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Department of Orthopedics and Physical Rehabilitation, University of Massachusetts Medical Center, Worcester, Massachusetts 01655
Address all correspondence and requests for reprints to: Dr. Moira Milne, Department of Orthopedics and Physical Rehabilitation, University of Massachusetts Medical Center, 55 Lake Avenue North, Worcester, Massachusetts 01655.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The importance of T3 for normal cell function is well
established, but the molecular and cellular mechanisms of
T3 action in bone are unclear. The molecular effects of
T3 are mediated by the binding of T3 to nuclear
thyroid hormone receptors (TRs) that bind to response elements on
specific genes. A total of four TR isoforms have been identified to
date, TR
1, TR2, TRß1, and
TRß2, with TR2
(c-erbA2) being a nonligand-binding variant
(7, 8). The TR isoforms have been identified in rat and human
osteosarcoma cell lines (9, 10) as well as in primary osteoblast
cultures (11, 12).
In the present study, we describe a reproducible in vitro system in which osteoprogenitors present in bone marrow derived from adult female rat femurs and vertebrae differentiate into osteoblasts capable of mineralizing their extracellular matrix. Specifically in this study, we investigated whether chronic T3 treatment has similar or different effects on osteoblast-related gene expression in cell populations derived from the two skeletal sites. Previously, it was reported that T3 stimulates the production of insulin-like growth factor I (IGF-I) in rat osteoblasts in vitro (13) and increases IGF-I mRNA levels in a mouse osteoblastic clonal cell line (14). This growth factor is highly abundant in bone and acts in an autocrine and paracrine manner to support growth and differentiation of osteoblasts (15). These observations prompted us to examine the effect of T3 on IGF-I mRNA levels in bone marrow cell cultures derived from femurs and vertebrae. We also compared the effect of chronic vs. short term T3 treatment on AP-positive colony formation in femoral and vertebral cultures.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell culture
The animals were killed by exsanguination after injection with
xylazine/ketamine. The experimental protocol was approved by the
institutional animal care and use committee at the University of
Massachusetts Medical School in Worcester, and the animals were
maintained in accordance with NIH Guidelines for the Care and Use of
Laboratory Animals.
For each cell culture preparation, the femurs and lumbar vertebrae were
aseptically removed from five 100- to 125-g Sprague-Dawley female rats
and immediately placed in cold Tyrode’s solution containing 50 ml
antibiotic solution (10,000 IU/ml penicillin G, 10 mg/ml streptomycin,
and 25 mg/ml amphotericin B; Life Technologies, Grand Island, NY)
per liter. The bones were cleaned of all muscle and connective tissue.
The marrow was harvested as follows. Femurs were split open down the
middle with a scalpel, and all marrow found within the trabecular
regions of the proximal and distal femur as well as marrow from the
cortical shaft was flushed out using a 21-gauge needle attached to a
syringe. Vertebrae were split open down the middle, and all marrow
within the trabecular regions was removed. The marrow was collected
into two separate tubes (femur and vertebrae) using the following
medium: MEM containing 10 ml antibiotic solution/liter, vitamin K
(10-8 M), and 20% heat-inactivated FBS
(Atlanta Biologicals, Norcross, GA). The cells were pelleted for 10 min
at 1000 x g, resuspended in fresh medium, filtered to
obtain a single cell suspension, and seeded at 5 x
106 total marrow cells/ml (9 x 105
cells/cm2) onto 100-mm uncoated culture dishes (Corning,
Cambridge, MA).
Femoral and vertebral cultures were maintained in a humidified
atmosphere (95% air-5% CO2) at 37 C. On day 1,
dexamethasone (dex; 10-8 M) and freshly
prepared ascorbic acid (50 µg/ml) were added to all dishes. Cells
were allowed to attach for 4 days, at which time the culture medium was
replaced with fresh medium containing MEM, 20% FBS, ascorbic acid,
and dex. Cells were fed every 2 days and were maintained in primary
culture to monitor the osteoblast phenotype, as assessed by cell
morphology and bone nodule mineralization, or were passaged for gene
expression, mineralization, and thyroid hormone response studies. Cell
passage was performed on day 8, when femoral and vertebral cells were
60–80% confluent. These cells were detached with 0.25% trypsin in 1
mM EDTA (Life Technologies), resuspended in culture medium,
and established in secondary culture by seeding at 2.5 x
104 cells/ml (6.25 x 103
cells/cm2). The culture medium was as described above,
except that the dex concentration was increased to
10-7 M, and ß-glycerophosphate
(ß-GP; 10 mM) was added. Cells were maintained for up to
18 days in primary or secondary culture. For thyroid hormone response
studies, T3 (10-8 and 10-7
M) or vehicle (0.0001 N NaOH) was present
continuously in the medium beginning on day 0 of secondary culture.
Marrow cells were cultured in the continuous presence of dex. In this
study, we examined the long term effect of T3 on osteogenic
differentiation in culture. Glucocorticoids are essential for promoting
osteogenic differentiation in cell cultures derived from adult rat
femurs (17, 18) and from adult rat vertebrae (19). Culture conditions
are summarized in Table 1
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The establishment of osteoblast culture systems is notoriously serum dependent. To verify that osteoblast differentiation was reproducible by cells derived from both femurs and vertebrae, we compared different commercial serum preparations. Marrow was harvested from five 6-week-old female rats and used for the simultaneous testing of three separate serum lots (4002k, 5006i, and 6008b, Atlanta Biologicals). Primary cultures were examined for mineral deposition on day 18 or were subcultured on day 8, reseeded at 2.5 x 104 cells/ml, and maintained in their starting serum lots. The cells in secondary culture were examined for mineralization on day 10. All plates were examined in duplicate.
Northern analysis
Total RNA from control and T3-treated femoral and
vertebral bone marrow cells maintained in secondary culture on six-well
plates for various periods of time was isolated by the guanidinium
thiocyanate method (21). The RNA (5 µg/lane) was size-fractionated on
1.2% agarose-1.8% formaldehyde gels, transferred onto nitrocellulose
membranes (Duralose-UV, Stratagene, La Jolla, CA), and UV cross-linked
(Stratagene UV Crosslinker). The blots were hybridized with
[32P]deoxy-CTP (Amersham, Arlington Heights, IL) probes
labeled by the random primer method (22) (DECAprime II kit, Ambion,
Austin, TX).
The following complementary DNA (cDNA) probes were used to measure
osteocalcin (OC), collagen type I (coll I), IGF-I, and glyceraldehyde
3-phosphate dehydrogenase (GAPDH) mRNA levels, respectively: rat OC
(23), rat coll I (cDNA plasmid IRI) (24), and rat GAPDH (25),
provided by Drs. J. B. Lian and G. S. Stein, University of
Massachusetts Medical Center (Worcester, MA); and rat IGF-I (26),
provided by Dr. Ernesto Canalis, St. Francis Hospital and Medical
Center (Hartford, CT).
The blots were exposed to XAR film (Eastman Kodak, Rochester, NY) for detection of OC, coll I, and GAPDH or to BioMax MS film (Kodak) for the more sensitive detection required for IGF-I. For valid comparison, femoral and vertebral Northern blot membranes were always exposed side by side on the same piece of film. Identical film exposure times were used to compare femoral vs. vertebral data and control vs. T3-treated data. The autoradiograms were quantified by scanning laser densitometry (LKB 2400 Gel Scan XL). GAPDH was used as the control, housekeeping gene. The T3 response studies were repeated twice, with similar results obtained.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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When the cells were subcultured, they attached within 1 day and reached
100% confluence by days 5–6 for vertebral cells and by days 7–8 for
femoral cells. Cuboidal cell clusters were evident in both femoral and
vertebral secondary cultures by days 5–6 (Fig. 1, A and B). The nodules were much more
numerous, more uniformly distributed, and smaller than those formed in
primary culture. Femoral and vertebral cells displayed numerous opaque
nodules by day 10 in secondary culture. At this time the nodules
appeared brown to black in color when viewed with the phase contrast
microscope.
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, C and D. Many clusters of cuboidal cells
had mineralized, as shown by positive (black) von Kossa staining. Three
separate serum lots were tested for the ability to maintain
simultaneous osteogenic differentiation of femoral and vertebral marrow
cells. For each serum, duplicate culture dishes showed identical
patterns of nodule formation and mineral deposition in primary and
secondary cultures (data not shown). Both femoral and vertebral cells
in secondary culture showed extensive mineralization with all serum
lots. Thus, cell morphology and histochemistry demonstrated bone
formation in vitro by both femoral and vertebral osteoblasts
in primary and secondary culture.
Sequential osteoblast gene expression and effects of
T3
Total cellular RNA from femoral and vertebral marrow cells
was analyzed on days 3–12 of secondary culture. A representative
Northern blot is shown in Fig. 1E. Cells from these control cultures
had undetectable OC mRNA during the earliest time points examined. OC
expression was detectable beginning on day 9 in femoral cultures and on
day 6 in vertebral cultures, and was abundantly expressed on day 12 for
both cultures. Figure 2 confirms that
both untreated femoral and vertebral secondary cultures expressed OC
mRNA in a pattern consistent with the development of the osteoblast
phenotype. A single mRNA band was hybridized at 0.6 kilobase (kb) on
Northern blots of both cell populations. OC expression peaked on day 12
in femoral cultures and was not detectable by day 18. In vertebral
cultures, the OC transcript increased up to day 15 and continued to be
expressed, in contrast to that in femoral cells. The effects of
T3 supplementation on OC gene expression in femoral
vs. vertebral cells are shown in Fig. 2. T3
differentially affected OC steady state levels in cells derived from
the two skeletal sites. Although femoral OC mRNA decreased on day 15
and was undetectable by day 18 in the femoral untreated cells,
treatment with T3 (10-8 and 10-7
M) maintained OC expression. Vertebral cells did not
respond in a similar manner. The effect of T3 on vertebral
OC mRNA levels was complex. OC expression on day 12 in
T3-treated vertebral cells was increased by
10-8 M T3, but decreased by
10-7 M T3. On days 15 and 18,
T3 caused only a small dose-dependent increase in OC
expression.
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, coll I expression in
untreated femoral and vertebral cultures followed a pattern similar to
OC expression. Coll I expression was highest on day 12, decreased to
undetectable expression by day 18 in femoral cultures, and peaked on
day 12 with continued expression through day 18 in vertebral cells. In
each culture, two mRNA bands (5.8 and 4.7 kb) were detected. Coll I
gene expression was dose dependently enhanced by continuous exposure to
T3 on days 15 and 18 in femoral cells. Again, the effects
of T3 on coll I expression in vertebral cells were complex,
with slight stimulation on days 12 and 15 (10-8 and
10-7 M) and a decrease on day 18
(10-8 M).
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. The temporal
expression of the osteoblast-specific gene OC is also shown to compare
the appearance of differentiated osteoblasts in culture with the
expression of IGF-I. The results clearly indicate that IGF-I expression
is differentially regulated in femoral vs. vertebral
cultures. IGF-I gene expression was undetectable by Northern analysis
of control cultures of femoral cells. In contrast, IGF-I gene
expression was evident in vertebral bone marrow cultures, with the
highest mRNA levels observed on days 12, and 15, corresponding to peak
OC transcript levels.
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demonstrates the effects of
T3 on IGF-I mRNA isolated from femoral and vertebral
control and T3-treated cells. IGF-I expression was again
undetectable in femoral untreated cultures, whereas T3
caused a slight increase on day 12. However, in vertebral cultures,
T3 treatment markedly increased IGF-I mRNA levels in a
dose-dependent manner at each time point examined. Four IGF-I mRNA
species were apparent. A 6.5-kb band was predominant, with 4.1-, 1.7-,
and 0.9-kb bands also present. T3 caused a proportional
increase in these four IGF-I steady state transcripts. The effects of
T3 on gene expression in bone marrow cultures is summarized
in Table 2.
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) in cultures derived from both skeletal
sites. In two independent experiments, the presence of T3
for the first 6 days of culture decreased the extent of AP-positive
staining in femoral bone marrow cultures, but no visible differences
were seen in vertebral culture (Fig. 6A). The continuous presence of
T3 throughout the 18-day culture period resulted in a
further decrease in femur AP-positive colonies and a slight decrease in
vertebral AP-positive colonies (Fig. 6B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have established that osteoprogenitors present in bone marrow cell populations from adult female rat vertebrae and femurs differentiate into osteoblasts in vitro. Vertebral and femoral cells multilayer and form mineralized bone nodules when cultured in the presence of ascorbic acid, ß-GP, and dexamethasone. This cellular multilayering and nodule formation are consistent with results previously described for in vitro bone formation by rat femur bone marrow stromal cells (16). Osteoblast differentiation of both cell populations during secondary culture is associated with high AP enzyme activity and the expression of genes for the major bone proteins, coll I and OC. Thus, we have a suitable model to compare osteoblast responses to various factors as a function of skeletal location.
Our results obtained by Northern analysis of secondary cultures support the concept of a skeletal site heterogeneity in response to thyroid hormone. In untreated femoral cultures, OC and coll I steady state mRNA levels increased with time in culture and then decreased to undetectable levels. In the continuous presence of T3, femoral OC and coll I mRNA levels remained expressed throughout the culture period. Vertebral cells did not have the same gene expression pattern; OC and coll I mRNA levels remained elevated throughout the culture period for untreated cells, and T3 had only minimal effects on the expression of these genes. Previously, it was reported that T3 increased the level of OC mRNA in fetal rat calvarial osteoblast cultures (31). Here we have shown that T3 also increased OC mRNA levels in adult femoral osteoblastic cultures. Although multiple TR isoform genes are expressed in both rat femurs and vertebrae (12), thyroid hormone administration in vivo decreases femoral bone mass, but not vertebral bone mass, in rats (6, 28, 29). Our present findings indicate that OC gene expression in vertebral cultures is not dramatically altered in response to T3.
We also found that although cultures derived from both skeletal sites had high AP enzyme activity, T3 markedly depressed AP activity in femoral, but not vertebral, cultures. Furthermore, T3 decreased AP activity in femoral, but not vertebral, cells that had been exposed to the hormone for the first 6 days in culture only, a period during which the osteoblast marker gene OC was not yet expressed. This suggests that thyroid hormone can affect femoral osteoprogenitors at an early stage of differentiation, but its addition does not alter osteoblast differentiation of vertebral osteogenic cells.
To address whether continuous T3 treatment can alter expression of any of the known osteoblast-related genes in vertebral cultures, we studied the effects of T3 on IGF-I mRNA levels. This growth factor has been suggested to mediate some of the effects of thyroid hormone on bone formation (32), and osteoblasts secrete IGF-I, which acts in an autocrine and paracrine manner to regulate osteoblast function (15). In addition, T3 was reported to increase levels of IGF-I mRNA in a murine osteoblastic cell line (MC3T3 cells) (14). The results of our Northern analysis of total RNA showed that IGF-I expression was detectable in vertebral, but not femoral, untreated cultures, and that T3 increased IGF-I mRNA levels dose dependently in vertebral, but not femoral, cells. This skeletal site-dependent difference in the pattern of IGF-I gene expression is in agreement with a recent study indicating that production of the IGF system components by human bone cells is significantly different between the skeletal sites of their origin (33). It is possible that the IGF-I expression correlates with a greater abundance of this growth factor for the vertebral cells compared with the femoral cells, leading to a sustained increase in osteoblast phenotype markers and a prevention of bone loss in the spine. This is consistent with our data showing that chronic exposure to high levels of T3 (10-7 M) did not result in a decrease in AP-positive staining in vertebral cultures. This leads to the speculation that if IGF-I were added to femoral cultures, their gene expression and histochemistry profile might resemble those of vertebral cultures.
IGF-I gene expression was detected by Northern analysis in vertebral cultures despite the continuous presence of dexamethasone. The four transcript sizes detected, 6.5, 4.1, 1.7, and 0.9 kb, correspond to those previously reported in rat osteoblast cultures (34). The highest mRNA levels observed correspond to peak OC transcript levels. Glucocorticoids repress transcription of the IGF-I gene (35) and synthesis of IGF-I (36) in rat osteoblasts. The presence of dex was required for both vertebral and femoral cultures to maintain their osteoblastic phenotype with regard to OC and coll I temporal gene expression, mineralization potential, and AP activity, but the activities of the two osteogenic cultures differed in response to added T3; IGF-I gene expression was stimulated dramatically only in vertebral cultures. This suggests the presence of a factor(s) in vertebral cells that overrides gene suppression due to dex.
As the present studies were performed with continuous T3 treatment, it is uncertain whether the increase in IGF-I expression in vertebral cultures was a direct effect on IGF-I gene transcription or whether it was due to an increase in osteoblast differentiation as a consequence of T3 treatment. Also, the marrow culture system has a heterogeneous population of cell types (37), and in addition to osteoblasts, rat marrow stromal cells have been shown to synthesize IGF-I (38). In any case, the effects of T3 on IGF-I gene expression by vertebral cultures far exceeded those on gene expression by femoral cultures. We conclude that vertebral and femoral bone marrow cells populations are differentially regulated by T3in vitro. The simultaneous comparison of osteogenic cultures from different skeletal regions facilitates the examination of mechanisms by which thyroid hormone regulates osteoblast function. The effects of T3 have been studied previously in fetal rat calvaria cells (31, 39). It has been reported that T3 suppresses osteoprogenitor differentiation when cells are maintained in the continuous presence of added T3 (10–9–10-8 M) under culture conditions with 15% thyroid hormone-depleted FBS (31). The serum used in the present study was not stripped of hormones. The endogenous concentration of T3 in the 20% FBS culture medium was assayed to be 3.9 x 10-10 M. After adding T3 to make the final concentration, 10-8 M, the final total T3 concentration in the culture medium with 20% FBS was 1.13 x 10-8 M. This value compares well with that found in hyperthyroidism (31) and shows that the T3 responses in gene expression of femoral and vertebral osteoblasts were observed at hormone levels obtainable in vivo.
In conclusion, our results show that differences exist between bone cell populations derived from different skeletal sites. The varied responses of different regions of the skeleton to the hormonal environment is of clinical importance. The culture model presented in this report should prove useful for identifying the regulatory factors responsible for such skeletal site-specific heterogeneity.
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Acknowledgments |
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Footnotes |
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Received September 29, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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