1: Blood. 2005 Jan 15;105(2):689-96. Epub 2004 Jul 13. 

STAT3 regulates NF-kappaB recruitment to the IL-12p40 promoter in dendritic
cells.

Hoentjen F, Sartor RB, Ozaki M, Jobin C.

Center for Gastrointestinal Biology and Diseases, University of North Carolina
at Chapel Hill, USA.

Interleukin-10-deficient (IL-10(-/-)) mice develop an IL-12-mediated intestinal
inflammation in the absence of endogenous IL-10. The molecular mechanisms of the
dysregulated IL-12 responses in IL-10(-/-) mice are poorly understood. In this
study, we investigated the role of nuclear factor-kappa B (NF-kappaB) and signal
transducers and activators of transcription 3 (STAT3) in lipopolysaccharide
(LPS)-induced IL-12p40 gene expression in bone marrow derived-dendritic cells
(BMDCs) isolated from wild-type (WT) and IL-10(-/-) mice. We report higher
IL-12p40 mRNA accumulation and protein secretion in LPS-stimulated BMDCs
isolated from IL-10(-/-) compared with WT mice. LPS-induced NF-kappaB signaling
is similar in IL-10(-/-) and WT BMDCs as measured by IkappaBalpha
phosphorylation and degradation, RelA phosphorylation and nuclear translocation,
and NF-kappaB transcriptional activity, with no down-regulatory effects of
exogenous IL-10. Chromatin immunoprecipitation demonstrated enhanced NF-kappaB
(cRel, RelA) binding to the IL-12p40 promoter in IL-10(-/-) but not WT BMDCs.
Interestingly, LPS induced STAT3 phosphorylation in WT but not IL-10(-/-) BMDCs,
a process blocked by IL-10 receptor blocking antibody. Adenoviral gene delivery
of a constitutively active STAT3 but not control green fluorescence protein
(GFP) virus blocked LPS-induced IL-12p40 gene expression and cRel recruitment to
the IL-12p40 promoter. In conclusion, dysregulated LPS-induced IL-12p40 gene
expression in IL-10(-/-) mice is due to enhanced NF-kappaB recruitment to the
IL-12p40 promoter in the absence of activated STAT3.

PMID: 15251981 [PubMed - indexed for MEDLINE]


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2: J Biol Chem. 2004 Jun 4;279(23):24873-80. Epub 2004 Mar 26. 

PIAS3 suppresses NF-kappaB-mediated transcription by interacting with the
p65/RelA subunit.

Jang HD, Yoon K, Shin YJ, Kim J, Lee SY.

Division of Molecular Life Sciences and Center for Cell Signaling Research, Ewha
Womans University, Seoul 120-750, Korea.

Nuclear factor-kappaB (NF-kappaB) is a transcription factor critical for key
cellular processes, including immune response, apoptosis, and cell cycle
progression. A yeast two-hybrid screening, using the Rel homology domain (RHD)
of the p65 subunit (RelA) of NF-kappaB as bait, led to the isolation of PIAS3,
previously identified as a specific inhibitor of STAT3. We show that PIAS3 can
directly associate with p65 using an in vitro pull-down and in vivo
coimmunoprecipitation assays. When overexpressed, PIAS3 inhibits
NF-kappaB-dependent transcription induced by treatment with tumor necrosis
factor alpha (TNF-alpha) or interleukin-1beta or by overexpression of TNF family
receptors such as RANK, TNFR1, and CD30 or signal transducers of TNF
receptor-associated factors (TRAFs), including TRAF2, TRAF5, and TRAF6.
Downregulation of PIAS3 by RNA interference reverses its effect on
TNF-alpha-mediated NF-kappaB activation. We found that an N-terminal region of
PIAS3 is necessary for both the interaction with p65 and the transcriptional
suppression activity. In addition, we found that an LXXLL coregulator signature
motif located within the N-terminal region of PIAS3 is the minimal requirement
for the interaction with p65. Furthermore, we demonstrate that PIAS3 interferes
with p65 binding to the CBP coactivator, thereby resulting in a decreased
NF-kappaB-dependent transcription. Taken together, these data suggest that PIAS3
may function in vivo as a modulator in suppressing the transcriptional activity
of p65.

PMID: 15140884 [PubMed - indexed for MEDLINE]


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3: Mol Immunol. 2003 Oct;40(6):373-80. 

Transcription factor c-Rel enhances C-reactive protein expression by
facilitating the binding of C/EBPbeta to the promoter.

Agrawal A, Samols D, Kushner I.

Department of Pharmacology, East Tennessee State University, Johnson City, TN
37614, USA.

Induction of C-reactive protein (CRP) synthesis in hepatocytes by cytokines
occurs at the transcriptional level. In Hep3B cells, the transcription factors
C/EBPbeta, STAT3, and Rel p50 have been shown to participate in this process. A
C/EBP binding site centered at -53 and an overlapping nonconsensus kappaB site
on the promoter are critical for CRP expression. We have previously found that
an oligonucleotide containing a kappaB site diminished binding of C/EBPbeta to
the C/EBP site, suggesting that unidentified Rel proteins present in Hep3B
nuclei facilitate the formation of C/EBPbeta-complexes. The current studies were
undertaken to determine which of the five Rel proteins, p50/p65/p52/c-Rel/RelB,
play such a role. Mutation of the nonconsensus kappaB site did not abolish
binding of C/EBPbeta to its binding site, indicating that this site was not
necessary for the formation of C/EBPbeta-complexes. Depletion of Rel proteins
from Hep3B nuclei led to decreased formation of C/EBPbeta-complexes on a CRP
promoter-derived oligonucleotide that contained only the intact C/EBP binding
site but not the nonconsensus kappaB site. This finding indicates that Rel
proteins are involved in the binding of C/EBPbeta to its binding site by a
kappaB site-independent mechanism. Electrophoretic mobility shift assays (EMSAs)
revealed that it was c-Rel that facilitated formation of C/EBPbeta-complexes and
that c-Rel bound directly to C/EBPbeta-complexes formed on the C/EBP site.
Cotransfection of c-Rel enhanced the induction of CRP promoter-driven luciferase
activity and enhanced endogenous CRP expression in cells transfected with
C/EBPbeta. We conclude that c-Rel regulates CRP expression without the
requirement of binding to a kappaB site, and binds directly to C/EBPbeta to
facilitate the binding of C/EBPbeta to the CRP promoter.

PMID: 14522018 [PubMed - indexed for MEDLINE]


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4: Immunology. 2003 Apr;108(4):539-47. 

Overexpressed nuclear factor-kappaB can participate in endogenous C-reactive
protein induction, and enhances the effects of C/EBPbeta and signal transducer
and activator of transcription-3.

Agrawal A, Cha-Molstad H, Samols D, Kushner I.

Department of Biochemistry, Case Western Reserve University, Cleveland, OH, USA.

C-reactive protein (CRP), the prototypical human acute phase protein, is
produced primarily by hepatocytes. Its expression is modestly induced by
interleukin (IL)-6 in Hep3B cells while IL-1, which alone has no effect,
synergistically enhances the effects of IL-6. In previous studies of the
proximal CRP promoter, we found that signal transducer and activator of
transcription-3 (STAT3) and C/EBPbeta -mediated IL-6-induced transcription and
that Rel p50 acted synergistically with C/EBPbeta, in the absence of p65, to
enhance CRP transcription. Neither a requirement nor a binding site for the
classic nuclear factor (NF)-kappaB heterodimer p50/p65 were found. The current
studies were undertaken to determine whether similar novel transcription factor
interactions might regulate the endogenous CRP gene. Transiently overexpressed
p50 or p65 induced CRP mRNA accumulation in Hep3B cells. The heterodimer p50/p65
was markedly more effective than p50 or p65 homodimers. Co-overexpression of p50
or p65 with C/EBPbeta or STAT3 synergistically enhanced CRP expression. Maximal
expression was observed with overexpression of all four transcription factors;
comparable effects were observed with IL-1beta treatment of cells overexpressing
STAT3 + C/EBPbeta. Data from the Human Genome Project revealed 13 potential
kappaB sites in the first 4000 bases of the CRP promoter, only one of which,
centred at -2652, bound nuclear p50/p65 heterodimer activated by IL-1beta. Our
findings indicate that classical NF-kappaB activation can participate in
endogenous CRP induction, and that activated NF-kappaB may synergistically
enhance the effects of C/EBPbeta and STAT3. They raise the possibility, not as
yet established, that NF-kappaB activation may be responsible for the
synergistic effect of IL-1beta on IL-6-induced CRP expression.

PMID: 12667216 [PubMed - indexed for MEDLINE]


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5: Cell Immunol. 2002 May-Jun;217(1-2):47-57. 

IL-6 rescues the hyporesponsiveness of c-Rel deficient B cells independent of
Bcl-xL, Mcl-1, and Bcl-2.

Tumang JR, Hsia CY, Tian W, Bromberg JF, Liou HC.

Department of Medicine, Division of Immunology, Weill Medical College of Cornell
University, New York, NY 10021, USA.

The hematopoietically restricted member of the NF-kappaB/Rel family, c-Rel, is
essential for B cell survival and proliferation. Here we demonstrate that the
production of the interleukins 6, 10, and 15 (IL-6, IL-10, and IL-15) are
diminished in c-Rel(-/-) B lymphocytes. In a manner similar to that seen in
IL-6(-/-) B cells, resultant STAT activation is reduced in c-Rel(-/-) B cells
following B cell receptor (BCR) ligation. Addition of either exogenous IL-6 or
IL-10, but not IL-15, partially restores proliferation, and this occurs through
enhanced cell survival rather than promoting cell cycle progression. This
increase in viability occurs independently of Bcl-xL and Mcl-1 expression
though, two survival genes reported to be downstream of IL-6 signaling.
Nonetheless, transgenically expressed Bcl-xL, a direct c-Rel target gene in B
cells, corrects not only the survival defect of c-Rel deficiency, but also
partially ameliorates hypoproliferation. Together IL-6 and Bcl-xL are additive
but incomplete in the restoration of proliferation. Known deficits in the
induction of several key cell cycle components in c-Rel(-/-)B cells are not
corrected upon treatment with exogenous cytokine. Together, these data
demonstrate that IL-6 enhances B cell responses by employing multiple survival
factors.

PMID: 12426000 [PubMed - indexed for MEDLINE]


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6: J Immunol. 2001 Oct 1;167(7):3652-60. 

STAT5 induces macrophage differentiation of M1 leukemia cells through activation
of IL-6 production mediated by NF-kappaB p65.

Kawashima T, Murata K, Akira S, Tonozuka Y, Minoshima Y, Feng S, Kumagai H,
Tsuruga H, Ikeda Y, Asano S, Nosaka T, Kitamura T.

Division of Hematopoietic Factors, Institute of Medical Science, University of
Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

We recently demonstrated that STAT5 can induce a variety of biological functions
in mouse IL-3-dependent Ba/F3 cells; STAT5-induced expression of pim-1,
p21(WAF/Cip1), and suppressor of cytokine signaling-1/STAT-induced STAT
inhibitor-1/Janus kinase binding protein is responsible for induction of
proliferation, differentiation, and apoptosis, respectively. In the present
study, using a constitutively active STAT5A (STAT5A1*6), we show that STAT5
induces macrophage differentiation of mouse leukemic M1 cells through a distinct
mechanism, autocrine production of IL-6. The supernatant of STAT5A1*6-transduced
cells contained sufficient concentrations of IL-6 to induce macrophage
differentiation of parental M1 cells, and STAT3 was phosphorylated on their
tyrosine residues in these cells. Treatment of the cells with anti-IL-6 blocking
Abs profoundly inhibited the differentiation. We also found that the STAT5A1*6
transactivated the IL-6 promoter, which was mediated by the enhanced binding of
NF-kappaB p65 (RelA) to the promoter region of IL-6. These findings indicate
that STAT5A cooperates with Rel/NF-kappaB to induce production of IL-6, thereby
inducing macrophage differentiation of M1 cells in an autocrine manner. In
summary, we have shown a novel mechanism by which STAT5 induces its pleiotropic
functions. Cytokines

PMID: 11564778 [PubMed - indexed for MEDLINE]


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7: Curr Opin Oncol. 2000 Nov;12(6):543-9. 

Regulation of Bcl-xL: a little bit of this and a little bit of STAT.

Grad JM, Zeng XR, Boise LH.

Sylvester Comprehensive Cancer Center, Department of Microbiology and
Immunology, University of Miami School of Medicine, Florida, USA.

The Bcl-2 family of proteins are key regulators of apoptosis. Bcl-xL, is an
anti-apoptotic protein with a high degree of homology to Bcl-2; however, the
signals that regulate Bcl-xL and Bcl-2 appear to be different. Levels of Bcl-xL,
but not Bcl-2, are increased in response to various survival signals.
Furthermore, an inverse correlation between the levels of Bcl-2 and Bcl-xL has
been reported for a number of cancers. Although the precise molecules that
control Bcl-xL activity are unclear, the STAT, Rel/NF-kappaB, and Ets
transcription factor families have recently been reported to directly regulate
the bcl-x gene. Activated Ras, integrin, vitronectin, and hepatocyte growth
factor signaling cascades have also been linked to changes in Bcl-xL expression.
Bcl-xL can also be affected by post-translational mechanisms. Here we review
recent advances in identifying the signaling pathways and factors involved in
regulation of the bcl-x gene.

Publication Types:
    Review
    Review, Tutorial

PMID: 11085453 [PubMed - indexed for MEDLINE]


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8: J Immunol. 2000 Oct 15;165(8):4592-7. 

The Rel family member P50 mediates cytokine-induced C-reactive protein
expression by a novel mechanism.

Cha-Molstad H, Agrawal A, Zhang D, Samols D, Kushner I.

Department of Biochemistry, Case Western Reserve University, Cleveland, OH
44106, USA.

Transcription of C-reactive protein (CRP) in Hep 3B cells is induced by IL-6,
acting through C/EBP isoforms and STAT3. IL-1beta, which alone has no effect,
greatly enhances IL-6-induced transcription by unknown mechanisms. Because
IL-1beta activates the NF-kappaB system, we explored the effects of
overexpressed Rel family members on CRP expression. Unexpectedly,
transactivation assays in transiently transfected Hep 3B cells showed p50
overexpression to markedly induce CRP transcription, acting in a region 3' to
-86. In the presence of overexpressed p50, IL-1beta induced a 3-fold increase in
CRP expression, and responses to IL-6 and to IL-6 plus IL-1beta were 4-fold
greater than seen in cells without p50 overexpression. In contrast,
overexpressed p65 abolished CRP induction by p50 and by cytokines. EMSA studies
demonstrated that recombinant p50 bound to a nonconsensus kappaB site
overlapping the proximal C/EBP binding site on the CRP promoter. Mutation of a
polypyrimidine tract in the p50-binding site inhibited the transactivating
effect of cytokines. P50- but not p65-containing dimers were found in nuclei of
Hep 3B cells 18 h after stimulation with IL-1beta, when C/EBPbeta is greatly
activated, in the presence or absence of IL-6. These findings suggest that
IL-1beta induces nuclear translocation of p50-containing dimers and that p50
interacts with C/EBPbeta activated by both IL-6 and IL-1beta to induce CRP
expression.

PMID: 11035101 [PubMed - indexed for MEDLINE]


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9: Am J Pathol. 2000 Mar;156(3):997-1007. 

Prevention of hepatic apoptosis and embryonic lethality in RelA/TNFR-1 double
knockout mice.

Rosenfeld ME, Prichard L, Shiojiri N, Fausto N.

Department of Pathology, University of Washington, Seattle, Washington 98195,
USA.

Mice deficient in the nuclear factor kappaB (NF-kappaB)-transactivating gene
RelA (p65) die at embryonic days 14-15 with massive liver apoptosis. In the
adult liver, activation of the NF-kappaB heterodimer RelA/p50 can cause
hepatocyte proliferation, apoptosis, or the induction of acute-phase response
genes. We examined, during wild-type fetal liver development, the expression of
the Rel family member proteins, as well as other proteins known to be important
for NF-kappaB activation. We found these proteins and active NF-kappaB complexes
in the developing liver from at least 2 days before the onset of lethality
observed in RelA knockouts. This suggests that the timing of NF-kappaB
activation is not related to the timing of lethality. We therefore hypothesized
that, in the absence of RelA, embryos were sensitized to tumor necrosis factor
(TNF) receptor 1 (TNFR-1)-mediated apoptosis. Thus, we generated mice that were
deficient in both RelA and TNFR-1 to determine whether apoptotic signaling
through TNFR-1 was responsible for the lethal phenotype. RelA/TNFR-1 double
knockout mice survived embryonic development and were born with normal livers
without evidence of increased hepatocyte apoptosis. These animals became runted
shortly after birth and survived an average of 10 days, dying from acute
hepatitis with an extensive hepatic infiltration of immature neutrophils. We
conclude that neither RelA nor TNFR-1 is required for liver development and that
RelA protects the embryonic liver from TNFR-1-mediated apoptotic signals.
However, the absence of both TNFR-1 signaling and RelA activity in newborn mice
makes these animals susceptible to endogenous hepatic infection.

PMID: 10702415 [PubMed - indexed for MEDLINE]


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10: Oncogene. 1999 Feb 18;18(7):1401-9. 

Binding of c-Rel to STAT5 target sequences in HTLV-I-transformed T cells.

Sun SC, Maggirwar SB, Harhaj EW, Uhlik M.

Department of Microbiology and Immunology, Pennsylvania State University College
of Medicine, Hershey Medical Center 17033, USA.

The type I human T-cell leukemia virus (HTLV-I) induces abnormal growth and
subsequent transformation of T cells, which is associated with the development
of an acute T-cell malignancy termed adult T-cell leukemia. A characteristic of
HTLV-I-transformed T cells is the constitutive nuclear expression of
NF-kappaB/Rel family of transcription factors, which appears to be essential for
the growth of these transformed cells. Although NF-kappaB/Rel factors are known
to induce the expression of T-cell growth factor interleukin (IL)-2, it is
unclear how they participate in the IL-2-independent growth of
HTLV-I-transformed cells. In this study, we show that certain NF-kappaB/Rel
members, predominantly c-Rel, interact with enhancer sequences for STAT5, a key
transcription factor mediating IL-2-induced T-cell proliferation. Reporter gene
assays reveal that the binding of c-Rel to the STAT5 site present in the Fc
gammaR1 gene leads to potent transactivation of this enhancer. Binding of c-Rel
to the Fc gammaR1 STAT site also occurs in human peripheral blood T cells
immortalized with HTLV-I in vitro and is correlated with enhanced levels of
proliferation of these cells. These results raise the possibility that
NF-kappaB/Rel may participate in the growth control of HTLV-I-transformed T
cells by regulating genes driven by both kappaB and certain STAT enhancers.

PMID: 10050877 [PubMed - indexed for MEDLINE]


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