17-DMAG disrupted the autophagy flux leading to the apoptosis of acute lymphoblastic leukemia cells by inducing heat shock cognate protein 70.

AIMS: B-lineage acute lymphoblastic leukemia (B-ALL) is most common in children. We had reported heat shock protein 90 (Hsp90) over-expressed in high risk B-ALL children. 17-DMAG is a water soluble Hsp90 inhibitor, which was proved to be effective for advanced solid tumors and hematological malignancy. However, there is little research on its application in newly diagnosed B-ALL. And the detailed mechanism is seldom discussed. MAIN METHODS: Primary blast cells from 24 newly diagnosed B-ALL pediatric patients and two B-ALL cell lines were used in this study. Cell viability was measured by MTS assay. Apoptosis was evaluated by flow cytometry after annexin V-PI double staining. Protein expression was detected by immunoblotting analysis and immunofluorescence imaging. Cyto-ID autophagy detection assay was performed to show the autophagosomes and LysoTracker labeling to show the lysosomes. Gene knockdown was performed by RNA interference, and mRNA expression was measured by RT-qPCR. KEY FINDINGS: We showed 17-DMAG induced apoptosis in newly diagnosed B-ALL blasts and cell lines effectively. 17-DMAG induced heat shock cognate protein 70 (Hsc70) expression significantly. High expressed Hsc70 inhibited cathepsin D post-transcriptionally to impede the autophagic flux, which lead to the cell death. SIGNIFICANCE: Our work added new information towards understanding the molecular pharmacology of 17-DMAG, and suggested the newly diagnosed B-ALL pediatric patients might be benefited from 17-DMAG. Furthermore, we proved Hsc70 participated in the mechanism of cell death 17-DMAG leading in B-ALL.

Hsc70 Interacts with ß4GalT5 to Regulate the Growth of Gliomas.

Heat shock cognate protein 70 (Hsc70) is a key mediator for the maintenance of intracellular proteins and regulates cellular activities. And it is elevated in various tumor tissues including glioma, which is closely related to the malignancy and poor prognosis of the tumors. However, the effects of Hsc70 on gliomas and its regulatory mechanism have not yet been elucidated. In the present study, we found that Hsc70 was overexpressed in glioma tissues and cultured glioma cells. Furthermore, Hsc70 expression exhibited positive correlation with the grades of gliomas. Knockdown of Hsc70 could effectively inhibit cell proliferation and increase cell apoptosis. Furthermore, we identified that ß4GalT5 was a critical target for Hsc70-mediated anti-glioma effects. Blocking ß4GalT5 activity could effectively reverse the anti-tumor effect of Hsc70. Taken together, these data indicate that Hsc70 regulates ß4GalT5 levels, and possibly plays a role in cell proliferation and apoptosis of glioma.

Bisphenol A and Its Derivatives Induce Degradation of HIF-1alpha via the Lysosomal Pathway in Human Hepatocarcinoma Cell Line, Hep3B.

Bisphenol A (BPA, 2,2-bis(4-hydroxyphenyl)propane), one of the phenolic compounds widely used in the manufacture of plastic and epoxy resins, is known as an endocrine disruptor. In a previous study, we found that BPA induced hypoxia inducible factor-1alpha (HIF-1alpha) degradation by dissociation from heat shock protein 90 (Hsp90). In this study, to investigate the structural requirements for degradation of HIF-1alpha, we estimated the effect of BPA derivatives (BPE, BPF, BPB, Dimethyl butylidene diphenol (DMBDP), Ethyl hexylidene diphenol (EHDP), Bishydroxyphenyl cyclohexane (BHCH), and Methyl benzylidene bisphenol (MBBP)) on HIF-1alpha protein degradation, using human hepatocarcinoma cell line, Hep3B. BPB, DMBDP, BHCH, and MBBP decreased HIF-1alpha protein levels more efficiently than BPA, but BPE, BPF, and EHDP did not affect HIF-1alpha protein levels. BPA degraded HIF-1alpha even in the presence of MG132, a proteasome inhibitor. In this study, we found that ammonium chloride (NH4Cl), a lysosomal enzyme inhibitor, efficiently restored the decrease in HIF-1alpha protein levels by BPA. Recent studies indicated that HIF-1alpha is degraded by the lysosomal pathway as well as the proteasomal pathway. Therefore, we investigated the levels of heat shock cognate 70 kDa protein (HSC70) protein after treatment with BPA. We found that BPA induced HSC70 protein and overexpression of HSC70 enhanced HIF-1alpha degradation in Hep3B cells. These results suggested that BPA causes the degradation of HIF-1alpha by induction of HSC70, leading lysosomal degradation of HIF-1alpha.

Hsc70 chaperone activity underlies Trio GEF function in axon growth and guidance induced by netrin-1.

During development, netrin-1 is both an attractive and repulsive axon guidance cue and mediates its attractive function through the receptor Deleted in Colorectal Cancer (DCC). The activation of Rho guanosine triphosphatases within the extending growth cone facilitates the dynamic reorganization of the cytoskeleton required to drive axon extension. The Rac1 guanine nucleotide exchange factor (GEF) Trio is essential for netrin-1-induced axon outgrowth and guidance. Here, we identify the molecular chaperone heat shock cognate protein 70 (Hsc70) as a novel Trio regulator. Hsc70 dynamically associated with the N-terminal region and Rac1 GEF domain of Trio. Whereas Hsc70 expression supported Trio-dependent Rac1 activation, adenosine triphosphatase-deficient Hsc70 (D10N) abrogated Trio Rac1 GEF activity and netrin-1-induced Rac1 activation. Hsc70 was required for netrin-1-mediated axon growth and attraction in vitro, whereas Hsc70 activity supported callosal projections and radial neuronal migration in the embryonic neocortex. These findings demonstrate that Hsc70 chaperone activity is required for Rac1 activation by Trio and this function underlies netrin-1/DCC-dependent axon outgrowth and guidance.

E3 ligase CHIP and Hsc70 regulate Kv1.5 protein expression and function in mammalian cells.

Kv1.5 confers ultra-rapid delayed-rectifier potassium channel current (IKur) which contributes to repolarization of the atrial action potential. Kv1.5 proteins, degraded via the ubiquitin-proteasome pathway, decreased in some atrial fibrillation patients. Carboxyl-terminus heat shock cognate 70-interacting protein (CHIP), an E3 ubiquitin ligase, is known to ubiquitinate short-lived proteins. Here, we investigated the roles of CHIP in Kv1.5 degradation to provide insights into the mechanisms of Kv1.5 decreases and treatments targeting Kv1.5 for atrial fibrillation. Coexpression of CHIP with Kv1.5 in HEK293 cells increased Kv1.5 protein ubiquitination and decreased the protein level. Immunofluorescence revealed decreases of Kv1.5 proteins in the endoplasmic reticulum and on the cell membrane. A siRNA against CHIP suppressed Kv1.5 protein ubiquitination and increased its protein level. CHIP mutants, lacking either the N-terminal tetratricopeptide region domain or the C-terminal U-box domain, failed to exert these effects on Kv1.5 proteins. Immunoprecipitation showed that CHIP formed complexes with Kv1.5 proteins and heat shock cognate protein 70 (Hsc70). Effects of Hsc70 on Kv1.5 were similar to CHIP by altering interaction of CHIP with Kv1.5 protein. Coexpression of CHIP and Hsc70 with Kv1.5 additionally enhanced Kv1.5 ubiquitination. Kv1.5 currents were decreased by overexpression of CHIP or Hsc70 but were increased by knockdown of CHIP or Hsc70 in HEK 293 cells stably expressing Kv1.5. These effects of CHIP and Hsc70 were also observed on endogenous Kv1.5 in HL-1 mouse cardiomyocytes, decreasing IKur and prolonging action potential duration. These results indicate that CHIP decreases the Kv1.5 protein level and functional channel by facilitating its degradation in concert with chaperone Hsc70.

Bacterial lipopolysaccharide augments febrile-range hyperthermia-induced heat shock protein 70 expression and extracellular release in human THP1 cells.

Sepsis, a devastating and often lethal complication of severe infection, is characterized by fever and dysregulated inflammation. While infections activate the inflammatory response in part through Toll-like receptors (TLRs), fever can partially activate the heat shock response with generation of heat shock proteins (HSPs). Since extracellular HSPs, especially HSP70 (eHSP70), are proinflammatory TLR agonists, we investigated how exposure to the TLR4 agonist, bacterial lipopolysaccharide (LPS) and febrile range hyperthermia (FRH; 39.5°C) modify HSP70 expression and extracellular release. Using differentiated THP1 cells, we found that concurrent exposure to FRH and LPS as well as TLR2 and TLR3 agonists synergized to activate expression of inducible HSP72 (HSPA1A) mRNA and protein via a p38 MAP kinase-requiring mechanism. Treatment with LPS for 6 h stimulated eHSP70 release; levels of eHSP70 released at 39.5°C were higher than at 37°C roughly paralleling the increase in intracellular HSP72 in the 39.5°C cells. By contrast, 6 h exposure to FRH in the absence of LPS failed to promote eHSP70 release. Release of eHSP70 by LPS-treated THP1 cells was inhibited by glibenclamide, but not brefeldin, indicating that eHSP70 secretion occurred via a non-classical protein secretory mechanism. Analysis of eHSP70 levels in exosomes and exosome-depleted culture supernatants from LPS-treated THP1 cells using ELISA demonstrated similar eHSP70 levels in unfractionated and exosome-depleted culture supernatants, indicating that LPS-stimulated eHSP70 release did not occur via the exosome pathway. Immunoblot analysis of the exosome fraction of culture supernatants from these cells showed constitutive HSC70 (HSPA8) to be the predominant HSP70 family member present in exosomes. In summary, we have shown that LPS stimulates macrophages to secrete inducible HSP72 via a non-classical non-exosomal pathway while synergizing with FRH exposure to increase both intracellular and secreted levels of inducible HSP72. The impact of increased macrophage intracellular HSP70 levels and augmented secretion of proinflammatory eHSP70 in the febrile, infected patient remains to be elucidated.

Identification of a novel human LAP1 isoform that is regulated by protein phosphorylation.

Lamina associated polypeptide 1 (LAP1) is an integral protein of the inner nuclear membrane that is ubiquitously expressed. LAP1 binds to lamins and chromatin, probably contributing to the maintenance of the nuclear envelope architecture. Moreover, LAP1 also interacts with torsinA and emerin, proteins involved in DYT1 dystonia and X-linked Emery-Dreifuss muscular dystrophy disorder, respectively. Given its relevance to human pathological conditions, it is important to better understand the functional diversity of LAP1 proteins. In rat, the LAP1 gene (TOR1AIP1) undergoes alternative splicing to originate three LAP1 isoforms (LAP1A, B and C). However, it remains unclear if the same occurs with the human TOR1AIP1 gene, since only the LAP1B isoform had thus far been identified in human cells. In silico analysis suggested that, across different species, potential new LAP1 isoforms could be generated by alternative splicing. Using shRNA to induce LAP1 knockdown and HPLC-mass spectrometry analysis the presence of two isoforms in human cells was described and validated: LAP1B and LAP1C; the latter is putatively N-terminal truncated. LAP1B and LAP1C expression profiles appear to be dependent on the specific tissues analyzed and in cultured cells LAP1C was the major isoform detected. Moreover, LAP1B and LAP1C expression increased during neuronal maturation, suggesting that LAP1 is relevant in this process. Both isoforms were found to be post-translationally modified by phosphorylation and methionine oxidation and two LAP1B/LAP1C residues were shown to be dephosphorylated by PP1. This study permitted the identification of the novel human LAP1C isoform and partially unraveled the molecular basis of LAP1 regulation.

Antioxidant signaling involving the microtubule motor KIF12 is an intracellular target of nutrition excess in beta cells.

Beta cell injury due to oxidative stress is a typical etiology of diabetes caused by nutritional excess, but its precise mechanism remains largely elusive. Here, we demonstrate that the microtubule motor KIF12 mediates an antioxidant cascade in beta cells as an intracellular target of excess fat intake or "lipotoxicity." KIF12 knockout mice suffer from hypoinsulinemic glucose intolerance due to increased beta cell oxidative stress. Using this model, we identified an antioxidant signaling cascade involving KIF12 as a scaffold for the transcription factor Sp1. The stabilization of nascent Sp1 appeared to be essential for proper peroxisomal function by enhancing Hsc70 expression, and the pharmacological induction of Hsc70 expression with teprenone counteracted the oxidative stress. Because KIF12 is transcriptionally downregulated by chronic exposure to fatty acids, this antioxidant cascade involving KIF12 and Hsc70 is proposed to be a critical target of nutritional excess in beta cells in diabetes.

Ultrasound targeted microbubble destruction for novel dual targeting of HSP72 and HSC70 in prostate cancer.

The aim was to determine whether ultrasound targeted microbubble destruction (UTMD) promotes dual targeting of HSP72 and HSC70 for therapy of castration-resistant prostate cancer (CRPC), to improve the specific and efficient delivery of siRNA, to induce tumor cell specific apoptosis, and to find new therapeutic targets specific of CRPC.VCaP cells were transfected with siRNA oligonucleotides. HSP70, HSP90 and cleaved caspase-3 expression were determined by real-time quantitative polymerase chain reaction and Western blotting. Apoptosis and transfection efficiency were assessed by flow cytometry. Cell viability assays were used to evaluate safety. We found HSP72, HSC70 and HSP90 expression to be absent or weak in normal prostate epithelial cells (RWPE-1), but uniformly strong in prostate cancerous cells (VCaP). UTMD combined with dual targeting of HSP72 and HSC70 siRNA improve the efficiency of transfection, cell uptake of siRNA, downregulation of HSP70 and HSP90 expression in VCaP cells at the mRNA and protein level, and induction of extensive tumor-specific apoptosis. Cell counting kit-8 assays showed decreased cellular viability in the HSP72/HSC70-siRNA silenced group. These results suggest that the combination of UTMD with dual targeting HSP70 therapy for PCa may be most efficacious, providng a novel, reliable, non-invasive, safe targeted approach to improve the specific and efficient delivery of siRNA, and achieve maximal effects.

Uterine selection of human embryos at implantation.

Human embryos frequently harbor large-scale complex chromosomal errors that impede normal development. Affected embryos may fail to implant although many first breach the endometrial epithelium and embed in the decidualizing stroma before being rejected via mechanisms that are poorly understood. Here we show that developmentally impaired human embryos elicit an endoplasmic stress response in human decidual cells. A stress response was also evident upon in vivo exposure of mouse uteri to culture medium conditioned by low-quality human embryos. By contrast, signals emanating from developmentally competent embryos activated a focused gene network enriched in metabolic enzymes and implantation factors. We further show that trypsin, a serine protease released by pre-implantation embryos, elicits Ca(2+) signaling in endometrial epithelial cells. Competent human embryos triggered short-lived oscillatory Ca(2+) fluxes whereas low-quality embryos caused a heightened and prolonged Ca(2+) response. Thus, distinct positive and negative mechanisms contribute to active selection of human embryos at implantation.

Downregulation of Hsp70 inhibits apoptosis induced by sialic acid-binding lectin (leczyme).

Heat shock proteins (Hsps) are molecular chaperones that maintain homeostasis of organisms. In regards to the Hsps, many studies have investigated the structure, expression, localization and functions of Hsp70 and Hsc70 including expression in the glycosphingolipid-enriched microdomain (GEM) on the cell surface and involvement in cell death. Sialic acid-binding lectin (SBL) isolated from oocytes of Rana catesbeiana is a multifunctional protein which has lectin activity, ribonuclease activity and antitumor activity. SBL has potential as a new type of anticancer drug, since it causes cancer-selective induction of apoptosis by multiple signaling pathways in which RNA is its target; and the participation of the mitochondrial pathway and the endoplasmic reticulum (ER) stress-mediated pathway has been suggested. It has also been suggested that receptor(s) for SBL (SBLR) may exist in the GEM on the cell surface. In the present study, we studied the possible involvement of Hsp70 and Hsc70 in SBL-induced apoptosis. We showed that Hsp70 and Hsc70 were expressed on the P388 cell surface similar to SBLR, and their distribution in cells dramatically changed immediately prior to the execution of apoptosis following stimulation of SBL. Functional study of Hsp70 revealed that decreased expression of Hsp70 diminished the apoptosis induced by SBL. It is suggested that Hsp70 participates in the antitumor effect of SBL.

Electrophoretic and immunocytochemical analysis of Hsp72 and Hsp73 expression in heat-stressed mouse testis and epididymis.

OBJECTIVE: One of the most profound events in stressed cells is the synthesis of a highly conserved family of proteins, the 'heat shock proteins' (Hsp). The Hsp70 family is the most diverse, and includes constitutive as well as stress-inducible proteins with overlapping or unique functions in different cell compartments. Elucidation of Hsp70 expression during different stages of spermatogenesis and maturation of germ cells is of particular interest due to their high sensitivity to heat treatment. STUDY DESIGN: Expression of the main isoforms of the Hsp70 family (constitutive Hsp73 and stress-inducible Hsp72) was determined in normal and heat-stressed mouse testes and epididymis from sexually mature (60-day-old) mice during spermatogenesis and maturation of germ cells. Immunocytochemical analysis and one- and two-dimensional gel electrophoresis were used to separate mouse testicular and epididymal proteins from saline extracts, followed by Western blotting. RESULTS: Using a polyclonal anti-Hsp70 antibody that recognizes both isoforms, inducible Hsp72 expression, was demonstrated immunocytochemically only in heat-stressed tissues, while a high level of constitutive Hsp73 isoform expression was found in both normal and heat-stressed mouse male reproductive tissues. Morphological studies have shown that round and elongated spermatids in the testes, as well as all segments of the epididymis, are most sensitive to heat stress. In the epididymis, the reaction was localized in different cell compartments. CONCLUSION: In heat stress conditions, Hsp73 is mobilized to prevent apoptosis in the testes and epididymis, and assists Hsp72 in the repair of stress-altered protein conformations.

The PI3K/Akt signaling pathway regulates the expression of Hsp70, which critically contributes to Hsp90-chaperone function and tumor cell survival in multiple myeloma.

Despite therapeutic advances multiple myeloma remains largely incurable, and novel therapeutic concepts are needed. The Hsp90-chaperone is a reasonable therapeutic target, because it maintains oncogenic signaling of multiple deregulated pathways. However, in contrast to promising preclinical results, only limited clinical efficacy has been achieved through pharmacological Hsp90 inhibition. Because Hsp70 has been described to interact functionally with the Hsp90-complex, we analyzed the suitability of Hsp72 and Hsp73 as potential additional target sites. Expression of Hsp72 and Hsp73 in myeloma cells was analyzed by immunohistochemical staining and western blotting. Short interfering RNA-mediated knockdown or pharmacological inhibition of Hsp72 and Hsp73 was performed to evaluate the role of these proteins in myeloma cell survival and for Hsp90-chaperone function. Furthermore, the role of PI3K-dependent signaling in constitutive and inducible Hsp70 expression was investigated using short interfering RNA-mediated and pharmacological PI3K inhibition. Hsp72 and Hsp73 were frequently overexpressed in multiple myeloma. Knockdown of Hsp72 and/or Hsp73 or treatment with VER-155008 induced apoptosis of myeloma cells. Hsp72/Hsp73 inhibition decreased protein levels of Hsp90-chaperone clients affecting multiple oncogenic signaling pathways, and acted synergistically with the Hsp90 inhibitor NVP-AUY922 in the induction of death of myeloma cells. Inhibition of the PI3K/Akt/GSK3ß pathway with short interfering RNA or PI103 decreased expression of the heat shock transcription factor 1 and down-regulated constitutive and inducible Hsp70 expression. Treatment of myeloma cells with a combination of NVP-AUY922 and PI103 resulted in additive to synergistic cytotoxicity. In conclusion, Hsp72 and Hsp73 sustain Hsp90-chaperone function and critically contribute to the survival of myeloma cells. Translation of Hsp70 inhibition into the clinic is therefore highly desirable. Treatment with PI3K inhibitors might represent an alternative therapeutic strategy to target Hsp70.

Analysis of chaperone-assisted ubiquitylation.

Molecular chaperones are traditionally viewed as cellular protein folding and assembly factors. However, in recent years it became more and more evident that certain chaperones, i.e., members of the 70-kDa heat shock protein family (Hsp70s), participate very actively in protein degradation and in this way significantly contribute to protein homeostasis. Degradation is often initiated through a close cooperation of Hsp70s with chaperone-associated ubiquitin ligases. This results in the ubiquitylation of chaperone-bound client proteins and triggers client sorting toward the proteasome or the autophagosome-lysosome system. Here, we describe the in vitro reconstitution of chaperone-assisted ubiquitylation, which allows analyzing molecular details of this important proteostasis mechanism.

Responses of HSC70 expression in diencephalon to iron deficiency anemia in rats.

A powdered diet containing 100 or 3 ppm Fe was fed to rats starting at the age of 3 weeks. The voluntary activity level was checked using a wheel in the cage during the 17th week after the beginning of supplementation. Significantly less activity was seen in the 3 ppm Fe group during both light and dark periods. After 20 weeks, the blood and diencephalon were sampled from both groups. Lower hematocrit and blood hemoglobin content was observed in the 3 ppm Fe group. The level of 70 kDa heat shock cognate (HSC70) expression was greater in the diencephalon of the 3 ppm Fe group. In addition, the distribution of HSC70 was determined by proximity ligation assay. More HSC70-positive as well as total cells were noted in several areas of the diencephalon of the iron-deficient rats. The altered expression and distribution of HSC70 might play some role in the neurological changes.

Oxymatrine inhibits hepatitis B infection with an advantage of overcoming drug-resistance.

Oxymatrine (OMTR) is an anti-hepatitis drug used in China. Its mechanism of action is unknown. Recently, we found that OMTR inhibits hepatitis B virus (HBV) via down-regulating the expression of heat-stress cognate 70 (Hsc70), a host protein required for HBV DNA replication. Goal of this study was to assess the effect of OMTR on clinical HBV drug-resistance. OMTR monotherapy (oral, 12 months) reduced blood HBV DNA by 96% and HBeAg by 70% in the chronic hepatitis B (CHB) patients resistant to lamivudine (n = 17), equal to its efficacy in the naïve CHB cohort (n = 20). Liver biopsy study showed that OMTR treatment caused a decrease of Hcs70 mRNA in liver cells, parallel with a reduction of intracellular HBV DNA. Combination of lamivudine with OMTR (n = 15) (oral, 12 months) showed an enhanced anti-HBV effect as compared to lamivudine monotherapy (n = 25). The incidence of drug resistance against lamivudine in the combination group was significantly lower than that in the lamivudine group (1/15 vs 7/25; p<0.01). The results were further confirmed in vitro. Treatment of HBV(+) HepH2215 cells with sub-optimal dose of OMTR for 8 months suppressed HBV replication without inducing drug resistance, whereas lamivudine monotherapy caused drug-resistant mutation in 3 months. Combination of OMTR with lamivudine prevented HBV from developing drug resistance.

Neurons but not glial cells overexpress ubiquitin in the rat brain following focused ultrasound-induced opening of the blood-brain barrier.

Focused ultrasound-induced opening of the blood-brain barrier (BBB) in the presence of ultrasound contrast agents is a promising strategy for a targeted drug delivery to the brain. The aim of our study was to identify whether brain molecular stress pathways are targeted by ultrasound treatment. Using an upper level of acoustic pressures in combination with microbubbles, which have been previously reported as reliable for BBB opening without causing tissue damage, we found that ultrasound leads to an increased ubiquitinylation of proteins in neuronal (11+/-3 ubiquitin-overexpressing cells per optical field) but not glial cells 6 h post-insonation, increasing to 16 (+/-4) labelled cells after 24 h. No changes in the expression of Hsp70 and Hsc70 were detected over 24 h. Ultrasound treatment was followed by limited apoptosis after 24 h (32+/-6 cleaved-caspase 3-positive cells per optical field) in the insonated areas. Only neurons were identified in the apoptotic population. Although these observations may not be applicable for all acoustic parameters useful for BBB opening, they demonstrate that insonation of the rat brain with the parameters used in our experiments is a useful tool for BBB opening and induces specific cellular stress response restricted to neuronal cells.

Gene and protein expression of Drosophila Starvin during cold stress and recovery from chill coma.

In Drosophila melanogaster, the sole member of the Bcl-2-associated anthanogene (BAG)-family proteins, called Starvin (Stv), has only been recently described. BAG proteins regulate a large range of physiological processes including life/death cell balance and stress response. The role of Stv has been poorly studied in the context of abiotic stress and particularly during and after cold stress. In this study we investigated the temporal expression of Stv gene and protein in adult flies during both the cold stress (up to 9 h at 0 degrees C) and the subsequent recovery phase (up to 8 h at 25 degrees C). Because BAG proteins can regulate positively and negatively the function of Hsp70/Hsc70, we also checked whether Stv expression was related to Hsp70 and Hsc70. Stv mRNA and Stv protein both showed a similar expression pattern: no modulation during the cold period followed by a significant up-regulation during the recovery period. A coordinated response of Stv and Hsp70 mRNA was observed, but not for Hsc70. Our findings indicate that Stv expression is part of a stress-induced program in D. melanogaster. It probably acts as a co-chaperone modulating the activity of Hsp70 chaperone machinery during recovery from cold stress. Finally our results support the suggestion that Stv and human BAG3 may be functional homologs.

Severity of experimental autoimmune encephalomyelitis is unexpectedly reduced in mice born to vitamin D-deficient mothers.

Accumulating data indicate that vitamin D, a sun-induced hormone, plays a key role in multiple sclerosis (MS) etiology. Notably, it has been shown that there is a remarkable season of birth effect in MS. We surmised that gestational vitamin D deficiency is a risk factor for MS. To test this hypothesis, a vitamin D deficiency was induced in C57BL/6 female mice 6 weeks prior to conception and prolonged until offspring birth. Contrary to our prediction, we show here that adult offspring exposed to developmental vitamin D deficiency (DVD) developed a striking milder and delayed experimental autoimmune encephalomyelitis (EAE), when compared to control offspring. Using reverse transcription and quantitative real-time PCR, we measured the expression level of 22 candidate transcripts in the spleen, the cerebrum and the spinal cord, at Day0 and Day30 post-immunization. We report here that, at Day30 post-immunization, TNF, osteopontin, H2-Eb were over-expressed and IFN was under-expressed in the spinal cord of control mice and not in DVD mice. Another discrepancy between nervous and immune systems was observed: expression of IL4 was dysregulated exclusively in the spleen. Reduced symptom severity in DVD mice can partially be explained by a nervous system-restricted over-expression of vitamin D receptor (VDR), two heat shock proteins (HSP90, HSPa8) and FK506 binding protein 1a (FKBP1a), at Day0. Our clinical test and molecular findings converge to indicate that maternal hypovitaminosis D imprints the foetus and alters the susceptibility of the offspring to EAE. We propose a new hypothesis to explain our unexpected observations.

Transgenic expression of Hsc70 in pancreatic islets enhances autoimmune diabetes in response to beta cell damage.

Inflammation following tissue damage promotes lymphocyte recruitment, tissue remodeling, and wound healing while maintaining self tolerance. Endogenous signals associated with tissue damage and cell death have been proposed to initiate and instruct immune responses following injury. In this study, we have examined the effects of elevated levels of a candidate endogenous danger signal, heat shock cognate protein 70 (hsc70), on stimulation of inflammation and autoimmunity following cell damage. We find that damage to pancreatic beta cells expressing additional cytosolic hsc70 leads to an increased incidence of diabetes in a transgenic mouse model. Steady-state levels of activated APC and T cell populations in the draining lymph node were enhanced, which further increased following streptozotocin-induced beta cell death. In addition, proinflammatory serum cytokines, and lymphocyte recruitment were increased in hsc70 transgenic mice. Islet Ag-specific T cells underwent a greater extent of proliferation in the lymph nodes of mice expressing hsc70 following beta cell damage, suggesting elevated Ag presentation following release of Ag in the presence of hsc70. These findings suggest that an elevated content of hsc70 in cells undergoing necrotic or apoptotic cell death can increase the extent of sterile inflammation and increase the susceptibility to autoimmunity.