{alpha}v Integrin regulates TNF-{alpha}-induced nitric oxide synthesis in rat mesangial cells—possible role of osteopontin

Toshiki Nagasaki1, Eiji Ishimura2, Hidenori Koyama2, Atsushi Shioi2, Shuichi Jono2, Masaaki Inaba2, Tadayoshi Hasuma1, Minehiko Yokoyama3, Yoshiki Nishizawa2, Hirotoshi Morii2 and Shuzo Otani1

1 Second Department of Biochemistry and 2 Second Department of Internal Medicine,Osaka City University Medical School, Osaka and 3 Meiji Institute of Health Science,Odawara, Japan

Correspondence and offprint requests to: Eiji Ishimura MD PhD, Second Department of Internal Medicine, Osaka City University MedicalSchool, 1–4-3, Asahi-machi, Abeno-ku, Osaka, 545-8585, Japan.



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Tumour necrosis factor-{alpha} (TNF-{alpha}) induces nitric oxide (NO) synthesis in rat mesangial cells (MCs). We previously demonstrated that osteopontin (OP), a matrix protein that mainly interacts with the{alpha}v integrin family, increased time-dependently by TNF-{alpha} stimulation at gene and protein levels. The regulation of NO synthesis by integrins or matrixproteins is unclear.

Methods. We examined whether integrin, especially{alpha}v integrin, regulates NO synthesis in rat MCs and whether OP, an{alpha}v integrin ligand, has an effect on TNF-{alpha}-induced NO synthesis. Furthermore, OP and inducible NO synthase (iNOS) gene expression wasexamined by Northern blotting.

Results. TNF-{alpha} increased NO synthesis in MCs in a time-dependent manner. Synthetic GRGDSP peptide, whichis known to inhibit various integrins that interact with RGD-containing extracellular matrices,increased TNF-{alpha}-induced NO levels in a dose-dependent manner. Cyclical RGD peptide, the specific inhibitor of{alpha}v integrin, also exhibited a dose-dependent effect of increasing NO levels, while GRGESP peptide,which has very low affinity to integrins, had no effect. In addition, NO synthesis was found to besignificantly reduced when MCs were plated on OP-coated dishes compared to type I or IVcollagen-coated dishes. Furthermore, anti-OP antibody increased NO synthesis in MCs. iNOSmRNA levels were increased by TNF-{alpha}, and were abruptly diminished after OP mRNA was significantly induced.

Conclusions. The present study demonstrated the involvement of{alpha}v integrin in TNF-{alpha}-induced NO synthesis in rat MCs, and the possible role of OP was suggested in themechanism. TNF-{alpha} and extracellular matrices can co-operate to regulate the behaviour of MCs at least partlythrough NO synthesis, which may participate in the course of glomerular diseases.

Keywords: {alpha}v integrin; mesangial cell; nitric oxide; osteopontin; tumour necrosis factor-{alpha}



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Integrins are heterodimeric transmembrane glycoproteins that consists of varying combinations ofnon-covalently bound{alpha} andß chains, and are involved in adhesive interactions with the extracellular matrix. The{alpha}v chain mediates attachment of a variety of cells to diverse matrix proteins, and are critical forangiogenesis, migration, and cell proliferation [13].{alpha}v Integrin, the vitronectin receptor, is the most promiscuous member of integrin that interacts with awide variety of proteins including vitronectin, fibronectin, osteopontin (OP) and thrombospondin [1]. The expression of{alpha}v integrin has been confirmed in various kinds of cells, such as endothelial and smooth musclecells, fibroblasts and mesangial cells [46].

OP is a sialic-acid-rich, non-collagenous bone phosphoprotein that binds strongly to the calcium-phosphate-based bone matrix (hydroxylapatite). It possesses an RGD-cell attachment sequencecapable of interacting with{alpha}v integrin and facilitating cell attachment and spreading [7]. Furthermore, OP has been reported to be expressed in various kinds of cells, such asosteoblasts, vascular endothelial and smooth muscle cells [8,9]. OP has been reported to participate in chemotaxis, migration, pathological calcification, and nitricoxide synthesis, presumably in a paracrine manner [9,10].

We previously reported the presence of OP gene expression and protein synthesis in cultured ratMCs, and concluded that increased OP production under the stimulation of tumour necrosis factor-{alpha} (TNF-{alpha}) suggests that intraglomerular OP contributes to the development of various glomerular diseases [11]. TNF-{alpha} is an inflammatory cytokine that induces NO synthesis inin vitro studies on MCs, and is involved in the development of glomerulonephritisin vivo [12,13]. Therefore we examined whether{alpha}v integrin and one of its ligands, OP, regulate TNF-{alpha}-induced NO production in cultured rat MCs.



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Materials
Fetal calf serum (FCS) and various RGD peptides were purchased from Gibco BRL (Grand Island,NY). Mouse TNF-{alpha}, rat type IV collagen, and bovine type I collagen were purchased from Genzyme (Cambridge, MA),Sigma Chemical Co. (St Louis, MO), and Vitrogen (Palo Alta, CA) respectively. Synthetic rat OPwas kindly supplied by Dr Giachelli (University of Washington, Seattle, WA). Polyclonal anti-OPantibody (whole serum) was raised in rabbits by immunization with human OP which was isolatedfrom human milk, using methods similar to that described elsewhere [14]. This antibody was confirmed to cross-react with rat, mouse and human OP, byimmunohistochemistry and Western blotting [11].

Cells and cell culture
Glomeruli of the kidneys from Sprague–Dawley rats were isolated by the sieving method [15]. MCs, obtained from outgrowths of isolated glomeruli, were cultured in plastic flasks or plates inphenol-red-free Dulbecco's Modified Eagle Medium (DMEM, GIBCO) supplemented with 10%FCS (Gibco), penicillin (100 units/ml), and streptomycin (100 µg/ml) at 37°C in ahumidified atmosphere containing 5% CO2. MCs of the 4th to 9th passages were used for the experiments. MCs were serum-starved for 12 hby culturing in DMEM with ITS mixture (5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml sodiumselenite at a final concentration). Then MCs were treated with various RGD peptides or split ondishes coated with various matrix proteins, and further incubated for 12 h to harvest the medium orcellular proteins. Under the concentration of RGD peptides used in the present study, MCs were notdetached from the surface of the culture plates.

Measurement of NO
NO production by MCs was determined by measuring of NO2- in the culture medium. Five hundred µl of culture medium was mixed with an equal volume ofGriess reagent (1 part 0.1% naphthylethylenediamine dihydrochloride to 1 part 1%sulphanilamide in 5% phosphoric acid) [16]. Absorbance at 540 nm was measured and the NO concentration was determined from a curvecalibrated with sodium nitrite standards. RGD peptides supplemented in the culture medium atvarious concentration in the experiments did not affect the aborbance at 540 nm. To quantify thechanges in NO, protein contents were measured using a BCA protein assay kit (Pierce, IL), and theNO amount were adjusted by the amount of cellular protein.

Northern blotting.
Total RNA from MCs was extracted by using acid guanidinum thiocyanate-phenol-chloroformmethod. For Northern blot analysis, total RNA was denatured with 6% formaldehyde,fractionated by 1% agarose gel electrophoresis, and transferred to a nylon membrane(Hybond-N, Amersham, Buckinghamshire, UK). The membranes were hybridized in a buffercontaining 50% formamide, 3xSSC (1xSSC; 0.15 M NaCl and 0.015 M sodiumcitrate, pH 7.4), 50 mM Tris–HCl, 20 mg/ml tRNA, 20 mg/ml boiled salmon sperm DNA, 1mM EDTA, 0.02% bovine serum albumin, 0.2% polyvinylpyrolidone, and 0.02%Ficoll, with32P-labelled rat OP [11], or rat inducible NO synthase (iNOS) [12], or rat glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) c-DNA as a probe at 37°C for48 h. After washing, filter membranes were exposed to X-ray film at -70°C for detectionof hybridized RNA. The DNA probes used were electrophoretically purified from agarose gels andlabelled with [{alpha}-32P]dCTP by using a random priming method (Megaprime DNA labelling system, Amersham). Forquantification of the changes in OP and iNOS mRNA expression, OP/GAPDH and iNOS/GAPDHmRNA ratios were used after densitometric estimation of each band.

Statistical analysis.
All results are expressed as the mean±SE and statistical significance was tested usingone-way analysis of variance (ANOVA) and multiple comparisons (Scheffe's F type).



   Results
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
NO synthesis in MCs
The effect of TNF-{alpha} on NO synthesis in cultured MCs was examined. MCs were cultured for 12 h in serum free DMEMand the cells were treated with TNF-{alpha} (10 ng/ml) for the indicated number of hours. TNF-{alpha} increased the release of NO into the media in a time-dependent manner (Figure1Go). Until 48 h, NO levels in the culture media gradually increased by 4.2-fold, and were significantlyhigher than those without TNF-{alpha}.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 1. Effect of TNF-{alpha} on NO2- in the culture medium. Measurement of NO2- was performed as described in Subjects and methods. After MCs were cultured for 12 h in serum-free medium, TNF-{alpha} (10 ng/ml) (—•—) and vehicle (—{circ}—) were added, and themedium was collected before (0) and 4, 12, 24, 32 and 48 h after stimulation. The results are themean±SE of four separate experiments. TNF-{alpha} induced a time-dependent increase in NO synthesis, which progressively increased by 4.2-fold inthe culture medium for up to 48 h. The vehicle showed little increase in NO levels over the 48-hperiod. *P<0.05vs 0 h.

 
Effect of {alpha}v integrin inhibitors on NO synthesis in MCs
We next examined the effects of peptide inhibitors for integrins on TNF-{alpha}-induced NO synthesis in MCs. MCs were serum-starved and preincubated with variousconcentrations of peptides for 12 h before TNF-{alpha} stimulation. NO levels in the culture media were determined 12 h after the TNF-{alpha} stimulation. GRGDSP peptide, which is known to inhibit various integrins which interact with RGD-containing extracellular matrices [17], increased TNF-{alpha}-stimulated NO production dose-dependently (Figure2Go). The maximal effect was observed at 100 nM. In contrast, GRGESP peptide, which has very lowaffinity to all integrins [17], did not affect NO levels at the same concentrations (Figure3Go). Cyclical RGD peptide, a specific inhibitor of{alpha}v integrin [18], also dose-dependently increased the NO levels (Figure4Go).



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 2. Dose-response effects of GRGDSP peptides on TNF-{alpha}-induced NO levels. Measurement of NO2- was performed as described in Subjects and methods. After MCs were cultured for 12 h in serum-free medium with various concentrations of GRGDSP, TNF-{alpha} (10 ng/ml) was added, and the medium was collected 12 h after the stimulation. The results are themean±SE of four separate experiments. GRGDSP peptides from 50 to 100 nM increasedthe NO levels dose dependently up to 1.8-fold. *P<0.05vs control cells; **P<0.005vs control cells.

 


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 3. Dose-response effects of GRGESP peptide on TNF-{alpha}-induced NO levels. MCs were treated in the same way as forFig.2Go, except that the GRGESP peptide was used instead of the GRGDSP peptide. GRGESP peptidesdid not affect NO levels at the same concentrations. Data are the mean±SE of four separateexperiments. .

 


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 4. Dose response effects of cyclical RGD peptide on TNF-{alpha}-induced NO levels. MCs were treated in the same way as forFig.2Go, except that cyclical RGD peptide was used instead of the GRGDSP peptide. Cyclical RGDpeptide showed similar dose-dependent effects of increasing NO levels at concentrations above 100nM (C). Data are the mean±SE of four separate experiments. *P<0.05vs control cells; **P<0.005vs control cells.

 
Effect of OP on NO synthesis in MCs
We have previously demonstrated that TNF-{alpha} increases OP [11], which is known to mainly interact with the{alpha}v integrin family [18,10,19]. Therefore we next examined the effect of OP on NO production using matrix-coated platesprepared as previously described [20]. When MCs were cultured on OP-coated dishes, TNF-{alpha}-stimulated NO production was significantly suppressed as compared to cells cultured on type I ortype IV collagen-coated dishes (Figure5Go).



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 5. Effect of matrix protein coating of the culture plates on NO synthesis in MCs. The results are themean±SE of four separate experiments. After MCs, which were cultured for 9 h in serum-free medium, were split on each matrix protein-coated plate and left for 3 h, TNF-{alpha} was added and the medium was collected 12 h after the stimulation. NO2- levels of the medium obtained from OP-coated plates were significantly lower than those from type Ior IV collagen-coated plates. *P<0.05vs control cells. .

 
To examine the involvement of endogenously secreted OP on NO production in MCs, the effect of apolyclonal blocking anti-OP antibody (raised in rabbits) on NO levels in the media was determined.The antibody was used at a final concentration of 1:200 and non-immunized rabbit serum was usedat the same concentration as a control. TNF-{alpha}-stimulated NO production was significantly increased by the addition of anti-OP antibody,compared to the addition of non-specific rabbit serum (control) (Figure6Go).



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 6. Effect of anti-OP antibody addition on NO synthesis in MCs. The results are the mean±SEof three separate experiments. After MCs were cultured for 12 h in serum-free medium on non-coated plates, anti-OP antibodies were added at the time of TNF-{alpha} stimulation, and the medium was collected 12 h after the stimulation. Non immunized rabbit serumwas used at the same concentration as a control. Anti-OP antibody addition significantly increasedNO levels, compared to the control medium added non-immunized rabbit serum. *P<0.05vs control cells.

 
iNOS and OP gene expression in MCs
Northern blot analysis was performed to examine changes in mRNA levels of iNOS in TNF-{alpha}-stimulated MCs. MCs were cultured for 12 h in serum-free DMEM and the cells were treated withTNF-{alpha} (10 ng/ml) for 0, 2, 4, 6 and 12 h. As shown inFigure7Go, iNOS mRNA levels were increased time-dependently over a 6-h period and abruptly diminishedthereafter. As previously reported [11], TNF-{alpha} significantly increased the level of 1.7 kB transcript of OP as early as 6 h and the maximal effectwas observed at 12 h, suggesting a possible role for OP on NO synthesis.



View larger version (38K):
[in this window]
[in a new window]
 
Fig. 7. Effect of TNF-{alpha} on osteopontin (OP) and inducible NO synthase (iNOS) mRNA expression. Northern blotting wasconducted as described in Subjects and methods. After MCs were cultured for 12 h in serum-freemedium, TNF-{alpha} (10 ng/ml) was added. RNA was extracted before, and 2, 4, 6, 12 h after the stimulation. Eachautoradiogram shown in the upper panel is representative of three separate experiments. Lowerpanel shows OP/GAPDH mRNA ratio and iNOS/GAPDH mRNA ratio by densitometric analysis ofthe Northern blots (mean±SE of three separate experiments). iNOS mRNA levels wereincreased time-dependently over a 6-h period and abruptly diminished thereafter. TNF-{alpha} significantly increased the level of 1.7 kB transcript of OP as early as 6 h and the maximal effectwas observed at 12 h. *P<0.05vs 0 h.

 


   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The present study demonstrates the involvement of integrin, especially{alpha}v integrin, and one of its ligands, OP [18,10,19] in TNF-{alpha}-induced NO synthesis in cultured rat MCs.

TNF-{alpha} is known to stimulate the production of complements, monocyte adhesion and NO synthesis [12,21,22]. TNF-{alpha}, which is expressed and participates in glomerular diseases in either an autocrine or paracrinemanner, is also known to induce NO synthesis in MCs and macrophages [12,13,16]. In this study, TNF-{alpha} increased time-dependent NO synthesis at 10 ng/ml (Figure1Go), a concentration usually used inin vitro MC examination [16]. Although the role of TNF-{alpha}-stimulated NO synthesis in glomerular pathophysiology is still unclear, NO was reported toregulate matrix protein synthesis and afferent arteriolar tones, and thus may play a role in theprogression of glomerular diseases [12,13,23,24].

Evidence has recently been obtained concerning the importance of integrins and their ligands in theoutside-in signalling between cells and their environment [36]. Recently, Hafdiet al. [6] reported that{alpha}v integrin is present in human MCs. In the present study, we showed that cyclical RGD peptide [18], which is known to specifically inhibit{alpha}v integrins, and synthetic GRGDSP peptide [17], which inhibits broad RGD-recognizing integrins, increased TNF-{alpha}-induced NO levels in a dose-dependent manner, while control GRGESP peptide had no effect (Figures2,3 and4GoGoGo). The findings suggest that{alpha}v integrin is at least one of major integrins which regulates TNF-{alpha}-induced NO production in MCs. In the present study, the concentrations of the peptides used wereless than 1 µM, which is markedly below 100 µM, the amount typically needed for completeabrogation of cell-matrix interaction, in order to avoid a supraphysiological effect to cause celldetachment [17,18]. Considering the relatively low concentration of the peptides, the NO production pathway may besensitively regulated by{alpha}v integrins.

We previously reported that TNF-{alpha} increases OP gene expression and protein levels in rat MCs, while platelet-derived growth factor,transforming growth factor-ß, or interleukin-1ß have no effects [11]. OP is known to interact mainly with the{alpha}v integrin family, although OP is recently reported to also bind to CD44 and{alpha}8 integrin other than{alpha}v integrin [18,10,19,25]. Thus we examined the effect of OP, as a candidate for an{alpha}v integrin ligand which may be involved in TNF-{alpha}-induced NO synthesis. We showed here that OP, which was coated on culture plates, inhibitsTNF-{alpha}-stimulated NO production in cultured MCs (Figure5Go), but type I or type IV collagen does not. On the other hand, anti-OP antibody enhanced TNF-{alpha}-stimulated NO production by MCs (Figure6Go). These data suggest that endogenously produced OP has a suppressive role in NO production.Since OP added to the culture media in the soluble form did not have a significant effect (data notshown), immobilization of this ligand may be necessary for OP-mediated regulation of NO.Recently, Rollo and Denhaldt [26] reported that, in mouse macrophages, recombinant OP inhibits lipopolysaccharide-and interferon-{gamma}-induced NO synthesis and that anti-OP antibody or RGD peptide promotes it [26]. The RGD peptides, such as GRGDSP hexaptide, particulary in soluble form, also have beenreported to have an opposite effect of RGD-containing matrix protein, such as osteopontin [10,27]. To exert biological effect on cellular function, RGD-containing proteins are considered to bind tointegrin not only at RGD portion but also at the other part(s) of the molecule [10,27]. We showed here for the first time in mesenchymal cells that{alpha}v integrins and its ligand OP are involved in NO production. In the experiments of Northern blottingexamining the levels of mRNA expression for both iNOS and OP, the levels of iNOS mRNAincreased time-dependently after TNF-{alpha} stimulation over a 6-h period and abruptly diminished as those of OP mRNA significantly increasedin the cultured rat MCs. The results also suggest that OP production by MCs possibly suppressesiNOS expression which results in a decrease in NO production and secretion by the cells.

OP has been reported to participate in NO synthesis such as in macrophage, proximal tubularepithelial cells of the kidney, and cardiomyocytes, presumably in a paracrine manner [10,2629]. In the present study we also demonstrated the participation of OP in NO synthesis by amesenchymal cell, mesangial cell. Hwanget al. [28,29] demonstrated that OP synthesis inhibits the induction of nitric oxide synthesis and suggested thatOP synthesis is compensatory to an NO increase induced in ischaemic tubular cells. It may beconsidered that TNF-{alpha}-induced OP production by MCs is a compensatory response to attenuate the effect of increasedNO, possibly a cytotoxic agent as a reactive nitrogen intermediate, induced in glomerular diseases.A unique regulatory mechanism of NO production by OP may be present, which is different fromthat of other extracellular matrix proteins, such as type I and type IV collagen.

In summary, this study demonstrates the regulation of NO synthesis by{alpha}v integrin in cultured rat MCs and the possible role of OP, an{alpha}v integrin ligand, in the mechanism. Thus, TNF-{alpha} and extracellular matrices can co-operate to regulate the behaviour of MCs at least partly throughNO synthesis, which may participate in the course of glomerular diseases.



   Acknowledgments
 
We thank Dr Cecilia M. Giachelli (Pathology Department, School of Medicine, University ofWashington, Seattle, WA, USA) and Dr Eisuke Sato (Department of Biochemisty, Osaka CityUniversity Medical School) for generously providing the recombinant human OP and rat iNOS c-DNA, respectively.



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

  1. Felding-Habermann B, Cheresh DA. Vitronectin and its receptors.Curr Opin Cell Biol1993;5:864–868[Medline]
  2. Brooks PC, Clark RA, Cheresh DA. Requirement of vascular integrin{alpha}vß3 for angiogenesis.Science1994;264:569–571[ISI][Medline]
  3. Drake CJ, Cheresh DA, Little CD. An antagonist of integrin{alpha}vß3 prevents maturation of blood vessels during embryonic neovascularization.J Cell Sci1995;108:2655–2661[Abstract/Free Full Text]
  4. Liaw L, Skinner MP, Raines EWet al. The adhesive and migratory effects of osteopontin are mediated via distinct cell surface integrins.Role of{alpha}vß3 in smooth muscle cell migration to osteopontinin vitro.J Clin Invest1995;95:713–724[ISI][Medline]
  5. Gailit J, Clark RA. Studiesin vitro on the role of alpha v and beta 1 integrins in the adhesion of human dermal fibroblasts to provisionalmatrix proteins fibronectin, vitronectin, and fibrinogen.J Invest Dermatol1996;106:102–108[Abstract]
  6. Hafdi Z, Lesavre P, Tharaux PL, Bessou G, Baruch D, Mecarelli LH. Role of{alpha}v integrins in mesangial cell adhesion to vitronectin and von Willebrand factor.Kidney Int1997;51:1900–1907[ISI][Medline]
  7. Butler WT. The nature and significance of osteopontin.Connect Tissue Res1989;23:123–136[ISI][Medline]
  8. Johnson PT, Leach RJ. Regulation of osteoblast gene expression in intratypic osteosarcoma hybridcells.Exp Cell Res1995;221:370–376[ISI][Medline]
  9. Shioi A, Nishizawa Y, Jono S, Koyama H, Hosoi M, Morii H. Beta-glycerophosphate acceleratescalcification in cultured bovine vascular smooth muscle cells.Arterioscler Thromb Vasc Biol1995;15:2003–2009[Abstract/Free Full Text]
  10. Singh K, Balligand JL, Fischer TA, Smith TW, Kelly RA. Glucocorticoids increase osteopontinexpression in cardiac myocytes and microvascular endothelial cells.Role in regulation of inducible nitric oxide synthaseJ Biol Chem1995;270:28471–28478[Abstract/Free Full Text]
  11. Nagasaki T, Ishimura E, Shioi Aet al. Osteopontin gene expression and protein synthesis in cultured rat mesangial cells.Biochem Biopys Res Commun1996;233:81–85[ISI]
  12. Ikeda M, Ikeda U, Ohkawa T, Shimada K, Kano S. Nitric oxide synthesis in rat mesangial cellsinduced by cytokines.Cytokine1994;6:602–607[ISI][Medline]
  13. Kashgarian M, Sterzel RB. The pathobiology of the mesangium.Kidney Int1992;41:524–529[ISI][Medline]
  14. Senger DR, Perruzzi CA, Papadopoulos A. Tenen-DG purification of a human milk protein closelysimilar to tumor-secreted phosphoproteins and osteopontin.Biochem Biophys Acta1989;996:43–48[ISI][Medline]
  15. Ishimura E, Sterzel RB, Budde K, Kashgarian M. Formation of extracellular matrix by cultured ratmesangial cells.Am J Pathol1989;134:843–855[Abstract]
  16. Cattell V, Cook T, Saivador M. Glomeruli synthesize nitrite in experimental nephrotoxic nephritis.Kidney Int1990;38:1056–1060[ISI][Medline]
  17. Pytela R, Pierschbacher MD, Ginsberg MH, Plow EF, Ruoslahti E. Platelet membrane glycoproteinIIb/IIIa: member of a family of Arg–Gly–Asp-specific adhesion receptors.Science1986;231:1559–1561[ISI][Medline]
  18. Pierschbacher MD, Ruoslahti E. Influence of stereochemistry of the sequence Arg-Gly-Asp-Xaa onbinding specificity in cell adhesion.J Biol Chem1987;262:17294–17298[Abstract/Free Full Text]
  19. Weber GF, Ashkar S, Glimcher MJ, Cantor H. Receptor-ligand interaction between CD44 andOsteopontin (Eta-1).Science1996;271:509–512[Abstract]
  20. Koyama H, Raines EW, Bornfeldt KE, Roberts JM, Ross R. Fibrillar collagen inhibits arterialsmooth muscle proliferation through regulation of Cdk2 inhibitors.Cell1996;87:1069–1078[ISI][Medline]
  21. Timmerman JJ, Verweij CL, Gijilswijk Vet al. Cytokine-regulated production of the major histocompatibility complex class-III-encodedcomplement proteins factorß and C4 by human glomerular mesangial cells.Hum Immunol1995;43:19–28[ISI][Medline]
  22. Mene P, Fais S, Cinnoti GA, Pugliese F, Luttmann W, Thierauch KM. Regulation of U-937monocyte adhesion to cultured human mesangial cells by cytokines and vasoactive agents.Nephrol Dial Transplant1995;10:481–489[Abstract]
  23. Trachtman H, Futterweit S, Singhal P. Nitric oxide modulates the synthesis of extracellular matrixproteins in cultured rat mesangial cells.Biochem Biopys Res Commun1995;207:120–125[ISI][Medline]
  24. Ito S, Arima S, Ren YL, Juncos LA, Carretero OA. Endothelium-derived relaxing factor/nitric oxidemodulates angiotensin II action in the isolated microperfused rabbit afferent but not efferent arteriole.J Clin Invest1993;91:2012–2019[ISI][Medline]
  25. Denda S, Reichardt LF, Muller U. Identification of osteopontin as a novel ligand for the integrin{alpha}8ß1 and potential roles for this integrin-ligand interaction in kidney morphogenesis.Mol Biol Cell1998;9:1425–1435[Abstract/Free Full Text]
  26. Rollo EE, Denhaldt DT. Differential effects of osteopontin on the cytotoxic activity of macrophagesfrom young and old mice.J Immunol1996;88:642–647
  27. Teti A, Farina AR, Villanova Iet al. Activation of MMP-2 by human GCT23 giant cell tumor cells induce by osteopontin, bonesialoprotein and GRGDSP peptides is RGD and cell shape change dependent.Int J Cancer1998;77:82–93[ISI][Medline]
  28. Hwang SM, Wilson PD, Laskin JD, Denhaldt DT. Age and development-related changes inosteopontin and nitric oxide synthase mRNA levels in human kidney proximal tubule epithelial cells:contrasting responses to hypoxia and reoxygenation.J Cell Physiol1994;160:61–68[ISI][Medline]
  29. Hwang SM, Lopez CA, Heck DEet al. Osteopontin inhibits induction of nitric oxide synthase gene expression by inflammatory mediatorsin mouse kidney epithelial cells.J Biol Chem1994;268:711–715
Received for publication: 5.11.98
Accepted in revised form: 9. 4.99





This Article
Abstract
FREE Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (2)
Disclaimer
Request Permissions
Google Scholar
Articles by Nagasaki, T.
Articles by Otani, S.
PubMed
PubMed Citation
Articles by Nagasaki, T.
Articles by Otani, S.