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First published online 11 March 2008
doi: 10.1242/jcs.014456

Journal of Cell Science 121, 1025-1035 (2008)
Published by The Company of Biologists 2008
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Epithelial cell-cell contacts regulate SRF-mediated transcription via Rac-actin-MAL signalling

Stephan Busche1, Arnaud Descot1, Sylvia Julien1, Harald Genth2 and Guido Posern1,*

1 Department of Molecular Biology, AG Regulation of Gene Expression, Max-Planck-Institute of Biochemistry, 82152 Martinsried, Germany
2 Institute of Toxicology, Hannover Medical School, 30625 Hannover, Germany


Figure 1
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  		Fig. 1. Dissociation of epithelial junctions activates SRF-mediated transcription. (A) Dose-response curve of SRF activity. 1.5x106 MDCK cells were transiently transfected with SRF reporter p3DA-Luc (1.2 µg). Transfected cells were reseeded to form a confluent monolayer (600,000 per 1-cm diameter) and cultured in DMEM containing the physiological Ca2+ concentration (1.8 mM) for 24 hours. Then the medium was exchanged to the indicated amounts of Ca2+ and luciferase activity was measured 7 hours later. Shown is the relative luciferase activity normalised to protein amount. (B) Morphology and epithelial junction integrity of confluent monolayers after medium exchange to the Ca2+ amounts indicated in A. Cells were fixed and analysed by phase-contrast microscopy or immunostained for E-cadherin (DECMA-1). (C) SRF luciferase reporter activation on tissue culture plastic or in fully polarised cells grown on microporous membranes. Cells were either transfected with p3DA-Luc (3 x SRF) or a reporter containing SRF binding site mutations (mut.) and treated as in A. For comparison, cells were treated for 7 hours with 15% serum following 18 hours starvation (FCS) (left). To form a confluent monolayer 175,000 p3DA-Luc transfected cells were seeded on a Transwell filter (BD Falcon), and medium exchange was 24 hours later (right). (D) SRF activity in cells lacking E-cadherin expression. Rac V12 served as a positive control for the existence of a functional pathway in these cells. Transfection and stimulation was as before. E-cadherin expression (inset) in the three cell lines was determined by western blotting using anti-E-cadherin (clone 36, BD Transduction Laboratories). Mock, medium exchange with 1.8 mM Ca2+; –Ca2+, medium exchange with 0.02 mM Ca2+. Error bars indicate s.e.m. of three independent experiments. Stars indicate statistical significance at P<=0.05 according to Student's unpaired t-test.

 

Figure 2
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  		Fig. 2. Time course of epithelial disintegration and SRF induction. (A) Scheme of the reporter plasmid for life imaging. Three SRF-binding sites were inserted in front of an EGFP expression cassette containing a PEST domain for fast proteasomal degradation. (B) Time-lapse images of living cells harbouring the EGFP reporter. Confluent monolayers of stably transfected MDCK cells were microscopically analysed to monitor EGFP expression (upper panel) and cell shape changes (lower panel) upon Ca2+ switch to 0.02 mM for the indicated times. Mock indicates medium exchange to normal (1.8 mM) Ca2+ for 7 hours to exclude any potential alterations by addition of fresh medium or shear forces. (C) Induction profile showing the time course of EGFP expression in B. The arbitrary fluorescence intensity was calculated by subtracting the fluorescence at each time point in the mock experiment from the equivalent fluorescence value upon Ca2+ withdrawal.

 

Figure 3
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  		Fig. 3. Disrupted cell contacts activate SRF via monomeric actin. (A) MDCK cells were transfected with the SRF reporter p3DA-Luc (1.2 µg), pRL-TK (1 µg) and, if indicated, with 2.5 µg of wild-type actin (wt.) or the non-polymerisable actin mutant R62D and reseeded to form a confluent monolayer. Latrunculin B (LatB, 1 µM) was added 30 minutes before medium exchange. Shown is the mean luciferase activity, measured 7 hours after Ca2+ withdrawal. Error bars indicate s.e.m. (n=4). (B) Junction assembly and disassembly in cells expressing Flag-tagged actins. Cells were transfected with Flag-tagged actin wt., R62D or G15S, and seeded to form a confluent monolayer. 24 hours later, the medium was exchanged as indicated; after 7 hours incubation, cells were fixed and stained for Flag and E-cadherin (DECMA-1). Immunofluorescence micrographs are shown from cells maintained in physiological (upper panels) or low (lower panels) Ca2+. Transfected cells are marked by stars. (C) Correlation of F-actin reorganisation and Ca2+ level. Medium containing the indicated Ca2+ concentration was exchanged on a confluent monolayer of MDCK cells. After 7 hours, cells were fixed and F-actin was visualised with rhodamin-phalloidin. Mock, medium exchange with 1.8 mM Ca2+; –Ca2+, medium exchange with 0.02 mM Ca2+. Bars, 50 µm.

 

Figure 4
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  		Fig. 4. Dissociating epithelial junctions regulate SRF via MAL. (A) Effects of MAL constructs or the F-actin stabilising mutant actin G15S. 0.4 µg MAL (f.l.), 2.5 µg MAL {Delta}N{Delta}B1, 2.5 µg MAL {Delta}N{Delta}C, 0.4 µg MAL {Delta}N (scheme) or 2.5 µg actin G15S were co-transfected and Ca2+ exchange was carried out as in Fig. 3A. Luciferase activity is normalised to pRL-TK, with error bars indicating s.e.m. (n=3). (B) Dissociation of the actin-MAL complex upon Ca2+ withdrawal. MDCK cells were electroporated with 10 µg Flag-actin and 10 µg MAL-HA. 36 hours later, the medium was exchanged to low Ca2+ for the times indicated and cell lysates were immunoprecipitated with anti Flag. Proteins were visualised using antibodies against Flag and HA-tags. Latrunculin B (3 µM) or cytochalasin D (10 µM) was added 30 minutes before medium exchange. Relative association was calculated from densitometric analysis. Mock, medium exchange with 1.8 mM Ca2+; –Ca2+, medium exchange with 0.02 mM Ca2+.

 

Figure 5
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  		Fig. 5. SRF activation by dissociating epithelial junctions is dependent on Rac, but not on RhoA or Cdc42 activation. (A) Activity of endogenous RhoA, Rac1 and Cdc42 upon Ca2+ withdrawal. MDCK cells were seeded to form a confluent monolayer. 36 hours later, the medium was exchanged. After the indicated times, cells were lysed and GST-Rhotekin or GST-PAK-CRIB pull-down assays were employed. Uncoupled beads (control) or lysates incubated with GTP{gamma}S served as negative and positive controls, respectively. Precipitated proteins (upper panels) and total lysates (lower panels) were blotted using antibodies against RhoA, Rac1 or Cdc42. Pairwise densitometric analysis of the fold induction is shown below each panel. (B) Effect of the inhibition of Rac1 or RhoA on SRF. MDCK cells were transfected and treated as in Fig. 1A. Prior to medium exchange, cells were preincubated with equipotent concentrations of TcdBF (0.1, 0.25 or 0.75 µg/ml) or TcdB (0.3, 1 or 3 ng/ml) for 4 hours, and with Tat-C3 (0.3, 1 or 3 µM) for 15 hours. Inhibitor treatment was maintained throughout the assay. Shown is the mean luciferase activity normalised to protein. (C) Specificity of TcdBF, TcdB and Tat-C3 on Rac1 and RhoA. When indicated, cells were preincubated with TcdBF (0.25 µg/ml, 4 hours), TcdB (1 ng/ml, 4 hours) or Tat-C3 (1 µM, 15 hours). Activity of Rac1 and RhoA was measured as in A, either 5 or 2 minutes after medium exchange, respectively. (D) E-cadherin localisation of cells treated with TcdBF, Tat-C3, TcdB or vehicle. Confocal immunofluorescence micrographs of MDCK cells treated for 11 hours (TcdBF, TcdB) or 22 hours (Tat-C3) in normal medium, as before. Cells were fixed and stained for E-cadherin. (E) Effect of constitutive active Rac1 or RhoA on SRF activity. 2.5 µg RacV12 or RhoQ63L were co-transfected and cells were treated as in Fig. 3A. Shown is the relative SRF luciferase reporter activity normalised to pRL-TK. (F) Effect of the inhibition of RhoA on SRF in NIH3T3 fibroblasts. Cells (35,000 per 1-cm diameter) were transfected with p3DA-Luc (20 ng) and pRL-TK (50 ng). After transfection, cells were maintained in starvation medium (0.5% FCS) for 20 hours before stimulation with 15% FCS. Tat-C3 treatment of NIH3T3 cells was as in B for MDCK cells. Mock, medium exchange with 1.8 mM Ca2+; –Ca2+, medium exchange with 0.02 mM Ca2+. Error bars indicate s.e.m. (n=3). Stars indicate statistical significance at P<=0.05 according to the Student's unpaired t-test.

 

Figure 6
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  		Fig. 6. Induction of SRF and endogenous target genes during epithelial dissociation and EMT. (A) Relative SRF luciferase reporter activity in HGF-treated MDCK cells. Cells were transfected and 24 hours later HGF (40 ng/ml) was added for 24 hours prior to analysis of luciferase activity. Phase-contrast images of the cells prior to lysis are shown in B. (C) Effect of TGFβ on SRF activity in EpRas murine mammary epithelial cells. 24 hours after reseeding transfected EpRas cells, EMT was induced by TGFβ (5 ng/ml) for 72 hours. Shown is the relative luciferase activity. Phalloidin-stained cells are shown in D following EMT induction for 7 days. (E) Induction of the endogenous target genes for vinculin (vcl) and smooth muscle {alpha}-actin (acta2) upon junction disruption. RNA was isolated from confluent EpRas cells 3 hours after medium exchange and analysed by quantitative RT-PCR. Shown is the fold induction of the endogenous SRF target genes in 0.02 mM Ca2+, normalised to medium exchange with 1.8 mM Ca2+. (F) Effect of inhibiting Rac1 and RhoA on induction of acta2, determined as in E. Where indicated, cells were preincubated with Tat-C3 (1 µM, 15 hours), TcdB (0.3 ng/ml, 4 hours) or TcdBF (0.25 µg/ml, 4 hours). Error bars indicate s.e.m. (n=3). Stars indicate statistical significance at P<=0.01 according to the Student's unpaired t-test.

 

Figure 7
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  		Fig. 7. Actomyosin contractility is not sufficient to activate SRF. (A) Effect of Blebbistatin and Y-27632 on induction of SRF by Ca2+ withdrawal. MDCK cells were transfected and treated as before. Blebbistatin (100 µM, 2.5 hours pretreatment) and Y-27632 (20 µM, 30 minutes pretreatment) was added prior to medium exchange and maintained throughout the assay. (B) Rac1 and RhoA activity following Blebbistatin pretreatment. Pull-down assays were performed as in Fig. 5 for Rac1 or RhoA, either 5 or 2 minutes after medium exchange, respectively. (C) Blebbistatin effect on the actin cytoskeleton. Confluent monolayers were pretreated for 2.5 hours with Blebbistatin prior to medium exchange for 7 hours, as in A. Cells were fixed and stained for E-cadherin and F-actin (phalloidin). Shown are micrographs from cells maintained in normal (left) or low (right) Ca2+. (D) No effect of constitutively active ROCK{Delta}4 or the myosin-phosphatase inhibitor calyculin A on SRF activity. Calyculin A was added 30 minutes before medium exchange and maintained throughout the assay. (E) Influences of ROCK{Delta}4 and calyculin A on the actin cytoskeleton. Cells were transfected or treated as before and stained for myc (ROCK{Delta}4) and F-actin (phalloidin). Mock, medium exchange with 1.8 mM Ca2+; –Ca2+, medium exchange with 0.02 mM Ca2+; CalA, treated with calyculin A (5 nM). Error bars indicate s.e.m. (n=3). Bars, 50 µm.

 

Figure 8
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  		Fig. 8. Model of the regulation of MAL/SRF-dependent transcription by epithelial junctions. Disassembly of E-cadherin-mediated cell-cell contacts leads to transient activation of Rac and alterations in actin treadmilling. This releases the G-actin-mediated inhibition of MAL. GTP-loaded Rac is both required and sufficient for MAL and SRF activation upon epithelial disintegration, and subsequent transcription of endogenous target genes such as vinculin (vcl) and smooth muscle {alpha}-actin (acta2). Actomyosin contractility is a prerequisite for appropriate actin remodelling during this process. Blue bars represent transmembrane proteins.

 

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© The Company of Biologists Ltd 2008