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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.
Renal glomerular diseases such as glomerulosclerosis and diabetic nephropathy often result in the loss of glomerular function and consequently end-stage renal disease. The glomerulus consists of endothelial cells, mesangial cells and glomerular epithelial cells also referred to as podocytes. A fine-tuned crosstalk between glomerular cells warrants control of growth factor synthesis and of matrix production and degradation, preserving glomerular structure and function. Hydrogen sulfide (H2S) belongs together with nitric oxide (NO) and carbon monoxide (CO) to the group of gasotransmitters. During the last three decades, these higher concentration toxic gases have been found to be produced in mammalian cells in a well-coordinated manner. Recently, it became evident that H2S and the other gasotransmitters share common targets as signalling devices that trigger mainly protective pathways. In several animal models, H2S has been demonstrated as a protective factor in the context of kidney disorders, in particular of diabetic nephropathy. Here, we focus on the synthesis and action of H2S in glomerular cells, its beneficial effects in the glomerulus and its action in the context of the other gaseous signalling molecules NO and CO.
S1P and its receptors have been reported to play important roles in the development of renal fibrosis. Although S1P5 has barely been investigated so far, there are indications that it can influence inflammatory and fibrotic processes. Here, we report the role of S1P5 in renal inflammation and fibrosis. Male S1P5 knockout mice and wild-type mice on a C57BL/6J background were fed with an adenine-rich diet for 7 days or 14 days to induce tubulointerstitial fibrosis. The kidneys of untreated mice served as respective controls. Kidney damage, fibrosis, and inflammation in kidney tissues were analyzed by real-time PCR, Western blot, and histological staining. Renal function was assessed by plasma creatinine ELISA. The S1P5 knockout mice had better renal function and showed less kidney damage, less proinflammatory cytokine release, and less fibrosis after 7 days and 14 days of an adenine-rich diet compared to wild-type mice. S1P5 knockout ameliorates tubular damage and tubulointerstitial fibrosis in a model of adenine-induced nephropathy in mice. Thus, targeting S1P5 might be a promising goal for the pharmacological treatment of kidney diseases.
Sphingosine 1-phosphate (S1P) is a lipid mediator with numerous biological functions. The term ‘S1P’ mainly refers to the sphingolipid molecule with a long-chain sphingoid base of 18 carbon atoms, d18:1 S1P. The enzyme serine palmitoyltransferase catalyses the first step of the sphingolipid de novo synthesis using palmitoyl-CoA as the main substrate. After further reaction steps, d18:1 S1P is generated. However, also stearyl-CoA or myristoyl-CoA can be utilised by the serine palmitoyltransferase, which at the end of the S1P synthesis pathway, results in the production of d20:1 S1P and d16:1 S1P respectively. We measured these S1P homologues in mice and renal tissue of patients suffering from renal cell carcinoma (RCC). Our experiments highlight the relevance of d16:1 S1P for the induction of connective tissue growth factor (CTGF) in the human renal clear cell carcinoma cell line A498 and human RCC tissue. We show that d16:1 S1P versus d18:1 and d20:1 S1P leads to the highest CTGF induction in A498 cells via S1P2 signalling and that both d16:1 S1P and CTGF levels are elevated in RCC compared to adjacent healthy tissue. Our data indicate that d16:1 S1P modulates conventional S1P signalling by acting as a more potent agonist at the S1P2 receptor than d18:1 S1P. We suggest that elevated plasma levels of d16:1 S1P might play a pro-carcinogenic role in the development of RCC via CTGF induction.
Although the human immune response to cancer is naturally potent, it can be severely disrupted as a result of an immunosuppressive tumor microenvironment. Infiltrating regulatory T lymphocytes contribute to this immunosuppression by inhibiting proliferation of cytotoxic CD8+ T lymphocytes, which are key to an effective anti-cancer immune response. Other important contributory factors are thought to include metabolic stress caused by the local nutrient deprivation common to many solid tumors. Interleukin-33 (IL-33), an alarmin released in reaction to cell damage, and sphingosine-1-phosphate (S1P) are known to control cell positioning and differentiation of T lymphocytes. In an in vitro model of nutrient deprivation, we investigated the influence of IL-33 and S1P receptor 4 (S1P4) on the differentiation and migration of human CD8+ T lymphocytes. Serum starvation of CD8+ T lymphocytes induced a subset of CD8Low and IL-33 receptor-positive (ST2L+) cells characterized by enhanced expression of the regulatory T cell markers CD38 and CD39. Both S1P1 and S1P4 were transcriptionally regulated after stimulation with IL-33. Moreover, expression of the chemokine receptor CXCR4 was increased in CD8+ T lymphocytes treated with the selective S1P4 receptor agonist CYM50308. We conclude that nutrient deprivation promotes CD8Low T lymphocytes, contributing to an immunosuppressive microenvironment and a poor anti-cancer immune response by limiting cytotoxic effector functions. Our results suggest that S1P4 signaling modulation may be a promising target for anti-CXCR4 cancer immunotherapy.
Sphingosine 1 phosphate (S1P) lyase (Sgpl1) catalyses the irreversible cleavage of S1P and thereby the last step of sphingolipid degradation. Loss of Sgpl1 in humans and mice leads to accumulation of sphingolipids and multiple organ injuries. Here, we addressed the role of hepatocyte Sgpl1 for regulation of sphingolipid homoeostasis by generating mice with hepatocyte-specific deletion of Sgpl1 (Sgpl1HepKO mice). Sgpl1HepKO mice had normal body weight, liver weight, liver structure and liver enzymes both at the age of 8 weeks and 8 months. S1P, sphingosine and ceramides, but not glucosylceramides or sphingomyelin, were elevated by ~1.5–2-fold in liver, and this phenotype did not progress with age. Several ceramides were elevated in plasma, while plasma S1P was normal. Interestingly, S1P and glucosylceramides, but not ceramides, were elevated in bile of Sgpl1HepKO mice. Furthermore, liver cholesterol was elevated, while LDL cholesterol decreased in 8-month-old mice. In agreement, the LDL receptor was upregulated, suggesting enhanced uptake of LDL cholesterol. Expression of peroxisome proliferator-activated receptor-γ, liver X receptor and fatty acid synthase was unaltered. These data show that mouse hepatocytes largely compensate the loss of Sgpl1 by secretion of accumulating sphingolipids in a specific manner into blood and bile, so that they can be excreted or degraded elsewhere.
Experimental work on highly pathogenic viruses such as Ebola virus (EBOV) and severe acute respiratory syndrome coronavirus-2 requires high-level biosafety facilities. Here, we provide a detailed step-by-step protocol which details the production and application of replication-incompetent murine leukemia virus-based pseudotyped particles to monitor and quantify the viral entry efficiency in human cell lines under biosafety level-2 conditions. We describe the use of viral particles encoding luciferase gene and the quantification of transduction efficiency by measuring luciferase activity.
Sphingosine 1-phosphate (S1P) signaling influences numerous cell biological mechanisms such as differentiation, proliferation, survival, migration, and angiogenesis. Intriguingly, our current knowledge is based solely on the role of S1P with an 18-carbon long-chain base length, S1P d18:1. Depending on the composition of the first and rate-limiting enzyme of the sphingolipid de novo metabolism, the serine palmitoyltransferase, other chain lengths have been described in vivo. While cells are also able to produce S1P d20:1, its abundance and function remains elusive so far. Our experiments are highlighting the role of S1P d20:1 in the mouse central nervous system (CNS) and human glioblastoma. We show here that S1P d20:1 and its precursors are detectable in both healthy mouse CNS-tissue and human glioblastoma. On the functional level, we focused our work on one particular, well-characterized pathway, the induction of cyclooxygenase (COX)-2 expression via the S1P receptor 2 (S1P2). Intriguingly, S1P d20:1 only fairly induces COX-2 expression and can block the S1P d18:1-induced COX-2 expression mediated via S1P2 activation in the human glioblastoma cell line LN229. This data indicates that S1P d20:1 might act as an endogenous modulator of S1P signaling via a partial agonism at the S1P2 receptor. While our findings might stimulate further research on the relevance of long-chain base lengths in sphingolipid signaling, the metabolism of S1P d20:1 has to be considered as an integral part of S1P signaling pathways in vivo.
Colorectal cancer (CRC) is one of the most frequently diagnosed tumor in humans and one of the most common causes of cancer-related death worldwide. The pathogenesis of CRC follows a multistage process which together with somatic gene mutations is mainly attributed to the dysregulation of signaling pathways critically involved in the maintenance of homeostasis of epithelial integrity in the intestine. A growing number of studies has highlighted the critical impact of members of the tripartite motif (TRIM) protein family on most types of human malignancies including CRC. In accordance, abundant expression of many TRIM proteins has been observed in CRC tissues and is frequently correlating with poor survival of patients. Notably, some TRIM members can act as tumor suppressors depending on the context and the type of cancer which has been assessed. Mechanistically, most cancer-related TRIMs have a critical impact on cell cycle control, apoptosis, epithelial–mesenchymal transition (EMT), metastasis, and inflammation mainly through directly interfering with diverse oncogenic signaling pathways. In addition, some recent publications have emphasized the emerging role of some TRIM members to act as transcription factors and RNA-stabilizing factors thus adding a further level of complexity to the pleiotropic biological activities of TRIM proteins. The current review focuses on oncogenic signaling processes targeted by different TRIMs and their particular role in the development of CRC. A better understanding of the crosstalk of TRIMs with these signaling pathways relevant for CRC development is an important prerequisite for the validation of TRIM proteins as novel biomarkers and as potential targets of future therapies for CRC.
Emerging evidence suggests a complex relationship between sphingosine 1-phosphate (S1P) signaling and stroke. Here, we show the kinetics of S1P in the acute phase of ischemic stroke and highlight accompanying changes in immune cells and S1P receptors (S1PR). Using a C57BL/6 mouse model of middle cerebral artery occlusion (MCAO), we assessed S1P concentrations in the brain, plasma, and spleen. We found a steep S1P gradient from the spleen towards the brain. Results obtained by qPCR suggested that cells expressing the S1PR type 1 (S1P1+) were the predominant population deserting the spleen. Here, we report the cerebral recruitment of T helper (TH) and regulatory T (TREG) cells to the ipsilateral hemisphere, which was associated with differential regulation of cerebral S1PR expression patterns in the brain after MCAO. This study provides insight that the S1P-S1PR axis facilitates splenic T cell egress and is linked to the cerebral recruitment of S1PR+ TH and TREG cells. Further insights by which means the S1P-S1PR-axis orchestrates neuronal positioning may offer new therapeutic perspectives after ischemic stroke.