Frank Blaeser

JM2, encoding a fork head-related protein, is mutated in X-linked autoimmunity-allergic disregulation syndrome.

X-linked autoimmunity-allergic disregulation syndrome (XLAAD) is an X-linked recessive immunological disorder characterized by multisystem autoimmunity, particularly early-onset type 1 diabetes mellitus, associated with manifestations of severe atopy including eczema, food allergy, and eosinophilic inflammation. Consistent with the allergic phenotype, analysis of two kindreds with XLAAD revealed marked skewing of patient T lymphocytes toward the Th2 phenotype. Using a positional-candidate approach, we have identified in both kindreds mutations in JM2, a gene on Xp11.23 that encodes a fork head domain-containing protein. One point mutation at a splice junction site results in transcripts that encode a truncated protein lacking the fork head homology domain. The other mutation involves an in-frame, 3-bp deletion that is predicted to impair the function of a leucine zipper dimerization domain. Our results point to a critical role for JM2 in self tolerance and Th cell differentiation.

Ca2+-dependent Gene Expression Mediated by MEF2 Transcription Factors

Ca2+ induction of a subset of cellular and viral immediate-early activation genes in lymphocytes has been previously mapped to response elements recognized by the MEF2 family of transcription factors. Here, we demonstrate that Ca2+ activation of MEF2 response elements in T lymphocytes is mediated in synergy by two Ca2+/calmodulin-dependent enzymes, the phosphatase calcineurin, and the kinase type IV/Gr (CaMKIV/Gr), which promote transcription by the MEF2 family members MEF2A and MEF2D. Calcineurin up-regulates the activity of both factors by an NFAT-dependent mechanism, while CaMKIV/Gr selectively and independently activates MEF2D. These results identify MEF2 proteins as effectors of a pathway of gene induction in T lymphocytes which integrates diverse Ca2+ activation signals and may be broadly operative in several tissues.

Control of Cortical Axon Elongation by a GABA-Driven Ca2+/Calmodulin-Dependent Protein Kinase Cascade

Ca2+ signaling plays important roles during both axonal and dendritic growth. Yet whether and how Ca2+ rises may trigger and contribute to the development of long-range cortical connections remains mostly unknown. Here, we demonstrate that two separate limbs of the Ca2+/calmodulin-dependent protein kinase kinase (CaMKK)–CaMKI cascades, CaMKK–CaMKIα and CaMKK–CaMKIγ, critically coordinate axonal and dendritic morphogenesis of cortical neurons, respectively. The axon-specific morphological phenotype required a diffuse cytoplasmic localization and a strikingly α-isoform-specific kinase activity of CaMKI. Unexpectedly, treatment with muscimol, a GABAA receptor agonist, selectively stimulated elongation of axons but not of dendrites, and the CaMKK–CaMKIα cascade critically mediated this axonogenic effect. Consistent with these findings, during early brain development, in vivo knockdown of CaMKIα significantly impaired the terminal axonal extension and thereby perturbed the refinement of the interhemispheric callosal projections into the contralateral cortices. Our findings thus indicate a novel role for the GABA-driven CaMKK–CaMKIα cascade as a mechanism critical for accurate cortical axon pathfinding, an essential process that may contribute to fine-tuning the formation of interhemispheric connectivity during the perinatal development of the CNS.

Pathogenicity of a disease-associated human IL-4 receptor allele in experimental asthma

Polymorphisms in the interleukin-4 receptor α chain (IL-4Rα) have been linked to asthma incidence and severity, but a causal relationship has remained uncertain. In particular, a glutamine to arginine substitution at position 576 (Q576R) of IL-4Rα has been associated with severe asthma, especially in African Americans. We show that mice carrying the Q576R polymorphism exhibited intense allergen-induced airway inflammation and remodeling. The Q576R polymorphism did not affect proximal signal transducer and activator of transcription (STAT) 6 activation, but synergized with STAT6 in a gene target– and tissue-specific manner to mediate heightened expression of a subset of IL-4– and IL-13–responsive genes involved in allergic inflammation. Our findings indicate that the Q576R polymorphism directly promotes asthma in carrier populations by selectively augmenting IL-4Rα–dependent signaling.

Targeted Inactivation of the IL-4 Receptor α Chain I4R Motif Promotes Allergic Airway Inflammation

The insulin/interleukin-4 (IL-4) receptor (I4R) motif mediates the association of insulin receptor substrate (IRS)-2 with the interleukin-4 (IL-4)Rα chain and transduces mitogenic signals in response to IL-4. Its physiological functions were analyzed in mice with a germline point mutation that changed the motifs effector tyrosine residue into phenylalanine (Y500F). The Y500F mutation abrogated IRS-2 phosphorylation and impaired IL-4–induced CD4+ T lymphocyte proliferation but left unperturbed Stat6 activation, up-regulation of IL-4-responsive gene products, and Th cell differentiation under Th2 polarizing conditions. However, in vivo the Y500F mutation was associated with increased allergen-induced IgE production, airway responsiveness, tissue eosinophilia, and mucus production. These results define an important role for the I4R motif in regulating allergic inflammation.

Long-Term Memory Deficits in Pavlovian Fear Conditioning in Ca2+/Calmodulin Kinase Kinase α-Deficient Mice

ABSTRACT Signaling by the Ca2+/calmodulin kinase (CaMK) cascade has been implicated in neuronal gene transcription, synaptic plasticity, and long-term memory consolidation. The CaM kinase kinase α (CaMKKα) isoform is an upstream component of the CaMK cascade whose function in different behavioral and learning and memory paradigms was analyzed by targeted gene disruption in mice. CaMKKα mutants exhibited normal long-term spatial memory formation and cued fear conditioning but showed deficits in context fear during both conditioning and long-term follow-up testing. They also exhibited impaired activation of the downstream kinase CaMKIV/Gr and its substrate, the transcription factor cyclic AMP-responsive element binding protein (CREB) upon fear conditioning. Unlike CaMKIV/Gr-deficient mice, the CaMKKα mutants exhibited normal long-term potentiation and normal levels of anxiety-like behavior. These results demonstrate a selective role for CaMKKα in contextual fear memory and suggest that different combinations of upstream and downstream components of the CaMK cascade may serve distinct physiological functions.

Requirement for Ca2+/Calmodulin-Dependent Kinase Type IV/Gr in Setting the Thymocyte Selection Threshold

The outcome of thymocyte selection is influenced by the nature of Ca2+ signals transduced by the TCR. Robust Ca2+ responses characterize high-affinity, negatively selecting peptide/TCR interactions, while modest responses typify lower-affinity, positively selecting interactions. To elucidate mechanisms by which thymocytes decode distinct Ca2+ signals, we examined selection events in mice lacking Ca2+/calmodulin-dependent protein kinase type IV/Gr (CaMKIV/Gr), which is enriched in thymocytes. CaMKIV/Gr-deficient thymocytes exhibited impaired positive selection and defective Ca2+-dependent gene transcription. Significantly, CaMKIV/Gr deficiency raised the selection threshold of peptide/TCR interactions such that a peptide that normally induced weak negative selection instead promoted positive selection. These results demonstrate an important role for CaMKIV/Gr in sensitizing thymocytes to selection by low-affinity peptides.

Glucagon regulates ACC activity in adipocytes through the CAMKKβ/AMPK pathway

Glucagon is important for regulating lipid metabolism in part through its inhibition of fatty acid synthesis in adipocytes. Acetyl-CoA carboxylase 1 (ACC1) is the rate-limiting enzyme for fatty acid synthesis. Glucagon has been proposed to activate cAMP-dependent protein kinase A (PKA), which phosphorylates ACC1 to attenuate the lipogenic activity of ACC1. Because AMP-activated protein kinase (AMPK) also inhibits fatty acid synthesis by phosphorylation of ACC1, we examined the involvement of AMPK and its upstream kinase in the glucagon-elicited signaling in adipocytes in vitro and in vivo. LC-MS-MS analysis suggested that ACC1 was phosphorylated only at Ser(79), an AMPK-specific site, in glucagon-treated adipocytes. Pharmacological inhibitors and siRNA knockdown of AMPK or PKA in adipocytes demonstrate that glucagon regulates ACC1 and ACC2 activity through AMPK but not PKA. By using Ca(2+)/calmodulin-dependent protein kinase kinase-β knockout (CaMKKβ(-/-)) mice and cultured adipocytes, we further show that glucagon activates the CaMKKβ/AMPK/ACC cascade. Additionally, fasting increases the phosphorylation of AMPK and ACC in CaMKKβ(+/+) but not CaMKKβ(-/-) mice. These results indicate that CaMKKβ/AMPK signaling is an important molecular component in regulating lipid metabolism in adipocytes responding to glucagon and could be a therapeutic target for the dysregulation of energy storage.