Expression of a gene encoding a 16.9-kDa heat-shock protein, Oshsp16.9, in Escherichia coli enhances thermotolerance

A gene encoding the rice 16.9-kDa class I low-molecular-mass (LMM) heat-shock protein (HSP), Oshsp16.9, was introduced into Escherichia coli using the pGEX-2T expression vector to analyze the possible function of this LMM HSP under heat stress. It is known that E. coli does not normally produce class I LMM HSPs. We compared the survivability of E. coli XL1-Blue cells transformed with a recombinant plasmid containing a glutathione S-transferase (GST)–Oshsp16.9 fusion protein (pGST-FL cells) with the control E. coli cells transformed with the pGEX-2T vector (pGST cells) under heat-shock (HS) after isopropyl β-d-thiogalactopyranoside induction. The pGST-FL cells demonstrated thermotolerance at 47.5°C, a treatment that was lethal to the pGST cells. When the cell lysates from these two E. coli transformants were heated at 55°C, the amount of protein denatured in the pGST-FL cells was 50% less than that of the pGST cells. Similar results as pGST-FL cells were obtained in pGST-N78 cells (cells produced a fusion protein with only the N-terminal 78 aa in the Oshsp16.9 portion) but not in pGST-C108 cells (cells produced a fusion protein with C-terminal 108 aa in the Oshsp16.9 portion). The acquired thermotolerant pGST-FL cells synthesized three types of HSPs, including the 76-, 73-, and 64-kDa proteins according to their abundance at a lethal temperature of 47.5°C. This finding indicates that a plant class I LMM HSP, when effectively expressed in transformed prokaryotic cells that do not normally synthesize this class of LMM HSPs, may directly or indirectly increase thermotolerance.

Endogenous expression of Müllerian inhibiting substance in early postnatal rat Sertoli cells requires multiple steroidogenic factor-1 and GATA-4-binding sites

Müllerian inhibiting substance (MIS) is a key element required to complete mammalian male sex differentiation. The expression pattern of MIS is tightly regulated in fetal, neonatal, and prepubertal testes and adult ovaries and is well conserved among mammalian species. Although several factors have been shown to be essential to MIS expression, its regulatory mechanisms are not fully understood. We have examined MIS promoter activity in 2-day postnatal primary cultures of rat Sertoli cells that continue to express endogenous MIS mRNA. Using this system, we found that the region between human MIS−269 and −192 is necessary for full MIS promoter activity. We identified by DNase I footprint and electrophoretic mobility-shift analyses a distal steroidogenic factor-1 (SF-1)-binding site that is essential for full promoter activity. Mutational analysis of this new distal SF-1 site and the previously identified proximal SF-1 site showed that both are necessary for transcriptional activation. Moreover, the proximal promoter also contains multiple GATA-4-binding sites that are essential for functional promoter activity. Thus multiple SF-1- and GATA-4-binding sites in the MIS promoter are required for normal tissue-specific and developmental expression of MIS.

Chlorophyll precursors are signals of chloroplast origin involved in light induction of nuclear heat-shock genes

Coordination between the activities of organelles and the nucleus requires the exchange of signals. Using Chlamydomonas, we provide evidence that plastid-derived chlorophyll precursors may replace light in the induction of two nuclear heat-shock genes (HSP70A and HSP70B) and thus qualify as plastidic signal. Mutants defective in the synthesis of Mg-protoporphyrin IX were no longer inducible by light. Feeding of Mg-protoporphyrin IX or its dimethyl ester to wild-type or mutant cells in the dark resulted in induction. The analysis of HSP70A promoter mutants that do or do not respond to light revealed that these chlorophyll precursors specifically activate the light signaling pathway. Activation of gene expression was not observed when protoporphyrin IX, protochlorophyllide, or chlorophyllide were added. A specific interaction of defined chlorophyll precursors with factor(s) that regulate nuclear gene expression is suggested.

Unbalanced expression of the different subunits of elongation factor 1 in diabetic skeletal muscle

In studies using subtraction cloning to screen for alterations in mRNA expression in skeletal muscle from humans with Type 2 diabetes mellitus and control subjects, one of the most prominent differences was in the mRNA for elongation factor (EF)-1α. With Northern blot analysis, EF-1α expression was enhanced by 2- to 6-fold in both Types 1 and 2 human diabetics. In contrast, no changes in expression of EF-1β or -γ were noted. We observed similar results in animal models of Type 1 diabetes. EF-1α expression, but not EF-1β or -γ expression, was also enhanced in streptozotocin-induced diabetic rats, and this effect was reversed by insulin treatment. An increased level of EF-1α mRNA was also observed in nonobese diabetic mice. This unbalanced regulation of the expression of the different subunits of EF-1 may contribute to alterations not only in protein synthesis but also in other cellular events observed in the diabetic state.

Expression of Small Heat-Shock Proteins at Low Temperatures1 : A Possible Role in Protecting against Chilling Injuries

We previously reported that short exposure of tomato (Lycopersicon esculentum L.) fruits to high temperature protects them from chilling injury. To study the involvement of heat-shock proteins (HSPs) in the acquisition of low-temperature tolerance, we cloned two heat-shock-induced genes that are also expressed at low temperatures. The cloned cDNAs belong to the small HSP group. Sequence analyses of the clones showed perfect homology to the tomato-ripening gene tom66 and to the tomato chloroplastic HSP21 gene tom111. The expression of both genes was induced by high temperature in fruits, flowers, leaves, and stems, but not by low or ambient temperatures or by other stresses such as drought and anaerobic conditions. When the heated fruits were transferred to low temperature, tom66 and tom111 mRNA levels first decreased but were then reinduced. Induction was not observed in nonheated fruits at low temperature. Immunodetection of tom111-encoded protein indicated that this protein is present at low temperatures in the heated fruits. The results of this study show that the expression of tom66 and tom111 is correlated with protection against some, but not all, symptoms of chilling injury.

Macrophage-colony-stimulating factor regulates expression of the integrins alpha 4 beta 1 and alpha 5 beta 1 by murine bone marrow macrophages.

We observed that when monocyte/macrophage precursors derived from murine bone marrow were treated with macrophage-colony-stimulating factor (M-CSF), there was a dose-dependent increase in both the number of adherent cells and the degree to which the cells were highly spread. Attachment was supported by fibronectin, but not by vitronectin or laminin, suggesting that the integrins alpha 4 beta 1 and/or alpha 5 beta 1 might mediate this event. Binding to fibronectin was blocked partially by antibodies to either integrin, and inhibition was almost complete when the antibodies were used in combination. By a combination of surface labeling with 125I and metabolic labeling with [35S]methionine and [35S]cysteine, we demonstrated that M-CSF treatment led to increased synthesis and surface expression of the two beta 1 integrins. Since attachment to fibronectin and/or stromal cells plays an important role in the maturation of other hematopoietic lineages, we propose that the action of M-CSF in the differentiation of immature monocytes/macrophages includes stimulated expression of the integrins alpha 4 beta 1 and alpha 5 beta 1, leading to interactions with components of the marrow microenvironment necessary for cell maturation.

Heat shock protein hsp90 regulates dioxin receptor function in vivo.

The dioxin (aryl hydrocarbon) receptor is a ligand-dependent basic helix-loop-helix (bHLH) factor that binds to xenobiotic response elements of target promoters upon heterodimerization with the bHLH partner factor Arnt. Here we have replaced the bHLH motif of the dioxin receptor with a heterologous DNA-binding domain to create fusion proteins that mediate ligand-dependent transcriptional enhancement in yeast (Saccharomyces cerevisiae). Previously, our experiments indicated that the ligand-free dioxin receptor is stably associated with the 90-kDa heat shock protein, hsp90. To investigate the role of hsp90 in dioxin signaling we have studied receptor function in a yeast strain where hsp90 expression can be down-regulated to about 5% relative to wild-type levels. At low levels of hsp90, ligand-dependent activation of the chimeric dioxin receptor construct was almost completely inhibited, whereas the activity of a similar chimeric construct containing the structurally related Arnt factor was not affected. Moreover, a chimeric dioxin receptor construct lacking the central ligand- and hsp90-binding region of the receptor showed constitutive transcriptional activity in yeast that was not impaired upon down-regulation of hsp90 expression levels. Thus, these data suggest that hsp90 is a critical determinant of conditional regulation of dioxin receptor function in vivo via the ligand-binding domain.

Degradation of sigma 32, the heat shock regulator in Escherichia coli, is governed by HflB.

The heat shock response in Escherichia coli is governed by the concentration of the highly unstable sigma factor sigma 32. The essential protein HflB (FtsH), known to control proteolysis of the phage lambda cII protein, also governs sigma 32 degradation: an HflB-depleted strain accumulated sigma 32 and induced the heat shock response, and the half-life of sigma 32 increased by a factor up to 12 in mutants with reduced HflB function and decreased by a factor of 1.8 in a strain overexpressing HflB. The hflB gene is in the ftsJ-hflB operon, one promoter of which is positively regulated by heat shock and sigma 32. The lambda cIII protein, which stabilizes sigma 32 and lambda cII, appears to inhibit the HflB-governed protease. The E. coli HflB protein controls the stability of two master regulators, lambda cII and sigma 32, responsible for the lysis-lysogeny decision of phage lambda and the heat shock response of the host.

Characterization of heat shock protein-specific T cells in atherosclerosis

A role for infection and inflammation in atherogenesis is widely accepted. Arterial endothelium has been shown to express heat shock protein 60 (HSP60) and, since human (hHSP60) and bacterial (GroEL) HSP60s are highly conserved, the immune response to bacteria may result in cross-reactivity, leading to endothelial damage and thus contribute to the pathogenesis of atherosclerosis. In this study, GroEL-specific T-cell lines from peripheral blood and GroEL-, hHSP60-, and Porphyromonas gingivalis-specific T-cell lines from atherosclerotic plaques were established and characterized in terms of their cross-reactive proliferative responses, cytokine and chemokine profiles, and T-cell receptor (TCR) V beta expression by flow cytometry. The cross-reactivity of several lines was demonstrated. The cytokine profiles of the artery T-cell lines specific for GroEL, hHSP60, and P. gingivalis demonstrated Th2 phenotype predominance in the CD4 subset and Tc0 phenotype predominance in the CD8 subset. A higher proportion of CD4 cells were positive for interferon-inducible protein 10 and RANTES, with low percentages of cells positive for monocyte chemoattractant protein 1 and macrophage inflammatory protein la, whereas a high percentage of CD8 cells expressed all four chemokines. Finally, there was overexpression of the TCR V beta 5.2 family in all lines. These cytokine, chemokine, and V beta profiles are similar to those demonstrated previously for P. gingivalis-specific lines established from periodontal disease patients. These results support the hypothesis that in some patients cross-reactivity of the immune response to bacterial HSPs, including those of periodontal pathogens, with arterial endothelial cells expressing hHSP60 may explain the apparent association between atherosclerosis and periodontal infection.

Increased TG2 expression can result in induction of transforming growth factor β1, causing increased synthesis and deposition of matrix proteins, which can be regulated by nitric oxide

In fibrotic conditions increases in TG2 activity has been linked to an increase in the deposition of extracellular matrix proteins. Using TG2 transfected Swiss 3T3 fibroblasts expressing TG2 under the control of the tetracycline-regulated inducible promoter, we demonstrate that induction of TG2 not only stimulates an increase in collagen and fibronectin deposition but also an increase in the expression of these proteins. Increased TG2 expression in these fibroblasts led to NF-kappaB activation, resulting in the increased expression of transforming growth factor (TGF) beta(1). In addition, cells overexpressing TG2 demonstrated an increase in biologically active TGFbeta(1) in the extracellular environment. A specific site-directed inhibitor of TG abolished the NF-kappaB and TGFbeta1 activation and the subsequent elevation in the synthesis and deposition of extracellular matrix proteins, confirming that this process depends on the induction of transglutaminase activity. Treatment of TG2-induced fibroblasts with nontoxic doses of nitric oxide donor S-nitroso-N-acetylpenicillamine resulted in decreased TG2 activity and apprehension of the inactive enzyme on the cell surface. This was paralleled by a reduction in activation of NF-kappaB and TGFbeta(1) production with a subsequent decrease in collagen expression and deposition. These findings support a role for NO in the regulation of TG2 function in the extracellular environment.

Heat shock proteins in leukaemia cell differentiation and cell death

When HL60 cells were induced to differentiate to granulocyte-like cells with the agents N-methylformamide and tunicamycin an concentrations marginally below those which were cytotoxic, there was a decrease in the synthesis of the glucose- regulated proteins which preceded the expression of markers of a differentiated phenotype. There was a transient increase in the amount of hsp70 after 36 hours in NMF treated cells but in differentiated cells negligible amounts were detected. Inducers which were known to modulate hsp70 such as azetadine carboxylic acid did not induce differentiation suggesting early changes in the endoplasmic reticulum may be involved in the commitment to terminal differentiation of HL60 cells. These changes in group synthesis were not observed when K562 human chronic myelogenous leukemia cells were induced to differentiate to erythroid-like cells but there was a comparable increase in amounts of hsp70. When cells were treated with concentrations of drugs which brought about a loss in cell viability there was an early increase in the amount of hsp70 protein in the absence of any increase in synthesis. HL60 cells were treated with NMF (225mM), Adriamycin (1μM), or CB3717 (5μM) and there was an increase in the amounts of hsp70, in the absence of any new synthesis, which preceded any loss of membrane integrity and any significant changes in cell cycle but was concomitant with a later loss in viability of > 50% and a loss in proliferative potential. The amounts of hsp70 in the cell after treatment with any of the drugs was comparable to that obtained after a heat shock. Following a heat shock hsp70 was translocated from the cytoplasm to the nucleus, but treatment with toxic concentrations of drug caused hsp70 to remain localised in the cytoplasm. Changes in hsp70 turn-over was observed after a heat shock compared to NMF-treated cells. Morphological studies suggested that cells that had been treated with NMF and CB3717 were undergoing necrosis whereas the Adriamycin cells showed characteristics that were indicative of apoptosis. The data supports the hypothesis that an increase in amounts of hsp70 is an early marker of cell death.

Regulation of Pygopus (hPygo2) expression in prostate cancer by E74-Like factor 1 (Elf-1) transcription factor

Prostate cancer is a worldwide health concern. Pygopus2 (hPygo2) protein is required for growth in breast, ovarian, cervical and prostate cancer. hPygo2 expression is regulated by the Rb protein via the ETS factor Elf-1 in cervical and breast cancer. Additionally, the ETS family has confirmed roles in carcinogenesis and proliferation. The mechanism of hPygo2 expression has not been elucidated in prostate cancer. My hypothesis proposes that hPygo2 expression is regulated by Elf-1 bound to its promoter region. Prostate cancer cell lines were used to show protein levels of hPygo2, Elf-1 and ETS. ChIP assays confirmed varying binding capability of Elf-1 and ETS factors to the proximal promoter region between cell lines. Elf-1 knockdown experiments were performed, results show no change in hPygo2 protein levels but show reduction in 22Rv1 mRNA levels. These results suggest that Elf-1 might not be exclusively involved in the activation of Pygopus expression in prostate cancer.

The expression and prognostic significance of bcl-2-associated transcription factor 1 in rectal cancer following neoadjuvant therapy

Date of Acceptance: 12/07/2015 © 2015 John Wiley & Sons Ltd. Acknowledgements This study was supported by funding from the Encompass kick start and SMART:Scotland award schemes of Scottish Enterprise and Friends of Anchor. The Grampian Biorepository assisted with the immunohistochemical investigations.

The expression and prognostic significance of bcl-2-associated transcription factor 1 in rectal cancer following neoadjuvant therapy

Date of Acceptance: 12/07/2015 © 2015 John Wiley & Sons Ltd. Acknowledgements This study was supported by funding from the Encompass kick start and SMART:Scotland award schemes of Scottish Enterprise and Friends of Anchor. The Grampian Biorepository assisted with the immunohistochemical investigations.