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Departments of Medicine (A.R., W.L.S.-P.) and Pediatrics (K.S.), University of Alberta, Edmonton, Alberta, Canada T6G 2S2; and Department of Biochemistry and Molecular Biology, New Jersey Medical School (K.S., S.C.), Newark, New Jersey 07103
Address all correspondence and requests for reprints to: Alex Rabinovitch, M.D., 430 Heritage Medical Research Centre, University of Alberta, Edmonton, Canada T6G 2S2. E-mail: alex.rabinovitch{at}ualberta.ca
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, rat
calbindin-D28k was stably expressed in the pancreatic islet
ß-cell line, ßTC-3. Calbindin-D28k expression resulted
in increased cell survival in the presence of the cytotoxic combination
of the cytokines IL-1ß (30 U/ml), TNF (103 U/ml), and
interferon 
(103 U/ml). The greatest protection was
observed in the ßTC-3 cell clone expressing the highest concentration
of calbindin-D28k. Apoptotic cell death was detected by
annexin V staining and by the TdT-mediated dUTP-X nick end
labeling assay in vector-transfected ßTC-3 cells incubated with
cytokines (14–15% apoptotic cells). The number of apoptotic cells was
significantly decreased in calbindin-D28k-overexpressing
ßTC-3 cells incubated with cytokines (5–6% apoptotic cells). To
address the mechanism of the antiapoptotic effects of calbindin,
studies were done to examine whether calbindin inhibits free radical
formation. The stimulatory effects of the cytokines on lipid
hydroperoxide, nitric oxide, and peroxynitrite production were
significantly decreased in the calbindin-D28k-expressing
ßTC-3 cells. Our findings indicate that calbindin-D28k,
by inhibiting free radical formation, can protect against
cytokine-mediated apoptosis and destruction of ß-cells. These
findings suggest that calbindin-D28k may be an important
regulator of cell death that can protect pancreatic islet
ß-cells from autoimmune destruction in type 1 diabetes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,
and TNF can inhibit insulin biosynthesis and release and decrease
ß-cell viability (2, 3). In addition, studies in
vivo have indicated diabetogenic roles for these cytokines.
Transgenic expression of IFN (4) or TNF
(5) in islet ß-cells was found to lead to loss of
pancreatic islet tolerance; and, in nonobese diabetic (NOD) mice,
IFN mRNA expression was found to correlate with ß-cell destructive
insulitis (6, 7). Furthermore, the development of
autoimmune diabetes in NOD mice (8, 9) or
BioBreeding rats (10) can be prevented by treatment
with antibodies to IFN. Cytokine-induced ß-cell destruction has
been reported to involve production of oxygen free radicals
(11) and the nitric oxide radical (NO·)
(12). Cytotoxic actions of radicals include DNA damage and
islet cell death by apoptosis (13). Apoptosis has been
reported as a mechanism of cytokine-mediated islet ß-cell destruction
(14, 15, 16), and characteristics of apoptosis have been noted
in the islet ß-cells of NOD mice (17). Calbindin-D28k is a cytosolic calcium-binding protein expressed in high levels in avian intestine and in avian and mammalian kidney (where it is induced by vitamin D and thought to act as a facilitator of calcium diffusion) (18, 19). Calbindin-D28k is also expressed in many other tissues that are not primary regulators of serum calcium, including pancreas and brain (18, 19). Previous studies using islets isolated from calbindin-D28k knockout mice and islet ß-cell lines established a role for calbindin in the modulation of depolarization-stimulated insulin release (20). Calbindin has also been reported to prevent apoptotic cell death in different cell types (21), including motor neuron hybrid cells (22), glial cells (23), PC 12 cells (24), and lymphocytes (25). In a more recent study, calbindin-D28k was reported to suppress cytokine-mediated apoptotic cell death in osteoblasts (26). In the present study, we report that calbindin-D28k inhibits cytokine-induced free radical formation and protects against cytokine-mediated apoptosis and destruction of an islet ß-cell line. Our findings suggest that calbindin may be an important regulator of cell death that can protect pancreatic islet ß-cells from autoimmune destruction in type 1 diabetes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-CD28, was composed of pBSR (a gift from Dr. Michael Olszowy,
WA University School of Medicine, St. Louis, MO) that uses the
SV40 promoter to drive the expression of
calbindin-D28k cDNA isolated by PCR from cDNA
prepared from rat renal distal tubular mRNA (29). Ten
micrograms each of pBSR vector alone or pBSR-CD28 were mixed with
50 µl LipofectAMINE reagent (Life Technologies, Inc.),
then added to cells in Opti-MEM reduced serum medium (Life Technologies, Inc.) with 2% FBS, and incubated for 20 h at
37 C. Normal growth medium was added the next day. Three days after
transfection, selection began with increasing amounts of G418, to a
final concentration of 800 µg/ml. After 8 wk of G418 selection,
colonies were picked under sterile conditions and grown in T25 culture
flasks (VWR Canlab, Mississauga, Ontario, Canada) to allow 60–70%
confluency before cell dissociation for the different studies.
Western blot analysis
ßTC-3, ßHC-13, and RIN-m5F cells from vector- and
calbindin-transfected cell lines were washed twice with ice-cold PBS,
scraped off the T25 culture flasks, lysed in Laemmli sample buffer
(30), and boiled for 5 min. The supernatant solution
obtained after centrifugation at 14,000 x g for 15 min
at 4 C was used to measure total protein, by the bicinchoninic acid
(BCA) method, using a Micro BCA protein assay reagent kit
(Pierce Chemical Co., Rockford, IL).
Calbindin-D28k expression in the different cell
lines was determined by Western blot analysis (24),
modified to quantitate the levels of
calbindin-D28k protein expressed. Cell protein
extracts and calbindin-D28k protein standards
(0.1, 0.5, 1, 5, and 10 µg protein) were diluted in Lane
marker-reducing sample buffer (Pierce Chemical Co.) and
submitted to electrophoresis on a 4–20% precast SDS-PAGE gel
(Bio-Rad Laboratories, Inc., Hercules, CA) and then
transferred to a Hytrand C nitrocellulose sheet (Amersham Pharmacia Biotech, Baie d’Urfe, Québec, Canada). The
nitrocellulose sheet was blocked for 30 min with Tris-buffered saline
(pH 7.4) containing 5% dry milk, and then incubated for 1 h with
a 1:1000 dilution of a rabbit antirat antibody to
calbindin-D28k generated and characterized as
described previously (31). The sheet was washed with
Tris-buffered saline containing 0.06% Tween-20
(Sigma, St. Louis, MO) and then incubated for 30
min with peroxidase (POD)-labeled goat antirabbit antibody diluted
1:25,000 (Amersham Pharmacia Biotech). The
detection step was performed using Dura signal chemiluminescence
reagent (Pierce Chemical Co.), and the air-dried sheet was
exposed to Hyperfilm linearized BioMax-MR ECL (Amersham Pharmacia Biotech). Optical densities of the protein bands were analyzed
by using a Fluor-S Max MultiImager integrated to Quantity One software
(Bio-Rad Laboratories, Inc.).
Immunocytochemical studies
ßTC-3 cells were removed from the culture flasks by incubation
in PBS-based, enzyme-free cell dissociation buffer (Life Technologies, Inc.) at 37 C for 10 min, followed by syringe
injection through progressively narrower gauge needles (from size
16–22). The cells were washed twice in PBS, then fixed with 4%
paraformaldehyde in PBS for 10 min and washed twice in PBS. The fixed
cells (104 in 10 µl PBS) were placed on glass
slides coated with 3-aminopropyltriethoxysilane (Sigma),
and the slides were air-dried and stored at -70 C until cell staining
was performed, as described previously (32). Briefly, the
fixed cells were permeabilized with 1.5% saponin (Sigma)
in PBS (PBS-saponin). Endogenous cell POD was blocked by incubation in
PBS-saponin containing 1%
H2O2, followed by 20%
normal goat serum. The cells were incubated first with a rabbit
antibody to calbindin-D28k (31),
diluted 1:500 in PBS-saponin. Control incubations were done with rabbit
Igs (Cedarlane Laboratories, Hornby, Ontario, Canada). Next, the cells
were incubated with a secondary antibody, biotinylated goat antirabbit
IgG (Zymed Laboratories, Inc., South San Francisco, CA),
then streptavidin-POD conjugate (Zymed Laboratories, Inc.)
and substrate chromogen, 3-amino 9-ethylcarbazole (Biomeda, Foster
City, CA), which stained calbindin-containing cells red. The slides
were sealed by using Crystal/Mount and Clarion mounting medium
(Biomeda).
Cell incubations with cytokines
ßTC-3, ßHC-13, and RIN-m5F cells were removed from the
culture flasks by using cell dissociation buffer as described for the
immunocytochemical studies. The cells were then washed and resuspended
in phenol red-free RPMI-1640 medium (Life Technologies, Inc.) containing 0.3 mM L-arginine, 11
mM glucose, 10% heat-inactivated FCS, 100 U/ml penicillin,
100 µg/ml streptomycin, 0.25 µg/ml Amphotericin, and
12 mM HEPES (test medium). In the first study, the effects
of cytokines on viability of vector- and calbindin-transfected cells
were examined by trypan blue and MTT assays. Cells
(103/well) were seeded in 170 µl test medium in
96-well A/2 area nontissue culture-treated plates (Sarstedt, Montreal,
Québec, Canada) for incubations to be followed by trypan blue
assays, and 105 cells/well were seeded in 170
µl test medium in 96-well A/2 area tissue culture plates (Sarstedt)
for incubations to be followed by MTT assays. Cells were incubated in
test medium alone and test medium containing the cytokine combination
of IL-1ß (30 U/ml), TNF (103 U/ml), and
IFN (103 U/ml) for 72 h. Recombinant
human IL-1ß (2–4 x 107 U/mg) was
provided by the Upjohn Co. (Kalamazoo, MI); recombinant murine TNF
(1.2 x 107 U/mg) and recombinant murine
IFN (8 x 106 U/mg) were provided by
Genentech, Inc. (South San Francisco, CA). After 72 h
incubation, cells were collected for trypan blue and MTT assays. In the
second study, the effects of cytokines on apoptosis of vector-and
calbindin-transfected ßTC-3 cells were examined by annexin V and
TUNEL assays. For these experiments, 3 x
104 cells were seeded in 120 µl test medium in
8-well culture slides (Becton Dickinson and Co.,
Bedford, MA) and incubated in test medium alone and test medium
containing the cytokine combination of IL-1ß (30 U/ml), TNF
(103 U/ml), and IFN (103
U/ml) for 72 h. After the incubations, cells attached to the
slides were examined for apoptosis by annexin V and TUNEL assays. In
the third study, the effects of cytokines on free radical production in
vector- and calbindin-transfected ßTC-3 cells were examined. For
these experiments, 5 x 105 cells were
seeded in 1.6 ml test medium in 35 x 10-mm nontissue
culture-treated Falcon dishes (Becton Dickinson and Co.)
and incubated in test medium alone and test medium containing the
cytokine combination of IL-1ß (30 U/ml), TNF
(103 U/ml), and IFN (103
U/ml) for 72 h. After the incubations, cell media were collected
for assays of lipid hydroperoxide and nitrite, and cells were collected
for nitrotyrosine assay.
Trypan blue assay
Cell viability was determined by cellular exclusion of trypan
blue. The cells were washed in PBS, incubated for 1 min in 0.2% trypan
blue in PBS (pH 7.4), and counted using a hemacytometer at 40x
magnification. The percentage of cells that stained blue (nonviable
cells) was calculated as a percentage of the total.
MTT assay
Cell viability was also determined by a colorimetric assay that
detects the reduction of 3-(4,5-dimethylthiazolyl-2)
2,5-diphenyltetrazolium bromide (MTT, Sigma) into a blue
formazan product by viable metabolically active cells, as described by
Mosmann (33) and modified by Sladowski et al.
(34).
Annexin V assay
Cells undergoing early apoptosis were identified by using an
annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit
(Kamiya Biomedical Co., Seattle, WA). This method is based on the
binding of annexin V to phosphatidylserine that translocates to the
outer plasma membrane of the apoptotic cell (35). Cells
attached to the slides were washed in PBS, incubated in 100 µl
annexin-binding buffer for 45 min at 4 C; then, 1 µl annexin V-FITC
(final concentration 0.25 µg/ml) was added, and the incubation was
continued for 30 min in the dark at 4 C. All subsequent incubations
were done in the dark. The cells were washed in PBS, fixed in 4%
paraformaldehyde in PBS for 30 min at room temperature, and washed in
PBS. The slides were air-dried, then sealed by using Crystal/Mount and
Clarion mounting medium (Biomeda). Apoptotic cells were stained green,
as detected by fluorescence microscopy (annexin
V-FITC+). Cell preparations were stained in
duplicate, and 3000 cells were scored blindly by two independent
readings in which 60 different microscopic fields were scanned (oil
immersion 60x).
TUNEL assay
Cells undergoing early apoptosis were also identified by using a
POD in situ cell death detection kit
(Roche Diagnostics, Laval, Québec, Canada).
This method is based on the detection of single- and double-stranded
DNA breaks (nicks) by the TdT-mediated dUTP-X nick end labeling (TUNEL)
method (36). Cells attached to the slides were washed in
PBS, fixed in 4% paraformaldehyde, then washed in PBS. The cells were
then incubated in 3% H2O2
in PBS for 15 min at room temperature, followed by a permeabilization
step using 3% saponin in PBS (PBS-saponin) for 2 h at room
temperature. TUNEL POD detection antibody for the TUNEL reaction
(Roche Diagnostics) was added for 2 h at room
temperature, followed by washing in PBS-saponin for 30 min. POD
converter reagent was added for 20 min at 37 C, then the cells were
washed in PBS-saponin, and an aminoethylcarbazole chromogen was added.
Apoptotic cells with DNA breaks were identified by red nuclear staining
by using brightfield light microscopy. Cell preparations were scored as
described for the annexin V apoptosis study.
Lipid hydroperoxide assay
Lipid hydroperoxides were measured by using a Lipid
Hydroperoxide assay kit (Cayman Chemical, Ann Arbor, MI). This method
is based upon the oxidation of ferrous ions to chromogenic ferric ions
by hydroperoxides (37). Lipid hydroperoxides in cell
incubation media were first extracted into chloroform, followed by a
deproteination procedure to ensure that the sample lipid hydroperoxides
are extracted quantitatively into a small volume of
chloroform-methanol. The hydroperoxide concentration was determined
based on the absorbances of sample and standard at 504 nm in a
spectrophotometer microplate reader (Molecular Devices,
Menlo Park, CA).
Nitrite assay
Nitrite in cell incubation media was determined by the method of
Green et al. (38) using HPLC equipment,
modified as described previously (11).
Nitrotyrosine assay
Sample preparation was as described by Hensley et al.
(39). Cells were briefly sonicated in 400 µl sodium
acetate (10 mM, pH 6.5), then rapidly vortexed
for 1 h, and centrifuged for 10 min at 12,000 x
g. A 50-µl aliquot of the supernatant was removed for
protein assay using a Micro BCA protein assay reagent kit (Pierce Chemical Co.). To 150 µl of the supernatant was added 25 µl
sodium acetate buffer and 50 µl pronase (1 mg/ml in acetate buffer).
The solution was then heated at 50 C for 18 h and dried in a Speed
Vac system. The dried extract was dissolved in 100 µl
ethanol:H2O (70:30) by rapid vortexing and then
centrifuged at 12,000 x g for 10 min. The supernatant
was frozen at -20 C until derivatization and quantitation by HPLC, as
described by Kamisaki et al. (40).
Derivatization of nitrotyrosine was done by adding 10 µl sodium
borate, 0.1 M (pH 8.7), and 10 µl
4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole (10 mg/ml in ethanol) to 50
µl of the ethanol:H2O solution containing islet
extract and incubating at 60 C for 2 min. The reaction was terminated
by addition of 15 µl 0.1 M HCl, and an aliquot
(50–80 µl) was injected into the HPLC column. The chromatography
procedure was as described by Kamisaki et al.
(40). The detection limit for nitrotyrosine was
approximately 1 pmol at a signal-to-noise ratio of 5.
Statistical analysis
Data are expressed as means ± SE for five to
six separate experiments. The statistical significance of difference
between mean values was determined by one-way ANOVA and the post
hoc Bonferroni multiple-comparisons test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-CB28 was determined by
immunocytochemical staining and by quantitative Western blot analysis.
In vector-transfected ßTC-3 cells, immunocytochemical analysis
indicated no cell staining for calbindin-D28k
(Fig. 1A
), and
calbindin-D28k levels were below the limits of
detection (< 5 ng/mg protein by Western blot analysis).
Immunocytochemical staining was detected in the
low-calbindin-D28k-expressing clone (Fig. 1B, 87 ± 5 ng/mg protein by Western blot analysis) and most intense
in the high-calbindin-D28k-expressing clone
(Fig. 1C; 375 ± 15 ng/mg protein by Western blot analysis).
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, and IFN significantly decreased the
survival of mouse pancreatic ßTC-3 cells transfected with vector
alone (CaBP 0), as determined by an increase in trypan blue-positive
cells (Fig. 2A) and by decreased MTT
reduction by the cells (Fig. 2B). In contrast,
calbindin-D28k-overexpressing ßTC-3 cells
exhibited resistance to cytokine-mediated cell death, as indicated by
significantly smaller increases in trypan blue-positive cells in
response to cytokines in both the low- and
high-calbindin-D28k-expressing clones (Fig. 2A).
Also, the cytotoxic effect of cytokines, as determined in the MTT
assay, was significantly decreased in the
high-calbindin-D28k-expressing ßTC-3 clone
(Fig. 2B). In addition, we found that two other islet ß-cell lines,
ßHC-13 cells and RIN-m5F cells, were protected from cytokine-induced
destruction by overexpressing calbindin-D28k
(Table 1).
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, and IFN induced apoptosis of ßTC-3 cells, as
determined by both the annexin V method (Fig. 3A) and the TUNEL method (Fig. 3B). In
contrast, the proapoptotic effects of the cytokines in both assays were
significantly decreased in
calbindin-D28k-expressing ßTC-3 cells.
Therefore, calbindin-D28k-overexpressing
ßTC-3 cells were protected from both necrosis (trypan blue uptake)
and apoptosis (annexin V and TUNEL positivity) induced by
cytokines.
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, and IFN significantly increased
lipid peroxidation by ßTC-3 cells transfected with vector alone, and
this effect was significantly decreased in
calbindin-D28k-expressing ßTC-3 cells (Fig. 4A). Similar to studies in pancreatic
islets and in other ß-cell lines (12, 13, 14), the cytokine
combination significantly increased the amount of nitrite, the stable
end product of the NO·, produced by ßTC-3 cells (Fig. 4B).
Also, cytokines significantly increased the formation of nitrotyrosine,
which is a marker of peroxynitrite (ONOO-), the
reaction product of superoxide (O2·-)
and nitric oxide free radicals (NO·) (Fig. 4C). The effects of
the cytokines on nitrite production and nitrotyrosine formation were
significantly decreased in the
calbindin-D28k-expressing ßTC-3 cells (Fig. 4, B and C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and IFN. These cytokines, in combination,
are cytotoxic to islet ß-cells in vitro (2, 3), and cytokine-induced destruction of mouse islet ß-cells
involves both apoptotic and necrotic cell death (43).
Similarly, in this study, we found that the cytokine combination of
IL-1ß, TNF, and IFN induced both apoptosis and necrosis of
ßTC-3 cells from a mouse islet ß-cell line. Our findings
demonstrate, for the first time, that the calcium-binding protein,
calbindin-D28k, can protect against
cytokine-induced ß-cell destruction. We found that overexpression of
calbindin-D28k in ßTC-3 cells, by transfecting
the cells with a calbindin-D28k cDNA construct,
protected the cells from cytokine-induced necrosis and apoptosis. In
addition, overexpression of calbindin-D28k in two
other ß-cell lines, mouse ßHC-13 cells and rat RIN-m5F cells,
protected the ß-cells from cytokine-induced destruction. Calbindin-D28k has been shown, by immunocytochemistry, to be present in islet ß-cells, and concentrations of calbindin-D28k between 3 and 400 ng/mg protein have been reported for whole pancreas (18). In the present study, we found that the level of calbindin-D28k expressed in ßTC-3, ßHC-13, and RIN-m5F ß-cell lines was low (<5–50 ng/mg protein) compared with levels expressed in the ß-cell lines transfected with calbindin-D28k cDNA (375–1900 ng/mg protein) and thereby protected from cytokine-induced destruction. When we used the calbindin-D28k expression vector to transfect calbindin-D28k in other cell lines (3T3 osteoblasts, PC12 cells, and C6 glioma cells) (24, 26), protection against cell death was observed with levels of calbindin-D28k in the same range as in the high-calbindin-D28k-expressing ßTC-3 clone in the present study.
Nitric oxide and oxygen free radicals have been suggested to be
important mediators of the cytotoxic actions of cytokines in ß-cells
(11, 12, 13, 14). We found that the combination of IL-1ß,
TNF, and IFN increased production of nitric oxide and
nitrotyrosine, a marker of peroxynitrite, in ßTC-3 cells.
Peroxynitrite (ONOO-) is a highly reactive
oxidant produced by the combination of superoxide
(O2·-) and NO·s, and
peroxynitrite may be a more potent cytotoxic mediator than superoxide
or nitric oxide alone (44). Peroxynitrite is highly toxic
to rat and human islets in vitro (45), and this
oxidant has been detected in pancreatic islets in vivo in
conjunction with ß-cell destruction and autoimmune diabetes
development in NOD mice (31). In this study, we found that
cytokine-induced production of nitric oxide was significantly decreased
in ßTC-3 cells that overexpressed
calbindin-D28k. Inhibition of nitric oxide
production may explain the protective effect of
calbindin-D28k against cytokine-induced necrosis
(trypan blue uptake) of mouse ßTC-3 cells, because cytokine-induced
necrosis of mouse islet ß-cells is nitric oxide-dependent
(43). In addition, calbindin overexpression inhibited
cytokine-induced production of peroxynitrite even more than nitric
oxide. Because peroxynitrite is the reaction product of superoxide and
nitric oxide, our findings suggest that calbindin inhibited production
of the superoxide radical more than the NO·. Also,
cytokine-induced formation of another oxygen free radical product,
lipid hydroperoxide, was significantly decreased in
calbindin-expressing ßTC-3 cells.
Previous studies have shown that sustained increases of the cytoplasmic free Ca2+ concentration ([Ca2+]i) can result in an adverse effect on mitochondrial electron transport, leading to superoxide radical production and its conversion to further cytotoxic reactive oxygen species, such as hydrogen peroxide and peroxynitrite (24, 41, 42). Overexpression of calbindin-D28k was reported to protect against elevations of [Ca2+]i, generation of reactive oxygen species, and apoptosis of a neural cell line induced by amyloid ß-peptide and potentiated by mutant presenilin 1, proteins involved in Alzheimer’s disease (24). In other cell lines, cytokine-induced apoptosis has been reported to occur after a rise in [Ca2+]i (46). In addition, cytokines were found to induce a low voltage-activated Ca2+ current in mouse ßTC-3 cells, and an increase in [Ca2+]i in mouse islet cells that was associated with DNA fragmentation and cell death (47). Thus, it is possible that calbindin-D28k overexpression protected against cytokine-induced death of ßTC-3 cells in the present study by stabilizing cellular calcium, preventing calcium-mediated mitochondrial damage and consequent generation of oxygen free radicals. Previous studies showed that the protooncogene, bcl-2, also prevents cytokine-induced oxygen free radical production and ß-cell destruction (48, 49). Because Bcl-2 protein has been reported to inhibit mitochondrial release of cytochrome c (50) that can trigger apoptosis (51), it was suggested that Bcl-2 prevents the generation of the superoxide radical and protects ß-cells from apoptosis, in part, by retaining cytochrome c in the mitochondria (49). Further studies are needed to determine whether inhibition of release of cytochrome c may also be one mechanism whereby calbindin can protect against oxygen free radical production and ß-cell death.
Evidence for a protective role of calbindin-D28k
in the cell death process has been noted in different cell types. In
the nervous system, numerous studies have provided evidence that
calbindin, by buffering calcium, can regulate intracellular calcium
responses to physiological stimuli and can protect against
calcium-mediated cellular toxicity (21, 22, 23, 24). Studies in
neuronal cells suggest that calbindin can protect against toxins that
alter calcium homeostasis, either directly by activation of glutamate
receptors and calcium ionophore treatment (52, 53) or
indirectly by treatment with the antimetabolite, 3-acetylpyridine
(53). In lymphocytes, overexpression of calbindin has been
reported to protect against cell death in response to a variety of
insults that also involve calcium-dependent events, including exposure
to calcium ionophore, cAMP, and glucocorticoids (25).
However, in recent studies, we found that protection of
cytokine-mediated apoptotic cell death of osteoblasts by calbindin does
not result from calcium buffering but rather from the ability of
calbindin to bind to and inhibit caspase-3 (26). Caspase-3
is a member of the family of cytosolic aspartate-specific proteases and
a final effector of apoptosis in response to many signals, including
cytokines (54). The involvement of calcium-independent
mechanisms in ß-cell apoptosis is suggested by recent studies in the
MIN 6 ß-cell line, which showed that increases in intracellular
calcium in response to KCl or calcium ionophore was not sufficient to
induce apoptosis (55). The involvement of the caspase
pathway, and specifically caspase-3, in ß-cell apoptosis was noted in
TNF-induced apoptosis of NIT-1 cells, a ß-cell line derived from
NOD mice (16), and in Fas-induced apoptosis of ßTC cells
(56). Thus, it is possible that both calcium-dependent and
calcium-independent pathways are involved in calbindin’s ability to
protect against cytokine-mediated ß-cell apoptosis.
In conclusion, our results support a role for calbindin-D28k in protecting against cytokine-mediated ß-cell death by mechanisms involving inhibition of free radical formation. A further understanding of the exact mechanisms involved may have important therapeutic implications for the prevention of autoimmune destruction of ß-cells in type 1 diabetes.
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Footnotes |
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Abbreviations: BCA, Bicinchoninic acid; FITC, fluorescein isothiocyanate; IFN, interferon; MTT, 3-(4,5-dimethylthiazolyl-2)2,5-diphenyltetrazolium bromide; NO, nitric oxide radical; NOD, nonobese diabetic; POD, peroxidase; TUNEL, TdT-mediated deoxyuridine 5-triphosphate-X nick end labeling.
Received January 29, 2001.
Accepted for publication May 2, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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N. Allaman-Pillet, J. Storling, A. Oberson, R. Roduit, S. Negri, C. Sauser, P. Nicod, J. S. Beckmann, D. F. Schorderet, T. Mandrup-Poulsen, et al. Calcium- and Proteasome-dependent Degradation of the JNK Scaffold Protein Islet-brain 1 J. Biol. Chem., December 5, 2003; 278(49): 48720 - 48726. [Abstract] [Full Text] [PDF]
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S. Bertera, M. L. Crawford, A. M. Alexander, G. D. Papworth, S. C. Watkins, P. D. Robbins, and M. Trucco Gene Transfer of Manganese Superoxide Dismutase Extends Islet Graft Function in a Mouse Model of Autoimmune Diabetes Diabetes, February 1, 2003; 52(2): 387 - 393. [Abstract] [Full Text] [PDF]
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,
P < 0.05; 
, P <
0.01vs. CaBP 0 cytokines + group.
