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Gene Regulation, Bone and Inflammation Research Division (Y.L.M., R.L.C., D.L.H., X.Y., Q.Z., S.C., T.J.M., J.E.O.), Lilly Research Laboratories, Eli Lilly & Co., Indianapolis, Indiana 46285; and St. Vincent’s Institute of Medical Research (T.J.M.), Fitzroy, Victoria 3065, Australia
Address all correspondence and request for reprints to: Dr. Yanfei. L. Ma, Gene Regulation, Bone and Inflammation Research Division, Building 98C/B, DC 0403, Lilly Research Labs, Indianapolis, Indiana 46285. E-mail: ma_linda{at}lilly.com
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Introduction |
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The recent discoveries of members of the TNF receptor and TNF ligand families (OPG and RANKL) as powerful influences on osteoclast formation and activity have generated great interest in their role and regulation by calciotropic agents such as PTH. OPG is a TNF receptor family member that inhibits osteoclast formation at a late stage of its development (22, 23, 24, 25, 26, 27). Overexpression of OPG in transgenic mice resulted in osteopetrosis because of failure of osteoclast formation. Targeted deletion of the OPG gene resulted in severe, early-onset osteopenia (28). Furthermore, treatment of ovariectomized rats with OPG prevented bone loss (23). This same molecule was identified independently and called osteoclastogenesis inhibitory factor (22, 29). The ligand for OPG has been identified as osteoclast differentiation factor, also known as RANKL, or OPG ligand, a member of the TNF ligand family (24, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41). It is expressed on the osteoblast/stromal cell surface and promotes osteoclast formation in the presence of macrophage colony–stimulating factor and without any accompanying stromal/osteoblastic cells (24, 40). In vitro studies show that the mRNA levels of both RANKL and OPG in osteoblasts/stromal cells are influenced by factors such as cytokines that increase bone resorption (42, 43, 44, 45, 46, 47). However, it is not yet clear that OPG and RANKL expression are regulated in vivo by relevant bone active factors to stimulate bone resorption.
In the present study, we examined the role of OPG and RANKL in bone resorption induced by continuous infusion of PTH 1–38 in parathyroidectomized (PX) rats. PX animals were used to eliminate the confounding effects of endogenous PTH, and it is a well-established bone resorption model. We propose that the catabolic effects of PTH and the net decrease in bone mass after PTH infusion occur via rapid but sustained regulation of OPG and RANKL and a decrease in the osteoblast phenotype and bone formation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Serum-circulating OPG
Serum OPG level was detected by "Sandwich" ELISA with rabbit
polyclonal anti-hOPG IgG. Briefly, 96-well plates (DYNEX Tech., IMMULON 4HBX, Chantilly, VA) were coated with
0.5 µg/well of purified rabbit anti-OPG antibody (IgG) diluted in 100
µl of carbonate/bicarbonate buffer (Sigma, St. Louis, MO
C-3041), 4 C overnight, and blocked with 200 µl/well of blocker
casein in PBS (Pierce Chemical Co., Rockford, IL)
for 1 h at room temperature. The plate was incubated with 100 µl
of serum diluted in 1% BSA-TPBS for 1 h and three washes with
TPBS. To each well was added 100 µl of diluted biotinylated anti-OPG
IgG for 1 h and followed by 100 µl/well of Streptoavidin-HRP
conjugate (Zymed Laboratories, Inc., San Francisco,
CA) for 30 min with TPBS washing. ELAST ELISA amplification system
(NEN Life Science Products, Boston, MA) was applied before
color development according to the manufacturer’s instruction. Color
was developed with 100 µl/well of 3,3',5,5'-tetramethylbenzidine
substrate for 15 min and stopped with 100 µl/well of 1 N
phosphoric acid with reading OD at A450nm.
Quantitation of osteoclast number
Proximal tibiae were fixed in 10% buffered formalin for 2 days,
decalcified in decalcifier II (Surgipath, Richmond, IL)
for 3 days, and processed for embedding in paraffin. Longitudinal cut,
5-µm-thick, decalcified sections were stained by 0.1% Toluidine blue
(Sigma). Osteoclast number was measured on the entire
marrow region within the cortical shell between 0.67 and 2 mm distal to
the growth plate metaphyseal junction under 20x magnification.
Trabecular bone surface was measured by using a digitizing image
analyzing system named Osteomeasure (OsteoMetrics, Inc.,
Atlanta, GA). The osteoclast numbers were then normalized to trabecular
bone surface (48).
Isolation of poly A+RNA and Northern blotting
The mRNA expression was analyzed by Northern blotting. At
autopsy, femora were resected and all connective tissue, including
periosteum, completely removed. The distal epiphysis, including the
growth plate, was removed and a subjacent 3-mm-wide band of the
metaphyseal trabecular primary spongiosa was frozen in liquid nitrogen.
Bone samples were pooled into treated or control groups for each
indicated time point. Total RNA was extracted from bone by
homogenization in Ultraspec-II using an LS 10–35 Polytron homogenizer
(Brinkmann Instruments, Inc., Westbury, NY) as recommended
by the manufacturer. Poly A+ RNA was isolated from total RNA using
Oligotex (QIAGEN, Santa Clarita, CA) according to the
manufacturer’s protocol and quantitated by spectrophotometry. The
absorbance at 260 nm was determined and the 260/280-nm absorbance ratio
was calculated to ensure the absence of protein contamination. Samples
of poly A+ RNA (2 µg) were denatured in 0.04 M
3-(N-morpholine) propanesulfonic acid, pH 7.0, 10
mM sodium acetate, 1 mM
EDTA, 2.2 M formaldehyde, and 50% formamide at
60 C for 10 min, and size fractionated by electrophoresis through 1%
agarose gels in 2.1 M formaldehyde and 1x
3-[N-morpholino]propanesulfonic and transferred to nylon
membranes (Brightstar-Plus, Ambion, Inc., Austin, TX). The
membranes were air dried and the RNA samples cross-linked to the nylon
membrane by UV irradiation in a Stratalinker (Stratagene,
La Jolla, CA). Migration of 28S and 18S ribosomal RNAs were determined
by ethidium bromide staining. DNA probes were labeled by the random
primer method (Life Technologies, Inc., Grand Island, NY)
using 
32P-dCTP. Prehybridization and
hybridization were carried out at 48 C in NorthernMax buffers
(Ambion, Inc.). After hybridization, membranes were washed
for 30 min at room temperature in buffer containing 2x sodium citrate
and 0.1% SDS, then 30 min at 48 C in 0.2x sodium citrate and exposed
to Biomax MS x-ray film (Kodak, Rochester, NY) at -70 C.
Autoradiograms were quantitated by scanning laser densitometry (2400
Gel Scan XL, LKB, Piscataway, NJ). Labeled bands were
quantitated as densitometric units and the data were expressed as
percent change vs. untreated control samples. The
experiments were repeated two to four times for each time point to
confirm findings.
Radiolabeled probes
The mouse RANKL cDNA was PCR cloned from cDNA derived from
BALC stromal osteoblasts using the following primer pair, 5'atc
aga aga cag cac tca ct 3' and 5'atc tag gac atc cat gct aat gtt c 3' as
published (42). Mouse cDNA probe for osteoblast specific
transcription factor (cbfa-1) was obtained from Dr. Gerard Karsenty
(Houston, TX). The other cDNA probes were cloned using PCR and specific
primer pairs for the respective genes as published previously
(14, 47, 49, 50). The cloned cDNAs were confirmed by
restriction enzyme mapping and/or sequencing.
OPG immunohistochemistry
Five-µm-thick proximal tibial sections were prepared as
described for osteoclast number measurement. Anti-hOPG serum used for
immunohistochemistry was generated by immunization of a rabbit with
purified monomeric human OPG. Immunohistochemical stains were performed
using the Vectastain ABC kit (Vector Laboratories, Inc.,
Burlingame, CA). Tissue sections were deparaffinized in two successive
10-min washes of xylene, followed by two washes in ethanol, and a brief
wash in PBS containing 0.1% Tween 20, pH 7.4. Sections were then
immersed in 0.3% hydrogen peroxide in absolute methanol for 30 min to
quench endogenous peroxidase activity. Before the addition of the
primary antibody, nonspecific tissue binding was blocked by incubating
the tissue section for 30 min at room temperature by 10% normal goat
serum in PBS for 30 min. The primary antibodies were applied and
incubated overnight at 4 C in a humid environment. The sections were
then washed three times in 0.1% Tween 20. The secondary biotinylated
antibody and the Streptavidin-HRP conjugate complex were applied in a
humidified chamber for 60 and 30 min, respectively. After washing in
buffer, the chromogen diaminobenzidine was applied for 5 min followed
by a counterstain with Mayer’s hematoxylin. Negative controls included
substituting the primary antisera with preimmune sera from the same
species and omitting the primary antibody. All controls revealed the
expected negative results.
Statistical analysis
Serum calcium, osteoclast number, and serum OPG levels were
presented as mean ± SEM. Raw data of group
differences were assessed by ANOVA using Fisher’s protected least
significant difference for which the significance level was
P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) and by quantitation
of osteoclast number (Fig. 1B). A 6-h PTH infusion resulted in a
dose-dependent increase in serum-ionized calcium level in PX rats (Fig. 1A). At PTH 20 µg/100 g per 6-h dose, serum ionized calcium was 205%
of the vehicle-treated PX rats (0.65 ± 0.02 mmol/liter
vs. 1.33 ± 0.03 mmol/liter) and was no different from
sham rats (1.36 ± 0.03 mmol/liter vs. 1.33 ±
0.03 mmol/liter). The increase in serum calcium was correlated with a
corresponding dose-dependent increase in osteoclast number, which was
up to 300% of the vehicle-treated PX rats and 175% of the sham rats
at a 20-µg dose, indicating increased bone resorption (Fig. 1B).
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As shown in Fig. 2, in the
controls (sham and PX vehicle treated), both OPG and RANKL mRNA was
detectable with OPG more readily than RANKL. Six-hour PTH infusion
resulted in a 3-fold decrease in OPG mRNA and a 27-fold increase in
RANKL mRNA. To determine the effects of lower doses of PTH, we next
examined the expression of OPG and RANKL after 6 h of continuous
infusion of lower doses (0.1–10 µg) of hPTH 1–38. As shown in the
autogram in Fig. 3A and plotted in
Fig. 3B, PTH infusion resulted in a dose-dependent decrease in OPG mRNA
(optimal 3-fold) and reciprocal increase in RANKL (5-fold at 10 µg of
PTH). The effects on both OPG and RANKL were detectable and
reproducible with infusion as low as 1 µg of PTH. To further
ascertain the kinetics of this effect on both OPG and RANKL, we
examined the expression of mRNA for both OPG and RANKL at 0, 1, 3, 6,
and 24 h after the start of hPTH1–38 (20 µg) infusion.
Treatment of animals with hPTH 1–38 (20 µg/100 g per 6 h, or
3.3 µg/100 g per hour) caused a rapid and sustained decrease in OPG
and reciprocal increase in RANKL mRNA. The inhibition of OPG expression
was evident within 1–6 h and was sustained up to 24 h (7.5-fold).
The stimulation of RANKL was detected at 3 h, optimal at 6 h,
and still elevated at 24 h (5.5-fold) (Fig. 4). We next compared this effect of
continuous PTH to acute exposure of PTH. In this experiment, intact
(non-PX) animals were given a single normal calcium dose sc as
previously described (2, 12, 47). In contrast to the
effects of continuous PTH infusion, acute exposure to PTH 1–38
resulted in a rapid but transient change in OPG and RANKL expression.
The decrease in OPG mRNA (3.4-fold) and increase of RANKL mRNA
(3.8-fold) were observed at 1 h, and they both recovered to near
the control levels by 3 h (Fig. 5).
These results were similar and reproducible in multiple
experiments.
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). In bone, OPG protein was detected
predominantly in preosteoblasts, mature osteoblasts, and some newly
formed bone matrix in the primary and secondary metaphyseal spongiosa.
As we have noted previously, many areas of specific
immunohistochemistry staining for OPG were seen in bone matrix (Fig. 7). In diaphyseal bone sections, OPG
staining was less intense than that seen in the metaphysis and was
localized only to endosteal osteoblasts and occasional marrow cells
(data not shown). No staining was observed in tissue sections in which
the primary antibody was omitted. Treatment with PTH infusion resulted
in weaker staining and in loss of matrix staining, indicating PTH
infusion decreased the OPG expression in bone (Fig. 7).
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, mRNA expression was analyzed. As shown in
Fig. 8, the expressions of genes that
mark the osteoblast phenotype and bone formation were drastically
decreased [cbfa-1 (2.3-fold), OC (3-fold), BSP (2.8-fold), and COL1A1
(5-fold)]. This result is consistent with a decrease in osteoblast
activity and bone formation after continuous PTH infusion.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, we demonstrate that the expression of RANKL and OPG are rapidly and robustly altered in a well-validated in vivo model of PTH-induced bone resorption. Our results indicate that resorptive effects of PTH were marked by an increase in osteoclast number and serum calcium levels. Infusion of PTH induced a rapid and sustained increase in RANKL mRNA and decrease in both OPG mRNA and protein in the osteoblasts. The changes in RANKL and OPG were dose and time dependent and preceded peak increases in bone resorption. Additionally, a decreased expression of genes associated with the bone formation-osteoblast phenotype was found. Similar findings have been reported in several in vitro systems (42, 51, 52). Our results provide further evidence that the regulation of these molecules by calciotropic agents such as PTH may be an important regulatory mechanism linking osteoblast and osteoclast. The immunohistochemical and Northern blot studies presented here and elsewhere clearly establish that osteoblasts produce OPG and RANKL and that both are regulated in vivo upon PTH infusion. The increase in RANKL and decrease in OPG confirmed that the true balance of RANKL and OPG is altered to favor bone resorption. Further, OPG levels were also decreased in serum, highlighting the extent and magnitude of PTH effects to favor bone resorption. This is consistent with an important regulatory role for these molecules in the initiation and maintenance of the catabolic effect of PTH in normal physiology of osteoclast differentiation.
Our results show that the expression and regulation of these molecules in bone fulfill their proposed role as local regulators of bone resorption. As shown by immunohistochemistry, OPG was expressed predominantly in osteoblasts and their precursors. These are the target cells regulated by calciotropic hormones to elicit a resorptive response. The consistent findings of OPG in the bone matrix might reflect its availability from this site to exercise control over osteoclast formation. In the control state (untreated) there was a strong constitutive expression of OPG and relatively much lower RANKL mRNA. This balance favoring increased OPG is consistent with low basal resorption in normal physiology in the mature animals. The rapid and sustained alteration in this balance after continuous PTH infusion to create an increased RANKL/OPG ratio clearly would favor increased resorption and decreased bone mass. It was also noted that OPG was lost from the matrix under this condition. Perhaps this sustained resorptive signal is required to ensure a catabolic effect of PTH infusion. In support of this premise, acute exposure to a single anabolic normal calcemia dose of PTH triggered a rapid but transient alteration in the RANKL/OPG ratio (47). The decrease in OPG mRNA and increase in RANKL mRNA was detected in 1 h but recovered to control levels by 3 h.
It is interesting to note that a continuous PTH infusion also resulted in down-regulation of various genes that are associated with the osteoblast bone formation phenotype in vivo, such as cbfa-1, OC, BSP, and COL1A1. The observation was in contrast with the findings of a single injection or intermittent administration of PTH, which resulted in an increased bone formation and up-regulation of these genes (11, 13, 14, 15, 21 ; Onyia et al., unpublished data). On the basis of the response to these two different regimens of PTH, we propose a role for OPG and RANKL in both anabolic and catabolic effects of PTH in rat bone. The differential responsiveness can be explained by the differences in the magnitude and duration of resorptive signaling. Continuous PTH results in enhanced resorption and decreased bone mineral density via pronounced and sustained increases in osteoclast formation and activity. On the other hand, PTH effects on formation may require a subtle and/or transient increase in osteoclast formation and activity, perhaps to prepare the bone surface for the deposition of new matrix (44, 47).
In conclusion, these results provide in vivo evidence that OPG and RANKL are important regulators of bone homeostasis in PTH action. Our results demonstrate that PTH concurrently inhibits production of the antiresorptive cytokine receptor OPG and stimulates the levels of the bone-resorbing cytokine RANKL. In continuous infusion of PTH, the changes in OPG and RANKL were rapid and sustained and preceded peak effects on bone resorption. We also demonstrated that the resorptive effect of continuous PTH is associated with decreased expression of osteoblast bone- formation genes. Taken together, these findings provide a potential molecular explanation for PTH effects on bone resorption.
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Acknowledgments |
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Footnotes |
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Received February 14, 2001.
Accepted for publication May 3, 2001.
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