Circulating TSLP associates with decreased wheezing in non-atopic preschool children: data from the URECA birth cohort.

BACKGROUND: Mouse models of atopic march suggest that systemic, skin-derived thymic stromal lymphopoietin (TSLP) mediates progression from eczema to asthma. OBJECTIVE: We investigated whether circulating TSLP is associated with eczema, allergic sensitization, or recurrent wheezing in young children. METHODS: A prospective analysis of the relationship between plasma levels of TSLP to allergic sensitization and recurrent wheezing was conducted in the birth cohort from the Urban Environment and Childhood Asthma (URECA) study. Plasma TSLP levels were measured at 1, 2, and 3 years of age and analysed for correlation with clinical parameters in each of the three years. Only those children with consecutive samples for all three years were included in this analysis. RESULTS: We detected TSLP in 33% of 236 children for whom plasma samples were available for all three years. Overall, a consistently significant association was not found between TSLP and eczema or allergic sensitization. With regard to recurrent wheezing, children with detectable TSLP at one year of age were significantly less likely to experience recurrent wheezing by 3 years compared with those children without detectable TSLP, but this was only seen in children without aeroallergen sensitization at 3 years (P < 0.01). CONCLUSIONS AND CLINICAL RELEVANCE: Contrary to our expectations, circulating TSLP was not significantly associated with eczema, allergen sensitization, or recurrent wheezing during the first three years of life. Early presence of circulating TSLP was significantly associated with reduced incidence of recurrent wheeze in those children not sensitized to aeroallergen. These findings suggest a possible underlying distinction between pathogenesis of developing atopic vs. non-atopic recurrent wheeze.

Regulated intramembrane proteolysis takes another twist.

Rhomboid, a seven-transmembrane domain protein, has been shown genetically to potentiate EGFR signaling via the TGFalpha-like ligand Spitz. Here we discuss recently published papers that identify Rhomboid as a novel serine protease, cleaving Spitz within its transmembrane domain.

Mouse notch: expression in hair follicles correlates with cell fate determination.

Many vertebrate tissues, including skin, are known to develop as a consequence of epithelial-mesenchymal interactions. Much less is known about the role of cell-cell interaction within the epithelial or the mesenchymal compartments in morphogenesis. To investigate cell-cell interactions during skin development, and the potential role of the Notch homolog in this process, we cloned the mouse homolog of Notch (mNotch) and studied its expression pattern, starting as early as mesoderm formation. The novel application of double-labeled in situ hybridization in vertebrates allowed high resolution analysis to follow the fate of mNotch expressing cells directly. In comparison with the distribution of Id mRNA, analysis confirmed that in the hair follicle high levels of mNotch are expressed exclusively in the epithelial compartment. Hair follicle matrix cells start expressing mNotch as different cell types become distinguishable in the developing follicle. mNotch mRNA expression persists throughout the growth phase of the follicle and maintains the same expression profile in the second hair cycle. The cells in the follicle that undergo a phase of high level mNotch expression are in transition from mitotic precursors to several discreet, differentiating cell types. Our observations point out that both in time (during development) and in space (by being removed one cell layer from the dermal papilla) mNotch expression is clearly separated from the inductive interactions. This is a novel finding and suggests that mNotch is important for follicular differentiation and possibly cell fate selection within the follicle.

The use of retinoic acid to probe the relation between hyperproliferation-associated keratins and cell proliferation in normal and malignant epidermal cells.

When cells from normal human epidermis and from the human squamous cell carcinoma line SCC-13 were seeded on floating rafts of collagen and fibroblasts, they stratified and underwent terminal differentiation. Although the program of differentiation in SCC-13 cells was morphologically abnormal, the cultures resembled normal epidermal raft cultures by expressing the terminal differentiation-specific keratins, K1/K10, and by restricting their proliferative capacity to the basal-like cells of the population. In addition, the differentiating cells of both normal and SCC-13 raft cultures expressed keratins K6 and K16, which are not normally expressed in epidermis, but are synthesized suprabasally during wound-healing and in various epidermal diseases associated with hyperproliferation. While the behavior of normal and SCC-13 rafts was quite similar when they were cultured over normal medium, significant biochemical differences began to emerge when the cultures were exposed to retinoic acid. Most notably, while the SCC-13 cultures still stratified extensively, they showed a marked inhibition of both abnormal (K6/K16) and normal (K1/K10) differentiation-associated keratins, concomitantly with an overall disappearance of differentiated phenotype. Surprisingly, the reduction in K6/K16 in retinoid-treated SCC-13 cultures was not accompanied by a decrease in cell proliferation. Using immunohistochemistry combined with [3H]thymidine labeling, we demonstrate that while the expression of K6 and K16 are often associated with hyperproliferation, these keratins are only produced in the nondividing, differentiating populations of proliferating cultures. Moreover, since their expression can be suppressed without a corresponding decrease in proliferation, the expression of these keratins cannot be essential to the nature of the hyperproliferative epidermal cell.

Retinoids as important regulators of terminal differentiation: examining keratin expression in individual epidermal cells at various stages of keratinization.

When human epidermal cells were seeded on floating rafts of collagen and fibroblasts, they stratified at the air-liquid interface. The suprabasal cells synthesized the large type II (K1) and type I (K10/K11) keratins characteristic of terminal differentiation in skin. At earlier times in culture, expression of the large type II keratins appeared to precede the expression of their type I partners. At later times, all suprabasal cells expressed both types, suggesting that the accumulation of a critical level of K1 keratin may be a necessary stimulus for K10 and K11 expression. Expression of the terminal differentiation-specific keratins was completely suppressed by adding retinoic acid to the culture medium, or by submerging the cultures in normal medium. In submerged cultures, removal of vitamin A by delipidization of the serum restored the keratinization process. In contrast, calcium and transforming growth factor-beta did not influence the expression of the large keratins in keratinocytes grown in the presence of retinoids, even though they are known to induce certain morphological features of terminal differentiation. Retinoic acid in the raft medium not only suppressed the expression of the large keratins, but, in addition, induced the synthesis of two new keratins not normally expressed in epidermis in vivo. Immunofluorescence localized one of these keratins, K19, to a few isolated cells of the stratifying culture. In contrast, the other keratin, K13, appeared uniformly in a few outer layers of the culture. Interestingly, K13 expression correlated well with the gradient of retinoid-mediated disruptions of intercellular interactions in the culture. These data suggest that K13 induction may in some way relate to the reduction in either the number or the strength of desmosomal contacts between suprabasal cells of stratified squamous epithelial tissues.

Use of monospecific antisera and cRNA probes to localize the major changes in keratin expression during normal and abnormal epidermal differentiation.

We report here the isolation and characterization of three antisera, each of which is specific for a single keratin from one of the three different pairs (K1/K10, K14/K5, K16/K6) that are differentially expressed in normal human epidermis and in epidermal diseases of hyperproliferation. We have used these antisera in conjunction with monospecific cRNA probes for epidermal keratin mRNAs to investigate pathways of differentiation in human epidermis and epidermal diseases in vivo and in epidermal cells cultured from normal skin and from squamous cell carcinomas in vitro. Specifically, our results suggest that: (a) the basal-specific keratin mRNAs are down-regulated upon commitment to terminal differentiation, but their encoded proteins are stable, and can be detected throughout the spinous layers; (b) the hyperproliferation-associated keratin mRNAs are expressed at a low level throughout normal epidermis when their encoded proteins are not expressed, but are synthesized at high levels in the suprabasal layers of hyperproliferating epidermis, coincident with the induced expression of the hyperproliferation-associated keratins in these cells; and (c) concomitantly with the induction of the hyperproliferation-associated keratins in the suprabasal layers of the epidermis is the down-regulation of the expression of the terminal differentiation-specific keratins. These data have important implications for our understanding of normal epidermal differentiation and the deviations from this process in the course of epidermal diseases of hyperproliferation.

Expression of keratin K14 in the epidermis and hair follicle: insights into complex programs of differentiation.

Keratins K14 and K5 have long been considered to be biochemical markers of the stratified squamous epithelia, including epidermis (Moll, R., W. Franke, D. Schiller, B. Geiger, and R. Krepler. 1982. Cell. 31:11-24; Nelson, W., and T.-T. Sun. 1983. J. Cell Biol. 97:244-251). When cells of most stratified squamous epithelia differentiate, they downregulate expression of mRNAs encoding these two keratins and induce expression of new sets of keratins specific for individual programs of epithelial differentiation. Frequently, as in the case of epidermis, the expression of differentiation-specific keratins also leads to a reorganization of the keratin filament network, including denser bundling of the keratin fibers. We report here the use of monospecific antisera and cRNA probes to examine the differential expression of keratin K14 in the complex tissue of human skin. Using in situ hybridizations and immunoelectron microscopy, we find that the patterns of K14 expression and filament organization in the hair follicle are strikingly different from epidermis. Some of the mitotically active outer root sheath (ORS) cells, which give rise to ORS under normal circumstances and to epidermis during wound healing, produce only low levels of K14. These cells have fewer keratin filaments than basal epidermal cells, and the filaments are organized into looser, more delicate bundles than is typical for epidermis. As these cells differentiate, they elevate their expression of K14 and produce denser bundles of keratin filaments more typical of epidermis. In contrast to basal cells of epidermis and ORS, matrix cells, which are relatively undifferentiated and which can give rise to inner root sheath, cuticle and hair shaft, show no evidence of K14, K14 mRNA expression, or keratin filament formation. As matrix cells differentiate, they produce hair-specific keratins and dense bundles of keratin filaments but they do not induce K14 expression. Collectively, the patterns of K14 and K14 mRNA expression and filament organization in mitotically active epithelial cells of the skin correlate with their relative degree of pluripotency, and this suggests a possible basis for the deviation of hair follicle programs of differentiation from those of other stratified squamous epithelia.

An Epstein-Barr virus transforming protein associates with vimentin in lymphocytes.

The Epstein-Barr virus (EBV) latent infection membrane protein (LMP) is likely to be an important mediator of EBV-induced cell proliferation, since it is one of the few proteins encoded by the virus in latent infection and since production of this protein in Rat-1 cells results in their conversion to a fully transformed phenotype. LMP was previously noted to localize to patches at the cell periphery. In this paper we examine the basis of LMP patching in EBV-infected, transformed lymphocytes. Our data indicate that LMP is associated with the cytoskeletal protein vimentin. Although LMP is fully soluble in isotonic Triton X-100 buffer, only 50% of it is extracted from cells in this solution. The rest remains bound to the cytoskeleton. LMP undergoes phosphorylation, and phosphorylated LMP is preferentially associated with the cytoskeleton. As judged by both immunofluorescence and immunoelectron microscopy, the vimentin network in EBV-transformed lymphocytes or EBV-infected Burkitt tumor lymphocytes is abnormal. Vimentin and LMP often colocalize in a single patch near the plasma membrane. In response to Colcemid treatment of EBV-infected cells, vimentin reorganizes into perinuclear rings, as it does in uninfected cells. LMP is associated with these perinuclear rings. Vimentin (or a vimentin-associated protein) may be a transducer of an LMP transmembrane effect in lymphoproliferation.

The Notch pathway: democracy and aristocracy in the selection of cell fate.

Major advances in the past two years have increased our understanding of the molecular components of the Notch signal-transduction pathway in both invertebrates and vertebrates. Recent studies have begun to address the interaction of other signaling pathways with the Notch pathway. Of particular interest is the integration of signals from the Wingless pathway and from asymmetrically segregating determinants such as numb. Molecular models for Notch-mediated cell-fate selection within groups of developmentally equivalent cells are presented.

All good things must come to an end: how is Notch signaling turned off?

Turning off signaling pathways can be just as important for proper biological regulation as turning them on. The Notch signaling pathway controls development of the nervous system in Drosophila. Proteolysis of Notch appears to initiate signaling, but further proteolysis may also terminate signals from this pathway. Kopan discusses mechanisms that limit signaling by Notch, including recent evidence that degradation of specifically targeted proteins by the proteasome is required.

Cancer linked to Alzheimer disease but not vascular dementia.

OBJECTIVE: To investigate whether cancer is associated with Alzheimer disease (AD) and vascular dementia (VaD). METHODS: Cox proportional hazards models were used to test associations between prevalent dementia and risk of future cancer hospitalization, and associations between prevalent cancer and risk of subsequent dementia. Participants in the Cardiovascular Health Study-Cognition Substudy, a prospective cohort study, aged 65 years or older (n = 3,020) were followed a mean of 5.4 years for dementia and 8.3 years for cancer. RESULTS: The presence of any AD (pure AD + mixed AD/VaD; hazard ratio [HR] = 0.41, 95% confidence interval [CI] = 0.20-0.84) and pure AD (HR = 0.31, 95% CI = 0.12-0.86) was associated with a reduced risk of future cancer hospitalization, adjusted for demographic factors, smoking, obesity, and physical activity. No significant associations were found between dementia at baseline and rate of cancer hospitalizations for participants with diagnoses of VaD. Prevalent cancer was associated with reduced risk of any AD (HR = 0.72; 95% CI = 0.52-0.997) and pure AD (HR = 0.57; 95% CI = 0.36-0.90) among white subjects after adjustment for demographics, number of APOE epsilon4 alleles, hypertension, diabetes, and coronary heart disease; the opposite association was found among minorities, but the sample size was too small to provide stable estimates. No significant association was found between cancer and subsequent development of VaD. CONCLUSIONS: In white older adults, prevalent Alzheimer disease (AD) was longitudinally associated with a reduced risk of cancer, and a history of cancer was associated with a reduced risk of AD. Together with other work showing associations between cancer and Parkinson disease, these findings suggest the possibility that cancer is linked to neurodegeneration.

A new look into an old problem: keratins as tools to investigate determination, morphogenesis, and differentiation in skin.

We have investigated keratin and keratin mRNA expression during (1) differentiation of stem cells into epidermis and hair follicles and (2) morphogenesis of follicles. Our results indicate that a type I keratin K14 is expressed early in embryonal basal cells. Subsequently, its expression is elevated in the basal layer of developing epidermis but suppressed in developing matrix cells. This difference represents an early and major biochemical distinction between the two diverging cell types. Moreover, because expression of this keratin is not readily influenced by extracellular regulators or cell culture, it suggests a well-defined and narrow window of development during which an irreversible divergence in basal and matrix cells may take place. In contrast to K14, which is expressed very early in development and coincident with basal epidermal differentiation, a hair-specific type I keratin and its mRNA is expressed late in hair matrix development and well after follicle morphogenesis. Besides providing an additional developmental difference between epidermal and hair matrix cells, the hair-specific keratins provide the first demonstration that keratin expression may be a consequence rather than a cause of cell organization and differentiation.

Synthesis and possible role of proteoglycans during Volvox development.

During the embryonic development of Volvox the cells synthesize proteoglycans of relatively small molecular size up to 14 h after inversion. In the following stages there is a gradual transition towards larger forms, due to an increase in sulfated sugar moieties. The presence of sulfate in the medium was obligatory for this transition. None of these molecules could be dissociated in 4.0 M GuHCl, neither could they be digested by glycosidases. Of all proteases examined subtilisin, a bacterial protease, digested the proteoglycans. The possible role of proteoglycans during Volvox morphogenesis has been examined. Somatic proteoglycans caused spheroids to start their swimming earlier, although they inhibited their expansion. Spheroids cultured in sulfate-free medium start their swimming 24 h later than controls, but the addition of somatic proteoglycans to the sulfate-free medium could induce motility almost as well as in the normal forms. Embryonic weight proteoglycans caused aggregation of the spheroids, an effect that also appeared in the sulfate-free medium.

The intracellular domain of mouse Notch: a constitutively activated repressor of myogenesis directed at the basic helix-loop-helix region of MyoD.

We show that Myf-5 and mNotch mRNA are both present in the presomitic mesoderm before muscle cell commitment and before muscle structural gene activation. The failure of presomitic mesoderm to respond to Myf-5 and express myogenic properties implies that there may be a mechanism in presomitic mesoderm to suppress muscle differentiation. Here we show that ectopic expression of the intracellular domain of mNotch (mNotchIC) functions as a constitutively activated repressor of myogenesis both in cultured cells and in frog embryos. Mutagenesis experiments indicate that the target for inactivation by mNotch is the MyoD basic helix-loop-helix domain. mNotchIC contains a nuclear localization signal and localizes to the nucleus. Removal of the nuclear localization signal (NLS) reduces nuclear localization and diminishes the inhibition of myogenesis caused by Myf-5 or MyoD. Additional experiments show that the CDC10/SWI6/ankyrin repeats are also necessary for myogenic inhibition.

An activated Notch suppresses neurogenesis and myogenesis but not gliogenesis in mammalian cells.

P19 cells, a mouse embryonal carcinoma line, can be induced to differentiate into neurons. After induction, however, only a small subpopulation of cells develop as neurons, suggesting that equipotent cells adopt different cell fates. In invertebrate systems, the lin-12-Notch family of genes is thought to control the choice of cell fate. We have therefore asked whether activation of murine Notch (mNotch) regulates neuronal differentiation in P19 cells. We demonstrate that a dominant gain-of-function mutant of mNotch suppresses neurogenesis, as well as myogenesis in P19 cells. Overexpression of the full-length mNotch protein also suppresses neurogenesis. In contrast, the differentiation of glia is not affected by an activated mNotch homologue. These data indicate that mNotch may play a central role in the choice of cell fate in differentiating cells in culture and suggests that mNotch may play a similar role in the choice of fate in the developing mammalian embryo.

Notch-1 signalling requires ligand-induced proteolytic release of intracellular domain.

Notch proteins are ligand-activated transmembrane receptors involved in cell-fate selection throughout development. No known enzymatic activity is contained within Notch and the molecular mechanism by which it transduces signals across the cell membrane is poorly understood. In many instances, Notch activation results in transcriptional changes in the nucleus through an association with members of the CSL family of DNA-binding proteins (where CSL stands for CBF1, Su(H), Lag-1). As Notch is located in the plasma membrane and CSL is a nuclear protein, two models have been proposed to explain how they interact. The first suggests that the two interact transiently at the membrane. The second postulates that Notch is cleaved by a protease, enabling the cleaved fragment to enter the nucleus. Here we show that signalling by a constitutively active membrane-bound Notch-1 protein requires the proteolytic release of the Notch intracellular domain (NICD), which interacts preferentially with CSL. Very small amounts of NICD are active, explaining why it is hard to detect in the nucleus in vivo. We also show that it is ligand binding that induces release of NICD.

Activation of the Notch pathway in the hair cortex leads to aberrant differentiation of the adjacent hair-shaft layers.

Little is known about the mechanisms underlying the generation of various cell types in the hair follicle. To investigate the role of the Notch pathway in this process, transgenic mice were generated in which an active form of Notch1 (Notch(DeltaE)) was overexpressed under the control of the mouse hair keratin A1 (MHKA1) promoter. MHKA-Notch(DeltaE) is expressed only in one precursor cell type of the hair follicle, the cortex. Transgenic mice could be easily identified by the phenotypes of curly whiskers and wavy, sheen pelage hair. No effects of activated Notch on proliferation were detected in hair follicles of the transgenic mice. We find that activating Notch signaling in the cortex caused abnormal differentiation of the medulla and the cuticle, two neighboring cell types that did not express activated Notch. We demonstrate that these non-autonomous effects are likely caused by cell-cell interactions between keratinocytes within the hair follicle and that Notch may function in such interactions either by directing the differentiation of follicular cells or assisting cells in interpreting a gradient emanating from the dermal papilla.

A human homologue of the Drosophila developmental gene, Notch, is expressed in CD34+ hematopoietic precursors.

Members of the Notch gene family have been shown to mediate cell-fate decisions by multipotent precursors in a number of different systems. To determine whether members of this family might play a similar role in hematopoiesis, we asked if homologues of the Notch gene are expressed in human hematopoietic precursors. Using degenerate oligonucleotides corresponding to conserved amino acid sequences in known Notch homologues as primers for the polymerase chain reaction (PCR), we demonstrated that at least one Notch homologue is expressed in human bone marrow CD34+ cells, a population enriched for hematopoietic precursors. Cloning and sequencing of the PCR products identified this Notch homologue as TAN-1, a member of the Notch family previously cloned from a T-cell leukemia with a translocation involving this gene. Subsequent evaluation of bone marrow hematopoietic cells for TAN-1 expression using a reverse transcription-PCR assay confirmed the expression of TAN-1 in CD34+ hematopoietic precursors, including the immature subset that lacks expression of lineage-associated antigens (CD34+lin-). These findings, together with the known role of Notch homologues in other systems, suggest that members of the Notch family, including TAN-1, may be involved in mediating cell-fate decisions during hematopoiesis.