From the Geriatric Research, Education, and Clinical
Center (GRECC), and Research Service, Veterans Affairs Medical Center,
and the Departments of § Medicine, ¶ Biochemistry & Molecular Biology, and
Neurology,
University of Miami School of Medicine, and the
§§ Stein Gerontological Institute, Miami,
Florida 33125
Received for publication, December 8, 2000
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ABSTRACT |
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Osteoblasts and adipocytes are
thought to differentiate from a common stromal progenitor cell.
These two phenotypically mature cell types show a high degree of
plasticity, which can be observed when cells are grown under specific
culture conditions. Gap junctions are abundant among osteoblastic cells
in vivo and in vitro, whereas they are
down-regulated during adipogenesis. Gap junctional communication (GJC) modulates the expression of genes associated with the
mature osteoblastic phenotype. Inhibition of GJC utilizing
18- Osteoblasts and adipocytes are phenotypically distinct mature
cells thought to differentiate from a common mesenchymal progenitor cell (1-2). The molecular events that determine lineage progression toward a specific phenotype remain to be fully characterized. Either
cell type can be cultured in vitro under conditions that foster or maintain their respective phenotypes. Osteogenic conditions include the presence of ascorbic acid, Gap junctions (GJ) are aggregations of individual gated cell-to-cell
channels that allow the exchange of small molecules (up to 1 kDa)
between the cytoplasms of adjacent cells (7). Connexin43 (Cx43) has
been identified as the major component of gap junctions in osteoblastic
cells (8-11) and in bone (12). Cx43 expression and junctional
permeability increase during in vitro osteoblastic maturation and are regulated by osteotropic factors (8, 13, 14). The
responsiveness of osteoblastic cells to stimulation by parathyroid
hormone is modulated by gap junctional communication (GJC) (15, 16).
Moreover, inhibition of gap junctional communication interferes with
the maturation process of osteoblastic cells in culture (17, 18). In
contrast, murine marrow-derived preadipocytic cells undergoing in
vitro adipogenic differentiation down-regulate Cx43 and GJC (19).
Accordingly, these data suggest that GJC may play a critical role in
modulating the progression of progenitor cells toward a specific
phenotype in vivo. Moreover, we have reported that in the
rat, Cx43 is down-regulated as a function of age in most tissues
examined (20).
In this study we investigate whether modulation of GJC per
se can influence the phenotype of osteoblastic cells in culture. We have used 18- Cell Cultures and Treatments--
The cloned nontransformed
murine osteogenic cell line MC3T3-E1 (23) was kindly provided to us by
Dr. M. Kumegawa (Meikai University, Sakada, Japan) and subcultured in
Measurement of Gap Junctional Communication--
GJC was
assessed using a modification of the two-dye method (26) as previously
described (16). Briefly, cells grown in osteogenic medium in parallel
cultures identical to those to be assayed for GJC were loaded with the
dyes calcein-AM (Molecular Probes, Eugene, OR; 4 µM) and
DiI (Molecular Probes; 8-10 µM). Double-labeled donor
cells were trypsinized and ~100-200 single cells were gently
overlaid on unlabeled recipient cells growing in 6-well plates
(Costar). Transfer of GJ-permeable calcein (green fluorescence) from
the double-labeled donor into the recipient cells was monitored for
periods of up to 24 h. The GJ-impermeable DiI (red fluorescence)
was monitored to identify the donor cells. GJC was scored as 0 when no
transfer of calcein was observed, as 1 when calcein transferred from
the donor cell to an adjacent recipient cell, as 2 when the adjacent
cell transferred calcein to a subsequent adjacent cell in a direction
away from the donor cell, as 3 when transfer was to yet a third cell
away from the donor, and so on. Dye transfer to 5 or more cells away
from the donor was difficult to quantify and was scored as Isolation and Analysis of RNA--
For RNA isolation, cells were
grown in 60-mm or 100-mm dishes (Costar). At the indicated times the
medium was removed, and total RNA was extracted as previously described
(13) using the TriZol Reagent (Life Technologies, Inc., Manassas, VA)
according to the manufacturer's instructions. RNA was quantitated
spectrophotometrically. For Northern blot analysis, 10 to 20 µg of
total RNA was processed as previously described (13). A mouse bone
sialoprotein (BSP) plasmid was kindly provided by Dr. Marian Young, the
mouse osteocalcin (OC) probe was a 371-bp fragment obtained by RT-PCR
corresponding to positions 8-379 of GenBankTM/EBI locus
MMBGPR, and the mouse GAPDH fragment was from Ambion (Austin, TX).
For semiquantitative RT-PCR analysis, total RNA was treated with RQ1
RNase-free DNase (Promega, Madison, WI), RNA was precipitated in a 2 M LiCl (Sigma) solution, and quantitated
spectrophotometrically. Five micrograms of high molecular weight RNA
was reverse transcribed using MuLV reverse transcriptase, 200 pmol
random hexamer primer, and 50 pmol of oligo(dT) (16). Aliquots (4%) of
the total cDNA were amplified in each PCR in a 20-µliter reaction
mixture containing 10 pmol of 5' and 3' primers in a standard PCR
buffer supplemented with 0.5 µCi of [32P]dCTP (10 µCi/µl; PerkinElmer Life Sciences). Each sample was run in
duplicate. Controls lacking reverse transcriptase were treated in the
same fashion. All PCR reagents were from Perkin-Elmer (Norwalk, CT).
Amplifications were performed in a GeneAmp9600 thermal cycler
(Perkin-Elmer) for 17-28 cycles (typically: 94 °C for 30 s;
55 °C for 60 s; 72 °C for 60 s) after an initial denaturation of 94 °C for 2 min. PCR products in a 10-µl aliquot were size separated by electrophoresis in 6%
acrylamide/Tris-borate-EDTA gels. Quantitation of the
amplification reactions was performed by electronic autoradiography
using an InstantImager analyzer (Packard Instrument Co., Meriden, CT).
Semiquantitative analysis of gene expression was assessed from the
differences between amplified products normalized to the corresponding
elongation factor (EF-1a) PCR product. Primer Sequences: mouse
osteocalcin (371-bp product; GenBankTM/EBI locus: MMBGPR),
mOC-F (5'-CAAGTCCCACACAGCAGCTT-3') and mOC-R (5'-AAAGCCGAGCTGCCAGAGTT-3); human PPAR- Cytochemistry--
Cells were washed briefly in
phosphate-buffered saline and fixed in 2% formaldehyde, 0.2%
glutaraldehyde for 30 min at 4 °C. Lipid droplets were stained using
a 3% solution of Sudan IV (Sigma) or Oil-Red-O (Sigma) in isopropyl
alcohol/water (3:2) according to the manufacturer's specifications.
Matrix-bound mineral content was determined using alizarin red-S as
previously described (27).
Quantitation of Cytoplasmic Triglycerides--
Cellular lipids
were extracted directly from the tissue culture wells with
hexane/isopropyl alcohol (3:2; v/v) as described (28). Cellular
cholesterol and triglyceride were measured by published methods (29,
30). After the lipid extraction, cellular proteins were dissolved in
0.1 M NaOH, and protein concentration was determined by the
method of Lowry et al. (31) using bovine serum albumin as
standard. Cholesterol and triglyceride mass were expressed as µg
lipid per mg of cell protein.
Statistical Analysis--
Data are expressed as the mean ± S.E. for each group. Statistical differences among treatment groups
were evaluated with analysis of variance. p values less than
0.05 were considered to be significant.
Effect of AGRA on GJC during Long-Term Cultures of MC3T3-E1
Cells--
MC3T3-E1 cells were seeded as described under
"Experimental Procedures," and the following day (day 0) the medium
was replaced with medium supplemented with ascorbic acid and
Effect of Inhibition of GJC on the Expression of Osteoblastic
Markers in MC3T3-E1 Cells--
MC3T3-E1 cells were seeded and grown as
described under "Experimental Procedures." Parallel cultures were
grown in the presence or absence of vehicle (Me2SO), 100 µM GA, or 100 µM AGRA and used to determine
matrix-bound mineral or for RNA isolation. Growth medium was changed
every 3-4 days, and fresh test substances were added every time the
medium was changed. AGRA-mediated inhibition of GJC resulted in a
decrease in matrix mineral content at all times tested (Table I).
Northern blot analysis demonstrated that expression of BSP and OC
(Table I) mRNAs, assayed within 4 h after addition of
chemicals, were decreased only when GJC was inhibited. Semiquantitative
RT-PCR analysis of the same RNA used in the Northern blot
experiments confirmed the Northern blot data (not shown).
Because inhibition of GJC in long-term cultures of MC3T3-E1
preosteoblastic cells inhibited the development of the osteoblast phenotype, we investigated whether it would have an effect on the
appearance of the adipocytic phenotype. Sudan IV staining of cells
grown under osteogenic conditions for 28 days showed that lipid
droplets accumulated in the cytoplasm of some of the cells only when
GJC had been inhibited by AGRA (Fig.
1A).
Effect of AGRA, Oleamide, and GA on GJC in Human Osteoblastic
Cells--
Having found that inhibition of GJC not only inhibited the
development of an osteoblastic phenotype but stimulated adipocytic characteristics (cytoplasmic lipid droplets) in long-term cultures of
murine preosteoblastic MC3T3-E1 cells, we wanted to determine whether
inhibition of GJC for a relatively short term had an effect on the
capacity of phenotypically mature human osteoblastic cells to develop
an adipocytic phenotype. Human U-2 OS and bone-derived primary
osteoblastic cells were seeded as described under "Experimental Procedures" and the next day (day 0) exposed to osteogenic
conditions. The cells were grown for 7 days in osteogenic medium until
they were fully confluent. On day 8 the cells were incubated in the absence or presence of 50-90 µM AGRA, 20-90
µM oleamide, 20-90 µM GA, or vehicle
(Me2SO) in osteogenic medium. GJC was determined using
donor cells grown in parallel in the absence of any inhibitors. GJC was
evaluated every 2-6 h for up to 24 h after overlaying the
dye-loaded cells. AGRA and oleamide effectively inhibited GJC in a
dose-dependent fashion, whereas GA had no inhibitory effect
on GJC (Fig. 2). At a concentration of 90 µM, AGRA inhibited GJC without any detectable deleterious
effects on the cells (no change in cell numbers; not shown); however,
it was observed that after 24 h the cells recovered some of their
capacity to communicate via GJ. In a fashion similar to that
observed on day 8, AGRA inhibited GJC on days 9 and 10 when fresh
medium containing inhibitors was added (not shown). Oleamide
effectively inhibited GJC at a concentration of 70 µM in
U-2 OS and at 90 µM in primary osteoblasts. In a similar fashion, oleamide-treated cells also partially recovered their capacity
to communicate via GJ after 24 h. However, oleamide appeared to
inhibit GJC more effectively for longer periods of time compared with
AGRA.
Effect of Inhibition of GJC on the Expression of Adipocytic Markers
in Human Osteoblastic Cells--
Human U-2 OS cells express phenotypic
characteristics of mature osteoblasts (33). Also the bone-derived
primary osteoblastic (hOB) cells used in these studies showed alkaline
phosphatase activity and expressed bone sialoprotein mRNA (not
shown). Cells were seeded and grown under osteogenic conditions in
parallel cultures for 4, 8, or 15 days as described under
"Experimental Procedures." In each case the cells were exposed to
the GJC inhibitors AGRA or oleamide, the noninhibitory analog GA, or
vehicle for 3 consecutive days. Because AGRA and oleamide were
effective in inhibiting GJC for approximately a 24-h period, the medium
was replenished with fresh osteogenic medium (containing vehicle, AGRA,
oleamide, or GA) each consecutive day. On the fourth day after
beginning the treatments with the chemical agents, the cells were fixed
and stained for cytoplasmic lipid droplets with Sudan IV or Oil-Red-O.
Lipid staining showed the accumulation of cytoplasmic droplets only in
cells from cultures grown under osteogenic conditions for 8 days
(confluent cultures) after GJC was inhibited by AGRA or oleamide for 3 consecutive days (Fig. 3). No
accumulation of lipid droplets was detected in sparse U-2 OS cultures
grown for 4 days or in compact overconfluent cultures grown for 15 days in which GJC had been inhibited with either AGRA or oleamide for an
additional 3 consecutive days (not shown).
To ascertain that lipid droplet accumulation in the cytoplasm of
AGRA-treated cells was caused by inhibition of GJC and not uptake of
excess lipids in the growth medium or fatty acid-induced adipogenesis,
U-2 OS and hOB cells were grown for 7 days and exposed to the
nonmetabolizable fatty acid
To confirm that the observed results were not an isolated phenomenon
and that indeed inhibition of GJC could promote the
trans-differentiation of other human osteoblastic cells to an
adipocytic phenotype, the osteoblastic cell lines SaOS, HOS, and an
additional primary osteoblastic culture were exposed to the same
treatment. In all cases the osteoblastic cells accumulated lipid
droplets only when complete inhibition of GJC by either AGRA or
oleamide was achieved (Fig. 4). AGRA- and
oleamide-mediated inhibition of GJC in SaOS, HOS, and additional
primary osteoblastic cultures was time- and dose- dependent in a fashion similar to that observed for
U-2 OS (not shown).
Analysis of the lipid content in U-2 OS cells indicated that inhibition
of GJC resulted in a 3-5-fold increase in triglyceride levels in the
cytoplasm of the cells (Fig. 5), whereas
the cholesterol levels were not affected (not shown). In a similar
fashion, analysis of lipid content in hOB cells showed that inhibition
of GJC resulted in a 3-8-fold increase in cytoplasmic triglyceride
content without affecting cholesterol levels (not shown).
To further characterize the effect of inhibition of GJC on the
development of an adipogenic phenotype by human osteoblastic cells, the
level of expression of PPAR- The results demonstrate that the effects of AGRA-mediated
inhibition of GJC in MC3T3-E1 osteoblastic cells are very similar to
those obtained as a result of gene-mediated inhibition of GJC utilizing
dominant-negative mutants of Cx43 (18). AGRA-mediated inhibition of GJC has been utilized as a means to demonstrate other
biological events in which GJC was found to play a significant role
(34-36). We have previously reported that Cx43 (13) and GJC (16)
increase during in vitro osteoblastogenesis. Inhibition of
GJC in MC3T3-E1 cells with AGRA is reversible, and because the medium
containing the inhibitor is replaced every 3-4 days, it is likely that
GJC is at least partially recovered among the cells before the addition
of fresh inhibitor blocks it again. Long-term GJC inhibition was
sufficient to interfere with the ability of osteoblastic cells to
express two genes, BSP and OC, intimately
associated with the mature osteoblastic phenotype. GJC inhibition also
profoundly affected the capacity of the osteoblastic cells to produce a
mineralized extracellular matrix, the ultimate phenotypic hallmark of
an osteoblast. In addition, GJC inhibition not only blocked the
progression of preosteoblastic cells toward the mature osteoblastic
phenotype, but it appeared to stimulate cytoplasmic accumulation of
lipid droplets in a portion of the cells, a characteristic of
adipocytic cells.
Our results also demonstrate that cultured human bone-derived
osteoblastic cells, as well as widely used osteoblastic cell lines,
undergo trans-differentiation toward the adipogenic phenotype after 3 days of AGRA- or oleamide-mediated inhibition of GJC despite being
grown under osteogenic culture conditions. Adipogenesis was
characterized by the expression of functional as well as molecular markers. Lipid staining and subsequent analysis showed that indeed triglycerides accumulate in the cytoplasm of cells as a consequence of
GJC inhibition by either AGRA or oleamide. This phenotypic change took
place with a parallel increase in PPAR There is a possibility that AGRA may be inducing adipogenesis via a
mechanism different from inhibition of GJC. Previous studies have shown
that AGRA can inhibit calcium waves in addition to inhibiting GJC (38).
We have used oleamide, which does not inhibit intercellular calcium
signaling (38), instead of AGRA and found it to inhibit GJC in a
dose-dependent fashion and to have similar effects in
inducing trans-differentiation to adipocytes. We observed that complete
inhibition of GJC with either AGRA or oleamide was required in order
for osteoblastic cells to develop adipocytic characteristics within the
time frame described. The results obtained using AGRA and oleamide, two
inhibitors with different chemical structures, support a role for GJC
in determining lineage progression of skeletal cells with
multipotential capacity. A future direction is to determine the
adipogenic potential of marrow-derived progenitor cells from
Cx43-null mice, whose osteoblastic cells were reported to
have a decreased osteogenic potential (39).
Exposure of U-2 OS cell cultures grown for 4 days (sparsely populated)
or for 15 days (tightly compact) to the same GJC inhibitory conditions
(3 days) did not increase cytoplasmic lipid content. These results
suggest that there may be a window of time, during the culture of these
osteoblastic cells, during which inhibition of GJC will lead to
adipogenesis. Genetic approaches using inducible expression vectors
which will profoundly inhibit GJC, such as dominant-negative for
Cx43 and Cx45, now under development, will confirm the results obtained with the chemical inhibitors.
Our results support the concept that plasticity exists among cells of
the stromal lineage and that modulation of GJC may be a mechanism
involved in determining progression toward an osteoblastic or
adipocytic fully differentiated phenotype. Whereas cell-to-cell coupling via gap junctions would favor osteogenesis, uncoupling would
favor adipogenesis. Changes in GJC could contribute to a shift in
lineage progression by affecting the second-messenger systems activated
in response to external stimuli or the rate of transcription of
specific genes associated with the fully differentiated phenotypes. In
support of this concept, Vander Molen et al. (15) have
demonstrated that the responsiveness of osteoblasts to parathyroid hormone changes as a function of GJC, and Lecanda et al.
(40) have identified specific regulatory sequences in the osteocalcin promoter which are sensitive to GJC in osteoblastic cells.
Cells coupled via gap junctions may transfer second-messenger
molecules, such as calcium, inositol phosphates, or cyclic nucleotides, from the stimulated cell to neighboring cells. Alternatively, when
uncoupled, the relative concentration of the second messenger, e.g. calcium, could reach higher intracellular levels than
when the cell is coupled. Thus cell coupling may influence the
amplitude and the duration of the response to a stimulus, which may, in turn, regulate the cascade of events (e.g. protein kinases)
leading to transcription factor activity modulation. Inactivation of a particular set of transcription factors, such as those involved in the
regulation of OC or BSP expression, in parallel with activation of a
different set, such as PPAR Modulation of skeletal GJC may represent a pharmacological target by
which inhibition of marrow adipogenesis can take place with the
parallel enhancement of osteoblastogenesis. Such a scenario could
provide a novel therapeutic approach to the treatment of human
age-related osteopenic diseases and postmenopausal osteoporosis.
-glycyrrhetinic acid (AGRA) blocks the maturation of
pre-osteoblastic cells in vitro. Moreover, cytoplasmic
lipid droplets are detectable at the end of the culture period,
suggesting that GJC inhibition may favor an adipocytic phenotype. We
used several human osteoblastic cell lines, as well as bone-derived
primary osteoblastic cells, to show that confluent cultures of human
osteoblastic cells grown under osteogenic conditions developed an
adipocytic phenotype after 3 days of complete inhibition of GJC using
AGRA or oleamide, two dissimilar nontoxic reversible inhibitors.
Development of an adipogenic phenotype was confirmed by the
accumulation of triglyceride droplets and the increase in mRNA
expression of the adipocytic markers peroxisome proliferator-activated
receptor
2 and lipoprotein lipase. Glycyrrhizic acid, a
noninhibitory AGRA analog, or
-bromopalmitate, a nondegradable fatty
acid, had no effect. Modulation of skeletal GJC may represent a new
pharmacological target by which inhibition of marrow adipogenesis can
take place with the parallel enhancement of osteoblastogenesis,
thus providing a novel therapeutic approach to the treatment of human
age-related osteopenic diseases and postmenopausal osteoporosis.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glycerol phosphate, and dexamethasone or vitamin D3, depending on the maturational
stage of the osteoblast precursor, in the culture medium. In contrast, adipogenic conditions include the presence of
3-isobutyl-1-methylxanthine (IBMX),1 insulin, and
dexamethasone in the culture medium. Nutall et al. (3) have
shown that mature human osteoblastic cells are capable of
trans-differentiation to adipocytes when cultured in the presence of
dexamethasone and IBMX. On the other hand, Park et al. (4) have shown that cloned single human adipocytes can dedifferentiate into
fibroblast-like cells, which subsequently differentiate into osteoblasts or adipocytes under appropriate culture conditions. This
plasticity between the differentiation of osteogenic and adipogenic
human cells provides further support for a common precursor and has
significant physiological implications, because the decrease in bone
mass associated with age-related osteopenia and osteoporosis is
accompanied by an increase in bone marrow adipose tissue (5-6).
-glycyrrhetinic acid (AGRA), a reversible inhibitor of GJC (21, 22) and its noninhibitory analog glycyrrhizic acid (GA)
(21) to confirm that chemical inhibition of GJC interferes with the
timely development of osteoblastic phenotypic markers in the MC3T3-E1
preosteoblastic differentiation model (23) and to determine whether
AGRA-mediated inhibition of GJC can promote the development of
adipogenic phenotypic characteristics. To investigate if human
osteoblastic cells are susceptible to trans-differentiation toward an
adipocytic phenotype, under osteogenic culture conditions, we utilized
both osteoblasts derived from trabecular bone explants and the widely
used osteoblastic osteosarcoma U-2 OS. Inhibition of GJC was achieved
by the use of AGRA and oleamide (24), two inhibitors of GJC with
dissimilar chemical structures. Adipogenesis is characterized
phenotypically by the appearance of triglyceride droplets in the
cytoplasm of the osteoblastic cells U-2 OS, SaOS, HOS, and primary
cultures of vertebra-derived human osteoblasts, and genotypically by
the expression of mRNAs for the adipocytic lineage-specific
transcription factor peroxisome proliferator-activated receptor
2
(PPAR
2) and enzyme lipoprotein lipase (LPL).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-modified essential medium (
-MEM) supplemented with 5% fetal
bovine serum (FBS), 100 units/ml penicillin (pen), and 100 µg/ml
streptomycin (strep), with a change of medium every 3 to 4 days.
Primary cultures of human osteoblast-like cells were obtained from
trabecular bone explants isolated from a lumbar vertebra of a
10-year-old normal female (primary culture 1) or of a 67-year-old
normal male (primary culture 2) after fatal traumatic injury (25).
Primary human osteoblastic (hOB) cells were grown in Dulbecco's
modified essential medium supplemented with 10% FBS. Human
osteoblastic osteosarcoma U-2 OS cells (ATCC; Manassas, VA) and SaOS
cells (ATCC) were grown in McCoy's 5a medium with 1.5 mM
L-glutamine supplemented with 15% FBS, and HOS cells
(ATCC) in MEM with 10% FBS. Osteoblastic cells were expanded in their
respective growth media and when sufficient cells were obtained they
were seeded at 10,000 cells/cm2 in 6-well plates (for
staining and GJC assays; Costar, Cambridge, MA) or 60- to 100-mm dishes
(for RNA isolation; Costar). The following day (day 0) the respective
growth medium in each case was replaced with osteogenic medium, growth
medium supplemented with 50 µg/ml ascorbic acid (Sigma, St. Louis,
MO) and 10 mM
-glycerol phosphate (Sigma). Thereafter
the cells were grown in osteogenic medium for periods ranging from 7 to
28 days. The medium was replaced every 3-4 days or as specified in the
figure legends. All tissue culture media were from Life Technologies,
Inc. (Grand Island, NY). FBS was from Hyclone Laboratories (Logan, UT).
Glycyrrhizic acid (GA), 18-
-glycyrrhetinic acid (AGRA), and oleamide
were purchased from Sigma, and
-bromopalmitate was from Fluka
(Milwaukee, WI). These test reagents were prepared fresh immediately
before use as 30-mM stocks in Me2SO and
diluted as needed. In those experiments where cells were exposed to GA,
AGRA, oleamide,
-bromopalmitate, or vehicle (Me2SO), the
growth medium was supplemented with these agents every time it was
replaced, and the final Me2SO concentration was always kept
constant (0.3%). Treatment times are indicated in each figure legend.
Studies involving human cells were approved by the local VA
institutional review board for human tissues in research.
5. GJC was scored only when the donor was a single cell at least 10 cells away
from another single donor cell.
2 (419-bp product;
HSU79012): hPPAR-F (5'-ATTCTCCTATTGACCCAGAAAGCG-3') and hPPAR-R
(5'-AGCTTTATTCTCCACAGACACGACATT-3'); human LPL (276-bp product;
HSLPLR): hLPL-F (5'-GAGATTTCTCTGTATGGCACC-3') and hLPL-R
(5'-CTGCAAATGAGACACTTTCTC-3'); human EF-1a (235-bp product; HUMPTI1A):
hEF1a-F (5'-AGGTGATTATCCTGAACCATCC-3') and hEF1a-R
(5'-AAAGGTGGATAGTCTGAGAAGC-3').
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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-glycerol phosphate (osteogenic medium). Under these conditions,
these preosteoblastic cells undergo a maturation process similar to
that of primary osteoblastic cells (32). Parallel cultures were grown
in osteogenic medium in the presence or absence of 100 µM
AGRA, 100 µM GA, or vehicle (Me2SO) for 3 to
28 days. At this concentration, AGRA had no detectable deleterious
effect (i.e., no decrease in cell number) in these cells
(not shown). GJC was measured from 2 to 6 h after overlaying the
donor cells on those days in which the medium was changed. GJC
increased with time in culture in cells treated with either vehicle or
100 µM GA in a fashion similar to untreated cells at all
times tested. In contrast, AGRA inhibited GJC within the time frame in
which it was assayed (Table I).
Phenotypic markers in MC3T3-E1 osteoblastic cells
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Fig. 1.
Sudan IV staining of MC3T3-E1 treated with
100 µM GA
(left) or 100 µM AGRA
(right) for 28 days as described under "Experimental
Procedures." Cytoplasmic lipid droplets were detected only when
GJC was inhibited as a result of AGRA treatment.
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Fig. 2.
GJC in U-2 OS (top) and hOB
(bottom) cells. GJC was assayed as described
under "Experimental Procedures" in untreated cells or cells that
had been exposed to different treatments as a function of time. Both
cell types were very well coupled, with hOB cells showing a somewhat
higher basal level. Treatments were as follows: No Tx ( ),
untreated cells; Veh (
), vehicle; GA20 (+), 20 µM GA; GA90 (×), 90 µM GA;
AGRA20 (
), 20 µM AGRA; AGRA70
(
), 70 µM AGRA; AGRA90 (
), 90 µM AGRA. Oleamide treatment is represented as follows: in
U-2 OS cells (top), large triangles, 50 µM oleamide; and large inverted triangles, 70 µM oleamide; in hOB cells (bottom),
large triangles, 70 µM oleamide; and
large inverted triangles, 90 µM oleamide. Each
point represents the mean ± S.E. of n
20.
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Fig. 3.
Sudan IV staining of U-2 OS
(top) and Oil-Red-O staining of hOB
(bottom) cells. U-2 OS cells were treated with
vehicle (left), 90 µM GA (center),
or 90 µM AGRA (right) for 3 days as described
under "Experimental Procedures." hOB cells were treated with
vehicle (left), 90 µM -bromopalmitate
(center), or 90 µM oleamide
(right). Cytoplasmic lipid droplets were detected only when
GJC was inhibited as a result of AGRA or oleamide treatment.
-bromopalmitate (90 µM)
from day 8 to day 11 under the culture conditions described. Treatment with
-bromopalmitate did not induce accumulation of cytoplasmic lipid droplets (Fig. 3).
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Fig. 4.
Sudan IV staining of hOB2
(left), HOS (center), and SaOS
(right) cells exposed to noninhibitors of GJC
(top) or to GJC inhibitors (bottom)
at the indicated concentrations.
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Fig. 5.
Changes in triglyceride content in the
cytoplasms of U-2 OS cells as a result of inhibition of GJC.
Co, untreated; Veh, Me2SO;
GA, 90 µM GA; Palm, 90 µM -bromopalmitate; AGRA, 70 µM AGRA; Ole, 70 µM
oleamide-treated cells.
2, LPL, and EF-1
transcripts was
evaluated using semiquantitative RT-PCR in U-2 OS cells. Inhibition of
GJC with AGRA resulted in an increase in the expression of the
adipogenic-specific genes PPAR-
2 and
LPL, whereas expression of EF-1a was unaffected (Fig.
6A). Statistical analysis of
changes in the detectable levels of the transcripts are also shown
(Fig. 6B). Similar results were obtained in U-2 OS cells
treated with oleamide and in hOB cells where GJC had been inhibited
with either AGRA or oleamide (not shown).
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Fig. 6.
Changes in the expression of adipocytic gene
markers in U-2 OS cells as a result of inhibition of GJC. A,
RT-PCR analysis of the expression of PPAR -2, LPL, and EF-1
mRNAs in U-2 OS cells treated with 90 µM GA or 70 µM AGRA for 3 days as described under "Experimental
Procedures." Total RNA was treated in the presence of reverse
transcriptase (+RT) or in its absence (
RT). The
products of these reactions were amplified by PCR using specific
primers for 23 cycles to detect EF-1a transcripts or for 27 cycles to
detect PPAR
-2 and LPL transcripts. Inhibition of GJC by 70 µM AGRA increased the expression of PPAR
-2 (419-bp
product) and LPL (276-bp product) transcripts whereas it had no effect
on EF-1a (235-bp product) transcription. B, relative level
of expression of PPAR
-2 and LPL
genes as a function of treatment, determined from the actual number of
dpm obtained for each amplified band normalized to the counts obtained
for EF-1
. Mean ± S.E. of three independent experiments.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 and LPL transcripts, molecular markers of adipogenesis. In the studies described we use
AGRA, a polycyclical lipid, as well as oleamide, an amide of oleic
acid, as inhibitors of GJC. It has previously been reported that
certain lipids, in particular fatty acids, are capable of inducing
differentiation of preadipocytes to adipocytes (37). However, we have
found no evidence that lipids with a chemical structure resembling that
of AGRA or oleamide are capable of inducing adipogenesis. As a control
we have used GA, a glycoside of 18-
-glycyrrhetinic acid (21), which
does not inhibit GJC, at the same concentration and found to have no
effect on adipogenesis. 18-
-glycyrrhetinic acid also inhibited GJC
in osteoblastic cells; however, in our hands it had a significantly
higher toxicity than its
-isomer and was not used for that reason
(data not shown). To further exclude the possibility that the GJC
inhibitors may be inducing adipogenesis as a consequence of their lipid
nature, we used the nonmetabolizable fatty acid
-bromopalmitate and
found that at concentrations of up to 90 µM, it also
was unable to induce trans-differentiation of human bone-derived
osteoblastic cells to adipocytes, suggesting that lipids alone are not
sufficient to induce this process in these cells. Similarly, Nutall
et al. (3) reported that
-bromopalmitate by itself was
unable to induce expression of an adipocytic phenotype in treated osteoblasts.
2 or others involved in adipogenesis, may
cause a genotypic switch leading to a change in the phenotype of a
cell. The role of GJC in this lineage interrelationship may have
important consequences in terms of the progression of osteopenic diseases. The excess adipogenesis in postmenopausal women and in
age-related osteopenia may occur at the expense of osteogenesis and may
involve a decrease in GJC as a result of aging or endocrine changes. We
have recently demonstrated an age-related decrease in marrow
osteoblastogenesis in humans (25). Moreover, in the femurs of young
mice there are twice as many GJ (among osteoblasts, stromal, and
hematopoietic cells) than in those from older animals (41).
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ACKNOWLEDGEMENTS |
---|
We thank David Vazquez and Nubia Rodriguez for their expert technical assistance. We also thank Dr. Marian F. Young (National Institutes of Health) for the plasmid mBSP1.
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FOOTNOTES |
---|
* This work was supported in part by the Veterans Affairs Research Service and GRECC.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. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: University of
Miami School of Medicine, VA Medical Center, GRECC (11GRC), 1201 N. W.
16th St., Miami, FL 33125. Tel.: 305-324-3388; Fax: 305-324-3365; E-mail: pschille@med.miami.edu.
** Present address: Inst. of Pharmacological Sciences, University of Milan, 20133 Milan, Italy.
¶¶ Recipient of a Senior Research Career Scientist Award from the Department of Veterans Affairs.
Published, JBC Papers in Press, January 25, 2001, DOI 10.1074/jbc.M011055200
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ABBREVIATIONS |
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The abbreviations used are:
IBMX, 3-isobutyl-1-methylxanthine;
GJ, gap junction;
GJC, GJ communication;
FBS, fetal bovine serum;
bp, base pair(s);
RT-PCR, reverse
transcriptase-polymerase chain reaction;
LPL, lipoprotein lipase;
GA, glycyrrhizic acid;
PPAR, peroxisome proliferator-activated receptor;
AGRA, 18--glycyrrhetinic acid;
EF, elongation factor.
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