Figure 4. Activation of p38MAPK in ECFCs induces senescence and p16 expression.

(a) Overexpression of active MKK6 by lentiviral transduction in control ECFCs increases phospho-p38MAPK. Control ECFCs were also transduced by lentivirus expressing empty vector or kinase inactive MKK6. Western blotting of lysates confirmed increased phospho-p38MAPK by active MKK6, but not empty vector or inactive MKK6. Blot is representative of 3 independent experiments. (b) Effect of p38MAPK activation by active MKK6 on senescence in control and GDM ECFCs. Following transduction with either active or inactive MKK6, the percentage of SA-β–gal positive control and GDM ECFCs was assessed. White bars indicate control ECFCs, and black bars indicated GDM ECFCs. Expression of active MKK6 induced significantly increased senescence in both control and GDM ECFCs, by two-way ANOVA (*p<0.05). No differences between control and GDM were found by Sidak’s multiple comparisons. Results shown are from 3 independent experiments. (c) p16^INK4a expression is increased following expression of active MKK6, but not inactive MKK6. RNA was isolated from control and GDM ECFCs overexpressing active or inactive MKK6. Relative mRNA levels of p16^INK4a were quantified by real-time RTPCR, normalizing to HPRT expression. White bars indicate control ECFCs, and black bars indicate GDM ECFCs. Two-way ANOVA identified a significant increase in p16^INK4a mRNA in ECFCs overexpressing active MKK6 compared to ECFCs expressing inactive MKK6 (*p<0.05). No significant difference was found between control and GDM. Results represent 3 different cell lines from both control and GDM groups. (d) Western blotting confirmed increased expression of p16^INK4a with active MKK6 and p38MAPK activation (P-p38MAPK). Total p38MAPK is shown as a loading control. Blots are representative of 3 independent experiments.