Opposing Activities of Notch and Wnt Signaling Regulate Intestinal Stem Cells and Gut Homeostasis

SummaryProper organ homeostasis requires tight control of adult stem cells and differentiation through the integration of multiple inputs. In the mouse small intestine, Notch and Wnt signaling are required both for stem cell maintenance and for a proper balance of differentiation between secretory and absorptive cell lineages. In the absence of Notch signaling, stem cells preferentially generate secretory cells at the expense of absorptive cells. Here, we use function-blocking antibodies against Notch receptors to demonstrate that Notch blockade perturbs intestinal stem cell function by causing a derepression of the Wnt signaling pathway, leading to misexpression of prosecretory genes. Importantly, attenuation of the Wnt pathway rescued the phenotype associated with Notch blockade. These studies bring to light a negative regulatory mechanism that maintains stem cell activity and balanced differentiation, and we propose that the interaction between Wnt and Notch signaling described here represents a common theme in adult stem cell biology

The Ccr4-Not Complex Interacts with the mRNA Export Machinery

The Ccr4-Not complex is a key eukaryotic regulator of gene transcription and cytoplasmic mRNA degradation. Whether this complex also affects aspects of post-transcriptional gene regulation, such as mRNA export, remains largely unexplored. Human Caf1 (hCaf1), a Ccr4-Not complex member, interacts with and regulates the arginine methyltransferase PRMT1, whose targets include RNA binding proteins involved in mRNA export. However, the functional significance of this regulation is poorly understood.Here we demonstrate using co-immunoprecipitation approaches that Ccr4-Not subunits interact with Hmt1, the budding yeast ortholog of PRMT1. Furthermore, using genetic and biochemical approaches, we demonstrate that Ccr4-Not physically and functionally interacts with the heterogenous nuclear ribonucleoproteins (hnRNPs) Nab2 and Hrp1, and that the physical association depends on Hmt1 methyltransferase activity. Using mass spectrometry, co-immunoprecipitation and genetic approaches, we also uncover physical and functional interactions between Ccr4-Not subunits and components of the nuclear pore complex (NPC) and we provide evidence that these interactions impact mRNA export.Taken together, our findings suggest that Ccr4-Not has previously unrealized functional connections to the mRNA processing/export pathway that are likely important for its role in gene expression. These results shed further insight into the biological functions of Ccr4-Not and suggest that this complex is involved in all aspects of mRNA biogenesis, from the regulation of transcription to mRNA export and turnover

Basal and insulin-stimulated glucose uptake in cultured skeletal myotubes.

<p>Conditioned media (2% in final media volume) from <b>(a,</b> basal state; <b>b</b> insulin-stimulated<b>)</b> primary human and <b>(c,</b> basal state; <b>d</b> insulin-stimulated<b>)</b> THP-1 macrophages treated with vehicle (PBS), 75 µg/ml acLDL/10 µg/ml Sandoz compound with or without 50 µg/ml HDL for 18 hours was placed on human primary skeletal myotubes for 24 hours. Cells were treated with insulin (100 nM) or vehicle for 30 minutes before glucose uptake was measured. n = 6/group; data are presented as mean ± SEM; *indicates significantly different from Con and acLDL+HDL, P<0.05.</p

Myotube Akt phosphorylation, human primary macrophage cholesterol content and THP-1 macrophage JNK phosphorylation. (a)

<p>Phosphorylation of Akt at Serine-473 in human primary skeletal myotubes treated as for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056601#pone-0056601-g001" target="_blank">Figure 1</a>, n = 6/group. <b>(b)</b> Total intracellular cholesterol (free cholesterol and cholesteryl esters) in human primary macrophages after treatment with vehicle (PBS) or 75 µg/ml acLDL/10 µg/ml Sandoz with or without 50 µg/ml HDL for 18 hours, n = 6/group. <b>(c)</b> Phosphorylation of JNK in THP-1 macrophages treated with vehicle or 75 µg/ml acLDL/10 µg/ml Sandoz with or without 50 µg/ml HDL for 1, 2 and 4 hours. n = 9/group; data are presented as mean ± SEM; *indicates significantly different from Con at the corresponding time point, P<0.05.</p