Protective effect of Aquaphilus dolomiae extract-G1, ADE-G1, on tight junction barrier function in a Staphylococcus aureus-infected atopic dermatitis model.

BACKGROUND: Atopic dermatitis (AD) is a common skin disease characterized by recurrent pruritic inflammatory skin lesions and defects of the skin barrier. Bacterial infection with Staphylococcus aureus contributes to increased severity of AD by compromising the barrier further. A microorganism component of Avène Thermal Spring Water, Aquaphilus dolomiae, is thought to contribute to some of its beneficial effects to skin, eg AD alleviation. AIMS: Here, we have investigated the effects of an extract of A. dolomiae, A. dolomiae extract-G1 (ADE-G1), on the structural barrier function of keratinocytes, tight junction (TJ) protein expression and the expression of several genes altered in AD patients. METHODS: An epidermal cell culture model mimicking the AD environment and phenotype was used, in which S. aureus-infected cell cultures of normal human epidermal keratinocytes were exposed to a proinflammatory environment. Endpoints measured included the transepithelial electrical resistance (TER) and immunohistological staining of the epidermal TJ proteins, claudin and occludin. Additional analysis was made of several genes known to be differentially regulated in skin from AD patients (C-C motif chemokine ligand 20 (CCL20), interleukin-8 (IL-8), S100 calcium binding protein A7 (S100A7), defensin beta 4 (DEFB4) and filaggrin). RESULTS: Aquaphilus dolomiae extract-G1 strongly increased TER in non-infected cells and provided protection against infection by overcoming the decrease in TER induced by the infection with S. aureus. In infected cells exposed to a pro-inflammatory environment - depicting AD-like conditions - TER protection by ADE-G1 was still observed. Gene expression analysis of infected and pro-inflammatory stimulated cells indicated that ADE-G1 modulated the inflammatory response (induced IL-8 and attenuated CCL20 expression), increased antimicrobial activities (induced DEFB4 and A100A7) and strengthened barrier function (restored filaggrin expression). CONCLUSIONS: ADE-G1 reinforces barrier function and strongly protects TJ barrier disruption induced by bacterial infection and inflammation.

An ancient defense system eliminates unfit cells from developing tissues during cell competition.

Developing tissues that contain mutant or compromised cells present risks to animal health. Accordingly, the appearance of a population of suboptimal cells in a tissue elicits cellular interactions that prevent their contribution to the adult. Here we report that this quality control process, cell competition, uses specific components of the evolutionarily ancient and conserved innate immune system to eliminate Drosophila cells perceived as unfit. We find that Toll-related receptors (TRRs) and the cytokine Spätzle (Spz) lead to NFκB-dependent apoptosis. Diverse "loser" cells require different TRRs and NFκB factors and activate distinct pro-death genes, implying that the particular response is stipulated by the competitive context. Our findings demonstrate a functional repurposing of components of TRRs and NFκB signaling modules in the surveillance of cell fitness during development.

Loss of PI3K blocks cell-cycle progression in a Drosophila tumor model.

Tumorigenesis is a complex process, which requires alterations in several tumor suppressor or oncogenes. Here, we use a Drosophila tumor model to identify genes, which are specifically required for tumor growth. We found that reduction of phosphoinositide 3-kinase (PI3K) activity resulted in very small tumors while only slightly affecting growth of wild-type tissue. The observed inhibition on tumor growth occurred at the level of cell-cycle progression. We conclude that tumor cells become dependent on PI3K function and that reduction of PI3K activity synthetically interferes with tumor growth. The results presented here broaden our insights into the intricate mechanisms underling tumorigenesis and illustrate the power of Drosophila genetics in revealing weak points of tumor progression.

Outcome prediction in critical care: physicians' prognoses vs. scoring systems.

BACKGROUND AND OBJECTIVE: To compare the accuracy of prognoses made by intensive care physicians with the performance of two indicators, the original Simplified Acute Physiology Score (SAPS) II and a modified version optimized to the patient sample. METHODS: Data from 412 patients consecutively admitted to intensive care units of Göttingen University Hospital, Germany, were collected according to the original score criteria. Information necessary for the computation of SAPS II and the vital status on hospital discharge was recorded. To customize the original SAPS II in our cohort, the database was randomly divided into two subgroups. Logistic regression analysis with physiological values as explanatory variables was used. A bootstrap procedure completed the process. Furthermore, physicians were asked to indicate their prognostic judgement concerning the patients' hospital mortality. RESULTS: Discrimination analysis showed the following areas under receiver operating characteristic curves: physicians' prognoses 0.84 (confidence interval (CI): 0.79-89), SAPS II 0.75 (CI: 0.69-0.80) and customized SAPS 0.72 (CI: 0.66-0.78). The physician's forecast was significantly better, while the customized and the original SAPS were not substantially different as regards their accuracy. CONCLUSIONS: Prognoses made by physicians are superior to objective models. This may result from more extensive knowledge and other kinds of information available to clinicians. A clinician's action also depends on his/her prognosis at the beginning of the treatment, giving raise to a possible correlation between medical outcome and the clinician's prognosis. Our findings indicate that physicians do not limit their prognosis to the objective factors at their disposal, but indicate that they base their decisions on experience and individual observations.

Brinker requires two corepressors for maximal and versatile repression in Dpp signalling.

decapentaplegic (dpp) encodes a Drosophila transforming growth factor-beta homologue that functions as a morphogen in the developing embryo and in adult appendage formation. In the wing imaginal disc, a Dpp gradient governs patterning along the anteroposterior axis by inducing regional expression of diverse genes in a concentration-dependent manner. Recent studies show that responses to graded Dpp activity also require an input from a complementary and opposing gradient of Brinker (Brk), a transcriptional repressor protein encoded by a Dpp target gene. Here we show that Brk harbours a functional and transferable repression domain, through which it recruits the corepressors Groucho and CtBP. By analysing transcriptional outcomes arising from the genetic removal of these corepressors, and by ectopically expressing Brk variants in the embryo, we demonstrate that these corepressors are alternatively used by Brk for repressing some Dpp-responsive genes, whereas for repressing other distinct target genes they are not required. Our results show that Brk utilizes multiple means to repress its endogenous target genes, allowing repression of a multitude of complex Dpp target promoters.

Skinny hedgehog, an acyltransferase required for palmitoylation and activity of the hedgehog signal.

One of the most dominant influences in the patterning of multicellular embryos is exerted by the Hedgehog (Hh) family of secreted signaling proteins. Here, we identify a segment polarity gene in Drosophila melanogaster, skinny hedgehog (ski), and show that its product is required in Hh-expressing cells for production of appropriate signaling activity in embryos and in the imaginal precursors of adult tissues. The ski gene encodes an apparent acyltransferase, and we provide genetic and biochemical evidence that Hh proteins from ski mutant cells retain carboxyl-terminal cholesterol modification but lack amino-terminal palmitate modification. Our results suggest that ski encodes an enzyme that acts within the secretory pathway to catalyze amino-terminal palmitoylation of Hh, and further demonstrate that this lipid modification is required for the embryonic and larval patterning activities of the Hh signal.

An absolute requirement for Cubitus interruptus in Hedgehog signaling.

Hedgehog (Hh) proteins play diverse organizing roles in animal development by regulating gene expression in responding cells. Several components of the Hh signal transduction pathway have been identified, yet their precise role in mediating the various outputs of the pathway is still poorly understood. The Gli homolog Cubitus interruptus (Ci) is involved in controlling the transcription of Drosophila Hh target genes and thus represents the most downstream component known in this pathway. We address the question of whether the Hh pathway is distally branched or, in other words, whether the regulation of Ci activity is the sole output of Hh signaling. Putative Ci-independent branches of Hh signaling are explored by analyzing the behavior of cells that lack Ci but have undergone maximal activation of the Hh transduction pathway due to the removal of Patched (Ptc). The analysis of target gene expression and morphogenetic read-outs of Hh in embryonic, larval and adult stages indicates that Ci is absolutely required for all examined aspects of Hh outputs. We interpret this as evidence against the existence of Ci-independent branches in the Hh signal transduction pathway and propose that most cases of apparent Ci/Gli-independent Hh output can be attributed to the derepression of target gene expression in the absence of Ci/Gli repressor function.

Direct transcriptional control of the Dpp target omb by the DNA binding protein Brinker.

The gradient morphogen Decapentaplegic (Dpp) organizes pattern by inducing the transcription of different target genes at distinct threshold concentrations during Drosophila development. An important, albeit indirect, mode by which Dpp controls the spatial extent of its targets is via the graded downregulation of brinker, whose product in turn negatively regulates the expression of these targets. Here we report the molecular dissection of the cis-regulatory sequences of optomotor-blind (omb), a Dpp target gene in the wing. We identify a minimal 284 bp Dpp response element and demonstrate that it is subject to Brinker (Brk) repression. Using this omb wing enhancer, we show that Brk is a sequence-specific DNA binding protein. Mutations in the high-affinity Brk binding site abolish responsiveness of this omb enhancer to Brk and also compromise the input of an unknown transcriptional activator. Our results therefore identify Brk as a novel transcription factor antagonizing Dpp signalling by directly binding target genes and repressing their expression.

Schnurri mediates Dpp-dependent repression of brinker transcription.

Signalling by Decapentaplegic (Dpp), a member of the TGFbeta superfamily of signalling molecules, controls many aspects of Drosophila development by activating and repressing target genes. Several essential components of the Dpp signalling pathway have been identified, including the Dpp receptors Punt and Thick veins (Tkv) as well as the cytoplasmic mediators Mad and Medea. For target genes to be activated, Dpp signalling must suppress transcription of a repressor encoded by the brinker (brk) gene. Here we show that Schnurri (Shn), a large zinc-finger protein, is essential for Dpp-mediated repression of brk transcription; in contrast, Shn is not required for target-gene activation. Thus, the Dpp signalling pathway bifurcates, downstream of the signal-mediating SMAD proteins, into a Shn-dependent pathway leading to brk repression and a Shn-independent pathway leading to gene activation. The existence of several Shn-like proteins in vertebrates and the observation that Brk functions in BMP signalling in Xenopus indicates that a similar regulatory cascade may be conserved in higher organisms.

Suppressor of fused opposes hedgehog signal transduction by impeding nuclear accumulation of the activator form of Cubitus interruptus.

Hedgehog controls the expression of key developmental genes through the conversion of the transcription factor Cubitus interruptus (Ci) into either an activator (Ci[act]) or a repressor (Ci[rep]) form. Proteolytic cleavage of full-length Ci is important for the generation of Ci[rep], but little is known about how Ci[act] arises in response to Hh. Here we examine Hh signal transduction components for their role in the conversion of full-length Ci into either Ci[act] or Ci[rep]. We report that Cos2, PKA and Fused are necessary for the generation of Ci[rep], whereas the inhibition of either Cos2 or PKA activity is a prerequisite for Ci[act] formation. Fused (Fu) kinase stimulates a constitutively active form of Ci in a Hh-dependent manner, suggesting that Fu enhances the activity rather than the formation of Ci[act]. Su(fu) reduces the nuclear accumulation of the constitutively active form of Ci, arguing that Su(fu) can function subsequent to Ci[act] formation. We propose that Hh induces target gene expression by a two-step mechanism in which Ci[act] is first formed and then accumulates in the nucleus via Fu-induced neutralization of Su(fu) activity.

The repressor and activator forms of Cubitus interruptus control Hedgehog target genes through common generic gli-binding sites.

The Drosophila Gli homolog Cubitus interruptus (Ci) controls the transcription of Hedgehog (Hh) target genes. A repressor form of Ci arises in the absence of Hh signalling by proteolytic cleavage of intact Ci, whereas an activator form of Ci is generated in response to the Hh signal. These different activities of Ci regulate overlapping but distinct subsets of Hh target genes. To investigate the mechanisms by which the two activities of Ci exert their opposite transcriptional effect, we dissect here the imaginal disc enhancer of the dpp gene, which responds to both activities of Ci. Within a minimal disc enhancer, we identify the DNA sequences that are necessary and sufficient for the control by Ci, show that the same sequences respond to the activator and repressor forms of Ci, and demonstrate that their activities can be replaced by a single synthetic Gli-binding site. We further show that the enhancer sequences of patched, a gene responding only to the activator form of Ci, effectively integrate also the repressor activity of Ci if placed into a dpp context. These results provide in vivo evidence against the employment of distinct binding sites for the different forms of Ci and suggest that target genes responding to only one form must have acquired distant cis-regulatory elements for their selective behavior.

The Wingless target gene Dfz3 encodes a new member of the Drosophila Frizzled family.

Here we report the identification of Dfz3, a novel member of the Frizzled family of seven-pass transmembrane receptors. Like Dfz2, Dfz3 is a target gene of Wingless (Wg) signalling, but in contrast to Dfz2, it is activated rather than repressed by Wg signalling in imaginal discs. We show that Dfz3 is not required for viability but is necessary for optimal Wg signalling at the wing margin.

Opposing transcriptional outputs of Hedgehog signaling and engrailed control compartmental cell sorting at the Drosophila A/P boundary.

The wing imaginal disc is subdivided into two nonintermingling sets of cells, the anterior (A) and posterior (P) compartments. Anterior cells require reception of the Hedgehog (Hh) signal to segregate from P cells. We provide evidence that Hh signaling controls A/P cell segregation not by directly modifying structural components but by a Cubitus interruptus (Ci)-mediated transcriptional response. A shift in the balance between repressor and activator forms of Ci toward the activator form is necessary and sufficient to define "A-type" cell sorting behavior. Moreover, we show that Engrailed (En), in the absence of Ci, is sufficient to specify "P-type" sorting. We propose that the opposing transcriptional activities of Ci and En control cell segregation at the A/P boundary by regulating a single cell adhesion molecule.

Distinct and regulated activities of human Gli proteins in Drosophila.

In both vertebrates and Drosophila, limb development is organized by a posteriorly located source of the signalling protein Hedgehog (Hh) [1] [2] [3] [4]. In Drosophila, the expression of Hh target genes is controlled by two opposing activities of the transcriptional regulator Cubitus interruptus (Ci), which activates target genes in response to Hh signalling but is converted into a repressor form in the absence of Hh [5] [6] [7] [8] [9] [10]. Three homologs of Ci (Gli1, Gli2, and Gli3) have been implicated in mediating responses to Sonic hedgehog (Shh) in vertebrates [11] [12]. Much attention has been devoted to the expression pattern of GLI genes; GLI1 is induced by Shh, whereas GLI3 transcription appears to be repressed by Shh signalling [13] [14] [15]. The regulation of GLI gene expression is therefore one important mechanism by which GLI genes organize pattern. It is not well understood, however, whether Shh signalling also controls the activities of Gli proteins post-translationally and whether these activities have activating or repressing effects on target genes in vivo. Here, we have subjected the human proteins Gli1 and Gli3 to the precise and well-defined Hh signalling assay of Drosophila wing development and established that Gli1 functions as an activator and Gli3 as a repressor of Hh target genes; that the activating transcriptional activity of Gli1 and the repressing activity of Gli3 are both subject to Hh regulation in vivo; and that the combined activities of Gli1 and Gli3 can substitute for Ci in controlling Hh target gene expression during embryonic and larval development.

Compartment boundaries: at the edge of development.

Compartment boundaries have fascinated biologists for more than 25 years. We now know that these boundaries play important roles in pattern formation, yet how these boundaries are established during development remained a mystery. Here, we describe the exciting progress that has been made recently towards elucidating the mechanisms of boundary formation.

Hedgehog controls limb development by regulating the activities of distinct transcriptional activator and repressor forms of Cubitus interruptus.

Hedgehog (Hh) proteins play diverse organizing roles in development by regulating gene expression in responding cells. The Gli homolog Cubitus interruptus (Ci) is involved in controlling the transcription of Hh target genes. A repressor form of Ci arises in the absence of Hh signaling by proteolytic cleavage of intact Ci. We show that this cleavage is essential for limb patterning and is regulated by Hh in vivo. We provide evidence for the existence of a distinct activator form of Ci, which does not arise by mere prevention of Ci proteolysis, but rather depends on a separate regulatory step subject to Hh control. These different activities of Ci regulate overlapping but distinct subsets of Hh target genes. Thus, limb development is organized by the integration of different transcriptional outputs of Hh signaling.

The dominant mutation Glazed is a gain-of-function allele of wingless that, similar to loss of APC, interferes with normal eye development.

Dominant mutations have served as invaluable tools for Drosophila geneticists. Here we analyze the dominant eye mutation Glazed (Gla) that was described by T. H. Morgan more than 50 years ago. We show that Gla causes the loss of photoreceptor cells during pupal stages, in a process reminiscent of apoptosis, with a concomitant overproduction of eye pigment. This phenotype is very similar to that caused by the loss of D-APC, a negative regulator of Wingless (Wg) signal transduction. Genetic analyses reveal however that the Gla gain-of-function phenotype can be reverted to wild-type. By generating a P-element-induced revertant of Gla we demonstrate that Gla is allelic to wg. The molecular lesion in Gla indicates that the insertion of a roo retrotransposon leads to ectopic expression of wg during pupal stages. We show that the Gla phenotype is similar to that caused by ectopic expression of Wg driven by the sevenless (sev) enhancer. In both cases Wg exerts its effect, at least in part, by negatively regulating the expression of the Pax2 homolog sparkling (spa). Gla represents not only the first dominant allele of wg, but it may also be the first allele ever described for wg.

Dispatched, a novel sterol-sensing domain protein dedicated to the release of cholesterol-modified hedgehog from signaling cells.

Members of the Hedgehog (Hh) family of secreted signaling proteins function as potent short-range organizers in animal development. Their range of action is limited by a C-terminal cholesterol tether and the upregulation of Patched (Ptc) receptor levels. Here we identify a novel segment-polarity gene in Drosophila, dispatched (disp), and demonstrate that its product is required in sending cells for normal Hh function. In the absence of Disp, cholesterol-modified but not cholesterol-free Hh is retained in these cells, indicating that Disp functions to release cholesterol-anchored Hh. Despite their opposite roles, Disp and Ptc share structural homology in the form of a sterol-sensing domain, suggesting that release and sequestration of cholesterol-modified Hh may be based on related molecular pathways.