Fig 1.
Immunohistochemical examination of Slco2a1 in mouse lung.
(A-D) DAB immunohistochemistry was performed to examine Slco2a1 expression in mouse lungs. WT (A-C) and Slco2a1-/- (D) mouse lung cryosections (10 μm) incubated with anti-Slco2a1 antibody were stained brown by immunoenzymatic reaction with DAB in the absence (A, B, D) or presence of antigenic peptide (C). (E, F) Fluorescent immunostaining confirmed DAB staining of Slco2a1 expression. Sections were labeled with Alexa Fluor 594-conjugated secondary antibody and nuclei were stained blue with Hoechst 33342. (G) Electron-microscopic immunohistochemistry detected DAB staining of Slco2a1 in alveoli. (H) Semi-thin sections (4 μm) were also stained with DAB of Slco2a1. (I, J) DAB immunohistochemical analysis was performed to examine Pges (I) and 15-Pgdh (J) in the lung. Nuclei were counter-stained blue with hematoxylin (A-D, H-J). A, AE, AS, AW, B, BV, C and VE indicate alveoli, airway epithelial cells, alveolar sac, airway, bronchiole, blood vessel, capillary, and vascular endothelial cells, respectively.
Fig 2.
Expression of functional Slco2a1 in rat and mouse alveolar epithelial cells.
(A-D) Fluorescent immunostaining for pro-SPC and Slco2a1 was performed in rat AECs in primary culture. Expression of pro-SPC (green) and Slco2a1 (red) was immunohistochmically detected in ATII (A, C) and ATI-L cells (B, D) primarily cultured from lung tissue of rats. (E) Uptake of [3H]PGE2 (3 nM) by ATII (open circles) and ATI-L (closed circles) cells in primary culture from rats was measured over 20 min at 37°C and pH 7.4 (F, G). The effect of various compounds on [3H]PGE2 (1.5 nM) uptake by rat ATI-L cells in primary culture was measured using unlabeled PGE2 (100 μM), TGBz (25 μM), BSP (a known inhibitor of SLCO2A1, 25 μM), CAR (carnitine, 1000 μM) and TEA (tetraethylammonium, 100 μM) for 5 min (F) and MK571 (25 μM) for 20 min (G). Uptake of [3H]PGE2 was normalized by the value obtained without any inhibitors (Control). (H) Immunofluorescence for anti-Slco2a1 (red) was confirmed in ATII and ATI-L cells (on Day 6) from Slco2a1-/- (top) and WT (bottom) mice. (I) [3H]PGE2 (3 nM) uptake by ATI-L cells from WT and Slco2a1-/- mice was measured. Each point or bar represents the mean ± SEM (at least n = 3). Student’s t-test was used for statistical analysis (*; p < 0.05, **; p <0.01, and ***; p<0.001).
Fig 3.
Disposition of PGE2 in the lung and BALF from WT and Slco2a1-/- mice.
(A) Endogenous PGE2, PGF2α, and 15-keto PGE2 concentrations were analyzed using LC-MS/MS in lung homogenates of WT (open column) and Slco2a1-/- (closed column) mice. (B) Amounts of PGE2 and PGF2α recovered in BALF were quantified by LC-MS/MS. Concentration was normalized by wet weight of tissues. Each column represents the mean (n = 4) + SEM. Student’s t-test was used for statistical analysis (*, p < 0.05).
Fig 4.
BLM-induced pulmonary fibrosis in WT and Slco2a1-/- mice.
(A) Slco2a1 protein expression was confirmed by Western blot analysis in lung homogenates prepared from WT and Slco2a1-/- mice. (B) Slco2a1 protein expression was examined by Western blot analysis in lung homogenates from PBS- and BLM-treated WT mice. Western blot analysis was repeated at least three times using lung homogenates prepared from three mice, and a representative picture is shown. (C) Immunohistochemical analysis of Slco2a1 expression is shown in the lungs of BLM-treated WT mice. Figure shows a typical image of DAB staining with guinea pig anti-Slco2a1 antibodies. Nuclei were stained by hematoxylin. (D) Body weight of each WT (solid line, n = 4) or Slco2a1-/- (dotted line, n = 5) mouse is shown up to day 13. One Slco2a1-/- mouse died of severe fibrosis, and no other symptoms were observed. (E) Typical images of hematoxylin and eosin staining of lung sections are shown at low magnification (× 4); left panel shows Slco2a1-/- and right panel shows WT mice. (F) Typical images of Picrosirius Red staining of lung sections are presented at low (× 4, top panels) and high magnification (× 40, bottom panels). (G) The % of the area stained by Picrosirius Red was significantly increased in Slco2a1-/- (closed column), compared to the WT (open column) mice. Each bar represents the mean value of randomly selected 19 Picrosirius Red-stained images from at least 4 mice from each group. Student’s t-test was used for statistical analysis (**, p <0.01).
Fig 5.
PGE2 levels in lung tissue homogenates and BALF of BLM-treated mice.
(A) PGE2 concentrations were quantified in lung tissue homogenates of WT (open column) and Slco2a1-/- (closed column) mice. (B) Amounts of PGE2 in BALF were measured by means of LC-MS/MS as described in Material and Methods or S1 Table. Each column shows the mean of four individual determinants with SEM. Student’s t-test was used for statistical analysis (**, p<0.01).
Fig 6.
mRNA expression of fibrosis-related genes.
Fibrosis-related gene expression was evaluated by quantifying mRNA expression; Tgf-β1 (A), α-Sma (B), Fgf-2 (C), Col1a1 (D), Col1a2 (E), and Pai-1 (F). Each bar represents the mean of four individual determinants of WT (open column) or Slco2a1-/- (closed column) mice with SEM. Student’s t-test was used for statistical analysis (*, p < 0.05, **, p <0.01).
Fig 7.
Western blot analyses of PGE2-related proteins and key signaling molecules in fibrosis.
(A) Expression of proteins related to PGE2 metabolism and activation of signaling molecules were studied by Western blot analysis using lung homogenates prepared from WT and Slco2a1-/- mice (six per each group), and representative images are shown. (B) Quantitative analysis of each protein or phosphorylation was performed. Degree of expression of Cox2 and 15-Pgdh are shown by normalizing band intensity with that corresponding to Gapdh. Activation of phosphorylation of molecules was shown by normalizing band intensity for phosphorylated protein over the intensity for its total expression. Bands for phosphorylation of PKCδ and PKCθ were distinguished by molecular size on the blots. Phosphorylation of PKCα and βII was not distinguished by molecular size; therefore, we normalized their phosphorylation with total expression of PKCα and then the ratio was compared. Expression or degree of activation was compared between WT (open column) and Slco2a1-/- (closed column) mice, and each column represents the mean with SEM (lung tissues from 5 or 6 individual mice). Student’s t-test was used for statistical analysis (*, p < 0.05).