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Are Hsp70 proteins only activated in response to heat shock?

Are Hsp70 proteins only activated in response to heat shock?


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Hsp70 proteins are chaperones that assist in protein folding in my plant physiology textbook it says the Hsp70 proteins were discovered by inducing heat shock. But do they only work in response to heat shock stress?

I know these types of proteins are found in many organisms but I am interested in how they assist on protein folding or stress response in plants specifically.


Frontiers in Physiology

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    Role of heat shock proteins in gastric mucosal protection

    Heat shock proteins (HSP) are crucial for the maintenance of cellular homeostasis during normal cell growth and for survival during and after various cellular stresses. Gastric surface mucous cells are the first line of defence against insults derived from ingested foods and Helicobacter pylori infection. Primary cultures of gastric surface mucous cells from guinea-pig fundic glands exhibited a typical heat shock response after exposure to elevated temperature or metabolic insults, such as ethanol and hydrogen peroxide, and they were able to acquire resistance to these stressors. Restraint and water immersion stress rapidly activated heat shock factor 1 (HSF1) in rat gastric mucosa within 15 min and induced HSP70 mRNA expression and its protein accumulation. The extent of the induction inversely correlated with the severity of mucosal lesions, suggesting an important role of HSP70 in gastric mucosal defence. This heat shock response appeared to be mediated by the α1A-adrenoceptor. The HSP70 family functions as a molecular chaperone and reduces stress-induced denaturation and aggregation of intracellular proteins. In addition to its chaperoning activities, HSP70 has been suggested to exert its cytoprotective action by protecting mitochondria and by interfering with the stress-induced apoptotic programme. Recently, we introduced geranylgeranylacetone as a non-toxic HSP70 inducer. This compound weakly stimulated HSP70 induction in cultured gastric mucosal cells and gastric mucosa by directly activating HSF1 and markedly augmented HSP70 induction in response to subsequent exposure to stress. Thus, non-toxic HSP70 inducers may have a potential benefit for the prevention and treatment of stress ulcer.


    Chemistry

    Synthesis of O-alkylated quercetin derivatives

    To determine the importance of individual hydroxyl groups on the inhibition of heat-induced HSP70 expression and other biological activities, we synthesized the mono-methylated quercetin derivatives D2, D3, D4, D6, and D7 according to previously reported procedures. 45 Because we ultimately intend to attach affinity tags such as biotin to quercetin to help in identifying the protein target(s) of quercetin activity, we also synthesized a number of carboxymethylated derivatives by the same synthetic route ( Scheme 1 ). Thus, quercetin was benzylated to give 20% of the tribenzyl derivative 1 and 60% of the tetrabenzyl derivative 2. The tribenzyl derivative was selectively carbomethoxymethylated at the 3′-OH to give 4, whereas the tetrabenzyl derivative was carbomethoxymethylated at the 5-OH to give 3. The products were debenzylated with palladium hydroxide and hydrogen to afford the methyl esters D8 and D1 respectively.

    To carbomethoxymethylate positions 3 and 7, we utilized the 3′,4′-diphenylmethylene derivative of quercetin, compound 5 ( Scheme 2 ). This derivative reacted with methylbromoacetate under basic conditions at the 3-OH to give 6, which then afforded the carboxymethyl derivative D5 following removal of the diphenylmethylene group by refluxing with acetic acid, which also hydrolyzed the ester. To obtain the O7 derivative, the 3-OH of compound 5 was first benzylated to give 7 which was then carbomethoxymethylated at the 7 position. Removal of the benzyl and diphenylmethylene groups was carried out in two steps by hydrogenolysis with palladium hydroxide followed by hydrolysis with acetic acid and water which also unexpectedly hydrolyzed the ester to the carboxymethyl product D9.

    Many attempts to methylate D9 to produce D10 were unsuccessful and so other synthetic routes were investigated. The best of these involved a previously reported method for selectively methylating or benzylating the 7-OH of quercetin by treatment of quercetin pentaacetate with the alkylating agent in the presence of potassium carbonate and potassium iodide in acetone. 46 Thus treatment of quercetin pentaacetate 47 under these conditions with methyl bromoacetate yielded compound 10 which could be deacetylated in high yield under neutral conditions with N-methyl-2-dimethylaminoacetohydroxamic acid 48 to give D10 ( Scheme 3 ). This deacetylation method was chosen as more basic conditions, such as sodium methoxide and methanol at 0 ଌ, tend to result in air oxidation of the product, 49 and more acidic conditions lead to hydrolysis of the methyl ester.

    Structural characterization

    The sites of alkylation in all the compounds were confirmed by analysis of the coupling patterns in the 1D 1 H NMR spectra together with correlations in the COSY, HMQC and HMBC spectra. As an illustration, the B ring hydrogens H5′ and H6′ of D1 ( Figure 3 ) and D10 ( Figure 4 ) were assigned by the large ortho coupling, and the strong crosspeak in the COSY spectrum (crosspeak a, panel A). These assignments led to the assignment of H2′ through meta coupling with H6′ that could be detected in the 1D 1 H NMR spectrum. The carbons directly attached to these protons could then be assigned through the HMQC spectra (Panel B). The hydrogen signal at 10 ppm in D1 could then be assigned to the 4′-OH by the presence of a long range correlation to C5′ at 116 ppm ( Figure 3 , crosspeak e) and to a quaternary carbon signal at 146 ppm (crosspeak b) together with the absence of a correlation to C2′ in the HMBC spectrum (panel C). The signal at 146 ppm could therefore be assigned to C3′ which in turn showed a correlation to the α hydrogen of the carbomethoxymethylene group (crosspeak d, panel C). The position of the carbomethoxymethylene group in D10 could be assigned to O7 via a correlation between the α-methylene protons and a quaternary carbon signal at 162 ppm ( Figure 4 , crosspeak d in panel C). The signal at 162 ppm could be assigned to C7 via multiple bond correlations to the two meta coupled protons in the A ring in the HMBC spectrum (crosspeaks b & c in panel C).

    Structure assignment of D1. (A) COSY, (B) HMQC and (C) HMBC of D1 in DMSO-d6

    Structure assignment of D10. (A) COSY, (B) HMQC and (C) HMBC of D10 in DMSO-d6.


    FACTORS AND CONDITIONS THAT MODULATE HSP70 EXPRESSION

    Some stimuli and conditions that modulate HSP70 expression.

    It is well documented that multiple stimuli can induce the in vivo accumulation of HSP70 proteins. As pointed out in Fig. 1, these stimuli include hyperthermia (28, 50, 51, 92), ischemia-reperfusion (15, 69), hypoxia (22,30, 44), energy depletion (88), acidosis (104), and ROS formation (103). Because these stimuli are similar to the integrated metabolic changes associated with exercise, it is not surprising that exercise has been demonstrated to induce HSPs.

    In one of the initial animal experiments to address the issue of HSP induction with exercise, Locke et al. (67) observed that a single bout of exhaustive treadmill running in rats could increase Hsp70 synthesis in skeletal muscle, lymphocytes, and spleen. Subsequent studies by numerous investigators have confirmed that acute exercise produces increased Hsp70 levels in contracting skeletal muscle as well as critical organs such as the heart, kidney, and liver (50, 66,68, 85, 86, 92). It is difficult to delineate which of the many stimuli generated during acute exercise are contributing, and to what degree, to the observed increases in cellular Hsp70 accumulation, in part because exercise produces elevations in both Tc and tissue (e.g., contracting skeletal muscle) temperature concurrent with other physiological stimuli.

    To investigate whether exercise could increase HSP70 expression independent of changes in Tc, Skidmore et al. (92) designed experiments in which rats were exercised in a cool environment to prevent Tc from increasing above resting levels. HSP70 accumulation was increased in hindlimb locomotor muscles and cardiac tissue after an exercise bout, suggesting that factors other than heat stress may contribute to the accumulation of HSP70 during acute exercise. Although it is likely that the local temperatures of locomotor muscles such as the gastrocnemius and soleus were elevated during treadmill running, data from previous studies indicate that the temperature of contracting skeletal muscles in rats is comparable to Tc at the termination of moderate intensity treadmill exercise (10). Therefore, it is reasonable to assume that stimuli other than increased temperature also contribute to the augmented skeletal and cardiac muscle HSP70 levels that accompany exercise.

    Human studies evaluating Hsp70 responses to exercise are more infrequent and less insightful, likely due in part to the difficulties in sample acquisition and the challenging conditions for most experimental designs. These studies have focused primarily on stress protein expression in either skeletal muscle or circulating leukocytes. Two separate studies have demonstrated that a single bout of exercise in untrained subjects can elicit increased Hsp70 mRNA concentrations in vastus lateralis muscle (26, 80). Interestingly, Hsp70 protein levels were assessed 3 h after exercise in one of these studies and remained unchanged from control levels (80). In contrast, Liu et al. (65) found that expression of Hsp70 was increased in human vastus lateralis muscle after 1–4 wk of rowing training.

    There are a few additional studies that have evaluated the Hsp70 response to exercise in peripheral blood leukocytes. Ryan et al. (84) found only small increases in Hsp70 expression in blood obtained from young men who performed 2 h of treadmill exercise in warm conditions, whereas Fehrenbach et al. (27) determined that heavy-intensity endurance exercise was associated with an increase in Hsp70 expression in leukocytes. A unique aspect of this study was the observation that the exercise-induced Hsp70 response was blunted in subjects who were aerobically trained compared with untrained controls. In a study with contrasting results (16), exercise and estrogen replacement in young women was found to have no effect on leukocyte Hsp70 levels several hours after a moderate-intensity exercise bout.

    Overall, the limited number of studies available in humans does not permit a definitive conclusion to be drawn concerning the impact of exercise on Hsp70 responses. The differences in intensity and type of exercise protocol utilized, along with issues ranging from gender to training status, have likely contributed to the disparate findings in this area. Further research is necessary to address issues related to Hsp70 regulation in humans and to determine whether this regulation is consistent with results obtained in animals and in vitro cell systems.

    Aging and altered Hsp70 expression.

    As noted above, cells, tissues, and whole organisms have the ability to become resistant to stressors such as hyperthermia after a prior sublethal heat exposure (i.e., acquired thermotolerance), and HSPs appear to play a critical role in this process. One important and clinically relevant scenario in which tolerance to thermal stress is reduced is old age. In humans, the aging process is associated with elevated morbidity and mortality rates due to stressors such as heat exposure. For instance, age-related decrements in the stress response are thought to be linked to the increased incidence of death that has been reported for older individuals subjected to prolonged heat waves (39, 89). In our laboratory, we have noted that older animals are less thermotolerant and have higher mortality rates than their younger counterparts when presented with repeated heat challenges (37).

    There are several potential mechanisms that likely contribute to reduced stress tolerance with aging, including alterations in HSP70 accumulation and function. Investigators have been examining the stress protein response in aged organisms to determine whether a deficit in HSP70 is a potential explanation for the reduced thermotolerance in older populations (9, 25, 36, 64). In vitro studies have demonstrated that aged human (64) and rat (25) fibroblasts respond to heating with lower HSP70 mRNA and total protein levels. In vivo data, primarily from tissues extracted from intact animals (8, 37, 51, 77), suggest that the expression of HSP70 and its potential protective role in cellular stress may decline with advancing age. Moreover, studies taking advantage of cDNA expression array technology determined that aging results in altered gene expression in response to heat stress that is indicative of decreased stress protein expression (110).

    A recent study by Hall et al. (37) addressed this issue in more detail by investigating whether a reduction in cellular ability to mount an appropriate stress protein response in senescent animals following consecutive heating trials is associated with a decline in thermotolerance. Young and old Fischer 344 rats were heat stressed (Tc of 41°C) twice, separated by 24 h. Liver samples were obtained at several time points in the subsequent 48-h recovery period, and representative samples were then evaluated for Hsp70 expression. Several pieces of evidence from these studies suggested that there is a functional link between age-related decrements in Hsp70 expression and pathophysiological responses to heat stress. First, immunoblot and immunohistochemistry results demonstrated that the magnitude of the Hsp70 response was markedly reduced with age. In young animals, a robust Hsp70 response was present in the vicinity of the central vein, which is functionally associated with reduced blood flow and lower oxygen tensions. In contrast, senescent animals had relatively low Hsp70 expression in this region (Fig.2). Second, older animals showed extensive liver injury in the central vein region that corresponded to the diminished Hsp70 response in these regions and a reduced ability to survive consecutive heat stresses. Conversely, liver injury was markedly lower in the young cohort. Finally, a comparison of the stress-induced patterns of Hsp70 expression showed distinct differences in the two age groups. Young rats responded to heat stress with a strong pattern of nuclear and cytoplasmic Hsp70 expression that was maintained for 48 h in hepatocytes located in central vein regions (Fig. 2). In aged animals, nuclear and cytoplasmic Hsp70 expression was both delayed and reduced, although there was a transient increase in Hsp70 expression at 2 h of recovery that subsequently diminished at later time points. These results suggest that the blunted stress protein response in older animals may have functional consequences that correlate with the increased cellular injury and reduced stress tolerance that is associated with advancing age.

    Fig. 2.Cellular localization and zonal distribution of immunoreactive Hsp70 protein in the liver of young and old rats after heat stress (Ref. 37). Liver biopsies were collected from young (left) and old (right) Fischer 334 rats 12 h after a heat stress protocol, and sections were stained with a monoclonal antibody specific for Hsp70. There was no evidence of immunoreactive Hsp70 in hepatocytes from young or old euthermic control animals (not shown). However, at 12 h of recovery from heat stress, strong cytoplasmic (solid arrows) and nuclear (open arrows) staining for anti-Hsp70 was observed in hepatocytes surrounding the central vein region in young rats (A). Conversely, in old rats (B), only weak cytoplasmic expression (solid arrows) was observed in hepatocytes in proximity to the central vein. Magnification = ×40.


    Genomic approach - hsp genes and amino acid sequence of B. mori

    The HSP family consists of ubiquitous proteins, which are phylogenetically conserved from bacteria to mammals and plants ( Craig 1985). They have been divided into sub-families such as HSP110, HSP100, HSP90, HSP70, HSP60, HSP40, and HSP20 on the basis of their molecular weights ( Nover and Scharf 1997 Gething 1998). Although, expression of HSPs has been reported from different silkworm strains ( Table 1), only a few have been characterized in B. mori. Recently, Landais et al. (2001) characterized a cDNA encoding a 90 kDa HSP in B. mori and compared it with Spodoptera frugiperda (both lepidopteran insects). These two cDNAs encode 716 aa (amino acid) and 717 aa proteins in B. mori and S. frugiperda, respectively, with calculated molecular mass of 83 kDa which is similar to Drosophila.

    Protein profile derived from the fifth instar Bombyx mori larvae of CSR2 strain, heat shocked (HT) at 40° C and untreated control (C). Arrows indicate expression of 84, 60, 62, 47, 42, and 33 kDa heat shock proteins. M indicates molecular weight marker. High quality figures are available online.

    Protein profile derived from the fifth instar Bombyx mori larvae of CSR2 strain, heat shocked (HT) at 40° C and untreated control (C). Arrows indicate expression of 84, 60, 62, 47, 42, and 33 kDa heat shock proteins. M indicates molecular weight marker. High quality figures are available online.

    Unlike in vertebrates, hsp90 does not contain introns and is a unique gene both in the B. mori and S. frugiperda genomes. Comparison of aa sequences of B. mori and S. frugiperda with that of D.melanogaster,Homo sapiens, and S. cerevisiae revealed a high percentage of similarity and phylogenetic relationships (for details see Landais et al. 2001). Apparently, extensive study is required to determine their expression at different developmental stages of different silkworm strains as the HSP90 expression is found rather in early instars than late instars ( Vasudha et al. 2006) and expression of some hsp genes changes during development ( Craig 1985). In D. melanogaster, hsc70-4 (constitutive hsp gene family) was expressed at a high level in embryos, larvae, and adults, whereas the hsc70-1 and hsc70-2 expression was highest in adults but not detected in larvae. The hsc70-1 was expressed at a low level while no expression of hsc70-2 was observed in the embryo. In Chironomus tentans, hsc70 expression was evident at all developmental stages but slightly lower in the embryo than older stages ( Karouna-Renier et al. 2003).

    Small heat shock proteins (smHSPs or sHSPs) belong to a family of genes that are seemingly less conserved compared with those of major hsp gene families, but occur ubiquitously in a variety of organisms. These proteins are involved in apoptosis as well as protection against heat stress ( Arrigo 2005 Feder and Hofmann 1999). In B. mori (strain p50) six genes encoding sHSP19.9, sHSP20.1, sHSP20.4, sHSP20.8, sHSP21.4, and sHSP23.7 were reported ( Sakano et al. 2006) although their biological and commercial roles remain unknown. The deduced amino acid residues of these sHSPs ( Table 2) are quite similar to each other. CLUSTALW multiple alignments indicated 82, 80, and 80% identity between Pia25 and sHSP20.8, sHSP20.8, sHSP20.4, sHSP20.4, and sHSP19.9, respectively. Besides the ?-crystallin domain, the N-terminal XXLXDQXFG motifs are commonly conserved in the sequences of these HSPs ( Sakano et al. 2006). Further, reverse transcriptase–polymerase chain reaction (RT-PCR) analysis showed no difference in expression levels of smHSP genes in different organs ( Sakano et al. 2006), but indicated an increased amount of transcripts following heat shock in B. mori strains p50 ( Sakano et al. 2006), Nistari and NB4D2 ( Velu et al. 2008), which was found to be strain specific. BmHSPs (B. mori HSPs) with other organisms was computed using available data in National Center for Biotechnology Information (NCBI) data bank (http://www.ncbi.nlm.nih.gov) and presented in Table 3.

    Bombyx mori heat shock proteins (BmHSPs) accession numbers, protein IDs and their deduced amino acids.

    HSPs . Accession no. . Protein ID no. . Total no. of .
    amino acids .
    Hsp90 AB060275 BAB41209 716
    Hsp70 DQ311189 ABD36134.1 676
    Chaperonin(Hsp60) NM_001079879 NP_001073348 545
    Hsp40 AB206400 BAD90846.1 351
    Hsp23.7 AB195973 BAD74198.1 209
    Hsp21.4 AB195972 BAD74197.1 187
    Hsp20.8 AF315317 AAG30944.1 186
    Hsp20.8A AF315319 AAG30946 186
    Hsp20.4 AF315318 AAG30945.2 181
    Hsp20.1 AB195971 BAD74196.1 178
    Hsp19.9 AB195970 BAD74195.1 177
    Hsp1 DQ443370.1 ABF51459 198
    α-crystallin1 AF309497.1 AAK06407 122
    α-crystallin2 AF309499.1 AAK06409 90
    HSPs . Accession no. . Protein ID no. . Total no. of .
    amino acids .
    Hsp90 AB060275 BAB41209 716
    Hsp70 DQ311189 ABD36134.1 676
    Chaperonin(Hsp60) NM_001079879 NP_001073348 545
    Hsp40 AB206400 BAD90846.1 351
    Hsp23.7 AB195973 BAD74198.1 209
    Hsp21.4 AB195972 BAD74197.1 187
    Hsp20.8 AF315317 AAG30944.1 186
    Hsp20.8A AF315319 AAG30946 186
    Hsp20.4 AF315318 AAG30945.2 181
    Hsp20.1 AB195971 BAD74196.1 178
    Hsp19.9 AB195970 BAD74195.1 177
    Hsp1 DQ443370.1 ABF51459 198
    α-crystallin1 AF309497.1 AAK06407 122
    α-crystallin2 AF309499.1 AAK06409 90

    Bombyx mori heat shock proteins (BmHSPs) accession numbers, protein IDs and their deduced amino acids.

    HSPs . Accession no. . Protein ID no. . Total no. of .
    amino acids .
    Hsp90 AB060275 BAB41209 716
    Hsp70 DQ311189 ABD36134.1 676
    Chaperonin(Hsp60) NM_001079879 NP_001073348 545
    Hsp40 AB206400 BAD90846.1 351
    Hsp23.7 AB195973 BAD74198.1 209
    Hsp21.4 AB195972 BAD74197.1 187
    Hsp20.8 AF315317 AAG30944.1 186
    Hsp20.8A AF315319 AAG30946 186
    Hsp20.4 AF315318 AAG30945.2 181
    Hsp20.1 AB195971 BAD74196.1 178
    Hsp19.9 AB195970 BAD74195.1 177
    Hsp1 DQ443370.1 ABF51459 198
    α-crystallin1 AF309497.1 AAK06407 122
    α-crystallin2 AF309499.1 AAK06409 90
    HSPs . Accession no. . Protein ID no. . Total no. of .
    amino acids .
    Hsp90 AB060275 BAB41209 716
    Hsp70 DQ311189 ABD36134.1 676
    Chaperonin(Hsp60) NM_001079879 NP_001073348 545
    Hsp40 AB206400 BAD90846.1 351
    Hsp23.7 AB195973 BAD74198.1 209
    Hsp21.4 AB195972 BAD74197.1 187
    Hsp20.8 AF315317 AAG30944.1 186
    Hsp20.8A AF315319 AAG30946 186
    Hsp20.4 AF315318 AAG30945.2 181
    Hsp20.1 AB195971 BAD74196.1 178
    Hsp19.9 AB195970 BAD74195.1 177
    Hsp1 DQ443370.1 ABF51459 198
    α-crystallin1 AF309497.1 AAK06407 122
    α-crystallin2 AF309499.1 AAK06409 90

    DISCUSSION

    Since HSF1 is responsible for the stress response in mammalian cells (Morimoto et al., 45 , 46 70 ), levels of this transcription factor may influence the magnitude of hsp70 induction following thermal stress. In the 2-day-old rat, higher levels of HSF1 protein and HSF-HSE-binding activity are present in hyperthermic kidney compared with brain regions, consistent with the magnitude of induction of hsp70 protein after thermal stress in these tissues. In the adult rat, higher levels of

    HSF1 protein and HSF-HSE-binding activity are present in hyperthermic brain compared with kidney, consistent with the relative magnitude of induction of hsp70 protein in these two organs after thermal stress. There are high basal levels of hsp70 protein in kidney of control animals and therefore presumably high basal levels of hsp70 mRNA. Because heat shock markedly increases the stability of hsp70 mRNA ( 64 ), stabilization of the relatively large amount of hsp70 mRNA in the hyperthermic kidney may contribute to the level of hsp70 protein in that tissue. In brain, not all cell types induce hsp70 after a physiologically relevant increase in body temperature (Brown, 9 , 7 40 12 ). For example, oligodendrocytes and some microglia induce hsp70 mRNA after heat shock, whereas large neurons and glial fibrillary acidic protein-positive astrocytes in the forebrain do not ( 62 Foster and Brown, 23 , 24 ). This is despite the facts that abundant levels of HSF1 are present in forebrain neurons and that this HSF1 is localized to the nuclei of these neurons in both control and hyperthermic animals ( 11 ). After hyperthermia, dentate granule cells of the hippocampus induce high levels of hsp70 mRNA but little hsp70 protein, suggesting posttranscriptional regulation of the synthesis of the protein ( 36 ).

    Despite the very low levels of HSF1 in adult kidney, this organ can nevertheless induce a large amount of hsp70 after heat shock. Therefore, why is it necessary for adult brain regions to have such high levels of HSF1 ? Naively, we might question whether HSF1 is entirely responsible for the induction of hsp70 after heat shock. However, Xiao et al., ( 73 ) have shown that hsf1(-/-) mice cannot induce hsp70 after hyperthermia. Levels of hsp90 and hsc70 in adult rat brain are much higher than in kidney ( 19 present study, Fig. 7B), and those proteins may dampen the heat shock response in brain at low stress levels. A high level of HSF1 in the brain may be necessary to lower the threshold for the heat shock response in the presence of large amounts of hsc70 and hsp90. Another possibility is that high levels of neural HSF1 are necessary to repress non-hsp genes. There is evidence that Drosophila HSF binds to dozens of non-hsp genes, perhaps repressing their transcription after heat shock ( 68 ). In heat-shocked human monocytes, activated HSF1 binds to an HSE in the prointerleukin 1β gene and represses transcription of that gene ( 14 ). HSF1 may also inhibit expression of a wide spectrum of other cytokines ( 33 73 ). It may be that HSF1 is involved in the repression of a range of other non-heat shock genes. At all developmental stages, a higher percentage of nonrepeated DNA is transcribed in mammalian brain than in kidney ( 8 ). The complexity of transcription increases in brain from the newborn to the 2-week-old stage before leveling off at the 6-week-old stage ( 8 ). However, the complexity of RNA transcription in kidney appears to decrease with the development of the animal ( 8 ). It is interesting that this pattern matches the developmental expression of HSF1 protein in those tissues. Therefore, an additional function of HSF1 may be to shut off genes following heat shock.

    HSF1 may be performing other functions as well. For example, HSF1 may help to regulate DNA-dependent protein kinase ( 49 ), and it may play a role in cell cycle regulation ( 13 ). HSF1 may influence postnatal rat development in ways that do not involve the induction of heat shock genes. The developmental functions of Drosophila HSF are not mediated through the induction of heat shock genes ( 34 ). As mentioned previously, hsf1(-/-) knockout mice have multiple phenotypic abnormalities, despite the fact that basal hsp expression is not altered appreciably ( 43 73 ). This suggests that HSF1, like Drosophila HSF, might be involved in regulating other important genes or signaling pathways ( 73 ).

    The initial temperature of heat-shocked 2-day-old rats affected the duration of HSF-HSE-binding activity and the hyperphosphorylation of HSF1. Rats that were at 30°C before the 40°C heat shock experienced a longer duration of HSF-HSE-binding activity than those that were initially at 35°C. This result agrees with that of Abravaya et al. ( 1 ), who found that HeLa cells grown at 35°C experienced a greater magnitude and duration of HSF-HSE binding after heat shock than cells grown at 37°C. Also, 2-day-old rats that were initially at 30°C showed apparent stress-induced phosphorylation of HSF1, whereas rats that were initially at 35°C did not.

    HSF2 protein levels decline in brain and kidney during postnatal rat development. The fact that HSF2 levels are higher in the immature rat may suggest a developmental role of HSF2. However, there is some HSF2 in adult tissues, which indicates that HSF2 has a function in the mature animal. HSF2 levels are higher in brain than in kidney. Even though HSF2 levels are high in the 2-day-old rat, there is no constitutive HSF-HSE-binding activity in 2-day-old cerebellum or kidney, suggesting that HSF2 is not involved in gene transcription at this time. Furthermore, a previous study in our laboratory investigated developmental changes in basal levels of hsp90, hsp70, hsc70, and hsp60 in the rat brain and kidney ( 19 ), and these changes do not parallel the declining HSF2 levels observed in the present study. Similarly, Rallu et al. ( 54 ) found no obvious correlation between the expression patterns of the major hsps and that of HSF2 during mouse embryonic development. However, another heat shock factor, HSF4b, which could act as an activator of heat shock genes under normal conditions, has recently been found in mammals ( 63 ).

    As mentioned before, the smaller isoform of HSF2, HSF2-β, may act as a negative regulator of HSF2 activity during hemin-mediated erythroid differentiation of K562 cells ( 37 ). The HSF2-β isoform is present in higher amounts than the HSF2α isoform in the postnatal brain. This may explain the lack of any HSF2 activation in the 2-day-old brain, despite high levels of HSF2 protein. Immunocytochemistry of the rat brain shows that HSF2 is localized to the nuclei of neurons at day 2 and in the cytoplasm at day 30 ( 11 ).

    Recently, it was shown that HSF2 is activated when the ubiquitin-proteasome pathway is inhibited ( 41 ). It is known that many neurodegenerative diseases, as well as normal aging, are characterized by an accumulation of ubiquitinated proteins in neurons and some glia cells ( 2 ). Neurons may be especially sensitive to malfunction of the ubiquitin/ATP-dependent pathway ( 2 ). It is possible that the relatively high amounts of HSF2 protein in brain protect against the accumulation of ubiquitinated proteins. Also, HSF2 may influence rat development via its previously discussed influence on PP2A activity. Creation of an HSF2(-/-)-deficient mouse would clarify the function of HSF2.

    In summary, HSF1 levels increase in brain regions and decline in kidney during postnatal rat development. In both neonatal and adult rats, levels of HSF1 protein in brain and kidney are proportional to the levels of HSF-HSE-binding activity and the magnitude of hsp70 protein induction after thermal stress. There appears to be more HSF1 protein in adult brain needed for stressinduced expression of hsp70, suggesting that HSF1 may have other functions in addition to its role as a stressinducible activator of heat shock genes. HSF2 protein levels decline during postnatal rat development in brain regions and kidney, suggesting a role for HSF2 in development. However, gel mobility shift analysis shows that HSF2 is not in a DNA-binding form in the neonatal brain and kidney, suggesting that HSF2 may not be involved in the constitutive expression of hsps at that time. There is no apparent relationship between levels of HSF2 protein and basal levels of hsp90, hsp70, hsc70, and hsp60.


    Role of HSP in relation to commercial traits

    To date, the greatest emphasis has been given to HSP70 and HSP90 as molecular chaperons that help organisms to cope with stresses of internal and external nature. Recent approaches not only revealed the importance of HSP90 in normal growth and development of eukaryotes, and parasite (Plasmodium falciparum) growth in human erythrocytes ( Banumathy et al. 2003), but also elucidated the relationship between HSPs and life history traits focusing on the ecological and evolutionary relevance ( Sorensen et al. 2003 Sorensen and Loeschcke 2007). Concomitantly, the relationship between heat shock, HSPs expression, and commercial traits was studied in great detail in the case of B. mori ( Vasudha et al. 2006). Notably, an increased cocoon weight of 17.52 vs. 13.48%, and increase in shell weight of 19.44 vs. 13.45% in NB4D2 over its control was observed following heat shock at 35 and 40 o C, respectively. Concurrently, CSR2 also exhibited a 13.11 vs. 6.44% increase in cocoon weight and 16.26 vs. 5.03% increase in shell weight at 35 and 40 o C heat shock over their respective controls. The increased cocoon and shell weight observed in heat shock induced bivoltine silkworm strains compared to controls would be due to expression of HSPs at larval stage. While Joy and Gopinathan (1995) did not observe any heat shock effects on commercial traits, Lohmann and Riddiford (1992) reported that of the nine animals heat shocked at 44° C for 1 h, only 5 resumed feeding, while 3 spun cocoons. Commercial traits of these animals were not evaluated and compared with that of controls. Consequently, as a novel strategy, heat shocked larvae (whole organisms) were allowed to grow under natural environmental conditions and they spun better quality cocoons than the non heat shocked larvae reared in natural environmental conditions ( Vasudha et al. 2006). These investigations highlighted the fact that knowledge obtained from model organisms under normal laboratory conditions does not always reflect what happens out in the field, where conditions are continuously changing and unpredictably hostile. Interestingly, the increased cocoon weight and shell weight over control, reflects the positive correlation between heat shock responses and silk protein content in the cocoon. Abramova et al. (1991) reported suppression of fibroin synthesis in the silk gland following heat shock, but recently Zhang et al. (2006) identified HSP90, HSP70, and HSP60 in the silk glands of B. mori, offering the opportunity for further systematic investigation in different breeds of silkworm. None of the larvae recovered from heat shock at 45° C ( Vasudha et al. 2006) and 46° C ( Lohmann and Riddiford 1992), were able to spin cocoons. However, the observed differences between cocoon weight, shell weight, and shell ratio among various silkworm strains will require further investigations to determine species-specific responses to heat shock. Altogether, these observations clearly indicate that mild heat shock between 35 and 40° C for 2 h facilitates bivoltine silkworm larvae to respond and overcome the fluctuating natural environmental conditions in succeeding instars. The practical application of this phenomenon will need to be explored positively and systematically (using multivoltine and bivoltine silkworm strains) in laboratory and field conditions in order to achieve stabilized sericulture farming in tropical countries like India.


    Results

    Identification of conserved Hsp and cis-regulatory HSEs

    We recovered conserved Hsps from all of the major gene families (Hsp90, Hsp70, Hsp60, Hsp40, small Hsps Table 1). Three paralogues within the Hsp90 gene family (trap1, gp93, and hsp83) were found across all surveyed insects. We recovered five of the six Drosophila melanogaster Hsp70 homologues (CG2918, hsc70-3 (BIP), hsc70-4, hsc70-5, and hsp70CB Table 1) for Hymenoptera. With the exception of Nasonia vitripennis, the Hymenopteran taxa all lacked the heat-inducible orthologue hsp70 (Table 1). For all species, we recovered two paralogues of Hsp60 (Table 1). Hsp40 gene families are one of the most diverse Hsps, but we narrowed our search to DnaJ-1, which is the known heat-inducible paralogue of D. melanogaster (Table 1). We did not recover a DnaJ-1 paralogue from any of the insects surveyed and found the best BLAST match to be D. melanogaster CG5001 (Table 1). Forward BLAST searching for D. melanogaster sHsps (hsp22, hsp23, hsp26, hsp27) yielded no reciprocal BLAST hits instead, the closest match was lethal 2 essential for life (l(2)efl), for which there were 3–9 copies in the Hymenoptera, and 1–17 copies in other members of the outgroup (Table 1).

    Of the Hsp homologues, eight were quantifiable by qPCR and were subsequently searched for cis-regulatory HSEs (Table 1, indicated with asterisks). Local alignment of the promoter regions of hsp83, hsc70-4 (h1 and h2), and hsp40 across species indicated conserved location, conformation, and arrangement of cis-regulatory HSEs (Figs. 1, 2 and 3), whereas hsc70-3 (BIP), hsc70-5, hsp60, and l(2)efl had less conserved HSEs (Additional file 1: Figures S1, Additional file 2: Figures S2, Additional file 3: Figures S3 data not shown for l(2)efl). For Hsps with conserved HSEs, 193 HSE motifs were annotated, including 114 head types (‘nGAAn’) and 79 tail types (‘nTTCn’ Figs. 1, 2 and 3). Across all sampled insects, we found no consistent preference for head or tail motifs in hsp83 (exact binomial test, p = 0.055), significant preference for the head motif in hsc70-4 (p < 0.001), and significant preference for the tail motif in hsp40 (p < 0.05).

    Evolutionary gains and losses in hsp83 within Hymenoptera, followed by diversification in cis-regulatory HSEs. Relationships of homologous hsp83 were reconstructed with PhyML for 17 insect species (rooted with A. pisum) using a JTT substitution model with 1000 bootstrap replicates (>90 bootstrap support indicated left). Branches of the outgroup taxa are colored in blue and black, while well-supported paralogues of Hymenopteran branches are colored in orange (h1) and red (h2). Statistically significant episodic diversifying selection using Branch-Rel is indicated along the branch (+ corresponds to p < 0.05 * = p < 0.01 ** = p < 0.001). Cis-regulatory HSEs in the promoter region spanning 400 bps from the transcription start site (TSS right) are mapped onto the phylogeny and are annotated by their length and motif type

    Evolutionary conservation of two copies of hsc70-4 within Hymenoptera, but both copies harbor an extraordinary amount of diversity in cis-regulatory HSEs. Relationships of homologous hsc70-4 were reconstructed with PhyML for 17 insect species (rooted on A. pisum) using a JTT substitution model with 1000 bootstrap replicates (>90 bootstrap support indicated left). Branches of the outgroup taxa are colored in blue, while well-supported paralogues of Hymenopteran branches are colored in orange (h1) and red (h2). Statistically significant episodes of positive selection identified with Branch-Rel are indicated along the branch(+ corresponds to p < 0.05 * = p < 0.01 ** = p < 0.001). Cis-regulatory HSE elements in the promoter region spanning 570 bps from the transcription start site (TSS right side) are mapped onto the phylogeny and are annotated by their length and motif type

    Evolutionary conservation of hsp40 copy number and cis-regulatory HSEs. Relationships of homologous hsp40 were reconstructed with PhyML for 17 insect species (rooted on A. pisum) using a JTT substitution model with 1000 bootstrap replicates (>90 support indicated). The outgroup and Hymenopteran branches are indicated in blue and red, respectively. Statistically significant episodes of positive selection using Branch-Rel are indicated along the branch (+ corresponds to p < 0.05 * = p < 0.01 ** = p < 0.001). Cis-regulatory HSE elements in the promoter region spanning 370 bps from the transcription start site (TSS right side) are mapped onto the phylogeny and are annotated by their length and motif type. S. invicta did not provide enough sequence information for the identification of cis-regulatory HSEs

    Heat shock protein (Hsp) and cis-regulatory heat shock element (HSE) evolution

    Hsp83

    Phylogenetic reconstruction of hsp83 revealed multiple duplications and losses in both the outgroup and Hymenoptera (Fig. 1). An early duplication event in a common ancestor of the Hymenoptera generated two paralogues of hsp83 (h1 and h2 in Fig. 1). Although both paralogues are present in bees and wasps, only one paralogue (h2) exists in ants, indicating a secondary loss. A second duplication of the h2 orthologue occurred in Linepithema humile. Selection analysis along the length of the gene sequence indicated that most sites (608/714 and 625/714, Single likelihood ancestor counting (SLAC) and Relative effects likelihood (REL) analyses, respectively, Table 2) identified purifying selection there was no evidence for episodic diversifying selection in branches leading to Hymenopteran paralogues (Branch-REL, p > 0.5 Fig. 1).

    In spite of overall sequence conservation, Hymenopteran hsp83 h2 differs in genomic structure and cis-regulation from Hymenopteran hsp83 h1 and from outgroup species in three ways. First, Hymenopteran hsp83 h1 and most outgroup species completely lack introns, whereas hsp83 h2 has two introns Apis mellifera hsp83 h1 is the exception, with one intron in hsp83 h1 (Additional file 4: Figure S4). Second, Hymenopteran hsp83 h2 has a split HSE arrangement (4–6 and 3 HSE motifs), whereas both hsp83 Hymenopteran h1 and the outgroup have a contiguous HSE arrangement (6–9 HSE motif length) at the proximal end of the molecule (30–100 bps upstream TSS Fig. 1). Third, there is a preference in head-type motifs only in Hymenopteran hsp83 h2 (Fisher’s Exact Test, p <0.001 Fig. 1).

    Hsc70-4

    Phylogenetic reconstruction of hsc70-4 indicates multiple duplication events both within species (C. quinquefasciatus and A. pisum) and in a common ancestor of the Hymenoptera, leading to two paralogues (h1 and h2 Fig. 2). Each paralogue forms a strongly supported clade, with the exception of the two Bombus species, in which the h1 paralogue is nested within the h1 clade but the second copy does not group with either Hymenopteran paralogue (Fig. 2). There is evidence of episodic diversifying selection along the branch preceding the hsc70-4 duplication in the Hymenoptera and also in the Hymenopteran hsc70-4 h2 lineage (Branch-REL, p <0.001 in both cases Fig. 2), even though most individual sites (608/710 and 610/710, SLAC and REL analyses, respectively) were under purifying selection (Table 2).

    Hymenopteran hsc70-4 differs in genomic structure and cis-regulatory HSEs from that of D. melanogaster. The orthologue of hsc70-4 in D. melanogaster lacks introns and cis-regulatory HSEs (Additional file 5: Figure S5 Fig. 2). In contrast, Hymenopteran hsc70-4 h1 has one intron, with the exception of N. vitripennis, which has two introns. Hymenopteran hsc70-4 h2 also has two introns, with the exception of Bombus (Additional file 5: Figure S5). Compared to the hsc70-4 in members of the outgroup (Fig. 2, right), both Hymenopteran hsc70-4 paralogues showed high diversification in cis-regulatory HSEs, particularly at the more distal positions ( >120 bps upstream TSS). At the proximal position (30–115 bps upstream TSS), however, HSEs of Hymenopteran hsc70-4 aligned locally with the inducible D. melanogaster hsp70 gene (data not shown).

    Hsp40

    Both sequence and copy number of hsp40 were phylogenetically conserved across all insect species (Fig. 3). Most sites were under purifying selection (Table 2), and there was no evidence of episodic diversifying selection along branches leading to the Hymenoptera (Fig. 3). Cis-regulatory HSEs of hsp40 were concentrated in one conserved proximal block of 3–7 HSE subunits that were located 35–100 bps upstream of the TSS, although in D. melanogaster HSEs were located 255–285 bps upstream (Fig. 3). However, the genetic structure appears less conserved, ranging from zero to three introns (Additional file 6: Figure S6).

    Inducible Hsp expression

    We tested whether the presence or absence of conserved cis-regulatory HSEs successfully predicted Hsp gene induction in response to experimental heat shock. The four Hsp genes with conserved HSEs were all significantly up-regulated in response to increasing temperature treatments (hsp83 (F5,12 = 8.48 p < 0.01), hsc70-4 h1 (F5,12 = 3.74 p < 0.05), hsc70-4 h2 (F5,12 = 10.6 p < 0.001), and hsp40 (F5,12 = 6.97, p < 0.01) Fig. 4a–d). The other four Hsps, which lacked conserved HSEs, were not significantly up-regulated after heat shock (hsc70-5 (F5,12 = 2.17 p = 0.13), hsc70-3 (F5,12 = 1.91 p = 0.17), hsp60 (F5,12 = 2.86 p = 0.063), and l(2)efl (F5,12 = 0.223 p = 0.946) Fig. 5a–d).

    Relative fold increase in gene expression (+/− SD) for four inducible HSPs in A. picea and P. barbatus across different temperature treatment. Relative expression of hsp83 (a), hsc70-4 h1 (b), hsc70-4 h2 (c), and hsp40 (d) were normalized to the 18 s rRNA and β-actin, 18 s rRNA and GAPDH in A. picea (N = 4 per treatment) and P. barbatus (N = 3 per treatment), respectively. Significant up-regulation from 25 °C (A. picea) and 30 °C (P. barbatus) is denoted by ‘*’ from post hoc Tukey tests (p < 0.05)

    Relative fold change in gene expression (+/− SD) for four non-inducible Hsps in A. picea and P. barbatus across different temperature treatment. Relative expression of hsc70-5 (a), hsc70-3 (b), hsp60 (c), and l(2)efl (d) were normalized to the 18 s rRNA and β-actin and 18 s rRNA and GAPDH for A. picea (N = 4 per treatment) and P. barbatus (N = 3 per treatment), respectively

    Species comparisons

    We then tested whether variation in thermal tolerances between two ant species was accompanied by changes in Hsp inducibility. The median lethal temperature 50 (LT50) of the warm-climate P. barbatus (median LT50 = 46.9 °C) was significantly higher than the LT50 of the cool-climate A. picea (median LT50 = 38.78 °C generalized linear model (GLM) with a binomial response variable: influence of species, p < 0.001 Additional file 7: Figure S7). These survivorship differences were matched by patterns of Hsp gene expression: P. barbatus shifted its expression profile toward higher temperatures than did A. picea for all inducible Hsps (Fig. 4a–d). For hsp83, hsc70-4 h1, and hsc70-4 h2, P. barbatus showed peak expression at 43 °C, whereas A. picea showed peak expression at 35–38.5 °C (Fig. 4a–c). For hsp40, peak expression was 40 and 35 °C for P. barbatus and A. picea, respectively (Fig. 4d). P. barbatus exhibited significantly higher constitutive expression of hsc70-4 h1 (ANOVA, F1,5 = 87.8, p < 0.01) and l(2)efl (F1,5 = 6.92, p < 0.05), and significantly lower constitutive expression of hsc70-3 (F1,5 = 596, p < 0.01), hsc70-5 (F1,5 = 24.3, p < 0.001), and hsp60 (F1,5 = 31.2, p < 0.01) than did A. picea (Fig. 6). Among the inducible Hsps, there was a positive relationship between relative basal expression levels and relative inducibility (linear regression, r 2 = 0.918, p < 0.05 Fig. 7).

    Relative basal heat shock gene (target) expression (+/− SD) between P. barbatus(N = 3) and A. picea (N = 4). Relative gene expression was normalized with the geometric mean of 18 s rRNA and β-actin as the calibrator (* = p < 0.05** = p < 0.01 *** = p < 0.001 levels of significance) and fold change was calculated as P. barbatus relative to A. picea was calculated as follows: 2 Target(Pbar-Apic) /2 Calibrator(Pbar-Apic) (Pbar = P. barbatus, Apic = A. picea). -1 was divided by values less than one to calculate negative relative basal expression. Significant up-regulation in P. barbatus and A. picea are colored in red and blue, respectively

    The positive relationship between the log ratios of basal expression levels (P. barbatus/A. picea) at rearing temperatures and max induction (β1 slope = 0.2398, r 2 = 0.918, p < 0.05)


    Discussion

    In the present study, we expanded the concept that HSP70 is a role player in vascular physiology, as we show, for the first time, that proper vascular (hbox ^<2+>) handling stimulated by PE requires this protein. Our findings are of utmost importance because (hbox ^<2+>) is the chief mediator of vascular contraction and fluctuations in its intracellular levels modulate the contractile phenotype of blood vessels 1 . Therefore, notwithstanding the limitations of our study, it fills in a knowledge gap in vascular biology and it will, in turn, guide future research in this field, particularly due to the emergent link between HSP70 and cardiovascular/renal diseases 25,26,27,28,29 .

    Throughout this study, we applied a well-established, yet indirect, protocol to investigate (hbox ^<2+>) changes in the presence of VER155008, which functions as an ATP-competitive inhibitor 30 . To overcome this limitation and strengthen our claims, a biochemical assay kit was also used to evaluate the free levels of this cation. In Fig. 1B, we specifically show that inhibition of HSP70 decreases the total levels of free (hbox ^<2+>) , which ultimately, reduces the force of contraction. Here, it is important to recognize some pitfalls of this method. In recent years, researchers have evaluated (hbox ^<2+>) fluctuations with a fluorescent indicator, such as fura-2, and since the concentration of (hbox ^<2+>) rapidly change in vascular structures, it is considered a more accurate measurement for this cation. Additionally, in order to perform the biochemical assay, samples need to be homogenized and it might include mitochondrial (hbox ^<2+>) , which does not affect vascular smooth muscle contraction. Still, we argue that the claim that blockade of HSP70 impairs vascular contraction by affecting (hbox ^<2+>) handling mechanisms are based on our collective findings, which include not only the indirect measurement of the free levels of (hbox ^<2+>) , but also an extensive and well-detailed set of functional studies as well as evidence from previous studies. The literature shows a complex interaction between HSP70 and (hbox ^<2+>) . In fact, the ATPase domain of HSP70 binds two (hbox ^<2+>) ions 9 and changes in the intracellular concentration of this cation modulates the expression of HSP70 10,11 . Additionally, the genetic deletion of this protein impairs (hbox ^<2+>) homeostasis in cardiac and skeletal muscle 7,8 . Thus, our data elegantly builds upon previous knowledge as it uncovers a new biological process where HSP70 interacts with (hbox ^<2+>) .

    It is known that phasic contraction in response to PE in the aorta involves IP3r-mediated (hbox ^<2+>) release from the SR 12,15 . We previously demonstrated that, in vessels stimulated with this (alpha -1) agonist when HSP70 is blocked, there is a reduction in the amplitude of the fast component 4 . However, it was yet-to-be-determined if a direct relationship exists with the IP3r. Here, we confirmed that blockade of HSP70 also weakens PE-induced phasic contraction in aorta under 0[ (hbox ^<2+>) ] Krebs’ solution (Fig. 2A). Similar results were also detected in samples incubated with 2-APB, an inhibitor of the IP3r (Fig. 2D). Interestingly, we found that the combination of VER155008 and 2-APB does not augment the latter’s inhibitory effect (Fig. 2E vs. D). Such findings indicate both inhibitors acting upon similar mechanisms. Corroborating this statement, we also detected that, if we block HSP70 after PE-mediated IP3r-induced phasic (hbox _>) , the impact of VER155008 is abolished (Fig. 2A,C), which strongly suggests that HSP70 contributes to PE-induced phasic contraction via IP3r-mediated (hbox ^<2+>) release. In a counterintuitive manner, it has been previously demonstrated that upregulation of HSP70 reduces IP3r protein levels following ischemia/reoxygenation in PC12 cells 31 . Here, it is important to consider that (hbox ^<2+>) overload can occur during ischemia/reoxygenation 32 , and as discussed by the authors, the ultimate outcome observed was that HSP70 contributes to maintaining (hbox ^<2+>) homeostasis in these cells 31 . In this sense, our results align with the previous literature as we also show that the precise control of (hbox ^<2+>) handling requires HSP70.

    Next, we turned our attention to try at understanding the mechanism(s) by which HSP70 affects vascular tonic contraction. A previous study demonstrated that PE-induced tonic contraction includes (hbox ^<2+>) influx via voltage-dependent and independent channels 12 . While there is limited information about an interaction between HSP70 and LTCC, it has been suggested that HSP70 might act by inhibiting voltage-gated (hbox ^<2+>) channel to prevent (hbox ^<2+>) overload, and consequently, apoptosis 33 . However, as highlighted by the authors experimental evidence was lacking. Here, we found that the HSP70 inhibitor potentiates the inhibitory effect of an LTCC blocker (Fig. 4B vs. A), but not of a non-selective inhibitor of voltage-independent (hbox ^<2+>) channels (Fig. 5B vs. A). Therefore, our data corroborate the idea that HSP70 contributes to tonic contraction by acting upon voltage-independent (hbox ^<2+>) channel-facilitated (hbox ^<2+>) influx. In support of this statement, we also confirmed that VER155008 reduces tonic contraction independently of the moment it is added to the chamber (i.e., whether it was before or after IP3r-mediated phasic contraction) (Fig. 2). Noteworthy, we used 2-APB to target voltage-independent (hbox ^<2+>) channels, which has an inhibitory effect towards NSCC and CRAC channels 12,34 . Consequently, we are unable to pinpoint the exact channel targeted by the HSP70 inhibitor. Another possibility one should consider in this context is the fact that a previous study has demonstrated that the constitutive HSP70 interacts with lipid membranes leading to the generation of a functional ATP-dependent cationic pathway 35 . Therefore, further studies are required to uncover the precise mechanism by which HSP70 targets (hbox ^<2+>) influx in PE-stimulated aorta.

    Subsequently, we focused on determining the contribution of Rho-kinase, which promotes (hbox ^<2+>) sensitization, and therefore, affects the contractile phenotype of vascular structures 19,20 . From our data, it is clear that the combination of VER155008 with Y27632 amplifies the hyporesponsive pattern observed in the aorta in comparison with blocking these proteins independently (Fig. 6). Given our findings regarding the role of HSP70 in (hbox ^<2+>) influx, one can argue that these results were to be expected, especially because Rho-kinase impacts vascular contraction by inhibiting the myosin light chain phosphatase, which, in turn, prevents relaxation 36,37 . Therefore, it appears that two different mechanisms were targeted in this set of experiments. Corroborating this statement, a previous study found that heat shock-mediated vascular hypercontractility does not directly involve Rho-kinase 38 .

    Finally, we investigated a potential interaction between HSP70 and the SERCA pump, which mediates (hbox ^<2+>) re-uptake by the SR 22 . In this study, we used the SERCA pump inhibitor, cyclopiazonic acid (CPA), which does not affect phasic contraction 15 . In fact, it has been demonstrated that it increases the intracellular concentration of (hbox ^<2+>) without changing the force of contraction 12 . Interestingly, in the presence of CPA, there is a shift in the contractile phenotype of vascular smooth muscle to rely mainly on (hbox ^<2+>) influx via voltage-independent (hbox ^<2+>) channels 12 . Here, as showed by others, we confirmed that CPA does not impact PE-induced phasic contraction (Fig. 7). More interestingly, we found that, under the conditions examined in this study, it disrupts the ability of the vessel to elicit tonic contraction following re-addition of (hbox ^<2+>) (Fig. 7). It is important to consider the importance of (hbox ^<2+>) re-uptake by the SR, as the influx of this ion is gated by its depletion from the SR 16 . When we combined the HSP70 inhibitor with CPA, we observed a complete impairment of the vessels’ ability to elicit contraction, which could be due to (a) the effects of CPA upon (hbox ^<2+>) re-uptake and/or (b) the effects of VER155008 on voltage-independent (hbox ^<2+>) channels, which was, under this condition, the main source of (hbox ^<2+>) influx. While, to the best of our knowledge, data regarding an interplay between HSP70 and the SERCA pump in vascular structures are nonexistent, the literature shows that this chaperone has a protective role towards this protein 33 . For example, in cardiomyocytes, the deletion of HSP70 associates with a decrease in the expression of SERCA2a 7 whereas, in PC12 cells, overexpression of HSP70 increases the levels of SERCA2a and SERCA2b 31 . Importantly, it has been demonstrated that, in HEK-293 cells, HSP70 prevents thermal inactivation of SERCA2a, potentially by decreasing its oxidation and nitrosylation 39 . Likewise, HSP70 prevents the thermal inactivation of SERCA1a in fast-twitch skeletal muscle 40 . Nevertheless, these studies differ in many aspects from our work, especially the fact that we aimed at investigating this interaction in the absence of a pathological condition.

    In summary, we showed that blockade of HSP70 affects (hbox ^<2+>) handling mechanisms in aorta stimulated with PE via crosstalk with (a) IP3r-mediated (hbox ^<2+>) release from the SR (phasic) and (b) voltage-independent (hbox ^<2+>) channels-facilitated (hbox ^<2+>) influx (tonic) (Fig. 8). There are, however, many points to enlighten, including the molecular aspects guiding this process. Here, we took an indirect approach to evaluate the role of HSP70 in vascular contraction, and therefore, further studies employing in situ enhancement of HSP70 in vascular smooth muscle are still required, since they could shed much light on the exact mechanisms involved in HSP70-mediated arterial contraction. A striking question arising at this point is whether the interaction between HSP70 and (hbox ^<2+>) remains in vascular diseases, such as diabetes and hypertension. Nevertheless, such a development in our understanding shifts the way one might approach disease-associated vascular complications, especially because we provided evidence that, under the conditions evaluated in this study, HSP70 contributes to vascular (hbox ^<2+>) dynamics, and (hbox ^<2+>) is a key player in healthy and diseased states.


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