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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{alpha}). 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 {alpha}-subunit of Gq) suppressed apoptotic signaling by the PTHR, whereas the C-terminal fragment of GRK2 (an inhibitor of the activity of the ß{gamma}-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{alpha}. 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go). 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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Figure 1. 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 ) on the Number of HEK 293 Cells Expressing the Wt PTHR

Cells were plated at approximately 50 cells/mm2 in 12-well plates, and 24 h later (day 0) 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 at the concentrations indicated. In some wells, complete medium was replaced by serum-free medium on day 0 (No Serum). Random fields of cells were counted at 24-h intervals, and the bars indicate the SEM of triplicate determinations.

 
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. 2AGo). 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. 2BGo). 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. 2CGo). 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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Figure 2. 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 ) on Apoptosis of HEK 293 Cells Expressing the Wt PTHR

A, Apoptag staining of cells treated with or without 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 72 h. Positive peroxidase staining (brown) results from the labeling of DNA terminal fragments. B, Quantitation of TUNEL staining of 293 cells treated with or without 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 1 or 3 days, or after serum withdrawal for 3 days. All cells (including floating cells) were harvested and fixed before staining. Several hundred cells were counted for each data point: cells with brown staining were scored as positive, blue cells were scored as negative. Control cells at days 1 and 3 revealed less than 1% positive (apoptotic) staining. C, Agarose gel electrophoresis of DNA isolated from cells treated for 3 days in the following ways: maintained in the continuous presence of serum-containing medium (Ctrl); subject to serum withdrawal (NS); or exposed to PMA (400 nM), 1 µM 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 ) (a PTHR antagonist), or 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 ).

 
Differences in cell morphology after PTH treatment and serum withdrawal were observed. The morphological response to serum withdrawal was cell shrinkage/cell rounding (Fig. 3AGo), a response frequently associated with apoptosis (15). The initial morphological response to PTH treatment was cell spreading and flattening (Fig. 3AGo). 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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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. 3CGo). 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. 4AGo) and apoptosis as determined by TUNEL (Fig. 4BGo). 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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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. 5Go). 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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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. 6Go). Second, activation of adenylyl cyclase via isoproteronol-induced activation of endogenous ß2-adrenergic receptors failed to reduce cell number or induce apoptosis (Fig. 6Go). 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. 6Go).



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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. 6Go). 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. 6BGo). 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. 6AGo). 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. 7Go). However, calbindin overexpression did not protect cells from the effects of serum withdrawal.



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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{gamma}- and {alpha}q-subunit function. RGS4 accelerates the GTPase activity of {alpha}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. 8Go). 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{gamma}-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. 9Go). Overexpression of CtGRK2 blocked ß-adrenergic receptor-mediated activation of MAP kinase, a process known to be dependent upon the ß{gamma}-subunits of Gi (28). This demonstrates that sufficient CtGRK2 was expressed to inhibit the functional activity of ß{gamma}-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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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).

 


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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. 10Go. 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. 11Go). Also shown is the effect of tumor necrosis factor-{alpha} (TNF{alpha}) (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{alpha}. 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{alpha}, whereas others (such as clone 22) remained fully sensitive to TNF{alpha}.



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Figure 10. Effects of PTH, TNF{alpha}, 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{alpha} (10 ng/ml), or serum withdrawal.

 


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Figure 11. Model Showing Putative Pathways Leading to Apoptosis Induced by PTHR Activation, TNF{alpha}, 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 {alpha}-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{alpha} 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{alpha} 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{alpha} receptor (TNFR). Most cells express the TNFR (30), and TNF{alpha} was found in the present study to be a potent inducer of apoptosis in HEK293 cells (Fig. 10Go). 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. 11Go), 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{alpha}, 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. 5Go). 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. 11Go). 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{gamma}-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 {alpha}-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{alpha}, 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. 11Go). Other cell lines were resistant to TNF{alpha} 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{alpha}.

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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{alpha} 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.


    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.


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