Conditional deletion of Zeb1 in Csf1r+ cells reduces inflammatory response of the cornea to alkali burn.

ZEB1 is an essential factor in embryonic development. In adults, it is often highly expressed in malignant tumors with low expression in normal tissues. The major biological function of ZEB1 in developing embryos and progressing cancers is to transdifferentiate cells from an epithelial to mesenchymal phenotype; but what roles ZEB1 plays in normal adult tissues are largely unknown. We previously reported that the reduction of Zeb1 in monoallelic global knockout (Zeb1+/-) mice reduced corneal inflammation-associated neovascularization following alkali burn. To uncover the cellular mechanism underlying the Zeb1 regulation of corneal inflammation, we functionally deleted Zeb1 alleles in Csf1r+ myeloid cells using a conditional knockout (cKO) strategy and found that Zeb1 cKO reduced leukocytes in the cornea after alkali burn. The reduction of immune cells was due to their increased apoptotic rate and linked to a Zeb1-downregulated apoptotic pathway. We conclude that Zeb1 facilitates corneal inflammatory response by maintaining Csf1r+ cell viability.

Zeb1 facilitates corneal epithelial wound healing by maintaining corneal epithelial cell viability and mobility.

The cornea is the outmost ocular tissue and plays an important role in protecting the eye from environmental insults. Corneal epithelial wounding provokes pain and fear and contributes to the most ocular trauma emergency assessments worldwide. ZEB1 is an essential transcription factor in development; but its roles in adult tissues are not clear. We identify Zeb1 is an intrinsic factor that facilitates corneal epithelial wound healing. In this study, we demonstrate that monoallelic deletion of Zeb1 significantly expedites corneal cell death and inhibits corneal epithelial EMT-related cell migration upon an epithelial debridement. We provide evidence that Zeb1-regulation of corneal epithelial wound healing is through the repression of genes required for Tnfa-induced epithelial cell death and the induction of genes beneficial for epithelial cell migration. We suggest utilizing TNF-α antagonists would reduce TNF/TNFR1-induced cell death in the corneal epithelium and inflammation in the corneal stroma to help corneal wound healing.

Sphere-induced reprogramming of RPE cells into dual-potential RPE stem-like cells.

BACKGROUND: The retinal pigment epithelium (RPE) has the potential to regenerate the entire neuroretina upon retinal injury in amphibians. In contrast, this regenerative capacity has been lost in mammals. The reprogramming of differentiated somatic cells into induced pluripotent stem cells (iPSCs) by viral transduction of exogenous stem cell factors has triggered a revolution in regenerative medicine. However, the risks of potential mutation(s) caused by random viral vector insertion in host genomes and tumor formation in recipients hamper its clinical application. One alternative is to immortalize adult stem cells with limited potential or to partially reprogram differentiated somatic cells into progenitor-like cells through non-integration protocols. METHODS: Sphere-induced RPE stem cells (iRPESCs) were generated from adult mouse RPE cells. Their stem cell functionality was studied in a mouse model of retinal degeneration. The molecular mechanism underlying the sphere-induced reprogramming was investigated using microarray and loss-of-function approaches. FINDINGS: We provide evidence that our sphere-induced reprogramming protocol can immortalize and transform mouse RPE cells into iRPESCs with dual potential to differentiate into cells that express either RPE or photoreceptor markers both in vitro and in vivo. When subretinally transplanted into mice with retinal degeneration, iRPESCs can integrate to the RPE and neuroretina, thereby delaying retinal degeneration in the model animals. Our molecular analyses indicate that the Hippo signaling pathway is important in iRPESC reprogramming. INTERPRETATION: The Hippo factor Yap1 is activated in the nuclei of cells at the borders of spheres. The factors Zeb1 and P300 downstream of the Hippo pathway are shown to bind to the promoters of the stemness genes Oct4, Klf4 and Sox2, thereby likely transactivate them to reprogram RPE cells into iRPESCs. FUND: National Natural Science Foundation of China and the National Institute of Health USA.

Vav proteins are necessary for correct differentiation of mouse cecal and colonic enterocytes.

In the mammalian cecum and colon, a single layer of absorptive, mature enterocytes are a crucial element of the physical barrier to the contents of the lumen. Enterocytic differentiation involves expansion of cytoplasmic cytoskeletal networks, which have been proposed to maintain structural integrity of individual cells and thus the entire epithelial barrier. We sought molecular tools to test this hypothesis in vivo, because in vitro systems displaying full intestinal epithelial differentiation have not yet been developed. Vav proteins are RhoGEFs that modulate cytoskeletal networks in immune cells. We found that Vav proteins were preferentially expressed in terminally differentiating cecal and colonic enterocytes. Loss of Vav protein expression in triple-knockout mice (Vav1(-/-);Vav2(-/-);Vav3(-/-)) resulted in defective expansion of microtubule cytoskeletons, a significant decrease in cell height and diminished expression of differentiation markers. Despite these changes, enterocytes in the triple-mutant mice did not contain measurable alterations in actin cytoskeleton, apical cell-cell junctions, nuclear position or global polarized delivery of proteins involved in terminal differentiation. Aged triple-mutant mice spontaneously developed ulcerative lesions that were, in part, a result of defective wound repair. These studies show that Vav proteins are required for enterocytic differentiation and colonic epithelial barrier integrity.

A key role for autophagy and the autophagy gene Atg16l1 in mouse and human intestinal Paneth cells.

Susceptibility to Crohn's disease, a complex inflammatory disease involving the small intestine, is controlled by over 30 loci. One Crohn's disease risk allele is in ATG16L1, a gene homologous to the essential yeast autophagy gene ATG16 (ref. 2). It is not known how ATG16L1 or autophagy contributes to intestinal biology or Crohn's disease pathogenesis. To address these questions, we generated and characterized mice that are hypomorphic for ATG16L1 protein expression, and validated conclusions on the basis of studies in these mice by analysing intestinal tissues that we collected from Crohn's disease patients carrying the Crohn's disease risk allele of ATG16L1. Here we show that ATG16L1 is a bona fide autophagy protein. Within the ileal epithelium, both ATG16L1 and a second essential autophagy protein ATG5 are selectively important for the biology of the Paneth cell, a specialized epithelial cell that functions in part by secretion of granule contents containing antimicrobial peptides and other proteins that alter the intestinal environment. ATG16L1- and ATG5-deficient Paneth cells exhibited notable abnormalities in the granule exocytosis pathway. In addition, transcriptional analysis revealed an unexpected gain of function specific to ATG16L1-deficient Paneth cells including increased expression of genes involved in peroxisome proliferator-activated receptor (PPAR) signalling and lipid metabolism, of acute phase reactants and of two adipocytokines, leptin and adiponectin, known to directly influence intestinal injury responses. Importantly, Crohn's disease patients homozygous for the ATG16L1 Crohn's disease risk allele displayed Paneth cell granule abnormalities similar to those observed in autophagy-protein-deficient mice and expressed increased levels of leptin protein. Thus, ATG16L1, and probably the process of autophagy, have a role within the intestinal epithelium of mice and Crohn's disease patients by selective effects on the cell biology and specialized regulatory properties of Paneth cells.

Cotyledonoid leiomyoma: a benign uterine tumor with alarming gross appearance.

Cotyledonoid leiomyoma or "grapelike" leiomyoma is a very rare tumor among the ever-expanding repertoire of growth variants described in benign uterine leiomyoma. We report a case of cotyledonoid leiomyoma in a 55-year-old woman who presented with menorrhagia and uterine prolapse. A large multinodular fungating tumor adhering to the right posterolateral wall of the uterus and extending to the broad ligament was discovered at vaginal hysterectomy. With a provisional diagnosis of sarcoma, total hysterectomy and bilateral salpingo-oophorectomy were performed. Postoperatively, the patient was well with no evidence of recurrence at 14 months. Pathologic examination revealed a 10-cm, red-brown tumor that comprised multiple bulbous processes protruding over the uterine surface, in continuity with a dissecting intramyometrial component. It was composed of fascicles and nodules of bland-looking smooth muscle cells with prominent perinodular hydropic degeneration. Coagulative necrosis, mitoses, and nuclear atypia were absent. Cotyledonoid leiomyoma apparently results from a combination of several uncommon growth patterns operating together, including subserosal growth, dissecting growth, and perinodular hydropic degeneration. Increased awareness of this grossly alarming variant of benign uterine leiomyoma can help avoid overtreatment.