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HSP90 and fibronectin fragments colocalize in cells. Colocalization analysis of mCherry-HSP90β (red) and full-length FN-YPET, FN30-EGFP, and FN70-EGFP (green) by confocal microscopy. Scale bar is 10 μm and data shown are representative of independent biological triplicate experiments. The Rr shows the average Pearson's correlation coefficient (±SD, n ≥ 3) between HSP90 and FN signals.
Source publication
Heat shock protein 90 (HSP90) is an evolutionarily conserved chaperone protein that controls the function and stability of a wide range of cellular client proteins. Fibronectin (FN) is an extracellular client protein of HSP90, and exogenous HSP90 or inhibitors of HSP90 alter the morphology of the extracellular matrix. Here, we further characterized...
Contexts in source publication
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... we assessed the colocalization of HSP90α and FL-FN, FN30, and FN70 in cells. HEK293T cells were co-transfected with fluorescent fusion proteins of FN and HSP90β (Figure 3). mCherry-HSP90 colocalized in cells expressing all the FN proteins, with the FN30-EGFP fragment showing the highest degree of colocalization (Pearson's correlation coefficient, Rr = 0.9 ± 0.07), followed by FN70-EGFP (Rr = 0.71 ± 0.12) and FN-YPET (yellow fluorescent protein variant) (Rr = 0.5 ± 0.10). ...
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... obtained a strong signal in BiFC from VC-FN70 and VC-FN30 with either VN-HSP90α or HSP90αM which was above the background signals ( Figure 4G-J). The distribution of the GFP signal in the HSP90α and FN30/FN70 transfections was similar to that observed in colocalization analyses ( Figure 3). Clear puncta were observed around the nucleus and near the cell membrane in cells transfected with VC-FN30 and VN-HSP90αM or HSP90α. ...
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... we assessed the colocalization of HSP90α and FL-FN, FN30, and FN70 in cells. HEK293T cells were co-transfected with fluorescent fusion proteins of FN and HSP90β (Figure 3). mCherry-HSP90 colocalized in cells expressing all the FN proteins, with the FN30-EGFP fragment showing the highest degree of colocalization (Pearson's correlation coefficient, Rr = 0.9 ± 0.07), followed by FN70-EGFP (Rr = 0.71 ± 0.12) and FN-YPET (yellow fluorescent protein variant) (Rr = 0.5 ± 0.10). ...
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... obtained a strong signal in BiFC from VC-FN70 and VC-FN30 with either VN-HSP90α or HSP90αM which was above the background signals ( Figure 4G-J). The distribution of the GFP signal in the HSP90α and FN30/FN70 transfections was similar to that observed in colocalization analyses ( Figure 3). Clear puncta were observed around the nucleus and near the cell membrane in cells transfected with VC-FN30 and VN-HSP90αM or HSP90α. ...
Citations
... This is the result of increased protein quality control necessary to assist cells during the cellular signaling and production of extracellular matrix proteins observed in pulmonary fibrosis [40]. Indeed, HSP90 binds extracellular fibronectin through its N-terminal fragment, thus regulating its expression [41]. HSP90 is similarly involved in promoting TGF-β signaling via direct stabilization of the TGFβ receptor [42], assistance to ERK, via CDC37, and regulating the nuclear translocation of Smad proteins [43,44]. ...
Exposure to hydrochloric acid (HCl) can provoke acute and chronic lung injury. Because of its extensive production for industrial use, frequent accidental exposures occur, making HCl one of the top five chemicals causing inhalation injuries. There are no Food and Drug Administration (FDA)-approved treatments for HCl exposure. Heat shock protein 90 (HSP90) inhibitors modulate transforming growth factor-β (TGF-β) signaling and the development of chemical-induced pulmonary fibrosis. However, little is known on the role of Heat Shock Protein 70 (HSP70) during injury and treatment with HSP90 inhibitors. We hypothesized that administration of geranylgeranyl-acetone (GGA), an HSP70 inducer, or gefitinib (GFT), an HSP70 suppressant, alone or in combination with the HSP90 inhibitor, TAS-116, would improve or worsen, respectively, HCl-induced chronic lung injury in vivo and endothelial barrier dysfunction in vitro. GGA, alone, improved HCl-induced human lung microvascular endothelial cells (HLMVEC) barrier dysfunction and, in combination with TAS-116, improved the protective effect of TAS-116. In mice, GGA reduced HCl toxicity and while TAS-116 alone blocked HCl-induced chronic lung injury, co-administration with GGA, resulted in further improvement. Conversely, GFT potentiated HCl-induced barrier dysfunction and impaired the antidotal effects of TAS-116. We conclude that combined treatments with HSP90 inhibitors and HSP70 inducers may represent a novel therapeutic approach to manage HCl-induced chronic lung injury and pulmonary fibrosis.
... Although both isoforms possess identical functional activities in chaperone complexes, Hsp90α are often overexpressed and secreted during cellular stress events [72], and they exhibit significantly different behavior with respect to substrate interactions under stress conditions [73]. One interesting link, is the fact that exogenous extracellular Hsp90 has been shown to increase incorporation of FN fragments into fibrils within the ECM, suggesting that Hsp90 may regulate FN matrix assembly through its interaction with N-terminal FN domains [74]. These observations merit further investigation to understand how the expression of Hsp90 isoforms would be crucial in evaluating diseases progression. ...
The discovery of extracellular vesicles (EVs) as efficient exogenous biotransporters of therapeutic agents into cells across biological membranes is an exciting emerging field. Especially the potential of EVs as targeted delivery systems for diseases with selective treatments, such as fibrosis, whose treatment causes side effects in other organs not involved in the disease. Methods: In this study, we collected embryonic fibroblast-derived EVs from two different centrifugation fractions, 10 K g and 100 K g fractions from a NIH-3T3 cell line loaded with an experimental drug. Mice with fibrotic hearts and lungs were obtained by administration of angiotensin II. We generated fluorescent EVs and bioluminescent drug to observe their accumulation by colocalization of their signals in fibrotic heart and lung. The biodistribution of the drug in various organs was obtained by detecting the Au present in the drug nanostructure. Results: The drug-loaded EVs successfully reduced fibrosis in pathological fibroblasts in vitro, and modified the biodistribution of the experimental drug, enabling it to reach the target organs in vivo. We described the pre-analytical characteristics of EVs related to physical variables, culture and harvesting conditions, crucial for their in vivo application as nanotransporters using a previously validated protein-based antifibrotic drug. The results showed the colocalization of EVs and the experimental drug in vivo and ex vivo and the efficient reduction of fibrosis in vitro. This work demonstrates that 10K-EVs and 100K-EVs derived from fibroblasts can act as effective biotransporters for targeted drug delivery to profibrotic fibroblasts, lungs, or heart.
Conclusion: We observed that fibroblast-derived 10K-EVs and 100K-EVs are useful biotransporters encapsulating a new generation drug leading to a reduction of fibrosis in profibrotic fibroblasts in vitro. In addition, drug containing EVs were shown to reach fibrotic heart and lungs in vivo, enhancing free drug biodistribution.
... Since this discovery, eHsp90 has been shown to interact with multiple extracellular proteins. Interestingly, a large subset of Hsp90's interacting proteins in the TME are ECM proteins (fibronectin) [23][24][25], or ECM-modifying proteins, such as MMP-2 [3,8,10,11,18,26], MMP-9 [18], Tissue Plasminogen Activator (tPA) [27], and LOXL2 [28]. Through these interactions and those with other membrane-bound proteins, eHsp90 has been shown to increase the invasiveness of several types of cancer cells, including breast (MB231, SUM159, MCF-7, SKBR3, MB453, MB435, MB361 MB468, BT474, and T47D) [3,11,19,[27][28][29][30], fibrosarcoma (HT1080) [3], melanoma (B16) [9,17], colorectal (HCT-8) [29], prostate (PC3) [15,19], bladder (T24) [19], and glioblastoma (A172) [27]. ...
... The active MMP2 is implicated in cancer invasion by degrading the ECM surrounding the tumor cells [79][80][81]. Subsequent studies have not only substantiated the critical role of eHsp90 in enhancing invasion, but also discovered other extracellular interacting proteins that are proinvasive when in active state [3,5,8,10,24,25,27,82,83]. ...
... The eHsp90 was found to interact with extracellular proteins that that primarily are ECM proteins or ECM-modifying proteins, such as MMP-2, MMP-9, tPA, and LOXL2 [3,5,8,10,18,24,25,27,28,84]. Consequently, eHsp90 can be considered a pro-invasive hub that activates a cohort of proteins outside cancer cells to enhance invasion and metastasis, and its inhibition may be of benefit in treating metastatic cancers. ...
Simple Summary
Breast cancer cells secrete Hsp90, a protein that, inside of cells, regulates the function of hundreds of proteins, but outside of cells, extracellular Hsp90 (eHsp90) can activate a subset of proteins that promote invasion, the first step of metastasis. Blocking eHsp90 in mouse models inhibits metastasis, and we sought to understand how this occurs. Prior studies have predominantly focused on eHsp90 in cancer invasion within the immediate vicinity of the primary tumor, specifically its role in invading outside the epithelial compartment. However, eHsp90’s role in cancer invasion across the extended connective tissue after the cells have crossed the boundary of the epithelial compartment remains unknown. We show here that eHsp90 directly binds to and aligns Collagen-1 fibers, a major structural component of connective tissues, which, when aligned, form highways that allow efficient cancer migration. Our study suggests that the Hsp90 dimer, in its open state, binds to Collagen-1 molecules to align the fibers, which results in enhanced breast cancer invasion through the Collagen-1 matrix. Knowing this could help us propose experiments to test eHsp90 inhibitors for therapeutically targeting metastatic breast cancer.
Abstract
Cancer cell-secreted eHsp90 binds and activates proteins in the tumor microenvironment crucial in cancer invasion. Therefore, targeting eHsp90 could inhibit invasion, preventing metastasis—the leading cause of cancer-related mortality. Previous eHsp90 studies have solely focused on its role in cancer invasion through the 2D basement membrane (BM), a form of extracellular matrix (ECM) that lines the epithelial compartment. However, its role in cancer invasion through the 3D Interstitial Matrix (IM), an ECM beyond the BM, remains unexplored. Using a Collagen-1 binding assay and second harmonic generation (SHG) imaging, we demonstrate that eHsp90 directly binds and aligns Collagen-1 fibers, the primary component of IM. Furthermore, we show that eHsp90 enhances Collagen-1 invasion of breast cancer cells in the Transwell assay. Using Hsp90 conformation mutants and inhibitors, we established that the Hsp90 dimer binds to Collagen-1 via its N-domain. We also demonstrated that while Collagen-1 binding and alignment are not influenced by Hsp90’s ATPase activity attributed to the N-domain, its open conformation is crucial for increasing Collagen-1 alignment and promoting breast cancer cell invasion. These findings unveil a novel role for eHsp90 in invasion through the IM and offer valuable mechanistic insights into potential therapeutic approaches for inhibiting Hsp90 to suppress invasion and metastasis.
... Here we utilised this assay to evaluate the subcellular location and interaction of the HSP90-CDK4 complex in cells as an indicator of HSP90-client protein interactions [24]. ...
Protein-protein interactions (PPI) in cells play a pivotal role in cellular function and dynamics. Cellular proteostasis is maintained by PPI networks between molecular chaperones, co-chaperones, and client proteins. Consequently, strategies to visualize and analyze PPI in cells are useful in understanding protein homeostasis regulation. The Bimolecular Fluorescence Complementation (BiFC) assay has emerged as a useful tool for studying PPI between proteins in live or fixed cells. BiFC is based on the detection of fluorescence generated when interacting protein pairs, produced as fusion proteins with either the N- or C-terminal fragment of a fluorescent protein, are in sufficient proximity to permit reconstitution of the split fluorophore. Here, we describe the application of the BiFC assay to a model of chaperone-client interactions using Hsp90 and the validated client protein CDK4. This assay allows for the distribution and spatiotemporal analysis of HSP90-CDK4 complexes in live or fixed cells and is amenable to studying the effects of inhibitors and mutations on chaperone-client protein networks.
... Furthermore, Studies have indicated that the administration of exogenous HSP90 can induce changes in the structure and composition of the ECM [22]. Furthermore, HSP90 plays a critical role in the activation and survival promotion of hepatic stellate cells (HSCs), the primary contributors to ECM production in liver fibrosis. ...
Liver fibrosis is a progressive condition characterized by the build-up of fibrous tissue resulting from long-term liver injury. Although there have been advancements in research and treatment, there is still a need for effective antifibrotic medication. HSP90 plays a crucial role in the development of fibrosis. It acts as a molecular chaperone that assists in the proper folding and stability of TβRII, potentially regulating the signaling of TGF-β1. It has been established that TβRII can be degraded through the proteasome degradation system, either via ubiquitination-dependent or -independent pathways. In the present study, STA9090 demonstrated promising effects in both in vitro and in vivo models. It reduced LDH leakage, prolonged the survival rate of hepatocytes in rats with liver fibrosis, and improved liver function. Importantly, STA9090 exerted pleiotropic effects by targeting proteins involved in limiting collagen production, which resulted in improved microscopic features of the rat livers. Our findings suggest that STA9090-induced inhibition of HSP90 leads to the degradation of TβRII, a fibrogenic client protein of HSP90, through the activation of the 20S proteasomal degradation system. We also revealed that this degradation mechanism is not dependent on the autophagy–lysosomal pathway. Additionally, STA9090 was found to destabilize HIF-1α and facilitate its degradation, leading to the reduced transcription of VEGF. Moreover, STA9090’s ability to deactivate the NFκB signaling pathway highlights its potential as an anti-inflammatory and antifibrotic agent. However, further research is necessary to fully elucidate the underlying mechanisms and fully capitalize on the therapeutic benefits of targeting HSP90 and associated pathways.
... for interaction with the type I FN motif (FNI) (Hunter et al. 2014;Chakraborty et al. 2020). The N-terminal 30 kDa fragment of FN (FN30) showed the highest affinity for HSP90 (Chakraborty et al. 2020) and is required for FN matrix assembly (Sottile et al. 1991;Maurer et al. 2016). ...
... for interaction with the type I FN motif (FNI) (Hunter et al. 2014;Chakraborty et al. 2020). The N-terminal 30 kDa fragment of FN (FN30) showed the highest affinity for HSP90 (Chakraborty et al. 2020) and is required for FN matrix assembly (Sottile et al. 1991;Maurer et al. 2016). Exogenous HSP90 increased FN matrix assembly (Hunter et al. 2014;Chakraborty et al. 2020), whereas HSP90β inhibition or knockdown resulted in significant FN matrix turnover and decreased matrix stability in Hs578T cells (Hunter et al. 2014;Boel et al. 2020). ...
... The N-terminal 30 kDa fragment of FN (FN30) showed the highest affinity for HSP90 (Chakraborty et al. 2020) and is required for FN matrix assembly (Sottile et al. 1991;Maurer et al. 2016). Exogenous HSP90 increased FN matrix assembly (Hunter et al. 2014;Chakraborty et al. 2020), whereas HSP90β inhibition or knockdown resulted in significant FN matrix turnover and decreased matrix stability in Hs578T cells (Hunter et al. 2014;Boel et al. 2020). In prostate cancer explants, HSP90 inhibitor AUY922 also reduced FN secretion (Armstrong et al. 2018), while FN expression was stimulated by HSP90 inhibitor geldanamycin in an HSF1-dependent manner (Dhanani et al. 2017). ...
HSP90 is a ubiquitously expressed chaperone protein that regulates the maturation of numerous substrate proteins called 'clients'. The glycoprotein fibronectin (FN) is an important protein of the extracellular matrix (ECM) and a client protein of HSP90. FN and HSP90 interact directly, and the FN ECM is regulated by exogenous HSP90 or HSP90 inhibitors. Here, we extend the analysis of the HSP90 - FN interaction. The importance of the N-terminal 70-kDa fragment of fibronectin (FN70) and FN type I repeat was demonstrated by competition for FN binding between HSP90 and the functional upstream domain (FUD) of the Streptococcus pyogenes F1 adhesin protein. Furthermore, His-HSP90α mutations F352A and Y528A (alone and in combination) reduced the association with full-length FN (FN-FL) and FN70 in vitro. Unlike wild type His-HSP90α, these HSP90 mutants did not enhance FN matrix assembly in the Hs578T cell line model when added exogenously. Interestingly, the HSP90 E353A mutation, which did not significantly reduce the HSP90 - FN interaction in vitro, dramatically blocked FN matrix assembly in Hs578T cell-derived matrices. Taken together, these data extend our understanding of the role of HSP90 in FN fibrillogenesis and suggest that promotion of FN ECM assembly by HSP90 is not solely regulated by the affinity of the direct interaction between HSP90 and FN.
... This led us to believe that FN1 associates itself with AR through the NTD of AR [26]. Fibronectin interacts with integrin receptors through the central binding domain (FNIII [1][2][3][4][5][6][7][8][9][10][11][12] [27,28]. On the other hand, HSP90 a chaperone protein interacts with FN1 via the N-terminal regions to aid in matrix assembly [29]. ...
MicroRNAs play an important role in modulating normal cellular functions through protein-protein interaction in addition to regulating gene expression via mRNA degradation or translational repression. PVT1 is a non-protein coding gene that encodes six annotated microRNAs (miRNAs), including miR-1207-3p, a demonstrated modulator in PCa. MiR-1207-3p is significantly underexpressed in PCa cells when compared to normal prostatic cells and directly targets fibronectin type III domain containing 1 (FNDC1), which was found to be overexpressed in PCa cells along with concomitant overexpression of fibronectin (FN1)/androgen receptor (AR)/c-MYC. To better understand the role of FNDC1/FN1/AR/c-MYC in PCa progression we examined the interaction between FNDC1/FN1/AR/c-MYC. Coimmunoprecipitation study showed direct physical interaction between FNDC1/FN1/AR/c-MYC in PCa cells and FN1/AR/cMYC in normal prostatic cell. Knockdown of FN1, AR and cMYC not only confirmed FN1/FNDC1/AR/cMYC interaction but also gave rise to the possibility of a complex. We also examined the spatial localization of the FNDC1/FN1/AR/c-MYC pathway by performing immunofluorescence staining in PCa cells. Single staining analysis revealed that FNDC1, AR and c-MYC localize to the nucleus and cytoplasm while FN1 localizes only to the cytoplasm in PCa cells but not in the non-tumorigenic prostate cells. However, our findings show that miR-1207-3p has no direct effect on the FNDC1/FN1/AR/cMYC interaction. Understanding this molecular interaction can reveal additional insights into the role of FNDC1/FN1/AR/cMYC interaction in PCa progression.
... Consistent with this, C-terminal but not N-terminal Hsp90 inhibition promoted matrix turnover. This turnover required the catabolic FN receptor LRP1, which is also a receptor for extracellular Hsp90, potentially suggesting the presence of an extracellular Hsp90-FN-LRP1 ternary complex (Chakraborty et al. 2020). Together, these data identify Hsp90 in the extracellular environment as an important regulator of matrix stability (Chakraborty and Edkins 2021). ...
The Second International Symposium on Cellular and Organismal Stress Responses took place virtually on September 8–9, 2022. This meeting was supported by the Cell Stress Society International (CSSI) and organized by Patricija Van Oosten-Hawle and Andrew Truman (University of North Carolina at Charlotte, USA) and Mehdi Mollapour (SUNY Upstate Medical University, USA). The goal of this symposium was to continue the theme from the initial meeting in 2020 by providing a platform for established researchers, new investigators, postdoctoral fellows, and students to present and exchange ideas on various topics on cellular stress and chaperones. We will summarize the highlights of the meeting here and recognize those that received recognition from the CSSI.
... Additionally, this promoting ability was observed for all individual domains. Although HSP90 has no RGD motif, its activation of the plasmin-plasminogen pathway might be due to its direct binding property on the crystal surface and/ or its potential interaction with fibronectin [69], which is an abundant component of ECM [70]. Moreover, our finding was consistent with the previous report demonstrating that α-enolase secreted from RTECs in response to COM crystals could promote the crystal invasion [53]. ...
Human heat-shock protein 90 (HSP90) has four functional domains, including NH2-terminal (N), charged linker region (LR), middle (M) and COOH-terminal (C) domains. In kidney stone disease (or nephrolithiasis/urolithiasis), HSP90 serves as a receptor for calcium oxalate monohydrate (COM), which is the most common crystal to form kidney stones. Nevertheless, roles of HSP90 and its four domains in kidney stone formation remained unclear and under-investigated. We thus examined and compared their effects on COM crystals during physical (crystallization, growth and aggregation) and biological (crystal–cell adhesion and crystal invasion through extracellular matrix (ECM)) pathogenic processes of kidney stone formation. The analyses revealed that full-length (FL) HSP90 obviously increased COM crystal size and abundance during crystallization and markedly promoted crystal growth, aggregation, adhesion onto renal cells and ECM invasion. Comparing among four individual domains, N and C domains exhibited the strongest promoting effects, whereas LR domain had the weakest promoting effects on COM crystals. In summary, our findings indicate that FL-HSP90 and its four domains (N, LR, M and C) promote COM crystallization, crystal growth, aggregation, adhesion onto renal cells and invasion through the ECM, all of which are the important physical and biological pathogenic processes of kidney stone formation.
Graphical abstract
... HER2, LRP1, TGFβR, TLR4) (Stellas et al., 2007;Cheng et al., 2008;Sidera et al., 2008;Stellas et al., 2010;Cheng et al., 2011;El Hamidieh et al., 2012;Garcia et al., 2016;Secli et al., 2021a;Secli et al., 2021b), in complex with components and remodelers of the extracellular matrix (eg. Fibronectin, LOXL2) (Hunter et al., 2014;Edkins, 2016;de la Mare et al., 2017;Chakraborty et al., 2020;Chakraborty and Edkins, 2021), associated with cochaperones or client proteins following cell release (eg. TIMP2, AHA1, MMPs, Morgana) (McCready et al., 2010;Baker-Williams et al., 2019;Secli et al., 2021a), or attached on or encapsulated in tumor released extracellular vesicles (Li et al., 2012;Tang et al., 2019;Eguchi et al., 2020) (Figure 2). ...
... 04 4 Functions of eHsp90 4.1 ECM assembly Extracellular Hsp90 helps to regulate extracellular matrix (ECM) assembly and stability, important for normal processes including embryogenesis, wound healing and cell migration. One extracellular client protein is fibronectin (FN1), a structural component of the ECM, and eHsp90 was shown to assist in incorporation of fibronectin into fibrils (Hunter et al., 2014;Chakraborty et al., 2020). Additionally, fibronectin is stress inducible; Hsp90 N-domain inhibitor treatment-induced heat shock response led to an increase in fibronectin secondary to HSF1 activity at heat shock elements in its promoter (Dhanani et al., 2017). ...
The molecular chaperone Heat Shock Protein-90 (Hsp90) is known to interact with over 300 client proteins as well as regulatory factors (eg. nucleotide and proteins) that facilitate execution of its role as a chaperone and, ultimately, client protein activation. Hsp90 associates transiently with these molecular modulators during an eventful chaperone cycle, resulting in acquisition of flexible structural conformations, perfectly customized to the needs of each one of its client proteins. Due to the plethora and diverse nature of proteins it supports, the Hsp90 chaperone machinery is critical for normal cellular function particularly in response to stress. In diseases such as cancer, the Hsp90 chaperone machinery is hijacked for processes which encompass many of the hallmarks of cancer, including cell growth, survival, immune response evasion, migration, invasion, and angiogenesis. Elevated levels of extracellular Hsp90 (eHsp90) enhance tumorigenesis and the potential for metastasis. eHsp90 has been considered one of the new targets in the development of anti-cancer drugs as there are various stages of cancer progression where eHsp90 function could be targeted. Our limited understanding of the regulation of the eHsp90 chaperone machinery is a major drawback for designing successful Hsp90-targeted therapies, and more research is still warranted.
