QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 3 January 2006
doi: 10.1242/jcs.02723

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Miñambres, R. Articles by Guerri, C. Search for Related Content PubMed PubMed Citation Articles by Miñambres, R. Articles by Guerri, C. Social Bookmarking                         What's this?

The RhoA/ROCK-I/MLC pathway is involved in the ethanol-induced apoptosis by anoikis in astrocytes

Department of Cellular Pathology, Centro de Investigación Príncipe Felipe, c/EP Autopista del Saler 16-3, 46013-Valencia, Spain

View larger version (23K): [in a new window]   Fig. 1. Ethanol exposure induces apoptosis in astrocytes. Astrocytes were exposed to 100 and 200 mM ethanol concentrations for 3, 6, 14 and 24 hours. (A) The sub-G0/G1 astrocyte population, quantified by flow cytometry, increases after ethanol exposure. Representative histograms are shown for control and 200 mM ethanol-treated samples (for 24 hours). (B) Inhibition of the sub-G0/G1 population with the pan-caspase inhibitor z-VAD-fmk (100 µM), at two different ethanol concentrations (100 and 200 mM). (C) Ethanol increases the caspase-3 activity. (D) Preincubation with z-VAD-fmk (50 and 100 µM) abolishes ethanol-induced caspase-3 activation (300 mM for 24 hours). (E) Quantification of early apoptosis using annexin-V together with 7-AAD and detected by flow cytometry. Values are mean ±s.d. of five different experiments. *P<0.05 and **P<0.001 versus control values in one-way ANOVA.

View larger version (58K): [in a new window]   Fig. 2. Astrocytes exposed to ethanol show morphological changes associated with apoptosis. (A) Cells were incubated with 100 mM ethanol for 1 hour. Actin (a,d,g,j), myosin (b,e,h,k) and nuclei (c,f,i,l) were analyzed by immunofluorescence. Cultured astrocytes show F-actin (a) and myosin (b) colocalization, running throughout the cytoplasm. Ethanol induces an actin-myosin reorganization into a peripheral ring (d,e) that leads to membrane blebbing (g,h,j,k) and chromatin condensation (i), as well as nuclear fragmentation (l). (B) Quantification of morphological apoptotic features such as actin reorganization, membrane blebbing and chromatin condensation at 1, 3, 6 and 14 hours in alcohol-exposed cells (100 mM). The values shown are means ±s.d. from four different primary cultures and more than 300 cells were counted for each experiment. Asterisks indicate significant differences when referred to control samples (*P<0.01 and **P<0.001 by a one-way ANOVA test). Bars, 10 µm.

View larger version (54K): [in a new window]   Fig. 3. Ethanol reorganizes focal adhesions and decreases their levels. Astrocytes were exposed to 100 mM ethanol for an hour. (A) Actin (a,c,e) and paxillin (b,d,f) were detected by immunofluorescence. Control astrocytes show a punctuate pattern of paxillin (b). Ethanol treatment causes a paxillin reorganization at the cell periphery (d) and dramatically decreases its content when cells are densely packed (f). (B) A representative western blot of paxillin from control- and ethanol-treated cells, at different times of alcohol exposure. (C) Densitometric quantification of paxillin staining at two ethanol concentrations (100 and 200 mM for 24 hours). Equal amount of protein was used (50 µg/well). The values represent means ±s.d. from four different cultures and they are expressed as percentages of the control value. Asterisks indicate significant differences with control sample (**P<0.001). (D) Astrocytes were double-stained for F-actin (a,c,e) and phosphotyrosine (b,d,f). Ethanol-exposed cells show a cortical reorganization (d) and a reduction in phosphotyrosine staining of the focal adhesion proteins (f). Bars, 10 µm.

View larger version (53K): [in a new window]   Fig. 4. RhoA and ROCK-I induce membrane blebbing, chromatin condensation and DNA framentation. (A,D) Cultured astrocytes were grown for seven days and then cotransfected with plasmids expressing either active proteins (V14RhoA and ROCKI-1) or inactive proteins (V14A37RhoA and ROCKI-5) together with a plasmid expressing GFP. Astrocytes transfected with either RhoA (A) or ROCK-I (D) were analyzed by immunofluorescence (actin, GFP and nuclear condensation). (B,E) Active proteins (V14RhoA and ROCKI-1) induce DNA fragmentation as revealed by TUNEL staining (green) in astrocytes transfected with myc-tagged expression vectors of either V14RhoA (B) or ROCK-I (E). (C) Quantification of blebbing cells, as well as chromatin condensation (Hoechst staining), in astrocytes overexpressing either RhoA (C) or ROCK-I (F). Values are mean ±s.d. of five different experiments. *P<0.001 versus control values in one-way ANOVA. Bars, 10 µm.

View larger version (43K): [in a new window]   Fig. 5. Ethanol-induced membrane blebbing requires RhoA-dependent, caspase-3-independent, ROCK-I activation. (A) In vitro kinase assay (measured by Histone H1 phosphorylation), shows that ethanol (100 mM) induces activation of ROCK-I in both situations: when ROCK-I was immunoprecipitated with either H-85 or C-19 antibodies. (B) Activated Rho, from lysates of astrocytes (control or exposed to ethanol), was pulled down by GST-TRBD (Rho-binding domain from Rhotekin) on glutathione beads, and analyzed by immunoblotting. (C) Lysates of astrocytes were subjected to immunoprecipitation with anti-ROCK-I (C-19) and then immunoblotted with RhoA and ROCK-I (C19) antibodies. (D) Inhibition of ROCK-I kinase activity using C3 exoenzyme in control- and ethanol-exposed astrocytes (100 mM) (see Materials and Methods). *P<0.05 versus ethanol-treated cells in a one-way ANOVA. (E-a) A representative western blotting of ROCK-I using H-85 antibody is shown, in control and ethanol-treated astrocytes. (E-b) A representative western blot analysis of PARP cleavage in astrocytes exposed to ethanol (100 mM) for 6 and 14 hours. (F) Ethanol-induced blebbing is abolished when cells are pretreated with either Y-27632 (a ROCK inhibitor) or C3 exoenzyme (a Rho inhibitor). **P<0.001 versus ethanol-treated experiments in a one-way ANOVA. (G) Preincubation of astrocytes with either Y-27632 or C3 before ethanol treatment (3, 6, 14 and 24 hours) decreases the caspase-3 activity. Percent of caspase-3 inhibition was calculated by comparing the activity in the presence and absence of the inhibitor, at each time-point analyzed. Results are mean ±s.d. of three different experiments. (H) Quantification of annexin-V positive cells (7-AAD–) of ethanol-exposed astrocytes, in the presence and absence of the ROCK inhibitor Y-27632. Results are the average of around 500 cells from three different cultures. Values are mean ±s.d. of five different experiments. *P<0.05 and **P<0.001 versus control values in one-way ANOVA.

View larger version (36K): [in a new window]   Fig. 6. Ethanol-induced MLC phosphorylation correlates with ROCK-I kinase activity. Cultured astrocytes were treated with 100 mM alcohol for 3, 6, 14 and 24 hours. Lysates of control and ethanol-treated astrocytes were subjected to immunoprecipitation with the anti-ROCK-I antibody C-19. These immunoprecipitates were used for A and B. (A) ROCK-I kinase activity measured by phosphorylated Histone H1. A representative autoradiograph is shown. (B) Western blot analysis of ROCK-I, as a control of protein content. (C) A representative western blotting of phosphorylated MLC from lysates of astrocytes (control and ethanol-treated) that were immunoprecipitated with anti-MLC-pp antibody. (D) Inhibition of the ethanol-induced MLC phosphorylation when astrocytes are pretreated with either Y27632 or C3. Bars represent the mean ±s.d. of the densitometric quantification obtained from three different immunoblots. Percent of MLCpp inhibition was calculated by comparing the densitometric values in the presence and absence of the inhibitor, at each time-point analyzed.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter