1. TNF enhancement of IL-6/STAT3 signaling in HEK-Blue IL-6 cells proceeds through TAK1
Vinita Doshi, Shivani Krovvidi, David Wu, Michelle Nguyen, Trung Nguyen, Ting Wu,
Pranitha Naiki, Dhaval Oza, Ralph Loring
Dept. Pharmaceutical Sci., Northeastern Univ., Boston, MA
Abstract Results
Conclusions & Future Directions
References
TNF (Tumor necrosis factor alpha) and Interleukin 6 (IL-6) are cytokines with important inflammatory roles
exhibiting different interactions depending on cell context, e.g. TNF sometimes induces IL-6 secretion, but
TNF may also alter IL-6 signaling and vice versa (via crosstalk). IL-6 activates STAT3 (Signal transducer and
activator of transcription 3) through JAK (Janus Kinase 1 & 2) in HEK-Blue® IL-6 cells (Invivogen, San Diego).
When activated in these cells, STAT3 stimulates a reporter gene, secreted alkaline phosphatase (SEAP). Co-
application of 0.3-10 ng/ml TNF increases IL-6-induced SEAP secretion 2-5 fold. However, TNF alone has no
effect on basal STAT3 signaling, suggesting that TNF increases IL-6 sensitivity without causing IL-6 secretion in
these cells. Confirming this, TNF had no effect on IL-6 secretion from HEK- Blue® cells measured by enzyme-
linked immunosorbent assays (ELISAs). TNF enhancement of IL6 signaling causes an increase in both STAT3
and phospho-STAT3 ELISAs, corroborating the SEAP results. TAK1 (TGF activated kinase 1) is downstream
from TNF receptors. The TAK1 inhibitor 5Z-7-oxozeaenol (1 mM) blocks TNF-enhanced SEAP secretion
without affecting basal IL-6 signaling. 5Z-7-oxozeaenol completely blocks “nuclear factor kappa-light-chain-
enhancer of activated B cells” (NFkB) driven SEAP secretion in a different HEK line with an NFKB reporter
system. In contrast, JAK inhibitors AG490 and AZD1480 and STAT3 inhibitor S3I-201 completely block both
basal and TNF-enhanced IL-6 induced SEAP secretion at expected concentrations. Further work is required to
see if TNF-driven TAK1 signaling through NFkB is solely responsible for increasing STAT3 translation and
thereby increasing cell sensitivity to IL-6. Since NFkB and STAT3 signaling can be synergistic or antagonistic
depending on cell context, the HEK reporter cells line may help determine the molecular basis of one version
of TNF and IL-6 crosstalk.
Background and Methods
Model of potential TNF and IL-6 crosstalk between NFkB and STAT3
The cytokines Tumor Necrosis Factor a (TNF) and Interleukin 6 (IL-6) interact in both cancer
and inflammation (e.g. Kiu et al., 2007, Al-Shanti et al., 2008, Patra et al., 2012). Shown above are some possible interactions
between the major transcription factor signaling pathways for the two cytokines. TNF binding
to its receptor activates NFkB (or other pathways shown above), while IL-6 activates
JAK2/STAT3 signaling measured with SEAP.
1) Depending on context, STAT3 can either enhance or inhibit NFkB signaling.
(e.g. Yu & Kone, 2003; Hosur & Loring, 2011)
2) Also depending on context, NFkB can either enhance or inhibit STAT3 (Squarize et al., 2006)
3) TNF also leads to the transcription of IL-6 mRNA through NFkB and secretion of IL-6 protein
which can then bind to the IL-6 receptor. (e.g. Rotter et al. 2008)
4) Possible interactions exist between other signaling pathways that we are starteing to
investigate.
We investigated the interaction between TNF and IL-6 signaling in a commercially-available cell
line (HEK-Blue® IL-6 cells from Invivogen). Addition of IL-6 (Invivogen) activates STAT3 via
JAK1/2/TYK2, and STAT3 causes transcription of the reporter gene, secreted alkaline
phoshatase (SEAP). We also used HEK cells transfected with a NFkB-driven SEAP reporter.
Modified from http://www.invivogen.com/hek-blue-il6
JNK p38
C-JUN? ELK1?
CREB?
(5Z)-7-Oxozeaenol?
(5Z)-Zeaenol (inactive)
JNK inhibitor II?
SB 203580?
Secreted SEAP is
the readout,
SEAP acts on
Quanti-Blue®
substrate
(Invivogen) to
form a dye
absorbing at 640
nm.IL-6
1?
2?
Extracellular space
JAK Inhibitor 1
AG490
AZD1480
Stattic
S3I-201
Z-Vad-FMK?
Apoptosis,
Cell Death
Parthenolide?
PDTC?
BAY 11-7082?
TAK1
(MAP3K7)
Cytoplasm
Nucleus
4?
3?
Modified from http://www.invivogen.com/hek-blue-il6
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 5 10 15
OD640
ng/mL IL-6
TNF effects on IL-6 response
TNFa=0
TNFa=0.1ng/mL
TNFa=0.3ng/mL
TNFa=1ng/mL
TNFa=3ng/mL
TNFa=10ng/mL
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 5 10 15
OD640
ng/mL TNFa
TNF enhancement of IL-6
response
IL-6=0
IL-6=0.1ng/mL
IL-6=1ng/mL
IL-6=10ng/mL
TNF enhances IL-6 evoked STAT3 responses. Left, IL-6 dose response curves in increasing
concentrations of TNF. Right, the same data showing the effect of varying TNF concentration
on SEAP secretion evoked by four concentrations of IL-6. In this experiment, TNF increased IL-6
responses 3-5 fold (in some experiments only 2 fold) over the concentration range of 0.1-10
ng/ml TNF. Note that TNF had no effect in the absence of IL-6 (1x), suggesting TNF does not
induce IL-6 secretion, but instead increases IL-6 sensitivity.
1x
3x
5x
Initial Observations of TNF-IL6 crosstalk:
TNF does not cause IL-6 secretion in HEK-Blue cells
0
1
2
3
4
5
6
7
IL-6concentrationng/ml
IL-6 concentration in cell medium after 24 H
no TNFa, no IL-6
10ng/ml TNFa, no IL-6
no TNFa, 10ng/ml IL-6
10ng/ml TNFa, 10ng/ml IL-6
no cells, 10ng/ml IL-6
A commercial ELISA kit for human IL-6 (BD Bioscience) revealed that treating cells with TNF
in the absence of IL-6 (red arrow) or in the presence of 10 ng/ml IL-6 (purple arrow) did not
change the IL-6 concentration in the cell medium 24 h later compared with controls (blue &
green arrows). The presence of cells significantly decreased IL-6 in the medium, suggesting
that the cells metabolize IL-6.
• HEK-Blue® IL-6 cells show TNF-induced enhancement of IL-6 signaling.
• TNF has no effect on basal IL-6 secretion in HEK-Blue® IL-6 cells.
• The mechanism by which TNF increases IL-6 sensitivity remains to be determined
but so far, does not appear to involve the p38 or JNK signaling pathways (not
shown).
• Western blotting will establish whether TNF increases the expression of IL-6
receptors.
• TNF–induced changes in JAK or STAT3 expression will be determined by Western
blots and/or ELISAs.
• The TAK1 inhibitor 5z-7-oxozeaeonol blocks the TNF-induced enhancement of IL-6
signaling, and will be a useful tool to evaluate the contribution of the NFkB pathway
in this process.
•Al-Shanti et al., 2008, Beneficial synergistic interactions of TNF-a and IL-6 in C2 skeletal
myoblasts-Potential cross-talk with IGF system, Growth Factors, 26: 61–73
• Hosur & Loring, 2011, a4b2 Nicotinic Receptors Partially Mediate Anti-Inflammatory Effects
through Janus Kinase 2-Signal Transducer and Activator of Transcription 3 but Not Calcium or
cAMP Signaling, Mol Pharmacol 79:167–174
• Kiu et al., 2007, Mechanism of crosstalk inhibition of IL-6 signaling in response to LPS and
TNFa, Growth Factors, 25: 319–328
• Lin et al., 1995, Inhibition of translocation of transcription factor NF-kB by a synthetic peptide
containing a cell-permeable motif and nuclear localization sequence. J. Biol. Chem. 270: 14255-
14258.
• Patra et al., 2012, Integrative role of neuropeptides and cytokines in cancer anorexia-cachexia
syndrome. Clin. Chim. Acta 413: 1025-34
• Rotter et al., 2003, Interleukin-6 (IL-6) Induces Insulin Resistance in 3T3-L1 Adipocytes and Is, Like
IL-8 and Tumor Necrosis Factor-a, Overexpressed in Human Fat Cells from Insulin-resistant
Subjects. J. Biol. Chem. 278:45777–45784
• Squarize et al., 2006, Molecular Cross-Talk between the NFKB and STAT3 Signaling Pathways in
Head and Neck Squamous Cell Carcinoma, Neoplasia 8: 733 – 746
• Yu & Kone, 2003, The STAT3 DNA-Binding Domain Mediates Interaction with NF-kB p65 and
Inducible Nitric Oxide Synthase Transrepression in Mesangial Cells, J Am Soc Nephrol 15: 585–591
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 2 4 6 8 10 12
OD640
5-7-Oxozeaeonol concentration (mM)
TNF+ oxozeaenol
Control
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0.001 0.01 0.1 1 10
OD640
5z-7-Oxozeaeonal Concentration (mM)
Block of TNF-enhanced IL6 signaling by oxozeaeonal
(STAT3 driven signaling)
IL6+TNF+Oxo
IL6+ Oxo
Untreated control
//
//
//
0
Oxozeaeonol Blocks TNF-Driven NFkB SEAP
NFkB-driven signaling
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
OD450
STAT3 ELISA
Phospho-STAT3 (PY705) Total STAT3
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0 0.2 0.4 0.6 0.8 1
OD640
Concentration AZD1480 (mM)
IL6+TNF
IL6
Control
0
0.5
1
1.5
2
2.5
0 100 200 300 400 500 600 700
OD640
Concentration S3I-201 (mM)
IL6 alone
IL6 + TNF
Left Panel: TAK1 inhibitor Oxozeaeonol completely blocks NFkB-SEAP in TNF-treated NFkB-SEAP
HEK cells.
Right Panel: Oxozeaeonal blocks only the TNF-enhanced portion of IL6-driven STAT3-SEAP
secretion in HEK-Blue® IL6-SEAP cells. These data suggest that TNF enhancement effects may
occur through NFkB signaling.
Left Panel: JAK2 inhibitor AZD1480 completely blocks both IL6 and TNF-enhanced IL6 signaling,
indicating that all HEK cells.
Right Panel: STAT3 inhibitor S3I-201 blocks STAT3 signaling in HEK-Blue® IL6-SEAP cells. These
data suggest that all IL6 signaling in these cells proceeds through the JAK2/STAT3 pathway.
These results are part of capstone projects by David Wu and Michelle Nguyen.
JAK2 inhibitor AZD1480 blocks STAT3 signaling in
HEK-Blue IL6 –SEAP cells
STAT3 inhibitor S3I-201 blocks STAT3 signaling in
HEK-Blue IL6 –SEAP cells
TNF enhances Phospho-STAT3 and total STAT3 production by IL-6
STAT3 ELISA data indicates that overnight treatment with TNF increases the
amount of both total and phospho-STAT3 induced by 30 min treatment by IL6.
However, a 60 minute TNF treatment doesn’t cause the same effect. We will be
investigating whether TNF acts to increase the total amount of STAT3 and other
components of the JAK/STAT signaling pathway using PCR and Western blots.