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Division of Endocrinology (G.Z., T.N., J.W.P., J.A.F., T.L.C.), Departments of Medicine and Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267; Research Service (T.L.C.), Veterans Administration Medical Center, Cincinnati, Ohio 45220; Division of Nephrology, Bone and Mineral Metabolism (M.-C.M.-F., M.C.L., Z.G., H.H.M.), University of Kentucky School of Medicine, Lexington, Kentucky 40536; Division of Endocrinology (S.D.C.), Children’s Hospital, Cincinnati, Ohio 45229; Maine Center for Osteoporosis Research and Education (C.R., L.-R.D.), St. Joseph Hospital, Bangor, Maine 04401
Address all correspondence and requests for reprints to: Thomas L. Clemens, Ph.D., Division of Endocrinology, University of Cincinnati College of Medicine, Vontz Center for Molecular Studies, 3125 Eden Avenue, Cincinnati, Ohio 45267. E-mail: Clementl{at}uc.edu
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Numerous in vitro studies have documented the ability of IGF-I to influence osteoblast growth and differentiation (2). IGF-I exerts its actions through the type I IGF receptor, a tyrosine kinase receptor structurally related to the insulin receptor (4). IGF-I also exerts distinct activities on differentiated osteoblast function. For example, during the initial phases of differentiation of fetal rat calvarial osteoblasts in vitro, IGF-I promotes the progression of a preosteoblast to mature osteoblast (5). IGF-I expression declines with the appearance of the differentiated osteoblast phenotype but rises again during the terminal phases of osteoblast differentiation. Moreover, IGF-I can induce type I collagen expression and inhibit collagen degradation in differentiated fetal rat osteoblasts (6, 7, 8).
In addition to its growth promoting effects, IGF-I is increasingly recognized as an important survival factor for many cell types including neurons (9), skeletal myoblasts (10), fibroblasts (11), and osteoblasts (12). IGF-I also appears to regulate bone resorption, but whether this occurs by a direct action on osteoclasts is still uncertain. IGF-I promotes the formation of osteoclast-like cells from mononuclear precursors and can stimulate the resorptive activity of preexist osteoclasts (13). However, both of these actions likely require the presence of osteoblasts. Thus IGF-I appears to play a fundamental role in bone remodeling and influence osteoblast differentiation, matrix production, and mineralization, as well as osteoclastic activity.
Despite many in vitro studies supporting a role for IGF-I in regulating bone formation and turnover, precisely how this growth factor functions in its normal paracrine setting in vivo is less well understood. This is largely due to the fact that each tissue environment has a specific set of IGFBPs and IGFBP-specific proteases that modulate the bioavailability of IGF-I. Recent studies in genetically altered mice have confirmed the importance of IGF-I in the development of a normal skeleton in vivo. For example, mice lacking the IGF-I gene (14) appear to develop normally but are smaller than wild-type mice and frequently die in the postnatal period. By contrast, IGF-I receptor (-/-) mice demonstrate organ hypoplasia, delayed skeletal calcification, severe growth retardation, and invariably die postnatally. Thus, while these models suggest that IGFs play important roles in embryogenesis, severe disturbances in organ development and frequent lethality prohibit the use of these models for examination of the physiologic role of IGF-I in the mature animal. On the other hand, overexpression of growth factors and cytokines has provided additional insight into their biology. For example, transgenic mice that overexpress IGF-I ubiquitously demonstrate selective organomegaly but without an increase in skeletal growth (15).
To provide a more physiological context to study the skeletal actions of IGF-I, we examined the impact of overexpression of IGF-I in osteoblasts of transgenic mice. Quantitative histomorphometric analysis revealed a dramatic increase in trabecular bone volume at the distal femur. This anabolic action occurred without a significant increase in the numbers of osteoblasts, indicating that locally produced IGF-I functions primarily to increase the metabolic activity of resident osteoblasts.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Total RNA was isolated from tissues by a single step acid guanidinium thiocyanate-phenol-chloroform extraction method. Northern blots were performed as described (17). Briefly, 10 µg of tissue total RNA were gel-separated, transferred to a nylon membrane, and then hybridized with random primed rat IGF-I cDNA. For standardization, blots were rehybridized with either human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA, when comparing within the same tissue type, or 18S rRNA, for comparisons between different tissues.
Measurement of serum IGF-I concentrations
Serum concentrations of IGF-I were determined using an IGF-I RIA
(Diagnostics Systems Laboratories, Inc. Webster, TX)
following extraction to remove IGF binding proteins. The limit of
detection of the assay is 21 ng/ml.
In situ hybridization
In situ hybridization of IGF-I messenger RNA (mRNA)
was performed on undecalcified sections of mouse femur. Three
micron-thick sections were pretreated as follows: Sections were
deplasticized in 2-methoxy ethylacetate, rinsed in PBS, incubated in
0.1 M HCl for 5 min, and then rinsed in PBS.
Slides were then incubated in proteinase K for 15 min at 37 C, washed
in fresh 0.2% glycine, incubated in 0.1M
triethanolamine (TEA) with 0.25% acetic anhydride for 10 min, washed
in 0.2 x SSC, and dehydrated in graded EtOH solutions. Sections
were rinsed in chloroform and then in 100% EtOH. An antisense cRNA
probe for rat IGF-I was labeled with 33P-rUTP,
using a commercially available kit (Stratagene, La Jolla,
CA). For generation of the antisense IGF-I riboprobe, the prIGF-I
plasmid was linearized with BamHI, and transcribed with T7
RNA polymerase. The 733-bp product encodes 678-bp complementary to
IGF-I mRNA. The sense IGF-I 732-bp riboprobe was obtained after
linearizing the same vector with EcoRI, and transcribing
with T3 RNA polymerase. Hybridization was performed with either
antisense or sense rat IGF-I riboprobes at a concentration of 1 x
106 cpm per section in 200 µl of hybridization
buffer (deionized formamide, 50% dextran sulfate, 5
M NaCl, 1 M Tris, 0.5
M EDTA, 50 x Denhardt’s solution, 1
M DTT, and DEPC-treated water) at 55 C overnight.
Subsequently, slides were washed in 2 x SSC, incubated in RNase A
at 37 C, and briefly rinsed in 1 x SSC. Slides were then
incubated in 1 x SSC at 67 C for 30 min, rinsed in 1 x SSC
at room temperature, regularly rinsed in graded EtOH solutions, and air
dried before being dipped in NTB emulsion and exposed for 4 weeks.
After development, slides were counterstained with hematoxylin,
dehydrated in graded EtOH and xylenes, and then coverslipped.
Measurement of bone mineral density
Bone mineral density was measured at the femur and spine using
the PIXImus small animal DEXA system (Lunar Corp.,
Madison, WI), software version 1.43.036.008. Data were acquired from
isolated specimens that included the lumbar vertebrae, the pelvic
girdle and both hind legs. The resolution of the PIXImus is 0.18
x 0.18 mm pixels with a usable scanning area of 80 x 65 mm,
allowing for measurement of single whole mice and collections of
isolated specimens. Calibrations were performed with a phantom using
known values, and quality assurance measurements were performed daily
with this same phantom. The precision for BMD is less than 1% for
whole body and approximately 1.5% for specialized regions.
Bone mineral density of isolated femurs was also measured by pQCT with a Stratec XCT 960M (Norland Medical Systems, Inc., Ft. Atkinson, WI) as previously described (19). Femurs were scanned at 2-mm intervals over their entire lengths, using an x-ray attenuation threshold of 2.000 U to define high density bone, a threshold of 1.300 to define low density bone, and a unit volume for measurement of mineral set at 0.1 mm3. The precision of this instrument for densitometry of mouse bones has been determined to be 1.2% by repeated placement and measurement of a single femur. Calibration of the densitometer was done with a set of hydroxyapatite standards and yielded a correlation of 0.997 between standards and pQCT estimation of bone density. In this paper, femoral BMD is defined as total femoral mineral divided by total femoral volume.
Mineralized bone histology and bone morphometry
Distal femora and calvaria were fixed in 100% ethanol. After
dehydration, bone samples were embedded in methyl methacrylate (20) and
4-µm sections were cut with a heavy-duty microtome (Microm,
C.Carl Zeiss, Thornwood, NY). This thickness allows
analysis of the same histologic features in slides prepared for light
and fluorescent microscopy. Four bone sections were stained using the
modified Masson-Goldner trichrome technique (21), and four others
serial to the stained sections were left unstained for fluorescent
microscopy.
Static and dynamic parameters of bone structure, formation, and resorption were measured at a standardized site below the growth plate using a semiautomatic method (Osteoplan II, Kontron Instruments Ltd. Munich, Germany) (22). All parameters comply with the recommendations of the Histomorphometry Nomenclature Committee of the American Society of Bone and Mineral Research (23).
Statistical analysis
Results are expressed as mean ± SEM. All
statistical tests were two-sided. An assigned significance level of
0.05 was used. Comparability of the two groups at any given time was
assessed using the Mann-Whitney U test. Comparability of
data from a group at different time points was done using the
Kruskal-Wallis H test. All computations were performed using the
SPSS, Inc. software package for Windows release 7.5
(SPSS, Inc. Chicago, IL). Data for bone density obtained
by DEXA and pQCT were analyzed by Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Two mouse lines with
high levels of mRNA expression (36, 32) were selected for more
extensive analysis. The distribution of expression of the IGF-I
transgene in tissues dissected from a 6-week-old OC-IGF-I transgenic
female mouse of line 36 is shown in Fig. 2B. Endogenous IGF-I mRNA was
detectable in the liver, whereas the IGF-I transgene was detected at
high levels in calvaria, femur, and vertebrae. The greater size of the
transgenic IGF-I mRNA likely reflects differences in the composition of
the 3' UT and polyA region of the transgenic message.
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). In the transgenic mice, the abundance
of transcript varied among individual osteoblasts possibly reflecting
heterogeneity of differentiation status of the osteoblast. Signals were
also observed in osteocytes (Fig. 3F) and occasionally in bone marrow
(not shown) likely due to OC-IGF-I transgene expression by
megakaryocytes, which are known to express osteocalcin (24). IGF-I mRNA
signals were also observed in osteoblasts of control mice but at lower
intensity than that in the transgenic mice (Fig. 3E).
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, there is no significant difference in
total body weight in transgenic mice compared with their nontransgenic
littermates. Serum IGF-I levels were also not significantly different
in transgenic vs. control mice (Fig. 4, inset).
These results demonstrate that locally overexpressed IGF-I does not
escape into plasma.
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). There was no
significant change in the length of the femur or its width at the
diaphysis (not shown). Measurement of BMD by pQCT revealed a
significant increase in both cortical and trabecular BMD in the
OC-IGF-I transgenic mice compared with controls. PIXI measurements on
femora from a second line (no. 32) of OC-IGF-I transgenic mice also
showed a significant increase in BMD compared with controls (Table 1).
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). Trabecular bone volume was also
significantly increased in 6-week-old OC-IGF-I transgenic mice from a
second line (no. 32) (9.11 ± 0.55 vs. 7.23 ± 0.56% for
transgenic and control mice, respectively, P = 0.039).
In mice from line no. 36, cortical bone volume was not significantly
altered in the OC-IGF-I mice (not shown). No significant changes in any
histomorphometric parameter were seen at 3 weeks or 24 weeks of age
(Table 2). This suggested that the
greater bone volume and density in the 6-week-old transgenic mice
resulted from an increased rate of bone formation at an earlier time.
As shown in Fig. 6, 3
-week-old
OC-IGF-I mice had a dramatic increase in bone formation rate and
decrease in mineralization lag time. Significant changes were also
observed in mineral apposition rate, and osteoid maturation time in
transgenic mice compared with controls at 3 weeks (Table 2). When
viewed under polarized light, the newly formed bone had normal lamellar
architecture with no evidence of woven bone. By contrast, no changes
were observed in total calvarial thickness in the OC-IGF-I mice
compared with controls at any time. However, in 3-week-old transgenic
mice, calvarial porosity was reduced as shown in Fig. 7.
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). However, at 3 weeks,
the bone formation rate per osteoblast was higher in the OC-IGF-I
transgenic mice compared with controls (19.25 + 5.26 vs.
8.13 + 1.23 mm3/ob/yr), suggesting that IGF-I
increased the activity of individual osteoblasts. There were no
significant differences in bone formation rate or any other dynamic
measures in 6- and 24-week-old OC-IGF-I mice compared with controls
(Table 2).
Overexpression of IGF-I in osteoblasts increases osteocytic lacunae
occupancy
Osteocytes are former osteoblasts that have become encased in bone
and are believed to function as mechanosensors (25). As the osteocalcin
promoter is transcriptionally active in osteocytes we compared the
total number of osteocyte lacunae and osteocyte occupancy frequency in
OC-IGF-I transgenic mice to controls. At 3 weeks, osteocyte number and
occupancy frequency were similar in transgenic and wild-type mice (Fig. 8A). There was also a significant decline
in the number of osteocytic lacunae per bone area in both transgenic
and nontransgenic mice at 6 weeks compared with 3 weeks (Fig. 8A).
However, at 6 weeks, the percent of occupied osteocytic lacunae in
wild-type mice had dropped, whereas in transgenic mice the proportion
remained the same and was significantly higher than that in wild-type
(Fig. 8B). Thus, IGF-I overexpression in osteoblasts increased the
osteocytic lacunae occupancy.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The skeletal phenotype of our transgenic mice with osteoblast-specific overexpression of IGF-I enables new insights into the cellular mechanisms by which locally produced IGF-I exerts anabolic actions on the skeleton. As mentioned above, previous studies in cultured osteoblasts have shown that IGF-I exerts multiple actions including promoting proliferation and prolonging life span by decreasing the frequency of apoptosis. In addition, IGF-I has been shown to increase the formation of osteoclast-like cells from mononuclear precursors (13). However, the increase in bone formation rate in the OC-IGF-I transgenic mice took place in the absence of any change in the number of either osteoblasts or osteoclasts. Moreover, when bone formation rate was expressed per osteoblast, it was greater in OC-IGF-I mice than controls. This suggests that the primary effect of IGF-I is to increase the work per unit time of resident osteoblasts. One caveat to this interpretation is that IGF-I may have exerted a more dramatic effect on the mature, well differentiated osteoblast owing to greater expression of the OC-IGF-I transgene by these cells. While we do not exclude this possibility, it seems likely that the preosteoblasts, which also express osteocalcin (31) and are in close spatial proximity to mature osteoblasts, would therefore be also exposed to high levels of IGF-I. Despite this, however, osteoblast number was unchanged in the IGF-I overexpressing mice consistent with the notion that IGF-I functions primarily to enhance the activity of existing osteoblasts rather by promoting replication of osteoblast progenitors. This observation is in keeping with results from in vitro studies demonstrating that IGF-I is a relatively weak mitogen for osteoblasts (32) but has profound effects on cellular metabolic activities including enhanced ascorbic acid transport which are required for collagen formation (33).
In theory, antiapoptotic actions of IGF-I may have also contributed to its anabolic effect by prolonging osteoblast life span and thereby extending the time spent in matrix formation. However, a significant decrease in the rate of osteoblast apoptosis should have resulted in increased osteoblast and osteocyte number as the population of the latter cell is determined by input from the osteoblast pool (34). For example, in mice given intermittent doses of PTH, a 10- to 20-fold decrease in the rate of osteoblast apoptosis was accompanied by a marked (40–60%) increase in osteoblast numbers and osteocyte density with no apparent change in osteoblast proliferation rate (34). By contrast, neither osteoblast nor osteocyte number was altered in the OC-IGF-I mice, suggesting that antiapoptotic effects of IGF-I on osteoblasts, if present, were minimal. The increased osteocyte lacunae occupancy in the OC-IGF-I mice is intriguing and at first glance suggested that IGF-I might be prolonging the life-span of the osteocyte. However, in this scenario, one would again expect an increase in osteocyte density unless the entrance of osteoblasts into the osteocyte pool was also diminished. Therefore, it is possible that the overexpression of IGF-I both interfered with osteocyte generation and prolonged their life span.
The effects of IGF-I overexpression were more pronounced in trabecular
than in cortical bone. Moreover, cranial thickness was influenced in
the OC-IGF-I transgenic mice. This shows that the anabolic activity of
IGF-I is dependent on the skeletal site. Expression of transgenic IGF-I
mRNA was greater in calvaria than in femur (Fig. 2), excluding the
possibility that the site specificity was due to a difference in
expression of the transgene. Levels of IGFs and IGFBPs vary
significantly among different skeletal sites (35), and it is possible
that the abundance of IGF-I relative to inhibitory or stimulatory
binding proteins are important determinants of site-specific actions of
IGFs. However, it seems unlikely that these considerations are central
to the differences observed because the levels of transgene expression
were high at all bone sites. Trabecular bone at the distal femur is
known to be a site of very active modeling in the pubertal mouse
(Faugere, M.-C., unpublished observations). Therefore, it is more
likely that the effects of IGF-I on cellular activity within this
compartment are more pronounced. It is interesting to note that
targeted expression of calcitonin gene-related peptide (CGRP) to
osteoblasts of transgenic mice also increased trabecular bone formation
rate at the distal femur but not in calvaria (36). IGF-I mRNA abundance
in bone from the CGRP transgenic mice was increased suggesting that
IGF-I may have contributed to the accelerated bone formation rate seen
in this model.
Interestingly, the increase in trabecular bone volume observed in
the OC-IGF-I mice at 6 weeks had returned to control levels by 24 weeks
(Table 2). It is unlikely that an age-related decrease in the activity
of the osteocalcin promoter accounted for the waning of the anabolic
effect because bone IGF-I mRNA levels were similar in 3, 6, and
24-week-old mice (not shown). It is more likely that overexpression of
IGF-I triggered counter-regulatory events such as down-regulation of
the IGF-I receptor and/or up-regulation of expression of inhibitory
IGFBPs. For instance, an up-regulation of IGFBP-4, an abundant IGF
binding protein in bone (37), would likely reduce the growth factors
access to its receptor. However, the exact mechanisms responsible for
the transient nature of the anabolic effect remain unclear at present
and are the subject of ongoing investigations.
In summary, targeted overexpression of IGF-I to osteoblasts of transgenic mice increases cancellous bone formation rate and volume without any change in osteoblast number. We conclude that locally delivered IGF-I exerts its anabolic actions primarily, if not exclusively, by increasing the activity of resident osteoblasts. The effects are most pronounced during the pubertal period, a time when peak bone mass is achieved in the mouse. The signaling pathways through which IGF-I exerts its anabolic actions represent attractive targets for new therapeutic agents to optimize bone mass.
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Acknowledgments |
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Footnotes |
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Received February 22, 2000.
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) and wild-type mice (
) at 3 and 6 weeks. n = 12
(WT) and n = 14 (TG) at 3 weeks, n = 8 (WT) and n = 12
(TG) at 6 weeks. Data represent mean ± SEM. *,
P < 0.05.