Absence Of Heat Shock Research Articles

The filamentous γ-prefoldin chaperone is not essential for growth and thermal adaptation in Methanocaldococcus jannaschii.

Elucidating the role of molecular chaperones in extremely thermophilic archaea, including the gamma prefoldin (γPFD) in the deep-sea methanogen Methanocaldococcus jannaschii, is integral to understanding microbial adaptation to hot environments. This study focuses on genetically engineered knock-out and overexpression strains to evaluate the importance of γPFD in the growth and thermal tolerance of M. jannaschii. An in-depth analysis of cell growth, morphology and transcriptional responses to heat stress revealed that although the gene encoding γPFD is substantially upregulated in response to heat shock, the γPFD is not indispensable for high-temperature survival. Instead, its absence in the knock-out strain is compensated for by the upregulation of several proteolytic proteins in the absence of heat shock, nearly matching the corresponding transcription profile of selected transcripts for proteins involved in protein synthesis and folding in the wild-type strain following heat shock, using quantitative reverse-transcription PCR (RT-qPCR). These findings bridge environmental adaptation with molecular biology, underscoring the versatility of extremophiles and providing a deeper mechanistic understanding of how they cope with stress.

Chaperones Hsc70 and Hsp70 Bind to the Protein PGK Differently inside Living Cells.

Differences in the physical interactions between proteins, such as binding equilibria, can provide clues about the differences in their function. The binding of heat shock proteins to substrate proteins in living cells is one such example. Eukaryotic cells have evolved many homologues in the Hsp70 family of heat shock proteins, each of which is specialized for a specific function. We previously showed that Hsp70, which is upregulated during heat shock, binds to the model substrate phosphoglycerate kinase (PGK) in human cells before PGK completely unfolds. We dubbed this the "preemptive holding" mechanism. Here, we studied the homologue Hsc70 (heat shock cognate protein), which is constitutively expressed in human cells even in the absence of heat shock. Recent literature has demonstrated the multiple functions performed by Hsc70 in cells under normal conditions. Despite the name "heat shock cognate", very few studies have shown whether Hsc70 is actually involved in the heat shock response. Here we corroborate the existence of the in-cell heat shock response of Hsc70. We show that Hsc70 binds to PGK in human cells in a cooperative manner that directly correlates with protein thermal unfolding. This "unfolded state holding" mechanism differs from the Hsp70 "preemptive holding" mechanism. We rationalize this difference by protein evolution; unlike Hsp70, which is upregulated in order to bind proteins specifically during heat shock, the finite amount of Hsc70 in cells cannot afford to bind to still-folded proteins without compromising its multiple other functions.

Heat shock responsive drug delivery system based on mesoporous silica nanoparticles coated with temperature sensitive gatekeeper

Mesoporous silica nanoparticles (MSN) have several advantages as carriers for drug delivery, including high drug loading capacity, good biocompatibility, excellent stability, and easily tailorable surface properties. This study describes the design of an MSN-based carrier for use in a heat shock responsive drug delivery system. MSN were functionalized with the temperature-sensitive PEG/PCL multiblock copolymer, as gatekeepers, allowing the release of entrapped drugs in response to heat shock stimuli (MBC-MSN). In the absence of heat shock, doxorubicin (Dox)-loaded MBC-MSN showed very low cytotoxicity, as PEG/PCL inhibited the premature release of Dox from MBC-MSN. In response to heat-shock stimuli, however, these Dox@MBC-MSN showed significant cytotoxicity, similar to that of free Dox. Dox release was due to the loosening of the structure of the gatekeeper, PEG/PCL, in response to the heat shock stimuli. Taken together, these findings suggest that MBC-MSN are a strong candidate to act as a carrier in heat shock responsive drug delivery.

Heat Shock Enhances the Expression of the Human T Cell Leukemia Virus Type-I (HTLV-I) Trans-Activator (Tax) Antigen in Human HTLV-I Infected Primary and Cultured T Cells

The environmental factors that lead to the reactivation of human T cell leukemia virus type-1 (HTLV-I) in latently infected T cells in vivo remain unknown. It has been previously shown that heat shock (HS) is a potent inducer of HTLV-I viral protein expression in long-term cultured cell lines. However, the precise HTLV-I protein(s) and mechanisms by which HS induces its effect remain ill-defined. We initiated these studies by first monitoring the levels of the trans-activator (Tax) protein induced by exposure of the HTLV-I infected cell line to HS. HS treatment at 43 °C for 30 min for 24 h led to marked increases in the level of Tax antigen expression in all HTLV-I-infected T cell lines tested including a number of HTLV-I-naturally infected T cell lines. HS also increased the expression of functional HTLV-I envelope gp46 antigen, as shown by increased syncytium formation activity. Interestingly, the enhancing effect of HS was partially inhibited by the addition of the heat shock protein 70 (HSP70)-inhibitor pifithlin-μ (PFT). In contrast, the HSP 70-inducer zerumbone (ZER) enhanced Tax expression in the absence of HS. These data suggest that HSP 70 is at least partially involved in HS-mediated stimulation of Tax expression. As expected, HS resulted in enhanced expression of the Tax-inducible host antigens, such as CD83 and OX40. Finally, we confirmed that HS enhanced the levels of Tax and gp46 antigen expression in primary human CD4+ T cells isolated from HTLV-I-infected humanized NOD/SCID/γc null (NOG) mice and HTLV-I carriers. In summary, the data presented herein indicate that HS is one of the environmental factors involved in the reactivation of HTLV-I in vivo via enhanced Tax expression, which may favor HTLV-I expansion in vivo.

Whi3, an S. cerevisiae RNA-Binding Protein, Is a Component of Stress Granules That Regulates Levels of Its Target mRNAs

RNA binding proteins (RBPs) are vital to the regulation of mRNA transcripts, and can alter mRNA localization, degradation, translation, and storage. Whi3 was originally identified in a screen for small cell size mutants, and has since been characterized as an RBP. The identification of Whi3-interacting mRNAs involved in mediating cellular responses to stress suggested that Whi3 might be involved in stress-responsive RNA processing. We show that Whi3 localizes to stress granules in response to glucose deprivation or heat shock. The kinetics and pattern of Whi3 localization in response to a range of temperatures were subtly but distinctly different from those of known components of RNA processing granules. Deletion of Whi3 resulted in an increase in the relative abundance of Whi3 target RNAs, either in the presence or absence of heat shock. Increased levels of the CLN3 mRNA in whi3Δ cells may explain their decreased cell size. Another mRNA target of Whi3 encodes the zinc-responsive transcription factor Zap1, suggesting a role for Whi3 in response to zinc stress. Indeed, we found that whi3Δ cells have enhanced sensitivity to zinc toxicity. Together our results suggest an expanded model for Whi3 function: in addition to its role as a regulator of the cell cycle, Whi3 may have a role in stress-dependent RNA processing and responses to a variety of stress conditions.

Molecular cloning and expression of the Streptomyces coniferyl alcohol dehydrogenase gene in Escherichia coli

Coniferyl alcohol dehydrogenase (CADH) is a key enzyme in catabolism of lignin-related aromatic compounds in bacteria. In Streptomyces sp. NL15-2K, CADH is a tetramer of identical subunits with an individual molecular mass of 39kDa. This work describes the cloning and sequencing of the CADH gene from Streptomyces sp. NL15-2K, optimization of a protocol for high-level active CADH expression, and purification of recombinant CADH. A BLAST search and motif analyses of the predicted CADH amino acid sequence indicated the enzyme belongs to the medium-chain zinc-dependent alcohol dehydrogenase group. Cell density at heat-shock treatment, temperatures for heat shock and culture, duration of heat shock, concentration of isopropyl-β-d-thiogalactopyranoside (IPTG) as an inducer, and culture time after induction were adjusted for optimal CADH expression. Expression of active CADH under optimized conditions was approximately 4-fold higher than in the absence of heat shock. CADH purified from recombinant Escherichia coli was in the tetrameric form, as was natural CADH from NL15-2K.

NF-κB signaling pathway is inhibited by heat shock independently of active transcription factor HSF1 and increased levels of inducible heat shock proteins

NF-κB transcription factor regulates numerous genes important for inflammation, immune responses and cell survival. HSF1 is the primary transcription factor activated under stress conditions that is responsible for induction of genes encoding heat shock proteins. Previous studies have shown that the NF-κB activation pathway is blocked by heat shock possibly involving heat shock proteins. Here, we investigate whether active HSF1 inhibited this pathway in the absence of stress conditions. Activation of the NF-κB pathway and expression of NF-κB-dependent genes were analyzed in TNFα-stimulated U-2 OS human osteosarcoma cells that were either heat-shocked or engineered to express a constitutively active form of HSF1 in the absence of heat shock. As expected, heat shock resulted in a general blockade in the degradation of the IκBα inhibitor, nuclear translocation of NF-κB and expression of NF-κB-dependent target genes. In marked contrast, the presence of constitutively active HSF1 did not block TNFα-induced activation of the NF-κB pathway or expression of a set of the NF-κB-dependent genes. We conclude that in the absence of heat shock, the NF-κB activation pathway is inhibited by neither active HSF1 transcription factor nor by increased levels of HSF1-induced heat shock proteins.

Evidence for the possible involvement of calmodulin in regulation of steady state levels of Hsp90 family members (Hsp87 and Hsp85) in response to heat shock in sorghum

Pharmacological studies, using Ca2+ channel blockers (LaCl3 and verapamil) and calmodulin (CaM) antagonists (CPZ and W7), were carried out to understand the role of Ca2+/CaM in the regulation of heat shock-induced expression of Hsp90 (Hsp87 and Hsp85) and Hsp70 (Hsp75 and Hsp73) members in sorghum. It was observed that the expression of both Hsp87 and Hsp85 proteins was decreased in presence of Ca2+ channel blockers and CaM antagonists, under both control and heat stress conditions, as contrary to the steady state levels of Hsp75 and Hsp73, which were not affected significantly under similar conditions. Further, the exposure of sorghum seedlings to geldanamycin, a specific inhibitor of Hsp90, resulted in induction of Hsp87 and Hsp85 in the absence of heat shock also. This study provides the first evidence suggesting that in plants, the in vivo expression of Hsp90 (Hsp87 and Hsp85) is likely to be modulated by Ca2+/CaM under normal and thermal stress conditions. The likely implications of these findings are discussed.

Prostaglandin E2 potentiates heat shock-induced heat shock protein 72 expression in A549 cells

The heat shock (HS) response is an important cytoprotective response comprising the expression of heat shock proteins (HSPs) and orchestrated by the heat/stress-induced transcription factor, heat shock factor-1 (HSF-1). Previous studies suggest that the activation threshold and magnitude of the HS response may be modified by treatment with arachidonic acid (AA). We analyzed the effect of exogenous AA and its metabolites, PGE 2, LTD 4, and 15-HETE on HSF-1-dependent gene expression in A549 human respiratory epithelial-like cells. When added at 1 μM, PGE 2 much more than LTD 4, but not 15-HETE increased activity of a synthetic HSF-1-dependent reporter after HS exposure (42 °C for 2 h), but had no effect in the absence of HS. Exposing A549 cells to HS stimulated the release of PGE 2 and treatment with the cyclooxygenase inhibitor, ibuprofen, reduced HS-induced HSF-1-dependent transcription. PGE 2 increased HS-induced HSP72 mRNA and protein expression but EMSA and Western blot analysis failed to show an effect on HSF-1 DNA binding activity or post-translational modification. In summary, we showed that HS stimulates the generation of PGE 2, which augments the generation of HSPs. The clinical consequences of this pathway have yet to be determined.

Role of the heat shock transcription factor, Hsf1, in a major fungal pathogen that is obligately associated with warm-blooded animals.

All organisms have evolved mechanisms that protect them against environmental stress. The major fungal pathogen of humans, Candida albicans, has evolved robust stress responses that protect it against human immune defences and promote its pathogenicity. However, C. albicans is unlikely to be exposed to heat shock as it is obligatorily associated with warm-blooded animals. Therefore, we examined the role of the heat shock transcription factor (Hsf1) in this pathogen. We show that C. albicans expresses an evolutionarily conserved Hsf1 (orf19.4775) that is phosphorylated in response to heat shock, induces transcription via the heat shock element (HSE), contributes to the global transcriptional response to heat shock, and is essential for viability. Why has Hsf1 been conserved in this obligate animal saprophyte? We reasoned that Hsf1 might contribute to medically relevant stress responses. However, this is not the case, as an Hsf1-specific HSE-lacZ reporter is not activated by oxidative, osmotic, weak acid or pH stress. Rather, Hsf1 is required for the expression of essential chaperones in the absence of heat shock (e.g. Hsp104, Hsp90, Hsp70). Furthermore, Hsf1 regulates the expression of HSE-containing genes in response to growth temperature in C. albicans. Therefore, the main role of Hsf1 in this pathogen might be the homeostatic modulation of chaperone levels in response to growth temperature, rather than the activation of acute responses to sudden thermal transitions.

Cytosolic heat shock protein 90 regulates heat shock transcription factor in Arabidopsis thaliana

Plant survival requires the ability to acclimate to heat, which is involves the expression of heat-inducible genes. We found cytosolic heat shock protein (HSP) 90 serves as a negative regulator of heat shock transcription factor (HSF), which is responsible for the induction of heat-inducible genes in plant. Transient inhibition of HSP90 induces heat-inducible genes and heat acclimation in Arabidopsis thaliana seedlings. Most of upregulated genes by heat shock and HSP90 inhibitor treatments carry heat shock response element (HSE) in their promoter, which suggests that HSF participates in the response to HSP90 inhibition. A. thaliana HSP90.2 interacts with AtHsfA1d, which is one of the constitutively expressed HSFs in A. thaliana. Heat shock depleted cytosolic HSP90 activity, as shown by the activity of exogenously expressed glucocorticoid receptor (GR), which is a substrate of cytosolic HSP90. Thus, it appears that in the absence of heat shock, cytosolic HSP90 negatively regulates HsfA1. Upon heat shock, cytosolic HSP90 is transiently inactivated, and this may lead to the activation of HsfA1.

Cytosolic HSP90 Regulates the Heat Shock Response That Is Responsible for Heat Acclimation in Arabidopsis thaliana

Plant survival requires the ability to acclimate to heat. When plants are subjected to heat shock, the expression of various genes is induced, and the plants become tolerant of higher temperatures. We found that transient treatment with geldanamycin and radicicol, two heat shock protein 90 (HSP90) inhibitors, induced heat-inducible genes and heat acclimation in Arabidopsis thaliana seedlings. Heat shock reduced the activity of exogenously expressed glucocorticoid receptor (GR). Since GR activity depends on HSP90, this suggests that heat shock reduces cytosolic HSP90 activity in vivo. Microarray analysis revealed that many of the genes that are up-regulated by both heat shock and HSP90 inhibitors are involved in protein folding and degradation, suggesting that the activation of a protein maintenance system is a crucial part of this response. Most of these genes have heat shock response element-like motifs in their promoters, which suggests that heat shock transcription factor (HSF) is involved in the response to HSP90 inhibition. Several HSF genes are expressed constitutively in A. thaliana, including AtHsfA1d. Recombinant AtHsfA1d protein recognizes the heat shock response element motif and interacts with A. thaliana cytosolic HSP90, HSP90.2. Overexpression of a dominant negative form of HSP90.2 induced the heat-inducible gene. Thus, it appears that in the absence of heat shock, cytosolic HSP90 negatively regulates heat-inducible genes by actively suppressing HSF function. Upon heat shock, cytosolic HSP90 is transiently inactivated, which may lead to HSF activation.

Circadian Transcription Depends on Limiting Amounts of the Transcription Co-activator nejire/CBP

The circadian clock orchestrates physiological and behavioral activities, including metabolism, neuronal activity, and cell proliferation in synchrony with the environmental cycle of day and night. Here we show that the Drosophila ortholog of the CBP/p300 family of transcription co-activators, nejire (nej), is an intrinsic component of the circadian clock that performs regulatory functions for circadian controlled transcription. Screening of overexpression mutants revealed that gain of nej function was associated with a loss of behavioral and molecular rhythms. Overexpression of NEJ suppresses the long period phenotype of a mutation in the clock gene period (per). NEJ physically interacts through two binding sites with CLOCK and the CLOCK. CYCLE (CLK.CYC) complex. Induction of CLK.CYC-dependent transcripts upon induction of nej expression from a heat-shock promoter showed that NEJ is limiting. Reduced CLK.CYC-mediated transcription in a nej hypomorphic mutant indicates an essential function of NEJ/CBP for CLK.CYC activity and a regulation of circadian transcription by availability of the co-activator. Competition for recruitment of NEJ/CBP provides a potential mechanism for cross-talk between circadian transcription and other CBP-dependent physiological processes.

The Gα12-RGS RhoGEF-RhoA signalling pathway regulates neurotransmitter release in C. elegans

In Caenorhabditis elegans adults, the single Rho GTPase orthologue, RHO-1, stimulates neurotransmitter release at synapses. We show that one of the pathways acting upstream of RHO-1 in acetylcholine (ACh)-releasing motor neurons depends on Galpha12 (GPA-12), which acts via the single C. elegans RGS RhoGEF (RHGF-1). Activated GPA-12 has the same effect as activated RHO-1, inducing the accumulation of diacylglycerol and the neuromodulator UNC-13 at release sites, and increased ACh release. We showed previously that RHO-1 stimulates ACh release by two separate pathways-one that requires UNC-13 and a second that does not. We show here that a non-DAG-binding-UNC-13 mutant that partially blocks increased ACh release by activated RHO-1 completely blocks increased ACh release by activated GPA-12. Thus, the upstream GPA-12/RHGF-1 pathway stimulates only a subset of RHO-1 downstream effectors, suggesting that either the RHO-1 effectors require different levels of activated RHO-1 for activation or there are two distinct pools of RHO-1 within C. elegans neurons.

Inducible hkRASG12D Expression in Zebrafish Hematopoietic Cells Leads to Myeloproliferative Disease.

Activating mutations in RAS family members are common in myeloproliferative disease (MPD) and acute myeloid leukemia (AML). Because the zebrafish has proven to be an excellent leukemia model and can be used in forward genetic and chemical screens to identify modulators of disease pathways, we developed a transgenic zebrafish model of RAS-induced myeloproliferative disease. Stable transgenic zebrafish lines were created in which the ubiquitous B-actin promoter drives expression of a loxed GFP transgene. Upon CRE-mediated recombination, the loxed GFP cassette is excised and the human kRASG12D transgene is expressed. When mated to hsp70-CRE transgenic zebrafish that express CRE recombinase when the animals are heated to 37C for 1 hour, a cohort of 300 double transgenic embryos was generated. In the heat-shocked group, the medial survival time was 25 days, suggesting that oncogenic RAS expression is lethal in developing zebrafish larvae. Of those animals that survived past this time window, most developed sarcomatous muscle tumors. In contrast, most of the double transgenic zebrafish that did not receive heatshock survived to adulthood. Because the hsp70 promoter is active in development and can be induced by stress, the hsp70-CRE transgenic zebrafish line exhibits low levels of CRE expression even in the absence of heatshock. In the non-heat shocked cohort, 10 of 120 double transgenic zebrafish developed MPD by 66 days of life. Histological examination and fluorescence cytometry analysis revealed an expansion of myeloid cell populations within the kidney, comprising granulocytic and monocytic cells in various stages of differentiation, mimicking myeloproliferative diseases seen in both human and mouse. To specifically induce kRASG12D expression in hematopoietic cells, kidneys were dissected from healthy double transgenic animals; heat shocked ex vivo, and then transplanted into sub-lethally irradiated recipient fish. Upon analysis of transplant animals at three months of age, flow cytometry confirmed that MPD had developed in the recipient fish, however other tumor subtypes were not observed. When kidney marrow from transplant animals having MPD were introduced into sub-lethally irradiated secondary recipients, transplant efficiency was greatly reduced. Taken together these results suggest that, as is seen in MPD in mammals, myeloid cells are not fully transformed in our model. In summary, we show that inducible hkRASG12D expression in zebrafish hematopoietic cells leads to myeloproliferative disease and suggest that this model will allow for the identification of novel pathways responsible for full transformation leading to AML.

Overexpression of CaHSP26 in transgenic tobacco alleviates photoinhibition of PSII and PSI during chilling stress under low irradiance

A sweet pepper cDNA clone, CaHSP26 encoding the chloroplast (CP)-localized small heat shock protein (sHSP), was isolated and characterized with regard to its sequence, response to various temperatures and function in transgenic tobacco plants. The deduced amino acid sequence contained three highly conserved regions, showing high identities with other plant sHSPs. Expression of the CaHSP26 gene showed that the mRNA accumulation of CaHSP26 was induced by heat stress. Higher transcript levels were observed when sweet pepper leaves were treated at 42 degrees C for 3h. However, the expression of the CaHSP26 gene was not induced by chilling stress (4 degrees C) in the absence of heat shock (HS). But the transcripts were still detected at 48h at 4 degrees C after HS while not at 25 degrees C. The photochemical efficiency of PSII (Fv/Fm) and the oxidizable P700 in transgenic tobacco overexpressing CaHSP26 were higher than that in wild type tobacco during chilling stress under low irradiance. These results suggest that CP sHSP protein plays an important role in protection of PSII and PSI during chilling stress under low irradiance.

Heat Shock-Independent Induction of Multidrug Resistance by Heat Shock Factor 1

The screening of two different retroviral cDNA expression libraries to select genes that confer constitutive doxorubicin resistance has in both cases resulted in the isolation of the heat shock factor 1 (HSF1) transcription factor. We show that HSF1 induces a multidrug resistance phenotype that occurs in the absence of heat shock or cellular stress and is mediated at least in part through the constitutive activation of the multidrug resistance gene 1 (MDR-1). This drug resistance phenotype does not correlate with an increased expression of heat shock-responsive genes (heat shock protein genes, or HSPs). In addition, HSF1 mutants lacking HSP gene activation are also capable of conferring multidrug resistance, and only hypophosphorylated HSF1 complexes accumulate in transduced cells. Our results indicate that HSF1 can activate MDR-1 expression in a stress-independent manner that differs from the canonical heat shock-activated mechanism involved in HSP induction. We further provide evidence that the induction of MDR-1 expression occurs at a posttranscriptional level, revealing a novel undocumented role for hypophosphorylated HSF1 in posttranscriptional gene regulation.

Inhibition of Hsp90 function delays and impairs recovery from heat shock

The induction of the heat shock response as well as its termination is autoregulated by heat shock protein activities. In this study we have investigated whether Hsp90 functional protein levels influence the characteristics and duration of the heat shock response. Treatment of cells with several benzoquinone ansamycin inhibitors of Hsp90 (geldanamycin, herbimycin A) activated a heat shock response in the absence of heat shock, as reported previously. Pretreatment of cells with the Hsp90 inhibitors significantly delayed the rate of restoration of normal protein synthesis following a brief heat shock. Concurrently, the rate of Hsp synthesis and accumulation was substantially increased and prolonged. The cessation of heat shock protein synthesis did not occur until the levels of Hsp70 were substantially elevated relative to its standard threshold for autoregulation. The elevated levels of HSPS 22-28 (the small HSPS) and Hsp70 are not able to promote thermotolerance when Hsp90 activity is repressed by ansamycins; rather a suppression of thermotolerance is observed. These results suggest that a multicomponent protein chaperone complex involving both Hsp90 and Hsp70 signals the cessation of heat shock protein synthesis, the restoration of normal translation, and likely the establishment of thermotolerance. Impaired function of either component is sufficient to alter the heat shock response.

Ectoine from halophilic microorganisms induces the expression of hsp70 and hsp70B′ in human keratinocytes modulating the proinflammatory response

The heat shock proteins (Hsps) have an important role in the cytoprotection and repair of cells and tissues. One potential mechanism of protection is the ability of Hsp to inhibit genetic expression of proinflammatory cytokines, the transcription of which is dependent on nuclear factor-kappa B (NF-kappaB) activation. In this study, we evaluated the ability of ectoine, a novel natural biomolecule produced by halophilic microorganisms, to activate the hsp70 and hsp70B'. By reverse transcriptase-polymerase chain reaction and Western blot analysis, we demonstrated increased hsp70B' gene expression in human keratinocytes treated with ectoine and heat stressed. In contrast, in the absence of heat shock, ectoine was unable to induce hsp70B' but had the ability to induce another member of the Hsp family, the hsp70. The latter is not only elevated in response to stress but is also present at basal level in unstressed cells. In addition, ectoine had no effect on proinflammatory cytokines interleukin (IL)-1alpha, IL-6, IL-8, and tumor necrosis factor-alpha and on NF-kappaB and IkappaB-alpha pathway, whereas it downregulated the expression of cited proinflammatory cytokines, in lipopolysaccharides-treated keratinocytes. These results highlighted the ability of ectoine to protect cells from stress conditions and to prevent cell damage by maintaining an elevated level of the Hsp70. Overall, these data might suggest the use of this compatible solute in cosmetic and even pharmaceutical preparations aiming to activate a cytoprotective heat shock response in human cells.

Sphingosine 1-phosphate protects bovine oocytes from heat shock during maturation.

Sphingosine 1-phosphate (S1P) is a sphingolipid metabolite that can block apoptosis by counteracting the proapoptotic effects of ceramide. Experiments were performed to evaluate whether S1P blocks the disruption in oocyte developmental competence caused by heat shock. Cumulus-oocyte complexes (COCs) were placed in maturation medium and cultured at 38.5 or 41 degrees C for the first 12 h of maturation. Incubation during the last 10 h of maturation, fertilization, and embryonic development were performed at 38.5 degrees C. Heat shock during the first 12 h of maturation reduced cleavage rate, the number of oocytes developing to the blastocyst stage, and the percentage of cleaved embryo that subsequently developed to blastocysts. Addition of 50 nM S1P to maturation medium had no effect on oocytes matured at 38.5 degrees C but blocked effects of thermal stress on cleavage and subsequent development. The blastocysts formed at Day 8 did not differ between S1P and control groups in caspase activity, total cell number, or percentage of cells that were apoptotic. Blocking endogenous generation of S1P by addition of 50 nM N1N-dimethylsphingosine, a sphingosine kinase inhibitor, reduced or tended to reduce cleavage rate and blastocyst development regardless of whether maturation of COCs was at 38.5 or 41 degrees C. Results demonstrate that S1P protects oocytes from a physiologically relevant heat shock and affects oocyte maturation even in the absence of heat shock. The S1P-treated oocytes that survived heat shock and became blastocysts had a normal developmental potential as determined by caspase activity, total cell number, and percentage of apoptotic cells. Thus, modulation of developmental competence of oocytes using S1P may be a useful approach for enhancing fertility in situations where developmental competence of oocytes is compromised.