help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2006-0970
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Arafat, H. A.
Right arrow Articles by Dafoe, D. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Arafat, H. A.
Right arrow Articles by Dafoe, D. C.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene
*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*NITRIC OXIDE
Endocrinology Vol. 148, No. 2 575-584
Copyright © 2007 by The Endocrine Society

Osteopontin Protects the Islets and ß-Cells from Interleukin-1 ß-Mediated Cytotoxicity through Negative Feedback Regulation of Nitric Oxide

Hwyda A. Arafat, Anand K. Katakam, Galina Chipitsyna, Qiaoke Gong, Ajith R. Vancha, Jagadeesh Gabbeta and Donald C. Dafoe

Department of Surgery, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

Address all correspondence and requests for reprints to: Hwyda A. Arafat, M.D., Ph.D., Department of Surgery, Thomas Jefferson University, 1015 Walnut Street, Philadelphia, Pennsylvania 19107. E-mail: hwyda.arafat{at}jefferson.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Osteopontin (OPN), a phosphorylated glycoprotein that binds to an integrin-binding motif, has been shown to regulate nitric oxide (NO) production via inhibition of induced NO synthase (iNOS) synthesis. In the transplanted islets, iNOS and toxic amounts of NO are produced as a result of islets infiltration with inflammatory cells and production of proinflammatory cytokines. Here, we demonstrate that addition of OPN before IL-1ß in freshly isolated rat islets improved their glucose stimulated insulin secretion dose-dependently and inhibited IL-1ß-induced NO production in an arginine-glycine-aspartate-dependent manner. Transient transfection of OPN gene in RINm5F ß-cells fully prevented the toxic effect of IL-1ß at concentrations that reduced the viability by 50% over 3 d. OPN prevention of IL-1ß-induced toxicity was accompanied by inhibited transcription of iNOS by 80%, resulting in 50% decreased formation of the toxic NO. In OPN-transfected cells, the IL-1ß-induced nuclear factor-{kappa}B activity was significantly reduced. Islets exposed to IL-1ß revealed a naturally occurring early up-regulated OPN transcription. OPN promoter activity was increased in the presence of IL-1ß, IL-1ß-induced NO, and an inducer of NO synthesis. These data suggest the presence of a cross talk between the IL-1ß and OPN pathways and a unique trans-regulatory mechanism in which IL-1ß-induced NO synthesis feedback regulates itself through up-regulation of OPN gene transcription. Our data also suggest that influencing OPN expression represents an approach for affecting cytokine-induced signal transduction to prevent or reduce activation of the cascade of downstream devastating effects after islet transplantation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TRANSPLANTATION OF ISLETS of Langerhans has long been considered a potential curative treatment for diabetes (1). However, the results of several clinical trials showed that most transplant recipients failed to achieve complete insulin independence. The immunosuppressive regimen (2, 3) in Edmonton, Canada, has resulted in unprecedented success in achieving insulin independence with transplanted islets. Despite this success, some problems still persist and influence the outcome of islet transplantation. Transplanted islets are exquisitely susceptible to the injurious effect of mediators elicited by very early host inflammatory response (4). This results in early islet cell dysfunction and possibly death of the transplanted tissue. Host cytokine production is part of the early host inflammatory response (5). Proinflammatory cytokines and macrophage-derived byproducts such as IL-1ß, TNF{alpha}, and nitric oxide (NO) perturb insulin secretion from transformed ß-cell lines and whole islets (6, 7, 8, 9). Cytokine-treated rodent (and human) pancreatic islets demonstrate increased expression of the induced NO synthase (iNOS) gene that in turn leads to NO production (10). NO is hypothesized to deleteriously affect ß-cell function by inducing apoptosis and suppressing glucose stimulated insulin release (11).

Osteopontin (OPN) is an integrin- and calcium-binding phosphoprotein produced by a limited set of normal cells, including cells of mineralized tissue, epithelial cells, activated cells of the immune system, and bladder smooth muscle cells (12, 13, 14, 15). Classical mediators of acute inflammation such as TNF-{alpha} and IL-1ß strongly induce OPN expression (16, 17). The function of OPN in the normal and pathological contexts in which it is expressed remains poorly understood. However, many of its effects appear to be mediated by interaction of OPN, via its conserved RGD (arginine-glycine-aspartic acid) amino acid sequence, with integrin molecules (especially {alpha}vß3) (18, 19, 20).

The relationship between NO and OPN has been examined in many cell types. Studies in murine macrophages (19), ventricular myocytes, and cardiac microvascular endothelial cells (21, 22), and primary mouse kidney proximal tubule epithelial cells (18) showed a role for exogenous and endogenous OPN in the regulation of NO production and signaling. OPN has been suggested as a negative feedback regulator of iNOS synthesis in murine macrophages (19). Recent data from our lab have demonstrated that OPN improves the function of diabetic islets via reduction of NO and iNOS levels (23). The aim of the present work was therefore to investigate the potential role of OPN in protection of the islets and ß-cells against the IL-1ß-mediated cytotoxicity and dysfunction and to investigate the mechanisms involved. The role of IL-1ß in the regulation of endogenous OPN as an intracellular negative feedback mechanism that regulates IL-1ß action and thus the cellular fate after IL-1ß exposure was also evaluated.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Islet isolation, culture, and treatment
Male Wistar rats (Charles River Laboratories) weighing 250–300 g were housed on a 12-h light-dark cycle and were allowed free access to water and standard laboratory chow. All animal studies were performed in accordance with guidelines set forth by the Animal Care Committee of Thomas Jefferson University. Pancreatic islets were isolated as described previously (24) with modification. Briefly, rats were anesthetized by ip injection of ketamine hydrochlorides (100 mg/kg) and Rompun xylazine (20 mg/kg); the pancreas was exposed and injected at multiple sites with a total of 20 ml cold Hanks’ buffer/type IV collagenase solution. Animals were killed by surgical pneumothorax and the inflated pancreas was removed, finely chopped with scissors, and digested in a shaker-type water bath at 37 C for 10 min. The digestion was terminated by addition of 10 ml ice-cold Hanks’ balanced salt solution (HBSS), the contents dispersed by pipette, and tissue collected by centrifugation at 1000 x g for 10 min. Two washes with ice-cold HBSS were performed, with collection by centrifugation at 450 and 250 x g, respectively. Digested pancreas was then washed with HBSS and stained with dithiazone (Sigma Chemical Co., St. Louis, MO), which specifically stains the islets red. Islets were then separated by applying density separation medium lympholyte 1.1 (Cedarlane, Ontario, Canada) and handpicked under a microscope. Islets were aliquoted and cultured in RPMI-1640 medium containing 11 mmol/liter glucose and supplemented with 10 mmol/liter HEPES, 1% L-glutamine and penicillin/streptomycin, and were allowed to equilibrate for 3 h. Native rat OPN, a kind gift from Dr. William Butler (University of Texas, Houston, TX) was added 2 h before addition of IL-1ß (0.1–10 ng/ml) (R&D Systems, Inc., Minneapolis, MN).

Glucose-stimulated insulin secretion (GSIS)
To evaluate the protective effect of OPN in the islets, we tested its effect on IL-1ß-mediated cytotoxic effects. Immediately after islet isolation, 10 rat islets per experiment were cultured for 3 h in 24-well dishes containing 750 µl of RPMI medium with 5 mmol/liter glucose plus 10% fetal calf serum (FCS), 10 mmol/liter HEPES, penicillin G 100 U/ml, and streptomycin 100 µg/ml. Rat islets were pretreated with OPN (0.15–15 nM) for 2 h before addition of IL-1ß (0.1–10 ng/ml) and maintained overnight at 37 C. The next morning, islets were gently transferred to RPMI medium with 3 mmol/liter glucose and incubated for 1 h, at which time the medium was sampled for insulin measurement. The medium glucose concentration was then increased to 17 mmol/liter and the islets incubated for an additional 1 h. Insulin assay was performed using rat-specific ultra sensitive insulin ELISA kit (DRG Diagnostics, Mountainside, NJ). Using more than 10 islets for these studies gave insulin concentrations that were higher than the upper range measured by the kit.

To evaluate whether OPN binding to the RGD-integrin binding domain results in reduction of NO production and iNOS mRNA synthesis, islets were incubated for 1 h with either GRGDNP [Gly-Arg-Gly-Asp-Asn-Pro (glycine-arginine-glycine-aspartate-asparagine-proline)] peptide (1 mM) or GRADNP [Gly-Arg-Ala-Asp-Asn-Pro (glycine-arginine-alanine-aspartate-asparagine-proline)] (control) peptide (1 mM) (Biomol, Plymouth Meeting, PA), followed by a 2-h treatment with OPN (15 nM). IL-1ß (0.1 ng/ml) was added for 24 h, after which time media and islets were harvested for NO assay and iNOS mRNA analysis. All concentrations were used according to our preliminary concentration studies with references nitrite levels.

Transient transfection
To further understand the mechanisms of OPN action in ß-cells, we used the ß-cell line, RIN, clone 5F (RINm5F), an insulinoma cell line derived from the NEDH (New England Deaconess Hospital) rat islet cell tumor (American Type Culture Collection, Manassas, VA). RINm5F cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 1 mmol/liter sodium pyruvate, 10% FCS, and 50 µmol/liter ß-mercaptoethanol and antibiotics (100 U/ml penicillin and 100 U/ml streptomycin), and maintained at 37 C in humidified air containing 5% CO2. pGEM4–2rcDNA plasmid containing the full-length cDNA sequence that encodes mouse OPN was a generous gift form Dr. David Denhardt (Rutgers University, New Brunswick, NJ). RINm5F cells were plated at a concentration of 2 x 105/ml. At approximately 80% confluence, cells were depleted in a medium containing 0.1% BSA for 18 h. Cells were transfected using cationic liposome reagent TransFast (Promega, Madison, WI), with 10 µg of pGEM4–2rcDNA or with the empty vector. In addition, we performed parallel transfection experiments with a pGEM4/enhanced green fluorescent protein (EGFP) plasmid and counted EGFP-expressing cells vs. total cell number to obtain an estimate of transfection efficiency (data not shown). We estimated our transfection efficiency at approximately 80%. After 3 h, the medium volume was increased to 2 ml with RMPI-1640 plus 10% FCS. The RINm5F cells were cultured for 18 h overnight, at which time the medium was changed to RPMI-1640 with 11 mmol/liter D-glucose plus 0.1% BSA and incubated for an additional 18–24 h. IL-1ß was added on the following day, and the cells and media were harvested after 18–24 h. We performed RT-PCR and ELISA to compare the OPN mRNA and protein levels in control and OPN-transfected cells.

ELISA
OPN levels in the media from OPN-transfected (OPN+) and control cells were measured using rat-specific ELISA kit (Assay Design, Ann Arbor, MI). Spectrophotometric evaluation of OPN levels were made by Synergy HT multidetection microplate reader (BioTeck, Winooski, VT)

[3-(4,5-Dimethylthiazolyl-2)-2,5-dimethyl tetrazolium bromide] (MTT) assay
OPN+ and control RINm5F cells were depleted in a medium containing 0.1% BSA for 18 h. The cells were then gently washed in PBS, and depletion medium was added back. At that time, IL-1ß at 1–10 ng/ml was added. Cell viability was examined using MTT assay (Sigma). The concentrations used were according to our preliminary concentration studies with reference to cell viability.

NO determination
In aqueous solution, NO is rapidly converted to nitrate and nitrite. The commercial kit we used (Calbiochem, La Jolla, CA) includes a nitrate reductase step that converts nitrate to nitrite before quantitation using Griess reagent. Nitrite measurement was performed as an indirect measure of NO production in rat pancreatic islets and ß-cells. Spectrophotometric evaluation of nitrite levels was made by Synergy HT multidetection microplate reader (BioTeck).

RNA isolation and semiquantitative RT-PCR
Total RNA was isolated using the spin or vacuum total RNA isolation system (Promega) according to the manufacturer’s protocol. Oligo(deoxythymidine)15 (Promega) primed cDNA was synthesized from 3.5 µg of total RNA using murine Moloney leukemia virus reverse transcriptase (Invitrogen, Carlsbad, CA) at 37 C for 60 min. Samples were incubated at 90 C for 5 min to terminate the reverse-transcription reaction. The cDNA mixtures (2 µl) were subjected to PCR using AmpliTaq gold DNA polymerase (PE Biosystems, Wellesley, MA). Upstream and downstream primers for iNOS: 5'-TCCGGGCAGCCTGTGAGACG3' and 5'GCTGGGTGGGAGGGGTAGTGATGT-3'.

Upstream and downstream primers that could anneal with the 3'-untranslated region of rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were included in the PCR as an internal standard: 5'-GCATGGCCTTCCGTGTTCCTACC-3' and 5'-GCCGCCTGCTTCACCACCTTCT-3'. The following conditions were used: 50 sec at 94 C, 90 sec at 55 C and 150 sec at 72 C, with a 7-min final extension at 72 C after 35 cycles.

OPN mRNA levels were analyzed by RT-PCR. OPN primers were designed according to the published sequence of rat OPN cDNA: 5'-AAGGCGCATTACAGCAAACACTCA-3' and 5'-CTCATCGGACTCCTGGCTCTTCAT-3'.

The linear range of amplification for each set of primers was determined to ensure that we used a number of cycles in the linear range. The densitometry used was ensured to provide a linear response. PCR products were electrophoresed on 2% agarose gels and band intensities were quantified using Kodak Electrophoresis Documentation and Analysis System 290 (EDAS 290; Kodak, New Haven, CT).

Sequence determination
PCR bands were purified from the agarose gel using the Geneclean II kit (BI 101 Inc., Carlsbad, CA) according to the manufacturer’s protocol. Purified products were directly sequenced after estimating the concentration of DNA Products. Sequences were aligned with published sequences using MegAlign sequence analysis software (DNASTAR, Inc., Madison, WI) to confirm their identity.

Promoter studies
Quiescent control and OPN+ cells were obtained after 18 h incubation in serum-free medium. The rat iNOS promoter (piNOS-1002luc) containing nucleotides –1002 to +132, which are required for maximal IL-1ß-induced iNOS activation in rat insulin-producing cells, was kindly provided by Dr. Decio Eizirik (Free University Brussels, Belgium) (25, 26). At approximately 80% confluence, cells were cotransfected by TransFast reagent (Promega) and 0.5 µg of the vector containing the rat luciferase-labeled iNOS promoter and 0.1 µg of green fluorescent protein as transfection control. Two hours later, serum-containing medium was be overlaid and the cells were incubated for additional 24 h. The cells then were incubated with serum-free medium for 16 h followed by addition of IL-1ß (1 ng/ml) for 3 h.

To assess the effect of NO on OPN transcription, transfection studies were conducted using the rat OPN promoter, (–1984luc) (GenBank accession no. AF017274) in a luciferase expression vector pGL2 basic (Promega), kindly provided by Dr. S. Mori (Chiba University, Chiba, Japan) (27, 28). We also used N-{omega}-nitro-L-arginine methyl ester (L-NAME), a pharmacological inhibitor of iNOS activity or the NO donor, S-nitroso-N-acetylpenicillamine (SNAP), or a combination of them to determine whether activation of OPN promoter is specific to IL-1ß-induced NO. Cells were treated with the following: IL-1ß plus L-NAME (40 µM), or L-NAME alone, or SNAP alone (50 µM), or IL-1ß plus L-NAME (40 µM) plus SNAP, or IL-1ß plus SNAP. Luciferase activities were assayed with the Dual-Luciferase Reporter Assay System (Promega) in a TD-20/20 Luminometer. Transfection efficiency was normalized using the total protein concentration of the cell lysates. Relative luciferase activity was calculated after deduction of the activity levels with the vector alone.

Western immunoblotting
Western blot analysis was performed essentially as described previously (15). Islets and ß-cells from the different studies were lysed in modified RIPA lysis buffer, and the protein concentrations in the supernatant were determined using the bicinchoninic acid protein assay reagent (Pierce, Rockford, IL). Equal protein concentrations (50 µg) were denatured in a gel loading buffer at 100 C for 5 min and then loaded onto 10% sodium dodecyl sulfate-polyacrylamide slab gels and transferred to polyvinylidene difluoride membranes and incubated at 4 C overnight with primary antibodies diluted in PBS-Tween 20 (PBST): anti-OPN, anti-I{kappa}B{alpha} (Santa Cruz Biotechnology, Santa Cruz, CA), and antiactin (Chemicon, Temecula, CA). The blots were washed and incubated with horseradish peroxidase-conjugated secondary antibodies. The protein bands were visualized with enhanced chemiluminescence reagents (ECL Plus Western Blotting Detection System; Amersham Pharmacia Biotech, Piscataway, NJ).

Immunofluorescent staining
RINm5F cells were grown onto cover glass (Fisher Scientific, Pittsburgh, PA) at a density of 5 x 104 cells/cover glass. To study the localization of p65 nuclear factor (NF)-{kappa}B subunit, serum-starved OPN+ or control cells were incubated with IL-1ß (1 ng/ml) for 1 h. The immunofluorescent staining was performed as described previously (15). The cells were washed, fixed in 2% paraformaldehyde in PBS for 15 min, and were then treated with 0.1% Triton X-100 for 30 sec to permeabilize nuclear membranes. After blocking nonspecific reaction with normal donkey serum, the cells were incubated overnight with antirabbit p65 IgG (400 ng/ml) 4 C. Subsequently, Texas Red-conjugated goat antirabbit IgG was applied on the cells as a secondary antibody. Fluorescence was visualized by Nikon fluorescence microscope (Nikon Co., Tokyo, Japan). Negative controls were stained with nonimmune serum or with the secondary antibody alone. Stained nuclei that were clearly seen were counted and the total number of nuclei per field was determined using ImagePro Image analysis software. The number of stained nuclei in 10 fields was averaged, and the data were calculated as the percentage of nuclear staining/total number of nuclei.

NF-{kappa}B activation assay
NF-{kappa}B activation was quantified using the StressXpress NF-{kappa}B kits to detect the active form of the p65 subunit (Stressgen, Victoria, Canada). Briefly, whole-cell extracts were prepared from 5 x 105 OPN-transfected or control cells that were subjected to IL-1ß (1 ng/ml) for 1 h, according to the manufacturer’s instructions. Protein concentrations of cell extracts were determined using bicinchoninic acid protein assay. Ten micrograms/well of cell extracts were incubated in a 96-well plate on which have been immobilized double-stranded oligonucleotides containing the consensus NF-{kappa}B DNA binding site (5-CACAGTTGAGGGGACTTTCCCAGGC-3). The primary antibody used in the kit to detect NF-{kappa}B recognized an epitope on p65 subunit that is accessible only when NF-{kappa}B is activated and bound to its target DNA. After incubation with a horseradish peroxidase-conjugated secondary antibody and the developing solution, absorbance was read at 450 nm with a reference wavelength of 655 nm using a Synergy HT multidetection luminometer.

Statistical analysis
All experiments were performed four to six times. Data were analyzed for statistical significance by ANOVA with post hoc Student’s t test analysis. These analyses were performed with the assistance of a computer program (JMP 5 Software; SAS, Cary, NC). Differences were considered significant at P ≤ 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
OPN prevents IL-1ß-induced rat islet dysfunction
We first established IL-1ß-mediated dose-dependent islet dysfunction. Untreated rat islets showed an approximately 7-fold increase in insulin secretion with 17 mmol/liter glucose, whereas IL-1ß-treated islets showed dose-dependent ß-cell dysfunction and lowered insulin surge at 17 mM (data not shown). Then the islets were pretreated with different doses of OPN for 2 h before the addition of IL-1ß (0.1 ng/ml). As shown in Fig. 1AGo, at 17 mM glucose, control untreated islets secreted 20.16 ± 7 µg/liter insulin · h–1 · 10 islets–1, whereas IL-1ß-treated islets secreted 4.86 ± 2.54 µg/liter insulin · h–1 · 10 islets–1 (P < 0.002). Islets treated with IL-1ß-plus OPN showed a dose-dependent increase in insulin secretion between 8.5 ± 2.9 µg/liter at 0.15 nM OPN and 30.1 ± 8.7 µg/liter insulin · h–1 · 10 islets–1 at 15 nM OPN (P < 0.02 vs. IL-1ß-treated islets), whereas islets treated with OPN alone did not show any alteration in GSIS compared with control islets (18.9 ± 2 µg insulin/liter · h–1 · 10 islets–1). Therefore, OPN was able to restore GSIS in cytokine-treated rat islets to 40–60% of their control value.


Figure 1
View larger version (24K):
[in this window]
[in a new window]

  		FIG. 1. A, OPN dose-dependently improves IL-1ß-induced rat islet dysfunction. GSIS in rat islets treated with IL-1ß (0.1 ng/ml) plus or minus OPN (0.15–15 nM). Untreated rat islets and ß-cells demonstrated an approximately 7-fold increase in insulin secretion with 17 mmol/liter glucose, whereas IL-1ß-treated islets had deficient insulin secretion. Addition of OPN showed a dose-dependent (40–60%) restoration of control values, whereas islets treated with OPN alone did not show any alteration in GSIS compared with control islets. Data are expressed as mean ± SEM. Each experiment was performed three times and repeated three times for reproducibility. *, P < 0.05 vs. 17 mmol/control islets; #, P < 0.05 vs. 17 mmol/liter IL-1ß-treated islets using one-way repeated ANOVA with subsequent all pairwise comparison procedure by Student’s t test. B, OPN-mediated regulation of IL-1ß-induced NO production regulation is RGD dependent. Cytokine induced NO production in the presence of exogenous GRGDSP (1 mM), GRADSP (1 mM), and OPN (15 nM). GRGDSP was added before addition of OPN. Islets were incubated with IL-1ß for a period of 24 h. GRGDSP (1 mM) inhibits OPN-mediated NO regulation. *, P < 0.05 vs. IL-ß-induced induction; #, P < 0.05 vs. OPN + GRGDSP-treated islets. Values are expressed as mean ± SEM of three experiments. RAD, Arginine-alanine-aspartate-control peptide. C, PCR analysis of iNOS and GAPDH mRNA transcripts from islets pretreated with GRGDSP or GRADSP with or without OPN. Addition of the GRGDSP peptide to the islets blocked OPN-mediated reduction of iNOS mRNA. The 492- and 109-bp bands correspond to the amplified iNOS and GAPDH, respectively. Values are expressed as mean ± SEM of three experiments. *, P < 0.05 vs. untreated cells; #, P < 0.05 vs. IL-1ß-treated islets using one-way repeated ANOVA with subsequent all pairwise comparison procedure by Student’s t test.

 
OPN causes an RGD-dependent inhibition of IL-1ß-induced NO and iNOS production
OPN contains an RGD integrin binding domain (13, 19). The hexapeptide, GRGDSP, which blocks binding of OPN to cell surface integrins (23), was used to determine the receptor-mediated effects of OPN. Islets were pretreated with GRGDSP-hexa peptide or control peptide (1 mM) for 1 h before addition of OPN for 2 h. In comparison to islets treated with control peptide, GRGDSP was found to significantly inhibit OPN-mediated decrease of nitrite production (IL-1ß, 3.4 ± 0.1; IL-1ß + OPN, 1.3 ± 0.1; RGD+ IL-1ß+ OPN, 3.0 ± 0.1-fold) (Fig. 1BGo). Similarly, OPN-mediated reduction of iNOS synthesis in the islets was inhibited by pretreatment with GRGDSP-hexa peptide (Fig. 1CGo), suggesting that OPN-integrin receptor binding is required for OPN-mediated NO and iNOS regulation.

To understand the mechanisms of OPN action, we transiently transfected RINm5F cells with the pGEM4–2rcDNA plasmid containing mouse OPN gene. OPN mRNA by RT-PCR (Fig. 2Go, top) and protein levels by ELISA (Fig. 2Go, bottom) shows about 5-fold increase in OPN expression levels. IL-1ß (1–10 ng/ml) was added to control, OPN-transfected, and empty vector quiescent cells for 24 h, at which time the cells were harvested and analyzed for cell viability, nitrite levels, and iNOS mRNA. From here on, OPN-transfected cells will be called OPN+ cells.


Figure 2
View larger version (40K):
[in this window]
[in a new window]

  		FIG. 2. Transient transfection of OPN in RINm5F cells. Top, Total RNA was extracted from control and transiently transfected RINm5F cells (OPN+). RT-PCR was performed using specific OPN and GAPDH primers. PCR products were analyzed on 2% agarose gel and identified with ethidium bromide staining. Bottom, Medium OPN levels were examined from cultures of control and OPN+ RINm5F cells using rat-specific ELISA kit. OPN+ cells express approximately 5-fold higher OPN levels. *, P < 0.05 vs. control cells.

 
OPN prevents IL-1ß-impaired ß-cell viability
The viability, determined by the MTT assay, of the RINm5F cells exposed to IL-1ß (1–10 ng/ml) for 72 h was significantly reduced by approximately 50% compared with the control condition (P < 0.003; Fig. 3AGo). The toxic effect of IL-1ß at these concentrations was fully prevented in OPN+ cells. OPN expression per se did not influence the viability of the RINm5F cells as seen from the control conditions. Parallel control experiments performed on the parental RINm5F cell line demonstrated that the empty vector control by itself did not influence the IL-1ß-induced toxicity (data not shown).


Figure 3
Figure 3
View larger version (34K):
[in this window]
[in a new window]

  		FIG. 3. OPN improves the viability and reduces NO and iNOS production in RINm5F cells. OPN+ and control RINm5F cells were treated with IL-1ß at 1–10 ng/ml for 72 h. A, Cell viability was examined using MTT assay. Significant improvement of islet viability can be seen in OPN+ cells. *, P < 0.05; **, P < 0.01 vs. control + IL-1ß values. B, Nitrite measurement was performed as an indirect measure of NO production in the media collected from the cells. Significant reduction in NO levels can be seen in OPN+ cells. C, PCR analysis of iNOS and GAPDH mRNA transcripts. Significant inhibition of iNOS synthesis can be seen in OPN+ cells treated with IL-1ß. The 492- and 109-bp bands correspond to the amplified iNOS and GAPDH, respectively. *, P ≤ 0.05 vs. control + IL-1ß values. D, A dose-dependent IL-1ß-induced iNOS promoter activity is significantly inhibited in OPN+ cells. Data are expressed as mean ± SEM. Each experiment was performed in duplicate and repeated four times for reproducibility. *, P < 0.01 vs. control + IL-1ß values, using one-way repeated ANOVA with subsequent all pair-wise comparison procedure by Student’s t test.

 
OPN overexpression reduces IL-1ß-mediated NO production
As shown in Fig. 3BGo, control RINm5F cells generated 1.3 ± 0.02 µmol of nitrite per 106 cells, whereas control cells treated with IL-1ß generated 2.6 ± 0.04 µmol of nitrite in 48 h (P = 0.0006 vs. control). The toxic effect of both IL-1ß at this concentration was fully prevented in OPN+ cells. In OPN+ cells, the high nitrite levels induced by IL-1ß were reduced to 1.1 ± 0.03 µmol nitrite, which was significantly less than IL-1ß-treated control cells. Overexpression of OPN by itself did not show any difference in nitrite production when compared with control cells (1.2 ± 0.02 µmol · 106 cells –1 · 48 h–1).

OPN reduces IL-1ß-induced iNOS gene expression
We performed semiquantitative PCR analysis to further confirm whether OPN-mediated cytoprotection involves iNOS gene expression. The IL-1ß (1 ng/ml) for 24-h-mediated induction of iNOS gene expression was significantly reduced in OPN+ cells (Fig. 3CGo). OPN expression per se did not influence iNOS gene expression as seen from the control conditions.

OPN reduces IL-1ß-induced iNOS promoter activity
INOS promoter activity was determined in control and OPN+ cells after their exposure to different concentrations of IL-1ß. As shown in Fig. 3DGo, after 24 h, the dose-dependent IL-1ß-mediated induction of iNOS promoter activity was significantly inhibited in OPN+ cells: (IL-1ß 0.1, 1, 10 ng/ml) 100% vs. 12% ± 1%, 8% ± 1%, and 5% ± 1% P < 0.01 in the control or OPN+ cells, respectively, suggesting a direct inhibition of IL-1ß signaling. The empty vector alone did not influence the IL-1ß-induced promoter activity induced in the parental RINm5F cell-line (data not shown). OPN expression per se did not significantly reduce the promoter activity.

OPN inhibits NF-{kappa}B signaling in RINm5F cells
Activation of transcription factor NF-{kappa}B is an essential step for the IL-1ß-induced iNOS expression. We investigated whether OPN mediates its NO-regulatory effect through inhibition of IL-1ß-induced NF-{kappa}B activation. First, the degradation of I{kappa}B{alpha} protein should be requisite for the binding of NF-{kappa}B to {kappa}B sites in the promoter region. We evaluated p65 nuclear translocation in control and OPN+ after their treatment with IL-1ß by three independent assays: Western analysis, nuclear immunolocalization, and p65 NF-{kappa}B activity assay. OPN inhibited the IL-1ß-induced I{kappa}B{alpha} protein degradation (4A). Cultured cells were visualized by fluorescence microscopy (Nikon) and images were analyzed with Image Pro analysis Image analysis software. We localized the p65 NF-{kappa}B subunit in RINm5F cells (Fig. 4BGo). The number of clearly stained nuclei in 10 fields was averaged, and the data were calculated as the percentage of nuclear staining/total number of nuclei. In the control untreated and OPN+ cells, a diffuse cytoplasmic staining was observed, whereas cells treated with IL-1ß had a clear nuclear staining, indicating nuclear translocation of p65. Presence of OPN in the cells significantly (P < 0.05) prevented the IL-1ß-induced nuclear translocation of p65 in ß-cells (Fig. 4BGo) (OPN+ cells + IL-1ß, 7 ± 0.7 stained nuclei/105 nuclei; Control + IL-1ß 95 ± 21 stained/105 nuclei. This inhibitory effect of OPN on the IL-1ß-mediated activation of NF-{kappa}B was verified in lysates from the RINm5F ß-cells subjected to IL-1ß for 1 h, with or without OPN. Cells were analyzed for the presence of the active forms of NF-{kappa}B p65 using the StressXpress ELISA kits. The assay uses streptavidin-coated plates with bound NF-{kappa}B biotinylated-consensus sequence to capture only the active form of NF-{kappa}B. IL-1ß-stimulated RINm5F 6302 ± 271 chemiluminescence arbitrary units, whereas control and IL-1ß plus OPN (15 nM)-treated RINm5F cells demonstrated 2895 ± 87 U and 2910 ± 117 U, respectively (P < 0.0001 vs. IL-1ß-treated RINm5F cells) (Fig. 4CGo). OPN+ cells showed chemiluminescence levels similar to control untreated cells (2732 ± 76 chemiluminescence arbitrary units). These findings suggest that one mechanism by which OPN prevents the IL-1ß-induced iNOS expression in RINm5F cells is through the inhibition of the IL-1ß-mediated NF-{kappa}B activation and nuclear translocation.


Figure 4
View larger version (22K):
[in this window]
[in a new window]

  		FIG. 4. OPN inhibits IL-1ß-mediated activation of NF-{kappa}B. OPN+ and control RINm5F cells were treated with IL-1ß at 1 ng/ml for 1 h. A, Total cellular proteins (25 µg) were separated on a 8% SDS-PAGE and the levels of I{kappa}B{alpha} protein (37 kDa) were measured by Western blot analysis. Equal loading of protein was verified by probing the same blot for actin (43 kDa). B, Immunofluorescent staining for p65 NF-{kappa}B subunit showing exclusive cytoplasmic staining in the control and OPN+ cells. Control cells treated with IL-1ß show intense nuclear staining, whereas OPN+ cells show inhibition of the IL-1ß-induced nuclear translocation in most of the cells (white arrows), whereas few cells retained their nuclear staining (black arrows). C, Lysates from the RINm5F ß-cells subjected to IL-1ß for 1 h, with or without OPN, were analyzed for the presence of the active forms of NF-{kappa}B p65 using the StressXpress ELISA kits. OPN+ cells demonstrated significant down-regulation of the IL-1ß-mediated NF-{kappa}B p65 activation. Values are expressed as mean ± SEM of three experiments. *, P ≤ 0.05 vs. control + IL-1ß values, using one-way repeated ANOVA with subsequent all pair-wise comparison procedure by Student’s t test.

 
IL-1ß-induced OPN mRNA and protein expression in rat islets and RINm5F cells
To explore whether OPN might be a component in the native islet defense system against the toxic effect of NO, as demonstrated in other systems (19), isolated rat pancreatic islets were exposed for 24 h to IL-1ß (0.1 ng/ml). The level of OPN mRNA steady-state expression as percent of the internal standard GAPDH was evaluated. In response to IL-1ß a statistically significant induction of OPN mRNA expression was observed after a 24-h IL-1ß exposure (ANOVA, P = 0.001; Fig. 5AGo). Western blotting of the islets treated with the same dose of IL-1ß revealed significant increase in OPN protein expression (Fig. 5BGo). In RINm5F cells, exposure to IL-1ß (1 ng/ml) induced a time-dependent induction of OPN mRNA expression (between 3 and 6 h) (Fig. 5CGo).


Figure 5
View larger version (65K):
[in this window]
[in a new window]

  		FIG. 5. Effect of IL-1ß on OPN expression. A, OPN mRNA expression in rat islets exposed IL-1ß (0.1 ng/ml) for 24 h. Semiquantitative analysis of OPN mRNA expression relative to the internal standard GAPDH was performed. Data are mean ± SEM of four separate experiments. *, P < 0.05 vs. control at 24 h. B, Representative Western immunoblot of protein extracts from islets treated with IL-1ß. Islet OPN protein is expressed as one band at approximately 65 kDa. IL-1ß induces significant increase in OPN protein expression in the islets. Blots were stripped and reprobed with actin antibody to control for loading errors. Average densitometry values of the samples were multiplied to obtain the arbitrary levels. Data are means ± SEM of n = 4 in each group. *, P < 0.05 vs. untreated islets. C, Semiquantitative analysis of OPN mRNA expression relative to the internal standard GAPDH was performed in RINm5F cells exposed to IL-1ß showing early up-regulation of OPN mRNA between 3 and 6 h. Data are mean ± SEM of three separate experiments. *, P < 0.05 vs. control at 24 h.

 
IL-1ß and IL-1ß-mediated NO production induces OPN transcription
To determine whether increased OPN gene expression is the result of increased OPN promoter activity, transient transfection analysis was performed in RINm5F cells using a plasmid construct in which OPN promoter was cloned upstream from a luciferase reporter gene (Fig. 6Go). In the presence of IL-1ß, normalized luciferase activity was approximately 6.5-fold greater than that of unstimulated controls (P < 0.01). In IL-1ß plus the iNOS inhibitor L-NAME-treated cells, OPN promoter activity was not significantly different from that of control or L-NAME-treated cells. Restoration of NO levels with the NO donor SNAP resulted in luciferase activity that was 9-fold greater than that of controls (P < 0.001). These results suggest that IL-1ß-induced NO production increases OPN promoter activity with resultant increases in OPN mRNA and protein levels.


Figure 6
View larger version (22K):
[in this window]
[in a new window]

  		FIG. 6. Effect of IL-1ß-mediated NO production on activity of OPN promoter plasmid transfected into RINm5F cells. Relative luciferase activity was calculated after deduction of the activity levels with pGL2 vector alone. Results represent mean ± SEM of triplicate determinations. All experiments were repeated at least three times to confirm the reproducibility (*, P < 0.01 IL-1ß vs. control, IL-1ß plus L-NAME, and L-NAME; *, P < 0.01 IL-1ß-plus L-NAME plus SNAP vs. control, IL-1ß plus L-NAME, and L-NAME; #, P < 0.001 IL-1ß-plus SNAP vs. control by Student’s t test).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Several mechanisms may regulate the ß-cell response to cytokines, including antagonists such as specific binding proteins and soluble receptors, other cytokines, growth factors, and hormones (29, 30). In this study, we show for the first time that OPN might represent an endogenous, regulated, negative feedback system for IL-1ß signaling.

OPN is a highly hydrophilic and negatively charged sialoprotein of approximately 298 amino acids that contains a Gly-Arg-Gly-Asp-Ser sequence. OPN exists both intra- and extracellularly. Recently, data from our lab have shown that OPN is expressed in the pancreatic islets of the rat and its pancreatic levels are acutely up-regulated in response to streptozotocin-induced diabetes. We also demonstrated that OPN protects the islets from streptozotocin-induced NO production (23).

In ß-cells, proinflammatory cytokines induce iNOS expression and NO production, leading to impairment of secretory function and cell death. IL-1ß has been shown to inhibit mitochondrial aconitase activity and the oxidation of glucose to CO2, leading to reduction of cellular ATP levels. NO mediates the inhibitory effects of IL-1ß on insulin release through this mitochondrial dysfunction (31, 32, 33). Our data show that OPN restored, although not completely, insulin secretion from islets treated with IL-1ß (Fig. 1AGo). We show also that prior addition of OPN to the islets provided protection against IL-1ß cytotoxic effects partly by reducing NO production and inhibition of iNOS mRNA synthesis. The anti-iNOS effect of OPN appears to be mediated by a membrane-bound integrin receptor because this effect was reversed when a peptide that blocks the integrin receptor was added (Fig. 1BGo). In ß-cell lines transiently transfected with OPN the activation of iNOS promoter by IL-1ß was inhibited and iNOS mRNA levels were reduced resulting in approximately 50% decreased formation of toxic NO in ß-cells (Fig. 3Go). These data are in line with similar results in murine macrophages, where exogenous recombinant OPN protein was effective in blocking NO production and cytotoxicity toward the NO-sensitive mastocytoma cells (22). Their work suggested that OPN in extracellular fluid might protect certain tumor cells from macrophage-mediated destruction by inhibiting the synthesis of NO. Singh et al. (21, 22) reported that a synthetic 20-amino acid OPN peptide analog decreased iNOS mRNA and protein levels in ventricular myocytes and cardiac microvascular endothelial cells. In primary mouse kidney proximal tubule epithelial cells, OPN suppressed NO synthesis induced by interferon-{gamma} and lipopolysaccharide, suggesting a regulatory role for OPN in the NO signaling pathway (18).

Because intracellular OPN up-regulation protected from IL-1ß-mediated impairment of cell viability (Fig. 3AGo) and inhibited iNOS transcription (Fig. 3Go, C and D), we studied the transcription factor NF-{kappa}B as a possible target for OPN-mediated effects. NF-{kappa}B comprises a collection of dimers composed of various combinations of members of the Rel family. Five mammalian Rel proteins have been identified: p50, p52, c-Rel, p65 (RelA), and RelB. NF-{kappa}B is sequestered in the cytoplasm by binding to inhibitor protein {kappa} B{alpha} (I{kappa}B{alpha}). After cytokine exposure, I{kappa}B{alpha} is phosphorylated, ubiquitinated and degraded by the proteasomal complex exposing NF-{kappa}B nuclear recognition site to translocate to the nucleus and bind {kappa}B consensus sequences in promoter regions of numerous proinflammatory genes such as iNOS (34). IL-1ß activates NF-{kappa}B in rodent (35) and human (36) islet cells, and blocking NF-{kappa}B activation prevents cytokine-induced apoptosis in these cells (37). Our data demonstrate, for the first time, that OPN inhibits the IL-1ß-induced activation of the NF-{kappa}B (Fig. 4CGo). OPN inhibited the degradation of I{kappa}B{alpha} (Fig. 4AGo) and the consequent translocation of NF-{kappa}B to the nucleus (Fig. 4BGo). These effects could explain the anticytotoxic state induced by OPN when IL-1ß is present. Alternatively, OPN-induced inhibition of NF-{kappa}B activity may prevent transcription of NF-{kappa}B-dependent iNOS gene transcription.

Although the regulation of iNOS has been examined at many levels, little is known of its negative feedback regulatory systems. In the mouse macrophages, studies have shown that endogenously produced NO inhibits posttranslational assembly of dimeric iNOS by down-regulating heme insertion and availability (38). Additional studies have shown that NO inhibits DNA binding of NF-{kappa}B and down-regulates iNOS gene transcription (39). NO can also directly inhibit catalysis by binding to the iNOS heme iron to form an inactive iron-nitrosyl complex (40). Furthermore, NO S-nitrosylates a key active site cysteine residue in the NF-{kappa}B p50 DNA binding domain and inhibits subsequent DNA binding and iNOS promoter activity in macrophages (40). Results of our studies suggest that NO itself could regulate itself by induction of OPN transcription.

However, the details of the mechanism by which IL-1ß and NO up-regulate OPN promoter activity are still unclear. Analysis of the murine OPN promoter demonstrated the presence of potential binding sites whose corresponding transcription factor activities are modified by NO, such as activating protein-1 (41). NO may induce binding of NO-sensitive transcription factors to the promoter or an enhancer region. Alternatively, NO and Il-1ß may induce changes in the secondary and tertiary structure of the promoter. Studies addressing transcription factor binding are ongoing in our laboratory.

Our study demonstrates that OPN inhibits NO production and improves islet function in the setting of IL-1ß stimulation. OPN mediates its effects through binding to {alpha}vß3 integrins, and deactivation of IL-1ß-induced NF-{kappa}B activity. OPN promoter activity and gene transcription are significantly up-regulated in the presence of IL-1ß-mediated NO production.

The existence of OPN as a potential endogenous negative feedback protective factor against IL-1ß-induced cytotoxicity in the islets is unique and suggests potential targets for modulation of the NO-dependent and other components of the inflammatory response. Because inflammatory cells release cytotoxic cytokines during the immune response in islet transplantation, influencing OPN expression may represent an early intervention for affecting cytokine-induced signal transduction to prevent or reduce activation of the cascade of downstream devastating effects during islet transplantation.


   Acknowledgments
 
We thank Dr. William Butler (University of Texas, Houston, TX) for providing native OPN protein, Dr. David Denhardt (Rutgers University, New Brunswick, NJ) for providing the murine OPN cDNA, Dr. Decio Eizirik (Free University, Brussels, Belgium) for providing the rat iNOS promoter construct, and Dr. Seijiro Mori (Chiba University, Chiba, Japan) for providing the OPN promoter construct.


   Footnotes
 
This work was supported by grants from the American Diabetes Association 1-05-JF-01, Diabetes Transplant Fund, and Diabetes Action, Research and Education Foundation.

The authors have nothing to declare.

First Published Online November 16, 2006

Abbreviations: FCS, Fetal calf serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GRADNP, Gly-Arg-Ala-Asp-Asn-Pro (glycine-arginine-alanine-aspartate-asparagine-proline); GRGDNP, Gly-Arg-Gly-Asp-Asn-Pro (glycine-arginine-glycine-aspartate-asparagine-proline); GSIS, glucose-stimulated insulin secretion; HBSS, Hanks’ balanced salt solution; I{kappa}B{alpha}, inhibitor protein {kappa} B{alpha}; iNOS, induced NO synthase; L-NAME, N-{omega}-nitro-L-arginine methyl ester; MTT, [3-(4,5-dimethylthiazolyl-2)-2,5-dimethyl tetrazolium bromide]; NF, nuclear factor; NO, nitric oxide; OPN, osteopontin; PBST, PBS-Tween 20; RGD, arginine-glycine-aspartate; SNAP, S-nitroso-N-acetylpenicillamine.

Received July 20, 2006.

Accepted for publication November 3, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Bretzel RG, Alejandro R, Hering BJ, van Suylichem PT, Ricordi C 1994 Clinical islet transplantation: guidelines for islet quality control. Transplant Proc 26:388–392[Medline]
  2. Shapiro AM, Lakey JR, Ryan EA, Korbutt GS, Toth E, Warnock GL, Kneteman NM, Rajotte RV 2000 Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 343:230–238[Abstract/Free Full Text]
  3. Ryan EA, Lakey JR, Paty BW, Imes S, Korbutt GS, Kneteman NM, Bigam D, Rajotte RV, Shapiro AM 2002 Successful islet transplantation: continued insulin reserve provides long-term glycemic control. Diabetes 1:2148–2157
  4. Kaufman DB, Platt JL, Rabe FL, Dunn DL, Bach FH, Sutherland DE 1990 Differential roles of Mac-1+ cells, and CD4+ and CD8+ T lymphocytes in primary nonfunction and classic rejection of islet allografts. J Exp Med 172:291–302[Abstract/Free Full Text]
  5. Gysemans CA, Waer M, Valckx D, Laureys JM, Mihkalsky D, Bouillon R, Mathieu C 2000 Early graft failure of xenogeneic islets in NOD mice is accompanied by high levels of interleukin-1 and low levels of transforming growth factor-ß mRNA in the grafts. Diabetes 49:1992–1997[Abstract/Free Full Text]
  6. Eizirik DL, Flodstrom M, Karlsen AE, Welsh N 1996 The harmony of the spheres: inducible nitric oxide synthase and related genes in pancreatic ß cells. Diabetologia 39:875–890[Medline]
  7. Bendtzen K, Mandrup-Poulsen T, Nerup J, Nielsen JH, Dinarello CA, Svenson M 1986 Cytotoxicity of human pI 7 interleukin-1 for pancreatic islets of Langerhans. Science 232:1545–1547[Abstract/Free Full Text]
  8. Southern C, Schulster D, Green IC 1990 Inhibition of insulin secretion by interleukin-1ß and tumour necrosis factor-{alpha} via an L-arginine-dependent nitric oxide generating mechanism. FEBS Lett 276:42–44[CrossRef][Medline]
  9. Thomas HE, Darwiche R, Corbett JA, Kay TW 2002 Interleukin-1 plus {gamma}-interferon-induced pancreatic ß-cell dysfunction is mediated by ß-cell nitric oxide production. Diabetes 51:311–316[Abstract/Free Full Text]
  10. Corbett JA, Sweetland MA, Wang JL, Lancaster Jr JR, McDaniel ML 1993 Nitric oxide mediates cytokine-induced inhibition of insulin secretion by human islets of Langerhans. Proc Natl Acad Sci USA 90:1731–1735[Abstract/Free Full Text]
  11. Eizirik DL, Pavlovic D 1997 Is there a role for nitric oxide in ß-cell dysfunction and damage in IDDM? Diabet Metab Rev 13:293–307[CrossRef][Medline]
  12. Denhardt DT, Noda M, O’Regan AW, Pavlin D, Berman JS 2001 Osteopontin as a means to cope with environmental insults: regulation of inflammation, tissue remodeling, and cell survival. J Clin Invest 107:1055–1061[Medline]
  13. Denhardt DT, Guo X 1993 Osteopontin: a protein with diverse functions. FASEB J 7:1475–1482[Abstract]
  14. Patarca R, Saavedra RA, Cantor H 1993 Molecular and cellular basis of genetic resistance to bacterial infection: the role of the early T-lymphocyte activation-1/osteopontin gene. Crit Rev Immunol 13:225–246[Medline]
  15. Arafat HA, Wein AJ, Chacko S 2002 Osteopontin gene expression and immunolocalization in the rabbit urinary tract. J Urol 167:746–752[CrossRef][Medline]
  16. Yu XQ, Fan JM, Nikolic-Paterson DJ, Yang N, Mu W, Pichler R, Johnson RJ, Atkins RC, Lan HY 1999 IL-1 up-regulates osteopontin expression in experimental crescentic glomerulonephritis in the rat. Am J Pathol 154:833–841[Abstract/Free Full Text]
  17. Attur, M G, Dave M N, Clancy RM, Patel IR, Abramson SB, Amin AR 2000 Functional genomic analysis in arthritis affected cartilage: yin-yang regulation of inflammatory mediators by integrins. J Immunol 164:2684–2691[Abstract/Free Full Text]
  18. Hwang SM, Lopez CA, Heck DE, Gardner CR, Laskin DL, Laskin JD, Denhardt DT 1994 Osteopontin inhibits induction of nitric oxide synthase gene expression by inflammatory mediators in mouse kidney epithelial cells. J Bi Chem 269:711–715
  19. Guo H, Cai CQ, Schroeder RA, Kuo PC 2001 Osteopontin is a negative feedback regulator of nitric oxide synthesis in murine macrophages. J Immunol 166:1079–1086[Abstract/Free Full Text]
  20. Rollo EE, Laskin DL, Denhardt DT 1996 Osteopontin inhibits nitric oxide production and cytotoxicity by activated RAW264.7 macrophages. J Leukocyte Biol 60:397–404[Abstract]
  21. Singh K, Sirokman G, Communal C, Robinson KG, Conrad CH, Brooks WW, Bing OHL, Colucci WS 1999 Myocardial osteopontin expression coincides with the development of heart failure. Hypertension 33:663–670[Abstract/Free Full Text]
  22. Singh K, Balligand JL, Fischer TA, Smith TW, Kelly RA 1995 Glucocorticoids increase osteopontin expression in cardiac myocytes and microvascular endothelial cells. J Biol Chem 270:28471–28478[Abstract/Free Full Text]
  23. Katakam AK, Chipitsyna G, Gong Q, Vancha AR, Gabbeta J, Arafat HA 2005 Streptozotocin (STZ) mediates acute upregulation of serum and pancreatic osteopontin (OPN): a novel islet-protective effect of OPN through inhibition of STZ-induced nitric oxide production. J Endocrinol 187:237–247[Abstract/Free Full Text]
  24. Hill DJ, Petrik J, Arany E, McDonald TJ, Delovitch TL 1999 Insulin-like growth factors prevent cytokine-mediated cell death in isolated islets of Langerhans from pre-diabetic non-obese diabetic mice. J Endocrinol 16:153–165
  25. Darville MI, Eizirik DL 1998 Regulation by cytokines of the inducible nitric oxide synthase promoter in insulin-producing cells. Diabetologia 41:1101–1108[CrossRef][Medline]
  26. Darville MI, Ho YS, Eizirik DL 2000 NF-{kappa}B is required for cytokine-induced manganese superoxide dismutase expression in insulin-producing cells. Endocrinology 141:153–162[Abstract/Free Full Text]
  27. Takemoto M, Yokote K, Nishimura M, Shigematsu T, Hasegawa T, Kon S, Uede T, Matsumoto T, Saito Y, Mori S 2000 Enhanced expression of osteopontin in human diabetic artery and analysis of its functional role in accelerated atherogenesis. Arterioscler ThrombVasc Biol 3:624–628
  28. Asaumi S, Takemoto M, Yokote K, Ridall AL, Butler WT, Fujimoto M, Kobayashi K, Kawamura H, Take A, Saito Y, Mori S 2003 Identification and characterization of high glucose and glucosamine responsive element in the rat osteopontin promoter. J Diabetes Complications 17:34–38[CrossRef][Medline]
  29. Mandrup-Poulsen T, Nerup J, Reimers JI, Pociot F, Andersen HU, Karlsen A, Bjerre U, Bergholdt R 1995 Cytokines and the endocrine system. I. The immunoendocrine network. Eur J Endocrinol 133:660–671[Abstract/Free Full Text]
  30. Dinarello CA 1998 Interleukin-1, interleukin-1 receptors and interleukin-1 receptor antagonist. Int Rev Immunol 16:457–499[Medline]
  31. Bossy-Wetzel E, Lipton SA 2003 Nitric oxide signaling regulates mitochondrial number and function. Cell Death Differ 10:757–760[CrossRef][Medline]
  32. Delaney CA, Tyrberg B, Bouwens L, Vaghef H, Hellman B, Eizirik DL 1996 Sensitivity of human pancreatic islets to peroxynitrite-induced cell dysfunction and death. FEBS Lett 394:300–306[CrossRef][Medline]
  33. Delaney CA, Eizirik DL 1996 Intracellular targets for nitric oxide toxicity to pancreatic ß-cells. Braz J Med Biol Res 29:569–579[Medline]
  34. Baldwin AS 1996 The NF-{kappa}-b and I-{kappa}-b proteins—new discoveries and insights. Annu Rev Immunol 14:649–683[CrossRef][Medline]
  35. Saldeen J, Welsh N 1994 Interleukin-1ß induced activation of NF-{kappa}B in insulin producing RINm5F cells is prevented by the protease inhibitor N {alpha}-p-tosyl-L-lysine chloromethylketone. Biochem Biophys Res Commun 203:149–155[CrossRef][Medline]
  36. Flodstrom M, Welsh N, Eizirik DL 1996 Cytokines activate the nuclear factor {kappa}B (NF-{kappa}B) and induce nitric oxide production in human pancreatic islets. FEBS Lett 385:4–6[CrossRef][Medline]
  37. Giannoukakis N, Rudert WA, Trucco M, Robbins PD 2000 Protection of human islets from the effects of interleukin-1ß by adenoviral gene transfer of an I{kappa}B repressor. J Biol Chem 275:36509–36513[Abstract/Free Full Text]
  38. Albakri, QA, Stuehr DJ 1996 Intracellular assembly of inducible NO synthase is limited by nitric oxide mediated changes in heme insertion and availability. J Biol Chem 271:5414–5421[Abstract/Free Full Text]
  39. Park SK, Lin H L, Murphy S 1997 Nitric oxide regulates nitric oxide synthase-2 gene expression by inhibiting NF-{kappa}B binding to DNA. Biochem J 322:609–613
  40. DelaTorre A, Schroeder R A, Punzalan C, Kuo PC 1999 Endotoxin mediated S-nitrosylation of p50 alters NF-{kappa}B dependent gene transcription in ANA-1 murine macrophages. J Immunol 162:4101–4108[Abstract/Free Full Text]
  41. Guo X, Zhang YP, Mitchell DA, Denhardt DT, Chambers A F 1995 Identification of a ras-activated enhancer in the mouse osteopontin promoter and its interaction with a putative ETS-related transcription factor whose activity correlates with the metastatic potential of the cell. Mol Cell Biol 15:476–487[Abstract]



This article has been cited by other articles:


Home page
 IOVSHome page
G. Chidlow, J. P. M. Wood, J. Manavis, N. N. Osborne, and R. J. Casson
Expression of Osteopontin in the Rat Retina: Effects of Excitotoxic and Ischemic Injuries
Invest. Ophthalmol. Vis. Sci., February 1, 2008; 49(2): 762 - 771.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
K. X. Wang, Y. Shi, and D. T. Denhardt
Osteopontin regulates hindlimb-unloading-induced lymphoid organ atrophy and weight loss by modulating corticosteroid production
PNAS, September 11, 2007; 104(37): 14777 - 14782.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Arafat, H. A.
Right arrow Articles by Dafoe, D. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Arafat, H. A.
Right arrow Articles by Dafoe, D. C.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene
*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*NITRIC OXIDE

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals