A Short ERAP2 That Binds IRAP Is Expressed in Macrophages Independently of Gene Variation.

The M1 zinc metalloproteases ERAP1, ERAP2, and IRAP play a role in HLA-I antigen presentation by refining the peptidome either in the ER (ERAP1 and ERAP2) or in the endosomes (IRAP). They have also been entrusted with other, although less defined, functions such as the regulation of the angiotensin system and blood pressure. In humans, ERAP1 and IRAP are commonly expressed. ERAP2 instead has evolved under balancing selection that maintains two haplotypes, one of which undergoing RNA splicing leading to nonsense-mediated decay and loss of protein. Hence, likewise in rodents, wherein the ERAP2 gene is missing, about a quarter of the human population does not express ERAP2. We report here that macrophages, but not monocytes or other mononuclear blood cells, express and secrete an ERAP2 shorter form independent of the haplotype. The generation of this "short" ERAP2 is due to an autocatalytic cleavage within a distinctive structural motif and requires an acidic micro-environment. Remarkably, ERAP2 "short" binds IRAP and the two molecules are co-expressed in the endosomes as well as in the cell membrane. Of note, the same phenomenon could be observed in some cancer cells. These data prompt us to reconsider the role of ERAP2, which might have been maintained in humans due to fulfilling a relevant function in its "short" form.

Phosphatidylinositol 4-phosphate 5-kinase α and Vav1 mutual cooperation in CD28-mediated actin remodeling and signaling functions.

Phosphatidylinositol 4,5-biphosphate (PIP2) is a cell membrane phosphoinositide crucial for cell signaling and activation. Indeed, PIP2 is a pivotal source for second messenger generation and controlling the activity of several proteins regulating cytoskeleton reorganization. Despite its critical role in T cell activation, the molecular mechanisms regulating PIP2 turnover remain largely unknown. In human primary CD4(+) T lymphocytes, we have recently demonstrated that CD28 costimulatory receptor is crucial for regulating PIP2 turnover by allowing the recruitment and activation of the lipid kinase phosphatidylinositol 4-phosphate 5-kinase (PIP5Kα). We also identified PIP5Kα as a key modulator of CD28 costimulatory signals leading to the efficient T cell activation. In this study, we extend these data by demonstrating that PIP5Kα recruitment and activation is essential for CD28-mediated cytoskeleton rearrangement necessary for organizing a complete signaling compartment leading to downstream signaling functions. We also identified Vav1 as the linker molecule that couples the C-terminal proline-rich motif of CD28 to the recruitment and activation of PIP5Kα, which in turn cooperates with Vav1 in regulating actin polymerization and CD28 signaling functions.

Phosphatidylinositol 4-phosphate 5-kinase α activation critically contributes to CD28-dependent signaling responses.

CD28 is one of the most relevant costimulatory receptors that deliver both TCR-dependent and TCR-independent signals regulating a wide range of signaling pathways crucial for cytokine and chemokine gene expressions, T cell survival, and proliferation. Most of the CD28-dependent signaling functions are initiated by the recruitment and activation of class IA PI3Ks, which catalyze the conversion of phosphatidylinositol 4,5-biphosphate (PIP2) into phosphatidylinositol 3,4,5-triphosphate, thus generating the docking sites for key signaling proteins. Hence, PIP2 is a crucial substrate in driving the PI3K downstream signaling pathways, and PIP2 turnover may be an essential regulatory step to ensure the activation of PI3K following CD28 engagement. Despite some data evidence that CD28 augments TCR-induced turnover of PIP2, its direct role in regulating PIP2 metabolism has never been assessed. In this study, we show that CD28 regulates PIP2 turnover by recruiting and activating phosphatidylinositol 4-phosphate 5-kinases α (PIP5Kα) in human primary CD4(+) T lymphocytes. This event leads to the neosynthesis of PIP2 and to its consumption by CD28-activated PI3K. We also evidenced that PIP5Kα activation is required for both CD28 unique signals regulating IL-8 gene expression as well as for CD28/TCR-induced Ca(2+) mobilization, NF-AT nuclear translocation, and IL-2 gene transcription. Our findings elucidate a novel mechanism that involves PIP5Kα as a key modulator of CD28 costimulatory signals.

Characterization of a proteasome and TAP-independent presentation of intracellular epitopes by HLA-B27 molecules.

Nascent HLA-class I molecules are stabilized by proteasome-derived peptides in the ER and the new complexes proceed to the cell surface through the post-ER vesicles. It has been shown, however, that less stable complexes can exchange peptides in the Trans Golgi Network (TGN). HLA-B27 are the most studied HLA-class I molecules due to their association with Ankylosing Spondylitis (AS). Chimeric proteins driven by TAT of HIV have been exploited by us to deliver viral epitopes, whose cross-presentation by the HLA-B27 molecules was proteasome and TAP-independent and not restricted to Antigen-Presenting Cells (APC). Here, using these chimeric proteins as epitope suppliers, we compared with each other and with the HLA-A2 molecules, the two HLA-B*2705 and B*2709 alleles differing at residue 116 (D116H) and differentially associated with AS. We found that the antigen presentation by the two HLA-B27 molecules was proteasome-, TAP-, and APC-independent whereas the presentation by the HLA-A2 molecules required proteasome, TAP and professional APC. Assuming that such difference could be due to the unpaired, highly reactive Cys-67 distinguishing the HLA-B27 molecules, C67S mutants in HLA-B*2705 and B*2709 and V67C mutant in HLA-A*0201 were also analyzed. The results showed that this mutation did not influence the HLA-A2-restricted antigen presentation while it drastically affected the HLA-B27-restricted presentation with, however, remarkable differences between B*2705 and B*2709. The data, together with the occurrence on the cell surface of unfolded molecules in the case of C67S-B*2705 mutant but not in that of C67S-B*2709 mutant, indicates that Cys-67 has a more critical role in stabilizing the B*2705 rather than the B*2709 complexes.

Corrigendum: Bivalent binding of staphylococcal superantigens to the TCR and CD28 triggers inflammatory signals independently of antigen presenting cells.

[This corrects the article DOI: 10.3389/fimmu.2023.1170821.].

Bivalent binding of staphylococcal superantigens to the TCR and CD28 triggers inflammatory signals independently of antigen presenting cells.

Staphylococcus aureus superantigens (SAgs) such as staphylococcal enterotoxin A (SEA) and B (SEB) are potent toxins stimulating T cells to produce high levels of inflammatory cytokines, thus causing toxic shock and sepsis. Here we used a recently released artificial intelligence-based algorithm to better elucidate the interaction between staphylococcal SAgs and their ligands on T cells, the TCR and CD28. The obtained computational models together with functional data show that SEB and SEA are able to bind to the TCR and CD28 stimulating T cells to activate inflammatory signals independently of MHC class II- and B7-expressing antigen presenting cells. These data reveal a novel mode of action of staphylococcal SAgs. By binding to the TCR and CD28 in a bivalent way, staphylococcal SAgs trigger both the early and late signalling events, which lead to massive inflammatory cytokine secretion.

CD28 costimulation regulates FOXP3 in a RelA/NF-κB-dependent mechanism.

The molecular mechanisms whereby CD28 alone or associated with TCR can regulate FOXP3 expression are not understood, although the importance of CD28 as a pivotal regulator of CD4(+) CD25(+) FOXP3(+) T cells is well recognized. We previously demonstrated that unique CD28-induced, NF-κB-dependent signals were sufficient to activate FOXP3 transcription in human CD4(+) CD25(-) T cells; however, the exact mechanisms are currently unknown. In this study, we have identified novel κB-binding sites on FOXP3 gene and demonstrated that CD28 signals mediated FOXP3 trans activation by nuclear translocation of RelA/NF-κB and not of c-Rel. The occupancy of FOXP3 κB-binding sites by RelA dimers that correlated with histone acetylation and recruitment of Pol II were required both to initiate FOXP3 transcription and to control the promoter occupancy by NFAT. Interestingly, knockdown of RelA in CD4(+) CD25(-) T cells stimulated through TCR and CD28 significantly affected FOXP3 expression, confirming that also the transcriptional activation of FOXP3 gene by TCR in the presence of CD28-costimulatory signals is RelA-dependent. In conclusion, these data suggest a new mechanism by which FOXP3 is activated and supports the critical role of CD28 in the regulation of peripheral tolerance.

Binding of Staphylococcal Enterotoxin B (SEB) to B7 Receptors Triggers TCR- and CD28-Mediated Inflammatory Signals in the Absence of MHC Class II Molecules.

The inflammatory activity of staphylococcal enterotoxin B (SEB) relies on its capacity to trigger polyclonal T-cell activation by binding both T-cell receptor (TCR) and costimulatory receptor CD28 on T cells and MHC class II and B7 molecules on antigen presenting cells (APC). Previous studies highlighted that SEB may bind TCR and CD28 molecules independently of MHC class II, yet the relative contribution of these interactions to the pro-inflammatory function of SEB remained unclear. Here, we show that binding to MHC class II is dispensable for the inflammatory activity of SEB, whereas binding to TCR, CD28 and B7 molecules is pivotal, in both human primary T cells and Jurkat T cell lines. In particular, our finding is that binding of SEB to B7 molecules suffices to trigger both TCR- and CD28-mediated inflammatory signalling. We also provide evidence that, by strengthening the interaction between CD28 and B7, SEB favours the recruitment of the TCR into the immunological synapse, thus inducing lethal inflammatory signalling.

CD28 Autonomous Signaling Orchestrates IL-22 Expression and IL-22-Regulated Epithelial Barrier Functions in Human T Lymphocytes.

IL-22 is a member of the IL-10 cytokine family involved in host protection against extracellular pathogens, by promoting epithelial cell regeneration and barrier functions. Dysregulation of IL-22 production has also frequently been observed in acute respiratory distress syndrome (ARDS) and several chronic inflammatory and autoimmune diseases. We have previously described that human CD28, a crucial co-stimulatory receptor necessary for full T cell activation, is also able to act as a TCR independent signaling receptor and to induce the expression of IL-17A and inflammatory cytokines related to Th17 cells, which together with Th22 cells represent the main cellular source of IL-22. Here we characterized the role of CD28 autonomous signaling in regulating IL-22 expression in human CD4+ T cells. We show that CD28 stimulation in the absence of TCR strongly up-regulates IL-22 gene expression and secretion. As recently observed for IL-17A, we also found that CD28-mediated regulation of IL-22 transcription requires the cooperative activities of both IL-6-activated STAT3 and RelA/NF-κB transcription factors. CD28-mediated IL-22 production also promotes the barrier functions of epithelial cells by inducing mucin and metalloproteases expression. Finally, by using specific inhibitory drugs, we also identified CD28-associated class 1A phosphatidylinositol 3-kinase (PI3K) as a pivotal mediator of CD28-mediated IL-22 expression and IL-22-dependent epithelial cell barrier functions.

CD28 Individual Signaling Up-regulates Human IL-17A Expression by Promoting the Recruitment of RelA/NF-κB and STAT3 Transcription Factors on the Proximal Promoter.

CD28 is an important co-stimulatory receptor for T lymphocytes that, in humans, delivers TCR-independent signal leading to the up-regulation of pro-inflammatory cytokines. We have recently reported that CD28 autonomous signaling induces the expression of IL-17A in peripheral CD4+ T lymphocytes from healthy donors, multiple sclerosis, and type 1 diabetes patients. Due to the relevance of IL-17A in the pathophysiology of several inflammatory and autoimmune diseases, we characterized the mechanisms and signaling mediators responsible for CD28-induced IL-17A expression. Here we show that CD28-mediated up-regulation of IL-17A gene expression depends on RelA/NF-κB and IL-6-associated STAT3 transcriptions factors. In particular, we found that CD28-activated RelA/NF-κB induces the expression of IL-6 that, in a positive feedback loop, mediates the activation and nuclear translocation of tyrosine phosphorylated STAT3 (pSTAT3). pSTAT3 in turn cooperates with RelA/NF-κB by binding specific sequences within the proximal promoter of human IL-17A gene, thus inducing its expression. Finally, by using specific inhibitory drugs, we also identified class 1A phosphatidylinositol 3-kinase (PI3K) as a critical upstream regulator of CD28-mediated RelA/NF-κB and STAT3 recruitments and trans-activation of IL-17A promoter. Our findings reveal a novel mechanism by which human CD28 may amplify IL-17A expression in human T lymphocytes and provide biological bases for immunotherapeutic approaches targeting CD28-associated class 1A PI3K to dampen IL-17A-mediated inflammatory response in autoimmune/inflammatory disorders.

A non-conserved amino acid variant regulates differential signalling between human and mouse CD28.

CD28 superagonistic antibodies (CD28SAb) can preferentially activate and expand immunosuppressive regulatory T cells (Treg) in mice. However, pre-clinical trials assessing CD28SAbs for the therapy of autoimmune diseases reveal severe systemic inflammatory response syndrome in humans, thereby implying the existence of distinct signalling abilities between human and mouse CD28. Here, we show that a single amino acid variant within the C-terminal proline-rich motif of human and mouse CD28 (P212 in human vs. A210 in mouse) regulates CD28-induced NF-κB activation and pro-inflammatory cytokine gene expression. Moreover, this Y209APP212 sequence in humans is crucial for the association of CD28 with the Nck adaptor protein for actin cytoskeleton reorganisation events necessary for CD28 autonomous signalling. This study thus unveils different outcomes between human and mouse CD28 signalling to underscore the importance of species difference when transferring results from preclinical models to the bedside.

miR-135a Regulates Synaptic Transmission and Anxiety-Like Behavior in Amygdala.

MicroRNAs are a class of non-coding RNAs with a growing relevance in the regulation of gene expression related to brain function and plasticity. They have the potential to orchestrate complex phenomena, such as the neuronal response to homeostatic challenges. We previously demonstrated the involvement of miR-135a in the regulation of early stress response. In the present study, we examine the role of miR-135a in stress-related behavior. We show that the knockdown (KD) of miR-135a in the mouse amygdala induces an increase in anxiety-like behavior. Consistently with behavioral studies, electrophysiological experiments in acute brain slices indicate an increase of amygdala spontaneous excitatory postsynaptic currents, as a result of miR-135a KD. Furthermore, we presented direct evidences, by in vitro assays and in vivo miRNA overexpression in the amygdala, that two key regulators of synaptic vesicle fusion, complexin-1 and complexin-2, are direct targets of miR-135a. In vitro analysis of miniature excitatory postsynaptic currents on miR-135a KD primary neurons indicates unpaired quantal excitatory neurotransmission. Finally, increased levels of complexin-1 and complexin-2 proteins were detected in the mouse amygdala after acute stress, accordingly to the previously observed stress-induced miR-135a downregulation. Overall, our results unravel a previously unknown miRNA-dependent mechanism in the amygdala for regulating anxiety-like behavior, providing evidences of a physiological role of miR-135a in the modulation of presynaptic mechanisms of glutamatergic neurotransmission.

Transcriptional regulation of IL-8 by Staphylococcus aureus in human conjunctival cells involves activation of AP-1.

PURPOSE: To identify signal transduction pathways involved in interleukin (IL)-8 expression by human conjunctival cells challenged with Staphylococcus aureus. METHODS: Conjunctival cells were cultured in the presence of live or heat-killed S. aureus. IL-8 protein and mRNA were determined by ELISA and RT-PCR, respectively. Activation of mitogen-activated protein kinases (MAPKs) and NF-kappaB was analyzed by Western blot analysis with phosphospecific antibodies. Conjunctival cells were transfected with wild-type (wt) or mutated IL-8 promoters (IL-8-97, lacking the AP-1 site; IL-8-97 mutant C/EBP; IL-8-97 mutant NF-kappaB; IL-8/AP-1 double mutant for C/EBP and NF-kappaB) or c-Jun-NH(2)-terminal kinase (JNK)-responsive GAL-c-Jun. In further experiments, cells were cotransfected with wt IL-8 promoter and expression plasmids for p38MAPK-responsive C/EBP homologous protein (CHOP) or wt or dominant negative transactivation domain mutant (TAM-67) c-Jun. A protein-DNA binding study was performed by electrophoretic mobility shift assay (EMSA), to identify the transcription factors bound to the IL-8 promoter. RESULTS: S. aureus induced significant IL-8 expression and synthesis in human conjunctival epithelial cells by activating c-Jun phosphorylation and transactivation potential via JNK. The IL-8 promoter activation was NF-kappaB- and p38MAPK-independent. Transfection and EMSA experiments suggested that only AP-1 transcription factors were necessary for optimal IL-8 expression. CONCLUSIONS: Human conjunctival epithelial cells possess the ability to respond to Gram-positive S. aureus and to activate the innate immune response by the IL-8 gene expression. These results are the first to delineate the transcription factors involved in S. aureus-induced IL-8 release by conjunctival epithelium.

Estrogens and progesterone promote persistent CCND1 gene activation during G1 by inducing transcriptional derepression via c-Jun/c-Fos/estrogen receptor (progesterone receptor) complex assembly to a distal regulatory element and recruitment of cyclin D1 to its own gene promoter.

Transcriptional activation of the cyclin D1 gene (CCND1) plays a pivotal role in G(1)-phase progression, which is thereby controlled by multiple regulatory factors, including nuclear receptors (NRs). Appropriate CCND1 gene activity is essential for normal development and physiology of the mammary gland, where it is regulated by ovarian steroids through a mechanism(s) that is not fully elucidated. We report here that CCND1 promoter activation by estrogens in human breast cancer cells is mediated by recruitment of a c-Jun/c-Fos/estrogen receptor alpha complex to the tetradecanoyl phorbol acetate-responsive element of the gene, together with Oct-1 to a site immediately adjacent. This process coincides with the release from the same DNA region of a transcriptional repressor complex including Yin-Yang 1 (YY1) and histone deacetylase 1 and is sufficient to induce the assembly of the basal transcription machinery on the promoter and to lead to initial cyclin D1 accumulation in the cell. Later on in estrogen stimulation, the cyclin D1/Cdk4 holoenzyme associates with the CCND1 promoter, where E2F and pRb can also be found, contributing to the long-lasting gene enhancement required to drive G(1)-phase completion. Interestingly, progesterone triggers similar regulatory events through its own NRs, suggesting that the gene regulation cascade described here represents a crossroad for the transcriptional control of G(1)-phase progression by different classes of NRs.

Distinct signaling pathways mediate stimulation of cell cycle progression and prevention of apoptotic cell death by estrogen in rat pituitary tumor PR1 cells.

Estrogens control cell growth and viability in target cells via an interplay of genomic and extragenomic pathways not yet elucidated. Here, we show evidence that cell proliferation and survival are differentially regulated by estrogen in rat pituitary tumor PR1 cells. Pico- to femtomolar concentrations of 17beta-estradiol (E2) are sufficient to foster PR1 cell proliferation, whereas nanomolar concentrations of the same are needed to prevent cell death that occurs at a high rate in these cells in the absence of hormone. Activation of endogenous (PRL) or transfected estrogen-responsive genes occurs at the same, higher concentrations of E2 required to promote cell survival, whereas stimulation of cyclin D3 expression and DNA synthesis occur at lower E2 concentrations. Similarly, the pure antiestrogen ICI 182,780 inhibits estrogen response element-dependent trans-activation and cell death more effectively than cyclin-cdk activity, G1-S transition, or DNA synthesis rate. In antiestrogen-treated and/or estrogen-deprived cells, death is due predominantly to apoptosis. Estrogen-induced cell survival, but not E2-dependent cell cycle progression, can be prevented by an inhibitor of c-Src kinase or by blockade of the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase signaling pathway. These data indicate the coexistence of two distinguishable estrogen signaling pathways in PR1 cells, characterized by different functions and sensitivity to hormones and antihormones.

Forkhead transcription factor FOXP3 upregulates CD25 expression through cooperation with RelA/NF-κB.

Considerable evidence supports the prediction that CD25 is directly regulated by the forkhead transcription factor FOXP3. However, given that CD25 is normally upregulated in activated T cells, regardless of whether they express FOXP3, this issue has still to be definitively demonstrated. Here we describe that FOXP3, induced by CD28 signals in human CD4(+)CD25(-) T lymphocytes, synergizes with RelA on a regulatory region of Cd25 promoter to mediate the transcriptional activation of Cd25 gene. We found that a striking feature of this regulatory region is the presence of a κB site and of two tandem copies of a non-consensus FOXP3 binding site separated at 5' ends by 19 nucleotides that allow FOXP3 and RelA binding to DNA and their physical interaction. The occupancy of the two FOXP3 binding sites in conjunction with RelA binding site occupancy allows FOXP3 to function as a positive activator of Cd25 gene. Indeed mutations of both FOXP3 binding sites such as mutation of κB site on Cd25 promoter abolished FOXP3 activatory functions. Moreover, FOXP3 mutation ΔE251, that compromises FOXP3 homotypic interactions, failed to trans activate Cd25 promoter, suggesting that both FOXP3 DNA binding and dimerization are required to trans activate Cd25 promoter. These findings identify a novel mechanism by which RelA and FOXP3 cooperate to mediate transcriptional regulation of target genes and characterize a region on Cd25 promoter where FOXP3 dimer could bridge intramolecularly two DNA sites and trans activate Cd25 gene.

Interaction pattern of Arg 62 in the A-pocket of differentially disease-associated HLA-B27 subtypes suggests distinct TCR binding modes.

The single amino acid replacement Asp116His distinguishes the two subtypes HLA-B*2705 and HLA-B*2709 which are, respectively, associated and non-associated with Ankylosing Spondylitis, an autoimmune chronic inflammatory disease. The reason for this differential association is so far poorly understood and might be related to subtype-specific HLA:peptide conformations as well as to subtype/peptide-dependent dynamical properties on the nanoscale. Here, we combine functional experiments with extensive molecular dynamics simulations to investigate the molecular dynamics and function of the conserved Arg62 of the α1-helix for both B27 subtypes in complex with the self-peptides pVIPR (RRKWRRWHL) and TIS (RRLPIFSRL), and the viral peptides pLMP2 (RRRWRRLTV) and NPflu (SRYWAIRTR). Simulations of HLA:peptide systems suggest that peptide-stabilizing interactions of the Arg62 residue observed in crystal structures are metastable for both B27 subtypes under physiological conditions, rendering this arginine solvent-exposed and, probably, a key residue for TCR interaction more than peptide-binding. This view is supported by functional experiments with conservative (R62K) and non-conservative (R62A) B*2705 and B*2709 mutants that showed an overall reduction in their capability to present peptides to CD8+ T cells. Moreover, major subtype-dependent differences in the peptide recognition suggest distinct TCR binding modes for the B*2705 versus the B*2709 subtype.

A novel association between filamin A and NF-κB inducing kinase couples CD28 to inhibitor of NF-κB kinase α and NF-κB activation.

CD28 costimulatory molecule plays a critical role in the activation of NF-κB. Indeed, while stimulation of T cells with either professional APCs or anti-TCR plus anti-CD28 antibodies efficiently activates NF-κB, TCR alone fails to do that. Moreover, CD28 stimulation by B7 in the absence of TCR may activate IκB kinase α (IKKα) and a non-canonical NF-κB2-like pathway, in human primary CD4(+) T cells. Despite its functional relevance in NF-κB activation, the molecules connecting autonomous CD28-mediated signals to IKKα and NF-κB activation remain still unknown. In searching for specific upstream activators linking CD28 to the IKKα/NF-κB cascade, we identify a novel constitutive association between filamin A (FLNa) and the NF-κB inducing kinase (NIK), in both Jurkat and human primary T cells. Following CD28 engagement by B7, in the absence of TCR, FLNa-associated NIK is activated and induces IKKα kinase activity. Both proline (P(208)YAP(211)P(212)) and tyrosine residues (Y(206)QPY(209)APP) within the C-terminal proline-rich motif of CD28 are involved in the recruitment of FLNa/NIK complexes to the membrane as well as in the activation of NIK and IKKα.

Characterization of a new cancer-associated mutant of p53 with a missense mutation (K351N) in the tetramerization domain.

Inactivation of the tumor suppressor p53 is central to carcinogenesis and acquisition of resistance to drug-induced apoptosis. The majority of alterations are missense mutations and occur within the DNA-binding domain. However, little is known about the point mutations in the tetramerization domain (TD). Here we investigated the properties of a new p53 mutant (Lys 351 to Asn) in the TD identified in a cisplatin-resistant ovarian carcinoma cell line (A2780 CIS). We found that K351N substitution significantly reduces the thermodynamic stability of p53 tetramers without affecting the overall half-life of the protein. Moreover, p53 K351N has a reduced ability to bind DNA and to trans-activate its specific target gene promoters, such as bax. Data obtained from the analysis of p53 subcellular localization revealed that K351N mutation inhibits the nuclear export of p53 and accumulation in the cytoplasm induced by cisplatin treatment. These results identify p53 K351N as a new cancer associated mutant with reduced tumor suppressor activity and altered functions in response to apoptotic stimuli.

Prostaglandin E2 induces interleukin-8 gene transcription by activating C/EBP homologous protein in human T lymphocytes.

The effect of prostaglandin E(2) (PGE(2)) in regulating the synthesis of the pro-inflammatory chemokine interleukin-8 (IL-8) in T lymphocytes is not yet defined, even though it may reduce or enhance IL-8 synthesis in other cell types. Here, we demonstrate that, in human T cells, PGE(2) induced IL-8 mRNA transcription through prostaglandin E(2) receptors 1- and 4-dependent signal transduction pathways leading to the activation of the transcription factor C/EBP homologous protein (CHOP), never before implicated in IL-8 transcription. Several kinases, including protein kinase C, Src family tyrosine kinases, phosphatidylinositol 3-kinase, and p38 MAPK, were involved in PGE(2)-induced CHOP activation and IL-8 production. The transactivation of the IL-8 promoter by CHOP was NF-kappaB-independent. Our data suggest that PGE(2) acts as a potent pro-inflammatory mediator by inducing IL-8 gene transcription in activated T cells through different signal transduction pathways leading to CHOP activation. These findings show the complexity with which PGE(2) regulates IL-8 synthesis by inhibiting or enhancing its production depending on the cell types and environmental conditions.