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Introduction: Intestinal epithelial barrier dysfunction results from a wide variety of pathologic conditions such as infection, trauma, inflammation and malignancy. At the gastrointestinal mucosal layer cells must be capable of maintaining their integrity, and do this through the interplay of multiple active processes including cell proliferation, migration, differentiation, and apoptosis. Previous reports from our lab have shown that Sphingosine-1-phophate (S1P) promotes intestinal epithelial barrier function through regulation of these processes. Sphingosine kinase-1(SphK-1) critically regulates S1P production, as it phosphorylates sphingosine to form S1P, which in turn can function as a second messenger or be secreted extracellularly for autocrine and paracrine effects through signaling via S1P receptors. In the current study we hypothesized that SphK-1 overexpression would increase S1P production and lead to augmented cellular proliferation through enhancement of c-Myc mRNA by Chk2-dependent HuR phosphorylation. Methods: SphK-1 overexpression stable cell lines were selected in rat intestinal epithelial cells (IECs). SphK-1 activity and S1P production were measured by radioactive isotope assay. pGL3-Luc-c-Myc 3’UTR was generated to determine translational efficiency of c-Myc 3’UTR by reporter system. c-Myc protein synthesis was measured by L-[35S] methionine and L-[35S]cysteine incorporation assays. The interaction of HuR and c-Myc mRNA was confirmed by biotin labeled c-Myc 3’UTR pull-down assays. Results: SphK-1 activity and S1P production was increased ~5 fold compared to control vector cells. Cell cycle analysis showed that cell proliferation in SphK1 overexpression cells was enhanced, as G1 to S phase transition was increased versus vector cells. C-myc protein levels were increased by ~ 10 fold. This effect was due to increased translation, as there was no observed change in levels of c-myc mRNA, and silencing c-myc mRNA reversed the effects. Overexpression of SphK-1 promoted the translocation of HuR from nucleus to cytoplasm. HuR phosphorylation was increased by ~12 fold, and this was accompanied similarly by increase (~5 fold) in Chk2 phosphorylation, Chk2 silencing reduced this HuR phosphorylation.Conclusions: Our findings demonstrate that SphK-1 regulates cell proliferation by enhancing c-Myc translation in IECs, the enhanced c-Myc expression was modulated though HuR phosphorylation by Chk2 and provided new insight into the molecular functions of SphK1.