Gene regulation by Sphingosine kinase.

Abstract

Sphingosine kinases (SKs) are lipid kinases that catalyse the phosphorylation of sphingosine to form sphingosine-1-phosphate (S1P), a bioactive phospholipid that plays important roles in a wide variety of cellular processes, including calcium mobilisation, proliferation, apoptosis, angiogenesis, inflammatory responses and cytoskeletal rearrangement. Two SK isoforms exist in mammals, termed SK1 and SK2, which originate from different genes, but possess a high degree of sequence similarity. Although the two enzymes utilise the same substrate, sphingosine, to generate S1P, surprisingly, studies have suggested that SK1 and SK2 may have opposing cellular functions, with SK1 inducing cell survival and SK2 appearing to promote apoptosis. However, the molecular mechanisms mediating these apparently divergent roles for the two SKs have not been extensively examined at present. Furthermore, mouse knockout studies have suggested the two enzymes may have at least some overlapping functions. There is strong evidence implicating SK1 in crucial role(s) in the development and progression of tumourigenesis. However, the mechanism whereby this enzyme induces tumourigenic processes is less clear and remains an important question to be answered in the field. Although high levels of intracellular S1P appears to have a role in regulation of cell proliferation and survival, various observations also suggest a role for extracellular S1P in cell surface G protein-coupled receptor-mediated cell proliferation and survival. However, the specific downstream pathways mediating this oncogenic signalling by SK1 are still poorly defined. In attempts to answer these questions, studies to date have mainly focused on elucidating the cellular signalling pathways that are transiently modulated following SK1 activation. Considerable evidence suggests that SK1 is transcriptionally upregulated in many human cancers and also that its product, S1P, can induce activation of various transcription factors to regulate transcription of other genes. While this type of cellular regulation by SK1 is likely to play an important role in tumourigenesis, no studies have yet been published that systematically examined the molecular mechanisms whereby enhanced SK1 levels lead to oncogenesis. Thus, the main aim of the studies outlined in this thesis was to elucidate the genes regulated by increased cellular SK activity that may be important for normal and pathological cellular regulation. In order to do this, we generated cell lines with tight doxycycline-inducible expression of SK1 and SK2 via a novel approach that involves the incorporation of AU-rich mRNA destabilizing elements (AREs) into the 3’ untranslated regions of the tetracycline-inducible constructs. Use of these tightly controlled SK inducible systems allowed us to perform DNA microarrays and microRNA arrays to elucidate genes and microRNAs regulated soon after a moderate increase in cellular SK levels (approximately 10- and 6-fold overexpression of SK1 and SK2, respectively). This was done to maximise the likelihood of observing direct downstream effects of physiologically relevant increased SK expression that may have been missed by very high constitutive SK expression. While no microRNA regulation was observed following SK1 expression, screening of the Compugen human 19,000-oligonucleotide library, lead to the identification of various genes that were regulated by either SK1 or SK2 or by both enzymes. Of the various SK-regulated genes identified, transferrin receptor 1 (TFR1) was examined in greater detail in this study since its upregulation has been reported in various human cancers, and implicated in tumourigenic progression. Here, we demonstrate a novel mechanism whereby SK1 regulates cell survival, proliferation and neoplastic transformation through upregulation of TFR1 expression. We show that elevated levels of SK1 enhanced total as well as cell-surface TFR1 expression resulting in increased transferrin (Tf) uptake into the cells. We also found that SK1 phosphorylation and/or translocation to the plasma membrane, which have been shown previously to be critical for SK1-mediated oncogenic effects, are necessary for regulation of TFR1 expression. Furthermore, we also demonstrated that S1P receptor 2 (S1P₂) is essential for SK1-induced TFR1 expression through the use of a S1P₂-specific inhibitor and siRNA knock-down of S1P₂. Finally, we show that blocking TFR1 function with a neutralizing antibody attenuated SK1-induced cell proliferation, survival and transformation. Together, these findings suggest that TFR1 plays an important role in oncogenesis mediated by SK1.