Christian Schmedt

Essential role of Src-family protein tyrosine kinases in NF-κB activation during B cell development

The nature of signals that govern the development of immunoglobulin heavy chain-dependent B cells is largely unknown. Using mice deficient for the B cell-expressed Src-family protein tyrosine kinases (SFKs) Blk, Fyn and Lyn, we show an essential role of these kinases in pre-B cell receptor (pre-BCR)– mediated NF-κB activation and B cell development. This signaling defect is SFK specific, as a deficiency in Syk, which controls pre-B cell development, does not affect NF-κB induction. Impaired NF-κB induction was overcome by the activation of protein kinase C (PKC)-λ, thus suggesting the involvement of PKC-λ in pre-BCR–mediated SFK-dependent activation of NF-κB. Our data show the existence of a functionally distinct SFK signaling module responsible for pre-BCR–mediated NF-κB activation and B cell development.

Oxysterols direct immune cell migration via EBI2.

Epstein–Barr virus-induced gene 2 (EBI2, also known as GPR183) is a G-protein-coupled receptor that is required for humoral immune responses; polymorphisms in the receptor have been associated with inflammatory autoimmune diseases. The natural ligand for EBI2 has been unknown. Here we describe the identification of 7α,25-dihydroxycholesterol (also called 7α,25-OHC or 5-cholesten-3β,7α,25-triol) as a potent and selective agonist of EBI2. Functional activation of human EBI2 by 7α,25-OHC and closely related oxysterols was verified by monitoring second messenger readouts and saturable, high-affinity radioligand binding. Furthermore, we find that 7α,25-OHC and closely related oxysterols act as chemoattractants for immune cells expressing EBI2 by directing cell migration in vitro and in vivo. A critical enzyme required for the generation of 7α,25-OHC is cholesterol 25-hydroxylase (CH25H). Similar to EBI2 receptor knockout mice, mice deficient in CH25H fail to position activated B cells within the spleen to the outer follicle and mount a reduced plasma cell response after an immune challenge. This demonstrates that CH25H generates EBI2 biological activity in vivo and indicates that the EBI2–oxysterol signalling pathway has an important role in the adaptive immune response.

Inducible Ablation of Melanopsin-Expressing Retinal Ganglion Cells Reveals Their Central Role in Non-Image Forming Visual Responses

Rod/cone photoreceptors of the outer retina and the melanopsin-expressing retinal ganglion cells (mRGCs) of the inner retina mediate non-image forming visual responses including entrainment of the circadian clock to the ambient light, the pupillary light reflex (PLR), and light modulation of activity. Targeted deletion of the melanopsin gene attenuates these adaptive responses with no apparent change in the development and morphology of the mRGCs. Comprehensive identification of mRGCs and knowledge of their specific roles in image-forming and non-image forming photoresponses are currently lacking. We used a Cre-dependent GFP expression strategy in mice to genetically label the mRGCs. This revealed that only a subset of mRGCs express enough immunocytochemically detectable levels of melanopsin. We also used a Cre-inducible diphtheria toxin receptor (iDTR) expression approach to express the DTR in mRGCs. mRGCs develop normally, but can be acutely ablated upon diphtheria toxin administration. The mRGC-ablated mice exhibited normal outer retinal function. However, they completely lacked non-image forming visual responses such as circadian photoentrainment, light modulation of activity, and PLR. These results point to the mRGCs as the site of functional integration of the rod/cone and melanopsin phototransduction pathways and as the primary anatomical site for the divergence of image-forming and non-image forming photoresponses in mammals.

Protein Kinase C β Controls Nuclear Factor κB Activation in B Cells Through Selective Regulation of the IκB Kinase α

Activation of the nuclear factor (NF)-κB transcription complex by signals derived from the surface expressed B cell antigen receptor controls B cell development, survival, and antigenic responses. Activation of NF-κB is critically dependent on serine phosphorylation of the IκB protein by the multi-component IκB kinase (IKK) containing two catalytic subunits (IKKα and IKKβ) and one regulatory subunit (IKKγ). Using mice deficient for protein kinase C β (PKCβ) we show an essential role of PKCβ in the phosphorylation of IKKα and the subsequent activation of NF-κB in B cells. Defective IKKα phosphorylation correlates with impaired B cell antigen receptor–mediated induction of the pro-survival protein Bcl-xL. Lack of IKKα phosphorylation and defective NF-κB induction in the absence of PKCβ explains the similarity in immunodeficiencies caused by PKCβ or IKKα ablation in B cells. Furthermore, the well established functional cooperation between the protein tyrosine kinase Brutons tyrosine kinase (Btk), which regulates the activity of NF-κB and PKCβ, suggests PKCβ as a likely serine/threonine kinase component of the Btk-dependent NF-κB activating signal transduction chain downstream of the BCR.

Csk controls antigen receptor-mediated development and selection of T-lineage cells

The development and function of αβT lymphocytes depend on signals derived from pre-T and αβT cell receptors (preTCR and αβTCR) (reviewed in refs 1, 2). The engagement of these receptors leads to the activation of Lck and Fyn,, which are protein tyrosine kinases (PTKs) of the Src family. It remains unclear to what extent the activation of Src-family PTKs can direct the differentiation steps triggered by preTCR and αβTCR. Here we show that the inactivation of the negative regulator of Src-family PTKs, carboxy-terminal Src kinase (Csk), in immature thymocytes abrogates the requirement for preTCR, αβTCR and major histocompatibility complex (MHC) class II for the development of CD4+8+ double-positive and CD4+ single-positive thymocytes as well as peripheral CD4 αβT-lineage cells. These data show that Csk and its substrates are required to establish preTCR/αβTCR-mediated control over the development of αβT cells.

Protein kinase C beta controls nuclear factor kappaB activation in B cells through selective regulation of the IkappaB kinase alpha.

Activation of the nuclear factor (NF)-κB transcription complex by signals derived from the surface expressed B cell antigen receptor controls B cell development, survival, and antigenic responses. Activation of NF-κB is critically dependent on serine phosphorylation of the IκB protein by the multi-component IκB kinase (IKK) containing two catalytic subunits (IKKα and IKKβ) and one regulatory subunit (IKKγ). Using mice deficient for protein kinase C β (PKCβ) we show an essential role of PKCβ in the phosphorylation of IKKα and the subsequent activation of NF-κB in B cells. Defective IKKα phosphorylation correlates with impaired B cell antigen receptor–mediated induction of the pro-survival protein Bcl-xL. Lack of IKKα phosphorylation and defective NF-κB induction in the absence of PKCβ explains the similarity in immunodeficiencies caused by PKCβ or IKKα ablation in B cells. Furthermore, the well established functional cooperation between the protein tyrosine kinase Brutons tyrosine kinase (Btk), which regulates the activity of NF-κB and PKCβ, suggests PKCβ as a likely serine/threonine kinase component of the Btk-dependent NF-κB activating signal transduction chain downstream of the BCR.

TLE3 Is a Dual-Function Transcriptional Coregulator of Adipogenesis

PPARγ and Wnt signaling are central positive and negative regulators of adipogenesis, respectively. Here we identify the groucho family member TLE3 as a transcriptional integrator of the PPARγ and Wnt pathways. TLE3 is a direct target of PPARγ that participates in a feed-forward loop during adipocyte differentiation. TLE3 enhances PPARγ activity and functions synergistically with PPARγ on its target promoters to stimulate adipogenesis. At the same time, induction of TLE3 during differentiation provides a mechanism for termination of Wnt signaling. TLE3 antagonizes TCF4 activation by β-catenin in preadipocytes, thereby inhibiting Wnt target gene expression and reversing β-catenin-dependent repression of adipocyte gene expression. Transgenic expression of TLE3 in adipose tissue in vivo mimics the effects of PPARγ agonist and ameliorates high-fat-diet-induced insulin resistance. Our data suggest that TLE3 acts as a dual-function switch, driving the formation of both active and repressive transcriptional complexes that facilitate the adipogenic program.

The Ubiquitously Expressed Csk Adaptor Protein Cbp Is Dispensable for Embryogenesis and T-Cell Development and Function

ABSTRACT Regulation of Src family kinase (SFK) activity is indispensable for a functional immune system and embryogenesis. The activity of SFKs is inhibited by the presence of the carboxy-terminal Src kinase (Csk) at the cell membrane. Thus, recruitment of cytosolic Csk to the membrane-associated SFKs is crucial for its regulatory function. Previous studies utilizing in vitro and transgenic models suggested that the Csk-binding protein (Cbp), also known as phosphoprotein associated with glycosphingolipid microdomains (PAG), is the membrane adaptor for Csk. However, loss-of-function genetic evidence to support this notion was lacking. Herein, we demonstrate that the targeted disruption of the cbp gene in mice has no effect on embryogenesis, thymic development, or T-cell functions in vivo. Moreover, recruitment of Csk to the specialized membrane compartment of “lipid rafts” is not impaired by Cbp deficiency. Our results indicate that Cbp is dispensable for the recruitment of Csk to the membrane and that another Csk adaptor, yet to be discovered, compensates for the loss of Cbp.

C-Terminal Src Kinase Controls Acute Inflammation and Granulocyte Adhesion

To establish whether the widely expressed regulator of Src family kinases Csk contributes to the control of acute inflammation in vivo, we inactivated csk in granulocytes by conditional mutagenesis (Cre/loxP). Mutant mice (Csk-GEcre) developed acute multifocal inflammation in skin and lung. Animals were protected from the disease in a microbiologically controlled environment, but remained hypersensitive to LPS-induced shock. Csk-deficient granulocytes showed enhanced spontaneous and ligand-induced degranulation with hyperinduction of integrins. This hyperresponsiveness was associated with hyperadhesion and impaired migratory responses in vitro. Hyperphosphorylation of key signaling proteins such as Syk and Paxillin in mutant granulocytes further supported breakdown of the activation threshold set by Csk. By enforcing the need for ligand engagement Csk thus prevents premature granulocyte recruitment while supporting the motility of stimulated cells through negative regulation of cell adhesion.

Deletion of the potassium channel Kv12.2 causes hippocampal hyperexcitability and epilepsy

We found the voltage-gated K+ channel Kv12.2 to be a potent regulator of excitability in hippocampal pyramidal neurons. Genetic deletion and pharmacologic block of Kv12.2 substantially reduced the firing threshold of these neurons. Kv12.2−/− (also known as Kcnh3−/−) mice showed signs of persistent neuronal hyperexcitability including frequent interictal spiking, spontaneous seizures and increased sensitivity to the chemoconvulsant pentylenetetrazol.