S1P Stimulates Erythropoietin Production in Mouse Renal Interstitial Fibroblasts by S1P1 and S1P3 Receptor Activation and HIF-2α Stabilization

Abstract

Erythropoietin (Epo) is the critical hormone for erythropoiesis. In adults, Epo is mainly produced by a subset of interstitial fibroblasts in the kidney, with minor amounts being produced in the liver and the brain. In this study, we used the immortalized renal interstitial fibroblast cell line FAIK F3-5 to investigate the ability of the bioactive sphingolipid sphingosine 1-phosphate (S1P) to stimulate Epo production and to reveal the mechanism involved. Stimulation of cells with exogenous S1P under normoxic conditions (21% O2) led to a dose-dependent increase in Epo mRNA and protein levels and subsequent release of Epo into the medium. S1P also enhanced the stabilization of HIF-2α, a key transcription factor for Epo expression. S1P-stimulated Epo mRNA and protein expression was abolished by HIF-2α mRNA knockdown or by the HIF-2 inhibitor compound 2. Furthermore, the approved S1P receptor modulator FTY720, and its active form FTY720-phosphate, both exerted a similar effect on Epo expression as S1P. The effect of S1P on Epo was antagonized by the selective S1P1 and S1P3 antagonists NIBR-0213 and TY-52156, but not by the S1P2 antagonist JTE-013. Moreover, inhibitors of the classical MAPK/ERK, the p38-MAPK, and inhibitors of protein kinase (PK) C and D all blocked the effect of S1P on Epo expression. Finally, the S1P and FTY720 effects were recapitulated in the Epo-producing human neuroblastoma cell line Kelly, suggesting that S1P receptor-dependent Epo synthesis is of general relevance and not species-specific. In summary, these data suggest that, in renal interstitial fibroblasts, which are the primary source of plasma Epo, S1P1 and 3 receptor activation upregulates Epo under normoxic conditions. This may have a therapeutic impact on disease situations such as chronic kidney disease, where Epo production is impaired, causing anemia, but it may also have therapeutic value as Epo can mediate additional tissue-protective effects in various organs.

Sphk1 and Sphk2 Differentially Regulate Erythropoietin Synthesis in Mouse Renal Interstitial Fibroblast-like Cells

“…Based on our data, we propose the following mechanism of Epo regulation by Sphk2: inhibition or downregulation of Sphk2 leads to an immediate accumulation of sphingosine, which feeds into the Sphk1 system to produce more iS1P which is exported by the S1P transporter Spns2 or another transporter. Exported S1P will then act as an autocrine ligand of S1P 1 and S1P 3 receptors to promote HIF-2α stabilization and increase Epo synthesis, like previously described [ 44 ]. This proposed mechanism is underlined by the findings that: (i) the enhanced Epo synthesis in Sphk2−/− is abolished by the combined addition of the selective S1P 1 and S1P 3 antagonists NIBR-0213 [ 49 ] and TY52156 [ 50 ]; (ii) the addition of exogenous Sph also enhances HIF-2α and Epo, which is antagonized by NIBR-0213 and TY52156; (iii) blockade of both Sphks prevents iS1P generation and export, and decreases HIF-2α and Epo; and (iv) blockade of both kinases also prevents exogenous Sph-increased HIF-2α and Epo.…”

“…For hypoxia exposures, a hypoxia chamber (Whitley H35 HEPA Hypoxystation; Don Whitley Scientific) was used at 1% oxygen and 5% CO 2 . Cell stimulation and homogenization was done exactly as previously described [ 44 ]. Protein lysates containing equal amounts of protein were separated by SDS-PAGE, transferred to nitrocellulose membranes by wet blotting using a buffer containing 25 mM Tris-HCl pH 8.3, 190 mM glycine and 20% ( v / v ) methanol, or by semi-dry Fast blotting (Bio-Rad Laboratories Inc., Hercules, CA, USA) with the recommended buffer.…”

“…RNA extraction was done exactly as previously described [ 44 ]. First strand cDNA was synthesized using 2 µg of total RNA as template for reverse transcription (RT).…”

“…In a recent study, we showed that in the immortalized mouse REPC-derived cell line FAIK F3-5, extracellular S1P (eS1P) stimulated Epo synthesis by enhancing HIF-2α stabilization [ 44 ]. However, the role of iS1P on Epo production is still not addressed.…”

Fount, fate, features, and function of renal erythropoietin-producing cells

“…Therefore, we reasoned that isolation of cells acutely tagged for an "open" Epo locus by conditional Cre induction may enhance the chance of obtaining functional REP cells. Following immortalization with a large T antigen, we could reproducibly generate such cell lines [46,50], but the Epo mRNA and protein levels were still quite low when compared to the human hepatoma and neuroblastoma models. Therefore, we generated additional REP-derived (REPD) cell lines from Epo-Cre ERT2 reporter mice, but used a heat-sensitive SV40 large T antigen for the immortalization [4,5].…”