Input:
Calcium-dependent immediate-early gene induction in lymphocytes is negatively regulated by p21Ha-ras.
The induction of immediate-early (IE) response genes, such as egr-1, c-fos, and c-jun, occurs rapidly after the activation of T lymphocytes. The process of activation involves calcium mobilization, activation of protein kinase C (PKC), and phosphorylation of tyrosine kinases. p21(ras), a guanine nucleotide binding factor, mediates T-cell signal transduction through PKC-dependent and PKC-independent pathways. The involvement of p21(ras) in the regulation of calcium-dependent signals has been suggested through analysis of its role in the activation of NF-AT. We have investigated the inductions of the IE genes in response to calcium signals in Jurkat cells (in the presence of activated p21(ras)) and their correlated consequences. The expression of activated p21(ras) negatively regulated the induction of IE genes by calcium ionophore. This inhibition of calcium-activated IE gene induction was reversed by treatment with cyclosporin A, suggesting the involvement of calcineurin in this regulation. A later result of inhibition of this activation pathway by p21(ras) was down-regulation of the activity of the transcription factor AP-1 and subsequent coordinate reductions in IL-2 gene expression and protein production. These results suggest that p2l(ras) is an essential mediator in generating not only positive but also negative modulatory mechanisms controlling the competence of T cells in response to inductive stimulations.

reductions

gene expression

induction

induction

c-fos

induction

c-jun

occurs

induction

activated

expression of activated

gene expression


Input:
Apoptosis signaling pathways in normal T cells: differential activity of Bcl-2 and IL-1beta-converting enzyme family protease inhibitors on glucocorticoid- and Fas-mediated cytotoxicity.
Fas-mediated apoptosis plays an important role in regulating the immune response in peripheral T cells. Restimulation of T cell blasts up-regulates Fas and Fas ligand expression, with subsequent interaction leading to cell death. Overexpression of Bcl-2 in tumor cells blocks apoptosis induced by many stimuli, but inhibition of Fas-mediated killing has not been consistently observed. To examine the behavior of Bcl-2 in normal cells, T cell blasts were transiently transfected with Bcl-2 and related gene products to determine the effect on apoptotic signaling. Transient overexpression of Bcl-2 in mouse and human T cell blasts did not block Fas-mediated apoptosis, whereas etoposide- and glucocorticoid-induced cytotoxicity was potently inhibited. Expression of Bcl-xL and adenovirus E1B 19K did not interfere with anti-Fas killing. In contrast, interleukin-1beta-converting enzyme family protease inhibitors Ac-DEVD-CHO and CrmA blocked Fas-mediated apoptosis. These results suggest that peripheral T cells use distinct apoptosis signaling pathways with differential sensitivity to Bcl-2 and interleukin-1beta-converting enzyme family protease inhibitors. Since T cells normally express Bcl-2 and Bcl-xL following activation, their inability to block Fas-mediated apoptosis may allow for the elimination of self-reactive cells and the appropriate regulation of immune responses.

interaction

Fas

Fas ligand

expression

Fas

expression

Fas ligand

Overexpression

transfected

overexpression

Expression

Bcl-xL

Expression

E1B 19K

express

express

Bcl-xL

up-regulates

expression

Overexpression

Overexpression

transfected

transfected

overexpression

overexpression

following

express


Input:
Interferons induce normal and aberrant retinoic-acid receptors type alpha in acute promyelocytic leukemia cells: potentiation of the induction of retinoid-dependent differentiation markers.
Treatment of the acute promyelocytic (APL) cell line NB4 with interferon alpha (IFN(alpha)), as well as IFN(beta) and gamma, results in an increased expression of the transcripts coding for retinoic-acid receptor type alpha (RAR(alpha)) and the leukemia-specific retinoic acid receptor PML-RAR. Transcriptional induction of the RAR(alpha) and PML-RAR mRNAs is rapid and it is parallelled by an increase in the corresponding proteins. Up-regulation of RAR(alpha) and PML-RAR gene expression by IFN(alpha) is accompanied by a strong potentiation in the induction of 2 retinoid-dependent granulocytic markers, i.e., granulocyte-colony-stimulating factor receptor mRNA and leukocyte alkaline phosphatase. However, IFN(alpha) does not have any effects on the retinoid-dependent regulation of the myeloid surface markers CD11b and CD33. The IFN-dependent increase in RAR(alpha) levels and the enhancing effect of the cytokine on retinoid-dependent granulocytic markers expression may be a characteristic of PML-RAR positive cells, since the phenomena are not observed in HL-60 promyelocytes. Interferons as well as retinoids inhibit the growth of NB4 cells, although the 2 classes of compounds do not significantly interact in terms of anti-proliferative activity. These results suggest the possible use of combinations between IFNs and retinoic acid in the cyto-differentiating treatment of APL patients.

expression

RAR(alpha)

expression

PML-RAR

levels

RAR(alpha)

induce

retinoic-acid receptors type alpha

results in an increased

expression

Transcriptional induction

RAR(alpha)

Transcriptional induction

PML-RAR

parallelled

Transcriptional induction

increase

increase

RAR(alpha)

increase

PML-RAR

Up-regulation

expression

induction

granulocyte-colony-stimulating factor receptor

induction

leukocyte alkaline phosphatase

dependent

granulocyte-colony-stimulating factor receptor

dependent

leukocyte alkaline phosphatase

increase

levels

observed

RAR(alpha)

effects

regulation

regulation

regulation

expression

RAR(alpha)

expression

PML-RAR


Input:
An IL-2 response element in the human IL-2 receptor alpha chain promoter is a composite element that binds Stat5, Elf-1, HMG-I(Y) and a GATA family protein.
Expression of the human interleukin-2 (IL-2) receptor alpha chain gene is potently upregulated by its own ligand, IL-2. In this study, we characterize an essential upstream IL-2 response element that contains both consensus and non-consensus GAS motifs, two putative Ets binding sites (EBS), one of which overlaps the consensus GAS motif, and a GATA motif, which overlaps the non-consensus GAS motif. We demonstrate that although the individual components of this element do not respond to IL-2, together they form a composite element capable of conferring IL-2 responsiveness to a heterologous promoter. Multiple factors including Stat5, Elf-1, HMG-I(Y) and GATA family proteins bind to the IL-2 response element and mutation of any one of these binding sites diminishes the activity of this element. An unidentified Ets family protein binds to the EBS overlapping the consensus GAS motif and appears to negatively regulate the human IL-2R alpha promoter. Thus, IL-2-induced IL-2R alpha promoter activity requires a complex upstream element, which appears to contain binding sites for both positive and negative regulatory factors.

binds

binds

binds

HMG-I(Y)

bind

bind

bind

HMG-I(Y)

Expression

interleukin-2 (IL-2) receptor alpha chain

negatively regulate

IL-2R alpha

promoter

upregulated

Expression

induced

IL-2R alpha

promoter

requires

induced


Input:
Active suppression of the class II transactivator-encoding AIR-1 locus is responsible for the lack of major histocompatibility complex class II gene expression observed during differentiation from B cells to plasma cells.
In this study the genetic control of major histocompatibility complex (MHC) class II gene expression during the transition from B cell to plasma cell has been analyzed. Class II molecules are not expressed in plasma cells because of an active suppression resulting in the abrogation of class II gene transcription. We show here that the plasma cell-specific repressor function, designated SIR (suppressor of immune response genes), does not act directly on the transcription of class II genes, but instead on the transcription of the AIR-1 gene, whose product, the class II transactivator (CIITA), is fundamental for the regulation of the constitutive and inducible expression of MHC class II genes. This was unambiguously demonstrated by the fact that plasmacytoma x B cell hybrids carrying an AIR-1 locus derived from CIITA-expressing cells do not express CIITA-specific transcripts. Transfection of a cDNA containing the human CIITA coding sequence under the control of an heterologous promoter restores expression of human MHC class II genes in the hybrids and is responsible for de novo expression of mouse MHC class II genes in both the mouse plasmacytoma cell line and the hybrids. These results confirm and extend the notion of the functional conservation of the AIR-1 gene product across species barriers. Interestingly, in CIITA-transfected cell hybrids, cell surface expression of the human HLA-DQ heterodimer was not observed. This result was not attributable to lack of HLA-DQ alpha or -DQ beta transcription, because both transcripts were present in the CIITA-transfected hybrids, although at reduced levels. These findings further support our previous observations on the distinct regulation of expression of the human HLA-DQ class II subset, which may be thus controlled at the posttranscriptional level by a CIITA-independent mechanism.

Transfection

CIITA

transfected

CIITA

present

HLA-DQ alpha

present

-DQ beta

transfected

CIITA

suppression

class II transactivator

act

transcription

lack

transcription

reduced levels

transcription

transfected

Active

suppression

express

transcripts

Transfection

under the control

transfected

transfected

transfected

transfected

under the control

Transfection

attributable

lack

transcription

CIITA

transcripts

CIITA

transcription

HLA-DQ alpha

transcription

-DQ beta


Input:
Silencing of human fetal globin expression is impaired in the absence of the adult beta-globin gene activator protein EKLF.
Globin genes are subject to tissue-specific and developmental stage-specific regulation. A switch from human fetal (gamma)-to adult (beta)-globin expression occurs within erythroid precursor cells of the adult lineage. Previously we and others showed by targeted gene disruption that the zinc finger gene, erythroid Kruppel-like factor (EKLF), is required for expression of the beta-globin gene in mice, presumably through interaction with a high-affinity binding site in the proximal promoter. To examine the role of EKLF in the developmental regulation of the human gamma-globin gene we interbred EKLF heterozygotes (+/-) with mice harboring a human beta-globin yeast artificial chromosome transgene. We find that in the absence of EKLF, while human beta-globin expression is dramatically reduced, gamma-globin transcripts are elevated approximately 5-fold. Impaired silencing of gamma-globin expression identifies EKLF as the first transcription factor participating quantitatively in the gamma-globin to beta-globin switch. Our findings are compatible with a competitive model of switching in which EKLF mediates an adult stage-specific interaction between the beta-globin gene promoter and the locus control region that excludes the gamma-globin gene.

interaction

EKLF

interaction

beta-globin

promoter

expression

fetal globin

expression

human fetal (gamma)-

expression

adult (beta)-globin

expression

beta-globin

expression

beta-globin

expression

gamma-globin

Silencing

expression

impaired

Silencing

absence

absence

EKLF

switch

expression

reduced

expression

Impaired

silencing

silencing

expression

switch

gamma-globin

excludes

mediates

gamma-globin

switch

expression

required

EKLF

expression

through

required

interaction

role

EKLF

regulation

elevated

gamma-globin

participating

EKLF

switch

switch

beta-globin

mediates

EKLF

interaction

regulation

gamma-globin


Input:
Nasal NK- and T-cell lymphomas share the same type of Epstein-Barr virus latency as nasopharyngeal carcinoma and Hodgkin's disease.
Nasal T/NK-cell lymphomas can be further separated into those of natural killer (NK) cell lineage or of T-cell lineage, with differences in cellular phenotype, T-cell receptor (TcR) gene rearrangement and TcR transcript expression. Both NK- and T-cell subtypes are closely associated with Epstein-Barr virus (EBV). In this study, EBV gene expression was determined in 23 cases of nasal lymphoma (NL) by in situ hybridisation (ISH), reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry (IH). Of the 23 cases, 19 were classified as NK-cell and 4 as T-cell tumours. ISH for EBV-encoded small non-polyadenylated RNAs showed that all cases, whether NK or T, harboured EBV in virtually all tumour cells. RT-PCR demonstrated that NL of both subtypes expressed EBNAI of the QUK splice pattern, the latent membrane proteins, LMP1 and 2 and the BamHI A rightward transcripts in the absence of EBNA2 mRNAs, compatible with the latency type II pattern. In addition, analysis of EBV protein expression by IH revealed a heterogeneous pattern of EBV gene expression at the single-cell level consisting of both LMP1+ and LMP1- tumour cells, suggesting a mixture of latency I and II. Although 2 early lytic transcripts, BZLF1 and BHRF1, were also detected in 13 and 10 cases, respectively, the lack of ZEBRA staining in any case indicates that these lytic transcripts are most likely expressed by rare cells in the biopsies entering lytic cycle. The viral transcriptional pattern similar to that of nasopharyngeal carcinoma and Hodgkin's disease suggests that EBV can exploit common regulatory mechanisms for gene transcription in diverse host cell types. Down-regulation of immunogenic proteins (EBNA2-EBNA6) in nasal lymphoma may enable tumour cells to evade host cytotoxic T-cell surveillance.

expressed

EBNAI

expressed

expressed

Down-regulation

Down-regulation

absence

detected

detected

lack

ZEBRA

expressed

expressed


Input:
Identification and characterization of a leukocyte-specific component of the nuclear body.
The nuclear body (NB) is a cellular organelle that is involved in the pathogenesis of acute promyelocytic leukemia and viral infection. The NB is also a target of antibodies in the serum of patients with the autoimmune disease primary biliary cirrhosis. In this study, serum from a patient with primary biliary cirrhosis was used to identify a cDNA encoding a novel component of the NB, a 140-kDa protein designated Sp140. The predicted amino acid sequence of the amino-terminal portion of Sp140 was similar to Sp100, a previously identified NB protein. The carboxyl portion of Sp140 contained a zinc-finger domain and a bromodomain, motifs that are present in proteins regulating gene transcription. High levels of Sp140 mRNA were detected in human spleen and peripheral blood leukocytes, but not other human tissues. The level of SP140 mRNA in myeloid precursor cell lines HL60 and NB4 markedly increased in response to chemically induced cellular differentiation. Immunohistochemical techniques were used to demonstrate that SP140 localized to the NB in differentiated HL60 and NB4 cells. The location of Sp140 in the NB, and expression of this gene in cells involved in host defense, suggest that Sp140 may be involved in the pathogenesis of acute promyelocytic leukemia and viral infection.

expression

localized

NB

location

NB

High levels

detected

increased

level

detected

level


Input:
Potent gene regulatory and antiproliferative activities of 20-methyl analogues of 1,25 dihydroxyvitamin D3.
The biological active form of vitamin D3, 1,25-dihydroxyvitamin D3 (VD), regulates cellular growth and differentiation. This provides the hormone with an interesting therapeutic potential. However, hypercalcemia is a side effect, which is caused by VD's classical action, the regulation of calcium homeostasis. This made the need for VD analogues with selectively increased cell regulatory properties. Studies with 20-epi analogues pointed out the importance of the carbon-20 position and led to the development of 20-methyl derivatives of VD. In this report the biological properties of the compounds ZK161422 and ZK157202, which are 20-methyl- and 20-methyl-23-eneanalogues, respectively, have been analyzed in comparison with VD. Both compounds show about 2-fold lower affinity to the VD receptor (VDR) than VD. However, compared to VD, their antiproliferative effect is up to 30-fold higher on human peripheral blood mononuclear cells and even up to 300-fold higher on human breast cancer MCF-7 cells. Whereas the hypercalcemic effect for ZK157202 is also increased 10-fold, ZK161422 has the same calcium-mobilizing potency as VD. Moreover, ZK161422, but not ZK157202, showed preference for gene activation from a promoter carrying a VD response element with a palindromic arrangement of two hexameric receptor binding sites spaced by 9 nucleotides (IP9) rather than for activation from a response element formed by a direct repeat spaced by 3 nucleotides (DR3). This observation supports a model, in which promoter selectivity reflects the selectively increased antiproliferative effect of VD analogues.

lower affinity

VDR


Input:
Second messenger up-regulation of androgen receptor gene transcription is absent in androgen insensitive human prostatic carcinoma cell lines, PC-3 and DU-145.
A theoretical pathway of transcriptional regulation of the androgen receptor (AR) gene is via a cAMP response element (CRE) present in its promoter region (-508 to -501). After 20 h of stimulation with 8-bromo-cAMP, AR mRNA was upregulated in LNCaP but not in either PC-3 or DU-145 cell lines. We have demonstrated that the level of CRE binding protein (CREB) was the same in all cell lines and that the putative AR-CRE forms specific and compatible protein interactions with CREB. The ability to regulate AR gene transcription via the second messenger pathway is lost in the PC-3 and DU-145 cell lines. This may be an important primary mechanism of androgen insensitivity in prostate cancer.

interactions

CREB

lost

regulate

via

transcriptional regulation

upregulated

AR

transcriptional regulation

AR

regulate

transcription

transcription

AR


Input:
Activation of the NF-kappaB transcription factor in a T-lymphocytic cell line by hypochlorous acid.
Reactive oxygen species (ROS) such as hydrogen peroxide serve as second messengers in the induction of the transcription factor NF-kappaB, and hence in the activation and replication of human immunodeficiency virus type 1 (HIV-1) in human cells. During inflammatory reactions, many oxidative species are produced, one of which is hypochlorous acid (HOCl), which is responsible for the microbicidal effects of activated human polymorphonuclear leukocytes. Treatment of a T-lymphocytic cell line with micromolar concentrations of HOCl promoted the appearance of transcription factor NF-kappaB (the heterodimer p50/p65) in the nucleus of the cells, even in the absence of de novo protein synthesis. Western blot analysis of the NF-kappaB inhibitory subunits (IkappaB) demonstrated that both IkappaB-alpha proteolysis and p105 processing were induced by the treatment. NF-kappaB activation was very effective when cells were subjected to hyperthermia before being treated with HOCl. Various antioxidants, such as pyrrolidine dithiocarbamate, p-bromophenacyl-bromide and nordihydroguaiaretic acid could strongly reduce NF-kappaB translocation, demonstrating the importance of oxidative species in the transduction mechanism. Moreover, ACH-2 cells treated with HOCl or H2O2 released tumour necrosis factor-alpha (TNF-alpha) in the supernatants. The importance of TNF-alpha release in NF-kappaB induction by HOCl or H2O2 was demonstrated by the fact that: (1) the nuclear appearance of NF-kappaB was promoted in untreated cells; and (2) synergism between TNF-alpha and HOCl was detected. Collectively, these results suggest that HOCl should be considered as an oxidative species capable of inducing NF-kappaB in a T-lymphocytic cell line through a transduction mechanism involving ROS, and having a long-distance effect through subsequent TNF-alpha release.

appearance

nucleus

appearance

nucleus

released

TNF-alpha

release

TNF-alpha

release

TNF-alpha

promoted

appearance

induced

proteolysis

treated with

released

subsequent

release

proteolysis

IkappaB-alpha


Input:
Thymocytes control the CD4 gene differently from mature T lymphocytes.
We analyzed the activity of the enhancer, the promoter and the silencer of the human CD4 gene during T cell development using transgenic mice. Immunofluorescence studies on thymic populations of mice carrying transgenes in various combinations of these regulatory DNA elements revealed that thymocytes control the CD4 gene in a different manner than mature peripheral T lymphocytes. The 5'-positive regulatory unit, consisting of the promoter and the 5' enhancer, is already active at the CD4-CD8-double-negative (DN) stage of development. However, its activity becomes lower in the double-positive and a fraction of the CD4+ CD8int/- cell population, indicating that an additional enhancer, located in either the first or the third intron of the CD4 gene, is required for CD4 gene expression in this population. The other studied regulatory element is the minimal CD4 silencer which inhibits CD4 gene expression in peripheral CD8 T lymphocytes. This silencer is inactive in the most immature DN thymocytes, which probably use a distinct silencer mechanism to down-regulate CD4 gene expression. Unexpectedly, the CD4 silencer is also active in CD4+ CD8int/- cells of the thymus, implying that an anti-silencer may be required to resume CD4 expression in this cell population. Altogether, the CD4 gene is regulated by several positive and negative regulatory mechanisms which come into play in a developmentally coordinated manner.

expression

expression

expression

expression

double-negative

inhibits

expression

down-regulate

expression

resume

expression

regulated

double-positive

required

expression

required

resume

regulated

control

control


Input:
Triggering of HLA-DR antigens differentially modulates tumor necrosis factor alpha release by B cells at distinct stage of maturation.
Triggering of HLA class II antigens by the anti-HLA-DR monoclonal antibody (mAb) L243 significantly (P < 0.05) and differentially enhanced the release of tumor necrosis factor alpha (TNF-alpha) by the non-Hodgkin's lymphoma cells Ri-I, Ci-I, and Sc-I, which are at a distinct stage of B-cell differentiation, and by the more mature Burkitt lymphoma cell Raji; in contrast, it did not induce TNF-alpha release by the pre-B leukemia cells Nalm-6 and BV173. TNF-alpha release peaked at 24 h and decreased thereafter, and it was dose dependent and preceded by an increase of TNF-alpha mRNA detectable after 3 h of stimulation with mAb L243. Secreted TNF-alpha mediated the enhancement of nuclear factor kappa B (NF-kappa B) and activator protein-1 (AP-1) binding activity; in fact, the triggering of HLA-DR antigens in the presence of antihuman TNF-alpha-neutralizing antibodies did not upregulate NF-kappa B and AP-1. In contrast, released TNF-alpha was not responsible for the homotypic aggregation of Ri-I, Ci-I, Sc-I, and Raji cells induced by mAb L243, and it did not affect the proliferation of B cells investigated. Altogether, our data demonstrate that: (a) the ability of B cells to release TNF-alpha after triggering of HLA-DR antigens depends on their stage of differentiation; (b) levels of released TNF-alpha seem to correlate with the stage of B-cell maturation but do not correlate with the amounts of cell surface HLA-DR antigens; (c) secreted TNF-alpha regulates the levels of expression of NF-kappa B and AP-1 by an autocrine loop; and (d) intracellular signals mediating TNF-alpha release by B cells are distinct from those regulating homotypic aggregation and proliferation.

release

tumor necrosis factor alpha

release

TNF-alpha

release

TNF-alpha

release

TNF-alpha

Secreted

TNF-alpha

released

TNF-alpha

release

TNF-alpha

levels of released

TNF-alpha

secreted

TNF-alpha

release

TNF-alpha

enhanced

release

induce

release

peaked

release

preceded

release

increase

increase

TNF-alpha

after

release

mediating

release

modulates

release


Input:
Cytomegalovirus immediate early genes upregulate interleukin-6 gene expression.
BACKGROUND: The immediate early genes (IE) of human cytomegalovirus (CMV) can be expressed in monocytic cells and are known to regulate viral and cellular genes. Interleukin-6 (IL-6) plays a central role in numerous inflammatory and immune processes. Interleukin-6 levels are increased in lung transplant patients clinically diagnosed with CMV pneumonitis. The regulation of IL-6 is dependent on various stimuli that include lipopolysaccharide (LPS), viruses, and other cytokines. These studies examined the ability of CMV IE gene products to modulate IL-6 production. METHODS: THP-1 cells, a monocytic cell line, were transfected with the CMV IE genes. Interleukin-6 protein and IL-6 mRNA were measured in control and CMV immediate early transfected cells. Cotransfection of CMV IE genes and IL-6 chloramphenicol acetyl transferase (CAT) or IL-6 luciferase constructs were used to study IL-6 promoter activity. RESULTS: Interleukin-6 protein and mRNA production were significantly increased in cells transfected with the CMV IE genes and stimulated with LPS compared to LPS-stimulated control cells. Cytomegalovirus IE gene products significantly enhanced LPS stimulation of IL-6 promoter activity in both IL-6 CAT and IL-6 luciferase assays. A deletion construct that contains a NF-kappa B site but is missing the multiple response region demonstrated a continued increase in IL-6 luciferase activity in LPS-stimulated CMV transfected cells. CONCLUSION: Cytomegalovirus immediate early gene products significantly enhanced expression of IL-6 in LPS-stimulated cells. The increase in IL-6 luciferase activity occurs in the absence of the multiple response region, the area of the IL-6 promoter responsive to IL-1, TNF alpha, cyclic amp, and phorbol 12-myristate 13-acetate. The ability of CMV IE gene products to enhance IL-6 production may play an important role in immune inflammatory states associated with CMV infection.

expression

production

expression

production

upregulate

expression

increased

increased

production

enhanced

stimulation

stimulation

promoter

enhanced

expression

enhance

production

regulation

modulate

production

responsive

promoter

TNF alpha

responsive

promoter

production


Input:
Homodimerization of the human interleukin 4 receptor alpha chain induces Cepsilon germline transcripts in B cells in the absence of the interleukin 2 receptor gamma chain.
The cytokines interleukin (IL)-4 and IL-13 play a critical role in inducing Cepsilon germline transcripts and IgE isotype switching in human B cells. The IL-4 receptor (IL-4R) in B cells is composed of two chains, the IL-4-binding IL-4Ralpha chain, which is shared with the IL-13R, and the IL-2Rgamma (gammac) chain, which is shared with IL-7R, IL-9R, and IL-15R. IL-4 induces Cepsilon germline transcripts and IgE isotype switching in B cells from patients with gammac chain deficiency. Induction of Cepsilon germline transcripts by IL-4 in B cells that lack the gammac chain may involve signaling via the IL-13R. Alternatively, the IL-4Ralpha chain may transduce intracellular signals that lead to Cepsilon gene transcription independently of its association with other chains. We show that ligand-induced homodimerization of chimeric surface receptors consisting of the extracellular and transmembrane domains of the erythropoietin receptor and of the intracellular domain of IL-4Ralpha induces Janus kinase 1 (Jak1) activation, STAT6 activation, and Cepsilon germline transcripts in human B cell line BJAB. Disruption of the Jak1-binding proline-rich Box1 region of IL-4Ralpha abolished signaling by this chimeric receptor. Furthermore, B cells transfected with a chimeric CD8alpha/IL-4Ralpha receptor, which is expressed on the cell surface as a homodimer, constitutively expressed Cepsilon germline transcripts. These results suggest that homodimerization of the IL-4Ralpha chain is sufficient to transduce Jak1-dependent intracellular signals that lead to IgE isotype switching.

Homodimerization

interleukin 4 receptor alpha chain

binding

IL-4Ralpha chain

as a homodimer

chimeric CD8alpha/IL-4Ralpha receptor

homodimerization

IL-4Ralpha chain

lack

gammac chain

transfected

chimeric CD8alpha/IL-4Ralpha receptor

expressed

chimeric CD8alpha/IL-4Ralpha receptor

deficiency

gammac chain

induces

activation

activation

transfected

transfected


Input:
Characterization of peripheral blood T-lymphocytes transduced with HTLV-I Tax mutants with different trans-activating phenotypes.
Tax1, a transcriptional trans-activator of the Human T-cell leukemia virus type I (HTLV-I), induces the expression of many cellular genes through interaction with at least three distinct cellular transcription factors; CREB/ATF, NF-kappaB, and SRF. This Tax1-induced activation of cellular genes is considered to be a critical event in T-cell transformation by HTLV-I. To elucidate the role of each Tax1-inducible transcriptional pathway in T-cell transformation, we introduced Tax1 mutants with different trans-activating phenotypes into peripheral blood lymphocytes (PBL) by retroviral vectors. Analysis of these PBLs revealed that activation of the NF-kappaB pathway is sufficient to promote the growth response to IL-2. However, for the clonal expansion of CD4+ T-cells, which is a characteristic result of HTLV-I infection, activation of the CREB/ATF and SRF pathways is also required.

interaction

SRF

interaction

transduced

Tax

introduced

transduced

transduced

introduced

introduced


Input:
Involvement of the N-terminal region of the human mineralocorticoid receptor hormone-binding domain in agonist and antagonist binding as revealed by a new monoclonal antibody.
To gain a better understanding of the mechanism of binding to the human mineralocorticoid receptor (hMR), we developed a new monoclonal antibody (mAb) raised against the hormone-binding domain (HBD). For this purpose, mice were immunized with a fusion protein including the sequence Thr729-Lys984 of hMR. After ELISA screening, mAb 18C7 was selected for its specificity towards the HBD. This antibody recognized both the denatured and native MR forms, as well as the hetero-oligomeric MR form and the transformed MR state. By using several HBD subfragments, the mAb 18C7 epitope was located in the N-terminal region of the HBD from Thr729 to Leu765. We then studied the effect of the antibody on aldosterone and progesterone binding to the hMR. When 18C7 was incubated with liganded MR, it was able to partly displace (20%) the hormone from its binding site. When 18C7 was incubated with MR before aldosterone or progesterone, the antibody inhibited 75-80% of the binding. The effect of 18C7 on the binding was similar with both hormones. A sucrose gradient analysis indicated the simultaneous presence of two kinds of receptor complexes: the steroid-MR complex and the antibody-MR complex. After its associated proteins, especially the heat-shock protein hsp90, had been cross-linked with the hMR by dimethylpimelimidate, 18C7 was still able to react with the receptor. Our results indicated that the epitope recognized by 18C7 was directly implicated in hormone binding. The lack of steroid binding of HBD mutants with the Thr729-Leu765 sequence deleted [Jalaguier, Mesnier, Leger and Auzou (1996) J.Steroid Biochem.Mol.Biol.57, 43-50] supports this hypothesis. Because of the similar behaviours of aldosterone and progesterone, we conclude that the N-terminal Thr729-Leu765 region of the HBD is similarly involved in the binding of both hormones.

binding

mineralocorticoid receptor

binding

MR

recognized

MR

recognized

MR

binding

MR

liganded

MR

binding

MR

binding

MR

complex

MR

complex

MR

cross-linked

hMR

binding

MR

binding

MR

inhibited

binding

still able to

cross-linked

react

effect

binding

effect

binding

react

hMR


Input:
Abnormal T lymphocyte development induced by targeted overexpression of IkappaB alpha.
A role in thymic maturation for factors of the NF-kappaB family has long been suspected, but not yet proven. Transgenic mice with a lymphocyte-specific defect in NF-kappaB activation were produced by targeted expression of human IkappaB alpha. The thymic cellularity of these mice was significantly decreased. The proportion of mature, TCRhigh thymocytes of the alphabeta lineage was reduced, and the remaining TCRhigh population contained an unusually high proportion of double-positive cells. This defect in maturation resulted in a transgene dose-dependent reduction in peripheral T lymphocytes, with the CD8 lineage being more severely affected. These data provide direct evidence for the involvement of NF-kappaB/Rel family proteins in late stages of T lymphocyte development, coincident with positive and negative selection.

expression

IkappaB alpha

overexpression

IkappaB alpha


Input:
Sequential development of structural and functional alterations in T cells from tumor-bearing mice.
The TCR alpha beta or -gamma delta chains bind the peptide ligand, whereas the associated CD3 deltaepsilongamma and TCR zeta subunits couple the TCR to intracellular signal transduction components. Recently, several groups have described marked alterations in signal transduction elements in T cells from cancer patients or in mice bearing tumor for a few weeks (>26 days). The sequence in which these alterations develop is unknown. The aim of this study was to explore the kinetics of the development of alterations in signal transduction molecules (TCR zeta chain, NF kappaB family proteins, and tyrosine kinase p56(lck)) in mice bearing MC38 colon adenocarcinoma. The results demonstrate that alterations in NF kappaB family proteins, specifically the failure of p65 translocation to the nucleus, occur earlier and more frequently than the decrease in zeta-chain. These defects are paralleled by an impaired ability to produce Th1 cytokines (IL-2 and IFN-gamma). These initial changes are followed by the eventual loss of TCR zeta chain and p56(lck) and a marked decrease in cytotoxic function. An increased rate of lysosomal degradation is one of the mechanisms responsible for the loss of zeta-chain.

produce

produce

IFN-gamma

translocation

nucleus

failure

translocation

decrease

TCR zeta chain

impaired ability

produce

loss

TCR zeta chain

loss

occur

failure

paralleled

failure

impaired ability

followed

impaired ability

loss

followed

failure

loss


Input:
An isotype-specific activator of major histocompatibility complex (MHC) class II genes that is independent of class II transactivator.
Patients with one type of major histocompatibility complex class II combined immunodeficiency have mutations in a gene termed class II transactivator (CIITA), which coordinately controls the transcription of the three major human class II genes, HLA-DR, -DQ, and -DP. However, the experimentally derived B-lymphoblastoid cell line, clone 13, expresses high levels of HLADQ in the absence of HLA-DR and HLA-DP, despite its mapping by complementation analysis to this group. It was possible that one of the clone 13 CIITA alleles bore a mutation that allowed HLA-DQ, but not HLA-DR or -DP transcription. Alternatively, another factor, distinct from CIITA, might control HLA-DQ expression. We report here that ectopic expression of CIITA cDNAs derived by reverse transcriptase polymerase chain reaction from clone 13 do not restore expression of HLA-DQ in another CIITA-deficient cell line, RJ2.2.5. In addition, no CIITA protein is detectable in clone 13 nuclear extracts. In contrast, somatic cell fusion between clone 13 and RJ2.2.5 restored expression of the HLA-DQ haplotype encoded by the RJ2.2.5 DQB gene. Taken together, these data demonstrate the existence of an HLA-DQ isotype-specific trans-acting factor, which functions independently of CIITA.

expression

CIITA

detectable

CIITA


Input:
Induction of cytokine expression in leukocytes by binding of thrombin-stimulated platelets.
BACKGROUND: Activated platelets tether and activate myeloid leukocytes. To investigate the potential relevance of this mechanism in acute myocardial infarction (AMI), we examined cytokine induction by leukocyte-platelet adhesion and the occurrence of leukocyte-platelet conjugates in patients with AMI. METHODS AND RESULTS: We obtained peripheral venous blood samples in 20 patients with AMI before and daily for 5 days after direct percutaneous transluminal coronary angioplasty (PTCA) and in 20 patients undergoing elective PTCA. Throughout the study period, CD41 immunofluorescence of leukocytes (flow cytometry) revealed increased leukocyte-platelet adhesion in patients with AMI compared with control patients (mean +/- SE of fluorescence [channels] before PTCA: 77 +/- 16 versus 35 +/- 9; P = .003). In vitro, thrombin-stimulated fixed platelets bound to neutrophils and monocytes. Within 2 hours, this resulted in increased mRNA for interleukin (IL),1 beta, IL-8, and monocyte chemoattractant protein (MCP)-1 in unfractionated leukocytes. After 4 hours, IL-1 beta and IL-8 concentration of the cell-free supernatant had increased by 268 +/- 36% and 210 +/- 7%, respectively, and cellular MCP-1 content had increased by 170 +/- 8%. Addition of activated platelets to adherent monocytes had a similar effect and was associated with nuclear factor-kappa B activation. Inhibition of binding by anti-P selectin antibodies reduced the effect of activated platelets on cytokine production. CONCLUSIONS: In patients with AMI, leukocyte-platelet adhesion is increased. Binding of activated platelets induces IL-1 beta, IL-8, and MCP-1 in leukocytes. Our findings suggest that leukocyte-platelet adhesion contributes to the regulation of inflammatory responses in AMI.

resulted in increased

interleukin (IL),1 beta

resulted in increased

resulted in increased

monocyte chemoattractant protein (MCP)-1

increased

IL-1 beta

increased

increased

induces

IL-1 beta

induces

induces


Input:
The class II trans-activator CIITA interacts with the TBP-associated factor TAFII32.
The class II trans- activator (CIITA) is the main transcriptional co-activator for the expression of MHC class II proteins. Its N-terminal 125 amino acids function as an independent transcriptional activation domain. Analyses of the primary amino acid sequence of the activation domain predict the presence of three alpha-helices, each with a high proportion of acidic residues. Using site-directed mutagenesis, we found that two of these predicted alpha-helices are required for full transcriptional activation by CIITA. Moreover, a CIITA protein in which both functional alpha-helices have been deleted displays a dominant negative phenotype. This activation domain of CIITA interacts with the 32 kDa subunit of the general transcription complex TFIID, TAFII32. Decreased transcriptional activation by N-terminal deletions of CIITA is correlated directly with their reduced binding to TAFII32. We conclude that interactions between TAFII32 and CIITA are responsible for activation of class II genes.

interacts

CIITA

interacts

activation domain

CIITA

binding

CIITA

interactions

CIITA

displays a dominant negative phenotype

CIITA

reduced

binding


Input:
Lipopolysaccharide induction of the tumor necrosis factor-alpha promoter in human monocytic cells. Regulation by Egr-1, c-Jun, and NF-kappaB transcription factors.
Biosynthesis of tumor necrosis factor-alpha (TNF-alpha) is predominantly by cells of the monocytic lineage. This study examined the role of various cis-acting regulatory elements in the lipopolysaccharide (LPS) induction of the human TNF-alpha promoter in cells of monocytic lineage. Functional analysis of monocytic THP-1 cells transfected with plasmids containing various lengths of TNF-alpha promoter localized enhancer elements in a region (-182 to -37 base pairs (bp)) that were required for optimal transcription of the TNF-alpha gene in response to LPS. Two regions were identified: region I (-182 to -162 bp) contained an overlapping Sp1/Egr-1 site, and region II (-119 to -88) contained CRE and NF-kappaB (designated kappaB3) sites. In unstimulated THP-1, CRE-binding protein and, to a lesser extent, c-Jun complexes were found to bind to the CRE site. LPS stimulation increased the binding of c-Jun-containing complexes. In addition, LPS stimulation induced the binding of cognate nuclear factors to the Egr-1 and kappaB3 sites, which were identified as Egr-1 and p50/p65, respectively. The CRE and kappaB3 sites in region II together conferred strong LPS responsiveness to a heterologous promoter, whereas individually they failed to provide transcriptional activation. Furthermore, increasing the spacing between the CRE and the kappaB3 sites completely abolished LPS induction, suggesting a cooperative interaction between c-Jun complexes and p50/p65. These studies indicate that maximal LPS induction of the TNF-alpha promoter is mediated by concerted participation of at least two separate cis-acting regulatory elements.

bind

c-Jun

binding

c-Jun

binding

binding

binding

Biosynthesis

TNF-alpha

induction

tumor necrosis factor-alpha

promoter

induction

TNF-alpha

promoter

required

in response to

in response to

transcription

increased

binding

induction

TNF-alpha

promoter

Regulation

induction

Regulation

induction

c-Jun

role

induction

mediated

induction

transcription

TNF-alpha


Input:
Involvement of Rel, Fos, and Jun proteins in binding activity to the IL-2 promoter CD28 response element/AP-1 sequence in human T cells.
CD28 is an important costimulatory molecule in the activation of human T cells. Costimulation of T cells through both the Ag receptor and CD28 leads to high level IL-2 production, which is vital to the development of an immune response in vivo. Previous reports have suggested the CD28 stimulation contributes to the activation of the IL-2 promoter by up-regulating the activity of several transcription factors, including AP-1 and nuclear factor-kappaB (NF-kappaB)/Rel family members as well as an uncharacterized transcription factor called CD28 response complex. While several lines of investigation have suggested that NF-kappaB/Rel family members make up the CD28 response complex transcription factor, other work has not supported this conclusion. Recent studies suggest that the CD28 response element (CD28RE) does not function independently but works instead in conjunction with the adjacent promoter proximal AP-1-binding site and this hypothesis is confirmed here. Also in the current study, binding activity to the CD28RE/AP-1 sequence of the IL-2 promoter is evaluated. Although four specific complexes can be detected binding to this sequence, only one of these complexes is specific for both the CD28RE and the adjacent AP-1 site. Of the NF-kappaB/Rel family members tested, this CD28RE/AP-1-specific complex contains predominantly c-Rel, despite the fact that both p50 and RelA can efficiently bind to the CD28RE. c-Fos and c-Jun are also found in this CD28RE/AP-1-specific complex. These data indicate that functional complexes encompassing both the CD28RE and the AP-1-binding sites influence IL-2 promoter activity in CD28-costimulated T cells.

binding activity

CD28RE/AP-1 sequence

binding

CD28RE/AP-1 sequence

bind

bind

RelA

production

leads

high level

high level

production

contributes

activation

activation

promoter

influence

promoter


Input:
c-Myb and Ets proteins synergize to overcome transcriptional repression by ZEB.
The Zfh family of zinc finger/homeodomain proteins was first identified in Drosophila where it is required for differentiation of tissues such as the central nervous system and muscle. ZEB, a vertebrate homolog of Zfh-1, binds a subset of E boxes and blocks myogenesis through transcriptional repression of muscle genes. We present evidence here that ZEB also has an important role in controlling hematopoietic gene transcription. Two families of transcription factors that are required for normal hematopoiesis are c-Myb and Ets. These factors act synergistically to activate transcription, and this synergy is required for transcription of at least several important hematopoietic genes. ZEB blocks the activity of c-Myb and Ets individually, but together the factors synergize to resist this repression. Such repression imposes a requirement for both c-Myb and Ets for transcriptional activity, providing one explanation for why synergy between these factors is important. The balance between repression by ZEB and transcriptional activation by c-Myb/Ets provides a flexible regulatory mechanism for controlling gene expression in hematopoietic cells. We demonstrate that one target of this positive/negative regulation in vivo is the alpha4 integrin, which play a key role in normal hematopoiesis and function of mature leukocytes.

binds

ZEB

blocks

ZEB

c-Myb

synergize to resist

blocks

c-Myb

positive/negative regulation

alpha4 integrin


Input:
GABP factors bind to a distal interleukin 2 (IL-2) enhancer and contribute to c-Raf-mediated increase in IL-2 induction.
Triggering of the T-cell receptor-CD3 complex activates two major signal cascades in T lymphocytes, (i) Ca2+-dependent signal cascades and (ii) protein kinase cascades. Both signal cascades contribute to the induction of the interleukin 2 (IL-2) gene during T-cell activation. Prominent protein kinase cascades are those that activate mitogen-activated protein (MAP) kinases. We show here that c-Raf, which is at the helm of the classic MAP-Erk cascade, contributes to IL-2 induction through a distal enhancer element spanning the nucleotides from positions -502 to -413 in front of the transcriptional start site of the IL-2 gene. Induction of this distal IL-2 enhancer differs from induction of the proximal IL-2 promoter-enhancer, since it is induced by phorbol esters alone and independent from Ca2+ signals. In DNA-protein binding studies, we detected the binding of transcription factors GABP alpha and -beta to a dyad symmetry element (DSE) of the distal enhancer, which is formed by palindromic binding sites of Ets-like factors. Introduction of point mutations suppressing GABP binding to the DSE interfered with the induction of the distal enhancer and the entire IL-2 promoter-enhancer, while overexpression of both GABP factors enhanced the IL-2 promoter-enhancer induction. Overexpression of BXB, a constitutive active version of c-Raf, and of further members of the Ras-Raf-Erk signal cascade exerted an increase of GABP-mediated promoter-enhancer induction. In conjunction with previously published data on c-Raf-induced phosphorylation of GABP factors (E.Flory, A. Hoffmeyer, U.Smola, U.R.Rapp, and J.T.Bruder, J.Virol.70:2260- 2268, 1996), these results indicate a contribution of GABP factors to the Raf-mediated enhancement of IL-2 induction during T-cell activation.

bind

interleukin 2

enhancer

binding

GABP alpha

binding

-beta

overexpression

GABP alpha

overexpression

-beta

Overexpression

BXB

interfered

induction

increase

c-Raf

induction

induction

induction

induction

induction

promoter-enhancer

induced

promoter-enhancer

independent

promoter-enhancer

induction

promoter-enhancer

overexpression

overexpression

enhanced

overexpression

induction

induction

promoter-enhancer

Overexpression

Overexpression

enhancement

induction

induction

contribute

increase

contribute

induction

contribution

enhancement


Input:
Genomic organization, sequence, and transcriptional regulation of the human eotaxin gene.
Eotaxin is an eosinophil specific beta-chemokine assumed to be involved in eosinophilic inflammatory diseases such as atopic dermatitis, allergic rhinitis, asthma and parasitic infections. Its expression is stimulus- and cell-specific. We here describe the genomic organisation (3 exons of 132, 112 and 542 bp and 2 introns of 1211 and 378 bp) and sequence including 3 kb of DNA from the immediate 5' upstream region of the human eotaxin gene. Among the regulatory promoter elements potentially regulating eotaxin gene expression and/or mediating the effects of anti-inflammatory drugs we identified consensus sequences known to interact with nuclear factors like NF-IL6, AP-1, a NF-kappa-B like consensus sequence and gamma-interferon- as well as glucocorticoid response elements.

interact

expression

Eotaxin

expression

eotaxin

Genomic organization

eotaxin

transcriptional regulation

eotaxin

regulating

expression


Input:
Dysregulation of monocytic nuclear factor-kappa B by oxidized low-density lipoprotein.
Nuclear factor-kappa B (NF-kappa B)/Rel transcription factors may be involved in atherosclerosis, as is suggested by the presence of activated NF-kappa B in human atherosclerotic lesions. The aim of the present study was to investigate the effects of oxidized LDL (oxLDL) on the NF-kappa B system in human THP-1 monocytic cells as well as adherent monocytes. Our results demonstrate that short-term incubation of these cells with oxLDL activated p50/p65 containing NF-kappa B dimers and induced the expression of the target gene IL-8. This activation of NF-kappa B was inhibited by the antioxidant and H2O2 scavenger pyrrolidine dithiocarbamate and the proteasome inhibitor PSI. The oxLDL-induced NF-kappa B activation was accompanied by an initial depletion of I kappa B-alpha followed by a slight transient increase in the level of this inhibitor protein. In contrast, long-term treatment with oxLDL prevented the lipopolysaccharide-induced depletion of I kappa B-alpha, accompanied by an inhibition of both NF-kappa B activation and the expression of tumor necrosis factor-alpha and interleukin-1 beta genes. These observations provide additional evidence that oxLDL is a potent modulator of gene expression and suggest that (dys)regulation of NF-kappa B/Rel is likely to play an important role in atherogenesis.

expression

tumor necrosis factor-alpha

expression

interleukin-1 beta

depletion

I kappa B-alpha

prevented

depletion

depletion

I kappa B-alpha

inhibition

expression

increase

I kappa B-alpha


Input:
Suppression of MHC class II expression by human class II trans-activator constructs lacking the N-terminal domain.
The class II trans-activator (CIITA) is a bi- or multi-functional domain protein which plays a critical role in the expression of MHC class II genes. We report that removal of the N-terminal 151 amino acids, encompassing all of the acidic domain but leaving intact the proline/serine/threonine-rich domain, results in a mutant protein with potent suppressive properties for MHC class II expression. HeLa cells stably or transiently transfected with mutant CIITA constructs showed up to 99% suppression of MHC class II antigen induction by IFN-gamma and marked suppression of HLA-DRA mRNA expression. Transient transfection of a B lymphoma line resulted in up to 89% reduction of constitutive MHC class II expression within 5 days and suppression of HLA-DRA mRNA synthesis.

transfected

CIITA

transfection

CIITA

transfected

transfected

transfection

transfection


Input:
CD30-dependent degradation of TRAF2: implications for negative regulation of TRAF signaling and the control of cell survival.
CD30 is a cell-surface receptor that can augment lymphocyte activation and survival through its ability to induce the transcription factor NF-kappaB. CD30, however, has also been implicated in the induction of apoptotic cell death of lymphocytes. Here we show that one of the effects of CD30 signal transduction is to render cells sensitive to apoptosis induced by the type 1 tumor necrosis factor receptor (TNFR1). This sensitization is dependent on the TRAF-binding sites within the CD30 cytoplasmic domain. One of the proteins that binds to these sites is TRAF2, a signal transduction molecule that is also utilized by TNFR1 to mediate the activation of several downstream kinases and transcription factors. During CD30 signal transduction, we found that binding of TRAF2 to the cytoplasmic domain of CD30 results in the rapid depletion of TRAF2 and the associated protein TRAF1 by proteolysis. These data suggest a model in which CD30 limits its own ability to transduce cell survival signals through signal-coupled depletion of TRAF2. Depletion of intracellular TRAF2 and its coassociated proteins also increased the sensitivity of the cell to undergoing apoptosis during activation of death-inducing receptors such as TNFR1. Consistent with this hypothesis, expression of a dominant-negative form of TRAF2 was found to potentiate TNFR1-mediated death. These studies provide a potential mechanism through which CD30, as well as other TRAF-binding members of the TNFR superfamily, can negatively regulate cell survival.

binds

TRAF-binding sites

binding

results

binding

proteolysis

activation

degradation

proteolysis

dependent

degradation


Input:
TNFalpha cooperates with the protein kinase A pathway to synergistically increase HIV-1 LTR transcription via downstream TRE-like cAMP response elements.
Activating protein-1 (AP-1) binding TPA responsive elements (TRE) are located downstream of the transcription initiation site in the U5 region of the HIV-1 long terminal repeat (LTR). These downstream sequence elements, termed DSE, can bind both AP-1 and CREB/ATF transcription factors. Recently, we demonstrated that the DSE are also cAMP-responsive elements (CRE), since they mediated activation signals elicited by cholera toxin (Ctx), a potent activator of the cAMP-dependent protein kinase A (PKA) signal transduction pathway. In the present study, we demonstrate that the HIV-1 DSE can mediate the transcriptional synergy elicited by the combination of Ctx and TNFalpha. Ctx combined with TNFalpha or IL-1beta to produce a synergistic increase in p24 antigen production in U1 promonocytic cells. Transfection studies of LTR reporter constructs indicated that mutation of the DSE sites abrogated the LTR-mediated synergy induced by Ctx and TNFalpha, whereas the synergy induced by Ctx and IL-1beta was unaffected, suggesting TNFalpha and IL-1beta cooperate differently with the cAMP/PKA activation pathway to induce HIV-1 expression in U1 cells. Because the DSE are also TRE sites, we assessed the effect of the agonist combinations on AP-1-dependent transcription. TNFalpha as well as IL-1beta cooperated with Ctx to produce a synergistic activation of AP-1-mediated transcription. These data indicate that the TRE-like cAMP-responsive DSE sites within the 5'-untranslated leader can mediate the transcriptional cooperativity between TNFalpha and the cAMP/PKA pathway. Since the DSE and TRE sites cannot bind CREB/ATF homodimers, we propose a mechanism in which the HIV-1 DSE bind heterodimers composed of both AP-1 and CREB/ATF proteins. Copyright 1997 Academic Press.

production

p24 antigen

to produce a synergistic increase

TNFalpha

production

to produce a synergistic increase

production


Input:
The Epstein-Barr virus oncogene product latent membrane protein 1 engages the tumor necrosis factor receptor-associated death domain protein to mediate B lymphocyte growth transformation and activate NF-kappaB.
The Epstein-Barr virus latent membrane protein 1 (LMP1) is essential for the transformation of B lymphocytes into lymphoblastoid cell lines. Previous data are consistent with a model that LMP1 is a constitutively activated receptor that transduces signals for transformation through its carboxyl-terminal cytoplasmic tail. One transformation effector site (TES1), located within the membrane proximal 45 residues of the cytoplasmic tail, constitutively engages tumor necrosis factor receptor-associated factors. Signals from TES1 are sufficient to drive initial proliferation of infected resting B lymphocytes, but most lymphoblastoid cells infected with a virus that does not express the 155 residues beyond TES1 fail to grow as long-term cell lines. We now find that mutating two tyrosines to an isoleucine at the carboxyl end of the cytoplasmic tail cripples the ability of EBV to cause lymphoblastoid cell outgrowth, thereby marking a second transformation effector site, TES2. A yeast two-hybrid screen identified TES2 interacting proteins, including the tumor necrosis factor receptor-associated death domain protein (TRADD). TRADD was the only protein that interacted with wild-type TES2 and not with isoleucine-mutated TES2. TRADD associated with wild-type LMP1 but not with isoleucine-mutated LMP1 in mammalian cells, and TRADD constitutively associated with LMP1 in EBV-transformed cells. In transfection assays, TRADD and TES2 synergistically mediated high-level NF-kappaB activation. These results indicate that LMP1 appropriates TRADD to enable efficient long-term lymphoblastoid cell outgrowth. High-level NF-kappaB activation also appears to be a critical component of long-term outgrowth.

engages

latent membrane protein 1

interacting

TRADD

interacted

TRADD

associated

TRADD

associated

TRADD

associated

TRADD

activated


Input:
Transcription factor NF-kappaB regulates inducible Oct-2 gene expression in precursor B lymphocytes.
The POU transcription factors Oct-1 and Oct-2 regulate the activity of octamer-dependent promoters, including those that direct transcription from rearranged immunoglobulin genes. Unlike Oct-1, which is constitutively expressed in many cell types, Oct-2 expression is restricted primarily to B lymphocytes and can be induced in precursor B cells by stimulation with bacterial lipopolysaccharide (LPS). However, the precise factors that mediate this induction mechanism remain unknown. In the present study, we monitored Oct-2 expression in cells arrested for the activation of NF-kappaB, an LPS-responsive member of the Rel transcription factor family. Despite stimulation with LPS, disruption of the NF-kappaB signaling pathway in precursor B cells led to the loss of inducible Oct-2 DNA binding activity in vitro and the suppression of Oct-2-directed transcription in vivo. This biochemical defect correlated with a specific block to Oct-2 gene expression at the level of transcription, whereas the expression of Oct-1 was unaffected. The finding that Oct-2 is under NF-kappaB control highlights an important cross-talk mechanism involving two distinct transcription factor families that regulate B lymphocyte function.

binding

gene expression

expressed

expression

expression

expression

Despite

loss

loss

binding

block

expression at the level of transcription

unaffected

expression

inducible

gene expression

restricted

expressed

restricted

expression

induced

expression

mediate

induced

inducible

binding

regulates

gene expression

control

expression at the level of transcription


Input:
Paternal expression of WT1 in human fibroblasts and lymphocytes.
The Wilms' tumor suppressor gene ( WT1 ) was previously identified as being imprinted, with frequent maternal expression in human placentae and fetal brains. We examined the allele-specific expression of WT1 in cultured human fibroblasts from 15 individuals. Seven of 15 fibroblast lines were heterozygous for polymorphic alleles, and the expression patterns were variable, i.e., equal, unequal or monoallelic paternal expression in three, two and two cases, respectively. Exclusive paternal expression of WT1 was also shown in non-cultured peripheral lymphocytes from the latter two individuals. The allele-specific expression profiles of other imprinted genes, IGF2 and H19, on human chromosome 11 were constant and consistent with those in other tissues. Our unexpected observations of paternal or biallelic expression of WT1 in fibroblasts and lymphocytes, together with the previous findings of maternal or biallelic expression in placentae and brains, suggest that the allele-specific regulatory system of WT1 is unique and may be controlled by a putative tissue- and individual-specific modifier.

Paternal expression

maternal expression

allele-specific expression

expression

paternal expression

allele-specific expression

allele-specific expression

biallelic expression

expression

biallelic expression

expression

allele-specific regulatory

controlled

allele-specific regulatory


Input:
Extinction of immunoglobulin gene expression in B cells upon fusion with HeLa cells is preceded by rapid nuclear depletion of essential transcription factors and is accompanied by widespread inactivation of genes expressed in a B cell-specific manner.
When immunoglobulin (Ig) expressing B cells are fused with non-B cells, Ig expression is rapidly suppressed at the level of transcription, a phenomenon termed extinction. Here we demonstrate that fusion of HeLa cells with either diploid or tetraploid B cells (Daudi) results in widespread extinction of several other B cell-encoded genes that are expressed in a B cell-specific manner. In contrast, expression of B cell-expressed genes that are not dependent on cell-specific controls is unaffected. We show that the molecular mechanism(s) underlying Ig gene extinction can be explained, at least in part, by a lack of transcription factors that are essential for Ig gene transcription. These transcription factors are either not produced due to block of transcription of their respective genes (Oct-2, OBF-1, PU.1), or are rendered inactive posttranslationally (NF-kappa B, E47). By isolating Daudi x HeLa heterokaryons a few hours after fusion, we have studied the initial fate of two B cell-specific transcription factors involved in Ig gene transcription, Oct-2 and NF-kappa B. This report provides the first demonstration that upon fusion with HeLa cells, the nuclear contents of B cell-expressed transcription factors are depleted within a few hours with kinetics that are as fast or faster than that of Ig gene extinction. Thus, the extinguishing mechanism is effective very early after fusion. We suggest that extinction of Ig genes is part of a global mechanism that suppresses the differentiation program foreign to the HeLa phenotype.

block

transcription

inactive

transcription

transcription

transcription


Input:
Regulation of Id3 cell cycle function by Cdk-2-dependent phosphorylation.
The functions of basic helix-loop-helix (bHLH) transcription factors in activating differentiation-linked gene expression and in inducing G1 cell cycle arrest are negatively regulated by members of the Id family of HLH proteins. These bHLH antagonists are induced during a mitogenic signalling response, and they function by sequestering their bHLH targets in inactive heterodimers that are unable to bind to specific gene regulatory (E box) sequences. Recently, cyclin E-Cdk2- and cyclin A-Cdk2-dependent phosphorylation of a single conserved serine residue (Ser5) in Id2 has been shown to occur during late G1-to-S phase transition of the cell cycle, and this neutralizes the function of Id2 in abrogating E-box-dependent bHLH homo- or heterodimer complex formation in vitro (E.Hara, M.Hall, and G.Peters, EMBO J.16:332-342, 1997). We now show that an analogous cell-cycle-regulated phosphorylation of Id3 alters the specificity of Id3 for abrogating both E-box-dependent bHLH homo- or heterodimer complex formation in vitro and E-box-dependent reporter gene function in vivo. Furthermore, compared with wild-type Id3, an Id3 Asp5 mutant (mimicking phosphorylation) is unable to promote cell cycle S phase entry in transfected fibroblasts, whereas an Id3 Ala5 mutant (ablating phosphorylation) displays an activity significantly greater than that of wild-type Id3 protein. Cdk2-dependent phosphorylation therefore provides a switch during late G1-to-S phase that both nullifies an early G1 cell cycle regulatory function of Id3 and modulates its target bHLH specificity. These data also demonstrate that the ability of Id3 to promote cell cycle S phase entry is not simply a function of its ability to modulate bHLH heterodimer-dependent gene expression and establish a biologically important mechanism through which Cdk2 and Id-bHLH functions are integrated in the coordination of cell proliferation and differentiation.

phosphorylation

phosphorylation

phosphorylation

dependent

phosphorylation

dependent

cyclin E

phosphorylation

dependent

phosphorylation

dependent

cyclin A

phosphorylation

dependent

phosphorylation

Regulation

phosphorylation

regulated

phosphorylation


Input:
Cytokine rescue from glucocorticoid induced apoptosis in T cells is mediated through inhibition of IkappaBalpha.
We previously reported that dexamethasone (DEX), a synthetic glucocorticoid, causes apoptosis in mature Th cell lines, and that this induction of cell death is prevented by specific cytokines, namely, by IL-2 in Th1 cells and by IL-4 in Th2 cells. We now show that this differential rescue by specific cytokines in Th cells correlates with the level of IkappaBalpha that is regulated by DEX and cytokines. In both cell types the cellular levels of IkappaBalpha mRNA and protein were evaluated by DEX treatment. Interestingly, the DEX-mediated IkappaBalpha induction was completely inhibited by IL-2, but not IL-4, in Th1 cells, while the reverse profile was seen in Th2 cells. In both cell types, the cytokine that inhibits the induction of IkappaBalpha by DEX, also rescues these cells from DEX-induced apoptosis, although the rescue cytokine is different in Th1 and Th2 cells. Our results imply that T cells need to maintain a certain level of NF-kappaB transcriptional activity in order to survive; up- or down-regulation of nuclear NF kappaB through modulation of IkappaBalpha expression by cytokines or DEX may lead to cell survival or cell death, respectively.

level

IkappaBalpha

levels

IkappaBalpha

expression

IkappaBalpha

inhibition

IkappaBalpha

inhibited

induction

inhibited

induction

inhibits

induction

induction

IkappaBalpha

induction

IkappaBalpha

regulated

level

modulation

expression

levels

IkappaBalpha


Input:
Xenogeneic human serum promotes leukocyte adhesion to porcine endothelium under flow conditions, possibly through the activation of the transcription factor NF-kappa B.
Endothelial cell activation and leukocyte infiltration are a consistent feature of discordant xenograft rejection. Here we evaluated whether xenogeneic serum, as a source of xenoreactive natural antibodies and complement, induced endothelial cell activation with consequent leukocyte adhesion under flow conditions. Porcine aortic endothelial cells (PAEC) were incubated for 1 hr 30 min or 5 hr with 10% homologous porcine serum (control) or 10% xenogeneic human serum and then perfused with total human leukocytes in a parallel plate flow chamber under laminar flow (1.5 dynes/cm2). Adherent cells were counted by digital image analysis. Xenogeneic human serum significantly (P < 0.01) increased the number of adherent leukocytes as compared with porcine serum. A similar adhesive response was elicited by TNF alpha (100 U/ml), one of the most potent inducers of endothelial cell adhesive properties, here used as positive control. In order to elucidate possible mechanisms underlying endothelial cell activation by xenogeneic serum, we focussed on transcription factor NF-kappa B, a central regulator for the induction of different genes, including adhesive molecules and chemoattractants. By confocal fluorescence microscopy, we observed a positive staining for NF-kappa B (p65 subunit) in the nuclei of PAEC exposed for 1 hr 30 min to human serum, which indicated NF-kappa B activation in this setting. At variance, in PAEC incubated with the homologous serum, NF-kappa B was strictly localized in the cell cytoplasm. Treatment of PAEC exposed to xenogeneic serum with the NF-kappa B inhibitors pyrrolidinedithiocarbamate (PDTC, 25 microM) and tosyl-phechloromethylketone (TPCK, 25 microM) significantly (P < 0.01) reduced leukocyte adhesion in respect to PAEC treated with human serum alone. Findings that xenogeneic serum promotes leukocyte-endothelium interaction possibly through NF-kappa B activation might be relevant for designing future therapeutic strategies aimed at prolonging xenograft survival.

positive staining


Input:
IL-2-induced growth of CD8+ T cell prolymphocytic leukemia cells mediated by NF-kappaB induction and IL-2 receptor alpha expression.
The binding of interleukin-2 (IL-2) to its receptor on normal T cells induces nuclear expression of nuclear factor kappaB (NF-kappaB), activation of the IL-2 receptor (IL-2R) alpha chain gene, and cell proliferation. In the present study, the role of IL-2R signaling in the growth of CD8+ T cell prolymphocytic leukemia (T-PLL) cells has been investigated. Flow cytometry revealed that primary leukemia cells from a patient with CD8+ T-PLL expressed IL-2Ralpha and beta chains, and the cells showed a proliferative response and an increase in IL-2Ralpha expression on culture with exogeneous IL-2. Northern blot analysis failed to detect IL-2 mRNA, suggesting that IL-2 may act in a paracrine manner in vivo. Electrophoretic mobility-shift assays revealed that recombinant IL-2 increased NF-kappaB binding activity in nuclear extracts of the leukemia cells, and Northern blot analysis showed that IL-2 increased the abundance of mRNAs encoding the NF-kappaB components c-Rel and KBF1 in these cells. IL-2 binding analysis demonstrated that IL-2 markedly increased the number of low affinity IL-2Rs on the leukemia cells, without an effect on the number of high-affinity IL-2Rs. These results show that IL-2 is capable of inducing the nuclear expression of NF-kappaB in primary CD8+ T-PLL cells, and that this effect is mediated, at least in part, at a pretranslational level.

binding

binding

expression

IL-2 receptor alpha

expressed

expressed

beta

expression

induces

binding

activation

activation

IL-2 receptor (IL-2R) alpha chain

increase

expression

increased

abundance of mRNAs

detect

abundance of mRNAs

c-Rel

abundance of mRNAs


Input:
Molecular and cellular analysis of human immunodeficiency virus-induced apoptosis in lymphoblastoid T-cell-line-expressing wild-type and mutated CD4 receptors.
We have previously shown that the presence of the CD4 cytoplasmic tail is critical for human immunodeficiency virus (HIV)-induced apoptosis (J.Corbeil, M.Tremblay, and D.D.Richman, J.Exp.Med.183:39-48, 1996). We have pursued our investigation of the role of the CD4 transduction pathway in HIV-induced apoptosis. To do this, wild-type and mutant forms of the CD4 cytoplasmic tail were stably expressed in the lymphoblastoid T-cell line A2.01. Apoptosis was prevented when CD4 truncated at residue 402 was expressed; however, cells expressing mutated receptors that do not associate with p56(lck) (mutated at the dicysteine motif and truncated at residue 418) but which conserved proximal domains of the cytoplasmic tail underwent apoptosis like wild- type CD4. The differences between wild-type and mutated receptors in the induction of apoptosis were not related to levels of p56(lck) or NF- kappaB activation. Initial signaling through the CD4 receptor played a major role in the sensitization of HIV-infected T cells to undergo apoptosis. Incubation of HIV-infected cells with monoclonal antibody (MAb) 13B8-2, which binds to CD4 in a region critical for dimerization of the receptor, prevented apoptosis without inhibiting HIV replication. Moreover, the apoptotic process was not related to Fas-Fas ligand interaction; however, an antagonistic anti-Fas MAb (ZB-4) enhanced apoptosis in HIV-infected cells without inducing apoptosis in uninfected cells. These observations demonstrate that CD4 signaling mediates HIV-induced apoptosis by a mechanism independent of Fas-Fas ligand interaction, does not require p56(lck) signaling, and may involve a critical region for CD4 dimerization.

associate

binds

interaction

Fas

Fas ligand

interaction

Fas

Fas ligand

dimerization

expressing

CD4 receptor

expressed

expressed

expressing


Input:
NF-kappaB only partially mediates Epstein-Barr virus latent membrane protein 1 activation of B cells.
The latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is required for EBV-induced immortalization of human B cells and causes tumorigenic transformation of cell lines. LMP1 expression induces phenotypic changes resembling B cell activation, such as cell size increase and up-regulation of cell surface activation markers. LMP1 contains two domains that activate the transcription factor NF-kappaB, one through interactions with TRAF proteins and the other with the TRADD protein. The purpose of the present study was to investigate the importance of NF-kappaB induction in the up-regulation of the B cell activation markers ICAM-1 and CD71 by LMP1. This study shows that expression of LMP1 activates transcription from p50/p65- and c-Rel- responsive promoters, and that this activity can be completely inhibited by expression of a dominant inhibitory IkappaB mutant. ICAM-1 and CD71 are nevertheless up-regulated by LMP1 in primary B cells and cell lines expressing the dominant IkappaB. Furthermore, LMP1-induced cell size increase of primary B cells was unaffected by IkappaB expression. It was concluded that even when LMP1 is unable to activate NF-kappaB, it is still capable of inducing certain characteristics of activated B cells, strongly suggesting that LMP1 can also activate cells independently of NF-kappaB.

expression

expression

up-regulation

up-regulation

up-regulated

up-regulated

importance

up-regulation


Input:
Relationship between IkappaBalpha constitutive expression, TNFalpha synthesis, and apoptosis in EBV-infected lymphoblastoid cells.
In order to understand the role of NF-kappaB in EBV transformation we have established stably transfected IkappaBalpha into lymphoblastoid cells. Two clones were obtained in which the loss of NF-kappaB binding activity correlated with the constitutive expression of the transgenic IkappaBalpha. Protein latency expression was determined by immunocytochemistry. Expression of surface markers, intracytoplasmic content of cytokines cell cycle analysis after BrdU incorporation and DNA staining with propidium iodide were studied by flow cytometry. Percentage of apoptotic cells was determined by in-situ labelling of DNA strand breaks. No significative changes in EBV latency nor in cell surface marker expression was found. In contrast, intracytoplasmic TNFalpha levels were strongly reduced in transfected clones. Furthermore, 30% of IkappaBalpha transfected cells were apoptotic after 8 h of TNFalpha treatment. This correlated with a strong reduction of BrdU incorporation after 24 h of TNFalpha treatment. No effect was seen with non transfected cells or with cells transfected with a control plasmid. Our results suggest that the TNFalpha gene could be one of the targets of NF-kappaB in EBV infected cells and that NF-kappaB protects EBV-infected cells from apoptosis induced by TNFalpha, which may favour the proliferative effect of this cytokine.

expression

IkappaBalpha

synthesis

TNFalpha

stably transfected

IkappaBalpha

constitutive expression

IkappaBalpha

transfected

IkappaBalpha

reduced

TNFalpha

stably transfected

stably transfected

transfected

transfected

targets

TNFalpha


Input:
Uncoupling activation-dependent HS1 phosphorylation from nuclear factor of activated T cells transcriptional activation in Jurkat T cells: differential signaling through CD3 and the costimulatory receptors CD2 and CD28.
CD3, CD2, and CD28 are functionally distinct receptors on T lymphocytes. Engagement of any of these receptors induces the rapid tyrosine phosphorylation of a shared group of intracellular signaling proteins, including Vav, Cbl, p85 phosphoinositide 3-kinase, and the Src family kinases Lck and Fyn. Ligation of CD3 also induces the tyrosine phosphorylation of HS1, a 75-kDa hematopoietic cell-specific intracellular signaling protein of unknown function. We have examined changes in HS1 phosphorylation after differential stimulation of CD3, CD2, and CD28 to elucidate its role in T cells and to further delineate the signaling pathways recruited by these receptors. Unlike ligation of CD3, stimulation with anti-CD28 mAb or CHO cells expressing the CD28 ligands CD80 or CD86 did not lead to tyrosine phosphorylation of HS1 in Jurkat T cells. Additionally, no tyrosine phosphorylation of HS1 was induced by mitogenic pairs of anti-CD2 mAbs capable of activating the transcription factor NFAT (nuclear factor of activated T cells). Costimulation through CD28 and/or CD2 did not modulate the CD3-dependent phosphorylation of HS1. In vivo studies indicated that CD3-induced HSI phosphorylation was dependent upon both the Src family tyrosine kinase Lck and the tyrosine phosphatase CD45, did not require MEK1 kinase activity, and was regulated by protein kinase C activation. Thus, although CD3, CD28, and CD2 activate many of the same signaling molecules, they differed in their capacity to induce the tyrosine phosphorylation of HSI. Furthermore, activation-dependent tyrosine phosphorylation of HS1 was not required for NFAT transcriptional activation.

Engagement

Engagement

ligands

ligands

expressing

expressing

phosphorylation

phosphorylation

tyrosine

Vav

phosphorylation

tyrosine

Cbl

phosphorylation

tyrosine

Lck

phosphorylation

tyrosine

Fyn

phosphorylation

tyrosine

phosphorylation

phosphorylation

tyrosine

phosphorylation

tyrosine

phosphorylation

phosphorylation

HSI

phosphorylation

tyrosine

HSI

phosphorylation

tyrosine

dependent

phosphorylation

induces

Engagement

phosphorylation

induces

phosphorylation

stimulation

stimulation

lead

phosphorylation

induced

phosphorylation

dependent

phosphorylation

induced

phosphorylation

dependent

induced

Lck

dependent

induced

require

induced

induce

phosphorylation

induce

phosphorylation

dependent

phosphorylation

changes

phosphorylation

stimulation

modulate

dependent

regulated

induced


Input:
IL-7 reconstitutes multiple aspects of v-Abl-mediated signaling.
The mechanism by which early lymphoid cells are selectively transformed by v-Abl is currently unknown. Previous studies have shown constitutive activation of IL-4 and IL-7 signaling pathways, as measured by activation of Janus protein kinase (JAK)1, JAK3, STAT5, and STAT6, in pre-B cells transformed by v-Abl. To determine whether activation of these cytokine signaling pathways by v-Abl is important in the cellular events induced by the Abelson murine leukemia virus, the effects of IL-4 and IL-7 on pre-B cells transformed with a temperature-sensitive v-Abl mutant were examined. Whereas IL-4 had little or no effect, IL-7 delayed both the apoptosis and cell cycle arrest that occur upon v-Abl kinase inactivation. IL-7 also delayed the decreases in the levels of c-Myc, Bcl-2, and Bcl-xL that occur upon loss of v-Abl kinase activity. IL-7 did not maintain v-Abl-mediated differentiation arrest of the pre-B cells, as activation of NF-kappaB and RAG gene transcription was unaffected by IL-7. These results identify a potential role for IL-7 signaling pathways in transformation by v-Abl while demonstrating that a combination of IL-4 and IL-7 signaling cannot substitute for an active v-Abl kinase in transformed pre-B cells.

delayed

decreases

decreases

c-Myc

decreases

decreases

Bcl-xL

reconstitutes

signaling

signaling

v-Abl

activation

Janus protein kinase (JAK)1

activation

activation


Input:
p21ras initiates Rac-1 but not phosphatidyl inositol 3 kinase/PKB, mediated signaling pathways in T lymphocytes.
p21ras is activated by the T cell antigen receptor (TCR) and then co-ordinates important signaling pathways for T lymphocyte activation. Effector pathways for this guanine nucleotide binding protein in T cells are mediated by the serine/threonine kinase Raf-1 and the Ras-related GTPase Rac-1. In fibroblasts, an important effector for the Ras oncogene is Phosphatidylinositol 3-kinase (PtdIns 3-kinase). Activation of this lipid kinase is able to induce critical Rac-1 signaling pathways and can couple p21ras to cell survival mechanisms via the serine/threonine kinase Akt/PKB. The role of PtdIns 3-kinase in Ras signaling in T cells has not been explored. In the present study, we examined the ability of PtdIns 3-kinase to initiate the Rac-1 signaling pathways important for T cell activation. We also examined the possibility that Akt/PKB is regulated by Ras signaling pathways in T lymphocytes. The results show that Ras can initiate a Rac-1 mediated pathway that regulates the transcriptional function of AP-1 complexes. PtdIns 3-kinase signals cannot mimic p21ras and induce the Rac mediated responses of AP-1 transcriptional activation. Moreover, neither TCR or Ras activation of AP-1 is dependent on PtdIns 3-kinase. PKB is activated in response to triggering of the T cell antigen receptor; PtdIns 3-kinase activity is both required and sufficient for this TCR response. In contrast, p21ras signals are unable to induce Akt/PKB activity in T cell nor is Ras function required for Akt/PKB activation in response to the TCR. The present data thus highlight that PtdIns 3-kinase and Akt/PKB are not universal Ras effector molecules. Ras can initiate Rac-1 regulated signaling pathways in the context of T cell antigen receptor function independently of PtdIns 3-kinase activity.

activated

mediated

Effector pathways

mediated

Effector pathways

couple

Akt

activated

PKB

required and sufficient

activated

induce

Akt

required

activation

activation

Akt

Effector pathways

regulated

Akt


Input:
Signal transduction abnormalities in T lymphocytes from patients with advanced renal carcinoma: clinical relevance and effects of cytokine therapy.
Studies have demonstrated abnormalities of the CD3/T-cell antigen receptor (TCR) and pathways of signal transduction in T lymphocytes from animals and patients with advanced malignancy. Diminished expression of TCRzeta and p56(lck) that are associated with the TCR and reduced nuclear localization of RelA containing nuclear factor kappaB (NFkappaB) complexes have been noted. These defects have been described in T cells from patients with malignant melanoma, renal cell carcinoma (RCC), ovarian cancer, and colorectal cancer. Preliminary observations also indicate possible correlation with clinical variables such as stage in selected instances. To further characterize altered expression of TCRzeta, p56(lck), and impaired activation of NFkappaB, T lymphocytes were obtained from 65 patients with RCC, the majority of whom were receiving combination cytokine therapy [interleukin (IL)-2, IFN alpha-containing regimens] and 37 control individuals. In 29 of these patients, levels of TCRzeta and p56(lck) were determined by Western blots of T-cell lysates and semiquantitated using densitometry. Relative levels were then correlated with a series of clinical variables including response to therapy, performance status, survival, disease sites, age, and others. In another group of 28 patients (three individuals from the first group), the frequency of abnormal NFkappaB activation was studied using electrophoretic mobility shift assays after activation of T cells with phorbol myristate acetate/ionomycin or anti-CD3 monoclonal antibody. Changes in these signaling molecules during cytokine treatment were also investigated. TCRzeta and p56(lck) were detected in the peripheral blood T cells in 27 of 29 patients, and overall, reduced levels were noted visually in 12 of 29 (41%) and 13 of 29 (45%) individuals, respectively. When levels were semiquantitated using densitometry, significant decreases of TCRzeta (P = 0.029) and p56(lck) (P = 0.029) but not CD3epsilon (P = 0.131), compared with control levels, were found. In patients treated with IL-2/IFN alpha-based therapy, relative levels of TCRzeta increased significantly (P = 0.002) on day 15 of cycle one compared with the baseline. Correlations of TCRzeta or p56(lck) levels with response or disease variables, except for lower TCRzeta levels (P < 0.001) in the presence of bone metastases, were not found. Abnormal NFkappaB activation after stimulation with phorbol myristate acetate/ionomycin and/or anti-CD3 monoclonal antibody was found in 59% of patients (17 of 28) and was not accounted for by the advanced age of the study cohort. Activation of NFkappaB in peripheral blood T cells was inducible during cytokine therapy in four of six individuals who displayed impaired NFkappaB activity prior to therapy. Moreover, impaired activation of NFkappaB does not appear linked to a reduction of TCRzeta expression, because in five patients, normal TCRzeta levels were present although kappaB binding was not inducible. In the majority of patients with advanced RCC, peripheral blood T cells express TCRzeta and p56(lck), and in a subset, reduced levels of these TCRzeta associated molecules are seen that may increase during cytokine-based therapy. Abnormal activation of NFkappaB is also present in >50% of patients and may also revert to normal during IL-2/IFN alpha-based treatment. This alteration in NFkappaB activation occurred in the presence of normal expression of TCRzeta-associated signaling elements. The clinical significance of these findings remains unclear.

associated

TCRzeta

associated

expression

TCRzeta

expression

expression

TCRzeta

expression

levels

TCRzeta

levels

detected

TCRzeta

detected

expression

TCRzeta

express

TCRzeta

express

localization

nuclear

RelA

Diminished

expression

reduced

localization

defects

Diminished

defects

reduced

altered

expression

reduced

detected

decreases

TCRzeta

decreases

decreases

lower

TCRzeta

reduction

expression

reduced levels

TCRzeta

reduced levels

Changes

TCRzeta

Changes

increased

TCRzeta

increase

reduced levels


Input:
Transcriptional regulation of lysosomal acid lipase in differentiating monocytes is mediated by transcription factors Sp1 and AP-2.
Human lysosomal acid lipase (LAL) is a hydrolase required for the cleavage of cholesteryl esters and triglycerides derived from plasma lipoproteins. It is shown here that during monocyte to macrophage differentiation, the expression of LAL-mRNA is induced. This induction is dependent on protein kinase C activity and protein synthesis. The cell type-specific increase in LAL expression is further investigated in the THP-1 cell line with respect to transcriptional regulation. The human monocytic leukemia cell line THP-1 differentiates into macrophage-like cells when treated with phorbol esters. In order to determine the cis-acting elements necessary for both basal and phorbol 12-myristate-13 acetate (PMA)-enhanced promoter activity, we performed deletion analysis and reporter gene assays. A PMA responsive element has been identified between -182 bp and -107 bp upstream of the major transcription start site. Gel mobility shift assays demonstrated that binding of Sp1 and AP-2 to the LAL promoter is increased by PMA in THP-1 cells. Co-transfections with expression plasmids for Sp1 and AP-2 further emphasized the important role of these transcription factors in both basal and PMA-enhanced LAL expression. Our data suggest that differentiation dependent increase of lysosomal acid lipase (LAL) expression in THP-1 cells is mediated by a concerted action of Sp1 and AP-2.

binding

LAL

promoter

binding

LAL

promoter

expression

LAL

expression

LAL

expression

LAL

induced

expression

dependent

induced

increase

expression

increased

binding

enhanced

expression

increase

expression

Transcriptional regulation

lysosomal acid lipase

mediated

Transcriptional regulation

transcriptional regulation

LAL

important role

enhanced

important role

expression

dependent

increase

mediated

dependent

expression

LAL


Input:
A nongenomic mechanism for progesterone-mediated immunosuppression: inhibition of K+ channels, Ca2+ signaling, and gene expression in T lymphocytes.
The mechanism by which progesterone causes localized suppression of the immune response during pregnancy has remained elusive. Using human T lymphocytes and T cell lines, we show that progesterone, at concentrations found in the placenta, rapidly and reversibly blocks voltage-gated and calcium-activated K+ channels (KV and KCa, respectively), resulting in depolarization of the membrane potential. As a result, Ca2+ signaling and nuclear factor of activated T cells (NF-AT)-driven gene expression are inhibited. Progesterone acts distally to the initial steps of T cell receptor (TCR)-mediated signal transduction, since it blocks sustained Ca2+ signals after thapsigargin stimulation, as well as oscillatory Ca2+ signals, but not the Ca2+ transient after TCR stimulation. K+ channel blockade by progesterone is specific; other steroid hormones had little or no effect, although the progesterone antagonist RU 486 also blocked KV and KCa channels. Progesterone effectively blocked a broad spectrum of K+ channels, reducing both Kv1.3 and charybdotoxin-resistant components of KV current and KCa current in T cells, as well as blocking several cloned KV channels expressed in cell lines. Progesterone had little or no effect on a cloned voltage-gated Na+ channel, an inward rectifier K+ channel, or on lymphocyte Ca2+ and Cl- channels. We propose that direct inhibition of K+ channels in T cells by progesterone contributes to progesterone-induced immunosuppression.

reducing


Input:
Role of IKK1 and IKK2 in lipopolysaccharide signaling in human monocytic cells.
Mononuclear phagocytes play a major role in immune and inflammatory responses. Bacterial lipopolysaccharide (LPS) induces monocytes to express a variety of genes by activating the NF-kappaB/Rel transcription factor family. Recently, we have reported that the tumor necrosis factor and interleukin 1 signaling pathways activate two kinases, IKK1 and IKK2. Phosphorylation of the IkappaB cytoplasmic inhibitors, IkappaBalpha, IkappaBbeta, and IkappaBepsilon, by these kinases triggers proteolytic degradation and the release of NF-kappaB/Rel proteins into the nucleus. At present, the role of the IKKs in LPS signaling has not been investigated. Here, we report that LPS induces IKK activity in human monocytes and THP-1 monocytic cells. The kinetics of activation of kinase activity in monocytic cells are relatively slow with maximal activity observed at 60 min, which coincides with the degradation of IkappaBs and the nuclear translocation of NF-kappaB. In transfection experiments, overexpression of wild type IKK1, a dominant negative mutant IKK1 (K44M), or wild type IKK2 did not affect LPS-induced kappaB-dependent transcription in monocytic cells. In contrast, a dominant negative mutant of IKK2 inhibited LPS induction of kappaB-dependent transcription in a dose-dependent manner. These results indicate that LPS induction of kappaB-dependent gene expression in human monocytic cells requires activation of IKK2.

overexpression

overexpression

Phosphorylation

IkappaBalpha

Phosphorylation

IkappaBbeta

Phosphorylation

IkappaBepsilon

activate

activate

by

Phosphorylation

by

Phosphorylation

triggers

by

proteolytic degradation

overexpression

overexpression

activation

proteolytic degradation

IkappaBalpha

proteolytic degradation

IkappaBbeta

proteolytic degradation

IkappaBepsilon


Input:
Effects of overexpression of IL-1 receptor-associated kinase on NFkappaB activation, IL-2 production and stress-activated protein kinases in the murine T cell line EL4.
The association and activation of the IL-1 receptor-associated protein kinase (IRAK) to the IL-1 receptor complex is one of the earliest events detectable in IL-1 signal transduction. We generated permanent clones of the murine T cell line EL4 6.1 overexpressing human (h)IRAK to evaluate the role of this kinase in IL-1 signaling. Overexpression of hIRAK enhanced IL-1-stimulated activation of the transcription factor NFkappaB, whereas a truncated form (N-IRAK) specifically inhibited IL-1-dependent NFkappaB activity. In clones stably overexpressing hIRAK a weak constitutive activation of NFkappaB correlated with a low basal IL-2 production which was enhanced in an IL-1-dependent manner. Compared to the parental cell line the dose-response curve of IL-1-induced IL-2 production was shifted in both potency and efficacy. These results demonstrate that IRAK directly triggers NFkappaB-mediated gene expression in EL4 cells. Qualitatively different effects were observed for the IL-1-induced activation of stress-activated protein (SAP) kinases: permanent overexpression of IRAK did not affect the dose dependence but prolonged the kinetics of IL-1-induced activation of SAP kinases, suggesting that this signaling branch may be regulated by distinct mechanisms.

production

production

production

enhanced

production

induced

production

Effects

production


Input:
Decreased IL-12 production and Th1 cell development by acetyl salicylic acid-mediated inhibition of NF-kappaB.
IL-12 is a 75-kDa heterodimeric cytokine composed of two covalently linked p35 and p40 chains. This pro-inflammatory cytokine plays a prominent role in the development of Th1 cell-mediated immune responses. Th1 cell-mediated immune responses have been implicated in the pathogenesis of chronic inflammatory autoimmune diseases. Thus, IL-12 appears to be a critical factor in the generation and maintenance of chronic inflammatory conditions. In this study, we investigated the effects of a commonly prescribed anti-inflammatory drug, acetyl salicylic acid (ASA), on IL-12 production and Th1 cell development. ASA was found to inhibit secretion of the IL-12 heterodimer as well as p40 monomer by human monocytic cells. This was associated with the down-regulation of IL-12p40 mRNA expression. Analysis of the regulation of the p40 gene promoter revealed that ASA inhibited NF-kappaB activation and binding to the p40-kappaB site in the p40 promoter, leading to transcriptional repression of the p40 gene. Addition of ASA to an in vitro T helper cell differentiation system, at concentrations compatible with plasma levels reached during anti-inflammatory therapy, resulted in reduced development of Th1 cells. These results suggest that the inhibition of NF-kappaB activation by ASA leads to down-regulation of IL-12 production and inhibition of Th1 cell development.

secretion

inhibit

secretion

down-regulation

mRNA expression

leading to transcriptional repression

regulation

promoter

mRNA expression


Input:
Membrane-associated lymphotoxin on natural killer cells activates endothelial cells via an NF-kappaB-dependent pathway.
BACKGROUND: Inhibition of complement in small animal models of xenotransplantation has demonstrated graft infiltration with natural killer (NK) cells and monocytes associated with endothelial cell (EC) activation. We have previously demonstrated that human NK cells activate porcine EC in vitro, which results in adhesion molecule expression and cytokine secretion. In this study, we used the NK cell line NK92 to define the molecular and cellular basis of NK cell-mediated EC activation. METHODS: EC were transfected with either reporter constructs containing the luciferase gene driven either by E-selectin or interleukin (IL)-8 promoters or a synthetic NF-kappaB-dependent promoter. In addition, a dominant-negative mutant tumor necrosis factor receptor I (TNFRI) expression vector was co-transfected in inhibition studies. Forty-eight hours after transfection, EC were stimulated with NK cells or NK cell membrane extracts for 7 hr and activation was measured by a luciferase assay. RESULTS: Co-culture of NK cells with transfected EC enhanced E-selectin, IL-8, and NF-kappaB-dependent promoter activity. NK cell membrane extracts retained the capacity to activate EC and induced nuclear translocation of NF-kappaB (p50 and p65). Western blotting of NK cell and membrane extracts detected the presence of Lymphotoxin-alpha (LTalpha) but not tumor necrosis factor-alpha. Furthermore, LTalpha was secreted in NK:EC co-cultures. Co-transfection with dominant-negative mutant TNFRI inhibited EC activation by NK cell membrane extracts and by NK cells by 80% and 47%, respectively. The same pattern of inhibition was observed using anti-human LT sera. CONCLUSIONS: Human NK cell membrane-bound LT signals across species via TNFRI, leading to NF-kappaB nuclear translocation and transcription of E-selectin and IL-8, which results in EC activation. The discrepancy in the degree of inhibition by membrane extracts and NK cells with mutant TNFRI suggests that additional pathways are utilized by NK cells to activate EC.

co-transfected

tumor necrosis factor receptor I

presence

LTalpha

presence

tumor necrosis factor-alpha

Co-transfection

TNFRI

translocation

nuclear

translocation

nuclear

secreted

LTalpha

co-transfected

co-transfected

induced

translocation

Co-transfection

Co-transfection

leading

transcription

transcription

E-selectin

transcription


Input:
The involvement of multiple tumor necrosis factor receptor (TNFR)-associated factors in the signaling mechanisms of receptor activator of NF-kappaB, a member of the TNFR superfamily.
Receptor activator of NF-kappaB (RANK) is a recently identified member of the tumor necrosis factor receptor superfamily and is expressed on activated T cells and dendritic cells. Its cognate ligand (RANKL) plays significant roles in the activation of dendritic cell function and osteoclast differentiation. We demonstrate here the interaction of RANK with tumor necrosis factor receptor-associated factors (TRAFs) 1, 2, 3, 5, and 6 both in vitro and in cells. Mapping of the structural requirements for TRAF/RANK interaction revealed multiple TRAF binding sites clustered in two distinct domains in the RANK cytoplasmic tail. These TRAF binding domains were shown to be functionally important for the RANK-dependent induction of NF-kappaB and c-Jun NH2-terminal kinase activities. Site-directed mutagenesis demonstrated that these TRAF binding sites exhibited selective binding for different TRAF proteins. In particular, TRAF6 interacted with membrane-proximal determinants distinct from those binding TRAFs 1, 2, 3, and 5. When this membrane-proximal TRAF6 interaction domain was deleted, RANK-mediated NF-kappaB signaling was completely inhibited while c-Jun NH2-terminal kinase activation was partially inhibited. An NH2-terminal truncation mutant of TRAF6 inhibited RANKL-mediated NF-kappaB activation, but failed to affect constitutive signaling induced by receptor overexpression, revealing a selective role for TRAF6 in ligand-induced activation events.

interaction

RANK

tumor necrosis factor receptor-associated factors (TRAFs) 1

interaction

RANK

interaction

RANK

interaction

RANK

interaction

RANK

interaction

RANK

interacted

binding

TRAFs 1

binding

binding

binding

expressed

RANK