The ERK5 mitogen-activated protein kinase (MAPK) differs from other MAPKs in possessing a potent transcriptional activation domain. ERK5؊/؊ embryos die from angiogenic defects, but the precise physiological role of ERK5 remains poorly understood. To elucidate molecular functions of ERK5 in the development of vasculature and other tissues, we performed gene profile analyses of erk5 ؊/؊ mouse embryos and erk5 ؊/؊ fibroblast cells reconstituted with ERK5 or ERK5(1-740), which lacks the transactivation domain. These experiments revealed several potential ERK5 target genes, including a proapoptotic gene bnip3, known angiogenic genes flt1 and lklf (lung Krüppel-like factor), and genes that regulate cardiovascular development. Among these, LKLF, known for its roles in angiogenesis, T-cell quiescence, and survival, was found to be absolutely dependent on ERK5 for expression in endothelial and T cells. We show that ERK5 drives lklf transcription by activating MEF2 transcription factors. Expression of erk5 short hairpin or a dominant-negative form of the ERK5 upstream activator, MEK5, in T cells led to downregulation of LKLF, increased cell size and upregulation of activation markers. Thus, through its kinase and transcriptional activation domains, ERK5 regulates transcriptional responses of cell survival and quiescence critical for angiogenesis and T-cell function.Cellular differentiation programs are regulated through molecular mechanisms that guide signals generated in response to external stimuli, to ultimately induce changes at the gene level. The members of the mitogen-activated protein kinase (MAPK) family regulate a wide range of cellular responses. The MAPK pathways are induced via sequential phosphorylation and activation of a canonical three-kinase cascade, MAPK kinase kinase (MAPKKK)-MAPK kinase (MAPKK)-MAPK (28, 57). Conventionally, activated MAPKs catalyze phosphorylation of substrates at serine or threonine residues N terminally adjacent to a proline (Pro at ϩ1 position), a change that induces a variety of responses including gene transcription, apoptosis, and proliferation. We previously described an alternative mode of activation utilized by the ERK5 MAPK. ERK5 possesses a unique transcriptional coactivator domain, which mediates protein-protein interactions with the myocyte enhancer factor 2 (MEF2) transcription factors and provides a potent coactivator function toward MEF2-driven transcription (22). In immature T lymphocytes, activation of ERK5 induces immediate-early transcription of the nur77 orphan steroid receptor gene via MEF2 proteins, an event that ultimately triggers apoptosis (6, 53, 54). Interestingly, ectopic expression of the C-terminal coactivator domain of ERK5 is sufficient on its own to induce MEF2-dependent transcription (22), demonstrating that this domain represents a functionally independent module. Other studies have shown that ERK5 also activates MEF2 proteins by the conventional mechanism of phosphorylation (23,24,33,56), indicating that ERK5 may utilize diverse mechanisms to affe...

T cell antigen receptor (TCR) signaling is necessary but not sufficient to promote the positive selection of CD4 ؉ CD8 ؉ thymocytes into CD4 ؉ or CD8 ؉ mature T cells. Notch signaling has also been implicated as a potential regulator of both CD4͞CD8 T cell development and TCR signaling. However, the relationship between positive selection, TCR signaling, and Notch remains unclear. Here we use DNA microarray analysis to compare gene expression changes in CD4 ؉ CD8 ؉ double-positive thymocytes undergoing positive selection, TCR stimulation, and Notch activation. We find that the genes induced during positive selection can be resolved into two distinct sets. One set, which we term ''TCR-induced,'' is also induced by in vitro TCR stimulation and contains a large proportion of transcription factors. A second set, which we term ''positive-selection-induced,'' is not induced by in vitro TCR simulation and contains a large proportion of genes involved in signal transduction pathways. Genes induced by Notch activity overlap substantially with genes induced during positive selection. We also find that Notch activity potentiates the effects of TCR stimulation on gene expression. These results help to identify TCR-and positive-selection-specific transcriptional events and help to clarify the relationship between positive selection and Notch.microarray ͉ gene expression profile ͉ negative selection

Signaling by the hedgehog (hh)-class gene pathway is essential for embryogenesis in organisms ranging from Drosophila to human. We have isolated a hh homolog (Hrohh) from a lophotrochozoan species, the glossiphoniid leech, Helobdella robusta, and examined its expression by reverse transcription polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization. The peak of Hrohh expression occurs during organogenesis (stages 10-11). No patterned expression was detected within the segmented portion of the germinal plate during the early stages of segmentation. In stage 10-11 embryos, Hro-hh is expressed in body wall, foregut, anterior and posterior midgut, reproductive organs and in a subset of ganglionic neurons. Evidence that Hro-hh regulates gut formation was obtained using the steroidal alkaloid cyclopamine, which specifically blocks HH signaling. Cyclopamine induced malformation of both foregut and anterior midgut in Helobdella embryos, and no morphologically recognizable gonads were seen. In contrast, no gross abnormalities were observed in the posterior midgut. Segmental ectoderm developed normally, as did body wall musculature and some other mesodermal derivatives, but the mesenchymal cells that normally come to fill most of the coelomic cavities failed to develop. Taken with data from Drosophila and vertebrates, our data suggest that the role of hh-class genes in gut formation and/or neural differentiation is ancestral to the bilaterians, whereas their role in segmentation evolved secondarily within the Ecdysozoa.