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R I Morimoto

Publications and source records attributed to R I Morimoto.

At least 73 records · Page 4Linked to original sources
PubMed1994

Arachidonate is a potent modulator of human heat shock gene transcription.

Cell and tissue injury activate the inflammatory response through the action(s) of arachidonic acid and its metabolites, leading to the expression of acute-phase proteins and inflammatory cytokines. At the molecular level, little is known how arachidonic acid regulates the inflammatory response. As inflammation is also associated with local increase in tissue temperatures, we examined whether arachidonic acid was directly involved in the heat shock response. Extracellular exposure to arachidonic acid induced heat shock gene transcription in a dose-dependent manner via acquisition of DNA-binding activity and phosphorylation of heat shock factor 1 (HSF1). In addition, exposure of cells to low concentrations of arachidonic acid, which by themselves did not induce HSF1 DNA-binding activity, reduced the temperature threshold for HSF1 activation from elevated temperatures which are not physiologically relevant (> 42 degrees C) to temperatures which can be attained during the febrile response (39-40 degrees C). These results indicate that elevated heat shock gene expression is a direct consequence of an arachidonic acid-mediated cellular response.

Arachidonic Acid↗
PubMed1994

Heat shock factor 2-like activity in mouse blastocysts.

The expression of heat shock genes is induced in all living cells by a series of proteotoxic treatments. Heat shock genes are also activated spontaneously during different phases of embryonic development. HSP89 alpha and HSC70 are expressed at a high level in the mouse blastocyst. A family of factors, called HSFs, are able to bind to the promoters of heat shock genes on upstream conserved elements (HSEs). HSF1 is unable to bind to HSE sequences in absence of stress. It is activated after a stress by post-translational modifications and conformational change. HSF2 shows common structural domains with HSF1; however, it is active at normal temperatures. Recently we showed the presence of an abundant HSE-binding activity in nonshocked blastocysts. We demonstrate here by using polyclonal antibodies that HSF2 is the major constituent of this constitutive HSE-binding activity. HSF2 might be involved in the control of heat shock gene expression during early mammalian embryogenesis.

Animals↗
PubMed1994

Expression of heat shock factor 2 in mouse testis: potential role as a regulator of heat-shock protein gene expression during spermatogenesis.

We have examined the expression and function of heat shock transcription factor 2 (HSF2) in spermatogenic cells of mouse testis. The results of in situ RNA hybridization analysis, RNA filter hybridization, and reverse transcription-polymerase chain reaction (RT-PCR) analysis indicate that HSF2 mRNA expression in testis is subject to developmental and cell type-dependent, as well as stage-dependent, regulation. Localized expression of HSF2 mRNA in testis first appears between Day 14 and Day 21 of postnatal development. In adult testis, HSF2 mRNA is found at highest levels in spermatocytes and round spermatids. Immunocytochemical staining and gel mobility shift analysis demonstrate that HSF2 protein is localized to the nuclei of spermatocytes and round spermatids and that this transcription factor exists in testis in a constitutively active DNA-binding state. We further demonstrate that the constitutive HSF2 DNA-binding activity present in testis is able to interact with promoter sequences of the hsp70.2 gene, a testis-specific member of the hsp70 gene family. Taken together, our results show that the expression and functional properties of HSF2 are regulated in spermatogenic cell types of the mouse testis, supporting a role for this transcription factor as a regulator of hsp gene expression during spermatogenesis.

Animals↗
PubMed1994

Selection of new HSF1 and HSF2 DNA-binding sites reveals difference in trimer cooperativity.

Multiple heat shock transcription factors (HSFs) have been discovered in several higher eukaryotes, raising questions about their respective functions in the cellular stress response. Previously, we had demonstrated that the two mouse HSFs (mHSF1 and mHSF2) interacted differently with the HSP70 heat shock element (HSE). To further address the issues of cooperativity and the interaction of multiple HSFs with the HSE, we selected new mHSF1 and mHSF2 DNA-binding sites through protein binding and PCR amplification. The selected sequences, isolated from a random population, were composed primarily of alternating inverted arrays of the pentameric consensus 5'-nGAAn-3', and the nucleotides flanking the core GAA motif were nonrandom. The average number of pentamers selected in each binding site was four to five for mHSF1 and two to three for mHSF2, suggesting differences in the potential for cooperative interactions between adjacent trimers. Our comparison of mHSF1 and mHSF2 binding to selected sequences further substantiated these differences in cooperativity as mHSF1, unlike mHSF2, was able to bind to extended HSE sequences, confirming previous observations on the HSP70 HSE. Certain selected sequences that exhibited preferential binding of mHSF1 or mHSF2 were mutagenized, and these studies demonstrated that the affinity of an HSE for a particular HSF and the extent of HSF interaction could be altered by single base substitutions. The domain of mHSF1 utilized for cooperative interactions was transferable, as chimeric mHSF1/mHSF2 proteins demonstrated that sequences within or adjacent to the mHSF1 DNA-binding domain were responsible. We have demonstrated that HSEs can have a greater affinity for a specific HSF and that in mice, mHSF1 utilizes a higher degree of cooperativity in DNA binding. This suggests two ways in which cells have developed to regulate the activity of closely related transcription factors: developing the ability to fully occupy the target binding site and alteration of the target site to favor interaction with a specific factor.

Animals↗
PubMed1994

Human heat shock factors 1 and 2 are differentially activated and can synergistically induce hsp70 gene transcription.

Two members of the heat shock transcription factor (HSF) family, HSF1 and HSF2, both function as transcriptional activators of heat shock gene expression. However, the inducible DNA-binding activities of these two factors are regulated by distinct pathways. HSF1 is activated by heat shock and other forms of stress, whereas HSF2 is activated during hemin-induced differentiation of human K562 erythroleukemia cells, suggesting a role for HSF2 in regulating heat shock gene expression under nonstress conditions such as differentiation and development. To understand the distinct regulatory pathways controlling HSF2 and HSF1 activities, we have examined the biochemical and physical properties of the control and activated states of HSF2 and compared these with the properties of HSF1. Our results reveal that the inactive, non-DNA-binding forms of HSF2 and HSF1 exist primarily in the cytoplasm of untreated K562 cells as a dimer and monomer, respectively. This difference in the control oligomeric states suggests that the mechanisms used to control the DNA-binding activities of HSF2 and HSF1 are distinct. Upon activation, both factors acquire DNA-binding activity, oligomerize to a trimeric state, and translocate into the nucleus. Interestingly, we find that simultaneous activation of both HSF2 and HSF1 in K562 cells subjected to hemin treatment followed by heat shock results in the synergistic induction of hsp70 gene transcription, suggesting a novel level of complex regulation of heat shock gene expression.

Cell Compartmentation↗
PubMed1994

Stress induction of the mammalian GRP78/BiP protein gene: in vivo genomic footprinting and identification of p70CORE from human nuclear extract as a DNA-binding component specific to the stress regulatory element.

GRP78, also known as BiP, is one of the better-characterized molecular chaperones. It has been implicated in protein folding and also calcium sequestration in the endoplasmic reticulum. When the cells are subjected to endoplasmic reticulum stress, in particular the depletion of stored calcium and/or the accumulation of abnormal proteins, the rate of transcription of grp78 is enhanced. Previous studies have shown that the core region of the rat grp78 promoter (-170 to -135), which is 95% conserved with the human grp78 core (-133 to -98), is one of the key regulatory elements. Using ligation-mediated PCR, we have found that there are specific changes in factor occupancy after stress induction and the major changes occur within a cluster of bases located in the 3' half of the grp core, whereas other regulatory elements are constitutively occupied. This inducible binding to the 3' half of the human grp78 core region is observed under diverse stress signals, suggesting a common mechanism for the grp stress response. Nonetheless, the lack of constitutive in vivo protection at this region is not due to the absence of a binding factor in nuclear extracts. Using in vitro gel mobility shift assays, we detected a constitutive binding activity which exhibits specificity and affinity to the stress-inducible region. Through sodium dodecyl sulfate-polyacrylamide gel electrophoresis size fractionation and renaturation analysis, the activity is found in polypeptides with molecular sizes of 65 to 75 kDa. After a three-step purification scheme including core affinity column chromatography, we purified p70CORE, which is about 70 kDa in its monomeric form. The purified p70CORE is sufficient to form a complex specific to the stress-inducible region.

Base Sequence↗
PubMed1993

Investigations of mechanisms of drug-induced changes in gene expression: N-methylformamide-induced changes in synthesis of the M(r) 72,000 constitutive heat shock protein during commitment of HL-60 cells to granulocyte differentiation.

HL-60 cells were treated with the differentiating agent N-methylformamide and early changes in gene expression and protein content were investigated. Analysis of protein synthesis had previously shown an early (< 12 h) fall in the synthesis of M(r) 70,000 heat shock proteins (F. M. Richards, A. Watson, and J. A. Hickman. Cancer Res., 48: 6715-6720, 1988). The changes have now been characterized in detail and their kinetics compared to those of the expression of the c-myc protein. Immunoanalysis, using antibodies to either the stress-inducible heat shock protein hsp70 (4G4) or a pan-M(r) 70,000 heat shock protein antibody (3A3), showed that there was a striking reduction in the levels of the constitutive heat shock protein hsc70 when cells were incubated continuously with 170 mM N-methylformamide. A reduction in the level of hsc70 RNA was observed within 3 h and continued thereafter. In contrast, transcription of the hsc70 gene was induced within 1-2 h, after which the rate returned to basal level. There were no significant changes in the rate of transcription of the stress-inducible heat shock proteins hsp70 or hsp90. When N-methylformamide was removed from the cells, prior to commitment to differentiation, the levels of hsc70 were reestablished, whereas after 36 h of treatment there was no recovery. Western blotting with an antibody to the c-myc protein showed this to fall to virtually undetectable levels by 3 h under the same conditions. The results suggest that the loss of hsc70, which may perform a protein chaperoning role, was mediated at both transcriptional and posttranscriptional levels of regulation and was an early event closely associated with the commitment of HL-60 cells to differentiation. The fall in hsc70 was not associated with alterations in the cell cycle, nor were the kinetics of the change suggestive of a relationship with the decrease in content of c-myc protein.

Cell Differentiation↗
PubMed1993

Activation of heat shock gene transcription by heat shock factor 1 involves oligomerization, acquisition of DNA-binding activity, and nuclear localization and can occur in the absence of stress.

The existence of multiple heat shock factor (HSF) genes in higher eukaryotes has promoted questions regarding the functions of these HSF family members, especially with respect to the stress response. To address these questions, we have used polyclonal antisera raised against mouse HSF1 and HSF2 to examine the biochemical, physical, and functional properties of these two factors in unstressed and heat-shocked mouse and human cells. We have identified HSF1 as the mediator of stress-induced heat shock gene transcription. HSF1 displays stress-induced DNA-binding activity, oligomerization, and nuclear localization, while HSF2 does not. Also, HSF1 undergoes phosphorylation in cells exposed to heat or cadmium sulfate but not in cells treated with the amino acid analog L-azetidine-2-carboxylic acid, indicating that phosphorylation of HSF1 is not essential for its activation. Interestingly, HSF1 and HSF2 overexpressed in transfected 3T3 cells both display constitutive DNA-binding activity, oligomerization, and transcriptional activity. These results demonstrate that HSF1 can be activated in the absence of physiological stress and also provide support for a model of regulation of HSF1 and HSF2 activity by a titratable negative regulatory factor.

3T3 Cells↗
PubMed1993

Characterization of a novel chicken heat shock transcription factor, heat shock factor 3, suggests a new regulatory pathway.

We have cloned three avian heat shock transcription factor (HSF) genes corresponding to a novel factor, HSF3, and the avian homologs of mammalian HSF1 and HSF2. The predicted amino acid sequence of HSF3 is approximately 40% related to the sequence of HSF1 and HSF2. The sequences for all three factors exhibit extensive identify in the DNA binding motifs and the heptad repeats of hydrophobic amino acids which are common to all eukaryotic HSFs. Despite these overall similarities, each avian HSF exhibits distinct DNA binding properties. HSF2 when expressed in vitro binds constitutively to the heat shock element promoter sequence, whereas neither HSF1 nor HSF3 expressed in vitro binds to DNA. HSF1 DNA binding is induced upon heat shock or treatment with nonionic detergents, whereas the DNA binding properties of HSF3 are not induced by these conditions in vitro. These results suggest that HSF3 activation may involve an induction pathway distinct from the traditional forms of heat shock gene induction. HSF3 DNA binding activity, however, is obtained when the carboxyl-terminal region including the distal heptad repeat is deleted, indicating the presence of negative cis-regulatory sequences. The HSF3 message, like HSF1 and HSF2 messages, is coexpressed during development and in most tissues, which suggests a general role for the regulatory pathway involving HSF3.

Age Factors↗
PubMed1993

Mouse heat shock transcription factors 1 and 2 prefer a trimeric binding site but interact differently with the HSP70 heat shock element.

To understand the function of multiple heat shock transcription factors in higher eukaryotes, we have characterized the interaction of recombinant mouse heat shock transcription factors 1 and 2 (mHSF1 and mHSF2) with their binding site, the heat shock element (HSE). For our analysis, we utilized the human HSP70 HSE, which consists of three perfect 5'-nGAAn-3' sites (1, 3, and 4) and two imperfect sites (2 and 5) arranged as tandem inverted repeats. Recombinant mHSF1 and mHSF2, which exist as trimers in solution, both bound specifically to this HSE and stimulated transcription of a human HSP70-CAT construct in vitro. Footprinting analyses revealed differential binding of mHSF1 and mHSF2 to the HSP70 HSE. Specifically, mHSF1 bound all five pentameric sites, whereas mHSF2 failed to interact with the first site of the HSE but bound to sites 2 to 5. Missing-nucleoside analysis demonstrated that the third and fourth nGAAn sites were essential for mHSF1 and mHSF2 binding. The binding of the initial mHSF1 trimer to the HSE exhibited preference for sites 3, 4, and 5, and then binding of a second trimer occurred at sites 1 and 2. These results suggest that HSF may recognize its binding site through the dyad symmetry of sites 3 and 4 but requires an adjacent site for stable interaction. Our data demonstrate that mHSF1 and mHSF2 bind specifically to the HSE through major groove interactions. Methidiumpropyl-EDTA footprinting revealed structural differences in the first and third repeats of the HSE, suggesting that the DNA is distorted in this region. The possibility that the HSE region is naturally distorted may assist in understanding how a trimer of HSF can bind to what is essentially an inverted repeat binding site.

Animals↗
PubMed1992

Transcriptional regulation of heat shock genes. A paradigm for inducible genomic responses.

The heat shock response offers an ideal paradigm to understand how the cell recognizes and responds to acute and chronic exposures to environmental and physiological stress. Of the numerous inducible genomic responses, the heat shock response has contributed fascinating insights into the molecular and cellular mechanisms of adaptation, ranging from the regulation of heat shock gene expression to the function of stress proteins. The recent cloning of multiple heat shock transcription factor (HSF) genes in higher eukaryotes and studies on the biochemical and cellular properties of HSFs have revealed several novel features of the transcriptional response.

Animals↗
PubMed1992

Antiproliferative prostaglandins activate heat shock transcription factor.

Treatment of human K562 erythroleukemia cells with the antiproliferative prostaglandin A1 results in the elevated transcription of two heat shock genes, HSP70 and HSP90. Parallel with increased heat shock gene transcription is the activation of heat shock transcription factor. Heat shock transcription factor levels are induced within 60 min after prostaglandin A1 addition to levels similar to that achieved during heat shock. The requirement for protein synthesis for prostaglandin A1 activation of heat shock transcription factor suggests that effects on nascent protein synthesis may be involved in the signaling mechanism. Although it is unclear whether the activation of a heat shock response by prostaglandins is relevant to the biochemical properties of these natural substances, cells pretreated with prostaglandin A1 are protected against a subsequent heat shock, indicative of a thermotolerant state.

DNA-Binding Proteins↗
PubMed1992

Effect of sodium salicylate on the human heat shock response.

Sodium salicylate, an anti-inflammatory agent, was examined for its effects on the heat shock response in cultured human cells. Salicylate activation of DNA binding by the heat shock transcription factor (HSF) was comparable to activation attained during heat shock. However, sodium salicylate did not induce heat shock gene transcription even though the HSF was bound in vivo to the heat shock elements upstream of the heat shock protein 70 (Hsp 70) gene. These results reveal that activation of the heat shock transcriptional response is a multistep process. Modulation of extracellular pH augments sensitivity to salicylate-induced activation of HSF.

DNA↗
PubMed1992

Transcription factors: positive and negative regulators of cell growth and disease.

Recent advances in our understanding of transcription factors that regulate RNA polymerase II transcribed genes are described. This review considers the role of alternate protein-protein interactions to yield positive or negative regulation, the consequences of deregulated factor activity on cell function and approaches to establish factor-binding specificities.

Animals↗
PubMed1992

The human heat shock protein hsp70 interacts with HSF, the transcription factor that regulates heat shock gene expression.

Transcriptional regulation of the human hsp70 gene in response to heat shock and other forms of physiological stress occurs through the activation of heat shock transcription factor (HSF). Exposure of cells to a heat shock temperature of 42 degrees C results in transient activation of HSF; its DNA-binding activity increases rapidly, plateaus, and attenuates, during which the intracellular levels of hsp70 increase. In an effort to understand whether HSF is regulated negatively by hsp70, we have examined whether HSF associates with hsp70. We show that activated HSF associates with hsp70 and that the interaction is detected as the levels of hsp70 increase in the cell. Addition of ATP and other hydrolyzable nucleotides results in the dissociation of hsp70 from HSF while nonhydrolyzable nucleotide analogs do not disrupt the complex. We demonstrate that exogenous recombinant wild-type hsp70 can associate with activated HSF, whereas no association is observed with an amino-terminal or a carboxy-terminal deletion mutant of hsp70. We also show that hsp70 blocks the in vitro activation of HSF from its cryptic non-DNA-binding state to a DNA-binding form; this inhibitory effect of hsp70 is abolished by ATP. We suggest that hsp70 may negatively regulate the activation of HSF.

Adenosine Triphosphate↗
PubMed1992

Activation of heat shock factor 2 during hemin-induced differentiation of human erythroleukemia cells.

Hemin induces nonterminal differentiation of human K562 erythroleukemia cells, which is accompanied by the expression of certain erythroid cell-specific genes, such as the embryonic and fetal globins, and elevated expression of the stress genes hsp70, hsp90, and grp78/BiP. Previous studies revealed that, as during heat shock, transcriptional induction of hsp70 in hemin-treated cells is mediated by activation of heat shock transcription factor (HSF), which binds to the heat shock element (HSE). We report here that hemin activates the DNA-binding activity of HSF2, whereas heat shock induces predominantly the DNA-binding activity of a distinct factor, HSF1. This constitutes the first example of HSF2 activation in vivo. Both hemin and heat shock treatments resulted in equivalent levels of HSF-HSE complexes as analyzed in vitro by gel mobility shift assay, yet transcription of the hsp70 gene was stimulated much less by hemin-induced HSF than by heat shock-induced HSF. Genomic footprinting experiments revealed that hemin-induced HSF and heat shock-induced HSF, HSF2, and HSF1, respectively, occupy the HSE of the human hsp70 promoter in a similar yet not identical manner. We speculate that the difference in occupancy and/or in the transcriptional abilities of HSF1 and HSF2 accounts for the observed differences in the stimulation of hsp70 gene transcription.

Base Sequence↗
PubMed1991

Selective activation of human heat shock gene transcription by nitrosourea antitumor drugs mediated by isocyanate-induced damage and activation of heat shock transcription factor.

Treatment of cultured human tumor cells with the chloroethylnitrosourea antitumor drug 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) selectively induces transcription and protein synthesis of a subset of the human heat shock or stress-induced genes (HSP90 and HSP70) with little effect on other stress genes or on expression of the c-fos, c-myc, or beta-actin genes. The active component of BCNU and related compounds appears to be the isocyanate moiety that causes carbamoylation of proteins and nucleic acids. Transcriptional activation of the human HSP70 gene by BCNU is dependent on the heat shock element and correlates with the level of heat shock transcription factor and its binding to the heat shock element in vivo. Unlike activation by heat or heavy metals, BCNU-mediated activation is strongly dependent upon new protein synthesis. This suggests that BCNU-induced, isocyanate-mediated damage to newly synthesized protein(s) may be responsible for activation of the heat shock transcription factor and increased transcription of the HSP90 and HSP70 genes.

Actins↗