Abstract
Although the ability of bioactive lipid sphingosine-1-phosphate (S1P) to positively regulate anti-apoptotic/pro-survival responses by binding to S1P1 is well known, the molecular mechanisms remain unclear. Here we demonstrate that expression of S1P1 renders CCL39 lung fibroblasts resistant to apoptosis following growth factor withdrawal. Resistance to apoptosis was associated with attenuated accumulation of pro-apoptotic BH3-only protein Bim. However, although blockade of extracellular signal-regulated kinase (ERK) activation could reverse S1P1-mediated suppression of Bim accumulation, inhibition of caspase-3 cleavage was unaffected. Instead S1P1-mediated inhibition of caspase-3 cleavage was reversed by inhibition of phosphatidylinositol-3-kinase (PI3K) and protein kinase C (PKC), which had no effect on S1P1 regulation of Bim. However, S1P1 suppression of caspase-3 was associated with increased expression of anti-apoptotic protein Mcl-1, the expression of which was also reduced by inhibition of PI3K and PKC. A role for the induction of Mcl-1 in regulating endogenous S1P receptor-dependent pro-survival responses in human umbilical vein endothelial cells was confirmed using S1P receptor agonist FTY720-phosphate (FTY720P). FTY720P induced a transient accumulation of Mcl-1 that was associated with a delayed onset of caspase-3 cleavage following growth factor withdrawal, whereas Mcl-1 knockdown was sufficient to enhance caspase-3 cleavage even in the presence of FTY720P. Consistent with a pro-survival role of S1P1 in disease, analysis of tissue microarrays from ER+ breast cancer patients revealed a significant correlation between S1P1 expression and tumour cell survival. In these tumours, S1P1 expression and cancer cell survival were correlated with increased activation of ERK, but not the PI3K/PKB pathway. In summary, pro-survival/anti-apoptotic signalling from S1P1 is intimately linked to its ability to promote the accumulation of pro-survival protein Mcl-1 and downregulation of pro-apoptotic BH3-only protein Bim via distinct signalling pathways. However, the functional importance of each pathway is dependent on the specific cellular context.
Highlights
We demonstrate that endogenous and recombinant S1P1 is able to suppress levels of proapoptotic Bcl-2 homology domain 3 (BH3)-only protein Bim via a mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK1,2 pathway, and enhance the accumulation of anti-apoptotic protein Mcl-1 via a PI3Kmediated and protein kinase C (PKC)-mediated pathway
Receptors were localised to the cell surface (Figure 1a) and no staining was detectable in control CCL39 cells, confirming the specificity of the immunostaining for S1P1
Treatment of these cells with FTY720P was able to sustain a concentration-dependent activation of the ERK1,2 pathway (Figure 1b) with a pEC50 1⁄4 10.52±0.32 (n 1⁄4 3)
Summary
S1P1 expression enhances survival of CCL39 cells following growth factor withdrawal. As enhanced survival, or a resistance to apoptosis, is a key aspect of. Consistent with previous studies,[21] the appearance of cleaved caspase-3 in growth factor-deprived control CCL39 cells was preceded by the accumulation of pro-apoptotic protein Bim. In parallel with the effect on caspase-3 activation, Bim expression following serum withdrawal was reduced in S1P1-expressing cells. Mcl-1 and endogenous S1P receptor regulation of apoptosis in vascular ECs. To examine the potential significance of Mcl-1 regulation in cells expressing endogenous S1P1, we utilised human umbilical vein ECs (HUVECs) as several studies have demonstrated S1P1 activation of PKC and PI3K pathways in these cells.[32,33,40] Growth factor removal resulted in a time-dependent increase in levels of cleaved caspase-3 that peaked at 3 h and was sustained for up to 6 h (Figure 8a). The ability to evade apoptosis is one of the hallmarks of cancer[41] and increased expression of pro-survival proteins can promote resistance of mammary tumours to chemo- and radiotherapies.[42,43] As enhanced plasma membrane expression of S1P1 in ER þ breast cancer is associated with poor prognosis in patients treated with tamoxifen,[44] we examined
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