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1.
2.
Zhemin Zhou Yoshiteru Hashimoto Michihiko Kobayashi 《The Journal of biological chemistry》2009,284(22):14930-14938
3.
Matthias Gralle Michelle Gralle Botelho Fred S. Wouters 《The Journal of biological chemistry》2009,284(22):15016-15025
The amyloid precursor protein (APP) is implied both in cell growth and
differentiation and in neurodegenerative processes in Alzheimer disease.
Regulated proteolysis of APP generates biologically active fragments such as
the neuroprotective secreted ectodomain sAPPα and the neurotoxic
β-amyloid peptide. Furthermore, it has been suggested that the intact
transmembrane APP plays a signaling role, which might be important for both
normal synaptic plasticity and neuronal dysfunction in dementia. To understand
APP signaling, we tracked single molecules of APP using quantum dots and
quantitated APP homodimerization using fluorescence lifetime imaging
microscopy for the detection of Förster resonance energy transfer in
living neuroblastoma cells. Using selective labeling with synthetic
fluorophores, we show that the dimerization of APP is considerably higher at
the plasma membrane than in intracellular membranes. Heparan sulfate
significantly contributes to the almost complete dimerization of APP at the
plasma membrane. Importantly, this technique for the first time structurally
defines the initiation of APP signaling by binding of a relevant physiological
extracellular ligand; our results indicate APP as receptor for neuroprotective
sAPPα, as sAPPα binding disrupts APP dimers, and this disruption
of APP dimers by sAPPα is necessary for the protection of neuroblastoma
cells against starvation-induced cell death. Only cells expressing reversibly
dimerized wild-type, but not covalently dimerized mutant APP are protected by
sAPPα. These findings suggest a potentially beneficial effect of
increasing sAPPα production or disrupting APP dimers for neuronal
survival.The amyloid precursor protein
(APP)4 is known both
for its important role in the development and plasticity of the nervous system
(1–6)
and for its involvement in Alzheimer disease (AD)
(7,
8). Despite intensive research
efforts, the initial events that lead to the prevalent sporadic, i.e.
non-familial, forms of AD are still unclear. Furthermore, although a higher
gene dose of APP (9) or the
presence of pathological APP mutations is sufficient to induce familial AD
(for review, see Ref. 10), the
exact pathological mechanism that is triggered by APP is still under
debate.Some fragments of APP, such as the β-amyloid peptide (Aβ), are
thought to contribute to synaptic dysfunction and neurotoxicity
(11,
12). On the other hand, the
α-secretase-derived extracellular fragment of APP (sAPPα), which
is present at lower levels in AD patients than in controls
(13), has been shown to be
beneficial for memory function, to possess neuroprotective properties, and to
counteract the effects of Aβ
(14–18).Signaling by transmembrane APP may directly contribute to neurodegeneration
in AD
(19–24);
however, the signal transduction pathway for transmembrane APP remains
unknown, although several potential regulatory proteins, glycosaminoglycans,
and metal ions are known to bind with high affinity to APP and sAPPα
(25,
26). The most common form of
signal transduction for single-pass transmembrane proteins is the
ligand-induced perturbation of a monomer/dimer equilibrium. Indeed, the
dimerization of transmembrane APP has been implied several times in the past.
Several studies have investigated the effects of presumed dimer-breaking
perturbations on biological read-outs, such as the production of Aβ
(27,
28), but without directly
measuring the APP aggregation state, or have investigated the aggregation
state of APP subdomains, often reconstituted in cell-free systems
(27–32).
Dimerization interfaces in both the extracellular and the transmembrane domain
have been suggested.In the studies investigating the aggregation state of full-length APP, most
of the employed methods, such as chemical cross-linking and
co-immunoprecipitation, do not lend themselves readily to a rigorous
quantitative analysis of the abundance of potentially instable dimers
(31,
33), whereas in other cases
the use of chimeras may have influenced the dimerization potential or
precluded the search for a natural stimulus
(23,
34). The only previously
reported direct observation of APP dimerization by Förster resonance
energy transfer (FRET) microscopy uses an assay in which the FRET efficiency
varies with the level of overexpression
(35). Therefore, a
concentration-dependent FRET component due to nonspecific stochastic
encounters cannot be excluded in this study.Most importantly, as none of the published procedures permitted the
selective detection of APP dimers on the surface of live cells, where they
would encounter ligands, they could not differentiate between subpopulations
of APP. This may be one reason why no natural ligand of APP has ever been
shown to signal via modulation of its monomer/dimer equilibrium.Another elusive goal is the identity of the receptor for neuroprotective
sAPPα
(36–39).
The ligand-dependent dimerization of sAPPα in solution
(40) and its origination from
transmembrane APP suggest that APP might serve as receptor for sAPPα,
but this binding has never been experimentally shown. 相似文献
4.
5.
Benjamin E. L. Lauffer Stanford Chen Cristina Melero Tanja Kortemme Mark von Zastrow Gabriel A. Vargas 《The Journal of biological chemistry》2009,284(4):2448-2458
Many G protein-coupled receptors (GPCRs) recycle after agonist-induced
endocytosis by a sequence-dependent mechanism, which is distinct from default
membrane flow and remains poorly understood. Efficient recycling of the
β2-adrenergic receptor (β2AR) requires a C-terminal PDZ
(PSD-95/Discs Large/ZO-1) protein-binding determinant (PDZbd), an intact actin
cytoskeleton, and is regulated by the endosomal protein Hrs (hepatocyte growth
factor-regulated substrate). The PDZbd is thought to link receptors to actin
through a series of protein interaction modules present in NHERF/EBP50
(Na+/H+ exchanger 3 regulatory factor/ezrin-binding phosphoprotein
of 50 kDa) family and ERM (ezrin/radixin/moesin) family proteins. It is not
known, however, if such actin connectivity is sufficient to recapitulate the
natural features of sequence-dependent recycling. We addressed this question
using a receptor fusion approach based on the sufficiency of the PDZbd to
promote recycling when fused to a distinct GPCR, the δ-opioid receptor,
which normally recycles inefficiently in HEK293 cells. Modular domains
mediating actin connectivity promoted receptor recycling with similarly high
efficiency as the PDZbd itself, and recycling promoted by all of the domains
was actin-dependent. Regulation of receptor recycling by Hrs, however, was
conferred only by the PDZbd and not by downstream interaction modules. These
results suggest that actin connectivity is sufficient to mimic the core
recycling activity of a GPCR-linked PDZbd but not its cellular regulation.G protein-coupled receptors
(GPCRs)2 comprise the
largest family of transmembrane signaling receptors expressed in animals and
transduce a wide variety of physiological and pharmacological information.
While these receptors share a common 7-transmembrane-spanning topology,
structural differences between individual GPCR family members confer diverse
functional and regulatory properties
(1-4).
A fundamental mechanism of GPCR regulation involves agonist-induced
endocytosis of receptors via clathrin-coated pits
(4). Regulated endocytosis can
have multiple functional consequences, which are determined in part by the
specificity with which internalized receptors traffic via divergent downstream
membrane pathways
(5-7).Trafficking of internalized GPCRs to lysosomes, a major pathway traversed
by the δ-opioid receptor (δOR), contributes to proteolytic
down-regulation of receptor number and produces a prolonged attenuation of
subsequent cellular responsiveness to agonist
(8,
9). Trafficking of internalized
GPCRs via a rapid recycling pathway, a major route traversed by the
β2-adrenergic receptor (β2AR), restores the complement of functional
receptors present on the cell surface and promotes rapid recovery of cellular
signaling responsiveness (6,
10,
11). When co-expressed in the
same cells, the δOR and β2AR are efficiently sorted between these
divergent downstream membrane pathways, highlighting the occurrence of
specific molecular sorting of GPCRs after endocytosis
(12).Recycling of various integral membrane proteins can occur by default,
essentially by bulk membrane flow in the absence of lysosomal sorting
determinants (13). There is
increasing evidence that various GPCRs, such as the β2AR, require
distinct cytoplasmic determinants to recycle efficiently
(14). In addition to requiring
a cytoplasmic sorting determinant, sequence-dependent recycling of the
β2AR differs from default recycling in its dependence on an intact actin
cytoskeleton and its regulation by the conserved endosomal sorting protein Hrs
(hepatocyte growth factor receptor substrate)
(11,
14). Compared with the present
knowledge regarding protein complexes that mediate sorting of GPCRs to
lysosomes (15,
16), however, relatively
little is known about the biochemical basis of sequence-directed recycling or
its regulation.The β2AR-derived recycling sequence conforms to a canonical PDZ
(PSD-95/Discs Large/ZO-1) protein-binding determinant (henceforth called
PDZbd), and PDZ-mediated protein association(s) with this sequence appear to
be primarily responsible for its endocytic sorting activity
(17-20).
Fusion of this sequence to the cytoplasmic tail of the δOR effectively
re-routes endocytic trafficking of engineered receptors from lysosomal to
recycling pathways, establishing the sufficiency of the PDZbd to function as a
transplantable sorting determinant
(18). The β2AR-derived
PDZbd binds with relatively high specificity to the NHERF/EBP50 family of PDZ
proteins (21,
22). A well-established
biochemical function of NHERF/EBP50 family proteins is to associate integral
membrane proteins with actin-associated cytoskeletal elements. This is
achieved through a series of protein-interaction modules linking NHERF/EBP50
family proteins to ERM (ezrin-radixin-moesin) family proteins and, in turn, to
actin filaments
(23-26).
Such indirect actin connectivity is known to mediate other effects on plasma
membrane organization and function
(23), however, and NHERF/EBP50
family proteins can bind to additional proteins potentially important for
endocytic trafficking of receptors
(23,
25). Thus it remains unclear
if actin connectivity is itself sufficient to promote sequence-directed
recycling of GPCRs and, if so, if such connectivity recapitulates the normal
cellular regulation of sequence-dependent recycling. In the present study, we
took advantage of the modular nature of protein connectivity proposed to
mediate β2AR recycling
(24,
26), and extended the opioid
receptor fusion strategy used successfully for identifying diverse recycling
sequences in GPCRs
(27-29),
to address these fundamental questions.Here we show that the recycling activity of the β2AR-derived PDZbd can
be effectively bypassed by linking receptors to ERM family proteins in the
absence of the PDZbd itself. Further, we establish that the protein
connectivity network can be further simplified by fusing receptors to an
interaction module that binds directly to actin filaments. We found that
bypassing the PDZ-mediated interaction using either domain is sufficient to
mimic the ability of the PDZbd to promote efficient, actin-dependent recycling
of receptors. Hrs-dependent regulation, however, which is characteristic of
sequence-dependent recycling of wild-type receptors, was recapitulated only by
the fused PDZbd and not by the proposed downstream interaction modules. These
results support a relatively simple architecture of protein connectivity that
is sufficient to mimic the core recycling activity of the β2AR-derived
PDZbd, but not its characteristic cellular regulation. Given that an
increasing number of GPCRs have been shown to bind PDZ proteins that typically
link directly or indirectly to cytoskeletal elements
(17,
27,
30-32),
the present results also suggest that actin connectivity may represent a
common biochemical principle underlying sequence-dependent recycling of
various GPCRs. 相似文献
6.
7.
Kelvin B. Luther Hermann Schindelin Robert S. Haltiwanger 《The Journal of biological chemistry》2009,284(5):3294-3305
The Notch receptor is critical for proper development where it orchestrates
numerous cell fate decisions. The Fringe family of
β1,3-N-acetylglucosaminyltransferases are regulators of this
pathway. Fringe enzymes add N-acetylglucosamine to O-linked
fucose on the epidermal growth factor repeats of Notch. Here we have analyzed
the reaction catalyzed by Lunatic Fringe (Lfng) in detail. A mutagenesis
strategy for Lfng was guided by a multiple sequence alignment of Fringe
proteins and solutions from docking an epidermal growth factor-like
O-fucose acceptor substrate onto a homology model of Lfng. We
targeted three main areas as follows: residues that could help resolve where
the fucose binds, residues in two conserved loops not observed in the
published structure of Manic Fringe, and residues predicted to be involved in
UDP-N-acetylglucosamine (UDP-GlcNAc) donor specificity. We utilized a
kinetic analysis of mutant enzyme activity toward the small molecule acceptor
substrate 4-nitrophenyl-α-l-fucopyranoside to judge their
effect on Lfng activity. Our results support the positioning of
O-fucose in a specific orientation to the catalytic residue. We also
found evidence that one loop closes off the active site coincident with, or
subsequent to, substrate binding. We propose a mechanism whereby the ordering
of this short loop may alter the conformation of the catalytic aspartate.
Finally, we identify several residues near the UDP-GlcNAc-binding site, which
are specifically permissive toward UDP-GlcNAc utilization.Defects in Notch signaling have been implicated in numerous human diseases,
including multiple sclerosis
(1), several forms of cancer
(2-4),
cerebral autosomal dominant arteriopathy with sub-cortical infarcts and
leukoencephalopathy (5), and
spondylocostal dysostosis
(SCD)3
(6-8).
The transmembrane Notch signaling receptor is activated by members of the DSL
(Delta, Serrate, Lag2) family of ligands
(9,
10). In the endoplasmic
reticulum, O-linked fucose glycans are added to the epidermal growth
factor-like (EGF) repeats of the Notch extracellular domain by protein
O-fucosyltransferase 1
(11-13).
These O-fucose monosaccharides can be elongated in the Golgi
apparatus by three highly conserved
β1,3-N-acetylglucosaminyltransferases of the Fringe family
(Lunatic (Lfng), Manic (Mfng), and Radical Fringe (Rfng) in mammals)
(14-16).
The formation of this GlcNAc-β1,3-Fuc-α1,
O-serine/threonine disaccharide is necessary and sufficient for
subsequent elongation to a tetrasaccharide
(15,
19), although elongation past
the disaccharide in Drosophila is not yet clear
(20,
21). Elongation of
O-fucose by Fringe is known to potentiate Notch signaling from Delta
ligands and inhibit signaling from Serrate ligands
(22). Delta ligands are termed
Delta-like (Delta-like1, -2, and -4) in mammals, and the homologs of Serrate
are known as Jagged (Jagged1 and -2) in mammals. The effects of Fringe on
Drosophila Notch can be recapitulated in Notch ligand in
vitro binding assays using purified components, suggesting that the
elongation of O-fucose by Fringe alters the binding of Notch to its
ligands (21). Although Fringe
also appears to alter Notch-ligand interactions in mammals, the effects of
elongation of the glycan past the O-fucose monosaccharide is more
complicated and appears to be cell type-, receptor-, and ligand-dependent (for
a recent review see Ref.
23).The Fringe enzymes catalyze the transfer of GlcNAc from the donor substrate
UDP-α-GlcNAc to the acceptor fucose, forming the GlcNAc-β1,3-Fuc
disaccharide
(14-16).
They belong to the GT-A-fold of inverting glycosyltransferases, which includes
N-acetylglucosaminyltransferase I and β1,4-galactosyltransferase
I (17,
18). The mechanism is presumed
to proceed through the abstraction of a proton from the acceptor substrate by
a catalytic base (Asp or Glu) in the active site. This creates a nucleophile
that attacks the anomeric carbon of the nucleotide-sugar donor, inverting its
configuration from α (on the nucleotide sugar) to β (in the
product) (24,
25). The enzyme then releases
the acceptor substrate modified with a disaccharide and UDP. The Mfng
structure (26) leaves little
doubt as to the identity of the catalytic residue, which in all likelihood is
aspartate 289 in mouse Lfng (we will use numbering for mouse Lunatic Fringe
throughout, unless otherwise stated). The structure of Mfng with UDP-GlcNAc
soaked into the crystals (26)
showed density only for the UDP portion of the nucleotide-sugar donor and no
density for two loops flanking either side of the active site. The presence of
flexible loops that become ordered upon substrate binding is a common
observation with glycosyltransferases in the GT-A fold family
(18,
25). Density for the entire
donor was observed in the structure of rabbit
N-acetylglucosaminyltransferase I
(27). In this case, ordering
of a previously disordered loop upon UDP-GlcNAc binding may have contributed
to increased stability of the donor. In the case of bovine
β1,4-galactosyltransferase I, a section of flexible random coil from the
apo-structure was observed to change its conformation to α-helical upon
donor substrate binding (28).
Both loops in Lfng are highly conserved, and we have mutated a number of
residues in each to test the hypothesis that they interact with the
substrates. The mutagenesis strategy was also guided by docking of an
EGF-O-fucose acceptor substrate into the active site of the Lfng
model as well as comparison of the Lfng model with a homology model of the
β1,3-glucosyltransferase (β3GlcT) that modifies O-fucose on
thrombospondin type 1 repeats
(29,
30). The β3GlcT is
predicted to be a GT-A fold enzyme related to the Fringe family
(17,
18,
29). 相似文献
8.
9.
10.
Ruben K. Dagda Salvatore J. Cherra III Scott M. Kulich Anurag Tandon David Park Charleen T. Chu 《The Journal of biological chemistry》2009,284(20):13843-13855
Mitochondrial dysregulation is strongly implicated in Parkinson disease.
Mutations in PTEN-induced kinase 1 (PINK1) are associated with familial
parkinsonism and neuropsychiatric disorders. Although overexpressed PINK1 is
neuroprotective, less is known about neuronal responses to loss of PINK1
function. We found that stable knockdown of PINK1 induced mitochondrial
fragmentation and autophagy in SH-SY5Y cells, which was reversed by the
reintroduction of an RNA interference (RNAi)-resistant plasmid for PINK1.
Moreover, stable or transient overexpression of wild-type PINK1 increased
mitochondrial interconnectivity and suppressed toxin-induced
autophagy/mitophagy. Mitochondrial oxidant production played an essential role
in triggering mitochondrial fragmentation and autophagy in PINK1 shRNA lines.
Autophagy/mitophagy served a protective role in limiting cell death, and
overexpressing Parkin further enhanced this protective mitophagic response.
The dominant negative Drp1 mutant inhibited both fission and mitophagy in
PINK1-deficient cells. Interestingly, RNAi knockdown of autophagy proteins
Atg7 and LC3/Atg8 also decreased mitochondrial fragmentation without affecting
oxidative stress, suggesting active involvement of autophagy in morphologic
remodeling of mitochondria for clearance. To summarize, loss of PINK1 function
elicits oxidative stress and mitochondrial turnover coordinated by the
autophagic and fission/fusion machineries. Furthermore, PINK1 and Parkin may
cooperate through different mechanisms to maintain mitochondrial
homeostasis.Parkinson disease is an age-related neurodegenerative disease that affects
∼1% of the population worldwide. The causes of sporadic cases are unknown,
although mitochondrial or oxidative toxins such as
1-methyl-4-phenylpyridinium, 6-hydroxydopamine
(6-OHDA),3 and
rotenone reproduce features of the disease in animal and cell culture models
(1). Abnormalities in
mitochondrial respiration and increased oxidative stress are observed in cells
and tissues from parkinsonian patients
(2,
3), which also exhibit
increased mitochondrial autophagy
(4). Furthermore, mutations in
parkinsonian genes affect oxidative stress response pathways and mitochondrial
homeostasis (5). Thus,
disruption of mitochondrial homeostasis represents a major factor implicated
in the pathogenesis of sporadic and inherited parkinsonian disorders (PD).The PARK6 locus involved in autosomal recessive and early-onset PD
encodes for PTEN-induced kinase 1 (PINK1)
(6,
7). PINK1 is a cytosolic and
mitochondrially localized 581-amino acid serine/threonine kinase that
possesses an N-terminal mitochondrial targeting sequence
(6,
8). The primary sequence also
includes a putative transmembrane domain important for orientation of the
PINK1 domain (8), a conserved
kinase domain homologous to calcium calmodulin kinases, and a C-terminal
domain that regulates autophosphorylation activity
(9,
10). Overexpression of
wild-type PINK1, but not its PD-associated mutants, protects against several
toxic insults in neuronal cells
(6,
11,
12). Mitochondrial targeting
is necessary for some (13) but
not all of the neuroprotective effects of PINK1
(14), implicating involvement
of cytoplasmic targets that modulate mitochondrial pathobiology
(8). PINK1 catalytic activity
is necessary for its neuroprotective role, because a kinase-deficient K219M
substitution in the ATP binding pocket of PINK1 abrogates its ability to
protect neurons (14). Although
PINK1 mutations do not seem to impair mitochondrial targeting, PD-associated
mutations differentially destabilize the protein, resulting in loss of
neuroprotective activities
(13,
15).Recent studies indicate that PINK1 and Parkin interact genetically
(3,
16-18)
to prevent oxidative stress
(19,
20) and regulate mitochondrial
morphology (21). Primary cells
derived from PINK1 mutant patients exhibit mitochondrial fragmentation with
disorganized cristae, recapitulated by RNA interference studies in HeLa cells
(3).Mitochondria are degraded by macroautophagy, a process involving
sequestration of cytoplasmic cargo into membranous autophagic vacuoles (AVs)
for delivery to lysosomes (22,
23). Interestingly,
mitochondrial fission accompanies autophagic neurodegeneration elicited by the
PD neurotoxin 6-OHDA (24,
25). Moreover, mitochondrial
fragmentation and increased autophagy are observed in neurodegenerative
diseases including Alzheimer and Parkinson diseases
(4,
26-28).
Although inclusion of mitochondria in autophagosomes was once believed to be a
random process, as observed during starvation, studies involving hypoxia,
mitochondrial damage, apoptotic stimuli, or limiting amounts of aerobic
substrates in facultative anaerobes support the concept of selective
mitochondrial autophagy (mitophagy)
(29,
30). In particular,
mitochondrially localized kinases may play an important role in models
involving oxidative mitochondrial injury
(25,
31,
32).Autophagy is involved in the clearance of protein aggregates
(33-35)
and normal regulation of axonal-synaptic morphology
(36). Chronic disruption of
lysosomal function results in accumulation of subtly impaired mitochondria
with decreased calcium buffering capacity
(37), implicating an important
role for autophagy in mitochondrial homeostasis
(37,
38). Recently, Parkin, which
complements the effects of PINK1 deficiency on mitochondrial morphology
(3), was found to promote
autophagy of depolarized mitochondria
(39). Conversely, Beclin
1-independent autophagy/mitophagy contributes to cell death elicited by the PD
toxins 1-methyl-4-phenylpyridinium and 6-OHDA
(25,
28,
31,
32), causing neurite
retraction in cells expressing a PD-linked mutation in leucine-rich repeat
kinase 2 (40). Whereas
properly regulated autophagy plays a homeostatic and neuroprotective role,
excessive or incomplete autophagy creates a condition of “autophagic
stress” that can contribute to neurodegeneration
(28).As mitochondrial fragmentation
(3) and increased mitochondrial
autophagy (4) have been
described in human cells or tissues of PD patients, we investigated whether or
not the engineered loss of PINK1 function could recapitulate these
observations in human neuronal cells (SH-SY5Y). Stable knockdown of endogenous
PINK1 gave rise to mitochondrial fragmentation and increased autophagy and
mitophagy, whereas stable or transient overexpression of PINK1 had the
opposite effect. Autophagy/mitophagy was dependent upon increased
mitochondrial oxidant production and activation of fission. The data indicate
that PINK1 is important for the maintenance of mitochondrial networks,
suggesting that coordinated regulation of mitochondrial dynamics and autophagy
limits cell death associated with loss of PINK1 function. 相似文献
11.
Isabel Molina-Ortiz Rub��n A. Bartolom�� Pablo Hern��ndez-Varas Georgina P. Colo Joaquin Teixid�� 《The Journal of biological chemistry》2009,284(22):15147-15157
Melanoma cells express the chemokine receptor CXCR4 that confers high
invasiveness upon binding to its ligand CXCL12. Melanoma cells at initial
stages of the disease show reduction or loss of E-cadherin expression, but
recovery of its expression is frequently found at advanced phases. We
overexpressed E-cadherin in the highly invasive BRO lung metastatic cell
melanoma cell line to investigate whether it could influence CXCL12-promoted
cell invasion. Overexpression of E-cadherin led to defective invasion of
melanoma cells across Matrigel and type I collagen in response to CXCL12. A
decrease in individual cell migration directionality toward the chemokine and
reduced adhesion accounted for the impaired invasion. A p190RhoGAP-dependent
inhibition of RhoA activation was responsible for the impairment in
chemokine-stimulated E-cadherin melanoma transfectant invasion. Furthermore,
we show that p190RhoGAP and p120ctn associated predominantly on the plasma
membrane of cells overexpressing E-cadherin, and that E-cadherin-bound p120ctn
contributed to RhoA inactivation by favoring p190RhoGAP-RhoA association.
These results suggest that melanoma cells at advanced stages of the disease
could have reduced metastatic potency in response to chemotactic stimuli
compared with cells lacking E-cadherin, and the results indicate that
p190RhoGAP is a central molecule controlling melanoma cell invasion.Cadherins are a family of Ca2+-dependent adhesion molecules that
mediate cell-cell contacts and are expressed in most solid tissues providing a
tight control of morphogenesis
(1,
2). Classical cadherins, such
as epithelial (E) cadherin, are found in adherens junctions, forming core
protein complexes with β-catenin, α-catenin, and p120 catenin
(p120ctn). Both β-catenin and p120ctn directly interact with E-cadherin,
whereas α-catenin associates with the complex through its binding to
β-catenin, providing a link with the actin cytoskeleton
(1,
2). E-cadherin is frequently
lost or down-regulated in many human tumors, coincident with morphological
epithelial to mesenchymal transition and acquisition of invasiveness
(3-6).Although melanoma only accounts for 5% of skin cancers, when metastasis
starts, it is responsible for 80% of deaths from skin cancers
(7). Melanocytes express
E-cadherin
(8-10),
but melanoma cells at early radial growth phase show a large reduction in the
expression of this cadherin, and surprisingly, expression has been reported to
be partially recovered by vertical growth phase and metastatic melanoma cells
(9,
11,
12).Trafficking of cancer cells from primary tumor sites to intravasation into
blood circulation and later to extravasation to colonize distant organs
requires tightly regulated directional cues and cell migration and invasion
that are mediated by chemokines, growth factors, and adhesion molecules
(13). Solid tumor cells
express chemokine receptors that provide guidance of these cells to organs
where their chemokine ligands are expressed, constituting a homing model
resembling the one used by immune cells to exert their immune surveillance
functions (14). Most solid
cancer cells express CXCR4, a receptor for the chemokine CXCL12 (also called
SDF-1), which is expressed in lungs, bone marrow, and liver
(15). Expression of CXCR4 in
human melanoma has been detected in the vertical growth phase and on regional
lymph nodes, which correlated with poor prognosis and increased mortality
(16,
17). Previous in vivo
experiments have provided evidence supporting a crucial role for CXCR4 in the
metastasis of melanoma cells
(18).Rho GTPases control the dynamics of the actin cytoskeleton during cell
migration (19,
20). The activity of Rho
GTPases is tightly regulated by guanine-nucleotide exchange factors
(GEFs),4 which
stimulate exchange of bound GDP by GTP, and inhibited by GTPase-activating
proteins (GAPs), which promote GTP hydrolysis
(21,
22), whereas guanine
nucleotide dissociation inhibitors (GDIs) appear to mediate blocking of
spontaneous activation (23).
Therefore, cell migration is finely regulated by the balance between GEF, GAP,
and GDI activities on Rho GTPases. Involvement of Rho GTPases in cancer is
well documented (reviewed in Ref.
24), providing control of both
cell migration and growth. RhoA and RhoC are highly expressed in colon,
breast, and lung carcinoma
(25,
26), whereas overexpression of
RhoC in melanoma leads to enhancement of cell metastasis
(27). CXCL12 activates both
RhoA and Rac1 in melanoma cells, and both GTPases play key roles during
invasion toward this chemokine
(28,
29).Given the importance of the CXCL12-CXCR4 axis in melanoma cell invasion and
metastasis, in this study we have addressed the question of whether changes in
E-cadherin expression on melanoma cells might affect cell invasiveness. We
show here that overexpression of E-cadherin leads to impaired melanoma cell
invasion to CXCL12, and we provide mechanistic characterization accounting for
the decrease in invasion. 相似文献
12.
Andrés Norambuena Claudia Metz Lucas Vicu?a Antonia Silva Evelyn Pardo Claudia Oyanadel Loreto Massardo Alfonso González Andrea Soza 《The Journal of biological chemistry》2009,284(19):12670-12679
Galectins have been implicated in T cell homeostasis playing complementary
pro-apoptotic roles. Here we show that galectin-8 (Gal-8) is a potent
pro-apoptotic agent in Jurkat T cells inducing a complex phospholipase
D/phosphatidic acid signaling pathway that has not been reported for any
galectin before. Gal-8 increases phosphatidic signaling, which enhances the
activity of both ERK1/2 and type 4 phosphodiesterases (PDE4), with a
subsequent decrease in basal protein kinase A activity. Strikingly, rolipram
inhibition of PDE4 decreases ERK1/2 activity. Thus Gal-8-induced PDE4
activation releases a negative influence of cAMP/protein kinase A on ERK1/2.
The resulting strong ERK1/2 activation leads to expression of the death factor
Fas ligand and caspase-mediated apoptosis. Several conditions that decrease
ERK1/2 activity also decrease apoptosis, such as anti-Fas ligand blocking
antibodies. In addition, experiments with freshly isolated human peripheral
blood mononuclear cells, previously stimulated with anti-CD3 and anti-CD28,
show that Gal-8 is pro-apoptotic on activated T cells, most likely on a
subpopulation of them. Anti-Gal-8 autoantibodies from patients with systemic
lupus erythematosus block the apoptotic effect of Gal-8. These results
implicate Gal-8 as a novel T cell suppressive factor, which can be
counterbalanced by function-blocking autoantibodies in autoimmunity.Glycan-binding proteins of the galectin family have been increasingly
studied as regulators of the immune response and potential therapeutic agents
for autoimmune disorders (1).
To date, 15 galectins have been identified and classified according with the
structural organization of their distinctive monomeric or dimeric carbohydrate
recognition domain for β-galactosides
(2,
3). Galectins are secreted by
unconventional mechanisms and once outside the cells bind to and cross-link
multiple glycoconjugates both at the cell surface and at the extracellular
matrix, modulating processes as diverse as cell adhesion, migration,
proliferation, differentiation, and apoptosis
(4–10).
Several galectins have been involved in T cell homeostasis because of their
capability to kill thymocytes, activated T cells, and T cell lines
(11–16).
Pro-apoptotic galectins might contribute to shape the T cell repertoire in the
thymus by negative selection, restrict the immune response by eliminating
activated T cells at the periphery
(1), and help cancer cells to
escape the immune system by eliminating cancer-infiltrating T cells
(17). They have also a
promising therapeutic potential to eliminate abnormally activated T cells and
inflammatory cells (1). Studies
on the mostly explored galectins, Gal-1, -3, and -9
(14,
15,
18–20),
as well as in Gal-2 (13),
suggest immunosuppressive complementary roles inducing different pathways to
apoptosis. Galectin-8
(Gal-8)4 is one of the
most widely expressed galectins in human tissues
(21,
22) and cancerous cells
(23,
24). Depending on the cell
context and mode of presentation, either as soluble stimulus or extracellular
matrix, Gal-8 can promote cell adhesion, spreading, growth, and apoptosis
(6,
7,
9,
10,
22,
25). Its role has been mostly
studied in relation to tumor malignancy
(23,
24). However, there is some
evidence regarding a role for Gal-8 in T cell homeostasis and autoimmune or
inflammatory disorders. For instance, the intrathymic expression and
pro-apoptotic effect of Gal-8 upon CD4highCD8high
thymocytes suggest a role for Gal-8 in shaping the T cell repertoire
(16). Gal-8 could also
modulate the inflammatory function of neutrophils
(26), Moreover Gal-8-blocking
agents have been detected in chronic autoimmune disorders
(10,
27,
28). In rheumatoid arthritis,
Gal-8 has an anti-inflammatory action, promoting apoptosis of synovial fluid
cells, but can be counteracted by a specific rheumatoid version of CD44
(CD44vRA) (27). In systemic
lupus erythematosus (SLE), a prototypic autoimmune disease, we recently
described function-blocking autoantibodies against Gal-8
(10,
28). Thus it is important to
define the role of Gal-8 and the influence of anti-Gal-8 autoantibodies in
immune cells.In Jurkat T cells, we previously reported that Gal-8 interacts with
specific integrins, such as α1β1, α3β1, and
α5β1 but not α4β1, and as a matrix protein promotes cell
adhesion and asymmetric spreading through activation of the extracellular
signal-regulated kinases 1 and 2 (ERK1/2)
(10). These early effects
occur within 5–30 min. However, ERK1/2 signaling supports long term
processes such as T cell survival or death, depending on the moment of the
immune response. During T cell activation, ERK1/2 contributes to enhance the
expression of interleukin-2 (IL-2) required for T cell clonal expansion
(29). It also supports T cell
survival against pro-apoptotic Fas ligand (FasL) produced by themselves and by
other previously activated T cells
(30,
31). Later on, ERK1/2 is
required for activation-induced cell death, which controls the extension of
the immune response by eliminating recently activated and restimulated T cells
(32,
33). In activation-induced
cell death, ERK1/2 signaling contributes to enhance the expression of FasL and
its receptor Fas/CD95 (32,
33), which constitute a
preponderant pro-apoptotic system in T cells
(34). Here, we ask whether
Gal-8 is able to modulate the intensity of ERK1/2 signaling enough to
participate in long term processes involved in T cell homeostasis.The functional integration of ERK1/2 and PKA signaling
(35) deserves special
attention. cAMP/PKA signaling plays an immunosuppressive role in T cells
(36) and is altered in SLE
(37). Phosphodiesterases
(PDEs) that degrade cAMP release the immunosuppressive action of cAMP/PKA
during T cell activation (38,
39). PKA has been described to
control the activity of ERK1/2 either positively or negatively in different
cells and processes (35). A
little explored integration among ERK1/2 and PKA occurs via phosphatidic acid
(PA) and PDE signaling. Several stimuli activate phospholipase D (PLD) that
hydrolyzes phosphatidylcholine into PA and choline. Such PLD-generated PA
plays roles in signaling interacting with a variety of targeting proteins that
bear PA-binding domains (40).
In this way PA recruits Raf-1 to the plasma membrane
(41). It is also converted by
phosphatidic acid phosphohydrolase (PAP) activity into diacylglycerol (DAG),
which among other functions, recruits and activates the GTPase Ras
(42). Both Ras and Raf-1 are
upstream elements of the ERK1/2 activation pathway
(43). In addition, PA binds to
and activates PDEs of the type 4 subfamily (PDE4s) leading to decreased cAMP
levels and PKA down-regulation
(44). The regulation and role
of PA-mediated control of ERK1/2 and PKA remain relatively unknown in T cell
homeostasis, because it is also unknown whether galectins stimulate the PLD/PA
pathway.Here we found that Gal-8 induces apoptosis in Jurkat T cells by triggering
cross-talk between PKA and ERK1/2 pathways mediated by PLD-generated PA. Our
results for the first time show that a galectin increases the PA levels,
down-regulates the cAMP/PKA system by enhancing rolipram-sensitive PDE
activity, and induces an ERK1/2-dependent expression of the pro-apoptotic
factor FasL. The enhanced PDE activity induced by Gal-8 is required for the
activation of ERK1/2 that finally leads to apoptosis. Gal-8 also induces
apoptosis in human peripheral blood mononuclear cells (PBMC), especially after
activating T cells with anti-CD3/CD28. Therefore, Gal-8 shares with other
galectins the property of killing activated T cells contributing to the T cell
homeostasis. The pathway involves a particularly integrated signaling context,
engaging PLD/PA, cAMP/PKA, and ERK1/2, which so far has not been reported for
galectins. The pro-apoptotic function of Gal-8 also seems to be unique in its
susceptibility to inhibition by anti-Gal-8 autoantibodies. 相似文献
13.
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
14.
15.
Although agonist-dependent endocytosis of G protein-coupled receptors
(GPCRs) as a means to modulate receptor signaling has been widely studied, the
constitutive endocytosis of GPCRs has received little attention. Here we show
that two prototypical class I GPCRs, the β2 adrenergic and M3 muscarinic
receptors, enter cells constitutively by clathrin-independent endocytosis and
colocalize with markers of this endosomal pathway on recycling tubular
endosomes, indicating that these receptors can subsequently recycle back to
the plasma membrane (PM). This constitutive endocytosis of these receptors was
not blocked by antagonists, indicating that receptor signaling was not
required. Interestingly, the G proteins that these receptors couple to,
Gαs and Gαq, localized together with their
receptors at the plasma membrane and on tubular recycling endosomes. Upon
agonist stimulation, Gαs and Gαq remained
associated with the PM and these endosomal membranes, whereas β2 and M3
receptors now entered cells via clathrin-dependent endocytosis. Deletion of
the third intracellular loop (i3 loop), which is thought to play a role in
agonist-dependent endocytosis of the M3 receptor, had no effect on the
constitutive internalization of the receptor. Surprisingly, with agonist, the
mutated M3 receptor still internalized and accumulated in cells but through
clathrin-independent and not clathrin-dependent endocytosis. These findings
demonstrate that GPCRs are versatile PM proteins that can utilize different
mechanisms of internalization depending upon ligand activation.G protein-coupled receptors
(GPCRs)2 belong to a
superfamily of seven transmembrane-spanning proteins that respond to a diverse
array of sensory and chemical stimuli
(1–4).
Activation of GPCRs through the binding of specific agonists induces
conformational changes that allow activation of heterotrimeric guanine
nucleotide-binding proteins (G proteins)
(5,
6). To ensure that the signals
are controlled in magnitude and duration, activated GPCRs are rapidly
desensitized through phosphorylation carried out by G protein-coupled receptor
kinases (GRKs) (7). This
facilitates β-arrestin binding and promotes receptor uncoupling from the
G protein (8,
9). In addition to its role in
GPCRs desensitization, β-arrestins promote the translocation of the
receptor to the endocytic machinery involving clathrin and adaptor protein-2
(AP-2), thereby facilitating receptor removal from the plasma membrane
(10–15).
Once internalized, some GPCRs may even continue to signal from endosomes
(16).Although GPCR internalization is generally considered to be an
agonist-dependent phenomenon, some evidence suggests that GPCRs can be
endocytosed even in the absence of agonist, a process known as constitutive
internalization
(17–20).
The role of constitutive internalization of GPCRs is not clear. One
interesting study on cannabinoid CB1 receptors in neurons has shown that
constitutive internalization from the somatodendritic and not axonal membrane
is responsible for the overall redistribution of receptors from the
somatodentritic to the axonal membrane
(17). Another study on the
melanocortin MC4 receptor raised the possibility that constitutive endocytosis
could be a consequence of the basal activity of the receptor
(18).Even less is known about the potential trafficking of the transducer of
GPCR signaling, the G protein
(21). Generally, the binding
of the agonist to the GPCR promotes the exchange of GDP on the Gα
protein for GTP and allows the dissociation of the trimeric G protein into
Gα-GTP and Gβγ dimer subunits
(5,
22). Then, the activated G
proteins target different effectors
(23,
24). G proteins are localized
primarily to the PM where they interact with GPCRs; however, it is not known
whether G proteins always remain at the PM or whether they might move into
cells along endocytic pathways. Previous work showed that Gαs
does not colocalize with β2 receptor on internal compartments after
agonist stimulation, but the cellular distribution of Gαs was
not examined (25).In general, cargo proteins at the plasma membrane (PM) enter the cell
through a variety of endocytic mechanisms that can be divided into two main
groups: clathrin-dependent endocytosis (CDE) and clathrin-independent
endocytosis (CIE). CDE is used by PM proteins such as the transferrin receptor
(TfR) that contain specific cytoplasmic sequences recognized by adaptor
proteins allowing a rapid and efficient internalization through
clathrin-coated vesicles (26,
27). In contrast, CIE is used
by PM proteins that lack adaptor protein binding sequences including cargo
proteins such as the major histocompatibility complex class I protein (MHCI),
the glycosylphosphatidylinositol-anchored protein CD59, and integrins
(28–30).
In HeLa cells CIE is independent of, and CDE dependent on, clathrin and
dynamin and thus the two different endocytic pathways are distinct and well
defined (31). After
internalization in separate vesicles, MHCI-containing vesicles from CIE and
transferrin receptor-containing vesicles from CDE subsequently fuse with the
early endosomal compartment that is associated with Rab5 and the early
endosomal antigen 1 (EEA1)
(32). TfR is recycled back out
to the PM in Rab4- and Rab11-dependent processes. In contrast, some MHCI is
trafficked on to late endosomes and lysosomes for degradation, and some is
recycled back out to the PM along tubular endosomes that lack TfR and emanate
from the juxtanuclear area. Recycling of MHCI back to the PM requires the
activity of Arf6, Rab22, and Rab11
(33,
34).In this study, we analyzed the trafficking of GPCRs and their G proteins in
the presence and absence of agonist in HeLa cells. We examined the trafficking
of two prototypical class I GPCRs: the β2 adrenergic receptor (coupled to
Gαs) and the M3 acetylcholine muscarinic receptor (coupled to
Gαq). We find that β2 and M3 receptors traffic
constitutively via CIE, and then, in the presence of agonist, they switch to
the CDE pathway. We also examined the role of the third intracellular loop of
the M3 receptor in this process. To our knowledge, this study represents the
most comprehensive analysis of constitutive trafficking of class I GPCRs and
related Gα proteins. We demonstrate that GPCRs are versatile PM cargos
that utilize different mechanisms of internalization depending upon ligand
activation. Considering the high level of homology between class I GPCRs, this
evidence could be applicable to the other members of this family. 相似文献
16.
17.
Ivana I. Knezevic Sanda A. Predescu Radu F. Neamu Matvey S. Gorovoy Nebojsa M. Knezevic Cordus Easington Asrar B. Malik Dan N. Predescu 《The Journal of biological chemistry》2009,284(8):5381-5394
It is known that platelet-activating factor (PAF) induces severe
endothelial barrier leakiness, but the signaling mechanisms remain unclear.
Here, using a wide range of biochemical and morphological approaches applied
in both mouse models and cultured endothelial cells, we addressed the
mechanisms of PAF-induced disruption of interendothelial junctions (IEJs) and
of increased endothelial permeability. The formation of interendothelial gaps
filled with filopodia and lamellipodia is the cellular event responsible for
the disruption of endothelial barrier. We observed that PAF ligation of its
receptor induced the activation of the Rho GTPase Rac1. Following PAF
exposure, both Rac1 and its guanine nucleotide exchange factor Tiam1 were
found associated with a membrane fraction from which they
co-immunoprecipitated with PAF receptor. In the same time frame with
Tiam1-Rac1 translocation, the junctional proteins ZO-1 and VE-cadherin were
relocated from the IEJs, and formation of numerous interendothelial gaps was
recorded. Notably, the response was independent of myosin light chain
phosphorylation and thus distinct from other mediators, such as histamine and
thrombin. The changes in actin status are driven by the PAF-induced localized
actin polymerization as a consequence of Rac1 translocation and activation.
Tiam1 was required for the activation of Rac1, actin polymerization,
relocation of junctional associated proteins, and disruption of IEJs. Thus,
PAF-induced IEJ disruption and increased endothelial permeability requires the
activation of a Tiam1-Rac1 signaling module, suggesting a novel therapeutic
target against increased vascular permeability associated with inflammatory
diseases.The endothelial barrier is made up of endothelial cells
(ECs)4 connected to
each other by interendothelial junctions (IEJs) consisting of protein
complexes organized as tight junctions (TJs) and adherens junctions (AJs). In
addition, the focal adhesion complex located at the basal plasma membrane
enables firm contact of ECs with the underlying basement membrane and also
contributes to the barrier function
(1-3).
The glycocalyx, the endothelial monolayer, and the basement membrane all
together constitute the vascular barrier.The structural integrity of the ECs along with their proper functionality
are the two most important factors controlling the tightness of the
endothelial barrier. Changes affecting these factors cause loss of barrier
restrictiveness and leakiness. Therefore, defining and understanding the
cellular and molecular mechanisms controlling these processes is of paramount
importance. Increased width of IEJs in response to permeability-increasing
mediators (4) regulates the
magnitude of transendothelial exchange of fluid and solutes. Disruption of
IEJs and the resultant barrier leakiness contribute to the genesis of diverse
pathological conditions, such as inflammation
(5), metastasis
(6,
7), and uncontrolled
angiogenesis (8,
9).Accumulated evidence demonstrated that IEJs changes are responsible for
increased or decreased vascular permeability, and the generally accepted
mechanism responsible for them was the myosin light chain (MLC)-mediated
contraction of ECs (5,
10). However, published
evidence showed that an increase in vascular permeability could be obtained
without a direct involvement of any contractile mechanism
(11-16).The main component of the vascular barrier, the ECs, has more than 10% of
their total protein represented by actin
(17), which under
physiological salt concentrations subsists as monomers (G-actin) and assembled
into filaments (F-actin). A large number of actin-interacting proteins may
modulate the assembly, disassembly, and organization of G-actin and of actin
filaments within a given cell type. Similar to the complexity of
actin-interacting proteins found in other cell types, the ECs utilize their
actin binding proteins to stabilize the endothelial monolayer in order to
efficiently function as a selective barrier
(11). In undisturbed ECs, the
actin microfilaments are organized as different networks with distinctive
functional and morphological characteristics: the peripheral filaments also
known as peripheral dense band (PDB), the cytoplasmic fibers identified as
stress fibers (SF), and the actin from the membrane cytoskeleton
(18). The peripheral web,
localized immediately under the membrane, is associated with (i) the luminal
plasmalemma (on the apical side), (ii) the IEJ complexes on the lateral
surfaces, and (iii) the focal adhesion complexes on the abluminal side (the
basal part) of polarized ECs. The SF reside inside the endothelial cytoplasm
and are believed to be directly connected with the plasmalemma proper on the
luminal as well as on the abluminal side of the cell. As described, the
endothelial actin cytoskeleton (specifically the SF) seems to be a stable
structure helping the cells to remain flat under flow
(19). It is also established
that the actin fibers participate in correct localization of different
junctional complexes while keeping them in place
(20). However, it was
suggested that the dynamic equilibrium between F- and G-actin might modulate
the tightness of endothelial barrier in response to different challenges
(13).Mediators effective at nanomolar concentrations or less that disrupt the
endothelial barrier and increase vascular permeability include C2 toxin of
Clostridium botulinum, vascular permeability factor, better known as
vascular endothelial growth factor, and PAF
(21). C2 toxin increases
endothelial permeability by ribosylating monomeric G-actin at Arg-177
(22). This results in the
impairment of actin polymerization
(23), followed by rounding of
ECs (16) and the disruption of
junctional integrity. Vascular permeability factor was shown to open IEJs by
redistribution of junctional proteins
(24,
25) and by interfering with
the equilibrium of actin pools
(26). PAF
(1-O-alkyl-2-acetyl-sn-glycero-3-phosphocoline), a naturally
synthesized phospholipid is active at 10-10 m or less
(27). PAF is synthesized by
and acts on a variety of cell types, including platelets
(28), neutrophils
(29), monocytes
(30), and ECs
(31). PAF-mediated activation
of ECs induced cell migration
(32), angiogenesis
(7), and vascular
hyperpermeability (33)
secondary to disassembly of IEJs
(34). The effects of PAF on
the endothelium are initiated through a G protein-coupled receptor (PAF-R)
localized at the plasmalemma, in a large endosomal compartment inside the cell
(34), and also in the nuclear
membrane (35). In ECs, PAF-R
was shown to signal through Gαq and downstream activation of
phospholipase C isozymes (PLCβ3 and PLCγ1),
and via cSrc (32,
36). Studies have shown that
PAF challenge induced endothelial actin cytoskeletal rearrangement
(37) and marked vascular
leakiness (38); however, the
signaling pathways have not been elucidated.Therefore, in the present study, we carried out a systematic analysis of
PAF-induced morphological and biochemical changes of endothelial barrier
in vivo and in cultured ECs. We found that the opening of endothelial
barrier and the increased vascular leakiness induced by PAF are the result of
a shift in actin pools without involvement of EC contraction, followed by a
redistribution of tight junctional associated protein ZO-1 and adherens
junctional protein VE-cadherin. 相似文献
18.
Cristian A. Droppelmann Jaime Guti��rrez Cecilia Vial Enrique Brandan 《The Journal of biological chemistry》2009,284(20):13551-13561
Matrix metalloproteinase-2 (MMP-2) is an important extracellular matrix
remodeling enzyme, and it has been involved in different fibrotic disorders.
The connective tissue growth factor (CTGF/CCN2), which is increased in these
pathologies, induces the production of extracellular matrix proteins. To
understand the fibrotic process observed in diverse pathologies, we analyzed
the fibroblast response to CTGF when MMP-2 activity is inhibited. CTGF
increased fibronectin (FN) amount, MMP-2 mRNA expression, and gelatinase
activity in 3T3 cells. When MMP-2 activity was inhibited either by the
metalloproteinase inhibitor GM-6001 or in MMP-2-deficient fibroblasts, an
increase in the basal amount of FN together with a decrease of its levels in
response to CTGF was observed. This paradoxical effect could be explained by
the fact that the excess of FN could block the access to other ligands, such
as CTGF, to integrins. This effect was emulated in fibroblasts by adding
exogenous FN or RGDS peptides or using anti-integrin αV
subunit-blocking antibodies. Additionally, in MMP-2-deficient cells CTGF did
not induce the formation of stress fibers, focal adhesion sites, and ERK
phosphorylation. Anti-integrin αV subunit-blocking antibodies
inhibited ERK phosphorylation in control cells. Finally, in MMP-2-deficient
cells, FN mRNA expression was not affected by CTGF, but degradation of
125I-FN was increased. These results suggest that expression,
regulation, and activity of MMP-2 can play an important role in the initial
steps of fibrosis and shows that FN levels can regulate the cellular response
to CTGF.Extracellular proteolysis is an essential physiological process that
controls the immediate cellular environment and thus plays a key role in
cellular behavior and survival
(1). The members of the matrix
metalloproteinase
(MMP)2 family of
zinc-dependent endopeptidases are major mediators of extracellular proteolysis
by promoting the degradation of extracellular matrix (ECM) components and cell
surface-associated proteins (2,
3). Each one of these enzymes
is negatively regulated by tissue inhibitors of metalloproteinases (TIMPs)
(4) and is secreted as a
zymogen (pro-MMPs) that is activated in the extracellular space
(5–7).
This mechanism is an important form of regulation of gelatinase activity and
in consequence, highly significant for ECM homeostasis. Among the members of
the MMP family, the metalloproteinase type 2 (MMP-2 or gelatinase A) is known
to be a key player in many physiological and pathological processes, such as
cell migration, inflammation, angiogenesis, and fibrosis
(8–11).Fibrotic disorders are typified by excessive connective tissue and ECM
deposition that precludes normal healing of different tissues. ECM
accumulation can be explained in two ways: increasing expression and
deposition of connective tissue proteins and/or decreasing degradation of ECM
proteins (12). Transforming
growth factor type β, a multifunctional cytokine, is strongly
overexpressed, and it is associated to the pathogenesis of these diseases
(13,
14). It stimulates the
expression of connective tissue growth factor (CTGF/CCN2)
(15), a cytokine that is
responsible for transforming growth factor type β fibrotic activity
(16,
17). The role of CTGF in
fibrosis has gained attention in recent years
(16,
18–22).
CTGF overexpression is known to occur in a variety of fibrotic skin disorders
(23,
24), renal
(25), hepatic
(26), and pulmonary fibrosis
(27) and in muscles from
patients with Duchenne muscular dystrophy
(28).On the other hand, several pathologies involving fibrosis show an increase
in MMP expression, including gelatinase A. Augmented expression of MMP-2 was
found in submucous (29), skin
(30), liver
(31), and lung fibrosis
(32,
33) and dystrophic myotubes
from fibrotic muscles of Duchenne muscular dystrophy
(34). It has been shown that
transforming growth factor type β induces an increase in the amount of
MMP-2 in fibroblasts (35) and
that CTGF induces MMP-2 expression in cultured renal interstitial fibroblasts
(36). The putative role
assigned to MMP-2 in fibrotic disorders is related to tissue regeneration
because of the capacity of this enzyme to degrade basal lamina
(37–39).
Because MMP-2 expression is up-regulated in these pathologies but still a high
ECM deposition is observed, we propose that this accumulation could be
explained by a diminution of the MMP-2 enzymatic activity.In this article, we demonstrate that CTGF increases fibronectin (FN)
amount, MMP-2 expression, and gelatinase activity in 3T3 fibroblasts. More
significantly, we show that MMP-2-deficient cells have an increased basal
amount of FN and show a response to CTGF that is opposite to that of control
cells. This paradoxical effect could be explained by the increase in the FN
amount that blocks the integrins (at least integrins with αV
subunit), which can act like CTGF receptors. 相似文献
19.
20.
Yang Wang Dan Li Roza Nurieva Justin Yang Mehmet Sen Roberto Carre?o Sijie Lu Bradley W. McIntyre Jeffrey J. Molldrem Glen B. Legge Qing Ma 《The Journal of biological chemistry》2009,284(19):12645-12653
The activation of LFA-1 (lymphocyte function-associated antigen) is a
critical event for T cell co-stimulation. The mechanism of LFA-1 activation
involves both affinity and avidity regulation, but the role of each in T cell
activation remains unclear. We have identified antibodies that recognize and
block different affinity states of the mouse LFA-1 I-domain. Monoclonal
antibody 2D7 preferentially binds to the low affinity conformation, and this
specific binding is abolished when LFA-1 is locked in the high affinity
conformation. In contrast, M17/4 can bind both the locked high and low
affinity forms of LFA-1. Although both 2D7 and M17/4 are blocking antibodies,
2D7 is significantly less potent than M17/4 in blocking LFA-1-mediated
adhesion; thus, blocking high affinity LFA-1 is critical for preventing
LFA-1-mediated adhesion. Using these reagents, we investigated whether LFA-1
affinity regulation affects T cell activation. We found that blocking high
affinity LFA-1 prevents interleukin-2 production and T cell proliferation,
demonstrated by TCR cross-linking and antigen-specific stimulation.
Furthermore, there is a differential requirement of high affinity LFA-1 in the
activation of CD4+ and CD8+ T cells. Although
CD4+ T cell activation depends on both high and low affinity LFA-1,
only high affinity LFA-1 provides co-stimulation for CD8+ T cell
activation. Together, our data demonstrated that the I-domain of LFA-1 changes
to the high affinity state in primary T cells, and high affinity LFA-1 is
critical for facilitating T cell activation. This implicates LFA-1 activation
as a novel regulatory mechanism for the modulation of T cell activation and
proliferation.LFA-1 (lymphocyte function-associated antigen), an integrin family member,
is important in regulating leukocyte adhesion and T cell activation
(1,
2). LFA-1 consists of the
αL (CD11a) and β2 (CD18) heterodimer. The
ligands for LFA-1, including intercellular adhesion molecule
ICAM3-1, ICAM-2, and
ICAM-3, are expressed on antigen-presenting cells (APCs), endothelial cells,
and lymphocytes (1). Mice that
are deficient in LFA-1 have defects in leukocyte adhesion, lymphocyte
proliferation, and tumor rejection
(3–5).
Blocking LFA-1 with antibodies can prevent inflammation, autoimmunity, organ
graft rejection, and graft versus host disease in human and murine
models
(6–10).LFA-1 is constitutively expressed on the surface of leukocytes in an
inactive state. Activation of LFA-1 is mediated by inside-out signals from the
cytoplasm (1,
11). Subsequently, activated
LFA-1 binds to the ligands and transduces outside-in signals back into the
cytoplasm that result in cell adhesion and activation
(12,
13). The activation of LFA-1
is a critical event in the formation of the immunological synapse, which is
important for T cell activation
(2,
14,
15). The active state of LFA-1
is regulated by chemokines and the T cell receptor (TCR) through Rap1
signaling (16). LFA-1 ligation
lowers the activation threshold and affects polarization in CD4+ T
cells (17). Moreover,
productive LFA-1 engagement facilitates efficient activation of cytotoxic T
lymphocytes and initiates a distinct signal essential for the effector
function
(18–20).
Thus, LFA-1 activation is essential for the optimal activation of T cells.The mechanism of LFA-1 activation involves both affinity (conformational
changes within the molecule) and avidity (receptor clustering) regulation
(21–23).
The I-domain of the LFA-1 αL subunit is the primary
ligand-binding site and has been proposed to change conformation, leading to
an increased affinity for ligands
(24–26).
The structural basis of the conformational changes in the I-domain of LFA-1
has been extensively characterized
(27). Previously, we have
demonstrated that the conformation of the LFA-1 I-domain changes from the low
affinity to the high affinity state upon activation. By introducing disulfide
bonds into the I-domain, LFA-1 can be locked in either the closed or open
conformation, which represents the “low affinity” or “high
affinity” state, respectively
(28,
29). In addition, we
identified antibodies that are sensitive to the affinity changes in the
I-domain of human LFA-1 and showed that the activation-dependent epitopes are
exposed upon activation (30).
This study supports the presence of the high affinity conformation upon LFA-1
activation in cell lines. It has been demonstrated recently that therapeutic
antagonists, such as statins, inhibit LFA-1 activation and immune responses by
locking LFA-1 in the low affinity state
(31–34).
Furthermore, high affinity LFA-1 has been shown to be important for mediating
the adhesion of human T cells
(35,
36). Thus, the affinity
regulation is a critical step in LFA-1 activation.LFA-1 is a molecule of great importance in the immune system, and its
activation state influences the outcome of T cell activation. Our previous
data using the activating LFA-1 I-domain-specific antibody MEM83 indicate that
avidity and affinity of the integrin can be coupled during activation
(37). However, whether
affinity or avidity regulation of LFA-1 contributes to T cell activation
remains controversial (23,
38,
39). Despite the recent
progress suggesting that conformational changes represent a key step in the
activation of LFA-1, there are considerable gaps to be filled. When LFA-1 is
activated, the subsequent outside-in signaling contributes to T cell
activation via immunological synapse and LFA-1-dependent signaling. It is
critical to determine whether high affinity LFA-1 participates in the
outside-in signaling and affects the cellular activation of T cells.
Nevertheless, the rapid and dynamic process of LFA-1 activation has hampered
further understanding of the role of high affinity LFA-1 in primary T cell
activation. The affinity of LFA-1 for ICAM-1 increases up to 10,000-fold
within seconds and involves multiple reversible steps
(23). In addition, the
activation of LFA-1 regulates both adhesion and activation of T cells, two
separate yet closely associated cellular functions. When LFA-1 is
constitutively expressed in the active state in mice, immune responses are
broadly impaired rather than hyperactivated, suggesting the complexity of
affinity regulation (40).
Therefore, it is difficult to dissect the mechanisms by which high affinity
LFA-1 regulates stepwise activation of T cells in the whole animal system.In the present study, we identified antibodies recognizing and blocking
different affinity states of mouse LFA-1. These reagents allowed us to
determine the role of affinity regulation in T cell activation. We found that
blocking high affinity LFA-1 inhibited IL-2 production and proliferation in T
cells. Furthermore, there is a differential requirement of high affinity LFA-1
in antigen-specific activation of CD4+ and CD8+ T cells.
The activation of CD4+ T cells depends on both high and low
affinity LFA-1. For CD8+ T cell activation, only high affinity
LFA-1 provides co-stimulation. Thus, affinity regulation of LFA-1 is critical
for the activation and proliferation of naive T cells. 相似文献