Katherine J. Rowland

The “cryptic” mechanism of action of glucagon-like peptide-2

Glucagon-like peptide-2 (GLP-2) is a peptide hormone with multiple beneficial effects on the intestine, including expansion of the mucosal surface area through stimulation of crypt cell proliferation, as well as enhancement of nutrient digestion and absorption. Recent advances in clinical trials involving GLP-2 necessitate elucidation of the exact signaling pathways by which GLP-2 acts. In particular, the GLP-2 receptor has been localized to several intestinal cell types that do not include the proliferating crypt cells, and the actions of GLP-2 have thus been linked to a complex network of indirect mediators that induce diverse signaling pathways. The intestinotropic actions of GLP-2 on the colon have been shown to be mediated through the actions of keratinocyte growth factor and insulin-like growth factor (IGF)-2, whereas small intestinal growth has been linked to IGF-1, IGF-2, and ErbB ligands, as well as the IGF-1 receptor and ErbB. The cellular source of these mediators remains unclear, but it likely includes the intestinal subepithelial myofibroblasts. Conversely, the anti-inflammatory and blood flow effects of GLP-2 are dependent on vasoactive intestinal polypeptide released from submucosal enteric neurons and nitric oxide, respectively. Finally, recent studies have suggested that GLP-2 not only modulates intestinal stem cell behavior but may also promote carcinogenesis in models of sporadic colon cancer. Further consideration of the molecular cross-talk and downstream signaling pathways mediating the intestinotropic effects of GLP-2 is clearly warranted.

Loss of Glucagon-Like Peptide-2–Induced Proliferation Following Intestinal Epithelial Insulin-Like Growth Factor-1–Receptor Deletion

BACKGROUND & AIMS Glucagon-like peptide-2 (GLP-2) is an intestinal hormone that promotes growth of the gastrointestinal tract. Although insulin-like growth factor (IGF)-1 and the IGF-1 receptor (IGF-1R) are required for GLP-2-induced proliferation of crypt cells, little is known about localization of the IGF-1R which mediates the intestinotropic actions of GLP-2. METHODS We examined intestinal growth and proliferative responses in mice with conditional deletion of IGF-1R from intestinal epithelial cells (IE-igf1rKO) after acute administration (30-90 min) of GLP-2, in response to 24-hour fasting and re-feeding (to induce GLP-2-dependent adaptation), and after chronic exposure (10 days) to GLP-2. RESULTS IE-igf1rKO mice had normal small intestinal weight, morphometric parameters, proliferative indices, and distribution of differentiated epithelial cell lineages. Acute administration of GLP-2 increased nuclear translocation of β-catenin in non-Paneth crypt cells and stimulated the crypt-cell proliferative marker c-Myc in control but not IE-igf1rKO mice. Small intestinal weight, crypt depth, villus height, and crypt-cell proliferation were decreased in control and IE-igf1rKO mice after 24-hour fasting. Although re-feeding control mice restored all of these parameters, re-fed IE-igf1rKO mice had reductions in adaptive regrowth of the villi and crypt-cell proliferation. Control mice that were given chronic GLP-2 had increases in small intestinal weight, mucosal cross-sectional area, crypt depth, villus height, and crypt-cell proliferation. However, the GLP-2-induced increase in crypt-cell proliferation was not observed in IE-igf1rKO mice, and growth of the crypt-villus axis was reduced. CONCLUSIONS The proliferative responses of the intestinal epithelium to exogenous GLP-2 administration and conditions of GLP-2-dependent adaptive re-growth require the intestinal epithelial IGF-1R.

Mechanism of Action of Glucagon-Like Peptide-2 to Increase IGF-I mRNA in Intestinal Subepithelial Fibroblasts

IGF-I, a known secretory product of intestinal subepithelial myofibroblasts (ISEMFs), is essential for the intestinotropic effects of glucagon-like peptide-2 (GLP-2). Furthermore, GLP-2 increases IGF-I mRNA transcript levels in vitro in heterogeneous fetal rat intestinal cultures, as well as in vivo in the rodent small intestine. To determine the mechanism underlying the stimulatory effect of GLP-2 on intestinal IGF-I mRNA, murine ISEMF cells were placed into primary culture. Immunocytochemistry showed that the ISEMF cells appropriately expressed α-smooth muscle actin and vimentin but not desmin. The cells also expressed GLP-2 receptor and IGF-I mRNA transcripts. Treatment of ISEMF cells with (Gly2)GLP-2 induced IGF-I mRNA transcripts by up to 5-fold of basal levels after treatment with 10(-8) m GLP-2 for 2 h (P < 0.05) but did not increase transcript levels for other intestinal growth factors, such as ErbB family members. Immunoblot revealed a 1.6-fold increase in phospho (p)-Akt/total-(t)Akt with 10(-8) m GLP-2 treatment (P < 0.05) but no changes in cAMP, cAMP-dependent β-galactosidase expression, pcAMP response element-binding protein/tcAMP response element-binding protein, pErk1/2/tErk1/2, or intracellular calcium. Furthermore, pretreatment of ISEMF cells with the phosphatidylinositol 3 kinase (PI3K) inhibitors, LY294002 and wortmannin, abrogated the IGF-I mRNA response to GLP-2, as did overexpression of kinase-dead Akt. The role of PI3K/Akt in GLP-2-induced IGF-I mRNA levels in the murine jejunum was also confirmed in vivo. These findings implicate the PI3K/Akt pathway in the stimulatory effects of GLP-2 to enhance intestinal IGF-I mRNA transcript levels and provide further evidence in support of a role for IGF-I produced by the ISEMF cells in the intestinotropic effects of GLP-2.

The intestinal epithelial insulin-like growth factor-1 receptor links glucagon-like peptide-2 action to gut barrier function.

Glucagon-like peptide-2 (GLP-2) is an intestinal growth-promoting hormone used to treat short bowel syndrome. GLP-2 promotes intestinal growth through a mechanism that involves both IGF-1 and the intestinal-epithelial IGF-1 receptor (IE-IGF-1R). GLP-2 also enhances intestinal barrier function, but through an unknown mechanism. We therefore hypothesized that GLP-2-enhanced barrier function requires the IE-IGF-1R and is mediated through alterations in expression and localization of tight junction proteins. Conditional IE-IGF-1R-null and control mice were treated with vehicle or degradation-resistant Gly(2)-GLP-2 for 10 days; some animals also received irinotecan to induce enteritis. Mice were then examined for gastrointestinal permeability to 4-kDa fluorescein isothiocyanate-dextran, jejunal resistance using Ussing chambers, tight junction structure by electron microscopy, and expression and localization of tight junction proteins by immunoblot and immunohistofluorescence, respectively. GLP-2 treatment decreased permeability to 4-kDa fluorescein isothiocyanate-dextran and increased jejunal resistance (P <.05-.01), effects that were lost in IE-IGF-1R-null mice. Electron microscopy did not reveal major structural changes in the tight junctions in any group of animals. However, the tight junctional proteins claudin-3 and -7 were upregulated by GLP-2 in control (P <.05-.01) but not null mice, whereas IE-IGF-1R deletion induced a shift in occludin localization from apical to intracellular domains; no changes were observed in expression or distribution of claudin-15 and zona occludins-1. Finally, in irinotecan-induced enteritis, GLP-2 normalized epithelial barrier function in control (P < .05) but not knockout animals. In conclusion, the effects of GLP-2 on intestinal barrier function are dependent on the IE-IGF-1R and involve modulation of key components of the tight junctional complex.

Inhibition of Dopamine Receptor D4 Impedes Autophagic Flux, Proliferation, and Survival of Glioblastoma Stem Cells

Glioblastomas (GBM) grow in a rich neurochemical milieu, but the impact of neurochemicals on GBM growth is largely unexplored. We interrogated 680 neurochemical compounds in patient-derived GBM neural stem cells (GNS) to determine the effects on proliferation and survival. Compounds that modulate dopaminergic, serotonergic, and cholinergic signaling pathways selectively affected GNS growth. In particular, dopamine receptor D4 (DRD4) antagonists selectively inhibited GNS growth and promoted differentiation of normal neural stem cells. DRD4 antagonists inhibited the downstream effectors PDGFRβ, ERK1/2, and mTOR and disrupted the autophagy-lysosomal pathway, leading to accumulation of autophagic vacuoles followed by G0/G1 arrest and apoptosis. These results demonstrate a role for neurochemical pathways in governing GBM stem cell proliferation and suggest therapeutic approaches for GBM.

GLP-1R agonists promote normal and neoplastic intestinal growth through mechanisms requiring Fgf7.

Glucagon-like peptide-1 (GLP-1) secreted from enteroendocrine L cells promotes nutrient disposal via the incretin effect. However, the majority of L cells are localized to the distal gut, suggesting additional biological roles for GLP-1. Here, we demonstrate that GLP-1 receptor (GLP-1R) signaling controls mucosal expansion of the small bowel (SB) and colon. These actions did not require the epidermal growth factor (EGF) or intestinal epithelial insulin-like growth factor (IGF1) receptors but were absent in Glp1r(-/-) mice. Polyp number and size were increased in SB of exendin-4-treated Apc(Min/+) mice, whereas polyp number was reduced in SB and colon of Glp1r(-/-):Apc(Min/+) mice. Exendin-4 increased fibroblast growth factor 7 (Fgf7) expression in colonic polyps of Apc(Min/+) mice and failed to increase intestinal growth in mice lacking Fgf7. Exogenous exendin-4 and Fgf7 regulated an overlapping set of genes important for intestinal growth. Thus, gain and loss of GLP-1R signaling regulates gut growth and intestinal tumorigenesis.

Life in the crypt: A role for glucagon-like peptide-2?

The epithelial layer of the intestinal tract serves as a model to study the mechanisms regulating tissue renewal. Central to this process is the intestinal stem cell and, thus, both the intrinsic and extrinsic factors that modulate the function of these cells must be understood. Amongst the intrinsic regulators, both the canonical wnt and bone morphogenic protein (bmp) signaling pathways have been shown to be essential determinants of stem cell dynamics and intestinal homeostasis. The intestinotrophic hormone, glucagon-like peptide-2 (GLP-2), has also recently been demonstrated to exert a variety of effects on the intestinal crypt cells, including enhancement of the putative stem cell marker, musashi-1, as well as stimulating intestinal proliferation. As the GLP-2 receptor is not expressed by the crypt cells, these actions have been hypothesized to be mediated indirectly, through other gut peptides and/or growth factors. Of these, recent studies have demonstrated a requirement for insulin-like growth factor-1 in the proliferative effects of GLP-2, through a pathway that involves activation of the canonical wnt signaling pathway. This extrinsic pathway represents a novel mechanism by which intestinal stem cell dynamics may be regulated.

Abstract B14: Wnt signaling circuits in glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most common malignant tumour in the central nervous system with a prevalence of 2-3 cases per 100 000 people. Although the standard treatment of surgery, chemotherapy and radiotherapy improve survival, the median survival continues to remain at only 15 months with a 5-year survival rate of under 10%. Glioma neural stem-like (GNS) cells have been identified in GBM and have the capability of regenerating the tumour. Treatment strategies that target the majority of the tumour may be incapable of also targeting GNS cells and thus characterization of GNS cells may provide insight into additional treatment options. The Wnt signalling pathway has been linked to several cancers including GBM. Wnt signalling involves the secretion of Wnt ligand proteins that bind to specific Frizzled (FZD) receptor complexes on the cell surface of Wnt-responding cells to activate intracellular signalling cascades. The transcriptional and epigenetic regulation in GNS cells is the focus of several recent studies. The transcription factor ASCL1 was identified to be overexpressed and to lead to Wnt signalling activation by repressing Dickkopf (DKK1, Wnt inhibitor). Using microarray data we analyzed the expression of FZD receptors and Wnt target genes in over 50 primary GNS cell lines cultured in serum free conditions in order to maintain the GIC population and identified a subgroup of glioma lines with activated Wnt signalling. To determine the requirement of autocrine Wnt signalling for GNS cell renewal we inhibited Wnt secretion, using the porcupine inhibitor LGK974, and measured self-renewal using a limited dilution assay. There was a significant reduction in GNS cell frequency with LGK974 (1uM) treatment in four out of eight lines tested (G432NS, G472NS, G511NS and G523NS). Furthermore, a secondary sphere assay with G523NS cells also showed a significant reduction in GNS cell frequency. When G511NS and G523NS cells were treated with LGK974 (1uM) over a two week period, there was a significant increase in the percentage of GFAP (astrocytic marker) expressing cells whereas a two week treatment with Bio (1uM, Wnt activator), significantly increased the percentage of Tuj1 (neuronal marker) expressing cells. This finding suggest that these cells require a specific amount of Wnt signalling for self-renewal and Wnt inhibition or activation may lead to differentiation. RNAseq analysis comparing the four LGK974 responsive lines with the four LGK974 unresponsive lines identified ASCL1 to be highly expressed in the responsive lines. Gene set enrichment analysis identified four gene sets significantly enriched in the responsive group including the Glioblastoma Proneural gene set whereas genes from the Glioblastoma Mesenchymal gene set were significantly enriched in the unresponsive group. We have identified a subset of GNS cells that are dependent on Wnt secretion for self-renewal and RNAseq analysis suggests that GBMs that fall under the Proneural subtype may be sensitive to Wnt inhibition. Citation Format: Nishani Rajakulendran, Hayden Selvadurai, Katherine Rowland, Nicole Park, Nizar Batada, Peter Dirks, Stephane Angers. Wnt signaling circuits in glioblastoma multiforme. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr B14.