RhoGTPases and Non-Viral Gene Delivery Figure 6. Effect of expression of constitutively active mutants of RhoGTPases on gene transfer for cells cultured on collagen I substrates. (A) mMSCs were cultured on amine functionalized PDMS strips and serum starved overnight before exposing to collagen I coated surfaces for 20 minutes to assess Rho-GTP and 1 hour to assess Rac and Cdc42-GTPases. The cells were subsequently lysed and respective GLISA assays were performed to quantify RhoA, Rac1,2,3 and Cdc42. Statistical analysis was done using the unpaired t-test (two tail p value). (B) mMSCs transiently transfected with constitutively active negative of RhoA, Rac1 or Cdc42 conjugated with GFP were cultured on collagen I coated plates for 16 hours prior to bolus transfection with polyplexes. The transgene expression was analyzed 48 hours post transfection using luciferase assay and normalized with total protein analyzed using Peirce BCA assay. Statistical analysis was done using a one-way Anova followed by the Dunnett Multiple Comparison test. The Anova p value was 0.0203. The symbols *, ** and *** represents a significant change to the level of p,0.05, p,0.01 and p,0.001, respectively. doi:10.1371/journal.pone.0035046.g006 EGF activates Rac and Cdc42, EGF also stimulates macropino- cytosis [39]. Therefore, the role of activated RhoA, Rac1 and Cdc42 was specifically studied by transiently expressing constitu- tively active RhoGTPase mutants. Transfection with constitutively active RhoGTPases resulted in increased amounts of active RhoA, Rac and Cdc42. However, like CP and EGF treatment, the expression of constitutively active RhoA, Rac1 and Cdc42 did not influence transgene expression or uptake for mMSCs plated on fibronectin. These results indicate that a further activation of RhoGTPases does not increase transgene expression in mMSCs cultured on fibronectin-coated dishes. As mentioned we previously observed that gene transfer to mMSCs plated on collagen I was inhibited [6]. Thus, we investigated if the activation of RhoGTPases could rescue the transfection ability of mMSCs plated on collagen I. For mMSCs exposed to collagen I coated dishes, only Rho was slightly activated, while both Rac and cdc42 were inhibited. The activation of Rac1 and Cdc42 with constitutively active genes increased the transgene expression by 4.1 and 6.7 fold, respectively, for cells plated on collagen I coated surfaces. As was the case for cells seeded on fibronectin, the further activation of RhoA by transfection with a constitutively active RhoA gene does not further stimulate gene transfer. On the other hand, Rac and Cdc42 were inhibited on Collagen I coated surfaces, and thus the fact that transfection with constitutively active Cdc42 and Rac1 increased transgene expression indicates that these RhoGT- Pases can promote transgene expression in cells with less active RhoGTPases. The small RhoGTPases employ downstream effectors to mediate their effects on cell morphology, focal adhesion and migration. RhoGTPase binds to serine/threonine kinases [40] namely Rho Kinase, ROK and the related p160ROCK; and elevate their activity. The role of Rho downstream effector ROCK in non-viral gene transfer was also investigated. ROCK inhibition using Y27632 significantly decreased the polyplex internalization and transgene expression. This suggests that cell contractility mediated by ROCK contributes to the regulation of polyplex uptake by RhoGTPase. Besides ROCK, there are other Rho effector molecules like Phosphatidyl inositol 4-phosphate 5-kinase (PIP5-kinase) and p140mDia that also regulate actin polymeriza- tion [41,42] and could play a role in mediating gene transfer and would be interesting to study in future. Besides integrins, syndecans also mediate cell-Fn interactions [43] and activate PKC, which acts upstream to activate RhoGTPases [44] and mediate cell contractility. Syndecan-4 directly recruits PKC-a and facilitates focal adhesion formation on fibronectin [45]. To determine the function of PKC in non-viral gene transfer, it was inhibited using staurosporine. A decrease in transgene expression was seen on PKC inhibition as a resultant of decreased internalization and proliferation. PKC stimulation has been shown to induce clathrin mediated endocytosis of a5b1 [46] and actin dependent uptake of cationic DNA complexes [47]. These results indicate RhoGTPase activity regulated by PKC [44] is required for effective transgene expression. In conclusion, our results indicate that RhoGTPases modulate non-viral gene transfer for mMSCs plated on fibronectin-coated surfaces. Our results show that the engagement of the ECM dictates the RhoGTPase activation level, which in turn dictates the efficiency of transgene expression. Activation of RhoGTPases for cells with inactive RhoGTPases leads to enhanced transgene expression, but activation of RhoGTPases for cells with already active RhoGTPases does not result in further enhancement. Since only RhoA, Rac1 and Cdc42 were studied we cannot offer conclusions about the involvement of RhoGTPases (e.g. RhoB/ C)nin non-viral gene transfer. We believe that a molecular understanding of the process of non-viral gene transfer is necessary to engineer systems that achieve efficient gene delivery. Materials and Methods Cell Culture Mouse bone marrow cloned mesenchymal stem (D1, CRL12424) were purchased from ATCC (Manassas, VA, USA). Cells were maintained in Dulbecco’s modified eagle’s medium PLoS ONE | www.plosone.org 7 April 2012 | Volume 7 | Issue 4 | e35046