The expression of total RCAN1, RCAN1-1 and RCAN1-4 specific transcripts was analyzed by reverse transcription and quantitative real-time PCR of ethanol vehicle or 100 nM Dex treated CEM-C7-14 and CEM-C1-15 cells using exon 5-, 1- and 4-specific forward primers, respectively (Table 1). Representative raw amplification profiles (Figure 1A) and gels corresponding to total RCAN1, RCAN1-1 and RCAN 1.4 (Figure 1B) are shown. To normalize between samples, β-actin was amplified using specific primers shown in Table 1, and used as a reference. Relative expression of each transcript was calculated using a modification of the Pfaffl method 30 (Figure 1C). Total RCAN1 and RCAN1-1 transcript levels were induced 15-and 13-fold in CEM-C1-14 cells, but were not significantly altered in CEM-C1-15 cells after 24 h treatment with Dex (Figure 1C). Using a two sample t-test, the Dex response in CEM-C7-14 cells was significant compared to CEM-C1-15 cells (p < 0.015) for both total RCAN1 and RCAN1-1. Basal expression of RCAN1-4 was minimal in both cell types, and was not affected by Dex treatment (Figure 1).

Various reports suggest that RCAN1 expression is regulated by intracellular calcium ([Ca²⁺]_i) levels 1721. Since GCs upregulate [Ca²⁺]_i levels in CEM cells in correlation with apoptosis 31, we determined whether Dex-dependent upregulation of RCAN1-1 was modulated by calcium signaling. CEM-C7-14 cells were treated for 24 h with either Dex or thapsigargin (TG; inducer of [Ca²⁺]_i levels) in the presence or absence of the calcium ionophore A23187, or the calcineurin inhibitor cyclosporin A (CsA), and expression of transcripts corresponding to total RCAN1, RCAN1-1 and RCAN1-4 was evaluated by RT-QPCR. RCAN1-4 is a minor transcript compared to RCAN1-1; therefore expression profiles of total RCAN1 (Figure 2A) correlated mostly with those of RCAN1-1 (Figure 2B) and any regulation of RCAN1-4 did not influence total RCAN1 profiles. Dex-induced upregulation of total RCAN1 and RCAN1-1 was partially inhibited by A23187 (p < 0.02 and 0.03 respectively), suggesting that Dex-dependent induction of these transcripts was inhibited by increases in [Ca²⁺]_i levels (Figures 2A and 2B). CsA enhanced Dex-evoked induction of total RCAN1, but the effect was not statistically significant. Expression of RCAN1-4 was induced 200 and 18-fold, respectively by 500 nM TG and 150 nM A23187 (Figure 2C), suggesting that RCAN1-4 expression was regulated via the calcium signaling pathway. This was confirmed by the observation that 600 nM CsA blunted TG-dependent induction of RCAN1-4 (Figure 2C) (p < 0.003).

Cells treated with ethanol vehicle or 100 nM Dex for 24 h were analyzed for RCAN1 protein expression by Western blotting using an antibody that recognizes the carboxyl terminal of RCAN1, and therefore detects both RCAN1-1 and RCAN1-4. Accordingly, a fast migrating minor band around 29 kDa representing RCAN1-4, and a 41 kDa band representing RCAN1-1 were detected (Figure 3A). One or two additional bands recognized by the antibody in various experiments were found to be nonspecifically interacting proteins, as demonstrated through the use of a blocking peptide (Abgent, Cat # BP6315c) specific for the antibody (Figure 3B). Preincubation of the antibody with the blocking peptide competed out the two RCAN1 bands, while not affecting the ability of the antibody to recognize the two nonspecific bands. The induction of RCAN1-1 was minimal in CEM-C1-15 cells (Figure 3A).

To determine whether RCAN1-1 was pohsphorylated, cell lysates were treated with lambda phosphatase prior to Western blotting (Figure 3B) to detect either RCAN1 or CREB using appropriate antibodies. CREB shifted to a faster migrating band, demonstrating a loss of phosphate residues, and confirming successful phosphatase treatment of the sample. The migration of RCAN1-1, or the relative abundance of the band was not affected by the phosphatase treatment, suggesting that phosphatase treatment did not alter its phosphorylation state. It is possible that the gel is not capable of resolving small differences in migration of phosphorylated and unphosphorylated RCAN1-1. To test this hypothesis, lysates of ethanol or Dex treated CEM-C7-14 cells were immunoprecipitated with phospho-serine, phospho-threonine or phospho-tyrosine specific antibodies coupled to Agarose beads. None of these antibodies pulled down RCAN1-1(data not shown), supporting the hypothesis that RCAN1-1 was not phosphorylated.

CEM-C7-14 cells were simultaneously treated with various kinase inhibitors in the presence of either ethanol or Dex, and processed for QPCR analysis (Figure 4A) or Western blotting (Figure 4B) to determine whether GC-mediated upregulation of RCAN1-1 transcript or protein was kinase dependent. The concentration for each inhibitor was close to its IC50 value: PD98059 and LY294002 were at 5 μg/ml, SB202190 was at 1 μg/ml and staurosporine was at 40 nM 32. A strong correlation was observed between transcript and protein levels, with the exception of the MEK inhibitor, PD98059, which was shown to enhance Dex-mediated upregulation of RCAN1-1 transcript (p < 0.001) while not significantly affecting RCAN1-1 protein levels. The PI3 kinase inhibitor LY294002 did not significantly alter RCAN1-1 transcript upregulation by Dex, but reduced the intensity of RCAN1-1 protein in Western blotting to about sixty percent of Dex levels, as estimated by densitometric scanning of two independent blots (Figure 4B). The p38 MAP kinase inhibitor SB202190 partially blunted Dex-mediated upregulation of both RCAN1-1 transcript (p < 0.1) and protein (to 60%). Staurosporine significantly curtailed Dex-dependent expression of RCAN1-1 transcript (p < 0.001) and reduced protein expression to basal levels. These data suggest that Dex-dependent upregulation of RCAN1-1 is at least partially p38 MAP kinase dependent. Figure 4C demonstrates the specificity of the inhibitors used in inhibiting the respective kinase. Lysates prepared from CEM-C7-14 cells treated with each kinase inhibitor in the presence of either ethanol or Dex were subjected to Western blotting with an antibody specific for the PI3 kinase product phospho-Akt in Figure 4Ci. The PI3 kinase inhibitor LY294002 partially inhibited Akt phosphorylation, as depicted by the reduced intensity of bands in lysates of cells treated with 5 μg/ml of LY294002. To evaluate the specificity of p38 MAP kinase inhibition by SB202190, lysates were subjected to a p38 MAP Kinase assay using a kit (Cell Signaling Technology, cat # 9820) (Figure 4Cii). p38 MAP kinase from appropriately treated CEM-C7-14 cell lysates was immunoprecipitated with an immobilized phospho-p38 MAP kinase antibody followed by an in vitro kinase assay using the substrate ATF-2. Extent of phospho ATF-2 product was subsequently detected by Western blotting using a specific antibody. Figure 4Cii shows that Dex treatment induces p38 MAP kinase activity, which is blunted in the presence of 1 μg/ml SB202190.

Lysates from CEM-C7-14 cells treated for 24 h with either ethanol vehicle or 100 nM Dex were adsorbed to Protein A-Agarose conjugated to an antibody specific for calcineurin A, the beads were washed with RIPA buffer to remove non-specifically bound proteins, and subjected to Western blot analysis using an antibody specific for RCAN1-1. (Figure 5A). Since Dex treated samples had greater abundance of RCAN1-1, a greater fraction bound to and coimmunoprecipitated with calcineurin.

Interactions between RCAN1 and calcineurin have been shown to inhibit calcineurin activity 19. The abundance of RCAN1-1, and hence the proportion of calcineurin bound to RCAN1 increases in response to GC treatment of CEM-C7-14 cells (Figure 5A). In correlation, calcineurin phosphatase activity, measured by estimating the phosphate released from a phosphopeptide substrate, also decreases in a dose-dependent manner in response to Dex in CEM-C7-14 cells (Figure 5B). Calcineurin activity in cells treated with 1 μM Dex was 36% of that in ethanol treated control CEM-C7-14 cells (p < 0.03). A dose-dependent decrease in calcineurin activity in response to Dex was not apparent in CEM-C1-15 cells, however, a small decrease at 1 μM Dex (67% of control) was observed, but was not found to be statistically significant. These data suggest that RCAN1-1 binding may inactivate calcineurin. Calcineurin has been shown to protect T-cells from GC-evoked apoptosis 9, and promote T cell survival 33, hence loss of calcineurin activity may contribute to GC-evoked apoptosis.

Dexamethasone (Dex), the calcium ionophore A23187, Cyclosporin A (CsA) and thapsigargin (TG) were purchased from EMD Biosciences (Madison, WI). Reagents for reverse transcription (RT) and Real-time QPCR, including M-MLV reverse transcriptase, oligo(dT)₁₅ primer, RNasin^® Ribonuclease inhibitor, dNTP mix, and Taq DNA polymerase were purchased from Promega Life Sciences (Madison, WI). SYBR^® JumpStart™ Taq ReadyMix was from Sigma-Aldrich (St. Louis, MO). A polyclonal rabbit antibody (Cat # AP6315c) directed against the C-terminal region RCAN1 that recognizes both RCAN1-1 and RCAN 1-4 as 41 kDa and 29 kDa bands respectively, was from Abgent (San Diego, CA), phosphor-Akt(Ser473) antibody (Cat # 9271) was from Cell Signaling Technology (Beverly MA), calcineurin A antibody (Cat. #C1956) was from Sigma-Aldrich (St. Louis, MO), CREB antibody (Cat # sc-186), secondary horseradish peroxidase (HRP) conjugated anti-rabbit IgG and anti-mouse IgG were from Santa Cruz Biotechnology (Santa Cruz, CA), the ECL Western Blotting substrate was from Pierce Biotechnology (Rockford, IL). P38 MAP kinase assay kit (cat # 9820) was from Cell Signaling Technology (Beverly MA). Calcineurin Cellular Assay Kit PLUS was from BIOMOL International, Plymouth Meeting, PA. Lambda Phosphatase (Cat. # 14405) was from Upstate Biotechnology, Temecula, CA. Other reagent grade chemicals were purchased from Fisher Scientific (Pittsburgh, PA) or Sigma-Aldrich (St. Louis, MO).

Tissue culture media and components, including fetal bovine serum (FBS) were purchased from Mediatech (Washington D.C.). CEM-C7-14 and CEM-C1-15 cells were kindly provided by Dr. E. B. Thompson (UTMB, Galveston, TX), and are derivatives of the parental line CCRF-CEM, obtained from a patient with acute lymphoblastic leukemia 39. Cells were cultured in RPMI 1640 medium supplemented with 5% FBS at 37°C in a humidified 5% CO₂ incubator, and were maintained in log phase by passaging every 3 days. Cell treatments were for 24 h in RPMI supplemented with 5% FBS. All treatment agents were prepared as 1000× stock solutions in either ethanol or DMSO. A vehicle alone (0.1% ethanol or 0.1% DMSO) control was used in all experiments.

Cells were treated at a density of 4 × 10⁵ cells/ml for 24 h with the appropriate agent, and RNA was extracted from approximately 1 × 10⁷ cells using the TRIzol reagent (Invitrogen Life Technologies, La Jolla, CA). For first-strand DNA synthesis, 5 μg of total RNA was reverse transcribed for 3 h at 42°C in the presence of 0.5 μg of oligo(dT)₁₅, 1 μl (~200 U) of M-MLV reverse transcriptase, 0.5 mM dNTP mix, and 100 U of RNase inhibitor. PCR amplification of transcripts specific for total RCAN1 (RCAN1 All) and RCAN1-4 was accomplished using forward primers corresponding to sequences within exon 5 and exon 4, respectively, and a reverse primer corresponding to exon 7. RCAN1-1 was amplified using forward and reverse primers corresponding to sequences within exon 1 and 5 respectively. Primer sequences are shown in Table 1. Sequences for RCAN1 transcripts were extracted from the GenBank database (Accession #s NM-004414 and NM-203418). For real time QPCR, 1 μl of reverse transcription product was amplified in a Cephid SmartCycler with 125 pmoles of each primer and 12.5 μl of the SYBR^® JumpStart™ Taq ReadyMix (Sigma, cat # S-4438) in a 25 μl reaction under the following conditions: 30 sec at 94°C, 45 sec at 50°C (55°C for RCAN1-4), 1 min at 72°C. In duplicate reactions PCR was terminated during the exponential phase and PCR products were resolved on a 1% Agarose gel in Tris-Borate-EDTA buffer, as per conventional procedures. To normalize between samples, β-actin was used as a control (primers are shown in Table 1). To quantitate relative expression levels, the crossing point (CP; or cycle threshold Ct) values for each sample were used to calculate fold inductions using the Pfaffl method formula30: (E_target)^ΔCPtarget/(E_ref)^ΔCPref where ΔCPtarget = (CP_ethanol-CP_sample) for RCAN1 and ΔCPref = (CP_ethanol-CP_sample) for β-actin, and the software LinRegPCR.

Cells plated at a density of 4 × 10⁵ cells/ml were treated for 24 h with the appropriate agents, and approx. 8 × 10⁶ cells were harvested, washed and lysed in buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, plus a protease and phosphatase inhibitor cocktail. The amount of protein in each sample was estimated using the Bradford assay, and 50 μg of each sample was boiled in SDS-PAGE sample buffer (final composition: 120 mM Tris, 4% SDS, 20% glycerol, 5% 2-mercaptoethanol, 0.05% bromophenol blue, pH 6.8) Samples were resolved on a 10% polyacrylamide-SDS gel, and electoblotted on to PVDF membranes. Membranes were blocked in 10% non-fat dry milk and incubated sequentially with a RCAN1-specific polyclonal antibody, AP6315c, and a HRP-coupled anti-rabbit secondary antibody. Membranes were developed using an Enhanced Chemiluminescence (ECL) kit from Pierce. To detect the phosphorylation state of RCAN1-1, cell lysates were treated with 400 U of lambda phophatase for 20 min prior to loading the gel.

Appropriately treated CEM-C7-14 cells were washed with phosphate buffered saline (PBS, pH 7.4) and lysed with RIPA buffer (final concentration: 150 mM NaCl, 50 mM Tris pH 8.0, 1% NP-40). Calcineurin antibody was added in Protein G-Agarose (50% suspension) and incubated overnight at 4°C on a rotor. Protein G-Agarose was washed three times with RIPA buffer and cell lysate corresponding to 500 μg of proteins were added and incubated overnight at 4°C on a rotor. Protein G-Agarose was washed three times with RIPA buffer before 15.0 μL of 2× SDS PAGE reagent was added.

The p38 MAP kinase assay kit from Cell Signaling Technology was used to estimate the specificity of kinase inhibitors in inhibiting MAP kinase activity. Immobilized phospho-p38 MAP kinase antibody was used to pull down p38MAP kinase from 500 ug of appropriately treated CEM-C7-14 cell lysates. The Agarose beads were washed and resuspended in 25 μl kinase buffer supplemented with 200 μM ATP and 20 μg/mL of the p38 MAP kinase substrate, ATF-2 fusion protein. After incubation at 30°C for 1 h, the reaction was stopped by addition of 5× SDS-PAGE sample buffer. Western blotting was performed using ATF-2 specific antibody provided in the kit.

The Calcineurin cellular assay kit Plus (AK-816) from Biomol International (Plymouth Meeting, PA) was used for measurement of calcineurin activity in CEM cells in response to 0 to 1 μM Dex. Cells seeded at a density of 5 × 10⁵/ml were treated with Dex for 24 h were lysed in the manufacturer's lysis buffer containing protease inhibitors. Cell lysates were passed through size-exclusion micro Bio-Spin 30 columns (Bio-Rad, Hercules, CA) to remove free phosphate, and were incubated with RII phosphopeptide, a known substrate for calcineurin. The phosphate released was measured by a Malchite Green assay. As per guidelines provided in the kit, PP1 and PP2A activity was inhibited by okadaic acid, while EGTA and okadaic acid both were used to measure PP2C activity. Phosphate released from these two conditions was subtracted from total phosphate released in the absence of any inhibitor, to reveal PP3C activity.