The role of sphingosine kinases and dihydroceramide desaturase in cell survival/apoptosis and inflammation pathways

Rights statement
Awarding institution
  • University of Strathclyde
Date of award
  • 2020
Thesis identifier
  • T15534
Person Identifier (Local)
  • 201580557
Qualification Level
Qualification Name
Department, School or Faculty
  • The literature details significant controversy regarding the role of dihydroceramide desaturase (Degs1) in regulating cell survival/apoptosis and therefore this study primarily examined the molecular basis of the reported opposing roles of Degs1. The sphingosine kinase inhibitor SKi [2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole] or the Degs1 inhibitor fenretinide promoted the polyubiquitination of Degs1 (Mr = 40-140 kDa) through a mechanism involving p38 mitogen-activated protein kinase (MAPK), oxidative stress, and Mdm2 (E3 ligase) in HEK293T cells.;The polyubiquitinated forms of Degs1 acquire a 'gain of function' and activate pro-survival pathways, p38 MAPK/c-Jun N-terminal kinase (JNK), and X-box protein 1s (XBP-1s) in HEK293T cells. In contrast, the sphingosine kinase inhibitor, ABC294640 [3-(4- chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide] (25 to 50 μM), did not promote formation of polyubiquitinated Degs1 forms and induced apoptosis of HEK293T cells via a mechanism involving native Degs1. Native Degs1 appears to function in this context via substrate induction to increase de novo synthesis of apoptotic ceramides.;These results were achieved using siRNA transfections, western protein analysis of protein expression, immunoprecipitation, [3H]-Thymidine incorporation assay, and mass spectrometry. These novel findings are the first to reveal that the polyubiquitinated forms of Degs1 exhibit opposing function compared with the native form, which could explain the controversy concerning the role of this enzyme in apoptosis versus cell survival described within literature.;The study next investigated the role of sphingosine kinases (SK1, SK2) in regulating p53, stress activated protein kinases, and XBP-1s in HEK293T cells since SK inhibitors are known to promote p53-dependent cell death. SKi stimulated polyubquitination of p53 to form two higher molecular mass proteins (63 and 90 kDa), which might represent inactive forms of p53. The formation of p63/p90 in response to SKi was enhanced by completely eliminating SK1 from HEK293T cells using a combination of SK1 siRNA and SKi.;In addition, sphingolipids measurements showed a decrease in the levels of S1P and increase in the levels of sphingosine under these conditions. However, SK2 or Degs1 had no role in regulating the formation of p63/p90. In addition, the complete elimination of SK1 enhanced the activation of p38 MAPK/JNK pro-survival pathways in response to SKi.;Taken together with the effect on p53, these findings suggest that SK1 opposes pro-survival signalling pathways in HEK293T cells. The proteasome inhibitor, MG132 also induced expression of the pro-survival protein XBP-1s and this was enhanced when HEK293T cells were treated with SKi. SK2 siRNA reduced XBP-1s levels in response to MG132/SKi while p53 siRNA promoted this effect. These findings were achieved using siRNA and transient plasmid transfections, western protein analysis of protein expression, immunoprecipitation, [3H]-Thymidine incorporation assay, mass spectrometry, and immunofluorescence microscopy which suggest that SK2 opposes the death promoting function of p53.;Lastly, the role of SK1 and SK2 in regulating inflammation-based transcriptional factors in keratinocytes was investigated using western protein analysis of protein expression and luciferase reporter assays. SK2 inhibitors, such as ABC294640 or SKi or K145 or (R)-methylether FTY720 (ROMe) were shown to reverse the degradation of inhibitor kappa B (IκB) and transcriptional regulation of nuclear factor kappa B (NF-κB) in response to TNFα. In contrast, the potent SK1 inhibitor, PF-543 did not reverse IκB degradation and only weakly inhibited transcriptional regulation of NF-κB at a concentration that is 50-fold higher than the Ki for inhibition of SK1 activity.;Thus SK2 and not SK1 is proposed to regulate NF-κB signalling and transcription. The effect of the sphingosine kinases on transcriptional regulation of Activator Protein-1 (AP-1) was also investigated. SK2 inhibitors, ABC294640 or K145 reduced phorbol myristate acetate (PMA) stimulated phosphorylation of JNK and ERK-1/2 and transcriptional activity of AP-1. In contrast, other SK inhibitors including PF-543, SKi and ROMe did not inhibit JNK and ERK-1/2 signalling and only produced a minimal reduction in AP-1 transcriptional activity, thereby suggesting that the effects of SK2 inhibitors on JNK/ERK signalling and AP-1 transcriptional activity are likely to be 'off-target'.;These novel significant findings provide improved understanding of the role of Degs1, SK1 and SK2 in regulating cell survival and inflammation and this might ultimately aid in the identification of novel signalling networks and therapeutic targets for treatment of various diseases, including cancer.
Advisor / supervisor
  • Pyne, Nigel
  • Pyne, Susan
Resource Type
Date Created
  • 2020
Former identifier
  • 9912881693302996