Bile Acids Signal via TGR5 to Activate Intestinal Stem Cells and Epithelial Regeneration

BACKGROUND & AIMS: Renewal and patterning of the intestinal epithelium is coordinated by intestinal stem cells (ISCs); dietary and metabolic factors provide signals to the niche that control ISC activity. Bile acids (BAs), metabolites in the gut, signal nutrient availability by activating the G protein-coupled bile acid receptor 1 (GPBAR1, also called TGR5). TGR5 is expressed in the intestinal epithelium, but it is not clear how its activation affects ISCs and regeneration of the intestinal epithelium. We studied the role of BAs and TGR5 in intestinal renewal, and regulation of ISC function in mice and intestinal organoids. METHODS: We derived intestinal organoids from wild-type mice and Tgr5(-/-) mice, incubated them with BAs or the TGR5 agonist INT-777, and monitored ISC function by morphologic analyses and colony-forming assays. We disrupted Tgr5 specifically in Lgr5-positive ISCs in mice (Tgr5ISC(-/-) mice) and analyzed ISC number, proliferation, and differentiation by flow cytometry, immunofluorescence, and organoid assays. Tgr5ISC(-/-) mice were given cholecystokinin; we measured the effects of BA release into the intestinal lumen and on cell renewal. We induced colitis in Tgr5ISC(-/-) mice by administration of dextran sulfate sodium; disease severity was determined based on body weight, colon length, and histopathology analysis of colon biopsies. RESULTS: BAs and TGR5 agonists promoted growth of intestinal organoids. Administration of cholecystokinin to mice resulted in acute release of BAs into the intestinal lumen and increased proliferation of the intestinal epithelium. BAs and Tgr5 expression in ISCs were required for homeostatic intestinal epithelial renewal and fatespecification, and for regeneration after colitis induction. Tgr5ISC(-/-) mice developed more severe colitis than mice without Tgr5 disruption in ISCs. ISCs incubated with INT-777 increased activation of yes-associated protein 1 (YAP1) and of its upstream regulator SRC. Inhibitors of YAP1 and SRC prevented organoid growth induced by TGR5 activation. CONCLUSIONS: BAs promote regeneration of the intestinal epithelium via activation of TGR5 in ISCs, resulting in activation of SRC and YAP and activation of their target genes. Release of endogenous BAs in the intestinal lumen is sufficient to promote ISC renewal and drives regeneration in response to injury.

Impaired expression of peroxisome proliferator-activated receptor gamma in ulcerative colitis

Johan Auwerx, Mehdi Karoui

BACKGROUND & AIMS: The peroxisome proliferator-activated receptor gamma (PPAR gamma) has been proposed as a key inhibitor of colitis through attenuation of nuclear factor kappa B (NF-kappa B) activity. In inflammatory bowel disease, activators of NF-kappa B, including the bacterial receptor toll-like receptor (TLR)4, are elevated. We aimed to determine the role of bacteria and their signaling effects on PPAR gamma regulation during inflammatory bowel disease (IBD). METHODS: TLR4-transfected Caco-2 cells, germ-free mice, and mice devoid of functional TLR4 (Lps(d)/Lps(d) mice) were assessed for their expression of PPAR gamma in colonic tissues in the presence or absence of bacteria. This nuclear receptor expression and the polymorphisms of gene also were assessed in patients with Crohn's disease (CD) and ulcerative colitis (UC), 2 inflammatory bowel diseases resulting from an abnormal immune response to bacterial antigens. RESULTS: TLR4-transfected Caco-2 cells showed that the TLR4 signaling pathway elevated PPAR gamma expression and a PPAR gamma-dependent reporter in an I kappa kappa beta dependent fashion. Murine and human intestinal flora induced PPAR gamma expression in colonic epithelial cells of control mice. PPAR gamma expression was significantly higher in the colon of control compared with Lps(d)/Lps(d) mice. Although PPAR gamma levels appeared normal in patients with CD and controls, UC patients displayed a reduced expression of PPAR gamma confined to colonic epithelial cells, without any mutation in the PPAR gamma gene. CONCLUSIONS: These data showed that the commensal intestinal flora affects the expression of PPAR gamma and that PPAR gamma expression is considerably impaired in patients with UC.

2003

Role of canonical Wnt signaling in mammary gland development

Renuga Devi Rajaram

The female reproductive hormones estrogens, progesterone, and prolactin control postnatal breast development and are important to breast carcinogenesis. In particular, exposure to progesterone is tightly linked to breast cancer risk. Using the mouse as a model system to study the mechanisms through which hormones elicit morphogenetic changes in the mammary gland in vivo, we demonstrated that progesterone acts by a paracrine mechanism and identified Wnt4 as a target of progesterone important in mediating side branch formation. The mechanisms underlying Wnt4 function remains poorly understood. To understand the pattern of canonical Wnt signaling activation during mammary gland development and to address which cellular compartment of the mammary gland is activated by canonical Wnt signaling, a reporter mouse model was utilized in which the lacZ gene is under the control of endogenous Axin2 promoter, classical target promoter of canonical Wnt signaling. Analysis of reporter mice at different developmental stages of mammary gland development showed the highest reporter activity during early pregnancy which correlated with the time of side branch formation. At this stage, β-galactosidase activity was found exclusively in myoepithelial cells. Overexpression of Wnt1 from MMTV promoter increases reporter activity in the myoepithelium. Gene expression analysis on FACS sorted luminal and myoepithelial cells shows that known canonical Wnt signaling pathway components such as receptors and co receptors as well as targets are specifically expressed in the myoepithelial compartment further supporting the notion that myoepithelial cells are the target cells for Wnt1 and Wnt4 secreted by luminal cells. Consistent with a model whereby progesterone induced Wnt4 secretion results in canonical Wnt signaling activation, reporter activity was induced by progesterone treatment and lacZ expression increased during diestrus when serum progesterone levels are high under physiological conditions. Analysis of contralateral mammary glands engrafted with PR-/-.Axin2 lacZ and PR+/-.Axin2 lacZ epithelium during pregnancy revealed that intact PR signaling is required to activate canonical Wnt signaling. Similar experiments with Wnt4-/-.Axin2 lacZ and Wnt4+/+.Axin2 lacZ mammary epithelium showed that reporter activation relies on Wnt4. Histological analysis on mammary glands in which β-catenin was ablated using MMTV-Cre revealed that β-catenin deleted cells were present only in the luminal epithelial compartment and showed that β-catenin deleted myoepithelial cells cannot participate in side branch formation. Similarly, ectopic expression of dominant negative β-catenin in wt MECs reduced side branching indicating that canonical Wnt signaling mediated by β-catenin is required for side branching. Furthermore, constitutive activation of β-catenin in the mammary epithelium was sufficient to induce side branch formation in virgin mice. In Wnt4 mutant epithelium, proliferation of progesterone receptor positive and negative cells were significantly reduced in response to progesterone treatment. Wnt signaling has been implicated in stem/progenitor activation in several tissues including mammary gland. Consistent with Wnt4 activating stem/progenitors, serial transplantation assays revealed that Wnt4-/- epithelium failed to reconstitute already at 2nd to 3rd generation when the wt has the ability to reconstitute up to seven generations indicating that Wnt4 is required for stem cell function while Wnt4 is dispensable for embryonic and rudimentary mammary gland formation. Histology and gene expression analysis in late pregnancy showed that Wnt4 mutant epithelia were able to differentiate and produce milk proteins, further supporting the thought that Wnt4 function is important for stem/progenitor cell activity and not required for differentiation. Taken together, I report that Wnt4 induces canonical Wnt signaling in myoepithelial cells and activation of canonical Wnt signaling is required and sufficient for side branching. Moreover, Wnt4 function is essential for mammary stem/progenitor cells.

EPFL2010

What signals operate in the mammary niche?

Cathrin Brisken, Tamara Tanos

Adult stem cells reside in a specialized microenvironment, the niche, which controls their behavior. As mammary stem cells, and consequently their niches, are still poorly defined, we look at better-characterized adult mammalian stem cell niches in the hematopoietic system and the skin. We attempt to define the mammary stem cell niche functionally, based on the widely used mammary fat pad reconstitution assay. We note that the concept of the niche needs to be extended from the specialized microenvironment described in the hematopoietic system, to a model that takes into account the macroenviroment, as recently shown in the skin, and systemic clues as we will illustrate for the mammary gland where the reproductive hormones are major determinants of stem cell activation. In fact, in the mammary gland a special type of stem cells is determined only during pregnancy. Reproductive hormones act on hormone receptor positive cells, sensor cells, in the mammary epithelium to induce paracrine signaling that leads to activation of stem cells. Some of the downstream mediators are in common with other niches such as Wnt and possibly Notch signaling. Other signals are specific to the mammary gland such as amphiregulin and RANKL.

2008

Role of canonical Wnt signaling in mammary gland development

Renuga Devi Rajaram

EPFL2010