Increased isoform-specific membrane translocation of conventional and novel protein kinase C in human neuroblastoma SH-SY5Y cells following prolonged hypoxia.

Abstract

Several studies have suggested that protein kinase C (PKC) plays a key role in the mechanism of cerebral ischemic/hypoxic preconditioning (I/HPC). However, detailed information regarding PKC isoforms in response to brain ischemia/hypoxia and their potential role in neuroprotection is unclear. Previous studies in our laboratory have demonstrated that the levels in membrane translocation of conventional PKC (cPKC) betaII, gamma, and novel PKCepsilon (nPKC), but not cPKCalpha, betaI, nPKCdelta, eta, mu, theta, and atypical PKC (aPKC) zeta and iota/lambda, were increased significantly in the hippocampus and cortex of intact mice with hypoxic preconditioning. To further detect cPKC and nPKC isoforms activation following prolonged hypoxia in vitro, we tested the membrane translocation (an indicator of PKC activation) of cPKCalpha, betaI, betaII, and gamma, and nPKCdelta, epsilon, eta, mu, and theta in a human neuroblastoma SH-SY5Y cell line following sustained hypoxic exposure (1% O(2)/5% CO(2)/94% N(2)). Using Western blot and immunocytochemistry methods, we found that the levels of cPKCalpha, betaI, betaII, and nPKCepsilon, but not nPKCdelta, eta, mu, and theta, membrane translocation were increased significantly (P < 0.05, n = 8) in a time-dependent manner (from 0.5 to 24 h) following sustained hypoxic exposure. Similarly, the immunostaining experiment also showed a noticeable translocation of cPKCalpha, betaI, betaII, and nPKCepsilon from the cytosol to the perinuclear or membrane-related areas after 6 h posthypoxic exposure. In addition, no cPKCgamma was detected in this cell line under either a normoxic or hypoxic condition. These results suggested that prolonged hypoxia may induce the activation of cPKCalpha, betaI, betaII, and nPKCepsilon by triggering their membrane translocation in SH-SY5Y cells.

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Short Communication
Increased isoform-specific membrane translocation of
conventional and novel protein kinase C in human
neuroblastoma SH-SY5Y cells following prolonged hypoxia
Junfa Lia,⁎, Yanming Qua, Pengyu Zua, Song Hana, Ge Gaoa, Qunyuan Xua, Li Fangb,⁎
aInstitute for Biomedical Science of Pain, Beijing Key Laboratory for Neural Regeneration and Repairing, Department of Neurobiology,
Capital University of Medical Sciences, #10 You An Men Wai Xi Tou Tiao, Beijing 100054, China
bDivision of Neurosurgery, Department of Surgery, Neuroscience and Cell Biology, The University of Texas Medical Branch, Galveston,
TX 77555-0517, USA
A R T I C L E I N F OA B S T R A C T
Article history:
Accepted 23 March 2006
Available online 8 May 2006
Several studies have suggested that protein kinase C (PKC) plays a key role in the
mechanism of cerebral ischemic/hypoxic preconditioning (I/HPC). However, detailed
information regarding PKC isoforms in response to brain ischemia/hypoxia and their
potential role in neuroprotection is unclear. Previous studies in our laboratory have
demonstrated that the levels in membrane translocation of conventional PKC (cPKC) βII, γ,
and novel PKCε (nPKC), but not cPKCα, βI, nPKCδ, η, μ, θ, and atypical PKC (aPKC) ζ and ι/l,
were increased significantly in the hippocampus and cortex of intact mice with hypoxic
preconditioning. To further detect cPKC and nPKC isoforms activation following prolonged
hypoxia in vitro, we tested the membrane translocation (an indicator of PKC activation) of
cPKCα, βI, βII, and γ, and nPKCδ, ε, η, μ, and θ in a human neuroblastoma SH-SY5Y cell line
following sustained hypoxic exposure (1% O2/5% CO2/94% N2). Using Western blot and
immunocytochemistry methods, we found that the levels of cPKCα, βI, βII, and nPKCε, but
not nPKCδ, η, μ, and θ, membrane translocation were increased significantly (P < 0.05, n = 8)
in a time-dependent manner (from 0.5 to 24 h) following sustained hypoxic exposure.
Similarly, the immunostaining experiment also showed a noticeable translocation of cPKCα,
βI, βII, and nPKCε from the cytosol to the perinuclear or membrane-related areas after 6 h
posthypoxic exposure. In addition, no cPKCγ was detected in this cell line under either a
normoxic or hypoxic condition. These results suggested that prolonged hypoxia may induce
the activation of cPKCα, βI, βII, and nPKCε by triggering their membrane translocation in SH-
SY5Y cells.
© 2006 Elsevier B.V. All rights reserved.
Keywords:
Sustain hypoxia
PKC isoform
Membrane translocation
SH-SY5Y neuroblastoma cell
Protein kinase C (PKC), a family of serine/threonine kinases, is
a critical component of intracellular signal transduction
pathways regulating a number of cellular events, including
homeostasis, migration, proliferation, apoptosis, remodeling
of the actin cytoskeleton, and modulation of ion channels
(Kanashiro and Khalil, 1998; Nishizuka, 1984). The PKC
family includes at least 12 isoforms: α, βI, βII, γ, δ, ε, η, θ, μ,
ν, ζ, and ι/l. To date, there three subgroups have been
B R A I N R E S E A R C H 1 0 9 3 ( 2 0 0 6 ) 2 5 – 3 2
⁎ Corresponding authors. J. Li is to be contacted at fax: +86 10 8391 1491. L. Fang, fax: +1 409 772 4687.
E-mail addresses: junfali@cpums.edu.cn (J. Li), lfang@utmb.edu (L. Fang).
0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.brainres.2006.03.110
available at www.sciencedirect.com
www.elsevier.com/locate/brainres

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identified according to their structures and activation modes
in mammalian tissues. They are recognized as (1) conven-
tional PKC (cPKCα, βI, βII, γ), which requires diacylglycerol,
phosphatidylserine, and Ca2+influx for their activation; (2)
novel PKC (nPKCδ, ε, η, θ, and μ) whose activation is not
dependent on influx Ca2+but does require diacylglycerol;
and (3) atypical PKC (aPKCζ, ι/l) whose regulation is not fully
understood and probably does not require either Ca2+or
diacylglycerol for activation (Hayashi et al., 1999; Newton,
1997; Nishizuka, 1992; Nishizuka, 1995).
PKC membrane translocation from the cytosolic fraction
to the membrane fraction is now widely accepted as a form
of enzyme activation. This PKC activation event undergoes
translocation from one intracellular compartment to another
following a series of stimulation factors such as stress,
neurotransmitters, hormones, growth factors, and ischemic/
hypoxic stimulus (Harada et al., 1999; Kurkinen et al., 2001a;
Li et al., 2005; Liu, 1996; Mochly-Rosen et al., 1990; Niu et al.,
2005; Wu et al., 2002). PKC is present in high concentrations
in neuronal tissues and previous studies have suggested that
PKC plays a critical role in the development of cerebral
ischemic/hypoxic preconditioning (I/HPC) (Cardell et al. 1990;
Harada et al., 1999; Katsura et al., 2001; Kurkinen et al.,
2001a; Mizukami et al., 1997). However, little is known about
which PKC isoforms may contribute to the response
elements in the mechanism of I/HPC. We previously
demonstrated that membrane translocation levels of
cPKCβII, γ, and nPKCε, but not the isoforms of cPKCα, βI,
nPKCδ, η, μ, θ, and aPKCζ, ι/l, were significantly increased in
the hippocampus and cortex of intact mice with hypoxic
preconditioning (Li et al., 2005; Niu et al., 2005). To further
detect the cPKC and nPKC isoforms activation status in an in
vitro environment with prolonged hypoxia, we used a
human neuroblastoma cell line, SH-SY5Y, and Western blot
and immunostaining techniques to test the levels of cPKCα,
βI, βII, γ, and nPKCδ, ε, η, μ, θ membrane translocation
following prolonged hypoxic stimulation.
Human neuroblastoma SH-SY5Y cells (obtained from Cell
Center of Peking Union Medical College, China) were main-
tained as reported (Bando et al., 2003; Webster et al., 2001). SH-
SY5Y cells were plated at a concentration of 2.0 × 106cells/
100 mm for immunocytochemistry examination or at a
concentration of 5.0 × 106cells/100 mm per dish for Western
blot analysis. Cells were incubated at 37 °C in humidified 95%
air/5% CO2for 2 days before the experiment. For prolonged
hypoxia conditioning, plates were placed in a modular
incubator chamber (2000 mm3) for 0–24 h at 37 °C. As a
control, air components were regulated in a humidified mixer
of 95% air/5% CO2or hypoxia condition with mixed 1% O2/5%
CO2/94% N2. Posthypoxic exposures were recognized at 0, 0.5,
2, 4, 6, 8, 12, and 24 h for each time point of the experiment
(Webster et al., 2001). Cultured cells were washed with cool
PBS three times before harvest and were stored at −70 °C for
later analysis after we recorded the cell viability by counting
the cell numbers in 10–20 random microscopic fields per
condition per experiment.
The Western blot study was conducted as our previous
reports (Li et al., 2005; Niu et al., 2005; Wu et al., 2002). Briefly,
frozen samples were homogenized at 4 °C in 100 μl per dish of
buffer A (50 mM Tris–Cl, containing 2 mM dithiothreitol, 2 mM
EDTA, 1 mM EGTA, 50 μM 4-(2-aminoethyl)-benzenesulfonyl-
fluoride hydrochloride, 5 mg/ml each of leupeptin, aprotinin,
pepstatin A, chymostatin, 50 mM KF, 50 μM okadaic acid, and
5 mM sodium pyrophosphate). Homogenates werecentrifuged
at 100,000 × g for 30 min at 4 °C in an Optima™ TLX ultra-
centrifuge to yield the cytosol. Pellet fractions were resus-
pended in 100 μl of buffer A containing 0.5% Nonidet P-40,
sonicated, and centrifuged again. The resulting supernatants
were used as particulate fractions. Twenty micrograms of
protein from the cytosolic or particulate fractions was loaded
for SDS–PAGE (10%) and transferred to a nitrocellulose
membrane (Schleicher & Schell, USA) at 4 °C. The membrane
was washed for 10 min with TTBS (20 mM Tris–Cl, pH 7.5,
containing 0.15 M NaCl and 0.05% Tween 20) and blocked with
10% nonfat dried milk in TTBS for 1 h following washing 3
times with TTBS. Next, the membrane was incubated with
primary and secondary antibodies in TTBS for 3 and 1 h,
respectively. After extensively washing in TTBS, membrane
was visualized by using an enhanced chemiluminescence
reagent (ECL kit, Perkin-Elmer Life Science, USA).
To detect the cPKC and nPKC isoform-specific membrane
translocation as our previous reports (Li et al., 2005; Niu et al.,
2005), different primary polyclonal antibodies were used
against cPKCα, βI, βII, γ, and nPKCδ, ε, η, μ, θ isoforms (Santa
Cruz Biotechnology Inc., USA) at a 1:1000 dilution. The
horseradish peroxidase-conjugated goat anti-rabbit IgG
(Amersham Company, IL, USA) were used as second anti-
bodies at a 1:3000 dilution. In order to determine any
variation during the loading process, an immunoblot for β-
actin was performed at the same time as an internal control
and the individual bands were quantified. Those membranes
blotted with antibodies against the PKC isoforms were
stripped and reprobed with primary monoclonal antibody
against β-actin (Sigma, USA). To quantitatively analyze
membrane translocation, we normalized the ratio of the
density of the PKC isoform-specific band (band density in
particulate/band densities in both particulate and cytosol) in
the normoxic control group (0) to 100%, and the data of the
groups of 0.5–24 h postexposure were expressed as a
percentage of the value.
In the immunocytochemistry analysis, coverslips were
fixed with 4% paraformaldehyde for 30 min and quenched in
10% methanol with 10% hydrogen peroxide for 5 min followed
by 1% goat serum with 0.1% Triton for blocking. Primary rabbit
polyclonal antibodies of cPKCα, βI, βII, γ, and nPKCδ, ε, η, μ, θ
isoforms (Santa Cruz Biotechnology Inc., USA) at 1:300 dilution
were incubated for 12 h. After washing 3 times with PBS,
coverslips were incubated in horseradish peroxidase-conju-
gated goat anti-rabbit (1:300) for 2 h. Finally, coverslips were
developed in 0.04% hydrogen peroxide and 0.05% 3,3′-diami-
nobenzidine (Sigma, USA) for 5 min. Slides were dehydrated
and mounted with resin. Stained cells were visualized and
recorded under a light microscope (C-35-AD-4, Olympus,
Japan).
Quantitative analysis for the immunoblotting experiment
was performed after scanning X-ray film with Quantitative-
One software (version-4.2.3, Gel Doc 2000 imagine system, Bio-
Rad Company, CA, USA). Presented values are mean ± SE from
at least 8 independent experiments. Statistical analysis was
conducted by a one-way analysis of variance followed by all
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pairwise multiple comparison procedures using a Bonferroni
test. Significance was regarded as P < 0.05.
Results from the immunoblots showed that cPKCα, βI, βII,
and nPKCε, but not nPKCδ, η, μ, and θ, isoforms were gradually
translocated from the cytosolic to the particulate fraction
following sustained hypoxic exposure (Figs. 1A and 2A).
Quantitative analysis of changes in the 4 isozymes from
8 independent experiments demonstrated that membrane
translocation levels of cPKCα, βI, βII, and nPKCε increased
significantly (P < 0.05, n = 8) in a time-dependent manner
following prolonged hypoxic exposure. Statistical significance
for bothα andβI cPKC isoformtranslocation could be observed
at 0.5 h posthypoxia (P < 0.05, n = 8) and at 2 h posthypoxic
induction for the cPKCβII and nPKCε isoform (P < 0.05, n = 8;
Fig. 1 – Membrane translocation of cPKCα, βI, and βII isoforms were increased in SH-SY5Y cells following prolonged hypoxic
exposure. (A) Representative results of Western blot analysis showed that the cPKCα, βI, and βII isoforms gradually
translocated fromthe cytosolicfractiontothe particulatefractionin response to sustained hypoxic exposure starting from 0.5 to
24 h. (B) Quantitative analysis demonstrated that the levels in membrane translocation of cPKCα , βI, and βII were significantly
increased in a time-dependent manner compared to the value of the control (0 group). (C) Changes in cell viability during
sustained hypoxic exposure. *P < 0.05 vs. 0 group, n = 8.
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Figs. 1B and 2B). In addition, the maximum translocation
levels of 4 activated forms could be reached without signifi-
cant cell lost after 6 h sustained hypoxic exposure (Figs. 1B, C,
and 2B). However, no cPKCγ isoform was detected in this cell
line (data not shown). These results suggested that 6 h of
hypoxic exposure might be the proper duration to induce
hypoxic preconditioning in cultured SH-SY5Y cells.
To characterize the cellular localization of cPKC and nPKC
isoforms in response to prolonged hypoxic exposure, we used
immunocytochemical analysis to detect subcellular distribu-
tion of cPKC α, βI, βII, and nPKCδ, ε, η, μ, θ isoforms in cultured
SH-SY5Y cells. Similar to the results from the Western blot
analysis, immunostaining showed that cPKCα (Fig. 3A), βI (Fig.
3C), βII (Fig. 3E), and nPKCε (Fig. 3G) localized mainly in the
cytosolic apartment of SH-SY5Y cells in the normoxic
condition. However, there was a translocation from the
cytosol to the perinuclear or membrane-related areas after
6 h posthypoxic exposure (Figs. 3B, D, F, and H). The immu-
nolabeled cPKCδ was shown as a representative of negative
result (Figs. 3I and J). These results provide histological
evidence that hypoxic stimulation may trigger the membrane
translocation of cPKC α, βI, βII, and nPKCε isoforms in SH-
SY5Y cell lines.
I/HPC is an endogenous strategy in which brief periods of
sublethal ischemia or hypoxia render neurons more resistant
to a subsequent ischemic or hypoxic damaging insult. Murry
Fig. 2 – Membrane translocation of nPKCε isoform was increased in SH-SY5Y cells following prolonged hypoxic exposure.
(A) Typical results of Western blot analysis showed the changes in membrane translocation of nPKCδ, ε, η, μ, and θ in SH-SY5Y
cells following sustained hypoxic exposure. (B) quantitative analysis demonstrated that membrane translocation levels of the
nPKCε, but not nPKCδ, η, μ, and θ, was significantly enhanced in a time-dependent manner compared to the value of the
control (0 group). *P < 0.05 vs. 0 group, n = 8.
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Fig. 3 – Effect of prolonged hypoxic exposure on the subcellular distribution of cPKCα, βI, and βII and nPKCε and δ isoforms in
SH-SY5Y cells. Immunostaining showed that immunolabeled cPKCα (A, ×400), βI (C, ×400), and βII (E, ×400) and nPKCε (G, ×400)
and δ (I, ×400) predominantly localized in the cytosol at the normoxic condition. After 6 h of posthypoxic exposure, cPKCα
(B, ×400), βI (D, ×400), and βII (F, ×400) and nPKCε (H, ×400), but not nPKCδ (J, ×400), translocated to the perinuclear or the
membrane-related areas.
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