Functional Analysis Of Extracelluar S100A14 Protein

S100 belongs to a multigenic family of Ca2+-regulated proteins of the EF-hand type. S100 proteins have been implicated in intracellular and extracellular regulatory activities. It has been investigated that S100 proteins play important roles on the development and progress of various tumors. Our previous study showed that S100A14, a newly discovered member, was significantly deregulated in Esophageal Squamous Cell Carcinoma (ESCC). Up-regulation of S100A14 expression in ESCC tissue was correlated with lymph node metastasis. Overexpression of S100A14 promoted cell motility and invasion in EC9706 cells. To further investigate the role of S100A14 in human esophageal squamous cell carcinoma cells, we explored the extracellular functions of S100A14 with ESCC cell lines.First of all, expression and purification of the human recombinant S100A14 in Escherichia coli were performed. Treatment of KYSE180 cells with low dose of S100A14 stimulated cell proliferation and increased S phase proportion of cells as shown by MTT assay and FACS analysis. Low concentration of S100A14 added to cells also protected cells from injuries induced by Dox and H2O2. Moreover, we found that addition of exogenous S100A14 stimulated both ERK1/2 phosphorylation and NF-κB activity. U0126, an MEK/ERK inhibitor can inhibit the effects.RAGE (receptor for advanced glycation end products), a multiligand receptor of the immunoglobulin superfamily, has been identified as a signal transducing receptor for S100/calgranulin proteins. Pull-down assays were performed to demonstrate the interaction between S100A14 and RAGE receptor, and antagonism of RAGE blocked this interaction. Furthermore, the effects of S100A14 on cell signaling, proliferation, and survival were blocked by interfering with RAGE including addition of AmphP, siRNA molecules for RAGE, and a dominant negative RAGE expression. We can conclude that low dose of exogenous S100A14 activates p-ERK and NF-κB signaling which further stimulates cell proliferation and promotes cell survival via RAGE ligation. Further studies indicated that key amino acid mutations of N-EF hand (E39A, E45A) significantly attenuated low dose of S100A14 induced cell proliferation and p-ERK activation.On the other hand, we found that high dose of S100A14 protein induced cell injury. Further study indicated that S100A14 induced cell apoptosis in the mitochondrial pathway. The cytotoxic activity of S100A14 at relatively high concentration is also RAGE-dependent because cells transfected with RAGE siRNA or RAGE-Δcyto expression significantly inhibited the toxic effects of S100A14 protein. However, other mechanisms or receptors may also exist since inhibition of RAGE function can only block the effects partially. Reactive oxygen species (ROS) production likely contributes to the apoptotic effect of S100A14 protein.Next, we also found that low dose of S100A14 protein promoted cell invasion, up-regulated the expression of MMP-2, but did not change cell motility and wound healing capability. Extracellular S100A14 also stimulated the expression of endogenous S100A14 in a positive feedback manner.Taken together, these data indicate that extracellular S100A14 exerts trophic and toxic effects on ESCC cells depending on its concentration through interacting with RAGE activating different signaling pathways. This work would therefore help us understand more about the biological functions of S100A14 on ESCC and shed new light on the role of S100 proteins in carcinogenesis.