Fig. 1.

Among ECM proteins, VTN uniquely activates LIF and IL-6 expression in C6 astroglioma cells. Serum contains a variety and various abundances of integrin-binding ECM proteins. We plated C6 cells for 24 h in 10% serum, then replaced the medium with a low serum formulation (1% v/v; LS) for 24 h, which reduced LIF (A) and IL-6 (B) mRNA expression, as measured by RT-qPCR, relative to that seen in control 10% serum (Ctrl). (C) In the same experiment as in A and B, CNTF mRNA was increased, likely due to inhibitory activity of serum ECMs, such as VTN, on CNTF expression which is negatively regulated by integrin–FAK signaling. In further experiments, we seeded C6 cells and maintained them for 24 h before serum was removed for a further 24 h. Then, VTN was ‘spiked’ into the medium (10 μg/ml concentration), where it rapidly (within 4 h) and robustly induced LIF (D) and IL-6 (E) mRNA relative to what was seen upon addition of PBS control (vehicle, no ECM substrate). Fibronectin (FN1), fibrinogen (FGN), laminin-111 (LM) or collagen-I (COL) addition had no effect. VTN increased LIF (F) and IL-6 (G) in a dose-dependent fashion. NS, no serum; concentrations of VTN are μg/ml. VTN treatment for 4 h caused increased release of LIF (H) and IL-6 (I) protein in conditioned medium, as shown by a dot blot assay (representative of three independent experiments). Plasma from VTN^−/− mice induced LIF (J) and IL-6 (K) gene expression by less than plasma from VTN^+/+ littermates when added for 4 h to C6 cells that had been serum-deprived for 24 h. Data are means±s.e.m. of three or four independent experiments (as denoted in columns). **P<0.01, ***P<0.001.