Structure and expression of the three MHC-linked HSP70 genes

A duplicated locus encoding the major heat shock-induced protein HSP70 is located in the major histocompatibility complex (MHC) class III region 92 kilobases (kb) telomeric to the C2 gene. Nucleotide sequence analysis of the two intronless genes, HSP70-1 and HSP70-2, has shown that they encode an identical protein product of 641 amino acids. A third intronless gene, HSP70-Hom, has also been identified 4 kb telomeric to the HSP70-1 gene. This encodes a more basic protein of 641 amino acids which has 90% sequence similarity with HSP70-1. In order to investigate the expression of the three (MHC)-linked HSP70 genes individually by northern blot analysis, we have isolated locus-specific probes from the 3 untranslated regions of the genes. The HSP70-1 and HSP70-2 genes have been shown to be expressed at high levels as a 2.4 kb mRNA in cells heat-shocked at 42°C. HSP70-1 is also expressed constitutively at very low levels. The HSP70-Hom gene, which has no heat shock consensus sequence in its 5 flanking sequence, is expressed as a 3 kb mRNA at low levels both constitutively and following heat shock.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M34267-9. Address correspondence and offprint requests to: R. D. Campbell.     

Polymorphic analysis of the three MHC-linked HSP70 genes

Three genes encoding members of the M _r 70 000 heat shock protein family (HSP70) are known to lie in the class III region of the human major histocompatibility complex. IN order to determine whether these genes or their protein products exhibit any polymorphism the three genes have been specifically amplified from genomic DNA and sequenced. The HSP70-1 and HSP70-2 genes encode the major heat-inducible HSP70. A comparison of the nucleotide sequences of these genes from B8, SC01, DR3, B18, F1C30, DR3, and B7, SC30, DR2 haplotypes has revelad only very limited sequence variation which is not associated with any amino acid polymorphism. The HSP70-Hom gene encodes a protein that is highly related to HSP70-1, but which is not heat-inducible. Nucleotide sequence analysis of this gene from different haplotypes has revealed a Met Thr amino acid substitution at residue 493 in a number of the haplotypes tested. This variable amino acid lies in the proposed peptide-binding site of the HSP70-Hom protein. Address correspondence and offprint requests to: R. D. Campbell.     

Specific activation of the human HSP70 promoter by copper sulfate in mosaic transgenic zebrafish

Heat shock proteins (HSPs) play a central role in cell protection and repair upon stresses, such as that caused by heat and heavy metals. Copper sulfate inducibility of a pHhsp70 construct expressing the enhanced green fluorescent protein (EGFP) gene under the control of the exogenous human hsp70 promoter was tested in transfected CHSE 214 cells and transgenic zebrafish (Danio rerio). We developed a transient expression system, using mosaically transgenic zebrafish, which allows rapid analysis of transgenic expression. Transfected CHSE 214 cells which had been exposed to 250 nM and 2.5 microM copper sulfate for up to 24h showed increased EGFP expression in a dose-dependent manner. The 1.5 microM copper sulfate caused stronger EGFP fluorescence than the 1.0 microM copper sulfate in transgenic zebrafish. Most of the expression was spotty and was detected in the gills, dorsal and ventral retina, myotubes of the trunk, and skin epithelium. Transgenic zebrafish exposed to copper sulfate exhibited gross dysmorphogenesis, edema and trunk abnormalities, such as spinal lordosis, in vertebral development 5 days after fertilization. This transgenic zebrafish system was sensitive enough to detect copper sulfate at doses below the median lethal concentration (the LC50 was calculated to be 1.2 microM (95% confidence interval of 0.6-1.9 microM)). These results indicate that zebrafish could be useful transgenic biosensor systems for the detection of xenobiotic toxicants in the environment.     

Repression of the aroP gene of Escherichia coli involves activation of a divergent promoter.

The repression of aroP expression which is mediated by the TyrR protein with phenylalanine, tyrosine, or tryptophan has been shown to be primarily a direct result of TyrR-mediated activation of a divergent promoter, P3, which directs the RNA polymerase away from promoter P1. Evidence which has been presented to support this conclusion is as follows. Repression of P1 does not occur either in vitro or in vivo if wild-type TyrR protein is substituted by the activation-negative mutant RQ10 (with an R-to-Q change at position 10). Repression of P1 is greatly diminished if the P3 promoter is inactivated or if a 5-bp insertion is made between the P3 promoter and the binding sites for TyrR. Repression is also abolished if the promoter strength of P1 is increased or a putative UP element associated with P3 is altered. Repression of the second promoter, P2, still occurs if the wild-type TyrR protein is substituted with RQ10 or EQ274. The tryptophan-mediated repression of aroP does not involve the TrpR protein.     

Differential regulation of HSC70, HSP70, HSP90 alpha, and HSP90 beta mRNA expression by mitogen activation and heat shock in human lymphocytes

A subset of heat shock proteins, HSP90 alpha, HSP90 beta, and a member of the HSP70 family, HSC70, shows enhanced synthesis following mitogenic activation as well as heat shock in human peripheral blood mononuclear cells. In this study, we have examined expression of mRNA for these proteins, including the major 70-kDa heat shock protein, HSP70, in mononuclear cells following either heat shock or mitogenic activation with phytohemagglutinin (PHA), ionomycin, and the phorbol ester, tetradecanoyl phorbol acetate. The results demonstrate that the kinetics of mRNA expression of these four genes generally parallel the kinetics of enhanced protein synthesis seen following either heat shock or mitogen activation and provide clear evidence that mitogen-induced synthesis of HSC70 and HSP90 is due to increased mRNA levels and not simply to enhanced translation of preexisting mRNA. Although most previous studies have focused on cell cycle regulation of HSP70 mRNA, we found that HSP70 mRNA was only slightly and transiently induced by PHA activation, while HSC70 is the predominant 70-kDa heat shock protein homologue induced by mitogens. Similarly, HSP90 alpha appears more inducible by heat shock than mitogens while the opposite is true for HSP90 beta. These results suggest that, although HSP70 and HSC70 have been shown to contain similar promoter regions, additional regulatory mechanisms which result in differential expression to a given stimulus must exist. They clearly demonstrate that human lymphocytes are an important model system for determining mechanisms for regulation of heat shock protein synthesis in unstressed cells. Finally, based on kinetics of mRNA expression, the results are consistent with the hypothesis that HSC70 and HSP90 gene expression are driven by an IL-2/IL-2 receptor-dependent pathway in human T cells.     

Heterologous activation of the Porphyra tenera HSP70 promoter in Bangiophycean algal cells

Porphyra has attracted great attention for its biological and industrial importance. However, establishment of a stable nuclear transformation has not yet been achieved in these organisms, which impedes the molecular biological study and the development of a molecular breeding method for them. Toward establishing the stable transformation, we have recently developed an efficient transient gene expression system in Bangiophycean algae, in which the HSP70 promoter from P. tenera (PtHSP70 promoter) was activated heterologously in P. yezoensis cells. Since heterologous promoters are required for homologous recombination-based stable transformation, the identification of heterologously activated promoters is important in establishing a stable transformation system in individual Bangiophycean alga. We here examined the activation of the PtHSP70 promoter using the GC-rich PyGUS reporter system in additional Porphyra and Bangia species. The results indicated that this promoter drove expression of the PyGUS gene efficiently in all examined algae, whereas there was quite low expression of PyGUS by the cauliflower mosaic virus 35S promoter that is widely used as a heterologous promoter in the transformation of green land plants. Therefore, heterologous activation of the PtHSP70 promoter could promote the establishment of the stable transformation system in various kinds of Bangiophycean algae.     

Functional analysis of the HS185 regulatory element in the rice HSP70 promoter

A series of HSP70 promoter deletion constructs was established. Analysis of beta-glucuronidase activities from the promoter deletion constructs in transient expression assays identified a cis-element, located from −493 to −308 bp upstream of the ATG start site. This element was designated as HS185 and has a crucial role in HSP70 promoter activity. HS185 has some characteristics of a miniature inverted-repeat transposable element (MITE), such as terminal inverted repeats (TIRs) (GGTCCCACA) and a putative target site duplication. There are 362 copies of homologous sequences of HS185 in the rice genome, which are preferentially distributed to non-coding regions. Based on these sequence features, we propose that HS185 is an uncharacterized rice MITE, possibly derived from the rice transposon Mutator-like element VIII family. Further transient expression assays showed that HS185 inhibited the enhancer activity of the cauliflower mosaic virus 35S promoter. These results demonstrate that not only is HS185 necessary for HSP70 promoter activity, but it also has a functional role as an insulator. This study explored new regulatory functions of non-coding repeat sequences in rice.     

Expression of Ku70 and Ku80 mediated by NF-kappa B and cyclooxygenase-2 is related to proliferation of human gastric cancer cells

Cyclooxygenase-2 (COX-2) expression is mediated by constitutive NF-kappaB and regulates human gastric cancer cell growth and proliferation. Inactivating Ku70 or Ku80 suppresses cell growth and induces apoptosis. It has been hypothesized that Ku70 and Ku80 expression may be associated with NF-kappaB activation and COX-2 expression and is involved in cell proliferation. In this study, we found that inhibition of constitutive NF-kappaB (by transfecting a mutated IkappaBalpha gene) and of COX-2 (by treatment with indomethacin and NS-398) suppressed Ku70 and Ku80 expression in cells. Treatment with prostaglandin E(2) adenocarcinoma gastric (AGS) increased expression of these Ku proteins in cells with low constitutive NF-kappaB levels. Inhibition of the Ku DNA end-binding activity by transfection with the C-terminal Ku80 expression gene suppressed cell proliferation. Ku70 or Ku80 overexpression by transfection with the Ku70 or Ku80 expression gene, respectively, enhanced proliferation of cells with low NF-kappaB levels. These results demonstrate that Ku70 and Ku80 expression is mediated by constitutively activated NF-kappaB and constitutively expressed COX-2 in gastric cancer cells and that the high Ku DNA end-binding activity contributes to cell proliferation. Ku70 and Ku80 expression may be related to gastric cell proliferation and carcinogenesis.     

Sec6 enhances cell migration and suppresses apoptosis by elevating the phosphorylation of p38 MAPK,MK2, and HSP27

The signaling axis of p38 mitogen-activated protein kinase (p38 MAPK) and MAPK-activated protein kinase 2 (MK2) is the dominant pathway that leads to heat shock protein 27 (HSP27) phosphorylation. After activation of MK2 by p38 MAPK, HSP27 is phosphorylated and depolymerized by MK2, thereby increasing the cell migration and directly interfering with the apoptotic signaling cascades. Sec6 is one of the components of the exocyst complex that is an evolutionarily conserved 8-protein complex. Even though several studies have demonstrated that Sec6 is involved in various cellular physiological functions, the relationship between Sec6 and HSP27 or p38 MAPK during cell migration and apoptosis remains unclear. In the present study, we observed that Sec6 increased the phosphorylation of p38 MAPK through the activation of MAPK kinase 3/6 (MKK3/6). Moreover, Sec6 knockdown suppressed the phosphorylation of HSP27 at Ser⁷⁸ and Ser⁸² sites via suppression of activated MK2. Furthermore, the reduction of phosphorylated HSP27 or p38 MAPK by Sec6 knockdown suppressed cell migration and promoted apoptosis after treatment with tumor necrosis factor-α and cycloheximide. The present study suggested that Sec6 is involved in the enhancement of cell migration and suppression of apoptosis through the activation of HSP27 or p38 MAPK phosphorylation.     

Specific interaction of IP6 with human Ku70/80, the DNA-binding subunit of DNA-PK

In eukaryotic cells, DNA double-strand breaks can be repaired by non-homologous end-joining, a process dependent upon Ku70/80, XRCC4 and DNA ligase IV. In mammals, this process also requires DNA-PK(cs), the catalytic subunit of the DNA-dependent protein kinase DNA-PK. Previously, inositol hexakisphosphate (IP6) was shown to be bound by DNA-PK and to stimulate DNA-PK-dependent end-joining in vitro. Here, we localize IP6 binding to the Ku70/80 subunits of DNA- PK, and show that DNA-PK(cs) alone exhibits no detectable affinity for IP6. Moreover, proteolysis mapping of Ku70/80 in the presence and absence of IP6 indicates that binding alters the conformation of the Ku70/80 heterodimer. The yeast homologue of Ku70/80, yKu70/80, fails to bind IP6, indicating that the function of IP6 in non-homologous end-joining, like that of DNA-PK(cs), is unique to the mammalian end-joining process.     

HSP70, a Novel Regulatory Molecule in B Cell-Mediated Suppression of Autoimmune Diseases

B cells have recently emerged as playing regulatory role in autoimmune diseases. We have previously demonstrated that human peripheral blood CD19⁺ CD24^hiCD27⁺ B cells have regulatory function both in healthy donors and in patients with autoimmune disease. However, the mechanism of this regulation is still not fully understood. In this study, microarrays were utilized to compare gene expression of CD19⁺ CD24^hiCD27⁺ B cells (regulatory B cells, Bregs) with CD19⁺ CD24^loCD27⁻ B cells (non-Bregs) in human peripheral blood. We found that heat shock protein 70 (HSP70) expression was significantly upregulated in Bregs. In vitro studies explored that HSP70 inhibition impaired the regulatory function of peripheral blood Bregs. In mouse models of autoimmune disease, using HSP70-deficient mice or HSP70 inhibitors, Bregs suppressed effector cells and rescued disease-associated phenotypes that were dependent on HSP70. Mechanistically, Bregs secreted HSP70, directly suppressing effector cells, such as T effect cells. These findings reveal that HSP70 is a novel factor that modulates Breg function and suggest that enhancing Breg-mediated production of HSP70 could be a viable therapy for autoimmune disease.