Abstract
S100B is a Ca(2+)-binding protein of the EF-hand type that is abundantly expressed in astrocytes and has been implicated in the regulation of several intracellular activities, including proliferation and differentiation. We show here that reducing S100B levels in the astrocytoma cell line GL15 and the Müller cell line MIO-M1 by small interference RNA technique results in a rapid disassembly of stress fibers, collapse of F-actin onto the plasma membrane and reduced migration, and acquisition of a stellate shape. Also, S100B-silenced GL15 and MIO-M1 Müller cells show a higher abundance of glial fibrillary acidic protein filaments, which mark differentiated astrocytes, compared with control cells. These effects are dependent on reduced activation of the phosphatidylinositol 3-kinase (PI3K) downstream effectors, Akt and RhoA, and consequently elevated activity of GSK3beta and Rac1 and decreased activity of the RhoA-associated kinase. Also, rat primary astrocytes transiently down-regulate S100B expression when exposed to the differentiating agent dibutyryl cyclic AMP and re-express S100B at later stages of dibutyryl cyclic AMP-induced differentiation. Moreover, reducing S100B levels results in a remarkably slow resumption of S100B expression, suggesting the S100B might regulate its own expression. Finally, we show that S100B interacts with Src kinase, thereby stimulating the PI3K/Akt and PI3K/RhoA pathways. These results suggest that S100B might contribute to reduce the differentiation potential of cells of the astrocytic lineage and participate in the astrocyte activation process in the case of brain insult and in invasive properties of glioma cells.
Highlights
S100B, a member of a multigenic family of Ca2ϩ-binding protein of the EF-hand type, has been implicated in the regulation of both intracellular and extracellular activities [1,2,3]
We found that inhibition of S100B expression in GL15 cells by small interference RNA techniques resulted in stress fiber disassembly, acquisition of a stellate morphology, reduced migration and, to a lesser extent, proliferation, and GFAP filament formation and that these effects were dependent on reduced activity of the Src kinase/phosphatidylinositol 3-kinase (PI3K) module
As control and S100B small interference RNA (siRNA)-treated cells despite the reduced levinvestigated by Western blotting at post-transfection day 1, lev- els of phosphorylated Akt and GSK3 (Fig. 3, A and D) and the els of phosphorylated (Tyr527) Src were significantly higher in significantly lower S100B levels in S100B siRNA-treated cells
Summary
Cell Culture Conditions and Transfections—GL15 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Invitrogen), 100 IU/ml penicillin G, 100 g/ml streptomycin, 2 mM glutamine. Measurement of Cell Cycle—Cells were washed in PBS, resuspended, centrifuged (400 ϫ g, 7 min), and processed for cell cycle analysis by propidium iodide staining and flow cytometry. For the wound healing assay, control and S100B siRNA-transfected cells were seeded onto glass coverslips at the end of the transfection procedure, grown for 24 h in DMEM containing 10% FBS, scratched with a plastic tip, and cultivated in DMEM. Control and S100B siRNA-treated cells were washed with ice-cold Trisbuffered saline and lysed in cold buffer (50 mM Tris, pH 7.2, containing 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 0.5 M NaCl, 10 mM MgCl2, 10 g/ml each of leupeptin and aprotinin, and 1 mM phenylmethylsulfonyl fluoride).
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