José Alberola-Ila

Regulation of the helix-loop-helix proteins, E2A and Id3, by the Ras-ERK MAPK cascade

Activation of mitogen-activated protein kinase (MAPK) pathways leads to cellular differentiation and/or proliferation in a wide variety of cell types, including developing thymocytes. The basic helix-loop-helix (bHLH) proteins E12 and E47 and an inhibitor HLH protein, Id3, play key roles in thymocyte differentiation. We show here that E2A DNA binding is lowered in primary immature thymocytes consequent to T cell receptor (TCR)-mediated ligation. Whereas expression of E2A mRNA and protein are unaltered, Id3 transcripts are rapidly induced upon signaling from the TCR. Activation of Id3 transcription is regulated in a dose-dependent manner by the extracellular signal-regulated kinase (ERK) MAPK module. These observations directly connect the ERK MAPK cascade and HLH proteins in a linear pathway.

GATA-3 Expression Is Controlled by TCR Signals and Regulates CD4/CD8 Differentiation

GATA-3 is expressed at higher levels in CD4 than in CD8 SP thymocytes. Here we show that upregulation of GATA-3 expression in DP thymocytes is triggered by TCR stimulation, and the extent of upregulation correlates with the strength of the TCR signal. Overexpression of GATA-3 or a partial GATA-3 agonist during positive selection inhibits CD8 SP cell development but is not sufficient to divert class I-restricted T cell precursors to the CD4 lineage. Conversely, expression of the GATA-3 antagonist ROG or of a GATA-3 siRNA hairpin markedly enhances development of CD8 SP cells and reduces CD4 SP development. We propose that GATA-3 contributes to linking the TCR signal strength to the differentiation program of CD4 and CD8 thymocytes.

Lck Activity Controls CD4/CD8 T Cell Lineage Commitment

Thymocytes carrying MHC class I-restricted TCRs differentiate into CD8 T cells, while those recognizing MHC class II become CD4 T cells. The mechanisms underlying how MHC class recognition, coreceptor expression, and effector function are coordinated are not well understood. Since the tyrosine kinase Lck binds with more affinity to CD4 than CD8, it has been proposed as a candidate to mediate this process. By using transgenic mice with altered Lck activity, we show that thymocytes carrying a class II-restricted TCR develop into functional CD8 T cells when Lck activity is reduced. Conversely, thymocytes carrying a class I-restricted TCR develop into functional CD4 T cells when Lck activity is increased. These results directly show that quantitative differences in the Lck signal control the CD4/CD8 lineage decision.

Disruption of T cell signaling networks and development by Grb2 haploid insufficiency.

The developmental processes of positive and negative selection in the thymus shape the T cell antigen receptor (TCR) repertoire and require the integration of multiple signaling networks. These networks involve the efficient assembly of macromolecular complexes and are mediated by multimodular adaptor proteins that permit the functional integration of distinct signaling molecules. We show here that decreased expression of the adaptor protein Grb2 in Grb2+/− mice weakens TCR-induced c-Jun N-terminal kinase (JNK) and p38, but not extracellular signal–regulated kinase (ERK), activation. In turn, this selective effect decreases the ability of thymocytes to undergo negative, but not positive, selection. We also show that there are differences in the signaling thresholds of the three mitogen-activated protein kinase (MAPK) families. These differences may provide a mechanism by which quantitative differences in signal strength can alter the balance of downstream signaling pathways to induce the qualitatively distinct biological outcomes of proliferation, differentiation or apoptosis.

The Ras/MAPK cascade and the control of positive selection

Immature double positive (DP) thymocytes bearing a T cell receptor (TCR) that interacts with self‐major histocompatibility complex (MHC) molecules receive signals that induce either their differentiation (positive selection) or apoptosis (negative selection). Furthermore, those cells that are positively selected develop into two different lineages, CD4 or CD8, depending on whether their TCRs bind to MHC class II or I, respectively. Positive selection therefore involves rescue from the default fate (death), lineage commitment, and progression to the single positive (SP) stage. These are probably temporally distinct events that may require both unique and overlapping signals. Work in the past several years has started to unravel the signaling networks that control these processes. One of the first pathways identified as important for positive selection was Ras and its downstream effector, the Erk mitogen‐activated protein kinase (MAPK) cascade. In this review we examine the factors that connect the TCR to the Ras/Erk cascade in DP thymocytes, as well as what we know about the downstream effectors of the Ras/Erk cascade important for positive selection. We also consider the possible role of this cascade in CD4/CD8 lineage development, and the possible interactions of the Ras/Erk cascade with Notch during these cell fate determination processes.

Distinct signals mediate maturation and allelic exclusion in lymphocyte progenitors.

Successful in-frame rearrangement of immunoglobulin heavy chain genes or T cell antigen receptor (TCR) beta chain genes in lymphocyte progenitors results in formation of pre-BCR and pre-TCR complexes. These complexes signal progenitor cells to mature, expand in cell number, and suppress further rearrangements at the immunoglobulin heavy chain or TCRbeta chain loci, thereby ensuring allelic exclusion. We used transgenic expression of a constitutively active form of c-Raf-1 (Raf-CAAX) to demonstrate that activation of the Map kinase pathway can stimulate both maturation and expansion of B and T lymphocytes, even in the absence of pre-TCR or pre-BCR formation. However, the same Raf signal did not mediate allelic exclusion. We conclude that maturation of lymphocyte progenitors and allelic exclusion require distinct signals.

Conspiracy Theory: RAS and RAF Do Not Act Alone

Several recent papers have added new proteins that conspire with RAS, RAF, and receptors to transduce signals. These new findings raise many more questions than they answer and suggest that we need to reevaluate RAS and RAF signaling and its regulation. Pasadena, California 91125 SUR-8/SOC-2 Helps RAS EGF receptor signaling in C. elegans has been most extensively analyzed in the context of vulval develop-Introduction ment, which provides a facile assay for RAS activity. In the early 1990s, genetic analysis in D. melanogaster Mutants of RAF, MEK, MAP kinase, and KSR-1 suppress and C. elegans helped define a signaling pathway from the effects of activated RAS in C. elegans. Recently, a cell surface receptors to the nucleus. Cell surface recep-new protein, SUR-8 (Suppressor of RAS), was identified tors with intrinsic tyrosine kinase activity (receptor tyro-in this way (Sieburth et al., 1998). This protein was also sine kinases or RTKs) respond to peptide ligands, identified as acting downstream of the C. elegans FGFR, growth factors, and inductive signals in development. which when constitutively active leads to a phenotype Activation of RTKs often leads to activation of RAS, called Clear (hence soc-2, Suppressor of clear; Selfors which in its GTP-bound state activates effectors, the et al., 1998). Mutants of SEM-5 or SOC-2 disrupt sig-proteins that exert its biological effect. The identification naling by activated FGFR. Because SUR-8/SOC-2 is of SOS (Son of sevenless) as a guanine nucleotide ex-necessary for the action of activated FGFR and of RAS, change factor for RAS, and the adaptor protein GRB2 SUR-8/SOC-2 is likely to be a positive regulator in RAS (in mammals)/SEM-5 (in C. elegans)/DRK (in Drosophila), signaling. coupled with the finding that these two proteins act SUR-8/SOC-2 and a human homolog have 18 leucine-downstream of RTKs and upstream of RAS, allowed rich repeats (LRRs), a relatively common protein–protein the biochemical linking of RTKs to RAS activation. The interaction motif (Kobe and Deisenhofer, 1994) and one adaptor GRB2 binds to proteins phosphorylated on tyro-that is found in adenylyl cyclase, an effector for S. cere-sine by RTKs and thereby recruits SOS to the membrane, visiae RAS. Indeed, SUR-8/SOC-2 binds to RAS but not allowing it to activate RAS. Similarly, the finding that the RAF, MEK, MAPK, or KSR-1 by yeast two-hybrid assays. serine/threonine protein kinase RAF acts downstream In vitro, SUR-8/SOC-2 binds the effector domain of RAS. of RAS led to its identification as a bona fide effector …

Estrogen receptor signaling promotes dendritic cell differentiation by increasing expression of the transcription factor IRF4

During inflammation, elevated granulocyte macrophage-colony-stimulating factor (GM-CSF) directs the development of new dendritic cells (DCs). This pathway is influenced by environmental factors, and we previously showed that physiologic levels of estradiol, acting through estrogen receptor alpha (ERalpha), promote the GM-CSF-mediated differentiation of a CD11b(+) DC subset from myeloid progenitors (MPs). We now have identified interferon regulatory factor 4 (IRF4), a transcription factor induced by GM-CSF and critical for CD11b(+) DC development in vivo, as a target of ERalpha signaling during this process. In MPs, ERalpha potentiates and sustains GM-CSF induction of IRF4. Furthermore, retroviral delivery of the Irf4 cDNA to undifferentiated ERalpha(-/-) bone marrow cells restored the development of the estradiol/ERalpha-dependent DC population, indicating that an elevated amount of IRF4 protein substitutes for ERalpha signaling. Thus at an early stage in the MP response to GM-CSF, ERalpha signaling induces an elevated amount of IRF4, which leads to a developmental program underlying CD11b(+) DC differentiation.

The transcription factor c-Myb primes CD4+CD8+ immature thymocytes for selection into the iNKT lineage.

Type I invariant NKT cells (iNKT cells) are a subset of αβ T cells characterized by the expression of an invariant α-chain variable region 14–α-chain joining region 18 (Vα14Jα18) T cell antigen receptor (TCR) α-chain. The iNKT cells derive from CD4+CD8+ double-positive (DP) thymocytes, and their generation requires a long half-life of DP thymocytes to allow Vα14-Jα18 rearrangements, expression of glycolipid-loaded CD1d on DP thymocytes, and signaling through the signaling-activation molecule SLAM–adaptor SAP pathway. Here we show that the transcription factor c-Myb has a central role in priming DP thymocytes to enter the iNKT lineage by simultaneously regulating CD1d expression, the half-life of DP cells and expression of SLAMF1, SLAMF6 and SAP.

Egr2 Is Required for Bcl-2 Induction during Positive Selection

The repertoire of TCR specificities is established by a selection process in the thymus, during which precursor survival and maturation is dictated by the nature of the TCR signals. The differences in signals that determine whether precursors will survive and mature or be induced to die remain poorly understood. Among the molecular effectors involved in executing the differentiation process initiated by TCR-ligand interactions is a family of Zn-finger transcription factors termed early growth response genes (Egr). Indeed, ablation of the Egr1 gene impairs ligand-induced maturation (positive selection) but not ligand-induced deletion (negative selection). The partial impairment of positive selection by Egr1 deficiency is not enhanced by simultaneous deletion of another Egr family member, Egr3. Accordingly, we asked whether this results from compensation by another family member, Egr2. In this manuscript, we demonstrate that deletion of Egr2 impairs positive selection of both CD4 and CD8 single-positive thymocytes. Interestingly, many of the genes involved in positive selection and T cell differentiation are up-regulated normally in the Egr2-deficient thymocytes. However, Bcl-2 up-regulation is not sustained during late stages of positive selection. This defect is at least partially responsible for the developmental blockade in Egr2-deficient thymocytes, as enforced expression of Bcl-2 rescues T cell development in Egr2−/− thymocytes. Taken together, these data suggest that Egr2 plays a central role in the up-regulation of the survival molecule Bcl-2 during positive selection.