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Division of Endocrinology and Metabolism (L.G., X.H., P.F., M.K., J.R., M.S.N.), Department of Medicine and Department of Orthopedics (S.B.), Emory University School of Medicine and Veterans Affairs Medical Center, Atlanta, Georgia 30033
Address all correspondence and requests for reprints to: Mark S. Nanes, M.D., Ph.D., VA Medical Center (mail code 111), 1670 Clairmont Road, Decatur, Georgia 30033. E-mail: mnanes{at}emory.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(TNF-) has a key role in skeletal disease
in which it promotes reduced bone formation by mature osteoblasts and
increased osteoclastic resorption. Here we show that TNF inhibits
differentiation of osteoblasts from precursor cells. TNF- treatment
of fetal calvaria precursor cells, which spontaneously
differentiate to the osteoblast phenotype over 21 days, inhibited
differentiation as shown by reduced formation of multilayered,
mineralizing nodules and decreased secretion of the
skeletal-specific matrix protein osteocalcin. The effect of TNF was
dose dependent with an IC50 of 0.6 ng/ml, indicating a high
sensitivity of these precursor cells. Addition of TNF- from days
2–21, 2–14, 7–14, and 7–10 inhibited nodule formation but addition
of TNF after day 14 had no effect. Partial inhibition of
differentiation was observed with addition of TNF on only days 7–8,
suggesting that TNF could act during a critical period of phenotype
selection. Growth of cells on collagen-coated plates did not prevent
TNF inhibition of differentiation, suggesting that inhibition of
collagen deposition into matrix by proliferating cells could not,
alone, explain the effect of TNF. Northern analysis revealed that TNF
inhibited the expression of insulin-like growth factor I (IGF-I). TNF
had no effect on expression of the osteogenic bone morphogenic proteins
(BMPs-2, -4, and -6), or skeletal LIM protein (LMP-1), as determined by
semiquantitative RT-PCR. Addition of IGF-I or BMP-6 to fetal calvaria
precursor cell cultures enhanced differentiation but could not overcome
TNF inhibition, suggesting that TNF acted downstream of these proteins
in the differentiation pathway. The clonal osteoblastic cell line,
MC3T3-E1–14, which acquires the osteoblast phenotype spontaneously in
postconfluent culture, was also studied. TNF inhibited differentiation
of MC3T3-E1–14 cells as shown by failure of mineralized matrix
formation in the presence of calcium and phosphate. TNF was not
cytotoxic to either cell type as shown by continued attachment and
metabolism in culture, trypan blue exclusion, and Alamar Blue
cytotoxicity assay. These results demonstrate that TNF- is a potent
inhibitor of osteoblast differentiation and suggest that TNF acts
distal to IGF-I, BMPs, and LMP-1 in the progression toward the
osteoblast phenotype. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is one of
several cytokines produced in excess in postmenopausal osteoporosis and
within the joint space in rheumatoid arthritis (1, 2, 3, 4).
TNF- has been shown to reduce bone formation by inhibiting the
production of matrix proteins by phenotypically mature osteoblasts and
to promote osteoclastic resorption. The loss of bone after estrogen
withdrawal can be abrogated by sequestration of TNF with soluble TNF
receptors, revealing a key role for TNF in postmenopausal osteoporosis
(5, 6). Work from several laboratories has revealed an
inhibitory effect of TNF- on the synthesis of type I collagen and
induction of osteoblast resistance to vitamin D, as shown by inhibition
of 1,25-dihydroxyvitamin D3 stimulated production
of osteocalcin (7, 8, 9, 10, 11, 12, 13, 14). These actions of TNF shift the
formation/resorption balance in the skeleton toward resorption, which
leads to fractures in postmenopausal osteoporosis and periarticular
bone loss in inflammatory arthritis. Although the suppressive effects
of TNF- on the function of mature osteoblasts has been described,
little is known about the effects of TNF- on the differentiation of
osteoblasts from their precursor cell pool. We considered that TNF
might also inhibit the recruitment of osteoblasts from their stromal
progenitor cells.
Osteoblasts derive from a pool of pluripotent stem cells capable of
differentiating toward a number of phenotypes (15, 16, 17, 18).
Stem cells that are destined to become osteoblasts must achieve an
osteoblastic trajectory rather than proceed along an adipocytic,
myocytic, or fibroblastic path. A number of secreted and intracellular
mediators have been suggested to promote the differentiation and
survival of osteoblasts. These factors promote a succession of cellular
events that include precursor cell proliferation, growth arrest,
phenotype selection, and finally, osteoblast-specific gene expression
(19). Paracrine factors suggested to support osteoblast
differentiation include bone morphogenic proteins-2, -4, and -6,
and IGF-I as well as nuclear protein transcription factors
(20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30). TNF- could potentially regulate any of these
cellular events in the differentiation pathway. In addition, TNF has
been suggested to regulate apoptosis of osteoblasts, a mechanism that
could accelerate the exit of osteoblasts or their precursors from their
functional pool (31, 32, 33, 34, 35).
We studied the effect of TNF- on spontaneous differentiation of
precursor cells toward the osteoblast phenotype and on enhancement of
differentiation by IGF-I and BMP-2. To do this, we used two models of
osteoblast differentiation, fetal rat calvaria preosteoblasts and a
murine calvaria clonal osteoblastic cell line, MC3T3-E1–14. Fetal
calvaria cells acquire the osteoblast phenotype in postconfluent
culture in the presence of ascorbate. Over a 3-week period, precursor
cells grow to confluence and form multilayered nodules that mineralize
and secrete the osteoblast-specific matrix protein, osteocalcin. The
clonal MC3T3-E1–14 cells also spontaneously differentiate under these
conditions and secrete a matrix competent for mineralization. Here we
show that TNF- is a potent suppressor of osteoblast differentiation
in these experimental models at the point of phenotype selection in the
differentiation pathway.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IGF-I were purchased from PeproTech, Inc. (Rocky Hill, NJ), human
BMPs-2, -4, and -6 were generous gifts from Genetics Institute, Inc. (Cambridge, MA), human PTH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32) from
Peninsula Laboratories, Inc. (Belmont, CA), Types I and II
collagenase from Worthington Biochemical Corp. (Lakewood,
NJ), and Earle’s minimum essential medium (MEM) from Life Technologies, Inc. (Grand Island, NY). Heat-inactivated FBS was
purchased from HyClone Laboratories, Inc. (Logan, UT),
Dulbecco’s PBS (without calcium and magnesium), trypsin/Versene,
sodium bicarbonate solution, HEPES, and penicillin/streptomycin were
purchased from BioWhittaker, Inc. (Walkersville, MD). BGJb
(Fitton-Jackson modification) was from either Life Technologies, Inc. (liquid medium) or Sigma (powdered medium, St.
Louis, MO). Other cell culture reagents were purchased from
Sigma. Cell culture plates coated with rat tail collagen I
were purchased from Becton Dickinson and Co. (Bedford,
MA), and TRIzol Reagent from Life Technologies, Inc.
Actinomycin-D-Mannitol was purchased from
Sigma and Alamar Blue from BioSource International, Inc. (Camarillo, CA). Primers for RT-PCR and
probes for Northern analysis were synthesized by the Emory University
Microchemical Facility (Atlanta, GA). Dr. Lawrence Phillips (Emory
University, Atlanta, GA) kindly provided a full-length IGF-I
complementary DNA (cDNA). Zeta-Probe GT Genomic Tested Blotting
Membranes were purchased from Bio-Rad Laboratories, Inc.
(Hercules, CA) and 32P-dCTP was purchased from
Amersham Pharmacia Biotech (Piscataway, NJ). The probe for
GAPDH was prepared by RT-PCR. GeneAmp RNA PCR Core Kits were purchased
from PE Biosystems (Foster City, CA).
Fetal rat calvaria cultures
The Emory University and VA Medical Center animal use committee
approved all procedures. Timed pregnant Sprague Dawley rats were
obtained from Charles River Laboratories, Inc.
(Wilmington, MA). Cultures of primary and secondary fetal rat calvaria
cells were prepared as previously described with the exception that the
primary culture of digested fetal calvaria cells was allowed to
incubate for 8 days rather than 7 (3 days postconfluent)
(36). Briefly, frontal and parietal bones were dissected
from day 22 fetal rat calvaria and subjected to four sequential, 20 min
digestions with a mixture of Types I and II collagenase. Cells in
fractions three and four were washed, combined, and cultured at
0.8–1.0 x 106 cells per 75
cm2 flask in Earle’s MEM + 10% FBS. After 8
days, during which the medium was replaced twice, the primary cells
were subcultured using trypsin/Versene and plated at
105 cells/2 ml per well in 6-well plates. These
secondary cultures were grown in MEM + 10% FBS until confluent (7
days), then switched to MEM + 10% FBS + 50 µg/ml
L-ascorbic acid for the next 7 days. On day 14 after
plating, the medium was switched to BGJb (Fitton-Jackson modification)
+ 10% FBS + 5 mM ß-glycerophosphate for the final 7 days
of culture. The medium was changed on days 4, 11, and 18.
Mineralized nodules were fixed with 70% ethanol on day 21, stained
with the von Kossa technique (37), and counted using an
Optomax V HR image analyzer (Hollis, NH). Total nodule number,
including nonmineralized nodules, was assessed by counting each culture
5 times after staining with hematoxylin. The addition of TNF-,
BMP-6, or IGF-I to the culture medium is described for each experiment.
The assessment of TNF inhibition of unmineralized and mineralized
nodule formation was done relative to control cultures grown in the
absence of TNF.
MC3T3-E1–14 clonal osteoblastic cultures
The clonal osteoblastic cell line, MC3T3-E1, clone 14, was
kindly provided by Dr. Rene Franceschi (University of Michigan, Ann
Arbor, MI). Stock cultures were grown in MEM + 10% FBS. For
experiments, cells were plated in MEM + 10% FBS (1.9 x
105 cells/ml/well in 12-well plates) and switched to
-MEM + 10% FBS + 50 µg L-ascorate the next day.
Mineralization was induced by adding 10 mM
ß-glycerophosphate to this medium on day 8 after plating. Because
these cells are clonal, differentiation occurs throughout the culture,
unlike the fetal calvaria cells that form discrete nodules. Von Kossa
staining was done as indicated on day 16 of culture, by which time
control cells were uniformly mineralized.
Osteocalcin assay
Culture supernatants were collected and stored at -70 C until
assayed for osteocalcin levels by the Biomedical Technologies competitive rat osteocalcin RIA (Stoughton,
MA).
Cytotoxicity assay
The cytotoxicity assay was adapted from the method of Ahmed
et al. (38). Serial dilutions of TNF- and
Actinomycin D were prepared in MEM + 10% FBS in duplicate rows of
96-well plates (100 µl/well). A suspension of MC3T3-E1–14 cells
(104 cells/100 µl/well in MEM + 10% FBS + 200
U/ml penicillin + 200 µg/ml streptomycin) was added to the plates.
The 96-well plates were incubated for 4 days in a humidified incubator
at 37 C with 5% CO2. Alamar Blue (40 µl/well,
diluted 1:2 in MEM + 10% FBS) was added and the plates were incubated
for an additional 6 h. Cell growth was measured as the absorbance
at 570 nm minus the absorbance at 620 nm using a 96-well plate reader
(Bio-Tek Instruments, Inc., Model EL311).
Northern analysis and RT-PCR
Total cellular RNA was prepared from fetal rat calvaria cultures
by adding TRIzol (1 ml per well of a 6-well plate) to lyse the cells.
Chloroform was added (0.2 ml/sample) to separate the aqueous and
organic phases, followed by precipitation of the RNA from the aqueous
phase with isopropanol (0.5 ml per sample). Northern analysis for IGF-I
was carried out by fractionating total RNA in a 2.2 M
formaldehyde gel followed by capillary transfer to Zeta-Probe GT
Genomic Tested Blotting Membrane. IGF-I messenger RNA (mRNA) species
were detected using a full-length rat IGF-I cDNA after random primer
labeling with 32P-dCTP. Membranes were stripped
and rehybridized with a human GAPDH cDNA probe. mRNA band intensity was
quantitated using a Molecular Dynamics, Inc.
phosphorimager (Sunnyvale, CA) and results were calculated as
IGF-I/GAPDH. Semiquantitative RT-PCR was carried out using 0.5 µg
total cellular RNA per reaction. Preliminary experiments showed that 22
cycles were well within the linear range of amplification for each gene
being measured. The primers used are shown in Table 1
. Primers were end labeled with
32P-[ATP-
] using T4
kinase (39). Results were quantitated using a
phosphorimager (Molecular Dynamics, Inc., Sunnyvale, CA)
and corrected for 18S RNA amplified from the same samples in the PCR
reaction.
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0.05. |
Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows that addition
of TNF- to fetal rat calvaria preosteoblasts dramatically reduces
the number of multilayered, mineralized nodules. Control cultures show
typical nodules and internodular areas filled with a cobblestone
pattern of confluent cells. After TNF- treatment, however, nodules
fail to appear, but the remaining confluent cells appear intact.
TNF- treated cells remain attached to the culture plate. Trypan blue
staining of control and TNF--treated cultures shows no uptake of
stain, confirming viability. To determine the time course of
sensitivity to TNF- by the differentiating cultures, TNF- (100
ng/ml) was added and maintained during days 2–21, 7–21, or 14–21.
Figure 1B shows that addition of TNF- day 7–14 is sufficient to
produce maximal inhibition at the 100 ng/ml dose. Control cultures and
cultures treated with TNF- (100 ng/ml) beginning on day 14 show no
significant difference in the number of total nodules by day 21. The
apparent increase in nodules after treatment with TNF days 14–21, seen
in Fig. 1C, was not observed consistently (total nodules observed in
repeat experiment, TNF 10 ng/ml days 14–21: control 100 ± 5%,
TNF 107 ± 1.1%, n = 3 wells/group, P >
0.05 by Student’s t test). Nodule counts were similar on
day 17, indicating no loss of nodules once they were formed (day 17
counts of total nodules: control 329 ± 8.5, TNF, 340 ± 7.7;
day 21 counts of total nodules: control 315 ± 10.7, TNF 316
± 5.8 nodules/well; n = 6/group, P > 0.05).
These results suggest that TNF- inhibits entry of cells into the
differentiation pathway but does not cause loss of osteoblastic nodules
once they are formed. Treatment of cultures with TNF- inhibited
mineralized as well as total nodules as shown in Fig. 1C.
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shorter exposures to TNF- were used. Figure 2 shows that a 24-hour exposure between
days 7 and 8 produces significant dose-dependent reduction in nodule
numbers compared with those observed in control cultures. Additional
dose-dependent reductions are observed if the exposure time is
lengthened to 4 days (days 7–11) or 7 days (days 7–14). As in the
previous experiment, no greater decrease in nodule formation is
observed when cultures are treated past day 14 (days 7–21). Figure 3A (solid line) shows the dose
response effect of TNF- and reveals an IC50 of
0.6 ng/ml when TNF- is present continuously during days 7–21. We
also measured the osteoblast specific protein, osteocalcin, as an index
of differentiation. The level of secreted osteocalcin, which is
directly proportional to the number of nodules, is also
dose-dependently inhibited by TNF- (Fig. 3A, dashed
line). The presence of TNF- between days 7 and 21 also prevents
full maturation of nodules as measured by a dose-dependent reduction in
the percentage of nodules that undergo mineralization (Fig. 3B).
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is known to inhibit the production of type I
collagen, an essential constituent of this competent matrix
(41). To determine if TNF- inhibition of type I
collagen synthesis is sufficient to explain suppression of
differentiation, fetal calvaria cells were grown on rat tail collagen
I-coated tissue culture plates and the effect of TNF- was
determined. Figure 4 shows that
osteoblasts grown on collagen-coated plates produce the same number of
nodules as those grown on uncoated plates. Despite the presence of type
I collagen on the coated plates, the addition of TNF- from days
2–14 suppresses osteoblast differentiation and prevents nodule
formation. Thus, TNF- acts as an inhibitor even in the presence of a
type I collagen matrix.
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was also studied in MC3T3-E1 cells, which
differentiate in culture. We used the Clone 14 cell line (MC3T3-E1–14)
that forms a mineralized matrix after 16 days in culture in the
presence of ascorbate and ß-glycerophosphate (42).
Figure 5A shows that continuous exposure
of these cells to TNF- (0.01–10 ng/ml, days 1–16) completely
prevents mineralization in a dose-dependent manner. Undifferentiated
cells remained attached and viable as shown in Fig. 5B. The inhibition
of differentiation in MC3T3-E1–14 cells is not associated with
increased cell death, as shown by cytotoxicity assay using the
metabolic indicator dye, Alamar Blue. Figure 6 shows that doses of TNF- up to 5000
ng/ml did not cause a reduction in cell viability compared with control
cultures. Actinomycin D, a positive control, decreased viability as
expected. Although this experiment was done after 4 days of TNF
treatment, a repeat experiment done after 16 days of TNF (10 ng/ml)
showed no cytotoxicity.
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suppresses expression of these factors. Northern analysis was
used to measure steady-state IGF-I mRNA species and semiquantitative
RT-PCR was used to measure mRNA levels of BMP-2, -6, and LMP-1 in fetal
rat calvaria cultures. Figure 7 shows a
representative Northern analysis from day 7 fetal calvaria cell
cultures treated with TNF (100 ng/ml). TNF potently decreases the
steady-state level of IGF-I mRNA species as measured 16 h after
addition of TNF (T) compared with control cultures (C) as previously
reported (43). Table 2
quantitates the inhibition of IGF-I mRNA species as % GAPDH control.
Table 2 also shows the effect of TNF- on BMP steady-state mRNA.
Semiquantitative RT-PCR does not reveal any effect of TNF on BMPs -2,
-6, or LMP-1, shown as % control after correction for 18S ribosomal
RNA (Table 2). To determine if suppression of IGF-I by TNF- explains
the inhibition of differentiation, cultures were treated with TNF-,
IGF-I, or both. IGF-I was added to calvaria cultures on day 7 and
maintained until day 21. Figure 8 shows
that IGF-I alone stimulates nodule formation well above the numbers
produced by control cultures. Despite the positive effect of IGF-I on
differentiation, TNF- (100 ng/ml) is still able to suppress
differentiation in IGF-I treated cultures. Submaximal concentrations of
TNF-, capable of causing a 50% suppression of nodule number, cause
a 50% suppression of nodules in the presence of IGF-I (not shown).
Thus, IGF-I is not able to reverse the effect of TNF-.
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, we considered that
TNF could cause resistance to the action of BMPs. To test this, BMP-6
was added to calvaria cultures from day 7 to day 14 with or without
TNF-. Figure 9 shows that BMP-6
treatment is osteogenic but is unable to reverse the inhibition by
TNF- (10 ng/ml). In addition, treatment of cultures with BMP-2 or -4
also results in a robust osteogenic response that is inhibited by TNF.
In this experiment, TNF (10 ng/ml) was added simultaneously with BMP-2
or -4 (50 ng/ml) from days 7–14. Results were: Control 100 ±
2%, TNF 5.6 ± 1%; BMP-2 185 ± 3%, BMP-4 173 ± 2%,
TNF+BMP-2 30 ± 3%, TNF+BMP-4 15 ± 2%) (n =
5–6/group, all groups differ from control, P <
0.05).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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dose-dependently inhibits
differentiation of osteoblasts from their precursor cells in both a
fetal rat calvaria model and in clonal MC3T3-E1–14 cells. This effect
of TNF- is supported by the inhibition of mineralized nodule
formation and inhibition of expression of the skeletal-specific gene,
osteocalcin. Low doses of TNF- are able to suppress differentiation,
suggesting that osteoblast precursors are quite sensitive to the action
of this cytokine. The rat calvaria model also allowed us to determine
that a restricted time of TNF- sensitivity occurs between days 7–10
of culture, when cells are postconfluent. This time period corresponds
to a critical window of phenotype selection in these cultures of
pluripotent precursor cells. In the postconfluent culture,
osteoblast-specific factors allow the selection of the osteoblast over
that of adipocytic, myocytic, or fibroblastic phenotypes. The
inhibitory action of TNF- is not associated with cytotoxicity of
fetal calvaria cells or of MC3T3-E1–14 cells as determined by trypan
blue exclusion and Alamar Blue reduction. In addition, careful counting
of bone nodules in culture following TNF- treatment did not reveal a
loss of nodules once they are formed. Thus, TNF- appears to block
entrance into the osteoblast differentiation pathway rather than cause
decreased survival of mature osteoblasts, once formed. We cannot
completely exclude the possibility that TNF- increases apoptosis of
osteoblast progenitors as previously described (31).
However, the lack of cell death in our cultures makes an apoptotic
mechanism less likely.
We considered that TNF- could inhibit differentiation by suppression
of type I collagen synthesis, an important constituent of a competent
skeletal matrix. The importance of type I collagen has been
demonstrated with ascorbate depletion in this model (17, 40, 41, 44). Osteoblast differentiation, which is normally blocked by
ascorbate depletion, will occur in the absence of ascorbate if cells
are grown on plates precoated with type I collagen. In our hands,
provision of a matrix replete with collagen (coated plates) does not
prevent TNF- inhibition of differentiation. Thus, TNF- inhibition
of collagen synthesis alone cannot explain inhibition of
differentiation.
We determined whether factors known to augment differentiation were
inhibited by TNF-. In this report, we studied IGF-I expression,
which is potently inhibited by TNF-, and the osteogenic proteins
BMP-2, BMP-6, and LMP-1. Addition of IGF-I to the medium in
postconfluent cultures increases the formation of nodules, but TNF-
continues to inhibit this process. Similarly, response to osteogenic
BMPs -2, -4, and -6 is inhibited by TNF-. Thus, TNF- blocks
differentiation at a site distal to the action of IGF-I and BMP-6. We
have previously shown that BMP-6 is one of the earliest BMPs to be
expressed during differentiation and we cannot completely exclude an
inhibitory effect of TNF on the response to BMP-2 or -4, which follow
BMP-6 expression (27). In addition, TNF- could inhibit
the expression or response to osteogenic transcription factors induced
by BMPs (SMADS) or to factors that select a skeletal specific path of
differentiation (Cbfa-1, OSF-1 factor) (28, 29, 45, 46).
Further work will be needed to address the possible actions of TNF-
at these levels.
It is possible that TNF- could select precursor cells for
differentiation along an adipocytic, fibroblastic, or skeletal muscle
pathway, thus shunting cells away from an osteoblastic direction.
However, the doses of TNF- (1–10 ng/ml, days 7–14) that can
completely suppress nodule formation in the fetal calvaria cell model
are not associated with a change in cell morphology. There are
currently no reports of TNF- induction of adipocyte, skeletal
muscle, or fibroblastic differentiation; indeed, TNF- has been shown
to inhibit adipocyte differentiation (47, 48, 49). Thus, it is
unlikely that TNF shunts precursor cells toward an alternate mature
phenotype. We favor the hypothesis that TNF arrests differentiation by
blocking transition of precursor cells into the differentiation
pathway, perhaps by eliminating responsiveness to skeletal specific
stimuli that are important at a stage later than BMP expression.
In summary, we have shown that TNF- inhibits osteoblast
differentiation. Suppression of osteoblast differentiation is likely to
be an important mechanism of decreased bone formation in many
circumstances where excess TNF- is produced in the bone
microenvironment. These include chronic inflammatory disease, estrogen
deficiency, and some types of malignancy (50, 51, 52, 53).
Up-regulation of the TNF-stimulated transcription factor, NF
B, may
also influence expression of additional regulators of both
osteoblastogenesis and osteoclastogenesis (54, 55). The
inhibitory action of TNF- may occur at a point in the
differentiation pathway distal to IGF-I, BMP-6, or LMP-1 expression.
Further work will be needed to determine the specific mechanism of
TNF- action.
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Footnotes |
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Received March 24, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 K. Redlich, B. Gortz, S. Hayer, J. Zwerina, N. Doerr, P. Kostenuik, H. Bergmeister, G. Kollias, G. Steiner, J. S. Smolen, et al. Repair of Local Bone Erosions and Reversal of Systemic Bone Loss Upon Therapy with Anti-Tumor Necrosis Factor in Combination with Osteoprotegerin or Parathyroid Hormone in Tumor Necrosis Factor-Mediated Arthritis Am. J. Pathol., February 1, 2004; 164(2): 543 - 555. [Abstract] [Full Text] [PDF]
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 R. Sitcheran, P. C. Cogswell, and A. S. Baldwin Jr. NF-{kappa}B mediates inhibition of mesenchymal cell differentiation through a posttranscriptional gene silencing mechanism Genes & Dev., October 1, 2003; 17(19): 2368 - 2373. [Abstract] [Full Text] [PDF]
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 A. Moralez, W. H. Busby Jr., and D. Clemmons Control of Insulin-Like Growth Factor Binding Protein-5 Protease Synthesis and Secretion by Human Fibroblasts and Porcine Aortic Smooth Muscle Cells Endocrinology, June 1, 2003; 144(6): 2489 - 2495. [Abstract] [Full Text] [PDF]
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J. Pfeilschifter, R. Koditz, M. Pfohl, and H. Schatz Changes in Proinflammatory Cytokine Activity after Menopause Endocr. Rev., February 1, 2002; 23(1): 90 - 119. [Abstract] [Full Text] [PDF]
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K. Kurokouchi, C. R. Jacobs, and H. J. Donahue Oscillating Fluid Flow Inhibits TNF-alpha -induced NF-kappa B Activation via an Ikappa B Kinase Pathway in Osteoblast-like UMR106 Cells J. Biol. Chem., April 13, 2001; 276(16): 13499 - 13504. [Abstract] [Full Text] [PDF]
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L. Gilbert, X. He, P. Farmer, J. Rubin, H. Drissi, A. J. van Wijnen, J. B. Lian, G. S. Stein, and M. S. Nanes Expression of the Osteoblast Differentiation Factor RUNX2 (Cbfa1/AML3/Pebp2alpha A) Is Inhibited by Tumor Necrosis Factor-alpha J. Biol. Chem., January 18, 2002; 277(4): 2695 - 2701. [Abstract] [Full Text] [PDF]
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