Siegfried Ussar

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 (13)C(6)-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 beta1 integrin, beta-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.

Kindlin-3 is essential for integrin activation and platelet aggregation

Integrin-mediated platelet adhesion and aggregation are essential for sealing injured blood vessels and preventing blood loss, and excessive platelet aggregation can initiate arterial thrombosis, causing heart attacks and stroke. To ensure that platelets aggregate only at injury sites, integrins on circulating platelets exist in a low-affinity state and shift to a high-affinity state (in a process known as integrin activation or priming) after contacting a wounded vessel. The shift is mediated through binding of the cytoskeletal protein Talin to the β subunit cytoplasmic tail. Here we show that platelets lacking the adhesion plaque protein Kindlin-3 cannot activate integrins despite normal Talin expression. As a direct consequence, Kindlin-3 deficiency results in severe bleeding and resistance to arterial thrombosis. Mechanistically, Kindlin-3 can directly bind to regions of β-integrin tails distinct from those of Talin and trigger integrin activation. We have therefore identified Kindlin-3 as a novel and essential element for platelet integrin activation in hemostasis and thrombosis.

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.

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.

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.

Interactions between Gut Microbiota, Host Genetics and Diet Modulate the Predisposition to Obesity and Metabolic Syndrome

Obesity, diabetes, and metabolic syndrome result from complex interactions between genetic and environmental factors, including the gut microbiota. To dissect these interactions, we utilized three commonly used inbred strains of mice-obesity/diabetes-prone C57Bl/6J mice, obesity/diabetes-resistant 129S1/SvImJ from Jackson Laboratory, and obesity-prone but diabetes-resistant 129S6/SvEvTac from Taconic-plus three derivative lines generated by breeding these strains in a new, common environment. Analysis of metabolic parameters and gut microbiota in all strains and their environmentally normalized derivatives revealed strong interactions between microbiota, diet, breeding site, and metabolic phenotype. Strain-dependent and strain-independent correlations were found between specific microbiota and phenotypes, some of which could be transferred to germ-free recipient animals by fecal transplantation. Environmental reprogramming of microbiota resulted in 129S6/SvEvTac becoming obesity resistant. Thus, development of obesity/metabolic syndrome is the result of interactions between gut microbiota, host genetics, and diet. In permissive genetic backgrounds, environmental reprograming of microbiota can ameliorate development of metabolic syndrome.

Sirt3 Regulates Metabolic Flexibility of Skeletal Muscle through Reversible Enzymatic Deacetylation

Sirt3 is an NAD+-dependent deacetylase that regulates mitochondrial function by targeting metabolic enzymes and proteins. In fasting mice, Sirt3 expression is decreased in skeletal muscle resulting in increased mitochondrial protein acetylation. Deletion of Sirt3 led to impaired glucose oxidation in muscle, which was associated with decreased pyruvate dehydrogenase (PDH) activity, accumulation of pyruvate and lactate metabolites, and an inability of insulin to suppress fatty acid oxidation. Antibody-based acetyl-peptide enrichment and mass spectrometry of mitochondrial lysates from WT and Sirt3 KO skeletal muscle revealed that a major target of Sirt3 deacetylation is the E1α subunit of PDH (PDH E1α). Sirt3 knockout in vivo and Sirt3 knockdown in myoblasts in vitro induced hyperacetylation of the PDH E1α subunit, altering its phosphorylation leading to suppressed PDH enzymatic activity. The inhibition of PDH activity resulting from reduced levels of Sirt3 induces a switch of skeletal muscle substrate utilization from carbohydrate oxidation toward lactate production and fatty acid utilization even in the fed state, contributing to a loss of metabolic flexibility. Thus, Sirt3 plays an important role in skeletal muscle mitochondrial substrate choice and metabolic flexibility in part by regulating PDH function through deacetylation.

MEK1 and MEK2, Different Regulators of the G1/S Transition

The ERK cascade is activated by hormones, cytokines, and growth factors that result in either proliferation or growth arrest depending on the duration and intensity of the ERK activation. Here we provide evidence that the MEK1/ERK module preferentially provides proliferative signals, whereas the MEK2/ERK module induces growth arrest at the G1/S boundary. Depletion of either MEK subtype by RNA interference generated a unique phenotype. The MEK1 knock down led to p21cip1 induction and to the appearance of cells with a senescence-like phenotype. Permanent ablation of MEK1 resulted in reduced colony formation potential, indicating the importance of MEK1 for long term proliferation and survival. MEK2 deficiency, in contrast, was accompanied by a massive induction of cyclin D expression and, thus, CDK4/6 activation followed by nucleophosmin hyperphosphorylation and centrosome over-amplification. Our results suggest that the two MEK subtypes have distinct ways to contribute to a regulated ERK activity and cell cycle progression.

Colocalization of kindlin-1, kindlin-2, and migfilin at keratinocyte focal adhesion and relevance to the pathophysiology of Kindler syndrome.

Kindler syndrome (KS) results from pathogenic loss-of-function mutations in the KIND1 gene, which encodes kindlin-1, a focal adhesion and actin cytoskeleton-related protein. How and why abnormalities in kindlin-1 disrupt keratinocyte cell biology in KS, however, is not yet known. In this study, we identified two previously unreported binding proteins of kindlin-1: kindlin-2 and migfilin. Co-immunoprecipitation and confocal microscopy studies show that these three proteins bind to each other and colocalize at focal adhesion in HaCaT cells and normal human keratinocytes. Moreover, loss-of-function mutations in KIND1 result in marked variability in kindlin-1 immunolabeling in KS skin, which is mirrored by similar changes in kindlin-2 and migfilin immunoreactivity. Kindlin-1, however, may function independently of kindlin-2 and migfilin, as loss of kindlin-1 expression in HaCaT keratinocytes by RNA interference and in KS keratinocytes does not affect KIND2 or FBLIM1 (migfilin) gene expression or kindlin-2 and migfilin protein localization. In addition to identifying protein-binding partners for kindlin-1, this study also highlights that KIND1 gene expression and kindlin-1 protein labeling are not always reduced in KS, findings that are relevant to the accurate laboratory diagnosis of this genodermatosis by skin immunohistochemistry.

ASC-1, PAT2, and P2RX5 are cell surface markers for white, beige, and brown adipocytes

The cell surface markers ASC-1, PAT2, and P2RX5 can be used to mark and identify brown, beige, and white adipocytes in both rodents and humans. Fat Cells Gain New Identities There’s “good fat” and there’s “bad fat.” Good fat is considered to be brown adipose tissue (BAT), which burns calories. Bad fat can be white adipose tissue (WAT), which stores lipids as energy and, in excess, contributes to obesity. When brown fat cells, or adipocytes, develop within white fat, they are called “beige.” Sorting out these different adipocyte subtypes within the human body has been challenging but will be important in uncovering the underlying mechanisms for obesity and its comorbidities, such as type 2 diabetes. To this end, Ussar and colleagues have now identified three new surface markers of white, beige, and brown fat cells. These markers—ASC-1, PAT2, and P2RX5—were first selected in silico, then confirmed in mouse WAT and BAT, and lastly verified in human adipose tissue biopsies. ASC-1, PAT2, and P2RX5 are located in the plasma membrane of adipocytes, thus making them prime targets for imaging fat locations within the body and for directing therapeutics toward particular fat depots. White, beige, and brown adipocytes are developmentally and functionally distinct but often occur mixed together within individual depots. To target white, beige, and brown adipocytes for diagnostic or therapeutic purposes, a better understanding of the cell surface properties of these cell types is essential. Using a combination of in silico, in vitro, and in vivo methods, we have identified three new cell surface markers of adipose cell types. The amino acid transporter ASC-1 is a white adipocyte–specific cell surface protein, with little or no expression in brown adipocytes, whereas the amino acid transporter PAT2 and the purinergic receptor P2RX5 are cell surface markers expressed in classical brown and beige adipocytes in mice. These markers also selectively mark brown/beige and white adipocytes in human tissue. Thus, ASC-1, PAT2, and P2RX5 are membrane surface proteins that may serve as tools to identify and target white and brown/beige adipocytes for therapeutic purposes.