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Originally published In Press as doi:10.1074/jbc.M611522200 on March 8, 2007

J. Biol. Chem., Vol. 282, Issue 18, 13351-13362, May 4, 2007
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Cyclosporin A and FK506 Inhibit IL-12p40 Production through the Calmodulin/Calmodulin-dependent Protein Kinase-activated Phosphoinositide 3-Kinase in Lipopolysaccharide-stimulated Human Monocytic Cells*

Wei Ma{ddagger}12, Sasmita Mishra{ddagger}13, Katrina Gee§4, Jyoti P. Mishra{ddagger}3, Devki Nandan||, Neil E. Reiner||, Jonathan B. Angel{ddagger}§5, and Ashok Kumar{ddagger}**6

From the Departments of **Pathology and Laboratory Medicine, and {ddagger}Biochemistry, Microbiology and Immunology, Division of Virology and Molecular Immunology, Research Institute, Children's Hospital of Eastern Ontario, §Ottawa Health Research Institute and the Division of Infectious Diseases, Ottawa Hospital General Campus, University of Ottawa, Ottawa, Ontario K1H 8L1, Canada and the ||Division of Infectious Diseases, Departments of Medicine, and Microbiology and Immunology, University of British Columbia, Faculties of Medicine and Science, and Vancouver Coastal Health Research Institute, Vancouver, V5Z 3J5 British Columbia, Canada

Received for publication, December 15, 2006 , and in revised form, February 26, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cyclosporine-A (CyA) and FK506 are potent immunosuppressive agents because of their ability to suppress the production of Th1 cytokines including interleukin (IL)-12. However, the mechanisms underlying the inhibitory effects of CyA and FK506 on the production of IL-12p40, a critical component of IL-12, remain unknown. Both CyA and FK506 are potent inhibitors of calcineurin in the calcium signaling pathway. Interestingly, calcium and phosphoinositide 3-kinase (PI3K) signaling pathways have been shown to negatively regulate lipopolysaccharide (LPS)-induced murine IL-12p40 production. Contrary to these observations, we show that LPS-induced IL-12p40 production in human monocytic cells is positively regulated by the calcium pathway and in particular by calmodulin-(CaM) and CaM-dependent protein kinase-II (CaMK-II)-activated PI3K. Furthermore, LPS-induced IL-12p40 production was regulated by the p110{alpha} catalytic subunit of PI3K. Moreover, LPS induced IL-12p40 production through the CaM/CaMK-II-activated NF{kappa}B and AP-1 transcription factors. LPS-induced IL-12p40 production is known to be regulated by the c-Jun N-terminal kinase (JNK) pathway. Importantly, both CyA and FK506 down-regulated LPS-induced IL-12p40 transcription by inhibiting CaM/CaMK-II-activated PI3K and their downstream transcription factors NF{kappa}B and AP-1 independent of the JNK pathway.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cyclosporine (CyA)7 and FK506 (Tacrolimus) are immunosuppressive agents and have been widely used in a variety of disease conditions such as clinical post-organ transplantation and autoimmune diseases (1, 2). CyA is a cyclic polypeptide produced as a fungal metabolite and consists of 11 amino acids, whereas FK506 is a macrolide lactone (2). CyA and FK506 bind to cyclophilin A and the 12-kDa FK506-binding protein, FKBP12, respectively, and suppress activation of NFAT in T cells (3, 4). CyA and FK506 are potent inhibitors of Ca2+-dependent T cell activation, and the CyA-cyclophilin A and FK506-FKBP12 complexes bind to the same target, calcineurin (3–5), thereby inhibiting the T cell antigen receptor-mediated signal transduction pathway. FK506 and CyA modulate the biological functions of T cells, B cells, macrophages, and dendritic cells (DCs) (5–9) and have been shown to inhibit Th1 responses by interfering with cytokine production including IL-12 by murine DCs (1, 6–9).

IL-12 is produced by monocytic cells, DCs, B cells, and other accessory cells and plays a key role in the development of Th1 responses (10–14). It is a potent inducer of interferon-{gamma} either alone or in synergy with other inducers and causes proliferation of T and NK cells and lymphokine-activated killer cells (11–14). IL-12 is a heterodimeric 70-kDa protein composed of a disulfide linked 40-kDa and 35-kDa subunits (15, 16). Although the IL-12p40 subunit is secreted in excess over IL-12p70, only the heterodimer is biologically active. Many cell types, including those that are not known to produce IL-12, express mRNA-encoding IL-12p35. However, mRNA-encoding IL-12p40 seems to be restricted to cells that produce the biologically active IL-12 (15, 16). Therefore, IL-12p40 expression is generally considered as an indicator of IL-12p70 production. Moreover, the IL-12p40 subunit is also shared by another Th1 cytokine, IL-23, which makes it a highly significant cytokine for determination of Th1 type responses (17).

There is relatively little information on the role of intracellular signaling molecules that regulate IL-12p40 synthesis in human monocytic cells following LPS stimulation. LPS-induced cell signaling involves activation of tyrosine and serine/threonine protein kinases including PKC and the mitogen-activated protein kinases (18–21). We and others (20, 22) have previously demonstrated the involvement of c-Jun-N-terminal kinase (JNK) in IL-12p40 production in LPS-stimulated human monocytic cells. Multiple transcription factors including interferon-{gamma} regulatory factors, NF{kappa}B, Ets-2, AP-1, C/EBP, and PU.1 transcription factors, and their complexes have been suggested to regulate IL-12p40 transcription in LPS-stimulated murine and human monocytic cells (20, 23–28).

The molecular mechanism by which CyA and FK506 inhibit IL-12p40 production in monocytic cells remains unknown. Since both FK506 and CyA are potent inhibitors of calcineurin (3, 4), we hypothesized that these agents may inhibit IL-12p40 production through interference with calcium signaling. However, several studies have reported the negative regulatory role of calcium and the phosphoinositide 3-kinase (PI3K) signaling in LPS-induced IL-12p40 production in murine macrophages and DCs (29–36). Surprisingly, and in contrast to the regulation of murine IL-12p40 production, our results suggest that LPS-induced IL-12p40 production is positively regulated by the PI3K as well as the calcium signaling pathway, in particular calmodulin (CaM) and the calmodulin-dependent protein kinase-II (CaMK-II). Furthermore, both CyA and FK506 inhibited LPS-induced IL-12p40 production by inhibiting the activation of CaM and CaMK-II and their downstream NF{kappa}B and AP-1 transcription factors without affecting the JNK pathway.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation of Monocytes from PBMCs, Cell Lines, Cell Culture, and Reagents—Monocytes from PBMCs of healthy blood donors were isolated as described previously (20). Briefly, PBMCs were isolated by density gradient centrifugation over Ficoll-Hypaque (Amersham Biosciences). Monocytes were isolated by negative selection by depletion of T and B cells using magnetic polystyrene Dynabeads coated with Abs specific for CD2 (T cells) and CD19 (B cells) (Dynal Biotech, Oslo, Norway). The monocytes obtained contained <1% CD2+ T cells and CD19+ B cells as determined by flow cytometric analysis. The human promonocytic cell line, THP-1, was obtained from the American Type Culture Collection (Manassas, VA). Cells were cultured in IMDM (Sigma) supplemented with 10% FBS (Invitrogen), 100 units/ml penicillin, 100 µg/ml gentamicin, 10 mM HEPES, and 2 mM glutamine.

IL-12p40 ELISA—IL-12p40 was measured by using two different mAbs that recognize distinct epitopes as described previously (20). Briefly, IL-12p40 was detected by using a primary anti-IL-12p40 mAb and a second biotinylated anti-IL-12p40 mAb (4 µg/ml and 350 ng/ml, respectively, R&D Systems). Streptavidin-peroxidase (Jackson ImmunoResearch Laboratories, West Grove, PA) was used at a final dilution of 1/1000. rIL-12p40 (R&D Systems) was used as a standard. The sensitivity of the ELISA for IL-12p40 was 16 pg/ml.

RNA Isolation and RT-PCR—Total RNA was extracted as described using a monophasic solution containing guanidine thiocyanate and phenol (Tri Reagent solution; Molecular Research Center, Cincinnati, OH) (20). RNA (1 µg) was reverse transcribed by using Moloney murine leukemia virus reverse transcriptase (PerkinElmer Life Sciences). Equal aliquots (5 µl) of cDNA equivalent to 100 ng of RNA were subsequently amplified for IL-12p40 (373 bp) and beta-actin (663 bp) using their respective primers as described (20). Real-time RT-PCR was performed with Universal PCR Master Mix using beta-actin and IL-12p40 primers (Applied Biosystems). Data were analyzed on 7500 System SDS software, version 1.3 (Applied Biosystems).

Calcium Influx—Changes in concentrations of intracellular free calcium were measured by flow cytometry as described previously (37). Cells were pretreated with FK506 and CyA for 2 h followed by washing with Ca2+-free phosphate-buffered saline and were resuspended in buffer A containing 1 mM Fluo-3/AM (Molecular Probes). The reaction was stopped by adding an equal volume of buffer B (buffer A containing 5% FBS, pH 7.4). After washing, cells were analyzed for Ca2+ levels in the presence and the absence of LPS by a FACScan flow cytometer (BD Biosciences) equipped with CellQuest software, version 3.2.1 fl. Ca2+ ionophore A23187 [GenBank] (20 mM) and 5 mM EGTA (Sigma) were used as controls.

Western Blot Analysis—Briefly, phosphorylation of Akt, p85 PI3K, p110{alpha} PI3K, JNK, CaMK-II, and NFAT were determined using anti-phospho-Akt (New England Biolabs), anti-phospho-p85, anti-phospho-p110 PI3K, anti-phospho-JNK, anti-phospho-CaMKII, or anti-NFAT (Cell Signaling) antibodies, respectively. To control for protein loading, the membranes were stripped and reprobed with antibodies directed against their respective unphosphorylated proteins (Cell Signaling). The immunoblots were visualized by ECL as described (20).

Plasmid Constructs—A series of deletion of hIL-12p40 promoter fragments –880 to +108 were generated by PCR and subcloned into the NheI/NcoI site of the pGL3B luciferase reporter plasmid as described previously (20). To generate mutations in AP-1 nd NF{kappa}B binding sites on IL-12p40 promoter (–232 to –116), site-directed mutagenesis was performed by PCR using mutagenic primers (20). All sequences were confirmed by the Biotechnology Research Institute, University of Ottawa.

Transient Transfection—DNA transfection was performed by FuGENE 6 reagent (Roche Diagnostics) as described previously (20, 38). Briefly, 1 µg of the test plasmid and 0.5 µg of the pSV-beta-galactosidase (Promega, Madison, WI) were incubated for 5 min in 50 µl of IMDM. The mixture was then incubated with 3 µl of FuGENe 6 in 50 µl of IMDM to allow formation of DNA-liposome complexes. These complexes were added to the cell suspension in each well. After 24 h, cells were stimulated with LPS for another 24 h followed by analysis of luciferase and beta-galactosidase activity by using luciferase and beta-galactosidase assay kits (Promega). Luciferase activities were normalized by measuring beta-galactosidase value (20).


Figure 1
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  		FIGURE 1.
FK506 and CyA inhibit LPS-induced IL-12p40 expression in human monocytes and THP-1 cells. A, monocytes and THP-1 cells (1 x 106) were treated with 1 µM FK506 and 2 µM CyA for 2 h and stimulated with 1 µg/ml LPS for 0–12 h for analysis of IL-12p40 mRNA expression by semiquantitative RT-PCR. The results shown are from one of three independent experiments with similar results. B, monocytes and THP-1 cells pretreated with CyA and FK506 at various concentrations for 2 h were stimulated with LPS for 24 h followed by analysis of IL-12p40 production by ELISA. The results shown are a mean ± S.D. of three experiments.

 
Transfection of p85 PI3K siRNA—Cells (106) were transfected with siRNA SMARTpool PI3K p85{alpha} and nonspecific control pool (siRNA control) using DharmaFECTTM 2 transfection reagent as per the manufacturer's instructions (Dharmacon). Following transfection, cells were stimulated with LPS (1 µg/ml) for 8 h for real-time PCR and for 30 min for Western blotting for p85 PI3K. The supernatants were collected after 24 h of stimulation for IL-12p40 ELISA.

Electrophoretic Mobility Shift Assay—Briefly, cells (107) were harvested in Tris-EDTA-saline buffer, pH 7.8, and lysed for 10 min at 4 °C with buffer A containing 0.1% Non-idet P-40. The pellets containing the nuclei were suspended in buffer B at 4 °C for 15 min. Both buffers A and B contained the proteolytic inhibitors and have been described (20). Nuclear proteins (5 µg) were mixed for 20 min at room temperature with either 32P-labeled AP-1 or NF{kappa}B oligonucleotide probes (20), and the complexes were subjected to non-denaturing 5% PAGE. To illustrate specificity of binding for NF{kappa}B and AP-1 probes, the wild type and mutant oligonucleotide sequences corresponding to the NF{kappa}B and AP-1 binding sites in the IL-12p40 promoter were used as cold competitors as described earlier (20). The gel was dried and exposed to x-ray film (Eastman Kodak Co.).

Statistical Analysis—Means were compared by two-tailed Student's t test. The results are expressed as mean ± S.E.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
FK506 and CyA Inhibit LPS-induced IL-12p40 Production by Inhibiting a Calcium-regulated Pathway in Human Monocytic Cells—LPS stimulation of primary human monocytes and THP-1 cells induced maximum expression of IL-12p40 at 6 h post-stimulation. Treatment of cells with either CyA or FK506 for 2 h prior to stimulation with LPS significantly inhibited IL-12p40 expression as determined by semiquantitative RT-PCR (Fig. 1A) and ELISA (Fig. 1B) in both primary monocytes and THP-1 cells. Since FK506 and CyA exert their biological effects by inhibiting the calcium-dependent signaling pathway (3, 4), we determined the role of calcium signaling in LPS-induced IL-12p40 production. Stimulation of monocytes and THP-1 cells with LPS increased the levels of intracellular calcium at 8 and 12 min, respectively, that was inhibited following treatment of cells with EGTA (Fig. 2A). FK506 and CyA inhibited LPS-induced increases in intracellular calcium in both monocytes (from a mean channel fluorescence of 128 to the basal levels of 32) and THP-1 cells (from a mean channel fluorescence of 64 to the basal levels of 32) in a dose-dependent manner (Fig. 2A). To confirm that FK506 and CyA are biologically active, Jurkat cells were treated with these agents for 2 h prior to stimulation with PMA/ionomycin for another 5 min followed by analysis of NFAT-4 expression by Western blotting. NFAT-4 is constitutively expressed in resting Jurkat cells. PMA/ionomycin stimulation induces its translocation into the nuclei that is inhibited by prior treatment of cells with either CyA or FK506 PMA and ionomycin treatment caused disappearance of the NFAT-4 band, which was restored by pretreatment of cells with FK506 or CyA (Fig. 2B). To determine a direct link for LPS-induced increases in intracellular free calcium and IL-12p40 production, cells were incubated with EGTA prior to LPS stimulation for 24 h. EGTA inhibited LPS-induced IL-12p40 production in both cell types as measured by ELISA (Fig. 2C) and semiquantitative RT-PCR analysis (Fig. 2D).

Calmodulin/CaMK-II Regulates LPS-induced IL-12p40 Production—To further delineate the role of calcium signaling, we used pharmacological inhibitors specific for members of the calcium signaling pathway. Increases in cytoplasmic calcium concentrations occur by stimuli that activate voltage or ligand-gated calcium channels in the plasma membrane or following release of calcium present in intracellular stores, mainly in the endoplasmic reticulum (ER) (39, 40). The role of receptor-mediated entry of extracellular Ca2+ was studied by using SKF-96365 (41). To determine whether calcium release from the ER regulates IL-12p40 expression, we employed 2-APB, an inhibitor of the inositol triphosphate receptor which blocks calcium release from the ER by blocking inositol triphosphate receptorgated channels (42). Prior treatment of cells with either SKF-96365 or 2-APB significantly inhibited LPS-induced IL-12p40 production in both cell types as determined by ELISA (Fig. 3A) and semiquantitative RT-PCR analysis (Fig. 2D). It may be noted that monocytes were more sensitive to the effects of EGTA, SKF-96365, and 2-APB than were THP-1 cells. These results suggest that receptor-mediated Ca2+ entry as well as the Ca2+ release from the ER may be involved in LPS-induced IL-12p40 expression.


Figure 2
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  		FIGURE 2.
Inhibition of IL-12p40 production by FK506 and CyA is calcium-dependent in LPS-activated monocytes and THP-1 cells. A, FK506 and CyA inhibit LPS-induced increase in the levels of intracellular calcium in monocytes and THP-1 cells. Cells (0.5 x 106) were pretreated with varying doses of FK506 (2–5 µM) and CyA (2–5 µM) for 2 h and loaded with Fluo-3/AM followed by LPS stimulation for 0–15 min. The levels of intracellular calcium were then measured by flow cytometric analysis. B, the biological activity of FK506 and CyA was confirmed by their effect on NFAT-4 expression in Jurkat T cells. Jurkat T cells (2 x 106/ml) were treated with FK506 and CyA for 2 h and stimulated with PMA plus ionomycin for 5 min. Cell lysats were analyzed for NFAT-4 expression by Western blotting using anti-NFAT-4 antibody (top panel). To ensure equal protein loading, membranes were stripped and reprobed with anti-CaMK-II Abs (bottom panel). C, THP-1 cells and monocytes were pretreated with EGTA at concentrations ranging from 5 to 25 mM for 2 h followed by LPS stimulation for another 24 h. IL-12p40 production in the supernatant was measured by ELISA. Results shown are a mean ± S.D. of three experiments. D, IL-12p40 mRNA expression is regulated by CaM/CaMK-II and PI3K pathways. Monocytes and THP-1 cells (5 x 106/ml) were treated with various inhibitors at the indicated concentrations for 2 h prior to stimulation with 1 µg/ml LPS for 4 h followed by analysis for IL-12p40 mRNA expression by semiquantitative RT-PCR analysis using beta-actin as an internal control. The results shown are representative of three independent experiments.

 
CaM, a major calcium receptor, is present in both cytoplasmic and nuclear compartments. The calcium/CaM complex regulates several downstream targets including protein kinases and phosphatases (43, 44). To determine the role of CaM, we used a specific calmodulin inhibitor, W-7 (37, 45). W-7 inhibited LPS-induced IL-12p40 production in both cell types as determined by ELISA (Fig. 3B) and semiquantitative RT-PCR analysis (Fig. 2D). One major family of CaM effectors is the calmodulin-dependent protein kinase (CaMK), which includes a multifunctional kinase, CaMK-II. To gain further insight into the role of calcium/CaM, we examined the role of CaMK-II by employing the CaMK-II-specific inhibitor, KN93 (46, 47), which significantly inhibited LPS-induced IL-12p40 production in both cell types as determined by ELISA (Fig. 3B) and semiquantitative RT-PCR analysis (Fig. 2D). We also confirmed that LPS induced CaMK-II phosphorylation was inhibited by W-7 and KN93 in both cell types (Ref. 37 and Fig. 3C).

The p110{alpha} Isoform of Class IA PI3K Positively Regulates LPS-induced IL-12p40 Expression—The PI3K pathway negatively regulates IL-12p40 expression in murine DCs and monocytic cells (32–36). Therefore, it was of interest to determine whether PI3K regulates human IL-12p40 production in a manner similar to the calcium pathway. Therefore, we first demonstrated that LPS induced phosphorylation of Akt, a downstream substrate for PI3K, and LY294002 inhibited this phosphorylation in both monocytes and THP-1 cells (Fig. 4A). Interestingly, Ly294002 significantly inhibited LPS-induced IL-12p40 expression in both cell types (Figs. 2D and 4B).

Mammalian class-IA PI3K are heterodimers consisting of a regulatory subunit (p85{alpha}, p85beta, p55, or other splice variants) and a p110 ({alpha}, beta, or {gamma} isoforms) catalytic unit (48–50). Herein, we investigated the role of the p110{alpha} isoform by employing THP-1 cells stably deficient in the p110{alpha} isoform (51). Cells were stably transduced with a lentiviral vector expressing short hairpin RNA for the p110{alpha} subunit (HR-p110{alpha}3). These cells exhibited complete inhibition of the PI3K p110{alpha} isoform expression without affecting the expression of p85 (Fig. 4C) or that of p110beta and p110{gamma} isoforms (Ref. 51 and data not shown). Cells transduced with the control lentiviral vector HRp110{alpha}1 exhibited normal levels of the p110{alpha} isoform (Fig. 4C, inset, and Ref. 51). To determine the involvement of the PI3K p110{alpha} isoform, normal THP-1 cells and THP-1 cells stably expressing HR-p110{alpha}3 and HR-p110{alpha}1 were stimulated with LPS for 24 h and analyzed for IL-12p40 expression. The results show that IL-12p40 production was significantly reduced in THP-1 cells transfected with HR-p110{alpha}3 compared with the control (Fig. 4C) suggesting a key role for p110{alpha} isoform of PI3K in LPS-induced IL-12p40 production. The role of p85 subunit of PI3K was confirmed by transfecting THP-1 cells with vectors containing p85-specific or a control siRNA. Transfection with p85-specific siRNA significantly reduced LPS-induced p85 phosphorylation (Fig. 5A) and IL-12p40 expression as determined by real-time PCR (Fig. 5B) and ELISA (Fig. 5C).

FK506 and CyA Inhibit LPS-induced IL-12p40 Production by Inhibiting CaM/CaMK-II-activated PI3K Independent of the JNK Pathway—We have previously demonstrated a key role for JNK in LPS-induced IL-12p40 production (20). Since PI3K is involved in TLR-4-mediated signaling and Akt has been shown to regulate c-Raf (ERK), p38, and JNK mitogen-activated protein kinases (52, 53), we examined whether activation of calmodulin/CaMK-II, PI3K, and JNK by LPS constitute distinct pathways in IL-12p40 regulation. This was accomplished by pretreating cells for 2 h with various concentrations of inhibitors specific for calcium-dependent signaling followed by LPS stimulation and analysis of Akt and JNK phosphorylation. EGTA, 2-APB, SKF, KN93, W7, FK506, and CyA suppressed phosphorylation of Akt without any effect on JNK phosphorylation in THP-1 cells and monocytes (Fig. 6). These results suggest that LPS-induced IL-12p40 production is regulated by two distinct pathways, namely PI3K activated upstream by CaM/CaMK-II and the JNK pathway, independent of the latter. Moreover, CyA and FK506 inhibited LPS-induced IL-12p40 production through the selective inhibition of pathways involving calcium (Fig. 2A) and PI3K without affecting the JNK pathway (Fig. 6, A and B).

FK506 and CyA Regulate LPS-induced IL-12p40 Expression by NF{kappa}B and AP-1 through the Activation of CaM/CaMK-II and PI3K Pathways—We have previously shown that LPS-induced IL-12p40 production is regulated by JNK through the activation of NF{kappa}B and AP-1 (20). To determine whether LPS-induced IL-12p40 production is regulated by CaM/CaMK-II-activated NF{kappa}B and/or AP-1, we confirmed our earlier results by transfecting cells with a series of 5'-deletion constructs containing IL-12p40 promoter (–880/+118 bp) sequences (Fig. 7A) linked with the luciferase reporter plasmid, pGL3B. Subsequent LPS-induced luciferase activity analysis showed that transfection with the –880 to +118 construct induced significant luciferase activity compared with the cells transfecetd with the control vector, whereas transfection with the –116 vector abrogated luciferase activity (Fig. 7B). Transfection with vectors containing mutant NF{kappa}B in the presence of PU.1 binding sequence (–128, pIL-12p40.PU.1, NF{kappa}Bmut) abrogated the luciferase activity, while transfection with vectors containing wild type AP-1 and mutant NF{kappa}B sites (–232, pIL-12p40.AP-1, Ets-2, PU.1, NF{kappa}Bmut) still exhibited an induction of luciferase activity after LPS stimulation. However, when both AP-1 and NF{kappa}B sites were mutated (–232, pIL-12p40.AP-1mut, Ets-2, PU.1, NF{kappa}Bmut), LPS-induced luciferase activity was significantly decreased (Fig. 7B, left panel). These results suggest that both AP-1 and NF{kappa}B regulate LPS-induced IL-12p40 production and that either one or the other are required to induce IL-12p40 transcription.


Figure 3
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  		FIGURE 3.
LPS-induced IL-12p40 expression in monocytes and THP-1 cells is regulated by the CaM/CaMK-II pathway. A and B, monocytes (left panel) and THP-1 cells (right panel) at a concentration of 1 x 106/ml were treated with varying concentrations of SKF, 2-APB, W7, or KN-93 for 2 h prior to stimulation with LPS (1 µg/ml) for 24 h. The cell supernatants were analyzed for IL-12p40 production by ELISA. Results shown are a mean ± S.D. of three experiments. C, cells (1 x 106) were treated with W-7 and KN93 for 2 h prior to stimulation with LPS for 12 min. Cells were harvested for determining the phosphorylation of CaMK-II by Western blot analysis using anti-phospho-CaMK-II antibody (top panel). To ensure equal protein loading, the membrane were stripped and reprobed with anti-CaMK-II Abs (bottom panel). The relative fold increase in phosphorylation intensity is shown. Results shown are a representative of three experiments performed.

 


Figure 4
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  		FIGURE 4.
LPS-induced IL-12p40 expression is positively regulated by the p110{alpha} isoform of PI3K in human monocytic cells. A, monocytes and THP-1 cells (1 x 106/ml) were stimulated with LPS for 0–60 min (left panel). Monocytes and THP-1 cells (1 x 106/ml) were cultured with various concentrations of Ly294002 ranging from 0 to 50 µM for 2 h followed by stimulation with LPS for 30 min. Total proteins were subjected to Western blot analysis for Akt phosphorylation using anti-phospho-Akt Abs. The equal loading of protein was confirmed with anti-Akt Abs. The blot shown is a representative of three independent experiments. B, monocyes and THP-1 cells were pretreated with Ly294002 at indicated concentrations for 2 h followed by LPS (1 µg/ml) stimulation for 24 h. IL-12p40 production in the supernatants was measured by ELISA. Results shown are a mean ± S.D. of three experiments. C, normal THP-1 cells and THP-1 cells transduced with HR-p110{alpha}3 or HR-p110{alpha}1(1 x 106) were stimulated with LPS (1 µg/ml) for 24 h followed by analysis for IL-12p40 production by ELISA. The expression of PI3K p110{alpha} and p85 isoforms in the above cells was determined by Western blotting by employing anti-p110{alpha} and anti-p85 antibodies, respectively (inset). The results shown are representative of three independent experiments.

 
CaMK-II has been shown to act as a mediator of I{kappa}B kinase activation in response to CD3/T cell receptor stimulation (54). To determine whether CaM/CaMK-II regulated LPS-induced IL-12p40 expression through NF{kappa}B activation, cells transfected with the full-length IL-12p40 promoter and the deletion constructs were treated with various pharmacological inhibitors of the calcium and PI3K pathways (EGTA, W7, 2-APB, SKF-96365, KN-93, CyA, FK506, and Ly294002) prior to LPS stimulation followed by measurement of luciferase activity. All these agents significantly decreased the LPS-induced luciferase activity in cells transfected with the full length (–880 to +112), the –232 (pIL-12p40.AP-1, Ets-2, PU.1, NF{kappa}Bmut), and –128 (pIL-12p40. PU.1, NF{kappa}B) promoter constructs (Fig. 7B). These results suggest that CyA- and FK506-mediated inhibition of LPS-induced IL-12p40 transcription in human monocytic cells involves CaM/CaMK-II- and PI3K-dependent activation of NF{kappa}B and/or AP-1.

FK506 and CyA Inhibit Binding of NF{kappa}B and AP-1 to Their Binding Sites in the IL-12p40 Promoter through the CaM/CaMKII and PI3K Pathways—We confirmed our earlier results showing that NF{kappa}B and AP-1 regulate LPS-induced IL-12p40 transcription (20). LPS stimulation induced significant binding of NF{kappa}B and AP-1. The specificity of NF{kappa}B and AP-1 binding was demonstrated by competition with specific and nonspecific oligonucleotides and by supershift analysis with anti-p50 and anti-p65 NF{kappa}B, and anti-c-Jun and anti-c-Fos antibodies for AP-1 (Fig. 8, A and B). To determine whether binding of NF{kappa}B and AP-1 to their respective binding sites in the IL-12p40 promoter was regulated by CaM/CaMK-II and/or PI3K, cells were treated with inhibitors of the calcium and PI3K signaling pathways before stimulation with LPS. As before, EGTA, W7, SKF-96365, KN-93, FK506, CyA, and LY294002 inhibited the binding of NF{kappa}B and AP-1 to their respective probes in LPS-stimulated cells (Fig. 8, A and B). These results suggest that LPS-induced IL-12p40 transcription is regulated by CaM/CAMK-II as well as PI3K through NF{kappa}B and AP-1 activation. Moreover, CyA and FK506 inhibited IL-12p40 transcription by inhibiting AP-1 and NF{kappa}B activity.


Figure 5
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  		FIGURE 5.
LPS-induced IL-12p40 expression is positively regulated by p85 PI3K in human monocytic cells. THP-1 cells were transfected with the vectors containing either the p85 or the control siRNA for 24 h followed by stimulation with LPS (1 µg/ml) either for 30 min for the determination of phosphorylated p85 by Western blot analysis (A), for 8 h for measurement of IL-12p40 RNA by real-time RT-PCR (B), or for 24 h for measurement of IL-12p40 by ELISA (C).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
CyA and FK506 are potent inhibitors of calcium-regulated signaling and exert their biological effects through calcineurin (3, 4). PI3K and calcium pathways were shown to negatively regulate LPS-induced IL-12p40 expression in murine macrophages and DCs (32–36). In contrast, our results show that calcium signaling through CaM and CaMK-II, as well as through PI3K positively regulated LPS-induced IL-12p40 production in human monocytic cells. Moreover, both CyA and FK506 inhibited LPS-induced IL-12p40 production by inhibiting NF{kappa}B and AP-1 activity through the upstream CaM/CaMK-II-activated PI3K and independent of the JNK pathway.


Figure 6
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  		FIGURE 6.
LPS-induced Akt phosphorylation is regulated by the calcium signaling pathway. THP-1 cells (A) and monocytes (B)(2 x 106/ml) were treated with EGTA, 2-APB, SKF, W-7, CyA, or FK506 at indicated concentrations for 2 h prior to stimulation with LPS (1 µg/ml) for 30 min. Total proteins were subjected to SDS-PAGE analyzed for the phosphorylation of Akt and JNK using anti-phospho-Akt and anti-phospho-JNK Abs, respectively. For determining equal protein loading, membranes were reprobed with anti-Akt Abs. The results shown are representative of three independent experiments.

 
Inhibition of calcium signaling has been shown to enhance IL-12p40 production in LPS-stimulated murine monocytic cells. Ligation of Fc receptors on murine monocytic cells inhibited LPS-induced IL-12p40 production that was reversed by pretreatment with the calcium ion chelator, EGTA (29–31). In contrast, our results show that inhibition of LPS-induced intracellular free calcium by EGTA significantly reduced IL-12p40 production in human monocytic cells. A positive role for calcium was further supported by employing specific inhibitors for CaM/CaMK-II. CaM is a key signaling protein responsible for integrating Ca2+ signals to transcription factors (44). Two important downstream targets of CaM are calcineurin and CaMK-II (55, 56). As with other kinases, CaMK-II undergoes autophosphorylation on a threonine residue contained in a phosphopeptide common to its {alpha} and beta subunits thereby converting it into a Ca2+/CaM-independent enzyme (57). The results obtained by employing specific inhibitors for CaM, calcineurin, and CaMK-II suggested that LPS-induced IL-12p40 production is regulated by the CaM/CaMK-II pathway. The observation that both FK506 and CyA inhibited LPS-induced increase in intracellular free calcium, CaMK-II phosphorylation, and IL-12p40 production in monocytic cells suggests that this may be a mechanism by which these agents inhibit Th1 type responses in these cells. The observed enhanced sensitivity of primary monocytes to the calcium inhibitors compared with the THP-1 cells may be due to inherent differences between the primary versus the leukemic cells.


Figure 7
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  		FIGURE 7.
LPS-induced expression of IL-12p40 is regulated by NF{kappa}B and AP-1 transcription factors through the activation of calcium and PI3K pathways. A, the nucleotide sequence of 5'-flanking region of the human IL-12 promoter (GenBankTM accession number U89323). The numbers indicated nucleotide positions relative to the transcription factors, AP-1, NF{kappa}B, and others, whose binding sequences are shown in capital letters, whereas the coordinated mutants are shown in lowercase letters. B, pharmacological inhibitors specific for either calcium- or PI3K-regulated signaling inhibit LPS-induced activation of NF{kappa}B and AP-1 in THP-1 cells. THP-1 cells (2 x 106) were pretreated with various inhibitors specific for either calcium (EGTA, SKF, 2-APB, W7, KN-93, CyA, and FK-506) or PI3K (Ly294002) at indicated concentrations for 2 h followed by transient transfection with 1 µg of various hIL-12p40 promoter constructs containing either wild type or mutant AP-1 and NF{kappa}B binding sequences, or control reporter vector, plus 0.5 µg of beta-galactosidase plasmid for 24 h. Cells were then stimulated with LPS for another 24 h and harvested for measuring luciferase and beta-galactosidase activity. Luciferase activity was normalized with the beta-galactosidase activity. The results shown are mean ± S.D. of four experiments each performed in triplicate.

 
Class Ia PI3K consist of a 85-kDa regulatory subunit ({alpha}, beta, and {gamma} isoforms) and a 110-kDa catalytic unit ({alpha}, beta, and {delta} isoforms). Interaction of the p85 subunit with phosphorylated YxxM motifs in transmembrane receptors or adaptor molecules results in the recruitment of p85-p110 heterodimers to its substrate phosphatidylinositol 4,5-bisphosphate (49, 50, 58–60) that leads to the release of phosphatidylinositol 3,4,5-trisphosphates and eventual phosphorylation of a number of substrates including Akt, protein kinase A, and protein kinase C isoforms (48, 59). There is considerable evidence to suggest that PI3K plays a key role in the negative feedback regulation of IL-12p40 production by murine DCs and monocytic cells (32, 33). PI3K was shown to negatively regulate IL-12p40 production by human monocytic cells stimulated by the TLR-2 ligand, Porphyromonas gingivalis LPS (34) and following ligation of complement receptors C1q and C5a in LPS-stimulated human and murine monocytic cells and DCs (35, 61). In contrast, we show that LPS-induced IL-12p40 production by primary human monocytes as well as THP-1 cells is regulated positively through PI3K activation.

Understanding the role and functions of PI3K family members has been difficult because of their resistance to genetic manipulation, broad biological activity of PI3K inhibitors to inhibit nearly all classes of PI3K isoforms (62), and embryonically lethal PI3K p110{alpha} or p110beta gene knockouts (63, 64). There is evidence to suggest that the p85 subunit independent of the p110{alpha} can regulate the expression of several genes including IL-2 (65, 66). Recently, using an RNA interference-based approach, the p110{alpha} subunit of PI3K was shown to regulate vitamin D3-induced adherence and up-regulation of CD11b in monocytic cells (51). Interestingly, herein, we have demonstrated that THP-1 cells deficient in the expression of the p110{alpha} catalytic unit of PI3K produced significantly reduced levels of IL-12p40 in response to LPS without any effect on the expression of IL-10, CD44, B7.1, B7.2, and CD11b (data not shown). These results suggest that p85/p110{alpha} PI3K selectively regulates LPS-induced IL-12p40 production.

The results of this study in concert with our earlier observations showing the regulation of LPS-induced IL-12p40 production through the JNK pathway (20) suggest that LPS-induced IL-12p40 production is regulated by two distinct signaling pathways namely the CaM/CaMKII-activated PI3K pathway and the JNK pathway. Interestingly, both pathways regulated IL-12p40 production through the activation of NF{kappa}B and AP-1 transcription factors. It is not clear why the JNK pathway failed to compensate for LPS-induced IL-12p40 production in the presence of inhibitors specific for the calcium pathway. In addition to signal transduction pathways, molecular mechanisms controlling gene expression involve chromatin remodeling and DNA methylation (67–69). Understanding the status of promoter methylation and chromatin remodeling may elucidate the mechanisms underlying the involvement of distinct signaling pathways controlling IL-12p40 transcription.

Herein, we have shown that IL-12p40 production is regulated by NFkB and AP-1 through the activation of upstream calcium and PI3K pathways. NF{kappa}B and AP-1 are known to be activated by the calcium/CaMKs (54, 70), although it is not clear if Akt and CaM/CaMK-II can directly interact with the members of the NF{kappa}B and AP-1 family members. It is also not clear that in the presence of an intact promoter whether both of these transcription factors can synergize to induce IL-12p40 transcription. Although deletion mutation studies conducted to define the role of individual transcription factors did not reveal a synergistic effect at the level of luciferase activity, the potential cooperation between these two transcription factors cannot be ruled out. It is possible that the level of cooperation between these transcription factors is modulated following interaction with other signaling molecules activated by different external stimuli. Further studies are needed to elucidate the precise cooperation between NF{kappa}B and AP-1 and possibly other factors involved in IL-12p40 transcription.


Figure 8
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  		FIGURE 8.
Binding of AP-1 and NF{kappa}B to their binding sites in the human IL-12p40 promoter is inhibited by pharmacological inhibitors specific for calcium and PI3K-regulated pathways in LPS-stimulated THP-1 cells. THP-1 cells were stimulated with LPS for 30 min. Nuclear proteins (5 µg) were incubated for 1 h with 32P-labeled oligonucleotide corresponding to the NF{kappa}B or AP-1 sequences located in the IL-12p40 promoter. To determine the specificity of NF{kappa}B or AP-1 transcription factor binding, the nuclear extracts were incubated with 100 fold excess of unlabeled specific (CC) or nonspecific (NS CC) probes for 30 min before addition of labeled probes (A and B, left panel). The nuclear extracts were also incubated with anti-p50 and anti-p65 NF{kappa}B antibodies (A, left panel) or anti-c-Jun and anti-c-Fos antibodies along with their respective oligonucleotide probes for supershift analysis. THP-1 cells were incubated with various inhibitors specific for the calcium (EGTA, SKF, 2-APB, W7, KN-93, CyA, and FK-506) and PI3K (Ly294002) pathways at the indicated concentrations for 2 h followed by stimulation with LPS for 30 min (middle and right panels, A and B). Nuclear extracts were analyzed for binding to 32P-labled oligonucleotide probes corresponding to the NF{kappa}B(A) and AP-1 (B) binding sites in IL-12p40 promoter. The results shown are representative of three independent experiments.

 
The unique role of IL-12p40 in the regulation of IL-12 and IL-23 suggests that it may be critically involved in the immunopathogenesis of Th1-mediated inflammatory and autoimmune disorders. Our investigations regarding the molecular mechanism underlying the inhibitory effects of CyA and FK506 on IL-12p40 production have revealed for the first time a positive role of calcium and PI3K and in particular a selective involvement of p110{alpha} subunit of PI3K in regulating IL-12 production by human monocytes. These findings raise the possibility of developing novel agents capable of inhibiting calcium and PI3K pathways as potential therapeutics for disorders related to excessive IL-12 production. The observations that calcium and PI3K signaling pathways positively regulate human IL-12p40 production in contrast to their negative regulatory role in murine IL-12p40 production highlight the importance of understanding in detail the mechanism involved in regulating the production of critical cytokines IL-12 and IL-23 in human settings.


   FOOTNOTES
 
* This work was supported by grants from the Canadian Institute of Health Research (to A. K. and N. E. R. (MOP-8633)) and by Michael Smith Foundation for Health Research Research Unit Infrastructure Grant RUA042031 (to N. E. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 These two authors contributed equally to this manuscript. Back

2 Supported by a fellowship from the Natural Sciences and Engineering Research Council, Canada. Back

3 Both authors were supported by fellowships from the Ontario Graduate Scholarship program and the Ontario Graduate Scholarships in Science and Technology program. Back

4 Supported by a fellowship from the Ontario HIV Treatment Networks (OHTN). Back

5 Recipient of a Career Scientist Award from the OHTN. Back

6 Recipient of a Career Scientist Award from the OHTN. To whom correspondence should be addressed: Division of Virology, Dept. of Pathology and Laboratory Medicine, Research Institute, Children's Hospital of Eastern Ontario, 401 Smyth Rd., Ottawa, Ontario K1H 8L1, Canada. Tel.: 613-737-7600 (ext. 3920); Fax: 613-738-4825; E-mail: akumar{at}uottawa.ca.

7 The abbreviations used are: CyA, cyclosporine; CaM, calmodulin; CaMK-II, calmodulin-dependent protein kinase-II; DC, dendritic cell; ER, endoplasmic reticulum; JNK, c-Jun N-terminal kinase; PI3K, phosphoinositide 3-kinase; LPS, lipopolysaccharide; PBMC, peripheral blood mononuclear cell; IMDM, Iscove's modified Dulbecco's medium; FBS, fetal bovine serum; ELISA, enzyme-linked immunosorbent assay; Ab, antibody; mAb, monoclonal antibody; RT, reverse transcription; NFAT, nuclear factor of activated T cells; siRNA, small interfering RNA; PMA, phorbol 12-myristate 13-acetate. Back



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2007 by the American Society for Biochemistry and Molecular Biology.
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