1: J Immunol. 2005 Jul 1;175(1):469-77. 

ERK activation following macrophage FcgammaR ligation leads to chromatin
modifications at the IL-10 locus.

Lucas M, Zhang X, Prasanna V, Mosser DM.

Department of Cell Biology and Molecular Genetics, University of Maryland,
College Park, MD 20742, USA.

We have previously demonstrated that macrophages stimulated in the presence of
immune complexes produce high levels of IL-10. We now examine the mechanism of
IL-10 superinduction. We report that the enhanced production of IL-10 correlates
with a rapid and enhanced activation of two MAPKs, ERK and p38. The inhibition
of either ERK or p38 prevented IL-10 induction, indicating that both MAPKs were
required for IL-10 synthesis. By chromatin immunoprecipitation assay, we
demonstrate that activation of ERK leads to the phosphorylation of serine 10 on
histone H3 at the il-10 gene, making the promoter more accessible to
transcription factors generated in response to p38 activation. Inhibition of ERK
activation prevented histone modifications, and decreased the binding of Sp1 and
STAT3 to the IL-10 promoter. We conclude that the activation of ERK following
FcgammaR ligation leads to a remodeling of the chromatin at the il-10 locus,
making it more accessible to transcription factors. The rapid and transient
regulation of transcription factor accessibility to the IL-10 promoter by MAPK
activation represents a novel way that the production of this cytokine is
regulated.

PMID: 15972681 [PubMed - indexed for MEDLINE]


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2: Gene. 2005 Jun 20;353(1):107-17. 

Characterization of the infection-responsive bovine lactoferrin promoter.

Zheng J, Ather JL, Sonstegard TS, Kerr DE.

Lactation and Mammary Gland Biology Group, Department of Animal Science, 213
Terrill Hall, University of Vermont, Burlington, VT 05405, USA.

The concentration of lactoferrin in bovine milk is dramatically increased in
response to infection. The high levels of lactoferrin may have a role in the
prevention of microbial infection of the mammary gland. However, molecular
mechanisms of how the lactoferrin gene is regulated in the mammary gland in
response to infection remain unknown. In this study, we isolated and
characterized the 5' flanking region of the bovine lactoferrin gene. An 8.2
kilobase (kb) fragment of the bovine lactoferrin gene, containing 4.4 kb of 5'
flanking region, exon 1, intron 1, and exon 2, was isolated from a bovine
genomic library on two overlapping bacterial artificial chromosome (BAC) clones.
Sequence analysis of the isolated lactoferrin gene revealed that the promoter
region contains a high GC content, a non-canonical TATA box, multiple
stimulating protein 1 (SP1)/GC elements, and other putative binding sites for
transcription factors including nuclear factor-kappaB (NF-kappaB), activator
protein 1 (AP1), signal transducer and activator of transcriptions 3 and 5
(STAT3 and STAT5), and steroid hormone receptors. To demonstrate that the
isolated promoter is functional, 4.4 kb of 5' flanking region was inserted
upstream from the firefly luciferase gene and the chimeric construct was
transiently transfected into murine mammary epithelial cells. Transfection
studies showed that the basal promoter activity is quite potent, being similar
in strength to that of the simian virus 40 (SV40) promoter/enhancer. In
addition, a 24-h treatment with Escherichia coli lipopolysaccharide (LPS)
significantly stimulated its activity up to 2.3-fold in a dose-dependent manner.
Furthermore, promoter deletion analysis indicated that the sequence up to -543
was sufficient for basal activity, whereas the sequence up to -1029 was required
for maximal basal activity. The basal activity of the promoter is affected by
both positive regulatory regions (-2462/-1879 and -1029/-75) and a negative
regulatory region (-1407/-1029). LPS-responsive regions of the promoter were
localized to the region from -1029 to -543 containing one STAT3 site and two
NF-kappaB sites, and the region from -4355 to -2462 containing three AP1 sites
and six NF-kappaB sites. Taken together, our findings suggested that the
lactoferrin promoter responds to infection via the NF-kappaB pathway.

PMID: 15935571 [PubMed - indexed for MEDLINE]


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3: Arterioscler Thromb Vasc Biol. 2005 Jul;25(7):1395-400. Epub 2005 Apr 28. 

Signal transducer and activator of transcription 3alpha and specificity protein
1 interact to upregulate intercellular adhesion molecule-1 in
ischemic-reperfused myocardium and vascular endothelium.

Yang XP, Irani K, Mattagajasingh S, Dipaula A, Khanday F, Ozaki M, Fox-Talbot K,
Baldwin WM 3rd, Becker LC.

Cardiology Division, Halsted 500, Johns Hopkins Hospital, 600 N Wolfe St,
Baltimore, MD 21287-5500. lbecker@mail.jhmi.edu.

OBJECTIVE: Intercellular adhesion molecule-1 (ICAM-1) is upregulated rapidly on
endothelial cells during ischemia-reperfusion (I-R) and mediates tissue
leukocyte accumulation. The ICAM-1 proximal promoter contains a signal
transducer and activator of transcription (Stat) binding motif (gamma-interferon
activation site [GAS] sequence), which flanks a specificity protein 1 (Sp1)
binding site. We examined the roles of Stat and Sp1 in the regulation of ICAM-1
after myocardial I-R. METHODS AND RESULTS: Open-chest anesthetized rats
underwent coronary artery occlusion for 35 minutes and reperfusion for 0 to 240
minutes. Stat became activated within 15 minutes after reperfusion, primarily in
vascular endothelial cells; the activated Stat protein was identified as Stat3
(alpha-isoform). After phosphorylation on serine 727 (p-S727), Stat3alpha was
found in association with the transcriptional regulator Sp1, and the complex
bound to an ICAM-1-GAS probe. ICAM-1 expression increased after I-R and lagged
shortly behind Stat3alpha activation. In cultured human umbilical vein
endothelial (HUVE) cells, activation of Stat3alpha after hypoxia-reoxygenation
(H-R) was dependent on the small GTPase Rac1. Transfection of a
dominant-negative Stat3 (Y705F) adenovirus or a GAS decoy oligonucleotide
reduced ICAM-1 mRNA expression after H-R. Using a reporter gene transfected into
HUVE cells, mutation of the GAS element in the ICAM-1 promoter resulted in
reduced transcriptional activity after H-R. Sp1 coimmunoprecipitated with p-S727
Stat3 during H-R, and Sp1 or Stat3alpha interfering RNA markedly reduced ICAM-1
mRNA expression. CONCLUSIONS: The Sp1-Stat3 complex appears to play an important
role in the upregulation of ICAM-1 transcription after reoxygenation or
reperfusion.

PMID: 15860735 [PubMed - in process]


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4: Int J Cancer. 2005 Jun 10;115(2):202-13. 

Interleukin-6 induces transcriptional activation of vascular endothelial growth
factor (VEGF) in astrocytes in vivo and regulates VEGF promoter activity in
glioblastoma cells via direct interaction between STAT3 and Sp1.

Loeffler S, Fayard B, Weis J, Weissenberger J.

Division of Neuropathology, Institute of Pathology, University of Bern, Bern,
Switzerland.

Interleukin-6 (IL-6) expression is strongly correlated with the degree of human
glioma malignancy and necessary for tumor formation in a mouse model of
spontaneous astrocytomas. Yet, exactly how IL-6 contributes to malignant
progression of these brain tumors is still unclear. We have scrutinized the
mechanism of transcriptional activation of vascular endothelial growth factor
(VEGF) expression by IL-6 in the mouse brain and in glioblastoma cells. We
demonstrate here that IL-6 drives transcriptional upregulation of VEGF in
astrocytes in vivo using glial fibrillary acidic protein (GFAP)-IL-6/VEGF-green
fluorescent protein (GFP) double transgenic mice. We further show that
IL-6-induced VEGF transcription and VEGF secretion by human glioblastoma cells
is dependent on signal transducer and activator of transcription 3 (STAT3). By
progressive 5'-deletion analysis we defined the minimal VEGF promoter region for
IL-6-responsiveness to nucleotides -88/-50. Surprisingly, this promoter region
is rich in GC-boxes and does not contain STAT3 binding elements. Electrophoretic
mobility shift and supershift assays revealed binding of Sp1 and Sp3 to the
-88/-50 element upon IL-6 stimulation. Interestingly, preincubation with STAT3
antibody prevented the binding of Sp1 and Sp3 to the -88/-50 element, indicating
that STAT3 is involved in IL-6-driven Sp1/Sp3 protein-DNA complex formation.
Physical interaction of STAT3 and Sp1 was demonstrated by coimmunoprecipitation.
The functional relevance of the STAT3/Sp1 association was corroborated by
transient transfection experiments, which showed that overexpression of
constitutively active STAT3 increased the minimal VEGF promoter activity. Taken
together, our study suggests that IL-6 promotes tumor angiogenesis in gliomas
and describes a novel transcriptional activation mechanism for STAT3 in the
context of a STAT3 binding element (SBE)-free promoter. Copyright 2005
Wiley-Liss, Inc

PMID: 15688401 [PubMed - indexed for MEDLINE]


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5: Leuk Lymphoma. 2005 Jan;46(1):21-7. 

Activation of the Jak3 pathway and myeloid differentiation.

Mangan JK, Reddy EP.

Fels Institute for Cancer Research and Molecular Biology, Temple University
School of Medicine, Philadephia, PA 19140, USA.

Although the role of Jak3 in lymphoid development has been well-characterized,
increasing evidence demonstrates that activation of the Jak3 pathway plays an
important role in myeloid differentiation as well. Overexpression of Jak3 in
murine myeloid 32Dcl3 cells has been shown to result in an acceleration of
granulocytic differentiation induced by G-CSF. Early onset of G1 cell cycle
arrest along with upregulation of the cyclin dependent kinase inhibitor p27Kip1
and downregulation of Cdk2, Cdk4, Cdk6, and Cyclin E has also been observed in
Jak3-overexpressing 32Dcl3 cells. In addition, Jak3 overexpression in normal
mouse bone marrow cells results in accelerated granulocytic and monocytic
differentiation in response to GM-CSF, while pharmacological inhibition of Jak3
results in a block to GM-CSF-induced colony formation in normal mouse bone
marrow cells. Jak3 is unique among the members of the Jak kinase family in that
it is inducibly expressed and is a target for regulation at the level of
transcription. Recent studies have demonstrated that upregulation of Jak3 during
myeloid differentiation is achieved through the cooperative action of Sp1 and
STAT3, consistent with evidence indicative of a crucial role for STAT3 in
myeloid differentiation. These results suggest that cytokine-inducible
activation of Jak3 plays a critical role in integrating the processes of growth
arrest and differentiation of myeloid cells.

Publication Types:
    Review
    Review, Tutorial

PMID: 15621777 [PubMed - indexed for MEDLINE]


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6: J Cell Biochem. 2004 Nov 1;93(4):844-56. 

CCAAT/Enhancer binding protein delta (c/EBPdelta) regulation and expression in
human mammary epithelial cells: II. Analysis of activating signal transduction
pathways, transcriptional, post-transcriptional, and post-translational control.

Sivko GS, Sanford DC, Dearth LD, Tang D, DeWille JW.

Department of Veterinary Biosciences and Division of Molecular Biology and
Cancer Genetics, Ohio State Comprehensive Cancer Center, The Ohio State
University, Columbus, Ohio, USA.

CCAAT/enhancer binding protein delta (C/EBPdelta) plays a key role in mammary
epithelial cell G0 growth arrest. C/EBPdelta gene expression is down-regulated
in rodent mammary tumorigenesis and in human breast cancer, suggesting that
"loss of function" alterations in C/EBPdelta gene expression are common in
mammary gland malignancies. The goal of this study was to systematically
investigate the mechanisms controlling C/EBPdelta gene expression in MCF-10A and
MCF-12A human mammary epithelial cell lines. The results demonstrate that G0
growth arrest conditions (i.e., serum and growth factor withdrawal or Oncostatin
M (OSM) treatment) result in the activation of JAK1, JAK2, and Tyk 2, members of
the Janus kinase family of non-receptor tyrosine kinases, in MCF-10A and MCF-12A
cells. Growth arrest or OSM treatment also specifically increases activated
(phosphorylated) signal transduction and activators of transcription 3 (STAT3)
levels, demonstrating that STAT3, not STAT1 or STAT5, is the downstream target
of the activated Janus kinases in MCF-10A and MCF-12A cells. Whole cell lysates
from G0 growth arrested (GA) and OSM-treated MCF-12A cells exhibit increased
acute phase response element (APRE) binding compared to lysates from growing
(GR) MCF-12A cells. Transient transfection using C/EBPdelta promoter-luciferase
constructs demonstrated that the APRE (STAT3) consensus binding site is
essential for growth arrest or OSM induction of the C/EBPdelta promoter.
Mutation of the C/EBPdelta promoter STAT3 site or expression of a dominant
negative STAT3 construct (STAT3delta) reduces C/EBPdelta promoter activity in
response to growth arrest conditions. The human C/EBPdelta promoter also
contains an Sp1 site at -61 bp (relative to the transcriptional start site)
which is required for basal transcriptional activation. Mutation or deletion of
the Sp1 site decreases promoter activity in response to growth arrest
conditions. Treatment with the transcriptional inhibitor actinomycin D
demonstrated that the C/EBPdelta mRNA exhibits a relatively short half-life
(approximately 40 min). Similarly, treatment with the translational inhibitor
anisomysin demonstrated that the C/EBPdelta protein half-life was also
relatively short (approximately 160 min). These results indicate that the human
C/EBPdelta gene is controlled at multiple levels, consistent with a role for
C/EBPdelta in cell cycle control and/or cell fate determination.

PMID: 15389878 [PubMed - indexed for MEDLINE]


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7: Shock. 2004 Oct;22(4):333-9. 

Impaired induction of IL-10 expression in the lung following hemorrhagic shock.

Khadaroo RG, Fan J, Powers KA, Fann B, Kapus A, Rotstein OD.

Departments of Surgery, University Health Network and University of Toronto,
Toronto, Ontario, Canada.

The balance between pro- and anti-inflammatory cytokines is considered to be an
important determinant of the magnitude of inflammation in a number of disease
states. We previously showed that resuscitated hemorrhagic shock augmented
LPS-induced release of proinflammatory molecules by alveolar macrophages (AM).
In the present studies, we evaluated the expression and regulation of the
counter inflammatory cytokine IL-10 in the lung using this model. We
hypothesized that impaired up-regulation of IL-10 in shock/resuscitated animals
might serve as a mechanism contributing to accentuated lung inflammation. In a
rodent model, animals exposed to LPS alone exhibited enhanced IL-10 mRNA levels
in lung tissue as well as in AM, but antecedent shock/resuscitation delayed and
attenuated the LPS-induced IL-10 mRNA levels. The ability of shock to attenuate
LPS-stimulated IL-10 was also seen in the protein levels. This effect correlated
with an augmented expression of cytokine-induced neutrophil chemoattractant
(CINC) mRNA. Shock/resuscitated animals given exogenous IL-10 had reduced
proinflammatory response, as shown by decreased expression of CINC mRNA and
decreased neutrophil sequestration in the lung. Shock/resuscitation plus LPS
markedly reduced the transcription rate of IL-10 mRNA compared to LPS alone but
did not affect IL-10 mRNA stability. Reduced IL-10 transcription was not caused
solely by impaired nuclear translocation of STAT3 and Sp1/Sp3 transcription
factors because LPS-induced nuclear translocation of these factors was augmented
by antecedent shock. Considered together, these findings show that
shock/resuscitation suppresses LPS-induced IL-10 expression by AM in the lung by
inhibiting IL-10 gene transcription. Failed up-regulation of counter
inflammatory cytokines may contribute to augmented organ dysfunction in trauma
patients.

PMID: 15377888 [PubMed - indexed for MEDLINE]


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8: J Mol Cell Cardiol. 2004 Apr;36(4):547-59. 

Comment in:
    J Mol Cell Cardiol. 2004 Jul;37(1):1-3.

Angiogenic signal triggered by ischemic stress induces myocardial repair in rat
during chronic infarction.

Fukuda S, Kaga S, Sasaki H, Zhan L, Zhu L, Otani H, Kalfin R, Das DK, Maulik N.

Molecular Cardiology Laboratory, University of Connecticut Medical School,
Farmington, Connecticut 06030, USA.

In the present study, we examined a novel method of stimulating myocardial
angiogenesis through ischemic preconditioning (IP) in the form of in vivo four
repetitive cycles of coronary artery occlusion each followed by reperfusion.
Rats divided into 4 groups: Control+Sham surgery (CS), Control+ Left anterior
descending coronary artery (LAD) occlusion (CMI), IP+ Sham surgery (IPS) and
IP+LAD occlusion (IPMI). For cardiac function, rats were subjected to stress
testing with dobutamine after 2, 4, 7, 14 and 21 operative days. Capillary
density (CD) and arteriolar density (AD) were evaluated by immunohistochemistry.
Western blot was performed to examine the expression pattern for VEGF and
anti-death candidates, Bcl-2 and survivin. Blood flow and the extent of
endothelial and cardiomyocyte cell death were examined. The protein/DNA array
was performed to determine the status of various transcription factor related to
stress signal. Left ventricular functional reserve was better preserved in IPMI
compared to the CMI group. The infarct size and apoptotic cell death were
reduced in IPMI group significantly. Left ventricular regional blood flow,
perfused capillary density and AD increased significantly in the IPMI group.
VEGF, Bcl-2 and survivin expression were increased in IPMI compared to CMI. VEGF
mediated vascular permeability was controlled in the IPMI due to suppression of
c-Src in the infarcted myocardium. Our study documented first time the ability
of IP to induce angiogenesis in the infarcted myocardium along with the
activation of several transcription factors such as Stat3, Pax-5, NF kappa B,
TFIID, SP1 and reduction of VEGF mediated vascular permeability by inhibition of
c-Src in IPMI group thereby reducing ischemic injury in rat MI model.

PMID: 15081314 [PubMed - indexed for MEDLINE]


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9: Oncogene. 2004 Apr 29;23(20):3530-40. 

Rituximab inhibits p38 MAPK activity in 2F7 B NHL and decreases IL-10
transcription: pivotal role of p38 MAPK in drug resistance.

Vega MI, Huerta-Yepaz S, Garban H, Jazirehi A, Emmanouilides C, Bonavida B.

Department of Microbiology, Immunology, and Molecular Genetics, Jonsson
Comprehensive Cancer Center, University of California, 10833 Le Conte Ave.
A2-060 CHS, Los Angeles, CA 90095, USA.

We have recently reported that Rituximab (anti-CD20) sensitizes drug-resistant
2F7 and 10C9 B Non-Hodgkin's lymphoma (NHL) cell lines to the apoptotic effects
of various chemotherapeutic drugs by downregulation of IL-10 and Bcl-2
expression. The mechanism by which Rituximab induces downregulation of IL-10 was
examined. We hypothesized that Rituximab may inhibit p38 MAPK activity that
regulates IL-10 expression via Sp1. Treatment of 2F7 cells with Rituximab or the
p38 inhibitor SB203580 inhibited the constitutive p38 MAPK activity and resulted
in the inhibition of Sp1, IL-10, STAT3, and Bcl-2. Inhibition of the Src-family
PTKs, Lyn, and Src-family PTKs upstream signaling molecules of the p38MAPK
pathway, by PP2, a specific Src-family kinase inhibitor, resulted in the
inhibition of p38MAPK and IL-10 expression. In addition to p38 MAPK, Rituximab
also inhibited NF-kappaB activity. Inhibition of the Src PTKs, MAPK, and
NF-kappaB activities by Rituximab or by specific chemical inhibitors sensitized
the cells to CDDP-mediated apoptosis. The above signaling-mediated effects by
Rituximab were observed with similar kinetics beginning at 1 h following
treatment. Thus, altogether, these results demonstrate that signaling by
Rituximab results in the inhibition of the p38MAPK pathway, which in turn
inhibits the transcription of IL-10 via Sp1. Inhibition of the IL-10
autocrine/paracrine loop results in the inhibition of STAT3 activity and,
consequently, inhibition of Bcl-2 expression and sensitization to
drugs-apoptosis. Further, Rituximab-mediated signaling identifies several new
intracellular targets in NHL that may be of potential therapeutic interest for
the development of new drugs in the treatment of drug-refractory NHL tumor
cells.

PMID: 15077178 [PubMed - indexed for MEDLINE]


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10: J Mol Endocrinol. 2004 Apr;32(2):533-45. 

Aromatase expression in the human fetal osteoblastic cell line SV-HFO.

Watanabe M, Simpson ER, Pathirage N, Nakajin S, Clyne CD.

Prince Henry's Institute of Medical Research, PO Box 5152,Clayton, VIC 3168,
Australia.

A number of clinical studies have highlighted the importance of estrogen in bone
growth and maintenance in men and postmenopausal women. In these instances,
estrogen is synthesized locally within bone tissue by aromatase, encoded by the
CYP19 gene. The mechanisms regulating aromatase expression in bone, however, are
unclear. In this work we characterized the expression of aromatase activity and
gene transcripts in the human fetal osteoblastic cell line, SV-HFO. Aromatase
activity and gene transcript expression were stimulated by dexamethasone.
Oncostatin M strongly stimulated aromatase expression in synergy with
dexamethasone. These factors induced CYP19 transcripts that included the
sequence of exon I.4 in their 5'UTR. Consistent with this, a reporter construct
harboring the genomic sequence of the promoter region of exon I.4 (promoter I.4)
was also activated by dexamethasone and oncostatin M. 5' deletion and mutation
analysis revealed important roles for a glucocorticoid response element, an
interferon gamma activating sequence and a putative binding site for Sp1.
Transfection of exogenous glucocorticoid receptor, STAT3 or Sp1 increased
promoter activity, indicating a potential role for these transcription factors
in regulating aromatase expression in SV-HFO cells. These data suggest that the
SV-HFO cell line is a valuable model with which to elucidate the mechanisms
regulating local estrogen synthesis in osteoblasts.

PMID: 15072557 [PubMed - indexed for MEDLINE]


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11: Blood. 2004 Jun 1;103(11):4093-101. Epub 2004 Feb 19. 

Mechanisms associated with IL-6-induced up-regulation of Jak3 and its role in
monocytic differentiation.

Mangan JK, Rane SG, Kang AD, Amanullah A, Wong BC, Reddy EP.

Temple University School of Medicine, Fels Institute for Cancer Research and
Molecular Biology, 3307 N Broad St, AHP Room 154, Philadelphia, PA 19140, USA.

We report here that Janus kinase 3 (Jak3) is a primary response gene for
interleukin-6 (IL-6) in macrophage differentiation, and ectopic overexpression
of Jak3 accelerates monocytic differentiation of normal mouse bone marrow cells
stimulated with cytokines. Furthermore, we show that incubation of normal mouse
bone marrow cells with a JAK3-specific inhibitor results in profound inhibition
of myeloid colony formation in response to granulocyte-macrophage
colony-stimulating factor or the combination of stem cell factor, IL-3, and
IL-6. In addition, mutagenesis of the Jak3 promoter has revealed that Sp1
binding sites within a -67 to -85 element and a signal transducer and activator
of transcription (Stat) binding site at position -44 to -53 are critical for
activation of Jak3 transcription in murine M1 myeloid leukemia cells stimulated
with IL-6. Electrophoretic mobility shift assay (EMSA) analysis has demonstrated
that Sp1 can bind to the -67 to -85 element and Stat3 can bind to the -44 to -53
STAT site in IL-6-stimulated M1 cells. Additionally, ectopic overexpression of
Stat3 enhanced Jak3 promoter activity in M1 cells. This mechanism of activation
of the murine Jak3 promoter in myeloid cells is distinct from a recently
reported mechanism of activation of the human JAK3 promoter in activated T
cells.

PMID: 14976041 [PubMed - indexed for MEDLINE]


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12: Science. 2004 Jan 16;303(5656):383-7. Epub 2003 Dec 4. 

RNA leaching of transcription factors disrupts transcription in myotonic
dystrophy.

Ebralidze A, Wang Y, Petkova V, Ebralidse K, Junghans RP.

Biotherapeutics Development Lab, Harvard Institute of Human Genetics, Harvard
Medical School and Division of Hematology-Oncology, Beth Israel Deaconess
Medical Center, 4 Blackfan Circle, Boston, MA 02215, USA.

Myotonic dystrophy type 1 (DM1) is caused by a CUGn expansion (n approximately
50 to 5000) in the 3' untranslated region of the mRNA of the DM protein kinase
gene. We show that mutant RNA binds and sequesters transcription factors (TFs),
with up to 90% depletion of selected TFs from active chromatin. Diverse genes
are consequently reduced in expression, including the ion transporter CIC-1,
which has been implicated in myotonia. When TF specificity protein 1 (Sp1) was
overexpressed in DM1-affected cells, low levels of messenger RNA for CIC-1 were
restored to normal. Transcription factor leaching from chromatin by mutant RNA
provides a potentially unifying pathomechanistic explanation for this disease.

PMID: 14657503 [PubMed - indexed for MEDLINE]


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13: Biol Chem. 2003 Aug;384(8):1147-54. 

Cloning and characterization of the promoter region of the mouse
frizzled-related protein 4 gene.

Wong VK, Yam JW, Hsiao WL.

Department of Biology, The Hong Kong University of Science and Technology, Clear
Water Bay, Kowloon, Hong Kong, China.

Frizzled-related protein (Frp) is a newly identified family of secreted proteins
involved in the Wnt signaling pathway. To date, little is known about the
underlying mechanisms regulating Frp expression. In this study the promoter
region of mouse frizzled related protein 4 (sFrp4) gene was cloned, sequenced,
and analyzed using transient reporter assays along with site-directed
mutagenesis. Two clusters of cis-acting elements, STAT3/Lyf-1/MZF1 (site 1) and
C/EBP-beta/ GATA-1/CREB (site 2) located in the promoter region from -238 to
-144 were found to be essential for the promoter activity of sFrp4. In addition
to sites 1 and 2, putative transcriptional factor binding sites for TFIID,
SP1/GC and ATF/CREB exhibited positive, while the site for NRSE exhibited
negative regulatory functions, as determined by the alkaline phosphatase
activities of the reporter assay. We also demonstrate that the ATF/CREB site may
cooperatively interact with the NRSF-like element in regulating sFrp4 promoter
activity. The data of our study, which is the first promoter analysis of mouse
Frp genes, provide the basis for understanding the functions and the regulation
of Frp and its role in regulating Wnt signals.

PMID: 12974383 [PubMed - indexed for MEDLINE]


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14: J Leukoc Biol. 2002 Dec;72(6):1198-205. 

Interleukin-10 differently regulates monocyte chemoattractant protein-1 gene
expression depending on the environment in a human monoblastic cell line, UG3.

Ikeda T, Sato K, Kuwada N, Matsumura T, Yamashita T, Kimura F, Hatake K, Ikeda
K, Motoyoshi K.

Third Department of Internal Medicine, National Defense Medical College,
Saitama, Japan.

Interleukin (IL)-4, IL-10, and IL-13 affect monocyte/macrophage functions
including regulation of cytokine production. We analyzed the regulatory effects
of these cytokines on cytokine production using a human monoblastic cell line,
UG3. It is interesting that IL-10 up-regulated, whereas IL-4 and IL-13
down-regulated monocyte chemoattractant protein-1 (MCP-1) production by
unstimulated UG3 cells. IL-10-induced expression of MCP-1 mRNA occurred without
de novo protein synthesis at transcriptional and post-transcriptional levels.
The enhancement of binding activity of nuclear factor Sp1 (Sp-1) and signal
transducer and activators of transcription (STAT)1 and 3 but not nuclear factor
kappaB (NF-kappaB) was associated with this IL-10-induced MCP-1 expression.
Furthermore, IL-10 suppressed lipopolysaccharide (LPS)-induced NF-kappaB binding
but not Sp-1. The present results suggest IL-10 has two contrasting actions on
the MCP-1 production of monocytes/macrophages, between the resting and activated
conditions. The combination of activated Sp-1 and STATs is important for
IL-10-induced MCP-1 expression in resting monocytes/macrophages, and the
inhibition of LPS-induced NF-kappaB binding is crucial for down-regulation of
MCP-1 by IL-10 in stimulated monocytes/macrophages.

PMID: 12488502 [PubMed - indexed for MEDLINE]


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15: Gastroenterology. 2002 Apr;122(4):1020-34. 

Interferon-alpha activates multiple STAT signals and down-regulates c-Met in
primary human hepatocytes.

Radaeva S, Jaruga B, Hong F, Kim WH, Fan S, Cai H, Strom S, Liu Y, El-Assal O,
Gao B.

Section on Liver Biology, Laboratory of Physiologic Studies, National Institute
on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda,
Maryland 20892, USA.

BACKGROUND & AIMS: Interferon (IFN)-alpha therapy is currently the primary
choice for viral hepatitis and a promising treatment for hepatocellular
carcinoma (HCC). Primary mouse and rat hepatocytes respond poorly to IFN-alpha
stimulation. Thus, it is very important to examine the IFN-alpha signal pathway
in primary human hepatocytes. METHODS: The IFN-alpha-activated signals and genes
in primary human hepatocytes and hepatoma cells were examined by Western
blotting and microarray analyses. RESULTS: Primary human hepatocytes respond
very well to IFN-alpha stimulation as shown by activation of multiple signal
transducer and activator of transcription factor (STAT) 1, 2, 3, 5, and multiple
genes. The differential response to IFN-alpha stimulation in primary human and
mouse hepatocytes may be caused by expression of predominant functional
IFN-alpha receptor 2c (IFNAR2c) in primary human hepatocytes vs. expression of
predominant inhibitory IFNAR2a in mouse hepatocytes. Microarray analyses of
primary human hepatocytes show that IFN-alpha up-regulates about 44 genes by
over 2-fold and down-regulates about 9 genes by 50%. The up-regulated genes
include a variety of antiviral and tumor suppressors/proapoptotic genes. The
down-regulated genes include c-myc and c-Met, the hepatocyte growth factor (HGF)
receptor. Down-regulation of c-Met is caused by IFN-alpha suppression of the
c-Met promoter through down-regulation of Sp1 binding and results in attenuation
of HGF-induced signals and cell proliferation. CONCLUSIONS: IFN-alpha directly
targets human hepatocytes, followed by activation of multiple STATs and
regulation of a wide variety of genes, which may contribute to the antiviral and
antitumor activities of IFN-alpha in human liver.

PMID: 11910354 [PubMed - indexed for MEDLINE]


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16: Mol Cell Biol. 2001 Dec;21(24):8301-17. 

Influenza virus infection induces metallothionein gene expression in the mouse
liver and lung by overlapping but distinct molecular mechanisms.

Ghoshal K, Majumder S, Zhu Q, Hunzeker J, Datta J, Shah M, Sheridan JF, Jacob
ST.

Department of Molecular and Cellular Biochemistry, College of Medicine, The Ohio
State University, 333 Hamilton Hall, 1645 Neil Ave., Columbus, OH 43210, USA.

Metallothionein I (MT-I) and MT-II have been implicated in the protection of
cells against reactive oxygen species (ROS), heavy metals, and a variety of
pathological and environmental stressors. Here, we show a robust increase in
MT-I/MT-II mRNA level and MT proteins in the livers and lungs of C57BL/6 mice
exposed to the influenza A/PR8 virus that infects the upper respiratory tract
and lungs. Interleukin-6 (IL-6) had a pronounced effect on the induction of
these genes in the liver but not the lung. Treatment of the animals with RU-486,
a glucocorticoid receptor antagonist, inhibited induction of MT-I/MT-II in both
liver and lung, revealing a direct role of glucocorticoid that is increased upon
infection in this induction process. In vivo genomic footprinting (IVGF)
analysis demonstrated involvement of almost all metal response elements, major
late transcription factor/antioxidant response element (MLTF/ARE), the STAT3
binding site on the MT-I upstream promoter, and the glucocorticoid responsive
element (GRE1), located upstream of the MT-II gene, in the induction process in
the liver and lung. In the lung, inducible footprinting was also identified at a
unique gamma interferon (IFN-gamma) response element (gamma-IRE) and at Sp1
sites. The mobility shift analysis showed activation of STAT3 and the
glucocorticoid receptor in the liver and lung nuclear extracts, which was
consistent with the IVGF data. Analysis of the newly synthesized mRNA for
cytokines in the infected lung by real-time PCR showed a robust increase in the
levels of IL-10 and IFN-gamma mRNA that can activate STAT3 and STAT1,
respectively. A STAT1-containing complex that binds to the gamma-IRE in vitro
was activated in the infected lung. No major change in MLTF/ARE DNA binding
activity in the liver and lung occurred after infection. These results have
demonstrated that MT-I and MT-II can be induced robustly in the liver and lung
following experimental influenza virus infection by overlapping but distinct
molecular mechanisms.

PMID: 11713267 [PubMed - indexed for MEDLINE]


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17: J Biol Chem. 2001 Apr 13;276(15):12395-403. Epub 2001 Jan 17. 

Co-operative effect of c-Src tyrosine kinase and Stat3 in activation of
hepatocyte growth factor expression in mammary carcinoma cells.

Hung W, Elliott B.

Cancer Research Laboratories, Botterell Hall, Queen's University, Kingston,
Ontario, K7L 3N6 Canada.

We have previously shown coexpression of hepatocyte growth factor (HGF) and its
receptor Met in the invasive tumor front of human breast carcinomas. We have
also demonstrated secretion of HGF, constitutive activation of Met, and
increased invasion in a murine breast carcinoma cell line, SP1. These
observations suggest the presence of an HGF autocrine loop in some breast
carcinoma cells, which confers increased survival, growth, and invasiveness
during tumor progression and metastasis. c-Src tyrosine kinase, which is
critical in regulating the expression of many genes, is activated in SP1
carcinoma cells, as well as in most human breast cancers. We therefore examined
the role of c-Src kinase in HGF expression in breast carcinoma cells. Expression
of activated c-Src in SP1 cells increased transcription from the HGF promoter
and expression of HGF mRNA and protein, while dominant negative c-Src had the
opposite effect. Using deletion analysis, we showed that the region between -254
and -70 base pairs was required for c-Src responsiveness of the HGF promoter.
This region contains two putative consensus sequences (at -110 and -149 base
pairs) for the Stat3 transcription factor, which bind protein complexes
containing Stat3 (but not Stat1, -5A, or -5B). Coexpression of activated c-Src
and Stat3 synergistically induced strong HGF promoter activity in SP1 cells, as
well as in a nonmalignant epithelial cell line, HC11 (HGF negative). c-Src
kinase activity correspondingly increased the tyrosine 705 phosphorylation and
DNA binding affinity of Stat3 (but not Stat1, -5A, or -5B). Collectively, our
data indicate a cooperative effect of c-Src kinase and Stat3 in the activation
of HGF transcription and protein expression in breast carcinoma cells. This
process may be important in overriding the strong repression of HGF expression
in nonmalignant epithelium, and thereby promote tumorigenesis.

PMID: 11278729 [PubMed - indexed for MEDLINE]


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18: J Mol Cell Cardiol. 2000 Jan;32(1):23-33. 

Angiotensin blockade inhibits SIF DNA binding activities via STAT3 after
myocardial infarction.

Omura T, Yoshiyama M, Takeuchi K, Kim S, Iwao H, Yamagishi H, Toda I, Teragaki
M, Akioka K, Yoshikawa J.

First Department of Internal Medicine, Osaka City University Medical School,
Osaka, Japan.

The in vivo activation of transcription factors, which is important for cell
regulation by gene expression, has not been well examined in myocardial
infarcted heart. The purpose of this study was to determine whether myocardial
signal transducer and activator of transcription (STAT) pathway is activated as
sis-inducing factor (SIF) in infarcted heart, and to assess the angiotensin
blockade on SIF activity in ischemic and non-ischemic myocardium of rat.
Myocardial infarction was made by ligation of the coronary artery in Wistar
rats. In electrophoretic mobility shift assay, myocardial SIF DNA binding
activities gradually increased and reached to peak at 1 week in infarcted and
non-infarcted regions after myocardial infarction. Imidapril and candesartan
cilexitil significantly prevented the increase in SIF DNA binding activity in
infarcted and non-infarcted regions. This increased SIF DNA complex was
supershifted by specific anti-STAT3 antibody, indicating that increased SIF
complex at least contained activated STAT3 proteins in myocardial infarcted
heart. Furthermore, immunoprecipitation-Western blot analysis revealed that
STAT3 of infarcted and non-infarcted regions were tyrosine-phosphorylated at 1
week after myocardial infarction. Imidapril and candesartan cilexitil prevented
the increase in phosphorylated STAT3. Thus, the transcriptional activation of
STAT3 through AT1 receptor may be partially involved in cardiac remodeling after
myocardial infarction. Copyright 2000 Academic Press.

PMID: 10652187 [PubMed - indexed for MEDLINE]


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19: Mol Cell Biol. 1998 Apr;18(4):2108-17. 

Interleukin-6-specific activation of the C/EBPdelta gene in hepatocytes is
mediated by Stat3 and Sp1.

Cantwell CA, Sterneck E, Johnson PF.

Eukaryotic Transcriptional Regulation Group, ABL-Basic Research Program,
NCI-Frederick Cancer Research and Development Center, Maryland 21702-1201, USA.

C/EBPdelta (CCAAT/enhancer binding protein delta) has been implicated as a
regulator of acute-phase response (APR) genes in hepatocytes. Its expression
increases dramatically in liver during the APR and can be induced in hepatic
cell lines by interleukin-6 (IL-6), an acute-phase mediator that activates
transcription of many APR genes. Here we have investigated the mechanism by
which C/EBPdelta expression is regulated by IL-6 in hepatoma cells. C/EBPdelta
promoter sequences to -125 bp are sufficient for IL-6 inducibility of a reporter
gene and include an APR element (APRE) that is essential for IL-6
responsiveness. DNA binding experiments and transactivation assays demonstrate
that Stat3, but not Stat1, interacts with this APRE. Two Sp1 sites, one of which
is adjacent to the APRE, are required for IL-6 induction and transactivation by
Stat3. Thus, Stat3 and Sp1 function cooperatively to activate the C/EBPdelta
promoter. Replacement of the APRE with Stat binding elements (SBEs) from the
ICAM-1 or C/EBPbeta promoter, both of which recognize both Stat1 and Stat3,
confers responsiveness to gamma interferon, a cytokine that selectively
activates Stat1. Sequence comparisons suggest that the distinct Stat binding
specificities of the C/EBPdelta and C/EBPbeta SBEs are determined primarily by a
single base pair difference. Our findings indicate that the cytokine specificity
of C/EBPdelta gene expression is governed by the APRE sequence.

PMID: 9528783 [PubMed - indexed for MEDLINE]


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20: Mol Cell Endocrinol. 1997 Apr 25;129(1):7-16. 

Induction of Sp1 activity by prolactin and interleukin-2 in Nb2 T-cells:
differential association of Sp1-DNA complexes with Stats.

Too CK.

Department of Biochemistry, Dalhousie University, Halifax, Nova Scotia, Canada.
CTOO@IS.DAL.CA

The induction of the transcription factor Sp1 by prolactin (PRL) and
interleukin-2 (IL-2) was investigated in the PRL- and IL-2 responsive rat Nb2
T-cell line. Western analysis showed a rapid increase in Sp1 synthesis in Nb2
cells in response to PRL or IL-2. Elevation of Sp1 protein levels occurred
within 15 min following PRL or IL-2 stimulation, reached a maximum by 1 h and
was inhibited by cycloheximide, indicating de novo protein synthesis.
Interestingly, dilution of confluent, growth-arrested Nb2 cells to low density
also caused a rapid elevation in Sp1 suggesting that growth arrest may
down-regulate Sp1 synthesis. Electrophoretic mobility shift assays using an Sp1
consensus oligonucleotide as probe showed a rapid but transient formation of a
single PRL-inducible complex at 30 min. In contrast, three IL-2-inducible
complexes were formed at 30 min and persisted to at least 60 min. Mobility shift
interference assays using specific Stat antibodies failed to detect Stat1alpha,
Stat3 or Stat5 in the 30 min PRL-inducible complex. In contrast, the IL-2
induced complexes contained Stat3 alone at 30 min and both Stat3 and Stat5 at 60
min. The PRL- and IL-2-inducible complexes did not contain the tumor suppressor
protein, p53. The time dependent association of the Stat proteins with the
IL-2-inducible complexes, but not with the PRL-inducible complex, suggests that
the two mitogens may selectively utilize specific promoter elements for
transcriptional activation of PRL- and IL-2-responsive genes. Alternatively, the
two mitogens may be activating different genes with Sp1-binding promoter
elements for their mitogenic action in Nb2 cells.

PMID: 9175624 [PubMed - indexed for MEDLINE]


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21: J Biochem (Tokyo). 1997 Apr;121(4):731-8. 

CCAAT/enhancer-binding protein delta gene expression is mediated by APRF/STAT3.

Yamada T, Tobita K, Osada S, Nishihara T, Imagawa M.

Department of Environmental Biochemistry, Faculty of Pharmaceutical Sciences,
Osaka University, Suita.

The CCAAT/enhancer-binding protein delta (C/EBP delta) transcription factor is
known to be rarely expressed but sharply induced at an early stage of the acute
phase response. To investigate the regulation mechanisms for this induction, the
5'-flanking region of the rat C/EBP delta gene was isolated. Functional analyses
involving transfection and footprinting indicated that the upstream region up to
- 175 bp is sufficient for the full basal activity in rat fibroblast 3Y1 cells.
At least three cis-elements including a GC box are involved in this activity.
When HepG2 cells were treated with interleukin-6 (IL-6), C/EBP delta mRNA was
rapidly induced. Transfection and gel shift analyses identified the binding site
for the acute phase response factor/signal transducers and activators of
transcription (APRF/STAT3). These findings strongly indicate that C/EBP delta
gene expression is mediated by APRF/STAT3, which is phosphorylated for the
activation through the IL-6 receptor when cells are treated with IL-6, and
trans-activates the other acute phase response genes.

PMID: 9163525 [PubMed - indexed for MEDLINE]


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22: J Biol Chem. 1995 Jul 7;270(27):16449-57. 

Aromatase P450 gene expression in human adipose tissue. Role of a Jak/STAT
pathway in regulation of the adipose-specific promoter.

Zhao Y, Nichols JE, Bulun SE, Mendelson CR, Simpson ER.

Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of
Texas Southwestern Medical Center, Dallas 75235-9051, USA.

In the present report we describe a heretofore unrecognized role for a Jak/STAT
signaling pathway, namely the stimulation of expression of the aromatase P450
(CYP19) gene, and hence of estrogen biosynthesis, in human adipose tissue.
Expression of this gene in adipose tissue as well as in adipose stromal cells
maintained in the presence of serum and glucocorticoids is regulated by a distal
TATA-less promoter, I.4, which contains a glucocorticoid response element, an
Sp1 binding site, and an interferon-gamma activation site (GAS) element. The
stimulatory action of serum (in the presence of dexamethasone) can be replaced
by interleukin (IL)-11, leukemia inhibitory factor, and oncostatin-M, as well as
by IL-6, providing the IL-6 soluble receptor is also present. Stimulation of the
cells by these factors led to rapid phosphorylation of Jak1, but not Jak2 or
Jak3, on tyrosine residues. STAT3 but not STAT1 was also phosphorylated and
bound to the GAS element in the I.4 promoter region. When regions of this
promoter were fused upstream of the chloramphenicol acetyltransferase reporter
gene and transfected into the cells, mutagenesis or deletion of the GAS element
led to complete loss of reporter gene expression. Since adipose tissue is the
major site of estrogen biosynthesis in men and in postmenopausal women, this
pathway involving a Jak/STAT signaling mechanism acting together with
glucocorticoids and Sp1 appears to be the principal means whereby estrogen
biosynthesis is regulated in the elderly.

PMID: 7608217 [PubMed - indexed for MEDLINE]


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