１）In alveolar epithelial cells
２）In central nervous systems
３）In colorectal cancer
１）SLC25A3
３ Transporters Associated with Prostate Cancer
４ MRP4-mediated Uric acid and PGs Transport
１) Functional expression of SLCO2A1 in alveolar epithelial cells
The lung is one of the tissues with the highest PGE2 production capacity. PGE2 is synthesized by PGES from PGH2 generated from arachidonic acid via COX1/2. There are three isoforms of PGES: cPGES, which is present in the cytoplasm, and mPGES1 and mPGES2, which are bound to microsomal membranes. PGE2 is the major metabolite of the COX2/mPGES1 pathway, which is induced in pathological conditions such as inflammation and cancer. Therefore, mPGES1 is regarded as a target molecule for anti-inflammatory effects (1). On the other hand, PGE2 is known to exhibit anti-inflammatory effects in various tissues. Under physiological conditions, the concentration of PGE2 in the bronchial epithelium is higher than that in plasma (2) and plays an important role in the rapid repair of the airway epithelium (3). Furthermore, it has been shown to be effective in preventing asthma by suppressing the activation of immune cells and limiting the production of pro-inflammatory cytokines, histamine, and leukotrienes (4). PGE2 is thought to inhibit IgE production probably because it suppresses Th2 differentiation (5). Furthermore, when lung epithelium is damaged, PGE2 plays a role in normal tissue repair by regulating epithelial cell and fibroblast proliferation. PGE2 also suppresses fibroblast proliferation and myofibroblast differentiation by limiting the production of pro-inflammatory cytokines such as IL-8, IL-12, MCP-1, and GM-CSF, thereby it is strongly involved in tissue remodeling in pathological conditions such as asthma and fibrosis.
Functional SLCO2A1 Expression in the lungs
Thus, our group has analyzed the expression and function of membrane transporters for PGs in order to clarify PG dynamics in the lower respiratory tract and alveolar epithelium. In type 1 alveolar epithelial cell (e.g., AT1-like cells; AT1L), which transdifferentiated from primary cultured rat type 2 alveolar epithelial cells (AT2), the expression of membrane transporters that accept PG as a substrate was determined by RT-PCR (Fig. 1A). The expression of Slco2a1 was the highest, and MRP4 (Abcc4), which excretes PGE2 from the cells, was also found to be expressed (6). In addition, immunohistochemistry detected strong murine Slco2a1 immunoreactivity in the lower airway epithelium, AT1 alveolar epithelial cells, and vascular endothelial cells (Fig. 1B & C), and confirmed its high RNA expression (7).
Interestingly, AT2 primary cultured cells did not express Slco2a1 protein expression in the cell membrane, but AT1L, which were cultured for additional 4 days, showed a strong Slco2a1 immunoreactivity localized along the cell membranes (Fig. 2A). Furthermore, to confirm the PG transport function of the SLCO2A1 protein, we compared [3H]PGE2 uptake between AT1L cells prepared from wild-type (Wt) and systemic Slco2a1 KO mice. Approximately 80% of the uptake activity disappeared (Fig. 2B); therefore, these results revealed that SLCO2A1 expressed in alveolar epithelial cells functions as an uptake transporter of PG from the alveolar space into cells.
Pulmonary fibrosis in Slco2a1 KO mice
In order to if SLCO2A1 is involved in tissue remodeling, bleomycin (BLM) was administered intratracheally to Wt and Slco2a1 KO mice to create fibrotic lungs. In the phosphate-buffered saline (PBS)-administered group, no structural changes in lung tissue due to Slco2a1 deficiency were observed (Fig. 3A). Two weeks after BLM administration, HE-stained images of Slco2a1 KO mouse lungs showed more extensive thickening of the alveolar walls and collapse of the alveolar region, compared to Wt mice, and strong Sirius red staining indicating the accumulation of collagen fibers (red) was observed in the entire regions of the lungs (7). Furthermore, the deletion of Slco2a1 increased significantly the mRNA expression of genes encoding Tgf-β1, which is strongly associated with tissue fibrosis, and mediator molecules located downstream of its signal, such as α-Sma and Pai-1 (Fig. 3B). Collectively, it was thought that BLM-induced tissue remodeling (fibrosis) progressed due to Slco2a1 deficiency.
Next, regarding PGE2 dynamics, we measured the PGE2 concentration in lung tissues and bronchoalveolar lavage fluids (BALF). As shown in Fig. 4A, the concentration of PGE2 in the lung tissue after BLM administration tended to decrease in KO mice. In the PBS-treated group, no significant difference was observed in the amount of PGE2 in BALF between Wt and KO mice, but 2 weeks after BLM administration, the amount of PGE2 was significantly increased in KO mice. Furthermore, metabolomic analysis targeting 48 types of eicosanoids showed that only 7 eicosanoids were detected in BLF and only PGE2 was significantly increased in KO mice (7)
Transcellular transport of PGE2 across the monolayers of AT1L cells.
To examine whether SLCO2A1 is involved not only in the accumulation of PGE2 but also in its membrane permeation, we evaluated the transcellular transport of PGE2 across the monolayers of rat AT1L cells (6). After culturing AT1L in transwells, membrane permeation of [3H]PGE2 from the apical (AP, e.g., alveolar space) side to the basolateral (BL, e.g., lung interstitial tissue) side increased in proportion to time, and decreased significantly in the presence of BSP, an inhibitor of SLCO2A1 (Fig. 5A). The membrane permeability coefficient (Pc) of PGE2 was about 20 times higher than that of the intercellular space marker mannitol, and this value decreased to about 1/4 in the presence of BSP. Interestingly, the membrane permeation from the BL side to the AP side was about 1/4 of the membrane permeation from the AP side to the BL side, and no inhibitory effect on the transport in the direction from the AP to the BL was observed in the presence of BSP (Fig. 5B). The membrane permeation of PGE2 is concentration dependent, and the Km value was estimated to be 118 nM from fitting to the Michaels-Menten equation by non-linear regression, which was comparable to the Km value for PGE2 uptake by rat AT1L cells (Fig. 5C). Since Fig. 1A suggested that rat AT1L expresses Mrp4, which is thought to play a role in PGE2 excretion, we evaluated the effect of Ceefourin 1, a selective inhibitor of MRP4 (Fig. 5D). The permeation rate of PGE2 from the AP side to the BL side was partially but significantly inhibited in the presence of Ceefourin 1. The above observation results indicate that SLCO2A1-mediated membrane permeation of PGE2 occurs selectively from the alveolar space toward the tissue, suggesting that MRP4 contributes to its excretion from cells.
Conclusion
In conclusion, SLCO2A1 was shown to play a dominant role in PGE2 uptake from the alveoli and airways. Therefore, a decrease in its function and expression leads to stasis of PGE2 in the airways or alveolar spaces, and positive feedback may occur due to excessive signal input of PGE2, which increases PGE2 production by COX2 and keeps inflammatory cells to infiltrate into the alveolar spaces. Collectively, loss of SLCO2A1 function stimulates inflammation and contributes to the chronicity of inflammation. In other words, PG membrane transporter is an important site of action for toxicity in the lung/respiratory region if there is something that inhibit or reduce the function of PGE2. Since many atmospheric xenobiotics (such as cigarette smoke) and drugs are known to exhibits pulmonary toxicity, clarifying their interactions with SLCO2A1 may help us to identify a novel mechanism of the toxicity.
References
-
Trebino, C. E., Stock, J. L., Gibbons, C. P., Naiman, B. M., Wachtmann, T. S., Umland, J. P., Pandher, K., Lapointe, J. M., Saha, S., Roach, M. L., Carter, D., Thomas, N. A., Durtschi, B. A., McNeish, J. D., Hambor, J. E., Jakobsson, P. J., Carty, T. J., Perez, J. R., and Audoly, L. P. (2003) Impaired inflammatory and pain responses in mice lacking an inducible prostaglandin E synthase. Proc Natl Acad Sci U S A 100, 9044-9049
-
Ozaki, T., Rennard, S. I., and Crystal, R. G. (1987) Cyclooxygenase metabolites are compartmentalized in the human lower respiratory tract. J Appl Physiol 62, 219-222
-
Savla, U., Appel, H. J., Sporn, P. H., and Waters, C. M. (2001) Prostaglandin E(2) regulates wound closure in airway epithelium. Am J Physiol Lung Cell Mol Physiol 280, L421-L431
-
Vancheri, C., Mastruzzo, C., Sortino, M. A., and Crimi, N. (2004) The lung as a privileged site for the beneficial actions of PGE2. Trends Immunol 25, 40-46
-
Gavett, S. H., Madison, S. L., Chulada, P. C., Scarborough, P. E., Qu, W., Boyle, J. E., Tiano, H. F., Lee, C. A., Langenbach, R., Roggli, V. L., and Zeldin, D. C. (1999) Allergic lung responses are increased in prostaglandin H synthase-deficient mice. J Clin Invest 104, 721-732
-
Nakanishi, T., Takashima, H., Uetoko, Y., Komori, H., and Tamai, I. (2019) Experimental evidence for resecretion of PGE2 across rat alveolar epithelium by OATP2A1/SLCO2A1-mediated transcellular transport. J Pharmacol Exp Ther 368, 317-325
-
Nakanishi, T., Hasegawa, Y., Mimura, R., Wakayama, T., Uetoko, Y., Komori, H., Akanuma, S., Hosoya, K., and Tamai, I. (2015) Prostaglandin transporter (PGT/SLCO2A1) protects the lung from bleomycin-induced fibrosis. PloS one 10, e0123895
2) Functional expression of SLCO2A1 in alveolar epithelial cells
一方、PGは脳の機能と密接関わります。世界保健機関WHOによれば、世界のうつ病患者は3億人前後、認知障患者は5000 万人程度と推計されています。近年、うつ病およびアルツハイマー病などの中枢神経系疾患の患者数は増加の一途を辿り、未曽有の高齢化社会を迎える今後はさらに増加すると予想されています。したがって、これらの精神疾患の予防や治療法を確立することは喫緊の課題と言えます。近年、中枢や循環血中のPG濃度が中枢神経系疾患患者で変動していることが分かってきました。ところが、中枢のPG濃度が調節される仕組みや、中枢神経系疾患におけるPGの役割は十分に解明されていません。私たちはこれまでに、SLCO2A1が脳内のミクログリアや血管内皮細胞に発現し、体温調節を担う視床下部におけるPGE2量の調節に関わることを発見しました(J Neurosci, 2018)。結果的に、SLCO2A1ノックアウトマウスでは、炎症性の発熱が抑制されることを報告しています。現在、SLCO2A1が脳内のPGE2の作用を調節するkey regulatorであると位置づけ、SLCO2A1以外のPG膜輸送体も含め、中枢のPG調節機構を明らかにすべく研究を展開しています(図2)。
