participation of the conventional calpains in apoptosis

Participation of the conventional calpains in apoptosis

Tao Lu, Ying Xu, Maura T. Mericle, Ronald L. Mellgren*

The Department of Pharmacology and Therapeutics, Medical College of Ohio, 3035 Arlington Avenue, Toledo, OH 43614-5804, USA

Received 4 December 2001; received in revised form 29 January 2002; accepted 7 March 2002

Abstract

The conventional calpains, m- and A-calpain, are suggested to be involved in apoptosis triggered by many different mechanisms.

However, it has not been possible to definitively associate calpain function with apoptosis, largely because of the incomplete selectivity of the

cell permeable calpain inhibitors used in previous studies. In the present study, Chinese hamster ovary (CHO) cell lines overexpressing A-calpain or the highly specific calpain inhibitor protein, calpastatin, have been utilized to explore apoptosis signals that are influenced by

calpain content. This approach allows unambiguous alteration of calpain activity in cells. Serum depletion, treatment with the endoplasmic

reticulum (ER) calcium ATPase inhibitor thapsigargin, and treatment with calcium ionophore A23187 produced apoptosis in CHO cells,

which was increased in calpain overexpressing cells and decreased by induced expression of calpastatin. Inhibition of calpain activity

protected h-spectrin, but not a-spectrin, from proteolysis. The calpains seemed not to be involved in apoptosis triggered by a number of other

treatments. Calpain protected against TNF-a induced apoptosis. In contrast to previous studies, we found no evidence that calpains

proteolyze InB-a in TNF-a-stimulated cells. These studies indicate that the conventional calpains participate in some, but not all, apoptotic

signaling mechanisms. In most cases, they contributed to apoptosis, but in at least one case, they were protective. D 2002 Published by

Elsevier Science B.V.

Keywords: Calpain; Apoptosis; Thapsigargin; A23187; TNF-alpha; CHO cell

1. Introduction

Cell death can either be the consequence of a passive,

degenerative process, or the consequence of an active

process. The former type of cell death is termed necrosis,

the latter apoptosis, or programmed cell death (PCD).

Apoptosis was originally identified morphologically [1]. It

represents a mode of death that is actively driven by the cell.

Necrosis represents a passive consequence of gross injury to

the cell. It is morphologically distinct from apoptosis, and

its physiological consequences at the tissue, organ and

organism levels are also different.

Calpains are non-lysosomal cysteine proteases that are

present in the cells of many different eukaryotic single-

celled and multicellular organisms [2,3]. While there are a

number of members of the calpain gene family, the best

characterized have been named ‘‘conventional calpains’’: m-

and A-calpain, one or both of which is present in all

mammalian cells. The possible involvement of calpains in

apoptosis was first suggested in 1993 [4,5], and has been

reported for several cells including thymocytes, hippocam-

pal cells and hepatocytes [6–9]. Both a-spectrin and h-spectrin breakdown by caspase-3 and calpain were reported

during apoptosis in neural cells [10,11]. The spectrin break-

down product (SBDP) SBDP120 of caspase-3 is a very

typical hallmark of apoptosis, on the other hand, calpain-

mediated SBDPs are generally found in necrotic as well as

in apoptotic neuronal death [12,13]. And the relative con-

tribution to SBDP from calpain is quite cell type specific

[14].

Although it is generally accepted that calpains are

involved in cell death processes, their exact role is

unknown, and little has been done to sort out their degree

of participation in any of the more common apoptotic

models. Moreover, the specificity of the calpain inhibitors

used for such experiments is crucial in interpreting results,

and to date, no satisfactorily selective calpain inhibitors

have been developed. Thus, there is still some confusion as

to the true targets of these tools vis-a-vis apoptosis, and it is

difficult to rule out effects on proteasome and other intra-

cellular proteases.

In this study, CHO cell-derived clones were developed

that overexpressed either active A-calpain or calpastatin.

Unlike the synthetic calpain inhibitors, calpastatin specifi-

0167-4889/02/$ - see front matter D 2002 Published by Elsevier Science B.V.

PII: S0167 -4889 (02 )00193 -3

* Corresponding author. Tel.: +1-419-383-5307; fax: +1-419-383-2871.

E-mail address: [email protected] (R.L. Mellgren).

www.bba-direct.com

Biochimica et Biophysica Acta 1590 (2002) 16–26

cally inhibits activity of the conventional calpains in vivo.

By unambiguously increasing or decreasing calpain activity

in the cells, it has been possible to demonstrate a clear-cut

difference in participation of the conventional calpains in

the various apoptosis models in a single cell type: CHO

cells.

2. Materials and methods

2.1. Materials

SHI cells were selected from a CHO cell line by chal-

lenging with the calpain inhibitor, ZLLY-DMK, as previ-

ously described [15]. They possess one-third to one-half of

the A-calpain content of the parental CHO cells. Thapsigar-

gin, A23187, staurosporine, H2O2, EGTA and (3-[4,5-yl]-

2,5-diphenyltetrazolium bromide) (MTT) were obtained

from Sigma. EcR-CHO cells, ponasterone A, zeocin, and

the pIND (SP1)/V5-His C vector for ecdysone-inducible

expression from EcR-CHO cells were purchased from

Invitrogen. The calpain small subunit cDNAwas a gift from

Dr. John S. Elce at Queen’s University, Kingston, Ontario,

Canada. The full-length human calpastatin cDNAwas a gift

from Dr. Masatoshi Maki, Nagoya University, Japan. Pro-

teinase K, Rnase A were purchased from Promega. Mouse

anti-spectrin (nonerythroid) monoclonal antibody was

obtained from Chemicon. Mouse anti-h-spectrin monoclo-

nal antibody and the calpain inhibitor SJA6017 were gifts

from Dr. Kevin K.W. Wang at Pfizer, Ann Arbor Laborato-

ries, USA. Anti-mouse IgG, biotinylated species-specific

whole antibody (from sheep), and streptavidin-alkaline

phosphatase were purchased from Amersham Pharmacia

Biotech. InB-a polyclonal antibody was obtained from

Santa Cruz. Mouse anti-human A-calpain large subunit

monoclonal antibody, 1A-11; mouse anti-human calpastatin

monoclonal antibody 5-8A; mouse anti-calpain small sub-

unit monoclonal antibody, P-1; and a rabbit immune serum

against bovine m-calpain, which also recognized the hamster

antigen, were made in-house. Fibroblasts derived from

calpain small subunit knockout [Capn4 (� /� )] mouse

embryos, or from wild-type littermates [16], were kindly

provided by Dr. John S. Elce.

2.2. Human calpain overexpressing clones

Human A-Calpain large subunit cDNA, and rat p21

truncated calpain small subunit cDNA [17], were separately

inserted into pSBC-B and pSBC-A plasmids, respectively.

These plasmids were cut and ligated as previously described

[18], to form a dicistronic construct, pSBC-S/L, having the

calpain small subunit upstream of the large subunit, and

separated by an internal ribosomal entry sequence. SHI cells

were co-transfected with pSBC-S/L and NEO vectors, and

G418 was used for selection. A4 cells were derived by

transfection with pSBC-B containing A-calpain large sub-

unit alone. However, a heterodimeric A-calpain appeared to

be expressed in A4 clone, because the human A-calpainimmunoreactivity could be co-immunoprecipitated from A4

cell lysates using P-1 small subunit antibody (not shown).

Several human calpain producing cell lines were estab-

lished, and the two that expressed high levels of active

human A-calpain, SL18 and SL225, were chosen for this

study. Twenty cell lines with no detectable human A-calpainexpression by Western blot were pooled together randomly,

and early passages of this cell pool were used as mock-

transfected cells, SHI-NEO.

2.3. Human full-length calpastatin overexpressing clones

The full-length human calpastatin cDNA was inserted

into the polylinker EcoRI site of pIND (SP1)/V5-His C

vector. EcR-CHO cells, which already constitutively express

ecdysone receptor, were transfected with the calpastatin

construct using electroporation, and selected in the presence

of G418 (1.4 mg/ml) and zeocin (0.25 mg/ml). We identified

two stable cell lines, clone 83 and 106, which overexpressed

human calpastatin when induced with ponasterone. Clone

83 was chosen for most of our studies, because its morpho-

logic and growth characteristics were very similar to the

parental EcR-CHO cells. Clone 106 displayed a slightly

larger size and elongated appearance. Mock-transfected

cells, which did not express the human calpastatin, were

obtained by transfecting the EcR-CHO cells with empty

pIND (SP1)/V5-His C vector.

2.4. Protease and inhibitor assays

Calpain activity was determined by measuring the release

of trichloroacetic acid (TCA)-soluble fragments from 14C-

methylcasein [19]. CHO cells or clones were lysed in a

hypotonic buffer: 50 mM imidazole–HCl, 1 mM EGTA, 1

mM dithiothreitol, 70 mM NaCl, pH 7.4. Under these

conditions, calpains are extracted, but much of the calpas-

tatin remains sedimentable [20, unpublished observations];

allowing assay of calpain activity in 10,000� g super-

natants of the lysate. Calpastatin activity was determined

by its ability to inhibit a defined amount of purified human

erythrocyte A-calpain (1 ng/Al) in the standard 14C-casein-

olytic assay. The inhibition activity was calculated by

subtracting the percentage of calpain activity remaining

from 100%. Proteasome was assayed fluorometrically, using

Succinyl-LLVY-MCA as the substrate, as previously

described [21].

2.5. Cell culture and treatments

SL clone cells were cultured in 5% CO2 and 95%

humidified air atmosphere at 37 jC in complete IMDM

medium supplemented with heat-inactivated 10% bovine

serum, 100 units/ml penicillin, and 100 Ag/ml streptomycin.

Calpastatin overexpressing clones were cultured in complete

T. Lu et al. / Biochimica et Biophysica Acta 1590 (2002) 16–26 17

Ham’s F12 medium containing 10% heat-inactivated fetal

bovine serum and the same antibiotics. For induction of

calpastatin expression, clone 83 cells were cultured until

95–100% confluent, and pre-treated for 3 days with 5 AMponasterone A before any other treatments. Mock-trans-

fected EcR-CHO cells were subjected to the same pre-

treatment protocol. Cells were either further treated with

100 nM thapsigargin for 3 days, 5 AM A23187 for 2 days, 1

AM staurosporine for 24 h, 3 mM EGTA for 24 h, or 3–10

mM H2O2 in serum-free medium for 2 h, then changed back

to normal medium for another 24 h before harvest.

2.6. MTT assay

Cell viability was estimated by using the MTT assay

method [22]. Cells were cultured in 96-well plates until 90–

100% confluent, and treated by different cell death stimuli

for various durations. Adding 0.5 mM MTT to the plates,

followed by incubation at 37 jC for at least 4 h. Isopropa-

nol—0.1 N HCl, 100 Al, was added to dissolve the reduced

tetrazolium dye. OD595 was determined using the Softmax

software provided with a Thermomax microplate reader

from Molecular Device Corp. For serum deprivation experi-

ments, cells were cultured in the absence of serum for 24 h

before assay. UV light irradiation experiments were per-

formed as described [23]. Briefly, culture medium was

exchanged for a PBS/Mg2 + solution, and the cells were

exposed to 254 nm radiation at 5 mJ/cm2 for different times.

They were then changed back to the usual growth medium,

and further cultured for another 2 days before performing

MTT assays. Irradiation was carried out by using the

Stratagene product StratalinkerR UV cross-linker.

2.7. Extraction of proteins, immunoblots, and densitometric

analysis

Cells were cultured in 12-well plates until about 100%

confluent, and treated by different stimuli at different con-

centrations for different times as described in Section 2.5.

Cells were collected, washed with ice-cold PBS, and lysed in

the cell lysis buffer (1% Triton X-100, 20 mM Tris, 150 mM

NaCl, 5 mM EDTA, 5 mM EGTA, 100 AM Na3VO4, 100

Fig. 1. Characterization of CHO cell clones that overexpress A-calpain. SHI clone of CHO cells was transfected with a dicistronic vector containing human

A-calpain large subunit and rat small subunit cDNAs, as described in Materials and methods. Clone A4 was transfected with A-calpain large subunit alone, as

described in Materials and methods. Cell lysates were assayed for calpain activity at 200 AM Ca2 + , to specifically detect the A-calpain isozyme (panel A).

Other samples were subjected to protein immunoblot analysis for heterologously expressed human A-calpain large subunit (panel B). Hamster m-calpain

large subunit (panel C), and calpastatin (panel D) were detected using antibodies with broad species specificities. Duplicate lanes for each cell line were

loaded in panels B through D.

T. Lu et al. / Biochimica et Biophysica Acta 1590 (2002) 16–2618

nMmicrocystin-LR, 200 AM phenylmethylsulfonyl fluoride,

10 AM pepstatin A, and 1 mM dithiothreitol, pH 7.4) on ice

for 10 min, then homogenized for about eight strokes, and

centrifuged at 4 jC, 10,000� g for 10 min. Quantitation of

supernatant protein was carried out with the BCA reagent

[24]. Supernatants were treated with SDS-PAGE sample

buffer, and equal amounts of sample protein were subjected

to 7.5% polyacrylamide SDS-PAGE. The gels were subse-

quently transferred to polyvinylidene difluoride membranes

by electroblotting at 4 jC for 3 h at 70 V. Immunoblotting

was performed with the following first antibodies: mouse

anti-spectrin (nonerythroid) monoclonal antibody (1:2000);

mouse anti-h-spectrin monoclonal antibody (1:700); rabbit

Fig. 3. Calpastatin overexpression inhibited calpain autoproteolysis. Clone

83 cells or mock-transfected EcR-CHO cells were cultured for 3 days with

our without 5 AM ponasterone A in the culture medium. Where indicated,

100 nM thapsigargin or 3 AM A23187 were then added to the medium.

After 2 more days in culture (A23187) or 3 more days (thapsigargin), cell

lysates were prepared and subjected to protein immunoblot analysis for

calpain small subunit.

Fig. 2. Regulated expression of human calpastatin in clone 83 CHO cells.

Clone 83 cells stably transfected with pIND vector expressing human full-

length calpastatin were cultured minus or plus 5 AM ponasterone A for 3

days. Cell lysates were subjected to protein immunoblot analysis using a

human-specific calpastatin antibody (panel A), or heat-treated as described

in Materials and methods and assayed for calpastatin activity (panel B).

Fig. 4. Calpastatin overexpression increased viability of clone 83 cells

exposed to thapsigargin or A23187. Clone 83 cells were cultured plus (filled

bars) or minus ponasterone, and then exposed to 100 nM thapsigargin (panel

A) or 3 AM A23187 (panel B). After 1, 2 or 3 days, cell survival was

measured by MTT reductase activity as described in Materials and methods.

Where indicated, values were significantly different from the minus

ponasterone sample at the same time point; * =P< 0.01, ** =P < 0.001.


anti-m-calpain large subunit (1:1000); mouse anti-calpain

small subunit monoclonal antibody, P-1, (2 Ag/ml); mouse

anti-human A-calpain large subunit monoclonal antibody,

1A-11 (2 Ag/ml); mouse anti-human calpastatin monoclonal

antibody 5-8A (2 Ag/ml). Immunodetection was carried out

with alkaline phosphatase second antibody, and BCIP sub-

strate with nitro-blue tetrazolium development [25]. Densi-

tometry was performed with a Bio-Rad bioscanner and

Molecular Analyst software.

2.8. Cell morphology

Cells were cultured until about 100% confluence, and

treated by the indicated stimuli for the indicated times as

described in Section 2.5. Phase-contrast photomicrographs

were taken using a Nikon N 5005 inverted phase-contrast

microscope and camera at 100� magnification.

2.9. DNA fragmentation assay

Cells were cultured in 60 mm petri dishes, and treated by

different stimuli at the indicated conditions. Cells were lysed

in 500 Al of DNA extraction solution (50 mM Tris–HCl, 10

mM EDTA, 0.5% Triton, and 100 Ag/ml proteinase K, pH

8.0). After incubating overnight, Dnase-free Rnase A was

added to 100 Ag/ml. The samples were incubated at 37 jCfor 2 h, extracted with an equal volume of phenol/chloro-

form (1:1 v/v), and centrifuged at 20,000� g for 10 min at

4 jC. The aqueous phase was re-extracted with chloroform.

DNAwas collected by precipitating the aqueous supernatant

Fig. 5. Calpastatin overexpression attenuated cell rounding associated with thapsigargin and A23187-induced cell death. Clone 83 and mock-transfected cells

were treated with 100 nM thapsigargin or 3 AM A23187 as described in Materials and methods, then observed by phase contrast microscopy at 100�magnification.


with 2 volumes of cold absolute ethanol in the presence of

1/10 volume of 3 M NaAc. After washing with 70%

ethanol, the DNA pellet was dissolved in 40 Al of 10 mM

Tris–HCl and 1 mM EDTA, pH 8.0. DNAwas separated on

1.8% agarose gel containing 0.5 Ag/ml ethidium bromide,

and DNA fragments were visualized by exposing the gels to

UV light.

2.10. Statistical analysis

Where indicated, samples were analysed by Students

unpaired t-test. Results are expressed as meanF standard

deviation.

3. Results

3.1. Characterization of clones overexpressing calpain or

calpastatin

Calpain overexpressing clones were established by co-

transfecting Neo vector and the pSBC-S/L construct, which

had both the A-calpain large subunit and calpain small

subunit, into SHI cells. Seven cell lines expressing human

A-calpain were established (Fig. 1). S/L18 and S/L225 cells

were chosen for further study, because they expressed

intermediate and high levels of A-calpain, respectively

(Fig. 1A). Moreover, they displayed morphologies and

growth characteristics comparable to the mock-transfected

Fig. 6. Calpastatin overexpression decreased DNA ladder formation in

A23187 and thapsigargin-treated cells. Clone 83 and mock-transfected cells

were exposed to thapsigargin or A23187 as described in Materials and

methods. DNA was prepared and subjected to electrophoretic analysis for

fragmentation as described in Materials and methods.

Fig. 7. Calpastatin overexpression inhibited h-spectrin proteolysis in

thapsigargin-treated cells. Clone 83 and mock-transfected cells were treated

with thapsigargin as described in Materials and methods. Cell lysates were

subjected to SDS-PAGE and protein immunoblotting using anti-a-spectrin

antibody (panel A) or anti-h-spectrin antibody (panel B), as described in

Materials and methods. The asterisk in panel A shows the location of the

immunoreactive band at slightly higher mobility than a-spectrin that was

consistently protected by calpastatin overexpression.

Table 1

Effect of calpastatin overexpression on loss of h-spectrin following

exposure to thapsigargin or A23187

Drug Cells Ponasterone Degradation of

h-spectrinF SD

relative to untreated

samples (%)

Thapsigargin mock � 72F 1

mock + 70F 5

clone 83 � 74F 1

clone 83 + 42F 6*

A23187 mock � 65F 10

mock + 67F 11

clone 83 � 76F 8

clone 83 + 62F 7**

* P< 0.01 vs. minus ponasterone sample.

** P< 0.05 vs. minus ponasterone sample.


(SHI-NEO) cells. In contrast, S/L115 cells, which expressed

between 3 and 4 units of A-calpain/mg protein (Fig. 1A),

displayed an elongated cell shape, and a slower growth rate

than the other clones (data not shown). All clones expressed

comparable levels of m-calpain (Fig. 1C) and calpastatin

(Fig. 1D).

EcR-CHO cells were transfected with the full-length

calpastatin construct pIND (SP1)/V5-His C vector and

selected in the presence of G418 (1.4 mg/ml) and Zeocin

(0.25 mg/ml) antibiotics. Clone 83 derived from these

studies stably expressed human calpastatin when exposed

to micromolar ponasterone A in the culture medium (Fig.

2A). Direct assay of calpastatin activity in clone 83 lysate

demonstrated a 2- to 3-fold increase in calpastatin activity

upon induction with ponasterone A (Fig. 2B).

To determine whether expressed human calpastatin

could inhibit calpains in intact cells, clone 83 cells were

cultured in the presence of A23187 or thapsigargin, both

of which activated calpains, as evidenced by increased

autoproteolysis of calpain small subunit (Fig. 3; lanes 1, 2,

5, and 6). Overexpression of calpastatin decreased autopro-

teolysis (Fig. 3; lanes 3, 4, 7, and 8). No protection was

observed when mock-transfected cells were exposed to

ponasterone A.

3.2. Effect of altering calpain or calpastatin content on

survival of cells exposed to A23187- or thapsigargin-

induced apoptosis

Initial studies of the effect of calpain content on cell

survival were carried out utilizing the calpastatin-overex-

pressing clone 83. The divalent cation ionophore A23187,

and thapsigargin, an inhibitor of the endoplasmic reticulum

calcium pump, were used as apoptogenic agents. As pre-

viously noted, both of these reagents activate calpains in

CHO cells (Fig. 3). Treatment with either A23187 or

thapsigargin resulted in time-dependent cell death, which

was inhibited by calpastatin overexpression (Fig. 4). Light

microscopic examination of cells exposed to A23187 or

thapsigargin confirmed that calpastatin overexpression

inhibited cell damage, as evidenced by decreased rounding

and lifting from the culture surface (Fig. 5). No protection of

apoptosis was observed in mock-transfected cells treated

with ponasterone (Fig. 5; top).

The MTT reductase assay utilized to measure cell sur-

vival cannot distinguish between apoptosis and necrosis.

Therefore, further studies were carried out to confirm that

A23187 and thapsigargin trigger apoptosis in the cell lines

utilized in the present investigation. Both treatments pro-

duced DNA fragmentation into a multi-nucleosomal ‘‘DNA

ladder’’ (Fig. 6), typical of apoptosis. Overexpression of

calpastatin attenuated DNA breakdown (Fig. 6; panel B).

Spectrin proteolysis has been utilized as a marker for

apoptosis, especially generation of the 120 kDa a-spectrin

fragment by caspase 3 [26]. Exposure of clone 83 cells to

either thapsigargin or A23187 resulted in a substantial

Fig. 8. Clone 83 cells exposed to H2O2 undergo necrotic cell death. Clone 83

cells were cultured in the presence of H2O2 as described in Materials and

methods. Panel A DNA was isolated and electrophoresed to detect frag-

mentation. Panel B shows the H2O2 concentration dependence of spectrin

degradation.

Table 2

The effects of different stimuli on the cell death of calpastatin overexpressing clone #83 and calpain overexpressing S/L clones

Percent cell survival

Calpastatin overexpressing Calpain overexpressing

Mock Clone 83 Mock S/L18 S/L225

� Ponasterone + Ponasterone � Ponasterone + Ponasterone

Treatment

� Serum 62.1F 2.5 58.3F 3.7 60.0F 3.1 78.1F 5.4 * 53.1F1.4 50.7F 2.1 29.4F 1.8#

A23187 26.6F 1.4 25.0F 0.8 23.6F 1.5 37.9F 2.4 * 51.9F 1.4 50.8F 2.5 41.3F 0.7#

Thapsigargin 66.2F 2.4 68.0F 2.4 65.0F 2.6 86.9F 3.3 * 49.9F 2.0 42.0F 0.8# 40.6F 1.1#

Staurosporine 35.7F 4.4 37.0F 1.3 35.3F 6.8 39.9F 1.5 36.2F 1.1 34.2F 1.0 37.0F 0.3

EGTA 66.0F 1.7 65.4F 1.4 68.0F 1.6 66.8F 2.5 64.2F 1.7 65.5F 0.8 64.6F 1.7

H2O2 30.3F 6.7 29.9F 4.4 31.3F 3.3 30.2F 4.0 47.4F 1.5 47.1F 3.3 43.7F 1.7

UV 54.7F 2.7 53.6F 2.0 56.1F 2.6 53.1F 2.3 46.6F 0.6 44.7F 0.9 45.4F 2.0

*P< 0.001 compared with � ponasterone samples.# P < 0.001 compared with mock-transfected cells.


increase in spectrin immunoreactivity at 120 kDa on protein

blots (Fig. 7; panel A). However, increased expression of

calpastatin had no apparent effect on generation of the 120

kDa fragment. In contrast, a minor immunoreactive band

slightly smaller than a-spectrin was consistently spared

(Fig. 7; panel A, asterisk). Utilization of a selective antibody

demonstrated that this band was h-spectrin, and that its

proteolysis was reduced upon calpastatin overexpression

(Fig. 7; panel B). Overexpression of calpastatin in clone

83 cells resulted in a significant reduction of h-spectrinproteolysis induced by thapsigargin or A23187 treatment,

under the conditions of our studies (Table 1).

3.3. Contribution of the conventional calpains to cell death

produced by other apoptotic models

The availability of CHO cells overexpressing A-calpainor calpastatin allowed a systematic survey of calpain

involvement in other commonly studied apoptotic models

(Table 2). The MTT reductase assay demonstrated that cell

death produced upon serum withdrawal appeared to have a

calpain component, as evidenced by its exacerbation by

calpain overexpression, and the protective effect of calpas-

tatin overexpression. Of the other apoptotic triggers studied,

none except A23187 and thapsigargin appeared to be

influenced by calpain/calpastatin balance. Further experi-

ments were carried out to determine the mechanism of cell

death in these studies. DNA fragmentation and spectrin

proteolysis analysis confirmed that the conditions in Table

Fig. 9. Calpain overexpression protects against TNF-a-induced apoptosis of

SHI cells. Clones S/L18, S/L225 and mock-transfected SHI cells (Neo)

were cultured in the presence of TNF-a for 20 h. Panel A: cell survival was

determined by MTT reductase assay as described in Materials and methods.

Panel B: DNA was prepared and analyzed for fragmentation.

Fig. 10. Apoptosis of calpain small subunit knockout mouse fibroblasts.

Fibroblasts isolated from Capn4 (� /� ) mouse embryos or a Capn4 (+/+)

littermate were subjected to several of the apoptotic signals previously

utilized in experiments with the calpain and calpastatin overexpressing

CHO cell lines. Survival was determined by the MTT reductase assay, as

described in Materials and methods. N= 8 for each bar; * =P< 0.01 vs.

Capn4 (+/+), ** =P< 0.05 vs. Capn4 (+/+).


2, except exposure to H2O2, resulted in apoptosis (data not

shown). H2O2 produced necrotic cell death at all concen-

trations studied (Fig. 8), with accompanying non-distinct

DNA fragmentation, and proteolysis of a-spectrin without

production of the distinct 120 kDa fragment.

3.4. Calpain protected against TNF-a triggered apoptosis

In contrast with the results presented in Table 2, calpain

overexpression was found to protect against cell death

signaling by TNF-a (Fig. 9A). Assessment of DNA ladder

formation demonstrated apoptosis as the mechanism for

cell death, and confirmed the protective effect of calpain

overexpression (Fig. 9B). To assess the generality of

calpain protection against TNF-a-stimulated apoptosis,

fibroblasts derived from calpain small subunit knockout

[Capn4 (� /� )] mouse embryos were utilized. Compared

with wild-type mouse fibroblasts, the Capn4 (� /� ) fibro-

blasts were more sensitive to TNF-a induced apoptosis

(Fig. 10). In contrast, the knockout fibroblasts were less

sensitive to apoptosis produced by addition of A23187 to

the culture medium, or serum deprivation.

Previous studies in isolated hepatocytes have indicated

that calpains can proteolyze InB-a, thereby releasing NF-

nB [27]. This is consistent with a protective effect of

calpain in apoptosis, because it would allow expression of

anti-apoptotic genes by NF-nB. However, in the present

investigation, there was no detectable change in InB-astability in the calpain overexpressing cell lines (data not

shown). To further investigate calpain participation in InB-a degradation, the effects of lactacystin and SJA6017 were

explored. The former is a selective proteasome inhibitor

[28,29], while the latter is a potent calpain inhibitor having

no detectable inhibitory activity on proteasome (Fig. 11A).

InB-a degradation in response to TNF-a was nearly com-

pletely abolished by lactacystin pre-treatment (Fig. 11B),

while SJA6017 did not noticeably protect InB-a (Fig. 11C).

Therefore, the protective influence of calpain did not appear

to be related to InB-a proteolysis.

4. Discussion

By manipulating expression levels of A-calpain and

calpastatin, it has been possible to alter the sensitivity of

CHO cells to apoptotic stimuli. These results indicate that

the conventional calpains (A- and m-calpain), which are

inhibited by calpastatin, participate in some apoptotic

pathways. Preliminary studies with calpain small subunit

knockout mouse fibroblasts confirm this observation (Fig.

10). Notably, of the different systems studied, only apop-

tosis associated with A23187, thapsigargin, or serum

depletion was shown to be increased by a positive cal-

pain/calpastatin balance (Table 2). Cell death following

exposure of CHO cells to staurosporine, EGTA, or ultra-

violet light was not detectably influenced by calpain con-

tent. Calpain did not influence cell death upon exposure to

H2O2. In some cell types, H2O2 produces apoptotic cell

death [30–32]. However, it only produced necrotic death

of CHO cells over a range of concentrations. Therefore,

the influence of calpain on apoptosis produced by H2O2

could not be addressed by the present studies. Other well-

characterized apoptotic stimuli could not be studied in the

CHO cell system, because significant cell death was not

observed. For example, silica fibers, which produce apop-

tosis in some cells [33,34], simply did not kill the CHO

cells (data not shown).

Calpain protected against apoptosis produced by TNF-a.

Other investigators have found that calpain can cleave

InB-a in hepatocytes stimulated with TNF-a [27]. In

principle, this would lead to increased levels of free NF-

nB transcription factor, which could translocate to the

nucleus and increase expression of anti-apoptosis genes.

Fig. 11. Calpains do not appear to proteolyze InB-a after exposure of CHO

cells to TNF-a. Panel A: SJA6017 was assayed for inhibitory activity

against purified human m-calpain (filled circles) or human proteasome

(hollow circles). In other studies, the effect of various concentrations of

lactacystin (panel B) or SJA6017 (panel C) on degradation of InB-a was

explored utilizing protein immunoblot experiments and anti-InB-a anti-

body.


However, InB-a did not appear to be a calpain target in the

CHO cells used in the present studies. Very recently, it has

been reported that TNF-a-dependent NF-nB activation in

dermal fibroblasts is TRAF2- and NIK-independent, but

requires sphinomyelinase and calpain activity [35]. Thus,

calpain appears to act through a mechanism separate from

the NIK:InB-a phosphorylation pathway. Further investiga-

tion will be required to elucidate the mechanism by which

calpains protect against TNF-a-stimulated apoptosis.

A further surprising result from the present studies was

the apparent lack of effect of calpain on a-spectrin proteol-

ysis. Although calpastatin overexpression partially protected

against apoptosis secondary to A23187 or thapsigargin

treatment, a-spectrin was not noticeably protected from

proteolysis (Fig. 7). Furthermore, the predominant break-

down product was the 120 kDa band expected from caspase

cleavage of a-spectrin [26], suggesting that caspases are

responsible for the majority of a-spectrin breakdown in

these studies. Interestingly, there was significant protection

of h-spectrin proteolysis upon calpastatin overexpression

(Fig. 7B and Table 1). Although calpainolysis of a-spectrin

has been most extensively studied, both a- and h-spectrinshave been shown to be calpain substrates [36,37], and

cleavage of both is required to fully dismantle the cortical

cytoskeleton [11]. It was previously shown that in eryth-

rocytes, only h-spectrin is cleaved by calpains [38]. If

a-spectrin is predominantly proteolyzed by caspases in

some cell types, while calpains degrade h-spectrin, this

would provide a rational framework for utilizing combina-

tions of caspase and calpain inhibitors to inhibit apoptotic

death in these cell types.

In conclusion, the present studies demonstrate that direct

alteration of the calpain/calpastatin balance in CHO cells

influences some apoptotic systems, but not others. In no

case did abrogation of conventional calpain activity lead to

complete protection against apoptosis. Therefore, these

calpains do not appear to be indispensable for this process.

For a complex function like programmed death, it would be

surprising if compromising one part of the signaling system

would completely abolish the overall event. It is important

to note that the changes in calpain and calpastatin expres-

sion levels displayed by the CHO cell clones described in

this study are within the physiologic range observed in

various cell types. For example, the amount of A-calpain in

the S/L 225 clone is similar to the level measured in blood

platelets [39], and the maximum content of calpastatin in

clone 83 cells after 3 or more days of induction with

ponasterone A is only 2 or 3-times the level of endogenous

hamster calpastatin (Fig. 2). Some of the apoptotic proto-

cols utilized in these studies were clearly pharmacological

in nature. However, two of the calpain-responsive apoptotic

stimuli, TNF-a treatment, and abrogation of the serum

survival pathway, represent physiologic apoptotic path-

ways. It will be important to determine which signal trans-

duction events in these latter pathways are influenced by

calpains.

Acknowledgements

This work was supported in part by NIH grant HL36573.

The authors gratefully acknowledge Dr. Kevin K.W. Wang

for providing valuable advice during these studies, as well

as making available SJA6017 calpain inhibitor and anti-

bodies against spectrins. We thank Dr. John Elce for the

kind gift of Capn4 (� /� ) and Capn4 (+/+) fibroblasts, and

Dr. Masatoshi Maki for providing calpastatin cDNA.

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participation of the conventional calpains in apoptosis

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