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-Mediated Apoptosis Is Initiated in Caveolae-Like Domains1
Ilchun Molecular Medicine Institute Medical Research Center, Cancer Research Center, and Department of Biochemistry, Seoul National University College of Medicine, Seoul, Korea
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Abstract |
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, Raf-1, phospholipase C
1, and
tyrosine phosphoproteins. Immunoblotting and immunofluorescent data
showed that TNF receptor 1 colocalized with CD36 in CLDs, suggesting
that TNF--initiated apoptosis occurs in CLDs. When cells were
incubated with lipoprotein-deficient medium, the cholesterol
concentration was greatly decreased in CLDs but not in other fractions,
implying that the CLDs were selectively disrupted. In the CLD-disrupted
cells, the surface expression of TNF receptor 1 and CD36 was
significantly reduced. Analysis of cellular morphology, percent DNA
fragmentation, DNA laddering, and caspase-3 activity showed that
TNF--mediated apoptosis was blocked in CLD-disrupted cells, whereas
anti-Fas-mediated apoptosis was not. Since Fas was not found in
CLDs of Jurkat cells, apoptosis by Fas ligation might not require CLDs.
Taken together, these data strongly imply that TNF--mediated
apoptosis is initiated in CLDs. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is known to trigger apoptosis by binding to a specific
receptor, the TNF receptor
(TNFR)4 (1, 2, 3). The TNFR
superfamily also includes Fas, nerve growth factor receptor, CD40,
CD30, and others (4, 5). Whereas all these receptors have sequence
homology in cysteine-rich pseudorepeats in their extracellular domains,
the C-terminals of TNFR1 and Fas also share a sequence homology,
defined as the death domain, that is required for apoptosis signaling
(4). Using the yeast two-hybrid system, other death domain-containing
proteins, such as TNFR-associated death domain protein (TRADD) and
Fas-associated death domain protein (FADD), have also been identified
(6, 7). Upon cellular activation by TNF- or Fas ligand (FasL), TRADD
and FADD are recruited to TNFR1 and Fas, respectively (6, 7). In
addition, TRADD interacts directly with FADD (8), implying that TNF and
FasL signaling might converge at FADD for the purpose of cell death. Ceramide, which is generated from sphingomyelin by sphingomyelinase (SMase) activation, is known to be a second messenger inducing apoptosis (9, 10). Although it is unclear whether the ceramide produced upon TNF stimulation is directly involved in apoptosis, the ceramide produced by neutral SMase mediates the Raf/MEK/MAP kinase pathway (MEK = MAP/extracellular signal-related kinase kinase) (11, 12, 13, 14, 15). The site of ceramide formation and the possible compartmentalization of signaling pools of sphingomyelin remain poorly understood. However, detergent-insoluble complexes or caveolae have been suggested to be the plasma membrane compartments producing ceramide, since they are enriched in sphingomyelin (16, 17) and mediate IL-1ß- or nerve growth factor-activated conversion of sphingomyelin to ceramide (18, 19).
Caveolae are flask-shaped invaginations of plasma membrane coated by a 22-kDa structural protein called caveolin (20). Caveolae have been isolated from other membrane components on the basis of their detergent insolubility and low density (21, 22). The detergent-insoluble complexes are enriched in caveolin, glycosylphosphatidylinositol (GPI)-anchored proteins, cholesterol, and glycosphingolipid (21). However, caveolin-rich domains could be separated from GPI-anchored protein-rich domains by coating endothelial cells with silica beads (23). In addition, neuroblastoma cells and lymphocytes present detergent-insoluble complexes without caveolin expression and morphologic plasma membrane invaginations, i.e., caveolae, implying that caveolin is not necessary to form a detergent-insoluble complex (24, 25). The detergent-insoluble complexes from these cells and unicellular organisms such as Saccharomyces cerevisiae (26) and Dictyostelium discoideum (27) have the same biochemical properties as caveolae and, therefore, have been called caveolae-like domains (CLDs) (28).
Caveolae or CLDs have been implicated in the range of signaling events. Specific signaling events originating in caveolae or CLDs were reported as follows: epidermal growth factor-stimulated Raf-1 recruitment (29), caveolin-1 phosphorylation by Src kinase (30), the platelet-derived growth factor receptor kinase cascade (17, 31, 32), TCR signal transduction cascade (33), turnover of phosphatidylinositol 4,5-bisphosphate (34), and conversion of cellular prion protein to scrapie prion protein (28).
To address whether CLDs are major sites mediating apoptosis signals we
investigated the presence of TNFR1 in CLDs of U937 cells. We
demonstrated that TNFR1 is predominantly localized in CLDs, and that
apoptosis by TNF is blocked in CLD-disrupted cells, indicating that
TNF--mediated apoptosis is initiated in the specific plasma membrane
compartments, CLDs.
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Materials and Methods |
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[6-3H]Thymidine (25.0 Ci/mmol) and an enhanced
chemiluminescence substrate kit were obtained from Amersham (Aylesbury,
U.K.). TNF- and anti-Fas used for apoptosis induction were
purchased from Upstate Biotechnology (Lake Placid, NY), the cholesterol
test kit was obtained from Boehringer Mannheim (Mannheim, Germany), and
lipoprotein-deficient serum was obtained from Sigma (St. Louis, MO).
The CPP32/caspase-3 colorimetric protease assay kit was obtained from
Medical & Biological Laboratories Co. (Nagoya, Japan).
Antibodies for immunoblotting or immunofluorescence were obtained from
the following sources. Anti-caveolin-1, anti-Fyn, anti-PKC,
anti-Raf-1, anti-Fas, and anti-tyrosine phosphoprotein were
purchased from Transduction Laboratories (Lexington, KY). Anti-CD36 was
purchased from SeroTech (Oxford, U.K.), and anti-TNFR1 was obtained
from R & D Systems (Minneapolis, MN). Anti-PLC1 was obtained from
Dr. P.-G. Seo (PosTech University, Pohang, Korea).
Cell culture
U937 and Jurkat cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated FBS and 50 µg/ml of streptomycin and penicillin in a 5% CO2 incubator at 37°C. NIH-3T3 cells were grown in DMEM (high glucose) supplemented with 10% heat-inactivated calf serum. For cholesterol-depleting conditions, cells grown in 10% FBS-containing medium were washed twice with serum-free medium and incubated for 24 h in medium with 10% lipoprotein-deficient serum.
Isolation of CLDs and alkaline phosphatase assay
CLDs were isolated according to the method of Pike and Casey (34) with little modification. Briefly, 8 x 108 cells were rapidly chilled in liquid nitrogen and washed twice with cold PBS by centrifugation (600 x g, 5 min, 4°C). The collected cells were incubated with 1 ml of HEPES buffer (25 mM HEPES (pH 7.4), 150 mM NaCl, and 1% Triton X-100) containing protease inhibitors (1 mM PMSF, aprotinin, 10 µg/ml soybean trypsin inhibitor, 10 µM leupeptin, and 1 µg/ml pepstatin A) and phosphatase inhibitors (1 mM NaF and 1 mM Na3VO4) for 20 min, and homogenized 20 times with a tight Dounce homogenizer (Kontes, Vinland, NJ). The extract was mixed with 1 ml of 2.5 M sucrose, transferred to an SW41 centrifuge tube, and overlaid with 6 ml of 30% sucrose solution and 4 ml of 5% sucrose solution containing 25 mM HEPES (pH 6.5) and 150 mM NaCl. The discontinuous sucrose gradients were centrifuged for 21 h at 4°C in an SW41 rotor at 39,000 rpm. The gradient was fractionated into 10 fractions from the bottom to the top. Cholesterol and protein concentrations were determined using a cholesterol test kit and the bicinchoninic acid method (Pierce, Rockford, IL), respectively, according to the manufacturer’s protocols.
Alkaline phosphatase activity was measured according to the method of Liu and Anderson (18). PBS-washed filter paper was placed in a Bio-Rad Bio-Dot apparatus (Hercules, CA) and overlaid with nitrocellulose membrane that was washed with PBS. The excess PBS was removed by suction, and 50 µl of sample was loaded in each well. A vacuum was applied to the apparatus to transfer proteins in the sample on a nitrocellulose membrane. The membrane was then washed with 50 ml of PBS and developed using 50 ml of substrate from Bio-Rad alkaline phosphatase substrate kit. The reaction was stopped by membrane washing with water.
SDS-PAGE and Western blotting
Proteins were concentrated by TCA/acetone precipitation and resuspended in 300 µl of 2x SDS sample buffer. After 35 µl of proteins were run in SDS-polyacrylamide gels and transferred to a nitrocellulose membrane, Ags were visualized by sequential treatment with specific Abs, HRP-conjugated secondary antibodies, and an enhanced chemiluminescence substrate kit.
Immunofluorescence
Cells were fixed in 10% formaldehyde in PBS for 20 min and permeabilized in 0.5% Triton X-100 in PBS for 5 min at room temperature. The cells were incubated with blocking buffer (5% BSA in PBS), and primary Abs (goat anti-TNFR1 and mouse anti-CD36 or goat anti-TNFR1 and mouse anti-caveolin-1). The primary antibodies were detected with fluorescein-conjugated anti-goat IgG and rhodamine-conjugated anti-mouse IgG. Cells were observed with a Bio-Rad confocal microscope (MRC 1024).
Apoptosis assay
Cells (2 x 106 cells) were lysed in 500 µl of lysis buffer (100 mM Tris-HCl (pH 8.0), 0.5 mM EDTA, 0.2 M NaCl, 0.2% SDS, and 0.1 mg/ml proteinase K). After overnight incubation at 37°C, DNA was precipitated by adding an equal volume of isopropanol, dissolved in 20 µl of Tris-HCl, pH 8.0, containing 1 mM EDTA and 0.1 mg/ml RNase A, and incubated at 37°C for 30 min. The DNA was analyzed by 2% agarose gel electrophoresis with 0.5 µg/ml ethidium bromide.
The quantitative apoptosis assay was followed according to the method
of Duke and Cohen (35). Briefly, 1 x 106/ml cells
were incubated with [3H]thymidine (1 µCi/ml) for 2
h to label DNA, then washed with serum-free medium. After treatment
with TNF- or anti-Fas, cells were transferred to a
microcentrifuge tube labeled B and microcentrifuged at 4000 rpm for 10
min at 4°C, and the supernatant was transferred to a tube labeled S.
The cell pellet was lysed with TTE solution (10 mM Tris (pH 7.4), 1 mM
EDTA, and 0.2% Triton X-100) for 10 min and microcentrifuged at 14,000
rpm for 10 min at 4°C, and the supernatant was transferred to a tube
labeled T. The pellet after microcentrifugation was resuspended with
0.5 ml of STE solution (1% SDS, 10 mM Tris (pH 7.4), and 1 mM EDTA).
The radioactivity from each tube was determined by liquid scintillation
counting, and the percent DNA fragmentation was calculated as
following: % DNA fragmentation = 100 x (S + T)/(S + T + B).
Measurement of caspase-3 activity
Caspase-3 activity was measured according to the manufacturer’s protocol. Cells (7 x 106) were lysed with 300 µl of chilled cell lysis buffer. After microcentrifugation (10,000 x g, 1 min, 4°C), the clear supernatant was used for caspase-3 colorimetric protease assay. The protein (170 µg) was mixed with 50 µl of 2 x reaction buffer (containing 10 mM DTT) and 5 µl of 4 mM Asp-Glu-Val-Asp-p-nitroanilide (200 µM final concentration). After incubation at 37°C for 2 h, samples were read at 405 nm in a microtiter plate reader.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using their detergent insolubility and low density, CLDs were
isolated from U937 cells. After ultracentrifugation, there was an
opaque band at a density of the boundary between 30 and 5% sucrose,
which contained the CLDs. The sucrose gradient was fractionated into 10
fractions for further analysis. To certify whether the Triton
X-100-insoluble fraction contained the signaling molecules reported to
be present in CLDs, we analyzed each fraction for these species by
immunoblotting. Caveolin-1, a caveolar marker protein, was not present
in any fraction (data not shown), suggesting that U937 cells do not
have morphologic caveolar structures. Fig. 1
shows that alkaline phosphatase, a
GPI-anchored protein, as well as Fyn and phosphotyrosine proteins were
predominantly present in the CLDs. In addition, small quantities of
PKC, Raf-1, and PLC1 were also found in the CLDs. Especially,
PLC1 found in the CLDs could activate the turnover of
phosphatidylinositol 4,5-biphosphate, which is mediated by the Triton
X-100-insoluble complex (34). The presence of signaling molecules in
the CLDs suggests that the CLDs of U937 cells might be involved in
various signaling events.
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To address the possibility that CLDs mediate the apoptotic
pathway, we analyzed TNFR1 and CD36 in CLDs by immunoblotting and
immunofluorescence with anti-TNFR1; since CD36 is enriched in the
Triton X-100-insoluble complex (21, 36), CD36 was used as a marker of
CLDs. Immunoblotting data (Fig. 2A) showed that TNFR1 exists
predominantly in CLDs, strongly suggesting that TNF--mediated
apoptosis could be initiated in CLDs. Exactly the same pattern was seen
when the samples were immunoblotted with anti-CD36.
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B). In optical sections
through the middle of the cell, staining of CD36 and TNFR1 appeared in
small, bright patches around the cell periphery (Fig. 2B).
This pattern was quite distinct from the faint, nonspecific
fluorescence seen in cells stained with secondary antibody alone. Also,
the presence of CD36 and TNFR1 was visualized by double
immunofluorescence to demonstrate that these molecules are colocalized.
Fig. 2B indicates that these two proteins are indeed
colocalized, showing the same patched and punctate staining pattern.
We used NIH-3T3 cells to verify the location of TNFR1. Anti-TNFR1 IgG
fluorescence staining was concentrated in patches on the cell surface
(Fig. 2C). These patches were appeared as stretches along
the margin of the cell or irregularly shaped regions over the cell
body. The same cell processed using an anti-caveolin-1 IgG had
nearly an identical staining pattern. A comparison of both images
showed extensive colocalization of the two Ags. This suggests that
TNFR1 is concentrated in caveolin-rich domains. Since molecules in
caveolae or caveolae-like domains appeared colocalized in indirect
immunofluorescence, we conclude that TNFR1 is indeed localized in
caveolae or caveolae-like domains.
The disruption of CLDs reduces the surface expression of TNFR1 and CD36
Cholesterol is a building block of caveolae or CLDs and appears to be required for the maintenance of their structure (22). Caveolae disappeared in cells that were depleted of cholesterol or exposed to sterol-binding agents, such as filipin and nystatin (22). Disassembly of caveolae means disruption of signal transduction via caveolae (37). MDCK cells incubated in low density lipoprotein-deficient medium showed the reduced surface expression of gD1-DAF (38), a GPI-anchored protein that is predominantly localized in detergent-insoluble complexes. In addition, cells treated with lovastatin, a cholesterol synthesis inhibitor, lost their ability to convert cellular prions to scrapie prions (39), both forms of which are enriched in CLDs (28). These results indicate that cholesterol depletion inhibits the cell surface expression of molecules present in caveolae or CLDs and blocks signaling events through caveolae or CLDs.
When cells were stained with trypan blue after incubation with
lipoprotein-deficient medium for 24 h to disrupt CLDs, they still
appeared morphologically healthy and viable, although the total
cellular cholesterol concentration was 62.8% that found in normal
cells. The cholesterol concentrations were 13.6 and 8.1 µg/g protein,
respectively, in normal and lipoprotein-deficient conditions. Fig. 3A shows the concentrations of
cholesterol and protein, respectively, in isolated CLDs. Approximately
53% of the cholesterol in normal cells was recovered in CLDs. There
was a significant decrease in cholesterol in the lipoprotein-deficient
condition (30% of normal condition) considering the cholesterol amount
only in CLDs (fractions 4 and 5). However, the amount of cholesterol in
the bottom fractions (fractions 9 and 10) did not change. The protein
concentration in CLDs, accounting for <1% of the total proteins,
remained constant in both conditions (Fig. 3A). From these
results, it was concluded that cells grown in lipoprotein-deficient
medium have selectively disrupted CLDs.
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B) showed that the surface
expression of TNFR1 and CD36 was greatly reduced in cells grown under
lipoprotein-deficient conditions. When the relative signal densities of
TNFR1 and CD36 were measured by confocal microscopy, cells incubated in
lipoprotein-deficient medium lost 64.7 and 64.6% of their signal
density, respectively. When the amount of immunofluorescence of TNFR1
was measured by FACS scanning, cells grown in lipoprotein-deficient
medium also showed a great decrease in TNFR1 fluorescence compared with
cells grown under normal conditions (data not shown). With confocal
microscopic and FACS scanning data, it is concluded that the surface
expression of TNFR1 is greatly reduced in cells grown in
lipoprotein-deficient medium.
Disruption of CLDs blocks TNF--mediated apoptosis, but not
Fas-mediated apoptosis
To address whether CLDs are required for TNF- signaling,
apoptosis by TNF- was measured in cells grown in normal or
lipoprotein-deficient medium. The cells were preincubated with
serum-free medium for 2 h and treated with 5 nM TNF- or 0.5
µg/ml anti-Fas for 3 h, and cell morphology was observed
under the inverted microscope. Following treatment with TNF-,
blebbing morphology appeared in cells grown under normal conditions,
but not under lipoprotein-deficient conditions (data not shown).
However, there was no difference in the blebbing morphology under both
conditions after treatment with anti-Fas (data not shown). The same
results were obtained with respect to intranucleosomal DNA laddering
and quantitative DNA fragmentation (Fig. 4, A and B).
Following treatment with TNF-, DNA fragmentation appeared in cells
grown under normal conditions, but only a small amount was observed
under lipoprotein-deficient conditions. However, there was no
difference in the DNA fragmentation under both conditions after
treatment with anti-Fas, suggesting that Fas-mediated apoptosis is
not affected in CLD-disrupted cells.
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(5 nM) or anti-Fas (1
µg/ml) for 3 h under normal and lipoprotein-deficient conditions
(Fig. 4C). Following treatment of TNF-, caspase-3
activity was highly increased (4.3-fold increase over untreated cells)
in cells grown under normal conditions, but its activity under
lipoprotein-deficient conditions was 39% of normal. Meanwhile, there
was little difference in caspase-3 activity under both conditions after
treatment with anti-Fas. Its activity under normal conditions was
increased 3.4-fold over that in untreated cells, whereas under
lipoprotein-deficient conditions it was 94% of normal. These data
suggest that the membrane compartments for TNF and Fas signaling could
be spatially separated. Since Fas was not detected in U937 cells by
immunoblotting, we selected Jurkat cells expressing high level of Fas
to investigate whether Fas is localized on CLDs. Fig. 5 shows that CLDs of Jurkat cells
harbored exclusively alkaline phosphatase. However, Fas was found in
only the bottom fraction, not in the CLD fraction, explaining why
apoptosis by Fas ligation was not influenced in cells grown in
lipoprotein-deficient medium, which disrupted CLDs. Taken together, we
conclude that the disruption of CLDs blocked TNF--mediated
apoptosis, but not anti-Fas-mediated apoptosis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been reported that Raf-1 is recruited to the plasma membrane,
especially to caveolae, after epidermal growth factor stimulation (29).
Raf-1 in caveolae is able to phosphorylate MAP kinase kinase,
suggesting that the MAP kinase pathway (from receptor to MAP kinase)
might be mediated via caveolae (32). If that is true, TNF-- or
ceramide-mediated MAP kinase pathway might also be mediated in caveolae
or CLDs, since Raf-1 is also phosphorylated and activated by TNF or
ceramide (11, 12). In addition, we cannot exclude the possibility that
CLDs are major membrane compartments for recruiting FAN and neutral
SMase, which are known to be localized in the plasma membrane (10, 12),
and for converting sphingomyelin to ceramide after TNF-
stimulation.
The fate of any T cell, whether it is developing in the thymus or
functioning in the peripheral immune system, is dependent on TCR
specificity for Ags presented by MHC molecules and on the consequences
of TCR-generated intracellular signaling pathways that lead to
activation, anergy, or apoptosis (42, 43). Upon TCR stimulation, TCRs,
phospho-ZAP70, and phosphotyrosine proteins are recruited to the
detergent-insoluble microdomain (31). Apo2L/TRAIL (TNF-related
apoptosis-inducing ligand) is one TNF family member and induces
apoptosis by its ligation (2). Apo2L/TRAIL-mediated apoptosis is
blocked by transfecting decoy receptors (44), which are GPI-anchored
proteins, and might be localized in CLDs. CD20 is a nonglycosylated 33-
to 37-kDa phosphoprotein involved in B cell signaling that subserves
important functions in the regulation of B cell proliferation and
differentiation. Extensive cross-linking of CD20 with murine
anti-CD20 mAbs in the presence of either goat anti-mouse IgG or
Fc receptor-expressing cells directly inhibits B cell proliferation,
induces nuclear DNA fragmentation, and leads to cell death by apoptosis
(45). Binding of CD20 Abs to B cells induces the rapid redistribution
of up to 95% of CD20 molecules to low density, detergent-insoluble
microdomains and induces the appearance of an approximately 50-kDa
phosphorylated tyrosine protein in the same compartment (46, 47). With
these findings for TCR, Apo2L, and CD20 and our data for TNF-, we
strongly suggest that CLDs might be a specific plasma membrane
compartment regulating apoptosis.
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Acknowledgments |
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Footnotes |
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2 Current address: Center for ARS Network, National Creative Research Initiatives, Sung Kyun Kwan University, 300 Chunchun-Dong, Jangan-Ku, Suwon, Kyunggi-Do 440-746, Korea. E-mail address:
3 Address correspondence and reprint requests to Dr. Jeong-Sun Seo, Ilchun Molecular Medicine Institute Medical Research Center and Department of Biochemistry, Seoul National University College of Medicine, 28 Yongon-Dong, Jongno-Ku, Seoul 110-799, Korea. E-mail address:
4 Abbreviations used in this paper: TNFR1, TNF receptor 1; TRADD, TNFR-associated death domain protein; FADD, Fas-associated death domain protein; FasL, Fas ligand; SMase, sphingomyelinase; MAP, mitogen-activated protein; GPI, glycosylphosphatidylinositol; CLDs, caveolae-like domains; PKC, protein kinase C; PLC, phospholipase C; FAN, factor associated with neutral SMase activation.
Received for publication December 29, 1998. Accepted for publication April 5, 1999.
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References |
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B activation. Cell 81:495.[Medline]
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J. E. S. Doan, D. A. Windmiller, and D. W. H. Riches Differential Regulation of TNF-R1 Signaling: Lipid Raft Dependency of p42mapk/erk2 Activation, but Not NF-{kappa}B Activation J. Immunol., June 15, 2004; 172(12): 7654 - 7660. [Abstract] [Full Text] [PDF]
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D. J. Powell, E. Hajduch, G. Kular, and H. S. Hundal Ceramide Disables 3-Phosphoinositide Binding to the Pleckstrin Homology Domain of Protein Kinase B (PKB)/Akt by a PKC{zeta}-Dependent Mechanism Mol. Cell. Biol., November 1, 2003; 23(21): 7794 - 7808. [Abstract] [Full Text] [PDF]
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 F. C. Bender, J. C. Whitbeck, M. Ponce de Leon, H. Lou, R. J. Eisenberg, and G. H. Cohen Specific Association of Glycoprotein B with Lipid Rafts during Herpes Simplex Virus Entry J. Virol., September 1, 2003; 77(17): 9542 - 9552. [Abstract] [Full Text] [PDF]
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 C. Rodemer, T.-P. Thai, B. Brugger, T. Kaercher, H. Werner, K.-A. Nave, F. Wieland, K. Gorgas, and W. W. Just Inactivation of ether lipid biosynthesis causes male infertility, defects in eye development and optic nerve hypoplasia in mice Hum. Mol. Genet., August 1, 2003; 12(15): 1881 - 1895. [Abstract] [Full Text] [PDF]
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K. A. Cho, S. J. Ryu, J. S. Park, I. S. Jang, J. S. Ahn, K. T. Kim, and S. C. Park Senescent Phenotype Can Be Reversed by Reduction of Caveolin Status J. Biol. Chem., July 18, 2003; 278(30): 27789 - 27795. [Abstract] [Full Text] [PDF]
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N. Harper, M. Hughes, M. MacFarlane, and G. M. Cohen Fas-associated Death Domain Protein and Caspase-8 Are Not Recruited to the Tumor Necrosis Factor Receptor 1 Signaling Complex during Tumor Necrosis Factor-induced Apoptosis J. Biol. Chem., July 3, 2003; 278(28): 25534 - 25541. [Abstract] [Full Text] [PDF]
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H. Ha, H. B. Kwak, S. K. Lee, D. S. Na, C. E. Rudd, Z. H. Lee, and H.-H. Kim Membrane Rafts Play a Crucial Role in Receptor Activator of Nuclear Factor kappa B Signaling and Osteoclast Function J. Biol. Chem., May 9, 2003; 278(20): 18573 - 18580. [Abstract] [Full Text] [PDF]
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C. Munoz-Pinedo, C. Ruiz-Ruiz, C. Ruiz de Almodovar, C. Palacios, and A. Lopez-Rivas Inhibition of Glucose Metabolism Sensitizes Tumor Cells to Death Receptor-triggered Apoptosis through Enhancement of Death-inducing Signaling Complex Formation and Apical Procaspase-8 Processing J. Biol. Chem., April 4, 2003; 278(15): 12759 - 12768. [Abstract] [Full Text] [PDF]
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 R. Barsacchi, C. Perrotta, S. Bulotta, S. Moncada, N. Borgese, and E. Clementi Activation of Endothelial Nitric-Oxide Synthase by Tumor Necrosis Factor-alpha : A Novel Pathway Involving Sequential Activation of Neutral Sphingomyelinase, Phosphatidylinositol-3' kinase, and Akt Mol. Pharmacol., April 1, 2003; 63(4): 886 - 895. [Abstract] [Full Text] [PDF]
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T. Garofalo, R. Misasi, V. Mattei, A. M. Giammarioli, W. Malorni, G. M. Pontieri, A. Pavan, and M. Sorice Association of the Death-inducing Signaling Complex with Microdomains after Triggering through CD95/Fas. EVIDENCE FOR CASPASE-8-GANGLIOSIDE INTERACTION IN T CELLS J. Biol. Chem., February 28, 2003; 278(10): 8309 - 8315. [Abstract] [Full Text] [PDF]
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V. Cottin, J. E. S. Doan, and D. W. H. Riches Restricted Localization of the TNF Receptor CD120a to Lipid Rafts: A Novel Role for the Death Domain J. Immunol., April 15, 2002; 168(8): 4095 - 4102. [Abstract] [Full Text] [PDF]
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 P. E. Bickel Lipid rafts and insulin signaling Am J Physiol Endocrinol Metab, January 1, 2002; 282(1): E1 - E10. [Abstract] [Full Text] [PDF]
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 B. Krishnamoorthy, B. Darnay, B. Aggarwal, D. H. Dinh, G. Kouraklis, W. C. Olivero, M. Gujrati, and J. S. Rao Glioma Cells Deficient in Urokinase Plaminogen Activator Receptor Expression Are Susceptible to Tumor Necrosis Factor-{alpha}-related Apoptosis-inducing Ligand-induced Apoptosis Clin. Cancer Res., December 1, 2001; 7(12): 4195 - 4201. [Abstract] [Full Text] [PDF]
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 J. Liu, P. Lee, F. Galbiati, R. N. Kitsis, and M. P. Lisanti Caveolin-1 expression sensitizes fibroblastic and epithelial cells to apoptotic stimulation Am J Physiol Cell Physiol, April 1, 2001; 280(4): C823 - C835. [Abstract] [Full Text] [PDF]
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R. F. Thorne, J. F. Marshall, D. R. Shafren, P. G. Gibson, I. R. Hart, and G. F. Burns The Integrins alpha 3beta 1 and alpha 6beta 1 Physically and Functionally Associate with CD36 in Human Melanoma Cells. REQUIREMENT FOR THE EXTRACELLULAR DOMAIN OF CD36 J. Biol. Chem., November 3, 2000; 275(45): 35264 - 35275. [Abstract] [Full Text] [PDF]
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S. Bulotta, R. Barsacchi, D. Rotiroti, N. Borgese, and E. Clementi Activation of the Endothelial Nitric-oxide Synthase by Tumor Necrosis Factor-alpha . A NOVEL FEEDBACK MECHANISM REGULATING CELL DEATH J. Biol. Chem., February 23, 2001; 276(9): 6529 - 6536. [Abstract] [Full Text] [PDF]
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X. Feng, M. L. Gaeta, L. A. Madge, J.-H. Yang, J. R. Bradley, and J. S. Pober Caveolin-1 Associates with TRAF2 to Form a Complex That Is Recruited to Tumor Necrosis Factor Receptors J. Biol. Chem., March 9, 2001; 276(11): 8341 - 8349. [Abstract] [Full Text] [PDF]
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H. Grassme, A. Jekle, A. Riehle, H. Schwarz, J. Berger, K. Sandhoff, R. Kolesnick, and E. Gulbins CD95 Signaling via Ceramide-rich Membrane Rafts J. Biol. Chem., June 1, 2001; 276(23): 20589 - 20596. [Abstract] [Full Text] [PDF]
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A. Cremesti, F. Paris, H. Grassme, N. Holler, J. Tschopp, Z. Fuks, E. Gulbins, and R. Kolesnick Ceramide Enables Fas to Cap and Kill J. Biol. Chem., June 22, 2001; 276(26): 23954 - 23961. [Abstract] [Full Text] [PDF]
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W.-Y. Park, J.-S. Park, K.-A Cho, D.-I. Kim, Y.-G. Ko, J.-S. Seo, and S. C. Park Up-regulation of Caveolin Attenuates Epidermal Growth Factor Signaling in Senescent Cells J. Biol. Chem., June 30, 2000; 275(27): 20847 - 20852. [Abstract] [Full Text] [PDF]
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