Emerging concepts suggest that macrophage functional phenotype is regulated by transcription factors that define alternative activation states. We found that RBP-J, the major nuclear transducer of Notch signaling, augmented TLR4-induced expression of key mediators of classically activated M1 macrophages and thus innate immune responses to L. monocytogenes. Notch-RBP-J signaling controlled expression of the transcription factor IRF8 that induced downstream M1-specific genes. RBP-J promoted IRF8 protein synthesis by selectively augmenting IRAK2-dependent TLR4 signaling to the MNK1 kinase and downstream translation initiation control through eIF4E. These results define a signaling network in which Notch-RBP-J and TLR signaling are integrated at the level of IRF8 protein synthesis and identify a mechanism by which heterologous signaling pathways can regulate TLR-induced inflammatory macrophage polarization.

The metalloproteinase and major amyloid precursor protein (APP) ␣-secretase candidate ADAM10 is responsible for the shedding of proteins important for brain development, such as cadherins, ephrins, and Notch receptors. Adam10 ؊/؊ mice die at embryonic day 9.5, due to major defects in development of somites and vasculogenesis. To investigate the function of ADAM10 in brain, we generated Adam10 conditional knock-out (cKO) mice using a Nestin-Cre promotor, limiting ADAM10 inactivation to neural progenitor cells (NPCs) and NPC-derived neurons and glial cells. The cKO mice die perinatally with a disrupted neocortex and a severely reduced ganglionic eminence, due to precocious neuronal differentiation resulting in an early depletion of progenitor cells. Premature neuronal differentiation is associated with aberrant neuronal migration and a disorganized laminar architecture in the neocortex. Neurospheres derived from Adam10 cKO mice have a disrupted sphere organization and segregated more neurons at the expense of astrocytes. We found that Notch-1 processing was affected, leading to downregulation of several Notch-regulated genes in Adam10 cKO brains, in accordance with the central role of ADAM10 in this signaling pathway and explaining the neurogenic phenotype. Finally, we found that ␣-secretasemediated processing of APP was largely reduced in these neurons, demonstrating that ADAM10 represents the most important APP ␣-secretase in brain. Our study reveals that ADAM10 plays a central role in the developing brain by controlling mainly Notch-dependent pathways but likely also by reducing surface shedding of other neuronal membrane proteins including APP.

SummaryProteolytic enzymes belonging to the A Disintegin And Metalloproteinase (ADAM) family are able to cleave transmembrane proteins close to the cell surface, in a process referred to as ectodomain shedding. Substrates for ADAMs include growth factors, cytokines, chemokines and adhesion molecules, and, as such, many ADAM proteins play crucial roles in cell-cell adhesion, extracellular and intracellular signaling, cell differentiation and cell proliferation. In this Review, we summarize the fascinating roles of ADAMs in embryonic and adult tissue development in both vertebrates and invertebrates. Key words: ADAM, Notch, Ectodomain shedding, Cell fate determination, DifferentiationIntroduction Proteins belonging to the 'A Disintegrin And Metalloproteinase' (ADAM) family are membrane-anchored proteases that are able to cleave the extracellular domains of membrane-bound proteins in a process known as 'ectodomain shedding'. Typical substrates of ADAM proteases are growth factors, cytokines, chemokines and their receptors, as well as cell adhesion molecules and differentiation factors (Reiss and Saftig, 2009). ADAMs were initially discovered as novel type I transmembrane proteins with homology to snake venom integrin ligands and that played a functional role during guinea-pig sperm-egg fusion (Blobel et al., 1992). Subsequently, approximately half of the ADAM family members were predicted and then shown to possess zinc-dependent protease activity related to that exhibited by adamalysins metallopeptidases (Wolfsberg et al., 1993; Huxley-Jones et al., 2007). These active 'sheddases ' (ADAM8, 9, 10, 12, 15, 17, 19, 20, 21, 28, 30 and 33) share a typical consensus sequence (HEXGHXXGXXHD) that is present in zinc-binding proteases (Bode et al., 1993). So far, 40 family members have been identified in the mammalian genome (Puente and Lopez-Otin, 2004; Edwards et al., 2008), of which 37 are expressed in mice (most of them in a testis-specific manner) and 22 are thought to be expressed in humans (Table 1). In both mouse and human, intronless coding sequences probably representing pseudogenes (e.g. human ADAM5P and ADAM6) have also been described. The expression of ADAM or ADAM-related proteins has also been described in many different species, including the yeast Schizosaccharomyces pombe, the nematode worm Caenorhabditis elegans, the frog Xenopus laevis, the zebrafish Danio rerio and the fruitfly Drosophila melanogaster (Alfandari et al., 1997;Nakamura et al., 2004; Huxley-Jones et al., 2007; Iida et al., 2010). It was quickly realized that ADAMs are widely expressed and play fundamental roles during developmental processes, by regulating cell-cell and cell-matrix interactions and by modulating differentiation, migration, receptor-ligand signaling or repulsion (Becherer and Blobel, 2003).Following studies of the catalytically active ADAMs, ectodomain shedding emerged as a central biological event. This proteolytic process primarily affects type I and type II transmembrane proteins, although glycosylphosphatidylinisot...

SUMMARYThe disintegrin and metalloproteinase Adam10 has been implicated in the regulation of key signaling pathways that determine skin morphogenesis and homeostasis. To address the in vivo relevance of Adam10 in the epidermis, we have selectively disrupted Adam10 during skin morphogenesis and in adult skin. K14-Cre driven epidermal Adam10 deletion leads to perinatal lethality, barrier impairment and absence of sebaceous glands. A reduction of spinous layers, not associated with differences in either proliferation or apoptosis, indicates that loss of Adam10 triggers a premature differentiation of spinous keratinocytes. The few surviving K14-Adam10-deleted mice and mice in which Adam10 was deleted postnatally showed loss of hair, malformed vibrissae, epidermal hyperproliferation, cyst formation, thymic atrophy and upregulation of the cytokine thymic stromal lymphopoetin (TSLP), thus indicating non cell-autonomous multi-organ disease resulting from a compromised barrier. Together, these phenotypes closely resemble skin specific Notch pathway loss-of-function phenotypes. Notch processing is indeed strongly reduced resulting in decreased levels of Notch intracellular domain fragment and functional Notch signaling. The data identify Adam10 as the major Site-2 processing enzyme for Notch in the epidermis in vivo, and thus as a central regulator of skin development and maintenance.

A disintegrin and metalloproteinase10 (ADAM10) has been implicated as a major sheddase responsible for the ectodomain shedding of a number of important surface molecules including the amyloid precursor protein and cadherins. Despite a well-documented role of ADAM10 in health and disease, little is known about the regulation of this protease. To address this issue we conducted a split-ubiquitin yeast two-hybrid screen to identify membrane proteins that interact with ADAM10. The yeast experiments and co-immunoprecipitation studies in mammalian cell lines revealed tetraspanin15 (TSPAN15) to specifically associate with ADAM10. Overexpression of TSPAN15 or RNAi-mediated knockdown of TSPAN15 led to significant changes in the maturation process and surface expression of ADAM10. Expression of an endoplasmic reticulum (ER) retention mutant of TSPAN15 demonstrated an interaction with ADAM10 already in the ER. Pulse-chase experiments confirmed that TSPAN15 accelerates the ER-exit of the ADAM10-TSPAN15 complex and stabilizes the active form of ADAM10 at the cell surface. Importantly, TSPAN15 also showed the ability to mediate the regulation of ADAM10 protease activity exemplified by an increased shedding of N-cadherin and the amyloid precursor protein. In conclusion, our data show that TSPAN15 is a central modulator of ADAM10-mediated ectodomain shedding. Therapeutic manipulation of its expression levels may be an additional approach to specifically regulate the activity of the amyloid precursor protein alpha-secretase ADAM10.

This study is the first to functionally investigate the role of PP1/PP2A for Ca homeostasis in diseased human myocardium. Our data indicate that a modulation of phosphatase activity potently impacts Ca cycling properties. An activation of PP1 counteracts increased kinase activity in heart failure and successfully seals the arrhythmogenic SR Ca leak. It may thus represent a promising future antiarrhythmic therapeutic approach.

Background Considerable evidence suggests that CaMKII overactivity plays a crucial role in the pathophysiology of heart failure (HF), a condition characterized by excessive β-adrenoceptor (β-AR) stimulation. Recent studies indicate a significant crosstalk between β-AR signaling and CaMKII activation presenting CaMKII as a possible downstream mediator of detrimental β-AR signaling in HF. In this study we investigated the effect of chronic β-AR blocker treatment on CaMKII activity in human and experimental HF. Methods and Results Immunoblot analysis of myocardium from end stage HF patients (n=12) and non-HF subjects undergoing cardiac surgery (n=12) treated with β-AR blockers revealed no difference in CaMKII activity when compared to non-β-AR-blocker-treated patients. CaMKII activity was judged by analysis of CaMKII expression, autophosphorylation and oxidation and by investigating the phosphorylation status of CaMKII downstream targets. To further evaluate these findings, CaMKIIδC transgenic mice were treated with the β1-AR blocker metoprolol (270 mg/kg*d). Metoprolol significantly reduced transgene-associated mortality (n≥29, p<0.001), attenuated the development of cardiac hypertrophy (−14±6% heart weight/tibia length, p<0.05) and strongly reduced ventricular arrhythmias (−70±22% PVCs, p<0.05). On a molecular level, metoprolol expectedly decreased PKA dependent phospholamban (PLN) and ryanodine receptor 2 (RyR2) phosphorylation (−42±9% for P-PLN-S16 and −22±7% for P-RyR2-S2808, p<0.05). However, this was neither paralleled by a reduction in CaMKII autophosphorylation, oxidation and substrate binding nor a change in the phosphorylation of CaMKII downstream target proteins (n≥11). The lack of CaMKII modulation by β-AR blocker treatment was confirmed in healthy wildtype mice receiving metoprolol. Conclusions Chronic β-AR blocker therapy in patients and in a mouse model of CaMKII-induced HF is not associated with a change in CaMKII activity. Thus, our data suggests that the molecular effects of β-AR blockers are not based on a modulation of CaMKII. Directly targeting CaMKII may therefore further improve HF therapy in addition to β-AR blockade.

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