The ceramide-1-phosphate analogue PCERA-1 modulates tumour necrosis factor-alpha and interleukin-10 production in macrophages via the cAMP-PKA-CREB pathway in a GTP-dependent manner

Abstract

The synthetic phospho-ceramide analogue-1 (PCERA-1) down-regulates production of the pro-inflammatory cytokine tumour necrosis factor-alpha (TNF-alpha) and up-regulates production of the anti-inflammatory cytokine interleukin-10 (IL-10) in lipopolysaccharide (LPS) -stimulated macrophages. We have previously reported that PCERA-1 increases cyclic adenosine monophosphate (cAMP) levels. The objective of this study was to delineate the signalling pathway leading from PCERA-1 via cAMP to modulation of TNF-alpha and IL-10 production. We show here that PCERA-1 elevates intra-cellular cAMP level in a guanosine triphosphate-dependent manner in RAW264.7 macrophages. The cell-permeable dibutyryl cAMP was able to mimic the effects of PCERA-1 on cytokine production, whereas 8-chloro-phenylthio-methyladenosine-cAMP, which specifically activates the exchange protein directly activated by cAMP (EPAC) but not protein kinase A (PKA), failed to mimic PCERA-1 activities. Consistently, the PKA inhibitor H89 efficiently blocked PCERA-1-driven cytokine modulation as well as PCERA-1-stimulated phosphorylation of cAMP response element binding protein (CREB) on Ser-133. Finally, PCERA-1 activated cAMP-responsive transcription of a luciferase reporter, in synergism with the phosphodiesterase (PDE)-4 inhibitor rolipram. Our results suggest that PCERA-1 activates a G(s) protein-coupled receptor, leading to elevation of cAMP, which acts via the PKA-CREB pathway to promote TNF-alpha suppression and IL-10 induction in LPS-stimulated macrophages. Identification of the PCERA-1 receptor is expected to set up a new target for development of novel anti-inflammatory drugs.

Figure 1

Synergistic interleukin-10 (IL-10) production by lipopolysaccharide (LPS) and phospho-ceramide analogue-1 (PCERA-1) is associated with a PCERA-1-induced GTP-dependent cAMP increase. (a) Mouse macrophage RAW264.7 cells were incubated at 37° for 2 hr with LPS (100 ng/ml) and/or the indicated concentrations of PCERA-1. IL-10 release to the medium was measured by enzyme-linked immunosorbent assay. Each data point represents mean ± SD (n = 6). Background IL-10 level was 25 ± 6 pg/ml. P<0·003 for cells treated with PCERA-1 and LPS, compared with LPS only. (b) The cells were pre-incubated with the phosphodiesterase (PDE) inhibitor isobutylmethylxanthine (IBMX; 0·5 mm) for 10 min at 37° before the addition of either PCERA-1 at the indicated concentrations, prostaglandin E₂ (PGE₂; 0·1 μm), or LPS (100 ng/ml) for an additional period of 10 min. Intra-cellular cAMP was then measured by enzyme immunoassay. Each data point represents mean ± SD (n = 6) following reduction of control value (64 pmol cAMP/mg protein). P<0·005 for cells treated with PCERA-1 or PGE₂, compared with vehicle. (c) Membranes of RAW264.7 cells were prepared and adenylyl cyclase (AC) activation by PCERA-1 (1 μm) was assayed for 30 min at 25° in the presence of increasing concentrations of GTP, as indicated. A detailed protocol of the LANCE-cAMP method is described in the Materials and methods. As controls, AC activation by forskolin (10 μm) or vehicle was similarly measured in the presence or absence of GTP (10 μm). The β-AR antagonist, propranolol (1 μm), was added to all treatments including control to reduce the background of constitutive β-AR activity. Each data point represents mean ± SD (n = 5). *P<0·02, **P<0·003, ***P<0·0002.

Figure 2

Modulation of lipopolysaccharide (LPS) -induced cytokine production by phospho-ceramide analogue-1 (PCERA-1) is mimicked by dibutyryl (db-) cAMP and not by an exchange protein directly activated by cAMP (EPAC) agonist. RAW264.7 macrophages were incubated at 37° for 2 hr with LPS (100 ng/ml) and with either a specific EPAC activator – 8CPT-2Me-cAMP (0·3 mm), db-cAMP (0·3 mm) or PCERA-1 (1 μm). Tumour necrosis factor-α (TNF-α) and interleukin-10 (IL-10) release to the medium was measured by enzyme-linked immunosorbent assay. Each data point represents mean ± SD (n = 6). The cytokines were undetectable (< 20 pg/ml) in the absence of LPS. *P<0·0002, **P<0·004, ***P<0·05.

Figure 3

Phospho-ceramide analogue-1 (PCERA-1) modulates tumour necrosis factor-α (TNF-α) and interleukin-10 (IL-10) production via protein kinase A (PKA). RAW264.7 macrophages were pre-incubated for 30 min at 37° with the PKA inhibitor H89 (30 μm, solid bars) or with vehicle (open bars). The cells were then stimulated with lipopolysaccharide (LPS; 100 ng/ml) and with either PCERA-1 (1 μm), prostaglandin E₂ (PGE₂; 0·1 μm) or dexamethasone (1 μm), and further incubated for 2 hr. TNF-α and IL-10 release to the medium was measured by enzyme-linked immunosorbent assay. Each data point represents mean ± SD (n = 6). The cytokines were undetectable (< 20 pg/ml) in the absence of LPS. *P<0·0002.

Figure 4

Phospho-ceramide analogue-1 (PCERA-1) stimulates protein kinase A (PKA) -mediated phosphorylation of cAMP response element binding protein (CREB). (a) RAW264.7 macrophages were stimulated with PCERA-1 (1 μm) for the indicated time at 37°. The proteins in cell lysates (30 μg) were subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred to Immobilon-FL poylvinylidene difluoride. The membrane was probed with antibodies against phospho Ser-133 CREB, and against α-tubulin (for normalization). Quantitative Western blot analysis is shown as the ratio of intensities of phospho-CREB and tubulin, relative to unstimulated control cells. The treatments had no effect on the total level of CREB (data not shown). The results are representative of five independent experiments. (b) Pre-incubation of RAW264.7 macrophages for 30 min at 37° with either H89 (PKA and MSK1 inhibitor, 30 μm), Ro318220 (‘Ro’, MSK1 inhibitor, 5 μm), SB203580 (‘SB’, p38 inhibitor, 30 μm) or vehicle, was followed by the addition of PCERA-1 (1 μm) or vehicle for an additional 15 min. Cell lysates were analysed as in (a). The results are representative of three independent experiments.

Figure 5

Phospho-ceramide analogue-1 (PCERA-1) induces CRE-luciferase reporter gene activation. RAW264.7 macrophages were transiently transfected for 24 hr at 37° with a reporter gene construct which codes for firefly luciferase under the regulation of CRE, and with a renilla luciferase construct for normalization. Luciferase activity assay was performed as described in the Materials and methods. Each data point represents mean ± SD (n = 6) of values normalized against renilla luciferase activity, and relative to unstimulated control cells. (a) The cells were washed and incubated with PCERA-1 (1 μm), dibutyryl (db-) cAMP (0·1 mm) or vehicle, for 3 hr at 37°. P<0·004 for cells treated with PCERA-1 or db-cAMP, compared with control. (b) The cells were washed and pre-incubated for 20 min at 37° with the specific PDE-4 inhibitor rolipram at the indicated concentrations, before PCERA-1 (1 μm, upper bars) or vehicle (lower bars) was added for an additional 3 hr. P<0·001 for cells treated with rolipram, compared with control (either with or without PCERA-1).

Figure 6

Phospho-ceramide analogue-1 (PCERA-1) regulates transcriptional activation in a protein kinase A (PKA) -dependent manner. RAW264.7 macrophages were pre-incubated for 30 min at 37° with H89 (PKA inhibitor, 1-30 μm), Ro318220 (‘Ro’, MSK1 inhibitor, 5 μm), SB203580 (‘SB’, p38 inhibitor, 30 μm) or vehicle, and then further incubated with PCERA-1 (1 μm) and/or lipopolysaccharide (LPS; 100 ng/ml) for 3 hr (a) or 2 hr (b, c). (a) Before the the above treatment, the cells were transiently transfected for 24 hr at 37° with a reporter gene construct which codes for firefly luciferase under the regulation of CRE, and with a renilla luciferase construct for normalization. Following treatment, luciferase activity assay was performed as described in the Materials and methods. Each data point represents mean ± SD (n = 6) of values normalized against renilla luciferase activity, and relative to unstimulated control cells. (b, c) TNF-α and IL-10 release to the medium were measured by enzyme-linked immunosorbent assay. Each data point represents the mean ± SD (n = 6). The cytokines were undetectable (< 20 pg/ml) in the absence of LPS. *P<0·02, **P<0·005, ***P<0·0007.