Osteopontin Inhibits Induction of Nitric Oxide Synthase Gene ...

Vol. 269, No. 1, Issue of January 7, pp. 711-715, 1994

Tm JOURNAL OF B I O ~ I C C AL m m y

Printed in U.S.A.

Q 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Osteopontin Inhibits Inductionof Nitric Oxide Synthase Gene Expression by Inflammatory Mediatorsin Mouse Kidney Epithelial Cells* (Received for publication, August 23,

1993, and in revised form, September 28,

1993)

Shiaw-minHwang$$, CeciliaA. Lopez$, DianeE. Hecm Carol R. Gardned, Debra L. Laskinll, Jeffrey D. Laskinn and DavidT.Denhardt$** From the Departments of $Biological Sciences and ll'harmacology and Tbxicology, Rutgers University and the Wepartment of Environment and Community Medicine, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, New Jersey 08855

We report that osteopontin (OPN), a secreted, kg-Gly- ducer its best understood effector is guanylyl cyclase, which it activates; increased intracellular cGMP in turn impacts on a Asp-containing phosphoprotein expressed at high levels (NO) synthesis in- variety of biochemical pathways, for example those involving in the kidney, suppresses nitric oxide duced by the inflammatory mediators y-interferon and cGMP-dependent protein kinases. Nitric oxide inhibits ironlipopolysaccharide in primary mouse kidney proximal containing enzymes importantin respiration andDNA synthetubule epithelial cells. Northern blot and immunofluo- sis. I t combines with superoxide to form peroxynitrite, which rescenceanalyses of induciblenitricoxidesynthase decomposes to the reactive NOz and hydroxyl radicals, and it (iNOS) expression revealed that the inflammatoryme- stimulates ADP-ribosylation of variousproteins,including diators increased iNOS mRNA and protein levels. Re- glyceraldehyde-3-phosphatedehydrogenase, with consequent combinant humanOPN (purifiedfrom both mammalian inactivation. cells and fromEscherichia coli) inhibited this response OPN (osteopontin) is an -44-kDa secreted phosphoprotein by a process that was blocked by anti-OPN antiserum that is highly negatively charged and frequently associated and by the peptide GRGDS, but not GRGES. The data of NO synthesis by OPN in these with mineralization processes (2).I t is produced by many episuggest that inhibition thelial cell types and found both in normal plasma and in a kidney cellsis mediated by an integrin, possibly the a,& integrin, which is known to be an OPN receptor. NO is variety of body secretions including urine, milk, and bile (3). believed to control blood flow through the glomerulus, Transformed cells, particularly ras-transformed cells, express to be important OPN at eievated levels (4).Via a n interaction of a n Arg-Gly-Asp regulating salt and water balance, and as a defense against tumor cells and infecting microor-(RGD) sequence in OPN with an integrin,likely the a& integanisms. The ability of OPN to inhibit the inductionof grin, OPN is able to promote cell adhesion and to activate a iNOS suggests thatOPN may be an important regulator signal transductionpathway. One consequence of OPN signalin cellular [Ca2+li levels; both a decrease ( 5 ) ing is an alteration of the NO signaling pathway and NO-mediated cytotoxic processes. and an increase (6)in osteoclasts have been observed. In the mouse kidney, high level focal expression is observed in a subset of the nephrons, mostly in the epithelialcells of the thick Nitric oxide (NO), synthesized by nitric oxide synthase ascending limb of the long loop of Henle, and in sclerosing (NOS)l from arginine andoxygen, is an important signal trans- glomeruli (7). NO, becauseit contributes toblood flow regulation by setting ducing molecule in various cell types (1).In the brain, NO the degree of relaxation of vascular smooth muscle cells, is mediates the excitatory effect of glutamate. In the vascular critical to the maintenance of normal kidney function. I t asendothelium, NO was characterized as endothelium-derived sures adequate oxygenation of the renal medulla( 8 ) and regurelaxing factor because it promoted vascular smooth muscle relaxation. In mouse macrophages, it has assumed under cer- lates glomerular capillary pressure(9). NO produced by endotain situations therole of a cytotoxic agent, a reactive nitrogen thelial cells can inhibit sodium transport by cortical collecting intermediate that together with reactiveoxygen metabolites is duct cells (10).Tubule epithelial cells appear to containboth a lethal to cancer cells and microorganisms. As a signal trans- constitutive form of NOS (11)and a form that can be inducedby tumor necrosis factor-a and IFN-y in both proximal tubule cells (12).Since NO can and inner medullary collecting duct cells * This research was supported inpart by funds from the Charles and Johanna Buschendowment and by National Institutes of Health exert a cytotoxic action both on cells that produce it and on GrantsAG07972 and DC01295(toD. T. D.), ES04738 and GM34310 (to neighboring cells, it is importantthat its production be subject D. L. L.), andES 03647 and ES 05022 (to J. D. L.). The costsof publi- to fairly tight negative regulation (for example by OPN). cation of this article were defrayed in part by the payment of page charges. This article must thereforebe hereby marked "advertisement" EXPERIMENTALPROCEDURES in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. 8 Supported by a graduate fellowshipfrom the NationalScience Cell Culture-Primary proximal tubule epithelial ( R E ) cells were Council of Taiwan. prepared from the kidneys of 6-8-week-old CD1 mice using culture ** To whom correspondence should be addressed: Nelson Biological conditions shown to be selective for tubule epithelial cells (13). Briefly, Laboratories,Rutgers University, Piscataway,NJ 08855-1059. "el.: 908- slices of cortex from decapsulated kidneys were washed in unsupple932-4569; Fax: 908-932-0104. The abbreviations used are: NOS, nitric oxide synthase; iNOS, in- mented RPMI 1640 andcutintol-2-mm3 pieces, which were then ducibleNOS; I F N , interferon; IL, interleukin; L - N M A , NG-methyl-L- incubated for 20 min at 37 "C in Krebs-Henseleit buffer (Sigma) supplemented with 10 m~ HEPES (pH 7.5), 1 giliter o-glucose, 200 mgAiter arginine; LPS,lipopolysaccharide;OPN,osteopontin,FTE,proximal tubule epithelial; TGF, tumor growth factor; OPP, OPNpeptide (PTVD- sodium pyruvate, 0.125 mdml collagenase (melA, Sigma), and 0.6 unit/ml dispase (Boehringer Mannheim). The digestedcortex was cenVPDGRGDSLAYRLRSK); GST,glutathione S-transferase.

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Osteopontin Inhibits Induction of NO Synthase Gene Expression

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FIG.1. SDSPAGE and Western blot analysis of purified OPN. Approximately 5 pgof purified OPN was electrophoresed in two lanes ( b and c ) of a 12% polyacrylamide-SDSgel, each of which was blotted onto nitrocellulose. Lane a, protein markers, stained with Coomassie Blue. Lane b, total protein stained with ISS Pro-BlueTMand Silver StainDaichi (Integrated Separation Systems, Natick, MA). The black and white photograph does not do justice to the silver-stained gel, which showed a very intense blue-green color a t the position of OPN (indicated by the asterisk), suggesting that thepurity was even better than evident here. Lane c, protein immunoreactive with the polyclonal anti-OPN antiserum LF-7 detected with anti-rabbit IgG (Bio-Rad) conjugated to horseradish peroxidase and visualized with enhanced chemiluminescence (ECL,Amersham Corp.). OPN doesnot stain well with Coomassie Blue. trifuged a t 70 x g for 3 min and the cells resuspended in RPMI 1640 supplemented with 10m~ HEPES (pH7.5), 5 pg/d human transferrin, 50 IW dexamethasone, 5 p g / d insulin (Life Technologies, Inc.),3%fetal bovine serum, 100 unitdml penicillin, and 100 pg/ml streptomycin. For NO measurements, cells were subcultured into 24-well culture plates (Linbro, Hamden, CT) a t 5 x lo4 celldl6-mm well and incubated overnight. After removal of the medium, the cells were washed three times with Dulbecco's phosphate-buffered saline (Sigma D5652) and refed with phenol red-free RPMI 1640 medium containing 2 m~ L-arginine and 10 pg/ml tetrahydrobiopterin (ICN Biochemicals) in thepresence or absence of 100 ng/ml lipopolysaccharide (Sigma) and 100 unitdd recombinant mouse y-interferon (kindly provided by Dr. s. Pestka, Robert Wood Johnson Medical School, Piscataway, NJ). P-Methyl-L-arginine ( L - N M A ) was obtained from BIOMOL Research Laboratory Inc. (Plymouth Meeting, PA). Northern Blot AnaZysis-'Ibtal RNA was isolated using the TRI reagent (Molecular Research Center, Cincinnati, OH). The RNA ( 5 pg) was fractionated on formaldehyde-agarose gels, blotted, and probed using standard procedures (14). The mouse cDNA corresponding to the macrophage-inducible nitric oxide synthase (iNOS) was provided byDr. James Cunningham, Brigham and Women's Hospital, Boston, MA (15). Measurement of NO (as NO,-) Production-Nitric oxide is a shortlived reactive nitrogen intermediate that in aqueous solution, in the absence of oxyhemoproteins or superoxide, is oxidized primarily to nitrite (16). Under such conditions, NO production can be assessed by the accumulation of nitrite in the medium. Nitrite was determined in our experiments using the Griess reagent, which is generated by mixing equal volumes of 60 m~ sulfanilamide in 50% H3P04 and 4 m~ N-lnaphthylethylenediamine dihydrochloride in H20. Incubation of one volume of medium first with one volume of sulfanilamide, then 5 min later with one volume of N-1-naphthylethylenediaminedihydrochloride, results in the formation of a purple azo dye, indicative of the presence of nitrite, thatcan be quantified spectrophotometrically after 30 min a t 540 nm (17). Preparation of Reconbinant OPN-A human opn cDNA (OP10, kindly provided by Dr. Larry Fisher, National Institutes of Health) was cloned into the mammalian expression vector pNMH under the control of the mouse metallothionein-1 promoter (18).The plasmid was transfected into into MH2 cells (a cell line obtained from human embryonic kidney cells by immortalization with the Adl2Ela gene) that did not express endogenous OPN. From the resulting G418' clones, one was isolated that had multiple copies of the expression cassette integrated into the chromosome and could be induced with 0.5 p~ Cd2+to express high levels of OPN. OPN was purified from serum-free, Cdz+-containing a-minimal essential medium conditioned by cells for 18-20 h.2 Fig. 1 illustrates the quality and purity of the OPN preparation used in this work. Protein concentrations were determined with the Pierce bicinchoninic acid reagent. GST-OPN, a fusion protein of human osteopontin with glutathione S-transferasepurified from Escherichia coli, was genC. A. Lopez and D. T. Denhardt, manuscript in preparation.

LNMAhM)

FIG.2. The arginine analog L - N M A inhibits production of nitrite by primary tubule epithelial cells. Cells were cultured and stimulated with LPS + y-IFN as described under "Experimental Procedures." L - N M A was added simultaneously with the inducing agents in fresh medium a t the indicated concentration. U,unstimulated cells, no L - N M A . Nitrite in themedium was measured a t 24 h. The values are the mean 2 standard error from three separate plates. erously made available by Drs. A. Chambers and J. Xuan, London Regional Cancer Center, Ontario, Canada (19). OPP is the peptide AcPro-Thr-Val-Asp-Val-Pro-Asp-Gly-Arg-Gly-Asp-Ser-~u-~a-~-GlyLeu-Arg-Ser-Lys-NH2and was synthesized by American Peptide Company, Inc., Sunnyvale, CA; it represents a consensus sequence around the Arg-Gly-Asp sequence in OPN. RESULTS

Generation of NO by n b u l e Epithelial Cells-Cultures of primary mouse kidney epithelial cells accumulated nitrite in the medium in a time- and dose-dependent manner when stimulated with LPS and y-IFN, or y-IFN alone, but not when stimulated with LPS aloneor not stimulated (datanot shown, but see below). Inhibition of nitrite accumulation by the arginine analog p-methyl-L-arginine ( L - N M A ) (Fig. 2) strongly suggests that nitrite synthesis is dependent upon a nitric oxide synthase. Confirmation that the nitriteis indeed indicative of NO synthesis was provided by the fact that stimulated cells had a substantially enhanced level of the mRNA encoding iNOS (Fig. 3 A , lane 2). An immunofluorescence analysis revealed increased amounts of iNOS protein, largely in a perinuclear location, in essentially all of the stimulated cells (Fig. 4). These experiments establish that inflammatory mediators induce NO synthesis in thesecells by increasing expression of the iNOS gene. Inhibition of NO Production by OPN--Recombinant human OPN was found to lower iNOS mRNA induction (measured at 8 h, Fig. 3 A ) and nitrite production (measured at 24 h, Fig. 3B) in subconfluent PTE cells treated with LPS + y-IFN. Concentrations of 1 and 100 PM OPN, respectively, added simultaneously with the inducers, reduced the level of the 4-kilobase mRNA species to 54 and 31% (normalized to 18 S rRNA) and nitrite production to 45 and 24% (relative to the LPS + y-IFNstimulated cells). The two larger mRNA species detected in the Northern blot may result from the utilization of alternative polyadenylation sites (11). The diminished concentration of iNOS protein in theOPN-treated stimulated cells is obvious in Fig. 4. The results shown in Fig. 5A indicate that OPN added 60 min prior to the addition of y-IFN + LPS was more inhibitory than when added simultaneously withthe inducers. Inhibition of NOproduction decreased as the time between the addition of LPS + y-IFN and OPN increased. Fig. 5B shows that an antiserum raised againstOPN purified from human bone blocked the ability of OPN to suppress theinduction of NOsynthesis by these cells. The preparation of OPN used in this experiment (and Fig. 6) was not quite as active as that used in theexperiments described in Figs. 2 and 3 because of some loss of activity during storage. Eviknce That an Zntegrin ReceptorMediates OPN's Effect on NO Induction-'Ib identify the receptor for OPN we investigated the ability of certain peptides to mimic, or inhibit, the

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Osteopontin Inhibits Induction of NO Synthase Gene Expression a

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FIG. 3. Inhibition of N O S gene induction by OPN. a , Northern blot analysis of MOS mRNA and 18 S rRNA levels in primary mouse kidneyepithelial cells 8 h after treatment. b, accumulation of nitrite in themedium 24 h after treatment. Values are the mean * standard error from three separate plates. Lane I , control; lanes 2,4, and 6, stimulated with 100 ndml LPS plus 100 unitdml y-IFN; lanes 3 and 4, treated with 1 PM OPN; lanes 5 and 6, treated with 100 PM OPN.

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suppression of iNOS protein in PTE cells. Cells were fixed and permeabilized by incubation for 30 min with 0.4 m~ lysophosphatidylcholine in 1%buffered formalin. After washing three times with phosphate-buffered saline, cells were blocked with 0.1% bovine serum albumin in phosphate-buffered saline and then incubated for 4 h, first with a 1:10,000dilution of rabbit peptide antibody to the C-terminal 10 amino acids of iNOS (C. Nathan, Cornel1 University), and then, after washing, with fluorescein isothiocyanate-conjugated goat anti-rabbit secondary antibody (Cappel, West Chester, PA). After an additional 30 min, the cells were washed and analyzed for fluorescence intensity using a Meridian ACAS anchored cell analysis system. Upper left, untreated control cells;upper right, cells treated for 48 h with LF'S (100 nglml) and y-IFN (100 unitdml); lower left, cells treated for 48 h with OPN (100 PM);lower right, cells treated for 48 h with LPS, y-IFN, and OPN. The color bar represents relative fluorescence intensity on a four-decade log scale with white being the most intense. FIG.4.OPN-induced

action of OPN on induced NO production. Recent studies have indicated that the cell adhesion properties of OPN are mediated via a GRGDS sequence in theprotein that interactswith the avP3integrin (20, 21). We therefore investigated whether an RGD-dependent integrin might also mediate the ability of OPN to inhibit NO production. Fig. 6u shows that both GSTOPN (a fusion protein between glutathione S-transferase and human OPN made in E. coli) and OPP (a synthetic 20 aminoacid peptide that represents the sequence centered on the RGD sequence of OPN) were also effective at reducing LPS + y-IFN-induced NO production. GRGDS and GRGES did not show any effect on NO production up to 10 nM. Fig. 6b shows that GRGDS, but not GRGES, was able to reverse the OPNmediated suppression of y-IFN-induced NO production. Nitrite production was less in this experiment compared to that

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FIG.5. Time dependence and antiserum sensitivity of OPNmediated down-regulationof NO production. Values are the mean * standard error from three separate plates. The asterisks indicate a statistically significant difference from the stimulated cells at a c o d dence levelp c 0.05. a , OPN (100 PM)was added at the indicated time prior to or after stimulation with of LPS + y-IFN;nitrite was measured after 24 h. b, treatment with a Y105 dilution of the anti-OPN antiserum LF7. Antiserum and OPN were incubated together at 25 "C for 10 min prior to adding to the medium.

shown in panel a because the cells were stimulated only with y-IFN. A 100-fold molar excess of GRGDS blocked the action of OPN, whereas the same concentration of GRGES had no effect. These results are in harmony with the idea that OPN is acting via an integrin, presumably the a& integrin, to inhibit the induction of iNOS. The avP3integrin has been localized to the basolateral surface of tubule epithelial cells in the mouse kidney (7).

Osteopontin Inhibits Induction of NO Synthase Gene Expression molo et al. (613 have shown that OPN (purified from urine) at

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FIG.6. Effect of OPN, GST-OPN, OPP, GRGDS,and GRGES on nitrite production. Cells were stimulated and nitrite measured as describedabove.Values are the mean standard error from three separate plates. a,effect of each agent alone on nitrite production by the stimulated cells. AI1 concentrations are expressed as nanomolar. b, ability of GRGDS but not GRGES to reverse the O P ~ - m e d i a ~inhibition d of nitrite production by stimulated cells. Concentrations are as indicated. The asterisks indicate a statistically significant difference from the stimulated cells at a confidence level p 0.05.

*

DISCUSSION

The above data establish 1)that y-IFN + LPS stimulate NO production in renal epithelial cells by inducing expression of the iNOS gene, and 2) thatpurified recombinant OPN inhibits the elevation ofNO synthesis that is caused by these inflammatory mediators by suppressing iNOS gene induction. The experiments described in Figs. 5 and 6 showed that an antiOPN antiserum and the GRGDS peptide, but not GRGES, could reverse the OPN-mediated suppression of NO induction. They also showed that both a peptide homologous to a 20-amino acidsegment including the RGD site inOPN and a GSTOPN fusion protein purified from E. coli were also able to inhibit NO synthesis. This is interesting notonly because it strengthens our conclusions about the action of OPN on NO production but also because it suggests that the post-translational m ~ f i c a t i o n scharacteristic of the protein made in mammalian cells are unnecessary for this integrin-mediated down-regulation. OPN was effective on the subconfluent PTE cells used in this work at surprisingly low concentrations (-10 PM); effects on confluent cells required higher concentrations, a situation that may more closely reflect the situation in the intact organ. Zi-

concentrations as low as 1PM could raise the intracellular free Ca2+level in osteoclasts. (For reasons unknown, this conflicts with an earlier report (5)that much higher concentrations of OPN purified from bone lowered CaZ+levels.) If one assumes that a GRGDS peptide is 10-fold less effective than the functional protein at interacting with the target integrin, then it could be argued from the data inFig. 6b (where an apparent equimolar concentration of GRGDSpartially reversed the OPN inhibition) that the concentration of functional OPN in our protein preparations is some 10-fold less than the measured protein concentration, reinforcing the conclusion that the protein is effective at low concentrations. We have some insight into the signal transduction pathway that might be responsible for the observed effect of OPN on iNOS gene expression. Integrins, upon ligand binding, have the capacity to interact with cytoskeletal components, and C. Lopez4has preliminary evidence indicating phosphorylation of the focal adhesion kinase upon OPN binding. Thus it may be that a cascade of phosphorylation events results in thedownregulation of iNOS expression. It is possible that changes in the OPN level in the renal interstitial fluid, for example during inflammation, could modulate NO production and thereby control blood flow through the kidney; NO is an important vasodilator in the kidney (8-12). OPN expression in the kidney is curiously focal in that only a subset of nephrons express it at high level at any one time (7). Giachelli et al. 122)have observed that,in angiotensin 11-induced tubulointerstitialnephritis, there arefocal increases in OPN levels that precede overt pathological changes in regions that correlate with sites of macrophage accumulation. There are several ways in which the OPN-mediated downregulation of NO biosynthesis could be of physiological significance in therenal tubuleepithelium. For example, NO production is important in regulatingblood flow (8)and ion transport (lo), and consequently blood volume and osmolality. NOS is abundant in the macula densa, consistent with the belief that these tubular epithelial cells adjacent to the afferent arteriole of cortical nephrons regulate via NO production glomerular capillary pressureand the tubuloglomerular feedback response, including renin release (9). Localization of the OPN receptor on the basolateral surface of the tubule cells is consistent with their being subject to regulation by interstitial OPN. Glomerulonephritis, induced forexample by the accumulation of immune complexes and the consequent infiltration of neutrophils and activated macrophages, is characterized by the enhanced production of NO, which may contribute to the tissue injury (23,241. During inflammation or stress-related processes such as reperfusion injury, kidney cells are exposed to an array of inflammatory mediators and paracrine factors derived not only from the epithelial tissue but alsofrom infiltrating macrophages, granulocytes and lymphocytes. Many of these factors, in particular y-IFN, IL-lp, and tumor necrosis factor-a, stimulate NO production in mesangial cells (11, 25). Excessive NO and related reactive nitrogen intermediates, as well as reactive oxygen intermediates, are cytotoxic, damaging the epithelium and compromising kidney function. Lipid peroxidation reactions generated by the reactive oxygen and nitrogen intermediates are known to damage cell membranes. Thus, OPN localized in the kidney may serve not only to regulate renal homeostatic processes, but also to protect the organ against NO-induced cell injury. Enhanced NO production is a serious

Z. Zimolo, personal communication. C . A. Lopez, personal communication.

Osteopontin Inhibits Induction

o f N 0 Synthase Gene Expression

715

consequence of uremia (26),and it will be important to ascerREFE~NCES tain theextent to which OPNcan amelioratrt this patholo~cal 1. Nathan, C. (1992) FASEB J. 6,3051-3064 2. Denhardt, D. T., and Guo, X. (1993) FASEB J., inpress condition. 3. Brown, L. E, Beme, B., Van De Water, L., Papadopoulos-Sergiou,A., P e m z i , OPN is not the first protein to be reported to inhibit NO C. A., Manseau, E. J.,Dvorak, H. F., and Senger, D. R. (1992) Mol. Biol. Cell 3, 1169-1180 production.Macrophage deactivating factor and TGFp par4. Chambers, A. F., Behrend, E. I., Wilson, S. M., and Denhardt, D.T. (1992) tially block NO release by macrophages activated with y-IFN + Anticancer Res. 12,4348 TGFa, but not when activated by y-IFN + LPS (27);epidermal 5. Miyauchi, A., Alvarez, J., Greenfield, E. M., Teti, A,, Grano, M., Colucei, S., Zambonin-Zallone, A,, Ross, F.P., Teitelbaum, S . L., Cheresh, D., and growth factor can suppress both NO and Hz02 production by Hruska, K A. (1991) J. Biol. Chem. 266,20369-20374 keratinocytes (28); incubation of LPS-activated peritoneal neu6. Zimolo, Z., Wesolowski, G.,Tanaka, H., Hyman, J.,Hoyer, J. R., and Rodan, G. A. (1993) Am. J. Physiol. 69,355-363 trophils with IL-8blocks both the release ofNO and NOS 7. Lopez, C. A,, Hoyer, J. R., Wilson, P. D., Waterhouse, P., and Denhardt, D.T. induction at the transcriptional level (29); TGFp1 and 12-0(1993) Lab. Inuest. 69,355-363 tetradecanoy1phorbol-13-acetateinhibit LPS + y-IFN-induced 8. Brezis, M., Heyman, S. N., Dinour, D., Epstein, F. H., and Rosen, S. (1991) J. Clin. Invest. 68,390-395 NO synthesis inmouse bonemarrow cells (30);both acidic and 9. Wilcox, C. S., Welch, W. J., Murad, F., Gross, S. S., Taylor, G., Levi, R., and basic fibroblast growth factor inhibit (andTGFB increases) niSchmidt, H. H. H. W. (1992)Proc.Natl. A d . Sci. U. S. A. 89,11993-11997 trite production a t t r i b u ~ b l eto LPS plus y-IFN treatment of 10. stoas, B. A, Carretern, 0. A,. Farhy, R. D., scicli, G., and Garvin, J. L. (2992) J. Clin. Invest. 89,761-765 bovine retinal pigmented epithelial cells, likely by inhibiting 11. Ishii, K., Chang, B., Kerwin, J. F., Wagenaar, F. L., Huang, Z.-J., and Murad, the induction of NOS mRNA at the transcriptional level (31); F. (1991) J. Pharmncol. Erp. Thex 256,3843 12. Markewitz, B. A, Michael, J. R., and Kohan, D. E. (1993) J. Clin. Inuest. 91, and insulin-like growth factor I reduces the amount of NO 2136-2143 produced by the action of IL-lp on vascular smooth muscle cells 13. Elliget, K. A., and h p , B. F. (1991) In Vitro CeZl. Deu. Biol. 27A, 739-748 (32). The fact that so many agents can affect NO production 14. Sambmok, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, suggests that NO production is important in many different NY 15. Lyons, C. R., Orloff, G. J., and Cunningharn, J. M. (1992) J. Biol. Chem. 267, contexts. 6370-6374 We have reported elsewhere5 that NOS mRNA abundance 16. Ignarm, L. J., Fukuto, J. M., Griscavage, J. M., Rogers, N. E., and Byma, R.E. increases during hypoxia but then decreases upon subsequent (1993) Pmc. Natl. Acad. Sci. U. S. A. BO, 810343107 repefision of primary human PTE cells in culture. How a tran- 17. Green, L. C., Wagner, D. A., Glogowski, J., Skipper, P.L., Wishnok, J. F., and Tannenbaum, S. R. (1982) A w l . Bioekem. 126,131-138 sient increment in NO production impacts on the survival of the 18. Khokha, R., and Denhardt, D. T.(1987) Anticancer Res. 7,653-660 hypoxic cellis not known, but one possibilityis that,by means 19. Xuan, J.-W., Hob, C., and Chambers, A. F.(1994) J. CeZZ. Biochem., in press M.E., no^^, M., HeinepHrd, D., ReinhoIt, F. P., and Andemson, G. of guanylyl cyclase activation, beneficial changes in Ca2+levels 20. Flores, (19923 Exp. Cell Res. 201,526&30 and gene expression are effected. Giventhe known cytobxicity 21. Ross, F. P., Chappel, J., Alvarez, J. I., Sander, D., Butler, W. T., Farach-Carson, M. C., Mintz, K. A,, Robey, P.G., Teiklbaum, S. L., and Cheresh, D. A. (1993) of NO, it is likely that rapid and effective down-regulationof NO J. Biol. Chem. 268.9901-9907 production is necessary for the survival of the tubulecell. Thus 22. Giachelli, C.M., Pichler, R., Lombardi, D., Denhardt, D.T., Alpers, C. E., Schwartz, S., and Johnson, R. J. (1994) Kidney int., in press it isinteresting that OPN expression was augmented not only Cattell, V., Cook, T., and Moncada, S . (1990) Kidney Int. 38,1056-1060 during hypoxia but also, and even more so, during subsequent 23. 24. Cook, H. T., and Sullivan, R. (1991) Am. J. Pathol. 139, 1047-1052 reperfusion. Elevated expression of OPN, and consequent down- 25. Nicolson, A. G., Haites, N. E., McKay, N. G . , Wilson, H. M., MacLeod, A. M., and Benjamin, N. (1993) Biochem. Biophys. Res. Commun. 193,1269-1274 regulation of NO production, may be necessary for minimizing 26. Nons, M., Benigni, A,, Boccardo, P.,Aiello, S., Gaspari, F., lbdeschini, M., ischemic injury in the kidney and other organs. Figliuzzi, M., and Remuzzi, G.(1993) Kidney Int. 44,445-450 Acknowledgments-We are grateful to Larry Fisher and Marian Young for the gif€ of the human OPNlO cDNA clone and LF7 antiserum. We also appreciate the generosity of Ann Chambers and Jim Xuan in providing the GST-OPN protein. We thank John Hoyer and Cecilia Giachelli for useful comments on the manuscript.

S.-m. Hwang, P.D. Wilson, J. D. Laskin, and D. T.Denhardt, submitted for publication.

27. Ding, A., Nathan, C. F., Graycar, J., Derynck, R.. Stuehr, D. J., and Srirnal, S. (1990) J. Immunol. 146,940-944 28. Heck, D.E., Laskin, D. L., Gardner, C. R., and Laskin, J. D. (1992) J. Biol. Chem. 267,21277-21280 29. McCalI, T. B., Palmer, R. M. J., and Moncada, S. (1992)Biochem. Biophys. Res. Commun. 186,680485 30. Punjabi, C. J.. Laskin, D. L., Heck, D. E.,and Laskin, 3. D. (1992)J.Immuol. 149,2179-2184 31. Goureau, O.,Lepoivre, M., Becquet, F., and Courtois, Y. (1993) Pme. Natl. Acad. Sci. U. S.A. 90,427€-4280 32. Schini, V. B., Catovsky, S., and Vanhoutte, P. M. (1993) FASEB ESP.Bioi. 7,

A243 (abstr.)