Transforming growth factor 1 (TGF-1) is a potent fibrotic factor responsible for the synthesis of extracellular matrix. TGF-1 acts through the TGF- type I and type II receptors to activate intracellular mediators, such as Smad proteins, the p38 mitogen-activated protein kinase (MAPK), and the extracellular signal-regulated kinase pathway. We expressed the kinase domain of the TGF- type I receptor [activin receptor-like kinase (ALK)5] and the substrate, Smad3, and determined that SB-431542 is a selective inhibitor of Smad3 phosphorylation with an IC 50 of 94 nM. It inhibited TGF-1-induced nuclear Smad3 localization. The p38 mitogen-activated protein kinase inhibitors SB-203580 and SB-202190 also inhibit phosphorylation of Smad3 by ALK5 with IC 50 values of 6 and 3 M, respectively. This suggests that these p38 MAPK inhibitors must be used at concentrations of less than 10 M to selectively address p38 MAPK mechanisms. However, the p38 MAPK inhibitor SB-242235 did not inhibit ALK5. To evaluate the relative contribution of Smad signaling and p38 MAPK signaling in TGF-1-induced matrix production, the effect of SB-431542 was compared with that of SB-242235 in renal epithelial carcinoma A498 cells. All compounds inhibited TGF-1-induced fibronectin (FN) mRNA, indicating that FN synthesis is mediated in part via the p38 MAPK pathway. In contrast, SB-431542, but not the selective p38 MAPK inhibitor SB-242235, inhibited TGF-1-induced collagen I␣1 (col I␣1). These data indicate that some matrix markers that are stimulated by TGF-1 are mediated via the p38 MAPK pathway (i.e., FN), whereas others seem to be activated via ALK5 signaling independent of the p38 MAPK pathway (i.e., col I␣1).
SB-525334 (6-[2-tert-butyl-5-(6-methyl-pyridin-2-yl)-1H-imidazol-4-yl]-quinoxaline) has been characterized as a potent and selective inhibitor of the transforming growth factor-1 (TGF-1) receptor, activin receptor-like kinase (ALK5). The compound inhibited ALK5 kinase activity with an IC 50 of 14.3 nM and was ϳ4-fold less potent as an inhibitor of ALK4 (IC 50 ϭ 58.5 nM). SB-525334 was inactive as an inhibitor of ALK2, ALK3, and ALK6 (IC 50 Ͼ 10,000 nM). In cell-based assays, SB-525334 (1 M) blocked TGF-1-induced phosphorylation and nuclear translocation of Smad2/3 in renal proximal tubule cells and inhibited TGF-1-induced increases in plasminogen activator inhibitor-1 (PAI-1) and procollagen ␣1(I) mRNA expression in A498 renal epithelial carcinoma cells. In view of this profile, SB-525334 was used to investigate the role of TGF-1 in the acute puromycin aminonucleoside (PAN) rat model of renal disease, a model of nephritis-induced renal fibrosis. Orally administered doses of 1, 3, or 10 mg/kg/day SB-525334 for 11 days produced statistically significant reductions in renal PAI-1 mRNA. Also, the compound produced dose-dependent decreases in renal procollagen ␣1(I) and procollagen ␣1(III) mRNA, which reached statistical significance at the 10-mg/kg/day dose when compared with vehicle-treated PAN controls. Furthermore, PAN-induced proteinuria was significantly inhibited at the 10-mg/kg/day dose level. These results provide further evidence for the involvement of TGF-1 in the profibrotic changes that occur in the PAN model and for the first time, demonstrate the ability of a small molecule inhibitor of ALK5 to block several of the markers that are predictive of fibrosis and renal injury in this model.
Embryonic dorsal root ganglion (DRG) neurons die after axonal damage in vivo, and cultured embryonic DRG neurons require exogenous neurotrophic factors that activate the neuroprotective transcription factor nuclear factor-kappaB (NF-kappaB) for survival. In contrast, adult DRG neurons survive permanent axotomy in vivo and in defined culture media devoid of exogenous neurotrophic factors in vitro. Peripheral axotomy in adult rats induces local accumulation of the cytokine tumor necrosis factor alpha (TNFalpha), a potent activator of NF-kappaB activity. We tested the hypothesis that activation of NF-kappaB stimulated by endogenous TNFalpha was required for survival of axotomized adult sensory neurons. Peripheral axotomy of lumbar DRG neurons by sciatic nerve crush induced a very rapid (within 2 h) and significant elevation in NF-kappaB-binding activity. This phenomenon was mimicked in cultured neurons in which there was substantial NF-kappaB nuclear translocation and a significant rise in NF-kappaB DNA-binding activity after plating. Inhibitors of NF-kappaB (SN50 or NF-kappaB decoy DNA) resulted in necrotic cell death of medium to large neurons (> or =40 microm) within 24 h (60 and 75%, respectively), whereas inhibition of p38 and mitogen-activated protein/extracellular signal-regulated kinase did not effect survival. ELISA revealed that these cultures contained TNFalpha, and exposure to an anti-TNFalpha antibody inhibited NF-kappaB DNA-binding activity by approximately 35% and killed approximately 40% of medium to large neurons within 24 h. The results show for the first time that cytokine-mediated activation of NF-kappaB is a component of the signaling pathway responsible for maintenance of adult sensory neuron survival after axon damage.
In this report female and male CD-1 mice were treated with a neurotoxic regimen of methamphetamine (MA) to compare gender differences in striatal dopamine depletion and concordant changes in mRNA markers of the transforming growth factor-β injury response associated with neurodegeneration. Striatal dopamine concentrations of MA-treated female mice were less depleted and significantly greater than that of identically treated males. Associated with this gender difference in striatal dopamine depletion were significantly decreased mRNA levels of plasminogen activator inhibitor-1 and a trend for increased (p = 0.06) mRNA levels of glial fibrillary acidic protein within females. No statistically significant differences between MA-treated female and male mice were obtained in mRNA levels for transforming growth factor-β, transforming growth factor-β type 2 receptor, activin-like kinase-5 or fibronectin. These data demonstrate the presence of changes in two specific molecular markers of the transforming growth factor-β injury response which are in accordance with gender differences in MA-induced striatal dopamine depletion. The results suggest that the neuroprotective advantage displayed by females may in part be related to reductions in the transforming growth factor-β injury response as indicated by decreased mRNA plasminogen activator inhibitor-1 and an increased response of reactive astrocytes which promote neuronal survival as indicated by augmented glial fibrillary acidic protein mRNA levels.
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