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J. Biol. Chem., Vol. 277, Issue 19, 17308-17314, May 10, 2002

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Effect of Hyaluronan Oligosaccharides on the Expression of Heat Shock Protein 72^*

Heping Xu^§, Tomomi Ito, Akira Tawada, Hiroshi Maeda, Hiroko Yamanokuchi, Kyoko Isahara^¶, Keiichi Yoshida, Yasuo Uchiyama^¶, and Akira Asari

From the  Seikagaku Corporation, Tateno 3-1253, Higashiyamato-shi, Tokyo 207-0021, Japan and the ^¶ Department of Cell Biology and Anatomy, Osaka University Medical School, Suita-shi, Osaka 565-0871, Japan

Received for publication, December 26, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have previously shown that intraarticular treatment with a hyaluronan (HA) preparation (840 kDa), HA84, up-regulates heat shock protein 72 (Hsp72) expression and suppresses degeneration of synovial cells in an arthritis model. In that study, the HA84 administered was degraded into HA oligosaccharides in the synovial tissue, suggesting that HA84 or degradation products of HA may up-regulate Hsp72 expression. Thus, in the present study, we examined the effects of HA of various molecular sizes on Hsp72 expression and cell death in stressed cells. Western blotting analysis showed that treatment of K562 cells with HA tetrasaccharides up-regulated Hsp72 expression after exposure to hyperthermia. On the other hand, treatment of the cells with HA of other sizes (di-, hexa-, deca-, dodecasaccharides), HA84, or tetrasaccharides of keratan sulfate did not elicit any change in expression of the Hsp72 protein. Treatment of the cells with tetrasaccharides of HA up-regulated not only expression of the Hsp72 protein but also Hsp72 mRNA expression and enhanced activation of HSF1, a transcription factor controlling Hsp72 expression, after exposure to hyperthermia. Because the level of Hsp72 protein was not affected by tetrasaccharides of HA when the K562 cells were kept at 37 °C without any stress, it is evident that tetrasaccharides of HA did not act as a stress factor. In addition, tetrasaccharides of HA suppressed cell death in the case of K562 cells exposed to hyperthermia and of PC12 cells under serum deprivation. These results suggest that a certain size of oligosaccharides, i.e. the tetrasaccharides of HA, up-regulates Hsp72 expression by enhancing the activation of HSF1 under stress conditions and suppresses cell death.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Heat shock proteins (Hsps)¹ are induced to suppress cell damage when cells are exposed to environmental insult (1, 2). Hsp70 suppresses apoptosis by preventing processing of caspase 3 (3, 4). It is well known that brief ischemia induces tolerance to subsequent ischemia in hippocampal neurons as a result of the induction of Hsp70 expression (5). We have previously shown that intraarticular treatment with hyaluronan (HA) preparation (840 kDa), HA84, suppresses degeneration of synovial cells in a canine arthritis model and up-regulates Hsp72 expression (6, 7). In that study, we also injected fluorescent-labeled HA84 in synovial tissues and found that some labeled HA particles could not be detected by means of an HA-binding protein that binds specifically to HA molecules larger than decasaccharides (7). These observations suggested that HA oligosaccharides formed through degradation of HA84 in the tissue might suppress cell damage by up-regulating Hsp72 expression. In the present study, we prepared HA oligosaccharides of various molecular sizes and treated cultured cells with them under stress conditions in an effort to understand the appropriate size of HA oligosaccharides required to up-regulate Hsp72 expression or to suppress cell death. Effects of HA molecules on Hsp72 were investigated by examining both Hsp72 protein levels and Hsp72 mRNA levels, and the activation of heat shock factor 1 (HSF1), a transcription factor controlling Hsps expression, in K562 cells exposed to the stress of hyperthermia. HSF1 is known to be transferred to the nucleus from the cytoplasm, and it binds to a heat shock element (HSE) in the DNA (8, 9). Moreover, HSF1 is phosphorylated, and its molecular weight thereby increases when activated soon after heat shock treatment (9). In addition to Hsp72 expression and HSF1 activation, the effects of HA molecules on cell death were investigated using K562 cells exposed to hyperthermia and PC12 cells under conditions of serum deprivation in the present study.

It has been reported that low molecular weight fragments of HA induce angiogenesis (10) and/or induce the expression of genes involved in the inflammatory response, e.g. genes for chemokines and cytokines (11). In addition to these activities, we show here a novel activity of HA oligosaccharides, the acceleration of Hsp72 expression through activation of HSF1 under stress conditions and its suppressive effect on cell death.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- HA, chondroitin sulfate C type, keratan sulfate, chondroitinase ACI, and chondroitinase ACII were obtained from Seikagaku Corporation (Tokyo, Japan). Other reagents and chemicals were obtained from commercial sources.

Preparation of Oligosaccharides-- Glycosaminoglycan oligosaccharides were prepared by the modified method of Inoue and Nagasawa (12). Saturated HA tetra-(HA₄), hexa- (HA₆), octa-(HA₈), deca-(HA₁₀), and dodeca- (HA₁₂) saccharides were prepared from the degradation products generated by treatment of HA with testicular hyaluronidase (Biozyme Laboratory, Gwent, UK) in 0.1 M sodium phosphate buffer, pH 5.3, containing 150 mM NaCl at 37 °C. Saturated disaccharides of HA (HA₂) were prepared from the degradation products generated by treatment of HA with dimethyl sulfoxide containing 10% of 0.1 M HCl for 16 h at 95 °C. Unsaturated HA di- (HA₂), tetra-(HA₄), and hexa-(HA₆) saccharides were generated by enzymatic digestion of HA with chondroitinase ACI in 0.1 M acetate buffer, pH 6.0, at 30 °C and separated by the same chromatography method as mentioned above. Chondroitin tetrasaccharides (Ch0₄) were generated by treatment of chondroitin sulfate C type with the dimethyl sulfoxide containing HCl. Chondroitin sulfate C type tetrasaccharides (ChS₄) were prepared by testicular hyaluronidase (Biozyme) digestion of chondroitin sulfate C type. Keratan sulfate oligosaccharides, Gal(SO₃)-GlcNAc(SO₃) [L4] and Gal(SO₃)-GlcNAc(SO₃)-Gal(SO₃)-GlcNAc(SO₃) [L4L4], were prepared from a keratanase II (Seikagaku Corporation) digest of keratan sulfate (shark fin, Seikagaku Corporation) through sequential steps of gel filtration and anion exchange adsorption column chromatography. The degraded oligosaccharides were divided into fractions of each size by sequential steps of anion exchange chromatography.

All oligosaccharides were checked by Limulus amebocyte lysate assays using Toxicolor LS Set (Seikagaku Corporation). HA₄ contains 0.03 pg/mg endotoxins, and similar results were obtained in other oligosaccharides.

Sizes and purity of HA oligosaccharides were determined by HPLC, fluorophore-assisted carbohydrate electrophoresis, and mass spectrometry.² Assignments of ¹H and ¹³C NMR spectroscopy and of element analysis were obtained for each HA oligosaccharide.² Oligosaccharides of chondroitin, chondroitin sulfate C type, and keratan sulfate were analyzed by HPLC and/or capillary electrophoresis (data not shown). In addition, keratan sulfate oligosaccharides have been analyzed by mass spectrometry (13).

Culture of K562 Cells for Detection of Hsp72 and HSF1-- Western blotting was done with K562 cells that were incubated in the presence of 0, 1, 10, or 100 ng/ml HA₂, HA₄, HA₆, HA₁₀, HA₁₂, HA84, or L4L4 at 43 °C for 20 min followed by further incubation at 37 °C for 2 h. K562 cells incubated at 37 °C for 2 h and 20 min without any treatment were used as the "no heat shock" normal control. Moreover, K562 cells incubated in the presence of HA₄ at 37 °C for 2 h and 20 min were examined by Western blotting to investigate whether Hsp72 expression is induced by HA₄ under non-stress conditions. For the detection of Hsp72 protein expression by flow cytometry, K562 cells were incubated in the presence or absence of 1 ng/ml HA₄ at 43 °C for 20 min followed by further incubation at 37 °C for 2 or 4 h.

To detect Hsp72 mRNA expression by Northern blotting, K562 cells were incubated in the presence or absence of 1 ng/ml HA₄ at 43 °C for 20 min with or without further incubation at 37 °C for 30 min, 1 h, or 2 h. To evaluate HSF1 activation by Western blotting, K562 cells were incubated with 0, 1, 10, or 100 ng/ml HA₄, HA₄, HA₆, HA₈, or HA84. The K562 cells were stressed at 42 or 43 °C for 20 min. K562 cells incubated at 37 °C for 20 min without any treatment were used as the no heat shock normal control.

To analyze HSF1 retained in the nuclear fraction by flow cytometry and to observe the results of immunostaining specific for HSF1 by confocal laser scanning microscopy, K562 cells were incubated in the presence or absence of 1 ng/ml HA₄ at 43 °C for 20 min with or without further incubation at 37 °C for 2 h. Moreover, to investigate the effect of HA₄ on activation of HSF1 under non-stress conditions, K562 cells were incubated in the presence or absence of 1 ng/ml HA₄ for 2 h and 20 min at 37 °C.

Antibodies Used in Immunostaining for Hsp72 and HSF1-- For the detection of Hsp72, monoclonal anti-Hsp72 antibody (Amersham Biosciences) was used as the first antibody, and horseradish peroxidase- or FITC-conjugated goat anti-mouse IgG (Jackson Laboratory, West Grove, PA) was used as the second antibody. For the detection of HSF1, rabbit anti-HSF1 polyclonal antibody (Stressgene, Victoria, British Columbia, Canada) was used as the first antibody, and horseradish peroxidase- or FITC-conjugated goat anti-rabbit IgG (Jackson Laboratory) was used as the second antibody.

Western Blotting Analysis of Hsp72 and HSF1-- K562 cells were surveyed by antibodies against Hsp72 or HSF1 described above. After electrophoresis, the proteins were electroblotted onto a nitrocellulose membrane. To reduce nonspecific interactions, the membrane was blocked by incubation with 0.3% skim milk in Tris-buffered saline (TBS) at 37 °C for 1 h. Following incubation with the first antibodies at 4 °C overnight, the membrane was washed three times with 0.1% Tween 20 in TBS and incubated with the secondary antibodies described above at 37 °C for 1 h. Color development was performed with 0.05% diaminobenzidine solution in TBS containing 0.01% H₂O₂.

Northern Blotting Analysis of Hsp72-- Total RNA was prepared from control K562 cells and each culture of HA₄-treated cells. Each sample was fractionated by electrophoresis on a 1% agarose-formaldehyde gel and transferred to a nylon membrane. For hybridization, the membrane was incubated overnight at 42 °C in the presence of a denatured ³²P-labeled human hsp72 oligonucleotide probe (Oncogene Science, Inc., Cambridge, MA) added to the prehybridization solution. A labeled cDNA probe specific for glyceraldehyde-3-phosphate dehydrogenase was used as a hybridization control. The membrane was washed at room temperature in saline/sodium phosphate/EDTA and then subjected to autoradiography.

Confocal Laser Scanning Microscopy-- K562 cells were fixed with 4% paraformaldehyde for 15 min at 4 °C, washed with PBS, and then permeabilized by incubation in 0.01% Tween 80 in PBS for 1 h at 4 °C. The cells were incubated with 1% bovine serum albumin in PBS and then with 1:200 diluted rabbit anti-HSF1 polyclonal antibody (Stressgene) overnight at 4 °C followed by FITC-conjugated goat anti-rabbit IgG (1:100, Jackson Laboratory) for 1 h at room temperature. Then they were observed using a confocal laser scanning microscope (Leica, Heidelberg, Germany).

Flow Cytometry-- For detection of intracellular Hsp72 in K562 cells, we used the monoclonal anti-Hsp72 antibody as described above and FITC-conjugated goat anti-mouse IgG (Jackson Laboratory). The cells were fixed with 4% paraformaldehyde for 15 min at 4 °C, washed with PBS, and then permeabilized by incubation in a solution of 0.01% Tween 80 in PBS for 1 h at 4 °C. The cells were incubated with 1% bovine serum albumin in PBS for 1 h at 4 °C and then with anti-Hsp72 antibody (1:200) overnight at 4 °C followed by FITC-conjugated goat anti-mouse IgG (1:100) for 1 h at room temperature.

To detect nuclear HSF1 in K562 cells, we used rabbit anti-HSF1 polyclonal antibody and FITC-conjugated goat anti-rabbit IgG (Jackson Lab.). Before immunostaining, the nuclear fraction was obtained by mincing the cells in a HEPES buffer (10 mM HEPES-KOH, 10 mM KCl, 0.1 mM EDTA) using a Dounce homogenizer followed by centrifugation at 1300 rpm for 5 min. Because activated HSF1 binds HSE in the nucleus of heat shocked cells, HSF1 is retained in the nucleus even after fractionation (14). HSF1 retained in the nuclear fraction was measured by FACScan (Becton Dickinson, Franklin Lake, NJ) after immunostaining for HSF1. Before immunostaining, the nuclear fraction was incubated in HEPES buffer for 30 min to remove any HSF1 not bound to HSE. The nuclear fraction was fixed with 4% paraformaldehyde for 15 min at 4 °C and washed with PBS, and then the nuclei were permeabilized by incubation in a solution of 0.01% Tween 80 in PBS for 1 h at 4 °C. The samples were incubated with 1% bovine serum albumin in PBS for 1 h at 4 °C and then with anti-HSF1 antibody (1:200) overnight at 4 °C followed by FITC-conjugated goat anti-rabbit IgG (1:100) for 1 h at room temperature.

After immunostaining for Hsp72, HSF1, or Annexin V staining, the cells were analyzed by flow cytometry (FACScan, Becton-Dickinson) using an instrument equipped with a 15-mA ion laser and with filter settings for FITC. Ten thousand cells from each sample were computed in list mode, and data analysis was done with a commercial software program (CELLQuest, Becton-Dickinson). Analysis gates were set on leukocyte, according to forward and side scatter properties.

Detection of Cell Death-- To evaluate the effect of HA₄ on cell death induced by hyperthermia, K562 cells were incubated with 1 ng/ml HA₄ for 20 min at 43 °C followed by incubation for 2 or 4 h at 37 °C. K562 cells incubated for 4 h at 37 °C without any treatment were used as a no heat shock normal control. Then these cells were incubated with Annexin V (Bender Medsystems, Vienna, Austria), which binds to phosphatidylserine exposed on the outer surface of the cell membrane of dead cells, just after cell culture as described above. Cell death was analyzed by flow cytometry (FACScan, Becton-Dickinson).

It has been reported that serum deprivation induces apoptosis in PC12 cells (15). PC12 cells were cultured under conditions of serum deprivation in the presence of HA oligosaccharides, HA84, L4, L4L4, Ch0₄, and ChS₄ at 100 ng/ml. The cell death assay was done by the trypan blue exclusion method, 24 h after the start of culture. The survival rate of cells cultured in the absence of serum but in the presence of 100 ng/ml nerve growth factor was taken to be 100%.

Digestion of HA₄-- One mg of HA₄ was digested with 0.01 units of chondroitinase ACII in 0.1 M sodium acetate buffer, pH 6.0, at 37 °C for 20 h. The reaction was stopped by boiling 3 min. Boiled chondroitinase ACII was added to HA₄ as a negative control. These samples were ultrafiltrated with Centricon Plus-20 10K (Millipore Co., Bedford, MA) to remove endotoxins and separated by anion exchange chromatography. The separated oligosaccharides were identified by HPLC. These products of HA₄ were applied in the cell death assay using PC12 cells described above.

HPLC Procedures-- Normal phase HPLC was performed using YMC-pack NH₂ (4.0 × 250 mm, YMC, Kyoto, Japan). Oligosaccharides were eluted with a linear gradient of 2-100% 0.8 M NaH₂PO₄ at a flow rate of 1.0 ml/min at 40 °C. Absorbance was monitored at 210 nm.

Statistical Analysis-- Comparisons were analyzed by using the unpaired Student's t test or Dunnet multiple comparison test.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Effects of HA Oligosaccharides on Hsp72 Expression-- Hsp72 protein expression was detected even in non-treated K562 cells not exposed to hyperthermia (Fig. 1, A and B). The results showed that treatment of the K562 cells with HA₄ up-regulated Hsp72 expression 2 h after exposure to hyperthermia (Fig. 1A). The same result was obtained in the case of HA₄-treated cells (data not shown). The Hsp72 protein level was not changed by treatment with HA₂, HA₆, HA84, L4L4 (Fig. 1A), HA₁₂ (data not shown), or HA₁₀ (data not shown) in the case of K562 cells exposed to hyperthermia. Hsp72 expression was not affected by HA₄ treatment in the case of cells not exposed to hyperthermia (Fig. 1B). Flow cytometry showed that treatment with 1 ng/ml HA₄ up-regulated and down-regulated Hsp72 expression 2 and 4 h after exposure to hyperthermia, respectively (Fig. 2). Northern blotting analysis showed that Hsp72 mRNA expression in K562 cells was up-regulated 30 min and 1 h after exposure to hyperthermia as a result of treatment with HA₄ (Fig. 3).

View larger version (52K): [in this window] [in a new window]   Fig. 1.   Western blotting of Hsp72. Hsp72 was detected even in K562 cells incubated at 37 °C (A, B). Hyperthermia treatment up-regulated the Hsp72 expression (A). Treatment with HA4, but not with HA2, HA6, HA84, or L4L4, up-regulated more of the Hsp72 expression in the K562 cells exposed to hyperthermia (A). Hsp72 expression was not changed by the treatment with the HA4 treatment in the K562 cells incubated at 37 °C (B). bH, bacterial Hsp72 used as a control.

View larger version (32K): [in this window] [in a new window]   Fig. 2.   Flow cytometric analysis of Hsp72 expression. Treatment with 1 ng/ml HA4 up-regulated and down-regulated the Hsp72 expression in K562 cells 2 and 4 h after the exposure to hyperthermia, respectively. **, p < 0.01; *, p < 0.05 (Student's t test).

View larger version (46K): [in this window] [in a new window]   Fig. 3.   Northern blotting of Hsp72. K562 cells were incubated in the presence (+) or absence () of 1 ng/ml HA4 after exposure to hyperthermia. The treatment with HA4 up-regulated the Hsp72 mRNA expression in K562 cells 30 min and 1 h after the exposure to hyperthermia. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Effects of HA Oligosaccharides on HSF1 Activation-- Western blotting analysis showed that treatment of the K562 cells with HA₄ up-regulated the level of phosphorylated HSF1 (~80 kDa), i.e. an activated form of HSF1, and diminished the level of non-phosphorylated HSF1 (~70 kDa) in cells exposed to hyperthermia at 42 °C (Fig. 4A). In addition, HA₄ or HA₄ increased the levels of both phosphorylated and non-phosphorylated HSF1 when the cells were exposed to hyperthermia at 43 °C (Fig. 4B). Activation of HSF1 was little influenced by HA84 (Fig. 4B) or HA₆ (data not shown). Treatment with HA₈ up-regulated the level of non-phosphorylated HSF1 but not of phosphorylated HSF1 (Fig. 4B).

View larger version (62K): [in this window] [in a new window]   Fig. 4.   Western blotting of HSF1. K562 cells were treated with HA4, HA4, HA8, or HA84 immediately after exposure to hyperthermia at 42 °C (A), or 43 °C (B) for 20 min. The treatment with HA4 or HA4 up-regulated the level of an activated form of HSF1 (A, B). bHF, bacterial HSF1 used as a control. 70 kDa, non-phosphorylated HSF1. 80 kDa, phosphorylated (activated) HSF1.

The treatment with HA₄ did not alter the retention of HSF1 in the nucleus of cells cultured at 37 °C (Fig. 5). The level of HSF1 retained in the nucleus was found to be elevated immediately after and 2 h after exposure to hyperthermia (Fig. 5). The level of HSF1 retained in the nucleus was even more elevated in the presence of HA₄ immediately after exposure to hyperthermia (Fig. 5). However, in cells treated with HA₄, the level of HSF1 retained in the nucleus was slightly diminished 2 h after exposure to hyperthermia (Fig. 5).

View larger version (29K): [in this window] [in a new window]   Fig. 5.   Flow cytometric analysis of the levels of HSF1 retained in the nucleus fraction of K562 cells. K562 cells were incubated in the presence or absence of 1 ng/ml HA4 incubated at 37 °C for 2 h and 20 min (left column), at 43 °C for 20 min (center column), and at 43 °C for 20 min followed by incubation at 37 °C for 2 h (right column). The treatment with HA4 up-regulated and down-regulated the level of nuclear HSF1 in the K562 cells immediately and 2 h after the exposure to hyperthermia, respectively. **, p < 0.01; *, p < 0.05 (Student's t test).

Immunodeposits of HSF1 were detected in HA₄-treated (Fig. 6B) as well as non-treated (Fig. 6A) K562 cells not exposed to hyperthermia. After exposure to hyperthermia, immunodeposits of HSF1 were detected as aggregated granular structures in the K562 cells incubated in the absence of HA₄ (Fig. 6, C and E). The HSF1-positive granules in the cells incubated at 37 °C for a further 2 h (Fig. 6E) were slightly larger in size than those observed immediately after exposure to hyperthermia (Fig. 6C). The immunodeposits of HSF1 in the K562 cells incubated in the presence of HA₄ (Fig. 6, D and F) were finer than those in the cells incubated in the absence of HA₄ (Fig. 6, C and E) after exposure to hyperthermia.

View larger version (106K): [in this window] [in a new window]   Fig. 6.   Immunostaining of HSF1 in K562 cells incubated in the presence (HA4 (+)) or absence (HA4 ()) of 1 ng/ml HA4. K562 cells were incubated at 37 °C for 2 h and 20 min (A, B), at 43 °C for 20 min (C, D), or at 43 °C for 20 min followed by incubation at 37 °C for 2 h (E, F). The hyperthermia treatment induced the formation of aggregated granules of HSF1 (C, E). The treatment with HA4 suppressed the formation of aggregated granules of HSF1 (D, F). Bar = 10 µm.

Effects of Tetrasaccharides of HA on Cell Death-- Treatment with HA₄ suppressed cell death in the case of K562 cells as determined 2 and 4 h after exposure to hyperthermia (Fig. 7). Apoptosis of PC12 cells under conditions of serum deprivation was prevented by treatment of the cells with tetrasaccharides of HA (Fig. 8). On the other hand, treatment with the other HA oligosaccharides, HA84, L4, L4L4, Ch0₄, or ChS₄ faintly suppressed the cell death (Fig. 8).

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Flow cytometric analysis of cell death in the case of K562 cells. K562 cells were incubated in the presence or absence of 1 ng/ml HA4 for 2 and 4 h at 37 °C after exposure to hyperthermia. The treatment with HA4 suppressed the cell death of K562 cells induced by the hyperthermia both 2 and 4 h after the exposure to hyperthermia. **, p < 0.01; *, p < 0.05 (Student's t test).

View larger version (42K): [in this window] [in a new window]   Fig. 8.   Survival rates of PC12 cells under conditions of serum deprivation in the presence or absence (None) of several kinds of saccharides (100 ng/ml). The survival rate of PC12 cells treated with nerve growth factor (NGF) was taken to be 100%. The treatment with HA4 or HA4 suppressed the cell death of PC12 cells. **, p < 0.01 (Dunnet multiple comparison test).

Effects of Digestion Product of HA₄ on Cell Death-- To confirm the effect of HA₄ on the cell death of PC12 cells, chondroitinase ACII digestion of HA₄ was examined. HPLC analysis showed that HA₄ was depolymerized into disaccharides (HA₂ plus HA₂) by the treatment with chondroitinase ACII (Fig. 9). HA₄ was not depolymerized by the treatment with boiled chondroitinase ACII (Fig. 9). Apoptosis of PC12 cells under conditions of serum deprivation was not prevented by treatment of the cells with the digestion products of HA₄, i.e. HA₂ plus HA₂ (Fig. 10), whereas apoptosis of PC12 cells was suppressed by HA₄ treated with boiled chondroitinase ACII (Fig. 10).

View larger version (21K): [in this window] [in a new window]   Fig. 9.   HPLC analyses of the digestion product of HA4. A, intact HA4. B, HA4 treated with boiled chondroitinase ACII. C, HA4 treated with chondroitinase ACII. HA4 was degraded into HA2 plus HA2. D, mixture of HA oligosaccharides before isolating into each size of HA oligosaccharides.

View larger version (17K): [in this window] [in a new window]   Fig. 10.   Effects of digestion product of HA4 on cell death on the survival rates of PC12 cells under conditions of serum deprivation. The apoptosis of PC12 cells was not prevented by the treatment with HA4 digestion, that is HA4 treated with chondroitinase ACII, i.e. HA2 plus HA2. HA4+ boil, HA4 treated with boiled chondroitinase ACII. NGF, nerve growth factor.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Our experiment show that a critical size of HA oligosaccharides, i.e. tetrasaccharides of HA, is required to up-regulate Hsp72 expression including HSF1 activation in K562 cells exposed to the stress of hyperthermia and to suppress cell death in the case of PC12 cells under conditions of serum deprivation. High molecular weight HA, HA84, and other kinds of GAG oligosaccharides, i.e. L4, L4L4, Ch0₄, or ChS₄, showed little effect on Hsp72 expression and/or cell death. HA₆ oligomers have been used as a tool for probing the cell surface in a study of HA receptor function (16) and was shown to be the minimum size required to effectively compete with native HA in binding to chondrocytes via CD44 surface receptors (17). K562 cells were used as CD44-negative cell lines (18, 19). We also confirmed that CD44 expression on the K562 cell surface is very weak by flow cytometry (data not shown). The treatment with HA₄ as well as HA₄ up-regulated the Hsp72 expression in the present study, suggesting the possibility that there may be as yet unidentified receptors for HA tetrasaccharides in the cells that induce the signal transduction to up-regulate the Hsp72 expression. As shown by Western blotting, the treatment with HA₄ as well as HA₄ activated HSF1, indicating that the carboxyl group of the non-reducing end of HA tetrasaccharides is not essential to up-regulate the activation of HSF1. Hsp72 expression was not affected by HA₄ treatment in the case of cells not exposed to hyperthermia, suggesting that HA₄ treatment does not induce Hsp72 expression under non-stress conditions but up-regulates it under stress conditions.

Sistonen et al. have shown that HSF1 is transferred from the cytoplasm to the nucleus in K562 cells after exposure to hyperthermia as demonstrated by a biochemical method examining nuclear and cytoplasmic fractions (20). It has been reported that there is a significant decrease in the level of HSF in the nuclear fraction prepared from unshocked cells, whereas nuclei from heat-shocked cells retain a high level of HSF (14). Because activated HSF1 binds to HSE in the nucleus of heat shocked cells, the activated HSF1 is retained in the nucleus even after fractionation (14). Activation of HSF1, which is reflected by nuclear HSF1 levels, was up-regulated immediately after exposure to hyperthermia and down-regulated 2 h after exposure to hyperthermia in the HA₄-treated cells as compared with non-treated cells. In the HA₄-treated cells, Hsp72 expression was up- and down-regulated 2 and 4 h after exposure to hyperthermia, respectively. Moreover, Hsp72 mRNA expression was up-regulated 30 min and 1 h but not 2 h after exposure to hyperthermia in the HA₄-treated cells. These results suggest that the treatment with HA₄ accelerates not only HSF1 activation followed by the acceleration of Hsp72 expression but also the feedback regulation that controls the disappearance of Hsp72 (Fig. 11).

View larger version (28K): [in this window] [in a new window]   Fig. 11.   Proposed mechanism of the effect of HA4 on Hsp72 expression and cell death. The difference in thickness of arrows between A and B indicates intensities of activation of HSF1, transcription and translation of Hsp72, and suppression of cell death. The treatment with tetrasaccharides of HA up-regulates the activation of HSF1 to enhance the level of Hsp72 expression and suppression of cell death.

Activation of HSF1 involves the conversion of HSF1 from a latent cytoplasmic monomer to a trimeric nuclear protein complex that controls the transcription of heat shock genes (21, 22). Nuclear localization and DNA binding, which occur as the first step in activation of HSF1, are not sufficient for the full transcriptional competence of HSF1 (8, 9). Phosphorylation is required as the second step in HSF1 activation to stimulate transcription (9). Localization and retention of HSF1 in the nucleus, which means DNA binding of HSF1, was found to be accelerated by the HA₄ treatment in the present study. In addition, we showed that phosphorylation of HSF1 were accelerated by the treatment with HA₄. Non-steroidal anti-inflammatory drugs such as salicylate are known to enhance thermotolerance in K562 cells by prolonging Hsp70 expression (23). Such drugs induce HSF binding to HSE even under non-stress conditions, but they are unable to induce Hsp70 transcription (24). In the present study, retention of HSF1 in the nucleus was not changed by HA₄ treatment under non-stress conditions. This suggests that the mechanism of the effect of non-steroidal anti-inflammatory drugs on Hsp70 expression differs from that of HA₄. When cells were exposed to severe hyperthermia (43 °C), treatment of HA₄ or HA₄ up-regulated both non-activated (non-phosphorylated) and activated (phosphorylated) HSF1 levels. These results indicate that the treatment of HA₄ or HA₄ induces not only the activation but also the synthesis of HSF1.

The treatment with HA₄ suppressed the formation of aggregated granules of HSF1 in cells exposed to hyperthermia. Sarge et al. have shown that the kinetics of appearance of HSF1 granules in the nuclei of HeLa cells during heat shock is very well correlated with the kinetics of HSF DNA binding and heat shock gene transcription (20). Alternatively, they noted the possibility that the granules observed may represent large aggregated particles of inactive HSF1. This coincides well with our present findings that the HA₄ treatment suppresses HSF1 formation of aggregated granules of HSF1 and up-regulates the HSF1 activation as well as Hsp72 expression after exposure to hyperthermia. It seems likely that the finer the HSF1 particles, the sooner the HSF1 can move between nucleus and cytoplasm. This coincides well with the result that disappearance as well as expression of Hsp72 protein were also accelerated by the treatment with HA₄ (Fig. 11). Further studies, however, are required to elucidate the precise mechanism involved in the activation of HSF1 by HA₄.

Cell death was suppressed in the case of cells treated with HA₄ as determined both 2 and 4 h after exposure to hyperthermia in the present study. The suppression of cell death observed 4 h after exposure to hyperthermia may be due to the prior up-regulation of Hsp72 expression in cells treated with HA₄ because the Hsp72 level was lower in the HA₄-treated cells than non-treated cells 4 h after exposure to hyperthermia (Fig. 11).

PC12 cells undergo apoptosis when cultured under conditions of serum deprivation (15, 25). To confirm the effect of HA oligosaccharides on cell death in a cell type except for K562 cells under stress conditions other than hyperthermia, we treated PC12 cells with HA oligosaccharides under conditions of serum deprivation in the present study. In this experiment, tetrasaccharides of HA were found to be more effective in suppressing cell death than the other HA oligosaccharides tested. Further experiments are required to elucidate the relationship between cell death and Hsp72 expression in PC12 cells under conditions of serum deprivation. After digestion of HA₄ by chondroitinase ACII, the product did not suppress cell death of PC12 cells, confirming the specificity of the suppressive effect of HA₄ on the cell death.

Hyaluronidase activity is known to be elevated in tumors (26) and inflammatory tissues (27) and to depolymerize HA. Free radicals also depolymerize HA in inflammatory tissues (28). It has been reported that hyaluronic acid with a molecular mass about 1.2 MDa inhibits the advanced glycation endproducts-induced activation of the transcription factor nuclear factor-B (NF-B) and the NF-B-regulated cytokines interleukin-1, interleukin-6, and tumor necrosis factor- (29). In addition to it, expression of interleukin-1 mRNA in synovium has been suppressed in the mild grades of osteoarthritis by the intraarticular treatment with high molecular mass HA (about 1.0 MDa) (30). Moreover, high molecular mass HA is known to suppress the proliferation of endothelial cells (31). On the contrary, it is well known that low molecular weight HA induces angiogenesis (10) and inflammation (11). When HA is natively depolymerized by hyaluronidases or radicals in vivo, HA may acquire novel functions. One of them is the up-regulation of Hsp72 expression (Fig. 12).

View larger version (28K): [in this window] [in a new window]   Fig. 12.   Cell biological activities of hyaluronan depending on its molecular sizes. See "Discussion" for details.

In conclusion, our results show that tetrasaccharides of HA up-regulate Hsp72 expression by enhancing the activation of HSF1 under stress conditions and suppress cell death (Fig. 12).

	ACKNOWLEDGEMENT

We thank Dr. Hascall at the Cleveland Clinic Foundation for review of this manuscript.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Present address: Dept. of Ophthalmology, University of Aberdeen Medical School, Foresterhill, Aberdeen AB25 2ZD, United Kingdom.

To whom correspondence should be addressed. Tel.: 81-42-563-5830; Fax: 81-42-563-5847; E-mail: asari@seikagaku.co.jp.

Published, JBC Papers in Press, February 25, 2002, DOI 10.1074/jbc.M112371200

² A. Tawada, T. Masa, Y. Oonuki, A. Watanabe, Y. Matsuzaki, and A. Asari, submitted for publication.

	ABBREVIATIONS

The abbreviations used are: Hsp(s), heat shock proteins; HA, hyaluronan; HSF, heat shock factor; HSE, heat shock element; HA₄, HA tetrasaccharides; HA₆, HA hexasaccharides; HA₈, HA octaasaccharides; HA₁₀, HA decaasaccharides; HA₁₂, HA dodecaaccharides; HA₂, HA disaccharides; HA₂, unsaturated HA diasaccharides; HA₄, unsaturated HA tetrasaccharides; HA₆, unsaturated HA hexasaccharides; Ch0₄, chondroitin tetrasaccharides; ChS₄, chondroitin sulfate C type tetrasaccharides; HPLC, high pressure liquid chromatography; FITC, fluorescein isothiocyanate; TBS, Tris-buffered saline; PBS, phosphate-buffered saline.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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