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Keywords:

  • cytokines;
  • knock-out;
  • osteoblast;
  • osteoclast;
  • gp130

Abstract

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

IL-6 and -11 regulate bone turnover and have been implicated in estrogen deficiency-related bone loss. In this study, deletion of IL-11 signaling, but not that of IL-6, suppressed osteoclast differentiation, resulting in high trabecular bone volume and reduced bone formation. Furthermore, IL-11 signaling was not required for the effects of estradiol or estrogen deficiency on the mouse skeleton.

Introduction: Interleukin (IL)-6 and -11 stimulate osteoclastogenesis and bone formation in vitro and have been implicated in bone loss in estrogen deficiency. Because of their common use of the gp130 co-receptor signaling subunit, the roles of these two cytokines are linked, and each may compensate for the absence of the other to maintain trabecular bone volume and bone cell differentiation.

Materials and Methods: To determine the interactions in bone between IL-11 and IL-6 in vivo and whether IL-11 is required for normal bone turnover, we examined the bone phenotype of mature male and female IL-11 receptor knockout mice (IL-11Rα1−/−) and compared with the bone phenotype of IL-6−/− mice and mice lacking both IL-6 and IL-11Rα. To determine whether IL-11 is required for the effects of estrogen on trabecular bone, mature IL-11Rα1−/− mice were ovariectomized and treated with estradiol.

Results: In both male and female IL-11Rα1−/− mice, trabecular bone volume was significantly higher than that of wildtype controls. This was associated with low bone resorption and low bone formation, and the low osteoclast number generated by IL-11Rα1−/− precursors was reproduced in ex vivo cultures, whereas elevated osteoblast generation was not. Neither trabecular bone volume nor bone turnover was altered in IL-6−/− mice, and compound IL-6−/−:IL-11Rα1−/− mice showed an identical bone phenotype to IL-11Rα1−/− mice. The responses of IL-11Rα1−/− mice to ovariectomy and estradiol treatment were the same as those observed in wildtype mice.

Conclusions: IL-11 signaling is clearly required for normal bone turnover and normal trabecular bone mass, yet not for the effects of estradiol or estrogen deficiency on the skeleton. In the absence of IL-11Rα, increased trabecular bone mass seems to result from a cell lineage-autonomous reduction in osteoclast differentiation, suggesting a direct effect of IL-11 on osteoclast precursors. The effects of IL-11Rα deletion on the skeleton are not mediated or compensated for by changes in IL-6 signaling.


INTRODUCTION

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

OSTEOCLASTS (BONE-RESORBING cells) and osteoblasts (bone-forming cells) on trabecular bone surfaces continually remodel bone, making calcium available to the circulation and replacing old bone with new. The differentiation of these two cell types is normally regulated by intercellular communication pathways with the effect that a change in the level of bone resorption (i.e., an increase or decrease) is usually associated with a similar change in bone formation. Interleukin (IL)-6 and IL-11 are two cytokines known to rely on osteoblast-osteoclast communication for their effects on osteoclast differentiation.(1) Cells of the osteoblast lineage express IL-11 and IL-6 in response to a wide range of stimuli, and osteoblast differentiation is stimulated by these cytokines through specific receptors on the cell surface.(2–5) Although these receptors have also been described on osteoclasts, their function in these cells is not understood, because IL-6 and IL-11 promote osteoclast formation by stimulating RANKL expression by cells of the osteoblast lineage rather than acting directly on the osteoclast itself,(5–7) although some RANKL-independent stimulation has been claimed recently.(8) Whereas IL-6, but not IL-11, seems to regulate both estradiol and progesterone levels in human granulosa cells,(9,10) the circulating levels of both cytokines seem to depend on estrogen status in human subjects.(11) This, coupled with the stimulatory effects of IL-6 and IL-11 on osteoblast and osteoclast differentiation,(2–7) has led to the hypothesis that the effects of estrogen and estrogen deficiency may be mediated by changes in IL-6 and/or IL-11 expression,(12) but this proposal remains controversial.(13–15) It has also been proposed that these cytokines may also be involved in the pathogenesis of Paget's disease,(16) hyperparathyroidism,(17) rheumatoid arthritis,(18) and other bone diseases.(19) An inverse relationship between circulating levels of IL-11 and IL-6 has been shown in healthy and hyperparathyroid patients, suggesting compensatory roles in vivo.(17) Furthermore, elevated circulating IL-11 in IL-6−/− mice may compensate for the absence of IL-6 and be responsible for the mildness of the bone phenotype in these mice.(20)

The similarity of the effects of IL-6 and IL-11 on bone cells and their potential to compensate for one another might be traced to their common gp130-linked signaling pathway.(21) Whereas these cytokines bind to ligand-specific transmembrane receptors (IL-6Rα and IL-11Rα1) and, in the case of IL-6, a naturally occurring soluble IL-6 receptor (sIL-6R), none of these α subunits are capable of intracellular signaling themselves. Rather, the ligand-bound α subunits induce homodimerization between two common β receptor subunits, gp130, before activation of a number of intracellular signaling pathways.(22) The two major intracellular pathways downstream of gp130, SHP2/ERK/MAPK and STAT1/3, have been shown to regulate specifically either bone growth or the maintenance of trabecular bone structure.(23)

To study whether IL-11 and IL-6 are required for normal bone turnover, we have undertaken a bone histomorphometric analysis of IL-11Rα1−/− and IL-6−/− mice. Furthermore, to determine unique and redundant actions of IL-11 and IL-6 in bone, we compared the skeletons of mice rendered null for the IL-11Rα and IL-6 genes with those of compound IL-11Rα1−/−:IL-6−/− mice. Finally, the role of IL-11 in the response of bone to estrogen and estrogen deficiency was studied in IL-11Rα1−/− mice.

MATERIALS AND METHODS

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

Knockout mice

IL-11Rα1−/− and IL-6−/− mice were generated as previously described.(24–26) The IL-11Rα1−/− mouse expresses neither isoform of the IL-11R (IL-11Rα1 nor IL-11Rα2), and this targeted deletion ablates both the increase in granulocyte-macrophage colony forming unit (GM-CFU) formation(26) and the increased STAT1/3 and Erk1/2 signaling induced by IL-11 treatment of bone marrow cells (Jenkins and Ernst, unpublished data, 2003). Compound IL-11Rα1−/−:IL-6−/− mice were generated by crossing IL-6−/− mice with IL-11Rα1+/− mice. For initial phenotype analysis, all mice were on a mixed C57Bl6/129J background, and littermate controls were used. For ovariectomy and estradiol treatment studies, IL-11Rα1−/− were backcrossed to a C57Bl/6 background for at least 10 generations, and C57Bl/6 mice were used as wildtype controls. All animal handling procedures were approved by animal ethics committees at the Ludwig Institute for Cancer Research/The University of Melbourne Department of Surgery, The Walter and Eliza Hall Institute or St. Vincent's Health.

Histomorphometry

Tibias were collected at 12-16 weeks or 12 months of age, fixed in 3.7% formaldehyde in PBS, and embedded in methylmethacrylate.(27) Double fluorochrome labeling was performed as described previously.(27) Five-micrometer sections were stained with Toluidine blue for standard histomorphometry or Xylenol orange for fluorochrome analysis.(28) Adipocyte volume was measured as previously described.(27) Histomorphometry was carried out according to standard procedures in the secondary spongiosa of the proximal tibia using the Osteomeasure system (OsteoMetrics, Decatur, GA, USA). Tibial cortical thickness (Ct.Th) and periosteal mineral appositional rates were measured as described previously.(27) Cartilage volume as a percentage of trabecular bone volume was measured in the same region using sections stained with Safranin O (specific stain for cartilage) and a Fast Green counterstain. Femoral length and width were determined from contact X-rays that were scanned and measured using NIH Image 1.62 as described previously.(27)

Serum biochemistry

Serum was collected from 10-week-old mice. Intact PTH was measured by a mouse-specific ELISA (Immunotopics, San Clemente, CA, USA). Serum calcium was measured by reaction with o-cresolphthalein (Sigma Diagnostics, St Louis, MO, USA).

Cell culture systems

Primary cell culture systems were used to determine whether alterations in osteoclast and osteoblast number in the IL-11α1−/− mice were autonomous to that cell lineage rather than requiring the presence of other cell types in the bone microenvironment.

Osteoclastogenic potential of bone marrow and of bone marrow macrophages (BMM precursors) was determined as described previously(29) by stimulating bone marrow cell preparations flushed from the femora and tibias of male and female mice (plated at 105 cells per 10-mm-diameter well) with soluble recombinant glutathion S transferase (GST)-RANKL protein(30) and macrophage-colony stimulating factor (M-CSF) (R&D Systems, Minneapolis, MN, USA). TRACP+ multinucleated cells (MNCs) were counted at day 7.

Osteoblast differentiation was assessed in ex vivo osteoblast cultures derived from bone marrow precursors as described previously.(31) Briefly, tibias and femora from 8- to 10-week-old mice of each genotype were dissected free of adhering muscle. The ends were removed, and the marrow cavity was flushed with α-MEM. Cells were plated at 2 × 106 cells/well in 24-well plates in α−MEM containing 10% FCS, 10 nM dexamethasone, 10 mM β-glycerophosphate, and 50 μg/ml ascorbic acid. Cells were fixed at days 14, 21, and 28 and stained for alkaline phosphatase to detect osteoblast colony formation or by a von Kossa technique to detect mineralization.(32)

Ovariectomy and estradiol treatments

To determine the contribution of IL-11 to ovariectomy-induced bone loss and effects of estrogen on bone, 12-week-old wildtype and IL-11Rα1−/− mice were either sham operated, subjected to bilateral ovariectomy (OVX), or ovariectomized and supplied with a subcutaneous controlled release estrogen pellet, releasing 0.1 μg/day (Innovative Research America, Sarasota, FL, USA) as previously described.(33) Four weeks after the operation, mice were killed, and serum and tissues were collected as described above.

RESULTS

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

Skeletons of IL-11Rα1−/−, IL-6−/−, and IL-11Rα1−/−:IL-6−/− mice were grossly normal (Fig. 1A). Among males, femoral length was significantly reduced only in IL-11Rα1−/− mice, whereas femurs were significantly shorter in females of all three lines of knockout mice compared with wildtype controls (Table 1). These changes were not associated with any significant alterations in the width of the growth plate or the proliferating or hypertrophic zones within the growth plate at 14-16 weeks of age (data not shown). Regulation of bone length or chondrocyte differentiation by IL-11 and IL-6 have not been reported previously; however, we have shown that gp130 signaling through the STAT1/3 pathway is required for normal longitudinal bone growth, yet signaling through the MAPK pathway downstream of gp130 is dispensable for that function.(23) This indicates that the regulation of longitudinal bone growth by IL-11 and IL-6 is likely to be mediated by the gp130-STAT1/3 pathway.

Table Table 1.. Changes in Bone Size and Cortical Growth in the Absence of IL-11 and IL-6 Signaling in Male and Female Mice
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Figure FIG. 1.. High bone mass in male mice in the absence of IL-11Rα. (A) Representative X-rays of femurs from 16-week-old male wildtype, IL-11Rα−/− (IL-11R), IL-6−/− (IL-6), and IL-11Rα−/−:IL-6−/− (IL-11R:IL-6) mice, showing an increase in BMD, particularly in the distal metaphysis (denoted by the white box), in IL-11Rα−/− mice; differences in trabecular bone volume; all images are from the same X-ray film; white bar = 5 mm. (B) Histomorphometric measures of trabecular bone volume (BV/TV) and trabecular number (Tb.N) were significantly higher and trabecular spacing (Tb.Sp) was significantly lower in IL-11Rα−/− and IL-11Rα−/−:IL-6−/− mice at 14-16 weeks of age. (C) In 12-month-old male mice, BV/TV and TbN are still significantly higher in IL-11Rα−/− compared with wildtype littermates. All values are mean ± SE from 6-12 mice per group. *p < 0.05; **p < 0.01; ***p < 0.001 vs. wildtype of the same sex.

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Femoral width was significantly reduced in male and female IL-11Rα1−/− and IL-6−/− mice (Table 1), but not in IL-11Rα1−/−:IL-6−/− mice. In male IL-11Rα1−/− and IL-6−/− mice, this seemed to relate to reduced periosteal appositional rate (PsMAR), indicating an early cessation or slower progression of transverse bone growth (Table 1). Cortical thickness (Ct.Th) was lower only in male IL-11Rα1−/− mice, probably because of the lower level of bone formation in the endosteal compartment in these mice. PsMAR and CtTh were not significantly altered in female mice of any genotype (Table 1).

High trabecular bone mass in the absence of IL-11Rα in male and female mice

X-ray analysis of male mice revealed an elevation in trabecular bone mass in the femoral distal metaphysis in both IL-11Rα1−/− and IL-11Rα1−/−:IL-6−/− mice (Fig. 1A). Quantification by bone histomorphometry in adult male mice revealed that trabecular bone volume (BV/TV) was significantly greater in IL-11Rα1−/− mice compared with wildtype controls (Fig. 1B). This high bone volume was associated with high trabecular number (Tb.N), low trabecular separation (Tb.Sp), but no change in trabecular thickness (Tb.Th; Fig. 1B). A very similar alteration in trabecular bone structure was observed in IL-11Rα1−/−:IL-6−/− mice, indicating no further effect of IL-6 deletion on the skeleton when IL-11Rα signaling has already been deleted. As previously reported,(20) no markers of trabecular bone structure were significantly altered in male IL-6−/− mice.

In wildtype, both male and female mice trabecular bone loss usually occurs during aging, and on the C57Bl/6 background used in this study, there is very little trabecular bone remaining by the age of 12 months (Fig. 1C).(34) In the absence of IL-11Rα, whereas some age-related reduction in bone mass was observed by 12 months of age, BV/TV and TbN at this age were still well above levels seen in wildtype mice, and the proportion of bone mass lost in IL-11Rα1−/− mice between 14-16 weeks and 12 months of age was much less compared with wildtype controls (Fig. 1C). In contrast to the preservation of the trabecular bone, Ct.Th was significantly lower in the absence of IL-11Rα, as observed in the younger adult mice (Fig. 1C compared with Table 1), although the relative growth-related increase in Ct.Th between these two ages was the same for each genotype. The higher level of bone formation at the periosteal surface in the IL-11Rα1−/−:IL-6−/− males (Table 1) suggests an interaction between IL-6 and IL-11 on osteoblasts or their precursors in this microenvironment that does not exist in the marrow. Osteoblasts at the trabecular (endosteal) and periosteal surfaces are derived from different precursors, and a difference in effect of genetic manipulation in these compartments has been reported previously.(35) It is unlikely that this effect is mediated by an alteration in gp130 itself, because no effect on periosteal growth was detected in mice unable to elicit either gp130-dependent SHP2/ERK/MAPK or STAT1/3 signaling.(23)

In female mice, a very similar phenotype was observed in the absence of IL-11Rα. von Kossa-stained sections from 16-week-old tibias (Fig. 2A) indicated a distinct increase in trabecular bone volume in the absence of IL-11Rα. This was confirmed by histomorphometry (Fig. 2B). The magnitude of the effect of IL-11Rα deletion was more pronounced in female mice than in the males; in female IL-11Rα1−/− mice, BV/TV was almost double (190%) that of wildtype, whereas in male mice, BV/TV was increased by one-third (134%). This may relate to the high bone turnover and low trabecular bone volume normally observed in female wildtype mice compared with males.(34) In female IL-6−/− mice, there was a slight, but significant elevation in trabecular number (Fig. 2B).

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Figure FIG. 2.. High bone mass in female mice in the absence of IL-11Rα. (A) Representative images of von Kossa-stained proximal tibias of female 16-week-old wildtype, IL-11Rα−/− (IL-11R), IL-6−/− (IL-6), and IL-11Rα−/−:IL-6−/− (IL-11R:IL-6) mice, showing differences in trabecular bone volume (white box shows secondary spongiosa region used for histomorphometric measurements); bar = 500 μm. (B) Histomorphometric measures of trabecular bone volume (BV/TV) and trabecular number (Tb.N) were elevated and trabecular spacing (Tb.Sp) was reduced in IL-11Rα−/− and IL-11Rα−/−/IL-6−/− mice. Trabecular number was also mildly elevated in IL-6−/− mice. All values are mean ± SE from 7-12 mice per group, 14-16 weeks of age. *p < 0.05; **p < 0.01; ***p < 0.001 vs. wildtype of the same sex.

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Reduced bone formation in the absence of IL-11Rα1

Deletion of IL-11Rα was associated with a low level of bone formation in both male and female mice (Figs. 3A-3C). All histomorphometric markers of bone formation, including osteoblast surface (ObS/BS), osteoid volume (OV/BV), osteoid thickness (O.Th), and the calcein label-derived parameter, bone formation rate (BFR/BS), were significantly reduced in the absence of IL-11Rα, irrespective of the presence of IL-6. No changes in any markers of bone formation were observed when IL-6 alone was deleted (Figs. 3A and 3B). However, when osteoblast precursors were grown ex vivo from wildtype and IL-11Rα1−/− bone marrow cells, we did not observe any significant difference in the ability of these precursors to differentiate into alkaline phosphatase-positive osteoblasts or any change in their ability to form mineralized nodules compared with wildtype controls (data not shown). This indi-cated that the low level of bone formation in the absence of IL-11Rα was not cell autonomous but was dependent on communication from other cells within the bone microenvironment.

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Figure FIG. 3.. Reduced bone formation in the absence of IL-11Rα in both male and female mice. Histomorphometric indices of bone formation including tibial osteoblast surface (ObS/BS) and bone formation rate (BFR/BS) were significantly higher in male (A) and female (B) in IL-11Rα−/− (IL-11R) and IL-11Rα−/−:IL-6−/− (IL-11R:IL-6) mice compared with wildtype and IL-6−/− (IL-6) mice. All values are mean ± SE from 6-12 mice per group, 14-16 weeks of age. *p < 0.05; **p < 0.01; **p < 0.001 vs. wildtype of the same sex. (C) Representative images of calcein label incorporation in trabecular bone of the secondary spongiosa stained with Xylenol orange. Note the reduction in green calcein labeling in the IL-11Rα−/− (IL-11R) sample compared with wildtype. White bar = 250 μm.

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Because osteoblasts and adipocytes are derived from the same precursor pool, and IL-11 has been reported to simultaneously inhibit adipogenesis and stimulate osteoblast formation from their common stromal cell precursors,(36) we also measured marrow adipocyte volume as a percentage of marrow volume (AV/MV) in sections from 12-month-old males. AV/MV was significantly reduced in IL-11Rα1−/− mice (mean ± SE: wildtype, 8.59 ± 2.35%; IL-11Rα1−/−, 0.137 ± 0.08%; p < 0.005). In 16-week-old animals, a similar trend was observed in IL-11Rα−/− and IL-11Rα1−/−:IL-6−/− mice, but not IL-6−/− mice (data not shown); this did not reach statistical significance, probably because very few adipocytes are present in the marrow at this age (AV/MV in wildtype mice at 16 weeks was 1.24 ± 0.44). This effect of IL-11Rα is in contrast to known effects of IL-11 as an inhibitor of adipogenesis in vitro(37) and may indicate the presence of compensatory mechanisms in its absence in vivo. This reduction in the numbers of differentiated osteoblasts and adipocytes is not associated with any reduction in their precursor populations or any change in body weight in the IL-11Rα1−/− mice.(26)

Reduced osteoclastogenesis in the absence of IL-11Rα

Bone resorption was also low in male and female mice in the absence of the IL-11Rα. In female mice, osteoclast surface (OcS/BS) and osteoclast number (data not shown) were significantly lower in IL-11Rα1−/− and IL-11Rα1−/−: IL-6−/− mice (Fig. 4A). To determine whether this observation was associated with a low level of bone resorption, we stained wildtype and IL-11Rα1−/− sections with Safranin O, which stains cartilage orange. Indeed, we observed an increase in cartilage remnants, derived from the growth plate, within trabecular bone in the secondary spongiosa (Fig. 4B), suggesting a functional reduction in bone resorption.(34) We next measured these cartilage remnants, and expressed this as a percentage of trabecular bone volume (CtgV/BV). This parameter correlated inversely with osteoclast surface in wildtype mice (CtgV/BV = 0.912 − 0.133 × OcS/BS; p < 0.05), and was significantly greater in both IL-11Rα1−/− and IL-11Rα1−/−:IL-6−/− mice compared with wildtype controls (Fig. 4A).

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Figure FIG. 4.. Reduced bone resorption in the absence of IL-11Rα in both male and female mice. Tibial trabecular osteoclast surface (OcS/BS) was significantly increased in female (B) IL-11Rα−/− (IL-11R) and IL-11Rα−/−:IL-6−/− (IL-11R:IL-6) mice compared with wildtype and IL-6−/− (IL-6) mice, but not in males (A). However, calcified cartilage remnants within the trabecular bone (CtgV/BV) were increased in both (A) male and (B) female IL-11Rα−/− (IL-11R) and IL-11Rα−/−:IL-6−/− (IL-11R:IL-6) mice compared with wildtype and IL-6−/− (IL-6) mice. All values are mean ± SE from 6-12 mice per group, 14-16 weeks of age. **p < 0.01; ***p < 0.001 vs. wildtype of the same sex. (C) Representative images of Safranin O/Fast green secondary spongiosa (top of image is 600 μm below the growth plate) in wildtype and IL-11Rα−/− clearly show the increase in cartilage remnants deep within the mature trabecular bone in the absence of IL-11Rα. White bar = 250 μm. (D) In vitro osteoclastogenesis assays showing reduced TRACP+ MNCs (TRACP+ MNCs/well) generated from RANKL-induced primary bone marrow cultures from IL-11Rα−/− mice compared with wildtype mice. All values are mean ± SE of a minimum of quadruplicate cultures from three separate mouse bone marrows of mixed sexes. ***p < 0.005 vs. wildtype culture.

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In male mice, whereas we did not detect a significant alteration in osteoclast surface in IL-11Rα1−/− or IL-11Rα1−/−:IL-6−/− mice, the volume of cartilage remnants within trabecular bone was altered in a similar manner to that seen in female mice in the absence of IL-11Rα (Fig. 4C). IL-6 deletion on its own did not alter bone resorption or osteoclast surface in male or female mice (Figs. 4A and 4C).

To determine whether the low level of osteoclastogenesis was cell lineage autonomous, we assessed osteoclast formation in ex vivo cultures of RANKL-stimulated bone marrow from wildtype and IL-11Rα1−/− mice. We observed a significantly lower level of osteoclast formation from precursors isolated from IL-11Rα1−/− bone marrow (Fig. 4D), indicating that the low level of osteoclastogenesis in these mice was cell autonomous and resulted from the absence of IL-11 signaling in the hematopoietic lineage.

Serum biochemistry

Despite the low level of bone turnover, serum calcium levels were not significantly altered in IL-11Rα1−/− mice (mean: wildtype, 7.27 ± 0.38 mg/dl versus IL-11Rα1−/−, 7.57 ± 0.32 mg/dl; values are from nine female mice per group at 20 weeks of age). Similarly, serum PTH levels were also unchanged (mean: wildtype, 19.0 ± 4.0 pg/ml versus IL-11Rα1−/−, 17.7 ± 8.4 pg/ml; values are from nine female mice per group at 20 weeks of age).

IL-11Rα is not required for ovariectomy-induced bone loss or for the protective effects of estradiol on bone

Because of the low bone turnover resulting from IL-11Rα ablation, a phenotype closely related to that observed in the ERα−/− mouse,(34) and the hypothesis that IL-11 may mediate the effects of estradiol on bone,(12–14) we set out to determine whether IL-11Rα is required for the effects of estradiol on bone or in estrogen deficiency bone loss. Ovariectomy and replacement estradiol treatment were carried out in adult wildtype and IL-11Rα1−/− mice.

The effects of ovariectomy and estradiol treatment were identical in wildtype and IL-11Rα1−/− mice (Figs. 5A and 5B). In both wildtype and IL-11Rα1−/− mice, ovariectomy was associated with a significant reduction in BV/TV and TbN (Fig. 5B), as well as a significant increase in bone turnover (Fig. 5C). Estradiol infusion at 0.1 μg/kg/day, which we have previously shown to prevent bone loss associated with ovariectomy,(34) was associated with a significant increase in both BV/TV and TbN (Fig. 5B) because of a reduction in bone turnover, particularly reduced bone resorption (Fig. 5C). These data indicate that neither the loss of trabecular bone associated with ovariectomy nor the osteoprotective effects of estradiol is dependent on IL-11R signaling.

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Figure FIG. 5.. Ovariectomy-induced bone loss does not require IL-11Rα. (A) Representative images of von Kossa-stained proximal tibias of female 20-week=old wildtype or IL-11Rα−/− mice, 4 weeks after sham operation (sham), bilateral ovariectomy (ovx), or ovariectomy with estrogen implants (ovx + E2), showing differences in trabecular bone volume; grey bar = 500 μm. (B) Histomorphometric measures of trabecular bone volume (BV/TV) and trabecular number (Tb.N) were significantly reduced by ovariectomy (diagonal striped bars), and this was prevented by estradiol treatment (stippled bars) in both wildtype and IL-11Rα−/− mice compared with sham-operated littermates (white bars). All values are mean ± SE from 7-12 mice per group. #p < 0.05; ##p < 0.01; ###p < 0.001 vs. wildtype of the same sex; *p < 0.05 vs. sham-operated mice of the same genotype; +p < 0.05, ++p < 0.01, +++p < 0.001 vs. ovariectomized mice of the same genotype.

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DISCUSSION

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

In the absence of IL-11Rα, we observed a significant elevation in trabecular bone volume caused by repression of both bone formation and bone resorption. The IL-11Rα1−/− mouse expresses neither isoform of the IL-11R (IL-11Rα1 nor IL-11Rα2).(26) In this model, IL-11 signaling was disrupted such that the IL-11-induced increases in both GM-CFU formation(26) and STAT1/3 and Erk1/2 signaling (Jenkins and Ernst, unpublished data, 2003) have been blocked. The bone phenotype observed indicates that IL-11 signaling is needed for maintenance of normal bone turnover and trabecular bone volume. Furthermore, IL-11Rα deletion has the same effect on trabecular bone both in the presence and absence of IL-6, indicating that, whereas IL-6 and IL-11 signal through the same gp130 co-receptor subunit, their roles in trabecular bone are distinct and separable. This difference may relate to differences in IL-11 and IL-6 receptor expression and the requirement of participation of sIL-6R for IL-6 action in the osteoblast, which itself is susceptible to control.(38)

It has been shown previously that the promotion of osteoclast formation by IL-6 requires participation of the osteoblast lineage.(38) IL-6 stimulates production of RANKL by osteoblasts and their precursors, and this is mediated by STAT1/3 signaling downstream of gp130 dimerization, rather than activation of the SHP2/ERK/MAPK pathway.(39) Although it is likely that IL-11 promotion of osteoclastogenesis uses an identical signaling pathway,(5) others have presented evidence that IL-11 can stimulate osteoclast formation directly.(8) It seems from this evidence that the effect of IL-11 deletion on osteoclast differentiation can also occur without the presence of osteoblasts, supporting a second, direct pathway by which IL-11 stimulates osteoclast formation. Furthermore, in bone marrow precursors from mice displaying enhanced activation of gp130-dependent SHP2/ERK/MAPK signaling in the absence of the STAT1/3 pathway, IL-11 stimulates osteoclast differentiation,(40) suggesting that this direct stimulatory effect of IL-11 on the osteoclast precursor is mediated through this pathway rather than the STAT1/3 pathway used by IL-11-stimulated osteoblasts.

The osteoclast-driven bone phenotype observed in the absence of IL-11Rα contrasts with the osteoblast-specific phenotype observed in transgenic mice overexpressing IL-11 in a wide range of tissues including bone.(41) In those mice, bone formation was increased by IL-11, yet osteoclast numbers remained normal, despite ample evidence that STAT1/3 signaling through osteoblasts both stimulates osteoblast formation and mediates the RANKL-induced promotion of osteoclast formation in response to IL-11.(5,39) The surprising lack of effect of the IL-11 transgenic on osteoclasts may relate to the continuous elevation in IL-11 expression, the levels of protein expressed, and the cell types in which IL-11 has been overexpressed, which has not yet been reported.

The increase in trabecular bone volume caused by low levels of both resorption and formation in IL-11Rα1−/− mice resembles the effects of estrogen receptor-α (ERα) deletion in male and female mice(34) and can be described as a mild osteopetrosis, where a low level of resorption is responsible for an increase in cartilage remnants within the trabecular bone as growth progresses. Although bone formation is also low in both knockouts, the increased trabecular bone volume implies that the physiological effect of the low level of bone resorption in the ERα and IL-11Rα1 knockouts is greater than the effect of lowered bone formation. This is likely to relate to the absence of either receptor from birth, including the major period of longitudinal bone growth, where osteoclasts would normally resorb calcified cartilage from the growth plate. In contrast, a low level of bone turnover in adult bone may result in low trabecular bone volume, as is commonly observed in human osteoporosis. In mice, low bone turnover has been associated with low trabecular bone volume in the absence of insulin receptor substrate-1 (IRS-1).(42) In contrast to the ERα and IL-11Rα knockout mice, the IRS-1 knockouts showed a much greater reduction in bone formation, which was also cell autonomous. In the IL-11Rα1−/− mice, it seems likely that the more mild, and non-cell autonomous, reduction in bone formation is a consequence of the low level of osteoclastogenesis, reflecting the lesser activity of the coupling process in the IL-11Rα1−/− mice.

ERα−/− mice, which have a similar trabecular bone phenotype to the IL-11Rα1−/−, also showed dramatically elevated circulating testosterone and estradiol levels and were not responsive to estradiol treatment.(34) It has been suggested that effects of estrogen and estrogen deficiency may be mediated by changes in IL-6 and/or IL-11.(11–14) Previous work in the IL-6−/− mouse did not show bone loss or increased bone turnover after ovariectomy, indicating that IL-6 may play an important role in the loss of bone associated with estrogen deficiency.(20) However, in this study, circulating estradiol and testosterone levels were below the level of detection in assays used, and we observed normal response to both ovariectomy and estradiol treatment in the absence of IL-11Rα. This finding contrasts with the recent report suggesting a requirement for IL-11 in ovariectomy-induced bone loss, because an IL-11-neutralizing antibody, which impairs osteoclast formation, has been shown to partially inhibit ovariectomy-induced bone loss.(14)

In conclusion, we have shown that IL-11 is essential for normal bone turnover and bone structure and that the increased trabecular bone volume in the absence of IL-11Rα is caused by a cell autonomous reduction in osteoclast differentiation. Although IL-11 is required for normal bone structure, we observed that IL-11 signaling does not play an essential role in the bone loss associated with estrogen deficiency or the osteoprotective effects of estradiol on trabecular bone.

Acknowledgements

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

The authors thank Ingrid Kriechbaum for excellent histological assistance, Jan Elliott for carrying out osteoblast primary cell cultures, and Marilyn Ibrahim and Bette Borobokas for skilled technical assistance. This work was supported by an Early Career Research Grant from the University of Melbourne, a Program Grant (003211), and Project Grant (247909) from the National Health and Medical Research Council Australia (NHMRC). NAS is supported by an RJ Gleghorn Research Fellowship from the University of Melbourne and an NHMRC Career Development Award. MTG and ME are Research Fellows of the NHMRC.

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