Mounting evidence suggests that Hsp90 is present and functionally active in the extracellular space. The biological function of extracellular Hsp90 (eHsp90) remains relatively uncharacterized compared to that of intracellular Hsp90. eHsp90 has been shown to interact with a finite number of extracellular proteins, however, despite the identification of eHsp90 interacting proteins, the function of eHsp90 in these complexes is unknown. Several reports suggest a role for eHsp90α in cell migration and invasion. Reported targets for eHsp90 stimulated cell migration include MMPs, LRP-1, tyrosine kinase receptors and possible others unidentified. Limited studies report a role for eHsp90β. Recently, Hsp90α and Hsp90β were isolated in a complex containing fibronectin (FN) on the surface of MDA-MB-231 breast cancer cells. Herein, we report direct binding of Hsp90α and Hsp90β to FN using a solid phase binding assay and surface plasmon resonance (SPR) spectroscopy. SPR spectroscopy showed that Hsp90β bound the 70 kDa amino-terminal fragment of FN (FN70), but that binding of FN to Hsp90β was not limited to FN70. Confocal microscopy showed regions of colocalization of Hsp90 with extracellular FN matrix fibrils in Hs578T breast cancer cell lines. Treatment of Hs578T breast cancer cells with novobiocin (an Hsp90 inhibitor) and an LRP-1 blocking antibody resulted in a loss of FN matrix and FN endocytosis (novobiocin treated). Addition of exogenous Hsp90β was able to recover such effect after both treatments. FN was shown to colocalize with intracellular LRP-1 in novobiocin treated Hs578T cells. Immunoprecipitation of an LRP-1 containing complex showed the presence of Hsp90 and 70 and 120+ kDa FN fragments. Treatment of Hs578T cells with novobiocin increased the level of FN120+ bound in LRP-1 immunoprecipitate. Exogenous Hsp90β decreased the level of low and high molecular weight FN fragments in a complex with LRP-1, despite the fact that higher levels of lower molecular weight FN fragments were detected in this cell lysate compared to the other treatments. We report FN as a novel interacting protein of eHsp90. Taken together, we provide evidence for a direct role of eHsp90β in FN matrix remodeling. We suggest that Hsp90 plays a direct role in FN matrix dynamics through interaction with FN and LRP-1. The identification of FN as a novel interacting protein of eHsp90 suggests a role for Hsp90 in FN matrix remodeling, which is important for a number of fundamental cellular processes including cell migration and metastasis.

Current dogma suggests that the Heat Shock Protein (Hsp) molecular chaperones and associated co-chaperones function primarily within the cell, although growing evidence suggests a role for these proteins on the plasma membrane of cancer cells. Hsp90 does not function independently in vivo, but instead functions with a variety of partner chaperones and co-chaperones, that include Hsp70 and Hsp90/Hsp70 organising protein (Hop), which are thought to regulate ATP hydrolysis and the binding of Hsp90 to its client proteins. Hsp90 on the plasma membrane appears to have distinct roles in pathways leading to cell motility, invasion and metastasis. We hypothesised that Hsp90 on the plasma membrane is present as part of a multi-chaperone complex that participates in the chaperone-assisted folding of client membrane proteins in a manner analogous to the intracellular chaperone complex. This study characterised the membrane expression of Hsp90, Hsp70 and Hop in different cell models of different adhesive and migratory capacity, namely MDA-MB-231 (metastatic adherent breast cancer cell line), MCF-7 (non-metastatic adherent breast cancer cell line), U937 and THP1 (monocytic leukemia suspension cell lines). Membrane expression of the Hsps was analysed using a combination of subcellular fractionation, biotin-streptavidin affinity purification and immunofluorescence. This study provided evidence to suggest that Hsp90, Hsp70 and Hop are membrane associated in MDA-MB-231 and MCF-7 breast cancer cells. Hsp90, Hsp70 and Hop associated with the plasma membrane such that at least part of the protein is located extracellularly. Immunofluorescence analysis showed that Hsp90, Hsp70 and Hop at the leading edge may localize to membrane ruffles in MDA-MB-231 cells, in accordance with the published role of Hsp90 in migration. An increase in this response was seen in cells stimulated to migrate with SDF-1. By immunoprecipitation, we isolated a putative extracellular membrane associated complex containing Hsp90, Hsp70 and Hop. Using soluble Hsp90 and antibodies against membrane associated Hsp90, we suggested roles for soluble extracellular Hsp90 in mediating migration by wound healing assays and inducing actin reorganisation and vinculin-based focal adhesion formation. The effects of extracellular Hsp90 are mediated by signalling through an ERK1/2 dependent pathway. An anti-Hsp90 antibody against an N-terminal epitope in Hsp90 appeared to be able to overcome the death inducing effects of a combination of SDF-1 and AMD3100, while soluble Hsp90 could not overcome this effect. We propose that this study provides preliminary evidence that extracellular Hsp90 functions as part of a multi-chaperone complex that includes Hsp70 and Hop. The extracellular Hsp90 chaperone complex may mediate cell processes such as migration by modulating the conformation of cell surface receptors, leading to downstream signalling.

To date, a significant portion of research has been devoted to understanding the biological role of the molecular chaperone, heat shock protein 90 (Hsp90), in cancer development and metastasis. Studies have alluded to over 300 clients for intracellular Hsp90, many of which are involved in oncogenic signaling pathways, making Hsp90 a bone fide drug target with several inhibitors already in clinical trials. In recent years, a limited number of extracellular Hsp90 clients have been elucidated with roles in cancer cell migration and invasion. Examples of such clients include matrix metalloproteinase-2 (MMP-2), LRP-1/CD91 and HER-2. Inhibition of extracellular Hsp90 using cellimpermeable inhibitors has been shown to reduce cancer cell migration and metastasis by a hitherto undefined mechanism. Using surface biotinylation and an enzyme linked immunosorbent assay, we provided evidence to support that Hsp90 was found extracellularly in cancers of different origin, cell type and malignancy. Next, we isolated extracellular Hsp90-containing complexes from MDA-MB-231 breast cancer cells using a cell impermeable crosslinker followed by immunoprecipitation and identified by mass spectrometry that the extracellular matrix protein, fibronectin, co-precipitated with Hsp90β. This interaction between Hsp90β and fibronectin was confirmed using pull down assays and surface plasmon resonance spectroscopy with the purified proteins. The ability of exogenous Hsp90β to increase the insoluble fibronectin matrix in Hs578T breast cancer cells indicated a role for Hsp90 in fibronectin matrix stability or fibrillogenesis. Hsp90 knockdown by RNA interference or inhibition with the small molecule inhibitor, novobiocin, resulted in a dose and time-dependent reduction of the extracellular fibronectin matrix. Furthermore, novobiocin was shown to cause the internalization of a fluorescently-labeled exogenous fibronectin matrix incorporated into the extracellular matrix by Hs578T cells. This suggested endocytosis as a possible mechanism for fibronectin turnover. This was supported by the colocalization of fibronectin with key vesicular trafficking markers (Rab-5 and LAMP-1) in small, intracellular vesicles. Furthermore, treatment with the vesicular trafficking inhibitor, methyl-β-cyclodextrin, resulted in a dose-dependent recovery in the extracellular fibronectin matrix following treatment with novobiocin. Taken together, these data provided the first evidence to suggest fibronectin as a new client of Hsp90 and that Hsp90 was involved in regulating extracellular fibronectin matrix dynamics.

Lung tumor progression is regulated by proangiogenic factors. Heat shock protein A12B (HSPA12B) is a recently identified regulator of expression of proangiogenic factors. However, whether HSPA12B plays a role in lung tumor growth is unknown. To address this question, transgenic mice overexpressing HSPA12B (Tg) and wildtype littermates (WT) were implanted with Lewis lung cancer cells to induce lung tumorigenesis. Tg mice showed significantly higher number and bigger size of tumors than WT mice. Tg tumors exhibited increased angiogenesis and proliferation while reduced apoptosis compared with WT tumors. Interestingly, a significantly enhanced upregulation of Cox-2 was detected in Tg tumors than in WT tumors. Also, Tg tumors demonstrated upregulation of VEGF and angiopoietin-1, downregulation of AKAP12, and increased eNOS phosphorylation compared with WT tumors. Celecoxib, a selective Cox-2 inhibitor, suppressed the HSPA12B-induced increase in lung tumor burden. Moreover, celecoxib decreased angiogenesis and proliferation whereas increased apoptosis in Tg tumors. Additionally, celecoxib reduced angiopoietin-1 expression and eNOS phosphorylation but increased AKAP12 levels in Tg tumors. Our results indicate that HSPA12B stimulates lung tumor growth via a Cox-2-dependent mechanism. The present study identified HSPA12B as a novel facilitator of lung tumor growth and a potential therapeutic target for the treatment of lung cancer.

Cardiac dysfunction is a major consequence of septic shock and may be responsible for the high mortality of sepsis. We have reported that transgenic mice with cardiac-specific overexpression of heat shock protein 27 (Hsp27 Tg) exhibited the protection against doxorubicin-induced cardiac dysfunction. We hypothesized that overexpression of Hsp27 will attenuate cardiac dysfunction during endotoxemia. Hsp27 Tg and age-matched wild-type (WT) mice were injected with LPS. Cardiac function was evaluated by echocardiography, survival rate was carefully monitored, and activities of signaling pathways were determined by immunoblot. LPS administration significantly decreased cardiac function in WT mice. In Hsp27 Tg mice, LPS-induced cardiac dysfunction was significantly attenuated as evidenced by increased ejection fraction (27.3%) and fractional shortening (37.1%), respectively, compared with LPS-treated WT mice. Heat shock protein 27 Tg mice were more resistant to LPS-induced mortality than WT. The levels of phospho-Akt and phospho-glycogen synthase kinase 3β (phospho-GSK-3β) in the myocardium were significantly increased in Hsp27 Tg mice compared with WT after LPS administration. Nuclear factor κB-binding activity was significantly decreased in Hsp27 Tg mice compared with WT mice after LPS challenge. Similar results were observed in in vitro studies using Hsp27-transfected rat cardiomyoblasts. Importantly, phosphoinositide 3-kinase inhibition abolished the protective effect of Hsp27 in LPS-induced cardiac dysfunction and mortality of endotoxemia. Our results suggest that Hsp27 plays an important role in attenuation of cardiac dysfunction and mortality in endotoxemia and that the mechanisms of the protection may involve activation of the PI3K/Akt signaling pathway.

Heat shock protein A12A (HSPA12A) is a newly discovered member of the Hsp70 family. The biological characteristics and functional roles of HSPA12A are poorly understood. This study investigated the effects of HSPA12A on ischaemic stroke in mice. Ischaemic stroke was induced by left middle cerebral artery occlusion for 1 h followed by blood reperfusion. We observed that HSPA12A was highly expressed in brain neurons, and neuronal HSPA12A expression was downregulated by ischaemic stroke and stroke-associated risk factors (aging, obesity and hyperglycaemia). To investigate the functional requirement of HSPA12A in protecting ischaemic brain injury, HSPA12A knockout mice (Hspa12a−/−) were generated. Hspa12a−/− mice exhibited an enlarged infarct volume and aggravated neurological deficits compared to their wild-type (WT) littermates after stroke. These aggravations in Hspa12a−/− mice were accompanied by more apoptosis and severer hippocampal morphological abnormalities in ischaemic hemispheres. Long-term examination revealed impaired motor function recovery and neurogenesis in stroke-affected Hspa12a−/− mice compared to stroke-affected WT controls. Significant reduced activation of GSK-3β/mTOR/p70S6K signalling was also observed in ischaemic hemispheres of Hspa12a−/− mice compared to WT controls. Administration with lithium (non-selective GSK-3β inhibitor) activated GSK-3β/mTOR/p70S6K signalling in stroke-affected Hspa12a−/− mice. Notably, lithium administration attenuated the HSPA12A deficiency-induced aggravation in infarct size, neurological deficits and neuronal death in Hspa12a−/− mice after stroke. Altogether, the findings suggest that HSPA12A expression encodes a critical novel prosurvival pathway during ischaemic stroke. We identified HSPA12A as a novel neuroprotective target for stroke patients.

Small heat shock proteins (hsp) have been implicated in mediation of classic preconditioning in the rabbit. Hsp27 is a terminal substrate of the p38 MAPK cascade. One and 2D gel electrophoresis and immunoblotting of cell fractions was used to determine p38 MAPK and hsp27 phosphorylation levels, respectively, during in vitro ischemia in control, calyculin A (Cal A)-treated (protein phosphatase inhibitor), SB203580-treated (p38MAPK inhibitor) and preconditioned (IPC) isolated adult rabbit cardiomyocytes. The dual phosphorylation of p38 MAPK was increased by early ischemia (30-60 min), after which there was a loss of total cytosolic p38 MAPK. The ischemic increase of p38 MAPK dual phosphorylation was enhanced by IPC. Cal A strongly activated dual phosphorylation of p38 MAPK in oxygenated cells and this was maintained into early ischemia. SB203580 inhibited the dual phosphorylation of p38 MAPK and attenuated the loss of total cytosolic p38 MAPK. In each protocol, ischemia translocated hsp27 from the cytosolic fraction to the cytoskeletal fraction at similar rates and extents. Hsp27 phosphorylation was quantitated as the fraction of diphosphorylated hsp27, based on IEF mobility shifts of hsp27 phosphorylation isoforms. In oxygenated control cells, cytosolic and cytoskeletal hsp27 was highly phosphorylated. After 90 min ischemia, cytoskeletal hsp27 was markedly dephosphorylaled. Cal A slightly increased control cytoskeletal hsp27 phosphorylation. During ischemic incubation, Cal A blocked ischemic dephosphorylation. SB203580 accelerated ischemic hsp27 dephosphorylation and injury. IPC insignificantly decreased the initial rate of ischemic dephosphorylation of hsp27, but not the extent of dephosphorylation in later ischemia. Phosphorylation is regulated by both kinase and phosphatase activities. IPC protection was not correlated with a significant increase in cytosolic or cytoskeletal hsp27 phosphorylation levels during prolonged (> 60-90 min) ischemia.

AB-type protein toxins such as cholera toxin (CT) consist of a catalytic A subunit and a cell-binding B subunit. CT proceeds through the secretory pathway in reverse, termed retrograde trafficking, and is delivered to the endoplasmic reticulum (ER). In order for the catalytic A1 subunit to become active it must separate from the rest of the holotoxin, and this dissociation event occurs in the ER lumen. CTA1 assumes an unfolded conformation upon dissociation from the holotoxin and is recognized by ERassociated degradation (ERAD), a quality control system that recognizes and exports misfolded proteins to the cytosol for degradation by the 26S proteasome. CTA1 is not degraded by the 26S proteasome because it has few sites for poly-ubitiquination, which is recognized by the cap of the 26S proteasome for degradation. Thus, CTA1 escapes the degradation of ERAD while at the same time using it as a transport mechanism into the cytosol. It was originally proposed that CTA1 is thermally stable and that ER chaperones actively unfolded CTA1 for translocation to the cytosol. In contrast, we hypothesized that the dissociated CTA1 subunit would unfold spontaneously at 37°C. This study focused on the three conditions linked to CTA1 instability and translocation: (i) CTA1 dissociation from the holotoxin, (ii) the translocation-competent conformation of CTA1, and the extraction of CTA1 from the ER into the cytosol. Disruption of any of these events will confer resistance to the toxin. The original model suggested that PDI actively unfolds CTA1 to allow for translocation. However, Fourier transform infrared iv spectroscopy (FTIR) and surface plasmon resonance (SPR) data we have gathered demonstrated that PDI dislodges CTA1 from the rest of the holotoxin without unfolding CTA1. Once released by the holotoxin, CTA1 spontaneously unfolds. PDI is thus required for the toxicity of CT, but not as an unfoldase as originally proposed. CTA1 must maintain an unfolded conformation to keep its translocation-competent state. Based on our model, if CTA1 is stabilized then it will not be able to activate the ERAD translocation system. Our SPR and toxicity results demonstrated that treatment with 4- phenylbutyrate (PBA), a chemical chaperone, stabilizes the structure of CTA1. This stabilization resulted in a decrease in translocation from the ER to the cytosol and a block of intoxication, which makes it a viable candidate for a therapeutic. Because CTA1 exits the ER in an unfolded state, there must be a driving force for this translocation. We hypothesized that Hsp90, a cytosolic chaperone, is responsible for the translocation of CTA1 across the membrane. Previous research had shown Hsp90 to be present on the cytosolic face of the ER and had also shown that Hsp90 will refold exogenously added proteins that enter the cytosol. Using drug treatments and RNAi, we found that Hsp90 is required for the translocation of CTA1 from the ER lumen to the cytosol, a brand new function for this chaperone. We have provided evidence to support a new, substantially different model of CTA1 translocation. CTA1 does not masquerade as a misfolded protein in order to utilize ERAD for entry into the cytosol; it actually becomes misfolded and is treated as any other ERAD substrate. The spontaneous unfolding of CTA1 is the key to its v recognition by ERAD and ultimately its translocation into the cytosol. Host factors play very important roles in intoxication by AB toxins and are targets for blocking intoxication.

Poor emergence of sorghum [(Sorghum bicolor (L) Moench] affects the stand and potential yields. The major objective of this research was to find correlations among field emergence data and laboratory seed vigor tests. Thirty-two and 30 hybrids of sorghum were planted at three Virginia locations in 1995 and 1996, respectively. Field emergence was subsequently compared with results from laboratory tests that included: 1) standard germination; 2) osmotic-stress using polyethylene glycol 8000 (mw); 3) heat-shock using 2 hr at 50°C stress; 4) electrical conductivity of steep water of germinating seeds; and 5) seed mass. Field emergence of grain sorghum differed among hybrids, years and locations. Mean emergence across years and locations was 67.5%, whereas mean germination in the laboratory was 87.8%. There were interaction between hybrid and location and between hybrid and year. Germination under optimal conditions (standard germination test) and with osmotic or heat-shock stress differed among hybrids. Regression analyses showed a weak correlation between laboratory germination (with or without stress) and field emergence in both years. The fresh weight of seedlings whether from standard germination or stress tests also differed among hybrids in both years, and the associations with field emergence were weakly correlated in 1996. Hybrids showed significant differences in radicle length when grown under laboratory stress in both years following standard germination. There was a weak correlation with field emergence and radicle length following heat-shock treatment in 1996. The measurement of electrical conductivity in the seed steep water showed significant differences among hybrids. A weak correlation with field emergence was seen in 1996. Conductivity values per gram of seed and per cm² of seed area revealed differences among hybrids. The correlation of these parameters with field emergence was higher than conductivity per seed. Seed mass varied among hybrids in both years , but was no correlation between seed mass and emergence. Of the laboratory parameters examined, germination proved to be the most consistent predictor of variations in field emergence of sorghum hybrids. / Ph. D.

No description available.