Physiological and molecular assessment of altered expression of Hsc70-1 in Arabidopsis. Evidence for pleiotropic consequences

Hsp70s function as molecular chaperones. The protective chaperone activities of hsp70 help to confer tolerance to heat, glucose deprivation, and drought. Overexpression of hsp70s in many organisms correlates with enhanced thermotolerance, altered growth, and development. To better understand the roles of hsp70 proteins in Arabidopsis, the molecular and physiological consequences of altered expression of the major heat shock cognate, Hsc70-1, were analyzed. Extensive efforts to achieve underexpression of Hsc70-1 mRNA using a full-length antisense cDNA resulted in no viable transgenic plants, suggesting that reduced expression is lethal. Constitutive overexpression of Hsc70-1 also appeared to be deleterious to viability, growth, and development because fewer transformants were recovered, and most were dwarfed with altered root systems. Despite being dwarfed, the overexpression plants progressed normally through four selected developmental stages. Heat treatment revealed that Hsc70-1 overexpression plants were more tolerant to heat shock (44 degrees C for 10 min). The elevated basal levels of HSC70-1 in transgenic plants led to delayed heat shock response of several heat shock genes. The data in this study suggest that tight regulation of Hsc70-1 expression is critical for the viability of Arabidopsis and that the functions of HSC70-1 contribute to optimum growth, development, thermotolerance, and regulation of the heat shock response.     

Functional analysis of the Hikeshi-like protein and its interaction with HSP70 in Arabidopsis

Heat shock proteins (HSPs) refold damaged proteins and are an essential component of the heat shock response. Previously, the 70 kDa heat shock protein (HSP70) has been reported to translocate into the nucleus in a heat-dependent manner in many organisms. In humans, the heat-induced translocation of HSP70 requires the nuclear carrier protein Hikeshi. In the Arabidopsis genome, only one gene encodes a protein with high homology to Hikeshi, and we named this homolog Hikeshi-like (HKL) protein. In this study, we show that two Arabidopsis HSP70 isoforms accumulate in the nucleus in response to heat shock and that HKL interacts with these HSP70s. Our histochemical analysis revealed that HKL is predominantly expressed in meristematic tissues, suggesting the potential importance of HKL during cell division in Arabidopsis. In addition, we show that HKL regulates HSP70 localization, and HKL overexpression conferred thermotolerance to transgenic Arabidopsis plants. Our results suggest that HKL plays a positive role in the thermotolerance of Arabidopsis plants and cooperatively interacts with HSP70.     

Differential gene expression of HSC70/HSP70 in yellowtail cells in response to chaperone-mediated autophagy

A cell line derived from the tailfin of the marine teleost yellowtail fish Seriola quinqueradiata was established to examine cellular temperature regulation in an ectothermic animal. Three cytosolic members of the HSP70 family, heat-shock cognate proteins HSC70-1, HSC70-2 and heat-shock protein HSP70, were isolated from cultured yellowtail cells as stress-responsive biomarkers. Expression of hsp70 was heat-inducible, in contrast to the hsc70-1 gene product, which was expressed constitutively. In addition, expression of hsc70-2 was only induced under severe heat-shock conditions. Subcellular fractionation and immunocytochemistry showed localization of HSC70/HSP70 in the lysosomes, indicating that chaperone-mediated autophagy is induced by heat shock. Thus, chaperone-mediated autophagy is assisted by HSC70/HSP70, and heat-inducible expression of the genes encoding these proteins may be responsible for survival and adaptation under heat-shock conditions in fish cells.     

Quantitative evidence that both Hsc70 and Hsp70 contribute to thermal adaptation in hybrids of the livebearing fishes Poeciliopsis

The 70-kilodalton heat shock protein family is composed of both environmentally inducible (Hsp) and constitutively expressed (Hsc) family members. While the role of the constitutively expressed stress proteins in thermotolerance is largely unknown, de novo expression stress proteins in response to elevated temperatures has been associated with increased thermotolerance in many cell lines, developing embryos and adult organisms. Distinct, hemiclonal hybrids between the livebearing fish species Poeciliopsis monacha and P. lucida varied in their abilities to survive temperature stress, with survival being greatest when rates of temperature increase to 40°C were slowest and when P. monacha genomes were combined with a sympatric P. lucida genome. Quantification of Hsp70 under heat shock conditions and Hsc70 under normal physiological conditions indicated that variation in survival among hemiclones was best explained by the combined effects of these two proteins. Similar complex interactions between maternal and paternal genomes and rate of temperature increase were found to underline patterns of survival, Hsp70 accumulation and Hsc70 abundance. These data suggest that the relationship between Hsps and thermotolerance is more intricate than previously thought and that Hsps contribute to thermal adaptation in these fishes through genetic interactions specific to particular environments.     

Heat shock protein 25 or inducible heat shock protein 70 activates heat shock factor 1: dephosphorylation on serine 307 through inhibition of ERK1/2 phosphorylation

The expression of heat shock proteins (HSPs) is known to be increased via activation of heat shock factor 1 (HSF1), and excess expression of HSPs exerts feedback inhibition of HSF1. However, the molecular mechanism to modulate such relationships between HSPs and HSF1 is not clear. In the present study, we show that stable transfection of either Hsp25 or inducible Hsp70 (Hsp70i) increased expression of endogenous HSPs such as HSP25 and HSP70i through HSF1 activation. However, these phenomena were abolished when the dominant negative Hsf1 mutant was transfected to HSP25 or HSP70i overexpressed cells. Moreover, the increased HSF1 activity by either HSP25 or HSP70i was found to result from dephosphorylation of HSF1 on serine 307 that increased the stability of HSF1. Either HSP25 or HSP70i inhibited ERK1/2 phosphorylation because of increased MKP1 phosphorylation by direct interaction of these HSPs with MKP1. Treatment of HOS and NCI-H358 cells, which showed high expressions of endogenous HSF1, with small interfering RNA (siRNA) of either HSP27 (siHSP27)or HSP70i (siHSP70i) inhibited both HSP27 and HSP70i proteins; this was because of increased ERK1/2 phosphorylation and serine phosphorylation of HSF1. The results, therefore, suggested that when the HSF1 protein level was high in cancer cells, excess expression of HSP27 or HSP70i strongly facilitates the expression of HSP proteins through HSF1 activation, resulting in severe radio- or chemoresistance.     

Changes in hsp70 alter thermotolerance and heat-shock regulation in Drosophila.

To test the role of the heat shock protein hsp70 in induced thermotolerance and in the regulation of the heat-shock response, we established cell lines with altered expression of the Hsp70 gene. Underexpressing cells were created by transformation with antisense Hsp70 genes, and overexpressing cells by transformation with extra copies of the wild-type gene. Expression at normal temperatures was achieved by placing Hsp70 coding sequences under the control of the metallothionein promoter. Cells that expressed mutant hsp70s were created by transforming cells with deletion and frameshift mutations. The results indicate that hsp70 plays a major role in both thermotolerance and regulation. Surprisingly, they also indicate that these functions can be separated. Overexpression affected thermotolerance more than regulation; underexpression affected regulation more than thermotolerance. A carboxyl-terminal deletion of Hsp70 had a severe dominant-negative effect on thermotolerance but only a minor effect on regulation; an amino-terminal deletion strongly affected regulation but not thermotolerance. A model that explains these observations is presented.     

Inducible and constitutive heat shock gene expression responds to modification of Hsp70 copy number in Drosophila melanogaster but does not compensate for loss of thermotolerance in Hsp70 null flies

Background  

The heat shock protein Hsp70 promotes inducible thermotolerance in nearly every organism examined to date. Hsp70 interacts with a network of other stress-response proteins, and dissecting the relative roles of these interactions in causing thermotolerance remains difficult. Here we examine the effect of Hsp70 gene copy number modification on thermotolerance and the expression of multiple stress-response genes in Drosophila melanogaster, to determine which genes may represent mechanisms of stress tolerance independent of Hsp70.     

Regulation of heat shock protein 70 synthesis by heat shock in the preimplantation murine embryo

Induced thermotolerance in murine embryos occurs at the 8-cell stage when embryos are maintained in vitro but not until the blastocyst stage if development proceeds in vivo. Present results indicate that ability of embryos to undergo induced thermotolerance is not limited by heat shock protein 70 (HSP70) synthesis. Exposure of 8-cell embryos to 40 degrees C enhanced synthesis of 2 constitutive HSP70 proteins (HSC70 and HSC72) and induced another protein, HSP68; exposure of 43 degrees C was required to induce similar responses in expanded blastocysts. Unlike induced thermotolerance, increased synthesis of HSP70 molecules did not depend on whether embryos were cultured or developed in vivo. Thus, other biochemical mechanisms in addition to HSP70 confer thermotolerance in the preimplantation-stage murine embryo. The observation that the temperature threshold for induction of HSP70 synthesis increased from the 8-cell to the blastocyst stage is indicative of these other biochemical processes.