Validation of highly selective sphingosine kinase 2 inhibitors SLM6031434 and HWG-35D as effective anti-fibrotic treatment options in a mouse model of tubulointerstitial fibrosis

Schwalm, Stephanie; Beyer, Sandra; Hafizi, Redona; Trautmann, Sandra; Geißlinger, Gerd; Adams, David H.; Pyne, Susan; Pyne, Nigel J.; Schaefer, Liliana; Huwiler, Andrea; Pfeilschifter, Josef

doi:10.1016/j.cellsig.2020.109881

Cited by 12 publications

(7 citation statements)

References 30 publications

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“…To investigate whether the Epo-enhancing effect by loss of Sphk2 can be mimicked by a Sphk2 inhibitor, we treated wildtype primary fibroblasts for 24 h with the two highly selective Sphk2 inhibitors SLM6031434 and HWG-35D. Both inhibitors were previously shown to block Sphk2 activity in vitro [ 46 , 47 ], as well as in vivo [ 48 ]. Both inhibitors, at a concentration of 3 µM, enhanced HIF-2α protein and upregulated Epo protein ( Figure 5 A) and mRNA expression ( Figure 5 B).…”

Section: Resultsmentioning

confidence: 99%

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

Hafizi

Imeri

Tanturovska

et al. 2022

IJMS

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Erythropoietin (Epo) is a crucial hormone regulating red blood cell number and consequently the hematocrit. Epo is mainly produced in the kidney by interstitial fibroblast-like cells. Previously, we have shown that in cultures of the immortalized mouse renal fibroblast-like cell line FAIK F3-5, sphingosine 1-phosphate (S1P), by activating S1P1 and S1P3 receptors, can stabilize hypoxia-inducible factor (HIF)-2α and upregulate Epo mRNA and protein synthesis. In this study, we have addressed the role of intracellular iS1P derived from sphingosine kinases (Sphk) 1 and 2 on Epo synthesis in F3-5 cells and in mouse primary cultures of renal fibroblasts. We show that stable knockdown of Sphk2 in F3-5 cells increases HIF-2α protein and Epo mRNA and protein levels, while Sphk1 knockdown leads to a reduction of hypoxia-stimulated HIF-2α and Epo protein. A similar effect was obtained using primary cultures of renal fibroblasts isolated from wildtype mice, Sphk1−/−, or Sphk2−/− mice. Furthermore, selective Sphk2 inhibitors mimicked the effect of genetic Sphk2 depletion and also upregulated HIF-2α and Epo protein levels. The combined blockade of Sphk1 and Sphk2, using Sphk2−/− renal fibroblasts treated with the Sphk1 inhibitor PF543, resulted in reduced HIF-2α and Epo compared to the untreated Sphk2−/− cells. Exogenous sphingosine (Sph) enhanced HIF-2α and Epo, and this was abolished by the combined treatment with the selective S1P1 and S1P3 antagonists NIBR-0213 and TY52156, suggesting that Sph was taken up by cells and converted to iS1P and exported to then act in an autocrine manner through S1P1 and S1P3. The upregulation of HIF-2α and Epo synthesis by Sphk2 knockdown was confirmed in the human hepatoma cell line Hep3B, which is well-established to upregulate Epo production under hypoxia. In summary, these data show that sphingolipids have diverse effects on Epo synthesis. While accumulation of intracellular Sph reduces Epo synthesis, iS1P will be exported to act through S1P1+3 to enhance Epo synthesis. Furthermore, these data suggest that selective inhibition of Sphk2 is an attractive new option to enhance Epo synthesis and thereby to reduce anemia development in chronic kidney disease.

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Section: Resultsmentioning

confidence: 99%

“…Confluent cells in 60-mm-diameter dishes were scraped in methanol and taken for lipid extraction and quantification of the different sphingolipids as previously described [ 48 ].…”

Section: Methodsmentioning

confidence: 99%

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

Hafizi

Imeri

Tanturovska

et al. 2022

IJMS

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“…Targeting enzymes that generate ceramide and S1P to restore a rheostat of ceramide-S1P have been considered as a therapeutic strategy in various diseases (reviewed in [ 283 , 284 ]). In the same line, the application of compounds that can reduce the ratio of ceramide/S1P by targeting the enzymes involved in ceramide production and SphKs would be beneficial to prevent oxidant-induced kidney injury ( Table 4 ) [ 27 , 43 , 62 , 66 , 71 , 74 , 75 , 76 , 78 , 85 , 94 , 198 , 200 , 207 , 219 , 226 , 260 , 264 , 281 , 284 , 285 , 286 , 287 , 288 , 289 , 290 , 291 , 292 , 293 ]. Among these compounds, fumonisin B1, for the treatment of acute rejection, where ROS are involved, and fingolimod (FTY720) have only been used in kidney transplant recipients [ 294 , 295 ].…”

Section: A Rheostat Of Ceramide-s1p In the Regulation Of Oxidative St...mentioning

confidence: 99%

A Rheostat of Ceramide and Sphingosine-1-Phosphate as a Determinant of Oxidative Stress-Mediated Kidney Injury

Ueda

2022

IJMS

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Reactive oxygen species (ROS) modulate sphingolipid metabolism, including enzymes that generate ceramide and sphingosine-1-phosphate (S1P), and a ROS-antioxidant rheostat determines the metabolism of ceramide-S1P. ROS induce ceramide production by activating ceramide-producing enzymes, leading to apoptosis, while they inhibit S1P production, which promotes survival by suppressing sphingosine kinases (SphKs). A ceramide-S1P rheostat regulates ROS-induced mitochondrial dysfunction, apoptotic/anti-apoptotic Bcl-2 family proteins and signaling pathways, leading to apoptosis, survival, cell proliferation, inflammation and fibrosis in the kidney. Ceramide inhibits the mitochondrial respiration chain and induces ceramide channel formation and the closure of voltage-dependent anion channels, leading to mitochondrial dysfunction, altered Bcl-2 family protein expression, ROS generation and disturbed calcium homeostasis. This activates ceramide-induced signaling pathways, leading to apoptosis. These events are mitigated by S1P/S1P receptors (S1PRs) that restore mitochondrial function and activate signaling pathways. SphK1 promotes survival and cell proliferation and inhibits inflammation, while SphK2 has the opposite effect. However, both SphK1 and SphK2 promote fibrosis. Thus, a ceramide-SphKs/S1P rheostat modulates oxidant-induced kidney injury by affecting mitochondrial function, ROS production, Bcl-2 family proteins, calcium homeostasis and their downstream signaling pathways. This review will summarize the current evidence for a role of interaction between ROS-antioxidants and ceramide-SphKs/S1P and of a ceramide-SphKs/S1P rheostat in the regulation of oxidative stress-mediated kidney diseases.

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“…For example, SPHK2 is important for resolving inflammatory vascular lung injury (Joshi et al, 2020). However, pharmacologically inhibiting SPHK2 leads to activation of anti-inflammatory mechanisms in models of Crohn's disease Maines et al (2010) and renal inflammation (Schwalm et al, 2021). Conversely, in a model of collagen-induced arthritis, siRNA-mediated SPHK2 downregulation leads to higher levels of proinflammatory cytokines and more aggressive disease than in control mice (Lai et al, 2009).…”

Section: Sphk2 and Inflammationmentioning

confidence: 99%

The Functional Role of Sphingosine Kinase 2

Escarcega¹,

McCullough²,

Tsvetkov

2021

Front. Mol. Biosci.

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Sphingosine-1-phosphate (S1P) is a bioactive lipid molecule that is present in all eukaryotic cells and plays key roles in various extracellular, cytosolic, and nuclear signaling pathways. Two sphingosine kinase isoforms, sphingosine kinase 1 (SPHK1) and sphingosine kinase 2 (SPHK2), synthesize S1P by phosphorylating sphingosine. While SPHK1 is a cytoplasmic kinase, SPHK2 is localized to the nucleus, endoplasmic reticulum, and mitochondria. The SPHK2/S1P pathway regulates transcription, telomere maintenance, mitochondrial respiration, among many other processes. SPHK2 is under investigation as a target for treating many age-associated conditions, such as cancer, stroke, and neurodegeneration. In this review, we will focus on the role of SPHK2 in health and disease.

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Validation of highly selective sphingosine kinase 2 inhibitors SLM6031434 and HWG-35D as effective anti-fibrotic treatment options in a mouse model of tubulointerstitial fibrosis

Cited by 12 publications

References 30 publications

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

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

A Rheostat of Ceramide and Sphingosine-1-Phosphate as a Determinant of Oxidative Stress-Mediated Kidney Injury

The Functional Role of Sphingosine Kinase 2

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