Effect of hypophysectomy on the proliferation and
differentiation of rat bone marrow stromal cells
James K.
Yeh1,2,
Jodi F.
Evans1,
Meng-Meng
Chen1, and
John F.
Aloia1,2
1 Department of Medicine,
Winthrop-University Hospital, Mineola 11501; and
2 The Health Sciences Center,
State University of New York, Stony Brook, New York 11794
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ABSTRACT |
Conditions such as
estrogen deficiency, skeletal unloading, and aging have all been
demonstrated to have various effects on the proliferation and
differentiation of bone marrow stroma-derived osteoprogenitor cells.
Here we have sought to examine the effects of pituitary hormone
deficiency on the proliferation and the differentiation of these
osteoprogenitor cells using the hypophysectomized (HX) rat as a model.
In the present study, we use an in vitro culture system to examine the
effects of HX on the osteogenic potential of rat bone marrow stroma.
With the intact animal as a control, we used
[3H]thymidine
incorporation and cell number as indexes of proliferation. We also
measured alkaline phosphatase enzyme activity, relative levels of
osteocalcin expression with RT-PCR, and osteopontin and bone
sialoprotein steady-state levels by Northern blot to delineate the
effect on differentiation. Our results indicate that osteoprogenitor
cells exposed to a pituitary hormone-deficient environment in vivo
demonstrate an enhanced proliferative capacity and also exhibit an
augmented expression of differentiation markers when exposed to an
optimal environment in vitro.
pituitary hormone; osteoblasts; osteogenesis; alkaline phosphatase; calcium
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INTRODUCTION |
THE SKELETON OF VERTEBRATES is capable of extensive
regeneration (modeling and remodeling), which serves to maintain its
health and strength. To retain this capacity, it must possess precursor cell populations capable of differentiating into fully functional osteoblasts and osteoclasts, and the quantity of cells in these precursor pools must be maintained through proliferation (5). Results
of studies performed on cells from animals are consistent with the
hypothesis that the renewal of the osteoblast population at the bone
surface is dependent on the recruitment, proliferation, and
differentiation of osteoprogenitor cells (18, 31). It has been
determined that cells of the osteogenic lineage, which includes both
osteoblasts and chondroblasts, are derived from multipotential
mesenchymal stem cells present in the bone marrow stroma (2, 15, 19).
In vitro culture systems have been established in an effort to
characterize the stromal system of bone. Conditions permissive for
osteoblast differentiation from marrow stromal cells have been tested
extensively. These cells, when grown in the presence of
-glycerophosphate, ascorbic acid, and dexamethasone, have been
proven to form mineralized bone nodules (4, 6, 22, 28). Three stages of
development have been delineated for these primary derived osteoblasts:
proliferation, extracellular matrix (ECM) maturation, and ECM
mineralization (2, 26, 28). As the osteoprogenitor develops into the
preosteoblast and osteoblast under these culture conditions, it
expresses various bone-specific markers in a temporal manner. For
example, type I collagen and alkaline phosphatase (AP) expression is
relatively high at early stages but then decreases as mineralization
occurs; osteopontin (OPN) is the first matrix protein to appear
followed by bone sialoprotein (BSP) and osteocalcin (OCN), with
BSP detected first and OCN appearing with mineralization (2).
Conditions such as estrogen deficiency, skeletal unloading, and aging
have all been demonstrated to have various effects on the proliferation
and differentiation of osteoprogenitor cells derived from bone marrow
stroma. Here we have sought to examine the effects of pituitary hormone
deficiency on the proliferation and the differentiation of bone
marrow-derived osteoprogenitor cells with the hypophysectomized (HX)
rat as a model. The HX rat has been used as a model for the effects of
pituitary hormone deficiency on bone for many years. Systemically, it
results in a suppression of weight gain, a reduction in lean mass, and
decreased skeletal growth (8, 14, 30, 33). One of the most striking effects of HX demonstrated in vivo is a cessation of longitudinal growth (14, 30, 33). Hypophysectomy has also been shown to result in
cancellous bone loss because of the suppression of longitudinal
growth-dependent bone gain and the inhibition of tissue-based bone
turnover, with more suppression occurring in bone formation than in
resorption (8, 33). In cortical bone, HX results in suppressed radial
growth-dependent bone gain, which is associated with a decrease in
modeling-dependent bone formation (8). Studies focusing on de novo bone
differentiation have yielded similar results. Not only does HX markedly
reduce and delay osteogenesis, but also the quality of new bone formed
is deficient in the absence of hypophysial hormones (21, 29). On examination of the bone surface of the HX animal, we have discovered a reduction in the osteoblast and osteoclast population compared with
an intact age-matched control.
Currently, it is not known whether the reduction in osteogenesis at the
bone surface can be attributed to a reduction in the capacity of
osteoprogenitors to proliferate and develop into mature osteoblasts. In
the present study, we use an in vitro culture system to examine the
effects of HX on the osteoprogenitor potential of rat bone marrow
stroma. With the intact animal as a control, we used
[3H]thymidine
incorporation and cell number as indexes of proliferation. We also
measured AP enzyme activity, relative levels of OCN expression with
RT-PCR, and OPN and BSP steady-state levels by Northern blot to
delineate the effect on differentiation. Our results indicate that
osteoprogenitor cells exposed to a pituitary hormone-deficient environment in vivo demonstrate an enhanced proliferative and differentiative capacity in vitro when their environment is again optimal.
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MATERIALS AND METHODS |
Animal care. Age-matched HX and intact
control female, 8-wk-old Sprague-Dawley rats were purchased from
Hilltop where the hypophysectomy surgeries were performed. On arrival,
3 days postoperatively, and throughout the experiment, the HX rats were
given 3% sucrose water and allowed free access to a standard pelleted
chow diet (Rodent Laboratory Chow 5001, Ralston Purina, St. Louis, MO). Animals were maintained in accordance with the National Institutes of
Health Guide for the Care and Use of Laboratory
Animals, and animal protocols were approved by the
Laboratory Animal Care Committee of Winthrop University Hospital.
Histomorphometric analysis. Rats were
labeled with 8 mg/kg of calcein subcutaneously (Sigma, St. Louis, MO)
at 3 days before being killed. The left and right tibiae
were removed and cut with an Isomet saw (Buehler, Lake Bluff, IL). The
proximal section of each right tibia was stained by Villanueva
osteochrome bone stain (Polysciences, Warrington, PA) for 5 days and
then processed for methyl methacrylate embedding without
decalcification (33). The specimens were destained and dehydrated with
sequential changes (70, 95, and 100%) of ethanol solution and xylene
and then embedded in methyl methacrylate (Eastman Organic Chemicals,
Rochester, NY). Proximal tibiae were frontal-cut longitudinally with a
diamond wire histo-saw (Delaware Diamond Knives, Wilmington, DE) at a thickness of 40 µm and then coverslipped with Eukitt
(Calibrated Instruments, Hawthorne, NY).
Histomorphometric parameters of cancellous bone of the proximal tibia
were measured with a digitizing morphometry system (OsteoMetrics, Atlanta, GA) and the nomenclature standard (20) as described previously
(33). Briefly, the measured parameters included total tissue area,
trabecular bone area and perimeters, and calcein labeled perimeters in
the secondary spongiosa of the metaphysis in the region 1-4 mm
proximal to the growth plate-metaphysial junction. These parameters
were then used to calculate cancellous bone volume, total volume, and
labeled surface tissue area.
The left tibia was removed and decalcified for 5 wk at 4°C in 10%
EDTA containing 7.0% sucrose (pH 7.2). After a thorough wash in PBS solution, the decalcified bones were dehydrated in graded
alcohol and embedded in 2-hydroxyethyl methacrylate (JB4-Plus embedding
kit, Polysciences). Polymerization was carried out at 4°C in a cold
room overnight. Polymerized blocks were kept in a desiccator at 4°C
until sectioning. Sections of 4-µm thickness were cut with a
microtome (2040 Reichert-Jung, Cambridge Instruments, Buffalo, NY) and
then mounted on glass slides. Tartrate-resistant acid phosphatase was
localized with a commercially available kit with
-naphthyl phosphate
disodium salt as substrate (Sigma)
(33).
Cell culture. Both femoral and tibial
bones were removed from the rats at 6 wk post-HX. Age-matched intact
rats served as the control group. Under aseptic conditions, the bones
were dissected free of soft tissue, and the epiphysial cartilage was
scraped away with a scalpel and discarded. The distal end of the bones was then cut open, and bone marrow was flushed out with 10 ml
-MEM
medium (without phenol red) supplemented with 10% FBS,
antibiotic-antimycotic, 50 µg/ml ascorbic acid, and 2 mM
L-glutamine (basal medium) with an 18-gauge needle for the femur and a 22-gauge needle for the tibia.
The bone marrow cells were pooled from each of the four bones of each
animal, and a single cell suspension was created by repeated flushing
through an 18-gauge needle followed by a 22-gauge needle. For the
initial proliferation experiments, cells were seeded at a density of
3.5 × 105/cm2
and allowed to attach for 3 days before the removal of nonadherent cells. Differentiation experiments were carried out in the above medium
supplemented with 10 mM
-glycerophosphate and
10
8 M dexamethasone
(differentiation medium), and initial plating was at the same density
as in the proliferation experiments. Cell numbers were determined with
a hemocytometer.
Adherent and nonadherent cell
fractions. Bone marrow cells were plated at a density
of 3.5 × 105/cm2.
After a 1-day initial attachment period, the nonadherent cells in the
medium were removed and cultured in separate dishes, and either basal
or differentiation medium was replaced in the adherent culture. The
first medium change for the adherent cultures took place 4 days later,
and medium was changed every second day thereafter. The nonadherent
cultures were allowed a 4-day attachment period before the medium was
replaced with fresh basal or differentiation medium. Thereafter, the
medium was changed every second day. Another set of dishes was seeded
at the same density and allowed to attach for 5 days before the
nonadherent cells were removed and the medium was replaced with either
fresh basal or differentiation medium. Cell number was determined after
trypsinization with a hemocytometer.
[3H]thymidine
incorporation. Bone marrow cells were plated as stated
above and cultured in basal medium. The medium was replaced after 72 h,
the adherent cells were cultured for another 4 days, and the medium was
changed every 2 days. One microcurie of
[3H]thymidine (37 Ci/mmol, Amersham, Arlington Heights, IL) was added to each well, and
the cells were cultured for another 24 h. The medium was then removed,
and the cells were washed with PBS and detached with 0.05% trypsin in
PBS. The cells were collected by centrifugation, lysed with 0.1 N
sodium hydroxide, and
[3H]thymidine
incorporation was measured with a scintillation counter and expressed
in counts per minute.
AP activity. On
day
14 of culture, the cells were washed
with 1× PBS and fixed with ice-cold 95% ethanol (EtOH). The
cells were then washed with distilled
H2O and allowed to
equilibrate in 20 mM bicarbonate buffer, pH 8.8, followed by a 20-min
incubation at room temperature with 1 mg/ml
p-nitrophenylphosphate in bicarbonate buffer with continuous agitation.
p-Nitrophenol in the supernatant was
then measured by spectrophotometry at 405 nm, and the results were
expressed as nanomoles per minute per
105 cells. Histochemical staining
for AP was performed with the Sigma 85L-1 kit according to the
directions of the manufacturer. Cell number for the AP cultures was
obtained by the method of Currie (9). After the AP activity of the
cells was measured, the cells were fixed again in 10% Formalin for
further mechanical stability. They were then washed once with tap water
and twice with borate buffer (10 mM, pH 8.8), stained with 1%
methylene blue in borate buffer for 15 min, and then rewashed several
times with tap water followed by borate buffer. Methylene blue-positive
colony-forming units (CFU) were used as a measure of total
CFU-fibroblast (f) colony numbers. Bound methylene blue was then eluted
with 1% HCl, and the absorbance was read at 650 nm. Cell number was
then determined by comparison with a standard curve.
Calcium. At
day
21 of culture, calcium in the cell
layers was extracted with 1% HCl, and calcium content was measured
with atomic absorbance. Mineralized nodules were histochemically
stained at day
18 of culture with alizarin red, pH
6.2, and nodules were counted manually (24).
RT-PCR for semiquantitation of OCN
expression. Bone marrow cells were initially plated at
a density of 5 × 105/cm2
in 60-mm dishes and allowed to adhere to the dish for 5 days before the
nonadherent cells were removed and the basal medium was replaced with
differentiation medium. The cells were then extracted for RNA at 7, 9, 12, 14, and 16 days of culture with Trizol Reagent (Life Technologies)
following the recommendations of the manufacturer. RNA (1 µg) was
used in each reaction and was first treated with DNase I (Life
Technologies). The RNA was then reverse transcribed under the following
conditions: 50 mM Tris · HCl (pH 8.3), 75 mM KCl, 20 mM DTT, 3 mM MgCl2, 0.5 mM each
dGTP, dATP, dTTP, and dCTP, 100 pg
oligo(dT)12-18, 10 U RNase inhibitor (Life Technologies), and 50 U M-MLV reverse transcriptase (Life Technologies). The reaction was incubated at 37°C
for 2 h followed by 100°C for 10 min and then immediately placed on
ice. Aliquots (10 ml) of the reverse transcription reaction were used
in the simultaneous PCR reactions for rat OCN and rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The reactions were
carried out under the following conditions: 20 mM
Tris · HCl (pH 8.0), 50 mM KCl, 0.2 mM each dATP,
dCTP, dGTP, and dTTP, 2.5 mM MgCl, 1.25 U
Taq polymerase (Life Technologies),
and 50 pmol each of forward and reverse primers. Primer sequences were as published in Fleet and Hock (12). Reactions were carried out with a
Gene-Amp PCR system 2400 thermocycler (Perkin-Elmer, Norwalk, CT).
Cycling parameters were as follows: 95°C for 5 min, followed by 20 cycles of 95°C for 15 s, 55°C for 1 min 28 s, 72°C for 45 s, with a final 72°C hold for 7 min. Twenty cycles of PCR were
determined to be within the linear range for both OCN and GAPDH.
Quantitation of PCR products and confirmation of sequence identity were
performed by Southern blot analysis followed by chemiluminescent detection with a modification of the method of Fleet and Hock (12).
Briefly, the Genius nonradioactive detection kit (Boehringer Mannheim,
Indianapolis, IN) was utilized with slight modification. The PCR
reactions were separated on 2.5% agarose gels. After electrophoresis, the gels were denatured, neutralized, and transferred to Biodyne B
membrane (Life Technologies) with 10× standard sodium citrate (SSC) overnight. DNA was fixed to the membrane by baking at 80°C for 1 h. Membranes were prehybridized in 50% formamide, 5× SSC, 5% blocking reagent (Boehringer Mannheim), 0.1%
N-laurylsarcosine, and 0.02% SDS for
at least 4 h at room temperature. Approximately 15 pmol of 3'-end
digoxigenin-linked ddUTP-labeled probe (nonradioactive 3'-end
labeling kit from Boehringer Mannheim) per 100 cm2 membrane were added to the
prehybridization buffer and allowed to hybridize overnight. OCN was
hybridized at 31.8°C and GAPDH at 23°C as described in Fleet
and Hock (12). The sequences of the nested oligonucleotide probes were
1) rat OCN,
5'-GTCTATTCACCACCTTACTGCCCTC-3' and
2) rat GAPDH,
5'-CTAAGCAGTTGGTGGTGCA-3' (Midland Certified Reagent).
After hybridization, the membranes were washed twice at room
temperature in 2× SSC-0.1% SDS for 30 min followed by one 15-min
wash in 0.1× SSC-0.1% SDS. Detection of the digoxigenin probe
was carried out at room temperature. Membranes were washed in
buffer
1 (0.15 M NaCl and 0.1 M maleic acid,
pH 7.5) for 5 min followed by a 1-h incubation in
buffer
2 (buffer
1 with 1% blocking reagent). A
1:5,000 dilution of the anti-digoxigenin-AP conjugate was
then made in buffer
2, and the membrane was incubated in
this solution for 30 min followed by two washes, 15 min each, in
buffer
1 and a 5-min wash in
buffer
3 (0.1 M Tris, 0.15 M NaCl, and 50 mM
MgCl2, pH 9.5). A final incubation
in a 1:100 dilution of CDP-Star in
buffer
3 for 5 min was performed, and the
membranes were exposed to Fuji NIF film for 25 s to 5 min. Band
intensities were measured with the SigmaGel computer program (Jandel
Scientific Software, San Rafael, CA) and an HP Desk Scan II scanner.
OCN expression was normalized to GAPDH.
Northern analysis. Total cellular RNA
was extracted from cultures as stated above, and 15-µg aliquots were
fractionated on 1.2% agarose-formaldehyde gels. The RNA was then
transferred to Biodyne B membrane (Life Technologies) with 20×
SSC overnight. Membranes were prehybridized in Hybrisol I (Oncor,
Gaithersburg, MD) and 0.5 mg/ml sonicated salmon sperm DNA at 42°C
for >2 h. The following cDNA probes were used: a 1.0-kb mouse OPN
EcoR I fragment, a 1.0-kb mouse BSP
EcoR I fragment (generous gifts of Dr. David
Rowe, University of Connecticut, Farmington, CT), and a 1.2-kb
EcoR I human GAPDH fragment (Clonetech, Palo
Alto, CA). Probes were labeled with
[32P]dCTP (NEN,
Boston, MA) with the RadPrime DNA labeling system (Life Technologies).
Membranes were hybridized overnight at 42°C followed by two room
temperature washes in 2× SSC-0.1% SDS for 20 min each, one room
temperature wash with 0.2× SSC-0.1% SDS for 15 min, and a final
wash with 0.2× SSC-0.1% SDS at 42°C for 15 min. The
membranes were then exposed to Fuji NIF film overnight with
intensifying screens at
80°C. Between hybridizations, bound probes were removed with 0.1% SDS at 80°C. Band intensities were measured with the SigmaGel computer program (Jandel Scientific Software) and an HP Desk Scan II scanner. Expression levels were normalized to GAPDH expression.
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RESULTS |
Body weight and histomorphometric
data. At 6-wk postsurgery, as previously demonstrated,
the body weight of the HX rats remained within a 5% range of their
initial presurgery weight, whereas the age-matched intact control rats
experienced a 32% gain in weight (Table
1). The
cancellous bone volume of the proximal tibia was significantly
decreased by 6-wk post-HX compared with the intact control group. Using
calcein labeling to examine the osteoblast population on the bone
surface, we found that the osteoblast number was decreased by HX.
Furthermore, the osteoclast number per millimeter of growth plate was
also decreased to less than one-half of that of the intact animals.
Characterization of bone marrow stromal cell
proliferation under basal and differentiating
conditions. Total marrow stromal cells recovered from
the HX animals were significantly less than total numbers recovered
from the intact animals (3.35 ± 0.57 × 108 cells HX vs. 4.57 ± 0.77 × 108 cells control). These
data represent the total number of cells recovered from two femora and
two tibiae (n = 10). However, initial plating densities were the same in both groups throughout the experiment. After 7 days of incubation in basal medium, the 24-h [3H]thymidine
incorporation was 88% greater in the HX group, but the cell number per
dish did not differ from the intact control (Table
2). However, by 14 days
of culture in basal medium, the cell number and total CFU-f were both
higher (81 and 70%, respectively) in the HX group compared with the
intact control. When the initial 3-day attachment period was followed
by the addition of differentiation medium, cell number was still
greater in the HX group. By day 8 under differentiating conditions, it
was already 67% more than the intact control group, and by
day
14 it was 90% greater (Table 3). CFU-AP and CFU-f colony numbers, after
14 days of incubation in differentiation medium, were also greater in
the HX vs. intact control group by 58 and 19%, respectively. The
percentage of AP-positive colonies was also greater in the HX group.
Sixty percent of the total colonies in control cultures were AP
positive, whereas 80% of the total colonies were AP positive in the HX
cultures.
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Table 2.
Proliferation characteristics of bone marrow stromal cells from intact
control and HX rats under basal culture conditions
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Table 3.
Proliferation characteristics of bone marrow stromal cells from intact
control and HX rats under differentiation culture conditions
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Recruitment of noncommitted precursor
cells. To investigate whether the increased cell number
in the HX group is because of an enhanced recruitment of noncommitted
precursors, we separated the stromal cells into adherent and
nonadherent fractions. The cell fractions were separated and cultured
as stated in MATERIALS AND METHODS.
Cell numbers were counted at days
8 and
14 in the basal cultures and at
day
14 for cultures grown in
differentiation medium. At day
8 of basal culture, the cell number in
the 1-day adherent fraction of the HX group was only 44% of the intact
control, but by day
14 there was no significant difference
in cell number (Fig. 1). This implies that
the initial committed mesenchymal precursor population in the HX
animals is less than that of the intact control, but after exposure to
optimal culture conditions, the HX cells surpass the controls in
proliferative capacity. In the nonadherent fractions by
day
14, the HX group had a 35% greater cell number than the intact control. Moreover, the HX cultures had
greater cell numbers at both time points when cells were allowed to
attach and recruit other noncommitted mesenchymal precursor cells for 5 days. Therefore, we conclude that there is an enhanced recruitment of
noncommitted stromal cells in the HX cultures, and the proliferative
capacity of the committed precursor population surpasses control levels
after 14 days of optimal culture conditions. In differentiation medium,
the results were similar to those with the basal medium in the 5-day
adherent fractions, but in the 1-day adherent and nonadherent
fractions, the HX cultures had a much higher cell number (+268 and
+218%, respectively) than the intact control cultures. This is
possibly an indication that both the committed and noncommitted stromal
cells of the HX animals are more responsive to differentiating
conditions.

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Fig. 1.
Effect of hypophysectomy (HX) on proliferation of committed and
noncommitted stromal cells under basal and differentiating conditions.
Cell fractions were separated and cultured in basal (bas) or
differentiation (diff) medium as described in
MATERIALS AND METHODS,
and cell numbers were measured at days
8 and
14. Values are means ± SD of 3 replicate cultures from each of 6 different animals/group. Hypox,
hypophysectomy; adh, adherent; nadh, nonadherent. * Significant
difference between HX and intact control cultures as determined by
Student's t-test.
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Characterization of bone marrow stroma-derived
osteoprogenitor cell differentiation.
CELLULAR INDEXES.
When AP activity was measured at day
14 of culture under differentiating
conditions, the HX cultures demonstrated a greater activity (at least
+110%) in all cell fractions (Fig. 2).
Mineralized nodules were counted at
day
18, and calcium content was measured at day
21 of incubation. These
differentiation parameters were also greater in the HX cultures vs. the
intact control cultures in all fractions analyzed. The most significant
difference was found with the 1-day adherent fraction, where the intact
control cultures had developed <10% the number of nodules and only
15% of the level of calcium as the HX group (Fig.
3). Again these results indicate that both
the committed preosteoblasts and the noncommitted stromal cells in the
HX cultures have an enhanced sensitivity to differentiating conditions.

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Fig. 2.
Effect of HX on alkaline phosphatase activity on both committed and
noncommitted osteoblasts. Cell fractions were separated and cultured in
differentiation medium as described in MATERIALS AND
METHODS. Alkaline phosphatase activity was measured on
day
14 of culture. Open bars, intact
control cultures; filled bars, HX cultures. Values are means ± SD
of 3 replicate cultures from each of 6 different animals/group.
* Significant difference between HX and intact control cultures
as determined by Student's t-test.
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Fig. 3.
Effect of HX on mineralization of both committed and noncommitted
osteoblasts. Cell fractions were separated and cultured in
differentiation medium as stated in MATERIALS AND
METHODS. A: cultures
were stained with alizarin red at day
18 of culture, and colony-forming
units staining positive for calcium (CFU-Ca+) were counted
manually. B: cultures were extracted
for calcium with 1% HCl at day
21 of culture, and calcium was
measured with atomic absorption. Open bars, intact control cultures;
filled bars, HX cultures. * Significant difference between intact
control and HX cultures as determined by Student's
t-test.
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MOLECULAR INDEXES.
Once we found that the cellular markers of osteoblast differentiation
were enhanced in the HX bone marrow stromal cell cultures, we sought to
examine a few osteogenic markers to determine whether they too would be
enhanced. RT-PCR was used to compare relative levels of OCN mRNA
levels. Initially, we analyzed OCN expression levels in 1-day adherent
and 5-day adherent cultures after 12 days of incubation. The 12-day
time point was used because it has been demonstrated to be near the
beginning of matrix maturation and the point at which OCN expression
can first be detected in stromal cell-derived osteoblast cultures (16,
32). In both the 1-day and 5-day adherent cultures, the HX group
demonstrated higher relative OCN expression at
day
12 (3× and 1.7×,
respectively) (Fig.
4A). The
nonadherent fractions were not analyzed for OCN expression. The
significant difference in OCN expression between the control and HX
cultures prompted us to investigate temporal differences in expression.
After a 5-day initial seed, total RNA was extracted from HX and intact
control cultures at days
7, 9, 12,
14, and
16 and analyzed for relative OCN
expression (Fig. 4B). No significant
difference was seen until day
12, and by
day 16, the difference had increased to
the point at which HX culture OCN mRNA levels were 2.7 times those of
the intact control cultures.

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Fig. 4.
Effect of HX on osteocalcin (OC) expression under differentiation
conditions as measured by RT-PCR. Relative expression levels were
quantitated with Southern blot followed by nonradioactive detection.
A: representative Southern blot
demonstrating the effect of HX on OC expression in bone marrow stromal
cells subjected to different seeding times. Cells were allowed to
attach for either 1 day or 5 days before nonadherent cells were removed
and basal medium was replaced with differentiation medium. Cultures
were incubated for 12 days before total cellular RNA was extracted.
B: densitometric evaluation of OC
expression in both intact control and HX cultures after 7, 9, 12, 14, and 16 days of incubation. Stromal cells were allowed to attach for 5 days before nonadherent cells were removed and basal medium was
replaced with differentiation medium. GAPDH, glyceraldehyde-3-phosphate
dehydrogenase; C, control.
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Duplicate cultures were also analyzed by Northern blot to compare
steady-state expression levels of OPN and BSP. Levels of OPN expression
at day
9 were already 1.5 times intact
control levels in the HX cultures (Fig.
5A); BSP
levels, however, did not surpass the control until
day
12, when they reached a level 3.2 times greater than the intact control cultures (Fig.
5B). In the HX cultures, expression
levels of both of these markers seem to reach peak levels earlier than
intact controls, but, by day
16, there is no significant difference
between expression levels. Figure 5C
is a representative Northern blot depicting steady-state levels of OPN
and BSP transcripts in the HX and intact control cultures at
days
12 and
14.

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Fig. 5.
Effect of HX on the osteogenic markers osteopontin (OP) and bone
sialoprotein (BSP) under differentiating conditions as measured by
Northern blot. A and
B: densitometric evaluation of OPN and
BSP expression in HX (filled bars) and intact control cultures (open
bars) after 9, 12, 14 and 16 days of incubation. Stromal cells were
allowed a 5-day attachment period before the nonadherent cells were
removed and the basal medium was replaced with differentiation
medium. C: representative Northern
blot of OPN and BSP expression in HX and intact control cultures at
days
12 and
14.
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DISCUSSION |
Hypophysectomy is known to result in osteopenia, and the
histomorphometric data from these experiments confirm previous
conclusions that this osteopenia is, in part, due to a decrease in the
osteoblast population on the bone surface (23, 33). Hypophysectomy has also been demonstrated to have a negative effect on hematopoiesis and
to result in anemia, leukopenia, thrombocytopenia, and impaired DNA and
RNA synthesis in the bone marrow (17). The total number of marrow
stromal cells recovered from the HX animals is in parallel with these
findings. Total stroma cells recovered from HX animals were
significantly less than totals recovered from the intact controls. This
study was designed to determine whether or not the reduction in
osteogenesis at the bone surface is due to a reduction in the capacity
of osteoprogenitors present in the bone marrow to proliferate and
develop into mature osteoblasts.
In our initial proliferation studies using a 3-day attachment period,
[3H]thymidine
incorporation in the HX cultures is significantly greater than in the
intact control cultures, but there is no significant difference in cell
number at day
8 of culture under nondifferentiating conditions. By day
14, however, the HX group has a
significantly greater cell number than the control, demonstrating that
the HX committed mesenchymal precursor population, when exposed to an optimal microenvironment in vitro, shows a proliferative capacity that
surpasses that of the intact animal. Under differentiating conditions
and a 3-day initial attachment period, the HX cultures show greater
cell numbers after only 8 days of culture, indicating a possibly
greater sensitivity to dexamethasone. When examining total CFU-f under
nondifferentiating and differentiating conditions, we again found
significantly greater numbers in the HX cultures; when comparing the
percentage of total fibroblastic colonies positive for alkaline
phosphatase we also found a greater percentage in the HX cultures vs.
the control. This implies that the osteoblastic precursors of the HX
marrow are a definite contributor to the enhanced proliferative
capacity of the marrow stroma. The HX stromal cells also appear to have
superior recruitment potential, as evidenced by the greater total
number of CFU-f colonies under both conditions. Through visual
observation, we also noted that the area of CFU-f and CFU-AP per dish
was also greater in the HX cultures, which supports our hypothesis of
enhanced proliferative capacity of both mesenchymal precursors and the
preosteoblast population.
To investigate the recruitment potential of the two groups we separated
the bone marrow stromal cells into adherent and nonadherent fractions
as described in MATERIALS AND METHODS
and cultured them under nondifferentiating and differentiating
conditions. Under nondifferentiating conditions, we discovered that the
initial committed mesenchymal precursor population (1-day adherent
fraction) of the HX animal appears to be reduced compared with the
intact control. However, after 14 days of optimal culture conditions, this fraction of cells from the HX cultures has greater cell numbers, indicating a greater proliferative capacity. The proportion of nonadherent cells in the two groups cannot be deduced in this manner
because other factors like recruitment potential contribute to final
cell numbers. We also did not measure the proportion of cells in the
different fractions and therefore cannot attribute greater cell numbers
in the nonadherent fraction of the HX group to greater starting
numbers. If our assumption that the initial committed fraction of cells
is less in the HX group than when comparing the 5-day adherent
fractions, we can say that the committed progenitor population of the
HX animal possesses recruitment potential superior to the intact counterparts.
We have also demonstrated that there is a greater induction of the
osteoblast phenotype from the noncommitted progenitor population in the
marrow stroma by separating these two fractions of progenitor cells and
culturing them in differentiation medium. Under these conditions, all
fractions from the HX animal showed greater proliferative and
differentiation capacities than the intact control cultures. By 14 days
of culture in differentiation medium, cell number in the HX cultures
was very significantly greater in all fractions, indicating an extreme
sensitivity of these cells to differentiating conditions. After
examining differentiation markers at the cellular level in the
different fractions, we found that AP activity per cell was greater in
all fractions of the HX cultures, confirming a greater sensitivity of
these cells to differentiating conditions. Because AP activity is not
exclusively a marker of osteoblasts, we examined calcium-containing
colony-forming units (CFU-Ca) colony numbers and total
calcium content as well. These measurements are indicators of
mineralization potential. CFU-Ca colony numbers were significantly
greater, and calcium content was at significantly increased levels, in
all fractions of the HX cultures. These data correlate with the
increases in cell number and AP activity of these fractions under
differentiating conditions compared with the intact control. Several
factors could be at work here. First, the nonadherent and initial
adherent progenitors from the pituitary-deficient animal appear to be
more sensitive to the inductive properties of the dexamethasone in the
medium. Second, the recruitment potential of the adherent precursor
population could be augmented as discussed earlier. It has been
hypothesized that when the adherent fraction of precursors is exposed
to anabolic stimuli present in the culture medium, they secrete factors
such as prostaglandin E2, which
has been shown to mediate the transition between nonadherent and
adherent preosteoblast cells (24, 25). Because the adherent
preosteoblasts from the HX animals have an enhanced response to
anabolic stimuli as demonstrated through our proliferation experiments,
perhaps they are secreting greater amounts of recruiting factors into the medium and therefore augmenting osteoprogenitor recruitment.
Our examination of molecular markers of differentiation has revealed an
earlier progression toward mineralization and enhanced levels of
expression in HX cultures. We examined gene expression of two
noncollagenous matrix proteins, OPN and BSP. OPN expression has been
detected during both the proliferative and differentiation stages of
osteoblast development (2, 28, 32), but the role it plays in
mineralization has not been clearly defined (32). It has been
speculated, however, that its function during the proliferative stage
of development is related to the mediation of cell attachment and it
conditions the surface of the culture dish in preparation for
mineralization (28, 32). As in these other osteoblastic development
models, we have also detected this matrix protein during the
proliferation stage with considerably higher levels detected in the HX
cultures. The second matrix protein of which gene expression levels
were measured, BSP, has been implicated as an influence in the initial
formation of mineralized nodules (32). When temporal patterns and peak
expression of BSP between the two groups are compared, HX cells not
only reach maximal expression earlier than control cells, but peak
levels are also greater than those of control cells as well. Using
RT-PCR coupled to Southern blot, we also analyzed relative OCN mRNA
levels in the two groups. This osteoblastic marker is not only specific
to bone but is also highly correlated with the calcification of the ECM
(28). By day
12 of culture, there were considerably
greater mRNA levels of OCN in the HX cultures, and this difference
remained through day
16.
It has been postulated that proliferation is functionally related to
the synthesis of the ECM, and the maturation and organization of the
ECM contribute to the shutdown of proliferation. This, in turn,
promotes the expression of genes that prepare the matrix for
mineralization. ECM mineralization or events during the early stages of
mineralization are possibly responsible for downregulating genes
expressed during ECM maturation and organization (28). Therefore, in
this model of osteoblast development, the formation of the ECM is
completely related to the stages of differentiation. If this model is
applied to the current study, it may help to explain the phenomenon
that is occurring in the differentiation of the HX osteoprogenitors.
During the first stage, proliferation is elevated, leading to an
enhanced deposition of matrix proteins during the ECM maturation stage,
as evidenced by increased OPN levels at the end of this stage. This
perhaps led to an early shutdown of proliferation and an early and
enhanced preparation of the matrix indicated by a temporal shift to the
left and greater peak levels of BSP. Ultimately, these events sparked
the increased expression levels of OCN and subsequently mineralization.
This explanation still leaves us to explain the causative mechanism behind the initial increase in proliferation of the HX osteoblast lineage cells. Why do the in vivo and in vitro results seem in such
stark contrast?
The elevated response of the bone marrow stromal cells of the HX animal
when placed in vitro could be because of a hypersensitivity of these
cells to a factor(s) present in the fetal bovine serum and lacking for
an extended period of time in vivo. The most obvious of these is growth
hormone (GH), a major systemic growth regulator and mitogenic factor
that exerts its effects on bone both directly and indirectly via
insulin-like growth factor (IGF)-I (3, 10). Circulating GH interacts
with certain receptors located mainly in hepatic tissue, resulting in
the stimulation of the synthesis and secretion of IGF-I (1). IGF-I also
has paracrine-autocrine properties and is locally secreted by
osteoblasts in response to GH (10). Perhaps these osteoprogenitors
experience an upregulation of the IGF-I receptor because of the acute
lack of GH and, subsequently, the lack of systemic and local IGF-I. It
has been demonstrated that human patients with reduced serum levels of
IGF-I experienced a compensatory upregulation of lymphocytic IGF-I
receptor gene expression in response to low circulating IGF-I levels
(11). If this were also true with osteoblasts, it would explain why, when these cells are placed in culture with fetal bovine serum, of
which bovine GH is a definite component, they are stimulated to divide
at an elevated rate. These receptors initiate the
Ras/Raf/MEK/mitogen-activated protein kinase (MAPK) signaling cascade
and MAPKs have been indicated as transcriptional regulators of
proliferation in osteoblasts (27). This is only one possibility,
however, because the HX animal lacks more than just GH. Many other
hormones with anabolic properties in bone are also lacking in vivo,
such as parathyroid hormone and estrogen. Moreover, GH replacement in
HX rats cannot completely restore trabecular bone formation (7),
indicating the involvement of other pituitary and/or local
growth factors in bone formation.
In conclusion, we have observed that once bone marrow stromal cells
from a HX animal are cultured in vitro, they experience an enhanced
proliferative and differentiative capacity compared with those of the
intact animal. The cellular and molecular mechanisms behind this
phenomenon have yet to be elucidated. An understanding of these
regulatory mechanisms is critical for gaining insight into the
pathogenesis of pituitary deficiency-related osteoporosis and other
bone-related diseases, as well as for the development of new treatment regimens.
 |
ACKNOWLEDGEMENTS |
The authors are grateful to Nancy Ling and Amy Ching-Man Chu for
expert technical assistance.
 |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: J. K. Yeh, Metabolism Laboratory, Dept.
of Medicine, Winthrop-Univ. Hospital, Mineola, NY 11501.
Received 20 April 1998; accepted in final form 8 September
1998.
 |
REFERENCES |
1.
Adamo, M. L.,
M. A. Bach,
C. T. Roberts,
and
D. LeRoith.
Regulation of insulin, IGF-I, and IGF-II gene expression.
In: Insulin-Like Growth Factors: Molecular and Cellular Aspects, edited by D. LeRoith. Boca Raton, FL: CRC, 1991, p. 271-303.
2.
Aubin, J. E., F. Liu, L. Malaval, and A. K. Gupta. Osteoblast and chondroblast differentiation.
Bone 17, Suppl.: 77S-83S, 1995.
3.
Barnard, R.,
T. J. Martin,
and
M. J. Waters.
Growth hormone (GH) receptors in clonal osteoblast-like cells mediate a mitogenic response to GH.
Endocrinology
128:
1459-1464,
1991[Abstract].
4.
Bellows, C. G.,
J. E. Aubin,
and
J. N. M. Heersche.
Physiological concentrations of glucocorticoids stimulate formation of bone nodules from isolated rat calvarial cells in vitro.
Endocrinology
121:
1985-1992,
1987[Abstract].
5.
Beresford, J. N.
Osteogenic stem cells and the stromal system of bone and marrow.
Clin. Orthop.
240:
270-280,
1989[Medline].
6.
Cheng, S.-L.,
J. W. Yang,
L. Rifas,
S.-F. Zhang,
and
L. V. Avioli.
Differentiation of human bone marrow osteogenic stromal cell in vitro: induction of the osteoblast phenotype by dexamethasone.
Endocrinology
134:
277-286,
1994[Abstract].
7.
Chen, M.-M.,
J. K. Yeh,
and
J. F. Aloia.
Histologic evidence: growth hormone completely prevents reduction in cortical bone gain and partially prevents cancellous osteopenia in the tibia of hypophysectomized rats.
Anat. Rec.
249:
163-172,
1997[Medline].
8.
Chen, M.-M.,
J. K. Yeh,
and
J. F. Aloia.
Skeletal alterations in hypophysectomized rats. II. A histomorphometric study on tibial cortical bone.
Anat. Rec.
241:
513-518,
1995[Medline].
9.
Currie, G. A.
Platelet-derived growth factor requirements for in vitro proliferation of normal and malignant cells.
Br. J. Cancer
43:
335-343,
1981[Medline].
10.
Ernst, E.,
and
E. R. Froesch.
Growth-hormone dependent stimulation of osteoblast-like cells in serum-free cultures via local synthesis of insulin-like growth factor I.
Biochem. Biophys. Res. Commun.
151:
142-147,
1988[Medline].
11.
Eshet, R.,
H. Werner,
B. Klinger,
A. Silbergeld,
Z. Laron,
D. LeRoith,
and
C. T. Roberts.
Up-regulation of insulin-like growth factor-I (IGF-I) receptor gene expression in patients with reduced serum IGF-I levels.
J. Mol. Endocrinol.
10:
115-120,
1993[Abstract].
12.
Fleet, J. C.,
and
J. M. Hock.
Identification of osteocalcin mRNA in non-osteoid tissue of rats and humans by reverse transcription-polymerase chain reaction.
J. Bone Miner. Res.
9:
1565-1573,
1994[Medline].
13.
Hu, J. L.,
X. J. Guan,
and
E. R. Sanchez.
Enhancement of glucocorticoid receptor-mediated gene expression by cellular stress: evidence for the involvement of a heat shock-initiated factor or process during recovery from stress.
Cell Stress Chaperones
1:
197-205,
1996.[Medline]
14.
Kember, N. F.
Growth hormone and cartilage cell division in hypophysectomized rats.
Cell Tissue Kinet.
4:
193-199,
1971[Medline].
15.
Maniatopoulos, C.,
J. Sodek,
and
A. H. Melcher.
Bone formation in vitro by stromal cells obtained from bone marrow of young adult rats.
Cell Tissue Res.
254:
317-330,
1988[Medline].
16.
Mathieu, E.,
and
J. Merregaert.
Characterization of the stromal osteogenic cell line MN7: mRNA steady-state level of selected osteogenic markers depends on cell density and is influenced by 17
-estradiol.
J. Bone Miner. Res.
9:
183-192,
1994[Medline].
17.
Nagy, E.,
and
I. Berczi.
Pituitary dependence of bone marrow function.
Br. J. Haematol.
71:
457-462,
1989[Medline].
18.
Nijweide, P. J.,
E. H. Burger,
and
J. H. Feyen.
Cells of bone: proliferation, differentiation and hormonal control.
Physiol. Rev.
66:
855-886,
1986[Free Full Text].
19.
Owen, M.
Marrow stromal cells.
J. Cell Sci. Suppl.
10:
63-76,
1988[Medline].
20.
Parfitt, A. M.,
M. K. Drezner,
F. H. Glorieux,
J. A. Kanis,
H. Malluche,
P. J. Meunier,
S. M. Ott,
and
R. R. Recker.
Bone histomorphometry: standardization of nomenclature, symbol, and units.
J. Bone Miner. Res.
2:
595-610,
1987[Medline].
21.
Reddi, A. H.,
and
N. E. Sullivan.
Matrix-induced endochondral bone differentiation: influence of hypophysectomy, growth hormone, and thyroid-stimulating hormone.
Endocrinology
107:
1291-1299,
1980[Abstract].
22.
Rickard, D. J.,
T. A. Sullivan,
B. J. Shenker,
P. S. Leboy,
and
I. Kazhdan.
Induction of rapid osteoblast differentiation in rat bone marrow stromal cell cultures by dexamethasone and BMP-2.
Dev. Biol.
161:
218-228,
1994[Medline].
23.
Schmidt, I. U.,
H. Dobnig,
and
R. T. Turner.
Intermittent parathyroid hormone treatment increases osteoblast number, steady state messenger ribonucleic acid levels for osteocalcin, and bone formation in tibial metaphysis of hypophysectomized female rats.
Endocrinology
136:
5127-5134,
1995[Abstract].
24.
Scutt, A.,
and
P. Bertram.
Bone marrow cells are targets for the anabolic actions of prostaglandin E2 on bone: induction of a transition from nonadherent to adherent osteoblast precursors.
J. Bone Miner. Res.
10:
474-487,
1995[Medline].
25.
Scutt, A.,
P. Bertram,
and
M. Brautigam.
The role of glucocorticoids and prostaglandin E2 in the recruitment of bone marrow mesenchymal cells to the osteoblastic lineage: positive and negative effects.
Calcif. Tissue Int.
59:
154-162,
1996[Medline].
26.
Scutt, A.,
H. Mayer,
and
E. Wingender.
New perspectives in the differentiation of bone forming cells.
Biofactors
4:
1-13,
1992[Medline].
27.
Siddhanti, S. R.,
and
L. D. Quarles.
Molecular to pharmacologic control of osteoblast proliferation and differentiation.
J. Cell. Biochem.
55:
310-320,
1994[Medline].
28.
Stein, G. S.,
and
J. B. Lian.
Molecular mechanisms mediating proliferation/differentiation interrelationships during progressive development of the osteoblast phenotype.
Endocr. Rev.
14:
424-442,
1993[Medline].
29.
Syftestad, G. T.,
and
M. R. Urist.
Growth-hormone dependent matrix-induced heterotropic bone formation.
Proc. Soc. Exp. Biol. Med.
163:
411-415,
1980.
30.
Thorngren, K. G.,
L. I. Hansson,
K. Menander-Sellman,
and
A. Stenstrom.
Effect of hypophysectomy on longitudinal bone growth in the rat.
Calcif. Tissue Res.
11:
281-300,
1973[Medline].
31.
Wlodarski, K. H.
Properties and origins of osteoblasts.
Clin. Orthop.
252:
276-293,
1990[Medline].
32.
Yao, K.-L.,
R. Todescan,
and
J. Sodek.
Temporal changes in matrix protein synthesis and mRNA expression during mineralized tissue formation by adult rat bone marrow cells in culture.
J. Bone Miner. Res.
9:
231-240,
1994[Medline].
33.
Yeh, J. K.,
M.-M. Chen,
and
J. F. Aloia.
Skeletal alterations in hypophysectomized rats. I. A histomorphometric study on tibial cancellous bone.
Anat. Rec.
241:
505-512,
1995[Medline].
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