237 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

A K Katakam, G Chipitsyna, Q Gong, A R Vancha, J Gabbeta and H A Arafat Departments of Surgery, Pathology, Anatomy & Cell Biology, Thomas Jefferson University, 1015 Walnut Street, Philadelphia, PA 19107, USA (Requests for offprints should be addressed to H Arafat; Email: [email protected])

Abstract Osteopontin (OPN) is a secreted acidic phosphoprotein isolated mildly diabetic islets (blood glucose <300 mg/dl) that binds to a cell-surface integrin-binding motif and is significantly improved their glucose-stimulated insulin involved in many inflammatory and immune-modulating secretion and reduced their NO levels. Next we investi- disorders. There is compelling evidence that soluble OPN gated the regulation of OPN in
-cells. When STZ can in a variety of situations help cells survive an otherwise (5 mM) was added to the
-cell line RINm5F it signifi- lethal insult. In this study we show that OPN is localized cantly increased OPN mRNA levels within 6 h. To in the rat pancreatic islets and ducts. Staining of pancreatic distinguish between the effect of STZ and high glucose on serial sections with islet hormone antibodies showed that OPN transcription, RINm5F cells were transfected with all islet cells express OPN. Rats treated with a single dose luciferase-labeled rat OPN promoter and treated with of streptozotocin (STZ; 50 mg/kg) showed acute upregu- STZ (0·05–5 mM) or with glucose (5–25 mM). STZ lation of serum OPN levels and pancreatic OPN mRNA induced upregulation of OPN promoter activity within and protein. Serum OPN dropped by the end of day 7 but 3 h, while high glucose induced upregulation of OPN was still higher than prediabetic levels. Pancreatic mRNA promoter activity after 48 h. Our data introduce OPN as and protein showed a similar pattern. Twenty-four hours a novel islet protein that is differentially regulated by STZ after STZ injection, the intensified OPN expression was and glucose in the islets. OPN initial upregulation after localized towards the periphery of the islets and sur- diabetes induction was probably due to STZ-induced rounded the remaining insulin-positive cells. To explore toxicity, while maintenance of the high OPN levels the significance of OPN acute upregulation, freshly iso- might be due to hyperglycemia. The acute induction of lated islets were pretreated with OPN (0·15–15 nM) OPN after STZ-induced diabetes might represent an before addition of STZ. OPN significantly reduced the endogenous mechanism to protect the islets against STZ- STZ-induced NO levels in the islets through an Arg-Gly- induced cytotoxicity, partly via an RGD-dependent NO Asp (RGD)-dependent reduction of inducible NO syn- regulatory mechanism. thase (iNOS) mRNA levels. Addition of OPN to freshly Journal of Endocrinology (2005) 187, 237–247

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

Journal of Endocrinology (2005) 187, 237–247 DOI: 10.1677/joe.1.06411 0022–0795/05/0187–237  2005 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org

Downloaded from Bioscientifica.com at 09/27/2021 08:27:17PM via free access 238 A K KATAKAM and others · Osteopontin in STZ-induced diabetes

with increased expression of proinflammatory cytokines, USA). Spectrophotometric evaluation of OPN levels were increased expression of the inducible NO synthase (iNOS) made by Synergy HT multi-detection microplate reader gene, and subsequent increased NO production. NO is (BioTeck, Winooski, VT, USA) hypothesized to deleteriously affect
-cell function by inducing apoptosis and suppressing glucose-stimulated insulin release (Corbett et al. 1993, Eizirik & Pavlovic RNA extraction and reverse transcriptase (RT)-PCR 1997). Total RNA was isolated from pancreata of the different Many investigators have shown that both endogenous groups and from islets, using the SV total RNA isolation and exogenous OPN can inhibit induction of iNOS and system (Promega, Madison, WI, USA) according to the have defined OPN as an important regulator of the NO manufacturer’s protocol. Oligo (dT)15 (Promega)-primed signaling pathway (Singh et al. 1995, Rollo et al. 1996, cDNA was synthesized from 3·5 g total RNA using Singh et al. 1999). It is not clear whether the reported Moloney murine leukemia virus RT (Gibco BRL) at OPN upregulation in diabetes is related to its role in 37 C for 60 min. Samples were incubated at 90 Cfor regulation of NO production. 5 min to terminate the reverse transcription reaction. The In the current study we examined the early temporal cDNA mixtures (2 µl) were subjected to PCR using changes of serum OPN levels and the associated changes AmpliTaq Gold DNA polymerase (PE Biosystems, Welle- in its mRNA and protein expression in the pancreas in an sley, MA, USA) and the following OPN primers were experimental STZ-diabetic rat model. We investigated designed according to the published sequence of rat OPN the functional significance of these changes in vitro using an cDNA (Oldberg et al. 1986): forward primer, 5-AAGG STZ-induced cytotoxicity model. We also explored the CGCATTACAGCAAACACTCA-3; reverse primer, regulation of OPN promoter by STZ and glucose in a rat 5’-CTCATCGGACTCCTGGCTCTTCAT-3. iNOS
-cell line. primers were used to study the effect of OPN treatment of STZ-treated islets: forward primer, 5-TCCGGGCAGC CTGTGAGACG-3; reverse primer, 5-GCTGGGTG Research design and methods GGAGGGGTAGTGATGT-3. Upstream and down- stream primers that could anneal with the 3-untranslated Diabetes induction region of rat GAPDH were included in the PCR reaction as an internal standard. GAPDH forward primer, 5-GCA All animal studies were performed in accordance with TGGCCTTCCGTGTTCCTACC-3; reverse primer, guidelines set forth by the Animal Care Committee of   ff 5 -GCCGCCTGCTTCACCACCTTCT-3 . The fol- Thomas Je erson University, Philadelphia, PA, USA. lowing conditions were used: 50 s at 94 C,90sat55C, Adult male (200–250 g) Wistar rats (Harlan, Indianapolis, and 150 s at 72 C, with a 7-min final extension at 72 C IN, USA; n=24) were injected intraperitoneally with a after 35 cycles. PCR products were electrophoresed on 2% single dose of STZ (Biomol; 50 mg/kg body weight in agarose gels and band intensities were quantified using 10 mM sodium citrate buffer, pH 4·5). Control animals ff the Kodak Electrophoresis Documentation and Analysis (n=18) received only the vehicle bu er. Animals were System 290 (EDAS 290). 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 Sequence determination on days 1, 3, and 7 after STZ injection. Blood glucose PCR bands were purified from the agarose gel using the was monitored using a glucometer (accu-check; Roche, Geneclean II kit (BI 101, Carlsbad, CA, USA) according Indianapolis, IN, USA). Animals were considered diabetic > to the manufacturer’s protocol. Purified products were when fasting blood glucose was 250 mg/dl. Each experi- sequenced directly after estimating the concentration of mental group comprised eight animals. Tissues from five DNA products. Sequences were aligned with published animals were fixed in neutral formaline, covered with sequences using MegAlign sequence analysis software optimum cutting temperature (OCT) compound (Sakura (DNASTAR, Madison, WI, USA) to confirm their identity. 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 Protein isolation and Western blot analysis for islet isolation and culture. Pancreata from the different groups were lysed in modified RIPA lysis buffer (Gould & Hunter 1988), and the pro- tein concentrations in the supernatant were determined ELISA using the BCA protein assay reagent (Pierce, Rockford, Fasting OPN serum levels prior to induction of diabetes IL, USA). Equal protein concentrations were loaded and in control and diabetic animals were measured using on 10% SDS/polyacrylamide slab gels and transferred rat-specific ELISA kit (Assay Design, Ann Arbor, MI, to Immobilon-P membranes (Millipore, Bedford, MA,

Journal of Endocrinology (2005) 187, 237–247 www.endocrinology-journals.org

Downloaded from Bioscientifica.com at 09/27/2021 08:27:17PM via free access Osteopontin in STZ-induced diabetes · A K KATAKAM and others 239

USA). Blotted proteins were reacted with primary mouse were harvested. To evaluate whether OPN binding to the monoclonal OPN antibody (1:150 diluted in Tween/PBS; Arg-Gly-Asp (RGD) integrin-binding domain results in (0·2% Tween in PBS, pH 7·4) Santa Cruz Biotechnology, reduction of iNOS mRNA synthesis, cells were incubated Santa Cruz, CA, USA). Specificity of the antibody was with STZ+OPN (15 nM) +Gly-Arg-Gly-Asp-Asn-Pro evaluated by Western blot analysis with recombinant (GRGDNP) peptide (1 mM), or with STZ+OPN OPN protein containing a C-terminal His tag (Chemicon, (1·5 nM) +Gly-Arg-Ala-Asp-Ser-Pro (GRADSP; con- Temecula, CA, USA). The protein bands were visual- trol) peptide (1 mM; Biomol, Plymouth Meeting, PA, ized with enhanced chemiluminescence reagents (ECL USA). All concentrations were used according to our Plus Western Blotting Detection System; Amersham preliminary concentration studies with references to the Biosciences), analyzed, and intensity-quantified using values of nitrite release. the Kodak Electrophoresis Documentation and Analysis Diabetic islets were treated with OPN (0·15–15 nM) System 290 (EDAS 290). 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 Immunohistochemistry vehicle-treated healthy animals, whereas from the diabetic To localize OPN in the pancreas and study the changes in animals (at day 7), and according to the severity of its expression after diabetes induction, formaline-fixed, diabetes, we were able to isolate 0–200 islets/pancreas. 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 NO determination antibody against OPN (2A1; 1:100; Santa Cruz Biotech- In aqueous solution, NO is rapidly converted to nitrate nology), ready-to-use antibodies against rat insulin, gluca- and nitrite. The commercial kit we used (Calbiochem, gon, and pancreatic polypeptide (BioGenex; San Ramon, La Jolla, CA, USA) includes a nitrate reductase step that CA, USA) and somatostatin (1:100; Accurate Chemical, converts nitrate to nitrite prior to quantitation using Westbury, NY, USA). A vectastain universal elite ABC Griess reagent. Nitrite measurement was performed as an kit and diaminobenzedine chromogenic substrate (Vector indirect measure of NO production. Spectrophotometric Laboratories) were used according to the manufacturer’s evaluation of nitrite levels was made by Synergy HT protocol to visualize the tissue reaction to the antibodies. multi-detection microplate reader (BioTeck). In the diabetic pancreata, double immunostaining was employed to visualize OPN and insulin in paraffin sec- tions. Double immunostaining was also carried out in GSIS studies cryostat-cut 5 µm frozen sections to visualize OPN and Immediately after islet isolation, 5–10 rat islets per experi- infiltrating macrophages using antibody against CD68 ment were cultured for 3 h in insert-containing 24-well (1:200; Dako, Carpinteria, CA, USA). Insulin and plates (Corning, Corning, NY, USA) with 750 µl RPMI CD68 were visualized by alkaline phosphatase reaction medium with 5 mM glucose, 10% fetal calf serum, 10 mM (red), whereas diaminobenzedine was used to visualize HEPES, 100 U/ml penicillin G, and 100 µg/ml strepto- OPN (brown). mycin. 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 main- Islet isolation, culture, and STZ treatment tained overnight at 37 C. The next morning, the insert- Islets were isolated as mentioned elsewhere (Hill et al. containing islets were removed and transferred to RPMI 1999). Briefly, 20 ml cold Hank’s buffer/type IV colla- medium with 3 mM glucose and incubated for 1 h, at genase solution was infused into the rat pancreatic which time the medium was sampled for insulin measure- parynchema after its dissection. The inflated pancreas was ment. The medium glucose concentration was then cleaned from the surrounding fat and lymph nodes, increased to 17 mM and the islets incubated for an minced, and digested in a shaker-type water bath at 37 C. additional hour. Insulin assay was performed using Islets were recognized and handpicked under the stereo- rat-specific ultrasensitive insulin ELISA kit (DRG microscope after their staining with dithiazone. Islets were Diagnostics, Mountainside, NJ, USA). aliquoted and cultured in RPMI medium containing 5 mM glucose and supplemented with 10 mM HEPES, 1% -glutamine, and penicillin/streptomycin. Native rat OPN promoter studies OPN, a kind gift from Dr W. Butler, University of Texas, To explore the regulation of OPN in
-cells, RIN, clone was used for these studies. Islets were allowed to equili- 5F (RINm5F), an insulinoma cell line derived from the brate for 3 h before their treatment. Healthy islets with or NEDH rat islet cell tumor, were used. Cells were pur- without prior addition of OPN (0·15–15 nM) were treated chased from American Type Culture Collection and  with 0·5 mM STZ for 24 h after which islets and media grown at 37 C under a humidified, 5% CO2 atmosphere www.endocrinology-journals.org Journal of Endocrinology (2005) 187, 237–247

Downloaded from Bioscientifica.com at 09/27/2021 08:27:17PM via free access 240 A K KATAKAM and others · Osteopontin in STZ-induced diabetes

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 ampho- tericin 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 Figure 1 Serum OPN levels. Serum levels of OPN were examined (5–25 mM). Luciferase activities were assayed with the in STZ- and control vehicle-treated animals using rat-specific ELISA kit. Serum levels of OPN showed an initial 5-fold upregulation, Dual-Luciferase Reporter Assay System (Promega) in a 24 h after injection of STZ (day 1). Levels then came down but TD-20/20 Luminometer (Turner Designs, Sunnyvale, were still higher than prediabetic levels by the end of day 7. n=8 CA, USA). Transfection efficiency were normalized using in STZ-treated and n=6 in vehicle-treated groups. *P<0·05 versus the total protein concentration of the cell lysates. control prediabetic values.

Statistical analysis minimal changes. On days 3 and 7 the 50 kDa band was All experiments were performed between four and six downregulated but was still significantly higher than the times. Data were analyzed for statistical significance by prediabetic levels. analysis of variance (ANOVA) with post-hoc Student’s Semiquantitative PCR of OPN mRNA and correction t test analysis. These analyses were performed with the of the band intensity with GAPDH showed the same assistance of a computer program (JMP 5 software; SAS, pattern seen with the protein (Fig. 2B). These data Cary, NC, USA). Differences were considered significant indicate that the pancreas might be an active source of at Pc0·05. serum OPN.

OPN distribution in the control and diabetic pancreas Results Clear OPN immunoreactivity could be seen in vehicle- OPN serum levels treated rats. OPN-reactive cells were seen in the islets and in the pancreatic ducts (Fig. 3iB and D). Some islets Temporal changes in serum OPN levels after diabetes showed more OPN reactivity than others. Staining of induction was examined using a rat-specific ELISA kit. serial sections with islet hormone antibodies revealed > Serum OPN levels showed an initial 5-fold upregulation, the expression of OPN by other hormone-secreting cells  24 h after induction of diabetes (Fig. 1), from 19·49 0·5 (Fig. 3ii). Twenty-four hours after STZ injection, double   to 109·80 43·14 ng/ml (means S.E.M.). By day 3 levels immunostaining showed a more peripheral distribution of   dropped to 79·79 5·9 ng/ml, and then to 37·66 intensely stained OPN-reactive cells surrounding few 6·0 ng/ml by day 7. insulin-positive cells (Fig. 3 iiiB). OPN-intensified reac- tivity at this early time point was not associated with infiltrating macrophages (Fig. 3 iiiA), indicating that this is Expression of OPN protein and mRNA in control and a constitutive expression of OPN by islet cells and not by diabetic pancreas infiltrating macrophages that are reported to secret OPN Equal concentrations of the isolated protein from control (Masutani et al. 2003). and diabetic pancreata at the specified time points were electrophoresed using 10% SDS/PAGE. OPN protein molecular-mass isoforms were detected at 65 and OPN inhibits NO production in STZ-treated healthy islets 50 kDa. The 50 kDa band showed acute upregulation Next we investigated the functional significance of the by day 1 (Fig. 2A), while the 65 kDa band showed early high levels of OPN in the islets. NO production was

Journal of Endocrinology (2005) 187, 237–247 www.endocrinology-journals.org

Downloaded from Bioscientifica.com at 09/27/2021 08:27:17PM via free access Osteopontin in STZ-induced diabetes · A K KATAKAM and others 241

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 meansS.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 meansS.E.M. for control vehicle-treated (n=6) and STZ-treated (n=8) groups. *P<0·05 versus control prediabetic values. measured in rat islets exposed to STZ (0·5 mM) plus or glucose >400 mg/dl) produced low levels of NO and had minus pretreatment with OPN (0·15–15 nM). Nitrite defective GSIS. Addition of OPN to those islets did measurement was performed as an indirect measure of NO not significantly affect their nitrite production and did production in the media collected from the islets. The not improve their GSIS (data not shown). However, significantly elevated STZ-induced NO levels were dose- islets isolated from less severely diabetic animals (blood dependently reduced in the presence of OPN (Fig. 4A). glucose <300 mg/dl) produced high levels of NO. Islets that were treated with OPN alone did not show Addition of OPN (0·15–15 nM) significantly reduced significant changes when compared with untreated their NO levels (Fig. 5A). islets. OPN contains an RGD integrin-binding domain Healthy rat islets showed a 10-fold increase in insulin (Denhardt & Guo 1993, Guo et al. 2001). The hexapep- secretion with 17 mM glucose, whereas diabetic islets tide GRGDSP, which blocks binding of OPN to cell- showed
-cell dysfunction and significantly lower insulin surface integrins (Singh et al. 1995), was used to determine surge at 17 mM. Diabetic islets showed a dose-dependent the receptor-mediated effects of OPN. Cells were pre- significant improvement in GSIS after treatment with treated with GRGDSP hexapeptide (1 mM). In compari- OPN and a 40–60% restoration of control values (Fig. 5B), son to islets treated with GRADSP control peptide, showing that OPN is capable of improving the islet GRGDSP (1 mM) was found to significantly inhibit the function only in islets that are not severely dysfunctional. OPN-mediated decrease in iNOS synthesis (Fig. 4B), suggesting a requirement for OPN-integrin receptor bind-
ing for OPN-mediated NO regulation. Induction of OPN mRNA by STZ in RINm5F -cells To evaluate the expression of OPN in the RINm5F
-cell line and its regulation by STZ, we treated the cells with OPN inhibits NO production and improves the GSIS in STZ (5 mM). OPN mRNA was induced only with diabetic islets the highest dose of STZ (5 mM) after 6 h (Fig. 6A). To test whether addition of OPN could benefit the The concentration and duration of STZ treatment was diabetic islets, we isolated the islets from 1-week diabetic determined by dose- and time-response studies (data not rats. Islets isolated from severely diabetic animals (blood shown). www.endocrinology-journals.org Journal of Endocrinology (2005) 187, 237–247

Downloaded from Bioscientifica.com at 09/27/2021 08:27:17PM via free access 242 A K KATAKAM and others · Osteopontin in STZ-induced diabetes

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 con- centrations 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 microenviron- ment 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, atheroscler- osis, 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

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.

Journal of Endocrinology (2005) 187, 237–247 www.endocrinology-journals.org

Downloaded from Bioscientifica.com at 09/27/2021 08:27:17PM via free access Osteopontin in STZ-induced diabetes · A K KATAKAM and others 243

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 meansS.E.M. Each experiment was performed in duplicate and repeated three times for reproducibility. *Pc0·05 versus control values and #Pc0·05 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 meanS.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.

1996), kidney (Verhulst et al. 2002), bladder (Arafat et al. suggesting an autocrine/paracrine role for endogenous 2002), placenta (Johnson et al. 2003), and brain cells pancreatic OPN. The enhanced OPN reactivity was not (Iczkiewicz et al. 2004). In vivo expression of OPN has associated with accumulation of inflammatory cells in the previously been analyzed in several diabetic animal models islets (Fig. 3 iii), implicating islet OPN in the early islet in different tissues. Towler et al. (1998) demonstrated the response to STZ-induced cytotoxicity. upregulation of OPN expression in the aortas of high-fat One of the important pathogenetic mechanisms of diet-induced diabetic mice. Fischer et al. (1998) reported
-cell damage during experimental STZ-induced diabetes that the upregulation of OPN expression in the renal in rats and probably also in human insulin-dependent cortex of STZ-induced diabetic rats was mediated by diabetes mellitus is the cytokine-induced overproduction bradykinin. Aspord et al. (2004) reported OPN among the of NO by iNOS, with subsequent increase of local genes specifically activated in the islets and lymph nodes in oxidative stress in the pancreatic islets (Kwon et al. 1994, non-obese diabetic (NOD) mice. However, data concern- Haluzik & Nedvidkova 2000). We show here that ing the early changes in OPN expression after diabetes addition of STZ to isolated islets induced a signifi- induction and its functional significance have not been cant upregulation of NO levels (Fig. 4A). Interestingly, reported previously. In the present study, we demonstrate the same treatment induced significant upregulation of for the first time that as early as 24 h, OPN expression endogenous OPN mRNA and promoter activity (Fig. 6A levels are enhanced in the serum and in diabetic pancreas, and B). It would be of interest to know whether a more suggesting that diabetes-induced upregulation of OPN classical NO donor compound than STZ regulated OPN, expression is a general phenomenon observed across the and whether NO mediated the cytokine-induced increase different tissues. We also show that OPN is constitutively in OPN expression. Studies addressing these questions are expressed in the rat pancreatic islets and its expression is ongoing in our laboratory. intensified after STZ-diabetes induction. However, when The relationship between NO and OPN has been OPN serum levels were substantially reduced by day 7, examined by a number of investigators. Rollo et al. (1996) the pancreas continued to produce OPN (Fig. 2A and B), demonstrated that exogenous recombinant OPN protein www.endocrinology-journals.org Journal of Endocrinology (2005) 187, 237–247

Downloaded from Bioscientifica.com at 09/27/2021 08:27:17PM via free access 244 A K KATAKAM and others · Osteopontin in STZ-induced diabetes

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 meansS.E.M. Each experiment was performed in duplicate and repeated three times for reproducibility. *Pc0·05 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 meansS.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.

was effective in blocking RAW264·7 murine macrophage dose-dependent manner, but did it dose-dependently NO production and cytotoxicity toward the NO-sensitive restore the GSIS in the diabetic islets, suggesting the mastocytoma cells. Their work suggested that OPN in involvement of other mechanisms that mediate OPN extracellular fluid might protect certain tumor cells from protective effects. macrophage-mediated destruction by inhibiting the syn- Our data show that the anti-iNOS effect of OPN thesis of NO. However, these authors did not attempt to appears to be mediated by a membrane-bound integrin localize a potential cellular source for OPN in this setting. receptor because this effect was reversed when a peptide Singh et al. (1999) reported that a synthetic 20-amino acid that blocks the integrin receptor was added (Fig. 4B). OPN peptide analogue decreased iNOS mRNA and Nevertheless, a number of features of this OPN-NO protein levels in ventricular myocytes and cardiac micro- regulatory system remain to be clarified and the specific vascular endothelial cells. Transfection of cardiac micro- signaling pathway by which OPN ultimately modulates vascular endothelial cells with an antisense OPN cDNA iNOS synthesis in STZ-treated islets requires further increased iNOS mRNA in response to interleukin-1
and studies. For example, it would be important to determine interferon-, suggesting that endogenous OPN inhibits whether OPN plays a role in the regulation of nuclear NO production (Singh et al. 1995). Hwang et al. (1994) factor-B, which is a primary transcription factor necessary found that OPN suppressed NO synthesis induced by for iNOS synthesis (Flodstrom et al. 1996, Cardozo et al. interferon and lipopolysaccharide in primary mouse 2001), and whether OPN regulates additional signaling kidney proximal tubule epithelial cells, suggesting a regu- cascades that are activated by STZ. latory role for OPN in the NO signaling pathway. Data In this study, we also showed that OPN promoter from our study clearly demonstrate that OPN inhibits the was regulated by STZ and glucose. STZ induced an early STZ-mediated induction of NO in the islets through (3 h) upregulation, while glucose induced a late (48 h) downregulation of iNOS synthesis and suggest that OPN upregulation. This is suggestive that the early rise in is an important regulator of the NO signaling pathway and pancreatic OPN after diabetes induction is a response to NO-mediated cytoregulatory processes in the islets. In STZ-induced cytotoxicity, whereas the late rise is addition OPN reduced the NO levels in mildly diabetic hyperglycemia-induced. It is not known, however, islets and improved their GSIS. However, NO levels were whether there are STZ specific cis-element on the OPN surprisingly low in the severely diabetic islets and were not promoter and whether or not the STZ-induced OPN affected by exogenous OPN treatment, indicating that upregulation is mediated through NO, which has been OPN could be used to rescue islets that are not severely reported to induce OPN transcription in macrophages dysfunctional. High doses of OPN did not reduce STZ- (Guo et al. 2001). Studies into this question are currently derived nitrite or nitrite from STZ-diabetic islets in a ongoing in our laboratory.

Journal of Endocrinology (2005) 187, 237–247 www.endocrinology-journals.org

Downloaded from Bioscientifica.com at 09/27/2021 08:27:17PM via free access Osteopontin in STZ-induced diabetes · A K KATAKAM and others 245

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 meansS.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 meansS.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 meansS.E.M. from triplicate determinations. All experiments were repeated at least three times to confirm the reproducibility of the observations. www.endocrinology-journals.org Journal of Endocrinology (2005) 187, 237–247

Downloaded from Bioscientifica.com at 09/27/2021 08:27:17PM via free access 246 A K KATAKAM and others · Osteopontin in STZ-induced diabetes

The presence of a system of OPN-mediated regulation Eizirik DL & Pavlovic D 1997 Is there a role for nitric oxide in
-cell of NO in the islets and
-cells suggests potential targets for dysfunction and damage in IDDM? Diabetes/Metabolism Reviews 13 293–307. modulation of the NO-dependent components of the Fierabracci A, Biro PA, Yiangou Y, Mennuni C, Luzzago A, inflammatory response. The existence of OPN as a poten- Ludvigsson J, CorteseR&Bottazzo GF 1999 Osteopontin is an tial endogenous negative-feedback protective factor autoantigen of the somatostatin cells in human islets: identification against STZ-induced NO in the islets is unique. A better by screening random peptide libraries with sera of patients with understanding of OPN regulation and action during the insulin-dependent diabetes mellitus. Vaccine 18 342–354. Fischer JW, Tschope C, Reinecke A, Giachelli CM & Unger T 1998 early stages of diabetes and the factors involved in regula- Upregulation of osteopontin expression in renal cortex of tion of NO pathways will help to unravel the patho- streptozotocin-induced diabetic rats is mediated by bradykinin. physiology of diabetes and its associated complications. Diabetes 9 1512–1518. 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. Acknowledgements Gould KL & Hunter T 1988 Platelet-derived growth factor induces multisite phosphorylation of pp60c-src and increases its This study was supported by grants from the American protein-tyrosine kinase activity. Molecular Cell Biology 8 3345–3356. Guo H, Cai CQ, Schroeder RA & Kuo PC 2001 Osteopontin is a Diabetes Association 1–05-JF-01, Diabetes Trust negative feedback regulator of nitric oxide synthesis in murine Foundation, Diabetes Transplant Fund, and Diabetes macrophages. Journal of Immunology 166 1079–1086. Action Research & Education Foundation. The authors Haluzik M & Nedvidkova J 2000 The role of nitric oxide in the wish to thank Dr W. Butler, University of Texas, for development of streptozotocin-induced diabetes mellitus: experimental and clinical implications. Physiological Research 49 S37–S42. providing native OPN protein and Dr S. Mori, Chiba Hill DJ, Petrik J, Arany E, McDonald TJ & Delovitch TL 1999 University, Japan, for providing the OPN promoter. The Insulin-like growth factors prevent cytokine-mediated cell death in authors declare that there is no conflict of interest that isolated islets of Langerhans from pre-diabetic non-obese diabetic would prejudice the impartiality of this scientific work. mice. Journal of Endocrinology 16 153–165. 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 References kidney epithelial cells. Journal of Biological Chemistry 269 711–715. Iczkiewicz J, Rose S & Jenner P 2004 Osteopontin (Eta-1) is present Arafat HA, Wein AJ & Chacko S 2002 Osteopontin gene expression in the rat basal ganglia. Brain Research Molecular Brain Research 132 and immunolocalization in the rabbit urinary tract. Journal of Urology 64–72. 167 746–752. Johnson GA, Burghardt RC, Bazer FW & Spencer TE 2003 Asaumi S, Takemoto M, Yokote K, Ridall AL, Butler WT, Fujimoto Osteopontin: roles in implantation and placentation. Biology of M, Kobayashi K, Kawamura H, Take A, SaitoY&MoriS2003 Reproduction 69 1458–1471. Identification and characterization of high glucose and glucosamine Kohri K, Nomura S, Kitamura Y, Nagata T, Yoshioka K, Iguchi M, responsive element in the rat osteopontin promoter. Journal of Yamate T, Umekawa T, Suzuki Y, SinoharaH&Kurita T 1993 Diabetes Complications 17 34–38. Structure and expression of the mRNA encoding urinary stone Aspord C, Rome S & Thivolet C 2004 Early events in islets and protein (osteopontin). Journal of Biological Chemistry 268 pancreatic lymph nodes in autoimmune diabetes. Journal of 15180–15184. Autoimmunity 23 27–35. KwonNS,LeeSH,ChoiCS,KhoT&LeeHS1994Nitric oxide Cantor H 1995 The role of Eta-1/osteopontin in the pathogenesis of generation from streptozotocin. FASEB Journal 8 529–533. immunological disorders. Annals of the New York Academy of Sciences Liaw L, Birk DE, Ballas CB, Whitsitt JS, Davidson JM & Hogan BL 21 143–150. 1998 Altered wound healing in mice lacking a functional Cardozo AK, Heimberg H, Heremans Y, Leeman R, Kutlu B, osteopontin gene (spp1). Journal of Clinical Investigation 101 KruhofferM,OrntoftT&Eizirik DL 2001 A comprehensive 1468–1478. analysis of cytokine-induced and nuclear factor-kappa B-dependent Lukic ML, Stosic-GrujicicS&ShahinA1998Effector mechanisms in genes in primary rat pancreatic beta-cells. Journal of Biological low-dose streptozotocin-induced diabetes. Developmental Immunology Chemistry 276 48879–48886. 6 119–128. Corbett JA, Sweetland MA, Wang JL, Lancaster JR Jr & McDaniel Masutani K, Tokumoto M, Nakashima H, Tsuruya K, Kashiwagi M, ML 1993 Nitric oxide mediates cytokine-induced inhibition of Kudoh Y, Fukuda K, Kanai H, Akahoshi M, Otsuka T et al. 2003 insulin secretion by human islets of Langerhans. PNAS 90 Strong polarization toward Th1 immune response in ANCA- 1731–1735. associated glomerulonephritis. Clinical Nephrology 59 395–405. Craig AM, Bowden GT, Chambers AF, Spearman MA, Greenberg McKee MD & Nanci A 1996 Osteopontin at mineralized tissue AH, Wright JA, McLeodM&Denhardt DT 1990 Secreted interfaces in bone, teeth, and osseointegrated implants: phosphoprotein mRNA is induced during multi-stage ultrastructural distribution and implications for mineralized tissue carcinogenesis in mouse skin and correlates with the metastatic formation, turnover, and repair. Microscopy Research and Technique 33 potential of murine fibroblasts. International Journal of Cancer 46 141–164. 133–137. Oldberg A, FranzenA&Heinegard D 1986 Cloning and sequence Denhardt DT & Guo X 1993 Osteopontin: a protein with diverse analysis of rat bone sialoprotein (osteopontin) cDNA reveals an functions. FASEB Journal 7 1475–1482. Arg-Gly-Asp cell-binding sequence. PNAS 83 8819–8823. Denhardt DT, Noda M, O’Regan AW, PavlinD&BermanJS2001 Patarca R, Saavedra RA & Cantor H 1993 Molecular and cellular Osteopontin as a means to cope with environmental insults: basis of genetic resistance to bacterial infection: the role of the early regulation of inflammation, tissue remodeling, and cell survival. T-lymphocyte activation-1/osteopontin gene. Critical Reviews in Journal of Clinical Investigation 107 1055–1061. Immunology 13 225–246.

Journal of Endocrinology (2005) 187, 237–247 www.endocrinology-journals.org

Downloaded from Bioscientifica.com at 09/27/2021 08:27:17PM via free access Osteopontin in STZ-induced diabetes · A K KATAKAM and others 247

Rabinovitch A 1998 An update on cytokines in the pathogenesis of its functional role in accelerated atherogenesis. Arteriosclerosis insulin-dependent diabetes mellitus. Diabetes/Metabolism Reviews 14 Thrombosis and Vascular Biology 3 624–628. 129–151. Takemoto M, Yokote K, Yamazaki M, Ridall AL, Butler WT, Rollo EE, Laskin DL & Denhardt DT 1996 Osteopontin inhibits MatsumotoT,TamuraK,SaitoY&MoriS2000b Enhanced nitric oxide production and cytotoxicity by activated RAW264·7 expression of osteopontin by high glucose. Involvement of macrophages. Journal of Leukocyte Biology 60 397–404. osteopontin in diabetic macroangiopathy. Annals of the New York RydgrenT&SandlerS2002Efficacy of 1400 W, a novel inhibitor of Academy of Sciences 902 357–363. inducible nitric oxide synthase, in preventing interleukin-1 Towler DA, Bidder M, Latifi T, ColemanT&Semenkovich CF beta-induced suppression of pancreatic islet function in vitro and 1998 Diet-induced diabetes activates an osteogenic gene regulatory multiple low-dose streptozotocin-induced diabetes in vivo. European program in the aortas of low density lipoprotein receptor-deficient Journal of Endocrinology 147 543–551. mice. Journal of Biological Chemistry 273 30427–30434. Senger DR, Perruzzi CA & Papadopoulos A 1989 Elevated expression Verhulst A, Persy VP, Van Rompay AR, Verstrepen WA, Helbert of secreted phosphoprotein I (osteopontin, 2ar) as a consequence of MF & De Broe ME 2002 Osteopontin synthesis and localization neoplastic transformation. Anticancer Research 9 1291–1299. along the human nephron. Journal of the American Society of Singh K, Balligand JL, Fischer TA, Smith TW & Kelly RA 1995 Nephrology 13 1210–1218. Glucocorticoids increase osteopontin expression in cardiac myocytes and microvascular endothelial cells. Journal of Biological Chemistry Yu XQ, Fan JM, Nikolic-Paterson DJ, Yang N, Mu W, Pichler R, 270 28471–28478. Johnson RJ, Atkins RC & Lan HY 1999 IL-1 up-regulates Singh K, Sirokman G, Communal C, Robinson KG, Conrad CH, osteopontin expression in experimental crescentic Brooks WW, Bing OHL & Colucci WS 1999 Myocardial glomerulonephritis in the rat. American Journal of Pathology 154 osteopontin expression coincides with the development of heart 833–841. failure. Hypertension 33 663–670. Takemoto M, Yokote K, Nishimura M, Shigematsu T, Hasegawa T, Kon S, Uede T, Matsumoto T, SaitoY&MoriS2000a Enhanced Received 1 July 2005 expression of osteopontin in human diabetic artery and analysis of Accepted 26 August 2005

www.endocrinology-journals.org Journal of Endocrinology (2005) 187, 237–247

Downloaded from Bioscientifica.com at 09/27/2021 08:27:17PM via free access Downloaded from Bioscientifica.com at 09/27/2021 08:27:17PM via free access