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(Hypertension. 1996;28:53-57.)
© 1996 American Heart Association, Inc.

Articles

Activation of Heat Shock Transcription Factor 1 in Rat Aorta in Response to High Blood Pressure

Qingbo Xu; Timothy W. Fawcett; Robert Udelsman; Nikki J. Holbrook

the Section on Gene Expression and Aging, National Institute on Aging, National Institutes of Health, and Division of Endocrine Surgery (R.U.), The Johns Hopkins Hospital, Baltimore, Md.

	Abstract

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We have previously demonstrated that acute hypertension induces heat shock protein gene expression in rat arterial wall. Here we provide evidence that this induction is mediated through the activation of heat shock transcription factor 1 in response to high blood pressure. Rats subjected to restraint or immobilization stress displayed an acute elevation in systolic pressure accompanied by an increase in heat shock protein 70 mRNA expression. Consistent with the rapid time course of mRNA induction, an increase in binding activity to an oligonucleotide encompassing a consensus heat shock element sequence was seen in protein extracts from aorta of restrained rats as assessed with gel mobility shift assays. A similar increase in DNA binding activity was also observed in aortic extracts from rats treated with various hypertensive agents, including phenylephrine, angiotensin II, and vasopressin. That the DNA binding activity was attributed to heat shock factor 1 was shown through use of antibodies to the transcription factor that retarded the DNA-protein complexes in gel mobility supershift assays. Western blot analysis of heat shock factor 1 protein expression in aortic extracts showed a slower mobility form of the protein in hypertensive rats, indicative of an activated, presumably phosphorylated, form of the transcription factor. These findings support the view that heat shock factor 1 is responsible for induction of heat shock protein 70 in the arterial wall during acute hypertension, a response that is likely to play an important role in protecting arteries during hemodynamic stress.

Key Words: gene expression • heat shock proteins • stress • transcription, genetic

	Introduction

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The hsp's are a group of highly conserved proteins that are induced in response to heat and other stresses. They are subdivided into multimember families on the basis of the molecular weights of the proteins encoded (eg, hsp27, hsp60, hsp70, and hsp90), with hsp70 being the most extensively studied hsp in mammalian cells.^{1 2 3} Functioning as a chaperone, hsp70 plays a role in both the assembly and transport of newly synthesized proteins within the cells as well as the removal of denatured proteins. Thus, it appears to play a role in preventing damage as well as in cellular repair processes after injury.^{4 5 6} Indeed, increased hsp70 expression has been shown to protect cells against a broad range of stresses, including oxidative stress, heat shock, ethanol, and cellular damage after ischemia or sepsis-induced injury.^{7 8 9}

The heat shock response is primarily regulated at the level of transcription and is mediated by one or more of a family of HSFs that interact with a specific regulatory element, the HSE, present in the promoters of hsp genes.^{6 10} Present constitutively in the cell in a non–DNA binding state, HSFs are activated in response to various stresses to a DNA binding form. The activation process is poorly understood, but based on studies with cultured cells, it appears to involve the oligomerization of HSF from a monomeric or dimeric form to a trimeric state and is associated with its hyperphosphorylation.^{11 12 13} Two distinct HSFs have been shown to exist in mammalian species.^{14 15} Recent studies have suggested that functional differences exist between HSF1 and HSF2 and that the signals that activate one or the other to a DNA binding state are specific. HSF1 has been shown to be involved in the regulation of hsp expression following such inducers as elevated temperature, heavy metals, and amino acid analogues, and HSF2 appears to be involved in hsp expression during hemin-induced differentiation of human erythroleukemia cells.^{14 15 16}

Although most of our knowledge concerning the regulation of hsp expression has come from studies with cultured cells, we and others have provided evidence that induction of hsp's also occurs in vivo in response to physiologically relevant stresses.^{17 18 19 20} In particular, we have demonstrated that hsp70 is induced in the arterial wall in response to acute hypertension brought about by restraint or hypertensive agents.^{21 22} In the present study, we provide evidence that this induction is associated with enhanced DNA binding to a consensus HSE, indicative of HSF activation. We demonstrate that the major component of these HSE binding complexes in rat aortic extracts is HSF1 and show that HSF1 is modified, possibly phosphorylated, during hypertension.

	Methods

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Animals and Restraint Model
Four-month-old male Wistar rats were obtained from Hilltop Lab Animals, Inc (Scottdale, Pa) and acclimated in individual cages for 1 week before experimentation. Rats were maintained on a 12-hour light/dark cycle at 24°C and received food and water ad libitum. All procedures were performed according to protocols approved by the National Institute on Aging Committee for the Use and Care of Laboratory Animals in accordance with guidelines established by the National Institutes of Health. For restraint experiments, individual rats were placed in clear, ventilated Plexiglas chambers as described previously.²⁰

BP Measurements
Rats underwent light anesthesia with thiopental (40 mg/kg IM) followed by insertion of polyethylene catheters via the common femoral artery and vein into the abdominal aorta and inferior vena cava, respectively.²¹ The aortic catheter was connected to a pressure transducer (COBE) and a BP analyzer (Micro-MED, Inc). BP measurements were made every 30 seconds up to 60 minutes.

Chronic Catheterization Procedure and Drug Administration
Polyethylene catheters were inserted via the common femoral vein into the inferior vena cava with rats under thiopental (40 mg/kg IM) anesthesia.²¹ The catheters were tunneled through the subcutaneous tissue to exit from the back where they were connected to a swivel device (Rodent Multi-fluid Channel Swivel, Stoelting Co). This model allows for complete animal mobility so that subsequent experiments could be performed in conscious, unstressed rats. Saline (0.4 mL) was injected through the catheter daily for 3 days after catheter insertion. Phenylephrine (140 µg/kg), angiotensin II (2 µg/kg), and vasopressin (2 µg/kg) (Sigma Chemical Co) were administered via the catheter into the vena cava.

RNA Extraction and Northern Analysis
Freshly harvested tissues were homogenized and the RNA extracted with RNA Stat-60 (Tel-Test "B," Inc). Total RNA (10 µg per lane) was fractionated by electrophoresis on formaldehyde-agarose gels and transferred to nylon membranes (GeneScreen Plus, DuPont). Hybridizations were performed with an -³²P–labeled hsp70 cDNA probe as previously described.^{20 21 22} Accuracy of loading and transfer, as well as RNA integrity, was confirmed by quantitative analysis of 18S levels on the same blots. Autoradiographs of the blots were obtained in the linear range of detection and were quantified for levels of specific expression by scanning laser densitometry (Molecular Dynamics) of autoradiographs.

Protein Extractions
Fresh or rapidly thawed tissues were homogenized with a Polytron homogenizer (PT1200, Kinematica AG) at the number 6 setting for 30 seconds on ice in buffer containing 20 mmol/L HEPES (pH 7.5), 1.5 mmol/L MgCl₂, 0.2 mmol/L EDTA, 0.4 mol/L NaCl, 0.2 mmol/L dithiothreitol, 1 mmol/L phenylmethylsulfonyl fluoride SC (Boehringer Mannheim), 20% glycerol, and 1 µg/mL leupeptin. Supernatants were collected and protein concentrations measured by the Bradford assay (Bio-Rad). Aliquots were frozen in liquid nitrogen and stored at -80°C.

Gel Mobility Shift Assays
The procedure for gel mobility shift assays has been described previously.²⁰ In short, DNA binding was determined after incubation of 20 µg of aortic protein extract with 10 fmol of an oligonucleotide containing the HSE sequence from the Drosophila hsp70 promoter (5'-GCCTCGAATGTTCGCGAAGTTT-3') labeled with [³²P]dCTP. Reaction buffer contained 10 mmol/L HEPES (pH 7.9), 1 mmol/L dithiothreitol, 1 mmol/L EDTA, 80 mmol/L KCl, 4% Ficoll, and 2 µg poly(dIdC) as a nonspecific competitor. Supershift assays were performed with antibodies generated against and specific to rodent HSF1 and HSF2 (gifts from Dr R.I. Morimoto, Northwestern University, Evanston, Ill^{14 15} ). The antibodies were added to samples after the initial binding reactions between protein extracts, and oligonucleotides were allowed to occur (see above). Samples were subjected to nondenaturing polyacrylamide gel electrophoresis in 4% gels, after which the gels were dried onto DE81 paper and exposed to PhosphorImager screens for 24 to 48 hours. The images were analyzed with the Image-Quant software package (Molecular Dynamics).

Western Blot Analysis
Total protein extracts (50 µg adrenal tissue; 100 µg vessel tissue) were separated by electrophoresis through a 10% sodium dodecyl sulfate (SDS)–polyacrylamide gel and transferred to an Immobilon-P transfer membrane (Millipore). The membranes were processed with rabbit antisera to mammalian HSF1 or HSF2.^{14 15} Specific antibody–HSF1 complexes were detected with the ECL Western Blot Detection Kit (Amersham Co).

	Results

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hsp70 Gene Induction in Response to Acute Hypertension
We have shown previously that restraint, considered to be a moderate behavioral stress, results in elevated BP accompanied by hsp70 gene expression in vasculature and adrenal glands.^{20 21} To confirm this relationship between BP elevation and aortic hsp70 expression, arterial BP was measured and aortic hsp70 mRNA analyzed during restraint stress. As shown in Fig 1A, restraint resulted in a rapid rise (within 2 minutes) in systemic BP (systolic, from 120 to 150 to 160 mm Hg) that was maintained for the entire period of restraint (60 minutes). The effect of restraint stress on hsp70 mRNA levels is shown in Fig 1B. Restraint resulted in the rapid induction of hsp70 with maximum levels (>10-fold elevation over those of untreated controls) achieved within 30 minutes of restraint. Despite the fact that BP remained elevated, the level of hsp70 mRNA declined with longer periods of restraint.

View larger version (34K): [in this window] [in a new window]   Figure 1. Effect of restraint on blood pressure and hsp70 expression in rat aorta. A, Arterial systolic pressure is increased in rats subjected to restraint. B, Representative Northern blot (10 µg of RNA per lane) showing hsp70 mRNA induction in aorta of restrained rats; the blot was hybridized sequentially with an hsp70 cDNA probe and oligonucleotide probe to 18S rRNA. Rats were killed at the indicated times during 60 minutes of restraint. For the 120-minute time point, rats were returned to their cages for 60 minutes after restraint. The aorta was freed of adventitia and homogenized, and total RNA was extracted.

Activation of HSF1 by High BP
In most in vitro model systems studied, stress-induced hsp70 induction has been shown to be mediated via activation of HSF1, which in turn leads to enhanced transcription of the hsp70 gene.^{6 10} To determine whether a similar situation occurs in vivo in the arterial wall in response to hemodynamic stress, we examined protein extracts prepared from aorta of either control or restrained rats for binding activity to an oligonucleotide encompassing a consensus HSE site. Fig 2A shows the results of gel mobility shift assays examining the levels of DNA binding activity to the HSE in extracts prepared from rats restrained for various lengths of time. Two regions of DNA binding activity were apparent. The faster migrating band designated NS is presumed to represent nonspecific or constitutive interactions, as this binding activity was present in comparable amounts in aortic extracts from both treated and untreated rats. The broad, slower migrating region indicated by the arrow was found to represent specific binding. Fig 2B shows the results of a gel mobility shift assay in the presence or absence of either an unlabeled HSE or nuclear factor-B (NF-B) binding element. The restraint-induced increase in binding activity was specific for the HSE, as increased concentrations of unlabeled HSE effectively competed for binding to the factor, whereas the NF-B binding element did not. It is worth noting that the level of HSE binding activity seen here is relatively low compared with that we have observed in other rat tissues after heat stress. However, the amount of HSE binding activity in the aorta after heat stress is in fact similar to that observed with restraint (data not shown).

View larger version (248K): [in this window] [in a new window]   Figure 2. HSE binding activity in aortic extracts of restrained rats. A, Gel mobility shift assays were performed with whole tissue extracts (20 µg protein per lane) from aortas of unrestrained rats or rats restrained for the indicated times up to 1 hour. At 3 and 6 hours, rats were returned to their cages after 1 hour of restraint and killed at the indicated times. Extracts were incubated with a radiolabeled oligonucleotide encompassing a 24-bp HSE of the hsp70 promoter. B, Specificity of HSE binding activity in aortic extracts. Tissue lysates from rats restrained for 20 minutes were incubated with 32P-labeled HSE oligonucleotide in the presence or absence of unlabeled oligonucleotides containing either an HSE or nuclear factor-B (NF-kB) binding element. Note the competition by the HSE but not the nuclear factor-B site containing oligonucleotide. Arrows indicate specific HSF binding complexes; NS, nonspecific binding; and FP, free probe.

Consistent with the rapid and transient induction of hsp70 mRNA, increased HSE binding activity was evident within 20 minutes of restraint and declined after 30 minutes (Fig 2A). As expected, treatment of rats with the three different hypertensive agents (phenylephrine, angiotensin II, and vasopressin), all of which have been previously shown to induce hsp70 expression in the aorta, also resulted in increased HSE binding activity (Fig 3).

View larger version (110K): [in this window] [in a new window]   Figure 3. Effect of hypertensive agents on HSE binding activity in aortic extracts. Rats were treated with phenylephrine (140 µg/kg), angiotensin II (2 µg/kg), vasopressin (2 µg/kg), or saline (control). Aortic tissue extracts prepared from rats 20 minutes after treatment were incubated with the radiolabeled oligonucleotide HSE. Arrow indicates specific HSE binding complexes; NS, nonspecific binding. Numbers above the lanes identify individual rats tested with each agent.

Next, we examined the composition of the DNA binding complexes using antibodies to HSF1, HSF2, and c-fos. Addition of the anti-HSF1 antibody to the binding reaction resulted in a complete shift of the binding complexes to a slower migrating species, whereas the anti-HSF2 and anti–c-fos antibodies were without effect (Fig 4A). Curiously, however, in the binding reactions with HSF1, the nonspecific band also shifted up. This suggests either that the bands we presume to be nonspecific do indeed contain HSF1 protein or that their shift up is an artifact, perhaps caused by the high amounts of antisera used. To address this issue, we performed additional mobility shift assays using greater dilutions of HSF1 antiserum and in the presence or absence of cold HSE. As shown in Fig 4B, these experiments showed a shift up of the higher molecular weight species but not the lower complexes attributed to nonspecific interactions. These results indicate the presence of HSF1 but not HSF2 or c-fos proteins in the stress-inducible HSE binding complexes in aortic extracts.

View larger version (224K): [in this window] [in a new window]   Figure 4. HSE binding complexes in rat aortic extracts contain HSF1. A, Aortic extracts from restrained rats were incubated with radiolabeled HSE oligonucleotide with no addition (-) or in the presence of (+) various antibodies specific to HSF1 (undiluted), HSF2, or c-fos. The bracket indicates specific protein-DNA complexes; NS, nonspecific DNA binding. B, Supershift assays performed with 5- and 20-fold dilutions of HSF1 antisera (lanes 2 and 5, and 1 and 6, respectively) in the presence or absence of 50-fold molar excess cold HSE oligonucleotide.

Phosphorylation of HSF1 in Response to Acute Hypertension
In unstressed cells, HSF1 normally migrates on SDS-polyacrylamide gels as an approximately 70-kD protein. In cultured cells, heat and other stresses known to activate HSF1 result in an apparent increase in its size (slower mobility) due to enhanced phosphorylation.^{14 15} In view of the above noted increase in DNA binding activity of aortic HSF1 in restrained rats, it was of interest to examine the relative mobility of HSF1 from these rats on an SDS-polyacrylamide gel. Fig 5 shows the results of Western blot analysis comparing the amount and mobility of HSF1 in aortas of untreated rats and rats restrained for various times. As evidenced by the weak signal seen on the Western blot, HSF1 is present in very low levels in the aorta. This is consistent with the relatively low DNA binding activity noted above in gel mobility shift assays. Similar to previous reports in cultured cells indicating that HSF1 levels are not regulated in response to stress, the amount of HSF1 protein did not differ significantly between treated and untreated rats. In the absence of restraint, HSF1 consisted of multiple bands, presumably reflecting variable phosphorylation of the protein. However, restraint treatment resulted in a rapid shift to higher molecular weight species, indicative of an increase in phosphorylation, followed by a return to the initial pattern within 3 hours after restraint (Fig 5A). This transient shift in the mobility of HSF1 is consistent with the kinetics of both DNA binding activity and mRNA induction (Figs 1 and 2).

View larger version (63K): [in this window] [in a new window]   Figure 5. Western analysis of HSF1 protein in control and restrained rats. A, Aortic extracts prepared from rats restrained for the indicated times were analyzed by Western immunoblotting for HSF1 protein. Arrow indicates the position of slower migrating forms of HSF1 protein. B, -Actin expression on the same blot as used for analysis of HSF1 protein in A. C, HSF1 protein expression in adrenal tissue of control, restrained, and heat-stressed rats.

Because the amount of HSF1 protein in aortic extracts appeared to be much lower than that seen in other tissues or cell types, we performed additional experiments to verify the techniques being used and the ability of the antibodies to recognize the HSF1 protein. Fig 5B shows a Western blot examining the amount of -actin on the same blot. As expected, -actin expression was strong and the band was of the expected size, verifying the integrity of the protein extracts. Using the same procedure, we likewise analyzed adrenal extracts from control, restrained, and heat-shocked rats for HSF1 expression. We had previously demonstrated that although restraint leads to the induction of hsp70 mRNA associated with increased DNA binding activity of HSF1, unlike that seen above in vessel, it results in little change in the mobility of HSF1 protein.¹⁵ The results shown for restrained rats in Fig 5C are consistent with these prior studies. On the other hand, heat stress, which leads to greater induction of the hsp70 mRNA, results in a significant shift in the protein to a higher molecular weight form (Fig 5C). These studies demonstrate our ability to detect HSF1 protein on Western analysis and demonstrate that although levels are low in the aorta, in response to restraint, HSF1 does show a transient increase in phosphorylation.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Many factors, such as physical exertion, drug toxicity, noise, and emotional stress, lead to a rise in BP,^{23 24} which in certain circumstances can lead to severe damage to the vessel wall or even rupture.^{25 26} We and others have recently provided evidence that acute hypertension results in the induction of hsp70 expression, suggesting a likely role for this hsp in the host defense against hemodynamic stress.^{21 22 27 28 29}

The mechanisms contributing to hsp induction in response to heat and other classic stresses have been extensively studied in cultured cells,^{6 10} but few studies have explored the mechanisms regulating hsp expression in vivo in physiologically relevant models of stress other than heat.^{21 22 27 28 29} Here we provide evidence that the induction of hsp70 in the arterial wall of hypertensive rats is mediated at least in part through the activation of HSF1. How acute hypertension actually triggers the activation of the transcription factor, however, is an important question that remains unanswered. We speculate that elevated BP results in stretching of the arterial wall, leading to dislocation, denaturation, or unfolding of proteins in smooth muscle cells, which in turn serve as a signal for initiating the hsp70 gene transcription.

A somewhat curious finding of our studies was the extremely low levels of HSF1 present in the aorta relative to other tissues of the rat. This was particularly surprising given the high level of hsp70 mRNA induction that occurs in the aorta of rats that is equivalent to or exceeds the induction occurring in other tissues in response to heat stress. This suggests that additional regulatory factors contribute to the expression in this tissue and/or that the absolute levels of HSF1 in most tissues are present in vast excess and have little to do with determining the magnitude of transcriptional activation (ie, HSF1 levels are unlikely to be a limiting factor in hsp expression). Further understanding of how acute hypertension leads to activation of HSF1, as well as knowledge concerning other factors that contribute to the regulation of hsp70 expression in the aorta, could provide valuable information that might be beneficial for enhancing the expression of this stress response protein, thereby imparting protection to the arterial wall.

Recent studies have focused on the protective role of hsp70 in cardiovascular disorders.³⁰ For example, transgenic mice overexpressing hsp70 show enhanced resistance to ischemic injury,^{31 32} and increased expression of hsp70 in atherosclerotic lesions may be beneficial for arterial smooth muscle cell survival.³³ Our studies raise additional questions with respect to the role of hsp's in vascular tissue; ie, would overexpression of hsp70 in the arterial wall confer resistance to hemodynamic stress? And what if any relationship exists between hsp70 expression and the development of chronic hypertension? The search for answers to these questions poses current and future challenges for us and other researchers in the field.

	Selected Abbreviations and Acronyms

 

BP = blood pressure HSE = heat shock element HSF = heat shock transcription factor hsp = heat shock protein

	Footnotes

 
Reprint requests to Dr Nikki J. Holbrook, Section on Gene Expression and Aging, National Institute on Aging, National Institutes of Health, Box 31, 4940 Eastern Ave, Baltimore, MD 21224.

Received October 10, 1995; first decision November 6, 1995; first decision February 27, 1996;

	References

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