Prostaglandin E2 stimulates amyloid precursor protein gene expression: inhibition by immunosuppressants

Abstract

Amyloid plaques that accumulate in the brains of patients with Alzheimer's disease (AD) are primarily composed of aggregates of amyloid peptides that are derived from the amyloid precursor protein (APP). Overexpression of APP in cell cultures increases the formation of amyloidogenic peptides and causes neurodegeneration and cognitive dysfunction in transgenic mice. We now report that activation of prostaglandin E2 (PGE2) receptors increases cAMP formation and stimulates overexpression of APP mRNA and holoprotein in primary cultures of cortical astrocytes. Levels of glial fibrillary acidic protein were also increased by PGE2 treatment, suggesting that these cultured astrocytes resemble reactive astrocytes found in vivo. The stimulation by PGE2 of APP synthesis was mimicked or blocked by activators or inhibitors, respectively, of protein kinase A. Actinomycin D or cycloheximide also inhibited the increase in APP holoprotein stimulated by PGE2. Treatment of astrocytes with 8-Bromo-cAMP or forskolin for 24 hr also stimulated APP overexpression in cultured astrocytes. The immunosuppressants cyclosporin A and FK-506 inhibited the increase in APP mRNA and holoprotein levels caused by PGE2 or by other treatments that elevated cellular cAMP levels; the inhibitory effect of FK-506 but not of cyclosporin A was attenuated by rapamycin. These results suggest that prostaglandins produced by brain injury or inflammation can activate APP transcription in astrocytes and that immunosuppressants may be used to prevent APP overexpression and possibly the pathophysiological processes underlying AD.

Fig. 1.

Effect of PGE₂ on cellular and secreted APP and on cAMP production in cultured astrocytes.A, Representative immunoblots show that 0.1, 1, 10, and 100 μm PGE₂ treatment for 24 hr stimulated dose-dependent increases in cellular APP holoprotein. The levels of APP holoprotein as measured by mAb 22C11, antisera R37, or antisera R98 did not differ significantly. B, The graph represents the means and SEM of APP holoprotein levels stimulated by different concentrations of PGE₂ (*p < 0.05). Densitometric analysis of APP levels using mAb 22C11 (n = 2), antiserum R37 (n = 2), or antiserum R98 (n = 1) were expressed as arbitrary values and normalized to the levels obtained from untreated, control cells. C, Representative immunoblots showing the levels of APP released into the media, as detected by mAb 22C11 or antiserum C8. Both immunoblots revealed increased amounts of secreted APP after PGE₂ treatment for 24 hr compared with control cells. Similar results were obtained from a subsequent experiment.D, Dose-dependent increases in cellular cAMP levels obtained by PGE₂ treatment of astrocytes. Graph represents means and SEM from a representative experiment performed on duplicate dishes (*p < 0.05).

Fig. 2.

Effect of actinomycin D (Act D) or cycloheximide (Cyclohex) on the increase in APP holoprotein stimulated by PGE₂. Actinomycin D or cycloheximide (both 2.5 μm) effectively inhibited the increase in APP holoprotein stimulated by PGE₂ (10 μm) but had no significant effect on basal APP levels. Graph represents means and SEM from four independent experiments (*p < 0.05).

Fig. 3.

Effect of 8Br-cAMP or forskolin on cAMP production and APP synthesis in cultured astrocytes. A, Cellular cAMP levels in astrocytes stimulated by 8Br-cAMP (250 μm) or forskolin (50 μm). Graph represents means and SEM from a representative experiment performed on duplicate dishes (*p < 0.05). B, Representative Northern and Western blots show that APP mRNA, APP holoprotein (Holo APP), and GFAP levels are increased by 8Br-cAMP (250 μm) or forskolin (50 μm). The amount of RNA loaded for each lane on Northern blots was not different as measured by G3PDH mRNA levels.

Fig. 4.

Effect of PKA activation and inhibition on APP synthesis in cultured astrocytes. A, APP holoprotein (Holo APP) levels are increased by activation of PKA with Sp-cAMPS triethylamine. Graph represents means and SEM from three independent experiments (*p < 0.05).B, APP mRNA and APP holoprotein increases stimulated by PGE₂ (10 μm) are blocked by the PKA inhibitor H-89 (10 μm). APP was detected with antiserum R98 directed at the KPI motif of APP. These results were replicated in subsequent experiments using mAb 22C11 or R37 directed at the N and C termini of APP, respectively.

Fig. 5.

Effect of the immunosuppressant cyclosporin A or FK-506 on APP synthesis caused by PGE₂ or forskolin treatment of cultured astrocytes. A, Increases in APP mRNA caused by PGE₂ (10 μm) are inhibited by the immunosuppressant cyclosporin A (CsA) or FK-506 (both 50 μm). Graph and SEM represent data collected from three independent experiments (*p < 0.05).B, Increases in APP holoprotein caused by forskolin (50 μm) are inhibited by the immunosuppressant cyclosporin A (CsA) or FK-506 (both 50 μm). Graph and SEM represent data collected from three independent experiments (*p < 0.05). C, Representative Northern and Western blots show that the increases in APP mRNA and APP holoprotein stimulated by PGE₂ (10 μm), but not the increases in GFAP levels, were inhibited by cyclosporin A (CsA) or FK-506 (both 50 μm).

Fig. 6.

Effect of the immunosuppressant cyclosporin A or FK-506 on cAMP production caused by PGE₂ treatment of cultured astrocytes. Cellular cAMP levels stimulated by PGE₂ are not inhibited by the immunosuppressant cyclosporin A (CsA) or by FK-506 (both 50 μm). Graph represents means and SEM from a typical experiment conducted on duplicate dishes (*p < 0.05).

Fig. 7.

Effect of rapamycin on the FK506- or cyclosporin A-mediated inhibition of APP increases in astrocytes treated with PGE₂. Rapamycin (1 μm) antagonized the inhibitory effect of FK-506 (0.1 μm) but not of cyclosporin A (1 μm) on PGE₂-mediated increases in APP holoprotein. Graph represents means and SEM from three independent experiments (*p < 0.05).