Mary Ann Stevenson

HSP70 stimulates cytokine production through a CD14-dependant pathway, demonstrating its dual role as a chaperone and cytokine

Here, we demonstrate a previously unknown function for the 70-kDa heat-shock protein (HSP70) as a cytokine. HSP70 bound with high affinity to the plasma membrane, elicited a rapid intracellular calcium flux, activated nuclear factor (NF)-κB and upregulated the expression of pro-inflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 in human monocytes. Furthermore, two different signal transduction pathways were activated by exogenous HSP70: one dependent on CD14 and intracellular calcium, which resulted in increased IL-1β, IL-6 and TNF-α; and the other independent of CD14 but dependent on intracellular calcium, which resulted in an increase in TNF-α but not IL-1β or IL-6. These findings indicate that CD14 is a co-receptor for HSP70-mediated signaling in human monocytes and are indicative of an previously unrecognized function for HSP70 as an extracellular protein with regulatory effects on human monocytes, having a dual role as chaperone and cytokine.

Novel Signal Transduction Pathway Utilized by Extracellular HSP70 ROLE OF Toll-LIKE RECEPTOR (TLR) 2 AND TLR4

Recent studies have initiated a paradigm shift in the understanding of the function of heat shock proteins (HSP). It is now clear that HSP can and do exit mammalian cells, interact with cells of the immune system, and exert immunoregulatory effects. We recently demonstrated that exogenously added HSP70 possesses potent cytokine activity, with the ability to bind with high affinity to the plasma membrane, elicit a rapid intracellular Ca2+ flux, activate NF-κB, and up-regulate the expression of pro-inflammatory cytokines in human monocytes. Here for the first time, we report that HSP70-induced proinflammatory cytokine production is mediated via the MyD88/IRAK/NF-κB signal transduction pathway and that HSP70 utilizes both TLR2 (receptor for Gram-positive bacteria) and TLR4 (receptor for Gram-negative bacteria) to transduce its proinflammatory signal in a CD14-dependent fashion. These studies now pave the way for the development of highly effective pharmacological or molecular tools that will either up-regulate or suppress HSP70-induced functions in conditions where HSP70 effects are desirable (cancer) or disorders where HSP70 effects are undesirable (arthritis and arteriosclerosis).

Sequential Phosphorylation by Mitogen-activated Protein Kinase and Glycogen Synthase Kinase 3 Represses Transcriptional Activation by Heat Shock Factor-1

Mammalian heat shock genes are regulated at the transcriptional level by heat shock factor-1 (HSF-1), a sequence-specific transcription factor. We have examined the role of serine phosphorylation of HSF-1 in the regulation of heat shock gene transcription. Our experiments show that mitogen-activated protein kinases (MAPKs) of the ERK-1 family phosphorylate HSF-1 on serine residues and repress the transcriptional activation of the heat shock protein 70B (HSP70B) promoter by HSF-1 in vivo. These effects of MAPK are transmitted through a specific serine residue (Ser-303) located in a proline-rich sequence within the transcriptional regulatory domain of human HSF-1. However, despite the importance of Ser-303 in transmitting the signal from the MAPK cascade to HSP70 transcription, there was no evidence that Ser-303 could be phosphorylated by MAPK in vitro, although an adjacent residue (Ser-307) was avidly phosphorylated by MAPK. Further studies revealed that Ser-303 is phosphorylated by glycogen synthase kinase 3 (GSK3) through a mechanism dependent on primary phosphorylation of Ser-307 by MAPK. Secondary phosphorylation of Ser-303 by GSK3 may thus repress the activity of HSF-1, and its requirement for priming by MAPK phosphorylation of Ser-307 provides a potential link between the MAPK cascade and HSF-1. Our experiments thus indicate that MAPK is a potent inhibitor of HSF-1 function and may be involved in repressing the heat shock response during normal growth and development and deactivating the heat shock response during recovery from stress.

Heat shock factor 1 represses transcription of the IL-1β gene through physical interaction with the nuclear factor of interleukin 6

Heat shock factor (HSF) 1 is the major heat shock transcription factor that regulates stress-inducible synthesis of heat shock proteins and is also essential in protection against endotoxic shock. Following our previous study, which demonstrated the transcriptional repression of the IL-1β gene by HSF1 (Cahill, C. M., Waterman, W. R., Xie, Y., Auron, P. E., and Calderwood, S. K. (1996) J. Biol. Chem. 271, 24874–24879), we have examined the mechanisms of transcriptional repression. Our studies show that HSF1 represses the lipopolyliposaccharide-induced transcription of theIL-1β promoter through direct interaction with the nuclear factor of interleukin 6 (NF-IL6, also known asCCAAT enhancer bindingprotein (C/EBPβ), an essential regulator inIL-1β transcription. We show for the first time that HSF1 binds directly to NF-IL6 in vivo and antagonizes its activity. The HSF1/NF-IL6 interaction involves a sequence of HSF1 containing the trimerization and regulatory domains and the bZip region of NF-IL6. HSF1 has little effect on IL-1β promoter activity stimulated by the essential monocytic transcription factor Spi.1 but is strongly inhibitory to transcriptional activation by NF-IL6 and to the synergistic activation by NF-IL6 and Spi.1. Because of its ability to bind to specific C/EBP elements in the promoters of multiple genes and its ability to interact with other transcription factors, NF-IL6 is involved in transcriptional regulation of a wide range of genes. Interaction between HSF1 and NF-IL6 could thus be an important mechanism in HSF1 regulation of general gene transcription during endotoxin stress.

Extracellular HSP70 binding to surface receptors present on antigen presenting cells and endothelial/epithelial cells

Extracellular HSP70 has been found to participate in both innate and adaptive immune responses. However, little is known about the molecular mechanisms that mediate this process. Previous reports suggest that HSP70 interacts with antigen presenting cells (APC) through a plethora of surface receptors. In this study, we have examined the relative binding of potential HSP70 receptors and found high affinity binding to LOX‐1 but not other structures with a role in HSP70–APC interactions such as LRP/CD91, CD40, TLR2, TLR4 or another c‐type lectin family member (DC‐SIGN) closely related to LOX‐1. In addition to APC, HSP70 can avidly bind to non‐APC cell lines, especially those from epithelial or endothelial background.

Transcriptional Activity of Heat Shock Factor 1 at 37 oC Is Repressed through Phosphorylation on Two Distinct Serine Residues by Glycogen Synthase Kinase 3α and Protein Kinases Cα and Cζ

Heat shock factor 1 (HSF1) is the key transcriptional regulator of the heat shock genes that protect cells from environmental stress. However, because heat shock gene expression is deleterious to growth and development, we have examined mechanisms for HSF1 repression at growth temperatures, focusing on the role of phosphorylation. Mitogen-activated protein kinases (MAPKs) of the ERK family phosphorylate HSF1 and represses transcriptional function. The mechanism of repression involves initial phosphorylation by MAP kinase on serine 307, which primes HSF1 for secondary phosphorylation by glycogen synthase kinase 3 on a key residue in repression (serine 303).In vivo expression of glycogen synthase kinase 3 (α or β) thus represses HSF1 through phosphorylation of serine 303. HSF1 is also phosphorylated by MAPK in vitro on a second residue (serine 363) adjacent to activation domain 1, and this residue is additionally phosphorylated by protein kinase C. In vivo, HSF1 is repressed through phosphorylation of this residue by protein kinase Cα or -ζ but not MAPK. Regulation at 37 °C, therefore, involves the action of three protein kinase cascades that repress HSF1 through phosphorylation of serine residues 303, 307, and 363 and may promote growth by suppressing the heat shock response.

HSP70 peptide-bearing and peptide-negative preparations act as chaperokines

Abstract We recently elucidated a novel function for the 70-kDa heat shock protein (HSP70) as a chaperone and a cytokine, a chaperokine in human monocytes. Here we show that peptide-bearing and peptide-negative HSP70 preparations isolated from EMT6 mammary adenocarcinoma cells (EMT6-HSP70) act as chaperokines when admixed with murine splenocytes. EMT6-HSP70 bound with high affinity to the surface of splenocytes recovered from naive BALB/c mice. The [Ca2+]i inhibitor BAPTA dose dependently inhibited HSP70- but not LPS-induced NF-κB activity and subsequent augmentation of proinflammatory cytokine TNF-α, IL-1β, and IL-6 production. Taken together, these results suggest that presence of peptide in the HSP70 preparation is not required for spontaneous activation of cells of the innate immune system.

Clinically significant cardiac disease in patients with Hodgkin lymphoma treated with mediastinal irradiation

This study assessed the cumulative incidence of clinically significant cardiac disease in 1279 Hodgkin lymphoma patients treated with mediastinal irradiation and quantified the standard incidence ratios (SIRs) and absolute excess risks of cardiac procedures compared with a normal matched population. Cox regression analysis was used to explore factors associated with cardiac complications. Poisson regression analysis of SIRs was used to estimate the excess risk of cardiac interventions from mediastinal irradiation. After a median follow-up of 14.7 years, 187 patients experienced 636 cardiac events and 89 patients required a cardiac procedure. 5-, 10-, 15-, and 20-year cumulative incidence rates of cardiac events were 2.2%, 4.5%, 9.6%, and 16%. SIRs for cardiac procedures were increased for coronary artery bypass graft (3.19), percutaneous intervention (1.55), implantable cardioverter defibrillator or pacemaker placement (1.9), valve surgery (9.19), and pericardial surgery (12.91). Absolute excess risks were 18.2, 19.3, 9.4, 14.1, and 4.7 per 10 000 person-years, respectively. Older age at diagnosis and male sex were predictors for cardiac events. However, younger age at diagnosis was associated with excess risk specifically from radiation therapy compared with the general population. These results may help guideline development for both the types and timing of cardiac surveillance in survivors of Hodgkin lymphoma.

Simultaneous Planck, Swift, and Fermi observations of X-ray and γ-ray selected blazars

We present simultaneous Planck, Swift, Fermi, and ground-based data for 105 blazars belonging to three samples with flux limits in the soft X-ray, hard X-ray, and -ray bands, and we compare our results to those of a companion paper presenting simultaneous Planck and multi-frequency observations of 104 radio-loud northern active galactic nuclei selected at radio frequencies. While we confirm several previous results, our unique data set has allowed us to demonstrate that the selection method strongly influences the results, producing biases that cannot be ignored. Almost all the BL Lac objects have been detected by Fermi Large Area Telescope (LAT), whereas 30 to 40% of the flat-spectrum radio quasars (FSRQs) in the radio, soft X-ray, and hard X-ray selected samples are still below the -ray detection limit even after integrating 27 months of Fermi-LAT data. The radio to sub-millimetre spectral slope of blazars is quite flat, withh i 0 up to about 70 GHz, above which it steepens toh i 0:65. BL Lacs have significantly flatter spectra than FSRQs at higher frequencies. The distribution of the rest-frame synchrotron peak frequency ( S ) in the spectral energy distribution (SED) of FSRQs is the same in all the blazar samples withh S i = 10 13:1 0:1 Hz, while the mean inverse-Compton peak frequency,h IC i, ranges from 10 21 to 10 22 Hz. The distributions of S and of IC of BL Lacs are much broader and are shifted to higher energies than those of FSRQs; their shapes strongly depend on the selection method. The Compton dominance of blazars ranges from less than 0.2 to nearly 100, with only FSRQs reaching values larger than about 3. Its distribution is broad and depends strongly on the selection method, with -ray selected blazars peaking at 7 or more, and radio-selected blazars at values close to 1, thus implying that the common assumption that the blazar power budget is largely dominated by high-energy emission is a selection e ect. A comparison of our multi-frequency data with theoretical predictions shows that simple homogeneous SSC models cannot explain the simultaneous SEDs of most of the -ray detected blazars in all samples. The SED of the blazars that were not detected by Fermi-LAT may instead be consistent with SSC emission. Our data challenge the correlation between bolometric luminosity and S predicted by the blazar sequence.

Expression of heat shock proteins and heat shock protein messenger ribonucleic acid in human prostate carcinoma in vitro and in tumors in vivo

Abstract Heat shock proteins (HSPs) are thought to play a role in the development of cancer and to modulate tumor response to cytotoxic therapy. In this study, we have examined the expression of hsf and HSP genes in normal human prostate epithelial cells and a range of prostate carcinoma cell lines derived from human tumors. We have observed elevated expressions of HSF1, HSP60, and HSP70 in the aggressively malignant cell lines PC-3, DU-145, and CA-HPV-10. Elevated HSP expression in cancer cell lines appeared to be regulated at the post–messenger ribonucleic acid (mRNA) levels, as indicated by gene chip microarray studies, which indicated little difference in heat shock factor (HSF) or HSP mRNA expression between the normal and malignant prostate cell lines. When we compared the expression patterns of constitutive HSP genes between PC-3 prostate carcinoma cells growing as monolayers in vitro and as tumor xenografts growing in nude mice in vivo, we found a marked reduction in expression of a wide spectrum of the HSPs in PC-3 tumors. This decreased HSP expression pattern in tumors may underlie the increased sensitivity to heat shock of PC-3 tumors. However, the induction by heat shock of HSP genes was not markedly altered by growth in the tumor microenvironment, and HSP40, HSP70, and HSP110 were expressed abundantly after stress in each growth condition. Our experiments indicate therefore that HSF and HSP levels are elevated in the more highly malignant prostate carcinoma cells and also show the dominant nature of the heat shock–induced gene expression, leading to abundant HSP induction in vitro or in vivo.