ORIGINAL ARTICLE

Journal of
1044
Cellular
Physiology
Blockade of TNFR1 Signaling:
A Role of Oscillatory Fluid Shear
Stress in Osteoblasts
HAIFANG WANG,1,2 SUZANNE R. YOUNG,1 RITA GERARD-O’RILEY,1 JULIA M. HUM,1
ZHOUQI YANG,1,2 JOSEPH P. BIDWELL,3 AND FREDRICK M. PAVALKO1*
1
Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, Indiana
2
Key Laboratory for Space Bioscience and Biotechnology, Faculty of Life Sciences, Northwestern Polytechnical University,
Shaanxi, China
3
Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, Indiana

Fluid shear stress protects cells from TNF-a-induced apoptosis. Oscillatory fluid shear stress (OFSS) is generally perceived as
physiologically relevant biophysical signal for bone cells. Here we identify several cellular mechanisms responsible for mediating the
protective effects of OFSS against TNF-a-induced apoptosis in vitro. We found that exposure of MC3T3-E1 osteoblast-like cells to as little
as 5 min of OFSS suppressed TNF-a-induced activation of caspase-3, cleavage of PARP and phosphorylation of histone. In contrast, H2O2-
induced apoptosis was not inhibited by OFSS suggesting that OFSS might not be protecting cells from TNF-a-induced apoptosis via
stimulation of global pro-survival signaling pathways. In support of this speculation, OFSS inhibition of TNF-a-induced apoptosis was
unaffected by inhibitors of several pro-survival signaling pathways including pI3-kinase (LY294002), MAPK/ERK kinase (PD98059 or
U0126), intracellular Ca2þ release (U73122), NO production (L-NAME), or protein synthesis (cycloheximide) that were applied to cells
during exposure to OFSS and during TNF-a treatment. However, TNF-a-induced phosphorylation and degradation of IkBa was blocked
by pre-exposure of cells to OFSS suggesting a more specific effect of OFSS on TNF-a signaling. We therefore focused on the mechanism of
OFSS regulation of TNF-receptor 1 (TNFR1) signaling and found that OFSS (1) reduced the amount of receptor on the cell surface, (2)
prevented the association of ubiquitinated RIP in TNFR1 complexes with TRADD and TRAF2, and (3) reduced TNF-a-induced IL-8
promoter activity in the nucleus. We conclude that the anti-apoptotic effect of OFSS is not mediated by activation of universal pro-survival
signaling pathways. Rather, OFSS inhibits TNF-a-induced pro-apoptotic signaling which can be explained by the down-regulation of
TNFR1 on the cell surface and blockade of TNFR1 downstream signaling by OFSS.
J. Cell. Physiol. 226: 1044–1051, 2011. ß 2010 Wiley-Liss, Inc.

Mechanical loading of the skeleton plays a key role in regulating
the health of bone tissue by maintaining a proper balance
between resorption of existing bone and deposition of new
bone (Turner and Pavalko, 1998). A critical component in
mechanoregulation of bone is the movement of interstitial fluid
through the spaces within bone that generates fluid shear stress
(FSS) across the surfaces of bone cells (Piekarski and Munro,
1977). FSS functions as an external mechanical stimulus that can
activate cytoplasmic signaling molecules to produce changes in
bone metabolism through a process known as cellular
mechanotransduction, recently reviewed in (Huang and
Ogawa, 2010). Many in vitro models use unidirectional or
steady fluid shear stress (SFSS) to study the response of cells to
fluid movement. While this is appropriate for endothelial cells
that normally experience unidirectional flow in blood vessels,
given the cyclic nature of normal skeletal loading, we and others
have suggested that oscillatory fluid shear stress (OFSS) may be
a more physiological biophysical signal than SFSS for bone cells
(Jacobs et al., 1998; Kurokouchi et al., 2001; Ponik et al., 2007).
Our previous work indicated that exposure of osteoblasts to
SFSS protected cells from TNF-a-induced apoptosis (Pavalko
et al., 2003a). To better understand the molecular mechanisms
responsible for the anti-apoptotic effects of FSS, we have
extended these studies to include consideration of the effect of
OFSS on TNF-a-induced apoptosis in osteoblasts.
We have proposed that mechanical loading of cells regulates
the formation and function of membrane-associated multi-
protein complexes, referred to as mechanosomes, that affect
cellular responses to external mechanical stimuli (Pavalko et al.,
2003b). This hypothesis predicts the integration of diffusion-
controlled signaling pathways with a solid-state scaffold directly
linking extracellular mechanical stimuli at the cell membrane to
the genes. These multi-protein membrane complexes may be
linked to the cytoskeleton and can be mobilized from
membrane structures such cadherin-mediated cell–cell
junctions or integrin-containing focal adhesions (Pavalko et al.,
1998; Ponik and Pavalko, 2004) and may involve kinases such as
focal adhesion kinase (FAK) (Young et al., 2009),
nucleocytoplasmic shuttling molecules, and transcriptional
regulators such as Nmp4, NFkB, and b-catenin (Chen et al.,
2003; Norvell et al., 2004; Yang et al., 2010; Young et al., 2010).
The in vitro application of FSS induces a number of responses in
osteoblastic cells, including intracellular calcium mobilization
(Hung et al., 1995; Jacobs et al., 1998; Donahue et al., 2001) and
activation of MAPKs (You et al., 2001), PI3K/AKT activation
(Pavalko et al., 2003a), NF-kB activation (Chen et al., 2003;
Young et al., 2010), production of nitric oxide (Klein-Nulend
et al., 1995; Johnson et al., 1996), and cytoskeleton
reorganization (Pavalko et al., 1998; Chen et al., 2000). These
are all generally associated with anti-apoptotic signaling. Our
studies demonstrate for the first time that these traditional
anti-apoptotic pathways are not responsible for mediating the
beneficial effects of OFSS against TNF-a-induced apoptosis.
Contract grant sponsor: NIH;
Contract grant numbers: R01AR052682, AR056188, DK053796.
*Correspondence to: Fredrick M. Pavalko, Department of Cellular
and Integrative Physiology, Indiana University School of Medicine,
635 Barnhill Dr., MS346A, Indianapolis, IN 46202. E-mail:
fpavalko@iupui.edu
Received 27 August 2010; Accepted 31 August 2010
Published online in Wiley Online Library
(wileyonlinelibrary.com), 20 September 2010.
DOI: 10.1002/jcp.22427

ß 2 0 1 0 W I L E Y - L I S S , I N C .
 SHEAR STRESS INDUCED BLOCK OF TNFR1 SIGNALING
Instead, OFSS, rather than simply generating pro-survival
signals, appears to inhibit several processes specifically involved
in TNF-a signaling via TNF receptor 1 (TNFR1) including down-
regulation of surface expression of TNFR1 and inhibition of
signaling downstream of TNFR1 including activation of the
TNF-a target gene, IL-8. Thus, these results suggest blockade of
death-induced signaling via TNFR1 by OFSS may represent
another example of the ability of a mechanical stimulus to affect
the assembly and function of an important cell surface-
associated multi-protein signaling complex, or mechanosome,
which ultimately results in modulation of genes.
Materials and Methods
Cell culture
The mouse osteoblast-like MC3T3 cell line was obtained from the
American Type Culture Collection and cultured in minimal
essential media alpha (a-MEM, Gibco, Life Technologies, Carlsbad,
CA) supplemented with 10% fetal calf serum (FCS) and 1%
penicillin/streptomycin (Gibco, Life Technologies). Cells were
maintained in 5% CO2 at 378C. For the induction of apoptosis, cells
were treated with 10 ng/ml of TNF-a (Calbiochem, San Diega, CA)
and 10 mg/ml of cyclohexamide (CHX, Sigma–Aldrich, St. Louis,
MO) for 4 h. Apoptosis was evaluated by observing the cellular
morphologic changes under light microscope and Western blotting
analyses of caspase-3 processing, PARP cleavage, and histone
phosphorylation.
Oscillatory fluid flow
Before flow experiments, MC3T3 cells were passed onto sterile
glass slides at a density of 2.5  105 cells per slide, grown for 24 h
and serum-starved in a-MEM media supplemented with 0.1% FCS
and 1% penicillin/streptomycin for 18–22 h. The a-MEM media
supplemented with 0.1% FCS and 1% penicillin/streptomycin was
also used for flow experiments. Oscillatory fluid flow was
performed in a parallel plate flow chamber at 378C using a
previously described fluid flow device. This system subjected cells
to 10–12 dyn/cm2 of OFSS at a frequency of 1 Hz. Hard-walled PVC
tubing was used to connect the chamber to the pump. Static
control cells were held in a-MEM containing 0.1% serum at 378C
with 5% CO2.
Western blotting analysis and antibodies
Cells extracts were collected directly in SDS sample buffer and
protein concentrations were determined using the amido black
method. Equal amounts of protein (20 mg) were loaded onto SDS–
PAGE gels for separation and transferred to nitrocellulose for
immunoblotting. The following primary antibodies were used:
caspase-3/cleaved caspase-3 (Cell Signaling Technology, Danvers,
MA), Parp (Santa Cruz Biotechnology, Santa Cruz, CA), ph-histone
and vinculin (Sigma–Aldrich), ph-IkBa/IkBa (Cell Signaling
Technology), TNFR1 (Abcam, Cambridge, MA), cadherin-11 (Life
Technologies, Carlsbad, CA), receptor-interacting protein (RIP,
BD Biosciences, San Jose, CA), TRADD (Santa Cruz
Biotechnology), ph-Erk1/2/Erk1/2 (Santa Cruz Biotechnology).
The appropriate anti-mouse Ig and anti-rabbit Ig peroxidase-
conjugated secondary antibodies (Jackson Immunoresearch
Laboratories, West Grove, PA) and anti-goat Ig (Santa Cruz
Biotechnology) were used. The second antibody signals were
detected and quantified using a Luminescent Image Analyzer LAS-
3000 system (Fujifilm Life Science, Stamford, CT).
Analysis of cell surface TNFR1 protein
Cell surface protein biotinylation and purification were performed
using the Pierce Cell Surface Protein Isolation Kit, and the amount
of TNFR1 protein was quantified by Western blotting analysis.
MC3T3 cells grown in 10 cm dishes or on glass slides were
subjected to static or flow condition for 2 h, then the cells were
JOURNAL OF CELLULAR PHYSIOLOGY
1045
washed twice with ice-cold PBS and incubated with 1 mg/ml of EZ
link NHS-Sulfo-SS-biotin in PBS for 30 min at 48C. The reaction was
quenched by two washes with cold quenching buffer. The cells
were then lysed in 800 ml of lysis buffer (50 mM Tris–HCl, pH 7.4,
500 mM NaCl, 1 mM EDTA/EGTA, 0.5% SDS, 1% Triton X-100,
and 0.1% NP-40) containing fresh protease inhibitors (1 mM
pepstatin A, 250 mM phenylmethylsulfonyl fluoride, 1 mg/ml
leupeptin, and 1 mg/ml aprotinin) and disrupted by sonicating for
40 s on ice. Lysates were centrifuged at 10,000g for 10 min at 48C,
and supernatants were incubated with Neutravidin beads (100 ml)
for 1.5 h at 48C. Beads were washed three times with fresh lysis
buffer, and bound proteins were eluted with 80 ml of 2 Western
blotting sample buffer for 6 min at 1008C. Aliquots from total cell
lysates (40 ml) and Neutravidin-bound samples (40 ml) were
analyzed by Western blotting analysis. Biotinylated TNFR1 protein
(representing the surface TNFR1) was normalized using levels of
cadherin on the cell surface, and values were averaged across three
experiments. To analyze TNF-a-induced TNFR1 internalization,
cells were subjected to 2 h of static culture or flow condition and
then treated with 10 ng/ml TNF-a for 1 and 5 min at 378C, or 4 and
10 min in 48C cold media at room temperature to allow for
internalization of TNFR1. Then cells were cooled rapidly to 48C to
inhibit additional internalization, washed twice with ice-cold PBS
and subjected to surface protein isolation.
Analysis of TNFR1–TRADD–RIP complex by co-
immunoprecipitation
The complexes initiated upon TNF-a stimulation were analyzed by
use of a rabbit TRADD antibody (Santa Cruz) or a mouse TNFR1
antibody. MC3T3 cells (1  107 cells) grown in culture dishes or on
glass slides were subjected to 2 h static or flow condition,
stimulated for the indicated times with 10 ng/ml TNF-a and then
lysed in 1 ml co-IP lysis buffer (50 mM Tris–HCl, pH 7.4, 150 mM
NaCl, 0.5% NP40, 10% glycerol, 1 mM EDTA/EGTA) containing
fresh protease inhibitors for 25 min on ice. Detergent-insoluble
material was removed by centrifugation at 10,000g for 10 min at
48C. The supernatants were pre-cleared with 40 ml Protein A
beads (Sigma) for 1 h at 48C and incubated with rabbit anti-TRADD
or mouse anti-TNFR1 antibody for 2 h. The immune complexes
were then precipitated with 40 ml Protein A beads (Sigma) for
3 h at 48C. Beads were recovered by centrifuging at 10,000g
for 3 min and washed four times with 500 ml of lysis buffer. The
beads-bound proteins were eluted with 80 ml of Western blotting
sample buffer for 6 min at 1008C and subjected to immunoblotting
analysis.
IL-8 reporter assay
Osteoblasts grown to 80% confluency in 10 cm dishes were
transfected with 6 mg of the IL-8 promoter-firefly luciferase
construct using FuGENE per manufacture’s protocol. Transfected
cells were incubated over night and the next day were plated for
TNF-a treatment or exposure to OFFS. Luciferase activities were
determined using at Luciferase Assay Kit (Promega, Madison, WI)
according to the manufacture’s protocol. Data are presented as
fold change in activity relative to untreated static controls.
Graphical data is representative of three independent trials.
Statistical analysis
Statistical significance was assessed by one-tailed t-tests or analysis
of variance as indicated and a P-value of <0.05 was interpreted as
statistically significant.
Results
Oscillatory fluid shear stress inhibits TNF-a-induced, but
not H2O2-induced apoptosis
To evaluate the relevant molecular mechanisms involved in
mediating the effect of OFSS on TNF-a-induced apoptosis in
1046 WANG ET AL.

osteoblasts in vitro, MC3T3 cells were subjected to OFSS for a
very brief period (5 min) to longer periods (2 h), followed by
treatment with 10 ng/ml TNF-a and 10 mg/ml CHX for 4 h.
Apoptosis was evaluated by observing the cellular morphologic
changes under light microscope and Western blotting analyses
of caspase-3 activation, PARP cleavage, and histone
phosphorylation. This OFSS time-course study indicated that
OFSS inhibits TNF-a-induced caspase-3 activation in a time-
dependent manner. A significant inhibitory effect was achieved
with very brief exposure to OFSS (5 min) and almost complete
inhibition was achieved with 2 h of flow (Fig. 1A). We found that
typical apoptotic morphologic signs induced by TNF-a,
including cell shrinkage and rounding, surface blebbing, and
apoptotic bodies were completely abolished by pre-exposing
cells to 2 h of OFSS (Fig. 1B). Western blotting analyses showed
that cleavage of caspase-3 and PARP were almost completely
abolished and phosphorylation of histone H2A.X was
significantly reduced by 2 h of OFSS (Fig. 1C). In contrast, when
apoptosis was induced by 250 mM H2O2, 2 h of OFSS had no
inhibitory effect on apoptosis (Fig. 1D). These results suggested
a protective effect of OFSS specifically against TNF-a-induced
cell apoptosis. We also examined the effect of OFSS on
activation of caspase-8, which is the initiator of TNFR1-
mediated apoptosis and is upstream of caspase-3. We found
that cleavage of caspase-8 was almost completely abolished by
application of OFSS suggesting that initiation of TNFR1 signaling
might be blocked by OFSS (Fig. 1C).
Inhibition of MEK/ERKs pathway, pI3K/AKT pathway,
cytoskeleton reorganization, intracellular calcium
mobilization, new protein synthesis, or nitric oxide
production did not attenuate OFSS-induced anti-
apoptosis
To evaluate the potential role of several well-established pro-
survival signaling pathways in mediating the protective effect of
OFSS against TNF-a-induced apoptosis, MC3T3 cells were
treated with an effective dose of selective inhibitors of ERK
(50 mM PD98059 or 20 mM U1026), or PI3K/AKT signaling
(50 mM LY294002), cytoskeleton reorganization (10 mM
colchicine or nocodazole for tubulin cytoskeleton and 4 mM
cytochalasin B or D for actin filaments), phospholipase C (PLC)-
mediated intracellular Ca2þ release (5 mM U73122), new
protein synthesis (10 mg/ml CHX), or nitric oxide production

Fig. 1. Comparison of effects of OFSS on TNF-a (A–C) and H2O2 (D)-induced apoptosis in MC3T3 cells. A: Time-dependent inhibition of OFSS on
TNF-a-induced activation of caspase-3. B: Prevention of TNF-a-induced apoptotic morphological changes by 2 h of OFSS. C: Inhibition of TNF-a-
induced activation of caspase-3, cleavage of Parp, phosphorylation of histone, and activation of caspase-8 by 2 h of OFSS. D: H2O2-induced histone
phosphorylation was not affected by 2 hof OFSS. MC3T3 cells under static condition or exposed to 2 hof OFSS were treated with 10 ng/ml TNF-a for
4 h or 250 mM H2O2 for 2 h, cell pictures were taken and then samples were collected and subjected to Western blot analysis (aP < 0.05, bP < 0.01 vs.
static plus TNF-a treatment).

JOURNAL OF CELLULAR PHYSIOLOGY

 SHEAR STRESS INDUCED BLOCK OF TNFR1 SIGNALING 1047

(250 mM L-NAME) during the 2 h period of time cells were
exposed to OFSS and during subsequent treatment with TNF-a
for 4 h. Surprisingly, none of these inhibitors showed any ability
to block the protective effect of OFSS against TNF-a-induced
apoptosis as assessed by morphological changes (not shown) or
as measured by activation of caspase-3 (Fig. 2).
Blockade of TNF-a-induced IkBa signaling by 2 h of
OFSS
In addition to activating a pro-apoptotic signals, TNF-a also
stimulates NF-kB signaling which is mediated by
phosphorylation and subsequent degradation of inhibitor-kB
(IkBa) (Chen and Goeddel, 2002). As expected, and as we have
shown previously (Young et al., 2010), application of OFSS
resulted in IkBa degradation within 30 min of the onset of flow,
with IkBa levels recovering to pre-treatment values after 2 h of
continuous flow (not shown). Because IkBa levels returned to
normal after 2 h of OFSS, we were able to assess the effect of
OFSS on TNF-a-activation of the NF-kB signaling pathway.
MC3T3 cells were first subjected to 2 h of OFSS, and then
treated with TNF-a for 5, 10, 15, and 30 min. Phosphorylation
and degradation of IkBa, which represent activation of NF-kB
signaling pathway, were analyzed by Western blotting. Under
static culture conditions, phosphorylation, and degradation of
IkBa induced by TNF-a was observed as early as 5 min post-
stimulation. In contrast, phosphorylation of IkBa and
degradation of IkBa were inhibited by pre-exposure of the
MC3T3 cells to 2 h of OFSS (Fig. 3). These results suggest that
OFSS may inhibit the initiation and progression of TNF-a-
induced TNFR1-mediated signaling.
Two hours of OFSS reduced expression level of TNFR1
on cell surface but did not prevent TNF-a-induced
internalization of TNFR1
We next investigated the effect of 2 h of OFSS on upstream
initiation of TNFR1 signaling by TNF-a by measuring cell surface
expression of TNFR1 in response to OFSS and by assessing
TNF-a-induced receptor internalization. First, MC3T3 cells

Fig. 2. Inhibitory effect of 2 h OFSS on TNF-a-induced caspase-3 activation was not reduced by treating cells with inhibitors of ph-ERKs pathway
(50 mM PD98059 or 20 mM U1026, A), PI3K/ph-AKT pathway (50 mM LY294002, B), or cytoskeleton reorganization (10 mM colchicine or
nocodazole for tubulin cytoskeleton and 4 mM cytochalasin B or D for actin filaments, C), intracellular calcium mobilization (5 mM U73122 for
effective inhibition of phospholipase C, D), new protein synthesis (10 mg/ml cyclohexymide, E), or NO synthesis (250 mM L-NAME, E) during the
whole flow period. MC3T3 cells were treated with individual inhibitor under static condition or during the 2 h of flow experiment, then cells were
treated with 10 ng/ml TNF-a for 4 h and samples were subjected to Western blot analysis.

JOURNAL OF CELLULAR PHYSIOLOGY

1048 WANG ET AL.

maintained in static culture and subjected to OFSS, the level of

Fig. 3. Blockade of TNF-a-induced phosphorylation and
degradation of IkBa by 2 h of OFSS. MC3T3 cells under static
condition or subjected to 2 h of OFSS were treated with 10 ng/ml
TNF-a for 5, 10, 15, or 30 min. IkBa and phosphorylated IkBa were
analyzed by Western blot. Vinculin was analyzed as a loading control.
were exposed to 2-h OFSS, or maintained in static culture. The
amount of total TNFR1 in cell lysates and the amount of TNFR1
specifically expressed on the cell surface was analyzed by
Western blotting. While we found no significant difference in
the total levels of TNFR1 in cell lysates between cells
TNFR1 on the cell surface was significantly reduced (35%) by
OFSS (Fig. 4A). We next treated cells at 378C with TNF-a to
induce receptor internalization. As expected, treatment with
TNF-a resulted in rapid loss of TNFR1 from the cell surface that
was significant within 5 min as the receptor was rapidly
internalized. Importantly, TNFR1 surface expression did not
recover during the following 60 min (Fig. 4B). Because TNF-a
induced such a rapid internalization of TNFR1 receptors, we
attempted a similar experiment at 48C. We found that TNF-a-
induced internalization of TNFR1 was completely blocked at
10 min when cells were maintained at 48C demonstrating that
the rate of internalization of TNFR1 could be effectively
reduced by treating cells with TNF-a in cold media (Fig. 4B). We
took advantage of this fact to measure the effect of OFSS on
TNF-a-induced receptor internalization as shown in
Figure 4C. The upper part (378C) shows that, at 1 and 5 min
post-TNF-a treatment, surface expression of TNFR1 appeared
lower in cells pre-exposed to OFSS than in cells held in static
culture, although this difference was not statistically significant.
To better demonstrate this difference, we slowed down the
receptor internalization by treating MC3T3 cells with TNF-a in
cold (48C) media. As shown in the bottom part of
Figure 4C (48C), a trend towards an increased rate of TNF-a-
induced TNFR1 internalization between the two groups (static
vs. OFSS) was seen at both 5 and 10 min post-treatment,

Fig. 4. Effect of 2 h of OFSS on TNFR1 protein level at cell surface. A: Down-regulation of TNFR1 level on cell surface by 2 h of OFSS. MC3T3 cells
under static condition or exposed to 2 h of OFSS were immediately subjected to cell surface protein extraction and Western blot analysis for
TNFR1. B: TNF-a-induced internalization of TNFR1. Cells treated with 10 ng/ml TNF-a for 5, 10, 15, 30, or 60 min were subjected to cell surface
protein extraction and Western blot analysis. C: Western blot analysis of effects of OFSS on TNF-a-stimulated internalization of TNFR1. Cells
under static condition or pre-exposed to 2 h of OFSS were treated with 10 ng/ml TNF-a in warm (37-C, upper bands) or ice-cold (4-C) media at 37-C
as indicated time and then subjected to cell surface protein extraction and Western blot analysis. D: Effects of OFSS on TNF-a-induced
internalization of TNFR1. Cells under static condition or pre-exposed to 2 h of OFSS were treated with 10 ng/ml TNF-a in ice-cold (4-C) media at 37-
C, and then subjected to cell surface protein extraction and Western blot analysis. Cadherin-11 expressed on cell surface was used as a loading
control (aP < 0.05 vs. static plus TNF-a treatment).

JOURNAL OF CELLULAR PHYSIOLOGY

 SHEAR STRESS INDUCED BLOCK OF TNFR1 SIGNALING
although we were unable to demonstrate that this difference
was statistically significant. Taken together, these results
indicated that TNF-a-induced TNFR1 internalization was
certainly not inhibited by pre-exposure to OFSS (Fig. 4D) and,
although not statistically significant, actually trended towards a
more rapid rate of internalization following pre-exposure to
OFSS.
Inhibition of ubiquitinated RIP in TNF-a-induced
membrane complexes by 2 h of OFSS
Next we evaluated the effect of OFSS on TNF-a-induction of
NFkB-mediated signaling by assessing association of
ubiquitinated RIP, an important component of the TNF-a-
induced pro-survival signaling complex I that forms with
TNFR1, TRADD, and TNF-associated factor 2 (TRAF2). We
used co-immunoprecipitation experiments to pull down
signaling complex I components using either an anti-TRADD
antibody or an anti-TNFR1 antibody and then blotted for RIP
and ubiquitinated RIP, which migrates more slowly on SDS–
PAGE. MC3T3 cells were first subjected to 2 h of OFSS or
maintained in static culture and then stimulated with TNF-a
(10 ng/ml) for 5 min or left untreated. Cells were then lysed,
TRADD or TNFR1 were immunoprecipitated, and the
presence of RIP and ubiquitinated RIP in the immune complex
was assayed by Western blotting. As shown in Figure 5, TNFa-
induced association of ubiquitinated RIP in complexes pulled
down with either TRADD or TNFR1 antibody was observed at
5 min post-TNF-a treatment in cells maintained in static
culture. However, the amount of ubiquitinated RIP that was
pulled down in co-immunoprecipitated complexes was
significantly reduced in cells pre-exposed to 2 h of OFSS. Taken
together with the observation that the amount of non-
ubiquitinated RIP co-immunoprecipitating in complexes
following pre-exposure to 2 h of OFSS was unchanged, these
results suggest that OFSS specifically inhibited the association of
ubiquitinated RIP with TNF-a-induced TNFR1/TRADD/
TRAF2 complexes. TRAF2-mediated ubiquitination of RIP has
been shown to be required for TNF-a-induced complex I
formation and NF-kB activation (Li et al., 2006). Therefore,
these results suggest that OFSS may inhibit TNF-a-induced
complex I formation and thereby prevent recruitment of Fas-
associated DD protein (FADD) and caspase-8 which would
ultimately prevent the formation of the death-inducing signaling
complex (DISC).
Fig. 5. Prevention of ubiquitination RIP by 2 h of OFSS. MC3T3 cells
under static condition or exposed to 2 h of OFSS were left untreated
or stimulated with TNF-a for 10 min. Cells were lyzed, TRADD or
TNFR1 was immunoprecipitated and the presence of RIP and its
modified form in the immune complex was detected by Western
blotting analysis.
JOURNAL OF CELLULAR PHYSIOLOGY
1049
Inhibition of TNF-a-induced IL-8 promoter activity by
OFSS
The results described above predict that OFSS protects cells
from TNF-a-induced apoptosis by down-regulating TRNR1
signaling. To test this directly, we used an IL-8 promoter activity
assay to determine the effect of OFSS on TNF-a-induced IL-8
transcriptional activity in the nucleus of MC3T3-E1 cells. We
found that pre-exposure of cells to OFSS significantly reduced
TNF-a-induced IL-8 activation compared to cells that were
maintained in static culture (Fig. 6). As expected, and as we have
reported previously (Young et al., 2010), OFSS alone induced a
significant increase in IL-8 reporter activity. This result strongly
supports the hypothesis that OFSS regulates the assembly and
activity of the multi-protein TNFR1 complex and that this
regulation by OFSS at the cell membrane ultimately affects gene
regulatory activity in the nucleus.
Discussion
In this study we have made several novel observations and
identified multiple specific mechanisms through which
mechanical stimulation of osteoblast-like MC3T3-E1 cells by
OFSS may promote cell survival. These specific mechanisms
include (1) down-regulation of surface expression of TNFR1,
(2) inhibition of the association of ubiquitinted RIP in TRNR1/
TRADD/TRAF2 complexes, (3) inhibition of NFkB signaling by
blockade of TNF-aIkBa phosphorylation and subsequent
degradation, and (4) inhibition of caspase-8 activation
downstream of DISC formation (summarized in Fig. 7). Among
several novel observations, we show for the first time the
protective effect of a brief period of oscillatory (OFSS), as
opposed to continuous steady (SFSS) or unidirectional shear
stress, against TNF-a-induced apoptosis in osteoblastic cells.
Furthermore, and we think very importantly, we show that
multiple classical pro-survival signaling pathways, including
MAPK, AKT, PLC, and NO-dependent pathways, are
apparently not involved in mediating the anti-apoptotic effect of
OFSS. Further, we found that inhibition of TNF-a-induced
Fig. 6. Quantification of IL-8 promoter reporter assay expressed as
fold activation. MC3T3-E1 osteoblasts transfected with an IL-8
promoter-firefly luciferase construct were maintained in static
culture conditions (static) or exposed to 2 h of OFFS followed by a 4-h
post-flow incubation period during which either TNF-a or vehicle
alone was added. aSignificant difference OFFS versus static;
b
significant difference static versus static R TNF-a; csignificant
difference static RTNF-a versus OFSS R TNF-a by ANOVA
( P < 0.05). Error bars represent SEM.
1050 WANG ET AL.
Fig. 7. Model of blockade of TNFR1-mediated signaling by OFSS.
apoptosis did not require an intact microtubule or actin
cytoskeleton.
It is generally accepted that binding of TNF-a to TNFR1 on
the plasma membrane rapidly triggers the recruitment of the
intracellular adapter protein TNFR1-associated death domain
containing protein (TRADD), RIP and TRAF2 to form an active
complex I and signals activation of NF-kB. This is followed by
dissociation of TRADD from TNFR1 and recruitment of FADD
and caspase-8 to form a complex II, or the DISC which leads to
cell apoptosis if new gene transcription induced by NF-kB is
inhibited (Micheau and Tschopp, 2003).
TNF-a-induced apoptosis is inhibited if the interactions
between TNFR1 and its downstream molecules are blocked
(He and Ting, 2002). Yang et al. (2005) reported that the
activation of TNF-a/IkBa/NF-kB signaling pathway was
inhibited by cardiac glycosides through a mechanism blocking
recruitment of TRADD, RIP, and TRAF2 to the TNFR1.
Gururaja et al. (2007) showed that a class of small-molecule
triazoloquinoxalines were able to block ligand-dependent
internalization of the TNFR1 and the association of TNFR1 with
TRADD and RIP1, thus inhibiting both survival and death
pathways induced by TNFa. Yamawaki et al. (2003) reported
the inhibition of TNF-a-induced MAPKs (JNK, p38, and ERK) by
chronic physiological FSS in rabbit aorta endothelial cells
through blocking the association of TRAF2 with TNFR1. Here,
we show that OFSS down-regulates the signaling capability of
TNFR1 membrane complexes and that this ultimately affects
transcriptional regulation of the IL-8 promoter, a well-
characterized target of TNF-a signaling in the nucleus. This
result is consistent with a previous report in which OFSS
inhibited TNF-a-induced NF-kB activation and intercellular
adhesion molecule-1 (ICAM-1) mRNA expression
(Kurokouchi et al., 2001). This study, using UMR106 cells, also
found that inhibition was shear stress-dependent indicating that
OFSS has potent effects on mechanotransduction pathways.
Our results provide further evidence that TNF-a-induced
apoptosis is inhibited by OFSS in osteoblasts via blockade of
signaling through membrane-associated protein complexes.
These findings are consistent with the mechanosome concept
in that mechanical signals detected at the cell surface are
translated into biochemical responses inside the cell through
regulation of membrane-associated multi-protein signaling
complexes that ultimately affect gene expression in the nucleus
JOURNAL OF CELLULAR PHYSIOLOGY
(Pavalko et al., 2003b). Interestingly, down-regulation of growth
factor/cytokine receptor signaling activity does not appear to
be a universal response to FSS. In fact, our group has recently
reported up-regulation of IGF-1 receptor signaling in response
to SFSS in MC3T3-E1 cells (Triplett et al., 2007) indicating that
the response of membrane receptors can differ depending on
the specific receptor, and perhaps, based on the type of
mechanical stimulus applied. This raises the question as to
whether there are ‘‘stop’’ mechanosomes that carry
information for attenuating bone formation, ‘‘go’’
mechanosomes that enhance bone growth, and finally whether
there is cross-talk between them within a network.
Studies using TRADD-deficient mice (Ermolaeva et al., 2008;
Pobezinskaya et al., 2008) indicate that, as predicted by
biochemical data, TRADD is absolutely necessary for TNF-
induced apoptosis and is also required for the recruitment of
TRAF2 to TNFR1 (Pobezinskaya et al., 2008).
Upon TNF-a stimulation, TRADD, RIP, and TRAF2 were
sequentially recruited to activated TNFR1 and formed a plasma
membrane bound complex (complex I), leading to activation of
NF-kB (Micheau and Tschopp, 2003). RIP is a serine/thronine
kinase that plays an essential role in TNF-a-induced NF-kB
activation through interaction with downstream signaling
components such as NEMO, the regulator subunit of the IKK
complex, and other molecules (Devin et al., 2000). TRAF2
catalyzed ubiquitination of RIP is required for TNF-a-induced
NF-kB activation (Li et al., 2006). Therefore, the failure of TNF-
a-induced NF-kB activation can be caused by the inhibition of
RIP ubiquitination. The loss of uniquitinated RIP from TNFR1
complexes (complex I) following OFSS suggests that fluid shear
may be affecting the ability of TRAF2 to associate with the
complex. Previous studies in endothelial cells showed that FSS
inhibited TNF-a-induced activation of MAPK by destroying
association of TRAF2 with TNFR1 (Yamawaki et al., 2003).
Our group showed that the PI3-kinase inhibitor LY294002
suppressed SFSS-induced inhibition of caspase-3 activation and
blocked the ability of SFSS to rescue osteoblasts from apoptosis
(Pavalko et al., 2003a). However, LY294002 treatment did not
affect inhibition of caspase-3 activation and anti-apoptosis
induced by OFSS in MC3T3 cells. Notably, AKT phosphorylation
induced by OFSS was effectively inhibited by LY294002 but
SFSS-induced phosphorylation of Akt was not affected,
suggesting different mechanisms may be involved in activation of
signaling pathways by the two patterns of fluid flow.
Importantly, OFSS inhibits TNF-a-induced IKK activation,
leading to a decrease in phosphorylation and degradation of
IkBa, which in turn resulted in decreased TNF-a-induced NF-
kB activation (Kurokouchi et al., 2001).
Plotkin et al. (2005) have reported that mechanical
stimulation by stretching rescued osteocytes from etoposide-
or glucocorticoid-induced apoptosis via activation of ERKs.
ERKs promote cell survival by phosphorylating transcription
factors that regulate the expression of apoptosis-related genes
(Kousteni et al., 2003; Huang et al., 2007). OFSS also activates
ERKs (You et al., 2001; Young et al., 2009) and that may result in
synthesis of new proteins, leading to the pro-survival effect. In
this study we found that these signaling pathways were not
involved in the anti-apoptotic effects of OFSS. Taken together,
our results suggest that OFSS protected MC3T3 cells from
TNF-a-induced apoptosis using mechanisms different from
those involved in the anti-apoptotic response of stretching.
We evaluated whether OFSS altered the amount of TNFR1
expressed on the cell surface and whether OFSS affected the
ability of TNF-a to induce internalization of the receptor. Since
TNF-a binds TNFR1 and triggers a signaling cascade leading to
the activation of NFkB transcription factors and the activation
of caspases, the effect of OFSS on TNFR1 surface expression
and internalization could impact cell survival. Previous studies
showed that the TNFR1-mediated death pathway is
 SHEAR STRESS INDUCED BLOCK OF TNFR1 SIGNALING
internalization-dependent (Micheau and Tschopp, 2003;
Schneider-Brachert et al., 2004) and inhibition of receptor
internalization selectively blocked TNF-mediated cytotoxicity
but did not affect NF-kB activation (Schutze et al., 1999;
Schneider-Brachert et al., 2006).
In conclusion, our results indicate that OFSS, generally
accepted as the most physiologically relevant fluid flow profile
for bone cells, inhibits TNF-a-induced apoptosis via a
mechanism that specifically blocks key steps in the TNF-a
signaling pathway that are mediated by TNFR1, RIP, and
caspase-8. Surprisingly, we found that rescue of osteoblasts
from TNF-a-induced apoptosis did not require an intact actin
cytoskeleton or any of several traditional ‘‘cell survival’’
pathways including MAPK, AKT, PLC, and NO. Finally, these
results are consistent with the concept that cellular regulation
of external mechanical signals is mediated by load bearing
mechanosomes which are regulated by OFSS at the cell
membrane and ultimately transmit information directly into the
nucleus to affect cell function.
Acknowledgments
This work was supported by NIH grants R01AR052682 to
F.M.P., DK053796 to J.P.B., and by post-doctoral fellowship
AR056188 to SRLY.
Literature Cited
Chen G, Goeddel DV. 2002. TNF-R1 signaling: A beautiful pathway. Science 296:1634–1635.
Chen NX, Ryder KD, Pavalko FM, Turner CH, Burr DB, Qiu J, Duncan RL. 2000. Ca(2þ)
regulates fluid shear-induced cytoskeletal reorganization and gene expression in
osteoblasts. Am J Physiol Cell Physiol 278:C989–C997.
Chen NX, Geist DJ, Genetos DC, Pavalko FM, Duncan RL. 2003. Fluid shear-induced
NFkappaB translocation in osteoblasts is mediated by intracellular calcium release. Bone
33:399–410.
Devin A, Cook A, Lin Y, Rodriguez Y, Kelliher M, Liu Z. 2000. The distinct roles of TRAF2 and
RIP in IKK activation by TNF-R1: TRAF2 recruits IKK to TNF-R1 while RIP mediates IKK
activation. Immunity 12:419–429.
Donahue SW, Jacobs CR, Donahue HJ. 2001. Flow-induced calcium oscillations in rat
osteoblasts are age, loading frequency, and shear stress dependent. Am J Physiol Cell
Physiol 281:C1635–C1641.
Ermolaeva MA, Michallet MC, Papadopoulou N, Utermohlen O, Kranidioti K, Kollias G,
Tschopp J, Pasparakis M. 2008. Function of TRADD in tumor necrosis factor receptor 1
signaling and in TRIF-dependent inflammatory responses. Nat Immunol 9:1037–1046.
Gururaja TL, Yung S, Ding R, Huang J, Zhou X, McLaughlin J, Daniel-Issakani S, Singh R,
Cooper RD, Payan DG, Masuda ES, Kinoshita T. 2007. A class of small molecules that inhibit
TNFalpha-induced survival and death pathways via prevention of interactions between
TNFalphaRI, TRADD, and RIP1. Chem Biol 14:1105–1118.
He KL, Ting AT. 2002. A20 inhibits tumor necrosis factor (TNF) alpha-induced apoptosis by
disrupting recruitment of TRADD and RIP to the TNF receptor 1 complex in Jurkat T cells.
Mol Cell Biol 22:6034–6045.
Huang C, Ogawa R. 2010. Mechanotransduction in bone repair and regeneration. FASEB J
24:1–8.
Huang D, Khoe M, Befekadu M, Chung S, Takata Y, Ilic D, Bryer-Ash M. 2007. Focal adhesion
kinase mediates cell survival via NF-kappaB and ERK signaling pathways. Am J Physiol Cell
Physiol 292:C1339–C1352.
Hung CT, Pollack SR, Reilly TM, Brighton CT. 1995. Real-time calcium response of cultured
bone cells to fluid flow. Clin Orthop 313:256–269.
Jacobs CR, Yellowley CE, Davis BR, Zhou Z, Cimbala JM, Donahue HJ. 1998. Differential
effect of steady versus oscillating flow on bone cells. J Biomech 31:969–976.
Johnson DL, McAllister TN, Frangos JA. 1996. Fluid flow stimulates rapid and continuous
release of nitric oxide in osteoblasts. Am J Physiol 271:E205–E208.
Klein-Nulend J, Semeins CM, Ajubi NE, Nijweide PJ, Burger EH. 1995. Pulsating fluid flow
increases nitric oxide (NO) synthesis by osteocytes but not periosteal fibroblasts—
JOURNAL OF CELLULAR PHYSIOLOGY
1051
Correlation with prostaglandin upregulation. Biochem Biophys Res Commun 217:640–
648.
Kousteni S, Han L, Chen JR, Almeida M, Plotkin LI, Bellido T, Manolagas SC. 2003. Kinase-
mediated regulation of common transcription factors accounts for the bone-protective
effects of sex steroids. J Clin Invest 111:1651–1664.
Kurokouchi K, Jacobs CR, Donahue HJ. 2001. 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 276:13499–13504.
Li H, Kobayashi M, Blonska M, You Y, Lin X. 2006. Ubiquitination of RIP is required for tumor
necrosis factor alpha-induced NF-kappaB activation. J Biol Chem 281:13636–13643.
Micheau O, Tschopp J. 2003. Induction of TNF receptor I-mediated apoptosis via two
sequential signaling complexes. Cell 114:181–190.
Norvell SM, Alvarez M, Bidwell JP, Pavalko FM. 2004. Fluid shear stress induces beta-catenin
signaling in osteoblasts. Calcif Tissue Int 75:396–404.
Pavalko FM, Chen NX, Turner CH, Burr DB, Atkinson S, Hsieh YF, Qiu J, Duncan RL. 1998.
Fluid shear-induced mechanical signaling in MC3T3-E1 osteoblasts requires cytoskeleton-
integrin interactions. Am J Physiol 275:C1591–C1601.
Pavalko FM, Gerard RL, Ponik SM, Gallagher PJ, Jin Y, Norvell SM. 2003a. Fluid shear
stress inhibits TNF-alpha-induced apoptosis in osteoblasts: A role for fluid shear stress-
induced activation of PI3-kinase and inhibition of caspase-3. J Cell Physiol 194:194–
205.
Pavalko FM, Norvell SM, Burr DB, Turner CH, Duncan RL, Bidwell JP. 2003b. A Model for
mechanotransduction in bone cells: The load-bearing mechanosomes. J Cell Biochem
88:104–112.
Piekarski K, Munro M. 1977. Transport mechanism operating between blood supply and
osteocytes in long bones. Nature 269:80–82.
Plotkin LI, Mathov I, Aguirre JI, Parfitt AM, Manolagas SC, Bellido T. 2005. Mechanical
stimulation prevents osteocyte apoptosis: Requirement of integrins, Src kinases, and ERKs.
Am J Physiol Cell Physiol 289:C633–C643.
Pobezinskaya YL, Kim YS, Choksi S, Morgan MJ, Li T, Liu C, Liu Z. 2008. The function of
TRADD in signaling through tumor necrosis factor receptor 1 and TRIF-dependent Toll-
like receptors. Nat Immunol 9:1047–1054.
Ponik SM, Pavalko FM. 2004. Formation of focal adhesions on fibronectin promotes fluid
shear stress induction of COX-2 and PGE2 release in MC3T3-E1 osteoblasts. J Appl Physiol
97:135–142.
Ponik SM, Triplett JW, Pavalko FM. 2007. Osteoblasts and osteocytes respond differently to
oscillatory and unidirectional fluid flow profiles. J Cell Biochem 100:794–807.
Schneider-Brachert W, Tchikov V, Neumeyer J, Jakob M, Winoto-Morbach S, Held-Feindt J,
Heinrich M, Merkel O, Ehrenschwender M, Adam D, Mentlein R, Kabelitz D, Schutze S.
2004. Compartmentalization of TNF receptor 1 signaling: Internalized TNF receptosomes
as death signaling vesicles. Immunity 21:415–428.
Schneider-Brachert W, Tchikov V, Merkel O, Jakob M, Hallas C, Kruse ML, Groitl P, Lehn A,
Hildt E, Held-Feindt J, Dobner T, Kabelitz D, Kronke M, Schutze S. 2006. Inhibition of TNF
receptor 1 internalization by adenovirus 14.7 K as a novel immune escape mechanism.
J Clin Invest 116:2901–2913.
Schutze S, Machleidt T, Adam D, Schwandner R, Wiegmann K, Kruse ML, Heinrich M, Wickel
M, Kronke M. 1999. Inhibition of receptor internalization by monodansylcadaverine
selectively blocks p55 tumor necrosis factor receptor death domain signaling. J Biol Chem
274:10203–10212.
Triplett JW, O’Riley R, Tekulve K, Norvell SM, Pavalko FM. 2007. Mechanical loading by fluid
shear stress enhances IGF-1 receptor signaling in osteoblasts in a PKCzeta-dependent
manner. Mol Cell Biomech 4:13–25.
Turner CH, Pavalko FM. 1998. Mechanotransduction and functional response of the skeleton
to physical stress: The mechanisms and mechanics of bone adaptation. J Orthop Sci 3:346–
355.
Yamawaki H, Lehoux S, Berk BC. 2003. Chronic physiological shear stress inhibits tumor
necrosis factor-induced proinflammatory responses in rabbit aorta perfused ex vivo.
Circulation 108:1619–1625.
Yang Q, Huang W, Jozwik C, Lin Y, Glasman M, Caohuy H, Srivastava M, Esposito D, Gillette
W, Hartley J, Pollard HB. 2005. Cardiac glycosides inhibit TNF-alpha/NF-kappaB signaling
by blocking recruitment of TNF receptor-associated death domain to the TNF receptor.
Proc Natl Acad Sci USA 102:9631–9636.
Yang Z, Bidwell JP, Young SR, Gerard-O’Riley R, Wang H, Pavalko FM. 2010. Nmp4/CIZ
inhibits mechanically induced beta-catenin signaling activity in osteoblasts. J Cell Physiol
223:435–441.
You J, Reilly GC, Zhen X, Yellowley CE, Chen Q, Donahue HJ, Jacobs CR. 2001. Osteopontin
gene regulation by oscillatory fluid flow via intracellular calcium mobilization and activation
of mitogen-activated protein kinase in MC3T3-E1 osteoblasts. J Biol Chem 276:13365–
13371.
Young SR, Gerard-O’Riley R, Kim JB, Pavalko FM. 2009. Focal adhesion kinase is important for
fluid shear stress-induced mechanotransduction in osteoblasts. J Bone Miner Res 24:411–
424.
Young SR, Gerard-O’Riley R, Harrington M, Pavalko FM. 2010. Activation of NF-kappaB by
fluid shear stress, but not TNF-alpha, requires focal adhesion kinase in osteoblasts. Bone
47:74–82.