{'fraction_non_alphanumeric': 0.04468283882606875, 'fraction_numerical': 0.03505041719016784, 'mean_word_length': 4.874716124148373, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
The Regulatory Mechanism of Extracellular Hsp90α on Matrix Metalloproteinase-2 Processing and Tumor Angiogenesis*

Heat shock protein 90α (Hsp90α) is a ubiquitously expressed molecular chaperone that 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 the Hsp90α middle domain and the MMP-2 C-terminal hemopexin domain. Moreover, this mechanism also applies to endothelial cells that 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) 2 is an ATP-dependent molecular chaperone that 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)(3)(4). Many of the 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 Hsp90␣ and Hsp90␤ (6). Intriguingly, the Hsp90␣ isoform also exists extracellularly (7). Recent studies indicate that extracellular Hsp90␣ is significantly correlated with tumor invasiveness and metastasis (8), and the antibody or impermeable inhibitor of Hsp90␣ can suppress tumor metastasis efficiently in mouse models (9 -11). Furthermore, Hsp90␣ can be detected in the blood of cancer patients, and the level of Hsp90␣ is positively associated with tumor malignancy (9). In addition to tumor cells, extracellular Hsp90␣ has also been identified in neuron cells, dermal fibroblasts, keratinocyte, macrophages, and epithelial cells and participates in neuronal cell migration, wound healing, and viral and bacteria infections (7).
Accumulating evidence indicates that extracellular Hsp90␣ plays important roles in both physiological and pathological processes, especially in tumor progression (7). However, the molecular mechanism of how extracellular Hsp90␣ functions is still largely unknown (12). Eustace et al. (8) reported that extracellular Hsp90␣ can interact with matrix metalloproteinase 2 (MMP-2) and that the impermeable inhibitor of Hsp90␣ (immobilized geldanamycin) inhibits MMP-2 proteolytic activity, but the regulatory molecular mechanism behind this phenomenon is still a mystery.
In the present study, to further elucidate the molecular mechanism of extracellular Hsp90␣ function, we have investigated the regulatory mechanism of extracellular Hsp90␣ on MMP-2 activity. We reveal that extracellullar Hsp90␣ stabilizes MMP-2 and protects it from processing and subsequent inactivation in tumor cells. The regulatory function of Hsp90␣ on MMP-2 processing is isoform-specific and ATPindependent. The interaction of Hsp90␣ and MMP-2 is mediated by the middle domain of Hsp90␣ and the C-terminal hemopexin domain of MMP-2. Moreover, we further confirm this mechanism in endothelial cells, which can secrete increasing amounts of Hsp90␣ upon the treatment of VEGF. The effects of recombinant human Hsp90␣ (rHsp90␣) and the Hsp90␣ antibody on angiogenesis in vitro and in vivo were also examined. The result shows that rHsp90␣ promotes whereas the antibody of Hsp90␣ suppresses angiogenesis in an MMP-2-dependent manner, suggesting that extracellular Hsp90␣ 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 and 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 (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 g/ml) from MCF-7 cells transfected with pcDNA3.1-MMP-2.
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 MgCl 2 , 20 mM KCl, 5 mM CaCl 2 , 2 mM p-aminophenylmercuric acetate, pH 7.4. The processing was terminated by electrophoresis sample buffer.
Endothelial Cell Transmigration Assay-This assay was performed using 6.5-mm millicell inserts (8-m pore size; Millipore) coated with Matrigel (50 g/insert) (Vigorous, Beijing, China) on the upper side of the filter membranes. 2 ϫ 10 5 HMECs/insert were inoculated. 0.5% FBS was added in the lower chamber. Then the cells were cultured for 24 h. The cells at the lower surface of the filter membranes were stained with hematoxylin and eosin. The images were visualized by microscope (Olympus IX71), and five random fields per insert were captured by CCD camera (Olympus DP71) for counting.
Tube Formation Assay-24-well plates were coated with 125 l/well of Matrigel. HMECs were seeded (1 ϫ 10 5 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 five random fields of view/well using Image-Pro Plus.
Tumor Growth Assay-B16/F10 cells (1 ϫ 10 6 , 100 l) were subcutaneously inoculated in the back of nude mice (6 -8 weeks old). Nonimmune mouse IgG (10 mg/kg), Hsp90␣ mAb (5 or 10 mg/kg, according to the previous report (10)), or Hsp90␤ mAb (5 or 10 mg/kg) was injected intraperitoneally every other day from the day after implantation. The experiment was performed with six mice in each group. After 25 days treatment, the mice were sacrificed, and the tumors were excised, weighed, fixed, and applied to immunofluorescence. All of the animal studies were performed with the approval of the Scientific Investigation Board of Tsinghua University.
Immunofluorescence Assay-Paraffin-embedded tumor tissues or Matrigel plugs were processed into 5-m-thick sections. The sections were rehydrated and blocked with 5% rabbit or goat serum and 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. The sections were analyzed by confocal microscopy (Nikon A1).

Hsp90␣ Prevents the Processing and Inactivation of MMP-2-
To investigate the regulatory mechanism of Hsp90␣ on MMP-2 activity, an MCF-7 stable cell line that overexpresses human MMP-2 with C-terminal tandem Myc and His tags was established (Fig. 1A). MMP-2 expression in the conditioned medium (CM) of this cell line treated with rHsp90␣ or its antibody was analyzed. As expected, activated MMP-2 was increased upon the treatment with increasing amounts of rHsp90␣ ( Fig. 1, B and C), which is consistent with the previous report (8). Intriguingly, the amount of ProMMP-2 was also increased, whereas some overprocessed fragments of MMP-2 (ϳ30 kDa), which were detected by Western blotting but not zymography (Fig. 1, B and C), were decreased in this process (Fig. 1B). Furthermore, the antibody of Hsp90␣ exhibited the opposite effect on MMP-2 processing (Fig. 1, D and E), whereas rHsp90␤ and anti-Hsp90␤ antibody showed no such effect (supplemental Fig. S1). These results indicate that Hsp90␣ 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 rHsp90␣ or anti-Hsp90␣ antibody also showed that Hsp90␣ was mainly involved in the inactivation processing but not the activation of MMP-2 ( Fig. 1, F and G).
We next sequenced the major processed fragment (ϳ30 kDa) observed above and identified its N terminus as LYGAS (supplemental Fig. S2), beginning from the amino acid residue 444 (Leu 444 ) of MMP-2 (Fig. 1H). Because this fragment can be detected by the anti-Myc antibody, it should end at the Myc-tagged C terminus of MMP-2 (Cys 660 ). Thus this fragment was identified as Leu 444 -Cys 660 comprised of both the hemopexin domain (Pro 466 -Cys 660 ) and 22 residues of the linker domain. The theoretical molecular mass of this fragment (Leu 444 -Cys 660 ) is 24.5 kDa. With the additional C-ter-minal tags, the molecular mass of the fragment is increased to 27.4 kDa, which is consistent with its migratory position on the SDS-PAGE (Fig. 1, B and D). According to the previous report, this fragment is a stable and inactive product of MMP-2 autocatalytic degradation (15,16). The cleavage at Glu 443 -Leu 444 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 Hsp90␣ can stabilize MMP-2 and protect it from autocatalytic cleavage at Glu 443 -Leu 444 , which subsequently leads to the complete inactivation of MMP-2.
Hsp90␣ Stabilizes MMP-2 in an ATP-independent and Isoform-specific Manner-To affirm the above hypothesis, we prepared the purified rHsp90␣, rHsp90␤ (supplemental Fig.  S3A), and rProMMP-2 (supplemental Fig. S3B) and investigated the influence of these chaperones on MMP-2 autocatalytic processing in an in vitro noncell system. Because Hsp90 is an ATP-dependent molecular chaperone in the cytosol (17,18), we first examined whether the stabilization effect of Hsp90␣ on MMP-2 is ATP-dependent. Purified rProMMP-2 was mixed with PBS, equal molar of Hsp90␣ or Hsp90␤ 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 Hsp90␣ but not Hsp90␤ can protect MMP-2 from autocatalytic processing and inactivation and that ATP exhibited no effect on the stabilization activity of both Hsp90␣ and Hsp90␤ (supplemental Fig. S3C), demonstrating that the stabilization of MMP-2 mediated by Hsp90␣ is ATP-independent.
Next the effect of Hsp90␣ and Hsp90␤ 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. 2, A, top left, and B), whereas with equal molar amounts of rHsp90␣, Ͼ90% MMP-2 were in their pro-or activated forms after 1 h of incubation at 37°C, and even after 20 h of incubation, more than 30% MMP-2 was still active (Fig. 2, A, top right, and B). This result is indicative that Hsp90␣ has the ability to remarkably  DECEMBER 17, 2010 • VOLUME 285 • NUMBER 51 stabilize MMP-2. Similar to the result shown in supplemental Fig. S3C, here Hsp90␤ also showed no obvious effect on the stabilization of MMP-2 ( Fig. 2, A, bottom, and B).

Extracellular Hsp90␣ and MMP-2 in Tumor Angiogenesis
To further confirm this result, the effects of Hsp90␣ and Hsp90␤ on MMP-2 stabilization were compared in a concentration gradient. The result showed that after incubation at 37°C for 1 h (without Hsp90␣ or Hsp90␤), 80% of pro-active or active MMP-2 was processed into small inactive fragments, whereas equal molar amounts of Hsp90␣ can inhibit the inactivation processing of MMP-2 completely (Fig. 2, C, top, and D). As for Hsp90␤, even after increasing its concentration to 40 M, which is 8-fold of the concentration of MMP-2, the inactivation processing of MMP-2 could not be inhibited completely (Fig. 2, C, bottom, and D). Based on the above results, we conclude that Hsp90␣ can stabilize MMP-2 directly and specifically.
Because Hsp90␣ and Hsp90␤ are closely related isoforms of Hsp90, which are 86% identical and 93% similar in their amino acid sequences (19,20), it seems contradictory that Hsp90␤ shows no effect on the stabilization of MMP-2. To investigate this mystery, the status of Hsp90␣ and Hsp90␤ during incubation with MMP-2 was explored. Interestingly, Hsp90␣ was found to be gradually and slowly degraded by equal molar amounts of MMP-2 within 20 h (supplemental Fig. S4A), whereas Hsp90␤ was rapidly degraded within 1 h and was found to be much more unstable than Hsp90␣ (supplemental Fig. S4B). This result provides an explanation for the different behaviors of Hsp90␣ and Hsp90␤ in the stabilization of MMP-2, which is determined not only by the interaction with MMP-2 but also by the individual stabilities of each chaperone. A, the processing of ProMMP-2 alone or the one co-incubated with rHsp90␣ or rHsp90␤ 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 ϳ30-kDa inactive fragment/total MMP-2 proteins) in 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 rHsp90␣ or rHsp90␤ 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 C was quantified using the same method as that in B. IB, immunoblot.
The Hemopexin Domain Is Essential for the Interaction of MMP-2 with Hsp90␣-To investigate the molecular mechanism of the stabilization of MMP-2 by Hsp90␣, the binding region of MMP-2 to Hsp90␣ was mapped. The different functional domains of MMP-2 are shown in the top panel of Fig. 3A. Mutants ⌬PEX (full-length MMP-2 lacking the hemopexin domain) and PEX (hemopexin domain with the signal peptide for secretion) were constructed, and their interactions with Hsp90␣ were examined by co-immunoprecipitation (Co-IP). Hsp90␣ was coprecipitated with both the MMP-2 full length and PEX but not ⌬PEX (Fig. 3A, bottom), demonstrating that MMP-2 binds to Hsp90␣ via the hemopexin domain.
The interaction of Hsp90␣ 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 g/ml) were added to the concentrated CM of MCF-7 stable cell line overexpressing MMP-2 (Fig. 3B, top left). The Co-IP of Hsp90␣ and MMP-2 was blocked by GST-PEX in a dose-dependent manner (Fig. 3B, top right and bottom left), whereas the  DECEMBER 17, 2010 • VOLUME 285 • NUMBER 51

JOURNAL OF BIOLOGICAL CHEMISTRY 40043
Co-IP of Hsp90␣ 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 Hsp90␣ and MMP-2.
Hsp90␣ Binds to MMP-2 via Its Middle Domain-Next the binding site of Hsp90␣ to MMP-2 was mapped. We constructed FLAG-tagged truncation mutants of Hsp90␣ according to its functional domains, the N-terminal, middle, and C-terminal 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 Hsp90␣ full length, middle, N-terminal/middle, and middle/C-terminal domains but not the N-terminal or Cterminal domain, demonstrating that Hsp90␣ interacts with MMP-2 via its middle domain (Fig. 3C, bottom left). Intriguingly, the MMP-2 proteins co-precipitated with Hsp90␣ were mainly in the activated form (Fig. 3C, bottom left), implicating the specific stabilization effect of Hsp90␣ on the activated MMP-2.
It is reported that the middle domain of Hsp90␣ is responsible for the binding of several Hsp90␣ 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 the binding of these substrates (21,22), we constructed point mutations of Hsp90␣ (Fig. 3C, top) and screened the binding sites of Hsp90␣ 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 Hsp90␣ to MMP-2 (Fig.  3C, bottom right), whereas another mutation, W320A, which disrupts the binding of PKB/AKT to Hsp90␣ (23), showed no such effect (Fig. 3C, bottom right).
Subsequently, we prepared the recombinant proteins of Hsp90␣ mutants F349A/L351A/F352A and R400A/E401K and then compared their abilities on MMP-2 stabilization with that of the WT Hsp90␣. As expected, these two non-MMP2-binding Hsp90␣ mutants cannot protect MMP-2 from inactivation processing (Fig. 3D), demonstrating that the protection of Hsp90␣ on MMP-2 requires physical interaction, and this further confirms that Hsp90␣ can specifically stabilize MMP-2.
Hsp90␣ Promotes the Transmigration and Tube Formation of Endothelial Cells-Because we demonstrated that Hsp90␣ 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 a high level of MMP-2 (24), which is an important positive regulator of angiogenesis (12); we thus considered whether Hsp90␣ can be secreted by endothelial cells, which would subsequently be involved in the modulation of angiogenesis.
To address this question, the secretion of Hsp90␣ from two kinds of endothelial cells, HMECs and HUVECs, was exam-ined. Without any stimulation, both HMECs and HUVECs secrete a low level of Hsp90␣ compared with tumor cells (Fig.  4A), whereas when endothelial cells were treated with increasing amounts of vascular endothelial growth factor (VEGF-A165), which can stimulate the proliferation and induce the angiogenic responses of endothelial cells, the secretion of Hsp90␣ from both HMECs and HUVECs was increased remarkably (Fig. 4, B and C), suggesting that the secretion of Hsp90␣ is correlated with the angiogenic status of endothelial cells.
Subsequently, we examined the influence of rHsp90␣ and Hsp90␣ mAb on MMP-2 processing and angiogenesis using endothelial cells. rHsp90␤ and Hsp90␤ mAb were taken as the control proteins. As expected, rHsp90␣ inhibited whereas Hsp90␣ mAb promoted the inactivation processing of MMP-2 in HMECs (Fig. 5A). rHsp90␤ and Hsp90␤ 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 rHsp90␣ and decreased with the treatment of Hsp90␣ mAb ( Fig. 5B and supplemental Fig. S5B). Moreover, with the knockdown of MMP-2 in HMECs (supplemental Fig. S5A), the pro-transmigration effect of rHsp90␣ was almost completely abrogated (Fig. 5B and supplemental 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 Hsp90␣ and MMP-2 in promoting angiogenesis, a Matrigel plug assay with lentivirus delivered MMP-2 shRNA was employed to examine the role of Hsp90␣ in angiogenesis in vivo. The knockdown and infectious efficiency of the lentivirus delivered MMP-2 shRNA was examined and shown in supplemental Fig. S6, A and B. The tube formation in Matrigel plug was detected by immunofluorescence staining with anti-CD31 antibody. The result showed that Hsp90␣ but not Hsp90␤ promoted angiogenesis in an MMP-2-dependent manner ( Fig. 5D and supplemental Fig. S6C), whereas the antibody of Hsp90␣ suppressed angiogenesis remarkably ( Fig.  5D and supplemental Fig. S6C). Taken together, these results demonstrate that Hsp90␣ can promote angiogenesis via stabilizing MMP-2.
Hsp90␣ Promotes Tumor Angiogenesis and Growth in Vivo-Because we found that Hsp90␣ can be secreted by endothelial cells and promotes endothelial cell transmigration and tube formation in vitro and in vivo, we proposed that Hsp90␣ may also be involved in the angiogenesis of tumors in vivo. We next examined the effect of Hsp90␣ mAb on tumor growth and angiogenesis using the B16/F10 melanoma mouse model. Hsp90␤ mAb was taken as the control protein. The results indicate that Hsp90␣ mAb but not Hsp90␤ mAb can suppress tumor growth in a dose-dependent manner (Fig. 6, A and B). Furthermore, the blood vessel density of the different groups was assessed using CD31 staining, which showed a significant decrease upon the treatment of Hsp90␣ mAb but not Hsp90␤ mAb (Fig. 6C). Collectively, the above results suggest that the antibody of Hsp90␣ is a potential therapeutic agent for the inhibition of not only tumor metastasis (8 -10) but also tumor angiogenesis and growth.
Because Hsp90␣ mAb remarkably inhibits tumor angiogenesis, we wondered whether the direct localization of Hsp90␣ 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 Hsp90␤ mAb, no specific staining was observed in the tumor tissues, whereas Hsp90␣ mAb was specifically localized on the tumor blood vessels and the periphery of the tumor cells (Fig. 6D), reflecting the localization of endogenous Hsp90␣ 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

Extracellular Hsp90␣ and MMP-2 in Tumor Angiogenesis
blotting. Consistently, the antibody of Hsp90␣ but not Hsp90␤ promoted the degradation of MMP-2. The total amounts of pro-activated and activated MMP-2 were decreased in the tumors treated with Hsp90␣ mAb but not Hsp90␤ mAb (Fig. 6E). These results further confirm that Hsp90␣ can actually stabilize and enhance the activity of MMP-2 in vivo, which subsequently promotes tumor angiogenesis and invasiveness.

DISCUSSION
The Regulatory Mechanism of Hsp90␣ 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 Hsp90␣ (Figs. 1 and 2). Many substrates or clients of Hsp90␣ are structurally labile signaling proteins (3). In the absence of Hsp90␣ 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 structure 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 Hsp90␣, which is Glu 443 -Leu 444 and locates in the linker domain of MMP-2 (supplemental 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 Hsp90␣, 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 showing how Hsp90␣ mediated the stabilization of MMP-2 is shown in supplemental Fig. S7B.
The Interaction of Hsp90␣ and MMP-2-Previous studies have shown that Hsp90␣ interacts with MMP-2 and that the impermeable inhibitor of Hsp90␣ (immobilized geldanamycin) decreases the activity of MMP-2 (8). In this study, we demonstrate that Hsp90␣ interacts with the C-terminal hemopexin domain of MMP-2, and this binding protects MMP-2 from inactivation processing (Figs. 1 and 2). Furthermore, Hsp90␣ antibody promotes the inactivation processing of MMP-2 (Figs. 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 Hsp90␣, which is attributed to the stabilization effect of Hsp90␣ on the processing of MMP-2.
In addition, we observed that the stabilization effect of Hsp90␣ on MMP-2 is ATP-independent (supplemental Fig.  S3C). Hsp90␣ contains an ATPase domain, and the hydrolysis of ATP is essential for Hsp90␣ 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 Hsp90␣ 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 Hsp90␣ chaperoning machinery that requires the interplay of Hsp90␣ with its cochaperones, regulators, and clients (2). On the other hand, extracellular Hsp90␣ was identified to be hyperacetylated, which attenuates its binding to ATP but not to MMP-2 (35). Very recently, our group reported that extracellular Hsp90␣ is phosphorylated at Thr 90 , which is located in the ATP-binding pocket and may also influence the binding of ATP to Hsp90␣ (9). These reports all indicate that the function of extracellular Hsp90␣ is ATP-independent.
Besides, although ATP is not essential for the activity of extracellular Hsp90␣, geldanamycin and its derivatives, the inhibitors of Hsp90␣ that act by blocking ATP binding (17,19), are still able to bind with extracellular Hsp90␣ (35). Then their inhibitory effects on MMP-2 activity and tumor invasiveness (8) may be attributed to the attenuation of client binding.
Extracellular Hsp90␣ Is a Substrate of MMP-2-As an exosite, the hemopexin domain determines the interaction of several substrates to MMP-2 (37). Extracellular Hsp90␣, which is identified to be a hemopexin domain-binding protein here (Fig. 3, A and B), is a highly probable substrate of MMP-2. In fact, the cleavage of Hsp90␣ by MMP-2 was detected previously by analyzing the substrate degradome of MMP-2 (38). In our work, the degradation of Hsp90␣ upon the treatment of MMP-2 was also observed (supplemental Fig.  S4A), and the major degradation products (ϳ80 and ϳ50 kDa) were consistent with the previous report (38). More interestingly, Hsp90␤, which is quite similar to Hsp90␣ (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 (supplemental Fig. S4B), whereas Hsp90␣ is degraded much more slowly under the same condition (supplemental Fig. S4A). These results indicate that the proteolysis mechanisms of MMP-2 on Hsp90␣ and Hsp90␤ are quite different, which may be determined by both the amino acid sequences and the secondary structures of these two isoforms. Although we have detected the cleavage of Hsp90␤ by MMP-2 by an in vitro noncell 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, Hsp90␣ stabilizes MMP-2 and prevents it from inactivation processing, but MMP-2 degrades Hsp90␣. This observation once again illustrates that the biological system is nothing if not complex. Extracellular Hsp90␣, Angiogenesis, and Tumor Microenvironment-Because intracellular Hsp90␣ is essential for the stability of diverse cell signaling pathways of cancer cells (40), the inhibitors of Hsp90 can suppress tumor progression by targeting almost all hallmarks of cancer cells, including angiogenesis (41)(42)(43). On the other side, extracellular Hsp90␣ was considered to play a unique role in tumor metastasis (7). In our study, extracellular Hsp90␣ is demonstrated for the first time to be a positive regulator of tumor angiogenesis (Figs. 5 and 6), signifying the potential role of Hsp90␣ antibody on the inhibition of tumor angiogenesis and growth.
In addition, along with the discovery of additional extracellular Hsp90␣ client proteins (44 -46), more functions of extracellular Hsp90␣ will be revealed. We propose that Hsp90␣ is not only a biochemical buffer for the genetic lesions of tumor cells in the cytosol but is also essential for the homeostasis of entire tumor microenvironment. Consequently, the applications of Hsp90␣ antibody or impermeable inhibitors of Hsp90␣ 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 (36,47,48) or other components of tumor microenvironment (7). The applications of these extracellular chaperones as therapeutic targets in cancer treatment all merit further investigation.
In summary, our results demonstrate for the first time that extracellular Hsp90␣ stabilizes MMP-2 via the interaction of the middle domain of Hsp90␣ and the hemopexin domain of MMP-2, providing novel mechanistic explanations for both the function of extracellular Hsp90␣ and the regulatory mechanism of MMP-2 activity. Furthermore, we reveal that Hsp90␣ can be secreted by endothelial cells and promotes angiogenesis in vitro and in vivo, whereas the antibody of Hsp90␣ is a potential anti-tumor drug targeting not only metastasis but also angiogenesis.