Heat Shock Protein 70B Research Articles

TurboID reveals the proxiomes of Chlamydomonas proteins involved in thylakoid biogenesis and stress response.

In Chlamydomonas (Chlamydomonas reinhardtii), the VESICLE-INDUCING PROTEIN IN PLASTIDS 1 and 2 (VIPP1 and VIPP2) play roles in the sensing and coping with membrane stress and in thylakoid membrane biogenesis. To gain more insight into these processes, we aimed to identify proteins interacting with VIPP1/2 in the chloroplast and chose proximity labeling (PL) for this purpose. We used the transient interaction between the nucleotide exchange factor CHLOROPLAST GRPE HOMOLOG 1 (CGE1) and the stromal HEAT SHOCK PROTEIN 70B (HSP70B) as test system. While PL with APEX2 and BioID proved to be inefficient, TurboID resulted in substantial biotinylation in vivo. TurboID-mediated PL with VIPP1/2 as baits under ambient and H2O2 stress conditions confirmed known interactions of VIPP1 with VIPP2, HSP70B, and the CHLOROPLAST DNAJ HOMOLOG 2 (CDJ2). Proteins identified in the VIPP1/2 proxiomes can be grouped into proteins involved in the biogenesis of thylakoid membrane complexes and the regulation of photosynthetic electron transport, including PROTON GRADIENT REGULATION 5-LIKE 1 (PGRL1). A third group comprises 11 proteins of unknown function whose genes are upregulated under chloroplast stress conditions. We named them VIPP PROXIMITY LABELING (VPL). In reciprocal experiments, we confirmed VIPP1 in the proxiomes of VPL2 and PGRL1. Our results demonstrate the robustness of TurboID-mediated PL for studying protein interaction networks in the chloroplast of Chlamydomonas and pave the way for analyzing functions of VIPPs in thylakoid biogenesis and stress responses.

Quantum dots induce heat shock-related cytotoxicity at intracellular environment

Quantum dots (QDs) are semiconductor nanocrystals with unique optical properties. Different proteins or polymers are commonly bound to their surfaces to improve biocompatibility. However, such surface modifications may not provide sufficient protection from cytotoxicity due to photodegradation and oxidative degradation. In this study, the cytotoxic effects of QDs, CdTe, and CdSe/ZnS were investigated using cadmium-resistant cells. CdTe QDs significantly reduced cell viability, whereas, CdSe/ZnS treatment did not markedly decrease the cell number. CdTe QDs were cytotoxic in cadmium-resistant cells suggesting that internalized QDs degraded and cadmium ions contributed to the cytotoxic effects. CdTe QDs were consistently more cytotoxic than CdSe/ZnS QDs, but both QDs as well as cadmium ions activated heat shock protein 70B' promoter. QDs themselves are likely to contribute to HSP70B' promoter activation in cadmium-resistant cells, because CdSe/ZnS QDs do not release sufficient cadmium to activate this promoter.

Pharmacokinetics of combined gene therapy expressing constitutive human GM-CSF and hyperthermia-regulated human IL-12

BackgroundAn adenovirus that expresses both interleukin (IL)-12 and granulocyte-macrophage colony-stimulating-factor (GM-CSF) has been proven to be very effective in treating several tumors, but causes serious normal tissue toxicities.MethodsIn this study, a novel adenoviral vector was constructed by placing the human GM-CSF gene under the control of the CMV-IE promoter and human IL-12 gene under the control of heat shock protein 70B gene promoter. Both hGM-CSF and hIL-12 expressions in virus-infected tumor cells were analyzed in vitro and in vivo when underlying single or multiple rounds of hyperthermia.ResultsWe observed constitutive high expression of human GM-CSF and heat-induced expression of human IL-12 after a single round of hyperthermia post viral infection. The heat-induced hIL-12 expression exhibited a pulse-like pattern with a peak at 24 hrs followed by a decline 48 hrs post heat stress. Repeated heat treatment was more effective in inducing hIL-12 expression than a one-time heat treatment. Interestedly, we also observed that constitutive expression of hGM-CSF could be stimulated by heat stress in tested tumor cells.ConclusionOur study provided a novel strategy for combined gene therapy that allows constitutive expression of a non-toxic gene such as GM-CSF and heat-induced expression of a toxic gene such as IL-12. In addition, our study also showed that hyperthermia can be used to trigger gene expression in temporal and special manner.

Heat-inducible transgene expression with transcriptional amplification mediated by a transactivator

Purpose: Control of therapeutic gene expression in tumours is a major goal of gene therapy research, as it can restrict cytotoxic gene expression in cancer cells. In addition, the combination of hyperthermia with gene therapy through the application of heat-inducible vectors can result in considerable improvements in therapeutic efficiency. In this study, to combine heat-inducibility with high-level transgene expression, we developed a heat-inducible transgene expression system with transcriptional amplification mediated by a tetracycline-responsive transactivator.Materials and methods: A hybrid promoter was generated by placing the heat shock protein (HSP) 70B′ promoter under the tetracycline-repressor responsive element sequence, and a reporter/therapeutic gene expression plasmid was constructed by placing a reporter/therapeutic gene under the control of this hybrid promoter.Results: When the transactivator expression plasmid harbouring an expression cassette of the tetracycline-responsive transactivator gene was co-transfected with a reporter gene expression plasmid, the reporter gene expression was controlled by heat treatment. With this system, high levels of heat-induced transgene expression were observed compared to that from the HSP promoter alone without the transactivator. Evaluation of in vitro therapeutic effects using cancer cell lines revealed that therapeutic gene expression effectively caused cell death in a greater percentage of the cells.Conclusion: These findings indicate that this strategy improves the efficacy of cancer gene therapy.

Polydatin, A Natural Precursor of Resveratrol, Induces β-Defensin Production and Reduces Inflammatory Response

It is well known that human keratinocytes produce the anti-microbial peptide β-defensin 2. Its production is enhanced by pathogenic microorganisms or other environmental stressors. In this study, we evaluated the effect of resveratrol, a polyphenol found in several dietary source as grape seed, and its natural precursor, polydatin on heat-stressed human keratinocytes. By reverse transcription-polymerase chain reaction and enzyme-linked immunoadsorbent assay, we demonstrated that resveratrol used in combination with polydatin was able to modulate interleukin (IL)-6, IL-8 and tumor necrosis factor-alpha gene expression. In addition, our data show that resveratrol and polydatin increased the heat shock protein (Hsp)70B' gene expression, a Hsp that plays an important role in the cytoprotection and repair of cells and tissues. Worthy of note, polydatin used alone or in combination with resveratrol, increased the release of human β-defensin 2. These results highlighted the ability of polydatin and resveratrol to reinforce cytoprotective response in stress conditions and suggest their use in cosmetic or pharmaceutical preparations.

Detection of cellular response to titanium dioxide nanoparticle agglomerates by sensor cells using heat shock protein promoter

Nanotechnology is becoming increasingly important for products used in our daily lives, such as the masses of titanium dioxide nanoparticle agglomerates (TiO(2) NPs) used in the pharmaceutical industry, for cosmetic products, or for pigments. Meanwhile, a serious lack of detailed information concerning the interaction between the nanomaterials and cells limits their biological and medical applications. Sensing technology is very important for understanding these interactions. We have shown that TiO(2) NPs induce heat shock protein 70B' (HSP70B') mRNA [Okuda-Shimazaki et al., 2010. Int J Mol Sci 11:2383-2392]. In the current work, sensor cells for detection of cellular responses to NPs were prepared by transfecting an HSP70B' promoter-reporter plasmid. First, to find suitable cells for detection, five different mammalian cell lines were chosen as potential sensor cells. The results showed TiO(2) NP response in some cell lines, although different sensor cells had different TiO(2) NP response levels, as heat shock response ability is important for the detection. Then, we studied the TiO(2) NP time-course response and dose response. The results indicated that our sensor cells can detect TiO(2) NP cellular responses. Our work should aid in understanding the interactions between bio-nanomaterials and cells.

Heat-inducible transgene expression system incorporating a positive feedback loop of transcriptional amplification for hyperthermia-induced gene therapy

One of the major goals of gene therapy is to regulate the expression of therapeutic genes in desired cells or tissues. For this purpose, heat-inducible vectors have been exploited for cancer gene therapy combined with hyperthermia, which can result in considerable improvement of therapeutic effects. In the present study, we constructed a novel heat-inducible gene expression system incorporating a transactivation system with a positive feedback loop of transcriptional amplification. The target gene expression mediated by the transactivator under the control of a heat shock protein 70B' promoter is enhanced by self-promoted transactivator gene expression. This expression system showed tight control of target gene expression together with high-level expression; enhanced expression of the reporter gene was observed in transfected cells upon heat treatment, while negligible gene expression was detected in non-heated cells. When a therapeutic gene was used as the target gene, a considerable cytotoxic effect was observed after heat treatment of cancer cells transfected with the plasmids. The heat-induced transgene expression system is a promising new approach for the development of both a safe and effective vector for hyperthermia-based cancer gene therapy.

Differential regulation of acid sphingomyelinase in macrophages stimulated with oxidized low‐density lipoprotein (LDL) and oxidized LDL immune complexes: role in phagocytosis and cytokine release

Oxidized low-density lipoprotein (oxLDL) and oxLDL-containing immune complexes (oxLDL-IC) contribute to the formation of lipid-laden macrophages (foam cells). Fcγ receptors mediate uptake of oxLDL-IC, whereas scavenger receptors internalize oxLDL. We have previously reported that oxLDL-IC, but not free oxLDL, activate macrophages and prolong their survival. Sphingomyelin is a major constituent of cell membranes and lipoprotein particles and acid sphingomyelinase (ASMase) hydrolyses sphingomyelin to generate the bioactive lipid ceramide. ASMase exists in two forms: lysosomal (L-ASMase) and secretory (S-ASMase). In this study we examined whether oxLDL and oxLDL-IC regulate ASMase differently, and whether ASMase mediates monocyte/macrophage activation and cytokine release. The oxLDL-IC, but not oxLDL, induced early and consistent release of catalytically active S-ASMase. The oxLDL-IC also consistently stimulated L-ASMase activity, whereas oxLDL induced a rapid transient increase in L-ASMase activity before it steadily declined below baseline. Prolonged exposure to oxLDL increased L-ASMase activity; however, activity remained significantly lower than that induced by oxLDL-IC. Further studies were aimed at defining the function of the activated ASMase. In response to oxLDL-IC, heat-shock protein 70B' (HSP70B') was up-regulated and localized with redistributed ASMase in the endosomal compartment outside the lysosome. Treatment with oxLDL-IC induced the formation and release of HSP70-containing and IL-1β-containing exosomes via an ASMase-dependent mechanism. Taken together, the results suggest that oxLDL and oxLDL-IC differentially regulate ASMase activity, and the pro-inflammatory responses to oxLDL-IC are mediated by prolonged activation of ASMase. These findings may contribute to increased understanding of mechanisms mediating macrophage involvement in atherosclerosis.

Effect of Polyethylene Glycol Modification of TiO2 Nanoparticles on Cytotoxicity and Gene Expressions in Human Cell Lines

Nanoparticles (NPs) are tiny materials used in a wide range of industrial and medical applications. Titanium dioxide (TiO2) is a type of nanoparticle that is widely used in paints, pigments, and cosmetics; however, little is known about the impact of TiO2 on human health and the environment. Therefore, considerable research has focused on characterizing the potential toxicity of nanoparticles such as TiO2 and on understanding the mechanism of TiO2 NP-induced nanotoxicity through the evaluation of biomarkers. Uncoated TiO2 NPs tend to aggregate in aqueous media, and these aggregates decrease cell viability and induce expression of stress-related genes, such as those encoding interleukin-6 (IL-6) and heat shock protein 70B’ (HSP70B’), indicating that TiO2 NPs induce inflammatory and heat shock responses. In order to reduce their toxicity, we conjugated TiO2 NPs with polyethylene glycol (PEG) to eliminate aggregation. Our findings indicate that modifying TiO2 NPs with PEG reduces their cytotoxicity and reduces the induction of stress-related genes. Our results also suggest that TiO2 NP-induced effects on cytotoxicity and gene expression vary depending upon the cell type and surface modification.

Detection of DNA Damage Response Caused by Different Forms of Titanium Dioxide Nanoparticles using Sensor Cells

Titanium dioxide nanoparticles (TiO2 NPs) are generally considered to be biologically inert. However, TiO2 occurs in several crystalline forms, the two most common being rutile and anatase. Although both forms are tetragonal, the different crystalline forms give rise to different physical and chemical characteristics such as hardness, refractive index and photocatalytic ability. We hypothesized that the two forms of TiO2 NPs would also elicit different cellular responses. Three cell-based biosensors, using B-cell Translocation Gene 2 (BTG2), heat shock protein70B' (HSP70B') and nuclear factor kappa B (NF-AÂoB) sensor cells, were used to determine if the different forms of TiO2 NPs cause different cellular responses. The cellular responses induced by TiO2 NPs were detected using HSP70B' and NF-AÂoB sensor cells; we found that the different forms of TiO2 NPs resulted in the same HSP70B' and NF-AÂoB response. BTG2 expression is up-regulated by DNA damage via p53 activation. A cellular DNA damage response stimulated by different forms of TiO2 NPs was detected by our cell-based DNA damage biosensor. The results showed that an increased DNA damage response is elicited by the anatase form compared to the rutile or mixed rutile/anatase forms. Our work indicates that the crystalline form of NPs is an important point to investigate when studying the interaction between nanomaterials and cells.

Combination of Cell Delivery and Thermoinducible Transcription for in Vivo Spatiotemporal Control of Gene Expression: A Feasibility Study

To demonstrate the feasibility of combining in situ delivery of genetically modified cells into the rat kidney, to induce expression of a reporter gene under transcriptional control of a heat-inducible promoter activated with magnetic resonance (MR)-guided focused ultrasonography (US), and to demonstrate in vivo the local expression of the synthesized protein. Experiments were conducted in agreement with the European Commission guidelines and directives of the French Research Ministry. C6 cells were genetically modified by incorporating the firefly luciferase (LucF) gene under transcriptional control of a heat-sensitive promoter (human heat shock protein 70B). Engineered cells were injected in the renal artery of a superficialized left kidney (15 rats). Two days later, intrarenal LucF expression was induced noninvasively by local hyperthermia in 15 renal locations in nine rats with focused US and was controlled with MR temperature imaging. Six hours after heating, LucF activity was detected in vivo with bioluminescence imaging. The genetically engineered C6 cell line was characterized in vitro for LucF expression related to the heating parameters. Changes in renal morphology and hemodynamic parameters as a result of rat kidney superficialization were not significant. Intrarenal temperature measurement at the focal point followed the scheduled temperature in 13 of 15 cases. On bioluminescence images, LucF activity was present only in heated regions. The level of LucF expression was also dependent on heating parameters. Substantial tissue damage was noted at histologic analysis in only the two cases in which temperature control was inadequate. A strategy combining cell delivery of a transgene and a thermosensitive promoter that can be locally activated with MR-guided focused US is able to induce in vivo gene expression controlled in space and time. http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.10100767/-/DC1.

Localisation of Neuregulin 1-β3 to different sub-nuclear structures alters gene expression

Neuregulins are growth factors that signal via the ErbB3 and ErbB4 receptors. Here we show using immunohistochemistry that they are often expressed in the nucleus of a range of tumour types including soft tissue and breast. The Neuregulin 1 type I-β3 (NRG1-β3) isoform localises to two sub-nuclear compartments in animal cells, nucleoli and spliceosomes. We used NRG1-β3 tagged with photoactivatable GFP and demonstrated that this re-localised from nucleoli to spliceosomes over 90 min. Tyrosine kinase activity was not required for retaining the NRG1-β3 within the nucleus. Mutation of the lysines 14 and 16 or 15 and 16 together prevented nucleolar uptake while four positively charged residues were identified which were required for spliceosome uptake. Molecular modelling suggests that three of these may form a binding site. We showed using a kinome array that NRG1-β3 and a mutant exclusively localising to spliceosomes increased phosphorylation and/or expression of the HER4 and HER2 receptors. Using a transcriptomic analysis the same two constructs induced expression of several messenger RNAs and we confirmed the increased expression at the protein level of the most highly induced, Heat Shock Protein 70B′. These results suggest that Neuregulin activates receptor signalling in spliceosomes leading to altered gene expression.

Heat Shock Protein 70B′ (HSP70B′) Expression and Release in Response to Human Oxidized Low Density Lipoprotein Immune Complexes in Macrophages

Heat shock proteins (HSPs) have been implicated in the activation and survival of macrophages. This study examined the role of HSP70B', a poorly characterized member of the HSP70 family, in response to oxidatively modified LDL (oxLDL) and immune complexes prepared with human oxLDL and purified human antibodies to oxLDL (oxLDL-IC) in monocytic and macrophage cell lines. Immunoblot analysis of cell lysates and conditioned medium from U937 cells treated with oxLDL alone revealed an increase in intracellular HSP70B' protein levels accompanied by a concomitant increase in HSP70B' extracellular levels. Fluorescence immunohistochemistry and confocal microscopy, however, demonstrated that oxLDL-IC stimulated the release of HSP70B', which co-localized with cell-associated oxLDL-IC. In HSP70B'-green fluorescent protein-transfected mouse RAW 264.7 cells, oxLDL-IC-induced HSP70B' co-localized with membrane-associated oxLDL-IC as well as the lipid moiety of internalized oxLDL-IC. Furthermore, the data demonstrated that HSP70B' is involved in cell survival, and this effect could be mediated by sphingosine kinase 1 (SK1) activation. An examination of regularly implicated cytokines revealed a significant relationship between HSP70B' and the release of the anti-inflammatory cytokine interleukin-10 (IL-10). Small interfering RNA knockdown of HSP70B' resulted in a corresponding decrease in SK1 mRNA levels and SK1 phosphorylation as well as increased release of IL-10. In conclusion, these findings suggest that oxLDL-IC induce the synthesis and release of HSP70B', and once stimulated, HSP70B' binds to the cell-associated and internalized lipid moiety of oxLDL-IC. The data also implicate HSP70B' in key cellular functions, such as regulation of SK1 activity and release of IL-10, which influence macrophage activation and survival.

Chloroplast DnaJ-like proteins 3 and 4 (CDJ3/4) from Chlamydomonas reinhardtii contain redox-active Fe–S clusters and interact with stromal HSP70B

In the present study we report on the identification and characterization of three novel chloroplast-targeted DnaJ-like proteins CDJ3-5, which in addition to their J-domains contain bacterial-type ferredoxin domains. In sequence databases we could identify homologues of CDJ3-5 in green algae, moss and higher plants, but not in cyanobacteria. Phylogenetic analyses allowed us to distinguish two clades containing CDJ3/4 and CDJ5 that must have diverged early in the ancestor of the 'green lineage' and have further diversified later on. Molecular and biochemical analysis of CDJ3 and CDJ4 from Chlamydomonas reinhardtii revealed that both proteins are weakly expressed and appear to be localized to the stroma and to thylakoid membranes respectively. The low transcript levels of the CDJ3 and CDJ4 genes declined even further in the initial phase of heat shock, but CDJ3 transcript levels strongly increased after a dark-to-light shift. Accordingly, the Arabidopsis orthologue of CDJ5 was also found to be light-inducible and to be under strong circadian control. CDJ3 and CDJ4 proteins could both be expressed in Escherichia coli and had redox-active Fe-S clusters. In vitro cross-linking studies demonstrated that CDJ3 and CDJ4 interact with chloroplast ATP-bound HSP70B (heat-shock protein 70B), presumably as dimers, and immunoprecipitation studies showed that CDJ3/4 were also in a complex with HSP70B in Chlamydomonas cell extracts. Finally, CDJ3 was found in complexes with apparent molecular masses of approx. 550-2800 kDa, which appeared to contain RNA. We speculate that the CDJ3-5 proteins might represent redox switches that act by recruiting HSP70B for the reorganization of regulatory protein complexes.

DNA damage sensible engineered promoter for cellular biosensing of cytotoxicity

We have established a cytotoxic sensor cell line by transfecting HepG2 cells with a luciferase protein plasmid derived from the heat shock protein 70B' (HSP70B') promoter, which is induced by cytotoxic reagents. HSP70B genes are up-regulated by a wide-range of cytotoxic stimulators, in particular, those that denature proteins. However, the HSP70B genes do not respond to DNA damage. We used a PCR array to detect marker genes of DNA damage-related cytotoxic stimulation and found the BTG2 gene to be one such gene. Analysis of the BTG2 gene functional promoter region by transfection of various deletion constructs into HepG2 cells indicated that the p53 and NFY biding sites on BTG2 are important for the response to DNA damage. We then constructed HepG2 sensor cells using the functional BTG2 promoter, and found that these sensor cells can specifically detect the cytotoxicity accompanied by DNA strand breaks with high sensitivity.

The Chloroplast DnaJ Homolog CDJ1 of Chlamydomonas reinhardtii Is Part of a Multichaperone Complex Containing HSP70B, CGE1, and HSP90C

We report on the molecular and biochemical characterization of CDJ1, one of three zinc-finger-containing J-domain proteins encoded by the Chlamydomonas reinhardtii genome. Fractionation experiments indicate that CDJ1 is a plastidic protein. In the chloroplast, CDJ1 was localized to the soluble stroma fraction, but also to thylakoids and to low density membranes. Although the CDJ1 gene was strongly heat shock inducible, CDJ1 protein levels increased only slightly during heat shock. Cellular CDJ1 concentrations were close to those of heat shock protein 70B (HSP70B), the major HSP70 in the Chlamydomonas chloroplast. CDJ1 complemented the temperature-sensitive phenotype of an Escherichia coli mutant lacking its dnaJ gene and interacted with E. coli DnaK, hence classifying it as a bona fide DnaJ protein. In soluble cell extracts, CDJ1 was found to organize into stable dimers and into complexes of high molecular mass. Immunoprecipitation experiments revealed that CDJ1 forms common complexes with plastidic HSP90C, HSP70B, and CGE1. In blue native-polyacrylamide gel electrophoresis, all four (co)chaperones migrated at 40% to 90% higher apparent than calculated molecular masses, indicating that greatest care must be taken when molecular masses of protein complexes are estimated from their migration relative to standard native marker proteins. Immunoprecipitation experiments from size-fractioned soluble cell extracts suggested that HSP90C and HSP70B exist as preformed complex that is joined by CDJ1. In summary, CDJ1 and CGE1 are novel cohort proteins of the chloroplast HSP90-HSP70 multichaperone complex. As HSP70B, CDJ1, and CGE1 are derived from the endosymbiont, whereas HSP90C is of eukaryotic origin, we observe in the chloroplast the interaction of two chaperone systems of distinct evolutionary origin.

Live cells‐based cytotoxic sensorchip fabricated in a microfluidic system

We report here a live cells-based sensorchip fabricated in microfluidic channels in which several laminar flows were achieved. In addition, we established a cytotoxic sensor cell line, which was transfected with a green fluorescence protein (GFP) plasmid derived from the heat shock protein 70B' (HSP70B') promoter, which is induced by cytotoxic reagents. The fluorescence in the sensor cells increased in a CdCl(2) dose-dependent manner in the microfluidic channels. In this system, cytotoxic reagents can be quantitatively detected in a quick, sensitive and high-throughput manner. The combination of sensor cells and microfluidic systems will provide an important basis for the development of micro-total analysis systems (micro-TAS) technology, and can be applied to toxicology, environmental assessment and drug screening.

The Effect of Metal Materials on Heat Shock Protein 70B' Gene Expression

To avoid the toxic effect of released nickel ions and compounds from conventional stainless steels, nickel-free austenitic stainless steels have been developed. We previously established a new manufacturing process to produce nickel-free austenitic stainless steel that involves nitrogen adsorption treatment. Although the cytocompatibility of nickelfree austenitic stainless steel produced using this method has been evaluated using two viability assay, molecular level analysis, such as gene expression analysis, has not been previously performed. In the present study, the cytotoxicity of our nickel-free austenitic stainless steel, as well as of commercially available metal materials, was evaluated by analysis of heat shock protein 70B’ (HSP70B’) gene expression as a stress response marker. Furthermore, to investigate the effect of metal materials on cytotoxicity, HSP70B’ gene expression was quantified using human osteoblast-like SaOS-2 cells, human monocyte THP-1 cells and the mouse macrophage cell line J774A.1. We found no significant differences in HSP70B’ expression among the various metal materials, including the nickel-free austenitic stainless steel, indicating that the nickel-free austenitic stainless steel produced using our nitrogen adsorption method has the same cytocompatibility as commercially available metal materials.

The Effect of Metal Materials on Heat Shock Protein 70B’ Gene Expression

To avoid the toxic effect of released nickel ions and compounds from conventional stainless steels, nickel-free austenitic stainless steels have been developed. We previously established a new manufacturing process to produce nickel-free austenitic stainless steel that involves nitrogen adsorption treatment. Although the cytocompatibility of nickelfree austenitic stainless steel produced using this method has been evaluated using two viability assay, molecular level analysis, such as gene expression analysis, has not been previously performed. In the present study, the cytotoxicity of our nickel-free austenitic stainless steel, as well as of commercially available metal materials, was evaluated by analysis of heat shock protein 70B’ (HSP70B’) gene expression as a stress response marker. Furthermore, to investigate the effect of metal materials on cytotoxicity, HSP70B’ gene expression was quantified using human osteoblast-like SaOS-2 cells, human monocyte THP-1 cells and the mouse macrophage cell line J774A.1. We found no significant differences in HSP70B’ expression among the various metal materials, including the nickel-free austenitic stainless steel, indicating that the nickel-free austenitic stainless steel produced using our nitrogen adsorption method has the same cytocompatibility as commercially available metal materials.

Hsp70B′ regulation and function

Heat shock protein (Hsp) 70B' is a human Hsp70 chaperone that is strictly inducible, having little or no basal expression levels in most cells. Using siRNAs to knockdown Hsp70B' and Hsp72 in HT-29, SW-480, and CRL-1807 human colon cell lines, we have found that the two are regulated coordinately in response to stress. We also have found that proteasome inhibition is a potent activator of hsp70B'. Flow cytometry was used to assay hsp70B' promoter activity in HT-29eGFP cells in this study. Knockdown of both Hsp70B' and Hsp72 sensitized cells to heat stress and increasing concentrations of proteasome inhibitor. These data support the conclusion that Hsp72 is the primary Hsp70 family responder to increasing levels of proteotoxic stress, and Hsp70B' is a secondary responder. Interestingly ZnSO4 induces Hsp70B' and not Hsp72 in CRL-1807 cells, suggesting a stressor-specific primary role for Hsp70B'. Both Hsp70B' and Hsp72 are important for maintaining viability under conditions that increase the accumulation of damaged proteins in HT-29 cells. These findings are likely to be important in pathological conditions in which Hsp70B' contributes to cell survival.