Bone Vol. 28, No.
2
Ethanol Activates NF␬B DNA Binding and p56lck Protein
Tyrosine Kinase in Human Osteoblast-like Cells
Z. YAO,1,2 J. ZHANG,3 J. DAI,3 and E. T. KELLER1,3,4,5
1
Unit for Laboratory Animal Medicine, University of Michigan, Ann Arbor, MI, USA
2
Department of Immunology, Tianjin Medical University, Tianjin, China
3
Department of Pathology, the 4Institute of Gerontology, and the 5Program in Cellular and Molecular Biology, University of Michigan, Ann Arbor,
MI, USA
Alcoholics frequently suffer from moderate to severe bone
loss that results in bone fractures. Both decreased bone
production and increased bone resorption have been postu-
lated to contribute to ethanol (ETOH)-mediated bone loss.
Bone resorption is induced by several proinflammatory cy-
tokines such as interleukin-1 and -6. The expression of these
cytokines is induced by the transcription factor NF␬B,
which, in turn, is activated by several kinases. It follows that
protein kinase and NF␬B activation may contribute to
ETOH-induced bone loss. Accordingly, we sought to deter-
mine if ETOH activates protein tyrosine kinases (PTK) and
NF␬B DNA binding in a human osteoblast-like cell line
(HOBIT). Ethanol at 50 and 100 mmol/L (reflective of blood
ethanol levels reached in chronic alcoholics) for 24 h did not
alter HOBIT cell viability. In contrast, 200 mmol/L ethanol
decreased cell viability by 40%. Treatment of HOBIT cells
with 100 mmol/L ETOH induced nuclear NF␬B:DNA com-
plex formation and NF␬B activity. Incubation of HOBIT
cells with ETOH at 50 and 100 mmol/L for 30 min induced a
2.5- and 4.2-fold increase in PTK activity, respectively. Pre-
incubation of HOBIT cells with damnacanthal (DAM), which
inhibits p56lck, blocked ETOH-mediated PTK activity;
whereas, preincubation with herbimycin A, which inhibits
pp60src, did not. DAM inhibited both ethanol-induced NF␬B
activation in HOBIT cells and interleukin-6 expression in
primary human osteoblasts. Finally, preincubation with the
protein kinase C inhibitor, bisindolylmaleimide I HCl (BIS),
diminished ETOH-mediated PTK activity; whereas, preincu-
bation with the protein kinase A inhibitor, H89, did not.
These data demonstrate that ETOH induces NF␬B nuclear
translocation through p56lck in HOBIT cells. BISⴕ inhibition
of PTK activation suggests that ETOH activates PTK
through a protein kinase C-dependent pathway. These data
suggest that ETOH may contribute to bone loss through
activation of signal transduction that results in production of
an osteoclastogenic cytokine (i.e., interleukin-6) in
osteoblasts. (Bone 28:167–173; 2001) © 2001 by Elsevier
Science Inc. All rights reserved.
Address for correspondence and reprints: Evan T. Keller D.V.M., Ph.D.,
RM 5304 CCGCB, 1500 E. Medical Center Drive, Ann Arbor, MI
48109-0940. E-mail: etkeller@umich.edu
© 2001 by Elsevier Science Inc.
All rights reserved.
February 2001:167–173
Key Words: Alcohol; Osteoblast; Signal transduction; Kinase;
Interleukin-6; Cytokine.
Introduction
Excessive consumption of alcoholic beverages is associated with
an increased risk for developing osteoporosis.24,44,47 The mech-
anisms through which alcohol induces bone loss are currently not
well understood, but there is evidence for both direct and indirect
actions. Alcohol ingestion alters systemic markers of bone ho-
meostasis, including producing a transitory hypoparathyroidism
followed by rebound hyperparathyroidism, hypocalcemia, hyper-
calciuria, hypermagnesemia, hypermagnesuria, low vitamin D
serum levels, and decreased osteocalcin levels.1,3,20,27
In addition to decreased osteocalcin levels, there are several
other lines of evidence that ethanol inhibits bone production.
Specifically, there is histomorphometric evidence of decreased
osteoblast number8,43 and bone formation rate4,5,10,30,43 in alco-
holics, and several studies have demonstrated that ethanol inhib-
its proliferation of human osteoblasts.6,13,15,25,26 However, in
addition to its inhibition of bone production, ethanol increases
bone resorption, as seen in chick tibiae7,13 and rat trabecular
bone.2 Furthermore, chronic alcoholics, compared with nonalco-
holics, have histomorphometric evidence of increased osteoclast
numbers in iliac crest biopsies.18 Ethanol’s ability to promote
bone resorption may occur through inducing expression of cyto-
kines, such as interleukin-6 (IL-6) and interleukin-1 (IL-1),
which are important mediators of bone resorption (reviewed in
references 31, 33). These cytokines are secreted from osteoblasts
and induce osteoclastogenesis.
NF␬B is a transcription factor that induces expression of
IL-6.22 NF␬B can be activated by a variety of kinases including
I␬B kinase (IKK) and protein tyrosine kinases (PTK). Thus, it
follows that if ethanol induces the PTK that induce NF␬B, this
may lead to expression of the osteoclastogenic cytokines. Ac-
cordingly, the objective of the present study was to explore
ethanol’s ability to induce NF␬B DNA binding activity and
activate protein kinases.
Materials and Methods
Cell Culture and Treatments
The HOBIT cell line (generously provided by B. L. Riggs, Mayo
Foundation, Rochester, MN) was established by transfecting
normal adult human osteoblast-like cells with pSV3 neo, a
167 8756-3282/01/$20.00
PII S8756-3282(00)00425-7
168 Z. Yao et al.
Ethanol induces NF␬B and p56lck in HOBIT cells
plasmid encoding SV40 small and large-T antigen.21 HOBIT
cells approximate the phenotype of mature osteoblasts, includ-
ing: (1) 1,25-dihydroxy vitamin D-inducible expression of os-
teocalcin and alkaline phosphatase; (2) expression of ␣(I)procol-
lagen, osteopontin 1a, transforming growth factor ␤, IL-1␤, IL-6,
androgen receptor, and estrogen receptor; and (3) production of
a mineralizable matrix. HOBIT cells were maintained in F12/
DMEM supplemented with 15% fetal bovine serum (FBS).
Primary human osteoblasts were obtained from femoral heads
resected from hip-replacement patients and isolated as follows.
Trabeculae were scraped from the metaphysis and washed three
times in PBS and 1% penicillin/streptomycin. The trabeculae
were then ground into small particles using a sterile orthopedic
quality grinder (Mini-mill; Biocomp, Ventura, CA) and washed
three times in Hanks balanced salt solution (HBSS). The bone
particles were incubated in collagenase-P (1 mg/mL) in HBSS at
37°C for 1–1.5 h with shaking. The digested material was then
placed in DMEM/F12 with 20% FBS. Osteoblast phenotype was
confirmed by measuring the cells’ ability to synthesize serum
alkaline phosphatase, osteocalcin, and mineral deposition (data
not shown).
Cells were incubated the indicated levels of ethanol in cell
culture dishes that were wrapped with parafilm and taped closed
to minimize loss of ethanol. Cell viability was assessed by cell
count and trypan blue exclusion.
The following kinase inhibitors were purchased from Calbio-
chem (San Diego, CA): damnacanthal, which inhibits p56lck;
herbimycin A, which inhibits pp60src; bisindolylmaleimide I HCl
(BIS), which inhibits protein kinase C; and H89, which inhibits
protein kinase A. The kinase inhibitors were used at 2⫻ their
IC50 based on the manufacturer’s data. These levels were non-
lethal to the cells, which could proliferate upon removal of the
inhibitors and replacement with fresh media (data not shown).
Nuclear Extracts
Nuclear extracts were obtained by standard methods as we
previously described.22 Briefly, cells were scraped with a rubber
policeman into 5 mL of ice-cold phosphate-buffered saline (PBS)
and centrifuged at 250 g for 10 min at 4°C twice. The resulting
cell pellet was resuspended in 5 volumes of buffer A (10 mmol/L
Hepes [pH 7.9], 10 mmol/L KCl, 1.5 mmol/L MgCl2, 0.5
mmol/L dithiothreitol [DTT], 0.5 mmol/L phenylethylsulphonyl
fluoride [PMSF]), incubated on ice for 10 min, centrifuged at 250
g for 10 min, and resuspended in 3 volumes of buffer A with
Nonidet P-40 (NP40) added to 0.05% (v/v). Nuclei were then
released by homogenizing the cells with 30 strokes in a tight-
fitting Dounce homogenizer. Successful lysis was confirmed by
phase-contrast microscopy. Nuclei were pelleted by centrifuga-
tion at 250 g for 10 min, resuspended in 500 ␮L buffer C (5
mmol Hepes [pH 7.9], 26% glycerol [v/v], 1.5 mmol/L MgCl2,
300 mmol/L NaCl, 0.2 mmol/L EDTA, 0.5 mmol/L DTT, 0.5
mmol/L PMSF), and incubated on ice for 30 min. After incuba-
tion, the lysed nuclei were centrifuged at 24,000 g for 20 min at
4°C and the supernatant was snap-frozen in dry ice and ethanol
bath and maintained at ⫺80°C until used. Protein concentration
was determined with the BCA protein assay kit (Pierce Chemical
Co., Rockville, IL).
Electrophoretic Mobility Shift Assay (EMSA)
Two oligomers consisting of the sequences 5⬘-TCGACATGT-
GGGATTTTCCCATGAC-3⬘ and 5⬘-TCGAGTCATGGGAAAA-
TCCCACATG-3⬘ were annealed to form the NF␬B response
element present within the IL-6 promoter from ⫺43 to ⫺61 bp. The
probe was 32P-end-labeled with T4 kinase and purified on a Chro-
Bone Vol. 28, No. 2
February 2001:167–173
maspin 10 column (Clontech, Palo Alto, CA). Probe (10,000 cpm)
was incubated with 10 ␮g of LNCaP nuclear extract in EMSA
binding buffer 1 (10 mmol/L Hepes [pH 7.9], 50 mmol/L KCL, 0.2
mmol/L EDTA, 2.5 mmol/L DTT, 10% glycerol [v/v], 0.05% NP40
[v/v]) for 30 min at room temperature. For competition studies,
before addition of labeled probe, nuclear extracts were incubated for
10 min at room temperature with 100⫻ unlabeled specific probe
consisting of the consensus NF␬B response element (Promega,
Madison, WI), with nonspecific probe consisting of consensus Oct
1 response element (Promega). Protein/DNA complexes without the
addition of loading dye were resolved on a 7% nondenaturing
polyacrylamide gel (37.5:1 acrylamide/bisacrylamide) run in TG
buffer (5 mmol/L Tris, 38 mmol/L glycine). The gels were then
imaged on a Phosphorimager (Molecular Dynamics, Sunnyvale,
CA).
Transfection
The reporter vector pNF␬Bx2, which contains two NF␬B re-
sponse elements in tandem with a thymidine kinase minimal
promoter upstream of luciferase cDNA, has been described37 and
was generously provided by Dr. S. Ghosh (National Institutes of
Health, Bethesda, MD). The internal control vector, pRL-CMV,
containing the cytomegalovirus (CMV) promoter driving Renella
luciferase, was purchased from Promega (Madison, WI). pBlue-
script, an empty vector used to add optimize the amount of DNA
in the transfection reaction, was obtained from Promega. Trans-
fection was performed using SuperFect (Qiagen, Valencia, CA)
as recommended by the manufacturer. Briefly, 106 HOBIT cells
were plated in 2 mL of media/well in six-well plates 24 h before
transfection. A fresh transfection solution containing pNF␬Bx2
(1 ␮g), pRL-CMV (50 ng), and pBluescript (950 ng) per 10 ␮L
Superfect was made. Then, 1 mL of media was removed and 10
␮L of transfection solution was added, and the cells were
incubated for 2 h at 37°C, followed by addition of 1 mL of fresh
media. Media was replaced the next day and the cells were
incubated for an additional 24 h. Cells were then treated with
ethanol as indicated for 24 h. Total protein was then collected
using lysis buffer, and luciferase activity was measured using the
Dual-Luciferase System (Promega) and the TD-20/20 Luminom-
eter (Turner Designs, Sunnyvale, CA). Luciferase activities were
normalized to the internal Renalla luciferase activity and re-
ported as the mean ⫾ SE of two separate experiments.
Cell Extract
Cells were cultured in a 10-cm tissue culture plate until ⬃75%
confluent in F12/DMEM supplemented with 15% FBS. Cells
were then washed with PBS and incubated for 12 h in F12/
DMEM in the absence of FBS. Ethanol was then added at the
indicated concentrations and the cells were incubated for 30 min
at 37°C. Cells were then washed with PBS and solubilized by
addition of 2 mL of PTK extraction buffer (20 mmol/L Hepes
[pH 7.4], 1% Triton X-100, 5 mmol/L EDTA, 50 mmol/L NaCl,
30 ␮mol/L ␤-glycerophosphate, 50 mmol/L sodium fluoride, 50
␮g/mL aprotinin, 10 ␮mol/L leupeptin, 1 mmol/L AEBSF) for
10 min on ice. The extract was collected and centrifuged at
100,000 g at 4°C for 1 h. The supernatant was collected and
frozen at ⫺80°C until assayed.
PTK Assay
PTK activity was measured using the SignaTECT PTK Assay
System (Promega) as directed by the manufacturer. Briefly, a
biotinylated PTK target peptide was incubated with the cell
extract in the presence of [␥-32P]ATP for 5 min. The reaction
Bone Vol. 28, No. 2
February 2001:167–173
Figure 1. Ethanol-induced toxicity in HOBIT cells. HOBIT cells (5 ⫻
106/10-cm plate) were plated in DMEM ⫹ 20% FBS for 24 h, then were
incubated at the indicated concentrations of ethanol for 24 h. Cells were
then collected using trypsin digestion and assessed for viability using
trypan blue exclusion. Results are reported as mean (⫾ SEM) for
triplicate cultures. *p⬍0.05 compared with 0 mmol/L ethanol treatment.
was terminated with 7.5 mol/L guanidine hydrochloride. A
12.5-␮L aliquot of the reaction was spotted onto a strepavidin-
coated membrane, which is then washed. The amount of radio-
activity bound to the membrane was then determined using a
scintillation counter or Phosphorimager.
IL-6 Measurement
IL-6 was measured by B9 cell bioassay as we have previously
described.51
Statistical Analysis
Data were analyzed using analysis of variance, followed by
Fisher’s least significant difference for post-hoc analysis. Statis-
tical significance was determined as pⱕ0.05.
Results
Ethanol at ⱕ 100 mmol/L Does Not Alter the Viability of
HOBIT Cells
Consumption of ethanol may result in blood ethanol concentra-
tions up to approximately 90 mmol/L before lethal toxicity.35
Accordingly, to determine the cellular toxicity of in vitro expo-
sure to ethanol, HOBIT cells were incubated with a range of
ethanol concentrations that reflected levels attainable in
vivo.16,48 HOBIT cell viability was not decreased by 50 and 100
mmol/L ethanol (Figure 1). In contrast, viability was decreased
by 40% in HOBIT cells exposed to 200 mmol/L ethanol (Figure
1). These results suggest that at levels attainable in vivo, ethanol
will not cause osteoblast death. In contrast, at higher levels (i.e.,
200 mmol/L), osteoblast death will occur.
Ethanol Activates NF␬B
We and others have demonstrated that IL-6 is induced through
activation of NF␬B.22,39 Because ethanol induces IL-6 expres-
Z. Yao et al. 169
Ethanol induces NF␬B and p56lck in HOBIT cells
sion in bone marrow cells,23 we sought to determine if ethanol
activates NF␬B. Accordingly, HOBIT cells were incubated with
ethanol (100 mmol/L) for 30 min, then nuclear NF␬B DNA-
binding activity was assessed. Ethanol induced nuclear NF␬B
DNA-binding activity (Figure 2A). This activity was competed
away by unlabeled excess NF␬B response element, thus demon-
strating specificity for NF␬B DNA binding. The presence of
increased nuclear DNA binding does not necessarily correlate
with activity. Accordingly, we determined if ethanol activated a
minimal promoter driven by NF␬B response elements. Ethanol
(100 mmol/L) induced a 10-fold increases of promoter activity
(Figure 2B). Taken together, these data demonstrate that ethanol
induced NF␬B activity in HOBIT cells.
Ethanol Induces PTK Activity
PTK have been implicated in activation of NF␬B.42 Accordingly,
we determined if ethanol activated PTK. We observed that 50
and 100 mmol/L ethanol induced a 2.5-fold and 4.2-fold increase
in PTK activity, respectively (Figure 3A). Induction of PTK
activity was observed as early as 5 min after the addition of
ethanol and peaked approximately 25 min after ethanol was
added (Figure 3B). To begin to dissect which PTK mediates this
process, we preincubated cells with specific PTK inhibitors
before addition of ethanol. Preincubation of HOBIT cells with
Herb A (a pp60src inhibitor) had no effect on ethanol-induced
PTK activity (Figure 4). In contrast, preincubation with DAM (a
p56lck inhibitor) inhibited ethanol-induced PTK activity by ap-
proximately 50% (Figure 4), suggesting that ethanol induces
p56lck.
Ethanol Induces NF␬B Nuclear Translocation and IL-6
Expression Through p56lck
To confirm if ethanol activation of p56lck induces NF␬B nuclear
translocation, we preincubated HOBIT cells with DAM, then
added ethanol and measured nuclear NF␬B levels. Inhibition of
p56lck with DAM diminished ethanol-induced NF␬B nuclear
translocation (Figure 5A). Furthermore, to determine if p56lck
inhibition prevents ethanol-mediated upregulated IL-6 expres-
sion, we preincubated primary human osteoblasts with DAM,
added ethanol, then measured IL-6 levels in the cell culture
supernatant. Inhibition of p56lck with DAM diminished ethanol-
induced IL-6 expression (Figure 5B). These results demonstrate
that ethanol-induced NF␬B nuclear translocation and IL-6 ex-
pression requires p56lck activity.
Ethanol Induces PTK Activity Through Protein Kinase C and
Not Protein Kinase A
To determine if ethanol induces PTK through other classes of
kinases, we preincubated the cells with a PKA inhibitor (H89)
and a PKC (BIS). Preincubation of HOBIT cells with H89 had no
effect on ethanol-induced PTK activity (Figure 6). In contrast,
preincubation with BIS inhibited ethanol-induced PTK activity
by approximately 50% (Figure 6). These results suggest that
ethanol induces PTK through PKC and independently of PKA.
Discussion
In the current report, we demonstrate a putative molecular
mechanism through which ethanol induced bone resorption.
Specifically, ethanol induces PTK, which in turn induces NF␬B
and IL-6. Although we did not directly demonstrate the NF␬B
induction accounts for IL-6 expression in these cells, the ability
of ethanol to induce nuclear NF␬B DNA binding in HOBIT cells
170 Z. Yao et al.
Ethanol induces NF␬B and p56lck in HOBIT cells
Figure 2. Ethanol induces NF␬B activity. (A) HOBIT cells were
plated in T750 flasks and left untreated or treated with ethanol (100
mmol/L) for 1 h. Cells were then harvested and nuclear extract was
isolated. The nuclear extract was preincubated with no oligomer, a
100⫻ concentration NF␬B response element oligomer, or a 100⫻
concentration of Oct-1 response element oligomer. Then, a 32P-end-
labeled NF␬B consensus oligomer at 1⫻ concentration was added to
the extract and incubated for 30 min. The reaction was subjected to
electrophoresis and the gel was imaged using a Phosophorimager.
Lane 1 indicates the probe alone. (B) HOBIT cells were cotransfected
with pNF␬BX2, pRL-CMV, and pBluescript. Twenty-four hours after
transfection, the media were changed and the indicated amount of
ethanol was added. Cells were incubated for an additional 24 h, total
protein extract was collected, and luciferase levels were determined
using a luminometer. Luciferase levels were normalized to Renella
luciferase levels. Data are reported as the mean (⫾SD) lux activity
relative to the basal activity, which was set as 100%, from two
independent experiments. *p ⬍ 0.01 compared with basal activity.
Bone Vol. 28, No. 2
February 2001:167–173
Figure 3. Ethanol induces PTK activity in HOBIT Cells. HOBIT cells
(5 ⫻ 106/10-cm plate) were plated in DMEM ⫹ 20% FBS for 24 h, then
changed to DMEM ⫹ 0.5% FBS and the indicated concentration of
ethanol. (A) At 30 min after the addition of ethanol, total cell extracts
were then harvested and evaluated for PTK activity by their ability to
phosphorylate a PTK-specific substrate using the Signal TECT system
(Promega). (B) The HOBIT cells were treated with 100 mmol/L ethanol
for the indicated times. PTK activity was then determined. Results are
reported as mean (⫾ SEM) from duplicates of two experiments. *p ⱕ
0.01 compared with 0 mmol/L ethanol treatment (A) or 0 time point (B).
suggests ethanol activates genes that are regulated by NF␬B-
responsive promoters, such as the il-6 gene.22 These data are
consistent with the previous demonstration that ethanol induces
NF␬B DNA binding in monocytes.32 However, in that study,
ethanol preferentially induced the inhibitory p50/p50 ho-
modimer, and resulted in no induction of the p65/p50 het-
erodimer. This observation suggests that ethanol inhibits NF␬B
activity because the p50/p50 homodimer is inhibitory on NF␬B
response elements.32 Our observation that ethanol activated a
minimal promoter downstream of NF␬B response elements dem-
onstrates that ethanol induces active NF␬B in HOBIT cells.
The observation that 50 and 100 mmol/L ethanol induced
PTK suggests that PTK is induced at levels attainable by ethanol
ingestion.16,48 In contrast, the observation that 200 mmol/L
ethanol did not stimulate PTK activity is consistent with the
observation that this level of ethanol had a direct toxic effect on
the cells. Ethanol induced PTK rapidly (starting at 5 min and
peaking at 25 min), which is consistent with a PTK response.
The observation that ethanol activated NF␬B through PTK in
HOBIT cells is consistent with previous reports on PTK-medi-
ated activation of NF␬B. For example, IL-1 activation of NF␬B
Bone Vol. 28, No. 2 Z. Yao et al. 171
February 2001:167–173 Ethanol induces NF␬B and p56lck in HOBIT cells

Figure 4. Ethanol-induces p56lck activity. Cells were plated as described

in Figure 3 and treated with 100 mmol/L ethanol. However, herbimycin
A (24 mmol/L) or damnacanthal (34 mmol/L) was added 30 min before
ethanol addition as indicated. Results are reported as mean (⫾ SEM)
from duplicates of two experiments. *p ⱕ 0.02 compared with no
treatment or damacanthal pretreatment.

in melanoma cells was dependent on PTK activity.19 Addition-

ally, Raf-1 protein kinase directly phosphorylated I␬B␣, and its
overexpression in cells leads to elevated levels of nuclear NF␬B
and activation of an NF␬B-responsive promoter.29 Finally, mi-
togen-activated protein kinase/ERK kinase-1 (MEKK1), a dual
tyrosine/threonine kinase, has been demonstrated to induce site-
specific phosphorylation of I␬B␣.28 In addition to these PTK,
several I␬B kinase (IKK) subunits have been identified.12,34,40
CHUK (also called IKK␣ and IKK1) is a serine kinase that
induces phosphorylation of I␬B␣ at Ser32 and Ser36. Additional
kinase subunits of the IKK complex, IKK␣ and IKK␥, have been
identified.34,41,49,50 Future studies to evaluate ethanol-mediated
modulation of IKK may provide further clues as to ethanol’s
ability to induce NF␬B.
That the pp60src PTK inhibitor HERB did not modulate
ethanol-induced PTK activity demonstrates that ethanol induces
PTK activity through a mechanism that does not involve pp60src.
In contrast, that the p56lck inhibitor DAM reduced ethanol-
induced PTK activity suggests that p56lck, at least in part,
accounts for ethanol-induced PTK activity. These results are
consistent with the previous reports that oxidants induce p56lck
activity and NF␬B activity in human lymphocytes42 and COS
cells.17 Furthermore, the oxidant pervanadate could not activate
NF␬B in lck-deficient COS cells, whereas transfection of cells
with lck restored pervanadate’s ability to activate NF␬B.17 These
data suggest that oxidants induce NF␬B through a mechanism
dependent on p56lck. This observation is consistent with etha-
nol’s ability to induce oxidative stress.14,38
The ability of the PKC inhibitor BIS to reduce ethanol-
induced PTK activity suggests that ethanol induces PTK activity,
in part, through PKC. Taken together, these data suggest that
ethanol induces PTK activity, in part, through p56lck activation,
which in turn depends on activation of PKC. These findings are
consistent with the previous report that ethanol induces PKC in
murine thymoctes.45,46 The evidence for PKC-induced phos-
phorylation of I␬B␣ is inconsistent. Inhibition of PMA-inducible
PKC isoforms did not inhibit NF␬B binding to DNA.9,36 How-
ever, inhibition of a PKC isoform that does not bind PMA,
PKC␨, with a dominant negative PKC␨ inhibited NF␬B activa-
Figure 5. Ethanol induces NF␬B nuclear translocation and IL-6 expres-
sion. (A) HOBIT cells were pretreated with damnacanthal (34 mmol/L)
for 30 min, then 100 mmol/L ethanol was added for 1 h. Cells were then
harvested and nuclear extract was isolated. The nuclear extract was
preincubated with no oligomer, a 100⫻ concentration NF␬B response
element oligomer, or a 100⫻ concentration of Oct-1 response element
oligomer. Then, a 32P-end-labeled NF␬B consensus oligomer at 1⫻
concentration was added to the extract and incubated for 30 min. The
reaction was subjected to electrophoresis and the gel was imaged using
a Phosophorimager. Lane 1 indicates probe alone. (B) Primary human
osteoblasts were pretreated with damnacanthal (34 mmol/L) for 30 min
followed by the addition of ethanol. Cells were then incubated for 24 h,
at which time, supernatants were collected and subjected to B9 cell
bioassay for IL-6. Results are shown as mean (⫾ SEM) from three
experiments.
tion. Furthermore, PKC␨ induced I␬B␣ phosphorylation in tumor
necrosis factor-stimulated murine fibroblasts.11
That DAM diminished ethanol-mediated induction of IL-6 in
primary human osteoblasts provided evidence that p56lck con-
tributes to IL-6 induction. Ethanol-mediated induction of IL-6
can contribute to osteoclastogenesis33 and thus account, at least
in part, for ethanol-mediated bone loss.
In conclusion, the current study demonstrates that ethanol
induces NF␬B through p56lck in HOBIT cells and IL-6 in
primary osteoblasts. Furthermore, the data suggest that activation
of the PTK is dependent on PKC activation. These results
suggest that ethanol’s ability to activate signal transduction may
172 Z. Yao et al.
Ethanol induces NF␬B and p56lck in HOBIT cells
Figure 6. Ethanol induces PTK activity through PKC. HOBIT cells were
treated as described in Figure 3. However, bisindolylmaleimide I hydro-
chloride (4.9 ␮mol/L) or H-89 (0.1 ␮mol/L) was added 30 min before
ethanol addition. These concentrations are two times their IC50. Results
are reported as mean (⫾ SEM) from duplicated of two experiments.
contribute to its ability to induce osteoclastogenic cytokines from
osteoblasts that promote bone resorption.
Acknowledgments: The authors thank Dr. Riggs for the HOBIT cell line
and Dr. S. Ghosh for pNFkBX2. This work was supported by National
Institutes of Health grant R01 AG15904 and the program in Comparative
Integrative Biology and Genomics.
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Date Received: August 18, 2000
Date Revised: October 2, 2000
Date Accepted: October 2, 2000