Roles for neural stem cells in adult mammalian olfactory behaviour

Bibliography: p. 126-15

Regulation of Skeletal Muscle Formation and Regeneration by the Cellular Inhibitor of Apoptosis 1 (cIAP1) Protein

The inhibitor of apoptosis (IAP) proteins traditionally regulate programmed cell death by binding to and inhibiting caspases. Recent studies have uncovered a variety of alternate cellular roles for several IAP family members. The cellular inhibitor of apoptosis 1 (cIAP1) protein, for instance, regulates different axes of the NF-κB signalling pathway. Given the extensive functions of NF-κB signalling in muscle differentiation and regeneration, I asked if cIAP1 also plays critical roles in skeletal muscle myogenesis. In a primary myoblast cell-culture system, genetic and pharmacological approaches revealed that loss of cIAP1 dramatically increases the fusion of myoblasts into myotubes. NF-κB signalling occurs along a classical and an alternative pathway, both of which are highly active in cIAP1-/- myoblasts. Suppression of the alternative pathway attenuates myotube fusion in wildtype and cIAP1-/- myoblasts. Conversely, constitutive activation of the alternative pathway increases myoblast fusion in wildtype myoblasts. cIAP1-/- mice have greater muscle weight and size than wildtypes, as well as an increased number of muscle stem cells. These results identify cIAP1 as a regulator of myogenesis through its modulation of classical and alternative NF-κB signalling pathways. Loss of the structural protein dystrophin in the mdx mouse model of Duchenne muscular dystrophy leads to chronic degeneration of skeletal muscle. The muscle pathology is strongly influenced by NF-κB signaling. Given the roles demonstrated for cIAP1 in cell culture and in vivo, I asked whether loss of cIAP1 would influence muscle pathology in the mdx mouse. To address this question, double-mutant mice were bred lacking both cIAP1 and dystrophin (cIAP1-/-;mdx). Histological analyses revealed that double-mutant mice exhibited reduced indications of damage on several measures, as compared to single-mutant (cIAP1+/+;mdx) controls. Unexpectedly, these reductions were seen in the “slow-twitch” soleus muscle but not in the “fast-twitch” extensor digitorum longus (EDL) muscle. The improvements in pathology of double-mutant solei were associated with reductions in muscle infiltration by CD68-expressing macrophages. Finally, the double-mutant mice exhibited improved endurance and resistance to damage during treadmill-running exercise. Taken together, these results suggest that loss of cIAP1, through its multiple regulatory functions, acts to improve myogenesis and increase muscle resistance to damage

Role of the TWEAK-Fn14-cIAP1-NF-kB signaling axis in the regulation of myogenesis and muscle homeostasis

Mammalian skeletal muscle maintains a robust regenerative capacity throughout life, due largely to the presence of a stem cell population known as satellite cells in the muscle milieu. In normal conditions, these cells remain quiescent; they are activated upon injury to become myoblasts, which proliferate extensively and eventually differentiate and fuse to form new multinucleated muscle fibers. Recent findings have identified some of the factors, including the cytokine TNFα-like weak inducer of apoptosis (TWEAK), which govern these cells’ decisions to proliferate, differentiate, or fuse. In this review, we will address the functions of TWEAK, its receptor Fn14, and the associated signal transduction molecule, the cellular inhibitor of apoptosis 1 (cIAP1), in the regulation of myogenesis. TWEAK signaling can activate the canonical NF-κB signaling pathway, which promotes myoblast proliferation and inhibits myogenesis. In addition, TWEAK activates the noncanonical NF-κB pathway, which, in contrast, promotes myogenesis by increasing myoblast fusion. Both pathways are regulated by cIAP1, which is an essential component of downstream signaling mediated by TWEAK and similar cytokines. This review will focus on the seemingly contradictory roles played by TWEAK during muscle regeneration, by highlighting the interplay between the two NF-κB pathways under physiological and pathological conditions. We will also discuss how myogenesis is negatively affected by chronic conditions which affect homeostasis of the skeletal muscle environment

Identification of the SUMO E3 ligase PIAS1 as a potential survival biomarker in breast cancer.

Metastasis is the ultimate cause of breast cancer related mortality. Epithelial-mesenchymal transition (EMT) is thought to play a crucial role in the metastatic potential of breast cancer. Growing evidence has implicated the SUMO E3 ligase PIAS1 in the regulation of EMT in mammary epithelial cells and breast cancer metastasis. However, the relevance of PIAS1 in human cancer and mechanisms by which PIAS1 might regulate breast cancer metastasis remain to be elucidated. Using tissue-microarray analysis (TMA), we report that the protein abundance and subcellular localization of PIAS1 correlate with disease specific overall survival of a cohort of breast cancer patients. In mechanistic studies, we find that PIAS1 acts via sumoylation of the transcriptional regulator SnoN to suppress invasive growth of MDA-MB-231 human breast cancer cell-derived organoids. Our studies thus identify the SUMO E3 ligase PIAS1 as a prognostic biomarker in breast cancer, and suggest a potential role for the PIAS1-SnoN sumoylation pathway in controlling breast cancer metastasis

PIAS1 protein abundance analysis in reference TMA and breast cancer TMA.

<p>(A) PIAS1 and actin immunoblots of lysate of MDA-MB-231 cells expressing four increasing concentrations of PIAS1 (samples 1 to 4). (B) Bar graph of actin-normalized PIAS1 protein abundance in samples 1 to 4 shown in A and expressed relative to actin-normalized PIAS1 abundance in sample 3. (C) PIAS1 immunoblot of serially diluted lysate of MDA-MB-231 cells overexpressing PIAS1 in sample 4. (D) XY-graph plot of the lysate's total protein on the x-axis versus PIAS1 protein abundance, quantified from PIAS1immunoblot in C, on the y-axis. (E) Representative PIAS1 (red), and nuclei (blue) fluorescence micrographs of sections of Histogel-embedded MDA-MB-231 cells reference TMA expressing increasing abundance of PIAS1, corresponding to samples 1 to 4 in panel A, which were subjected to anti-PIAS1 indirect immunofluorescence and DAPI dye staining to visualize nuclei. (F) Bar graph depicts AQUA analysis software-quantified PIAS1 abundance in the reference TMA shown in E. (G) The XY-graph shows the relationship between Log of relative abundance of PIAS1 in samples 1 to 4 of MDA-MB-231 cell lysates quantified by immunoblotting on the x-axis versus Log of abundance of PIAS1 in MDA-MB-231 samples 1 to 4 of reference TMA quantified by AQUA analyses of immunocytochemistry on the y-axis. (H) Representative fluorescence microscopy micrographs of histogel-MDA-MB-231 cell reference TMA blocks. (I) Representative fluorescence microscopy micrographs of paraffin-embedded normal breast tissue and examples of three breast cancer tissues expressing different amounts of PIAS1. For both H and I, TMA were subjected to anti-PIAS1 (red) and anti-Pan cytokeratin (green) antibodies indirect immunofluorescence, and nuclear counterstaining with DAPI (blue). PIAS1-Cytokeratin-Nuclei merged fluorescence micrograph panels show relative abundance of PIAS1 in reference breast cancer TMA, normal breast and breast cancer tissue array.</p

Sumoylation of SnoN supresses TGFβ-induced invasiveness of breast cancer organoids.

<p>(A) SnoN and actin immunoblots of lysates of MDA-MB-231 cells expressing SnoN (WT), SnoN (KdR), SUMO-SnoN or transfected with a control vector. (B) Representative DIC light microscopy micrographs of untreated or 100pM TGFβ-treated 8-day old three dimensional organoids derived from MDA-MB-231 cells transfected as in A. (C) Bar graph represents mean ± SEM proportion of non-deformed organoids expressed as a percentage of total colonies counted for each experimental condition from four independent experiments including the one shown in B. Non-deformed organoids represents non-invasive growth phenotype. SnoN (KdR) promoted an invasive growth of breast cancer cell-derived organoids even in the absence of TGFβ. SUMO-SnoN suppressed TGFβ-induced invasive growth of breast cancer cell-derived organoids. Significant difference, ANOVA: ***p<0.001. Scale bar indicates 50 μm. Arrows and arrowheads indicate non-deformed and invasive organoids, respectively.</p

PIAS1 suppresses TGFβ-induced budding and disruption of breast cancer cell-derived organoids via sumoylation of SnoN.

<p>(A) Representative DIC light microscopy micrographs of untreated or 100pM TGFβ-treated 8-day old organoids derived from MDA-MB-231 cells stably expressing SUMO-SnoN or the control vector, and transfected with a pool of plasmids encoding short hairpin RNAs targeting distinct regions of PIAS1 mRNA, or a control RNAi plasmid. (B) Bar graph represents mean ± SEM proportion of non-deformed organoids expressed as a percentage of total colonies counted for each experimental condition from three independent experiments including the one shown in A. SUMO-SnoN suppressed the ability of PIAS1 knockdown to promote invasive growth of MDA-MB-231 cell-derived organoids in absence or presence of TGFβ. (C) Representative DIC light microscopy micrographs of untreated or 100pM TGFβ-treated 8-day old organoids derived from MDA-MB-231 cells stably expressing SUMO-SnoN or the control plasmid, and transfected with a vector expressing PIAS1 (CS) or the control vector. (D) Bar graph represents mean ± SEM proportion of non-deformed organoids expressed as a percentage of total colonies counted for each experimental condition from three independent experiments including the one shown in C. SUMO-SnoN suppressed PIAS1 (CS)-induction of invasive growth of MDA-MB-231 cell-derived organoids in absence or presence of TGFβ. (E) Representative DIC light microscopy micrographs of untreated or 100pM TGFβ-treated 8-day old organoids derived from MDA-MB-231 cells stably expressing SnoN (WT), SnoN (KdR) or a vector control, and transfected with PIAS1 (WT) or the control vector. (F) Bar graph depicts mean ± SEM proportion of non-deformed organoids expressed as a percentage of total colonies counted for each experimental condition from three independent experiments including the one shown in E. SnoN (KdR) antagonized the ability of PIAS1 (WT) to suppress the invasive growth of MDA-MB-231 cell-derived organoids in absence or presence of TGFβ. Significant difference, ANOVA: ***P≤0.001, **P≤0.01. Scale bar indicates 50 μm. Non-deformed organoids represents non-invasive growth phenotype. Arrows and arrowheads indicate intact non-deformed and invasive organoids, respectively.</p