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. 2002 Apr;7(2):156–166. doi: 10.1379/1466-1268(2002)007<0156:cafaoa>2.0.co;2

Characterization and functional analysis of a heart-enriched DnaJ/Hsp40 homolog dj4/DjA4

Khaleque Md Abdul 1, Kazutoyo Terada 1, Tomomi Gotoh 1, Rahman Md Hafizur 1, Masataka Mori 1,1
PMCID: PMC514813  PMID: 12380683

Abstract

DnaJ homologs are cochaperones of the heat shock protein 70 (hsp70) family. Homologs dj1 (hsp40/hdj-1/DjB1), dj2 (HSDJ/hdj-2/rdj-1/DjA1), and dj3 (cpr3/DNAJ3/HIRIP4/rdj2/DjA2) have been identified in the mammalian cytosol and characterized. In this paper we characterized newly found dj4 (DjA4) and compared it with other chaperones. The dj4 messenger ribonucleic acid (mRNA) and protein were expressed strongly in heart and testis, moderately in brain and ovary, and weakly in other tissues in mice. Dj4 constituted about 1% of the total protein in heart. Testis gave extraspecies of dj4 mRNA and protein in addition to those seen in other tissues. On subcellular fractionation of the mouse heart, dj4 was recovered mostly in the cytosol fraction. In immunocytochemical analysis of the H9c2 heart muscle cells, dj4 and heat shock cognate 70 (hsc70) colocalized in the cytoplasm under normal conditions, whereas they colocalized in the nucleus after heat shock. When H9c2 cells were differentiated by culturing for up to 28 days with a lowered serum concentration, dj4 was increased markedly, dj3 was increased moderately, and dj1 and dj2 were little changed. The homolog dj4 as well as hsp70, dj1, and dj2 were induced in H9c2 cells by heat treatment at 43°C for 30 minutes, whereas hsc70 and dj3 were not induced. Heat pretreatment promoted survival of cells after severe heat shock at 47°C for 90 minutes or 120 minutes. H9c2 cells overexpressing hsp70 were more resistant to severe heat shock, and a better survival was obtained when dj4 or dj2 was co-overexpressed with hsp70. Taking a high concentration of dj4 in heart into consideration, these results suggest that the hsc70/hsp70-dj4 chaperone pair protects the heart muscle cells from various stresses.

INTRODUCTION

Heat shock proteins (Hsps) are induced in response to various stresses and protect cells from such stresses (Samali and Orrenius 1998; Smith et al 1998). Cells that have been subjected to a priming heat shock become less susceptible to the following challenging heat shock. This transient thermotolerant state is accompanied by a temporary increase in the expression of Hsps. Several lines of evidence indicates that the induction of Hsps is responsible for protecting cells against severe heat shock (Heads et al 1994; Nollen et al 1999). Overexpression of 1 Hsp, especially hsp70, can protect cells against stresses (Angelidis et al 1991; Li et al 1991). Some Hsps are constitutively expressed and act as molecular chaperones. In the mammalian cytosol, 2 hsp70 family members, hsp70 and the 70-kDa heat shock cognate protein (hsc70), are thought to participate in many biological processes, including folding and assembly, intracellular transport, and degradation of proteins (Hartl 1996; Rassow et al 1997).

Activities of the Hsp70 family members are regulated by partner chaperones including DnaJ/Hsp40 cochaperones (Cyr et al 1994; Kelley 1998). Nearly 30 DnaJ homologs have been identified in mammals (Ohtsuka and Hata 2000). DnaJ proteins have 3 distinct domains, a highly conserved J-domain of approximately 70 amino acid residues that is often found near the amino terminus and interacts with Hsp70 members, a glycine and phenylalanine–rich region (G/F domain) possibly acting as a flexible linker, and a cysteine-rich region (C domain) containing 4 [CXXCXGXG] motifs resembling a zinc-finger domain (Bork et al 1992). Major DnaJ homologs in the mammalian cytosol are dj1 (hsp40/hdj-1/DjB1) (Raabe and Manley 1991; Ohtsuka 1993), dj2 (HSDJ/hdj-2/rdj-1/DjA1) (Chellaiah et al 1993; Oh et al 1993), dj3 (cpr3/DNAJ3/HIRIP4/rdj2/DjA2) (Andres et al 1997), and the recently identified dj4 (DjA4) (Hata and Ohtsuka 2000). These members of the DnaJ family are classified into 3 groups according to their domain structures (Cheetham and Caplan 1998; Ohtsuka and Hata 2000). Type I members have all 3 domains (J, G/F, and C), type II members have the J- and G/F-domains but no C-domain, and type III members have only the J-domain. Type I contains 4 members, 3 in the cytosol and 1 in the mitochondria (Ohtsuka and Hata 2000). Homologs dj2, dj3, and dj4 correspond to the 3 cytosolic members and have the “CAAX” prenylation motif at their COOH termini. In fact, dj2 and dj3 have been shown to be farnesylated (Andres et al 1997; Kanazawa et al 1997). On the other hand, dj1 belongs to type II and has no farnesylation motif. Previously, we found that dj2 and dj3, not dj1, in combination with hsc70, facilitate mitochondrial protein import and luciferase refolding (Terada et al 1997; Terada and Mori 2000). However, little is known on the effect of DnaJ homologs on stress-induced cell death.

In the present study we investigated the properties of the newly found dj4 and its function in comparison with those of dj1, dj2, and dj3, including tissue distribution, intracellular localization, and heat inducibility. The homolog dj4 was increased when the H9c2 heart muscle cells were differentiated. Overexpression of hsp70 protected H9c2 cells from severe heat shock–induced cell death, and coexpression of dj4 or dj2 with hsp70 gave better protection.

MATERIALS AND METHODS

Plasmid construction

The mouse dj4 complementary deoxyribonucleic acid (cDNA) (GenBank, accession number AB032401) was amplified by polymerase chain reaction (PCR) using mouse heart cDNA and cloned into the HincII site of pGEM-3zf(+) (Promega, Madison, WI, USA) to generate pGEM-mdj4. The PCR oligonucleotides were 5′-AGAGGAGCAGACTTCAGAAG-3′ (sense) and 5′-CATTCATCATGTACTAGAGTCC-3′ (antisense), giving a fragment of 1275 base pairs. The nucleotide sequence was verified by sequencing. A second PCR was done to construct the expression plasmid using 5′-ATGGTGAAGGAGACCCAGTACTATG-3′ (sense) and 5′-GTCATAGCTGTTTCCTG-3′ (antisense) as the primers and pGEM-mdj4 as the template. A PCR product of 1321 base pairs containing a full-length dj4 cDNA was directly inserted into the BamHI site of pQE32 (Qiagen, Chatworth, CA, USA) to generate an expression plasmid pQE-mdj4. pCAGGS-dj4, a mammalian expression plasmid for mouse dj4, was constructed by inserting the full-length cDNA fragment for mouse dj4, used earlier, into the XhoI site of the plasmid pCAGGS (Niwa et al 1991). pCAGGS-hsp70 (Gotoh et al 2001) and pCAGGS-dj2 (Kanazawa et al 1997), human hsp70 and human dj2 expression plasmids were described. cDNA for rat dj1 was isolated by PCR, using the messenger ribonucleic acid (mRNA) from the lung of a male Wistar rat. PCR was carried out using dj1 primers corresponding to nucleotides 226–775 (GenBank, accession number AB028272, mouse). The PCR product was inserted into the HincII site of pGEM-3zf(+), yielding pGEM-rdj1. cDNA for rat dj2 was isolated by PCR, using mRNA from rat liver. PCR was carried out using dj2 primers corresponding to nucleotides 390–989 (GenBank, accession number U53922). The PCR product was inserted into the HincII site of pGEM-3zf(+), yielding pGEM-rdj2. cDNA for rat dj3 was isolated by PCR using mRNA from rat liver. PCR was carried out using dj3 primers corresponding to nucleotides 336–991 (GenBank, accession number U95727). The PCR product was inserted into the HincII site of pGEM-3zf(+), yielding pGEM-rdj3. cDNA for rat hsc70 was isolated by PCR using the mRNA from rat liver. PCR was carried out using hsc70 primers corresponding to nucleotides 1630–1979 (GenBank, accession number M19141). The PCR product was inserted into the HincII site of pGEM-3zf(+), yielding pGEM-rhsc70. cDNA for rat hsp70 was isolated by PCR using mRNA from rat liver. PCR was carried out using hsp70 primers corresponding to nucleotides 2894–3911 (GenBank, accession number X74271). The PCR product was inserted into the BamHI site of pGEM-3zf(+), yielding pGEM-rhsp70.

Protein purification and antibody production

The recombinant plasmid pQE-mdj4 was transformed into Escherichia coli M15[pREP4] (Qiagen), and hexahistidine-tagged mdj4 (H6mdj4) was induced with 1 mM isopropyl-1-thio-β-d-galactopyranoside. H6mdj4 was recovered in inclusion bodies and purified by Ni2+-NTA-Sepharose (Amersham Pharmacia Biotech, Buckinghamshire, UK) column chromatography under denaturing conditions. Briefly, the insoluble H6mdj4 protein was solubilized in buffer A (20 mM Tris-HCl, pH 7.9, 0.5 M NaCl, 6 M guanidine hydrochloride) containing 5 mM imidazole and was subjected to a Ni2+-NTA-Sepharose column. The column was washed with buffer A containing 30 mM imidazole, and then H6mdj4 was eluted with buffer A containing 1 M imidazole. The eluted protein was dialyzed against 50 mM Tris-HCl, pH 7.9, containing 0.75 M NaCl and was further purified by using the same column under native conditions. Finally, the eluate was again dialyzed and concentrated with Centricon-30 (Amicon, Beverly, MA, USA).

Antibodies

Rabbit antisera against human dj1 (Terada et al 1997), human dj2 (Kanazawa et al 1997), and jelly fish green fluorescent protein (GFP) (Yano et al 1997) and rat 1B5 monoclonal antibody specific to hsc70 (Terada et al 1995) were used. Anti-mouse dj4 antiserum was raised in a rabbit by injecting the purified dj4. Other antibodies were obtained from the following sources: monoclonal antibodies against hsp70 (C92), hsp60, and BiP/Grp78 from Stressgen Biotechnologies Corp (Victoria, Canada); monoclonal antibody against glyceraldehyde-3-phosphate dehydrogenase from Chemicon International (Temecula, CA, USA); monoclonal antibody against nucleoporin from Transduction Laboratories (Lexington, KY, USA); monoclonal antibody against dj2 from NeoMarkers (Fremont, CA, USA); Cy3-labeled goat anti-rabbit IgG from Amersham Pharmacia Biotech; and Alexa fluor 488–labeled goat anti-rat IgG from Molecular Probes (Eugene, OR, USA).

Cell culture

H9c2 cells, a clonal line derived from rat heart (ATCC CRL-1446; American Type Culture Collection, Rockville, MD, USA), were cultured in Dulbecco modified Eagle medium (DMEM), containing 25 mM glucose, 44 mM NaHCO3, 4 mM l-glutamine, and 1 mM sodium pyruvate, supplemented with 10% fetal calf serum at 37°C under an atmosphere of 5% CO2 and 95% air to 70–80% confluency. The medium was changed every 3 days. For heat shock treatment, culture dishes were sealed with parafilm and floated on a water bath at 43°C for 30 minutes. The cells were then returned to a CO2 incubator at 37°C and recovered for various time periods. Severe heat treatment was done by transferring cells to the DMEM containing 20 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES)-NaOH buffer, pH 7.4, preheated to 47°C. Sealed dishes were transferred to a water bath at 47°C and maintained for 90 minutes or 120 minutes. To promote differentiation of H9c2 cells, the serum concentration was reduced to 1%, and cells were cultured up to day 28 (Kimes and Brandt 1976).

RNA blot analysis

Total RNA was isolated from mouse tissues and cultured H9c2 cells using the acid guanidium thiocyanate–phenol-chloroform extraction procedure (Chomczynski and Sacchi 1987). RNA (2 μg per lane) was electrophoresed in denaturing formaldehyde-agarose (1%) gels and was blotted onto nylon membranes (Schleicher & Schuell, Dassel, Germany). Hybridization was performed with the digoxigenin-labeled antisense RNA probes, synthesized from cDNAs under the control of the T7 or SP6 promoter, using a transcription kit (Roche Diagnostic GmbH, Mannheim, Germany). cDNAs for the following proteins were used for the synthesis of probes: pGEM-mdj4 for mouse dj4, pGEM-rdj1 for rat dj1, pGEM-rdj2 for rat dj2, pGEM-rdj3 for rat dj3, pGEM-rhsc70 for rat hsc70, and pGEM-rhsp70 for rat hsp70. Chemiluminescence signals derived from hybridized probes were detected using a DIG luminescence detection kit (Roche Diagnostic GmbH), and the intensity of chemiluminescence was quantitated using a LAS1000plus chemiluminescence imager (Fuji Photo Film Co Ltd, Tokyo, Japan).

Immunoblot analysis

Mouse tissues were homogenized in 9 volumes of 20 mM HEPES-KOH buffer, pH 7.4, containing 0.5% Triton X-100, 20% glycerol, 1 mM dithiothreitol, 50 μM antipain, 50 μM leupeptin, 50 μM chymostatin, and 50 μM pepstatin. The homogenates were centrifuged at 25 000 ×g for 30 minutes at 4°C, and the supernatants were used as tissue extracts. H9c2 cells were trypsinized, washed twice with cold phosphate-buffered saline (PBS), and homogenized in 10 mM Tris-HCl, pH 7.4, containing 5 mM MgCl2, 1 mM dithiothreitol, 1 mM phenylmethyl sulfonyl fluoride, 5 μM antipain, 5 μM leupeptin, 5 μM chymostatin, and 5 μM pepstatin. After sonication and centrifugation at 15 000 × g for 10 minutes, the supernatant served as cell extracts. The tissue or cell extracts were subjected to sodium dodecyl sulfate (SDS)-10% polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes using a semidry transfer apparatus (Bio-Rad Laboratories, Hercules, CA, USA). Immunodetection was performed by using an ECL detection kit (Amersham Pharmacia Biotech). Intensity of chemiluminescence was quantitated using a LAS1000plus chemiluminescence imager.

Heart fractionation

The fractionation procedure of mouse heart was performed essentially as described by Green et al (1948) and Schneider (1948). Briefly, mouse heart was homogenized in 9 volumes of the homogenization buffer (0.25 M sucrose containing 50 mM potassium HEPES, pH 7.4) using a glass-Teflon homogenizer (5–6 strokes). The homogenate was centrifuged at 600 × g for 10 minutes at 4°C, and the precipitate was washed 3 times with 0.25 M sucrose to obtain the nuclear fraction. The supernatant was centrifuged at 5000 × g for 15 minutes at 4°C, and the precipitate was washed 3 times with 0.25 M sucrose to obtain the mitochondrial fraction. The second supernatant was then centrifuged at 100 000 × g for 1 hour at 4°C, and the supernatant and the precipitate were used as the cytosolic and microsomal fractions.

Immunofluorescent staining

H9c2 cells were grown on coverslips. After appropriate treatment, cells were placed on ice, washed 3 times with PBS and twice with acetone-methanol (1:1), and fixed in acetone-methanol (1:1) for 3 minutes at −20°C. Fixed cells were blocked with 10% fetal calf serum in PBS for 1 hour at 4°C. After washing 3 times with PBS, cells were incubated with primary antibodies for 1 hour at 4°C. After washing 4 times with PBS, cells were reincubated with fluorescent-conjugated secondary antibodies for 1 hour at 4°C. Finally, cells were washed 4 times with PBS. Immunostained cells were immediately analyzed using an Olympus BX50 fluorescent microscope equipped with appropriate filters, and images were taken using a C5810 color-chilled 3CCD video camera system (Hamamatsu Photonics, Hamamatsu, Japan).

Immunodetection of dj4 in the mouse heart was performed essentially as described by Koshiyama et al (2000). Briefly, the mouse was deeply anesthesized and perfused with ice-cold PBS followed by 4% paraformaldehyde. The heart was desected and soaked in ice-cold PBS for a few minutes and in 4% paraformaldehyde for 4 hours. Then the tissue was kept overnight in PBS at 4°C. The tissue was dehydrated through a graded series of ethanol and xylene and embedded into a paraffin block. Sections (8 μm) were cut, deparaffinized, and digested with 0.05% trypsin at 37°C for 1 hour to improve the penetration of antibodies. The section was incubated for 1 hour with anti-dj4 (1:200 dilution) and then washed 3 times with PBS for 10 minutes. Nonimmune rabbit serum was used as a negative control. The sections were incubated with fluorescent conjugated Cy3-labeled goat anti-rabbit IgG (1:200 dilution) for 1 hour. After washing 3 times with PBS, immunostained tissues were embedded in PBS and immediately analyzed as described previously.

Other methods

Survival or death of H9c2 cells was quantified by a trypan blue exclusion method. The protein concentration was determined with the protein assay reagent (Bio-Rad Laboratories) using bovine serum albumin as standard.

RESULTS

Expression of dj4 mRNA in mouse tissues

We studied the distribution of dj4 mRNA in mouse tissues (Fig 1). The dj4 mRNA of about 3.3 kb was expressed most strongly in heart and testis, moderately in brain and ovary, and weakly in other tissues. In testis, a smaller form of about 1.8 kb was expressed very strongly.

Fig. 1.

Fig. 1.

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 Ribonucleic acid (RNA) blot analysis for dj4 in mouse tissues. Total RNAs (2.0 μg) from mouse tissues were subjected to RNA blot analysis, using digoxigenin-labeled antisense RNAs as probes. The positions of 28S and 18S ribosomal RNAs (rRNAs) are shown on the right. Lower panel shows ethidium bromide staining of 28S and 18S rRNAs.

Tissue distribution and intracellular concentration of the dj4 protein

We prepared a polyclonal antibody against dj4 and checked its specificity and cross-reactivity with other DnaJ homologs. In immunoblot analysis this antibody gave a major single band of 46 kDa in many tissues, except the testis (Fig 2B). In the cross-reactivity test, anti-dj4 and anti-dj3 were specific to each chaperone, whereas anti-dj2 cross-reacted weakly with dj3 and dj4 (Fig 2A). The dj4 protein of 46 kDa was expressed most strongly in heart and testis, moderately in brain and uterus, and weakly in other tissues (Fig 2B). Two additional larger species were obtained from the testis. The tissue distribution of the dj4 protein was similar to that of the dj4 mRNA. Extraspecies of the dj4 mRNA and protein in testis remain to be characterized.

Fig. 2.

Fig. 2.

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 Tissue distribution and intracellular concentration of the dj4 protein. (A) Cross-reactivity among the DnaJ antibodies. Purified hexahistidine-tagged human dj2 (H6hdj2), human dj3 (H6hdj3), and mouse dj4 (H6mdj4) (each 30 ng) were subjected to immunoblot analysis using antisera against human dj2, human dj3, or mouse dj4. Amino acid sequence identities between human and mouse dj2 and dj3 are 99% (from database). Those between mouse DnaJ homologs are 55% between dj2 and dj3, 51% between dj3 and dj4, and 67% between dj2 and dj4 (Hata and Ohtsuka 2000; Ohtsuka and Hata 2000). (B) Tissue distribution of dj4 and other chaperones. Tissue extracts (2.0 μg of protein for dj2 and 5.0 μg of protein for dj4, dj1, dj3, hsp70, and hsc70) were subjected to immunoblot analysis using rabbit antisera against dj4, dj1, dj2 (1:1000), and dj3 (1:200), monoclonal anti-hsp70 (0.2 μg IgG/mL), and monoclonal anti-hsc70 (0.5 μg IgG/mL) as primary antibodies. Protein molecular mass markers (Rainbow-colored markers; Amarsham Pharmacia Biotech) were myosin (220 kDa), phosphorylase b (97 kDa), serum albumin (66 kDa), ovalbumin (46 kDa), and carbonic anhydrase (30 kDa). (C) Content of dj4 in mouse heart. Heart homogenate (2.0 μg of protein) and purified hexahistidine-tagged mouse dj4 (H6mdj4) (3 ng, 6 ng, 12 ng, and 24 ng) were subjected to immunoblot analysis.

The distribution of dj4 was compared with those of other cytosolic chaperones. Whereas hsp70 was expressed in many tissues to different degrees, hsc70 was expressed uniformly in all tissues. dj1, dj2, and dj3 were expressed ubiquitously, but their tissue distribution was not uniform and differed among DnaJ homologs. Taking these results together, 1 specific feature of dj4 was that this protein, but not the other DnaJ homologs, was expressed most strongly in the heart.

The concentration of dj4 in the mouse heart was measured by immunoblot analysis, using purified H6mdj4 as the standard (Fig 2C). H6mdj4 migrated as a polypeptide of 47 kDa in an SDS-polyacrylamide gel. The concentration of mdj4 was calculated to represent about 1% of total heart protein. Accordingly, the concentration of mdj4 in other tissues (except the testis) was much lower.

Subcellular localization of dj4

In immunofluorescent staining of dj4 in the mouse heart, the cytoplasm of the muscle cells was stained (Fig 3A). The mouse heart was fractionated into nuclear, mitochondrial, microsomal, and cytosol fractions (Fig 3B). Fractionation was assessed with marker proteins. dj4 was recovered mostly from the cytosol fraction and partly from the nuclear fraction. On the other hand, hsc70 was recovered from the nuclear and cytosol fractions, and slightly from the microsomal fraction. Taking the distribution of the total protein into consideration (see the caption of Fig 3B), the amount of hsc70 in the cytosol fraction was much higher than that in the nuclear fraction. Therefore, the distribution of dj4 was similar, although not identical, to that of hsc70.

Fig. 3.

Fig. 3.

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 Immunofluorescence staining and subcellular distribution of dj4. (A) Immunostaining of mouse heart for dj4. Mouse heart paraffin sections were immunostained with anti-dj4 antiserum (1:200 dilution) or with nonimmune rabbit serum (1:200) as the negative control. Secondary antibody used was Cy3-labeled goat anti-rabbit IgG. Fluorescence images were taken under identical conditions. (B) Subcellular distribution of dj4 in the heart. The mouse heart was subfractionated as described in the section “Materials and Methods”. Total extract, nuclear, mitochondrial, microsomal, and cytosol fractions (5 μg of protein) were subjected to immunoblot analysis. Distribution of the total protein in nuclear, mitochondrial, microsomal, and cytosolic fractions was 9.1%, 8.4%, 32.1%, and 50.4%, respectively. (C) Distribution of dj4 in H9c2 cells before and after heat shock. Cells were grown on coverslips at 37°C (control) or heat shocked at 45°C for 15 minutes and allowed to recover at 37°C for 1 hour. Cells were double stained with rabbit anti-dj4 antiserum and rat monoclonal antibody against hsc70. Secondary antibodies used were Cy3-labeled goat anti-rabbit IgG and Alexa fluor 488–labeled goat anti-rat IgG

We next examined the localization of dj4 in H9c2 cells and compared it with that of hsc70, using double immunofluorescence microscopy (Fig 3C). Under normal conditions, dj4 was localized almost exclusively in the cytoplasm and colocalized with hsc70. When cells were heat shocked, dj4 migrated from the cytoplasm to the nucleus and roughly colocalized again with hsc70. All these results suggest that dj4 is able to work as a cochaperone of hsc70 (and hsp70, see “Discussion”) under both normal and stressed conditions.

Changes in dj4 and other chaperones during differentiation of the H9c2 cells

H9c2 cells were differentiated by culturing for up to 28 days with a decreased serum concentration. Under these conditions, extensive cell fusion into multinuclear tubular cells was observed (data not shown). The level of dj4 appeared to decrease slightly at day 7 and then increased up to day 28 (Fig 4). Induction at day 28 was about 3.2-fold over the level at day 3. In contrast, dj1 remained little changed up to day 21 and decreased at day 28, and dj2 remained little changed up to day 28. The level of dj3 increased up to day 21 or day 28, but less markedly than dj4. hsc70 remained unchanged during the period.

Fig. 4.

Fig. 4.

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 Changes in dj4 and other chaperones during the differentiation of H9c2 cells. Cells were cultured in Dulbecco modified Eagle medium (DMEM) containing 10% fetal calf serum. After 7 days of plating (near 100% confluent), the medium was changed to DMEM containing 1% serum to promote differentiation. The medium was changed every 3 days, and cells were collected at indicated times. Whole cell extracts (5.0 μg of protein) were subjected to immunoblot analysis for DnaJ proteins and hsc70

Heat shock induction of dj4 and other chaperones in H9c2 cells

Effects of heat shock on mRNAs and proteins for dj4 and other chaperones in H9c2 cells are shown in Figure 5. Cells were heat shocked at 43°C for 30 minutes and returned for recovery to 37°C for 2 hours and 4 hours for mRNAs (Fig 5A) and up to 6 hours for proteins (Fig 5B). The dj4 mRNA was induced by about 11-fold at 2 hours and remained high at 4 hours. The dj4 protein level was somewhat decreased at 0 hour and 1 hour, and was then increased up to 6 hours. The increase at 6 hours over the control was about 2.4-fold. The dj1 mRNA level was markedly increased at 2 hours and decreased at 4 hours. The dj1 protein was increased gradually up to 4–6 hours. The dj2 mRNA and protein were markedly increased. In contrast, dj3 mRNA was decreased by heat shock, and its protein remained little changed. The hsp70 mRNA and protein were not detected before heat treatment and were markedly increased after the treatment. The hsc70 mRNA was moderately increased, whereas its protein remained little changed.

Fig. 5.

Fig. 5.

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 Heat shock induction of messenger ribonucleic acids (mRNAs) and proteins of dj4 and other chaperones in H9c2 cells. (A) Cells were maintained at 37°C (control) or heated at 43°C for 30 minutes and allowed to recover at 37°C for 2 hours or 4 hours. Total RNAs (2.0 μg) were subjected to RNA blot analysis, using digoxigenin-labeled RNAs as probes. The position of 18S ribosomal RNA (rRNA) is shown. The bottom panel shows ethidium bromide staining of 28S and 18S rRNAs. (B) Cells were heat shocked as in (A) and recoverd at 37°C for the indicated time periods. Whole cell extracts (5.0 μg of protein) were subjected to immunoblot analysis for hsp70, hsc70, and DnaJ proteins using their respective antibodies

Preheat treatment enhances survival of H9c2 cells against severe heat treatment

H9c2 cells were heat treated at 43°C for 30 minutes and then subjected to severe heat treatment at 47°C for 90 minutes or 120 minutes. Without preheat treatment, cell survival decreased to 35% and 23% after severe heat treatment for 90 minutes and 120 minutes, respectively (Fig 6). When cells were preheat treated, survival increased to 63% and 42% after severe heat treatment for 90 minutes and 120 minutes, respectively. These results, along with results in Figure 5, suggest that the chaperone pair of hsp70 and dj4 or hsp70 and dj2 (or both) is responsible for increased cell survival.

Fig. 6.

Fig. 6.

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 Effect of heat pretreatment on thermotolerance of H9c2 cells. Control cells and heat-pretreated cells (43°C for 30 minutes and recovery at 37°C for 6 hour; pre-HS) were subjected to severe heat treatment (severe HS) at 47°C for 90 minutes or 120 minutes. Cell survival was assessed as described in “Materials and Methods.” Results from 3 independent experiments were expressed as means ± standard deviation

Coexpression of hsp70 and dj4 or dj2 enhances the survival of H9c2 cells against severe heat treatment

H9c2 cells were cotransfected with a GFP expression plasmid with the hsp70, dj4, or dj2 plasmid alone, or in combination, and cell survival after severe heat treatment was assessed by the number of GFP-positive cells (Fig 7A) and by the level of GFP protein (Fig 7 B,C). In control cells the number of GFP-positive cells was markedly decreased after severe heat treatment. When the GFP and the hsp70 were coexpressed, the decrease was moderately prevented. Expression of dj4 or dj2 alone had a tendency to increase GFP-positive cells, but the effects were not significant. In contrast, when hsp70 and dj4 along with GFP were coexpressed, a much better prevention was obtained. Coexpression of hsp70 and dj2 was even more effective in enhancing cell survival. These results were confirmed by immunoblot analysis of GFP (Fig 7 B,C). When cell survival was assessed by trypan blue exclusion, similar results were obtained (Fig 7D). These results indicate that hsp70 can enhance the survival of H9c2 cells after severe heat treatment and that dj4 and dj2 can further improve the survival.

Fig. 7.

Fig. 7.

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 Effect of overexpression of hsp70, dj4, or dj2 on thermotolerance of H9c2 cells. (A) Cells grown on 35-mm dishes were transfected with pEGFP (2.0 μg) alone or with hsp70 (0.2 μg), dj4 (0.67 μg), or dj2 (0.67 μg) chaperone plasmids in pCAGGS as indicated, using the GenePORTER transfection reagent (Gene Therapy Systems, California, USA). The amount of total plasmid was adjusted to 2.0 μg with pCAGGS. Transfection efficiency was 35%. Forty-eight hours after transfection, cells were subjected to severe heat shock by transferring sealed dishes to a water bath at 47°C for 2 hours. Green fluorescent protein (GFP) fluorescence was observed before and after severe heat shock. (B) Cell extracts (5.0 μg of protein) were subjected to immunoblot analysis for GFP, hsp70, dj4, dj2, and hsc70 proteins. The larger species of dj4 and dj2 are unfarnesylated precursor forms. (C) The results for GFP in (B) and 2 other independent experiments were quantified and are shown as means ± standard deviation (SD) (n = 3). (D) Cells were transfected and subjected to severe heat shock as in (A). Cell viability was monitored by trypan blue exclusion. Results are expressed as means ± SD (n = 3)

DISCUSSION

The dj4 cDNA was recently isolated based on a cDNA database, and its mRNA was reported to be highly expressed in the heart and the testis of mice (Hata and Ohtsuka 2000). In the present study using mouse tissues, we confirmed this at both mRNA and protein levels and further showed that dj4 is also expressed at lower levels in many other tissues. dj4, as well as dj2 and dj3, belongs to type I of the DnaJ family and has a C-terminal prenylation motif. In fact, overexpression of dj4 and dj2 gave larger species in SDS-polyacrylamide gel that apparently correspond to unprenylated precursor forms (Fig 7B). Farnesylation of dj2 and dj3 was demonstrated (Andres et al 1997; Kanazawa et al 1997). In contrast, dj1 belongs to type II and has no prenylation motif. The level of dj4 was calculated to represent about 1.0% of the total protein in heart; this value is much higher than those of dj1, dj2, and dj3 in mouse heart. dj4 was also present in heart muscle cell–derived H9c2 cells but was undetectable in COS-7 and PC12 neuronal cells (data not shown). dj4, but not the other DnaJ homologs, was markedly induced in H9c2 cells during differentiation. All these results suggest that dj4 has a special role in heart muscle cells.

hsp70 protects cells from various stresses (Samali and Orrenius 1998). It is thought to play a critical role in thermotolerance of mammalian cells, presumably because of its chaperone activity. Several studies showed that the overexpression of hsp70 protects cells from severe heat treatment (Li et al 1991; Heads et al 1994; Nollen et al 1999). Overexpression of hsp70 in a transgenic mouse increases the resistance of the heart to ischemic injury (Marber et al 1995; Plumier et al 1995; Hutter et al 1996; Radford et al 1996). hsp70 has been reported to protect cells from apoptosis at the level of cytochrome c release (Mosser et al 2000) or by preventing formation of an active apoptosome (Beere et al 2000; Saleh et al 2000). However, the effect of hsp70 cochaperones has little been studied. We found that the hsp70/hsc70-dj1 or -dj2 chaperone pair prevents nitric oxide–mediated apoptosis in macrophages (Gotoh et al 2001). hsp70/hsc70 alone was little effective. Here, we showed that in H9c2 cells hsc70 and dj4 are colocalized in the cytoplasm under normal conditions and in the nucleus after heat shock. hsp70 also migrates from the cytoplasm to the nucleus after heat shock (Nollen et al 1999). When H9c2 cells were heat pretreated, dj4 and dj2, in addition to hsp70, were markedly induced, and cells became resistant to severe heat shock. Transfection experiments showed that hsp70 alone enhances the survival of H9c2 cells after severe heat shock and that coexpression of dj4 or dj2 with hsp70 further enhances survival. Because dj4 is much more abundant than dj2 and dj3 in heart muscle cells, it is likely that the hsp70/hsc70-dj4 chaperone pair is mainly responsible for the thermotolerance. The effect of this chaperone pair on protection of the heart muscle cells against other stresses such as ischemia remains to be studied.

Acknowledgments

We thank Seiichi Oyadomari and other colleagues for suggestions and discussion. This work was supported in part by Grant-in-Aid (09276103 to M.M.) from the Ministry of Education, Science, Sports, and Culture of Japan.

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