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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
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). 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 |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 Jansen’s 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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). 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
48–72 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).
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).
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).
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), 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.
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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.
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). 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).
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). 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).
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). 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.
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- 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.
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. 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.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 manufacturer’s 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.
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FOOTNOTES |
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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.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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