Protein kinase A-mediated CREB phosphorylation is an oxidant-induced survival pathway in alveolar type II cells

Oxidant stress plays a role in the pathogenesis of pulmonary diseases, including fibrotic lung disease and cancer. We previously found that hydrogen peroxide (H2O2) initiates an increase in Ca2+/cAMP-response element binding protein (CREB) phosphorylation in C10 alveolar type II cells that requires activation of extracellular regulated kinases 1/2 (ERK1/2). Here, we investigated the role of crosstalk between protein kinase A (PKA) and epidermal growth factor receptor (EGFR) in oxidant-induced signaling to ERK1/2 and CREB in C10 cells. Application of H2O2 increased nuclear accumulation of PKA, and inhibition of PKA with H89 reduced oxidant-mediated phosphorylation of both CREB and ERK1/2. Single cell measurements of cAMP and redox status, using a FRET-based biosensor and a redox-sensitive GFP, respectively, indicated that H2O2 increases production of cAMP that correlates with redox state. Inhibition of EGFR activity decreased both H2O2-induced CREB phosphorylation and translocation of PKA to the nucleus, suggesting that crosstalk between PKA and EGFR underlies the oxidant-induced CREB response. Furthermore, knockdown of CREB expression using siRNA led to a decrease in bcl-2 and an increase in oxidant-induced apoptosis. Together these data reveal a novel role for crosstalk between PKA, ERK1/2 and CREB that mediates cell survival during oxidant stress.
Introduction
Lung epithelial cells, a target cell of inhaled oxidants, are subjected to a variety of environmental stresses, including oxidizing gases, particulates, and airborne microorganisms. Although the formation of oxidants is normally well regulated, excessive production may cause inflammation and cellular injury. Numerous studies suggest a role for oxidant stress in the pathogenesis of pulmonary diseases, including asthma, pulmonary fibrosis, and cancer [1].
Reactive oxygen species (ROS) are involved in multiple physiological processes through their capacity to regulate the expression of several growth factor receptors, protein kinases and phosphatases. In alveolar type II lung epithelial cells, hydrogen peroxide (H2O2) induces tyrosine phosphorylation of the epidermal growth factor receptor (EGFR) [2] and leads to activation of protein kinase C (PKC) [3] and the mitogen activated protein kinases (MAPK) p38, c-Jun amino-terminal kinase (JNK), and extracellular signal-regulated kinase 1/2 (ERK1/2) [4, 5]. H2O2 also activates the expression of transcription factors, including Ca2+/cyclic AMP-response element binding protein (CREB) [6] and members of the activator protein (AP-1) family, c-jun and c-fos [7], resulting in proliferation [4] and apoptosis [6, 8].
CREB is a 43 kDa transcription factor belonging to the basic-leucine zipper (bZIP) family and is regulated by phosphorylation at serine residue 133 [9, 10]. Phosphorylation of CREB initiates recruitment of co-factors to the Ca2+/cAMP-response element (CRE) that are necessary for transcriptional activation such as CREB-binding protein (CBP300) [11]. CREB activation is regulated by both Ca2+ and cAMP, which have been shown to regulate both ERK1/2- and protein kinase A (PKA)-mediated CREB phosphorylation [6, 12, 13]. Many genes important for regulation of proliferation and apoptosis, including c-fos and bcl-2, respectively, contain CREs in their promoter [11].
Our previous studies have shown that exposure of alveolar type II cells to bolus H2O2 or asbestos fibers results in CREB activation that is dependent on ERK1/2 activation [6, 14]. Furthermore, we found that preventing CREB activation promoted cell survival and enhanced bcl-2 transcription, revealing a potential role for CREB in H2O2-mediated apoptosis [6]. Because of known crosstalk between PKA, ERK1/2, and CREB in many cell types and the dose-dependent effects of H2O2, the goal of this study was to determine their relative importance in oxidant-induced signaling. The effects of transient bolus H2O2 as well as a peroxide generating system were tested to represent a range of oxidant concentrations that lead to cell proliferation and/or apoptosis. The data support the hypothesis that PKA and EGFR are central regulators of oxidant–induced ERK1/2 and CREB activation and demonstrate a link between PKA, EGFR, and CREB in cell survival following exposure to H2O2 in alveolar type II cells.
Materials and methods
Cell culture and treatments
C10 cells, a contact-inhibited, non-transformed murine alveolar type II epithelial cell line [15], were grown in CMRL 1066 medium supplemented with L-glutamine, penicillin/streptomycin, and 10% fetal bovine serum (FBS) (GIBCO BRL, Rockville, MD). Cells were grown to 90% confluence, and then complete medium was replaced with CMRL 1066 medium supplemented with l-glutamine, penicillin/streptomycin, and 0.5% FBS for 48 h before exposure to agents.
H2O2 (Sigma, St. Louis, MO) was added to the medium at concentrations from 100 μM to 300 μM. Recombinant glucose oxidase (GO; 5 mU/ml, 15 mU/ml, or 30 mU/ml) (Roche, Indianapolis, IN) was used to provide low level fluxes of H2O2 [16, 17]. Forskolin and epidermal growth factor (EGF) (Sigma, St. Louis, MO) (10 μM and 100 ng/ml, respectively) were used as positive controls for induction of phospho-CREB and phospho-EGFR, respectively. Tumor necrosis factor α (TNFα) (Calbiochem, LaJolla, CA) was used at 0.1 μg/ml to induce apoptosis. Control cultures received medium without agents and were treated identically.
The ERK1/2 inhibitor, U0126 (10 μM for 30 min prior to treatment), the EGFR tyrosine kinase inhibitor, Tyrphostin AG1478 (10 μM for 1 h prior to treatment), and the antioxidant enzyme, catalase (1000 U/ml 1 h prior to treatment) were obtained from Calbiochem. The PKA inhibitor, H89 (10 μM for 1 h pre-treatment) was obtained from Biomol (Plymouth Meeting, PA). N-acetyl-l-cysteine (NAC; 10 mM 16 h prior to treatment) and DL-Buthionine-(S,R)-sulfoximine (BSO; 10 μM 16 h prior to treatment) were purchased from Sigma (St. Louis, MO).
Western blot analysis
After C10 cells were exposed to agents as described above, the cells were washed twice with cold PBS and collected in 4X sample buffer (200 μM Tris, pH 6.8, 4% SDS, 4 mg/ml bromophenol blue, 0.04% β-mercaptoethanol, 40% glycerol, 2 μM pyronin-Y). The amount of protein in each sample was determined using the RC/DC protein assay (Bio-Rad). About 30 μg of protein was separated by a 10% SDS-PAGE and transferred to nitrocellulose. Western blots were performed as described previously [6] using antibodies specific to total and phosphorylated CREB (1:1000; rabbit polyclonal anti-CREB, Cell Signaling Technologies, Danvers, MA; 1:500; rabbit polyclonal anti-phospho-CREB, Cell Signaling Technologies), total and phosphorylated ERK1/2 (1:1000; rabbit polyclonal anti-ERK1/2, Cell Signaling Technologies; 1:500; rabbit polyclonal anti-phospho-ERK1/2, Cell Signaling Technologies). Antibody binding was detected using horse radish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (1:5000; Jackson ImmunoResearch Laboratories, Inc., West grove, PA), followed by chemiluminescence (Kirkgaard and Perry Laboratories, Gaithersburg, MD). QuantityOne (Bio-Rad, Hercules, CA) was used to quantify band density, and intensity of phospho-CREB bands were normalized to the intensity of the corresponding total CREB bands.
Live cell imaging of cAMP and redox potential
C10 cells grown on glass coverslips in 6-well tissue culture dishes were transiently transfected with 8 μg of a plasmid encoding a unimolecular fluoresecent resonance energy transfer (FRET)-based cAMP biosensor, Epac1-camps (provided by Dr. Martin Lohse, University of Würtzburg) [18, 19], or 1 μg of a plasmid encoding a redox-sensitive green fluorescent protein (GFP), roGFP2 (a gift from Dr. James Remington, University of Oregon) [20], using Lipofectamine 2000 (Invitrogen, Carlsbad, CA), following the manufacturer’s instructions. The transfection efficiency was approximately 50%.
Imaging experiments were conducted after 48–72 h of transfection, and the growth medium removed and replaced with Krebs-Ringer bicarbonate solution (KRB; 119 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1 mM MgCl2, 1 mM KH2PO4, 25 mM NaHCO3 or 10 mM HEPES-NaOH (pH 7.40), and 2 mM glucose). Coverslips were placed into a heated microperfusion chamber mounted on the specimen stage of an inverted fluorescence microscope (Nikon TE-2000U) equipped with a CARV spinning disk confocal system (Atto Bioscience Inc., Rockville, MD). Cells were continuously superfused with Krebs-Ringer bicarbonate solution (2–5 ml/min) at 37°C. Dithiothreitol (DTT) (Calbiochem, LaJolla, CA) was used to calibrate the redox minimum.
For imaging Epac1-camps FRET and roGFP2, cells were visualized with a Nikon Super Fluor 40× objective. MetaFluor/MetaMorph software was used for image acquisition and analysis (Universal Imaging). The Epac1-camps EYFP excitation wavelength was 440 nm. Dual emission ratio imaging at 485 nm and 535 nm for Epac1-camps and dual excitation ratio imaging at 400 nm and 490 nm for roGFP2 were accomplished using a computer-controlled high speed filter wheel (Lambda 10-2 optical filter changer with rotation every 60–80 ms, Sutter Instrument Co., Novato, CA). For roGFP2, a 505DRLP dichroic mirror and an emission filter, 535DF25, were used. Images (50–250 ms exposure) were captured every 10 s with a 16-bit Cascade 650 digital camera (Roper Instruments, Trenton, NJ) and background-corrected by manual selection of background regions [19].
The ratio values were normalized to the average baseline values measured 1-min prior to application H2O2. Epac1-camps FRET decreases with increasing cAMP concentration, thus increased [cAMP]c is correlated with an increase in the relative emission ratio of ECFP/EYFP (485/535) [18]. Data were thus expressed as relative ratio 485/535 for Epac1-camps and fold increase in relative ratio 400/490 for roGFP2.
Immunofluorescence in C10 cells
C10 cells were grown on glass coverslips for all experiments. After experimental exposures, immunofluorescence to detect the catalytic subunit of PKAα was performed as previously described [6, 21]. Briefly, cells were washed with phosphate buffered saline (PBS), fixed in 3.7% formaldehyde, permeablized with −20°C methanol and incubated with blocking solution containing 2% bovine serum albumin (BSA) in PBS. Cells were incubated with primary antibody (1:100; rabbit polyclonal PKAα catalytic subunit antibody, Santa Cruz, Santa Cruz, CA) diluted in 2% BSA plus 0.1% Triton X-100 in PBS (BSA/PBS-T) overnight at 4°C. Secondary antibody (1:400; AlexaFlour 568 goat-anti-rabbit IgG, Molecular Probes) diluted in BSA/PBS-T was applied for 1 h at room temperature (RT), and followed by incubation with nuclear counterstain, YOYO-1 iodide (Molecular Probes, Carlsbad, CA; 1:10,000), 1 unit/ml RNase, and 0.1% sodium azide in BSA/PBS-T for 30 min at RT. Coverslips were mounted onto slides with AquaPolyMount (Polysciences, Inc. Warrington, PA). For each sample, confocal images were collected in fluorescence modes using a Bio-Rad MRC1024ES confocal scanning laser microscope (Bio-Rad, Hercules, CA). For quantification, a nuclear mask was generated in Corel Photopaint using the YOYO-1 image template, and pixel intensities for PKAα were determined within the nuclear mask area as previously described [22].
Transient transfections with small interfering RNA (siRNA)
The siCONTROL non-targeting siRNA #2 and SMARTpool mouse CREB siRNA (100 nM; Dharmacon, Lafayette, CO) were transfected into C10 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA), following the manufacturer’s instructions. After 4 h in transfection media, FBS was added to 10% and cells were incubated for 24 h. Cells were then transferred to reduced serum media (0.5% FBS) for 24 h prior to treatments.
Real time quantitative PCR (RT qPCR)
Total RNA was extracted from C10 cells using the RNeasy™ PLUS protocol for total RNA isolation from animal cells (Qiagen,Valencia, CA). cDNA was reverse transcribed from 500 ng total RNA using an Omniscript™ Reverse Transcriptase RNase free DNase kit (Qiagen), with an oligo dT primer, according to the manufacturer’s protocol. RT qPCR primers and probes for B-cell lymphoma-2 (bcl-2) and c-fos were obtained as Assays-on-Demand™ kits from Applied Biosystems (Foster City, CA). PCR products were detected by TaqMan qPCR, as previously described [23]. Expression levels of target genes were determined using hypoxanthine-guanine phosphoribosyl transferase (hprt) as the internal standard. Samples were run in duplicate from 3 independent experiments and the comparative Ct (cycle threshold) method for relative quantity (RQ value) was used to calculate relative mRNA expression among samples.
Detection and quantification of apoptosis
Apoptosis was detected by measuring single-stranded DNA using Apostain™ as previously described [24]. Briefly, cell monolayers grown on glass coverslips were treated with 30 mU/ml GO for 24 h, then fixed in methanol for 24 h at −20°C, boiled for 5 min in PBS containing 5 mM MgCl2, and then immersed in ice-cold PBS for 10 min. Cells were blocked with 40% FBS and then incubated with Apostain F7-26 (Alexis Biochemicals, San Diego, CA, 10 μg/ml) followed by HRP-conjugated secondary antibody (goat anti-mouse IgM; Jackson Labortories, West Grove, PA, 1:400). To visualize secondary antibody binding, the peroxidase substrate DAB (Sigma) was used. Coverslips were mounted onto slides with AquaPolyMount (Polysciences, Inc. Warrington, PA) for subsequent examination using bright field light microscopy. To determine the numbers of apoptotic cells and total cell numbers per field, 5 random fields were evaluated per experimental condition at x200 total magnification.
Statistical analysis
Statistical analyses were performed using non-normalized data, and pair-wise comparisons between treatment groups were achieved using Student’s t-test or Mann–Whitney Rank Sum Test method for unequal variances. ANOVA (Holm–Sidak method) was used for multiple comparisons. Differences were considered statistically significant at P < 0.05.
Results
CREB is phosphorylated after exposure to H2O2 and glucose oxidase
We have previously shown that H2O2 causes CREB phosphorylation in alveolar type II cells [6]. Here, we further show by Western blot that the CREB response to bolus addition of H2O2 (Fig. 1a) and H2O2 generated by glucose oxidase (GO) (Fig. 1b) is reduced when pretreated with the antioxidants NAC or catalase at effective concentrations documented previously in C10 cells [25].
Fig. 1Bolus and glucose oxidase generated H2O2 mediate CREB phosphorylation in an oxidant-dependent manner. (a) C10 cells, pre-incubated with 1,000 U/ml catalase (CAT) for 1 h, were treated with 10 μM forskolin or 100, 200, or 300 μM H2O2 for 10 min, and then analyzed by Western blot analysis for phospho-CREB (p-CREB). (b) C10 cells, pre-incubated with 10 mM NAC for 16 h or 1,000 U/ml catalase (CAT) for 1 h, were treated with 10 μM forskolin or 5, 15, or 30 mU/ml glucose oxidase (GO) for 4 h, and then analyzed by Western blot analysis for phospho-CREB (p-CREB). An antibody recognizing total CREB was used as a control for protein loading in A and B. Data are representative of 3 separate experiments
H2O2-induced CREB phosphorylation is decreased after the reduction of PKA activity
Because PKA is an important regulator of CREB [9], we investigated its role in H2O2-induced CREB phosphorylation. Cells were examined after exposure to lower concentrations previously associated with expression of cyclin D (100 μM) and higher concentrations known to induce apoptosis (300 μM) [6, 26]. Exposure to H2O2 led to CREB phosphorylation in a concentration-dependent manner that was significantly decreased after reduction of PKA activity using the specific inhibitor H89 (Fig. 2a, b). The pattern of CREB phosphorylation in response to H2O2 closely paralleled that seen in response to activation of PKA by forskolin. H89 also reduced H2O2-mediated ERK1/2 activation, suggesting that PKA may regulate CREB activation directly or indirectly through ERK1/2-mediated CREB activation.
Fig. 2H2O2-mediated CREB activation is inhibited by the PKA inhibitor, H89. (a) C10 cells, pre-incubated with 10 μM H89 for 1 h, were treated with 10 μM forskolin or indicated concentrations of H2O2 for 10 min, and then analyzed by Western blot analysis for phospho-CREB (p-CREB), total CREB, phospho-ERK1/2 (p-ERK1/2) and total ERK1/2. (b) Quantification of p-CREB band intensities from A corrected using the corresponding CREB band intensity and normalized to the untreated control. Data represent 4 separate experiments; *P < 0.05 when compared with corresponding condition without H89. (c) C10 cells, pre-incubated with 10 μM H89 for 1 h, were treated with 10 μM forskolin or indicated concentrations of glucose oxidase (GO) for 4 h, and then analyzed by Western blot for phospho-CREB (p-CREB), total CREB, phospho-ERK1/2 (p-ERK1/2), and total ERK1/2. (d) Quantification of p-CREB band intensities from C corrected using the corresponding CREB band intensity and normalized to the untreated control. Data represent mean ± SEM from 4 separate experiments; *P < 0.05 when compared with corresponding condition without H89
Similar to results with bolus addition of H2O2, H2O2 generated by GO stimulated CREB phosphorylation in a dose-dependent manner and the phosphorylation was significantly reduced by PKA inhibition at higher concentrations of GO (Fig. 2c, d). However, unlike bolus addition, H2O2 generated by GO led to a dose-dependent increase in ERK1/2 phosphorylation that was marginally sensitive to inhibition of PKA activity, suggesting that the duration of the stimulus may cause differences in signaling patterns.
H2O2 stimulates cAMP production in C10 cells
Single cell imaging was utilized to determine whether H2O2 exerts a direct effect on upstream regulation of PKA through production of cAMP. Epac1-camps, a FRET-based cAMP biosensor, was expressed in C10 cells. Epac1-camps senses changes in cytoplasmic cAMP levels ([cAMP]c) through a cAMP-dependent conformational change resulting in reduced FRET between ECFP (485 emission) and EYFP (535 emission) [18, 19]. Exposure to H2O2 caused a monophasic increase in [cAMP]c (Fig. 3a) that was comparable to the response elicited by direct activation of adenylyl cyclase with forskolin, but with a slower rise time (Fig. 3c). H2O2 generated by GO stimulated a rise in [cAMP]c similar in magnitude to bolus addition of H2O2, but with a longer lag-time presumably due to the slower generation of oxidant (Fig. 3b).
Fig. 3H2O2 stimulates an increase in cAMP levels. C10 cells were transiently transfected with the cAMP FRET indicator Epac1-camps and then exposed to 10 μM 250 μM H2O2 (a), 15 mU/ml glucose oxidase (GO) (b), or 10 μM forskolin (FSK) (c) for the times indicated. Data (means ± SEM) are expressed as the relative ratio (485/535) of the cAMP response from the live emission recording of a representative cell. Grey bars indicate time of addition and length of exposure. Results are representative of ≥9, 5, and 4 cells (A, B and C, respectively)
Single cell imaging was also used to measure redox status and correlate cAMP levels with redox status. The redox sensitive GFP variant, roGFP2, was expressed in C10 cells. Oxidation is detected by roGFP through a change in GFP excitation from 400 nm and 490 nm [20]. The time course of H2O2-mediated cAMP production correlated well with the level of intracellular oxidation measured by roGFP2 (Fig. 4a). Upon removal of H2O2, the redox state returned to baseline approximately 36 min after the initial exposure (Fig. 4b).
Fig. 4Redox status correlates with cAMP level in cells responding to H2O2. C10 cells were transiently transfected with the redox indicator, roGFP2 and then exposed to 250 μM H2O2 for 10 min followed by 1 mM DTT (a) or 15 mU/ml glucose oxidase (GO) for 65 min, followed by 200 μM H2O2, and then 10 mM DTT (b). Data (means ± SEM) are expressed as the fold change in the excitation ratio at 400/490 nm normalized to the average base-line ratio values measured 1 min before stimulation. Results are averages of ≥23 and 11 cells (A and B, respectively). Grey bars indicate time of addition and length of exposure, and WO indicates wash out with KREBs buffer
EGFR tyrosine kinase activity is important for signaling from H2O2 to PKA and CREB
It has been shown that the protein tyrosine activity of EGFR is important for EGF-mediated stimulation of adenylyl cyclase [27]. Thus to determine the contributions of EGFR in the observed H2O2-induced CREB phosphorylation, cells were evaluated with or without pretreatment with the specific EGFR tyrosine kinase inhibitor, AG1478. Application of AG1478 significantly reduced CREB phosphorylation in response to bolus addition H2O2 and to H2O2 generated by higher concentrations of GO (Fig. 5), suggesting that EGFR activity is involved in H2O2-induced CREB phosphorylation. As expected, reduction of EGFR activity also inhibited H2O2-induced ERK phosphorylation (Fig. 5).
Fig. 5Reduction of EGFR tyrosine kinase activity leads to the inhibition of both H2O2-induced CREB and ERK1/2. C10 cells, pre-incubated with the EGFR tyrosine kinase inhibitor AG1478 (10 μM) for 1 h, were treated with 10 μM forskolin or 100 ng/ml EGF for 5 min, the indicated concentrations of H2O2 for 10 min (a) or glucose oxidase (GO) for 4 h (c), and then analyzed by Western blot analysis for phospho-CREB (p-CREB), total CREB, phospho-ERK1/2 (p-ERK1/2), and total ERK1/2. (b, d) Quantification of p-CREB band intensities from A and C respectively corrected using the corresponding CREB band intensity and normalized to the untreated control. Data represent mean ± SEM of 3 separate experiments; *P < 0.05 when compared with corresponding condition without AG1478
Immunofluorescence evaluation of activated PKA translocation to the nucleus was used to more directly test the effects of H2O2 on PKA activation and to further evaluate a role for EGFR. Exposure to H2O2 led to an increase in the PKA free catalytic subunit that was significant in the nucleus at 300 μM and similar to the response to forskolin. Inhibition of EGFR tyrosine kinase activity with AG1478 prevented the H2O2-induced increases at both 200 μM and 300 μM (Fig. 6).
Fig. 6Reduction of EGFR tyrosine kinase activity inhibits the H2O2-induced nuclear translocation of activated PKA. C10 cells, pre-incubated with 10 μM AG1478 for 1 h, were exposed to 10 μM forskolin and H2O2 for the indicated concentrations for 10 min. (a) The PKA free catalytic subunit (red) was detected by immunofluorescence and nuclei were stained with YOYO-1 (green). (b) Quantification of PKA immunofluorescence pixel intensity within nuclei; *P < 0.05 compared to control, #P < 0.05 compared to treatment, n = 3 experiments, 5 fields/experiment
Direct activation of CREB by PKA is a minor pathway in EGF-induced CREB phosphorylation
Because of the known crosstalk between EGFR, ERK1/2, and PKA, we examined the role of PKA and ERK1/2 in forskolin- and EGF-induced CREB activation. Treatment with both forskolin and EGF resulted in a significant increase in CREB phosphorylation when compared to control cells (Fig. 7). Inhibition of PKA activity with H89 significantly decreased forskolin-induced CREB phosphorylation, but only blunted EGF-induced CREB phosphorylation. Inhibition of ERK1/2 phosphorylation with the MEK inhibitor, U0126, also considerably decreased CREB phosphorylation. The inhibitory effects of H89 and U0126 were not additive towards either forskolin- or EGF-induced CREB phosphorylation, suggesting that direct activation of CREB through PKA may play a minor role in the activation of CREB. Furthermore, both forskolin and EGF induced ERK1/2 phosphorylation, but only forskolin-mediated CREB phosphorylation was inhibited by H89, suggesting that PKA regulates CREB indirectly through the MAPK/ERK1/2 pathway.
Fig. 7Crosstalk between PKA and ERK1/2 signaling is important in regulating CREB phosphorylation. (a) C10 cells, pre-incubated with 10 μM H89, U0126, or H89 and U0126 for 1 h, were treated with 10 μM forskolin for 10 min or 100 ng/ml EGF for 5 min, and then analyzed by Western blot analysis for phospho-CREB (p-CREB), total CREB, phospho-ERK1/2 (p-ERK1/2), and total ERK1/2. (b) Quantification of phospho-CREB in A corrected using CREB intensities and phospho-ERK/12 in A corrected using ERK1/2 intensities; *P < 0.05 for DMSO treatment compared to AG1478 treatment; #P < 0.05 for DMSO control compared to treatment. Data represent mean ± SEM of 3 separate experiments
Knockdown of CREB using siRNA results in loss of oxidant-induced c-fos and bcl-2 transcription
To more directly assess the role of CREB in oxidant-mediated responses, an siRNA approach was used to reduce levels of CREB within C10 cells. Transfection with siCREB resulted in greater than 80% knockdown of both CREB mRNA and protein levels (Fig. 8a, b). CRE-containing genes previously shown to be upregulated by H2O2 including bcl-2 and c-fos were found to be dose-dependently induced by GO. Induction was significantly reduced in cells transfected with siCREB (Fig. 8c, d), suggesting that CREB is essential for the transcriptional response of these genes to H2O2.
Fig. 8Knockdown of CREB using siRNA results in loss of oxidant-induced c-fos and bcl-2 transcription. (a) C10 cells were transfected with 100 nM scrambled siRNA (siControl) or siCREB followed by Western blot analysis to detect CREB. β-actin was used as a loading control. (b) Cells were transfected as in A, followed by treatment with 5 or 15 mU/ml glucose oxidase (GO) for 6 h. Total RNA was extracted and cDNA was assessed by RT qPCR to quantify CREB, bcl-2 and c-fos mRNA levels. Shown are relative quantity (RQ) values normalized to the siControl sample. #P < 0.05 when compared to untreated siCon; *P < 0.05 when compared to treatment siCon
Knockdown of CREB significantly increases the number of cells undergoing apoptosis following exposure to glucose oxidase
Recently, we have shown that reduction of CREB activity, by introducing a phosphorylation-null CREB construct, led to a decrease in apoptosis [6]. To resolve whether CREB is essential for H2O2-induced apoptosis, C10 cells transfected with scrambled siRNA (siControl) or siCREB were treated with 30 mU/ml GO for 8 h and apoptotic cells were identified by Apostain (Fig. 9). H2O2 generated from GO induced a significant increase in the percentage of cells undergoing apoptosis compared to the control in both siControl and siCREB cells. Cells expressing siCREB had significantly higher levels of apoptosis than the siControl cells in both the untreated condition and after GO exposure, supporting a role for CREB in the regulation of apoptosis induced by H2O2. Interestingly, the apoptotic enhancing effect was not selective for H202-mediated apoptosis, as siCREB also enhanced the apoptotic effect of TNFα. Taken together, these results suggest that CREB plays an important role in the cellular response to oxidant stress including upregulation of survival genes and promoting cell survival.
Fig. 9CREB is protective against oxidant-induced apoptosis. (a) C10 cells, transfected with siControl or siCREB, were exposed to 30 mU/ml glucose oxidase (GO) or 0.1 μg/ml tumor necrosis factor α (TNFα) for 8 h followed by identification of apoptotic cells using Apostain. (b) Quantification of % Apostain positive cells was determined by scoring the average of 5 fields/condition; *P < 0.05 when compared to untreated siCon; #P < 0.05 when compared to treatment siCon. Data represent mean ± SEM of 4 separate experiments. Bar = 100 μm
Discussion
Exposure to various environmental stresses, such as oxidative gases, metals, and particulates, initiates multiple physiological processes in alveolar type II cells. Oxidants have been implicated in the pathogenesis of lung cancer, pulmonary fibrosis, and asthma through their capacity to regulate an intricate network of protein kinases. Yet the relationship between oxidants and the pathogenesis of lung disease is unclear, primarily because of the lack of understanding of the mechanisms by which oxidants function in both normal physiological and disease states. In this study, we show for the first time that H2O2 exposure leads to an increase in cytoplasmic cAMP levels, followed by PKA-dependent CREB and ERK1/2 phosphorylation in alveolar type II cells. Furthermore, we show that EGFR activity is necessary for H2O2-stimulated nuclear accumulation of activated PKA and for PKA-mediated CREB phosphorylation. Together these data suggest that PKA is a central hub for interplay between EGFR, ERK1/2, and CREB signaling pathways activated by oxidant stress.
Several studies have shown crosstalk between PKA and ERK1/2 signaling pathways. This crosstalk appears to be cell type specific, since an increase in cAMP-dependent PKA activation stimulates ERK1/2 in some cells, but suppresses it in others [28–30]. We have previously demonstrated that H2O2 exposure leads to ERK1/2-dependent CREB phosphorylation in C10 alveolar type II cells [6]. Studies here have dissected the underlying mechanisms to reveal that H2O2-induces cAMP formation and PKA activation that are important for both ERK1/2 and CREB phosphorylation.
EGFR is overexpressed and activated in response to epithelial injury [31]. Its activation is believed to play an integral role in the cellular responses of lung epithelium to injury and oxidant stress, possibly by signal amplification through MAPK phosphorylation cascades. Oxidative modification of a reduced cysteine residue in the EGFR reversibly affects its activation [32, 33]. Moreover, Goldkorn and colleagues have shown that in alveolar type II cells, H2O2 induces EGFR phosphorylation on tyrosine residues [2]. These findings suggest that even though there is no specific receptor for oxidants, their signaling may be efficiently transduced through interactions with the EGFR.
In the present study, we show that EGFR activity is necessary for both activation of PKA nuclear translocation and subsequent CREB phosphorylation in response to H2O2. Mitogenic signals transmitted through the EGFR have been shown to involve PKA-dependent signaling cascades. Tyrosine phosphorylation of the EGFR requires an increase in cAMP levels and PKA function in many, but not all cell types [34, 35]. In addition, tyrosine kinase activity of EGFR is required for growth factor stimulation of adenylyl cyclase activity, which leads to the activation of PKA [27, 36]. PKA activity has also been shown to promote downstream Raf-1 function and subsequent ERK activation [37]. Crosstalk has also been seen from EGFR to adenylyl cyclase activity and PKA in airway epithelial cells via beta adrenergic receptors [38, 39]. Thus, communication between EGFR and PKA is potentially bi-directional.
The H2O2-mediated generation of cAMP and the CREB activation response to H2O2 increased with oxidant concentration but, with minor exception, were not dependent on whether oxidant was added by bolus or generated by glucose/glucose oxidase. Although reduction of the CREB response by PKA or EGFR inhibition was only significant for the higher oxidant concentrations, a trend was observed with lower concentrations. While it is difficult to accurately predict the physiological significance of these oxidant levels, when put in context with previous findings, these observations suggest that CREB signaling through PKA and EGFR could participate in signaling that affects both oxidant-mediated proliferation and apoptosis.
Previously we demonstrated that expression of phosphorylation-incompetent CREB paradoxically increases bcl-2 transcription and reduces apoptosis following bolus H2O2 exposure [6]. Findings here show that siRNA-mediated knockdown of CREB results in a loss of bcl-2 mRNA and an increase in apoptosis in cells responding to an H2O2 generating system. The difference is likely due to the method of CREB inhibition, in that the dominant-negative CREB may have altered chromatin interactions with other transcription factors or caused quenching of the response. Putting these results in context with the findings of others, we propose that CREB activation through PKA signaling serves as a key survival pathway in cells responding to oxidant stress. PKA-and CREB-mediated gene expression may thus be important in regulating oxidant-mediated apoptosis resulting from lung pathologies, and future experiments will explore the importance of these signaling pathways in promoting changes in gene expression following lung epithelial cell injury or oxidant exposure.

oxidative stress
transcription factor
pulmonary epithelium
lung fibrosis
fret biosensor


The Importance of Preconception Care for Women With Disabilities

Overall estimates of the number of women with disabilities in the United States range from 16.8 to 28.6 million, or approximately one in every five women [1–3]. A large percentage of these women are in their reproductive years, and they often encounter greater obstacles to receiving health care than women without disabilities [4]. Some disabling conditions, such as spinal cord injury (SCI) and multiple sclerosis (MS), have low prevalence rates but are more common in the reproductive age group. Such conditions are often associated with a greater severity of disability and increased need for specialized services, including prenatal care [5]. Health care professionals need to be aware of the reproductive health care issues facing women with disabilities and take every opportunity to address them.
Sexuality and reproductive health issues have received inadequate attention for women with disabilities. Many women with disabilities desire children and are capable of conceiving, but face considerable pressure not to reproduce [6, 7]. Although a growing number of women with disabilities are becoming pregnant, anecdotal evidence suggests that many of these women encounter negative attitudes towards their pregnancies and report difficulty receiving comprehensive prenatal care [6].
Women with disabilities who become pregnant face similar issues and concerns as pregnant women without disabilities. Although many pregnant women experience problems with weight gain, fatigue, fluid retention, bladder dysfunction, and urinary tract infections, these problems may be more serious in women with disabilities [8]. All women are concerned about the health of their unborn child. Like other women, women with disabilities express concern about the possibility of giving birth to a child with disabilities and may seek genetic counseling. On the other hand, not all disabled women embrace the idea of genetic testing. To these women the idea of genetic counseling raises psychosocial issues that may be difficult for them to consider [6].
Some disabling conditions pose unique problems during or after pregnancy. Complications associated with specific conditions, such as systemic lupus erythematosus (SLE), SCI, or MS, can affect pregnancy and should be evaluated prior to conception [8–12]. For example, an increased risk of fetal loss, growth retardation, premature placental aging with thrombosis, and preeclampsia are described in women with lupus [9]. Given the seriousness of these complications, ongoing monitoring and interventions to reduce them are critical. Pregnant women with SCI above the level of T6 also require special attention [7, 8, 10, 11]. Such women have an increased risk of autonomic dysreflexia during all stages of pregnancy and, if it is not recognized and treated, autonomic dysreflexia can be fatal [8, 11]. Although women with stable MS can safely carry a pregnancy to term, postpartum exacerbations of MS are quite common and occur in up to 30% of women within one month of delivery [11, 12]. Pregnancy management in women with disabilities should therefore include preconception counseling and evaluation to identify these and other potential problems that can occur throughout the course of the pregnancy.
Health care professionals need to know that physical barriers, such as the lack of accessible scales or examination tables, present enormous and recurring obstacles to obtaining adequate prenatal care. Also, communication barriers, particularly for those who are deaf or hard of hearing, affect a women's ability to receive appropriate medical care. In addition, there are some aspects of gynecologic health care such as informed consent, sedation, and contraceptive issues that are unique for women with developmental disabilities. A recent publication from the American College of Obstetricians and Gynecologists highlights ways to improve care for this group of women [7].
Preconception care is associated with improved pregnancy outcomes. It is recommended that primary care providers assess all women of reproductive age for their preconception risk conditions and provide or refer individuals for interventions as appropriate [13]. When caring for a woman with a disability, a team approach involving the primary physician, obstetrician, anesthesiologist, neurologist, physiatrist, and other allied health professionals such as occupational and physical therapists, is recommended [6–8, 11]. This is particularly important, as many obstetricians are unfamiliar with disability-related complications. A multidisciplinary approach, which involves nursing and social work, can further improve pregnancy outcomes. This shared approach to pregnancy management can also increase the level of comfort for women with disabilities, by knowing that more than one health care provider is looking out for their well-being.
Preconception counseling for women with disabilities should address the medical, psychological, and social impact of a pregnancy [10]. During preconception counseling, it is particularly important to evaluate the prenatal and postpartum social support systems available to women with disabilities. Body changes such as decreased mobility or bladder dysfunction increase the need for personal assistance with routine activities of daily living. Many women with disabilities also benefit from an occupational therapy evaluation to assess environmental barriers and explore adaptive equipment and techniques that facilitate breast feeding and infant care. Health care providers might want to recommend that women with disabilities explore creative parenting strategies such as those offered by Through the Looking Glass, a national resource center on parenting with a disability, located in Berkeley, California [6, 14].
An increasing number of women with disabilities are becoming pregnant. Despite this little is known about the reproductive experiences of these women. Population-based data describing the reproductive experiences of women with disabilities is virtually non-existent. Currently, disability is not regularly included in national or statewide surveys and surveillance tools related to pregnancy. Consequently, the true magnitude of health concerns, including reproductive issues, facing women with disabilities is largely unknown. To improve our understanding of these issues, disability should be included as a demographic variable in all national and state-level surveys addressing reproductive health issues.
All women of childbearing age, including women with disabilities, should have access to preconception care that addresses the medical, psychological and social impacts of pregnancy. Women with disabilities can and do have healthy pregnancies. Accurate knowledge of disabling conditions and a shared multidisciplinary approach to pregnancy management are critical components for good pregnancy outcomes among women with disabilities.


Effectiveness of bisphosphonates on nonvertebral and hip fractures in the first year of therapy: The risedronate and alendronate (REAL) cohort study

Introduction Randomized clinical trials have shown that risedronate and alendronate reduce fractures among women with osteoporosis. The aim of this observational study was to observe, in clinical practice, the incidence of hip and nonvertebral fractures among women in the year following initiation of once-a-week dosing of either risedronate or alendronate.
Introduction
Osteoporosis, a common skeletal disease in older populations, leads to more than a million fractures annually in the United States [1]. Nonvertebral fractures represent 75% of osteoporotic fractures seen in clinical practice [2]. The incidence of nonvertebral fractures, especially at the hip, increases rapidly with age [3]. In order to prevent these fractures, US clinical guidelines recommend that candidates for osteoporosis therapy be identified by screening the bone mineral density of all woman ages 65 and over (age 60 for high risk populations) [4].
Oral bisphosphonates are currently the most common therapy for osteoporosis [5]. While the three most utilized bisphosphonates (alendronate, risedronate, ibandronate) approved by the Food and Drug Administration have been shown to reduce vertebral fractures in randomized clinical trials, it is unknown if these three are equally effective in reducing nonvertebral fractures of real-world patients in clinical practice. A comparison of the results from the randomized clinical trials of each bisphosphonate, though limited by methodological differences between trials, suggests potential differences in degree of fracture reduction across bisphosphonates. In the primary analyses of the trials that followed patients for at least 3 years, risedronate significantly reduced the incidence of nonvertebral fractures by up to 39% [6, 7]; alendronate reduced the incidence of nonvertebral fractures by up to 21% [8–10]; ibandronate did not reduce nonvertebral fractures [11]. Post-hoc analyses of these trial data suggest that there are differences in the onset of fracture reduction. In those analyses, reduction of nonvertebral fractures began at 6 months for 5 mg daily dosing of risedronate [12] and at either 12 months (when fractures were recorded as adverse events) for 10 mg daily dosing of alendronate [13] or 24 months for 5 mg daily dosing of alendronate [14]. These possible differences in both the amount and the onset of fracture reduction between the bisphosphonates could arise from their differences in structure, potency, and binding properties [15].
The only direct comparison of bisphosphonates in a randomized clinical trial is based on surrogate endpoints (e.g., changes in bone mineral density and markers of bone turnover) [16]. However, the association between changes in these surrogates and subsequent fracture reduction is not consistent across studies [17, 18]. Unlike randomized clinical trials based on surrogate endpoints, observational studies of large populations provide the opportunity to use major disease endpoints (e.g., hip fracture) as the outcome of interest. The limitation of observational studies can be misleading results from bias arising from non-randomized treatment groups. This bias can be accounted for in part by statistically adjusting for known risk differences between groups. Furthermore, when different therapies are available to be prescribed for the same indication, there is at least some expectation of similarity in prognostic factors between treatment groups occurring naturally [19]. For example, observational studies have compared both the many antihypertensive drug therapies and many statins for reducing the disease endpoint of myocardial infarctions [20, 21].
Since the once-a-week dosing regimens of both risedronate and alendronate have been available in the US since 2002, there is now an opportunity to observe their effect on reducing fractures in a large population of patients seen in clinical practice. Hence, we conducted an observational study across multiple US health plans to observe the incidence of hip and nonvertebral fractures among women ages 65 and over following initiation of therapy with once-a-week dosing of either risedronate or alendronate.
Methods
The RisedronatE and ALendronate (REAL) cohort study was a retrospective observation of bisphosphonate patients within healthcare utilization records in the United States. The analysis plan was based upon an earlier report [22]. All authors had access to the data. For assurance of reproducibility [23], the analyses were independently replicated by the respective organizations of the authors. The reporting of this study is consistent with the STROBE guidelines [24].
Data source
The data source was commercially available datasets of healthcare utilization from the 1 health plan within Ingenix Lab/Rx (Eden Prairie, MN; data through June 2004) and the 100 health plans of employers within MedStat Marketscan (Ann Arbor, MI; data through September 2004). These datasets contain a longitudinal history of patient-specific data including demographic information (sex, age, dates of dataset inclusion), clinical encounters (inpatient and outpatient services by associated procedures and diagnoses specified by the International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM)), and outpatient pharmaceutical dispensations (retail and mail order specified by the national drug code (NDC)). To maximize sample size, the two datasets were combined for all analyses. At the time of data extraction for the current study, the combined datasets contained 12 million persons across 34 states in the US.
Study population
Within the data source, candidates for study inclusion were all women aged 65 and older with any use of once-a-week dosing of risedronate (35 mg; Actonel, P&G Pharmaceuticals) or once-a-week dosing of alendronate (35 or 70 mg; Fosamax, MSD) after July 2002, a date corresponding with contemporaneous commercial availability of once-a-week versions of both therapies (Fig. 1). Patient exclusion criteria and rationale were: 1) less than 6 months (182 days) of health plan enrollment before their first bisphosphonate use after July 2002, a history period to measure baseline fracture risk; 2) less than 3 months (92 days) of health plan enrollment after their first bisphosphonate use, a minimum observation period with expectation of any fracture reduction (based on post-hoc analyses of clinical trials [12, 14]); 3) any bisphosphonate use (daily, weekly, or Paget's doses) during their 6 month history period, to include only patients who are new users of bisphosphonates; 4) diagnosis of malignant neoplasm, ICD-9-CM 140 - 208; or Paget's disease, ICD-9-CM 731.0 during either the 6 month history period or first 3 months of observation, to exclude patients with fracture risk not related to osteoporosis; and 5) discontinued therapy within the first 3 months, a minimum period of therapy adherence.
Fig. 1Identification of the study population
Length of observation
Observation for a subject was censored at one of following end points, whichever occurred first: date of fracture, 12 months after date of first bisphosphonate prescription, end date of health plan enrollment, date of switch between bisphosphonate therapies or end date of therapy adherence. Adherence was measured as a function of the gaps between refills, which provides the best available measure within datasets of medical claims [25]. Once the gap between the completion of a 30-day supply and the start of a new prescription exceeded 15 days, the end date of therapy adherence was the prescription date before gap plus 45 days. For a 90-day supply, a gap of 45 days was allowed between completion and a new prescription, the end date of therapy adherence was the prescription date before gap plus 135 days. For the last prescription, the end date of therapy adherence was the date of last prescription plus 45 days for a 30-day supply and date of last prescription plus 135 days for a 90-day supply.
Fracture outcomes
From the data source, two outcomes were identified: subjects with nonvertebral fractures collectively (hip, wrist, humerus, clavicle, pelvis, leg - sites previously specified [12]) and subjects with a hip fracture. Vertebral fractures were not included because the majority of them do not come to clinical attention and thus not systematically captured in the data source. In an attempt to identify incident fractures more likely related to osteoporosis, we used a series of exclusion criteria (Table 1). A total of 109 hip and 507 nonvertebral fracture patients were available for analyses.
Table 1Identification of fractures outcomes in the study population (n = 33,830) Hip fractureaNonvertebral fracturebSubjects with a medical claim for fracture during the observation period after initial bisphosphonate.135923Exclusion of medical claim if a fracture at the same site both before and after start of bisphosphonate therapy; in order to increase the likelihood of including only new fractures.−16−368Exclusion of medical claim if a fracture at an unspecified sitec before the start of bisphosphonate therapy; in order to increase the likelihood of including only new fractures.−2−6Exclusion of medical claim if an open fractured; in order to decrease the likelihood of including traumatic fractures. −3−11Exclusion of medical claim if documented cause (E-codes) of injury is other than an accidental falle; in order to decrease the likelihood of including traumatic fractures.−5−31Subjects with a fracture outcome109507aInpatient ICD-9-CM codes (820.x, 733.14)bIn addition to inpatient hip fractures, inpatient and outpatient ICD-9-CM codes for fracture of the wrist (813.x, 733.12), humerus (812.x, 733.11), clavicle (810.x), pelvis (808.x), and leg (821.x, 823.x, 733.15, 733.16)cICD-9-CM code (733.10 or 733.19)dICD-9-CM code that is not 733.1x or where the 4th digit is not = .1, .3, .5, .9eICD-9-CM code with “E” classification (E880 - E888) for accidental falls
Statistical analyses
To assess the comparability of baseline characteristics (see Table 2 for specific definitions of demographic characteristics and health history) between the risedronate and alendronate cohorts, the chi-square test was used for dichotomous variables and the Wilcoxon rank sum test was used for continuous variables.
Table 2Comparison of baseline characteristics between cohorts in study CohortsCharacteristicRisedronateAlendronatep-valueNumber of women subjects12,21521,615Duration of observation period Days (mean)226238< 0.001Age at study entryYears & months (mean)74 & 1074 & 7< 0.001 Ages 65 – 74 (%)53.552.4 Ages 75 – 84 (%)36.836.7 Ages 85 and over (%)9.711.0Medications – 6 month historya Concomitant medications (mean)b4.03.6< 0.001 Gastrointestinal medication use (%)c26.220.1< 0.001 Estrogen use (%)d17.216.50.08 Other non-estrogen anti-osteoporotic use (%)e15.611.0< 0.001 Glucocorticosteroid use (%)f10.38.5< 0.001Medical encounters – 6 month historya Office visits (mean)5.65.1< 0.001 Hospitalization (%)8.28.20.87 Osteoporosis diagnosis (%)g37.733.8< 0.001 Osteopenia diagnosis (%)h12.510.5< 0.001 Bone densitometry procedure (%)i47.441.5< 0.001 Gastrointestinal diagnosis (%)j15.412.3< 0.001 Rheumatoid arthritis diagnosis (%)k2.72.30.01aSix months before and including date of starting first bisphosphonate prescriptionbBased on number of therapeutic classes with a prescription [34]cBased on NDC codes for at least one prescription for either: H2 antagonists (ranitidine, cimetidine, famotidine, nizatidine); Proton pump inhibitors (omeprazole, esomeprazole, lansoprazole, pantoprazole); Cytoprotectives (misoprostol, sucralfate) [35]dBased on NDC codes for at least one prescription of estradiol, conjugated estrogen, esterified estrogen, or estropipateeBased on NDC codes for at least one prescription of calcitonin or raloxifenef Based on NDC codes for at least one prescription for triamcinolone, prednisone, prednisolone, methylprednisolone, dexamethasone, budesonide, betamethasone, cortisone, or hydrocortisonegICD-9 733.0xhICD-9 733.90 and no record of 733.0xiCPT 76070, 76075, 76076, 78350, 78351, ICD-9 88.98jMultiple ICD-9 codes [36]kICD-9 714.0
For the primary analysis, the main outcome measures were the 6 and 12 month incidence of nonvertebral fractures and hip fractures. Cox proportional hazard modeling (PROC PHREG, SAS Institute, Cary, NC) was used to compare the incidence of fractures between risedronate and alendronate cohorts, adjusting for potential differences in measurable risk factors for fractures. A parsimonious model for each outcome was developed to enhance precision of the parameter estimates and interpretation of results. The selection of variables to be included in the model was based on forward selection. These models were checked against models based on backward selection. The appropriateness of the proportional hazard assumption was assessed by graphical and numerical methods (ASSESS statement, SAS Institute).
In order to evaluate if the results of the primary analysis were dependent on methodology, sensitivity analysis were used to compare the incidence of fractures between risedronate and alendronate cohorts. These methods included: (1) an intent-to-treat analysis that observed all subjects for 12 months regardless of therapy adherence; (2) a proportional hazard model using the propensity score to adjust for differences in baseline fracture risk between cohorts; (3) use of different inclusion criteria for the study population; (4) use of different inclusion criteria for the study outcomes (see Fig. 5 for specifics).
Results
The risedronate cohort included 12,215 subjects on once-a-week dosing of 35 mg followed for a mean of 226 days on therapy. 37% of this cohort was censored before 12 months because of the end date of available data and 41% was censored for an end in therapy adherence. The alendronate cohort included 21,615 subjects on once-a-week dosing of 35 mg (8%) or 70 mg (92%) followed for a mean of 238 days on therapy. 33% of this cohort was censored before 12 months because of the end date of available data and 41% was censored for an end in therapy adherence. Upon start of bisphosphonate therapy, the two cohorts were different in several baseline characteristics (Table 2). Statistically, the risedronate cohort was older, had more concomitant medications, had more use of glucocorticoids, and had more patients with rheumatoid arthritis than the alendronate cohort - characteristics that may increase fracture risk. Conversely, the risedronate cohort also had greater past use of calcitonin or raloxifene - a characteristic that may decrease fracture risk. Within the 12 months prior to the initiation of bisphosphonate therapy, similar percentages of the two cohorts had a diagnosis for a nonvertebral fracture and a clinical vertebral fracture, while a statistically larger percentage of the risedronate cohort had a diagnosis for a hip fracture than the alendronate cohort (Fig. 2).
Fig. 2Percent of patients with a clinical diagnosis of fracture before initiation of bisphosphonate therapy. 1ICD-9 codes 808.x, 810.x, 812.x, 813.x, 820.x, 821.x, 823.x, 733.10, -.12, -.14, -.19; 2ICD-9 codes 820.x, 733.14; 3ICD-9 codes 805.x, 806.x, 733.13; 4Subset (81%) of study population with available 12 month history. *Statistical difference (p<0.05) between cohorts
During the 12 months of observation after the start of bisphosphonate therapy, 507 subjects had nonvertebral fractures. The site of nonvertebral fracture was wrist (30%), hip (21%), leg (17%), pelvis (15%), humerus (14%), and clavicle (3%). For the 109 women hospitalized with a hip fracture, the skeletal sites were intertrochanteric (46%), transcervical (28%), unspecified (20%), and trochanteric or subtrochanteric (6%).
For the primary analysis of nonvertebral fractures, the fracture incidence was similar between the risedronate and alendronate cohorts over the first 3 months of therapy (Fig. 3). After 6 months of therapy, the risedronate cohort had a 19% lower (95% CI 0% – 35%, p-value = 0.05) incidence of nonvertebral fracture than the alendronate cohort. After 12 months of therapy, the risedronate cohort had an 18% lower (95% CI 2% – 32%, p-value = 0.03) incidence of nonvertebral fracture than the alendronate cohort (Table 3).
Fig. 3Cumulative incidence of nonvertebral fractures in patients treated with alendronate or risedronate for up to 1 yearTable 3Cumulative incidence of fractures during therapyFracture typeCohort sizeNumber of women with a fracturePercent of women with a fractureaCrude rate ratiobAdjusted rate ratiob95% CIp-valueTime on therapy        Cohort       Nonvertebral6 Months Alendronate21,6152531.31–––– Risedronate12,2151231.140.870.810.65–1.000.0512 Months Alendronate21,6153432.30–––– Risedronate12,2151641.990.880.820.68–0.980.03Hip6 Months Alendronate21,615540.29–––– Risedronate12,215190.170.630.540.32–0.910.0212 Months Alendronate21,615800.58–––– Risedronate12,215290.370.680.570.37–0.870.01CI = confidence interval of adjusted rate ratioaProportion is based on Kaplan-Meier estimate of the survival function.bBased on Cox regression model.cBased on Cox regression model. Variables selected by forward stepwise selection where criteria for selection p < 0.1. Model for nonvertebral fractures included age, estrogen use, number of medications, rheumatoid arthritis diagnosis, and history of nonvertebral fractures. Model for hip fractures included age, estrogen use, number of medications, and history of hospitalization.
For the primary analysis of hip fractures, the fracture incidence was similar between the risedronate and alendronate cohorts over the first 3 months of therapy (Fig. 4). After 6 months of therapy, the risedronate cohort had a 46% lower (95% CI 9% – 68%, p-value = 0.02) incidence of hip fracture than the alendronate cohort. After 12 months of therapy, the risedronate cohort had a 43% lower (95% CI 13% – 63%, p-value = 0.01) incidence of hip fracture than the alendronate cohort (Table 3).
Fig. 4Cumulative incidence of hip fractures in patients treated with alendronate or risedronate for up to 1 year
These differences between the risedronate and alendronate cohorts in the incidence of fractures were consistent across other methods of analysis (Fig. 5). Depending on the method, the risedronate cohort had an estimated 6% to 23% lower incidence of nonvertebral fractures and an estimated 30% to 50% lower incidence of hip fractures than the alendronate cohort through 12 months of therapy. All of these other estimates were within the confidence intervals of the primary analyses.
Fig. 5Sensitivity analysis: Rate ratio for fracture in the first year of therapy between patients on risedronate and patients on alendronate; results of the primary analysis and 4 other methods of analyses
Discussion
In this observational study across multiple US health plans, we observed that patients on once-a-week dosing of risedronate had a lower incidence of hip and nonvertebral fractures than patients on once-a-week dosing of alendronate. Differences in fracture incidence between these two cohorts of patients were observed at 6 and 12 months after initiating therapy.
As with all observational studies, systematic errors (e.g., selection bias, measurement misclassification) may be the basis for the observed results [26]. In this study, systematic errors may arise from differences in fracture risk between the 2 cohorts of patients at initiation of therapy. Between the two cohorts, there were statistical differences in measurable fracture risk characteristics (Table 2), there are likely to be differences in known fracture risk characteristics not available within medical claims data (e.g., bone mineral density, family history, smoking history), and there are likely to be differences in unknown fracture risk characteristics (i.e., those that are controlled through randomized trials). The differences in measurable fracture risk characteristics, for which a greater percentage of the risedronate cohort has risk factors for fracture than the alendronate cohort suggesting bias towards higher fracture rates in the risedronate cohort, are inconsistent with observed results. The near unity in fracture incidence between the two cohorts during the first 3 months of therapy (Figs. 3 and 4) - a period for which there is also unity in fracture incidence between bisphosphonate therapy and placebo in clinical trials [12, 14] - suggest that both cohorts had similar risk for fracture at initiation of therapy. However, differences in fracture risk at initiation of therapy between the two cohorts cannot be excluded.
Within healthcare utilization data, which are collected for purposes other than research, misclassification of fracture events and of therapy use are inevitable. As a check on the data, the rate of fracture events and therapy use of these health utilization data are in agreement with other data sources. In the current study, the annual fracture rates following initiation of therapy (≈2.0% for nonvertebral fractures and ≈0.5% for hip fractures) are consistent with the annual rates in the treated population of clinical trials (between 2.0 and 2.3% for nonvertebral fractures and between 0.4% and 0.7% for hip fractures [6–10, 27]). In this study, risedronate patients constituted 25% [45,360/(45,360 + 137,412)] of the bisphosphonate users in the utilization data compared to 24% [13.6 million / (13.6 million + 44.1 million)] of all bisphosphonate prescriptions in the US during the same period [28]. A good method for evaluating misclassification within healthcare utilization data is through a medical chart review. In a prior study, the proportion of fracture claims confirmed by chart review to be a fracture was highest for the hip relative to other fracture sites [29]. Since the effect of misclassification at these other fracture sites is likely to be no different between cohorts (i.e., misclassified exposure does not depend on cohort status), the study results for nonvertebral outcomes are likely more attenuated by misclassification than results for the hip [30].
The strength of observational studies can be the generalizability of results. In contrast, the generalizability of results from randomized trials to a real world setting can be limited by differences between the two in relation to expertise of health care provider, quality of medical care, course of therapy, and types of patients [31]. For example, it has been observed that the majority of patients considered candidates for osteoporosis therapy by their physician would not meet the eligibility criteria for inclusion in the randomized trials [32]. Since the population within the current observational study is drawn from multiple health plans in many US states and consists of subjects with a mixture of health characteristics (e.g., prior gastrointestinal comorbidities), the results are likely to be generalizable. Furthermore, the length of observation of therapy adherence (≈232 days) in the current study was consistent to the previously reported average duration of adherence to bisphosphonate therapy (245 days) [33].
In conclusion, within this observational study of clinical practice, a cohort of patients receiving risedronate had lower rates of hip and nonvertebral fractures during their first year of therapy than a cohort of patients receiving alendronate. These results do not appear to be explained by baseline differences in fracture risk between cohorts. In addition, the observed rates of fracture were consistent with the fracture rates in clinical trials. Thus it appears, patients receiving risedronate are better protected from hip and nonvertebral fractures during their first year of therapy than patients receiving alendronate.

bisphosphonates
hip fractures
osteoporosis
nonvertebral fractures
epidemiology


Identification and characterization of the human inhibitor of caspase-activated DNase gene promoter

DNA fragmentation factor is a heterodimer complex of the nuclease CAD and its specific inhibitor ICAD, which can be activated during apoptosis to induce DNA fragmentation. Although ICAD expression levels have been quantified in a variety of human cancer cells, the mechanism of ICAD gene regulation remains unknown. In this study, we identified a 106-bp TATA-less region upstream of the transcription start site as a basal promoter of the human ICAD gene. An E-Box motif, which binds transcription factors of the basic helix-loop-helix/leucine zipper family, is responsible for transcriptional activity, as demonstrated using mutated promoter-reporters. A chromatin immunoprecipitation assay further demonstrated that Myc binds to an endogenous ICAD promoter. The functional importance of Myc in the regulation of ICAD transcription was also demonstrated by knock-down of c-Myc and N-Myc gene expression, as well as their ectopic expression. Structural analysis of the human ICAD promoter and identification of factors which regulate its activity might further our understanding of the biological role of ICAD with respect to regulation of apoptosis and cancer development.
Introduction
Apoptosis or programmed cell death ensures the elimination of unwanted cells during normal development and homeostasis [1]. This process is progressively inactivated during malignant development and loss of the capacity for apoptosis is a hallmark of malignant cells. Chromatin condensation and internucleasomal DNA fragmentation are typical nuclear features and well-recognized events in apoptosis. DNA fragmentation factor (DFF) is a heterodimer protein composed of a 40-kDa caspase-activated DNase (CAD) otherwise known as DFF40, and its cognate 45-kDa inhibitor (inhibitor of CAD: ICAD or DFF45 [2–8]). Both human genes map to 1p36 [9, 10]. CAD is thought to be responsible for the majority of nuclear activity resulting in chromosomal DNA fragmentation. When apoptosis is activated, ICAD is cleaved by executor caspases, mainly caspase-3, into three fragments, after which it dissociates from CAD, resulting in CAD activation.
Thus, the DFF complex may play a role in malignant transformation, and up- or down-regulation of ICAD/CAD expression might correlate with cancer aggression. Expression levels of ICAD have been examined in a variety of human cancers. For example, ICAD expression is down-regulated during the exponential growth phase of human colon carcinoma cells [11]. In some neuroblastomas, preferential ICAD expression is observed in low-stage, but not in their high grade [12]. Other research suggests that down-regulation of ICAD may contribute to tumor growth and lymph node metastasis in esophageal carcinoma [13], and that ICAD expression might serve as a marker of aggressive tumor behavior with an associated poor prognosis in ovarian cancer [14]. We have previously demonstrated that the hepatitis C virus core protein, which not only encodes the viral nucleocapsid but has a number of properties enabling persistent viral infection, induces gene expression of ICAD, thereby increasing steady-state levels of the ICAD protein [15]. To date, the mechanism of transcriptional regulation of the ICAD gene is not well understood. Analysis of the gene structure of mouse ICAD showed that a 118-bp flanking region of the ICAD gene is required for promoter activity [16]. In this study, we identified a functional promoter of the human ICAD gene and investigated the role of c-Myc and N-Myc in regulation of the ICAD promoter.
Materials and methods
Cell cultures
Human hepatoblastoma cells (Huh-7), human oral squamous carcinoma cells (HSC-2, HSC-3 and Ca22-9), mouse neuroblastoma cells (IMR-32 and GOTO) and mouse 3T3 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 100 units/ml of penicillin, and 100 mg/ml of streptomycin and kept at 37°C in a 5% CO2 incubator.
Cloning of the 5′-upstream region of the human ICAD gene
Human genomic DNA was isolated from Huh-7 cell lines using the QIAamp DNA Mini kit according to the manufacturer’s instructions (QIAGEN). A 5′-upstream region of the ICAD gene was amplified by PCR using primers based on the genomic DNA sequence of the human ICAD gene (GenBank accession No. NM_004401). Primers containing EcoRI sites were used as follows: forward (5′-GAATTCAGGCTGGTCTCAAACTACTG-3′) and reverse (5′-GAATTCGATCTCGCCAGATTCTGGTA-3′). The PCR reaction was carried out at 94°C for 1 min, 55°C for 1.5 min, and 72°C for 2 min, for total of 35 cycles. Each PCR product was purified and subcloned into a pGEM-T Easy vector (Promega), followed by sequencing using an ABI PRISM 310 automated DNA sequencer (Applied Biosystems).
5′ rapid amplification of cDNA ends (5′-RACE) assay
The 5′ ends of the ICAD transcript were cloned by 5′-RACE using a Gene Racer RACE-Ready cDNA kit in accordance with the manufacturer’s instructions (Invitrogen). Double-stranded cDNA molecules prepared from human liver poly(A)+ RNAs ligated by exposure to the adaptor were amplified in a primary PCR reaction using the adaptor primer 1 and the ICAD gene-specific antisense primer ICA1AS (5′-GTGCTGTTCGCGGCTGTAGTT-3′, nt −147 to −173), followed by a secondary PCR reaction using nested oligonucleotides, the adaptor primer 2, and the antisense ICAD specific primer ICA2AS (5′-CACGGTGACTGGTGTCAGGGACTTATC-3′, nt −228 to −254). PCR products were purified and cloned, followed by nucleotide sequencing as described above.
Plasmid constructions
The 1-kb sequence containing the human ICAD promoter was excised by digestion of the above-mentioned pGEM-T vector with SacI and HindIII, and then cloned into the firefly-luciferase-expressing reporter plasmid pGL3-Basic (Promega), resulting in pLUC1005. A series of constructs with 5′ end-deletions of the ICAD promoter were created by PCR amplification using the reverse primer (5′-CAAGCTTGCCTCCACAAGGTGGGACCTG-3′) and the following forward primers: 5′-GGCTAGCCAGTACCCATTTCTGAAGAAG-3′ (nt −936 to +71), 5′-GGCTAGCCCTCATTTGGGTCCATTTTCC-3′ (nt −622 to +71), 5′-GGCTAGCCAGCTTTTTCAGACAGAATGG-3′ (nt −272 to +71), 5′-GGCTAGCCAGCTTTTTCAGACAGAATGG-3′ (nt −205 to +71), 5′-GGCTAGCCAGCTTTTTCAGACAGAATGG-3′ (nt −145 to +71), 5′-GGCTAGCCAGCTTTTTCAGACAGAATGG-3′ (nt −106 to +71), 5′-GGCTAGCCTATTTAGTTTGGTTAGTAAT-3′ (nt −90 to +71), and 5′-GGCTAGCCCAGATGGTAAATATACACAA-3′ (nt −43 to +71). Each SacI/HindIII fragment was inserted into the pGL3-basic vector to yield pLUC(−936/+71), pLUC(−622/+71), pLUC(−272/+71), pLUC(−205/+71), pLUC(−145/+71), pLUC(−106/+71), pLUC(−90/+71) and pLUC(−43/+71).
Transfection and reporter assay
Huh-7 cells were seeded at 5 × 104 cells/well in 24-well plates and maintained at 37°C in a 5% CO2/95% atmosphere. DNA transfection of cells with each ICAD-promoter-luciferase construct (1 μg) with an internal control vector pRL-TK (0.1 μg) (Promega) was performed with Trans IT LT-1 (Mirus) during 6-h of incubation. Cells were then rinsed with phosphate-buffered saline (PBS) 48 h after transfection, and luciferase activity was measured in the cell lysate using dual luciferase assay reagents (Promega) [17]. Firefly luciferase activity was standardized according to Renilla luciferase activity.
Chromatin immunoprecipitation
Chromatin immunoprecipitation assays were performed using the ChIP assay kit (Upstate). Briefly, cells in 100-mm dishes were grown to 70% confluency over 48 h. The chromatin from formaldehyde-fixed cells was sonicated and immunoprecipitated using mouse monoclonal anti-c-Myc or anti-N-Myc antibodies (Santa Cruz). The chromatin immunoprecipitate was analysed by PCR with the following primer pairs: F1 (5′-CGAGCTCGGTATACATGCGTGTGCATCG-3′) and R1 (5′-CAAGCTTGCCTCCACAAGGTGGGACCTG-3′) for amplifying the region from nt −272 to −71 containing potential Myc-binding sites; and F2 (5′-GAGATCAAAACTGCAGTGAG-3′) and R2 (5′-CACTGTTGGAGATTGTTCAG-3′) for amplifying the region from nt −789 to −451 that does not contain Myc-binding sites.
Western blotting
Cells were washed with PBS and lysed in SDS sample buffer. Cell lysate samples were separated by 10% SDS-polyacrylamide gel electrophoresis and electrotransferred to a polyvinylidene difluoride membrane (Immobilion; Millipore). After blocking in nonfat milk solution (Blocking One; Nakaraitesk), the membranes were probed with monoclonal antibody against the ICAD protein (Santa Cruz), c-Myc (Sigma), or N-Myc (Santa Cruz), as the primary antibody for 1 h. After being washed, the membranes were incubated with horseradish peroxidase-conjugated anti-mouse immunoglobulin as the secondary antibody, followed by visualization with SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology).
siRNA experiments
siRNAs for human c-Myc, N-Myc and the negative control were purchased from B-Bridge International. Each siRNA consisted of three different target sequences as follows: 5′-GAGGAGACAUGGUGAACCA-3′, 5′-GAGAAUGUCAAGAGGCGAA-3′, and 5′-GAGAAUGUCAAGAGGCGAA-3′ for c-Myc siRNA; 5′-CGGAGAUGCUGCUUGAGAA-3′, 5′-CGGAGUUGGUAAAGAAUGA-3′, and 5′-CAGCAGUUGCUAAAGAAAA-3′ for N-Myc siRNA; and 5′-ATCCGCGCGATAGTACGTA-3′, 5′-TTACGCGTAGCGTAATACG-3′, and 5′-TATTCGCGCGTATAGCGGT-3′ for the negative control. Huh-7 cells were transfected with 100 pmol/ml siRNA using FuGENE6 (Roche) in Opti-MEM I (Invitrogen), after which the medium was replaced by standard DMEM after 6 h of transfection.
RNA preparation and real-time quantitative RT-PCR
Total RNA was extracted using Trizol reagent (Invitrogen) according to the manufacturer’s protocol. First-strand cDNAs were synthesized using the SuperScriptIII First-Strand Synthesis kit (Invitrogen) and then used as templates for real-time PCR. Quantitative PCR was performed using the ABI Prism 7700 sequence detection system using Taq-Man Gene Expression assays (Applied Biosystems). The standard curve was created using serially diluted total RNA obtained from Huh-7 cultures and β-actin was chosen as the internal standard to control for variability in amplification. Amplification was performed at 95°C for 10 min, followed by 40 cycles of amplification at 95°C for 15 s and 60°C for 60 s.
Results and discussion
Localization of transcription start sites of the human ICAD gene
Precise localization of transcription start sites of the ICAD gene was examined in human hepatoma Huh-7 cells by 5′-RACE analysis. A fragment of approximately 100 bp was obtained (Fig. 1a) and cloned, after which sequencing analysis of several cDNA clones identified two of 5′ termini (Fig. 1b). These were located 71 and 68 nucleotides upstream from the translation start codon of the ICAD gene. From a database search of the DBTSS [18], we found that these are two of multiple sites for putative transcription initiation for the human ICAD gene. Although transcription may be initiated at both sites, the upper site was designated position +1 in this study.
Fig. 1(a) Mapping of the transcription start sites by 5′ RACE analysis. Following nested PCR reaction, a PCR product of 100-bp was detected by 1.5% agarose gel electrophoresis (lane 2). Lane 1: no template control; lane M: 100-bp DNA ladder marker. (b) The 5′ flanking region of the human ICAD gene. The nucleotide sequence is numbered from the major transcription site, which is indicated by an arrow. Putative binding sites for transcription factors analyzed by the TRANSFAC database and search program were shown with underlines
Characterization of the ICAD gene promoter
To examine potential regulatory sequences involved in ICAD gene expression, a 1 kb 5′-upstream region of the ICAD gene was sequenced and analyzed. The overall GC content of the 1-kb genomic DNA fragment was 46%, while GC content among the 200 bp proximal to the ATG start codon was approximately 62%. A search of transcription consensus motifs using the TRANSFAC database [19] demonstrated that none of the major eukaryotic promoter elements, TATA-, CCAAT-, or GC box, were located within 200 bp upstream of the transcription start site, although a TATA-like element was found more than 400 bp upstream from the start sites, indicating that the human ICAD gene has a TATA-less promoter. No CpG island, which is frequently observed in the TATA-less promoter, was found by screening with the CpG island searcher (http://cpgislands.usc.edu/). Absence of a TATA-like sequence upstream of the transcription start site has also been reported for the mouse ICAD gene, suggesting that TATA-less promoters might be a common feature in transcriptional regulation of the ICAD gene.
As shown in Fig. 1b, the 1-kb sequence contains potential binding sites for several transcription factors. The ability of the 5′-upstream region of the ICAD gene to function as a promoter was assessed by its capacity to drive the expression of a luciferase reporter gene. A series of constructs in which genomic DNA fragments were fused to a promoterless firefly luciferase gene of the pGL3-basic vector were generated with the 3′ end always terminating +71 bp from the transcription start site. The 5′ ends began at bases −936, −662, −272, −205, −145, −106, −90, and −43 (Fig. 2). Promoter activity was assessed by measuring luciferase activity in transiently transfected Huh-7 cells. Luciferase activity was normalized to Renilla luciferase activity. As shown in Fig. 2 on the right, the region extending from −936 to +71 had promoter activity since luciferase activity of the reporter gene was 45-fold that of the empty vector. A 5′ deletion of the promoter sequence to −662(pLuc(−662/+71)) enhanced promoter activity, and successive removal of nucleotides from −272 to −106 also enhanced promoter activity, suggesting the possibility of negative regulatory elements within the −936/−622 and −272/−106 regions. When the sequence from −106 to −90 was removed (pLuc(−90/+71)), promoter activity fell to parallel that of the pGL3 basic vector. This suggests the presence of a critical element in the region extending from −106 to −90, thus, we have identified the region 106 bp upstream of the transcription start site as the basal promoter of the human ICAD gene.
Fig. 2Human ICAD promoter drives reporter gene expression in transiently transfected Huh-7 cells. Deletion constructs of the upstream region of the ICAD gene linked to the firefly luciferase reporter gene (LUC) are illustrated in the left. Huh-7 cells were cotransfected with a firefly luciferase reporter and a Renilla luciferase internal control (pRL-TK). The relative luciferase activity (RLU) was obtained by normalizing the firefly luciferase activity to the Renilla luciferase activity, and is expressed as a percentage of RLU of pLuc(−936/+71). The average values are shown with standard deviation (SD) (n = 4 per construct)
Myc and USF enhance ICAD promoter activity
As indicated in Fig. 1b, a computer-based sequence analysis revealed that the 106 bp region contains potential binding sites for GATA-1, deltaE, Myc, and various upstream stimulatory factors (USF). Within this region, there is a canonical E-box sequence (5′-CACGTG-3′) located between −103 and −98. E box motifs are known to bind to transcription factors of the basic helix-loop-helix/leucine zipper (bHLH LZ) family, including Myc and USF [20–23]. We examined the potential role of E-box-binding proteins, Myc and USF, in potentiating ICAD gene transcription. Myc is bound to a partner protein Max via a bHLH LZ domain and the Myc-Max heterodimer activates transcription by binding to an E-box sequence [24, 25]. Max is present in greater amounts than Myc since the Myc transcript and protein have shorter half-lives compared to Max [26, 27]. Therefore, it is highly likely that Myc is the limiting, regulated component of the heterodimer. To examine the potential role of E box binding proteins, Huh-7 cells were co-transfected with luciferase reporter plasmids and expression vectors for c-Myc, N-Myc, USF1, and USF2 (Fig. 3a). Luciferase activity of the −145/+71 promoter construct increased 2- to 5-fold, and 1.5- to 2-fold, when co-expressed with Myc or USF, respectively. The effect of c-Myc and N-Myc on activity of the −90/+71 construct was less than for the −145/+71 construct. USF expression did not enhance activity of the −90/+71 construct.
Fig. 3(a) ICAD promoter activity after transient expression of c-Myc, N-Myc, USF1 and USF2. Huh-7 cells were cotransfected with either expression vector for c-Myc, N-Myc, USF1 or USF2 driven by the CMV promoter and pLuc(−145/+71), pLuc(−90/+71) or pGL3 basic. Each firefly luciferase reporter plasmid (n = 4) was cotransfected in cells with pRL-TK for normalization of the reporter activity. RLU is expressed as a percentage of that of pLuc(−145/+71) in the absence of expressing plasmids for Myc and USF. (b) Effect of substitution mutation in the E-box element on the ICAD promoter activity. Cells were cotransfected with pLuc(−106/±71) or an E-box-mutant, pLuc(−106/±71)mt and either expression vector for c-Myc or N-Myc. pRL-TK was also used for normalization of the activity. RLU is expressed as a percentage of that of pLuc(−106/±71) without over-expression of Myc
To further investigate involvement of the E box sequence in ICAD promoter activity, a reporter construct with an E box mutation (CCCGCG) was constructed and luciferase activity was examined in transfected cells (Fig. 3b). The E box mutation resulted in marked down-regulation of reporter gene expression and the reporter activity expressed from the E-box mutant was little increased when co-expressed with Myc, suggesting a functionally important role of the −106/−90 region E box motif with regard to basal transcriptional activation of the ICAD promoter. These data indicates that E-box-binding proteins, especially Myc, actively participate in positive regulation of the human ICAD promoter.
Myc binds to the ICAD promoter in vivo
To obtain direct evidence of an interaction between Myc and the ICAD promoter in vivo, we next examined binding of c-Myc and N-Myc to the 5′-upstream region of the ICAD gene within the context of native chromatin in living cells by chromatin immunoprecipitation. An alignment of the sequence around putative Myc binding site of human ICAD promoter and the corresponding part of mouse ICAD sequence demonstrated conservation of Myc binding sequence in human and mouse (Fig. 4a) [16]. Proteins were cross-linked to genomic DNA isolated not only from human (Huh-7) but mouse (IMR-32 and GOTO) cells, followed by immunoprecipitation with normal rabbit IgG or polyclonal antibodies to either c-Myc or N-Myc. IMR-32 and GOTO are neuroblastoma cells with N-Myc gene amplification [28]. The precipitated DNA was then subjected to PCR utilizing primers designed to amplify a 343-bp fragment (−272/+71) or a 338 bp fragment (−789/−451) of the ICAD 5′ flanking region. As shown in Fig. 4b, the 343-bp DNA fragment was observed in the immunoprecipitate from Huh-7 cells following exposure to anti-c-Myc or anti-N-Myc, while amplification of the ICAD promoter fragment was not detected in the negative control immunoprecipitate. Under similar experimental conditions, we did not detect binding of c-Myc and N-Myc to the −789/−451 region, which is not thought to contain a Myc-binding site. Similar results were obtained from IMR-32 and GOTO cells, which were immunoprecipitated with anti-N-Myc antibody. These results demonstrate that Myc forms a complex with the human ICAD promoter in cells, presumably through binding to the E-box sequence.
Fig. 4(a) An alignment of the sequence around putative Myc binding site of human ICAD promoter and the corresponding part of mouse ICAD sequence. (b) Binding of Myc proteins in the endogenous ICAD promoter. Crosslinked chromatins isolated from Huh-7, IMR-32 and GOTO cells were immunoprecipitated with indicated antibodies (c-Myc, N-Myc) or an equivalent amount of mouse IgG. Recovered DNAs were purified and PCR-amplified with primers for nt −272/±71 region or for nt −789/−451 region. Input represents 1% of chromatin sample applied for immunoprecipitation
Myc-dependent expression of the ICAD protein
The above data suggests that the ICAD promoter is regulated by endogenous Myc proteins. This assumption is supported by the loss-of-function studies shown in Fig. 5a. We employed siRNA (small interference RNA) transfection to knock-down the expression of c-Myc or N-Myc in cells, after which we analyzed the effect of reduced Myc expression on steady state levels of the ICAD protein by Western blotting. Treatment with siRNA specific for either c-Myc or N-Myc markedly reduced ICAD expression without affecting the expression of β-actin. In contrast, control siRNA did not reduce ICAD levels. We further investigated the effect of over-expression of Myc on ICAD protein levels (Fig. 5b). Transfection of Huh-7 and mouse 3T3 cells with a c-Myc or N-Myc expression vector enhanced ICAD protein levels with an effect that was less pronounced than in the loss-of-function studies, but reproducible nonetheless. These results suggest an important role of Myc proteins in positive regulation of ICAD expression.
Fig. 5Myc-dependent expression of human ICAD. (a) Suppression of Myc expression decreases expression of ICAD protein. Huh-7 cells were transiently transfected with Myc siRNA (c-Myc, N-Myc) or the control siRNA (cont). Three days later, the cells were harvested and subjected to Western blotting. (b) Ectopic expression of Myc increases expression of ICAD protein. Cells (Huh-7 and mouse 3T3) were transfected with the expression vector for c-Myc or N-Myc, and after 3 days the cells were harvested for Western blotting. (c) Comparison of the mRNA expression of c-Myc and ICAD in Huh-7 cells and human oral squamous carcinoma cells (Ca22-9, HSC-2, and HSC-3). Levels of mRNA expression of ICAD and c-Myc were measured by quantitative real-time RT-PCR based on Taq Man chemistry. Results, relative to β-actin mRNA, are depicted as averages with SD (n = 3)
Finally, we examined if there might be a correlation between ICAD and Myc expression levels in cancer cell lines. The mRNA expression of ICAD and c-Myc in human oral squamous cell carcinomas (Ca22-9, HSC-2, and HSC-3), in which ICAD expression has not been investigated to date, as well as in Huh-7 cells during their late exponential phase of growth, was analyzed by quantitative RT-PCR (Fig. 5c). ICAD mRNA was readily detected in all cancer cells but the expression level was not consistent. A relatively high level of expression was observed in Ca22-9 and HSC-3 cells, while HSC-2 cells demonstrated the lowest. Interestingly, expression pattern of c-Myc mRNA in these cell lines showed a tendency similar to that of ICAD, suggesting an important role of Myc in ICAD expression in human cancers. A large scale analysis for investigating correlation between ICAD and Myc expression in a variety of cancer tissues obtained from patients is ongoing.
Several studies have shown up- or down-regulation of ICAD in a variety of human cancers [11–14, 29, 30]. Although transcriptional deregulation is presumably involved in this aberrant expression of ICAD, little is known about the transcriptional regulation of ICAD in human cells. This work was done to characterize the human ICAD promoter and to examine transcription factors which may be involved in its regulation. We experimentally determined putative transcription start sites by employing the 5′-RACE method, and identified a promoter region required for basal ICAD gene expression using promoter-reporter constructs with progressive deletions. A combined study of site-directed mutagenesis of a reporter construct and chromatin immunoprecipitation revealed the importance of an E box Myc-binding motif located at position −103 to −98 from the transcription start sites, as well as an in vivo interaction between c-Myc and N-Myc and the proximal ICAD promoter. Furthermore, we demonstrated the functional importance of Myc proteins with regard to transcriptional regulation of the ICAD gene from studies examining ectopic expression of c-Myc and N-Myc, as well as with RNAi technology. We showed that expression levels of ICAD and Myc correlate in some tumors.
c-Myc and N-Myc are transcription factors of the bHLH LZ family that bind to the E box sequence within promoters to control proliferation, cell differentiation, and apoptosis [25, 31, 32]. c-myc and N-myc genes are deregulated in a number of human cancers and influence proliferation and growth. A link between Myc and cancer is well established both in vivo and in vitro, and oncogenic activation of Myc has been observed to promote the development of a number of clinically significant cancers [25, 33]. However, the molecular and cellular mechanisms of Myc-mediated transformation are not fully understood. Although a variety of Myc target genes were identified and recruitment of Myc to the target promoters such as prothymosin and telomerase were shown [34], there has been no direct evidence on involvement of Myc in regulation of ICAD expression. It is known that c-Myc activation usually occurs during the later stages of carcinoma in humans. Conversely, in premalignant cells, c-Myc is a robust stimulator of apoptosis and programmed cell death [32]. One area of investigation into cancer cell death mechanisms centers on the mechanism by which c-Myc stimulates or suppresses apoptosis. For instance, Myc has been reported to potentiate apoptosis through both p53-dependent and -independent mechanisms [35, 36]. Myc controls the balance between pro- and anti-apoptotic factors at the level of the mitochondria, thereby regulating cytochrome C release and activation of downstream caspases [37, 38]. Involvement of Myc in up-regulation of ICAD expression as demonstrated in this study might present a novel mechanism of Myc-dependent inhibition of apoptosis. It is possible that elevated levels of ICAD in cells inhibit activation of endonuclear activity, thereby increasing the threshold for apoptosis signaling.
Kawane et al. have reported on the structure and analyzed the promoter of a murine ICAD gene, by which they demonstrated that a 118-bp flanking region of the ICAD gene is required for its transcription [16]. The mouse sequence shares approximately 82% homology with a corresponding upstream region of the human ICAD gene. The mouse ICAD promoter has a number of potential binding sites for transcription factors, such as Ikaros, c-Rel, Myc, and Gfi-1 [16]. Conservation of the Myc-binding motif among human and murine promoters suggests a functional significance of Myc in transcriptional regulation of ICAD expression.
In conclusion, this is the first report to identify a functional promoter of the human ICAD gene, and to demonstrate that Myc proteins are able to positively regulate ICAD gene expression. Extensive apoptosis research to date has shown that tumor aggression depends on various defects in apoptosis signaling [39]. Further investigation into the molecular events linking Myc expression with ICAD gene regulation may provide insight into Myc’s role in cell proliferation, transformation and apoptosis.