ORIGINAL PAPER

Calpain and caspase processing of caspase-12 contribute to the ERstress-induced cell death pathway in differentiated PC12 cells

Juan A. Martinez • Zhiqun Zhang •

Stanislav I. Svetlov • Ronald L. Hayes •

Kevin K. Wang • Stephen F. Larner

� Springer Science+Business Media, LLC 2010

Abstract Neuronal cell death after traumatic brain injury,

Alzheimer’s disease and ischemic stroke may in part be

mediated through endoplasmic reticulum (ER) stress and

unfolded protein response (UPR). UPR results in induction

of molecular chaperone GRP78 and the ER-resident cas-

pase-12, whose activation has been proposed to be medi-

ated by calpain and caspase processing, although their

relative contribution remains unclear. In this study we

induced ER stress with thapsigargin (TG), and determined

the activation profile of calpain-2, caspase-3, caspase-7,

and caspase-12 by analyses of protein levels, correspond-

ing substrates and breakdown products (BDP). Specific

calpain and caspase activity was assessed by analysis of

aII-spectrin BDP of 145 kDa (SBDP145), BDP of 150 kDa

(SBDP150) and BDP of 120 kDa (SBDP120). Decrease in

pro-calpain-2 protein and increased SBDP145 levels by 3 h

after TG treatment indicated early calpain activity. Active

caspase-7 (p20) increase occurred after 8 h, followed by

concomitant up-regulation of active caspase-3 and SBDP120

after 24 h. In vitro digestion experiments supported that

SBDP120 was exclusively generated by active caspase-3 and

validated that kinectin and co-chaperone p23 were calpain

and caspase-7 substrates, respectively. Pro-caspase-12 pro-

tein processing by the specific action of calpain and caspase-

3/7 was observed in a time-dependent manner. N-terminal

pro-domain processing of pro-caspase-12 by calpain

generated a 38 kDa fragment, while caspase-3/7 generated a

35 kDa fragment. Antibody developed specifically against

the caspase-3/7 C-terminal cleavage site D341 detected the

presence of large subunit (p20) containing 23 kDa fragment

that increased after 24 h of TG treatment. Significant cas-

pase-12 enzyme activity was only detected after 24 h of TG

treatment and was completely inhibited by caspase 3/7

inhibitor DEVD-fmk and partially by calpain inhibitor SNJ-

1945. ER-stress-induced cell death pathway in TG-treated

PC12 cells was characterized by up-regulation of GRP-78

and processing and activation of caspase-12 by the orches-

trated proteolytic activity of calpain-2 and caspase-3/7.

Keywords ER stress � Thapsigargin � Unfolded protein

response � Calpain � Caspase � PC12 cells

Abbreviations

ER Endoplasmic reticulum

UPR Unfolded protein response

GRP-78 Glucose responsive protein of 78 kDa

TG Thapsigargin

BDP Breakdown product

TBI Traumatic brain injury

Introduction

The endoplasmic reticulum (ER) specialized functions

include protein translation, folding and transport of pro-

teins for local cellular use (e.g. transmembrane recep-

tors and other integral membrane proteins), or export

(e.g., secreted digestive enzymes), calcium sequestration

for intracellular calcium homeostasis, post-translational

J. A. Martinez (&) � Z. Zhang � S. I. Svetlov �R. L. Hayes � K. K. Wang � S. F. Larner (&)

Center of Innovative Research, Banyan Biomarkers Inc,

12085 Research Drive, Alachua, FL 32615, USA

e-mail: jmartinez@banyanbio.com

S. F. Larner

e-mail: sflarner@banyanbio.com

123

Apoptosis

DOI 10.1007/s10495-010-0526-4

modification and quality control of proteins, and the pro-

duction and storage of glycogen, steroids, and other mac-

romolecules [1]. Physiological and pathological conditions

that disrupt protein folding and/or cause loss of calcium

homeostasis leading to ER dysfunction include glycosyla-

tion deficiency [2], elevated protein synthesis and secretion

[3], glucose deprivation [4], neurodegenerative diseases [5]

and traumatic brain injury (TBI) [6, 7]. TBI leads to the ER

accumulation of misfolded proteins that can cause ER

stress [6]. Cells react to ER stress by activation of the

unfolded protein response (UPR) pathway which activates

signal transduction reactions in both the nucleus and Golgi

apparatus in a coordinated manner to cope with cellular

demand [8]. The UPR enables the cell to reduce the ER

unfolded protein load by translational attenuation, by pro-

moting protein folding through induction of molecular

chaperones and foldases, and by increasing degradation of

unfolded and misfolded ER proteins. However, if ER

homeostasis is not restored in a timely manner its func-

tional capacity is rapidly exceeded and apoptosis is

induced.

The mammalian UPR pathway is mediated primarily

through the ER transmembrane proteins, ATF6, IRE1 and

PERK [8]. Under normal conditions, the luminal domains

of these sensor proteins are occupied by GRP-78/BiP

(glucose responsive protein of 78 kDa/immunoglobulin

binding protein), an ER-resident belonging to the heat-

shock 70 (HSP70) family. Induction of ER stress causes

accumulation of unfolded proteins that have high affinity

for GRP-78 causing its release from these sensor proteins

and thus up-regulating the UPR signal. Although the

cooperative effect of these three sensors serves to protect

the cell until ER function is restored, they also contribute

to the induction of apoptosis if ER homeostasis is not

restored.

The ER-resident caspase-12 has been shown to mediate

apoptosis signaling induced by ER stress [9]. Both cal-

pain(s) and caspase(s) have been proposed to mediate

processing and activation of caspase-12 after induction of

the unfolded protein response and ER stress. Calpain-

deficient mouse embryonic fibroblasts (MEFs) have been

shown to have decreased ER stress-induced activation of

caspase-12 and are resistant to ER stress-induced apoptosis

[10]. Increased cytoplasmic Ca2? levels caused by thapsi-

gargin in primary MEFs appear to lead to the accumulation

and activation of calpain at the ER membrane where it

presumably activates caspase-12. Another protease, cas-

pase-7, has also been implicated in the activation of cas-

pase-12 in response to ER stress [11] and has been reported

to translocate from the cytosol to the ER to interact with

caspase-12 leading to its activation [10, 12].

Although caspase-12 is activated during the ER stress-

induced UPR, the involvement of caspase-12 in ER stress-

induced apoptosis remains controversial. Initial reports on

caspase-12 knockout mice showed increased resistance to

ER stress-induced apoptosis [13], but later studies sug-

gested caspase-12 had a more prominent role during the

inflammatory response [14]. It has also been suggested that

ER-stress induced apoptosis is primarily mediated by cal-

pains and not caspases [15]. The aim of this study was to

determine the relative contribution of calpains and caspases

to caspase-12 activation and the ER stress-induced cell

death pathway in vitro before in vivo TBI studies. A

common mechanism associated with TBI and neurotoxic

shock is the transient loss of calcium homeostasis. To

mimic this loss, thapsigargin was used to induce ER stress

in a neuronal-like cell model using pheochromocytoma cell

line (PC12), differentiated with nerve growth factor (NGF).

Our hypothesis is that the processing of caspase-12 by both

calpains and caspases, are involved in the activation of

caspase-12 and this activation contributes to the ER stress-

induced cell death pathway.

Materials and methods

Rat pheochromocytoma (PC12) cell culture

Rat PC12 YH cell cultures were grown on modified poly-

styrene T-75 tissue culture flasks (Cell-T plus, Sarstedt,

North Carolina, USA) in B-27 supplemented Neurobasal

medium with 10% horse serum, 5% fetal bovine serum,

2 mM glutamine, 1% Fungizone, 100 units/ml penicillin

and 100 lg/ml streptomycin (Gibco/Invitrogen, Carlsbad,

CA, USA). Cultures were kept at 37�C in a humidified 5%

CO2 incubator. Medium was replaced every 3 days and

cells were passaged by standard trypsinization (0.05%

Trypsin-EDTA) (Gibco/Invitrogen, Carlsbad, CA, USA)

upon reaching confluency at a 1:3 split ratio. For experi-

ments, 3 9 104 cells were seeded onto 6-well tissue culture

clusters (Nunclone, NUNC, Denmark) and grown for 48 h.

PC12 cells were differentiated by incubating in low serum

Neurobasal medium (2% horse serum, 1% fetal bovine

serum) with 50 ng/ml nerve growth factor (NGF) (2.5S

subunit, EMD Biosciences, Gibbstown, NJ, USA) and

glutamine/penicillin/streptomycin/amphotericin B as indi-

cated above, for 4–5 days. Cell differentiation was defined

by[80% cells having two or more neurites of any length as

determined by light microscopy of two randomly selected

visual fields using a Zeiss Axiovert 135 inverted micro-

scope. Media was then removed, cells washed with phos-

phate buffered saline (PBS) and experimental conditions

were carried out in serum-free Neurobasal medium with

NGF and supplements mentioned above but with reduced

(1/3) antibiotic/antimycotic content.

Apoptosis

123

Cell death assessment by lactate dehydrogenase (LDH)

assay

Cell death was determined by measuring LDH released

into culture medium using Promega’s CytoTox 96TM

(Madison, WI, USA) assay kit following manufacturer’s

protocol. Briefly, a 50 ll conditioned-medium aliquot was

collected from each sample and transferred to a 96-well

plate. A background control (medium alone) and a maxi-

mum positive control (total cell lysate) were measured to

establish assay range. After adding assay reagents, plates

were incubated for 30 min in the dark at room temperature.

Reaction was then stopped and plates read at 490 nm in a

SpectraMax Plus 384 microplate reader (Molecular Devi-

ces, Ontario, CA).

PC12 cell lysate sample preparation

ER stress was induced in differentiated PC12 cells by

exposure to 1 lM thapsigargin (Sigma Chemical Co, St.

Louis, MO, USA) at various time points (3, 8, 16, 24 h)

applying a reverse time course protocol for simultaneous

cell sampling, in the absence or presence of calpain

inhibitor (SNJ-1945) [16] or caspase inhibitor DEVD-fmk

(R&D Systems, Minneapolis, MN, USA) [17]. Cells were

washed with phosphate buffered saline (PBS) prior to

collection. Total cell lysate was collected by direct in-well

lysis by adding 600 ll of Triton lysis buffer [20 mM Tris-

HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA,

1% Triton X-100, 1 mM NaF, 1 mM Na3VO4, 1 mM DTT

and protease inhibitor cocktail (Roche, Indianapolis, IN,

USA)]. Plates were incubated for 90 min at 4�C in orbital

shaker. Cell lysate was transferred to microcentrifuge tube

and sonicated on ice for 3 sec (Fisher Sonic Dismembrator

60, Fisher Scientific, Pittsburgh, PA, USA) to shear any

remaining DNA. Samples were then centrifuged at

15,0009g for 10 min at 4�C to clarify crude lysate. Super-

natant was snap frozen on dry ice and stored at -80�C. Sub

cellular and size fractionation of PC12 lysate was collected

by scraping cells into 300 ll of cell resuspension buffer

(CRB) (50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 2 mM

EGTA, 0.33 M sucrose, 1 mM DTT and protease inhibitor

cocktail). Cell suspension was mechanically disrupted by

passing through a 27G syringe needle (1 cc) on ice. The

suspension was then centrifuged at 8009g for 10 min at 4�C

to pellet membrane/nuclear fraction. Pellet was snap frozen

for future analysis of nuclear proteins and supernatant was

centrifuged at 12,0009g for 20 min at 4�C to pellet the

mitochondria. Supernatant (cytosol/ER fraction) was trans-

ferred to 1.5 ml microcentrifuge tube and snap frozen on dry-

ice. Sample preparation for immunodetection of cleaved

caspase-12 fragments required further processing by

enrichment of proteins in the 10–100 kDa range from either

total protein or cytoplasmic fractions using microconcen-

trators with 100 and 10 kDa molecular weight cutoff mem-

branes (Microcon-100, Microcon-10, Amicon, Bellerica,

MA, USA).

Caspase cleaved caspase-12 antibody development

The peptide sequence H2N–C-C9 linker-SITKAHVETD-OH

corresponding to amino acids 332–341 of murine caspase-

12, where D341 is a caspase-7 cleavage site, was synthesized

and purified by Proteos Inc. (Kalamazoo, MI), conjugated to

keyhole limpet hemocyanin (KLH) and injected into rabbit

host (Pocono Rabbit Farm and Lab, Canadensis, PA) with

subsequent booster injections in a 72-day antibody produc-

tion protocol. Antiserum was purified by peptide affinity

chromatography using disulfide linkage of peptide to agarose

resin (SulfoLink, Pierce/Thermo Scientific, Rockford, IL,

USA) following manufacturer protocol (2 ml columns). A

1:1 PBS-diluted antiserum solution was loaded onto column,

washed with 500 mM NaCl-containing PBS (pH 7.4) fol-

lowed by 5 column volume washes of PBS. The bound

IgG was eluted with 2 ml of 0.1 M glycine (pH 1.9) and

immediately neutralized with 100 ll 1 M Tris base (pH 9).

The eluted IgG’s were dialyzed against PBS (pH 7.4),

concentrated 5-fold with centrifugal microconcentrators

(Microcon-10, Amicon/Millipore, Bellerica, MA, USA) and

stored at -20�C in 50% glycerol.

Quantitative Western Blot Analysis (qWB)

The protein concentration of the PC12 lysates was deter-

mined by a modified Lowry method (DC Protein Assay Kit,

Bio-Rad Laboratories, Hercules, CA, USA) using bovine

serum albumin (BSA) standard curve reference in the

microplate format. Equal protein amounts were loaded onto

4–20% Tris/glycine gel (Invitrogen Life Technologies,

Carlsbad, CA, USA) for sodium dodecyl sulfate-poly-

acrylamide electrophoresis (SDS-PAGE). Samples were

then transferred onto a polyvinylidene fluoride (PVDF)

membrane (Bio-Rad Laboratories, Hercules, CA, USA) by

the semidry method in transfer buffer (39 mM glycine,

48 mM Tris and 5% methanol) at 20 V for 90 min at room

temperature (RT). Membranes were blocked with 5% non-

fat milk in Tween-20-containing Tris-buffered saline

(TBST) (20 mM Tris–HCl, pH 7.4, 150 mM NaCl, 0.05%

Tween-20) for 1 h at RT and then incubated with the pri-

mary antibody in 5% non-fat milk in TBST or Signal

BoostTM (EMD Biosciences, San Diego, CA, USA) at 4�C

overnight. After washing in TBST, the membranes were

then incubated for one hour at RT with corresponding

Apoptosis

123

peroxidase (1:20,000–1:40,000) or alkaline phosphatase

(1:5,000–1:10,000) conjugated secondary antibodies (EMD

Biosciences, San Diego, CA, USA). Alkaline phosphatase

conjugates were visualized using 5-bromo-4-chloro-

3-indolylphosphate/nitroblue tetrazolium (BCIP/NBT)

phosphatase substrate (Kirkegaard & Perry Laboratories,

Gaithersburg, MD, USA). Blots were scanned and analyzed

with NIH ImageJ software. Peroxidase conjugates were

visualized by chemiluminescent detection with SuperSig-

nalTM West Femto substrate (Pierce/Thermo Scientific,

Rockford, Il, USA) in a Kodak GL2200 CCD-based imag-

ing system and analyzed by Kodak Molecular Imaging

Software (Carestream Health, New Haven, CT, USA). For

multiple antigen probing, blots were stripped and reprobed

using Restore Plus reagent (Pierce/Thermo Scientific,

Rockford, Il, USA) following manufacturer specifications.

Graphical representation and statistics were performed with

Prism v.5 software (GraphPad, La Jolla, CA, USA).

Primary antibodies included: (1) mouse monoclonal anti-

a-fodrin (BioMol Intl, Plymouth Meeting, PA, USA; 1:5,000),

(2) caspase-12 (rabbit polyclonal, 1:1,000), (3) anti-active

caspase-7 (p20) (rabbit polyclonal, 1:1,000), (4) anti-cleaved

caspase-3 (Asp175) (rabbit polyclonal, 1:1,000), (5) anti-Grp-

78 (rabbit monoclonal, 1:2000), (#2–#5 from Cell Signaling,

Danvers, MA, USA), (6) pro-calpain-2 (in-house, rabbit

polyclonal, 1:500), (7) mouse monoclonal anti-p23 (Affinity

Bioreagents Ltd., Plymouth Meeting, PA, USA; 1:2,000), (8)

anti-kinectin-1 (rabbit polyclonal, Santa Cruz Biotechnology,

CA, USA 1:1000), (9) mouse monoclonal anti-b-actin (Sigma

Chemical Co, St. Louis, MO, USA, 1:10,000), (10) cleaved-

caspase-12 at D341 (C12D341, in-house, rabbit polyclonal,

1:300), (11) caspase-12 small fragment (rabbit polyclonal,

1 lg/ml, Anaspec, Fremont, CA, USA).

In vitro calpain and caspase substrate digestions

Differentiated PC12 cells were lysed in Triton X-100 lysis

buffer as previously described but without protease inhibi-

tors. For caspase enzyme digestions, 50 ll of cell lysate

(200 lg total protein) was incubated with (1) 20 U of

recombinant human caspase-3 (Chemicon/Millipore, Teme-

cula, CA, USA), or (2) 20 U of recombinant human caspase-

7 (EMD Biosciences, San Diego, CA, USA) in enzyme

buffer (EB) (100 mM Tris-HCl, pH 7.4 and 20 mM DTT) at

37�C for 4 h in rocking platform. Calpain digestion of PC12

lysate was performed by incubating 50 ll cell lysate (200 lg

total protein) with 1 lg recombinant rat calpain-2 (EMD

Biosciences, San Diego, CA, USA) at RT for 30 min in

rocking platform in EB with 10 mM CaCl2. Undigested

lysate served as control (30 min for calpain, 4 h for caspase-

3 and -7). After digestion samples were snap frozen in dry ice

and stored at -80�C until used.

Caspase-12 enzyme activity assay

Enzyme assay is based on the cleavage of the caspase-12-

specific substrate ATAD-AFC (MBL International, Woburn,

MA, USA). Total cell lysates from 8 and 24 h TG-treated

cultures were collected as previously described but in the

absence of protease and phosphatase inhibitors. Reactions

were carried in 100 ll volume with 5 lM substrate, enzyme

buffer, and 50 lg total protein sample from PC12 lysate in

duplicates. After 3 h of dark incubation at 37�C with shaking

in microplate incubator/shaker (Amersham, UK), fluores-

cence emission was measured using a SpectraMax Gemini

EM (Molecular Devices, Ontario, CA) set at Ex-400/Em-

505. Results were analyzed using SoftMax 5.0 software and

statistical analysis performed in Prism v5.0 (GraphPad, La

Jolla, CA).

Results

Induction of the unfolded protein response (UPR)

by thapsigargin precedes cytotoxicity in differentiated

PC12 cells

ER stress was induced in differentiated PC12 cells by

exposure to 1 lM thapsigargin (TG). Morphological

assessment during initial long-term time course studies

showed that[90% cell death occurred after 48 h and about

50% cell death was observed after 32 h (data not shown).

During the 24-h time course of TG treatment cellular mor-

phology appeared unremarkable under the light microscope.

Cell death analysis by LDH released into medium indicated a

significant level of cytotoxicity after 24 h of TG-treatment

(Fig. 1a). No significant release of LDH was observed at

earlier time points during the 24-h time course. However,

TG-induced stress activated the UPR after 8 h as evidenced

by an increase in protein levels of GRP-78 as determined by

qWB analysis (Fig. 1a, b). There was no significant increase

in the level of GRP-78, before 8 h but after that the levels

began to almost double every 8 h, with peak levels recorded

at the end of the 24-h time course. Results suggest that the

UPR precedes ER stress-induced cell death in PC12 cells.

Calpain- and caspase-3-mediated aII-spectrin

breakdown products induced by TG in PC12 cells

indicate calpain activity precedes caspase-3

We examined the processing of aII-spectrin during TG-

induced ER stress in PC12 cells to determine the level of

calpain and caspase activity. Distinct breakdown products

of aII-spectrin are generated as the result of the action of

calpain and caspase-3 [18–20]. Spectrin breakdown prod-

uct of 145 kDa (SBDP145) results from sequential calpain

Apoptosis

123

cleavage of aII-spectrin at Y1176 to generate SBDP150,

followed by cleavage at G1230, to remove an additional

5 kDa. Spectrin breakdown product of 120 kDa

(SBDP120) is generated by caspase-3 cleavage at D1478

[21]. Two products of almost identical mass (150 kDa) are

generated by the action of calpain and caspase that are

recognized by the total spectrin antibody. The abovemen-

tioned SBDP150 (without the second calpain cleavage) is

the product of calpain, but cleavage by caspase-3 at D1185

generates what has been labeled SBDP150i [22]. Analysis

of the levels of SBDP145, SBDP150/SBDP150i and

SBDP120 during TG-induced ER stress in PC12 cells

confirmed that the initial cellular response is primarily

calpain-mediated but then becomes predominantly a cas-

pase-mediated response. Protein levels of SBDP145 were

significantly increased as early as 3 h during TG exposure

time course indicating early calpain activity (Fig. 2a, d).

This significant increase in the level of SBDP145 was

progressively reduced during the experimental time course

and eventually disappeared after 24 h. In contrast, levels of

SBDP120 and SBDP 150/150i showed a small but signif-

icant increase after 16 h; but the largest increase was

observed by 24 h (Fig. 2b–d). Although we were unable to

distinguish SBDP150 from SBDP150i in these experi-

ments, the parallel increase of the 150 kDa SBDP to

SBDP120, and not to SBDP145 suggests that it is indeed

8 16 240

20

40

60

80

100

120

0.0

0.5

1.0

1.5

2.0LDH

BiP(GRP-78)

3

**

**

Time (hrs)

No

rmal

ized

BiP

leve

l(a

rbit

rary

un

its)

LD

H (O

.D. @

490nm

)

b

a

Fig. 1 Relationship between cell death and unfolded protein

response (UPR) induction in thapsigargin (TG)-treated NGF-differ-

entiated PC12 cells. a Cell death measured by lactate dehydrogenase

(LDH) released into culture medium by CytoToxTM LDH assay (righty-axis). Induction of UPR determined by qWB analyses of GRP78/

BiP normalized to level of the control (left y-axis). Each sample

represents the mean ± SEM, n = 3. * Statistical significance over

control by Student’s t test (P \ 0.05). b Representative immunoblot

of GRP-78 and b-actin (loading control) for a 24-h time course.

Control (-) and corresponding thapsigargin (TG)-treated (?) samples

indicate time and condition

d

c

ab

Fig. 2 Quantificated aII-spectrin levels and breakdown products

(SBDP) in NGF-differentiated PC12 cells induced by thapsigargin.

Graphs a, b and c represent qWB analyses of immunoblots probed for

aII- spectrin breakdown products (SBDP’s). Control (open circle),

thapsigargin (TG)-treated (filled circle) cells. a SBDP150 (caspase-

cleaved), b SBDP-145 (calpain-cleaved), c SBDP120 (caspase-3

cleaved). * Statistically significant difference (P \ 0.05, n = 3,

Student’s t test), d representative aII-spectrin immunoblot indicates

intact spectrin and breakdown products, SBDP150, SBDP145 and

SBDP120 for a 24 h time course. b-actin immunoblot shown for

loading control. Control (-) and corresponding thapsigargin (TG)-

treated (?) samples are indicated

Apoptosis

123

SBDP150i, the product of caspase-3 activity. Results of

spectrin breakdown product biomarker analysis indicate

early calpain activity (before 16 h) followed by increased

caspase activity (after 16 h) during TG-induced ER stress

in PC12 cells.

Calpain-2 and caspase-7 activation precedes

that of caspase-3 in TG-induced ER stress in PC12 cells

Quantitative analysis of pro-calpain-2 immunoblots showed

a significant decrease in protein levels observed as early as

3 h, and remained low throughout the 24-h time course

(Fig. 3a, c). Although decreased levels in pro-calpain-2 do

not necessarily indicate activation of calpain-2, the activa-

tion of calpains resulting from the intracellular calcium

elevation caused by TG in PC12 cells has been previously

described [23].

Caspase-3 and -7 have been classified as downstream

executioners of apoptosis regardless of the inducing stim-

ulus. Although both proteases are widely considered to

coordinate the demolition phase of apoptosis, their relative

contribution remains highly speculative [24]. During ER

stress induced by TG in PC12 cells a significant increase in

the level of active caspase-7 as early as 8 h with peak

levels at 16 h was observed (Fig. 3b, c). On the other hand,

activated caspase-3 levels were not significantly increased

until 24 h suggesting that caspase-7 activation precedes

that of caspase-3 in TG-treated PC12 cells. Results suggest

a functional difference between these two structurally

related proteases during TG-induced ER stress and it

potentially establishes calpain as an early mediator of the

unfolded protein response in PC12 cells.

Kinectin, aII-spectrin and co-chaperone p23 are

susceptible to caspase-7, caspase-3 and calpain-2

cleavage in vitro

To determine substrate specificity of the calpain and cas-

pase substrates kinectin and co-chaperone p23 in PC12

cells we performed in vitro digestions with exogenous

recombinant calpain-2, caspase-7 and caspase-3. Kinectin

and p23 have been suggested to be specific caspase-7

substrates [24, 25]. However, because caspase-7 and -3

share almost identical substrate specificity as a conse-

quence of their structural similarity (56% identity, 73%

similarity), it is possible that kinectin and p23 could also be

vulnerable to caspase-3 activity. Similarly, it has been well

documented that calpains and caspases share many similar

substrates [26–28]; thus it is conceivable that these two

proteins might be vulnerable to calpain proteolysis as well.

We used spectrin BDP biomarker analysis as positive

control for calpain (SBDP145) and caspase-3 (SBDP120)

activity. Under the same in vitro digestion conditions, both

kinectin isoforms were completely digested by calpain

(Fig. 4a). In fact, kinectin proved to be an excellent indi-

cator of calpain activity because cleavage by calpain

resulted in total loss of antigen recognition by immuno-

blotting without total protein degradation, evidenced by the

coomassie stain pattern of the transferred gel that showed

similar total protein content in all four lanes (data not

0 8 16 240

50

100

150Caspase-7

Caspase-3

3

*

* **

Time (h)No

rmal

ized

act

ive

casp

ase

leve

ls(a

rbit

rary

un

its)

ab

0 8 16 240

20

40

60

*

3

**

*

Control

TG

Time (h)

Pro

-Cal

pai

n-2

leve

ls(a

rbit

rary

un

its)

c

Fig. 3 Time course of the level

of pro-calpain-2, active caspase-

7 and active caspase-3 in

differentiated PC12 cells

induced by thapsigargin.

a Protein levels of pro-calpain-2

and b active caspase-3 and -7

were determined by qWB

analyses of cytoplasmic fraction

as described in ‘‘Methods’’.

Each point represents

normalized caspase activity

relative to control

(mean ± SEM, n = 3).

* Statistical significance over

corresponding control

(P \ 0.05) by Student’s t test.

c Representative immunoblots

of pro-calpain-2, active caspase-

3 and -7 time course. Control

(-) and corresponding

thapsigargin (TG)-treated (?)

samples are indicated

Apoptosis

123

shown). Full length kinectin (160 kDa) digestion by cas-

pase-7 and -3 showed a BDP of 120 kDa. This indicates

that the 160 kDa form of kinectin was susceptible to both

caspase-7 and -3 cleavage. But interestingly, the short form

of kinetin (80 kDa) was degraded to a truncated form of

60 kDa by caspase-3, but not caspase-7. We confirmed

activity of calpain and caspase-3 by spectrin BDP bio-

marker analysis of SBDP145, SBDP150i (see * in Fig. 4b)

and SBDP120 as we have reported previously [29].

Co-chaperone p23 was not susceptible to calpain cleavage

and was preferentially, although not exclusively, cleaved by

caspase-7 compared to caspase-3 (Fig. 4c). This demon-

strates that kinectin was a substrate preferentially processed

by calpain-2 and differentially processed by caspase-7 and -

3. Although kinectin could be used to determine activity of

the abovementioned proteases, its sensitivity to calpain-

mediated proteolysis makes it potentially useful for assess-

ment of calpain activity. Similarly, p23 was shown to be

preferentially processed by caspase-7 to generate p17 and

was not a calpain substrate, thus p17 could serve as indicator

of caspase-7 activity. The fact that SBDP120 could be pro-

duced only by the action of caspase-3 and not caspase-7

provides a unique marker to distinguish activity between

these two related proteases.

Temporal processing of kinectin and p23 support

that calpain activation precedes that of caspase-7

and -3 in TG-treated PC12 cells

Kinectin, the molecular motor kinesin receptor in ER,

although previously identified as a substrate for caspase-7

[25], as noted above, it was also a caspase-3, caspase-7 and

calpain substrate (Fig. 4a) and we decided to determine its

processing during TG-induced ER stress in PC12 cells.

Similarly, the processing of co-chaperone p23 protein, a

member of the heat shock protein 90 family (Hsp90),

shown to be a substrate with higher affinity for caspase-7

than caspase-3 [24], was examined. Analysis of the integ-

rity of these substrates during TG-induced ER stress in

PC12 cells showed different temporal profiles. Both the

native 160 kDa protein and the more abundant short form

of kinectin (80 kDa band) were immunodetected. It has

been previously reported that both are present in brain

tissue lysate [30]. Analysis of both immunoreactive species

showed a homologous temporal processing profile during

TG-induced ER stress. No breakdown products were

detected for either kinectin isoforms, but we did observe a

significant decrease in the protein level for both 160 kDa

and 80 kDa forms after 3 h of TG treatment (Fig. 5a, b, d).

The difference in the decreased level of the 80 kDa

kinectin band was more pronounced than that of the

160 kDa kinectin form. On the other hand, western blot

analysis of co-chaperone p23 showed a breakdown product

of 17 kDa (p17) that was significantly increased after 8 h

(Fig. 5c, d), indicating caspase-7 activity. Results of cal-

pain and caspase substrate processing during TG-induced

ER stress in PC12 cells again indicate that calpain activity

precedes that of caspase.

Processing and activation of caspase-12 by calpain

and caspase during ER stress produce distinct calpain

and caspase cleavage products

In TG-induced ER stress PC12 cells differential processing

of pro-caspase-12 by the action of calpain and caspase-3/7

was observed. After 8 h, TG produced a 38 kDa fragment

that was inhibitable by the calpain inhibitor SNJ-1945, but

not by the caspase-3/7 inhibitor DEVD. This suggests this

38 kDa fragment was the product of calpain-cleavage

(Fig. 6a, b). This 38 kDa calpain product was also observed

at 24 h but only with 24 h of TG-treatment did we began to

observe a 35 kDa fragment that was completely inhibited

a

b

c

Fig. 4 Calpain and caspase in vitro digestion of kinectin and

co-chaperone p23 in NGF-differentiated PC12 cells. PC12 lysate

was digested with active calpain-2, caspase-3 and caspase-7 (see

‘‘Methods’’). a Kinectin immunoblot shows 160 and 80 kDa bands.

Kinectin was completely digested by calpain-2. Both caspase-3 and -7

cleaved the 160 kDa kinectin form to generate a 120 kDa band, but

only caspase-3 cleaved the 80 kDa kinectin form to generate a

60 kDa band. b Immunodetection of intact aII-spectrin and break-

down products SBDP145 and SBDP120 confirmed calpain and

caspase-3 activity, respectively. Caspase-7 and -3 processing of aII-

spectrin produced a SBDP band of 150 kDa (*SBDP-150i). c Co-

chaperone p23 immunoblot did not show degradation by calpain-2,

however it showed almost complete digestion to p17 with caspase-7,

but was minimally digested by caspase-3

Apoptosis

123

by DEVD but not by SNJ-1945. This suggests that this

fragment was the product of caspase-3/7 cleavage (Fig. 6a,

c). The 35 kDa caspase-generated fragment was not present

before 24 h indicating that pro-caspase-12 processing by

calpain preceded that of caspase-3/7. The partial, but sig-

nificant inhibition of the 35 kDa caspase-12 band by SNJ-

1945 might suggest a role for calpain in the generation of

this fragment.

Using a commercially available caspase-12 antibody

(Casp12 small, Anaspec) that specifically recognized a

caspase-12 38 kDa fragment [11], we validated that the

38 kDa fragment is in fact the calpain cleavage product.

The 38 kDa caspase-12 fragment was increased by TG at

both 8 and 24 h and inhibited by the calpain inhibitor SNJ-

1945 but not significantly inhibited by the caspase-3/7

inhibitor DEVD (Fig. 7). We also developed an antibody

specific to caspase-12 cleaved after D341 by caspase-3/7 by

targeting the new C-terminal sequence from S332 to D341,

(see Fig. 10), to further study the caspase-3/7 processing of

caspase-12 during TG-induced ER stress. The antibody

recognized a 23 kDa band that was not detected prior to

24 h of TG-treatment. This 23 kDa fragment most likely

just precedes the final autolytic processing of caspase-12

(after D318) to the active P20 large subunit (Fig. 10). The

23 kDa fragment was induced by TG and inhibited by

DEVD but not by SNJ-1945, indicating that it is a caspase

3/7, and not a calpain cleavage-specific product (Figs. 8,

10). Taken together, these results suggest that during the

unfolded protein response in PC12 cells caspase-12 is

processed by the action of calpain before caspase-3/7 and

autolysis.

Effects of calpain and caspase 3/7 inhibitors

on caspase-12 activity and cell death following

thapsigarin challenge

Caspase-12 enzymatic activity determined by ATAD-AFC

cleavage (see ‘‘Methods’’) was not observed in TG-treated

PC12 cells before 24 h (data not shown). The catalytic

activity of caspase-12 was significantly increased by TG

and completely abolished by DEVD and partially by SNJ-

1945 (Fig. 9a). The presence of the 38 kDa calpain-gen-

erated fragment (Figs. 6, 7) did not have a direct correla-

tion with caspase-12 enzyme activity, but the caspase-

generated fragments of 35 kDa and 23 kDa (Figs. 6, 8) did

coincide with the increased caspase-12 enzyme activity

observed. This suggests that caspase-3/7 might have a more

direct role in final enzymatic activation of caspase-12,

while calpain contributes to the early pro-domain pro-

cessing of caspase-12, Finally, consistent with this, the

increased TG-induced cytotoxicity determined by LDH

release after 24 h was significantly reduced by caspase-3/7

inhibition but not by calpain inhibition suggesting that

partial suppression of caspase-12 processing by calpain

inhibitor was not sufficient to rescue cells from the ER

stress-induced cell death pathway (Fig. 9b).

0 8 16 240

50

100

150

*

*

*

3

Time (h)

Cle

aved

p23

leve

ls (

p17

)(a

rbit

rary

un

its)

a

0 8 16 240

50

100

150

* * * *3

*

Time (h)

Kin

ecti

n 1

60 k

Da

leve

ls (

arb

itra

ry u

nit

s)

b

d

0 8 16 240

50

100

150

200

** * *

3

Time (h)

Kin

ecti

n 8

0 kD

a le

vels

(ar

bit

rary

un

its)

c

Fig. 5 Kinectin and co-chaperone p23 protein levels in NGF-

differentiated PC12 cells induced by thapsigargin. a Kinectin

(160 kDa), b kinectin (80 kDa) and c cleaved-p23 product (p17)

protein levels were determined by qWB analysis. Each point

represents the mean ± SEM, n = 3. * Statistically significant

differences over control by Student’s t test (P \ 0.05), d represen-

tative immunoblot of kinectin (160 and 80 kDa) and p23 with cleaved

fragment (p17) in a 24 h time course. Control (-) and corresponding

thapsigargin (TG)-treated (?) samples are indicated

Apoptosis

123

Discussion

Growing evidence implicates ER stress in several diseases

and targeting ER function could provide future therapeutic

opportunities [31]. ER stress specifically appears to be

involved in a number of neurological conditions including

Parkinson’s, Alzheimer’s, prion, polyglutamine, and lyso-

somal storage diseases as well as ischemia and traumatic

brain injury [5, 6, 32]. Cerebral ischemia and reperfusion

(I/R) have also been shown to cause accumulation of

potentially toxic unfolded proteins and activation of the

UPR [33]. Although the UPR allows the cell to alleviate

ER stress by attenuating translation while increasing pro-

tein folding, secretion and degradation, if ER function is

not promptly restored, it can lead to the activation of an

intrinsic apoptotic cell death program [12, 34]. Thus,

understanding the mechanisms that control the UPR and

ER stress-induced apoptosis in neurons could provide a

target to enhance cell survival after brain injury.

The increased ER protein folding capacity is achieved

by the selective up-regulation of molecular chaperones and

foldases, such as the glucose regulated protein GRP-78, a

key indicator of ER stress. GRP-78 is normally bound on

the luminal side of the ER to all three UPR sensors, and it

serves as a ‘‘master switch’’ that regulates their activities.

Up-regulation of GRP-78 at the transcriptional and trans-

lational level is a hallmark of the UPR and has been

commonly used as a marker of ER stress [35, 36]. ER

stress, the UPR and the ER stress-induced apoptotic cell

death pathway can be induced by serum deprivation [35] as

well as by a variety of chemicals such as tunicamycin (an

asparagine-linked protein glycosylation inhibitor), brefel-

din A (an inhibitor of ER to Golgi transport), calcium

ionophore A23187 and thapsigargin (an inhibitor of endo-

plasmic reticulum Ca2? ATPase).

In our cell model 1 lM thapsigargin induced ER stress

in differentiated PC12 cells, activating the UPR within 8 h

(Fig. 1) without significant cytotoxicity. Others have

reported that lower doses of TG were sufficient to decrease

cell viability in undifferentiated PC12 cells after 24 h [29].

This discrepancy might be explained by the differentiated

state of our model system. Since an increase in cytosolic

8 h 24 hPro-caspase-12 (55 kDa)

38 kDa caspase-12 fragment (Calpain)

35 kDa caspase-12 fragment (Caspase 3/7)

Co

ntr

ol

TG

TG

+DE

VD

TG

+SN

J

Co

ntr

ol

TG

TG

+DE

VD

TG

+SN

J

a

Co

ntr

ol

TG

TG

+DE

VD

TG

+SN

J

Co

ntr

ol

TG

TG

+DE

VD

TG

+SN

J

0

20

40

60

*

#

*

#

Cal

pai

n-c

leav

ed C

asp

-12

frag

men

t(3

8 kD

a) le

vel (

arb

. un

its)

8 h 24 hC

on

tro

l

TG

TG

+DE

VD

TG

+SN

J

Co

ntr

ol

TG

TG

+DE

VD

TG

+SN

J

0

10

20

30

40

*

#

#

Cas

pas

e-cl

eave

d C

asp

-12

frag

men

t(3

5 kD

a) le

vel (

arb

. un

its)

8 h 24 h

b cCalpain Caspase

β-actin (43 kDa)

Fig. 6 Calpain and caspase-mediated processing of caspase-12

during TG-induced ER stress in PC12 cells. a Representative total

caspase-12 western blot show both pro-form (55 kDa) and cleaved

fragments (38 and 35 kDa) generated by TG treatment at 8 and 24 h,

and the effect of the inhibitors DEVD (caspase-3/7) and SNJ-1945

(calpain). b-Actin loading control shown below. b Quantitative

analysis of calpain-generated caspase-12 fragment (38 kDa) and

c caspase-generated caspase-12 fragment (35 kDa). Each column

represents mean ± SEM, n = 3. * Statistically significant increase

over control, # statistically significant change from ER stress-induced

(TG) (Student’s t test, P \ 0.05)

Apoptosis

123

Ca2? may be a key component of mitosis, disruption of

Ca2? homeostasis is more likely to have an adverse effect

on proliferating cells [37]. The apoptosis-inducing effect of

thapsigargin on PC12 cells has been clearly established

[38, 39]. Since the target for TG is the endoplasmic retic-

ulum Ca2? ATPase, the relative increase in the intracellular

calcium level will more likely depend on the extensive

nature or the ER network for a particular cell type, rather

than the dose of TG.

The analysis of aII-spectrin breakdown product levels

provided a robust method for assessment of calpain and

caspase-3 activity in vitro [18–20] (Fig. 2), indicating that

TG-induced ER stress in PC12 cells is initially a calpain-

dominated response with subsequent transition to a pre-

dominantly caspase-3-mediated apoptotic induction. These

results were consistent with western blot analyses of cal-

pain-2 and active caspase-3 (Fig. 3) and validated the use

of specific spectrin BDP’s for assessment of calpain and

caspase-3 activity. The early calpain activation observed is

also consistent with the documented rapid intracellular

calcium elevation resulting from TG activity [31, 36].

To confirm specificity of the suggested calpain-2, cas-

pase-3 and caspase-7 substrates aII-spectrin, kinectin and

co-chaperone p23, in vitro digestions of PC12 lysates were

performed with exogenous recombinant enzymes. Results

in Fig. 4 show differential processing of kinectin, aII-

spectrin and co-chaperone p23 by the different proteases.

First, although kinectin had been proposed to be a caspase-

7 substrate [17], it was also cleaved specifically by caspase-

3 and was most susceptible to calpain proteolysis. Second,

co-chaperone p23, also known as prostaglandin E synthase-

3, is not a calpain substrate and is preferentially cleaved by

caspase-7 over caspase-3.

It is important to point out that caspase-3 and -7 have

very similar substrate specificities (both recognize the tet-

rapeptide sequence DExD) resulting from their structural

homology that to date no known commercially available

substrate is able to distinguish the proteolytic activity of

one over the other. Here we show that we do have tools to

determine the relative action of both caspase-7 and -3 in

vitro. For example, caspase-3 specifically cleaves aII-spec-

trin at D1478 to generate SBDP120, a site not cleaved by

caspase-7 (Fig. 4b). We showed that kinectin has a caspase-

3-specific cleavage site that generates a 60 kDa form from

Co

ntr

ol

TG

TG

+DE

VD

TG

+SN

J

Co

ntr

ol

TG

TG

+DE

VD

TG

+SN

J

0

20

40

60

*

*

#

#

Cal

pai

n-c

leav

ed C

asp

-12

frag

men

t(3

8kD

a) le

vel (

arb

. un

its)

Calpain-cleaved Casp12 (38 kDa)

8 h 24 h

β-actin(43 kDa)

Fig. 7 Calpain-mediated processing of caspase-12 during TG-induced

ER stress in PC12 cells. Representative caspase-12 small (Anaspec, see

‘‘Methods’’) western blot shows a 38 kDa band inhibited by calpain

inhibitor SNJ-1945 but not by caspase-3/7 inhibitor DEVD. b-Actin

loading control shown below. Graph represents densitometric analysis

of 38 kDa caspase-12 fragment. Each column represents mean ±

SEM, n = 3. * Statistically significant increase over control, # statis-

tically significant change from ER stress-induced (TG) (Student’s t test,

P \ 0.05)C

on

tro

l

TG

TG

+ D

EV

D

TG

+ S

NJ

0

5

10

15

*

#

Cas

pas

e-cl

eave

d C

asp

-12

frag

men

t (

23 k

Da)

leve

l (ar

b.u

nit

s)

Caspase-cleaved Casp12 (23 kDa)

24 h

β-actin(43 kDa)

Fig. 8 Caspase-mediated processing of caspase-12 to active large

subunit (p20) after 24 h of TG-induced ER stress in PC12 cells.

Representative immunoblot of anti-cleaved caspase-12 after D319

(made in-house (C12D319), see ‘‘Methods’’) shows a 20 kDa band

that is totally inhibited by the caspase inhibitor DEVD but only

partially by the calpain inhibitor SNJ-1945. No significant increase

was observed after 8 h (data not shown). b-Actin loading control

shown below. Graph represents densitometric analysis of 20 kDa

caspase-12 fragment. Each column represents mean and SEM, n = 3.

* Statistically significant increase over control, # statistically signif-

icant change from ER stress-induced (TG) (Student’s t test, P \ 0.05)

Apoptosis

123

the 80 kDa form (Fig. 4a). In the rat sequence (XP341306,

XP001073656) this corresponds to the tetrapeptide

DELD178. Co-chaperone p23 contains several caspase-3/7

(DExD) tetrapeptide sequences near the C-terminus, but the

consensus is that D150 is the caspase-7 cleavage site that is

less efficiently cleaved by caspase-3. The differential pro-

cessing of aII-spectrin, kinectin and co-chaperone p23 by

caspase-3 and -7 provide further evidence for the function-

ally distinct nature of these two executioner protease families

that has been previously reported [24, 40–42].

Homologous to the use of specific spectrin BDP’s for the

assessment of calpain and caspase-3 activity we selected

previously reported specific substrates of caspase-7 to val-

idate results in Fig. 3b. In our ER stress cellular model,

kinectin western blot provided support to the observed early

calpain activation indicated by SBDP145 analysis, although

processing by other proteases present in cell lysate could

not be ruled out. Co-chaperone p23 breakdown was an

excellent marker for caspase-3/7 activity in this model

because it was not susceptible to calpain cleavage. The

observed preferential cleavage of p23 by caspase-7 (Fig. 5c,

d) was consistent with active caspase-7 western blot data

(Fig. 3b, c). The lack of a similar increase in active caspase-

7 level from 16 to 24 h (Fig. 3b) that corresponded to that

observed in the p17 level (Fig. 5c) suggests that the 24 h

increase was most likely due to caspase-3 activity.

Caspase-12 [43] processing and activation during ER

stress is thought to be mediated by the action of calpain and

caspase-7 but their relative contribution has remained

unclear [10]. Caspase-12 contains a CARD domain within

the N-terminus pro-domain that must be cleaved prior to

enzymatic activation [44]. It is possible that pro-domain

cleavage of caspase-12 may be the result of caspase-7

cleavage at D94 with subsequent processing at D341.

Figure 10 illustrates a schematic of caspase-12 with

calpain and caspase-3/7 and autolytic cleavage sites indi-

cated and size of fragments produced. The sequence of

murine caspase-12 was used because there was more

completely cleavage site information. The rat caspase-12

sequence is 87% identical to murine caspase-12 and all

cleavage sites indicated in Fig. 10 are conserved. Results

by us and others [45, 46] have shown that the rapid increase

in intracellular calcium resulting from the action of TG is

likely responsible for the early calpain activity (Figs. 2a,

3b), Calpain cleavage of caspase-12 at two potential sites

(T132, K158) has been suggested to be a prerequisite for

caspase-12 autoprocessing at D318 [13]. Our results do not

completely support this claim because calpain inhibition by

SNJ-1945 only partially abolished TG-induced caspase-12

activity (Fig. 9). This is likely due to alternative pathway

by caspase-3/7 processing at the adjacent D94 site (Fig. 10)

[38]. Until now, the only caspase-12 cleavage-specific

consensus sequence corresponds to that of the autoproteo-

lytic cleavage D318 site that contains the tetrapeptide ATAD

and is the basis for commercially available caspase-12

enzyme assays. Cleavage by caspase-3/7 at D318, producing

a 23 kDa caspase-12 fragment (Fig. 8) that contains the

whole large subunit P20 the intact catalytic C298 and thus is

potentially active or could lead to final autolytic formation of

active P20 large subunit (Fig. 10).

The most significant increase in caspase-3 activity was

observed at 24 h of ER stress (Figs. 2c, 3b), simultaneous

to increased caspase-12 enzymatic activity (Fig. 9). Cas-

pase-12 processing took place prior to the significant

increased activity observed for both caspase-12 and -3,

suggesting that these fragments might have a role in

apoptotic signaling besides the typical caspase catalytic

cascade. Early caspase-12 processing does not appear to

have an effect on apoptotic cell death via direct activation

of executioner caspase-3, but it could prepare caspase-12 to

Caspase-12 activity in PC12 (24 h)

Control TG TG+DEVD TG+SNJ0

2

4

6

8

*

#

#A

TA

D-A

FC

cle

avag

e (R

FU

)

LDH Assay in PC12 (24 h)

Control TG TG+DEVD TG+SNJ0.0

0.5

1.0

1.5

2.0

*#

LD

H-r

elea

sed

(O

.D. @

490

nm

)

a b

Fig. 9 Relationship between caspase-12 enzymatic activity and TG-

induced ER stress cell death pathway in PC12 cells. a Caspase-12

Enzyme activity determined by fluorescent measurement of cleaved

caspase-12-specific substrate ATAD-AFC (Relative Fluorescence

Units) as indicated in methods section. b The effect of calpain and

caspase-3/7 inhibition on TG-induced cell death was determined by

optical density (OD) measurement at 490 nm (OD at 490 nm) of LDH

released into cell culture medium by CytoToxTM LDH assay as

indicated in methods section. Each column represents the mean and

SEM, n = 5. * Statistically significant increase over control, # Sta-

tistically significant change from ER stress-induced (TG) (Student’s

t test, P \ 0.05)

Apoptosis

123

participate in activation of caspase-3 via caspase-9 in

presence or absence of apoptosome complex formation [12,

47]. Studies using the caspase-12 inhibitor z-ATAD-fmk

indicate a reduction in both ER stress induced apoptosis,

active caspase-9 and active caspase-3 in PC12 cells [48]. It

has been suggested that a 36 kDa caspase-12 fragment

detected by a caspase-12 cleaved at D341 antibody trans-

locates to nucleus during ER stress [49].

Taken the literature data and our results together, our ER

stress caspase-12 activation model suggests that (i) cas-

pase-12 is initially processed by calpain, removing the

CARD-containing pro-domain producing a 38 kDa frag-

ment; (ii) if calpain is not available, caspase-3/7 can also

remove the prodomain; (iii) Caspase-3/7 will also proceed

to release the p10 small subunit by C-terminal cleavage (a

critical step in the process), followed by (iv) the subsequent

caspase-3/7 processing to generate the large subunit con-

taining 23 kDa fragment, (v) which is further autolytically

processed to generate the fully active caspase-12 large

subunit(p20) (Fig. 10). It is possible that active caspase-12

can initiate a positive feedback loop that activates caspase-

3 through caspase-9 activation and thus potentiating

apoptosis induction. Both activation of caspase-12 by

caspase-3 and activation of caspase-9 by caspase-12 have

been previously established [12, 34, 39, 47]. Finally,

inhibition of caspase-3/7 activity caused a larger decrease

in caspase-12 activation than calpain inhibition and a more

robust rescue against thapsigarin-induced PC12 cell death

(Fig. 9). In conclusion, although both calpain and caspase

are involved in the processing of caspase-12 during ER

stress, our results indicate that caspase-3/7 activity is more

critical than calpain for caspase-12 enzyme activation and

the overall activation of ER stress-induced cell death

pathway.

Acknowledgements This work was supported by NIH Grant

R01NS051431 to R. L. H. Dr. K. K. W. Wang and Dr. R. L. Hayes

hold equity in Banyan Biomarkers, Inc. (Alachua, FL), a company

commercializing the brain injury biomarker technology.

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