Division of Endocrinology and Metabolism, Center for Osteoporosis and Metabolic Bone Diseases, and Central Arkansas Veterans Healthcare System, University of Arkansas for Medical Sciences, Little Rock, Arkansas
Submitted 11 June 2004 ; accepted in final form 30 April 2005
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
bone; mechanotransduction; osteoblastic cells; caveolae; stretching
Death of osteocytes is expected to compromise the mechanosensory function of the osteocyte network and to diminish the mechanical competence of the skeleton. In support of this notion, the increased bone fragility that results from glucocorticoid excess or sex steroid deficiency in animals and humans is associated with increased prevalence of osteocyte apoptosis (36, 61, 66). Conversely, bisphosphonates, intermittent parathyroid hormone (PTH) administration, and sex steroids all prevent osteocyte apoptosis, raising the possibility that preservation of osteocytes contributes to the antifracture efficacy of these agents (30, 36, 53). In support of this latter contention, blockade of glucocorticoid action on osteocytes in a transgenic mouse model preserves bone strength despite loss of bone mass, suggesting that osteocyte viability is indeed an independent determinant of bone strength (47).
The life span of osteocytes might be regulated by mechanical stimuli. Physiological levels of load imposed on bone in vivo appear to decrease the number of apoptotic osteocytes (46). On the other hand, a lack of mechanical stimulation induced by unloading of bone is associated with an increased number of hypoxic osteocytes, an effect that is reversed by loading, suggesting that mechanical forces facilitate oxygen diffusion and osteocyte survival (14).
How mechanical forces are transduced into biochemical signals in osteocytes is not understood. Osteocytes interact with the ECM in the pericellular space through discrete sites in their membranes, enriched in integrins and vinculin (2, 22), and through transverse elements that tether osteocytes to the canalicular wall (70). Therefore, fluid movement in the canaliculi resulting from mechanical loading might induce ECM deformation, shear stress, and/or tension in the tethering elements. The consequent changes in circumferential strain in the osteocyte membranes might be converted into intracellular signals by integrin clustering and their interaction with cytoskeletal and catalytic proteins at the focal adhesions (13, 20). However, the molecular machinery assembled in response to mechanical stimuli as well as the consequences of the generation of intracellular signaling for osteocyte life span have remained heretofore unknown. The findings of the present report are consistent with the transduction of mechanical forces by integrins and a signalsome comprising actin filaments, microtubules, the focal adhesion kinase FAK, and Src kinases, resulting in activation of the ERK pathway and attenuation of osteocyte apoptosis. This evidence provides the molecular basis for the profound role of mechanical forces, or lack thereof, in skeletal health and disease.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cells.
MLO-Y4 osteocytic cells derived from murine long bones and MLO-Y4 cells stably expressing GFP targeted to the nucleus (MLO-GFP) were cultured at 12 x 104 cells/cm2 in phenol red-free -MEM (GIBCO-BRL, Gaithersburg, MD) supplemented with 2.5% FBS and 2.5% bovine calf serum (BCS) (Hyclone, Logan, UT), 100 U/ml penicillin, and 100 µg/ml streptomycin on plates coated with calf skin collagen type I (Sigma) as previously described (31, 53).
Plasmids. The wild-type or dominant-negative (dn) constructs for HA-MEK, Src, GST-Shc, or HA-FAK were provided by N. G. Ahn (University of Colorado, Boulder, CO; Ref. 42), W. C. Horne (Yale University, New Haven, CT; Ref. 71), K. S. Ravichadran (University of Virginia, Charlottesville, VA; Ref. 63), and J.-L. Guan (Cornell University, Ithaca, NY; Ref. 73), respectively. Wild-type and cytoplasmic ERK2 fused to GFP (GFP-ERK2) were provided by R. Seger (Weizmann Institute of Sciences, Rehovot, Israel; Ref. 56). The constructs encoding nuclear GFP (nGFP) or nuclear red fluorescent protein (nRFP) were described previously (36, 53).
Mechanical stimulation of cell cultures. Cells were cultured for 24 h on flexible-bottom wells coated with collagen type I, poly-L-lysine, or poly-L-lysine plates further coated with activating anti-integrin antibodies by overnight incubation at 4°C with 10 µg/ml of each antibody in PBS. Subsequently, cultures were subjected to cycles of biaxial stretching with a FX-4000 Flexercell Strain Unit (Flexcell International) for 120 min. Each cycle consisted of 20 s of stretching at 2% or 5% cell elongation (corresponding to 0.02 and 0.05 strains, respectively), followed by 0.1 s of release.
Western blot analysis.
Cells were serum starved for 30 min and stretched for the last 120 min as indicated above. For experiments using inhibitors, cells were incubated for 20 min in medium without serum containing vehicle, 50 µM PD-98059, 0.4 µM cytochalasin D, 3 µM colchicine, 10 µM gadolinium chloride, 0.510 µM PP1, 15 mg/ml -cyclodextrin, 0.03 µM wortmannin, or 100 µM SB-203580, followed by stretching at 5% elongation for 10 min. Cells were lysed with (in mM) 20 Tris·HCl (pH 7.5), 150 NaCl, 1 EDTA, 10 NaF, 1 sodium orthovanadate, and 1 phenylmethylsulfonyl fluoride, with 5 mg/ml leupeptin, 0.14 U/ml aprotinin, and 1% Triton X-100. Proteins were separated on 7.5% SDS-polyacrylamide gels, electrotransferred to a polyvinylidene difluoride membrane, and detected by the appropriate antibodies. Blots were developed by enhanced chemiluminescence, and the intensity of the bands in the autoradiograms was quantified by scanning and densitometry. Both ERK bands (p42 and p44) were quantified together. Results are expressed as fold increase (mean ± SD) over the respective unstretched (basal) control group, which is designated as 1. For immunoprecipitation, cell lysates (700 µg of protein per condition) were precleared with 1 µg of normal rabbit IgG together with 20 µl of protein G agarose (Santa Cruz Biotechnology) and pelleted by centrifugation at 1,500 rpm at 4°C for 5 min. Supernatants were then incubated with anti-ERK or anti-caveolin-1 antibodies for 4 h, followed by precipitation of the complexes by addition of 20 µl of protein G agarose. Immunoprecipitates were dissolved with buffer for electrophoresis, separated on SDS-PAGE gels, and electrotransferred. Blots were probed with anti-caveolin-1,
1-integrin, or ERK antibodies.
Quantification of apoptosis.
MLO-GFP cells were pretreated with pharmacological inhibitors for 30 min and then stretched for 10 min at 2% or 5% elongation. Subsequently, 50 µM etoposide or 1 µM dexamethasone was added to the culture medium, and 6 h later cells were fixed and apoptosis was quantified by enumerating fluorescent cells exhibiting chromatin condensation and/or nuclear fragmentation as previously reported (53). More than 250 cells from fields selected by systematic random sampling (27) were examined for each experimental condition. The sample size required to detect differences with a 95% confidence level was calculated with a two-group continuity-corrected 2 test (15). Data are presented as percentage of etoposide- or dexamethasone-induced apoptosis in the absence of stretching. The percentage of apoptosis was calculated with the formula (%DCp+st %DCst)/(%DCp %DCv) x 100, where DC = dead cells, p = cultures treated with proapoptotic agent(s), st = stretched cultures, and v = vehicle-treated cultures, as previously published (6, 52). To appreciate the effect of FAK overexpression on unstretched cells, data in Fig. 5D are expressed as percentage of apoptotic cells instead of percentage of apoptosis induced by each proapoptotic agent. For the experiments using dn mutants, MLO-Y4 cells were transiently transfected with nGFP or nRFP along with the indicated constructs. Forty-eight hours later, cells were subjected to stretching and apoptosis was quantified as described above. Caspase 3 activity was determined by measuring the degradation of the fluorogenic substrate Ac-DEVD-AFC (Biomol) in lysates of cells stretched for 10 min at 5% elongation followed by addition of etoposide for 9 h, as previously reported (53). The units of caspase 3 activity per microgram of protein were calculated by using a standard curve prepared with recombinant caspase 3 assayed together with the samples.
|
Subcellular localization of ERK2. MLO-Y4 cells were transiently transfected with GFP-ERK2 to allow the visualization of ERK and cultured for 48 h. Cells were then incubated with medium without serum containing 2% bovine serum albumin for 30 min, subjected to cycles of 2% or 5% elongation for the last 120 min of the incubation period, and then immediately fixed. Alternatively, cells were stretched at 5% elongation for 10 min and fixed immediately or 30 min, 1 h, 3 h or 6 h after the termination of stretching. Nuclear accumulation of ERK2 was quantified in >250 cells/condition by enumerating the percentage of cells exhibiting increased GFP in the nucleus compared with the cytoplasm.
Image acquisition. Fluorescent images were collected on an inverted microscope (Axiovert 200, Carl Zeiss Light Microscopy, Gottingen, Germany) with a LD A-Plan, x32/0.40 lens and a low-light camera (Polaroid DMC Ie, Polaroid, Cambridge, MA), using a filter set for GFP. The acquisition software was Image-Pro Plus (Media Cybernetics, Silver Spring, MD).
Statistical analysis. Data were analyzed by one-way ANOVA, and the Student-Newman-Keuls method was used to estimate the level of significance of differences between means. The data of Fig. 2B were analyzed by Students t-test.
|
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Mechanically induced ERK activation requires intact actin and tubulin cytoskeletons and Src kinase activity and is abolished by disrupting caveolae with -cyclodextrin.
We next investigated how integrin engagement induced by stretching activates ERKs. Outside-in signaling mediated by integrins might involve cytoskeletal and catalytic proteins (4). Consistent with this evidence, we found that disruption of actin filaments with cytochalasin D or inhibition of microtubule formation with colchicine abolished stretch-induced ERK activation (Fig. 3A). As expected, the specific inhibitor of ERK activation PD-98059 also abolished the response. On the other hand, the inhibitor of stretch-activated channels gadolinium did not modify stretch-induced ERK activation. Inhibition of Src kinases with PP1 or disruption of caveolae with the cholesterol chelator
-cyclodextrin also abolished stretch-induced ERK phosphorylation (Fig. 3, B and C, respectively). PP1 or
-cyclodextrin also abolished ERK activation induced by stretching in cells plated on poly-L-lysine coated with anti-
5 and anti-
1 antibodies (Fig. 3D). Caveolin-1 is indeed expressed in MLO-Y4 cells and coimmunoprecipitated with both
1-integrin and ERKs from lysates treated with an anti-caveolin-1 antibody (Fig. 3E). Likewise, caveolin-1 and
1-integrin were both detected in immunoprecipitates obtained with an anti-ERK antibody. Together, these findings suggest that the transduction of mechanical signals into ERK activation in osteocytic cells involves integrins, cytoskeletal proteins, and Src kinases, perhaps assembled in caveolae. Consistent with our findings, mechanically induced ERK activation is blunted by caveolae-disrupting agents in osteoblastic cells (16).
|
|
Moreover, cells transfected with a dn Src mutant lacking the SH2 domain (SH2) also lost the responsiveness to stretching, whereas cells transfected with a dn Src mutant lacking the SH3 domain (
SH3) remained responsive (Fig. 5B). ERK activation initiated by integrins results from autophosphorylation of FAK in Tyr397 and the consequent recruitment of proteins containing SH2 domains, such as Src (18, 50). Therefore, we examined whether the survival effect induced by stretching requires FAK activation. Cells expressing wild-type FAK were protected from etoposide-induced apoptosis as effectively as cells transfected with vector. On the other hand, cells expressing the dn autophosphorylation-deficient mutant FAK Y397F were not protected (Fig. 5D). As was the case with all the constructs used in this study, transfection with the FAK constructs did not alter the effect of etoposide in unstretched cells. However, transfection of wild-type FAK or Y397F FAK abolished dexamethasone-induced apoptosis (Fig. 5D). For that reason, the requirement of FAK kinase activity for stretch-induced survival could be evaluated only by using etoposide as a proapoptotic stimulus. Together, the results of the experiments presented in Figs. 4 and 5 demonstrate that mechanical stimulation prevents apoptosis of osteocytic cells and that activation of FAK and the Src/Shc/ERK signaling pathway is required for this effect.
Antiapoptosis induced by mechanical signals requires nuclear accumulation of ERKs and new RNA and protein synthesis.
ERKs could promote survival by phosphorylating transcription factors that regulate the expression of apoptosis-related genes (7, 35). Because nuclear localization of ERKs is a prerequisite for this function, we sought to determine whether mechanical signals induce nuclear accumulation of ERKs, using MLO-Y4 cells transiently transfected with ERK2 fused to GFP. The percentage of cells plated on collagen I exhibiting ERK nuclear accumulation increased on stretching at 5% elongation for 10 min (Fig. 6A). As expected, blockade of ERK phosphorylation by the MEK inhibitor PD-98059 completely abolished stretching-induced ERK nuclear accumulation. Cultures stretched at 5% elongation for as little as 1 min exhibited a significant increase in the percentage of cells exhibiting ERK nuclear accumulation, and the magnitude of this effect increased with the duration of the mechanical stimulation, reaching a plateau at 5 min (Fig. 6B). The percentage of cells showing ERK nuclear accumulation induced by 20 min of mechanical stimulation was comparable to that induced by 5-min stimulation with estradiol, another inducer of ERK activation (35), in the same experiment (not shown). In contrast to the lack of an effect of 2% elongation on ERK phosphorylation shown in Fig. 1A, cultures subjected to 2% elongation also exhibited a significant increase in the percentage of cells with ERK nuclear accumulation. These results indicate that ERK nuclear accumulation is a more sensitive readout of ERK activity than ERK phosphorylation and are consistent with the demonstration that 2% elongation inhibited the proapoptotic action of etoposide or dexamethasone (Fig. 4E). In addition, and consistent with the results of ERK phosphorylation (Figs. 1 and 2), the percentage of cells exhibiting ERK nuclear accumulation under basal, that is, unstretched, conditions was higher in cells plated on poly-L-lysine than in cells plated on collagen I, and mechanical stimulation (either at 2% or 5% elongation) failed to induce further ERK nuclear accumulation (Fig. 6B). We next determined the time course of the reversal of nuclear ERK accumulation. In cultures stretched for 10 min at 5% elongation, the percentage of cells exhibiting ERK nuclear accumulation remained significantly elevated for up to 1 h after the termination of the mechanical stimulus, declining to levels similar to those observed in unstimulated cultures by 3 h (Fig. 6C). Importantly, cells expressing wild-type GFP-ERK2 were protected from etoposide- or dexamethasone-induced apoptosis by mechanical stimulation. On the other hand, cells expressing a GFP-ERK2 mutant that lacks the ability to translocate to the nucleus and is constitutively localized in the cytoplasm were not protected (Fig. 6D). These results indicate the requirement of nuclear localization of ERKs to exert their antiapoptotic effects on osteocytic cells in response to mechanical stimulation. The mechanism by which GFP-ERK2 abolishes the action of endogenous ERKs is unclear. However, earlier work demonstrated that homodimerization of ERKs on phosphorylation is required for their nuclear translocation (33). Therefore, it is likely that dimer formation of endogenous ERKs with cytoplasm-anchored GFP-ERK (largely in excess) inhibits the nuclear translocation of the endogenous kinases, thus abolishing their antiapoptotic effect. Nevertheless, consistent with the findings on cytoplasm-anchored GFP-ERK, the protein synthesis inhibitor cycloheximide and the RNA synthesis inhibitor actinomycin D abolished the antiapoptotic effect of mechanical stimulation at concentrations at which they effectively inhibited [3H]leucine and [3H]uridine incorporation, respectively, without affecting cell viability (Fig. 6E). Together, these results indicate that mechanical signals prevent osteocyte apoptosis via a mechanism that requires new gene transcription and strongly suggests the participation of ERK-activated transcription factors.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The findings reported here demonstrate that mechanical stimuli preserve osteocyte viability via activation of ERKs and new gene transcription. The transduction of mechanical signals into ERK activation requires integrin engagement, intact actin and microtubular cytoskeletons, FAK, Src kinase activity, and the adaptor protein Shc. This evidence is consistent with earlier reports demonstrating ERK activation by mechanical stimulation in osteoblastic cells (8, 16, 29, 67).
Convergence of systemic and mechanical survival signals on ERK activation. Similar to the findings reported here with mechanical stimulation, ERK activation is required for the antiapoptotic actions of sex steroids and bisphosphonates on osteocytes and osteoblasts (36, 53). Nonetheless, the molecular events that lead to ERK activation as well as the mechanisms by which ERKs promote survival in each case are distinct. Indeed, whereas sex steroids activate ERKs through the estrogen and androgen receptors (35, 36), bisphosphonates activate ERKs by opening connexin43 hemichannels (52). Furthermore, whereas bisphosphonates exert their antiapoptotic effect by cytoplasm-restricted ERK signals independent of transcription (51), survival induced by sex steroids (35) or by mechanical signals (this report) requires nuclear ERK accumulation and new gene transcription. Moreover, the antiapoptotic effect of sex steroids also requires activation of PI3K (35), but as shown here this kinase is not required for antiapoptosis by mechanical stimuli. Collectively, these findings support the notion that additive or synergistic effects on osteoblast and/or osteocyte survival could explain at least partially the advantages of therapeutic regimens in which sex steroids and bisphosphonates are combined with each other or with physical exercise (10, 23, 40).
The dependence of stretch-induced survival on the nuclear localization of ERKs and on new gene transcription strongly suggests the participation of nuclear ERK substrates. Consistent with this, activation of the transcription factors Runx2/Cbfa1, egr-1, and AP-1 by mechanical signals has been shown to depend on ERKs in osteoblastic cells (25, 26, 76). Furthermore, Runx2 is involved in the survival signals of PTH in osteoblasts, even though in this case PKA, but not ERKs, is required (6). The question of whether these or other transcription factors are indeed required for the antiapoptotic effect of mechanical stimulation will require further studies.
Requirement of integrins and Src for mechanically induced osteocyte survival.
Engagement of specific integrins with activating antibodies revealed that 1- (but not
2),
2-, and
5-integrins mediate stretch-induced ERK activation. This finding is consistent with the expression of high levels of
1-integrin, but very low levels of
2-integrin, in MLO-Y4 osteocytic cells (unpublished data) as well as with the expression of
1-integrin in osteocytes in vivo and its involvement in osteocyte adhesion to ECM proteins (2, 22, 28). Our results are also consistent with extensive evidence indicating that
1-integrin triggers antiapoptotic intracellular signaling. Thus ligation of
1-integrin abrogates anoikis of fibroblastic cells induced by collagen gel contraction (60). In addition, the ECM protein fibronectin, ligand of
5
1-integrin, induces survival of different cell types, including osteoblasts (5, 21, 72), and collagen I, which binds to
2
1-integrin, is also a survival factor (19, 60, 75).
The different basal levels of ERK activation observed in cells plated on poly-L-lysine compared with collagen I or with different integrin-activating antibodies is not unexpected, considering that engagement per se of some but not all integrins leads to modulation of the activity of ERKs themselves as well as other kinases that might influence ERK activation (24, 39, 54, 58). Nevertheless, independently of its basal levels, stretching induces an increase in ERK activation only when particular integrins are engaged. However, this effect is more modest than that observed in cells plated on collagen. This finding might reflect suboptimal conditions for the transduction of mechanical forces in cells in which only a set of receptors is engaged by the activating antibody(ies) and suggests that collaboration among different integrins is required to obtain the maximal cellular response to stretching.
Signaling through the integrin family of receptors requires their association with molecules capable of signal transduction because integrins lack intrinsic enzymatic activity. This association is facilitated by the close proximity of integrins to catalytic proteins localized in caveolae, specialized microdomains of the plasma membrane that are rich in the protein caveolin and in cholesterol. 1-Integrin, in particular, colocalizes and interacts with caveolin (64, 65), and caveolin, in turn, is required for the association of
1-integrin with Src kinases and the phosphorylation of the Src kinase substrate Shc (65). In the present study, we found that sequestering cholesterol with
-cyclodextrin abolishes stretch-induced ERK activation and antiapoptosis, suggesting that caveolae are involved in the response of osteocytes to mechanical stimulation. Although cyclodextrin might also interfere with noncaveolar related processes (55, 59), the fact that caveolin-1 is present in MLO-Y4 cells and coimmunoprecipitates with
1-integrin and ERKs suggests that a signalsome does indeed assemble in caveolin-rich membrane domains in these cells. This finding is consistent with previous evidence that caveolae are involved in mediating integrin-dependent ERK activation in endothelial cells (49) and that caveolin-3, the muscle-specific member of the caveolin family of proteins, is required for stretch-induced responses (32). However, further studies are required to confirm the requirement of caveolae in the survival effect of mechanical stimulation and to investigate the mechanism by which caveolin-1 controls mechanotransduction in osteocytes.
Both the kinase and the SH2 domain of Src are required for the antiapoptotic effect of stretching. This fact indicates that Src, besides playing a role in phosphorylating downstream substrates such as Shc, also plays a critical role in the assembly of the signalsome required for the transduction of mechanical signals via protein-protein interaction through its SH2 domain. Our findings show that one of these proteins, FAK, previously shown to become phosphorylated in Tyr397 on integrin engagement and to bind Src leading to ERK activation (38, 57), is required for antiapoptosis induced by stretching. Interestingly, even though phosphorylated FAK in Tyr397 also binds to the p85 subunit of the PI3K (11), only inhibition of ERKs, but not PI3K, abolishes the survival response induced by stretching.
Other proteins with affinity for the Src SH2 domain might also participate in the transduction of mechanical signals. Indeed, our recent findings (3) suggest that the estrogen receptor (ER) is also an essential component of the stretch-induced signaling cascade described here. Consistent with this, Src interacts through the SH2 domain with the ER (45). Furthermore, mice lacking ER exhibit a poor anabolic response to bone loading (37).
Regulation of osteocyte life span in vivo. The relevance of the present in vitro findings to the in vivo situation is a matter of conjecture at this time. Nevertheless, the strain applied to osteocytic cells in the current studies is of a magnitude similar to that to which osteocytes might be exposed to in vivo, according to the strain amplification model in which cell-level strains are at least one order of magnitude larger than bone tissue-level strains (69, 70). In addition, osteocytes have been shown to respond more readily than osteoblasts to mechanical stimulation (34), and osteoblastic cells in general appear to be more sensitive to fluid flow than to substrate stretching (44, 48), suggesting that the antiapoptotic responses reported here may be underestimated by the use of the latter approach. Furthermore, mounting evidence indicates that osteoblast and osteocyte survival is maintained not only by endocrine or autocrine/paracrine soluble factors but also by the ECM and that loss of antiapoptotic signals originated in the ECM causes osteoblastic cell apoptosis, a phenomenon referred to as "anoikis" (17). Thus neutralizing antibodies to the ECM protein fibronectin induce osteoblast apoptosis (21), and transgenic mice expressing collagenase-resistant collagen type 1 exhibit increased prevalence of osteoblast and osteocyte apoptosis compared with age-matched wild-type controls (75). Collectively, this evidence supports the notion that interactions between intact or cryptic sites of ECM proteins with cellular integrins result in "outside-in" signaling that preserves viability. Considering that osteocytes do not divide or differentiate in vivo, maintenance of osteocyte viability by promoting ECM-osteocyte interactions is likely to be one of the most relevant biological responses of these cells to mechanical forces.
In conclusion, in this report, we demonstrate that mechanical stimuli preserve osteocyte viability via activation of ERKs and new gene transcription. The evidence linking mechanical stimulation, activation of an integrin/cytoskeleton/Src/ERK signaling pathway, and osteocyte survival provides a mechanistic basis for the profound role of mechanical forces, or lack thereof, in skeletal health and disease. Furthermore, the fact that the survival responses triggered by strain activate distinct pools of the same intracellular kinases utilized by systemic stimuli provides evidence for the participation of osteocytes in the integrated response of the skeleton to both biochemical and physical stimuli.
![]() |
GRANTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
ACKNOWLEDGMENTS |
---|
Present address of I. Mathov: Cátedra de Inmunología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Instituto de Estudios de la Imunidad Humoral (Consejo Nacional de Investigaciones Científicas y Tecnicas, Universidad de Buenos Aires), Buenos Aires, Argentina.
![]() |
FOOTNOTES |
---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* L. I. Plotkin and I. Mathov contributed equally to this work.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Aarden EM, Nijweide PJ, Van Der Plas A, Alblas MJ, Mackie EJ, Horton MA, and Helfrich MH. Adhesive properties of isolated chick osteocytes in vitro. Bone 18: 305313, 1996.[CrossRef][ISI][Medline]
3. Aguirre JI, Plotkin LI, Strotman BA, McCauley LK, Gubrij I, Kousteni S, Manolagas SC, and Bellido T. The anti-apoptotic effects of mechanical stimulation in osteoblasts/osteocytes are transduced by the estrogen receptor (ER): a novel ligand-independent function of the ER (Abstract). J Bone Miner Res 18: S71, 2003.
4. Alenghat FJ and Ingber DE. Mechanotransduction: all signals point to cytoskeleton, matrix, and integrins. Sci STKE 2002: PE6, 2002.[Medline]
5. Almeida EAC, Ili D, Han Q, Hauck CR, Jin F, Kawakatsu H, Schlaepfer DD, and Damsky CH. Matrix survival signaling: from fibronectin via focal adhesion kinase to c-Jun NH2-terminal kinase. J Cell Biol 149: 741754, 2000.
6. Bellido T, Ali AA, Plotkin LI, Fu Q, Gubrij I, Roberson PK, Weinstein RS, O'Brien CA, Manolagas SC, and Jilka RL. Proteasomal degradation of Runx2 shortens parathyroid hormone-induced anti-apoptotic signaling in osteoblasts. A putative explanation for why intermittent administration is needed for bone anabolism. J Biol Chem 278: 5025950272, 2003.
7. Bonni A, Brunet A, West AE, Datta SR, Takasu MA, and Greenberg ME. Cell survival promoted by the Ras-MAPK signaling pathway by transcription-dependent and -independent mechanisms. Science 286: 13581362, 1999.
8. Boutahar N, Guignandon A, Vico L, and Lafage-Proust MH. Mechanical strain on osteoblasts activates autophosphorylation of FAK and PYK2 tyrosine sites involved in ERK activation. J Biol Chem 279: 3058830599, 2004.
9. Boyce BF, Xing L, Jilka RL, Bellido T, Weinstein RS, Parfitt AM, and Manolagas SC. Apoptosis in bone cells. In: Principles of Bone Biology, edited by Bilezikian JP, Raisz LG, and Rodan GA. San Diego: Academic, 2002, p. 151168.
10. Braith RW, Magyari PM, Fulton MN, Aranda J, Walker T, and Hill JA. Resistance exercise training and alendronate reverse glucocorticoid-induced osteoporosis in heart transplant recipients. J Heart Lung Transplant 22: 10821090, 2003.[CrossRef][ISI][Medline]
11. Chen HC, Appeddu PA, Isoda H, and Guan JL. Phosphorylation of tyrosine 397 in focal adhesion kinase is required for binding phosphatidylinositol 3-kinase. J Biol Chem 271: 2632926334, 1996.
12. Chen KD, Li YS, Kim M, Li S, Yuan S, Chien S, and Shyy JY. Mechanotransduction in response to shear stress. Roles of receptor tyrosine kinases, integrins, and Shc. J Biol Chem 274: 1839318400, 1999.
13. Clark EA and Brugge JS. Integrins and signal transduction pathways: the road taken. Science 268: 233239, 1995.[ISI][Medline]
14. Dodd JS, Raleigh JA, and Gross TS. Osteocyte hypoxia: a novel mechanotransduction pathway. Am J Physiol Cell Physiol 277: C598C602, 1999.
15. Elashoff JD. nQuery Advisor User's Guide (version 4.0). Los Angeles: Statistical Solutions, 2000.
16. Ferraro JT, Daneshmand M, Bizios R, and Rizzo V. Depletion of plasma membrane cholesterol dampens hydrostatic pressure and shear stress-induced mechanotransduction pathways in osteoblast cultures. Am J Physiol Cell Physiol 286: C831C839, 2004.
17. Frisch SM and Ruoslahti E. Integrins and anoikis. Curr Opin Cell Biol 9: 701706, 1997.[CrossRef][ISI][Medline]
18. Frisch SM, Vuori K, Ruoslahti E, and Chan-Hui PY. Control of adhesion-dependent cell survival by focal adhesion kinase. J Cell Biol 134: 793799, 1996.[Abstract]
19. Gendron S, Couture J, and Aoudjit F. Integrin 2
1 inhibits Fas-mediated apoptosis in T lymphocytes by protein phosphatase 2A-dependent activation of the MAPK/ERK pathway. J Biol Chem 278: 4863348643, 2003.
20. Giancotti FG and Ruoslahti E. Integrin signaling. Science 285: 10281032, 1999.
21. Globus RK, Doty SB, Lull JC, Holmuhamedov E, Humphries MJ, and Damsky CH. Fibronectin is a survival factor for differentiated osteoblasts. J Cell Sci 111: 13851393, 1998.
22. Gohel AR, Hand AR, and Gronowicz GA. Immunogold localization of 1-integrin in bone: effect of glucocorticoids and insulin-like growth factor I on integrins and osteocyte formation. J Histochem Cytochem 43: 10851096, 1995.
23. Going S, Lohman T, Houtkooper L, Metcalfe L, Flint-Wagner H, Blew R, Stanford V, Cussler E, Martin J, Teixeira P, Harris M, Milliken L, Figueroa-Galvez A, and Weber J. Effects of exercise on bone mineral density in calcium-replete postmenopausal women with and without hormone replacement therapy. Osteoporos Int 14: 637643, 2003.[CrossRef][ISI][Medline]
24. Goldschmidt ME, McLeod KJ, and Taylor WR. Integrin-mediated mechanotransduction in vascular smooth muscle cells: frequency and force response characteristics. Circ Res 88: 674680, 2001.
25. Granet C, Boutahar N, Vico L, Alexandre C, and Lafage-Proust MH. MAPK and SRC-kinases control EGR-1 and NF-B inductions by changes in mechanical environment in osteoblasts. Biochem Biophys Res Commun 284: 622631, 2001.[CrossRef][ISI][Medline]
26. Granet C, Vico AG, Alexandre C, and Lafage-Proust MH. MAP and src kinases control the induction of AP-1 members in response to changes in mechanical environment in osteoblastic cells. Cell Signal 14: 679688, 2002.[CrossRef][ISI][Medline]
27. Gundersen HJ, Bendtsen TF, Korbo L, Marcussen N, Moller A, Nielsen K, Nyengaard JR, Pakkenberg B, Sorensen FB, and Vesterby A. Some new, simple and efficient stereological methods and their use in pathological research and diagnosis. APMIS 96: 379394, 1988.[ISI][Medline]
28. Hughes DE, Salter DM, Dedhar S, and Simpson R. Integrin expression in human bone. J Bone Miner Res 8: 527533, 1993.[ISI][Medline]
29. Jessop HL, Rawlinson SC, Pitsillides AA, and Lanyon LE. Mechanical strain and fluid movement both activate extracellular regulated kinase (ERK) in osteoblast-like cells but via different signaling pathways. Bone 31: 186194, 2002.[CrossRef][ISI][Medline]
30. Jilka RL, Weinstein RS, Bellido T, Roberson P, Parfitt AM, and Manolagas SC. Increased bone formation by prevention of osteoblast apoptosis with parathyroid hormone. J Clin Invest 104: 439446, 1999.
31. Kato Y, Windle JJ, Koop BA, Mundy GR, and Bonewald LF. Establishment of an osteocyte-like cell line, MLO-Y4. J Bone Miner Res 12: 20142023, 1997.[ISI][Medline]
32. Kawamura S, Miyamoto S, and Brown JH. Initiation and transduction of stretch-induced RhoA and Rac1 activation through caveolae: cytoskeletal regulation of ERK translocation. J Biol Chem 278: 3111131117, 2003.
33. Khokhlatchev AV, Canagarajah B, Wilsbacher J, Robinson M, Atkinson M, Goldsmith E, and Cobb MH. Phosphorylation of the MAP kinase ERK2 promotes its homodimerization and nuclear translocation. Cell 93: 605615, 1998.[CrossRef][ISI][Medline]
34. Klein-Nulend J, Van Der Plas A, Semeins CM, Ajubi NE, Frangos JA, Nijweide PJ, and Burger EH. Sensitivity of osteocytes to biomechanical stress in vitro. FASEB J 9: 441445, 1995.
35. Kousteni S, Han L, Chen JR, Almeida M, Plotkin LI, Bellido T, and Manolagas SC. Kinase-mediated regulation of common transcription factors accounts for the bone-protective effects of sex steroids. J Clin Invest 111: 16511664, 2003.
36. Kousteni S, Bellido T, Plotkin LI, O'Brien CA, Bodenner DL, Han L, Han K, DiGregorio GB, Katzenellenbogen JA, Katzenellenbogen BS, Roberson PK, Weinstein RS, Jilka RL, and Manolagas SC. Nongenotropic, sex-nonspecific signaling through the estrogen or androgen receptors: dissociation from transcriptional activity. Cell 104: 719730, 2001.[ISI][Medline]
37. Lee K, Jessop H, Suswillo R, Zaman G, and Lanyon L. Endocrinology: bone adaptation requires oestrogen receptor-. Nature 424: 389, 2003.
38. Li S, Kim M, Hu YL, Jalali S, Schlaepfer DD, Hunter T, Chien S, and Shyy JY. Fluid shear stress activation of focal adhesion kinase. Linking to mitogen-activated protein kinases. J Biol Chem 272: 3045530462, 1997.
39. Liang F, Atakilit A, and Gardner DG. Integrin dependence of brain natriuretic peptide gene promoter activation by mechanical strain. J Biol Chem 275: 2035520360, 2000.
40. Lindsay R, Cosman F, Lobo RA, Walsh BW, Harris ST, Reagan JE, Liss CL, Melton ME, and Byrnes CA. Addition of alendronate to ongoing hormone replacement therapy in the treatment of osteoporosis: a randomized, controlled clinical trial. J Clin Endocrinol Metab 84: 30763081, 1999.
41. Manolagas SC. Birth and death of bone cells: basic regulatory mechanisms and implications for the pathogenesis and treatment of osteoporosis. Endocr Rev 21: 115137, 2000.
42. Mansour SJ, Matten WT, Hermann AS, Candia JM, Rong S, Fukasawa K, Vande Woude GF, and Ahn NG. Transformation of mammalian cells by constitutively active MAP kinase kinase. Science 265: 966970, 1994.[ISI][Medline]
43. Marotti G, Cane V, Palazzini S, and Palumbo C. Structure-function relationships in the osteocyte. Ital J Min Electrol Metab 4: 93106, 1990.
44. McGarry JG, Klein-Nulend J, Mullender MG, and Prendergast PJ. A comparison of strain and fluid shear stress in stimulating bone cell responsesa computational and experimental study. FASEB J 19: 482484, 2005.
45. Migliaccio A, Castoria G, Di Domenico M, de Falco A, Bilancio A, Lombardi M, Barone MV, Ametrano D, Zannini MS, Abbondanza C, and Auricchio F. Steroid-induced androgen receptor-oestradiol receptor -Src complex triggers prostate cancer cell proliferation. EMBO J 19: 54065417, 2000.
46. Noble BS, Peet N, Stevens HY, Brabbs A, Mosley JR, Reilly GC, Reeve J, Skerry TM, and Lanyon LE. Mechanical loading: biphasic osteocyte survival and targeting of osteoclasts for bone destruction in rat cortical bone. Am J Physiol Cell Physiol 284: C934C943, 2003.
47. O'Brien CA, Jia D, Plotkin LI, Bellido T, Powers CC, Stewart SA, Manolagas SC, and Weinstein RS. Glucocorticoids act directly on osteoblasts and osteocytes to induce their apoptosis and reduce bone formation and strength. Endocrinology 145: 18351841, 2004.
48. Owan I, Burr DB, Turner CH, Qiu J, Tu Y, Onyia JE, and Duncan RL. Mechanotransduction in bone: osteoblasts are more responsive to fluid forces than mechanical strain. Am J Physiol Cell Physiol 273: C810C815, 1997.
49. Park H, Go YM, Darji R, Choi JW, Lisanti MP, Maland MC, and Jo H. Caveolin-1 regulates shear stress-dependent activation of extracellular signal-regulated kinase. Am J Physiol Heart Circ Physiol 278: H1285H1293, 2000.
50. Parsons JT. Focal adhesion kinase: the first ten years. J Cell Sci 116: 14091416, 2003.
51. Plotkin LI, Aguirre JI, Kousteni S, Manolagas SC, and Bellido T. Bisphosphonates and estrogens inhibit osteocyte apoptosis via distinct molecular mechanisms downstream of ERK activation. J Biol Chem 280: 73177325, 2005.
52. Plotkin LI, Manolagas SC, and Bellido T. Transduction of cell survival signals by connexin-43 hemichannels. J Biol Chem 277: 86488657, 2002.
53. Plotkin LI, Weinstein RS, Parfitt AM, Roberson PK, Manolagas SC, and Bellido T. Prevention of osteocyte and osteoblast apoptosis by bisphosphonates and calcitonin. J Clin Invest 104: 13631374, 1999.
54. Reyes-Reyes M, Mora N, Gonzalez G, and Rosales C. 1 and
2 integrins activate different signalling pathways in monocytes. Biochem J 363: 273280, 2002.[CrossRef][ISI][Medline]
55. Rodal SK, Skretting G, Garred O, Vilhardt F, van Deurs B, and Sandvig K. Extraction of cholesterol with methyl--cyclodextrin perturbs formation of clathrin-coated endocytic vesicles. Mol Biol Cell 10: 961974, 1999.
56. Rubinfeld H, Hanoch T, and Seger R. Identification of a cytoplasmic-retention sequence in ERK2. J Biol Chem 274: 3034930352, 1999.
57. Sanders MA and Basson MD. Collagen IV-dependent ERK activation in human Caco-2 intestinal epithelial cells requires focal adhesion kinase. J Biol Chem 275: 3804038047, 2000.
58. Schwartz MA and Assoian RK. Integrins and cell proliferation: regulation of cyclin-dependent kinases via cytoplasmic signaling pathways. J Cell Sci 114: 25532560, 2001.[ISI][Medline]
59. Subtil A, Gaidarov I, Kobylarz K, Lampson MA, Keen JH, and McGraw TE. Acute cholesterol depletion inhibits clathrin-coated pit budding. Proc Natl Acad Sci USA 96: 67756780, 1999.
60. Tian B, Lessan K, Kahm J, Kleidon J, and Henke C. 1 Integrin regulates fibroblast viability during collagen matrix contraction through a phosphatidylinositol 3-kinase/Akt/protein kinase B signaling pathway. J Biol Chem 277: 2466724675, 2002.
61. Tomkinson A, Reeve J, Shaw RW, and Noble BS. The death of osteocytes via apoptosis accompanies estrogen withdrawal in human bone. J Clin Endocrinol Metab 82: 31283135, 1997.
62. Verborgt O, Gibson G, and Schaffler MB. Loss of osteocyte integrity in association with microdamage and bone remodeling after fatigue in vivo. J Bone Miner Res 15: 6067, 2000.[ISI][Medline]
63. Walk SF, March ME, and Ravichandran KS. Roles of Lck, Syk and ZAP-70 tyrosine kinases in TCR-mediated phosphorylation of the adapter protein Shc. Eur J Immunol 28: 22652275, 1998.[CrossRef][ISI][Medline]
64. Wary KK, Mainiero F, Isakoff SJ, Marcantonio EE, and Giancotti FG. The adaptor protein Shc couples a class of integrins to the control of cell cycle progression. Cell 87: 733743, 1996.[CrossRef][ISI][Medline]
65. Wei Y, Yang X, Liu Q, Wilkins JA, and Chapman HA. A role for caveolin and the urokinase receptor in integrin-mediated adhesion and signaling. J Cell Biol 144: 12851294, 1999.
66. Weinstein RS, Jilka RL, Parfitt AM, and Manolagas SC. 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: 274282, 1998.
67. Weyts FA, Li YS, van Leeuwen J, Weinans H, and Chien S. ERK activation and v
3 integrin signaling through Shc recruitment in response to mechanical stimulation in human osteoblasts. J Cell Biochem 87: 8592, 2002.[ISI][Medline]
68. Winkler DG, Sutherland MK, Geoghegan JC, Yu C, Hayes T, Skonier JE, Shpektor D, Jonas M, Kovacevich BR, Staehling-Hampton K, Appleby M, Brunkow ME, and Latham JA. Osteocyte control of bone formation via sclerostin, a novel BMP antagonist. EMBO J 22: 62676276, 2003.
69. You LD, Cowin SC, Schaffler MB, and Weinbaum S. A model for strain amplification in the actin cytoskeleton of osteocytes due to fluid drag on pericellular matrix. J Biomech 34: 13751386, 2001.[CrossRef][ISI][Medline]
70. You LD, Weinbaum S, Cowin SC, and Schaffler MB. Ultrastructure of the osteocyte process and its pericellular matrix. Anat Rec 278A: 505513, 2004.
71. Zhang Z, Baron R, and Horne WC. Integrin engagement, the actin cytoskeleton, and c-Src are required for the calcitonin-induced tyrosine phosphorylation of paxillin and HEF1, but not for calcitonin-induced Erk1/2 phosphorylation. J Biol Chem 275: 3721937223, 2000.
72. Zhang Z, Vuori K, Reed JC, and Ruoslahti E. The 5
1 integrin supports survival of cells on fibronectin and up-regulates Bcl-2 expression. Proc Natl Acad Sci USA 92: 61616165, 1995.
73. Zhao JH, Reiske H, and Guan JL. Regulation of the cell cycle by focal adhesion kinase. J Cell Biol 143: 19972008, 1998.
74. Zhao S, Zhang YK, Harris S, Ahuja SS, and Bonewald LF. MLO-Y4 osteocyte-like cells support osteoclast formation and activation. J Bone Miner Res 17: 20682079, 2002.[ISI][Medline]
75. Zhao W, Byrne MH, Wang Y, and Krane SM. Osteocyte and osteoblast apoptosis and excessive bone deposition accompany failure of collagenase cleavage of collagen. J Clin Invest 106: 941949, 2000.
76. Ziros PG, Gil AP, Georgakopoulos T, Habeos I, Kletsas D, Basdra EK, and Papavassiliou AG. The bone-specific transcriptional regulator Cbfa1 is a target of mechanical signals in osteoblastic cells. J Biol Chem 277: 2393423941, 2002.