Colonic healing requires WNT produced by epithelium as well as Tagln+ and Acta2+ stromal cells

While Wnt signaling is clearly important for the intestinal epithelial homeostasis, the relevance of various sources of Wnt ligands themselves remains incompletely understood. Wnt blockage in distinct stromal cell types suggested obligatory functions of several stromal cell sources and yielded different observations. The physiological contribution of epithelial Wnt to tissue homeostasis remains unclear. We show here that blocking epithelial Wnts affected colonic Reg4+ epithelial cell differentiation, and impaired colonic epithelial regeneration after injury. Single cell RNA analysis of intestinal stroma showed that the majority of Wnt-producing cells were contained in transgelin (Tagln+) and smooth muscle actin alpha 2 (Acta2+) expressing populations. We genetically attenuated Wnt production from these stromal cells using Tagln-Cre and Acta2-CreER drivers, and found that Wnt blockage from either epithelium or Tagln+ and Acta2+ stromal cells impaired colonic epithelial healing after chemical-induced injury. Aggregated Wnt blockage from both epithelium and Tagln+ or Acta2+ stromal cells drastically diminished epithelial repair, increasing morbidity and mortality. These results from two uncharacterized stromal populations suggested that colonic recovery from colitis-like injury depends on multiple Wnt-producing sources.

STEM-26. BLOOD-TUMOR BARRIER IS COMPOSED OF MECHANOSENSING TUMOR CELLS THAT MASK THERAPEUTIC VULNERABILITY

Two major obstacles in brain cancer treatment are the blood-tumor barrier (BTB), which restricts delivery of most therapeutic agents, and the quiescent brain tumor-initiating cells (BTICs), which evade cell cycle-targeting chemotherapy. Mechanosensation, the transduction of mechanical cues into cellular signaling, underlies physiological processes such as touch, pain, proprioception, hearing, respiration, epithelial homeostasis, and vascular and lymphatic development. We report that medulloblastoma (MB) BTICs are mechanosensing, a property conferred by force-activated ion channel Piezo2. In contrast to the prevailing view that astrocytes function as a physical barrier in BTB, BTICs project endfeet to ensheathe capillaries. MB develops a tissue stiffness gradient as a function of distance to capillaries. Piezo2 senses substrate stiffness to sustain local intracellular calcium, actomyosin tension, and adhesion at BTIC growth cones, which allow BTICs to mechanically interact with their substrate and sequester β-Catenin to prevent WNT/β-Catenin signaling in BTICs. Our single cell analysis uncovers a two-branched BTIC trajectory that progresses from a deep quiescent state to two cycling states. Tumor cell-specific Piezo2 knockout reverses the off-on WNT/β-Catenin signaling states in BTICs and endothelial cells, collapses the BTB, reduces quiescence depth of BTICs, and markedly enhances MB response to chemotherapy. Our study reveals that BTICs co-opt astrocytic mechanism to contribute to the BTB and provides the first evidence that BTB depends on mechanochemical signaling to mask tumor chemosensitivity. Targeting Piezo2 addresses BTB and BTIC properties that underlie therapy failures in brain cancer.

NF-κB determines Paneth versus goblet cell fate decision in the small intestine

ABSTRACT Although the role of the transcription factor NF-κB in intestinal inflammation and tumor formation has been investigated extensively, a physiological function of NF-κB in sustaining intestinal epithelial homeostasis beyond inflammation has not been demonstrated. Using NF-κB reporter mice, we detected strong NF-κB activity in Paneth cells, in ‘+4/+5’ secretory progenitors and in scattered Lgr5+ crypt base columnar stem cells of small intestinal (SI) crypts. To examine NF–κB functions in SI epithelial self-renewal, mice or SI crypt organoids (‘mini-guts’) with ubiquitously suppressed NF-κB activity were used. We show that NF-κB activity is dispensable for maintaining SI epithelial proliferation, but is essential for ex vivo organoid growth. Furthermore, we demonstrate a dramatic reduction of Paneth cells in the absence of NF-κB activity, concomitant with a significant increase in goblet cells and immature intermediate cells. This indicates that NF-κB is required for proper Paneth versus goblet cell differentiation and for SI epithelial homeostasis, which occurs via regulation of Wnt signaling and Sox9 expression downstream of NF-κB. The current study thus presents evidence for an important role for NF-κB in intestinal epithelial self-renewal.

The Balance between Differentiation and Terminal Differentiation Maintains Oral Epithelial Homeostasis

The oral epithelium is one of the fastest repairing and continuously renewing tissues. Stem cell activation within the basal layer of the oral epithelium fuels the rapid proliferation of multipotent progenitors. Stem cells first undergo asymmetric cell division that requires tightly controlled and orchestrated differentiation networks to maintain the pool of stem cells while producing progenitors fated for differentiation. Rapidly expanding progenitors subsequently commit to advanced differentiation programs towards terminal differentiation, a process that regulates the structural integrity and homeostasis of the oral epithelium. Therefore, the balance between differentiation and terminal differentiation of stem cells and their progeny ensures progenitors commitment to terminal differentiation and prevents epithelial transformation and oral squamous cell carcinoma (OSCC). A recent comprehensive molecular characterization of OSCC revealed that a disruption of terminal differentiation factors is indeed a common OSCC event and is superior to oncogenic activation. Here, we discuss the role of differentiation and terminal differentiation in maintaining oral epithelial homeostasis and define terminal differentiation as a critical tumour suppressive mechanism. We further highlight factors with crucial terminal differentiation functions and detail the underlying consequences of their loss. Switching on terminal differentiation in differentiated progenitors is likely to represent an extremely promising novel avenue that may improve therapeutic interventions against OSCC.

Grhl3 promotes retention of epidermal cells under endocytic stress to maintain epidermal architecture in zebrafish

Epithelia such as epidermis cover large surfaces and are crucial for survival. Maintenance of tissue homeostasis by balancing cell proliferation, cell size, and cell extrusion ensures epidermal integrity. Although the mechanisms of cell extrusion are better understood, how epithelial cells that round up under developmental or perturbed genetic conditions are reintegrated in the epithelium to maintain homeostasis remains unclear. Here, we performed live imaging in zebrafish embryos to show that epidermal cells that round up due to membrane homeostasis defects in the absence of goosepimples/myosinVb (myoVb) function, are reintegrated into the epithelium. Transcriptome analysis and genetic interaction studies suggest that the transcription factor Grainyhead-like 3 (Grhl3) induces the retention of rounded cells by regulating E-cadherin levels. Moreover, Grhl3 facilitates the survival of MyoVb deficient embryos by regulating cell adhesion, cell retention, and epidermal architecture. Our analyses have unraveled a mechanism of retention of rounded cells and its importance in epithelial homeostasis.

Intestinal epithelial IPMK protects mice from experimental colitis via governing colonic tuft cell development

As a pleiotropic signaling factor, inositol polyphosphate multikinase (IPMK) is involved in key biological events such as growth and innate immunity, acting either enzymatically to mediate the biosynthesis of inositol polyphosphates and phosphatidylinositol 3,4,5-trisphosphates, or noncatalytically to control key signaling target molecules. However, the functional significance of IPMK in regulating gut epithelial homeostasis remains largely unknown. Here we show that intestinal epithelial-specific deletion of IPMK aggravates dextran sulfate sodium (DSS)-induced colitis with higher clinical colitis scores and elevated epithelial barrier permeability. No apparent defects in PI3K-AKT signaling pathway and pro-inflammatory cytokine production were found in IPMK-deficient colons challenged by DSS treatment. RNA-sequencing and FACS analyses further revealed significantly decreased tuft cells in IPMK-deficient colons. Importantly, IPMK deletion in the gut epithelium was found to decrease choline acetyltransferase (ChAT) but not IL-25, suggesting selective loss of cholinergic signaling. Thus, these findings identify IPMK as a physiological determinant of tuft cell differentiation and highlight the critical function of IPMK in the control of gut homeostasis.

First person – Bhuminder Singh

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Bhuminder Singh is first author on ‘ Induction of apically mistrafficked epiregulin disrupts epithelial polarity via aberrant EGFR signaling’, published in JCS. Bhuminder conducted the research described in this article while a research fellow in Robert J. Coffey's lab at Vanderbilt University Medical Center, Nashville, TN, USA. He is now an assistant professor at Vanderbilt University Medical Center, investigating the role of EGFR signaling in epithelial homeostasis and disorders such as cancer.

The Candidate IBD Risk Gene CCNY Is Dispensable for Intestinal Epithelial Homeostasis

The CCNY gene, which encodes cyclin Y, has been implicated in the pathogenesis of inflammatory bowel disease (IBD). Cyclin Y promotes Wnt/β-catenin signaling and autophagy, which are critical for intestinal epithelial cell (IEC) homeostasis, and may thereby contribute to wound repair in colitis. However, whether cyclin Y has an essential function in IECs is unknown. We, therefore, investigated the epithelial injury response and mucosal regeneration in mice with conditional knock-out of Ccny in the intestinal epithelium. We observed that Ccny-deficient mice did not exhibit any differences in cell proliferation and disease activity compared to wild-type littermates in the dextran sulfate sodium (DSS) colitis model. Complementary in vitro experiments showed that loss of CCNY in model IECs did not affect Wnt signaling, cell proliferation, or autophagy. Additionally, we observed that expression of the cyclin-Y-associated cyclin-dependent kinase (CDK) 14 is exceedingly low specifically in IEC. Collectively, these results suggest that cyclin Y does not contribute to intestinal epithelial homeostasis, possibly due to low levels of specific CDKs in these cells. Thus, it is unlikely that CCNY mutations are causatively involved in IBD pathogenesis.