We have shown previously that over expression of RGC-32 induced cell cycle activation and progression through S and G2
/M phases (Badea et al., 2002
; Badea et al., 1998
) suggesting an important role of RGC-32 in cell cycle activation. This observation led us to investigate whether endogenous RGC-32 is required for cell cycle activation induced by C5b-9 or other growth factors. To assess the requirement for endogenous RGC-32 in activating cell cycle by C5b-9 and growth factors, we used siRNA to silence RGC-32 expression.
We were able to show for the first time that RGC-32 silencing effectively abolished C5b-9 and growth factors induced cell proliferation. These findings suggest that endogenous RGC-32 is an essential mediator of cell cycle activation induced by growth factors. Our data also show that RGC-32 silencing abolished C5b-9 induced CDC2 activation and suggesting that at least in part the effect on the cell cycle is mediated by down regulation of CDC2 activation. Since RGC-32 binds to and up regulates CDC2 activity we can speculate that RGC-32 is an essential regulator of CDC2 activation. CDC2 is known to be required during mitosis (Sherr and Roberts, 1999
) and accumulating evidence indicates that CDC2 also affects the G1
/S transition and S-phase. In human T-lymphocytes, CDC2 is expressed at G1
/S transition (Furukawa et al., 1990
) and CDC2 phosphorylation is also required for DNA replication (Dutta and Stillman, 1992
). CDC2-cyclin B1 complex, when expressed in nuclei, can phosphorylate S-phase substrates and promote S-phase entry (Moore et al., 2003
). In CDK2 knockout mice, cyclin E-CDC2 complex was found to induce S-phase entry (25).
Despite these evidences, in an inducible tet system we were not able to show that CDC2 plays a role in RGC-32 induced G1/S transition. These data support the model that CDC2 activation requires RGC-32 during mitosis and that both might be required for C5b-9 induced cell cycle activation. In addition CDK2 seems to play an important role in G1/S transition both in AEC and in stable overexpression of RGC-32 in HeLa cells.
C5b-9, like many other growth factors, achieves its pleotrophic biological activities by activating multiple signaling pathways in target cells (Niculescu and Rus, 2001
). Cell proliferation induced by C5b-9 is dependent on the activation of PI3K/Akt/FOXO1 signaling pathway (Fosbrink et al., 2006
). This signaling pathway also regulates the C5b-9 induced release of growth factors, such as FGF, PDGF (Benzaquen et al., 1994
), IL-8 (Rus et al., 1996b
) and PlGF (Fosbrink et al., 2006
). To assess if RGC-32 plays a role in growth factor release, we analyzed their release in the AEC supernatants in response to C5b-9. Interestingly, RGC-32 silencing caused an altered expression profile of growth factors in AEC by decreasing the release of Leptin, PlGF and RANTES, and increasing the release of ENA-78, IL-8, TIMP-1 and VEGF-D. These results clearly indicate that RGC-32 is involved in regulation of C5b-9 induced growth factors release. The fact that RGC-32 can affect PlGF and Leptin release may be significant since PlGF and Leptin induce cell proliferation, migration and neovascularization (Sierra-Honigmann et al., 1998
; Ziche et al., 1997
). In addition, local delivery of PlGF promotes neointima formation in hypercholesterolemic rabbits and macrophage accumulation in atherosclerotic lesions in ApoE-/- mice (Khurana et al., 2005
). Moreover, RANTES can bind to the surface of activated endothelium, where it triggers the firm arrest and transmigration of monocytes under flow conditions, which may be instrumental for its involvement in atherogenic processes, such as neointima formation after arterial injury (Schober et al., 2002
Since the release of many growth factors is Akt dependent, we have tested if the silencing of RGC-32 had any effect on Akt phosphorylation. It is reasonable to speculate that the role-played by RGC-32 in growth factor release is most probably mediated through binding and regulation of Akt phosphorylation. Since Akt and the release of growth factors play important roles in cell cycle activation induced by C5b-9, the inhibition of growth factors by RGC-32 knockdown might be responsible for the inhibition of cell cycle activation.
Our data thus indicate that endogenous RGC-32 plays a critical role in regulating cell cycle activation induced by C5b-9, in part by regulating phosphorylation of multiple cell cycle and signal transduction-related substrates. Further exploration of role played by RGC-32 in vivo is necessary to fully understand its role in cell cycle activation and proliferation.