Effect of PDGF BB and TGF B on the time course of FLS mRNA order inhibitor expression In order to determine whether the effect of 2GF on FLS protein secretion was observed at the mRNA expression level, a time course experiment was conducted and the expression of IL6, MIP1, and MMP3 mRNA in FLS was studied. TNF caused a rapid rise in IL6 and MIP1 mRNA expression, Inhibitors,Modulators,Libraries reaching a plateau at one hour and maintaining significant expression until the end of the experiment at 24 h. 2GF alone induced a small amount of IL6 mRNA at three and eight hours, but no MIP1. When 2GF and TNF was added in combina tion, significantly elevated IL6 levels were observed at three and eight hours. For MIP1, potentiation by 2GF of TNF induced chemokine was only observed at three hours. Similar results were obtained for IL8 expression.

In the case of MMP3, TNF alone induced a slow steady increase of mRNA levels evident from three hours and lasting until the end of the experiment Inhibitors,Modulators,Libraries at 24 h. The addition of 2GF in combination with TNF led to significantly elevated MMP3 levels at 8, 16 and 24 h. Thus, the syn ergistic effect of 2GF on TNF induced inflammatory mediator production by FLS is evident at the transcrip tional level. Effect of temporal separation of the addition of growth factors and TNF to FLS Next, the addition of 2GF and TNF was separated in time to determine whether the potentiating effect of 2GF would be maintained. PDGF and TGF B were added at various time points in relation to TNF, which was in turn allowed to stimulate the FLS for 24 h before super natants were analyzed for secreted proteins.

Under these conditions, 2GF was able to potentiate TNF induced IL6, IL8 and MMP3 secretion when added at any time between 2 h and 2 h in Inhibitors,Modulators,Libraries relation to a TNF addition. The extent of the potentiating effect was sim ilar to that observed when 2GF and TNF were added simultaneously. For IL6 and MMP3 secretion, potentiation by 2GF was also observed when added as much as six hours prior to TNF. In similar experiments studying the gene mRNA expression at three hours following TNF addition, 2GF synergistically potentiated TNF induced IL6 expression Inhibitors,Modulators,Libraries when added between 4 h and 2 h in relation to TNF addition. In separate experiments, FLS could be exposed to 2GF for as little as 15 minutes, even when added as early as four hours before TNF, and signifi cantly elevated IL6 expression could still be noted.

This suggests that the synergistic effect does not require continuous exposure to the 2GF, and that it involves signaling pathways that are maintained over the course of several hours. Sustained activation of Erk and Akt in FLS by growth factors For the purpose of elucidating the relevant signaling pathways causing the synergistic effect, FLS were treated with TNF, 2GF, or a combination Inhibitors,Modulators,Libraries for selleck 15 minutes to four hours, and cell extracts analyzed by Western blot.