SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

It is unclear how mechanical stress influences bone cells. Mechanical stress causes fluid shear stress (FSS) in the bone. Osteoblast lineage cells are thought to sense FSS and regulate bone remodeling. We therefore investigated the effects of FSS on human osteoblast-like osteosarcoma cells: SaOS-2 cells in vitro. The conditioned medium of the SaOS-2 cells after 24 h of FSS (24 h-FSS CM) showed such osteoclastic phenotype inductions as significantly increasing the number of tartrate-resistant acid phosphatase (TRAP) positive multinuclear cells in rat bone marrow cells and TRAP-positive cells in human preosteoclastic cells: FLG 29.1 cells. An enzyme-linked immunosorbent assay showed interleukin-11 (IL-11) protein to increase 7-fold in the 24 h-FSS CM. A Northern analysis showed that IL-11 mRNA increased 4-fold in the SaOS-2 cells after 6 h-FSS; however, no IL-6 mRNA expression was detected. Furthermore, the anti-human IL-11 antibody significantly neutralized the osteoclastic phenotype induction of the 24 h-FSS CM. The IL-11 mRNA up-regulation in SaOS-2 cells by the 6 h-FSS was not inhibited by the anti-human transforming growth factor-β1 antibody, but it was significantly inhibited by indomethacin. An enzymeimmunoassay showed prostaglandin E2 to increase 7-fold in the 1 h-FSS CM. These findings thus suggest that FSS induces osteoblasts to produce IL-11 (mediated by prostaglandins) and thus stimulates bone remodeling.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Bone is a tissue that responds to a mechanical load by changing its internal architecture.(1) The volume of bone and ultimately its strengths are determined by the balance between two opposing processes: osteoblastic bone formation and osteoclastic bone resorption (i.e., coupling).(2) The balance is shifted toward bone formation by increasing the mechanical stress and toward bone resorption either by disuse or by such chronic disease states as osteoporosis. Osteoblasts play an important role in modulating the differentiation of osteoclast progenitors into osteoclasts in two different ways: the production of soluble factors and cell-to-cell recognition between the osteoclast progenitors and osteoblasts.(2) Macrophage colony stimulating factor is supposed to be an important soluble factor.(3) Interleukin-6 (IL-6) is known to have a crucial effect on bone metabolism.(4) IL-11 also appears to be an important regulatory molecule in bone metabolism because it decreases the osteoblastic activity and stimulates osteoclast development.(5–7)

Recently, fluid shear stress (FSS) has been reported to play a significant role in the sensing of mechanical stress by osteocytic cells.(8) In fact the fluid flow in bone can occur as a result of imposed mechanical loads.(9) It is possible that the response to mechanical loading in vivo is caused by fluid flowing through the canalicular channels. Such FSS can act on the membranes of osteocytic cells (extending in the canaliculi) and may induce stress-related bone formation and resorption. However, there have so far been few reports on the influence of the FSS over the production of growth factors and cytokines by osteoblast-like cells.(8,10,11) We therefore applied FSS on human osteoblast-like osteosarcoma cells: SaOS-2 cells in vitro. We previously reported that FSS up-regulates the transforming growth factor (TGF)-β1 expression in the SaOS-2 cells.(12) Furthermore, we obtained the evidence that the conditioned medium of the SaOS-2 cells after 24 h of FSS (24 h-FSS CM) significantly increased the number of tartrate-resistant acid phosphatase (TRAP)-positive cells rather than the 24 h control CM (no FSS for 24 h), using rat bone marrow cells and human preosteoclastic cells (FLG 29.1 cells). By investigating the soluble factors capable of inducing an osteoclastic phenotype, we showed that FSS stimulates the production of IL-11 in SaOS-2 cells. In addition, anti-human IL-11 antibody significantly neutralized the osteoclastic phenotype induction of the 24 h-FSS CM. Because Elias et al. reported that both TGF-β1 and prostaglandin E2 (PGE2) stimulate the IL-11 expression in the SaOS-2 cells(13) and FSS is known to up-regulate both local factors in osteoblast-like cells,(11–12,14–16) we attempted to elucidate whether the IL-11 up-regulation in SaOS-2 cells by FSS is mediated by TGF-β1 or prostaglandins. We also verified the time course of PGE2 production that is transiently up-regulated by FSS.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

SaOS-2 cell cultures

The human osteosarcoma cell line, SaOS-2, was obtained from the Riken Cell Bank (Ibaraki, Japan). SaOS-2 cells have been shown to be osteoblastic based on the criteria of alkaline phosphatase activity, parathyroid hormone (PTH)-linked adenylate cyclase, osteonectin production, specific receptors for 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), and osteogenesis in diffusion chambers.(17) The cells were plated in 100-mm diameter culture dishes (Becton Dickinson Labware, Lincoln Park, NJ, U.S.A.) and then were maintained in Dulbecco's modified Eagle's medium (DMEM)/F12 (GIBCO BRL, Gaithersburg, MD, U.S.A.) containing 10% fetal calf serum (FCS) (GIBCO BRL), pH 7.4, and gentamicin (50 μg/ml) (Wako, Osaka, Japan). The cultures were maintained at 37°C in a humidified incubator with 5% CO2.

Shear experiments

The plated SaOS-2 cells were exposed to continuous laminar FSS in a cone viscometer in a serum-free medium as previously reported (Fig. 1).(18) The centrifugal forces cause a centripetal flow in the culture medium next to the stationary culture plate to which the cells are attached. The cone was positioned so that the central point was elevated from the culture plate by ∼1 mm, and the culture plate was thus prevented from mechanical friction. The speed of rotation of the cone was monitored continuously. FSS: θ, can be calculated at different values of the radius, r, as shown by Eqs. 1 and 2 below:

  • equation image(1)
  • equation image(2)

where R is the Reynolds number, μ is the viscosity, ν is the kinematic viscosity, ω is the rotational velocity, and α is the cone angle.(19) The viscosity of the medium, measured by using a viscometer, was 0.71 centipoise at 37°C. The 100 mm diameter cone with 0.5° was rotated at 200 rpm. In this case, the Reynolds number ranged from zero at the center of the dish to 0.468 at the edge of the dish. We drew a graph of the FSS: τ determined by R in Eq. 1 using a Macintosh computer (Apple Computer, Inc., Cupertino, CA, U.S.A.) and thus verified the range of the FSS ranged from 1.704 Pascal (Pa) (= N/m2) at the center of the dish to 2.045 Pa at the edge of the dish in our system. This level is considered to be physiological according to Weinbaum et al.(20) The cultured SaOS-2 cells were harvested after 1, 3, 6, 12, and 24 h of exposure to FSS, respectively. The control cells were harvested without any exposure to FSS. Cell culture supernatants after 1, 3, 6, 12, and 24 h of exposure to FSS were centrifuged to remove the cells. Most of each medium was snap-frozen and kept at −20°C until the bioassays and an ELISA were performed. The rest of each medium was immediately used for an enzymeimmunoassay (EIA). After each cells were trypsinized and harvested, the number of them was determined, respectively, using a Coulter counter. To investigate the mRNA expression, a similar experiment was performed and the cells were immediately lysed for RNA extraction.

thumbnail image

Figure FIG. 1. Schematic drawing of the cone-plate viscometer apparatus employing standard plastic tissue culture plates. A cone with a 0.5 angle is positioned at its tip 1 mm above the center of the tissue culture plate while rotating the cone constantly, thereby imparting fluid shear stress on the confluent monolayer of osteoblastic cells grown on the plate's surface. The fluid shear stress magnitude is then computed as described in the Materials and Methods.

Download figure to PowerPoint

Rat bone marrow cell cultures

Bone marrow cultures were performed according to the method of Hata et al.(21) Briefly, the Sprague-Dawley rats (5-week-old) were killed by ether anesthesia, and bone marrow cells were flushed out from femur and tibia after cutting off both ends. The cells were seeded into wells of 24-well culture plates (Becton Dickinson Labware) (106 cells/well) and cultured in alpha modified essential medium (α-MEM) (GIBCO BRL) (1 ml) containing 10% FCS (GIBCO BRL) in the presence of 10−8 M 1α,25(OH)2D3 (BIOMOL, Plymouth Meeting, PA, U.S.A.). The medium was changed with fresh medium (1 ml) on day 2. When the stromal cells were subconfluent on day 4, the medium was changed with fresh medium (1 ml) and the SaOS-2 CM either with or without 24 h FSS or vehicle (fresh DMEM/F12 with 10% of FCS) (200 μl) was added to each well. The medium was changed in the same way on day 6. On day 8, after fixation with acetone-citrate-formaldehyde, the cells were stained for TRAP activity using an acid phosphatase kit (Sigma, St. Louis, MO, U.S.A.). The TRAP-positive multinuclear cells were counted. All experiments were carried out five times.

FLG 29.1 cell cultures

The human preosteoclastic cell-line, FLG 29.1, was established from a 38-year-old female who suffered from acute monoblastic leukemia.(22) These cells are capable of differentiating toward the osteoclastic phenotype in the presence of 10−7 M 12-O-Tetradecanoylphorbol-13-Acetate and in a coculture with SaOS-2 cells.(22,23) FLG 29.1 cells were plated in culture flasks (Becton Dickinson Labware) and then cultured in an RPMI-1640 (GIBCO BRL), supplemented with 10% FCS (GIBCO BRL) and 100 μg/ml of gentamicin (Wako). The cultures were maintained at 37°C in a humidified incubator with 5% CO2.

Direct cell-to-cell contact between the osteoclast progenitors and osteoblasts is necessary for the differentiation of osteoclast progenitors.(2) Orlandini et al. established the coculture system of SaOS-2 cells and FLG 29.1 cells.(23) Briefly, 2 × 105 SaOS-2 cells were cultured in DMEM/F12 in 24-well dishes (Becton Dickinson Labware) for 24 h. After the medium was aspirated, FLG 29.1 cells (1.5 × 105 cells) were then added to SaOS-2 cells in a mixture of DMEM/F12 and RPMI-1640 medium (1:1) supplemented with 10% FCS (1 ml). Furthermore, SaOS-2 CM either with or without 24 h FSS or vehicle (fresh DMEM/F12 with 10% of FCS) (200 μl) was added to each well. After the 48 h coculture, TRAP staining was performed on FLG 29.1 and SaOS-2 cells. Namely, after fixation with acetone-citrate-formaldehyde, the cells were stained for TRAP activity by using an acid phosphatase kit (Sigma). The TRAP-positive FLG 29.1 cells were counted. All experiments were carried out five times.

ELISA analysis

An ELISA analysis was performed by using a human IL-11 ELISA kit (R&D Systems, Minneapolis, MN, U.S.A.). All experiments were carried out five times.

Northern blot analysis

RNA was extracted from the cells using a solution containing 4 M guanidine isothiocyanate (Sigma), 25 mM sodium citrate (Wako), 0.2% (w/v) n-lauroylsarcosine (Wako), and 0.7% (v/v) β-mercaptoethanol (Sigma). Ten micrograms of total RNA from each sample was separated on formaldehyde agarose gel and then transferred to a nylon membrane (Schleicher & Schuell, Dassel, Germany) by capillary blotting overnight. The cDNAs for human IL-11 was kindly donated by Dr. Paul Schendel (Genetics Institute, Cambridge, MA, U.S.A.) and the cDNAs for human IL-6 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was obtained from the American Type Culture Collection (ATCC) (Rockville, MD, U.S.A.). These cDNA fragments were32P-labeled by random priming (TaKaRa Shuzo Co., Ltd., Shiga, Japan). The blots were hybridized at 65°C for 2 h using the Quick hybrisolution (Stratagene Cloning Systems, La Jolla, CA, U.S.A.) and then washed at 55°C in 0.5× SSC (75 mM sodium chloride, 7.5 mM sodium citrate, pH 7) and 0.1% SDS (sodium dodecyl sulfate) for 30 minutes. The membranes were exposed to an imaging plate (Fuji Film Co., Tokyo, Japan) at room temperature overnight. To control any variability in the loading of RNA, ethidium bromide staining of the gel for ribosomal RNA was routinely assessed and confirmed by rehybridization and autoradiography with a32P-labeled oligonucleotide for the human GAPDH probe. All experiments were carried out five times.

Densitometer analysis

The autoradiographic results generated from Northern blots were quantitated using a Bas 1000 system (Fuji Film, Tokyo, Japan) to determine the relative levels of each mRNA signal. To calibrate the slight variability of the amount of mRNA, each mRNA signal of IL-11 was calibrated with that of GAPDH.

Human IL-11 blocking assay

A bioassay similar to the one described above was performed to elucidate whether the induction of the osteoclastic phenotype is mediated by IL-11.

Sprague-Dawley rats (5-week-old) were killed by ether anesthesia, and bone marrow cells were flushed out from femur and tibia after cutting off both ends. The cells were seeded into wells of 24-well culture plates (Becton Dickinson Labware) (106 cells/well) and cultured in α-MEM (GIBCO BRL) (1 ml) containing 10% FCS (GIBCO BRL) in the presence of 10−8 M 1α,25(OH)2D3 (BIOMOL). The medium was changed with fresh medium (1 ml) on day 2. When the stromal cells were subconfluent on day 4, the medium was changed with fresh medium (1 ml) and the SaOS-2 CM either with or without 24 h FSS or vehicle (fresh DMEM/F12 with 10% of FCS) (200 μl) was added to each well. Therefore, the SaOS-2 CM was diluted to six times. Anti-human IL-11 antibody (10 μg/ml) (R&D Systems) was then further added to one series. The Neutralization Dose50 of this anti-human IL-11 antibody is ∼10–20 μg/ml in the presence of 1.0 ng/ml of recombinant human IL-11 according to R&D Systems. The medium was changed in the same way on day 6. On day 8, TRAP staining was performed. The TRAP-positive multinuclear cells were counted. All experiments were carried out five times.

The 2 × 105 SaOS-2 cells were cultured in DMEM/F12 in 24-well dishes (Becton Dickinson Labware) for 24 h. After the medium was aspirated, FLG 29.1 cells (1.5 × 105 cells) were then added to SaOS-2 cells in a mixture of DMEM/F12 and RPMI-1640 medium (1:1) supplemented with 10% FCS (1 ml). Furthermore, the SaOS-2 CM with or without 24 h FSS or vehicle (fresh DMEM/F12 with 10% of FCS) (200 μl) was added to each well. Anti-human IL-11 antibody (10 μg/ml) (R&D Systems) was then further added to one series. After the 48 h coculture, TRAP staining was performed. The TRAP-positive FLG 29.1 cells were counted. All experiments were carried out five times.

Human TGF-β1 blocking assay

We previously elucidated that the FSS up-regulates SaOS-2 TGF-β1 protein production.(12) TGF-β1 is, however, detectable with ELISA only after the activation with acid. The FSS therefore seems to up-regulate almost latent TGF-β1. The secreted latent TGF-β1 does not seem to be activated by acid or protease in our system, therefore it is not likely to mediate the SaOS-2 IL-11 production in an autocrine manner. To rule out the possibility that some of the latent TGF-β1 are activated by unknown reasons in our system, we investigated whether the IL-11 up-regulation in SaOS-2 cells by FSS is mediated by the endogenous activated TGF-β1. About 300 pg/ml of latent TGF-β1 exists in the 24 h-FSS CM.(12) One microgram per milliliter of anti-human TGF-β1 neutralizing antibody (R&D Systems) is sufficient for neutralizing the 300 pg/ml of activated TGF-β1 according to the R&D Systems. Plated SaOS-2 cells were exposed to continuous laminar FSS in a cone viscometer in a serum-free DMEM/F12 either with vehicle (PBS) or anti-human TGF-β1 antibody (1 μg/ml) (R&D Systems). The cultured SaOS-2 cells were harvested after 1, 3, 6, 12, and 24 h of exposure to FSS, respectively. The control cells were harvested without any exposure to FSS. The cells were immediately lysed for RNA extraction. A Northern analysis was performed as described above. All experiments were carried out five times.

Prostaglandins blocking assay

We investigated whether the IL-11 up-regulation in SaOS-2 cells by FSS is mediated by prostaglandins. The plated SaOS-2 cells were exposed to continuous laminar FSS in a cone viscometer in a serum-free DMEM/F12 either with vehicle (PBS) or indomethacin (20 μM) (Sigma). The cultured SaOS-2 cells were harvested after 1, 3, 6, 12, and 24 h exposure to FSS, respectively. The control cells were harvested without any exposure to FSS. The cells were immediately lysed for RNA extraction. A Northern analysis was performed as described above. All experiments were carried out five times.

PGE2 EIA

An EIA for PGE2 was performed by using a PGE2 EIA kit (R&D Systems). All experiments were carried out five times.

Statistics

The results are presented as the means ± SEM of either the number of TRAP-positive multinuclear cells/well (Fig. 2), the number of TRAP-positive FLG 29.1 cells/well (Fig. 3), the IL-11 protein concentration (Fig. 4), or the IL-11 mRNA fold induction of control (Figs. 5B and 6B). Each experiment was repeated five times, and comparisons were made using Student's t-test after verifying the equal variance using the F-test. The values were considered to be significant if p < 0.05.

thumbnail image

Figure FIG. 2. The histogram of the number of TRAP-positive multinuclear cells from rat bone marrow cell cultures under different circumstances. (A) Cultured with the conditioned medium of the SaOS-2 cells without fluid shear stress for 24 h (24 h control CM). (B) Cultured with the conditioned medium of the SaOS-2 cells after 24 h of fluid shear stress (24 h-FSS CM). (C) Cultured with the 24 h control CM with 5 μg/ml of anti-human IL-11 antibody. (D) Cultured with the 24 h-FSS CM with 5 μg/ml of anti-human IL-11 antibody. Compared with the 24 h control CM, the 24 h-FSS CM further showed an increase in the number of TRAP-positive multinuclear cells of up to about 2-fold (335 ± 13 cells/well for the 24 h control CM vs. 573 ± 22 cells/well for the 24 h-FSS CM, p = 0.0008). This effect was then blocked by 5 μg/ml of anti-human IL-11 antibody (573 ± 22 cells/well for the 24 h-FSS CM vs. 359 ± 12 cells/well for the 24 h-FSS CM with anti-human IL-11 antibody, p = 0.001). The data shown are from five separate experiments, and the bars indicate SEM.

Download figure to PowerPoint

thumbnail image

Figure FIG. 3. The histogram of the number of TRAP-positive FLG 29.1 cells under different circumstances. (A) Cultured with the conditioned medium of the SaOS-2 cells without fluid shear stress for 24 h (24 h control CM). (B) Cultured with the conditioned medium of the SaOS-2 cells after 24 h of fluid shear stress (24 h-FSS CM). (C) Cultured with the 24 h control CM with 5 μg/ml of anti-human IL-11 antibody. (D) Cultured with the 24 h-FSS CM with 5 μg/ml of anti-human IL-11 antibody. Compared with the 24 h control CM, the 24 h-FSS CM further showed an increase in the number of TRAP-positive FLG 29.1 cells of up to about 2-fold (108 ± 6 cells/well for the 24 h control CM vs. 198 ± 10 cells/well for the 24 h-FSS CM, p = 0.0003). This effect was then blocked by 5 μg/ml of anti-human IL-11 antibody (**p < 0.001). The data shown are from five separate experiments, and the bars indicate SEM.

Download figure to PowerPoint

thumbnail image

Figure FIG. 4. Release of IL-11 protein in the SaOS-2 culture medium after 24 h of fluid shear stress (FSS). Samples of the medium with cultures of the SaOS-2 cells exposed to FSS and control samples (no FSS) were collected at 24 h. IL-11 protein, as determined by an ELISA, was prominent after 24 h of FSS (353.7 ± 10.0 pg/million cells). The control SaOS-2 cells released low levels of IL-11 over time (35.5 ± 7.3 pg/million cells). The data shown are from five separate experiments, and the bars indicate SEM (*p < 0.0001).

Download figure to PowerPoint

thumbnail image

Figure FIG. 5. (A) A Northern blot analysis of the IL-11 mRNA expression in the time course during exposure to fluid shear stress (FSS). The total RNA of SaOS-2 cells exposed to FSS was isolated at 1, 3, 6, 12, and 24 h. Control samples (no FSS) were harvested at 1, 3, 6, 12, and 24 h. The total RNA (10 μg/lane) was probed sequentially with the cDNAs for either human IL-11 or GAPDH. Note the significant increase in IL-11 mRNA at 3 h and 6 h in response to FSS and the absence of any such change in the GAPDH signal with FSS exposure. (B) A densitometric analysis of the Northern blot analysis findings hybridized with the IL-11 cDNA probe, normalized with the corresponding GAPDH signal. FSS significantly increased the IL-11 mRNA expression about 3-fold after 3 h (*p < 0.05) and further about 4-fold after 6 h (**p < 0.0001), and the levels of the IL-11 mRNA expression thereafter returned to control levels after 12 h of continuous exposure. The data shown are from five separate experiments, and the bars indicate SEM.

Download figure to PowerPoint

thumbnail image

Figure FIG. 6. (A) Northern blot analyzes of the IL-11 mRNA expression in the time course during exposure to fluid shear stress (FSS) with different circumstances. The total RNA of SaOS-2 cells exposed to FSS either with vehicle (PBS), anti-human TGF-β1 antibody (1 μg/ml), or indomethacin (20 μM) was isolated at 1, 3, 6, 12, and 24 h. Control samples (no FSS) were harvested at 1, 3, 6, 12, and 24 h. The total RNA (10 μg/lane) was probed sequentially with the cDNAs for either human IL-11 or GAPDH. Note the significant increase in IL-11 mRNA at 6 h in response to FSS both with vehicle (PBS), and anti-human TGF-β1 antibody (the upper and middle figures) and the absence of any such change with indomethacin (the lower figure). (B) A densitometric analysis of the Northern blot analysis findings hybridized with the IL-11 cDNA probe, normalized with the corresponding GAPDH signal. The FSS even with anti-human TGF-β1 antibody significantly increased the IL-11 mRNA expression about 4-fold after 6 h (*p < 0.0001); however, indomethacin significantly inhibited the up-regulation of the IL-11 mRNA (**p = 0.0023). The data shown are from five separate experiments, and the bars indicate SEM.

Download figure to PowerPoint

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

The effects of the 24 h-FSS CM on rat bone marrow cells

After the rat bone marrow cells were cultured in the presence of 10−8 M 1α,25(OH)2D3 for 8 days, osteoclast-like multinuclear cells with high TRAP activity were seen in all wells (Fig. 7). The effect of the 24 h-FSS CM on osteoclast-like cell formation was determined by counting the number of the TRAP-positive multinuclear cells formed in the bone marrow cultures treated with the 24 h-FSS CM. There was no significant difference in the formation of the osteoclast-like cells between the vehicle (fresh DMEM/F12 with 10% of FCS) and the 24 h control CM. Compared with the 24 h control CM, the 24 h-FSS CM significantly increased the number of TRAP-positive multinuclear cells by about two times (335 ± 13 cells/well for the 24 h control CM vs. 573 ± 22 cells/well for the 24 h-FSS CM) (Fig. 2).

thumbnail image

Figure FIG. 7. Microphotographs of representative TRAP-positive multinuclear cells from rat bone marrow cell cultures (original magnification ×50). TRAP staining is represented by the violet color.

Download figure to PowerPoint

The effects of the 24 h-FSS CM on the FLG 29.1 cells

After the FLG 29.1 cells were cocultured with SaOS-2 cells and the CM for 48 h, strong TRAP-positive reactions in the FLG 29.1 cells were seen in all wells (Fig. 8). In addition, multinuclear TRAP-positive FLG 29.1 cells were also observed (Fig. 8). Compared with the 24 h control CM (no FSS for 24 h), the 24 h-FSS CM significantly increased the number of TRAP-positive FLG 29.1 cells (108 ± 6 cells/well for the 24 h control CM vs. 198 ± 10 cells/well for the 24 h-FSS CM) (Fig. 3).

thumbnail image

Figure FIG. 8. Microphotographs of representative TRAP-positive FLG 29.1 cells (original magnification ×70). TRAP staining appears in red.

Download figure to PowerPoint

The FSS up-regulates IL-11 protein and mRNA in SaOS-2 cells

The application of the FSS resulted in an increase of IL-11 protein in the SaOS-2 CM. As assessed by ELISA, the 24 h control CM showed 35.5 ± 7.3 pg/million cells of IL-11, whereas the 24 h-FSS CM showed a 10-fold increase of IL-11 protein (353.7 ± 10.0 pg/million cells) (Fig. 4).

As assessed by a Northern blot analysis, confluent SaOS-2 cells expressed modest levels of IL-11 mRNA constitutively (Fig. 5A). FSS further increased the IL-11 mRNA expression about 4-fold after 6 h, and the levels of the IL-11 mRNA expression thereafter returned to control levels after 12 h of continuous exposure (Figs. 5A and 5B). No IL-6 mRNA expression was detected even after the SaOS-2 cells were exposed to FSS (data not shown).

The inhibition of the formation of TRAP-positive multinuclear cells by the anti-human IL-11 antibody

In view of the above evidence, we examined the ability of the anti-human IL-11 antibody to abolish the stimulatory effect of the 24 h-FSS CM on the formation of TRAP-positive multinuclear cells. No significant change in the number of TRAP-positive multinuclear cells was seen between the control CM (without FSS or anti-human IL-11 antibody) and the CM (without FSS) with anti-human IL-11 antibody (Fig. 2). The stimulatory effect of the 24 h-FSS CM was also significantly neutralized by anti-human IL-11 antibody (573 ± 22 cells/well for the 24 h-FSS CM vs. 359 ± 12 cells/well for the 24 h-FSS CM with anti-human IL-11 antibody) (Fig. 2).

The inhibition of the formation of TRAP-positive FLG 29.1 cells by the anti-human IL-11 antibody

We also examined the ability of the anti-human IL-11 antibody to inhibit the formation of the TRAP-positive FLG 29.1 cells. No significant change in the number of the TRAP-positive FLG 29.1 cells was seen in the SaOS-2 CM that had anti-human IL-11 antibody added to it (without FSS) in comparison with the control SaOS-2 CM (without FSS or anti-human IL-11 antibody) (Fig. 3). The stimulatory effect of the 24 h-FSS CM was also significantly neutralized by anti-human IL-11 antibody (198 ± 10 cells/well for the 24 h-FSS CM vs. 114 ± 9 cells/well for the 24 h-FSS CM with anti-human IL-11 antibody) (Fig. 3).

The inhibition of the FSS-induced IL-11 up-regulation by the anti-human TGF-β1 or indomethacin

To examine whether the FSS-induced IL-11 up-regulation in the SaOS-2 cells is mediated by TGF-β1 or prostaglandins, we added either anti-human TGF-β1 antibody or indomethacin to the serum-free DMEM/F12 before the plated SaOS-2 cells were exposed to continuous laminar FSS. As assessed by a Northern blot analysis, neither the anti-human TGF-β1 antibody nor indomethacin changed the basal levels of IL-11 mRNA (Fig. 6A). The anti-human TGF-β1 antibody did not change the FSS-induced IL-11 up-regulation in SaOS-2 cells (Figs. 6A and 6B). Indomethacin, however, significantly suppressed the FSS-induced IL-11 up-regulation in SaOS-2 cells (Figs. 6A and 6B).

PGE2 EIA

We also investigated the time course of PGE2 production. The application of the FSS resulted in a transient increase of PGE2 in the SaOS-2 CM. As assessed by EIA, control CMs showed 0.048 ± 0.005 pg/million cells of PGE2, whereas the 3 h-FSS CM showed a maximum increase of PGE2 (7.226 ± 0.166 pg/million cells) (Fig. 9). The timing of PGE2 production precedes that of IL-11 production. This evidence directly indicates that prostaglandins are involved in this process.

thumbnail image

Figure FIG. 9. The time course of PGE2 in the SaOS-2 culture medium after fluid shear stress (FSS). Samples of the medium with cultures of the SaOS-2 cells exposed to FSS and control samples (no FSS) were collected at 0, 1, 3, 6, 12, and 24 h. PGE2, as determined by an ELISA, was prominent after 3 h of FSS (7.226 ± 0.166 pg/million cells). The control SaOS-2 cells released low levels of PGE2 over time (0.048 ± 0.005 pg/million cells). The data shown are from five separate experiments, and the bars indicate SEM (*p < 0.0001).

Download figure to PowerPoint

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

This study was designed to further our understanding of the mechanisms by which osteoblasts sense mechanical stress, induce the differentiation of preosteoclastic cells, and promote bone remodeling. Osteoblast lineage cells (especially osteocytes) are exposed to FSS when the bone receives a mechanical load.(9) Weinbaum et al. calculated the level of the FSS at the cellular level in bone.(20) They assumed that the canalicular spaces around the osteocytic processes would be filled with glycosaminoglycans and thus predicted FSS in the canaliculi between 0.8 Pa and 3.0 Pa. Their results show that the physiological levels of FSS could easily be relatively large in these small channels. Judging from their data, the 1.7–2.0 Pa of FSS used in our study is considered to be within the normal physiological levels. There is also considerable evidence suggesting that osteoblasts respond to this range of FSS. The number of intracellular Ca2+ ions increases in the cultured monolayers of neonatal rat calvarial osteoblasts in the range of a 0.6–6.0 Pa FSS after 35 s.(24) Neonatal rat calvarial osteoblasts exposed to FSS in the range of 0.01–3.5 Pa for 0.5–15 minutes also exhibited increased levels of intracellular cAMP.(25) The exposure of these cells to 2.4 Pa for up to 2 h resulted in elevated levels of PGE2 and inositol triphosphate.(16)

These days, we know that a number of factors produced by osteoblasts modulate bone remodeling, including the differentiation of osteoclast precursors.(2) Many of these agents are growth factors and cytokines. However, there have so far been few reports on the influence of FSS over the osteoblast production of these agents.

At first we investigated whether the 24 h-FSS CM has a greater capacity to induce the formation of osteoclast-like TRAP-positive multinuclear cells than the 24 h control CM in rat bone marrow cell cultures. The TRAP-positive multinuclear cells in the rat bone marrow cell cultures are thought to be osteoclasts.(21) The 24 h-FSS CM had a greater capacity to induce the formation of TRAP-positive multinuclear cells than the 24 h control CM.

We further elucidated this effect on the human preosteoclastic cells: FLG 29.1 cells. Since osteoclast formation requires cell-to-cell recognition between the osteoclast progenitors and osteoblasts, the direct coculture of SaOS-2 cells and FLG 29.1 cells is necessary to clarify the effect of the soluble factors by which osteoblasts influence the differentiation of preosteoclast. Specifically, the interaction of osteoclast progenitors with osteoblasts is required for differentiation into cells expressing TRAP, a marker enzyme for identifying cells in osteoclast lineage.(2) Under these circumstances, the effect of soluble factors that are up-regulated by FSS can thus be quantitated. We therefore used the coculture system of SaOS-2 cells and FLG 29.1 cells established by Orlandini et al.(23) This system has also been used to quantitate the osteoclastic phenotype-inducing effect elsewhere.(26–28) The 24 h-FSS CM had more capacity to induce the expression of TRAP in FLG 29.1 cells than the 24 h control CM.

We next investigated the soluble factors that induce the osteoclastic phenotype. The significant increase of IL-11 protein was seen in the 24 h-FSS CM. Our study is the first evidence that the FSS directly increases the IL-11 mRNA expression about 4-fold after 6 h and then increases the level of protein production about 7-fold after 24 h in the SaOS-2 cells. IL-11 and IL-6 are osteotropic cytokines.(4–6) Hughes et al. showed that IL-11 inhibits bone nodule formation in a more potent manner than IL-6 in vitro, thus suggesting that IL-11 may be an important inhibitor of bone formation.(5) Girasole et al. reported IL-11 to indeed be a potent inducer of osteoclastogenesis and bone resorption at concentrations as low as 50 pM(6) They also reported that a monoclonal anti-human IL-11 antibody inhibited PTH-, 1α,25(OH)2D3-, IL-1–, or TNFα-mediated osteoclast formation by 50–100%. Recently, Hill et al. reported that IL-11 mainly shows the osteotropic effect by increasing the number of mature osteoclasts rather than promoting the ability of bone resorption by each osteoclast.(7) On the contrary, Bellido et al. showed that human IL-11 and other IL-6–type cytokines are capable of stimulating osteoblastogenesis and osteoblast differentiation.(29,30) Therefore, IL-11 (up-regulated by FSS) may impact both osteoclastogenesis and osteoblastogenesis.

Many investigators have also shown that other types of cells respond to FSS, thus resulting in an increase in growth factors and cytokines. Human endothelial cells respond to FSS and also increase the expression of platelet-derived growth factors, basic fibroblast growth factor, and TGF-β mRNA.(19,31) Mohtai et al. showed that human articular chondrocytes increase IL-6 production in response to FSS.(18) However, the articular chondrocytes did not show an up-regulation of TGF-β1 expression.(18) We therefore hypothesized that FSS may also stimulate osteoblast IL-6 production because both osteoblasts and chondrocytes originate from mesenchymal stem cells. However, FSS did not up-regulate the IL-6 mRNA in SaOS-2 cells. It is interesting to note that different cell types show different responses to FSS.

After obtaining evidence that the FSS increased SaOS-2 IL-11 production, we next tested the effect of anti-human IL-11 antibody on the osteoclastic phenotype induction of the 24 h-FSS CM. Anti-human IL-11 antibody significantly inhibited the formation of both TRAP-positive multinuclear cells in the rat bone marrow cell cultures and TRAP-positive FLG 29.1 cells further stimulated by the 24 h-FSS CM. This result suggests that IL-11 may thus be one of the main factors linking FSS to the differentiation of osteoclast progenitors.

TGF-β is the most abundant growth factor in the bone matrix. It can be produced by osteoblasts and is released from the matrix during tissue remodeling.(32) It is a potent stimulator of matrix production and has also been demonstrated to inhibit bone resorption and osteoclast-like cell formation in bone marrow cultures.(32) However, TGF-β is a potent stimulator of osteoblast IL-11 production.(13,33) Elias et al. showed that SaOS-2 cells produced IL-11 constitutively, and this production was further augmented by such cytokines as IL-1 and TGF-β1, such hormones as PTH and PTH-related peptide (PTHrP), and agents that activate protein kinase C and increase the levels of intracellular cAMP.(13) Morinaga et al. showed that TGF-β promotes the osteoblast production of IL-11 and increases bone resorption.(33) We therefore thought that it was important to clarify whether osteoblast IL-11 production may be mediated by TGF-β1 or not, because FSS increases the TGF-β1 mRNA expression and TGF-β1 protein production in osteoblast-like cells.(11–12,15) We previously elucidated that the FSS up-regulates SaOS-2 TGF-β1 protein production.(12) TGF-β1 is, however, detectable with ELISA only after the activation with acid. The FSS therefore seems to up-regulate almost latent TGF-β1. The secreted latent TGF-β1 does not seem to be activated by acid or protease in our system; therefore, it is not likely to mediate the SaOS-2 IL-11 production in an autocrine manner. To rule out the possibility that some of the latent TGF-β1 are activated by unknown reasons in our system, we investigated whether the IL-11 up-regulation in SaOS-2 cells by FSS is mediated by the endogenous activated TGF-β1. The anti-human TGF-β1 neutralizing antibody, however, did not inhibit the FSS-induced IL-11 up-regulation in the SaOS-2 cells. The FSS-induced up-regulation of IL-11 does not seem to be mediated by endogenous activated TGF-β1 in our system.

Many investigators have reported FSS to increase prostaglandins within 1 h.(1,8,11,14,16) We thus investigated whether the osteoblast IL-11 production is mediated by prostaglandins. Indomethacin significantly suppressed the FSS-induced IL-11 up-regulation in the SaOS-2 cells. The FSS-induced up-regulation of IL-11 seems to be mediated by prostaglandins. Rawlinson et al. reported the early responses of osteoblast lineage cells to mechanical strain (resulting in PGE2 production) could be mainly mediated by calcium ion channels.(34) Reich et al. reported that G proteins and calcium influx play an important role in the flow-induced PGE2 production in osteoblasts.(35) We also previously reported that cation (including calcium) channel could play an important role in the FSS-induced TGF-β1 up-regulation in the SaOS-2 cells.(12) We speculated on the cation channel involvement at the early stage in the FSS-induced IL-11 up-regulation. However, neither gadolinium (channel blocker against stretch activated cation nonselective channel) nor verapamil (voltage-dependent calcium channel blocker) inhibited the FSS-induced IL-11 up-regulation in our preliminary study, showing its prostaglandin-mediated pathway independent of cation channel. The mechanism of the FSS-induced IL-11 up-regulation is thus unclear and should be elucidated further.

This study is thus considered to provide intriguing evidence of a novel mechano-transduction pathway linking FSS, IL-11 gene transcription, and protein secretion by osteoblast-like cells and the induction of the osteoclastic phenotype. IL-11 may not be the sole cytokine or growth factor altered by physical force.(36) Therefore, the roles of other cytokines and growth factors in the process of bone remodeling caused by mechanical stress should be further defined. Further experiments are also required in normal human osteoblasts. Strictly speaking, this FSS model is presumed to reflect the FSS experienced by osteocyte in vivo. Therefore, some study of the effect of FSS on osteocytes, for example cultured chick osteocytes, is required.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

We thank Drs. H. Higaki and T. Murakami for assistance in this study, Yamanouchi Co. Ltd., Tokyo, Japan for kindly providing human IL-11 probe, and Dr. Brian Quinn for critically reading the manuscript. This work was supported in part by a Grant-in-Aid in Scientific Research (C: 8671665) from the Ministry of Education, Science, Sports and Culture of Japan.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
  • 1
    Duncan RL, Turner CH 1995 Mechanotransduction and the functional response of bone to mechanical strain Calcif Tissue Int 57:344358.
  • 2
    Suda T, Udagawa N, Nakamura I, Miyaura C, Takahashi N 1995 Modulation of osteoclast differentiation by local factors Bone 17:87S91S.
  • 3
    Felix R, Cecchini MG, Fleisch H 1990 Macrophage colony stimulating factor restores in vivo bone resorption in the op/op osteopetrotic mouse Endocrinology 127:25922594.
  • 4
    Jilka RL, Hangoc G, Girasole G, Passeri G, Williams DC, Abrams JS, Boyce B, Broxmeyer H, Manolagas SC 1992 Increased osteoclast development after estrogen loss: Mediation by interleukin-6 Science 257:8891.
  • 5
    Hughes FJ, Howells GL 1993 Interleukin-11 inhibits bone formation in vitro Calcif Tissue Int 53:362364.
  • 6
    Girasole G, Passeri G, Jilka RL, Manolagas SC 1994 Interleukin-11: A new cytokine critical for osteoclast development J Clin Invest 93:15161524.
  • 7
    Hill PA, Tumber A, Papaioannou S, Meikle MC 1998 The cellular actions of interleukin-11 on bone resorption in vitro Endocrinology 139:15641572.
  • 8
    Klein-Nulend J, van der Plas A, Semeins CM, Ajubi NE, Frangos JA, Nijweide PJ, Burger EH 1995 Sensitivity of osteocytes to biomechanical stress in vitro FASEB J 9:441445.
  • 9
    Dillaman RM, Roer RD, Gay DM 1991 Fluid movement in bone: Theoretical and empilical J Biomech 24:163177.
  • 10
    Klein-Nulend J, Semeins CM, Ajubi NE, Nijweide PJ, Burger EH 1995 Pulsating fluid flow increases nitric oxide (NO) synthesis by osteocytes but not periosteal fibroblasts— Correlation with prostaglandin upregulation Biochem Biophys Res Commun 217:640648.
  • 11
    Klein-Nulend J, Semeins CM, Burger EH 1996 Prostaglandin mediated modulation of transforming growth factor-beta metabolism in primary mouse osteoblastic cells in vitro J Cell Physiol 168:17.
  • 12
    Sakai K, Mohtai M, Iwamoto Y 1998 Fluid shear stress increases transforming growth factor beta 1 expression in human osteoblast-like cells: Modulation by cation channel blockades Calcif Tissue Int 63:515520.
  • 13
    Elias JA, Tang W, Horowitz MC 1995 Cytokine and hormonal stimulation of human osteosarcoma interleukin-11 production Endocrinology 136:489498.
  • 14
    Ajubi NE, Klein-Nulend J, Nijweide PJ, Vrijheid-Lammers T, Alblas MJ, Burger EH 1996 Pulsating fluid flow increases prostaglandin production by cultured chicken osteocytes—A cytoskeleton-dependent process Biochem Biophys Res Commun 225:6268.
  • 15
    Klein-Nulend J, Roelofsen J, Sterck JG, Semeins CM, Burger EH 1995 Mechanical loading stimulates the release of transforming growth factor-beta activity by cultured mouse calvariae and periosteal cells J Cell Physiol 163:115119.
  • 16
    Reich KM, Frangos JA 1991 Effect of flow on prostaglandin E2 and inositol trisphosphate levels in osteoblasts Am J Physiol 261:C428C432.
  • 17
    Rodan SB, Imai Y, Thiede MA, Wesolowski G, Thompson D, Bar-Shavit Z, Shull S, Mann K, Rodan GA 1987 Characterization of a human osteosarcoma cell line (Saos-2) with osteoblastic properties Cancer Res 47:49614966.
  • 18
    Mohtai M, Gupta MK, Donlon B, Ellison B, Cooke J, Gibbons G, Schurman DJ, Lane Smith R 1996 Expression of interleukin-6 in osteoarthritic chondrocytes and effects of fluid-induced shear on this expression in normal human chondrocytes in vitro J Orthop Res 14:6773.
  • 19
    Malek AM, Gibbons GH, Dzau VJ, Izumo S 1993 Fluid shear stress differentially modulates expression of genes encoding basic fibroblast growth factor and platelet-derived growth factor B chain in vascular endothelium J Clin Invest 92:20132021.
  • 20
    Weinbaum S, Cowin SC, Zeng Y 1994 A model for the excitation of osteocytes by mechanical loading-induced bone fluid shear stresses J Biomech 27:339360.
  • 21
    Hata K, Kukita T, Akamine A, Kukita A, Kurisu K 1992 Trypsinized osteoclast-like multinucleated cells formed in rat bone marrow cultures efficiently form resorption lacunae on dentine Bone 13:139146.
  • 22
    Gattei V, Bernabei PA, Pinto A, Bezzini R, Ringressi A, Formigli L, Tanini A, Attadia V, Brandi ML 1992 Phorbol ester induced osteoclast-like differentiation of a novel human leukemic cell line (FLG 29.1) J Cell Biol 116:437447.
  • 23
    Orlandini SZ, Formigli L, Benvenuti S, Lasagni L, Franchi A, Masi L, Bernabei PA, Santini V, Brandi ML 1995 Functional and structural interactions between osteoblastic and preosteoclastic cells in vitro Cell Tissue Res 281:3342.
  • 24
    Williams JL, Iannotti JP, Ham A, Bleuit J, Chen JH 1994 Effects of fluid shear stress on bone cells Biorheology 31:163170.
  • 25
    Reich KM, Gay CV, Frangos JA 1990 Fluid shear stress as a mediator of osteoblast cyclic adenosine monophosphate production J Cell Physiol 143:100104.
  • 26
    Benvenuti S, Petilli M, Frediani U, Tanini A, Fiorelli G, Bianchi S, Bernabei PA, Albanese C, Brandi ML 1994 Binding and bioeffects of Ipriflavone on a human preosteoclastic cell line Biochem Biophys Res Commun 201:10841089.
  • 27
    Bonucci E, Silvestrini G, Ballanti P, Masi L, Franchi A, Bufalino L, Brandi ML 1992 Cytological and ultrastructural investigation on osteoblastic and preosteoclastic cells grown in vitro in the presence of ipriflavone: Preliminary results Bone Miner 19:S15S25.
  • 28
    Gattei V, Aldinucci D, Quinn JM, Degan M, Cozzi M, Perin V, Iuliis AD, Juzbasic S, Improta S, Athanasou NA, Ashman LK, Pinto A 1996 Human osteoclasts and preosteoclast cells (FLG 29.1) express functional c-kit receptors and interact with osteoblast and stromal cells via membrane-bound stem cell factor Cell Growth Differ 7:753763.
  • 29
    Bellido T, Borba VZ, Roberson P, Manolagas SC 1997 Activation of the Janus kinase/STAT (signal transducer and activator of transcription) signal transduction pathway by interleukin-6-type cytokines promotes osteoblast differentiation Endocrinology 138:36663676.
  • 30
    Bellido T, O'Brien CA, Roberson PK, Manolagas SC 1998 Transcriptional activation of the p21 (WAF1,CIP1,SDI1) gene by interleukin-6 type cytokines: A prerequisite for their pro-differentiating and anti-apoptotic effects on human osteoblastic cells J Biol Chem 273:2113721144.
  • 31
    Ohno M, Cooke JP, Dzau VJ, Gibbons GH 1995 Fluid shear stress induces endothelial transforming growth factor beta-1 transcription and production: Modulation by potassium channel blockade J Clin Invest 95:13631369.
  • 32
    Baylink DJ, Finkelman RD, Mohan S 1993 Growth factors to stimulate bone formation J Bone Miner Res 8:S565S572.
  • 33
    Morinaga Y, Fujita N, Ohishi K, Tsuruo T 1997 Stimulation of interleukin-11 production from osteoblast-like cells by transforming growth factor-beta and tumor cell factors Int J Cancer 71:422428.
  • 34
    Rawlinson SC, Pitsillides AA, Lanyon LE 1996 Involvement of different ion channels in osteoblasts' and osteocytes' early responses to mechanical strain Bone 19:609614.
  • 35
    Reich KM, McAllister TN, Gudi S, Frangos JA 1997 Activation of G proteins mediates flow-induced prostaglandin E2 production in osteoblasts Endocrinology 138:10141018.
  • 36
    Rubin J, Biskobing D, Fan X, Rubin C, McLeod K, Taylor WR 1997 Pressure regulates osteoclast formation and MCSF expression in marrow culture J Cell Physiol 170:8187.