Chlamydia Pneumoniae Infection Induces Vascular Smooth Muscle Cell Migration By Activating Rac1

ObjectiveUsing the model of rat primary vascular smooth muscle cell (VSMC) infected with Chlamydia pneumoniae (C.pn) in vitro, to observe the role of Rac1activation in C.pn infection-induced VSMC migration, to investigate the possible link between Rac1activation, phosphoinositide3-kinase (PI3K) and IQGAP1in this process, to explore the related molecular mechanisms.Methods1. VSMCs were infected with C.pn in vitro after the culture and propagation of C.pn.2. GST pull-down was performed to detect the activities of Racl in the VSMCs at different time potins after C.pn infection.3. After Rac1activity was inhibited by a Rac1specific inhibitor NSC23766, GST pull-down assay was used to detect the changes in Racl activity in the infected VSMCs; Wound-healing assay and Transwell assay were performed to observe the changes in VSMC migration induced by C.pn infection.4. After PI3K activity was inhibited by a PI3K specific inhibitor LY294002, GST pull-down assay was performed to detect the changes in Racl activity in order to explore the role of PI3K in the Racl activation in the VSMC infected with C.pn.5. After the activity of Racl was inhibited by NSC23766, Western blot was performed to detect the IQGAP1expression in order to explore the role of Racl activation in IQGAP1expression in the VSMC infected with C.pn.Results1. The recombinant plasmid encoding the glutathione S-transferase (GST)-p21binding domain of p21activated kinase1(PBD)(GST-PBD) fusion protein was transformed into the competent bacteria and enough and biologically active fusion protein was induced to combine with GTP-Rac1. VSMCs were infected by C.pn at0h,0.5h,1h,2h,3h,6h,8h,12h and proteins were extracted. GST pull-down results showed that C.pn stimulated the activation of Racl as early as0.5h after the infection and Rac1activity increased gradually to reach a peak at3h post-infection and then began to decrease until8h after infection. No significant changes in the expressions of total Rac1protein were detected during the infection. 2. GST pull-down results showed that when VSMCs were treated with NSC23766, the activaton of Racl was significantly inhibited. The expression of GTP-Racl in the C.pn-infected VSMCs pretreated with NSC23766was decreased compared to C.pn infection group (P<0.05). No significant changes in the expressions of total Racl protein were detected during the infection.3. Wound-healing assay results showed that the migration area of C.pn-infected VSMCs pretreated with NSC23766was significantly smaller than that of C.pn infection group (P<0.05). In the Transwell assay, the infected VSMCs pretreated with NSC23766were found to migrate less than the VSMCs only infected with C.pn (P<0.05).4. GST pull-down results showed that the expression of GTP-Racl induced by C.pn infection significantly decreased after the activity of PI3K was inhibited by LY294002(25μM)(P<0.05). No significant changes in the expressions of total Rac1protein were detected during the infection.5. Western blot results showed that the expression of IQGAP1induced by C.pn infection significantly decreased after the activity of Rac1was inhibited by NSC23766(100μM)(P<0.05) but not NSC23766(50μM)(P>0.05).ConculusionC.pn infection induces VSMCs migration possibly by activating Racl. In this process the Racl activation was dependent on PI3K activity and could influence the expression of IQGAP1.