Prostaglandin E2 is a novel inducer of oncostatin-M expression in macrophages and microglia

Abstract

Oncostatin-M (OSM), a pluripotent cytokine of the interleukin-6 (IL-6) family, is produced in a number of inflammatory conditions. Known sources of OSM include monocytes-macrophages and T-cells. Here we present microglia, the resident macrophages of the brain, as a source of OSM in the CNS. In this context, we describe a novel inducer of OSM, prostaglandin E(2) (PGE(2)). PGE(2) induces OSM expression in microglia, monocytes, and macrophages of human and murine origin. PGE(2) induction of OSM is mimicked by cholera toxin, an activator of stimulatory G (G(s))-proteins; by forskolin, an activator of adenylate cyclase; and by the cAMP analog, dibutyryl-cAMP. PGE(2) induction of OSM gene expression is inhibited by the adenylate cyclase inhibitor 2',5'-dideoxyadenosine, by the protein kinase A (PKA) inhibitor H-89, and by a dominant-negative PKA construct. These data indicate that PGE(2) signals via G(s)-protein-coupled receptor(s), adenylate cyclase, and PKA to induce OSM expression. Accordingly, other activators of cAMP signaling such as norepinephrine and PGE(1) induce OSM. The ability of PGE(2) to induce OSM expression was tested under more physiological conditions, using cocultures of astrocytes and monocytes. Treatment of the cocultures with IL-1beta or tumor necrosis factor-alpha (TNF-alpha) results in production of PGE(2) and OSM. PGE(2) produced in the cocultures is responsible for OSM induction, because pretreatment with indomethacin, an inhibitor of prostaglandin synthesis, as well as depletion of PGE(2), abrogate OSM expression induced by IL-1beta or TNF-alpha. These data suggest that in the CNS, OSM may be produced through collaboration of astrocytes and macrophages-microglia.

Fig. 1.

PGE₂ induces OSM mRNA expression in cells of monocytic lineage. THP-1 monocytic cells (4 × 10⁶), plated in 60 mm dishes, were stimulated with PGE₂ (0.1 μm) for 1–3 hr. Total RNA was extracted and analyzed by RPA for OSM and GAPDH mRNA (A). Quantification of the RPA blot is shown inB, with OSM mRNA expression normalized to GAPDH levels, and plotted as fold induction over control. Results are representative of at least three experiments. BV-2 murine microglial cells and RAW264.7 murine macrophage cells, plated in 60 mm dishes (4 × 10⁶), were stimulated with PGE₂ (1–10 μm) for 1 hr. Total RNA was extracted and analyzed by RPA for OSM and GAPDH mRNA expression. Results are representative of at least two independent experiments (C).

Fig. 2.

Kinetics and dose-response of PGE₂-induced OSM protein expression. THP-1 cells, plated in six-well plates (2 × 10⁶/2 ml), were treated for 3–24 hr with PGE₂ (0.1 μm), and OSM concentrations in the supernatants were determined by ELISA (A). OSM concentrations were normalized to total protein levels. Data shown are the mean ± SD for three experiments. THP-1 cells (B) or primary human monocyte-derived macrophages (C) were plated in six-well plates and stimulated with PGE₂ (0.001–10 μm) for 24 hr, and OSM concentrations were determined as described above. Data shown are the mean ± SD for three experiments.

Fig. 3.

PGE₂ elevates cAMP levels in THP-1 cells. THP-1 cells (0.5 × 10⁶/ml) were treated with PGE₂ (0.1 μm) for 5–150 min. Cells were then lysed, and intracellular cAMP concentrations were determined by ELISA (A). THP-1 cells were treated for 30 min with PGE₂ (0.001–10 μm), and intracellular cAMP levels were determined by ELISA (B). Data shown are the mean ± SD of three experiments.

Fig. 4.

Agents that increase intracellular cAMP levels induce OSM. Primary human monocyte-derived macrophages were plated in six-well plates and treated with PGE₁ or norepinephrine (NE) (0.01–10 μm) for 24 hr. OSM concentrations in the supernatants were measured by ELISA and normalized to total protein. Data shown are the mean ± SD of three experiments (A). THP-1 cells plated in six-well plates were stimulated with the indicated doses of cholera toxin (CTX), forskolin (FSK), or dibutyryl-cAMP (dbcAMP) for 24 hr. OSM concentrations in the supernatants were determined by ELISA and normalized to total protein. Shown are the mean ± SD of three experiments (B).

Fig. 5.

PGE₂-induced OSM expression is partially inhibited by the adenylate cyclase inhibitor DDA. THP-1 cells plated in six-well plates were pretreated for 30 min with DDA (1–2 mm), before stimulation with PGE₂ (0.1 μm). After 6 hr, OSM concentrations in the supernatants were determined by ELISA. The OSM concentration in the sample without the inhibitor was set to 100%, and other concentrations represent a percentage fraction of this maximal induction. Data shown are the mean ± SD of three experiments. The difference between PGE₂-treated and inhibitor-treated samples was significant as determined by the Student's t test. *p < 0.05; n = 3 (A). THP-1 cells plated in 60 mm dishes were pretreated with DDA (2 mm) for 30 min and then treated with medium or PGE₂ (0.1 μm) for 1 hr. Total RNA was extracted and RPA analysis was performed for OSM and GAPDH mRNA (B). Quantification of the RPA blot is shown inC. Data shown are representative of three independent experiments.

Fig. 6.

Inhibition of protein kinase A abrogates PGE₂-induced OSM expression. THP-1 cells plated in six-well plates were pretreated with H-89 (5–20 μm) for 1 hr before stimulation with PGE₂ (0.1 μm) for 24 hr. OSM concentrations in the supernatants were measured by ELISA and normalized to total protein per well. Data shown are the mean ± SD of three experiments. The difference between PGE₂-treated and inhibitor-treated samples was significant as determined by the Student's t test. *p < 0.05; n = 3 (A). For RPA analysis of OSM and GAPDH mRNA, THP-1 cells plated in 60 mm dishes were pretreated with H-89 (5–10 μm) or the corresponding volume of solvent (DMSO) for 1 hr before stimulation with PGE₂(0.1 μm) for 1 hr (B). Quantification of the RPA blot is shown in C. Data shown are representative of three experiments.

Fig. 7.

Dominant-negative PKA abrogates PGE₂-induced activation of OSM promoter activation. BV-2 cells were transiently transfected with a 4.7 kb OSM promoter (1 μg) plus empty vector (0.5 μg) or expression vector for a dominant-negative regulatory subunit of PKA (DN-PKA) (0.5 μg). In addition, all cells were cotransfected with a β-galactosidase vector (0.1 μg). Cells were treated with ZnSO₄ (0.1 mm) for 3 hr before stimulation with PGE₂ (1 μm) for 3 hr. Cells were lysed, and luciferase activity was measured and normalized to β-galactosidase activity as described in Materials and Methods. Fold induction represents a ratio of normalized luciferase activity of PGE₂-treated sample over untreated control. Results are representative of at least three independent experiments performed in triplicate.

Fig. 8.

Endogenously produced PGE₂ mediates OSM induction in astrocyte–monocyte cocultures. THP-1 cells (1 × 10⁶) and CRT-MG astroglioma cells (5 × 10⁵) were plated in the upper and lower chambers, respectively, of six-well transwell plates and stimulated with IL-1β (0.001–10 ng/ml). After 24 hr, the supernatants were harvested, and PGE₂ and OSM concentrations were determined using respective ELISA kits. OSM concentrations, normalized to total cell protein, are plotted against the left y-axis, whereas similarly normalized PGE₂ concentrations are plotted against the right y-axis. Data shown are the mean ± SD of threeexperiments (A). THP-1 cells and CRT-MG cells plated as above in six-well transwells were pretreated with indomethacin (1 μm) for 30 min before treatment with IL-1β (0.1–1 ng/ml) for 24 hr. OSM concentrations in the supernatants were determined using ELISA. Data shown are the mean ± SD of three experiments. The difference between IL-1β-treated and indomethacin-treated samples was significant as determined by the Student's t test. *p < 0.05;n = 3 (B). CRT-MG cells (1 × 10⁵) were plated in six-well plates and stimulated with IL-1β (0.1 ng/ml) or TNF-α (50 ng/ml) for 24 hr. Aliquots (1 ml) of the supernatants were incubated with either anti-PGE₂-coated Sepharose beads or plain Sepharose beads for 1 hr at 37°C before being added to THP-1 cells (1 × 10⁶) for 24 hr. The concentration of OSM in the THP-1 supernatants was determined using ELISA. Data shown are the mean ± SD of three experiments. The difference between samples incubated with Sepharose beads and anti-PGE₂-coated beads was significant as determined by the Student's t test. *p < 0.05; n = 3 (C). Astrocytes (2 × 10⁵) and monocytes (3 × 10⁵) were cocultured in transwell plates and stimulated with IL-1β (0.1 ng/ml) for 12–24 hr. At indicated times, RNA was harvested and analyzed by RPA for OSM and GAPDH mRNA. Data shown are representative of two independent experiments (D).

Fig. 9.

Two-step model of OSM production. In the first step, IL-1β or TNF-α act on astrocytes to release PGE₂as a diffusible inducer of OSM synthesis. In the second step, this inducer stimulates macrophages or microglia to produce OSM.