INTRODUCTION

Top Abstract Introduction Materials and methods Results Discussion References

Studies of a variety of primary cell culture systems suggest that cell-to-cell contact interactions can modulate growth and differentiation in an antagonistic manner. Disengagement of cells, such as hepatocytes or glia, from the normal tissue structure and their transfer to a monodispersed cell culture result in stimulation of cell proliferation and repression of differentiation-specific gene expression. The opposite occurs when the cells are reaggregated or cultured in monolayers at a high cell density: specific gene expression resumes and cell proliferation ceases (5, 9, 19, 35, 37, 44, 60). The involvement of direct cell-to-cell contact interactions in this process is also evidenced by the finding that addition of plasma membrane preparations (26, 44) or purified adhesion molecules (13, 33, 57) to cells at low cell density is sufficient to inhibit cell growth and reactivate differentiation properties. The signals triggered by cell-to-cell contacts and the components involved in their intracellular transduction are largely unknown.

Contacts between neighboring cells are mediated by adhesion molecules, which are linked via their intracellular domains to the cytoskeletal network (11). In response to changes in cell contacts the cytoskeletal network undergoes massive rearrangements and assumes distinct structural patterns (20, 68). It seems reasonable to consider that these cytoskeletal changes might be sensed by internal signaling pathways and converted into changes in growth and differentiation. Several studies have indeed demonstrated that depolymerization of the cytoskeleton with drugs is sufficient to release confluent cells from density-dependent inhibition of growth, allowing entry into the S phase of the cell cycle (12, 18, 46, 59). While treatment with drugs causes general depolymerization of the actin or microtubule network, dissociation of cell-to-cell contacts might cause depolymerization of only a small and very specific subset of the cytoskeleton. Nevertheless, if depolymerization of the cytoskeleton by drugs constitutes a relevant cell contact signal, then such treatment should also affect the differentiation properties of the cell. We therefore decided to examine whether depolymerization of the cytoskeleton in cells of an intact tissue can mimic the effects of cell dissociation and cause changes in the control of differentiation-specific gene expression.

The neural retina of the chicken embryo offers important advantages for the molecular analysis of growth and differentiation. In this tissue, expression of the gene for the differentiation marker glutamine synthetase (L-glutamate-ammonia ligase [ADP forming]; EC 6.3.1.2) is restricted to Müller glial cells, regulated by glucocorticoids, and ultimately dependent on glia-neuron cell contacts (35, 37, 60). Glucocorticoids stimulate the transcription of the gene in intact retinal tissue, but not in dissociated retinal cells that are maintained in adherent monolayer cultures or in cell suspension; however, when the separated cells are reassembled into multicellular aggregates, restoring cell contacts, glutamine synthetase expression can again be induced.

Control of glutamine synthetase expression by cell contacts is mediated by changes in the transactivating capability of the glucocorticoid receptor (GR). This ligand-dependent transcription factor is a cytoplasmic protein that translocates into the nucleus upon binding to its ligand and activates the transcription of target genes, such as glutamine synthetase. The regulatory region of glutamine synthetase contains a glucocorticoid response element (GRE) that can bind the GR protein and confer responsiveness to glucocorticoids on an attached reporter gene (4, 71). Separation of retinal cells renders the GR molecules transcriptionally inactive. This was demonstrated by the use of a chloramphenicol acetyltransferase (CAT) construct that is regulated by a minimal GRE and a control construct that does not contain the GRE sequence. Glucocorticoids can induce CAT expression in the intact tissue but not in separated retinal cells. We have shown that GR becomes transcriptionally inactive by a mechanism that involves the transcription factor c-Jun (48). The c-Jun protein is a central component in the AP-1 complex of transcription factors and is involved in the control of a set of genes that regulate cell growth (2). It can also block the transcriptional activity of GR by forming a protein-protein complex with the receptor (29, 54, 69) or by competing for limiting amounts of a common coactivator in the cell (30). Cell separation causes a rapid and dramatic increase in c-Jun expression, and this increase is causally related to the decline in GR activity (48).

In this study we demonstrate that depolymerization of the actin or the microtubule network in cells of the intact retinal tissue inhibits the expression of glutamine synthetase and represses the transactivating capability of GR by activating a tyrosine kinase-dependent signaling pathway which induces c-Jun expression in glial cells only. These findings suggest that changes in the cytoskeletal structure might have a functional role in the transduction of cell contact signals to the nucleus.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and methods Results Discussion References

Reagents and plasmids. Nocodazole, vinblastine, colchicine, cytochalasin B, taxol, cortisol, and genistein were purchased from Sigma. Latrunculin A and latrunculin B were provided by Y. Kashman, Tel Aviv University, Tel Aviv, Israel. SU4984, SU5402, and SU65847 were provided by SUGEN, Inc., Redwood City, Calif. Fibroblast growth factor (FGF) was provided by G. Neufeld, Technion, Haifa, Israel. The reporter plasmid pG46TCO (4) was derived from pG46TCO (53) and contains two copies of a synthetic GRE sequence linked to the herpes thymidine kinase promoter-CAT gene. The RSVL(SEL) construct and the RSVCAT construct contain the luciferase reporter gene and the CAT reporter gene, respectively, under the transcriptional control of the Rous sarcoma virus (RSV) promoter (16, 21). The GR expression vector p6RGR (40) and the c-Jun expression vector pRSVc-jun (51) are under the transcriptional control of the RSV promoter. Plasmid pGS116-9 is a subclone of the chicken glutamine synthetase gene (60). Plasmid DNA was prepared by using the Qiagen plasmid preparation kit. The glutathione S-transferase (GST)-c-Jun expression vector, pGEX2T-c-Jun (27), was transformed into the XL1-blue strain of Escherichia coli. Protein induction and purification were done as described previously (55).

Tissue culture, glutamine synthetase induction, and transfection procedure. Retinal tissue was isolated under sterile conditions from eyes of chicken embryos (White Leghorn) at day 10 of embryonic development. The tissue was organ cultured in Erlenmeyer flasks in Dulbecco's modified Eagle's medium with 10% fetal calf serum on a gyratory shaker at 38°C. For induction of glutamine synthetase, cortisol was added to the medium to a final concentration of 0.33 µg/ml. Plasmid DNA was transfected into pieces of intact retinal tissue by electroporation with a gene pulser (Bio-Rad, Richmond, Calif.) with voltage and capacitance settings of 400 V and 960 µF, as described previously (6). Following electroporation, retinas transfected in the same cuvette were placed in two Erlenmeyer flasks and cultured for a further 24 h in the absence or presence of cortisol.

CAT, luciferase, and glutamine synthetase assays. CAT, luciferase, and glutamine synthetase activities were determined in tissue sonicates. CAT activity was determined as described previously (21). In all experiments, the CAT assay was adjusted to include an equal amount of luciferase activity originating from cotransfected RSVL(SEL). CAT activity was determined by scanning the thin-layer chromatographic plates (Merck) with a phosphorimager instrument (Fujix Bas 1000; Fuji) and TINA software (Raytest Isotopenme ger GmbH) and calculating the percentage of conversion of the substrate (chloramphenicol) to the acetylated products. Luciferase activity was assayed as described previously (16) and recorded by a luminometer (LKB, Rockville, Md.). The specific activity of glutamine synthetase was determined by the colorimetric assay (35) and expressed as micromolar -glutamylhydroxamate per hour per milligram of protein.

RNA preparation and analysis. Cellular RNA was prepared with the RNAzolTM B RNA isolation solvent (Biotecx Laboratories, Houston, Tex.). For Northern blot analysis, RNA was denatured by heating it at 60°C for 10 min in 2.2 M formaldehyde-50% formamide and fractionated by electrophoresis in 1.2% agarose gels containing 2.2 M formaldehyde and MOPS (morpholinepropanesulfonic acid) buffer. The fractionated RNA was transferred to a nitrocellulose filter and hybridized with pGS116-9 DNA (60) labeled with ³²P by nick translation. The levels of hybridization were visualized by autoradiography. As a loading control, the nitrocellulose filter was stained for rRNA by soaking it in a solution of 0.5 M sodium acetate (pH 5.2) and 0.04% methylene blue for 5 min at room temperature and rinsing it in water for 10 min.

Protein preparation and immunoblotting. GR was detected in total cellular extracts or in nuclear and cytoplasmic fractions as previously described (22). Briefly, total cellular extracts were prepared by suspension of the tissue in RIPA buffer (150 mM NaCl-1% Triton X-100-0.5% deoxycholate-0.1% sodium dodecyl sulfate [SDS]-50 mM Tris [pH 8]-5 mM EDTA-1 µg of leupeptin/ml-1 µg of aprotonin/ml-1 µg of pepstatin/ml-2 mM phenylmethylsulfonyl fluoride [PMSF]) on ice and homogenization in a Dounce homogenizer followed by centrifugation to clarify the cell lysates. Nuclear and cytoplasmic fractions were prepared by resuspension of the tissue in hypotonic HEN buffer (10 mM HEPES [pH 7.8]-1 mM EDTA-10 mM NaCl) on ice and homogenization in a Dounce homogenizer. Nuclei were separated from the cytoplasmic fraction by centrifugation at 1,500 × g for 10 min at 4°C. The nuclear pellet was resuspended and extracted in HEN buffer containing 0.5 M NaCl for 1 h on ice. Cytoplasmic supernatants and nuclear extracts were clarified by centrifugation at 140,000 × g for 1 h at 4°C. For the detection of c-Jun, total cell extracts were prepared by sonication of the tissue in HEPES buffer (25 mM HEPES-0.5% Nonidet P-40 [pH 7.8]) followed by centrifugation at 20,000 × g for 10 min at 4°C. For the detection of tyrosine-phosphorylated proteins, the tissue was sonicated in lysis buffer (50 mM HEPES [pH 7.5]-150 mM NaCl-10% glycerol-1% Triton X-100-1.5 mM MgCl₂-1 mM EGTA-50 µg of leupeptin/ml-1 mM PMSF-0.5 mM Na₃VO₄-100 mM NaF) and the extracts were clarified by centrifugation at 20,000 × g for 10 min at 4°C. Equal amounts of protein were separated on SDS-polyacrylamide gels (7 or 10% polyacrylamide) and electroblotted onto nitrocellulose filters in Tris-glycine buffer (48 mM Tris [pH 8.5]-39 mM glycine-0.037% SDS-20% methanol). To identify specific protein bands, the filters were incubated overnight in a blocking solution of 5% milk in TBS-T buffer (20 mM Tris [pH 7.6]-137 mM NaCl-0.05% Tween 20) and reacted with the anti-GR monoclonal antibody (MAb) GR49-4 (63), anti-carbonic anhydrase II (CAII)MAb (38), anti-c-Jun MAb (J31920; Transduction Laboratories, Lexington, Ky.), antiphosphotyrosine MAb (SC-508; Santa Cruz Biotechnology, Santa Cruz, Calif.), anti-c-Jun N-terminal kinase (JNK) polyclonal antibody; (Sigma), anti-p38 mitogen-activated protein kinase (MAPK) polyclonal antibody (anti MAPK-p38; Sigma), anti-ERK polyclonal antibody directed against the unphosphorylated form of the protein (anti MAPK-WS; Sigma) or anti-ERK MAb directed against the phosphorylated form of the protein (anti-MAPK activated; Sigma). Subsequently, the filters were reacted with anti-mouse or anti-rabbit immunoglobulin G coupled to horseradish peroxidase (HRP), and visualized by the enhanced-chemiluminescence (ECL) procedure (Amersham, Arlington Heights, Ill.), except for the filters reacted with anti-JNK or anti-p38 MAPK antibodies, which were developed with alkaline phosphatase-conjugated anti-rabbit Fab (Sigma).

Kinase assays. Solid-phase assays of JNK protein activity were performed as described by Hibi et al. (27). Retinal tissue was sonicated in lysis buffer (50 mM -glycerophosphate-1.5 mM EGTA-1 mM EDTA-0.1 mM Na₃VO₄-1 mM benzamidine-1 mM dithiothreitol [DTT]-1 µg of aprotinin/ml-1 µg of leupeptin/ml-2 µg of pepstatin/ml). Equal amounts of clarified cell extracts were mixed with 20 µl of glutathione-agarose suspension (Sigma) to which 10 µg of GST-c-Jun was bound. The mixture was rotated at 4°C for 2 h in a microcentrifuge tube and pelleted by a brief centrifugation. After four 1-ml washes in HEPES-Triton buffer (20 mM HEPES [pH 7.7]-25 mM MgCl₂-50 mM NaCl-0.1 mM EDTA-0.05% Triton X-100), the pellet was resuspended in 30 µl of kinase buffer (20 mM HEPES [pH 7.7]-20 mM MgCl₂-40 mM -glycerophosphate-0.1 mM Na₃VO₄-2 mM DTT) containing 20 µM cold ATP and 2 µCi of [-³²P]ATP. After 20 min at 30°C the reaction was terminated by the addition of gel sample buffer. The products were resolved on a SDS-10% polyacrylamide gel and visualized by autoradiography. Phosphorylation of GST-c-Jun was quantitated by scanning the autoradiogram with the LKB Ultrascan XL enhanced laser densitometer. For examination of p38 protein kinase activity, clarified cell extracts were prepared as described above and the p38 protein was immunoprecipitated from equal amounts of cell extracts with anti-p38 polyclonal antibodies (c-20; Santa Cruz Biotechnology) bound to protein A-Sepharose. The immunoprecipitates were washed twice with Li buffer (0.5 M LiCl-0.1 M Tris [pH 8.0]) and twice with lysis buffer. Kinase activity was then determined by incubation of the immune complexes at 30°C for 20 min with 30 µl of kinase buffer (25 mM -glycerophosphate-0.5 mM DTT-1.5 mM EGTA-0.15 mM Na₃VO₄-10 mM MgCl₂-0.1 mM ATP-2.5 µg of bovine serum albumin/ml) containing 2.5 µCi of [-³²P]ATP and 15 µg of myelin basic protein (MBP) as a substrate. Samples were separated on a SDS-15% polyacrylamide gel, and phosphorylation of MBP was quantitated as described above.

Immunohistochemistry. Retinal tissue was cultured for 2 h in the presence or absence of cytochalasin B or nocodazole. Samples were fixed with paraformaldehyde (3.5% paraformaldehyde-1% ethyldimethylaminopropyl carbodiimide-0.1 M cacodylate buffer [pH 7.4]) and frozen in OCT compound (Lab-Tek Products, Naperville, Ill.) in liquid nitrogen (17). Immunostaining was performed by the indirect-immunofluorescence method. The sections were washed with phosphate-buffered saline (pH 7.4) containing 2% bovine serum albumin and stained with anti-c-Jun antibody (Transduction Laboratories) or anti-GR antibody GR49-4 (63). The sections were then washed with phosphate-buffered saline and incubated with fluorescein isothiocyanate-conjugated anti-mouse antibody (Jackson Immuno-Research Laboratories, West Grove, Pa.). Sections of cytochalasin B-treated retina were also labeled with the fluorescent N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) phallacidin (Molecular Probes, Eugene, Oreg.). Antibody binding or phallacidin staining was detected by immunofluorescence with epi-illumination from a UV source. Untreated retina was fixed with Carnoy's fixative and embedded in paraffin. Paraffin sections were rehydrated and stained with hematoxylin-eosin.

	RESULTS

Top Abstract Introduction Materials and methods Results Discussion References

Depolymerization of the cytoskeleton inhibits glutamine synthetase expression. To examine whether depolymerization of the cytoskeletal network in cells of the intact retina affects the expression of glutamine synthetase, the tissue was treated with cytochalasin B (66), latrunculin A, or latrunculin B (56), which depolymerize the actin filament network, or with nocodazole, vinblastine, or colchicine, which inhibit tubulin polymerization (64). Treated or untreated retina was cultured in the presence of cortisol, and the level of the enzyme was measured. As shown in Fig. 1, all of the drugs caused a dose-dependent decrease in glutamine synthetase expression. Northern blot analysis revealed that this decrease was due to inhibition of glutamine synthetase mRNA accumulation (Fig. 2). Cortisol induced a major increase in the level of glutamine synthetase mRNA in untreated retina but not in retina treated with cytochalasin B or vinblastine. In the absence of cortisol the retinal tissue expressed a low basal level of glutamine synthetase, which was not affected by the addition of the various drugs (not shown). Thus, depolymerization of either the actin or the microtubule network in cells of the intact retinal tissue repressed the ability of cortisol to stimulate glutamine synthetase gene expression.

View larger version (24K): [in this window] [in a new window]   FIG. 1.   Depolymerization of the cytoskeleton causes a dose-dependent decline in glutamine synthetase expression. Retinal tissue was cultured in the presence of the indicated amounts of nocodazole (A), vinblastine (B), colchicine (C), cytochalasin B (D), latrunculin A (E), and latrunculin B (F). Cortisol was added 30 min after addition of the drugs, and the level of glutamine synthetase (GS) activity was measured 24 h later. GS activity is expressed as micromolar -glutamylhydroxamate per hour per milligram of protein. The results shown are the means plus standard errors of the mean of at least three independent experiments.

View larger version (62K): [in this window] [in a new window]   FIG. 2.   Inhibition of glutamine synthetase mRNA accumulation by cytochalasin B and vinblastine. Retinal tissue was cultured for 2 h in the presence (+) (lanes 2, 4, and 6) or absence () (lanes 1, 3, and 5) of cortisol. Cytochalasin B (50 µg/ml; lanes 3 and 4) or vinblastine (20 µg/ml; lanes 5 and 6) was added 30 min prior to the addition of cortisol. Total RNA was prepared, size fractionated (30 µg/lane) by electrophoresis, and transferred to a nitrocellulose filter. The filter was stained for rRNA (B) and subsequently probed with a 32P-labeled clone of the glutamine synthetase (GS) gene, pGS116-9, and visualized by autoradiography (A).

Depolymerization of the cytoskeleton inhibits GR transcriptional activity. Hormonal induction of glutamine synthetase expression is mediated by GR, which is a key factor in the transcription machinery of the gene and is directly involved in the cell contact-dependent control of glutamine synthetase expression (60). GR is transcriptionally active in the intact retina but becomes inactive upon dissociation of the tissue into separated cells (48). To investigate whether a similar mechanism underlies the cytoskeleton-dependent control of glutamine synthetase expression, we examined the transactivating capability of the receptor molecules in treated and untreated tissues. GR transcriptional activity was assayed by using the reporter plasmid pG46TCO, which contains the CAT gene under the transcriptional control of a minimal consensus GRE promoter (4). The plasmid was transfected into the intact retinal tissue, and the ability of cortisol to induce CAT expression was assayed. Treatment with either cytochalasin B or nocodazole caused a dramatic decline in CAT induction (Fig. 3A). This decline was not due to a general suppression of gene transcription, since disruption of the cytoskeleton did not affect the expression of the noninducible construct pRSVCAT (Fig. 3B). Moreover, overexpression of GR (by cotransfection of a GR expression vector) restored the inducibility of the CAT construct (Fig. 3C). This latter finding raised the possibility that the decline in GR activity might reflect a cytoskeleton-dependent decrease in the cellular level of the GR protein. We examined the level of the receptor protein by Western blotting with the CAII protein as an internal standard. This protein, like glutamine synthetase and GR, is expressed in Müller glial cells only; however, expression of CAII is not modulated by glucocorticoids and is not dependent on cell contacts (36, 48, 60). Analysis of total cellular extracts or of nuclear and cytoplasmic fractions revealed that treatment with cytochalasin B or colchicine did not cause a detectable decline in the cellular level of the receptor protein (Fig. 4A) nor did it impair the ability of the receptor molecules to translocate into the nucleus in response to hormonal induction (not shown). Depolymerization of the cytoskeletal network, therefore, appears to affect the inducibility of glutamine synthetase by directly repressing the transcriptional activity of the endogenous receptor molecules.

View larger version (22K): [in this window] [in a new window]   FIG. 3.   GR transcriptional activity is repressed by cytochalasin B, nocodazole, or c-Jun. Retinal tissue was precultured for 30 min in the presence of cytochalasin B (50 µg/ml) (bars 3 and 4) or nocodazole (40 µg/ml) (bars 5 and 6) or in their absence (bars 1 and 2) or transfected with the c-Jun expression vector RSVc-Jun (1 µg of DNA/8 × 106 cells) (bars 7 and 8). The tissue was transfected (bars 1 to 6) or cotransfected (bars 7 and 8) with the glucocorticoid-inducible CAT construct pG46TCO (1 µg of DNA/8 × 106 cells) (A) or the noninducible construct RSVCAT (1 µg of DNA/8 × 106 cells) (B) or cotransfected with pG46TCO (1 µg of DNA/8 × 106 cells) and the GR expression vector p6RGR (1 µg of DNA/8 × 106 cells) (C). In all cases, the luciferase reporter construct RSVL(SEL) (1 µg of DNA/8 × 106 cells) was cotransfected as a control. The transfected cultures were maintained for 24 h in the presence (solid bars) or absence (open bars) of cortisol. The CAT and luciferase activities were then examined. The CAT assays were adjusted to include an equal amount of luciferase activity. The percentage of CAT conversion was calculated by scanning the thin-layer chromatographic plates with a phosphorimager instrument. In each experiment the value of CAT conversion in the cortisol-treated control (bar 1) was used to normalize all other results. The data shown in A and B are the means plus standard errors of the mean of three separate experiments.

View larger version (57K): [in this window] [in a new window]   FIG. 4.   Depolymerization of the cytoskeleton does not alter the level of GR but induces a rapid and dose-dependent increase in the level of c-Jun. (A) Retinal tissue was cultured for 24 h in the presence of the indicated amounts of cytochalasin B (lanes 1 to 6) or colchicine (lanes 7 to 12). Protein extracts were prepared, and samples (30 µg/lane) were fractionated by electrophoresis on a SDS-7.5% polyacrylamide gel. The gel was electroblotted onto a nitrocellulose filter, and the blot was reacted with anti-GR MAb (which detects the 90-kDa isoform of GR), anti-CAII MAb (which detects the 30-kDa CAII protein), and HRP-conjugated anti-mouse antibody. Protein bands were visualized by the ECL procedure. (B) Retinal tissue was cultured for 24 h in the presence of the indicated amounts of cytochalasin B (lanes 1 to 6) or colchicine (lanes 7 to 12). Protein extracts were prepared, and samples (30 µg/lane) were fractionated by electrophoresis on a SDS-10% polyacrylamide gel. After electroblotting, the blots were reacted with anti-c-Jun MAb (which detects the 40-kDa c-Jun protein), anti-CAII MAb, and HRP-conjugated anti-mouse antibody. Protein bands were visualized by the ECL procedure. (C) Retinal tissue was cultured for the indicated periods of time in the presence of cytochalasin B (50 µg/ml) or colchicine (20 µg/ml). Protein extracts were prepared and analyzed by gel electrophoresis and immunoblotting, as described for panel B. (D) Retinal tissue was preincubated for 30 min in the presence (+) (lanes 3 and 5) or absence () (lanes 1, 2, and 4) of taxol (5 µg/ml) and cultured for an additional 2 h in the presence of vinblastine (20 µg/ml; lanes 2 and 3) or colchicine (10 µg/ml; lanes 4 and 5). Protein extracts were prepared and analyzed by gel electrophoresis and immunoblotting, as described for panel B.

Cytoskeletal control of the c-Jun signaling pathway. GR transcriptional activity can be effectively inhibited by the c-Jun protein. This mode of regulation has been shown to control GR activity in retinal cells during embryonic development (6) and in response to changes in cell contacts (48). Figure 3 demonstrates that the effect of cytoskeleton-disrupting agents on GR activity was similar to that of c-Jun. As with cytochalasin B or nocodazole treatment, elevation of c-Jun in cells of the intact retina (by transfection of the c-Jun expression vector pRSVc-Jun) repressed the transcriptional activity of GR, and cotransfection of the GR expression vector p6RGR restored the inducibility of the cells. Involvement of c-Jun in the cytoskeletal control of GR activity implies that disruption of the cytoskeletal network causes an increase in the cellular level of the c-Jun protein. Western blot analysis indeed revealed that treatment with cytochalasin B, colchicine (Fig. 4B), or the various other cytoskeleton-disrupting agents (not shown) caused a dose-dependent increase in the level of c-Jun expression. The increase was rapid and already apparent 30 min after addition of the drugs (Fig. 4C). In view of the fact that several cytoskeleton-disrupting agents that differ in their biochemical properties and in their sites of action induced a marked and rapid increase in c-Jun expression, it is conceivable that c-Jun induction was caused by their depolymerization activity. This notion was also supported by the finding that taxol, a microtubule-stabilizing agent (14), could inhibit the ability of colchicine or vinblastine to induce c-Jun expression (Fig. 4D). It did not, however, inhibit the induction of c-Jun by cytochalasin B, which depolymerizes the actin filament network (not shown). The effects of the drugs were reversible, and their removal, even after 24 h of treatment, caused a decline in the abundance of the c-Jun protein and restored the inducibility of glutamine synthetase (Fig. 5).

View larger version (36K): [in this window] [in a new window]   FIG. 5.   The effects of cytochalasin B and nocodazole on c-Jun expression and glutamine synthetase induction are reversible. Retinal tissue was cultured for 24 h in the presence of cytochalasin B (50 µg/ml; bar 2) and for an additional 24 h in its absence (bar 3) or for 24 h in the presence of nocodazole (40 µg/ml; bar 5) and for an additional 24 h in its absence (bar 6). Control tissue was cultured for 24 h in the absence of drugs (bars 1 and 4). Cortisol was added for the last 23 h, and the levels of glutamine synthetase (GS) activity (A) and c-Jun protein (B) were measured. The level of GS activity in the control tissue was given the arbitrary value of 100 and was used to normalize all other results. The level of c-Jun was analyzed as described in the legend to Fig. 4B and quantitated by scanning the autoradiogram with the LKB Ultrascan XL enhanced laser densitometer. The highest level of c-Jun was given the arbitrary value of 100 and used to normalize all other results.

View larger version (34K): [in this window] [in a new window]   FIG. 6.   Induction of tyrosine phosphorylation by depolymerization of the cytoskeleton and repression of c-Jun induction by tyrosine kinase inhibitors. (A) Retinal tissue was cultured for the indicated periods of time in the presence of cytochalasin B (50 µg/ml) or for 5 min in the presence of 1 mM peroxovanadate (VOOH). Protein extracts were prepared, and samples (50 µg/lane) were fractionated by electrophoresis on a SDS-10% polyacrylamide gel. After electroblotting, the blots were reacted with antiphosphotyrosine MAb and with HRP-conjugated anti-mouse antibody. Protein bands were visualized by the ECL procedure. (B) Retinal tissue was preincubated for 30 min in the absence () (lanes 1, 2, and 4) or presence (+) (lanes 3 and 5) of genistein (5 µM) and cultured for an additional 2 h in the presence of cytochalasin B (50 µg/ml; lanes 2 and 3) or nocodazole (40 µg/ml; lanes 4 and 5) or in their absence (lane 1). Protein extracts were prepared, and samples (50 µg/lane) were fractionated by electrophoresis on a SDS-10% polyacrylamide gel. After electroblotting, the blots were reacted with anti c-Jun MAb, anti-CAII MAb, and HRP-conjugated anti-mouse antibody. Protein bands were visualized by the ECL procedure. (C) Retinal tissue was preincubated for 10 min in the absence (lanes 1 and 2) or presence of the tyrosine kinase receptor (TKR) inhibitor SU4984 (40 µM; lane 3), SU65847 (30 µM; lane 4), or SU5402 (20 µM; lane 5). The tissue was cultured for an additional 2 h in the absence (lane 1) or presence (lanes 2 to 5) of FGF (5 ng/ml). Protein extracts were prepared and analyzed by gel electrophoresis and immunoblotting as described for panel B. (D) Retinal tissue was preincubated in the absence (lanes 1, 2, and 6) or presence of the TKR inhibitor SU4984 (40 µM; lanes 3 and 7), SU65847 (30 µM; lanes 4 and 8), or SU5402 (20 µM; lanes 5 and 9). The tissue was cultured for an additional 2 h in the presence of cytochalasin B (50 µg/ml; lanes 2 to 5) or nocodazole (40 µg/ml; lanes 6 to 9) or in their absence (lane 1). Protein extracts were prepared and analyzed by gel electrophoresis and immunoblotting as described for panel B.

View larger version (31K): [in this window] [in a new window]   FIG. 7.   Depolymerization of the cytoskeleton activates p38 MAPK and JNK pathways. Activation of p38 (A), JNK (B), and ERK (C) was determined after incubation of the retinal tissue with cytochalasin B (50 µg/ml) for the indicated periods of time. Control tissue was incubated for 5 min in the presence of 0.7 M NaCl, which activates the p38 MAPK pathway (A), or for 20 min with 1 mM peroxovanadate (VOOH), which activates the JNK and ERK pathways (B and C). (A) Equal amounts of tissue extracts (250 µg) were immunoprecipitated with anti-p38 polyclonal antibodies bound to protein A-Sepharose. A kinase assay was performed by incubation of the immune complex with MBP as a substrate. Samples were analyzed by electrophoresis on a SDS-15% polyacrylamide gel and autoradiography. The results of this assay are shown in the top gel. The bottom gel shows the total amount of p38 MAPK present in each cell extract (30 µg/lane) as measured by Western blotting. The autoradiograms were quantitated by scanning, and the highest level of MBP phosphorylation was used to normalize all other results. (B) Equal amounts of tissue extracts (150 µg) were incubated with GST-c-Jun bound to glutathione-agarose. After solid-phase kinase assay the products were analyzed by electrophoresis on a SDS-10% polyacrylamide gel and phosphorylated GST-c-Jun was visualized by autoradiography. The results of this assay are shown in the top gel. The bottom gel shows the total amount of JNK present in each cell extract (30 µg/lane) as measured by Western blotting. The autoradiograms were quantitated by scanning, and the highest level of phosphorylated GST-c-Jun was used to normalize all other results. (C) Equal amounts of tissue extracts (30 µg/lane) were analyzed by electrophoresis on a SDS-10% polyacrylamide gel and Western blotting with antibodies directed against the phosphorylated (P) form of ERK (top gel) or the unphosphorylated form of the protein (bottom gel). The autoradiograms were quantitated by scanning, and the level of phosphorylated ERK in the peroxovanadate-treated tissue was used to normalize all other results. Results similar to those shown were obtained in three independent experiments.

Induction of c-Jun expression is restricted to Müller glial cells. Avian retina consists of five different types of neurons and a single glial cell type, Müller glia. Hormonal induction of glutamine synthetase is always restricted to Müller glial cells (23, 35), which are the only cells in the tissue that express the GR protein (22). The involvement of c-Jun in inhibition of glutamine synthetase induction and GR transcriptional activity implies that depolymerization of the cytoskeleton induces c-Jun expression, inter alia in Müller glial cells. These cells are localized in an inner region of the intermediate nuclear layer of the retinal tissue, and their position can be visualized by immunostaining with antibodies against GR (Fig. 8D). Immunostaining with anti-c-Jun antibodies revealed that treatment with either cytochalasin B or nocodazole induced c-Jun expression in Müller glial cells only (Fig. 8B and C). This is in spite of the fact that the depolymerization activity of these drugs is not restricted to particular cell types, as evidenced by phalloidin staining of glia and neurons in the cytochalasin B-treated retina (not shown). In some sections, c-Jun expression could be observed in the ganglion cell layer as well, but this was also seen in some control sections of untreated tissue. Depolymerization of the actin or of the microtubule network therefore appears to elicit a signaling pathway that induces c-Jun expression in glial cells only. In view of the role of c-Jun in the control of cell growth, it is possible that this cell-type-specific activation of c-Jun is directly related to the fact that cell separation induces proliferation of glial cells but not of neurons (34). The cell type specificity of the cytoskeletal response and its effects on the transcription factors c-Jun and GR raised the possibility that the cytoskeletal network is involved in mediation of cell-to-cell contact control of growth and differentiation.

View larger version (64K): [in this window] [in a new window]   FIG. 8.   Induction of c-Jun is restricted to Müller glial cells. Retinal tissue was cultured for 2 h in the absence () (A, D, and E) or presence of cytochalasin B (Cyto B) (B) or nocodazole (NOC) (C). Cryostat sections were stained with anti-c-Jun MAb (A to C) or anti-GR MAb (D) and with fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin G. The figure shows paraffin sections of untreated retina stained with hematoxylin-eosin (H & E) (E). (Bar = 12.5 µm.)

	DISCUSSION

Top Abstract Introduction Materials and methods Results Discussion References

This study shows that depolymerization of the cytoskeletal network in cells of intact retinal tissue can mimic the effects of cell separation, i.e., it can inhibit the expression of the differentiation marker, glutamine synthetase, gene. The molecular basis for inhibition of glutamine synthetase expression involves, in both cases, the transcription factors GR and c-Jun. Depolymerization of the actin or microtubule network rendered the GR molecules transcriptionally inactive. This was demonstrated by use of the CAT reporter construct pG45TCO, which is controlled by a minimal GRE promoter, and a control construct that does not contain the GRE sequence. Depolymerization of the cytoskeleton markedly reduced the ability of the endogenous GR molecules to induce CAT expression. The decline in GR transcriptional activity occurred in spite of the fact that depolymerization of the actin or microtubule network did not cause a detectable decrease in the cellular level of the GR molecules or a change in the ability of the receptor molecules to translocate into the nucleus in response to hormonal induction. The possibility that the various cytoskeleton-disrupting agents had inhibitory side effects on cortisol transport across the plasma membrane and thus inhibited glutamine synthetase induction and GR transcriptional activity was excluded by the finding that the receptor molecules translocated into the nucleus in response to hormonal induction.

The finding that depolymerization of the cytoskeleton induced a rapid and marked increase in the cellular level of the c-Jun protein offers a possible mechanistic basis for the decline in GR activity. The c-Jun protein is a central component of the AP-1 complex of transcription factors and is involved in the control of a set of genes that regulate cell growth (2). This protein can also inhibit the transcriptional activity of GR by forming a protein-protein complex with GR (29, 54, 69) or by competing for limiting amounts of a common coactivator in the cell (30). We have shown that elevation of c-Jun expression in retinal cells, by induction of the endogenous c-jun gene or by transfection of a c-Jun expression vector, blocks the transcriptional activity of the GR molecules (6). This inhibitory interaction between c-Jun and GR plays an important role in the control of glutamine synthetase expression during growth and differentiation of the retinal tissue. At early embryonic stages, while retinal cells are still proliferating, the level of c-Jun is high, GR is transcriptionally inactive, and glutamine synthetase cannot be induced. As cell growth ceases with development, the level of c-Jun declines, GR becomes transcriptionally active, and expression of glutamine synthetase can be induced (6). Dissociation of the retinal tissue into separated cells reverses this process, reinducing an increase in c-Jun expression, repression of GR activity, and inhibition of glutamine synthetase expression (48). Our results show that depolymerization of the cytoskeleton in cells of the intact tissue triggered similar molecular events, namely, an increase in c-Jun expression, a decline in GR transcriptional activity, and inhibition of glutamine synthetase expression. In both cases overexpression of GR, by transfection of a GR expression vector, reversed the effects and restored the responsiveness of the cells to glucocorticoids.

There is another striking similarity between the cytoskeletal and cell separation responses. Dissociation of the retinal tissue into separated cells induces proliferation of glial cells but not of neurons (34). Likewise, depolymerization of the cytoskeleton induced c-Jun expression only in glial cells. In view of the involvement of c-Jun in the regulation of genes that control cell growth, it is possible that these two cell type-specific responses of c-Jun induction and cell proliferation are causally related. It is not clear why differentiated neurons lose the ability to proliferate whereas glial cells do not. Neuronal cell growth might be irreversibly blocked at the level of cell cycle progression or at a more upstream process, related to the transduction of growth signals. Our finding that depolymerization of the cytoskeleton can induce c-Jun expression only in glial cells might suggest that in neurons particular aspects of the c-Jun signaling pathway are impaired. This possibility is also supported by our preliminary results showing that other inducers of this pathway, such as growth factors and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate can also stimulate c-Jun expression in glial cells only. An understanding of the molecular basis for this cell type specificity of c-Jun induction might shed light on the mechanism that underlies the inability of neuronal cells to proliferate. Our finding that depolymerization of the cytoskeleton induces c-Jun expression might also be relevant for several other biological activities that are triggered by depolymerization of the cytoskeleton. These include the induction of genes, such as those for collagenase and urokinase-type plasminogen activator, both of which are regulated by AP-1 (7, 62), and stimulation of DNA synthesis (12, 18, 46, 59). Depolymerization of the microtubule has also been shown to activate the transcription factor NF-B and NF-B-dependent gene expression (49). Although the two transcription factors NF-B and c-Jun can be activated by different signaling pathways (32, 65, 70), it is possible that both pathways can sense and respond to similar cytoskeletal changes.

How does depolymerization of the cytoskeleton lead to induction of c-Jun? There is compelling evidence for cross-coupling between the cytoskeletal network and intracellular signaling pathways (67). The most extensively studied examples involve members of the Rho GTPase family, which appear to be key players in controlling the organization of the actin cytoskeleton (58). Rho, Rac, and Cdc42 have been shown to regulate a signal transduction pathway that links extracellular signals to the formation of stress fibers, lamellipodia, and filopodia, respectively (45). These GTP-binding proteins have also been implicated in the regulation of a wide range of other biological responses, including cell growth. Rac and Cdc42 are important regulators of JNK and p38 MAPK pathways, which are directly involved in the control of c-Jun expression (3, 8, 10, 24, 41, 72). These two signaling pathways regulate the abundance of c-Jun by controlling the transcription of the gene (8, 25, 31) and by modulating the stability of the protein (43). As with other inducers of c-Jun expression (8, 15, 27, 47), depolymerization of the cytoskeleton was shown here to activate the JNK and p38 MAPK pathways. That microtubule-disrupting agents activate the JNK pathway but not the ERK pathway was also recently reported by others (61). Activation of the JNK and p38 MAPK pathways depends on tyrosine kinase activity, since genistein, a general inhibitor of tyrosine kinase activity (1), prevented the induction of c-Jun. We found that depolymerization of the cytoskeleton stimulated tyrosine phosphorylation of a protein band with an apparent molecular mass of 38 kDa, which might represent the p38 MAPK. However, it is possible that the response is also mediated by other tyrosine-phosphorylated proteins, which were not detected in this assay. Possible candidates are tyrosine kinase receptors, which were shown to be the initial target for activation of the c-Jun signaling pathway by growth factors and by UV radiation (50, 52). The finding that c-Jun induction is repressed by SU4984 and SU5402, which block the kinase activity of the FGF receptor (42), but is not repressed by SU65847, an inhibitor of the platelet-derived growth factor receptor (39), might imply that the cytoskeletal response is initiated by activation of specific tyrosine kinase receptors. The exact biochemical process by which depolymerization of the cytoskeleton activates the c-Jun signaling pathway remains to be elucidated.

A plausible model for the interplay between cell contacts, the cytoskeletal network, and the transcription factors c-Jun and GR is presented schematically in Fig. 9. This model suggests that in the intact tissue the c-Jun signaling pathway is blocked. Cell-to-cell contacts render the growth factor receptors inactive and/or maintain the existence of a specific cytoskeletal architecture which locks components of the c-Jun signaling pathway in an inactive conformation (Fig. 9A). In these cells the level of c-Jun is low, GR is transcriptionally active, and glucocorticoids can induce the expression of glutamine synthetase. Since adhesion molecules are linked to the cytoskeletal network, changes in cell contacts cause the restructuring of the cytoskeleton and vice versa. Disruption of cell contacts or depolymerization of the cytoskeleton removes the restraint, thereby facilitating the transduction of the c-Jun signaling pathway (Fig. 9B). This results in an increase in c-Jun and repression of GR transcriptional activity. The ability of the c-Jun pathway to sense changes in both the cytoskeleton network and cell contacts and to transform them into changes in gene expression might provide a conceptual framework for investigating their roles in the control of growth and differentiation.

View larger version (23K): [in this window] [in a new window]   FIG. 9.   Interplay between cell contacts, the cytoskeletal network, and the transcription factors c-Jun and GR. (A) Cell-to-cell contacts within the intact tissue, which are mediated by adhesion molecules (black boxes), render the growth factor receptors inactive and/or maintain the existence of a specific cytoskeletal architecture (gray ovals) which locks components of the c-Jun signaling pathway (X) in an inactive conformation. Under these conditions the level of c-Jun is low (downward arrowhead) and GR activity is high (upward arrowhead), capable of inducing the transcription of target genes. (B) Disruption of cell contacts or depolymerization of the cytoskeleton removes the restraint, thereby facilitating signal transduction. This results in an increase in c-Jun and repression of GR transcriptional activity.