help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lea, C. K.
Right arrow Articles by Flanagan, A. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lea, C. K.
Right arrow Articles by Flanagan, A. M.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*ESTRADIOL
Endocrinology Vol. 140, No. 1 273-279
Copyright © 1999 by The Endocrine Society

ARTICLES

Macrophage Colony Stimulating-Factor Transcripts Are Differentially Regulated in Rat Bone-Marrow by Gender Hormones1

C. K. Lea, U. Sarma and A. M. Flanagan

Department of Histopathology, Imperial College School of Science, Technology and Medicine at St. Mary’s, London W2 1PG, United Kingdom

Address all correspondence and requests for reprints to: Adrienne M. Flanagan, Department of Histopathology, Imperial College School of Science, Technology and Medicine at St. Mary’s, Norfolk Place, London W2 1PG, United Kingdom. E-mail: a.flanagan{at}ic.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
There are at least three forms of macrophage colony-stimulating factor (M-CSF), a cytokine that is critical for osteoclast formation; and evidence exists that the membrane-bound form is involved in this process. We wished to test the hypothesis that the expression of the membrane form of M-CSF is modulated by the presence of gender steroids. This was achieved by analyzing M-CSF messenger RNA and protein in the bone-marrow of estrogen- and androgen-replete, and -deficient female rats. We found that the 1.4-kb M-CSF transcript was not detected in sham-operated rats but that the 4.6-kb transcript was expressed in abundance. In contrast, these transcripts were differentially expressed in ovariectomized rats, and this effect was reversed by 17ß-estradiol treatment. Administration of androstenedione to ovariectomized rats, so that androstenedione plasma levels were restored to just below that in sham-operated rats, also suppressed the expression of the 1.4-kb M-CSF transcript. This effect was abrogated by antiandrogen treatment, indicating that this was an androgen-mediated effect. The membrane-bound protein was detected in the bone-marrow of sham-operated rats and was elevated post ovariectomy, whereas ovariectomy had no effect on the soluble isoform. Our data support the hypothesis that the membrane form of M-CSF is modulated by gender hormones and that this isoform is involved in the estrogen- and androgen-mediated effects on the skeleton.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ESTROGEN IS A major regulator of bone metabolism (for review, see Ref. 1). In the event of a marked reduction in plasma levels of 17ß-estradiol (17ß-E2), which occurs most commonly as a consequence of the natural menopause, bone turnover increases. This results in a deficit in bone mass, because the increase in osteoclastic bone resorption (BR) is not matched by the increase in bone formation. This increase in bone turnover can be suppressed by estrogen (2, 3, 4) and androgen replacement (5, 6), but the mechanism(s) by which this occurs is not fully understood.

The majority of current data suggest that estrogen regulates osteoclast formation by altering the production of, or the response to, cytokines in the bone-marrow (BM) microenvironment, the site where osteoclasts are formed. The cytokines that have been most consistently implicated include tumor necrosis factor {alpha} (TNF{alpha}) (7, 8, 9, 10, 11), interleukin (IL)-1{alpha} (7, 8, 9, 12, 13), IL-6 (14, 15, 16, 17, 18), transforming growth factor ß (19, 20), and macrophage colony stimulating-factor (M-CSF) (21, 22). The most convincing evidence is based on data derived from in vivo experiments. In particular, it has been shown that simultaneously blocking the effects of TNF{alpha} and IL-1{alpha} and ß prevents bone loss as a result of post-ovariectomy estrogen deficiency. These data are supported by the ovariectomy experiments performed in transgenic mice that overexpress a soluble form of the TNF receptor that blocks TNF binding to membrane receptors (10) and in IL-1-receptor-deficient mice (12). However, IL-1{alpha} and TNF{alpha} messenger RNA (mRNA) in BM were not altered post ovariectomy (23), which suggests that if the rise in BR were attributed to increased production of these cytokines, their expression is regulated by estrogen posttranscriptionally. It has been proposed that the effect of TNF{alpha} and IL-1{alpha} is mediated by IL-6, but this view is not supported by work of others (8, 11). Furthermore, it has been reported that mRNA and protein for IL-6 are not increased in ovariectomized mice (23, 24).

Our hypothesis was that the synthesis of the membrane-bound form of M-CSF is increased in BM in estrogen-deficient states, and this accounts for the elevation in BR post ovariectomy. This theory was based on the knowledge: 1) that M-CSF is critically involved in osteoclast formation (25, 26); 2) that 17ß-E2 reduces osteoclast parameters in a dose-responsive manner in human BM cultures and that the inhibitory effect was reversed by the addition of high levels of soluble M-CSF (22); 3) that, in the same human BM cultures, protein and mRNA for the membrane form of M-CSF were specifically suppressed in the presence of 17ß-E2 (22); and 4) that a much higher concentration of the soluble (85-kDa) M-CSF was required to induce osteoclasts in op/op BM cultures than is found in the serum of wild-type op/op mice (27), and finally that this M-CSF isoform is regulated by osteotropic agents and is involved in osteoclast formation (28).

We also postulated that the expression of M-CSF mRNA in BM from ovariectomized rats would be suppressed by androstenedione treatment, given that we have recently reported that treatment of ovariectomized rats with physiological levels of androstenedione resulted in suppression of bone turnover (5, 6).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal experimentation
Thirteen-week-old (average weight, 231 g; range, 224–243 g), 11-week-old (average weight, 210 g; range, 198–219 g), and 8-week-old (average weight, 201 g; range, 163–214 g) female Sprague Dawley rats, purchased from Harlan Olac (Bicester, Oxon, UK), were used in the experiments. The animals were housed at 21 C with a 12-h light, 12-h dark cycle and were fed rat laboratory diet (Lillico, Betchworth, Surrey, UK). The animals were pair-fed. Water was available ad libitum. 17ß-E2 (Sigma Chemical Co. Chemicals, Dorset, UK), dissolved in 95% corn oil/5% benzyl alcohol, was administered as a daily sc injection either from the day of ovariectomy or from day 14 post ovariectomy. Androstenedione was administered as slow-release pellets (Innovative Research of America Toledo, OH), and these were implanted sc at the time of surgery or 14 days post ovariectomy. Bicalutamide, an antiandrogen, which mediates its effect by antagonizing androgen receptor binding (a kind gift from Dr. B. M. Vose, Zeneca Pharmaceuticals, Macclesfield, UK; 1 mg/kg·day) was dissolved in saline and 0.5% Tween and was given orally daily. At the end of the experiments, cardiac puncture was performed under anesthesia, and plasma samples were stored at -70 C until required. The animals were killed by cervical dislocation after periods between 3 and 180 days post ovariectomy. Bone histomorphometric analysis on the animals in the 180-day experiment has previously been published (5).

The uteri were removed after death and were weighed. Ovariectomy was confirmed by the absence of ovarian tissue. In the 19-day experiment, the right tibiae were used to perform histomorphometric analysis. In other experiments, the BM was taken for Western blot analysis. The BM from the left tibiae was used for Northern blot analysis (see below).

Histomorphometry
The right tibiae were fixed in 70% alcohol for 24 h, dehydrated through graded alcohols, and embedded without decalcification in London Resin (London Resin Co. Ltd., Basingstoke, Hants). Longitudinal sections of the proximal metaphysis were cut using a Reichart-Jung microtome (Leica Corp. Ltd., Wetzlar, Germany). Five-micrometer sections were stained with toluidine blue, and bone histomorphometry was performed using transmitted microscopy linked to a computer-assisted image analyzer (Seescan Ltd., Cambs. UK). Cancellous bone volume (BV/TV) was measured by tracing the relevant features with a cursor on the video screen at x40 magnification. All sections were analyzed without knowledge of the group from which they came. BV/TV at the proximal metaphyzeal cancellous bone was measured on two nonconsecutive sections. A standard area of 2 mm2 (at least 2 mm from the growth plate, to exclude the primary spongiosa) was measured.

The results were analyzed using Fisher’s least-significant difference for multiple comparisons in a one-way ANOVA and were expressed as means ± SEM. Significance was considered when P < 0.05. Statview 4.0 (Abacus Concepts, Cupertino, CA) was used to analyze the results.

RIAs for plasma hormone levels
Plasma levels of androstenedione (Diagostics Systems Laboratories, Inc., Great Hasley, Oxon, UK) and 17ß-E2 (INCSTAR Corp, Berkshire, UK) were measured by RIA, as instructed in the manufacturer’s guidelines. The RIAs had an intraassay coefficient of variation of less than 5% and an interassay coefficient of variation of less than 6%. The results were analyzed using the Student’s t test. P < 0.05 was considered significant.

Northern blot analysis
The BM was flushed from the left tibiae, and total RNA was extracted using RNAzol B (Biogenesis, Bournemouth, UK), according to the manufacturer’s instructions. The amount and purity of the RNA were assessed by spectrophotometry. The RNA was denatured by formaldehyde/formamide loading buffer (Sigma Chemical Co.). Fifteen micrograms total RNA was run on a 1% agarose formaldehyde denaturing gel, along with markers (Sigma Chemical Co.), for 6 h at 4 V/cm and was blotted onto Hybond+ nylon membrane (Amersham, Buckinghamshire UK). Hybridization was carried out at 55 C, according to Church and Gilbert (1984) for 24 h. The probe, generated from rat BM complementary DNA (cDNA), using RT-PCR, as described below, was labeled with {alpha}-32-P-deoxycycidine triphosphate, using an oligolabeling kit (Amersham). After hybridization, the blot was washed in buffers of successively lower saline-sodium citrate (SSC) concentrations, as follows: x2 SSC, 0.1% SDS for 30 min at room temperature; x1 SSC, 0.1% SDS for 30 min at 60 C; and 0.1% SDS for 5 min at 60 C. After development of the autoradiograph for M-CSF gene analysis, the membrane was stripped and reprobed for ß-actin, using similar conditions to those described above, for assessment of the amounts of RNA loaded in each well.

The M-CSF probe for the Northern blots was a 420-bp PCR product, which was generated using previously published primers for human M-CSF (22); however, in this case, the probe was generated from amplified rat cDNA. The primers were originally designed against the human sequence, and they contained three mismatches in both the sense and antisense primers, compared with the rat sequence (GenBank sequence g203640). The probe labeled both the 4.6-kb (secreted) and 1.4-kb (membrane) transcripts because the primers were designed so that the PCR product spanned the splice site where the 4.6-kb M-CSF mRNA-specific sequence is located (see Fig. 1Go). The sense primer sequence was 5'-TAATGGAGGACACCATGCGC-3', and the antisense primer was 5'-CTCTGAGGCTCTTGATGGCT-3'.



View larger version (14K):
[in this window]
[in a new window]
  		Figure 1. A schematic diagram of the M-CSF cDNA probe used in the Northern blot analyzes.

 
RT and PCR
PCR, using primers described above, was performed on cDNA from rat BM to detect the M-CSF 1.4-kb transcript in estrogen-replete animals. This was done using 75 mM Tris-HCl (pH 9 at 25 C), 20 mM (NH4)2SO4, 0.01% Tween, 1 mM MgCl2, 1.25 mM of each deoxynucleotide triphosphate, 25 pmol of each primer (see section above), 0.5 U Taq polymerase, and 50 ng total RNA-equivalent of cDNA. The reaction was carried out for 10 min at 94 C, followed by 35 cycles of 55 C for 1 min, 72 C for 30 sec, and 94 C for 30 sec. The 420-bp product was checked using the restriction enzymes BamHI and HaeIII.

Western blot analysis
After being flushed from the BM cavity, the cells were resuspended in either SDS-sample buffer for total protein or in lysis buffer for plasma membrane containing protease inhibitors (Boehringer Mannheim, East Sussex, UK). The former was boiled and collected after centrifugation. In the case of the latter, the cells were left to swell for 15 min on ice, after which they were homogenized using a Dounce tissue homogenizer and centrifuged at 1,000 x g for 10 min to remove nuclear and cellular debris. The supernatant was overlaid on a 35% sucrose solution and centrifuged for 60 min at 20,000 x g. The plasma membranes found in a single band at the interface were collected, resuspended in Tris-sucrose buffer [10 mM Tris-HCl (pH 7.4), 250 mM sucrose], and centrifuged for 60 min at 100,000 x g. The pellets were then boiled and collected in the same way as for total protein. The amount of protein extracted was determined by spectrophotometry, using a BCA protein assay kit (Pierce Chemical Co., Rockford, IL), and was frozen until required.

Fifty micrograms of protein (total or plasma membrane) were loaded per lane, along with prestained protein markers (New England Biolabs, Inc., Hertfordshire, UK), electrophoresed on a 10% SDS polyacrylamide gel at 8 V/cm, and electroblotted onto an Immobilon-P polyvinylidene difluoride membrane using a semidry electroblotter. Incubation was carried out with antirat M-CSF antiserum (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 4 C, overnight, in TBS-T buffer [20 mM Tris, 137 mM NaCl (pH 7.6) and 0.1% Tween 20] with 3% nonfat milk. After washing, blots were incubated with a peroxidase-labeled rabbit antigoat antiserum for 60 min and were developed using enhanced chemiluminescence with ECL hyperfilm (Amersham).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Sham-operated animals were significantly lighter than ovariectomized rats in all but the 3-day experiment, despite being pair-fed (data not shown). There was no difference in the body weights of the control and sham-operated animals. Uterine atrophy occurred in ovariectomized rats not treated with estrogen, at all times points except at 3 days post ovariectomy. Data derived from animals killed 180 days post ovariectomy have previously been published (6). In brief, this experiment showed that ovariectomy caused uterine atrophy and weight gain. The former was increased to a minor degree in response to androstenedione, an effect which was abrogated by Anastrozole (Arimidex), an aromatase inhibitor; the latter was not altered by androstenedione treatment.

Treatment with 10 and 20 µg 17ß-E2 daily to ovariectomized rats maintained uterine weight at the level of the sham animals, whereas 5 µg failed to do so. Androstenedione did not affect the uterine weights in the ovariectomized rats treated with or without bicalutamide (Table 1Go). Plasma levels of 17ß-E2 in the ovariectomized rats were significantly reduced, compared with the intact animals, at all time points (Table 2Go).


View this table:
[in this window]
[in a new window]
  		Table 1. Uterine weights (mg) from rats after various time points post ovariectomy, in response to treatment with 17ß-estradiol and androstenedione (adione)

 

View this table:
[in this window]
[in a new window]
  		Table 2. Plasma 17ß-E2 (E2) (pg/ml) in rats at various days post ovariectomy (Ovx)

 
We have found that the 1.4-kb M-CSF mRNA is not detected in the BM of rats with intact ovaries when assessed by Northern blot analyses, although it was present when assessed by RT-PCR (data not shown). In contrast, the 4.6-kb transcript is expressed in abundance in this hormonally replete state (Figs. 2Go, 3Go, and 4Go). The expression of these transcripts changed differentially as a result of ovariectomy, and this was detected as early as 3 days post ovariectomy (Fig. 2Go). Restoration of 17ß-E2 plasma levels to those in the sham-operated rats was brought about in response to 5 µg/kg·day of 17ß-E2; and this treatment was sufficient to suppress completely the detectable expression of the 1.4-kb transcript, and it virtually restored the expression of the 4.6-kb transcript to that in the intact animals (Fig. 2Go). The Western blot showed that there was no parallel reduction in expression of the soluble 43-kDa protein (Fig. 3Go). 17ß-E2 (5 µg/kg·day) also largely prevented the cancellous bone loss in the ovariectomized rats (Fig. 2Go); and therefore, further experiments were performed using this treatment.



View larger version (43K):
[in this window]
[in a new window]
  		Figure 2. Effect of increasing concentrations of 17ß-E2 on M-CSF mRNA expression and percentage of BV/TV in ovariectomized rats. A, Northern blot hybridization on total RNA from sham and ovariectomized rats, the latter treated with increasing amounts of 17ß-E2. The cDNA probe for M-CSF is described schematically in Fig. 1Go. B, Cancellous bone volume was determined at the proximal tibial metaphysis in the same animals. The rats were 13 weeks old when surgery was performed. The stated treatments were commenced on the day of surgery (day 1) and were given daily for 19 days, when the experiment was terminated by killing the animals. C, Northern blot analysis on total RNA from sham and ovariectomized rats, the latter treated with or without 17ß-E2. The rats were 11 weeks old when surgery was performed; 17ß-E2 or vehicle treatment was commenced on the day of surgery (day 1) and was continued for 3 days, when the experiment was terminated by killing the animals. In both experiments, n = 4/group. S, Sham; V, vehicle; Ovx, ovariectomy; E2, 17ß-E2.

 


View larger version (37K):
[in this window]
[in a new window]
  		Figure 3. The effect of 17ß-E2 and androgens on M-CSF gene expression. A, Northern blot was performed for M-CSF gene expression on total RNA from BM of control, sham-operated, and ovariectomized rats, which received treatments as indicated in the figure; B, Western blot analysis on total BM protein, from the opposite tibiae in the same animals, using the anti-M-CSF antibody, which identifies both the 43-soluble and the 22-kDa membrane-bound monomers of M-CSF. Surgery was performed on day 1, when the animals were 8 weeks old. The stated treatments were commenced on day 1 and continued for 7 days, when the experiment was terminated by killing the animals. n = 4/group. C, Control; P, placebo; A, androstenedione.

 


View larger version (44K):
[in this window]
[in a new window]
  		Figure 4. The effect of androgens and the antiandrogen, bicalutamide, on M-CSF gene expression. A, Northern blot analysis for M-CSF on total RNA from BM of control, sham-operated, and ovariectomized rats, which received various treatments as indicted in the figure. Surgery was performed on day 1, when the rats were 8 weeks old. Treatment was commenced 14 days after surgery and continued for 7 days, after which the experiment was terminated by killing the animals (21 days post surgery). n = 4/group. Bic, Bicalutamide. B, Northern blot analysis for M-CSF on BM from sham-operated and ovariectomized rats. Surgery was performed on day 1, when the rats were 13 weeks old; and the experiment was terminated 180 days later, when the animals were killed. Slow-release androstenedione and placebo pellets were inserted at the time of surgery, and a second pellet was inserted 90 days post surgery. n = 4/group.

 
As previously demonstrated (5), androstenedione plasma levels were significantly reduced post ovariectomy, and androstenedione treatment restored the plasma levels to just below that in the sham-operated rats (Table 3Go). Furthermore, androstenedione treatment of ovariectomized rats had the same effect on the expression of M-CSF mRNA in the BM as that induced by 17ß-E2 (Figs. 3Go and 4Go). The possibility that the androstenedione-mediated effect occurred as a result of peripheral conversion of androgens to estrogens was excluded because the suppression of the 1.4-kb M-CSF transcript did not occur in the presence of bicalutamide, an antiandrogen (Fig. 4Go). This is consistent with our report that P450 aromatase transcripts failed to be detected in bone or BM of female rats or humans, which were either estrogen replete or deficient (29). Figure 4Go also showed that the suppression of the membrane form of M-CSF mRNA in the ovariectomized rats, in response to either estrogen or androgen treatment, occurred when hormone treatment was delayed 14 days post ovariectomy (Fig. 4Go). Furthermore, the androstenedione-mediated effect was maintained for 180 days post ovariectomy (Fig. 4Go). Using the same animals, we have previously shown that androstenedione treatment significantly protected against cancellous bone loss post ovariectomy at this time point and that this skeletal-protective effect was attributed to suppression of bone turnover (5).


View this table:
[in this window]
[in a new window]
  		Table 3. Plasma androstenedione (adione) (ng/ml) in rats at 7 and 21 days post ovariectomy (Ovx)

 
The 22-kDa membrane bound M-CSF isoform could only be reproducibly detected when BM protein was enriched for plasma membranes. In a separate experiment, we demonstrated that the BM of ovariectomized rats expressed higher levels of the 22-kDa protein, compared with that of sham-operated animals, and that this increase was suppressed in response to 5 µg/kg·day of 17ß-E2 (Fig. 5Go).



View larger version (28K):
[in this window]
[in a new window]
  		Figure 5. The effect of estrogen status on the expression of membrane-bound M-CSF. Western blot analysis for M-CSF on plasma membrane protein from sham-operated and ovariectomized rat BM. Plasma membranes were partially purified from tibial BM of sham-operated and ovariectomized rats and immunoblotted with the same anti-M-CSF antibody used in Fig. 3Go. The animals were 8 weeks old at the time of surgery, which was performed on day 1. The stated treatments were commenced 14 days post ovariectomy and continued for a further 14 days, after which time (28 days after surgery), the experiment was terminated by killing the animals.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
M-CSF plays a critical role in osteoclast formation (22, 25, 26, 30, 31, 32, 33, 34); and although several splice variants of this gene exist (35), it is not known which of the various M-CSF isoforms exert an effect on osteoclast formation in vivo. Soluble M-CSF is capable of inducing osteoclast formation (25, 27, 31, 32, 33, 36), but there is growing evidence that the membrane-bound form of M-CSF is also implicated in this process (22, 37). However, it was Yao et al. (28) who convincingly demonstrated that this isoform is involved in osteoclast in vitro formation and that osteotropic hormones/cytokines induced its expression.

We report here, for the first time, that the 4.6-kb and 1.4- kb M-CSF transcripts are differentially expressed in the presence of gender hormones in vivo, which suggests that they exert different physiological effects. We have identified that the membrane-bound form of the protein is increased in estrogen and androgen-deficient rats, conditions which are associated with high bone turnover (2, 3, 6, 38). The finding that the 1.4-kb M-CSF transcript in the BM is only detected by RT-PCR indicates that relatively low levels of the mRNA are required in low bone turnover states. Because we have previously reported that 17ß-E2 suppresses the expression of the human BM 1.6-kb M-CSF transcript and relevant protein in our osteoclast-inductive in vitro assay (22), the current data substantiate our in vitro findings, and they indicate that the regulation of M-CSF mRNA expression in the presence of gender hormones is similar in both species. This study implicates increased expression of the membrane form of M-CSF as a mechanism by which osteoclast formation and BR occur in gender-hormone-deficient states.

This study does not investigate whether gender hormones mediate their effect directly or indirectly on M-CSF expression, nor does it identify the cell type through which the effect is exerted. However, because it is known that monocytes/macrophages produce M-CSF (39), it seems likely that the increase in this cell population, which occurs after ovariectomy (40, 41), largely accounts for the increase in M-CSF production in our experiments, and administration of estrogen or androstenedione prevents this. The alternative is that the enhanced expression of membrane-bound M-CSF is by the nonhemopoietic cell population (stromal/fibroblastic/osteoblastic BM cells); this is thought unlikely because these cells are in a minority, compared with the monocyte/macrophage population in the BM (Ref. 39 and references therein). These cells could be a contributory factor, however, and the recent report showing that membrane-bound M-CSF is regulated by dexamethasone, another steroid hormone involved in osteoclast formation, in ST2 osteoclast-supportive murine osteoblast-like cells (42) suggests that this is a possibility. Because M-CSF is critical in osteoclast formation, the finding that the membrane-bound form is specifically increased in gender-hormone deficiency, a condition associated with elevated osteoclast formation and BR, suggests that the production of this isoform is intricately involved in this process, irrespective of the cell type that produces this form of the protein in the BM. Further work is required to investigate this question.

There is now good evidence that the osteoclast derives from a cell with a monocyte/macrophage phenotype (33, 43, 44, 45, 46). Therefore, the expansion of the monocyte/macrophage BM population after ovariectomy (40, 41) provides an explanation for the increase in osteoclast population in gender-hormone-deficient states. The increase in the monocyte population post ovariectomy may therefore also account for the reported increase in the inflammatory cytokines, including IL-1{alpha}, TNF{alpha} (47), and IL-6 (48), which occur in estrogen-deficient states and which have been previously implicated in estrogen-deficiency-associated bone loss. Many of these ubiquitous pleiotropic cytokines exert an autocrine and paracrine effect on the monocyte/macrophage population; however, it is impossible to elucidate further, in this in vivo model, how these interact.

Factors other than M-CSF are clearly required to maintain bone volume in androgen-deficient states. This is apparent because, in our previously reported 180-day histomorphometric skeletal analysis of ovariectomized rats (in response to androstenedione), we found that, despite plasma levels of androstenedione and testosterone being restored in the ovariectomized rats to sham-ovariectomy levels, androgens offered a skeletal-protective effect that (although significant) was incomplete (see Ref. 6). In contrast, the expression of the BM 1.4-kb M-CSF transcript from those same animals, reported in this study, was completely restored to that in the sham-ovariectomized rat. We do not know, from our current study, whether the same holds true for estrogen; because, although we prevented bone loss by maintaining physiological plasma levels of estrogen in the ovariectomized rats, perhaps lower estrogen levels would fail to do so. However, it is possible that lower levels of estrogen are capable of maintaining the expression of the BM 1.4-kb M-CSF transcript at the same level as in the sham-operated rats. Alternatively, the mechanism by which the gender hormones exert their effect on the expression of M-CSF may differ.

Contrary to our data, the soluble form of M-CSF has been found by others (21) to be suppressed in the presence of estrogen. However, we were not surprised by these results, because we had previously noted that the expression of the soluble form of M-CSF was not altered, in response to 17ß-E2, in our human BM osteoclast cultures, at the time when estrogen exerted its effect on BR (22). However, we now postulate that it is the proteoglycan form of M-CSF that is suppressed in BM in the presence of estrogen, because this isoform of M-CSF was differentially expressed, compared with the membrane form, in response to estrogen, in our human osteoclast-forming assay (22). This isoform results from cotranslational glycosylation of the 4.6-kb full-length M-CSF transcript, which also produces the soluble form (glycoprotein) (43 kDa) (49). This theory cannot be tested, at present, because an antibody against the murine proteoglycan M-CSF is not available.

The menopause is associated with a dramatic change in the gender-hormone status of women; and, as a consequence, skeletal metabolism and the lipid profile alter, so that women are at increased risk of developing osteoporosis and cardiovascular disease. The importance of M-CSF in the formation of, and protection against, atheroma is well documented (50, 51, 52, 53); and it is possible that the proteoglycan form is involved in this process. It is therefore interesting to speculate that both these diseases, which are prone to develop in the presence of estrogen deficiency, are (at least partially) caused by altered regulation of the M-CSF gene, although via different splice variants.


   Footnotes
 
1 This work was supported by The Arthritis and Rheumatism Council, UK. Back

Received June 1, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Turner RT, Riggs BL, Spelsberg TC 1994 Skeletal effects of estrogen. Endocr Rev 15:275–300[CrossRef][Medline]
  2. Wronski TJ, Cintron M, Doherty AL, Dann LM 1988 Estrogen treatment prevents osteopenia and depresses bone turnover in ovariectomized rats. Endocrinology 123:681–686[Abstract]
  3. Wronski TJ, Cintron M, Dann LM 1988 Temporal relationship between bone loss and increased bone turnover in ovariectomized rats. Calcif Tissue Int 43:179–183[Medline]
  4. Raisz LG, Wiita B, Artis A, Bowen A, Schwartz S, Trahiotis M, Shoukri K, Smith J 1996 Comparison of the effects of estrogen alone and estrogen plus androgen on biochemical markers of bone formation and resorption in postmenopausal women. J Clin Endocrinol Metab 81:37–43[Abstract]
  5. Lea CK, Flanagan AM 1998 Physiological plasma levels of androgens reduce bone loss in the ovariectomized rat. Am J Physiol 274:E328–E335
  6. Lea CK, Moxham V, Reed MJ, Flanagan AM 1998 Androstenedione treatment reduces loss of cancellous bone volume in ovariectomised rats in a dose-responsive manner and the effect is not mediated by oestrogens. J Endocrinol 156:331–339[Abstract]
  7. Kitazawa R, Kimble R, Vannice JL, Kung VT, Pacifici R 1994 Interleukin 1-receptor antagonist and tumor necrosis binding protein decrease osteoclast formation and bone resorption in ovariectomised rats. J Clin Invest 94:2397–2406
  8. Kimble RB, Matayoshi AB, Vannice JL, Kung VT, Williams C, Pacifici R 1995 Simultaneous block of interleukin-1 and tumor necrosis factor is required to completely prevent bone loss in the early postovariectomy period. Endocrinology 136:3054–3061[Abstract]
  9. Rifas L, Kenney JS, Marcelli M, Pacifici R, Cheng SL, Daeson LL, Avioli LV 1995 Production of interleukin-6 in human osteoblasts and human bone marrow stromal cells: evidence that induction by interleukin-1 and tumour necrosis factor-alpha is not regulated by ovarian steroids. Endocrinology 136:4056–4067[Abstract]
  10. Ammann P, Rizzoli R, Bonjour J-P, Bourrin S, Meyer J-M, Vassalli P, Garcia I 1997 Transgenic mice expressing soluble tumour necrosis factor-receptor are protected against bone loss caused by estrogen deficiency. J Clin Invest 99:1699–1703[Medline]
  11. Kimble RB, Bain S, Pacifici R 1997 The functional block of TNF but not IL-6 prevents bone loss in ovariectomized mice. J Bone Miner Res 12:935–941[CrossRef][Medline]
  12. Lorenzo J, Naprta A, Rao Y, Alander C, Glaccum M, Widmer M, Gronowicz J, Kalinowski J, Pilbeam C 1998 Mice lacking the type 1 interleukin-1 receptor do not lose bone mass after ovariectomy. Endocrinology 139:3022–3025[Abstract/Free Full Text]
  13. Kawaguchi H, Pilbeam CC, Vargas SJ, Morse EE, Lorenzo JA, Raisz LG 1995 Ovariectomy enhances and estrogen replacement inhibits the activity of bone marrow factors that stimulate prostaglandin production in cultured mouse calvariae. J Clin Invest 96:539–548
  14. 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 257:88–91[Abstract/Free Full Text]
  15. Girasole G, Jilka RL, Passeri G, Boswell S, Boder G, Williams DC, Manolagas SC 1992 17ß-estradiol inhibits interleukin-6 production by bone marrow-derived stromal cells and osteoblasts in vitro: a potential mechanism for the antiosteoporotic effect of estrogens. J Clin Invest 89:883–891
  16. Passeri G, Girasole G, Jilka RL, Manolagas SC 1993 Increased interleukin-6 production by murine bone marrow and bone cells after estrogen withdrawal. Endocrinology 133:822–828[Abstract]
  17. Miyaura A, Kusano K, Masuzawa T, Chaki O, Onoe Y, Aoyagi M, Sasaki T, Tamura T, Koishihara Y, Ohsugi Y, Suda T 1995 Endogenous bone resorbing factors in estrogen deficiency: cooperative effects of IL-1 and IL-6. J Bone Miner Res 10:1365–1373[Medline]
  18. Most W, Schot L, Edervees A, van der WeepPals L, Papapoulos S, Lowik C 1995 In vitro and ex vivo evidence that estrogens suppress increased bone resorption induced by ovariectomy or PTH stimulation through and effect on osteoclastogenesis. J Bone Miner Res 10:1523–1530[Medline]
  19. Finkelman RD, Bell NH, Strong DD, Demers LM, Baylink DJ 1992 Ovariectomy selectively reduces the concentration of transforming growth factor ß in rat bone: implications for estrogen deficiency-associated bone loss. Proc Natl Acad Sci USA 89:12190–12193[Abstract/Free Full Text]
  20. Hughes DE, Dai A, Tiffee JC, Li HH, Mundy GR, Boyce BF 1996 Estrogen promotes apoptosis of murine osteoclasts mediated by TGF-ß. Nat Med 2:1132–1136[CrossRef][Medline]
  21. Kimble RB, Srivastava S, Ross FP, Matayoshi A, Pacifici R 1996 Estrogen deficiency increases the ability of stromal cells to support murine osteoclastogenesis via an interleukin-1- and tumor necrosis factor-mediated stimulation of macrophage colony-stimulating factor production. J Biol Chem 271:28890–28897[Abstract/Free Full Text]
  22. Sarma U, Edwards M, Motoyoshi K, Flanagan AM 1998 17ß-Estradiol inhibits human osteoclast formation in vitro. J Cell Physiol 175:99–108[CrossRef][Medline]
  23. Van Bezooijen RL, Farih-Sips HCM, Papapoulos SE, Lowick CWGM 1998 IL-1{alpha}, IL-1ß, IL-6, and TNF-{alpha} steady-state mRNA levels analyzed by reverse transcription-competitive PCR in bone marrow of gonadectomized mice. J Bone Miner Res 13:185–194[CrossRef][Medline]
  24. Vargas SJ, Naprta A, Lee S-K, Kalinowski H, Kawaguchi H, Pilbeam CC, Raisz LG, Lorenzo JA 1996 Lack of evidence for an increase in interleukin-6 expression in adult murine bone, bone-marrow, and marrow stromal cell cultures after ovariectomy. J Bone Miner Res 11:1926–1934[Medline]
  25. Felix R, Cecchini MG, Fleisch H 1990 Macrophage colony stimulating factor restores in vivo bone resorption in the op/op osteopetrotic mouse. Endocrinology 127:2592–2594[Abstract]
  26. Wiktor-Jedrzejczak W, Bartocci A, Ferrante Jr AW, Ahmed-Ansari A, Sell KW, Pollard JW, Stanley ER 1990 Total absence of colony-stimulating factor 1 in the macrophage-deficient osteopetrotic (op/op) mouse. Proc Natl Acad Sci USA 87:4828–4832[Abstract/Free Full Text]
  27. Takahashi N, Udagawa N, Akatsu T, Tanaka H, Isogai Y, Suda T 1991 Deficiency of osteoclasts in osteopetrotic mice is due to a defect in the local microenvironment provided by osteoblastic cells. Endocrinology 128:1792–1796[Abstract]
  28. Yao G-Q, Sun B-h, Hammond EE, Spencer EN, Horowitz MC, Insogna KL, Weir EC 1998 The cell-surface form of colony-stimulating factor-1 is regulated by osteotropic agents and supports formation of multinucleated osteoclast-like cells. J Biol Chem 273:4119–4128[Abstract/Free Full Text]
  29. Lea CK, Ebrahim H, Tennent S, Flanagan AM 1997 Aromatase cytochrome transcripts are detected in fractured human bone but not in normal skeletal tissue. Bone 21:433–440[Medline]
  30. Yoshida H, Hayashi S-I, Kunisada T, Ogawa M, Nishikawa S, Okamura H, Sudo T, Shultz LD, Nishikawa S-I 1990 The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene. Nature 345:442–444[CrossRef][Medline]
  31. Kodama H, Yamasaki A, Nose M, Niida S, Ohgame Y, Abe M, Kumegawa M, Suda T 1991 Congenital osteoclast deficiency in osteopetrotic (op/op) mice is cured by injections of macrophage colony-stimulating factor. J Exp Med 173:269–272[Abstract/Free Full Text]
  32. Sarma U, Flanagan AM 1996 Macrophage-colony stimulating factor (M-CSF) induces substantial osteoclast formation in human bone-marrow cultures. Blood 88:2531–2540[Abstract/Free Full Text]
  33. Fujikawa Y, Quinn JMW, Sabokbar A, McGee JOD, Athanasou NA 1996 The human osteoclast precursor circulates in the monocyte fraction. Endocrinology 137:4058–4060[Abstract]
  34. Lader CS, Flanagan AM 1998 Prostaglandin E2, interleukin 1{alpha} and tumor necrosis factor {alpha} increase human osteoclast formation and bone resorption in vitro. Endocrinology 139:3157–3164[Abstract/Free Full Text]
  35. Rajavashisth TB, Eng R, Shadduck RK, Waheed A, Ben-Avram CM, Shively JE, Lusis AJ 1987 Cloning and tissue-specific expression of mouse macrophage colony-stimulating factor mRNA. Proc Natl Acad Sci USA 84:1157–1161[Abstract/Free Full Text]
  36. Hattersley G, Owens J, Flanagan AM, Chambers TJ 1991 Macrophage colony stimulating factor (M-CSF) is essential for osteoclast formation in vitro. Biochem Biophys Res Commun 177:526–531[CrossRef][Medline]
  37. Sundquist KT, Cecchini MG, Marks SC 1995 Colony-stimulating factor-1 injections improve but do not cure skeletal sclerosis in the osteopetrotic (op) mouse. Bone 16:39–46[Medline]
  38. Christiansen C, Christiansen MS, Larsen N-E, Transbol I 1982 Pathophysiological mechanism of estrogen effect on bone metabolism: dose-response relationship in early postmenopausal women. J Clin Endocrinol Metab 55:1124–1130[Abstract]
  39. Temeles DS, McGrath HE, Kittler ELW, Shadduck RK, Kister VK, Crittenden RB, Turner B, Quesenberry PJ 1993 Cytokine expression from bone marrow derived macrophages. Exp Hematol 21:388–393[Medline]
  40. Jilka RL, Girasole G, Cooper S, Abrams J, Broxmeyer H 1995 Estrogen loss upregulates hematopoiesis in the mouse: a mediating role of IL-6. Exp Hematol 23:500–506[Medline]
  41. Erben RG, Raith S, Eberle J, Stangassinger M 1998 Ovariectomy augments B lymphopoiesis and generation of monocyte-macrophage precursors in rat bone marrow. Am J Physiol 274:E476–E483
  42. Rubin J, Biskobing DM, Jadhav L, Fan D, Nanes MS, Perkins S, Fan X 1998 Dexamethasone promotes expression of membrane-bound macrophage colony-stimulating factor in murine osteoblast-like cells. Endocrinology 139:1006–1012[Abstract/Free Full Text]
  43. Takahashi N, Udagawa N, Tanaka S, Murakami H, Owan I, Tamura T, Suda T 1994 Postmitotic osteoclast precursors are mononuclear cells which express macrophage-associated phenotypes. Dev Biol 163:212–221[CrossRef][Medline]
  44. Udagawa N, Takahashi N, Akatsu T, Tanaka H, Sasaki T, Nishihara T, Koga T, Martin TJ, Suda T 1990 Origin of osteoclasts: mature monocytes and macrophages are capable of differentiating into osteoclasts under a suitable microenvironment prepared by bone marrow-derived stromal cells. Proc Natl Acad Sci USA 87:7260–7264[Abstract/Free Full Text]
  45. Kerby JA, Hattersley G, Collins DA, Chambers TJ 1992 Derivation of osteoclasts from hematopoietic colony-forming cells in culture. J Bone Miner Res 7:353–362[Medline]
  46. Massey H, Rae A, Horton MA, Flanagan AM 1998 Characterisation of the human osteoclast phenotype. J Bone Miner Res 13:526
  47. Pacifici R 1996 Estrogen, cytokines and pathogenesis of postmenopausal osteoporosis. J Bone Miner Res 11:1043–1051[Medline]
  48. Manolagas SC, Jilka RL 1995 Bone marrow, cytokines, and bone remodeling. New Engl J Med 332:305–311[Free Full Text]
  49. Stanley ER 1994 The Cytokine Handbook, 2nd ed. Academic Press, Harcourt Brace & Company, London, pp 387–418
  50. Qiao J-H, Tripathi J, Mishra NK, Cai Y, Tripathi S, Wang X-P, Imes S, Fishbein MC, Clinton SK, Libby P, Lusis AJ, Rajavashisth TB 1997 Role of macrophage colony-stimulating factor in atherosclerosis. Am J Pathol 150:1687–1699[Abstract]
  51. Smith JD, Trogan E, Ginsberg M, Grigaux C, Tian J, Miyata M 1995 Decreased atherosclerosis in mice deficient in both macrophage colony-stimulating factor (op) and apolipoprotein E. Proc Natl Acad Sci USA 92:8264–8268[Abstract/Free Full Text]
  52. Flanagan AM, Lader CS 1998 Update on the biological effects of macrophage colony-stimulating factor. Curr Opin Hematol 5:181–185[Medline]
  53. Hayes-Donnelly L, Bree MP, Hunter SE, Keith JC, Schaub RG 1997 Immunoreactive macrophage colony-stimulating factor is increased in atherosclerotic lesions of Watanabe heritable hyperlipidemic rabbits after recombinant human macrophage colony-stimulating factor therapy. Mol Reprod Dev 46:92–95[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J. Appl. Physiol.Home page
C. P. Schneider, M. G. Schwacha, and I. H. Chaudry
Impact of sex and age on bone marrow immune responses in a murine model of trauma-hemorrhage
J Appl Physiol, January 1, 2007; 102(1): 113 - 121.
[Abstract] [Full Text] [PDF]

Home page
 J. Dent. Res.Home page
U.H. Lerner
Bone Remodeling in Post-menopausal Osteoporosis.
J. Dent. Res., July 1, 2006; 85(7): 584 - 595.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
T. M. Doherty, L. A. Fitzpatrick, D. Inoue, J.-H. Qiao, M. C. Fishbein, R. C. Detrano, P. K. Shah, and T. B. Rajavashisth
Molecular, Endocrine, and Genetic Mechanisms of Arterial Calcification
Endocr. Rev., August 1, 2004; 25(4): 629 - 672.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
E. F. Verdu, Y. Deng, P. Bercik, and S. M. Collins
Modulatory effects of estrogen in two murine models of experimental colitis
Am J Physiol Gastrointest Liver Physiol, July 1, 2002; 283(1): G27 - G36.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
J. Pfeilschifter, R. Koditz, M. Pfohl, and H. Schatz
Changes in Proinflammatory Cytokine Activity after Menopause
Endocr. Rev., February 1, 2002; 23(1): 90 - 119.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
X. Fan, D. Fan, H. Gewant, C. L. Royce, M. S. Nanes, and J. Rubin
Increasing membrane-bound MCSF does not enhance OPGL-driven osteoclastogenesis from marrow cells
Am J Physiol Endocrinol Metab, January 1, 2001; 280(1): E103 - E111.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
J. Rubin, T. Murphy, M. S. Nanes, and X. Fan
Mechanical strain inhibits expression of osteoclast differentiation factor by murine stromal cells
Am J Physiol Cell Physiol, June 1, 2000; 278(6): C1126 - C1132.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. Katagiri, Y. Hakeda, D. Chikazu, T. Ogasawara, T. Takato, M. Kumegawa, K. Nakamura, and H. Kawaguchi
Mechanism of Stimulation of Osteoclastic Bone Resorption through Gas6/Tyro 3, a Receptor Tyrosine Kinase Signaling, in Mouse Osteoclasts
J. Biol. Chem., March 2, 2001; 276(10): 7376 - 7382.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Srivastava, G. Toraldo, M. N. Weitzmann, S. Cenci, F. P. Ross, and R. Pacifici
Estrogen Decreases Osteoclast Formation by Down-regulating Receptor Activator of NF-kappa B Ligand (RANKL)-induced JNK Activation
J. Biol. Chem., March 16, 2001; 276(12): 8836 - 8840.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lea, C. K.
Right arrow Articles by Flanagan, A. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lea, C. K.
Right arrow Articles by Flanagan, A. M.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*ESTRADIOL

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals