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Lilly Y. W. Bourguignon Weiliang Xia Gabriel Wong 《The Journal of biological chemistry》2009,284(5):2657-2671
6.
Mikael K. Schnizler Katrin Schnizler Xiang-ming Zha Duane D. Hall John A. Wemmie Johannes W. Hell Michael J. Welsh 《The Journal of biological chemistry》2009,284(5):2697-2705
The acid-sensing ion channel 1a (ASIC1a) is widely expressed in central and
peripheral neurons where it generates transient cation currents when
extracellular pH falls. ASIC1a confers pH-dependent modulation on postsynaptic
dendritic spines and has critical effects in neurological diseases associated
with a reduced pH. However, knowledge of the proteins that interact with
ASIC1a and influence its function is limited. Here, we show that
α-actinin, which links membrane proteins to the actin cytoskeleton,
associates with ASIC1a in brain and in cultured cells. The interaction
depended on an α-actinin-binding site in the ASIC1a C terminus that was
specific for ASIC1a versus other ASICs and for α-actinin-1 and
-4. Co-expressing α-actinin-4 altered ASIC1a current density, pH
sensitivity, desensitization rate, and recovery from desensitization.
Moreover, reducing α-actinin expression altered acid-activated currents
in hippocampal neurons. These findings suggest that α-actinins may link
ASIC1a to a macromolecular complex in the postsynaptic membrane where it
regulates ASIC1a activity.Acid-sensing ion channels
(ASICs)2 are
H+-gated members of the DEG/ENaC family
(1–3).
Members of this family contain cytosolic N and C termini, two transmembrane
domains, and a large cysteine-rich extracellular domain. ASIC subunits combine
as homo- or heterotrimers to form cation channels that are widely expressed in
the central and peripheral nervous systems
(1–4).
In mammals, four genes encode ASICs, and two subunits, ASIC1 and ASIC2, have
two splice forms, a and b. Central nervous system neurons express ASIC1a,
ASIC2a, and ASIC2b
(5–7).
Homomeric ASIC1a channels are activated when extracellular pH drops below 7.2,
and half-maximal activation occurs at pH 6.5–6.8
(8–10).
These channels desensitize in the continued presence of a low extracellular
pH, and they can conduct Ca2+
(9,
11–13).
ASIC1a is required for acid-evoked currents in central nervous system neurons;
disrupting the gene encoding ASIC1a eliminates H+-gated currents
unless extracellular pH is reduced below pH 5.0
(5,
7).Previous studies found ASIC1a enriched in synaptosomal membrane fractions
and present in dendritic spines, the site of excitatory synapses
(5,
14,
15). Consistent with this
localization, ASIC1a null mice manifested deficits in hippocampal
long term potentiation, learning, and memory, which suggested that ASIC1a is
required for normal synaptic plasticity
(5,
16). ASICs might be activated
during neurotransmission when synaptic vesicles empty their acidic contents
into the synaptic cleft or when neuronal activity lowers extracellular pH
(17–19).
Ion channels, including those at the synapse often interact with multiple
proteins in a macromolecular complex that incorporates regulators of their
function (20,
21). For ASIC1a, only a few
interacting proteins have been identified. Earlier work indicated that ASIC1a
interacts with another postsynaptic scaffolding protein, PICK1
(15,
22,
23). ASIC1a also has been
reported to interact with annexin II light chain p11 through its cytosolic N
terminus to increase cell surface expression
(24) and with
Ca2+/calmodulin-dependent protein kinase II to phosphorylate the
channel (25). However, whether
ASIC1a interacts with additional proteins and with the cytoskeleton remain
unknown. Moreover, it is not known whether such interactions alter ASIC1a
function.In analyzing the ASIC1a amino acid sequence, we identified cytosolic
residues that might bind α-actinins. α-Actinins cluster membrane
proteins and signaling molecules into macromolecular complexes and link
membrane proteins to the actincytoskeleton (for review, Ref.
26). Four genes encode
α-actinin-1, -2, -3, and -4 isoforms. α-Actinins contain an
N-terminal head domain that binds F-actin, a C-terminal region containing two
EF-hand motifs, and a central rod domain containing four spectrin-like motifs
(26–28).
The C-terminal portion of the rod segment appears to be crucial for binding to
membrane proteins. The α-actinins assemble into antiparallel homodimers
through interactions in their rod domain. α-Actinins-1, -2, and -4 are
enriched in dendritic spines, concentrating at the postsynaptic membrane
(29–35).
In the postsynaptic membrane of excitatory synapses, α-actinin connects
the NMDA receptor to the actin cytoskeleton, and this interaction is key for
Ca2+-dependent inhibition of NMDA receptors
(36–38).
α-Actinins can also regulate the membrane trafficking and function of
several cation channels, including L-type Ca2+ channels,
K+ channels, and TRP channels
(39–41).To better understand the function of ASIC1a channels in macromolecular
complexes, we asked if ASIC1a associates with α-actinins. We were
interested in the α-actinins because they and ASIC1a, both, are present
in dendritic spines, ASIC1a contains a potential α-actinin binding
sequence, and the related epithelial Na+ channel (ENaC) interacts
with the cytoskeleton (42,
43). Therefore, we
hypothesized that α-actinin interacts structurally and functionally with
ASIC1a. 相似文献
7.
8.
Christopher P. Gayer Lakshmi S. Chaturvedi Shouye Wang David H. Craig Thomas Flanigan Marc D. Basson 《The Journal of biological chemistry》2009,284(4):2001-2011
The intestinal epithelium is repetitively deformed by shear, peristalsis,
and villous motility. Such repetitive deformation stimulates the proliferation
of intestinal epithelial cells on collagen or laminin substrates via ERK, but
the upstream mediators of this effect are poorly understood. We hypothesized
that the phosphatidylinositol 3-kinase (PI3K)/AKT cascade mediates this
mitogenic effect. PI3K, AKT, and glycogen synthase kinase-3β
(GSK-3β) were phosphorylated by 10 cycles/min strain at an average 10%
deformation, and pharmacologic blockade of these molecules or reduction by
small interfering RNA (siRNA) prevented the mitogenic effect of strain in
Caco-2 or IEC-6 intestinal epithelial cells. Strain MAPK activation required
PI3K but not AKT. AKT isoform-specific siRNA transfection demonstrated that
AKT2 but not AKT1 is required for GSK-3β phosphorylation and the strain
mitogenic effect. Furthermore, overexpression of AKT1 or an AKT chimera
including the PH domain and hinge region of AKT2 and the catalytic domain and
C-tail of AKT1 prevented strain activation of GSK-3β, but overexpression
of AKT2 or a chimera including the PH domain and hinge region of AKT1 and the
catalytic domain and C-tail of AKT2 did not. These data delineate a role for
PI3K, AKT2, and GSK-3β in the mitogenic effect of strain. PI3K is
required for both ERK and AKT2 activation, whereas AKT2 is sequentially
required for GSK-3β. Furthermore, AKT2 specificity requires its catalytic
domain and tail region. Manipulating this pathway may prevent mucosal atrophy
and maintain the mucosal barrier in conditions such as ileus, sepsis, and
prolonged fasting when peristalsis and villous motility are decreased and the
mucosal barrier fails.Mechanical forces are part of the normal intestinal epithelial environment.
Numerous different forces deform these cells including shear stress from
endoluminal chyme, bowel peristalsis, and villous motility
(1,
2). During normal bowel
function the mucosa is subjected to injury that must be repaired to maintain
the mucosal barrier (3,
4). Deformation patterns of the
bowel are altered in conditions such as prolonged fasting, post-surgical
ileus, and sepsis states, resulting in profoundly reduced mucosal deformation.
When such states are prolonged, proliferation slows, the mucosa becomes
atrophic, and bacterial translocation may ensue as the mucosal barrier of the
gut breaks down
(5–7).In vitro, repetitive deformation is trophic for intestinal
epithelial cells (8) cultured
on type I or type IV collagen or laminin. Human Caco-2 intestinal epithelial
cells (9), non-transformed rat
IEC-6 intestinal epithelial cells
(10), and primary human
intestinal epithelial cells isolated from surgical specimens
(11) proliferate more rapidly
in response to cyclic strain
(12) unless substantial
quantities of fibronectin are added to the media or matrix
(11) to mimic the acute phase
reaction of acute or chronic inflammation and injury. Cyclic strain also
stimulates proliferation in HCT 116 colon cancer cells
(13) and differentiation of
Caco-2 cells cultured on a collagen substrate
(9). This phenomenon has also
been observed in vivo
(14). Thus, repetitive
deformation may help to maintain the normal homeostasis of the gut mucosa
under non-inflammatory conditions. Previous work in our laboratory has
implicated Src, focal adhesion kinase, and the mitogen-activated protein
kinase (MAPK)2
extracellular signal-related kinase (ERK) in the mitogenic effect of strain
(10). Although p38 is also
activated in Caco-2 cells subjected to cyclic strain on a collagen matrix, its
activity is not required for the mitogenic effect of strain
(12).Although often the PI3K/AKT pathway is thought of as a parallel pathway to
the MAPK, this is not always the case. Protein kinase C isoenzymes
differentially modulate thrombin effect on MAPK-dependent retinal pigment
epithelial cell (RPE) proliferation, and it has been shown that PI3K or AKT
inhibition prevented thrombin-induced ERK activation and RPE proliferation
(15).PI3K, AKT, and glycogen synthase kinase (GSK), a downstream target of AKT
(16), have been implemented in
intestinal epithelial cell proliferation in numerous cell systems not
involving strain
(17–19)
including uncontrolled proliferation in gastrointestinal cancers
(20–22).
Mechanical forces activate this pathway as well. PI3K and AKT are required for
increased extracellular pressure to stimulate colon cancer cell adhesion
(23), although the pathway by
which pressure stimulates colon cancer cells in suspension differs from the
response of adherent intestinal epithelial cells to repetitive deformation
(24), and GSK is not involved
in this effect.3
Repetitive strain also stimulates vascular endothelial cell proliferation via
PI3K and AKT (25,
26), whereas respiratory
strain stimulates angiogenic responses via PI3K
(27). We, therefore,
hypothesized that the PI3K/AKT/GSK axis would be involved in the mitogenic
effects of repetitive deformation on a collagen matrix.To test this hypothesis, we used the Flexcell apparatus to rhythmically
deform Caco-2 intestinal epithelial cells. IEC-6 cells were used to confirm
key results. A frequency of 10 cycles per min was used, which is similar in
order of magnitude to the frequency that the intestinal mucosa might be
deformed by peristalsis or villous motility in vivo
(28,
29). Mechanical forces such as
repetitive deformation are likely cell-type and frequency-specific, as
different cell types respond to different frequencies. Vascular endothelial
cells respond to frequencies of 60–80 cycles/min
(25), whereas intestinal
epithelial cells may actually decrease proliferation in response to
frequencies of 5 cycles/min
(30). We characterized PI3K,
AKT, and GSK phosphorylation with strain, blocked these molecules
pharmacologically or by siRNA, and delineated the specificity of the AKT
effect using isozyme-specific siRNA and transfection of AKT1/2 chimeras. We
also characterized the interaction of this pathway with the activation of ERK
by strain, which has previously been implicated in the mitogenic response
(12). 相似文献
9.
Jacamo R Sinnett-Smith J Rey O Waldron RT Rozengurt E 《The Journal of biological chemistry》2008,283(19):12877-12887
Protein kinase D (PKD) is a serine/threonine protein kinase rapidly
activated by G protein-coupled receptor (GPCR) agonists via a protein kinase C
(PKC)-dependent pathway. Recently, PKD has been implicated in the regulation
of long term cellular activities, but little is known about the mechanism(s)
of sustained PKD activation. Here, we show that cell treatment with the
preferential PKC inhibitors GF 109203X or Gö 6983 blocked rapid
(1–5-min) PKD activation induced by bombesin stimulation, but this
inhibition was greatly diminished at later times of bombesin stimulation
(e.g. 45 min). These results imply that GPCR-induced PKD activation
is mediated by early PKC-dependent and late PKC-independent mechanisms.
Western blot analysis with site-specific antibodies that detect the
phosphorylated state of the activation loop residues Ser744 and
Ser748 revealed striking PKC-independent phosphorylation of
Ser748 as well as Ser744 phosphorylation that remained
predominantly but not completely PKC-dependent at later times of bombesin or
vasopressin stimulation (20–90 min). To determine the mechanisms
involved, we examined activation loop phosphorylation in a set of PKD mutants,
including kinase-deficient, constitutively activated, and PKD forms in which
the activation loop residues were substituted for alanine. Our results show
that PKC-dependent phosphorylation of the activation loop Ser744
and Ser748 is the primary mechanism involved in early phase PKD
activation, whereas PKD autophosphorylation on Ser748 is a major
mechanism contributing to the late phase of PKD activation occurring in cells
stimulated by GPCR agonists. The present studies identify a novel mechanism
induced by GPCR activation that leads to late, PKC-independent PKD
activation.A rapid increase in the synthesis of lipid-derived second messengers with
subsequent activation of protein phosphorylation cascades has emerged as a
fundamental signal transduction mechanism triggered by multiple extracellular
stimuli, including hormones, neurotransmitters, chemokines, and growth factors
(1). Many of these agonists
bind to G protein-coupled receptors
(GPCRs),4 activate
heterotrimeric G proteins and stimulate isoforms of the phospholipase C
family, including β, γ, δ, and ε (reviewed in Refs.
1 and
2). Activated phospholipase Cs
catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate to produce
the second messengers inositol 1,4,5-trisphosphate and diacylglycerol (DAG).
Inositol 1,4,5-trisphosphate mobilizes Ca2+ from intracellular
stores (3,
4) whereas DAG directly
activates the classic (α, β, and γ) and novel (δ,
ε, η, and θ) isoforms of PKC
(5–7).
Although it is increasingly recognized that each PKC isozyme has specific
functions in vivo
(5–8),
the mechanisms by which PKC-mediated signals are propagated to critical
downstream targets remain incompletely defined.PKD, also known initially as PKCμ
(9,
10), and two recently
identified serine protein kinases termed PKD2
(11) and PKCν/PKD3
(12,
13), which are similar in
overall structure and primary amino acid sequence to PKD
(14), constitute a new protein
kinase family within the Ca2+/calmodulin-dependent protein kinase
group (15) and separate from
the previously identified PKCs
(14). Salient features of PKD
structure include an N-terminal regulatory region containing a tandem repeat
of cysteine-rich zinc finger-like motifs (termed the cysteine-rich domain)
that confers high affinity binding to phorbol esters and DAG
(9,
16,
17), followed by a pleckstrin
homology (PH) domain that negatively regulates catalytic activity
(18,
19). The C-terminal region of
the PKDs contains its catalytic domain, which is distantly related to
Ca2+-regulated kinases.In unstimulated cells, PKD is in a state of low kinase catalytic activity
maintained by the N-terminal domain, which represses the catalytic activity of
the enzyme by autoinhibition. Consistent with this model, deletions or single
amino acid substitutions in the PH domain result in constitutive kinase
activity
(18–20).
Physiological activation of PKD within cells occurs via a
phosphorylation-dependent mechanism first identified in our laboratory
(21). In response to cellular
stimuli, PKD is converted from a low activity form into a persistently active
form that is retained during isolation from cells, as shown by in
vitro kinase assays performed in the absence of lipid co-activators
(21,
22). PKD activation has been
demonstrated in response to engagement of specific GPCRs either by regulatory
peptides
(23–30)
or lysophosphatidic acid (27,
31,
32); signaling through
Gq, G12, Gi, and Rho
(27,
31–34);
activation of receptor tyrosine kinases, such as the platelet-derived growth
factor receptor (23,
35,
36); cross-linking of B-cell
receptor and T-cell receptor in B and T lymphocytes, respectively
(37–40);
and oxidative stress
(41–44).Throughout these studies, multiple lines of evidence indicated that PKC
activity is necessary for rapid PKD activation within intact cells. For
example, rapid PKD activation was selectively and potently blocked by cell
treatment with preferential PKC inhibitors (e.g. GF 109203X or
Gö 6983) that do not directly inhibit PKD catalytic activity
(21,
22), implying that PKD
activation in intact cells is mediated, directly or indirectly, through PKCs.
In line with this conclusion, cotransfection of PKD with active mutant forms
of “novel” PKCs (PKCs δ, ε, η, and θ)
resulted in robust PKD activation in the absence of cell stimulation
(21,
44–46).
Many reports demonstrated the operation of a rapid PKC/PKD signaling cascade
in response to multiple GPCR agonists in a broad range of cell types,
including normal and cancer cells (reviewed in Ref.
14). Our previous studies
identified Ser744 and Ser748 in the PKD activation loop
(also referred as the activation segment or T-loop) as phosphorylation sites
critical for PKC-mediated PKD activation (reviewed in Ref.
14). Collectively, these
findings demonstrated the existence of rapidly activated PKC-PKD protein
kinase cascade(s) and raised the possibility that some PKC-dependent
biological responses involve PKD acting as a downstream effector.PKD has been reported recently to mediate several important cellular
activities and processes, including signal transduction
(30,
47–49),
chromatin modification (50),
Golgi organization and function
(51,
52), c-Jun function
(47,
53,
54), NFκB-mediated gene
expression (43,
55,
56), and cell survival,
migration, and differentiation and DNA synthesis and proliferation (reviewed
in Ref. 14). Thus, mounting
evidence indicates that PKD has a remarkable diversity of both its signal
generation and distribution and its potential for complex regulatory
interactions with multiple downstream pathways, leading to multiple responses,
including long term cellular events. Despite increasing recognition of its
importance, very little is known about the mechanism(s) of sustained PKD
activation as opposed to the well documented rapid, PKC-dependent PKD
activation.The results presented here demonstrate that prolonged GPCR-induced PKD
activation is mediated by sequential PKC-dependent and PKC-independent phases
of regulation. We report here, for the first time, that PKD
autophosphorylation on Ser748 is a major mechanism contributing to
the late phase of PKD activation occurring in cells stimulated by GPCR
agonists. The present studies expand previous models of PKD regulation by
identifying a novel mechanism induced by GPCR activation that leads to late,
PKC-independent PKD activation. 相似文献
10.
11.
12.
Christian Rosker Gargi Meur Emily J. A. Taylor Colin W. Taylor 《The Journal of biological chemistry》2009,284(8):5186-5194
Ryanodine receptors (RyR) are Ca2+ channels that mediate
Ca2+ release from intracellular stores in response to diverse
intracellular signals. In RINm5F insulinoma cells, caffeine, and
4-chloro-m-cresol (4CmC), agonists of RyR, stimulated Ca2+
entry that was independent of store-operated Ca2+ entry, and
blocked by prior incubation with a concentration of ryanodine that inactivates
RyR. Patch-clamp recording identified small numbers of large-conductance
(γK = 169 pS) cation channels that were activated by
caffeine, 4CmC or low concentrations of ryanodine. Similar channels were
detected in rat pancreatic β-cells. In RINm5F cells, the channels were
blocked by cytosolic, but not extracellular, ruthenium red. Subcellular
fractionation showed that type 3 IP3 receptors (IP3R3)
were expressed predominantly in endoplasmic reticulum, whereas RyR2 were
present also in plasma membrane fractions. Using RNAi selectively to reduce
expression of RyR1, RyR2, or IP3R3, we showed that RyR2 mediates
both the Ca2+ entry and the plasma membrane currents evoked by
agonists of RyR. We conclude that small numbers of RyR2 are selectively
expressed in the plasma membrane of RINm5F pancreatic β-cells, where they
mediate Ca2+ entry.Ryanodine receptors
(RyR)3 and inositol
1,4,5-trisphosphate receptors (IP3R)
(1,
2) are the archetypal
intracellular Ca2+ channels. Both are widely expressed, although
RyR are more restricted in their expression than IP3R
(3,
4). In common with many cells,
pancreatic β-cells and insulin-secreting cell lines express both
IP3R (predominantly IP3R3)
(5,
6) and RyR (predominantly RyR2)
(7). Both RyR and
IP3R are expressed mostly within membranes of the endoplasmic (ER),
where they mediate release of Ca2+. Functional RyR are also
expressed in the secretory vesicles
(8,
9) or, and perhaps more likely,
in the endosomes of β-cells
(10). Despite earlier
suggestions (11),
IP3R are probably not present in the secretory vesicles of
β-cells (8,
12,
13).All three subtypes of IP3R are stimulated by IP3 with
Ca2+ (1), and the
three subtypes of RyR are each directly regulated by Ca2+. However,
RyR differ in whether their most important physiological stimulus is
depolarization of the plasma membrane (RyR1), Ca2+ (RyR2) or
additional intracellular messengers like cyclic ADP-ribose. The latter
stimulates both Ca2+ release and insulin secretion in β-cells
(8,
14). The activities of both
families of intracellular Ca2+ channels are also modulated by many
additional signals that act directly or via phosphorylation
(15,
16). Although they commonly
mediate release of Ca2+ from the ER, both IP3R and RyR
select rather poorly between Ca2+ and other cations (permeability
ratio, PCa/PK ∼7)
(1,
17). This may allow
electrogenic Ca2+ release from the ER to be rapidly compensated by
uptake of K+ (18),
and where RyR or IP3R are expressed in other membranes it may allow
them to affect membrane potential.Both Ca2+ entry and release of Ca2+ from
intracellular stores contribute to the oscillatory increases in cytosolic
Ca2+ concentration ([Ca2+]i) that
stimulate exocytosis of insulin-containing vesicles in pancreatic β-cells
(7). Glucose rapidly
equilibrates across the plasma membrane (PM) of β-cells and its oxidative
metabolism by mitochondria increases the cytosolic ATP/ADP ratio, causing
KATP channels to close
(19). This allows an
unidentified leak current to depolarize the PM
(20) and activate
voltage-gated Ca2+ channels, predominantly L-type Ca2+
channels (21). The resulting
Ca2+ entry is amplified by Ca2+-induced Ca2+
release from intracellular stores
(7), triggering exocytotic
release of insulin-containing dense-core vesicles
(22). The importance of this
sequence is clear from the widespread use of sulfonylurea drugs, which close
KATP channels, in the treatment of type 2 diabetes. Ca2+
uptake by mitochondria beneath the PM further stimulates ATP production,
amplifying the initial response to glucose and perhaps thereby contributing to
the sustained phase of insulin release
(23). However, neither the
increase in [Ca2+]i nor the insulin release
evoked by glucose or other nutrients is entirely dependent on Ca2+
entry (7,
24) or closure of
KATP channels (25).
This suggests that glucose metabolism may also more directly activate RyR
(7,
26) and/or IP3R
(27) to cause release of
Ca2+ from intracellular stores. A change in the ATP/ADP ratio is
one means whereby nutrient metabolism may be linked to opening of
intracellular Ca2+ channels because both RyR
(28) and IP3R
(1) are stimulated by ATP.The other major physiological regulators of insulin release are the
incretins: glucagon-like peptide-1 and glucose-dependent insulinotropic
hormone (29). These hormones,
released by cells in the small intestine, stimulate synthesis of cAMP in
β-cells and thereby potentiate glucose-evoked insulin release
(30). These pathways are also
targets of drugs used successfully to treat type 2 diabetes
(29). The responses of
β-cells to cAMP involve both cAMP-dependent protein kinase and epacs
(exchange factors activated by cAMP)
(31,
32). The effects of the latter
are, at least partly, due to release of Ca2+ from intracellular
stores via RyR
(33–35)
and perhaps also via IP3R
(36). The interplays between
Ca2+ and cAMP signaling generate oscillatory changes in the
concentrations of both messengers
(37). RyR and IP3R
are thus implicated in mediating responses to each of the major physiological
regulators of insulin secretion: glucose and incretins.Here we report that in addition to expression in intracellular stores,
which probably include both the ER and secretory vesicles and/or endosomes,
functional RyR2 are also expressed in small numbers in the PM of RINm5F
insulinoma cells and rat pancreatic β-cells. 相似文献
13.
14.
15.
16.
17.
18.
Kuen-Feng Chen Pei-Yen Yeh Chiun Hsu Chih-Hung Hsu Yen-Shen Lu Hsing-Pang Hsieh Pei-Jer Chen Ann-Lii Cheng 《The Journal of biological chemistry》2009,284(17):11121-11133
Hepatocellular carcinoma (HCC) is one of the most common and aggressive
human malignancies. Recombinant tumor necrosis factor-related
apoptosis-inducing ligand (TRAIL) is a promising anti-tumor agent. However,
many HCC cells show resistance to TRAIL-induced apoptosis. In this study, we
showed that bortezomib, a proteasome inhibitor, overcame TRAIL resistance in
HCC cells, including Huh-7, Hep3B, and Sk-Hep1. The combination of bortezomib
and TRAIL restored the sensitivity of HCC cells to TRAIL-induced apoptosis.
Comparing the molecular change in HCC cells treated with these agents, we
found that down-regulation of phospho-Akt (P-Akt) played a key role in
mediating TRAIL sensitization of bortezomib. The first evidence was that
bortezomib down-regulated P-Akt in a dose- and time-dependent manner in
TRAIL-treated HCC cells. Second, , a PI3K inhibitor, also sensitized
resistant HCC cells to TRAIL-induced apoptosis. Third, knocking down Akt1 by
small interference RNA also enhanced TRAIL-induced apoptosis in Huh-7 cells.
Finally, ectopic expression of mutant Akt (constitutive active) in HCC cells
abolished TRAIL sensitization effect of bortezomib. Moreover, okadaic acid, a
protein phosphatase 2A (PP2A) inhibitor, reversed down-regulation of P-Akt in
bortezomib-treated cells, and PP2A knockdown by small interference RNA also
reduced apoptosis induced by the combination of TRAIL and bortezomib,
indicating that PP2A may be important in mediating the effect of bortezomib on
TRAIL sensitization. Together, bortezomib overcame TRAIL resistance at
clinically achievable concentrations in hepatocellular carcinoma cells, and
this effect is mediated at least partly via inhibition of the PI3K/Akt
pathway.Hepatocellular carcinoma
(HCC) LY2940022 is currently
the fifth most common solid tumor worldwide and the fourth leading cause of
cancer-related death. To date, surgery is still the only curative treatment
but is only feasible in a small portion of patients
(1). Drug treatment is the
major therapy for patients with advanced stage disease. Unfortunately, the
response rate to traditional chemotherapy for HCC patients is unsatisfactory
(1). Novel pharmacological
therapy is urgently needed for patients with advanced HCC. In this regard, the
approval of sorafenib might open a new era of molecularly targeted therapy in
the treatment of HCC patients.Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a
type II transmembrane protein and a member of the TNF family, is a promising
anti-tumor agent under clinical investigation
(2). TRAIL functions by
engaging its receptors expressed on the surface of target cells. Five
receptors specific for TRAIL have been identified, including DR4/TRAIL-R1,
DR5/TRAIL-R2, DcR1, DcR2, and osteoprotegerin. Among TRAIL receptors, only DR4
and DR5 contain an effective death domain that is essential to formation of
death-inducing signaling complex (DISC), a critical step for TRAIL-induced
apoptosis. Notably, the trimerization of the death domains recruits an adaptor
molecule, Fas-associated protein with death domain (FADD), which subsequently
recruits and activates caspase-8. In type I cells, activation of caspase-8 is
sufficient to activate caspase-3 to induce apoptosis; however, in another type
of cells (type II), the intrinsic mitochondrial pathway is essential for
apoptosis characterized by cleavage of Bid and release of cytochrome
c from mitochondria, which subsequently activates caspase-9 and
caspase-3 (3).Although TRAIL induces apoptosis in malignant cells but sparing normal
cells, some tumor cells are resistant to TRAIL-induced apoptosis. Mechanisms
responsible for the resistance include receptors and intracellular resistance.
Although the cell surface expression of DR4 or DR5 is absolutely required for
TRAIL-induced apoptosis, tumor cells expressing these death receptors are not
always sensitive to TRAIL due to intracellular mechanisms. For example, the
cellular FLICE-inhibitory protein (c-FLIP), a homologue to caspase-8 but
without protease activity, has been linked to TRAIL resistance in several
studies (4,
5). In addition, inactivation
of Bax, a proapoptotic Bcl-2 family protein, resulted in resistance to TRAIL
in MMR-deficient tumors (6,
7), and reintroduction of Bax
into Bax-deficient cells restored TRAIL sensitivity
(8), indicating that the Bcl-2
family plays a critical role in intracellular mechanisms for resistance of
TRAIL.Bortezomib, a proteasome inhibitor approved clinically for multiple myeloma
and mantle cell lymphoma, has been investigated intensively for many types of
cancer (9). Accumulating
studies indicate that the combination of bortezomib and TRAIL overcomes the
resistance to TRAIL in various types of cancer, including acute myeloid
leukemia (4), lymphoma
(10–13),
prostate
(14–17),
colon (15,
18,
19), bladder
(14,
16), renal cell carcinoma
(20), thyroid
(21), ovary
(22), non-small cell lung
(23,
24), sarcoma
(25), and HCC
(26,
27). Molecular targets
responsible for the sensitizing effect of bortezomib on TRAIL-induced cell
death include DR4 (14,
27), DR5
(14,
20,
22–23,
28), c-FLIP
(4,
11,
21–23,
29), NF-κB
(12,
24,
30), p21
(16,
21,
25), and p27
(25). In addition, Bcl-2
family also plays a role in the combinational effect of bortezomib and TRAIL,
including Bcl-2 (10,
21), Bax
(13,
22), Bak
(27), Bcl-xL
(21), Bik
(18), and Bim
(15).Recently, we have reported that Akt signaling is a major molecular
determinant in bortezomib-induced apoptosis in HCC cells
(31). In this study, we
demonstrated that bortezomib overcame TRAIL resistance in HCC cells through
inhibition of the PI3K/Akt pathway. 相似文献
19.
20.
The inhalation anesthetic desflurane induces caspase activation and increases amyloid beta-protein levels under hypoxic conditions 总被引:1,自引:0,他引:1
Zhang B Dong Y Zhang G Moir RD Xia W Yue Y Tian M Culley DJ Crosby G Tanzi RE Xie Z 《The Journal of biological chemistry》2008,283(18):11866-11875
Perioperative factors including hypoxia, hypocapnia, and certain
anesthetics have been suggested to contribute to Alzheimer disease (AD)
neuropathogenesis. Desflurane is one of the most commonly used inhalation
anesthetics. However, the effects of desflurane on AD neuropathogenesis have
not been previously determined. Here, we set out to assess the effects of
desflurane and hypoxia on caspase activation, amyloid precursor protein (APP)
processing, and amyloid β-protein (Aβ) generation in H4 human
neuroglioma cells (H4 naïve cells) as well as those overexpressing APP
(H4-APP cells). Neither 12% desflurane nor hypoxia (18% O2) alone
affected caspase-3 activation, APP processing, and Aβ generation.
However, treatment with a combination of 12% desflurane and hypoxia (18%
O2) (desflurane/hypoxia) for 6 h induced caspase-3 activation,
altered APP processing, and increased Aβ generation in H4-APP cells.
Desflurane/hypoxia also increased levels of β-site APP-cleaving enzyme in
H4-APP cells. In addition, desflurane/hypoxia-induced Aβ generation could
be reduced by the broad caspase inhibitor benzyloxycarbonyl-VAD. Finally, the
Aβ aggregation inhibitor clioquinol and γ-secretase inhibitor
L-685,458 attenuated caspase-3 activation induced by desflurane/hypoxia. In
summary, desflurane can induce Aβ production and caspase activation, but
only in the presence of hypoxia. Pending in vivo confirmation, these
data may have profound implications for anesthesia care in elderly patients,
and especially those with AD.An estimated 200 million patients worldwide undergo surgery each year.
Several reports have suggested that anesthesia and surgery may facilitate
development of Alzheimer disease
(AD)4
(1–3).
A recent study also reported that patients having coronary artery bypass graft
surgery under general anesthesia are at increased risk for AD as compared with
those having percutaneous transluminal coronary angioplasty under local
anesthesia (4).Genetic evidence, confirmed by neuropathological and biochemical findings,
indicates that excessive production and/or accumulation of amyloid
β-protein (Aβ) play a fundamental role in the pathology of AD
(reviewed in Refs. 5 and
6). Aβ is produced via
serial proteolysis of amyloid precursor protein (APP) by aspartyl protease
β-site APP-cleaving enzyme (BACE), or β-secretase,
andγ-secretase. BACE cleaves APP to generate a 99-residue
membrane-associated C terminus fragment (APP-C99). APP-C99 is further cleaved
by γ-secretase to release 4-kDa Aβ and β-amyloid precursor
protein intracellular domain
(7–9).
Presenilin and γ-secretase co-fractionate as a detergent-sensitive, high
molecular weight complex (10)
that includes at least three other proteins, nicastrin/APH-2, APH-1, and
PEN-2, all of which are necessary and sufficient for γ-secretase
activity
(11–13).
Increasing evidence indicates that apoptosis is associated with a variety of
neurodegenerative disorders, including AD (Refs.
14–17;
reviewed in Ref. 18). Aβ
has been shown to cause caspase activation and apoptosis, which can in turn
potentiate Aβ generation
(16,
19–28).
Finally, fibrillar aggregates of Aβ and oligomeric species of Aβ are
more neurotoxic
(29–37).Perioperative factors, including hypoxia
(38–42),
hypocapnia (43), and
anesthetics
(44–47),
have been reported to potentially contribute to AD neuropathogenesis. These
perioperative factors may also cause post-operative cognitive dysfunction, a
dementia associated with surgery and anesthesia, by triggering AD
neuropathogenesis.Isoflurane, sevoflurane, and desflurane are the most commonly used
inhalation anesthetics. It has been reported that isoflurane enhances the
oligomerization and cytotoxicity of Aβ
(44) and induces apoptosis
(48–51).
Our recent studies have shown that a clinically relevant concentration of
isoflurane can lead to caspase-3 activation, decrease cell viability, alter
APP processing, and increase Aβ generation in human H4 neuroglioma cells
overexpressing human APP
(45–47).
Loop et al. (49)
reported that isoflurane and sevoflurane, but not desflurane, can induce
caspase activation and apoptosis in human T lymphocytes. However, effects of
desflurane and desflurane plus other perioperative risk factors, e.g.
hypoxia, on APP processing and Aβ generation have not been assessed.In the present study, we set out to determine effects of desflurane,
hypoxia, and the combination of the two (desflurane/hypoxia) on caspase-3
activation, APP processing, and Aβ generation in H4 human neuroglioma
cells (H4 naïve cells) and H4 naïve cells stably transfected to
express full-length (FL) APP (H4-APP cells). We also investigated whether the
caspase inhibitor, Z-VAD, the γ-secretase inhibitor L-685,458, and the
Aβ aggregation inhibitor clioquinol could attenuate
desflurane/hypoxia-induced caspase-3 activation and Aβ generation. 相似文献