Development of Erf-Mediated Craniosynostosis and Pharmacological Amelioration.

ETS2 repressor factor (ERF) insufficiency causes craniosynostosis (CRS4) in humans and mice. ERF is an ETS domain transcriptional repressor regulated by Erk1/2 phosphorylation via nucleo-cytoplasmic shuttling. Here, we analyze the onset and development of the craniosynostosis phenotype in an Erf-insufficient mouse model and evaluate the potential of the residual Erf activity augmented by pharmacological compounds to ameliorate the disease. Erf insufficiency appears to cause an initially compromised frontal bone formation and subsequent multisuture synostosis, reflecting distinct roles of Erf on the cells that give rise to skull and facial bones. We treated animals with Mek1/2 and nuclear export inhibitors, U0126 and KPT-330, respectively, to increase Erf activity by two independent pathways. We implemented both a low dosage locally over the calvaria and a systemic drug administration scheme to evaluate the possible indirect effects from other systems and minimize toxicity. The treatment of mice with either the inhibitors or the administration scheme alleviated the synostosis phenotype with minimal adverse effects. Our data suggest that the ERF level is an important regulator of cranial bone development and that pharmacological modulation of its activity may represent a valid intervention approach both in CRS4 and in other syndromic forms of craniosynostosis mediated by the FGFR-RAS-ERK-ERF pathway.

Craniofacial, orofacial and dental disorders: the role of the RAS/ERK pathway.

Deviations from the precisely coordinated programme of human head development can lead to craniofacial and orofacial malformations often including a variety of dental abnormalities too. Although the aetiology is still unknown in many cases, during the last decades different intracellular signalling pathways have been genetically linked to specific disorders. Among these pathways, the RAS/extracellular signal-regulated kinase (ERK) signalling cascade is the focus of this review since it encompasses a large group of genes that when mutated cause some of the most common and severe developmental anomalies in humans. We present the components of the RAS/ERK pathway implicated in craniofacial and orodental disorders through a series of human and animal studies. We attempt to unravel the specific molecular targets downstream of ERK that act on particular cell types and regulate key steps in the associated developmental processes. Finally we point to ambiguities in our current knowledge that need to be clarified before RAS/ERK-targeting therapeutic approaches can be implemented.

Suppression of Fgf2 by ETS2 repressor factor (ERF) is required for chorionic trophoblast differentiation.

ETS2 repressor factor (ERF) is a ubiquitous transcriptional repressor regulated by Extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Homozygous deletion of Erf in mice blocks chorionic trophoblast differentiation, resulting in the failure of chorioallantoic fusion and subsequent embryo death. Fibroblast growth factor (FGF) signaling is important for proper trophoblast stem cell (TSC) differentiation and development of the hemochorial placenta. Lack of Fgf2 promotes TSC differentiation, while FGF4 or FGF2 is required for murine TSC maintenance. Here, we show that low in vivo Fgf2 mRNA abundance occurs in patches of placental chorion cells and ex vivo in TSCs. This expression is repressed via direct interaction of ERF with the Fgf2 transcription unit is increased in the absence of ERF, and is decreased in the presence of an ERF mutant resistant to ERK phosphorylation. Thus, FGF2 inhibition by ERF appears to be necessary for proper chorionic TSC differentiation, and may account for the block of chorionic trophoblast differentiation in Erf-knockout animals. The differentiation of ERF-overexpressing TSC lines also suggests that ERF may have an FGF2-independent effect during the commitment towards syncytiotrophoblasts.

EGR1 and the ERK-ERF axis drive mammary cell migration in response to EGF.

The signaling pathways that commit cells to migration are incompletely understood. We employed human mammary cells and two stimuli: epidermal growth factor (EGF), which induced cellular migration, and serum factors, which stimulated cell growth. In addition to strong activation of ERK by EGF, and AKT by serum, early transcription remarkably differed: while EGF induced early growth response-1 (EGR1), and this was required for migration, serum induced c-Fos and FosB to enhance proliferation. We demonstrate that induction of EGR1 involves ERK-mediated down-regulation of microRNA-191 and phosphorylation of the ETS2 repressor factor (ERF) repressor, which subsequently leaves the nucleus. Unexpectedly, knockdown of ERF inhibited migration, which implies migratory roles for exported ERF molecules. On the other hand, chromatin immunoprecipitation identified a subset of direct EGR1 targets, including EGR1 autostimulation and SERPINB2, whose transcription is essential for EGF-induced cell migration. In summary, EGR1 and the EGF-ERK-ERF axis emerge from our study as major drivers of growth factor-induced mammary cell migration.

ETS2 repressor factor (ERF) is involved in T lymphocyte maturation acting as regulator of thymocyte lineage commitment.

Thymocyte differentiation and lineage commitment is regulated by an extensive network of transcription factors and signaling molecules among which Erk plays a central role. However, Erk effectors as well as the molecular mechanisms underlying this network are not well understood. Erf is a ubiquitously expressed transcriptional repressor regulated by Erk-dependent phosphorylation. Here, we investigated the role of Erf in T cell maturation and lineage commitment, using a double-fluorescent Erf-floxed mouse to produce thymus-specific Erf knockouts. We observed significant accumulation of thymocytes in the CD4/CD8 DP stage, followed by a significant reduction in CD4SP cells, a trend for lower CD8SP cell frequency, and an elevated percentage of γδ expressing thymocytes in Erf-deficient mice. Also, an elevated number of CD69+ TCRß+ cells indicates that thymocytes undergoing positive selection accumulate at this stage. The expression of transcription factors Gata3, ThPOK, and Socs1 that promote CD4+ cell commitment was significantly decreased in Erf-deficient mice. These findings suggest that Erf is involved in T cell maturation, acting as a positive regulator during CD4 and eventually CD8 lineage commitment, while negatively regulates the production of γδ T cells. In addition, Erf-deficient mice displayed decreased percentages of CD4+ and CD8+ splenocytes and elevated levels of IL-4 indicating that Erf may have an additional role in the homeostasis, differentiation, and immunologic response of helper and cytotoxic T cells in the periphery. Overall, our results show, for the first time, Erf's involvement in T cell biology suggesting that Erf acts as a potential regulator during thymocyte maturation and thymocyte lineage commitment, in γδ T cell generation, as well as in Th cell differentiation.

Micro-CT for Biological and Biomedical Studies: A Comparison of Imaging Techniques.

Several imaging techniques are used in biological and biomedical studies. Micro-computed tomography (micro-CT) is a non-destructive imaging technique that allows the rapid digitisation of internal and external structures of a sample in three dimensions and with great resolution. In this review, the strengths and weaknesses of some common imaging techniques applied in biological and biomedical fields, such as optical microscopy, confocal laser scanning microscopy, and scanning electron microscopy, are presented and compared with the micro-CT technique through five use cases. Finally, the ability of micro-CT to create non-destructively 3D anatomical and morphological data in sub-micron resolution and the necessity to develop complementary methods with other imaging techniques, in order to overcome limitations caused by each technique, is emphasised.

Erf Affects Commitment and Differentiation of Osteoprogenitor Cells in Cranial Sutures via the Retinoic Acid Pathway.

ETS2 repressor factor (ERF) haploinsufficiency causes late-onset craniosynostosis (CRS) (OMIM entry 600775; CRS4) in humans, while in mice Erf insufficiency also leads to a similar multisuture synostosis phenotype preceded by mildly reduced calvarium ossification. However, neither the cell types affected nor the effects per se have been identified so far. Here, we establish an ex vivo system for the expansion of suture-derived mesenchymal stem and progenitor cells (sdMSCs) and analyze the role of Erf levels in their differentiation. Cellular data suggest that Erf insufficiency specifically decreases osteogenic differentiation of sdMSCs, resulting in the initially delayed mineralization of the calvarium. Transcriptome analysis indicates that Erf is required for efficient osteogenic lineage commitment of sdMSCs. Elevated retinoic acid catabolism due to increased levels of the cytochrome P450 superfamily member Cyp26b1 as a result of decreased Erf levels appears to be the underlying mechanism leading to defective differentiation. Exogenous addition of retinoic acid can rescue the osteogenic differentiation defect, suggesting that Erf affects cranial bone mineralization during skull development through retinoic acid gradient regulation.

Transcriptional repressor erf determines extraembryonic ectoderm differentiation.

Extraembryonic ectoderm differentiation and chorioallantoic attachment are fibroblast growth factor (FGF)- and transforming growth factor beta-regulated processes that are the first steps in the development of the placenta labyrinth and the establishment of the fetal-maternal circulation in the developing embryo. Only a small number of genes have been demonstrated to be important in trophoblast stem cell differentiation. Erf is a ubiquitously expressed Erk-regulated, ets domain transcriptional repressor expressed throughout embryonic development and adulthood. However, in the developing placenta, after 7.5 days postcoitum (dpc) its expression is restricted to the extraembryonic ectoderm, and its expression is restricted after 9.5 dpc in a subpopulation of labyrinth cells. Homozygous deletion of Erf in mice leads to a block of chorionic cell differentiation before chorioallantoic attachment, resulting in a persisting chorion layer, a persisting ectoplacental cone cavity, failure of chorioallantoic attachment, and absence of labyrinth. These defects result in embryo death by 10.5 dpc. Trophoblast stem cell lines derived from Erf(dl1/dl1) knockout blastocysts exhibit delayed differentiation and decreased expression of spongiotrophoblast markers, consistent with the persisting chorion layer, the expanded giant cell layer, and the diminished spongiotrophoblast layer observed in vivo. Our data suggest that attenuation of FGF/Erk signaling and consecutive Erf nuclear localization and function is required for extraembryonic ectoderm differentiation, ectoplacental cone cavity closure, and chorioallantoic attachment.

cAMP regulates IL-10 production by normal human T lymphocytes at multiple levels: a potential role for MEF2.

Signal transduction by the cAMP/cAMP-dependent protein kinase A (PKA) pathway is triggered through multiple receptors and is important for many processes in a variety of cells. In T cells, the engagement of the TCR-CD3 complex induces cAMP, a second messenger that controls immune response. IL-10, produced by a variety of lymphocyte subpopulations, is an important regulator of this response exerting a wide range of immunomodulatory actions. Elevation of cAMP has been shown to increase IL-10 production by monocytes. However, the mechanism of cAMP mediated regulation of IL-10 production by T lymphocytes remains unclear. In this study using normal peripheral T lymphocytes stimulated either through the TCR-CD3 complex or the TCR-CD3 and the CD28 molecule, we show that IL-10 is produced mainly by memory T lymphocytes after either way of stimulation and is drastically inhibited (70-90%) by cAMP elevating agents. cAMP mediated inhibition was reversed by the use of the specific PKA inhibitor Rp-8-Br-cAMP but not by the addition of exogenous rhIL-2, indicating that the inhibitory effect depends on PKA activation and is not secondary to IL-2 inhibition. Inhibition is taking place at both transcriptional and posttranscriptional level. Transfection of a luciferase reporter plasmid carrying the IL-10 promoter in T cells, revealed that TCR/CD28-induced activation was inhibited by 60% by cAMP elevation. The most sensitive part to cAMP mediated inhibition was a fragment of 135 bp upstream of TATA box, which contains multiple binding sites for MEF-2. Overexpression of MEF-2 in the same cells increased IL-10 promoter activity by 2.5-fold. Stimulation through TCR/CD28 increased MEF-2 binding in its corresponding binding sites which was inhibited by 80% in the presence of cAMP elevating agents. These results suggest that the inhibitory effect of cAMP on IL-10 production by normal peripheral T lymphocytes is cell type and stimulus specific, exerted on multiple levels and involves MEF2 transcription factor.

Ets2 is required for trophoblast stem cell self-renewal.

The Ets2 transcription factor is essential for the development of the mouse placenta and for generating signals for embryonic mesoderm and axis formation. Using a conditional targeted Ets2 allele, we show that Ets2 is essential for trophoblast stem (TS) cells self-renewal. Inactivation of Ets2 results in TS cell slower growth, increased expression of a subset of differentiation-associated genes and decreased expression of several genes implicated in TS self-renewal. Among the direct TS targets of Ets2 is Cdx2, a key master regulator of TS cell state. Thus Ets2 contributes to the regulation of multiple genes important for maintaining the undifferentiated state of TS cells and as candidate signals for embryonic development.

ERF nuclear shuttling, a continuous monitor of Erk activity that links it to cell cycle progression.

The ets domain transcriptional repressor ERF is an effector of the receptor tyrosine kinase/Ras/Erk pathway, which, it has been suggested, is regulated by subcellular localization as a result of Erk-dependent phosphorylation and is capable of suppressing cell proliferation and ras-induced tumorigenicity. Here, we analyze the effect of ERF phosphorylation on nuclear import and export, the timing of its phosphorylation and dephosphorylation in relation to its subcellular location, Erk activity, and the requirements for ERF-induced cell cycle arrest. Our findings indicate that ERF continuously shuttles between the nucleus and the cytoplasm and that both phosphorylation and dephosphorylation of ERF occur within the nucleus. While nuclear import is not affected by phosphorylation, ERF nuclear export and cytoplasmic release require multisite phosphorylation and dephosphorylation. ERF export is CRM1 dependent, although ERF does not have a detectable nuclear export signal. ERF phosphorylation and export correlate with the levels of nuclear Erk activity. The cell cycle arrest induced by nonphosphorylated ERF requires the wild-type retinoblastoma protein and can be suppressed by overexpression of cyclin. These data suggest that ERF may be a very sensitive and constant sensor of Erk activity that can affect cell cycle progression through G(1), providing another link between the Ras/Erk pathway and cellular proliferation.

Conditional up-regulation of IL-2 production by p38 MAPK inactivation is mediated by increased Erk1/2 activity.

The p38 mitogen-activated protein kinase regulates many cellular processes in almost all eukaryotic cell types. In T cells, p38 was shown to regulate thymic development and cytokine production. Here, the role of p38 on interleukin-2 (IL-2) production by human peripheral blood CD4+ T cells was examined. When T cells were stimulated under weak stimulation conditions, pharmaceutical and molecular p38 inhibitors induced a dramatic increase of IL-2 production. In contrast, IL-2 levels were not affected significantly when strong stimulation was provided to T cells. The increase in IL-2 production, following p38 inhibition, was associated with a strong up-regulation of extracellular signal-regulated kinase (Erk)1/2 activity. Furthermore the Erk inhibitor U0126 was able to counteract the effect of p38 inhibition on IL-2 production, supporting the conclusion that p38 mediates its effect through Erk. These results suggest that the p38 kinase, through its ability to control Erk activation levels, acts as a gatekeeper, which prevents inappropriate IL-2 production. Also, the finding that p38 acts in a strength-of-stimulation-dependent way provides an explanation for previously reported, contradictory results regarding the role of this kinase in IL-2 expression.

The Ets2 Repressor Factor (Erf) Is Required for Effective Primitive and Definitive Hematopoiesis.

Erf is a gene for a ubiquitously expressed Ets DNA-binding domain-containing transcriptional repressor. Erf haploinsufficiency causes craniosynostosis in humans and mice, while its absence in mice leads to failed chorioallantoic fusion and death at embryonic day 10.5 (E10.5). In this study, we show that Erf is required in all three waves of embryonic hematopoiesis. Mice lacking Erf in the embryo proper exhibited severe anemia and died around embryonic day 14.5. Erf epiblast-specific knockout embryos had reduced numbers of circulating blood cells from E9.5 onwards, with the development of severe anemia by E14.5. Elimination of Erf resulted in both reduced and more immature primitive erythroblasts at E9.5 to E10.5. Reduced definitive erythroid colony-forming activity was found in the bloodstream of E10.5 embryos and in the fetal liver at E11.5 to E13.5. Finally, elimination of Erf resulted in impaired repopulation ability, indicating that Erf is necessary for hematopoietic stem cell maintenance or differentiation. We conclude that Erf is required for both primitive and erythromyeloid progenitor waves of hematopoietic stem cell (HSC)-independent hematopoiesis as well as for the normal function of HSCs.

ERF, an ETS-related transcriptional repressor, can induce erythroid differentiation.

Ets-family genes have been implicated in leukemia, as well as in normal hematopoiesis. ERF is an ets-related gene that represses transcription and is regulated by MAPK phosphorylation through its effect on ERF sub-cellular localization. Using pluripotent human cell lines, we studied the effect of ERF on erythroid differentiation. K562 and HEL cells expressing ERF expressed elevated levels of the erythroid-specific markers CD71 and Glycophorin A, as well as hemoglobin and GATA1. Treatment with the Erk kinase inhibitor PD98058 further enhanced the erythroid phenotype in ERF-expressing cells and cells expressing a non-phosphorylatable ERF mutant exhibited an even more enhanced phenotype. These results are consistent with the fact that ERF function is regulated by MAPK, and suggest that the effect of the MAPK pathway in erythroid differentiation is partially mediated by ERF. The effect of ERF is similar to the one shown for ETS1 and opposite to the FLI1 function in these cells, suggesting that several ets genes may play key roles in hematopoietic differentiation.

Reduced dosage of ERF causes complex craniosynostosis in humans and mice and links ERK1/2 signaling to regulation of osteogenesis.

The extracellular signal-related kinases 1 and 2 (ERK1/2) are key proteins mediating mitogen-activated protein kinase signaling downstream of RAS: phosphorylation of ERK1/2 leads to nuclear uptake and modulation of multiple targets. Here, we show that reduced dosage of ERF, which encodes an inhibitory ETS transcription factor directly bound by ERK1/2 (refs. 2,3,4,5,6,7), causes complex craniosynostosis (premature fusion of the cranial sutures) in humans and mice. Features of this newly recognized clinical disorder include multiple-suture synostosis, craniofacial dysmorphism, Chiari malformation and language delay. Mice with functional Erf levels reduced to ∼30% of normal exhibit postnatal multiple-suture synostosis; by contrast, embryonic calvarial development appears mildly delayed. Using chromatin immunoprecipitation in mouse embryonic fibroblasts and high-throughput sequencing, we find that ERF binds preferentially to elements away from promoters that contain RUNX or AP-1 motifs. This work identifies ERF as a novel regulator of osteogenic stimulation by RAS-ERK signaling, potentially by competing with activating ETS factors in multifactor transcriptional complexes.

Semaphorin-7a reverses the ERF-induced inhibition of EMT in Ras-dependent mouse mammary epithelial cells.

Epithelial-to-mesenchymal transition (EMT) is a key process in cancer progression and metastasis, requiring cooperation of the epidermal growth factor/Ras with the transforming growth factor-ß (TGF-ß) signaling pathway in a multistep process. The molecular mechanisms by which Ras signaling contributes to EMT, however, remain elusive to a large extent. We therefore examined the transcriptional repressor Ets2-repressor factor (ERF)-a bona fide Ras-extracellular signal-regulated kinase/mitogen-activated protein kinase effector-for its ability to interfere with TGF-ß-induced EMT in mammary epithelial cells (EpH4) expressing oncogenic Ras (EpRas). ERF-overexpressing EpRas cells failed to undergo TGF-ß-induced EMT, formed three-dimensional tubular structures in collagen gels, and retained expression of epithelial markers. Transcriptome analysis indicated that TGF-ß signaling through Smads was mostly unaffected, and ERF suppressed the TGF-ß-induced EMT via Semaphorin-7a repression. Forced expression of Semaphorin-7a in ERF-overexpressing EpRas cells reestablished their ability to undergo EMT. In contrast, inhibition of Semaphorin-7a in the parental EpRas cells inhibited their ability to undergo TGF-ß-induced EMT. Our data suggest that oncogenic Ras may play an additional role in EMT via the ERF, regulating Semaphorin-7a and providing a new interconnection between the Ras- and the TGF-ß-signaling pathways.

Calcium/calmodulin-dependent protein kinase II regulates IL-10 production by human T lymphocytes: a distinct target in the calcium dependent pathway.

Calcium (Ca2+) plays an essential role in lymphocyte activation and differentiation by affecting signaling pathways leading to cytokine production. Among the enzymes responding to calcium increase, Ca2+/calmodulin-dependent protein kinase II (CaMKII) has been involved in anergy with a still poorly characterized role. IL-10 produced by different T lymphocyte subpopulations is critical mediator of tolerance. We tested the hypothesis that CaMKII may be involved in IL-10 production. We report that CaMKII upregulates IL-10 production by primary human T lymphocytes stimulated through the antigen receptor or bypassing that. Overexpression of constitutively active mutant forms of Calcineurin or CaMKII specifically increase IL-10 protein product and IL-10 mRNA accumulation in T lymphocytes. By cotransfecting constitutively active CaMKII with luciferase reporter plasmids carrying specific fragments or the whole IL-10 promoter, we show that CaMKII specifically activates IL-10 promoter activity, whereas it inhibits IL-2 and IL-4 promoter. This effect is mediated by the first 500 bp fragment, which contains binding sites for Myocyte Enhancer Factor-2 (MEF2). A constitutively active mutant of CaMKII activated a luciferase reporter plasmid under the control of MEF2, when cotransfected in T lymphocytes stimulated by Ionomycin and PMA, whereas its inhibitor KN-62 inhibited MEF2 binding in cell lysates of the same cells. Moreover, overexpression of MEF2 enhanced by 2.5-fold IL-10 promoter activity. Our data for the first time suggest a distinct role of CaMKII in the induction of anergy in T lymphocytes, by differential regulation of IL-10 and IL-2 gene transcription suggest MEF2 as a molecular target which can integrate different calcium signals.

The RAS-dependent ERF control of cell proliferation and differentiation is mediated by c-Myc repression.

The ERF transcriptional repressor is a downstream effector of the RAS/ERK pathway that interacts with and is directly phosphorylated by ERKs in vivo and in vitro. This phosphorylation results in its cytoplasmic export and inactivation, although lack of ERK activity allows its immediate nuclear accumulation and repressor function. Nuclear ERFs arrest cell cycle progression in G(1) and can suppress ras-dependent tumorigenicity. Here we provide evidence that ERF function is mediated by its ability to repress transcription of c-Myc. Promoter reporter assays indicate a DNA binding-dependent and repressor domain-dependent Myc transcriptional repression. Chromatin immunoprecipitations in primary cells suggest that ERF specifically binds on the c-Myc promoter in an E2F4/5-dependent manner and only under conditions that the physiological c-Myc transcription is stopped. Cellular systems overexpressing nuclear ERF exhibit reduced c-Myc mRNA and tumorigenic potential. Elimination of Erf in animal models results in increased c-Myc expression, whereas Erf(-)(/)(-) primary fibroblasts fail to down-regulate Myc in response to growth factor withdrawal. Finally, elimination of c-Myc in primary mouse embryo fibroblasts negates the ability of nuclear ERF to suppress proliferation. Thus Erf provides a direct link between the RAS/ERK signaling and the transcriptional regulation of c-Myc and suggests that RAS/ERK attenuation actively regulates cell fate.

The transcriptional ETS2 repressor factor associates with active and inactive Erks through distinct FXF motifs.

The transcriptional ETS2 repressor factor (ERF) is phosphorylated by Erks both in vivo and in vitro. This phosphorylation determines the subcellular localization and biological function of ERF. Here, we show that active and inactive Erk2 proteins bind ERF with high affinity through a hydrophobic pocket formed by the alphaF and alphaG helices and the activation loop of Erk2. We have identified two FXF motifs on ERF that mediate the specific interaction with Erks. One of these motifs is utilized only by active Erks, whereas the other mediates the association with inactive Erks but also contributes to interaction with active Erks. Mutation of the phenylalanines of these motifs to alanines resulted in decreased association and phosphorylation of ERF by Erks both in cells and in vitro. ERF proteins carrying these mutations exhibited increased nuclear accumulation and increased inhibition of cellular proliferation. Expression of ERF regions harboring these motifs could inhibit Erk activity in cells. Our data suggest that, in the proper context, FXF motifs can mediate a strong and specific interaction not only with active but also inactive Erks and that these interactions determine protein function in vivo.

IL-2 and IL-10 production by human CD4+T cells is differentially regulated by p38: mode of stimulation-dependent regulation of IL-2.

Antigenic stimulation of T cells initiates a complex series of intracellular signaling pathways that target and activate different cytokine genes. The participation of mitogen-activated protein kinases (MAPKs) in these processes has not been studied thoroughly and in some instances conflicting results have been reported. Here we have examined the role of p38 MAPK on IL-2 and IL-10 production following activation of human CD4+ T cells or of the leukemic cell line Hut-78, with either plate-bound anti-CD3 in the presence or absence of soluble anti-CD28 (plCD3, plCD3/sCD28), or with cross-linked anti-CD3 and anti-CD28 (crsCD3+CD28), or with PMA plus ionomycin. Pharmacological inhibition of the p38 pathway with either SB203580, SB202190, or SKF86002 strongly downregulated IL-10 production by T cells stimulated with any of the above treatments. In contrast the effect of p38 inhibition on IL-2 was stimulus dependent. Thus, p38 inhibition strongly upregulated IL-2 production (up to 10-fold) in the plCD3- and plCD3/sCD28-stimulated cultures while it had minimal or no effect in the other two stimulation protocols. Intracellular and mRNA levels of IL-2 and IL-10 were also upregulated and downregulated, respectively, by p38 inhibitors in the plCD3/sCD28-stimulated CD4+ T cells. Also, the induction of IL-2 and the parallel suppression of IL-10 by p38 inhibitors were independent of the balance between these two cytokines, as demonstrated by the addition of exogenous IL-10 or blocking anti-IL-10 antibody in CD4+ and Hut-78 cell cultures. These results show that p38 acts as a molecular switch that changes the balance between IL-2 and IL-10. This is especially important considering the opposing role of these cytokines in peripheral immune tolerance.