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Department of Orthodontics and Pediatric Dentistry, United Medical and Dental Schools of Guy’s and St. Thomas’ Hospitals, University of London, London, United Kingdom SE1 9RT
Address all correspondence and requests for reprints to: Dr. Peter A. Hill, Department of Orthodontics and Pediatric Dentistry, United Medical and Dental Schools of Guy’s and St. Thomas’ Hospitals, London Bridge, London, United Kingdom SE1 9RT.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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IR-3, an antibody to the type I
IGF receptor, whereas the effects of insulin were only partially
blocked. This antibody blocked the potentiating effects of
platelet-derived growth factor on IGF-I-mediated osteoblast survival,
but only partially blocked those of bFGF. Although a 100% survival of
osteoblasts was seen in the presence of 2% FCS, the highest level
attained by any of the above GF combinations was 
75%. The
monocyte-derived factor, tumor necrosis factor- (TNF) was the
only agent that enhanced PCD in this study. These results suggest that
osteoblast survival is promoted by those GFs sequestrated in bone
matrix and that the type I, but not the type II, IGF receptor is
involved in the response. Our data also indicate that other
unidentified GFs or components of the extracellular matrix may be
involved in promoting osteoblast survival and that TNF may abrogate
their effects in vivo. We propose that these GFs may be
released from bone matrix during phases of bone resorption and promote
osteoblast survival, thereby playing an important role in bone
remodeling, and that PCD induced by TNF may contribute to the bone
loss in inflammatory bone disease. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In many models of PCD, cells are induced to die as a result of changes in environmental stimuli (4, 5, 6). In general, these studies of PCD suggest that the absence of a survival factor, such as a particular hormone or growth factor, will induce a cell to initiate its own cell death.
Skeletal cells and other cells within the bone microenvironment,
synthesize a variety of growth factors (GFs) and cytokines (7). The
extracellular matrix of bone has been shown to be an abundant source of
several polypeptide factors, most notably transforming growth
factor-ß (TGFß) (8), insulin-like growth factors (IGF-I and -II),
platelet-derived growth factor (PDGF), and acidic and basic fibroblast
growth factors (FGFs) (9). When released and presented to responsive
cells during phases of bone resorption, these GFs influence bone
remodeling in conjunction with interleukin-1 (IL-1) and tumor necrosis
factor- (TNF), cytokines produced mainly by bone marrow
mononuclear cells. IGFs are important skeletal GFs not only because of
their abundance in bone, but also because they have important actions
on bone cell function and are expressed by skeletal cells (10). These
various GFs and cytokines influence the differentiated function of
osteoblasts and bone resorption by interacting with cell surface
receptors present on osteoblasts. These effects are critical to the
formation of new bone and to the maintenance of bone matrix.
Based on the importance of GFs and cytokines in bone remodeling we have
investigated their effects on osteoblast survival and apoptosis
in vitro. We report that IGF-I, IGF-II, and basic FGF (bFGF)
are the only GFs that enhanced the survival of osteoblasts in this
study and that the effect of the IGFs is mediated via an interaction
with the type I IGF receptor. Although PDGF had no effect on osteoblast
survival, this GF increased the survival-promoting activity of IGF-I,
IGF-2, and insulin. In contrast, the monocyte-derived product TNF is
the only factor that induced osteoblast apoptosis in this study.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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was purchased from R&D Systems
(Minneapolis, MN). IR-3, a monoclonal antibody to the type I IGF
receptor was obtained from Oncogene Sciences (Cambridge, UK). Terminal
deoxynucleotidyl transferase, biotinylated deoxy (d)-UTP, and
streptavidin fluorescein were purchased from Boehringer Mannheim
(Mannheim, Germany). Human recombinant IGF-I and II, insulin from
bovine pancreas, bFGF, epidermal growth factor (EGF), PDGF, TGFß,
3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT),
and all cell culture reagents were purchased from Sigma Chemical Co.
(St. Louis, MO). Macrophage colony-stimulating factor (M-CSF),
granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage
colony-stimulating factor (GM-CSF), interferon-
(IFN), and murine
leukemia inhibitory factor (LIF) were purchased from British
Biotechnology. 1,25-Dihydroxyvitamin D3
[1,25-(OH)2D3] was a gift from Roche (Welwyn
Garden City, UK). Deoxy-[5-3H]cytidine (SA, 888
gigabecquerels/mmol) was purchased from Amersham International
(Aylesbury, UK).
Preparation of osteoblasts from neonatal mouse calvariae
Calvarial osteoblasts were prepared and characterized as
previously described (11). Briefly, neonatal mouse calvariae were
dissected free from adherent soft tissue, washed in Ca2+-
and Mg2+-free Tyrode’s solution (10 min), and sequentially
digested with 1 mg/ml trypsin (10 min), 2 mg/ml dispase (30 min), and 2
mg/ml collagenase (twice, 30 min each time). Cells released by the last
two collagenase digestions were washed and grown in -modified MEM
(MEM) containing 10% FBS and antibiotics for 4 days before use. All
cultures were maintained at 37 C in a humidified atmosphere of 5%
CO2-95% air. For survival assays, osteoblasts were plated
in 100 µl serum-free CMRL-1066 medium containing 10-3
M thymidine to block cell proliferation, with or without
added cytokines.
Cell survival assays
MTT assay.
Mitochondrial function was assayed by the ability
of viable cells to convert soluble MTT into an insoluble dark blue
formazan reaction product (12). In the bulk cell photometric MTT assay,
the bulk conversion of MTT in the well of a 96-well plate was measured
photometrically as previously described (12). MTT was dissolved in PBS
at a concentration of 5 mg/ml and sterilized by passage through a
0.22-µm filter. This stock solution was added (one part to 10 parts
medium) to each well of a 96-well tissue culture plate, and the plate
was incubated at 37 C for 4 h. Acid-isopropanol (400 µl 10
M HCl in 100 ml isopropanol) was added to all wells and
mixed thoroughly to dissolve the dark blue crystals. After a few
minutes at room temperature, to ensure that all the crystals were
dissolved, the plates were read on a microplate reader at a wavelength
of 570–630 nm. A standard curve was set up using 200–50,000
cells/well, and the absorbance was directly proportional to the number
of cells over this range. The percent survival was defined as
[(experimentalabsorbance -
blankabsorbance)/controlabsorbance -
blankabsorbance)] x 100, where the
controlabsorbance was the value obtained for 10,000
cells/well, which is the number plated at the start of the experiment,
and blankabsorbance was the value obtained in wells
containing medium and MTT without cells. A linear relationship existed
between the absorbance values and the number of cells in the range of
1,000–50,000 cells/well.
Plasma membrane permeability.
Plasma membrane permeability
was assessed using the membrane-impermeant DNA dye, ethidium homodimer,
which labels dead cells. Live cells were labeled using the
membrane-permeant dye calcein AM. Calcein AM is a nonfluorescent dye
that is converted into the fluorescent dye, calcein, by intracellular
esterases and is retained only in cells with an intact plasma membrane.
The concentrated dyes were prepared according to the manufacturers
(Molecular Probes, Eugene, OR) instructions and were added to unwashed
cells in culture to final concentrations of 4 µM for
ethidium homodimer and 2 µM for calcein AM.
Cell proliferation assay
Primary osteoblasts were plated at a density of 1 x
104 cells/well in a 96-well plate and cultured for 48
h in CMRL-1066 medium with GFs or FCS in the presence or absence of
cold thymidine (10-3 M). The cells were pulsed
for the last 6 h with 1 µCi [3H]cytidine, and
DNA-associated radioactivity was performed at the end of the experiment
by fixing the cells with 5% trichloroacetic acid at 4 C for 10 min and
washing in PBS, and the cells were detached with trypsin-EDTA solution
(0.5%:0.02%).
Analysis of DNA fragmentation by agarose gel electrophoresis
DNA fragmentation was analyzed by agarose gel electrophoresis.
Primary mouse osteoblasts were cultured in serum free CMRL-1066 medium
in 75-cm2 flasks with or without TNF. Adherent cells
were lysed with 0.1 M NaCl, 10 mM Tris-HCl (pH
7.5), and 1 mM EDTA in 0.3% SDS and incubated with
proteinase K (500 µg/ml) at 55 C for 15 h. Samples were
extracted with an equal volume of phenol/chloroform, and the total DNA
contained in the aqueous phase was precipitated with 0.1 vol sodium
acetate (3 M; pH 6.6) and 2.5 vol ethanol at -80 C for
15 h. DNA pellets were obtained by centrifugation (13,000 x
g for 15 min) and resuspended in 50 µl 10 mM
Tris-HCl (pH 8.0) and 1 mM EDTA. Samples were then treated
with 10 U/ml deoxyribonuclease-free ribonuclease for 1 h at 37 C.
Electrophoresis was performed on a 1% agarose gel at 50 V for 1.5
h in the presence of 0.5 µg/ml ethidium bromide.
Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end
labeling
DNA cleavage was assessed by the terminal deoxynucleotidyl
transferase-mediated dUTP-biotin nick end labeling (TUNEL) reaction, as
described by Gavrieli et al. (13). Primary mouse osteoblasts
were cultured in Lab-Tek chamber slides (Nunc, Copenhagen, Denmark) in
serum free CMRL-1066 medium, containing thymidine (10-3
M) with or without GFs. After 48 h in culture, the
cells were fixed in 4% paraformaldehyde for 10 min, washed in 10
mM Tris-HCl, pH 8.0, and then permeabilized in 0.1% Triton
X-100 in 10 mM Tris-HCl, pH 8.0, for 5 min. After washing
in 10 mM Tris-HCl, pH 8.0, the cells were preincubated for
10 min at room temperature in the reaction buffer for terminal
deoxynucleotidyl transferase (200 mM potassium cacodylate,
0.22 mg/ml BSA, and 25 mM Tris-HCl, pH 6.6). After 10 min,
the preincubation buffer was removed, and reaction mixture containing
500 U/ml terminal deoxynucleotidyl transferase, 2.5 mM
CoCl2, and 40 µM biotinylated dUTP was added.
After 60 min at 37 C, the reaction was terminated by the addition of
300 mM NaCl and 30 mM sodium citrate. After 25
min at room temperature, cells were washed with PBS and incubated with
streptavidin fluorescein for 60 min at room temperature in the dark.
After extensive washing in PBS, the cells were examined in a Leica
fluorescence microscope.
Electron microscopy
Primary mouse osteoblasts were plated at 2 x
105 cells/ml on glass coverslips that had been previously
coated with poly-L-lysine. Cells were cultured for 48
h in serum-free CMRL-1066 medium with or without TNF. After 48
h, cells were fixed with 2.5% glutaraldehyde in 0.2 M
phosphate buffer (pH 7.3) at 4 C for 4 h. After washing in PBS,
the cells were postfixed in 1% osmium tetroxide in phosphate buffer at
4 C for 30 min. The cells were then dehydrated in ascending grades of
ethyl alcohol and embedded in resin. After removing the glass
coverslip, thin sections were cut on a Reichert ultramicrotome. The
sections were stained with a saturated solution of uranyl acetate and a
4% solution of lead citrate and examined using a Hitachi H7000
electron microscope (Tokyo, Japan).
Statistical analysis
Data are presented as the mean ± SEM of 6–12
cultures/group. Each experiment was repeated three times. Differences
between control and treatment groups were determined by the
Mann-Whitney U test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Survival of primary osteoblasts in culture
When osteoblasts were cultured in serum-free and insulin-free CMRL
1066 medium containing thymidine (10-3 M), it
was found that about 67% of cells survived after 24 h, whereas
only about 28% of cells survived in this medium after 48 h (Table 1
). However, when the osteoblasts were cultured with 2%
FCS in the presence of thymidine (10-3 M),
which blocks cell proliferation (14) and therefore permits the
assessment of factors on cell survival, there was a 100% survival of
the cells at both time intervals.
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, bFGF, IGF-I, IGF-II, or a concentration of insulin
(10-6 M) high enough to activate IGF-I
receptors increased the number of surviving osteoblasts after a 48-h
culture period, but had no effect on their survival after 24 h. In
contrast, the immunoregulatory cytokine TNF induced cell death in
the 24- and 48-h osteoblast cultures (Table 1). A variety of other GFs
and osteotropic factors that were tested had no effect on either
osteoblast survival or cell death, namely PDGF, EGF, TGFß, IL-1,
IL-3, IL-6, IL-11, LIF, IFN, M-CSF, and GM-CSF, G-CSF, and the
osteotropic hormones PTH and 1,25-(OH)2D3.
The effects of bFGF, IGF- I, IGF-II, and insulin on cell number were
due to effects on osteoblast survival rather than to those on cell
proliferation, as the addition of thymidine to the culture medium at a
10-3-M concentration effectively blocked the
proliferative effects of these factors. As shown in Table 2, these factors both increased the number of surviving
cells and proportionally decreased the number of dead cells, so that
the total numbers of cells in the factor-containing microwells were not
statistically different from the numbers in medium alone, thereby
confirming that thymidine had effectively blocked DNA synthesis. The
addition of thymidine did not induce cell death, as the proportion of
live/dead cells was similar in its presence/absence (Table 2).
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).
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(10-10 M) or a high concentration
(10-6 M) of the protein kinase inhibitor
staurosporine was consistent with their having died by PCD rather than
by necrosis. The cells exhibited the typical apoptotic morphology
described by Wyllie et al. (1), specifically, cell shrinkage
and membrane blebbing (Fig. 1). We next asked whether
the cells exhibited any of the other characteristics common to
apoptosis. One such feature is nuclear fragmentation (1), which can be
visualized by fluorescence microscopy using the membrane-impermeant dye
ethidium homodimer and the membrane-permeant dye calcein. As shown in
Fig. 2A cells cultured in the presence of 2% FCS for
48 h contained intact nuclei, which was identical to that in cells
that were cultured in the absence of serum for only 2 h. Cells
cultured in the absence of serum for 24 h, however, showed
extensive nuclear fragmentation (Fig. 2B). In many cases these
fragments were associated with apoptotic bodies, the small
membrane-bound vesicles released by apoptotic cells (1).
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, DNA isolated from osteoblasts cultured in the absence
of serum migrated as a single, high mol wt band (Fig. 3, lane 3). On
the other hand, in four separate experiments, DNA extracted from
osteoblasts that were cultured for 3, 6, 12, or 24 h in the
presence of either TNF or staurosporine, an agent that induces DNA
degradation in many cell types, showed no evidence of DNA degradation
into oligonucleosome fragments (Fig. 3, lanes 1 and 2, respectively),
which is often observed in PCD (1). In contrast, U937 leukemia cells
cultured under identical conditions exhibited the characteristic
pattern of DNA degradation in response to either TNF or
staurosporine (Fig. 3, lanes 4, and 5), whereas no degradation was
observed when the cells were cultured in serum (Fig. 3, lane 6).
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, osteoblasts cultured
in the presence of 2% FCS for 48 h demonstrated negligible DNA
fragmentation by TUNEL. However, TUNEL of osteoblasts cultured with
TNF for 24 h confirmed that substantial cell death (30%)
had taken place in accordance with the results obtained with the MTT
assay (Fig. 4B). The TUNEL method showed innumerable cells with
clear-cut staining indicative of chromatin condensation at the nuclear
membrane.
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induced PCD in osteoblasts,
as the chromatin was usually seen to be compacted and segregated into
sharply defined masses under the nuclear membrane (Fig. 5A), although margination of chromatin was observed in
about 50% of these cells (Fig. 5A); the cytoplasmic organelles, in
particular the mitochondria, remained largely intact. As a basis for
comparison, a swollen necrotic osteoblast is shown in Fig. 4B. In
contrast to apoptosis, there was a total disruption of the internal
structure of the cell undergoing necrosis, whereas the normal
morphology of the osteoblast was preserved when culture was performed
in the presence of IGF-I (Fig. 5C).
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induced apoptosis in mouse osteoblasts,
we investigated the effects of graded concentrations of this cytokine
on PCD in murine osteoblasts using the MTT cell survival assay. TNF
dose-dependently (10–14–10-9 M)
decreased the survival of primary mouse osteoblasts over a 24-h culture
period from a level of survival of 67 ± 2.8% at
10-14 M to 18 ± 1.1% at
10-9 M (Fig. 6).
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, a, b, c, and d,
respectively). The concentration that promoted half-maximal survival
(the ED50) was about 10-9 M for
IGF-I and 10-8 M for IGF-II, as expected if
both IGFs promote survival by binding to IGF-I receptors (15). The
ED50 for insulin-induced survival was about
10-7 M, and the dose-response curve was very
broad, extending over about 5 log units. The ED50 for bFGF
was about 5 ng/ml.
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, a–c), with a
maximum of about 75%. A similar effect was seen when a plateau
concentration of IGF-I was used in combination with graded
concentrations of bFGF (Fig. 7d). Surprisingly, although PDGF did not
promote survival on its own, when combined with insulin, IGF-I, or
IGF-II, survival was enhanced (Fig. 8, a–c). The
combination of bFGF and PDGF with IGF-I did not promote survival better
than that of bFGF with IGF-I (data not shown). A similar situation was
found with bFGF/PDGF/IGF-II and bFGF/PDGF/insulin combinations (data
not shown). There was also no additive effect on survival when several
of the other GFs, such as TGFß, EGF, IL-1, and IL-11, were used in
combination with the IGFs or insulin (data not shown).
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IR-3 on IGF-I- and IGF-II-promoted survival of mouse
osteoblastsIR-3, raised against the type I IGF
receptor. This antibody binds specifically to the type I receptor and
does not cross-react with either the type II IGF or insulin
receptor.
As shown in Fig. 9, IR-3 (1 µg/ml) was a potent
competitive inhibitor of both IGF-I- and IGF-II-mediated cell survival,
indicating that in serum-free culture, both IGF-I and IGF-II mediate
their effects on osteoblast survival by interacting with the type I IGF
receptor. However, IR-3 did not completely inhibit the
survival-promoting effects of insulin, which suggests that insulin may
be acting via an interaction with insulin receptors. IR-3 had no
effect on bFGF-mediated survival and only partially blocked the effect
of the bFGF/IGF-I combination on osteoblast survival, which suggests
that bFGF mediates its effects via an interaction with FGF receptors
(Fig. 9). Finally, IR3 blocked the action of PDGF/IGF-I on
osteoblast survival, which indicates that PDGF may be altering the
number and/or affinity of the type I IGF receptors on murine
osteoblasts (Fig. 9).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, G-CSF, M-CSF, GM-CSF, PTH, and
1,25-(OH)2D3 had no effect on either osteoblast
survival or apoptosis. In fact, the only factor that induced osteoblast
apoptosis in this study was the immunoregulatory cytokine, TNF. The effects of the IGFs on osteoblast survival is in agreement with their activity on the survival of oligodendrocytes (17) and rat Schwann cell precursors (18). However, a notable difference between these reports and the present study was that the IGFs were capable of promoting a 100% survival rate of neuron-derived cells as opposed to the 60–70% survival of osteoblasts achieved in the present study.
It would appear that insulin can promote survival of osteoblasts by
binding to its own receptor, because insulin had a significant effect
at a 10-8-M concentration, which is sufficient
to bind to insulin receptors but not IGF-I receptors (15). This is
supported by the fact that insulin receptors have been demonstrated on
rodent osteoblasts, and insulin can maintain the growth of these cells
at a similar concentration (19). Although osteoblasts express type I
and II IGF receptors (20, 21), the role of the type II receptor in
mediating the metabolic and proliferative activities of IGF-II is
controversial. It is known that the type I IGF receptor is recognized
by both IGF-I and -II and insulin (22), and several lines of evidence
from this study indicate that the type I IGF receptor is responsible
for mediating the effects of IGF-I and -II as well as high
concentrations of insulin. Firstly, the order of potency in stimulating
osteoblast survival (IGF-I > IGF-II > insulin) is similar
to the relative affinities of these hormones for binding to the type I
IGF receptor (23) and is consistent with a common mechanism involving
this receptor. Similar potencies have been reported for the effects of
the IGFs and insulin on other cellular activities, and the type I IGF
receptor seems to mediate the responses in these cell types (24).
Secondly, our findings that the combination of IGF-I and IGF-II did not
enhance the level of osteoblast survival over that produced by IGF-I
suggests that the type II receptor is not involved in the response.
Thirdly, IR-3, a monoclonal antibody specific for the type 1 IGF
receptor, inhibited the survival-promoting effects of both IGFs and
almost prevented those of insulin.
The finding in the present study that bFGF increased the survival of osteoblasts is similar to the situation for Schwann cell precursors (25). As osteoblasts may be derivatives of the neural crest cell lineage, of relevance to the results reported here is the observation that bFGF can rescue chick nonneuronal neural crest cell derivatives in vitro (26). Therefore, it seems likely that osteoblasts have retained the requirement of an FGF for survival displayed by their developmental ancestors.
Although PDGF has been shown to stimulate some cells to make IGF-I
(27), this GF inhibits the synthesis of IGF-I and IGF-II by osteoblasts
(28, 29). This may explain why PDGF was incapable of inducing
osteoblast survival on its own. The mechanism underlying the synergy
between IGFs and FGFs/PDGF may be due in part to the ability of these
GFs to modulate type-I IGF receptors. It has been shown, for example,
that bFGF and PDGF increase the number of type I IGF receptors
expressed by purified glial cells derived from hypothalamic cultures
without affecting receptor affinity (30). It is, therefore, conceivable
that in the experiments we report here, bFGF/PDGF may increase type I
IGF receptor number or affinity, resulting in an enhanced response to
IGF-I, IGF-II, and insulin. This is supported by the fact that the type
I receptor antibody, IR3, prevented the survival-enhancing effects
of both bFGF/IGF-I and PDGF/IGF-I on osteoblast survival.
The action of IGFs is known to be regulated by the synthesis and secretion of one of six IGF-binding proteins. Furthermore, bFGF has been reported to modulate the synthesis of one of the IGF-binding proteins in purified hypothalamic neural crest cell cultures. Modulation of these proteins by bFGF/PDGF, therefore, represents another way in which these GFs might alter cellular responses to IGFs (30, 31).
IGF-I is probably one of the most important regulators of bone mass because it is synthesized by bone cells, and it is present in substantial concentrations in bone tissue. Although bFGF, TGFß, PDGF, IGF-I, and IGF-II are all present in the bloodstream, it is apparent that the paracrine biosynthesis of GFs is more important in the modulation of cellular activity (32). In skeletal tissue, osteoblasts express messenger RNA transcripts for these GFs, and the proteins have been shown to influence osteoblast proliferation and bone matrix synthesis (33, 34, 35). Furthermore, studies on the quantification and characterization of GFs present in human bone have revealed that human bone matrix contains multiple GFs, including IGF-I, IGF-II, TGFß, bFGF, and PDGF. IGF-II and TGFß are the two most abundant GFs present in human bone, whereas bFGF, PDGF, and IGF-I are several-fold less abundant (36). It seems likely that GFs, released from the extracellular matrix and neighboring cells as well as osteoblasts, are responsible for promoting the survival of osteoblasts in bone as they do in vitro.
Interestingly, TGFß was the only GF present in bone matrix that did not have an effect on either osteoblast survival or PCD in this study. Osteoblast production of IGF-I and IGF-II is stimulated by TGFß (37); however, the absence of a survival-promoting effect suggests that this GF is unable to induce a single cell to produce enough IGF-I or IGF-II to save itself in microculture. The effects of TGFß on apoptosis and cell survival are variable and seem to depend upon cell phenotype. For example, although it induces PCD in a variety of epithelial and myeloid cells (38, 39), it prevents the process in synovial cells (40) and has no effect on the survival of teratocarcinoma cells (41), similar to its effect on osteoblasts in this study. A possible explanation for this is unclear, although it may relate to the density at which the cells are cultured, as Mathieu et al. (42) found that PCD in vitro exhibits a correlation with this parameter.
As complete survival of osteoblasts in this study could only be achieved using culture medium supplemented with FCS, this would suggest either that unidentified GFs or extracellular matrix (ECM) components are responsible for promoting osteoblast survival. Among cells that have been shown to require survival factors, we are not aware of any example where a single signaling molecule on its own permits long term survival in culture (43, 44, 45). The significance of the ECM in cell survival has recently been demonstrated for endothelial cells, which rapidly undergo PCD in the absence of integrin-mediated adhesion with components of the ECM (46). It seems possible that all cells require multiple survival factors for long term survival.
The effects of inflammatory cytokine TNF on osteoblast apoptosis in
this study is in accordance with its effects on several other mammalian
cell lines, including the human leukemia cell lines HL-60 and U937 (47)
and the murine fibrosarcoma cell lines L929 and WEHI (48).
Although we demonstrated biochemical and morphological features of
osteoblast apoptosis, we could not detect evidence of DNA fragmentation
by gel electrophoresis in dying osteoblasts, suggesting either that DNA
fragmentation is not an important part of the death mechanism in these
cells or that our methods were insufficiently sensitive to detect it.
DNA fragmentation, however, seems not to be an invariable feature of
PCD (16, 49), and condensation of the chromatin at the membrane of an
apoptotic nucleus is not always associated with activation of an
endonuclease with subsequent DNA degradation (50). TNF inhibits bone
formation and has been found to inhibit collagen synthesis and alkaline
phosphatase activity in osteoblasts, actions that contrast with those
GFs that promote osteoblast survival in this study. This suggests that
those factors exerting a catabolic action on osteoblasts may also
induce PCD while, conversely, agents with an anabolic action may
promote survival.
The present findings may contribute to our understanding of bone loss
induced by the inflammatory cytokine, TNF, in conditions such as
rheumatoid arthritis, tumor osteolysis, and periodontal disease and the
role of endogenous GFs in modulating bone turnover.
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Footnotes |
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Received February 20, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IR-1 and IR-3 with insulin
and somatomedin-C receptors. Endocrinology 118:223–226[Abstract]
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 A. M. Delany, D. Durant, and E. Canalis Glucocorticoid Suppression of IGF I Transcription in Osteoblasts Mol. Endocrinol., October 1, 2001; 15(10): 1781 - 1789. [Abstract] [Full Text] [PDF]
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 U. K. MESSMER, V. A. BRINER, and J. PFEILSCHIFTER Basic Fibroblast Growth Factor Selectively Enhances TNF-{alpha}--Induced Apoptotic Cell Death in Glomerular Endothelial Cells: Effects on Apoptotic Signaling Pathways J. Am. Soc. Nephrol., December 1, 2000; 11(12): 2199 - 2211. [Abstract] [Full Text]
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L. Gilbert, X. He, P. Farmer, S. Boden, M. Kozlowski, J. Rubin, and M. S. Nanes Inhibition of Osteoblast Differentiation by Tumor Necrosis Factor-{alpha} Endocrinology, November 1, 2000; 141(11): 3956 - 3964. [Abstract] [Full Text] [PDF]
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G. Zhao, M.-C. Monier-Faugere, M. C. Langub, Z. Geng, T. Nakayama, J. W. Pike, S. D. Chernausek, C. J. Rosen, L.-R. Donahue, H. H. Malluche, et al. Targeted Overexpression of Insulin-Like Growth Factor I to Osteoblasts of Transgenic Mice: Increased Trabecular Bone Volume without Increased Osteoblast Proliferation Endocrinology, July 1, 2000; 141(7): 2674 - 2682. [Abstract] [Full Text] [PDF]
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