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EXPERIMENTAL CELL RESEARCH 192,587-596 (1991)

Differential Regulation of HSC70, HSP70, HSPSOcw, nd HSPSOP mRNA

Expression by Mitogen Activation and Heat Shock in Human Lymphocytes

LINDA K. HANSEN,~ J. P. HOUCHINS,? AND JAMES J. OLEARY*

Department of Laboratory Medicine and Pathology, and tlmmunobiology Research Center, Box 198 UMHC,

IJniuersity of Minnesota, Minneapolis, Minnesota 55455

A

subset

of heat shock proteins, HSPSOcr, HSPSO@,

and a member of the HSP70 family, HSC70, shows en-

hanced synthesis following mitogenic activation as well

as heat shock in human peripheral blood mononuclear

cells. In this study, we have examined expression of

mRNA for these proteins, including the major 70-kDa

heat shock protein, HSP70, in mononuclear cells follow-

ing either heat shock or mitogenic activation with phy-

tohemagglutinin (PHA), ionomycin, and the phorbol es-

ter, tetradecanoyl phorbol acetate. The results demon-

strate that the kinetics of mRNA expression of these

four genes generally parallel the kinetics of enhanced

protein synthesis seen following either heat shock or

mitogen activation and provide clear evidence that mi-

togen-induced synthesis of HSC70 and HSPSO is due to

increased mRNA levels and not simply to enhanced

translation of preexisting mRNA. Although most

previous studies have focused on cell cycle regulation of

HSP70 mRNA, we found that HSP70 mRNA was only

slightly and transiently induced by PHA activation,

while HSC70 is the predominant 70-kDa heat shock

protein homologue induced by mitogens. Similarly,

HSPSOa appears more inducible by heat shock than mi-

togens while the opposite is true for HSP908. These re-

sults suggest that, although HSP70 and HSC70 have

been shown to contain similar promoter regions, addi-

tional regulatory mechanisms which result in differen-

tial expression to a given stimulus must exist. They

clearly demonstrate that human lymphocytes are an

important model system for determining mechanisms

for regulation of heat shock protein synthesis in un-

stressed cells. Finally, based on kinetics of mRNA ex-

pression, the results are consistent with the hypothesis

that HSC70 and HSPSO gene expression are driven by

an IL-2/IL-2 receptor-dependent pathway in human T

cells.

@ 1991 Academic Press, Inc.

INTRODUCTION

Physiologic stress, such as heat shock, preferentially

enhances the synthesis of a limited number of intracel-

1 Current address: Department of Surgical Research, The Chil-

drens Hospital 300 Longwood Avenue, Boston, MA 02115.

To whom correspondence and reprint requests should be ad-

dressed.

lular proteins (heat shock proteins or HSPS).~ The re-

sponse has been observed in all cells so far tested and

some of the HSPs a re highly conserved across species

(reviewed in [l]). The most strongly heat-inducible and

conserved HSPs found in eukaryotic cells are proteins

of about 90 kDa (HSPSO) and 70 kDa (HSP70). How-

ever, HSPSO is also constitutively expressed in un-

stressed cells, and all cell types that have been exam-

ined have constitutively expressed homologues of

HSP70, including the glucose-reactive protein, GRP76

in human cells, and a structural and functional homo-

logue of HSP70, which is less strongly induced by heat

shock and has been designated HSC70 in human cells.

HSPSO and these homologues of HSP70 are abundant

intracellular proteins and it is clear that these proteins

must have important roles in normal cell function in

addition to whatever role they may play in cellular adap-

tation to stress.

However, while the regulation of HSPSO and HSP70

synthesis and gene expression by heat shock has been

well studied, little is known of the regulation of these

gene products in unstressed cells. We have previously

reported [2] that mitogen ac tivation of human periph-

eral blood mononuclear cells in culture results in strong

and sustained preferential enhancement in the synthe-

sis of HSPSO and HSC70 during the prereplicative in-

terval, which is a period of about 24 h following mitogen

addition but prior to entry of the activated cells into S

phase. Human HSPSO actually consists of at least two

proteins, Q and 0, each encoded by its own gene [3] and

appears to be involved in a number of importan t intra-

cellular processes in unstressed cells including serving

as a cytoplasmic shuttle protein for growth-related mac-

romolecules such as steroid hormone receptors [4] and

the protooncogene product src-tyrosine kinase [5].

Compared to human HSP70, HSC70 is only moderately

heat inducible, but is 83% homologous at the amino acid

level with HSP70 [6]. In eukaryotes HSC70 appears to

be involved in a number of intracellular processes im-

portant for cell growth, such as uncoating of clathrin

3 Abbreviations used: PHA, phytohemagglutinin; PBLs, peripheral

blood lymphocytes; HSP, heat-shock protein; IL-2, interleukin-2;

TPA, tetradecanoyl phorbol acetate.

587

0014.4827/91 3.00

c

opynght Q 1991 by Academic Press, Inc.

All rights o f reproduction in any form reserved.


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588

HANSEN, HOUCHINS, AND OLEARY

triskelions from endocytotic vesicles [ 71 and transloca-

tion of nascent polypeptide chains across organelle

membranes [8, 91.

The enhanced expression of HSC70 and HSPSO ob-

served after mitogen activation of human lymphocytes

shows marked differences from the enhanced expres-

sion of these gene products associated with the heat

shock response. Heat shock of human cells in culture

leads to enhanced expression of HSP70 and HSPSO

mRN A and enhanced synthesis of the proteins which

can be detected within 15-30 min of the heat shock [3,

lo]. However, after stimulation of human peripheral

blood lymphocytes with the mitogen phytohemagglu-

tinin (PHA), which activates primarily T cells, preferen-

tial enhancement in HSC70 and HSPSO synthesis does

not occur until about 8 h following PHA addition reach-

ing a stable maximum by about 12-18 h of culture with

PHA [ 111. Unlike the heat shock response, we observed

no detectab le increase in HSP70 synthesis during the

prereplicative interval or even as the activated cells be-

gin to enter S phase. By contrast, a few studies have

reported enhanced expression of HSP70 in serum-

stimulated HeLa cells [lo, 121, but the enhancement

appears less than that which we observed for HSC70

and HSPSO in lymphocytes and these findings may not

be relevant for nontransformed cells. Some enhance-

ment in HSP70 synthesis and mRNA expression has

been seen in purified T lymphocytes activated with

PHA [13], but this study did not examine the HSC70

gene product, and again the enhancement appears small

compared to that observed for HSPSO and HSC70 in

PHA-activated mononuclear cells. One problem with

such studies of mitogen activation in human lympho-

cytes is that purified T cells lack the accessory cells, i.e.,

monocytes, required for optimal proliferative response

[14] and which are present at optimal levels for mito-

gen-induced proliferation in the mononuclear cell prepa-

rations used in our previous work [a].

In fact, serum- or growth factor-induced expression

of HSC70 mRNA has not been previously examined in

human cells, although similar genes have been studied

in Drosophila [15] and rat cells [16] which, like human

HSC70, are expressed in the absence of heat shock [6,

111. Studies thus far find evidence only for serum induc-

tion of the HSPSOcr gene product in transformed human

cells [ 171. However, it appeared from two-dimensional

gel electrophoresis in our studies of T cells that both

HSPSO cu and /3 are mitogen inducible [II]. Thus, the

goal of the current study was to begin to clarify the

mechanisms responsible for the marked enhancement

in HSC70 and HSPSO protein synthesis observed follow-

ing PHA activation of human mononuclear cells. The

basic question was whether the enhanced synthesis of

these proteins reflects enhanced levels of mRNA ex-

pression, implying growth factor-modulated enhance-

ment in gene expression, or is primarily due to enhanced

translation without an increase in steady-s tate mRNA

levels. In addition, we sought to clarify whether mitogen

activation might result in some enhancement in HSP70

mRNA expression as implied by the study of purified T

cells [13]. The results are consistent with a slight, but

transient up-regulation of HSP70 mRNA abundance

during the prereplicative interval of human T cells.

However, they demonstrate clearly that HSC70 is the

predominant 70-kDa HSP induced following mitogen

activation both at the level of mRNA abundance and

protein synthesis. Because the kinetics of expression of

HSC70 mRNA are dramatically different from that of

HSP70 following mitogen activation and heat shock, it

seems clear that despite great functional similarities the

genes for these products must contain quite different

regulatory elements. The results also show some degree

of differential modulation of HSPSOcu and HSPSOP

mRNA abundance in heat shock compared to mitogen

response, but both forms are clearly enhanced in the

two responses, implying a greater degree of regulatory

homology at the gene level. More importantly, these re-

sults are perhaps the strongest support so far reported

for the hypothesis that HSPSO and HSC70 expression

can be modulated in unstressed cells by a growth factor-

mediated pathway [ 181.

MATERIALS AND METHODS

Cell isolation and culture. Peripheral blood mononuclear cells

were isolated from healthy donor whole blood by centrifugation on

H&opaque (Sigma Chemical Co.), washed three times with Hanks

Balanced Salt Solution (GIBCO, Grand Island, NY), and resus-

pended at 2-5 X 10s cells/ml in 10% pooled hu man serum and RPM1

1640 medium (GIBCO). Phytohemagglutinin (PHA) (Wellcome

Diagnostics) was added at 1 pg/m l where indicated. Ionomyc in was

used at 5 fig/ml alone or 1 pg/m l when added with tetradecanoyl phor-

bol acetate (TPA). TPA alone was used at 50 or 100 rig/ml when

added with ionomycin. Heat stress consisted of incubating cell cul-

tures in 50.ml tubes in a water bath at 42 or 45C for 30 or 15 min,

respectively, followed by a recovery period at 37C. For protein label-

ing, cells were cultured in the presence of [3H]leucine (ICN) at a spe-

cific activity of 50 Ci/mmol and a dose of 100 &i/ml and [35S]methi-

onine (ICN) at a specific activity of 344 mCi/mmol and a dose of 50

pCi/ml.

cDNA probes. The HSP70 probe was a full-length cDNA con-

tained in plasmid, pH 2.3, a gracious gift from Dr. Richard Morimoto.

The HSC70 probe was a 500.bp fragment isolated from X-phage 7,

also a gift from Dr. Richard Morimoto. This 500.bp fragment was

derived from the 5 end of the HSC70 coding region, identified by in

uitro translation of larger cDNA. The HSC70 and HSP70 cDNA

probes exhibit no cross-reactivity under normal stringency condi-

tions. The HSPSOtu and B probes were the kind gift of Dr. Eileen

Hickey and were 900. and 800.bp cDNA fragments, respectively.

These cDNA probes are also non-cross-reac ting under normal strin-

gency conditions. The major histocompatibility Class I cDNA probe

was specific for the human B7 gene.

Subcellular f ractionation for protein synthesis. After cell culture,

cells were scraped f rom culture dishes with a rubber policeman and

washed three times with cold Hanks. The cell pellet was resuspended

in low detergent lysis buffer (0.1% Triton X-100, 150 mM KCl, 8 mM

MgCl,, 20 mM Tris, 1 mM PMSF, pH 7.5) at a concentration of 10

&lo6 cells. After a 15.min incubation on ice, the solution was spun

briefly in a microfuge to pellet nuclei. The supernatant was saved as

the cytoplasmic fraction.


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MITOGEN- AND HEAT-INDUCED EXPRESSION OF HSP mRNA

589

RNA isolation. RNA isolation was performed using a modifica-

tion of the Chirgwin method 1191. Briefly, after incubation , cells were

washed two times in cold Hanks, then lysed in 3 ml guanidinium

thiocyanate solution (4 M guanidinium thiocyanate [Fluka], 25 mM

sodium citrate, pH 7, 0.5% sarcosyl, 0.1 M 2-merceptoethanol). The

lysate was passed through a 22-gauge needle 6-10 times and layered

on a 1.5.m15.7 M CsCl cushion in Beckman polyallomer tubes. This

preparation was spun at 35,000 rpm for 18 h. The supernatant was

carefully removed and 100 ~1 TES (10 mM Tris-HCl, pH 7.6, 1 mM

EDTA, 5% sarkosyl) was added to the pellet which was allowed to

dissolve for 30 min. The buffer was removed and another 100 ~1 was

added to the tube for 30 min. The resulting 200.~1 sample was read at

A,,, and A,, to determine concentration and sample purity.

SDS-polyacrylamide ge:elelectrophoresis. Following cell fraction-

ation, counts per minute of label incorporated into the cytoplasmic

fractions were determined by scintillation counting . Equal counts per

minute of cytoplasmic samples were added to each lane in a given gel

and separated by SDSPAGE using the method of Laemmli [20].

Gels were dried on cellophane (Bio-Rad) on plastic frames (Idea Sci-

entific) and placed on Kodak X-Omat film for autoradiography.

Probe preparation. The cDNA sequences were cut from the plas-

mids with the appropriate restriction endonuclease and then electro-

phoresed in a known quantity on low melting point agarose (BRL)

gels. The cDNA fragment was excised from the gel, weighed, and

boiled 10 min. The amount of fragment present was estimated and

the solution was aliquoted into lo-fig samples and frozen at ~20C.

To label, aliquots were removed and boiled 2 min. The Multiprime

labeling kit (Amersham) was used to label the cDNA with [32P]dCTP.

A specific activity of 1 X lo9 cpmlwg was generally achieved.

Northern analysis. Northern analysis was performed as described

by Thomas 1211. Briefly, RNA samples were denatured 15 min at

60C in a 1X Mops, 50% formamide, 12% formaldehyde solution, and

then applied to formaldehyde-agarose denaturing gels (16% formalde-

hyde, 1X Mops, 1.5% agarose [BRL]). RNA ladder (BRL) was used

for molecular weight determina tions. After electrophoresis, the

RNA-containing gel was incubated at room temperature in a 0.05 N

NaOH solution for 30 min, followed with 2~ SSC for 30 min. The

RNA was transferred from the gel to GeneScreen (DuPont) using the

capillary blotting technique at least 20 h. The blot was washed briefly

in 1X SSC and then baked in V~CUOat 80C for 2 h.

Hybridization was performed as follows: B lots were prehybridized

in hybridization solution (1 M NaCl, 50% formamide, 15% dextran

sulfate, 5X Denhardts, and 1% SDS) for at least 4 h at 42C. Labeled

probe was boiled for 10 min in the presence of depurinated salmon

sperm DNA and added to the blot at 5 X lo cpm/ml hybridization

buffer. Hybridization took place at 42C for at least 16 h, after which

blots were washed two times in 2X SSC at room temperatu re, two

times in 2X SSC, 1% SDS at 6OC, and two times in 0.2~ SSC, 0.1%

SDS at room tempera ture. Blots were then resealed in a heat-sealable

bag with 1X SSC-saturated 3-mm Whatman paper and exposed to

Kodak X-Omat film with DuPont intensifying screens.

Blots were stripped by boiling for 2 min in 1% SDS followed by

cooling for 15 min. This was repeated, and autoradiography was per-

formed to assure that all the radioactive probe was removed. Blots

were reprobed as described above.

RESULTS

Specific mRN A Induction following PHA Activation

of

Human Mononuclear Cells

In Fig. 1, mRNA levels were compared by Northern

analysis of human mononuclear cells cultured for

various times after PHA addition using the cDNA

probes for HSC70, HSP70, HSPSOa, and HSPSOP de-

scribed above. cDNA for the major histocompatibility

HSC70

HSP9Oa

Class I

,. .-

-2.6 kb

-2.5

-2.7

-1.6

FIG. 1. Kinetics of HSP mRNA expression following mitogen ic

activation of mononuclear cells. Peripheral blood mononuclear cells

were PHA-activated and cytoplasmic RNA was isolated at the indi-

cated times following PHA addition. Levels of cellular HSC70,

HSP70, HSPSOtu, and HSP90fi mRNA were determined using the

cDNA probes described under Materials and Methods and the results

shown are for the same blot which was stripped and reprobed with

each cDNA. The major histocompatibility complex Class I probe was

used as a control to compare RNA loading in each lane. Molecular

weights (kilobases) of the bands are indicated to the right.

Class I gene, which is not mitogen-inducible in human T

lymphocytes , was used as a control. As shown in Fig. 1,

HSC70, HSPSOcY, and HSPSOP mRNA are detectable in

freshly isolated (0 h) cells. Enhanced HSC70, HSP90@,

and HSPSOB mRNA levels were observed between 4 and

8 h after PHA addition, increasing out to 24 h. HSP70

mRNA levels showed a slight enhancement only at 12 h

of culture, but return to baseline (0 h) levels by 24 h of

culture.

mRNA and Protein Induction following Heat Shock

To determine the kinetics of mRN A induction follow-

ing heat stress, mononuclear cells were heat shocked at

42C for 30 min, then returned to 37C (Fig. 2). Samples

were removed from the 37C water bath at the indicated

times for total cellular RNA isolation. While HSP70

mRNA is not detectable in the control cells incubated

continuously at 37C, immediately following the 30-min

heat shock a substantial amount of HSP70 mRNA is

present. E levated, but decreasing, levels are maintained

through 2 h post heat shock, and by 4 h HSP70 mRNA is


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590

HANSEN, HOIJCHINS, AND OLEARY

HSC70

-2.5

HSP90a

-2.95

HSP90P

-2.7

FIG. 2. Kinetics of HSP mRNA expression following heat shock.

The cells were heat shocked at 42C for 30 min and returned to 37C,

and total cellular RNA was isolated at the indicated times following

return to 37C. Northern analysis was performed as in Fig. 1 and the

results shown are for the same blot which was stripped and reprobed

with each cDNA. Although the data are not shown, mRNA levels

detected with the Class I gene probe were not affected by heat shock

and, relative to the induction seen with the heat shock gene probes,

reflected only slight differences in loading.

barely detectable. HSC70 mRNA is detectable in the

control cells, and slightly enhanced HSC70 mRNA lev-

els are seen immediately following the 30.min heat

shock, peaking at 30 min of incubation at 37C, and

decreasing to resting levels by l-2 h. Like HSC70,

HSPSOol mRNA is detectable in resting cells and

slightly enhanced immediately following the heat shock.

Maximal levels are achieved within 2 h after stress and

by 4 h, levels have returned to control. HSPSOP mRNA

shows a similar pattern of response to heat shock, al-

though maximal levels are seen slightly earlier.

The kinetics of heat shock-induced cytoplasmic pro-

tein synthesis are shown in Fig. 3. Following a 30 min

42C heat shock, the mononuclear cells were returned

to 37C and pulse labeled with radiolabeled amino acids

for the indicated intervals. After the labeling period,

cells were lysed and counts per minute of incorporated

label determined with equal counts per minute added to

each lane in the gel, followed by separation on SDS-

PAGE and autoradiography. As expected with addition

of equal amounts of label per lane the increases in the

heat shock bands are accompanied by decreased inten-

sity of bands corresponding to non-heat shock proteins,

particularly the major band corresponding to actin at

about 42 kDa. The exposure time for the autoradiogram

was adjusted to permit optimal visualization of the rela-

tive enhancement in heat shock protein synthesis.

Thus, the control lane is somewhat underexposed (No

HS).; however, as shown in Fig. 7 and previously [2,11],

HSP70 synthesis is undetectable in control cells even

with greater relative exposure of the autorad iograms.

As shown in Fig. 3, HSP70 synthesis is enhanced within

the first 30 min following stress. Synthesis reaches a

peak at l-2 h and returns to control levels by 446 h. By

contrast, HSC70 synthesis is detectab le in resting con-

trol cells and is enhanced 30-60 min following stress

and returns to control levels by 2-3 h. As with HSC70,

the HSPSO band, composed of both HSPSOtr and

HSPSOP, is detectably synthesized in resting cells. En-

hanced synthesis is observed 30-60 min following stress

with maximal synthesis seen at l-2 h. Baseline levels

are achieved by 4-6 h. Thus, the heat-induced enhance-

ment in protein synthesis parallels increases in specific

mRN A levels, although as expected the kinetics are

somewhat delayed.

Comparison of mRNA Induction following Heat Shock

and Mitogen Activation

The degree of specific mR NA induction following

heat stress or mitogen activation is compared in Fig. 4,

HSPSO-

HSC70-

HSP70

FIG. 3. Kinetics of protein synthesis in mononuc lear cells follow-

ing heat shock. Cells were heat shocked at 42C for 30 min, then

returned to 37C, and incubated with [3H]leucine and [%]methio-

nine for the indicated intervals prior to cell fractionation. Equal

counts per minute of incorporated label were loaded in each lane with

proteins separated by SDSPAGE followed by autoradiography. The

autoradiograph is shown. Molecular weight markers (kDa) are indi-

cated to the right and the positions of HSPSO (LYand B forms are not

distinguishable), HSC70, and HSP70 as shown in previous studies [2,

111 are indicated to the left.


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591

HSP70

-2.6 kb

HSP90a

HSPJN)P

-2.7

FIG. 4. Comparison of heat shock-induced and mitogen-induced

levels of HSP mRNA in mononuc lear cells. The cells were heat

shocked at 42C for 30 min or 45C for 15 min, and then placed at

37C for 1 h at which time total cellular RNA was isolated. In addi-

tion, RNA was isolated from lymphocytes which were incubated with

PHA for 1 2, 24, or 48 h. The results show a single blot which was

stripped and reprobed with each cDNA. Although not shown, varia-

tions in loading detected with the Class I gene probe were insignifi-

cant compared to the differences seen with the other probes.

which shows mitogen induction of HSP mRNA and the

induction of HSP mRNA after a 42 and 45C heat shock

with RNA isolated 1 h after the heat stress. In addition,

mRNA expression was examined out to 48 h after PHA

activation to see if the enhanced message levels are sus-

tained later in the cell cycle or only transiently ex-

pressed during the prereplicative, GO-S phase, transi-

tion. As shown previously [22], by 48 h following PHA

addition, most responding T lymphocytes have com-

pleted at least one round of division and are progressing

through the cell cycle with increasing asynchrony.

HSP70 mRNA is, as expected from the previous re-

sults, undetectable in resting cells. Heat stress at 42C

enhances abundant HSP70 mRNA, with even greater

enhancement at 45C. Consistent with Fig. 1, a very

faint HSP70 band is detectable at 12 h following PHA

activation, with the mRNA diminishing to undetectable

levels by 48 h. Again consistent with the previous re-

sults, HSC70 m RNA is detectable in resting cells and

less strongly induced than HSP70 mRNA by 42C heat

shock; however, the mRNA is strongly enhanced at

45C. PHA induced HSC70 levels at 12 h are signifi-

cantly enhanced over that seen in control (37C) cells or

42C heat-stressed cells. This increase in HSC70

mRNA is maintained out to 24 h, but is diminished at 48

h. The heat shock induction of HSC70 mRNA is compa-

rable to mitogen-induced levels only at the higher heat

shock temperature.

HSPSOn mRNA is detectab le in a very low quantity in

resting cells, and shows a lo- and 20-fold increase with

42 and 45C heat stress, respectively. After PHA addi-

tion, mRNA expression continues to increase out to 48

h. The level of 42C heat shock-induced mRNA is about

equivalent to that seen at 24 h after PHA addition, but

the amount of mRNA induced by severe heat shock ex-

ceeds that seen at any time point following mitogen ac-

tivation. HSP908 mRNA is also low in resting cells and

increases in response to heat stress, but its extent of

induction by heat stress is lower relative to that induced

by mitogen. Like HSPSOcu, mitogen-enhanced mRNA

levels persist at least 48 h following stimulation. Thus,

HSPSOcu appears to be more inducible by heat stress

than by mitogen, whereas HSPSOP shows greater mito-

gen enhancement.

Cytoplasm ic protein synthesis induced by different

degrees of heat stress is shown in Fig. 5. As in Fig. 3,

equal counts of incorporated label were added to each

lane. Prior to cell fractionation, aliquots of cells were

heat shocked followed by incubation with radiolabeled

amino acids for 2 h at 37C or the radiolabeled amino

acids were added and the cells were left at the heat

shock temperature for the 2-h labeling period. The 30-

min 42C heat stress followed by 37C recovery shows

the expected pattern of protein synthesis, with HSP70

showing greatest induction, along with HSPSO, HSC70,

and other minor heat shock proteins. A 2-h continuous

incubation at 42C shows essentially the same pattern.

In contrast, the 15-min 45C stress induces HSP70 and

HSC70 as expected, but the quantity of HSPSO protein

is diminished relative to that seen with the heat shock at

the lower temperature. Continuous incubation for 2 h at

45C inhibited any detectab le incorporation of radiola-

beled amino acids into proteins.

The HSP protein synthesis observed following the

15-min 45C heat shock stands in contrast to the

marked enhancement in mRNA levels observed with

this stress compared to the mRNA levels observed with

heat shock at 42C. Thus, while HSPSO mRNA levels

were more strongly induced by the 15.min 45C heat

shock, HSPSO protein synthesis declined, and HSP70

and HSC70 show about the same degree of enhanced

protein synthesis at 45C despite a marked increase in

mRN A levels with this heat shock condition.

Effects of Phorbol Ester and Ionomycin on HSP

Induction in Mononuclear Cells

Certain agents in addition to PHA can be mitogenic

for lymphocytes. Ionomycin, a calcium ionophore, and

TPA, a phorbol ester, can induce a partial mitogenic


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592


205kd

-116

96

66

-45

-29

FIG. 5. Protein synthesis following heat shock at different tem-

peratures. Mononuclear cells were heat shocked at 42C for 30 min or

45C for 15 min, and [H]leucine and [?S]methionine were added to

the culture medium. The cells were then either returned to 37C or

maintained at the heat shock temperature for the labeling interval.

The cells were then lysed and equal counts of incorporated label were

added per lane and separated by SDSPAGE followed by autoradiog-

raphy. The autoradiograph is shown: No heat shock, 37C for 2 h;

42C for 30 min, 37 for 2 h; 42C for 2.5 h; 45C for 15 min, 37C for 2

h: 45C for 2.25 h.

response alone or in combination by increasing intra-

cellular free calcium and activating protein kinase C,

respectively. mRNA induction by these agents was ex-

amined and compared to levels in resting cells and cells

activated by PHA, using concentrations of TPA (50 ng/

ml) and ionomycin (5 pg/ml) that separately induced

the greatest proliferative response, which was about 20-

25% of that seen with PHA. The optimal concentrations

when used together were 100 rig/ml and 1 pg/ml, respec-

tively, and this combination induced a proliferative re-

sponse about

55%

of the optimal PHA-induced re-

sponse (data not shown).

In these experiments, the cells were incubated with

the same concentrations of mitogens for 24 h in the pres-

ence or absence of serum, a required cofactor for the

proliferative response. In the presence of serum, HSP70

mRNA appears to show some slight enhancement in the

TPA- and PHA-activated cells (Fig. 6). In contrast,

HSC70 shows a similar, 5- to lo-fold induction, by iono-

mycin, TPA, or PHA, and both HSPSOo( and HSP90/3

also show strong induction with all treatments. The

pattern of protein induction in the presence of serum is

also similar with each stimulus (Fig. 7). As in Figs. 3 and

5, equal counts per minute of label incorporated into the

cytoplasmic factions was added per lane. Consistent

with our previous reports [2,11] the induction of HSC70

and HSPSO by PHA is not as striking as the relative

induction seen following heat shock . However, the pref-

erential enhancement with PHA activation occurs

against a general increase in protein synthesis during

the prereplicative interval, and synthesis of these pro-

teins accounts for more than 5% of the total protein

synthesized during this interval [2]. PHA added in the

absence of serum, although nonmitogenic [2], is suffi-

cient to induce the synthesis of both HSC70 and

HSPSO, while serum alone elicits no induction. TPA,

like PHA, is able to induce this set of proteins in both

the presence and the absence of serum as does the com-

bination of ionomycin and TPA. By contrast, ionomycin

added in the absence of serum gives a very different

pattern of protein synthesis with strong induction of a

band with molecular weight similar to another member

of the HSP70 family, BiP, or GRP76, which has been

shown to be induced by calcium ionophores in another

cell type [23]. However, the preferentially enhanced syn-

E g

2

%

= c

2 .P c .,

f

HSP70

-2.6 kb

HSC70

-2.5

HSP90u

HSP90P

-2.7

FIG. 6. HSP mRNA induction by ionomycin and phorbol ester.

Mononuclear cells were incubated in medium and 10% serum alone

(lane marked serum) or with ionomycin (5 pg/ml), TPA (50 rig/ml),

ionomycin (1 rg/ml) plus TPA (100 rig/ml), or PHA (1 Kg/ml). After

incubation for 18 h, total cellular RNA was isolated and Northern

analysis was performed. One blot was stripped and reprobed with

each cDNA. The Class I probe (data not shown) again revealed only

small differences in gel loading.


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593

-205kd

HSPSO-

HSC70-

FIG. 7. Protein synthesis induced by ionomycin and phorhol es-

ter. Mononuclear cells were placed in medium alone or medium p lus

10% serum and incubated with the indicated agents at concentrations

which gave the optimal proliferative responses, as described in the

text. lonomycin (i) was added at 5 pg/ml; TPA was added at 50

rig/ml; and when added together, the concentrations were 1 pg/ml

and 100 rig/ml, respectively, and PHA was used at 1 pg/ml. The cul-

tures containing serum are noted by +s. and serum = control

with no mitogens. The remaining cultures contained medium alone

with no serum added. Af ter 18 h in culture, cells were lysed and equal

counts from the cytoplasmic fractions were run on SDS-PAGE, fol-

lowed by autoradiography. The autoradiograph is shown.

thesis seen in Fig. 7 is clearly associated with a general

decrease in synthesis of most of the major bands seen in

the control lanes or in the lane with ionomycin in the

presence of serum. More severe forms of stress, e.g., the

more severe examples of heat shock shown in Fig. 5, also

induce a general suppression in protein synthesis and

thus, it seems likely that this result represen ts a toxic

effect due to an excessive free concentration of ionomy-

tin in serum-free medium which lacks serum proteins

which can bind this hydrophobic compound.

DISCUSSION

This paper is the first report to our knowledge to ex-

amine mitogen-induced HSC70, HSPSOcu, and HSPSOP

mRNA expression in human lymphocytes presented

with optimal conditions for cell growth and prolifera-

tion. The results show that HSC70 and HSP90a and /3

mRN A are significantly enhanced by mitogen activa-

tion in human periphera l blood mononuclear cells (Fig.

1). These enhanced mRNA levels correlate with the ki-

netics of enhanced HSC70 and HSPSO synthesis ob-

served following PHA activation (Fig. 7 and [2, 111). As

noted under Results, the preferential enhancement in

synthesis of the heat shock proteins appears more strik-

ing following heat shock (Figs. 3 and 5), but in part this

is due to the lower general level of protein synthesis in

quiescent cells which forms the background against

which this enhancement is visualized. Nevertheless,

with the major exception of the results following a brief

45C heat shock (F igs. 4 and 5), where protein synthesis

is not further enhanced (HSC70, HSP70) or declines

(HSPSO) despite greatly increased mRNA abundance,

differences in kinetics of protein synthesis with heat

shock are also generally mirrored by differences in ex-

pression at the level of mRNA. Taken together, these

results indicate that the preferentially enhanced pro-

tein synthesis is not simply due to enhanced transla-

tional activity of preexisting mRNA in mitogen-acti-

vated or heat-shocked mononuclear cells.

For HSC70 and HSPSO, enhanced mRNA levels were

observed within 8 h following mitogenic activation and

were sustained out to 24 h, which is just prior to entry

into S phase [22], and the increased levels are main-

tained beyond this prereplicative interval. By contrast,

HSP70 mRNA is only transiently increased at about 12

h after PHA addition. As expected, heat shock-induced

mRNA expression was seen for all four genes examined,

but unlike the PHA response enhanced expression is

sustained only 2-4 h following heat shock (Fig. 2). Thus,

even though HSP70 and HSC70 show 75% homology at

the DNA level and contain similar promoter regions

(See below and [6]), they exhibit very different kinetics

of expression to either stimulus.

Peripheral blood mononuclear cells consist of approx-

imately 5% monocytes, 10% B lymphocytes, and 85% T

lymphocytes, and the majority of the lymphocytes are in

a natural GO, or resting, stage of the cell cycle. Activa-

tion of the T cells by a mitogen like PHA in the presence

of adequate numbers of accessory cells initiates a com-

plex sequence of events as the cells progress through the

prereplicative interval. Perhaps the most well-charac-

terized events involve synthesis of the T-cell growth

factor, IL-2, and expression of IL-2 receptors on T cells.

Within 2-6 h after activation, CD4+ helper T lympho-

cytes in the presence of accessory cells begin to synthe-

size and secrete IL-2 and both the CD4+ and the CD8+

(cytotoxic/suppressor) T cells begin to express IL-2 re-

ceptors [24]. IL-2 then acts as a growth factor required

for lymphocyte progression from Gl into S phase [24,

251. As shown here, expression of HSC70 and HSPSO

mRNA appears to be up-regulated subsequent to this

initial increase in IL-2 and IL-2 receptor expression

(Fig. 1). Thus, the kinetics of enhanced HSC70 and

HSPSO expression are consistent with enhancement fol-

lowing IL-2/IL-2 receptor interaction.

This conclusion is also supported by the results of

Ferris

et

al. [ 131 using purified human T cells. As noted

above, isolated T cells lack the accessory cells which are


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594

HANSEN, HOUCHINS. AND OLEARY

required for IL-2 synthesis [ 141, and it is not surprising

that neither sustained synthesis of HSPSO nor induc-

tion of HSP7O mRNA was seen in primary response to

PHA by these investigators. Some increase in HSP70

protein synthesis was observed in response to PHA, but

they found no induction of HSP70 mRNA by PHA or

phorbol12-myristate 13-acetate. HSPSOprotein synthe-

sis (the (Yand /3 orms were not distinguished) was found

to be rapidly and transiently induced by PHA with ki-

netics of expression different from HSP70. However,

while IL-2 synthesis requires accessory cells, PHA can

induce IL-2 receptor expression in T cells depleted of

monocytes [22]. When IL-2 was added subsequent to

PHA addition, Ferris et al. [ 131 observed some enhanced

HSP70 mRNA and protein expression and a sustained

induction of HSPSO synthesis, similar to the kinetics of

HSPSO synthesis we have observed in PHA activated

mononuclear cells [ 111. Although HSC70 induction was

not examined by Ferris et al. [ 131, these results clearly

support the hypothesis that HSC70 and HSPSO synthe-

sis in T cells is driven by the IL-2/IL-2 receptor interac-

tion.

In contrast to these results with purified T cells, we

have been unable to demonstrate any increases in

HSP70 protein synthesis following PHA activation of

the T cells present in human mononuclear cells [2, 111.

All that we have been able to observe for HSP70 per se is

the very transient increase in mRN A levels at about 12

h after PHA addition shown in the current study (Fig.

1). In fact, Kaczmarek et al. [26] and Ida and Yahara

[27] demonstrated diminished HSP70 mRNA and pro-

tein expression, respectively, in PHA-activated lym-

phocytes. The different results observed in these studies

may be due at least in part to the existence of up to 10

genes in the HSP70 family [28] and lack of standardized

nomenclature or use of different gene probes for HSP70

may explain some of these apparently conflicting re-

sults. However, taken together it seems clear that

HSP70 synthesis and mRNA expression is only weakly

inducible by T-lymphocyte mitogens, and the constuiti-

vely synthesized homologue, HSC70, is the predomi-

nant form induced in this growth factor response.

HSP70 and HSPSO expression have also been exam-

ined in the growth response of other cell types. HSP70

and HSPSOcY mRNA expression have been shown to be

enhanced in HeLa cells following serum addition [lo,

121. c-myc can also induce HSP70 expression in mam-

malian cells [29] and Ela adenov irus infection, which

leads to increased expression of cellular gene products

involved in cell growth, has been reported to induce ex-

pression of HSP70 and HSPSOa, but not HSPSOP, in

HeLa cells [3, 171. On the other hand, Kao et al. [30]

report an increase in HSP70 protein synthesis following

S phase in HeLa cells rather than in the Gl interval

following serum addition. Regard less of this apparent

conflict, most studies with other human-derived cells

have focused on expression in transformed cell lines.

Such cell lines exhibit proliferation that is relatively in-

dependent of growth factors and serum deprivation gen-

erally does not lead to growth arrest in a true GO state.

Thus, responses of growth-arrested transformed cells to

serum addition may not accurately reflect the effects of

serum or growth factor addition on cell cycle progres-

sion in nontrans formed cell types.

By contrast, it is clear from the current results that

up-regulation of HSC70 and the HSPSO genes is part of

the sequence of events involved in the prereplicative

interval of human T cells and these findings suggest

that similar modulation may be a common feature of the

growth factor response of other nontransformed human

cell types. Unlike the studies of transformed cells, again

there is not much evidence for modulation of HSP70

levels during the proliferative cycle of T cells and, while

some studies have reported serum induction of

HSPSOa, but not HSPSOP, in HeLa cells [3, 171, our

results indicate that both the (Y and the /3 forms are mi-

togen inducible and in fact, HSPSOP mRNA exhibits

greater PHA-stimulated induction, relative to induction

by heat shock, than HSPSOn mRNA (Fig. 6).

HSC70, HSP70, HSPSOcu, and HSPSOB mRNA were

also induced by ionomycin and TPA in the current

study, both of which are only partial mitogens for pe-

ripheral blood lymphocytes [31]. Calcium ionophores

such as ionomycin or A23187 induce the release of in-

tracellular calcium stores, one of the events in the mito-

genie pathway. On the other hand, the phorbol esters,

such as TPA, activate protein kinase C [25]. In the

current results, ionomycin at an optimal concentration

induced a proliferative response that was 26% of that

with PHA, while TPA gave a 20% response, and with

both reagents the response was only 55% of that seen

with optimal PHA dose (data not shown). Although

these agents elicited only a partial mitogenic response

compared to PHA, both ionomycin and TPA induced

expression of the same set of heat shock mRNA seen

with PHA and expression was enhanced to about the

same extent with each stimulus (Fig. 6). An intriguing

finding was that enhanced synthesis of these proteins

by PHA and TPA does not require the presence of

serum in the culture medium. As shown previously [ll],

serum alone does not affect HSP synthesis, and serum

supplies transferrin, a required cofactor for T-cell prolif-

erative response. Thus, these results demonstrate that

induction of the heat shock gene products by PHA or

TPA in the absence of serum is not by itself sufficient to

induce the cells to enter the cell cycle. By contrast, iono-

mycin in the absence of serum induced the synthesis of a

protein with a molecular weight similar to that expected

for the glucose reactive HSP70 homologue, BiP or

GRP76, which has been shown to be induced by iono-

mycin in another cell type [23]. As noted under Results,

however, the enhanced synthesis of this protein was ac-

companied by marked suppression of other synthesized

bands, suggesting that in the absence of serum binding


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595

proteins the cells were subjec t to excessive concentra-

tions or the drug. Thus, it seem s likely that the appar-

ently enhanced synthesis of GRP76 in this experiment

may be an artifact of cell injury rather than due to spe-

cific induction of this gene product.

There is growing evidence for at least two distinct

promoters in the 5 untranslated region of some HSP70

genes which is consistent with the hypothesis that gene

activity may be modulated by growth factor ac tivation.

All heat shock genes contain a highly conserved heat

shock consensus element (HSE) which is responsible

for heat shock induction, and an additional region has

been identified as the serum-responsive element in the

HSP70 gene of HeLa cells along with a CCAAT box

which has been observed in a number of eukaryotic

other gene promoters [32, 331. An HSE and a CCAAT

box have also been identified in the 5 untranslated re-

gion of the human HSC70 [6]. Despite these indications

that HSC70 and HSP70 may have similar promotor re-

gions, as shown in the current study the kinetics and

levels of expression differ dramatically in response to

heat shock or PHA in human lymphocytes. Thus, there

must exist additional regulatory elements which are re-

sponsible for this differential expression, such as, as yet

undefined differences in the promotor regions, trans-

acting enhancer elements, or differences in mRNA sta-

bility. The existence of possible growth factor-respon-

sive promotors in the HSPSO genes has yet to be deter-

mined, but based on the current results it seems clear

that similar non-HSE promotor regions must be pres-

ent to explain the very significant and sustained in-

creases in HSPSO mRNA expression we observe, and

again additional regulatory elements must be involved

to explain the differential enhancement in HSPSOa and

HSPSOP mRNA, following mitogen activation and heat

shock.

Only two instances were observed in this study where

protein expression does not parallel mRNA expression

and which might indicate control of HSP synthesis at

some level other than mRNA abundance. First, in com-

paring mild (42C) to severe (45C) heat stress, mRNA

expression increases dramatically for each gene at the

higher temperature (Fig. 4), but protein synthesis is not

enhanced or diminished (Fig. 5). Similar to these re-

sults, the 90-kDa heat shock protein expressed in HeLa

cells is synthesized at 42C but not 45C [34]. It has

been proposed that one of the toxic effects of severe

heat stress is inhibition of mRNA translation [35]. On

the other hand, it has been reported that at tempera-

tures greater than 42.5C, mammalian cells are unable

to splice introns from unprocessed messages [36]. Be-

cause the HSP70 gene contains no introns, and introns

are present in HSC70 and both HSPSO genes [6,12,37],

it is possible that the diminished HSC70 and HSPSO

synthesis we observed at the higher heat shock tempera-

ture could be accounted for by this mechanism. How-

ever, our Northern blots do not show evidence for un-

processed message after the 45C heat shock and in fact

show enhanced levels of what appears to be mature

mRNA. Alternatively, studies of RNA metabolism fol-

lowing heat stress indicate that the processing and

transport to the cytoplasm of non-heat shock message

appears to be inhibited, while the transcription of these

genes remains unaffected [38]. As in our study, how-

ever, the accumulation of heat shock mRNA is ob-

served, and it has been postulated that heat shock

mRNA may represent a class of mRNA which main-

tains normal p rocessing during stress [38]. Thus, our

results are not consistent with inhibition of HSP pro-

tein synthesis due to inhibition of splicing or mRNA

transport and processing following severe heat shock

and the most likely mechanism leading to diminished

protein synthesis following severe heat shock in lym-

phocytes is direct inhibition of mRNA translation.

The second instance in which protein synthesis does

not parallel mRNA expression is in the later stages of

mitogen activation. Previous work has shown sustained

synthesis of HSC70 and HSPSO gene products as late as

48 h following PHA addition [ll]. However, HSC70

mRNA is diminished by 48 h after PHA addition, while

HSPSOa and fi mRNA remain enhanced at this time

point (Fig. 4). Thus, enhanced HSC70 m RNA transla-

tion or some other regulatory process may be required

to sustain HSC70 synthesis at later times and this result

indicates that there may be a separate down-regulatory

signal which operates on HSC70, but not HSPSOol or p,

during S phase.

We thank Drs. Richard Morimoto and Eileen Hickey for providing

us with cDNA probes, and Dr. Helen Hallgren for critical review of

the manuscript. This work was supported

by

NIH Grants AG 02338

and CA 39692 to James J. OLeary and a grant from the University of

Minnesota Graduate School to Linda K. Hansen. Portions of this

manuscript will be a part of the doctoral thesis of Linda K. Hansen to

fulfill the requirements of the University of Minnesota Graduate

School.

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Received July 2, 1990

Revised version received September 24, 1990

exp cell res- hansen

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