Extracellular Roles for the Molecular Chaperone, hsp90

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*Correspondence to: Daniel G. Jay; Department of Physiology; 136 Harrison .... Csermely P, Schnaider T, Soti C, Prohaszka Z, Nardai G. The 90-kDa molecular ...
[Cell Cycle 3:9, 1098-1100; September 2004]; ©2004 Landes Bioscience

Extracellular Roles for the Molecular Chaperone, hsp90 Extra Views

Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=1088

KEY WORDS heat shock protein, hsp90, chaperone, invasion, cancer, metastasis

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Organisms are challenged by a multitude of stresses throughout their lifespan which cause irreversible damage to proteins that in turn could impair cellular processes. To ameliorate these stresses, organisms have evolved proteins to protect the cells from damaged or improper proteins. Prominent among these are the heat shock proteins (hsps), first discovered by their increased expression in Drosophila exposed to high temperature.1 Hsps are among the most highly and ubiquitously expressed proteins in all cells from bacteria to mammals.2 As molecular chaperones for proteins, hsps are responsible for such diverse cellular processes as protein folding, activation, transport, and oligomeric assembly.2 These molecular chaperones not only maintain proper protein function, but also promote pathological protein function if improperly expressed.3 Hsp family members are thought to function primarily inside the cell, including the nucleus,4 lysosome,5 endoplasmic reticulum,6 cytoplasm,7 and mitochondria.8 However, there have been several reports that some hsp members are present and may function in the extracellular space.9-15 Recently, an extracellular role for the α isoform of hsp90 (heat shock protein 90 kDa) was demonstrated in the pathological process of tumor cell invasion and metastasis.16 This role was identified using a functional proteomic screen for targets located on the surface of tumor cells. Hsp90α, but not β, is expressed on the surface and in the conditioned media of HT-1080 fibrosarcoma and MDA-MD231 breast carcinoma cells. The inactivation of hsp90 (α but not β) function by Fluorophore-Assisted Light Inactivation (FALI) specifically on the surface of tumor cells led to a decreased invasiveness and a concomitant reduction of matrix metalloproteinase-2 (MMP2) activity in the extracellular space. The MMP2 protease is important for digestion of many of the major components of the extracellular matrix surrounding tumor masses (collagens, laminins, fibronectin, etc.), and for subsequent invasion of primary tumor cells.17 These results led to the conclusion that hsp90α assists in the maturation of pro-MMP2 to its active form by promoting propeptide cleavage; a role much like hsp90’s role in protein maturation and activation intracellularly. This study provides evidence for an important extracellular role for hsp90 and suggests that such chaperoning outside the cell is possible despite an environment that is distinct from hsp90’s intracellular environment. Reports of surface hsp90 were first published nearly 20 years ago when it was described as a tumor specific antigen on the surface of mouse cells, though specific functions were not described until very recently.18 The best studied examples of hsp90’s extracellular role

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heat shock protein heat shock protein 90 kDa heat shock protein 70 kDa hsp90 organizing protein hsp70 interacting protein matrix metalloproteinase 2

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hsp hsp90 hsp70 hop hip MMP2

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Received 07/13/04; Accepted 07/14/04

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*Correspondence to: Daniel G. Jay; Department of Physiology; 136 Harrison Avenue; M&V 709; Boston, Massachusetts 02111 USA; Tel.: 617.636.6714; Fax: 617.636.0445; Email: [email protected]

Heat shock proteins (hsps) are versatile molecular chaperones that are responsible for many cellular functions including proper folding, oligomeric assembly, activation, and transport of proteins. Most of the known roles for hsps involve intracellular proteins and processes. Mounting evidence suggests that hsps are present and function in the extracellular space. Hsp90α was recently found on the surface and in conditioned media of HT-1080 fibrosarcoma cells. Here it acts as a molecular chaperone that assists in the activation of matrix metalloproteinase-2 (MMP2), leading to increased tumor invasiveness. Few other extracellular substrates of hsp90 have been identified, but several independent observations of extracellular hsp90 suggest that this protein may be important for both normal physiology and disease states. Hsp90 typically works in a complex of associated proteins, and some of these proteins have also been observed extracellularly. Here we show that some of these components, including hsp90 organizing protein (hop) and p23, are also found in HT-1080 conditioned media supporting the notion that hsp90 complexes function in invasiveness. These findings suggest a wide-ranging phenomenon of extracellular molecular chaperoning that could have implications for biological processes and disease.

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Department of Physiology; Tufts University School of Medicine; Boston, Massacusetts USA

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ABSTRACT

Brenda K. Eustace Daniel G. Jay*

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EXTRACELLULAR ROLES FOR THE MOLECULAR CHAPERONE, HSP90

are in innate and adaptive immunity. Hsp90 has been documented as a key player in antigen processing and presentation during immune responses,19 and it is currently in clinical trials as a tumor immunogenicity vaccine.20 Another role for hsp90α is as a member of a cell surface signaling complex required for proper immune response to bacterial lipopolysaccharide.21 These examples from immune function provide established roles for extracellular hsp90 in physiology. Extracellular hsp90 has also been described as a key player in several pathophysiological states as well. A recent example described a novel interaction between extracellular hsp90α and annexin II on endothelial cells that modulates plasminogen activation, which may have implications for clotting abnormalities seen in diabetic states.11 Hsp90 also plays a role in the activation of the preallikrein-kininogen complex on the surface of human vascular endothelial cells, which is thought to play a role in the pathological development of bronchial hyper-reactivity in asthma.13 Both of these reports suggest novel interactions of hsp90 with extracellular proteins, but did not show bona fide chaperoning activities outside of the cell. It is tempting to speculate that hsp90 acts as a molecular chaperone for these extracellular proteins. There are other examples that suggest that hsp90 has unique extracellular roles. Hsp90α has been found to be secreted from vascular smooth muscle cells in response to oxidative stress, and application of hsp90α induces MAPK activity in the same cells.10 It has also been reported to induce neurite outgrowth of chick dorsal root ganglion cells in culture.22 Together these studies implicate hsp90 in extracellular processes and suggest that this role may be widespread. It will be important to investigate which other proteins, cellular functions, and pathologies may be influenced by hsp90’s extracellular chaperoning activity. Our study suggested that specifically inhibiting hsp90 outside the cell using impermeant inhibitors or neutralizing antibodies may have clinical benefit in limiting metastasis without affecting the many intracellular roles of hsp90. Establishing new roles for extracellular hsp90 in other pathologies could extend the utility of such reagents. Hsp90 has been shown to have some chaperoning activity alone,23-25 especially in prevention of aggregates and maintenance of proteins in a “folding-competent” state.26 However, it generally acts in concert with several chaperones, cochaperones and other binding proteins to promote folding or activation of target proteins. Some of the core proteins of this complex are hsp70 (heat shock protein 70 kDa), p23, cdc37 (p50), hip (hsp70 interacting protein), hop (hsp90 organizing protein), and immunophilins. Several of these cochaperones, including hsp70,27 cdc37,28 and several immunophilins,29 have themselves been found extracellularly, suggesting that a complex of molecular chaperoning could occur outside of the cell. We tested here if these components are present in HT-1080 conditioned media by immunoblotting (Fig. 1). Hsp90α (but not β) is readily detected in HT-1080 conditioned media as is the endoplasmic reticulum hsp90 family member, grp94 (glucose regulated protein 94 kDa). Grp94 is not known to complex with hsp90, but it has been implicated in extracellular peptide chaperoning to the immune system and could also play a role in chaperoning whole proteins.2 Hsp70 was not detected suggesting that this chaperone is not found extracellularly on HT-1080 cells. Among the hsp90 cochaperones, we observed that hop and p23 were present, but did not detect hip. Thus several components of the hsp90 complex (p23 and hop) but not the hsp70 complex (hsp70 and hip) are present extracellularly in a position to function with hsp90α in activating MMP-2. It remains to be investigated whether these hsp90 cochaperones are in a complex and are active extracellularly, but the accumulating evidence that a www.landesbioscience.com

Figure 1. HT-1080 cells secrete several chaperones and cochaperones. Serum-free conditioned media from HT-1080 cells was collected, concentrated, and immunoblotted with the antibodies as shown. Hsp90α, hsp90 familiy member grp94, hsp90 organizing protein (hop), and p23 are secreted by HT-1080 cells, while hsp90β, hsp70, and hsp70 interacting protein (hip) are absent.

wide variety of chaperones and cochaperones are secreted suggests this possibility. Many questions remain for extracellular chaperoning by hsp90, and future work will be guided by the known functions and activities of intracellular hsp90. How does hsp90 reach the extracellular environment? There are many examples of proteins that do not contain known signal sequences, but are nonetheless secreted efficiently.30 How does hsp90 function outside of the cell under conditions of low ATP concentration and oxidative environments?31 Is the extracellular role for hsp90 important for normal cellular functions, or is it simply a pathological condition of prolonged stress, such as infection or uncontrolled growth? Finally, will inhibition of extracellular hsp90 be an effective therapy to treat diseases such as cancer and diabetes? While molecular chaperones are among the most abundant proteins across all species and are well studied, our understanding of extracellular functions of these molecules is just beginning. As such, the impact of these proteins on biological processes has yet to be fully realized. Our increased understanding of how extracellular hsp90 and its chaperoning complex members function may lead to new therapeutics for cancer metastasis and other diseases. References 1. Holmgren R, Livak K, Morimoto R, Freund R, Meselson M. Studies of cloned sequences from four Drosophila heat shock loci. Cell 1979; 18:1359-70. 2. Csermely P, Schnaider T, Soti C, Prohaszka Z, Nardai G. The 90-kDa molecular chaperone family: Structure, function, and clinical applications. A comprehensive review. Pharmacol Ther 1998; 79:129-68. 3. Neckers L, Ivy SP. Heat shock protein 90. Curr Opin Oncol 2003; 15:419-24. 4. Schlatter H, Langer T, Rosmus S, Onneken ML, Fasold H. A novel function for the 90 kDa heat-shock protein (Hsp90): Facilitating nuclear export of 60 S ribosomal subunits. Biochem J 2002; 362:675-84. 5. Agarraberes FA, Dice JF. A molecular chaperone complex at the lysosomal membrane is required for protein translocation. J Cell Sci 2001; 114:2491-9. 6. Lee AS. The glucose-regulated proteins: Stress induction and clinical applications. Trends Biochem Sci 2001; 26:504-10. 7. Richter K, Buchner J. Hsp90: Chaperoning signal transduction. J Cell Physiol 2001; 188:281-90.

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8. Young JC, Hoogenraad NJ, Hartl FU. Molecular chaperones Hsp90 and Hsp70 deliver preporteins to the mitochondrial import receptor Tom70. Cell 2003; 112:41-50. 9. Triantafilou K, Triantafilou M, Dedrick RL. A CD14-independent LPS receptor cluster. Nat Immunol 2001; 2:338-45. 10. Liao DF, Jin ZG, Baas AS, Daum G, Gygi SP, Aebersold R, et al. Purification and identification of secreted oxidative stress-induced factors from vascular smooth muscle cells. J Biol Chem 2000; 275:189-96. 11. Lei H, Romeo G, Kazlauskas A. Heat shock protein 90α-dependent translocation of annexin II to the surface of endothelial cells modulates plasmin activity in the diabetic rat aorta. Circ Res 2004; 94:902-9. 12. DeLeo AB, Srivastava PK. Cell surface antigens of chemically induced sarcomas of murine origin. Cancer Surv 1985; 4:21-34. 13. Joseph K, Tholanikunnel BG, Kaplan AP. Heat shock protein 90 catalyzes activation of the prekallikrein-kininogen complex in the absence of factor XII. Proc Natl Acad Sci USA 2002; 99:896-900. 14. Kakimura J, Kitamura Y, Takata K, Umeki M, Suzuki S, Shibagaki K, et al. Microglial activation and amyloid-β clearance induced by exogenous heat-shock proteins. Faseb J 2002; 16:601-3. 15. Srivastava PK, Udono H, Blachere NE, Li Z. Heat shock proteins transfer peptides during antigen processing and CTL priming. Immunogenetics 1994; 39:93-8. 16. Eustace BK, Sakurai T, Stewart JK, Yimlamai D, Unger C, Zehetmeier C, et al. Functional proteomic screens reveal an essential extracellular role for hsp90α in cancer cell invasiveness. Nat Cell Biol 2004. 17. Itoh Y, Takamura A, Ito N, Maru Y, Sato H, Suenaga N, et al Homophilic complex formation of MT1-MMP facilitates proMMP-2 activation on the cell surface and promotes tumor cell invasion. EMBO J 2001; 20:4782-93. 18. Ullrich SJ, Robinson EA, Law LW, Willingham M, Appella E. A mouse tumor-specific transplantation antigen is a heat shock-related protein. Proc Natl Acad Sci USA 1986; 83:3121-5. 19. Basu S, Srivastava PK. Heat shock proteins: The fountainhead of innate and adaptive immune responses. Cell Stress Chaperones 2000; 5:443-51. 20. Castelli C, Rivoltini L, Rini F, Belli F, Testori A, Maio M, et al. Heat shock proteins: Biological functions and clinical application as personalized vaccines for human cancer. Cancer Immunol Immunother 2004; 53:227-33. 21. Triantafilou M, Triantafilou K. Lipopolysaccharide recognition: CD14, TLRs and the LPS-activation cluster. Trends Immunol 2002; 23:301-4. 22. Ishimoto T, Kamei A, Koyanagi S, Nishide N, Uyeda A, Kasai M, et al. HSP90 has neurite-promoting activity in vitro for telencephalic and spinal neurons of chick embryos. Biochem Biophys Res Commun 1998; 253:283-7. 23. Miyata Y, Yahara I. Interaction between casein kinase II and the 90-kDa stress protein, HSP90. Biochemistry 1995; 34:8123-9. 24. Jakob U, Lilie H, Meyer I, Buchner J. Transient interaction of Hsp90 with early unfolding intermediates of citrate synthase. Implications for heat shock in vivo. J Biol Chem 1995; 270:7288-94. 25. Wiech H, Buchner J, Zimmermann R, Jakob U. Hsp90 chaperones protein folding in vitro. Nature 1992; 358:169-70. 26. Freeman BC, Morimoto RI. The human cytosolic molecular chaperones hsp90, hsp70 (hsc70) and hdj-1 have distinct roles in recognition of a nonnative protein and protein refolding. EMBO J 1996; 15:2969-79. 27. Shin BK, Wang H, Yim AM, Le Naour F, Brichory F, Jang JH, et al. Global profiling of the cell surface proteome of cancer cells uncovers an abundance of proteins with chaperone function. J Biol Chem 2003; 278:7607-16. 28. Grammatikakis N, Grammatikakis A, Yoneda M, Yu Q, Banerjee SD, Toole BP. A novel glycosaminoglycan-binding protein is the vertebrate homologue of the cell cycle control protein, Cdc37. J Biol Chem 1995; 270:16198-205. 29. Coppinger JA, Cagney G, Toomey S, Kislinger T, Belton O, McRedmond JP, et al. Characterization of the proteins released from activated platelets leads to localization of novel platelet proteins in human atherosclerotic lesions. Blood 2004; 103:2096-104. 30. Cleves AE. Protein transports: The nonclassical ins and outs. Curr Biol 1997; 7:R318-20. 31. Picard D. Hsp90 invades the outside. Nat Cell Biol 2004; 6:479-80.

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