Identification and investigations of leucine-rich repeats and immunoglobulin-like domains protein 2 (LRIG2)

Receptor tyrosine kinases (RTKs) constitute a family of proteins controlling cell growth and proliferation and whose activities are tightly controlled in normal cells. LRIG1 is a negative regulator of RTK signaling and is a proposed tumor suppressor. The aim of this thesis was to identify and study possible paralogs of LRIG1. By using the basic local alignment search tool and cDNA cloning, a human mRNA sequence with similarity to LRIG1 was identified and named LRIG2. By fluorescence in situ hybridization analysis, LRIG2 was found to reside on chromosome 1p13. The LRIG2 amino acid sequence was 47% identical to LRIG1, and the predicted protein domain organization was the same as that of LRIG1. Antibodies against LRIG2 were developed and the apparent molecular weight of the protein was determined to be 132 kDa by SDS-polyacrylamide gel electrophoresis and Western blot analysis. The sub-cellular localization was studied by cell surface biotinylation experiments and confocal fluorescence laser microscopy, which revealed that LRIG2 resided at the cell surface and in the cytoplasm. The expression patterns of LRIG2 mRNA, during development and in adult tissues, were evaluated using whole-mount in situ hybridization and quantitative real-time RT-PCR, respectively. In E10.5, E11.5 and E12.5 mouse embryos, the Lrig2 expression domains were both overlapping and unique as compared to the expression domains of Lrig1 and the third family member, Lrig3. In adult human tissues, the most prominent LRIG2 mRNA expression was found in skin, uterus and ovary. To study the developmental and physiological role of LRIG2, Lrig2 knock-out mice were generated. The knock-out mice were born at Mendelian frequencies without any apparent morphological abnormalities. However, Lrig2 knock-out mice showed reduced body weight between 5 days and 12-15 weeks of age, increased mortality, and impaired reproductive capacity. To study the role of LRIG2 as a prognostic factor in oligodendroglioma, LRIG2 expression was analyzed in 65 human oligodendrogliomas by immunohistochemistry. Cytoplasmic LRIG2 expression was an independent prognostic factor associated with poor oligodendroglioma patient survival. The possible functional role of LRIG2 in oligodendroglioma biology was further investigated using the RCAS/tv-a mouse model. Tumors resembling human oligodendroglioma were induced by intracranial injection of PDGFB carrying RCAS retroviruses into newborn Ntv-a mice. Lrig2 wild-type animals developed tumors at a higher frequency and of higher malignancy than the Lrig2 knock-out mice. This result supports the notion that LRIG2 promotes PDGF-induced oligodendroglioma genesis. A possible molecular mechanism was revealed as LRIG2 overexpression increased PDGFRa levels in transfected cells. In summary, we identified a new gene named LRIG2, showed that it is expressed in a variety of tissues during development and in adulthood, knocked it out and found that it was required for proper animal growth, health, and reproduction. We also found that Lrig2 expression promoted PDGF-induced oligodendroglioma genesis and was associated with poor oligodendroglioma patient survival, possibly via a PDGFRa stabilizing function

Förskolors profilering : om att förskolor nischar sig mot Reggio Emiliafilosofin

Syfte med detta arbete är att få en djupare förståelse för varför många pedagoger väljer att nischa sin verksamhet och då framförallt mot Reggio Emiliafilosofin. Vi vill undersöka vad det är som lockar pedagogerna med Reggio Emiliafilosofin. Efter intervjuer med både pedagoger som arbetar på Reggio Emiliainspirerade förskolor och pedagoger som arbetar på förskolor som bara tagit till sig delar av filosofin fick vi reda på att det är främst barnsynen som lockar pedagogerna. Dessutom är miljön och pedagogisk dokumentation viktiga inslag i den pedagogiska filosofin. Framför allt är den stora skillnaden mellan den traditionella förskolan och den Reggio Emiliainspirerade förskolorna att man fokuserar på barns lärande och utveckling istället för omsorgen. Vi har även fått syn på att den största skillnaden mellan att arbeta på en nischad förskola jämfört med en som inte har en pedagogisk inriktning är att de på de icke profilerade förskolorna kan blanda olika teorier, om det är något positivt råder det delade meningar om

A protein interaction network centered on leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) regulates growth factor receptors

Leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) is a tumor suppressor and a negative regulator of several receptor tyrosine kinases. The molecular mechanisms by which LRIG1 mediates its tumor suppressor effects and regulates receptor tyrosine kinases remain incompletely understood. Here, we performed a yeast two-hybrid screen to identify novel LRIG1-interacting proteins and mined data from the BioPlex (biophysical interactions of ORFeome-based complexes) protein interaction data repository. The putative LRIG1 interactors identified in the screen were functionally evaluated using a triple co-transfection system in which HEK293 cells were co-transfected with platelet-derived growth factor receptor α, LRIG1, and shRNAs against the identified LRIG1 interactors. The effects of the shRNAs on the ability of LRIG1 to down-regulate platelet-derived growth factor receptor α expression were evaluated. On the basis of these results, we present an LRIG1 protein interaction network with many newly identified components. The network contains the apparently functionally important LRIG1-interacting proteins RAB4A, PON2, GAL3ST1, ZBTB16, LRIG2, CNPY3, HLA-DRA, GML, CNPY4, LRRC40, and LRIG3, together with GLRX3, PTPRK, and other proteins. In silico analyses of The Cancer Genome Atlas data sets revealed consistent correlations between the expression of the transcripts encoding LRIG1 and its interactors ZBTB16 and PTPRK and inverse correlations between the transcripts encoding LRIG1 and GLRX3. We further studied the LRIG1 function–promoting paraoxonase PON2 and found that it co-localized with LRIG1 in LRIG1-transfected cells. The proposed LRIG1 protein interaction network will provide leads for future studies aiming to understand the molecular functions of LRIG1 and the regulation of growth factor signaling

Health‐related quality of life in patients with heart failure eligible for treatment with sacubitril–valsartan

Aim: To describe and compare self‐reported health‐related quality of life between younger and older patients with severe heart failure eligible for treatment with sacubitril–valsartan and to explore the association between health‐related quality of life and age, NYHA classification, systolic blood pressure and NT‐proBNP level. Design: Cross‐sectional study. Methods: A total of 59 patients, eligible for treatment with sacubitril–valsartan were consecutively included and divided into a younger (≤75 years) and older group (>75 years). Health‐related quality of life was assessed using the Kansas City Cardiomyopathy Questionnaire and the EuroQol 5‐dimensions. Data were collected between June 2016 and January 2018. The STROBE checklist was used. Results: There were no differences in overall health‐related quality of life between the age groups. The older patients reported lower scores in two domains measured with the Kansas City Cardiomyopathy Questionnaire, namely self‐efficacy (67.0 SD 22.1 vs. 78.8 SD 19.7) and physical limitation (75.6 SD 19.0 vs. 86.3 SD 14.4). Higher NYHA class was independently associated with lower Kansas City Cardiomyopathy Questionnaire Overall Summary Score

Lrig1 is a haploinsufficient tumor suppressor gene in malignant glioma

Recently, a genome-wide association study showed that a single nucleotide polymorphism (SNP) —rs11706832—in intron 2 of the human LRIG1 (Leucine-rich repeats and immunoglobulin-like domains 1) gene is associated with susceptibility to glioma. However, the mechanism by which rs11706832 affects glioma risk remains unknown; additionally, it is unknown whether the expression levels of LRIG1 are a relevant determinant of gliomagenesis. Here, we investigated the role of Lrig1 in platelet-derived growth factor (PDGF)-induced experimental glioma in mice by introducing mono-allelic and bi-allelic deletions of Lrig1 followed by inducing gliomagenesis via intracranial retroviral transduction of PDGFB in neural progenitor cells. Lrig1 was expressed in PDGFB-induced gliomas in wild-type mice as assessed using in situ hybridization. Intriguingly, Lrig1-heterozygous mice developed higher grade gliomas than did wild-type mice (grade IV vs. grade II/III, p = 0.002). Reciprocally, the ectopic expression of LRIG1 in the TB107 high-grade human glioma (glioblastoma, grade IV) cell line decreased the invasion of orthotopic tumors in immunocompromised mice in vivo and reduced cell migration in vitro. Concomitantly, the activity of the receptor tyrosine kinase MET was downregulated, which partially explained the reduction in cell migration. In summary, Lrig1 is a haploinsufficient suppressor of PDGFB-driven glioma, possibly in part via negative regulation of MET-driven cell migration and invasion. Thus, for the first time, changes in physiological Lrig1 expression have been linked to gliomagenesis, whereby the SNP rs11706832 may affect glioma risk by regulating LRIG1 expression

The effect of <i>Lrig2</i> on the induction of immediate-early gene expression.

<p>Wild-type (<i>Lrig2E12+/+</i>), heterozygous (<i>Lrig2E12+/-</i>), or <i>Lrig2</i>-deficient (<i>Lrig2E12-/-</i>) MEFs were serum starved for 24 hours followed by stimulation with PDGF-BB for 0, 10, 20, 40, 60, or 120 minutes. Thereafter, total RNA was prepared, and gene expression was quantified using real-time RT-PCR. Samples were run in triplicate, and the specific mRNA levels were normalized to respective <i>Rn18s</i> levels. Shown are the specific mRNA/<i>Rn18s</i> ratios on arbitrary scales. (<b>A</b>) Kinetics of relative <i>Fos</i> expression in cells stimulated with 50 ng/ml PDGF-BB. Shown are the means from three independent experiments, including wild-type (n=8), heterozygous (n=9), and <i>Lrig2</i>-deficient (n=6) cell lines from three different litters, with standard error of the means indicated by error bars. (<b>B</b>) Kinetics of relative <i>Egr2</i> expression levels in cells stimulated with 10 ng/ml PDGF-BB. Shown are the means from two independent experiments, including wild-type (n=6), heterozygous (n=6), and <i>Lrig2</i>-deficient (n=4) cell lines from two different litters, with standard error of the means indicated by error bars. Compared with wild-type cells, the <i>Lrig2</i>-deficient cells showed altered and faster kinetics of induction of both <i>Fos</i> and <i>Egr2</i> expression in response to PDGF-BB stimulation. Significant differences compared with the wild-type cell lines are indicated with asterisks (*p<0.05 and **p<0.01).</p

Genomic organization of the mouse <i>Lrig2</i> gene and the generation and molecular analyses of <i>Lrig2-</i>deficient mice.

<p>(<b>A</b>) Schematic drawing of the wild-type, conditional, and disrupted <i>Lrig2</i> alleles. A PKG-neo selection cassette was inserted downstream of exon 12. Exon 12 and the PKG-neo cassette were flanked by <i>loxP</i> sites and were deleted together, in a single step, by mating with OzCre mice. Color coding is as in <b>C</b>. (<b>B</b>) Southern blot using tail DNA isolated from the offspring of an Lrig2E12+/- × Lrig2E12+/- mating. The expected sizes are as follows: wild-type (<i>Lrig2E12</i>+) allele, 11.0 kb; and <i>Lrig2</i> exon 12-ablated (<i>Lrig2E12</i>-) allele, 6.6 kb. Lane B13 is from <i>Lrig2E12-/</i>-, lanes B10-12 and B14-15 are from <i>Lrig2E12+/-</i>, and the remaining lanes are from <i>Lrig2E12+/+</i> mice. (<b>C</b>) Genomic organization of the mouse <i>Lrig2</i> gene. Gene structure of <i>Mus musculus Lrig2</i> is shown. Gene organization was deduced from the sequence of the mouse genome and the <i>Lrig2</i> mRNA (GenBank accession number NM_001025067). Exons are indicated with boxes and are drawn to scale. Exon numbers are indicated with numbers. The <i>Lrig2</i> gene is approximately 58 kb, and it is located on mouse chromosome 3 F2. Color coding depicts the encoded protein domains, including a signal peptide (red), a leucine-rich repeats domain (yellow), three immunoglobulin-like domains (blue), a transmembrane domain (orange), and a cytosolic domain (green). (<b>D</b>) Western blot of MEF cell lines of different <i>Lrig2</i> genotypes. Top, anti-Lrig2 polyclonal; Bottom, anti-actin. (<b>E</b>) <i>Lrig</i> mRNA levels in mice of different genotypes. The mRNA levels of <i>Lrig1</i>, exon 12-containing <i>Lrig2</i> (<i>Lrig2E12</i>), exon 17-18 boundary-containing <i>Lrig2</i> (<i>Lrig2E17-18</i>), and <i>Lrig3</i> in brains of 3-week old mice were analyzed using quantitative real-time RT-PCR. The <i>Lrig/Rn18s</i> ratio was calculated and normalized to the corresponding ratio in reference RNA from Stratagene. Shown are the means of wild-type (n=6), <i>Lrig2E12+/-</i> (n=5), and <i>Lrig2E12-/</i>- (n=8) mice, with error bars indicating the standard deviations. Significant differences compared with the wild-type mice are indicated with asterisks (*p<0.01 and **p<0.001).</p

PDGF induced phosphorylation events in cells of different <i>Lrig2</i> genotypes.

<p>Wild-type, heterozygous, or <i>Lrig2</i>-deficient MEFs were serum starved for 24 hours followed by stimulation with 50 ng/ml PDGF-BB for different times. (<b>A</b>–<b>N</b>) Cells were untreated or stimulated with 50 ng/ml PDGF-BB for 10 minutes followed by cell fixation and analysis of the phosphorylation status of respective Pdgfr by <i>in situ</i> proximity ligation assay (PLA). Phosphorylated Pdgfr was visualized using fluorescence (red spots). Cell nuclei were counter-stained with DAPI (blue). (<b>A</b>–<b>F</b>) Representative PLA images of phosphorylated Pdgfrα (red spots) in un-stimulated (<b>A</b>–<b>C</b>) or PDGF-BB stimulated (<b>D</b>–<b>F</b>) cells of the indicated <i>Lrig2</i> genotypes. (<b>G</b>–<b>L</b>) Representative PLA images of phosphorylated Pdgfrβ (red spots) in non-stimulated (<b>G</b>–<b>I</b>) or PDGF-BB-stimulated (<b>J</b>–<b>L</b>) cells of the indicated <i>Lrig2</i> genotypes. (<b>M</b>) Quantification of PLA spots for phosphorylated Pdgfrα. Shown are the means from three independent experiments, including wild-type (<i>Lrig2E12+/+</i>, n=8), heterozygous (<i>Lrig2E12+/-</i>, n=9), and <i>Lrig2</i>-deficient (<i>Lrig2E12-/</i>-, n=6) cell lines from three different litters, with standard deviations indicated by error bars. (<b>N</b>) Quantification of PLA spots for phosphorylated Pdgfrβ. Shown are the means from three independent experiments, including wild-type (<i>Lrig2E12+/+</i>, n=8), heterozygous (<i>Lrig2E12+/-</i>, n=9), and <i>Lrig2</i>-deficient (<i>Lrig2E12-/</i>-, n=5) cell lines from three different litters, with standard deviations indicated by error bars. There were no differences observed in the levels of activated Pdgfrα or Pdgfrβ between cells of different genotypes. (<b>O</b>–<b>Q</b>) Cell lysates from cells that had been untreated or treated with 50 ng/ml PDGF-BB for 15 minutes were analyzed through Western blotting with antibodies against the indicated proteins. (<b>O</b>) A representative Western blot is shown of Akt, Erk1/2, phosphorylated Akt (pAkt), and phosphorylated Erk1/2 (pErk1/2) using cell lysates from cells of the indicated <i>Lrig2</i> genotypes that had been untreated (-) or treated (+) with PDGF-BB. (<b>P</b>) Quantification of pAkt/Akt-ratios for non-stimulated and stimulated cells, respectively. Shown are the means from three independent experiments, including wild-type (n=8), heterozygous (n=9), and <i>Lrig2</i>-deficient (n=5) cell lines from three different litters, with standard deviations indicated by error bars. (<b>Q</b>) Quantification of pErk1/2/Erk1/2-ratios for non-stimulated and stimulated cells, respectively. Shown are the means and corresponding standard deviations as for <b>P</b>.</p

Expression level analyses of co-transfected LRIG1, LRIG2, and PDGFRα.

<p>HEK-293T cells were co-transfected with expression vectors encoding myc-LRIG1 or FLAG-LRIG2 and PDGFRα. The numbers indicate amount of plasmid (μg) used in the respective transfection. Empty <i>pcDNA 3.1</i> and <i>p3XFLAG-CMV-13</i>, respectively, were used to bring the total amount of plasmid DNA to the same amount (2 µg) in each transfection. Cell extracts were analyzed by Western blotting with antibodies against LRIG1, PDGFRα, FLAG (recognizing FLAG-LRIG2), or, as a loading control, actin. (<b>A</b>, <b>C</b>) Representative Western blots. Two specific PDGFRα bands were observed, here called PDGFRα-upper and PDGFRα-lower, respectively. (<b>B</b>, <b>D</b>) Quantification of PDGFRα immunoblots. Shown are the means of four independent co-transfection experiments, with error bars indicating the standard deviations. Significant differences compared with the empty vector control are indicated with asterisks (*p<0.05).</p