Goat polyclonal antibody to swiprosin-1 was from Imgenex (San Diego, CA). Antibodies to protein kinase C (PKC)-α, PKC-βI, PKC-η, PKC-ζ, actin, and I-κBα were from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). Antibodies to PKC-δ, and PKC-θ were from Cell Signaling Technology, Inc (Beverly, MA). Antibody to human CD3 (OKT3) was purified from hybridomas ATCC CRL-8001. Anti-human CD28 antibody was purchased from R&D Systems, Inc. (Minneapolis, MN). HRP-conjugated anti-goat, anti-rabbit, and anti-mouse IgGs were from GE Healthcare (Chalfont St. Giles, United Kingdom). Phorbol 12-myristate 13-acetate (PMA), A23187, phytohemagglutinin A (PHA), BAPTA-AM, ionomycin, SB203580, PD098059, SP600125, caffeic acid phenethyl ester (CAPE), and cyclosporine A (CsA) were purchased from Sigma Chemical Co (St. Louis, MO). Gö6983, Gö6976, rottlerin, and staurosporine were purchased from Calbiochem-Behring (La Jolla, CA). Total RNA isolation reagent was from WelPrep™ Join Bio Innovation (Daegu, South Korea). Maxime RT Premix (oligo dT primer), Maxime PCR PreMix, and a plasmid purification kit were from iNtRON Biotechnology (Daejon, South Korea). SYBR premix Ex Taq was from Takara Bio Inc (Shiga, Japan). The dual-luciferase reporter assay system was from Promega Corporation (Madison, WI). Small interfering RNA (siRNA) targeting PKC isotypes and a scrambled siRNA were obtained as a pool of four or more siRNA duplexes from Dharmacon (Chicago, IL).

Jurkat T cells (ATCC TIB-152, Manassas, VA) were maintained in RPMI 1640 medium (GIBCO, Gaitherburg, MD) supplemented with 10% (v/v) FBS (GIBCO, Invitrogen). Exponentially growing cells were seeded at 0.5-2×10⁶ per six-well plate, and used for various experimental purposes. After written informed consent, human primary PBLs were isolated from healthy donors by dextran sedimentation and centrifugation through a discontinuous Ficoll gradient (Amersham Biosciences, Piscataway, NJ). The cell lines and human PBLs mentioned above were cultured at 37℃ in a humidified incubator containing 5% CO₂ and 95% air. All experiments using human PBLs were approved by Ethics Committee of the School of Life Sciences, GIST.

Jurkat T cells (1.5×10⁶) or human primary PBLs were stimulated with either plate-bound anti-CD3 (OKT3 for human, 10 µg/ml)/CD28 (2 µg/ml), phytohemagglutinin A (PHA) and/or PMA (200 nM)/A23187 (1 µM). In some case, the cells were pretreated for 30 min with the various reagents that modulate intracellular signalings.

Cells from the in vitro cultures were harvested and total RNA was isolated using the WelPrep™ JBI method (iNtRON Biotechnology, Daejon, Korea) according to the manufacturer's instructions. Reverse transcription of the RNA was performed using oligo dT primer Maxime RT-PCR PreMix (iNtRON Biotechnology, Daejon, Korea). Two micrograms of RNA was transferred to an oligo dT primer mixture tube. The reaction volume was 20 µl. cDNA synthesis was performed at 45℃ for 60 min, followed by RT inactivation at 95℃ for 5 min. Thereafter, the RT-generated DNA was diluted to 40 µl volume with distilled water. The diluted RT-generated DNA (2 µl) was amplified using Maxime PCR PreMix (iNtRON Biotechnology, Daejon, Korea). The primers used for cDNA amplification were as follows: Swip-1, sense 5'-ATCTTCCGCAAGGCGGCGGCCGGGGAG-3' and antisense 5'-GACTGCAGCTCCTTGAAGGCCGCTTTC-3'; hIL-2, 5'-CACGTCTTGCACTTGTCAC-3' and antisense 5'-CCTTCTTGGGCATGTAAAACT-3'; hIL-3, sense 5'-CTTTGCCTTTGCTGGACTTC-3' and antisense 5'-CGAGGCTCAAAGTCGTCTG-3'; GAPDH, sense 5'-CGGAGTCAACGGATTTGGTCGTAT-3' and antisense 5'-AGCCTTCTCCATGGTGGTGAAGAC-3'. Amplification conditions were denaturation at 94℃ for 30 s, annealing at 58~68℃ for 20 s, and extension at 72℃ for 40 s for 30~35 cycles. The PCR products were resolved and visualized on a 1 or 1.5% agarose gel and stained with ethidium bromide.

In all the experiments, the expression levels of the examined genes were evaluated by real-time RT-PCR, unless otherwise indicated. PCR amplification was performed in DNA Engine Opticon for a continuous fluorescence detection system (MJ Research, Waltham, MA) in a total volume of 20 µl containing 2 µl of cDNA/control and gene specific primers using the SYBR premix Ex Taq kit (Takara, Shiga, Japan). The PCR was performed under the following conditions: 94℃ for 30 s, 58 ~68℃ for 30 s, 72℃ for 30 s, plate read (detection of fluorescent product) for 40 cycles, followed by 7 min of extension at 72℃. A melting curve analysis was done to characterize the dsDNA product by slowly raising the temperature (0.2℃/s) from 65℃ to 95℃ with fluorescence data collected at 0.2℃ intervals. The levels of expression (of swiprosin-1, IL-3, and IL-8) that were normalized by GAPDH were expressed as a relative value (i.e., percentage) of the maximum. The result of the maximum level in each experiment was considered as 100%.

For the analysis of swiprosin-1, and PKC isoforms (including PKC-α PKC-βI, PKC-θ, PKC-δ, PKC-η, and PKC-ζ. Jurkat T cells were rinsed twice with ice-cold PBS and then lysed in ice-cold lysis buffer (10 mM Tris-HCl, pH 7.4, containing 50 mM NaCl, 1% Triton X-100, and a protease inhibitor cocktail tablet). Cell lysates were centrifuged at 14,000 rpm for 20 min at 4℃ and equal amounts of protein supernatant were mixed with a one-fourth volume of 4X SDS sample buffer, boiled for 5 min, and then separated through an 8 or 10% sodium dodecyl sulfate-polyacrylamide gel. After electrophoresis, proteins were transferred to a nitrocellulose membrane by means of the Trans-Blot SD semidry transfer cell (Bio-Rad, Hercules, CA). The membrane was blocked in 5% skim milk (1 h), rinsed, and incubated overnight at 4℃ with primary antibodies in TBS containing 0.1% Tween 20 (TBS-T) and 3% skim milk. Excess primary Ab was then removed by washing the membrane three times in TBS-T, and the membrane was incubated with 0.1 µg/ml horseradish peroxidase-labeled secondary Ab (against goat, rabbit, or mouse) for 2 h. Following three washes in TBS-T, bands were visualized by ECL western blotting detection reagents and exposed to x-ray film.

Jurkat T cells (1.5×10⁶) were transfected with 100 µl of Amaxa's Nucleofector solution (Amaxa, Cologne, Germany) containing 1 µg of swiprosin-1/pEGFP-C1 or pEGFP-C1 with 1 µg of pRL-TK renilla and 1 µg of pGL3/NF-κB or pGL3/NF-AT, and then the cells were immediately transferred to 2.0 ml of complete medium and cultured in 6-well plates at 37℃. After 24 h of transfection, the medium was replaced with IMDM medium containing 10% FBS and antibiotics. Cells were treated with reagents as described above and incubated at 37℃ for 12 h. Cell lysates were prepared and assayed for luciferase activity using the Dual Luciferase Assay System (Promega, Madison, WI), according to the manufacturer's instructions.

The mean values were calculated from data taken from at least three (usually four or more) separate experiments conducted on separate days. Where significance testing was performed, an independent t test (Student's; two populations) was used. p-value of less than 0.05 was considered an indicator of statistical significance.

We first examined whether the level of swiprosin-1 is regulated in human T cells. To this end, Jurkat T cells were stimulated with anti-CD3/28 antibodies, and then the expression of swiprosin-1 and IL-2 was determined by RT-PCR. As shown in Fig. 1A, swiprosin-1 mRNA was significantly over-induced in Jurkat T cells and the maximum level was reached at the 6 h of stimulation. As a control, IL-2 expression was assessed (Fig. 1A). The up-regulation of swiprosin-1 expression was also achieved by treatment of cells with other stimuli such as PMA, PHA, PMA+A23187, and PMA+PHA+A23187 (Fig. 1B). Interestingly, however, we found that the up-regulation of swiprosin-1 is independent from the activation status of T cells as single PMA treatment significantly up-regulated swiprosin-1 mRNA expression while it did not induce cytokine expression including IL-2 and IL-3 (Fig. 1B). On the contrary, full activation of T cells by PMA+A23187 or PMA+PHA+A23187 did not significantly induce swiprosin-1 expression (Fig. 1B). Western blot analysis also revealed that single PMA treatment up-regulates the levels of swiprosin-1 protein in a time-dependent manner (Fig. 1C), suggesting that PKC may involve in the pathway of swiprosin-1 expression in T cells.

Interestingly, we found that single treatment with calcium ionophore A23187 had no effect on the expression of swiprosin-1 (Fig. 2A) in Jurkat T cells. This result led us to next examine whether PMA-induced swiprosin-1 expression is modulated by calcium signals. To this end, Jurkat T cells were pretreated with calcium modulators such as BAPTA (calcium blocker), A23187 (calcium ionophore), and ionomycin (calcium ionophore). Interestingly, the expression of swiprosin-1 was significantly reduced by treatment with A23187 or ionomycin but not by BAPTA, suggesting that calcium signal acts as a negative signal for swiprosin-1 expression in T cells (Fig. 2A, left). The effect of A23187 was also confirmed by Western blot analysis (Fig. 2A, right). We also tested the involvement of other signaling modules such as MAP kinase pathway. However, the expression of swiprosin-1 was not changed by treatment with any of three MAP kinase inhibitors (SB203580, p38 kinase inhibitor; PD098059, ERK inhibitor; SP600125, JNK inhibitor) (Fig. 2B).

As PKCs are primary targets for PMA, we next examined which PKC-isoforms could be responsible for swiprosin-1 expression in Jurkat T cells. Initially, we used pharmacologic agents that can modulate specific PKC isoforms. To this end, Jurkat T cells were pretreated with various concentrations of diverse PKC inhibitors, and then the cells were stimulated with PMA. As shown in Fig. 2C, Gö6976 (inhibitor of PKCα and PKCβI) and rottlerin (inhibitor of PKCδ, and PKCι) revealed no significant effects on PMA-induced swiprosin-1 expression. Gö6983 (inhibitor of PKCs α, βI, βII, δ and ζ) showed medium blocking effect. In contrast, staurosporine, a non-specific PKC inhibitor, significantly blocked PMA-induced swiprosin-1 expression (Fig. 2C). The data to this point demonstrated a requirement for PKC(s) in PMA-induced swiprosin-1 expression, but did not establish which PKC isoform (s) is (are) specifically involved in this process. We therefore used siRNAs to knockdown target PKC isoforms. Western blot analysis revealed that Jurkat T cells express PKCα, β, η, ζ, θ and δ forms (Fig. 3A) and that transfection of siRNA significantly down-regulated each target PKC in a time-dependent manner, showing maximal inhibition at 72 h of transfection (Fig. 3B). Among the six PKC isoforms examined, targeted knockdown of PKC-θ significantly inhibited PMA-induced swiprosin-1 expression (Fig. 3C), suggesting that PKC-θ plays a major function in regulating swiprosin-1 expression. Knockdown of PKC-θ also significantly reduced anti-CD3/28-induced swiprosin-1 up-regulation in both Jurkat T cells (Fig. 4A) and human peripheral blood lymphocytes (Fig. 4B).

Since a central event in T cell activation is the pathway involving the nuclear factors (13), we therefore tested whether swiprosin-1 induction is regulated by the transcriptional activities of the NF-κB and/or NF-AT. Both NF-κB and NF-AT promoter activities were highly induced in response to the PMA/A23187 (Fig. 5A). PMA significantly induced NF-κB promoter activity while it has no effect on NF-AT (Fig. 5A). In contrast, A23187 induced only NF-AT activity (Fig. 5A), suggesting that NF-AT promoter activity is dependent on the calcium signaling. Interestingly, the up-regulation of swiprosin-1 expression was significantly blocked by the NF-κB inhibitor CAPE (Fig. 5B). The effect of CAPE was also mimicked by the proteasome inhibitor MG132, a known indirect inhibitor of NF-κB (Fig. 5B). However, swiprosin-1 expression was not blocked by the NF-AT inhibitor CsA. These data further suggest that NF-κB signaling is critical for the swiprosin-1 induction in T cells.