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

in Journal of Endocrinology
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  • Departments of Surgery, Pathology, Anatomy & Cell Biology, Thomas Jefferson University, 1015 Walnut Street, Philadelphia, PA 19107, USA

Osteopontin (OPN) is a secreted acidic phosphoprotein that binds to a cell-surface integrin-binding motif and is involved in many inflammatory and immune-modulating disorders. There is compelling evidence that soluble OPN can in a variety of situations help cells survive an otherwise lethal insult. In this study we show that OPN is localized in the rat pancreatic islets and ducts. Staining of pancreatic serial sections with islet hormone antibodies showed that all islet cells express OPN. Rats treated with a single dose of streptozotocin (STZ; 50 mg/kg) showed acute upregulation of serum OPN levels and pancreatic OPN mRNA and protein. Serum OPN dropped by the end of day 7 but was still higher than prediabetic levels. Pancreatic mRNA and protein showed a similar pattern. Twenty-four hours after STZ injection, the intensified OPN expression was localized towards the periphery of the islets and surrounded the remaining insulin-positive cells. To explore the significance of OPN acute upregulation, freshly isolated islets were pretreated with OPN (0.15–15 nM) before addition of STZ. OPN significantly reduced the STZ-induced NO levels in the islets through an Arg-Gly-Asp (RGD)-dependent reduction of inducible NO synthase (iNOS) mRNA levels. Addition of OPN to freshly isolated mildly diabetic islets (blood glucose <300 mg/dl) significantly improved their glucose-stimulated insulin secretion and reduced their NO levels. Next we investigated the regulation of OPN in β-cells. When STZ (5 mM) was added to the β-cell line RINm5F it significantly increased OPN mRNA levels within 6 h. To distinguish between the effect of STZ and high glucose on OPN transcription, RINm5F cells were transfected with luciferase-labeled rat OPN promoter and treated with STZ (0.05–5 mM) or with glucose (5–25 mM). STZ induced upregulation of OPN promoter activity within 3 h, while high glucose induced upregulation of OPN promoter activity after 48 h. Our data introduce OPN as a novel islet protein that is differentially regulated by STZ and glucose in the islets. OPN initial upregulation after diabetes induction was probably due to STZ-induced toxicity, while maintenance of the high OPN levels might be due to hyperglycemia. The acute induction of OPN after STZ-induced diabetes might represent an endogenous mechanism to protect the islets against STZ-induced cytotoxicity, partly via an RGD-dependent NO regulatory mechanism.

Abstract

Osteopontin (OPN) is a secreted acidic phosphoprotein that binds to a cell-surface integrin-binding motif and is involved in many inflammatory and immune-modulating disorders. There is compelling evidence that soluble OPN can in a variety of situations help cells survive an otherwise lethal insult. In this study we show that OPN is localized in the rat pancreatic islets and ducts. Staining of pancreatic serial sections with islet hormone antibodies showed that all islet cells express OPN. Rats treated with a single dose of streptozotocin (STZ; 50 mg/kg) showed acute upregulation of serum OPN levels and pancreatic OPN mRNA and protein. Serum OPN dropped by the end of day 7 but was still higher than prediabetic levels. Pancreatic mRNA and protein showed a similar pattern. Twenty-four hours after STZ injection, the intensified OPN expression was localized towards the periphery of the islets and surrounded the remaining insulin-positive cells. To explore the significance of OPN acute upregulation, freshly isolated islets were pretreated with OPN (0.15–15 nM) before addition of STZ. OPN significantly reduced the STZ-induced NO levels in the islets through an Arg-Gly-Asp (RGD)-dependent reduction of inducible NO synthase (iNOS) mRNA levels. Addition of OPN to freshly isolated mildly diabetic islets (blood glucose <300 mg/dl) significantly improved their glucose-stimulated insulin secretion and reduced their NO levels. Next we investigated the regulation of OPN in β-cells. When STZ (5 mM) was added to the β-cell line RINm5F it significantly increased OPN mRNA levels within 6 h. To distinguish between the effect of STZ and high glucose on OPN transcription, RINm5F cells were transfected with luciferase-labeled rat OPN promoter and treated with STZ (0.05–5 mM) or with glucose (5–25 mM). STZ induced upregulation of OPN promoter activity within 3 h, while high glucose induced upregulation of OPN promoter activity after 48 h. Our data introduce OPN as a novel islet protein that is differentially regulated by STZ and glucose in the islets. OPN initial upregulation after diabetes induction was probably due to STZ-induced toxicity, while maintenance of the high OPN levels might be due to hyperglycemia. The acute induction of OPN after STZ-induced diabetes might represent an endogenous mechanism to protect the islets against STZ-induced cytotoxicity, partly via an RGD-dependent NO regulatory mechanism.

Introduction

Osteopontin (OPN) is an integrin- and calcium-binding multifunctional phosphoprotein produced by epithelial cells (Denhardt & Guo 1993), activated cells of the immune system (Patarca et al. 1993), cells of mineralized tissue (Denhardt et al. 2001), and bladder smooth muscle cells (Arafat et al. 2002). Overexpression of OPN has been reported in several physiological and pathological conditions, including immunologic disorders (Cantor 1995), neoplastic transformation (Senger et al. 1989), progression of metastasis (Craig et al. 1990), formation of urinary stones (Kohri et al. 1993), and wound healing (Liaw et al. 1998). Classical mediators of acute inflammation such as tumor necrosis factor α and interleukin-1β strongly induce OPN expression (Denhardt & Guo 1993, Patarca et al. 1993, Yu et al. 1999). OPN protein is selectively upregulated in the serum of type 1 diabetic patients (Fierabracci et al. 1999), in diabetic vascular walls (Takemoto et al. 2000a), and in diabetic kidneys (Fischer et al. 1998). Reports have shown that high glucose concentrations stimulate OPN expression in cultured smooth muscle cells (Takemoto et al. 2000b), partly through activation of high glucose and glucosamine-responsive elements in the OPN promoter (Asaumi et al. 2003).

β-Cell destructive insulitis in type I (Rabinovitch 1998) and streptozotocin (STZ)-induced experimental diabetes (Lukic et al. 1998, Rydgren & Sandler 2002) is associated with increased expression of proinflammatory cytokines, increased expression of the inducible NO synthase (iNOS) gene, and subsequent increased NO production. NO is hypothesized to deleteriously affect β-cell function by inducing apoptosis and suppressing glucose-stimulated insulin release (Corbett et al. 1993, Eizirik & Pavlovic 1997).

Many investigators have shown that both endogenous and exogenous OPN can inhibit induction of iNOS and have defined OPN as an important regulator of the NO signaling pathway (Singh et al. 1995, Rollo et al. 1996, Singh et al. 1999). It is not clear whether the reported OPN upregulation in diabetes is related to its role in regulation of NO production.

In the current study we examined the early temporal changes of serum OPN levels and the associated changes in its mRNA and protein expression in the pancreas in an experimental STZ-diabetic rat model. We investigated the functional significance of these changes in vitro using an STZ-induced cytotoxicity model. We also explored the regulation of OPN promoter by STZ and glucose in a rat β-cell line.

Research design and methods

Diabetes induction

All animal studies were performed in accordance with guidelines set forth by the Animal Care Committee of Thomas Jefferson University, Philadelphia, PA, USA. Adult male (200–250 g) Wistar rats (Harlan, Indianapolis, IN, USA; n=24) were injected intraperitoneally with a single dose of STZ (Biomol; 50 mg/kg body weight in 10 mM sodium citrate buffer, pH 4.5). Control animals (n=18) received only the vehicle buffer. Animals were allowed to free access to standard diet and water. Fasting blood from the tail vein was collected from animals prior to the experiment (day 0) and before death of the animals on days 1, 3, and 7 after STZ injection. Blood glucose was monitored using a glucometer (accu-check; Roche, Indianapolis, IN, USA). Animals were considered diabetic when fasting blood glucose was >250 mg/dl. Each experimental group comprised eight animals. Tissues from five animals were fixed in neutral formaline, covered with optimum cutting temperature (OCT) compound (Sakura Finetek, Torrance, CA, USA), snap-frozen in liquid nitrogen, or incubated in RNA Later (Ambion; Austin, TX, USA). Three pancreata from each group were used for islet isolation and culture.

ELISA

Fasting OPN serum levels prior to induction of diabetes and in control and diabetic animals were measured using rat-specific ELISA kit (Assay Design, Ann Arbor, MI, USA). Spectrophotometric evaluation of OPN levels were made by Synergy HT multi-detection microplate reader (BioTeck, Winooski, VT, USA)

RNA extraction and reverse transcriptase (RT)-PCR

Total RNA was isolated from pancreata of the different groups and from islets, using the SV total RNA isolation system (Promega, Madison, WI, USA) according to the manufacturer’s protocol. Oligo (dT)15 (Promega)-primed cDNA was synthesized from 3.5 g total RNA using Moloney murine leukemia virus RT (Gibco BRL) 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, USA) and the following OPN primers were designed according to the published sequence of rat OPN cDNA (Oldberg et al. 1986): forward primer, 5′-AAGG CGCATTACAGCAAACACTCA-3′; reverse primer, 5′-CTCATCGGACTCCTGGCTCTTCAT-3′. iNOS primers were used to study the effect of OPN treatment of STZ-treated islets: forward primer, 5′-TCCGGGCAGC CTGTGAGACG-3′; reverse primer, 5′-GCTGGGTG GGAGGGGTAGTGATGT-3′. Upstream and downstream primers that could anneal with the 3′-untranslated region of rat GAPDH were included in the PCR reaction as an internal standard. GAPDH forward primer, 5′-GCA TGGCCTTCCGTGTTCCTACC-3′; reverse primer, 5′-GCCGCCTGCTTCACCACCTTCT-3′. The following conditions were used: 50 s at 94 °C, 90 s at 55 °C, and 150 s at 72 °C, with a 7-min final extension at 72 °C after 35 cycles. PCR products were electrophoresed on 2% agarose gels and band intensities were quantified using the Kodak Electrophoresis Documentation and Analysis System 290 (EDAS 290).

Sequence determination

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

Protein isolation and Western blot analysis

Pancreata from the different groups were lysed in modified RIPA lysis buffer (Gould & Hunter 1988), and the protein concentrations in the supernatant were determined using the BCA protein assay reagent (Pierce, Rockford, IL, USA). Equal protein concentrations were loaded on 10% SDS/polyacrylamide slab gels and transferred to Immobilon-P membranes (Millipore, Bedford, MA, USA). Blotted proteins were reacted with primary mouse monoclonal OPN antibody (1:150 diluted in Tween/PBS; (0.2% Tween in PBS, pH 7.4) Santa Cruz Biotechnology, Santa Cruz, CA, USA). Specificity of the antibody was evaluated by Western blot analysis with recombinant OPN protein containing a C-terminal His tag (Chemicon, Temecula, CA, USA). The protein bands were visualized with enhanced chemiluminescence reagents (ECL Plus Western Blotting Detection System; Amersham Biosciences), analyzed, and intensity-quantified using the Kodak Electrophoresis Documentation and Analysis System 290 (EDAS 290).

Immunohistochemistry

To localize OPN in the pancreas and study the changes in its expression after diabetes induction, formaline-fixed, paraffin-embedded tissue blocks were prepared from pancreata of the different groups. Serial sections at 5 μm were stained with the following antibodies: a monoclonal antibody against OPN (2A1; 1:100; Santa Cruz Biotechnology), ready-to-use antibodies against rat insulin, glucagon, and pancreatic polypeptide (BioGenex; San Ramon, CA, USA) and somatostatin (1:100; Accurate Chemical, Westbury, NY, USA). A vectastain universal elite ABC kit and diaminobenzedine chromogenic substrate (Vector Laboratories) were used according to the manufacturer’s protocol to visualize the tissue reaction to the antibodies. In the diabetic pancreata, double immunostaining was employed to visualize OPN and insulin in paraffin sections. Double immunostaining was also carried out in cryostat-cut 5 μm frozen sections to visualize OPN and infiltrating macrophages using antibody against CD68 (1:200; Dako, Carpinteria, CA, USA). Insulin and CD68 were visualized by alkaline phosphatase reaction (red), whereas diaminobenzedine was used to visualize OPN (brown).

Islet isolation, culture, and STZ treatment

Islets were isolated as mentioned elsewhere (Hill et al. 1999). Briefly, 20 ml cold Hank’s buffer/type IV collagenase solution was infused into the rat pancreatic parynchema after its dissection. The inflated pancreas was cleaned from the surrounding fat and lymph nodes, minced, and digested in a shaker-type water bath at 37 °C. Islets were recognized and handpicked under the stereo-microscope after their staining with dithiazone. Islets were aliquoted and cultured in RPMI medium containing 5 mM glucose and supplemented with 10 mM HEPES, 1% l-glutamine, and penicillin/streptomycin. Native rat OPN, a kind gift from Dr W. Butler, University of Texas, was used for these studies. Islets were allowed to equilibrate for 3 h before their treatment. Healthy islets with or without prior addition of OPN (0.15–15 nM) were treated with 0.5 mM STZ for 24 h after which islets and media were harvested. To evaluate whether OPN binding to the Arg-Gly-Asp (RGD) integrin-binding domain results in reduction of iNOS mRNA synthesis, cells were incubated with STZ+OPN (15 nM) +Gly-Arg-Gly-Asp-Asn-Pro (GRGDNP) peptide (1 mM), or with STZ+OPN (1.5 nM) +Gly-Arg-Ala-Asp-Ser-Pro (GRADSP; control) peptide (1 mM; Biomol, Plymouth Meeting, PA, USA). All concentrations were used according to our preliminary concentration studies with references to the values of nitrite release.

Diabetic islets were treated with OPN (0.15–15 nM) for 18 h after which glucose-stimulated insulin secretion (GSIS) studies and NO measurement were performed. We were able to isolate 800–1000 islets/pancreas from the vehicle-treated healthy animals, whereas from the diabetic animals (at day 7), and according to the severity of diabetes, we were able to isolate 0–200 islets/pancreas.

NO determination

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

GSIS studies

Immediately after islet isolation, 5–10 rat islets per experiment were cultured for 3 h in insert-containing 24-well plates (Corning, Corning, NY, USA) with 750 μl RPMI medium with 5 mM glucose, 10% fetal calf serum, 10 mM HEPES, 100 U/ml penicillin G, and 100 μg/ml streptomycin. Healthy islets were pretreated with OPN (0.15–15 nM) for 2 h before addition of STZ (0.5 mM). Diabetic islets were treated with OPN (0.15–15 nM) and maintained overnight at 37 °C. The next morning, the insert-containing islets were removed and transferred to RPMI medium with 3 mM 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 mM and the islets incubated for an additional hour. Insulin assay was performed using rat-specific ultrasensitive insulin ELISA kit (DRG Diagnostics, Mountainside, NJ, USA).

OPN promoter studies

To explore the regulation of OPN in β-cells, RIN, clone 5F (RINm5F), an insulinoma cell line derived from the NEDH rat islet cell tumor, were used. Cells were purchased from American Type Culture Collection and grown at 37 °C under a humidified, 5% CO2 atmosphere in RPMI 1640 medium (Gibco BRL) supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and 2.5 μg/ml amphotericin B. For gene-reporter assay, quiescent cells were obtained after 18 h incubation in serum-free medium. The rat OPN promoter (–1984 luc; GenBank accession number AF017274) in a luciferase expression vector pGL2 basic (Promega) was kindly provided by Dr S Mori, Chiba University, Japan (Takemoto et al. 2000a, Asaumi et al. 2003). Cells were seeded into 24-well culture plates (105). At ~80% confluence they were cotransfected with Trans-Fast reagent (Promega), 0.5 μg pGL2 vectors containing the rat luciferase-labeled OPN promoter and 0.1 μg GFP as transfection control. Two hours later, serum-containing medium was overlaid and the cells incubated for an additional 24 h. The cells were then incubated with serum-free medium for 16 h followed by addition of STZ (0.05–5 mM) or different concentrations of glucose (5–25 mM). Luciferase activities were assayed with the Dual-Luciferase Reporter Assay System (Promega) in a TD-20/20 Luminometer (Turner Designs, Sunnyvale, CA, USA). Transfection efficiency were normalized using the total protein concentration of the cell lysates.

Statistical analysis

All experiments were performed between four and six times. Data were analyzed for statistical significance by analysis of variance (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, USA). Differences were considered significant at P ≤0.05.

Results

OPN serum levels

Temporal changes in serum OPN levels after diabetes induction was examined using a rat-specific ELISA kit. Serum OPN levels showed an initial >5-fold upregulation, 24 h after induction of diabetes (Fig. 1), from 19.49 ± 0.5 to 109.80 ± 43.14 ng/ml (means ± s.e.m.). By day 3 levels dropped to 79.79 ± 5.9 ng/ml, and then to 37.66 ± 6.0 ng/ml by day 7.

Expression of OPN protein and mRNA in control and diabetic pancreas

Equal concentrations of the isolated protein from control and diabetic pancreata at the specified time points were electrophoresed using 10% SDS/PAGE. OPN protein molecular-mass isoforms were detected at ~65 and ~50 kDa. The 50 kDa band showed acute upregulation by day 1 (Fig. 2A), while the 65 kDa band showed early minimal changes. On days 3 and 7 the 50 kDa band was downregulated but was still significantly higher than the prediabetic levels.

Semiquantitative PCR of OPN mRNA and correction of the band intensity with GAPDH showed the same pattern seen with the protein (Fig. 2B). These data indicate that the pancreas might be an active source of serum OPN.

OPN distribution in the control and diabetic pancreas

Clear OPN immunoreactivity could be seen in vehicle-treated rats. OPN-reactive cells were seen in the islets and in the pancreatic ducts (Fig. 3iB and D). Some islets showed more OPN reactivity than others. Staining of serial sections with islet hormone antibodies revealed the expression of OPN by other hormone-secreting cells (Fig. 3ii). Twenty-four hours after STZ injection, double immunostaining showed a more peripheral distribution of intensely stained OPN-reactive cells surrounding few insulin-positive cells (Fig. 3 iiiB). OPN-intensified reactivity at this early time point was not associated with infiltrating macrophages (Fig. 3 iiiA), indicating that this is a constitutive expression of OPN by islet cells and not by infiltrating macrophages that are reported to secret OPN (Masutani et al. 2003).

OPN inhibits NO production in STZ-treated healthy islets

Next we investigated the functional significance of the high levels of OPN in the islets. NO production was measured in rat islets exposed to STZ (0.5 mM) plus or minus pretreatment with OPN (0.15–15 nM). Nitrite measurement was performed as an indirect measure of NO production in the media collected from the islets. The significantly elevated STZ-induced NO levels were dose-dependently reduced in the presence of OPN (Fig. 4A). Islets that were treated with OPN alone did not show significant changes when compared with untreated islets. OPN contains an RGD integrin-binding domain (Denhardt & Guo 1993, Guo et al. 2001). The hexapeptide GRGDSP, which blocks binding of OPN to cell-surface integrins (Singh et al. 1995), was used to determine the receptor-mediated effects of OPN. Cells were pre-treated with GRGDSP hexapeptide (1 mM). In comparison to islets treated with GRADSP control peptide, GRGDSP (1 mM) was found to significantly inhibit the OPN-mediated decrease in iNOS synthesis (Fig. 4B), suggesting a requirement for OPN-integrin receptor binding for OPN-mediated NO regulation.

OPN inhibits NO production and improves the GSIS in diabetic islets

To test whether addition of OPN could benefit the diabetic islets, we isolated the islets from 1-week diabetic rats. Islets isolated from severely diabetic animals (blood glucose >400 mg/dl) produced low levels of NO and had defective GSIS. Addition of OPN to those islets did not significantly affect their nitrite production and did not improve their GSIS (data not shown). However, islets isolated from less severely diabetic animals (blood glucose <300 mg/dl) produced high levels of NO. Addition of OPN (0.15–15 nM) significantly reduced their NO levels (Fig. 5A).

Healthy rat islets showed a ~10-fold increase in insulin secretion with 17 mM glucose, whereas diabetic islets showed β-cell dysfunction and significantly lower insulin surge at 17 mM. Diabetic islets showed a dose-dependent significant improvement in GSIS after treatment with OPN and a 40–60% restoration of control values (Fig. 5B), showing that OPN is capable of improving the islet function only in islets that are not severely dysfunctional.

Induction of OPN mRNA by STZ in RINm5F β-cells

To evaluate the expression of OPN in the RINm5F β-cell line and its regulation by STZ, we treated the cells with STZ (5 mM). OPN mRNA was induced only with the highest dose of STZ (5 mM) after 6 h (Fig. 6A). The concentration and duration of STZ treatment was determined by dose- and time-response studies (data not shown).

Regulation of OPN promoter by STZ and glucose

Previous studies have shown that OPN promoter contains glucose-responsive elements (Asaumi et al. 2003). To investigate whether the induced rise of OPN levels is due to STZ or glucose, RINm5F cells were transfected with rat OPN promoter/luciferase gene construct. After 24 h of transfection, the cells were incubated with different concentrations of STZ (0.05–5 mM) and glucose (1–50 mM) for 3, 6, 24, and 48 h. Relative luciferase activity was calculated after deduction of the activity levels with pGL2 vector alone. High doses of STZ (5 mM) increased OPN promoter activity 1.5-fold after 3 h (Fig. 6B). Glucose induced a dose-dependent upregulation of OPN promoter activity after 48 h. These data show that the OPN promoter responds differentially, time-wise, to STZ and glucose.

Discussion

Whereas pathological changes within the pancreas, and in particular the islets, have been a major focus in diabetes, molecular changes in the islets and their microenvironment represent a major feature of diabetic islet injury. In this study, we introduce a new player, OPN, in the islet response to STZ-diabetes induction.

OPN is a highly hydrophilic and negatively charged sialoprotein of ~298 amino acids that contains a Gly-Arg-Gly-Asp-Ser (GRGDS) sequence. It is a secreted protein with diverse regulatory functions, including cell adhesion and migration, tumor growth and metastasis, atherosclerosis, aortic valve calcification, and repair of myocardial injury. Its expression is tissue specific and subject to regulation by many factors (Denhardt & Guo 1993, Patarca et al. 1993, Denhardt et al. 2001). Constitutive expression of OPN is found in bone (McKee & Nanci 1996), kidney (Verhulst et al. 2002), bladder (Arafat et al. 2002), placenta (Johnson et al. 2003), and brain cells (Iczkiewicz et al. 2004). In vivo expression of OPN has previously been analyzed in several diabetic animal models in different tissues. Towler et al.(1998) demonstrated the upregulation of OPN expression in the aortas of high-fat diet-induced diabetic mice. Fischer et al.(1998) reported that the upregulation of OPN expression in the renal cortex of STZ-induced diabetic rats was mediated by bradykinin. Aspord et al.(2004) reported OPN among the genes specifically activated in the islets and lymph nodes in non-obese diabetic (NOD) mice. However, data concerning the early changes in OPN expression after diabetes induction and its functional significance have not been reported previously. In the present study, we demonstrate for the first time that as early as 24 h, OPN expression levels are enhanced in the serum and in diabetic pancreas, suggesting that diabetes-induced upregulation of OPN expression is a general phenomenon observed across the different tissues. We also show that OPN is constitutively expressed in the rat pancreatic islets and its expression is intensified after STZ-diabetes induction. However, when OPN serum levels were substantially reduced by day 7, the pancreas continued to produce OPN (Fig. 2A and B), suggesting an autocrine/paracrine role for endogenous pancreatic OPN. The enhanced OPN reactivity was not associated with accumulation of inflammatory cells in the islets (Fig. 3 iii), implicating islet OPN in the early islet response to STZ-induced cytotoxicity.

One of the important pathogenetic mechanisms of β-cell damage during experimental STZ-induced diabetes in rats and probably also in human insulin-dependent diabetes mellitus is the cytokine-induced overproduction of NO by iNOS, with subsequent increase of local oxidative stress in the pancreatic islets (Kwon et al. 1994, Haluzik & Nedvidkova 2000). We show here that addition of STZ to isolated islets induced a significant upregulation of NO levels (Fig. 4A). Interestingly, the same treatment induced significant upregulation of endogenous OPN mRNA and promoter activity (Fig. 6A and B). It would be of interest to know whether a more classical NO donor compound than STZ regulated OPN, and whether NO mediated the cytokine-induced increase in OPN expression. Studies addressing these questions are ongoing in our laboratory.

The relationship between NO and OPN has been examined by a number of investigators. Rollo et al.(1996) demonstrated that exogenous recombinant OPN protein was effective in blocking RAW264.7 murine macrophage NO production and cytotoxicity toward the NO-sensitive mastocytoma cells. Their work suggested that OPN in extracellular fluid might protect certain tumor cells from macrophage-mediated destruction by inhibiting the synthesis of NO. However, these authors did not attempt to localize a potential cellular source for OPN in this setting. Singh et al.(1999) reported that a synthetic 20-amino acid OPN peptide analogue decreased iNOS mRNA and protein levels in ventricular myocytes and cardiac microvascular endothelial cells. Transfection of cardiac microvascular endothelial cells with an antisense OPN cDNA increased iNOS mRNA in response to interleukin-1β and interferon-γ, suggesting that endogenous OPN inhibits NO production (Singh et al. 1995). Hwang et al.(1994) found that OPN suppressed NO synthesis induced by interferon and lipopolysaccharide in primary mouse kidney proximal tubule epithelial cells, suggesting a regulatory role for OPN in the NO signaling pathway. Data from our study clearly demonstrate that OPN inhibits the STZ-mediated induction of NO in the islets through downregulation of iNOS synthesis and suggest that OPN is an important regulator of the NO signaling pathway and NO-mediated cytoregulatory processes in the islets. In addition OPN reduced the NO levels in mildly diabetic islets and improved their GSIS. However, NO levels were surprisingly low in the severely diabetic islets and were not affected by exogenous OPN treatment, indicating that OPN could be used to rescue islets that are not severely dysfunctional. High doses of OPN did not reduce STZ-derived nitrite or nitrite from STZ-diabetic islets in a dose-dependent manner, but did it dose-dependently restore the GSIS in the diabetic islets, suggesting the involvement of other mechanisms that mediate OPN protective effects.

Our data show that 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. 4B). Nevertheless, a number of features of this OPN-NO regulatory system remain to be clarified and the specific signaling pathway by which OPN ultimately modulates iNOS synthesis in STZ-treated islets requires further studies. For example, it would be important to determine whether OPN plays a role in the regulation of nuclear factor-κB, which is a primary transcription factor necessary for iNOS synthesis (Flodstrom et al. 1996, Cardozo et al. 2001), and whether OPN regulates additional signaling cascades that are activated by STZ.

In this study, we also showed that OPN promoter was regulated by STZ and glucose. STZ induced an early (3 h) upregulation, while glucose induced a late (48 h) upregulation. This is suggestive that the early rise in pancreatic OPN after diabetes induction is a response to STZ-induced cytotoxicity, whereas the late rise is hyperglycemia-induced. It is not known, however, whether there are STZ specific cis-element on the OPN promoter and whether or not the STZ-induced OPN upregulation is mediated through NO, which has been reported to induce OPN transcription in macrophages (Guo et al. 2001). Studies into this question are currently ongoing in our laboratory.

The presence of a system of OPN-mediated regulation of NO in the islets and β-cells suggests potential targets for modulation of the NO-dependent components of the inflammatory response. The existence of OPN as a potential endogenous negative-feedback protective factor against STZ-induced NO in the islets is unique. A better understanding of OPN regulation and action during the early stages of diabetes and the factors involved in regulation of NO pathways will help to unravel the pathophysiology of diabetes and its associated complications.

Figure 1
Figure 1

Serum OPN levels. Serum levels of OPN were examined in STZ- and control vehicle-treated animals using rat-specific ELISA kit. Serum levels of OPN showed an initial 5-fold upregulation, 24 h after injection of STZ (day 1). Levels then came down but were still higher than prediabetic levels by the end of day 7. n=8 in STZ-treated and n=6 in vehicle-treated groups. *P<0.05 versus control prediabetic values.

Citation: Journal of Endocrinology 187, 2; 10.1677/joe.1.06411

Figure 2
Figure 2

(A) OPN protein expression. Representative Western immunoblot of protein extracts from control vehicle- and STZ-treated pancreata on days 1, 3, and 7. Pancreatic OPN protein is expressed as one major band at ~65 kDa and one minor band at ~50 kDa. Significant upregulation of the 50 kDa isoform is detected after one day of STZ injection, whereas the 65 kDa band did not show significant changes. Levels were lower on days 3 and 7 but still higher than control levels. Average densitometry values of the samples were multiplied to obtain the arbitrary levels. Data are means ± s.e.m. from control vehicle-treated (n=6) and STZ-treated (n=8) groups. *P<0.05 versus control prediabetic values. (B) OPN mRNA expression. PCR analysis of OPN mRNA transcripts revealed the presence of considerable amount of OPN mRNA in control vehicle-treated pancreas. Visible upregulation of OPN mRNA levels is detected at day 1 and then levels were moderately reduced by days 3 and 7 but were still higher than the control vehicle-treated levels (468 and 109 bp bands correspond to the amplified OPN and GAPDH, respectively). The OPN mRNA contents are expressed as optical densities corrected for GAPDH. Data are means ± s.e.m. for control vehicle-treated (n=6) and STZ-treated (n=8) groups. *P<0.05 versus control prediabetic values.

Citation: Journal of Endocrinology 187, 2; 10.1677/joe.1.06411

Figure 3
Figure 3

(i) OPN distribution in the normal pancreas. Paraffin-embedded vehicle-treated rat pancreatic serial sections were stained with OPN antibody. OPN-positive cells (B, D) were detected in the islets (black arrows) and ducts (white arrows). Negative control samples (A, C) where the primary antibody was omitted did not show non-specific reaction. A, B, × 40; C, D × 200 original magnifications. (ii) Colocalization of OPN with the rest of islet hormones. To identify which cells in the islets express OPN, paraffin-embedded vehicle-treated rat pancreatic serial sections were stained with OPN antibody and with antibodies against insulin, glucagon, pancreatic polypeptide (PP), and somatostatin. It appears that most islet cells express OPN; × 200 original magnification. H&E, hematoxylin and eosin stain. (iii) OPN distribution in the islets of the diabetic pancreas. In frozen sections of STZ-treated rat pancreata double-immunostained with antibody against OPN and CD68 (A), no infiltrating macrophages could be detected. In paraffin embedded sections, double-immunostaining with OPN and insulin antibodies (B) shows a more peripheral distribution of intensely stained OPN-reactive cells (brown) surrounding the remaining insulin positive cells (red). Original magnification, × 200.

Citation: Journal of Endocrinology 187, 2; 10.1677/joe.1.06411

Figure 4
Figure 4

(A) OPN effect on STZ-induced nitrite formation in healthy islets. Healthy islets were pretreated with OPN (0.15–15 nM) for 2 h before the addition of STZ (0.5 mM) for an additional 24 h. Nitrite measurement was performed as an indirect measure of NO production in the media collected from the islets. Significant reduction in NO levels can be seen in islets treated with OPN when compared with STZ alone. OPN at high doses did not induce significant NO reduction. Data are expressed as means ± s.e.m. Each experiment was performed in duplicate and repeated three times for reproducibility. *P ≤0.05 versus control values and #P ≤0.05 versus STZ values, using one-way repeated ANOVA with subsequent all pairwise comparison procedure by Student’s t test. (B) PCR analysis of iNOS and GAPDH mRNA transcripts from islets treated with STZ (0.5 mM; lane 2) plus or minus pretreatment with OPN (1.5 nM; lane 3) with prior addition of exogenous GRGDSP (1 nM; lane 4) or GRADSP (1 nM; lane 5). Addition of the GRGDSP peptide to the islet blocked OPN-mediated reduction of iNOS mRNA. 492 and 109 bp bands correspond to the amplified iNOS and GAPDH, respectively. Values are expressed as mean ± s.e.m. of three experiments. *P<0.05 versus untreated islets; #P<0.05 versus STZ-treated islets using one-way repeated ANOVA with subsequent all pairwise comparison procedure by Student’s t test.

Citation: Journal of Endocrinology 187, 2; 10.1677/joe.1.06411

Figure 5
Figure 5

(A) OPN inhibits NO production in diabetic islets. NO production was measured in rat islets isolated from animals where blood glucose was less than 300 mg/dl plus or minus OPN (0.15–15 nM) for 18 h. Nitrite measurement was performed as an indirect measure of NO production in the media collected from the islets. Significant reduction in NO levels is seen in islets treated with OPN when compared with diabetic islets. Data are expressed as means ± s.e.m. Each experiment was performed in duplicate and repeated three times for reproducibility. *P ≤0.05 versus diabetic values, using one-way repeated ANOVA with subsequent all pairwise comparison procedure by Student’s t test. (B) OPN improves GSIS production in diabetic islets. Healthy rat islets showed a ~7-fold increase in insulin secretion with 17 mM glucose, while diabetic islets were dysfunctional and showed defective GSIS. Addition of OPN (0.15, 1.5, 15 nM) showed a dose-dependent (40–60%) restoration of control values. Data are expressed as means ± s.e.m. Each experiment was performed three times and repeated three times for reproducibility. *P<0.05 versus 17 mM healthy islets; #P<0.05 versus 17 mM diabetic islets using one-way repeated ANOVA with subsequent all pairwise comparison procedure by Student’s t test.

Citation: Journal of Endocrinology 187, 2; 10.1677/joe.1.06411

Figure 6
Figure 6

(A) OPN mRNA expression in RINm5F cells. Cells were cultured for 6 h under control conditions (lane 1) or with STZ (5 mM; lane 2). The concentration and duration of STZ treatment was determined by dose- and time-response studies (data not shown). OPN and GAPDH mRNA content were analyzed by RT-PCR. The OPN mRNA contents are expressed as optical densities corrected for GAPDH. (468 and 109 bp bands correspond to the amplified OPN and GAPDH, respectively). Values are expressed as means ± s.e.m. from three experiments. *P<0.05 versus untreated cells using one-way repeated ANOVA with subsequent all pairwise comparison procedure by Student’s t test. (B) Induction of luciferase activity with STZ in RINm5F cells transfected with rat OPN promoter/luciferase gene construct. After 24 h of transfection, the cells were incubated with different concentrations of STZ, as shown, for 3 h. After incubation, the luciferase activity in the cell lysates was measured. STZ causes a dose-dependent increase in OPN promoter activity. Relative luciferase activity was calculated after deduction of the activity levels with pGL2 vector alone. Results represent means ± s.e.m. from triplicate determinations. All experiments were repeated at least three times to confirm the reproducibility of the observations. (C) Induction of luciferase activity with glucose in RINm5F cells transfected with rat OPN promoter/luciferase gene construct. After 24 h of transfection, the cells were incubated with different concentrations of glucose, as shown, for 48 h. After incubation, the luciferase activity in the cell lysates was measured. Glucose induces a dose-dependent increase in OPN promoter activity. Relative luciferase activity was calculated after deduction of the activity levels with pGL2 vector alone. Results represent means ± s.e.m. from triplicate determinations. All experiments were repeated at least three times to confirm the reproducibility of the observations.

Citation: Journal of Endocrinology 187, 2; 10.1677/joe.1.06411

This study was supported by grants from the American Diabetes Association 1–05-JF-01, Diabetes Trust Foundation, Diabetes Transplant Fund, and Diabetes Action Research & Education Foundation. The authors wish to thank Dr W. Butler, University of Texas, for providing native OPN protein and Dr S. Mori, Chiba University, Japan, for providing the OPN promoter. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

References

  • Arafat HA, Wein AJ & Chacko S 2002 Osteopontin gene expression and immunolocalization in the rabbit urinary tract. Journal of Urology 167 746–752.

    • Search Google Scholar
    • Export Citation
  • 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. Journal of Diabetes Complications 17 34–38.

    • Search Google Scholar
    • Export Citation
  • Aspord C, Rome S & Thivolet C 2004 Early events in islets and pancreatic lymph nodes in autoimmune diabetes. Journal of Autoimmunity 23 27–35.

    • Search Google Scholar
    • Export Citation
  • Cantor H 1995 The role of Eta-1/osteopontin in the pathogenesis of immunological disorders. Annals of the New York Academy of Sciences 21 143–150.

    • Search Google Scholar
    • Export Citation
  • Cardozo AK, Heimberg H, Heremans Y, Leeman R, Kutlu B, Kruhoffer M, Orntoft T & Eizirik DL 2001 A comprehensive analysis of cytokine-induced and nuclear factor-kappa B-dependent genes in primary rat pancreatic beta-cells. Journal of Biological Chemistry 276 48879–48886.

    • Search Google Scholar
    • Export Citation
  • 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. PNAS 90 1731–1735.

    • Search Google Scholar
    • Export Citation
  • Craig AM, Bowden GT, Chambers AF, Spearman MA, Greenberg AH, Wright JA, McLeod M & Denhardt DT 1990 Secreted phosphoprotein mRNA is induced during multi-stage carcinogenesis in mouse skin and correlates with the metastatic potential of murine fibroblasts. International Journal of Cancer 46 133–137.

    • Search Google Scholar
    • Export Citation
  • Denhardt DT & Guo X 1993 Osteopontin: a protein with diverse functions. FASEB Journal 7 1475–1482.

  • 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. Journal of Clinical Investigation 107 1055–1061.

    • Search Google Scholar
    • Export Citation
  • Eizirik DL & Pavlovic D 1997 Is there a role for nitric oxide in β-cell dysfunction and damage in IDDM? Diabetes/Metabolism Reviews 13 293–307.

    • Search Google Scholar
    • Export Citation
  • Fierabracci A, Biro PA, Yiangou Y, Mennuni C, Luzzago A, Ludvigsson J, Cortese R & Bottazzo GF 1999 Osteopontin is an autoantigen of the somatostatin cells in human islets: identification by screening random peptide libraries with sera of patients with insulin-dependent diabetes mellitus. Vaccine 18 342–354.

    • Search Google Scholar
    • Export Citation
  • Fischer JW, Tschope C, Reinecke A, Giachelli CM & Unger T 1998 Upregulation of osteopontin expression in renal cortex of streptozotocin-induced diabetic rats is mediated by bradykinin. Diabetes 9 1512–1518.

    • Search Google Scholar
    • Export Citation
  • 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 Letters 385 4–6.

    • Search Google Scholar
    • Export Citation
  • Gould KL & Hunter T 1988 Platelet-derived growth factor induces multisite phosphorylation of pp60c-src and increases its protein-tyrosine kinase activity. Molecular Cell Biology 8 3345–3356.

    • Search Google Scholar
    • Export Citation
  • Guo H, Cai CQ, Schroeder RA & Kuo PC 2001 Osteopontin is a negative feedback regulator of nitric oxide synthesis in murine macrophages. Journal of Immunology 166 1079–1086.

    • Search Google Scholar
    • Export Citation
  • Haluzik M & Nedvidkova J 2000 The role of nitric oxide in the development of streptozotocin-induced diabetes mellitus: experimental and clinical implications. Physiological Research 49 S37–S42.

    • Search Google Scholar
    • Export Citation
  • 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. Journal of Endocrinology 16 153–165.

    • Search Google Scholar
    • Export Citation
  • 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. Journal of Biological Chemistry 269 711–715.

    • Search Google Scholar
    • Export Citation
  • Iczkiewicz J, Rose S & Jenner P 2004 Osteopontin (Eta-1) is present in the rat basal ganglia. Brain Research Molecular Brain Research 132 64–72.

    • Search Google Scholar
    • Export Citation
  • Johnson GA, Burghardt RC, Bazer FW & Spencer TE 2003 Osteopontin: roles in implantation and placentation. Biology of Reproduction 69 1458–1471.

    • Search Google Scholar
    • Export Citation
  • Kohri K, Nomura S, Kitamura Y, Nagata T, Yoshioka K, Iguchi M, Yamate T, Umekawa T, Suzuki Y, Sinohara H & Kurita T 1993 Structure and expression of the mRNA encoding urinary stone protein (osteopontin). Journal of Biological Chemistry 268 15180–15184.

    • Search Google Scholar
    • Export Citation
  • Kwon NS, Lee SH, Choi CS, Kho T & Lee HS 1994 Nitric oxide generation from streptozotocin. FASEB Journal 8 529–533.

  • Liaw L, Birk DE, Ballas CB, Whitsitt JS, Davidson JM & Hogan BL 1998 Altered wound healing in mice lacking a functional osteopontin gene (spp1). Journal of Clinical Investigation 101 1468–1478.

    • Search Google Scholar
    • Export Citation
  • Lukic ML, Stosic-Grujicic S & Shahin A 1998 Effector mechanisms in low-dose streptozotocin-induced diabetes. Developmental Immunology 6 119–128.

    • Search Google Scholar
    • Export Citation
  • Masutani K, Tokumoto M, Nakashima H, Tsuruya K, Kashiwagi M, Kudoh Y, Fukuda K, Kanai H, Akahoshi M, Otsuka T et al. 2003 Strong polarization toward Th1 immune response in ANCA-associated glomerulonephritis. Clinical Nephrology 59 395–405.

    • Search Google Scholar
    • Export Citation
  • McKee MD & Nanci A 1996 Osteopontin at mineralized tissue interfaces in bone, teeth, and osseointegrated implants: ultrastructural distribution and implications for mineralized tissue formation, turnover, and repair. Microscopy Research and Technique 33 141–164.

    • Search Google Scholar
    • Export Citation
  • Oldberg A, Franzen A & Heinegard D 1986 Cloning and sequence analysis of rat bone sialoprotein (osteopontin) cDNA reveals an Arg-Gly-Asp cell-binding sequence. PNAS 83 8819–8823.

    • Search Google Scholar
    • Export Citation
  • 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. Critical Reviews in Immunology 13 225–246.

    • Search Google Scholar
    • Export Citation
  • Rabinovitch A 1998 An update on cytokines in the pathogenesis of insulin-dependent diabetes mellitus. Diabetes/Metabolism Reviews 14 129–151.

    • Search Google Scholar
    • Export Citation
  • Rollo EE, Laskin DL & Denhardt DT 1996 Osteopontin inhibits nitric oxide production and cytotoxicity by activated RAW264.7 macrophages. Journal of Leukocyte Biology 60 397–404.

    • Search Google Scholar
    • Export Citation
  • Rydgren T & Sandler S 2002 Efficacy of 1400 W, a novel inhibitor of inducible nitric oxide synthase, in preventing interleukin-1 beta-induced suppression of pancreatic islet function in vitro and multiple low-dose streptozotocin-induced diabetes in vivo. European Journal of Endocrinology 147 543–551.

    • Search Google Scholar
    • Export Citation
  • Senger DR, Perruzzi CA & Papadopoulos A 1989 Elevated expression of secreted phosphoprotein I (osteopontin, 2ar) as a consequence of neoplastic transformation. Anticancer Research 9 1291–1299.

    • Search Google Scholar
    • Export Citation
  • Singh K, Balligand JL, Fischer TA, Smith TW & Kelly RA 1995 Glucocorticoids increase osteopontin expression in cardiac myocytes and microvascular endothelial cells. Journal of Biological Chemistry 270 28471–28478.

    • Search Google Scholar
    • Export Citation
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  • Takemoto M, Yokote K, Yamazaki M, Ridall AL, Butler WT, Matsumoto T, Tamura K, Saito Y & Mori S 2000b Enhanced expression of osteopontin by high glucose. Involvement of osteopontin in diabetic macroangiopathy. Annals of the New York Academy of Sciences 902 357–363.

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    Serum OPN levels. Serum levels of OPN were examined in STZ- and control vehicle-treated animals using rat-specific ELISA kit. Serum levels of OPN showed an initial 5-fold upregulation, 24 h after injection of STZ (day 1). Levels then came down but were still higher than prediabetic levels by the end of day 7. n=8 in STZ-treated and n=6 in vehicle-treated groups. *P<0.05 versus control prediabetic values.

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    (A) OPN protein expression. Representative Western immunoblot of protein extracts from control vehicle- and STZ-treated pancreata on days 1, 3, and 7. Pancreatic OPN protein is expressed as one major band at ~65 kDa and one minor band at ~50 kDa. Significant upregulation of the 50 kDa isoform is detected after one day of STZ injection, whereas the 65 kDa band did not show significant changes. Levels were lower on days 3 and 7 but still higher than control levels. Average densitometry values of the samples were multiplied to obtain the arbitrary levels. Data are means ± s.e.m. from control vehicle-treated (n=6) and STZ-treated (n=8) groups. *P<0.05 versus control prediabetic values. (B) OPN mRNA expression. PCR analysis of OPN mRNA transcripts revealed the presence of considerable amount of OPN mRNA in control vehicle-treated pancreas. Visible upregulation of OPN mRNA levels is detected at day 1 and then levels were moderately reduced by days 3 and 7 but were still higher than the control vehicle-treated levels (468 and 109 bp bands correspond to the amplified OPN and GAPDH, respectively). The OPN mRNA contents are expressed as optical densities corrected for GAPDH. Data are means ± s.e.m. for control vehicle-treated (n=6) and STZ-treated (n=8) groups. *P<0.05 versus control prediabetic values.

  • View in gallery

    (i) OPN distribution in the normal pancreas. Paraffin-embedded vehicle-treated rat pancreatic serial sections were stained with OPN antibody. OPN-positive cells (B, D) were detected in the islets (black arrows) and ducts (white arrows). Negative control samples (A, C) where the primary antibody was omitted did not show non-specific reaction. A, B, × 40; C, D × 200 original magnifications. (ii) Colocalization of OPN with the rest of islet hormones. To identify which cells in the islets express OPN, paraffin-embedded vehicle-treated rat pancreatic serial sections were stained with OPN antibody and with antibodies against insulin, glucagon, pancreatic polypeptide (PP), and somatostatin. It appears that most islet cells express OPN; × 200 original magnification. H&E, hematoxylin and eosin stain. (iii) OPN distribution in the islets of the diabetic pancreas. In frozen sections of STZ-treated rat pancreata double-immunostained with antibody against OPN and CD68 (A), no infiltrating macrophages could be detected. In paraffin embedded sections, double-immunostaining with OPN and insulin antibodies (B) shows a more peripheral distribution of intensely stained OPN-reactive cells (brown) surrounding the remaining insulin positive cells (red). Original magnification, × 200.

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    (A) OPN effect on STZ-induced nitrite formation in healthy islets. Healthy islets were pretreated with OPN (0.15–15 nM) for 2 h before the addition of STZ (0.5 mM) for an additional 24 h. Nitrite measurement was performed as an indirect measure of NO production in the media collected from the islets. Significant reduction in NO levels can be seen in islets treated with OPN when compared with STZ alone. OPN at high doses did not induce significant NO reduction. Data are expressed as means ± s.e.m. Each experiment was performed in duplicate and repeated three times for reproducibility. *P ≤0.05 versus control values and #P ≤0.05 versus STZ values, using one-way repeated ANOVA with subsequent all pairwise comparison procedure by Student’s t test. (B) PCR analysis of iNOS and GAPDH mRNA transcripts from islets treated with STZ (0.5 mM; lane 2) plus or minus pretreatment with OPN (1.5 nM; lane 3) with prior addition of exogenous GRGDSP (1 nM; lane 4) or GRADSP (1 nM; lane 5). Addition of the GRGDSP peptide to the islet blocked OPN-mediated reduction of iNOS mRNA. 492 and 109 bp bands correspond to the amplified iNOS and GAPDH, respectively. Values are expressed as mean ± s.e.m. of three experiments. *P<0.05 versus untreated islets; #P<0.05 versus STZ-treated islets using one-way repeated ANOVA with subsequent all pairwise comparison procedure by Student’s t test.

  • View in gallery

    (A) OPN inhibits NO production in diabetic islets. NO production was measured in rat islets isolated from animals where blood glucose was less than 300 mg/dl plus or minus OPN (0.15–15 nM) for 18 h. Nitrite measurement was performed as an indirect measure of NO production in the media collected from the islets. Significant reduction in NO levels is seen in islets treated with OPN when compared with diabetic islets. Data are expressed as means ± s.e.m. Each experiment was performed in duplicate and repeated three times for reproducibility. *P ≤0.05 versus diabetic values, using one-way repeated ANOVA with subsequent all pairwise comparison procedure by Student’s t test. (B) OPN improves GSIS production in diabetic islets. Healthy rat islets showed a ~7-fold increase in insulin secretion with 17 mM glucose, while diabetic islets were dysfunctional and showed defective GSIS. Addition of OPN (0.15, 1.5, 15 nM) showed a dose-dependent (40–60%) restoration of control values. Data are expressed as means ± s.e.m. Each experiment was performed three times and repeated three times for reproducibility. *P<0.05 versus 17 mM healthy islets; #P<0.05 versus 17 mM diabetic islets using one-way repeated ANOVA with subsequent all pairwise comparison procedure by Student’s t test.

  • View in gallery

    (A) OPN mRNA expression in RINm5F cells. Cells were cultured for 6 h under control conditions (lane 1) or with STZ (5 mM; lane 2). The concentration and duration of STZ treatment was determined by dose- and time-response studies (data not shown). OPN and GAPDH mRNA content were analyzed by RT-PCR. The OPN mRNA contents are expressed as optical densities corrected for GAPDH. (468 and 109 bp bands correspond to the amplified OPN and GAPDH, respectively). Values are expressed as means ± s.e.m. from three experiments. *P<0.05 versus untreated cells using one-way repeated ANOVA with subsequent all pairwise comparison procedure by Student’s t test. (B) Induction of luciferase activity with STZ in RINm5F cells transfected with rat OPN promoter/luciferase gene construct. After 24 h of transfection, the cells were incubated with different concentrations of STZ, as shown, for 3 h. After incubation, the luciferase activity in the cell lysates was measured. STZ causes a dose-dependent increase in OPN promoter activity. Relative luciferase activity was calculated after deduction of the activity levels with pGL2 vector alone. Results represent means ± s.e.m. from triplicate determinations. All experiments were repeated at least three times to confirm the reproducibility of the observations. (C) Induction of luciferase activity with glucose in RINm5F cells transfected with rat OPN promoter/luciferase gene construct. After 24 h of transfection, the cells were incubated with different concentrations of glucose, as shown, for 48 h. After incubation, the luciferase activity in the cell lysates was measured. Glucose induces a dose-dependent increase in OPN promoter activity. Relative luciferase activity was calculated after deduction of the activity levels with pGL2 vector alone. Results represent means ± s.e.m. from triplicate determinations. All experiments were repeated at least three times to confirm the reproducibility of the observations.

  • Arafat HA, Wein AJ & Chacko S 2002 Osteopontin gene expression and immunolocalization in the rabbit urinary tract. Journal of Urology 167 746–752.

    • Search Google Scholar
    • Export Citation
  • 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. Journal of Diabetes Complications 17 34–38.

    • Search Google Scholar
    • Export Citation
  • Aspord C, Rome S & Thivolet C 2004 Early events in islets and pancreatic lymph nodes in autoimmune diabetes. Journal of Autoimmunity 23 27–35.

    • Search Google Scholar
    • Export Citation
  • Cantor H 1995 The role of Eta-1/osteopontin in the pathogenesis of immunological disorders. Annals of the New York Academy of Sciences 21 143–150.

    • Search Google Scholar
    • Export Citation
  • Cardozo AK, Heimberg H, Heremans Y, Leeman R, Kutlu B, Kruhoffer M, Orntoft T & Eizirik DL 2001 A comprehensive analysis of cytokine-induced and nuclear factor-kappa B-dependent genes in primary rat pancreatic beta-cells. Journal of Biological Chemistry 276 48879–48886.

    • Search Google Scholar
    • Export Citation
  • 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. PNAS 90 1731–1735.

    • Search Google Scholar
    • Export Citation
  • Craig AM, Bowden GT, Chambers AF, Spearman MA, Greenberg AH, Wright JA, McLeod M & Denhardt DT 1990 Secreted phosphoprotein mRNA is induced during multi-stage carcinogenesis in mouse skin and correlates with the metastatic potential of murine fibroblasts. International Journal of Cancer 46 133–137.

    • Search Google Scholar
    • Export Citation
  • Denhardt DT & Guo X 1993 Osteopontin: a protein with diverse functions. FASEB Journal 7 1475–1482.

  • 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. Journal of Clinical Investigation 107 1055–1061.

    • Search Google Scholar
    • Export Citation
  • Eizirik DL & Pavlovic D 1997 Is there a role for nitric oxide in β-cell dysfunction and damage in IDDM? Diabetes/Metabolism Reviews 13 293–307.

    • Search Google Scholar
    • Export Citation
  • Fierabracci A, Biro PA, Yiangou Y, Mennuni C, Luzzago A, Ludvigsson J, Cortese R & Bottazzo GF 1999 Osteopontin is an autoantigen of the somatostatin cells in human islets: identification by screening random peptide libraries with sera of patients with insulin-dependent diabetes mellitus. Vaccine 18 342–354.

    • Search Google Scholar
    • Export Citation
  • Fischer JW, Tschope C, Reinecke A, Giachelli CM & Unger T 1998 Upregulation of osteopontin expression in renal cortex of streptozotocin-induced diabetic rats is mediated by bradykinin. Diabetes 9 1512–1518.

    • Search Google Scholar
    • Export Citation
  • 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 Letters 385 4–6.

    • Search Google Scholar
    • Export Citation
  • Gould KL & Hunter T 1988 Platelet-derived growth factor induces multisite phosphorylation of pp60c-src and increases its protein-tyrosine kinase activity. Molecular Cell Biology 8 3345–3356.

    • Search Google Scholar
    • Export Citation
  • Guo H, Cai CQ, Schroeder RA & Kuo PC 2001 Osteopontin is a negative feedback regulator of nitric oxide synthesis in murine macrophages. Journal of Immunology 166 1079–1086.

    • Search Google Scholar
    • Export Citation
  • Haluzik M & Nedvidkova J 2000 The role of nitric oxide in the development of streptozotocin-induced diabetes mellitus: experimental and clinical implications. Physiological Research 49 S37–S42.

    • Search Google Scholar
    • Export Citation
  • 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. Journal of Endocrinology 16 153–165.

    • Search Google Scholar
    • Export Citation
  • 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. Journal of Biological Chemistry 269 711–715.

    • Search Google Scholar
    • Export Citation
  • Iczkiewicz J, Rose S & Jenner P 2004 Osteopontin (Eta-1) is present in the rat basal ganglia. Brain Research Molecular Brain Research 132 64–72.

    • Search Google Scholar
    • Export Citation
  • Johnson GA, Burghardt RC, Bazer FW & Spencer TE 2003 Osteopontin: roles in implantation and placentation. Biology of Reproduction 69 1458–1471.

    • Search Google Scholar
    • Export Citation
  • Kohri K, Nomura S, Kitamura Y, Nagata T, Yoshioka K, Iguchi M, Yamate T, Umekawa T, Suzuki Y, Sinohara H & Kurita T 1993 Structure and expression of the mRNA encoding urinary stone protein (osteopontin). Journal of Biological Chemistry 268 15180–15184.

    • Search Google Scholar
    • Export Citation
  • Kwon NS, Lee SH, Choi CS, Kho T & Lee HS 1994 Nitric oxide generation from streptozotocin. FASEB Journal 8 529–533.

  • Liaw L, Birk DE, Ballas CB, Whitsitt JS, Davidson JM & Hogan BL 1998 Altered wound healing in mice lacking a functional osteopontin gene (spp1). Journal of Clinical Investigation 101 1468–1478.

    • Search Google Scholar
    • Export Citation
  • Lukic ML, Stosic-Grujicic S & Shahin A 1998 Effector mechanisms in low-dose streptozotocin-induced diabetes. Developmental Immunology 6 119–128.

    • Search Google Scholar
    • Export Citation
  • Masutani K, Tokumoto M, Nakashima H, Tsuruya K, Kashiwagi M, Kudoh Y, Fukuda K, Kanai H, Akahoshi M, Otsuka T et al. 2003 Strong polarization toward Th1 immune response in ANCA-associated glomerulonephritis. Clinical Nephrology 59 395–405.

    • Search Google Scholar
    • Export Citation
  • McKee MD & Nanci A 1996 Osteopontin at mineralized tissue interfaces in bone, teeth, and osseointegrated implants: ultrastructural distribution and implications for mineralized tissue formation, turnover, and repair. Microscopy Research and Technique 33 141–164.

    • Search Google Scholar
    • Export Citation
  • Oldberg A, Franzen A & Heinegard D 1986 Cloning and sequence analysis of rat bone sialoprotein (osteopontin) cDNA reveals an Arg-Gly-Asp cell-binding sequence. PNAS 83 8819–8823.

    • Search Google Scholar
    • Export Citation
  • 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. Critical Reviews in Immunology 13 225–246.

    • Search Google Scholar
    • Export Citation
  • Rabinovitch A 1998 An update on cytokines in the pathogenesis of insulin-dependent diabetes mellitus. Diabetes/Metabolism Reviews 14 129–151.

    • Search Google Scholar
    • Export Citation
  • Rollo EE, Laskin DL & Denhardt DT 1996 Osteopontin inhibits nitric oxide production and cytotoxicity by activated RAW264.7 macrophages. Journal of Leukocyte Biology 60 397–404.

    • Search Google Scholar
    • Export Citation
  • Rydgren T & Sandler S 2002 Efficacy of 1400 W, a novel inhibitor of inducible nitric oxide synthase, in preventing interleukin-1 beta-induced suppression of pancreatic islet function in vitro and multiple low-dose streptozotocin-induced diabetes in vivo. European Journal of Endocrinology 147 543–551.

    • Search Google Scholar
    • Export Citation
  • Senger DR, Perruzzi CA & Papadopoulos A 1989 Elevated expression of secreted phosphoprotein I (osteopontin, 2ar) as a consequence of neoplastic transformation. Anticancer Research 9 1291–1299.

    • Search Google Scholar
    • Export Citation
  • Singh K, Balligand JL, Fischer TA, Smith TW & Kelly RA 1995 Glucocorticoids increase osteopontin expression in cardiac myocytes and microvascular endothelial cells. Journal of Biological Chemistry 270 28471–28478.

    • Search Google Scholar
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