Notch signaling in the integrated control of keratinocyte growth/differentiation and tumor suppression.

Oncogenesis is closely linked to abnormalities in cell differentiation. Notch signaling provides an important form of intercellular communication involved in cell fate determination, stem cell potential and differentiation. Here we review the role of this pathway in the integrated growth/differentiation control of the keratinocyte cell type, and the maintenance of normal skin homeostasis. In parallel with the pro-differentiation function of Notch1 in keratinocytes, we discuss recent evidence pointing to a tumor suppressor function of this gene in both mouse skin and human cervical carcinogenesis. The possibility that Notch signaling elicits signals with a duality of growth positive and negative function will be discussed.

MsrR contributes to cell surface characteristics and virulence in Staphylococcus aureus.

MsrR, a factor contributing to methicillin resistance in Staphylococcus aureus, belongs to the LytR-CpsA-Psr family of cell envelope-associated proteins. Deletion of msrR increased cell size and aggregation, and altered envelope properties, leading to a temporary reduction in cell surface hydrophobicity, diminished colony-spreading ability, and an increased susceptibility to Congo red. The reduced phosphorus content of purified cell walls of the msrR mutant suggested a reduction in wall teichoic acids, which may explain some of the observed phenotypes. Microarray analysis of the msrR deletion mutant revealed only minor changes in the global transcriptome, suggesting that MsrR has structural rather than regulatory functions. Importantly, virulence of the msrR mutant was decreased in a nematode-killing assay as well as in rat experimental endocarditis. MsrR is therefore likely to play a role in cell envelope maintenance, cell separation, and pathogenicity of S. aureus.

CD8beta endows CD8 with efficient coreceptor function by coupling T cell receptor/CD3 to raft-associated CD8/p56(lck) complexes.

The extraordinary sensitivity of CD8+ T cells to recognize antigen impinges to a large extent on the coreceptor CD8. While several studies have shown that the CD8beta chain endows CD8 with efficient coreceptor function, the molecular basis for this is enigmatic. Here we report that cell-associated CD8alphabeta, but not CD8alphaalpha or soluble CD8alphabeta, substantially increases the avidity of T cell receptor (TCR)-ligand binding. To elucidate how the cytoplasmic and transmembrane portions of CD8beta endow CD8 with efficient coreceptor function, we examined T1.4 T cell hybridomas transfected with various CD8beta constructs. T1.4 hybridomas recognize a photoreactive Plasmodium berghei circumsporozoite (PbCS) peptide derivative (PbCS (4-azidobezoic acid [ABA])) in the context of H-2K(d), and permit assessment of TCR-ligand binding by TCR photoaffinity labeling. We find that the cytoplasmic portion of CD8beta, mainly due to its palmitoylation, mediates partitioning of CD8 in lipid rafts, where it efficiently associates with p56(lck). In addition, the cytoplasmic portion of CD8beta mediates constitutive association of CD8 with TCR/CD3. The resulting TCR-CD8 adducts exhibit high affinity for major histocompatibility complex (MHC)-peptide. Importantly, because CD8alphabeta partitions in rafts, its interaction with TCR/CD3 promotes raft association of TCR/CD3. Engagement of these TCR/CD3-CD8/lck adducts by multimeric MHC-peptide induces activation of p56(lck) in rafts, which in turn phosphorylates CD3 and initiates T cell activation.

BAFF mediates survival of peripheral immature B lymphocytes.

B cell maturation is a very selective process that requires finely tuned differentiation and survival signals. B cell activation factor from the TNF family (BAFF) is a TNF family member that binds to B cells and potentiates B cell receptor (BCR)-mediated proliferation. A role for BAFF in B cell survival was suggested by the observation of reduced peripheral B cell numbers in mice treated with reagents blocking BAFF, and high Bcl-2 levels detected in B cells from BAFF transgenic (Tg) mice. We tested in vitro the survival effect of BAFF on lymphocytes derived from primary and secondary lymphoid organs. BAFF induced survival of a subset of splenic immature B cells, referred to as transitional type 2 (T2) B cells. BAFF treatment allowed T2 B cells to survive and differentiate into mature B cells in response to signals through the BCR. The T2 and the marginal zone (MZ) B cell compartments were particularly enlarged in BAFF Tg mice. Immature transitional B cells are targets for negative selection, a feature thought to promote self-tolerance. These findings support a model in which excessive BAFF-mediated survival of peripheral immature B cells contributes to the emergence and maturation of autoreactive B cells, skewed towards the MZ compartment. This work provides new clues on mechanisms regulating B cell maturation and tolerance.

Delta-like 4 is the essential, nonredundant ligand for Notch1 during thymic T cell lineage commitment.

Thymic T cell lineage commitment is dependent on Notch1 (N1) receptor-mediated signaling. Although the physiological ligands that interact with N1 expressed on thymic precursors are currently unknown, in vitro culture systems point to Delta-like 1 (DL1) and DL4 as prime candidates. Using DL1- and DL4-lacZ reporter knock-in mice and novel monoclonal antibodies to DL1 and DL4, we show that DL4 is expressed on thymic epithelial cells (TECs), whereas DL1 is not detected. The function of DL4 was further explored in vivo by generating mice in which DL4 could be specifically inactivated in TECs or in hematopoietic progenitors. Although loss of DL4 in hematopoietic progenitors did not perturb thymus development, inactivation of DL4 in TECs led to a complete block in T cell development coupled with the ectopic appearance of immature B cells in the thymus. These immature B cells were phenotypically indistinguishable from those developing in the thymus of conditional N1 mutant mice. Collectively, our results demonstrate that DL4 is the essential and nonredundant N1 ligand responsible for T cell lineage commitment. Moreover, they strongly suggest that N1-expressing thymic progenitors interact with DL4-expressing TECs to suppress B lineage potential and to induce the first steps of intrathymic T cell development.

Inhibition of toxic epidermal necrolysis by blockade of CD95 with human intravenous immunoglobulin.

Toxic epidermal necrolysis (TEN, Lyell's syndrome) is a severe adverse drug reaction in which keratinocytes die and large sections of epidermis separate from the dermis. Keratinocytes normally express the death receptor Fas (CD95); those from TEN patients were found to express lytically active Fas ligand (FasL). Antibodies present in pooled human intravenous immunoglobulins (IVIG) blocked Fas-mediated keratinocyte death in vitro. In a pilot study, 10 consecutive individuals with clinically and histologically confirmed TEN were treated with IVIG; disease progression was rapidly reversed and the outcome was favorable in all cases. Thus, Fas-FasL interactions are directly involved in the epidermal necrolysis of TEN, and IVIG may be an effective treatment.

An exquisite cross-control mechanism among endothelial cell fate regulators directs the plasticity and heterogeneity of lymphatic endothelial cells.

Arteriovenous-lymphatic endothelial cell fates are specified by the master regulators, namely, Notch, COUP-TFII, and Prox1. Whereas Notch is expressed in the arteries and COUP-TFII in the veins, the lymphatics express all 3 cell fate regulators. Previous studies show that lymphatic endothelial cell (LEC) fate is highly plastic and reversible, raising a new concept that all 3 endothelial cell fates may co-reside in LECs and a subtle alteration can result in a reprogramming of LEC fate. We provide a molecular basis verifying this concept by identifying a cross-control mechanism among these cell fate regulators. We found that Notch signal down-regulates Prox1 and COUP-TFII through Hey1 and Hey2 and that activated Notch receptor suppresses the lymphatic phenotypes and induces the arterial cell fate. On the contrary, Prox1 and COUP-TFII attenuate vascular endothelial growth factor signaling, known to induce Notch, by repressing vascular endothelial growth factor receptor-2 and neuropilin-1. We show that previously reported podoplanin-based LEC heterogeneity is associated with differential expression of Notch1 in human cutaneous lymphatics. We propose that the expression of the 3 cell fate regulators is controlled by an exquisite feedback mechanism working in LECs and that LEC fate is a consequence of the Prox1-directed lymphatic equilibrium among the cell fate regulators.

Recruitment of TNF receptor 1 to lipid rafts is essential for TNFalpha-mediated NF-kappaB activation.

Engagement of TNF receptor 1 by TNFalpha activates the transcription factor NF-kappaB but can also induce apoptosis. Here we show that upon TNFalpha binding, TNFR1 translocates to cholesterol- and sphingolipid-enriched membrane microdomains, termed lipid rafts, where it associates with the Ser/Thr kinase RIP and the adaptor proteins TRADD and TRAF2, forming a signaling complex. In lipid rafts, TNFR1 and RIP are ubiquitylated. Furthermore, we provide evidence that translocation to lipid rafts precedes ubiquitylation, which leads to the degradation via the proteasome pathway. Interfering with lipid raft organization not only abolishes ubiquitylation but switches TNFalpha signaling from NF-kappaB activation to apoptosis. We suggest that lipid rafts are crucial for the outcome of TNFalpha-activated signaling pathways.

The long form of FLIP is an activator of caspase-8 at the Fas death-inducing signaling complex.

Death receptors, such as Fas and tumor necrosis factor-related apoptosis-inducing ligand receptors, recruit Fas-associated death domain and pro-caspase-8 homodimers, which are then autoproteolytically activated. Active caspase-8 is released into the cytoplasm, where it cleaves various proteins including pro-caspase-3, resulting in apoptosis. The cellular Fas-associated death domain-like interleukin-1-beta-converting enzyme-inhibitory protein long form (FLIP(L)), a structural homologue of caspase-8 lacking caspase activity because of several mutations in the active site, is a potent inhibitor of death receptor-induced apoptosis. FLIP(L) is proposed to block caspase-8 activity by forming a proteolytically inactive heterodimer with caspase-8. In contrast, we propose that FLIP(L)-bound caspase-8 is an active protease. Upon heterocomplex formation, a limited caspase-8 autoprocessing occurs resulting in the generation of the p43/41 and the p12 subunits. This partially processed form but also the non-cleaved FLIP(L)-caspase-8 heterocomplex are proteolytically active because they both bind synthetic substrates efficiently. Moreover, FLIP(L) expression favors receptor-interacting kinase (RIP) processing within the Fas-signaling complex. We propose that FLIP(L) inhibits caspase-8 release-dependent pro-apoptotic signals, whereas the single, membrane-restricted active site of the FLIP(L)-caspase-8 heterocomplex is proteolytically active and acts on local substrates such as RIP.

A gating mutation in the internal pore of ASIC1a.

Using a substituted cysteine accessibility scan, we have investigated the structures that form the internal pore of the acid-sensing ion channel 1a. We have identified the amino acid residues Ala-22, Ile-33, and Phe-34 in the amino terminus and Arg-43 in the first transmembrane helix, which when mutated into cysteine, were modified by intracellular application of MTSET, resulting in channel inhibition. The inhibition of the R43C mutant by internal MTSET requires opening of the channel. In addition, binding of Cd2+ ions to R43C slows the channel inactivation. This indicates that the first transmembrane helix undergoes conformational changes during channel inactivation. The effect of Cd2+ on R43C can be obtained with Cd2+ applied at either the extracellular or the intracellular side, indicating that R43C is located in the channel pore. The block of the A22C, I33C, and F34C mutants by MTSET suggests that these residues in the amino terminus of the channel also participate to the internal pore.

O-mannosyl phosphorylation of alpha-dystroglycan is required for laminin binding.

Alpha-dystroglycan (alpha-DG) is a cell-surface glycoprotein that acts as a receptor for both extracellular matrix proteins containing laminin-G domains and certain arenaviruses. Receptor binding is thought to be mediated by a posttranslational modification, and defective binding with laminin underlies a subclass of congenital muscular dystrophy. Using mass spectrometry- and nuclear magnetic resonance (NMR)-based structural analyses, we identified a phosphorylated O-mannosyl glycan on the mucin-like domain of recombinant alpha-DG, which was required for laminin binding. We demonstrated that patients with muscle-eye-brain disease and Fukuyama congenital muscular dystrophy, as well as mice with myodystrophy, commonly have defects in a postphosphoryl modification of this phosphorylated O-linked mannose, and that this modification is mediated by the like-acetylglucosaminyltransferase (LARGE) protein. These findings expand our understanding of the mechanisms that underlie congenital muscular dystrophy.

The control of microtubule stability in vitro and in transfected cells by MAP1B and SCG10.

In neurons, the regulation of microtubules plays an important role for neurite outgrowth, axonal elongation, and growth cone steering. SCG10 family proteins are the only known neuronal proteins that have a strong destabilizing effect, are highly enriched in growth cones and are thought to play an important role during axonal elongation. MAP1B, a microtubule-stabilizing protein, is found in growth cones as well, therefore it was important to test their effect on microtubules in the presence of both proteins. We used recombinant proteins in microtubule assembly assays and in transfected COS-7 cells to analyze their combined effects in vitro and in living cells, respectively. Individually, both proteins showed their expected activities in microtubule stabilization and destruction respectively. In MAP1B/SCG10 double-transfected cells, MAP1B could not protect microtubules from SCG10-induced disassembly in most cells, in particular not in cells that contained high levels of SCG10. This suggests that SCG10 is more potent to destabilize microtubules than MAP1B to rescue them. In microtubule assembly assays, MAP1B promoted microtubule formation at a ratio of 1 MAP1B per 70 tubulin dimers while a ratio of 1 SCG10 per two tubulin dimers was needed to destroy microtubules. In addition to its known binding to tubulin dimers, SCG10 binds also to purified microtubules in growth cones of dorsal root ganglion neurons in culture. In conclusion, neuronal microtubules are regulated by antagonistic effects of MAP1B and SCG10 and a fine tuning of the balance of these proteins may be critical for the regulation of microtubule dynamics in growth cones.

Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule.

Cell death is achieved by two fundamentally different mechanisms: apoptosis and necrosis. Apoptosis is dependent on caspase activation, whereas the caspase-independent necrotic signaling pathway remains largely uncharacterized. We show here that Fas kills activated primary T cells efficiently in the absence of active caspases, which results in necrotic morphological changes and late mitochondrial damage but no cytochrome c release. This Fas ligand-induced caspase-independent death is absent in T cells that are deficient in either Fas-associated death domain (FADD) or receptor-interacting protein (RIP). RIP is also required for necrotic death induced by tumor necrosis factor (TNF) and TNF-related apoptosis-inducing ligand (TRAIL). In contrast to its role in nuclear factor kappa B activation, RIP requires its own kinase activity for death signaling. Thus, Fas, TRAIL and TNF receptors can initiate cell death by two alternative pathways, one relying on caspase-8 and the other dependent on the kinase RIP.

Apoplastic polyesters in Arabidopsis surface tissues--a typical suberin and a particular cutin.

Cutinized and suberized cell walls form physiological important plant-environment interfaces as they act as barriers limiting water and nutrient loss and protect from radiation and invasion by pathogens. Due to the lack of protocols for the isolation and analysis of cutin and suberin in Arabidopsis, the model plant for molecular biology, mutants and transgenic plants with a defined altered cutin or suberin composition are unavailable, causing that structure and function of these apoplastic barriers are still poorly understood. Transmission electron microscopy (TEM) revealed that Arabidopsis leaf cuticle thickness ranges from only 22 nm in leaf blades to 45 nm on petioles, causing the difficulty in cuticular membrane isolation. We report the use of polysaccharide hydrolases to isolate Arabidopsis cuticular membranes, suitable for depolymerization and subsequent compositional analysis. Although cutin characteristic omega-hydroxy acids (7%) and mid-chain hydroxylated fatty acids (8%) were detected, the discovery of alpha,omega-diacids (40%) and 2-hydroxy acids (14%) as major depolymerization products reveals a so far novel monomer composition in Arabidopsis cutin, but with chemical analogy to root suberin. Histochemical and TEM analysis revealed that suberin depositions were localized to the cell walls in the endodermis of primary roots and the periderm of mature roots of Arabidopsis. Enzyme digested and solvent extracted root cell walls when subjected to suberin depolymerization conditions released omega-hydroxy acids (43%) and alpha,omega-diacids (24%) as major components together with carboxylic acids (9%), alcohols (6%) and 2-hydroxyacids (0.1%). This similarity to suberin of other species indicates that Arabidopsis roots can serve as a model for suberized tissue in general.