Esophageal Expression of Active IκB Kinase-ß in Mice Up-Regulates Tumor Necrosis Factor and Granulocyte-Macrophage Colony-Stimulating Factor, Promoting Inflammation and Angiogenesis.

BACKGROUND & AIMS: IκB kinase-ß (IKKß) mediates activation of the nuclear factor-κB, which regulates immune and inflammatory responses. Although nuclear factor-κB is activated in cells from patients with inflammatory diseases or cancer, little is known about its roles in the development and progression of esophageal diseases. We investigated whether mice that express an activated form of IKKß in the esophageal epithelia develop esophageal disorders. METHODS: We generated ED-L2-Cre/Rosa26-IKK2caSFL mice, in which the ED-L2 promoter activates expression of Cre in the esophageal epithelia, leading to expression of a constitutively active form of IKKß (IKKßca) in epithelial cells but not in inflammatory cells or the surrounding stroma (IKKßca mice). Mice lacking the Cre transgene served as controls. Some mice were given intraperitoneal injections of neutralizing antibodies against granulocyte-macrophage colony-stimulating factor (GM-CSF) or tumor necrosis factor (TNF), or immunoglobulin G1 (control), starting at 1 month of age. Epithelial tissues were collected and analyzed by immunofluorescence, immunohistochemical, and quantitative real-time polymerase chain reaction assays. Transgenes were overexpressed from retroviral vectors in primary human keratinocytes. RESULTS: IKKßca mice developed esophagitis and had increased numbers of blood vessels in the esophageal stroma, compared with controls. Esophageal tissues from IKKßca mice had increased levels of GM-CSF. Expression of IKKßca in primary human esophageal keratinocytes led to 11-fold overexpression of GM-CSF and 200-fold overexpression of TNF. Incubation of human umbilical vein endothelial cells with conditioned media from these keratinocytes increased endothelial cell migration by 42% and promoted formation of capillary tubes; these effects were blocked by a neutralizing antibody against GM-CSF. Injections of anti-GM-CSF reduced angiogenesis and numbers of CD31+ blood vessels in esophageal tissues of IKKßca mice, but did not alter the esophageal vasculature of control mice and did not alter recruitment of intraepithelial leukocytes to esophageal tissues of IKKßca mice. Injections of anti-TNF prevented the development of esophagitis in IKKßca mice. CONCLUSIONS: Constitutive activation of IKKß in the esophageal epithelia of mice leads to inflammation and angiogenesis, mediated by TNF and GM-CSF, respectively.

Drug-Induced Liver Injury Secondary to Enobosarm: A Selective Androgen Receptor Modulator.

Selective androgen receptor modulators (SARMs) are compounds that bind to androgen receptors and have similar anabolic properties to anabolic steroids. Unlike anabolic steroids, which bind to androgen receptors in many tissues all over the body, individual SARMs selectively bind androgen receptors in certain tissues, but not in others. This selectivity has attracted researchers due to the possibility of using SARMs for the potential benefits of androgen receptor stimulation, such as increased muscle mass and increased bone density, while minimizing the adverse effects, such as erythrocytosis and hepatotoxicity. Enobosarm, a SARM, has been studied for use in treatment of cachexia, osteoporosis, breast and prostate cancers, and stress urinary incontinence. Enobosarm can be found in some over-the-counter muscle-building supplements. We report a 31-year-old man with no significant personal or family medical history who presented with itching and dark-colored urine for 1 week. Three weeks prior to presentation, he had begun using a muscle-building supplement containing enobosarm. Diagnostic workup concluded a drug-induced hepatocellular liver injury secondary to enobosarm, which subsequently improved after discontinuation of enobosarm-containing muscle-building supplement use. As enobosarm and other SARMs are increasingly found in the over-the-counter supplements and being studied for other clinical applications, it is important to recognize their potential for liver toxicity.

The microRNA-30 family is required for vertebrate hepatobiliary development.

BACKGROUND & AIMS: The function of microRNA (miRNA) in liver development is unknown. To address this issue, we characterized miRNA expression in the embryonic mouse liver, performed functional miRNA analysis in zebrafish larvae, and identified novel hepatic miRNA targets. METHODS: Hepatic RNA isolated from mice at embryonic days 15.5, 18.5, and postnatal day 2 was hybridized to a mouse miRNA microarray. The microarray results were confirmed by Northern blot hybridization and quantitative reverse-transcription polymerase chain reaction. The spatial distribution of selected miRNAs was determined by in situ hybridization. Functional analysis of miR-30a was performed in zebrafish using antisense-mediated miRNA knockdown. Targets of miR-30a were identified by microarray analysis of gene expression following knockdown in cultured cells. RESULTS: A set of 38 differentially expressed fetal hepatic miRNAs was identified. Several of these miRNAs were found to exhibit distinct temporal and spatial patterns of expression in hepatocytes, cholangiocytes, and nonepithelial cells within the liver. Two (miR-30a and miR-30c) are the first examples of ductal plate and bile duct-specific hepatic miRNAs. Knockdown of miR-30a in the zebrafish larva results in defective biliary morphogenesis. Several newly identified targets of miR-30a are known regulators of liver development and function. CONCLUSIONS: We have identified miRNAs whose spatial and temporal patterns of expression are suggestive of functional roles in hepatic development and/or function. One of these, the biliary miRNA miR-30a, is required for biliary development in zebrafish. This is the first demonstration of a functional role for miRNA in hepatic organogenesis.

KLF4 activates NFκB signaling and esophageal epithelial inflammation via the Rho-related GTP-binding protein RHOF.

Understanding the regulatory mechanisms within esophageal epithelia is essential to gain insight into the pathogenesis of esophageal diseases, which are among the leading causes of morbidity and mortality throughout the world. The zinc-finger transcription factor Krüppel-like factor (KLF4) is implicated in a large number of cellular processes, such as proliferation, differentiation, and inflammation in esophageal epithelia. In murine esophageal epithelia, Klf4 overexpression causes chronic inflammation which is mediated by activation of NFκB signaling downstream of KLF4, and this esophageal inflammation produces epithelial hyperplasia and subsequent esophageal squamous cell cancer. Yet, while NFκB activation clearly promotes esophageal inflammation, the mechanisms by which NFκB signaling is activated in esophageal diseases are not well understood. Here, we demonstrate that the Rho-related GTP-binding protein RHOF is activated by KLF4 in esophageal keratinocytes, leading to the induction of NFκB signaling. Moreover, RHOF is required for NFκB activation by KLF4 in esophageal keratinocytes and is also important for esophageal keratinocyte proliferation and migration. Finally, we find that RHOF is upregulated in eosinophilic esophagitis, an important esophageal inflammatory disease in humans. Thus, RHOF activation of NFκB in esophageal keratinocytes provides a potentially important and clinically-relevant mechanism for esophageal inflammation and inflammation-mediated esophageal squamous cell cancer.

KLF4 transcriptionally activates non-canonical WNT5A to control epithelial stratification.

Epithelial differentiation and stratification are essential for normal homeostasis, and disruption of these processes leads to both injury and cancer. The zinc-finger transciption factor KLF4 is a key driver of epithelial differentiation, yet the mechanisms and targets by which KLF4 controls differentiation are not well understood. Here, we define WNT5A, a non-canonical Wnt ligand implicated in epithelial differentiation, repair, and cancer, as a direct transcriptional target that is activated by KLF4 in squamous epithelial cells. Further, we demonstrate functionally that WNT5A mediates KLF4 control of epithelial differentiation and stratification, as treatment of keratinocytes with WNT5A rescues defective epithelial stratification resulting from KLF4 loss. Finally, we show that the small GTPase CDC42 is regulated by KLF4 in a WNT5A dependent manner. As such, we delineate a novel pathway for epithelial differentiation and stratification and define potential therapeutic targets for epithelial diseases.

De novo formation of insulin-producing "neo-ß cell islets" from intestinal crypts.

The ability to interconvert terminally differentiated cells could serve as a powerful tool for cell-based treatment of degenerative diseases, including diabetes mellitus. To determine which, if any, adult tissues are competent to activate an islet ß cell program, we performed an in vivo screen by expressing three ß cell "reprogramming factors" in a wide spectrum of tissues. We report that transient intestinal expression of these factors-Pdx1, MafA, and Ngn3 (PMN)-promotes rapid conversion of intestinal crypt cells into endocrine cells, which coalesce into "neoislets" below the crypt base. Neoislet cells express insulin and show ultrastructural features of ß cells. Importantly, intestinal neoislets are glucose-responsive and able to ameliorate hyperglycemia in diabetic mice. Moreover, PMN expression in human intestinal "organoids" stimulates the conversion of intestinal epithelial cells into ß-like cells. Our results thus demonstrate that the intestine is an accessible and abundant source of functional insulin-producing cells.