Arx is required for normal enteroendocrine cell development in mice and humans

AbstractEnteroendocrine cells of the gastrointestinal (GI) tract play a central role in metabolism, digestion, satiety and lipid absorption, yet their development remains poorly understood. Here we show that Arx, a homeodomain-containing transcription factor, is required for the normal development of mouse and human enteroendocrine cells. Arx expression is detected in a subset of Neurogenin3 (Ngn3)-positive endocrine progenitors and is also found in a subset of hormone-producing cells. In mice, removal of Arx from the developing endoderm results in a decrease of enteroendocrine cell types including gastrin-, glucagon/GLP-1-, CCK-, secretin-producing cell populations and an increase of somatostatin-expressing cells. This phenotype is also observed in mice with endocrine-progenitor-specific Arx ablation suggesting that Arx is required in the progenitor for enteroendocrine cell development. In addition, depletion of human ARX in developing human intestinal tissue results in a profound deficit in expression of the enteroendocrine cell markers CCK, secretin and glucagon while expression of a pan-intestinal epithelial marker, CDX2, and other non-endocrine markers remained unchanged. Taken together, our findings uncover a novel and conserved role of Arx in mammalian endocrine cell development and provide a potential cause for the chronic diarrhea seen in both humans and mice carrying Arx mutations

Contractile force is enhanced in Aortas from pendrin null mice due to stimulation of angiotensin II-dependent signaling.

Pendrin is a Cl-/HCO3- exchanger expressed in the apical regions of renal intercalated cells. Following pendrin gene ablation, blood pressure falls, in part, from reduced renal NaCl absorption. We asked if pendrin is expressed in vascular tissue and if the lower blood pressure observed in pendrin null mice is accompanied by reduced vascular reactivity. Thus, the contractile responses to KCl and phenylephrine (PE) were examined in isometrically mounted thoracic aortas from wild-type and pendrin null mice. Although pendrin expression was not detected in the aorta, pendrin gene ablation changed contractile protein abundance and increased the maximal contractile response to PE when normalized to cross sectional area (CSA). However, the contractile sensitivity to this agent was unchanged. The increase in contractile force/cross sectional area observed in pendrin null mice was due to reduced cross sectional area of the aorta and not from increased contractile force per vessel. The pendrin-dependent increase in maximal contractile response was endothelium- and nitric oxide-independent and did not occur from changes in Ca2+ sensitivity or chronic changes in catecholamine production. However, application of 100 nM angiotensin II increased force/CSA more in aortas from pendrin null than from wild type mice. Moreover, angiotensin type 1 receptor inhibitor (candesartan) treatment in vivo eliminated the pendrin-dependent changes contractile protein abundance and changes in the contractile force/cross sectional area in response to PE. In conclusion, pendrin gene ablation increases aorta contractile force per cross sectional area in response to angiotensin II and PE due to stimulation of angiotensin type 1 receptor-dependent signaling. The angiotensin type 1 receptor-dependent increase in vascular reactivity may mitigate the fall in blood pressure observed with pendrin gene ablation

Pancreatic α-Cell Specific Deletion of Mouse Arx Leads to α-Cell Identity Loss

<div><p>The specification and differentiation of pancreatic endocrine cell populations (α-, β-, δ, PP- and ε-cells) is orchestrated by a combination of transcriptional regulators. In the pancreas, <i>Aristaless-related homeobox</i> gene (<i>Arx</i>) is expressed first in the endocrine progenitors and then restricted to glucagon-producing α-cells. While the functional requirement of <i>Arx</i> in early α-cell specification has been investigated, its role in maintaining α-cell identity has yet to be explored. To study this later role of <i>Arx</i>, we have generated mice in which the <i>Arx</i> gene has been ablated specifically in glucagon-producing α-cells. Lineage-tracing studies and immunostaining analysis for endocrine hormones demonstrate that ablation of <i>Arx</i> in neonatal α-cells results in an α-to-β-like conversion through an intermediate bihormonal state. Furthermore, these <i>Arx</i>-deficient converted cells express β-cell markers including <i>Pdx1, MafA,</i> and <i>Glut2</i>. Surprisingly, short-term ablation of <i>Arx</i> in adult mice does not result in a similar α-to-β-like conversion. Taken together, these findings reveal a potential temporal requirement for <i>Arx</i> in maintaining α-cell identity.</p></div

Arx is specifically ablated in YFP⁺ α-cells of GKO;Rosa-YFP mice.

<p>P5 pancreatic sections were stained for glucagon (blue), Arx (red), and YFP (green). (<b>A</b>): Arx is expressed in all glucagon⁺ cells in control;Rosa-YFP pancreata. A subset of glucagon⁺Arx⁺ cells is YFP⁺. (<b>B</b>): In GKO;Rosa-YFP animals, there is a subset of glucagon⁺ cells that express YFP. These YFP⁺ cells have lost Arx expression. Scale bar represents 25 µm. (<b>C</b>): Quantitative analysis of Arx and YFP expressing cells within glucagon⁺ population in P5 animals. Over 500 total glucagon⁺ cells were counted with three mice per group used. Error bars represent standard error of the mean with <i>p-value</i> indicated. N.S: not significant. (<b>D</b>): Quantitative PCR analysis for <i>Arx</i> mRNA in total pancreata at P5 and islets from P21 control and GKO animals. Control mRNA level was set at one fold ± standard error of the mean. Male and female control and GKO animals (n≥3) were sex-matched for all analyses.</p

Lineage tracing studies demonstrate that Arx ablated α-cells become glucagon⁺insulin⁺ at P5 then insulin expressing at P21.

<p>(<b>A–D</b>): Triple immunostaining for glucagon (red), insulin (blue), and YFP (green) in control;Rosa-YFP and GKO;Rosa-YFP pancreata at P5 and P21. YFP⁺ cells in P5 or P21 control;Rosa-YFP animals are positive for glucagon (A and C;^▪). “*” denote insulin cells that are negative for glucagon or YFP expression (A and C; *) Glucagon⁺insulin⁺YFP⁺ cells are found in P5 GKO animals (B; ^▪), but rarely in controls (A). YFP⁺ cells are positive for glucagon in control P21 pancreata (C; ^▪) but insulin⁺ in P21 GKO pancreata (D; ^▪). (<b>E</b>): Schematic outlining cell populations resulting from lineage-tracing and immunostaining analysis. (<b>F, G</b>): Quantification of hormone expression in YFP⁺ cells at P5 (F) and P21 (G). At P5 and P21, over a total of 10,000 cells were counted from 3–5 animals per group. Out of the 10,000 cells counted, approximately 1,000 cells were YFP⁺. Each category was calculated and presented as a percentage of total YFP⁺ cells per animal and then averaged. Error bars are denoted as standard error of the mean with significance (p≤0.05) between each color denoted with “*”, “^▪”, and “•”. Male and female GKO mice (n≥3) were used for all analysis and compared to their sex-matched controls. Scale bar represents 25 µm.</p

Rosiglitazone Attenuates Chronic Hypoxia–Induced Pulmonary Hypertension in a Mouse Model

Chronic hypoxia contributes to pulmonary hypertension through complex mechanisms that include enhanced NADPH oxidase expression and reactive oxygen species (ROS) generation in the lung. Stimulation of peroxisome proliferator–activated receptor γ (PPARγ) reduces the expression and activity of NADPH oxidase. Therefore, we hypothesized that activating PPARγ with rosiglitazone would attenuate chronic hypoxia–induced pulmonary hypertension, in part, through suppressing NADPH oxidase–derived ROS that stimulate proliferative signaling pathways. Male C57Bl/6 mice were exposed to chronic hypoxia (CH, FiO2 10%) or room air for 3 or 5 weeks. During the last 10 days of exposure, each animal was treated daily by gavage with either the PPARγ ligand, rosiglitazone (10 mg/kg/d) or with an equal volume of vehicle. CH increased: (1) right ventricular systolic pressure (RVSP), (2) right ventricle weight, (3) thickness of the walls of small pulmonary vessels, (4) superoxide production and Nox4 expression in the lung, and (5) platelet-derived growth factor receptor β (PDGFRβ) expression and activity and reduced phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression. Treatment with rosiglitazone prevented the development of pulmonary hypertension at 3 weeks; reversed established pulmonary hypertension at 5 weeks; and attenuated CH-stimulated Nox4 expression and superoxide production, PDGFRβ activation, and reductions in PTEN expression. Rosiglitazone also attenuated hypoxia-induced increases in Nox4 expression in pulmonary endothelial cells in vitro despite hypoxia-induced reductions in PPARγ expression. Collectively, these findings indicate that PPARγ ligands attenuated hypoxia-induced pulmonary vascular remodeling and hypertension by suppressing oxidative and proliferative signals providing novel insights for mechanisms underlying therapeutic effects of PPARγ activation in pulmonary hypertension

Loss of Arx in glucagon⁺ cells results in the appearance of a glucagon⁺insulin⁺ population.

<p><b>(A–H):</b> P5 control and GKO pancreata were stained for glucagon (green), insulin (red; A, B), somatostatin (Sst; red; C, D), PP (red; E, F), and Ghrelin (red; G, H). Glucagon⁺insulin⁺ cells are the only bihormonal population unique to GKO animals (B, D, F). Glucagon/ghrelin coexpressing cells are both found in control and GKO animals (G, H). Male and female control and GKO animals (n≥3) were sex-matched for all analyses. Scale bar denotes 25 µm. (<b>I–L</b>): Quantitative PCR analysis examining glucagon (I), insulin (J), somatostatin (K), and PP (L) gene expression in P5 control and GKO animals. Control mRNA level was set at one fold ± standard error of the mean. For all GKO and control groups, at least 3 biologic replicates were performed.</p