Prostaglandin Transporter (PGT/SLCO2A1) Protects the Lung from Bleomycin-Induced Fibrosis

<div><p>Prostaglandin (PG) E₂ exhibits an anti-fibrotic effect in the lung in response to inflammatory reactions and is a high-affinity substrate of PG transporter (SLCO2A1). The present study aimed to evaluate the pathophysiological relevance of SLCO2A1 to bleomycin (BLM)-induced pulmonary fibrosis in mice. Immunohistochemical analysis indicated that Slco2a1 protein was expressed in airway and alveolar type I (ATI) and II (ATII) epithelial cells, and electron-microscopic immunohistochemistry further demonstrated cell surface expression of Slco2a1 in ATI cells in wild type (WT) C57BL/6 mice. PGE₂ uptake activity was abrogated in ATI-like cells from <i>Slco2a1</i>-deficient (<i>Slco2a1</i>^-/-) mice, which was clearly observed in the cells from WT mice. Furthermore, the PGE₂ concentrations in lung tissues were lower in <i>Slco2a1</i>^-/- than in WT mice. The pathological relevance of SLCO2A1 was further studied in mouse BLM-induced pulmonary fibrosis models. BLM (1 mg/kg) or vehicle (phosphate buffered saline) was intratracheally injected into WT and <i>Slco2a1</i>^-/- mice, and BLM-induced fibrosis was evaluated on day 14. BLM induced more severe fibrosis in <i>Slco2a1</i>^-/- than in WT mice, as indicated by thickened interstitial connective tissue and enhanced collagen deposition. PGE₂ levels were higher in bronchoalveolar lavage fluid, but lower in lung tissues of <i>Slco2a1</i>^-/- mice. Transcriptional upregulation of TGF-β1 was associated with enhanced gene transcriptions of downstream targets including plasminogen activator inhitor-1. Furthermore, Western blot analysis demonstrated a significant activation of protein kinase C (PKC) δ along with a modest activation of Smad3 in lung from <i>Slco2a1</i>^-/- mice, suggesting a role of PKCδ associated with TGF-β signaling in aggravated fibrosis in BLM-treated <i>Slco2a1</i>^-/- mice. In conclusion, pulmonary PGE₂ disposition is largely regulated by SLCO2A1, demonstrating that SLCO2A1 plays a critical role in protecting the lung from BLM-induced fibrosis.</p></div

Expression of functional Slco2a1 in rat and mouse alveolar epithelial cells.

<p>(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 <i>Slco2a1</i>^-/- (top) and WT (bottom) mice. (I) [³H]PGE₂ (3 nM) uptake by ATI-L cells from WT and <i>Slco2a1</i>^-/- 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).</p

BLM-induced pulmonary fibrosis in WT and <i>Slco2a1</i>-/- mice.

<p>(A) Slco2a1 protein expression was confirmed by Western blot analysis in lung homogenates prepared from WT and <i>Slco2a1</i>^-/- 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 <i>Slco2a1</i>^-/- (dotted line, n = 5) mouse is shown up to day 13. One <i>Slco2a1</i>^-/- 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 <i>Slco2a1</i>^-/- 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 <i>Slco2a1</i>^-/- (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).</p

Western blot analyses of PGE₂-related proteins and key signaling molecules in fibrosis.

<p>(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 <i>Slco2a1</i>^-/- 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 <i>Slco2a1</i>^-/- (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).</p

Disposition of PGE₂ in the lung and BALF from WT and <i>Slco2a1</i>-/- mice.

<p>(A) Endogenous PGE₂, PGF_2α, and 15-keto PGE₂ concentrations were analyzed using LC-MS/MS in lung homogenates of WT (open column) and <i>Slco2a1</i>^-/- (closed column) mice. (B) Amounts of PGE₂ and PGF_2α 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).</p

Immunohistochemical examination of Slco2a1 in mouse lung.

<p>(A-D) DAB immunohistochemistry was performed to examine Slco2a1 expression in mouse lungs. WT (A-C) and <i>Slco2a1</i>^-/- (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.</p

mRNA expression of fibrosis-related genes.

<p>Fibrosis-related gene expression was evaluated by quantifying mRNA expression; <i>Tgf-β1</i> (A), <i>α-Sma</i> (B), <i>Fgf-2</i> (C), <i>Col1a1</i> (D), <i>Col1a2</i> (E), and <i>Pai-1</i> (F). Each bar represents the mean of four individual determinants of WT (open column) or <i>Slco2a1</i>^-/- (closed column) mice with SEM. Student’s t-test was used for statistical analysis (*, p < 0.05, **, p <0.01).</p