Figure 5. Role of PI3K and Akt in apoptotic cell-induced HGF expression.

RAW 264.7 cells were stimulated with apoptotic (A) or viable Jurkat cells (B) for the times indicated. (C) RAW 264.7 cells were pretreated with the PI3K inhibitor wortmannin (Wort) at the indicated concentrations for 1 h and then stimulated with apoptotic Jurkat cells for 15 min. RAW 264.7 cells were pretreated with the PI3K inhibitor and then stimulated with apoptotic Jurkat cells for 2 h to detect HGF mRNA expression (D) or for 24 h to detect secreted HGF (E). (D) HGF mRNA levels were analyzed using semiquantitative RT-PCR and normalized to β-actin mRNA levels. (E) HGF levels in the conditioned medium were measured by ELISA. RAW 264.7 cells were pretreated with C3 transferase for 20 h (F) or a Rho kinase inhibitor Y27632 for 1 h (G) and were transfected with RhoA siRNA or control vehicle (siRNA-GFP) for 24 h (H). (I–L) RAW 264.7 cells were pretreated with the PI3K inhibitor (100 nM wortmannin) or with inhibitors of p38 MAPK, ERK, or JNK (10 μM SB 203580, 30 μM PD 98059, or 30 μM JNK inhibitor II, respectively) for 1 h. RAW cells were then stimulated with apoptotic Jurkat cells for 15 min to detect phosphorylation of MAPKs or Akt. Total cell lysates were immunoblotted for phospho-Akt/Akt, phospho-p38 MAPK/p38 MAPK, phospho-ERK/ERK, or phospho-JNK/JNK. Relative values for phosphorylated kinase versus unphosphorylated kinase are indicated below the gel. Values represent means ± se of three separate experiments; *P < 0.05.