Insulin-induced Expression of Human Heat-shock Protein Gene hsp70*

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc,

Vol. 264, No. 6 , Issue of February 25, pp. 3404-3408, 1989

Printed in U.S.A.

Insulin-induced Expressionof Human Heat-shock Protein Gene hsp70* (Received for publication, October 17, 1988)

Ling-Pai TingS9, Chia-Ling Tu+, and Chen-KungChouSV From the $Graduate Institute of Microbiology and Immunology, National Yang-Ming Medical College and the VDepartment of Medical Research, Veterans General Hospital, Shih-Pai, Taipei, Taiwan

In human hepatoma cell line Hep3B/T2, the human heat-shock-inducible gene hsp7O could be induced by insulin. The dose-dependent insulin effect correlates very well with the dissociation constant of the insulin receptor, indicating that the insulin effect is mediated by the insulin receptor. The expression of hsp70 gene was neither significantly induced by .growth factors of epidermal and platelet-derived growth factors, nor by tumor promoter, calcium ionophore, CAMP, and glucocorticoids. These results indicate that the induction of expression of hsp70 geneby insulin is veryspecific and not cell cycle-related. Furthermore, the insulininduced expression of hsp7O gene is transient, occurring specifically from 4 to 8 h after insulin addition.

whether the heat-shock protein can be induced by hormones. The metabolism of human hepatoma cell line, Hep3B cells, which retained many differential functions of hepatocytes (121, could be regulated by a variety of hormones such as insulin. Here we report that insulin induces the expression of hsp70 gene. The effect of insulin is mediated by the insulin receptor and is probably not DNA synthesis-dependent. The expression of hsp70 gene is not induced by PDGF, EGF, TPA, A23187, CAMP, and glucocorticoids, suggesting that the induction is highly insulin-specific. EXPERIMENTALPROCEDURES

Cell Culture"Hep3B/T2 cell was a clonal derivative of human hepatoma cell line Hep3B (12). Hep3B/T2 cells were cultured in the Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum (Flow Laboratories, Staumor, Australia), 2 mM glutamine, mixtures of nonessential amino acids, 100 pg/ml streptomycin, 100 To a variety of metabolic stresses such as abnormally high IU/ml penicillin, and 2.5 pg/ml fungizone at 37 "C in a humidified temperatures, a general response of all organisms is the rapid atmosphere of 5% COz. and dramatic increase in the initiation rate of transcription Serum Starvation and Stimulation with Different Hormones or of several specific genes encoding heat-shock proteins (1). Factors"Hep3B/T2 cells were seeded in DMEM containing 10% Among heat-shock proteins, the hsp70l gene family which fetal calf serum, replaced with DMEM without fetal calf serum in the includes both heat-inducible genes (hsp70) and noninducible next day, and the same medium was renewed every other day for a total of 6 days. Cells were then treated with DMEM as the serumcognates (hsc7O) is the most abundant and generally con- free control or 10% fetal calf serum, PDGF, EGF, TPA, A23187, served. Upon heat shock of mammalian cells, both hsp7O and dexamethasone, 8-Br-cAMP, and insulin with different doses as hsc7O migrate to thenucleus and concentratein the nucleolus indicated. After treatment, cells were harvested, and total RNA was where they bind to partially assembled ribosomes (2), Over- isolated at theindicated time. Isolation of RNA and Northern Hybridization-Total cellular RNA production of HSP7O in cells transfected with the inducible hsp70gene accelerates the recoveryfrom heat shock (3). was isolated as described by Glisin et al. (13), and the Northern blot hybridization was carried out according to the procedure described Therefore, hsp70 gene has been speculated to participate in by Alwine et al. (14). phsp70 containing the hsp70 gene (15), pTWSl the scaffolding of protein complexes as well as in their disso- containing the hepatitis B surface antigen (HBsAg) gene (16, 171, ciation driven by ATP hydrolysis (4-6). GRP78 containing the glucose regulatory protein GRP78 gene (proRecently, it was found that the expression of hsp7O gene vided by Dr. R. Morimoto, Northwestern University), and pmribl.9 was induced by 20%. serum in a DNA synthesis-dependent containing the mouse 18 S rRNA gene (18)were used as the probes. Hybridization was done with 5 X lo6 cpm/ml 32P-labeledprobe (2-4 manner in HeLa cells, suggesting that it is regulated as a X 10' cpmlpg) in 5 X SSPE, 2 X Denhardt's solution, 0.1% SDS, 150 function of the cell cycle (7-11). These results suggested that pg/ml denatured salmon sperm DNA, and 50% formamide at 42 "C hsp7O may be induced during the changes of in vivo physio- for 2 days. The hybridized filters were washed with 2 X SSPE, 0.1% logical conditions. The most frequent environmental change SDS for 5 min at room temperature twice, and 0.2 X SSPE, 0.1% encountered by mammalian cells in vivo is probably caused SDS for 30 min at 55 "C, twice. The blots were then air-dried and by hormones. Therefore, we are interested in investigating autoradiographed with Fuji RX films. Protein Labeling and Two-dimensional Gel Electrophoresis-Cells This research was supported by Research Grant NSC 77-0412- in the 6-well plate were cultured in the serum-free medium for 6 days, BOIO-41 from the National Science Council, R. 0. C. The costs of then treated with DMEM or 10" M insulin for 5 h. Cells werewashed of ["SI pubIication of this article were defrayed in part by the payment of with phosphate-buffered saline and labeled with25FCi page charges. This article must therefore be hereby marked "aduer- methionine (1000 Ci/mmol; Du Pont-New England Nuclear) in 500 tiement" in accordance with 18U.S.C. Section 1734 solelyto indicate p1 of methionine-free medium or methionine-free medium containing 10" M insulin for 1h for serum-free control and insulin-treated cells, this fact. respectively. The heat-shock control cells were treated with DMEM § To whom correspondence and reprint requests should be adfor 4 h, followed by heat-shock at 43 "C for 1 h, and then labeling at dressed. ' The abbreviations used are: hsp70, heat-shock protein 70-kDa 37 "C for 1 h. After labeling, cells were washed with phosphategene; HSP, heat-shock protein; hsc70, heat-shock protein 70-kDa buffered saline three timesand lysed with 300 pl of lysis buffer (0.57% cognate gene; IGF-I, insulin-like growth factor I; IGF-11, insulin-like urea, 2% Nonidet NP-40, 0.01% Bio-Lyte 3/10, and 0.04% Bio-Lyte growth factor 11; GRP78, glucose-regulated protein 78 kDa; DMEM, 5/7). Two-dimensional gel electrophoresis was carried out as deDulbecco's modified Eagle's medium;PDGF, platelet-derived growth scribed by O'Farrell (19) except that a constant concentration of factor; EGF, epidermal growth factor; TPA, 12-0-tetradecanoyl phor- acrylamide (10%)rather than gradient was usedin the second dimenbol-13-acetate; ara-C, cytosine p-D-arabinofuranoside; SDS, sodium sion. Fluorographs were prepared by impregnating with En3HANCE (Du Pont-New England Nuclear). dodecyl sulfate; kb, kilobase pair.

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Insulin-induced h p 7 0 Expression C Y SAF I N

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Imp70

2.6kb-

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2.lkb-

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D

4.okb-

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FIG.1. Induction of hsp70 gene expression by insulin in human hepatoma cell line Hep3B/T2. Cells were serum-starved for 6 days and treated with either DMEM as the serum-free control ( l a n e S F ) or 10” M insulin in DMEM ( l a n e I N ) . Cells were also incubated in the complete medium (DMEM with 10% fetal calf serum) for 6 days and replaced with the fresh complete medium as the complete medium control (lane C M ) . After 6 h of treatments, cells were harvested, and total RNA was extracted. Ten pg of total RNA of each sample was used for Northern blot hybridization as described under “Experimental Procedures.” In A, B, C, and D, phsp70, pTWSl containing HBsAg gene, GRP78, and pmribl.9 containing mouse 18 S rRNA gene were usedas theprobes, respectively. PHIThymidine Incorporation-Cells in the individual well of a 24well plate were washed with phosphate-buffered saline and labeled with 6 pCi/ml [3H]thymidinefor 1 h. Then, cells were washed with phosphate-buffered saline and lysed in 125 pl of 0.1 N NaOH. The lysates were precipitated with 10% trichloracetic acid on GF/A (Millipore) filter paper and counted in liquid scintillation fluid Aquasol.

a

0 .

FIG.2. Analysisof protein synthesisof insulin-treated Hep3B/T2 cells by two-dimensional gel electrophoresis. Serum-starved cells were treated with either DMEM as the serumfree control or M insulin in DMEM for 5 h and labeled with [D5S] methionine in methionine-free medium alone ( S F )or in methioninefree medium containing 10” M insulin (SF+IN) for 1 h. The heatshock control (SF+HS) was prepared as follows: serum-starved cells were treated with DMEM for 4 h, heat shock at 43 “C for 1 h, back to 37 “C,and labeled with [35S]methioninein methionine-free medium for 1 h. Two-dimensional gel electrophoresis was carried outas described under “Experimental Procedures.”

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RESULTS

Inductive Expression of hsp70 mRNA and Protein by Insulin-We have known that the human hepatoma Hep3B/T2 cells incubating in the serum-starved medium for 6 days will reach the resting state and strongly enhance the amount of 4.0-kb transcript (20) of endogenously integrated hepatitis B surface antigen (HBsAg)gene.However, its levelis suppressed by M insulin.*Therefore, the HBsAg mRNA level is used to indicate the cell state. As shown in Fig. lB, the levelofHBsAggene transcript is increased in the serumstarved cells and suppressed by M insulin. Fig. lA shows that the levelof hsp70 mRNA is decreased in the serumstarved cells. However, when these cells were treated with M insulin for 6 h, the steady state level of hsp70 mRNA is greatly enhanced. As shown in Fig. 1, C and D , the level of GRP78 mRNA and the18 S rRNA has no significant change in all three conditions, indicating that neither the serum starvation nor insulin changes the ratio of mRNA to total RNA. The analysis of protein synthesis by two-dimensional gel electrophoresis shows that the synthesis of several heat shock proteins with molecular weight of70,000 is strongly enhanced at 6 h postinsulin stimulation (Fig. 2).These results clearly indicate that insulin can induce the expression of hsp7O gene at both the mRNA and protein levels. Insulin Dose Dependence of hsp70 Gene Expression-Different doses of insulin were used to treat serum-starved Hep3B/T2 cells. As shown inFig. 3,the mRNA levelof hsp70 gene is induced in an insulin concentration-dependent manner. The minimal induction occurs at 10”’ M, and the maximal induction is achieved at lo-’ M. This result suggests that *C.-K. Chou, T.-S. Su, C. Chang, C.-P. Hu, M.-Y. Huang, C.3. Suen, N.-W. Chou, and L.-P. Ting, manuscript submitted for publication.

2.6kb.

-hsp70

FIG. 3. Dose-dependent effect of insulin on hsp7OmRNA induction. Hep3B/T2 cells were serum-starved for 6 days and stimulated with DMEM alone as the serum-free control ([amS F ) or increasing insulin concentrations (lO-L1-lO” M) in DMEM for 6 h a t 37 “C. Ten pg of total RNA of each sample was used for Northern blot analysis with phsp70 as theprobe.

the insulin effect onthe induction of hsp70 mRNA of Hep3B/ T2 cells is mediated by the insulin receptor, since the apparent dissociation constant ( K d ) of the insulin receptor on this cell line was previously measuredas lo-’ M (21). Kinetic Change of Induction of hsp70 mRNA by InsulinThe serum-starved Hep3B/T2 cells were stimulated by M insulin, then cells were harvested at hour 0, 2, 4, 6, 8, 10, 14,18, and22; and the totalRNA wasextracted. As shown in Fig. 4, the increase of specific hsp70transcript starts from 2 h poststimulation, reaches the maximum at 6 h, and rapidly declines after 8 h. Time Course of Induction of DNA Synthesis by InsulinThe serum-starved Hep3B/T2 cells were stimulated with M insulin, then the DNA synthesis was measured every 4 h. As shown in Fig. 5A, the DNA synthesis occurs at 14-18 h postinsulin addition. In thepresence of DNA synthesis inhibitor, araC, 90% of the insulin-stimulated DNA synthesis is blocked. However, the increase of hsp7O mRNA induced by insulin is not inhibited by araC (Fig. 5B).We have always observedthat araC-treated cells have higher amounts of hsp70 mRNA detected, although the reason is not clear. However, araC has been known to have other effects on the cellular

Insulin-induced h p 7 0 Expression

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A

HR POST INSULIN CM 0-~ 2 4 . 6 _8 .10 ~ 14 18 22

HS

-hsp70

FIG.4. Kinetic change of hsp7O mRNA induced by insulin. Hep3B/T2 cells were serum-starved for 6 days and stimulated with 10" M insulin. At hour 0, 2, 4, 6, 8, 10, 14, 18, and 22, RNA was extracted and analyzed by Northern hybridization using phsp70 as the probe. For control, Hep3B/T2 cells were either cultured in the complete medium for 6 days (lane C M ) or followed by heat shock a t 43 "C for 2 h (lane H S ) . Ten p g of total RNA of each sample was used for Northern hybridization with phsp70 as theprobe.

B

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FIG.6. Northern analysis of effect of cycloheximide on insulin-induced expression of hsp7O gene transcript (lanes 58 ) .The serum-starved Hep3B/T2 cells were preincubated for 15 min with 30 pg/ml cycloheximide prior to the addition of 10" M insulin (lane 7).The serum-starved cells were incubated with DMEM (lane 5),lo" M insulin (lane 6 ) . or 30 pg/ml cycloheximide (lane 8). RNA was extracted a t 6 h after insulin stimulation. Northern analysis of insulin-induced expressionof hsp7Otranscript onexponentially growing cells (lanes 1-4). Hep3B/T2 cells were cultured in the complete medium until 70% confluence, then treatedwith the complete medium alone (lanes 1 and 4 ) or containing lo-' (lane 2 ) or 10" M ( l a n e 3 ) insulin in the complete medium for 6 h. Ten pg of total RNA was used for each sample. phsp70 in A and pTWSl in E were used as probes.

HR

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FIG.5. Effect of araC on the DNA synthesis and hsp70 mRNA in insulin-stimulatedHep3B/T2 cells. A, DNA synthesis was measured by [3H]thymidine incorporation in insulin-stimulated Hep3B/T2 cells in the absence (solid line) or presence (doshed line) of araC (20 pg/ml). B, level of hsp70 mRNA was measured by Northern hybridization in insulin-stimulated Hep3B/T2 cells in the absence or presence of araC (20 pglml). Ten pgof total RNA was used for each sample.

metabolism (22). Therefore, the induction of hsp70 mRNA by insulin in Hep3B/T2 does not seem to be DNA synthesisdependent. This result differs from the induction of hsp70 mRNA by 20% fetal calf serum in HeLa cells, in which the

induction is DNA synthesis-dependent (11). Effect of Cycloheximide on Insulin Effect-Cycloheximide at theconcentration of 30 pg/ml previously had been shown to completely inhibit the protein synthesis in human hepatoma cells.3 To determine whether the induction of hsp70 mRNA by insulin was dependent upon the synthesis of new protein(s), Hep3B/T2 cells were preincubated for 15 min with 30 pg/ml cycloheximide prior to insulin addition. As shown in lanes 5-8 of Fig. 6A, the hsp7O mRNA level is not affected by cycloheximide alone. However, the insulin-stimulated increase of hsp70 mRNA is inhibited by cycloheximide. For control, the suppression of HBsAg transcript by insulin shown in lanes 5-8 of Fig. 6B is not blocked by cycloheximide, which is consistent with the previous finding. Insulin Effect on Exponentially Growing Cells-The exponentially growing Hep3B/T2 cells cultured in the medium containing 10%fetal calf serum were stimulated with lo-' or M insulin. As shown in lanes 1-4 of Fig. 6A, the level of M hsp7O mRNA can be increased by insulin at lo-' or concentration. For control, as shown in lanes 1-4 of Fig. 6B, the HBsAg transcript is slightly inhibited by insulin in the complete medium condition, which is consistent with the previous finding. The kinetic change of induction of hsp70 mRNA by insulin in exponentially growing cells has a pattern very similar to that inserum-starved cells (data notshown). Induction of h p 7 0 mRNAby Other Hormones or Growth Factors-The serum-starved Hep3B/T2cells were stimulated by serum,insulin, TPA, EGF, PDGF, calcium ionophore L.-P. Ting, unpublished results.

Insulin-induced h p 7 0 Expression A

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regulatory factor is required to be newly synthesized for the insulin-inducedactivation ofhsp7O gene since the insulin effect is blocked by the protein synthesis inhibitor,cycloheximide. It is in contrast to the heat-shock-induced activation of hsp70 gene in which the regulatory protein is pre-existing and notrequired to be newly synthesized. Thus, the induction of hsp70 by insulin is apparently through different activation mechanism from the heat shock. It has been shown that the hsp7O gene is regulated as a function of the cell cycle in HeLa cells (9, 11).Since insulin can induce theproliferation of Hep3B/T2 cells (21),the B expression of hsp70 gene induced by insulin may link to the proliferation. However, three observations suggest that the insulin-induced expression of hsp70 gene is not cell cycledependent. First, the insulin effect is observed in the expoHBsAg nentially growing Hep3B/T2 cells, which are not synchronized. In addition, the kinetic change of insulin-induced hsp70 mRNA level in these cells is almost the same as that in the serum-starved cells. Second, the expression of hsp70 gene is not induced by EGF which also stimulates the proliferation FIG. 7. Induction of hsp70 mRNA in human hepatoma cell of Hep3B/T2. Third, the hsp70 mRNA expression induced line Hep3B/T2 by hormones,growthfactors,andcalcium by insulin is independent of the DNA synthesis, which is ionophore. For the completemedium control, cells were cultured in distinct from the serum-induced hsp7O mRNA expression in the complete medium for 6 days and replaced with fresh medium for HeLa cells. 6 h (lune C M ) . Six-day serum-starvedcells were treated with DMEM During heat shock, HSP70 may have the role in preventing alone as the medium control (lane D M E M ) or DMEM containing the aggregations of cellular proteins (28). In unstressed cells, 10% fetal calf serum (lane FBS), IO-' M insulin (lane I N ) , 0.5 p~ TPA (lune T P A ) , 1 p~ A23187 (lune Ca IONO), 1 p~ dexamethasone the HSP70 protein is probably involved in regulating mam( l a n e D E X ) , 10 pg/ml PDGF (lane PDGF), 10" M 8-bromo-CAMP malian cell growth (10, 29). However, since the insulin-in(lane 8-BrcAMP),and 0.53 pg/ml EGF (lane ECF) for 6 h. Ten pg duced hsp70 expression is very specific and does not relate to of total RNA of each samplewas analyzed by Northern hybridization the stressresponse or cell proliferation, the induction of hsp70 A and the samefirter was reprobed with with phsp70 as the probe in a new dimension in considering gene by insulin may add pTWSl in B . functions of HSP70. It is possible that in insulin-responding A23187, 8-Br-cAMP,anddexamethasone, respectively. As cells, the HSP70 may specifically play the important role in the signal transducing pathway of insulin action and modushown in Fig. 7, the amount of hsp70 mRNA can be greatly lation of gene expression. M insulin, as well increased by 10%fetal calf serum and as barely affected by either 10 pg/ml PDGF or 0.53 pg/ml Acknowledgments-We thank Dr. Richard I. Morimoto, NorthEGF, whereas TPA, calcium ionophore A23187, dexamethawestern University, for kindly providing us the phsp70 and GRP78 sone, and 8-Br-CAMP have no effect on hsp70 gene expres- plasmids. We also thankDr. Chungming Chang, National Yang-Ming sion. Medical College, for critical discussion and evaluation of the manuC M DMEM FBS

IN

C. 8-81 TPA ION0 L E X A)GFcAMP EGF

script, and toLing-Yu Ting for preparation of the manuscript. DISCUSSION

In this report, insulin hasbeen demonstrated to be able to induce the expression of hsp7O gene in human hepatoma cell line Hep3B/T2 cells at both mRNA and protein levels. The presence of bothinsulinreceptorandinsulin-like growth factor (IGF-I and IGF-11) receptors on this cell line make it difficult to determine which receptor mediates the insulin effect, since insulin can bind to all these .receptors (23). However, insulin binds to these two kinds of receptors with different dissociation constants, i.e. lo-' M for insulin M for IGF receptors (20).4The dose dependreceptor and ence of the insulin effect correlates very well with the dissociation constant between insulin and insulin receptor, indicating that the insulin-induced expression of hsp70 gene is mediated by the insulin receptor rather than by the IGF receptors. The actions of insulin are initiatedby the insulindependent tyrosine-specific proteinkinaseactivity of the insulin receptor (24-26). Whether the receptor switches to a conformation capable of interacting with a mediator generation system to generate second messengers to transduce the signals and the subsequent biochemical events are not completelyclear(27). Therefore, with current knowledge, it is difficult to analyze themolecular mechanism involved in the activation of hsp70 gene expression by insulin. However, a

' C.-K. Chou, unpublished results.

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