During angiogenesis, endothelial cells undergo proliferation, reorganization, and stabilization to establish a mature vascular network. This process is critical for establishing a functional circulatory system during development and contributes to the pathological process of tumor growth. Here we report that embryos deficient for the ERK5 MAPK die between embryonic days 10.5 and 11.5 with angiogenic failure and cardiovascular defects. We show that ERK5 deficiency leads to an increased expression of the vascular endothelial growth factor (VEGF), dysregulation of which has been shown to impede angiogenic remodeling and vascular stabilization. Our data also reveal that ERK5 negatively regulates transcription from the vegf locus during hypoxic responses. Importantly, ERK5 is required at an earlier developmental stage than p38␣, and p38␣ does not compensate for ERK5 deficiency. These results demonstrate that ERK5 plays a specific role in the regulation of early angiogenesis.Vasculogenesis and angiogenesis are two critical processes of establishing the cardiovascular system in vertebrate embryos. Vasculogenesis involves generation of the primitive vascular plexus from angioblasts that have recently differentiated from mesodermal tissues. This process leads to the formation of the early heart tube and major blood vessels such as the dorsal aorta (reviewed in Refs.

FADD is an adaptor known to transmit apoptotic signals from members of the tumor necrosis factor receptor family. We show here that FADD has a domain implicated in cell proliferation. Mice bearing the Asp mutation in the serine 191 phosphorylation site are runted and anemic and display splenomegaly. Apoptosis is unimpaired in these mice, but they exhibit many immune developmental problems indicative of proliferative defects. Mutant FADD T cells are defective in cell cycle progression, suggesting that regulation of phosphorylation at serine 191 is essential for growth/proliferation. Remarkably, serine 191 is conserved among mammalian FADD proteins, but this C-terminal region is absent in lower organisms, suggesting that FADD acquired a domain during evolution, rendering it a "proliferation-apoptosis coupler" that balances cell proliferation and apoptosis.

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.

TYK2 is a JAK family protein tyrosine kinase activated in response to multiple cytokines, including type I IFNs, IL-6, IL-10, IL-12, and IL-23. Extensive studies of mice that lack TYK2 expression indicate that the IFN-α, IL-12, and IL-23 pathways, but not the IL-6 or IL-10 pathways, are compromised. In contrast, there have been few studies of the role of TYK2 in primary human cells. A genetic mutation at the tyk2 locus that results in a lack of TYK2 protein in a single human patient has been linked to defects in the IFN-α, IL-6, IL-10, IL-12, and IL-23 pathways, suggesting a broad role for TYK2 protein in human cytokine responses. In this article, we have used a panel of novel potent TYK2 small-molecule inhibitors with varying degrees of selectivity against other JAK kinases to address the requirement for TYK2 catalytic activity in cytokine pathways in primary human cells. Our results indicate that the biological processes that require TYK2 catalytic function in humans are restricted to the IL-12 and IL-23 pathways, and suggest that inhibition of TYK2 catalytic activity may be an efficacious approach for the treatment of select autoimmune diseases without broad immunosuppression.

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...