Figure 8. Full-length PAI-1 down-regulates FN14 in an LRP1-dependent manner and protects against TWEAK-stimulated apoptosis.

(A) HuCASMCs were treated with 40 nM FL- PAI-1 or CL-PAI-1 for 24 h. Surface FN14 expression was assessed by FACS. Graphed in (A) is a plot of the mean ± SEM; asterisks=p<0.05, n=4. Below the graph is a representative FACS histogram. n/s indicates no statistical significance. (B) PAI-1 induction of pAkt is LRP1-dependent. RASMCs were serum-starved for 48 hours before a 5 minute pretreatment with 5 μg/ml RAP prior to PAI-1 stimulation for 30 minutes. Cells were lysed and pAkt/Erk levels assessed by western blot analysis; pAkt levels were normalized to Erk1 and represented as a mean ± SEM, n=3. (C) LRP1^−/− cells were treated with vehicle alone or 40 nM PAI-1 for 30 minutes; lysates were analyzed by western blot analysis. pAkt levels were normalized to total Erk1 levels. Data is represented as the mean ± SEM, n=3. Asterisks = p <0.05. n/s indicates no statistical significance. (D) RASMCs were incubated with 40 nM R76E-PAI-1 for 24 h, a recombinant PAI-1 mutant deficient in LRP1-binding. FACS was used to quantify surface FN14 expression. Graphed in (D) is a plot of the mean ± SEM; asterisks=p<0.05, n=4. n/s indicates no statistical significance. Below the graph is a representative FACS histogram. (E) HuCASMC were treated with 40 nM FL- PAI-1, CL-PAI-1 or 40 nM R76E-PAI-1 and 250 ng/ml soluble TWEAK, or TWEAK alone, for 6 h. Cells were collected, lysed and an aminomethylcoumarin (AMC)-tagged caspase-3 target (Z-DEVD) added. Fluorescence emission was monitored using a microplate reader every hour for 24 h. Data plotted represents the mean fluorescent intensity over a 24-h period ± SEM; asterisks=p<0.05 using a repeated-measures ANOVA. TPX=tiplaxtinin; FL-PAI-1=full length PAI-1; CL-PAI-1=cleaved PAI-1; R76E-PAI-1=LRP1-binding deficient mutant recombinant PAI-1 protein