help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Grey, A.
Right arrow Articles by Cornish, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Grey, A.
Right arrow Articles by Cornish, J.
Endocrinology Vol. 143, No. 12 4755-4763
Copyright © 2002 by The Endocrine Society

ARTICLE

The Phospholipids Sphingosine-1-Phosphate and Lysophosphatidic Acid Prevent Apoptosis in Osteoblastic Cells via a Signaling Pathway Involving Gi Proteins and Phosphatidylinositol-3 Kinase

Andrew Grey, Qi Chen, Karen Callon, Xin Xu, Ian R. Reid and Jill Cornish

Department of Medicine, University of Auckland, Private Bag 92019, Auckland, New Zealand

Address all correspondence and requests for reprints to: Dr. Grey, University of Auckland, Department of Medicine, School of Medicine, Second Floor, Park Avenue, Private Bag 92019, Auckland, New Zealand. E-mail: a.grey{at}auckland.ac.nz.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The naturally occurring phospholipids lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) have recently emerged as bioactive compounds that exert mitogenic effects in many cell types, including osteoblasts. In the current study, we examined the ability of each of these compounds to influence osteoblast survival. Using terminal deoxynucleotidyl transferase-mediated deoxyuridine 5'-triphosphate nick-end labeling and DNA fragmentation assays, we found that both LPA and S1P dose-dependently inhibited (by at least 50% and 40%, respectively) the apoptosis induced by serum withdrawal in cultures of primary calvarial rat osteoblasts and SaOS-2 cells. The antiapoptotic effects were inhibited by pertussis toxin, wortmannin, and LY294002, implicating Gi proteins and phosphatidylinositol-3 kinase (PI-3 kinase) in the signaling pathway that mediates phospholipid-induced osteoblast survival. Specific inhibitors of p42/44 MAPK signaling did not block LPA- or S1P-induced osteoblast survival. LPA and S1P induced PI-3 kinase-dependent activation of p70 S6 kinase, but rapamycin, a specific inhibitor of p70 S6 kinase activation, did not prevent phospholipid-induced osteoblast survival. LPA and S1P also inhibited apoptosis in Swiss 3T3 fibroblastic cells in a Gi protein-dependent fashion. In fibroblastic cells, however, the antiapoptotic effects of S1P were sensitive to inhibition of both PI-3 kinase and p42/44 MAPK signaling, whereas those of LPA were partially abrogated by inhibitors of p42/44 MAPK signaling but not by PI-3 kinase inhibitors. These data demonstrate that LPA and S1P potently promote osteoblast survival in vitro, and that cell-type specificity exists in the antiapoptotic signaling pathways activated by phospholipids.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT IS CURRENTLY BELIEVED that up to 65% of osteoblasts undergo apoptosis during the bone remodeling cycle (1). The majority of surviving osteoblasts become osteocytes, buried within the bone matrix, which probably play a role in coordinating further remodeling events, including repair of skeletal microdamage (2). An increase in osteoblast and/or osteocyte apoptosis would therefore be expected to compromise skeletal integrity and may contribute to the pathogenesis of the bone loss and fragility fractures associated with sex hormone deficiency and glucocorticoid excess (3, 4, 5, 6, 7). Conversely, interventions that suppress osteoblast apoptosis, such as intermittent administration of parathyroid hormone (8), increase bone mass, and reduce skeletal fragility (9). Thus, identifying the factors that regulate osteoblast survival and the molecular mechanisms by which such factors act is important to our understanding of bone remodeling in physiological and pathophysiological conditions.

In recent years, the naturally occurring phospholipids lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) have emerged as important bioactive compounds in their own right (10, 11, 12). LPA and S1P are present in the systemic circulation at high nanomolar to low micromolar concentrations and are synthesized and released by platelets, adipocytes, and fibroblasts in vitro (13, 14, 15). Thus, each of these compounds appears to exert endocrine and paracrine actions, and a growing body of evidence suggests that S1P also has important intracrine effects (16, 17). A family of specific G protein-coupled receptors (edg or lp) has been identified, members of which probably mediate some of the diverse cellular actions of LPA and S1P (18). Thus, LPA and S1P have been reported to induce proliferation and cytoskeletal reorganization in several cell types (16, 19), including osteoblasts (20, 21, 22). Recent evidence suggests that phospholipids also promote survival in macrophages (23), Schwann cells (24), T lymphocytes (25), fibroblasts (26), and endothelial cells (27).

In the current paper, we report that LPA and S1P prevent apoptosis in osteoblastic cells, and that this effect is in each case sensitive to inhibitors of Gi proteins and PI-3 kinase, but not of p42/44 MAPK signaling. Neither of the PI-3 kinase effectors protein kinase B (PKB)/Akt or p70 S6 kinase appears to be required for phospholipid-induced osteoblast survival. We also demonstrate cell-type specificity in the survival pathways activated by phospholipids because p42/44 MAPK activation appears to be important for the antiapoptotic effects of LPA and S1P in fibroblastic cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Fetal calf serum and tissue culture media were from Life Technologies, Inc. (Grand Island, NY). L-{alpha}-Oleoyl LPA, rapamycin, collagenase, leupeptin, pepstatin, aprotinin, and sodium orthovanadate were from Sigma (St. Louis, MO). PD-98059, U-0126, wortmannin, and LY294002 were from BIOMOL (Plymouth Meeting, PA). S1P was from BIOMOL or Matreya (State College, PA). [3H]-Thymidine was from Amersham Pharmacia Biotech (Little Chalfont, UK). Pertussis toxin was from List Biological Laboratories (Campbell, CA).

The antibodies to phosphorylated PKB/Akt (Ser473), phosphorylated p42/44 MAPK and total PKB/Akt were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The antibody to phosphorylated p70 S6 kinase (Thr389) was from Cell Signaling Technology (Beverly, MA). The antibodies to total p70 S6 kinase were from Cell Signaling Technology and Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence reagents were from Amersham Pharmacia Biotech.

Cell culture
Primary rat osteoblastic cells were prepared as previously described (28). In brief, osteoblasts from collagenase digests three to four of 20-d fetal rat calvariae were pooled, centrifuged, and then washed in DMEM with 10% fetal calf serum (FCS), resuspended in DMEM/10% FCS, and placed in 75 cm2 flasks. After 48 h, the medium was changed to MEM containing 10% FCS. Confluence of the primary cultures was reached within 5–6 d, at which time the cells were subcultured. Only cells passaged on one to two occasions were used in the experiments detailed below. The osteoblast-like character of these cells has been established by demonstration of alkaline phosphatase staining in more than 95% of cells, osteocalcin production, and a sensitive adenylyl cyclase response to PTH and prostaglandin E2 (28).

The human osteoblastic SaOS-2 cell line and the murine Swiss 3T3 fibroblastic cell line were grown in MEM containing 10% FCS and subcultured into six-well plates for assessment of apoptosis as described below.

Apoptosis assays
Terminal deoxynucleotidyl transferase-mediated deoxyuridine 5'-triphosphate nick end labeling (TUNEL) assay.
Apoptosis in cultures of primary rat osteoblasts was assessed using the TUNEL method (DeadEnd, Promega Corp., Madison, WI), according to the manufacturer’s instructions. Cells were seeded in eight-well chamber slides at 5 x 104 cells/ml in MEM containing 5% FCS. Twenty-four hours later, the medium was changed to MEM/0.1%BSA and the cells incubated overnight. The following morning, test substances were added in fresh MEM/0.1% BSA for 18 h. At the end of the treatment period, cells were fixed in 2% paraformaldehyde for 15 min, then permeabilized with 1% Triton in PBS for 5 min. Thereafter, biotinylated nucleotides were added in the presence of terminal deoxynucleotidyl transferase for 1 h at 37 C, and the reaction terminated with 2x SSC. Endogenous peroxidases were blocked with 0.3% H2O2 for 5 min, streptavidin-horseradish peroxidase added for 30 min, and apoptotic nuclei colorized by addition of diaminobenzidine/H2O2 mixture. After counterstaining with hematoxylin, the number of apoptotic nuclei per microscopic field was counted and expressed as a proportion of that observed in the cells exposed to serum starvation alone throughout the entire treatment period. Each experiment was performed at least twice and involved assessment of at least six chambers per treatment condition.

DNA fragmentation.
Apoptosis in cultures of primary rat osteoblasts, SaOS-2 cells and Swiss 3T3 cells was assessed in the presence and absence of signaling inhibitors using a DNA fragmentation assay (26, 29, 30, 31). Cells were seeded in six-well plates at 5 x 104/ml, grown to semiconfluence and labeled with 3[H]-thymidine (0.5 µCi/well) for 4 h in MEM containing 5–10% FCS. Thereafter, the medium containing unincorporated [3H]-thymidine was removed and the cells incubated in MEM/0.1%BSA with or without phospholipid and in the presence or absence of inhibitors for a further 24 h. Inhibitors were added 30 min before phospholipid. After extensive washing, cells were lysed in TE buffer (10 mM Tris-HCl; 1 mM EDTA, pH 7.4; 0.2% Triton X-100) and the lysates centrifuged at 13,000 rpm for 15 min. Radioactivity was measured in the supernatant and pellet by scintillation counting and the proportion of fragmented DNA, representing apoptotic cells, was calculated by the following formula: % apoptosis = 3[H]supernatant/3[H]supernatant + 3[H]pellet.

Preliminary time-course experiments confirmed that there was a progressive increase in the radioactivity measured in the cell lysate supernatant during the period of serum starvation, confirming that the supernatant counts are derived from degradation of prelabeled DNA. Each experiment included triplicate measurements under each treatment condition. In each experiment involving the use of pharmacological inhibitors, an inhibitor-only treatment was performed to control for independent effects on cell survival of the inhibitor or its vehicle. Apoptosis as a percentage of control values was calculated by expressing the values obtained in cultures of cells exposed to phospholipid or phospholipid and inhibitor as a proportion of the values obtained in cultures of cells exposed to serum starvation or inhibitor alone, respectively.

Immunoblotting
Primary rat osteoblastic cells were grown to near-confluence in MEM 5% FCS in six-well plates, serum-starved in MEM 0.1% BSA overnight, and treated with phospholipids at the indicated concentrations for the periods of time indicated in the figure legends. Inhibitors were added 30 min before phospholipid. Whole-cell lysates were prepared by scraping the cells in ice-cold HNTG lysis buffer (50 mM HEPES, pH 7.5; 150 mM NaCl; 1% Triton; 10% glycerol; 1.5 mM MgCl2; 1 mM EDTA) containing a cocktail of protease and phosphatase inhibitors (1 mM phenylmethylsulfonylfluoride, 1 µg/ml pepstatin, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM sodium vanadate, 500 mM NaF), and analyzed by immunoblotting as previously described (20). Detection of proteins of interest was by enhanced chemiluminescence.

Statistical analyses
All data were analyzed using GraphPad Software, Inc. (San Diego, CA) Prism. Data from individual experiments were expressed as treatment to control ratios. Data from at least three separate experiments under each treatment condition were collated and analyzed by ANOVA to determine the effects of LPA and S1P on osteoblast survival, in the presence and absence of the indicated inhibitor. All data shown are mean ± SEM unless otherwise stated.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
LPA and S1P prevent apoptosis in osteoblastic cells
Both LPA (10 µM) and S1P (10 µM) decreased the apoptosis observed in response to serum deprivation in cultures of primary rat osteoblastic cells, as judged by number of TUNEL-positive cells (Fig. 1Go). This effect was dose dependent (Fig. 2AGo) and apparent at concentrations as low as 10 nM of LPA and 100 nM of S1P. The antiapoptotic effect of LPA and S1P in osteoblastic cells was confirmed in primary rat osteoblasts (Fig. 2BGo, left panel) and SaOS-2 cells (Fig. 2BGo, right panel) using a DNA fragmentation assay. In each in vitro assay, LPA (10 µM) and S1P (5–10 µM) reduced osteoblast apoptosis induced by serum withdrawal by at least 50% and 40%, respectively. In primary rat osteoblasts, the combination of 10 µM LPA and 5 µM S1P produced slightly greater reduction in apoptosis than either phospholipid alone (mean ± SEM % apoptosis; LPA + S1P 27.0 ± 1.5%, LPA 42.3 ± 0.7%, S1P 35.7 ± 2.3%). These data demonstrate that phospholipids are potent survival factors in osteoblastic cells.



View larger version (60K):
[in this window]
[in a new window]
  		Figure 1. LPA and S1P prevent apoptosis in primary rat osteoblastic cells. Primary rat osteoblastic cells were grown in eight-well chamber slides, then subjected to serum withdrawal for 24 h in the absence (control, upper panel) or presence of phospholipid (LPA 10 µM, middle panel; S1P 10 µM, lower panel). Apoptosis was assessed using the TUNEL method as described in Materials and Methods. Arrowheads indicate TUNEL-positive nuclei. The bar represents 50 µm.

 


View larger version (21K):
[in this window]
[in a new window]
  		Figure 2. LPA and S1P prevent apoptosis in osteoblastic cells A, Summary data from the TUNEL assay in primary rat osteoblastic cells, pooled from at least two experiments for each concentration of phospholipid (LPA, left panel; S1P, right panel), each including at least six replicate measurements. Data are mean ± SEM; *, P < 0.05 vs. control; **, P < 0.01 vs. control. B, Primary rat osteoblasts (left panel) or SaOS-2 cells (right panel) were seeded in six-well plates and grown to 60% confluence. After labeling cellular DNA with [3H]-thymidine, the cells were serum-starved for 24 h in the presence of a range of concentrations of the indicated phospholipid. Apoptosis was quantified by determining the proportion of fragmented DNA, as described in Materials and Methods. Data are pooled from at least three experiments for each treatment condition, and are shown as mean ± SEM. FCS, Fetal calf serum; *, P < 0.05 vs. control; **, P < 0.01 vs. control.

 
Phospholipid-induced osteoblast survival requires Gi proteins and PI-3 kinase, but not p42/44 MAPKs
Pretreatment of primary rat osteoblastic cells with the Gi protein inhibitor pertussis toxin (10 ng/ml) completely reversed the antiapoptotic effect of both LPA and S1P (Fig. 3AGo). In contrast, the pan-PKC inhibitor calphostin C had no effect on phospholipid-induced osteoblast survival (Fig. 3AGo). Because signaling through PI-3 kinases has been implicated in the antiapoptotic effects of many growth factors (32), we examined the effect of the specific inhibitors of PI-3 kinase, LY294002 and wortmannin, on phospholipid-induced osteoblast survival. As shown in Fig. 3BGo, LY294002 dose dependently inhibited the antiapoptotic effect of each phospholipid. Wortmannin also inhibited LPA- and S1P-induced osteoblast survival. In contrast, neither of the specific inhibitors of p42/44 MAPK signaling, PD 98059 and U-0126, influenced osteoblast survival induced by either LPA or S1P (Fig. 3CGo). These data demonstrate that Gi proteins and PI-3 kinases, but not p42/44 MAPKs, are required for phospholipid-induced survival in osteoblastic cells.



View larger version (14K):
[in this window]
[in a new window]
  		Figure 3. Phospholipid-induced osteoblast survival requires Gi proteins and PI-3 kinase, but not p42/44 MAPKs. Primary rat osteoblasts were seeded in six-well plates, grown to 60% confluence, and cellular DNA labeled with [3H]-thymidine. Cells were then serum-starved with or without 10 µM LPA or 5 µM S1P in the presence or absence of pertussis toxin or calphostin C (A), PI-3 kinase inhibitors (B), or inhibitors of p42/44 MAPK signaling (C). Treatments were for 24 h, following which apoptosis was quantified by determining the proportion of fragmented DNA as described in Materials and Methods. *, P < 0.05 vs. phospholipid alone; **, P < 0.01 vs. phospholipid alone.

 
Neither Akt/PKB nor p70 s6 kinase mediate phospholipid-induced PI-3 kinase-dependent survival in osteoblastic cells
Akt/PKB is considered to be an important downstream mediator of PI-3 kinase-dependent survival signaling (33). We were unable to convincingly demonstrate activation of Akt (as judged by phosphorylation at the Ser473 moiety) by either LPA or S1P in osteoblastic cells (Fig. 4Go, top panel), although each phospholipid activated p42/44 MAPKs in the same experiments (Fig. 4Go, middle panel). Similar results were obtained in 6 independent experiments. In contrast, low concentrations of FCS (Fig. 4Go, top panel, lanes 6 and 12) or IGF-1 (data not shown) did consistently induce phosphorylation of Akt in our cells, confirming that Akt phosphorylation is inducible in osteoblasts.



View larger version (37K):
[in this window]
[in a new window]
  		Figure 4. Phospholipids do not activate Akt/PKB in osteoblastic cells. Primary rat osteoblasts were grown to near-confluence in six-well tissue culture plates, serum starved overnight, then treated with vehicle (lanes 1 and 7), 10 µM LPA (lanes 2–5), 5 µM S1P (lanes 8–11), or 5% FCS (lanes 6 and 12) for the indicated times. Cells were lysed in HNTG lysis buffer and 50 µg of clarified whole cell lysate was analyzed by immunoblotting with antibodies to phospho-Akt (top panel), phospho-p42/44 MAPK (middle panel), or total Akt (lower panel), as described in Materials and Methods. The depicted immunoblots were performed on the same nitrocellulose membrane, after division of the membrane at a position between the molecular weights of Akt and p42/44 MAPK.

 
Among many other putative intracellular targets of PI-3 kinase activation is the ribosomal protein kinase p70 S6 kinase (34), activation of which is strongly PI-3 kinase-dependent (35). Recent evidence has implicated this kinase in survival signaling (36). As shown in Fig. 5AGo, top panel, both LPA and S1P induced phosphorylation of p70 S6 kinase in osteoblastic cells at Thr389, a critical event for p70 S6 kinase activation. This effect was PI-3 kinase dependent because it was prevented by pretreatment with LY294002 (Fig. 5AGo, lanes 5 and 6). As expected, phospholipid-induced activation of p70 S6 kinase was also sensitive to rapamycin, a specific inhibitor of p70 S6 kinase activation (Fig. 5BGo, lanes 2 and 3). We therefore tested the ability of rapamycin to block phospholipid-induced osteoblast survival. As is shown in Fig. 5CGo, rapamycin at concentrations of up to 50 ng/ml had no effect on the antiapoptotic actions of either phospholipid. Taken together, these data demonstrate activation of PI-3 kinase signaling in osteoblastic cells by LPA and S1P, but suggest that neither Akt/PKB nor p70 S6 kinase is the important downstream mediator of the PI-3 kinase-dependent antiapoptotic effects of LPA and S1P.



View larger version (24K):
[in this window]
[in a new window]
  		Figure 5. Phospholipids induce PI-3 kinase-dependent phosphorylation of p70 S6 kinase, but their effects on osteoblast survival are not dependent on p70 S6 kinase. A, Primary rat osteoblasts were grown to near-confluence in six-well tissue culture plates, serum starved overnight, then treated for 20 min with vehicle (lanes 1 and 4), 10 µM LPA (lanes 2 and 5), or 5 µM S1P (lanes 3 and 6) in the presence (lanes 4–6) or absence (lanes 1–3) of 10 µM LY294002. Cells were lysed in HNTG lysis buffer and 50 µg of clarified whole cell lysate was analyzed by immunoblotting with antibodies to phospho-p70 S6 kinase (Thr389) (top panel) or total p70 S6 kinase (lower panel). B, Primary rat osteoblasts were grown to near-confluence in six-well tissue culture plates, serum starved overnight, then treated for 20 min with vehicle (lane 1), or 10 µM LPA (lanes 3 and 5), or 5 µM S1P (lanes 2 and 4) in the presence (lanes 2 and 3) or absence (lanes 4 and 5) of 20 ng/ml rapamycin. Cells were lysed in HNTG lysis buffer and 50 µg of clarified whole cell lysate was analyzed by immunoblotting with either antibodies to phospho-p70 S6 kinase (Thr389) (top panel) or total p70 S6 kinase (lower panel). C, Primary rat osteoblasts were seeded in six-well plates, grown to 60% confluence, and cellular DNA labeled with [3H]-thymidine. Cells were then treated with vehicle, 10 µM LPA, or 5 µM S1P in the presence or absence of rapamycin 50 ng/ml. Pooled data (mean ± SEM) from three separate experiments are shown as treatment to control ratios for each phospholipid in the presence and absence of inhibitor.

 
Survival signals activated by S1P and LPA differ between osteoblastic and fibroblastic cells
Recent evidence suggests that LPA promotes survival in fibroblastic cells independently of PI-3 kinase (26). We therefore compared the proximal signaling events required for phospholipid-induced survival in osteoblastic and fibroblastic cells. As shown in Fig. 6AGo, 5% FCS reduced apoptosis in Swiss 3T3 fibroblastic cells to a level 80% below that observed in cells exposed to serum starvation. LPA and S1P each prevented apoptosis induced by serum withdrawal in Swiss 3T3 fibroblastic cells, and the magnitude of the effect (50–60% reduction) was comparable to that which we observed in osteoblastic cells (compare with Fig. 2B). As was observed in osteoblastic cells, the antiapoptotic effect of each phospholipid in Swiss 3T3 cells was blocked by pertussis toxin (Fig. 6BGo). In contrast to our findings in osteoblastic cells, however, there was evidence of ligand-specific survival signaling in fibroblastic cells. Thus, LPA-induced fibroblast survival was insensitive to either of the PI-3 kinase inhibitors LY294002 and wortmannin (Fig. 6CGo), but partially inhibited by each of the p42/44 MAPK signaling inhibitors U-0126 and PD 98059 (Fig. 6DGo), whereas S1P-induced fibroblast survival was blocked by inhibitors of both PI-3 kinase and p42/44 MAPK signaling (Fig. 6Go, C and D). Taken together, these data demonstrate cell-type and ligand-type specificity in survival signaling initiated in response to LPA and S1P.



View larger version (21K):
[in this window]
[in a new window]
  		Figure 6. Survival signals activated by S1P and LPA differ between osteoblastic and fibroblastic cells. A, Swiss 3T3 cells were seeded in six-well plates and grown to 60% confluence. After labeling cellular DNA with [3H]-thymidine, the cells were serum-starved for 24 h in the absence or presence of 5% FCS or a range of concentrations of the indicated phospholipid. Apoptosis was quantified by determining the proportion of fragmented DNA, as described in Materials and Methods. Data are pooled from at least three experiments for each treatment condition, and are shown as mean ± SEM. **, P < 0.01 vs. control. B–D, Swiss 3T3 cells were seeded in six-well plates, grown to 60% confluence, and cellular DNA labeled with [3H]-thymidine. Cells were then serum-starved in the presence or absence of 10 µM LPA or 5 µM S1P and pertussis toxin (B), PI-3 kinase inhibitors (C), or inhibitors of p42/44 MAPK signaling (D). Treatments were for 24 h, following which apoptosis was quantified by determining the proportion of fragmented DNA as described in Materials and Methods. *, P < 0.05 vs. phospholipid alone; **, P < 0.01 vs. phospholipid alone.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
There is a growing awareness of the importance of osteoblast apoptosis in skeletal homeostasis, and in the therapeutics of osteoporosis (1, 2). The majority of osteoblastic cells are believed to suffer an apoptotic fate, and several hormones and cytokines that are known to regulate skeletal integrity do so in part by altering osteoblast and/or osteocyte survival (3, 4, 5, 6, 7). Therefore, identifying compounds that influence osteoblast apoptosis, and characterizing the mechanism(s) by which they exert their effects, is likely to significantly advance our understanding of skeletal physiology and pathophysiology. The current paper demonstrates that the naturally occurring phospholipids LPA and S1P exert potent antiapoptotic effects on osteoblastic cells in vitro, at concentrations that are comparable to those reported in the systemic circulation (15, 37). Taken together with recent evidence that each of these compounds is a potent osteoblast mitogen (20, 21, 22), the current data support the notion that LPA and S1P may be physiological regulators of bone metabolism.

Phospholipid signaling is a complex process. LPA and S1P activate intracellular signaling by binding to members of the recently characterized family of edg/lp G protein-coupled receptors, which currently includes eight members (38, 39). Edg 1, 3, 5, 6, and 8 are specific S1P receptors, whereas edg 2, 4, and 7 are specific LPA receptors. Several of these receptors are expressed in a variety of tissues, such that more than one receptor for either phospholipid may be expressed in the same tissue (36, 37). In addition, there is substantial overlap between the phospholipid receptors in the intracellular signaling pathways activated following ligand binding (11, 40). Further, some evidence suggests that some of the growth factor-like effects of LPA and S1P are not transduced through edg/lp receptors (41). Our data demonstrate that the ability of LPA and S1P to prevent osteoblast apoptosis is dependent upon functional Gi proteins and the phospholipid kinase PI-3 kinase. The former observation is consistent with a large body of evidence that attests to the role of Gi proteins in mediating the proliferative and antiapoptotic effects of each compound in a variety of tissues (16, 42). Thus, in osteoblasts Gi proteins are now implicated in phospholipid-induced proliferation and survival (20) (current paper). The near-complete inhibition of the proliferative and antiapoptotic effects of both LPA and S1P in osteoblastic cells by pertussis toxin strongly suggests that specific G protein-coupled receptors transduce these effects. Receptors for both S1P and LPA are expressed in osteoblastic cells (43) (Hill, B., and A. Grey, submitted), although which of these receptors mediate the mitogenic and/or antiapoptotic effects of these agents is currently unknown.

While Gi proteins are important for both the mitogenic and antiapoptotic effects of LPA and S1P in osteoblastic cells, the role of PI-3 kinase appears to be restricted to survival signaling, since specific inhibitors of PI-3 kinase do not prevent the potent proliferative actions of LPA or S1P in osteoblastic cells (17) (Xu, X., and A. Grey, submitted). PI-3 kinase signaling is believed to play an important role in cell survival in a diverse range of tissues in response to a broad range of extracellular stimuli (33). Many growth factors, acting through either G protein-coupled receptors or receptor tyrosine kinases, promote cell survival by activating one of the PI-3 kinase isoforms (44). Our data demonstrate that the antiapoptotic effect of each phospholipid in osteoblastic cells is PI-3 kinase dependent, because in each case it was blocked by the specific PI-3 kinase inhibitors LY294002 and wortmannin. This observation is consistent with studies of survival signaling activated by LPA in renal tubular cells (45), Schwann cells (24), and macrophages (23), and by S1P in hepatocytes (46), which implicate PI-3 kinase in the pathway subserving cell survival. However, PI-3 kinase is not universally a mediator of phospholipid-induced cell survival. Our studies in Swiss 3T3 fibroblastic cells indicate that the LPA-induced antiapoptotic effects are not dependent on this enzyme. This finding is consistent with those of Fang et al. (26), who reported that PI-3 kinase does not transduce the LPA survival signal in NIH 3T3 cells. Thus, there appears to be cell-type specificity in the mechanisms by which LPA prevents apoptosis. In addition, there is ligand specificity in phospholipid-induced fibroblast survival because the antiapoptotic effect of S1P, but not that of LPA, is PI-3 kinase dependent. The mechanism(s) underlying the cell-type and ligand specificity we observed are uncertain.

The identity of the PI-3 kinase-dependent effector(s) that mediate phospholipid-induced osteoblast survival remains uncertain. It is currently believed that the lipid products generated by activation of PI-3 kinase enable the kinase activity of 3-phosphoinositide-dependent kinases toward a number of potential downstream effectors (47). Some data suggest that PKB/Akt is a critical component of the survival signal downstream of PI-3 kinase/phosphoinositide-dependent kinase-1 (33, 48). However, we were unable to detect activation of PKB/Akt in osteoblastic cells in response to either LPA or S1P, as assessed by immunoblotting for phosphorylation at the critical Ser473 site. In contrast, low concentrations of FCS and IGF-1 reproducibly induced PKB/Akt phosphorylation in the same cells (Fig. 4Go, and data not shown). Thus, we believe it unlikely that PKB/Akt plays a major role in phospholipid-induced osteoblast survival. PI-3 kinase-dependent signaling was activated by each of the phospholipids in osteoblastic cells, as evidenced by LPA- and S1P-induced phosphorylation of p70 S6 kinase on Thr389, which was abrogated by the specific PI-3 kinase inhibitor LY294002. Activation of the ribosomal enzyme p70 S6 kinase has been implicated as a mediator of growth factor-induced survival (36). Although we observed activation of p70 S6 kinase in response to both LPA and S1P, specific inhibition of p70 S6 kinase activity using rapamycin did not prevent the antiapoptotic effect of either phospholipid. This finding again illustrates the occurrence of cell-type specificity in phospholipid signaling because rapamycin inhibits LPA-induced survival in macrophages (23). Other potential PI-3 kinase-dependent mediators of survival signaling include protein kinase C isoforms (49), the recently described serum- and glucocorticoid-induced kinases (50, 51), which are structurally homologous to Akt/PKB, and protein kinase A (52).

In summary, the current paper reports for the first time that the naturally occurring phospholipid compounds LPA and S1P potently prevent apoptosis in osteoblastic cells. This effect is likely to be mediated by a member(s) of the edg/lp family of G protein-coupled receptors because it was in each case completely inhibited by the Gi protein inhibitor pertussis toxin. Activation of PI-3 kinase is also required for phospholipid-induced osteoblast survival, whereas p42/44 MAPK signaling is not. The downstream components of the PI-3 kinase-dependent survival signal activated by LPA and S1P in osteoblasts are uncertain, but neither PKB/Akt nor p70 S6 kinase appears to be involved. The intracellular pathways by which LPA and S1P prevent apoptosis in osteoblastic cells differ from those required for prevention of fibroblast apoptosis. Phospholipids may contribute to the regulation of osteoblast cell fate in vivo.


   Acknowledgments
 
The authors thank Maureen Watson for technical assistance, and the Biomedical Imaging Research Unit at the Auckland University School of Medicine for assistance in producing the TUNEL images.


   Footnotes
 
This work was supported by grants from the Health Research Council of New Zealand, the Auckland Medical Research Foundation, the NZ Lotteries Board and the Royal Australasian College of Physicians.

Abbreviations: FCS, Fetal calf serum; LPA, lysophosphatidic acid; PI-3 kinase, phosphatidylinositol-3 kinase; PKB, protein kinase B; S1P, sphingosine-1-phosphate; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine 5'-triphosphate nick-end labeling.

Received March 25, 2002.

Accepted for publication August 5, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Manolagas SC 2000 Birth and death of bone cells: basic regulatory mechanisms and implications for the pathogenesis and treatment of osteoporosis. Endocr Rev 21:115–137[Abstract/Free Full Text]
  2. Weinstein RS, Manolagas SC 2000 Apoptosis and osteoporosis. Am J Med 108:153–164[Medline]
  3. Tomkinson A, Reeve J, Shaw RW, Noble BS 1997 The death of osteocytes via apoptosis accompanies estrogen withdrawal in human bone. J Clin Endocrinol Metab 82:3128–3135[Abstract/Free Full Text]
  4. Weinstein RS, Jilka RL, Parfitt AM, Manolagas SC 1998 Inhibition of osteoblastogenesis and promotion of apoptosis of osteoblasts and osteocytes by glucocorticoids. Potential mechanisms of their deleterious effects on bone. J Clin Invest 102:274–282[Medline]
  5. Weinstein RS, Nicholas RW, Manolagas SC 2000 Apoptosis of osteocytes in glucocorticoid-induced osteonecrosis of the hip. J Clin Endocrinol Metab 85:2907–2912[Abstract/Free Full Text]
  6. Gohel A, McCarthy MB, Gronowicz G 1999 Estrogen prevents glucocorticoid-induced apoptosis in osteoblasts in vivo and in vitro. Endocrinology 140:5339–5347[Abstract/Free Full Text]
  7. Weinstein RS, Jilka RL, Parfitt AM, Manolagas SC 1997 The effects of androgen deficiency on murine bone remodeling and bone mineral density are mediated via cells of the osteoblastic lineage. Endocrinology 138:4013–4021[Abstract/Free Full Text]
  8. Jilka RL, Weinstein RS, Bellido T, Roberson P, Parfitt AM, Manolagas SC 1999 Increased bone formation by prevention of osteoblast apoptosis with parathyroid hormone. J Clin Invest 104:439–446[Medline]
  9. Neer RM, Arnaud CD, Zanchetta JR, Prince R, Gaich GA, Reginster JY, Hodsman AB, Eriksen EF, Ish-Shalom S, Genant HK, Wang O, Mitlak BH 2001 Effect of parathyroid hormone (1–34) on fractures and bone mineral density in postmenopausal women with osteoporosis. New Engl J Med 344:1434–1441[Abstract/Free Full Text]
  10. Tigyi G 2001 Physiological responses to lysophosphatidic acid and related glycero-phospholipids. Prostaglandins Other Lipid Mediat 64:47–62
  11. Goetzl EJ 2001 Pleiotypic mechanisms of cellular responses to biologically active lysophospholipids. Prostaglandins Other Lipid Mediat 64:11–20[CrossRef][Medline]
  12. Spiegel S, Milstien S 2000 Sphingosine-1-phosphate: signaling inside and out. FEBS Lett 476:55–57[CrossRef][Medline]
  13. Yatomi Y, Ozaki Y, Ohmori T, Igarashi Y 2001 Sphingosine 1-phosphate: synthesis and release. Prostaglandins Other Lipid Mediat 64:107–122[CrossRef][Medline]
  14. Pages C, Simon MF, Valet P, Saulnier-Blache JS 2001 Lysophosphatidic acid synthesis and release. Prostaglandins Other Lipid Mediat 64:1–10[CrossRef][Medline]
  15. Eichholtz T, Jalink K, Fahrenfort I, Moolenaar WH 1993 The bioactive phospholipid lysophosphatidic acid is released from activated platelets. Biochem J 291:677–680
  16. Pyne S, Pyne NJ 2000 Sphingosine 1-phosphate signalling in mammalian cells. Biochem J 349:385–402[CrossRef][Medline]
  17. Spiegel S 1999 Sphingosine 1-phosphate: a prototype of a new class of second messengers. J Leuk Biol 65:341–344[Abstract]
  18. Goetzl EJ, An S 1998 Diversity of cellular receptors and functions for the lysophospholipid growth factors lysophosphatidic acid and sphingosine 1-phosphate. FASEB J 12:1589–1598[Abstract/Free Full Text]
  19. Moolenaar WH 1999 Bioactive lysophospholipids and their G protein-coupled receptors. Exp Cell Res 253:230–238[CrossRef][Medline]
  20. Grey A, Banovic T, Naot D, Hill B, Callon K, Reid I, Cornish J 2001 Lysophosphatidic acid is an osteoblast mitogen whose proliferative actions involve G(i) proteins and protein kinase C, but not P42/44 mitogen-activated protein kinases. Endocrinology 142:1098–1106[Abstract/Free Full Text]
  21. Caverzasio J, Palmer G, Suzuki A, Bonjour JP 2000 Evidence for the involvement of two pathways in activation of extracellular signal-regulated kinase (Erk) and cell proliferation by Gi and Gq protein-coupled receptors in osteoblast-like cells. J Bone Miner Res 15:1697–1706[CrossRef][Medline]
  22. Carpio LC, Stephan E, Kamer A, Dziak R 1999 Sphingolipids stimulate cell growth via MAP kinase activation in osteoblastic cells. Prostaglandins Leukot Essent Fatty Acids 61:267–273[CrossRef][Medline]
  23. Koh JS, Lieberthal W, Heydrick S, Levine JS 1998 Lysophosphatidic acid is a major serum noncytokine survival factor for murine macrophages which acts via the phosphatidylinositol 3-kinase signaling pathway. J Clin Invest 102:716–727[Medline]
  24. Weiner JA, Chun J 1999 Schwann cell survival mediated by the signaling phospholipid lysophosphatidic acid. Proc Natl Acad Sci USA 96:5233–5238[Abstract/Free Full Text]
  25. Goetzl EJ, Kong Y, Mei B 1999 Lysophosphatidic acid and sphingosine 1-phosphate protection of T cells from apoptosis in association with suppression of Bax. J Immunol 162:2049–2056[Abstract/Free Full Text]
  26. Fang X, Yu S, LaPushin R, Lu Y, Furui T, Penn LZ, Stokoe D, Erickson JR, Bast Jr RC, Mills GB 2000 Lysophosphatidic acid prevents apoptosis in fibroblasts via G(i)-protein-mediated activation of mitogen-activated protein kinase. Biochem J 352:135–143
  27. Kwon YG, Min JK, Kim KM, Lee DJ, Billiar TR, Kim YM 2001 Sphingosine 1-phosphate protects human umbilical vein endothelial cells from serum-deprived apoptosis by nitric oxide production. J Biol Chem 276:10627–10633[Abstract/Free Full Text]
  28. Cornish J, Callon KE, Lin CQ, Xiao CL, Mulvey TB, Coy DH, Cooper GJ, Reid IR 1998 Dissociation of the effects of amylin on osteoblast proliferation and bone resorption. Am J Physiol 274:E827–E833
  29. Zhang R, DeGroot LJ 1997 A monoclonal antibody against rat calcitonin inhibits the growth of a rat medullary thyroid carcinoma cell line in vitro. Endocrinology 138:1697–1703[Abstract/Free Full Text]
  30. Isik FF, Gibran NS, Jang YC, Sandell L, Schwartz SM 1998 Vitronectin decreases microvascular endothelial cell apoptosis. J Cellular Physiol 175:149–155[CrossRef][Medline]
  31. Hsieh CC, Yen MH, Liu HW, Lau YT 2000 Lysophosphatidylcholine induces apoptotic and non-apoptotic death in vascular smooth muscle cells: in comparison with oxidized LDL. Atherosclerosis 151:481–491[CrossRef][Medline]
  32. Vanhaesebroeck B, Leevers SJ, Panayotou G, Waterfield MD 1997 Phosphoinositide 3-kinases: a conserved family of signal transducers. Trends Biochem Sci 22:267–272[CrossRef][Medline]
  33. Datta SR, Brunet A, Greenberg ME 1999 Cellular survival: a play in three Akts. Gene Dev 13:2905–2927[Free Full Text]
  34. Volarevic S, Thomas G 2001 Role of S6 phosphorylation and S6 kinase in cell growth. Prog Nucleic Acids Res Mol Biol 65:101–127[Medline]
  35. Blume-Jensen P, Hunter T 2001 Oncogenic kinase signalling. Nature 411:355–365[CrossRef][Medline]
  36. Harada H, Andersen JS, Mann M, Terada N, Korsmeyer SJ 2001 p70S6 kinase signals cell survival as well as growth, inactivating the pro-apoptotic molecule BAD. Proc Natl Acad Sci USA 98:9666–9670[Abstract/Free Full Text]
  37. Yatomi Y, Ruan F, Ohta J, Welch RJ, Hakomori S, Igarashi Y 1995 Quantitative measurement of sphingosine 1-phosphate in biological samples by acylation with radioactive acetic anhydride. Anal Biochem 230:315–320[CrossRef][Medline]
  38. Fukushima N, Ishii I, Contos JJ, Weiner JA, Chun J 2001 Lysophospholipid receptors. Ann Rev Pharmacol Toxicol 41:507–534[CrossRef][Medline]
  39. Chun J, Contos JJ, Munroe D 1999 A growing family of receptor genes for lysophosphatidic acid (LPA) and other lysophospholipids (LPs). Cell Biochem Biophys 30:213–242[Medline]
  40. Fukushima N, Chun J 2001 The LPA receptors. Prostaglandins Other Lipid Mediat 64:21–32[CrossRef][Medline]
  41. Hooks SB, Santos WL, Im D-S, Heise CE, Macdonald TL, Lynch KR 2001 Lysophosphatidic acid-induced mitogenesis is regulated by lipid phosphate phosphatases and is edg-receptor independent. J Biol Chem 276:4611–4621[Abstract/Free Full Text]
  42. Moolenaar WH 1995 Lysophosphatidic acid, a multifunctional phospholipid messenger. J Biol Chem 270:12949–12952[Free Full Text]
  43. Lyons JM, Karin NJ 2001 A role for G protein-coupled lysophospholipid receptors in sphingolipid-induced Ca2+ signaling in MC3T3-E1 osteoblastic cells. J Bone Miner Res 16:2035–2042[CrossRef][Medline]
  44. Cantrell DA 2001 Phosphoinositide 3-kinase signalling pathways. J Cell Sci 114:1439–1445[Abstract]
  45. Levine JS, Koh JS, Triaca V, Lieberthal W 1997 Lysophosphatidic acid: a novel growth and survival factor for renal proximal tubular cells. Am J Physiol 273:F575–F585
  46. Osawa Y, Banno Y, Nagaki M, Brenner DA, Naiki T, Nozawa Y, Nakashima S, Moriwaki H 2001 TNF-{alpha}-induced sphingosine 1-phosphate inhibits apoptosis through a phosphatidylinositol 3-kinase/Akt pathway in human hepatocytes. J Immunol 167:173–180[Abstract/Free Full Text]
  47. Vanhaesebroeck B, Alessi DR 2000 The PI3K-PDK1 connection: more than just a road to PKB. Biochem J 346:561–576
  48. Leevers SJ, Vanhaesebroeck B, Waterfield MD 1999 Signalling through phosphoinositide 3-kinases: the lipids take centre stage. Curr Opin Cell Biol 11:219–225[CrossRef][Medline]
  49. Le Good JA, Ziegler WH, Parekh DB, Alessi DR, Cohen P, Parker PJ 1998 Protein kinase C isotypes controlled by phosphoinositide 3-kinase through the protein kinase PDK1. Science 281:2042–2045[Abstract/Free Full Text]
  50. Brunet A, Park J, Tran H, Hu LS, Hemmings BA, Greenberg ME 2001 Protein kinase SGK mediates survival signals by phosphorylating the forkhead transcription factor FKHRL1 (FOXO3a). Mol Cell Biol 21:952–965[Abstract/Free Full Text]
  51. Mikosz CA, Brickley DR, Sharkey MS, Moran TW, Conzen SD 2001 Glucocorticoid receptor-mediated protection from apoptosis is associated with induction of the serine/threonine survival kinase gene, sgk-1. J Biol Chem 276:16649–16654[Abstract/Free Full Text]
  52. Cheng X, Ma Y, Moore M, Hemmings BA, Taylor SS 1998 Phosphorylation and activation of cAMP-dependent protein kinase by phosphoinositide-dependent protein kinase. Proc Natl Acad Sci USA 95:9849–9854[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
N. Panupinthu, L. Zhao, F. Possmayer, H. Z. Ke, S. M. Sims, and S. J. Dixon
P2X7 Nucleotide Receptors Mediate Blebbing in Osteoblasts through a Pathway Involving Lysophosphatidic Acid
J. Biol. Chem., February 2, 2007; 282(5): 3403 - 3412.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
R. DeKroon, J. B. Robinette, A. B. Hjelmeland, E. Wiggins, M. Blackwell, M. Mihovilovic, M. Fujii, J. York, J. Hart, C. Kontos, et al.
APOE4-VLDL Inhibits the HDL-Activated Phosphatidylinositol 3-Kinase/Akt Pathway via the Phosphoinositol Phosphatase SHIP2
Circ. Res., October 13, 2006; 99(8): 829 - 836.
[Abstract] [Full Text] [PDF]

Home page
 J EndocrinolHome page
P J D Delhanty, B C J van der Eerden, M van der Velde, C Gauna, H A P Pols, H Jahr, H Chiba, A J van der Lely, and J P T M van Leeuwen
Ghrelin and unacylated ghrelin stimulate human osteoblast growth via mitogen-activated protein kinase (MAPK)/phosphoinositide 3-kinase (PI3K) pathways in the absence of GHS-R1a
J. Endocrinol., January 1, 2006; 188(1): 37 - 47.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
V. Limaye, X. Li, C. Hahn, P. Xia, M. C. Berndt, M. A. Vadas, and J. R. Gamble
Sphingosine kinase-1 enhances endothelial cell survival through a PECAM-1-dependent activation of PI-3K/Akt and regulation of Bcl-2 family members
Blood, April 15, 2005; 105(8): 3169 - 3177.
[Abstract] [Full Text] [PDF]

Home page
 Pharmacol. Rev.Home page
I. Tegeder and G. Geisslinger
Opioids As Modulators of Cell Death and Survival--Unraveling Mechanisms and Revealing New Indications
Pharmacol. Rev., September 1, 2004; 56(3): 351 - 369.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
D. Dadke, B. H. Fryer, E. A. Golemis, and J. Field
Activation of p21-Activated Kinase 1-Nuclear Factor {kappa}B Signaling by Kaposi's Sarcoma-Associated Herpes Virus G Protein-Coupled Receptor during Cellular Transformation
Cancer Res., December 15, 2003; 63(24): 8837 - 8847.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
A. Grey, Q. Chen, X. Xu, K. Callon, and J. Cornish
Parallel Phosphatidylinositol-3 Kinase and p42/44 Mitogen-Activated Protein Kinase Signaling Pathways Subserve the Mitogenic and Antiapoptotic Actions of Insulin-Like Growth Factor I in Osteoblastic Cells
Endocrinology, November 1, 2003; 144(11): 4886 - 4893.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Grey, A.
Right arrow Articles by Cornish, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Grey, A.
Right arrow Articles by Cornish, J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals