Insulin signaling regulates a functional interaction between adenomatous polyposis coli and cytoplasmic dynein.

Diabetes is linked to an increased risk for colorectal cancer, but the mechanistic underpinnings of this clinically important effect are unclear. Here we describe an interaction between the microtubule motor cytoplasmic dynein, the adenomatous polyposis coli tumor suppressor protein (APC), and glycogen synthase kinase-3ß (GSK-3ß), which could shed light on this issue. GSK-3ß is perhaps best known for glycogen regulation, being inhibited downstream in an insulin-signaling pathway. However, the kinase is also important in many other processes. Mutations in APC that disrupt the regulation of ß-catenin by GSK-3ß cause colorectal cancer in humans. Of interest, both APC and GSK-3ß interact with microtubules and cellular membranes. We recently demonstrated that dynein is a GSK-3ß substrate and that inhibition of GSK-3ß promotes dynein-dependent transport. We now report that dynein stimulation in intestinal cells in response to acute insulin exposure (or GSK-3ß inhibition) is blocked by tumor-promoting isoforms of APC that reduce an interaction between wild-type APC and dynein. We propose that under normal conditions, insulin decreases dynein binding to APC to stimulate minus end-directed transport, which could modulate endocytic and secretory systems in intestinal cells. Mutations in APC likely impair the ability to respond appropriately to insulin signaling. This is exciting because it has the potential to be a contributing factor in the development of colorectal cancer in patients with diabetes.

GSK-3ß Phosphorylation of Cytoplasmic Dynein Reduces Ndel1 Binding to Intermediate Chains and Alters Dynein Motility.

Glycogen synthase kinase 3 (GSK-3) has been linked to regulation of kinesin-dependent axonal transport in squid and flies, and to indirect regulation of cytoplasmic dynein. We have now found evidence for direct regulation of dynein by mammalian GSK-3ß in both neurons and non-neuronal cells. GSK-3ß coprecipitates with and phosphorylates mammalian dynein. Phosphorylation of dynein intermediate chain (IC) reduces its interaction with Ndel1, a protein that contributes to dynein force generation. Two conserved residues, S87/T88 in IC-1B and S88/T89 in IC-2C, have been identified as GSK-3 targets by both mass spectrometry and site-directed mutagenesis. These sites are within an Ndel1-binding domain, and mutation of both sites alters the interaction of IC's with Ndel1. Dynein motility is stimulated by (i) pharmacological and genetic inhibition of GSK-3ß, (ii) an insulin-sensitizing agent (rosiglitazone) and (iii) manipulating an insulin response pathway that leads to GSK-3ß inactivation. Thus, our study connects a well-characterized insulin-signaling pathway directly to dynein stimulation via GSK-3 inhibition.

Differential activation of catalase expression and activity by PPAR agonists: implications for astrocyte protection in anti-glioma therapy.

Glioma survival is dismal, in part, due to an imbalance in antioxidant expression and activity. Peroxisome proliferator-activated receptor (PPAR) agonists have antineoplastic properties which present new redox-dependent targets for glioma anticancer therapies. Herein, we demonstrate that treatment of primary cultures of normal rat astrocytes with PPAR agonists increased the expression of catalase mRNA protein, and enzymatic activity. In contrast, these same agonists had no effect on catalase expression and activity in malignant rat glioma cells. The increase in steady-state catalase mRNA observed in normal rat astrocytes was due, in part, to de novo mRNA synthesis as opposed to increased catalase mRNA stability. Moreover, pioglitazone-mediated induction of catalase activity in normal rat astrocytes was completely blocked by transfection with a PPARγ-dominant negative plasmid. These data suggest that defects in PPAR-mediated signaling and gene expression may represent a block to normal catalase expression and induction in malignant glioma. The ability of PPAR agonists to differentially increase catalase expression and activity in normal astrocytes but not glioma cells suggests that these compounds might represent novel adjuvant therapeutic agents for the treatment of gliomas.

Characteristics of autoimmune pancreatitis based on serum IgG4 level.

BACKGROUND: Autoimmune pancreatitis is categorized as an IgG4-related autoimmune disease, mostly associated with serological alterations, however characteristics of autoimmune pancreatitis based on serum markers have not been fully evaluated. METHODS: We evaluated demographics, symptoms, imaging and therapeutic outcome in 27 cases of autoimmune pancreatitis stratified by serum IgG4 level. RESULTS: Twenty patients (74%) had elevated serum IgG4 and 7 (26%) had normal IgG4 levels. Compared to patients with normal serum IgG4 levels, patients with elevated IgG4 had higher incidence of jaundice at onset (14.3% vs. 80%, respectively; P=0.002), more frequent diffuse pancreatic enlargement at imaging (14.3% vs. 60%, respectively; P=0.04), significantly higher 18F-2-fluoro-2-deoxy-d-glucose uptake of pancreatic lesions (SUV max: 4.0 vs. 5.7, respectively; P=0.02), more frequent extrapancreatic lesions (42.9% vs. 85%, respectively; P=0.03). Response to steroids was recognized regardless of serum IgG4 level, however maintenance therapy was required more frequently amongst patients with elevated compared to normal IgG4 (85.7% vs. 33.3%, respectively; P=0.04). CONCLUSIONS: Clinical features of autoimmune pancreatitis are different based on level of serum IgG4. Further studies are needed to clarify if normal serum IgG4 cases are a precursor of active type 1 or type 2 autoimmune pancreatitis.

Lis1 and Ndel1 influence the timing of nuclear envelope breakdown in neural stem cells.

Lis1 and Ndel1 are essential for animal development. They interact directly with one another and with cytoplasmic dynein. The developing brain is especially sensitive to reduced Lis1 or Ndel1 levels, as both proteins influence spindle orientation, neural cell fate decisions, and neuronal migration. We report here that Lis1 and Ndel1 reduction in a mitotic cell line impairs prophase nuclear envelope (NE) invagination (PNEI). This dynein-dependent process facilitates NE breakdown (NEBD) and occurs before the establishment of the bipolar spindle. Ndel1 phosphorylation is important for this function, regulating binding to both Lis1 and dynein. Prophase cells in the ventricular zone (VZ) of embryonic day 13.5 Lis1(+/-) mouse brains show reduced PNEI, and the ratio of prophase to prometaphase cells is increased, suggesting an NEBD delay. Moreover, prophase cells in the VZ contain elevated levels of Ndel1 phosphorylated at a key cdk5 site. Our data suggest that a delay in NEBD in the VZ could contribute to developmental defects associated with Lis1-Ndel1 disruption.

Genetic enhancement of the Lis1+/- phenotype by a heterozygous mutation in the adenomatous polyposis coli gene.

Hemizygous Lis1 mutations cause type 1 lissencephaly, a neuronal migration disorder in humans. The Lis1+/- mouse is a model for lissencephaly; mice exhibit neuronal migration defects but are viable and fertile. On an inbred genetic background, 20% of Lis1+/- mice develop hydrocephalus and die prematurely. Lis1 functions with the microtubule motor cytoplasmic dynein. Because dynactin, a dynein regulator, interacts with end-binding protein 1 (EB1) and beta-catenin, two known binding partners of the adenomatous polyposis coli (APC) protein, we looked for a genetic interaction between Lis1 and APC. Mice with a heterozygous truncating mutation in APC (Min mutation) do not exhibit neuronal migration defects or develop hydrocephalus. However, the presence of the APC mutation increases the migration deficit and the incidence of hydrocephalus in Lis1+/- animals. Lis1 and dynein distribution is altered in cells derived from Min mice, and both Lis1 and dynein interact with the C terminus of APC in vitro. Together, our findings point to a previously unknown interaction between APC and Lis1 during mammalian brain development.

Regulation of cytoplasmic dynein ATPase by Lis1.

Mutations in Lis1 cause classical lissencephaly, a developmental brain abnormality characterized by defects in neuronal positioning. Over the last decade, a clear link has been forged between Lis1 and the microtubule motor cytoplasmic dynein. Substantial evidence indicates that Lis1 functions in a highly conserved pathway with dynein to regulate neuronal migration and other motile events. Yeast two-hybrid studies predict that Lis1 binds directly to dynein heavy chains (Sasaki et al., 2000; Tai et al., 2002), but the mechanistic significance of this interaction is not well understood. We now report that recombinant Lis1 binds to native brain dynein and significantly increases the microtubule-stimulated enzymatic activity of dynein in vitro. Lis1 does this without increasing the proportion of dynein that binds to microtubules, indicating that Lis1 influences enzymatic activity rather than microtubule association. Dynein stimulation in vitro is not a generic feature of microtubule-associated proteins, because tau did not stimulate dynein. To our knowledge, this is the first indication that Lis1 or any other factor directly modulates the enzymatic activity of cytoplasmic dynein. Lis1 must be able to homodimerize to stimulate dynein, because a C-terminal fragment (containing the dynein interaction site but missing the self-association domain) was unable to stimulate dynein. Binding and colocalization studies indicate that Lis1 does not interact with all dynein complexes found in the brain. We propose a model in which Lis1 stimulates the activity of a subset of motors, which could be particularly important during neuronal migration and long-distance axonal transport.