Microscòpia de superresolució

Des de fa més de tres-cents anys, la biologia cel·lular ha fet servir imatges obtingudes mitjançant microscopis òptics per entendre l’organització i el funcionament de les cèl·lules. Tanmateix, des de l’època de Z. Jansen, H. Jansen, R. Hooke i A. van Leeuwenhoek hi ha hagut una barrera en la microscòpia òptica que els físics, de la mà dels enginyers i dels biòlegs, no han pogut sobrepassar: el límit de resolució dels microscopis. O, per ser més acurats, no fins avui.Els darrers anys s’han dissenyat i desenvolupat nous microscopis i metodologies que, basats en diferents aproximacions, permeten sobrepassar aquest límit a partir del fet de treure avantatge de les característiques físiques de les ones de llum i dels "uoròfors

Microscòpia de superresolució: més enllà de la física

Des de fa més de tres-cents anys, la biologia cel·lular ha fet servir imatges obtingudes mitjançant microscopis òptics per entendre l’organització i el funcionament de les cèl·lules. Tanmateix, des de l’època de Z. Jansen, H. Jansen, R. Hooke i A. van Leeuwenhoek hi ha hagut una barrera en la microscòpia òptica que els físics, de la mà dels enginyers i dels biòlegs, no han pogut sobrepassar: el límit de resolució dels microscopis. O, per ser més acurats, no fins avui.Els darrers anys s’han dissenyat i desenvolupat nous microscopis i metodologies que, basats en diferents aproximacions, permeten sobrepassar aquest límit a partir del fet de treure avantatge de les característiques físiques de les ones de llum i dels "uoròfors

Inhibition of Mitogen-Activated Protein Kinase Erk1/2 Promotes Protein Degradation of ATP Binding Cassette Transporters A1 and G1 in CHO and in HuH7 cells.

Signal transduction modulates expression and activity of cholesterol transporters. We recently demonstrated that the Ras/mitogen-activated protein kinase (MAPK) signaling cascade regulates protein stability of Scavenger Receptor BI (SR-BI) through Proliferator Activator Receptor (PPARα) -dependent degradation pathways. In addition, MAPK (Mek/Erk 1/2) inhibition has been shown to influence liver X receptor (LXR) -inducible ATP Binding Cassette (ABC) transporter ABCA1 expression in macrophages. Here we investigated if Ras/MAPK signaling could alter expression and activity of ABCA1 and ABCG1 in steroidogenic and hepatic cell lines. We demonstrate that in Chinese Hamster Ovary (CHO) cells and human hepatic HuH7 cells, extracellular signal-regulated kinase 1/2 (Erk1/2) inhibition reduces PPARα-inducible ABCA1 protein levels, while ectopic expression of constitutively active H-Ras, K-Ras and MAPK/Erk kinase 1 (Mek1) increases ABCA1 protein expression, respectively. Furthermore, Mek1/2 inhibitors reduce ABCG1 protein levels in ABCG1 overexpressing CHO cells (CHO-ABCG1) and human embryonic kidney 293 (HEK293) cells treated with LXR agonist. This correlates with Mek1/2 inhibition reducing ABCG1 cell surface expression and decreasing cholesterol efflux onto High Density Lipoproteins (HDL). Real Time reverse transcriptase polymerase chain reaction (RT-PCR) and protein turnover studies reveal that Mek1/2 inhibitors do not target transcriptional regulation of ABCA1 and ABCG1, but promote ABCA1 and ABCG1 protein degradation in HuH7 and CHO cells, respectively. In line with published data from mouse macrophages, blocking Mek1/2 activity upregulates ABCA1 and ABCG1 protein levels in human THP1 macrophages, indicating opposite roles for the Ras/MAPK pathway in the regulation of ABC transporter activity in macrophages compared to steroidogenic and hepatic cell types. In summary, this study suggests that Ras/MAPK signaling modulates PPARα- and LXR-dependent protein degradation pathways in a cell-specific manner to regulate the expression levels of ABCA1 and ABCG1 transporters

Inhibition of Mitogen-Activated Protein Kinase Erk1/2 Promotes Protein Degradation of ATP Binding Cassette Transporters A1 and G1 in CHO and in HuH7 cells.

Signal transduction modulates expression and activity of cholesterol transporters. We recently demonstrated that the Ras/mitogen-activated protein kinase (MAPK) signaling cascade regulates protein stability of Scavenger Receptor BI (SR-BI) through Proliferator Activator Receptor (PPARα) -dependent degradation pathways. In addition, MAPK (Mek/Erk 1/2) inhibition has been shown to influence liver X receptor (LXR) -inducible ATP Binding Cassette (ABC) transporter ABCA1 expression in macrophages. Here we investigated if Ras/MAPK signaling could alter expression and activity of ABCA1 and ABCG1 in steroidogenic and hepatic cell lines. We demonstrate that in Chinese Hamster Ovary (CHO) cells and human hepatic HuH7 cells, extracellular signal-regulated kinase 1/2 (Erk1/2) inhibition reduces PPARα-inducible ABCA1 protein levels, while ectopic expression of constitutively active H-Ras, K-Ras and MAPK/Erk kinase 1 (Mek1) increases ABCA1 protein expression, respectively. Furthermore, Mek1/2 inhibitors reduce ABCG1 protein levels in ABCG1 overexpressing CHO cells (CHO-ABCG1) and human embryonic kidney 293 (HEK293) cells treated with LXR agonist. This correlates with Mek1/2 inhibition reducing ABCG1 cell surface expression and decreasing cholesterol efflux onto High Density Lipoproteins (HDL). Real Time reverse transcriptase polymerase chain reaction (RT-PCR) and protein turnover studies reveal that Mek1/2 inhibitors do not target transcriptional regulation of ABCA1 and ABCG1, but promote ABCA1 and ABCG1 protein degradation in HuH7 and CHO cells, respectively. In line with published data from mouse macrophages, blocking Mek1/2 activity upregulates ABCA1 and ABCG1 protein levels in human THP1 macrophages, indicating opposite roles for the Ras/MAPK pathway in the regulation of ABC transporter activity in macrophages compared to steroidogenic and hepatic cell types. In summary, this study suggests that Ras/MAPK signaling modulates PPARα- and LXR-dependent protein degradation pathways in a cell-specific manner to regulate the expression levels of ABCA1 and ABCG1 transporters

Mek1/2 inhibition reduces ABCA1 and ABCG1 protein stability.

<p>(A) 1 µg of RNA extracted from HEK293 cells treated ± LXR agonist (1 µM T0901317) and Mek1/2 inhibitor (10 µM U0126) was reverse transcribed and Real Time RT-PCR to amplify ABCA1, ABCG1, and TATA Box Binding Protein (TBP) cDNA fragments was performed as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062667#pone.0062667-Wood1" target="_blank">[28]</a>. Relative expression from 2 independent experiments with duplicate samples is given and was calculated by normalization to the housekeeper mRNA (TBP). (B–D) HuH7 (B) and CHO-ABCG1 (C) treated with 20 µM Wy-14643 or 50 µM GW53965 overnight and PMA-differentiated THP1 monocytes (D) were incubated with 20 ng/ml cycloheximide (CHX)±10 µM PD98059 (PD) for 0–4 (B) and 0–8 h (C, D) as indicated. Western blot analysis of ABCA1, ABCG1 and β-actin in each lysate of a representative experiment is shown. The mean values ± S.D. of ABCA1 and ABCG1 expression levels (n = 2) are given. *, p<0.05 for Student’s <i>t</i> test.</p

Mek1/2 inhibition reduces ABCA1 expression in HuH7 hepatocytes.

<p>(A) HuH7 cells were incubated for 24 h ±20 µM Wy-14643 with or without 10 µM PD98059 or CI-1040 as indicated. Cell lysates were analyzed by Western blotting for expression levels of ABCA1. ABCA1 levels in each lysate were quantified and normalized to the amount of β-actin. The mean values ± S.D. of 3 independent experiments are given. *and **, p<0.05 and p<0.01 for Student’s <i>t</i>-test, respectively. (B) HuH7 hepatocytes were transfected with control RNAi (−) or RNAi targeting PPARα (see Material and Methods). 72 h after transfection, cells were treated for an additional 24 h ±20 µM Wy-14643 and 10 µM PD98059 as indicated. Expression of ABCA1, PPARα, and β-actin was analyzed. Blots are representative for 2 independent experiments. (C) THP1 monocytes were differentiated with 2 nM TPA for 24 h, pre-incubated for 60 min ±10 µM PD98059 (PD), stimulated ± HDL₃ (50 µg/ml) for 3 min and lysates were analyzed for activated Mek1/2 (P-Mek1/2), Erk1/2 (P-Erk1/2), Total Mek1/2 and Total Erk1/2. Blots are representative for 3 independent experiments. (D) TPA-differentiated THP1 monocytes were incubated ±10 µM PD98059 (PD) for 24 h as indicated. Western blot analysis of ABCA1, SR-BI and β-actin was quantified. *p<0.05 for Student’s <i>t</i>-test. Molecular weight markers are shown.</p

Ras/MAPK inhibition reduces ABCG1 expression in CHO cells.

<p>(A–C) CHO-ABCG1 (A), HEK293 (B) and THP1 macrophages (C) cells were incubated ±50 µM GW3965 and 10 µM PD98059 for 24 h as indicated. Cell lysates were analyzed for ABCG1, activated Erk1/2 (P-Erk1/2), Total Erk1/2 and β-actin. ABCG1 levels (see arrow) were quantified and normalized to β-actin. The arrowhead points at an additional GW3685-inducible and PD98059-sensitive protein (lane 3). Representative western blots for 3 independent experiments per cell line are shown. Molecular weight markers are shown. *, p<0.05 for Student’s <i>t</i> test.</p

Ras/MAPK signaling modulates ABCG1 activity in CHO-ABCG1 cells.

<p>(A) CHO-ABCG1 cells were incubated ±10 µM PD98059 for 24 h as indicated and subjected to subcellular fractionation through Percoll gradients. Ras-containing plasma membrane fractions (PM) in the middle of the gradient were isolated and analyzed for the amount of ABCG1, and markers for cytosol (GAPDH) and plasma membrane (Ras). For comparison, western blot analysis of ABCG1, GAPDH (cytosol) and Ras from whole cell lysates (WCL) is shown. (B) CHO-ABCG1 cells were starved, pre-incubated for 60 min ±10 µM PD98059 (PD) and stimulated ± HDL₃ (50 µg/ml) for 3 min at 37°C as indicated. Western blot analysis of activated Mek1/2 (P-Mek1/2), Erk1/2 (P-Erk1/2), Total Mek1/2 and Erk1/2 from each lysate is shown. (C) CHO-ABCG1 cells were incubated with [³H]-Cholesterol (2×10⁶ cpm/ml) for 24 h, washed with PBS, and incubated with HDL₃ (50 µg/ml) or apoA-I (30 µg/ml) for 6 h±10 µM PD98059. The ratio of released and cell-associated radioactivity was determined, normalized to total cell protein and the amount of efflux is given in (%). The background efflux obtained from CHOwt was equivalent to 1.5–3.0×10⁵ cpm/mg cell protein. *and **, p<0.05 and p<0.01 for Student’s <i>t</i> test.</p

Ras/MAPK signaling regulates ABCA1 expression.

<p>(A+B) CHOwt cells were transfected with empty vector (−), PPARα (+), constitutively active H-Ras (HRasG12V), K-Ras (GFP-KRasG12V) or Mek1 (Mek215-DD) as indicated. (A) After 24 h cells were treated±20 µM Fenofibrate (FF), 20 µM Wy-14643 and±10 µM PD98059 as indicated. Cell lysates were analyzed for ABCA1, PPARα, activated Mek/Erk1/2 (P-Mek1/2, P-Erk1/2), Total Mek1/2 and Erk1/2, Ras and β-actin. Molecular weight markers are shown. ABCA1 levels were quantified, normalized to actin and represent the mean ± S.D. from 3 independent experiments. * and **, p<0.05 and p<0.01 for Student’s <i>t</i>-test, respectively.</p