Iron Deficiency Increases Phosphorylation of SP1 to Upregulate SPNS2 Expression in Hepatocellular Carcinoma

The sphingosine-1-phosphate (S1P) transporter spinster homolog 2 (SPNS2) promotes tumor progression by modulating tumor immunity and enhancing tumor cells migration and invasion. Previously we found that iron deficiency in hepatocellular carcinoma upregulated SPNS2 expression to increase tumor metastasis. The present study aimed to identify the underlying mechanism of SPNS2 upregulation. Since the mRNA of SPNS2 was significantly increased, we used a transcription factor activity microarray to find the transcription factor responsible for this. The results showed that iron deprivation in hepatoma cells increased the transcriptional activities of 14 transcription factors while only 2 were decreased. Among these, 3 transcription factors, HIF1α, SP1, and YY1, were predicted to bind with the transcription promoter region of SPNS2. But only HIF1α and SP1 transcriptional activities on SPNS2 were increased by iron deficiency, and the increase of SP1 transcriptional activity was stronger than HIF1α. The protein level of HIF1α was increased by iron deficiency, while SP1 was not changed at the protein level but the phosphorylation level was increased. The inhibitor of HIF1α, PX478, and the inhibitor of SP1, Mithramycin A, reversed the increased mRNA and protein expressions of SPNS2 by iron deficiency, with a more significant effect by Mithramycin A. These results provided a comprehensive view of changes in transcriptional activities by iron deficiency and identified that SP1 was the main regulator of iron deficiency-inducing SPNS2 expression in hepatoma cells.