Tang, Y., Fiskus, W., Nechtman, J., Atadja, P., and Bhalla, K. (2008) Cancer
Res 68, 4833-4842
36. Stebbins, C. E., Russo, A. A., Schneider, C., Rosen, N., Hartl, F. U., and Pavletich, N. P. (1997) Cell 89,
239-250
37. Piccard, H., Van den Steen, P. E., and Opdenakker, G. (2007) Journal of Leukocyte Biology 81, 870-892
38. Dean, R. A., and Overall, C. M. (2007) Mol Cell Proteomics 6, 611-623
39. Csermely, P., Schnaider, T., Soti, C., Prohaszka, Z., and Nardai, G. (1998) Pharmacol Ther 79, 129-168
40. Xu, W., and Neckers, L. (2007) Clin Cancer Res 13, 1625-1629
41. Kaur, G., Belotti, D., Burger, A. M., Fisher-Nielson, K., Borsotti, P., Riccardi, E., Thillainathan, J.,
10
by guest on August 29, 2018
http://ww
w.jbc.org/
Dow
nloaded from
11
Hollingshead, M., Sausville, E. A., and Giavazzi, R. (2004) Clin Cancer Res 10, 4813-4821
42. Eccles, S. A., Massey, A., Raynaud, F. I., Sharp, S. Y., Box, G., Valenti, M., Patterson, L., de Haven
Brandon, A., Gowan, S., Boxall, F., Aherne, W., Rowlands, M., Hayes, A., Martins, V., Urban, F.,
Boxall, K., Prodromou, C., Pearl, L., James, K., Matthews, T. P., Cheung, K. M., Kalusa, A., Jones, K.,
McDonald, E., Barril, X., Brough, P. A., Cansfield, J. E., Dymock, B., Drysdale, M. J., Finch, H.,
Howes, R., Hubbard, R. E., Surgenor, A., Webb, P., Wood, M., Wright, L., and Workman, P. (2008)
Cancer Res 68, 2850-2860
43. Okawa, Y., Hideshima, T., Steed, P., Vallet, S., Hall, S., Huang, K., Rice, J., Barabasz, A., Foley, B.,
Ikeda, H., Raje, N., Kiziltepe, T., Yasui, H., Enatsu, S., and Anderson, K. C. (2009) Blood 113, 846-855
44. Sidera, K., Gaitanou, M., Stellas, D., Matsas, R., and Patsavoudi, E. (2008) J Biol Chem 283,
2031-2041
45. Lei, H. T., Romeo, G., and Kazlauskas, A. (2004) Circulation Research 94, 902-909
46. Cheng, C. F., Fan, J., Fedesco, M., Guan, S., Li, Y., Bandyopadhyay, B., Bright, A. M., Yerushalmi, D.,
Liang, M., Chen, M., Han, Y. P., Woodley, D. T., and Li, W. (2008) Mol Cell Biol 28, 3344-3358
47. Kern, J., Untergasser, G., Zenzmaier, C., Sarg, B., Gastl, G., Gunsilius, E., and Steurer, M. (2009) Blood
114, 3960-3967
48. Tramentozzi, E., Montopoli, M., Orso, G., Pagetta, A., Caparrotta, L., Frasson, M., Brunati, A. M., and
Finotti, P. (2008) Mol Immunol 45, 3639-3648
49. Futagami, S., Hiratsuka, T., Shindo, T., Hamamoto, T., Horie, A., Ueki, N., Kusunoki, M., Gudis, K.,
Miyake, K., Tsukui, T., and Sakamoto, C. (2008) J Gastroenterol Hepatol 23 Suppl 2, S222-228
FOOTNOTES
*This study was supported in part by the General Programs of the National Natural Science
Foundation of China (No. 30670419 and No. 30771083), the Major Program of National Natural
Science Foundation of China (No. 30490171), the National High Technology Research and
Development Program of China (No. 2007AA02Z155), and the State Key Development Program for
Basic Research of China (No. 2006CB91030).
We thank the members of the Luo lab greatly for their insightful discussions and suggestions on the
manuscript. We are also grateful to Prof. Duanqing Pei (GIBH, Chinese Academy of Sciences) for his
generosity in providing the plasmid of MMP-2. Special appreciation is extended to Bipo Sun for her
contribution as the lab manager.
The abbreviations used are: MMP-2, matrix metalloproteinase-2; Hsp90α, Heat shock protein 90 α;
PEX, hemopexin-domain of MMP-2; CM, conditioned media; CL, cell lysate; HMEC, human dermal
microvascular endothelial cell line; HUVECs, human umbilical venous endothelial cells.
FIGURE LEGENDS
Fig. 1. Hsp90α prevents the inactivation processing of MMP-2. A. Establishment of MMP-2
overexpression stable cell line. The overexpression was examined by anti-MMP-2 and anti-Myc
antibodies both in cell lysate (CL) and conditioned media (CM). B-C. The processing of MMP-2 in
CM was examined upon the treatment of rHsp90α on cells for 24 h by Western blotting (B) and
zymography (C). Actin in CL and BSA in CM was used as loading control. D-E. The effect of Hsp90α
antibody (Hsp90α Ab) on MMP-2 processing was assessed using the same procedure as that in panel
B and C. F-G. The intensity of the bands in panel B and D was quantified by Gel-Pro Analyzer
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software. The percentage of MMP-2 activation (activated MMP-2 / ProMMP-2) and inactivation
processing (MMP-2 ~30 kDa inactive fragment / total MMP-2 proteins) was analyzed. H. The
N-terminal sequence of the ~30 kDa inactive fragment was identified to be LYGAS, shown within the
full length of MMP-2. S, signal peptide; Pro, propeptide; Cat, catalytic site; FN, fibronectin-like
domain; Zn+, Zinc binding domain; L, linker domain; PEX, Hemopexin-like domain.
Fig. 2. Hsp90α stabilizes MMP-2 directly. A. The processing of ProMMP-2 alone or the one
co-incubated with rHsp90α or rHsp90β at 37°C was examined after different incubation periods. The
processing products were detected by Western blotting using anti-Myc antibody. B. The percentage of
MMP-2 inactivation processing (MMP-2 ~30 kDa inactive fragment / total MMP-2 proteins) in panel
A was calculated according to the intensity of the bands, which was quantified by Gel-Pro Analyzer
software. C. The effect of different concentrations of rHsp90α or rHsp90β on MMP-2 processing was
explored after co-incubation at 37°C for 1 h. The processing product was detected by anti-Myc
antibody. D. The percentage of MMP-2 inactivation processing in panel C was quantified using the
same method as that in panel B.
Fig. 3. The interaction domains of Hsp90α and MMP-2. A. The Co-IP of Hsp90α and MMP-2
fragments from the CM of MCF-7 cells transfected with MMP-2 FL or deletion mutants
(schematically represented on top of the panel). B. Competitive Co-IP of PEX and MMP-2 with
Hsp90α. Hsp90α and MMP-2 were co-precipitated by the anti-Hsp90α or anti-Myc antibodies with
Protein A agarose beads from the CM containing increasing amount of GST-PEX. Glutathione
sepharose 4B beads were used to pull down the complex of GST-PEX and Hsp90α. C. The Co-IP of
Hsp90α fragments or mutants (schematically shown on top of the panel) with MMP-2. D. The
processing of ProMMP-2 alone or the one co-incubated with Hsp90α WT or non-binding mutants was
examined at 37°C.
Fig. 4. Endothelial cells secrete Hsp90α. A. MCF-7, MDA-MB-231, HMEC, HUVEC and B16/F10
cells were plated in 6 cm dishes. When the cells grew to 80% confluence, media were replaced with
fresh one without FCS. After 8 h, the CM were collected and concentrated by 10 folds. The amounts
of Hsp90α in CM and CL were analyzed by Western blotting. Actin in CL was used as loading
controls. B. HMECs were firstly serum starved overnight, then the media were changed into fresh
ones and the cells were stimulated by VEGF for 8 h. The CM were collected and concentrated by 10
folds. Hsp90α in CM and CL were analyzed by Western blotting. C. The secretion of Hsp90α from
HUVECs was explored by the same method used in the panel B.
Fig. 5. Hsp90α stabilizes MMP-2 and promotes the transmigration and tube formation of
endothelial cells in vitro and in vivo. A. rHsp90α, rHsp90β, mouse IgG, Hsp90α mAb or Hsp90β mAb
(5 μg/ml) was added to the culture media (without FCS) of HMECs. After 24 h treatment, the CM was
collected and concentrated by 10 folds. MMP-2 and its fragments were detected by anti-MMP-2
antibody. B. The transmigration of HMECs (transfected with MMP-2 shRNA or not) treated by Hsp90
recombinant proteins or antibodies (5 μg/ml) were examined by Matrigel transmigration assay in
Millicell chambers. The transmigrated cells were stained by H&E and counted. Error bars represent
SD (n=5). P value: Student’s t-test. *, P<0.05; **, P<0.01; #, P> 0.05. C. The tube formation of
HMECs (transfected with MMP-2 shRNA or not) treated by Hsp90 recombinant proteins or
12
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13
antibodies (5 μg/ml) were examined by Matrigel model in vitro. Error bars represent SD (n=5). P
value: Student’s t-test. *, P<0.05; **, P<0.01; #, P> 0.05. D. The effects of Hsp90 recombinant
proteins or antibodies (50 μg/ml) on tube formation in Matrigel plug containing lentivirus delivered
MMP-2 shRNA or not were examined in nude mice. The tube formation was detected by
immuno-fluorescence using CD31 staining. The density of the tube was calculated by NIS-Elements
C, the software of Nikon A1 confocal. The Error bars represent SD (n=5). P value: Student’s t-test. *,
P<0.05; **, P<0.01; #, P> 0.05.
Fig. 6. The effect of Hsp90α antibody on tumor growth and angiogenesis in the B16/F10 melanoma
mouse model. Mouse IgG, Hsp90α mAb (5, 10 mg/kg) or Hsp90β mAb (5, 10 mg/kg) was injected i.p.
for 25 days. A. The tumor growth was monitored every 5 days from the 5th day after the tumor
implantation. B. The tumor weight was measured after the sacrifice. C. The blood vessel density in
tumor tissue was assessed by immuno-fluorescence using anti-CD31 antibody. Representitive images
were shown in the left. Scale bar: 100 μm. Quantified result was analyzed by NIS-Elements C,
software of Nikon A1 confocal. Error bars represent SD (n=5). P value: Student’s t-test. *, P<0.05; **,
P<0.01. D. The localization of administered Hsp90α mAb or Hsp90β mAb in tumor tissues was
detected by FITC-conjugated anti-mouse IgG. Anti-CD31 antibody was used for detecting blood
vessels. Scale bar: 20 μm. E. The processing of MMP-2 in tumor tissue lysate from the mice treated
with Hsp90 recombinant proteins or antibodies was detected by Western blotting.
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Yan Fu, Daifu Zhou and Yongzhang LuoXiaomin Song, Xiaofeng Wang, Wei Zhuo, Hubing Shi, Dan Feng, Yi Sun, Yun Liang,
(MMP-2) processing and tumor angiogenesis on matrix metalloproteinase-2αThe regulatory mechanism of extracellular Hsp90
published online October 11, 2010J. Biol. Chem.
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