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Effect of the tyrosine kinase inhibitor, genistein, on interleukin-1 stimulated PGE2 production in mesangial cells
Vo1.173, No. 2,1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 718-724
December14,1990
EFFECT OF THE TYROSINE KINASE INHIBITOR, GENISTEIN, ON STIMULATED PGE 2 PRODUCTION IN MESANGIAL CELLS
INTERLEUKIN-1
Daniel W. Coyne and Aubrey R. Morrison Washington University School of Medicine Departments of Medicine and Pharmacology St. Louis, Missouri 63110 Received October 16, 1990
SUMMARY: Prostaglandin production and cAMP formation are two signaling pathways identified for I L - I , though neither adequately account for the multitude of effects of IL-1. To investigate the role of tyrosine phosphorylation in IL-1 signaling, we used the tyrosine kinase inhibitor, genistein. At 10-30 #g/ml, genistein blocked IL-I stimulated prostaglandin production and induction of prostaglandin endoperoxide synthase (PES) in glomerular mesangial cells maintained in 10% serum. Addition of genistein hours after IL-1 addition also halted further PGE 2 synthesis. Genistein failed to block PES activity in vitro, indicating it was not acting as a PES inhibitor. Overall these data suggest that tyrosine phosphorylation may be a required event for IL-1 stimulation of PGE 2 and PES activity, either directly as part of IL-1 signaling, or indirectly as part of a serum/PDGF competence effect on mesangial cells. ©1990 AcademicPress, Inc.
Interleukin-lfl (IL-1) is a cytokine of 17,500 Mr synthesized by a variety of cells, though in highest concentration by activated macrophages (1).
During inflammation in the glomerulus
IL-1 may be released by activated macrophages and mesangial cells.
In cultured glomerular
mesangial cells, IL-1 stimulates proteinase release (2), proliferation (3),
and prostaglandin
production (4), and thus may function as a progression factor in glomerular disease.
IL-1
stimulated prostaglandin production in mesangial ceils occurs after a 4 to 6 hour lag, and is dependent on protein synthesis (4,5). In part, the increase in prostaglandin production is due to increased phospholipase A 2 (PLA2) (5), with consequent increased arachidonate availability. An increase in prostaglandin endoperoxide synthase (PES) activity (6) occurs and contributes to increased prostaglandin production. Two main signaling pathways for IL-I have been identified, prostaglandin production and stimulation of cAMP (4,7,8).
While increases in diacylglycerol have been detected in response
to IL-1, there is not clear evidence it actually activates protein kinase C (7,9).
Certain actions
of IL-1 are clearly mediated by prostaglandin production, including fever, sodium excretion, and hypotension.
Other effects, such as collagenase induction and stimulation of endothelial
procoagulant, are not blocked by cyclooxygenase inhibition (7). The multitude of effects of IL-I are not fully explained by the present signaling pathways identified. 0006-291X/90 $1.50 Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
718
Vol. 173, No. 2, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
In search of other signaling pathways,
IL-1
has recently been shown to stimulate
phosphorylation of several proteins in mesangial cells, and other cell lines (10,11), including tyrosine phosphorylation, although the IL-1 receptor(s) is not known to possess intrinsic tyrosine kinase activity. Additionally, tyrosine phosphorylation appears to be a unifying signal among the growth factors which stimulate mesangial proliferation (PDGF, IGF-1, EGF, etc.) (12). Further, these growth factors may induce mitogenesis in mesangial cells in part by induction of PDGF
03). Genistein, an isoflavonoid, is an inhibitor of tyrosine kinases. It appears to compete for the ATP binding site of the kinases (14).
It does not inhibit all tyrosine kinases, but is known to
be active against the EGF and PDGF receptors. at least 20-100 fold less.
Its activity against protein kinases A and C is
It has the advantage of acting within minutes of addition.
Tyrphostins have been demonstrated to inhibit tyrosine kinases, but require long-term exposure to cells for optimal inhibition and are unstable (15). In order to investigate the role of tyrosine phosphorylation
in serum induction of IL-1
responsiveness and IL-1 signaling, we studied the effects of genistein on IL-1 stimulated PGE 2 production and PES activity in 10% serum. production by genistein.
We found potent inhibition of stimulated PGE 2
Genistein also blocked induction of PES activity by IL-1. IL-I
stimulation of PGE 2 synthesis was reduced by 75% when performed in 10% platelet poor serum, which has markedly less PDGF than normal bovine serum. Taken together, these data suggest than a tyrosine phosphorylation event, stimulated by IL-1 or a serum component, is crucial for IL-1 induction of PGE 2 synthesis in mesangial cells. Activation of PLA e may be dependent on tyrosine phosphorylation.
MATERIALS AND METHODS
Materials Tissue culture media and additives were obtained from the tissue culture support center at Washington University. Bovine calf serum (FCS) was purchased from Baxter; genistein from ICN, platelet-poor serum (PDS) from Collaborative Research, and recombinant murine IL-1B was the gift of Dr. Brad Rovin at Washington University. Other supplies for PGE z and prostaglandin endoperoxide synthase (PES) activity were purchased from Sigma, St. Louis, MO. Mesangial cell culture Mesangial cells were prepared from rats as described and used between passage 2-6 (16). Cultures were maintained in RPMI-1640, 10% FCS, insulin, 2 mM L-glutamine, penicillin, streptomycin, fungizone, and 15 mM HEPES. PDS was substituted for FCS where indicated. PGE 2 and PES activity determinations PGE 2 in the media was determined by radioimmunoassay as previously described (17). PES activity was determined by washing the monolayer twice with buffered saline solution, then overlaying with RPMI containing 30 #M arachidonate and 1 mg/ml of bovine serum albumin for 719
Vol. 173, No. 2, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
fifteen minutes. An aliquot was then removed for PGE z determination. PGE 2 and PES activity were corrected for protein, as determined by the method of Bradford (18).
Determination of in vitro PES activity Rabbit medullary microsomes were used as a source of PES. Microsomes were used at a concentration of 100 /zg of protein, and mixed with 10 # g / m l of arachidonate in the presence of 0, I0, 20, and 50 /~g/ml of genistein. Samples were incubated at 37 C for 15 min, in 200 #1 volume and PGE z determined by G C / M S using Zd4 PGE 2 as internal standard.
RESULTS Production of PGE z by IL-1 and inhibition by genistein Because of the uncertainty in the signaling pathways employed by IL-1, and the reports of stimulation of tyrosine phosphorylation (10,11), we examined the effect of the tyrosine kinase
inhibitor, genistein, on IL-I stimulated PGE 2 production.
Rat mesangial cells were grown to
near confluence, washed and fresh media containing 10% FCS added.
Genistein, at the indicated
concentration, or 0.1% DMSO, was added 15-30 min prior to IL-1 (10 U / m l ) addition.
Figure I
demonstrates the dose-dependent inhibition of I L - l - s t i m u l a t e d PGE z production, determined 24 hrs after IL-1 addition. control cells.
Morphologically, genistein-treated cells were indistinguishable from
The large standard errors in the I L - I stimulated data (solid circles) reflects the
inter-experimental variability in maximal I L - l - s t i m u l a t e d PGE z.
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Fi$. I. Inhibition of IL-I stimulated PGE 2 production by genistein. Mesangial cells in multiwell plates were placed in fresh medium and 10% FCS, containing genistein or 0.1% DMSO. After 15-30 min, IL-I (i0 U/ml) (-o-) or media (-o-) was added. 24 hrs after IL-I addition, medium was removed for RIA, and well protein determined. Data are the average of 4 experiments in triplicate or duplicate ± SEM. * is p<.05 versus IL-I stimulated. Fig. 2. Effect of genistein on IL-I induction of were treated exactly as in Fig. i. After 24 hrs, (-0-) ± genistein, were washed with warm HBSS and are the average of three experiments performed in
720
PES activity. Mesangial cells IL-I (-°-) or control wells PES activity determined. Data duplicate or triplicate ± SEM.
Vol. 173, No. 2, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Inhibition of IL-l-stimulated PES activity induction IL-1 has been demonstrated in other cell types (19, 20) to increase PES activity and mass. We determined PES activity after 24 hrs of IL-1 treatment, and noted an approximate doubling over control values (Figure 2).
As with PGE 2 production, genistein inhibited PES activity
induction in a dose dependent manner.
Genistein also reduced control (IL-I untreated) PES
activity, though to a much lesser degree. Inhibition of IL-1 stimulated PES activity by genistein parallelled the reduction in PGE2, although substantial activity (~200 pg/#g protein) remained in the mesangial cell monolayer even at 30 #g/ml of genistein. Effect of genistein added after IL-1 addition To determine the effects of genistein after initial IL-1 cell stimulation, 10 U / m l of IL-1 was added to mesangial cell monolayers and genistein (20 #g/ml) added at various time points prior to and afterward.
Figure 3 demonstrates that genistein interrupts IL-l-stimulated PGE z
synthesis even when added 12 hrs after IL-1.
Whether added prior to or after IL-I, genistein
appears to act in a rapid and sustained manner.
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Fig. 3. Effect of genistein added after IL-I addition. Mesangial ceiis in fresh media containing 10~o FCS, were incubated in the presence (-.-) or absence (-o-) of IL-I (i0 U/ml) beginning at time 0. To IL-I containing wells, genistein (20 pg/ml) was added at -i hr (-[]-), + i hr (-m-) , and +12 hr (-.-). PGE 2 was determined at 24 hr after IL-I addition. Data are the average of two experiments in triplicate. Fig. 4. Effect of PDS and serum-free conditions on IL-l-stimulated PGE 2 production. Mesangial cells maintained in 10% FCS, 10% PDS, or RPMI alone for 48 hrs, were stimulated in fresh media with IL-I (i0 U/ml) for 24 hrs, then PGE 2 determined and corrected for well protein. Data are the average of two experiments in triplicate.
721
Vol. 173, No. 2, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Effect of platelet-poor serum and serum free conditions on PGE 2 production
Growth
in
serum-containing
medium
responsiveness to various agonists (21, 22).
appears
to
be
important
for
mesangial
cell
Because PDGF is a major growth factor of serum
(23), signals in part through tyrosine phosphorylation (24), and plays an important role in mesangial cell proliferation (25), we examined whether PDS (which has little PDGF activity), or serum-free conditions also blocked or reduced IL-I responsiveness. Figure 4 demonstrates the effect of 10% PDS and serum-free conditions on PGE 2 production compared to control cells in 10% FCS.
Cells were maintained in their respective media for 48
hrs prior to and during IL-I stimulation. PGE 2 production was corrected for well protein. PDS reduced IL-I responsiveness by 75%, while PGE 2 production was virtually absent in the serumfree wells. Effect of genistein on in vitro P G E 2 production by PES
Genistein has been reported to stimulate arachidonate-driven 02 consumption by PES in vitro (26, 27).
One explanation for the effects of genistein observed is that it inhibits PES
activity directly, by becoming a preferential substrate, and thus maintaining 02 consumption while blocking PGE 2 synthesis.
In order to exclude this possibility, we examined in vitro the
PGE 2 synthesis by PES in the presence of various concentrations of genistein.
As shown in
Table I, genistein slightly stimulated PGE 2 production at 10 #g/ml, and reduced PES activity by less than 10% even at 50 #g/ml.
DISCUSSION IL-1B is a cytokine with multiple biologic actions, and unclear signalling pathways (7). IL-I has been reported to induce tyrosine phosphorylation in the plasma membranes of mesangial cells and a tumor cell line (10, 11). However, stimulation of tyrosine phosphorylation by IL-1 has not been demonstrated in intact cells.
T a b l e I. M e d u l l a r y r e n a l m i c r o s o m e s w e r e incubated with arachidonate and g e n i s t e i n for 15 m i n u t e s at 37°C, and t h e n PGE 2 d e t e r m i n e d . C o n t r o l = 216 n g / 1 0 0 ~ g p r o t e i n / 1 5 min. Genistein (~g/ml)
% control PES activity
0 i0
i00 112
2O
95
50
91
722
Vol. 173, No. 2, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
In mesangial cells multiple growth factors, which are known phosphorylation, induce proliferation (25). the major growth factor in serum (24).
to stimulate tyrosine
The most potent of these growth factors is PDGF,
(23), and whose receptor possesses tyrosine kinase activity
PDGF has been demonstrated to increase IL-1 receptor number and gene expression within
3 hrs in BALB/c/3T3 fibroblasts (28, 29).
Genistein, at a concentration of 27 #g/ml, has been
demonstrated to block PDGF-induced tyrosine phosphorylation and phospholipase C activation
(30). Therefore, blockade of IL-1 -stimulated PGE z production by genistein could reflect interruption of the competence effect of serum/PDGF signaling, rather than direct IL-1 signaling.
This interpretation is consistent with the reduction observed in PGE z production in
platelet-poor serum, and the lack of PGE z production under serum-free conditions. Indirectly against this mechanism of action for genistein is the ability of genistein to block PGE z production hours after IL-1 addition. While removal of IL-1 from the medium does lead to a return to control PGE 2 production, this process is slow, with substantial PGE z produced in the 24 hrs after IL-1 removal.
Acute interruption of signaling by genistein could conceivably shut
off PGE z synthesis faster. Alternatively, genistein could block PES activity directly. However, in vitro, genistein has been demonstrated to actually stimulate arachidonate-driven O z consumption at a concentration of 16 #g/ml (26, 27).
Further, as demonstrated in Table I, genistein showed no in vitro
inhibition of PGE 2 production at the concentrations employed in these studies.
Additionally,
induction of PES activity by IL-1 (as determined by arachidonate saturation after genistein washout) should have occurred, and this was not observed. Overall these data indicate that genistein, employed at concentrations capable'of blocking tyrosine phosphorylation, is able to block IL-1-stimulated PGE 2 production and induction of PES activity in serum-fed mesangial cells. These data suggest that tyrosine phosphorylation may be a required event for IL-1 stimulation of PGE a and PES activity, either directly as part of IL-1 signaling, or indirectly as part of a serum/PDGF competence effect on mesangial cells.
Acknowledgments:
This research was supported by United States Public Health Service Award
DK 09926 and DK 3811.
Dr. Morrison is the recipient of a Burroughs Wellcome Clinical
Pharmacology Scholar Award. 723
Vol. 173, No. 2, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES 1. 2.
Le, J. and Vilcek, J. (1987) Lab. Invest. 56, 234-248. Martin, J., Lovett, D.H., Gemsa, D., Sterzel, R.B., and Davies, M. (1986) J. Immunol. 137, 525-529. 3. Lovett, D.H., Ryan, J.L., and Sterzel, R.B. (1983) J. Immunol. 131, 2830-2836. 4. Lovett, D.H., Resch, K., and Gemsa, D. (1987) Am. J. Pathol. 129, 543-551. 5. Pfeilschifter, J., Pignat, W., Vosbeck, F., and Marki, F. (1989) Biochem. Biophys. Res. Comm. 159, 385-394. 6. Topley, N., Floege, J., Wessel, K., Hass, R., Radeke, H.H., Kaever, V., and Resch, K. (1989) J. Immunol. 143, 1989-1995. 7. Dinarello, C.A., and Savage, N. (1989) Crit. Rev. Immunol. 9, 1-20. 8. Flad, H.D., Kirchner, H., and Resch, K. (1989) Lymphokine Res. 8, 227-233. 9. Kester, M., Simonson, M.S., Mene, P., and Sedor, J.R. (1989) J. Clin. Invest. 83, 718-723. 10. Lovett, D.H., Martin, M., Bursten, S., Szamel, M., Gemsa, D., and Resch, K. (1988) Kid. Int. 34, 26-35. 11. Martin, M., Lovett, D.H., Szamel, M., and Resch, K. (1989) Eur. J. Biochem. 180, 343-350. 12. Yardin, Y., and Ullrich, A. (1988) Annu. Rev. Biochem. 57, 443-478. 13. Silver, B.J., Jaffer, F.E., and Abboud, H.E. (1989) Proc. Natl. Acad. Sci. (USA) 86, 1056-1060. 14. Akiyama, T., Ishida, J., Nakagawa, S., Ogawara, H., Watanabe, S., Itok, N., Shibuya, M., and Fukami, Y. (1987) J. Biol. Chem. 262, 5592-5595. 15. Lyall, R.M., Zilberstein, A., Gazit, A., Gilow, C., Levitzki, and Schlessinger, J. (1989) J. Biol. Chem. 264, 14503-14509. 16. Lovett, D.H., Sterzel, R.B., Kashgarian, M., and Ryan, J. (1983) Kid. Int. 23, 342-349. 17. Lefkowith, J.B., Flippo, V., Sprechen, H., and Needleman, P. (1985) J. Biol. Chem. 260, 15736-15744. 18. Bradford, M. (1976) Anal. Biochem. 72:248-254. 19. Raz, A., Wyche, A., Siegel, N., and Needleman, P. (1988) J. Biol. Chem. 263, 3022-3028. 20. O'Neill, L.A., Barrett, M.L., and Lewis, G.P. (1981) FEBS Lett. 212, 35-39. 21. Simonson, M.S., Culp, L.A., and Dunn, M.J. (1990) Exp. Cell Res. 184, 484-488. 22. Baud, L., Perez, J., Friedlander, G., and Ardaillou, R. (1988) FEBS Lett. 239, 50-54. 23. Westermark, B., and Heldin, C.H. (1986) Acta Med. Scand. Suppl. 715, 19-23. 24. Williams, L.T. (1989) Science 243, 156,1-1570. 25. Schultz, P.J., Corleto, P.E., Silver, B.J., and Abboud, H.E. (1988) Amer. J. Physiol. 255, F674-F684. 26. Degen, G.H. (1986) Proceeding of Eur. Food Tox. II, pp. 85-91, University of Zurich, Zurich, Switzerland. 27. Degen, G.H. (1990) J. Steroid Biochem. 35, 473-479. 28. Chiou, W.J., Bonin, P.D., Harris, P.K., Carter, D.B., and Singh, J.P. (1989) J. Biol. Chem. 264, 21442-21445. 29. Bonin, P.D., and Singh, J.P. (1990) J. Biol. Chem. 263, 11053-11055. 30. Hill, T.D., Dean, N.M., Mordan, L.J., Lau, A.F., Kanemitsu, M.Y., and Boynton, A.L. (1990) Science 248, 1660-1663.
724
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 718-724
December14,1990
EFFECT OF THE TYROSINE KINASE INHIBITOR, GENISTEIN, ON STIMULATED PGE 2 PRODUCTION IN MESANGIAL CELLS
INTERLEUKIN-1
Daniel W. Coyne and Aubrey R. Morrison Washington University School of Medicine Departments of Medicine and Pharmacology St. Louis, Missouri 63110 Received October 16, 1990
SUMMARY: Prostaglandin production and cAMP formation are two signaling pathways identified for I L - I , though neither adequately account for the multitude of effects of IL-1. To investigate the role of tyrosine phosphorylation in IL-1 signaling, we used the tyrosine kinase inhibitor, genistein. At 10-30 #g/ml, genistein blocked IL-I stimulated prostaglandin production and induction of prostaglandin endoperoxide synthase (PES) in glomerular mesangial cells maintained in 10% serum. Addition of genistein hours after IL-1 addition also halted further PGE 2 synthesis. Genistein failed to block PES activity in vitro, indicating it was not acting as a PES inhibitor. Overall these data suggest that tyrosine phosphorylation may be a required event for IL-1 stimulation of PGE 2 and PES activity, either directly as part of IL-1 signaling, or indirectly as part of a serum/PDGF competence effect on mesangial cells. ©1990 AcademicPress, Inc.
Interleukin-lfl (IL-1) is a cytokine of 17,500 Mr synthesized by a variety of cells, though in highest concentration by activated macrophages (1).
During inflammation in the glomerulus
IL-1 may be released by activated macrophages and mesangial cells.
In cultured glomerular
mesangial cells, IL-1 stimulates proteinase release (2), proliferation (3),
and prostaglandin
production (4), and thus may function as a progression factor in glomerular disease.
IL-1
stimulated prostaglandin production in mesangial ceils occurs after a 4 to 6 hour lag, and is dependent on protein synthesis (4,5). In part, the increase in prostaglandin production is due to increased phospholipase A 2 (PLA2) (5), with consequent increased arachidonate availability. An increase in prostaglandin endoperoxide synthase (PES) activity (6) occurs and contributes to increased prostaglandin production. Two main signaling pathways for IL-I have been identified, prostaglandin production and stimulation of cAMP (4,7,8).
While increases in diacylglycerol have been detected in response
to IL-1, there is not clear evidence it actually activates protein kinase C (7,9).
Certain actions
of IL-1 are clearly mediated by prostaglandin production, including fever, sodium excretion, and hypotension.
Other effects, such as collagenase induction and stimulation of endothelial
procoagulant, are not blocked by cyclooxygenase inhibition (7). The multitude of effects of IL-I are not fully explained by the present signaling pathways identified. 0006-291X/90 $1.50 Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
718
Vol. 173, No. 2, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
In search of other signaling pathways,
IL-1
has recently been shown to stimulate
phosphorylation of several proteins in mesangial cells, and other cell lines (10,11), including tyrosine phosphorylation, although the IL-1 receptor(s) is not known to possess intrinsic tyrosine kinase activity. Additionally, tyrosine phosphorylation appears to be a unifying signal among the growth factors which stimulate mesangial proliferation (PDGF, IGF-1, EGF, etc.) (12). Further, these growth factors may induce mitogenesis in mesangial cells in part by induction of PDGF
03). Genistein, an isoflavonoid, is an inhibitor of tyrosine kinases. It appears to compete for the ATP binding site of the kinases (14).
It does not inhibit all tyrosine kinases, but is known to
be active against the EGF and PDGF receptors. at least 20-100 fold less.
Its activity against protein kinases A and C is
It has the advantage of acting within minutes of addition.
Tyrphostins have been demonstrated to inhibit tyrosine kinases, but require long-term exposure to cells for optimal inhibition and are unstable (15). In order to investigate the role of tyrosine phosphorylation
in serum induction of IL-1
responsiveness and IL-1 signaling, we studied the effects of genistein on IL-1 stimulated PGE 2 production and PES activity in 10% serum. production by genistein.
We found potent inhibition of stimulated PGE 2
Genistein also blocked induction of PES activity by IL-1. IL-I
stimulation of PGE 2 synthesis was reduced by 75% when performed in 10% platelet poor serum, which has markedly less PDGF than normal bovine serum. Taken together, these data suggest than a tyrosine phosphorylation event, stimulated by IL-1 or a serum component, is crucial for IL-1 induction of PGE 2 synthesis in mesangial cells. Activation of PLA e may be dependent on tyrosine phosphorylation.
MATERIALS AND METHODS
Materials Tissue culture media and additives were obtained from the tissue culture support center at Washington University. Bovine calf serum (FCS) was purchased from Baxter; genistein from ICN, platelet-poor serum (PDS) from Collaborative Research, and recombinant murine IL-1B was the gift of Dr. Brad Rovin at Washington University. Other supplies for PGE z and prostaglandin endoperoxide synthase (PES) activity were purchased from Sigma, St. Louis, MO. Mesangial cell culture Mesangial cells were prepared from rats as described and used between passage 2-6 (16). Cultures were maintained in RPMI-1640, 10% FCS, insulin, 2 mM L-glutamine, penicillin, streptomycin, fungizone, and 15 mM HEPES. PDS was substituted for FCS where indicated. PGE 2 and PES activity determinations PGE 2 in the media was determined by radioimmunoassay as previously described (17). PES activity was determined by washing the monolayer twice with buffered saline solution, then overlaying with RPMI containing 30 #M arachidonate and 1 mg/ml of bovine serum albumin for 719
Vol. 173, No. 2, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
fifteen minutes. An aliquot was then removed for PGE z determination. PGE 2 and PES activity were corrected for protein, as determined by the method of Bradford (18).
Determination of in vitro PES activity Rabbit medullary microsomes were used as a source of PES. Microsomes were used at a concentration of 100 /zg of protein, and mixed with 10 # g / m l of arachidonate in the presence of 0, I0, 20, and 50 /~g/ml of genistein. Samples were incubated at 37 C for 15 min, in 200 #1 volume and PGE z determined by G C / M S using Zd4 PGE 2 as internal standard.
RESULTS Production of PGE z by IL-1 and inhibition by genistein Because of the uncertainty in the signaling pathways employed by IL-1, and the reports of stimulation of tyrosine phosphorylation (10,11), we examined the effect of the tyrosine kinase
inhibitor, genistein, on IL-I stimulated PGE 2 production.
Rat mesangial cells were grown to
near confluence, washed and fresh media containing 10% FCS added.
Genistein, at the indicated
concentration, or 0.1% DMSO, was added 15-30 min prior to IL-1 (10 U / m l ) addition.
Figure I
demonstrates the dose-dependent inhibition of I L - l - s t i m u l a t e d PGE z production, determined 24 hrs after IL-1 addition. control cells.
Morphologically, genistein-treated cells were indistinguishable from
The large standard errors in the I L - I stimulated data (solid circles) reflects the
inter-experimental variability in maximal I L - l - s t i m u l a t e d PGE z.
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Fi$. I. Inhibition of IL-I stimulated PGE 2 production by genistein. Mesangial cells in multiwell plates were placed in fresh medium and 10% FCS, containing genistein or 0.1% DMSO. After 15-30 min, IL-I (i0 U/ml) (-o-) or media (-o-) was added. 24 hrs after IL-I addition, medium was removed for RIA, and well protein determined. Data are the average of 4 experiments in triplicate or duplicate ± SEM. * is p<.05 versus IL-I stimulated. Fig. 2. Effect of genistein on IL-I induction of were treated exactly as in Fig. i. After 24 hrs, (-0-) ± genistein, were washed with warm HBSS and are the average of three experiments performed in
720
PES activity. Mesangial cells IL-I (-°-) or control wells PES activity determined. Data duplicate or triplicate ± SEM.
Vol. 173, No. 2, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Inhibition of IL-l-stimulated PES activity induction IL-1 has been demonstrated in other cell types (19, 20) to increase PES activity and mass. We determined PES activity after 24 hrs of IL-1 treatment, and noted an approximate doubling over control values (Figure 2).
As with PGE 2 production, genistein inhibited PES activity
induction in a dose dependent manner.
Genistein also reduced control (IL-I untreated) PES
activity, though to a much lesser degree. Inhibition of IL-1 stimulated PES activity by genistein parallelled the reduction in PGE2, although substantial activity (~200 pg/#g protein) remained in the mesangial cell monolayer even at 30 #g/ml of genistein. Effect of genistein added after IL-1 addition To determine the effects of genistein after initial IL-1 cell stimulation, 10 U / m l of IL-1 was added to mesangial cell monolayers and genistein (20 #g/ml) added at various time points prior to and afterward.
Figure 3 demonstrates that genistein interrupts IL-l-stimulated PGE z
synthesis even when added 12 hrs after IL-1.
Whether added prior to or after IL-I, genistein
appears to act in a rapid and sustained manner.
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~o
20
(Do
3o
Time (hrs)
.
control
!iiiiiiiiiiiiii!!!
IL-I
Fig. 3. Effect of genistein added after IL-I addition. Mesangial ceiis in fresh media containing 10~o FCS, were incubated in the presence (-.-) or absence (-o-) of IL-I (i0 U/ml) beginning at time 0. To IL-I containing wells, genistein (20 pg/ml) was added at -i hr (-[]-), + i hr (-m-) , and +12 hr (-.-). PGE 2 was determined at 24 hr after IL-I addition. Data are the average of two experiments in triplicate. Fig. 4. Effect of PDS and serum-free conditions on IL-l-stimulated PGE 2 production. Mesangial cells maintained in 10% FCS, 10% PDS, or RPMI alone for 48 hrs, were stimulated in fresh media with IL-I (i0 U/ml) for 24 hrs, then PGE 2 determined and corrected for well protein. Data are the average of two experiments in triplicate.
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Effect of platelet-poor serum and serum free conditions on PGE 2 production
Growth
in
serum-containing
medium
responsiveness to various agonists (21, 22).
appears
to
be
important
for
mesangial
cell
Because PDGF is a major growth factor of serum
(23), signals in part through tyrosine phosphorylation (24), and plays an important role in mesangial cell proliferation (25), we examined whether PDS (which has little PDGF activity), or serum-free conditions also blocked or reduced IL-I responsiveness. Figure 4 demonstrates the effect of 10% PDS and serum-free conditions on PGE 2 production compared to control cells in 10% FCS.
Cells were maintained in their respective media for 48
hrs prior to and during IL-I stimulation. PGE 2 production was corrected for well protein. PDS reduced IL-I responsiveness by 75%, while PGE 2 production was virtually absent in the serumfree wells. Effect of genistein on in vitro P G E 2 production by PES
Genistein has been reported to stimulate arachidonate-driven 02 consumption by PES in vitro (26, 27).
One explanation for the effects of genistein observed is that it inhibits PES
activity directly, by becoming a preferential substrate, and thus maintaining 02 consumption while blocking PGE 2 synthesis.
In order to exclude this possibility, we examined in vitro the
PGE 2 synthesis by PES in the presence of various concentrations of genistein.
As shown in
Table I, genistein slightly stimulated PGE 2 production at 10 #g/ml, and reduced PES activity by less than 10% even at 50 #g/ml.
DISCUSSION IL-1B is a cytokine with multiple biologic actions, and unclear signalling pathways (7). IL-I has been reported to induce tyrosine phosphorylation in the plasma membranes of mesangial cells and a tumor cell line (10, 11). However, stimulation of tyrosine phosphorylation by IL-1 has not been demonstrated in intact cells.
T a b l e I. M e d u l l a r y r e n a l m i c r o s o m e s w e r e incubated with arachidonate and g e n i s t e i n for 15 m i n u t e s at 37°C, and t h e n PGE 2 d e t e r m i n e d . C o n t r o l = 216 n g / 1 0 0 ~ g p r o t e i n / 1 5 min. Genistein (~g/ml)
% control PES activity
0 i0
i00 112
2O
95
50
91
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In mesangial cells multiple growth factors, which are known phosphorylation, induce proliferation (25). the major growth factor in serum (24).
to stimulate tyrosine
The most potent of these growth factors is PDGF,
(23), and whose receptor possesses tyrosine kinase activity
PDGF has been demonstrated to increase IL-1 receptor number and gene expression within
3 hrs in BALB/c/3T3 fibroblasts (28, 29).
Genistein, at a concentration of 27 #g/ml, has been
demonstrated to block PDGF-induced tyrosine phosphorylation and phospholipase C activation
(30). Therefore, blockade of IL-1 -stimulated PGE z production by genistein could reflect interruption of the competence effect of serum/PDGF signaling, rather than direct IL-1 signaling.
This interpretation is consistent with the reduction observed in PGE z production in
platelet-poor serum, and the lack of PGE z production under serum-free conditions. Indirectly against this mechanism of action for genistein is the ability of genistein to block PGE z production hours after IL-1 addition. While removal of IL-1 from the medium does lead to a return to control PGE 2 production, this process is slow, with substantial PGE z produced in the 24 hrs after IL-1 removal.
Acute interruption of signaling by genistein could conceivably shut
off PGE z synthesis faster. Alternatively, genistein could block PES activity directly. However, in vitro, genistein has been demonstrated to actually stimulate arachidonate-driven O z consumption at a concentration of 16 #g/ml (26, 27).
Further, as demonstrated in Table I, genistein showed no in vitro
inhibition of PGE 2 production at the concentrations employed in these studies.
Additionally,
induction of PES activity by IL-1 (as determined by arachidonate saturation after genistein washout) should have occurred, and this was not observed. Overall these data indicate that genistein, employed at concentrations capable'of blocking tyrosine phosphorylation, is able to block IL-1-stimulated PGE 2 production and induction of PES activity in serum-fed mesangial cells. These data suggest that tyrosine phosphorylation may be a required event for IL-1 stimulation of PGE a and PES activity, either directly as part of IL-1 signaling, or indirectly as part of a serum/PDGF competence effect on mesangial cells.
Acknowledgments:
This research was supported by United States Public Health Service Award
DK 09926 and DK 3811.
Dr. Morrison is the recipient of a Burroughs Wellcome Clinical
Pharmacology Scholar Award. 723
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