Nuclear localization of prostaglandin E2 receptors

Abstract

Prostaglandin E2 receptors (EP) were detected by radioligand binding in nuclear fractions isolated from porcine brain and myometrium. Intracellular localization by immunocytofluorescence revealed perinuclear localization of EPs in porcine cerebral microvascular endothelial cells. Nuclear association of EP1 was also found in fibroblast Swiss 3T3 cells stably overexpressing EP1 and in human embryonic kidney 293 (Epstein-Barr virus-encoded nuclear antigen) cells expressing EP1 fused to green fluorescent protein. High-resolution immunostaining of EP1 revealed their presence in the nuclear envelope of isolated (cultured) endothelial cells and in situ in brain (cortex) endothelial cells and neurons. Stimulation of these nuclear receptors modulate nuclear calcium and gene transcription.

Figure 1

Competitive displacement of [³H]PGE₂ binding to subcellular fractions from porcine adult myometrium and newborn brain by prostaglandins and analogs: fractions 1 (a) and 4 (b) from myometrium and fractions 1 (c) and 4 (d) from brain. Fraction 1 contained plasma membrane and fraction 4 contained nuclear membrane. Membranes were incubated with 10 nM [³H]PGE₂ and increasing concentrations of prostaglandins and analogs. ○, PGE₂; ■ AH6809; ♦, Butaprost; •, M&B 28,767; ▴, AH23848B. Each point is mean ± SE of three experiments, in duplicate.

Figure 2

EP₁ immunofluorescence of ectopically expressed human EP₁ receptor in HEK293(EBNA) cells. (a) Cells transfected with vector alone; note the absence of fluorescence. (b) Nuclear stain (DAPI) of cells from a. (c) An EP₁-overexpressing clone; note the perinuclear halo. (d) Nuclear stain (DAPI) of cells from c.

Figure 3

Confocal microscopic images of EP₁ immunostaining in porcine cerebromicrovascular endothelial cells. (a) Anti-EP₁ antibody and Texas red-conjugated IgG. (b) DiOC₆(3), intracellular membranes (mostly endoplasmic reticulum) stain. (c) superimposed images of a and b. (d) Anti-EP₁ in the presence of cognate peptide (10 μg/ml) and nuclear stain (propidium iodide); note the absence of immunostaining.

Figure 4

EP₁ immunofluorescence of ectopically expressed EP₁ receptor in murine Swiss 3T3 cells: (a) Cells transfected with vector alone. (b) EP₁-overexpressing clone; note the intense perinuclear halo. Confocal microscopic images of EP₁ overexpressing cells. (c) Anti-EP₁ antibody and Texas red-conjugated IgG. (d) DiOC₆(3), intracellular membranes (mostly ER) stain. (e) Superimposed images of c and d. (f) Anti-EP₁ antibody and Texas red-conjugated IgG. (g) Sytox green nucleus stain. (h) Superimposed images of f and g. (i) Z section of h.

Figure 5

GFP protein localization in transfected HEK293(EBNA) cells. a and b are optical fluorescent microscopic images and c and d are confocal images. (a) GFP alone; note the diffuse staining pattern. (b and c) EP₁–GFP fusion protein; perinuclear halo at higher confocal magnification (c). (d) Nuclear stain (propidium iodide) of cells from b.

Figure 6

Immunoperoxidase and immunogold localization of EP₁ in porcine endothelial cells detected by electron microscopy (arrows). (a) Immunoperoxidase-IgG alone; note absence of immunostaining when primary antibody is omitted. (b) A low magnification showing immunostaining in plasma membrane and nuclear envelope. (c) A higher magnification showing immunostaining in the nuclear envelope. (d) Immunogold-IgG alone; note absence of immunostaining. Specific immunostaining can be observed in the plasma membrane in e, nuclear envelope in f, and Golgi apparatus in g. (Bar = 0.5 μm, except in b = 2 μm.)

Figure 7

Immunoperoxidase localization of EP₁ in adult rat brain cortex by electron microscopy (arrows). Specific immunostaining observed in plasma membrane and inner and outer nuclear membranes of capillary endothelial cell (a) and nuclear membranes of neurons (b). Note the luminal space of capillary on right in a. (Bars = 0.5 μm.)

Figure 8

Effects of EP₁ agonist, 17-phenyltrinor PGE₂ (17-P) and AH6809 (EP₁ antagonist, 10 μM) on calcium levels in isolated myometrial nuclei loaded with fura-2 AM (a and b). (a) Typical tracings; arrow shows time of compound or vehicle administration; 17-P (1 μM). (b) Peak increases in intranuclear calcium concentrations [(Ca²⁺)_n] after addition of 17-P (filled bars) or 17-P and AH6809 (10 μM; open bars). Effects of 17-phenyltrinor PGE₂ (0.1 μM) on c-fos transcription in native and EP₁ over-expressing Swiss 3T3 cells as measured by dot blot hybridization of RNA. (c) Dot blot; Cont, control without drug. β-actin dot blot is of parent cells; its intensity was unaffected by EP₁ cDNA transfection. (d) Densitometry of dot blots; c-fos RNA abundance corrected for β-actin RNA is expressed as percentage of native untreated controls (open bar). Data are means ± SE of three experiments. ∗, P < 0.05 compared with the corresponding control.