The balance and distribution of epithelial cell types is required to maintain tissue homeostasis. A hallmark of airway diseases is epithelial remodeling, leading to increased goblet cell numbers and an overproduction of mucus. In the conducting airway, basal cells act as progenitors for both secretory and ciliated cells. To identify mechanisms regulating basal cell fate, we developed a screenable 3D culture system of airway epithelial morphogenesis. We performed a high-throughput screen using a collection of secreted proteins and identified inflammatory cytokines that specifically biased basal cell differentiation toward a goblet cell fate, culminating in enhanced mucus production. We also demonstrate a specific requirement for Notch2 in cytokine-induced goblet cell metaplasia in vitro and in vivo. We conclude that inhibition of Notch2 prevents goblet cell metaplasia induced by a broad range of stimuli and propose Notch2 neutralization as a therapeutic strategy for preventing goblet cell metaplasia in airway diseases.

SUMMARY Current anti-angiogenic agents used to treat cancer only partially inhibit neovascularization and cause normal tissue toxicities, fueling the need to identify therapeutic agents that are more selective for pathological angiogenesis. Tumor Endothelial Marker 8 (TEM8), also known as anthrax toxin receptor 1 (ANTXR1), is a highly conserved cell-surface protein overexpressed on tumor-infiltrating vasculature. Here, we show that genetic disruption of Tem8 results in impaired growth of human tumor xenografts of diverse origin including melanoma, breast, colon, and lung cancer. Furthermore, antibodies developed against the TEM8 extracellular domain blocked anthrax intoxication, inhibited tumor-induced angiogenesis, displayed broad anti-tumor activity and augmented the activity of clinically approved anti-cancer agents without added toxicity. Thus, TEM8 targeting may allow selective inhibition of pathological angiogenesis.

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Retinoic acid (RA) exerts its pleiotropic effects on cell growth and differentiation through the activation of a family of transcription factors-the RA receptors (RARs). Three subtypes of these receptors exist, RARa, RAR(3, and RARly. The receptors are differentially expressed in different cell types and stages of development, suggesting that they may regulate different sets of genes. We have identified a synthetic retinoid with the characteristics of a selective RARa antagonist. This antagonist counteracts RA effects on HL-60 cell differentiation and on B-lymphocyte polyconal activation.Beyond its potential practical relevance, this and other specific antagonists will be useful to dissect the RAR system and to assign to one given receptor each of the many RA-regulated functions.The natural retinol (vitamin A) derivative retinoic acid (RA) is known to have profound effects on cell growth and differentiation (1) and to be essential for normal embryonic development (2). While RA and some synthetic analogs (retinoids) are useful in the control of some tumors (3) as well as of nonmalignant hyperproliferative conditions of the skin (4), they are, at high concentrations, teratogenic (5).The pleiotropic effects of retinoids are mediated by two known families of nuclear receptors, both belonging to the steroid-thyroid hormone receptor superfamily of ligandinducible transcriptional regulators (6, 7). The RA receptor (RAR) gene family comprises three subtypes-RARa (8, 9), RAR,[8][9][10][11][12], and RAR'y (13, 14)-with each gene encoding a variable number of isoforms arising by differential splicing of two primary . All receptors of the RAR family bind RA with comparable affinity (18). The retinoid receptors of the second family (RXR) do not bind the major form of RA (all-trans-RA) (19). They bind instead the 9-cis stereoisomer of RA (20, 21).Transcription of some RAR genes themselves is RA sensitive (22-25). Also, the expression of some of the cellular retinol-or RA-binding proteins (CRBP and CRABP), putatively involved in the storage, transport, and/or metabolism of retinol and RA, is differentially regulated by RA in a receptor-specific manner (26-28). The RA-related molecules represent, therefore, an autoregulated system. RAR types and isoforms, as well as RXRa and RXRB, are differentially expressed both spatially and temporally (15-18, 29-32). They might therefore regulate different target genes during embryonic and adult life, as well as in specific cell types at different stages of differentiation. RARa is the most ubiquitously expressed, while RAR8 and RARy display a more restricted pattern of distribution, with RARy being predominantly expressed in the skin (31).It seems reasonable to assume that the multiple effects of RA could be dissociated by specific ligands for each of the known receptors, and/or by receptor-specific antagonists, so as to obtain the desired beneficial effects while limiting the unwanted side effects. Retinoids with a good degree of selectivity have been described (33), and we have o...

Surface plasmon resonance biosensor technology was used to directly measure the binding interactions of small molecules to the ligand-binding domain of human estrogen receptor. In a screening mode, specific ligands of the receptor were easily discerned from nonligands. In a high-resolution mode, the association and dissociation phase binding responses were shown to be reproducible and could be fit globally to a simple interaction model to extract reaction rate constants. On average, antagonist ligands (such as tamoxifen and nafoxidine) were observed to bind to the receptor with association rates that were 500-fold slower than agonists (such as estriol and ␤-estradiol). This finding is consistent with these antagonists binding to an altered conformation of the receptor. The biosensor assay also could identify subtle differences in how the same ligand interacted with two different isoforms of the receptor (␣ and ␤). The biosensor's ability to determine kinetic rate constants for small molecule͞protein interactions provides unique opportunities to understand the mechanisms associated with complex formation as well as new information to drive the optimization of drug candidates.S urface plasmon resonance (SPR) biosensor technology has advanced to the point where it is possible to measure directly small molecules interacting with immobilized macromolecular targets (1, 2). This development suggests that biosensor analysis will become an important secondary screening tool in drug discovery, confirming hits from primary screens and providing detailed kinetics for lead optimization (3, 4). To illustrate the utility of current SPR technology, the binding properties of small compounds (200-500 Da) interacting with human estrogen receptor (ER) were analyzed.Ligand binding to ER is responsible for controlling the basic biology of estrogen-sensitive tissues. Using selective agonists or antagonists to modulate this biology is the focus of significant activity in the pharmaceutical industry (5-9). To date, a ligand's binding properties for ER have mainly been determined by equilibrium binding assays that often employ radiolabeled compounds and require overnight incubations. Here, we demonstrate how optical biosensors may be used to determine both kinetic and equilibrium binding constants for compounds interacting with ER in real time without labeling either binding partner.SPR biosensor experiments require immobilizing one reactant on a surface and monitoring its binding to a second reactant in solution. An antibody-capturing method was used to study the dynamics of ER͞ligand interactions. This assay format created a chemically homogenous receptor surface and allowed us to determine rapidly the binding properties of a variety of compounds. We examined the binding of 12 compounds (shown in Fig. 1) having differing receptor activities: both estrogen and non-estrogen agonists, SERMs (selective ER modulators, which for this discussion are referred to as antagonists), and nonbinding control compounds that possess core structures sim...