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J. Biol. Chem., Vol. 280, Issue 20, 20163-20170, May 20, 2005
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From the
Musculoskeletal Disease Center, Jerry L. Pettis Memorial Veterans Affairs Medical Center, and
Departments of Medicine and Biochemistry, Loma Linda University, Loma Linda, California 92357
Received for publication, February 8, 2005 , and in revised form, March 14, 2005.
| ABSTRACT |
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60% and the shear stress-induced mitogenic response by
20%. It completely abolished the mitogenic effect of IGF-I and that of the combination treatment. Shear stress also significantly reduced the amounts of co-immunoprecipitated SHP-2 and -1 with IGF-IR, suggesting that the synergy between shear stress and IGF-I in osteoblast proliferation involves integrin-dependent recruitment of SHP-2 and -1 away from IGF-IR. | INTRODUCTION |
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Bone growth factors function as autocrine and paracrine mediators of bone formation (4, 5). The mechanism whereby mechanical loading stimulates osteoblast proliferation and activity could involve bone growth factors and corresponding signaling pathways. IGF-I1 is one of the most abundant growth factors in bone (4), produced by bone cells (47), and an important stimulator of bone formation (47). Loading increases bone cell production of IGF-I in vivo (8) and in vitro (9). The signaling pathway of IGF-I involves Erk1/2 activation, which is essential for mechanical stimulation of bone cell proliferation (10). It has been reported that the bone cell mitogenic response to mechanical strain is mediated by the IGF-IR (11). In addition, recent studies suggested that loading might have a permissive role in the IGF-I mitogenic action in bone, as skeletal unloading induces resistance to IGF-I with respect to bone formation. Accordingly, unloading blocked the ability of IGF-I to stimulate bone formation in the rat (12). IGF-I administration stimulates bone formation in the loaded bone, but not in unloaded bone in vivo (12) and in vitro (12, 13). There is evidence that unloading-related resistance to IGF-I is mediated by inhibiting the activation of IGF-I pathway through down-regulation of integrin expression (13). Because unloading blocked the osteogenic action of IGF-I, we postulated that increased loading enhances the osteogenic action of IGF-I. Accordingly, it has been suggested that loading enhances the anabolic effects of IGF-I on articular cartilage formation (14) and also in nasopremaxillary growth (15).
Recent studies in smooth muscle cells (1622) revealed that the ability of IGF-I to initiate its intracellular signals is regulated not only by its binding to its own transmembrane receptor (IGF-IR) but also by other transmembrane proteins, such as SHPS-1 and
v
3 integrin, to recruit essential signaling proteins, such as SHP-2 and Shc. The integrin recruitment of SHP-2 is essential for regulation of the overall IGF-IR phosphorylation level (18) and the propagation of downstream signaling events (19). Accordingly, ligand occupancy of
v
3 integrin results in phosphorylation of the
3 integrin subunit, which leads to Downstream of tyrosine kinase I (DOKI)-mediated recruitment of SHP-2 (20). Blocking ligand occupancy of
v
3 integrin inhibited IGF-I-dependent downstream signaling events, membrane recruitment of SHP-2, and cell migration and proliferation (21, 22). Expression of a dominant negative mutant of the
3 integrin subunit in smooth muscle cells completely abolished the mitogenic activity of IGF-I (16). Thus, integrin activation may have a permissive action in the IGF-IR signaling pathway.
Integrins, which consist of a large family of heterodimers of
- and
-subunits, function as cell surface adhesion receptors for extracellular matrices (23) and link extracellular matrix components with various intracellular signaling mechanisms (24). It is believed that mechanical strains and shear stresses are distributed to cells through extracellular matrix scaffolds that hold the cells together and that mechanical signals that propagate from the extracellular matrix converge on integrins (25). The interaction between specific bone matrix ligands and corresponding integrin receptors has been suggested to be involved in the signal transduction process linking the extracellular mechanical signals to changes in gene expression, cytoskeletal reorganization, and DNA synthesis in osteoblasts and/or osteocytes (26). Specific antibodies for several integrins blocked mechanical strain-induced cellular responses (27). The integrin-
-catenin signal pathway has also been suggested to be involved in the cellular responses of human articular chondrocytes to mechanical stimulation (28). Thus, integrin activation has an important role in the transduction of mechanical signals. Consequently, we postulate that the integrin-dependent regulation of the IGF-I mitogenic signaling pathway could, in part, be involved in the mechanical stimulation of bone formation.
This study investigated the potential relationship between the signaling mechanism of mechanical stimulation of osteoblast proliferation and that of IGF-I-induced osteoblast proliferation by testing two hypotheses: 1) increased mechanical strain in the form of fluid shear stress could synergistically enhance the osteogenic action of IGF-I, and 2) the synergy between IGF-I and fluid shear stress involves the integrin-dependent up-regulation of IGF-IR phosphorylation through an inhibition of SHP-mediated IGF-IR dephosphorylation.
| EXPERIMENTAL PROCEDURES |
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Fluid Shear Stress ExperimentsHuman TE85 osteosarcoma cells were plated on glass slides (75 x 38 mm) at 5 x 104 cells/slide in Dulbecco's modified Eagle's medium supplemented with 10% bovine calf serum. When the cells reached
80% confluency, the cells were serum-deprived for 24 h and subjected to a steady fluid shear stress of 20 dynes/cm2 for 30 min in Cytodyne flow chambers as previously described (3). The static controls were performed on cells grown in identical conditions in Cytodyne chambers but without exposing to the shear stress.
Cell Proliferation AssaysCell proliferation was assessed by [3H]thymidine incorporation into cell DNA as described previously (29). Briefly, after 30 min of the shear stress, the treated and corresponding static control cells were incubated with the indicated dosages of IGF-I (or FGF-2) for 24 h, and [3H]thymidine (1.5 µCi/ml) was added during the final 6 h of the incubation. Effects of a 2-h pretreatment with U0126 (10 µM) or a 24-h pretreatment with a disintegrin, echistatin (100 nM), on shear stress and/or IGF-I-induced cell proliferation were also tested.
Western Immunoblot Analyses and ImmunoprecipitationImmediately following the 30-min shear stress and 10-min IGF-I treatments, the treated cells and corresponding controls were washed with phosphate-buffered saline and lysed in radioimmune precipitation assay buffer as described previously (3). The protein concentration of each extract was assayed with the bicinchoninic acid method. Ten µg of extract protein from each extract was loaded onto 10% SDS-polyacrylamide gels and transblotted to polyvinylidene difluoride membrane for Western immunoblot analysis. Erk1/2 activation was assessed by pErk1/2 level using the anti-pErk1/2 antibody normalized against the total Erk1/2 level. The pIGF-IR level was determined with an antibody against pIGF-IR, normalized against the level of total IGF-IR.
The relative level of IGF-IR-bound SHP-1 and -2 was each measured by co-immunoprecipitation followed by Western immunoblot analyses. Briefly, 1 mg of cell extract protein each from treated cells and corresponding controls was incubated with 2 µg of anti-IGF-IR or anti-integrin
3 antibodies for 2 h at 4 °C. A predetermined amount of anti-rabbit IgG beads (eBiosciences, San Diego, CA) was added for an additional 1 h at 4 °C. The bead-bound complex was washed three times with ice-cold lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 10 mg/ml phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 1 mM sodium orthovanadate). The washed complex was then resuspended in 40 µl of 2x SDS sample buffer and boiled for 5 min. The relative amounts of co-immunoprecipitated SHP-1 or SHP-2 were analyzed by Western analysis using anti-SHP-1 or anti-SHP-2 antibodies, respectively.
IGF-IR Binding AssaysSpecific IGF-I binding to IGF-IR was measured by receptor-bound [125I]IGF-I in the presence of 100-fold "cold" IGF-I. Radio-iodination of IGF-I was performed by a modified chloramine T method (30). Aliquots were immediately stored at 70 °C until assay. Assays were performed within 1 week of iodination. For the IGF-IR binding assay, TE85 cells were plated on glass slides and subjected to fluid shear stress as described above. Immediately after the shear stress, the treated and corresponding static control cells were rinsed with Dulbecco's modified Eagle's medium containing 20 mM HEPES, pH 7.4, and 1 mg/ml bovine serum albumin (binding medium). Fresh binding medium was then added, and the cells were incubated with 5 x 104 to 2 x 105 counts/min of [125I]labeled IGF-I in the absence or presence of 25100 ng (i.e. 100-fold) of unlabeled IGF-I for total and specific binding, respectively. The cells were incubated at room temperature for 3 h, and the radioactive medium was removed and the slides rinsed five times with ice-cold binding medium. The cells were then lysed in the lysis buffer (10 mM Tris-HCl, pH 7.4, 5 mM EDTA, and 0.2% SDS). The amount of bound 125I-labeled IGF-I was then quantified by
counting.
Statistical AnalysesResults are shown as mean ± S.D. with at least six replicates. The statistical significance of the differences between independent groups was determined with the two-tailed Student's t test. The dose-dependent effects were assessed with one-way ANOVA, followed by Tukey post-hoc test. Interactions between two treatments (e.g. shear stress and IGF-I) were evaluated by two-way ANOVA. The difference was considered significant when p < 0.05.
| RESULTS |
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1.52.5-fold (Fig. 1A). The 30-min steady shear stress of 20 dynes/cm2 also significantly (p < 0.05) increased [3H]thymidine incorporation in TE85 cells by 70% compared with the corresponding static control cells. The combination of the 30-min shear stress and IGF-I treatment produced much greater than additive stimulations (3.55.5-fold) of each treatment alone (Fig. 1A). Two-way ANOVA indicates a highly significant (p < 0.01) interaction between the two treatments, suggesting a synergistic interaction between shear stress and IGF-I on bone cell proliferation.
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1.52.0-fold). The combined treatment of the shear stress and FGF-2 yielded no further enhancement (not significant, two-way ANOVA) than FGF-2 alone, indicating that the synergistic interaction between shear stress and IGF-I is not universal to all bone growth factors.
Effects of Fluid Shear Stress on the IGF-I-mediated Activation of the Erk1/2 Mitogenic Signaling Pathway in Human TE85 CellsBecause the mitogenic action of IGF-I is mediated through Erk1/2 activation and fluid shear stress also activates Erk1/2 in osteoblasts (3, 10), we investigated the effect of shear stress and/or IGF-I (or FGF-2) on Erk1/2 phosphorylation (an index of Erk1/2 activation). Fig. 2A confirms that IGF-I alone, at the test doses, significantly and dose-dependently (p < 0.01, one-way ANOVA) increased the pErk1/2 level (by
1.25-fold) in TE85 cells. The 30-min steady shear stress alone also significantly (p < 0.01) increased the pErk1/2 level (by
2.5-fold). The combination of shear stress and IGF-I treatment produced a synergistic (p < 0.01, two-way ANOVA) enhancement (up to 12-fold) in Erk1/2 phosphorylation. Fig. 2B indicates that the mitogenic doses of FGF-2 (0.1 and 1 ng/ml) alone also markedly and significantly increased the pErk1/2 levels in TE85 cells (p < 0.01, one-way ANOVA). In contrast to IGF-I, the combination treatment of shear stress and FGF-2 did not result in a further increase in the pErk1/2 level compared with the FGF-2 treatment alone (not significant, two-way ANOVA). These findings further support the conclusions that the synergistic interaction between shear stress and IGF-I on bone cell proliferation is mediated through synergistic enhancement of IGF-I-dependent activation of the Erk1/2 mitogenic signaling pathway and that the synergy between shear stress and IGF-I on human bone cell proliferation is not shared by FGF-2.
To further evaluate whether activation of the Erk1/2 mitogenic signaling pathway is essential for the synergy, we tested the effect of U0126 (a specific inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1) on the stimulation of cell proliferation and Erk1/2 phosphorylation induced by IGF-I with or without the shear stress. Fig. 3A shows that pretreatment with U0126 at 10 µM completely blocked the IGF-I-mediated as well as the shear stress-induced TE85 cell proliferation. It also completely abolished the synergistic enhancement of IGF-I and shear stress. Fig. 3B reveals that the U0126 pretreatment also completely eliminated the synergistic enhancement on Erk1/2 activation by shear stress and IGF-I. Thus, these results are consistent with the conclusion that the synergistic activation of Erk1/2 by IGF-I and shear stress is associated with the synergistic enhancement on osteoblast proliferation. These findings indicate that the synergy between shear stress and IGF-I leading to activation of bone cell proliferation occurs upstream to the Erk1/2 activation. Consistent with previous findings (3, 10), U0216 had no inhibitory effect on either basal proliferation or basal Erk1/2 activation, indicating that basal TE85 cell proliferation is mediated primarily through Erk1/2-independent pathways.
Effect of Shear Stress on the IGF-I-mediated Phosphorylation of IGF-IR in TE85 CellsWe next tested whether the synergy between IGF-I and shear stress occurs prior to or after the phosphorylation of IGF-IR receptor. As expected, IGF-I at the test mitogenic doses significantly increased the IGF-IR phosphorylation level in a dose-dependent manner by 23.5-fold (Fig. 4). The 30-min steady shear stress alone also significantly (p < 0.01) increased the IGF-IR phosphorylation by 2.5-fold. The combination treatment of shear stress and IGF-I yielded a highly significant synergistic (p < 0.01, two-way ANOVA) enhancement in IGF-IR phosphorylation level (up to 8-fold).
Effects of Fluid Shear Stress on the Specific Binding of IGF-I to IGF-IR in TE85 CellsBecause shear stress synergistically enhanced IGF-IR phosphorylation, which is initiated by the binding of IGF-I to IGF-IR, we next assessed whether the synergistic enhancement between IGF-I and shear stress was because of an increase in IGF-I binding to IGF-IR. Fig. 5 shows that the application of a 30-min fluid shear stress at 20 dyne/cm2 led to a relatively small, but statistically significant (p < 0.05, one-way ANOVA) enhancement in the specific binding of IGF-I to IGF-IR in TE85 cells. However, this increase appeared to be of additive nature, as the two binding curves (i.e. with or without shear stress) were parallel to each other.
Effects of Echistatin on the IGF-I- and/or Shear Stress-induced Proliferation of TE85 CellsBecause shear stress involves integrin activation in bone cells (3, 26, 27), we evaluated whether integrin activation is involved in the synergy between IGF-I and shear stress in TE85 cells by determining the effect of the disintegrin echistatin (a competitive integrin receptor antagonist) on IGF-I- and/or shear stress-mediated cell proliferation and IGF-IR phosphorylation. Fig. 6A shows that echistatin, not only reduced the basal (by
60%) and shear stress-induced TE85 cell proliferation (by
20%), but also completely abolished the increase in cell proliferation induced by IGF-I alone as well as that by the combination treatment. Similarly, echistatin also completely abolished the basal, shear-stress, or IGF-I-induced IGF-IR phosphorylation (Fig. 6B). These findings suggest that the synergy between IGF-I and shear-stress on the proliferation and that on IGF-IR phosphorylation level may involve integrin activation.
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3 or with IGF-IR, determined by co-immunoprecipitation (Fig. 7, IP) followed by immunoblotting (IB). This study focused on integrin
3, because this integrin subunit is one of the major subunits in osteoblasts (31) and also because integrin
v
3 has been implicated to be the essential integrin subunit in regulating the IGF-I-dependent cellular responses in smooth muscle cells (1622). The results of Fig. 7 suggest that fluid shear stress, IGF-I, and the combination treatment each significantly enhanced the recruitment of SHP-2 to integrin
3 and away from the IGF-IR. We also determined whether the synergistic interaction could also involve integrin-dependent recruitment of the related SHP-1 away from the IGF-IR. Fig. 8 shows that fluid shear stress, IGF-I, and the combination treatment each also significantly enhanced the recruitment of SHP-1 to integrin
3 and away from the IGF-IR. However, the effects of fluid shear stress on the recruitment of SHP-2 away from the IGF-IR appeared to be more pronounced than those on the SHP-1 recruitment. On the other hand, treatment of IGF-I alone, although it markedly reduced the amounts of IGF-IR-bound SHP-2, did not significantly affect the amounts of SHP-2 bound to integrin
3. | DISCUSSION |
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3 signaling. Consequently, it appears that mechanical loading not only plays a permissive role in the osteogenic actions of IGF-I, but also interacts synergistically with the IGF-I signaling pathway to promote bone formation.
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Bikle and co-workers (13) have also reported that mechanical unloading markedly diminished the ability of IGF-I to activate several members of its mitogenic signaling pathway (i.e. IGF-IR, Ras, Erk1/2) in osteoblasts. Accordingly, we found that shear stress potentiates the IGF-I-mediated Erk1/2 activation. To gain insights into the molecular mechanism whereby shear stress interacts with the IGF-I signaling pathway to promote osteoblast proliferation, we examined whether synergy between shear stress and IGF-I also occurred at Erk1/2 activation and IGF-IR phosphorylation, two important steps of the IGF-I mitogenic signaling pathway. We reasoned that if the point of interaction (i.e. cross-talk) occurs prior to a given step in a pathway, a synergy would be evident at and after that particular step of the pathway. Conversely, if the point of interaction happens after a given step, no synergy would be expected at or prior to that given step. Accordingly, our findings that shear stress also synergized with IGF-I on Erk1/2 activation and IGF-IR phosphorylation strongly suggest that the synergy between shear stress and IGF-I to promote bone cell proliferation occurs prior to or at the step of IGF-IR phosphorylation.
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The relatively low amounts of receptor-bound IGF-I (i.e. <2%) compared with the total amounts of added IGF-I, presumably because of the fact that bone cells (including TE85 cells) release a large amount of IGF binding proteins (41) that compete with IGF-IR for IGF-I binding, has precluded an accurate determination of receptor number and/or binding affinity by Scatchard analysis. Thus, it is not known whether the small increase in IGF-I binding in response to shear stress was because of an increase in the number of IGF-IR or to an increase in ligand binding affinity. However, the parallel binding curves in the absence or presence of shear stress suggest that the increase in IGF-I binding could be due to a small increase in IGF-IR number. The reason for shear stress to increase the IGF-IR number is not clear, but the main point of the concept of our work is that the synergy between shear stress and IGF-I in the stimulation of bone cell proliferation occurs after the ligand binding, but prior to or at the step of IGF-IR phosphorylation, and that activation of the integrin signaling is essential for the synergy.
Recent studies from Clemmons and co-workers (1622) in smooth muscle cells have disclosed important information about the nature of the cross-talk between the integrin signaling pathway and the IGF-I signaling pathway. Specifically, they found that activated integrin
3 serves to recruit SHP-2 from the cytosol and subsequently to transfer SHP-2 to SHPS-1 and IGF-IR for activating and terminating, respectively, the IGF-I signaling pathway. These findings have provided the basis that integrin activation is potentially relevant to the molecular mechanism whereby shear stress interacts with IGF-I to enhance the IGF-I signaling mechanism in bone cells. The IGF-I signaling pathway is initiated, not only by IGF-IR autophosphorylation induced by ligand binding, but also by the recruitment of activated SHP-2 to SHPS-1 from activated integrins. The transfer of activated SHP-2 to IGF-IR is responsible for the dephosphorylation of IGF-IR and the termination of the IGF-I signaling pathway (1622). Accordingly, we postulate that mechanical strains or shear stresses, which activate the integrin signaling pathways in bone cells, enhance SHP-2 recruitment to activated integrins and also to SHPS-1. At the same time, the integrin activation in response to shear stresses inhibits the transfer of activated SHP-2 to IGF-IR, resulting in a reduction in the dephosphorylation of IGF-IR and an overall increase in IGF-IR phosphorylation level. This model would explain the synergy between shear stress and IGF-I on the IGF-IR phosphorylation level, Erk1/2 activation, and bone cell proliferation.
Our findings that shear stress, IGF-I, and the combination treatment each increased the relative amount of SHP-2 that was associated with integrin
3 and that each also reduced the relative amount of SHP-2 co-immunoprecipitated with IGF-IR in TE85 cells are consistent with our hypothesis that the shear stress-mediated recruitment of SHP-2 to activated integrins and away from IGF-IR may be responsible for the synergy between shear stress and IGF-IR to promote bone cell proliferation. It should be noted that the IGF-I-mediated and shear stress-induced recruitment of SHP-2 to integrin
3 was relatively small (i.e. <2-fold). However, human osteoblasts, including TE85 cells, synthesize multiple members of the integrin family (42). There is evidence that mechanical loading also up-regulated and activated other members of integrins (such as integrin
1) in bone cells, including TE85 cells (26). Therefore, it is highly possible that shear stress and/or IGF-I also increased SHP-2 recruitment to other members of the integrin family, including
1, and that this may explain why the enhancement in SHP-2 recruitment to integrin
3 induced by IGF-I and/or shear stress was relatively low. More importantly, we found that shear stress, IGF-I, and the combination treatment each also increased the relative amount of the integrin
3-associated SHP-1 and reduced the relative amount of IGF-IR-associated SHP-1. This suggests that SHP-2 and the related SHP-1 are both involved in the IGF-I signaling mechanism as well as in the synergy between shear stress and IGF-I in enhancing the overall IGF-IR phosphorylation level.
The effect of unloading on recruitment of SHP-2 (or SHP-1) to integrins has not been assessed previously (13). Thus, it is unclear at this time whether or not the unloading-induced resistance to IGF-I may also involve a reduction of SHP-2 (and/or SHP-1) recruitment to integrins. However, because unloading down-regulated integrin expression in osteoblasts (13) and because SHP-2 recruitment to integrins is essential for IGF-I signaling (1622), it is likely that the reduced integrin recruitment of SHP-2 and/or SHP-1 in response to unloading-mediated down-regulation of the integrin pathway could also play a pivotal role in the permissive effect of mechanical loading on the IGF-I anabolic action in bone (12, 13).
In conclusion, this study provides the first evidence for a synergistic interaction between shear stress and IGF-I in the stimulation of osteoblastic proliferation. This study also provides strong circumstantial evidence that the synergy involves an integrin-dependent up-regulation of IGF-IR phosphorylation level through an inhibition of the recruitment of SHP-1 and/or SHP-2 to IGF-IR as well as an inhibition of the SHP-1 and/or SHP-2-mediated IGF-IR dephosphorylation. These findings not only confirm that the integrin activation is essential for the IGF-I mitogenic pathway, but also provide mechanistic insights into the cross-talk between the integrin and IGF-I signaling pathways in the underlying molecular mechanisms of enhanced bone formation in response to mechanical loading.
| FOOTNOTES |
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¶ To whom correspondence should be addressed: Musculoskeletal Disease Ctr. (151), Jerry L. Pettis Memorial Veterans Affairs Medical Ctr., 11201 Benton St., Loma Linda, CA 92357. Tel.: 909-825-7084 (ext. 2836); Fax: 909-796-1680; E-mail: William.Lau{at}med.va.gov.
1 The abbreviations used are: IGF-I, insulin-like growth factor-I; IGF-IR, insulin-like growth factor-I receptor; pIGF-IR, phosphorylated IGF-IR; FGF-2, basic fibroblast growth factor; Erk1/2, extracellular regulated kinase 1/2; pErk1/2, phosphorylated Erk1/2; SHP-1, Src-homology 2 domain-containing protein-tyrosine phosphatase 1; SHP-2, Src-homology 2 domain-containing protein-tyrosine phosphatase 2; SHPS-1, SHP substrate 1; ANOVA, analysis of variance. ![]()
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