β1-integrin protects keratinocyte stem cells (KSC) from cell-detachment apoptosis (`anoikis'). Here we show that caspase-8 active protein is detected in both young transit amplifying (TA) cells and TA cells, but not in KSC. On suspension, caspases are activated earlier in young TA than in KSC, whereas anti-β1-integrin neutralizing antibody accelerates caspase activation in both KSC and young TA. Caspases 8 and 10 are the first caspases to be activated whereas caspase-8 inhibitor zIETD-fmk delays the activation of Bid, caspase-9 and caspase-3. However, the caspase-9 inhibitor zLEDH-fmk does not block the activation of caspase-8, Bid, caspase-10 and caspase-3. Moreover, caspase-8, but not caspase-9 inhibitor partially prevents keratinocyte anoikis. As FLIP inhibits caspase-8 processing, we retrovirally infected HaCaT keratinocytes with c-FLIPL. Anti-β1-integrin fails to activate caspase-8, Bid, caspase-9 and to induce the release of cytochrome c in c-FLIPL overexpressing keratinocytes. Finally, overexpression of c-FLIPL partially prevents anoikis in both suspended and anti-β1 integrin-treated cells. Taken together, these results indicate that the extrinsic apoptotic pathway triggered by caspase-8 predominates in keratinocyte anoikis. However, the release of cytochrome c and the later activation of caspase-9 seem to suggest that the intrinsic mitochondrial pathway may intervene as a positive feedback loop of caspase activation.

p75 neurotrophin receptor (p75NTR) belongs to the TNF-receptor superfamily and signals apoptosis in many cell settings. In human epidermis, p75NTR is mostly confined to the transit-amplifying (TA) sub-population of basal keratinocytes. Brainderived neurotrophic factor (BDNF) or neurotrophin-4 (NT-4), which signals through p75NTR, induces keratinocyte apoptosis, whereas b-amyloid, a ligand for p75NTR, triggers caspase-3 activation to a greater extent in p75NTR transfected cells. Moreover, p75NTR co-immunoprecipitates with NRAGE, induces the phosphorylation of c-Jun N-terminal kinase (JNK) and reduces nuclear factor kappa B (NF-jB) DNA-binding activity. p75NTR also mediates pro-NGF-induced keratinocyte apoptosis through its co-receptor sortilin. Furthermore, BDNF or b-amyloid cause cell death in TA, but not in keratinocyte stem cells (KSCs) or in p75NTR silenced TA cells. p75NTR is absent in lesional psoriatic skin and p75NTR levels are significantly lower in psoriatic than in normal TA keratinocytes. The rate of apoptosis in psoriatic TA cells is significantly lower than in normal TA cells. BDNF or b-amyloid fail to induce apoptosis in psoriatic TA cells, and p75NTR retroviral infection restores BDNF-or b-amyloid-induced apoptosis in psoriatic keratinocytes. These results demonstrate that p75NTR has a pro-apoptotic role in keratinocytes and is involved in the maintenance of epidermal homeostasis.

Interleukin-17A, the prototypical member of the interleukin-17 cytokine family, coordinates local tissue inflammation by recruiting neutrophils to sites of infection. Dysregulation of interleukin-17 signalling has been linked to the pathogenesis of inflammatory diseases and autoimmunity. The interleukin-17 receptor family members (A-E) have a broad range of functional effects in immune signalling yet no known role has been described for the remaining orphan receptor, interleukin-17 receptor D, in regulating interleukin-17A-induced signalling pathways. Here we demonstrate that interleukin-17 receptor D can differentially regulate the various pathways employed by interleukin-17A. neutrophil recruitment, in response to in vivo administration of interleukin-17A, is abolished in interleukin-17 receptor D-deficient mice, correlating with reduced interleukin-17A-induced activation of p38 mitogen-activated protein kinase and expression of the neutrophil chemokine mIP-2. In contrast, interleukin-17 receptor D deficiency results in enhanced interleukin-17A-induced activation of nuclear factor-kappa B and interleukin-6 and keratinocyte chemoattractant expression. Interleukin-17 receptor D disrupts the interaction of Act1 and TRAF6 causing differential regulation of nuclear factorkappa B and p38 mitogen-activated protein kinase signalling pathways.

Toll-like receptors (TLRs) act as primary sensors of the immune system by recognizing specific microbial motifs and inducing proinflammatory genes that facilitate innate and adaptive immunity. TLRs regulate gene expression by activating transcription factors, such as NF-B and interferon-regulatory factors. Dysregulation of these pathways can lead to inflammatory diseases, and thus they are subject to stringent control by negative regulators of innate immune signaling. Cactin (Cactus interactor) was initially discovered as a novel interactor of Drosophila Cactus, a regulator of Drosophila Toll signaling. We now describe the first functional characterization of the human ortholog of Cactin (hCactin) and show that it acts as a negative regulator of TLRs. Overexpression of hCactin suppresses TLRinduced activation of NF-B and interferon-regulatory factor transcription factors and induction of TLR-responsive genes, whereas knockdown of endogenous hCactin augments TLR induction of these responses. hCactin also interacts with IBlike protein and targets other proteins that are encoded by genes in the MHC Class III region of chromosome 6. We demonstrate that hCactin localizes to the nucleus, and this nuclear localization is critical for manifesting its inhibitory effects on TLR signaling. This study thus defines hCactin as a novel negative regulator of TLR signaling and reveals its capacity to target MHC Class III genes at the molecular and functional level.

TLRs initiate immune responses by direct detection of molecular motifs that distinguish invading microbes from host cells. Five intracellular adaptor proteins, each containing a Toll/IL-1R (TIR) domain, are used by TLRs and play key roles in dictating gene expression patterns that are tailored to the invader. Such gene expression is mediated by transcription factors, and although TIR adaptor-induced activation of NF-κB and the IFN regulatory factors have been intensively studied, there is a dearth of information on the role of TIR adaptors in regulating CREB. In this paper, we describe a role for the TIR adaptor Mal in enhancing activation of CREB. Mal-deficient murine bone marrow-derived macrophages show a loss in responsiveness to TLR2 and TLR4 ligands with respect to activation of CREB. Mal-deficient cells also fail to express the CREB-responsive genes IL-10 and cyclooxygenase 2 in response to Pam2Cys-Ser-(Lys)4 and LPS. We reveal that Mal-mediated activation of CREB is dependent on Pellino3 and TNFR-associated factor 6, because CREB activation is greatly diminished in Pellino3 knockdown cells and TNFR-associated factor 6-deficient cells. We also demonstrate the importance of p38 MAPK in this pathway with the p38 inhibitor SB203580 abolishing activation of CREB in murine macrophages. MAPK-activated protein kinase 2 (MK2), a substrate for p38 MAPK, is the likely downstream mediator of p38 MAPK in this pathway, because Mal is shown to activate MK2 and inhibition of MK2 decreases TLR4-induced activation of CREB. Overall, these studies demonstrate a new role for Mal as a key upstream regulator of CREB and as a contributor to the expression of both pro- and anti-inflammatory genes.

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a b s t r a c tThe immune system of teleost zebrafish (Danio rerio) shows high similarity to mammalian counterparts sharing many innate immune components including Toll-Like Receptors (TLRs), cytokines, chemokines and complement molecules. As in mammals, zebrafish also contains the transcription factor NF-jB that plays dualist roles in innate immunity and early development. Indeed NF-jB members are expressed in different temporal patterns during the early stages of zebrafish embryogenesis indicating that each molecule is involved in specific developmental events. In the present study we employ zebrafish as a model to characterise the expression pattern and role of a novel NF-jB regulator, termed Cactin, in early development. Cactin was first characterised in Drosophila as a new member of the Rel pathway that could affect the generation of dorsal-ventral polarity. To explore the potential developmental role of Cactin in zebrafish, we initially investigated its expression pattern and functional role during early embryonic developmental stages. We detect Cactin expression at all stages of early development and knockdown of Cactin by specific morpholino antisense oligonucleotides causes developmental abnormalities manifested by an overall dysmorphic cellular organisation. These results indicate that Cactin has been highly conserved during evolution and plays a key role in early embryonic development.

The peripheral nervous system comprises the autonomic and sensory (afferent) nervous systems. Major advances in our understanding of the autonomic and sensory transmission and function include the recognition of the phenotypic expression of a variety of transmitters and modulators that often coexist in individual neurons, the concept of co-transmission and chemical coding, the evidence for local effector functions of primary afferent nerves, and the discovery of plasticity of both the autonomic and the sensory nervous system during development, aging, diseases states, and inflammation. Co-transmission or plurichemical transmission, which indicates the release of more than one chemical messenger from the same neuron, enables autonomic and sensory neurons to exert a fine and highly regulated control of various functions such as circulation and immune response. The concept of chemical coding, in which the combination of transmitters/modulators is established, allows the identification of functional classes of neurons with their projections and targets. In addition to transmitters and modulators, autonomic and sensory neurons express multiple receptors, including G-proteincoupled and ion-gated receptors, further supporting the complexity of autonomic and sensory transmission and function. Autonomic neurons regulate the internal environment and maintain multiple homeostatic functions, and sensory neurons act as receptive structures that activate their targets in response to stimulation but also exert effector functions including the control of blood flow and vascular permeability, maintenance of mineralized tissue, and regulation of gene expression. Neurophysiology of painThe nociceptive system supports two sensory functions, pain and itch. Itch has often been regarded as a minor form of pain. Recently, it has been shown, however, that the pruritic system is supported by its own peripheral and central neuronal pathways which are closely associated, although antagonistic in some POMC processing in human melanocytes has been widely documented, and the a-MSH/MC1R/cAMP cascade has been implicated in the control of pigmentation. Only very recently, a role of b-endorphin, one cleavage product of b-LPH, has been demonstrated to influence melanocyte growth, dendricity and melanin biosynthesis via the m-opiate receptor. However, much earlier, it was shown that b-MSH, the other cleavage product of b-LPH, controls melanogenesis and melanin transfer in amphibians. To date, a specific receptor for b-MSH has not been identified. Earlier POMC processing has been found in melanosomes. Therefore, an MC1R-independent role of a-MSH was postulated and demonstrated in control of 6-tetrahydrobiopterin (6BH 4 )inhibited tyrosinase. Utilizing the depigmentation disorder vitiligo, we were now able to follow the fate of epidermal POMC processing in the presence of mM levels of hydrogen peroxide (H 2 O 2 ). In vitiligo epidermal PC2 and 7B2 protein expression is increased, whereas a-MSH, b-MSH and b-endorphin are significantly decreased. Analys...