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.     

LPA modulates monocyte migration directly and via LPA-stimulated endothelial cells

Lysophosphatidic acid (LPA) is a bioactive lysophospholipid ligand present in oxidized low-density lipoprotein. The effects of LPA were investigated, first separately on endothelial cells (EC) and monocytes. Using Ki16425 (an LPA₁ and LPA₃ receptor antagonist), GW9662 [a peroxisome proliferator-activator receptor (PPAR) antagonist], and pertussis toxin (that inhibits G_i/o), we demonstrate that LPA enhances IL-8 and monocyte chemoattractant protein-1 expression through a LPA₁-, LPA₃-, G_i/o- and PPAR-dependent manner in the EAhy926 cells. The effect of LPA on chemokine overexpression was confirmed in human umbilical vein endothelial cells. LPA was able to enhance monocyte migration at concentrations <1 µM and to inhibit their migration at LPA concentrations >1 µM, as demonstrated by using a chemotaxis assay. We then investigated the effects of LPA on the cross-talk between EC and monocytes by evaluating the chemotactic activity in the supernatants of LPA-treated EC. At 1 µM LPA, both cell types respond cooperatively, favoring monocyte migration. At higher LPA concentration (25 µM), the chemotactic response varies as a function of time. After 4 h, the chemotactic effect of the cytokines secreted by the EC is counteracted by the direct inhibitory effect of LPA on monocytes. For longer periods of time (24 h), we observe a monocyte migration, probably due to lowered concentrations of bioactive LPA, given the induction of lipid phosphate phosphatase-2 in monocytes that may inactivate LPA. These results suggest that LPA activates EC to secrete chemokines that in combination with LPA itself might favor or not favor interactions between endothelium and circulating monocytes. lysophosphatidic acid; endothelial cells; monocytes; chemotaxis     

Regulation of HSP70 and HSP28 gene expression: absence of compensatory interactions

We have previously reported the lack of HSP28 gene expression during acute and chronic thermotolerance development in L929 cells (J Cell Physiol 152: 118–125, 1992; Cancer Res 52: 5787, 1992). In contrast to HSP28, an extremely high level of inducible HSP70 synthesis was observed. These results led us to investigate the possibility of compensatory interactions between HSP70 and HSP28. To test the hypothesis, L929 cells were transfected with the human HSP28 gene contained in plasmid pCMV27. Data from Western blot and two-dimensional gel electrophoresis of [³H] leucine and [³²P] orthophosphate-labeled proteins showed the synthesis and phosphorylation of HSP28 in transfected cells after heating at 45°C for 10 min. However, the expression of constitutive and inducible HSP70 genes, along with the synthesis of their proteins, was not decreased after heat shock. These results suggest an independent regulation of HSP28 and HSP70 gene expression.     

Cloning and expression of HSP70 gene of sipuncula Phascolosoma esculenta

In this study the gene encoding HSP70 was isolated from Phascoloma esculenta by homologous cloning and rapid amplification of cDNA ends (RACE). The full-length of cDNA (2520 bp) consists of a 5′-terminal untranslated region (UTR) (125 bp), a 3′-terminal UTR (421 bp) with a canonical polyadenylation signal sequence (AATAAA), a poly (A) tail, and an open reading frame (ORF) (1974 bp). The predicted molecular mass and isoelectric point for HSP70 is 71.6 kDa and 5.15, respectively. BLAST analysis showed that P. esculenta HSP70 gene shared high similarity. Classical HSP signature motifs, ATP/GTP-Binding Site Motif A, Bipartite Nuclear Targeting Sequence, the cytosolic HSP70 could be expressed in Escherichia coli BL21. After purification, the recombinant pET-HSP70 protein was used to produce the polyclonal antibody in mice and the specificity of the antibody was confirmed by Western blot analysis. Fluorescent real-time quantitative PCR analysis showed that expression of Hsp70 in sipuncula was increased significantly after exposure to 10 mM Zn for12 h, Cd for 24 h, Cu for 48 h, and was exposure to 37 °C for 24 h sea water.     

HSP 70 gene expression in Trypanosoma cruzi is regulated at different levels

The level of HSP 70 mRNA is altered in Trypanosoma cruzi cells incubated at supra-optimal temperatures: the total amount of this RNA per cell is increased at 37°C, and slightly decreased at 40°C relative to its level at 29°C. However, its amount is greater in the polysomes at either temperature. The relative increase of this RNA is larger in the polysomes fraction than it is in the total RNA. In addition the level of HSP 70 protein in heat-shocked cells is greater than would be expected from the recruitment of HSP 70 mRNA in the polysomal fraction. Taken together the data are interpreted as indicating that at 37°C and 40°C the HSP 70 gene regulation in T. cruzi involves both the selective accumulation of the HSP 70 mRNA in the polysomes and its preferential translation. At 37°C, in addition, an increase in the total amount of this template is observed in the cells.     

Chronic antioxidant enzyme mimetic treatment differentially modulates hyperthermia-induced liver HSP70 expression with aging.

One postulated mechanism for the reduction in stress tolerance with aging is a decline in the regulation of stress-responsive genes, such as inducible heat shock protein 72 (HSP70). Increased levels of oxidative stress are also associated with aging, but it is unclear what impact a prooxidant environment might have on HSP70 gene expression. This study utilized a superoxide dismutase/catalase mimetic (Eukarion-189) to evaluate the impact of a change in redox environment on age-related HSP70 responses to a physiologically relevant heat challenge. Results demonstrate that liver HSP70 mRNA and protein levels are reduced in old compared with young rats at selected time points over a 48-h recovery period following a heat-stress protocol. While chronic systemic administration of Eukarion-189 suppressed hyperthermia-induced liver HSP70 mRNA expression in both age groups, HSP70 protein accumulation was blunted in old rats but not in their young counterparts. These data suggest that a decline in HSP70 mRNA levels may be responsible for the reduction in HSP70 protein observed in old animals after heat stress. Furthermore, improvements in redox status were associated with reduced HSP70 mRNA levels in both young and old rats, but differential effects were manifested on protein expression, suggesting that HSP70 induction is differentially regulated with aging. These findings highlight the integrated mechanisms of stress protein regulation in eukaryotic organisms responding to environmental stress, which likely involve interactions between a wide range of cellular signals.     

Genome-wide expression analysis of HSP70 family genes in rice and identification of a cytosolic HSP70 gene highly induced under heat stress

The heat shock protein 70 (HSP70) gene family plays a key role in protecting plant cells or tissues from thermal or oxidative stress. Although many studies have elucidated the molecular functions of individual family members, genome-wide analysis of this family is still limited, especially for crop species. Our objective was to integrate various meta-profiling data into the context of a phylogenetic tree, which would enable us to perform fine evaluation of functional dominancy or redundancy within this family. Our data indicated that a loss-of-function mutant of a rice cytosolic HSP70 gene (OsctHSP70-1) did not show a clear defective phenotype in response to high temperature because of the existence of another gene family member that was closely clustered with OsctHSP70-1 and had similar expression patterns. Moreover, the second gene showed much stronger anatomical expression. We indirectly analyzed the function of OsctHSP70-1 by studying GUS activity under the control of the endogenous promoter. We also designed a probable interaction network mediated by OsctHSP70-1 and used co-expression analysis among its components to refine the network, suggesting more probable model to explain the function of OsctHSP70-1.     

ERK1/2 regulates heat stress-induced lactate production via enhancing the expression of HSP70 in immature boar Sertoli cells

Lactate produced by Sertoli cells plays an important role in spermatogenesis, and heat stress induces lactate production in immature boar Sertoli cells. Extracellular signaling regulated kinase 1 and 2 (ERK1/2) participates in heat stress response. However, the effect of ERK1/2 on heat stress-induced lactate production is unclear. In the present study, Sertoli cells were isolated from immature boar testis and cultured at 32 °C. Heat stress was induced in a 43 °C incubator for 30 min. Proteins and RNAs were detected by western blotting and RT-PCR, respectively. Lactate production and lactate dehydrogenase (LDH) activity were detected using commercial kits. Heat stress promoted ERK1/2 phosphorylation, showing a reducing trend with increasing recovery time. In addition, heat stress increased heat shock protein 70 (HSP70), glucose transporter 3 (GLUT3), and lactate dehydrogenase A (LDHA) expressions, enhanced LDH activity and lactate production at 2-h post-heat stress. Pretreatment with U0126 (1?×?10^?6 mol/L), a highly selective inhibitor of ERK1/2 phosphorylation, reduced HSP70, GLUT3, and LDHA expressions and decreased LDH activity and lactate production. Meanwhile, ERK2 siRNA1 reduced the mRNA level of ERK2 and weakened ERK1/2 phosphorylation. Additionally, ERK2 siRNA1 reduced HSP70, GLUT3, and LHDA expressions decreased LDH activity and lactate production. Furthermore, HSP70 siRNA3 downregulated GLUT3 and LDHA expressions and decreased LDH activity and lactate production. These results show that activated ERK1/2 increases heat stress-induced lactate production by enhancing HSP70 expression to promote the expressions of molecules related to lactate production (GLUT3 and LDHA). Our study reveals a new insight in reducing the negative effect of heat stress in boars.     

HSP70 expression in the hippocampal CA3 subfield in different chronic stress models

This study examined the heat shock protein 70 (HSP70) expression in hippocampal CA3 subfield of rats in different chronic stress models. The chronic restraint stress (CRS) and the chronic mild stress (CMS) models were used in this study. Observation of exploratory behavior in an open field test indicated stress level. The expression of HSP70 in hippocampal CA3 subfield was measured by immunohistochemical methods. The results showed that the number of quadrant crossing in both CRS and CMS groups decreased more than that of the control (P < 0.01). CRS group crossing decreased more than CMS group. Damage in the hippocampus of the CMS group occurred later and to a less extent than that of the CRS group. Compared with CMS group, the expression of HSP70 was greater in the CRS group. Moreover, increased stress duration enhanced these effects. These results show that the CRS model affects both exploratory behavior and HSP70 expression in the hippocampus more dramatically than the CMS model.