Apoptosis Mediated by Activation of the G Protein-Coupled Receptor for Parathyroid Hormone (PTH)/ PTH-Related Protein (PTHrP)
Paul R. Turner,
Suzanne Mefford,
Sylvia Christakos and
Robert A. Nissenson
Endocrine Unit (P.R.T., S.M., R.A.N.) Veterans Affairs Medical
Center and the Departments of Medicine and Physiology University
of California San Francisco San Francisco, California 94121
Department of Molecular Biology and Biochemistry (S.C.) New
Jersey Medical School Newark New Jersey 07103
 |
ABSTRACT
|
---|
The present studies were carried out to evaluate
the mechanisms by which PTH/PTHrP receptor (PTHR) activation influences
cell viability. In 293 cells expressing recombinant PTHRs, PTH
treatment markedly reduced the number of viable cells. This effect was
associated with a marked apoptotic response including DNA fragmentation
and the appearance of apoptotic nuclei. Similar effects were evidenced
in response to serum withdrawal or to the addition of tumor necrosis
factor (TNF
). Addition of caspase inhibitors or overexpression of
bcl-2 partially abrogated apoptosis induced by serum withdrawal.
Caspase inhibitors also protected cells from PTH-induced apoptosis, but
overexpression of bcl-2 did not. The effects of PTH on cell number and
apoptosis were neither mimicked by activators of the cAMP pathway
(forskolin, isoproterenol) nor blocked by an inhibitor (H-89). However,
elevation of Cai2+ by addition of thapsigargin
induced rapid apoptosis, and suppression of
Cai2+ by overexpression of the calcium- binding
protein, calbindin D28k, inhibited PTH-induced apoptosis. The protein
kinase C inhibitor GF 109203X partially inhibited PTH-induced
apoptosis. Regulator of G protein signaling 4 (RGS4) (an inhibitor of
the activity of the
-subunit of Gq)
suppressed apoptotic signaling by the PTHR, whereas the C-terminal
fragment of GRK2 (an inhibitor of the activity of the ß
-subunits
of G proteins) was without effect. Chemical mutagenesis allowed
selection of a series of 293 cell lines resistant to the apoptotic
actions of PTH; a subset of these were also resistant to TNF
. These
results suggest that 1) apoptosis produced by PTHR and TNF receptor
signaling involve converging pathways; and 2) Gq-mediated phospholipase
C/Ca2+ signaling, rather than Gs-mediated cAMP
signaling, is required for the apoptotic effects of PTHR activation.
 |
INTRODUCTION
|
---|
Apoptosis or programmed cell death is a process fundamental to
normal growth and development, immune response, and tissue remodeling
after injury or insult. The mammalian signal transduction pathways that
mediate apoptosis, although under intense scrutiny, remain incompletely
understood. Recently, it has become apparent that apoptosis is a
crucial process in skeletal development and homeostasis and that
signaling by the PTH /PTH-related protein (PTHrP) receptor
(PTHR) can either promote or suppress apoptosis depending on the
cellular context (1, 2). In addition, growth- suppressive effects of
PTHR activation have been reported in osteoblastic target cells (3, 4, 5).
The PTHR is known to be capable of signaling in response to PTH or
PTHrP via two G protein-coupled pathways: 1) Gq-mediated activation of
phospholipase C (PLC), resulting in increased
Cai2+ and activation of protein
kinase C (PKC); and 2) Gs-mediated activation of adenylyl cyclase
leading to cAMP production and protein kinase A (PKA) activation (6).
However, it is unclear whether either or both of these signaling
pathways are linked to changes in PTH-induced cell proliferation or
apoptosis.
Embryonic mice lacking expression of functional PTHrP or PTHR gene
products display severe abnormalities of endochondral bone formation
(7, 8). The acceleration of chondrocyte differentiation and
disorganization of the growth plate seen in these mice underscores the
important role that PTHR signaling and apoptosis play in normal
skeletal growth and differentiation (1, 9). In addition, the skeletal
abnormalities that are observed in Jansens metaphyseal
chondrodysplasia have been attributed to point mutations in the PTHR,
which result in constitutively active mutant PTHRs (10, 11). The
mechanisms by which PTHrP and PTHR signaling affect skeletal
development are not known, although feedback between PTHR signaling and
Indian hedgehog has been proposed to modulate chondrocyte
differentiation (12).
Terminal differentiation of chondrocytes is associated with apoptosis
(13), and PTHrP has been shown to increase expression of the
antiapoptotic gene bcl-2 coincident with suppressing terminal
chondrocyte differentiation (1). However, preliminary studies indicate
that PTH administration to young rats promotes the apoptosis of
osteoblasts and osteocytes in vivo (2). This suggests that
apoptosis can be initiated by activation of the PTHR, and that this is
likely to contribute to the spectrum of physiological responses to PTH
and/or PTHrP. In the present study, we report that PTH induces
apoptosis in human embryonic kidney (HEK) 293 cells stably expressing
the PTHR. These effects require the second messenger products of PLC
signaling, but are independent of adenylyl cyclase signaling.
 |
RESULTS
|
---|
Initial studies were carried out to determine the effects of PTHR
signaling on cell viability. The wild-type (Wt) opossum PTHR was stably
expressed in 293 cells, which lack endogenous PTHRs. Exposure of these
cells to a PTHR agonist, bovine (b) PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34), resulted in a time- and
dose-dependent decrease in cell number (Fig. 1
). As little as 1 nM bovine
(b)PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) produced a significant effect, and 1 µM
bPTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) reduced the number of cells by approximately 80% within
4872 h. Serum withdrawal, known to induce apoptosis in 293 cells
(14), resulted in decreased cell numbers after 72 h. Addition of
bPTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) had no effect on the number of control 293 cells
(transfected with vector alone), and addition of a PTHR antagonist
[bPTH(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34), 1 µM], did not alter the number of
PTHR-expressing 293 cells (not shown).
To determine whether inhibition of cell number by PTH was associated
with apoptosis, we obtained a quantitative index of the amount of DNA
fragmentation in response to either PTH treatment or serum withdrawal.
Cells were fixed at various time points after commencement of PTH
treatment or serum withdrawal, and terminal deoxynucleotidyl
transferase-mediated dUTP nick end labeling (TUNEL) assays were
used to label terminal DNA fragments. Positive staining was readily
detected in apoptotic cells (Fig. 2A
).
Analysis of TUNEL assay results revealed that both serum withdrawal and
PTH treatment induced apoptosis in more than 20% of cells after
72 h (Fig. 2B
). Similar results were obtained in two additional
clonal cell lines of 293 cells expressing the PTHR (not shown). The
percent of cells with apoptotic nuclei after serum withdrawal (33%)
was similar to that previously reported for serum-deprived 293 cells
(14). DNA fragmentation was also visualized using agarose gel
electrophoresis of DNA extracts from cells after exposure to PTH or
after serum withdrawal (Fig. 2C
). The classical DNA ladder of 128-bp
DNA fragments was not visible among a more general smear of degraded
DNA after treatment with bPTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34), the phorbol ester PMA, or serum
withdrawal. The PTHR antagonist [PTH(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34), 1 µM], did
not induce visible DNA degradation. Very little DNA fragmentation was
observed in control cells by either TUNEL staining or gel
electrophoresis. These results show that apoptosis occurs only
infrequently in proliferating 293 cells. PTH did not elicit an
apoptotic response in 293 cells in the absence of PTHR expression (not
shown).
Differences in cell morphology after PTH treatment and serum withdrawal
were observed. The morphological response to serum withdrawal was cell
shrinkage/cell rounding (Fig. 3A
), a
response frequently associated with apoptosis (15). The initial
morphological response to PTH treatment was cell spreading and
flattening (Fig. 3A
). Fluorescent labeling of the actin cytoskeleton
with rhodamine-conjugated phalloidin demonstrated major cytoskeletal
reorganization after PTHR activation (not shown). The relationship
between these morphological changes and the apoptotic response to PTH
is unclear.

View larger version (134K):
[in this window]
[in a new window]
|
Figure 3. Morphological Effects of PTH and Serum Withdrawal
on HEK 293 Cells Expressing the Wt PTHR
A, Light microscope brightfield images 1 h after addition of
normal growth media (control), 1 µM PTH(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ), or 1
h after removal of serum. For all fields, the white scale
bar = 25 µm. Cell flattening after PTH treatment and
cell rounding and shrinkage after serum withdrawal were
characteristically seen. B, Electron micrographs of cells grown as
described in panel A. The nucleolus is visible in the center of the
spherical control cell nucleus. White stars mark
fragments of the nucleus in the PTH-treated cell. Fragmented nuclei
were not seen in images of control cells. Mitochondrial morphology also
appears to be disrupted by PTH treatment. White stars
mark the condensations of chromatin visible in nuclei from cells
subject to serum withdrawal. Ruffling of the nuclear membrane was also
apparent in these cells, and whole-cell shrinkage was apparent.
White scale bars = 2.5 µm.
|
|
DNA fragmentation is one of the final cellular events after exposure of
cells to apoptotic stimuli (16). An earlier indicator of the activation
of apoptosis pathways is the translocation of phosphatidylserine from
the cytosolic to the extracellular face of the plasma membrane (17).
This translocation can be monitored due to the high affinity of annexin
V for phosphatidylserine. PTH treatment or serum withdrawal induced
phoshatidylserine translocation to the extracellular plasma membrane
surface within 5 h. The percent annexin V-stained cells increased
from 3.6 ± 1.8% to 19.6 ± 4.1% after 5 h of exposure
to bPTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) (1 µM) and to 44.7 ± 4.5% 5 h
after serum withdrawal.
Characteristic nuclear changes are known to occur in response to
apoptotic stimuli, including nuclear condensation and fragmentation
(15). Hoechst 33342 nuclear dye staining revealed increased nuclear
condensation and fragmentation of the nucleus in response to
bPTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) or serum withdrawal, whereas heat treatment (48 C, 2
h) resulted in swollen, distended nuclei, characteristic of necrosis
(not shown). These nuclear changes were readily apparent in electron
micrographs of 293 cells after PTH treatment (Fig. 3C
). Such
fragmentation of the nucleus was not seen in any of more than 400
control cells that were examined. Cell fragmentation was also evident
in electron micrographs after PTH treatment or serum deprivation (not
shown). Such fragments most likely are a result of the final stages of
apoptosis, which include loss of plasma membrane integrity and
cytolysis. Cells undergoing these final stages of apoptosis could be
visualized using a combination of a vital stain (Syto 13), together
with propidium iodide (18). These dyes revealed a progressive loss of
membrane integrity in response to both PTH and serum withdrawal, with a
time course similar to that seen for DNA fragmentation (not shown).
The downstream effectors of mammalian apoptosis pathways are thought to
be the caspase family of proteases (19). Preincubation of 293 cells for
3 h with cell-permeable inhibitors of caspases, YVAD (inhibitor of
caspase 1), and DEVD (inhibitor of caspases 3, 8), significantly
reduced the effects of PTH treatment on cell number (Fig. 4A
) and apoptosis as determined by TUNEL
(Fig. 4B
). The amount of inhibitor used in each case was 0.2
µM, a dose known to be maximally effective in other
systems (20, 21). The combination of both caspase inhibitors was more
effective than either inhibitor alone, indicating that multiple
caspases may participate in the apoptotic response. While the caspase
inhibitors did not modify the suppressive effect of serum withdrawal on
cell number, they did ameliorate the apoptotic response to serum
withdrawal, indicating that serum contains essential growth factors
that act independently of the apoptotic signaling pathway.

View larger version (23K):
[in this window]
[in a new window]
|
Figure 4. Effect of Inhibitors of Caspase 1 (YVAD) or
Caspases 3 and 8 (DEVD) on Growth Inhibition (A) and Apoptosis (B) of
HEK 293 Cells after 3 Days of Serum Withdrawal (No Serum) or Treatment
with bPTH(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ) (1 µM)
Caspase inhibitors were added on day 0 and were present continuously
(either separately or together) each at a concentration of 0.2
µM.
|
|
The oncogene product bcl-2 is known to inhibit apoptotic signaling in
response to a wide range of stimuli. In 293 cells, bcl-2 is reported to
partially inhibit apoptosis in response to serum deprivation (14). We
evaluated 293 cells stably overexpressing bcl-2 as well as the Wt PTHR
and found that bcl-2 partially prevented the effects of serum
withdrawal on cell number and apoptosis (Fig. 5
). However, overexpression of bcl-2 was
ineffective in inhibiting the corresponding effects of bPTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34).

View larger version (14K):
[in this window]
[in a new window]
|
Figure 5. Effect of Overexpression of bcl-2 on Cell Number
and Apoptosis in HEK 293 Cells Treated with PTH or Subject to Serum
Withdrawal
A, Western blot of bcl-2 in extracts of cells expressing the PTHR and
bcl-2 (+), compared with cells expressing only the PTHR (-). The
mobilities of the Mr markers are indicated. B, Effect of 3
days of treatment with bPTH(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ) (1 µM) or 3 days of
serum withdrawal on cell number in cells ± overexpression of
bcl-2. C, Effect of 3 days of treatment with bPTH(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ) (1
µM) or 3 days of serum withdrawal on apoptosis of
cells ± overexpression of bcl-2.
|
|
The PTHR is known to signal through both the adenylyl cyclase/cAMP and
PLC/Cai2+/PKC pathways. We
therefore investigated the role of these pathways in mediating the
effects of PTH on 293 cell number and apoptosis. cAMP is known to
induce apoptosis in certain cells such as T cells (22). However, two
lines of evidence indicate that this pathway is neither necessary nor
sufficient to produce apoptosis in 293 cells. First,
receptor-independent production of cAMP, induced by treatment with
forskolin, did not affect cell number or induce apoptosis (Fig. 6
). Second, activation of adenylyl
cyclase via isoproteronol-induced activation of endogenous
ß2-adrenergic receptors failed to reduce cell
number or induce apoptosis (Fig. 6
).
ß2-Adrenergic receptors are known to signal via
Gs-mediated activation of adenylyl cyclase, and not via Gq-mediated
PKC/PLC activation. Third, 30 µM H-89, a concentration
known to inhibit PKA in 293 cells (23), had no significant effect on
PTH suppression of cell number or PTH-induced apoptosis (Fig. 6
).

View larger version (23K):
[in this window]
[in a new window]
|
Figure 6. Effects of Activators and Inhibitors of Signal
Transduction Pathways on Cell Number (A) and Apoptosis (B) of HEK 293
Cells
Cells expressing the Wt PTHR were treated with forskolin (100
µM), the PKA inhibitor H-89 (30 µM), the
PKC inhibitor GF 109203X (6 µM) (Bis Indo), or
thapsigargin (0.4 nM). In some cases, cells were exposed to
bPTH(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ) (1 µM) for 3 days in the presence or absence
of pharmacological agents. Cell number and apoptosis were also
evaluated in HEK 293 cells overexpressing the
ß2-adrenergic receptor (ß2-R) after a 3-day
treatment with isoproterenol (10 µM). In panel A), the Wt
and PLC-deficient PTHR (C0) were compared with respect to reduction in
cell number in response to a submaximal dose of bPTH(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ) (1
nM).
|
|
The other major signaling pathway activated by the PTHR is the
Gq-mediated PLC/Cai2+/PKC
pathway. Several approaches were used to assess a possible role for
this pathway in mediating the effects of PTH on cell number and
apoptosis. Thapsigargin is a Ca2+ATPase inhibitor
that elevates intracellular calcium concentration
[Ca2+]i, by promoting its
release from intracellular stores, and induces apoptosis in certain
cells (24). Thapsigargin was potent in reducing 293 cell number and
inducing apoptosis (Fig. 6
). The bisindolylmaleimide inhibitor of PKC,
GF 109203X, at a maximally effective dose (6 µM) weakly
inhibited (by
20%) PTH-induced TUNEL staining, suggesting a minor
role for PKC in the apoptotic response (Fig. 6B
). Further support for a
role for PLC came from the use of a mutant PTHR defective in PLC
signaling. We have previously shown that alanine mutations of key
residues in the N-terminal region of the third cytoplasmic loop of the
PTHR (R377A,V378A, L379A), result in a receptor (termed C0) that
displays reduced PTH-stimulated PLC signaling with retention of
adenylyl cyclase signaling (25). Compared with 293 cells expressing the
Wt PTHR, cell expressing this mutant were not as susceptible to the
reduction in cell number elicited by a submaximal dose of bPTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)
(1 nM) (Fig. 6A
). At a maximal dose, however, bPTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)
(1 µM) reduced cell number to the same degree as with
cells expressing the Wt PTHR (data not shown). Further evidence for a
role of PLC/Ca2+ signaling came from studies of
293 cells stably expressing the calcium-binding protein,
calbindin-D28k. These cells were partially protected from both the
reduction in cell number and the induction of apoptosis in response to
PTH treatment (Fig. 7
). However,
calbindin overexpression did not protect cells from the effects of
serum withdrawal.

View larger version (16K):
[in this window]
[in a new window]
|
Figure 7. Effect of bPTH(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ) (1 µM) and
Serum Withdrawal on Cell Number (A) and Apoptosis (B) of HEK 293 Cells
Expressing the Wt PTHR with and without Overexpression of the
Calcium-Binding Protein Calbindin
Cells were subject to PTH treatment (+ PTH) or serum withdrawal (No
Serum) for 3 days. PTH-induced reduction in cell number and induction
of apoptosis were significantly suppressed in cells overexpressing
calbindin (P values <0.05 and <0.01, respectively).
|
|
To more precisely assess the role of specific G protein subunits in
mediating the apoptotic action of PTH, we used 293 cells transfected
with constructs encoding known inhibitors of ß
- and
q-subunit function. RGS4 accelerates the
GTPase activity of
q and thereby inhibits
receptor-mediated activation of effectors such PLC. A line of 293 cells
stably overexpressing RGS4 has previously been shown to display
suppressed receptor-stimulated PLC activity (26). We expressed the PTHR
in these cells (which were kindly provided to us by Dr. Susanne Mumby),
and assessed the ability of PTH to produce a reduction in cell number
and an increase in apoptosis (Fig. 8
).
Expression of RGS4 almost fully prevented the PTH-induced reduction in
cell number and inhibited the apoptotic response to PTH by about 75%.
A C-terminal fragment of G protein-coupled receptor kinase 2 (CtGRK2)
is known to bind G protein ß
-subunits and thus to inhibit their
ability to activate effectors (27). We evaluated the ability of the
PTHR to initiate apoptotic signaling in 293 lines overexpressing CtGRK2
(Fig. 9
). Overexpression of CtGRK2
blocked ß-adrenergic receptor-mediated activation of MAP kinase, a
process known to be dependent upon the ß
-subunits of Gi (28). This
demonstrates that sufficient CtGRK2 was expressed to inhibit the
functional activity of ß
-subunits after G protein activation.
However, these cells were fully responsive to PTH, both with respect to
the reduction in cell number and induction of apoptosis.

View larger version (17K):
[in this window]
[in a new window]
|
Figure 8. Effects of RGS4 Expression on Cell Number (A) and
Apoptosis (B) in HEK 293 Cells Expressing the Wt PTHR
A, Cells were maintained under normal growth conditions in the presence
of serum (Control), treated with 1 µM bPTH(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ) in the
presence of serum (+ PTH), or grown in the absence of serum (-Serum).
Daily counts of adherent cells were taken over a 3-day period. B, Cells
were treated with 1 µM bPTH(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ) for 3 days, and the
percent of apoptotic nuclei was determined by TUNEL staining, as
described in Materials and Methods. In both cases, cells
expressing RGS4 were compared with cells stably transfected with the
corresponding empty vector (pCB6).
|
|

View larger version (17K):
[in this window]
[in a new window]
|
Figure 9. Effects of CtGRK2 Expression on Cell Number (A),
Apoptosis (B), and MAP Kinase (C) in HEK 293 Cells Expressing the Wt
PTHR
A, Cells were maintained under normal growth conditions in the presence
of serum (Control), treated with 1 µM bPTH(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ) in the
presence of serum (+ PTH), or grown in the absence of serum (-Serum).
Daily counts of adherent cells were taken over a 3-day period. B, Cells
were treated with 1 µM bPTH(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ) for 3 days, and the
percent of apoptotic nuclei was determined by TUNEL staining, as
described in Materials and Methods. (C) Cells were
transfected with a MAP kinase-activated luciferase reporter plasmid, as
described in Material and Methods. Three days later,
cells were treated with 1 µM isoproterenol, and
luciferase activity was measured. In all three cases, cells expressing
CtGRK2 were compared with cells transfected with the corresponding
empty vector (pCEP4).
|
|
The ability of PTHR signaling to efficiently kill 293 cells made it
possible to select cells resistant to this action. To accomplish this,
293 cells expressing the Wt PTHR were exposed to the UV-sensitive
chemical mutagen, trimethylpsoralen (TMP), together with UV irradiation
(29). Cells were subsequently grown in the continual presence of 1
µM bPTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34). All cells exposed only to the mutagen or
only to UV irradiation died within 2 weeks in the presence of
bPTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34). However, cells resistant to the killing effect of PTH were
present in cultures treated with both TMP and UV irradiation. Twenty
four clonal lines of PTH-resistant cells were isolated, of which 22
were found to bind radiolabeled PTHrP at levels comparable to
nonmutagenized cells. The effects of PTH on cell number and apoptosis
in two mutagenized clonal cell lines are shown in Fig. 10
. These cell lines displayed markedly
reduced sensitivity to the effects of PTH, but were fully sensitive to
the effects of serum withdrawal. For these two clonal lines, and indeed
for all of the 24 PTH-resistant clones (not shown), serum withdrawal
induced apoptosis (Fig. 11
). Also shown
is the effect of tumor necrosis factor-
(TNF
) (10 ng/ml) on cell
number and apoptosis of these cells. Nonmutagenized 293 cells displayed
both reduction of cell numbers and apoptosis in response to TNF
.
However, the mutagenized, PTH-resistant clonal cell lines proved to be
heterogeneous, with several (such as clone 19) displaying resistance to
the effects of TNF
, whereas others (such as clone 22) remained fully
sensitive to TNF
.

View larger version (23K):
[in this window]
[in a new window]
|
Figure 10. Effects of PTH, TNF , and Serum Withdrawal on
Cell Number (A) and Apoptosis (B) of Control and Mutagen-Treated,
PTH-Resistant Clonal HEK 293 Cells
Cells were treated with the mutagen TMP together with UV irradiation,
and PTH-resistant clonal lines were isolated as described in
Materials and Methods. Two PTH-resistant clonal lines
were compared with non-mutagen-treated HEK cells expressing the Wt PTHR
(Wt). Shown are the effects of 3 days of exposure to bPTH(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ) (1
µM), TNF (10 ng/ml), or serum withdrawal.
|
|

View larger version (35K):
[in this window]
[in a new window]
|
Figure 11. Model Showing Putative Pathways Leading to
Apoptosis Induced by PTHR Activation, TNF , and Serum Withdrawal in
HEK 293 Cells
For the PTHR, binding of PTH at the cell surface results in activation
of Gs and Gq. The present results suggest that apoptosis is induced by
an increase in [Ca2+]i (which can be
inhibited by calbindin D28K), presumably resulting from PLC activation
mediated by the 34 -subunit of Gq. Increased
[Ca2+]i induces apoptosis via activation of
components of the caspase cascade, including caspases 1 and 3. Results
from other studies indicate that activated TNF receptors (TNFR1)
recruit TRADD to the membrane, which in turn initiates a caspase
cascade involving caspases 8 and 10 as well as caspases 1 and 3. Serum
deprivation also produces activation of these caspases, leading
ultimately to apoptosis. Bcl-2 is thought to act as an inhibitor of
caspases 8 and 10, and thus protects cells from apoptosis induced by
TNF and serum withdrawal, but not PTH. Inhibitors of caspases 1 and
3 only partially protect cells from apoptosis, suggesting the existence
of alternative downstream apoptotic pathways.
|
|
 |
DISCUSSION
|
---|
The elucidation of mammalian apoptosis pathways has lagged behind
that of Caenorhabditis elegans and is only partially defined
at present in one case, that of the FAS/TNF receptor family
(30). This highlights the importance of understanding other apoptosis
pathways. The objectives of the present study were to assess whether
PTHR activation can initiate apoptotic signaling and, if so, to
characterize the signaling pathways mediating such a response.
Current understanding of mammalian apoptosis pathways is derived, in
part, from the study of apoptosis induced by activation of the TNF
receptor (TNFR). Most cells express the TNFR (30), and TNF
was found
in the present study to be a potent inducer of apoptosis in HEK293
cells (Fig. 10
). Receptors in this TNFR superfamily contain a cytosolic
region required for cell death signal transduction, termed the "death
domain." After ligand binding and TNFR trimerization (Fig. 11
), the
death domain couples receptors to signaling molecules such as TRADD
(TNFR-associated death domain protein). TRADD is an adapter molecule
that couples receptors to caspase proteases. Recruitment of a
procaspase to the receptor/TRADD complex results in procaspase cleavage
and formation of an active dimer (20). The newly active caspase is then
able to cleave various "death substrates" such as other caspases.
More than 10 caspases have been identified thus far, and a variety of
substrates have been characterized, including calpains, nuclear
scaffold proteases, gelsolin, and signaling pathway components (19, 31). The end result of this caspase cascade is DNA fragmentation (19, 20) and the morphological criteria that distinguish apoptosis from
necrosis such as DNA condensation and the fragmentation of the nucleus
before cytolysis (18).
Evaluation of the amino acid sequence of the PTHR does not reveal the
presence of a cytosolic death domain, indicating that an alternative
mechanism likely to involve G protein activation initiates the
apoptotic response to PTHR activation. PTHR-mediated apoptosis, like
that induced by the TNFR, appears to require the activation of
caspases, since apoptosis was partially abrogated by caspase
inhibitors. That the inhibition was only partial may reflect the fact
that other caspases in the cascade were activated or that inhibition of
the caspases was incomplete. The combination of inhibitors was more
effective than each inhibitor individually, consistent with the notion
that PTHR-induced apoptosis is associated with the activation of
multiple caspases. PTHR activation induced DNA fragmentation, as did
serum withdrawal. Addition of PTH potentiated the effects of serum
withdrawal (data not shown), a result that suggests that 293 cells
possess at least two separate pathways by which apoptosis can be
induced. In addition, PTH treatment induced other markers of apoptosis
such as phosphatidyl serine translocation at an early time (5 h), and
the number of cells with fragmented nuclei and lost viability (assessed
by electron or light microscopy) was similar to the number of
cells with fragmented DNA as determined by TUNEL assay. These findings
confirm that the effect of PTH on 293 cells was to induce apoptotic
rather than necrotic cell death.
Mammalian cells can often be protected from apoptotic stimuli,
including TNF
, by overexpression of the protooncogene bcl-2 (32).
The mode of action of bcl-2 is at present unclear, although it may
protect mitochondrial membrane integrity, prevent the proapoptotic
activity of bcl-2 homologs such as bad or bax by forming inactive
heterodimers (32), or perhaps act by inhibiting a protein required for
caspase activation (33). Bcl-2 may exert its effect at the level of
caspases 8 and 10, but it does not inhibit caspase 3, which may
therefore be acting more downstream in the apoptosis cascade. In fact,
bcl-2 can itself be a substrate for caspase 3 (34). Overexpression of
bcl-2 has been found to repress transcription in response to serum
withdrawal in 293 cells (14) and to abrogate serum withdrawal-induced
apoptosis in PTHR expressing 293 cells (Fig. 5
). However, bcl-2
overexpression did not prevent PTH-induced apoptosis, consistent with
the utilization of a different pathway from that activated by serum
withdrawal (Fig. 11
). Thus, unlike the TNFR pathway, the PTHR-mediated
apoptosis pathway appears to be bcl-2 independent. Alternately, it is
possible that PTHR activation leads to a rapid degradation of bcl-2
even in cells overexpressing the protooncogene.
The PTHR-activated signaling pathway that induces apoptosis
appears to be the Gq-mediated PLC/Cai2+
pathway. Increases in cAMP, known to induce apoptosis in certain cells
(22), was neither necessary nor sufficient for PTH-induced apoptosis in
293 cells. PKC inhibition was only weakly effective at inhibiting
PTH-induced cell death, suggesting a small contribution of PKC
activation to apoptotic signaling, as has been observed in other
systems (35). Thapsigargin was a powerful inducer of apoptosis,
consistent with a role for calcium mobilization. A variety of studies
have implicated changes in calcium ion homeostasis in apoptosis
(36, 37, 38, 39), but the underlying mechanisms are unclear. Experimentally
induced calcium store depletion induced by stimulation of
inositol-1,4,5- trisphosphate (IP3) receptors or
by inhibition of Ca2+-ATPase activity can result
in apoptosis (24, 40). It is possible that calcium store depletion is
sensed by the cell and directly leads to an apoptotic response.
Alternatively, it has been suggested that an increase in plasma
membrane calcium permeability resulting from calcium store depletion
signals apoptosis (41). Possible targets for the resulting elevations
in [Ca2+]i include
proteases such as calpains or caspases, or protein kinases, which then
promulgate the apoptotic signal (21). A crucial role for
Cai2+ has been documented in
neuronal cells where overexpression of the calcium-binding protein
calbindin 28 kDa was found to rescue cells from apoptosis (41, 42),
presumably by buffering the cytosol against increases in
[Ca2+]i. In the present
study, calbindin overexpression was found to protect 293 cells from
PTH-induced apoptosis, but not from serum withdrawal-induced apoptosis.
This supports the hypothesis that a PLC-dependent increase in
[Ca2+]i mediates
PTHR-induced apoptosis, whereas the effect of serum withdrawal is
independent of
[Ca2+]i.
Of the two G protein pathways known to be activated by the PTHR, only
Gq is affected by RGS4. Thus the observation that
RGS4 markedly suppressed PTH-induced apoptosis strongly supports a role
of Gq signaling in mediating the apoptotic
response to PTH. Although the ß
-subunits of
Gs or Gq that are released
after PTHR activation could theoretically contribute to PLC activation
(43), the finding that overexpression of CtGRK2 failed to inhibit
apoptosis points to the central role of the
-subunit of
Gq in initiating apoptotic signaling via PLC in
this system.
The efficiency with which PTH treatment induced apoptosis of 293 cells
expressing the Wt PTHR made it possible to use chemical mutagenesis
with TMP together with UV irradiation to generate clonal cell lines
resistant to this effect of PTH. Nearly all of the PTH-resistant clonal
lines obtained displayed near Wt levels of PTHrP binding, ruling out
loss of expression of the PTHR as the basis of PTH resistance in these
lines. All of the PTH-resistant cell lines displayed apoptosis in
response to serum withdrawal, indicating that downstream components of
the apoptotic signaling pathway were intact. Some of the clonal lines
also remained responsive to apoptosis induced by treatment with TNF
,
suggesting that disruption of apoptotic signaling occurred relatively
upstream in the PTHR-mediated apoptotic pathway (e.g. at the
level of phospholipase C activation or
Cai2+ mobilization/action) (Fig. 11
). Other cell lines were resistant to TNF
as well as PTH,
indicating that TMP/UV-induced disruption occurs at more distal sites
that are common to the actions of both agents (e.g. at the
level of the caspase cascade. These PTH-resistant cell lines will be
helpful in defining the nature of the apoptotic signaling pathways used
by PTH and TNF
.
In conclusion, these results indicate that PTHR signaling elicits
an apoptotic response in 293 cells by a mechanism other than activation
of adenylyl cyclase. The present study provides evidence that apoptosis
is mediated by the Gq-PLC/Cai2+
signaling pathway. The apoptosis so produced differs from that induced
by serum withdrawal in that bcl-2 does not protect against PTHR
activation, whereas calbindin overexpression protects against apoptosis
elicited by PTH, but not serum withdrawal. In addition, the PTHR and
TNFR pathways appear to share downstream components of apoptotic
signaling. Thus, this model system will be useful in the further
characterization of the molecular mechanisms of PTH-induced apoptosis
and in the identification of novel components of the PTHR-mediated
apoptotic signaling pathway. Additional studies are in progress to
assess the relevance of the apoptotic signaling pathway identified here
to the diverse physiological responses initiated by the PTHR in
vivo.
 |
MATERIALS AND METHODS
|
---|
Materials
Synthetic bPTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) and bPTH(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) were from Bachem California, Inc. (Torrance, CA). TNF
was obtained from R&D
systems (Minneapolis, MN). The apoptag (TUNEL) assay kit was from Oncor
(Gaithersburg, MD). Enhanced green fluorescent protein (EGFP),
Annexin V Kit, and cell-permeable caspase inhibitors zYVAD and zDEVD
were obtained from CLONTECH Laboratories, Inc. (Palo Alto,
CA). Hoechst 33342 nuclear dye, TMP, methylgreen, forskolin, H-89, the
bisindolylmaleidmide GF 109203X, and paraformaldehyde were obtained
from Sigma (St. Louis, MO). Syto 13 vital dye was obtained
from Calbiochem (San Diego, CA). Thapsigargin was obtained
from Molecular Probes, Inc. (Eugene, OR). The monoclonal
antibody against bcl-2 was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Stable Cell Lines
HEK 293 cells were transfected with the cDNA encoding the
opossum kidney (OKO) PTH receptor (kindly provided by Drs. H.
Jüppner and G. Segre). subcloned into pCDNA3.1
(Invitrogen, Carlsbad, CA). After transfection using the
Ca2PO4 precipitation method
(44), clones were selected with G418 antibiotic (200 µg/ml) and
isolated using limiting dilution in 96 well plates. Receptor expression
was confirmed using Western blotting and ligand binding
(125I-PTHrP) techniques, as previously described
(23). Complementary DNA encoding the human
ß2-adrenergic receptor (kindly supplied by Dr.
M. von Zastrow) was subcloned into the HindIII and
NotI sites of the episomal vector pCEP4
(Invitrogen). Transfected cell pools were isolated by
selecting cells in the presence of 200 µg/ml hygromycin. Control 293
cells were selected after transfection with pCEP4 vector alone.
Complementary DNA encoding human bcl-2 (kindly supplied by Dr. S.
Massa) and the C-terminal fragment of GRK2 (CtGRK2, kindly supplied by
Dr. R. Lefkowitz) were subcloned into pCEP4; the rat calbindin 28 kDa
cDNA was in pREP4 (45). Each construct was transfected separately into
293 cells stably expressing the PTHR, and hygromycin selection was
carried out to generate transfected cell pools, as described above.
cDNAs encoding the OKO Wt and the mutant PTH receptor R377A,V378A,
L379A (C0) (25) were also subcloned into pCEP4 at the
HindIII and NotI sites. These cDNAs were
transfected into 293 cells, and selection was carried out with
hygromycin as described above. Scatchard analysis demonstrated that the
293 cells lines expressed comparable numbers of Wt and mutant (C0)
receptors (
500,000 receptors per cell). For studies of RGS4, 293
cells stably transfected with a cDNA encoding RGS4 (in the expression
plasmid pCB6), and control cells stably transfected with pCB6 alone,
were provided by Dr. S. Mumby (26). Each of these cell lines was
transfected with a cDNA encoding the Wt PTHR in pCEP4, and selection of
cell pools was carried out with hygromycin as described above.
Comparable levels of functional PTHR expression were obtained in these
cell lines.
Assessment of Cell Number
HEK 293 cells expressing the appropriate receptors were
subcultured using 0.25% trypsin, and plated at a density of
approximately 104 cells per well (50
cells/mm2 in 12-well plates). Twenty four hours
later, the medium (DMEM with 10% FCS, 1% penicillin/streptomycin) was
replaced with medium containing PTH or with serum-free medium (t =
0) and cells were cultured for a 72-h period. Adherent cells were
counted every 24 h. Cells did not approach confluence under these
conditions.
TUNEL Assay
After 24, 48, and 72 h in culture, cells were detached
using Ca2+/Mg2+ free PBS.
Cells were centrifuged at 300 rpm, the supernatant was removed, and
cells were suspended and immediately fixed in 4% paraformaldehyde for
10 min. Aliquots of fixed cells were allowed to dry on a coverslip
surface, and then washed in 10 mM Tris-HCl, pH 8.0, for 5
min. Cells were permeabilized with 0.1% Triton X-100 in 10
mM Tris-HCl, pH 8.0, for 5 min, and after washing with 10
mM Tris-HCl, pH 8.0, were preincubated with terminal
deoxynucleotidyl transferase. After 10 min, the reaction mixture
containing terminal deoxynucleotidyl transferase and biotinylated
dUTP was added. After 1 h at 37 C, the reaction was terminated.
Cells were washed with PBS and incubated with streptavidin peroxidase
for 30 min. After extensive washing and counterstaining with methyl
green, cells were examined and scored positive or negative for DNA
fragmentation.
DNA Fragmentation by Gel Electrophoresis
293 cells in 10-cm dishes were lysed with 0.1 M
NaCl, 10 mM Tris HCl, pH 7.4, and 1 mM EDTA
with 0.3% SDS, and incubated with proteinase K overnight at 55 C.
Samples were extracted with phenol/chloroform, and DNA was precipitated
and resuspended in Tris-EDTA, pH 8.0, and treated with
ribonuclease for 1 h at 37 C. Electrophoresis was performed on a
4% agarose gel at 50 V for 4 h, in the presence of 0.5 µg/ml
ethidium bromide.
Evaluation of MAP Kinase Activation
ß-Adrenergic stimulation of MAP kinase was assessed using an
Elk1 reporter system (PathDetect, Stratagene). In brief,
293 cells stably expressing the Wt PTHR were cotransfected (using the
calcium phosphate method) with two plasmids one encoding the
transactivation domain of Elk1 (fused to the DNA-binding domain of
GAL4) and other containing a luciferase reporter gene bearing tandem
repeats of a GAL4 binding sequence. Three days later, cells were
treated ± 1 µM isoproterenol for 6 h, and
luciferase activity was measured using the Promega Corp.
luciferase assay kit according to the manufacturers instructions.
Light/Fluorescence Microscopy
Light and fluorescence microscopy was carried out with an
inverted Nikon (Garden City, NY) fluorescent microscope,
equipped with 10x, 20x, and 40x objectives. For Annexin V staining,
vital stain Syto 13, and propidium iodide, a fluorescein
isothiocyanate/rhodamine filter set, was used. For Hoechst 33342
nuclear stain, a 340-nm excitation filter was used, and for EGFP
visualization fluorescent excitation was carried out at 390 nm and a
510-nm emission filter was used.
Electron Microscopy
After 24, 48, and 72 h growth in 10-cm culture dishes, 293
cells were fixed with 2.5% glutaraldehyde in ice-cold 0.2
M sodium cacodylate buffer (pH 7.4) for 4 h. Cells
were washed in PBS three times and postfixed in 1% osmium tetroxide
for 30 min. Cells were then dehydrated in ascending grades of ethyl
alcohol and embedded in resin. Ultrathin sections were cut and stained
with uranyl acetate and lead citrate (4%) and examined using a H7000
electron microscope (Hitachi Scientific Instruments, Inc.,
San Jose, CA).
TMP Mutagenesis
A 3 mg/ml stock solution of TMP in dimethylsulfoxide was
diluted with DMEM to a final concentration of 30 µg/ml. This TMP
solution was added to 293 cells expressing the Wt PTHR, and the flasks
were rocked in the dark for 15 min at room temperature. Cells were then
exposed to UV irradiation (Blak-Ray lamp, 350 µW/cm2,
Fisher Scientific, Pittsburgh, PA) for 60 sec.
Cells were allowed to grow for 16 h at 37 C, after which time 1
µM bPTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) was added to the medium. Subsequently,
cells were grown in the continuous presence of bPTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) until all
cells died or until surviving clones were of sufficient size to isolate
using a cloning ring (
14 days after UV treatment). These
PTH-resistant cells were expanded for further study.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Robert A. Nissenson, Ph.D., Endocrine Unit, Veterans Affairs Medical Center (111N), 4150 Clement Street, San Francisco, California 94121.
This work was supported by funds from the Medical Research Service of
the Department of Veterans Affairs (R.A.N.), by NIH Grant DK-35323
(R.A.N), and by a Research Evaluation and Allocation Committee
award from University of California San Francisco (P.R.T.). Dr.
Nissenson is a Research Career Scientist of the Department of
Veterans Affairs.
Received for publication December 28, 1998.
Revision received October 14, 1999.
Accepted for publication October 18, 1999.
 |
REFERENCES
|
---|
-
Amling M, Neff L, Tanaka S, Inoue D, Kuida K, Weir E,
Philbrick WM, Broadus AE, Baron R 1997 Bcl-2 lies downstream of
parathyroid hormone-related peptide in a signaling pathway that
regulates chondrocyte maturation during skeletal development. J
Cell Biol 136:205213[Abstract/Free Full Text]
-
Yang XLJ, Wolfe J, Cain RL, Onyia JE, Santerre RF, Hock JM 1997 Selective stimulation of apoptosis in trabecular osteoblasts and
osteocytes of young rats treated with once daily hPTH 134 to increase
bone mass. J Bone Miner Res 12[Suppl 1]:S316
-
Kano J, Sugimoto T, Kanatani M, Kuroki Y, Tsukamoto T, Fukase
M, Chihara K 1994 Second messenger signaling of c-fos gene induction by
parathyroid hormone (PTH) and PTH-related peptide in osteoblastic
osteosarcoma cells: its role in osteoblast proliferation and
osteoclast-like cell formation. J Cell Physiol 161:358366[Medline]
-
Sabatini M, Lesur C, Pacherie M, Pastoureau P, Kucharczyk
N, Fauchère JL, Bonnet J 1996 Effects of parathyroid hormone and
agonists of the adenylyl cyclase and protein kinase C pathways on bone
cell proliferation. Bone 18:5965[CrossRef][Medline]
-
Onishi T, Hruska K 1997 Expression of p27Kip1 in
osteoblast-like cells during differentiation with parathyroid hormone.
Endocrinology 138:19952004[Abstract/Free Full Text]
-
Jüppner H, Abou-Samra AB, Freeman M, Kong XF, Schipani
E, Richards J, Kolakowski Jr LF, Hock J, Potts Jr JT, Kronenberg HM,
Segre GV 1991 A G protein-linked receptor for parathyroid hormone and
parathyroid hormone-related peptide. Science 254:10241026[Medline]
-
Karaplis AC, Luz A, Glowacki J, Bronson RT, Tybulewicz VL,
Kronenberg HM, Mulligan RC 1994 Lethal skeletal dysplasia from targeted
disruption of the parathyroid hormone-related peptide gene. Genes Dev 8:277289[Abstract]
-
Lanske B, Karaplis AC, Lee K, Luz A, Vortkamp A, Pirro A,
Karperien M, Defize LHK, Ho C, Mulligan RC, Abou-Samra AB,
Jüppner H, Segre GV, Kronenberg HM 1996 PTH/PTHrP receptor in
early development and Indian hedgehog-regulated bone growth [see
comments]. Science 273:663666[Abstract]
-
Amizuka N, Warshawsky H, Henderson JE, Goltzman D, Karaplis
AC 1994 Parathyroid hormone-related peptide-depleted mice show abnormal
epiphyseal cartilage development and altered endochondral bone
formation. J Cell Biol 126:16111623[Abstract]
-
Schipani E, Kruse K, Jüppner H 1995 A constitutively
active mutant PTH-PTHrP receptor in Jansen-type metaphyseal
chondrodysplasia. Science 268:98100[Medline]
-
Schipani E, Langman CB, Parfitt AM, Jensen GS, Kikuchi S, Kooh
SW, Cole WG, Jüppner H 1996 Constitutively activated receptors
for parathyroid hormone and parathyroid hormone-related peptide in
Jansens metaphyseal chondrodysplasia [see comments]. N Engl
J Med 335:708714[Abstract/Free Full Text]
-
Vortkamp A, Lee K, Lanske B, Segre GV, Kronenberg HM, Tabin CJ 1996 Regulation of rate of cartilage differentiation by Indian hedgehog
and PTH-related protein [see comments]. Science 273:613622[Abstract]
-
Zenmyo M, Komiya S, Kawabata R, Sasaguri Y, Inoue A, Morimatsu
M 1996 Morphological and biochemical evidence for apoptosis in the
terminal hypertrophic chondrocytes of the growth plate. J Pathol 180:430433[CrossRef][Medline]
-
Grimm S, Bauer MK, Baeuerle PA, Schulze-Osthoff K 1996 Bcl-2
down-regulates the activity of transcription factor NF-
B induced
upon apoptosis. J Cell Biol 134:1323[Abstract]
-
Allen RT, Hunter WJ, 3rd, Agrawal DK 1997 Morphological and
biochemical characterization and analysis of apoptosis. J
Pharmacol Toxicol Methods 37:215228[CrossRef][Medline]
-
Peter ME, Heufelder AE, Hengartner MO 1997 Advances in
apoptosis research. Proc Natl Acad Sci USA 94:1273612737[Abstract/Free Full Text]
-
Walton M, Sirimanne E, Reutelingsperger C, Williams C,
Gluckman P, Dragunow M 1997 Annexin V labels apoptotic neurons
following hypoxia-ischemia. Neuroreport 8:38713875[Medline]
-
Eguchi Y, Shimizu S, Tsujimoto Y 1997 Intracellular ATP levels
determine cell death fate by apoptosis or necrosis. Cancer Res 57:18351840[Abstract]
-
Salvesen GS, Dixit VM 1997 Caspases: intracellular signaling
by proteolysis. Cell 91:443446[Medline]
-
Du Y, Bales KR, Dodel RC, Hamilton-Byrd E, Horn JW,
Czilli DL, Simmons LK, Ni B, Paul SM 1997 Activation of a caspase
3-related cysteine protease is required for glutamate-mediated
apoptosis of cultured cerebellar granule neurons. Proc Natl Acad Sci
USA 94:1165711662[Abstract/Free Full Text]
-
Gressner AM, Lahme B, Roth S 1997 Attenuation of
TGF-beta-induced apoptosis in primary cultures of hepatocytes by
calpain inhibitors. Biochem Biophys Res Commun 231:457462[CrossRef][Medline]
-
Ivanov VN, Lee RK, Podack ER, Malek TR 1997 Regulation of
Fas-dependent activation-induced T cell apoptosis by cAMP signaling: a
potential role for transcription factor NF-
B. Oncogene 14:24552464[CrossRef][Medline]
-
Blind E, Bambino T, Nissenson RA 1995 Agonist-stimulated
phosphorylation of the G protein-coupled receptor for parathyroid
hormone (PTH) and PTH-related protein. Endocrinology 136:42714277[Abstract]
-
Lin XS, Denmeade SR, Cisek L, Isaacs JT 1997 Mechanism and
role of growth arrest in programmed (apoptotic) death of prostatic
cancer cells induced by thapsigargin. Prostate 33:201207[CrossRef][Medline]
-
Huang Z, Chen Y, Pratt S, Chen TH, Bambino T, Nissenson RA,
Shoback DM 1996 The N-terminal region of the third intracellular loop
of the parathyroid hormone (PTH)/PTH-related peptide receptor is
critical for coupling to cAMP and inositol phosphate/Ca2+ signal
transduction pathways. J Biol Chem 271:3338233389[Abstract/Free Full Text]
-
Huang C, Hepler JR, Gilman AG, Mumby SM 1997 Attenuation of
Gi- and Gq-mediated signaling by expression of RGS4 or GAIP in
mammalian cells. Proc Natl Acad Sci USA 94:61596163[Abstract/Free Full Text]
-
Koch WJ, Hawes BE, Inglese J, Luttrell LM, Lefkowitz RJ 1994 Cellular expression of the carboxyl terminus of a G protein-coupled
receptor kinase attenuates G beta gamma-mediated signaling. J Biol
Chem 269:61936197[Abstract/Free Full Text]
-
Daaka Y, Luttrell LM, Lefkowitz RJ 1997 Switching of the
coupling of the beta2-adrenergic receptor to different G proteins by
protein kinase A. Nature 390:8891[CrossRef][Medline]
-
Yandell MD, Edgar LG, Wood WB 1994 Trimethylpsoralen induces
small deletion mutations in Caenorhabditis elegans. Proc
Natl Acad Sci USA 91:13811385[Abstract]
-
Darnay BG, Aggarwal BB 1997 Early events in TNF signaling: a
story of associations and dissociations. J Leukoc Biol 61:559566[Abstract]
-
Widmann C, Gibson S, Johnson GL 1998 Caspase-dependent
cleavage of signaling proteins during apoptosis. A turn-off mechanism
for anti-apoptotic signals. J Biol Chem 273:71417147[Abstract/Free Full Text]
-
Strasser A, Huang DC, Vaux DL 1997 The role of the bcl-2/ced-9
gene family in cancer and general implications of defects in cell death
control for tumourigenesis and resistance to chemotherapy. Biochim
Biophys Acta 1333:F151178
-
Giambarella U, Yamatsuji T, Okamoto T, Matsui T, Ikezu T,
Murayama Y, Levine MA, Katz A, Gautam N, Nishimoto I 1997 G protein
ß
complex-mediated apoptosis by familial Alzheimers disease
mutant of APP. EMBO J 16:48974907[Abstract/Free Full Text]
-
Grandgirard D, Studer E, Monney L, Belser T, Fellay I, Borner
C, Michel MR 1998 Alphaviruses induce apoptosis in Bcl-2-overexpressing
cells: evidence for a caspase-mediated, proteolytic inactivation of
Bcl-2. EMBO J 17:12681278[Abstract/Free Full Text]
-
Lucas M, Sánchez-Margalet V 1995 Protein kinase C
involvement in apoptosis. Gen Pharmacol 26:881887[CrossRef][Medline]
-
Althoefer H, Eversole-Cire P, Simon MI 1997 Constitutively
active G
q and G
13 trigger apoptosis through different pathways.
J Biol Chem 272:2438024386[Abstract/Free Full Text]
-
Chattopadhyay N, Vassilev PM, Brown EM 1997 Calcium-sensing
receptor: roles in and beyond systemic calcium homeostasis. Biol Chem 378:759768[Medline]
-
Furuya Y, Lundmo P, Short AD, Gill DL, Isaacs JT 1994 The role
of calcium, pH, and cell proliferation in the programmed (apoptotic)
death of androgen-independent prostatic cancer cells induced by
thapsigargin. Cancer Res 54:61676175[Abstract]
-
McConkey DJ, Orrenius S 1997 The role of calcium in the
regulation of apoptosis. Biochem Biophys Res Commun 239:357366[CrossRef][Medline]
-
Jayaraman T, Marks AR 1997 T cells deficient in inositol
1,4,5-trisphosphate receptor are resistant to apoptosis. Mol Cell Biol 17:30053012[Abstract]
-
Lee S, Christakos S, Small MB 1993 Apoptosis and signal
transduction: clues to a molecular mechanism. Curr Opin Cell Biol 5:286291[Medline]
-
Mattson MP, Cheng B, Baldwin SA, Smith-Swintosky VL, Keller J,
Geddes JW, Scheff SW, Christakos S 1995 Brain injury and tumor necrosis
factors induce calbindin D-28 k in astrocytes: evidence for a
cytoprotective response. J Neurosci Res 42:357370[Medline]
-
Katz A, Wu D, Simon MI 1992 Subunits ß
of heterotrimeric
G protein activate ß 2 isoform of phospholipase C. Nature 360:686689[CrossRef][Medline]
-
Chen C, Okayama H 1987 High-efficiency transformation of
mammalian cells by plasmid DNA. Mol Cell Biol 7:27452752[Medline]
-
Pollock AS, Santiesteban HL 1995 Calbindin expression in renal
tubular epithelial cells. Altered sodium phosphate co-transport in
association with cytoskeletal rearrangement. J Biol Chem 270:1629116301[Abstract/Free Full Text]