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Endocrinology Vol. 144, No. 11 4886-4893
Copyright © 2003 by The Endocrine Society

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

Andrew Grey, Qi Chen, Xin Xu, Karen Callon and Jill Cornish

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

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IGF-I is an endocrine and paracrine regulator of skeletal homeostasis, principally by virtue of its anabolic effects on osteoblastic cells. In the current study, we examined the intracellular signaling pathways by which IGF-I promotes proliferation and survival in SaOS-2 human osteoblastic cells. Inhibition of each of the phosphatidylinositol-3 kinase (PI-3 kinase), p42/44 MAPK, and p70s6 kinase pathways partially inhibited the ability of IGF-I to stimulate osteoblast proliferation and survival. Because activation of p70s6 kinase is downstream of both PI-3 kinase and p42/44 MAPK activation in osteoblasts treated with IGF-I, this ribosomal kinase represents a convergence point for IGF-I-induced PI-3 kinase and p42/44 MAPK signaling in osteoblastic cells. In addition, abrogation of PI-3 kinase-dependent Akt signaling, which does not inhibit IGF-I-induced p70s6 kinase phosphorylation, also inhibited the antiapoptotic effects of IGF-I in osteoblasts. Finally, interruption of Gß{gamma} signaling partially abrogated the ability of IGF-I to promote osteoblast survival, without inhibiting signaling through PI-3 kinase/Akt, p42/44 MAPKs, or p70s6 kinase. These data suggest that IGF-I signals osteoblast mitogenesis and survival through parallel, partly overlapping intracellular pathways involving PI-3 kinase, p42/44 MAPKs, and Gß{gamma} subunits.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IGF-I IS A POTENT growth factor in many tissues, exerting its actions by both endocrine and paracrine mechanisms. Skeletal tissue is enriched with IGF-I and its associated binding proteins (1). In skeletal tissue, a large body of in vitro and in vivo evidence attests to the actions of IGF-I as an endocrine and paracrine regulator of bone metabolism (1). In vitro, IGF-I promotes the proliferation (2, 3, 4), survival (5, 6), and differentiation (7) of osteoblastic cells in culture, acts as a paracrine mediator of the anabolic effects of PTH (8, 9) and thyroid hormone (10), and potently induces the development of osteoclasts (11, 12). Expression and production of IGF-I by osteoblastic cells is regulated by important systemic osteotropic factors such as estrogen (13), glucocorticoids (14), and active vitamin D metabolites (15). In vivo, transgenic murine experiments demonstrate that IGF-I (16, 17), the IGF-I receptor (18), and insulin receptor substrate (IRS)-1 (19), a key downstream effector of IGF-I signaling, are required for skeletal homeostasis.

The cellular actions of IGF-I are mediated by a receptor tyrosine kinase (IGF-IR) that is expressed in a diverse range of tissues. Upon ligand binding, the IGF-IR homodimerizes, and undergoes a series of autophosphorylation events that promote activation of at least two canonical intracellular signaling pathways, involving phosphatidylinositol-3 kinase (PI-3 kinase) and p42/44 MAPKs. Current evidence suggests that these signaling pathways regulate many of the growth factor-like actions of IGF-I (20). However, it is clear that the relative contributions of each pathway to the diverse cellular actions of IGF-I vary according to cell type (21). In some tissues, PI-3 kinase and p42/44 MAPK signals are independent of each other, whereas in other tissues activation of PI-3 kinase signaling is upstream of p42/44 MAPK (22, 23). Thus, it is necessary to study IGF-I signaling in individual tissues to determine the role(s) of each pathway in that cell type.

Because IGF-I is an important osteoblast growth factor, we sought to examine the intracellular mechanisms by which it exerts two of its pivotal effects, mitogenesis and survival. Using a combination of approaches (pharmacological inhibitors and transient transfection of dominant negatives), we found that the proliferative and antiapoptotic actions of IGF-I in SaOS-2 human osteoblastic cells are mediated by parallel PI-3 kinase and p42/44 MAPK signaling pathways that converge in part on the downstream effector p70s6 kinase. Activation of the PI-3 kinase target Akt and signaling through Gi proteins also contribute to the survival-promoting effects of IGF-I in osteoblasts.


   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.-BRL Laboratories (Grand Island, NY). Recombinant human IGF-I was from Kabi Pharmacia (Gothenburg, Sweden). Rapamycin, 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). Pertussis toxin was from List Biological Laboratories (Campbell, CA). [3H]-thymidine was from Amersham Pharmacia Biotech (Little Chalfont, UK).

The antibodies to phosphorylated Akt (Ser473), phosphorylated p42/44 MAPK, phosphorylated p70s6 kinase (Thr389), total p42/44 MAPK, total Akt, total p70s6 kinase, ß-adrenergic receptor kinase (ßARK)/GRK2 (G protein-coupled receptor kinase 2), and HA (hemagglutinin) were from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-conjugated secondary antibodies and ECL reagents were from Amersham Pharmacia Biotech (Little Chalfont, UK).

cDNA encoding the C-terminal fragment of ßARK/GRK2 (ßARK-ct) was kindly provided by Dr. R. Lefkowitz, Duke University Medical Center (Durham, NC) (24). cDNA encoding the dominant-negative, kinase-dead HA-Akt construct (HA-AktK179M) was kindly provided by Dr. M. Greenberg, Harvard University (Cambridge, MA) (25). FuGENE 6 transfection reagent was from Roche Diagnostics (Indianapolis, IN).

Cell culture
Human osteoblastic SaOS-2 cells were maintained in MEM containing 10% fetal calf serum (FCS) in T75 flasks before subculturing for experimental procedures. Primary rat osteoblastic cells were prepared as previously described (26). All experiments involving animals were conducted in accordance with University of Auckland guidelines and were approved by the institutional ethics committee.

Transient transfection was performed in subconfluent cultures of SaOS-2 cells in either six-well (apoptosis assay, immunoblotting experiments) or 24-well (mitogenesis assay) plates, in 5% FCS using FuGene transfection reagent (1 µg of plasmid DNA or empty vector/3 µl transfection reagent). Successful transfection was confirmed by immunoblotting lysates of transfected cells with antibodies to either HA or ßARK.

Osteoblast mitogenesis
Proliferation of osteoblastic cells was assessed as previously described (26). In brief, SaOS-2 cells were seeded in 24-well tissue culture plates at 50,000 cells/ml in MEM containing 10% FCS, grown overnight to semiconfluence, then growth arrested in MEM 0.1% BSA for 18 h before addition of test substances. Treatments were for 24 h. [3H]-thymidine incorporation was assessed by pulsing the cells with [3H]-thymidine (0.5 µCi/well) 2 h before the end of the experimental incubation. In experiments involving pharmacological inhibitors, the inhibitor was added 30 min before the IGF-I, and an inhibitor-only treatment was included. In the transient transfection experiments, transfection was performed on the day after cell seeding, following which growth arrest and cell treatments were performed as described above. Each experiment was performed at least three times using experimental groups consisting of at least six wells. [3H]-thymidine incorporation as a fold stimulation over control values was calculated by dividing the values obtained in cultures of cells exposed to IGF-I or IGF-I and inhibitor by the values obtained in cultures of cells exposed to vehicle or inhibitor alone, respectively.

Osteoblast apoptosis
Apoptosis in cultures of SaOS-2 cells or primary rat osteoblastic cells was assessed in the presence and absence of signaling inhibitors using a DNA fragmentation assay (27, 28, 29, 30, 31). We have previously validated this assay against the TUNEL assay (27). Cells were seeded in six-well plates at 5 x 104/ml, grown to semiconfluence and labeled with [3H]-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 IGF-I in the presence or absence of inhibitors for a further 24 h. Inhibitors were added 30 min before IGF-I. 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 formula:

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. The duration of the period of [3H]-thymidine labeling did not affect the results of the assay. Each experiment included triplicate measurements under each treatment condition. In each experiment involving the use of 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 IGF-I or IGF-I and inhibitor as a proportion of the values obtained in cultures of cells exposed to vehicle or inhibitor alone, respectively.

Immunoblotting
SaOS-2 cells were seeded in six-well tissue culture plates at an initial density of 5 x 104 cells/ml in MEM 5% FCS, and grown to 80–90% confluence. After serum starvation overnight, cells were treated at room temperature with 10 nM IGF-I in MEM 0.1% BSA. In experiments designed to determine the effect of inhibitors of signal transduction, the cells were pretreated with the inhibitor for 30 min before addition of IGF-I. The exception was pertussis toxin, which was added 18 h before IGF-I. After treatment for the indicated period of time, the treatment medium was aspirated, the cells were washed in ice-cold PBS and then scraped 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 phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM sodium vanadate, and 500 mM NaF). The lysates were briefly vortexed, clarified by centrifugation at 13,000 rpm at 4 C, then stored at -70 C until analyzed. Lysates were subjected to 8% SDS-PAGE, transferred to nitrocellulose membranes and immunoblotted overnight at 4 C with primary antibody. Incubation with the horseradish peroxidase-conjugated secondary antibody was for 1 h at room temperature, and the membranes were analyzed by ECL. As a control for protein loading, the same filters were stripped and reprobed with an antibody to the nonphosphorylated protein of interest. Immunoblots presented are representative of at least three separate experiments in each case.

Statistical analyses
All data were analyzed using GraphPad Prism (GraphPad Software, San Diego, CA). 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 IGF-I on osteoblast survival or proliferation, 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
 
The mitogenic and antiapoptotic effects of IGF-I in osteoblastic cells are mediated by signaling pathways involving PI-3 kinase, p42/44 MAPKs, and p70s6 kinase
In SaOS-2 osteoblastic cells, IGF-I dose-dependently (0.01–1 nM) stimulated proliferation, as judged by incorporation of [3H]-thymidine (Fig. 1AGo), and prevented apoptosis induced by serum withdrawal, as measured by DNA fragmentation (Fig. 1BGo). At a concentration of 1 nM, IGF-I induced a 2.2-fold increase in DNA synthesis, and a 45% reduction in apoptosis.



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  		FIG. 1. IGF-I dose-dependently promotes mitogenesis and survival in osteoblastic cells. A, SaOS-2 cells were seeded into 24-well plates, grown to semiconfluence, growth arrested, then treated with IGF-I at the indicated concentrations for 24 h. DNA synthesis was assessed by incorporation of [3H]-thymidine, as described in Materials and Methods. B, SaOS-2 cells were seeded into six-well plates, grown to semiconfluence, labeled with [3H]-thymidine, then treated with IGF-I at the indicated concentrations for 24 h. Apoptosis was measured by quantitating fragmented DNA as described in Materials and Methods. *, P < 0.05 vs. control; **, P < 0.01 vs. control.

 
To identify the intracellular signaling pathways subserving the mitogenic and antiapoptotic actions of IGF-I in osteoblastic cells, SaOS-2 cells were treated with IGF-I in the presence of pharmacological inhibitors of Gi protein (pertussis toxin), PI-3 kinase (LY294002), p42/44 MAPK (PD-98059, U-0126), or p70s6 kinase (rapamycin) signaling. Each of the inhibitors had been demonstrated in previous work in our laboratory to abrogate the targeted signaling pathway in osteoblastic cells at the concentration shown in Fig. 2Go (26, 27). As shown in Fig. 2AGo, the antiapoptotic effects of IGF-I were partially sensitive to inhibitors of PI-3 kinase, p42/44 MAPK kinase and p70s6 kinase. Each of these inhibitors also partially blocked the mitogenic effects of IGF-I (Fig. 2BGo). Pertussis toxin partially inhibited the antiapoptotic effects of IGF-I (Fig. 2AGo) but did not affect its proliferative action (Fig. 2BGo). The latter observation was supported by the finding that overexpression of the C-terminal fragment of ß-adrenergic receptor kinase (ßARK-ct), which binds and sequesters free Gß{gamma} subunits generated upon dissociation of heterotrimeric G proteins, also abrogated the antiapoptotic, but not the mitogenic, actions of IGF-I (Fig 3AGo). The pan-PKC inhibitor calphostin C did not inhibit either the mitogenic or the survival-promoting actions of IGF-I (data not shown). These data implicate PI-3 kinase, p42/44 MAPK, and p70s6 kinase in IGF-I-induced osteoblast proliferation and survival, and suggest that Gi proteins participate in antiapoptotic, but not mitogenic, signaling.



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  		FIG. 2. Signaling through PI-3 kinase, p42/44 MAPKs, p70s6 kinase, and Gi proteins mediates IGF-I-induced osteoblast survival and mitogenesis. A, Pharmacological inhibitors of signaling pathways were added 30 min before IGF-I and apoptosis assessed as described in Materials and Methods. Data are presented as the apoptosis observed in IGF-I-treated cells expressed as a percentage of the value in vehicle-treated cells (open bars), or as the apoptosis observed in cells treated with IGF-I + inhibitor expressed as a percentage of the value in inhibitor-treated cells (shaded bars). B, Pharmacological inhibitors of signaling were added 30 min before IGF-I and mitogenesis assessed as described in Materials and Methods. Data are presented as the fold stimulation of [3H]-thymidine incorporation induced by IGF-I treatment over vehicle treatment (open bars) or that induced by treatment with IGF-I + inhibitor over inhibitor treatment (shaded bars). *, P < 0.05 vs. IGF-I alone; **, P < 0.01 vs. IGF-I alone.

 


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  		FIG. 3. Signaling through Gß{gamma} subunits and Akt contribute to IGF-I-induced osteoblast survival. A, SaOS-2 cells were transiently transfected (see inset) with cDNA encoding the C-terminal fragment of ßARK/GRK2 (ßARK-ct) or empty plasmid vector (EV), then treated with 1 nM IGF-I and mitogenesis (left panel) or apoptosis (right panel) measured as described in Materials and Methods. B, SaOS-2 cells were transiently transfected (see inset) with cDNA encoding a dominant-negative, kinase-dead Akt (DN-Akt) or empty plasmid vector (EV), then treated with 1 nM IGF-I and mitogenesis (left panel) or apoptosis (right panel) measured as described in Materials and Methods. *, P < 0.05 vs. EV; **, P < 0.01 vs. EV.

 
Among the currently known effectors of PI-3 kinase activation, Akt is thought to play an important role in survival signaling (32). Expression of a dominant-negative, kinase-dead mutant Akt in SaOS-2 cells substantially inhibited the ability of IGF-I to prevent apoptosis, and less potently (mean reduction 25%) blocked osteoblast mitogenesis induced by IGF-I (Fig. 3BGo).

SaOS-2 cells are derived from a human osteogenic sarcoma and do not express the p53 tumor suppressor gene, the protein product of which plays role in cell survival. We therefore tested the effect of the pharmacological inhibitors of intracellular signaling on IGF-I-induced survival in cultures of primary neonatal rat osteoblasts. As shown in Table 1Go, LY294002, PD-98059, rapamycin, and pertussis toxin each partially inhibited the antiapoptotic effect of IGF-I in the primary cell cultures, suggesting that the results we obtained in SaOS-2 cells apply also to primary osteoblastic cells.


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  		TABLE 1. IGF-I-induced survival in primary rat osteoblastic cells involves p42/44 MAPKs, PI-3K, p70s6 kinase, and Gi proteins

 
Parallel PI-3 kinase and p42/44 MAPK signaling pathways mediate IGF-I-induced osteoblast proliferation and survival, through Akt and p70s6 kinase
The data described above suggest that the growth factor-like actions of IGF-I in osteoblastic cells are dependent upon several intracellular signaling molecules, at least in part. To determine the interrelationships among these signaling molecules, we examined the effects of pretreatment with specific inhibitors of activation of each signaling pathway. Binding of IGF-I to its receptor activates PI-3 kinase and p42/44 MAPK signaling via the adapter proteins Shc and IRS-1/2, respectively (20). In osteoblastic cells, IGF-I-induced phosphorylation of Akt is a PI-3 kinase-dependent event, because it is blocked by the PI-3 kinase inhibitor LY294002 (Fig. 4AGo, lanes 2 and 6). Phosphorylation of Akt in response to IGF-I treatment is not abrogated by inhibition of Gi protein signaling with either pertussis toxin (Fig 4AGo, lanes 2 and 4) or overexpression of ßARK-ct (Fig 4BGo), nor is it blocked by the MAPK kinase inhibitors PD-98059 and U-0126 (Fig. 4CGo, lanes 2, 4, and 6). These data demonstrate that PI-3 kinase-dependent signaling is independent of Gi proteins and activation of the p42/44 MAPK pathway in osteoblastic cells stimulated with IGF-I.



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  		FIG. 4. IGF-I-induced activation of Akt is PI-3 kinase dependent, but independent of G proteins and p42/44 MAPKs. A, SaOS-2 cells were treated for 10 min with 10 nM IGF-I (lanes 2, 4, and 6) or vehicle (lanes 1, 3, and 5) in the presence or absence of 200 ng/ml pertussis toxin (PTx, lanes 3 and 4), or 10 µM LY294002 (lanes 5 and 6). Whole cell lysates were subjected to SDS-PAGE and immunoblotted with antibodies to phospho-Akt (top panel) or total Akt (bottom panel). B, SaOS-2 cells were transiently transfected with empty vector (EV) or ßARK-ct, then treated for 10 min with 10 nM IGF-I (lanes 2 and 4) or vehicle (lanes 1 and 3). Whole cell lysates were subjected to SDS-PAGE and immunoblotted with antibodies to phospho-Akt (top panel) or total Akt (bottom panel). C, SaOS-2 cells were treated for 10 min with 10 nM IGF-I (lanes 2, 4, and 6) or vehicle (lanes 1, 3, and 5) in the presence or absence of 30 µM PD-98059 (lanes 3 and 4) or 10 µM U-0126 (lanes 5 and 6). Whole cell lysates were subjected to SDS-PAGE and sequentially immunoblotted with antibodies to phospho-Akt (top panel) and total Akt (bottom panel).

 
In some cell systems, IGF-I-induced activation of p42/44 MAPK signaling is downstream of Gi proteins (33) and/or PI-3 kinase (22). In osteoblastic cells, activation of p42/44 MAPK signaling is not abrogated by inhibiting Gi protein signaling with either pertussis toxin (Fig 5AGo, right middle panel, lanes 3 and 4) or overexpression of ßARK-ct (Fig 5AGo, right middle panel, lanes 6 and 8), nor is it affected by pretreatment with the PI-3 kinase inhibitors LY294002 and wortmannin (Fig 5AGo, left panel). These data demonstrate that activation of p42/44 MAPKs in osteoblastic cells after IGF-I treatment is independent of Gi proteins and PI-3 kinase.



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  		FIG. 5. IGF-I-induced activation of p70s6 kinase is PI-3 kinase and p42/44 MAPK dependent but independent of activation of Akt and G protein signaling. A, Left panel, SaOS-2 cells were treated for 10 min with 10 nM IGF-I (lanes 3, 4, and 6) or vehicle (lanes 1, 2, and 5) in the presence or absence of 500 nM wortmannin (WT, lanes 2 and 3), or 10 µM LY294002 (lanes 5 and 6). Whole cell lysates were subjected to SDS-PAGE and sequentially immunoblotted with antibodies to phospho-p42/44 MAPKs (top panel) and total p42/44 MAPK (bottom panel). Right panel, Nontransfected (lanes 1–4) or transiently transfected (empty vector [EV] lanes 5 and 6, ßARK-ct lanes 7 and 8) SaOS-2 cells were treated for 20 min with 10 nM IGF-I (lanes 3, 4, 6, and 8) or vehicle (lanes 1, 2, 5, and 7). Pertussis toxin (PTx, 200 ng/ml) was added to nontransfected cells as indicated (lanes 2 and 3). Whole cell lysates were subjected to SDS-PAGE, the nitrocellulose membrane divided at the 60-kDa marker, and the top portion immunoblotted with an antibody to phospho-p70s6 kinase (top panel), whereas the bottom portion was sequentially immunoblotted with antibodies to phospho-p42/44 MAPK (middle panel) and total p42/44 MAPK (bottom panel). B, SaOS-2 cells were treated for 20 min with 10 nM IGF-I (lanes 2, 4, 6, 8, and 10) or vehicle (lanes 1, 3, 5, 7, and 9) in the presence or absence of 10 µM LY294002 (lanes 3 and 4), 10 µM PD-98059 (lanes 5 and 6) or 50 ng/ml rapamycin (lanes 9 and 10). Whole cell lysates were subjected to SDS-PAGE and immunoblotted with antibodies to phospho-p70s6 kinase (top panel) or total p70s6 kinase (bottom panel). C, SaOS-2 cells were transiently transfected with empty vector (EV, lanes 1 and 2) or DN-Akt (lanes 3 and 4), then treated for 20 min with 10 nM IGF-I (lanes 2 and 4) or vehicle (lanes 1 and 3). Whole cell lysates were subjected to SDS-PAGE and immunoblotted with antibodies to phospho-p70s6 kinase (top panel) or total p70s6 kinase (bottom panel).

 
Phosphorylation and activation of the ribosomal kinase p70s6 kinase has been reported to be a consequence of activation of both PI-3 kinase and p42/44 MAPK signaling (34), and therefore represents a potential convergence point of IGF-I-dependent mitogenic and antiapoptotic signaling. Rapamycin, an inhibitor of p70s6 kinase activation (Fig. 5BGo, lanes 8 and 10), partially inhibits both the proliferative and the antiapoptotic actions of IGF-I in osteoblastic cells (Fig. 2Go, A and B). In osteoblastic cells, phosphorylation of p70s6 kinase is sensitive to inhibitors of PI-3 kinase (LY294002, Fig. 5BGo, lanes 2 and 4) and p42/44 MAPK signaling (PD-98059, Fig. 5BGo, lanes 2 and 6). However, the ability of Gi protein signaling inhibitors to block the antiapoptotic effects of IGF-I cannot be explained by effects on p70s6 kinase, because neither pertussis toxin nor the ßARK-ct construct prevented IGF-I-induced phosphorylation of p70s6 kinase (Fig. 5AGo, right top panel). To determine whether PI-3 kinase-dependent Akt signaling might in part explain the involvement of p70s6 kinase in the antiapoptotic actions of IGF-I in osteoblastic cells, we tested the effect of the dominant-negative Akt construct on IGF-I-induced phosphorylation of p70s6 kinase. As shown in Fig. 5CGo, the kinase-dead Akt mutant did not abrogate the phosphorylation of p70s6 kinase induced by IGF-I. These data suggest that activation of PI-3 kinase signaling by IGF-I promotes osteoblast survival via independent downstream pathways involving Akt and p70s6 kinase.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Accumulated evidence from both in vitro and in vivo studies over the past decade have clearly demonstrated that IGF-I signaling is necessary for normal skeletal development and homeostasis, although the relative contributions of local vis a vis systemic IGF-I remain unclear (1, 35). The available evidence suggests that the osteoblast is a critical IGF-I target in the skeleton. Thus, defining the molecular mechanisms by which IGF-I influences osteoblast function should further our understanding of this important skeletal growth factor. The findings of the current study suggest that two critical osteoblastic effects of IGF-I, proliferation and survival, are each subserved in part by activation of parallel PI-3 kinase and p42/44 MAPK signaling pathways, which converge on the downstream effector p70s6 kinase. Signaling through Gi proteins and Akt also contributes to IGF-I-activated survival signaling in osteoblastic cells, independent of p70s6 kinase. Taken together, our data suggest a degree of functional redundancy in the intracellular pathways by which IGF-I exerts its anabolic effects on osteoblasts (Fig. 6Go).



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  		FIG. 6. Schematic model of intracellular pathways subserving the proliferative and antiapoptotic actions of IGF-I in osteoblastic cells. Ligation of the IGF-I receptor stimulates signaling through parallel p42/44 MAPK and PI-3 kinase pathways, each of which promotes both survival and proliferation, in part by activating the common downstream mediator p70s6 kinase. PI-3 kinase signaling also promotes survival by an Akt-dependent, p70s6 kinase-independent mechanism. IGF-I-activated Gß{gamma} signaling promotes osteoblast survival via uncharacterized downstream mediators. Sites of action of the pharmacological inhibitors used in this study are indicated. Solid arrows indicate definite/probable pathways; dashed arrows indicate possible pathways. PDK, 3-Phosphoinositide-dependent protein kinase.

 
It is clear that there exists cell type specificity in the intracellular mechanisms by which IGF-I exerts its growth factor-like effects (21). Thus, proliferation of fibroblasts in response to IGF-I appears to depend upon a functional PI-3 kinase signaling pathway (36), the mitogenic effect of IGF-I in myoblasts and adipocytes is dependent upon p42/44 MAPK, but not PI-3 kinase, signaling (37, 38), and both pathways are involved in IGF-I-stimulated mitogenesis in intestinal smooth muscle cells (39). IGF-I-induced differentiation of adipocytes is mediated by PI-3 kinase signaling (38), whereas that of neuronal cells requires p42/44 MAPKs (40). The antiapoptotic actions of IGF-I in neuronal cells involve both PI-3 kinase and p42/44 MAPK signaling (23). In osteoblastic cells, the limited evidence available before the current study suggested that IGF-I-induced mitogenesis in MG63 cells is dependent on p42/44 MAPK activation (4). The molecular mechanisms underpinning IGF-I-stimulated osteoblast survival have not been studied. Osteoblast-specific deletion of the IGF-IR results in decreased osteoblast number, impaired bone formation, and decreased bone mass (18), but it is not yet clear whether this effect is attributable to decreased osteoblast proliferation, increased osteoblast apoptosis or both. Similarly, osteoblastic cells derived from IRS-1-null mice exhibit impaired proliferation and differentiation (19), but because IRS-1 couples the IGF-I receptor to both PI-3 kinase and p42/44 MAPK signaling, this in vivo experiment does not allow delineation of the importance of each individual pathway to osteoblast growth. Our data suggest that osteoblastic cells require both the PI-3 kinase and the p42/44 MAPK signaling pathways to be functional to respond maximally to the growth-promoting actions of IGF-I. We acknowledge that results generated from experiments performed in a cell line such as SaOS-2 might not be generalizable to the whole animal. However, the congruent data we obtained in survival assays in primary rat osteoblastic cells vis a vis SaOS-2 cells lends support to the notion that our observations apply to osteoblastic cells generally.

The ribosomal kinase p70s6 kinase is phosphorylated and activated in response to activation of both PI-3 kinase (41) and p42/44 MAPK signaling (34). It is likely that full activation of p70s6 kinase is dependent upon phosphorylation of serine and threonine residues that are distinct targets of p42/44 MAPK and PI-3 kinase signals (42). Our data confirmed that p70s6 kinase activation by IGF-I is downstream of both PI-3 kinase and p42/44 MAPK in osteoblasts, because specific inhibitors of each signaling pathway blocked IGF-I-induced p70s6 kinase phosphorylation. Recent evidence suggests that, in addition to transducing the mitogenic signal initiated by IGF-I (43), p70s6 kinase contributes to IGF-I-induced survival signaling (23), in part by phosphorylating the proapoptotic Bcl-2 family member Bad (44). Our data are broadly congruent with these findings in nonskeletal tissue because the p70s6 kinase inhibitor rapamycin partially abrogated the ability of IGF-I to promote osteoblast proliferation and survival. It is therefore likely that p70s6 kinase mediates, at least in part, the proliferative and antiapoptotic effects induced by IGF-I-activated PI-3 kinase and p42/44 MAPK signaling in osteoblasts.

Although p70s6 kinase contributes to the proliferative and antiapoptotic actions of IGF-I, other downstream mediators of both PI-3 kinase and p42/44 MAPK must also play a role, because rapamycin only partly abrogates the effects of IGF-I. PI-3 kinase-dependent survival signaling activated by IGF-I in osteoblasts also involves Akt independent of activation of p70s6 kinase because overexpression of a dominant-negative Akt construct, which substantially abrogates the antiapoptotic actions of IGF-I, did not inhibit phosphorylation of p70s6 kinase. Whereas the nature of the signaling intermediaries between PI-3 kinase activation and phosphorylation of p70s6 kinase remain unclear, most of the available data suggest that Akt signaling does not activate p70s6 kinase (43, 45), and that the 3-phosphoinositide-dependent protein kinase 1 phosphorylates both Akt and p70s6 kinase to initiate parallel PI-3 kinase-dependent signaling cascades (43, 46). Potential Akt downstream targets that signal cell survival include caspase 9, Bad, and forkhead proteins (32).

An intriguing aspect of the current work was our observation that inhibition of Gi protein signaling inhibited the ability of IGF-I to promote osteoblast survival. The finding that overexpression of the ßARK-ct peptide also inhibited IGF-I-induced osteoblast survival suggests that the Gß{gamma} component of the heterotrimeric G protein complex is involved in transducing the antiapoptotic signal. IGF-I-induced stimulation of Gß{gamma} signaling in osteoblasts is not coupled to either the PI-3 kinase or p42/44 MAPK pathways because neither pertussis toxin nor the ßARK-ct peptide blocked IGF-I-induced activation of either pathway. Previous studies have suggested a role for pertussis toxin-sensitive G proteins in some of the cellular actions of IGF-I, principally mitogenesis (39, 47), perhaps mediated by direct interactions between components of the G protein signaling complex and the IGF-I receptor (47, 48). In contrast to these earlier studies, we found no effect of either pertussis toxin or the ßARK-ct peptide on IGF-I-induced osteoblast proliferation.

In summary, the current work suggests that proliferation and survival, two of the crucial cellular responses induced by IGF-I in osteoblasts, are mediated by parallel signaling pathways involving PI-3 kinase and p42/44 MAPKs, which converge at a common downstream effector, p70s6 kinase. However, IGF-I-induced osteoblast survival is also mediated in part by PI-3 kinase signaling through Akt, independent of p70s6 kinase, and by Gß{gamma} signaling through as yet undefined effectors.


   Acknowledgments
 
We thank Dr. R. Lefkowitz (Duke University Medical Center, Durham, NC) for generously providing the ßARK-ct construct, and Dr. M. Greenberg (Harvard University, Cambridge, MA) for generously providing the dominant-negative Akt construct.


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

Abbreviations: ßARK, ß-Adrenergic receptor kinase; ßARK-ct, the C-terminal fragment of ßARK/GRK2; FCS, fetal calf serum; GRK, G protein-coupled receptor kinase; HA, hemagglutinin; IGF-IR, IGF-I receptor; IRS-1, insulin receptor substrate; PI-3 kinase, phosphatidylinositol-3 kinase.

Received March 20, 2003.

Accepted for publication July 18, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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