help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2007-0106
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wang, O.
Right arrow Articles by Le, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wang, O.
Right arrow Articles by Le, Y.
Endocrinology Vol. 149, No. 2 672-680
Copyright © 2008 by The Endocrine Society

Mechanisms of Glucose-Induced Expression of Pancreatic-Derived Factor in Pancreatic β-Cells

Oumei Wang, Kun Cai, Shanshan Pang, Ting Wang, Dongfei Qi, Quanfeng Zhu, Zimei Ni and Yingying Le

Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, and Graduate School of the Chinese Academy of Sciences, Shanghai 200031, People’s Republic of China

Address all correspondence and requests for reprints to: Yingying Le, M.D., Ph.D., Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 294 Taiyuan Road, Shanghai 200031, People’s Republic of China. E-mail: yyle{at}sibs.ac.cn.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Pancreatic-derived factor (PANDER) is a cytokine-like peptide highly expressed in pancreatic β-cells. PANDER was reported to promote apoptosis of pancreatic β-cells and secrete in response to glucose. Here we explored the effects of glucose on PANDER expression, and the underlying mechanisms in murine pancreatic β-cell line MIN6 and primary islets. Our results showed that glucose up-regulated PANDER mRNA and protein levels in a time- and dose-dependent manner in MIN6 cells and pancreatic islets. In cells expressing cAMP response element-binding protein (CREB) dominant-negative construct, glucose failed to induce PANDER gene expression and promoter activation. Treatment of the cells with calcium chelator [EGTA, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)ester (BAPTA/AM)], the voltage-dependent Ca2+ channel inhibitor (nifedipine), the protein kinase A (PKA) inhibitor (H89), the protein kinase C (PKC) inhibitor (Go6976), or the MAPK kinase 1/2 inhibitor (PD98059), all significantly inhibited glucose-induced PANDER gene expression and promoter activation. Further studies showed that glucose induced CREB phosphorylation through Ca2+-PKA-ERK1/2 and Ca2+-PKC pathways. Thus, the Ca2+-PKA-ERK1/2-CREB and Ca2+-PKC-CREB signaling pathways are involved in glucose-induced PANDER gene expression. Wortmannin (phosphatidylinositol 3-kinase inhibitor), ammonium pyrrolidinedithiocarbamate (nuclear factor-{kappa}B inhibitor and nonspecific antioxidant), and N-acetylcysteine (antioxidant) were also found to inhibit glucose-induced PANDER promoter activation and gene expression. Because there is no nuclear factor-{kappa}B binding site in the promoter region of PANDER gene, these results suggest that phosphatidylinositol 3-kinase and reactive oxygen species be involved in glucose-induced PANDER gene expression. In conclusion, glucose induces PANDER gene expression in pancreatic β-cells through multiple signaling pathways. Because PANDER is expressed by pancreatic β-cells and in response to glucose in a similar way to those of insulin, PANDER may be involved in glucose homeostasis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PANCREATIC-DERIVED FACTOR (PANDER), also named FAM3B, is a novel cytokine-like peptide that was recently identified during a search for novel four-helix-bundle cytokines using structure-based methods (1). The PANDER gene is strongly expressed in pancreatic islets, and slightly expressed in the prostate and small intestine. Both human and mouse PANDER contain 235 amino acids, with 78% homology and four conserved cysteines (1). PANDER protein is present in both {alpha}- and β-cells of pancreatic islets; it appears to colocalize with both glucagon and insulin (2). Recombinant PANDER protein induces apoptosis of {alpha}- and β-cells of mouse, rat, and human islets in a dose- as well as time-dependent manner (2, 3). Treatment with interferon (IFN)-{gamma} or a combination of IL-1β, TNF-{alpha}, and IFN-{gamma} up-regulates PANDER gene expression in mouse islets and in insulin secreting β-cell line in a time- and dose-dependent manner (4), suggesting that PANDER may function as a downstream signal in cytokine-mediated β-cell dysfunction and death. Recently, glucose was reported to induce PANDER secretion in rodent β-cell line and islets (5). In this study we show that glucose can induce PANDER expression in mouse pancreatic islets and β-cell line at both mRNA and protein levels, and demonstrate that the effect of glucose on PANDER gene expression is mediated by multiple signaling pathways.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Pancreatic islet preparation and cell culture
All experiments using animals were in accordance with the National Institutes of Heath Guide for the Care and Use of Laboratory Animals and were approved by the Biological Research Ethics Committee, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. Pancreatic islets were isolated from C57/BL6 mice (Shanghai SLAC Laboratory Animal Co., Shanghai, China) by type V collagenase (Sigma-Aldrich, S. Louis, MO) digestion, followed by Ficoll 400 (Amersham Pharmacia Biotech, Piscataway, NJ) gradient separation, as described previously (6). Islets were maintained in DMEM (Life Technologies, Inc., Burlington, Ontario, Canada) medium containing 5.6 mM glucose, 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin. A total of 100 islets was pooled as one sample for further experiments. Murine pancreatic β-cell line MIN6 cells were cultured in DMEM containing 5.6 mM glucose, 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin in a humidified atmosphere at 37 C with 5% CO2.

RNA extraction and real-time PCR
Total RNA was extracted from MIN6 cells or mouse pancreatic islets using the TRIZOL reagent (Invitrogen, Carlsbad, CA) and depleted of contaminating DNA with RNase-free DNase according to the manufacturers’ instructions. cDNA was synthesized from 2 µg RNA with M-MuLV reverse transcriptase and random hexamer according to the manufacturer’s instructions (Fermentas, Burlington, Ontario, Canada). Quantitative real-time PCR was performed by using an ABI Prism 7500 sequence detector (Applied Biosystems Inc, Foster City, CA). Briefly, reverse-transcribed cDNA in triplicate samples were checked for PANDER mRNA level with SYBR Green PCR master kit (TOYOBO Biotech, Osaka, Japan) according to the manufacturer’s instructions. Primers for murine PANDER were: 5'-GCGAC AAGTA TGCCA AGA (sense); and 5'-GACGA CAGCA ATGTTT ATCC (antisense). Mouse β-actin primers were: 5'-CAACG AGCGG TTCCG ATG (sense); and 5'-GCCAC AGGAT TCCAT ACCCA (antisense) (7). The assays were initiated with 5 min at 95 C, and then 40 cycles of 15 sec at 94 C, 1 min at 60 C. A threshold cycle, CT (cycle of gene amplification that exceeds the minimum level of fluorescent detection), was calculated for both PANDER and β-actin. Amplification of the PANDER cDNA was normalized to β-actin expression. Relative levels of PANDER mRNA expression were calculated using the 2–{Delta}{Delta}CT method (8).

Cell viability assay
The effect of different inhibitors on the viability of MIN6 cells was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method. Briefly, cells (1 x 103 per well) were plated in 96-well plate, incubated at 37 C for 24 h, and treated with the indicated concentrations of the compounds for 3 h. MTT reagent (Sigma-Aldrich) (0.2 µg/µl per well) was added and incubated at 37 C for 4 h. The intracellular purple formazan product was dissolved with hydrogen chloride/isopropanol (0.04 mM). The absorbance at 570 nm (reference at 630 nm) was determined by a microplate reader Multiskan JX (Thermo LabSystems, Franklin, MA). Each experiment was repeated three times in triplicate samples.

Western blotting assay
After a 2-h stabilization period at 37 C in buffer containing 124 mM NaCl, 5.6 mM KCl, 2.5 mM MgCl2, 2.5 mM CaCl2, 20 mM HEPES, and 1 mM glucose (pH 7.0) (9), either in the absence or the presence of inhibitors, MIN6 cells or mouse pancreatic islets were stimulated with glucose as indicated in the figure legends. The cells were then lysed with cold radioimmunoprecipitation assay lysis buffer containing 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.1% sodium dodecyl sulfate, 100 mM Tris (pH 8.0), 10 mM NaF, 1% deoxycholic acid, 1 mM phenylmethylsulfonylfluoride, 1 mM sodium vanadate, 1 mM dithiothreitol, 10 µM aprotinin, and 4 µM leupeptin. The cell lysates were centrifuged at 14,000 rpm for 20 min to remove insoluble materials, and the concentration of protein was determined by Bradford assay. Western blotting of PANDER or phosphorylated ERK1/2 (p-ERK) and cAMP response element-binding protein (CREB) were performed according to the manufacturers’ instructions. Briefly, protein were electrophoresed on 10% SDS-PAGE gel and transferred onto polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA). The membranes were blocked with 5% nonfat milk and then were incubated with primary antibodies overnight at 4 C. After incubation with a horseradish peroxidase-conjugated secondary antibody, the protein bands were detected with a Supersignal West Pico chemiluminescent substrate (Pierce, Rockford, IL) and X-Omat BT film (Eastman Kodak Co., Rochester, NY). For detection of total ERK and CREB, the membranes were stripped with Restore Western Blot Stripping Buffer (Pierce), followed by incubation with specific antibodies. Monoclonal antibody against p-ERK1/2 was bought from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against ERK1/2, phosphorylated-CREB (p-CREB), and CREB were obtained from Cell Signaling Technology (New England Biolabs, Beverly, MA). Antibody against mouse PANDER protein was purchased from R&D Systems Inc. (Minneapolis, MN).

Construction of the PANDER promoter luciferase plasmids and luciferase reporter assay
Mouse genomic DNA was used as a PCR template to create multiple PANDER promoter luciferase constructs. Different regions of the PANDER promoter spanning from 2-kb upstream to 491-bp downstream of the transcriptional start site were amplified by PCR and cloned into the pGL3-basic luciferase reporter plasmid (Promega Corp., Madison, WI) between the KpnI and MluI sites. Primers used for creation of PANDER promoter luciferase constructs are listed in Table 1Go. All constructs were confirmed by restriction enzyme digestion and sequencing. MIN6 cells (8 x 104 per well) were plated in a 24-well tissue culture plate. PANDER promoter luciferase plasmids were cotransfected with the pRL-TK Renilla plasmid with a 40:1 ratio using Lipofectamine 2000 (Invitrogen). Transfection of each plasmid was performed in triplicate in at least three independent experiments. Forty-two hours after the transfection, cells were cultured in DMEM containing different concentrations of glucose and 2% FBS for another 5 h. Inhibitors were added 2 h before harvesting the cells for luciferase assay. Luciferase activity of the promoter constructs and the pRL-TK construct was measured sequentially using the Dual-Luciferase Reporter Assay System (Promega). Variation in transfection efficiency was normalized by dividing the promoter construct activity by the respective cotransfected pRL-TK luciferase activity.


View this table:
[in this window]
[in a new window]

  		TABLE 1. Primers for creation of PANDER promoter luciferase constructs

 
Transient transfection
Transfection was performed in MIN6 cells or mouse pancreatic islets with SuperFect Transfection Reagent (QIAGEN, Valencia, CA) on six-well plates using 5 µg dominant-negative CREB expressing plasmid A-CREB or the control vector pRcCMV (10) (kindly provided by Dr. R. Goodman, Oregon Health Sciences University, Portland, OR). Forty hours after transfection, the cells were stimulated with glucose (16 mM) for 2 h, and PANDER expression was then examined by real-time PCR. In mouse pancreatic islets, enhanced green fluorescent protein (EGFP) expressing plasmid was cotransfected with A-CREB or pRcCMV for 42 h to check the transfection efficiency.

Statistical analysis
Data are presented as means ± SEM. Statistical significance of differences between groups was analyzed by the unpaired Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Glucose induces PANDER expression in MIN6 cells and mouse pancreatic islets
To evaluate the effects of glucose on PANDER gene expression, murine pancreatic β-cell line MIN6 was challenged with different concentrations of glucose for different lengths of time, and the mRNA levels of PANDER were detected by quantitative real-time PCR. MIN6 cells cultured in DMEM containing 5.6 mM glucose expressed transcripts for PANDER, which were significantly enhanced when the cells were exposed to higher concentrations of glucose. The maximal effect of glucose on PANDER gene expression was obtained at 16 mM with a 2-h incubation period (Fig. 1Go, A and B). To demonstrate the physiological relevance of glucose-simulated PANDER gene expression, we used isolated mouse pancreatic islets. Consistent with the results obtained from MIN6 cells, mRNA levels of PANDER in murine pancreatic islets were significantly enhanced by glucose after 2-h stimulation. PANDER expression at 16 mM glucose was 5.6-fold that of the control value at 5.6 mM glucose (Fig. 1CGo).


Figure 1
View larger version (14K):
[in this window]
[in a new window]

  		FIG. 1. Glucose induces PANDER gene expression in MIN6 cells and mouse pancreatic islets. MIN6 cells were incubated with 25 mM glucose for different time periods (A), or with different concentrations of glucose for 2 h (B), then total RNA was extracted and examined for PANDER gene expression by real-time PCR. C, Mouse pancreatic islets were treated with 16 mM glucose for 2 h; PANDER gene expression was examined by real-time PCR. The increase of PANDER mRNA quantities was calculated as described in Materials and Methods. *, P < 0.05; **, P < 0.01 vs. cells cultured with medium containing 5.6 mM glucose. D, MIN6 cells pretreated with 5.6 or 16 mM glucose for 2 h were cultured with 5 µg/ml actinomycin D for the indicated time intervals. PANDER mRNA levels were then examined by real-time PCR. Results represent the mean ± SEM of three independent experiments.

 
To determine whether glucose-induced increase of PANDER mRNA levels was due to the increase of PANDER mRNA stability, MIN6 cells pretreated with 5.6 or 16 mM glucose for 2 h were cultured with 5 µg/ml actinomycin D for 2, 4, and 6 h before PANDER mRNA levels were examined by real-time PCR. As shown in Fig. 1DGo, the half-life of PANDER mRNA in MIN6 cells in control medium with 5.6 mM glucose was 5 h. In the presence of 16 mM glucose, although the levels of PANDER mRNA were markedly increased, there was no significant difference in the curves of mRNA decay, suggesting that glucose mainly increases the PANDER transcription in MIN6 cells.

The effect of glucose on PANDER protein expression was also examined. Western blot assay showed that 16 mM glucose time dependently induced PANDER protein expression, and the maximal effect of glucose was obtained with 24-h incubation period (Fig. 2AGo). The enhancement of PANDER protein expression by glucose was dose dependent (Fig. 2BGo). Consistently, 16 mM glucose also induced PANDER protein level in mouse pancreatic islets (Fig. 2CGo).


Figure 2
View larger version (56K):
[in this window]
[in a new window]

  		FIG. 2. Glucose induces PANDER protein expression in MIN6 cells and mouse pancreatic islets. MIN6 cells (A and B) or mouse pancreatic islets (C) were cultured with control medium containing 5.6 mM glucose or indicated concentrations of glucose for different times (A), or with different concentrations of glucose for 24 h (B and C), then were examined for PANDER protein expression by Western blot assay. The representative results of three independent experiments are shown.

 
Glucose induces PANDER gene expression through activation of CREB
In the promoter region of PANDER gene, there are binding sites for different transcription factors, including CREB, a transcription factor crucial for β-cell biology. So, we tested if CREB activation was involved in glucose-induced PANDER gene expression. We observed that treatment of MIN6 cells with 16 mM glucose stimulated rapid and transient phosphorylation of CREB, with a maximum observed at 5-min stimulation (Fig. 3AGo). To assess whether CREB is responsible for the inducible effect of glucose on PANDER gene expression, we transfected MIN6 cells or mouse pancreatic islets with a control vector pRcCMV or a CREB dominant-negative construct A-CREB that was constructed by fusing a designed acidic amphipathic extension onto the N terminus of the CREB leucine zipper domain (10). The acidic extension of A-CREB interacts with the basic region of CREB, forming a coiled-coil extension of the leucine zipper and, thus, prevents the basic region of wild-type CREB from binding to DNA. In MIN6 cells and mouse pancreatic islets transiently overexpressing A-CREB, glucose-induced PANDER gene expression was significantly attenuated (Fig. 3Go, B and C), suggesting an essential role for CREB in mediating glucose-induced PANDER gene expression. The transfection efficiency was examined by cotransfection of pRcCMV or A-CREB plasmid with EGFP expressing plasmid. As shown in Fig. 3DGo, mouse pancreatic islets successfully expressed EGFP 42 h after transfection. Untransfected islets did not exhibit any EGFP expression, even under robust levels of fluorescent exposure.


Figure 3
View larger version (33K):
[in this window]
[in a new window]

  		FIG. 3. Glucose stimulates PANDER gene expression in MIN6 cells and mouse pancreatic islets through activation of CREB. A, MIN6 cells were stimulated with 16 mM glucose for various times and examined for CREB phosphorylation by Western blot assay. The experiments were performed at least three times, and the results presented are from representative experiment. MIN6 cells (B) or mouse pancreatic islets (C) were transiently transfected with A-CREB or control vector pRcCMV for 40 h, then were treated with control medium containing 5.6 mM glucose, or 16 mM glucose for another 2 h. PANDER gene expression was examined by real-time PCR. **, P < 0.01 vs. cells (with or without pRcCMV transfection) cultured with control medium containing 5.6 mM glucose. {dagger}{dagger}, P < 0.01 vs. cells (with or without pRcCMV transfection) stimulated with 16 mM glucose. Values represent the mean ± SEM of three independent experiments. D, Mouse pancreatic islets were isolated from C57BL/6 mice. EGFP expressing plasmid was cotransfected with pRcCMV or A-CREB for 42 h to check the transfection efficiency. Representative transfected islets are shown under bright field (left lane) and fluorescent microscope (right lane). Untransfected islets did not express EGFP. Magnification, x20.

 
Ca2+-protein kinase A (PKA)-ERK1/2-CREB and Ca2+-protein kinase C (PKC)-CREB pathways are involved in glucose-induced PANDER gene expression
It was reported that glucose-induced calcium influx activated the transcription factor CREB, which is required for glucose homeostasis and islet-cell survival by regulating β-cell gene expression (11). Glucose has induced ERK1/2 phosphorylation through activation of PKA in pancreatic β-cells, and calcium influx is required for glucose-induced ERK1/2 phosphorylation (12). Thus, we checked if glucose induced PANDER gene expression through activation of Ca2+-PKA-ERK1/2-CREB pathway. Pretreatment of MIN6 cells with EGTA and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)ester (BAPTA/AM), extracellular and intracellular calcium chelator, respectively, completely abolished glucose-induced PANDER gene expression (Fig. 4AGo). Nifedipine, a voltage-dependent Ca2+ channel inhibitor, partly but dose dependently inhibited glucose-induced PANDER gene expression (Fig. 4AGo). EGTA, BAPTA/AM, and nifedipine at tested concentrations had no effect on cell viability (Fig. 4BGo). These results suggest that cytosolic Ca2+ elevation consequent to glucose-promoted Ca2+ influx and release from internal stores are crucial for glucose-induced PANDER gene expression, glucose-induced Ca2+ influx is partly through the activation of voltage-dependent Ca2+ channel. Pretreatment of MIN6 cells with H89, a specific inhibitor of PKA, and PD98059, a MAPK (MEK1/2) inhibitor, both blocked glucose-induced PANDER expression in dose-dependent manner (Fig. 4Go, C and D). H89 and PD98059 at tested concentrations did not affect cell viability (Fig. 4FGo). These results indicate the involvement of PKA and ERK1/2 activation in regulation of PANDER gene expression by glucose. We observed that H89 at low and high concentration both inhibited glucose-induced PANDER mRNA expression (Fig. 4CGo). Because H89 at high concentration also inhibits PKC (13), we then examined whether PKC was involved in glucose-induced PANDER gene expression. Go6976, an inhibitor of Ca2+-dependent PKC{alpha} and PKCβ1, significantly inhibited 16 mM glucose-induced PANDER mRNA expression (Fig. 4EGo) and had no effect on cell viability (Fig. 4FGo), suggesting that Ca2+-dependent PKC{alpha} and PKCβ1 were also involved in glucose-stimulated PANDER gene expression.


Figure 4
View larger version (50K):
[in this window]
[in a new window]

  		FIG. 4. Ca2+, PKA, PKC, and ERK1/2 are involved in glucose-induced PANDER gene expression. MIN6 cells were incubated with different concentrations of EGTA (EG), nifedipine (Nif), BAPTA/AM (BAP) (A), H89 (C), PD98059 (PD) (D), or 1 µM Go6976 (Go) (E) for 1 h, then stimulated with 16 mM glucose for 2 h and examined for PANDER expression by real-time PCR. Equal concentration of DMSO (DM) or ethanol (Eth) was used as control dissolvent. The effect of EGTA, nifedipine, BAPTA/AM, PD98059, H89, and Go6976 on cell viability was examined by MTT assay (B and F). **, P < 0.01 vs. cells cultured with control medium containing 5.6 mM glucose. {dagger}, P < 0.05; {dagger}{dagger}, P < 0.01 compared with cells treated with 16 mM glucose in the presence or absence of DMSO or ethanol. Results represent the mean ± SEM of three independent experiments.

 
We next tested if glucose could induce CREB phosphorylation through the activation of Ca2+-PKA-ERK1/2 and Ca2+-PKC pathways. When mouse pancreatic islets were treated with 16 mM glucose for different times, ERK1/2 and CREB were transiently phosphorylated (Fig. 5AGo). The phosphorylation of ERK1/2 happened earlier than that of CREB. The maximal phosphorylation of ERK1/2 and CREB reached at 5 min after glucose stimulation (Fig. 5AGo). Pretreatment of MIN6 cells with EGTA, nifedipine, H89, or PD98059 significantly inhibited glucose-induced ERK1/2 phosphorylation (Fig. 5BGo). H89, PD98059, or Go6976 treatment completely inhibited glucose-induced CREB phosphorylation (Fig. 5CGo). These results suggest that CREB phosphorylation induced by glucose in β-cells relies on Ca2+-PKA-ERK1/2 and Ca2+-PKC pathways.


Figure 5
View larger version (18K):
[in this window]
[in a new window]

  		FIG. 5. Involvement of Ca2+, PKA, PKC and ERK1/2 in glucose-induced CREB activation. A, Serum-starved mouse pancreatic islets were treated with 16 mM glucose for different times and examined for ERK1/2 and CREB phosphorylation by Western blot assay. B, MIN6 cells were incubated with 1 mM EGTA, 10 µM nifedipine, 0.05 µM H89, or 50 µM PD98059 for 2 h and then stimulated with 16 mM glucose for 5 min, examined for ERK1/2 phosphorylation by Western blot. C, MIN6 cells were stimulated with 16 mM glucose for 5 min in the presence or absence of 0.05 µM H89, 50 µM PD98059, or 1 µM Go6976, then, checked for CREB phosphorylation by Western blot. The most representative blots of three independent experiments are shown.

 
A previous report indicates that murine PANDER promoter is responsive to glucose (14). We examined the involvement of Ca2+-PKA-ERK1/2 and Ca2+-PKC pathways in glucose-induced PANDER promoter activation by luciferase reporter assay. Compared with MIN6 cells transfected with pGL3-basic plasmid, cells transfected with PANDER promoter constructs –2338/+491, –1338/+491, –338/+491, or +1/+491 all responded to glucose. Glucose increases the luciferase expression in a dose-dependent manner in cells transfected with construct –338/+491 or +1/+491, with a higher potency of construct +1/+491 (Fig. 6AGo). EGTA, nifedipine, BAPTA/AM, H89, PD98059, and Go6976 all inhibited glucose induced-luciferase expression by cells transfected with the +1/+491 construct (Fig. 6BGo). Cotransfection of the CREB dominant-negative construct A-CREB with construct +1/+491 containing a CREB binding site at +21/+30 significantly inhibited glucose induced luciferase expression (Fig. 6CGo). These results confirmed the effects that Ca2+, PKA, ERK1/2, and PKC were involved in glucose-stimulated transcription factor, including CREB activation in PANDER promoter region.


Figure 6
View larger version (18K):
[in this window]
[in a new window]

  		FIG. 6. Involvement of Ca2+, PKA, PKC, and ERK1/2 in glucose-induced PANDER promoter activation. The pGL3- basic vector (basic) or PANDER promoter luciferase constructs (–2338/+491, –1338/+491, –338/+491, and +1/+491) were cotransfected with the pRL-TK renilla luciferase reporter plasmid into MIN6 cells in medium containing 5 mM glucose. After 42 h, medium containing 2 or 16 mM glucose was added, and luciferase activity was measured 5 h later (A). There was 0.1 mM EGTA (EG), 10 µM nifedipine (Nif), 2 µM BAPTA/AM (BAP), 0.05 µM H89, 10 µM PD98059 (PD), or 1 µM Go6976 (Go) added 2 h before harvesting the cells for luciferase assay for +1/+491 PANDER promoter luciferase construct (B). C, MIN6 cells were cotransfected with 0.3 µg PANDER promoter luciferase construct +1/+491 and 0.2 µg A-CREB or control vector pRcCMV for 42 h, then cultured with medium containing 2 or 16 mM glucose for another 5 h. Luciferase activity was measured and normalized to renilla expression of the pRL-TK plasmid, and is shown as fold over the activity of the pGL-3 basic (A) or +1/+491 construct (B and C) at 2 mM glucose. All data are shown as mean ± SEM from three independent experiments with each condition tested in triplicate. **, P < 0.01, normalized luciferase activity of PANDER promoter luciferase constructs vs. pGL3-basic vector (A) or +1/+491 construct (B and C) at 2 mM glucose. {dagger}{dagger}, P < 0.01, normalized luciferase activity at 16 vs. 2 mM glucose (A) or +1/+491 construct at 16 mM glucose (B and C).

 
Collectively, our results demonstrate that glucose up-regulates PANDER expression through both Ca2+-PKA-ERK1/2-CREB and Ca2+-PKC-CREB pathways. Other transcription factors downstream of Ca2+, PKA, ERK1/2, or PKC may also be involved in glucose-induced PANDER gene expression.

Glucose induces PANDER gene expression through phosphatidylinositol 3-kinase (PI3K)- and reactive oxygen species (ROS)-related pathways
PI3Ks are proteins coupled to a variety of cell surface receptors and play a key role in signal transduction cascade regulating fundamental cellular functions, such as transcription, proliferation, and survival. Glucose has regulated preproinsulin gene expression through activation of PI3K (15) and stimulated insulin secretion through activation of nuclear factor-{kappa}B (NF-{kappa}B) (16) in MIN6 cells. PANDER is reported to be localized to insulin-containing granules in β-cells and secret in response to glucose (4, 5), and our results showed that PANDER gene expression was up-regulated by glucose. These results suggest that the mechanisms involved in PANDER gene expression by glucose may be similar to that of insulin. To examine if PI3K and NF-{kappa}B were involved in glucose-induced PANDER expression, cells were treated with different concentrations of wortmannin (PI3K inhibitor), or ammonium pyrrolidinedithiocarbamate (PDTC) (NF-{kappa}B inhibitor) before glucose stimulation, then PANDER gene expression was checked by real-time PCR. Preincubation of cells with wortmannin (Fig. 7Go, A and B), or PDTC (Fig. 7Go, C and D) significantly inhibited 16 mM glucose-enhanced PANDER gene expression in MIN6 cells and mouse pancreatic islets. PDTC and wortmannin at tested concentrations did not affect cell viability (Fig. 7FGo). When we analyzed the promoter region of PANDER gene with the AliBaba2 program (17), no NK-{kappa}B binding site was found. Because PDTC is also a nonspecific antioxidant (18, 19), we tested if ROS was involved in glucose-induced PANDER gene expression. As shown in Fig. 7EGo, N-acetylcysteine (NAC), an antioxidant (20), significantly inhibited glucose-induced PANDER gene expression. NAC at tested concentration had no effect on cell viability (Fig. 7FGo). These results suggest that activation of PI3K and production of ROS are necessary for the up-regulation of PANDER gene expression by glucose, and the inhibitory effect of PDTC on glucose-induced PANDER gene expression may be mediated by its antioxidant activity. We further examined the effect of wortmannin, PDTC, and NAC on glucose-induced PANDER promoter activation in MIN6 cells. PDTC, wortmannin, or NAC significantly inhibited glucose-induced luciferase expression in cells transfected with PANDER promoter construct –338/+491 or +1/+491, respectively (Fig. 8Go), with more potent efficiency in cells expressing –338/+491 construct. These results confirmed that glucose regulated PANDER gene expression in MIN6 cells via PI3K- and ROS- related pathways, and suggest that PI3K- and ROS- related transcription modulators are present widely in the promoter region of PANDER gene.


Figure 7
View larger version (37K):
[in this window]
[in a new window]

  		FIG. 7. Involvement of PI3K and ROS in glucose stimulated PANDER gene expression in MIN6 cells and pancreatic islets. Cells were treated with wortmannin (Wor) (A, MIN6 cells; B, primary pancreatic islets), PDTC (C, MIN6 cells; D, primary pancreatic islets), or NAC (E) for 1 h before glucose (16 mM) stimulation, and then PANDER mRNA levels were analyzed by real-time PCR. **, P < 0.01 vs. cells cultured with control medium containing 5.6 mM glucose. {dagger}{dagger}, P < 0.01 vs. cells stimulated with 16 mM glucose alone. F, The effect of wortmannin, PDTC, or NAC on cell viability was examined by MTT assay. Values represent the mean ± SEM of three independent experiments.

 

Figure 8
View larger version (21K):
[in this window]
[in a new window]

  		FIG. 8. PI3K and ROS are involved in glucose-induced PANDER promoter activation. The –338/+491 (A) or +1/+491 (B) PANDER promoter luciferase construct was cotransfected with the pRL-TK renilla luciferase reporter plasmid into MIN6 cells in medium containing 5 mM glucose. After 42 h, medium containing 2 or 16 mM glucose was added, and luciferase activity was measured 5 h later. There was 1 µM PDTC, 100 nM wortmannin (Wor), or 1 mM NAC added 2 h before harvesting the cells for luciferase assay. Luciferase activity was normalized to renilla expression of the pRL-TK plasmid and is shown as fold over the activity of the construct at 2 mM glucose. **, P < 0.01, normalized luciferase activity at 16 mM glucose vs. 2 mM glucose. {dagger}, P < 0.05; {dagger}{dagger}, P < 0.01 vs. cells stimulated with 16 mM glucose alone. All data are shown as mean ± SEM from three independent experiments with each condition tested in triplicate.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this project we studied the effect of glucose on PANDER gene expression in both pancreatic β-cell line MIN6 and isolated mouse pancreatic islets. Our data show that glucose induces PANDER expression at mRNA and protein level but has no effect on PANDER mRNA stability. We demonstrate that glucose induces PANDER gene expression in pancreatic β-cells through multiple pathways.

PANDER is a novel gene that was recently discovered during a search for novel four-helix-bundle cytokines using structure-based methods (1). Northern blot analysis showed the greater expression of PANDER in human pancreas, and to a lesser extent in small intestine and prostate. Immunohistochemistry confirmed the presence of PANDER reactivity in the islets of Langerhans. A study with confocal microscopy and immunogold electron microscopy showed that PANDER was localized to the insulin-containing secretory granules in both murine β-cell line β-TC3 and rodent islet β-cells (4), suggesting that the regulation of PANDER expression and release may be similar to that of insulin.

Insulin biosynthesis and secretion from β-cells of islets of Langerhans are triggered by increased concentrations of circulating glucose, so we examined if PANDER gene expression was also regulated by glucose. We found that glucose induced PANDER gene expression in both MIN6 cells and murine primary islets, and the maximum effect of glucose reached at 2 h. We analyzed the sequence of the promoter region of PANDER gene with AliBaba2 program (17) and found that there is a binding site for transcription factor CREB at +21/+30. When MIN6 cells and murine pancreatic islets were transfected with dominant-negative CREB expression vector A-CREB, glucose failed to induce PANDER gene expression (Fig. 2Go, B and C). MIN6 cells expressing a luciferase reporter construct +1/+491 containing the CREB binding site in the promoter region of PANDER responded to glucose in a dose-dependent manner (Fig. 6BGo). Introduction of A-CREB attenuated glucose-induced luciferase expression (Fig. 6CGo). These results demonstrate that CREB is involved in glucose-induced PANDER gene expression. In support of our results, Wolf and colleagues (14) reported that murine β-TC3 cells and primary islets transfected with luciferase reporter plasmids constructed with the murine PANDER promoter region showed a strong response to glucose, with a robust increase in luciferase expression in a dose-dependent manner for +1/+491 constructs.

Previous studies showed that glucose-induced calcium influx activated the transcription factor CREB, which was required for glucose homeostasis and islet-cell survival by regulating β-cell gene expression, such as the insulin gene and the antiapoptotic gene bcl2 (11). Pancreatic β-cell ERK1/2 activation is required for the regulation of insulin gene expression by glucose (21, 22). PKA and ERK1/2 have induced the phosphorylation of CREB that is required for CREB-mediated stimulation of transcription of many genes considered essential for the glucose-responsive β-cell phenotype and survival (11). By using chelators for Ca2+ and inhibitors for Ca2+ channel, MEK1/2, PKA, and PKC, respectively, we demonstrate that the Ca2+-PKA-ERK1/2-CREB and Ca2+-PKC-CREB signaling pathways are involved in glucose-induced PANDER promoter activation and gene expression in pancreatic β-cells.

Glucose stimulated preproinsulin gene expression by activation of PI3K but not p38 MAPK/SAPK2 (15). We found that PI3K inhibitor could block glucose-enhanced PANDER promoter activation and gene expression in MIN6 cells and mouse pancreatic islets, but p38 MAP kinase inhibitor had no effect (Figs. 7Go and 8Go; data not shown). PI3Ks can be divided into three classes based on their structural characteristics and substrate specificity. Some class II PI3Ks use Ca2+ and ATP for their lipid kinase activity (23). In the present study, glucose induced Ca2+ influx may be involved in the activation of PI3K.

PKA has been reported to be upstream molecule of octamer-binding transcription factor (Oct-1) (24). PKC can activate activating protein 1 (AP-1) and early growth response 1 (Egr-1) (25, 26). ERK1/2 activates CREB, Ets-like protein (Elk), Egr-1, AP-1, Oct-1, and promoter-specific transcription factor 1 (Sp1) (27, 28, 29, 30, 31, 32, 33, 34, 35, 36). PI3K has been reported to be activator of CREB, Elk, Egr-1, hepatocyte nuclear factor-4 (HNF-4), and Sp1 (30, 32, 37, 38, 39). The binding sites of the aforementioned transcription factors all exist in the promoter region –338/+491 of PANDER gene (Table 2Go). Therefore, the activation of these transcription factors by their respective upstream molecules, such as PKA, PKC, ERK1/2, and PI3K, may also be involved in glucose-induced PANDER gene expression but need further investigation.


View this table:
[in this window]
[in a new window]

  		TABLE 2. Putative transcriptional factor (TF) binding sites in the promoter region (–338 /+491) of PANDER gene predicted by AliBaba2 program

 
Although PDTC, the NF-{kappa}B inhibitor significantly inhibited glucose-induced PANDER promoter activation and PANDER mRNA expression (Figs. 7Go, C and D, and 8Go) by MIN6 cells and primary pancreatic islets, we did not find NF-{kappa}B binding sites in the promoter region of PANDER gene. Because glucose can induce the production of ROS by pancreatic β-cells (40) and PDTC has been reported to be a potent antioxidant both in vitro and in vivo (18, 19), we tested whether ROS was involved in glucose-induced PANDER gene expression. Our results showed that NAC, which bolsters endogenous glutathione (a key free radical scavenger) synthesis and displays antioxidant, as well as free radical scavenging actions (20), significantly inhibited glucose-induced PANDER gene expression and promoter activation. Therefore, the inhibitory effect of PDTC on glucose-induced PANDER gene may be mediated by its antioxidant activity. ROS has mediated high glucose-induced activation of PKC, ERK, Egr-1, AP-1, and Sp1 (41, 42, 43, 44). Thus, these ROS downstream signaling molecules and transcription factors existing in the promoter region of PANDER gene may also be involved in glucose-induced PANDER gene expression.

Although the physiological role of PANDER is unknown, recombinant PANDER protein induces apoptosis of {alpha}- and β-cells of mouse, rat, and human islets in a dose-dependent as well as time-dependent manner (2), it is conceivable that PANDER may play role in β-cell death in the context of diabetes. PANDER gene expression is up-regulated by IFN-{gamma}, a cytokine implicated in the pathogenesis of type 1 diabetes (4), suggesting that PANDER may contribute to the pathogenesis of β-cell death. Recent studies showed that glucose stimulated PANDER secretion dose dependently in β-cell lines and primary islets but not in {alpha}-cells, and PANDER is likely cosecreted with insulin via the same regulatory mechanisms (5). Our present study showed that glucose induced PANDER gene expression in pancreatic β-cells through the Ca2+-PKA-ERK1/2-CREB, Ca2+-PKC-CREB, and PI3K-, ROS-related signaling pathways. An understanding of how PANDER is regulated along with the pivotal molecules involved is helpful to elucidate further the potential function of this novel cytokine. Because PANDER is expressed by pancreatic β-cells and response to glucose in a similar way to those of insulin, and is a pro-apoptotic factor, PANDER may be involved in glucose homeostasis and β-cell homeostasis. The effect of PANDER on glucose homeostasis is under investigation.


   Acknowledgments
 
We thank Ms. X. Jiang L. Ma and X. Yu for their technical assistance.


   Footnotes
 
This work was supported by research grants from One Hundred Talents Program of Chinese Academy of Sciences, and the Science & Technology Commission of Shanghai Municipality (04DZ14007) (to Y.L.).

Disclosure Summary: The authors have nothing to disclose.

First Published Online October 25, 2007

Abbreviations: AP-1, Activating protein 1; BAPTA/AM, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)ester; CREB, cAMP response element-binding protein; p-CREB, phosphorylated CREB; EGFP, enhanced green fluorescent protein; Egr-1, early growth response 1; Elk, EST-like protein; FBS, fetal bovine serum; IFN, interferon; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; NAC, N-acetylcysteine; NF-{kappa}B, nuclear factor-{kappa}B; Oct-1, octamer-binding transcription factor 1; PANDER, pancreatic-derived factor; PDTC, pyrrolidine dithiocarbamate; p-ERK, phosphorylated ERK; PI3K, phosphatidylinositol 3-kinase; PKA, protein kinase A; PKC, protein kinase C; ROS, reactive oxygen species; SP1, promoter-specific transcription factor 1.

Received January 24, 2007.

Accepted for publication October 15, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Zhu Y, Xu G, Patel A, McLaughlin MM, Silverman C, Knecht K, Sweitzer S, Li X, McDonnell P, Mirabile R, Zimmerman D, Boyce R, Tierney LA, Hu E, Livi GP, Wolf B, Abdel-Meguid SS, Rose GD, Aurora R, Hensley P, Briggs M, Young PR 2002 Cloning, expression, and initial characterization of a novel cytokine-like gene family. Genomics 80:144–150[CrossRef][Medline]
  2. Cao X, Gao Z, Robert CE, Greene S, Xu G, Xu W, Bell E, Campbell D, Zhu Y, Young R, Trucco M, Markmann JF, Naji A, Wolf BA 2003 Pancreatic-derived factor (FAM3B), a novel islet cytokine, induces apoptosis of insulin-secreting β-cells. Diabetes 52:2296–2303[Abstract/Free Full Text]
  3. Cao X, Yang J, Burkhardt BR, Gao Z, Wong RK, Greene SR, Wu J, Wolf BA 2005 Effects of overexpression of pancreatic derived factor (FAM3B) in isolated mouse islets and insulin-secreting βTC3 cells. Am J Physiol Endocrinol Metab 289:E543–E550
  4. Xu W, Gao Z, Wu J, Wolf BA 2005 Interferon-{gamma}-induced regulation of the pancreatic derived cytokine FAM3B in islets and insulin-secreting βTC3 cells. Mol Cell Endocrinol 240:74–81[CrossRef][Medline]
  5. Yang J, Robert CE, Burkhardt BR, Young RA, Wu J, Gao Z, Wolf BA 2005 Mechanisms of glucose-induced secretion of pancreatic-derived factor (PANDER or FAM3B) in pancreatic β-cells. Diabetes 54:3217–3228[Abstract/Free Full Text]
  6. McDaniel ML, Colca JR, Kotagal N, Lacy PE 1983 A subcellular fractionation approach for studying insulin release mechanisms and calcium metabolism in islets of Langerhans. Methods Enzymol 98:182–200[Medline]
  7. Vincent VA, DeVoss JJ, Ryan HS, Murphy Jr GM 2002 Analysis of neuronal gene expression with laser capture microdissection. J Neurosci Res 69:578–586[CrossRef][Medline]
  8. Livak KJ, Schmittgen TD 2001 Analysis of relative gene expression data using real-time quantitative PCR and the 2(-{Delta}{Delta}C(T)) method. Methods 25:402–408[CrossRef][Medline]
  9. Benes C, Roisin MP, Van Tan H, Creuzet C, Miyazaki J, Fagard R 1998 Rapid activation and nuclear translocation of mitogen-activated protein kinases in response to physiological concentration of glucose in the MIN6 pancreatic β cell line. J Biol Chem 273:15507–15513[Abstract/Free Full Text]
  10. Ahn S, Olive M, Aggarwal S, Krylov D, Ginty DD, Vinson C 1998 A dominant-negative inhibitor of CREB reveals that it is a general mediator of stimulus-dependent transcription of c-fos. Mol Cell Biol 18:967–977[Abstract/Free Full Text]
  11. Jhala US, Canettieri G, Screaton RA, Kulkarni RN, Krajewski S, Reed J, Walker J, Lin X, White M, Montminy M 2003 cAMP promotes pancreatic β-cell survival via CREB-mediated induction of IRS2. Genes Dev 17:1575–1580[Abstract/Free Full Text]
  12. Briaud I, Lingohr MK, Dickson LM, Wrede CE, Rhodes CJ 2003 Differential activation mechanisms of Erk-1/2 and p70(S6K) by glucose in pancreatic β-cells. Diabetes 52:974–983[Abstract/Free Full Text]
  13. Johannes FJ, Prestle J, Dieterich S, Oberhagemann P, Link G, Pfizenmaier K 1995 Characterization of activators and inhibitors of protein kinase C µ. Eur J Biochem 227:303–307[Medline]
  14. Burkhardt BR, Yang MC, Robert CE, Greene SR, McFadden KK, Yang J, Wu J, Gao Z, Wolf BA 2005 Tissue-specific and glucose-responsive expression of the pancreatic derived factor (PANDER) promoter. Biochim Biophys Acta 1730:215–225[Medline]
  15. Rafiq I, da Silva Xavier G, Hooper S, Rutter GA 2000 Glucose-stimulated preproinsulin gene expression and nuclear trans-location of pancreatic duodenum homeobox-1 require activation of phosphatidylinositol 3-kinase but not p38 MAPK/SAPK2. J Biol Chem 275:15977–15984[Abstract/Free Full Text]
  16. Norlin S, Ahlgren U, Edlund H 2005 Nuclear factor-{kappa}B activity in β-cells is required for glucose-stimulated insulin secretion. Diabetes 54:125–132[Abstract/Free Full Text]
  17. Grabe N 2002 AliBaba2: context specific identification of transcription factor binding sites. In Silico Biol 2:S1–S15
  18. Schreck R, Meier B, Mannel DN, Droge W, Baeuerle PA 1992 Dithiocarbamates as potent inhibitors of nuclear factor {kappa} B activation in intact cells. J Exp Med 175:1181–1194[Abstract/Free Full Text]
  19. Liu SF, Ye X, Malik AB 1999 Inhibition of NF-{kappa}B activation by pyrrolidine dithiocarbamate prevents in vivo expression of proinflammatory genes. Circulation 100:1330–1337[Abstract/Free Full Text]
  20. Wang AL, Wang JP, Wang H, Chen YH, Zhao L, Wang LS, Wei W, Xu DX 2006 A dual effect of N-acetylcysteine on acute ethanol-induced liver damage in mice. Hepatol Res 34:199–206[CrossRef][Medline]
  21. Benes C, Poitout V, Marie JC, Martin-Perez J, Roisin MP, Fagard R 1999 Mode of regulation of the extracellular signal-regulated kinases in the pancreatic β-cell line MIN6 and their implication in the regulation of insulin gene transcription. Biochem J 340:219–225[CrossRef][Medline]
  22. Khoo S, Griffen SC, Xia Y, Baer RJ, German MS, Cobb MH 2003 Regulation of insulin gene transcription by ERK1 and ERK2 in pancreatic β cells. J Biol Chem 278:32969–32977[Abstract/Free Full Text]
  23. Arcaro A, Volinia S, Zvelebil MJ, Stein R, Watton SJ, Layton MJ, Gout I, Ahmadi K, Downward J, Waterfield MD 1998 Human phosphoinositide 3-kinase C2β, the role of calcium and the C2 domain in enzyme activity. J Biol Chem 273:33082–33090[Abstract/Free Full Text]
  24. Diamond SE, Chiono M, Gutierrez-Hartmann A 1999 Reconstitution of the protein kinase A response of the rat prolactin promoter: differential effects of distinct Pit-1 isoforms and functional interaction with Oct-1. Mol Endocrinol 13:228–238[Abstract/Free Full Text]
  25. Payvandi F, Wu L, Naziruddin SD, Haley M, Parton A, Schafer PH, Chen RS, Muller GW, Hughes CC, Stirling DI 2005 Immunomodulatory drugs (IMiDs) increase the production of IL-2 from stimulated T cells by increasing PKC-è activation and enhancing the DNA-binding activity of AP-1 but not NF-{kappa}B, OCT-1, or NF-AT. J Interferon Cytokine Res 25:604–616[CrossRef][Medline]
  26. Gill PK, Gescher A, Gant TW 2001 Regulation of MDR1 promoter activity in human breast carcinoma cells by protein kinase C isozymes {alpha} and è. Eur J Biochem 268:4151–4157[Medline]
  27. Costes S, Broca C, Bertrand G, Lajoix AD, Bataille D, Bockaert J, Dalle S 2006 ERK1/2 control phosphorylation and protein level of cAMP-responsive element-binding protein: a key role in glucose-mediated pancreatic β-cell survival. Diabetes 55:2220–2230[Abstract/Free Full Text]
  28. Gille H, Kortenjann M, Thomae O, Moomaw C, Slaughter C, Cobb MH, Shaw PE 1995 ERK phosphorylation potentiates Elk-1-mediated ternary complex formation and transactivation. EMBO J 14:951–962[Medline]
  29. Bernal-Mizrachi E, Wen W, Srinivasan S, Klenk A, Cohen D, Permutt MA 2001 Activation of Elk-1, an Ets transcription factor, by glucose and EGF treatment of insulinoma cells. Am J Physiol Endocrinol Metab 281:E1286–E1299
  30. Zhang Q, Adiseshaiah P, Kalvakolanu DV, Reddy SP 2006 A phosphatidylinositol 3-kinase-regulated Akt-independent signaling promotes cigarette smoke-induced FRA-1 expression. J Biol Chem 28:110174–10181
  31. Biesiada E, Razandi M, Levin ER 1996 Egr-1 activates basic fibroblast growth factor transcription. Mechanistic implications for astrocyte proliferation. J Biol Chem 271:18576–18581[Abstract/Free Full Text]
  32. Gousseva N, Kugathasan K, Chesterman CN, Khachigian LM 2001 Early growth response factor-1 mediates insulin-inducible vascular endothelial cell proliferation and regrowth after injury. J Cell Biochem 81:523–534[CrossRef][Medline]
  33. Pulverer BJ, Kyriakis JM, Avruch J, Nikolakaki E, Woodgett JR 1991 Phosphorylation of c-jun mediated by MAP kinases. Nature 353:670–674[CrossRef][Medline]
  34. Chen RH, Abate C, Blenis J 1993 Phosphorylation of the c-Fos transrepression domain by mitogen-activated protein kinase and 90-kDa ribosomal S6 kinase. Proc Natl Acad Sci USA 90:10952–10956[Abstract/Free Full Text]
  35. Tong T, Fan W, Zhao H, Jin S, Fan F, Blanck P, Alomo I, Rajasekaran B, Liu Y, Holbrook NJ, Zhan Q 2001 Involvement of the MAP kinase pathways in induction of GADD45 following UV radiation. Exp Cell Res 269:64–72[CrossRef][Medline]
  36. Kuo L, Chang HC, Leu TH, Maa MC, Hung WC 2006 Src oncogene activates MMP-2 expression via the ERK/Sp1 pathway. J Cell Physiol 207:729–734[CrossRef][Medline]
  37. Roth U, Curth K, Unterman TG, Kietzmann T 2004 The transcription factors HIF-1 and HNF-4 and the coactivator p300 are involved in insulin-regulated glucokinase gene expression via the phosphatidylinositol 3-kinase/protein kinase B pathway. J Biol Chem 279:2623–2631[Abstract/Free Full Text]
  38. Jin HO, An S, Lee HC, Woo SH, Seo SK, Choe TB, Yoo DH, Lee SB, Um HD, Lee SJ, Park MJ, Kim JI, Hong SI, Rhee CH, Park I 2007 Hypoxic condition- and high cell density-induced expression of Redd1 is regulated by activation of hypoxia-inducible factor-1{alpha} and Sp1 through the phosphatidylinositol 3-kinase/Akt signaling pathway. Cell Signal 19:1393–1403[CrossRef][Medline]
  39. Niina I, Uchiumi T, Izumi H, Torigoe T, Wakasugi T, Igarashi T, Miyamoto N, Onitsuka T, Shiota M, Okayasu R, Chijiiwa K, Kohno K 2007 DNA topoisomerase inhibitor, etoposide, enhances GC-box-dependent promoter activity via Sp1 phosphorylation. Cancer Sci 98:858–863[CrossRef][Medline]
  40. Robertson R, Zhou H, Zhang T, Harmon JS 2007 Chronic oxidative stress as a mechanism for glucose toxicity of the β cell in type 2 diabetes. Cell Biochem Biophys 48:139–146[CrossRef][Medline]
  41. Lee HB, Yu MR, Yang Y, Jiang Z, Ha H 2003 Reactive oxygen species-regulated signaling pathways in diabetic nephropathy. J Am Soc Nephrol 14(8 Suppl 3):S241–S245
  42. Wung BS, Cheng JJ, Chao YJ, Hsieh HJ, Wang DL 1999 Modulation of Ras/Raf/extracellular signal-regulated kinase pathway by reactive oxygen species is involved in cyclic strain-induced early growth response-1 gene expression in endothelial cells. Circ Res 84:804–812[Abstract/Free Full Text]
  43. Nose K, Ohba M 1996 Functional activation of the egr-1 (early growth response-1) gene by hydrogen peroxide. Biochem J 316:381–383[Medline]
  44. Srinivasan S, Hatley ME, Bolick DT, Palmer LA, Edelstein D, Brownlee M, Hedrick CC 2004 Hyperglycaemia-induced superoxide production decreases eNOS expression via AP-1 activation in aortic endothelial cells. Diabetologia 47:1727–1734[CrossRef][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wang, O.
Right arrow Articles by Le, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wang, O.
Right arrow Articles by Le, Y.

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