High dose of lipoxin A4 induces apoptosis in rat renal interstitial fibroblasts

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Prostaglandins, Leukotrienes and Essential Fatty Acids 73 (2005) 127–137 www.elsevier.com/locate/plefa

High dose of lipoxin A4 induces apoptosis in rat renal interstitial fibroblasts Sheng-Hua Wu, Chao Lu, Ling Dong, Guo-Ping Zhou, Zha-Guang He, Zi-Qing Chen Department of Pediatrics, Central Laboratory, the First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing, Jiangsu 210029, People’s Republic of China Received 16 December 2004; accepted 23 February 2005

Abstract Studies have implicated that lipoxinA4 (LXA4) inhibited nuclear factor-kB (NF-kB), Akt/PKB and PI 3-kinase activity and proliferation of glomerular mesangial cells. It is speculated that LXA4 might serve as pro-apoptotic factor. Rat renal interstitial fibroblasts (NRK-49F cells) were exposed to LXA4 in 5% FCS for 24 h. LXA4 at 0.1 and 1 mM induced 9.83% and 33.82% apoptosis of the cells, respectively, upregulated the expression of calpain 10 and Smac, the levels of [Ca2+]i and activity of caspase-3, and downregulated the activity of STAT3 and threonine phosphorylated Akt1. Transfection of calpain 10 or Smac antisense oligodeoxynucleotide into the cells inhibited the LXA4-induced apoptosis, activity of caspase-3. Pretreatment of the cells with calcium inhibitor SK&F96365 inhibited the LXA4-induced apoptosis, levels of [Ca2+]i, expression of calpain 10 and Smac. In conclusion, LXA4 at high concentrations induced apoptosis of renal interstitial fibroblasts via [Ca2+]i-dependent upregulation of calpain 10 and Smac expression. r 2005 Published by Elsevier Ltd.

1. Introduction Renal interstitial fibrosis is the common pathway of chronic renal disease and characterized by increased numbers of fibroblasts resulted from the proliferation of fibroblasts, transformation of tubular epithelial cells into fibroblasts and insufficient rate of apoptosis of fibroblasts [1]. Cell death by apoptosis is one of the most important mechanisms of cell clearance under pathophysiological conditions and a failure in the clearance of renal fibroblasts may result in fibroblast accumulation during renal fibrosis [1,2]. Promoting the numbers of apoptotic fibroblasts may provide the basis for the therapeutic strategies in suppression of renal fibrosis. Studies have shown that lipoxinA4 (LXA4) and its analogues inhibited proliferation of glomerular mesangial cells induced by leukotriene D4 (LTD4) or plateletderived growth factor (PDGF) [3,4], blocked LTD4 stimulated phosphatidylinositol 3-kinase (PI 3-kinase) E-mail address: [email protected] (S.-H. Wu). 0952-3278/$ - see front matter r 2005 Published by Elsevier Ltd. doi:10.1016/j.plefa.2005.02.005

activity, and downregulated Akt/protein kinase B (PKB) activation stimulated by PDGF in mesangial cells [4] or nuclear factor-kB (NF-kB) activation by tumor necrosis factor-a in embryonic kidney cells [5]. LXA4 also inhibited NF-kB activation in human leukocytes and epithelial cells [6,7]. Since PI 3-kinase, NF-k and Akt/PKB play survival and anti-apoptotic role for cells [8–11], it can be speculated that LXA4 might serve as a pro-apoptotic factor. This study was therefore designed to examine whether LXA4 induced apoptosis of renal interstitial fibroblasts. The regulation of cell apoptosis has been investigated in a number of clinical disorders including renal fibrosis [1,2]. In renal cell injury, caspases play a crucial role in the execution or final phase of apoptosis [12]. The inhibitors of apoptosis proteins (IAPs) regulate apoptosis by inhibiting caspases. X chromosome-encoded IAP (XIAP), an active member of the IAP family, has been shown to directly inhibit caspase-3, -7, and -9 [13]. Recent studies have documented that mitochondrial protein Smac/DIABLO (second mitochondrial activator

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of caspases/direct IAPs binding protein with low pI) promotes apoptosis by eliminating the inhibitory effect of IAPs, especially of XIAP, on caspases [14,15]. During apoptosis, caspases can be also activated by calpain [16]. Calpains are calcium-dependent intracellular nonlysosomal proteases [17], and their activation played a role in apoptosis in mouse thymocytes [18]. Calpain 10 is the third ubiquitous calpain in tissues along with m- and mcalpains, and its expression level and subcellular distribution are influenced by calcium [19]. The role of intracellular calcium ([Ca2+]i) and calpain in cell death has been also demonstrated in experimental renal injury [20–22]. Calpain is also a signal transducer and activator of transcription (STAT)3 and STAT5 protease [23]. Various combinations of STATs by themselves or with other transcription factors activate distinct sets of genes, leading to different biological consequences [24]. STAT3, a member of the STAT protein family, played an anti-apoptotic role and mediates survival functions in most cases such as glomerular mesangial cell proliferation [25], survival of T lymphocytes and neurons [26]. Activation of STAT3 occurs in many solid and hematologic tumors and is correlated with growth stimulation and anti-apoptotic effects in malignancies [24] although STAT3 is pro-apoptotic in mammary development [26]. In the current studies, to explore the mechanisms of signal pathway of LXA4, we therefore assessed changes of caspase-3, [Ca2+]i, Smac, calpain 10, STAT3, Akt1 in LXA4-induced apoptosis of renal interstitial fibroblasts.

2. Materials and methods 2.1. Culture of rat renal interstitial fibroblasts Rat renal interstitial fibroblast lines (NRK-49F cells) were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences (originated from American Type Culture Collection, Rockville, MD, USA). These cells have been extensively characterized and express vimentin, desmin and fibroblast specific surface antigen but not keratin tested in this laboratory. The cells were incubated in RPMI-1640 medium (Gibco BRL, Grand Island, NY, USA) supplemented with 10% fetal calf serum (FCS, Sigma, St. Louis, MO, USA). After the cells reached 80% confluence, the cells were washed with serum-free media and left in serum free media for 6 h (to consist with antisense oligodeoxynucleotide treatment as described below). Subsequently, the cells were exposed to LXA4 (Calbiochem, La Jolla, CA, USA) at the concentrations of 10, 100 nM or 1 mM for 24 h. The vehicle of LXA4 was 1% ethanol in the media supplemented with 5% FCS. Cell viability was measured by trypan blue exclusion assay in pilot experiment and the percentage of viable cells was more

than 95% before exposure to LXA4 and after exposure to the vehicle of LXA4 for 24 h. 2.2. Studies of cell death For the morphological assessment of apoptosis, cells were grown in 6-well plates and stimulated with LXA4. The cells were trypsinized and suspended. The single cell suspension was adjusted to 107 cells/ml and 45 ml of cell suspension was added to slide and incubated with 5 ml of mixture of acridine orange and ethidium bromide (Sigma, St. Louis, MO, USA) at 10 mg/ml final concentration for 30 s, observed in a laser scanning confocal microscope (Axiovert LSM510, Carl Zeiss Co., Germany) and counted in a fluorescence microscope (Olympus, Japan) [11]. Apoptosis was quantified by flow cytometry following propidium iodide and annexin staining [27]. Cells were trypsinized and suspended, and washed with phosphatebuffered saline (PBS). Subsequently, cell pellets were fixed in 1 ml of ice-cold 75% ethanol and 1% Triton X100 for 2 h, and then washed with PBS, re-suspended in 0.1 mg/ml of RNase (Promega, Madison, WI, USA) for 15 min at room temperature. The single cell suspension was adjusted to 107 cells/ml and stained by propidium iodide (PI) and annexin (both 50 mg/ml, Sigma, St. Louis, MO, USA) at 4 1C for 30 min in the dark. Flow cytometric analysis was carried out at a flow rate of 100 events/s by using dual laser flow cytometer (Becton Dickinson, San Jose, CA, USA). A total 10,000 events were counted. Cell debris and clumps were excluded from the analysis by gating single cells in the forward and side light scatters. Propidium iodide was excited using the 488-nm ultraviolet line of the argon laser. The percentage of cells with decreased DNA staining (low PI uptake, A0 ), comprising apoptotic cells with fragmented nuclei, was counted. Acquired data were analyzed with Mac-based software (Tree Star, San Carlos, CA, USA). 2.3. Assay of caspase-3 activity Caspase-3 activity was assessed using ApoAlertTM Caspase-3 Colorimetric Assay kit (BD Biosciences, La Jolla, CA, USA) following the manufacturer’s instructions. The caspase-3 colorimetric substrate was DEVDp-nitroaniline (DEVD-pNA). The activity of caspase-3 was expressed as pNA (nM) based on the standard curve made between A405 nm and pNA products. 2.4. Assay of free cytosolic calcium The levels of free cytosolic calcium ([Ca2+]i) were analyzed in fura-2-loaded cells by laser scanning confocal microscopy [28]. The cells were washed and then loaded with 5 mM fura-2 acetoxy-methyl ester (Sigma, St. Louis, MO, USA). Subsequently, the cells

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were washed with HEPES-buffered Krebs–Hensleit solution. The coverslip covered with cell monolayer was mounted on the stage of a laser scanning confocal microscope (Axiovert LSM510, Carl Zeiss Co., Germany). The cell preparation was excited alternatively at 340 and 380 nm by excitation monochromater. The emission fluorescence was filtered at 510 nm. The fluorescence obtained at each excitation wavelength depended on the level of Ca2+ binding to fura-2, according to calibration performed using a range of EGTA-buffered Ca2+ solution of the fura-2-free acid. The ratio (R) between the fluorescence intensities at 340 and 380 nm excitation was converted to [Ca2+]i with Grynkiewicz Eq. (29): ½Ca2þ i ðnMÞ ¼ KdðR  Rmin Þ= ðRmax  RÞ. 2.5. RT-PCR analysis of calpain 10 and ALX Cells (2  106/well) were harvested and total RNA extraction was performed using Trizol reagent (Gibco BRL, Grand Island, NY, USA) followed by chloroform–isopropanol extraction and ethanol precipitation. RNA purity (A260 =A280 41:6) was checked by spectrophotometer of GeneQuant (Type II, Pharmacia Co., Sweden) and RNA integrity was confirmed by visualization of 28 and 18 s bands (2:1) on 1% agarose gel. Subsequently, 1 mg of RNA was reverse transcribed using the reverse transcription system (M-MLV-RT, Promega, Madison, WI, USA). Reverse transcriptionPCR (RT-PCR) analysis was performed with the following sets of primers for rat calpain 10,50 CTGTCGGATTTGGCAGTTTGG-30 (sense) and 50 GCCGCTGGCTTTCCTTATGT-30 (antisense) amplifying a 256-bp fragment. The sets of primers for rat LXA4 receptor (ALX) were 50 -ATGGAAGCCAACTATTCCATC-30 (sense) and 50 -TCATATTGCTTTTATATCAATGTT-30 (antisense), amplifying a 1053-bp fragment [30]. The rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as internal controls, 50 -ACCACAGTCCATGCCATCAC-30 (sense) and 50 TCCACCACCCTGTTGCTGTA-30 (antisense) generating a 452-bp fragment. The sets of calpain 10 and GAPDH primers had been selected by software-aided analysis (Primer Premier 5.0) and synthesized by Takara Co. (Japan). PCR reactions were performed in a 50 ml mixture containing 10  PCR buffer, MgCl2, dNTP (Promega, Madison, WI, USA) and Taq DNA polymerase (Promega, Madison, WI, USA). Amplification protocols for calpain 10 and GAPDH consisted of 30 repetitive cycles of pre-denaturing at 95 1C for 4 min, denaturing at 94 1C for 30 s, annealing at 62 1C for 40 s, extension at 72 1C for 30 s and final extension at 72 1C for 5 min. Amplification protocols for ALX consisted of 30 repetitive cycles of pre-denaturing at 95 1C for 4 min, denaturing at 94 1C for 30 s, annealing at 59 1C for 30 s, extension at 72 1C for 70 s and final extension at 72 1C

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for 5 min. Amplified cDNA was separated by 3% agarose gel electrophoresis and visualized with ethidium bromide. Semi-quantitative analysis was performed by using UVP-gel densitometry (UVP Co., San Gabriel, CA, USA). Arbitrary unit (AU) ¼ (Acalpain 10 or AALX/ AGAPDH)  100%. 2.6. Western blotting analysis of Smac and Akt1 The cells lysates were harvested and proteins were extracted using Protein Extraction kits (Active Motif, Carlsbad, CA, USA) following the manufacturer’s instructions. The 20 ml of lysates (40 mg of protein/well) were electrophoresed on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel for 4 h before blotting onto polyvinylidene difluoride membranes (Amersham, Arlington, IL, USA). Nonspecific sites on the membranes were blocked with 5% nonfat milk. The blots were incubated with primary goat anti-rat antibodies against Smac (C-20) or phosphorylated Akt1 (Thr308) or atubulin (Santa Cruz Biotechnology, CA, USA) at 1:1000 dilution for 1 h. The membranes were washed and incubated for 1 h with a horseradish peroxidase-conjugated rabbit anti-goat IgG antibodies (Santa Cruz Biotechnology, CA, USA) at 1:1000 final dilution. After washing, the membranes were incubated with an enhanced chemiluminance reagent system (Amersham, Arlington, IL, USA) and subsequently exposed to Kodak Biomax films. Semi-quantitative analysis was performed by using UVP-gel densitometry (UVP Co., USA). AU ¼ ðASmac =Aatubulin Þ or ðAAkt1 =Aa-tubulin Þ 100%. 2.7. EMSA of STAT3 activity Nuclear protein was extracted using Nuclear Protein Extraction Kit (Active motif, Carlsbad, CA, USA). Electrophoretic mobility shift assay (EMSA) was performed by using Gel Shift Assay kit (Promega, Madison, WI, USA) following the manufacturer’s instructions. The nuclear extracts containing 30 mg of total proteins were preincubated with gel shift binding buffer for 10 min, followed by the addition of 1 ml of g[32P]-labeled double-stranded oligonucleotide probe of STAT3 (Santa Cruz Bioechnology, CA, USA) and further incubation for 20 min. The oligonucleotide pairs of STAT3 were 50 -GATCCTTCTGGGAATTCCTAGATC-30 and 50 -GATCTAGGAATTCCCAGAAGGATC-30 , and radiolabeled with g-[32P]ATP (Promega, Madison, WI, USA) by incubation with 10 units of T4 polynucleotide kinase (Promega, Madison, WI, USA). Resulting nuclear protein–DNA complexes were resolved in 4% non-denaturing polyacrylamide gels and electrophoresis was performed under 90 V for 2 h. Gels were dried and exposed to Kodak Biomax films at 70 1C for 36 h. Semi-quantitative analysis was

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performed by using UVP-gel densitometry (UVP Co., USA). To assess the specificity of the reaction, competition assays were performed with 100-fold excess of unlabeled consensus oligonucleotide pairs of STAT3 (Santa Cruz Bioechnology, CA, USA). The unlabeled probes were added to the binding reaction mixture 10 min before the addition of the labeled probes. 2.8. Antisense oligodeoxynucleotide treatment The oligodeoxynucleotides had been selected by software-aided analysis (RNA structure 3.7) and synthesized by Takara Co. (Japan). The sequence of Smac antisense oligodeoxynucleotide was 50 0 GGCCTCCACACCGGAACATT-3 (NCBI ID: NM031356) phosphorothioated at both ends (underlined 3 bases) of the sequence to promote its hydrosoluble ability and resistance to nuclease. The target site of this sequence was 74–93 bases in Smac mRNA. The sequence of Smac sense and mismatch oligodeoxynucleotide was 50 -AATGTTCCGGTGTGGAGGCC-30 and 50 -TGCTGGATGGCTAGATCGCG-30 , respectively. The sequence of calpain 10 antisense oligodeoxynucleotide was 50 -GCCCGGACCGCCCGCATCTT30 (NCBI ID: AF227909) phosphorothioated at both ends (underlined 3 bases) of the sequence and its target site was 21–40 bases in calpain 10 mRNA. The sequence of calpain 10 sense and mismatch oligodeoxynucleotide was 50 -AAGATGCGGGCGGTCCGGGC-30 and 50 AGCTGGCGTCGGACGGGAGG-30 , respectively. The sequence of rat ALX antisense oligodeoxynucleotide was 50 -GATGGAATAGTTGGCTTCCA-30 phosphorothioated at both ends (underlined 3 bases) of sequence and its target site was 2–21 bases in ALX mRNA [30]. The sequence of the ALX sense and mismatch oligodeoxynucleotide was 50 -CTACCTTATCAACCGAAGGT-30 and 50 -GTAGTTATCGGTACGAGTAC-30 , respectively. To determine the concentration of oligodeoxynucleotide required to reduce the expression of Smac, calpain 10 or ALX, the oligodeoxynucleotides were diluted with opti-MEMI (Invitrogen, USA) to different concentrations of 0.1,1 and 10 mM. The oligodeoxynucleotides were then incubated with the lipofectamine 2000 (Invitrogen, USA) in opti-MEMI for 20 min to form DNA–liposome complexes. For transfection, the cells were plated in 10% FCS at a density of 2  105 cells/well and allowed to 80% confluence. The cells were then washed and the media was replaced with serum-free media (0.9 ml) mixed with 0.1 ml of DNA–liposome complexes followed by incubation at 37 1C for 6 h. Subsequently, the cells were washed and exposed to LXA4 in the media supplemented with 5% FCS for 24 h. To assess the cytotoxicity of each concentration of oligodeoxynucleotide, cell viability was measured by trypan blue exclusion assay following oligodeoxynucleotide transfection. To

determine the efficiency of oligodeoxynucleotide transfection, we used FITC-labeled oligodeoxynucleotide and measured the percentage of cell nuclei stained positive. 2.9. Calcium channel inhibition study To assess effect of the [Ca2+]i on LXA4-induced apoptosis of NRK-49F cells, activity of caspase-3 and and expression of Smac and calpain 10, an inhibitor of calcium channel SK&F96365 (1-b-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole, Miomol, USA) was employed in experiments [31]. The cells were pretreated with SK&F96365 at the concentrations of 10, 20 and 50 mM in serum-free media for 30 min before exposure to LXA4. 2.10. Statistical analysis Results are expressed as mean7standard error of the mean (SEM). Experimental data were analyzed using one-way analysis of variance (ANOVA) followed by S–N–K test (Q test) by Statistical Package for Social Sciences version 10.0 (SPSS, Chicago, IL, USA). Differences were considered to be statistically significant when the P value was less than 0.05.

3. Results 3.1. Apoptosis induced by high dose of LXA4 As shown in Fig. 1A, vehicle (1% ethanol in 5% FCS)-treated NRK-49F cells present the normal morphologic features of viable fibroblasts with shuttle or stellate appearance and green color stained by acridine orange. In Fig. 1B, NRK-49F cells showed morphologic features of apoptosis including condensed cytoplasm and pyknotic, shrunk nuclei, loss of microvilli around the cells following treatment with high dose of LXA4 (1 mM) for 24 h. The cells dyed yellow were in early stage of apoptosis and those dyed red by ethidium bromide were in later stage of apoptosis. Apoptosis was quantified by flow cytometer of permeabilized, propidium iodide and annexin stained fibroblasts. Flow cytometric analysis of DNA content demonstrated a distinct A0 peak among the exposed to high dose of LXA4 (Fig. 2). The subdiploid peak termed A0 consisted of cells with fragmented nuclei undergoing apoptosis. A0 cells were negligible in control fibroblasts grown in 5% FCS and in cells treated with low dose of LXA4 (10 nM). However, LXA4 at the concentrations of 100 nM or 1 mM induced 9.83% or 33.82% apoptosis of NRK-49F cells, respectively, reduced the cells of S and G2–M phase and increased the cells of G 0 2G 1 phase in a dosedependent manner (Fig. 2E). Effects of calpain 10 or Smac antisense oligodeoxynucleotide or calcium channel

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Fig. 1. LXA4-induced apoptosis of NRK-49F cells recognized by double staining using fluorescent dyes acridine orange and ethidium bromide, observed under laser scanning confocal microscopy. In A, vehicle-treated cells in 5% fetal calf serum (FCS) showed normal morphologic features of fibroblasts. In B, cells showed morphologic features of apoptosis including condensed cytoplasm and pyknotic nuclei, loss of microvilli around the cells following treatment with 1 mM LXA4 for 24 h in 5% FCS. The cells dyed yellow were in early stage of apoptosis and those dyed red by ethidium bromide were in later stage of apoptosis (  400).

inhibitor SK&F96365 on LXA4-induced apoptosis are shown in Fig. 3. Transfection of calpain 10 or Smac antisense oligodeoxynucleotide into the cells downregulated LXA4-induced apoptosis of the cells both in a dose-dependent manner. These effects were specific since transfection of calpain 10 or Smac sense or mismatch oligodeoxynucleotide failed to affect the LXA4 stimulated apoptosis. Pretreatment of the cells with SK&F96365 also inhibited LXA4-induced apoptosis of the cells in a dose-dependent manner. However, SK&F96365 alone did not induce apoptosis of the cells. Transfection of ALX antisense oligodeoxynucleotide into the cells partially but significantly reduced the LXA4-induced apoptosis.

[Ca2+]i from 0 to 10 min, the increment peaked at 3 min. Thus, NRK-49 cells were exposed to LXA4 for 3 min for measurement of [Ca2+]i. The [Ca2+]i of the cells was increased by stimulation with LXA4 at the dose of 100 nM or 1 mM. Pretreatment of the cells with calcium channel inhibitor SK&F96365 inhibited the LXA4induced increment of [Ca2+]i in a dose-dependent manner. 3.4. RT-PCR analysis of calpain 10 and ALX

As shown in Fig. 4, caspase-3 activities of NRK-49F cells were upregulated by LXA4 at the concentrations of 100 nM or 1 mM. However, LXA4 at the dose of l0 nM did not affect the activity of caspase-3 as compared to the vehicle-treated cells. The transfection of calpain 10 antisense oligodeoxynucleotide or Smac antisense oligodeoxynucleotide into the cells inhibited the LXA4 stimulated activity of caspase-3 both in a dose-dependent manner. These effects were specific since transfection of calpain 10 or Smac sense or mismatch oligodeoxynucleotide failed to affect the LXA4 stimulated activity of caspase-3.

ALX on human fibroblasts was documented by previous investigation [32]. In the current study, the ALX on NRK-49F cells was demonstrated by RT-PCR (data not shown) similar to the reported by Chiang N et al. [30]. As shown in Fig. 6A, treatment of the cells with LXA4 for 24 h increased the expression of calpain 10 in a dose-dependent manner. Fig. 6B demonstrated the inhibitory effects of transfection of cells with calpain 10 antisense oligodeoxynucleotide on LXA4 stimulated expression of calpain 10. These effects were specific as transfection of calpain 10 sense or mismatch oligodeoxynucleotide into the cells failed to affect the LXA4 stimulated expression of calpain 10. The effects of calcium channel inhibitor SK&F96365 on LXA4-induced expression of calpain 10 were shown in Fig. 6C. The enhanced expressions of calpain 10 triggered by LXA4 (1 mM) were downregulated by SK&F96365 in a dose-dependent manner.

3.3. Changes of [Ca2+]i levels

3.5. Western blotting analysis of Smac protein

Levels of free cytosolic calcium [Ca2+]i of NRK-49F cells is shown in Fig. 5. In pilot experiment, we performed time course of LXA4-induced changes of

Effects of LXA4 on expression of Smac in NRK-49F cells were shown in Fig. 7A. Smac expression of NRK-49F cells was upregulated by LXA4 at the

3.2. Changes of caspase-3 activity

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Fig. 2. Quantitative analysis of apoptosis by flow cytometry using propidium iodide and annexin staining. All NRK-49F cells were incubated in 5% FCS. In A, cells served as control, were treated with vehicle only. In B, C and D, cells were treated with 10, 100 nM, 1 mM LXA4 for 24 h, respectively. LXA4 at high concentration induced apoptosis shown as a subdiploid peak termed A0 peak, reduced the cells of S and G2–M phase and increased the cells of G0 2G 1 phase in a dose-dependent manner. In E, data were mean7SEM of six independent experiments. Po0:05 compared to the cells treated with vehicle alone.

concentrations of 100 nM or 1 mM. However, LXA4 at the dose of l0 nM failed to affect the expression of Smac. As indicated in Fig. 7B, transfection of Smac antisense oligodeoxynucleotide into the cells abolished the LXA4 stimulated expression of Smac. These effects were specific since transfection of Smac sense or mismatch oligodeoxynucleotide failed to affect the LXA4 stimulated expression of Smac. Fig. 7C demonstrated that pretreatment of the cells with calcium channel inhibitor SK&F96365 inhibited the LXA4-induced expression of Smac in a dose-dependent manner.

assessed by labeled probes as shown in Fig. 8. There was high DNA-binding activity of STAT3 of the cells incubated in 5% FCS alone. LXA4 inhibited the activity of STAT3 in a dose-dependent manner. As indicated in Fig. 8, transfection of calpain 10 antisense oligodeoxynucleotide into the cells ameliorated the decrement of activity of STAT3 induced by LXA4. These effects were specific since transfection of calpain 10 sense or mismatch oligodeoxynucleotide failed to affect the LXA4-downregulated activity of STAT3. 3.7. Western blotting analysis of Akt1

3.6. EMSA results of STAT3 Competition assays performed with unlabeled probes demonstrated the specificity of STAT3 measurement by EMSA since 100-fold excess of unlabeled consensus probes completely abolished the activity of STAT3

In preliminary experiment, we performed time course of 5% FCS-induced Akt1 phosphorylation from 0 to 60 min, the phosphorylation peaked at 15 min, similar to the time course of PDGF-induced Akt1 phosphorylation in mesangial cells [10]. Thus, the cells were exposed to

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Apoptotic cells %

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V eh ic le LX LX LX A A 4+ 4+ A li LX cal pa p o s 4 A om in 4+ 1 c e LX al pa 0 a O A in D 4+ N 10 ca lp sO LX ain D N 1 A 0 m 4+ L X Sm O D ac N A 4 + LX S m a O D N A ac 4+ sO S D LX ma cm N A 4+ SK OD N & F9 63 S LX K & 65 A F9 4+ LX AL 636 5 X A a LX 4 +A OD L A X N 4+ A sOD LX N m O D N

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Fig. 3. Quantitative analysis of apoptosis by flow cytometry using propidium iodide and annexin staining. All cells were incubated in 5% FCS. The cells were treated with or without LXA4 (1 mM) for 24 h or transfected with or without oligodeoxynucleotide (ODN) (10 mM), or treated with or without SK&F96365 (50 mM). Data were mean7SEM of six independent experiments. Po0:05 compared to the cells treated with LXA4 alone (lane 2). Abbreviations: aODN: antisense-oligodeoxynucleotide; sODN: sense-oligodeoxynucleotide; mODN: mismatcholigodeoxynucleotide; ALX: lipoxin A4 receptor.

Free cytosolic calcium (nM)

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Fig. 5. Changes of free cytosolic calcium [Ca ]i of NRK-49F cells assessed in fura-2-loaded cells by laser scanning confocal microscopy. All cells were incubated in 5% FCS. Data were mean7SEM of six independent experiments. Po0:05 compared to the cells treated with vehicle alone (lane 1). Po0:05 compared to the cells treated with LXA4 (1 mM) alone (lane 4).

Caspase-3 activity (nM pNA)

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Fig. 4. Caspase-3 activities of NRK-49F cells measured by colorimetric assay. All cells were incubated in 5% FCS. The cells were treated with or without LXA4 (1 mM) for 24 h, or transfected with or without oligodeoxynucleotide (ODN) (10 mM). Data were mean7 SEM of six independent experiments. Po0:05 compared to the cells treated with LXA4 alone (lane 2). Abbreviations: aODN: antisenseoligodeoxynucleotide; sODN: sense-oligodeoxynucleotide; mODN: mismatch-oligodeoxynucleotide.

LXA4 in the media supplemented with 5% FCS for 15 min. The results of Western blotting analysis of phosphorylated Akt1 (Thr308) are shown in Fig. 9. Threonine phosphorylated Akt1 proteins at 308 site stimulated by 5% FCS were significantly reduced by LXA4 in a dose-dependant manner.

Fig. 6. Expression of calpain 10 mRNA assessed by RT-PCR in NRK-49F cells. All cells were incubated in 5% FCS. The cells were treated with or without LXA4 for 24 h. In A, B and C, the lower panel shows RT-PCR products from glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal control for RNA expression. Results shown are representative of four independent experiments. In B, the cells were transfected with or without oligodeoxynucleotide (ODN). Abbreviations: aODN: antisense-ODN; sODN: sense-ODN; mODN: mismatch-ODN.

4. Discussion In the present studies, we report that 9.83–33.82% of NRK-49F cells become apoptotic cells following treatment with high dose of LXA4 (100 nM, 1 mM) for 24 h

(Figs. 1 and 2), as demonstrated by double staining using fluorescent dye acridine orange (Fig. 1). This assay has been extensively validated against biochemical and

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Fig. 7. Expression of Smac protein assessed by Western blotting analysis in NRK-49F cells. All cells were incubated in 5% FCS. The cells were treated with or without LXA4 for 24 h. In A, B and C, the upper panel shows a-tubulin protein served as a loading control. Results shown are representative of four independent experiments. In B, the cells were transfected with or without oligodeoxynucleotide (ODN) (10 mM). Abbreviations: aODN: antisense-ODN; sODN: sense-ODN; mODN: mismatch-ODN.

electron microscopic assays of apoptosis [11,33,34]. The apoptosis of NRK-49F cells induced by LXA4 also was supported by the results of caspase-3 assay (Fig. 4). LXA4 at high dose (10 nM, 1 mM) upregulated the activity of caspase-3, an execution phase of apoptosis. The LXA4 concentrations used in our experiments were higher than the usually used dose at 1 or 10 nM in the experiments of LXA4 actions on mesangial cell proliferation [3,4,35], and the time of treatment with LXA4 in our experiment was longer than the treatment time of 60 min reported in previous study [4]. High dose of LXA4 and long exposure time to LXA4 may contribute to LXA4-induced apoptosis. Transfection of ALX antisense oligodeoxynucleotide into the cells partially reduced the LXA4-induced apoptosis (Fig. 3) suggesting that ALX mediated the action of LXA4. However, LXA4 at high concentrations may activate the aryl hydrocarbon receptor, a ligand-activated transcription factor [36].

We have investigated the signaling pathways involving LXA4-induced apoptosis of NRK-49F cells. As indicated in Fig. 5, LXA4 increased the [Ca2+]i levels of NRK-49 cells. Our results were consistent with previous reports in which it was demonstrated that LXA4 stimulated cytosolic Ca2+ in human bronchial epithelium, human neutrophils, human monocytes and rainbow trout leukocytes [28,37,38]. In rat, mouse and human, LXA4 receptor is G-protein-coupled receptor (GPCR) [3,30]. Activation of LXA4 receptor was associated to increment of [Ca2+]i levels as the LXA4elevated [Ca2+]i was completely abolished following pretreatment of human bronchial epithelium with Gprotein inhibitor pertussis toxin [28]. LXA4 stimulated both external Ca2+ influx and its mobilization from internal stores in monocytes [37]. SK&F96365 used in the present studies is an inhibitor of receptor-operated Ca2+ channel (ROCC) and voltage-gated Ca2+ channel (VGCC) [29]. As shown in Fig. 5, pretreatment of the cells with SK&F96365 inhibited the LXA4-induced increment of [Ca2+]i, suggesting that LXA4 increased [Ca2+]i via GPCR in conjunction with ROCC and/or VGCC. Furthermore, SK&F96365 also reduced the numbers of apoptotic cells (Fig. 3), indicating that [Ca2+]i plays a critical role in LXA4-induced apoptosis of NRK-49F cells. It is well known that cytosolic Ca2+ is an important initiating agent during induction of apoptosis including activation of Ca2+-dependent enzymes such as calpains and endonucleases, and promotion of mitochondrial leakage of cytochrome C, which downregulates IAPs and activates the caspases [20,39]. Calcium-dependent calpains are non-lysosomal cysteine proteases and activate caspases during apoptosis [16]. Calpain 10 is the third ubiquitous calpain in tissues along with m- and m-calpains [19]. In the present studies, our data indicated that increased [Ca2+]i was necessary for activation of calpain 10 as pretreatment of the cells with SK&F96365 downregulated the expression of calpain 10 induced by LXA4 (Fig. 6C). Meanwhile, we demonstrated that calpain 10 plays a pivotal role in LXA4-induced apoptosis of NRK-49F cells. First, expression of calpain 10 was upregulated by LXA4 (Fig. 6A) in parallel to LXA4-induced apoptosis of the cells (Figs. 1 and 2). Furthermore, transfection of calpain 10 antisense oligodeoxynucleotide in to the cells downregulated both expression of calpain 10 (Fig. 6B) and the percentage of apoptotic cells induced by LXA4 (Fig. 3). Finally, transfection of calpain 10 antisense oligodeoxynucleotide also inhibited the activity of caspase-3 (Fig. 4). Calpain is also a STAT3 protease and STAT3 is a substrate for calpain in vivo and in vitro [23]. Enhanced expression of calpains might be associated with downregulation of activity of STAT3. Indeed, as indicated in Fig. 3, LXA4 downregulated the DNA binding activity of STAT3 stimulated by 5% FCS in parallel to the

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Fig. 8. DNA-binding activity of STAT3 assessed by EMSA in NRK-49F cells. All cells were incubated in 5% FCS. The cells were treated with or without LXA4 for 24 h, or transfected with or without oligodeoxynucleotide (ODN) (10 mM). Competition assays were performed with 100-fold excess of unlabeled oligonucleotide probe of STAT3 as shown in lanes 2 and 12. Results shown are representative of four independent experiments. Abbreviations: aODN: antisense-ODN; sODN: sense-ODN; mODN: mismatch- ODN.

Fig. 9. Effect of LXA4 on expression of phosphorylated Akt1 (Thr308) protein assessed by Western blotting analysis in NRK-49F cells. All cells were incubated in 5% FCS. The cells were treated with or without LXA4 for 15 min. The upper panel shows a-tubulin protein served as a loading control. Results shown are representative of four independent experiments.

enhanced expression of calpain 10 (Fig. 6A). In addition, transfection of calpain 10 antisense oligodeoxynucleotide into the cells inhibited the decrement of activity of STAT3 (Fig. 8). These data suggested that downregulation of activity of STAT3, a signaling molecule mediating survival functions [24,26], was involved in apoptosis of NRK-49F cells induced by LXA4. Enhanced cytosolic Ca2+ also promotes mitochondrial leakage of Smac/DIABLO concomitant with leakage of cytochrome C during apoptosis [40]. Smac/ DIABLO promotes cytochrome-C-dependent caspase

activation by eliminating the inhibitory effect of IAPs on caspase [40]. Consistent with above conclusion, we detected that enhanced [Ca2+]i (Fig. 5) and activity of caspase-3 (Fig. 4) were associated with upregulation of Smac proteins induced by LXA4 (Fig. 7A), and pretreatment of the cells with SK&F96365 downregulated the expression of Smac induced by LXA4 (Fig. 7C). In addition, our results demonstrated that Smac also plays a pivotal role in LXA4-induced apoptosis of NRK-49F cells. First, expression of Smac was upregulated by LXA4 in parallel to LXA4-induced apoptosis of the cells (Fig. 7A). Furthermore, transfection of Smac antisense oligodeoxynucleotide into the cells downregulated both expression of Smac (Fig. 7B) and the percentage of apoptotic cells induced by LXA4 (Fig. 3). Finally, transfection of Smac antisense oligodeoxynucleotide also inhibited the activity of caspase-3 (Fig. 4). Caspase proteases could be directly phosphorylated and inhibited by Akt/PKB [9]. Akt acts as a downstream effector of PI-3-kinase to prevent cells from undergoing apoptosis [8]. A previous report suggested that PI-3kinase-Akt pathway promoted survival and inhibited apoptosis of mesangial cells [10]. Since LXA4 blocked LTD4 stimulated PI-3-kinase activity [3] and inhibited PDGF-induced Akt/PKB activation in human mesangial cells [4], it can be speculated that PI-3-kinase-Akt pathway was downregulated in apoptosis of NRK-49F cells induced by high dose of LXA4. Indeed, as shown in Fig. 9, LXA4 inhibited the expression of phosphorylated

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Acknowledgments

LXA4

Ca2+

Support for this study was provided by 135 Medical Emphasis Grant (No. 135-45) from Government of Jiangsu Province, P.R. China.

ALX

References RO CC VG CC

SK&F96365

G protein

PI-3-K

Ca2+

Calpain 10

Mitochondria

Akt/PKB STAT3 Cyt C Smac

IAP

Caspase-3

APOPTOSIS

Fig. 10. Proposed mechanisms for high dose of LXA4-induced apoptosis in rat renal interstitial fibroblasts (NRK-49F cells). LXA4 binds to LXA4 receptor (ALX), a G-protein-coupled receptor (GPCR), inhibits PI-3-K-Akt/PKB activation and ameliorates inhibitory effects of Akt on caspase cascade. LXA4 increased [Ca2+]i via GPCR in conjunction with receptor-operated Ca2+ channel (ROCC) and/or voltage-gated Ca2+ channel (VGCC). Enhanced [Ca2+]i promotes mitochondrial leakage of Smac/DIABLO and cytochrome C (Cyt C). Upregulated Smac promotes caspase activation by eliminating IAP inhibition. Enhanced [Ca2+]i also promotes activity of calpain 10 which activates caspase-3 and downregulates STAT3, an anti-apoptotic signaling molecule in most cases (m, activation, ?, inhibition, y, abolished inhibition).

Akt1 induced by 5% FCS in parallel with LXA4-induced apoptosis of the cells (Figs. 1 and 2). In summary, we investigated the apoptosis of NRK49F cells induced by high dose of LXA4 and the signaling pathways of LXA4. LXA4 at high concentration can induce apoptosis of rat renal interstitial fibroblasts via [Ca2+]i-dependent upregulation of calpain 10 and Smac expression. Proposed mechanisms based on our and previous observations on signaling pathway of LXA4 [3,4,14] are summarized and illustrated in Fig. 10. Our data elaborate on the potential of LXA4 as an agent that is not only anti-inflammatory and anti-proliferative but may also act as an anti-fibrotic and pro-apoptotic agent at high dose. Increment of apoptotic fibroblasts by high dose of LXA4 may provide the basis for the therapeutic strategies in suppression of renal fibrosis [41].

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