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.

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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

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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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