Dirk Mielenz

Blockade of receptor activator of nuclear factor-κB (RANKL) signaling improves hepatic insulin resistance and prevents development of diabetes mellitus.

Hepatic insulin resistance is a driving force in the pathogenesis of type 2 diabetes mellitus (T2DM) and is tightly coupled with excessive storage of fat and the ensuing inflammation within the liver. There is compelling evidence that activation of the transcription factor nuclear factor-κB (NF-κB) and downstream inflammatory signaling pathways systemically and in the liver are key events in the etiology of hepatic insulin resistance and β-cell dysfunction, although the molecular mechanisms involved are incompletely understood. We here test the hypothesis that receptor activator of NF-κB ligand (RANKL), a prototypic activator of NF-κB, contributes to this process using both an epidemiological and experimental approach. In the prospective population-based Bruneck Study, a high serum concentration of soluble RANKL emerged as a significant (P < 0.001) and independent risk predictor of T2DM manifestation. In close agreement, systemic or hepatic blockage of RANKL signaling in genetic and nutritional mouse models of T2DM resulted in a marked improvement of hepatic insulin sensitivity and amelioration or even normalization of plasma glucose concentrations and glucose tolerance. Overall, this study provides evidence for a role of RANKL signaling in the pathogenesis of T2DM. If so, translation to the clinic may be feasible given current pharmacological strategies to lower RANKL activity to treat osteoporosis.

Orphan nuclear receptor NR4A1 regulates transforming growth factor-β signaling and fibrosis

Mesenchymal responses are an essential aspect of tissue repair. Failure to terminate this repair process correctly, however, results in fibrosis and organ dysfunction. Therapies that block fibrosis and restore tissue homeostasis are not yet available for clinical use. Here we characterize the nuclear receptor NR4A1 as an endogenous inhibitor of transforming growth factor-β (TGF-β) signaling and as a potential target for anti-fibrotic therapies. NR4A1 recruits a repressor complex comprising SP1, SIN3A, CoREST, LSD1, and HDAC1 to TGF-β target genes, thereby limiting pro-fibrotic TGF-β effects. Even though temporary upregulation of TGF-β in physiologic wound healing induces NR4A1 expression and thereby creates a negative feedback loop, the persistent activation of TGF-β signaling in fibrotic diseases uses AKT- and HDAC-dependent mechanisms to inhibit NR4A1 expression and activation. Small-molecule NR4A1 agonists can overcome this lack of active NR4A1 and inhibit experimentally-induced skin, lung, liver, and kidney fibrosis in mice. Our data demonstrate a regulatory role of NR4A1 in TGF-β signaling and fibrosis, providing the first proof of concept for targeting NR4A1 in fibrotic diseases.

Lipid Rafts Associate with Intracellular B Cell Receptors and Exhibit a B Cell Stage-Specific Protein Composition

Lipid rafts serve as platforms for BCR signal transduction. To better define the molecular basis of these membrane microdomains, we used two-dimensional gel electrophoresis and mass spectrometry to characterize lipid raft proteins from mature as well as immature B cell lines. Of 51 specific raft proteins, we identified a total of 18 proteins by peptide mass fingerprinting. Among them, we found vacuolar ATPase subunits α-1 and β-2, vimentin, γ-actin, mitofilin, and prohibitin. None of these has previously been reported in lipid rafts of B cells. The differential raft association of three proteins, including a novel potential signaling molecule designated swiprosin-1, correlated with the stage-specific sensitivity of B cells to BCR-induced apoptosis. In addition, MHC class II molecules were detected in lipid rafts of mature, but not immature B cells. This intriguing finding points to a role for lipid rafts in regulating Ag presentation during B cell maturation. Finally, a fraction of the BCR in the B cell line CH27 was constitutively present in lipid rafts. Surprisingly, this fraction was neither expressed at the cell surface nor fully O-glycosylated. Thus, we conclude that partitioning the BCR into lipid rafts occurs in the endoplasmic reticulum/cis-Golgi compartment and may represent a control mechanism for surface transport.

Fraternal twins: Swiprosin-1/EFhd2 and Swiprosin-2/EFhd1, two homologous EF-hand containing calcium binding adaptor proteins with distinct functions

Changes in the intracellular calcium concentration govern cytoskeletal rearrangement, mitosis, apoptosis, transcriptional regulation or synaptic transmission, thereby, regulating cellular effector and organ functions. Calcium binding proteins respond to changes in the intracellular calcium concentration with structural changes, triggering enzymatic activation and association with downstream proteins. One type of calcium binding proteins are EF-hand super family proteins. Here, we describe two recently discovered homologous EF-hand containing adaptor proteins, Swiprosin-1/EF-hand domain containing 2 (EFhd2) and Swiprosin-2/EF-hand domain containing 1 (EFhd1), which are related to allograft inflammatory factor-1 (AIF-1). For reasons of simplicity and concision we propose to name Swiprosin-1/EFhd2 and Swiprosin-2/EFhd1 from now on EFhd2 and EFhd1, according to their respective gene symbols. AIF-1 and Swiprosin-1/EFhd2 are already present in Bilateria, for instance in Drosophila melanogaster and Caenhorhabditis elegans. Swiprosin-2/EFhd1 arose later from gene duplication in the tetrapodal lineage. Secondary structure prediction of AIF-1 reveals disordered regions and one functional EF-hand. Swiprosin-1/EFhd2 and Swiprosin-2/EFhd1 exhibit a disordered region at the N-terminus, followed by two EF-hands and a coiled-coil domain. Whereas both proteins are similar in their predicted overall structure they differ in a non-homologous stretch of 60 amino acids just in front of the EF-hands. AIF-1 controls calcium-dependent cytoskeletal rearrangement in innate immune cells by means of its functional EF-hand. We propose that Swiprosin-1/EFhd2 as well is a cytoskeleton associated adaptor protein involved in immune and brain cell function. Pro-inflammatory conditions are likely to modulate expression and function of Swiprosin-1/EFhd2. Swiprosin-2/EFhd1, on the other hand, modulates apoptosis and differentiation of neuronal and muscle precursor cells, probably through an association with mitochondria. We suggest furthermore that Swiprosin-2/EFhd1 is part of a cellular response to oxidative stress, which could explain its pro-survival activity in neuronal, muscle and perhaps some malignant tissues.

The novel adaptor protein Swiprosin-1 enhances BCR signals and contributes to BCR-induced apoptosis.

B-cell receptor (BCR) signals are essential for B-cell differentiation, homeostasis and negative selection, which are regulated by the strength and quality of BCR signals. Recently, we identified a new adaptor protein, Swiprosin-1, in lipid rafts of B-cell lines that undergo apoptosis after BCR stimulation. During murine B-cell development, Swiprosin-1 exhibited highest expression in immature B cells of the bone marrow, but was also expressed in resting and activated splenic B cells and in non-lymphoid tissue, especially in the brain. Ectopic expression of Swiprosin-1 in the immature murine B-cell line WEHI231 enhanced spontaneous and BCR-induced apoptosis. In contrast, short hairpin RNA (shRNA)-mediated downregulation of Swiprosin-1 impaired specifically spontaneous and BCR-elicited apoptosis, but not BCR-induced G1 cell cycle arrest and upregulation of the cell cycle inhibitor p27Kip1. In accordance, Swiprosin-1 abundance regulated net cell growth of WEHI231 cell populations through reciprocal regulation of Bcl-xL, but not Bim, thereby controlling spontaneous apoptosis. Swiprosin-1-enhanced apoptosis was blocked through nuclear factor κB-activating stimuli, namely B-cell-activating factor of the TNF family, anti-CD40 and lipopolysaccharide (LPS). This correlated with enhanced BCR-induced IκB-α phosphorylation and degradation in cells expressing a Swiprosin-1-specific shRNA. Finally, ectopic Swiprosin-1 expression enhanced BCR-induced cell death in primary, LPS-stimulated splenic B cells. Hence, Swiprosin-1 may regulate lifespan and BCR signaling thresholds in immature B cells.

Analysis of the CCR7 expression on murine bone marrow-derived and spleen dendritic cells

About 40% of bone marrow‐derived dendritic cells (BM‐DCs) generated from stem cells of C57BL/6 (B6.WT) mice differentiate in the presence of granulocyte macrophage‐colony stimulating factor (GM‐CSF) without further stimuli to mature DCs. These cells are characterized by high levels of major histocompatibility complex class II, CD40, and CD86 on their surface. Recent studies have revealed that tumor necrosis factor (TNF) is crucial for maturation of BM‐DCs. However, once matured, the phenotype of mature TNF‐negative C57BL/6 (B6.TNF−/−) and B6.WT BM‐DCs is comparable. Both expressed high levels of CD40 and CD86 and were positive for mRNA of the chemokine receptor (CCR)7. To extend our studies, we generated a monoclonal antibody (mAb) specific for mouse CCR7. This mAb allowed us to analyze the surface expression of CCR7 during maturation of B6.WT and B6.TNF−/− BM‐DCs in the presence of GM‐CSF and stimulated with TNF or lipopolysaccharide (LPS) and to compare it with the CCR7 expression on ex vivo‐isolated splenic DCs with or without additional stimulation. Our results showed that CCR7 expression on murine BM‐DCs is an indication of cell maturity. Incubation with LPS induced the maturation of all BM‐DCs in culture but increased the number of mature CCR7+ splenic DCs only marginally.

Swiprosin-1/EFhd2 Controls B Cell Receptor Signaling through the Assembly of the B Cell Receptor, Syk, and Phospholipase C γ2 in Membrane Rafts

Compartmentalization of the BCR in membrane rafts is important for its signaling capacity. Swiprosin-1/EFhd2 (Swip-1) is an EF-hand and coiled-coil–containing adaptor protein with predicted Src homology 3 (SH3) binding sites that we identified in membrane rafts. We showed previously that Swip-1 amplifies BCR-induced apoptosis; however, the mechanism of this amplification was unknown. To address this question, we overexpressed Swip-1 and found that Swip-1 amplified the BCR-induced calcium flux in WEHI231, B62.1, and Bal17 cells. Conversely, the BCR-elicited calcium flux was strongly attenuated in Swip-1–silenced WEHI231 cells, and this was due to a decreased calcium mobilization from intracellular stores. Complementation of Swip-1 expression in Swip-1–silenced WEHI231 cells restored the BCR-induced calcium flux and enhanced spleen tyrosine kinase (Syk) tyrosine phosphorylation and activity as well as SLP65/BLNK/BASH and phospholipase C γ2 (PLCγ2) tyrosine phosphorylation. Furthermore, Swip-1 induced the constitutive association of the BCR itself, Syk, and PLCγ2 with membrane rafts. Concomitantly, Swip-1 stabilized the association of BCR with tyrosine-phosphorylated proteins, specifically Syk and PLCγ2, and enhanced the constitutive interaction of Syk and PLCγ2 with Lyn. Interestingly, Swip-1 bound to the rSH3 domains of the Src kinases Lyn and Fgr, as well as to that of PLCγ. Deletion of the predicted SH3-binding region in Swip-1 diminished its association and that of Syk and PLCγ2 with membrane rafts, reduced its interaction with the SH3 domain of PLCγ, and diminished the BCR-induced calcium flux. Hence, Swip-1 provides a membrane scaffold that is required for the Syk-, SLP-65–, and PLCγ2-dependent BCR-induced calcium flux.

Immunoglobulin μ Heavy Chains Do Not Mediate Tyrosine Phosphorylation of Igα from the ER-cis-Golgi

Signals delivered by Ig receptors guide the development of functional B lymphocytes. For example, clonal expansion of early μ heavy chain (μHC)-positive pre-B cells requires the assembly of a signal-competent pre-B cell receptor complex (pre-BCR) consisting of a μHC, a surrogate L chain, and the signal dimer Igαβ. However, only a small fraction of the pre-BCR is transported to the cell surface, suggesting that pre-BCR signaling initiates already from an intracellular compartment, e.g., the endoplasmic reticulum (ER). The finding that differentiation of pre-B cells and allelic exclusion at the IgH locus take place in surrogate L chain-deficient mice further supports the presence of a μHC-mediated intracellular signal pathway. To determine whether a signal-competent Ig complex can already be assembled in the ER, we analyzed the consequence of pervanadate on tyrosine phosphorylation of Igα in J558L plasmacytoma and 38B9 pre-B cells transfected with either a transport-competent IgL chain-pairing or an ER-retained nonpairing μHC. Flow cytometry, combined Western blot-immunoprecipitation-kinase assays, and confocal microscopy revealed that both the nonpairing and pairing μHC assembled with the Igαβ dimer; however, in contrast to a pairing μHC, the nonpairing μHC was retained in the ER-cis-Golgi compartment, and neither colocalized with the src kinase lyn nor induced tyrosine phosphorylation of Igα after pervanadate treatment of cells. On the basis of these findings, we propose that a signal-competent Ig complex consisting of μHC, Igαβ, and associated kinases is assembled in a post-ER compartment, thereby supporting the idea that a pre-BCR must be transported to the cell surface to initiate pre-BCR signaling.

Nanoparticles size-dependently initiate self-limiting NETosis-driven inflammation

Significance The current widespread exposure of humans to natural as well as man-made nanomaterials due to the deployment of nanoparticles (NPs) as food additives, as vaccine- or drug-delivery vehicles, and in diagnostic procedures encourages the evaluation of their interaction with the innate immune system. Understanding how organisms cope with hydrophobic and chemically inert particulate matter, which is excluded from metabolic processing, is of major importance for interpreting the responses associated with the use of NPs in the biosphere. The containment of NPs within neutrophil-derived aggregates locally orchestrates the resolution of inflammation. Overriding this mechanism bears the risk of inducing chronic inflammation and causing tissue damage. The critical size for strong interaction of hydrophobic particles with phospholipid bilayers has been predicted to be 10 nm. Because of the wide spreading of nonpolar nanoparticles (NPs) in the environment, we aimed to reveal the ability of living organisms to entrap NPs via formation of neutrophil extracellular traps (NETs). Upon interaction with various cell types and tissues, 10- to 40-nm-sized NPs induce fast (<20 min) damage of plasma membranes and instability of the lysosomal compartment, leading to the immediate formation of NETs. In contrast, particles sized 100–1,000 nm behaved rather inertly. Resulting NET formation (NETosis) was accompanied by an inflammatory reaction intrinsically endowed with its own resolution, demonstrated in lungs and air pouches of mice. Persistence of small NPs in joints caused unremitting arthritis and bone remodeling. Small NPs coinjected with antigen exerted adjuvant-like activity. This report demonstrates a cellular mechanism that explains how small NPs activate the NETosis pathway and drive their entrapping and resolution of the initial inflammatory response.

The B cell receptor-induced calcium flux involves a calcium mediated positive feedback loop

The B cell receptor (BCR)-elicited calcium flux results in activation of mature B cells. We have recently shown that the adaptor protein Swiprosin-1/EFhd2 (EFhd2) amplifies the BCR-induced calcium flux in B cell lines. EFhd2 is a calcium binding adaptor protein with two predicted EF-hands. Here we asked whether these domains are functional and control its function. Using a blot-overlay assay with radioactive calcium we show that both EF-hands of EFhd2 have an intrinsic capacity to bind calcium. Equilibrium centrifugation confirmed that EFhd2 binds 2 calcium ions, with an apparent Kd of 110 μM. Point mutations revealed that the conserved residues E116 and E152, which reside in the canonical calcium binding loop in EF-hands 1 and 2, are essential for calcium binding by EFhd2. These mutations as well as deletion of the EF-hands, in particular EF-hand 1, abolished the ability of EFhd2 to restore BCR-induced calcium signaling in EFhd2-deficient WEHI231 cells. N-terminal deletions, but not C-terminal deletions, acted similarly. Thus, the N-terminal part of EFhd2 as well as calcium binding to its EF-hands control the intracellular calcium concentration in response to BCR stimulation in WEHI231 cells. Hence, EFhd2 regulates the BCR-elicited calcium flux through a calcium-dependent positive feedback mechanism in WEHI231 cells.