Beroepenmobiliteit: Bruikbaarheid longitudinale gegevens Enquête Beroepsbevolking

Deze studie is een verslag van een gezamenlijk onderzoek van het Centraal Bureau voorde Statistiek (CBS) en het Researchcentrum voor Onderwijs en Arbeidsmarkt (ROA).De studie is een onderdeel van zowel het CBS-speerpunt Sociale dynamiek enuitbreiding Arbeidsrekeningen, in het bijzonder het project Onderwijs enberoepsloopbaan, als het Project Onderwijs-Arbeidsmarkt van het ROA. Het ProjectOnderwijs-Arbeidsmarkt wordt gefinancierd door het Ministerie van Onderwijs, Cultuuren Wetenschap (OCW), het Uitvoeringsinstituut Werknemersverzekeringen (UWV), hetUWV Werkbedrijf, het Ministerie van Landbouw, Natuur en Voedselkwaliteit (LNV), desamenwerkende kenniscentra voor beroepsonderwijs en bedrijfsleven COLO, RandstadNederland en de Raad voor Werk en Inkomen (RWI).De hoofdstukken 2, 3 en 4 zijn door het CBS samengesteld, hoofdstuk 5 door het ROA.De overige hoofdstukken zijn een gezamenlijk product. Bijlage 4 is door het ROAsamengesteld, de overige bijlagen door het CBS.De auteurs bedanken Henk-Jan Dirven, Wendy Smits, Johan van der Valk en de ledenvan de begeleidingscommissie van het Project Onderwijs-Arbeidsmarkt voor hetcommentaar op een eerdere versie.De in dit rapport weergegeven opvattingen zijn die van de auteurs en komen nietnoodzakelijk overeen met het beleid van het Centraal Bureau voor de Statistiek.education, training and the labour market;

Ribosomal Protein Mutations Induce Autophagy through S6 Kinase Inhibition of the Insulin Pathway

Mutations affecting the ribosome lead to several diseases known as ribosomopathies, with phenotypes that include growth defects, cytopenia, and bone marrow failure. Diamond-Blackfan anemia (DBA), for example, is a pure red cell aplasia linked to the mutation of ribosomal protein (RP) genes. Here we show the knock-down of the DBA-linked RPS19 gene induces the cellular self-digestion process of autophagy, a pathway critical for proper hematopoiesis. We also observe an increase of autophagy in cells derived from DBA patients, in CD34+ erythrocyte progenitor cells with RPS19 knock down, in the red blood cells of zebrafish embryos with RP-deficiency, and in cells from patients with Shwachman-Diamond syndrome (SDS). The loss of RPs in all these models results in a marked increase in S6 kinase phosphorylation that we find is triggered by an increase in reactive oxygen species (ROS). We show that this increase in S6 kinase phosphorylation inhibits the insulin pathway and AKT phosphorylation activity through a mechanism reminiscent of insulin resistance. While stimulating RP-deficient cells with insulin reduces autophagy, antioxidant treatment reduces S6 kinase phosphorylation, autophagy, and stabilization of the p53 tumor suppressor. Our data suggest that RP loss promotes the aberrant activation of both S6 kinase and p53 by increasing intracellular ROS levels. The deregulation of these signaling pathways is likely playing a major role in the pathophysiology of ribosomopathies

Insulin abolishes autophagy in cells with RPS19 loss.

<p>(<b>A</b>) Confocal microscopy analysis of GFP-LC3 HEK cells transfected with siScr or si<i>RPS19</i> and either untreated or stimulated with 350 nM insulin for 6 hours. Size bars = 10 µM. (<b>B</b>) Quantification of the average number of GFP-LC3 puncta per cell in (<b>A</b>). (<b>C</b>) Western blot analysis of LC3 expression in cells from (<b>A</b>). Densitometer analysis used to calculate the ratio of LC3II/actin.</p

DBA mutations induce autophagy.

<p>(<b>A</b>) Immunofluorescence with LC3 antibodies in LCLs derived from a normal control or DBA patients. Higher magnifications are represented in the lower panel. Arrows denote puncta indicative of LC3 recruitment to autophagosomes, or accumulation in autolysosomes. Size bars = 10 µM. (<b>B</b>) Quantification of the percent of cells revealing LC3 puncta compared to the total number of cells in the 60x shots. (<b>C</b>) Western blot analysis of LC3 in DBA LCLs compared to normal controls. The LC3II/actin ratio is determined by densitometer analysis. (<b>D</b>) Representative western blot analysis of p62 levels in normal control and DBA patient LCLs. (<b>E</b>) Densitometer analysis of p62 protein expression from western blots (N = 3) represented in (<b>D</b>). (<b>F</b>) Immunofluorescence with p62 antibodies of LCLs derived from a normal control or DBA patients. Size bars = 10 µM. (<b>G</b>) ImageJ measurements of p62 expression in (<b>F</b>) per total cell area. (<b>H</b>) Representative electron micrographs of LCLs derived from a normal control and <i>RPS17</i> cells. Control cells have small typically dense lysosomes (*). The much larger autolysosomes (A) are only detected in <i>RPS17</i> LCLs. The boxed area in the upper right panel is shown at higher magnification in the lower right panel. N = nucleus, ECS = extracellular space. Bars in top panels = 1 µM, bottom panels = 200 nM.</p

Graphic representation of how RP mutations result in autophagy and insulin pathway inhibition.

<p>In normal cells, insulin signals through the insulin receptor substrate (IRS) to the PI3-kinase/AKT pathway in a manner that inhibits autophagy and increases the expression of glucose transporters on the cell membrane. In cells with RP loss, an increase of ROS stabilizes p53 and induces phosphorylation of S6 kinase. This hyper-phosphorylation of S6 kinase results in degradation of IRS and a decrease of insulin signaling to PI3-kinase and AKT. This loss of AKT activation in turn releases the inhibition of autophagy and reduces glycolysis.</p

S6 kinase phosphorylation and autophagy is induced by reactive oxygen species (ROS).

<p>(<b>A</b>) Confocal microscopy analysis of GFP-LC3 HEK cells transfected with siScr or si<i>RPS19</i> and either untreated or treated with 10 mM Trolox overnight. Size bars = 10 µM. (<b>B</b>) Quantification of the average number of GFP-LC3 puncta per cell in (<b>A</b>). (<b>C</b>) Western blot analysis of phosphorylated S6 kinase expression in GFP-LC3 HEK transfected with siScr or si<i>RPS19</i> and either untreated or treated with 10 mM Trolox overnight. (<b>D</b>) Western blot analysis of phosphorylated S6 kinase and phosphorylated AKT substrate expression in 2 dpf zebrafish embryos untreated or treated with 10 mM Trolox overnight. (<b>E</b>) Survival rates of embryos treated overnight with 10 mM Trolox. The results are the compilation of three independent experiments with N = 25 embryos representing each mutation. The statistics shown are compared to wild type. (<b>F</b>) Analysis of p53 stabilization in 2 dpf wild type, rpS7-deficient embryos, <i>p53^M214K/M214K</i> mutant embryos, or double mutants (<i>rpS7;p53</i>) by western blotting. (<b>G</b>) Analysis of p53 stabilization by western blotting of zebrafish mutants treated overnight with 100 µM Trolox.</p

Reduction of rpS7 to haploinsufficient levels in zebrafish embryos induces autophagy in RBCs.

<p>(<b>A</b>) Western blot analysis of rpS7 protein levels in zebrafish carrying viral inserts in the <i>rpS7</i> gene at 1 and 2 dpf. The amount of rpS7 is calculated by densitometer analysis measuring the rpS7/actin ratio (N = 3). (<b>B</b>) Electron micrographs of 1 dpf embryos. Representative morphology of RBCs is shown in wild type (upper left panel) and rpS7-deficient embryos (upper right and lower panels). Arrowheads indicate double-membrane autophagosomes, arrows indicate autolysosomes. N = nucleus. Size bars upper and bottom left panels = 1 µM, upper right = 500 nM, lower right = 200 nM. (<b>C</b>) Quantification of double-membrane structures in the RBCs of micrographs in (<b>B</b>). In each case 28 cells were examined. (<b>D</b>) O-dianisidine staining of embryos at 2 dpf. Embryos (N>100) were scored in (<b>E</b>) and genotyped to confirm mutation status. (<b>F</b>) Representative mitochondrial morphology (m) displayed in wild type (upper left panel) and rpS7<i>-</i>deficient embryos at 2dpf (upper right). Typical Golgi complex (G) in wild type embryos shown (lower left panel) compared to Golgi complex in rpS7<i>-</i>deficient embryos (lower right). Arrows denote double membranes sequestering cytosolic material, an indication for autophagosome formation. *Denotes engulfed cytoplasmic material. Size bars = 200 nM.</p

RP loss results in a decrease of IRS1 and phosphorylated AKT substrates.

<p>(<b>A</b>) Western blot analysis of IRS1 in GFP-LC3 HEK cells transfected with siScr or si<i>RPS19</i>. (<b>B</b>) Western blot analysis of phosphorylated AKT substrates in GFP-LC3 HEK cells transfected with siScr or si<i>RPS19</i> and either untreated or stimulated with 350 nM insulin for 6 hours. (<b>C</b>) Densitometer analysis of the total expression level of phosphorylated AKT substrates in (<b>B</b>). (<b>D</b>) Western blot analysis of phosphorylated AKT substrates in 2 dpf zebrafish embryos.</p