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Glucocorticoid effect on human mesangial cell cytoskeletal proteins
Glucocorticoid effect on human mesangial cell cytoskeletal proteins SEVASTI B. KOUKOURITAKI and ELIAS A. LLANOS MILWAUKEE,WISCONSIN In human mesangial cells (HMCs), we assessed the effect of dexamethasone on the expression and levels of tyrosine phosphorylation of two cytoskeleton-associated proteins: focal adhesion kinase (FAK) and paxillin. Dexamethasone, 10-7 mol/L, increased levels of tyrosine phosphorylation of both proteins within 15 to 30 minutes without a change in protein levels. The exposure of HMCs to cytochalasin B, a disrupter of the cytoskeleton assembly, reduced basal tyrosine phosphorylation of both proteins. This effect was reversed by dexamethasone. These observations support a stabilizing effect of dexamethasone on the mesangial cell cytoskeleton. This may constitute a cytoprotective mechanism in the context of the anti-inflammatory action of the steroids in various glomerulopathies. (J Lab Clin Med 1999;I 33:378-83)
Abbreviations:CB = c y t o c h a l a s i n B; ECM = extracellular matrix; FAK = f o c a l a d h e s i o n kinase; HMC = h u m a n m e s a n g i a l cell; IgG = i m m u n o g l o b u l i n G; T-TBS = Tris-buffered saline solution (10 m m o l / L Tris [pH 7,5], 100 m m o l / L NaCI, 0.1% Tween-20)
G
lucocorticoids have long been and continue rto be used extensively in the treatment of various immune-mediated nephropathies involving glomeruli (glomerulonephritides) and in preventing or reversing kidney transplant rejection. Their beneficial effects have generally been attributed to the inhibition of the synthesis or release of mediators of inflammation and to the suppression of the immunocompetence of lymphocytes and macrophages. 1 A less well-known mechanism that may underlie the cytoprotective action of glucocorticoids is their stabilizing effect on the cell cytoskeleton. In this regard, glucocorticoids have been shown to stabilize the organization of actin microfilamerits, 2 an effect that can occur rapidly without the involvement of genomic pathways--that is, the regulation of genes encoding actin or other cytoskeletal pro-
From the CardiovascularResearchCenter, Nephrology,MedicalCollege of Wisconsin. Submitted for publicationJune 15, 1998; revision submitted October 8, 1998; acceptedDecember4, 1998. Reprint requests: Elias A. Lianos, MD, 9200 W Wisconsin Ave,Milwaukee, WI 53226. Copyright © 1999 by Mosby, Inc. 0022-2143/99 $8.00 + 0 511196326
378
teins. 3 Whether glucocorticoids have stabilizing effects on the cytoskeleton of intrinsic glomerular cells is unknown. In this study we explored the effect of dexamethasone on the levels of expression and the extent of tyrosine phosphorylation of two cytoskeleton-associated proteins that promote the assembly of actin microfilaments: FAK and paxillin. Our observations demonstrate that in HMCs, dexamethasone stimulates the tyrosine phosphorylation of FAK and paxillin. Moreover, it restores the tyrosine phosphorylation of these proteins after the disruption of the cytoskeletal assembly.
METHODS Reagents. Dexamethasone, cytochalasin B, and protein G Sepharose beads for immunoprecipitation experiments were obtained from Sigma Chemical Co, St Louis, MO. Monoclonal antibody against FAK or phosphotyrosine was from Upstate Biotechnology, Lake Placid, NY. Antibody against paxillin was from Transduction Laboratories, Santa Cruz, NM. The enhanced chemiluminescence Western blotting kit and horseradish peroxidase-conjugated sheep anti-mouse IgG were purchased from Amersham, Life Science Inc, Arlington Heights, IL. All other chemicals were obtained from the usual commercial sources at the purest grade available. HMC culture. A stable line of T-SV40 immortalized HMCs originating from the laboratory of Dr. J-D Sraer, Hospital
J Lab Clin Med Volume 133, Number 4
Koukouritaki and Lianos
EO
[o DEX 2'
15'
DEX
p
I
o' -
o
30' * - - FAK 125 KD
"O1 O
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~ 1
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IP: phosphotyrosine WB: FAK
Fig 1. Dexamethasone (DEX), 10-7 mol/L, rapidly stimulates tyrosine phosphorylation of FAK. HMCs were serum-starved for 24 hours and then exposed to dexamethasone for 2, 15, and 30 minutes. Cell lysates were immunoprecipitated with anti-phosphotyrosine antibody, and after separation of precipitates on a sodium dodecyl sulfate-polyacrylamide gradient (4% to 20%) gel, proteins were transferred to a nitrocellulose membrane and immunoblotted with anti-FAK antibody.
2 =o o
-. "-
2
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Paxillin 6 8 KD IgG (heavy chain)
* - - IgG (light chain)
IP: phosphotyrosine WB: paxillin Fig 3. Dexamethasone rapidly stimulates tyrosine phosphorylation of paxillin. HMCs were serum-starved for 24 hours and then exposed to dexamethasone, 10-7 mol/L, for 2, 15, and 30 minutes. Cell lysates were immunoprecipitated with anti-phosphotyrosine antibody, and after separation of precipitates on a sodium dodecyl sulfate-polyacrylamide gradient (4% to 20%) gel, proteins were transferred to a nitrocellulose membrane and immunoblotted with anti-paxillin antibody.
DEX
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15'
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1
379
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4 WB: FAK
Fig 2. Dexamethasone (DEX), 10-7 mol/L, has no effect on FAK levels. HMCs were serum-starved for 24 hours and then exposed to dexamethasone for 2, 15, and 30 minutes. Cell lysates were immunoprecipitated with anti-FAK antibody, and after separation of precipitates on a sodium dodecyl sulfate-polyacrylamide gradient (4% to 20%) gel, proteins were transferred to a nitrocellulose membrane and immunoblotted with anti-FAK antibody.
Tenon, Paris, France, 4 were used. Cultures were performed in a 5% CO2-95% air atmosphere at 37°C. Ceils were maintained in RPMI medium 1640 supplemented with 10% fetal calf serum, 1% L-glutamine, and 1% antibiotic/antimycotic solution (final concentrations, 100 U/ml penicillin and 100 mg/ml stretromycin). At near confluence, cells were washed twice and then serum-deprived for 24 hours before the experiment. For immunoprecipitation and Western blot experiments, cells were washed twice with cold phosphate-buffered saline solution and removed from plates with scrapers. Use of dexamethasone and experimental design. Dexamethasone was dissolved in absolute ethanol and introduced in cultured HMCs at 10-7 mol/L for defined time periods (2, 15, and 30 minutes). This concentration was chosen as one that is frequently achieved in the plasma of human patients undergoing steroid treatment. In control incubation experi-
ments, the dexamethasone vehicle (ethanol) was introduced for identical time periods. To explore whether an intact cytoskeleton is required for the tyrosine phosphorylation of FAK and paxillin by dexamethasone, cells were pretreated with CB, which specifically depolymerizes F-actin to Gactin. 5 In these experiments, CB was dissolved in absolute ethanol and introduced in cultured HMCs to a final concentration of 1.2 mmol/L for 2 hours. In control incubation experiments, the CB vehicle (absolute ethanol) was introduced for the same time period. Immunoprecipitation. Cells were lysed on ice with cold lysis buffer consisting of 50 mmol/L N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, pH 7.5; 150 mmol/L MgC12;
1 mmol/L ethyleneglycol-bis-(~-aminoethylether)-N,N,N',N'tetraacetic acid; 10% glycerol; 1% Triton X-100; I g g / m L aprotinin; 1 ~tg/mL leupeptin; 1 mmol/L phenyhnethylsulfonyl fluoride; and 20 gmol/L sodium orthovanadate. Cell lysates were scraped off the plates and allowed to lyse for an additional 30 minutes on an orbital shaker at 4°C. Samples were subsequently centrifuged at 14,000 g at 4°C, and supernatants were transferred to new centrifuge tubes. Protein concentrations were determined by using a calorimetric assay (Biorad Protein Assay). Cell lysates containing equal amounts of protein were incubated either with anti-FAK monoclonal IgG1 or with an anti-phosphotyrosine monoclonal IgG2bk at 4°C overnight. Antigen-antibody complexes formed were precipitated by agitation for 2 hours at 4°C with washed protein G Sepharose bead slurry. Beads were collected by pulse centrifugation (14,000 g, 5 seconds) and washed three times with lysis buffer. Immunoprecipitates were solubilized by dissolving in gel sample buffer and boiled for 5 minutes. Beads were collected by centrifugation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed with the supematant fraction.
380
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Koukouritaki and Lianos
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Fig 4. Dexamethasone (DEX)has no effect on paxiUin levels. HMCs were serum-starved for 24 hours and then exposed to dexamethasone, 10-7 mol/L, for 2, 15, and 30 minutes. Cell lysates were separated in a sodium dodecyl sulfate-polyacrylamide gel (12%). Proteins were transferred to a nitrocellulose membrane and immunoblotted with anti-paxillin antibody.
Immunoblotting. To resolve FAK-containing immunoprecipitates, the immunoprecipitates obtained as described above were run on 4% to 20% gradient polyacrylamide gels (100 V, constant voltage) and electrophoretically transferred to nitrocellulose paper. A 12% polyacrylamide gel was used for resolving paxillin-containing immunoprecipitates. Blots were blocked for 1 hour at room temperature with 5% non-fat dry milk in T-TBS before incubation with anti-FAK or anti-paxillin monoclonal antibody. Membranes were then probed for 1 hour at room temperature with one of the following primary antibodies: monoclonal anti-FAK antibody at 1 gg/mL diluted in T-TBS or monoclonal anti-paxillin antibody at 0.0625 ~tg/mL dilution in blocking buffer. After incubation with the primary antibody, membranes were extensively washed with several changes of T-TBS and incubated for 1 hour at room temperature with the appropriate secondary antibody. This was horseradish peroxidase-conjugated sheep anti-mouse IgG at a dilution of either 1:5000 (FAK) or 1:2500 (paxillin). Membranes were washed with several changes of T-TBS and processed for detection by enhanced chemiluminescence. In all blots, bands corresponding to the proteins of interest (paxillin or FAK) were identified by reference to protein standards (markers) run in parallel and were scanned with a UMAX supervista S-12 scanner. Three experiments were performed for each experimental condition described, and blots shown in Results represent one of the three experiments.
•
1
2
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IgG (light chain)
~
3 IP: phosphotyrosine WB: FAK
Fig 5. Dexamethasone (DEX)restores tyrosine phosphorylation of FAK after disruption of HMC cytoskeleton by CB. HMCs were serum-starved for 24 hours and then treated with CB (1.2 mmol/L) for 2 hours. Subsequently, dexamethasone was introduced at final concentration of 10-7 mol/L for 15 minutes. Controls were HMCs incubated with the CB vehicle (absolute ethanol) for 2 hours. Cell lysates were immunoprecipitated with anti-phosphotyrosine antibody, and after separation of precipitates on a sodium dodecyl sulfate-polyacrylamide gradient (4% to 20%) gel, proteins were transferred to a nitrocellulose membrane and immunoblotted with anti-FAK antibody.
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Fig 6.
RESULTS
CB has no effect on HMC FAK levels. HMCs were serumstarved for 24 hours and then treated with CB, 1.2 retool/L, for 2 hours. Dexamethasone, 10-7 moFL, was then introduced for 15 minutes. Cell lysates were immunoprecipitated with anti-FAK antibody, and after separation on a sodium dodecyl sulfate-polyacrylamide gradient (4% to 20%) gel, proteins were transferred to a nitrocellulose membrane and immunoblotted with anti-FAK antibody.
In Fig 1, changes in the tyrosine phosphorylation of F A K after 2-, 15-, and 30-minute incubations of HMCs with dexamethasone (10 -7 mol/L) are shown. Increased tyrosine phosphorylation was detectable after a 15minute incubation period. This increase was transient (not detectable at 30 minutes). Dexamethasone had no effect on F A K levels (Fig 2). Changes in the tyrosine phosphorylation of paxillin
after 2-, 15-, and 30-minute incubations of HMCs with dexamethasone are shown in Fig 3. An early transient increase in tyrosine phosphorylation of paxillin was detectable after 2- and 15-minute incubation periods. Dexamethasone had no effect on paxillin levels (Fig 4). Fig 5 shows the effect of dexamethasone on F A K tyrosine phosphorylation in the presence and absence
J Lab Clin Med Volume 133, Number 4
Koukouritaki and Lianos
38]
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Dexamethasone restores tyrosine phosphorylation of paxillin after disruption on HMC cytoskeleton by CB. The experiment was performed as described in the legend of Fig 5. Cell lysates were immunoprecipitated with anti-phosphotyrosine antibody, and after separation of precipitates on sodium dodecyl sulfate-polyacrylamide gradient (4% to 20%) gel, proteins were transferred to a nitrocellulose membrane and immunoblotted with anti-paxillin antibody.
of the cytoskeletal assembly disrupter CB. In cells incubated with CB for 2 hours there was a decrease in FAK tyrosine phosphorylation levels (Fig 5, compare lane 2 with lane 1). The addition of dexamethasone (10 -7 tool/L) into cells that had been pretreated with CB for 2 hours restored FAK tyrosine phosphorylation to control levels (Fig 5, compare lane 3 with lane 1). The 2hour incubation of HMCs with CB had no effect on FAK levels (Fig 6). Fig 7 shows the effect of dexamethasone on paxillin tyrosine phosphorylation in the presence and absence of CB. In cells incubated with CB for 2 hours, there was a decrease in paxillin tyrosine phosphorylation levels (Fig 7, compare lane 2 with lane 1). The addition of dexamethasone (10-7 mol/L) into cells that had been pretreated with CB for 2 hours restored paxillin tyrosine phosphorylation to control levels (Fig 7, compare lane 3 with lane 1). CB had no effect on paxillin levels (Fig 8). Fig 9 is a densitometric expression of changes in FAK and paxillin phosphorylation in response to dexamethasone (Fig 9, A and B) and to cytochalasin B in the absence and presence of dexamethasone (Fig 9, C and D). DISCUSSION
Our observations demonstrate that dexamethasone stimulates tyrosine phosphorylation of the cytoskeletal proteins FAK and paxillin in HMCs. Moreover, it
1
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Fig 8.
CB has no effect on HMC paxillin levels. HMCs were serumstarved for 24 hours and then treated with CB, 1.2 mmol/L, for 2 hours. Dexamethasone was then introduced at a final concentration of 10-7 mol/L for 15 minutes. Cell lysates were separated in a sodium dodecyl sulfate-polyacrylamide gel (12%). Proteins were transferred to a nitrocellulose membrane and immunoblotted with antipaxillin antibody.
restores tyrosine phosphorylation of these proteins after the disruption of the cytoskeletal assembly by CB. These observations strongly support a role for glucocorticoids in stabilizing the mesangial cell cytoskeleton. Tyrosine phosphorylation is essential for cytoskeleton microfilament assembly. 6 Tyrosine phosphorylation of specific cytoskeletal proteins is also a mechanism underlying the assembly of focal adhesions between cells and the ECM. 7 Focal adhesions are important structural links between the cell cytoskeleton and the ECM. Proteins that become phosphorylated on tyrosine in association with microfilament assembly and the formation of focal adhesion include FAK and paxillin, s Increases in FAK and paxillin tyrosine phosphorylation are accompanied by a profound alteration in the organization of the actin cytoskeleton and in the assembly of focal adhesions, the distinct areas of plasma membrane where FAK and paxillin are localized. 9 FAK and paxillin are frequently found to co-localize at the termini of F-actin fibers in focal adhesions. 1° FAK is composed of a highly conserved tyrosine kinase domain flanked by amino- and carboxyl- terminal non-catalytic domains. The carboxylterminal domain mediates the localization of FAK to focal adhesions and the binding of FAK to paxillin. 11 The phosphorylation of FAK on tyrosine residues occurs in response to the engagement of the integrin receptor with ECM proteins 12 as well as in response to extracellular signals such as hormones and growth factors. 13 FAK, in turn, phosphorylates paxillin. 14 Whether the glucocorticoid-induced tyrosine phosphorylation of FAK and paxillin as shown in the pre-
382
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Fig 9. Quantitative expression (densitometry units) of data presented in Figs 1 through 8. Each bar represents the ratio of phosphotyrosine band density over density of the band that reflects the level of protein (Western blot) whose tyrosine phosphorylation was assessed. Because protein (FAK or paxillin) levels did not change under various experimental conditions (Figs 2, 4, 6, and 8), this method of quantitative expression is appropriate. Each bar is the mean of densitometry values stained from these independent experiments. A, Time course of FAK phosphorylation in response to dexamethasone. B, Time course of paxillin phosphorylation in response to dexamethasone. C, Effect of CB on FAK phosphorylation and reversal of this effect by dexamethasone. D, Effect of CB on paxillin phosphorylation mad reversal of this effect by dexamethasone.
sent study promotes the formation of focal adhesions is unknown. Focal adhesions have been difficult to demonstrate in vivo. 15 Nevertheless, they have been localized at myotendinous junctions 16 and in developing blood vessels, including the glomerulus, where they are primarily found in the capillary loops and in the mesangial stalk. 17 It has been argued that interactions of mesangial cells with endothelial cells or with the glomerular basement membrane, either directly or via the ECM, resemble interactions within the myotendinous junction and other similar structures.~S It becomes possible, therefore, to envision the assembly of focal adhesions at sites of mesangial cell contact with
endothelial cells or with ECM. If the steroid treatment of various forms of immune-mediated glomerulopathies promotes the formation of focal adhesions at sites of mesangial cell contact with endothelial cells or the ECM, a stabilizing effect of steroids on the glomerular cytoskeleton and structure can be expected. This speculation, however, requires evidence. Finally, the dexamethasone-induced increase in tyrosine phosphorylation of FAK and paxillin observed in the present studies may constitute a part of wider signaling events. Recent work has shown an association of FAK with the cytosolic protein tyrosine kinases pp60src and pp59fyn, ]9 with phosphatidylinositol 3-
J Lab Clin Med Volume 133, Number 4
kinase, 2° and with the adapter protein GRB. 21 These associations point to important signaling roles for F A K and identify it as a step in steroid-induced signal transducfion in glomerular mesangial cells.
Koukouritaki and Llanos
11. 12.
REFERENCES
1. Boumpas DT, Chronsos JP, Wilder RL, Cupps TR, Balow JE. Glucocorficoid therapy for immune-mediated diseases: basic and clinicalcorrelates. Ann Intern Med 1993;119:11982008. 2. Korkouritaki SB, Margioris AN, Gravanis A, Hartig R, Stournaras C. Dexamethasone induces rapid actin assembly in human edometfial cells without affecting its synthesis. J Cell Biochem 1997;65:492-500. 3. Wehling M. Nongenomic actions of steroid hormones. Trends in Endocrinology and Metabolism 1994;5:347-53. 4. Sraer J-D, Delarue F, Hagege J, Feuntenn J, Pinet F, Nguyen G, et al. Stable cell lines of T-SV40 immortalized human glomerular mesangial cells. Kidney Int 1996;49:267-70. 5. McAllen-Fletcher S, Pollard TD. Mechanism of action of cytochalasin B on acfin. Cell 1980;20:329-41. 6. Melamed I, Downey GP, Roifman CM. Tyrosine phosphorylation is essential for microfilament assembly in B-lymphocytes. Biochem Biophys Res Commun 199] ;176:14249. 7. Chrzanowska-Wodnicka M, Burridge K. Tyrosine phosphorylation is involved in reorganization of the actin cytoskeleton in response to serum or LPA stimulation. J Cell Sci 1994;107:3643-54. 8. Burridge K, Turner CE, Romer LH. Tyrosine phosphorylation of paxillin and pp125FAKaccompanies cell adhesion to extracellular matrix: a role in cytoskeletal assembly. J Cell Biol 1992;119:893-903. 9. Seufferlein T, Rozengurt E. Sphingosylphosphorylcholine rapidly induces tyrosine phosphorylation of p125 FAKand paxillin, rearrangement of the acfin cytoskeleton and focal contact assembly. J Biol Chem 1995;270:24343-51. 10. Turner CE, Glenney JR, Burridge K. Paxillin: a new vin-
13.
14.
15.
16.
17.
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20.
21.
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culin-binding protein present in focal adhesions. J Cell Biol 1990;111:1059-68. Schaller MD, Parsons JT. Focal adhesion kinase and associated proteins. Curr Opin Cell Biol 1994;6:705-10. Lipfert L, Haimovich B, Schaller MD, Cobb BS, Parsons JT, Brugge JS. Integrin-dependent phosphorylation and activation of the protein tyrosine kinase pp125 FAK in platelets. J Cell Biol 1992;119:905-12. Rozengurt E. Convergent signaling in the action of integrins, neuropeptides, growth factors and oncogenes. Cancer Surveys 1995;24:81-96. Bellis SL, Miller JT, Turner CE. Characterization of tyrosine phosphorylation of paxillin in vitro by focal adhesion kinase. J Biol Chem 1995;270:17437-41. Sastry SK, Horwitz AF. Integrin cytoplasmic domains: mediators of cytoskeletal linkages and extra- and intracellular initiated transmembrane signaling. Curr Opin Cell Biol 1993;5:819-31. Baker LP, Daggett DF, Peng HB. Concentration of pp125 focal adhesion kinase (FAK) at the myotendinous junction. J Cell Sci 1994;107:1485-97. Polte TR, Naftilan AJ, Hanks SK. Focal adhesion kinase is abundant in developing blood vessels and elevation of its phosphotyrosine content in vascular smooth muscle cells in a rapid response to angiotensin II. J Cell Biochem 1994;55:106-19. Sakal T, Kriz W. The structural relationship between mesangial cells and basement membrane of the renal glomerulus. Anat Embryol 1987;176:378-86. Cobb BS, Schaller MD, Leu Th, Parsons JT. Stable association of pp60src and pp59fyn with the focal adhesionassociated protein tyrosine kinase, pp125FAK. Mol Cell Biol 1994;14:147-55. Chen HC, Guan IL. Association of focal adhesions kinase with its potential substrate phospbatidylinositol 3-kinase. Proc Natl Acad Sci USA 1994;91:10148-52. Schlaepfer DD, Hanks SK, Hunter T, Van der Greer P. Integrin-mediated signal transduction linked to Ras pathway by GRB2 binding to focal adhesion kinase. Nature 1994;372:786-91.
Abbreviations:CB = c y t o c h a l a s i n B; ECM = extracellular matrix; FAK = f o c a l a d h e s i o n kinase; HMC = h u m a n m e s a n g i a l cell; IgG = i m m u n o g l o b u l i n G; T-TBS = Tris-buffered saline solution (10 m m o l / L Tris [pH 7,5], 100 m m o l / L NaCI, 0.1% Tween-20)
G
lucocorticoids have long been and continue rto be used extensively in the treatment of various immune-mediated nephropathies involving glomeruli (glomerulonephritides) and in preventing or reversing kidney transplant rejection. Their beneficial effects have generally been attributed to the inhibition of the synthesis or release of mediators of inflammation and to the suppression of the immunocompetence of lymphocytes and macrophages. 1 A less well-known mechanism that may underlie the cytoprotective action of glucocorticoids is their stabilizing effect on the cell cytoskeleton. In this regard, glucocorticoids have been shown to stabilize the organization of actin microfilamerits, 2 an effect that can occur rapidly without the involvement of genomic pathways--that is, the regulation of genes encoding actin or other cytoskeletal pro-
From the CardiovascularResearchCenter, Nephrology,MedicalCollege of Wisconsin. Submitted for publicationJune 15, 1998; revision submitted October 8, 1998; acceptedDecember4, 1998. Reprint requests: Elias A. Lianos, MD, 9200 W Wisconsin Ave,Milwaukee, WI 53226. Copyright © 1999 by Mosby, Inc. 0022-2143/99 $8.00 + 0 511196326
378
teins. 3 Whether glucocorticoids have stabilizing effects on the cytoskeleton of intrinsic glomerular cells is unknown. In this study we explored the effect of dexamethasone on the levels of expression and the extent of tyrosine phosphorylation of two cytoskeleton-associated proteins that promote the assembly of actin microfilaments: FAK and paxillin. Our observations demonstrate that in HMCs, dexamethasone stimulates the tyrosine phosphorylation of FAK and paxillin. Moreover, it restores the tyrosine phosphorylation of these proteins after the disruption of the cytoskeletal assembly.
METHODS Reagents. Dexamethasone, cytochalasin B, and protein G Sepharose beads for immunoprecipitation experiments were obtained from Sigma Chemical Co, St Louis, MO. Monoclonal antibody against FAK or phosphotyrosine was from Upstate Biotechnology, Lake Placid, NY. Antibody against paxillin was from Transduction Laboratories, Santa Cruz, NM. The enhanced chemiluminescence Western blotting kit and horseradish peroxidase-conjugated sheep anti-mouse IgG were purchased from Amersham, Life Science Inc, Arlington Heights, IL. All other chemicals were obtained from the usual commercial sources at the purest grade available. HMC culture. A stable line of T-SV40 immortalized HMCs originating from the laboratory of Dr. J-D Sraer, Hospital
J Lab Clin Med Volume 133, Number 4
Koukouritaki and Lianos
EO
[o DEX 2'
15'
DEX
p
I
o' -
o
30' * - - FAK 125 KD
"O1 O
3
~ 1
4
IP: phosphotyrosine WB: FAK
Fig 1. Dexamethasone (DEX), 10-7 mol/L, rapidly stimulates tyrosine phosphorylation of FAK. HMCs were serum-starved for 24 hours and then exposed to dexamethasone for 2, 15, and 30 minutes. Cell lysates were immunoprecipitated with anti-phosphotyrosine antibody, and after separation of precipitates on a sodium dodecyl sulfate-polyacrylamide gradient (4% to 20%) gel, proteins were transferred to a nitrocellulose membrane and immunoblotted with anti-FAK antibody.
2 =o o
-. "-
2
~"~--"',,-3 4
Paxillin 6 8 KD IgG (heavy chain)
* - - IgG (light chain)
IP: phosphotyrosine WB: paxillin Fig 3. Dexamethasone rapidly stimulates tyrosine phosphorylation of paxillin. HMCs were serum-starved for 24 hours and then exposed to dexamethasone, 10-7 mol/L, for 2, 15, and 30 minutes. Cell lysates were immunoprecipitated with anti-phosphotyrosine antibody, and after separation of precipitates on a sodium dodecyl sulfate-polyacrylamide gradient (4% to 20%) gel, proteins were transferred to a nitrocellulose membrane and immunoblotted with anti-paxillin antibody.
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m
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1
2' ~
1
30'
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15'
2'
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1
379
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2
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3
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4 WB: FAK
Fig 2. Dexamethasone (DEX), 10-7 mol/L, has no effect on FAK levels. HMCs were serum-starved for 24 hours and then exposed to dexamethasone for 2, 15, and 30 minutes. Cell lysates were immunoprecipitated with anti-FAK antibody, and after separation of precipitates on a sodium dodecyl sulfate-polyacrylamide gradient (4% to 20%) gel, proteins were transferred to a nitrocellulose membrane and immunoblotted with anti-FAK antibody.
Tenon, Paris, France, 4 were used. Cultures were performed in a 5% CO2-95% air atmosphere at 37°C. Ceils were maintained in RPMI medium 1640 supplemented with 10% fetal calf serum, 1% L-glutamine, and 1% antibiotic/antimycotic solution (final concentrations, 100 U/ml penicillin and 100 mg/ml stretromycin). At near confluence, cells were washed twice and then serum-deprived for 24 hours before the experiment. For immunoprecipitation and Western blot experiments, cells were washed twice with cold phosphate-buffered saline solution and removed from plates with scrapers. Use of dexamethasone and experimental design. Dexamethasone was dissolved in absolute ethanol and introduced in cultured HMCs at 10-7 mol/L for defined time periods (2, 15, and 30 minutes). This concentration was chosen as one that is frequently achieved in the plasma of human patients undergoing steroid treatment. In control incubation experi-
ments, the dexamethasone vehicle (ethanol) was introduced for identical time periods. To explore whether an intact cytoskeleton is required for the tyrosine phosphorylation of FAK and paxillin by dexamethasone, cells were pretreated with CB, which specifically depolymerizes F-actin to Gactin. 5 In these experiments, CB was dissolved in absolute ethanol and introduced in cultured HMCs to a final concentration of 1.2 mmol/L for 2 hours. In control incubation experiments, the CB vehicle (absolute ethanol) was introduced for the same time period. Immunoprecipitation. Cells were lysed on ice with cold lysis buffer consisting of 50 mmol/L N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, pH 7.5; 150 mmol/L MgC12;
1 mmol/L ethyleneglycol-bis-(~-aminoethylether)-N,N,N',N'tetraacetic acid; 10% glycerol; 1% Triton X-100; I g g / m L aprotinin; 1 ~tg/mL leupeptin; 1 mmol/L phenyhnethylsulfonyl fluoride; and 20 gmol/L sodium orthovanadate. Cell lysates were scraped off the plates and allowed to lyse for an additional 30 minutes on an orbital shaker at 4°C. Samples were subsequently centrifuged at 14,000 g at 4°C, and supernatants were transferred to new centrifuge tubes. Protein concentrations were determined by using a calorimetric assay (Biorad Protein Assay). Cell lysates containing equal amounts of protein were incubated either with anti-FAK monoclonal IgG1 or with an anti-phosphotyrosine monoclonal IgG2bk at 4°C overnight. Antigen-antibody complexes formed were precipitated by agitation for 2 hours at 4°C with washed protein G Sepharose bead slurry. Beads were collected by pulse centrifugation (14,000 g, 5 seconds) and washed three times with lysis buffer. Immunoprecipitates were solubilized by dissolving in gel sample buffer and boiled for 5 minutes. Beads were collected by centrifugation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed with the supematant fraction.
380
J Lab Clin Med April 1999
Koukouritaki and Lianos
DEX
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Fig 4. Dexamethasone (DEX)has no effect on paxiUin levels. HMCs were serum-starved for 24 hours and then exposed to dexamethasone, 10-7 mol/L, for 2, 15, and 30 minutes. Cell lysates were separated in a sodium dodecyl sulfate-polyacrylamide gel (12%). Proteins were transferred to a nitrocellulose membrane and immunoblotted with anti-paxillin antibody.
Immunoblotting. To resolve FAK-containing immunoprecipitates, the immunoprecipitates obtained as described above were run on 4% to 20% gradient polyacrylamide gels (100 V, constant voltage) and electrophoretically transferred to nitrocellulose paper. A 12% polyacrylamide gel was used for resolving paxillin-containing immunoprecipitates. Blots were blocked for 1 hour at room temperature with 5% non-fat dry milk in T-TBS before incubation with anti-FAK or anti-paxillin monoclonal antibody. Membranes were then probed for 1 hour at room temperature with one of the following primary antibodies: monoclonal anti-FAK antibody at 1 gg/mL diluted in T-TBS or monoclonal anti-paxillin antibody at 0.0625 ~tg/mL dilution in blocking buffer. After incubation with the primary antibody, membranes were extensively washed with several changes of T-TBS and incubated for 1 hour at room temperature with the appropriate secondary antibody. This was horseradish peroxidase-conjugated sheep anti-mouse IgG at a dilution of either 1:5000 (FAK) or 1:2500 (paxillin). Membranes were washed with several changes of T-TBS and processed for detection by enhanced chemiluminescence. In all blots, bands corresponding to the proteins of interest (paxillin or FAK) were identified by reference to protein standards (markers) run in parallel and were scanned with a UMAX supervista S-12 scanner. Three experiments were performed for each experimental condition described, and blots shown in Results represent one of the three experiments.
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Fig 5. Dexamethasone (DEX)restores tyrosine phosphorylation of FAK after disruption of HMC cytoskeleton by CB. HMCs were serum-starved for 24 hours and then treated with CB (1.2 mmol/L) for 2 hours. Subsequently, dexamethasone was introduced at final concentration of 10-7 mol/L for 15 minutes. Controls were HMCs incubated with the CB vehicle (absolute ethanol) for 2 hours. Cell lysates were immunoprecipitated with anti-phosphotyrosine antibody, and after separation of precipitates on a sodium dodecyl sulfate-polyacrylamide gradient (4% to 20%) gel, proteins were transferred to a nitrocellulose membrane and immunoblotted with anti-FAK antibody.
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RESULTS
CB has no effect on HMC FAK levels. HMCs were serumstarved for 24 hours and then treated with CB, 1.2 retool/L, for 2 hours. Dexamethasone, 10-7 moFL, was then introduced for 15 minutes. Cell lysates were immunoprecipitated with anti-FAK antibody, and after separation on a sodium dodecyl sulfate-polyacrylamide gradient (4% to 20%) gel, proteins were transferred to a nitrocellulose membrane and immunoblotted with anti-FAK antibody.
In Fig 1, changes in the tyrosine phosphorylation of F A K after 2-, 15-, and 30-minute incubations of HMCs with dexamethasone (10 -7 mol/L) are shown. Increased tyrosine phosphorylation was detectable after a 15minute incubation period. This increase was transient (not detectable at 30 minutes). Dexamethasone had no effect on F A K levels (Fig 2). Changes in the tyrosine phosphorylation of paxillin
after 2-, 15-, and 30-minute incubations of HMCs with dexamethasone are shown in Fig 3. An early transient increase in tyrosine phosphorylation of paxillin was detectable after 2- and 15-minute incubation periods. Dexamethasone had no effect on paxillin levels (Fig 4). Fig 5 shows the effect of dexamethasone on F A K tyrosine phosphorylation in the presence and absence
J Lab Clin Med Volume 133, Number 4
Koukouritaki and Lianos
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Dexamethasone restores tyrosine phosphorylation of paxillin after disruption on HMC cytoskeleton by CB. The experiment was performed as described in the legend of Fig 5. Cell lysates were immunoprecipitated with anti-phosphotyrosine antibody, and after separation of precipitates on sodium dodecyl sulfate-polyacrylamide gradient (4% to 20%) gel, proteins were transferred to a nitrocellulose membrane and immunoblotted with anti-paxillin antibody.
of the cytoskeletal assembly disrupter CB. In cells incubated with CB for 2 hours there was a decrease in FAK tyrosine phosphorylation levels (Fig 5, compare lane 2 with lane 1). The addition of dexamethasone (10 -7 tool/L) into cells that had been pretreated with CB for 2 hours restored FAK tyrosine phosphorylation to control levels (Fig 5, compare lane 3 with lane 1). The 2hour incubation of HMCs with CB had no effect on FAK levels (Fig 6). Fig 7 shows the effect of dexamethasone on paxillin tyrosine phosphorylation in the presence and absence of CB. In cells incubated with CB for 2 hours, there was a decrease in paxillin tyrosine phosphorylation levels (Fig 7, compare lane 2 with lane 1). The addition of dexamethasone (10-7 mol/L) into cells that had been pretreated with CB for 2 hours restored paxillin tyrosine phosphorylation to control levels (Fig 7, compare lane 3 with lane 1). CB had no effect on paxillin levels (Fig 8). Fig 9 is a densitometric expression of changes in FAK and paxillin phosphorylation in response to dexamethasone (Fig 9, A and B) and to cytochalasin B in the absence and presence of dexamethasone (Fig 9, C and D). DISCUSSION
Our observations demonstrate that dexamethasone stimulates tyrosine phosphorylation of the cytoskeletal proteins FAK and paxillin in HMCs. Moreover, it
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Fig 8.
CB has no effect on HMC paxillin levels. HMCs were serumstarved for 24 hours and then treated with CB, 1.2 mmol/L, for 2 hours. Dexamethasone was then introduced at a final concentration of 10-7 mol/L for 15 minutes. Cell lysates were separated in a sodium dodecyl sulfate-polyacrylamide gel (12%). Proteins were transferred to a nitrocellulose membrane and immunoblotted with antipaxillin antibody.
restores tyrosine phosphorylation of these proteins after the disruption of the cytoskeletal assembly by CB. These observations strongly support a role for glucocorticoids in stabilizing the mesangial cell cytoskeleton. Tyrosine phosphorylation is essential for cytoskeleton microfilament assembly. 6 Tyrosine phosphorylation of specific cytoskeletal proteins is also a mechanism underlying the assembly of focal adhesions between cells and the ECM. 7 Focal adhesions are important structural links between the cell cytoskeleton and the ECM. Proteins that become phosphorylated on tyrosine in association with microfilament assembly and the formation of focal adhesion include FAK and paxillin, s Increases in FAK and paxillin tyrosine phosphorylation are accompanied by a profound alteration in the organization of the actin cytoskeleton and in the assembly of focal adhesions, the distinct areas of plasma membrane where FAK and paxillin are localized. 9 FAK and paxillin are frequently found to co-localize at the termini of F-actin fibers in focal adhesions. 1° FAK is composed of a highly conserved tyrosine kinase domain flanked by amino- and carboxyl- terminal non-catalytic domains. The carboxylterminal domain mediates the localization of FAK to focal adhesions and the binding of FAK to paxillin. 11 The phosphorylation of FAK on tyrosine residues occurs in response to the engagement of the integrin receptor with ECM proteins 12 as well as in response to extracellular signals such as hormones and growth factors. 13 FAK, in turn, phosphorylates paxillin. 14 Whether the glucocorticoid-induced tyrosine phosphorylation of FAK and paxillin as shown in the pre-
382
J Lab Clin M e d April 1999
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Fig 9. Quantitative expression (densitometry units) of data presented in Figs 1 through 8. Each bar represents the ratio of phosphotyrosine band density over density of the band that reflects the level of protein (Western blot) whose tyrosine phosphorylation was assessed. Because protein (FAK or paxillin) levels did not change under various experimental conditions (Figs 2, 4, 6, and 8), this method of quantitative expression is appropriate. Each bar is the mean of densitometry values stained from these independent experiments. A, Time course of FAK phosphorylation in response to dexamethasone. B, Time course of paxillin phosphorylation in response to dexamethasone. C, Effect of CB on FAK phosphorylation and reversal of this effect by dexamethasone. D, Effect of CB on paxillin phosphorylation mad reversal of this effect by dexamethasone.
sent study promotes the formation of focal adhesions is unknown. Focal adhesions have been difficult to demonstrate in vivo. 15 Nevertheless, they have been localized at myotendinous junctions 16 and in developing blood vessels, including the glomerulus, where they are primarily found in the capillary loops and in the mesangial stalk. 17 It has been argued that interactions of mesangial cells with endothelial cells or with the glomerular basement membrane, either directly or via the ECM, resemble interactions within the myotendinous junction and other similar structures.~S It becomes possible, therefore, to envision the assembly of focal adhesions at sites of mesangial cell contact with
endothelial cells or with ECM. If the steroid treatment of various forms of immune-mediated glomerulopathies promotes the formation of focal adhesions at sites of mesangial cell contact with endothelial cells or the ECM, a stabilizing effect of steroids on the glomerular cytoskeleton and structure can be expected. This speculation, however, requires evidence. Finally, the dexamethasone-induced increase in tyrosine phosphorylation of FAK and paxillin observed in the present studies may constitute a part of wider signaling events. Recent work has shown an association of FAK with the cytosolic protein tyrosine kinases pp60src and pp59fyn, ]9 with phosphatidylinositol 3-
J Lab Clin Med Volume 133, Number 4
kinase, 2° and with the adapter protein GRB. 21 These associations point to important signaling roles for F A K and identify it as a step in steroid-induced signal transducfion in glomerular mesangial cells.
Koukouritaki and Llanos
11. 12.
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