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Musculoskeletal Disease Center (N.M., Y.K., T.A.L., D.J.B., S.M.), J.L. Pettis VA Medical Center, Loma Linda, California 92357; Departments of Medicine (D.J.B., S.M.), Biochemistry (T.A.L., S.M.), Physiology (S.M.), and Pediatrics (T.A.L.), Loma Linda University, Loma Linda, California 92350
Address all correspondence and requests for reprints to: Subburaman Mohan, Ph.D., Musculoskeletal Disease Center (151), JLP Veterans Affairs Medical Center, 11201 Benton Street, Loma Linda, California 92357. E-mail: mohans{at}lom.med.va.gov
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The osteoblasts are the primary target cells for the anabolic effects of PTH on bone tissue. PTH effects on osteoblasts are known to be mediated via binding of PTH to the seven membrane-spanning G protein–coupled receptor and activation of both the cAMP PKA pathway and the phosphoinositide PKC pathway (15). PTH treatment in mice has been shown to increase the life span of mature osteoblasts by preventing their apoptosis (14). PTH effects on bone formation may involve direct action of PTH on osteoblast lineage cells or indirect action via modulation of local growth factor production. Based on the finding that PTH treatment causes an acute increase in several potential second messenger signaling molecules, such as c-fos, MAPK, and cyclin-dependent kinase, which are implicated in cell proliferation, it has been proposed that PTH effects on osteoblasts are direct (16, 17, 18).
In addition to direct effects of PTH, it has been shown that PTH treatment increases production of IGF-I in rat and mouse osteoblasts in vitro (19, 20, 21) and rat osteoblasts in vivo (22). In vitro studies using IGF-I–blocking antibody have shown that IGF-I antibody prevented the PTH stimulation of collagen synthesis in serum-free fetal rat calvaria cultures (23). Consistent with these data, neutralizing antibody against IGF-I has been shown to block the stimulatory effect on alkaline phosphatase (ALP) activity and the expression of osteocalcin mRNA induced by intermittent exposure of PTH in osteoblastic cells isolated from rat calvaria (24). In addition, a neutralizing antibody against IGF-I also inhibited the PTH-stimulated aggrecan synthesis in rat chondrocytes (25). These findings, together with the finding that intermittent in vivo administration of PTH in rats increased IGF-I mRNA levels more than 2-fold in 40% of osteoblasts (22), suggest that the stimulatory effect on bone by PTH may be, at least in part, mediated by an enhancement in the local production of IGF-I. Consistent with these findings, Hock and Fonseca (26) have shown that the anabolic effect of PTH was blunted in young male hypophysectomized rats, which was restored upon GH administration. However, subsequent studies revealed that the anabolic effect of PTH might be less dependent on GH in mature older rats (27, 28, 29).
In hypophysectomized rats, GH-dependent IGF-I expression is totally disrupted. In contrast, IGF-I expression in bone cells that is not dependent on GH remains intact. To determine the effect of total IGF-I absence (both GH-dependent and independent IGF-I) on PTH anabolic effects, we evaluated the effect of PTH on bone formation parameters in mice lacking functional IGF-I genes (IGF-I knockout) (30). In these studies, we found that PTH failed to stimulate bone formation and increase bone density in IGF-I knockout mice. To determine whether low basal IGF-I expression is sufficient to restore PTH anabolic effects in mice, we used an IGF-I midi mouse model (31), which express low levels of IGF-I, and found PTH increased bone formation and bone density in this model.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-MEM, DMEM, and calf serum were purchased from Life Technologies, Inc. (Gaithersburg, MD), Mediatech, Inc.
(Herndon, VA), and HyClone Laboratories, Inc. (Logan, UT),
respectively. BSA was purchased from Fluka (Buchs,
Switzerland). All other chemicals were enzyme grade and purchased from
Fisher Scientific (Tustin, CA) or Sigma (St.
Louis, MO). Human PTH (1–34) was synthesized as amides by SynPep (Dublin, CA). The purity of this analog was greater than 95% by amino acid analysis and mass spectrometry. Recombinant human IGF-I was a gift from UpJohn/Pharmacia (Stockholm, Sweden).
Animals
Breeder mice heterozygous for the IGF-I knockout (KO)
allele were kindly supplied by Dr. Argiris Efstratiadis (Columbia
University, New York, NY). IGF-I null pups were identified by the
characteristic small size at birth and failure to grow following birth
(30) as well as by PCR analysis using IGF-I forward primer
(5'-CCACAGGCTATGGCTCCAGCATTC-3'), IGF-I reverse primer
(5'-GTCAGTGTGGCGCTCGGCAC-3'), and neoreverse primer
(5'-ATCCATCTTGTTCAATGGCCGATCCC-3') yielding an approximately 450-bp
product for the disrupted IGF-I gene and 160-bp product for the
wild-type (WT) gene. Heterozygous littermates were genotyped and used
for breeding, and the WT littermates were used as control animals in
this study.
Homozygous breeder IGF-I midi mice were kindly provided by Lyn Powell-Braxton (Genentech, Inc., San Francisco, CA). During the generation of the IGF-I null mice, a single embryonic stem cell clone was obtained in which there had been a site-specific insertion of the targeting construct instead of a homologous recombination (31). The IGF-I allele in this clone contained the entire construct, including neomycin-interrupted exon 3, thymidine kinase gene, and vector sequences inserted 5' of the endogenous exon 3. If the mutant allele IGF-Im did not exhibit alternate splicing, this would result in a null allele and no IGF-I could be detected because there are multiple stop codons in all three reading frames in the neomycin cassette. However, alternate splicing is known to occur with exon 5 in the WT rodent IGF-I gene, and it has been shown that alternate splicing was occurring around the neomycin-containing exon, resulting in the production of a small amount of WT mRNA and protein in IGF-I midi mice (31).
In vitro experiments
Immortalized osteoblast clones were derived from bone cells
isolated from calvaria of newborn IGF-I KO and WT mice by established
procedures (24). The osteoblastic characteristics of
isolated cells were established by ALP staining and by osteocalcin
production (13, 24). Briefly, osteoblasts were cultured in
a 1:1 mixture of DMEM and Ham’s F12 media with 10% FCS. Cells were
grown to 50–80% confluence and split 1:3 for 26–32 passages, during
which cells spontaneously immortalized. Cells were then plated at
clonal densities in 10-cm culture dishes, and individual clones were
picked using glass cloning cylinders. Clones that proliferated well
were expanded and cells were stored cryogenically. Cells for
experiments were thawed and grown several passages in -MEM with 10%
FCS and then used for the in vitro experiments. When 80%
confluent cultures were incubated in serum-free media for 48 h,
the media of WT cells contained approximately 1 ng/ml of IGF-I
determined by RIA, and IGF-I was undetectable in media from IGF-I KO
cells. IGF-II levels were undetectable in the conditioned media derived
from osteoblasts isolated from IGF-I KO and WT mice.
The biological effect of PTH on osteoblasts was established by the AlamarBlue assay or by direct counting of cells. Immortalized osteoblast clones, as well as normal untransformed osteoblasts at passage 2, were used in these experiments. AlamarBlue is reduced by reactions innate to cellular metabolism and therefore provides indirect measure of viable cell number (AccuMed International, Inc., Westlake, OH). Briefly, cells were seeded into 96-well plates at 2,000 cells/well in 50 µl of DMEM containing 0.1% calf serum and 0.1% BSA. Twenty-four hours later, the media were removed, the cell layers were rinsed with PBS, and 100 µl of 0–100 ng/ml PTH and/or 0–10 ng/ml of IGF-I in DMEM containing 0.1% BSA were added to each well. The medium was replaced 48 h later with 100 µl of 10% AlamarBlue diluted in phenol red-free DMEM. The fluorescence was determined 4 h later using a fluorescent plate reader (Fluorolite 1000, Dynex Technologies, Inc., Chantilly, VA). For counting cell numbers, cells were seeded into 24-well plates at 20,000 cells/well in 500 µl of DMEM containing 0.1% calf serum and 0.1% BSA. Twenty-four hours later, the media was replaced with serum-free DMEM containing 0.1% BSA as described above and test agents added. After 72 h, cell cultures were fixed and stained with 10% ethanol containing 1% crystal violet and the number of cells counted in five random fields using a microscope eyepiece grid. Results were calculated as number of cells per well.
In vivo experiments
Five-week-old IGF-I(-/-) mice, IGF-I midi mice, and
their age-matched WT littermates were housed in a room maintained at 70
F on a 12-h light/12-h dark cycle and fed ad libitum with
standard rat/mouse diet containing normal calcium. The animals were
given daily sc injections of 160 µg/kg body weight of PTH (1–34) or
vehicle (PBS, PBS) for 10 d. The dose of PTH was selected based on
a dose-response study of the anabolic effects of PTH in a mouse model
(13). PTH was prepared in PBS before administration.
Twenty-four hours after the last injection, the mice were euthanized by
CO2 inhalation and decapitation; blood and right
femur were collected and stored at -70 C until biochemical
measurements were performed. The experimental procedures performed in
this study are in compliance with the NIH guide for the Care and Use of
Laboratory Animals and approved by the Animal Studies Subcommittee at
the Jerry L. Pettis Veterans Affairs Medical Center (Loma Linda,
CA).
Bone densitometry
Bone mineral density (BMD) of the left femur at the distal
metaphysis was measured by peripheral quantitative computer tomography
(pQCT) using a pQCT system, Stratec XCT Research M with software
version 5.20 (Norland Medical Systems, Atkinson, WI). Total
volumetric BMD (mg/cm3) was calculated at the
distal diaphysis as previously described (32, 33). Total
bone mineral content (BMC) and areal BMD (mg/cm2)
were determined for the entire femur using dual-energy x-ray
absorptiometry (DEXA) (PIXImus instrument, Lunar Corp.,
Madison, WI), according to the manufacturer’s instructions.
Mechanical testing
After pQCT measurements, the samples were applied to mechanical
testing using a Mechanical Tester 8841 (Instron, Canton,
MA). Three-point bending strength was measured at middiaphysis. The
bone was placed horizontally with the anterior surface upward, centered
on the supports, and the pressing force directed vertically to the
midshaft of the bone. Each bone was compressed with a constant speed of
2 mm/min until failure. Breaking force was defined as bending load at
failure. Stiffness was calculated as the slope of the linear (elastic)
part of the load-displacement curve. Stress 
and elastic or Young’s
modulus E was calculated as previously described (34).
Maximal stress was defined as stress at breaking force.
Biochemical assays
IGF-I RIA. IGF-I was measured by specific RIA using
rabbit polyclonal antiserum and recombinant IGF-I as standard and
tracer as previously described (35). The inter- and
intra-assay coefficient of variation was less than 10%. The
cross-reactivity of IGF-II in the IGF-I assay was less than 2%.
Osteocalcin RIA. Osteocalcin levels were determined by using rabbit antibody made against mouse osteocalcin synthetic peptide as described previously (36). The sensitivity of the assay was 19 ng/ml. The interassay and intraassay coefficient of variation was less than 10%.
ALP activity. The ALP activity of the serum and bone extracts was determined as previously described (37). ALP activity of the bone extracts was expressed as milliunits per milligram of protein or as milliunits per milligram dry weight of bone.
Total protein levels. Protein concentration was determined by Bradford assay using a commercial kit (Bio-Rad Laboratories, Inc., Richmond, CA)
Statistical analysis
Five to six animals were used for each treatment group. Values
are represented as means ± SEM. Statistical analysis
of the data were performed by an unpaired two-tailed t test
or Fisher’s protected least significant difference method (posthoc
test) for multiple comparisons in a one-way ANOVA as appropriate.
Results were considered significantly different at P <
0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). In WT animals, daily sc
injections of PTH caused a significant increase (113%) in serum
osteocalcin levels, compared with the control group. In contrast, PTH
treatment had no significant effect on serum osteocalcin levels in
IGF-I(-/-) mice. PTH treatment also caused a significant increase in
ALP activity in serum (48%) and in the femoral bone extract on the
basis of both bone weight (84%) and extractable protein (94%) in WT
animals (Figs. 2 and 3). However, in
IGF-I(-/-) mice, there was no significant effect of PTH on serum ALP
activity or ALP activity in bone extract.
PTH-induced increases in serum osteocalcin and bone ALP activity in WT
mice are consistent with the previous reports in humans and animals
that PTH treatment causes an acute increase in bone formation
(1, 2, 3, 4, 5, 6).
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We next performed BMD measurements using DEXA and pQCT to confirm the
lack of PTH anabolic effect, as reflected by serum biochemical marker
data, in the IGF-I KO mice. Table 1 shows
that PTH treatment did not increase total BMC or areal BMD, as
determined by DEXA in IGF-I KO mice, but both of these parameters were
significantly increased in the WT control mice. Furthermore, PTH
treatment had no significant effect on the total volumetric BMD
(vBMD), as measured by pQCT at the distal metaphysis of the
femur, in the IGF-I KO mice, but it increased total vBMD by 40% in the
corresponding WT mice (Fig. 4). Table 1 also
shows that both areal BMD and vBMD were 50% lower in the femur of
IGF-I KO mice, compared with WT mice.
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). Total
BMC and areal BMD, as measured by DEXA, were increased by 31% and
15%, respectively, in the PTH-treated group, compared with the control
group (Table 1
). Total BMD at the distal metaphysis of the femur, as
determined by pQCT, was increased by 32% in the PTH-treated group,
compared with the control group.
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shows that the load-bearing capacity of
the femur and area of moment of inertia were increased by 33% and
40%, respectively, after 10 d of treatment with PTH in midi mice,
compared with vehicle-treated IGF-I midi mice. PTH treatment had no
significant effect on the maximal stress or elastic modulus, compared
with the vehicle-treated control group in the IGF-I midi mice. Similar
results have been obtained on the effect of PTH (1–34) and PTH (1–31)
on mechanical strength parameters in Swiss/Webster mice
(13).
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shows that PTH treatment increased cell
numbers in osteoblasts derived from WT mice in a dose-dependent manner.
However, PTH treatment had no significant effect on number of
osteoblasts derived from IGF-I KO mice. In contrast to PTH, IGF-I
increased cell number equally well in osteoblasts derived from both
IGF-I KO and WT mice. To determine whether the lack of IGF-I signaling
could be responsible for the failure of PTH to increase cell number in
osteoblasts derived from IGF-I KO mice, we tested the PTH effects on
cell number in the presence of IGF-I. Table 4 shows that, in the presence of low
doses of IGF-I, PTH significantly increased cell number in osteoblasts
derived from IGF-I KO mice. To confirm the results obtained using the
AlamarBlue assay, which measures the metabolic activity of cells, cell
number was determined by direct counting of cells after treatment with
PTH and/or IGF-I. Fig. 7 shows that a single
addition of IGF-I at 1 ng/ml or PTH at 100ng/ml increased cell number
by more than 40% after 72-h treatment in osteoblasts derived from WT
mice. In contrast, IGF-I treatment but not PTH caused a significant
increase in cell number in osteoblasts derived from IGF-I KO mice. In
the presence of IGF-I at 1 ng/ml, however, PTH significantly increased
cell number in osteoblasts derived from IGF-I KO mice (Fig. 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The requirement of IGF-I for mediating the bone-forming effects of
systemic bone regulators may not be unique to PTH because past findings
from a number of laboratories, including ours, have revealed that many
of the major hormones that regulate the skeleton exert significant
effects on IGF-I expression. In this regard, it is well known that GH
effects on osteoblasts are mediated via increased production of IGF-I
(38). E2, another important regulator of skeletal
metabolism, has been shown to increase IGF-I production in rat
osteoblasts (39). In addition, IGF-I receptor-blocking
antibodies blocked the proliferative effect of E2 in ROS17/2.8
cells (40), thus suggesting that IGF-I is important in
mediating the E2 effect. Consistent with the role of IGF-I in mediating
steroid hormone effects on osteoblasts, Gori et al.
(41) have recently found that the 5DHT-induced increase
in proliferation of immortalized human fetal osteoblasts was blocked by
a monoclonal antibody specific for IGF-I. Furthermore, Huang et
al. (42) have shown that thyroid hormone increases
IGF-I production in osteoblasts and that inhibition of IGF-I action by
various approaches attenuated the stimulatory effects of thyroid
hormone in mouse osteoblasts. These in vitro studies
emphasize that many of the systemic hormones may recruit IGF-I to
mediate their effects on bone formation.
In this study, PTH effects on bone formation and bone density were evaluated in 5-wk-old growing mice. Our data on the lack of PTH anabolic effect on bone in growing mice are consistent with those obtained by Hock and Fonseca (26) in hypophysectomized young male rats lacking GH. In contrast to these studies, Schmidt et al. (27) have found that intermittent PTH treatment increased cancellous bone formation in adult hypophysectomized female rats and Hock and Wood (29) have found that GH did not further enhance the anabolic effect of PTH on bone mass in aged female rats. Future studies are needed to evaluate if the IGF-I requirement for PTH anabolic effect is specific to only growing mice or is similar in both growing and adult mice.
One potential explanation for the lack of PTH response in IGF-I KO mice could be the absence of target cells that are responsive to PTH. In this regard, it is known that IGF-I KO mice are much smaller and exhibit severe retardation in skeletal growth, compared with corresponding control mice (30). Thus, it is possible that the trabecular and endosteal surfaces of bones in IGF-I KO mice may have fewer osteoblasts, which may be inadequate to respond to PTH treatment. In this regard, the presence of significant levels of osteocalcin, a product of mature osteoblasts, in the serum of IGF-I KO mice argues against lack of osteoblasts in IGF-I KO mice. Furthermore, our recent finding that IGF-I treatment significantly increased bone formation parameters in IGF-I KO mice (43) suggest that the lack of PTH effect on bone formation may not be owing to inability of osteoblasts to respond to treatment with many anabolic agents. Future histomorphometric studies are needed to rule out the possibility that the lack of target cells is the primary reason for the inability of PTH to stimulate bone formation in mice lacking functional IGF-I.
Another explanation for the lack of PTH effect on bone formation is that absence of IGF-I signaling pathway may result in the failure of PTH to stimulate key target genes necessary for PTH to exert its biological effects on osteoblast lineage cells. Our finding that PTH increased cell number in osteoblasts derived from IGF-I KO mice in the presence of low doses of IGF-I in vitro is consistent with the later possibility. Other published reports also provide indirect evidence that IGF-I signaling may be necessary for PTH to mediate its effects on osteoblasts. In this regard, it is known that MAPKs are important regulators of cell growth and differentiation and that IGF-I effects on osteoblasts are mediated, in part, via activation of the MAPK pathway (44). PTH has also been shown to upregulate MAPK activity in opossum kidney cells, thus implicating a role for PTH-induced MAP pathway in mediating PTH cellular response (18). Both PTH and IGF-I have been shown to be involved in the activation of c-fos expression (16, 45) and cyclin-dependent kinase expression in osteoblasts (17, 44, 46). Thus, the PTH signaling pathway may interact with an IGF-I signaling pathway at one or more points, and the absence of an IGF-I signaling pathway in IGF-I KO mice may result in the failure of PTH to stimulate key target genes necessary for osteoblast proliferation and/or matrix synthesis. In this regard, previous findings that PTH increased bone mass in caloric-restricted rats in which IGF-I production is suppressed (28), and our data that PTH treatment significantly increased bone formation and bone density in midi mice deficient in IGF-I suggest that low level of basal IGF-I expression may be adequate to restore PTH anabolic effects.
The PTH-induced increase in bone formation may involve increased local IGF-I production in osteoblasts and/or circulating levels of IGF-I because IGF-I can act as an endocrine hormone as well as local growth factor (47). Although PTH treatment has been shown to increase production of IGF-I in osteoblasts, in vitro and in vivo in rats and mice (19, 20, 21, 22), PTH treatment had no significant effect on circulating levels of IGF-I in the present study. In this regard, it is now known that disruption of IGF-I genes specifically in liver decreased serum IGF-I by 80% but had little effect on postnatal growth (47, 48). Because the remaining 20% IGF-I in serum is probably derived from several tissues including bone, it is not surprising that PTH treatment did not increase circulating levels of IGF-I in mice. These findings and the findings that PTH effects on osteoblasts can be blocked by IGF-I neutralizing antibody in vitro (23, 24) are consistent with the idea that a PTH-induced increase in bone formation involves increased local production but not increased circulating levels of IGF-I.
In conclusion, our study provides the first direct evidence that the anabolic effects of PTH on bone require IGF-I action in growing mice. On the basis of this and other findings, we propose that IGF-I may play a critical role in mediating the anabolic effects of a number of systemic osteoregulatory agents on bone.
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Acknowledgments |
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Footnotes |
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Abbreviations: ALP, Alkaline phosphatase; BMC, bone mineral content; BMD, bone mineral density; DEXA, dual-energy x-ray absorptiometry; KO, knockout; pQCT, peripheral quantitative computer tomography; vBMD, volumetric BMD; WT, wild type.
Received February 26, 2001.
Accepted for publication June 26, 2001.
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