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Mechanisms of Ageing and Development 123 (2002) 1605 /1615 www.elsevier.com/locate/mechagedev
Paradoxical increase of heat-shock response with age in a substrain of F344 rats: comparison between F344/DuCrj and F344/Jcl Ryoya Takahashi a,, Emi Toyoda a, Yasunobu Aoki b, Kazuo T. Suzuki c, Sataro Goto a a
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan Research Center for Environmental Risk, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan c Graduate School of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi, Inage, Chiba 263-8522, Japan
b
Abstract The ability of hepatocytes isolated from young (7 /10 months) and old (31 months) male F344/Jcl and F344/DuCrj rats to express heat shock protein (hsp) 27, hsp70 and hsp90 was determined after a mild heat shock (42.5 8C for 30 min). The induction of these three mRNA levels by the heat shock was 50 /80% lower in hepatocytes isolated from old F344/Jcl rats than in those from young rats. However, the hepatocytes from old F344/DuCrj showed a marked increase (200 /250%) in the induction of hsp mRNAs by heat shock when compared to cells from young rats. Because heat shock transcription factor (HSF) plays a critical role in regulating the transcription of hsp genes, the effect of age on the binding activity HSF to heat shock element (HSE) was also studied. Again, the induction of binding activity of HSF to HSE was significantly increased with age in hepatocytes from F344/DuCrj rats while the reverse was true for the cells from F344/Jcl. The induced levels of hsp mRNAs were positively correlated with the binding activity of HSF to HSE in hepatocyte extracts from both F344 substrains, suggesting that the diverse age-related changes of heat-shock response in F344 substrains occurs in HSF activity. The contradictory age-related change in the heat-shock response is discussed with the differences in biochemical and genetic properties of substrains of F344 rats. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Substrain difference; F344 rat; Heat shock protein; Heat shock factor; Hepatocyte; Liver
1. Introduction Chemically and/or conformationally altered proteins accumulate in various tissues of aged
Corresponding author. Tel./fax: /81-47-472-1562 E-mail address: [email protected] (R. Takahashi).
animals (Stadtman, 2001; Goto et al., 2002; Hensley and Floyd, 2002). We have previously found that heat-labile enzymes accumulated in tissues of mice (Crj:BDF1 and ddY) and rats (F344/DuCrj and Wistar STD) with age (Takahashi et al., 1985a,b; Takahashi and Goto, 1987). Altered proteins may be harmful to cellular functions, especially when cells are exposed to
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elevated temperature that can further generate denatured proteins within cells. To maintain normal cellular functions, such proteins should be renatured or removed from cells (Goto et al., 2001, 2002; Tavernarakis and Driscoll, 2002). It is well known that a set of proteins called heat shock proteins (HSPs) is induced by hyperthermia and other stresses (ethanol, amino acid analogs, heavy metals, free radicals, etc.) (Lindquist and Craig, 1988; Fink, 1999). Most of these inducers have a common property of causing denaturation of proteins within cells. Hence, a function of HSPs has been thought to be to bind denatured or abnormal proteins and renature the proteins and/ or subsequent cellular deterioration. The major HSPs can be grouped into four major classes (HSP27, HSP70, HSP90 and HSP110) based on their approximate molecular weights and extent of homology (Lindquist and Craig, 1988; Fink, 1999). Some hsps, such as the 70-kDa cognate hsp (HSC70) and HSP90, are constitutively expressed in relatively high amounts in non-stressed cells and their synthesis is only moderately enhanced following heat shock. Constitutively expressed HSPs play an essential role in normal cellular growth and homeostasis. In contrast, the inducible HSPs play a crucial role in the acquisition of thermotolerance (Fink, 1999). Involvement in thermotolerance of HSP70, which is the most prominent HSP expressed after heat shock, is well characterized in various cells. For example, it was found that inactivation of HSP70 by injection of specific antibodies or reducing its expression by genetic means increased heat lability (Riabowol et al., 1988). Although the physiological importance of the induction of a set of HSPs by heat shock is not fully clear, recent studies suggested that the combined action of various HSPs is included in both protection against as well as better recovery from heat-induced cellular damage (Stege et al., 1995). Thus, the induction of a set of HSPs ensures survival under stressful conditions which can otherwise lead to irreversible damage and ultimate death of cells. An alteration in the capability of cells to express HSPs could be physiologically important in aging because all living organisms show a reduced ability to respond to stress with increasing age. Although
the induction of HSPs has been studied extensively in in vitro aged cells and various cells or tissues of aging animals, most of the studies have focused on hsp70. Studies from numerous laboratories have shown that the induction of hsp70 expression decreases with age in most tissues, e.g. hepatocytes, brain, heart, lung, skin, and lymphocytes from rat, lymphocytes from monkey and humans, and fibroblasts from humans (Heydari et al., 1994; Soti and Csermely, 2000). In addition, studies with cells in culture have shown that the induction of hsp70 expression by heat shock decreases with cell senescence in vitro (Heydari et al., 1994; Verbeke et al., 2000). At present, an age-related decline in the induction of hsp70 expression by hyperthermia appears to be a common phenomenon in most cells and tissues of mammals. Only limited information, however, is now available on age-related changes in the expression of other hsp genes such as hsp27 and hsp90 (Heydari et al., 1994; Soti and Csermely, 2000). In this work, the ability to express hsp27, hsp70 and hsp90 was investigated after a mild heat shock in hepatocytes isolated from young and old male F344/Jcl and F344/DuCrj rats. We found that the all these hsp mRNAs induced by heat shock were reduced in hepatocytes from F344/Jcl but increased in the cells from F344/DuCrj rats, even though they are phylogenical siblings (Tanaka et al., 2000). We discussed the relation between the diverse age-related changes in the heat-shock response and the other biochemical and genetic differences in these substrains of F344 rats.
2. Materials and methods 2.1. Animals and diets Male specific pathogen-free (SPF) rats (F344/ DuCrj) were obtained at 4 /5 weeks of age from Charles River Japan, and were maintained in the animal facility of Toho University under SPF conditions as described previously. The rats had a mean life span of about 29 months (Takahashi and Goto, 1987). This strain was established by Dr Dunning (Du), and passed to Charles River Laboratories (Wilmington, MA; CRL) and to
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Charles River Japan (CRJ) (see the latest CRJ catalogue for the animal origin). Male SPF F344/Jcl rats were purchased at the same age from Japan Clea (Tokyo, Japan), and maintained in the animal facility of the National Institute for Environmental Studies under SPF conditions as described previously (Sagai et al., 1984). The rats had a mean life span of about 27 months. The F344/Jcl strain originated from CRL, was passed to Yakult Central Institute (Tokyo, Japan), and then to Clea Japan (JCL) (see the latest JCL catalogue for the animal origin). 2.2. Isolation of hepatocytes and heat shock treatment Rat hepatocytes were isolated by in situ perfusion of the liver with collagenase as reported previously (Ishigami and Goto, 1990). A hepatocyte preparation with high (over 95%) viability was obtained by Percoll (Pharmacia) gradient centrifugation as described by Heydari et al. (Heydari et al., 1993). All procedures for handling the rats were performed in accordance with the guidelines for animal experiments of the Faculty of Pharmaceutical Sciences, Toho University, and were approved by the committee of the Faculty. We carried out individual heat shock experiments in parallel for young and old animals to minimize variations due to experimental conditions. For heat shock treatment, hepatocytes were suspended in Eagle’s minimal essential medium supplemented with 1% bovine serum albumin and incubated with shaking under an atmosphere of oxygen /carbon dioxide (95:5) at 42.5 8C for 30 min and were then returned to 37 8C. The control non-heat shocked cells were incubated at 37 8C throughout the experiment (Heydari et al., 1993). 2.3. RNA isolation and Northern blot hybridization Total RNA was isolated from hepatocytes using the acid guanidinium thiocyanate /phenol/chloroform method as described by Chomczynski and Sacchi (1987). For Northern blot analysis, the total RNA (10 mg) was electrophoresed on a 1.5% denaturing agarose gel and then transferred to Nylon 1 membrane (Gibco RBL) and fixed by UV
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irradiation. The relative level of mRNA was measured using an appropriate 32P-labeled cDNA or a synthesized anti-sense oligonucleotide (40 mer) as probes (see below). The Northern blot signals were visualized with an Imaging Plate (Fuji Film). The relative amounts of mRNAs were determined by scanning the signals in a Bioimaging Analyzer BAS 2000 (Fuji Film) (Semsei and Goto, 1997). 2.4. Gel mobility shift assay Whole-cell extracts containing 10 mg of protein were incubated with 50 pg of 5?-end-labeled, double-stranded heat shock element (HSE) oligonucleotide or SP-1 oligonucleotide, and subjected to a gel shift assay on a native 4% polyacrylamide gel essentially as described by Heydari et al. (1993). 2.5. Western blot analysis Proteins in hepatocyte homogenates were separated by SDS-polyacrylamide gel electrophoresis (10% gel), transferred to Immobilon PVDF transfer membrane, reacted with the anti-HSP70 (clone C92F3A-5, Stress Gen) or anti-HSC70 (clone 1B5, Stress Gen) monoclonal antibodies then with [125I]protein A (ICN). The immunological signals were visualized with the Imaging Plate (Fuji Film). The relative amounts of HSP70 and HSC70 were determined by scanning the signals in the Bioimaging Analyzer BAS 2000 (Fuji Film) (Nagai et al., 2000). 2.6. Molecular probes The cDNAs for hsp27 and hsp90 isolated from a Chinese hamster ovary cell line were kindly provided by Dr Albert J. Fornace, Jr. (National Cancer Institute, Bethesda, MD). The cDNAs for b-actin, albumin and 18S rRNA were provided by Dr Kiyoto Motojima (Meiji Pharmaceutical University, Japan). The anti-sense oligonucleotide sequences used for Northern blot analysis were the following: hsp70 mRNA (inducible): 5?CGCCTGGGCCCCGAAGCCCCCAGCCCCGGGAGCACCCGCA-3?; hsc70 mRNA (constitu-
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tively expressed hsp70), 5?-TATTCCATGTTACTTTTTGGGTCCCTGTGGAACAAAGCTA-3?. The sequence of a double-strand oligonucleotide probe for a gel shift assay was the following: HSE, 5?-CTAGAAGCTTCTAGAAGCTTCTAG-3? (Heydari et al., 1993), SP-1, 5?-ATTCGATCGGGGCGGGGCGAGC-3? (Promega Co.). 2.7. Statistical analysis All values were expressed as mean9/S.E.M. Statistical significance between means were determined using analysis of variance (ANOVA), followed by Fisher’s least significant difference test (Sokal and Rohlf, 1981). The significance level was set at P B/0.05.
3. Results Representative Northern blots of RNA isolated from the hepatocytes of young F344/DuCrj are presented in Fig. 1. No detectable signal for hsp27 or hsp70 mRNAs was observed in non-heat shocked cells. A mild heat shock of 42.5 8C for 30 min, which had no significant effect on the viability or rat hepatocytes (data not shown), resulted in a marked increase in the level of the hsp mRNAs (Fig. 1). The near maximum level of these hsp mRNAs was reached at about 2 h after heat shock (data not shown). Hsp90 is constitutively expressed in relatively high amounts and only slightly induced by hyperthermia. The level of hsp90 mRNA was increased by about two-fold. The levels of albumin and actin mRNA were not affected by the heat shock. Fig. 2 shows the time course of the induction of hsp27, hsp70 and hsp90 mRNAs by the heat shock in hepatocytes isolated from young and old F344/ Jcl and F344/DuCrj rats. The time courses for the induction of the three hsp mRNAs were similar for hepatocytes isolated from substrains of both young and old rats. In F344/Jcl rats, the level of these mRNAs induced by heat shock was significantly lower for the cells isolated from the old animals. For example, the level of hsp27 mRNA in hepatocytes from old rats was about 80% less than
those in cells from young ones. Similar age-related decline in the induction of hsp mRNA expression was observed for hsp70 mRNA. Although there was no significant difference in the level of constitutively expressed hsp90 mRNA in the liver of young and old rats, the induction of hsp90 mRNA was significantly reduced with age: e.g. about 3-fold and 1.7-fold increase in the cells of young and old rats, respectively. In contrast, the level of these hsp mRNAs induced by a similar heat shock in the cells from old F344/DuCrj was approximately 2-fold higher than that observed in hepatocytes from young rats. Age-associated relative induction of hsp27, hsp70 and hsp90 mRNAs was compared between hepatocytes isolated from F344/Jcl and F344/ DuCrj rats (Fig. 3). Even though the relative level of hsp27 mRNA in the cells from young F344/ DuCrj rats was slightly though not significantly lower compared to young F344/Jcl rats, the level in the cells from old F344/Jcl was still significantly lower (about 60%, P B/0.05) compared to young F344/DuCrj rats. A similar tendency was observed in the level of hsp70 mRNA. There was no significant difference in the level of hsp90 mRNA rats between young F344/DuCrj and F344/Jcl rats. The level of hsp90 mRNA in old F344/Jcl rats was significantly lower even in comparison with that in young F344/DuCrj rats. Furthermore, the level in old F344/DuCrj rats was significantly higher than that in young F344/Jcl rats. The age-related change in the induction of hsp mRNAs is most probably due to a change in the transcription of hsp genes because the expression of hsp by heat shock is regulated primarily at the level of transcription (Morimoto et al., 1992; Wu, 1995). The transcription factor HSF plays a critical role in regulating the transcription of heat shock gene. We therefore studied the effect of age on the HSF /HSE binding activity in the extracts of hepatocytes from young and old F344/Jcl and F344/DuCrj rats. Fig. 4 shows the time course of the change of HSF /HSE binding activity in whole cell extracts of hepatocytes isolated from young F344/DuCrj rats. No significant change of the activity was observed in the cells incubated at 37 8C although a heat shock of 42.5 8C for 30 min resulted in a
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Fig. 1. Effect of heat-shock on mRNA levels of hsps, actin and albumin in hepatocytes isolated from young male F344/DuCrj rats. (a) Representative Northern blot of total RNA in hepatocytes isolated from male F344/DuCrj rats. Hepatocytes were heated for 30 min at 42.5 8C and then incubated at 37 8C. The control, non-heat shocked cells were maintained at 37 8C throughout the experiment. The mRNA level was analyzed by Northern blot using an appropriate probe as described in Section 2. (b) The levels of mRNAs were quantified by a Bioimaging Analyzer BAS 2000 (Fuji Film). The data are expressed as the percentage of the maximum mRNA level. Each point represents mean9/S.D. of data obtained from five rats. HS, heat-shocked; C, control; Alb, albumin.
marked increase in the level of this activity. The maximum level of the activity was observed after about 30 min of heat shock. The heat shock treatment used in this study did not affect the level of SP1, another cis-acting element, binding activity in hepatocytes (Fig. 4). The activity of HSF/HSE binding in the extracts prepared from hepatocytes 30 min after heat shock in the two substrains is shown in Fig. 5. The HSE-binding activity was reduced by about 40% in hepatocytes isolated from old F344/Jcl rats compared to young animals of the same substrain, whereas it was increased by about 30% in F344/
DuCrj rats. Thus, the age-related change in the induction of hsp mRNAs was in parallel with a decline in HSF /HSE binding activity in both substrains. To ensure that the change in HSF /HSE binding alters the induction of all hsp mRNAs, we compared the HSF/HSE binding activity 30 min after heat shock and hsp mRNA level 2 h after heat shock in cells isolated from individual animals of both substrains. Significant linear correlation was found between the activity of HSF /HSE binding and the induction level of hsp27 mRNA as well as hsp70 and hsp90 mRNAs in F344/DuCrj
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Fig. 2. Effect of age on the induction of hsp27, hsp70 and hsp90 mRNAs by heat shock in hepatocytes from male F344/Jcl and F344/ DuCrj rats. Hepatocytes isolated from young and old F344/Jcl (left) and F344/DuCrj (right) rats were heated as described in the legend to Fig. 1. The relative levels of mRNA were analyzed by Northern blot as described in Section 2. The data are expressed as the percentage of the maximum mRNA level. Each point represents mean9/S.D. of data obtained from four to five rats. HS, heat-shocked (k, m); C, control (I, j); Y, young (7 /10 months old, k, I); O, old (31 months old, m, j).
rats (data not shown). No correlation was observed between the levels of hsp mRNAs and the activity of SP1 in the hepatocyte isolated from the individual rats (data not shown), suggesting that the change in HSF /HSE binding in the hepatocyte extracts was not an artifact of the preparation of the extract or the assay conditions. Similar results were obtained for F344/Jcl rats (data not shown). These results suggest that the age-related change in
the expression of the hsp genes by heat shock is primarily due to alteration of HSF /HSE binding activity. To determine whether hsp mRNA transcript increase with age in F344/DuCrj rats is accompanied by elevated levels of the corresponding proteins, we also measured the protein level of HSP70 by Western blot. Although HSP70 protein was undetectable in non-heat shocked hepato-
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Fig. 3. Comparison of the induction levels of hsp27, hsp70 and hsp90 mRNAs by heat shock in hepatocytes from male F344/DuCrj and F344/Jcl rats. The levels of induction of hsp27, hsp70 and hsp90 mRNAs 120 min after heat-shock (30 min, 42.5 8C) in hepatocytes isolated from young and old F344/DuCrj and F344/Jcl rats are compared. The data normalized to the average level of mRNA in young F344/Jcl rats as 100% represent mean9/S.D. of data obtained from four to five rats. (a) Significantly different from young F344/Jcl, (b) significantly different from old F344/Jcl, (c) significantly different from young F344/DuCrj (P B/0.05).
cytes, the level of the protein induced by heat shock was remarkably higher in hepatocytes from old rats than in the young counterparts (Fig. 6). Thus, the increased hsp70 mRNA level in hepatocytes by heat shock in old F344/DuCrj rats was further verified by the increase in amount of HSP70 protein.
4. Discussion Age-related change in the induction of hsps, mostly hsp70, has been studied extensively using cells or tissues from a variety of animals: fruit fly, rodent, monkey and human (Heydari et al., 1994). Most studies have shown that the induction of
Fig. 4. Induction of HSF /HSE binding activity by heat shock in hepatocytes from male F344/DuCrj rats. (a) Hepatocytes were heated as described in the legend to Fig. 1. HSF and SP1 oligonucleotide binding activities in whole-cell extracts of hepatocytes isolated from young F344/DuCrj rats was determined by a gel mobility shift assay using 32P-labeled oligonucleotides as described in Section 2. For the competition experiment, cell extracts were preincubated with a 250-fold molar excess of non-radioactive oligonucleotide at 25 8C for 5 min prior to the addition of 32P-labeled oligonucleotide. (b) Values for HSF and SP1 oligonucleotide binding activities were expressed as percentages of those observed in the extract of heat shocked hepatocytes. Each point represents mean9/S.D. of data obtained from four to five rats. HS, heat-shocked; C, control.
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Fig. 5. Effect of age on the induction of HSF /HSE binding activity by heat shock in hepatocytes from male F344/Jcl and F344/DuCrj rats. (a) Hepatocytes isolated from young and old F344/Jcl and F344/DuCrj rats were heated for 30 min at 42.5 8C. Whole cell extract obtained from the hepatocytes were analyzed by a gel mobility shift assay using 32P-labeled HSE oligonucleotide. (b) The data normalized to the average HSF /HSE binding activity in young F344/Jcl rats as 100% represent mean9/S.D. of data obtained from four to five rats. (a) Significantly different from young F344/Jcl, (b) significantly different from old F344/Jcl, (c) significantly different from young F344/DuCrj (P B/0.05).
Fig. 6. Effect of age on the induction of HSP70 by heat shock in hepatocytes from male F344/DuCrj. Hepatocytes isolated from young and old F344/DuCrj rats were heated for 30 min at 42.5 8C and then incubated for 2 h at 37 8C. The homogenates of hepatocytes were analyzed by a Western blot using anti-HSC70 and anti-HSP70 monoclonal antibodies as described in Section 2.
hsp70 expression decreases with increasing age in many cells and tissues except fruit fly (Drosophila ) (Fleming et al., 1992; Wheeler et al., 1999). So far, most studies on age-related changes in the induction of hsp expression by hyperthermia in rodents have been carried out for male F344 (Fischer) or Wistar rats obtained from colonies maintained by Harlan Sprague Dawley (HSD, Indianapolis, IN), Charles River Laboratory (CRL, Wilmington,
MA) for the National Institute on Aging (NIA) or the Gerontology Research Center at NIA. For example, in Richardson’s laboratory, age-related changes of the heat shock response have been intensively studied in hepatocytes (Heydari et al., 1993, 1995, 1996, 2000; Wu et al., 1993) and lymphocytes (Pahlavani et al., 1995) isolated from F344 rats obtained from HSD. They showed that the induction of hps70 expression by heat shock is significantly reduced in hepatocytes and lymphocytes from old rats and that the reduced expression is due to a deficit in the transcription of the hsp70 gene. Pardue et al. (1992) reported that F344 rats obtained from NIA showed a significant age-related decrease in induction of hsp70 mRNA in hippocampus when the rats were exposed to hyperthermia. A decline in the induction of hsp70 mRNA by hyperthermia has also been observed in primary cultures of fibroblasts isolated from the skin or lung of Wistar rats obtained from NIA (Fargnoli et al., 1990). Thus, previous studies from different laboratories on F344 and Wistar rats demonstrated that the induction hsp70 expression decreases generally with age in different tissues
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and cells. So far, the age-related decline in the induction of hsp70 expression by hyperthermia has appeared to be a common phenomenon in rodent tissues. In this study we investigated the effect of age on the heat induced expression of three heat shock protein genes (hsp27, hsc70 and hsp90) in hepatocytes isolated from two different F344 substrains: F344/Jcl and F344/DuCrj. We found that hepatocytes from F344/Jcl rats showed a marked decrease with age in the induced levels of these hsp mRNAs by the heat shock. Remarkably, however, the cells from F344/DuCrj rats showed an increase rather than decrease by the same treatment. Furthermore, we found that difference in age-related changes of the heat-shock response closely correlated with the change in HSF activity. To our knowledge, such an unusual heat shock response in rodent cells has not been reported previously. How the opposite response was developed in different F344 substrains during aging is unclear. However, such difference was also observed in antioxidant enzyme activities, such as superoxide dismutase (SOD) in the liver. For example, male F344 rats obtained from NIA showed the de-
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creases (40 /50%) in the activities of catalase and Cu/Zn-SOD in the liver with age (Semsei et al., 1989; Rao et al., 1990), whereas F344/DuCrj obtained from CRJ showed a decrease (about 40%) in the activity of catalase but a significant increase (about 20%) in the activity of Cu/Zn-SOD in this organ with age (Carrillo et al., 1992) (Table 1). Although the mechanism responsible for the age-related substrain differences in the heat shock response and SOD activity is unclear, it might be due to the difference in the genetic background between F344/DuCrj and other F344 substrains. To date, no information is available about the polymorphism of genes that might alter the expressions of hsps and SOD in different F344 substrains. Interestingly, it has been reported that F344/DuCrj rats maintained at CRJ specifically lack dipeptidyl peptidase IV (DPPIV) activity, whereas Fischer 344 (F344) rats from different sources within US (HSD, CRL and NIH) (Tiruppathi et al., 1990), F344/Jcl rats from CRL (Nagakura et al., 2001) and Wistar rats (IwakiEgawa et al., 1991) possess normal levels of the DPPIV activity (Table 1). Sequence analysis of the
Table 1 Effect of age on the induction of heat shock protein expression and the activities of anti-oxidant enzymes in the liver of different male F344 rats Strain
Animal source
Origin1 MLS (month)
DPPIV
Change with age Hsp70 mRNA le- Cu/Zn SOD acvel tivity
F344/ CRJ DuCrj F344/Jcl JCL Fischer 344 HSD (NIA)
Catalase activity
GPX activity
Du
292
Negative5 Inc7
Inc9
Dec9
No change9
Du N
273 274
Positive6 Positive5
/ Dec4,10
/ Dec4,10
/ No change4
Dec7 Dec8
CRJ, Charles River Japan Inc. (Japan); JCL, Clea Japan, Inc.(Japan); HSD, Harlan Sprague Dawley (Indianapolis, IN); NIA, the National Institute of Ageing; DPPIV, dipeptidy peptidase IV; SOD, superoxide dismutase; GPX, glutathione peroxidase. 1 Tanaka et al. (2000). 2 Takahashi and Goto (1987). 3 Data from JCL. 4 Rao et al. (1990). 5 Tiruppathi et al. (1990). 6 Nagakura et al. (2001). 7 Takahashi et al. (this study). 8 Heydari et al. 9 Carrillo et al. (1992). 10 Semsei et al. (1989).
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DPPIV gene revealed that a point mutation exists in F344/DuCrj that results in substitution of Gly633 to Arg at the active-site (Tsuji et al., 1992). DPPIV exists in plasma and on the surface of various types of cells, particularly in the liver, kidney and small intestine and plays an important role in cleavage and inactivation of biologically active peptides, many of which are implicated in regulation of immune, inflammatory, nervous and endocrine functions (Mentlein et al., 1993; Yaron and Naider, 1993). For example, DPPIV degrades glucagon-like peptide-1 (incretin) which induces glucose-dependent insulin secretion. The DPPIVdeficient F344/DuCrj strain shows lower blood glucose levels with enhanced insulin secretion than DPPIV-positive rats in an oral glucose tolerance test (Nagakura et al., 2001). How such altered glucose metabolism affects the aging process is not known. However, a previous study among a limited number of centenarians living in South Italy showed this group to have a more preserved glucose tolerance and insulin action than aged subjects (/75 years) (Paolisso et al., 1996). Low incidence of various tumors including leukemia and longer life span of the DPPIV-deficient F344/ DuCrj compared with DPPIV-positive rats (F344/ N, /DuCrl and /Jcl) (Tanaka et al., 2000) might be due to altered glucose metabolism via insulin secretion. We do not know whether the altered age-related changes of the heat shock response and SOD activity are consequences of compensatory changes in F344/DuCrj rats to adapt to the inherent DPPIV deficiency or other unidentified genetic polymorphisms. Further detailed genetic and biochemical studies will be required to determine the mechanism of the difference of agerelated parameters among different F344 substrains. Our present results and previous findings by others (Carrillo et al., 1992; Tanaka et al., 2000) suggest that the significance of various physiological and biochemical changes with age observed in F344 rats needs to be carefully evaluated even though they are phylogenical siblings. Comparative studies among different F344 substrains including F344/DuCrj may be useful to verify
what age-related changes are public (common) or private (unique) (Martin et al., 1996).
References Carrillo, M.C., Kanai, S., Sato, Y., Kitani, K., 1992. Agerelated changes in antioxidant enzyme activities are region and organ, as well as sex, selective in the rat. Mech. Ageing Dev. 65, 187 /198. Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolation by acid guanigium thiocyanate /phenol /chloroform extracts. Anal. Biochem. 162, 156 /159. Fargnoli, J., Kunisada, T., Fornace, A.J.J., Schneider, E.L., Holbrook, N.J., 1990. Decreased expression of heat shock protein 70 mRNA and protein after heat treatment in cells of aged rats. Proc. Natl. Acad. Sci. USA 87, 846 /850. Fink, A.L., 1999. Chaperone-mediated protein folding. Physiol. Rev. 79, 425 /449. Fleming, J.E., Reveillaud, I., Niedzwiecki, A., 1992. Role of oxidative stress in Drosophila aging. Mutat. Res. 275, 267 / 279. Goto, S., Takahashi, R., Kumiyama, A., Radak, Z., Hayashi, T., Takenouchi, M., Abe, R., 2001. Implications of protein degradation in aging. Ann. N.Y. Acad. Sci. 928, 54 /64. Goto, S., Takahashi, R., Araki, S., Nakamoto, H., 2002. Dietary restriction initiated in late adulthood can reverse age-related alteration of protein and protein metabolism. Ann. NY. Acad. Sci. 959, 50 /56. Hensley, K., Floyd, R.A., 2002. Reactive oxygen species and protein oxidation in aging: a look back, a look ahead. Arch. Biochem. Biophys. 397, 377 /383. Heydari, A.R., Wu, B., Takahashi, R., Strong, R., Richardson, A., 1993. Expression of heat shock protein 70 is altered by age and diet at the level of transcription. Mol. Cell. Biol. 13, 2909 /2918. Heydari, A.R., Takahashi, R., Gutsmann, A., You, S., Richardson, A., 1994. Hsp70 and aging. Experientia 50, 1092 /1098. Heydari, A.R., Conrad, C.C., Richardson, A., 1995. Expression of heat shock genes in hepatocytes is affected by age and food restriction in rats. J. Nutr. 125, 410 /418. Heydari, A.R., You, S., Takahashi, R., Gutsmann, A., Sarge, K.D., Richardson, A., 1996. Effect of caloric restriction on the expression of heat shock protein 70 and the activation of heat shock transcription factor 1. Dev. Genet. 18, 114 /124. Heydari, A.R., You, S., Takahashi, R., Gutsmann-Conrad, A., Sarge, K.D., Richardson, A., 2000. Age-related alterations in the activation of heat shock transcription factor 1 in rat hepatocytes. Exp. Cell Res. 256, 83 /93. Ishigami, A., Goto, S., 1990. Age-related change in the degradation rate of ovalbumin microinjected into mouse liver parenchymal cells. Arch. Biochem. Biophys. 277, 189 / 195. Iwaki-Egawa, S., Watanabe, Y., Fujimoto, Y., 1991. Identification of a rat liver dipeptidyl aminopeptidase IV with a liver
R. Takahashi et al. / Mechanisms of Ageing and Development 123 (2002) 1605 /1615 plasma membrane glycoprotein (gp110). A study using dipeptidyl aminopeptidase IV-deficient rats. FEBS Lett. 286, 167 /170. Lindquist, S., Craig, E.A., 1988. The heat-shock proteins. Annu. Rev. Genet. 22, 631 /677. Martin, G.M., Austad, S.N., Johnson, T.E., 1996. Genetic analysis of ageing: role of oxidative damage and environmental stresses. Nat. Genet. 13, 26 /34. Mentlein, R., Gallwitz, B., Schmidt, W.E., 1993. Dipeptidylpeptidase IV hydrolyses gastric inhibitory polypeptide, glucagon-like peptide-1(7-36)amide, peptide histidine methionine and is responsible for their degradation in human serum. Eur. J. Biochem. 214, 829 /835. Morimoto, R.I., Sarge, K.D., Abravaya, K., 1992. Transcriptional regulation of heat shock genes. A paradigm for inducible genomic responses. J. Biol. Chem. 267, 21987 / 21990. Nagai, M., Takahashi, R., Goto, S., 2000. Dietary restriction initiated late in life can reduce mitochondrial protein carbonyls in rat liver: Western blot studies. Biogerontology 1, 321 /328. Nagakura, T., Yasuda, N., Yamazaki, K., Ikuta, H., Yoshikawa, S., Asano, O., Tanaka, I., 2001. Improved glucose tolerance via enhanced glucose-dependent insulin secretion in dipeptidyl peptidase IV-deficient Fischer rats. Biochem. Biophys. Res. Commun. 284, 501 /506. Pahlavani, M.A., Harris, M.D., Moore, S.A., Weindruch, R., Richardson, A., 1995. The expression of heat shock protein 70 decreases with age in lymphocytes from rats and rhesus monkeys. Exp. Cell Res. 218, 310 /318. Paolisso, G., Gambardella, A., Ammendola, S., D’Amore, A., Balbi, V., Varricchio, M., D’Onofrio, F., 1996. Glucose tolerance and insulin action in healthy centenarians. Am. J. Physiol. 270, E890 /E894. Pardue, S., Groshan, K., Raese, J.D., Morrison-Bogorad, M., 1992. Hsp70 mRNA induction is reduced in neurons of aged rat hippocampus after thermal stress. Neurobiol. Aging 13, 661 /672. Rao, G., Xia, E., Richardson, A., 1990. Effect of age on the expression of antioxidant enzymes in male Fischer F344 rats. Mech. Ageing Dev. 53, 49 /60. Riabowol, K.T., Mizzen, L.A., Welch, W.J., 1988. Heat shock is lethal to fibroblasts microinjected with antibodies against hsp70. Science 242, 433 /436. Sagai, M., Ichinose, T., Kubota, K., 1984. Studies on the biochemical effects of nitorgen dioxide. Toxicol. Appl. Pharmacol. 73, 444 /456. Semsei, I., Goto, S., 1997. Expression of mRNAs of pancreatic and L type RNase inhibitors as a function of age in different tiddues of SAMP8 and BDF1 mice. Mech. Ageing Dev. 97, 249 /261. Semsei, I., Rao, G., Richardson, A., 1989. Changes in the expression of superoxide dismutase and catalase as a
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function of age and dietary restriction. Biochem. Biophys. Res. Commun. 164, 620 /625. Sokal, R.R., Rohlf, F.J., 1981. Biometry. Freeman, New York. Soti, C., Csermely, P., 2000. Molecular chaperones and the aging process. Biogerontology 1, 225 /233. Stadtman, E.R., 2001. Protein oxidation in aging and agerelated diseases. Ann. N.Y. Acad. Sci. 928, 22 /38. Stege, G.J., Brunsting, J.F., Kampinga, H.H., Konings, A.W., 1995. Thermotolerance and nuclear protein aggregation: protection against initial damage or better recovery? J. Cell. Physiol. 164, 579 /586. Takahashi, R., Goto, S., 1987. Age-associated accumulation of heat-labile aminoacyl tRNA synthetases in mice and rats. Arch. Gerontol. Geriatr. 6, 73 /82. Takahashi, R., Mori, N., Goto, S., 1985. Alteration of aminoacyl tRNA synthetases with age: accumulation of heat-labile enzyme molecules in rat liver, kidney and brain. Mech. Ageing Dev. 33, 67 /75. Takahashi, R., Mori, N., Goto, S., 1985. Accumulation of heatlabile elongation factor 2 in the liver of mice and rats. Exp. Gerontol. 20, 325 /331. Tanaka, S., Tamaya, N., Matsuzawa, K., Miyaishi, O., 2000. Differences in survivability among F344 rats. Exp. Anim. 49, 141 /145. Tavernarakis, N., Driscoll, M., 2002. Caloric restriction and lifespan: a role for protein turnover? Mech. Ageing Dev. 123, 215 /229. Tiruppathi, C., Miyamoto, Y., Ganapathy, V., Roesel, R.A., Whitford, G.M., Leibach, F.H., 1990. Hydrolysis and transport of proline-containing peptides in renal brushborder membrane vesicles from dipeptidyl peptidase IVpositive and dipeptidyl peptidase IV-negative rat strains. J. Biol. Chem. 265, 1476 /1483. Tsuji, E., Misumi, Y., Fujiwara, T., Takami, N., Ogata, S., Ikehara, Y., 1992. An active-site mutation (Gly6330/Arg) of dipeptidyl peptidase IV causes its retention and rapid degradation in the endoplasmic reticulum. Biochemistry 31, 11921 /11927. Verbeke, P., Clark, B.F., Rattan, S.I., 2000. Modulating cellular aging in vitro: hormetic effects of repeated mild heat stress on protein oxidation and glycation. Exp. Gerontol. 35, 787 /794. Wheeler, J.C., King, V., Tower, J., 1999. Sequence requirements for upregulated expression of Drosophila hsp70 transgenes during aging. Neurobiol. Aging 20, 545 /553. Wu, C., 1995. Heat shock transcription factors: structure and regulation. Annu. Rev. Cell. Dev. Biol. 11, 441 /469. Wu, B., Gu, M.J., Heydari, A.R., Richardson, A., 1993. The effect of age on the synthesis of two heat shock proteins in the hsp70 family. J. Gerontol. 48, B50 /B56. Yaron, A., Naider, F., 1993. Proline-dependent structural and biological properties of peptides and proteins. Crit. Rev. Biochem. Mol. Biol. 28, 31 /81.
Paradoxical increase of heat-shock response with age in a substrain of F344 rats: comparison between F344/DuCrj and F344/Jcl Ryoya Takahashi a,, Emi Toyoda a, Yasunobu Aoki b, Kazuo T. Suzuki c, Sataro Goto a a
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan Research Center for Environmental Risk, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan c Graduate School of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi, Inage, Chiba 263-8522, Japan
b
Abstract The ability of hepatocytes isolated from young (7 /10 months) and old (31 months) male F344/Jcl and F344/DuCrj rats to express heat shock protein (hsp) 27, hsp70 and hsp90 was determined after a mild heat shock (42.5 8C for 30 min). The induction of these three mRNA levels by the heat shock was 50 /80% lower in hepatocytes isolated from old F344/Jcl rats than in those from young rats. However, the hepatocytes from old F344/DuCrj showed a marked increase (200 /250%) in the induction of hsp mRNAs by heat shock when compared to cells from young rats. Because heat shock transcription factor (HSF) plays a critical role in regulating the transcription of hsp genes, the effect of age on the binding activity HSF to heat shock element (HSE) was also studied. Again, the induction of binding activity of HSF to HSE was significantly increased with age in hepatocytes from F344/DuCrj rats while the reverse was true for the cells from F344/Jcl. The induced levels of hsp mRNAs were positively correlated with the binding activity of HSF to HSE in hepatocyte extracts from both F344 substrains, suggesting that the diverse age-related changes of heat-shock response in F344 substrains occurs in HSF activity. The contradictory age-related change in the heat-shock response is discussed with the differences in biochemical and genetic properties of substrains of F344 rats. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Substrain difference; F344 rat; Heat shock protein; Heat shock factor; Hepatocyte; Liver
1. Introduction Chemically and/or conformationally altered proteins accumulate in various tissues of aged
Corresponding author. Tel./fax: /81-47-472-1562 E-mail address: [email protected] (R. Takahashi).
animals (Stadtman, 2001; Goto et al., 2002; Hensley and Floyd, 2002). We have previously found that heat-labile enzymes accumulated in tissues of mice (Crj:BDF1 and ddY) and rats (F344/DuCrj and Wistar STD) with age (Takahashi et al., 1985a,b; Takahashi and Goto, 1987). Altered proteins may be harmful to cellular functions, especially when cells are exposed to
0047-6374/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 4 7 - 6 3 7 4 ( 0 2 ) 0 0 0 9 6 - 9
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elevated temperature that can further generate denatured proteins within cells. To maintain normal cellular functions, such proteins should be renatured or removed from cells (Goto et al., 2001, 2002; Tavernarakis and Driscoll, 2002). It is well known that a set of proteins called heat shock proteins (HSPs) is induced by hyperthermia and other stresses (ethanol, amino acid analogs, heavy metals, free radicals, etc.) (Lindquist and Craig, 1988; Fink, 1999). Most of these inducers have a common property of causing denaturation of proteins within cells. Hence, a function of HSPs has been thought to be to bind denatured or abnormal proteins and renature the proteins and/ or subsequent cellular deterioration. The major HSPs can be grouped into four major classes (HSP27, HSP70, HSP90 and HSP110) based on their approximate molecular weights and extent of homology (Lindquist and Craig, 1988; Fink, 1999). Some hsps, such as the 70-kDa cognate hsp (HSC70) and HSP90, are constitutively expressed in relatively high amounts in non-stressed cells and their synthesis is only moderately enhanced following heat shock. Constitutively expressed HSPs play an essential role in normal cellular growth and homeostasis. In contrast, the inducible HSPs play a crucial role in the acquisition of thermotolerance (Fink, 1999). Involvement in thermotolerance of HSP70, which is the most prominent HSP expressed after heat shock, is well characterized in various cells. For example, it was found that inactivation of HSP70 by injection of specific antibodies or reducing its expression by genetic means increased heat lability (Riabowol et al., 1988). Although the physiological importance of the induction of a set of HSPs by heat shock is not fully clear, recent studies suggested that the combined action of various HSPs is included in both protection against as well as better recovery from heat-induced cellular damage (Stege et al., 1995). Thus, the induction of a set of HSPs ensures survival under stressful conditions which can otherwise lead to irreversible damage and ultimate death of cells. An alteration in the capability of cells to express HSPs could be physiologically important in aging because all living organisms show a reduced ability to respond to stress with increasing age. Although
the induction of HSPs has been studied extensively in in vitro aged cells and various cells or tissues of aging animals, most of the studies have focused on hsp70. Studies from numerous laboratories have shown that the induction of hsp70 expression decreases with age in most tissues, e.g. hepatocytes, brain, heart, lung, skin, and lymphocytes from rat, lymphocytes from monkey and humans, and fibroblasts from humans (Heydari et al., 1994; Soti and Csermely, 2000). In addition, studies with cells in culture have shown that the induction of hsp70 expression by heat shock decreases with cell senescence in vitro (Heydari et al., 1994; Verbeke et al., 2000). At present, an age-related decline in the induction of hsp70 expression by hyperthermia appears to be a common phenomenon in most cells and tissues of mammals. Only limited information, however, is now available on age-related changes in the expression of other hsp genes such as hsp27 and hsp90 (Heydari et al., 1994; Soti and Csermely, 2000). In this work, the ability to express hsp27, hsp70 and hsp90 was investigated after a mild heat shock in hepatocytes isolated from young and old male F344/Jcl and F344/DuCrj rats. We found that the all these hsp mRNAs induced by heat shock were reduced in hepatocytes from F344/Jcl but increased in the cells from F344/DuCrj rats, even though they are phylogenical siblings (Tanaka et al., 2000). We discussed the relation between the diverse age-related changes in the heat-shock response and the other biochemical and genetic differences in these substrains of F344 rats.
2. Materials and methods 2.1. Animals and diets Male specific pathogen-free (SPF) rats (F344/ DuCrj) were obtained at 4 /5 weeks of age from Charles River Japan, and were maintained in the animal facility of Toho University under SPF conditions as described previously. The rats had a mean life span of about 29 months (Takahashi and Goto, 1987). This strain was established by Dr Dunning (Du), and passed to Charles River Laboratories (Wilmington, MA; CRL) and to
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Charles River Japan (CRJ) (see the latest CRJ catalogue for the animal origin). Male SPF F344/Jcl rats were purchased at the same age from Japan Clea (Tokyo, Japan), and maintained in the animal facility of the National Institute for Environmental Studies under SPF conditions as described previously (Sagai et al., 1984). The rats had a mean life span of about 27 months. The F344/Jcl strain originated from CRL, was passed to Yakult Central Institute (Tokyo, Japan), and then to Clea Japan (JCL) (see the latest JCL catalogue for the animal origin). 2.2. Isolation of hepatocytes and heat shock treatment Rat hepatocytes were isolated by in situ perfusion of the liver with collagenase as reported previously (Ishigami and Goto, 1990). A hepatocyte preparation with high (over 95%) viability was obtained by Percoll (Pharmacia) gradient centrifugation as described by Heydari et al. (Heydari et al., 1993). All procedures for handling the rats were performed in accordance with the guidelines for animal experiments of the Faculty of Pharmaceutical Sciences, Toho University, and were approved by the committee of the Faculty. We carried out individual heat shock experiments in parallel for young and old animals to minimize variations due to experimental conditions. For heat shock treatment, hepatocytes were suspended in Eagle’s minimal essential medium supplemented with 1% bovine serum albumin and incubated with shaking under an atmosphere of oxygen /carbon dioxide (95:5) at 42.5 8C for 30 min and were then returned to 37 8C. The control non-heat shocked cells were incubated at 37 8C throughout the experiment (Heydari et al., 1993). 2.3. RNA isolation and Northern blot hybridization Total RNA was isolated from hepatocytes using the acid guanidinium thiocyanate /phenol/chloroform method as described by Chomczynski and Sacchi (1987). For Northern blot analysis, the total RNA (10 mg) was electrophoresed on a 1.5% denaturing agarose gel and then transferred to Nylon 1 membrane (Gibco RBL) and fixed by UV
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irradiation. The relative level of mRNA was measured using an appropriate 32P-labeled cDNA or a synthesized anti-sense oligonucleotide (40 mer) as probes (see below). The Northern blot signals were visualized with an Imaging Plate (Fuji Film). The relative amounts of mRNAs were determined by scanning the signals in a Bioimaging Analyzer BAS 2000 (Fuji Film) (Semsei and Goto, 1997). 2.4. Gel mobility shift assay Whole-cell extracts containing 10 mg of protein were incubated with 50 pg of 5?-end-labeled, double-stranded heat shock element (HSE) oligonucleotide or SP-1 oligonucleotide, and subjected to a gel shift assay on a native 4% polyacrylamide gel essentially as described by Heydari et al. (1993). 2.5. Western blot analysis Proteins in hepatocyte homogenates were separated by SDS-polyacrylamide gel electrophoresis (10% gel), transferred to Immobilon PVDF transfer membrane, reacted with the anti-HSP70 (clone C92F3A-5, Stress Gen) or anti-HSC70 (clone 1B5, Stress Gen) monoclonal antibodies then with [125I]protein A (ICN). The immunological signals were visualized with the Imaging Plate (Fuji Film). The relative amounts of HSP70 and HSC70 were determined by scanning the signals in the Bioimaging Analyzer BAS 2000 (Fuji Film) (Nagai et al., 2000). 2.6. Molecular probes The cDNAs for hsp27 and hsp90 isolated from a Chinese hamster ovary cell line were kindly provided by Dr Albert J. Fornace, Jr. (National Cancer Institute, Bethesda, MD). The cDNAs for b-actin, albumin and 18S rRNA were provided by Dr Kiyoto Motojima (Meiji Pharmaceutical University, Japan). The anti-sense oligonucleotide sequences used for Northern blot analysis were the following: hsp70 mRNA (inducible): 5?CGCCTGGGCCCCGAAGCCCCCAGCCCCGGGAGCACCCGCA-3?; hsc70 mRNA (constitu-
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tively expressed hsp70), 5?-TATTCCATGTTACTTTTTGGGTCCCTGTGGAACAAAGCTA-3?. The sequence of a double-strand oligonucleotide probe for a gel shift assay was the following: HSE, 5?-CTAGAAGCTTCTAGAAGCTTCTAG-3? (Heydari et al., 1993), SP-1, 5?-ATTCGATCGGGGCGGGGCGAGC-3? (Promega Co.). 2.7. Statistical analysis All values were expressed as mean9/S.E.M. Statistical significance between means were determined using analysis of variance (ANOVA), followed by Fisher’s least significant difference test (Sokal and Rohlf, 1981). The significance level was set at P B/0.05.
3. Results Representative Northern blots of RNA isolated from the hepatocytes of young F344/DuCrj are presented in Fig. 1. No detectable signal for hsp27 or hsp70 mRNAs was observed in non-heat shocked cells. A mild heat shock of 42.5 8C for 30 min, which had no significant effect on the viability or rat hepatocytes (data not shown), resulted in a marked increase in the level of the hsp mRNAs (Fig. 1). The near maximum level of these hsp mRNAs was reached at about 2 h after heat shock (data not shown). Hsp90 is constitutively expressed in relatively high amounts and only slightly induced by hyperthermia. The level of hsp90 mRNA was increased by about two-fold. The levels of albumin and actin mRNA were not affected by the heat shock. Fig. 2 shows the time course of the induction of hsp27, hsp70 and hsp90 mRNAs by the heat shock in hepatocytes isolated from young and old F344/ Jcl and F344/DuCrj rats. The time courses for the induction of the three hsp mRNAs were similar for hepatocytes isolated from substrains of both young and old rats. In F344/Jcl rats, the level of these mRNAs induced by heat shock was significantly lower for the cells isolated from the old animals. For example, the level of hsp27 mRNA in hepatocytes from old rats was about 80% less than
those in cells from young ones. Similar age-related decline in the induction of hsp mRNA expression was observed for hsp70 mRNA. Although there was no significant difference in the level of constitutively expressed hsp90 mRNA in the liver of young and old rats, the induction of hsp90 mRNA was significantly reduced with age: e.g. about 3-fold and 1.7-fold increase in the cells of young and old rats, respectively. In contrast, the level of these hsp mRNAs induced by a similar heat shock in the cells from old F344/DuCrj was approximately 2-fold higher than that observed in hepatocytes from young rats. Age-associated relative induction of hsp27, hsp70 and hsp90 mRNAs was compared between hepatocytes isolated from F344/Jcl and F344/ DuCrj rats (Fig. 3). Even though the relative level of hsp27 mRNA in the cells from young F344/ DuCrj rats was slightly though not significantly lower compared to young F344/Jcl rats, the level in the cells from old F344/Jcl was still significantly lower (about 60%, P B/0.05) compared to young F344/DuCrj rats. A similar tendency was observed in the level of hsp70 mRNA. There was no significant difference in the level of hsp90 mRNA rats between young F344/DuCrj and F344/Jcl rats. The level of hsp90 mRNA in old F344/Jcl rats was significantly lower even in comparison with that in young F344/DuCrj rats. Furthermore, the level in old F344/DuCrj rats was significantly higher than that in young F344/Jcl rats. The age-related change in the induction of hsp mRNAs is most probably due to a change in the transcription of hsp genes because the expression of hsp by heat shock is regulated primarily at the level of transcription (Morimoto et al., 1992; Wu, 1995). The transcription factor HSF plays a critical role in regulating the transcription of heat shock gene. We therefore studied the effect of age on the HSF /HSE binding activity in the extracts of hepatocytes from young and old F344/Jcl and F344/DuCrj rats. Fig. 4 shows the time course of the change of HSF /HSE binding activity in whole cell extracts of hepatocytes isolated from young F344/DuCrj rats. No significant change of the activity was observed in the cells incubated at 37 8C although a heat shock of 42.5 8C for 30 min resulted in a
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Fig. 1. Effect of heat-shock on mRNA levels of hsps, actin and albumin in hepatocytes isolated from young male F344/DuCrj rats. (a) Representative Northern blot of total RNA in hepatocytes isolated from male F344/DuCrj rats. Hepatocytes were heated for 30 min at 42.5 8C and then incubated at 37 8C. The control, non-heat shocked cells were maintained at 37 8C throughout the experiment. The mRNA level was analyzed by Northern blot using an appropriate probe as described in Section 2. (b) The levels of mRNAs were quantified by a Bioimaging Analyzer BAS 2000 (Fuji Film). The data are expressed as the percentage of the maximum mRNA level. Each point represents mean9/S.D. of data obtained from five rats. HS, heat-shocked; C, control; Alb, albumin.
marked increase in the level of this activity. The maximum level of the activity was observed after about 30 min of heat shock. The heat shock treatment used in this study did not affect the level of SP1, another cis-acting element, binding activity in hepatocytes (Fig. 4). The activity of HSF/HSE binding in the extracts prepared from hepatocytes 30 min after heat shock in the two substrains is shown in Fig. 5. The HSE-binding activity was reduced by about 40% in hepatocytes isolated from old F344/Jcl rats compared to young animals of the same substrain, whereas it was increased by about 30% in F344/
DuCrj rats. Thus, the age-related change in the induction of hsp mRNAs was in parallel with a decline in HSF /HSE binding activity in both substrains. To ensure that the change in HSF /HSE binding alters the induction of all hsp mRNAs, we compared the HSF/HSE binding activity 30 min after heat shock and hsp mRNA level 2 h after heat shock in cells isolated from individual animals of both substrains. Significant linear correlation was found between the activity of HSF /HSE binding and the induction level of hsp27 mRNA as well as hsp70 and hsp90 mRNAs in F344/DuCrj
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Fig. 2. Effect of age on the induction of hsp27, hsp70 and hsp90 mRNAs by heat shock in hepatocytes from male F344/Jcl and F344/ DuCrj rats. Hepatocytes isolated from young and old F344/Jcl (left) and F344/DuCrj (right) rats were heated as described in the legend to Fig. 1. The relative levels of mRNA were analyzed by Northern blot as described in Section 2. The data are expressed as the percentage of the maximum mRNA level. Each point represents mean9/S.D. of data obtained from four to five rats. HS, heat-shocked (k, m); C, control (I, j); Y, young (7 /10 months old, k, I); O, old (31 months old, m, j).
rats (data not shown). No correlation was observed between the levels of hsp mRNAs and the activity of SP1 in the hepatocyte isolated from the individual rats (data not shown), suggesting that the change in HSF /HSE binding in the hepatocyte extracts was not an artifact of the preparation of the extract or the assay conditions. Similar results were obtained for F344/Jcl rats (data not shown). These results suggest that the age-related change in
the expression of the hsp genes by heat shock is primarily due to alteration of HSF /HSE binding activity. To determine whether hsp mRNA transcript increase with age in F344/DuCrj rats is accompanied by elevated levels of the corresponding proteins, we also measured the protein level of HSP70 by Western blot. Although HSP70 protein was undetectable in non-heat shocked hepato-
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Fig. 3. Comparison of the induction levels of hsp27, hsp70 and hsp90 mRNAs by heat shock in hepatocytes from male F344/DuCrj and F344/Jcl rats. The levels of induction of hsp27, hsp70 and hsp90 mRNAs 120 min after heat-shock (30 min, 42.5 8C) in hepatocytes isolated from young and old F344/DuCrj and F344/Jcl rats are compared. The data normalized to the average level of mRNA in young F344/Jcl rats as 100% represent mean9/S.D. of data obtained from four to five rats. (a) Significantly different from young F344/Jcl, (b) significantly different from old F344/Jcl, (c) significantly different from young F344/DuCrj (P B/0.05).
cytes, the level of the protein induced by heat shock was remarkably higher in hepatocytes from old rats than in the young counterparts (Fig. 6). Thus, the increased hsp70 mRNA level in hepatocytes by heat shock in old F344/DuCrj rats was further verified by the increase in amount of HSP70 protein.
4. Discussion Age-related change in the induction of hsps, mostly hsp70, has been studied extensively using cells or tissues from a variety of animals: fruit fly, rodent, monkey and human (Heydari et al., 1994). Most studies have shown that the induction of
Fig. 4. Induction of HSF /HSE binding activity by heat shock in hepatocytes from male F344/DuCrj rats. (a) Hepatocytes were heated as described in the legend to Fig. 1. HSF and SP1 oligonucleotide binding activities in whole-cell extracts of hepatocytes isolated from young F344/DuCrj rats was determined by a gel mobility shift assay using 32P-labeled oligonucleotides as described in Section 2. For the competition experiment, cell extracts were preincubated with a 250-fold molar excess of non-radioactive oligonucleotide at 25 8C for 5 min prior to the addition of 32P-labeled oligonucleotide. (b) Values for HSF and SP1 oligonucleotide binding activities were expressed as percentages of those observed in the extract of heat shocked hepatocytes. Each point represents mean9/S.D. of data obtained from four to five rats. HS, heat-shocked; C, control.
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Fig. 5. Effect of age on the induction of HSF /HSE binding activity by heat shock in hepatocytes from male F344/Jcl and F344/DuCrj rats. (a) Hepatocytes isolated from young and old F344/Jcl and F344/DuCrj rats were heated for 30 min at 42.5 8C. Whole cell extract obtained from the hepatocytes were analyzed by a gel mobility shift assay using 32P-labeled HSE oligonucleotide. (b) The data normalized to the average HSF /HSE binding activity in young F344/Jcl rats as 100% represent mean9/S.D. of data obtained from four to five rats. (a) Significantly different from young F344/Jcl, (b) significantly different from old F344/Jcl, (c) significantly different from young F344/DuCrj (P B/0.05).
Fig. 6. Effect of age on the induction of HSP70 by heat shock in hepatocytes from male F344/DuCrj. Hepatocytes isolated from young and old F344/DuCrj rats were heated for 30 min at 42.5 8C and then incubated for 2 h at 37 8C. The homogenates of hepatocytes were analyzed by a Western blot using anti-HSC70 and anti-HSP70 monoclonal antibodies as described in Section 2.
hsp70 expression decreases with increasing age in many cells and tissues except fruit fly (Drosophila ) (Fleming et al., 1992; Wheeler et al., 1999). So far, most studies on age-related changes in the induction of hsp expression by hyperthermia in rodents have been carried out for male F344 (Fischer) or Wistar rats obtained from colonies maintained by Harlan Sprague Dawley (HSD, Indianapolis, IN), Charles River Laboratory (CRL, Wilmington,
MA) for the National Institute on Aging (NIA) or the Gerontology Research Center at NIA. For example, in Richardson’s laboratory, age-related changes of the heat shock response have been intensively studied in hepatocytes (Heydari et al., 1993, 1995, 1996, 2000; Wu et al., 1993) and lymphocytes (Pahlavani et al., 1995) isolated from F344 rats obtained from HSD. They showed that the induction of hps70 expression by heat shock is significantly reduced in hepatocytes and lymphocytes from old rats and that the reduced expression is due to a deficit in the transcription of the hsp70 gene. Pardue et al. (1992) reported that F344 rats obtained from NIA showed a significant age-related decrease in induction of hsp70 mRNA in hippocampus when the rats were exposed to hyperthermia. A decline in the induction of hsp70 mRNA by hyperthermia has also been observed in primary cultures of fibroblasts isolated from the skin or lung of Wistar rats obtained from NIA (Fargnoli et al., 1990). Thus, previous studies from different laboratories on F344 and Wistar rats demonstrated that the induction hsp70 expression decreases generally with age in different tissues
R. Takahashi et al. / Mechanisms of Ageing and Development 123 (2002) 1605 /1615
and cells. So far, the age-related decline in the induction of hsp70 expression by hyperthermia has appeared to be a common phenomenon in rodent tissues. In this study we investigated the effect of age on the heat induced expression of three heat shock protein genes (hsp27, hsc70 and hsp90) in hepatocytes isolated from two different F344 substrains: F344/Jcl and F344/DuCrj. We found that hepatocytes from F344/Jcl rats showed a marked decrease with age in the induced levels of these hsp mRNAs by the heat shock. Remarkably, however, the cells from F344/DuCrj rats showed an increase rather than decrease by the same treatment. Furthermore, we found that difference in age-related changes of the heat-shock response closely correlated with the change in HSF activity. To our knowledge, such an unusual heat shock response in rodent cells has not been reported previously. How the opposite response was developed in different F344 substrains during aging is unclear. However, such difference was also observed in antioxidant enzyme activities, such as superoxide dismutase (SOD) in the liver. For example, male F344 rats obtained from NIA showed the de-
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creases (40 /50%) in the activities of catalase and Cu/Zn-SOD in the liver with age (Semsei et al., 1989; Rao et al., 1990), whereas F344/DuCrj obtained from CRJ showed a decrease (about 40%) in the activity of catalase but a significant increase (about 20%) in the activity of Cu/Zn-SOD in this organ with age (Carrillo et al., 1992) (Table 1). Although the mechanism responsible for the age-related substrain differences in the heat shock response and SOD activity is unclear, it might be due to the difference in the genetic background between F344/DuCrj and other F344 substrains. To date, no information is available about the polymorphism of genes that might alter the expressions of hsps and SOD in different F344 substrains. Interestingly, it has been reported that F344/DuCrj rats maintained at CRJ specifically lack dipeptidyl peptidase IV (DPPIV) activity, whereas Fischer 344 (F344) rats from different sources within US (HSD, CRL and NIH) (Tiruppathi et al., 1990), F344/Jcl rats from CRL (Nagakura et al., 2001) and Wistar rats (IwakiEgawa et al., 1991) possess normal levels of the DPPIV activity (Table 1). Sequence analysis of the
Table 1 Effect of age on the induction of heat shock protein expression and the activities of anti-oxidant enzymes in the liver of different male F344 rats Strain
Animal source
Origin1 MLS (month)
DPPIV
Change with age Hsp70 mRNA le- Cu/Zn SOD acvel tivity
F344/ CRJ DuCrj F344/Jcl JCL Fischer 344 HSD (NIA)
Catalase activity
GPX activity
Du
292
Negative5 Inc7
Inc9
Dec9
No change9
Du N
273 274
Positive6 Positive5
/ Dec4,10
/ Dec4,10
/ No change4
Dec7 Dec8
CRJ, Charles River Japan Inc. (Japan); JCL, Clea Japan, Inc.(Japan); HSD, Harlan Sprague Dawley (Indianapolis, IN); NIA, the National Institute of Ageing; DPPIV, dipeptidy peptidase IV; SOD, superoxide dismutase; GPX, glutathione peroxidase. 1 Tanaka et al. (2000). 2 Takahashi and Goto (1987). 3 Data from JCL. 4 Rao et al. (1990). 5 Tiruppathi et al. (1990). 6 Nagakura et al. (2001). 7 Takahashi et al. (this study). 8 Heydari et al. 9 Carrillo et al. (1992). 10 Semsei et al. (1989).
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DPPIV gene revealed that a point mutation exists in F344/DuCrj that results in substitution of Gly633 to Arg at the active-site (Tsuji et al., 1992). DPPIV exists in plasma and on the surface of various types of cells, particularly in the liver, kidney and small intestine and plays an important role in cleavage and inactivation of biologically active peptides, many of which are implicated in regulation of immune, inflammatory, nervous and endocrine functions (Mentlein et al., 1993; Yaron and Naider, 1993). For example, DPPIV degrades glucagon-like peptide-1 (incretin) which induces glucose-dependent insulin secretion. The DPPIVdeficient F344/DuCrj strain shows lower blood glucose levels with enhanced insulin secretion than DPPIV-positive rats in an oral glucose tolerance test (Nagakura et al., 2001). How such altered glucose metabolism affects the aging process is not known. However, a previous study among a limited number of centenarians living in South Italy showed this group to have a more preserved glucose tolerance and insulin action than aged subjects (/75 years) (Paolisso et al., 1996). Low incidence of various tumors including leukemia and longer life span of the DPPIV-deficient F344/ DuCrj compared with DPPIV-positive rats (F344/ N, /DuCrl and /Jcl) (Tanaka et al., 2000) might be due to altered glucose metabolism via insulin secretion. We do not know whether the altered age-related changes of the heat shock response and SOD activity are consequences of compensatory changes in F344/DuCrj rats to adapt to the inherent DPPIV deficiency or other unidentified genetic polymorphisms. Further detailed genetic and biochemical studies will be required to determine the mechanism of the difference of agerelated parameters among different F344 substrains. Our present results and previous findings by others (Carrillo et al., 1992; Tanaka et al., 2000) suggest that the significance of various physiological and biochemical changes with age observed in F344 rats needs to be carefully evaluated even though they are phylogenical siblings. Comparative studies among different F344 substrains including F344/DuCrj may be useful to verify
what age-related changes are public (common) or private (unique) (Martin et al., 1996).
References Carrillo, M.C., Kanai, S., Sato, Y., Kitani, K., 1992. Agerelated changes in antioxidant enzyme activities are region and organ, as well as sex, selective in the rat. Mech. Ageing Dev. 65, 187 /198. Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolation by acid guanigium thiocyanate /phenol /chloroform extracts. Anal. Biochem. 162, 156 /159. Fargnoli, J., Kunisada, T., Fornace, A.J.J., Schneider, E.L., Holbrook, N.J., 1990. Decreased expression of heat shock protein 70 mRNA and protein after heat treatment in cells of aged rats. Proc. Natl. Acad. Sci. USA 87, 846 /850. Fink, A.L., 1999. Chaperone-mediated protein folding. Physiol. Rev. 79, 425 /449. Fleming, J.E., Reveillaud, I., Niedzwiecki, A., 1992. Role of oxidative stress in Drosophila aging. Mutat. Res. 275, 267 / 279. Goto, S., Takahashi, R., Kumiyama, A., Radak, Z., Hayashi, T., Takenouchi, M., Abe, R., 2001. Implications of protein degradation in aging. Ann. N.Y. Acad. Sci. 928, 54 /64. Goto, S., Takahashi, R., Araki, S., Nakamoto, H., 2002. Dietary restriction initiated in late adulthood can reverse age-related alteration of protein and protein metabolism. Ann. NY. Acad. Sci. 959, 50 /56. Hensley, K., Floyd, R.A., 2002. Reactive oxygen species and protein oxidation in aging: a look back, a look ahead. Arch. Biochem. Biophys. 397, 377 /383. Heydari, A.R., Wu, B., Takahashi, R., Strong, R., Richardson, A., 1993. Expression of heat shock protein 70 is altered by age and diet at the level of transcription. Mol. Cell. Biol. 13, 2909 /2918. Heydari, A.R., Takahashi, R., Gutsmann, A., You, S., Richardson, A., 1994. Hsp70 and aging. Experientia 50, 1092 /1098. Heydari, A.R., Conrad, C.C., Richardson, A., 1995. Expression of heat shock genes in hepatocytes is affected by age and food restriction in rats. J. Nutr. 125, 410 /418. Heydari, A.R., You, S., Takahashi, R., Gutsmann, A., Sarge, K.D., Richardson, A., 1996. Effect of caloric restriction on the expression of heat shock protein 70 and the activation of heat shock transcription factor 1. Dev. Genet. 18, 114 /124. Heydari, A.R., You, S., Takahashi, R., Gutsmann-Conrad, A., Sarge, K.D., Richardson, A., 2000. Age-related alterations in the activation of heat shock transcription factor 1 in rat hepatocytes. Exp. Cell Res. 256, 83 /93. Ishigami, A., Goto, S., 1990. Age-related change in the degradation rate of ovalbumin microinjected into mouse liver parenchymal cells. Arch. Biochem. Biophys. 277, 189 / 195. Iwaki-Egawa, S., Watanabe, Y., Fujimoto, Y., 1991. Identification of a rat liver dipeptidyl aminopeptidase IV with a liver
R. Takahashi et al. / Mechanisms of Ageing and Development 123 (2002) 1605 /1615 plasma membrane glycoprotein (gp110). A study using dipeptidyl aminopeptidase IV-deficient rats. FEBS Lett. 286, 167 /170. Lindquist, S., Craig, E.A., 1988. The heat-shock proteins. Annu. Rev. Genet. 22, 631 /677. Martin, G.M., Austad, S.N., Johnson, T.E., 1996. Genetic analysis of ageing: role of oxidative damage and environmental stresses. Nat. Genet. 13, 26 /34. Mentlein, R., Gallwitz, B., Schmidt, W.E., 1993. Dipeptidylpeptidase IV hydrolyses gastric inhibitory polypeptide, glucagon-like peptide-1(7-36)amide, peptide histidine methionine and is responsible for their degradation in human serum. Eur. J. Biochem. 214, 829 /835. Morimoto, R.I., Sarge, K.D., Abravaya, K., 1992. Transcriptional regulation of heat shock genes. A paradigm for inducible genomic responses. J. Biol. Chem. 267, 21987 / 21990. Nagai, M., Takahashi, R., Goto, S., 2000. Dietary restriction initiated late in life can reduce mitochondrial protein carbonyls in rat liver: Western blot studies. Biogerontology 1, 321 /328. Nagakura, T., Yasuda, N., Yamazaki, K., Ikuta, H., Yoshikawa, S., Asano, O., Tanaka, I., 2001. Improved glucose tolerance via enhanced glucose-dependent insulin secretion in dipeptidyl peptidase IV-deficient Fischer rats. Biochem. Biophys. Res. Commun. 284, 501 /506. Pahlavani, M.A., Harris, M.D., Moore, S.A., Weindruch, R., Richardson, A., 1995. The expression of heat shock protein 70 decreases with age in lymphocytes from rats and rhesus monkeys. Exp. Cell Res. 218, 310 /318. Paolisso, G., Gambardella, A., Ammendola, S., D’Amore, A., Balbi, V., Varricchio, M., D’Onofrio, F., 1996. Glucose tolerance and insulin action in healthy centenarians. Am. J. Physiol. 270, E890 /E894. Pardue, S., Groshan, K., Raese, J.D., Morrison-Bogorad, M., 1992. Hsp70 mRNA induction is reduced in neurons of aged rat hippocampus after thermal stress. Neurobiol. Aging 13, 661 /672. Rao, G., Xia, E., Richardson, A., 1990. Effect of age on the expression of antioxidant enzymes in male Fischer F344 rats. Mech. Ageing Dev. 53, 49 /60. Riabowol, K.T., Mizzen, L.A., Welch, W.J., 1988. Heat shock is lethal to fibroblasts microinjected with antibodies against hsp70. Science 242, 433 /436. Sagai, M., Ichinose, T., Kubota, K., 1984. Studies on the biochemical effects of nitorgen dioxide. Toxicol. Appl. Pharmacol. 73, 444 /456. Semsei, I., Goto, S., 1997. Expression of mRNAs of pancreatic and L type RNase inhibitors as a function of age in different tiddues of SAMP8 and BDF1 mice. Mech. Ageing Dev. 97, 249 /261. Semsei, I., Rao, G., Richardson, A., 1989. Changes in the expression of superoxide dismutase and catalase as a
1615
function of age and dietary restriction. Biochem. Biophys. Res. Commun. 164, 620 /625. Sokal, R.R., Rohlf, F.J., 1981. Biometry. Freeman, New York. Soti, C., Csermely, P., 2000. Molecular chaperones and the aging process. Biogerontology 1, 225 /233. Stadtman, E.R., 2001. Protein oxidation in aging and agerelated diseases. Ann. N.Y. Acad. Sci. 928, 22 /38. Stege, G.J., Brunsting, J.F., Kampinga, H.H., Konings, A.W., 1995. Thermotolerance and nuclear protein aggregation: protection against initial damage or better recovery? J. Cell. Physiol. 164, 579 /586. Takahashi, R., Goto, S., 1987. Age-associated accumulation of heat-labile aminoacyl tRNA synthetases in mice and rats. Arch. Gerontol. Geriatr. 6, 73 /82. Takahashi, R., Mori, N., Goto, S., 1985. Alteration of aminoacyl tRNA synthetases with age: accumulation of heat-labile enzyme molecules in rat liver, kidney and brain. Mech. Ageing Dev. 33, 67 /75. Takahashi, R., Mori, N., Goto, S., 1985. Accumulation of heatlabile elongation factor 2 in the liver of mice and rats. Exp. Gerontol. 20, 325 /331. Tanaka, S., Tamaya, N., Matsuzawa, K., Miyaishi, O., 2000. Differences in survivability among F344 rats. Exp. Anim. 49, 141 /145. Tavernarakis, N., Driscoll, M., 2002. Caloric restriction and lifespan: a role for protein turnover? Mech. Ageing Dev. 123, 215 /229. Tiruppathi, C., Miyamoto, Y., Ganapathy, V., Roesel, R.A., Whitford, G.M., Leibach, F.H., 1990. Hydrolysis and transport of proline-containing peptides in renal brushborder membrane vesicles from dipeptidyl peptidase IVpositive and dipeptidyl peptidase IV-negative rat strains. J. Biol. Chem. 265, 1476 /1483. Tsuji, E., Misumi, Y., Fujiwara, T., Takami, N., Ogata, S., Ikehara, Y., 1992. An active-site mutation (Gly6330/Arg) of dipeptidyl peptidase IV causes its retention and rapid degradation in the endoplasmic reticulum. Biochemistry 31, 11921 /11927. Verbeke, P., Clark, B.F., Rattan, S.I., 2000. Modulating cellular aging in vitro: hormetic effects of repeated mild heat stress on protein oxidation and glycation. Exp. Gerontol. 35, 787 /794. Wheeler, J.C., King, V., Tower, J., 1999. Sequence requirements for upregulated expression of Drosophila hsp70 transgenes during aging. Neurobiol. Aging 20, 545 /553. Wu, C., 1995. Heat shock transcription factors: structure and regulation. Annu. Rev. Cell. Dev. Biol. 11, 441 /469. Wu, B., Gu, M.J., Heydari, A.R., Richardson, A., 1993. The effect of age on the synthesis of two heat shock proteins in the hsp70 family. J. Gerontol. 48, B50 /B56. Yaron, A., Naider, F., 1993. Proline-dependent structural and biological properties of peptides and proteins. Crit. Rev. Biochem. Mol. Biol. 28, 31 /81.