Impaired LRP6-TCF7L2 Activity Enhances Smooth Muscle Cell Plasticity and Causes Coronary Artery Disease

SummaryMutations in Wnt-signaling coreceptor LRP6 have been linked to coronary artery disease (CAD) by unknown mechanisms. Here, we show that reduced LRP6 activity in LRP6R611C mice promotes loss of vascular smooth muscle cell (VSMC) differentiation, leading to aortic medial hyperplasia. Carotid injury augmented these effects and led to partial to total vascular obstruction. LRP6R611C mice on high-fat diet displayed dramatic obstructive CAD and exhibited an accelerated atherosclerotic burden on LDLR knockout background. Mechanistically, impaired LRP6 activity leads to enhanced non-canonical Wnt signaling, culminating in diminished TCF7L2 and increased Sp1-dependent activation of PDGF signaling. Wnt3a administration to LRP6R611C mice improved LRP6 activity, led to TCF7L2-dependent VSMC differentiation, and rescued post-carotid-injury neointima formation. These findings demonstrate the critical role of intact Wnt signaling in the vessel wall, establish a causal link between impaired LRP6/TCF7L2 activities and arterial disease, and identify Wnt signaling as a therapeutic target against CAD

Mechanosensing through talin 1 contributes to tissue mechanical homeostasis.

It is widely believed that tissue mechanical properties, determined mainly by the extracellular matrix (ECM), are actively maintained. However, despite its broad importance to biology and medicine, tissue mechanical homeostasis is poorly understood. To explore this hypothesis, we developed mutations in the mechanosensitive protein talin1 that alter cellular sensing of ECM stiffness. Mutation of a novel mechanosensitive site between talin1 rod domain helix bundles 1 and 2 (R1 and R2) shifted cellular stiffness sensing curves, enabling cells to spread and exert tension on compliant substrates. Opening of the R1-R2 interface promotes binding of the ARP2/3 complex subunit ARPC5L, which mediates the altered stiffness sensing. Ascending aortas from mice bearing these mutations show increased compliance, less fibrillar collagen, and rupture at lower pressure. Together, these results demonstrate that cellular stiffness sensing regulates ECM mechanical properties. These data thus directly support the mechanical homeostasis hypothesis and identify a novel mechanosensitive interaction within talin that contributes to this mechanism

Kinesin 5B (KIF5B) is required for progression through female meiosis and proper chromosomal segregation in mitotic cells.

The fidelity of chromosomal segregation during cell division is important to maintain chromosomal stability in order to prevent cancer and birth defects. Although several spindle-associated molecular motors have been shown to be essential for cell division, only a few chromosome arm-associated motors have been described. Here, we investigated the role of Kinesin 5b (Kif5b) during female mouse meiotic cell development and mitotic cell division. RNA interference (RNAi)-mediated silencing of Kif5b in mouse oocytes induced significant delay in germinal vesicle breakdown (GVBD) and failure in extrusion of the first polar body (PBE). In mitotic cells, knockdown of Kif5b leads to centrosome amplification and a chromosomal segregation defect. These data suggest that KIF5B is critical in suppressing chromosomal instability at the early stages of female meiotic cell development and mitotic cell division

Characterization of mouse KIF5B during cell division.

<p>A) Schematic diagram of KIF5B domains based <a href="http://www.ensembl.org" target="_blank">http://www.ensembl.org</a> sequence database. Blue, red, cyan and green boxes indicate motor, nucleotide binding, coiled-coil and globular domains respectively. B) Dynamic localization of KIF5B during different stages of cell division. Note that red and green represent DNA and KIF5B respectively.</p

KIF5B suppresses centrosome amplification.

<p>A–C) Control cells during different stages of cell division with one and two centrosomes. D–F) Representative images of cells with <i>Kif5b-shRNA-</i>mediated knockdown with more than two centrosomes. The DNA is stained with DAPI (blue), and centrosomes are stained with γ-tubulin (green). The arrow indicates the centrosome in each cell. G) The representative number of cells analyzed for centrosomes in control cells (n = 119) and <i>Kif5b-shRNA</i> knockdown cells (n = 175). Cells with greater than two centrosomes were significantly enriched by <i>Kif5b-shRNA-</i>mediated knockdown (*** P<0.0001). The Graph Pad Prism program was used for analysis of the data and employed the Mann Whitney test.</p

Downregulation of KIF5B leads to chromosome misalignment.

<p>A) Western blot showing the efficiency of <i>Kif5b-shRNA-</i>mediated knockdown with whole- cell extracts prepared from HeLa and control cells transfected with <i>control-shRNA</i>. B) Cell division-dependent localization of microtubules (green) and DNA arrangement (red) in <i>Kif5b-shRNA</i> versus control. Note that the arrow indicates examples of tripolar arrangement in microtubules. C) A statistically significant difference was observed for the estimated percentage of cells with multipolar arrangements of microtubules in <i>Kif5b-shRNA</i> knockdown cells (n = 110) versus wild type (n = 98)(*** p<0.0001). The Graph Pad Prism program was used for data analysis employing Mann Whitney statistical methods.</p

Y265C DNA Polymerase Beta Knockin Mice Survive Past Birth and Accumulate Base Excision Repair Intermediate Substrates

DNA is susceptible to damage by a wide variety of chemical agents that are generated either as byproducts of cellular metabolism or exposure to man-made and harmful environments. Therefore, to maintain genomic integrity, having reliable DNA repair systems is important. DNA polymeraseβis known to be a key player in the base excision repair pathway, and mice devoid of DNA polymerase beta do not live beyond a few hours after birth. In this study, we characterized mice harboring an impaired polβvariant. This Y265Cpolβ variant exhibits slow DNA polymerase activity but WT lyase activity and has been shown to be a mutator polymerase. Mice expressing Y265C polβ are born at normal Mendelian ratios. However, they are small, and 60% die within a few hours after birth. Slow proliferation and significantly increased levels of cell death are observed in many organs of the E14 homozygous embryos compared with WT littermates. Mouse embryo fibroblasts prepared from the Y265C polβ embryos proliferate at a rate slower than WT cells and exhibit a gap-filling deficiency during base ex-cision repair. As a result of this, chromosomal aberrations and single- and double-strand breaks are present at significantly higher levels in the homozygous mutant versus WT mouse embryo fibroblasts. This is study in mice is unique in that two enzymatic activities of polβ have been separated; the data clearly demonstrate that the DNA polymerase activity of polβ is essential for survival and genome stability

Sequences of RNAi and shRNA used for this study to knock down <i>Kif5b</i> and control.

<p>Sequences of RNAi and shRNA used for this study to knock down <i>Kif5b</i> and control.</p