expression to increased renal fibrosis is most likely to involve the central TGF-b1/Smad3 pro-fibrotic signalling pathway. We examined whether this pathway is altered in the setting of reduced or deleted eNOS expression. A clear increase in MRT-67307 web phosphorylation of the SSXS motif in the Smad3 C-terminal domain was evident in the obstructed kidney and presumably plays an important role in the fibrotic response to unilateral ureteric obstruction. However, the mechanism most closely associated with down-regulation of eNOS expression was phosphorylation of the Smad3 linker region. Within 6 hr of ureter obstruction there was increased Smad3 linker region phosphorylation in association with a marked reduction of eNOS expression and this preceded Smad3 C-terminal phosphorylation. Similarly, eNOS-/- mice showed enhanced Smad3 linker region phosphorylation without a change in C-terminal phosphorylation. Evidence of a direct link between these two events came from studies in which conditioned media from endothelial cells cultured in the presence of a nitric oxide synthase inhibitor caused a selective increase in Smad3 linker region phosphorylation in association with increased fibroblast proliferation and collagen production. This is likely to be an indirect action of secreted proteins by the treated endothelial cells since the media was extensively dialysed against a 10 kDa membrane 26148857 to remove L-NAME. The exact components in the L-NAME conditioned media that promoted fibroblast proliferation and collagen production are the focus of ongoing studies. Furthermore, our in vitro studies examining Smad3 driven collagen promoter activity verified that mutation of the phosphorylation sites in the linker region strongly inhibits TGF-b1 induced 21821671 collagen production, while previous studies have shown that mutation of phosphorylation sites in the linker region to glutamic acid augments Smad3 driven collagen production in vitro. Indeed, while increased Smad3 linker phosphorylation has been described in cancer, this study is the first report of increased Smad3 linker region phosphorylation in kidney fibrosis. It also 
provides in vivo and in vitro evidence for a pro-proliferative and profibrotic action of Smad3 linker region phosphorylation, which may help to clarify the contradictory findings described in different cancer cell lines. These findings also substantially extend a previous study showing that aortas from eNOS-/- mice exhibit enhanced basal and TGF-b1 induced collagen type I expression and that endothelial cells from these animals showed increased Smad2/3 phosphorylation. The function of Smad3 depends on extensive interactions with other signalling pathways. While the TGF-b-receptor I directly phosphorylates the SSXS motif in the Smad3 C-terminal domain, a range of other kinases such as mitogen-activated protein kinases, protein kinase B and cyclin-dependent kinases are involved in the phosphorylation of sites in the Smad3 linker region. Thus, there is considerable scope for a range of signalling pathways to positively or negatively regulate Smad3 phosphorylation and thus transcription activity. We identified JNK as a likely candidate for the increase Smad3 linker region phosphorylation in the obstructed kidney based on several findings. First, increased JNK activation was coincident with increased linker region phosphorylation at 6 hr following unilateral ureteric obstruction. Second, the increase in linker phosphorylation in eNOS-/- mice was also associa