Kindlin-2 controls bidirectional signaling of integrins.

Control of integrin activation is required for cell adhesion and ligand-induced signaling. Here we report that loss of the focal adhesion protein Kindlin-2 in mice results in peri-implantation lethality caused by severe detachment of the endoderm and epiblast from the basement membrane. We found that Kindlin-2-deficient cells were unable to activate their integrins and that Kindlin-2 is required for talin-induced integrin activation. Furthermore, we demonstrate that Kindlin-2 is required for integrin outside-in signaling to enable firm adhesion and spreading. Our findings provide evidence that Kindlin-2 is a novel and essential element of bidirectional integrin signaling

Loss of Kindlin-1 Causes Skin Atrophy and Lethal Neonatal Intestinal Epithelial Dysfunction

Kindler Syndrome (KS), characterized by transient skin blistering followed by abnormal pigmentation, skin atrophy, and skin cancer, is caused by mutations in the FERMT1 gene. Although a few KS patients have been reported to also develop ulcerative colitis (UC), a causal link to the FERMT1 gene mutation is unknown. The FERMT1 gene product belongs to a family of focal adhesion proteins (Kindlin-1, -2, -3) that bind several β integrin cytoplasmic domains. Here, we show that deleting Kindlin-1 in mice gives rise to skin atrophy and an intestinal epithelial dysfunction with similarities to human UC. This intestinal dysfunction results in perinatal lethality and is triggered by defective intestinal epithelial cell integrin activation, leading to detachment of this barrier followed by a destructive inflammatory response

Silac mouse for quantitative proteomics uncovers kindlin-3 as an essential factor for red blood cell function

Stable isotope labeling by amino acids in cell culture (SILAC) has become a versatile tool for quantitative, mass spectrometry (MS)-based proteomics. Here, we completely label mice with a diet containing either the natural or the 13C6-substituted version of lysine. Mice were labeled over four generations with the heavy diet, and development, growth, and behavior were not affected. MS analysis of incorporation levels allowed for the determination of incorporation rates of proteins from blood cells and organs. The F2 generation was completely labeled in all organs tested. SILAC analysis from various organs lacking expression of β1 integrin, β-Parvin, or the integrin tail-binding protein Kindlin-3 confirmed their absence and disclosed a structural defect of the red blood cell membrane skeleton in Kindlin-3-deficient erythrocytes. The SILAC-mouse approach is a versatile tool by which to quantitatively compare proteomes from knockout mice and thereby determine protein functions under complex in vivo conditions

Lessons on Conditional Gene Targeting in Mouse Adipose Tissue

Conditional gene targeting has been extensively used for in vivo analysis of gene function in adipocyte cell biology but often with debate over the tissue specificity and the efficacy of inactivation. To directly compare the specificity and efficacy of different Cre lines in mediating adipocyte specific recombination, transgenic Cre lines driven by the adipocyte protein 2 (aP2) and adiponectin (Adipoq) gene promoters, as well as a tamoxifen-inducible Cre driven by the aP2 gene promoter (iaP2), were bred to the Rosa26R (R26R) reporter. All three Cre lines demonstrated recombination in the brown and white fat pads. Using different floxed loci, the individual Cre lines displayed a range of efficacy to Cre-mediated recombination that ranged from no observable recombination to complete recombination within the fat. The Adipoq-Cre exhibited no observable recombination in any other tissues examined, whereas both aP2-Cre lines resulted in recombination in endothelial cells of the heart and nonendothelial, nonmyocyte cells in the skeletal muscle. In addition, the aP2-Cre line can lead to germline recombination of floxed alleles in ∼2% of spermatozoa. Thus, different “adipocyte-specific” Cre lines display different degrees of efficiency and specificity, illustrating important differences that must be taken into account in their use for studying adipose biology

Insulin Resistance in Vascular Endothelial Cells Promotes Intestinal Tumor Formation

The risk of several cancers, including colorectal cancer, is increased in patients with obesity and type 2 diabetes, conditions characterized by hyperinsulinemia and insulin resistance. Because hyperinsulinemia itself is an independent risk factor for cancer development, we examined tissue-specific insulin action in intestinal tumor formation. In vitro, insulin increased proliferation of primary cultures of intestinal tumor epithelial cells from ApcMin/+ mice by over 2-fold. Surprisingly, targeted deletion of insulin receptors in intestinal epithelial cells in ApcMin/+ mice did not change intestinal tumor number or size distribution on either a low or high-fat diet. We therefore asked whether cells in the tumor stroma might explain the association between tumor formation and insulin resistance. To this end, we generated ApcMin/+ mice with loss of insulin receptors in vascular endothelial cells. Strikingly, these mice had 42% more intestinal tumors than controls, no change in tumor angiogenesis, but increased expression of vascular cell adhesion molecule-1 (VCAM-1) in primary culture of tumor endothelial cells. Insulin decreased VCAM-1 expression and leukocyte adhesion in quiescent tumor endothelial cells with intact insulin receptors and partly prevented increases in VCAM-1 and leukocyte adhesion after treatment with tumor necrosis factor-α. Knockout of insulin receptors in endothelial cells also increased leukocyte adhesion in mesenteric venules and increased the frequency of neutrophils in tumors. We conclude that although insulin is mitogenic for intestinal tumor cells in vitro, its action on tumor cells in vivo is via signals from the tumor microenvironment. Insulin resistance in tumor endothelial cells produces an activated, proinflammatory state that promotes tumorigenesis. Improvement of endothelial dysfunction may reduce colorectal cancer risk in patients with obesity and type 2 diabetes